NZ735959B2 - Ophthalmic compositions and methods of use therefor - Google Patents

Ophthalmic compositions and methods of use therefor Download PDF

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Publication number
NZ735959B2
NZ735959B2 NZ735959A NZ73595916A NZ735959B2 NZ 735959 B2 NZ735959 B2 NZ 735959B2 NZ 735959 A NZ735959 A NZ 735959A NZ 73595916 A NZ73595916 A NZ 73595916A NZ 735959 B2 NZ735959 B2 NZ 735959B2
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New Zealand
Prior art keywords
corneal
composition
dexamethasone
cornea
eye
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NZ735959A
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NZ735959A (en
Inventor
Colin Green
Carol Ann Greene
Trevor Sherwin
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Auckland Unis Limitedphysical
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Priority claimed from PCT/NZ2016/050033 external-priority patent/WO2016140581A1/en
Publication of NZ735959A publication Critical patent/NZ735959A/en
Publication of NZ735959B2 publication Critical patent/NZ735959B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]

Abstract

The present disclosure encompasses use of a composition for preparing a medicament for treating or preventing a condition associated with a thinning or irregularity of a cornea; or treating or preventing a refractive error associated with a defect of a cornea, the composition comprising: a TGFfi3 polypeptide, and dexamethasone or any salts, esters, or hydrides thereof, and the TGFfi3 polypeptide comprising SEQ ID NO:1, or at least 100 amino acids of the amino acid sequence of SEQ ID NO:1, or sharing at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1. lypeptide, and dexamethasone or any salts, esters, or hydrides thereof, and the TGFfi3 polypeptide comprising SEQ ID NO:1, or at least 100 amino acids of the amino acid sequence of SEQ ID NO:1, or sharing at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1.

Description

OPHTHALMIC COMPOSITIONS AND METHODS OF USE THEREFOR RELATED APPLICATION This application claims the benefit of New Zealand provisional patent application number NZ 705727, filed 5 March 2015, the contents of which are hereby incorporated herein in their ty.
FIELD OF THE INVENTION The present disclosure relates to compositions and methods useful for the treatment and/or prevention of conditions of the eye. In particular, the disclosure relates to compositions and methods that can be used in augmenting and regenerating the cornea, and in correcting refractive errors of the eye.
OUND OF THE ION It was previously believed that differentiated cells relinquished their ability to regress to an r state. However, this View has been challenged by the induction of pluripotent stem cells (cell reprogramming) and evidence showing that differentiated cells can switch to another phenotype (Takahashi & ka 2006; Wemig et a1. 2007; Yarnanaka & Blau 2010; Gurdon & Melton 2008; Peran et a1. 2011). In addition, it is now believed that the microenvironment for cells, which includes the surrounding cells, extracellular matrix, and growth and differentiation factors, plays an important role in bringing about the redirection of cellular differentiation (Hakelien and Collas 2002).
With this information, researchers have begun to p therapeutics that utilise cell reprogramming and stem cell technologies.
The cornea of the eye accounts for more than two-thirds of the eye’s total refractive power ing power). Even small changes in corneal shape can have a dramatic effect on the clarity with which an image is brought to focus on the retina. The stromal layer of the cornea (the clear front surface of the eye) comprises the majority of the corneal tissue and is composed of highly organised lamellae which are made up of y packed collagen fibrils, mostly of collagen types I and V (Marshall et al. 1993).
The unique structure of the stromal layer as a result of the uniform alignment of the collagen fibrils confers the properties of ess and transparency on the cornea (Funderburgh 2000).
When stromal cells (the l keratocytes) are removed from the cornea and cultured in a monolayer they exhibit the logical teristics of fibroblasts W0 2016/140581 and switch from a stellate shaped cell to a multinucleate, fiJsiform shaped cell rburgh et al. 2001). Another commonly observed phenotype of cytes is the myofibroblast form that is seen in the cornea after injury (Jester et al. 1987). Changes in exogenous growth factors and nes are thought to bring about these phenotypic changes (Funderburgh et a1. 2001).
TGFB family of growth factors are known to be the most potent inducers of chondrogenic (cartilage) entiation (Heng, Cao, & Lee 2004; Johnstone et a1. 1998; Menetrey et al. 2000). TGFBI stimulates the sis of ens and fibronectin by chick embryo fibroblasts (Ignotz and Massague 1986). For keratocytes, TGFBl and TGFBZ are known to cause ECM deposition associated with scarring, possibly due to conversion of keratocytes into the myofibroblast phenotype (Funderburgh, Mann, Funderburgh, Corpuz, & Roth 2001). In contrast, TGFB3 has been shown to induce corneal fibroblasts to produce ECM depositions made up of collagen type 1 t fibrosis or scarring (Karamichos, Hutcheon, & Zieske 2011). Certain non—proteinaceous al compounds such as thasone (Johnstone, Hering, Caplan, Goldberg, & Yoo 1998), ascorbic acid (Farquharson, Berry, Barbara Mawer, Seawright, & Whitehead 1998), and ethanol (Kulyk & n 1996) are also known to promote chondrogenic differentiation in vitro.
There are a number of conditions affecting the cornea, including various defects, injuries, diseases, and degenerative conditions. Myopia results from excessive curvature of the cornea so that light entering the eye focuses in front of the retina. It is the most prevalent Vision impairment worldwide affecting the vision of 70 to 90% of people in some Asian countries and 30 to 40% in Europe and the United States (Frederick 2002). In most cases, myopia first occurs in school-age children and progresses until about the age of 20. It is also associated with increased prevalence of macular degeneration, retinal detachment, and glaucoma in adulthood (Ebenstein & Pruitt 2006).
Myopia is most commonly corrected by the use of prescription eye glasses or contact lenses. However, these s do not provide permanent treatment for the condition, and they are unsuitable for use during certain activities. Contact lenses are also ated with ophthalmic infections and more s ions, including corneal abrasions and ulcers. In certain circumstances, refractive surgery or orthokeratology is indicated for myopia. Still, these treatments provide only a temporarily correction for W0 2016/140581 mild to moderate myopia; they are not permanent treatments, and they are unsuitable for SCVCI‘C cases. conus is an ecstatic corneal dystrophy associated with stromal thinning and disruption of the portion of the cornea known as Bowman’s layer. The progressive thinning of the corneal stroma typically occurs over decades and s in the cornea developing a l shape. This results in an impairment of vision due to irregular astigmatism and . The pathogenesis of keratoconus is still unknown but has been associated with factors such as constant eye g and contact lens wear afiachmer, Feder, & Belin 1984; Sherwin & Brookes 2004). It can appear as early as puberty and continues to progress until the third or fourth decade of life.
The incidence of keratoconus has been estimated at approximately 1 in 2000 in the general population worldwide (Rabinowitz 1998), with no predilection for either gender. Since the onset of keratoconus is typically in early adulthood with continuation into prime earning and child-rearing years, the loss of quality of life and the economic burden of the treatment of keratoconus represent a significant public health n.
Keratoconus is a major indication for cornea transplantation in the Western world, determined by chers to constitute 28.8% of corneal transplantation in France (Legeais et a1. 2001) and from 11.4% to 15.4% in the United States (Cosar et a1. 2002; s et a1. 2000). There is an unusually high prevalence of keratoconus in New Zealand, with a disproportionately high incidence in Pasifika and Maori tions (Patel et a1. 2005; Patel & McGhee 2013). In New Zealand, approximately 50% of all corneal transplants performed are for keratoconus (Edwards et a1. 2002).
Despite several studies on keratoconus, the underlying biochemical process s poorly understood. The familial occurrence of conus suggests that one of the aetiological s is genetic (Ihalainen 1985). The condition has also been linked to certain biochemical and biomechanical factors. For example, it has been determined that the corneal thinning of keratoconus is a result of the loss of extracellular matrix (ECM) components. r, this could be due to their destruction, their defective formation, or a combination of these (Klintworth & Damms 1995; Klintworth 1999; Jhanji et a1. 2011). In the l stroma, changes associated with keratoconus include a decrease in the number of lamellae and keratocytes (Ku, Niederer, Patel, Sherwin, & McGhee 2008; Sherwin & Brookes 2004), and changes in organisation of the lamellae and distribution of collagen fibrillary mass (Meek et a1. 2005).
W0 2016/140581 It is thought that the degradation ofthe stromal layer might be due to aberrant proteolytic enzyme activity hi, Yue, Sugar, & Lam 1994). conus corneas are known to have decreased levels of enzyme inhibitors and an increased level of degradative enzymes (Kenney & Brown 2003). Biomechanical factors include thinning and decreased rigidity of the cornea due to oxidative damage caused by ultraviolet radiation and mechanical trauma (Kenney & Brown 2003). Biomechanical investigation of keratoconic corneas has revealed a decrease in elasticity and ess; r the reasons for this remain unknown (Edmund 1988). It has been suggested that a reduction in collagen cross—links could be a cause (Wollensak & Buddecke 1990). Currently there is no satisfactory animal model for keratoconus and investigations have been largely limited to an ex vivo setting.
Depending on the severity of the condition, attempts to slow progression of conus e the use of special spectacles and contact lens. In severe cases, corneal implants, intrastromal rings, or corneal transplants are ary (Jhanj i, Sharma, & Vajpayee 2011). Penetrating keratoplasty, a procedure in which the entire thickness of the cornea is removed and ed by donor corneal tissue, is the most commonly used surgical procedure used to treat advanced cases of conus (Rabinowitz 1998).
Keratoconus is the leading tion for corneal transplantation surgery worldwide, with about 12-20% ofthose affected by conus requiring a corneal transplant (Pramanik, Musch, n, & Farjo 2006).
Early treatment options for keratoconus, such as customised gas permeable lenses known as Rose K lenses, have been focussed on improving visual . Newer treatments aim to slow the progression of the disease. A ent known as corneal collagen cross-linking (CXL) looks at increasing corneal rigidity and biomechanical stability. In this procedure, the epithelium is debrided, topical riboflavin drops are administered, and the s are exposed to ultraviolet—A light at 370 nm for approximately 30 minutes (Ashwin & McDonnell 2010; G. Wollensak, Spoerl, & Seiler 2003). It is believed that the UV—A light activates the riboflavin thereby producing reactive oxygen species that induce the formation ofcovalent bonds between the collagen molecules in the corneal stroma (Spoerl, Huhle, & Seiler 1998; G. Wollensak et a1. 2003).
This ure, however, is not recommended for the treatment of corneas thinner than 400 um due to the possibility of endothelial cell damage. Although this treatment leads to a stiffer cornea, it does not address the problem of corneal thinning.
W0 2016/140581 Therefore, there is an ongoing need for therapeutic compositions and methods for addressing conditions of the eye, including conditions ing the cornea.
There is a particular need for therapies that are relatively non-invasive and readily administered.
SUMMARY OF THE INVENTION The inventors have ped compositions and methodsfor modulating corneal cells, to alter collagen expression and extracellular matrix formation in l tissue. These compositions and methods are useful for regenerating and/or augmenting the cornea, and thereby treating and/or preventing various conditions of the cornea and refractive errors of the eye.
In one aspect, the ion comprises a method of treating or preventing a ion associated with a thinning or irregularity of a cornea, comprising: contacting the cornea with a ition comprising a TGFB3 polypeptide or a variant or fragment thereof, and dexamethasone or derivative thereof or related dal agent, thereby treating or preventing the condition.
In s aspects: The TGFB3 polypeptide consists of the amino acid sequence of SEQ ID NO:1.
The dexamethasone is thasone phosphate.
The composition comprises 10 to 100 ng/ml of the TGFB3 polypeptide.
The composition comprises 40 to 4000 ng/ml dexamethasone.
The composition is formulated as an eye drop.
The composition is formulated with gellan gum.
The composition is administered once daily or twice daily.
The composition is co-administered with one or more additional agents for the eye.
The one or more additional agents for the eye are selected from the group consisting of: anaesthetic agents, anti-inflammatory agents, anti-microbial agents, and lubricants.
The composition is administered in conjunction with use of a t lens, corneal , corneal implant, or intrastromal ring.
W0 2016/140581 The contact lens, corneal insert, corneal t, or intrastromal ring is adapted for moulding or g corneal shape during and/or following treatment with the ition.
The contact lens, corneal insert, corneal implant, or intrastromal 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.
The administration of the composition is prior to and/or subsequent to crosslinking.
The condition is selected from the group consisting of: keratoconus, myopia, and astigmatism.
In an alternative aspect, the method comprises co-administration of a composition comprising the TGFB3 polypeptide or a variant or fragment thereof, and a ition comprising the dexamethasone or derivative thereof or related steroidal agent.
In one r aspect, the invention comprises a method of treating or preventing damage or injury of a cornea, comprising: contacting the cornea with a ition comprising a TGFB3 polypeptide or a variant or fragment thereof, and dexamethasone or tive thereof or related steroidal agent, thereby treating or preventing the damage or injury of the cornea.
In various aspects: The TGFB3 polypeptide consists of the amino acid ce of SEQ ID N021.
The dexamethasone is dexamethasone phosphate.
The ition comprises 10 to 100 ng/ml of the TGFB3 polypeptide.
The composition comprises 40 to 4000 ng/ml dexamethasone.
The composition is formulated as an eye drop.
The composition is formulated with gellan gum.
The composition is administered once daily or twice daily- The composition is co—administered with one or more additional agents for the eye.
W0 2016/140581 The one or more additional agents for the eye are selected from the group consisting of: anaesthetic agents, anti-inflammatory agents, anti-microbial agents, and lubricants.
The composition is administered in conjunction with use of a contact lens corneal insert, corneal implant, or intrastromal ring.
The t lens, l , corneal implant, or intrastromal ring is adapted for moulding or holding corneal shape during and/or following treatment with the composition.
The contact lens, l insert, corneal implant, or intrastromal ring is adapted to act a carrier for the composition or as a composition eluting device.
The damage or injury of the cornea is associated with one or more of: an abrasion, tear, ulcer, burn, puncture, and surgery.
In an alternative aspect, the method comprises co-administration of a composition comprising the TGFB3 polypeptide or a variant or fragment thereof, and a composition sing the dexamethasone or derivative thereof or related steroidal agent.
In yet a fithher aspect, the invention comprises a method of ng or preventing a refractive error of the eye, comprising: ting they eye with a composition sing a TGFB3 polypeptide or a variant or fragment thereof, and dexamethasone or derivative f or related steroidal agent, thereby treating or preventing the refractive error of the eye.
In various aspects: The TGFB3 polypeptide consists of the amino acid sequence of SEQ ID N021.
The dexamethasone is dexamethasone phosphate.
The ition comprises 10 to 100 ng/ml ofthe TGF[33 ptide.
The composition comprises 40 to 4000 ng/ml dexamethasone.
The composition is formulated as an eye drop.
The composition is formulated with gellan gum.
The composition is administered once daily or twice daily.
W0 2016/140581 The composition is co-administered with one or more onal agents for the eye.
The one or more additional agents for the eye are selected from the group consisting of: anaesthetic agents, anti-inflammatory agents, icrobial agents, and lubricants.
The composition is administered for use in conjunction with a contact lens, corneal insert, corneal implant, or intrastromal ring.
The contact lens, corneal insert, corneal implant, or intrastromal ring is d for moulding or holding corneal shape during and/or following treatment with the composition.
The contact lens, corneal insert, corneal implant, or intrastromal ring is adapted to act as a carrier for the composition or as a composition eluting device.
The method is performed preceding or following refractive surgery.
The refractive error of the eye is associated with one or more of: myopia, hyperopia, astigmatism, and presbyopia.
In an alternative aspect, the method comprises co-administration of a composition comprising the TGFB3 ptide or a variant or fragment f, and a composition comprising the dexamethasone or derivative f or related steroidal agent.
In still a timber aspect, the invention encompasses a kit comprising: a composition comprising a TGFB3 ptide or a variant or nt thereof, and dexamethasone or derivative thereof or related steroidal agent; and one or more contact lenses.
In various aspects: The contact lens, corneal , corneal implant, or intrastromal ring is adapted for moulding or holding corneal shape during and/or following treatment with the composition.
The contact lens, l insert, corneal implant, or intrastromal ring act as a carrier for the ition or as a composition eluting device.
The TGFB3 polypeptide ts of the amino acid sequence of SEQ ID NO:1.
W0 40581 The dexamethasone is dexamethasone phosphate.
The composition comprises 10 to 100 ng/ml ofthe TGFB3 polypeptide.
The composition comprises 40 to 4000 ng/ml dexamethasone.
The composition is formulated as an eye drop.
The composition is formulated with gellan gum.
The composition is ated for administration once daily or twice daily.
The composition is co-formulated with one or more additional agents for the eye.
The kit includes one or more additional agents for the eye.
The one or more additional agents for the eye are selected from the group consisting of: anaesthetic agents, nflammatory agents, icrobial agents, and lubricants.
The kit es a contact lens solution.
The kit includes instructions for use.
The kit is used for the treatment or prevention of a refractive error of the eye.
The kit is used for the treatment or prevention of a corneal condition selected from the group ting of: keratoconus, myopia, hyperopia, astigmatism, presbyopia, and stromal dystrophies.
The kit is used for the treatment of a l condition selected from the group consisting of: an abrasion, tear, ulcer, burn, puncture, corneal melt, and surgical injury.
In an alternative aspect, the kit ses as separate components a composition comprising the TGFB3 polypeptide or a variant or fragment thereof, and a composition comprising the dexamethasone or derivative thereof or related steroidal agent.
In even a r aspect, the invention comprises a method of inducing en type II expression in a keratocyte, comprising: contacting the keratocyte with a composition comprising a TGFB3 polypeptide or a variant or nt thereof, and dexarnethasone or derivative thereof or related steroidal agent, thereby inducing collagen type II expression in the keratocyte.
In various aspects: W0 2016/140581 The TGFB3 polypeptide consists of the amino acid sequence of SEQ ID NO:1.
The dexamethasone is dexamethasone phosphate.
The composition comprises 10 to 100 ng/ml of the TGFl33 polypeptide.
The composition comprises 40 to 4000 ng/ml dexamethasone.
The composition is formulated for administration via a contact lens, a corneal insert, a corneal implant, or an intrastromal ring.
The composition is formulated for administration as a solution, gel, cream, or emulsion.
The method is performed in viva.
The method is med ex vivo.
In an alternative aspect, the method comprises co-administration of a composition comprising the TGFB3 polypeptide or a variant or nt thereof, and a composition comprising the dexamethasone or derivative f or related steroidal agent.
The ing brief summary broadly describes the features and technical advantages ofcertain embodiments ofthe present invention. Further technical advantages will be described in the detailed description of the invention and examples that follows.
Novel features that are believed to be characteristic of the invention will be better understood from the detailed description of the invention when considered in connection with any accompanying figures and es. However, the figures and examples provided herein are intended to help illustrate the invention or assist with developing an tandingiofthe invention, and are not intended to limit the invention's scope.
BRIEF DESCRIPTION OF THE GS Figure 1A: Schematic showing myopia caused by an increased curvature of the cornea such that light entering the eye is not focussed onto the retina.
Figure 1B: View of a normal (A) and keratoconic (B) cornea, respectively.
(C): Scheimpflug image in severe keratoconus. cant corneal ng is appreciated in the l cornea.
Figure 2: Organotypic slice culture set up.
W0 40581 Figure 3: Growth factor eye drops led in the eye of adult male Wistar rat.
Figure 4: Phoenix Micron IV in vivo eye imaging system set up specific for imaging rat eyes.
Figure 5: tic of (A): a nanoindenter , (B): a typical load- displacement curve obtained during the indentation s which is used to calculate corneal elasticity and hysteresis. Pmax = maximum load d; hmax = penetration depth; he = contact depth (the height of the contact between the tip and the sample); hf = final depth; S = unloading stiffness.
Figure 6: Nanoindentation rigs designed to hold the human corneal button (A) and the rat globe (B). The central section of the cornea is located by using the microscope, (C), and once located the indenter probe is used, (D).
Figure 7: Corneal keratocytes seeded in chondrogenic differentiation medium. cytes cultured for 3 weeks in ogenic entiation medium containing TGF|33 and dexamethasone formed spheres, which were labelled with: (A) nestin around the periphery of the spheres; (B) collagen type II within the core. The culture medium was then switched to serum containing fibroblast proliferation medium for 1 week, causing cells from the spheres to spread out and populate the dish, (C). Cells in monolayer were negative for type II collagen whereas the cell clusters remained positive for collagen type II, (D).
Figure 8: Corneal keratocytes seeded in serum containing fibroblast proliferating medium. Keratocytes cultured in control fibroblast proliferation medium for 3 weeks were ve for nestin (A), and collagen Type II (B). Confluent asts were then cultured in chondrogenic differentiation medium containing TGFB3 and dexamethasone for 3 weeks, (C). The cells remained negative for collagen type II, (D).
(E): pellet culture of confluent fibroblasts in chondrogenic differentiation . After 3 weeks in culture the cell pellet was sectioned and labelled positive for the keratocyte marker keratocan, (F), and negative for the chondrocyte specific type II collagen, (G).
Figure 9: Human corneal slices 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 slices cultured for 1 week, (B) and (E), and 2 weeks, (C) and (F), in chondrogenic differentiation medium and ed for collagen type II, (B) and (C), and type I, (E) and (F). Strong labelling for type II collagen was seen in corneal slices treated W0 40581 for 2 weeks whereas slices treated for only 1 week were negative for type II collagen.
Slices ed in chondrogenic differentiation medium for both the time periods, although less strongly labelled when compared to the control treated slices, were positive for the native corneal collagen type I.
Figure 10: Human corneal slices cultured for 2 weeks in: (A) control medium and (B) chondrogenic differentiation medium and labelled for collagen type 11. Similar results were obtained as shown by Figure 9. In vivo experiments showing (C): untreated corneas; (D) and (E): treated corneas with a Widespread labelling of type II en in the TGFB3 and dexamethasone treated corneas of rats. er labelling was seen in the anterior ) part of the cornea, (D). Type II collagen appeared fibrillar and was evenly distributed throughout the ECM.
Figure 11: Keratoconic corneal button cultured in vitro in control medium, (A), (C) and (E), and chondrogenic differentiation medium, (B), (D) and (F), for 2 weeks and labelled for collagen type II (A) and (B), and vimentin (C)-(F), respectively.
Compared to the labelling in normal human corneas the labelling of type II en in treated conic corneas (B) was weaker. However, the deposition of type II collagen had a similar pattern to that previously Seen after in vitro and in vivo treatment of normal human and rat corneas. The fibroblast population in the treated half of the keratoconic button (D) and (F) increased in number and the keratocytes appeared healthier and intact with multiple, long cell processes (F), when compared to the untreated half of the keratoconic cornea, (C) and (E).
Figure 12: Ex vivo cultured human keratoconic cornea cultured for 3 weeks in control medium, (A) and (C), and chondrogenic differentiation media, (B) and (D), and ed for alpha smooth muscle actin (OLSMA), (A) and (B), and type III collagen, (C) and (D). There was stronger labelling for aSMA in stromal layer of corneas cultured in l medium (A), when compared to corneas cultured in ogenic entiation medium. Comeas cultured in either of the two media did not label positively for type III collagen.
Figure 13: Corneal arency of in viva treated comeas. Treated, (A) and (C), and untreated, (B) and (D), corneas. After 3 weeks the treated and ted corneas were indistinguishable from each other. The front view of the corneas (A) and (B) reveal a clear comea through which light easily passes to reveal the blood vessels of the back of the eye very clearly. At 8 weeks the in vivo imaging of the cross section of the cornea W0 2016/140581 reveals a clear, transparent cornea through which light easily passes. There were no signs of corneal opacity or scarring.
Figure 14: Quantitative gene expression of en type II (A), and collagen type I (B), in in viva treated corneas. There was an l se in type II collagen expression upon after 1 week of treatment. Upon withdrawal of the treatment there was a marked se in type II collagen expression, (A). Native corneal collagen type I sion was also initially upregulated, however upon long term treatment (up to 7 weeks) its expression was able to the control untreated , (B).
Figure 15: Comparison of 1 week in viva treated and untreated corneas does not reveal a significant difference in hardness (H) and reduced elastic modulus (Er).
Figure 16: Load deformation curves obtained for 3 week in viva d and untreated corneas from two rats. The corresponding graphs with the d values clearly show an increase in elastic modulus (Er) and hardness (H) in the treated corneas.
Figure 17: Comparison of elastic modulus and hardness of 8 week treated and control human keratoconic cornea reveals a marked increase in both parameters in the d cornea.
Figure 18: Reshaping of the cornea in the sheep eye by combining in viva cell reprogramming with a rigid contact lens to hold the desired corneal shape during treatment.
Figure 19: (A) The Phoenix Micron IV in viva eye imaging system. The imaging system enables measurement of corneal thickness, curvature, and transparency.
(B) An OCT attachment enables Visualisation of the anterior eye and measurement of corneal ess and integrity, similar to the image seen here. (C) A nanoindenter and (D) schematic representation of the set-up which will be used to assess l biomechanics ex viva in sheep. Output is shown as a load-displacement curve which can be analysed to obtain Young’s modulus of elasticity, and a measure ofhardness. In large animals such as sheep, corneal thickness (E) is indicated in microns and corneal curvature measurements (F) are obtained using a portable Pentacam®. For corneal curvature (F), widely spaced colour contours indicate a large radius of curvature; narrower contours te areas of r curvature. Numbers indicate the radius of curvature at each point.
Figure 20: Only TGFB3 combined with dexamethasone produces collagen type II deposition. Sheep corneal tissue were cultured in (A) BMP6, (B) BMP6 + W0 2016/140581 hydrocortisone, (C) TGFB3 + ortisone, (D) BMP6 + dexarnethasone, (E) TGFBS + prednisone, (F) TGFfi3 + Triesense®, (G) and (H) TGFB3 + thasone at 20X and 60X magnification, respectively, and labelled for cartilage specific collagen type II.
Figure 21: Dose response study for combinations of TGFB3 and dexamethasone. Sheep corneas were cultured for 3 weeks and labelled for collagen type II.
DETAILED DESCRIPTION OF THE INVENTION The following description sets forth numerous exemplary configurations, parameters, and the like. It should be recognised, however, that such description is not intended as a limitation on the scope of the present invention, but is instead provided as a description of exemplary embodiments.
Definitions In each instance herein, in descriptions, s, embodiments, and examples of the t invention, the terms “comprising’3 LC , including”, etc., are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an ive sense as to opposed to an exclusive sense, that is to say in the sense of “including but not limited to”.
As used herein, “augmenting” refers to s of increasing one or more of the thickness, hardness, elastic modulus, tensile strength, and regularity of the cornea, ing the corneal tissue (e.g., the stromal layer). Augmentation may be used to impose a particular shape to the , i.e., corneal curvature. Augmentation methods may be performed in the presence or absence of a particular condition of the eye, or of the cornea. Augmentation may involve the increase in components in the extracellular matrix of the cornea (e.g., collagen type II). tation may also involve increasing the number of cells (e.g., keratocytes) in the cornea. The number of cells may be increased, for example, by altering the proliferative state of such cells from quiescent to “Co-administration” or “co-administering” refers to the ed use of agents, for example, eutic agents for the eye, and includes the administration of co- formulations (i.e., combination formulations), as well as the simultaneous or sequential administration of te formulations. Similarly, “in conjunction” refers to the combined use ofa therapeutic composition and a therapeutic device/procedure.
This can W0 2016/140581 include use of the composition preceding use of the device/procedure, simultaneously with the device/procedure, and/or following use ofthe device/procedure.
A “condition” of the cornea refers to a state of disease, defect, damage, injury, degeneration, or ction of the cornea. The condition may affect the corneal tissue (e.g., the stromal layer) or corneal cells (e. g., keratocytes). The condition may be ' an acute condition, for example, an abrasion or ulceration, or may be a c condition, for e, keratoconus or myopia.
The “cornea” as used herein refers to the transparent front part ofthe eye that covers the iris, pupil, and anterior chamber of the eye. It includes the corneal epithelium, Bowman’s layer, corneal stroma, Descemet’s membrane, and the corneal epithelium. Of particular interest is the stromal layer (also called the substantia propria) ofthe cornea, which comprises an extracellular matrix of regularly ed collagen fibres along with keratocytes, A “derivative”, as relating to a chemical derivative, refers to a nd that has been chemically modified. The present sure encompasses each of the chemical compounds bed herein as well as any derivatives thereof, including chemically modified forms such as salts, hydrides, esters, and other modifications of the original compound.
“Isolated” as used herein, with particular nce to ptides, refers to a molecule that is separated from its natural environment. An isolated le may be obtained by any method or ation of methods as known and used in the art, including biochemical, recombinant, and synthetic ques. To obtain isolated components, the polypeptides 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 d to, synthetic chemistry, recombinant logy, purification ols, etc. Included are polypeptides isolated from natural, recombinant, or synthetic sources. Also included are polypeptides produced by chemical synthesis, or by plasmids, vectors, or other expression constructs that may be introduced into a cell or cell-free translation system. Such polypeptides are clearly distinguished fiom polypeptides as they naturally occur, without human intervention.
The terms “protein” or “polypeptide” (e.g., SEQ ID N021), and other such terms, for simplicity, refer to the molecules described herein. Such terms are not meant to provide the complete characterization of these molecules. Thus, a protein or W0 2016/140581 polypeptide may be characterised herein as having a particular amino acid sequence, a particular 2—dimensiona1 representation of the ure, but it is understood that the actual le claimed has other features, including 3—dimensional structure, mobility about certain bonds and other properties of the molecule as a whole. It is the les themselves and their properties as a whole that are encompassed by this sure. The terms “protein” and “polypeptide” are used interchangeably herein.
A TGFB3 “polypeptide” refers to ptides obtained from any source, and synthetic e.g., isolated naturally ing polypeptides, recombinant polypeptides, polypeptides, and to include polypeptides having the naturally occurring amino acid and fragments of sequence as well as polypeptides having variant amino acid sequences, such sequences, as described in detail herein. TGFB3 may also be referred to in the art as transforming growth factor-beta3, TGFB3, ARVD, and FL]16571.
Amino acid “sequence identity” refers to the amino acid to amino acid comparison oftwo or more polypeptides. A test sequence may be identical to a reference (i.e., share 100% identity), or may include one or more amino acid sequence substitutions. In preferred aspects, amino acid substitutions may possess r chemical and/or physical properties such as charge or hydrophobicity, as compared to the reference amino acid. Sequence identity may be typically determined by sequence alignments at the regions of highest homology. Sequence alignment algorithms, for example BLAST® in the art. Based on the ce alignment programs, are well known and widely used sequence alignment, the percent identity can be determined between the compared polypeptide sequences.
A ctive error” as used herein, refers to error in the focusing of light by the eye. Refractive errors may e spherical errors and cylindrical errors. Both lower order aberrations and higher order tions are included. Specifically included as refractive errors are the conditions of the eye noted as myopia, hyperopia, astigmatism, anisometropia, and presbyopia.
“Regeneration”, in relation to the cornea, refers to the restoration of one or more of the shape, thickness, regularity, hardness, elastic modulus, and tensile strength of the cornea, including that of the corneal tissue (e.g., the stromal layer). Methods of regeneration may be used to impose a particular shape to the cornea, i.e., l curvature. Regeneration methods may be med in the treatment of a ular ion of the eye, or of the cornea. Regeneration may involve the increase in W0 40581 components in the extracellular matrix ofthe cornea (e.g., collagen type II). Regeneration may also involve increasing the number of cells (e.g., keratocytes) in the cornea. The number of cells may be increased, for example, by altering the proliferative state of such cells from quiescent to active.
“Reprogramming” of cells, for example, for corneal cells (e. g., keratocytes) refers to changes in the state of differentiation. Reprogramming is associated with one or more s in cell morphology, ar gene expression (e.g., collagen expression, ing collagen type I and/or type II expression), or the cells erative state (e.g., quiescent or active).
The term “subject” refers to a human or non-human animal.
“Preventing” refers to stopping or delaying the onset of a condition, for example an eye condition, or particularly a corneal condition, such as a disorder or other defect of the cornea. A preventative measure will result in the stoppage or delay of one or more symptoms of the condition, or a lessening of ms if such do arise.
Prevention of a corneal condition may involve augmenting the cornea, as described in detail herein.
“Treating” refers to reducing, ameliorating, or resolving a condition, for example an eye condition, or particularly a corneal ion, such as a disorder or other defect of the cornea. A treatment will result in the ion, amelioration, or elimination of one or more symptoms of the condition. Treatment of a corneal condition may involve regeneration of the cornea, as detailed herein. The compositions and methods of the invention may be used for treating various conditions, for ting various conditions, or for both treating and preventing various conditions, as described in detail herein.
Cell and tissue regeneration Cell and tissue regeneration technologies hold considerable promise in eutic treatments. As disclosed herein, the inventors have developed compositions and methods for modulating cells using in situ cell reprogramming in order to affect collagen type II expression and extracellular matrix (ECM) deposition in l . This, in turn, is used to strengthen andfor augment the cornea of the eye. The inventors thereby provide a unique approach for the in situ / in vivo regeneration and augmentation ofthe corneal stromal Accordingly, the disclosed methods may be utilised in in vivo tissue engineering therapy for various conditions of the cornea, including myopia and keratoconus. As noted W0 2016/140581 above, myopia is characterised by the excessive curvature of the cornea (Figure 1A), while keratoconus is a progressive ectatic corneal dystrophy leading to a characteristic n of corneal thinning (Figure 1B; image adapted from Romero-Jimenez, Santodomingo-Rubido, & Wolffsohn 2010). l keratocytes are relatively ent and normally only produce large amounts of extracellular matrix (ECM) when they switch to a fibroblast or myofibroblast ype. ECM deposition associated with those ypes y leads to corneal fibrosis and loss of transparency (Kadler, Baldock, Bella, & andford 2007).
Chondrocytes, the cells that make up cartilage, secrete type II collagen which is a fibrillar collagen similar to type I found in the cornea. Type II collagen is also expressed by keratocytes during development ofthe chick cornea and it is only later replaced by type I in the mature chick stroma (Linsenmayer et a1. 1990).
The inventors have previously shown that stromal cells from adult human and rat corneas can be reprogrammed to e neuron specific proteins when treated with al lineage ying growth factors (Greene et a1. 2013). This data demonstrates that an adult cell population can be rarmned simply by the modulation of the growth factor environment both in vitro and in viva.
Now, as demonstrated herein, the inventors show that corneal stromal cells can be d in vitro and ex vivo to produce cartilage specific fibrillar collagen, collagen type II, by treating the cells with transforming growth factor B3 (TGFB3) and dexamethasone (Examples 8 and 9). In particular, the inventors have demonstrated that keratocytes in human keratoconic corneal biopsies express collagen type II when treated with these two compounds (Example 8). In addition, with animal studies, the inventors have demonstrated that the two compounds of TGFB3 and dexamethasone can be delivered in viva using eye drops to stimulate collagen II deposition le 9).
Notably, the deposition of collagen type II was uniform, improving the biomechanics of the cornea, with no fibrosis or scarring, and no effect on corneal transparency (Examples 11 and 13).
Without wishing to be bound by theory, it is esised that the collagen deposition is brought about by the reprogramming of cells within the stroma to a chondrocyte phenotype. It is known that chondrocytes secrete type II collagen which is not only a fibrillar collagen similar to type I found in the cornea, but is also expressed W0 2016/140581 during development of the chick cornea (Linsenmayer et al. 1990). It is only later replaced by collagen type I in the mature stroma (Linsenmayer et al. 1990).
In the results described herein, an initial se in collagen type I expression was observed upon ent of corneal keratocytes with TGFB3 and dexamethasone (Example 12). However, the inventors er that the observed level of en type I deposition would be insufficient to stiffen/reshape a cornea.
Furthermore, the tion of collagen type II is deemed more feasible as a treatment gy. It is noted that collagen type II is less susceptible to enzymatic degradation, for example, by enzymes present in a keratoconic cornea.
In accordance with the inventors’ s, it is le to use the reprogramming ofkeratocytes to e new ECM molecules as an effective treatment to improve the biomechanical characteristics of the cornea. This approach is ered advantageous, as it reduces susceptibility to degradation by corneal enzymes, as noted above. The disclosed treatment module aims not only to stabilise the cornea, but also to provide remedial aid for conditions of the eye, including various corneal conditions and refractive errors ofthe eye. Thus, the methods ofthe invention may be used, for example, for the treatment of keratoconic keratocytes in the ectatic cornea. Additionally, the methods of the invention may be used for the treatment of myopia and various other conditions of the cornea, as described in detail .
Conditions affecting the eye and cornea The itions described herein find particular use in regenerating augmenting the cornea (e.g., the stromal layer), as well as 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 irregularity. In this way, the compositions described herein may be utilised for various conditions affecting the eye, including ions involving corneal defects, disease, damage, injury, and/or degeneration, well as refractive errors of the eye.
In specific aspects, the invention encompasses s for treating defects of the cornea. In certain situations, the methods of the ion may also be used to prevent corneal defects. The defects may be associated with a particular ion of the cornea. ary conditions include keratoconus, as described in detail herein, and related conditions, which include corneal ectasias such as keratoglobus, pellucid marginal degeneration, and posterior keratoconus (see, e.g., Arffa 1997; Krachmer et al.
W0 2016/140581 1984; Rabonitz 2004; Jinabhai et a1. 2010). Specifically included as defects are , presbyopia, and also astigmatisms, which encompass regular and irregular astigmatisms.
Congenital defects of the cornea are also included. Amongst these are cornea plana and microcomea, the latter of which may be associated with fetal alcohol syndrome, Turner syndrome, Ehlers-Danlos syndrome, Weill-Marchesani syndrome, Waardenburg's syndrome, Nance—Horan syndrome, and Cornelia de Lange's syndrome. Included also is globus (mentioned above) that may be associated with Ehlers-Danlos syndrome type IV.
In further aspects, the invention encompasses methods for treating damage or degeneration of the cornea. In certain situations, the methods of the invention may be used to t l damage. Damage or degeneration may be associated with a particular condition of the cornea. Specifically included is corneal melt, for example, corneal melt associated with an inflammatory disorder, such as rheumatoid arthritis.
Other exemplary conditions include keratitis, such as marginal keratitis, stromal keratitis, exposure keratitis, neurotrophic keratitis, tary keratitis, rosacea keratitis, Viral keratifis including herpes keratitis, fiingal keratitis, protozoal keratitis, and other infectious keratitis, such as luetic interstitial keratitis, microsporidial tis, Thygeson's keratitis, and infectious crystalline keratopathy. Included also is ulcerative keratitis, also called peripheral ulcerative keratitis (PUK), which includes ulcerative keratitis that is ated with a systemic disease, such as toid arthritis Wegener's granulomatosis, systemic lupus erythematosus, relapsing ondritis, and polyarteritis nodosa. Endophthalmitis is also ed. Included as well are chronic corneal edema, Mooren's ulcer, dellen, phlyctenulosis, Terrien's ration, Salzman's degeneration, spheroidal degeneration, and Fuch’s phy. Such conditions are well known and well characterised in the art. See, e.g., Jackson 2008; Denniston 2009; and Willshaw et al. 2000. Additionally included are stromal dystrophies, for example, lattice corneal dystrophy (e.g., type I and type 2), granular corneal dystrophy (e.g., type 1 and type 2), macular l dystrophy, Schnyder l dystrophy, congenital stromal corneal dystrophy, and fleck corneal dystrophy.
] In still fiirther aspects, the invention encompasses methods for treating injury to the cornea. Included are injuries due to physical damage, chemical damage, radiation damage, and/or damage from particular medication. Injury may be associated with corneal abrasion, corneal erosion, corneal puncture, ne rupture, corneal scarring, W0 2016/140581 or l ulcers, including melting ulcers, indolent ulcers, and superficial ulcers.
Included also are injuries and other damage associated with eye surgery, including surgical wounds, corneal damage following radial keratectomy, and acute problems following plasty, which include persistent lial defects. Additionally included are injuries associated with corneal melt, for example, corneal melt ing surgery or other treatments of the eye (e.g., topical NSAID administration). l melting may be attributable to infectious, inflammatory, or trophic causes. Included also are injuries and damage of the cornea associated with aging.
In even further aspects, the invention asses methods for treating or preventing refractive errors of the eye. Such refractive errors may be associated with particular conditions, including myopia, hyperopia, presbyopia, anisometropia, higher order aberrations, and various astigmatisms. Higher order aberrations include, but are not limited to, comas, trefoils, quadrafoils, spherical aberrations, and aberrations identified by mathematical expressions (e.g., Zemike polynomials).
Conditions of the cornea may be diagnosed by s methods, including fluorescein staining, which may include a Seidel's test, specular microscopy, corneal topography, isometric tomography, pachymetry, ultrasound, slit lamps, corneal scrapes, and biopsies. Diagnosis may also e assessments for visual acuity and/or opacification. l conditions may be associated with one or more symptoms of: pain, photophobia, foreign body ion, reduced Visual acuity, oedema, white cell infiltrate, fluorescein uptake, vascularisation, redness, and systemic symptoms such as headaches, nausea, and fatigue. rly, symptoms of refractive errors may include but are not d to: reduced visual acuity as well as blurry , double vision, haziness of vision, visual fatigue, foreign body sensation, problematic glare or halos, starburst patterns, ghost images, impaired night Vision, ing, excessive staring, excessive blinking, headaches, eye rubbing, eye strain, eye surface dessication, eye tion, redness, and spasms of the eye.
Therapeutic compositions As noted above, the itions described herein may be ed for treating and/or preventing various conditions of the eye, ing conditions affecting the cornea and refractive errors of the eye. The compositions may include a TGFBS polypeptide, or variants or fragments thereof, along with dexamethasone, or derivatives thereof or related steroidal agents.
W0 2016/140581 In various aspects, the composition may be formulated to include the noted combination ofcomponents (a TGFB3 polypeptide (or variants or fragments thereof) plus dexamethasone (or derivatives for related steroidal agents)), or may be formulated to include a first component (a TGFB3 polypeptide (or ts or fragments thereof) or alternatively dexamethasone (or derivatives thereof or related steroidal agents)) with the second component to be added in prior to administration. atively, the ition may be formulated to include a first component (a TGFB3 polypeptide (or variants or nts thereof) or atively dexamethasone (or derivatives thereof or related steroidal agents)), which is used in simultaneous or sequential administration with a formulation that includes the second component.
In one aspect, the TGFB3 polypeptide may e at least the following amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ KSCK CS (SEQ ID NO:1) (GenBank Reference CAR70088.1). The TGFB3 polypeptide may include at least 112 amino acids shown above, and may have a molecular mass of 25.5 kDa. Alternatively, the TGFBS polypeptide may be derived from amino acids 644—850 (207 amino acids) ofthe precursor polypeptide sequence identified in k Reference CAA33024.1; GenBank Accession No. CAA33024; or NCBI Reference Sequence 230.1.
In other aspects, a TGFB3 variant or fragment may be utilised. For e, the variant or fragment may exhibit at least 75% sequence identity to SEQ ID NO:1, preferably at least 80% identity, more preferably at least 85%, 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 ty to SEQ ID N021, as described herein. It is ofparticular interest where the TGFB3 variant exhibits biological activity, for example, activity that is similar or improved compared to the non—variant polypeptide In further s, a particular fragment may be utilised. For example, the TGFB3 fragment may comprise at least 80 amino acids, at least 85 amino acids of SEQ ID NO:1, more ably 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, most preferably at least 100 amino acids, at least 101 amino acids, at least 102 W0 40581 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 1 11 amino acids ofSEQ ID NO:1. Ofparticular interest are functional fragments of TGFB3, for example, fragments that exhibit biological ty, for example, activity that is similar or improved compared to the reference polypeptide.
In a particular aspect, the TGFB3 polypeptide or the variant or fragment thereof may be provided as a recombinant ptide. For example, the polypeptide may be expressed in cell or cell-free expression systems as widely known and used in the art. Included amongst these are bacterial, fungal, plant, and mammalian expression systems. sion systems using E. coli cells, CHO cells, HEK cells, and Nicotiana benthamiana cells are specifically included. The TGFB3 polypeptide or the variant or fragment thereof may be provided as a human recombinant polypeptide expressed in human or non-human expression systems. The TGFB3 polypeptide or the variant or fragment thereof may be provided as a disulfide—linked homodimeric, non-glycosylated, polypeptide chain, in accordance with known methods.
The TGFB3 polypeptide or the variant or fragment thereof may be ed from recombinant expression systems by standard methods, including well known tographic techniques. The TGFB3 polypeptide or the variant or fragment thereof may include a sequence tag to facilitate cleavage, isolation, and/or localisation of the polypeptide. In accordance with the present invention, the TGFB3 polypeptide may be ed from various commercial sources. For example, recombinant human TGFB3 may be obtained from R&D Systems (Catalogue Nos. 243-B3-002; 243-B3-010), ion, Inc. ogue Nos. 4344-500; 4344-50; 4344-5), or c Protein Specialists (Catalogue Nos. CYT-113; CYT-319).
The biological activity of the TGFB3 polypeptide or the variant or fragment thereof may be measured in accordance with widely known and used methods. For example, biological activity may be measured in e by the polypeptides ability to inhibit the mink lung lial (leLu) cells proliferation (see, e.g., Premaraj et a1. 2006). Exemplary activity by this measurement is shown by an EDso of S 50 ng/ml.
Alternatively, biological activity may be ed by the dose-dependent inhibition of IL—4 induced proliferation of mouse HT—2 cells (BALB/c spleen ted by sheep erythrocytes in the presence of IL-2) (see, e.g., Tsang et al. . Exemplary activity by this measurement is typically 0.1 to 0.5 ng/ml. Alternatively, the composition that W0 2016/140581 2016/050033 includes the combination of agents described herein may be measured for biological activity using the methods noted below. For example, induction of collagen type II (e.g., collagen type II, alpha 1) in keratocytes can be assessed by one or more of: immunohistochemical assays, n assays, Western blot analysis, polymerase chain reaction (PCR) analysis, and quantitative PCR technologies.
As described herein, the composition may also include dexamethasone, a derivative f, and/or a related steroidal agent. Dexamethasone is characterised as having the following chemical structure: The trade names for dexamethasone include, for example, on®, Dexasone®, ®, Hexadrol®, Maxidex®, and Minims®.
In various s, derivatives of dexamethasone may be used, including any esters and salts thereof. Exemplary derivatives include but are not limited to: dexamethasone-l7-acetate (CAS RN: 11773), dexamethasone disodium phosphate (CAS RN: 2392—3 9—4), thasone valerate (CAS RN: 14899-3 6-6), dexamethasone- 21-isonicotinate (CAS RN: 22657), dexamethasone palmitate (CAS RN: 33755 3), dexamethasone propionate (CAS RN: 555415), dexamethasone acefurate (CAS RN: 83 880—70-0), dexamethasone—21-galactoside (CAS RN: 92901—23—0), thasone 21-thiopivalate, dexamethasone 21-thiopentan0ate, dexamethasone 21- thiolmethyl-butanoate, thasone 21—thiolmethyl-butanoate, thasone 21—thiohexanoate, dexamethasone 2l—thiolmethyl—pentanoate, dexamethasone 21— thiol-3,3 -dimethyl-butanoate, dexamethasone 21—thiolethy1—butanoate, dexamethasone 2 l ctan0ate, dexamethasone 2 l -thiolethyl-hexan0ate, dexamethasone 2 1 -thiononanoate, dexamethasone 2 l ecanoate, dexamethasone 2 1 - p-fluorothiobenzoate or a combination thereof. Specifically included are dexamethasone alcohol and dexamethasone sodium phosphate. Dexamethasone derivatives are also included, as described in US 4177268.
W0 2016/140581 The composition may include related steroidal agents, in lieu ofor in on to dexamethasone. For example, other corticoid steroids may be utilised, in replacement ofor along with dexamethasone. Preferred for use as related steroids are Group C steroids according to n classification, which includes thasone—type steroids, such as dexamethasone, thasone sodium phosphate, betamethasone, thasone sodium phosphate, and fluocortolone. Other related steroidal agents include but are not d to: fluoromethalone, lotoprendol, medrysone, prednisolone, prednisone, rimexolone, hydrocortisone, lodoxamide, or any tive or combination thereof.
Specifically included are fluoromethalone acetate, fluoromethalone alcohol, prednisolone acetate, predrn'solone sodium phosphate, lotoprendol etabonate, hydrocortisone e, and lodoxamide tromethamine. It is understood, for any of the chemicals of this sure, that the chemicals may be in various modified forms such as acetate forms, and sodium phosphate forms, sodium salts, and the like.
The composition may include, for example, 0.04 ng/ml to 4 ng/ml; or 0.04 ng/ml to 0.4 ng/ml; or 0.4 ng/ml to 4 ng/ml; or 4 to 40 ng/ml; or 40 ng/ml to 400 ng/ml, or 40 ng/ml to 4000 ng/ml thasone, or derivative f or related steroidal agent; or about 0.04 ng/ml, about 0.08 ng/ml, about 0.12 rig/ml, about 0.4 ng/ml, about 0.8 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 dexamethasone, or derivative thereof or related steroidal agent.
As further examples, the composition may include 0.4 pg/ml to 40 ug/ml; or 0.4 ug/ml to 4 ug/ml; or 4 ug/ml to 40 ug/ml dexamethasone, or derivative thereof or related steroidal agent; or about 0.4 ug/ml, about 0.8 ug/ml, about 1 , about 1-2 ug/ml, about 2 pig/ml, about 4 ug/ml, about 8 ug/ml, about 12 ug/ml, about 20 ug/rnl, or about 40 pg/ml dexamethasone, or derivative thereof or related steroidal agent.
As yet further examples, the composition may include 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; dexamethasone, or derivative thereof 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 dexamethasone, or derivative thereof or related steroidal agent.
W0 2016/140581 The composition may include, for example, 1 ng/ml to 1 pig/ml; or 1 ng/ml to 10 ng/ml; or 10 ng/ml to 100 ng/ml; or 100 ng/ml to l rig/ml TGFB3 ptide or variants or fragments 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 rig/ml TGFB3 polypeptide or variants or fragments thereof. In particular s, the composition may include at least 40 ng/ml dexamethasone, or derivative thereof or related steroidal agent, along with at least 4 ng/ml TGFB3 polypeptide, or variants or fragments thereof.
The composition may also include one or more nflammatory agents.
Exemplary anti-inflammatory agents include, at least, ketotifen fumarate, enac sodium, flurbiprofen sodium, ketorlac trometharnine, suprofen, celecoxib, naproxen, rofecoxib, or any derivative or combination thereof. Particularly included are non- steroidal anti-inflammatory drugs (NSAIDs). The composition may additionally include one or more anaesthetic agents. Exemplary hetics include, at least, topical anaesthetics such as proparacaine, lidocaine, and tetracaine, and any derivative or combination thereof. Other agents for the eye may be selected for inclusion with the ition; these may be chosen by the skilled artisan based on the ion and needs of the subject under treatment.
The compositions as described herein may be formulated for topical administration, as described herein and in accordance with known s. In certain circumstances, intraocular administration may be desirable. The composition may be provided in any form suitable for administration to the eye. Exemplary formulations include, at least, solutions, sions, ons (dispersions), gels, creams, or ointments in a suitable ophthalmic vehicle. For example, the composition may be provided in the form of eye drops, a semisolid gel, or a spray. In certain aspects, moulding contact lenses or other inserts/implants may be impregnated with the composition of the invention. In this manner, the composition can be delivered to the cornea continuously and in a time-release manner as the subject is wearing the contact .
For topical administration to the eye, the itions may be formulated with a pH range of 5.0 to 8.0. This pH range may be achieved by the addition of buffers to the solution. It is preferred that the formulations are stable in buffered solutions. That is, there is no e ction between the buffer and the active agents that would cause the composition to be le, e.g., by precipitation or aggregation. The W0 2016/140581 be hypertonic (5% to 40%, preferably approximately . composition may 10, 20, 30, or 40%) or hypotonic (0% to 5%, preferably approximately 1, 2, 3, or 4%) depending on the needs of the subject (e.g., working needs, rest hours, sleeping, etc.) A hypertonic composition (e.g., 40%) may be used when combined with moulding t lenses, as described in detail herein.
The compositions may e one or more suitable preservatives as optional ingredients. le preservatives may be added to t contamination, for example, bacterial contamination. Such agents may include, but are not limited to, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, EDTA, sorbic acid, Onamer® M, and other agents lmown to those skilled in the art, or any combination thereof. such preservatives may be typically employed at a level of 0.001% to 1.0% by weight of the composition.
] The itions may contain an optional co-solvent. The solubility of the components of the present compositions may be enhanced by a surfactant or other appropriate co—solvent in the composition. Such vents/surfactants include, for example, polysorbate 20, 60, and 80, p0lyoxyethylene/polyoxypropylene surfactants (e.g. Pluronic® F-68, F-84, and P-103), cyclodextrin, tyloxapol, and other agents known to those skilled in the art, and any combination thereof. Such co-solvents may be typically employed at a level of 0.01% to 2% by weight of the composition.
Penetration enhancing agents may be used to se uptake of the composition into the eye. Exemplary agents include, at least, cetylpyridinium chloride, ionophores such as lasalocid, benzalkonium chloride, Parabens, Tween 20, saponins, Brij , Brij 78, Brij 98, ethylenediaminetetraacetic acid, bile salts, and bile acids (such as sodium cholate, sodium taurocholate, sodium eoxycholate, sodium taurodeoxycholate, taurocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid), capric acid, azone, fusidic acid, hexamethylene lauramide, saponins, hexamethylene octanamide, and decylrnethyl sulfoxide.
In addition, bioadhesive polymers may be used to adhere to the mucin coat covering the eye, to prolong contact of the ition with the eye. Bioadhesive polymers may be macromolecular hydrocolloids with numerous hydrophilic functional , such as carboxyl-, hydroxyl-, amide, and sulphate capable of establishing electrostatic interactions. Exemplary agents include, at least, polyarylic acid (e.g., carbopol, hil, and rbophil) and carboxymethyl cellulose.
W0 2016/140581 Controlled release systems may also be used; such systems may involve in situ gels, colloidal particles, nanoparticles, and/or niosomes. Other drug delivery s include but are not limited to: non-erodible ocular inserts, erodible ocular inserts, hydrogels, collagen shields, liposomes, drug-loaded films (e.g., NOD®), and ionotophoresis.
The compositions may e, also, an al agent to increase viscosity.
Viscosity increased above that of simple aqueous solutions may be desirable to increase ocular absorption of the active compounds, to decrease ility in dispensing the formulation, to decrease physical separation of components of a suspension or emulsion of the ation and/or to otherwise improve the ophthalmic formulation. Such Viscosity builder agents e as examples polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, other agents known to those skilled in the art, or a combination thereof. Such agents may be typically employed at a level of 0.01% to 2% by weight of the composition.
In particular aspects, the compositions include a gelling agent, for example, high molecular weight water-soluble polysaccharides such as gellan gum. Gellan gum may be obtained from various commercial sources, for example, as sold under the trade name Kelcogel®. In particular, Kelcogel® LT100 may be used as a fine mesh, high acyl gellan, which forms soft, c, non-brittle gels. In specific aspects, the gellan gum based composition is formulated as a 0.5% eye drop ] Other agents may be used to further ise or otherwise enhance the composition. For example, one or more of EDTA, sodium chloride, tyloxapol, sodium sulfate, and/or hydroxyethylcellulose may have additional beneficial effects of further stabilising the composition.
Therapeutic methods As noted above, the compositions bed herein find particular use in regenerating or augmenting the cornea (e.g., the stromal layer), as well as corneal cells (e.g., cytes). In ular, the compositions may be used to provide enhanced shaping, thickness, regularity, hardness, elastic modulus, e th, or functionality (e. g., refraction) of the cornea. Thus, the compositions described herein may be used to address various conditions of the cornea and correct refractive errors ofthe eye, and may be used as adjunct therapy with other eye treatments.
W0 2016/140581 As previously noted, the composition may be formulated in any suitable means for administration to the eye. Included as formulations are ophthalmic solutions, creams, emulsions, nts, and gels. Specifically noted are formulations that are made as eye drops. In a particular aspect, the composition may be administered as an eye drop using any of the many types of eye drop dispensers on the market. As exemplifications, the container for the compositions ofthe invention may be clear, translucent, and opaque and may n other properties or combination of properties such as being glass lined, tamper proof, packaged in single or few dose aliquots, and any combination thereof.
The composition may be stered in therapeutically effective amounts to a subject to e a desired medical outcome. In particular, the composition may be administered in amounts to address an ophthalmic condition described herein, or at least te one or more symptoms of such condition. The precise dosage ofthe ition (i.e., amount and scheduling) may be determined by a clinician, based on the subject and the condition presented. Exemplary formulations (e. g., eye drops) may be administered 1 to 24 times per day, or 1 to 12 times per day, or 1 to 6 times per day, or 1 to 4 times per day, or 1 to 3 times per day, or 1 to 2 times per day, or 1, 2, 3 4, 6, 8, 12, 18, or 24 times per day. The composition may be topically applied as an eye drop by placing one drop in each eye to be treated. Alternatively, 2 to 3 drops may be applied to each eye.
For the described composition, the dosage range may be, for example, 0.2 pg to 2.4 ng; or 2 pg to 2.4 ng of dexamethasone, or tive f or related steroidal agent; 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 mg, or about 2.4 ng of thasone, or derivative thereof or related steroidal agent, per eye for one dose.
As other examples, the dosage range may be 12 ng to 1.3 ug; 0r 6 ng to 600 ng dexamethasone, or derivative thereof or related steroidal agent; or about 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, about 80 ng, about 90 ng, about 120 11g, 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, W0 2016/140581 about 900 ng, about 1.2 pg, about 1.3 pg of dexamethasone, or derivative thereof or related dal agent, per eye for one dose.
As still other examples, the dosage range may be 1.5 pg to 150 pg; 2.6 pg to 260 pg; or 6.5 pg to 650 pg of dexamethasone, or derivative thereof or related dal agent; or about 1.5 pg, about 2 pg, about 3 pg, about 4.5 pg, about 6 pg, about 6.5 pg, about 7.5 pg, about 10 pg, about 15 pg, about 22.5 pg, about 32.5 pg, about 20 pg, about 26 pg, about 30 pg, about 40 pg, about 45 pg, about 60 pg, about 65 pg, about 75 pg, about 80 pg, about 90 pg, about 100 pg, about 120 pg, about 130 pg, about 150 pg, about 180 pg, about 225 pg, about 240 pg, about 260 pg, about 200 pg, about 300 pg, about 325 pg, about 450 pg, about 600 pg, or about 650 pg dexamethasone, or derivative thereof or related dal agent, per eye for one dose. It will be recognised that specific formulations of dexamethasone are commercially available, and such may be utilised in accordance with accepted dosage amounts and scheduling.
Any of the above noted dosages of dexamethasone may be co-administered with a dosage range of, for example, 5 pg to 65 ng; or 0.5 ng to 65 ng of TGFB3 polypeptide, or variants or fragments thereof; 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 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 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 TGFB3 polypeptide, or variants or fragments thereof, per eye for one dose.
Dosage for one eye may be about one drop ofthe sed composition. One drop of composition may be 10 pl to 200 pl, 20 pl'and 120 pl, or 50 pl to 80 pl or any values in between. For example, dispensers such as pipettors can dispense drops from 1 pl to 300 p1 and any value in between. Preferably, the dispenser metes out about 15 pl, about 20 pl, about 30 pl, about 45 p1, about 60 pl, or about 65 pl per drop ofthe disclosed composition.
Where the composition is administered via a t lens or another /implant , the contact lens or insert/implant may include, for example, 0.01 mg to 10 mg of dexamethasone, or derivative thereof or related steroidal agent; or W0 2016/140581 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 tive thereof or related steroidal agent.
Alternatively, the contact lens or insert/implant may include 10 ng to 100 ng of thasone, or derivative thereof or related steroidal agent; or about 1 ng, about 5 ng, about 10 ng, about 20 ng, about 50 ng, about 80 ng, or about 100 ng dexamethasone, or derivative thereof or related steroidal agent. As further examples, the contact lens or insert/implant may include about 10 ng to 1 ug of TGFB3 polypeptide, or variants or fragments thereof; or about 10 ng, about 50 ng, about 100 ng, about 200 ng, about 500 ng, about 800 ng, or about 1 ug TGFB3 polypeptide, or variants or fragments thereof.
The compositions described herein may be used in conjunction with various al procedures or other treatments. For e, the compositions can be used along with surgical and non-surgical methods for the refractive correction of the eye.
Exemplary methods include but are not limited to: radial keratotomy (RK), including mini asymmetric radial tomy (MARK), hexagonal keratotomy (HK), photorefractive keratectomy (PRK), keratomilleusis, laser in situ keratomileusis (LASIK), e.g., intraLASIK®, laser epithelial keratomileusis (LASEK), e.g., Epi- LASEK, automated lamellar keratoplasty (ALK), laser thermal keratoplasty (LTK), conductive keratoplasty (CK), limbal relaxing incisions (LRI), astigmatic keratotomy (AK), epikeratophakia, anterior ciliary sclerotomy (ACS), scleral reinforcement surgery, presbyopia reversal, laser reversal of presbyopia (LRP), omeal rings (ICR), tromal corneal ring segments (e.g., INTACTS®), implantable contact lenses, scleral expansion bands (SEB), and M . Also included are thermokeratoplasty, orthokeratology, enzyme orthokeratology, and chemical orthokeratology.
The compositions may be used in conjunction with surgical correction of non—refractive conditions, for example, surgical correction of a corneal tear. In particular aspects, the compositions described herein may be used in ction with specific surgical methods performed on the cornea. ary methods include but are not limited to: corneal transplant surgery, ating keratoplasty (PK), phototherapeutic keratectomy (PTK), pterygium excision, corneal tattooing, keratoprosthesis insertion (e.g., KPro or Dohlman-Doane), and osteo-odonto—keratoprosthesis insertion (OOKP).
The compositions may be used in conjunction with corneal collagen crosslinking. Corneal crosslinking lly es the use of riboflavin solution W0 2016/140581 activated by exposure to UV-A light. Noted inking methods include but are not limited to: corneal inking with the epithelium removed (Dresden protocol, or epi- oft), transepithelial crosslinking (epi-on), and accelerated inking. Crosslinking procedures are generally available, and marketed as CXL, C3-R® CCL® and KXL® l crosslinking, amongst . Administration of the composition may be prior to, and/or subsequent to, the crosslinking procedure. It is proposed that the disclosed compositions can be used to avoid or counter the deleterious effects of crosslinking procedures, such as stromal haze and cell loss (described in more detail, below).
Moreover, corneal ration with the disclosed compositions can allow crosslinking to be performed on subjects who were previously ineligible for such procedures, e.g., those with l ess less than 400 um. Furthermore, the disclosed compositions can be used to slow or halt progressive corneal ng, which would not be addressed by the use of inking on its own.
The compositions described herein may be co-administered with one or more additional agents for the eye. In various aspects, co-administration may be by simultaneous or subsequent administration with such agents, or by co-formulation with such agents. Depending on the condition being treated or prevented, the compositions bed herein may be co—administered with one or more agents, which include but are not limited to: antihistamines, ‘sympathomimetics, beta receptor blockers, parasympathomimetics, parasympatholytics, glandins, nutrients, vasoconstrictors, lubricants, anti-microbials, and anaesthetics. Specifically included are various anti— inflammatory agents, including non-steroidal nflammatory drugs (NSAIDs). The compositions may also be co-administered with eye lubricating solutions and tearreplacing solutions.
Non-limiting examples of hetics include: benzocaine, bupivacaine, cocaine, etidocaine, lidocaine, mepivacaine, ine, prilocalne, chloroprocaine, procaine, acaine, ropicaine, and tetracaine. miting examples of anti— inflammatory agents 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, oxaproxin, mefenamic acid, meclofenamic acid, oxicams, piroxicam, tenoxicam, pyrazolidinediones, phenylbutazone, oxyphenthatrazone, pheniramine, antazoline, nabumetone, COX—2 W0 2016/140581 inhibitors rex®), apazone, nimesulide, and zileuton. Glucocorticoids such as ortisone, prednisolone, fluorometholone, and dexamethasone may also be used as anti-inflammatory agents.
Exemplary anti-microbial agents include but are not limited to: acin zinc, mphenicol, chlorotetracycline, ciprofloxacin, omycin, gentamicin, norfloxacin, sulfacetamide, sulfisoxazole, polymyxin B, tetracycline, tobramycin, idoxuridine, trifluridine, vidarabine, acyclovir, foscarnet, ganciclovir, natamycin, amphotericin B, mazole, econazole, fluconazole, ketoconazole, miconazole, flucytosine, clindamycin, pyrimethamine, folinic acid, sulfadiazine, and hoprim— sulfamethoxazole. Exemplary vasoconstrictors include but are net limited to: dipivefrin (Propine®), hrine, phenylephrine, apraclonidine, cocaine, hydroxyamphetamine, naphazoline, tetrahydrozoline, dapiprazole, betaxolol, carteolol, levobunolol, metipranolol, and timolol. Nutrients include vitamins, minerals, and other beneficial agents such as vitamin A, vitamin B1, vitamin B6, vitamin B12, vitamin C (ascorbic acid), vitamin E, vitamin K, and zinc.
] In specific s, the composition bed herein is formulated as eye drops, and such eye drops are used in conjunction with other eye drop formulations. Such other eye drops may include but are not limited to: rinse/lubricating eye drops, dry eye treatments, steroid and antibiotic eye drops, glaucoma eye drops, allergy/anti— inflammatory eye drops, and conjunctivitis eye drops.
The compositions may be used in conjunction with contact lenses, corneal inserts, corneal implants, or intrastromal rings, to assist in supporting or reshaping the subject’s cornea. Included amongst corneal inserts are l inlay and corneal onlay devices. For example, contact lenses, intrastromal rings, or other inserts/implants may be used for moulding or g corneal shape preceding, during, and/or ing treatment with the composition. It is noted that a corneal ‘insert’ typically refers to a temporary device inserted into the cornea, while a corneal ‘implant’ typically refers to a more permanent device. However, many well known devices are described interchangeably in the art as implants/inserts. Therefore, the terms insert/implant” as used herein are not to be deemed as strictly limiting based on time of usage.
The contact lens, corneal insert, l implant, or intrastromal ring may be used with the disclosed composition for treatment of corneal defects, diseases, damage, injury, and/or degeneration, as well as refractive errors of the eye. In various aspects, the W0 2016/140581 contact lens, corneal insert, corneal implant, or intrastromal ring may act as a carrier for the composition or as a composition eluting device. In other s, the contact lens, intrastromal ring, or other corneal insert/implant may be utilised with the composition that is suitable for administration to the eye, e.g., eye drops, as described in detail herein.
In certain aspects, computer software may be used to determine the contact lenses, corneal inserts, corneal implants, or intrastromal rings that are most suitable for the subject and/or to determine the formulationof the composition. In particular aspects, treatment utilising contact lenses, corneal inserts, corneal implants, or intrastromal rings along with the composition described herein is used preceding or following eye surgery, e.g., refractive or lant surgery.
The treatment may'involve assessing the subject (e.g., age, working needs of the t, eye defect or e, etc.), prescribing the use of moulding t lenses, corneal inserts, l implants, or intrastromal rings to assist with the needed changes in the radius of curvature of the or surface of the cornea, and ibing the composition described herein to be used in conjunction with the contact lenses or implants/inserts. The contact lenses or implants/inserts which are prescribed and utilised by the subject can be used exert a mechanical force on the cornea thereby inducing a change in shape, i.e., the refractive power, of the cornea.
In certain preferred aspects, the cornea may be supported or shaped by use of a corneal insert, corneal implant, or tromal ring in conjunction with the disclosed composition. Examples of commercially available devices include INTACS® and KeraRing intrastromal corneal rings. In another aspect, a moulding contact lens may be used in conjunction with the disclosed ition. The contact lens may be hard or rigid, or it may be a soft lens. atively, the contact lens may se both hard and soft portions. If a soft contact lens is used, more positive or negative curvature can be induce in the cornea, and the discomfort in the subject's eyes will diminish as he or she adapts to the contact lenses. If a hard contact lens is used, more mechanical pressure can be exerted on the . The contact lenses may be gas permeable. Moulding contact lenses may be ed from commercial s. Examples of commercially available lenses include, at least, DreamLite, OK Lens, EyeDream, MiracLens, DreamLens, i-GO OVC, GOV, Wake and See, CRT, Fargo/iSee, Emerald and Wave Contact Lens System lenses.
W0 2016/140581 Once the contact lens, corneal insert, corneal implant, or intrastromal ring is placed on/into the eye of the subject, 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 placement ofthe contact lens, l , corneal t, or intrastromal ring. Advantageously, the contact , intrastromal rings, or other inserts/implants and the composition may be used in ction to produce a change in the shape, and thereby the refractive power, of the cornea. The composition may be administered more frequently to allow the cornea to adopt the desired change in shape. In certain s, the composition is administered at least every 24, 12, or 8 hours.
In other aspects, the composition is administered every 6 hours. In certain other aspects, the composition is administered approximately every 3 hours. In yet other aspects, the composition is administered approximately every 2 hours. In still other aspects, the composition is administered every hour.
Without wishing to be bound by any particular theory, the combined use of contact lenses, corneal inserts, corneal implants, or intrastromal rings and the composition described herein may induce changes in the molecular structure of the cornea and may induce changes in the cells and proteins such as collagen (e.g., collagen type II) found in the corneal stroma. The surface of the cornea is thereby made more uniform. By ng irregularities in the surface ofthe cornea, the quality and clearness of all images (i.e., visual acuity) is improved.
For the calculation of the moulding contact lenses the flattest keratometry is taken. One of skill in this art could also use the steeper keratometry or an average ofboth and based on this corneal curvature make the necessary calculations to flatten or steepen the radius of curvature ofthe anterior surface of the cornea and thus correct the refractive defect of the eye. The base curve of the moulding contact lens may be calculated based on the change in the refractive power for each eye separately. In ular aspects, the base curve ofthe moulding contact lens may be calculated starting with one to four flatter or steeper rs, more preferably one to three flatter or steeper diopters, even more preferably one to two flatter or r diopters, ing on the tive error that is required. The peripheral base curve depends on the tion of the moulding contact lens and is calculated to be 0.5 mm of radius greater than the central zone, but can vary depending on the design.
W0 2016/140581 The diameter of the moulding contact lens used in accordance with the invention may be from 8.0 mm to 18.0 mm. Commercially available lenses are produced with such diameters. In certain aspects, the moulding contact lens may be a hard contact lens with a diameter ranging from 8.0 mm to 12.0 mm. In other aspects, the moulding contact lens may be a soft contact lens with a diameter ranging from 13.0 mm to 15.0 mm. Soft contact lenses may cover the entire cemea and go from sclera to sclera. In still other aspects, the moulding contact lens may be sed of hard and soft materials.
The contact lens may be hard in the , out to approximately 12.0 mm, 13.0 mm, 14.0 mm, or 15.0 mm, and then soft in the periphery out to 16.0 mm, 17.0 mm, and 18.0 mm.
A larger contact lens, preferably a soft contact lens, may be used at night as a moulding contact lens.
The power of the moulding contact lenses can be determined to the nearest possible tive power that the subject requires to see tably. During the adaptation process 'with the moulding contact lenses, if the vision is not adequate for the needs of the subject, the subject is prescribed sses while the subject is undergoing treatment. As the cornea is being reshaped or has been reshaped, various optometric measurements may be repeated to confirm that the treatment is progressing as planned and is adequate. Such measurements may include assessment of visual acuity for near and far vision, orthotypes, keratometry measurements, objective and subjective retinoscopy, diagrams of the adaptation of the moulding contact lens, movement of the moulding contact lens, and t of the moulding contact lens.
] After the measurements are taken, changes may be made to the ent program based on these measurements. With each tion, a on may be made whether to continue with the same moulding contact lens or whether a new contact lens should be used. In addition, the same on can be made with regard to the composition being used with the moulding contact lenses. Changes in the moulding contact lenses and/or in the composition can be made to induce the desired ing of the cornea over several weeks. In certain aspects, weekly periodic revisions are performed during the first 8 weeks after beginning ent.
The composition as described herein induces changes in the collagen content of the cornea (e.g., collagen type II). Other aspects of the anatomy, histology, and physiology of the cornea may also be affected by composition. In certain aspects, the composition may be onic or hypotonic to induce changes in l hydration. In W0 2016/140581 other aspects, the composition may be used to change the molecular structure of the cornea (e.g., the extracellular ) and in this way augment or repair the cornea, or reshape the cornea to the desired curvature.
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, keratanse, and stromelysin, which have been shown to work on various proteoglycan components of the cornea.
Included also are the enzyme collagenase, matrix metalloproteinase 1 (interstitial collagenase), and matrix metalloproteinase 2 (gelatinase). Where the composition is co- administered with any such s, it may be ble to include a vehicle such as a polymer (e.g., methylcellulose, polyvinyl alcohol, cellulose, etc.) in the composition to enhance the working of such enzymes. Additional agents may be included to activate metalloproteinase s, e.g., interleukin-la, tumour necrosis factor u/B and any subtypes thereof, monosodium urate drate, 4-amino mercuric acetate, human serum amyloid A, human B2 microglobin, and copper de. Also included may be carbamide (urea). Any combination of these agents may also be used.
The composition may also be co-administered with one or more enzymes that degrade other sugars or proteins found in the cornea. The composition may be co— administered with one or more anaesthetics used to reduce the irritation of the moulding contact lens or any corneal /implant to the cornea. The ition may be cc- administered with one or more lubricants to improve the comfort of the subject during the treatment. In other aspects, the composition may be co-administered with one or more anti-microbial agents such as anti-bacterial, anti-viral, and/or anti-fungal agents. The composition may also be co-administered with one or more vasoconstrictors. The person of skill in the art can determine the appropriate agents for co—administration to the subject based on the condition being treated.
In certain aspects, the composition may be provided in a kit. The kit include one or more of: moulding contact lenses, ating eye drops, cleaning or other ons for the contact lenses, a contact lens ng case, an extra pair of contact lenses, and instructions for wearing the contact lenses and using the ition. The composition provided with the kit may be formulated to include the noted combination of components (a TGFB3 polypeptide (or variants or nts thereof) plus dexamethasone (or derivatives thereofor related steroidal agents», or the kit may include W0 2016/140581 the components as te formulations, to be mixed together prior to administration, or to be administered together, i.e., by simultaneous or sequential stration.
EXAMPLES The examples described herein are provided for the purpose of illustrating c embodiments and aspects of the invention and are not intended to limit the invention in any way. Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments, aspects, and variations without undue experimentation. All such ments, aspects, and variations are considered to be part of this invention.
Example 1: Overview of experiments In previous experiments, the ors have shown that it is possible to direct keratocytes to differentiate down a neuronal lineage. The experiments described herein have aimed to investigate the potential of cytes to switch to a chondrocyte-like cells that secrete cartilage specific collagen type II. This type of cartilage is thought to be expressed during development (Linsenmayer et a1. 1990). A further aim has been to establish whether collagen type II deposition could be induced in vivo in the corneas of live rats and whether this treatment positively affected the optical properties of the corneas. A still further aim has been to determine whether keratocytes in keratoconic tissue could be amenable to this method of cell reprogramming and subsequent production of collagen type II rich ECM.
Finally, the experiments have aimed to evaluate the effect of type II collagen deposition on the biomechanical properties ofthe in vivo and ex vivo treated corneas using nanoindentation testing, a ineering approach that enables analysis of hardness and elastic s.
Example 2: Tissue samples Human tissue Cadaveric whole human corneas, keratoconic corneas ed at the time of lant surgery, human limbal rims and surgeon cut DSEK caps (excess stromal tissue from Descemet's stripping endothelial keratoplasty) were obtained from donors sourced h the New Zealand al Eye Bank (Auckland, New Zealand). Human limbal rims were collected after the central corneal button had been removed for corneal transplantation surgery leaving a 2 mm l margin from the limbal junction. Prior to the use oftissue, research ethics approval and t was obtained fiom the Northern X Regional Human Ethics Committee. All , until use, was stored inNew Zealand Eye Bank medium (2% FCS, 2 mM L-glutamine, 1 x Anti-Anti in Eagles MEM) and W0 2016/140581 transported in New Zealand Eye Bank transport medium (Eye Bank medium supplemented with 5% dextran).
Animal tissue Ethics al for animal s was obtained from the University of Auckland Animal Ethics Committee (application number R856). Eyes and cartilage from 6—8 week old adult male Wistar rats were obtained after euthanisation using a carbon dioxide chamber. The Whole eye was removed from the animal and the cornea was carefully dissected out using surgical scissors with the aid of a dissecting microscope.
The xiphoid process, which is part of the sternum that ns a thin, broad plate of cartilage at its end, was dissected out using a scalpel blade. The animal tissue was washed with povidone-iodine (PVP-I) and sodium thiosulphate. The excess fat and tissue covering the cartilage was scraped away with a blade. Freshly ted eyes and cartilage were stored for a minimal amount of time in phosphate ed saline solution until use.
Example 3: Histological analysis Tissue preparation and cryoseetioning Corneal and cartilage pieces (2 mm x 2 mm) were ed in l Cutting Temperature compound (OCT, Tissue-Tek, Sakura, The Netherlands) before being snap frozen in liquid nitrogen. Sections 10-15 pm thick were cut using a Microm HM550 Cryostat (Thermo-Scientific, USA) and mounted on SuperFrostTM Plus electrostatic slides (Menzel-Glenser, Germany). Cryosections were stored at —20°C until r use.
Cell and Tissue Culture Tissue digestion and cell ation from human and rat corneas Limbal rims were dissected to isolate stroma from sclera in a class II laminar flow hood. Following this, the corneal epithelium and endothelium was gently scraped off with a keratome and discarded. DSEK caps also received gentle scraping with a keratome to remove the epithelium. Remaining stromal tissue was then digested in 0.4% type II collagenase -Aldrich), in Hanks balanced salt solution ®, Life Technologies) at 37°C with gentle mixing on an orbital shaker. A variety of digestion times were used with 5 hours being the time required for optimal tissue digestion and cell Viability.
W0 2016/140581 ] Afier tissue digestion was complete the cells were ed by centrifiiging at 1200 rpm for seven minutes. The cells were then resuspended in a l amount ofan appropriate cell culture medium and counted using a Leica DM IL bench top inverted microscope and a Neubauer hemocytometer. A 1:1 ratio of cell suspension added to trypan blue solution (0.04% trypan blue stock in PBS) was used with a minimum ofthree counts per sample and the average value taken.
Cell culture of corneal keratocytes All cell manipulations were performed in a class II laminar flow hood using aseptic technique. Isolated keratocytes were cultured in either 12 or 24 well cluster plates (Falcon) on plastic or glass coverslips in 2-3 ml of cell culture media. Cells were kept in a humidified incubator at 37°C with 5% C02. Culture media was changed after 24 hours then every two days subsequently or more frequently if required. Cultures were viewed daily with a Leica DM IL bench top inverted microscope. For cell pellet culture, freshly obtained cells after tissue were pelleted by centrifuging at 300 g for 7 minutes at 20°C in a plastic conical tube. Appropriate culture media was added to the tubes. After 24 hours of incubation at 37°C, the cells had contracted and formed a pellet which did not adhere to the walls of the tube. The pellets were cultured in 2 m1 of media in a humidified atmosphere of 5% C02 at 37°C for three weeks. Media was d every other day.
Organotypic slice culture Human and rat corneal and cartilage tissue was thin-sliced (1-2 mm) in an anteroposterior plane with a blade and the slices were placed in an organotypic air-liquid hase culture system (Figure 2). , the explants ofhealthy tissue were ed on 0.4 pm pore size cell culture inserts (Millicell, France) at the interface between culture medium and a C02 rich environment. Corneal sections were placed epithelium side up on cell e plate inserts with 3 m1 of culture medium. The culture media was changed every other day.
Example 3: In vitro reprogramming Culture media Several custom made media were used as bed in the table below.
Table 1: Cell culture media used W0 2016/140581 Fibroblast Dulbecco's Modified Eagle 10% PBS, 1% Anti-anti (1OOX stock), 1% eration Medium (DMEM) (Life GlutaMAXTM (IOOX stock) medium lo ies, GIBCO® ogenic Advanced DMEM (Life 10 ng/ml TGF[33 (Abcam, ab52313), 10-7M reprogramming Technologies, GIBCO®) dexamethasone (Abcam, 43), medium 1% GlutaMAXTM (Life Technologies, GIBCO®) (100X stock), 1% Anti-Anti lOOX stock (Life Technolo ies, GIBCO® Control medium Dulbecco's Modified Eagle 1% GlutaMAXTM (100X stock), 1% Anti- Medium (DMEM) Anti lOOX stock Chondrogenic reprogramming of cytes Tissue slices were cultured in the chondrogenic differentiation medium for varying time intervals to determine the m time required for the growth factor treatment. Samples were collected for each time point (Table 2). For obtaining a monolayer of cells, keratocytes were seeded on glass coverslips at a density of 15 x 104 per cmz. The cells were allowed to attach to the coverslips for 24 hours and culture media was changed every other day. Cultures were maintained for up to 3 weeks.
Table 2: Experimental time points for l tissue slice culture Time point 1 Time point 1 Time point 1 Week 1 Week 2 Week 3 Sample 1 chondrogenic control medium control medium differentiation medium Sample 2 ogenic chondrogenic control medium differentiation medium differentiation medium Sample 3 chondrogenic chondrogenic chondrogenic differentiation medium differentiation medium differentiation medium Sample 4 control medium control medium control medium Example 4: In vivo ramming Gel eye drop formulation for growth factor delivery Eye drops were formulated using gellan gum which is a water soluble polysaccharide produced by the bacterium, Pseudomonas elodea. The use of gel base formulation allows a prolonged corneal residence time and increased ocular bioavailability of the therapeutic agent. Since polymeric gellan gum is an anionic polymer it undergoes in situ gelling in the presence of mono- and divalent cations such as Ca”, Mg“, K”, and Na+ (Bakliwal, & Pawar 2010) . The electrolytes present in the tear fluid cause the gelation of the polymer when it is instilled in the eye and this in turn results in a longer residence time and increased ilability of the drug (Ludwig W0 2016/140581 2005). Based on previous formulation studies, the polymer formulation is a non-irritant and safe for in vivo use (Rupenthal, Green, & Alany 2011).
A 0.5% solution was prepared by first heating distilled water to 80°C followed by the addition of gellan gum (KelcogelTM USA) with nt stirring. Once the powder was completely dissolved, the solution was cooled and stored at 4°C. The appropriate amounts of growth factors were added to the runny gel with constant stirring. A ten times higher concentration of growth factors than that used in the culture medium was used to make for the drug lost through naso—lacrymal drainage and blinking. The eye drop gel included a final concentration of 100 ng/ml TGFB3 and approximately 4 [Lg/ml dexamethasone. ent with neurogenic and chondrogenic factors The animals were manually restrained and approximately 15 uL ofthe eye drops were led in the right eye (Figure 3). The contra lateral eye was used as the control eye.
Thrice daily eye drops were administered for up to 5 days for neuronal specification and for up to 8 weeks for chondrogenic specification.
Example 5: histochemical (IHC) analysis Tissue harvesting and treatment At the end of the ent the animals were euthanised using a carbon dioxide chamber. The eyes were ted and rinsed in phosphate buffered saline. The corneas were then dissected out carefully and fixed in 4% paraforrnaldehyde (PFA) for 1 hour and treated with sucrose solution in order to cryoprotect the tissue before freezing and ning. Sucrose as a cryoprotection is a ant that prevents the formation of ice crystal artefact in frozen tissue sections. In the case of slow ng of the tissue cryoprotection is particularly important.
Briefly, the corneas were inunersed in 20% sucrose on for 5 hours at 4°C and then moved to a 30% sucrose solution and kept at 4°C until the tissue sinks (usually overnight). The corneas were then embedded in OCT nd and immersed in liquid nitrogen to bring about rapid freezing. The frozen blocks of tissue were stored at -80°C until further use. Approximately 10-15 pm thick cryostat sections were mounted on SuperFrostTM Plus slides and the slides were stored at -80°C until needed. In the case of cell cultures, the cells cultured on lips were rinsed with PBS and fixed with 4% PFA for 15 minutes. Coverslips were immersed in PBS until further use.
Immunohistochemistry W0 2016/140581 For tissue cryosections, before carrying out immunohistochemistry, the slides were kept at room temperature for 15-20 minutes. The OCT was washed offusing PBS and the zone around the tissue demarcated using a wax pen. The tissue slices were first incubated with a blocking solution of 10% normal goat serum for 1 hour followed by overnight incubation with the appropriate on of primary antibody at 4°C. The slides were then rinsed three times in PBS before incubation with the appropriate dilution of secondary antibody. The secondary antibody was left on for 2 hours at room temperature. Slices were counterstained with the nuclear marker 4’, 6’—diamidino phenylindol (DAPI) and d in Citifluor antifade agent (ProSciTech, Australia). An s FlroiewTM FV-lOOO confocal laser ng microscope (405 nm, 473 nm, and 559 nm wavelength ) and Leica DMRA fluorescence microscope were used for imaging.
Table 3: Antibodies used Antibod Su lier/Cat. No. Dilution used Prima antibodies Abcam/ab63080 12000 Mouse anti Collaen T ell Milliore/MAB8887 1:200 Mouse anti Colla_en Tpe III Bio_enesis/2150-0081 1:100 Mouse anti Vimentin iima/V6630 1 : 1000 '2Mouse anti0L Smooth muscle actin ovocastra/NCL-SMA 1:100 a dies Goat anti mouse Alexa 568 Molecular orobes®/A-11031 1:500 Goat anti rabbit Alexa 488 Molecular Probes®/A-11034 1:500 Goat anti mouse Alexa 488 Molecular Probes®/A-11001 1:500 e 6: Gene expression analysis RNA isolation and cDNA synthesis The mRNA extraction from samples was carried out using the PureLink® RNA MicroKit (Invitrogen). In brief, tissue s were mixed with 0.75 ml TRIzol® and carrier RNA and homogenised using a hand held homogeniser (PRO Scientific, Inc.).
The samples where 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 and was mixed with ethanol and then transferred to the collection column tube.
] The RNA was collected on the column by centrifuging at 12000 rpm for 1 minute. The flow-through was discarded and the extracted RNA treated with deoxyribonuclease ). The column was washed several times with the buffers W0 2016/140581 provided and the RNA was finally dispersed in ribonuclease (RNAse) free water. The tration was determined using a NanoDrop® (Thermo Scientific) and the mRNA was stored at -80°C.
The cript® VILOTM cDNA Synthesis Kit (InvitrogenTM, Life Technologies) was used to prepare cDNA. Briefly, 100 ng ofRNA was incubated at 25°C for 10 minutes with VILOTM Reaction Mix, SuperScript® Enzyme Mix, and RNAse free water. The samples were then incubated at 42°C for 120 minutes followed by 85°C incubation for 5 s. The cDNA was stored at -20°C.
Quantitative PCR using TaqMan® gene expression assays TaqMan® Gene Expression Assays for the genes of interest were ed.
In the PCR step, 10 uL of TaqMan® Universal Master Mix II was combined with 1 nL of the assay, approximately 25 ng of cDNA and 9 uL water to make up a volume of 20 uL. The tubes were vortexed and centrifuged briefly to spin down the contents. Each cDNA sample was prepared in triplicate and pipetted into a 3 84 well plate. 20 uL of each reaction mixture was loaded into each well of a MicroAmp® Optical 3 84-Well Reaction Plate (Applied Biosystems). The plate was then covered with a mp® Optical Adhesive Film (Applied Biosystems) and the plate was centrifuged briefly to eliminate air bubbles. The plate was transferred to the 7900HT Fast Real—Time PCR System and was run using the following l cycling parameters, 50°C for 2 min, 95°C for minutes followed by 40 cycles of 95°C at 15 sec and 60°C at 1 minute. Results were analysed as described in the previous section.
Table 4: TaqMan® gene assays used for QPCR Gene 5 mbol Gene name Assa 1]) Collal (Rat Collaen, me I, al-ha 1 Rn01463848 ml C012a1 Rat Collaen, e H, al-hal Rn01637085 ml C012a1 Rat Colla_en, eII, alhal Rn01637087 m1 Pop4 (Rat) Ribonuclease P protein subunit p29 Rn02347225_ml housekeeuin _ene en, ‘0611, al ha 1 H800264051 ml CDKN1A (Human) Cyclin—Dependent Kinase Inhibitor 1 H3003 55782_m1 kee in; _ene Example 7: g of biomechanical and optical properties of corneas following in situ stromal ECM protein deposition Examination of or segment (frontal structures) of the rodent eye W0 2016/140581 ] Corneal biomechanics have been shown to be relevant in the diagnosis and treatment of various corneal diseases and provide insight into the structure of the cornea and its relation to corneal physiological function. Comeas that have undergone treatment to bring about the deposition ofECM protein also need to be tested for l opacity as reduced arency would be undesirable.
The Phoenix Micron IV Rodent eye Imaging System (Phoenix Research Labs) was used to examine the corneas of treated rats. Rats were first sedated using an intra—peritoneal injection of ketamine and Domitor® (3:2). The slit-lamp attachment of the Micron IV imaging system was used to examine the layers ofthe cornea in detail and check corneal integrity and arency. Retinal imaging was also done to check corneal transparency. ing imaging, the rats were administered Antisedan® (atipamezole) for reversal of the sedative.
Nanoindentation measurements of in vitro and in vivo treated corneas ] Nanoindentation provides mechanical ements of materials of interest through the application ofultra-small forces perpendicular to the sample plane of interest and measurement of the ant sample indentation (Dias & Ziebarth 2013).
Nanoindentation has recently emerged as a powerful tool for measuring nano- and microscale mechanical properties in tissues and other biomaterials (Ebenstein & Pruitt 2006). The more recent advancement of in situ scanning probe microscopy (SPM) imaging, Where the nanoindenter tip is simultaneously used as a 3D imaging device combined with nanoindentation has enabled a new wave of novel materials research (Dickinson & Schirer 2009). Force, displacement, and time are ed simultaneously while a nanoindentation tip is pushed into the corneal tissue under a controlled load. The forces applied during nanoindentation can be as small as a few nanoNewtons or as large as several Newtons ng a range of size scales to be studied. Nanoindentation tests are output as a load-displacement curve which can be analysed using well defined equations to calculate the mechanical properties relating to rigidity, integrity, and city of the cornea.
Human conic corneas were put into organotypic culture either in control medium or in medium containing the specific ECM protein inducing reprogramming factors. Nanoindentation measurements were then taken at the end ofthe ent time. For the in vivo study, the animals were ly restrained and W0 2016/140581 approximately 15 uL of the gel eye drop formulation containing the ramming used factors were instilled in the right eye of each Wistar rat. The contra lateral eye was for up to seven weeks. as the control eye. Eye drops were administered thrice daily Nanoindentation measurements were ed after week 1, week 3, or week 7 of the treatment period on isolated eyes.
Nanoindentation testing was carried out at the Chemical and Materials Engineering lab at the University of Auckland. In order to test the cornea in its natural position was ed for nanoindentation. Previous studies have used a mould yrene and blue tack to hold the corneas in place. The effect ofthe mould deforming under the load was a potential source of error so a hard mould was decided on for testing.
The first material that was used to create a mould was conventional play dough. This was formed to the exact shape and curvature of human cornea s (Figure 6(A)). The play dough was then left to harden over the next two days before being used in testing.
The testing of the rat eyes was slightly different as the entire globe was used. To hold the globes in place a petri dish filled with a resin and having small indent to hold the globe was used (Figure 6(B)). PBS was used to keep the s from drying out.
Because the s are very soft biological samples a conospherical fluid tip was used for all nanoindentation testing. The indent load used for the human samples was 50 uN. For the rat globes a range of loads between 3 and 5 uN were used. The fibre optic light was switched on and the sample placed ly under the stream of light from the microscope. The central section of the cornea was placed directly in the stream of light as accurately as possible (Figure 6(C)). The sample was focused by adjusting the Z slider until the surface of the cornea could be observed in good resolution. To ensure that the focus was on the central highest point of the cornea sample, the View was moved in the x and y directions to e how the focus changed.
] Once the data collection point was focused on the centre of the cornea, the sample boundary was defined and a quick approach was performed. Before indenting the load function had to be set up correctly. The actual indentation process is automated by the Hysitron Triboindenter® (Figure 6(D)). The pre—defined load was placed on the indenter tip which penetrates the sample until it reaches a defined limit. The tip was then held for 10 seconds before the tip was unloaded from the sample. The hardness of the sample is determined by the area of residual indentation (Ar) after the tip is unloaded.
W0 2016/140581 m Load (P) Hardness =W Where Pmax is the maximum ation load and Area is the contact area of the conospherical tip with the sample. The reduced elastic modulus is a representation of the elastic modulus in both the sample and the indenter tip as shown by the following equation: 1 1 - vi2 1 - va ‘ + Er Ei Em Where 1' referrers to the er and m refers to the sample material. The reduced elastic modulus tells us how elastic a sample is. Because the same indenter tip is used for each test the reduced elastic modulus can be used to compare the city in each sample being tested.
Example 8: Adult human corneal keratocytes produce cartilage specific collagen type II upon treatment with exogenous TGFB3 and dexamethasone It is known that one growth factor may act on several types of cells With similar or varied effects whilst more than one growth factor may share r biological functions. When choosing growth factors, cytokines, and chemicals that might bring about en deposition in the corneal stroma it was important to consider the known effects of certain exogenous factors. In the present experiments, a combination treatment of TGFB3 and dexamethasone was utilised.
] Most ofthe evidence for the effects of TGFB3 and dexamethasone has been obtained by studies done on their effects on stem/progenitor cells (Schuldiner, Yanuka, itz-Eldor, Melton, & Benvenisty 2000; Worster, Nixon, Brower-Toland, & Williams 2000). A combination of TGFB and dexamethasone has been usly used to induce progenitor cells to differentiate into chondrocytes in vitro (Diekman, Rowland, Lennon, Caplan, & Guilak 2009; Johnstoner et al. 1998; Kolambkar, Peister, Soker, Atala, & Guldberg 2007; Winter et a1. 2003). Furthermore, dexamethasone, a synthetic d drug has been used to treat inflammatory eye conditions. Therefore a combination of TGFB3 and dexamethasone was used in the chondrogenic differentiation medium to drive the differentiation of keratocytes s a chondrocyte phenotype.
W0 2016/140581 In the present experiments, the expression of type I and type II en was specifically noted. It is known that fibrillar types of collagen such as types I and II self— assemble and crosslink to form highly crystalline fibres exhibit a very high stiffness, low extensibility and a remarkable elastic energy storage capacity (Wells 2003). It is the crosslinking which contributes towards the stiffness and tensile strength of the fibres.
The corneal stromal extracellular matrix (ECM) is composed of tightly packed heterotypic collagen fibrils made up mostly of collagen types I and V. r to corneal fibrils, cartilage fibrils are heterotypic (made up of types 11 and XI) and have a uniform diameter of 25 nm (slightly smaller than corneal ) (Mendler, ender, n, Winterhalter, & Bruckner 1989). Collagen II is the major fibril component of cartilage and is similar to en I in that the molecule essentially ts of a single uninterrupted helical domain 300 nm in length. Owing to their similarities, collagens II and XI are considered to be the cartilage analogues of collagens I and V (corneal stroma collagens) in other tissues.
In the present experiments, corneal keratocytes from adult corneas were seeded in either the chondrogenic differentiation medium containing TGFB3 and dexamethasone or a standard fibroblast proliferation medium. Within 2-3 days the keratocytes seeded in the chondrogenic differentiation media formed cell ations/spheres (Figure 7(A)) approximately 50—100 pm in diameter. The spheres ed for the chondrocyte c en type II in the central portion and nestin around the periphery (Figure 7(B)). Furthermore, once the s were placed in the fibroblast proliferation media cells from the spheres started spreading outwards (Figure 7(C)) to populate the culture dish thereby forming a cell monolayer. The regions where the cells had once been aggregated labelled for collagen type II whereas the cells in yer did not (Figure 7(D)).
Keratocytes seeded in the fibroblast proliferating medium formed an even monolayer of fibroblast-like cells (Figure 8(A)) which did not label for either nestin or collagen type II (Figure 8(B)). When the media was changed to chondrogenic differentiation medium there were no s in the appearance of the culture and cells remained collagen type 11 negative. These results suggest that cell aggregation appears to be important for cartilage-like ECM production. Keratocytes seeded into fibroblast proliferation medium failed to form the necessary cell aggregations. Therefore, in order to form fibroblast clusters, the confluent fibroblasts were dissociated from the culture W0 2016/140581 dish, ed, and grown as a pellet culture in chondrogenic differentiation medium for a further three weeks. Cell pellets labelled positive for the corneal stroma specific ECM n keratocan but not the cartilage specific ECM protein type II en (Figure 8(F) and (G)).
Example 9: Keratocytes in adult human corneas and adult rat corneas secrete collagen type 11 containing ECM when treated with TGF|33 and dexamethasone Slices of adult human cornea were placed in organotypic slice culture in either control medium or chondrogenic entiation medium for two weeks. The tissue slices were then ed for the ocyte specific ECM n collagen type II and the native corneal collagen type 1. Positive labelling was seen only in the TGFB3 and dexamethasone treated corneas (Figure 9(C) and 10(B)). It was found that a treatment period of two weeks resulted in deposition of type II collagen within the stroma] ECM of treated corneas (Figure 9(C)). Treatment for 1 week did not result in any e deposition of type II collagen in the stromal ECM (Figure 9(B)).
The amount and pattern of the native en type I appeared to be slightly altered in the treated corneas. In general, the intensity of the labelling was similar but the distribution was more extensive and the amount of labelling was higher in the untreated corneas (Figure 9(D)). Furthermore, the newly produced type II collagen was laid evenly and in an ordered fashion in the ECM without g any large masses or aggregates.
The labelling was clearly seen along the pre—existing collagen framework of the l stroma and was distributed across the entire thickness of the stroma] layer.
The in vitro human corneal tissue experiment was then extended to an in viva rodent study wherein the right corneas of male Wistar rats were d for two weeks with a thrice daily stration of 15 ul of a gellan gum based eye drop formulation of TGFl33 and dexamethasone. After two weeks the rats were euthanised and the corneas processed for immunohistochemistry. Only the treated corneas labelled positive for collagen type II with a higher degree of deposition observed in the anterior part of the cornea (Figure 10(D) and (E)). Thus, only corneal slices cultured in the chondrogenic differentiation medium were positive for type II collagen. Furthermore, type II collagen was laid down in uniform layers along the pre-existing collagen framework ofthe stroma.
W0 2016/140581 Example 10: Induction of collagen type II deposition in conic corneas The inventors next looked to confirm that the in viva reprogramming observed in their studies could be utilised in treatments for keratoconus. Experiments were carried out to affirm that keratocytes in conic corneas were amenable to the induction of collagen type II tion. Keratoconic corneal buttons obtained after corneal transplant surgery were placed into culture as soon as they were obtained. Half of each button was put into control medium and the other half placed in chondrogenic differentiation medium and maintained for 2 weeks. After 2 weeks the tissue was processed for either immunohistochemistry or mRNA extraction. The stromal ECM of only the d half of the cornea was ve for type II collagen (Figure 11(B)).
Although the intensity of the ing was lower in keratoconic tissue when compared to normal corneal tissue, the labelling pattern was similar and-followed an ordered arrangement along the backbone of isting collagen lamellae.
Vimentin ing revealed stark differences between keratocytes in the untreated and treated keratoconic corneas. In general the cyte density was lower in the untreated corneas with a scarcity of cells in the posterior part of the cornea (Figure 11(C)). Also, the cytes in treated corneas appeared more tous and complete in logy when compared to keratocytes in untreated corneas (Figure 11(E) and (F)). Keratocytes in treated corneas were longer and had a larger number ofcell processes which labelled strongly for Vimentin when ed to the keratocytes in the untreated corneas.
Example 11: TGFB3 and dexamethasone treatment does not induce deposition of fibrotic proteins or cause corneal opacity Human corneas cultured in the chondrogenic differentiation medium for up to three weeks were labelled for collagen type III and aSMA which are associated with fibrosis and scarring (Gabbiam' 2003; Karamichos et a1. 2012).
There was no evidence of any fibrotic matrix deposition, on the other hand there was a higher degree of aSMA labelling in the l tissue (Figure 12). These results confirm previous findings that, unlike TGFBI and TGFBZ, TGFl33 does not induce the differentiation of corneal keratocytes into myofibroblasts.
Slit lamp examination was performed on the live rats throughout the study period. Upon examination, treated and untreated corneas were indistinguishable with no signs of scarring or opacity. Back of the eye imaging to reveal the blood vessels showed W0 2016/140581 clear corneas which did not obstruct the passage of light (Figure 13(A) and (B)) and in vivo cross section imaging ofthe rat cornea using the Micron IV lens revealed transparent corneas through which light easily passed (Figure 13(C) and (D)). There was no Sign of any corneal y or cloudiness which would lead to the obstruction of light g through the cornea.
Example 12: Change in mRNA expression of collagen type II and type I upon treatment in vivo Rat s which were treated in vivo for 1 week, 7 weeks, and 3 weeks followed by a non-treatment period of 4 weeks were subjected to quantitative gene expression analysis. The aim was to determine whether type II collagen expression decreases again and/or permanently ceases after growth factor treatment is withdrawn.
The effect of the treatment on native corneal collagen type II was also investigated.
] When compared to the 7 weeks treated corneas, the 1 week d corneas expressed very high levels of type II collagen. The expression levels dropped considerably upon withdrawal of the treatment as indicated by the first graph in figure . For type I collagen expression, the 1 week and 7 week d corneas were each compared to their untreated corneas. It was found that there was an l spike in type I Collagen sion after 1 week treatment but by week 7 type I Collagen expression was significantly lower and comparable to its expression in the untreated cornea (Figure 14 (13)).
Example 13: Change in biomechanical properties of in vitro and in vivo treated C0rneas It was hypothesised that the laying down of type II en would affect the stiffness and elasticity of the corneas. In order to evaluate these changes, the in viva rat corneas and ex Vivo treated human corneas and their matching controls were subjected to nanoindentation testing.
When compared to the untreated controls the 1 week in vivo d rat corneas did not have a significant increase in either hardness or elasticity (Figure 15). In the 3 week in vivo treated corneas, there was a clear difference between the treated and control eye. Each of the corneas was tested up to eight times and the resulting load ation graphs obtained showed good reproducibility e 16). In the right eye exposed to the growth factor treatment, both the hardness and reduced elastic modulus were markedly higher. A matched pair toglobus corneas that were cultured ex vivo W0 2016/140581 in either the control medium or the chondrogenic differentiation medium for 6 weeks were also subjected to the same biomechanical testing. Once again, testing revealed a significant increase in hardness and elastic modulus in the treated cornea (Figure 17).
Example 14: Comparative combinations of growth factors and steroids An ex vivo study on sheep corneas was carried out in order to investigate the efficacy of other growth factor-steroid combinations in chondrogenic entiation of corneal keratocytes.
Fresh sheep eyes were obtained from Auckland Meat Processors. The corneas were immediately excised and washed with povidone-iodine (PVP-I) and sodium thiosulphate Solution. Then, 8 mm discs of sheep corneal tissue were cut using a trephine.
One sheep corneal disc was placed in each of the culture conditions (outlined in Table 5) for 3 weeks. The corneal discs were then placed in an organotypic air-liquid interphase culture system. , the explants of healthy tissue were cultured on 0.4 pm pore size cell culture inserts cell, France) at the interface between culture medium and a C02 rich environment. Corneal sections were placed epithelium side up on cell culture plate s with 3 ml of e . The culture media was changed every other day. The basal medium used was co's Modified Eagle Medium (DMEM) supplemented with 1% Anti-Anti (antibiotic-antimycotic solution) and 1% GlutaMAXTM (GIBCO®). At the end of 3 weeks, each corneal disc was fixed in 4% paraformaldehyde (PFA) for 1 hour and treated with sucrose solution in order to cryoprotect the tissue before freezing and sectioning.
] In brief, the corneas were immersed in 20% sucrose solution for 5 hours at 4°C and then moved to a 30% sucrose solution and kept at 4°C until the tissue sank (usually overnight). The corneas were then embedded in OCT (optimal cutting temperature) compound and immersed in liquid nitrogen to bring about rapid freezing.
The frozen blocks oftissue were stored at -80 °C until further use. imately 4—6 40 um thick cryostat ns were mounted on rostTM Plus slides and the slides were stored at —80 °C until needed. The corneal sections were then labelled for collagen type For immunohistochemistry, the slides were kept at room temperature for 15- s. The OCT was washed off using PBS and the zone around the tissue was demarcated using a wax pen. The tissue slices were first incubated with a blocking W0 2016/140581 solution of 10% normal goat serum for 1 hr followed by overnight tion with mouse anti en II antibody (Millipore/MAB8887) at 4°C. The slides were then rinsed three times in PBS before incubation with the appropriate on of goat anti mouse Alexa Fluor® 488 secondary antibody (Molecular Probes®/A—1 1001). The secondary dy was left on for 2 hours at room temperature. Slices were counterstained with the nuclear marker 4’, 6’—diamidinophenylindol (DAPI) and mounted in Citifluor antifade agent (ProSciTech, Australia). An Olympus FlroiewTM FV-1000 al laser scanning microscope (405 nm, 473 nm and 559 nm ngth ) and Leica DMRA fluorescence microscope were used to visualise labelling.
Table 5 s the findings from this study. Figure 20 shows representative images of collagen type II labelling in corneal sections, in each of the conditions.
Table 5: Tested combinations of growth factors and steroids Growth factor—steroid combination Collagen type II deposition (Y/N) Representative image BMP6 BMP6 + h drocortisone TGF33+ h drocortisone iii BMP6 + dexamethasone 2 TGF 33 + rednisone 2 TGF £3 + Triesense® TGF 33+ dexamethasone ii The results confirmed that the combination of TGFBB and dexamethasone is the only tested combination that ed the desired response from the target cells (Figure , panels G—H). The other growth factor-steroid combinations failed to produce the desired s 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- Previous studies have shown other growth factors and other steroid compounds to be unsuitable for corneal treatment and repair. TGFISI and TGFpZ both e fibrotic scarring ngton, Albon et a1. 2006; Desmouliere, Chaponnier et al. 2005; Jester, Huang et a1. 2002; Cowin et a1. 2001; Shah et a1. 1995). EGF negatively regulates chondrogenesis (Yoon 2000). Estrogen also negatively regulates chondrogenesis (Kato & Gospodarowicz 1985). Hydrocortisone has been shown to promote adipogenic rather than chondrogenic differentiation (Ghoniem et a1. 2015; Lee, Kuo et al. 2004). These earlier studies show the significance of the present findings on W0 2016/140581 TGFB3 and dexamethasone, which act together to promote chondrogenic entiation of corneal keratocytes and scar free corneal healing.
Example 15: Comparative dosages for TGFB3 and dexamethasone Prior to an in vivo study, experiments were performed to identify the various ive dosages for ex vivo treatments. A dose range study was carried out for TGFB3 and dexamethasone by culturing sheep corneas in culture media ning these two s in varying concentrations.
Fresh sheep eyes were obtained and corneas were excised and treated as noted in Example 14. One sheep corneal disc was placed in each of the 16 culture conditions (Figure 21) for 3 weeks. The corneal discs were cultured and then subjected to immunohistochemjcal and microscopic analysis as noted in e 14. Figure 21 shows representative images of collagen type II labelling in corneal sections in each of the conditions.
This study revealed that lower concentrations of TGFB3 (2—4 ng/mL) and dexamethasone (1-10 nM) had lower efficacy ex vivo (Figure 21, first and second rows).
Higher doses, i.e., 8—10 ng/mL TGFB3 and 100-1000 nM thasone were efficient in inducing collagen type II deposition (Figure 21, third and fourth rows).
These results confirmed the use of 100 nM thasone and 10 ng/mL TGFB3 as effective concentrations (Figure 21, third row). Higher concentrations of dexamethasone (1000 nM, i.e., 400 ng/mL) were also shown to be effective (Figure 21, fourth row). It was noted that the dexamethasone trations tested in this study were considerably lower than the concentrations used in cially ble eye drops (i.e., 1 mg/mL dexamethasone).
Example 16: Overview of experimental observations and results ] A combination of TGFBl and dexamethasone has been previously used to induce progenitor cells to differentiate into chondrocytes in vitro (Diekman et al. 2009; Johnstone et a1. 1998; Kolarnbkar et a1. 2007; Winter et a1. 2003). In other studies, a side population of l stromal cells has been shown to produce a matrix made up of the cartilage specific collagen II under similar chondrogenic differentiation conditions (Du, Funderburgh, Mann, SundarRaj, & Funderburgh 2005). It has also been reported that sclcral cells after four weeks in a chondrogenic differentiation medium containing TGFBI and BMP2 expressed cartilage specific markers including aggrecan, and collagen W0 2016/140581 type 11. Furthermore, human scleral cells have been shown to retain their chondrogenic potential in vivo after being transplanted into a rat cartilage defect (Seko et a1. 2008). It is known that the fibroblastic cells of the sclera and the corneal stroma share a common logical .
As shown , keratocytes seeded in e medium containing TGFB3 and dexamethasone and in the absence of serum spontaneously formed cell spheroids within 2-3 days by cell aggregation and by three weeks these cell clusters labelled positive for cartilage specific type II collagen. Initially upon treatment with TGFB3 and dexamethasone, type I collagen expression was also increased. When the medium was changed to a control medium containing fetal calf serum the cell clusters dispersed into a monolayer of cells. Cells growing in the yer no longer expressed type II en. These results suggest that cell aggregation or environment might be important in collagen type II induction.
Notably, keratocytes which were first proliferated as fibroblasts in serum ning medium did not secrete collagen type II when the medium was changed to the TGFB3 and dexamethasone containing chondrogenic differentiation medium. This suggests that once proliferated as fibroblasts the cells lose the ability to differentiate along a chondrogenic pathway. Further to this, fibroblasts grown in three-dimensional culture in chondrogenic differentiation medium as a pellet also failed to express cartilage specific collagen type 11. These results suggest that the ent keratocyte phenotype and cell aggregation are important to chondrogenic differentiation.
It is shown herein that ex vivo culture of normal and keratoconic corneas in chondrOgenic entiation media revealed uniform tion oftype II collagen along the stromal lamellae. Every keratocyte within the corneal stroma was associated with the collagen type II labelling, once again suggesting that the reprogramming into a chondrogenic ype is stochastic and confirming that results obtained from the in vitro cell culture were not as a result of proliferation of a side population of progenitor cells. Furthermore, in viva treatment of corneas in rats also caused the tion of type II collagen in a manner similar to that seen in ex vivo culture. r, stronger immunolabelling of type II collagen was seen in the anterior part of the cornea when treated in vivo, most probably reflecting easier diffusion of growth factors into the anterior layers of the stroma from the ocular surface.
W0 2016/140581 Studies looking at differences in keratocyte density in keratoconic corneas have reported an overall decrease in cell density. The s here also confirm this.
However, unlike other studies which have reported a marked decrease in cell density in the anterior part of the stroma (Hollingsworth, Efron, & Tullo 2005; Ku et a1. 2008; ci et a1. 2010; Niederer et a1. 2008), the results here indicate a marked decrease in cyte density in the posterior part of the stroma of the untreated keratoconic cornea also. In keratoconus there is a general thinning of the cornea. It is not known, however, whether this is due to the apoptosis of keratocytes and subsequent decreased tion of ECM or whether keratocyte apoptosis is secondary to the process of l thinning.
As shown herein, the treated half of the keratoconic cornea which was cultured in the chondrogenic medium containing TGFB3 and dexamethasone had an increased keratocyte density when compared to the control. Furthermore, the posterior region of the stroma appeared to be repopulated by keratocytes. The cytes in the treated half also appeared to look healthier with large prominent nuclei and several cell processes. This indicates that the treatment with the two factors have possibly caused keratocytes to proliferate and repopulate the stroma, in particular the ior part which was devoid of keratocytes.
Collagen crosslinking, one of the current treatments for keratoconus, s in an initial period of keratocyte apoptosis in the or part of the stroma. This is then followed by a period of repopulation of the stroma by the keratocytes. Keratocyte cell death is generally seen in response to an injury and in the case of inking is understood to be as a result ofUVA-induced cellular damage. This apoptotic se is thought to have d in order to t the cornea from further inflammation n, Netto, & Ambrosio 2003).
Stromal haze which can last up to several months is also observed after the crosslinking ent. It has been attributed to the increase in collagen diameter and spacing between the collagen fibrils which results in the modification of the corneal microstructure. Most studies have reported a decrease in corneal haze between 6—12 months after the treatment (Greenstein, Fry, Bhatt, & Hersh 2010; Mazzotta et a1. 2008).
Although there have been several clinical observations of the corneas carried out after the crosslinking treatment there is ambiguity regarding the cause ofthe corneal haze and other possible downstream effects of the treatment. The fact that it takes several months W0 2016/140581 for corneas to be repopulated and become clear suggests that the crosslinking might be triggering a wound healing response within the stroma.
] In this study, even upon long term (up to 8 weeks) in vitro and in vivo treatment there was no evidence of corneal opacity. This is probably due to the deposition of the collagen II in uniform layers along the pre-existing collagen lamellae. Deposition of collagen type III (associated with fibrosis) and alpha-smooth muscle actin (during oblast ion) leads to opacity and scarring. Both these are seen during corneal ng. Neither of these proteins was expressed in the treated corneas suggesting that wound healing cascades which could bring about ng were not being triggered.
As described herein, quantitative measurement of type II collagen mRNA expression showed that its expression was significantly lowered upon withdrawal of TGFB3 and dexamethasone. This suggests that the reprogramming of keratocytes is not irreversible and the subsequent deposition oftype II collagen in the ECM can potentially be controlled. This is important for the development of therapeutic methods, as it would not be desirable to induce irrepressible ECM deposition.
Nanoindentation has been employed in the assessment of postoperative therapeutic methods such as crosslinking for keratoconus (a corneal dystrophy) and post— LASIK ectasia in the eye. In one study done on human cadaver corneas it was found that en crosslinking caused a two-fold increase in the c modulus in the anterior corneal stroma while the posterior stroma was unaffected by the treatment (Dias, Diakonis, Kankariya, Yoo, & th 2013). In this study, anterior corneal elasticity was measured. In addition, the results in this study do te that posterior stroma keratocyte density was altered in the TGF03 and dexamethasone treated corneas.
While nanoindentation does not measure the properties of the individual collagen fibrils it can e the changes in the inherent elastic property of the cornea which will be altered on collagen II deposition with a subsequent increase in collagen crosslinking. Structural ences within the stroma are reflected in the corresponding differences in biomechanical properties. The results here show that there was almost a three-fold increase in c modulus and hardness in the growth factor treated rat s. These results te that the treatment results in a stiffer cornea with higher elasticity. The elastic modulus is a measure of a nce’s resistance to being deformed W0 2016/140581 elastically and therefore a higher elastic modulus indicates that a material is more ult to deform. In this study, a significant increase in hardness and c modulus in 3 week treated corneas when compared to 1 week treated corneas is consistent with the immunohistochemical labelling results that show at least 2-3 weeks of treatment is required for the laying down of detectable layers of type II collagen.
The immunohistochemical labelling results coupled with the gene expression studies and hanical testing show that keratocytes within an intact cornea are amenable to reprogramming along a chondrogenic pathway by treatment with TGF[33 and dexamethasone. The ramming by combined TGFB3 and dexamethasone treatment is stochastic and may be controlled via the modulation of the growth factor treatment period to result in stiffer, more elastic corneas. y, administration of both agents is ed; when TGFB3 and dexamethasone are tested separately, no collagen type II production in keratocytes is observed. A novel treatment is therefore proposed for conus and other eye conditions using in vivo tissue ering, by administration of TGFB3 and dexamethasone, as described herein.
Example 17: Large animal model to investigate reshaping of the cornea Reshaping the cornea whilst delivering the l regimen in a sheep model Additional experiments are carried out to use a large animal model to demonstrate reshaping ofthe cornea. For these experiments, a large animal model is used to allow placement of prescription contact lenses. Sheep are used as a model animal, as their eyes are comparable in size and physiology to that ofhumans. In addition, housing facilities are available at Lincoln University, Christchurch. It is noted also that sheep have a mild temperament, and are amenable to handling.
Sheep [are d in accordance with standard operating procedure in the housing facility. The eye drop formulation with optimal TGFB3 and thasone concentrations (volume scaled) based upon the rodent dose optimisation studies are instilled in the right eye followed by the placing of corneal ® (or similar l rings) to hold the desired curvature of the cornea during collagen deposition (Figure 18).
Eye drops are continued to be administered either once or twice daily (as determined in rodent sation studies) for a period ofthree weeks. The INTACS® are then removed and the animals are continued to be housed for a further three weeks or six months.
W0 2016/140581 ] Before ent and at the end of the treatment (when the ® are removed), corneal thickness and curvature measurements are taken. The le corneal pachymeter is used to detect changes in corneal thickness of treated versus control contralateral corneas in vivo. A portable Pentacam® is used to measure corneal curvature as well as corneal thickness of the sheep eyes before and after treatment (Figures 19(E) and (F)). Corneal measurements are repeated again at three weeks after lenses removal with the final (most accurate) Pentacam® measurements. These are taken after killing the animal but prior to eye removal for immunohistological and biomechanical analysis as described above for rodent s. In the unlikely event that the sheep are unable to tolerate a hard t lens (signs of infection, inflammation or irritability), the study is continued without lenses, which allows completion of key parameters such as type II en deposition and distribution, and hanical properties.
In view of the results, it is proposed to use in vivo tissue ering as described in detail herein, in combination with of a rigid gas permeable OrthoK contact lenses (or similar) to permanently reshape and stabilise the , providing treatment for common corneal defects, including .
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] All nces, including patents and patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Nor does discussion of any reference constitute an admission that such reference forms part ofthe common general knowledge in the art, in New Zealand or in any other country.

Claims (20)

What is claimed is:
1. Use of a composition for preparing a medicament for: (i) treating or preventing a condition associated with a thinning or irregularity of a cornea; or (ii) treating or preventing a refractive error associated with a defect of a cornea, the composition sing: a TGFβ3 polypeptide, and dexamethasone or any salts, esters, or hydrides thereof, wherein: the TGFβ3 polypeptide comprises SEQ ID NO:1, or the TGFβ3 polypeptide comprises at least 100 amino acids of the amino acid sequence of SEQ ID NO:1, or the TGFβ3 polypeptide shares at least 90% sequence ty to the amino acid sequence of SEQ ID NO:1.
2. The use of claim 1, wherein: (a) the TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1; and/or (b) the dexamethasone is dexamethasone phosphate.
3. The use of claim 1 or claim 2, wherein: (a) the ition comprises at least 8 ng/ml of the TGFβ3 polypeptide, or 10 to 100 ng/ml of the TGFβ3 polypeptide; and/or (b) the composition comprises at least 100 nM dexamethasone, or 40 to 4000 ng/ml thasone.
4. The use of any one of claims 1 to 3, wherein: (a) the composition is formulated as an eye drop; and/or (b) the composition is ated with gellan gum.
5. The use of any one of claims 1 to 4, wherein: (a) the composition is formulated for administration once daily or twice daily; and/or (b) the composition is ated for co-administration with one or more additional agents for the eye; and/or (c) the ition is formulated for administration in conjunction with use of one or more contact lenses, corneal s, corneal implants, or intrastromal rings.
6. The use of claim 5, wherein the one or more additional agents for the eye are selected from the group consisting of: anaesthetic agents, anti-inflammatory agents, antimicrobial agents, and lubricants.
7. The use of claim 5, wherein: (a) the one or more contact lenses, l inserts, corneal ts, or intrastromal rings are adapted to mould or hold corneal shape during and/or following treatment with the ition; and/or (b) the one or more contact lenses, corneal inserts, corneal implants, or intrastromal rings are adapted to act as a carrier for the composition or as a composition eluting device.
8. The use of any one of claims 1 to 7, wherein: for (i), the composition is formulated for administration in conjunction with corneal collagen crosslinking; or (ii), the composition is formulated for administration preceding or ing refractive surgery.
9. The use of claim 8, wherein the composition is formulated for administration prior to and/or subsequent to inking.
10. The use of any one of claims 1 to 9, wherein: (a) the condition is selected from the group ting of: keratoconus, myopia, astigmatism, and stromal dystrophies; and/or (b) the refractive error is associated with one or more of: myopia, hyperopia, astigmatism, or presbyopia.
11. Use of: (A) a TGFβ3 polypeptide, and (B) dexamethasone or any salts, esters, or hydrides thereof, for cture of ophthalmic medicament for: (i) treating or preventing a condition associated with a thinning or irregularity of a cornea; or (ii) treating or preventing a refractive error ated with a defect of a cornea, wherein: components (A) and (B) are adapted for sequential or simultaneous stration, and wherein: the TGFβ3 polypeptide comprises SEQ ID NO:1, or the TGFβ3 polypeptide comprises at least 100 amino acids of the amino acid sequence of SEQ ID NO:1, or the TGFβ3 polypeptide shares at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1.
12. The use of claim 11, wherein: (a) the TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1; and/or (b) t he dexamethasone is thasone phosphate.
13. The use of claim 11 or claim 12, wherein: (a) the composition of (A) comprises at least 8 ng/ml of the TGFβ3 polypeptide, or 10 to 100 ng/ml of the TGFβ3 polypeptide; and/or (b) the composition of (B) ses at least 100 nM dexamethasone, or 40 to 4000 ng/ml dexamethasone.
14. The use of any one of claims 11 to 13, wherein: (a) the administration is via an eye drop; and/or (b) the administration is via a ation with gellan gum.
15. The use of any one of claims 11 to 14, n: (a) the administration is once daily or twice daily; and/or (b) the administration includes co-administration with one or more additional agents for the eye; and/or (c) the stration is in conjunction with use of one or more contact lenses, corneal inserts, corneal implants, or intrastromal rings.
16. The use of claim 15, wherein the one or more additional agents for the eye are selected from the group ting of: anaesthetic agents, anti-inflammatory agents, crobial agents, and lubricants.
17. The use of claim 15, wherein: (a) the one or more contact lenses, corneal inserts, corneal implants, or intrastromal rings are adapted to mould or hold corneal shape during and/or following treatment with the composition; and/or (b) t he one or more contact lenses, corneal inserts, corneal implants, or intrastromal rings are adapted to act as a carrier for the composition or as a composition eluting device.
18. The use of any one of claims 11 to 17, n: for (i), the administration is in conjunction with corneal collagen crosslinking; or (ii), the administration is preceding or following refractive surgery.
19. The use of claim 18, wherein the administration is prior to and/or subsequent to crosslinking.
20. The use of any one of claims 11 to 19, wherein: (a) the ion is selected from the group consisting of: keratoconus, myopia, astigmatism, and stromal dystrophies; and/or (b) the tive error is associated with one or more of: myopia, hyperopia, astigmatism, or presbyopia.
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