WO2025043297A1 - Method of treating myopia - Google Patents

Abstract

Provided herein are methods of treating or preventing myopia in a subject by administering to the subject a therapeutically effective amount of an agent such as mefenamic acid that uncouples a retinal cell network.

Description

Method of treating myopia

Technical Field

[001] The invention relates to methods of treating or preventing myopia by administering an agent that uncouples a retinal cell network, for example a connexin inhibitor such as Mefenamic acid.

Cross reference to related application

[002] This application claims priority to Australian provisional patent application number 2023902799 flied 31 August 2023, which is incorporated by reference in its entirety.

Background

[003] Myopia or 'short-sightedness' is a visual impairment where distant objects appear blurred and hence unclear. It is a significant public health problem with 50% of the world population expected to be myopic by 2050 and up to 1 billion people expected to develop high myopia. High myopia (more than -5D) causes low vision and results in ocular pathologies in 70% of patients including detached retina, lacquer cracks and staphyloma which in one-third of patients can lead to profound blindness including from myopic macular degeneration.

[004] Myopia arises when light entering the eye come to focus in front of the retina instead of directly on it. This is due to elongation of the eyeball resulting in blurred vision. Myopia commonly develops from childhood, become progressively worse through adolescence so that correction lenses of increasing negative power are needed over time, and can progress to high myopia. Myopia and myopic progression are associated with a higher risk of myopic retinal degeneration, glaucoma, and retinal detachment. Thus there is a need to slow myopia development. There is also a need to stop the eye progressing from myopia to high myopia. Although standard single vision spectacle and contact lenses correct a myopic refractive error, they do not treat the underlying growth response of the eye. Novel multifocal spectacle and contact lens designs and corneal reshaping contact lenses have assisted with slowing myopia development, but none stop progression.

[005] Pharmaceutical approaches to myopia treatment include the use of atropine, a muscarinic antagonist, formulated as a 1% eye drop. This is known to strongly slow myopia development and while 1% atropine shows excellent efficacy, this concentration is associated with undesirable side effects such as blurred vision and cycloplegia, and when 1% atropine is discontinued rebound occurs. To avoid the side effects, much lower concentrations have been proposed as an alternative. However, efficacy tends to reduce with lower concentrations. For example, a small 10-year human study with daily 0.01% atropine eye drops, indicated that this concentration had limited efficacy in Australian children and it did not stop myopia development (Myles, Dunlop, and McFadden, 2021, The Effect of Long-Term Low-Dose Atropine on Refractive Progression in Myopic Australian School Children. J Clin Med, 10(7), 1444).

[006] Retinal coupling is mediated through gap junctions which allow electrical coupling between adjacent cells and facilitates rapid signal spread in a coupled network of cells. Gap junctions are composed of two hemiconnexons that dock together across adjoining cell membranes. Each hemiconnexon protein consists of six connexins arranged in a hexagonal pattern which spans the cell membrane. Each of the five subfamilies of connexins (a, p, y, 5 and E) mostly only interact with members of their own subfamily. Connexins (Cx) are therefore the constituent gap junction channel proteins and are ubiquitously expressed throughout body tissues with over 20 different types in humans.

[007] The inventors demonstrate that coupling between retinal networks is sensitive to the sign of imposed defocus, and that interfering with this coupling can be used to treat or prevent myopia.

[008] Any reference to or discussion of any document, act or item of knowledge in this specification is included solely for the purpose of providing a context for the present invention. It is not suggested or represented that any of these matters or any combination thereof formed, at the priority date, part of the common general knowledge, or was known to be relevant to an attempt to solve any problem with which this specification is concerned.

Summary

[009] In a first aspect, there is provided a method of treating or preventing myopia in a subject comprising administering to the subject a therapeutically effective amount of an agent that uncouples a retinal cell network.

[010] The agent may be a gap junction inhibitor, such as a non-specific inhibitor of retinal gap junctions.

[011] The gap junction inhibitor may block channels comprised of connexin and/or pannexin proteins. For example, the gap junction inhibitor may be a connexin inhibitor of one or more of 0x36, 0x43, 0x45, 0x50, 0x57, or 0x60, or any combination thereof.

[012] In one embodiment the retinal cell network comprises a Horizontal cell and/or an amacrine cell. The Horizontal cell may be an A-Type Horizontal cell, the amacrine cell may be a neuronal nitric oxide synthase (nNOS) amacrine cell. [013] In one embodiment the agent is a periodic exposure of an eye to short periods of darkness.

[014] In other embodiments the agent is one or more of Mefenamic acid (MFA), flufenamic acid, meclofenamic acid, tolfenamic acid, orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX), C8H17OH, tonabersat, Cx43 oligonucleotide (5-GTA-ATTGCG- GCA-GGA-GGAATT- GTT-TCT-GTC-3'),or a peptide selected from the group consisting of YPXGAG, a-Connexin carboxyl terminal RQPKIWFPNRRKPWKK - RPRPDDLEI, VDCFLSRPTEKT, YGRKKRRQRRRKQIEIKKFK, and LCLRPVGG -KQIEIKKFK.

[015] In a preferred embodiment the agent is Mefenamic acid (MFA), for example in a sustained release formulation or depot.

[016] The treatment may comprise inhibiting or slowing the progression of myopia, and/or inhibiting or slowing excessive eye growth or reducing eye elongation.

[017] The treatment may comprise reversing established myopia.

[018] The agent may be administered intraocularly or topically. For example, the intraocular administration may comprise intravitreal, retinal, conjunctival, subconjunctival, or scleral injection preferably the agent is administered intravitreal ly.

[019] The topical administration may comprise eye drops, eye wash solution, ointment, gel, suspension, emulsion, or via a contact lens.

[020] The myopia may be axial myopia, refractive myopia, myopic astigmatism, simple myopia, early- or late-onset myopia, progressive myopia, degenerative myopia or pathological myopia.

[021] In a second aspect there is provided use of an agent that uncouples a retinal cell network in the manufacture of a medicament for treating or preventing myopia. The agent may be a gap junction inhibitor. The agent may be one or more of Mefenamic acid (MFA), flufenamic acid, meclofenamic acid, tolfenamic acid, orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX), C8H17OH, tonabersat, Cx43 oligonucleotide (5 -GTA- ATTGCG- GCA-GGA-GGAATT- GTT-TCT-GTC-3'), or a peptide selected from the group consisting of YPXGAG, a-Connexin carboxyl terminal RQPKIWFPNRRKPWKK - RPRPDDLEI, VDCFLSRPTEKT, YGRKKRRQRRRKQIEIKKFK, and LCLRPVGG - KQIEIKKFK.

[022] Preferably the agent is Mefenamic acid (MFA), for example in a sustained release formulation or depot. [023] In one embodiment the agent is a gap junction inhibitor that blocks channels comprising connexin and/or pannexin proteins. For example the connexin may be Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof.

[024] In a third aspect there is provided mefenamic acid (MFA) for use in the treatment or prevention of myopia, preferably wherein the MFA blocks channels comprising connexin and/or pannexin proteins. For example the connexin may be Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof. The MFA may be in a sustained release formulation or depot.

Definitions

[025] The term "myopia" encompasses all forms of myopia, including but not limited to, axial myopia, refractive myopia, myopic astigmatism and simple myopia. Myopia may be classified by a number of different criteria, such as cause, degree, age of onset, and clinical appearance. Those skilled in the art will appreciate there are many types or forms of myopia to which methods of the invention are applicable. For example, the myopia may be induced myopia, such as lens- or instrument-induced myopia, form deprivation myopia, index myopia, simple myopia, early or late-onset myopia, progressive myopia, degenerative myopia, pathological myopia or pseudomyopia. The myopia may be, for example, low, medium or high myopia. The myopia may be, for example, congenital myopia, childhoodonset myopia or adult-onset myopia.

[026] The phrase "and/or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. As a non-limiting example, a reference to 'an agent that inhibits retinal gap junctions and/or uncouples retinal cell networks' can refer, in one embodiment, to an agent that inhibits retinal gap junctions only and, in another embodiment, to an agent that uncouples retinal cell networks only and in yet another embodiment, to an agent that inhibits retinal gap junctions and uncouples retinal cell networks.

[027] As used herein, the term "inhibitor" is used interchangeably with the term "antagonist" and refers to any agent such as a compound or molecule which results in a decrease in the magnitude of a biological activity of a protein. In certain embodiments, the presence of an antagonist or inhibitor blocks or dampens, i.e., results in complete or partial inhibition, of a biological activity of a protein. In certain descriptions, an antagonist or inhibitor may be referred to as a modulator. Thus, an agent that inhibits retinal gap junctions or retinal hemi-channels may refer to a compound or molecule that inhibits an activity or function of a gap junction protein, for example, an activity or function of one or more connexins, in whole or in part. In one embodiment the term 'gap junction inhibitor' relates to an inhibitor that interferes with connexin or pannexin proteins that constitute a gap junction so as to close, block, inhibit or partially close, block, or inhibit the ability of the hemichannel or the gap junction to pass signals across the cell membrane. Pannexin proteins may include but is not limited to Pannexinl (Panxl), Pannexin2 (Panx2) and Pannexin3 (Panx3).

[028] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[029] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the technology recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[030] In the context of the present specification, the terms "a" and "an" are used to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, reference to "an element" means one element, or more than one element.

[031] In the context of the present specification, the term "about" means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term "about" is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[032] The terms "treating", "treatment", "preventing" and "prevention" of myopia as used herein means: (1) preventing or delaying one or more symptoms of myopia from developing in a subject that may be predisposed to myopia but does not yet experience or display symptoms of myopia, (2) inhibiting myopia, i.e., arresting or reducing the development or progression of myopia or at least one or more symptoms thereof, or (3) relieving myopia, i.e., causing regression or reversing myopia or at least one of its symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient and/or to the physician. Thus, the term "treating", "treatment", "preventing", and "prevention and the like are to be considered in their broadest context and encompasses curing, ameliorating, or tempering the severity of myopia or one or more of its associated symptoms. [033] "Effective amount" and "therapeutically effective amount" refer to an amount of an agent or compound (e.g., MFA or another retinal gap junction inhibitor) sufficient to produce a desired therapeutic or pharmacological effect in the subject being treated. The terms are synonymous and are intended to qualify the amount of each agent that will achieve the goal of improvement in disease severity and/or the frequency of incidence over treatment of each agent by itself while preferably avoiding or minimizing adverse side effects, including side effects typically associated with other therapies. The "effective amount" or "therapeutically effective amount" will vary depending on the agent or compound, the disease severity, and the age, weight, physical condition and responsiveness of the individual to be treated.

[034] A "pharmaceutical carrier, diluent or excipient" includes, but is not limited to, any physiological buffered (i.e. , about pH 7.0 to 7.4) medium comprising a suitable water- soluble organic carrier, conventional solvents, dispersion media, gels, fillers, solid carriers, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.

[035] "Subject" as used herein refers to any animal including mammals (e.g., primates, mice, rabbits, guinea pigs, dogs, cats, sheep, pigs, cattle, horses and human) and is used interchangeably with the term "patient".

[036] In the context of this specification, the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering, or providing an agent or compound of the invention to a subject by any appropriate means.

[037] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all of the steps, features, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[038] In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

[039] Figure 1 is a comparison of the effect of low and high dose atropine on myopia development in young guinea pigs where atropine was administered as a daily eye drop in one eye concurrently with myopia induction in the same eye using form deprivation (FD) for two weeks. The difference between the untreated and treated eye in their spherical equivalent (SE) refractive error is shown. Open circles show the data for individual animals. Low dose (0.15%) atropine had reduced efficacy in inhibiting myopia compared to high dose (1%) atropine.

[040] Figure 2 is a schematic drawing showing example intracellular pathways that can close or block Cx36 and Cx45 connexin mediated gap junctions in retinal amacrine cells. Nitric Oxide (NO) can block 0x45 hemichannels via a cGMP-dependent kinase mechanism (protein kinase G (PKG)). Dopamine D1 receptor-driven uncoupling results from protein kinase A (PKA) activation of protein phosphatase 2A (PP2A) and subsequent dephosphorylation of 0x36.

[041] Figure 3 shows the effect of interrupting monocular negative lens wear (-6D) with regular daily exposure periods to different lighting conditions in young guinea pigs, a., b. Experiment 1a: During 2 x 1 hr daily periods, a negative lens was either left in place (-6D) or removed and animals were exposed to darkness (Dark), white light (03), cool white (06) or lime light, c., d. Experiment 1b: During a single 1 hr daily period, the negative lens was either left in place (-6D) or removed and animals were exposed to white light (White), darkness (Dark), or the -6D lens was substituted with an opaque black diffuser, a., c.

Show the effects of myopia interruption compared to uninterrupted myopia controls (-6D) on the difference in refractive error between the treated and untreated eye. White light was the most effective in inhibiting myopia, lime light or dark periods without vision also inhibited myopia, even when induced locally on one eye only (Black Diffuser in c shows the dark effect was not caused by a general circadian light interruption but was local to the treated eye), b., d. Show the effects of myopia interruption compared to uninterrupted myopia controls (-6D) on the difference in ocular length between the treated and untreated eye. For white light, two 1 hr periods were more effective than a single 1 hr light period in reducing ocular elongation. However, the dark effect was more rapid and inhibited ocular elongation even after a single 1 hr period of darkness during the day (d., rightmost bar).

[042] Figure 4 shows bidirectional change in Horizontal cell (HC)-HC coupling in the guinea pig superior retina, a., b. Show example sections of stained a-type horizontal cells (aHC) in control guinea pig retina loaded with neurobiotin dye demonstrating greater dye spread (meaning greater cell coupling) in the superior retina (b) compared to the inferior retina (a), c. The images in a. and b. were enlarged from c. which shows the entire guinea pig retina cut-loaded with neurobiotin dye along the superior, temporal and inferior retinal axes and counter labelled with streptavidin (488). d. shows a significantly greater coupling coefficient between a-type horizontal cells (aHC) in the superior retina relative to the inferior retina, e. no difference in the density of aHCs was observed in the superior and inferior retina meaning that the increased coupling in superior retina is not an artifact of cell spacing, f., g., h., i. Average coupling coefficient k, values in the inferior and superior retina (top graphs) and corresponding mean aHC fluorescence decay as a function of distance from the dye load position (at 0 cell separations) (bottom graphs) for four different myopic conditions (Experiment 2). These four conditions were: f. with myopia induced with a -6D lens worn on the right eye (RE) and animals raised under white light (700 lx), g. where the - 6D lens was removed for 3 days to induce recovery from myopia (-6D Recovery) and where animals were raised in white light (700 lx) h. with myopia induced using monocular formdeprivation (FD) under white light (700 lx) i. with myopia induced with a -6D lens worn on the right eye (RE) and animals raised under intense white light (3000lx). Retinae from left eyes (LE) were untreated controls (C). j. relative interocular difference (RE - LE) in aHC coupling coefficient in each of the four groups. Eyes recovering from myopia (second bar from the left in superior retina have reduced coupling while all other groups were myopic and show increased coupling in superior retina regardless of the method of myopia induction or the luminance level. In Figures 4f-i, each bar on the left of each pair of bars is the LE control, the right-hand bars of each pair are the RE Lens-wear myopia (Figures 4f, g and i) or the RE Form deprivation myopia (FD) (Figure 4h). In addition, in the lower panels of Figures 4f-i the solid line in each is the LE control eyes. In Figures 4f, g and I the dashed line is for the RE lens-wearing eyes and in Figure 4h the dashed line is for the RE form-deprivation eyes.

[043] Figure 5 shows differences in coupling between neuronal nitric oxide synthase (nNOS) amacrine cells (mediated by Cx45 and Cx36 connexins) in the guinea pig superior retina, a. difference between the two eyes (RE - LE) in displaced nNOS amacrine cell coupling in form-deprivation myopia compared to animals undergoing -6D lens-induced myopia with intermittent periods of free viewing in lime light or darkness. Eyes with inhibited myopia due to regular interruptions of negative lens wear by daily 1 hr periods without the lens while either in darkness (Myopia Recovery Dark, no vision available) or in lime light (Myopia Recovery lime, lime vision available) have reduced coupling while myopic form deprivation (FD) eyes have increased coupling, b., c., d. corresponding mean displaced nNOS amacrine cell fluorescence decay graphs from the superior retina for form-deprivation myopia guinea pigs, animals undergoing -6D lens myopia with intermittent periods of free viewing in lime light, or darkness respectively. Both lime light and darkness induce recovery from myopia (See Figure 3) and reduce the degree of coupling.

[044] Figure 6 demonstrates the effects of Mefenamic acid (MFA) in guinea pigs: a. MFA uncouples A-Type Horizontal cells in a dose dependent manner in vitro with MFA dissolved in the bath solution; b. In-vivo intravitreal injection of MFA (concentration in vitreous was 250pm) reduces coupling within 1 hr and the effects persist for at least another 2 hours; c. Difference in refractive error between the treated right eye (RE) and untreated left eye (LE) shows that daily intravitreal injections of MFA strongly inhibit myopia induced with form deprivation (FD, top line) compared to control animals receiving either vehicle injections and FD (middle line) or no injections and FD (bottom line) despite all-day long exposure to a myopiagenic stimulus and the short half-life of MFA (Experiment 3). d. Data for individual animals (filled circles) for their difference in refractive error between the treated and untreated eye at Day 14 in each of the three groups. Bars show the mean and standard error of the mean. e. Injections increase corneal power but there is no difference between vehicle or MFA injected animals and thus cannot explain the myopia inhibition induced by MFA shown in c. and d.; f.- j. Injection of MFA not only slows eye elongation but makes the eye’s axial length shrink (f.) relative to vehicle and untreated controls. This strong growth inhibition is not because of a smaller anterior chamber (g.), or smaller crystalline lens (h.), which do not significantly differ between vehicle and MFA injected eyes but is due to shrinkage in the vitreous chamber (i.), making MFA a true inhibitor of posterior elongation or growth and resulting in an overall relative reduction in ocular length (j.). In Figures 6d-6j the columns from left to right show form deprivation (FD), FD + vehicle, and FD + MFA.

Description of Embodiments

[045] The methods and uses disclosed herein relate to the treatment or prevention of myopia. Specifically, the invention relates to a method of treating or preventing myopia comprising administering to a subject in need thereof a therapeutically effective amount of an agent that uncouples a retinal cell network, for example a connexin inhibitor such as Mefenamic acid.

[046] The methods provided herein are based on the finding that strongly coupled specific cell networks characterise a myopic retina and a rapidly growing eye, while uncoupling in these specific networks is indicative of a significant reduction in accelerated eye elongation or growth. The inventors have found that interfering with retinal cell network coupling (mediated by gap junctions) can be used to treat or prevent myopia.

Agents

[047] The methods use an agent that inhibits retinal gap junctions and thereby interferes with retinal cell network coupling.

[048] Alternatively the agent may be in the form of a periodic exposure of an eye to short periods of darkness which results in uncoupling of a retinal cell network.

[049] The agent may be a specific or non-specific gap junction inhibitor. [050] Suitable inhibitors include but are not limited to, connexin inhibitors. A connexin inhibitor is a molecule that modulates or down regulates one or more functions, activities, or expressions of a connexin or a connexin hemichannel (connexon) comprising a connexin of interest. Connexin inhibitors may be a small molecule inhibitor, an antisense compound (e.g., antisense polynucleotides), RNA interference (RNAi) and small interfering RNA (siRNA) compounds, antibodies and binding fragments thereof, and peptides and polypeptides (including peptidomimetics and peptide analogs). Preferred connexin inhibitors are inhibitors of connexins found in retinal gap junctions. In one example, the connexin inhibitor is an inhibitor of Cx36 and/or CX45. In another example, the connexin inhibitor is an inhibitor of Cx57 and/or CX50. In yet another example, the connexin inhibitor is an inhibitor of one or more of Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof.

[051] In one embodiment, the connexin inhibitor uncouples retinal cell networks. For example, the connexin inhibitor uncouples A-Type Horizontal cells and nNOS amacrine cells. In one example, the connexin inhibitor uncouples A-Type Horizontal cells and nNOS amacrine cells in a dose dependent manner.

[052] Non-limiting examples of connexin inhibitors include Mefenamic acid (MFA) and other fenamates such as flufenamic acid, meclofenamic acid, and tolfenamic acid which are derivatives of orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX) and other synthetic glycyrrhetinic acid derivatives, long carbon chain n-alkanols such as heptanol and octanols (C8H17OH),Tonabersat (XiflamTM), Cx43 oligonucleotide (Nexagon®) (5 -GTA- ATTGCG- GCA-GGA-GGAATT- GTT-TCT-GTC-3'), Rotigaptide (YPXGAG), a-Connexin carboxyl terminal peptide (ACT1) (RQPKIWFPNRRKPWKK - RPRPDDLEI), Peptide5 (PeptagonTM) (VDCFLSRPTEKT), iNexin™ (aCT1), Gap19 (YGRKKRRQRRRKQIEIKKFK), and Xentry-Gap 19 (LCLRPVGG -KQIEIKKFK).

[053] An example of a non-specific gap-junction inhibitor is Mefenamic acid (MFA). MFA is a nonsteroidal anti-inflammatory drug (NSAID). Mefenamic acid oral capsules (e.g., Ponstel) are used to treat mild to moderate pain and dysmenorrhea (menstrual pain). The primary mode of action of MFA is as a non-selective cyclooxygenase (COX) inhibitor which acts on inflammatory pathways, and it can also be transformed into a selective COX-2 inhibitors via neutralization of the carboxylate moiety. However, MFA also acts as a non selective connexin inhibitor.

[054] MFA acts as a non-specific gap junction antagonist with high potency, water solubility, and relatively fast reversibility, with the time to complete block typically between 20 to 40 mins and maximum recovery typically >1 h when applied directly to extracted retinal slices. Those skilled in the art will appreciate that MFA is just one exemplary agent that may be employed in accordance with the present disclosure. Many other suitable agents are known to those skilled in the art and may be employed.

[055] In one embodiment the therapeutic agent uncouples retinal cell networks and therefore the invention may include any suitable agent that uncouples retinal cell networks. In one example the cell network is mediated by connexins. In one example, the agent of the present invention uncouples Horizontal cell networks and/or amacrine cell networks. In another example, the agent of the present invention uncouples A-Type Horizontal cell networks and/or nNOS amacrine cell networks. In one example, the agent of the present invention uncouples A-Type Horizontal cell networks and/or nNOS amacrine cell networks in a dose dependent manner. An exemplary agent that uncouples retinal cell networks is Mefenamic acid (MFA).

[056] The agent may be used alone or in combination with other therapeutic agents to treat or prevent myopia and an associated disease, disorder or condition, for example, a retinal disease, detached retina, cataract, glaucoma, staphyloma and myopic macular degeneration.

[057] Forms of the agent that inhibits retinal gap junctions and/or uncouples retinal cell networks include, but are not limited to, solutions, ointments, gels, emulsions, suspensions, gel shields, and the like. In one embodiment, the agent is formulated as an aqueous-based cream excipient, which can be applied to the eye. In another embodiment, the agent is formulated as a solution or suspension and is applied topically in the form of eye drops.

[058] The agent of the invention may be administered to the eye, for example, topically or intraocularly. Administration may be, for example, once per day, twice per day or multiple times per day. In one embodiment the agent may be conjugated to, or coated on, the surface of a contact lens or the contact lens may be impregnated with the agent. Alternatively, the agent may be administered by injection directly into a specific tissue or region of the eye, such as into the conjunctiva or sclera. Thus, the agent may be administered by, for example, intravitreal, conjunctival or scleral injection, or an intraocular implant or other slow-release delivery method. The agent may be administered topically such as in eye drops, an eye wash solution, an ointment or gel.

[059] Non-limiting examples of an agent that inhibits retinal gap junctions and/or uncouple retinal cell networks include Mefenamic acid (MFA) and other fenamates such as flufenamic acid, meclofenamic acid, and tolfenamic acid which are derivatives of orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX) and other synthetic glycyrrhetinic acid derivatives, long carbon chain n-alkanols such as heptanol and octanols (C8H17OH), Tonabersat (Xiflam™), Cx43 oligonucleotide (Nexagon®) (5-GTA-ATTGCG- GCA-GGA- GGAATT- GTT-TCT-GTC-3'), Rotigaptide (YPXGAG), a-Connexin carboxyl terminal peptide (ACT1) (RQPKIWFPNRRKPWKK - RPRPDDLEI), Peptide5 (Peptagon™) (VDCFLSRPTEKT), iNexin™ (aCT1), Gap19 (YGRKKRRQRRRKQIEIKKFK), and Xentry- Gap 19 (LCLRPVGG -KQIEIKKFK).

[060] As exemplified herein, MFA induces uncoupling of A-Type Horizontal cells and nNOS amacrine cells. This effect may be mediated by a number of retinal connexins such as one or more of 0x36, 0x43, 0x45, 0x50, 0x57, or 0x60, or any combination thereof.

Retinal gap junction

[061] Retinal coupling is mediated through gap junctions which allow electrical coupling between adjacent cells and facilitates rapid signal spread in a coupled network of cells. Connexins (Cx) are the constituent gap junction channel proteins and are ubiquitously expressed throughout the body with more than 20 different types in humans.

[062] In the retina, the most common connexin is 0x36, which supports gap junctions between photoreceptors, and specific types of bipolar cells, amacrine cells, and retinal ganglion cells. Alternatively, in mammalian retina, All amacrine cells integrate scotopic signals from the rod pathway with on-bipolar cells via gap junctions composed of 0x36 connexins in homologous gap junctions but with 0x36/0x45 connexins in heterologous gap junctions (Veruki and Hartveit, 2009, Meclofenamic acid blocks electrical synapses of retinal All amacrine and on-cone bipolar cells. (J Neurophysiol, 101(5), 2339-47). 0x36 coupling is modulated through D1 dopamine receptor activation. Specifically, 0x36 phosphorylation (which results in reduced coupling) is mediated by PKA activation following D1 receptor activation in rabbits and mice (Kothmann et al., 2009, Dopamine-stimulated dephosphorylation of connexin 36 mediates All amacrine cell uncoupling. J Neurosci, 29(47); Urschel et al., 2006, Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between All amacrine cells. J Biol Chem, 281(44), 33163-71). Evidence based on effects of dopamine antagonists implicate D1 receptors as underlying myopia inhibition, suggesting that dopamine acting via D1 receptors may interfere with CX36 coupling leading to myopia inhibition.

[063] Furthermore, the gene, GJD2, which codes the transmembrane connexin 0x36 has been implicated in human genome wide association studies of myopia where single nucleotide polymorphisms located near, although not in the GJD2 gene, have been consistently associated with refractive error. Changes in GJD2 expression have also been reported in myopic animal eyes (Zhu et al., 2020, Altered Expression of GJD2 Messenger RNA and the Coded Protein Connexin 36 in Negative Lens-induced Myopia of Guinea Pigs. Optom Vis Sci, 97(12), 1080-1088). There is some previous evidence that Cx36 is down regulated in myopic guinea pig retina, but this effect was only seen with form deprivation, a technique which also reduces retinal illumination (Zhi et al., 2021, The Role of Retinal Connexins Cx36 and Horizontal Cell Coupling in Emmetropization in Guinea Pigs. Invest Ophthalmol Vis Sci, 62(9), 27). These authors failed to detect changes in Cx36 or its phosphorylation when animals were made myopic with a minus lens (which has minimal effects on luminance exposure) but they did not study different parts of the retina. Furthermore, they reported that a non-specific gap junction uncoupling agent, 18-p- Glycyrrhetinic Acid (18-p-GA), given as daily subconjunctival injections to reduce retinal Cx36, had no effect on ocular length increases induced with negative lenses. Thus Cx36 has not been proven to be an effective target for myopia control to date.

[064] A-Type Horizontal cells (HCs) are strongly coupled via gap junctions composed of the connexins 0x50 and 0x57. Coupling between A-Type HCs is dependent on dopamine (via D1) as well as nitric oxide and all-trans retinoic acid. Retinal dopamine causes endogenous nitric oxide release, consequently reducing coupling between connexins other than 0x36 (e.g. 0x50, 0x57 and 0x45). Considering that inhibiting nitric oxide synthase ameliorates the protective effect of dopamine on the development of myopia in the chick (Nickla et al., 2013, Nitric oxide synthase inhibitors prevent the growth-inhibiting effects of quinpirole. Optom Vis Sci, 90(11), 1167-75), the failure of targeting 0x36 to mediate ocular growth may signify the importance of these secondary, nitric oxide-mediated, coupled cell networks in myopia development.

[065] Therefore there are several other connexins of interest (e.g., 0x50, 0x57 and 0x45) that may be relevant to myopia control. A previous study (Zhi et al., 2021, The Role of Retinal Connexins 0x36 and Horizontal Cell Coupling in Emmetropization in Guinea Pigs. Invest Ophthalmol Vis Sci, 62(9), 27) failed to find changes in HC-HC coupling and the authors concluded that HC-HC coupling in the outer plexiform layer (OPL) might not play a significant role in emmetropization and myopia development. In contrast, the present inventors show that coupling between A-Type HCs, which is mediated by 0x57/0x50, is strongly implicated in myopia control, since the degree of coupling is bidirectionally modulated by the sign of defocus.

Connexin inhibitors

[066] Most agents targeting connexins have focused on 0x43 as its inhibition can support injury responses including the spread of injury signals, extravasation of immune cells, granulation tissue formation and fibrosis and acceleration of wound healing. Some examples are shown in Table 1 (from Laird and Lampe, 2018, Therapeutic strategies targeting connexins. Nat Rev Drug Discov, 17, 905-921).

[067] iNexin™ (from Xequel Bio, Inc.) which is an alpha-Connexin carboxyl-Terminal 1 peptide (aCT1) ophthalmic solution based on the C-terminal sequence of Cx43, designed to selectively and reversibly inhibit protein binding of endogenous Cx43, is being trialled for the treatment of corneal injury after chemical or thermal injuries and/or surgery. OcuNexus also has an extensive focus on eye wounds and inflammation and has developed Nexagon® (AsODN, unmodified oligonucleotide that downregulates expression of Cx43), Xiflam® (tonabersat, reduces Cx26 expression, repurposed for treatment of macular degeneration and diabetic retinopathy) and Peptagon® (Peptide5, a synthetic peptide that is a specific inhibitor of Cx43 hemichannels and reported to enhance cell survival after inflammation and ischaemia). FirstString is also developing eye drop therapeutics to reduce inflammation and increase corneal wound healing and treatment of diabetic keratopathy and age-related macular degeneration. Connexin Therapeutics is also developing peptide based connexin inhibitors for glaucoma treatment. However none of these agents target myopia.

Table 1. Selected connexin inhibitors (from Laird, D. & Lampe, P., 2018, Therapeutic strategies targeting connexins. Nat Rev Drug Discov 17, 905-921).

AsODN, antisense oligodeoxynucleotide; Cx, connexin, GJIC, gap junction intercellular communication; NA, not available; Pre-IND, pre-investigational new drug application; ZO1 , zonula occludens 1.

Pharmaceutical formulations

[068] The agents described herein may be administered as a formulation comprising a pharmaceutically effective amount of a compound or molecule, in association with one or more pharmaceutically acceptable excipients including carriers, vehicles and diluents. The term "excipient" herein means any substance, not itself a therapeutic agent, used as a diluent, adjuvant, or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a solid dosage form such as a solution or suspension suitable for topical application. Excipients can include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, stabilizers, and substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition. Examples of excipients and their use is described in Remington's Pharmaceutical Sciences, 20th Edition (Lippincott Williams & Wilkins, 2000). The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[069] The compounds and pharmaceutical compositions of the invention may be formulated for oral, injectable, parenteral, subcutaneous, intravenous, topical, intravitreal, or intramuscular delivery. Non-limiting examples of particular formulation types include tablets, capsules, caplets, powders, granules, injectables, ampoules, vials, ready-to-use solutions or suspensions, lyophilized materials, creams, lotions, ointments, drops, suppositories and implants. Solid formulations such as the tablets or capsules may contain any number of suitable pharmaceutically acceptable excipients or carriers described above. The compounds of the invention may also be formulated for sustained delivery.

[070] Liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example, sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example, lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, oily esters such as glycerin, propylene glycol, or ethyl alcohol; preservatives, for example, methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring or colouring agents.

[071] In one or more preferred embodiments the compounds of the invention are formulated as an injectable solution, suspension or emulsion. In preferred embodiments, the compounds of the invention are formulated for intravitreal injection into the eye of a subject. Such formulations may be particularly preferred for administration to the eye.

[072] In preferred embodiments, ophthalmic formulations, including intravitreal formulations and other ophthalmic formulations, such as eye drops, typically may comprise one or more co-solvent(s), such as one or more organic co-solvents; one or more tonicity agent(s); a buffering system comprising one or more buffering agents; a stabilizing agent; pH between about 3-8. In preferred embodiments, the organic co-solvent may be polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for example, PEG 3350, or propylene glycol, or a combination thereof; the tonicity agent may be, for example, sodium chloride or potassium chloride; the stabilizing agent may be sucrose, sorbitol, glycerol, trehalose, or mannitol; and the buffering agent may be, for example, phosphate buffer, such as a sodium phosphate buffer.

[073] Intravitreal formulations are preferably sterile, isotonic and preferably have a pH within the range pH 3-8, preferably pH 5-7 or pH 3-5. Such formulations may contain one or more buffers as part of a buffer system, however, the concentration of buffers is preferably kept as low as possible. Buffer stressing studies may be carried out to select the minimal buffer amount needed to safely maintain the desired pH range. Exemplary intravitreal formulations are described in Shikari H, Samant PM. Intravitreal injections: A review of pharmacological agents and techniques. J Clin Ophthalmol Res 2016; 4:51-9.

[074] In preferred embodiments, ophthalmically acceptable formulations may be lyophilizable. Lyophilizable formulations can be reconstituted into solutions, suspensions, emulsions, or any other suitable form for administration or use. Lyophilizable formulations are typically first prepared as liquids, then frozen and lyophilized. The total liquid volume before lyophilization can be less, equal to, or more than, the final reconstituted volume of the lyophilized formulation. The lyophilization process is well known to those of ordinary skill In the art, and typically Includes sublimation of water from a frozen formulation under controlled conditions. Lyophilized formulations typically can be stored at a wide range of temperatures. For example, lyophilized formulations may be stored below 25°C, for example, refrigerated at 2-8°C, or at room temperature (e.g., approximately 25°C).

Preferably, lyophilized formulations are stored below about 25°C, more preferably, at about 4-20°C; below about 4°C; or below about 0°C. [075] Lyophilized formulations are preferably reconstituted with a solution consisting primarily of water (e.g., USP WFI, or water for injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl alcohol). Alternatively, solutions comprising buffers and/or excipients and/or one or more pharmaceutically acceptable carriers may be used. The liquid that is to undergo freeze-drying or lyophilization preferably comprises all components desired in a final reconstituted liquid formulation.

[076] The pharmaceutical compositions of the invention may be administered locally to an eye using a variety of routes including, but not limited to, topical, through an ocular implant or direct injection into the eye. In particular embodiments, the pharmaceutical composition of the invention is administered locally to the eye using intravitreal injection, subconjunctival injection, sub-tenon injection, retrobulbar injection, suprachoroidal injection, intrascleral injection, intracorneal injection, subretinal injection or intracameral injection; especially intravitreal injection. In some embodiments, the composition is administered using a microneedle, for example, through intrascleral or intracorneal injection.

[077] In some embodiments, the composition is administered using an ocular implant, for example, a biodegradable implant such as those made from, for example, polylactic acid (PLA), polyglycolic acid, poly(lactide-co-glycolide) (PLGA), cross-linked gelatin derivatives, hypromellose, polyesters and/or polycaprolactones; or a non-biodegradable implant such as those made from, for example, polyvinyl alcohol, ethylene vinyl acetate, silicon and/or polysulfone capillary fiber.

[078] In some embodiments, the composition of the invention is formulated in a sustained release formulation or depot. Exemplary sustained release formulations or depots include a microsphere; matrix; emulsion; lipid-based; polymer-based; nanomicelle; micelle; nanovesicle such as a liposome, noisome, transfersome, discome, pharmacosome, emulsome or spanlastic, especially a liposome; microparticle; nanoparticle such as a nanocapsule or nanosphere composed of e.g. lipids, proteins, natural or synthetic polymers such as albumin, sodium alginate, chitosan, PLGA, PLA and/or polycaprolactone; or in situ gel such as an in situ hydrogel drug delivery system.

[079] In some embodiments, the composition of the invention is formulated for topical administration to the eye. Thus, the composition may be in the form of an eye drop, gel or ointment; especially an eye drop. The composition may be in a single unit dose or multiple unit dose form.

[080] The amount of therapeutically effective compound that is administered and the dosage regimen for treating a disease condition with the compounds and/or pharmaceutical compositions of the present invention depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, the particular compound employed, as well as the pharmacokinetic properties (e.g., adsorption, distribution, metabolism, excretion) of the individual treated, and thus may vary widely. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the compound to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain an active ingredient in the range of about 0.001 mM (1 pM) to about 1 mM (1000pM), from about 0.01 mM to about 0.5 mM, or from about 0.15 mM to about 0.25 mM. For example, MFA may be administered in an amount of about 0.001 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM, 0.55 mM, 0.6 mM, 0.65 mM, 0.7 mM, 0.75 mM, 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM or 1 mM, which is preferably administered by intravitreal, conjunctival or scleral injection to the subject in need thereof. The daily dose will typically be administered in one or multiple, e.g., two, three or four, doses per day.

[081] The agents of the present invention may be administered along with a pharmaceutical carrier, diluent or excipient as described above. Alternatively, or in addition, the compounds may be administered in combination with other agents, for example, antidiabetic therapeutic agents, or VEGF inhibitor drugs.

[082] In accordance with various embodiments of the present invention the agent that uncouples a retinal cell network, for example a connexin inhibitor such as Mefenamic acid may be formulated or administered in combination with one or more other therapeutic agents. Thus, in accordance with various embodiments of the present invention, the agent may be included in combination treatment regimens with surgery and/or other known treatments or therapeutic agents, and/or adjuvant or prophylactic agents. In one embodiment the agent is a prodrug or a potential prodrug and the like.

[083] A number of agents are available in commercial use, in clinical evaluation and in pre-clinical development, or are listed, for example, in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 12th Ed., 1996, and subsequent editions, the entire contents of which are incorporated herein by reference.

[084] In other embodiments, the agent that uncouples a retinal cell network as described herein may be administered in combination with a growth factor inhibitor. Suitable growth factor inhibitors include, but are not limited to, a vascular endothelial growth factor (VEGF) inhibitor, such as ranibizumab, aflibercept, bevacizumab, pegaptanib, conbercept, abicipar pegol (MP0112) and MP0250; a platelet derived growth factor (PDGF) inhibitor, such as E10030 (anti-PDGF PEGylated aptamer), trapidil and pegpleranib; and pharmaceutically acceptable salts and combinations thereof. In some embodiments, the one or more pharmaceutically active agent is a VEGF inhibitor selected from the group consisting of ranibizumab, aflibercept, bevacizumab, pegaptanib, conbercept and pharmaceutically acceptable salts and combinations thereof.

[085] In other embodiments, the agent that uncouples a retinal cell network as described herein may be administered in combination with agents that increase the levels of neuronal nitric oxide synthase, such as L-arginine.

[086] Combination regimens may involve the active agents being administered together, sequentially, or spaced apart as appropriate in each case. Combinations of active agents including compounds of the invention may be synergistic.

[087] The co-administration of the gap-junction inhibitors may be affected by being in the same unit dose as another active agent, or the inhibitors and one or more other active agent(s) may be present in individual and discrete unit doses administered at the same, or at a similar time, or at different times according to a dosing regimen or schedule. Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial compound to be current when the second or later compound is administered, especially where a cumulative or synergistic effect is desired.

[088] Embodiments of the invention will now be discussed in more detail with reference to specific examples which are provided for exemplification only and which should not be considered as limiting the scope of the invention in any way.

Methods

[089] As exemplified herein a gap junction inhibitor such as MFA injected intravitreally into the eye not only slows myopia development but reverses the direction of eye growth indicating that inhibitors of gap junctions such as MFA can slow, prevent, or reverse myopia.

[090] Accordingly, provided herein is a method of treating or preventing myopia by administering to a subject in need thereof an effective amount of an agent that inhibits retinal gap junctions and/or uncouples retinal cell networks. In one embodiment, the agent is a non-specific inhibitor of gap junctions such as MFA. In another embodiment, the agent is an inhibitor of one or more connexins. For example, 0x36, 0x43, 0x45, 0x50, 0x57, or 0x60, or any combination thereof. The myopia may be induced myopia, such as lens- or instrument-induced myopia, form deprivation myopia, index myopia, simple myopia, early or late-onset myopia, progressive myopia, degenerative myopia, pathological myopia or pseudomyopia. The myopia may be, for example, low, medium or high myopia. The myopia may be, for example, congenital myopia, childhood-onset myopia or adult-onset myopia.

[091] In one embodiment, agents such as MFA and related specific inhibitors that target Horizontal cell networks and/or connexins and/or amacrine cell networks, in particular nNOS amacrine cells, are used to treat or prevent myopia. For example, MFA and the related specific inhibitors may be used to slow myopia development and/or treat excessive eye elongation.

[092] Various routes of administration may be employed in connection with the methodology of the present invention, including but not limited to, intravitreal injection, intraocular injection, topical administration, or nasal administration. In an embodiment of the invention the agent is administered by intravitreal injection to a subject in need thereof. In another embodiment disclosed herein the agent is administered by topical administration to a subject in need thereof (e.g., by eye drop).

[093] Administration may be, for example, once per day, twice per day or multiple times per day. By way of example, an agent that inhibits retinal gap junctions and/or uncouples retinal cell networks may be administered by implantation. In one embodiment the agent may be conjugated to, or coated on, the surface of a contact lens or the contact lens may be impregnated with the agent. Alternatively, the agent may be administered by injection directly into a specific tissue or region of the eye, such as into the conjunctiva or sclera. Thus, the agent may be administered by, for example, intravitreal, conjunctival or scleral injection, or an intraocular implant or other slow-release delivery method. The agent may be administered topically such as in eye drops, an eye wash solution, an ointment or gel. Preferably, the agent is administered in an eye drop formulation, via a contact lens, or via injection (e.g., intravitreal injection).

[094] The agent of the invention can be formulated for intraocular injection, topical administration to the eye, or oral administration. Agents may be administered in the form of pharmaceutical compositions where the compositions may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. In an exemplary embodiment, the compositions may be administered as injectable solutions or in a form or vehicle suitable for topical administration (such as eye drops, eye wash, ointment, gel or contact lenses). Examples

Animals and Housing

[095] Tri-coloured domestic guinea pig pups (Cavia porcellus) were reared in opaque boxes with wire lids (590 x 410 x 210mm) illuminated by overhead light emitting diodes diffused through a 3mm thick Perspex screen positioned 200 mm above the lid. Lights operated on a 12-hour light-dark cycle and food and water was provided ad libitum.

Experimental Design

Experiment 1a

[096] Guinea pigs were divided into one of five groups. Group 1 wore a -5D or -6D lens on one eye for 2 weeks uninterrupted (n=19); Groups 2-5 were treated the same as in Group 1 , but the negative lens was removed each day for two x 1 hr periods to interrupt myopia development. During the interrupt periods, animals were exposed to darkness (Group 2, n=9), broad white light (Group 3, n=7), cool white light (Group 4, n=10), or lime light (Group 5, n=11). Data corresponding to Experiment 1a is represented in Figure 3a. and b. and Figure 5.

Experiment 1b

[097] Guinea pigs were divided into one of four groups. Group 1 (n=9) wore a -6D lens on one eye for 2 weeks uninterrupted and raised under cool white light. Groups 2-4 were treated the same as in Group 1, but the lens was removed each day for a single 1 hr period to interrupt myopia development. During the interrupt periods, animals were exposed to broad white light (Group 2, n=8), darkness (Group 3, n=9), or the lens was substituted by a black diffuser which blocked all light to that eye while animals were exposed to broad white light (Group 4, n=8). Data corresponding to Experiment 1b is represented in Figure 3c. and d.

Experiment 2

[098] Guinea pigs were divided into one of four groups. Group 1 (n=12) wore -6D lenses for 14 days in white light at a moderate illuminance (Luxeon Star, LXHL-MW1 D, 700lx). Group 2 (n=10) wore -6D lenses for 14 days in the same white light at a moderate illuminance (700lx) after which the lens was removed for three days. Group 3 (n=9) wore white diffusers for 14 days in moderate white light (700lx). From day 7-14 a thin white elastic hood was positioned over the diffuser of the experimental eye. Group 4 (n=9) wore - 6D lenses for 14 days in bright white light (composite of five monochromatic LEDs with total illuminance of 3000lx). Data corresponding to Experiment 2 is represented in Figure 4.

Experiment 3

[099] Guinea pigs were reared in bright (3000lx) cages and assigned to one of three treatment groups: Form deprivation (FD) only (n=11), FD + vehicle (n=11), and FD + Mefenamic acid (MFA) (n=13). All animals wore diffusers (days 1-14) and elastic hoods (days 7-14) over the right eye. FD + vehicle and FD + MFA animals also received daily intravitreal injection in the FD eye. Left eyes were untreated. Data corresponding to Experiment 3 is represented in Figure 6.

Myopia induction

[0100] A -6D negative lens (Gelflex, Perth WA, base curve 8.5mm, diameter 14 mm) or white diffuser molded from Perspex was mounted onto a Velcro® backing and attached to matching Velcro® arcs glued above and below the eye as previously described (Howlett and McFadden, 2009, Spectacle lens compensation in the pigmented guinea pig. Vision Research 49, 219-227). After one week, a thin white elasticized hood (made from latex) was worn over the diffuser of form-deprived animals.

Intravitreal injections

[0101] In Experiment 3, each day, a 10pl injectate of 3mM polysorbate-60, 0.6% saline (vehicle solution) or 10pl of a 3.73mM meclofenamic acid sodium salt dissolved in vehicle solution (MFA solution), was injected into the vitreous via the pars plana using a 4mm, 32g needle (Steriject, TSK, Japan) attached to a 10OpI syringe (1710 TLL, Hamilton). The volume of the vitreous is approximately 150 pl making the concentration of the MFA in the vitreous approximately 1/16th of the injectate (0.25 mM) with lower concentrations expected to penetrate the retina. Injections were performed under gaseous isoflurane. Local anesthetic (0.5% proxymetacaine hydrochloride, Alcaine, Alcon) was applied to the eye for one minute prior to injection and one drop of Conoptal (0.5% fuscidic acid, Dechra) was applied following injection. Injections were performed in a 90 mins window starting 90 mins into the daily light cycle.

Optical and Biometry Measures

[0102] A custom infrared keratometer was used to measure cornea power as previously described. Refractive error was measured using a Nidek autorefractor (AR-20Nidek; Gamagori, Aichi) following cycloplegia, induced using 1% cyclopentolate hydrochloride eye drops (Minims™, Bausche and Lomb) applied to each eye for three mins. On axis ocular biometry was measured using high-frequency (20MHz) A-scan ultrasonography under gaseous isoflurane (5% in 1.5L/min O2 until induction of anesthesia then reduced to 3% in 1.5L/min O2).

Euthanasia

[0103] Euthanasia was performed under far-red LED light following one hour of dark adaptation. Briefly, animals were deeply anaesthetized using gaseous isoflurane (5% in 1.5 L/min O2) and euthanized via intracardial injection of Lethabarb (160mg/kg, Pentobarbitone Sodium).

Cut loading

[0104] Cut loading was performed as previously detailed (Myles and McFadden, 2022, Analytical methods for assessing retinal cell coupling using cut-loading. PLoS One 17). Briefly, eyes were enucleated in Ames solution (Sigma-Aldrich) at room temperature (21 °C). The cornea and limbus were removed followed by the crystalline lens and vitreous. Eye cups were transferred to a well plate containing 10mL of Ames solution (36°C) bubbled with carbonox (95% 02/5% CO2). The retina was separated from the underlying choroid using a blunt dental spatula and photoreceptor was mounted down onto 0.22pm pore size Millipore membrane filter paper (Merck & Co.). Tissues were acclimatized for 15 mins in darkness in the Ames solution and then briefly removed from solution and cut along the superior and inferior axes with a size 11 scalpel blade dipped in 3% biotin derivative, N-(2-aminoethyl) biotinamide hydrochloride (Neurobiotin™ Tracer, Vector Laboratories) diluted in Ames solution. The tissue was returned to the Ames solution for 25 mins before being removed from the bath solution and fixed in 4% paraformaldehyde (4% wt/v, diluted in 0.1 M phosphate buffer) at room-temperature (for 30 mins). Retinas were washed in 1 x PBS (for 30 mins) and reacted with Alexa-Fluor 488 conjugated streptavidin (ThermoFisher

Scientific, diluted 1 :100 in 0.5% Triton-x in PBS) overnight at 4°C. Retinas were washed in 1 x PBS and mounted, photoreceptor side down, onto microscope slides using anti-fade Vectashield aqueous mounting medium (Vector Laboratories).

Coupling Analysis

[0105] Coupling strength of aHC networks was assessed as described previously (Myles and McFadden, 2022, Analytical methods for assessing retinal cell coupling using cutloading. PLoS One 17). Briefly, the mean fluorescence (C) of each aHC soma and the perpendicular distance (x) to the cut were measured using the oval tool in Fiji. The x- distance was divided by the mean distance from aHC soma-soma to determine distance (n), or the number of cell-separations from the cut. This series was fitted with the following differential equation series: etc.

[0106] Where kj is a rate constant describing dye transfer between coupled cells within a network (cells²/s), k_s is sequestration or loss of dye as it passes through the tissue (cells²/s) and V is the relative volume of the cell (equal to 1 as all HC are assumed to have equal volume). These equations were solved in MATLAB (mathworks) by first fitting a 2-parameter Gaussian curve to the original data to calculate the mean relative fluorescence at discrete intervals (C_n).

Results

[0107] The inventors found in a mammalian animal model of myopia (form deprivation) that low concentration of atropine (0.15%) had reduced efficacy in inhibiting myopia compared to high concentration of atropine (1%) (Figure 1). Given the undesirable side effects of high dose atropine and the limited efficacy of low dose atropine, a safe alternative to atropine is urgently needed for the treatment of myopia.

[0108] While it is well known that bright light inhibits myopia, the inventors showed that dark periods also inhibit myopia development (Figure 3).

Coupling of retinal cell networks and myopia development

[0109] The inventors investigated the relationship between coupling of specific cell networks and the development of myopia, and the effects of uncoupling these specific networks.

[0110] It was found that coupling between A-Type Horizontal cells, which is mediated by 0x57 and 0x50, is strongly implicated in myopia control as the degree of coupling is bidirectionally modulated by the sign of defocus. Specifically, animals developing myopia from form deprivation or from -6D lens wear showed an increased cell coupling in the superior retina, which is reversed in animals experiencing inhibition of myopia development or recovery from myopia (Figure 4). [0111] Furthermore, it was found that coupling between nNOS amacrine cells, which is mediated by C36 and Cx45 (see Figure 2 illustration), was also affected in myopic retina. Specifically, coupling is enhanced in the superior retina in form-deprived eyes but relatively reduced in eyes recovering from myopia because of being exposed to 1 hr daily periods where the -6D lens was removed and animals were exposed to either darkness or lime light during these periods. (Figure 5). In nNOS amacrine cells, nitric oxide modulates Cx45 connexins, while dopamine modulates Cx36 connexins. One of the inventors has previously shown that nNOS expression in these cells is reduced in myopic eyes and a specific substrate of nNOS can inhibit myopia progression when formulated as an eye drop.

Inhibition of retinal gap junctions and uncoupling of retinal cell networks

[0112] Here, the inventors found that a non-specific inhibitor of retinal gap junctions Mefenamic acid (MFA) is effective in uncoupling both A-Type Horizontal cells (Figure 6a.) and displaced nNOS amacrine cells in a dose dependent manner. The inventors demonstrated that MFA injected intravitreally into the eye not only uncouples cell networks but acts to slow the development of myopia (Figure 6c.). Importantly, the data indicate that MFA acts to reverse the direction of eye growth (Figure 6 f., i., j.) and thus it could be used to slow, prevent and reverse myopic eye elongation. Furthermore, this effect is only weakly mediated via dopamine and by implication, Cx36, and therefore the effect may act via other retinal connexins including Cx45, Cx60, Cx57. The half-life of MFA when injected into the vitreous of the eye was shown to be approximately 2 hours (Figure 6b.). Daily intravitreal injections of MFA strongly inhibited myopia compared to control animals (Figure 6c.), despite all-day long exposure to a myopiagenic stimulus and the short half-life of MFA (Figure 6b.).

[0113] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:

1 . A method of treating or preventing myopia in a subject comprising administering to the subject a therapeutically effective amount of an agent that uncouples a retinal cell network.

2. The method of claim 1 , wherein the agent is a gap junction inhibitor.

3. The method of claim 2, wherein the gap junction inhibitor is a non-specific inhibitor of retinal gap junctions.

4. The method of any one of claims 1 to 3, wherein the gap junction inhibitor blocks channels comprised of connexin and/or pannexin proteins.

5. The method of claim 4, wherein the gap junction inhibitor is a connexin inhibitor of one or more of Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof.

6. The method of any one of claims 1 to 5, wherein the retinal cell network comprises a Horizontal cell and/or an amacrine cell.

7. The method of claim 6, wherein the Horizontal cell is an A-Type Horizontal cell.

8. The method of claim 6, wherein the amacrine cell is neuronal nitric oxide synthase (nNOS) amacrine cells.

9. The method of claim 1 , where the agent is in the form of a periodic exposure of an eye to short periods of darkness.

10. The method of any one of claims 1 to 8, wherein the agent is one or more of Mefenamic acid (MFA), flufenamic acid, meclofenamic acid, tolfenamic acid, orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX), CsHnOH, tonabersat, Cx43 oligonucleotide (5'-GTA-ATTGCG- GCA-GGA-GGAATT- GTT-TCT-GTC-3'),or a peptide selected from the group consisting of YPXGAG, a-Connexin carboxyl terminal RQPKIWFPNRRKPWKK - RPRPDDLEI, VDCFLSRPTEKT, YGRKKRRQRRRKQIEIKKFK, and LCLRPVGG -KQIEIKKFK.

11 . The method of claim 9, wherein the agent is Mefenamic acid (MFA).

12. The method of claim 11 , wherein the MFA is in a sustained release formulation or depot.

13. The method of any one of claims 1 to 12, wherein the treatment comprises reversing, inhibiting or slowing the progression of myopia.

14. The method of any one of claims 1 to 13, wherein the treatment comprises reversing, inhibiting or slowing excessive eye elongation, or eye growth.

SUBSTITUTE SHEET (RULE 26)

15. The method of any one of claims 1 to 12, wherein the treatment comprises reversing established myopia.

16. The method of any one of claims 1 to 15, wherein the agent is administered intraocularly or topically.

17. The method of 15, wherein the intraocular administration comprises intravitreal, retinal, conjunctival, subconjunctival, or scleral injection.

18. The method of claim 17, wherein the agent is administered intravitreally.

19. The method of 16, wherein the topical administration comprises eye drops, eye wash solution, ointment, gel, suspension, emulsion, or via a contact lens.

20. The method of any one of claims 1 to 19, wherein the myopia is axial myopia, refractive myopia, myopic astigmatism, simple myopia, early- or late-onset myopia, progressive myopia, degenerative myopia or pathological myopia.

21 . Use of an agent that uncouples a retinal cell network in the manufacture of a medicament for treating or preventing myopia.

22. The use of claim 21 , wherein the agent is a gap junction inhibitor.

23. The use of claim 21 or claim 22, wherein the agent is one or more of Mefenamic acid (MFA), flufenamic acid, meclofenamic acid, tolfenamic acid, orthoaminobenzoic acid, palmitoleic acid, Carbenoxolone (CBX), CsHnOH, tonabersat, Cx43 oligonucleotide (5- GTA-ATTGCG- GCA-GGA-GGAATT- GTT-TCT-GTC-3'),or a peptide selected from the group consisting of YPXGAG, a-Connexin carboxyl terminal RQPKIWFPNRRKPWKK - RPRPDDLEI, VDCFLSRPTEKT, YGRKKRRQRRRKQIEIKKFK, and LCLRPVGG - KQIEIKKFK.

24. The use of claim 22, wherein the agent is Mefenamic acid (MFA).

25. The use of claim 24, wherein the MFA is in a sustained release formulation or depot.

26. The use of any one of claims 22 to 25, wherein the gap junction inhibitor blocks channels comprising connexin and/or pannexin proteins.

27. The use of any one of claims 21 to 25, wherein the agent is a connexin inhibitor of one or more of Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof.

28. Mefenamic acid (MFA) for use in the treatment or prevention of myopia, preferably wherein the MFA blocks channels comprising connexin and/or pannexin proteins.

29. Mefenamic acid (MFA) for use according to claim 28 wherein the MFA is in a sustained release formulation or depot.

SUBSTITUTE SHEET (RULE 26)

30. The use of claim 28 or 29, wherein the connexin is one or more of Cx36, Cx43, Cx45, Cx50, Cx57, or Cx60, or any combination thereof.

SUBSTITUTE SHEET (RULE 26)