WO2005079781A1

WO2005079781A1 - Therapeutic and/or prophylactic method

Info

Publication number: WO2005079781A1
Application number: PCT/AU2005/000264
Authority: WO
Inventors: Sheila Crewther; David Crewther
Original assignee: La Trobe University
Priority date: 2004-02-25
Filing date: 2005-02-25
Publication date: 2005-09-01

Abstract

The present invention relates to a method for the treatment or prevention of myopia, which involves the administration of an agent which modulates the fluid content of the subretinal space, such as a diuretic or an ion channel agonist or antagonist. In other embodiments there is also provided the use of such an agent in the preparation of a medicament for use in the prevention or treatment of myopia and pharmaceutical or veterinary compositions for use in the prevention or treatment of myopia.

Description

Therapeutic and/or Prophylactic Method

Field of the invention The invention relates to a method for the treatment and/or prevention of myopia .

Background of the Invention Myopia or short-sightedness refers to a refractive error which causes the parallel rays of light entering the eye to come to a focus in front of the retina, necessitating optical correction for clear distance vision. "Short-sighted" eyes are usually abnormally axially elongated, with correlation and comparison studies in humans and animal models showing that in most cases myopia is primarily due to an enlargement of the vitreous chamber . Myopia affects the health and quality of life of more than one half of the world's adult population. In particular, there are many recent reports suggesting an increase over the last few decades in the prevalence and severity of myopia in young adults from several Asian countries . As higher degrees of myopia (greater than -6 Dioptres (D) of refractive error) are accompanied by significant visual impairment and greater risk of blindness from secondary complications, there are important issues relating not only to quality of life and individual health, but also to the impact upon public health management. Individuals with clinically significant myopia (greater than -ID) require optical correction and ongoing clinical monitoring. Recent estimates suggest that in the US alone, the cost of continuous monitoring and optical appliances required by patients with lower degrees of refractive error is approximately US $4.5 billion per year. Population statistics for western countries suggest that approximately 1% of 6 year olds are myopic, with the incidence increasing to 6 to 10% by 9 years and rising to between 15 and 25% by end of schooldays and to 35% of those over 25. Recent evidence suggests that myopia also continues to develop well into adulthood, especially among those with a life style requiring prolonged near work. Genetic factors appear to contribute a predisposition towards myopia, for instance with some Asian ethnic groups reporting a much higher incidence of myopia than similar groups in western countries. To date, however, research has not been able to clearly elucidate the process (es) which drive normal ocular growth nor the abnormal axial elongation which results in myopia. The refractive state of the eye depends on the balance between the axial length of the eyeball and focal length of the series of optical elements which are provided by the eye. At birth, the human eye is very much smaller than adult size and is typically hyperopic in refraction, with light from a distant object focusing behind the retina. Within the first two years of life the normal human eye grows in a coordinated fashion to 90% of the adult size. By this stage the main refractive elements of the eye, the cornea, the crystalline lens and the vitreous, are matched to the axial length of the eye to produce an image at the photoreceptors of the retina; the eye therefore has negligible refractive error and is referred to as being in a state of "emmetropia" . Refractive status in the adult human population is a highly leptokurtotic distribution around emmetropia, suggesting that emmetropization is an active process. The process of emmetropization is not well understood; however, the most significant determinant of any refractive error which may occur is the axial length of the vitreous chamber. Evidence from both humans and from animal models suggests that a perturbation of the clarity of the image at the retina induces abnormal axial elongation of the eye, particularly of the vitreous chamber, and results in refractive myopia. Most vertebrate species studied to date appear to be susceptible to persistent disruption the visual environment early in life by demonstrating axial elongation. Such observations have led to the development of several experimental animal models of refractive error which utilise primates, chicks, cats and tree shrews. The chick model has become the most popular due to the rapidity and reliability of its ocular growth response and refractive compensation to an experimentally-applied defocus . Primate studies, while confirming the results of the chick model, have generally added little new information. The most used paradigm for experimental refractive error research is associated with form deprivation by eyelid suture or the use of opaque translucent occluders, and is referred to as "Form Deprivation Myopia" (FDM) (Raviola and Wiesel, Neural control of eye growth and experimental myopia in primates. Ciba Found Symp . (1990) ,-155 :22-38; discussion 39-44). Myopic shifts in refraction and axial elongation which parallel those seen in animal models are also seen in young children following clinical form deprivation occasioned by corneal opacification, hemangiomas or extreme ptosis or phylctenular keratitis. It has been estimated that approximately 95% of human myopia is "axial" in nature. Monocularly-induced optical defocus induced by contact lens or spectacle wear in young animals, particularly chickens, also provokes refractive compensation and alteration in the axial length of the vitreal cavity of the eye receiving blurred vision. The size and direction of the compensatory shift in the axial dimensions and the refraction of the eye is dependent on the magnitude and sign of defocus . For example, one week of exposure to negatively defocusing lenses of -10D in chicks will induce an almost complete compensatory increase in vitreal chamber depth and shift in refraction (defocus induced myopia or "DIM") . Similarly, optical blur of +10D within the same time frame will inhibit normal axial growth of the eye and the vitreous chamber, and induce an almost complete compensatory shift in refraction (defocus induced hyperopia or "DIH" ) . FDM-mediated ocular growth and refractive compensation to lens-induced optical blur is not abolished after procedures which block conduction of visual information from the retina to the brain, such as optic nerve section and/or retinal ganglion cell blockade with tetrodotoxin. Similarly, the elimination of the accommodation response following lesion of the Edinger- Westphal nucleus or section of the ciliary nerve does not abolish FDM-induced refractive changes, suggesting that the detection of blur and its "sign" occurs primarily within the eye. It is likely, however, that at least some fine control of the response is centrally driven. A number of studies have used agents to perturb retinal function and disrupt the formation of refractive errors in the FDM, DIM and DIH models. Neurotransmitters and their agonists and antagonists, excitotoxins and gliotoxins have all been used to dissect retinal pathways, but in general have not clearly delineated the elements essential to either normal or abnormal ocular growth. Several studies have proposed the use of neuroactive compounds to interfere with the formation of myopia. Atropine for example, which antagonises muscarinic acetylcholine receptors, has been reported to retard the development of myopia in FDM models from several species. In US 5,461,052 the use of tricyclic muscarinic M2 antagonists was proposed for controlling abnormal postnatal growth of the eye, and muscarinic antagonists have entered clinical trials for treating myopia. In WO 01/52832 (PCT/US01/01692) it was proposed that during postnatal development therapeutic dosages of a nicotinic acetylcholine receptor antagonist could be used to inhibit the abnormal equatorial expansion of the eye and the development of myopia. In US 4,942,161, the use of drugs having beta-blocker activity, such as levobunolol, carteolol, befunolol and betaxolol, was proposed to treat the progression of infantile axial myopia. Despite the clear and long-felt need for a prophylactic or therapeutic non-surgical intervention for myopia, the above-mentioned approaches do not appear to have been widely accepted by practitioners in the field. Despite the FDM experimental model being available for over 20 years, no evidence has emerged which conclusively explains how defocus or form deprivation is able to increase or retard the growth of the vitreous chamber. Crewther (2000) provided a discussion of this long-standing problem and suggested possible models for the mechanism of refractive error correction. The contents of this citation are incorporated herein by cross-reference . Crewther (2000) suggested a variety of possible vectors which may be involved in the control of ocular shape, including ion species, proteins, cytokines and chemokines released from the retina or Retinal Pigment Epithelium (RPE) , the neurotransmitter dopamine or even water. Crewther indicated that several putative mechanisms had been advanced to explain the induction of myopia, although none had been proven. In the "choroidal push" model, the choroid is thought to actively push the retina into focus. Similarly, when the optical image is behind the retina (as in hyperopia) , the choroid is proposed to be responsible for pulling back the retina towards a situation of better focus. In the "scleral sculpting" model, defocus in the visual stimulus has the effect of reducing responses in the retinal neurons which code for contrast. The reduced retinal signal passes through the RPE and choroid (or via central connection) to reach the sclera. A third model suggested was that the RPE acts as a controller of fluid between two reservoirs, the vitreous chamber and the choroid. Growth of the eye, or inhibition of growth, was proposed to result from stimulation via factors either transported across the RPE or generated by intrinsic responses to biomechanical stress. Other hypotheses regarding growth control were also reviewed in Crewther (2000) , including the hypothesis that the "ciliary muscle-choroid layer behaves like a solid sheet of smooth muscle, so that it is able to resist part of the intraocular pressure and to regulate scleral stretch in the growing eye" . To date, however, there has been no convincing evidence published that any one of these proposed models is more correct. Recent reviews of the role of ocular tissues in the formation of myopia (see for example McBrien and Gentle, 2003, Prog Ret. Eye Res 22:307-338; Saw et al., 2002, Br J Ophthalmol 86:1306-1311; and Schaeffel et al . , 2003, Clin Exp Optom 86: 295-307) have concentrated on the "scleral sculpting" model or the use of muscarinic receptor antagonists, and emphasize that the mode of action of proposed therapies is not well understood. None of these reviews discuss other models proposed by Crewther (2000) . An aim of the preferred embodiment of the invention is to overcome a problem associated with the prior art, whether referred to herein or otherwise. It is a particular aim to provide a method for the treatment and/or prevention of myopia, which method is preferably non-surgical .

Summary of the Invention In a first aspect, the invention provides a method for the treatment and/or prevention of myopia, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent which modulates the fluid content of the subretinal space. In one embodiment the method comprises administering a diuretic or a pharmaceutically-acceptable derivative thereof. In a particular embodiment, the diuretic is selected from the group consisting of a loop diuretic, a distal tubule diuretic, a potassium sparing diuretic, and an osmotic diuretic. In an alternative aspect, the invention provides the use of an agent which modulates the fluid content of the subretinal space for the treatment and/or prevention of myopia . In another aspect, the invention provides a use of an agent which modulates the fluid content of the subretinal space in the preparation of a medicament for use in the treatment and/or prevention of myopia. Also provided is a pharmaceutical or veterinary agent for treating and/or preventing myopia, which comprises an agent which modulates the fluid content of the subretinal space . Further provided is a pharmaceutical or veterinary composition for the treatment and/or prevention of myopia comprising an agent which modulates the fluid content of the subretinal space, together with a pharmaceutically- acceptable carrier.

Detailed Description of the Invention Without wishing to be bound by any theoretical mechanism, it is proposed that fluid in the subretinal space (the extracellular region that lies between the outer segments of the retinal photoreceptors and the retinal pigment epithelium) contributes to a vector for axial growth of the vitreal chamber, and that accordingly agents which modulate the fluid content of the subretinal space, and possibly the vitreal chamber, may be able to prevent or treat myopia. To modify the fluid content of the subretinal space, the inventors have employed diuretics, for example bumetanide, or ion channel blockers, such as BaCl₂, and have demonstrated the ability of such agents to modulate changes in the axial dimensions of the vitreal chamber and hence, modulate refraction of the eye in response to a myopia-inducing stimulus. It is proposed that other agents which are capable of modulating the fluid content of the subretinal space may also find utility in the method of the invention. In the present specification, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It must be noted that, as used in the present specification, the singular forms "a" , "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a diuretic" includes a single diuretic, as well as two or more diuretics; and so forth. All references, including any patents or patent applications, cited in this specification are hereby incorporated by cross reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. The passage "an agent which modulates the fluid content of the subretinal space" is used herein in its broadest sense and refers to agents or drugs which are able to modulate any one or more of : the volume of the subretinal space, the passage of water into and/or out of the subretinal space, and the passage of one or more ion or other solute species into and/or out of the subretinal space. In particular, it is contemplated that agents which modulate the potassium and/or chloride ion levels in subretinal fluid will influence fluid movement into and out of the subretinal space, as experimental evidence suggests that the levels of these ions are elevated in the subretinal space during myopia-inducing compensatory growth of the eye. Such an agent may act locally, for example on ion and/or water channels, transporters, exchangers or pumps which are present in the tissues of the eye and in particular tissues surrounding the subretinal space. A detailed description of the ion transport mechanisms of the retinal pigment epithelium is described in Gallemore, et al . (1997, Prog. Retin. Eye Res. 16:509-566, the contents of which are hereby incorporated by cross reference) . In addition, or alternatively, the agent may act systemically to alter the fluid balance of the body, for example an agent which promotes excretion of water from the body and which has the effect of reducing tissue edema. Particular targets for an agent which modulates the fluid content of the subretinal space may include ion and/or water channels, transporters, exchangers or pumps found in Mύller cells of the retina, on the apical and/or basolateral surfaces of the cells of the RPE. An agent which may demonstrate activity could be an agonist or antagonist of any one of such targets, or may control a number of such targets. These targets may also or alternatively be found on other tissues which control the movement of water between the vitreal chamber of the eye and the choroidal vasculature. In a preferred embodiment, the agent is able to modulate myopia-forming ocular growth, such as the increase in axial length of the vitreal chamber, but does not modulate ocular growth which reduces myopia or is hyperopia-forming. In another embodiment, the agent is able to modulate myopia-forming growth of the eye and is also able to modulate hyperopia-forming growth. A particular example of an agent is a diuretic. The term "diuretic" is used herein in its broadest sense and refers to an agent or drug which facilitates the excretion of water from the body. Most commonly, such agents or drugs increase the rate at which urine is created. A wide variety of diuretics are known and used for indications other than myopia (see, for example, Chapter 15, "Diuretic Agents" from "Basic & Clinical Pharmacology", 8th Edition, Katzung ed. , Lange Medical Books/McGraw Hill (2001) , the contents of which are incorporated herein by cross-reference) . The diuretic may be selected from the class of diuretics which is characterised by their action at the loop of Henle to inhibit NaCl reabsorption (also known as "loop diuretics"), such as bumetanide, furosemide, ethacrynic acid, torsemide or an organic mercurial; the class of diuretics which is characterised by ability to inhibit NaCl reabsorption in the distal convoluted tubule "a distal tubule diuretic" , such as a "thiazide" diuretic such as hydrochlorothiazide, bendroflumethazide, benthiazide, chlorothiazide, chlorthalidone, hydroflumeth^'iazide, idapamide, methyclothiazide, metolazone, polythiazide, quinethazone, or trichlormethiazide; from the class of "potassium-sparing" diuretics, which include agents which antagonize the effects of aldosterone at the cortical collecting tubule and at the distal tubule, for example spironolactone, triamterene, amiloride, or eplerenone; from the class of "osmotic diuretics" which are characterised by their ability to inhibit water uptake at the proximal tubule and descending limb of Henle' s loop, for example mannitol, isosorbide or glycerine; or combinations of diuretics from one or more of such classes. Particular combinations are also contemplated and include one or more "loop agents" and one or more "thiazides" , or one or more potassium sparing agents and one or more "loop agents" or thiazides. In another embodiment the diuretic may be selected from the class of "proximal tubule diuretics" which is characterised by activity at the proximal tubule of the renal tubule, for example a carbonic anhydrase inhibitor such as acetazolamide . While the above description characterizes a diuretic by its activity or target tissue at the level of the kidney, a person skilled in the art will recognize that many of such compounds also have similar activities at other sites of the body. The carbonic anhydrase inhibitor acetazolamide, for instance, is known to also have application in treating glaucoma and acute altitude sickness, by virtue of its ability to decrease the rate of formation of aqueous humor and cerebrospinal fluid respectively. Accordingly, it is contemplated that a myopic therapeutic or prophylactic activity of a diuretic may be a result of the ability of the diuretic to facilitate excretion of water from the body, for instance to decrease fluid buildup in the body as a whole and thereby influence the water balance of a particular tissue, and/or as a result of the ability of the diuretic to directly influence the movement of water in a tissue. In one embodiment the diuretic has activity, either directly or indirectly, to modulate the water balance in one or more regions of the eye, such as the vitreal chamber or the subretinal space. Specific diuretics may be selected from organomercurials such as chlormerodrin, meralluride, mercaptomerin sodium, mercumatilin sodium, mercurous chloride or mersalyl ,- purines such as pamabrom, protheobromine or t eobromine; steroids, such as canrenone, oleandrin or spironolactone; sulfonamide derivatives such as ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexalone, disulfamide, ethoxzolamide, furosemide, mefruside, piretanide, torsemide, tripamide or xipamide; thiazides or thiazide analogues such as althiazide, bendroflumethiazide , benzthiazid , benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclothiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, indapamide, methyclothiazide, paraflutizide, polythiazide, quinethazone, teclothiazide or trichloromethiazide; or uracils such as aminometradine; amiloride; chlozanil; ethacrinic acid; etozolin; isosorbide; mannitol; muzolimide; perhexiline; ticrynafen; triamterene; and urea. In a particular embodiment, the diuretic is a sulfonamide derivative such as bumetanide. In another embodiment the diuretic is acetazolamide or methazolamide . Where an agent, such as a diuretic is used or provided by the invention, it is also contemplated that pharmaceutically-acceptable derivatives of an agent may be used or provided. By "pharmaceutically-acceptable derivative" is meant any pharmaceutically acceptable salt, hydrate, ester, amide, active metabolite, analogue, residue, solvates or any other compound which is not biologically or otherwise undesirable and induces the desired pharmacological and/or physiological effect. In particular, care should be taken to select a "pharmaceutically-acceptable derivative" which does not negate the ability of the diuretic to modulate the fluid content of the subretinal space. For example, care should be taken to avoid the cationic portion of a "pharmaceutically-acceptable derivative" salt acting as an antagonist for an ion channel, thereby inhibiting ion movement through that channel, if the anionic half of the salt acts as a agonist for the channel. The salts of the agent, such as a diuretic, which may be used in the method or compositions of the present invention are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic , toluenesulphonic , benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Generally, the terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease. "Treating" as used herein covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes: inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition. "Prophylaxis" or "prophylactic" or "preventative" therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it. The term "myopia" refers to the resultant refractive error which occurs when the length of the eye is such that the refractive elements of the eye, such as the cornea and lens cause light from a distant object to be focused in a plane in the vitreous before the retina. Myopia in approximately 95% of human cases is associated with abnormal axial elongation of the eye, and in particular axial elongation of the vitreal chamber. Refractive myopia may be assessed clinically by retinoscopy or using an automated refracto eter. The axial dimensions of individual components of the eye may be conveniently measured with a-scan ultrasonography. The term "subject" as used herein refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a non-human primate or non-primates such as used in animal model testing. While it is particularly contemplated that the agents according to the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as Galliformes, Anseriformes, horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. Suitable mammals include members of the Orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Members of the Orders Perissodactyla and Artiodactyla are particularly preferred because of their similar biology and economic importance. For example, the Order Artiodactyla comprises approximately 150 living species distributed through nine families: pigs (Suidae) , peccaries (Tayassuidae) , hippopotamuses (Hippopotamidae) , camels (Camelidae) , chevrotains (Tragulidae) , giraffes and okapi (Giraffidae) , deer (Cervidae) , pronghorn (Antilocapridae) , and cattle, sheep, goats and antelope (Bovidae) . Many of these animals are used as feed animals in various countries. More importantly, many of the economically important animals such as goats, sheep, cattle and pigs have very similar biology and share high degrees of genomic homology. The Order Perissodactyla comprises horses and donkeys, which are both economically important and closely related. Pharmaceutical or veterinary compositions of the present invention or usable in the methods of the present invention comprise at least one agent, such as a diuretic, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents. Each carrier, diluent, adjuvant and/or excipient must be pharmaceutically ^acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for ocular (including tear film, anterior chamber, posterior chamber or subretinal administration) , oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the agent with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the agent with liquid carriers, diluents, adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product. The agent may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically-active agents, as long as the combination does not eliminate the activity of the agent. It will be appreciated that the agent and the other medicament may be administered separately, sequentially or simultaneously. As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the agent to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Each carrier must be pharmaceutically "acceptable" in the sense of being not biologically or otherwise undesirable i.e. the carrier may be administered to a subject along with the agent without causing any or a substantial adverse reaction. The agent may be administered orally, topically, or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous injections, aerosol for administration to lungs or nasal cavity, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques. The present invention also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in the methods of treatment of the present invention. The agent may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. The tablets may contain the agent in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets . These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for control release. Agents useful in the method of the invention, such as diuretics, can be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intraarterial, intraperitoneally, intramuscularly, subcutaneously, subconjunctivally, intracavity, transdermally or infusion by, for example, osmotic pump. For in vitro studies the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like. It may prove advantageous to utilize ocular administration routes in the method of the present invention, for example to avoid side-effects which may result from a systemic administration of an agent with diuretic activity. Administration may be by transcorneal route using drops or ointments or through the use of a corneal bandage lens or "depots" which are held in the inferior or superior conjunctival fornix, by intraocular administration including slow release, crystalline or encapsulated formulations provided subconjunctivally (see for example the use of a slow release formulation of nano- and micro-particles of a drug provided subconjunctivally in Kompella, Bandi and Ayalasomoyajula (2003) Invest. Ophthalmol . Vis. Sci. 44:1192-1201, the contents of which are incorporated by cross-reference) or to the aqueous or vitreous chambers or by subretinal administration. Intraocular administration may prove beneficial for maintaining agent concentrations at a higher level in the relevant tissues than for the rest of the body or for addressing particular physiologically active structures. One method for administering an agent to the vitreal chamber is described, for instance, in US patent application 2003/0060763, the contents of which are incorporated by cross-reference. The agent may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for: (a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions) ; tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue; (b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat; (c) topical applications, e.g. as a cream, ointment or spray applied to the skin; or •^• (d) intravaginally, e.g. as a pessary, cream or foam. As used herein, the term "therapeutically effective amount" is meant an amount of a an agent, such as a diuretic, effective to yield a desired therapeutic response, for example, to prevent or treat myopia. A "prophylactically effective amount" has a similar definition. The specific "therapeutically effective amount" will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the agent or its derivatives. Dosage levels of a diuretic, for example, are of the order of about lOμg to about 20 mg per kilogram body weight, with a preferred dosage range between about 0.5 mg to about 10 mg per kilogram body weight per day (from about 0.5 mg to about 3 g per patient per day) . The amount of diuretic that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to lg of a diuretic with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 0.5 mg to 500 mg of diuretic . Optionally the agent is administered in a divided dose schedule, such that there are at least two administrations in total in the schedule. Administrations are given preferably at least every two hours for up to four hours or longer; for example a diuretic may be administered every hour or every half hour. In one preferred embodiment, the divided-dose regimen comprises a second administration of the agent after an interval from the first administration sufficiently long that the level of agent in the blood has decreased to approximately from 5-30% of the maximum plasma level reached after the first administration, so as to maintain an effective content of agent in the blood. Optionally one or more subsequent administrations may be given at a corresponding interval from each preceding administration, preferably when the plasma level has decreased to approximately from 10-50% of the immediately-preceding maximum.

Brief Description of the Figures In the examples hereinafter, reference will be made to the accompanying figures as follows: Figure 1 represents the effect of the diuretic bumetanide compared to a saline control on the refractive compensation to lens-induced defocus of powers +10D, 0D and -10D in chickens. Figure 2 represents the effect of diuretics from different functional classes on the refractive compensation to lens-induced defocus of powers +10D, 0D and -10D in chickens. Bumetanide and furosemide are "loop diuretics" , while amiloride is a "potassium sparing diuretic" . Figure 3 represents the effect of the potassium channel blocking agent Ba²⁺ compared to a saline control on the refractive compensation to lens-induced defocus of powers +10D, 0D and -10D in chickens. Figure 4 represents the effect of the potassium channel blocking agent Ba²⁺ compared to a saline control on the depth of the vitreous chamber during the refractive compensation to lens-induced defocus of powers +10D, 0D and -10D in chickens. Figure 5 is a graph which represents the relative abundance of sodium, potassium and chloride ions as measured by X-ray microanalysis in the subretinal space of chickens as they recover from 9 days of occluder-induced FDM. The filled symbols represent ion abundance in the form-deprived eye, while the open figures represent the ion abundance in the non-form deprived eye. Figure 6 is a graph which represents the relative abundance of potassium ions as measured by X-ray microanalysis in the different layers of the retina and subretina of chickens as they recover from 9 days of occluder-induced FDM. The level of potassium is elevated principally in the subretinal space (SRS) of the FDM eye, when compared with the choroid (Chor) or the inner segments (IS) , the inner nuclear layer (INL) or the ganglion cell layer (GCL) of the retina.

Examples The invention will now be described in detail by way of reference only to the following non-limiting examples.

Example 1 Creation of FDM, DIM and DIH in Animal Models Day old male cockerel chicks of constant strain Gallus domesticus obtained from commercial hatcheries were raised in special light-tight ventilated boxes (size: height 0.5m, length 1.0m, width 0.75m), kept at a temperature of 34 °C for the first week and thereafter at 32 °C. Strict control of the 12hr day/l2hr night lighting cycle was employed. Unlimited food and water was available at all times. Individual chickens were fasted for 2 hours prior to anaesthesia. Monocular form and/or light deprivation was effected through the use of occluders molded from white or black styrene sheet affixed to a ring of Velcro fastening material, the mating surface of which has been glued to the periocular feathers using cyanoacrylate glue. Optical defocus was induced through positive, negative (+10D) or piano (0D) lenses. Modified human PMMA contact lenses were attached to the chicks in a similar fashion to the translucent occluders. These methods are described in detail in Crewther and Crewther (2003) Neuroreport 14 : 1233-7, the contents of which are incorporated herein by cross reference) . Animals were monitored twice a day for health and to ensure that goggles remained attached and that the lenses were always clean and free of dust . For experiments using hatchling chicks one week of monocular defocus or 1 week of form deprivation was sufficient to create measurable refractive and ocular growth changes . Refractive changes were measured by Retinoscopy (streak or spot) . This technique measures refractive state whereby the direction of motion of the reflection of a line or spot of light moved across the pupil is adjudged to be either in the same or opposite direction as the physical stimulus motion. The neutralization point is attained by application of a lens of sufficient power that the light source and that part of the retina providing the reflection are optically conjugate. Ocular growth changes were assessed with A-scan ultrasonography. In this technique, a beam of ultrasound (typically of frequency range 7 - 20 MHz) is directed into the eye through a probe coupled by a sound conducting gel to the cornea. Reflected pulses measured as time-of- flight are converted to distances through knowledge of the speed of sound in the various ocular compartments. Where minor surgical procedures were required or where hatchling chickens were assessed biometrically, animals were lightly sedated using a ketamine :xylazine mixture of lOmg/kg : lmg/kg. The day the observations were carried out varied depending on the particular type of experiment . Two weeks of Form Deprivation (FD) by using translucent occlusion or one week of negative (-D) lens rearing induced dramatic refractive myopia and significant ultrastructural modifications which were greatest in outer retina, retinal pigment epithelium (RPE) and choroid. Both FD and -D lens rearing induced abnormal axial growth and in particular vitreous elongation and DIM. This was accompanied by significant thinning of the retina and choroid and thickening of the cartilaginous sclera. Positive (+D) lens rearing for a week induced DIH, a relatively shorter axial length, a thickened choroid, but little ultrastructural change in the retina or sclera. Rapid refractive recovery following occluder removal in FDM was accompanied by dramatic swelling of the choroid - seen within 30 minutes of removal of FD, with concurrent changes in choroidal thickness and changes in the infoldings of the basal lamina of the RPE, indicative of fluid movement across the RPE to the lymphatic vessels.

Example 2

Effects of bumetanide on refractory compensation The effect of the diuretic bumetanide on refractive compensation to lens-induced defocus of powers +10D, 0D and -10D was examined in chickens. The drug was injected intravitreally in one eye as a single bolus of 5μl (representing approximately l/lOOth the volume of the vitreal chamber) of 10^~3M bumetanide dissolved in dimethylsulphoxide or in 2M NaOH, diluted with saline and brought back to pH 7.4 with 1 M HCl, on day 4 post-hatching, at the time of application of the lenses. Controls were represented by chicks injected with the same volume of physiological saline. The eyes of the animals were refracted by retinoscopy and the axial dimensions of the components of the eye were measured by ultrasonography (as described above) on day 9. The results of these experiments are illustrated in Figure 1. The results indicate a significant inhibition by bumetanide relative to saline injection of the compensatory change in refraction to negative lens defocus but surprisingly not to positive lens defocus (Student t- test, t(16)= -3.04, p=0.008). It is the inventors' understanding that bumetanide 's "univalent" activity in inhibiting the myopia-forming refractory compensation of the eye, but not hyperopia-forming compensation, has not been previously demonstrated for any compound investigated. This may represent a novel and unexpected mode of action for such a drug. Further experiments were carried out to determine whether other diuretics also possess the ability to inhibit refractive compensation in response to DIM or DIH stimuli. Groups of chickens were given a +10D,

-10D or no lens in one eye and 5μl of 10^"3M of either bumetanide or furosemide or amiloride dissolved in dimethylsulphoxide, with the DMSO vehicle alone as a control, on day 4 post-hatching. The results of these experiments are shown in Figure

2. Error bars represent the Standard Error of the Mean. Both Bumetanide and Amiloride were able to reduce the refractory compensation in response to a -10D lens. Different dosage regimens may be used to determine the optimal and minimal effective intravitreal dose of these and other agents, and this information may be used to calculate the equivalent dosage required for topical ocular administration or other routes of administration, such as oral administration.

Example 3 Effects of BaCl₂ on refractory compensation The non-selective potassium channel-blocking agent Ba²⁺ ion was used under similar conditions to that of Example 2. Intravitreal injections of BaCl₂ diluted in saline to give an effective vitreal concentration of 5mM were made, with control chicks injected with the same volume of the physiological saline diluent only. The results of these experiments on the compensatory refractive change in the eye are illustrated in Figure 3. The use of the potassium channel-blocking agent BaCl₂ prevented full refractive compensation to induced defocus for both the DIM and DIH models. The results of these experiments on the axial length of the vitreal chamber are illustrated in Figure 4. While saline injection resulted in longer and shorter vitreous chamber lengths for -10D and +10D rearing conditions respectively, the effect of Ba²⁺ ion was to decrease the effect of induced defocus on eye growth dramatically. Other agents, such as other diuretics, are selected on the basis of their actions on the ionic transport channels of the tissues influencing the ionic concentrations in the subretinal space. In the first instance the action of classical diuretic compounds on ocular response to applied defocus is examined. The choice of these compounds is influenced by the fact that these drugs have already been shown to be safe for prolonged chronic use in humans. These compounds may include • Osmotic diuretics such as Mannitol, isosorbide and glycerine; • Loop diuretics such as Furosemide which inhibit chloride transport and water movement across the RPE from the retina to the choroid via the Cl-channel of the RPE basolateral membrane; • Potassium-sparing diuretics such as amiloride which inhibit the Na/H exchanger at the apical membrane of the RPE and decrease acidification of the RPE cell; • Carbonic Anhydrase Inhibitors such as Benzolamide accelerate the rate of sub-retinal fluid absorption and enhance retinal adhesion; • Ouabain, a metabolic inhibitor which is used to block the Na/K pump of the RPE and PR; • 4,4' -diisothiocyanostilbene-2 , 2 ' -disulfonate (DIDS) which is used intravitrally to block the apical membrane NaHC0₃ cotransporter and basally to block the RPE basal membrane chloride channel; and • Mercury-derived antagonists to aquaporin channels on the RPE and Mύller cells. The drug dose and frequency of injection regime for such compounds are initially designed on the basis of published studies. However, dose response functions for the promising diuretics (in terms of refractive treatment efficacy) are established using standard dilution series. Evidence of efficacy following administration is in terms of reduction in refractive change following a defocus challenge and applicable to either myopia or hyperopia. Confirmatory evidence is through axial length changes, particularly vitreous chamber depth and (in the animal models) anatomical evidence of changes in the thickness of the choroid as evidence of changes in fluid flow.

Example 4

Effects of form deprivation and recovery from form deprivation on ion distribution

Experiments were carried out to test hypothesis that prolonged form deprivation would lead to increased K in the subretinal space (SRS) . The relative abundance of biologically important elements, potassium (K) , sodium (Na) , chlorine (Cl) , phosphorus (P) and sulphur (S) were measured in the ionic microenvironments of five regions across the entire retina/choroid of freeze dried tissue. The relative abundance of K was investigated as SRS volume changes during changes in illumination have been suggested to be regulated by hydrogen and K ions . Changes in the levels of these ions are thought to contribute to changes in the pH of the outer retina/SRS. The relative abundances of Na and Cl were measured to give an indication of relative salt content and osmolarity of the tissue. The measurement of P was used to assess cellular and in particular cell wall metabolism and is commonly used in magnetic resonance spectroscopy to assess the integrity of metabolic function. Elemental S abundance gives an indication of any change in proteoglycan constituents in the extravascular choroid and interphotoreceptor matrix of the SRS, as well as in the more mobile form of sulphates. Groups of chickens were subjected to 9 days monocular form deprivation from 5 to 14 days post hatching using an occluder as described above . The occluder was then removed, and the form deprived and control fellow eye removed at a variety of time points and processed for scanning electron microscopy based X-ray microanalysis according to standard techniques. The following methods were used in the preparation of specimens for Scanning Electron Microscopy (SEM) . Specimens were examined using either the SEM freeze-dried or bulk-frozen techniques. The former method is routinely utilised for relative abundance of elements in cellular tissue. Freeze-drying inevitably results in the redistribution of solutes in fluid filled vessels and hence is less suitable for large fluid-filled vessels. For freeze-dried tissue, samples which included retina/choroid/sclera were obtained as 5 mm buttons, trephined 3.5 mm from the end of the pecten at the central posterior region of the eyecup to ensure consistent localization for morphological analyses. The small tissue blocks were immediately fixed in liquid nitrogen slush and freeze-dried for 12 hours. The dry tissue was then cracked transversely and carbon coated (~20nm thick) for routine SEM. The relative abundance of sodium, potassium and chloride ions in the subretinal space of eyes as they recovered from form deprivation was assessed and compared to the non-form deprived eye. These results are presented in Figure 5. In addition, a comparison of the relative abundance of potassium throughout different regions of the retina is presented in Figure 6. At the time of occluder removal the relative abundance of K was significantly higher in the RPE/SRS area of the form deprived retina than in the fellow eye.

Potassium abundance was not significantly different in any other region of the retinae of the freeze-dried eyes at occluder removal. By comparison, the relative abundance of Na was significantly higher in all areas sampled, with Cl abundance significantly higher in the outer three areas

- choroid, SRS and Inner Segment regions, but not in the regions of the inner nuclear and ganglion cell layers. No significant differences were seen in P or S counts between the form deprived and fellow eyes in any region of the retina. While the levels of all three ions were elevated in the subretinal space of the FDM eyes at the time of occluder removal, and these levels decreased as the eye recovered from form deprivation. The level of potassium ions in the subretinal space fell during recovery to below the levels seen in the control eyes. As sodium ions are only passively shunted into and out of the subretinal space, these results suggest that changes in levels of potassium and/or chloride ions, which are actively transported, in the subretinal space may be a driving force in the formation of refractive compensation, and that accordingly, agents which modulate the levels of these ions or the ion-driven fluid movements which result from these changes may be useful in modulating myopia- creating refractive compensation.

Example 5 Primate models are used to extend the results of the chick model . In this example infant marmosets are reared with helmets containing defocusing lenses of powers appropriate to the primate (±5D, 0D in the first instance) . Diuretics from the classes listed above are administered via intravitreal injection, employing techniques similar to those used in the chick model . Given the slower response to deprivation and defocus of the primate when compared with the chick models, weekly injections of the diuretic or control solution are administered in order to maintain diuresis in the eye. In the second instance, primates (marmosets) are tested for topical and oral administration using candidate diuretics from the first experiment . In the third instance, the effects of defocus challenge as a function of age and the efficacy of diuretic treatment as a function of age are tested. In addition the effectiveness of slow release formulations as described above are tested. In such demonstrations, the evidence informing decisions regarding whether to administer diuretics prophylactically or only in response to the development of myopia is accumulated. In the fourth instance, human clinical trials are established on the basis of the results of primate experiments. The design of clinical studies which have examined the efficacy of other pharmaceutical compounds on the progression of myopia may be used as a basis for the design of clinical trials (see for example Bartlett, J.D., Niemann, K. , Houde, B., Allred, T., Edmondson, M.J. (2000) . Safety and tolerability of pirenzepine ophthalmic gel in pediatric myopic patients. Investigative Ophthalmology & Visual Science, 41(4), S303, Abstract No. 1598.) Without wishing to be bound by a proposed mechanism, it is suggested that the most effective target site for an agent such as a diuretic is the RPE. The RPE is a polarised epithelium which lies between and in close apposition to the photoreceptors of the retina and the choroid, the specialised vascular bed which serves the retina. One function of the RPE is to control the continuous absorption of fluid across the retina into the choroid vasculature. This fluid transport has been measured in a variety of species to be from 2.7μl/cm²/hr to llμl/cm²/hr, with humans at the high end of the measured range. The movement of fluid across the choroid is mediated by both passive and active transport components; the active transport component including ionic channels which are present in the apical (retinal) and basal (choroidal) membrane surfaces of the RPE and the passive transport component including the movement of water and ions through the tight junctions between individual RPE cells . The interface between the outer segments of the retinal photoreceptors and the apical membrane of the RPE forms the subretinal space. Ionic fluxes within this space and the volume of the space itself are known to alter in response to even minute transitions between light and dark. These ionic channels, together with water channels play a role in chorio-retinal homeostasis and disorders which are associated with fluid imbalance in the retina. Normal ion movements arising from the retina during light-dark transitions initiate fluid movement across the retinal pigment epithelium into the choroid and across the Mύller glia of the retina into the vitreous. It is proposed that a diuretic is able to reduce abnormal build up of fluid in the subretinal space by stimulating the passage of fluid across RPE cells to the choroidal vasculature. By modifying the pool of fluid residing in the subretinal space and possibly posterior chamber, it is proposed that a stimulus for axial growth of the posterior chamber is removed or reduced. Because the RPE is a polarised epithelium the type and number of ion channels may not be the same in both the apical and basal surfaces. The diuretic may act on channels found in one or more of these membranes in order to provide the proposed activity. 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

1. A method for the treatment and/or prevention of myopia, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent which modulates the fluid content of the subretinal space.

2. The method according to claim 1, wherein the agent is able to modulate myopia-forming ocular growth and not hyperopia-forming ocular growth.

3. The method according to claim 1 or claim 2, wherein the agent which modulates the fluid content of the subretinal space is a diuretic or a pharmaceutically- acceptable derivative thereof.

4. The method according to claim 3, wherein the diuretic is selected from the group consisting of a loop diuretic, a distal tubule diuretic, a potassium-sparing diuretic, and an osmotic diuretic.

5. The method according to claim 4, wherein the loop diuretic is selected from the group consisting of bumetanide, furosemide, ethacrynic acid, torsemide and an organic mercurial .

6. The method according to claim 4, wherein the distal tubule diuretic is a thiazide diuretic.

7. The method according to claim 6, wherein the thiazide diuretic is selected from the group consisting of hydrochlorothiazide, bendroflumethazide, benthiazide, chlorothiazide, chlorthalidone, hydroflumethiazide, idapamide, methyclothiazide, metolazone, polythiazide, quinethazone, and trichlormethiazide .

8. The method according to claim 4, wherein the potassium sparing diuretic is selected from the group consisting of spironolactone, triamterene, amiloride, and eplerenone .

9. The method according to claim 4, wherein the osmotic diuretic is selected from the group consisting of mannitol, isosorbide and glycerine.

10. The method according to claim 3, wherein the diuretic is a proximal tubule diuretic.

11. The method according to claim 10, wherein the proximal tubule diuretic is a carbonic anhydrase inhibitor.

12. The method according to claim 11, wherein the carbonic anhydrase inhibitor is acetazolamide.

13. The method according to claim 1 or claim 2, wherein the agent which modulates the fluid content of the subretinal space is an agonist or antagonist of an ion channel, ion transporter or pump, or a pharmaceutically- acceptable derivative thereof.

14. The method according to claim 13, wherein the antagonist is a potassium channel antagonist.

15. Use of an agent which modulates the fluid content of the subretinal space as defined in any one of claims 1 to

14 for the treatment and/or prevention of myopia.

16. Use of an agent which modulates the fluid content of the subretinal space as defined in any one of claims 1 to 14 in the preparation of a medicament for use in the treatment and/or prevention of myopia.

16. A pharmaceutical or veterinary agent for treating and/or preventing myopia, which comprises an agent which modulates the fluid content of the subretinal space as defined in any one of claims 1 to 14.

17. A pharmaceutical or veterinary composition for the treatment and/or prevention of myopia, which comprises an agent which modulates the fluid content of the subretinal space as defined in any one of claims 1 to 14, together with a pharmaceutically-acceptable carrier.