The present invention provides a new therapeutic method for treating myopia progression by blocking cell growth in the retina using alpha-2-adrenergic receptor agonists or antagonists. The blockage will stop the progression of the myopia that is caused by growth and elongation of the eye- globe. The present invention specifically relates to treatment of juvenile myopia or infantile axial myopia. The present invention also relates to a pharmaceutical composition comprising an alpha-2- adrenergic receptor agonist or antagonist. Treatment is for example performed by using eye-drops on children and young adults comprising said pharmaceutical composition.

Background

The present invention relates generally to methods and systems for the treatment of myopia progression. Myopia or 'near-sightedness/short-sightedness' is one of the more prevalent human visual disorders, affecting up to 25% of European and American population. The associated cost of correction and management has been estimated to be several billion dollars per year. Myopia is a rapidly growing disorder and affects some 1.6 billion people in the world, and the prevalence is estimated to increase to 2.5 billion affected people by the year 2020. Myopia typically develops during the early school years and tends to progress more rapidly in pre-teens than in older teenagers. The early onset myopia is also called juvenile myopia or infantile axial myopia.

Treatments that control the progression of myopia early would have a widespread benefit because severe myopia has been associated with serious eye problems later in life, such as early cataract, myopic macular degeneration or even blindness from retinal detachment. It is also important to slow the progression of myopia because it may keep children from developing severe side effects or complications from myopia correction.

It is believed that environmental factors contribute to the onset and severity of the progression of myopia. Particularly, intensive and excessive work during extended time in front of computer- or smart phone screens have been identified. Specific hereditary or genetic factors in individuals or in whole populations may also be factors and increase the risk for developing severe myopia.

Juvenile myopia is generally believed to be a result of eye globe/eye-axis elongation. The refraction focal point will therefore fall in front of the retina (Fig. 1). Even a minor reduction of the elongation of the eye globe may relieve the need for refractive correction and thus reduces the risk for future severe complications due to progressive juvenile myopia.

Manifest myopia is commonly treated by correction of the light refraction of the eye either based on eyeglasses, contact-lenses or by LASIK "laser-assisted in situ keratomileusis". Lasik is corneal laser surgery that leads to reshaping of the cornea. Because the development of myopia is progressive in children a number of techniques have been developed to control or prevent myopia progression. The techniques are: pharmacological delivery of low-dose Atropine, multifocal contact lenses, multifocal eyeglasses or orthokeratology (Ortho-K).

Atropine is an anti-cholinergic drug that produce cycloplegia and mydriasis by inhibition of the parasympathetic system. Both the sympathetic and parasympathetic systems play roles in our ability to adapt successfully to sustained periods of near-vision tasks by regulating accommodation and vergence. Given the association between sustained near-vision and the onset and development of myopia, an aetiological role in myopia of sympathetic and parasympathetic regulation, has been suggested. Effects of anti-cholinergic and beta-adrenergic drugs, such as atropine and the non- selective beta-adrenergic receptor blocker, Timolol, have been studied. Clinical trials in Hong-Kong and Japan showed that the rate of progression of myopia is lower in children given atropine eye drops than those given placebo. The effect of Timolol was less pronounced. Atropine is associated with short term and possible long term adverse events.

US 2009/0156606 show that combinations of sympathetic agonists e.g. Brimonidine or sympathetic antagonists with parasympathetic agonists or antagonists have also been suggested to improve vision by ameliorating the effects of manifest presbyopia, myopia, hypermetropia or astigmatism. The treatment-rationale is however based on regulation of long term adaptive accommodation and vergence.

Brimonidine is also used to treat glaucoma and to reduce the incidence of complications in connection with eye surgery or LASIK, such as subconjunctival hemorrhage, hyperemia, and photophobia and "flap" complications. Summary

The present invention relates to prevention of the development of progressive myopia by a pharmacological approach that inhibits or stops the eye-axis elongation of the eye globe. This elongation process is very prominent in infants and juveniles.

The present invention pharmacologically targets and inhibits cell proliferation and growth of specific cells in the retina. The target is active cells with a potential for proliferation in the retina such as retinal progenitor cells located within the neural retina or in the peripheral rim of the retina called in the ciliary marginal zone. The active cells may be MLiller cells, retinal progenitor cells derived from Muller cells or progenitor cells in the ciliary marginal zone of the retina. The intent is to negatively regulate how prone the Muller cells are to de-differentiate and therefore to proliferate. The negative regulation attenuates the cell proliferation and thereby growth of the eye.

The invention is to pharmacologically treat the eye with the aim to block or inhibit the cells to initiate proliferation using receptor antagonists or agonists. The invention utilizes the fact that different G- protein coupled receptors (GPC s) may positively or negatively regulate signaling pathways, which are directly associated to cell proliferation.

According to a first aspect of the present invention, there is provided a G-protein coupled receptor (GPCR) agonist or antagonist for use in the treatment of juvenile myopia or infantile axial myopia.

According to one embodiment of said use, the GPCR agonist or antagonist it is selected from an alpha 2-adrenergic receptor agonist or antagonist, an endothelin receptor agonist or antagonist or other GPCR agonist or antagonist that activate signaling molecules regulating cell proliferation. According to another embodiment, the GPCR agonist or antagonist is selected from alpha 2- adrenergic receptor agonist is selected from Brimonidine, 7-Me-marsanidine, Agmatine,

Apraclonidine, Cannabigerol, Clonidine, Detomidine, Dexmedetomidine, Fadolmidine, Guanabenz, Guanfacine, Lofexidine, Marsanidine, Medetomidine, Methamphetamine, Mivazerol, Rilmenidine, Romifidine, Talipexole, Tiamenidine, Tizanidine, Tolonidine, Xylazine, and Xylometazoline. According to a specific embodiment, the GPCR agonist or antagonist is Brimonidine.

According to a further embodiment, the GPCR agonist or antagonist is selected from alpha 2- adrenergic receptor antagonist is selected from Aripiprazole, Asenapine, Atipamezole, Cirazoline, Clozapine, Efaroxan, Idazoxan, Lurasidone, Melperone, Mianserin, Mirtazapine, Napitane,

Olanzapine, Paliperidone, Phenoxybenzamine, Phentolamine, Piribedil, Rauwolscine, Quetiapine, Norquetiapine, Setiptiline, Tolazoline, Yohimbine, Ziprasidone, and Zotepine.

According to a still further embodiment, the GPCR agonist or antagonist is selected from endothelin receptor agonist or antagonist is selected from endothelin receptor type A antagonists Ambrisentan, Atrasentan, BQ-123, BMS 182874, CL1020, Sitaxentan, and Zibotentan.

According to yet another embodiment, the GPCR agonist or antagonist is selected from endothelin receptor agonist or antagonist is selected from endothelin receptor type B agonists IRL1620, BQ- 3020, [Alal,3,ll,15]-endothelin, and Sarafotoxin S6c. According to a specific embodiment, the GPCR agonist or antagonist is IRL1620.

According to yet another embodiment, the GPCR agonist or antagonist is selected from endothelin receptor agonist or antagonist is selected from endothelin receptor type B antagonists BQ 788, IRL- 2500, and IRL 1038.

In the medical use mentioned above, the GPCR agonist or antagonist is suitably administered topically to the eye, incorporated in for example a liquid, a gel, an ointment, and other suitable pharmaceutical vehicle, and combinations thereof. In one embodiment, said GPCR agonist or antagonist is administered incorporated in an eye drop solution.

According to second aspect of the present invention, there is provided a method for treatment of juvenile myopia or infantile axial myopia, comprising administering a G-protein coupled receptor (GPCR) agonist or antagonist to a juvenile patient (5 - 20 years). Initiation of treatment may vary, but preferably it should be commenced in early phases of myopia development, which is probably between ages 6 and 15, for example 9-11 years.

In one embodiment of said method, the GPCR agonist or antagonist is selected from an alpha 2- adrenergic receptor agonist or antagonist, an endothelin receptor agonist or antagonist or other GPCR agonist or antagonist that activate signaling molecules regulating cell proliferation.

In a specific embodiment, the alpha 2-adrenergic receptor agonist is selected from Brimonidine, 7- Me-marsanidine, Agmatine, Apraclonidine, Cannabigerol, Clonidine, Detomidine, Dexmedetomidine, Fadolmidine, Guanabenz, Guanfacine, Lofexidine, Marsanidine, Medetomidine, Methamphetamine, Mivazerol, Rilmenidine, Romifidine, Talipexole, Tiamenidine, Tizanidine, Tolonidine, Xylazine, and Xylometazoline. In a further specific embodiment, the alpha 2-adrenergic receptor agonist is selected from Brimonidine.

In another specific embodiment, the alpha 2-adrenergic receptor antagonist is selected from Aripiprazole, Asenapine, Atipamezole, Cirazoline, Clozapine, Efaroxan, Idazoxan, Lurasidone, Melperone, Mianserin, Mirtazapine, Napitane, Olanzapine, Paliperidone, Phenoxybenzamine, Phentolamine, Piribedil, Rauwolscine, Quetiapine, Norquetiapine, Setiptiline, Tolazoline, Yohimbine, Ziprasidone, and Zotepine. In a further specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type A antagonists Ambrisentan, Atrasentan, BQ-123, BMS 182874, CL1020, Sitaxentan, and Zibotentan.

In yet another specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type B agonists IRL1620, BQ-3020, [Alal,3,ll,15]-endothelin, and Sarafotoxin S6c. In a more specific embodiment, the endothelin receptor agonist or antagonist is selected from IRL1620.

In still further specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type B antagonists BQ 788, IRL-2500, and IRL 1038

In the medical treatment mentioned above, the GPCR agonist or antagonist is suitably administered topically to the eye, incorporated in for example a liquid, a gel, an ointment, and other suitable pharmaceutical vehicle, and combinations thereof. In one embodiment, said GPCR agonist or antagonist is administered incorporated in an eye drop solution.

According to a third aspect of the invention, there is provided a pharmaceutical composition for treatment of juvenile myopia or infantile axial myopia, comprising a G-protein coupled receptor (GPCR) agonist or antagonist.

In one embodiment of the pharmaceutical composition, the GPCR agonist or antagonist is selected from an alpha 2-adrenergic receptor agonist or antagonist, an endothelin receptor agonist or antagonist or other GPCR agonist or antagonist that activate signaling molecules regulating cell proliferation.

In another specific embodiment, the alpha 2-adrenergic receptor agonist is selected from

Brimonidine, 7-Me-marsanidine, Agmatine, Apraclonidine, Cannabigerol, Clonidine, Detomidine, Dexmedetomidine, Fadolmidine, Guana benz, Guanfacine, Lofexidine, Marsanidine, Medetomidine, Methamphetamine, Mivazerol, Rilmenidine, Romifidine, Talipexole, Tiamenidine, Tizanidine, Tolonidine, Xylazine, and Xylometazoline. In a further specific embodiment, the alpha 2-adrenergic receptor agonist is selected from Brimonidine.

In another specific embodiment, the alpha 2-adrenergic receptor antagonist is selected from Aripiprazole, Asenapine, Atipamezole, Cirazoline, Clozapine, Efaroxan, Idazoxan, Lurasidone, Melperone, Mianserin, Mirtazapine, Napitane, Olanzapine, Paliperidone, Phenoxybenzamine, Phentolamine, Piribedil, Rauwolscine, Quetiapine, Norquetiapine, Setiptiline, Tolazoline, Yohimbine, Ziprasidone, and Zotepine.

In further specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type A antagonists Ambrisentan, Atrasentan, BQ-123, BMS 182874, CL1020, Sitaxentan, and Zibotentan.

In yet another specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type B agonists IRL1620, BQ-3020, [Alal,3,ll,15]-endothelin, and Sarafotoxin S6c. In a more specific embodiment, the endothelin receptor agonist or antagonist is selected from IRL1620.

In a still further specific embodiment, the endothelin receptor agonist or antagonist is selected from endothelin receptor type B antagonists BQ 788, IRL-2500, and IRL 1038. In a more specific embodiment, the endothelin receptor agonist or antagonist is selected from IRL1620.

The pharmaceutical composition may be a liquid, a gel, an ointment, and other suitable

pharmaceutical vehicle, and combinations thereof. The composition may also comprise suitable adjuvants. In one embodiment, the composition is adapted for topical application, such as an eye drop solution.

Brief description of the drawings

Fig 1. Schematic diagram showing an elongated eye. Fig 2. Bar graph showing the dose response for the effect of N-methyl-D-aspartate (NMDA) on cell survival in the chicken retina. The x-axis shows the time of measurement (14 days) and the y-axis degree of cell survival in percent. All solutions used comprised saline.

Fig 3. Image showing the eye-axis of a chicken eye.

Fig 4. Representative images of (A) NMDA-treated and (B) control eye, and (C) a bar graph showing effect of Brimonidine (BMD) on eye-axis elongation triggered by NMDA.

Fig 5. Western blot and bar graph showing phospho-ERKl/2 levels in chicken retina after endothelin receptor stimulation.

Fig 6. Western blot and bar graph showing phospho-ERKl/2 levels in primary chicken Miiller cells after endothelin receptor stimulation. Detailed description

Experimental model and data Animal model

We have used the chicken eye as a model to study progressive eye-axis elongation. Acute elongation was induced by administration of the excitotoxin NMDA.

Much of the current knowledge about eye-axis elongation, are built on studies of eyes treated with excitatory amino acids (NMDA or kainic acid, KA). Excitotoxins triggers eye-axis elongation and development of progressive myopia. These experimental treatments have been performed on chicken eyes. The chicken eye has a robust response with an increase of the eye-axis length with formation of myopia after exposure to excitotoxins. Studies of the progressive elongation of the young eye-axis in the chicken eye after NMDA treatment is therefore a good model for studies of progressive myopia.

We studied the effect of alpha 2-adrenergic receptor stimulation on the chicken eye-axis elongation after NMDA treatment. Alpha 2-adrenergic receptor stimulation was achieved by treatment with an alpha 2-adrenergic receptor agonist. NMDA, saline or Brimonidine solutions were intraocularly injected at embryonic day 18 and the eye axis length was studied after 14 days.

Fertilized White Leghorn eggs from a local breed were incubated at 38°C in a humidified incubator. After hatching, chicks were marked with a numbered plastic ring in the leg and moved to a poultry farm. The chicken were fed ad libitum and kept under standard conditions according to experimental animal guidelines.

Eye treatment and NMDA-triggered elongation of the eye-axis:

To determine the dose of NMDA, a pilot experiment was carried out. A single intraocular injection of 10 μΙ was made in embryonic day 18 chick embryos, with four different doses of NMDA, 0.1, 1, 10, and 100 μg. Eggs were opened at the blunt end and a hole was opened in the eggshell. The head was pulled and intraocular injection was performed through the amniotic membranes in the right eye with a 27-gauge needle in the center of the eye. After injection, the egg was closed with an adhesive tape and incubated. The effect of NMDA was analyzed on cross-sections at 14 days post-injection (0.1, 1, 10, 100 μg NMDA) and the effect was monitored by the extent of tissue damage and loss of cells. A concentration was selected that produced a minimum amount of damage as analysed by surviving cells, but with a response of the eye-axis elongation. Injection of 1 μg NMDA was used for further analysis (Fig. 2).

Determination of eye-axis length:

The eyes were dissected and the eye-axis length was determined as the perpendicular length from the tip of the cornea to the posterior-most aspect of the eye globe, as indicated in (Fig. 3).

Examples

Example 1 - Stimulation of alpha 2-adrenergic receptors using Brimonidine: Alpha 2-adrenergic receptor stimulation was achieved by treatment using the alpha 2-adrenergic receptor agonist Brimonidine (80 μg Brimonidine in 10 μΙ sterile saline, 0.15 M NaCI, UK 14,304 tartrate; Tocris Bioscience). NMDA-injured (1 μg NM DA in 10 μΙ) eyes were treated one hour before NMDA injection. Saline- and Brimonidine-only-treatment was also analyzed.

Axis length of treated eyes were measured and the NMDA treated eyes that were treated with saline had a longer eye-axis than the eyes that were treated with Brimonidine or the ones that had not received any NMDA (Fig. 4 and table 1). The axis length of eyes treated with only Brimonidine were similar to the eyes that were not treated with NMDA.

Table 1 - Eye axis length after NMDA and Brimonidine (BMD) treatment

Control (saline + saline)

NMDA (Saline + NMDA)

BMD + NMDA (Brimonidine + NMDA)

BM D (Brimonidine + saline)

Length in mm

The results show that Brimonidine can inhibit the elongation of the eye axis that was triggered by the NMDA treatment.

Example 2 - Stimulation of endothelin receptors

We analyzed if stimulation of endothelin receptors could trigger a growth regulation response in retina and Miiller cells. The Miiller cell contributes to eye growth and eye-axis elongation.

ERKl/2 is a major intracellular regulator of cell proliferation and growth. We analyzed the activation of ERKl/2 as a monitor for the growth regulation response in retina and Miiller cells after stimulation of endothelin receptors. Stimulation of endothelin receptors was achieved by using the endothelin receptor agonist IRL1620.

Treatment of the eye:

Injection of endothelin receptor agonists was performed as described for injection of NMDA. ERKl/2 was studied using western blot analysis after injections of endothelin receptor agonist IRL1620. ERKl/2 was analyzed 2, 4, 6 and 24 h after stimulation.

Treatment of Miiller cells:

Primary chicken Miiller cell cultures were established from E14 chick eyes. Retinas were dissected, dissociated and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% NCS, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C. Cultures were 4 weeks.

Primary cultures were ready to use when all neurons were gone and the cultures only contained MLiller cells. Prior to cell cultures treatments, chick primary MLiller cells were serum-starved for 5 h. Serum-starved Muller cells were supplemented with IRL1620 (5 μΜ). For control experiments, cells were treated with vehicles.

Western blot analysis of IRL1620-stimulated chicken retina increased Phosho-ERK (P-ERK) levels (Fig. 5). The results showed increased P-ERK levels 2 hours after IRL1620 treatment. Bar graph with densitometry of P-ERK1/2 levels normalized to total ERK levels. Bar graph is mean ± SEM, n=3 (**P<0.01, ***P<0.0001) analyzed by one-way ANOVA with Tukey's post-hoc test.

Western blot analysis of IRL1620-stimulated chicken Muller cells in culture increased P-ERK levels (Fig. 6). The results showed increased P-ERK levels 10 min after IRL1620 treatment. Bar graph with densitometry of P-ERK1/2 levels normalized to total ERK levels. Bar graph is mean ± SEM, n=3 (**P<0.001, ***P<0.0001) analyzed by one-way ANOVA with Tukey's post-hoc test.

The result showed that endothelin receptors stimulation elicits a growth regulatory response in retina and in Muller cells.