HK1075211A - Modulation of ocular growth and myopia by gaba drugs - Google Patents

Description

Modulation of eye growth and myopia by GABA drugs

Reference to related applications:

this application claims priority from U.S. provisional patent application No.60/329.655, filed on 16/10/2001, the contents of which are incorporated herein by reference.

Government rights:

the present invention is supported, in part, by foundation No. ey-07354 from the national ministry of health. The government has certain rights in the invention.

The invention belongs to the field of the following:

the present invention relates to the control of postnatal eye growth and myopia. In particular, the present invention relates to the effects of gamma-aminobutyric acid (GABA) on retinal mechanisms that affect eye development, and the effects of drugs and compositions that interact with GABA receptors on eye growth and refractive development.

Background of the invention:

it is a function of the fact that about 1 in every 4 individuals worldwide has myopia (commonly referred to as myopic eye). At least half of these cases are due to axial myopia, i.e., the eye is elongated along the visual axis. At birth, the eyes of a person tend to be relatively short in the direction of the visual axis, resulting in a tendency for the child to have hyperopia (commonly referred to as presbyopia). During infancy, as the eye develops, the cornea and lens length increases and the optical properties change. Ideally, no sharp vision correction is required at distance, and the eye is normal. However, if the visual axis of the eye extends too far, the distant image is focused in front of the retinal plane, resulting in axial myopia. On the other hand, if the visual axis length of the eye is still too short, the near image is focused behind the retinal plane, resulting in hyperopia.

Various treatments and drugs have been proposed for the mechanism of focusing in front of the eye. Most of them attempt to either block the focusing ability of the eye by topical application of a drug (called accommodation), or to eliminate the need for near focusing by using a positive lens, which will actually perform the near focusing function. Topical drugs that relax the eye's focus muscle ciliary muscle, known as ciliary muscle anesthetics, have been used for nearly a hundred years.

Significant progress has recently been made indicating that myopia can cause the eye to suffer from a decline in the retinal image. Axial myopia has been shown to be experimentally induced in birds or primates, where the retina of the eye is lost as an image, for example by suturing the eyelids or wearing image diffusion goggles (Weisel and Raviola, nature 266: 66 (1977)). Experimental myopia induced in primates, such as monkeys, closely resembles normal axial myopia in humans. Thus, the visual process apparently contributes to a feedback mechanism by which postnatal eye growth can be normally regulated and refractive error of the animal determined, suggesting that this mechanism is neurological and likely originates in the retina.

Convincing evidence has revealed that the retina plays a dominant role in correlating postnatal eye growth with visual input (Wallman, research on retina 12: 133-. And several retinal neurotransmitters have been suggested to be involved in the development of refraction and pathogenesis of myopia (Stone, 1997; Fischer et al, compare journal of neurology 393: 1-15 (1998); Fischer et al, Nature neuroscience 2: 706-. Various retinal neurotransmitters localize to one or another subset of retinal amacrine cells. Given the complex, but still insufficiently well-defined, nature of ocular growth regulated by visual image (Schaeffel et al, Vision study 39: 1585-. U.S. patent nos. 5,055,302; 5,122,522 and 5,356,892(Laties and Stone) disclose methods of controlling abnormal postnatal eye growth in mature animals using Vasoactive Intestinal Peptide (VIP), PHI or analogs of these peptides or nitroglycerin, respectively.

In contrast to numerous neuropharmacological drugs which affect experimental myopia, it was discovered that relatively few drugs can alter the growth and refractive development of an eye with intact vision input, probably because the vision-dependent mechanisms governing eye growth determine the effect of the drugs. For example, dopamine agonists, opiates and basic fibroblast growth factors, each inhibit shape-loss myopia (form-deprivation myopia), but none alter the growth or refractive effects of non-occluded eyes (Stone et al, 1989; Rohrer et al, Experimental eye research 58: 553-. In only one study, the developmental role of muscarinic antagonists to block the growth of non-occluded eyes and induce refractive changes in the distance-vision direction was observed (Cottriall et al, Experimental eye study 74: 103-111 (2002)). Other drugs reported to have an effect on the growth and refractive effects of the unscented eyes of chicks include neurotoxins such as kainic acid, N-methyl-D-aspartic acid and tetrodotoxin etc. (Stone et al 2001; Fischer et al 1998; Wildsoet et al, ophthalmology research and Vision 29: 311-.

GABA (gamma-aminobutyric acid) is a widely distributed inhibitory amino acid neurotransmitter, localized in the central nervous system and in the retina. In the retina of vertebrates, GABA is localized to large distinct populations of nerve cells (Nguyen-Legros et al, microscopic research techniques 36: 26-42: (1997) And related to signaling of amacrine and horizontal cells (Kolb, 1997; barnstable, a modern view of neurobiology 3: 520- (1993); slauguer, retinal and ocular studies progress 14: 293-312(1995). U.S. Pat. Nos. 5,385,939 and 5,567,731(Laties and Stone) disclose a composition for inhibiting abnormal axial growth of eye birth in mature animals comprising GABA_BReceptor antagonists and a method for slowing and controlling the progression of amblyopia in a primate by administering a gamma aminobutyric acid antagonist are disclosed. However, it has not been reported so far that GABA or its receptor is present in peripheral nerves of the eye, or non-retinal tissues of the eye.

Like many other vertebrates, chickens contain in their retina a number of GABA-based cells: the amacrine cells in the inner nuclear layer, the horizontal cells in the ganglion cell layer and some neurons, which are likely to be replaced, the inner and outer reticular layers have many nerve fibers (Fischer et al 1998; Agardh et al, ophthalmology and Vision 27: 674-. Thus, chicken has become a recognized model animal for retinal studies in the art, and several findings have demonstrated that it can represent other vertebrates, including humans and other mammals.

GABA receptors have traditionally been classified into three major subtypes: GABA_A，GABA_BAnd BABA_CThe receptor (Chebib et al, clinical laboratory Pharmacology and physiology 26: 927-940 (1999)). GABA_AAnd GABA_cReceptors are each composed of ligand-gated chloride channels. It is believed that most of GABA_AThe receptor is composed of 5 subunits from the multi-subunit class (. alpha.1-6,. beta.1-4,. gamma.1-3,. delta.,. epsilon.,. theta.and/or. pi.) (Barnard et al, Pharmacology review: 50: 291-313 (1998); Barnard, in "pharmacology of GABA and glycine neurotransmission" (edited by M * hler), Berlin, Springer, pp79-99 (2001)). GABA_CThe receptor consists of one or several three different rho subunits and it is not known whether this receptor is complexed with proteins of other subunit classes (Barnard et al 1998; Bormann et al, in "pharmacology of GABA and glycine neurotransmission" (edited by M * hler), Berlin, Springer, pp271-296 (2001)). Despite its unique pharmacology, structure, genetics and function (Bormann et al 2001), GABA has recently been introduced_CReclassification of receptors as GABA_AGABA of the receptor family_A0rSubtype (Barnard et al 1998). Accordingly, the general term "GABA" will be used herein_AThe receptor "for the large family of bicolor-sensitive GABA receptors, and" GABA_A0rReceptor "use for what was previously referred to as" GABA_CReceptor's, bixilin-insensitive p-containing GABA_AA receptor.

GABA_BThe receptor being coupled to adenylyl cyclase or Ca⁺⁺And K⁺A metabotropic G-protein linked receptor of a channel. GABA_BOne of the functions of the receptors is to regulate the release of neurotransmitters and neuropeptides (Bormann, trends in pharmacology sciences 21: 16-19 (2000); Bowery, in "the pharmacology of GABA and glycine neurotransmission" (M * hler, eds.), Berlin, Springer. pp. 311-328 (2001)).

GABA_A、GABA_A0rAnd GABA_BEach of the receptor subtypes is widely expressed in the retina of vertebrates (Lukasiewicz et al, cell developmental biology 9: 293-299 (1998)). In fact, GABA_A0rThe primary localization of receptors in brain tissue is the neural retina. Among the various types of retinal neurons, GABA_AReceptors are present in both pre-and post-synaptic positions. Discovery of GABA_A0rReceptors are mainly present in bipolar cells, but not absolutely. GABA_BReceptors tend to localize postsynaptic to amacrine and ganglion cells. The data obtained with chickens are in accordance with these general laws.

GABA has been shown by means of immunohistochemistry_AReceptors are present in the inner and outer retinal layers and of different typesIn the cell body of the retina without the apophyses (Yazulla et al, J.Compare. neurology 780: 15-26 (1989)). GABA_A0rReceptors have also been localized to two layers of reticulum apparently corresponding to the processes of bipolar cells (Koulen et al, J. Compare neurology 380: 520-532 (1997). in situ hybridization in chick retinas, GABA has been identified at retinal levels corresponding to horizontal cells, bipolar cells, amacrine cells and perhaps ganglion cell bodies_A0rmRNA (Albrecht et al, neuroscience Commission 189: 155- & 158 (1995)). However, heretofore, GABA has not been obtained_BBiochemical characterization of the receptors, nor their localization at the cellular level was found in the retina.

In the retina, GABA co-localizes and/or interacts with other neurotransmitters that may be involved in the control of eye growth (Stone, 1997; Stone et al, Proc. Natl. Acad. Sci. USA 85: 257- -260 (1988); Guo et al, Current Ocular Studies 14: 385 (1995)), which include dopamine (Stone et al 1989; Nguyen-Legros et al 1997; Kazule et al, Visio 10: 621-. U.S. Pat. Nos. 5,385,939 and 5,567,731(Laties and Stone) first demonstrated that GABA receptors are involved in ocular development and disclose a composition for inhibiting abnormal postnatal axial growth of the eyes of mature animals comprising GABA_BReceptor antagonists and a method for treating a condition by administering GABA_BThe antagonist slows down and controls the development of amblyopia (lazy eye) in the eyes of the primate. In subsequent reports, a number of GABA-containing retinal neurons were found that were relatively resistant to quisquilic acid toxicity, and experimental myopia still developed after administration of this neurotoxin to the eye. It was then proposed that retinal GABA might be somehow associated with the formation of myopic eyes (Fischer et al, 1998), but in addition to the inventors' findings described aboveGABA_BBeyond the preliminary studies of receptor antagonists, this has hitherto only been an as yet unproven assumption. Thus, until the present invention, with respect to GABA_BThe role of the receptor in postnatal eye growth control, refractive development or myopia development, without any direct supplementary evidence, nor further elucidation of any other GABA receptor subtype or GABA pharmacological mechanism involved, thus constituting an unpredictable property of the biological system, indicating that the function of retinal GABA is far from being recognized, remains an unmet need in the art. There is also a need for compositions and methods for affecting the development of postnatal developing eyes in both the axial and equatorial dimensions.

SUMMARY

The present invention provides direct evidence that agents that interact with gamma aminobutyric acid (GABA) receptors in the retina have an effect on eye development, and includes several compositions and methods for controlling eye growth and refractive formation during postnatal development, including control of myopia. In a controlled assay, the eyes of subjects were somewhat worn with one-sided goggles for the induction of myopia and received daily intravitreal injections of agonists or antagonists to the major GABA receptor subtype and the effect of these drugs on eye development was tested by means of refractometry, as well as ultrasound and caliper measurements.

Discovery of GABA_AOr GABA_A0rAntagonists of the receptor inhibit shape-loss myopia. GABA_AThe antagonist shows a greater inhibition of myopia formation in the equatorial direction than in the axial direction, GABA_A0rThe antagonists exhibit parallel inhibition in the axial and equatorial directions. During the test, GABA is found_A0rAgonists, but not any GABA_AAgonists, alter the myopic refraction of the test animal's eye wearing the goggles. GABA_BReceptor antagonists also are compared to GABA_BThe receptor agonist can more delay the development of myopia, and can inhibit axial growth than inhibit the expansion of the equator direction when wearing gogglesIt is much more efficient. The retinal GABA content in the eye wearing the goggles showed a slight decrease.

When administering drugs to the eye without goggles, GABA_AAnd GABA_A0rAgonists and antagonists may also alter eye growth, more so stimulating it. However, there is only one kind of GABA_AAgonists have been shown to induce myopic refraction. Several of these agents stimulate eye growth in the axial direction, but not in the equatorial direction. GABA_BAgonists and GABA_BAntagonists also stimulate eye growth, but do not alter the refractive effect.

Thus, according to the findings of the present invention, GABA is influenced_A，GABA_A0rAnd GABA_BA receptor agent that modulates the growth and refractive formation of the postnatal eye. The anatomical effects of these drugs on the eye further suggest that, rather than simply adjusting the size of the eye, the shape of the eye is adjusted. The retinal location of action, in the eye with the loss of shape, coincides with GABA and its receptors, and with the known ocular localisation of changes in retinal biochemistry.

It is therefore an object of the present invention to provide a GABA receptor agonist or antagonist, or other compound, effective in altering eye growth and refractive formation in young animals or children. The change may be inhibition or reversal of myopia, for example by inhibiting axial elongation or equatorial expansion in a myopic eye with the aid of a suitable agent. Such modification may also include stimulating eye growth and reducing hyperopia with the aid of suitable medicaments in order to inhibit or reverse hyperopia. Also provided is a method for controlling postnatal ocular growth and the development of ocular errors in a mature eye of a subject comprising modulating retinal levels of GABA in the mature eye of the subject by administering to the eye a therapeutically effective amount of at least one GABA drug or compound, or other class of drug.

It is a further object to provide several GABA receptor types affecting the intracorneal GABA receptor type in the maturing eye_A，GAGA_BOr GABA_A0rThe composition of (1); and to provide several methods, whichPreferably such a composition is administered in a therapeutically effective amount of at least one agonist of at least one type of GABA receptor in the retina of the eye. In another preferred embodiment, provided is the administration of a drug or compound comprising a therapeutically effective amount of at least one antagonist of at least one type of GABA receptor in the retina of the eye.

It is also an object to provide several of the methods and compositions described above wherein the modulating step comprises inhibiting or reversing myopia of the eye of the postnatal subject. It is preferable to reduce the axial length or depth of the vitreous cavity with a corresponding reduction in myopic refraction. In yet another preferred embodiment, a therapeutically effective amount of GABA is administered to the maturing eye in a carrier or diluent_AA receptor agonist or antagonist, such carrier or diluent having been buffered to a pH suitable for ocular administration. This GABA_AExamples of receptor antagonists are SR95531 or bicolor. In a complementary preferred embodiment, a therapeutically effective amount of GABA in a carrier or diluent is administered to the maturing eye_A0rA receptor agonist or antagonist, such carrier or diluent having been buffered to a pH suitable for ocular administration. One such GABA_A0rThe receptor agonist is CACA, one such GABA_A0rThe receptor antagonist is TPMPA. In another preferred embodiment, a therapeutically effective amount of GABA is administered to the maturing eye in a carrier or diluent_BA receptor agonist or antagonist, such carrier or diluent having been buffered to a pH suitable for ocular administration. One such GABA_BThe receptor antagonist is baclofen, one such GABA_BThe receptor antagonist is CGP 46381.

It is also an object to provide several of the methods and compositions described above, wherein the step of modulating is directed to inducing eye growth and reducing hyperopia (in the latter case by stimulating refractive displacement of myopia) in the postnatal subject, or a combination thereof. It is preferable to increase the axial length or depth of the glass cavity, correspondingly reducing the distance vision refraction (or increasing the near vision refraction), and reducing the tendency to become distance vision. In a preferred embodiment, it is administered toThe eye in maturity is exposed to a therapeutically effective amount of a GABA receptor agonist or antagonist in a carrier or diluent which has been buffered to a pH suitable for ocular administration. One such GABA_AThe agonist is muscimol (muscimol), one such GABA_A0rThe antagonist is TPMPA.

It is still another object of the present invention to provide a method for determining the effect of GABA agents used in controlling postnatal eye growth and the formation of ocular errors in the maturation of animals.

Additional objects, advantages and novel features of the invention will be set forth in part in the detailed description, examples and figures which follow, and in part will be obvious from the description, or may be learned by practice of the invention, all by way of illustration only, and not by way of limitation.

Brief Description of Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

FIGS. 1A-1C are graphical depictions of the effect of a drug on the refraction of an eye wearing goggles, i.e., the anti-myopia activity of the drug. FIG. 1A shows the results for GABA_ADrugs with receptor selective effect, FIG. 1B shows the effect on GABA_A0rDrugs with receptor selective effect, and FIG. 1C shows on GABA_BThe effect of a receptor-selective agent on refraction. Bars marked with cross-hatching indicate that the vehicle-treated, goggle-worn chicks were given only to compare the control group in order to distinguish the control group from the other groups, n being the number of chicks in each cohort. The data represents the difference of the eye wearing the goggles minus the contralateral control eye. P-value is used to represent the results of using one-way analysis of variance (ANOVA) on the difference between drug-treated, goggle-worn eyes and contralateral excipient-treated, non-goggle-worn eyes. n.s is not significant.

FIG. 2 is a schematic representation ofDescription of GABA_AAnd GABA_A0rThe effect of selective drugs (agonists and antagonists) on the eye size of the wearer, i.e. the activity of the drug to inhibit ocular overgrowth in myopia. The number of chicks per experimental group is shown in figure 1. The bars marked with cross-hatching indicate that the vehicle-treated, goggle-worn chicks were given vehicle alone to compare the control group in order to distinguish the control group from the other groups. The data represents the difference of the eye wearing the goggles minus the contralateral control eye. P-values are used to represent the results of using one-way anova for the difference between drug-treated, goggle-worn eyes and contralateral excipient-treated, non-goggle-worn eyes. n.s is not significant.

FIG. 3 is a schematic representation of GABA_BThe effect of a receptor-selective agent on the size of the eye wearing the goggles, i.e. the activity of the agent in inhibiting ocular overgrowth in myopia. The number of chicks per experimental group is shown in figure 1. The bars marked with cross-hatching indicate that the vehicle-treated, goggle-worn chicks were given vehicle alone to compare the control group in order to distinguish the control group from the other groups. The data represents the difference of the eye wearing the goggles minus the contralateral control eye. P-values are used to represent the results of using one-way anova for the difference between drug-treated, goggle-worn eyes and contralateral excipient-treated, non-goggle-worn eyes. n.s is not significant.

Figure 4 graphically depicts the effect of indicated drugs on refraction of an eye without goggles. Three drugs were identified in the anova of the population as exhibiting an effect on refraction, however, only muscimol induced statistically significant refractive changes in drug-treated eyes compared to contralateral vehicle-treated eyes. The bars are shaded to distinguish the effects of different doses from each other, however, in each picture in fig. 4, the shading coincides with the respective dose level. The P-values shown are used to represent the statistical strength of the drug effect tested using two-way repeated measures anova (single factor repeat, factor with eye as repeat). n.s-drug treated eyes were not significantly different than contralateral eyes treated with vehicle alone. + achieved statistical significance in the dose comparison, but did not achieve statistically significant effects in the treated eyes compared to the eyes treated with vehicle alone.

Figure 5 is a graphical depiction of the effect of drugs on the size of an eye without goggles, which affect at least one of the parameters. The number of chicks in each queue is shown in figure 4. The bars are shaded to distinguish the effects of different doses from each other, however, in each picture in fig. 5, the shading coincides with the respective dose level. The P-values shown are used to represent the statistical strength of the drug effect tested using two-way repeated measures anova (single factor repeat, factor with eye as repeat). n.s is not significant. The onium ions achieved statistical significance only in dose-eye interactions, but not in drug-treated eyes compared to contra-lateral eyes treated with only excipients.

Description of the preferred embodiments of the invention

The usual visual function of an animal or human eye is that the light forming the image passes through the lens, is received by the retina, which transmits this information to the optic nerve, which then transmits it to the brain. Retinal neurochemicals (i.e., neuroactive compounds) are key components in this visual process. Specifically, the light forming the image is sensed by the photoreceptors of the retina, i.e., the rods and cones. In the regular process of transmitting image information to the brain, retinal nerve cells associated with photoreceptors release neurochemicals and transmit electrical signals, conveying the information to adjacent retinal cells that are part of the cell network in the retina, thereby directing the composition and characteristics of the signals to the optic nerve. These photoreceptors function as transducers that convert light energy into electrical and/or chemical signals.

When the animal's eye is made to lose vision during postnatal growth (e.g. by fixing translucent or image-distorting goggles over the eye), or the eye is made to undergo retinal image degradation, the result is often abnormal eye growth leading to myopia. During the time that the image is lost or faded, it has been found that the metabolism of certain retinal neurochemicals has been altered, resulting in altered concentrations of these substances within the retina. In particular, it has been noted that during the time that mature avian or primate visual images are lost, intraretinal chemical changes occur simultaneously with ocular overgrowth leading to myopia.

The present invention includes several methods for controlling postnatal ocular growth and the development of refractive error by administering to the eye of a young, mature animal or human an ocular drug or composition that interacts with GABA receptors. The active drug acts by modulating retinal levels of GABA, which is shown to be reduced in the case of myopia. Although the growth response process of the eye to GABA drugs is complex, the evidence provided herein suggests that GABA receptor agonists or antagonists may alter eye growth, affecting both the development of shape-loss myopia and the growth of eyes with normal visual input. Although the change in retinal GABA concentration in shape-losing myopic eyes is marginal, the consistency of this change in a variety of test animals supports the idea that GABA-based retinal neurons are associated with ocular growth control. Given that GABA is expressed by different retinal neurons and that the localization of GABA and its receptors in the eye is also known, combined with these data and the fact that the findings of the present invention are further supportive of the following principles: the retina can regulate eye growth, and retinal GABA can regulate refractive formation.

In general terms, the formation or development of a disorder of visual error, such as myopia, hyperopia or amblyopia, can be inhibited in the eye of a post-natal maturing animal by controlling the presence of a neurochemical in the eye after birth, or by virtue of an agonist or antagonist of such a neurochemical, including environmental factors that alter such a neurochemical under conditions which normally cause myopia in the course of maturation of the eye of a young animal. Prevention or treatment of myopia may be achieved by administration of a neurochemical, agonist or antagonist thereof, or other composition that affects eye growth and refractive development. This can also be achieved in another way by affecting the tissue level and/or bioavailability of naturally occurring neurochemicals by administering drugs that otherwise interact with the synthesis, storage, release, receptor interaction, resorption or degradation processes of such naturally occurring neurochemicals, wherein the neurochemicals and agonists or antagonists thereof can affect the growth and refractive development of myopic or hyperopic eyes.

Despite the significant anatomical differences between primate and avian eyes, the image-loss induced myopia (shape-loss myopia) of chickens, as shown by studies on chickens and young monkeys, is very similar to that of primates. There is evidence for both animals that control of postnatal ocular growth is substantially confined to the eye, apparently from the retina. Newborn chicks were widely used in the studies related to the present invention because the chickens matured rapidly.

One useful chicken model is a loss of shape model in which the vision of one eye is blurred by means of goggles or eyelid sutures, resulting in the ipsilateral eye being dilated and becoming myopic. In this case, shape-losing myopia is used to identify agents that have the potential effect of preventing myopia in children. For the chicken model as described herein, some chickens were made to wear a single goggle for myopia induction and received daily intravitreal injections of agonists or antagonists against the major GABA receptor subtype. The eye was then studied by means of refractometry, as well as ultrasound and caliper measurements, and the effect of these drugs on eye development was tested. For comparison purposes, other chicks were also fitted with one-sided goggles and the GABA content was measured.

As the term is used in the present invention, an agonist or antagonist of a neurochemical is a compound that has an effect on the action of this neurochemical in the retinal tissue. Agonists are drugs that activate receptors, resulting in an intracellular response. Thus, agonists mimic the effects of endogenous regulatory compounds. For the purposes of the present invention, an antagonist of a neurochemical is a compound which antagonizes or blocks the action of the neurochemical on retinal tissue and which effectively inhibits the action of an agonist, thereby effectively inhibiting postnatal excessive or abnormal axial growth of the eye of a mature animal. The antagonists are effective under conditions that generally result in excessive or abnormal axial growth and/or equatorial expansion. Although intraocular administration is described herein and is generally preferred, systemic administration may also be employed where appropriate.

Action of GABA drugs on shape-losing myopia：

In various embodiments of the invention, agents from each type of GABA drug significantly alter the progression of myopia. Against GABA_AAntagonists of the receptor, but not agonists, show significant inhibitory activity against shape-loss myopia. In a preferred embodiment of the invention, both the receptor antagonists bichalloline and SR95531 significantly reduce the enlargement of the vitreous cavity of the eye wearing the goggles in the equatorial direction. Neither antagonist significantly changed the axial dimension of the eye wearing the goggles, and only SR95531 caused a slight reduction in myopic refraction. In any event, GABA_AAntagonists are all the first-choice representative drugs reported to primarily inhibit the growth of the eye wearing the eyewear in the equatorial direction.

However, in another preferred embodiment of the present invention, GABA_A0rReceptor antagonist GABA_AReceptor antagonists, have been shown to be more potent against experimental myopia. As shown by means of ultrasound measurements, TPMPA can substantially eliminate myopic refractive offsets and significantly reduce the axial length of the eye and the depth of the vitreous cavity. It also prevents the eye from expanding in the direction of the equator. GABA_A0rThe receptor agonist, CACA, has a modest effect on refraction of the eye wearing goggles, which may be biphasic, but CACA does not alter the measured size of the eye.

Selective drugs for GABA, both agonists and antagonists, exhibit some degree of anti-myopia activity. The antagonist CGP46318 is the most effective of these drugs, inhibiting myopia and limiting axial, vitreous and equatorial enlargement of the eye.

Effect of GABA drugs on eyes without goggles：

As with the eyewear-worn eye, agents from each type of GABA drug can affect the development of the non-eyewear-worn eye. In certain embodiments, with GABA_AAnd GABA_A0rDrugs with receptor subtype interactions proved to be the most potent, but on GABA_ASelective agents of the receptor exhibit less potent stimulatory effects. In a preferred embodiment of the invention, mixed GABA_AThe agonist muscimol (muscimol) has the strongest effect on the eye, not only increasing axial and vitreous chamber length, but also enlarging the equator diameter. Muscimol was the only drug tested that induced statistically significant myopic refractive shifts. Presumably, the non-goggle-wearing eye receiving the other medication may still maintain normal vision because the ocular optics compensate for the elongation of the axial component.

In another embodiment, GABA_AThe receptor antagonist SR-95531 also increased axial and vitreous cavity length, but its effect on refraction was not statistically significant, and it did not change the equatorial dimension of the unaided eye. For GABA_A0rDrugs whose receptors are active may also stimulate eye growth, where there is a selective increase in axial size. In another embodiment, the agonist CACA is shown to moderately stimulate axial growth without changing refraction or affecting the equator diameter.

In another embodiment, the comparison shows GABA_A0rThe receptor antagonist TPMPA stimulates axial elongation and depth of the vitreous cavity without refractive changes. The geometry of the TPMPA effect is significant because the dimension of the median line is actually reduced for the non-goggle-mounted eye treated with TPMPA.

The ability of GABA drugs to stimulate eye growth and induce refractive errors tending to myopia without goggles indicates that this agent may also be used to treat hyperopia. In the case of hyperopia, the eye tends to be relatively short, but stimulating eye growth corrects the problem. With the aid of GABA drugs, hyperopia (or "+") refractive errors of myopic eyes can also be reduced or corrected when the myopic (or "-") refractive offset subtracts or neutralizes the hyperopic refractive error. Because the growth and optical effects of hyperopia and myopia are opposite, the "induction of myopia" in an open eye establishes the possibility of treating hyperopia. This is another use of the invention, since hyperopia in children can lead to strabismus (cross-eye) and/or amblyopia (inert eye).

Basic pharmacological mechanisms

Since the action of GABA drugs is complex, their basic pharmacological mechanism cannot be clearly explained. In some cases, agonists and antagonists exhibit similar effects on growth. Even dose-response curves tend to coincide because some of the best effective drug dose is in the middle of the range measured. During the biological response to drugs, the U-shaped or inverted U-shaped dose-response curve (known as toxicant stimulation) has been increasingly recognized, Calabrese et al, pharmacologic scientific trend 22: 285-291(2001). In addition to the problems of bioavailability and other pharmacokinetic factors, and not intended to be bound by a hypothesis, the present inventors have suggested that the visual response may reflect the diversity of GABA receptor subtypes in the retina (Barnard et al, 1998; Barnard, 2001; Bormann et al 2001).

Also, in some cases, biochemical changes to the eye as a result of treatment according to the compositions and/or methods of the present invention may not be detectable by means of existing methods. Nevertheless, this change still occurs and is sufficient to achieve control of the eye, or to change the growth and/or refraction of the eye.

The molecular subunit composition of retinal GABA receptors has not been widely characterized, and within the major GABA receptor subpopulation, drugs currently being studied may interact with multiple receptor subtypes. Thus, ocular growth in response to GABA drugs may reflect a complex distribution of GABA receptors in the retina, a particular type of GABA receptor subunit in the retina, GABA-based neuronal interactions with other retinal cells involved in ocular growth control, and/or different drug affinities for specificity or multiple GABA receptor subunits. The knowledge of the mechanism underlying the present invention has no effect on the present invention itself.

GABA drugs and eye shapes

The effects of neurotoxins on the vitreous cavity (Wildsoet et al, 1998; Calabrese et al 2001), including the bulging of the vitreous cavity in the eye with a single visor (Wallman, 1993, Wallman et al, science 237: 73-77(1987)), the effects of dopaminergic and muscarinic agents in inhibiting axial elongation but not in inhibiting equatorial enlargement in experimental myopia (Stone 1997; Stone 1989; Stone et al, Experimental eye Studies 52: 755 758-. However, since it is speculated that the growth effects disclosed herein may reflect retinal signaling, the ocular response to GABA drugs is equally related to the control of ocular morphology by the retina. Depending on the particular drug and the presumed corresponding receptor subtype, and depending on whether visual input was previously impaired or intact, GABA agents have been shown to have a generalized effect on the shape of the eye, either selectively in the axial or selective equatorial direction.

For example, GABA_AAnd GABA_A0rReceptors may have different roles in modulating the shape of the eye. For eyes wearing goggles, GABA_AEither agonists or antagonists have primarily an inhibitory effect on the mesopic enlargement, but GABA_A0rThe antagonist TPMPA has similar growth inhibitory effects on the axial and equatorial directions. For eyes without goggles, GABA_AThe agonist muscimol enlarges the vitreous chamber in both the axial and latitudinal directions, however, GABA_A0rAgonists cause only a slight axial elongation. Also for eyes without goggles, GABA antagonists can stimulate axial growth, but GABA_A0rAntagonists stimulate both axial growth and inhibit the equatorial expansion.

With respect to the clinical view of eye shape, selective axial elongation of the vitreous chamber is considered a morphological feature of many (but not all) myopic eyes (Cheng et al, ophthalmic Vision science 69: 698-1030 (1992); Mutti et al, ophthalmic Vision science research 41: 1022-1030 (2000)). For interruptions 12 with continuous illumination: the initial eye growth response of the dark phase of the 12 hour light dark cycle (Stone et al 1995), as well as the GABA drug response comprising the present invention, are the only conditions known or published to date that induce selective axial elongation of the vitreous cavity.

To inhibit axially-elongated myopia during maturation in animals, treatment may be administered by intravitreal injection with an effective amount of the agent, but for therapeutic purposes, it is preferably an eye drop, ointment or gel for topical administration, or an orally administered pill, tablet or liquid. Indeed, in most cases, the therapeutic agent is administered to the human eye by topical administration of a drug, usually as an eye drop, ointment or gel, but other topical modes of administration can be achieved with the present invention, with eye drops having an active agent concentration of about 0.1% to 4% in an ophthalmic base being generally preferred. For example, although not intended as a limitation, a 1% solution of the GABA agonist muscimol admixed in a delivery vehicle suitable for the eye may be a good clinically useful concentration.

By "effective amount" or "therapeutically effective amount" is meant an amount of GABA drug, alone or in combination with a carrier, diluent, another agonist or antagonist, and/or other synergistic components, which when administered to an animal, preferably a human, is effective in treating or preventing refractive errors such as myopia or hyperopia, the effect of which can be confirmed by means of caliper or ultrasound measurements as disclosed herein, or by means of standard ocular examinations of the human which may include refractive eye, ultrasound or related techniques. The compositions of the present invention provide the possibility of achieving significant therapeutic effects on myopia using, for example, smaller doses of drugs such as GABA, thereby reducing the side effects and possible pain or toxicity that may result from the use of some other treatment.

The terms "induce", "stimulate", "enhance", "increase", "inhibit" and "prevent" and the like mean their ordinary dictionary meanings for eye growth and myopia. For example, "enhance" refers to increase and/or induce growth. More specifically, "potentiation" refers to the ability of an agent that acts on GABA receptors to cause or induce prolonged growth of the eye of an animal in the axial or equatorial direction. By "reversal" of the visual error is meant either a decrease in the relative size of the eye for at least one parameter, in the case of myopia of the eye, thereby causing less myopia (or more hyperopia) of the eye, or an increase in size or growth stimulation for at least one parameter, in the case of hyperopia of the eye, thereby causing less hyperopia (or more myopia) of the eye.

There are certain limitations associated with pH, storage and/or stability during formulation. As eye drops, a pH of about 6.5 is expected to be acceptable. Buffering agents are common to eye drops and to GABA_A，GABA_BOr GABA_A0rReceptor agonists or antagonists are required. Other additives and ingredients may also be present, such as those disclosed in Chiou, U.S. Pat. No.4,865,599 (incorporated herein by reference).

A typical regimen for eye drop administration may be once a day to four times a day, with the average dispensing interval being the entire time of waking. More effective agents require less use times or are capable of applying dilute solutions to the eye. On the other hand, the knowledge of ointments, gels, solid inserts of powders and topical precipitants or other formulations is now increasingly enhanced by clinical practice. These methods of administration avoid the problem of drug breakdown and can improve compliance when it is desired to administer prescribed doses of the drug simultaneously. Of course, it is also possible to administer the active agents described above in therapeutically effective amounts by systemic administration in the form of pills, capsules, liquids, tablets or other formulations.

By "subject" is meant any bird or animal for which the present invention may be used, or for which the present invention is effective to regulate or prevent visual error. By "animal" is meant any recognized animal, including wild or commercially valuable species and livestock, as well as primates and humans. Still further included are neonatal, juvenile, adolescent or adult animals, although the eye that is in development or maturation is preferably an eye of a neonatal or juvenile animal of any animal species.

In experiments using animals, axial myopia has been experimentally induced by images formed by retinal loss as described herein, and others have noted that amblyopia can also be experimentally induced simultaneously in primates. Amblyopia is evidenced by poor visual acuity in the eye leading to poor visual performance. Generally, visual acuity improves with it during maturation. It is known that amblyopia can occur in humans, particularly in hyperopic children with small eyes, for unknown reasons or partly due to strabismus (e.g. inert eyes). It is likely that the administration of therapeutically effective amounts of GABA drugs to adults will also prevent, inhibit or reverse the development of permanent or persistent amblyopia. By performing similar therapeutic treatments with the above-mentioned agents, it is also likely that people who have developed amblyopia for other, or even unknown, reasons will be helped.

GABA agonists or antagonists can be assayed for GABA by methods known in the art_A，GABA_BOr GABA_A0rAffinity and relative affinity of the receptor.

Axial elongation and/or equatorial expansion can be demonstrated for chickens and humans by comparing the matched eye of one animal with the eye of another, or by treating one eye of an animal with the drug or compound to be tested unilaterally, while the other eye is treated with the drug excipient alone or without treatment as a control. In particular, methods for determining the effect of a GABA drug for inducing or inhibiting axial growth in an animal's eye comprise contacting one or both eyes of the animal with GABA_A，GABA_BOr GABA_A0rAgonist or antagonist contact of the receptor and detecting the axial direction of the eye and-Or equatorial growth, then contacting the other eye or both eyes of the control animal with a control agent or vehicle alone for drug delivery, and measuring the axial and/or equatorial growth of the eyes. The growth of the treated eye or the eye of the animal treated with the drug in the axial and/or equatorial direction is then compared to the control eye or the eye of the animal treated with the vehicle alone or the eye of the animal treated with the control agent. The refractive effect can likewise be assessed. These comparisons can be further evaluated by obtaining a comparison of the effect of the open eye with and without the eyewear.

It is possible that the same neurochemical processes described herein may be involved in different directions and/or degrees during the process of reduced axial growth of the eye after birth leading to hyperopia. It has therefore been proposed that a similar excess or deficiency of retinal neurochemicals is involved in the progression of hyperopia. Therefore, in order to treat hyperopia, a method of administering an effective amount of GABA drugs can be adopted.

The present invention is based on the following findings: topical ocular positioning of a compound to normal vision in a young chick promotes ocular growth to a degree that is sensitive to the modulating effects of other agents. By co-administration of GABA_A，GABA_BOr GABA_A0rAgonists or antagonists of the receptor may inhibit the effect of GABA drugs on growth as shown by the effect on the open-eye model of the present invention.

The invention will be further described in the following examples. These examples should not be construed as limiting the scope of the appended claims.

Examples

The following experiments were performed to provide direct evidence for the role of retinal GABA mechanism in controlling postnatal eye growth.

As described by Stone et al 2001, at 12 hours light: white naive chicks were bred under dark-cycle conditions (trulow Farms, chestetown. Chicks were anesthetized by ether inhalation throughout the application of the goggles and the drug injection. The experimental eyes were the right eye of one half of the chicks and the left eye of the other half of the chicks, numbered in alternating order in each group. Shape-loss myopia was induced by adhering a single-sided translucent white plastic goggle to periorbital feathers with cyanoacrylate glue. The entire experimental procedure was consistent with the ARVD analysis in "use of animals in ophthalmology and vision studies".

Intraocular administration

The administration of goggles and/or medication injections are all initiated when the chicks reach one week of age. Approximately 4 hours into the light phase, 10 μ l of drug plus vehicle or vehicle alone was injected intravitreally and vehicle was injected simultaneously into all contralateral eyes under sterile conditions to either the goggle-wearing eye or the non-goggle-wearing eye.

After 4 days of treatment, the chicks were anesthetized with an intramuscular injection of a mixture of ketamine (200mg/kg) and xylazine (5mg/kg) for ocular examination. The animals did not receive intraocular injections on this day. In Stone et al, "visual study 35: 1195-. The head of the chick is removed while still under general anesthesia and the dimensions in the axial and equatorial directions of the removed eye are measured with a digital caliper. Since the coronal profile of a small corn is elliptical, the dimension in the filling direction is expressed as the average of the shortest and longest filling direction dimensions.

Table 1 lists the drugs studied, their characteristic affinities for GABA receptor subtypes, the suppliers, and the daily dose (. mu.g) range. The daily dose administered for a particular experiment is given in the figure and in the description of the results below.

TABLE 1 drugs, Activity and dosage Range

	Pharmacological Activity#	Chemical name, and drug supplier+	Dose range of injected drug (μ g) calculated peak intravitreal concentration (μ M)*
				GABAAMedicine
Muscol	Mixed GABAAAnd GABAA0rAgonists	Muscarine hydrobromideR	5-200μg；320-6410μM
				TACA	Mixed GABAAAnd GABAA0rAgonists	Trans-4-aminocrotonic acid1	10-100μg；618-6180μM
Bihuoling (buckling medicine)	Antagonists	(-) -Bifenxylum bromideR	0.01-50μg；0.135-676μM
				SR95531	Antagonists	6-imino-3- (4-methoxy-phenyl) -1(6H) -pyridazine butyric acid hydrobromideT	1-100μg；17.0-1700μM
GABAA0rMedicine
				CACA	Agonists	Cis-4-aminocrotonic acidR	10-200μg；618-12360μM
TPMPA	Antagonists	(1, 2,5, 6-tetrahydro-pyridin-4-yl) methylphosphonic acidR	0.1-200μg；3.89-7760μM
				GABABMedicine
Baclofen	Agonists	R (+) -baclofenR	10-100μg；250-2500μM
				CGP46381	Antagonists	(3-aminopropyl) (cyclohexylmethyl) phosphonic acidT	1-200μg；28.5-5701μM
SCH50911	Antagonists	(+)-(2S) -5, 5-dimethyl-2-morpholineacetic acidT	10-200μg；361-7217μM
				2 OH-Shaclofen (saclofen)	Antagonists	2-HydroxysarofenR	10-200μg；235-4700μM
CGP35348	Antagonists	(3-aminopropyl) (diethoxymethyl) phosphonic acidT	1-500μg；27.8-13880μM

^#Chebib et al 1999; bormar et al 2001; bormann 2000; bowey, annual pharmaco-toxicology score 33: 109-147 (1993); bolser et al, J Experimental therapeutics 274: 1393-; froestl et al, in "receptor research prospects", Giardina et al, eds, Amsterdam: elsevier science B.V pp 253-270 (1996); johnston, pharmacology scientific trend 17: 319, 323 (1996); uchida et al, european journal of pharmacology 307: 89-96(1996).

^*The calculated maximum post-injection intravitreal concentration assumes that the initial concentration of drug with vehicle is distributed in 150 μ l of vitreous humor (Rohrer et al, Vision neuroscience 10: 447-453 (1993)).

⁺Compound supplier:^RRBI/Sigma(Natick，Ma)；^TTocris Cookson(Ballwin，MO)。

table 1 also provides an estimate of the maximum drug concentration in μ M that may be achieved in the vitreous humor, based on the assumption that the drug is rapidly and uniformly distributed into the vitreous humor, which has a volume of 150 μ l (Rohrer et al, 1993). Thus, the dosage of similar eye drops can be calculated. The number of chicks tested at each drug dose is shown in figures 1 and 4 and is illustrated in the following results.

Biochemical assay

To determine retinal GABA (Allison et al, analytical chemistry 56: 1089-.

For the assay, each frozen retina was placed at 0.5ml of 0.1M HCIO at 4 ℃ with 0.3mM 5-aminopentanoic acid HCl as internal standard₄And homogenizing. This homogenate was centrifuged at 14000rpm for 15 minutes at 4 ℃ and the supernatant was filtered using an Acrodisc 13mm syringe filter equipped with a 0.2 μm nylon membrane (Gelman, Sciences, Ann Arbor, MI).

For derivatization, 0.02ml of the filtered supernatant was incubated for 6 minutes at room temperature with 0.18ml of 15% carbonate buffer (pH9.6) containing 5mM o-phthalaldehyde (OPA, Sigme-Aldrich, St. Louis, Mo.), 50% methanol and 5mM 2-methyl-propanethiol (Sigme-Aldrich). Using a high pressure liquid chromatography system (Bioanalytical Systems, West Lafayette, IN) equipped with an LC-4C electrochemical detector, IN Beckman Ultrasphere C₁₈25 μ l of derivatized sample was separated on a reversed phase (ODS, 5 μm, 4-6 mm. times.25 cm) column. The column was eluted with mobile phase 58% 0.1M sodium acetate (pH5.0) and 42% acetonitrile at a flow rate of 1.0 ml/min and measured by means of a detector equipped with a +0.7V glassy carbon working electrode and an Ag/AgCl reference electrode.

For protein determination, the centrifuged pellet was dissolved in 1.0ml of 1.0M NaOH, and 10. mu.l of the solution was assayed using the Bio-Rad protein assay kit (Bio-Rod laboratories, Hercules, Calif.) using bovine serum albumin as a standard according to the manufacturer's instructions. The concentration of GABA is expressed as μ g/mg protein.

Data analysis

For the goggle-worn chicks, the primary measure was the effect of the drug on the ocular response of the goggle, and the individual doses were compared to each other, each drug also compared to vehicle-treated controls. Differences in refraction and various measurements between the eyes wearing the goggles and the contralateral eye were examined by one-way analysis of variance (ANOVA). For the non-goggle-worn chicks, the primary measurements of the drug-treated eye and contralateral vehicle-treated eye were compared using two-way repeated measurements anova (one factor repeated with the eye as the repeated factor) for both refraction and measurements.

The results of the statistical analysis of the drug-treated eyes were described briefly in a two-way analysis of variance by comparing them to the contralateral eye. In the case where this comparison did not reach statistical significance, but p < 0.05 was reached in terms of overall dose or eye-dose interaction, additional such comparisons may be considered as another indication of potential activity of the drug. When the normal or equal variance analysis of variance assumption is not met, then the corresponding rank analysis of variance is applied. If analysis of variance determines a therapeutic effect, Tukey's test can be used on post hoc multiple comparisons to identify specific treatment groups, assuming p < 0.05 is statistically significant (Glantz, "Biometrics entry" fourth edition, New York, McGraw-Hill, pp97-98 (1997)). When the global analysis of variance reaches a significance level of at least p < 0.05, its p-value is shown in the figure and post-hoc tests for intra-group comparisons are shown in the table below.

Retinal GABA levels in the eye with goggles were compared to retinal GABA concentrations in the contralateral eye without goggles by the student's paired t-test. Most experiments did not report the depth of the anterior chamber and the thickness of the crystals, as these parameters were unaffected in almost all cohorts. The only exceptions to be mentioned below are the crystal and anterior chamber of the non-goggle-worn chick receiving the CACA, and the crystal of the non-goggle-worn chick receiving CGP 46381. Results from different groups of chicks tested with the same drug, together with the results of the corresponding vehicle-treated controls, were pooled for analysis, data are presented as mean ± standard error, and analyzed using Sigmastat (spss, inc., chicago, IL).

TABLE 2 Post Hoc pairwise comparison of drug effect on eyewear-Tukey test

Medicine	Specificity of	Diopter	Axial length ultrasonic diameter measuring instrument		Vitreous cavity depth (ultrasonic)	Diameter of the equator (Caliper gauge)
							Bihuoling SR95531	GABAAGABA antagonistsAAntagonists	Not significant 50 μ g to control	Is not significant and significant	Is not significant and significant	Is not significant and significant	10 ug to control 10 ug to 0.01 ug to control 100 ug to 1 ug to 50 ugTo control and 1. mu.g to 10. mu.g to control and 1. mu.g to
CACA	GABAA0rAgonists	200 ug vs 50 ug and 10 ug	Is not significant	Is not significant	Is not significant	Is not significant
							TPMPA	GABAA0rAntagonists	200 ug vs control 100 ug vs control 50 ug vs control 10 ug vs control	100 ug vs control 10 ug vs control	Is not significant	200 ug vs control 100 ug vs control 10 ug vs control 1 ug vs control	200 μ g to control, 1 and 0.1 μ g to 100 μ g to control, 10.1 and 0.1 μ g to 50 μ g to control

Baclofen CGP46381	GABABAgonists GABABAntagonists	10 ug vs control 200 ug vs control 100 ug vs control and 1 ug vs control 50 ug vs control	Not significant 100 μ g to control	Not significantly 100. mu.g to control 10. mu.g to control	No significant 200 ug to control ratio 100 ug to control and 1 ug ratio	No significant 200 μ g to control ratio 100 μ g to control ratio
							SCH50911	GABABAntagonists	50 μ g to control	Is not significant	Is not significant	Is not significant	Is not significant
Dihydroxy-sablofen	GABABAntagonists	100 μ g to control and 10 μ g ratio	Is not significant	Is not significant	Is not significant	Is not significant

Not significant: analysis of variance p is more than or equal to 0.05

Shown is a statistically significant Post hoc pairwise comparison (p < 0.05 as determined by Tukey's test) between treatment groups for each drug for which treatment effects were identified by one-way anova (see figures 1-3, results of anova overall).

TABLE 3 Post Hoc pairwise crossing-Tukey test of drug effect on eyes without goggles

Medicine	Specificity of	Diopter	Axial length vitreous chamber equator depth diameter ultrasonic diameter gauge (ultrasonic) (diameter measuring line)
				Muscarine	GABAA/GABAA0rAgonists	50 and 10. mu.g	200, 50, 200, 50200, 50 and 200, 50 and 10 and 5 mug and 10 mugg 10μg 10μg
SR95531	GABAAAntagonists	*	Onium 100 μ g100 and 50 μ g are not significant
				CACA	GABAA0rAgonists	Is not significant	50 ug is not significant and not significant
TPMPA	GABAA0rAntagonists	§	10 μ g 200 and 100 μ g
				Baclofen	GABABAgonists	Is not significant	Not significant 100 μ g not significant
CGP46381	GABABAntagonists		Not significant and 10 mug significant

^*Analysis of variance between drug-treated and contralateral eyes p < 0.05, but no specific composition was identified by Tukey's testAnd (6) comparing.

Analysis of variance of global dose effects p < 0.05, but no significant effects were identified for drug-treated and contralateral eyes; the Tukey test identified that both 5g and 100g doses differed from each other in overall effect and effect on the eyes in the treated groups.

Analysis of variance for interaction of onium ions only on ocular and dosage effects p < 0.05; the Tukey test identified that the drug-treated eye was significantly different from the contralateral eye for the 100 μ g dose.

Analysis of variance for overall dose effects p < 0.05, but no significant effect was identified for drug-treated versus contralateral eyes; specific pairwise comparisons were identified by Tukey test.

And the analysis of variance p is more than or equal to 0.05.

Statistically significant Post hoc pairwise comparisons between drug-treated and contralateral vehicle-treated eyes (p < 0.05 as determined by Tukey's test) were identified by using each drug dose for which treatment was identified by anova with two-way repeated measures (see figures 4 and 5 for overall anova results).

Results

Eye wearing goggles, GABA acting agent: GABA agonists have no effect on shape-loss myopia. Mixed GABA_APartial GABA_A0rThe agonist muscarinic did not affect refractive, ultrasonic or caliper measurements with the eye on goggles (10, 50, 100 or 500 μ g were administered daily, n-9-10/group, data not shown). When administering a drug to the eye wearing goggles, GABA is mixed_A/GABA_A0rThe agonist TACA also had no statistically significant effect on refractive or size measurements (10 or 100 μ g administered daily, n-10-13/group, data not shown).

GABA_AThe antagonist primarily limits the eye's enlargement in the equatorial direction when wearing the goggles, as measured by a caliper. Typical GABA is shown by means of ultrasound or calipers_AAntagonist buttonThe daily dose of Ling up to 50 μ g had no effect on myopic refraction or axial measurements of the eye wearing the goggles (FIGS. 1A and 2). However, it does reduce the equator diameter of the eye wearing the goggles (fig. 2, table 2). The higher daily dose of bicuculline was not tested because 100 μ g or 200 μ g caused retinal whitening, believed to be due to edema or other toxicity throughout the retina.

Similarly, GABA_AAntagonist SR95531 resulted in a significant and dose-dependent inhibition of equator enlargement in the eye wearing the goggles (fig. 2, table 2). SR95531 also reduced myopic refraction in the eye wearing goggles, with a daily dose of 50 μ g being most effective (fig. 1A, table 2). While SR95531 was consistent with a reduction in myopic refraction in the direction that caused the tendency to reduce axial length or vitreous cavity depth (fig. 2), none of the length measurements were statistically significant (p 0.1 or greater). And unlike bicuculine, SR95531 did not cause any detectable toxicity to the eye at the dose used, neither during in vivo ocular testing nor by examining the dissected eye.

Eye wearing goggles, GABA_A0rActing agent: selective GABA_A0rThe agonist CACA has a biphasic effect on the refractive response of the eye to the goggle. The effect of CACA on refraction was relatively slight compared to the magnitude of refractive change in shape-loss myopia (fig. 1B), and was not accompanied by any statistically identifiable change in axial measurements detected by ultrasound or caliper (data not shown). Perhaps because the corresponding minor changes in axial dimension are masked by the measured variations. Also, the CACA does not cause the equator size to change for the eye wearing the goggles (data not shown).

Comparison shows that GABA_A0rThe antagonist TPMPA had potent anti-myopia effects (fig. 1, 2, table 2). For the eye wearing the goggles, it is shown by ultrasonic measurements to reduce myopic refraction, prevent axial and vitreous chamber elongation, and by caliper measurements to prevent equatorial expansion. Axial growth measured by caliperNone of the effects of (a) was statistically significant.

Eye wearing goggles, GABA_BActing agent: GABA_BThe agonist baclofen has only weak anti-myopia effect. It partially reduced myopic refractive error in the eye wearing the goggles (figure 1C, table 2), but at both doses tested, neither ultrasound nor caliper measurements showed statistically significant growth inhibition (figure 3).

Comparison shows that GABA with high affinity_BThe antagonist CGP46381 had potent anti-myopia effects (fig. 1C and 3, table 2). It can inhibit myopic refractive deviation, axial and vitreous cavity elongation, and equatorial expansion of the eye wearing the goggles. For the eye wearing goggles, the other two GABA_BBoth antagonists, SCH50911 and dihydroxy-sablofen, reduced myopic refraction, but neither drug reduced eye size to a degree that was statistically significant by ultrasound or caliper measurements (FIGS. 1C and 3). Detection and display of GABA by ultrasonic wave or diameter measuring instrument_BThe antagonist CGP 35348 had no statistically significant effect on refraction or overgrowth of the goggle eye (n-9-18/group, data not shown).

Eyes without goggles, GABA_AActing agent: GABA is significantly different from the case of its eye-deficiency effect on wearing goggles_AAmazing muscimol shifts refraction of the unaided eye towards myopia (figure 4, table 3), with a 50 μ g dose having the greatest effect. Consistent with this effect on refraction, muscimol also stimulates axial growth and deepens the vitreous cavity as measured by ultrasound or calipers. It also increased the equator diameter of the eyes without goggles (fig. 5, table 3). For the chick without goggles, the GABA is detected_AThe effect of the agonist muscimol on vehicle-treated contralateral eyes (ANOVA; p < 0.05) demonstrated its ability to induce myopic refractive shifts. Vehicle-treated refractive of the contralateral eye varied with the administered dose of muscimol: 5. mu.g (0.86. + -. 0.58D), 10. mu.g (+ 0.11. + -. 0.82D), 50. mu.g (1.78. + -. 1.21D), 200. mu.g (3.82. + -. 0.8D)3D) Wherein the dose of 10 μ g and 200 μ g are statistically different from each other as indicated by Tukey's test ("D" ═ diopters, i.e. units of refraction, where "-" values indicate myopia and "+" values indicate hyperopia). Presumably, because the displayed data has been corrected for the contralateral eye (FIG. 4), this degree of myopia in the contralateral eye can be considered to be a significant loss of myopic refractive offset at the 200 μ g dose. In the non-goggle-worn nebulitol-treated chicks, there was no evidence of growth effects of the drug as shown by either contralateral eye ultrasonics or caliper measurements, and no loss of growth effects of the treatment eye by nebulitol at the 200 μ g dose (FIG. 5), which supports the following conclusions: muscimol stimulates eye growth and induces myopia without goggles.

However, it was found that for drug-treated eyes, the combined GABA was found to be present when compared to their contralateral control eyes treated with vehicle only_AAgonist TACA did not affect its refraction nor the measured size of the eye (10 μ g or 100 μ g daily dose, n is 10/group, data not shown).

GABA_AThe antagonist SR95531 also stimulates eye growth (fig. 5, table 3), but slightly less so than the effect of muscial alcohol. SR95531 promoted axial growth as shown by caliper measurements and elongated the vitreous cavity to comparable amounts compared to vehicle treated contralateral eyes. While the drug-treated eye was not significant compared to the contralateral eye, the ultrasound measured axial length did show significant elongation of the eye when compared in dose (p ═ 0.02). The Tukey test identified significant differences between the eyes treated at the 100 μ g drug dose and the control eyes. For refraction, a global dose effect was found (p 0.046), but no statistically significant myopic shift was achieved to 1-2 diopters when the drug-treated eye was compared to the contralateral eye (p 0.07). SR95531 had no significant effect on the equator diameter of the unaided eye. In view of its possible toxic effects on the retina (see above), there was no effect of bichalmorn on the eye without goggles.

Non-goggle-wearing limits (including effects on the anterior chamber and crystals), GABA_A0rActing agent: selective GABA_A0rAgonist CACA, although not affecting refractive effect (fig. 4, or data not shown), showed a slight stimulation of axial length by ultrasound measurements (fig. 5, table 3, n-9-10/group) and had a tendency to increase the length of the vitreous cavity (fig. 5, p-0.056). Comparing the primary results for the drug-treated and vehicle-treated eyes (see methods above) shows that CACA does not affect crystal thickness or anterior chamber depth (0.22 for crystal p and 0.80 for anterior chamber p), but in the overall dose comparison, CACA has a statistically significant effect on both crystal and anterior chamber (0.001 for crystal p and 0.001 for anterior chamber p). In this regard, the chickens receiving the 10 μ g dose had a relatively thin crystal and a relatively deep anterior chamber, unlike the chickens receiving the other doses. For the crystals, the Turkey test identified that the thickness of the overall eye crystals of the chickens receiving the 10 μ g dose was statistically different from the thickness of the crystals of the chickens receiving either the 100 μ g or 200 μ g dose. For chicks receiving a 10 μ g dose, the individual drug-treated and vehicle-treated ocular crystals measured 2.22 ± 0.02 mm. The chick eye crystals treated with the 10 μ g dose of drug were 0.16mm thinner than the chick crystals dosed at either 100 μ g or 200 μ g, and the chick contralateral eye crystals treated with the 10 μ g dose of vehicle were 0.10mm thinner than the chick contralateral eye crystals treated with the other two cohorts of vehicle.

For intraocular comparisons, the Tukey test identified the difference in drug-treated eyes as statistically significant, but not excipient-treated eyes. For anterior chamber, Tukey test identification indicated that the anterior chamber of the eye of chickens receiving the 10 μ g dose was statistically different from the anterior chamber of chickens receiving the 50, 100, or 200 μ g dose. In the 10 + -g dose group, the drug-treated and vehicle-treated anterior chambers measured 1.32 + -0.03 and 1.31 + -0.03 mm, respectively, while the anterior chambers of the drug-treated and vehicle-treated chicks receiving the 10-g dose were approximately 0.08-0.14mm deeper than the anterior chambers of the eyes treated with the higher dose. For comparison in the eye, Tukey test identification showed that in drug-treated eyes, the 10 μ g dose was statistically different from the 100 μ g and 200 μ g doses, and that eyes treated with the 10 μ g dose of the chick contralateral excipient were statistically different from the contralateral eye that received the 200 μ g dose. None of the other growth measurements reached statistical significance (figure 5).

When administering drugs to the eye without goggles, GABA_A0rThe antagonist TPMPA stimulated axial growth and vitreous cavity length to a moderate degree (fig. 5, table 3). Unlike the effect on axial dimension, TPMPA reduces the equator diameter of an unaided eye. As with the effect of the various drugs administered on the eyes without goggles, the resulting slight myopic refractive shift achieved statistical significance when administered (p 0.02), but no such shift was observed when the drug-treated eyes were compared to the contralateral eyes (p 0.14).

Eyes without goggles, GABA_BActing agent: when GABA is given to eyes without goggles_BThe agonist baclofen, causes a modest deepening of the vitreous cavity. Comparable amounts of axial elongation achieved a level of statistical significance when measured with a caliper, but did not achieve a level of statistical significance when measured by ultrasound (fig. 5, table 3, n-8 for each dose administered). For eyes without goggles, including the effect on refraction (data not shown), no other effect of baclofen was achieved at a statistically significant level.

The most effective anti-myopia GABA was also tested in 2 daily administrations_BAntagonist CGP46381, whose effect on eyes without goggles was determined (n ═ 10/group). For eyes without goggles, the CGP46381 may slightly increase the length of its vitreous cavity (fig. 5, table 3). This comparable increase in axial length, measured by means of ultrasound or calipers, did not reach a statistically significant level.

Although CGP46381 had no effect on crystal thickness as shown by comparing drug treated eyes with vehicle treated eyes (ANOVA: p ═ 0.58), it did have a statistically significant effect on crystals in dose comparisons (p ═ 0.03), indicating that overall crystal thickness of chicks receiving the 10 μ g dose was statistically different from that of chicks receiving the 100 μ g dose. The drug-treated eye crystals and the excipient-treated eye crystals were measured to be 2.27. + -. 0.04mm and 2.26. + -. 0.03mm, respectively, in chickens receiving a 10. mu.g dose. These measurements were 0.07mm and 0.06mm thick, respectively, compared to crystals from drug-treated eyes and crystals from excipient-treated eyes receiving a 100 μ g dose.

For comparison in the eye, the Tukey test identified these differences as statistically significant in the drug-treated eyes but not in the vehicle-treated eyes. Neither CGP46381 caused statistically identifiable changes in refraction (data not shown), anterior chamber depth (data not shown) or equatorial line diameter (fig. 5).

Retinal biochemistry: the level of unscored intraocular GABA was determined to be 10.8. + -. 0.2. mu.g/mg protein by HPLC-ED assay, which is consistent with published values in chick retinas (Nistico et al, communication of pathological and pharmacological chemistry 40: 29-39 (1983)). GABA levels were determined to be 10.3. + -. 0.2. mu.g/mg protein in eyes with goggles on the contralateral side. Although the magnitude of this difference is small, the decrease in retinal GABA in myopic eyes does reach a statistically significant level (N ═ 23 for eyes, p < 0.02, paired t-test with bicuspids).

Small knot: GABA drugs both inhibit shape-loss myopia and affect the growth of eyes with normal visual input, and are therefore involved in the recognition of GABA receptors in the mechanisms that regulate eye growth and refractive formation. Ion channel-gated receptors (GABA) are involved in response to these drugs_AAnd GABA_A0rReceptors) and also to G-protein linked receptors (GABA)_BReceptor). The complex anatomical effects of these drugs strongly support the following facts: the retinal mechanism regulates the shape of the developing eye and not just the entire size of the eye. The sites of action on the neural retina are consistent with: the localization of GABA and its receptors in the eye, the small but consistent reduction in retinal GABA levels in the eye with loss of shape, and the response of eye development to these drugs are known. Although not explicitly disclosedThe action mechanism of GABA drugs, however, the action characteristics of GABA drugs on the growth of the eyes with and without goggles (open), and the enrichment of GABA in the retina_A0rThe fact of the receptor suggests that a potent pharmacological factor of GABA has been added in the study of retinal mechanisms that regulate eye growth and geometry. Moreover, since eyes receiving prolonged amanitol become myopic while eyes near drugs with different specificities maintain normal vision, GABA drugs appear to be useful for the in-depth study of the mechanism of retinal refraction normalization.

However, the present invention is not so limited and is intended to include several methods and compositions for controlling the growth of the postnatal eye and the development of ocular errors in the mature eye of a subject, including altering the refraction and/or growth of the mature eye of a subject by administering to the eye a therapeutically effective amount of at least one GABA drug or compound, including agonists or antagonists (alone or in combination with other compounds), and any other drug or compound of the kind having the effect of altering the refractive development and/or growth of the eye. Because the concentration of retinal GABA is altered in myopia and retinal GABA affects refractive formation and eye growth of the eye, in another aspect the present invention also contemplates an alternative method of altering refractive formation and eye growth of the eye by administering to the eye in the subject's maturity a therapeutically effective amount of at least one GABA drug or compound, including agonists or antagonists (alone or in combination with other compounds), as well as any kind of other drug or compound having the effect of correcting retinal GABA abnormalities, to modulate the level of retinal GABA in the eye.

The disclosures of each patent, patent application, and published paper cited or discussed in this specification are hereby incorporated by reference in their entirety.

While the foregoing specification has been described in terms of certain preferred embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that various modifications, and other embodiments of the invention can be made within the spirit and scope of the invention, and that certain details described herein can be modified considerably in accordance with the basic principles of the invention. It is also intended that such modifications and complementary embodiments be included within the scope of the appended claims.

Claims (36)

1. A method of controlling postnatal growth of the eye of a maturing animal or human, the method comprising administering GABA or at least one agonist or antagonist thereof.

2. A method of controlling refractive formation in a maturing animal or human eye comprising administering GABA or at least one agonist or antagonist thereof.

3. The method of claim 1 or 2, further comprising altering GABA or its concentration in the eye.

4. The method of any one of claims 1-3, wherein the postnatal growth of the eye further comprises abnormal growth.

5. The method of any one of claims 1-4, wherein postnatal growth of the eye results in abnormal refraction.

6. The method of any one of claims 1-5, further comprising administering to the eye a therapeutically effective amount of a GABA drug or composition.

7. The method of claim 6, wherein the administering affects GABA in the eye_A、GABA_BOr GABA_A0rGABA drugs or compositions of GABA receptors.

8. The method of claim 6 or 7, wherein the administered drug or composition comprises at least one agonist of at least one type of GABA receptor.

9. The method of claim 6 or 7, wherein the administered drug or composition comprises at least one antagonist of at least one type of GABA receptor.

10. The method of any one of claims 1-9, further comprising inhibiting or reversing myopia or the onset of myopia, or the progression of myopia, of the eye of the postnatal animal or human.

11. The method of claim 10, further comprising inhibiting or reducing the growth of axial length or vitreous cavity depth of the eye, or the enlargement of the eye in the equatorial direction, thereby preventing, inhibiting or reducing myopic refraction or the onset or progression of myopia.

12. The method of claim 10, further comprising inhibiting or reducing growth of axial length or vitreous cavity depth of the eye and enlargement of the eye in the equatorial direction, thereby preventing, inhibiting or reducing myopic refraction.

13. The method of any one of claims 1-9, further comprising inhibiting or reversing hyperopia or the onset of hyperopia, or slowing the progression of hyperopia in the eye of the post-natal animal.

14. The method of claim 13, further comprising stimulating or enhancing growth of axial length or vitreous cavity depth of the eye, or enlargement of the eye in the equatorial direction, thereby preventing, inhibiting or reducing hyperopia refraction or slowing progression of hyperopia.

15. The method of claim 13, further comprising stimulating or enhancing axial growth or vitreous cavity depth growth of the eye, and enlargement of the eye in the equatorial direction, thereby preventing inhibition or reduction of hyperopia refraction or slowing progression of hyperopia.

16. The method of any one of claims 1-9, further comprising inhibiting or reversing ocular amblyopia or the onset of amblyopia in a postnatal animal or human, or slowing the progression of amblyopia.

17. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_AThe receptor agonist, such carrier or diluent has been buffered to a pH suitable for ocular administration.

18. The method of claim 17, wherein GABA is_AThe receptor agonist is muscimol or TACA.

19. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_AThe receptor antagonist, such carrier or diluent, has been buffered to a pH suitable for ocular administration.

20. The method of claim 19, wherein GABA is_AThe receptor antagonist is SR95531 or bicolor.

21. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_A0rThe receptor agonist, such carrier or diluent has been buffered to a pH suitable for ocular administration.

22. The method of claim 21, wherein GABA is_A0rThe receptor agonist is CACA.

23. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_A0rThe receptor antagonist, such carrier or diluent, has been buffered to a pH suitable for ocular administration.

24. The method of claim 23, wherein GABA is_A0rThe receptor antagonist is TPMPA.

25. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_BThe receptor agonist, such carrier or diluent has been buffered to a pH suitable for ocular administration.

26. The method of claim 25, wherein GABA is_BThe receptor agonist is baclofen.

27. The method of any one of claims 1-16, further comprising administering to the maturing eye a therapeutically effective amount of GABA in a carrier or diluent_BThe receptor antagonist, such carrier or diluent, has been buffered to a pH suitable for ocular administration.

28. The method of claim 27, wherein GABA is GABA_BThe receptor antagonist is CGP4638, SCH50911 or 2 hydroxy-sablofen.

29. The method for the in vivo treatment of the eye of a subject according to any one of claims 1-28, wherein the subject has myopia, hyperopia or amblyopia.

30. A method according to any one of claims 1 to 28 for preventing myopia, hyperopia or amblyopia in a subject's eye in vivo.

31. A method of controlling postnatal growth of the eye of a maturing animal or human comprising administering to the eye of the animal or human an effective amount of a neurochemical, or an agonist or antagonist thereof, thereby modulating the presence of GABA or an agonist or antagonist thereof.

32. A method of detecting the effect of one or more GABA drugs or compounds that affect ocular growth in postnatal animal maturation comprising:

administering to the eye of the first animal a therapeutically effective amount of a retinal GABA receptor agonist or antagonist in a carrier or diluent which has been buffered to a pH suitable for ocular administration;

detecting a change in growth in the first eye axial or equatorial direction, or both;

an ocular control agent administered to the second animal comprising a carrier or diluent for use with the retinal GABA receptor agonist or antagonist in the first eye;

detecting the effect of the control agent on the second eye; and is

Comparing the change in growth of the first eye to the effect of the control agent on the second eye;

wherein the first eye is open or covered, such as by stitching, closed or by wearing goggles, and wherein the second eye is the same as the first eye (open or covered).

33. The method of claim 32, wherein the eye of the first animal and the eye of the second animal are in the same animal.

34. The method of claim 32, wherein the eye of the first animal and the eye of the second animal are in different animals.

35. The method of any one of claims 1-34, wherein the animal or subject is selected from the group consisting of birds and mammals, including primates, and wherein primates include humans.

36. The composition of matter for controlling postnatal growth of the eye of a maturing animal of any one of claims 1 to 35 wherein GABA is altered during the postnatal maturation of the eye.