KR20020081260A - Ocular Growth and Nicotinic Antagonists - Google Patents

Abstract

The present invention is directed to a method of controlling postnatal eye growth or inhibiting myopia development comprising ocularly administering a therapeutically effective amount of a nicotine antagonist.

Description

Ocular Growth and Nicotinic Antagonists

Visual input controls the regulation of postnatal eye growth and the development of refractive errors. Eye growth appears to be regulated locally, mainly in the eye, perhaps through the retina, and the specific role of other components of the nervous system, such as the brain or peripheral nervous system, is unclear (Stone, 1997, Myopia Updates: Proceedings of the 6th International Conference). on Myopia, pp. 241-254; Wallman, 1993, Progress in Retinal Research, 12: 133-153). The complex nature of visual images, such as blurring, affects eye growth, and it is reasonable to believe that nerve cells adjacent to the retina may constitute an element of local regulatory mechanisms. Indeed, much evidence now exists that suggests that amacrine cells of various types of retina are pathways linking visual information to eye growth regulation. The data most strongly support the role of dopamine-based amacrine cells (Stone, 1997, Myopia Updates: Proceedings of the 6th International Conference on Myopia, pp. 241-254; Stone et al., 1989, Proc. Natl. Acad. Sci USA, 86: 704-6). There is evidence suggesting that other retinal neurons are less fully developed or vice versa, while other subtypes of retinal amacrine cells that are supposed to affect refractive development include the vascular functional intestinal peptides (Pickett Seltner and Stell, 1995, Vision Res., 35: 1265-1270; Stone et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 257-60), Glucagon (Fischer et al., 1999a, Nature Neuroscience, 2: 706-12) , Nitric oxide (Fujikado et al., 1997, Curr. Eye Res., 16: 992-6), enkephalin (Pickett Seltner et al., 1997, Vis. Neurosci., 14: 801-809) and acetylcholine (Stone et al., 1991, Exp. Eye Res., 52: 755-8).

Choline mechanisms acting through muscarinic receptors appear to be involved in the regulation of eye growth, in which muscarin antagonist atropine is the chick (Stone et al., 1991, Exp. Eye Res., 52: 755-8), tree shrew (McKanna and Casagrande, 1981, Documenta Ophthalmologica Proceedings Series, 28: 187-192), monkeys (Raviola and Wiesel, 1985, N. Engl. J. Med., 312: 1609-15; Tigges et al., 1999, Optometry & Vision Science, 76: 397-407) and humans (Brodstein et al., 1984, Ophthalmol., 91: 1373-1379) to inhibit myopia development. However, it has been found difficult to identify specific choline neurons that contribute to the regulation of eye growth.

Because of the clinical relationship between myopia development and near-field work, it has been hypothesized that perspective regulation is the basis of the mechanisms leading to myopia, and that ciliary muscle palsy accounts for the anti-myopia activity of atropine, and thus mousse of choline nerve cells and ciliary muscles of ciliary ganglion Present the role of the carin receptor. However, despite the many interesting features of the hypothesis, little experimental work supports the role of perspective control in myopia development. Cleavage of ciliary nerves (Shih et al., 1994, Invest.Ophthalmol. Vis. Sci., 35: 3691-701) Ceniotomy (Lin et al., 1996, Curr. Eye Res., 15: 453-60; Raviola and Wiesel , 1985, N. Engl. J. Med., 312: 1609-15) or pre-ganglionic input injury from the Eddinger-Westphal nucleus to the ciliary ganglion (Troilo, 1990, The Ciba Foundation Symposium, 155: 89-102; discussion 102-14, respectively, failed to have a major impact on experimental myopia development. Pharmacological evidence also counters the role of perspective control in myopia development. M1-selective muscarinic antagonists with minimal ciliary palsy activity are at least as effective as atropine for experimental myopia in rhesus monkeys (Tigges et al., 1999, Optometry & Vision Science, 76: 397-407). It was also observed that the ciliary body contains striated muscle rather than smooth muscle, and bird's perspective control is controlled by nicotine mechanism rather than muscarin mechanism, and atropine has failed to paralyze chick's perspective control (Stone et al., 1991). , Exp.Eye Res., 52: 755-8). In addition, it is opposed to the perspective control mechanism that atropine blocks chick myopia (Stone, 1997, Myopia Updates: Proceedings of the 6th International Conference on Myopia, pp. 241-254).

Another choline mechanism has been considered to explain refractive development. Retinal choline neurons and muscarinic receptors have been suggested to be based on the activity of M1-selective muscarinic receptor antagonist subtypes in chick myopia (Stone et al., 1991, Exp. Eye Res., 52). : 755-8). This concept has also been described in the tree mole (Cottriall and McBrien, 1996, Invest.Ophthalmol. Vis. Sci., 37: 1368-79) and the rhesus monkey (Tigges et al., 1999, Optometry & Vision Science, 76: 397-407). Is also supported by the antimyopic activity of M1-selective muscarinic antagonists. However, studies conducted to confirm the involvement of choline neurons revealed that the activity of the biosynthetic enzyme (choline acetyltransferase) for acetylcholine was not altered in the retina of myopic chick eye, but decreased in ciliary ganglia ( Pendrak et al., 1995, Exp.Eye Res., 60: 237-43). Responsiveness of cultured chick sclera to retinal toxins damaging cholinergic amacrine cells (Lind et al., 1998, Invest.Ophthalmol. Vis. Sci., 39: 2217-2231) and chick eye responses (Fischer et al., Based on 1998b, Brain Res., 794: 48-60, it has been suggested that muscarinic antagonists can inhibit myopia development by acting at separate retinal sites such as sclera or choroid.

Further limiting the inventors' understanding of the role of choline neurons in the regulation of eye growth is the lack of research on the cholinergic nicotine mechanism. Both intravitreal and subconjunctival nicotine induce chick perspective control (Reiner et al., 1995, Vision Res., 35: 1227-1245). Injecting nicotine into the vitreous of chicks twice a day for 2 weeks resulted in myopic shifts of about 2 diopters in refraction compared to the contralateral eye, but intravenous saline injections had the same effect (Reiner et al. 1995, Vision Res., 35: 1227-1245). Injecting nicotine daily under the conjunctiva of the chick resulted in a slight myopic refraction shift of 0.75 diopters compared to the untreated eye, but no response was observed in the subconjunctival saline solution (Reiner et al., 1995, Vision Res., 35: 1227-1245), this degree of refractive shift in chicks may be of little biological significance because it is close to the focal depth of the chick's eye (Schmid and Wildsoet, 1997, Ophthal. Physiol. Opt., 17: 61-7). . The high fat affinity of nicotine can allow for rapid diffusion from the eye, so the potential action at separate eye sites further limits the mechanical interpretation of these results. The application of myocardial blockers and nicotine antagonists, brominated becuronium, to the cornea of the chicks paralyzed perspective control but failed to affect eye height following eye-induced primitive focal blurring. Against the regulatory mechanism (Schwahn and Schaeffel, 1994, Invest. Ophthalmol. Vis. Sci., 35: 3516-24). Antagonists with circular d-tubocurin loaded into myocardial nodules typically inadequately penetrate into the central nervous system and bind with low nicotine to all nicotine receptor subtypes (Gotti et al., 1997, Progress in Neurobiology, 53: 199). -237). Because brominated becuronium is also heavily loaded, it readily spreads and blocks the muscle nerve nodules in the intraocular muscles, while it may not be able to access receptor sites in adipose tissues such as the neural retina that are potentially involved in eye growth regulation. have.

The eyes of the chicks include the retina (Hamassaki-Britto et al., 1994a, Vis. Neurosci., 11: 63-70; Hamassaki-Britto et al., 1994b, J. Comp. Neurol., 347: 161-170; Keyser et al., 1993, J. Neurosci., 13: 442-454; Vailate et al., 1999, Mol. Pharmacol., 56: 11-19) and ciliary ganglia (Berg et al., 1998, Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities, pp. 187-196; Conroy and Berg, 1995, J. Biol. Chem., 270: 4424-4431; Halvorsen and Berg, 1990, J. Neuroses., 10: 1711-1718 Horch and Sargent, 1995, J. Neurosci., 15: 7778-7795; Pugh et al., 1995, Mol. Pharmacol., 47: 717-725).

Summary of the Invention

The present invention relates to the use of nicotine antagonists that have suitable solubility to penetrate into associated targets that regulate postnatal eye growth and inhibit postnatal eye growth and myopia development.

The present invention provides a method for controlling postnatal eye growth by ocularly administering a therapeutically effective amount of nicotine antagonist. Also provided is a method of inhibiting abnormal postnatal axis growth of host animal eye by administering a therapeutically effective amount of nicotine antagonist during postnatal development. The invention also provides a method of inhibiting abnormal equator swelling of a host animal eye by administering a therapeutically effective amount of nicotine antagonist during postnatal development. The present invention also provides a method of inhibiting abnormal vitreous swelling of a host animal eye by administering a therapeutically effective amount of nicotine antagonist during postnatal development. Another aspect of the invention provides a method of preventing or inhibiting the development of myopia by ocularly administering a therapeutically effective amount of a nicotine antagonist.

The present invention provides the use of nicotine antagonists in the manufacture of a medicament suitable for ocular administration for the control of postnatal eye growth. The invention also relates to the manufacture of a medicament for use such as inhibiting abnormal postnatal axis growth of a host animal eye during postnatal development, inhibiting abnormal equatorial swelling of the host animal eye during postnatal development, and inhibiting abnormal vitreous cavity expansion of the host animal eye during postnatal development. Provides the use of nicotine antagonists. The invention also provides the use of nicotine antagonists in the manufacture of a medicament suitable for ocular administration for the prevention or treatment of myopia.

In one embodiment of the invention, the nicotine antagonist may be a competitive nicotine antagonist such as methyllicaconitine or dihydro-β-erythroidine. In another embodiment, the nicotine antagonist may be a channel-blocking nicotine antagonist such as chlorisondamine or mecamylamine. In another embodiment of the invention, the nicotine antagonist may be a non-competitive nicotine antagonist such as sertraline, paroxetine, nefaxodon, venlafaxine, fluorcetin, buproprione, penciclidine and ibogaine. In another embodiment of the invention, the nicotine antagonist may be an antibody that inhibits nicotine receptor function. In another embodiment of the invention, the nicotine antagonist may be an agent that acts like a nicotine antagonist.

The present invention contacts a host animal's eye with a therapeutically effective amount of nicotine antagonist, detects growth changes in the eye exposed to a therapeutically effective amount of nicotine antagonist, and then applies a known control agent to the second eye. Observe the results of the control agent in the change in eye growth of 2 and compare the change in growth of the first eye exposed to the therapeutically effective amount of nicotine antagonist with that of the second eye exposed to the known control agent. Thereby confirming that nicotinic antagonists have the ability to regulate postnatal eye growth, thereby providing a method of detecting nicotine antagonist's postnatal eye growth regulation ability.

The present invention provides a method of incubating cells expressing nicotine receptors in the presence and absence of a test compound, determining whether the test compound binds to one or more nicotine receptors, and selecting a test compound that binds to one or more nicotine receptors. Administering the selected test compound to the test animal, determining whether the test compound altered myopia development of the test animal, and selecting a compound that alters the myopia development of the test animal. Provided are methods for identifying compounds present.

The present invention relates to the regulation of eye growth by nicotine receptor antagonists, and more particularly to the inhibition of postnatal eye growth and prevention of myopia of host animals by ocular administration of nicotine receptor antagonists.

1 shows drug effects on refraction of eye wearing goggles. Chlorisondamine (CHL; P <0.001), Mecamylamine (MEC; P <0.001) and Methylcaconitine (MLA; P = 0.04) each affected myopic refraction underneath the goggles as measured by ANOVA. Dihydro-β-erythroidine (DHBE) did not affect the refraction of the blinded eye. See Table 1 for the results of the pair comparison by Tukey's test. The number of chicks in each experimental group is shown in the table. Data is expressed as the difference between the goggles wearing eye and the contralateral open eye (mean ± S.E.M.). To facilitate the comparison, the bars of each control were hatched.

2, A to D show the effect of the drug on the ocular dimensions of the eye wearing goggles. 2A shows the drug effect on the axial length measured by ultrasound. 2B shows the drug effect on the vitreous cavity length measured by ultrasound. 2C shows drug effect on axial length measured by digital caliper. 2D shows drug effects on equatorial diameter measured by calipers. Chlorisondamine (CHL), mecamylamine (MEC) and methyllicaconitine (MLA) each reduced excessive eye growth under goggles and were statistically significant by direct ANOVA results for each data set. One effect (P <0.05) and suggestive trends were identified. The effect of dihydro-β-erythroidine (DHBE) was weaker because it showed statistically significant drug effects only by axial measurements by calipers. See Table 1 for the pair comparison results by the Tuki test. The number of chicks in each experimental group is provided in FIG. 1. Data are expressed as the difference between the goggles wearing eye and the contralateral open eye (mean ± S.E.M.). To facilitate the comparison, the bars of each control were hatched.

3 shows the effect of chlorisondamine on goggles unworn eyes. Ipsilateral administration of Chlorisondamine to the eye of a non-goggling chick's eye shifted the total refraction towards the primordial (ANOVA for grade: P = 0.004), reduced axial length (ANOVA for grade: ultrasound, P = 0.03; caliper, P = 0.03), inhibited axial expansion of the vitreous steel (ANOVA for grade: P = 0.01). See Table 1 for the pair comparison results by the Tuki test. N per group is 9-20 chicks. Data are presented as the difference (mean ± S.E.M.) between drug treated eyes and contralateral vehicle treated eyes.

Table A shows a summary of nicotine receptor subtypes.

Table 1 shows a post hoc pair comparison of drug effects using the Tuki assay.

Table 2 shows the long term effects of single dose Chlorisondamine (200 μg) on form loss myopia.

The nervous system regulates postnatal eye growth through many partial retinas, and the induction of refractive errors (requires eyeglasses) appears to be primarily due to neural mechanisms. Clinically the most common refractive error is myopia. The present specification includes a class of nicotine antagonist pharmaceutical drugs that are active against myopia experimental models but also inhibit "normal" eye growth.

In the present invention, we tested the effects of various nicotine antagonists with desirable drug properties on chick eye growth using intravitreal injection, which is an effective delivery route. The four nicotine antagonists selected were chlorisondamine (CHL), mecamylamine (MEC), methyllicaconitine (MLA) and dihydro-β-erythroidine (DHBE). Animal models using chicks provide good optical advantages as models for the human eye. We provide evidence for the actual role of the nicotine receptor (possibly the central role) in the regulation of eye growth using antagonists that have established profiles for neuronal nicotine receptors and have comparable local affinity to diffusion into nerve tissue. Said.

The present invention relates to the use of nicotine antagonists that have solubility suitable for penetration into obvious target sites that regulate postnatal eye growth and inhibit postnatal eye growth or myopia induction. All drug classes previously identified as regulating eye growth show activity only against a form loss myopia model (the eye starts at about 1 week of age and remains covered by goggles). The present invention has identified a drug class that is also active against open eyes (eyesight is not lost by goggles during maturation), which is the first class of agents that have been shown to exhibit this activity. This activity on open eyes is advantageous for applications in human myopia where form loss is not usually the environment. Although chicks use nicotine receptors for control of pupil size and perspective regulation, while mammalian eyes use a subclass of muscarinic receptors for control of pupil size and perspective regulation, nicotine antagonists in chickens after topical application to human eyes. It does not induce pupil dilation and perspective paralysis and is expected to be tolerated.

Section 1. Nicotine Receptor Subtypes

The nicotine acetylcholine receptor subtype consists of five homologous subunits that form acetylcholine-gate cation channels [Lindstrom, 1997, Mol. Neurobiology, 15: 193-222. Seventeen nicotine receptor subunits are known (α1-10, β1-4, γ, δ and ε). Each subunit has four transmembrane domains. The acetylcholine binding site is formed by at least three peptide loops (main component) on the α subunit and two peptide loops (supplementary component) on adjacent subunits. All α subunits have two tandem cysteine residues close to the site associated with the acetylcholine bonds, and subunits that are not α are deficient in tandem cysteine. Other sliced forms of α4 subunits (α4-1 and α4-2; rats) and α1 subunits (humans) have been identified [Mandelzys, A. et al., (1995) J. Neurophysiol. 74, 1212-1221; Papke, R.L. Et al. (1996) Neurosci. Lett. 213, 201-204; Albuquerque, E. X. Et al. (1997) J. Pharmacol. Exp. Ther. 280, 1117-1136; deFiebre, C.M. Et al., (1995) Mol. Pharmacol. 47, 164-171.

These receptors fall into three general classes: one muscle class and two nerve classes. Muscle types exist in only two forms-fetal and adult forms (each with an α1 subunit and other subunits specific for muscle receptors). One class of neuronal receptors binds α-branch toxins and often consists of α7, α8, α9 or α10 subunits as homologous receptors. Other classes of neuronal receptors do not bind to the α- branching toxin and are formed from a combination of β2 or β4 subunits and α2, α3, α4 or α6 subunits. Rapid desensitization and limited utilization of selective drugs suitable for in vivo studies undermine the definition of physiological function for these biochemically defined receptor subtypes. Nicotine receptor subtypes are summarized in Table A.

The Nicotine Receptor Subcommittee of NC-IUPHAR has developed a nomenclature and taxonomy for nicotine acetylcholine (nACh) receptors based on subunit compositions of known naturally-expressing nACh receptor subtypes and / or subtypes formed by heterologous expression. Recommended by Lukas et al., (1999) IUPHAR XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharm. Rev. 51, 397-401]. The title in Table A reflects a summary of nACh receptor subtype naming based on the major α subunits included in the receptor subtype. The following asterisk denoted by the α subunit means that other subunits are known or assembled with the indicated α subunit to form the named nACh receptor subtype (s). The absence of an asterisk indicates that the indicated subunits are known to be assembled into homologous nACh receptor subtypes. If subunit stoichiometry is known, the number of specific subunits of a specific nACh receptor subtype is indicated by subscripts below the subunit in parentheses. All subunits except α8 (algae) are of mammalian origin.

Section 1.1. Nicotine Receptor Subtype and Eye

Chick retinas include several classes of choline neurons (Miller et al., 1987, Neurosci., 21: 725-743). Several subtypes of muscarinic acetylcholine receptors [Fischer et al., 1998a, J. Comp. Neurol., 392: 273-84, the chick retina complexes express nicotine acetylcholine receptor subunits, including α3, α6, α7, α8 and β2, β3, β4. The cellular pattern of the neuronal location of nicotine receptors in the chick retina is complex [Hamassaki-Britto et al., 1994a, Vis. Neurosci., 11: 63-70; Hamassaki-Britto et al., 1994b, J. Comp. Neurol., 347: 161-170; Keyser et al., 1993, J. Neurosci., 13: 442-454; Vailate et al., 1999, Mol. Pharmacol., 56: 11-19. The ciliary ganglia of chicks are similarly rich in all of the delivery, synaptic and extrasynaptic positions of nicotine receptor subtypes including α3 subunits, α5, α7, β2 or β4 subunits representing autonomic ganglia [Berg et al., 1998, Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities, pp. 187-196; Conroy and Berg, 1995, J. Biol. Chem., 270: 4424-4431; Horch and Sargent, 1995, J. Neurosci., 15: 7778-7795; Pugh et al., 1995, Mol. Pharmacol., 47: 717-725].

Section 2. Nicotine Antagonists

The action of nicotine acetylcholine receptor (nicotine receptor or NR) can be antagonized by various compounds. The action of these compounds on NR may be complex and may involve one or more antagonistic mechanisms, but all details may not be fully characterized. Certain drugs for the purposes discussed are classified into mechanisms of action that are currently best understood. Nicotine antagonists are defined as compounds that inhibit, block, compete, prevent or interfere with any effect of nicotine agonists on the target. We claim the use of competitive nicotine antagonists in the present invention. Competitive nicotine antagonists are defined as compounds that appear to compete for agonist binding sites on nicotine receptors, where competitive antagonists appear to inhibit receptor action by preventing activation of the receptor by the agent. Examples of competitive antagonists include, but are not limited to, peptide hydrotoxins derived from snails, including dihydro-β-erythroidine, bungarot toxin, tubokurarin, methyllicaconitin, MI, EI, GI, SI, SIA, SII, as well as other Synthetic peptide antagonists derived from naturally occurring peptide antagonists and expression libraries [Lindstrom, 1997, Mol. Neurobiology, 15: 193-222, but is not limited thereto.

We claim the use of channel-blocking nicotine antagonists in the present invention. Channel-blocking nicotine antagonists are defined as compounds that appear to block the ion channels of nicotine receptors (NR) and thereby prevent transmembrane ion influx required for nicotine receptor action. Examples of channel-blocking nicotine antagonists include chlorisondamine, mecamylamine, hexamethonium, amantadine, memantine, dizocilpin [(+)-MK-801], 8 (dethylamino) octyl-3,4,5 -Trimethoxybenzoate (TMB-8) and zinc [Bencherif et al., 1995, J. Pharmacol. and Exper. Therapeutics, 275: 1418-1426; Buisson and Bertrand, 1998, Mol. Pharmacol., 53: 555-563, but is not limited thereto. These compounds appear to act preferentially to open channels, but [Peng et al., 1997, Mol. Pharmacol., 51: 776-784; Buisson and Bertrand, 1998, Mol. Pharmacol., 53: 555-563, compounds that block closed channels are also suitable for use in the present invention.

We claim the use of noncompetitive nicotine antagonists in the present invention. Noncompetitive nicotine antagonists are defined as compounds that antagonize the action of nicotine receptors (NR) but do not appear to block ligand binding sites or directly block ion channels. Functional blocking of ion influx through ion channels of nicotine receptors by noncompetitive nicotine antagonists cannot be overcome by increasing the concentration of the agent [Fryer and Lukas, 1999a, J. Pharmacol. and Exper. Therapeutics, 288: 88-92; Fryer and Lukas, 1999b, J. Neurochem., 72: 1117-1124]. Volatile anesthetics, including ethanol, and tetracaine and procaine, are noncompetitive action nicotine antagonists against other nicotine receptor subtypes [Bencherif et al., 1995, J. Pharmacol. and Exper. Therapeutics, 275: 1418-1426; Lindstrom, 1997, Mol. Neurobiology, 15: 193-222. Unexpected noncompetitive nicotine antagonists include psychoactive compounds such as buproprion, penciclidine, ibogaine, sertraline, paroxetine, nefaxodon, venlafaxine and fluoxetine [Fryer and Lukas, 1999a, J. Pharmacol. and Exper. Therapeutics, 288: 88-92; Fryer and Lukas, 1999b, J. Neurochem., 72: 1117-1124. Other noncompetitive nicotine antagonists may act through allosteric sites used by known positive allosteric effectors, such as ivermectin, or act as negative allosteric effectors that act on other sites on the receptor. Krause et al., 1998, Mol. Pharmacol., 53: 283-294]. Certain voltage-dependent mechanisms, including voltage-sensitive Mg ²⁺ blockade of nicotine receptor ion channels, voltage-dependent channel-blocking by intracellular spermine, and nicotine receptor inactivation induced by membrane depolarization are also noncompetitive. Can act as an antagonist.

We claim the use of antibodies that act as nicotine antagonists in the present invention. Antibodies can also act as nicotine antagonists; For example, the monoclonal antibody mAb 319 blocks the action of nicotine receptors (NR) upon intracellular injection [Cuevas and Berg, 1998, J. Neurosci. 18: 10335-10344. Antibodies include polyclonal antibodies, monoclonal antibodies, humanized or chimericized antibodies, single chain antibodies, FAb fragment F (Ab) ′ ₂ fragments, fragments prepared by FAb expression libraries, anti-ID antibodies, and Epitope-binding fragments of any of the above are included.

We claim the use of agents that act as nicotine antagonists in the present invention. The agent may also act as a nicotine antagonist under certain circumstances, for example based on time-average antagonist effect. Reversible desensitization is observed after stimulation with all agents including but not limited to acetylcholine, nicotine, epivatidine, cyticin, methylcarbamylcholine and DMPP. Some compounds have a dual action effect that acts as an agonist for some nicotine receptor subtypes and as an antagonist for others. Heterocyclic substituted pyridine derivatives (+/-)-2- (3-pyridinyl) -1-azabicyclo [2.2.2] octane (also known as RJR-2429) are human muscle nicotine receptors and putative α3β4 Selectively activates the containing receptors, but inhibits nicotine receptors in rat thalamus specimens. This compound is a partial agonist for nicotine receptors that mediates dopamine release from rat synaptic cell samples [Bencherif et al., 1998, J. Pharmacol. and Exper. Therapeutics, 284: 886-894. At sufficiently high concentrations, any agent can cause reversible and irreversible desensitization, and / or inactivation of nicotine receptors. Exposure to high concentrations of the agent results in time-averaged antagonist action on the exposed cells. For example, this phenomenon is observed after chronic nicotine exposure at concentrations comparable to the circulating nicotine levels found in human cigarette smokers, and may cause inactivation of certain nicotine receptor subtypes [Lindstrom, 1997, Mol. Neurobiology, 15: 193-222.

Section 3. Screening Assays for Compounds Modulating Nicotine Receptor Activity

The following assay was designed to identify compounds that interact with nicotine receptors (NR) that regulate ocular growth, compounds that interfere with NR, and compounds that modulate the activity of NR genes or modulate NR levels. In addition, assays can be used to identify compounds that can bind to and regulate NR levels.

Compounds that can be screened according to the invention include those that bind to NR to mimic activity induced by natural ligands (eg, agonists), or inhibit activity induced by natural ligands (eg, antagonists). Peptides, antibodies and fragments thereof, and other organic compounds (eg, peptide mimetics), as well as peptides, antibodies that mimic domains of NR (or portions thereof) to bind and “neutralize” natural ligands, and Fragments thereof, and other organic compounds, including but not limited to.

Such compounds include naturally occurring peptides such as contotoxins, for example random peptide libraries (see, eg, Lam et al., 1991, Nature, 354: 82-84; Houghten et al., 1991, Nature, 354: 84-86). ) And synthetic peptides (random or partially altered designated phosphates), such as soluble peptides, including but not limited to components of permutant chemically derived molecular libraries prepared from D- and / or L-sequencing amino acids, phosphopeptides. Components of, but not limited to, popopeptide libraries (see, eg, Songyang et al., 1993, Cell, 72: 767-778), antibodies (polyclonal, monoclonal, humanized, anti Individual, chimeric or single chain antibodies, and FAb, F (Ab ') 2 and FAb expression library fragments, and epitope-binding fragments thereof, and organic or inorganic small molecules, This is not restrictive.

Other compounds that may be screened in accordance with the present invention include NR genes or NRs that cross the blood-retinal or blood-aqueous humoral barrier and enter the appropriate cells (e.g., by interacting with regulatory regions or transcription factors related to gene expression). Organic small molecules that can affect the expression of several other genes involved in signal transduction pathways; Or compounds that affect the activity of NR or the activity of some other intracellular factor associated with the NR signal transduction pathway.

Computer modeling and retrieval techniques enable the identification of compounds capable of modulating NR activity, or the improvement of already identified compounds. When identifying such compounds or compositions, active sites or regions are identified. Such active sites can typically be ligand binding sites, such as a ligand's interaction domain with the NR itself. Active sites can be identified using methods known in the art, for example, from amino acid sequences of peptides, from nucleotide sequences of nucleic acids, or from studies of complexes of compounds or compositions apparent with natural ligands. In the case of complex studies, active sites can be determined by using chemical or X-ray crystallographic methods to find cases where complexed ligands are found on the factor. Next, the three-dimensional conformation of the active site is determined. This can be done by known methods which can determine the complete molecular structure, for example X-ray crystallography. On the other hand, solid state or liquid phase NMR may be used to determine specific intramolecular distances. Experimental methods of any other structural determination can be used to obtain partial or complete conformational structures. The conformational structure can be measured by natural or artificial complexed ligands, which can increase the accuracy of the determined active site structure.

Once an incomplete or less accurate structure is determined, computer-based numerical modeling methods can be used to complete the structure or improve its accuracy. Use any known modeling method, including a parameterization model specific for a particular biopolymer, such as a protein or nucleic acid, a molecular dynamics model based on molecular kinetic computing, a statistical mechanical model based on a set of columns, or a combined model Can be. For most types of models, a standard molecular force field representing the force between constituent atoms and groups is required, which can be selected from force fields known in the physicochemical field (Bencherif et al., 1998, J Pharmacol. And Exper.Therapeutics, 284: 886-894]. Incomplete or less accurate experimental structures can be a constraint on the complete and more accurate structures computed by these modeling methods.

Finally, once the structures of the active sites have been determined experimentally by modeling or combination method, candidate modulating compounds can be identified by examining a database containing compounds according to information about their molecular structure. This investigation finds compounds that interact with the groups that match the defined active site structure and define the active site. The survey may be passive, preferably with the aid of a computer. These compounds found through this investigation are potential NR modulating compounds.

Alternatively, these methods can be used to identify improved regulatory compounds from known regulatory compounds or ligands. The composition of known compounds can be modified, and the structural effects of the modification can be measured using experimental and computer modeling methods, such as the methods described above applied to new compositions. The altered structure is then compared to the active site structure of the compound to determine whether the fit or interaction is improved. In this way, one can quickly assess whether the composition is systematically altered by changing the side groups to yield modified regulatory compounds or ligands with improved specificity or activity.

Those skilled in the art will be familiar with additional experimental and computer modeling methods useful for identifying regulatory compounds, and related transduction and transcription factors, based on the identification of active sites of NR.

Examples of molecular modeling systems are the CHARMM and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM performs energy minimization and molecular dynamics functions. QUANTA performs the construction, graphical modeling and analysis of molecular structures. QUANTA allows for interaction configuration, modification, visualization, and analysis of molecular interactions.

Numerous literature on computer modeling of drug interactions with specific proteins (see, for example, Rotivinen et al. [1988, Acta Pharmaceutical Fennica 97: 159-166]; Ripka et al. Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann (1989, Annu. Rev. Pharmacol. Toxiciol. 29: 111-122); Perry and Davis Davides, Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989, Proc. Soc. Lond. 236: 125-140 and 141-162) and for model receptors for nucleic acid components (see, eg, Askew et al., 1989, J. Am. Chem. Soc. 111). : 1082-1090). Other computer programs for screening and graphically describing chemicals are biodesign, Inc. (BioDesign, Inc., Pasadena, CA), Allelix, Inc. (Allelix, Inc., Mississauga, Ontario, Canada) and HyperCube, Inc. (Hypercube, Inc., Cambridge, Ontario). They are primarily designed to apply to drugs specific for a particular protein, but if a region is identified, it can be adapted to design a drug specific for a region of DNA or RNA encoding NR.

Although described above for designing and producing compounds that can alter binding, a library of known compounds, including natural products or synthetic chemicals, and bioactive materials, including proteins, can be screened for compounds that are inhibitors or activators. It may be.

Compounds identified through assays as described herein may be useful, for example, to detail the biological function of NR and to improve myopia. Assays for testing the effects of compounds identified by techniques such as, for example, those described in sections 3.1 to 3.3 are discussed in section 3.4 below.

Section 3.1. In vitro screening assay for compounds that bind to NR

In vitro systems can be devised to identify compounds capable of interacting with NR (eg, capable of binding to NR). Identified compounds can, for example, modulate wild-type and / or mutant NR gene products, detail the biological function of NR, screen for identifying compounds that cleave normal NR interactions, or perform such interactions on their own. It may be useful for cleavage.

The principle of the assay used to identify compounds that bind to NR is that complexes that can be removed and / or detected in the reaction mixture by preparing a reaction mixture of the two components under sufficient time and conditions for the NR and test compound to interact and bind. It is related to forming The NR species used may vary depending on the purpose of the screening assay. For example, when looking for an agent of a natural ligand, full length NR or truncated NR, peptides corresponding to extracellular domains, or assay systems that provide advantages (eg, labeling of the resulting complex, Isolation and the like) can be used a fusion protein containing an NR ligand binding site fused to a protein or polypeptide. If one wants to identify a compound that interacts with the cytoplasmic domain (CD), a fusion protein containing a peptide corresponding to NR CD and NR CD can be used.

Screening assays can be performed in a variety of ways. For example, one method is to perform an assay that involves fixing an NR protein, polypeptide, peptide or fusion protein or test substance to the solid phase and detecting the NR / test compound complex immobilized on the solid phase at the end of the reaction. In one embodiment of this method, the NR reactant can be immobilized on a solid surface and the unimmobilized test compound can be labeled directly or indirectly.

In fact, microquantitative plates can be conventionally used as the solid phase. The immobilized component can be immobilized by noncovalent or covalent attachment. Non-covalent attachment can be achieved by simply coating the solid surface with a protein solution and drying. Alternatively, the protein can be immobilized on a solid surface using an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized. Surfaces can be manufactured and stored in advance.

To carry out the assay, the unimmobilized component is added to the coated surface containing the immobilized component. After the reaction is complete, the unreacted component is removed (eg, by washing) under conditions such that any complexes formed remain immobilized on the solid surface. Detection of complexes immobilized on solid surfaces can be accomplished in a number of ways. In the case of labeling a component that has not been immobilized beforehand, detection of the immobilized label on the surface indicates that a complex has been formed. If the components that have not been previously immobilized are not labeled beforehand, an indirect label may be used, e.g., a labeled antibody specific for the component that has not been previously immobilized (this antibody may also be directly or with a labeled anti-Ig antibody. May be indirectly labeled) to detect complexes immobilized on the surface.

Alternatively, the reaction can be carried out in the liquid phase to separate the reaction product from the unreacted components and detect the complex, for example an immobilized antibody specific for the NR protein, polypeptide, peptide or fusion protein, or test compound. Any complex formed in the solution is fixed and the fixed complex is detected using a labeled antibody specific for the other components of the possible complex.

Alternatively, cell based assays can be used to identify compounds that interact with NR. To this end, cell lines expressing NR, or cell lines genetically engineered to express NR (eg, transfection or transduction of NR DNA) can be used. The interaction of the test compound with the heterologous NR expressed by, for example, the host cell can be measured by comparing or competing with the original ligand.

Section 3.2. Assays for Intracellular Proteins Interacting with NR

Any method suitable for detecting protein-protein interactions can be used to identify transmembrane proteins or intracellular proteins that interact with NR. Typical methods that can be used include coimmunoprecipitation, crosslinking, and gradient or chromatographic columns of proteins and NR obtained from cell lysates or cell lysates to identify proteins in lysates that interact with NR. There is a co-purification method. For these assays, the NR component used is full-length NR, a soluble derivative lacking a membrane anchoring region (e.g., a terminal containing an extracellular domain in which a truncated molecule is fused with a cell domain that is deleted by a transmembrane region). This cleaved NR), a peptide corresponding to the cell domain, or a fusion protein containing the cell domain of the NR. Once isolated, these intracellular proteins can be identified, and then the proteins interacting with them can be identified using standard techniques. For example, at least a portion of the amino acid sequences of intracellular proteins that interact with NR are well known to those skilled in the art, such as Edman degradation techniques (see, eg, Creighton, [1983] , Proteins: Structures and Molecular Principles, pp. 34-39]. The obtained amino acid sequence can be used as a guide for the generation of oligonucleotide mixtures that can be used for screening for gene sequences encoding intracellular proteins. Screening can be performed, for example, by standard hybridization or PCR techniques. Techniques for the generation and screening of oligonucleotide mixtures are well known (see, for example, PCR Protocols: A Guide to Methods and Applications, 1990).

In addition, a method of simultaneously identifying genes encoding transmembrane or intracellular proteins that interact with NR can be used. Such methods include, for example, labeled NR proteins, or NR polypeptides, peptides or fusion proteins, such as markers (eg, enzymes, phosphors, luminescent proteins or dyes) in a manner similar to antibody probing techniques in the well known λgt11 library. Expression library probing using an NR polypeptide or an NR domain, or an IgG-Fc domain, fused thereto.

A two-hybrid system, one method of detecting protein interactions in vivo, is described in detail for illustrative purposes only and is not intended to be limiting. One variation of this system is described in Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 9578-9582, available from Clontech, Palo Alto, CA.

Briefly, the system is used to construct a plasmid encoding two hybrid proteins, one of which is a nucleotide encoding a DNA binding domain of a transcriptional activator protein fused to an NR coding nucleotide sequence, an NR polypeptide, a peptide or a fusion protein. And other plasmids, as part of the cDNA library, consist of nucleotides encoding the activation domain of a transcriptional activator protein fused to a cDNA that encodes an unknown protein recombined with this plasmid. DNA binding domain fusion plasmids and cDNA libraries were transformed into yeast Saccharomyces cerevisiae strains in which the regulatory region contains a reporter gene (eg, HBS or lacZ) containing the binding site of a transcriptional activator. Let's do it. One hybrid protein alone cannot activate transcription of the reporter gene. A hybrid containing DNA binding domains cannot activate transcription because it does not provide an activation function, and the activation domain containing hybrids can concentrate on the binding site of the active agent. It is not possible to activate transcription. The interaction of the two hybrid proteins reconstructs the functional activator protein to express the reporter gene, which is detected by assay for the reporter gene product.

Bi-hybrid systems or related methods can be used to screen the activation domain libraries for proteins that interact with the "bait" gene product. As a non-limiting example, NR can be used as a bait gene product. The entire genomic sequence and cDNA sequence are fused to the activation domain coding DNA. This library and the plasmid encoding the hybrid of the bait gene product fused to the DNA binding domain are co-transformed with an enzyme reporter strain and the resulting transformants are screened for reporter gene expression. As a non-limiting example, a bait NR gene sequence, such as an open reading frame, can be cloned into a vector to express the protein by fusion to DNA encoding the DNA binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. Next, DNA sequencing is used to identify the protein encoded by the library plasmid.

CDNA libraries of cell lines used to detect proteins that interact with the bait NR gene product can be prepared using methods commonly practiced in the art. According to certain systems described herein, cDNA fragments can be inserted into a vector to fuse to the transcriptional activation domain of GAL4 to express the protein. This library can be co-transformed with the bait NR gene-GAL4 fusion plasmid into a yeast strain containing the lacZ gene induced by a promoter containing a GAL4 activating sequence. The cDNA coded protein interacting with the bait NR gene product and fused to the GAL4 transcriptional activation domain will reconstruct the active GAL4 protein to induce the expression of the HIS3 gene. Colonies expressing HIS3 can be detected by growth on Petri dishes containing histidine deficient semisolid agar based medium. CDNA can then be purified from these strains, which can be used to prepare and isolate proteins that interact with the bait NR gene product using techniques commonly practiced in the art.

Section 3.3. Assays for compounds that interfere with NR / intracellular or NR / transmembrane macromolecular interactions

Macromolecules that interact with NR are referred to as "binding partners" for purposes of discussion. These binding partners seem to be related to the NR signal transduction pathway, and thus the role of NR in the regulation of postnatal eye growth. Thus, it is desirable to identify compounds that interfere with or disrupt the interaction of the binding partner with NR, which may be useful for the regulation of NR activity and for the regulation of postnatal eye growth associated with NR activity.

The basic principle of an assay system used to identify compounds that interfere with the interaction between NR and its binding partner or partners is that the binding partner interacts with the NR protein, polypeptide, peptide or fusion protein as described in sections 3.1 and 3.2 above. It involves the preparation of a reaction mixture of two materials under sufficient time and conditions to act and bind to form a complex. To test compounds for inhibitory activity, reaction mixtures are prepared in the presence and absence of test compounds. The test compound may be included in the reaction mixture from the beginning, or the test compound may be added at a predetermined time after adding the NR residue and its binding partner. The control reaction mixture is incubated without the test compound or with the placebo. Next, the formation of any complex between the NR residue and the binding partner is detected. The formation of complexes in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interfered with the interaction of the NR with the inactive binding partner. In addition, complex formation in the reaction mixture containing the test compound and the normal NR protein can be compared to complex formation in the reaction mixture containing the test compound and the mutant NR. This comparison may be important when it is desirable to identify compounds that disrupt the interaction of non-normal mutant NRs.

Analysis of compounds that interfere with the interaction of NR with the binding partner can be performed in a heterogeneous or uniform fashion. Heterogeneous analysis involves immobilizing the NR residue product or binding partner on the solid phase and detecting the complex immobilized on the solid phase at the end of the reaction. In homogeneous analysis, the entire reaction is carried out in the liquid phase. Either way, the order of addition of the reactants can be varied to give different information about the compound to be tested. For example, a test compound that interferes with interaction by competition can be identified by conducting the reaction in the presence of the test substance, ie by adding the test substance to the reaction mixture prior to or simultaneously with the addition of the NR residue and the interaction binding partner. have. Alternatively, a test compound that cleaves a previously formed complex, such as a compound with a larger binding constant, that substitutes for one of the complex components, can be tested by adding the test compound to the reaction mixture after the complex is formed. Various formats are briefly described below.

In heterogeneous assay systems, NR residues or interaction binding partners are immobilized on a solid surface while directly or indirectly labeling unfixed species. In practice, microquantitative plates are conveniently used. Immobilized species can be immobilized by noncovalent or covalent attachment. Non-covalent attachment can be accomplished simply by coating the solid surface with NR or binding partner solution and drying. Alternatively, immobilized antibodies specific for the species to be immobilized can be used to immobilize the species on a solid surface. The surface can be prefabricated and stored.

To perform this assay, the partner of the immobilized species is exposed to the coated surface in the presence or absence of the test compound. After completion of the reaction, unreacted components are removed (eg, by washing) and any complexes formed will remain immobilized on the solid surface. Detection of complexes immobilized on a solid surface can be performed by various methods. When pre-labeling non-immobilized species, detection of immobilized label on the surface indicates that a complex has formed. In the absence of pre-labeling non-immobilized species, indirect labeling can be used to detect complexes immobilized on the surface, e.g. labeled antibodies that are initially specific for non-immobilized species (antibody labeled -Ig antibodies can be labeled directly or indirectly). Depending on the order of addition of the reaction components, test compounds that inhibit the formation of the complex or cleave the previously formed complex can be detected.

Alternatively, immobilized antibodies that are specific for any of the binding components to carry out the reaction in the liquid phase in the presence or absence of the test compound, to separate the unreacted components from the reaction product, and to fix any complexes formed in the solution, for example In order to detect an immobilized complex, the complex can be detected using a labeled antibody specific for the other partner. In addition, according to the order in which the reactants are added in the liquid phase, it is possible to identify test compounds which inhibit the complex or cleave the previously formed complex.

In another embodiment of the present invention, a homogeneous assay can be used. In this method, a preformed complex of NR residues and interaction binding partners is prepared, wherein the NR or its binding partner is labeled, but the signal generated by the label due to the complex formation is quenched (e.g., in immunoassays See Rubenstein, US Pat. No. 4,109,496 using the method). The addition of a test substance that competes with a preformed complex to replace any of the species of the complex will result in a signal greater than the background value. In this way, it is possible to identify test substances that cleave the interaction of NRs with intercellular binding partners.

In certain embodiments, NR fusions can be prepared for passivation. For example, a fusion vector such as pGEX-5X-1 can be used to fuse the NR or peptide fragment into the glutathione-S-transferase (GST) gene in such a way that its binding activity is maintained in the resulting fusion protein. The interaction binding partner can be purified and used to generate monoclonal antibodies using methods commonly practiced in the art. Such antibodies can be labeled with radioisotopes ¹²⁵ I, for example by methods commonly used in the art. In heterogeneous assays, for example, GST-NR fusion proteins can be immobilized to glutathione-agarose beads. The interaction binding partner can then be added in a way that can cause interaction and binding, in the presence or absence of the test compound. At the end of the reaction period, unbound material can be washed away and labeled monoclonal antibodies can be added to this system to bind to the complexing component. Interaction with NR (as Gene Product) The binding partner can be detected by measuring the amount of radioactivity that remains bound with glutathione-agarose beads. Successful inhibition of interaction by the test compound will reduce the measured radioactivity.

Alternatively, the GST-NR fusion protein and the interaction binding partner can be mixed together in the liquid phase in the absence of solid glutathione-agarose beads. Test compounds may be added during or after the species interacts. This mixture can then be added to glutathione-agarose beads, and the unbound material is washed off. Again, labeled antibodies can be added and the radioactivity bound to the beads can be measured to detect the degree of inhibition of NR / binding partner interaction.

In another embodiment of the present invention, the same technique can be used in place of one or both of the full-length proteins using peptide fragments corresponding to the binding domains of the NR and / or interacting binding partner (if the binding partner is a protein). Can be. Methods commonly practiced in the art can be used to identify and isolate binding sites. Such methods include, without limitation, mutagenesis of genes encoding one of the proteins and screening for binding cleavage in co-immunoprecipitation assays. Subsequently, a compensation mutation in the gene encoding another species in the complex can be selected. Sequence analysis of the gene encoding each protein will reveal mutations corresponding to the protein regions involved in the interaction binding. Alternatively, one of the methods described above can be used to immobilize a protein on a solid surface and interact with its labeled binding partner, treated with a proteolytic enzyme such as trypsin. After washing, labeled short peptides comprising a binding domain can remain bound to a solid material, which can be isolated and identified by amino acid sequencing. In addition, once a gene encoding an intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity, purified or synthesized.

For example, without limitation, a GST-NR fusion protein can be prepared and immobilized to the solid material described above, for example by binding to glutathione agarose beads. The interaction binding partner is a radioisotope such as ³⁵ S. Labeling can cleave proteins such as trypsin with enzymes. The cleavage product can then be added to bind the immobilized GSR-NR fusion protein. After washing off the unbound peptide, labeled binding substances representing intercellular binding partner binding domains can be eluted, purified, and analyzed for amino acid sequences by known methods. The peptides thus identified can be prepared synthetically or fused to suitable promoter proteins using recombinant DNA technology.

3.4. Assays to identify compounds that improve abnormal postnatal eye growth

Without limitation, compounds including binding compounds identified by analytical techniques such as those described in 3.1 to 3.3 above can be tested for their ability to improve abnormal postnatal eye growth, including myopia. Compounds affecting NR activity (e.g., compounds that bind to NR and inhibit the binding of natural ligands to activate signal agonists (agonists) or prevent activation (antagonists), and bind to natural ligands of NR) To neutralize ligand activity); Or compounds that affect NR gene activity (molecules that can modulate expression of full-length or truncated forms of NR by affecting NR gene expression, for example by affecting or interfering with splicing events, such as Protein or small organic molecules). However, the assays described may affect downstream signaling events such as compounds that modulate NR signal transduction (eg, tyrosine kinases or inhibitors or enhancers of phosphatase activity that participate in delivering signals activated by ligands that bind NR). Compounds) can also be confirmed. Identification of these compounds that act on another step in the NR signal transduction pathway in which the NR gene and / or NR gene product is involved and that, by acting on this same pathway, can modulate the effect of NR on the development of abnormal postnatal eye growth. And use is within the scope of the present invention. These compounds can be used as part of a therapeutic method to treat other diseases resulting from myopia and abnormal postnatal eye growth.

The present invention encompasses cellular and animal model-based assays for identifying compounds that exhibit the ability to improve myopia symptoms, signs or characteristics. Such cell-based assay systems can also be used as "standards" for analyzing the purity and potency of natural ligands, including ligands prepared by recombinant or synthetic methods.

Cell line systems can be used to identify compounds that may act to ameliorate myopia symptoms, signs or characteristics. Such cellular systems may include recombinant or non-recombinant cells such as, for example, cell lines producing NR. For example, retinal cells or cell lines derived from the retina can be used. It also expresses functional NR, as measured, for example, by chemical or phenotypic changes, induction of another host cell gene, changes in ion flux (eg, Na ⁺ , K ⁺ ), and for activation by natural ligands. Expression host cells genetically engineered to respond (eg, COD cells, CHO cells, fibroblasts) can be used as endpoints in this assay.

Using such a cellular system, the cells are exposed to a compound that is expected to exhibit the ability to ameliorate myopia symptoms in an intact eye, and at a concentration sufficient for sufficient time to induce an improvement in myopia-associated cell phenotype or cell function in the cells that are exposed. May be exposed. After exposure, cells can be analyzed to determine alterations in gene expression, for example, by analyzing mRNA transcripts (eg, by Northern assay) or cell lysates for NR proteins expressed in the cells. Compounds that modulate or modulate the expression of the NR gene are good candidates for therapeutic use. Alternatively, cell testing determined whether one or more myopia-associated cell phenotypes or cell functions were altered to resemble a more normal or more wild type, non-myopia phenotype, or phenotype that is less likely to reduce the incidence or severity of myopia symptoms. It is also possible to analyze the expression and / or activity of the components of the signal transduction pathway in which NR is part of it, or the activity of the NR signal transduction pathway itself.

For example, after exposure, cell lysates can be analyzed for the presence of tyrosine phosphorylation of host cell proteins as compared to lysates derived from unexposed control cells. The ability of a test compound to inhibit tyrosine phosphorylation of host cell proteins in these assay systems indicates that the test compound inhibits signal delivery initiated by NR activation. Cell lysates can be readily analyzed using Western blot format, ie, host cell proteins are digested by gel electrophoresis and antiphosphotyrosine detection antibodies (e.g., radiolabels, phosphors, enzymes, etc.). Import and probe using antiphosphotyrosine antibodies labeled with the same signal generating compound (see, eg, Glenney et al., 1988, J. Immunol. Methods, 109: 277-285; Frackelton et al., 1983, Mol). Cell. Biol., 3: 1343-1352). Alternatively, the ELISA format can be used to immobilize specific host cell proteins involved in the NR signal transduction pathway using immobilized antibodies specific for the target host cell protein, and immobilized hosts using labeled antiphosphotyrosine antibodies. The presence of phosphotyrosine on cellular proteins is detected (see King et al., 1993, Life Sciences, 53: 1465-1472). In another method, ion flux, such as sodium or potassium ion flux, can be measured as an endpoint for NR stimulating signal delivery. Membrane depolarization can also be measured as an endpoint for NR stimulation effects.

In addition, animal-based myopia models can be used to identify compounds that may improve myopic-like symptoms. Such animal models can be used as test substrates to identify drugs, drugs, treatments and interventions that may be effective in treating the disease. For example, a compound that is expected to exhibit the ability to ameliorate myopia symptoms may be exposed to the animal model for a sufficient time and at a concentration sufficient to induce improvement of myopia symptoms in exposed animals. Animal response to exposure was monitored to assess the conduction of features, signs or symptoms associated with myopia. With respect to intervention, any therapy that can invert any of the myopic-like features, signs, or symptoms will be considered as a candidate for the intervention of myopia in humans. Dosages of test agents can be determined by deriving a dose-response curve.

Section 4. Pharmaceutical Compositions

The nicotine antagonists of the present invention have been found to have useful pharmacological properties. Nicotine antagonists regulate the postnatal growth of the eye, particularly with the desirable effect of inhibiting postnatal eye growth and preventing myopia development. Such effects can be confirmed, for example, using the methods described in the Examples below.

Thus, these compounds regulate postnatal growth of the eye, inhibit postnatal eye growth, prevent myopia, regulate abnormal postnatal eye growth, inhibit abnormal postnatal axial growth of the eye, inhibit abnormal equatorial swelling of the eye, It can be used to inhibit vitreous cavity expansion, suppress the progression of myopia, inhibit the onset of myopia and invert myopia. These compounds are particularly useful for suppressing the development of myopia.

The compounds of the present invention are generally administered to animals, including but not limited to birds, monopores, reptiles or fish, as well as mammals including humans.

The pharmacologically active compounds of the present invention can be treated according to conventional herbal preparation methods for the preparation of pharmaceutical preparations for administration to a patient, for example a mammal, including humans. The compounds of the present invention can be used in combination with conventional excipients, ie, pharmaceutically acceptable organic or inorganic carrier materials suitable for parenteral, digestive (oral) or topical ophthalmic applications that do not adversely react with the active compound. Suitable pharmaceutically acceptable carriers include water, salt solutions, alcohols, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and di Glycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, or any other suitable carrier. Pharmaceutical formulations may be sterilized and, if desired, mixed with auxiliary agents which do not adversely react with the active compound, for example lubricants, preservatives, stabilizers, wetting agents, emulsifiers, osmotic induction salts, buffers and the like. They can also be mixed with other active agents, for example vitamins, if desired.

For parenteral applications, in particular sterile solutions for injection, preferably aqueous or oily solutions as well as suspensions, emulsions or implants are suitable.

For digestive tract applications, tablets, dragees, solutions, drops, suppositories or capsules are particularly suitable. Syrup, elixir, etc., in which a sweetened vehicle is used, can be used.

For topical application, liquid to viscous to semisolid or solid forms can be used, including carriers suitable for topical application, preferably having a dynamic viscosity greater than water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, gels, powders, coatings, plasters, aerosols, etc., if desired, sterilized, or auxiliary agents such as preservatives, stabilizers, wetting agents, Mixed with buffers or osmotic induction salts and the like. For topical application, sprayable aerosol formulations are also suitable, in which the active ingredient, preferably the active ingredient in combination with a solid or liquid inert carrier material, is filled in a compressive bottle or otherwise sprayed through a vehicle capable of aerosolizing the preparation. .

The compounds of the present invention are useful for treating or preventing the development of myopia. Inhibition of axial elongation or equator dilatation, control of postnatal growth of eyes, suppression of postnatal eye growth, myopia prevention, control of abnormal postnatal eye growth, suppression of abnormal postnatal axis growth of eyes, suppression of abnormal equatorial dilation of eyes, suppression of vitreous cavity expansion, myopia progression Therapies for inhibition, myopia initiation inhibition, and reversal of myopia can be administered by using the formulation in eye drops. Eye drops typically consist of an active agent concentration of about 0.005% to 10%, advantageously about 0.01% to 5%, preferably about 0.1% to 2% in the eye medium. A pH of about 3.5 to 8.5, advantageously about 4.0 to 8.0, and preferably about 4.5 to 7.5 can be predicted as an ophthalmic drop. Phosphate Buffers Conventional but other buffers may also be used in eye drops. Typical regimens for the application of eye drops are 1 to 4 times a day at uniform intervals over the waking time. More effective formulations may require less application or may make it possible to use more dilute solutions.

It will be appreciated that the actual preferred amount of active compound in specific cases will vary depending upon the specific compound employed, the particular composition formulated, the form of application, and the particular location and organ to be treated. Dosages for a given host can be determined, for example, by appropriate conventional pharmacological protocols, using conventional considerations by, for example, the conventional activity of known activities and the distinct activity of the present compounds.

Way

A 12-day-old White Leghorn chick (Truslow Farms, Chestertown, Md.) Was used for 12 hours using a General Electric chroma 50 fluorescent lamp with roughly 50 mW / cm2 illuminance at the chick's eye level. It was raised in a chick nursery box on a cancer cycle. The chicks were freely supplied with Purina Chick Chow (registered trademark) feed and water.

The experiment started at 1 week of age. For some chicks, as a commonly studied experimental model that induces ipsilateral myopia in newly hatched chicks and nearly mature ones (Papastergiou et al ., 1998, Vision Res. , 38: 1883-8), one eye translucent white plastic The goggles adhered well to the periorbital wing using cyanoacrylate glue to induce loss of shape myopia. At this time, under sterile conditions, 10 μl of intravitreal injection of drug or saline vehicle was applied to the goggles eye. Other chicks were not wearing goggles, but likewise applied intravitreal injection of drug or vehicle to one eye. In most experimental groups, drugs and vehicles were administered by intraocular infusion daily or every other day at approximately 4 hours into the light phase. Following each, the experimental eyes alternated left and right, and all contralateral eyes were infused with saline vehicle simultaneously with the injection into the experimental eye. The chicks were anesthetized by inhaling ether for all goggles and drug injection.

After 1 week and 2 weeks of treatment, chicks were anesthetized with an intramuscular mixture of ketamine (20 mg / kg) and xylazine (5 mg / kg) and described by Stone et al. , 1995, Vision Res. , 35: 1195-202, and were subjected to ocular refraction and A-scan ultrasonography. No intraocular infusion was administered on the test day. Under still general anesthesia, the chicks were beheaded and the axis and equator dimensions of the extracted eye were measured with a digital caliper. The observation profile of the chick eye is elliptical, and the equatorial diameter is recorded as the average of the shortest and longest equatorial dimensions of the eye.

Data are presented as mean ± S.E.M. And analyzed by SigmaStat (SPSS, Inc., Chicago, IL). Neither vision loss or drug treatment for these eyes affected lens thickness, and these data are not reported for goggles wearing chicks. One-way ANOVA was performed using the difference between the blinded and contralateral eyes of the goggles wearing chicks to confirm drug effects on experimental myopia. Since the ultrasound data for the axis length after mecamylamine treatment for goggles wearing eyes do not meet the normal conditions, these data are summarized for Kruskal-Wallis for ranking of differences between experimental and contralateral eyes. Evaluated by one-way ANOVA. Data from different cohorts of chicks tested with the same drug with each vehicle treated control were collected for analysis (FIG. 1). Since drug effects on chicks without goggles were also not normally distributed, drug treated beagle goggles and vehicle treated contralateral eyes were compared by Friedman repeated measures ANOVA for ranking. Then, when ANOVA confirmed the test effect, post-multiple pair comparisons of the treatment groups were done with a Tukey test using statistical significance level values P <0.05. In assessing the sharp drug effect on ocular refraction and ultrasound, pre and post drug infusion measurements were compared by Student's paired t-test. The experiment was consistent with the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research.

<Example 1>

The drugs dihydro-β-erythroidine hydrobromide (RBI / Sigma; Natick, Mass.), Mecamylamine (RBI / Sigma) and methyllicaconitine citrate (RBI / Sigma) were administered daily. Since Chlorisondamine Diiodide (Tocris Cookson, Volwyn, Missouri, USA) is a long-acting nicotine antagonist in the mammalian brain (El-Bizri and Clarke, 1994, Br. J. Pharmacol. , 113: 917-925), It was administered every other day, usually by intravitreal injection, in most experiments.

Goggles wearing chick

As predicted in this study, a cohort of vehicle treated control chicks wearing one eye goggles was developed with ipsilateral myopia of about -7 to -12 diopters and compared with the contralateral beagle goggles. The axial length of the goggles wearing eye increased by 0.4 to 0.6 mm compared to the contralateral eye. In general, the axial length difference between the goggles wearing eye and the open eye was greater in the measurement by the ultrasound recording on the inner limiting membrane than in the measurement by the caliper recording on the outer sclera surface. In addition to the large variables in caliper measurements, this imbalance can be at least partially physiological, such as thinning both the choroid and the retina of a young chick while wearing goggles. The vitreous cavity of the goggles wearing eye expanded both the axial and equator dimensions as the vitreous steel elongated, which is a large cause of the increase in the overall axial length of the eye. Wearing goggles alone did not induce any significant effect on the anterior chamber depth in most cohorts of vehicle-treated chicks.

Two relatively non-selective nicotine antagonists, chlorisondamine and mecamylamine, were tested. Chlorisondamine reduced myopic refractive abnormalities (FIG. 1; ANOVA: P <0.001) and inhibited excessive axial elongation developing under goggles (FIGS. 2A, 2B and 2C ANOVA: ultrasound, P <0.001; calipers, P = 0.008), axial (ANOVA: P <0.001) and equator (ANOVA: P = 0.001) dimensions all reduced vitreous cavity expansion. Chlorisondamine had no statistically significant effect on the anterior chamber depth. Post pair comparison by the Tuki test (Table 1) showed significant drug effects on vehicle treated controls with respect to refractive, axial length and vitreous cavity depth measurements for comparison between several different groups.

The effect of mecamylamine on goggles wearing eyes was more complicated with different multifaceted responses between low drug doses and high drug doses. It had a maximum anti-myopia effect at intermediate doses, and at the lowest doses it tended to stimulate myopic refractive shift and growth of the goggles wearing eye. Overall, mecamylamine changed the reflection of goggles wearing eyes (FIG. 1; ANOVA: P <0.001). Although all three higher drug doses reduced induced myopia, only the 50 μg dose was significantly different from the control by the post pair comparison test (Table 1) and substantially eliminated the induced myopia. Post-mortem testing showed that the refraction at 1 and 10 μg doses differed from the control, respectively, while significant differences occurred between 10 and 50 μg doses as well as between 1 μg and 50, 100 and 200 μg doses, respectively. (Table 1). The anatomical effect of mecamylamine on goggles wearing eyes tends to follow the refraction effect: eyes are enlarged at doses that do not reduce myopia, and eyes are reduced at doses that decrease (FIG. 2). For axial length (FIGS. 2A and 2C), there was a tendency to drug effect only by ultrasound (FIG. 2A ANOVA: P = 0.07); There was no statistical effect on the axis length by caliper measurement. Mecamylamine affected the ultrasonic measurement of the vitreous steel length (FIG. 2B ANOVA: P = 0.007); Post hoc comparison assays showed that the 1 μg dose differed from the 50 μg dose, but not the control (Table 1). Likewise, the overall equator diameter of the goggles wearing eye was affected by mecamylamine (FIG. 2D ANOVA: P = 0.003); Post hoc comparison assays did not confirm that any individual doses differed from the control, but the 1 and 50 μg doses differed from each other and the 10 μg doses differed from both the 50 and 200 μg doses. (Table 1).

In addition, the anterior chamber could be affected from mecamylamine (ANOVA: P = 0.009), but no individual differences were identified in the post pair assay test. Examining the data on the anterior chamber depth, the vehicle treated goggles wearing eyes had a slightly shallower anterior chamber depth than the contralateral beagles wearing eyes in the mecamylamine experiment (1.22 ± 0.04 mm vs. goggles wearing eyes 1.34 ± 0.04 mm). The anterior chamber depth difference between the goggles wearing eye and the contralateral eye was similar for low mecamylamine doses; For the 100 and 200 μg doses, the anterior chamber depth of the drug treated goggles wearing eye was no longer reduced compared to the contralateral control, and was instead the same (data not shown).

Of the antagonists with some subtype selectivity, methyllicaconitine showed greater efficacy of the two drugs, similar to mecamylamine, with the best effect occurring at intermediate drug doses. Methyllicaconitine was applied to myopic refractive eyes (ANOVA: P = 0.04), axial length (ANOVA: ultrasound, P = 0.05 (FIG. 2A); calipers, P = 0.002 (FIG. 2C) ) And equatorial expansion of vitreous steel (ANOVA: P = 0.02 (FIG. 2D)). There was no noticeable effect on vitreous cavity length with this drug (FIG. 2B, ANOVA: P = 0.09). Post hoc multiple pair comparisons by the Tuki assay showed significant differences from the control when only 5 μg of the dose of equator swelling under the goggles was inhibited. In addition, 5 μg dose reduced the axial length (calipers) compared to doses 0.05, 0.5 and 50 μg (Table 1). Methylcaconitine did not affect the anterior chamber depth of the eye wearing goggles.

Dihydro-β-erythroidine showed only a minor effect on experimental myopic eyes (FIGS. 1, 2). The drug induces a significant reduction only in the axial length as measured by calipers (ANOVA: P = 0.02 (FIG. 2C)), but each drug dose was not confirmed by a post-multiple pair comparison test. Otherwise, the difference in refraction, ultrasonic measurements, or caliper measurements of the equatorial diameter did not have statistical significance by ANOVA.

Goggles without chicks : Chicks were bred without goggles and treated as described in the method section above. Under sterile conditions, 10 μl of the drug or saline vehicle was injected intravitreally into goggles without or “opened” chick eyes. All sagittal eyes were injected with saline vehicle at the same time as the experimental eyes. Drugs and vehicles are injected about 4 hours daily or every other day on the intraocular neck.

Ipsilateral intravitreal administration of chlorisondamine reduced axial growth in drug treated eyes of chicks without goggles at all (FIG. 3; ANOVA for grade: ultrasound, P = 0.03; calipers, P = 0.03). Growth reduction is limited to the vitreous steel (ANOVA for grade: P = 0.01) and reflected in the primitive change in refraction (ANOVA for grade: P = 0.004). The effect of equator expansion on the vitreous steel was not statistically significant. The paired comparison of the Tuki assay confirmed the refraction of the eyes treated with 200 μg and 10 μg, and the vitreous depths of the eyes were treated with 100 μg and 50 μg differently from each other (Table 1). In addition, the effect on the lens thickness was described in comparison with an increase of about 0.1 mm in the group administered with 10 μg in both eyes compared to the chicks administered with 50, 100 or 200 μg and saline injected in both eyes. For ANOVA: P <0.001), pair comparison of lenses by Tukey's test did not confirm significance.

In contrast to the chlorisondamine effect on open eyes, daily infusion of mecamylamine into the vitreous can be performed at 50 μg (dose with the best effect on shape loss myopia), or 10 or 1 μg (myopia for goggles). Doses, which tend to promote sexual response, had no effect on the growth or refraction of the eye without wearing goggles after one week.

<Example 2>

Acute drug effects : To assess acute drug effects, doses selected for different 2 week-old chicks (n = 5 / group) based on drug effects on eye growth with goggles (Chlorisondamine 200 μg, mecamyl Nicotine antagonists of 50 μg and 1 μg of amine, 5 μg of methyllicaconitine or 50 μg of dihydro-β-erythroidine) were injected into the ipsilateral vitreous single injection. After 0, 2 and 24 hours of injection, both eyes were The refractive index and the ultrasonic graph were examined by the method. Since the chicks of the eye growth study were not administered on the day of measurement, the irradiation time point after 24 hours was specifically selected to identify potential residual drug effects on intraocular muscles when appropriate for eye growth measurement.

The average baseline pupil diameter was 2.4 ± 0.5 mm. After 2 hours of infusion, each drug induced some swelling of the pupil (baseline change: 200 μg of chlorisondamine, 0.8 ± 0.1 mm, P <0.01; 50 μg of mecamylamine, 0.30 ± 0.1 mm, not significantly changed ; 1 μg mecamylamine, 0.8 ± 0.2 mm, P <0.05; 5 μg methylicaconitine, 0.4 ± 0.2 mm, not significantly changed; 50 μg of dihydro-β-erythroidine, 1.0 ± 0.1 mm, P < 0.01). The pupils of each group were expanded, but still contracted in response to light, while being inactive. By 24 hours, the pupils all returned to normal (baseline change: Chlorisondamine, 0.7 ± 0.2 mm, P <0.05; Mecamylamine 50 μg, 0.4 ± 0.1 mm, P <0.05). Drug use did not significantly affect refraction after 2 or 24 hours. According to the ultrasound graph, Chlorisondamine induces a 0.16 ± 0.04 mm (P <0.05) decrease in axial length after 2 hours and a 0.20 ± 0.07 mm (P <0.05) decrease in posterior ovary depth, each of which is at baseline after 24 hours. Regressed. In addition, Chlorisondamine reduced lens thickness by 0.12 ± 0.04 mm (P <0.05) after 2 hours and decreased 0.16 ± 0.05 mm (P <0.05) after 24 hours. Other drugs did not affect ultrasound measurements.

In these experiments, eyes were examined 2 and 24 hours after administration to see what acute effects the selected dose of nicotine antagonist had on the condition of intraocular muscles. Each drug induced some pupillary bands (pupilar dilation) that could regress to light. In addition, there was probably some control paralysis (perspective control inhibition). Based on the results, these nicotine antagonist drugs did not change their refraction acutely under irradiation conditions or showed any perspective control trends. Only Chlorisondamine changed the size of the eye acutely by ultrasound and temporarily reduced the axial and vitreous lengths after 2 hours of reading, but not 24 hours after reading. In addition, only chlorisondamine seemed to affect lens thickness, reducing lens thickness and increasing focal length. If any increase in lens focal length regulates the development of open eyes receiving this drug daily, it will promote eye growth (Schaeffel F, Glasser A, Howland HC, 1988, Vision Res., 28: 639-). 657), did not inhibit eye growth (FIG. 3). Since drug effects on eye growth are measured 24 hours after the last dose, observations of growth or refraction development shown in Examples 1 and 3 indicate acute drug effects on measurements of refractive or ocular components, otherwise acute drug effects on muscles. Can not be explained. This means that the effects of nicotine antagonists (Examples 1 and 3) on eye refraction and eye growth developed the eye, and the eye's refraction and / or dimensions consisted of developmental changes.

<Example 3>

Long-term effects of single doses of chlorisondamine : In separate experiments, 200 μg of chlorisondamine was administered to a single vitreous eye in the shape loss eyes of different groups of goggles wearing chicks, and saline was administered to the contralateral eye only with goggles. These chicks were compared with the ipsilateral goggles wearing chicks with a single saline injection in both eyes only when using goggles. Both groups were not subsequently injected intraocularly. The refractive index and ultrasound of these chicks were evaluated after 4 days using ketamine / xylazine anesthesia. One week after the use of goggles and a single drug infusion, the refractive index, ultrasound graph and caliper measurements of the extracted eyes of the same chick were again evaluated as described above.

A single dose of 200 μg of chlorisondamine with goggles significantly blunted the response to loss of shape over the next week (Table 2). The effect was evident after 4 days by significant attenuation of the refraction (p <0.05) and axial length (p <0.01) by the ultrasound graph of the goggles wearing eye. The effect on myopic change in refraction decreases after one week, while statistically significant attenuation is measured by ultrasonic measurements of shaft length (p <0.05) and vitreous depth (p = 0.05), and caliper measurements of shaft length (p < 0.05) and equatorial diameter (p <0.005) became evident after one week. The single-dose action of Chlorisondamine on myopic eye growth seemed to be the same after 4 days and 1 week because the ratio of the axial and vitreous lengths of drug-treated vehicle eyes was equal after 4 days and 1 week (Table 2). ).

<Example 4>

Histopathological effects : To identify potential pathological effects in other groups of chicks at 1 week of age with monocular loss or no goggles at all, Chlorisondamine (200 or 100 μg every other day; n = 5-6 / Group), mecamylamine (200 or 50 μg daily; n = 5-8 / group) or saline vehicle (n = 3-9 / group) with goggles open in both eyes, or in chicks without goggles at all Intravitreal injections were made in either eye (administering the vehicle to the contralateral eye) using the same method as above. One week after the treatment, the refractive, ultrasonic and caliper measurements of the method were examined. The eyes were then immersed in 3% glutaraldehyde / 0.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The posterior sections were deeply immersed in paraffin and cut to 5 μm thick, stained with hematoxylin and eosin, or deeply immersed in historesin, cut to 3 μm or 5 μm thick and 0.5% very in 1% borate Stained with LES II / 0.5% methylene blue.

A bi-day administration of 200 μg of chlorisondamine showed that a full examination of the eye cups of both the goggles wearing eye (4/5) and the eyes without wearing goggles at all (n = 5) was performed at the mid-peripheral base. It showed mild, labeled spots and discoloration. The central basal region of geographical regions of non-uniform dimensions was relatively poor or normal. The tissue compartment showed labeled rupture and aggregation of retinal pigment epithelial (RPE) cells in the region corresponding to the peripheral bleaching region. Pigment-containing cells, possibly macrophages, sometimes appear in the outer retina and rupture as the outer sections sometimes cover the ruptured epithelium. Otherwise, the retina appeared intact. The putative inflammatory cells infiltrate the peripheral choroidal capillaries below the most relevant area of the RPE, but otherwise did not affect the choroid. The central area of these eyes showed normal tissue or less labeled changes. In addition, eyes treated with goggles, treated with 200 μg of chlorisondamine and subjected to general full examination, showed normal tissues. Whole irradiation of goggle-eye or eye fold without any goggles, treated with 100 μg of Chlorisondamine every other day, showed a normal base or only minor peripheral pigment changes. Some eyes showed normal tissue, some eyes showed only one large and flexible hyperpigment content in one RPE cell, with only one perceived tissue change, and some labeled RPE / choroids as described above Small, isolated peripheral fragments were seen in capillary pathology. Importantly, it is not clear whether the growth and refraction response of goggles wearing or open eyes to chlorisondamine is related to histopathological extent of the retina.

Eyes with goggles treated daily with 200 or 50 μg of mecamylamine and the retina of the eye without any goggles could not be distinguished globally or histologically from vehicle treated control eyes.

All documents cited herein, whether research papers, patent documents or other cited documents, are hereby incorporated by reference in their entirety as if individually incorporated by reference. It will be appreciated by those skilled in the art that many and various variations can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the text of the present invention is merely illustrative, and is not intended to limit the scope of the present invention.

Postmortem Comparison of Drug Effects Using the Tuki Test Ultrasonic measurement Caliper measurement refraction Shaft length Vitreous Steel Depth Shaft length Equator Diameter Conditions and Drugs Goggles wearing chick Chlorisondamine 200 μg versus control, 50, 10 & 1 μg 100 μg versus control, 50, 10 & 1 μg 200 μg vs. control, 50, 10 & 1 μg 100 μg vs 50 & 1 μg 200 μg vs. control, 50, 10 & 1 μg 100 μg vs 50 & 1 μg 200 μg vs control, 50 & 10 μg 200 μg vs. 1 μg100 μg vs. 1 μg Mecamylamine 200 μg vs 1 μg 100 μg vs 1 μg 50 μg vs Control, 10 & 1 μg - 50 μg vs 1 μg - 200 μg vs 10 μg 50 μg vs 10 & 1 μg Methyllicaconitine - - - 5 μg vs 50, 0.5 & 0.05 μg 5 μg vs control Dihydro-β-erythroidine - - - - - Goggles without chicks Crorysondamine tested eye 200 μg vs 10 μg - 100 μg vs 50 μg - -

A statistically significant (defined as P <0.05) post pair comparison by the Tuki test was shown for each condition and drug, confirming the treatment effect with ANOVA (see text and FIGS. 1-3).

Long-term effect of single-dose chlorisondamine (200 μg) on form loss myopia Time after drug administration 4 days 1 week Refraction difference Vehicle n = 7 -8.82 ± 1.07 diopters -11.72 ± 1.19 diopters Chlorisondamine n = 10 -2.98 ± 1.88 diopters ^** -8.58 ± 2.06 diopters Ultrasonic measurement Shaft length difference Vehicle 0.43 ± 0.04 mm 0.61 ± 0.06 mm Chlorisondamine 0.20 ± 0.07 mm ^*** 0.33 ± 0.11 mm ^** Chlorisondamine / Vehicle B 0.47 0.54 Vitreous Steel Length Difference Vehicle 0.40 ± 0.04 mm 0.69 ± 0.06 mm Chlorisondamine 0.28 ± 0.06 mm 0.49 ± 0.08 mm ^* Chlorisondamine / Vehicle B 0.70 0.71 Caliper measurement Shaft length difference Vehicle - 0.51 ± 0.06 mm Chlorisondamine - 0.55 ± 0.10 mm ^** Equatorial Diameter Difference Vehicle - 0.51 ± 0.06 mm Chlorisondamine - 0.17 ± 0.07 mm ^{**** *} p = 0.05; ^** p <0.05; ^*** p <0.01; ^**** p <0.005, comparing pharmacotherapy group with vehicle group using one-way analysis of variance between goggles and contralateral eyes. The number of chicks in each group is for refraction.

Claims (33)

Use of nicotine antagonist in the manufacture of a medicament suitable for ocular administration for the control of postnatal eye growth. Use of nicotine antagonists in the manufacture of a medicament for inhibiting abnormal axial growth of host animal eyes during postnatal development. Use of a nicotine antagonist in the manufacture of a medicament for the inhibition of abnormal equatorial swelling of a host animal eye during postnatal development. Use of nicotine antagonists in the manufacture of a medicament for inhibiting abnormal vitreous swelling of a host animal eye during postnatal development. Use of a nicotine antagonist in the manufacture of a medicament suitable for ocular administration for the prevention or treatment of myopia. Use according to any one of claims 1 to 5, wherein the nicotine antagonist is a competitive nicotine antagonist. 7. Use according to claim 6, wherein said competitive nicotine antagonist is methyllicaconitin. 7. Use according to claim 6, wherein said competitive nicotine antagonist is dihydro-β-erythroidine. Use according to any one of claims 1 to 5, wherein said nicotine antagonist is a channel-blocking nicotine antagonist. 10. The use according to claim 9, wherein said channel-blocking nicotine antagonist is mecamylamine. 10. The use according to claim 9, wherein said channel-blocking nicotine antagonist is chlorisondamine. 6. Use according to any one of claims 1 to 5, wherein the nicotine antagonist is an uncompetitive nicotine antagonist. 13. The use according to claim 12, wherein said noncompetitive nicotine antagonist is a component of the group consisting of sertraline, paroxetine, nefaxodon, venlafaxine, fluorocetin, bupropion, penciclidine and ibogaine. Use according to any one of claims 1 to 5, wherein the nicotine antagonist is an antibody that inhibits nicotine receptor function. 6. Use according to any one of claims 1 to 5, wherein the nicotine antagonist is an agent that acts like a nicotine antagonist. A method of regulating postnatal eye growth comprising administering a therapeutically effective amount of a nicotine antagonist to the eye to control postnatal eye growth. Inhibiting abnormal axial growth of the eye by administering a therapeutically effective amount of nicotine antagonist to the eye of the host animal during postnatal development. Inhibiting abnormal equatorial swelling of the eye by administering a therapeutically effective amount of nicotine antagonist to the eyes of a host animal during postnatal development. Inhibiting abnormal vitreous cavity expansion of the eye by administering a therapeutically effective amount of nicotine antagonist to the eye of the host animal during postnatal development. Inhibiting the development of myopia by ocularly administering a therapeutically effective amount of nicotine antagonist. 21. The method of any one of claims 16-20, wherein the nicotine antagonist is a competitive nicotine antagonist. The method of claim 21, wherein said competitive nicotine antagonist is methyllicaconitin. The method of claim 21, wherein said competitive nicotine antagonist is dihydro-β-erythroidine. 21. The method of any one of claims 16-20, wherein said nicotine antagonist is a channel-blocking nicotine antagonist. The method of claim 24, wherein said channel-blocking nicotine antagonist is mecamylamine. The method of claim 24, wherein the channel-blocking nicotine antagonist is chlorisondamine. 21. The method of any one of claims 16-20, wherein the nicotine antagonist is an uncompetitive nicotine antagonist. The method of claim 27, wherein said noncompetitive nicotine antagonist is a component of the group consisting of sertraline, paroxetine, nefaxodon, venlafaxine, fluorocetin, bupropion, penciclidine, and ibogaine. 21. The method of any one of claims 16-20, wherein said nicotine antagonist is an antibody that inhibits nicotine receptor function. 21. The method of any one of claims 16-20, wherein the nicotine antagonist is an agent that acts like a nicotine antagonist. Contacting the first eye of the host animal with a therapeutically effective amount of nicotine antagonist, Detecting a change in growth of the first eye of the animal, Applying a known control agent to the second eye of the animal, Observing the result of the control agent in the growth change of the second eye, Comparing the growth change of the first eye with the growth change of the second eye to confirm that the nicotinic antagonist has the ability to regulate eye growth after birth A method of detecting postnatal eye growth control capacity of a host animal of a nicotine antagonist comprising a. A method of making a pharmaceutical composition comprising ascertaining that a nicotine antagonist has the ability to control postnatal eye growth as an active agent and combining the active agent with a pharmaceutical excipient. (a) incubating cells expressing nicotine receptors with and without test compounds, (b) determining whether the test compound binds to the nicotine receptor, (c) selecting a test compound that binds to the nicotine receptor, (d) administering said selected test compound of step (c) to a test animal, (e) determining whether the test compound altered myopia development of the test animal, and (f) selecting a compound that alters the myopia development of the test animal Method for identifying a compound that can be used for myopia adjustment, including.