WO2002024191A1

WO2002024191A1 - Therapeutic and prophylactic drugs of myopia

Info

Publication number: WO2002024191A1
Application number: PCT/CN2000/000286
Authority: WO
Inventors: George C. Y. Chiou; Tricia T. S. Cheng
Original assignee: Yong Guang Pharmaceutical Co., Ltd.
Priority date: 2000-09-20
Filing date: 2000-09-20
Publication date: 2002-03-28
Also published as: AU2000275035A1; CN1390124A

Abstract

The invention relates to the use of NO synthase substrate and/or NO donor, or inducible NO synthase induce inhibitor and/or inducible NO synthase activity inhibitor for the preparation of drugs for treating or preventing myopia; the pharmaceutical compositions containing one or more of the compound(s) mentioned above and pharmaceutically acceptable excipients; new arginine derivatives, the method for treating or preventing myopia, and the commercial kits.

Description

Technical Field of Prevention and Treatment of Myopia

The present invention relates to a preventive and therapeutic drug for myopia. Specifically, it relates to a substrate of NO synthase (hereinafter referred to as NOS), a NO donor, an immune NOS (hereinafter referred to as iNOS) induction inhibitor and an iNOS activity inhibitor in the preparation of treatment or Use in a medicament for preventing myopia and a pharmaceutical composition containing the same, a method for treating or preventing myopia, and a new arginine derivative.

Background technique

Myopia is a common visual condition or disease. Although the etiology of myopia can be attributed to a variety of factors, including genetics, environment, and weak light, it can be attributed to common factors, namely the extension of the central axis of the eyeball, and the failure of the eye. Due to proper refractive power or a combination of both. (Chen, W.C, et al., Myopia Updates II. Lin L. et al., Ed. Pp. 39-42, Springer, New York (1998)). Kinky to severe can cause retinopathy, age-related speckle degeneration (AMD), and cataracts.

One of the most commonly mentioned factors that cause myopia is initially the long-term excessive contraction of the ciliary muscles due to watching nearby objects (such as reading books, visual display terminals (VDT), watching TV at close range, working on fine assembly lines). . One of the theories of the occurrence and development of refractive myopia is that when working too close, it becomes rigid due to excessive contraction of the ciliary muscles, resulting in intermittent refractive power of the lens. Myopia ciliary muscles cannot relax properly to focus the lens on distant images (Tokoro, T. Nippon Ganka Gakkai Zasshl. 102: 796-812 (1998)). The contraction of the ciliary muscle is affected by several types (Including muscarinic receptors, prostaglandin receptors and endothelin receptors) (Erickson-Larry, K. et al., Invest. Ophthalmol. Vis. Sci 32: 492-495 (1991)). Ciliary muscle relaxation is regulated by two independent mechanisms: a cAMP-dependent mechanism, which includes β-adrenergic and prostaglandin receptor-regulated relaxation, and a cAMP-independent mechanism (Goh, Y., Et al., Invest. Ophthalmol. Vis. Sci. 36: 1188-1192 (1995)). Ciliary muscle relaxation as a pharmacological intervention for myopia has been used in human and animal models with anticholinergic drugs such as atropine (Shih, Y, -F. Et al., J. Ocular Pharmacol. Ther. 15: 85-90 (1999)) and Pie Lenzepine (Cottriall, C 丄 and McBrien, NA, Invest. Ophthalmol. Vis. Sci. 37: 1368-1379 (1996)), a ciliary muscle paralytic agent Tests such as cyclopentolate and topocamide, and sympathomimetic agents such as phenylephrine and epinephrine (Kamikavatoko, S. In Myopia Updates, Tokoro, T., ed., Proc. 6th Int. Conf Myopia, Tokyo, pp. 147-154 (1998)). However, the side effects of these drugs on vegetative nerves make them unsuitable for long-term use. In the study of possible drug treatment for myopia, the mechanism of cAMP-independent non-vegetative ciliary muscle relaxation has recently received considerable attention.

Another cause of myopia is due to scleral lesions at the base of the eye, such as inflammation, causing cell necrosis, resulting in softening or reduced stiffness of the sclera. As a result of the intraocular pressure, the sclera deforms, such as protruding backwards, which causes the axis of the eye to become longer and cause myopia. . There is no cure or prevention for this type of myopia.

The role of nitric oxide (NO) is relatively recent in pharmacology (since 1979). Generally, NO interacts with NO synthase (NOS) as a synthetase through L-arginine as a substrate Produced. Isoform NOS includes neuronal NOS (nNOS) or brain NOS (bNOS) in the central and peripheral nervous systems, and endothelial NOS (eNOS or ecN0S) in neurons and non-neuronal cells. nNOS and eNOS produce NO shortly after being activated by calcium / calmodulin to produce different biological responses. Immune NOS (iNOS) is the third isoform NOS induced by lipopolysaccharide and certain cytokines (such as interleukin-1, interleukin-6, tumor necrosis factor and interferon γ). They produce large amounts of NO for a period of time, causing pathophysiological responses (Szabo, C. et al., Nitric Oxide, Biochemistry, Molecular Biology, and Therapeutic Implication. Ignarro, L. and Mur d, F., pp. 113-153, Academic Press, New York (1995)).

Under normal conditions, small amounts of picomolar levels of NO are produced by the interaction of L-arginine as a substrate with two types of NOS (nNOS or eNOS). Then NO activates guanylate cyclase to generate cGMP, which in turn induces many biological responses, including vasodilation, increased ocular blood flow, reduced intraocular pressure (IOP), prevents platelet aggregation, and reduces polymorphonuclear cells ( PMN) chemical toxins, signal transduction of the central nervous system and peripheral nervous system, and inhibition of tumor cells (Ignarro, LJ Nitric Oxide, Principles and Actions. Lancaster J., Ed. Pp. 111-137 Academic Press, New York ( 1996)).

However, under abnormal conditions, a large amount of NO at the nanomolar level is produced by L-arginine through iNOS, and NO will be further oxidized to NO _2- , nitrite, peroxy Nitrate (0N00_) and free radicals interact with thiols, iron-sulfur centers of various enzymes, cytochrome oxidase, glycolytic enzymes, liver cells, macrophages, calmodulin, etc., as a result, the cells are changed Its biological functions damage the DNA and cause apoptosis, neurotoxicity, neurological degradation of Burgundy, and many ophthalmic diseases. iNOS is only induced by lipopolysaccharides, endotoxins, and cytokines under pathophysiological conditions (such as inflammation). The rate-limiting step in NO production from iNOS is the availability of L-arginine, which is recycled by arginine succinate synthase, which is simultaneously induced when iNOS is induced. In addition, the induction of iNOS also triggered the membrane carrier system of L-arginine to enhance the utilization of iNOS by L-arginine. As a result, a large amount of N0 was generated. Therefore, inhibition of iNOS is a major strategy to eliminate the pathophysiology response of NQ ( Dawson, VL et al., Nitric Oxide, Biochemistry, Molecular Biology, and Therapeutic Implication. Ignarro, L. and Murad, F., Ed. Pp. 323-349, Academic Press, New York (1995)).

NO has been linked to eye diseases such as glaucoma (Nathanson, J. A. J. Pharmacol. Exp. Ther. 956-965 (1992)), but the relationship between NO and myopia has not yet been shown. After careful research, the present inventors have unexpectedly discovered that oversupply of NO and oversupply of NO can occur in ocular tissues, which respectively cause the above-mentioned dysfunction of the eye and cause diseases such as the occurrence of myopia. Therefore, ocular dysfunction and diseases, especially the prevention or treatment of myopia, can reduce the oversupply of NO by making up for the deficiency of NO, or by inhibiting iNOS activity or inhibiting induction. As a result, it is possible to revolutionize the basic concepts of the treatment of myopia and eventually involve regulating the level of NO in ocular tissues.

In addition, in the existing methods for treating or preventing myopia, in addition to medication, physical therapies such as low-frequency treatment and ultrasonic treatment have been tried, but none of them can achieve satisfactory results. Therefore, there is an urgent need to develop an effective medicine for preventing or treating myopia.

In order to solve the above problems, an effective preventive or therapeutic agent for myopia is provided. After intensive research, the inventors have successfully discovered that NOS substrates and NO donors can relax through cAMP-independent non-vegetative ciliary muscles. The mechanism of action increases the excessively low NO concentration in the eye, thereby relaxing the ciliary muscles; iNOS induction inhibitors and iNOS activity inhibitors can reduce excessively high NO concentrations in the eyes and inhibit the protrusion of the central sclera; both effects of the epilepsy can ultimately Inhibit the elongation of the eye axis to achieve the effect of preventing or treating myopia, Thus, the present invention has been completed.

Summary of invention

A first aspect of the present invention relates to the use of a NOS substrate and / or a NO donor, or an iNOS induction inhibitor and / or an iNOS activity inhibitor in the manufacture or prevention of a medicament for myopia.

Another aspect of the present invention relates to a pharmaceutical composition containing a NOS substrate and / or a NO donor, or an iNOS induction inhibitor and / or an iNOS activity inhibitor as an active ingredient.

Another aspect of the invention relates to a new arginine ester derivative.

Another aspect of the present invention relates to a method for preventing and treating myopia, which comprises administering to a patient a therapeutically effective amount of the above-mentioned compound or composition.

Finally, the present invention relates to a commercial kit comprising a therapeutically effective amount of the above-mentioned compound or composition, and recording that the drug compound or composition can be used or should be used in the treatment or prevention of myopia. Information.

That is, the present invention is as follows.

(1) Application of a NOS substrate and / or a NO donor, or an iNOS induction inhibitor and / or an iNOS activity inhibitor in the manufacture of a medicament for treating or preventing myopia.

Among them, the NOS substrate is preferably 4-phenyl-3-3-N-oxodioxadicarbonitrile, arginine, cannosine, Nα-substituted arginine and a pharmaceutically acceptable salt or ester thereof; more preferably 4- Benzyl-3-N-oxodioxadicarbonitrile, L-arginine, L-cannoline, Not-substituted L-arginine and pharmaceutically acceptable salts or esters thereof; further preferred 4- Phenyl-3 -N-oxodioxazolonitrile, L-arginine, L-canardine, Not-acyl-substituted L-arginine, Net-alkoxycarbonyl-substituted L-arginine And pharmaceutically acceptable salts or esters thereof; 4-phenyl-3-3-N-oxodioxadicarbonitrile, L-arginine, L-cannoline, Nα-benzoyl-L-arginine are particularly preferred Ethyl ester, di-0- (Not-benzoyl-L-arginine) glycol ester, tri-0- (No-benzyl-L-arginine) glyceride; most preferred L- Arginine, L-canisine, Noc-benzyl-L-arginine ethyl ester.

The NO donor is preferably minoxidil, hydralazine, nitroglycerin, isosorbide and its esters, nitroprusside, nitrite, glutathione derivatives and N-nitropyrazole derivatives; more preferably rice Nordil, hydralazine, nitroglycerin, isosorbide nitrate, nitroprusside, sodium nitrite, glutathione, S-nitrosoglutathione, 3, 5-dimethyl-N-nitrate Base pyrazole, N-nitropyrazole or 3-methyl-N-nitropyrazole; most preferred is hydrazine. 1N0S-inducing inhibitors are preferably polyamines, calcium antagonists, platelet-derived growth factors, platelet activating factor antagonists, tumor necrosis factor antibodies (TNFab), interleukins, protein kinase inhibitors, Tyrokine kinase inhibitors; more preferably sperminealdehyde, Dihydropyridine, w- (N, N, -diethylamino) -n-alkyltrimethoxybenzoate (TMB), platelet-derived growth factor, platelet activating factor antagonist, TNFab, interleukin-4 , Interleukin-8, interleukin-10, interleukin-13, interleukin-lra, protein kinase inhibitor, chloroclonal; most preferred are spermine and chloroclonal.

The 1N0S activity inhibitor is preferably L-arginine analog, methylene blue, aminoguanidine, S-methylisothiourea; more preferably N ^G -methyl-L-arginine, N ^G -amino-hematospermine Amino acid, N ^G -amino-L-arginine, N ^G -nitro-L-arginine, dimethylarginine, methylene blue, aminoguanidine, S-methylisothiourea, most Aminoguanidine is preferred.

(2) A pharmaceutical composition containing one or more compounds selected from a NOS substrate and / or a NO donor, or an iNOS induction inhibitor and / or an iNOS activity inhibitor, and a pharmaceutically acceptable carrier or excipient.

Among them, the NOS substrate is preferably 4-benzyl-3 ~ N-oxoxadiazolonitrile, arginine, cananosine, Net-substituted arginine and a pharmaceutically acceptable salt or ester thereof; more preferably 4- Phenyl-3-N-oxodioxadicarbonitrile, L-arginine, L-cannosine, Not-substituted L-arginine and pharmaceutically acceptable salts or esters thereof; further preferably 4-benzyl -3-N-oxodioxazolonitrile, L-arginine, L-canine, Net-fluorenyl-substituted L-arginine, Nα-hydrocarbyloxycarbonyl-substituted L-arginine And pharmaceutically acceptable salts or esters thereof; acyl-substituted L-arginine and pharmaceutically acceptable salts or esters thereof; particularly preferred are 4-benzyl-3-N-oxodioxadicarbonitrile, L-arginine, L-canardine, Not-benzyl-L-arginine ethyl ester, di-0- (Nα-benzoyl-L-arginine) glycol ester, tri-0- (Noc- Benzoyl-L-arginine) glycerides; most preferred are L-arginine, L-canisine, Nα-benzoyl-L-arginine ethyl ester.

The NO donor is preferably minoxidil, hydralazine, nitroglycerin, isosorbide and its esters, nitroprusside, nitrite, glutathione derivatives and N-nitropyrazole derivatives; more preferably rice Nordil, hydralazine, nitroglycerin, isosorbide nitrate, nitroprusside, sodium nitrite, glutathione, S-nitrosoglutathione, 3, 5-dimethyl-N-nitrate Pyridazole, N-nitropyrazole or 3-methyl-N-nitropyrazole; most preferred is hydralazine.

iNOS-inducing inhibitors are preferably selected from polyamines, calcium antagonists, platelet-derived growth factors, platelet activating factor antagonists, tumor necrosis factor antibody (TNFab), and white interleukin , Protein kinase inhibitors, Tyrokine kinase inhibitors; more preferably spermine, dihydropyridine, w- (N, N, -diethylamino) -n-alkyltrimethoxybenzoate (TMB) , Platelet-derived growth factor, platelet-activating factor antagonist, TNFab, interleukin-4, interleukin-8, interleukin-10, interleukin-13, interleukin-lra, protein kinase inhibitors, clozamate; most preferably arginaldehyde , Clomacron.

The iNOS activity inhibitor is preferably L-arginine analog, methylene blue, aminoguanidine, S-methylisothiourea; more preferably N ^G -methyl-L-arginine, N ^G -amino-hematospermine Amino acid, N ^G -amino-L-arginine, N ^G -nitro-L-arginine, dimethylarginine, methylene blue, aminoguanidine, S-methylisothiourea; most Aminoguanidine is preferred.

The above pharmaceutical composition may further contain an anticholinergic agent, a ciliary muscle paralysis agent, and / or a sympathomimetic agent.

Among them, anticholinergic drugs are preferably atropine and pirenzepine, ciliary muscle paralyzing agents are preferably cyclopentolate and topocamide, and sympathomimetic agents are preferably benzylline and epinephrine; more preferably, atropine.

(3) A compound which is a di-0- (Nα-benzoyl-L-arginine) ethylene glycol ester, and a tri-0- (Net-benzoyl-L arginine) glyceride.

(4) A method for preventing or treating myopia, which comprises administering to a patient a therapeutically effective amount of the composition described in (2) or the compound of (3).

5. A commercial kit comprising a therapeutically effective amount of the above-mentioned composition of (2) and the compound of (3), and recording that the composition or compound can be used or should be used in the treatment or prevention of myopia Information about a substance or compound.

Brief description of the drawings

Fig. 1 illustrates the inhibition of iNOS induced by iNOS and speraldehyde by interleukin-1P.

Figure 2 illustrates the relaxation effect of L-cannoline on muscles.

Figure 3 illustrates the relaxation effect of hydralazine on muscles

Detailed description of the invention

Specific examples of myopia in the present invention include curvature myopia, malignant myopia, progressive myopia, proactive myopia, simple myopia, median myopia, and high myopia and low myopia classified by the degree of myopia. The classification of true myopia and pseudo myopia from the stage of myopia. In addition, it also includes related diseases caused by myopia, such as retinopathy, age-related speckle degeneration (AMD), and cataracts. Unless otherwise stated, the following terms used throughout the present specification should be understood to have the following meanings:

"Acyl" refers to a group represented by RC0-, wherein R represents an alkyl group or an aryl group, wherein the alkyl group is preferably a CI-C6 alkyl group, and refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. Examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl Group, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, etc .; aryl refers to benzene , Naphthalene and so on. Among them, R is preferably ethyl or phenyl. The above-mentioned group R can also be substituted with the following groups, for example, lower alkenyl (such as vinyl, butenyl, and propenyl C2-C6 alkenyl, etc.), cycloalkyl (such as cyclopropyl, cyclobutyl, etc.) , C3-C7 cycloalkyl, such as cyclopentyl, cyclohexyl, cycloheptyl, etc.), aryl (such as C6-C10 aryl such as benzyl, naphthyl), aromatic heterocyclic groups (such as furyl, thienyl, etc.) , Pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, amino, mono- or di-lower alkylamino (e.g. methylamino, ethylamino, dimethylamino, etc.) Or di-CI-C6 alkylamino group, etc.), tri-lower alkylammonium group (such as trimethyl-C6 alkylammonium group, such as trimethylammonium group, triethylammonium group, tripropylammonium group, etc.), the substituent of The number is preferably 1-3, and in the case of each substituent, they may be the same or different.

"Hydoxycarbonyl" is a group represented by the formula R0C0-, where R represents an alkyl group, a fluorenyl group, or an aryl group; wherein the alkyl group may be CI-C6 alkyl group, which means a straight chain having 1 to 6 carbon atoms Or a branched alkyl group, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl Methyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl Examples of the alkenyl group include C2-C6 alkenyl groups such as vinyl, butenyl, and propenyl; aryl refers to benzene, naphthalene, and the like. Among them, R is preferably methyl, ethyl, t-butyl or phenyl. The above-mentioned group R can also be substituted with the following groups, for example, lower alkenyl (such as vinyl, butenyl, and propenyl C2-C6 alkenyl, etc.), cycloalkyl (such as cyclopropyl, cyclobutyl, etc.) , C3-C7 cycloalkyl, such as cyclopentyl, cyclohexyl, cycloheptyl, etc.), aryl (such as C6-C10 aryl such as phenyl and naphthyl), aromatic heterocyclic groups (such as furyl, thienyl, etc.) , Pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, amino, mono- or di-lower alkylamino (e.g. methylamino, ethylamino, dimethylamino, etc.) Or two C1 -C6 alkylamino, etc.), tri-lower alkylammonium (such as trimethylammonium, triethylammonium, tripropylammonium, etc. tri-CI-C6 alkylammonium, etc.), the number of the substituents, preferably The number is 1 to 3, and when there are several substituents, they may be the same or different.

In this specification, "patient" includes humans and other mammals.

In this specification, the term "high myopia" refers to myopia above 500 degrees, preferably myopia above 600 degrees, and "low myopia" refers to myopia below 500 degrees.

In this specification, a NOS substrate is a substance that can be converted into NO by the action of a NO synthetase. Examples thereof include 4-phenyl-3 -N-oxidoxadiazolonitrile, cananosine, and compounds having a basic arginine structure. The so-called "having a basic arginine structure" refers to an arginine compound, an arginine derivative having different substitutions on the nitrogen of arginine, or other compounds that are metabolites of arginine such as N _α -hydroxyl and the particular arginine derivative Ν ω ^- hydroxy arginine and its various Ν α _- protected ester form. Also included are "prodrug" compounds that can be hydrolyzed to arginine. The above cannosine, and the compound having a basic arginine structure may be in the form of an ester, a salt or an amidate thereof.

In the present invention, canolaine has a L_isomer configuration. Substituted L - - arginine derivative compound preferred has a substantially arginine having configuration L - isomer configuration, preferably L - arginine and Ν _α. Suitable N _tt -substituents include the aforementioned acyl, benzoyl or acetyl groups; and the above-mentioned hydrocarbyloxycarbonyl groups, preferably tert-butoxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

Among them, some NOS substrates contain hydroxyl groups, and suitable esters containing hydroxyl compounds are, for example, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propyl Acid esters, succinates, fumarates, maleates, methylene-di-β-hydroxynaphthoate, 2, 5-dihydroxy stearates, isethionates, di -P-methylbenzyl tartrate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.

The upper NOS substrate having a carboxyl group can form a base addition salt, an ester, or an amidate. The bases that can be used in the preparation of the base addition salt preferably include those that can form a pharmaceutically acceptable salt when mixed with a free acid, the pharmaceutically acceptable salt means a salt whose cation is non-toxic to the patient at the dosage of the salt Therefore, the beneficial inhibitory effect of free base is not disturbed by the side effects caused by cations. The pharmaceutically acceptable salts within the scope of the present invention include salts derived from alkali metal and alkaline earth metal salts, including salts prepared from the following bases: sodium hydride, Sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl-glucosamine, lysine, arginine , Ornithine, choline, N, N, -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenylethylamine, diethylamine, piperazine, tris (Hydroxymethyl) aminomethane, tetramethylammonium hydroxide and the like. The carboxylic acid ester formed may be an ester that is non-toxic to the patient at the dose administered. Suitable esters include methyl ester, ethyl ester, isopropyl ester, butyl ester, isobutyl ester, tert-butyl ester, amyl ester, and isopropyl ester. Amyl esters, glycol esters, glycerides and benzyl esters are preferred, and ethyl esters, glycol esters and glycerides are preferred, and ethyl esters are more preferred. The amidate formed may be non-toxic to the patient at the administered dose, and suitable amidates include formamide, formylethylamine, formylpropylamine, formylbutylamine, formamide and the like.

Some of the aforementioned NOS substrates are basic, and these compounds can be used in the form of a free base or in the form of a pharmaceutically acceptable acid addition salt thereof. Acids that can be used in the preparation of acid addition salts include those acids that, when mixed with a free base, can form a pharmaceutically acceptable salt, the pharmaceutically acceptable salt means that its anion is non-toxic to the patient at the dosage of salt The beneficial inhibitory effect of the salt, thus the free base, is not disturbed by the side effects caused by the anions. The pharmaceutically acceptable salts within the scope of the present invention include salts made from inorganic and organic acids, and thus include hydroionates such as hydrochlorides and hydrobromides, sulfates, phosphates, nitrates, sulfamic acids Salt, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, sub Methyl-di-β-hydroxynaphthoate, 2, 5-dihydroxybenzoate, isethionate, di-p-toluoyl tartrate, mesylate, ethanesulfonate, Benzene sulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Hydrochloride is preferred.

In the present specification, the term "NO donor" is not limited as long as it is a compound that can release NO to exert its pharmacological effect. For example, the vasodilator nitroglycerin exerts its pharmacological effect by releasing nitrogen oxide. Examples include minoxidil, hydralazine, nitroglycerin, isosorbide and its esters, nitroprusside, nitrite, glutathione derivatives, and N-nitropyrazole derivatives. The "ester" and "salt" are the same as above. The glutathione derivative is a glutathione which may have a substituent, and examples thereof include glutathione and S-nitrosoglutathione. The "N-nitropyrazole derivative" is an N-nitropyrazole which may have a substituent, and examples thereof include 3,5-dimethyl-N-nitropyrazole, N-nitropyrazole, and 3-methyl-N-nitropyrazole. Better minoxidil, hydrazine, nitroglycerin, isosorbide nitrate, nitroprusside, sodium nitrite, glutathione, S-nitroso valley Glutathione, 3,5-dimethyl-N-nitropyrazole, N-nitropyrazole and 3-methyl-N

-Fluorenylpyrazole; hydralazine is most preferred.

The iNOS induction inhibitor is not limited as long as it is a substance that inhibits the induction of iNOS. Preferred are polyamines, calcium antagonists, platelet-derived growth factor, platelet activating factor antagonists, tumor necrosis factor antibody (TNFab), interleukins, protein kinase inhibitors, and Tyrokine kinase inhibitors. Examples of "polyamine" include: spermine. Examples of the "calcium antagonist" include dihydropyridine and W- (N, N, -diethylamino) -n-alkyltrimethoxybenzoate (TMB). "Interleukin" includes Interleukin_4, Interleukin-8, Interleukin-10, Interleukin-13, Interleukin_lra.

Examples of the "Tyrokine kinase inhibitor" include clomacromone.

More preferred are spermine, dihydropyridine, w- (N, N, -diethylamino) -n-alkyltrimethoxybenzoate (TMB), platelet-derived growth factor, platelet activating factor antagonist , TNFab, interleukin-4, interleukin-8, interleukin-10, interleukin-13, interleukin-lra, protein kinase inhibitor, chloroclonal; most preferred are spermine and chloroclonal.

The iNOS activity inhibitor is a substance having an inhibitory effect on iNOS activity, and there is no other limitation. L-arginine analogs, methylene blue, aminoguanidine, and S-methylisothiourea are preferred. Wherein "L-arginine analogue" may include: N ^G - Methyl - L-arginine, N ^G - amino - blood arginine, Ν ⁶ - amino - L-arginine, r - nitrate -L-arginine, dimethylarginine.

More preferred-methyl-L-arginine, N ^G -amino-blood arginine, N ^G -amino-L-arginine, N ^G -nitro-L-arginine, dimethylarginine Amino acid, methylene blue, aminoguanidine, S-methylisothiourea, aminoguanidine is most preferred.

The NOS substrate and the NO donor can relax the ciliary muscle by increasing the excessively low NO concentration in the eye; the iNOS induction inhibitor and the iNOS activity inhibitor can inhibit the protrusion of the central sclera by reducing the excessively high NO concentration in the eye; Both effects can shorten the central axis of the eyeball and achieve the purpose of treating or preventing myopia. Among them, the above iNOS induction inhibitors and iNOS activity inhibitors are particularly effective for high myopia.

In the present invention, di-0- (Nα-benzyl-L-arginine) ethylene glycol ester and tri-0- (Nα-benzoyl-L-arginine) glyceride are new compounds. Other compounds are known compounds, of which 4-phenyl-3-N-oxodioxadicarbonitrile is disclosed in W099 / 30716, and the rest are commercially available. Among them, Minoxidil, Hydrazine Drapazine and nitroprusside are antihypertensive drugs; nitroglycerin, isosorbide and its esters are antianginal drugs; calcium antagonists are antihypertensive and vasodilator drugs; platelet-derived growth factors are blood drugs; platelet activating factors Antagonists are anticoagulants; TNFab, interleukins, protein kinase inhibitors, and Tyrokine kinase inhibitors are immunomodulators; methylene blue is an antidote; aminoguanidine and S-methylisothiourea are commonly used agents.

The compound of the present invention has effective pharmacological activity, and therefore can be incorporated into a pharmaceutical composition. The present invention also includes a drug containing at least one compound of the present invention, a pharmaceutically acceptable carrier or excipient, and / or other drugs. combination.

Other drugs are other drugs used to treat or prevent myopia, such as anticholinergics, ciliary muscle paralysers and / or sympathomimetics.

Among them, anticholinergic drugs are preferably atropine and pirenzepine, ciliary muscle paralysing agents are preferably cyclopentalate and topicamide, and sympathomimetic agents are preferably phenylephrine and epinephrine. Most preferred is atropine.

The above-mentioned compound or composition can be administered by a suitable method. In practice, it can usually be administered parenterally, topically, rectally, orally, by inhalation or by implantation. Such as treatment of degenerative retinal damage in addition to local administration also requires systemic administration.

Depending on the mode of administration, the composition can be solid such as tablets, pills, powders, granules, capsules, dragees, etc., semi-solid such as ointments, gels, etc., liquid formulations such as injections, solutions, suspensions Liquid formulations, syrups, eye drops, etc., and gas dosage forms such as inhalants, etc., are preferably unit dose forms of precise doses suitable for single administration. In consideration of the effects on other circulatory systems and their effects, it is preferably used in the form of topical administration of the eye, and particularly preferred are eye drops, ophthalmic ointments, gels, and ophthalmic compositions, which are suitable for inserting into the scale Dosage form for the ocular area of a blind tube (pocket-like area formed with the conjunctiva) (such as a drug-impregnated solid carrier inserted into the eye)

In the preparation of the above administration form, necessary additives such as commonly used carriers, excipients, binders, and stabilizers can be added to the present compound, and the preparation can be prepared according to a conventional method. For example, the compound of the present invention is mixed with a carrier (excipient, binder, disintegrant, flavoring agent, flavoring agent, emulsifier, diluent, isotonicity agent, dissolution aid, etc.) that are allowed on the preparation. , Made into tablets, pills, powders, granules, capsules, dragees, ointments, gels, injections, solutions, suspensions, syrups, suppositories, inhalants, transdermal absorbents, Eye drops or ocular administration components are in a form suitable for oral administration, parenteral, topical, rectal, inhalation or implantation. When preparing a solid preparation, additives such as sucrose, lactose, cellulose, D-mannitol, maltitol, dextran, starch, agar, arginine ester, chitin, chitosan, pectin, yellow Tannin, acacia, gelatin, collagen, casein, albumin, calcium phosphate, sorbitol, glycine, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, glycerin, Polyethylene glycol, sodium bicarbonate, magnesium stearate, talc, etc. Moreover, the tablets can be made into tablets that are usually coated, such as sugar-coated tablets, enteric-coated tablets, film-coated tablets or double-layer tablets, multilayer tablets, if necessary.

For semi-solid preparations, animal and vegetable oils (olive oil, corn oil, castor oil, etc.), mineral oils (petrolatum, white petrolatum, solid paraffin, etc.), waxes (Simond oil, carnauba wax, beeswax, etc.) can be used. ), Polyethylene mineral oil gel, partially or fully synthesized glycerin fatty acid esters (lauric acid, myristic acid, palmitic acid, etc.).

When the preparation is an ointment, an ointment base (petrolatum, lanolin, plastic base), a preservative (benzyl ammonium chloride, parabens, chlorobutanol, etc.), etc. can be appropriately selected for preparation.

When preparing a liquid preparation, additives such as sodium chloride, glucose, sorbitol, glycerol, olive oil, propylene glycol, ethanol, and the like are used.

Examples of liquids are injections, eye drops and the like.

When injectable, sterile aqueous solutions (such as physiological saline), isotonic solutions, and oily solutions (such as sesame oil, soybean oil) can be used. In addition, if necessary, an appropriate suspending agent can also be used at the same time, such as sodium carboxymethyl 纡 avidin sodium, a nonionic surfactant, a dissolution aid (such as benzyl benzoate, benzyl alcohol), and the like.

When preparing eye drops, an aqueous liquid or solution can be used, especially a sterile aqueous solution for injection. To the eye drops, various additives such as a buffer, a stabilizer, a wetting agent, an emulsifier, a suspending agent, a surfactant, an isotonicity agent, a preservative, and a tackifier may be appropriately added.

As the buffering agent, for example, a tablet buffer, a borate buffer, a citrate buffer, a tartaric acid buffer, an acetate buffer, an amino acid, and the like can be used.

As the stabilizer, for example, sodium ethylenediamine tetraacetate, citric acid, and the like can be used.

Wetting agents such as glycerin.

An emulsifier is, for example, polyvinylpyrrolidone.

Examples of the suspending agent include hydroxypropyl methyl cellulose and methyl cellulose. Examples of the surfactant include Tween 80 and polyoxyethylene hydrogenated castor oil.

As the isotonicity agent, for example, sugars such as sorbitol, glucose, and mannitol, polyhydric alcohols such as glycerin and propylene glycol, and salts such as sodium chloride can be used.

As the preservative, for example, quaternary ammonium salts such as benzylammonium chloride and benzylmethoxyammonium chloride, parabens such as methyl paraben and ethyl paraben, benzyl alcohol, phenylethanol, and sorbitan can be used. Acids and their salts, thimerosal, chlorobutanol, etc.

pH adjusting agents such as sodium hydroxide, hydrochloric acid or sulfuric acid.

As the thickener, for example, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, and a salt thereof can be used.

If a suspension is applied, its particle size should be less than 10 μm to minimize eye irritation. When used as an eye drop, the pH is usually adjusted to about 3 to 9. 5, preferably to about 6 to 8. 5, most preferably the pH value close to tears, that is, pH 7.4 or so.

If a liposome preparation is used, it is added to various liposomes by mixing the compound or composition described above with a suitable lipid compound or phospholipid. Such formulations are a particularly useful form for NOS substrates and NO donors. Certain compounds arginine (such as tris _- (Ν α - benzoyl - L - arginyl) - glycerol) in itself can form spontaneously under appropriate conditions Nang bubbles results provide modified release and having And / or in the form of metabolic rate compounds.

If an ophthalmic composition is used, various polymers can be used as a formulated ophthalmic drug carrier. The following literature describes this type of polymer: (Saettone, MF, et al., J. Pharm. Pharmocol (1984) 36: 229, and Park, K. et al., In Recent Advances in Drug Delivery Systems. Anderson et al., Eds. , Plenum Press (1984) 163-183), which is incorporated herein by reference in its entirety. Drug release is generally through a process of dissolution or biological digestion of the polymer, infiltration, or a combination thereof. The composition should be formulated so that the rate of drug release does not significantly interfere with tear tension.

More specifically, several matrix type delivery systems can be used in the present invention. These systems are described in detail in the literature below. (Ocular Pharm. Of Drug Release Devices, in Controlled Drug Delivery. Bruck, ed., Vol. II, Chap 4, CRC Press Inc (1983)), which is incorporated herein by reference in its entirety. The system includes a hydrophilic soft contact spectacle lens impregnated or impregnated with a desired drug, and a biodegradable or soluble structure which does not need to be removed after being placed in the eye. The dissolvable ocular insert may consist of any degradable substance, it They can be tolerated by the eyes and are compatible with the drugs given. Substances include but are not limited to poly (vinyl alcohol), polymers and copolymers of polyacrylamide, ethyl acrylate and vinylpyrrolidone, and cross-linked polypeptides or polysaccharides, such as chitin.

A capsule-type delivery system can also be used in the present invention. These systems use polymer membranes to control the release of the drug. The composition is particularly useful for the delivery of hydrophilic drugs. Hydrophobic drugs can be administered via a silicone rubber construct.

The amount of the above compound or composition in the prophylactic or therapeutic agent for myopia is the preparation

0. 01-100% by weight, suitably 0.1-10% by weight. The dosage and number of administrations vary depending on the symptoms, number of administrations, age, weight, and mode of administration. Usually for adults, for example, when used as eye drops, NOS substrates can be administered containing 0. 001-10w / v%, preferably 0. 01-1. 0 ¥ ^% of the formulation; for NO donors, it can be administered containing 0. 001-10w / v%, preferably 0. 03- 0. 3w / v% formulation; for iNOS-inducing inhibitors or iNOS activity inhibitors; can be administered to a formulation containing 0.0001-10w / v%, preferably 0.1-1. (^%); several times a day, preferably 1- 6 times, each time, preferably 1-3 drops. When used as an injection, the administration range can be 0.1-20 mg / kg, preferably 8-15 mg / kg, most preferably about 10 mg / kg, several times a day, It is preferably 1-6 times. As an implanted sustained release method, the effective dose for controlling myopia is usually in the range of 0.1-20 mg / kg / day. This system can be implanted after surgery to prevent myopia. In order to treat myopia, every New implants are inserted from 3 to 10 days until day 60. Youyi implants new implants every 5 to 7 days until day 30.

The amount of anticholinergic drugs in the prevention or treatment of myopia is 0.03-0.25% by weight of the preparation, and the concentration is too low to achieve the corresponding effect; but if the concentration is too high, it will cause vegetative effects. Nerve side effects.

The present invention also includes a method for preventing or treating myopia, which method comprises administering a therapeutically effective amount of the above-mentioned compound or composition to a patient.

In the present invention, di-0- (Not-benzyl-L-arginine) ethylene glycol vinegar, tri-0- (Noc-benzoyl-L-arginine) glyceride can be passed through It is prepared by combining Net-benzylidene-L-arginine with ethylene glycol or glycerol.

The acid addition salts of the present invention can be prepared by reacting a free base with a suitable acid using known methods. For example, the acid addition salt of a compound of the present invention can be prepared by dissolving the free base in water or an aqueous alcohol solution or other suitable solvent containing a suitable acid and evaporating the solution to separate the salt, Reaction in organic solvents Should be prepared, in which case salts can be isolated directly or salts can be obtained by concentrating the solution.

The base addition salt of the present invention can be prepared by reacting a free acid with a suitable base by a known method. For example, the base addition salt of a compound of the present invention can be prepared by dissolving the free acid in water or an aqueous alcohol solution or other suitable solvent containing a suitable base and evaporating the solution to separate the salt, or by free acid and It is prepared by reaction in an organic solvent, in which case the salt can be isolated directly or the salt can be obtained by concentrating the solution.

The ester of the present invention can be obtained by a known method for producing a free acid or a hydroxyl-containing compound.

The amidate of the present invention can be obtained by combining a free acid and an amine by a known preparation method.

Hereinafter, the present invention will be described in detail through examples and test examples, but the present invention is not limited by the examples and test examples.

Examples

Example 1 Eye drops 1

The compound of Example 1 was dissolved in distilled water for injection, the pH was adjusted to 7 with sodium hydroxide, and eye drops were prepared according to the following formulation.

L-Arginine 0.5 g

Sodium dihydrogen phosphate

Sodium chloride 0.9 g

Distilled water for injection

100ml in full amount Example 2 Eye drops 2

According to the same method as in Example 1, eye drops containing L-canaline at a concentration of 1.0 _g / 100 ml were prepared.

Example 3 Eye drops 3

In the same manner as in Example 1, eye drops containing N-benzoyl-L-arginine ethyl ester at a concentration of 0.1 g / 100 ml were prepared.

Example 4 Eye drops 4

According to Example 1, eye drops containing hydralazine at a concentration of 0.1 g of 80 m 1 were prepared. Example 5 Drop of Agent 5

According to Example 1, eye drops containing aminoguanidine at a concentration of 0.1 g / 100 ml were prepared.

Example 6 Eye drops 6

In the same manner as in Example 1, eye drops containing arginaldehyde were prepared at a concentration of 0.7 g / 100 ml.

Example 7 Eye drops 7

In the same manner as in Example 1, eye drops containing chloroclonalone at a concentration of 1.0 g / 100 ml were prepared.

Example 8 Tablet

The compound of Example 1, lactose, corn starch, and crystalline cellulose were mixed, kneaded with a polyvinylpyrrolidone K30 paste, and granulated through a 20-mesh sieve. After drying at 50 ° C for 2 hours, it was passed through a 24 mesh sieve, mixed with talc and magnesium stearate, and a punch having a diameter of 7 mm was used to prepare 120 mg tablets.

No benzoyl-L-arginine ethyl ester 10. 00mg

Atropine 0. 04mg

Lactose 50. OOrng

Corn starch 20. OOrng

Crystalline cellulose 28. 66mg

Polyvinylpyrrolidone K30 5. OOrng

Talc 5. OOrng

Magnesium stearate 0.30mg

120. OOrng

Example 8 Capsules

The compound of Example 1, lactose and corn starch were mixed, kneaded with polyvinylpyrrolidone K30 paste, and granulated through a 20-mesh sieve. After drying at 50 ° C for 2 hours, it was passed through a 24 mesh sieve, mixed with talc and magnesium stearate, and filled into a hard gelatin shell (No. 4) to obtain 120 mg capsules. OOα-benzoyl-L-arginine ethyl ester 5. 00mg

Glutathione 3.OOmg

Lactose 72. OOmg

Corn starch 35.OOmg

Polyvinylpyrrolidone K30 2.00mg

Talc 2.70mg

Magnesium stearate 0.30mg

120. OOmg test example

Test Example 1 This example shows that the NO donor and NOS substrate can effectively relax the contraction of the ciliary muscle caused by carbachol and endothelin-1.

Method

Material

Carbachol (formylcholine chloride US P) was purchased from City Chemical Corp (New York-NY), endothelin-1 (ET-1, human and pig recombinant), hydralazine hydrochloride, and nitrite Sodium was purchased from Sigma Chemical Co (St. Louis. MO). L-arginine hydrochloride, L-cannoline sulfate, and N-benzoyl-L-arginine ethyl acetate (BAEE) were purchased from Pfales. & Bauer (Water bury CT). Improved Krebs solution (composition, mM: NaCl, 142.0; NaHC0 ₃ , 18.0; Glucose, 11.0; KC1,5.4; CaCl ₂ , 2.0; MgCl ₂ , 1.2) .

Tissue preparation

Fresh eyeballs were obtained from the local slaughterhouse and immediately carried on ice to the laboratory. Separate the ciliary body from the eyeball within 3 hours of removal. Under the operating microscope, carefully dissect the longitudinal ciliary muscle from the ciliary body and cut into muscle strips with a length of about 3-6 mm. The two ends of the muscle strip were tied with 5--0 silk sutures and immediately placed at 34 ° C in an organ bath with 10 ml of modified Krebs buffer.

Organ bath and recording device

Install the ciliary muscle strips vertically in a 10ml double-layer glass organ bath with sutures. One suture is connected to the positioning pestle in the bath, and the other suture is connected to the Grass mounted on the adjustable clamp FT-03 on isometric tension converter. The bath is full A non-circulating Krebs buffer was used, and a 95% 0 ₂ r 5% 0) ₂ gas mixture was continuously bubbled. The pH of the Krebs solution was constantly monitored, and the pH was maintained at 7.4 ± 0.4 by adding IN NaOH or IN HC1. Keep the bath and Krebs solution at 34 ° C with circulating water. The recording device consists of Grass 7D1 low-level D.C preamplifier, Grass 74DA amplifier, and Grass 79D four-lead platform paper-ink recorder. An upward shift of the pen records an increase in contraction force, and a downward shift records a decrease in contraction force. The recording device is calibrated before each recording.

Drug treatment program

In randomized manner, five test compounds used in this study were added to the ciliary muscle strips for in vitro testing. Before applying the drug, the muscle specimen was first equilibrated in an organ bath with a resting tension of 300 mg for at least 1 hour, and the resting tension baseline was closely monitored and adjusted by a vertical micromanipulator. Once the medication is added to the bath, the tension is no longer adjusted manually. To determine the test compound on carbachol contraction stimulatory effect of some muscle specimens bath was added the carbachol, to a final concentration of 1x10- ⁶ M, and the contractile response recorded. 1x10- ⁶ M will be determined as the lowest concentration of carbachol induced cholinergic bovine maximum longitudinal contraction of the ciliary muscle (data not shown). After about 5 minutes of maximum contraction, the test compound was added to the bath, and the change in tension was measured. Each of the test compound-treated muscle strips was treated with a series of concentrations from 0.01% to 3% by weight. Before adding subsequent concentrations, allow the tension to change to a relatively stable state, usually between about 5-10 minutes between the two concentrations. The control muscle strips were contracted with carbachol to keep them contracted during the experiment, or at least 60 minutes. If the tissue does not respond to carbachol, the specimen is removed.

To determine the test compound to ET - 1 stimulatory effect contraction, following the same test procedure carbachol-induced close to ET - 1 instead of carbachol, final bath concentration of 1x10- ⁷ M. The 1x10- ⁷ M ET is determined to be the largest causing bovine ciliary longitudinal contraction of 1 - lowest concentration (data not shown). Use ET-1 to cause control muscle strips to contract, keeping them contracted during the experiment, or for at least 60 minutes. If the tissue does not respond to ET-1, discard the specimen.

Statistical Analysis

All data are provided as mean ± standard error (SE). A combined Student's t-test was performed to analyze the significance of the difference between the two means. The difference was considered significant at P <0.05. result

Effects of carbachol and ET-1 on isolated ciliary muscle.

For all samples, 1x10- ⁶ M carbachol from baseline resting tension of 300mg, generated over the baseline average maximum shrinkage tension of 552 ± 43mg. For all specimens, lxl (T ⁷ M ET-1 from a baseline resting tension of 300 mg produced an average maximum contractile tension above the baseline of 408 ± 35 mg. For drug-treated tissues, the muscle was allowed to reach its maximum after carbachol was added. The value is about 5 to 10 minutes, and then the test drug is added. For the control tissue, allow the muscle to contract for 60 minutes, and apply the final tension after 60 minutes as the maximum contraction data point. For the carbachol control muscle strip, 60 minutes After the average final contraction is 560 ± 90mg tension above the baseline, or a maximum contraction of 96.0 ± 2.5%. For ET-1 control muscle strips, the average final contraction after 60 minutes is 340 ± 21mg tension above the baseline, or 71.7 ± 5.4% maximum contraction. Effect of NOS substrate

For carbachol-treated muscle, 0.3% by weight and 1% by weight of L-concanine and 0.1% by weight of BAEE produced significant relaxation effects compared to control (Table 1). Lower concentrations of L-canisine as low as 0.1% and lower concentrations of BAEE as low as 0.03% by weight did not produce significant relaxation effects compared to photographs. For carbachol-treated muscle, L-arginine produced a relaxing effect at 3% by weight compared to control (Table 1).

For ET-1 treated muscle, compared with the control, all concentrations of L-concanine produced significant relaxation effects (Table 2). Compared with the control, BAEE is only 0.01% -0. Iy. The lower concentrations produced significant relaxation effects (Table 2). Compared with the control, L-arginine produced a significant relaxation effect at a concentration of 3% by weight (Table 2).

Comparing the effects of various drug concentrations between carbachol-treated muscles and ET-1 treated muscles, the results were found to be different (Tables 1 and 2). For ET-1 treated muscle, compared to carbachol-treated muscle, L-cannoline is the only NOS substrate that produces a significant relaxation effect at all concentrations tested (Figure 2 ). For ET-1 treated muscles, BAEE produced a significantly stronger relaxation effect at lower concentrations of 0.01% to 0.1% by weight (P≤0.05), but for carbachol-treated muscles, 0.3% by weight And a higher concentration of 1% by weight actually produced a significantly stronger relaxation effect. L-arginine is shown at 3% by weight in measured concentrations of carbachol-treated muscle and ET-1 treated muscle There are significant differences (Tables 1 and 2).

Table 1

Effect of NOS Substrate on 1 × 10_ ⁶ M Carbachol-treated Ciliary Muscle

Kaba «^% of maximum contraction

Drug / concentration 0.01% 0.03% 0.1% 0.3% 1% 3%

L "^ Amino acid 102.342.3 102.942.3 101. +2.7 100.8 ± 3.4 98. d3.3 90. .0

103.3 + 2.6 100.8 + 2.6 95.3 ± 6.0 89.6 + 3.8 * 60.5 ± 14.4 *-

BAEE 101.2 ± 3.4 94.6 + 4.0 56.3 + 4.2 * 35.3 ± 3.6 * 28.5 + 6.5 * 21. mo.0 * All data are expressed as N = 6 average number SE,

* Indicates significant difference from 100%, P≤0.05

Table 2

Ciliary muscle action 1 treated - NOS substrate of 1x10- ⁷ M ET

Endothelin-1% of maximum contraction stimulated

Drug / 0.01% 0.03% 0.1% 0.3% 1% 3%

L "^ City 98.6 £ L 8 96.2 ± 3.6 87.3 ± 5.2 ^b 89.7 ± 6.2 85. .4 70.6 + 13.5 * L -knife 88.3 ± 3.4 * 75. .3 * 59.7 + 4.3 * 32.9 ± 5.6 * 8. r

BAEE 87.4άβ.6 * 74.6db6.6 * 77.. 96. .1 96.3ϋ2.2 27.7 + 8.4 * All data are expressed as N = 6 mean ± SE, (except ^a N = 5, ^b N = 8 )

* Indicates significant difference from 100%, P≤0.05

Role of Nitric Oxide Donors

For carbachol-treated muscle, 0.1% by weight of hydralazine and all concentrations of sodium nitrite produced a significant relaxation effect compared to control (Table 3). For ET-1 treated muscle, compared with the control, all concentrations of hydralazine and all concentrations of sodium nitrite produced a significant relaxation effect (Table 4).

Compared to carbachol-treated muscle, ET-1 treated muscle has been shown to produce significantly stronger relaxation at a concentration of 0.01% and 0.03% by weight (Figure 3). The relaxation effect of 0.1% hydralazine on ET-1 treated muscle and carbachol-treated muscle is close to 100% relaxation. At a concentration of 0.03% by weight and 0.1% by weight, sodium nitrite produced a significantly stronger relaxation effect on ET-1 treated muscles (P≤0.05), but at a concentration of 0.3% by weight, it produced significant effects on carbachol-treated muscles.箸 Strong relaxation effect (P≤0.05). 0.01% in sodium nitrite There were no significant differences in the effects of ET-1 and carbachol-treated muscle at the lowest and highest concentrations of 1% by weight (Tables 3 and 4).

table 3

Ciliary muscle acting NO donor to 1x10- ⁶ M carbachol treatment

Percentage of maximum contraction of Kaba M 'j

o

Drug / Concentration 0.01% 0.03% 0.1% 0.3% 1% Hydrazine 101.8 ± 1. 9 97. 8 + 2. 7 -1. 1 + 1. 8 * N / AN / A

Sodium 80. 8 + 4. 9 ^a * 75. 7 + 6. 2 ^a * 63. 4 + 5. 9 ^a * 22. 1 ± 3. 5 ^a * -15. 6 + 3. * O

All data are expressed as N = 6 o mean ± SE, (except ^a N = 5)

* Indicates a significant difference from 100%, P≤0.05

Table 4

1 ciliary muscle action process - NO donor on endothelin 1x10- ⁷ M

Endothelin-1 stimulated maximum contraction percentage

Drug / Concentration 0.3% 1% Hydrazine 57.5 + 4. 0 * 18. 9 ± 4. 1 * 2. 8 + 4. 5 * N / A N / A

75. 3 + 3. 1 * 58. 5 + 2. 1 * 47. 3 + 3. 9 * 38. 2. 2 * -8. 5i3. 6 * All data are expressed as the mean of N = 6. SE,

* Indicates significant difference from 100%, P≤0.05

(Result)

The NOS substrate and NO donor of the present invention can produce a significant ciliary muscle relaxation effect. Test example 2 cGMP measurement

(Method)

To measure intracellular cGMP production in RPE cells (purchased from ATCC) after administration, we will use a cyclic GMP enzyme immunoassay kit (purchased from Cayman Chemical). This assay is based on a competitive binding between free cGMP and acetylcholinesterase-linked cGMP tracer. When Ellman's reagent was added, the cGMP tracer turned yellow and had a strong absorbance at 412 nm. After giving the NO donor or NOS substrate and the control sample containing the carrier separately, the cells will be lysed according to the instructions of the kit, and a lysate will be prepared to measure cGMP.

(Result)

The results showed that the amount of cGMP was significantly higher than that of the control sample using the NO donor or NOS substrate of the present invention compared to the control vector. Test Example 3 Detection of iNOS by Western Blot

(Method)

Retinal pigment epithelial cells (RPE cells, purchased from American Tissue and Cell Collection) and interleukin-1β (100ng / ml, Sigma Chemical) or carrier or interleukin-1β (100ng / ml, Sigma Chemical) and iNOS induction inhibitor (100ng / ml) After 12 hours of incubation, remove the culture medium, and add 0.5ml of boiling lysis buffer (ImM Tris pH7.4, 1% sodium dodecyl sulfate (SDS)) to each well. For one half of the wells, in order to prevent the volatilization of NO, the production of nitrogen oxide in the culture medium was immediately measured (see Test Example 2). Remove the contents of each well and transfer to a 1.5 ml centrifuge tube. The lysed cell contents, along with the buffer, were passed 6 times through a 26 gauge needle to split the nuclear contents apart. Samples can be stored at -70. C. The BCA detection method (Pierce) was used to determine the protein concentration of each sample using a standard curve of bovine serum albumin prepared in 0.1% SDS. This measurement was performed in a 96-well microplate using a Packard Spectra Cont spectrophotometer. In order to reduce the interference of SDS on the BCA detection reagent (purchased from Pierce), the sample must be diluted 1:10 with water to reduce the concentration of SDS to 0.1%. Each sample was measured and the volume of 40μ ₈ contain total protein.

Samples were mixed with 10 ° / «staining buffer and 3% reduction buffer (purchased from Novex), and NuPAGE Bis-Tris minigels (purchased from Novex) were pre-stained with standard protein gradients (Gibco). Company) Add sample. This gel was electrophoresed in a MOPS-SDS running buffer (available from Novex) at a constant voltage of about 200V. After gel electrophoresis, proteins were transferred to the Immobilon-P PVDF transfer membrane (microwell) using a Bio-Rad Trans-Blot transfer device. After the transfer was completed, the membrane was blocked for 60 minutes by co-incubation with a 5% blocking solution (purchased from Amersham) prepared in Tris-buffered saline (TBS-T) containing 0.1% Tween-20 at room temperature. After the blocking solution was washed away in TBS-T, the assayed concentrated state was co-incubated at room temperature by co-incubation with an anti-iNOS polyclonal antiserum (from cytokine-induced murine phagocytes, Cayman Chemical) at room temperature. Minutes to probe the iNOS protein on the membrane. After the primary antibody solution was washed away, this membrane was bound with a peroxide-labeled goat anti-rabbit secondary antibody (purchased from Bio-Rad) to incubate the measured concentrated state for 60 minutes at room temperature. ECL Western Blot Detection Kit (purchased from Amersham Company), and the film was exposed to X-ray film by chemiluminescence method, and the protein band on the film was observed.

Statistical analysis of the data

All data will be provided as mean ± standard deviation (SE). The combined Stndent's t-test was used to analyze the significance between the two means.

(Result)

The results showed that the sample with the carrier only had no iNOS protein bands; the sample with the interleukin-1β had iNOS protein bands; and the sample with the interleukin-1β and speraldehyde had no iNOS protein bands . The iNOS induction inhibitor of the present invention, especially spermine, shows a significant blocking effect on IL- 1β induced iNOS, and the production of iNOS protein is inhibited by spermine (Figure 1).

Test Example 4 Determination of Nitric Oxide

(Method)

RPE cells (purchased from American Tissue and Cel l Collection) with interleukin-1β (100ng / ml, Sigma Chemical) or carrier or interleukin 1 (100ng / ml, Sigma Chemical) and iNOS induction inhibitor (100ng / ml) After 12 hours of incubation, the medium was removed, and the nitrate / nitrite colorimetric detection kit (purchased from Cayman Chemical) was used to measure the content of nitrogen oxide in the medium according to the instructions of the instruction manual.

Statistical analysis of the data

Same as test example 1

(Result)

The results showed that compared with the control interleukin-1β, using iNOS induction inhibitors, especially spermine, the amount of NO was significantly less than the amount of NO produced by using interleukin-1β alone, which was equivalent to the amount of NO produced by using only the carrier ( table 5 ) . Inhibition of interleukin-1β-induced NO production by speraldehyde in RPE cells

* Significantly increased compared to control vehicle P <0.05.

** Compared with interleukin-1β alone, the simultaneous use of sperminealdehyde significantly inhibited the production of N0 P <0.05.

From the above results, it can be seen that the N0 donor or N0S substrate can significantly increase the amount of cGMP produced by the activation of guanylate cyclase by N0, relax the ciliary muscle, and have a good effect on myopia with too low N0. The iNOS induction inhibitor has a significant blocking effect on iNOS induction, and this blocking effect can continuously eliminate a large amount of NO production for a long time. iNOS activity inhibitors can reduce iNOS activity and significantly reduce the amount of NO. In this way, both have good effects on myopia, especially high myopia, caused by excessive NO production. In addition, they are effective for retinopathy caused by myopia, age-related speckle degeneration (AMD), and cataracts.

Industrial application possibilities

The NOS substrate and the NO donor of the present invention can increase the too low NO concentration in the eye through the mechanism of cAMP-independent non-vegetative ciliary muscle relaxation, thereby relaxing the ciliary muscle; iNOS induction inhibitor and iNOS The activity inhibitor can reduce the excessively high NO concentration in the eye, and inhibit the protrusion of the central sclera. Both of the above effects can ultimately suppress the elongation of the axis of the eye and achieve the effect of preventing or treating myopia.

Therefore, the compound or composition of the present invention can be effectively used as a preventive or therapeutic agent for various myopia or diseases related to myopia, such as curvature myopia, malignant myopia, progressive myopia, precursor myopia, simple myopia, and medium myopia And high myopia, low myopia, true myopia, false myopia, and so on. In addition, it also includes retinopathy from myopia to severe, age-related speckle degeneration (AMD), and cataracts.

Claims

Rights request

1. Use of a NO synthase substrate and / or a NO donor, or an immune NO synthase induction inhibitor and / or an immune NO synthase activity inhibitor in the manufacture of a medicament for treating or preventing myopia.

2. The use according to claim 1, wherein the NO synthase substrate is selected from the group consisting of 4-benzyl-3 -N-oxidoxadiazolonitrile, arginine, canardine, Να-acyl substituted arginine, and It is a pharmaceutically acceptable salt or ester.

3. The use according to claim 2, wherein the NO synthase substrate is selected from the group consisting of 4-phenyl-3-N-oxidoxadiazolonitrile, L-arginine, L-cannoline, Net-acyl substituted L-Arginine and its pharmaceutically acceptable salts or esters.

4. The use according to claim 3, wherein the substrate of NO synthase is selected from the group consisting of 4-benzyl-3 -N-oxodioxadicarbonitrile, L-arginine, L-cannoline, Net-benzoyl -L-arginine ethyl ester, di-O- (Nα-benzoyl-L-arginine) ethylene glycol ester, tri-0- (Να-benzyl-L-arginine) glyceride .

5. The use according to claim 4, wherein the NO synthase substrate is selected from the group consisting of L-arginine, L-canisine, Nα-benzyl-L-arginine ethyl ester.

6. The use according to any one of claims 1 to 5, wherein the NO donor is selected from the group consisting of minoxidil, hydralazine, nitroglycerin, isosorbide and its esters, nitroprusside, nitrite, glutathione Derivatives and N-nitropyrazole derivatives.

7. The use of claim 6, wherein the NO donor is selected from the group consisting of minoxidil, hydralazine, nitroglycerin, isosorbide nitrate, nitroprusside, sodium nitrite, glutathione, S-nitrosoglutamine Glutathione, N-nitropyrazole, 3,5-dimethyl-N-nitropyrazole, 3-methyl-N-nitropyrazole: ■

8. The use of claim 7, wherein the NO donor is hydralazine.

9. The use of any one of claims 1 to 8, wherein the immune NO synthase induction inhibitor is selected from the group consisting of polyamines, calcium antagonists, platelet-derived growth factors, platelet activating factor antagonists, tumor necrosis factor antibodies, interleukins, proteins Kinase inhibitor, Tyrokine kinase inhibitor.

10. The use according to claim 9, wherein the immune NO synthase-inducing inhibitor is selected from the group consisting of spermine, dihydropyridine, w- (N, N, -diethylamino) -n-alkyltrimethoxybenzylcarboxylic acid Salt, platelet-derived growth factor, platelet activating factor antagonist, tumor necrosis factor antibody, Interleukin-4, Interleukin-8, Interleukin-10, Interleukin-13, Interleukin-lra, Protein Kinase Inhibitor, Clomacromone.

The use according to claim 10, wherein the immune NO synthase-inducing inhibitor is spermine aldehyde or chloroclonal.

12. The use according to any one of claims 1 to 11, wherein the immune NO synthase activity inhibitor is selected from the group consisting of L-arginine analog, methylene blue, aminoguanidine, and S-methylisothiourea.

13. The use according to claim 12, wherein the immune NO synthase activity inhibitor is selected from the group consisting of N ^G -methylarginine, N ^G -amino-blood arginine, N ^G -amino-L-arginine, N ^G -Nitro-L-arginine, dimethylarginine, methylene blue, aminoguanidine, S-methylisothiourea.

14. The use according to claim 13, wherein the inhibitor of immune NO synthase activity is aminoguanidine.

15. A pharmaceutical composition comprising one or more compounds selected from the group consisting of a NO synthase substrate and / or a NO donor, or an immune NO synthase induction inhibitor and / or a sputum-free NO synthase activity inhibitor, and Pharmaceutical carrier or excipient.

16. The composition of claim 15, wherein the NO synthase substrate is selected from the group consisting of 4-phenyl-3 -N-oxidation. -Azole carbonitrile, arginine, cancanine, Nα-acyl substituted arginine and pharmaceutically acceptable salts or esters thereof.

17. The composition of claim 16, wherein the NO synthase substrate is selected from the group consisting of 4-phenyl-3 oxazotricarbonitrile, L-arginine, L-canardine, Να-acyl-substituted L-quinine Amino acids and their pharmaceutically acceptable salts or esters.

18. The composition according to claim 17, wherein the NO synthase substrate is selected from the group consisting of 4-phenyl-3-N-oxoxazole nitrile, L-arginine, L-cannoline, Να-benzoyl -L-arginine ethyl ester, di-0- (Nα-benzoyl-L-arginine) glycol ester, tri-0- (Nα-benzoyl-L-arginine) glyceride .

19. The composition of claim 18, wherein the NO synthase substrate is selected from the group consisting of L-arginine, L-canavaline, Not-benzoyl-L-arginine ethyl ester.

20. The composition of any of claims 15-19, wherein the NO donor is selected from the group consisting of minoxidil, hydralazine, nitroglycerin, isosorbide and its esters, nitroprusside, nitrite, glutathione Peptide derivatives and N-nitropyrazole derivatives.

21. The composition of claim 20, wherein the NO donor is selected from the group consisting of minoxidil, hydralazine, nitroglycerin, isosorbide nitrate, nitroprusside, sodium nitrite, glutathione, S- Nitrosyl glutathione, N-nitropyrazole, 3,5-dimethyl-N-nitropyrazole, 3-methyl-N-nitropyrazole.

22. The composition of claim 21, wherein the NO donor is hydralazine.

23. The composition of any of claims 15-22, wherein the immune NO synthase-inducing inhibitor is selected from the group consisting of polyamines, calcium antagonists, platelet-derived growth factors, platelet activating factor antagonists, tumor necrosis factor antibodies, interleukins, Protein kinase inhibitors, Tyrokine kinase inhibitors.

24. The composition of claim 23, wherein the immune NO synthase induction inhibitor is selected from the group consisting of spermine, dihydropyridine, w- (N, N, -diethylamino) -n-alkyltrimethoxybenzyl Acid salt, platelet-derived growth factor, platelet-activating factor antagonist, tumor necrosis factor antibody, interleukin-4, interleukin-8, interleukin-10, interleukin-13, interleukin-lra, protein kinase inhibitor, chlorocromone.

25. The composition of claim 24, wherein the immune NO synthase-inducing inhibitor is spermine or chloroclonal.

26. The composition of any of claims 15-25, wherein the inhibitor of immune NO synthase activity is selected from the group consisting of L-arginine analogs, methylene blue, aminoguanidine, and S-methylisothiourea.

27. The composition of claim 26, wherein the inhibitor of immune NO synthase activity is selected from the group consisting of N ^G -methyl-L-arginine, N ^G -amino-blood arginine, N ^G -amino-L-arginine Acid, ^NG -nitro-L-arginine, dimethylarginine, methylene blue, aminoguanidine, S-methylisothiourea.

28. The composition of claim 27, wherein the inhibitor of immune NO synthase activity is aminoguanidine.

The pharmaceutical composition according to any one of claims 15 to 28, further comprising an anticholinergic agent, a ciliary muscle paralyzing agent, and / or a sympathomimetic agent.

30. The pharmaceutical composition of claim 29, wherein the anticholinergic agent is selected from the group consisting of atropine and pirenzepine, the ciliary muscle palsy agent is selected from the group consisting of cyclopentolate and topicamide, and the sympathomimetic agent is selected from the group consisting of phenylephrine and epinephrine.

31. The pharmaceutical composition of claim 29, further comprising atropine.

32. A compound, which is di-0- (Nα-benzyl-L-arginine) ethylene glycol ester, and tri-0- (Nα-benzoyl-L-arginine) glyceride.

33. A method for preventing or treating myopia, comprising administering a pharmacologically effective amount of the pharmaceutical composition according to any one of claims 15 to 31 or the compound according to claim 32 to a patient.

34. A commercial kit comprising the composition or compound according to any one of claims 15 to 32, and it is described that the pharmaceutical composition or compound can be used or should be used in the treatment or prevention of myopia. Or compound information.