KR20210107394A - Composition for treatment of corneal damage comprising 5-Amino-4-Imidazole Carboxamide Riboside-1-β-D-Ribofuranosideas an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating corneal damage, including 5-amino-4-imidazole carboxamide riboside-1-beta-D-ribofuranoside (AICAR) as an active ingredient. Activation of AMPK caused by AICAR significantly inhibits expression of TGF-β1 and ECM protein in vivo and in vitro. In addition, the anti-fibrosis effect of AICAR is AMPK-dependent, given that it is offset by the treatment with AMPK inhibitor and Compound C. Therefore, it can be seen that activation of AMPK caused by AICAR inhibits myofibroblast differentiation and ECM synthesis occurring after alkaline damage in corneal fibroblasts, which suggests the applicability of AICAR to inhibition of fibrosis after corneal damage.

Description

Composition for treatment of corneal damage comprising 5-amino-4-imidazole carboxamide riboside-1-β-D-ribofuranoside as an active ingredient Carboxamide Riboside-1-β-D-Ribofuranosideas an active ingredient}

The present invention relates to a composition for treating corneal damage containing 5-amino-4-imidazole carboxamide riboside-1-β-D-ribofuranoside as an active ingredient.

The ocular surface is a special body surface composed of conjunctival epithelial cells and corneal epithelial cells. These cells, together with tears secreted by the main and epithelial glands, make up a healthy ocular surface, and these healthy ocular surfaces are essential for the optimal functioning of the eye. am.

Among them, the cornea is a transparent, non-vascular tissue occupying 1/6 of the anterior surface of the eyeball. It not only protects the eye from the outside, but also plays a major role in refraction and transmission of light and has developed nerves. The cornea is composed of five layers, such as corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, and corneal endothelium. It consists of 7 layers of cells, and the bottom cell of the bottom layer proliferates, pushes out, and falls off after 7 to 14 days.

The avascular structure, which is one of the many features of the cornea, must always maintain transparency to preserve vision. However, the cornea is a thin tissue with a central thickness of about 0.5 mm and can be easily ruptured by severe impact.

In particular, in the case of corneal damage, which is a symptom of damage to the corneal tissue of the eye, corneal burns are best known among various causes. Corneal burns account for about 11 to 21% of total eye trauma and are known to be caused by various causes, such as chemical substances, heat damage, and radiation exposure. Most of the time it is worn

In this case, the cornea's ability to keep itself clear may be impaired, resulting in loss of transparency and whitening. In addition, corneal burns cause extensive tissue damage and reduced visual acuity, resulting in a serious deterioration in quality of life. In addition, although it is essential to control inflammation in the early stages of corneal burns, corneal opacity and new blood vessels occurring in the late stages not only cause ulcers and interstitial edema, but also decrease visual acuity and induce immune cell circulation, thereby evading corneal immunity and after corneal transplantation. It can also reduce transplant survival and, in severe cases, lead to permanent visual loss.

In the actual treatment process for corneal burns, the most problematic is the inflammatory response following corneal damage caused by corneal burns. Inflammation induced by corneal damage increases reactive oxygen species (ROS) and inflammatory cascade reactions to interfere with the restoration of corneal epithelium, thereby sustaining pain, and vascular endothelial growth factor (vascular endothelial growth factor). By inducing the synthesis of VEGF), basic fibroblast growth factor (bFGF), and matrix metallopreoteinases (MMPs), it causes abnormalities in the collagen structure in the corneal tissue and induces the creation of new blood vessels. It causes a dark opacity of the cornea.

Therefore, as described above, corneal damage and loss of vision due to an inflammatory reaction are a representative disease that has become a global problem, and there is a need to develop an effective and safe therapeutic agent for corneal damage.

AMPK is well known as a sensor and regulator of cellular energy homeostasis (Hardie, DG and Hawley, SA, "AMP-activated protein kinase: the energy charge hypothesis revisited", Bioassays, 23, 1112, (2001), Kemp, BE et al, "AMP-activated protein kinase, super metabolic regulator", Biochem Soc Transactions, 31, 162 (2003)).

Allosteric activation of these kinases due to elevated AMP levels occurs in a state of cellular energy depletion. Through serine/threonine phosphorylation of the resulting target enzyme, cellular metabolism is adapted to a low energy state. The net effect of AMPK activation-induced changes is inhibition of ATP consumption processes and activation of ATP production pathways, and thus regeneration of ATP stores. Examples of AMPK substrates include acetyl-CoA-carboxylase (ACC) and HMG-CoA-reductase (Carling D et al., "A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis ", FEBS Letters, 223, 217 (1987)) Through phosphorylation and thus inhibition of ACC, fatty acid synthesis is reduced (ATP-consumption) and at the same time fatty acid oxidation is increased (ATP-producing) HMG-CoA Cholesterol synthesis is reduced through phosphorylation of ductase and the resulting inhibition. Other substrates of AMPK include hormone-sensitive lipase (Garton, AJ et al, "Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase A possible antilipolytic mechanism", Eur J Biochem, 179, 249, (1989)) ), glycerol-3-phosphate acyltransferase (Muoio DM et al., "AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle:evidence that sn-glycerol-3-phosphate acyltransferase is a novel target ", Biochem J, 338, 783, (1999)), malonyl-CoA decarboxylase (Saha AK et al, "Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofura-noside", J Biol. Chem, 275, 24279, (2000)), and hepatocyte nuclear factor-4α (Leclerc I et al. , "Hepatocyte nuclear factor-4α involved in type-1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase", Diabetes, 50, 1515, (2001)), some of which are metabolically It is a potential drug target for the components of the syndrome. Additional processes that are thought to be regulated through AMPK activation, but for which the exact AMPK substrate has not been identified, include stimulation of glucose transport in skeletal muscle and regulation of expression of key genes in fatty acids and glucose metabolism in the liver. (Hardie DG and Hawley SA, "AMP-activated protein kinase: the energy charge hypothesis revisited", Bioassays, 23, 1112, (2001), Kemp BE et al, "AMP-activated protein kinase, supermetabolic regulator", Biochem Soc Transactions) , 31, 162, (2003), Musi N and Goodyear LJ, "Targeting the AMP-activated protein kinase for the treatment of type 2 diabetes", Current Drug Targets-Immune, Endocrine and Metabolic Disorders, 2, 119, (2002) ) following AMPK stimulation, e.g., glucose-6-phosphatase (Lochhead PA et al, "5-aminoimidazole-4-carbo-xamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase", Diabetes, 49, 896, (2000)), an important enzyme in hepatic glucose production, and SREBP-1c (Zhou G et al, "Role of AMP-activated protein kinase in mechanism of metformin action") , The J of Clin Invest, 108, 1167, (2001)), an important lipogenic transcription factor (key lipogenic tr anscription factor) was found to be reduced.

More recently, it has become apparent that AMPK is involved in the regulation of cellular as well as systemic energy metabolism. It has been shown that the adipocyte-derived hormone leptin stimulates AMPK and thus increases fatty acid oxidation in skeletal muscle (Minokoshi Y et al., "Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase", Nature). , 415, 339, (2002)) adiponectin, another adipocyte-derived hormone that improves carbohydrate and lipid metabolism, has been demonstrated to stimulate AMPK in liver and skeletal muscle (Yamauchi T et al, "Adiponectin stimulates glucose utilization and fatty acid oxidation by activating AMP-activated protein kinase", Nature Medicine, 8, 1288, (2002), Tomas E et al, "Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: Acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation", PNAS, 99, 16309, (2002)) Activation of AMPK in this environment is independent of an increase in cellular AMP levels, but rather one or more, phospho-phosphorus by being identified as an upstream kinase in the future. It appears to be due to the reel.

Based on the knowledge of the consequences of AMPK activation described above, a very advantageous effect is expected from the in vivo activation of AMPK. In the liver, reduced expression of gluconeogenic enzymes leads to reduced hepatic glucose production and improved overall glucose homeostasis, and direct inhibition and/or reduced expression of key enzymes in lipid metabolism, leading to fatty acid and cholesterol synthesis decreases and fatty acid oxidation will increase. AMPK stimulation in skeletal muscle will increase glucose uptake and fatty acid oxidation, decrease intra-myocyte triglyceride accumulation and enhance insulin action, resulting in improved glucose homeostasis. Finally, with increased energy expenditure, weight will be reduced. The combination of these effects in the metabolic syndrome would be expected to significantly reduce the risk of developing cardiovascular disease.

Some studies in rodents support this hypothesis (Bergeron R et al, "Effect of 5-aminoimidazole-4-carboxamide-1(beta)-D-ribofuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats", Diabetes, 50, 1076, (2001), Song SM et al, "5-Aminoimidazole-4- dicarboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabeted (ob/ob) mice", Diabetologia, 45, 56, (2002) , Halseth AE et al, "Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations", Biochem and Biophys Res Comm, 294, 798,(2002), Buhl ES et al., "Long -term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome", Diabetes, 51, 2199, (2002)) Until recently, most in vivo studies have focused on the AMPK activator AICAR, a cell-permeable precursor of ZMP. depended on

ZMP acts as an intracellular AMP mimic and, when accumulated at highly sufficient levels, can stimulate AMPK activity (Corton JM et al, "5-Aminoimidazole-4-carboxamide ribonucleoside, a specific method for activating AMP-activated protein kinase in intact cells?", Eur J Biochem., 229, 558, (1995))

However, ZMP also acts as an AMP analogue in the regulation of other enzymes, and thus is not a specific AMPK activator (Musi N and Goodyear LJ, "Targeting the AMP-activated protein kinase for the treatment of type 2 diabetes", Current Drug Targets- Immune, Endocrine and Metabolic Disorders, 2, 119 (2002)) Some in vivo studies have demonstrated beneficial effects of both acute and chronic AICAR administration in rodent models of obesity and type 2 diabetes. (Bergeron R et al., "Effect of 5-aminoimidazole-4-carboxamide-1β-Dribofuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats", Diabetes, 50,1076, (2001), Song SM et al. , "5-Amino- imidazole- 4-darboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabeted (ob/ob) mice", Diabetologia, 45, 56, (2002), Halseth AE et al., "Acute and chronic treatment" of ob/ob and db/db mice with AICAR decreases blood glucose concentrations", Biochem and Biophys Res Comm, 294, 798, (2002), Buhl ES et al., "Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displayin g feature of the insulin resistance syndrome", Diabetes, 51, 2199, (2002)) For example, 7 weeks of AICAR administration to obese Zucker (fa/fa) rats reduced plasma triglycerides and free fatty acids, and reduced HDL cholesterol is increased, and glucose metabolism is normalized as assessed by an oral glucose tolerance test (Minokoshi Y et al, "Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase", Nature, 415). , 339, (2002)) in both ob/ob and db/db mice, 8-day AICAR administration reduces blood glucose by 35% (Halseth AE et al., "Acute and chronic treatment of ob/ob and db/db") mice with AICAR decreases blood glucose concentrations", Biochem and Biophys Res Comm, 294, 798, (2002)) In addition to AICAR, and more recently, it should be determined to what extent the antidiabetic action of metformin depends on this activation. However, it has been found that the diabetic drug metformin can activate AMPK in vivo at high concentrations (Zhou G et al., "Role of AMP-activated protein kinase in mechanism of metformin action", The J of Clin Invest, 108, 1167, (2001), Musi N et al., "Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes", Diabetes, 51, 2074, (2002) ) As in the case of leptin and adiponectin, the stimulatory effect of metformin is indirect through activation of upstream kinases (Zhou G et al., "Role of AMP-activated protein kinase in mechanism of metformin action", The J of Clin Invest, 108, 1167, (2001)) In addition to pharmacologic interventions, several transgenic mouse models have been developed over the past few years, and early results have become available. Expression of dominant negative AMPK in skeletal muscle of transgenic mice, the effect of AICAR on stimulation of glucose transport depends on AMPK activation (Mu J et al, "A role for AMP-activated protein kinase in contraction and hypoxia-regulated glucose transport in skeletal muscle", Molecular Cell, 7,1085, (2001)), and thus presumably not attributable to non-specific ZMP effects. Similar studies in other tissues help to further clarify the consequences of AMPK activation. Pharmacological activation of AMPK is expected to be beneficial in metabolic syndrome while improving glucose and lipid metabolism and reducing body weight. To qualify a patient for metabolic syndrome, three of the following five criteria must be met: elevated blood pressure greater than 130/85 mmHg, fasting blood glucose greater than 110 mg/dl, waist circumference 40 defined by abdominal obesity greater than "(male) or 35" (female), and triglyceride elevation greater than 150 mg/dl or decreased HDL cholesterol less than 40 mg/dl (male) or 50 mg/dl (female) Changes in blood lipids as they become. Thus, with the combined effect that can be achieved through AMPK activation in patients diagnosed with metabolic syndrome, the benefit of these targets will be enhanced.

Stimulation of AMPK has been shown to stimulate the expression of uncoupling protein 3 (UCP3) in skeletal muscle (Zhou M et al., "UCP-3 expression in skeletal muscle: effects of exercise, hypoxia, and AMPactivated protein kinase", Am J Physiol Endocrinol Metab, 279, E622, (2000)) and thus may be a method to prevent damage from reactive oxygen species. Endothelial NO synthase (eNOS) has been shown to be activated through AMPK-mediated phosphorylation (Chen ZP, et al, "AMP-activated protein kinase phosphorylation of endothelial NO synthase", FEBS Letters, 443). , 285, (1999)), thus AMPK activation can be used to improve the local circulation.

[Prior Patent Literature]

Korean Patent Publication No. 10-2008-0034491

The present invention has been devised in response to the above needs, and an object of the present invention is to provide a composition for treating corneal damage.

In order to achieve the above object, the present invention provides a composition for preventing or treating corneal damage comprising 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. do.

In one embodiment of the present invention, the corneal damage is preferably caused by a chemical substance, heat damage or radiation, but is not limited thereto, and the chemical substance is more preferably alkaline, but is not limited thereto.

In another embodiment of the present invention, the corneal damage is Stevenson-Johnson syndrome, Sjogren's syndrome, dry eye syndrome, trauma, eye trauma due to eye surgery, uveitis (infectious or non-infectious), immune rejection after corneal transplant surgery, or It is preferable, but not limited to, keratoconjunctival epithelial damage caused by an extrinsic disease induced by wearing hard contact lenses.

Accordingly, the composition containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (hereinafter referred to as 'AICAR') of the present invention as an active ingredient is a composition for the treatment of corneal damage; An eye drop composition for the treatment of corneal damage, a composition for cleaning or preserving contact lenses, a composition for preserving an intraocular lens, or a pharmaceutical for treatment of any one or more diseases selected from the group consisting of glaucoma, optic nerve disease, macular degeneration, retinal degeneration and retinal edema It can be usefully used for the red composition.

In addition, the present invention includes all of AICAR, as well as pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates, or stereoisomers prepared therefrom.

The present invention can be used in the form of AICAR or a pharmaceutically acceptable salt thereof, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as doates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving the AICAR compound in an excess aqueous acid solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be manufactured by It can also be prepared by heating the same amount of the above compound and an aqueous acid solution or alcohol, followed by evaporating the mixture to dryness, or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, and such solid preparations include one or more compounds of the present invention with at least one excipient, for example, starch, calcium carbonate, water It is prepared by mixing sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like.

Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 81, cacao butter, taurine paper, glycerol, gelatin, etc. may be used.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the patient's age, sex, and weight, and generally 0.01 mg to 100 mg, preferably 0.05 mg to 10 mg per kg of body weight, is administered daily or every other day. Or, it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides an ophthalmic composition for preventing or treating corneal damage containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient.

The composition containing AICAR or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient may be formulated as an eye drop by a conventional method by mixing with a carrier commonly acceptable in the pharmaceutical field. The ophthalmic composition is preferably an isotonic aqueous solution or suspension, and the compositions mentioned are sterile and/or contain adjuvants (eg, preservatives, stabilizers, wetting agents or salts/or buffers for regulating the osmotic pressure). In addition, they may contain other therapeutically useful substances.

It has been known that anionic polymers such as hyaluronic acid and carboxymethylcellulose, or pharmaceutically acceptable salts thereof, are known to have moisturizing and lubricating effects in eye drops, and may contain a pharmaceutically acceptable carrier in addition to these components. can Examples of the pharmaceutically acceptable carrier include isotonic agents, buffers, stabilizers, pH adjusters and solvents. The tonicity agent plays a role in controlling the isotonicity of the eye drops, and sodium chloride or potassium chloride may be selected as representative. Buffers perform the function of adjusting the acidity or alkalinity of eye drops. Buffers commonly used for preparing eye drops include aminocaproic acid, sodium monohydrogen phosphate and sodium dihydrogen phosphate. The stabilizer serves to stabilize the eye drop, and sodium edetate and/or sodium perborate may be used as the stabilizer. The pH adjusting agent adjusts the pH of the eye drop composition, such as hydrochloric acid and/or sodium hydroxide.

It is preferable to use sterile purified water or distilled water for injection as the solvent. The eye drop according to the present invention is preferably in a liquid formulation. Preservatives, preservatives, etc. may be added to the eye drop composition as needed.

The recommended amount and frequency of use of the eye drop composition according to the present invention is 1 to 3 drops at a time, 5 to 6 times a day, but may be appropriately increased or decreased according to symptoms. The dosage level for a specific patient may be changed according to the patient's weight, age, sex, health status, administration time, administration frequency, severity of disease, and the like.

The present invention also provides a composition for cleaning or preserving contact lenses containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient.

In the case of a composition for cleaning contact lenses, a surfactant may be included in addition to AICAR as a main component. Surfactants having a cleaning action may include various surfactants known in the art, including anionic, cationic, nonionic and amphoteric surfactants, as primary cleaning agents. In addition, it may further include a wetting agent, an antibacterial agent, a stabilizer, an isotonic agent, a solubilizing agent, a viscosity modifier or a buffer.

In the case of the contact lens preservation composition, in addition to the AICAR compound or a pharmaceutically acceptable salt thereof, an aqueous solution for storing the contact lens may generally include saline, other buffered solutions or deionized water, and preferably, boric acid, Boron-based buffers such as borax, acetate-based buffers such as acetic acid, sodium acetate and potassium acetate, phosphate-based buffers such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and carbonate-based buffers such as sodium carbonate and sodium hydrogen carbonate , citric acid, a citrate-based buffer such as sodium citrate, or a trometamol buffer.

More preferably, a salt containing brine comprising sodium chloride, sodium borate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate or the corresponding potassium salt thereof may be included. In addition, a wetting agent, a surfactant, a stabilizer, a viscosity modifier, a tonicity agent, a solubilizing agent, an antioxidant, an antiseptic, a refreshing agent, a chelating agent, or an emollient may be further included.

In addition, the present invention provides a treatment for any one or more diseases selected from the group consisting of retinal degeneration and retinal edema containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. A composition for prevention or treatment is provided.

The present invention also provides a composition for inhibiting corneal fibrosis containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient.

In addition, the present invention treats corneal fibroblasts after corneal injury with 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) to differentiate myofibroblasts occurring after corneal injury from corneal fibroblasts. and methods of inhibiting ECM synthesis.

Hereinafter, the present invention will be described.

Increased TGF-β synthesis after corneal alkali injury is implicated in corneal fibrosis and promotes differentiation of keratosites into myofibroblasts. Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) by 5-amino-4-imidazolecarboxamide riboside-1-β ribofuranoside (AICAR) has fibrotic effects in corneal fibroblasts after alkali injury suppression was investigated.

The present invention also relates to the molecular mechanisms involved in the anti-fibrotic effect of AICAR in the cornea.

As can be seen from the present invention, alkali injury induced upregulation of TGF-β expression and increased α-SMA and fibronectin synthesis in vitro and in vivo, and AMPK activation by AICAR was inhibited in vivo and in vitro. TGF-β and ECM protein expression was significantly inhibited, and the anti-fibrotic effect of AICAR was AMPK-dependent, as it was offset by treatment with an AMPK inhibitor, compound C. Therefore, it can be seen that AMPK activation by AICAR inhibits myofibroblast differentiation and ECM synthesis that occur after alkali injury in corneal fibroblasts, suggesting the possibility of using AICAR for the purpose of inhibiting fibrosis after corneal injury.

1 is a diagram showing the time-lapse result of TGF-β synthesis induced by alkali damage,
Representative Western blot and its quantitative analysis showed a significant increase in TGF-β protein synthesis 18 hours after treatment with 0.01 N NaOH, and results that persisted during the 48 hour test period (** denotes vehicle-treated control cells) showed P < 0.01 compared with ).
2 is a diagram showing the effect of AICAR on alkaline burn-induced ECM protein and TGF-β expression in vivo and in vitro.
Representative Western blots and their quantitative analyzes show the expression induced by alkali exposure of TGF-β fibronectin and α-SMA, and AICAR treatment (1 mM) as can be seen from the figure (#, ##, ### are These represent P <0.05, 0.01, 0.001, respectively, compared to vehicle-treated control cells; *, **, *** represent P <0.05, 0.01, 0.001, respectively, compared to cells treated with NaOH only). decreased expression of the protein;
3 is a diagram showing the effect of AICAR on TGF-β-induced ECM protein expression and myofibroblast differentiation;
Representative Western blots and their quantitative analyzes show a significant anti-fibrotic effect of AICAR on TGF-β induced expression of α-SMA, EDA-fibronectin and fibronectin proteins in corneal fibroblasts, and assay mRNA transcription levels also -SMA, fibronectin, collagen and EDA- show a significant anti-fibrotic effect of AICAR on TGF-β induced transcription of fibronectin (##, ### respectively P <0.01 compared to vehicle-treated control cells, 0.001, *, **, *** indicate P <0.05, 0.01, 0.001, respectively, compared to TGF-β-treated cells only).
Figure 4 is a figure showing the collagen gel matrix contraction image,
(A) Representative gel images and (B) Quantitative analysis of gel area percentages showed no significant difference in gel shrinkage between vehicle-treated control cells and TGF-βAICAR (1 mM) treated cells during the test period. However, vehicle-treated control cells and TGF-β only-treated cells showed significant differences in gel shrinkage from day 2 of the test (*, **, *** respectively P <0.05 compared to vehicle-treated control cells). , representing 0.01, 0.001),
Figure 5 is a figure showing that the anti-fibrotic effect of AICAR is shown through the AMPK signal inhibitory effect,
Representative Western blots and their quantitative analyzes show a significant increase in AMPK activation after AICAR treatment (A, C), and the blot also shows that AICAR treatment significantly reduced NaOH- or TGF-β-induced expression of α-SMA and fibronectin. , which was reversed by compound C treatment, which subsequently resulted in a significant decrease in AMPK levels (B, D). (#, ## indicate P <0.05, 0.01, respectively, compared to vehicle-treated control cells; *, **, *** indicate P <0.05, 0.01, respectively, compared to NaOH or TGF-β treated cells only; , represents 0.001),
6 is a diagram showing the effect of AICAR on the mTOR signal,
Representative Western blots of p-mTOR and p-p70-S6K and their quantitative analysis showed the inhibitory effect of AICAR on their signaling pathways after treatment with 0.01 N NaOH(A) and 5 ng/mL TGF-β (#, ### indicates P <0.05, 0.001, respectively, compared to vehicle-treated control cells; *, **, *** indicate P <0.05, 0.01, and 0.001, respectively, compared to NaOH or TGF-β treated cells only; indicated),
7 is a diagram showing an optical coherence tomography (OCT) image of a rat cornea,
After alkali injury, OCT images show increased interstitial opacity (corneal opacity) and irregular corneal thickness suggesting corneal epithelial damage and neovascularization. After the combination treatment of AICAR and steroids, there was no corneal epithelial damage, the cornea showed a constant thickness, and corneal opacity was greatly reduced.
8 is a diagram showing immunohistochemical images of mouse corneas. Histopathology of mouse corneal tissues in which the expression of fibronectin and α-SMA was significantly reduced in the group treated with AICAR compared to the untreated group at 3 weeks after alkali injury. Represents an enemy analysis, magnification is 200x.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

The present invention has been approved for animal research by Yonsei University Wonju Medical School (YWC-170410-1). All experiments were conducted in accordance with the provisions of the Declaration of Helsinki in accordance with guidelines published by the Animal Protection Ethics Committee of Wonju Medical University, Yonsei University, and were performed in compliance with ARVO guidelines for the use of research in ophthalmic and vision research animals.

Example 1: Isolation and Culture of Primary Human Corneal Fibroblasts

Primary human corneal fibroblasts were obtained from the corneal limbus after removal of the central corneal button for corneal transplantation. After removing excess tissue including corneal epithelium and Descemet membrane, 0.5 mg/mL hyaluronidase (Sigma- Aldrich) and 2 mg/mL collagenase and 0.5 mg/mL hyaluronidase (Sigma-Aldrich).

After treatment, the isolated cells were collected by centrifugation (12000 × g for 5 min), resuspended in DMEM/F12 medium supplemented with 10% FBS and 1% antibiotic/antifungal agent (Gibco Life technologies) at 37°C and humidified. Incubated in 5% CO2 in an incubator. The cells were passaged with 0.25% trypsin and cultured in DMEM (Welgene, Gyeongsan, Korea) supplemented with 10% FBS and 1% antibiotic/antifungal agent. Culture medium was changed every 2-3 days until passaged to 80% confluence. Cells at passages 3-10 were stored at -80°C for subsequent use.

Example 2: In vitro alkali injury and Western blotting

For in vitro protein analysis, primary human corneal fibroblasts were seeded and adhered to culture plates. Already established (Hyun Sun Jeon, Kayoung Yi, Tae Young Chung, Joon Young Hyon, Won Ryang Wee, Young Joo Shin. Chemically injured keratocytes induce cytokine release by human peripheral mononuclear cells. Cytokine . 2012; 59 (2): 280) -285.), cells were treated with 0.01N NaOH in serum-free DMEM to generate an in vitro model of alkaline burn corneal injury and cultured for 1 h. Cells were then treated with or without 0.5 mM and 1 mM AICAR (Abcam) dissolved in DMSO without change of medium for 48 h to determine their effect on ECM protein synthesis and to determine their effect on signal proteins. Incubated for 24 hours.

Confirmatory in vitro experiments were performed by direct stimulation of corneal fibroblasts with TGF-β. Fibroblasts were pretreated with 0.5 mM and 1 mM AICAR for 2-3 hours, then treated with 5 ng/mL TGF-β (PeproTech, Seoul, Korea) and incubated. To determine whether the observed effect was AMPK dependent, cells were pretreated with compound C (Merck, Darmstadt, Germany), a 2.5 μM and 5 μM AMPK antagonist, for 2 h, followed by 1 mM AICAR for 2 h and then 5 ng Treated with /mL TGF-β. Cells were incubated for 8 hours for detection of signal protein and 48 hours for detection of ECM protein.

Total protein was extracted using RIPA buffer (Thermo Fisher Scientific, Rockford, USA) with protease and phosphatase inhibitors (Sigma-Aldrich Corporation, St. Louis, MO). Equal amounts of protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Milan, Italy). Protein blots were analyzed with a Pierce ™ enhanced chemiluminescence (ECL) western blotting substrate (Thermo Fisher Scientific, Rockford, USA). Images were captured by a UVP Bio-Spectrum 600 imaging system (Ultra-Violet Products Ltd., Cambridge, UK). Relative expression of target proteins was assessed by densitometry using ImageJ software (http://imagej.nih.gov/ij/index.html). Antibody information is provided in Table 1.

Example 3: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction

For quantitative real-time PCR analysis, fibroblasts were seeded and treated with AICAR and TGF-β. After 24 h incubation, cells were harvested and total RNA was extracted using RNA Easy Mini Extraction Kit (Qiagen, Germany) according to the manufacturer's protocol. The quality and quantity of RNA extracts were evaluated spectrophotometrically. RNA was reverse transcribed into cDNA using LaboPass™ cDNA synthesis kit (Cosmo Genetech, Seoul, Korea) according to the manufacturer's protocol. Q-PCR was performed using the primer pairs of the target genes listed in Table 2. Reactions were performed with SYBR Green PCR Mix (Applied Biosystems Life Technologies, Foster City, CA) in a 10 μL reaction volume.

Example 4: Collagen Gel Matrix Shrinkage Analysis

Cells were resuspended in serum-free DMEM at a concentration of 1.5 x 10 ^{5 cells/mL.} 400 μL of cell suspension was mixed with 200 μL of type I collagen derived from rat tail tendon solution (Gibco Life Technologies). The mixture was neutralized by adding 5 N of 1 N NaOH. The gel solution was added to a 24-well plate (600 μL per well) and incubated at 37° C. for 30 min for polymerization. Then, 600 μL of DMEM mixed with vehicle, 5 ng/mL TGF-β5 ng/mL TGF-β+1 was added to the gel. Gels were removed from the wells and incubated at 37° C. and 5% CO 2 in a humidified incubator. A digital camera was used at a fixed distance to capture shrinkage images of the gel at different time points. The extent of shrinkage was quantified by measuring the surface area using Image J software. Experiments were performed in triplicate for each treatment group.

Example 5: Animal Experiments

Healthy 8-week-old male BALB/C mice used in the present invention were acclimatized by maintaining them in standard animal room conditions (temperature, 24 ± 1 °C; humidity, 50 ± 5%) for 1 week. After acclimatization, alkali burns were applied to the right eye of the mice using the following protocol: First, the mice were randomly divided into 4 groups of 6 mice. Grade I-II burns were then induced by anesthesia with isoflurane followed by 2 μL of 0.1N NaOH solution immersed in 2 μL of 0.1N NaOH solution for 30 seconds. The eyes were thoroughly washed with sterile physiological saline. Mice were housed in plastic cages fed a normal diet with a 12 h/12 h day and night cycle. Topical application of the drug was carried out 4 times daily. Group 1 mice were not treated with test drugs as they served as positive controls. Group 2 mice were treated with 1 mM AICAR. Group 3 mice were treated with steroids (0.1% Flumetholone, Santen). Group 4 mice were treated with 1 mM AICAR and steroids.

To prevent bacterial infection, all damaged eyes were treated with topical antibiotics (0.5% levofloxacin, santhene). The intact left eye cornea was used as a negative control. Mice were euthanized after 21 days by cervical dislocation after anesthesia with isoflurane. The central cornea greater than 3.0 mm2 was scanned by the anterior segment 5-line raster method using a cirrus OCT (Carl Zeiss Meditec, AG, Jena, Germany).

Corneas were harvested and proteins were extracted for western blotting. In summary, tissue was excised and shown in previous studies (Patel N, Solanki E, Picciani R, Cavett V, Caldwell-Busby JA Bhattacharya SK Strategies to recover proteins from ocular tissues for proteomics. Proteomics. 2008; 8:1055-1070). Tissue Lyser II (Qiagen, MD) in a suitable lysis buffer (125 mM Tris-HCl (pH 7.0), 0.1 % Triton X-100, 0.1 % Tween 20, 100 mM NaCl, 0.1 % sodium dodecyl sulfate) for optimal protein extraction , USA) was used for homogenization. The homogenate was centrifuged at 10,000 x g for 5 min at 4 °C, and the supernatant with the extracted protein was collected and stored at -20 °C. Harvested corneal tissues were fixed in 10% phosphate buffered formalin for histopathological analysis.

Example 6: Immunohistochemistry

Paraffin sections of corneal tissue were deparaffinized, rehydrated, incubated overnight at 4 °C using monoclonal antibodies to α-SMA (1:500) and fibronectin (1:500) and by immunohistochemistry. analyzed.

Target proteins were detected using a biotinylated secondary antibody and streptavidin-biotin-peroxidase (1:200, incubated at 37° C. for 1 hour). Sections were counterstained with hematoxylin, dehydrated and mounted.

The data of the present invention were analyzed using Prism 5.0 software. Statistical comparisons were performed using ANOVA, and post-test analyzes were performed using Tukey's multiple comparisons. Statistical significance was defined as P <0.05 in all cases.

The results of the above examples are as follows.

AICAR inhibits TGF-β and ECM protein accumulation in corneal fibroblasts after alkali injury

To investigate possible mechanisms that may be involved in the antifibrotic effect of AICAR, we first looked at TGF-β protein synthesis in response to alkali burn. To determine the time course of TGF-β synthesis after alkali injury and to confirm its role in the fibrotic response, we incubated cells with 0.01 N NaOH for 6 hours, 12 hours, 18 hours, 24 hours and 48 hours. Significant TGF-β protein synthesis was observed for 18 h of incubation, which was sustained for the remainder of the test period as shown ( FIG. 1 ) (P = 0.0005).

An in vitro alkaline burn model of primary human corneal fibroblasts generated by exposing cells cultured as described above to 0.01 N NaOH was analyzed by Western blotting with or without AICAR treatment. Corneal tissues from mouse models of alkaline burns treated with or without AICAR and intact control corneal tissues were analyzed by Western blotting. The results showed that both in vivo and in vitro alkali injury resulted in a significant increase in TGF-β protein synthesis. However, treatment with AICAR significantly reduced TGF-β protein synthesis in vivo (P = 0.0091) and in vitro (P = 0.0178) (Fig. 2).

OCT images of mouse corneas showed increased stromal thickness with irregular epithelium and high hyperreactivity, and excessive neovascularization and fibrosis in the untreated group. Conversely, mice treated with AICAR showed more homogeneous, low reflectance, and regular corneal thickness indicating a decrease in ECM protein synthesis, which is consistent with the Western blotting results showing the fibrosis inhibitory effect induced by AICAR treatment. (Fig. 7).

Immunohistochemical images of mouse corneal tissues showed that the expression of fibronectin and α-SMA was significantly reduced in the AICAR-treated group compared to the untreated group (Fig. 8).

Induction of fibrosis by TGF-β in vitro also significantly increased the expression of α-SMA, fibronectin and EDA-fibronectin compared to vehicle-treated control cells. AICAR significantly inhibited the expression of these proteins (P = 0.024 for α-SMA, P <0.0001 for fibronectin and EDA-fibronectin). Western blot results were confirmed by real-time PCR results, which also showed a significant decrease in the relative mRNA transcription of these proteins as well as collagen 1 (P = 0025 for α-SMA, and P < 0.0001 for fibronectin, EDA- P = 0.0368 for fibronectin and P = 0.0014 for collagen 1 (Figure 3).

AICAR inhibits fibroblast contraction

Collagen contractility is characteristic of activated fibroblasts. Collagen gel matrix shrinkage assay was used to confirm the effect of AICAR on activated fibroblasts and consequently myofibroblast differentiation. As a result of the present invention using primary human corneal fibroblasts, the shrinkage of the gel treated with AICAR was significantly reduced compared to that treated with TGF-β alone, which was comparable to the control group not treated with TGF-β. That is, AICAR effectively inhibited TGF-β-induced fibroblast activation-contraction of collagen gel ( FIG. 4 ).

The antifibrotic effect of AICAR depends on AMPK signaling.

To investigate the molecular mechanism of AICAR in the inhibition of corneal fibrosis, we pre-cultured primary human corneal fibroblasts with compound C, an AMPK-specific signal transduction inhibitor. First, the activation of AMPK signal by AICAR was confirmed. The results showed a significant increase in p-AMPK protein synthesis in primary human corneal fibroblasts in both 0.01 N NaOH treatment (P = 0.0031) and TGF-β treatment (P = 0.0007).

As shown in the results of the present invention, compound C treatment was treated with NaOH (α-SMA: P = 0.0499; fibronectin: P = 0.0027) and TGF-β treatment (α-SMA: P = 0.0003; fibronectin: P = 0.0041) It significantly blocked the anti-fibrotic effect of AICAR in both groups, and significantly inhibited AMPK signaling in the NaOH-treated group (P = 0.0031) and TGF-β-stimulated cells (P < 0.0001). That is, this result suggests that the effect of AICAR is dependent on AMPK signaling (FIG. 5).

AICAR inhibits mTOR signaling

AMPK activation is known to inhibit mTOR signaling, thereby reducing protein synthesis in cells. Therefore, the effect of AICAR on the mTOR pathway in corneal fibroblasts was studied.

The results showed that AMPK activation by AICAR caused a significant decrease in p-mTOR (P = 0.0192) and p70-S6K protein (P = 0.0075) expression compared to only NaOH-treated cells. The results of using TGF-β to induce fibrosis also showed a decrease in p-mTOR (P = 0.0004) and p-p70-S6K (P = 0.0001) expression in the AICAR-treated group compared to the TGF-β-treated group. (Fig. 6)

Claims (9)

A composition for preventing or treating corneal damage comprising 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. The composition for preventing or treating corneal damage according to claim 1, wherein the corneal damage is caused by chemicals, heat damage, or radiation. The composition for preventing or treating corneal damage according to claim 2, wherein the chemical is alkali. The method of claim 1, wherein the corneal damage is Stevenson-Johnson syndrome, Sjogren's syndrome, dry eye syndrome, trauma, eye trauma due to eye surgery, uveitis (infectious or non-infectious), immunorejection after corneal transplant surgery, or hard contact A composition for preventing or treating corneal damage, characterized in that it is caused by corneal epithelial damage caused by extrinsic diseases caused by wearing lenses. An eye drop composition for preventing or treating corneal damage, comprising 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. A composition for cleaning or preserving contact lenses comprising 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. For the prevention or treatment of any one or more diseases selected from the group consisting of retinal degeneration and retinal edema containing 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient composition. A composition for inhibiting corneal fibrosis comprising 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) as an active ingredient. 5-amino-4-imidazole carboxamide riboside-1-β ribofuranoside (AICAR) was treated with corneal fibroblasts after corneal injury to inhibit myofibroblast differentiation and ECM synthesis from corneal fibroblasts. How to suppress.

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