KR20200093394A - Animal model having increased intraocular pressure, method of producing the same and uses thereof - Google Patents

Abstract

The present invention relates to an animal model with an elevated intraocular pressure, a manufacturing method thereof, and a screening method of a glaucoma therapeutic agent using the same. According to one embodiment of the present invention, the animal model with the elevated intraocular pressure using riboflavin and ultraviolet light may be manufactured, and the elevated intraocular pressure of the animal model manufactured thereby lasts for equal to or more than 12 weeks. Accordingly, the present invention is useful for studies on pathogenesis and treatment of glaucoma, and can be used to screen for a drug for preventing or treating glaucoma. The method for manufacturing the animal model with the elevated intraocular pressure comprises: administering a composition comprising riboflavin to an anterior chamber of an animal; and irradiating eyes of the animal to which the composition is administered with the ultraviolet light.

Description

Animal model with increased intraocular pressure, a method for manufacturing the same, and a screening method for glaucoma treatment using the same{Animal model having increased intraocular pressure, method of producing the same and uses thereof}

The present invention relates to a method for preparing an animal model with increased intraocular pressure, an animal model with increased intraocular pressure produced by the method, and a method for screening for a drug for preventing or treating glaucoma using an animal model with increased intraocular pressure. .

Glaucoma is a major cause of blindness with cataracts and diabetic retinopathy worldwide. As one of the most common ophthalmic diseases, degeneration of retinal ganglion cells and their axons occurs due to various causes, including increased intraocular pressure. It is an optic neuropathy in which defects of the body appear. Of the various risk factors for glaucoma development, intraocular pressure is the only major risk factor we can control. In addition, even in patients with glaucoma with normal intraocular pressure, dropping intraocular pressure is known to slow the progression of glaucoma.

Therefore, lowering intraocular pressure is important for the prevention and treatment of glaucoma. For the study of glaucoma, the method of raising the intraocular pressure in a laboratory animal model is to close the episcleral vein, perform laser fiber surgery, or insert a small bead to close the waterproof runway, or administer steroids. It is known to do. However, according to these methods, an increase in intraocular pressure cannot be maintained for a long period of time, or side effects of the procedure may occur.

Accordingly, the present inventors have completed the present invention by discovering that an animal model capable of stably maintaining an increase in intraocular pressure for a sufficient period of time can be screened for a substance that reduces intraocular pressure using the model.

Republic of Korea Patent Publication No. 10-2009-0125209

An object of the present invention is to provide a method for producing an animal model with elevated intraocular pressure using riboflavin and ultraviolet light.

Another object of the present invention is to provide an animal model with elevated intraocular pressure prepared using riboflavin and ultraviolet light.

Another object of the present invention is to provide a screening method of a drug for preventing or treating glaucoma, comprising selecting an agent that lowers intraocular pressure using an animal model with increased intraocular pressure.

One aspect of the present invention

Administering a composition comprising riboflavin to the anterior chamber of the animal; And

Provided is a method for preparing an animal model having elevated intraocular pressure, comprising irradiating ultraviolet rays to the eyes of an animal to which a composition comprising riboflavin has been administered.

Typically, the average range of normal intraocular pressure is 10 to 21 mmHg, and if the intraocular pressure rise exceeds 21 mmHg or 2.8 kPa, glaucoma may develop due to loss of retinal ganglion cells and damage to the optic nerve.

According to one embodiment of the present invention, the animal model having an increased intraocular pressure may have an increased intraocular pressure of 5 to 20 mmHg or more, 5 to 15 mmHg or more, 10 to 15 mmHg or more, or 10 mmHg or more than the control group not performing the method.

According to one embodiment of the present invention, the animal model with increased intraocular pressure is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% compared to a control group without performing the method. Or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 30% to 200%, 30% to 180%, 50% to 180%, 60% to 180%, 70% to 180%, 80% to 180%, 90% to 180%, or 100% to 180% intraocular pressure may be increased.

According to one embodiment of the present invention, the intraocular pressure increase rate is 30% to 200%, 30% to 180%, 50% to 180%, 60% to 180% compared to a control group not performing the above method in an animal model having elevated intraocular pressure. , 70% to 180%, 80% to 180%, 90% to 180%, 100% to 180%, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or It may be 100% or more.

According to an embodiment of the present invention, the rate of increase in intraocular pressure may be calculated by Equation 1 below.

According to one embodiment of the invention, for 3 to 20 weeks, 4 to 20 weeks, 5 to 20 weeks, 6 to 15 weeks, 8 to 15 weeks in an animal model with elevated intraocular pressure , 10 weeks to 12 weeks, 8 weeks, 10 weeks, 12 weeks, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks Elevated intraocular pressure may be maintained above, above 11 weeks, or above 12 weeks.

According to one embodiment of the invention, the step of administering a composition comprising the riboflavin may be performed by injection or injection into the anterior chamber of the animal.

According to one embodiment of the present invention, the composition comprising the riboflavin may be in various forms suitable for any route of administration including, but not limited to, injections, eye drops, ophthalmic ophthalmic formulations, ocular inserts, and is usually used when formulating them. Can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, or surfactants.

According to an embodiment of the present invention, the step of administering the composition comprising riboflavin may be performed by injecting a composition containing riboflavin directly into the anterior chamber of an animal using a syringe.

According to one embodiment of the present invention, when a composition containing riboflavin is administered as an injection, conventional additives such as a solubilizer, isotonic agent, suspending agent, emulsifier, stabilizer, or preservative may be included.

According to one embodiment of the present invention, suitable carriers that can be used in the composition comprising riboflavin include, for example, water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof, and / Or it may be a solvent or a dispersion medium containing vegetable oil, but is not limited thereto. According to one embodiment of the invention, suitable carriers include Hanks' solution, Ringer's solution, Phosphate-Buffered Saline (PBS) or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Isotonic solutions such as can be used.

According to one embodiment of the invention, the composition comprising riboflavin may be sterile.

According to one embodiment of the present invention, when a composition comprising riboflavin is administered as an injection, it may be a fluid that can be easily injected.

According to one embodiment of the invention, the composition comprising riboflavin can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. According to an embodiment of the present invention, in order to protect the composition containing riboflavin from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be further included. In addition, the composition may further include isotonic agents such as sugars or sodium chloride in most cases.

According to one embodiment of the present invention, riboflavin (riboflavin) is a water-soluble vitamin produced by plants and many microbial communities, and is a natural photoinitiator having various properties such as non-toxicity, biosynthesis, and biodegradability. According to one embodiment of the present invention, riboflavin can be used as an initiator that induces various photoreactions because it exhibits absorption spectra in the ultraviolet and visible regions.

According to one embodiment of the present invention, when the riboflavin reacts with ultraviolet rays, free radicals are formed, and the free radicals induce covalent bonding between amino groups of collagen fibril, thereby releasing the aqueous humor. Can interfere.

According to one embodiment of the invention, the composition comprising riboflavin is 0.1 to 0.9% (w/v), 0.2 to 0.8% (w/v), 0.3 to 0.7% (w/v), 0.4 to 0.6% ( w/v), 0.5 to 0.6% (w/v) or 0.5% (w/v) riboflavin. When a composition containing 1.0% (w/v) or more of riboflavin is administered in front of the eye, there are side effects such as corneal loss, corneal abnormality and/or ocular swelling in the eye of the animal model, and an increase in intraocular pressure may be maintained continuously. It is not suitable as a model to examine the mechanism of optic nerve damage or glaucoma that is caused by sustained high pressure.

In the present invention, "weight/volume percent" or "% (w/v)" refers to the mass percent of the solute in the solution calculated as the mass of the solute relative to the solution volume, for example 1% (w/v) The composition means that 1 g of solute is dissolved in the 100 L composition. For example, a composition containing 0.5% (w/v) of lipoflavin means that 0.5 g of riboflavin is dissolved in 100 L of the composition.

According to one embodiment of the invention, the composition comprising riboflavin is 0.1 to 0.9% by weight (w/v %), 0.2 to 0.8% by weight (w/v %), 0.3 to 0.7% by weight based on the total composition volume. (w/v %), 0.4 to 0.6% by weight (w/v %), 0.5 to 0.6% by weight (w/v %) or 0.5% by weight (w/v %) riboflavin.

According to one embodiment of the invention, ultraviolet irradiation may be performed at a wavelength of 100nm to 400nm, 150nm to 400nm, 200nm to 400nm, or 315nm to 400nm. The photopolymerization reaction may be realized by reacting with riboflavin by irradiating ultraviolet rays at a wavelength in the above range.

According to an embodiment of the present invention, the intensity of ultraviolet rays irradiated to the eyeball is 1 to 100 mW/cm ² , 1 to 80mW / cm ^2, 3 to 80mW / cm ^2, 3 to 70mW / cm ^2, 3 to 60mW / cm ^2, 3 to 50mW / cm ^2, 3 to 30mW / cm ^2, 3 to 20mW / cm ^2, or It may be 9 to 18mW/cm ² .

According to one embodiment of the invention, the step of irradiating ultraviolet rays is 2 to 100J/cm ² , 2 to 90J/cm ² , 2 to 80J/cm ² , 2 to 70J/cm ² , 2 to 60J/cm ² , 5 to 60J / cm ^2, from 5 to 50J / cm ^2, from 5 to 50J / cm ^2, 10 to 50 J/cm ² , 10 to 45 J/cm ² , 10 to 40 J/cm ² , 10 to 30J / cm ^2, can be carried out by irradiation with ultraviolet rays with energy amount of 15 to 30J / cm ^2, 16 to 27J / cm ² or 16.2 to about 27J / cm ^2.

According to one embodiment of the invention, the step of irradiating ultraviolet rays is 5 to 100J, 10 to 80J, 10 to 60J, 12 to 50J, 12 to 45J, 12 to 40J, 15 to 40J, 15 to 35J, 15 to 30J , It may be performed by irradiating ultraviolet rays with an energy amount of 16 to 27J or 16.2 to 27J.

According to one embodiment of the present invention, the amount of light (energy amount) of ultraviolet light per unit area irradiated to the eyeball of the animal model may be 15 to 30 J/cm ² or 16.2 to 27 J/cm ² .

According to one embodiment of the invention, the step of irradiating the ultraviolet rays may be performed when the riboflavin injected into the front of the eyeball of the animal is discharged through a trabecular meshwork.

According to an embodiment of the present invention, the step of irradiating the ultraviolet rays is 5 minutes to 20 minutes, 5 minutes to 18 minutes, 7 minutes to 18 minutes, 5 minutes to 15 minutes, 10 minutes to 5 minutes after the step of administering the riboflavin. It may be performed when 20 minutes or 10 to 15 minutes have elapsed.

When ultraviolet light is irradiated immediately after the riboflavin is administered to the front of the eye, photopolymerization occurs before the riboflavin is discharged from the front of the eye, and only the intraocular pressure cannot be increased without bulging and inflammation of the eye, and the effect of intraocular pressure elevation is negligible. In addition, when the riboflavin is administered and irradiated with ultraviolet rays after more than 20 minutes, photopolymerization occurs after the riboflavin is completely discharged, so it is not possible to effectively prevent the discharge of an aqueous humor, so the effect of intraocular pressure increase is negligible.

According to one embodiment of the present invention, the method may further include anesthetizing the animal before administering the composition comprising the riboflavin. The anesthesia step may include, for example, respiratory anesthesia using isoflurane, intramuscular injection using anesthetics such as ketamine and/or xylazine, or eye drop anesthetics such as proparacaine, benoxynate tetracaine, and alkaine. Using anesthesia may be used, but is not limited thereto, and may be performed according to a method commonly performed in the art.

According to one embodiment of the invention, the animal may be a monkey, rat, mouse, or rabbit.

According to one embodiment of the present invention, the increase in intraocular pressure generated by the above method can be usefully used to study the mechanism of glaucoma.

Another aspect of the present invention provides an animal model with elevated intraocular pressure prepared by the above method.

According to one embodiment of the present invention, the animal model having an increased intraocular pressure may have an increased intraocular pressure of 5 to 20 mmHg or more, 5 to 15 mmHg or more, 10 to 15 mmHg or more, or 10 mmHg or more than the control group not performing the method.

According to one embodiment of the present invention, the animal model with increased intraocular pressure is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% compared to a control group without performing the method. Or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 30% to 200%, 30% to 180%, 50% to 180%, 60% to 180%, 70% to 180%, 80% to 180%, 90% to 180%, or 100% to 180% intraocular pressure may be increased.

According to one embodiment of the present invention, the intraocular pressure increase rate is 30% to 200%, 30% to 180%, 50% to 180%, 60% to 180% compared to a control group not performing the above method in an animal model having elevated intraocular pressure. , 70% to 180%, 80% to 180%, 90% to 180%, 100% to 180%, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or It may be 100% or more.

According to an embodiment of the present invention, the rate of increase in intraocular pressure may be calculated by Equation 1 above.

According to one embodiment of the invention, for 3 to 20 weeks, 4 to 20 weeks, 5 to 20 weeks, 6 to 15 weeks, 8 to 15 weeks in an animal model with elevated intraocular pressure , 10 weeks to 12 weeks, 8 weeks, 10 weeks, 12 weeks, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks Elevated intraocular pressure may be maintained above, above 11 weeks, or above 12 weeks.

According to one embodiment of the invention, the animal may be a monkey, rat, mouse, or rabbit.

Another aspect of the invention,

Administering a candidate substance to the eyeball of the animal model with increased intraocular pressure;

Measuring the intraocular pressure of the animal model after administration of the candidate substance; And

It provides a screening method of a drug for the prevention or treatment of glaucoma, comprising the step of selecting a substance that lowers intraocular pressure compared to a control group to which the candidate substance is not administered.

Another aspect of the invention,

Administering a candidate substance to the eyeball of the animal model with increased intraocular pressure;

Measuring the intraocular pressure of the animal model after administration of the candidate substance; And

It provides a screening method for a drug that lowers the intraocular pressure, comprising the step of selecting a substance that lowers the intraocular pressure compared to the control group not administered with the candidate substance.

According to one embodiment of the invention, the animal may be a monkey, rat, mouse, or rabbit.

In the present invention, "glaucoma" refers to an ophthalmic disease in which the intraocular pressure is higher than normal (10 to 21 mmHg), compressing the optic nerve, thereby deteriorating or losing vision by damaging or destroying the optic nerve. Therefore, a substance that lowers intraocular pressure according to an embodiment of the present invention may be used as a drug for the prevention or treatment of glaucoma. Glaucoma can be divided into congenital glaucoma, primary glaucoma without other causative diseases, and secondary glaucoma due to trauma or side effects of drugs. Congenital glaucoma is a glaucoma that occurs due to developmental dysfunction in the right angle where the water is discharged by nature, and this prevents the discharge of the water. In the case of primary glaucoma, it is classified into open-angle glaucoma and closed-angle glaucoma again, depending on whether the waterproof angle is opened or closed from the eyeball, and the symptoms are different.

Open angle glaucoma is a type of glaucoma that shows symptoms of an increase in intraocular pressure due to an increase in resistance of the fiber line through which the anterior angle is open, but the waterproofing escapes, thereby causing an increase in intraocular pressure. Closed-angle glaucoma is a symptom of an increase in intraocular pressure due to the absence of waterproof leakage due to the closing of the anterior angle. At this time, the intraocular pressure rises sharply, causing severe eye pain, headache, nausea, and decreased vision.

According to an embodiment of the present invention, "candidate material" is a material used in screening to examine whether there is an activity that lowers intraocular pressure, various natural substances that may or may be expected to exhibit efficacy of lowering intraocular pressure, Compound libraries, gene or protein libraries, and the like.

According to an embodiment of the present invention, the candidate substance to be evaluated can be administered topically to the eye, usually as an eye drop. However, any route of administration of the drug or drug can be evaluated and is not particularly limited. Candidate substances administered parenterally or orally can be evaluated. Examples of the medicament administered parenterally include eye ointments, injections, external preparations and suppositories in addition to the eye drops described above. The dosage of the drug is comprehensively judged based on the properties of the drug, the type of the target to be administered, and age or weight, and the optimal amount should be appropriately determined and is not particularly limited.

According to one embodiment of the present invention, the administration conditions, such as the administration timing and the number of administrations, can be set to be appropriately optimal according to the properties of the drug, test and evaluation purposes, and are not particularly limited.

According to one embodiment of the present invention, as a method for selecting a substance that lowers intraocular pressure, immediately after preparation of an animal model, 72 hours after production, 1 week, 3 weeks, 5 weeks, 7 weeks, and 10 weeks , After 12 weeks, after 15 weeks, or after 20 weeks, it is administered several times every hour, and the tonometer can be measured at each time using a tonometer.

According to an embodiment of the present invention, the step of selecting a substance that lowers intraocular pressure may be any method as long as it achieves a desired purpose, and may be performed using, for example, an tonometer measuring intraocular pressure.

According to an embodiment of the present invention, as a tonometer for measuring intraocular pressure, a Goldman Applanation Tonometer, a rebound tonometer, or Tonopen or the like, or a non-contact tonometer (Non Contact typed) Tonometer) can be used.

According to one embodiment of the present invention, as a tonometer for measuring intraocular pressure, a rebound tonometer manufactured in consideration of corneal elasticity may be used to measure intraocular pressure in rodents.

In an animal model with elevated intraocular pressure according to an embodiment of the present invention, the intraocular pressure may be continued and the optic nerve may be damaged, which is similar to the process of developing open-angle glaucoma, and according to one embodiment of the present invention, prevention of glaucoma Alternatively, a material capable of lowering intraocular pressure may be selected using an animal model having elevated intraocular pressure for screening of a drug for treatment.

Therefore, according to an embodiment of the present invention, the glaucoma may be open-angle glaucoma.

According to one embodiment of the present invention, an animal model with increased intraocular pressure may be prepared using riboflavin and ultraviolet light, and the elevated intraocular pressure of the animal model manufactured according to one embodiment of the present invention is continued for 12 weeks or more. , It is useful for research on the development and treatment of glaucoma.

In addition, since a substance that lowers intraocular pressure can be selected using an animal model with increased intraocular pressure, a drug for preventing or treating glaucoma can be easily screened.

1 shows a state in which ultraviolet rays (UV) are irradiated to the eyes of a rat injected with a composition containing riboflavin in front of the eye according to one embodiment of the present invention.
2A and 2B show the eye state of a rat 1 week after administration of riboflavin (RF) and ultraviolet (UV) irradiation. Specifically, Figure 2a is PBS or 0.5% (w/v), 1% (w/v), or 2% (w/v) of riboflavin administered to the front of the eye and rat (Diameter 7mm, 18mW/cm) ² shows the ocular state of rats 1 week after irradiation for 5 minutes (5.4 J/cm ² ), 10 minutes (10.8 J/cm ² ) or 15 minutes (16.2 J/cm ² ) Figure 2B shows administration of riboflavin Concentration (0.5% (w/v), 1% (w/v), or 2% (w/v)) and UV irradiation time (amount of ultraviolet energy) (5 minutes (5.4 J/cm ² ), 10 minutes ( 10.8J/cm ² ), or 15 minutes (16.2J/cm ² )) to show the eye state of rats 1 week after administration of riboflavin and UV irradiation.
3A and 3B show the eye state of rats 3 weeks after the administration of riboflavin (RF) and ultraviolet (UV) irradiation. Specifically, Figure 3a is PBS or 0.5% (w/v), 1% (w/v), or 2% (w/v) of riboflavin administered to the front of the eye and rat (18mW/cm ² ) Represents the eye state of rats 3 weeks after irradiation for 5 minutes (5.4 J/cm ² ), 10 minutes (10.8 J/cm ² ), or 15 minutes (16.2 J/cm ² ). 3B shows the dose concentration of riboflavin (0.5% (w/v), 1% (w/v), or 2% (w/v)) and ultraviolet irradiation time (ultraviolet energy amount) (5 minutes (5.4 J/cm) ² ), 10 minutes (10.8J/cm ² ), or 15 minutes (16.2J/cm ² )) is changed to show the eye state of rats 3 weeks after riboflavin administration and ultraviolet irradiation.
Figure 4 shows the eye with adverse effects in rats administered 1% (w/v) or 2% (w/v) riboflavin in front of the eye.
FIG. 5 shows the eye state of rats 3 weeks after administration of PBS (0% RF) or 0.5% (w/v) riboflavin (0.5% RF) and ultraviolet (UV) irradiation.
6 is administered with 1% (w/v) or 2% (w/v) of riboflavin (RF), or 1% (w/v) or 2% (w/v) of riboflavin and ultraviolet (UV) ) For 5 minutes (5.4 J/cm ² ) to 15 minutes (16.2 J/cm ² ). PBS was administered to the front of the eyeball, and the rate of intraocular pressure increase (%) was calculated in each experimental group based on the measured intraocular pressure after 72 hours had elapsed.
7A to 7E, 0.5% (w/v) of riboflavin (0.5% R) was administered to the front of the eye, and ultraviolet (18mW/cm ² ) was applied to the eye for 5 minutes (FIGS. 7A and 5.4J/cm ² ), 10 minutes (Figure 7b, 10.8J/cm ² ), 15 minutes (Figure 7c, 16.2J/cm ² ), 20 minutes (Figure 7d, 21.6J/cm ² ), or 25 minutes (Figure 7e, 27J/cm ²⁾ ) Indicates the intraocular pressure measured in the rat irradiated.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of an animal model with elevated intraocular pressure

31G insulin syringe (BD Becton, Dickinson ultra-FineTMII short needle insulin Syringe 0.25mm (0.2 mm) in the anterior chamber of the male Sprague Dawley (SD) rat (Orient Bio, Inc.) with a weight of 1.5 to 2.0 kg 31G) × 8mm) dissolved in phosphate buffered saline (Phosphate-Buffered Saline; PBS), 0.5% (weight/volume, hereinafter w/v), 1% (w/v) or 2% (w/v) ) Was administered by injection of a composition comprising riboflavin (hereinafter, 0.5% (w/v), 1% (w/v) or 2% (w/v) riboflavin).

5 minutes to 20 minutes after administration of riboflavin or PBS, ultraviolet rays (7 mm diameter, 18 mW/cm ² ) at 365±5 nm were irradiated to the eyeballs of rats using an ultraviolet irradiation device (intacx XL, NanoSigma) for 0 minutes, 5 minutes ( 5.4J/cm ² ), 10 minutes (10.8J/cm ² ), 15 minutes (16.2J/cm ² ), 20 minutes (21.6J/cm ² ), or 25 minutes (27J/cm ² ) for photopolymerization Induced.

To prevent corneal damage and inflammation, ophthalmic ointments containing polymyxin B, neomycin and dexamethasone were treated in the eyes of all rats.

When a composition containing riboflavin is administered to the front of the eyeball and irradiated with ultraviolet rays within 5 minutes, photopolymerization occurs before riboflavin is discharged from the front of the eyeball. Could not be induced. In addition, when the composition containing riboflavin was administered and irradiated with ultraviolet light for more than 20 minutes, photopolymerization occurred after riboflavin was discharged, and thus effectively prevented the discharge of waterproofing, so the effect of intraocular pressure elevation was minimal.

1 shows a state in which ultraviolet rays (UV) are irradiated to the eyes of a rat injected with a composition containing riboflavin in front of the eye according to one embodiment of the present invention.

Example 2. Conditions for stably inducing an increase in intraocular pressure

Example 2-1. Eye observation in animal models

Riboflavin (0.5% (w / v) , 1% (w / v), or 2% (w / v)) only administration or ultraviolet (7mm diameter, 5 minute intensity of ^{18mW / cm 2 (5.4J / cm} 2 ), 10 minutes (10.8 J/cm ² ), or 15 minutes (16.2 J/cm ² )) of the rats irradiated and the eyes of the rats prepared in Example 1 were observed. In the same manner as in Example 1, ophthalmic ointment containing polymyxin B, neomycin and dexamethasone was treated in the eye of the rat to prevent corneal damage and inflammation in rats that were administered only riboflavin or irradiated with only ultraviolet rays.

2A and 2B show the eye state of rats 1 week after administration of riboflavin (described in RF on the drawing) and ultraviolet (UV) irradiation. Specifically, Figure 2a is PBS or 0.5% (w/v), 1% (w/v), or 2% (w/v) of riboflavin administered to the front of the eye and rat (7mm diameter, 18mW/cm) ² ) is irradiated for 5 minutes (5.4J/cm ² ), 10 minutes (10.8J/cm ² ), or 15 minutes (16.2J/cm ² ), and indicates the eye state of the rats 1 week old. 2B shows the concentration of riboflavin (0.5% (w/v), 1% (w/v), or 2% (w/v)) and UV irradiation time (amount of ultraviolet energy) (5 minutes (5.4 J/cm ² ), 10 minutes (10.8J/cm ² ), or 15 minutes (16.2J/cm ² )) to indicate the eye state of rats 1 week after the riboflavin administration and ultraviolet irradiation.

As shown in Figures 2a and 2b, in the group administered with 1% (w/v) and 2% (w/v) riboflavin, side effects such as corneal loss, ocular swelling, and corneal abnormality were irrespective of whether ultraviolet irradiation was applied. It was confirmed that it occurred.

3A and 3B show the eye state of rats 3 weeks after administration of riboflavin (described in RF on the drawing) and ultraviolet (UV) irradiation. Specifically, Figure 3a is PBS or 0.5% (w/v), 1% (w/v), or 2% (w/v) of riboflavin administered to the front of the eye and rat (18mW/cm ² ) Shows the eye status of rats 3 weeks after irradiation for 5, 10, or 15 minutes. 3B shows the concentration of riboflavin (0.5% (w/v), 1% (w/v), or 2% (w/v)) and UV irradiation time (amount of ultraviolet energy) (5 minutes (5.4 J/cm ² ), 10 minutes (10.8J/cm ² ), or 15 minutes (16.2J/cm ² )) to show the eye state of rats 3 weeks after the riboflavin administration and ultraviolet irradiation.

As shown in FIGS. 3A and 3B, side effects such as corneal loss, ocular swelling, and corneal abnormalities are irrespective of whether ultraviolet irradiation is performed in the group administered with 1% (w/v) and 2% (w/v) riboflavin. It was confirmed that it appeared.

Figure 4 shows the eye with side effects of corneal loss, corneal abnormality, or ocular dilation in rats administered 1% (w/v) or 2% (w/v) riboflavin.

As described above, the individual with ocular abnormalities had a problem in survival for a long period of time, and euthanasia was performed because it was impossible to proceed with the experiment for 4 to 6 weeks or more. When side effects such as corneal loss, corneal abnormality, or ocular swelling appeared in the eye of the animal model, it was impossible to stably induce an increase in intraocular pressure for a long period of time.

5 is administered with PBS (described as 0% RF on the drawing) or 0.5% (w/v) riboflavin (described as 0.5% RF on the drawing) and irradiated with ultraviolet light (UV) (18 mW/cm ² ) Intensity 5 minutes (5.4J/cm ² ), 10 minutes (10.8J/cm ² ), 15 minutes (16.2J/cm ² ), 20 minutes (21.6J/cm ² ), or 25 minutes (27J/cm ²⁾ ) Shows the eye condition of the rat 3 weeks after irradiation). As shown in FIG. 5, it was confirmed that there were no side effects such as corneal loss, corneal abnormality, and bulging of the eyes of all the rats to which the composition containing PBS or 0.5% riboflavin was administered.

Example 2-2. Intraocular pressure measurement

As described in Example 2-1, individuals with ocular abnormalities had problems with survival for a long period of time, and were unable to proceed with the experiment for 4 to 6 weeks or more, and euthanized. Before euthanasia, 1% (w/v) or 2% (w/v) of riboflavin is administered, or 1% (w/v) or 2% (w/v) of riboflavin is administered and ultraviolet light is irradiated. Intraocular pressure in rats (irradiated for 5 min (5.4 J/cm ² ), 10 min (10.8 J/cm ² ), or 15 min (16.2 J/cm ² ) at an intensity of 18 mW/cm ² ) Was measured using a tonometer (tonometer, Icare ^® TONOLAB rebound tonometer, ICARE FINLAND).

6 is administered with 1% (w/v) or 2% (w/v) of riboflavin (RF), or 1% (w/v) or 2% (w/v) of riboflavin and ultraviolet (UV) ) Represents the rate of increase in intraocular pressure (%) in rats irradiated for 5 minutes (5.4 J/cm ² ), 10 minutes (10.8 J/cm ² ), or 15 minutes (16.2 J/cm ² ). PBS was administered to the front of the eye, and after 72 hours, the measured intraocular pressure was used as the intraocular pressure of the control group, and the rate of intraocular pressure increase in each experimental group was calculated by Equation 1 below.

[Equation 1]

As shown in FIG. 6, an increase in intraocular pressure was observed in the other experimental group compared to the control group administered with PBS after 72 hours, but this was due to expansion of the eyeball, and it was confirmed that the increased intraocular pressure decreased with time. In addition, it was confirmed that the intraocular pressure did not increase in the control group administered with PBS.

In addition, as described in Example 2-1, rats administered 1% (w/v) or 2% (w/v) riboflavin had side effects such as corneal loss, corneal abnormality, and eyeball swelling. The number of euthanized individuals increased with the passage of time.

In animal models, intraocular pressure is highly influenced by stress, and side effects such as corneal loss, corneal abnormality, or bulging of the eyes of the animal model cannot be stably induced for a long period of time.

Therefore, when administering riboflavin at a concentration of 1% (w/v) or 2% (w/v) from Example 2-1 and the results, it causes damage to the eyeball of an individual and cannot effectively and stably raise intraocular pressure. Since it can be seen, a subsequent experiment was conducted using riboflavin at a concentration of 0.5% (w/v).

Example 3 Measurement of intraocular pressure in an animal model with increased intraocular pressure

As described above, a composition containing 0.5% (w/v) riboflavin (hereinafter, 0.5% (w/v) riboflavin) is administered to the front of the eye, and in the rats irradiated with ultraviolet rays for 5 to 25 minutes, the cornea There were no side effects such as loss, corneal abnormality, or bulging of the eye (FIG. 5). Therefore, to prepare an animal model with increased intraocular pressure using 0.5% (w/v) riboflavin, just prior to manufacturing (initial), 72 hours after production to see whether the elevated intraocular pressure is maintained stably for a long time. The intraocular pressure was measured in an animal model over a period of 12 to 12 weeks.

As in the method of Example 1, 0.5% (w/v) of riboflavin was administered to the front of the eye, and the eye of the rat was irradiated with ultraviolet light (5 minutes at an intensity of 18 mW/cm ² (5.4 J/cm ² ), 10 minutes. (10.8J/cm ² ), 15 minutes (16.2J/cm ² ), 20 minutes (21.6J/cm ² ) or 25 minutes (irradiated for 27J/cm ² ) to prepare an animal model with elevated intraocular pressure, Intraocular pressure was measured using a tonometer (tonometer, icare ^® TonoLab tonometer).

Tables 1 to 10 and FIGS. 7A to 7E are administered 0.5% (w/v) of riboflavin in front of the eye, and ultraviolet (18mW/cm ² ) is applied to the eye for 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 5.4J/cm ² (Fig. 7A and Table 1), 10.8J/cm ² (Fig. 7B and Table 3), 16.2J/cm ² (Fig. 7C and Table 5), and 21.6J/cm on the eyes after irradiation for 25 minutes, respectively. ² (Fig. 7d and Table 7) or 27J/cm ² (Fig. 7e and Table 9) shows the intraocular pressure (mmHg) measured in a rat irradiated with ultraviolet rays or a rat irradiated with only ultraviolet rays, and Table 2 (5.4J/cm ² ), 4(10.8J/cm ² ), 6(16.2J/cm ² ), 8(21.6J/cm ² ), and 10(27J/cm ² ) represent the rate of intraocular pressure increase (%) calculated under each condition.

Since intraocular pressure is highly influenced by stress, in order to confirm whether the elevated intraocular pressure is caused by stress, intraocular pressure measured on the opposite eye without administration of riboflavin and ultraviolet light was measured as a control.

Using Equation 1, the intraocular pressure increase rate (%) was calculated using the riboflavin dose and the intraocular pressure (10.1 mmHg) initially measured in the opposite eye not irradiated with ultraviolet light as the intraocular pressure of the control group in Equation 1. The results shown in Tables 1, 3, 5, 7, 9, and FIGS. 7A to 7E represent average values (unit: mmHg) obtained by measuring intraocular pressure three or more times in three or more subjects.

As shown in Tables 1 to 10 and FIGS. 7A to 7E, it was confirmed that riboflavin was administered to a rat irradiated with ultraviolet rays only, and the rate of intraocular pressure increase was large in the rat irradiated with ultraviolet rays, and the intraocular pressure elevation was maintained for a long period of time. This tendency is more pronounced when ultraviolet rays of 16.2 J/cm ² or more are irradiated (irradiation time of ultraviolet rays is 15 minutes or more), and 80 to 100% of the control group in rats prepared according to an embodiment of the present invention from Week 2 It was confirmed that abnormal intraocular pressure was elevated, and the elevated intraocular pressure was maintained until 12 weeks. In addition, 0.5% (w/v) riboflavin was administered according to one embodiment of the present invention, and when ultraviolet rays of 16.2 J/cm ² or more were irradiated, it was confirmed that an average rate of intraocular pressure increase of 100% or more was observed.

Typically, when the intraocular pressure rises, the loss of retinal ganglion cells and damage to the optic nerve can lead to glaucoma, and the animal model produced according to one embodiment of the present invention has a large intraocular pressure rise and stably stabilizes intraocular pressure. Since the rise continues, there is a high possibility of damage to the optic nerve due to a cumulative increase in intraocular pressure, which can be easily used for research on the mechanism and treatment of glaucoma.

Claims (9)

Administering a composition comprising riboflavin to the anterior chamber of the animal; And
A method of manufacturing an animal model having elevated intraocular pressure, comprising irradiating ultraviolet rays to the eyeballs of an animal administered with a composition comprising riboflavin.
The method of claim 1, wherein the composition contains 0.1 to 0.9% (w/v) riboflavin.
The method according to claim 1, wherein the step of irradiating ultraviolet rays is performed by irradiating ultraviolet rays with an energy amount of 15 to 30 J/cm ² .
The method according to claim 1, wherein the step of irradiating the ultraviolet rays is performed 5 to 20 minutes after the step of administering the composition comprising the riboflavin.
The method of claim 1, wherein the animal is a monkey, rat, mouse, or rabbit.
An animal model having elevated intraocular pressure produced by the method of claim 1.
The method of claim 6, wherein the animal model is that the elevated intraocular pressure is maintained for more than 12 weeks.
Administering a candidate substance to the eyes of the animal model having elevated intraocular pressure according to claim 6;
Measuring the intraocular pressure of the animal model after administration of the candidate substance; And
Comprising the step of selecting a substance that lowers intraocular pressure compared to the control group to which the candidate substance is not administered, screening method of a drug for the prevention or treatment of glaucoma.
The method according to claim 8, wherein the glaucoma is open-angle glaucoma.

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