WO2003028442A1

WO2003028442A1 - Method of screening remedy for glaucoma

Info

Publication number: WO2003028442A1
Application number: PCT/JP2002/008789
Authority: WO
Inventors: Takashi Ota; Yukiko Sugihara; Fumio Nakazawa; Mitsuaki Kuwano; Hideaki Hara
Original assignee: Santen Pharmaceutical Co., Ltd.
Priority date: 2001-08-30
Filing date: 2002-08-30
Publication date: 2003-04-10

Abstract

A method of pharmacologically screening a remedy for glaucoma with the use of an experimental animal wherein ocular hypertension and optic nerve injury have been induced by injecting a solution containing a crosslinked polymer into the protomerite. As the crosslinked polymer, crosslinked carboxyvinyl polymer or crosslinked polyvinyl alcohol is preferable. Using the method, ocular hypertension can be induced by injecting into the protomerite of the experimental animal the crosslinked polymer-containing solution in an amount corresponding to about 10% of the aqueous humor without replacing the aqueous humor. That is to say, the experimental animal can be constructed by a simplified procedure and the stimulus to the animal can be lessened. The method of screening a remedy for glaucoma with the use of the model animal is useful means of developing remedies for glaucoma.

Description

Description Screening method for glaucoma drugs Technical field

The present invention relates to a method for screening a therapeutic agent for glaucoma using an animal in which ocular hypertension and an optic nerve disorder have been induced, and a method for producing an experimental animal thereof. Background art

Glaucoma is an intractable eye disease in which the optic nerve is damaged by high intraocular pressure exceeding normal intraocular pressure, causing visual impairment such as loss of visual field or reduced visual acuity, and leading to blindness.

Glaucoma patients have chronic ocular hypertension symptoms, which are considered to be one of the causes of ganglion cell death. Therefore, screening of drugs using chronic ocular hypertension model animals is extremely important for research and development of glaucoma therapeutics. .

The anterior chamber of the eye is filled with aqueous humor, and aqueous humor production and aqueous humor outflow resistance contribute to the determination of intraocular pressure. The aqueous humor produced by the ciliary process passes between the iris and the lens and reaches the anterior chamber via the pupil. Most of the aqueous humor in the anterior chamber flows out of the trabecular meshwork into the superior scleral vein.

As an animal model for inducing ocular hypertension, a model in which a polymer solution is injected into the anterior chamber is known. Aust. New Zealand J. Ophthalmol., 20, 225-234 (1992) reports a model in which chondroitin sulfate, hydroxypropylmethylcellulose, carboxymethylcellulose, or methylcellulose was injected into the anterior chamber of the egret. . This model reduces the outflow of aqueous humor and increases intraocular pressure by clogging the meshwork of the trabecular meshwork, which is the outflow channel of aqueous humor, with a polymer. Since these polymers are linear, they pass through the mesh of the trabecular meshwork. Therefore, it is necessary to inject a large amount of polymer solution in order to maintain a high level of visibility with linear polymers. There is. Therefore, the polymer solution had to be injected after all the aqueous humor had been drained.

In such a method of replacing aqueous humor with a polymer solution, the aqueous humor is completely drained, so that new aqueous humor is produced, and at the same time, there is a problem that inflammation is caused by prosiglandins produced at the same time. In addition, the total replacement method requires two steps of draining the aqueous humor and then injecting the polymer, which complicates the work. If the polymer to be injected is irritating, it is desirable to reduce the amount of injected polymer as much as possible. Disclosure of the invention

The present inventors have conducted intensive studies on polymers capable of solving the above-mentioned problems. It has been found that intraocular pressure can be induced with a small amount of a crosslinked polymer-containing solution without requiring replacement.

The present invention relates to a pharmacological screening method for a therapeutic agent for glaucoma using an experimental animal in which ocular hypertension and optic nerve damage have been induced by injecting a solution containing a crosslinked polymer into the anterior chamber.

The present invention also relates to a method for producing an experimental animal in which ocular hypertension and optic nerve damage have been induced by injecting a solution containing a crosslinked polymer into the anterior chamber. In the screening method and the method for preparing an experimental animal, the crosslinked polymer is preferably a crosslinkable lipoxyvinyl polymer or a crosslinked polyvinyl alcohol.

When the crosslinked polymer of the present invention is used, the polymer is crosslinked to form a three-dimensional structure, so that it is harder to pass through the mesh of the trabecular meshwork than a linear polymer, and the effect of causing clogging is increased. Therefore, only a small amount of polymer is injected, and there is no need to drain the aqueous humor prior to injecting the polymer solution.

The animal used in the present invention is usually used as an experimental animal. .

There is no particular limitation as long as it is a mouse, a rat, a guinea pig, a rabbit, a dog, a cat, a monkey, a pig, and the like, but a rabbit and a monkey are preferable in terms of handling and the like.

The cross-linked polymer used in the present invention refers to a polymer obtained by cross-linking a linear polymer with a cross-linking agent, radiation irradiation or the like. These crosslinked polymers can be produced by a known method. Examples of linear polymers are carboxyvinyl polymer (hereinafter abbreviated as CVP), polymethacrylic acid, polyglutamic acid, polyvinyl alcohol (abbreviated as PVA), hyaluronic acid, polyallylamine, polyethyleneimine, collagen and polyvinylpyridine. And the like. Methods for cross-linking these linear polymers include a method using a cross-linking agent, a method of irradiating light, and a method of irradiating radiation, and a method suitable for each polymer may be selected. Examples of the cross-linking agent include ethylene glycol, glycerin, ethylene diamine, polyallylphenol erythryl, divinyl diol, dartalaldehyde, glutaric acid, and dibumohexane, and the like.The cross-linking agent depends on the functional group of the polymer. You can choose

Certain crosslinked polymers are commercially available, sold under the trade name Kyrupopol by Goodlitsch Chemical Co., Ltd., a CVP crosslinked with polyallylpenyl erythritol, and a commercial product of Hibis Co., Ltd. by Wako Pure Chemical Industries, Ltd. CVP, etc. cross-linked with divinyl dalicol, sold under the product name, is available.

Crosslinked polymers are preferably crosslinked CVP and crosslinked PVA. The crosslinked CVP can be obtained as a commercial product as described above. The crosslinked PVA is described in detail in the Examples section, and can be obtained by crosslinking commercially available PVA by irradiation.

The concentration of the crosslinked polymer in the liquid containing the crosslinked polymer used in the present invention can be appropriately increased or decreased, but is preferably 0.01% to 5%.

The crosslinked polymer dissolves or disperses it in a base that can be administered to a living body.

It can be injected in the form of a liquid containing a crosslinked polymer. Purified as base Water is preferred. An anti-inflammatory agent or an antibacterial agent can be added to the crosslinked polymer-containing liquid in order to prevent inflammation and infection accompanying the injection of the crosslinked polymer-containing liquid. Also, in order to adjust the environment in the anterior chamber after injection, a buffer such as disodium hydrogen phosphate, an isotonic agent such as sodium chloride or glycerin

PH adjusters such as hydrochloric acid, sodium hydroxide and the like can be added. It is characterized in that the crosslinked polymer-containing liquid can be injected into the anterior chamber without draining the aqueous humor. As a method for injecting the liquid containing the crosslinked polymer, a known method for injecting into the anterior chamber may be used. For example, the liquid may be injected into the anterior chamber from the limbus of the cornea using an injection needle. Although the number of injections of the crosslinked polymer-containing liquid is not particularly limited, it can be injected one to several times in consideration of the intraocular pressure and the state of inflammation of the eye.

The amount of the crosslinked polymer-containing liquid to be injected is preferably 5 to 15%, more preferably 1.0%, of the aqueous humor of the animal to be injected. The amount to be injected differs depending on the animal to be injected. For example, in the case of a heron, the amount of aqueous humor is 200 1 ^, so that 10 to 30 L is preferable, and about 20 L is particularly preferable. In the case of monkeys, the amount of aqueous humor is 100 / zL, so that it is preferably 5 to 15 L, particularly preferably 10 L.

The model animal used in the present invention can be bred under the same general breeding conditions as the breeding conditions before the injection of the liquid containing the crosslinked polymer.

Screening of a therapeutic agent for glaucoma using the model animal of the present invention is performed by administering a test agent to a model animal and determining whether a decrease in intraocular pressure is observed or a decrease in retinal ganglion cells is suppressed. To do. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relationship between RGC and AUC in a cross-linked CVP-injected ゥ sagi ocular hypertension model. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples. It is not limited to this.

1. Injection of cross-linked CVP into a heron ocular hypertension model and test for confirming its effect Intraocular pressure measurements and retinal ganglion cell counts were performed.

(Experimental method)

1— A. Injection of cross-linked CVP: Construction of a heron high intraocular pressure

1) Add 0.04 g of cross-linked (¥? (Hibis Co., Ltd. 105, manufactured by Wako Pure Chemical Industries)) to purified water 1 01111 ^ and stir with a mixer for 15 minutes to disperse (dispersion liquid 1). To 1 OmL, 0.02 g of diclofenac, 0.18 g of sodium chloride, and 0.24 mL of a 10% sodium hydroxide solution were added and stirred (solution 2) .Dispersion solution 1 and solution 2 were mixed, and mixed with a mixer. After stirring for 1 minute, the pH was adjusted to 7.3 with 1 N hydrochloric acid to give an injection.

2) One eye of awake egret was instilled with 0.4% oxybupro force-in ophthalmic solution. In the anterior chamber of the eye to which 0.4% oxybupro force-in ophthalmic solution was administered,

The injection solution was injected at 2 O L using a micro syringe connected to a 30 G injection needle.

3) 0.1% diclofenac ophthalmic solution was instilled after cross-linking CVP injection. Further, 0.1% diclofenac ophthalmic solution was instilled twice a day from the day after the injection of cross-linked CVP until the enucleation.

1— B. Intraocular pressure measurement in cross-linked CVP-injected heron ocular hypertension model

Using the egret ocular hypertension model prepared as described above, 1 day before cross-linking CVP injection, 6 hours after cross-linking CVP injection, 1, 2, 3, '4, 7, 9, 11, 1, 13, 15 Tonometry was taken after 17, 19, 22, 22, 25 and 28 days. The measurement was also performed on the eye to which the cross-linked CVP was not injected (contralateral eye).

11-C. Measurement of retinal ganglion cell counts in the cross-linked CVP-injected ギ heron ocular hypertension model

1) Using the heron ocular hypertension model prepared as described above, cross-link CVP 28 days after the injection, the eyes were enucleated, immersed in PBS containing 2% paraformaldehyde and 2.5% glutaraldehyde, and fixed in a low-temperature room for 0.1 mm.

2) The cornea and lens were removed from the fixed eyeball, the eyeball was washed in PBS, and then dehydrated in 70% and 90% ethanol for 1 hour each. 3) Paraffin replacement was performed using an automatic paraffin replacement machine.

4) The eyeball was paraffin-embedded using an embedding device, and four 3-thick sections were prepared using a microtome along a vertical line passing through the optic papilla and attached to a slide glass.

5) After drying the slide glass, hematoxylin-eosin staining was performed.

6) Select 3 slices from one eyeball (4 slices) arbitrarily, and use a retinal microscope photograph (20x objective lens, 20x camera lens 3.) up to 6 visual fields (0-3mm) below the optic disc from the optic disc. 3x) was taken.

7) The number of cells in the retinal ganglion cell layer on the photograph was counted, and the number of cells per 1 mm of the retina was calculated. As a control, the same treatment was carried out using a separately prepared untreated egret eyeball (normal eye), and the number of cells per lmm of the retina was calculated. The area under the curve (hereinafter abbreviated as AUC) and the maximum intraocular pressure were calculated from the measured intraocular pressure, and the correlation between the number of cells in the ganglion cell layer and AUC was examined.

(Result)

1— B. Intraocular pressure measurement results in a cross-linked CVP-injected ギ heron ocular hypertension model. The injection of cross-linked CVP increased intraocular pressure, which lasted 28 days after injection compared to the contralateral eye. The average intraocular pressure up to 28 days after injection was 35.2 mmHg, and the maximum intraocular pressure (average of the maximum intraocular pressure of each individual) was 65. OmmHg.

1— Results of measurement of retinal ganglion cell numbers in C. cross-linked CVP-injected ギ heron ocular hypertension model

Table 2 shows the number of retinal ganglion cells. The number of cells in the ganglion cell layer is

41. 9 / Z vs 23.4 / mm in cross-linked CVP injected eyes Decreased to. Fig. 1 shows the correlation between the number of cells in the ganglion cell layer (hereinafter abbreviated as RGC) and .AUC. AUC showed a good correlation with ganglion cell loss, indicating that ganglion cell death was dependent on sustained increases in intraocular pressure.

table 1

The values in the table indicate the average value of 9 eyes.Table 2

Normal eye values indicate the average of two eyes

The value of eyes with cross-linked CVP is the average of 9 eyes

2. Preparation of cross-linked PVA-injected heron high intraocular pressure model and effect confirmation test. . Furthermore, in order to evaluate the usefulness as an ocular hypertension model, a compound having an intraocular pressure lowering effect was administered. An experiment was performed to determine whether an intraocular pressure lowering effect was observed. (Experimental method)

2-A. Fabrication of cross-linked PVA injection ゥ egret high intraocular pressure model

1) Add 1.5 g of polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) to 1.5 L of purified water, dissolve by autoclaving (121 ° C for 15 minutes), and obtain a solution. Was stirred with a mixer until the temperature of the solution dropped. This was dispensed into a 45 OmL vial and irradiated with 18 kGy cobalt-60 gamma ray to crosslink polyvinyl alcohol to obtain an injection solution.

2) One eye of awake egret was instilled with 0.4% oxybupro force-in ophthalmic solution. 20 L of the infusion solution was injected into the anterior chamber of an eye to which 0.4% oxybupro hydrochloride was administered using a microsyringe connected to a 30 G injection needle.

2— B. Intraocular pressure measurement in cross-linked PV A-injected heron ocular hypertension model

Using the heron ocular hypertension model prepared as described above, just before cross-linking PVA injection, 2, 4, 6 hours after cross-linking PVA injection, (5, 8, 8 days as the first day) 3, 5, 8, And on day 11 at 10:30, intraocular pressure measurements were taken. The measurement was also performed on the eye to which the cross-linked PVA was not injected (contralateral eye).

2-C. Injection of 0.01% prostaglandin F _{2 a-} isopropyl ester (PGF _{2 (I} —IE) in a heron ocular hypertension model with cross-linked PVA injection On day 7 after injection of cross-linked PVA, apply 0.1% PGF _2a — IE and 50 L of physiological saline, immediately before and 1, 2, 4, 6, and 8 hours after instillation Later, intraocular pressure was measured.

(Result)

2—B. Intraocular pressure measurement results in cross-linked PVA-injected ゥ heron ocular hypertension model The injection of cross-linked PVA increased intraocular pressure, which persisted for 11 days after injection compared to the contralateral eye. After injection The average intraocular pressure up to 11 days was 40.9 mmHg, and the maximum intraocular pressure (average of the maximum intraocular pressure of each individual) was 55. OmmHg.

2-C. Cross-linked PVA injection 0.01% in the heron ocular hypertension model and the effect of rostaglandin F _2a isopropyl ester (PGF.I. IE) Table IV shows the intraocular pressure at each measurement time point. The intraocular pressure increased by the infusion of cross-linked PVA decreased by administering PGF _2a -IE, which has an intraocular pressure-lowering effect. Table 3

The values in the table indicate the average of three eyes

(However, the average of two eyes is shown for 6 hours after injection)

Table 4

Industrial applicability

According to the present invention, high intraocular pressure can be induced by injecting a bridge polymer-containing solution of about 10% of the aqueous humor into the anterior chamber of an experimental animal without replacing aqueous humor. Therefore, the procedure for preparing an experimental animal can be simplified and the irritation to the animal can be reduced. The screening method for a glaucoma drug using this model animal is a useful tool in the development of a glaucoma drug. .

Claims

The scope of the claims

1-A pharmacological screening method for a glaucoma drug using an experimental animal in which ocular hypertension and optic nerve damage have been induced by injecting a solution containing a crosslinked polymer into the anterior chamber.

2. The screening method according to claim 1, wherein the crosslinked polymer is a crosslinkable lipoxyvinyl polymer or crosslinked polyvinyl alcohol.

3. A method for producing an experimental animal in which ocular hypertension and optic nerve damage have been induced by injecting a solution containing a crosslinked polymer into the anterior chamber.

4. The method according to claim 3, wherein the crosslinked polymer is a crosslinked carboxyvinyl polymer or a crosslinked polyvinyl alcohol.