JP2000264847A - Therapeutic agent for corneal disorder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To examine the action of a compound having a Rho-activating action on corneal disorders, especially corneal epithelium extension, and to provide a therapeutic agent which is used for corneal disorders and contains the compound having the Rho-activating action as an active ingredient. SOLUTION: This therapeutic agent for corneal disorders, such as an agent for stimulating the extension of corneal epithelium, contains a compound having a Rho-activating action. The compound having a Rhoactivating action includes lysophosphatidic acid and its acyl derivatives. The corneal disorders includes corneal ulcers, corneal epithelium ablation, corneitis and dry eye.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a therapeutic agent for corneal disorders having a promoting action on corneal epithelial extension, comprising a compound having a Rho activating action such as lysophosphatidic acid or an acyl derivative thereof as an active ingredient.

[0002]

2. Description of the Related Art The cornea is a transparent, avascular tissue having a diameter of about 1 cm and a thickness of about 1 mm. The transparency of the cornea has an important effect on visual function, and various physiological and biochemical phenomena in the cornea function mainly for the purpose of maintaining the transparency of the cornea.

[0003] Corneal epithelial defects caused by various diseases such as corneal ulcer, corneal epithelial detachment, keratitis or dry eye are naturally repaired without concurrent infection. However, if the epithelial defect is prolonged for some reason or the repair is delayed or not performed, it not only adversely affects the normal construction of the epithelium but also impairs the structure and function of the parenchyma and endothelium. The principle of conventional treatment is passive, in which the epithelium expands spontaneously by protecting the corneal surface from external stimuli to re-cover the defect.
In recent years, with the development of cell biology, factors involved in cell division, migration, adhesion, and extension have been elucidated, and compounds that promote corneal epithelial extension play an important role in repairing corneal epithelial defects. It has been reported to be responsible for (臨眼, 46, 738-
743 (1992), ophthalmic surgery, 5 , 719-727 (1992)).

[0004] By the way, cells respond to an external signal to dynamically change a cytoskeleton and a cell adhesion device to adapt to an external environment. The main constituent components forming the cytoskeleton are three types of fiber structures: microfilaments composed of actin or the like, microtubules composed of tubulin or the like, and intermediate filaments composed of keratin or the like. While closely related to each other, they are responsible for higher-order functions such as cell adhesion, cell morphology, cytokinesis, and cell polarity formation.

Among them, it is thought that the actin-microfilament cytoskeleton is controlled.
The Rho family is one of a subfamily of low molecular weight GTP binding proteins. The Rho family is R
It is composed of members such as ho, Rac, and Cdc42, and acts downstream of extracellular signals such as cell growth factors. Recently, a target protein specific for Rho was identified and its cytoskeletal and adhesion regulatory mechanisms (Experimental Medicine, 16 , 1782-1
788 (1998)) and the control mechanism of cell motility (Experimental Medicine, 16 , 20)
32-2039 (1998)) and other mechanisms for controlling cellular phenomena are being elucidated.

On the other hand, lysophosphatidic acid or an acyl derivative thereof is known as a compound that specifically activates Rho (Cell, 70 , 389-399 (1992)). Various actions have been reported for lysophosphatidic acid or its acyl derivative. For example, to promote the binding of cells to fibronectin to regulate cell morphology (J.
Cell Biol., 127 , 1447-1459 (1994)), promotes the binding of fibronectin to epithelial cells and endothelial cells at the time of skin wound (US Pat. No. 5,480,877), and has a glycosaminoglycan production promoting action. It is a skin activator and is useful as a cosmetic and an external preparation for preventing skin aging (International Patent Publication WO95 / 3509)
No. 0), suppressing hyperproliferation of epithelial cells caused by psoriasis or the like (US Pat. No. 5,565,439), activating macrophages to suppress cell necrosis in tumors (US Pat. No. 5,149,527), suppressing apoptosis. Inhibition to maintain or restore cell function (International Patent Publication WO98 / 41213).

In the field of ophthalmology, promoting the growth of retinal pigment epithelial cells (Curr. Eye Res., 16 , 698-702 (1
997)), promoting Ca ion influx in cultured lens epithelial cells (Cell. Signal., 9 , 609-616 (199)
7)), an increase in the amount of lysophosphatidic acid or its acyl derivative in aqueous humor during corneal injury and promotion of normal keratocyte proliferation (Am. J. Physiol., 2
74, C1065-C1074 (1998)).

However, the relationship between Rho and corneal epithelial cells has not been reported, and the effect of a compound having a Rho activating effect on the movement mechanism of corneal epithelium, which is closely related to the repair of corneal epithelial defects, is of course unknown. Not.

[0009]

As described above, a compound having a Rho activating effect has been studied through studies on the involvement of Rho, a low molecular weight GTP-binding protein involved in the control mechanism of cellular phenomena, in the movement mechanism of corneal epithelium. It has been a very interesting task to examine the effect of keratin on corneal disorders, particularly on corneal epithelial extension.

[0010]

DISCLOSURE OF THE INVENTION The present inventors first studied Rh in order to examine the involvement of Rho in the movement mechanism of corneal epithelium.
o The effect of the inhibitor on the corneal epithelium was examined. As a result, it was clarified that the extension of the corneal epithelium was completely inhibited by the Rho inhibitor, and the movement mechanism of the corneal epithelium was involved in the regulation of the intracellular skeletal system protein by Rho, a low molecular weight GTP-binding protein. Was.

Next, the effect of the compound having a Rho activating action on the corneal epithelium was examined, and it was found that the compound had an excellent promoting action on corneal epithelial extension.

Furthermore, when a compound having a Rho activating effect and a Rho inhibitor are used in combination, the promotion of corneal epithelial extension is almost completely suppressed, and the promoting effect on corneal epithelial extension is based on the Rho activating effect. Was confirmed.

From the above, it can be seen that a compound having a Rho activating effect has an excellent corneal epithelial extension promoting effect, and corneal ulcer, corneal epithelial detachment, keratitis or corneal corneal in which the cornea is damaged by various factors. It became clear that it was useful as a therapeutic agent for corneal disorders such as dry eye.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The compound having a Rho activating action in the present invention refers to a compound that enhances the control mechanism of a cellular phenomenon involving Rho.

In the present invention, lysophosphatidic acid or an acyl derivative thereof is a compound represented by the following general formula [I].

[0016]

Embedded image

[In the formula, R represents a hydrogen atom or an acyl group. The acyl group in the present invention refers to a saturated or unsaturated aliphatic carbonyl group or aromatic carbonyl group,
It is preferably a saturated or unsaturated aliphatic carbonyl group, more preferably a higher aliphatic carbonyl group having 6 or more carbon atoms, and particularly preferred examples are an oleoyl group and a stearoyl group.

The corneal disorder in the present invention refers to corneal ulcer, corneal epithelial detachment, keratitis, dry eye and the like in which the cornea is damaged by various factors.

In order to examine the involvement of Rho in the movement mechanism of corneal epithelium, the effects of Rho inhibitors and compounds having Rho activating action on corneal epithelium were examined. The details will be described in the section of pharmacological test described below, but C3, which is an extracellular enzyme of Clostridium botulinum known as a Rho inhibitor, is used.
Enzyme (hereinafter referred to as Exoenzyme C3) (Cell, 70 , 389-3
99 (1992)) showed that corneal epithelial extension was almost completely suppressed.

From the above, it has been clarified that Rho, which is a low molecular weight GTP-binding protein, is involved in the movement mechanism of the corneal epithelium.

Next, the effect of lysophosphatidic acid or an acyl derivative thereof, which is a typical compound having a Rho activating effect, on corneal epithelial extension was examined. It was found to promote corneal epithelium extension in culture. Furthermore, it was recognized that the corneal epithelial extension promoting action was almost completely suppressed by Exoenzyme C3, confirming that the corneal epithelial extension promoting action of lysophosphatidic acid or its acyl derivative was based on the Rho activating action. Was done. From these facts, compounds having a Rho activating effect have an excellent corneal epithelial extension promoting effect, and corneal disorders, that is, corneal ulcers in which the cornea is damaged by various factors, corneal epithelial detachment, It became clear that it was useful for treating inflammation and dry eye.

The compound having a Rho activating effect can be administered orally or parenterally. Dosage forms include tablets, capsules, granules, powders, injections, eye drops and the like, with eye drops being particularly preferred. These can be formulated using commonly used techniques. For example, eye drops include isotonic agents such as sodium chloride and concentrated glycerin, buffering agents such as sodium phosphate and sodium acetate, polyoxyethylene sorbitan monooleate, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil and the like. Surfactants, stabilizers such as sodium citrate and sodium edetate, and preservatives such as benzalkonium chloride and paraben can be used as needed. The pH may be within the range acceptable for ophthalmic preparations, but is preferably in the range of 4 to 8. An eye ointment can be prepared using a commonly used base such as white petrolatum or liquid paraffin. In addition, oral preparations such as tablets, capsules, granules, powders, and the like include lactose, crystalline cellulose, starch, vegetable oil and other bulking agents, magnesium stearate, talc and other lubricants, hydroxypropyl cellulose, polyvinylpyrrolidone, and the like. Agent, carboxymethylcellulose
Calcium, disintegrators such as low-substituted hydroxypropylmethylcellulose, hydroxypropylmethylcellulose,
It can be prepared by adding a coating agent such as macrogol or silicon resin, a film agent such as a gelatin film and the like as needed.

The dose can be appropriately selected depending on the condition, age, dosage form, etc., but in the case of eye drops, 0.0001 to 1% (w
/ V), preferably 0.001 to 1% (w / v) may be instilled once or several times a day. In the case of an oral preparation, it is generally 0.1 to 5000 mg per day, preferably 1 to 1 mg.
000 mg may be administered once or in several divided doses.

In the following, preparation examples and results of pharmacological tests are shown, but these examples are for better understanding of the present invention and do not limit the scope of the present invention.

[0025]

EXAMPLES [Formulation Examples] Representative formulations using oleoyl lysophosphatidic acid (hereinafter referred to as oleoyl LPA) as a compound having a Rho activating effect are shown below.

1. Eye drops An eye drop having the following formulation was prepared using a commonly used method.

Formulation Example 1 (Ophthalmic solution) Oleoyl LPA 1 mg in 100 ml Sodium chloride 900 mg Sodium hydroxide qs Hydrochloric acid qs Sterile purified water qs

In the same manner as in Formulation Example 1, if necessary, a surfactant and a stabilizer were added to make oleoyl LPA 100%.
5 mg, 10 mg, 50 mg, 100 mg, 50 mg / ml
Ophthalmic solutions containing 0 mg and 1000 mg can be prepared.

Formulation Example 2 (eye ointment) In 100 g of oleoyl LPA 100 mg White petrolatum 90 g Liquid paraffin

In the same manner as in Formulation Example 2, oleoyl LPA
Ophthalmic ointment containing 1 mg, 5 mg, 10 mg and 50 mg can be prepared.

Formulation Example 3 (tablets) Oleoyl LPA 10 mg lactose 59.4 mg corn starch 20 mg carboxymethylcellulose calcium 6 mg hydroxypropylcellulose 4 mg magnesium stearate 0.6 mg in 100 mg

The tablets having the above formulation are coated with 2 mg of a coating agent such as hydroxypropylcellulose to obtain the desired coated tablets.

In the same manner as in Formulation Example 3, oleoyl LPA
0.1mg, 0.5mg, 1mg, 5m in 100mg
g, a tablet containing 50 mg can be obtained.

[Pharmacological test] Effect on corneal epithelial extension (in vitro) Using corneas of male Japanese white rabbits, corneal fragments were obtained according to the method of Nishida et al. (J. Cell Biol., 97 , 1653-1657 (1983)). The effect of the following test compounds on corneal epithelial extension was examined using the corneal epithelial extension length in the tissue culture system of Example 1 as an index.

(Experimental method) A corneal block cut out from a rabbit corneal piece was placed in a culture solution containing a test compound (TC-19).
In 9), the cells were cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ . After the culture, the corneal block was fixed in a mixed solution of ethanol and glacial acetic acid (volume ratio: 95: 5), and embedded in paraffin to prepare a section. After the sections were deparaffinized, they were stained with hematoxylin-eosin, and the extension length of the epithelial cell layer was measured under a microscope.

As a control, a culture which had been similarly cultured in a culture solution containing no test compound was used.

(Result 1) Exoenzyme, a Rho inhibitor
Table 1 shows the results obtained when the cells were cultured in a culture solution containing C3 as a test compound.

[0038]

[Table 1]

As can be seen from Table 1, Rho inhibitors
When cultured in a culture solution containing Exoenzyme C3, the extension of the corneal epithelium was almost completely suppressed, and it was revealed that Rho is involved in the extension of the corneal epithelium.

(Result 2) Table 2 shows the results when the cells were cultured in a culture solution containing oleoyl LPA at a concentration of 0.02 μM, 0.2 μM and 2 μM as a test compound.

[0041]

[Table 2]

As can be seen from Table 2, it was found that when cultured in a culture solution containing oleoyl LPA, extension of the corneal epithelium was remarkably promoted in a concentration-dependent manner.

(Result 3) Exoenzyme C was used as a test compound.
Table 3 shows the results when 3 was added to the culture solution together with oleoyl LPA.

[0044]

[Table 3]

As can be seen from Table 3, Rho inhibitors
When Exoenzyme C3 was added to the culture solution together with oleoyl LPA, the extension of the corneal epithelium was almost completely suppressed.

From these results, it was confirmed that the corneal epithelium extension promoting action was based on the activating action of Rho.

[0047]

According to the above pharmacological test, the compound having Rho activating action has an excellent corneal epithelial extension promoting action,
Through the promotion of wound healing by the corneal epithelium, the corneal epithelium was found to be useful as a therapeutic agent for corneal disorders such as corneal ulcer, corneal epithelial detachment, keratitis or dry eye in which the cornea is damaged by various factors. .

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Masatane Nakamura 2-12-3-205 Mimatsu, Nara City, Nara Prefecture F-term (reference) 4C084 AA17 NA14 ZA332 4C086 AA01 AA02 DA34 NA14 ZA33

Claims (5)

[Claims] 1. A therapeutic agent for corneal disorders comprising a compound having a Rho activating effect as an active ingredient. 2. The therapeutic agent for corneal disorders according to claim 1, wherein the compound having a Rho activating action is lysophosphatidic acid or an acyl derivative thereof. 3. The method according to claim 2, wherein the lysophosphatidic acid acyl derivative is oleoyl lysophosphatidic acid. 4. The therapeutic agent for corneal disorder according to claim 1, wherein the corneal disorder is corneal ulcer, corneal epithelial detachment, keratitis or dry eye. 5. A corneal epithelium extension promoter comprising a compound having a Rho activating effect as an active ingredient.