THERAPEUTIC AGENT FOR CORNEAL LESION

Abstract: A therapeutic agent for corneal lesion, containing lactoferrin and/or lactoperoxidase as the active ingredient. Lactoferrin and lactoperoxidase are proteins contained in the milk and tear of man, bovine, etc., and having an antibacterial activity and a pharmacological activity such as promotion of the growth of lymphocites. However they have been scarcely applied to the ophthalmological field. The invention is based on the finding that they have a new pharmacological activity applicable to this field and are useful as a therapeutic agent for corneal lesion, because they have an excellent activity of promoting the growth of corneal parenchymal cells.

Description: TECHNICAL FIELD This invention relates to the use of lactoferrin and/or lactoperoxidase as (an) active ingredient(s) for preparing pharmaceutical compositions for the treatment of corneal disorders. BACKGROUND ART Lactoferrin and lactoperoxidase are proteins existing in milk or tears of human being, bovine, etc. and are known to have pharmacological effects such as antibacterial effect and proliferating effect of lymphocytes. (Japanese Unexamined Patent Publication 48534/1990, etc.) International Application WO 87/07838 discloses compositions suitable for ophthalmic antibacterial use comprising lactoperoxidase as a bacterial inhibitor producer and possibly lactoferrin as a bacteriostatic agent. GB 2 0252 979 Patent application discloses eye lotions made from colostrum and therefore comprising a variety of compounds, lactoferrin included. The specific effect(s) of said compounds is/are neither determined nor deducible from this Patent application. Indeed, lactoferrin sole disclosed function in this Patent application is an antiseptic one. However, there are few reports on lactoferrin and lactoperoxidase pharmacological effects in ophthalmology. Therefore, the inventors studied to find new pharmacological effects of lactoferrin and lactoperoxidase and to apply them in ophthalmological field. As the result, the inventors found that these compounds have stimulative effects on proliferation of corneal keratocytes and are useful for a treatment of corneal disorders. DISCLOSURE OF THE INVENTION This invention relates to the use of lactoferrin and/or lactoperoxidase as (an) active ingredient(s) for preparing pharmaceutical compositions intended for the treatment of corneal disorders, particularly corneal injuries, by stimulating keratocyte proliferation. Lactoferrin and lactoperoxidase can be obtained generally from secretions, for example, milk and tears, of human being or animals such as bovine. There is, therefore, no problem with safety of these. The inventors studied to find new pharmacological effects of lactoferrin and lactoperoxidase and to apply them in ophthalmological field. As the result, the inventors found that these compounds have stimulative effects on proliferation of corneal keratocytes and are useful for a treatment of corneal disorders. To examine the stimulative effects of lactoferrin and lactoperoxidase on proliferation of corneal keratocytes, the inventors tested these compounds using cultured rabbit corneal keratocytes in vitro and rabbit corneal alkali burn injury model in vivo. As shown in the pharmacological test in detail, lactoferrin and lactoperoxidase significantly stimulated the proliferation of keratocytes. The results indicate that lactoferrin and lactoperoxidase are useful for a treatment of various corneal disorders such as corneal injury caused by ulceration, inflammation or ophthalmological surgery, etc. Lactoferrin and/or lactoperoxidase can be administered orally or parenterally, but the preferable dosage form is eye drops. For the purpose of this invention, lactoferrin or lactoperoxidase is administered alone or they are administered together. The dosage of lactoferrin and/or lactoperoxidase is adjusted depending on symptom, age, dosage form, etc. In case of eye drops, the concentration (W/W) of lactoferrin and/orlactoperoxidase is 0.01 - 3.0% preferably. Pharmaceutical preparations of lactoferrin and/or lactoperoxidase can be prepared by the known methods. In eye drops of lactoferrin and/or lactoperoxidase, usual excipient(s) such as isotonic agents, buffer, preservatives or pH adjusting agents can be combined. BEST MODE FOR CARRYING OUT THE INVENTION Examples of formulations are shown below. (FORMULATION EXAMPLES) formulation 1 lactoferrin 0.5 g sodium chloride 0.9 g sterile purified water q.s. total 100 ml formulation 2 lactoperoxidase 0.5 g sodium chloride 0.9 g sterile purified water q.s. total 100 ml formulation 3 lactoferrin 0.25g lactoperoxidase 0.25g sodium chloride 0.9 g sterile purified water q.s. total 100 ml (PHARMACOLOGICAL TEST) To examine the stimulative effects of lactoferrin and lactoperoxidase on proliferation of corneal keratocytes, the inventors tested these compounds using cultured rabbit corneal keratocytes in vitro and rabbit corneal alkali burn injury model in vivo. 1. Effect on cultured rabbit corneal keratocytes Using cultured rabbit corneal keratocytes, the stimulative effects of lactoferrin and lactoperoxidase were examined by counting uptake of 3 H-thymidine into keratocytes. (Experimental Method) Cultured rabbait corneal keratocytes ( 1 x 10 4 cells) were suspended in TC-199 medium (produced by GIBCO) containing 5 vol. % fetal calf serum. The keratocytes were cultured in a well of 96-well flat bottom culture plate with lactoferrin or lactoperoxidase at 37 o C with 5% CO 2 in a CO 2 incubator. After 24 hr, the reaction mixture was plussed with 3 H-thymidine (produced by AMERSHAM) and cultured at 37 o C for 24 hr. The proliferation of keratocytes were measured by liquid scintillation counter by counting radio activity of 3 H-thymidine uptake to keratocytes. (Result) The results were shown in Table 1. Effects of lactoferrin and lactoperoxidase on the proliferation of cultured keratocytes in vitro Samples 3 H-thymidine uptake ( x10 4 dpm) stimulation(%) Control 13.16 - Lactoferrin 30 µg/ml 25.98 97.4 100 µg/ml 35.41 169.1 300 µg/ml 38.78 194.7 1000 µg/ml 42.37 222.0 Lactoperoxidase 30 µg/ml 19.05 44.8 100 µg/ml 23.87 81.4 300 µg/ml 26.53 101.6 1000 µg/ml 26.57 101.9 As shown in Table 1, lactoferrin and lactoperoxidase significantly stimulated the proliferation of keratocytes in a dose-dependent manner. 2. Effect on corneal alkali burn injury To confirm the effects of lactoferrin and lacotperoxidase in vivo, the inventors examined the effects of the compounds on rabbit corneal alkali burn injury. (Experimental Method) Filter disc (diameter : 5 mm) soaked in 1N NaOH was placed on the rabbit cornea for 1.5 minutes to elicit inflammation, and the cornea was washed with physiological saline. Just after the elicitation of inflammation, 0.5 wt. % eye drops of lactoferrin or lactoperoxidase dissolved in physiological saline was instilled 10 times per day at one hour intervals. After the continuous treatment for 13 days, the corneal keratocytes were examined histopathologicaly. As the control, physiological saline was instilled. (result) The evaluation was made according to the following score table. Score Table 0 No regenearation was observed. 0.5 Very slight regeneration was observed. 1.0 Slight regeneration was observed. 2.0 Moderate regeneration was observed. 3.0 Great regenereation was observed. The results were shown in Table 2, in which each score was represented by total scores of eight eyes. test compound score control 9.5 lactoferrin 12.5 lactoperoxidase 16.0 The results proves that lactoferrin and lactoperoxidase significantly accelerate the regeneration of corneal keratocytes in vivo test. INDUSTRIAL APPLICABILITY This invention provides excellent therapeutic agents for corneal disorders, which contain lactoferrin and/or lactoperoxidase as (an) active ingredient(s).

Claims: Use of lactoferrin and / or lactoperoxidase, as active principles, for preparing pharmaceutical compositions for the treatment of corneal disorders, particularly corneal injuries, by stimulating keratocyte proliferation. Use according to claim 1, wherein the pharmaceutical compositions are instilled into eyes. Use according to claim 1 or 2, wherein concentration(s) of said active principles(s) in said composition(s) range(s) from 0.01 to 3.0 % (w/w).

Inventors: DEGRE MICHEL FRANCOIS, HIKIDA MITSUSHI, MITA SHIRO, DEGRE, MICHEL, FRANCOIS, HIKIDA, MITSUSHI, MITA, SHIRO, DEGRE, Michel, François, HIKIDA, MITSUSHI, 15-1-417, KITAOUCHICHO, MITA, SHIRO, 26-304, HIGASHIYAMACHO 7-CHOME
Applicants: SANTEN PHARMA CO LTD, SANTEN PHARMACEUTICAL CO., LTD.
Application year: 1991
Main classification: A61K38
IPC codes: A61K38, A61K9