Method for the healing of wounds caused by corneal injury

A method for the healing of wounds caused by corneal injury, which includes administering to a mammalian patient in need thereof an effective wound healing amount of lactoferrin, lactoperoxidase or a combination of lactoferrin and lactoperoxidase as active ingredients, either alone or in admixture with at least one excipient.

TECHNICAL FIELD 
This invention relates to therapeutic agents for corneal disorders, which 
contain lactoferrin and/or lactoperoxidase as (an) active ingredient(s). 
BACKGROUND ART 
Lactoferrin and lactoperoxidase are proteins existing in milk or tears of a 
human being, bovine, etc. and are known to have pharmacological effects 
such as an antibacterial effect and a proliferating effect of lymphocytes. 
(Japanese Unexamined Patent Publication 48534/1990, etc.) 
However, there are few reports on their pharmacological effects in 
ophthalmology. 
Therefore, the inventors studied to find new pharmacological effects of 
lactoferrin and lactoperoxidase and to apply them in the ophthalmological 
field. As the result, the inventors found that these compounds have 
stimulative effects on the proliferation of corneal keratocytes and are 
useful for treatment of corneal disorders. 
DISCLOSURE OF THE INVENTION 
This invention provides therapeutic agents for corneal disorders, which 
contain lactoferrin and/or lactoperoxidase as (an) active ingredient(s). 
Lactoferrin and lactoperoxidase can be obtained generally from secretions, 
for example, milk and tears, of a human being or animals such as bovine. 
There is, therefore, no problem with the safety of these. 
The inventors studied to find new pharmacological effects of lactoferrin 
and lactoperoxidase and to apply them in the ophthalmological field. As 
the result, the inventors found that these compounds have stimulative 
effects on the proliferation of corneal keratocytes and are useful for 
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 (hereinbelow) in detail, lactoferrin 
and lactoperoxidase significantly stimulated the proliferation of 
keratocytes. 
The results indicate that lactoferrin and lactoperoxidase are useful for 
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/or lactoperoxidase 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, buffers, 
preservatives or pH adjusting agents can be combined.

BEST MODE FOR CARRYING OUT THE INVENTION 
Examples of formulations are shown below. 
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(FORMULATION EXAMPLES) 
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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 0.25 g 
lactoperoxidase 0.25 g 
sodium chloride 0.9 g 
sterile purified water q.s. 
total 100 ml 
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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 the uptake of 
.sup.3 H-thymidine into keratocytes. 
Experimental Method 
Cultured rabbit corneal keratocytes (1.times.10.sup.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 a 96-well flat bottom culture 
plate with lactoferrin or lactoperoxidase at 37.degree. C. with 5% 
CO.sub.2 in a CO.sub.2 incubator. After 24 hr, the reaction mixture was 
plussed with .sup.3 H-thymidine (produced by AMERSHAM) and cultured at 
37.degree. C. for 24 hr. The proliferation of keratocytes were measured by 
liquid scintillation counter by counting radio activity of .sup.3 
H-thymidine uptake to keratocytes. 
Result 
The results were shown in Table 1. 
TABLE 1 
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Effects of lactoferrin and lactoperoxidase on the 
proliferation of cultured keratocytes in vitro 
.sup.3 H-thymidine 
uptake 
Samples (.times. 10.sup.4 dpm) 
stimulation (%) 
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Control 13.16 -- 
Lactoferrin 
30 .mu.g/ml 
25.98 97.4 
100 .mu.g/ml 
35.41 169.1 
300 .mu.g/ml 
38.78 194.7 
1000 .mu.g/ml 
42.37 222.0 
Lactoperoxidase 
30 .mu.g/ml 
19.05 44.8 
100 .mu.g/ml 
23.87 81.4 
300 .mu.g/ml 
26.53 101.6 
1000 .mu.g/ml 
26.57 101.9 
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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 
A 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. 
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Score Table 
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0 No regeneration was observed. 
0.5 Very slight regeneration was observed. 
1.0 Slight regeneration was observed. 
2.0 Moderate regeneration was observed. 
3.0 Great regeneration was observed. 
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The results were shown in Table 2, in which each score was represented by 
total scores of eight eyes. 
TABLE 2 
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test compound score 
control 9.5 
lactoferrin 12.5 
lactoperoxidase 16.0 
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The results prove that lactoferrin and lactoperoxidase significantly 
accelerate the regeneration of corneal keratocytes in the 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).