Corneal therapeutic agent

A corneal therapeutic agent comprises at least one of a hexapeptide and a pharmacologically acceptable salt thereof, represented by formula (I) shown below, and a pharmacologically acceptable carrier, EQU A-B-Pro-C-D-E (I) where A is L- or D-arginine or lysine whose N-terminal amino group is deaminated, alkylated or acylated; B is L- or D-arginine, lysine or hystidine; Pro is L- or D-proline; C is L- or D-tyrosine, tryptophane, or phenylalanine; D is L- or D-valine, isoleucine, or leucine having an amino group whose hydrogen atom may be substituted with an alkyl group having 1 to 4 carbon atoms; E is L- or D-valine, isoleucine or leucine having a C-terminal carboxylic group which is unsubstituted or substituted with --COOR, --CH.sub.2 OR or --CONHR, where R represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

This application is the U.S. National stage of PCT/JP95/01549 filed Aug. 4, 
1995. 
TECHNICAL FIELD 
The present invention relates to a corneal therapeutic agent, and more 
particularly, to a corneal therapeutic agent which accelerates the healing 
of the cornea after photon rendering keratectomy using an ultraviolet 
laser, especially, an excimer laser, and which prevents corneal opacity. 
BACKGROUND ART 
In the ophthalmic field, it has been recently studied to cure paropsia by 
geometrical incision of an eye. The corneal incision is employed to cure, 
for example, ametropia such as myopia, hyperopia, and astigmatism and 
corneal disorders such as corneal opacity. The surgical treatment 
mentioned above is quite advantageous in respect that the permanent 
correction can be realized, compared to conventional eyesight-correction 
methods using compensating lenses such as glasses or contact lenses. 
As a representative example of the surgical treatment for paropsia, there 
is a myopia therapy by means of keratectomy. In particular, radial 
keratotomy, one of the surgical treatment methods for ametropia, is used 
for correcting myopia caused by excessive corneal arcuation. In this 
method, cutting is made along the radial lines extending outwardly from a 
center of the cornea, usually with a surgical knife. The depth of the 
cutting is generally about 90 to 95% of the cornea thickness. The number 
of the cutting lines is possibly in the range of 4-16, generally, 8-12. 
The corneal incision mentioned above makes the cornea relaxed and slightly 
flattened, thereby mitigating or overcoming the myopia. 
Besides this, photon rendering keratectomy (PRK) using an excimer laser is 
known. In the PRK method, the central portion of a corneal front face is 
cut off to form a depressed portion. The cornea is cut off in the form of 
a meniscus. In an infrared laser such as a carbonate gas laser or a YAG 
laser, molecules constituting an irradiated object absorb a laser beam, 
causing molecular vibration. Heat generated by the molecular vibration 
melts and cuts the object. In the excimer laser, however, photon energy 
cuts the bonds of the molecules constituting the irradiated object. As a 
result, the cornea is rarely denatured with heat. In addition, the laser 
can process the object precisely, so that only required region of the 
cornea can be cut off accurately to a necessary depth. 
However, if the cornea is not completely cured after any surgery, the 
phenomena called white corneal opacity, namely keratoleukoma, will occur 
in some cases. The keratoleukoma is caused by random secretion of collagen 
to the corneal surface during a wound-healing stage. The random collagen 
secretion is induced by the stimulation of the cornea with an excimer 
laser or the like during a surgical operation. 
DISCLOSURE OF THE INVENTION 
It is an object of the present invention to provide a corneal therapeutic 
agent capable of preventing keratoleukoma by accelerating the curing of a 
cornea after the surgical operation of ametropia. 
The present invention provides a corneal therapeutic agent comprising at 
least one of a hexapeptide and a pharmacologically acceptable salt thereof 
(hereinafter, referred to as "HP") represented by formula (I) shown below, 
and a pharmacologically acceptable carrier, 
EQU A-B-Pro-C-D-E (I) 
where A is L- or D-arginine or lysine whose N-terminal amino group is 
deaminated, alkylated or acylated; B is L- or D-arginine, lysine or 
hystidine; Pro is L- or D-proline; C is L- or D-tyrosine, tryptophane, or 
phenylalanine; D is L- or D-valine, isoleucine, or leucine having an amino 
group whose hydrogen atom may be substituted with an alkyl group having 1 
to 4 carbon atoms; E is L- or D-valine, isoleucine or leucine having a 
C-terminal carboxylic group which is unsubstituted or substituted with 
--COOR, --CH.sub.2 OR or --CONHR, where R represents a hydrogen atom or an 
alkyl group having 1 to 4 carbon atoms. 
The corneal therapeutic agent is developed for curing a corneal wound and 
preventing corneal opacity, i.e., keratoleukoma, and is used in these 
uses. 
The corneal therapeutic agent may further contain at least one of an 
epithelial cell growth factor (hereinafter, referred to as "EGF") and 
fibronectin (hereinafter, referred to as "FN"), as an active ingredient. 
In this case, a more excellent effect can be expected. 
It is another object of the present invention to provide a corneal 
therapeutic kit comprising Agent A containing HP and Agent B containing at 
least one of EGF and FN. The agent A and the agent B may be administered 
separately or simultaneously. 
It is still another object of the present invention to provide a corneal 
therapeutic kit comprising Agent A containing HP, Agent B containing EGF, 
and Agent C containing FN. The Agents A, B, and C may be administered 
separately or simultaneously.

BEST MODE OF CARRYING OUT THE INVENTION 
Hereinbelow, the present invention will be described in detail. 
Amino acids described in the specification will be abbreviated according to 
the IU-IUB biochemical nomenclature (CBN). The amino acids are 
expressed as follows: 
Arg: L-arginine 
Ile: L-isoleucine 
Lys: L-lysine 
Pro: L-proline 
Trp: L-tryptophan 
D-Arg: D-arginine 
D-Ile: D-isoleucine 
D-Lys: D-lysine 
D-Pro: D-proline 
D-Trp: D-tryptophan 
His: L-histidine 
Leu: L-leucine 
Phe: phenylalanine 
Tyr: L-tyrosine 
Val: L-valine 
D-His: D-histidine 
D-Leu: D-leucine 
D-Phe: D-phenylalanine 
D-Tyr: D-tyrosine 
D-Val: D-valine 
As six amino acids constituting HP, either D- or L-amino acids may be used. 
The active agent HP of the corneal therapeutic agent of the present 
invention has been already proposed as a novel hexapeptide by the 
applicants of the present invention (Jpn. Pat. Appln. KOKAI Publication 
No. 5-194590). 
In formula (I), the first amino acid A of the N-terminal of the hexapeptide 
is arginine or lysine whose N-terminal amino group is deaminated, 
alkylated, or acylated. 
The deamination of the N-terminal amino group, for example, arginine, is 
achieved by dissolving 5-amino valeric acid in 2N aqueous sodium hydroxide 
solution and then adding S-methylthiocarbamide thereto. 
Examples of the alkyl group introduced into the N-terminal amino group of 
amino acid A are methyl, ethyl, propyl, and butyl. The alkylation of the 
N-terminal amino group can be carried out by reacting amino acid A with 
the corresponding alkylbromide, such as methyl bromide or ethyl bromide, 
in an organic solvent such as methylene chloride or pyridine. 
Examples of the acyl group introduced into the N-terminal amino group of 
amino acid A are formyl, acetyl, propionyl, benzoyl, and 
p-toluenesulfonyl. 
The acylation of the N-terminal amino group can be performed by reacting 
amino acid A with an acid anhydride such as acetic anhydride, or an acid 
chloride such as acetyl chloride, in an organic solvent such as methylene 
chloride or pyridine. 
In formula (I), the peptide bond between fourth amino acid C and fifth 
amino acid D from the N-terminal of the hexapeptide is represented by 
formula (II) shown below. 
EQU --CO--NX-- (II) 
where X is hydrogen atom or a C.sub.1 to C.sub.4 - alkyl group. 
In formula (II), the C.sub.1 to C.sub.4 - alkyl group is, for example, 
methyl, ethyl, n-propyl, t-propyl, n-butyl, i-butyl, or t-butyl. 
In formula (I), amino acid E, the sixth amino acid from the N-terminal of 
the hexapeptide, is valine, leucine or isoleucine. The C-terminal carboxyl 
group of sixth amino acid E may be substituted or unsubstituted with 
--COOR, --CH.sub.2 OR, or --CONHR, where R is a hydrogen atom or an alkyl 
group having 1 to 4 carbon atoms. Examples of the alkyl group are methyl, 
ethyl, n-propyl, t-propyl, n-butyl, i-butyl, or t-butyl. 
More specific examples of the aforementioned HP following hexapeptides No. 
1 to 12. 
______________________________________ 
Peptide No. 
Sequence 
______________________________________ 
1 Desamino-Arg--Arg--Pro--Tyr--Ile--Leu--OH 
2 Desamino-Arg--Arg--Pro--Tyr--Ile-Leucinol 
3 Desamino-Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2 
4 Desamino-Arg--Arg--Pro--Trp--Ile--Leu--OEt 
5 N--acetyl--Arg--Arg--Pro--Tyr--Ile--Leu--OH 
6 N--acetyl--Arg--Arg--Pro--Tyr--Ile-Leucinol 
7 N--acetyl--Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2 
8 N--actyl--Arg--Arg--Pro--Trp--Ile--Leu--OEt 
9 N--butyl--Arg--Arg--Pro--Tyr--Ile--Leu--OH 
10 N--butyl--Arg--Arg--Pro--Tyr--Ile-Leucinol 
11 N--butyl--Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2 
12 N--butyl--Arg--Arg--Pro--Trp--Ile--Leu--OEt 
______________________________________ 
The aforementioned HP can be synthesized by either a conventionally-known 
liquid-phase synthesizing method or a solid-phase synthesizing method 
which is generally employed for peptide synthesis. The details of the 
solid-phase HP synthesizing method are described in Jpn. Pat. Appln. KOKAI 
Publication No. 5-194590. 
The active agent of the corneal therapeutic agent of the present invention 
also includes salts of HP. Examples of pharmacologically acceptable 
nonpoisonous salts include a salt of HP with an alkaline metal such as 
sodium or potassium; an alkaline earth metal such as calcium or magnesium; 
an acid-added salt such as a salt of an inorganic acid including 
hydrochloric acid, sulfuric acid, phosphoric acid, or carbonic acid; and 
an acid-added salt such as a salt of an organic acid including acetic 
acid, propionic acid, tartaric acid, succinic acid, malic acid, aspartic 
acid, or glutamic acid. 
The corneal therapeutic agent of the present invention may be used as 
ophthalmic local preparations. Examples of the ophthalmic local 
preparations are eye perfusion liquids, eye drops, eye ointments and the 
like. The corneal therapeutic agent of the present invention for use in 
the eye perfusion liquid can be prepared by dissolving HP in sterilized 
and purified water. In order to make the composition of the eye perfusion 
liquid closer to that of aqueous humor, pharmacologically acceptable 
additives such as an isotonizing agent and a buffer can be added as 
necessary. Examples of the additives include glucose, sodium chloride, 
potassium chloride, calcium chloride, magnesium sulfate, sodium 
bicarbonate, glutathion, and the like. 
The corneal therapeutic agents of the present invention used as an eye drop 
include an aqueous eye drop, a non-aqueous eye drop, an opthalmic 
suspension, and an opthalmic emulsion. The eye drop is prepared by 
dissolving or suspending HP in an aqueous solvent such as sterilized and 
purified water or saline; or in a non-aqueous solvent, for example, a 
vegetable oil such as cotton seed oil, soybean oil, sesame oil, or peanut 
oil. In this case, pharmacologically acceptable additives such as an 
isotonizing agent, pH controlling agent, viscosity improver, suspending 
agent, emulsifying agent, and preservative may be added as necessary. 
Specific examples of the isotonizing agent includes sodium chloride, boric 
acid, sodium nitride, potassium nitride, D-mannitol, glucose, and the 
like. Examples of the pH controlling agent include boric acid, sodium 
sulfite anhydride, hydrochloric acid, citric acid, sodium citrate, acetic 
acid, potassium acetate, sodium carbonate, borax, and the like. Examples 
of the viscosity improver include methyl cellulose, hydroxypropyl 
cellulose, polyvinyl alcohol, sodium chondroitin sulfate, 
polyvinylpyrrolidone, and the like. Examples of the suspending agent are 
polysorbate 80, polyoxyethylene hardened castor oil 60, polyoxy hardened 
castor oil, and the like. Examples of the emulsifying agent are egg-yolk 
lecithin, polysorbate 80, and the like. Examples of the preservative 
include benzalkonium chloride, benzetonium chloride, chlorobutanole, 
phenylethyl alcohol, paraoxy benzoate, and the like. 
The corneal therapeutic agent of the present invention for the eye drop 
contains HP in the range of 10 ng/ml to 100 .mu.g/ml. The eye drop is 
administered 1 to 6 times per day and applied 1 to 3 droplets per time. 
An eye ointment is also included in the corneal therapeutic agent of the 
present invention. The corneal therapeutic agent of the present invention 
used as the eye ointment can be appropriately prepared by employing a base 
material generally used in an eye ointment. 
The corneal therapeutic agent for the eye ointment contains HP in the range 
of 10 ng/ml to 100 .mu.g/ml. The eye ointment may be applied to the rear 
side of an eye lid by means of an eye-drop stick 1 to 6 times per day and 
100 .mu.g per time. 
The corneal therapeutic agent of the present invention can be applied by 
attaching it onto contact lenses. In this case, the holding period of the 
agent is increased, improving the efficacy thereof. 
The corneal therapeutic agent of the present invention mentioned above, 
accelerates healing of a corneal wound given bye, e.g., an ametropia 
operation and shortens the time required for the recovery of the cornea 
after the operation by virtue of the corneal wound-healing accelerating 
effect. Furthermore, the corneal therapeutic agent of the present 
invention has a corneal-opacity preventing effect, so that corneal 
opacity, i.e., keratoleukoma, can be cured and prevented. 
In the radial keratotomy, the cornea is cut with a surgical knife to form a 
dissected portion having a V-shape cross section extending from the 
epithelium to the parenchyma via the Bowman's membrane, viewing from the 
surface side of the cornea. It takes several days to cure the dissection 
after the surgery. In the initial stage, the epithelial cells are grown 
along the surface of the dissected wall downwardly from the epithelium, 
covering the entire wall surface of the dissected portion. Then, the 
parenchymatous tissue is regenerated at the bottom of the dissected 
portion. As the parenchymatous tissue is healed from the bottom of the 
dissected portion upwardly as mentioned above, the epithelial cells, which 
have been formed on the wall of the dissected portion in the initial 
stage, are gradually protruded upward. 
However, the portion which has been formed of the parenchymatous tissue 
before the surgical operation is not regenerated completely only by the 
parenchymatous tissue and partly recouped by the epithelial cells formed 
at the initial stage of the healing step. Therefore, the cornea after the 
surgical operation is not completely recovered and does not regain the 
normal condition. Since the cornea is not completely healed, the cornea is 
mechanically weakened, generating keratoleukoma in some cases. 
In contrast, in the PRK method using an excimer laser, the corneal 
epithelium is cut off with a surgical knife, and thereafter, the corneal 
parenchymatous layer is cut off by an excimer laser. Hence, only the 
surface portion of the parenchymatous tissue can be cut off. In this 
manner, the wound due to the surgical operation can be healed only in the 
initial epithelial cell formation stage. As a result, the portion cut off 
by the excimer laser in the surgical operation is covered by the 
epithelial cells, thereby regaining almost the normal and healthy 
condition. However, the epithelial cells of the healed cornea are 
sometimes nonuniformly formed, so that the surface of the corneal 
epithelium becomes rough. In addition, when the recovery of the epithelial 
cells is delayed, keratoleukoma may be developed even after the PRK 
surgery, similarly to the case of the radial keratotomy. 
In the foregoing, the PRK method using an excimer laser has been explained. 
In the PRK method, an ultraviolet laser other than an excimer laser can be 
used. The ultraviolet laser is referred to a laser having a wavelength 
within the ultraviolet range of about 1 to 400 nm. 
When the corneal therapeutic agent of the present invention is applied to 
the cornea after the radial keratotomy or the PRK surgery, the healing of 
the surgical wound can be accelerated while inflammation of the surgical 
dissection and the cut-off portion and the secretion of collagen are being 
suppressed. Therefore, keratoleukoma is prevented. 
As the active gradient of the corneal therapeutic agent of the present 
invention, HP may be used alone or in combination with a 
conventionally-known compound having a corneal therapeutic activity. For 
example, the corneal therapeutic agent of the present invention may 
contain an epithelium growth factor (hereinafter, referred to as "EGF") 
which is disclosed in Jpn. Pat. Appln. KOKAI Publication Nos. 63-5745 and 
59-65020, and may contain fibronectin described in Jpn. Pat. Appln. KOKAI 
Publication No. 63-5745. In this case, more excellent corneal healing 
effect can be attained. 
Alternatively, both Agent A containing HP and Agent B containing either EGF 
or FN are prepared and administered separately or simultaneously. Agents A 
and B, each containing an active agent are applied, for example in the 
form of ophthalmic local preparations such as an eye drop or an eye 
ointment. Agents A and B may be administered separately or simultaneously. 
The effective amount of EGF used in an eye drop falls within the range of 
0.01 to 50 .mu.g/ml. The effective amount of FN used in an eye drop falls 
within the range of 10 ng/ml to 1 mg/ml. 
Similarly, Agent A containing HP, Agent B containing EGF, and Agent C 
containing FN are prepared and also administered separately or 
simultaneously. 
When HP, EGF, and FN are used together as mentioned above, the initial 
stage of the corneal healing is accelerated by EGF having an epithelial 
regeneration growth activity. In addition, FN has a activity concerning an 
intercellular adhesiveness. Hence, the cornea healing activity of HP can 
be accelerated by EGF and FN. As a result, keratoleukoma can be prevented 
and the cornea can be recovered quickly. 
EXAMPLES 
Hereinbelow, examples of the present invention will be described in detail. 
The following tests were performed with respect to the accelerating 
activity for the corneal wound healing after excimer laser treatment. 
1. Sample Preparation 
The following substances were used in this test. 
HP 1: N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leucinol 
HP 2: N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leu-OH 
EGF: manufactured by Bio Medical Technologies 
FN: manufactured by Bio Medical Technologies 
HP 1 and HP 2 were synthesized in accordance with Examples 31 and 32 
described in Jpn. Pat. Appln. KOKAI Publication No. 5-194590. 
HP 1 and HP 2 were diluted with 1.2 ml of an oxyglutathion 
eye-perfusion/washing solution (trade name: BSS plus, manufactured by 
Santen Seiyaku) to obtain a diluted solution in a final concentration of 
0.1 mg/ml. 1000 .mu.g/ml of EGF was diluted with 1 ml of the oxyglutathion 
eye-perfusion/washing solution to obtain a diluted solution in a final 
concentration of 0.1 mg/ml. FN was diluted with 10 ml of the oxyglutathion 
eye-perfusion/washing solution to obtain a diluted solution in a final 
concentration of 0.1 mg/ml. 
2. Corneal Incision of Test Animal 
A colored rabbit was used as a test animal. The following surgical 
operation was applied to the rabbit. 
An excimer laser was radiated to the corneas of both eyes of the rabbit 
under the condition of 180 mj/10 nsec (10 Hz) per pulse. By this 
radiation, the surface of central corneal parenchyma was cut off by 
abrasion in the form of a meniscus having a diameter of 4.5 mm (9.9 
diopter) and a maximum center-depth of 70 .mu.m. The excimer laser was 
radiated by using Omunimed (trade name) manufactured by Summit (U.S.A.). 
3. Sample Administration 
Each of the aforementioned samples was administered to both eyes twice a 
day. A single droplet (about 50 .mu.l) was administrated once in the 
morning and the afternoon for 5 to 7 days. 
TABLE 1 
______________________________________ 
Conc. Number Number of 
Example Sample (.mu.g/ml) 
of animals 
animal eyes 
______________________________________ 
control no treatment 1 2 
1 HP 1 100 1 2 
2 HP 2 100 1 2 
3 FN + HP1 100 1 2 
4 FN + HP2 100 1 2 
5 EGF + HP1 100 3 6 
6 EGF + FN 100 3 6 
HP1 
7 HP1 + HP2 100 1 2 
FN + EGF 
______________________________________ 
As Comparative examples, eye drops were prepared by dissolving the 
aforementioned samples in the oxyglutathion eye perfusion/washing solution 
in accordance with Table 2 and administered to both eyes of the rabbit. 
As a control, an excimer laser was radiated to the rabbit, and thereafter, 
no treatment was provided thereto. 
TABLE 2 
______________________________________ 
conc. Number Number of 
Example Sample (.mu.g/ml) 
of animals 
animal eyes 
______________________________________ 
control no treatment 1 2 
1 FN 100 1 2 
2 EGF 100 1 2 
3 FN + EGF 100 3 6 
______________________________________ 
After completion of droplet administration, right and left eyes were taken 
out from the rabbit and fixed with a glutathion aldehyde/formalin fixing 
solution. After the fixation, each cornea including a laser irradiated 
portion and a non-irradiated portion were excised out. Then, paraffin 
blocks were prepared in accordance with a customary method and thin-film 
slices of 5 .mu.m thick were prepared by means of a microtome. The 
obtained corneal slices were stained with H.E. (hematoxylin-eosin) and 
then subjected to special staining such as PAS or trichrome. Thereafter, 
the slices were observed with an optical microscope. 
The observation results are as follows: 
In the control, no corneal epithelium regeneration was observed at the 
center of the excimer-laser irradiated portion. 
In the FN group of Comparative Example 1, epithelium regeneration was 
observed in the entire irradiated portion. However, the thickness of the 
regenerated epithelium was nonuniform and inflammatory changes were 
observed mainly ascribed to inflammatory cellular infiltration. In 
addition, vacuolation and fibroblast growth were relatively significant at 
a portion immediately under the epithelium. 
In the EGF group of Comparative Example 2, epithelium regeneration was 
observed in the entire irradiated portion. The thickness of the 
regenerated epithelium is nearly uniform. However, vacuolation and 
fibroblast growth were observed in the tunica propria under the 
epithelium. Furthermore, inflammatory changes were observed. 
In the FN+EGF group of Comparative Example 3, epithelium regeneration was 
observed in the entire irradiated portion. However, the thickness of the 
regenerated epithelium was nonuniform. Vacuolation and fibroblast growth 
were observed in the tunica propria under the epithelium. In addition, 
inflammatory changes were observed. 
In the HP1 group of Example 1 and the HP2-group of Example 2, the 
regeneration of the epithelium was observed in the entire irradiated 
portion. However, the thickness of the regenerated epithelium was 
nonuniform. Vacuolation and fibroblast growth were observed in the tunica 
propria. However, inflammatory changes were not observed. 
In the FN+HP1 group of Example 3, FN+HP2 group of Example 4, EGF+HP1 group 
of Example 5, epithelium regeneration was observed in the entire 
irradiated portion. However, the thickness of the regenerated epithelium 
was nonuniform. Vacuolation and fibroblast growth were low in the tunica 
propria. However, inflammatory changes were not observed. 
In the EGF+FN+HP1 group of Example 6, epithelium regeneration was observed 
in the entire irradiated portion. The thickness of the regenerated 
epithelium was almost uniform. Slight vacuolation were observed under the 
epithelium. Inflammatory changes were not observed. 
In the HP1+HP2+FN+EGF group of Example 7, epithelium regeneration was 
observed in the entire irradiated portion. The thickness of the 
regenerated epithelium was normal. Vacuolation and fibroblast growth were 
rarely observed under the epithelium. Inflammatory changes were not 
observed. 
From the results above, since the cornea was regenerated in the HP1 group 
of Example 1 and HP2 group of Example 2, the samples of Examples 1 and 2 
were confirmed to have an corneal wound healing accelerating effect, as 
the same as in the FN group of Comparative Example 1 and the EGF group of 
Comparative Example 2 which are conventionally known as a corneal 
therapeutic agent. Furthermore, although inflammatory changes were 
observed in the FN group of Comparative Example 1 and the EGF group of 
Comparative Example 2, no inflammatory changes were observed in the HP1 
group of Example 1 and the HP2 group of Example 2. Hence, it was found 
that the occurrence of inflammation can be prevented. As a result, it was 
demonstrate that HP1 and HP2 can be used as a corneal therapeutic agent 
although the thickness of the regenerated epithelium is nonuniform. 
On the other hand, in the case where HP1 or HP2 is used together with FN or 
EGF, as are in the cases of the FN+HP1 group of Example 3, FN+HP2 group of 
example 4 and EGF+HP1 group of Example 5, it was found not only that the 
cornea was regenerated but also that vacuolation and fibroblast growth can 
be suppressed. In addition, occurrence of inflammation was prevented. 
In the case HP, FN and EGF were used together, as are in the cases of the 
EGF+FN+HP1 group of Example 6 and the HP1+HP2+FN+EGF group of Example 7, 
the corneal regeneration was observed. In addition, the thickness of the 
epithelium thereof was uniform. It was further confirmed that vacuolation 
and fibroblast growth were suppressed significantly. Furthermore, 
occurrence of inflammation can be prevented. In particular, the 
HP1+HP2+FN+EGF group of Example 7, it was demonstrated that the 
regenerated epithelium had a normal thickness, that vacuolation and 
fibroblast growth were rarely observed, and that a quite excellent corneal 
therapeutic effect was exhibited. As described, the corneal therapeutic 
effect can be enhanced by using HP, FN, and EGF together. It was 
successful to bring the state of the cornea after a surgical operation 
back closer to the state before operation. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 13 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ArgArgProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /product="Leucinol" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ArgArgProTyrIleXaa 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ArgLysProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ArgArgProTrpIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ArgArgProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /product="Leucinol" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ArgArgProTyrIleXaa 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ArgLysProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ArgArgProTrpIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ArgArgProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /product="Leucinol" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ArgArgProTyrIleXaa 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
ArgLysProTyrIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ArgArgProTrpIleLeu 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /product="Arg or Lys" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /product="Arg, Lys or His" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /product="Tyr, Trp or Phe" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /product="Val, Ile or Leu" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /product="Val, Ile or Leu" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
XaaXaaProXaaXaaXaa 
15 
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