U.S. patent application number 10/540446 was filed with the patent office on 2006-10-19 for ophthalmic therapeutic composition.
This patent application is currently assigned to Nihon Tenganyaku Kenkyusho Co., LTD.. Invention is credited to Hiroaki Iwata, Teruo Nishida, Yorihisa Uetake.
Application Number | 20060234945 10/540446 |
Document ID | / |
Family ID | 32708470 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234945 |
Kind Code |
A1 |
Nishida; Teruo ; et
al. |
October 19, 2006 |
Ophthalmic therapeutic composition
Abstract
An object is to find the minimum activity expression site of
fibronectin, clarify the actions of this minimum unit in relation
to ophthalmological fields, and provide a ophthalmological
composition having this minimum unit as an effective component.
This invention provides an ophthalmological composition, in
particular, a corneal disorder treatment agent containing the
peptide, PHSRN (SEQ ID NO: 1)(Pro-His-Ser-Arg-Asn (SEQ ID NO: 1)),
or Ac-Pro-His-Ser-Arg-Asn-NH.sub.2, which is a derivative thereof,
or a salt thereof that is allowable as a medical drug as an
effective component. The preferred dosage form is an ophthalmic
formulation.
Inventors: |
Nishida; Teruo; (Yamaguchi,
JP) ; Uetake; Yorihisa; (Aichi, JP) ; Iwata;
Hiroaki; (Aichi, JP) |
Correspondence
Address: |
BURR & BROWN
PO BOX 7068
SYRACUSE
NY
13261-7068
US
|
Assignee: |
Nihon Tenganyaku Kenkyusho Co.,
LTD.
76, Nishisakura-cho, Minami-ku Nagoya-shi
Aichi
JP
457-0039
|
Family ID: |
32708470 |
Appl. No.: |
10/540446 |
Filed: |
December 24, 2003 |
PCT Filed: |
December 24, 2003 |
PCT NO: |
PCT/JP03/16514 |
371 Date: |
May 8, 2006 |
Current U.S.
Class: |
435/6.13 ;
435/6.16; 514/20.8 |
Current CPC
Class: |
A61K 38/08 20130101;
C07K 14/78 20130101; A61K 9/0048 20130101; A61P 27/02 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
514/017 |
International
Class: |
A61K 38/08 20060101
A61K038/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2002 |
JP |
2002-381131 |
Claims
1. An ophthalmological composition, containing, as an effective
component, the peptide, PHSRN (SEQ ID NO: 1), or a salt of this
peptide that is allowable as a medical drug.
2. Either or both of a corneal disorder preventive agent and a
corneal disorder treatment agent, having, as an effective
component, the peptide, PHSRN (SEQ ID NO: 1), or a salt of this
peptide that is allowable as a medical drug.
3. Either or both of the corneal disorder preventive agent and the
corneal disorder treatment agent according to claim 2, with which
the corneal disorder is corneal ulcer, corneal epithelial erosion,
keratitis, or dry eye.
4. Either or both of the corneal disorder preventive agent and the
corneal disorder treatment agent according to claim 2, wherein the
dosage form is an ophthalmic formulation.
5. Either or both of the corneal disorder preventive agent and the
corneal disorder treatment agent according to claim 3, wherein the
dosage form is an ophthalmic formulation.
6. A corneal epithelium migration promoting agent having, as an
effective component, the peptide, PHSRN (SEQ ID NO: 1), or a salt
of this peptide that is allowable as a medical drug.
7. The corneal epithelium migration promoting agent according to
claim 6, wherein the dosage form is an ophthalmic formulation.
8. Usage of an effective amount of the peptide, PHSRN (SEQ ID NO:
1), or a salt of this peptide that is allowable as a medical drug
and a corneal disorder treatment method based on such usage.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a National Phase of International
Application No. PCT/JP2003/016514 having an international filing
date of Dec. 24, 2003, published in Japanese on Jul. 22, 2004,
which claims the benefit of Japanese Application 2002-381131, filed
Dec. 27, 2002, the entirety of which is incorporated herein by
reference.
FIELD OF THE ART
[0002] This invention concerns an ophthalmological composition.
BACKGROUND ART
[0003] This invention concerns the amino acid sequence,
proline-histidine-serine-arginine-asparagine (SEQ ID NO: 1), which
is an activity expression site of fibronectin, and a chemical
substance, with which both terminals of the above amino acid
sequence are modified (these shall be referred to hereinafter as
PHSRN (SEQ ID NO: 1) and Ac-Pro-His-Ser-Arg-Asn-NH.sub.2). This
invention also concerns either or both of an ophthalmological
treatment composition and a preventive composition having a salt of
the above-mentioned substance that is allowable as a medical drug
as an effective component thereof. This composition particularly
concerns either or both of a preventive agent and a treatment agent
for corneal disorder that exhibit wound healing promotion actions
for corneal epithelium.
[0004] The cornea is a thin tissue of 0.52 mm to 11.0 mm thickness.
The cornea is positioned at the foremost portion of the eyeball and
is a highly differentiated tissue having a transmitting property
and an appropriate refractive power for guiding light from the
exterior to the receptors of the retina. The cornea has extremely
important physiological functions. The structure of the cornea is
comparatively simple. That is, the cornea has a highly ordered,
microscopic, five-layer structure comprising the epithelial layer,
Bowman's membrane, corneal stroma, Descemet's membrane, and
endothelial cell layer.
[0005] Fibronectin is a glycoprotein with a molecular weight of
approximately 440 thousand that is involved in cell adhesion and
spreading and serves an important role in wound healing actions as
well as in morphogenesis, development, and other biological
phenomena. This fibronectin is a dimer in which two subunits of a
molecular weight of 220 to 250 thousand each are bonded.
Fibronectin has a domain structure. Fibronectin is involved in cell
adhesion and bonds specifically with various extracellular matrices
and bridges to fibronectin receptors (integrins) on cell
surfaces.
[0006] Corneal disorder is induced by corneal ulcer, corneal
epithelial erosion, keratitis, dry eye, and various other desease.
Such disorder is repaired naturally if there is no concurrent mixed
infection. The principles of repair are: (1) the appearance of
fibronectin at exposed corneal stroma portions at defective
epithelial portions resulting from corneal disorder; (2) the
binding of this fibronectin onto a matrix; and (3) the spreading
and moving of epithelial cells onto this matrix. As the cornea is
cured, fibronectin disappears from the damaged corneal
portions.
[0007] Due to some reason, the repair process may be delayed or the
epithelial defect may persist without being repaired. In such a
case, the normal structuring of the epithelium is affected
adversely and even the structures and functions of the stroma and
endothelium may become impaired. Conventional treatment methods are
passive methods in which the corneal surface is protected from
external irritation in order to allow the epithelium to spread
naturally and resurface the defective portions. With recent
developments in cell biology, factors involved in the division,
movement, adhesion, spreading, etc., of cells have become
clarified. In regard to the repair of corneal epithelial defects,
compounds that promote the spreading of the corneal epithelium have
come to be emphasized.
[0008] Components, such as fibronectin, EGF (Epidermal Growth
Factor), and hyaluronic acid, are known as treatment agents for
corneal epithelial wounds. Fibronectin, which exists in human
plasma, can be purified and used as blood product for instillation.
Such an ophthalmic formulation is known to promote the resufacing
of corneal epithelial defects and the healing of epithelial
wounds.
[0009] However presently, fibronectin must be purified from a
patient's autologous plasma using a special purifying kit. Extreme
trouble is thus taken to obtain fibronectin and a large burden is
placed on the patient. Due to this reason, fibronectin is not put
to adequate use even though it is clinically effective.
[0010] EGF (Epidermal Growth Factor) is a polypeptide with a
molecular weight of 6000 that is known for its actions as a
mitosis-promoting growth factor for corneal epithelium. It is known
that when factors that inhibit the mitosis of the epithelium are
present, the effects of EGF cannot be exhibited readily. In
addition, in cases accompanying inflammation and in cases of
diabetic keratopathy, angiogenesis occurs as a side-effect of
EGF.
[0011] Hyaluronic acid is a glucosaminoglycan with a molecular
weight of several million that has N-acetyl-D-glucosamine and
D-glucuronic acid as constituent sugars. Hyaluronic acid is known
to exhibit significant treatment effects as a treatment agent for
dry eye. In regard to actions, hyaluronic acid acts on the
adhesion, spreading, and movement of epithelial cells but is low in
terms of epithelial cell prolification effects. Hyaluronic acid has
the disadvantage of being difficult to use as an ophthalmic
formulation due to increasing in viscosity at high
concentration.
[0012] The peptide, PHSRN (SEQ ID NO: 1), is a pentapeptide
disclosed in International Publication WO98/22617 and in "The PHSRN
sequence induces extracellular matrix invasion and accelerates
wound healing in obese diabetic mice," The Journal of Clinical
Investigation, 105(11), pp. 1537-1545, 2000. In these prior-art
literatures, the peptide, PHSRN (SEQ ID NO: 1), is indicated as
exhibiting external wound healing effects as well as invasion and
prolification suppressive effects against cancer cells. However,
reports that concern the peptide, PHSRN (SEQ ID NO: 1), in relation
to ophthalmological fields are not known.
[0013] Satisfactory corneal disorder treatment compositions are
thus not known and better compositions have been desired
strongly.
[0014] As mentioned above, fibronectin is recognized to be
clinically effective in ophthalmological fields. However, due to
problems particular to blood product (for example, sanitation
problems, the large burden of having to sample a patient's
autologous blood, the troublesomeness of purified fibronectin from
plasma, etc.), fibronectin is not used widely. In addition, since
the active site of fibronectin had not been clarified adequately,
there was further room for research and development towards using
fibronectin as an effective component of a corneal disorder
treatment agent.
[0015] This invention has been made in view of the above
circumstances and an object thereof is to find the activity
expression site of fibronectin and another object thereof is to
provide a composition, with which the activity expression site of
fibronectin can be used as either or both of an ophthalmological
treatment drug and a preventive drug.
DISCLOSURE OF THE INVENTION
[0016] In order to achieve the above objects, the present inventors
focused on the peptides contained in fibronectin and examined their
actions on corneal disorder. As a result, the present inventors
found that PHSRN (SEQ ID NO: 1), which is the activity expression
site of fibronectin, promotes the healing of corneal epithelial
wounds.
[0017] That is, the present inventors (1) found a new application
of PHSRN (SEQ ID NO: 1) in an ophthalmological composition and (2)
found that a composition, using PHSRN (SEQ ID NO: 1) or a salt
thereof that is allowable as a medical drug, is available as either
or both of a preventive agent and treatment agent for corneal
ulcer, corneal epithelial erosion, keratitis, dry eye, and other
corneal disorder wherein the cornea is in a damaged state and have
thereby come to basically complete the present invention.
[0018] This invention thus provides a new ophthalmological
composition that exhibits strong treatment effects against corneal
disorder at small amounts, is low in molecular weight, and
excellent in terms of safety.
[0019] More Specifically, the present invention provides the
following:
[0020] (1) An ophthalmological composition, containing, as an
effective component, the peptide, PHSRN (SEQ ID NO: 1), or a salt
of this peptide that is allowable as a medical drug.
[0021] (2) Either or both of a corneal disorder preventive agent
and a corneal disorder treatment agent, having, as an effective
component, the peptide, PHSRN (SEQ ID NO: 1), or a salt of this
peptide that is allowable as a medical drug.
[0022] (3) Either or both of the corneal disorder preventive agent
and the corneal disorder treatment agent according to (2), with
which the corneal disorder is corneal ulcer, corneal epithelial
erosion, keratitis, or dry eye.
[0023] (4) Either or both of the corneal disorder preventive agent
and the corneal disorder treatment agent according to (3), wherein
the dosage form is an ophthalmic formulation.
[0024] (5) A corneal epithelium migration promoting agent having,
as an effective component, the peptide, PHSRN (SEQ ID NO: 1), or a
salt of this peptide that is allowable as a medical drug.
[0025] (6) The corneal epithelium migration promoting agent
according to (5), wherein the dosage form is an ophthalmic
formulation.
[0026] (7) Usage of an effective amount of the peptide, PHSRN (SEQ
ID NO: 1), or a salt of this peptide that is allowable as a medical
drug and a corneal disorder treatment method based on such
usage.
[0027] In the present Specification, the following abbreviations
shall be used for amino acid residues. That is Asn or N shall
indicate asparagine, Arg or R shall indicate arginine, His or H
shall indicate histidine, Pro or P shall indicate proline, and Ser
or S shall indicate serine. Also, Ac shall indicate the acetyl
group and NH.sub.2 shall indicate the amino group.
[0028] The peptide, PHSRN (SEQ ID NO: 1), is the pentapeptide that
is the active site of fibronectin and has the structure,
Pro-His-Ser-Arg-Asn (SEQ ID NO: 1). In the case where these amino
acids can generate a number of enantiomers, all such enantiomers
and mixtures thereof are included within this invention.
Compositions formed with the peptide, PHSRN (SEQ ID NO: 1), as a
motif should be interpreted as being within the scope of
equivalents and thus being within the scope of the claims of this
invention. Also, a substance, with which the N terminal of the
peptide, PHSRN (SEQ ID NO: 1), is acetylated and the C terminal is
amidated, that is, Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 is
preferable.
[0029] With the present invention, either or both of prevention and
treatment refers to either or both of the prevention of the
occurrence of a desease in advance (prevention) and the curing of a
patient affected by a desease (therapeutics) by administration to
an animal, including a human being.
[0030] With this invention, corneal disorder refers to corneal
ulcer, corneal epithelial erosion, keratitis, dry eye, etc.,
wherein the cornea is in a damaged state due to any of various
causes.
[0031] Examples of the salts of the peptide, PHSRN (SEQ ID NO: 1),
that are allowable as a medical drug include chlorides, sulfates,
phosphates, lactates, maleates, fumarates, oxalates,
methanesulfonates, paratoluenesulfonates, etc.
[0032] The peptide, PHSRN (SEQ ID NO: 1), or a salt of this peptide
that is allowable as a medical drug may be administered orally or
non-orally. Dosage forms include pills, capsules, granules,
powders, injectable solutions, ophthalmic formulations, etc. Among
these, an ophthalmic formulation, such as an eye drop, eye
ointment, etc., is preferable. These can be prepared by
generally-used arts. For example, in the case of pills, capsules,
granules, powders, and other oral agents, an excipient, such as
lactose, crystalline cellulose, starch, or vegetable oil, a
lubricant, such as magnesium stearate or talc, a binder, such as
hydroxypropylcellulose or polyvinylpyrrolidone, a disintegrator,
such as carboxymethylcellulose calcium or lowly-substituted
hydroxypropylmethylcellulose, a coating agent, such as
hydroxypropylmethylcellulose, macrogol, or silicon resin, a
film-forming agent, such as a gelatin membrane, etc., may be added
as necessary. An eye drop may be prepared using a tonicity agent,
such as sodium chloride, a buffer agent, such as sodium phosphate,
a preservative, such as benzalkonium chloride, etc. Though it is
sufficient that the pH of such a medical drug be within a range
allowable for an ophthalmological formulation, the pH is preferably
within the range of 4 to 8. An eye ointment may be prepared using a
generally-used base material, such as white petrolatum or liquid
paraffin.
[0033] This invention's corneal disorder treatment agent is
preferably administered topically and especially preferably
administered as an ophthalmic formulation. Though the concentration
of the peptide, PHSRN (SEQ ID NO: 1), in the ophthalmic formulation
may be set in accordance with the symptoms, age, etc., and is not
restricted in particular, it is preferably in the range of 0.00001%
to 1%. In the case of an eye drop, one to several drops at a time
is administered once to several times a day. Besides a normal eye
drop, the ophthalmic formulation may take on the form of a
dissolve-on-use type eye drop or an eye ointment. For formulation,
known arts may be employed, that is, an ophthalmic formulation may
be prepared using a normally-used method and adding a tonicity
agent, such as sodium chloride or potassium chloride, a buffer
agent, such as sodium hydrogenphosphate, or sodium
dihydrogenphosphate, a stabilizer, such as edetate sodium, a
preservative, such as ethylparaben, butylparaben, or benzalkonium
chloride, a pH adjuster, such as sodium hydroxide or dilute
hydrochloric acid, an eye ointment base, such as white petrolatum
or liquid paraffin, and other additives as necessary.
[0034] The peptide, PHSRN (SEQ ID NO: 1), of this invention can be
produced simply and inexpensively by a solid phase method of
growing the peptide chain from the C terminal on a insoluble
polymer carrier, a liquid phase method that does not use a carrier,
or other method that is normally used for peptide synthesis.
[0035] The use of the peptide, PHSRN (SEQ ID NO: 1), of this
invention for commercially producing this invention's
ophthalmological preparation and treatment methods of using and
administering the peptide, PHSRN (SEQ ID NO: 1), to a patient are
also included within the scope of this invention.
BRIEF DESCRIPTION OF THE DRAWING(S)
[0036] FIG. 1 is a graph showing the effects of the peptide, PHSRN
(SEQ ID NO: 1), on the migration of corneal epithelial cells. The
abscissa indicates the concentration of the peptide, PHSRN (SEQ ID
NO: 1) (0 (control), 51.2 nM, 102.5 nM, 153.7 nM, 256.2%, and 512.3
nM) and the ordinate indicates the migration length (.mu.m) of the
corneal epithelium.
BEST MODE FOR CARRYING OUT THE INVENTION
[0037] In order to examine the availability of the peptide, PHSRN
(SEQ ID NO: 1), the present inventors examined the effects of the
peptide, PHSRN (SEQ ID NO: 1), on corneal disorder. The details are
indicated in the subsequent section on pharmacological tests. The
present inventors have found that ophthalmic instillation of the
peptide, PHSRN (SEQ ID NO: 1), provides (1) a corneal epithelium
migration effect in a corneal organ culture system and (2) an
effect of promoting wound healing after corneal epithelial
abrasion. It has thereby become clear that the peptide, PHSRN (SEQ
ID NO: 1), is available for the treatment of corneal disorder (that
is, corneal ulcer, corneal epithelial erosion, keratitis, dry eye,
and other disorder wherein the cornea is damaged due to various
causes and especially corneal epithelial erosion) and dry eye.
[0038] Though preparation examples of this invention and the
results of pharmacological tests shall now be described, the scope
of the art of this invention is not limited to the embodiments
described below and various modifications are possible without
changing the gist of the invention. The scope of the art of this
invention covers the scope of equivalents.
1. Preparation Examples
[0039] 1) Eye Drops
[0040] As Formulation 1, an eye drop, containing 0.01 g of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2, 0.9 g of sodium chloride, and a
suitable amount of sterilized purified water in a total amount of
100 ml, was prepared. In the same manner as Formulation 1, eye
drops, respectively containing 0.00001 g, 0.00003 g, 0.0001 g,
0.0005 g, 0.001 g, 0.005 g, 0.05 g, and 0.1 g of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 in a total amount of 100 ml, were
prepared.
[0041] As Formulation 2, an eye drop, containing 0.1 g of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2, 0.8 g of sodium chloride, 0.1 g of
sodium hydrogen phosphate, a suitable amount of sodium dihydrogen
phosphate, and a suitable amount of sterilized purified water in a
total amount of 100 ml, was prepared. In the same manner as
Formulation 2, eye drops, respectively containing 0.00001 g,
0.00003 g, 0.0001 g, 0.0005 g, 0.001 g, 0.005 g, 0.05 g, and 0.1 g
of Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 in a total amount of 100 ml,
were prepared.
2) Eye Ointment
[0042] As Formulation 3, an eye ointment, containing 0.05 g of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2, 90 g of white petrolatum, and a
suitable amount of liquid paraffin in a total amount of 100 g, was
prepared. In the same manner as Formulation 3, eye ointments,
respectively containing 0.00001 g, 0.00003 g, 0.0001 g, 0.0005 g,
0.001 g, 0.005 g, 0.05 g, and 0.1 g of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 in a total amount of 100 g, were
prepared.
2. Examples
[0043] Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 was synthesized by a solid
phase method. Using this compound, (1) the in vitro corneal
epithelium migrating action and (2) in vivo corneal wound healing
promotion action were examined. The detailed data are indicated in
the section on pharmacological tests.
[0044] In comparison to the control groups, migration of corneal
epithelial cell layers and quick healing of corneal wounds were
exhibited clearly by the groups to which
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 was added. It was thus proved that
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 is effective as a treatment agent
for corneal disorder.
3. Pharmacological Tests
[0045] (1) Action on Corneal Epithelium Migration (In Vitro)
[0046] Using the cornea of male Japanese white rabbits, the effects
on corneal epithelium migration were examined using the migrating
length of the corneal epithelium in a corneal organ culture system
as an index in accordance with the method of Nishida et al. (J.
Cell. Biol., 97, pp. 1653-1657 (1983)).
[0047] (Experimental Method)
[0048] Corneal blocks (three per group) were cut out from rabbit
corneal tissue. These corneal blocks were incubated for 20 hours
under the conditions of 37.degree. C. and 5% CO.sub.2 in a culture
medium (Medium-199) containing the test compound. After incubation,
the corneal blocks were fixed in a mixed solution of ethanol and
glacial acetic acid (volume ratio of 95:5), embedded in paraffin,
and prepared as sections. After deparaffination, the sections were
stained with hematoxylin-eosin and the migrating length of the
epithelial cell layer was measured under a microscope. As a
control, corneal blocks incubated in a culture medium that does not
contain the test compound was used.
[0049] (Results)
[0050] As shown in FIG. 1, incubation in a medium containing
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 exhibited significant promotion of
the migration of corneal epithelium.
[0051] (2) Corneal Wound Healing Promotion Action 1 (In Vivo)
[0052] Using a male Japanese white rabbit, a wound of approximately
6 mm in diameter was produced in the cornea by inducing corneal
epithelial abrasion in accordance with the method of Cintron et al.
(Ophthalmic Res., 11, pp. 90-96 (1979)). The wound area was
measured using the fluorescein-stained area as an index. The
effects of the test compound on corneal wound healing were
examined.
[0053] (Experimental Method)
[0054] The eye drops containing the respective concentrations of
the test compound were instilled (30 .mu.l at a time) at 0, 3, 6,
9, 12, 18, 24, 27, 30, 33, 36, 42, and 48 hours after inducing
corneal epithelial abrasion. In measuring the wound area,
fluorescein staining was carried out and a photograph of the cornea
was measured. The fluorescein-stained area of the photographed
cornea was computed using an image analysis processing system. As a
control, a rabbit instilled with the base agent (PBS) that does not
contain the test compound was used.
[0055] (Results)
[0056] Tables 1 and 2 below show the post-treatment effects of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 (PHSRN (SEQ ID NO: 1)) on a rabbit
corneal wound model in the form of healing ratio. As shown in
Tables 1 and 2, the instillation of the peptide, PHSRN (SEQ ID NO:
1), exhibited significant promotion of wound healing.
[0057] In Tables 1 and 2, the respective values indicate the mean
value.+-.standard deviation (n=6). Statistical analysis was carried
out using Dunnett's multiple comparison with respect to PBS with
the area of the corneal wound portion immediately after (0 hours
after) corneal epithelial abrasion being set to 100% (*p<0.05,
** p<0.01; vs. control). TABLE-US-00001 TABLE 1
<Post-treatment effects (healing ratio) of PHSRN (SEQ ID NO: 1)
on a rabbit corneal wound model> Healing ratio(%) 0 hr 6 hr 12
hr 24 hr PBS 0 4.96 .+-. 3.28 19.14 .+-. 5.04 54.36 .+-. 9.00 2
.mu.M PHSRN (SEQ ID NO: 1) 0 9.66 .+-. 2.21 * 26.71 .+-. 2.62 **
61.91 .+-. 3.08 20 .mu.M PHSRN (SEQ ID NO: 1) 0 10.73 .+-. 3.21 **
28.01 .+-. 1.92 ** 64.07 .+-. 3.98 * 200 .mu.M PHSRN (SEQ ID NO: 1)
0 11.06 .+-. 3.50 ** 28.69 .+-. 4.10 ** 67.70 .+-. 5.37 ** 2000
.mu.M PHSRN (SEQ ID NO: 1) 0 11.15 .+-. 1.25 ** 28.99 .+-. 2.65 **
69.49 .+-. 3.17 ** 5 .mu.M EGF 0 14.49 .+-. 3.42 ** 31.63 .+-. 1.29
** 70.78 .+-. 6.91 **
[0058] TABLE-US-00002 TABLE 2 <Post-treatment effects (healing
ratio) of PHSRN (SEQ ID NO: 1) on a rabbit corneal wound model>
Healing ratio (%) 36 hr 48 hr PBS 84.49 .+-. 11.60 97.09 .+-. 5.98
2 .mu.M PHSRN (SEQ ID NO: 1) 88.08 .+-. 4.03 99.14 .+-. 1.90 20
.mu.M PHSRN (SEQ ID NO: 1) 89.09 .+-. 6.42 99.15 .+-. 1.52 200
.mu.M PHSRN (SEQ ID NO: 1) 94.97 .+-. 4.63 * 100.00 .+-. 0.00 2000
.mu.M PHSRN (SEQ ID NO: 1) 95.39 .+-. 3.06 * 100.00 .+-. 0.00 5
.mu.M EGF 96.41 .+-. 3.97 * 99.58 .+-. 1.03
[0059] (3) Corneal Wound Healing Promotion Action 2 (In Vivo)
[0060] Using the same method as (2) above, corneal epithelial
abrasion was induced in a Japanese white rabbit to produce a wound
of approximately 8 mm in diameter in the cornea. The wound area was
measured using the fluorescein-stained area as an index to examine
the effects on corneal wound healing.
[0061] (Experimental method)
[0062] The eye drops containing the respective concentrations of
the test compound were instilled (25 .mu.l at a time) at 0, 6, 12,
18, 24, 30, 36, 42, 48, and 54 hours after induction corneal
epithelial abrasion. In measuring the wound area, fluorescein
staining was carried out and a photograph of the cornea was
measured. The fluorescein-stained area of the photographed cornea
was computed using an image analysis processing system. As a
control, a rabbit instilled with the base agent (physiological
saline) that does not contain the test compound was used.
[0063] (Results)
[0064] Tables 3 and 4 below show the post-treatment effects of
Ac-Pro-His-Ser-Arg-Asn-NH.sub.2 (PHSRN (SEQ ID NO: 1)) on a rabbit
corneal wound model in the form of healing ratio. As shown in
Tables 3 and 4, the instillation of the peptide, PHSRN (SEQ ID NO:
1), exhibited significant promotion of wound healing.
[0065] In Tables 3 and 4, the respective values indicate the mean
value.+-.standard deviation (n=6). Statistical analysis was carried
out using Dunnett's multiple comparison with respect to
physiological saline with the area of the corneal wound portion
immediately after (0 hours after) corneal epithelial abrasion being
set to 100% (*p<0.05, ** p<0.01; vs. control). TABLE-US-00003
TABLE 3 <Post-treatment effects (healing ratio) of PHSRN (SEQ ID
NO: 1) on a rabbit corneal wound model> Healing ratio (%) 0 hr
12 hr 18 hr 24 hr 30 hr Physiological saline 0 8.27 .+-. 3.38 23.12
.+-. 4.05 52.71 .+-. 5.36 52.71 .+-. 5.36 0.3% hyaluronic acid 0
13.98 .+-. 4.88 * 28.45 .+-. 3.37 * 60.78 .+-. 8.07 ** 60.78 .+-.
8.07 * 0.04% PHSRN (SEQ ID NO: 1) 0 13.57 .+-. 3.74 * 29.45 .+-.
3.29 ** 58.71 .+-. 6.32 ** 58.71 .+-. 6.32 **
[0066] TABLE-US-00004 TABLE 4 <Post-treatment effects (healing
ratio) of PHSRN (SEQ ID NO: 1) on a rabbit corneal wound model>
Healing ratio (%) 36 hr 48 hr 54 hr 72 hr Physiological saline
65.63 .+-. 6.69 87.06 .+-. 7.72 94.46 .+-. 6.50 99.72 .+-. 0.80
0.3% hyaluronic acid 71.62 .+-. 11.02 90.19 .+-. 9.51 95.35 .+-.
6.44 99.92 .+-. 0.22 0.04% PHSRN (SEQ ID NO: 1) 70.29 .+-. 8.38
88.27 .+-. 8.74 94.24 .+-. 6.62 98.93 .+-. 2.16
EFFECTS OF THE INVENTION
[0067] The above pharmacological tests show that the peptide, PHSRN
(SEQ ID NO: 1), which is the minimum activity expression site of
fibronectin, exhibits a wound healing promotion action on corneal
epithelim and is available as either or both of a preventive agent
and a treatment agent for corneal ulcer, corneal epithelial
erosion, keratitis, dry eye, etc., wherein the cornea is subject to
damage due to any of various causes.
Sequence CWU 1
1
1 1 5 PRT artificial partial sequence in Fibronectin 1 Pro His Ser
Arg Asn 1 5
* * * * *