U.S. patent application number 15/941443 was filed with the patent office on 2018-10-11 for agent for applying to mucosa and method for the production thereof.
This patent application is currently assigned to SEIKAGAKU CORPORATION. The applicant listed for this patent is SEIKAGAKU CORPORATION. Invention is credited to Kenji MIYAMOTO, Yuuji SHIMOJIMA, Katsuya TAKAHASHI.
Application Number | 20180289730 15/941443 |
Document ID | / |
Family ID | 37942916 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180289730 |
Kind Code |
A1 |
MIYAMOTO; Kenji ; et
al. |
October 11, 2018 |
AGENT FOR APPLYING TO MUCOSA AND METHOD FOR THE PRODUCTION
THEREOF
Abstract
An agent for applying to mucosa capable of persistently exerting
a therapeutic effect on disorders such as inflammation and lesions
in the mucosa even by a lower frequency of administration because
the agent can stay at a diseased site for a long period of time by
exhibiting a high staying property in a mucosal epithelial layer is
provided, said agent for application to mucosa containing
glycosaminoglycan (GAG) into which a hydrophobic group is
introduced via a binding chain, as an active ingredient.
Inventors: |
MIYAMOTO; Kenji; (Tokyo,
JP) ; TAKAHASHI; Katsuya; (Tokyo, JP) ;
SHIMOJIMA; Yuuji; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEIKAGAKU CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
SEIKAGAKU CORPORATION
Tokyo
JP
|
Family ID: |
37942916 |
Appl. No.: |
15/941443 |
Filed: |
March 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14600558 |
Jan 20, 2015 |
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15941443 |
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13070210 |
Mar 23, 2011 |
8969319 |
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14600558 |
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12083189 |
Apr 4, 2008 |
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PCT/JP2006/320801 |
Oct 12, 2006 |
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13070210 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/728 20130101;
A61K 9/0048 20130101; A61P 1/02 20180101; A61P 1/04 20180101; A61K
9/006 20130101; A61K 31/726 20130101; A61P 15/02 20180101; A61K
49/0054 20130101; A61P 1/00 20180101; A61P 11/02 20180101; A61P
27/02 20180101; A61P 27/00 20180101; A61P 43/00 20180101; A61P
17/02 20180101; A61K 47/61 20170801; A61K 9/0034 20130101; A61K
49/0043 20130101; A61P 17/00 20180101; A61P 13/10 20180101; A61K
31/7008 20130101 |
International
Class: |
A61K 31/7008 20060101
A61K031/7008; A61K 49/00 20060101 A61K049/00; A61K 31/728 20060101
A61K031/728; A61K 31/726 20060101 A61K031/726; A61K 9/00 20060101
A61K009/00; A61K 47/61 20060101 A61K047/61 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 12, 2005 |
JP |
2005-297170 |
Jun 9, 2006 |
JP |
2006-160478 |
Claims
1-18. (canceled)
19. A method for producing an eye drop containing a
glycosaminoglycan into which a hydrophobic group is introduced via
the spacer chain as an active ingredient, the method comprising:
introducing the spacer chain and the hydrophobic group to a
hyaluronic acid or pharmaceutically acceptable salt thereof, with
the proviso that the method not comprising a photodimerization
reaction or a photopolymerization reaction to cross link the
glycosaminoglycan with one another, wherein the eye drop is liquid
preparation in which concentration of the glycosaminoglycan is 0.1
to 0.3% by weight, wherein the glycosaminoglycan having a repeat
unit of a structural unit represented by chemical formula (I):
##STR00002## wherein, R represents R.sub.1 or R.sub.2; Ac
represents an acetyl group; R.sub.1 represents ONa or OH; R.sub.2
represents (1) Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m--NH--; (2)
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--NH--; (3)
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--NH--; (4)
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2)n-NH--; (5)
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m--O--; (6)
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--O--; (7)
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--O--; or (8)
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2).sub.n--O--; wherein, Ph
represents phenyl group, m and n represent integers of 1 to 18,
respectively, and 1 represents 0 or an integer of 1 to 18, as a
basic skeleton, wherein the ratio of the above structural unit
wherein R represents R.sub.2 is 5 to 30% in molar equivalent
relative to the disaccharide repeat unit in molar equivalent of the
glycosaminoglycan.
20. The method according to claim 1, wherein the glycosaminoglycan
into which the hydrophobic group is introduced via the spacer chain
blocks transmission of the ultraviolet ray at a wavelength of 200
to 300 nm by 70 to 100%, when the glycosaminoglycan into which the
hydrophobic group is introduced via the spacer chain is made into
an aqueous solution of 0.1% by weight.
Description
TECHNICAL FIELD
[0001] The present invention relates to an agent for applying to
mucosa containing a hydrophobic group binding type
glycosaminoglycan as an active ingredient, and a method for
production thereof.
BACKGROUND ART
[0002] Conventionally, as substances having healing effects on
mucosal disorders such as inflammation and lesions, hyaluronic acid
which is a representative glycosaminoglycan (hereinafter described
as "GAG") has been known (e.g., Patent Document 1). However, in
mucosa in the cornea, oral cavity and nasalcavity, and conjunctiva
which contacts with the external world, and mucosa in the urinary
bladder, the mucosal surfaces are washed with secretions and
excretions such as tear fluid, salivary fluid and urine, and
foreign substances are removed. Thus derivatives of GAG which keep
medicinal effects inherent in GAG and exert a high staying property
in these mucosal tissues have been demanded.
[0003] Meanwhile, photoreactive hyaluronic acid whose
water-solubility has been increased by binding a photocrosslinking
group such as cinnamic acid to hyaluronic acid and further giving
an alkali treatment thereto has been known (e.g., Patent Document
2). This photoreactive hyaluronic acid has been provided by binding
a photocrosslinking group such as cinnamic acid to hyaluronic acid
in order to provide medical materials such as anti-adhesion
materials by giving the photocrosslinking, and does not aim at
enhancing the staying property in the mucosal tissue.
[0004] [Patent Document 1] Japanese Published Unexamined Patent
Publication No. Hei-1-238530
[0005] [Patent Document 2] Japanese Published Unexamined Patent
Publication No. 2002-249501
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
[0006] The present invention aims at providing an agent for
applying to mucosa, which exerts an excellent staying property and
pharmacological effects in mucosa.
Means for Solving the Problem
[0007] As a result of an extensive study for solving the above
problem, the present inventors have found that "hydrophobic group
binding type GAG" obtained by binding a hydrophobic group to GAG
via a binding chain can be used as an extremely excellent active
ingredient in an agent for applying to mucosa because this GAG
keeps healing effects inherent in GAG on mucosal disorders such as
inflammation and lesions and exhibits a high staying property when
applied thereto, and have completed the present invention.
[0008] The present invention relates to an agent for applying to
mucosa which contains glycosaminoglycan (GAG) into which the
hydrophobic group is introduced via the binding chain.
Effects of the Invention
[0009] The agent for applying to mucosa of the present invention
can exert the persistent healing effect on the mucosal disorder
such as inflammation and lesions even by low frequent
administration because this can stay at a diseased site for a long
period of time by exhibiting high staying property in the
mucosa.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a view showing a spectrum of light
transmittance;
[0011] FIG. 2 is a view showing healed area percentages;
[0012] FIG. 3 is a view showing healed areas;
[0013] FIG. 4 is a view showing healing rate;
[0014] FIG. 5 is a view showing healed areas;
[0015] FIG. 6 is a view showing healing rate;
[0016] FIG. 7 is a view showing healed areas;
[0017] FIG. 8 is a view showing healing rate;
[0018] FIG. 9 is a view showing healed areas;
[0019] FIG. 10 is a view showing healing rate;
[0020] FIG. 11 is a, view showing staying property at peeled sites
in rabbit corneal epithelia;
[0021] FIG. 12 is a view showing staying property at peeled sites
in rabbit corneal epithelia;
[0022] FIG. 13 is a view showing photographs of an eyeball after
irradiation with ultraviolet rays;
[0023] FIG. 14 is a view showing amounts of water evaporation in a
removed cornea;
[0024] FIG. 15 is a view showing amounts of water evaporation in a
removed cornea; and
[0025] FIG. 16 is a view showing changes in water evaporation
amount ratio in model hamsters for xerostomia.
[0026] FIG. 17 is a view showing healed areas;
[0027] FIG. 18 is a view showing healing rate;
[0028] FIG. 19 is a view showing healed areas;
[0029] FIG. 20 is a view showing healing rate.
BEST MODES FOR CARRYING OUT THE INVENTION
[0030] The present invention will be described in more detail below
by the best modes for carrying out the invention.
[0031] Herein, an alkyl group refers to a straight or branched
aliphatic hydrocarbon group having. a described number of carbon
atoms. An alkenyl group refers to a straight or branched aliphatic
hydrocarbon group having a described number of carbon atoms, having
at least one double bond. An alkynyl group refers to a straight or
branched aliphatic hydrocarbon group having a described number of
carbon atoms, having at least one triple bond.
[0032] An aryl group refers to a monocyclic or polycyclic aromatic
hydrocarbon group having 6 to 20 carbon atoms as ring-constituting
atoms. A heteroaryl group refers to a monocyclic or polycyclic
aromatic hydrocarbon group having 3 to 20 carbon atoms and one or
heteroatoms selected from nitrogen, sulfur and oxygen atoms as the
ring-constituting atoms.
[0033] An arylalkyl group refers to the alkyl group defined above
substituted with the aryl group defined above. An arylalkynyl group
refers to the alkenyl group defined above substituted with the aryl
defined above. An arylalkynyl group is the alkynyl group defined
above substituted with the aryl group defined above.
[0034] An amino acid group refers to a group derived by losing a
carboxyl group, an amino group or a hydroxyl group by a chemical
bond from a natural or synthetic amino acid.
[0035] Herein, the term "treatment" includes prevention, control of
progression (prevention of deterioration), improvement (reduction)
and cure of the mucosal disorder. The "mucosal disorder" means a
condition where morphology, properties and functions to be inherent
in the mucosa are disordered in some form. For example, the mucosal
disorder can include the conditions such as lesions, defects,
erosion, inflammation, ulcers and dryness.
[0036] GAG into which the hydrophobic group is introduced via the
binding chain, which is contained as the active ingredient in the
agent for applying to mucosa of the present invention can be any
GAG as long as the GAG binds the group having hydrophobicity
derived from a hydrophobic compound having a water, insoluble and
oil soluble nature. This hydrophobic group is bound to GAG via the
binding chain. As described later, it is not necessary that all
constitutive units of GAG bind the hydrophobic groups.
1) GAG
[0037] GAG in GAG into which the hydrophobic group is introduced
via the binding chain contained in the agent for applying to mucosa
of the present invention is an acidic polysaccharide having a
repeated long chain structure of disaccharide composed of amino
sugar and uronic acid (or galactose). Examples of such GAG include
hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparan
sulfate, dermatan sulfate and keratan sulfate, and among them,
hyaluronic acid is preferable. These GAG may be pharmaceutically
acceptable salts thereof. Examples of such salts include sodium
salts, potassium salts, magnesium salts and calcium salts, and
among them, the sodium salt is preferable. Therefore, it is the
most preferable that GAG in the agent for applying to mucosa of the
present invention is sodium hyaluronate. An origin of GAG is not
particularly limited, and GAG may be derived from an animal or a
microorganism or chemically synthesized. For example, when using
sodium hyaluronate, those derived from cock's comb can be
exemplified. The molecular weight of GAG is not particularly
limited, but its weight average molecular weight is preferably
200,000 to 3,000,000, more preferably 500,000 to 2,000,000 and most
preferably 600,000 to 1,200,000. When hyaluronic acid or the
pharmaceutically acceptable salt thereof is used, its weight
average molecular weight is preferably 200,000 to 3,000,000, more
preferably 500,000 to 2,000,000 and most preferably 600,000 to
1,200,000.
2) Hydrophobic Group
[0038] The hydrophobic group in GAG into which the hydrophobic
group is introduced via the binding chain contained in the agent
for applying to mucosa of the present invention is any group as
long as the hydrophobic group is derived from the compound having
the water insoluble and oil soluble nature. Examples of such a
group can include alkyl groups having 2 to 18 carbon atoms, alkenyl
groups having 2 to 18 carbon atoms, alkynyl groups 15 having 2 to
18 carbon atoms, aryl groups, heteroaryl groups, arylalkyl groups,
arylalkenyl groups, arylalkynyl groups and amino acid groups.
[0039] The alkyl groups having 2 to 18 carbon atoms can include
methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl,
n-pentyl, t-pentyl, isopentyl, neopentyl, n-heptyl, 5-methylhexyl,
4,4-dimethyl-pentyl, 1,1-dimethyl-pentyl and n-octyl. Among them,
the alkyl groups such as n-butyl having 2 to 6 carbon atoms such as
n-butyl can be preferably included.
[0040] The alkenyl groups having 2 to 18 carbon atoms can include
vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl,
1-methyl-1-propenyl, 1-pentenyl, 3-methyl-2-butenyl, 1-heptene-1-yl
and 2-heptene-1-yl. Among them, the alkenyl groups such as
1-butenyl having 2 to 6 carbon atoms such as 1-butenyl can be
preferably included.
[0041] The alkynyl groups having 2 to 18 carbon atoms can include
ethynyl, 1-propinyl, 2-propinyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-heptynyl, 2-heptynyl and 3-heptynyl. Among them, the alkynyl
groups such as 1-butynyl having 2 to 6 carbon atoms such as
1-butynyl can be preferably included.
[0042] The aryl groups can include groups such as phenyl, naphthyl,
anthryl and phenanthryl.
[0043] The heteroaryl groups can include groups such as furyl,
thionyl, thiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidinyl and
indolyl.
[0044] The arylalkyl groups can include groups such as benzyl,
phenethyl, naphthylmethyl and naphthylethyl.
[0045] The arylalkenyl groups can include groups such as
2-phenyl-ethenyl and p-aminophenylethenyl.
[0046] The arylalkynyl groups can include groups such as
2-phenyl-ethynyl and p-aminophenylethynyl.
[0047] The amino acid groups can include groups derived from
aliphatic amino acids such as glycine, alanine and 3-alanine;
branched aliphatic amino acids such as leucine, isoleucine and
valine; aromatic amino acids such as phenylalanine and tyrosine;
and heterocyclic amino acids such as tryptophan and histidine.
[0048] These hydrophobic groups may be monosubstituted or
polysubstituted with groups such as hydroxyl, carboxyl, cyano,
amino (which may be monosubstituted or disubstituted with the above
alkyl), nitro, oxo and alkylcarbonyloxy.
[0049] Among the above hydrophobic groups, aryl groups, arylalkyl
groups, arylalkenyl groups and arylalkynyl groups which are the
hydrophobic groups containing the aryl group can be preferably
included, and the arylalkenyl group and the aryl group substituted
with the alkylcarbonyloxy group can be particularly preferably
included. As such an arylalkenyl group, it is possible to
specifically use phenylethenyl and p-aminophenylethenyl. As the
aryl group, it is possible to preferably use the groups such as
CH3-(CH01-000-phenyl (wherein 1 represents or an integer of 1 to
18).
[0050] These hydrophobic groups may also have a function such as
ultraviolet ray absorption ability due to having a double bond in
the hydrophobic group as shown by a functional group such as
phenyl-ethenyl exemplified above contained in the hydrophobic
group. For example, when the agent for applying to mucosa of the
present invention is used as eye drops described later, it is
possible to make the eye drops having the function effectively
absorbing the harmful ultraviolet rays by the use of the group
having the ultraviolet ray absorption ability as the hydrophobic
group. Furthermore, for example, when the agent for applying to
mucosa of the present invention is used for the treatment of
corneal epithelial layer disorders such as corneal xerosis (dry
eye), keratoconjunctivitis, superficial punctate keratitis (SFK),
corneal epithelial erosion, corneal epithelial loss and corneal
tumor, it is possible to make the agent for applying to mucosa
which has pharmacological effects on the above disorders in
combination with the function effectively absorbing the harmful
ultraviolet rays by the use of the group having the ultraviolet ray
absorption ability as the hydrophobic group. As the group having
the ultraviolet ray absorption ability, for example, arylalkenyl
group having the conjugation double bond which is exemplified by
2-phenyl-ethenyl and p-aminophenylethenyl described above is
preferable. When the agent for applying to mucosa of the present
invention is used for the corneal disorder, it is preferable that
"GAG into which the hydrophobic group is introduced" which is the
active ingredient of the agent for applying to mucosa of the
present invention is made into an aqueous solution of 0.1% by
weight, which blocks 70 to 100% transmission of the ultraviolet
rays at a wavelength of 200 to 300 nm when an ultraviolet ray
transmittance is measured by the method described in the Example
described later. Such a hydrophobic group having the ultraviolet
ray absorption ability can preferably include the arylalkenyl
groups such as 2-phenyl-ethenyl and p-aminophenylethenyl.
3) Binding Chain
[0051] In GAG into which the hydrophobic group is introduced via
the binding chain contained in the agent for applying to mucosa of
the present invention, the above GAG is bound to the above
hydrophobic group via the binding chain. GAG has the functional
group which is a carboxyl, hydroxyl or sulfonate (--S0.sub.3H)
group as the side chain. Thus, the hydrophobic group can be bound
to GAG via the binding chain obtained by forming an ether bond,
carboxylate ester bond, sulfate ester bond, carboxylic acid amide
bond or sulfonate amide bond together with these functional groups.
Such a binding chain can specifically include --CONH--, --COO--,
--O--, --SO.sub.3, --and --SO.sub.2NH--. Among them, the carboxylic
acid amide bond of --CONH-- and the carboxylate ester bond of
--COO-- can be preferably used, and the carboxylic acid amide bond
of --CONH-- can be particularly preferably used.
4) Spacer Chain
[0052] In GAG into which the hydrophobic group is introduced via
the binding chain contained in the agent for applying to mucosa of
the present invention, the hydrophobic group is bound to GAG via
the above binding chain, and a spacer chain may further exist
between the binding chain and the hydrophobic group. As such a
spacer chain, any chain group can be used as long as the spacer
group does not completely lose the pharmacological effects which
GAG has. Specifically, --(CH.sub.2).sub.m-- and
--(CH.sub.2)--(OCH.sub.2).sub.n-- (wherein m and n are integers of
1- to 18, respectively) can be included.
[0053] These spacer chains can further have the binding chains such
as --CONH--, --COO--, --O--, --SO.sub.3-- and --SO.sub.2NH-- which
are the same as above at the hydrophobic group side. Such a spacer
chain having the binding chain at the hydrophobic group side can
specifically include --COO--(CH.sub.2).sub.m--,
--COO--CH.sub.2)--(OCH.sub.2).sub.n-1, --CONH--(CH.sub.2).sub.m--
and --CONH--(CH.sub.2)--(OCH.sub.2).sub.n--.
5) Ratio Having Hydrophobic Group
[0054] In GAG into which the hydrophobic group is introduced via
the binding chain contained in the agent for applying to mucosa of
the present invention, it is not necessary that all of GAG
constitutive units respectively have the hydrophobic groups. A
ratio of, the bound hydrophobic group in molar equivalent relative
to a disaccharide repeat unit in molar equivalent of GAG
(hereinafter, referred to as an "introduction ratio") can be
optionally determined depending on the type of the hydrophobic
group, the degree of required hydrophobicity, the type of the
mucosal disorder administered with the agent for applying to mucosa
and the administration site, etc. For example, when using a
phenylethenyl group which may be substituted as the hydrophobic
group, preferably 5 to 30% and more preferably 10 to 20% of a
hydrophobic group in molar equivalent is introduced relative to the
disaccharide repeat unit in molar equivalent of GAG (in the case
where the crosslinking bond described later is not formed).
6) Crosslink Forming Group
[0055] In GAG into which the hydrophobic group is introduced via
the binding chain contained in the agent for applying to mucosa of
the present invention, the hydrophobic group may form a
crosslinking bond between GAG molecules by the functional group
contained in the group. As the hydrophobic group capable of forming
the crosslinking bond, any group can be used as long as the
hydrophobic group produces a photodimerization reaction or a
photopolymerization reaction by irradiation of ultraviolet rays and
is the same as defined above. The hydrophobic group capable of
forming the crosslinking bond includes, for example, phenylethenyl,
p-aminophenylethenyl, ethenyl, 2-carboxyethenyl and
pentane-1,3-dienyl. It is desirable that these groups are bound to
GAG via the binding chain which contains the carbonyl group. Among
these hydrophobic groups, phenylethenyl or, p-aminophenylethenyl
which is bound to GAG via the binding chain which contains the
carbonyl group can be particularly preferably used.
[0056] In such GAG into which the hydrophobic group is introduced
capable of forming the crosslinking bond, GAG molecules can be
crosslinked with one another by being subjected to the
photodimerization reaction or the photopolymerization reaction by
standard methods. For example, according to the methods described
in Japanese Published Unexamined Patent Publication No.
2002-249501, the photodimerization reaction or the
photopolymerization reaction can be given.
7) Preferable GAG Constitutive Unit
[0057] Representatives of GAG into which the hydrophobic group is
introduced via the binding chain contained in the agent for
applying to mucosa of the present invention can specifically
include the agents for applying to mucosa containing GAG into which
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m--NH--;
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--NH--;
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--NHCO--;
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2).sub.n--NHCO--;
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m;
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--O;
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--O;
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2).sub.n--O;
CH.sub.3--(CH.sub.2).sub.1--COO-Ph-CONH--(CH.sub.2).sub.m--NH or
CH.sub.3--(CH.sub.2).sub.1--COO-Ph-CONH--CH.sub.2--(OCH.sub.2)n-NH
(wherein Ph represents phenyl group, m and n represent integers of
1 to 18, respectively, and 1 represents 0 or an integer of 1 to 18)
is introduced, as the active ingredient.
[0058] The following GAG can be included as the representative.
[0059] GAG having the repeat unit of the structural unit
represented by the Chemical formula 1, as a basic skeleton:
##STR00001##
wherein, R represents R.sub.1 or R.sub.2; Ac represents an acetyl
group; R.sub.1 represents ONa or OH; R.sub.2 represents (1)
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m--NH--; (2)
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--NH--; (3)
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--NH; (4)
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2), --NH--; (5)
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m, --O--; (6)
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--O--; (7)
Ph-CH.dbd.CH--CONH--(CH.sub.2).sub.m--O--; (8)
Ph-CH.dbd.CH--CONH--CH.sub.2--(OCH.sub.2).sub.n--O--; (9)
CH.sub.3--(CH.sub.2).sub.1--COO-Ph-CONH--CH.sub.2).sub.m--NH; or
(10) CH.sub.3--(CH.sub.2),
--COO-Ph-CONH--CH.sub.2--(OCH.sub.2).sub.n--NH--; wherein, Ph
represents phenyl group, m and n represent integers of 1 to 18,
respectively, and 1 represents 0 or an integer of 1 to 18, as a
basic skeleton, wherein the ratio of the above structural unit
wherein R represents R.sub.2 is 5 to 30% in molar equivalent
relative to the disaccharide repeat unit in molar equivalent of the
GAG. 8) Method for Producing GAG into which Hydrophobic Group is
Introduced Via Binding Chain
[0060] To obtain GAG into which the hydrophobic group is introduced
via the binding chain, GAG is reacted with a hydrophobic compound
in which the above hydrophobic group has been bound to the
functional group such as hydroxyl, carboxyl, amino or sulfonate
group which can form an ether bond, carboxylate ester bond, sulfate
ester bond, carboxylic acid amide bond or sulfonate amide bond
together with the carboxyl, hydroxyl or sulfonate (--S0.sub.3H)
group in GAG. Specifically, when the bond is the carboxylic acid
amide bond, GAG having the carboxyl group is reacted with the
hydrophobic compound having an amino group to bind the carboxyl
group in GAG to the amino group in the hydrophobic compound. In the
case of the carboxylate ester bond, GAG is reacted with the
hydrophobic compound having hydroxyl or carboxyl group to bind the
carboxyl group in GAG to the hydroxyl group in the hydrophobic
compound or bind the hydroxyl group in GAG to the carboxyl group in
the hydrophobic compound. In the case of the ether bond, GAG having
the hydroxyl group is reacted with the hydrophobic compound having
the hydroxyl group to react the hydroxyl group in GAG with the
hydroxyl group in the hydrophobic compound. In the case of the
sulfonate ester bond, GAG is reacted with the hydrophobic compound
having the hydroxyl group or sulfonate group to bind the hydroxyl
group in GAG to the sulfonate group in the hydrophobic compound or
bind the sulfonate group in GAG to the hydroxyl group in the
hydrophobic compound. These reactions can be performed by common
standard methods, and reaction conditions can be optionally
selected by those skilled in the art.
[0061] When the spacer chain is present between the binding chain
and the hydrophobic group, the order in introducing the spacer
chain and the hydrophobic group to GAG is not particularly limited.
For example, either the method in which a spacer compound having
the functional group such as the hydroxyl, carboxyl, amino or
sulfonate group, which can form the ether bond, carboxylate ester
bond, sulfate ester bond, carboxylic acid amide bond or sulfonate
amide bond together with the functional group in GAG at one end of
the above spacer chain is reacted with GAG, and subsequently, the
other end of the spacer compound is reacted with the hydrophobic
compound which is bound to the functional group such as the
hydroxyl, carboxyl, amino, or sultanate group, or the method in
which the spacer compound having the functional group such as the
hydroxyl, carboxyl, amino or sultanate group which can form an
ether bond, carboxylate ester bond; sulfate ester bond, carboxylic
acid amide bond or sulfonate amide bond together with the
functional group in the hydrophobic compound at one end is reacted
with the hydrophobic compound in which the hydrophobic group has
been bound to the functional group such as the hydroxyl, carboxyl,
amino or sulfonate group, and subsequently the other end of the
spacer compound is reacted with GAG may be used. In particular, the
method in which the spacer compound is reacted with the hydrophobic
compound followed by being reacted with GAG can be preferably
used.
[0062] The above-described method can be appropriately carried out
by publicly known methods, and preferably performed in the presence
of a condensing agent. Such a condensing agent can preferably
include water soluble carbodiimide such as
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCI.HCl), condensing agents such as dicyclohexylcarbodiimide
(DCC) and N-hydroxysuccinate imide (HOSu). For example, when
hyaluronic acid is used as GAG and the cinnamate derivative such as
Ph-CH.dbd.CH--COO--(CH.sub.2).sub.m--NH.sub.2 or
Ph-CH.dbd.CH--COO--CH.sub.2--(OCH.sub.2).sub.n--NH.sub.2 (wherein m
and n are integers of 1 to 18, respectively) is used as the
hydrophobic compound which is bound to the spacer compound, the
condensation method using water soluble carbodiimide such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI.HCl) and N-hydroxysuccinate imide can be preferably used. The
reaction can be accomplished using a mixed solvent of water and a
water soluble organic solvent such as dioxane, dimethylformamide or
ethanol. The hyaluronate derivative which is highly soluble in an
aqueous vehicle can be obtained by treating with a base such as
Sodium hydrogen carbonate after the completion of the reaction.
[0063] When the thus produced GAG into which the hydrophobic group
is introduced via the binding chain is subjected to the
photodimerization reaction or the photopolymerization reaction to
cross link the GAG molecules with one another, for example, the
method described in Japanese Published Unexamined Patent
Publication No. 2002-249501 can be used. Specifically, in the case
of the compound in which the phenylethenyl group as the hydrophobic
group is bound to GAG via --COO--(CH.sub.2).sub.m--NHCO--, the
crosslink can be formed by irradiating light, to the solution
containing them using an ultraviolet lamp.
9) Agent for Applying to Mucosa of the Present Invention
[0064] The agent for applying to mucosa of the present invention
contains one or more GAG into which the hydrophobic group is
introduced via the binding chain as the active ingredient, and may
also further include other medically, pharmaceutically or
biologically acceptable substances other than the GAG into which
the hydrophobic group is introduced via the binding chain. Such
substances include but are not limited to, salts such as sodium
chloride, potassium chloride, disodium hydrogen phosphate, sodium
dihydrogen phosphate and monopotassium hydrogen phosphate, and
preservatives such as paraoxybenzoate esters, benzalkonium
chloride; chlorobutanol and chlorhexidine glucoriate, and other
pharmacologically active ingredients.
[0065] The agent for applying to mucosa of the present invention
can be made into any publicly known formulation. forms (e.g., solid
preparations such as granules and powder, liquid preparations such
as aqueous solutions, suspension and emulsion, and gel
preparations) as the pharmaceutical for applying to the mucosa. In
the agent for applying to mucosa of the present invention, the form
thereof upon formulating and distributing and the form thereof upon
applying to the mucosa may be the same or different. For example,
the agent for applying to mucosa of the present invention may be
formulated in the form of solution and may be applied directly to
the mucosa as it is. Also, the agent for applying to mucosa of the
present invention may be formulated and distributed in the solid
form, and may be made into solution or gel when being applied to
the mucosa. Thus, the agent for applying to mucosa of the present
invention can be made into the formulation form for being prepared
when used.
[0066] When being made into the liquid agent by dissolving in
water, the amount of GAG into which the hydrophobic group is
introduced via the binding chain is preferably 0.02 to 5% by
weight, more preferably 0.1 to 3% by weight, extremely preferably
0.1 to 1% by weight and most preferably 0.1 to 0.6% by weight.
<Applied Subjects>
[0067] The agent for applying to mucosa of the present invention
aims at applying to the mucosa. Animals to which the agent for
applying to mucosa of the present invention is applied are not
particularly limited as long as they have the mucosa, and mammalian
animals are preferable. The mammalian animals include, but are not
limited to, humans, horses, cattle, dogs, cats, rabbits, hamsters,
guinea pigs and mice. The agent for applying to mucosa of the
present invention may be of course made into the pharmaceuticals
for humans, and can also be made into the pharmaceuticals for the
animals. Among them, it is preferable to be made into the
pharmaceuticals for humans.
[0068] The mucosa to which the agent for applying to mucosa of the
present invention can be applied is not particularly limited as
long as the mucosa is the mucosa present in the animal. Such mucosa
include mucosal tissues present in organs and tissues exemplified
by the gastrointestinal system such as the stomach and intestines,
the cardiovascular system, the respiratory system, the excretion
system such as the urinary bladder, rectum and anus, the genital
system such as the vagina, and organs such as eyes, nose and oral
cavity which contact with the external world. Among them, the agent
for applying to mucosa of the, present invention can be preferably
applied to the cornea, conjunctiva, oral cavity mucosa and urinary
bladder mucosa.
<Applied Diseases>
[0069] The agent for applying to Mucosa of the present invention
can be widely applied to such mucosa. The purpose of the
application is not particularly limited, and for example, the
purposes such as protection of the mucosal tissue (e.g., prevention
of snow blindness by ultraviolet rays, pterygium and cataract),
prevention of mucosal dryness and the treatment of mucosal disorder
Can be exemplified. Thus, the agent for applying to mucosa of the
present invention can be applied to not only the mucosa in the
abnormal state (e.g., mucosa where the disorder has occurred) but
also the mucosa in the normal state. However, since the agent for
applying to mucosa of the present invention exerts excellent
pharmacological effects in the mucosa where the disorder has
occurred, it is possible to preferably use for the treatment of the
mucosal disorders, e.g., disorders in the cornea, conjunctiva, oral
cavity mucosa and urinary bladder mucosa.
[0070] Since the agent for applying to mucosa, of the present
invention exerts excellent pharmacological effects particularly on
disorders in the mucosal epithelia among the mucosal disorders, it
is possible to be preferably used for the treatment of the
disorders in the mucosal epithelia.
[0071] Examples of such disorders in the mucosal epithelia include
corneal epithelial layer disorders such as corneal xerosis (dry
eye); keratoconjunctivitis, superficial punctate keratitis (SPK),
corneal epithelial erosion corneal epithelial loss and corneal
tumor; oral cavity` mucosal disorders such as xerostomia (dry
mouth), aphthous ulcer, stomatitis and glossitis; dryness and,
pruritus of nasal mucosa; urinary bladder mucosal disorders such as
interstitial cystitis; ulcerative proctitis, and dryness of the
rectum or vagina. Also dryness and lesions of organ mucosa upon
surgical operation can be exemplified. Among them, it is, possible
to be preferably used for the treatment of the corneal epithelial
layer disorders, the oral cavity mucosal epithelial disorders and
the urinary bladder mucosa epithelial layer disorders.
Application Method and Amount
[0072] The agent for applying to mucosa of the present invention
can be applied to the mucosal tissues exemplified above, and its
application method and application formulation can be appropriately
determined by those skilled in the art depending on the position,
morphology, property and function of the mucosa to be applied, and
the purpose of the application. However, it is preferable that the
agent for applying to mucosa of the present invention is applied to
the mucosa in the liquid form such as solution in use. In that
case, upon producing (formulating) or applying the agent for
applying to mucosa of the present invention, the liquid can be
obtained by dissolving GAG into which the hydrophobic group is
introduced via the binding chain in the solvent. The solvent is not
particularly limited as long as the solvent can dissolve the GAG
into which the hydrophobic group is introduced via the binding
chain and is the pharmaceutically acceptable solvent. For example,
a buffer such as a phosphate buffer or saline can be used, but the
solvent is not limited thereto. In this case, the concentration of
the GAG into which the hydrophobic group is introduced via the
binding chain in the liquid agent is not particularly limited, and
can be appropriately determined depending on the type of the mucosa
to be applied and the degree of the mucosal disorder. When the
agent for applying to mucosa of the present invention is the eye
drops, when the agent for applying to mucosa of the present
invention is applied to the oral cavity mucosa or the urinary
bladder mucosa, for example, the concentration is preferably 0.02
to 5% by weight, more preferably 0.1 to 3% by weight, still more
preferably 0.1 to 1% by, weight, still more preferably 0.1 to 0.6%
by weight, extremely preferably 0.1 to 0.5% by weight and most
preferably 0.1 to 0.3% by weight.
[0073] When the agent for applying to mucosa of the present
invention is applied to the mucosa in the stomach as the liquid as
above, an oral administration or the administration using a
catheter can be selected. When applied to the mucosa in the eye,
the nose or the oral cavity, for example, she administration method
such as instillation of drop, nasal instillation or oral inclusion
can be selected. For example, when the agent for applying to mucosa
of the present invention is applied to the mucosa mucosa in the
urinary bladder, rectum or vagina, or the mucosa of organs where
the dryness is concerned upon surgical operation, the method of
administering by injecting, spraying or applying the agent for
applying to mucosa of the present invention to a lumen or a surface
of these organs or tissues can be selected, but the methods are not
limited thereto.
[0074] The amount, the number of times and the frequency of the
application, (administration of) of the agent for applying to
mucosa of the present invention is not particularly limited, and
should be determined depending on the mucosa subjected, to the
application, the purpose of the application, the, type, age, body
weight, gender, and degree of mucosal disorder in the animal to be
applied.
[0075] Specifically, when the agent for applying to mucosa of the
present invention is used for the purpose of treating the human
corneal epithelial layer disorder, the agent for applying to mucosa
of the present invention at the above-described concentration as
the liquid formulation for the instillation of drops (eye drops)
containing GAG into which the hydrophobic group is introduced via
the binding chain can be administered by instilling 1 to 3 drops
per administration 1 to 5 times per day, and may be administered by
instilling 1 to 3 drops per administration 1 to 3 times per
day.
[0076] When the agent for applying to mucosa of the present
invention is used for the purpose of treating the human oral cavity
mucosal disorder, the agent for applying to mucosa of the present
invention at the above-described concentration as the liquid
containing GAG into which the hydrophobic group is introduced via
the binding chain can be administered by putting the agent for
applying to mucosa of the present invention in the oral cavity 1 to
5 times per day and rinsing for approximately several tens of
seconds (preferably approximately 20 to 30 seconds) followed by
spitting it out.
[0077] When the agent for applying to mucosa of the present
invention is applied to the urinary bladder mucosal disorder, this
is preferably used for the treatment of the urinary bladder mucosal
disorders exemplified by non-bacterial refractory cystitis
exemplified by interstitial cystitis, eosinophilic cystitis and
hemorrhagic cystitis which do not respond to anti-bacterial agents
although symptoms similar to those of acute bacterial cystitis are
exhibited. In this case, the agent for applying to mucosa of the
present invention at the above-described concentration as the
liquid containing GAG into which the hydrophobic group is
introduced via the binding chain can be administered by
administering the agent for applying to mucosa of the present
invention directly to the urinary bladder at the amount of 50 mL
per administration 1 to 7 times per week or administering with a
catheter in the urinary bladder.
[0078] The agent for applying to mucosa of the present invention
can stay at the diseased site for a longer period of time because
the active ingredient contained in the agent exhibits the high
staying property in the mucosa, compared with the conventional
drugs containing hyaluronic acid as the active ingredient in which
no hydrophobic group has been bound. Therefore, the agent for
applying to mucosa of the present invention can also exert the
treating effect persistently even at the low administration
frequency on the disorders such as inflammation and lesions in the
mucosa. However, the agent for applying to mucosa of the present
invention is not limited by its administration frequency.
[0079] The present invention will be described below by
Examples.
Example 1
(1-1) Preparation of Cinnamate Derivative-Introduced Sodium
Hyaluronate
[0080] A 172 mg/5 mL aqueous solution of N-hydroxysuccinimide
(HOSu: Watanabe Chemical Industries, Ltd.), a 143 mg/5 mL aqueous
solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCI.HCl) (Watanabe Chemical Industries, Ltd.), and
a 181 mg/5 mL aqueous solution of 3-aminopropyl cinnatate
hydrochloride (Tokyo Chemical Industry Co., Ltd.) were added to a
solution of sodium hyaluronate (1.06 g, 2.7 mmol/disaccharide unit,
weight-average molecular weight 900,000; derived from cock's comb,
Seikagaku Corporation) in water (115 mL)/dioxane (144 mL). The
mixture was stirred for 3 hours and a 750 mg/10 mL aqueous solution
of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added.
After stirring further 2 h 30 min., the reaction was quenched with
acetic acid (214 mg) and sodium chloride (1.0 g). Ethanol (300 mL)
was added and the resulting precipitation was filtered off and
washed twice successively by 80% ethanol, 95% ethanol. The solid
was dried in vacuo at 40.degree. C. over night to afford white
solid (1.06 g)(hereinafter in Examples, "cinnamate
derivative-introduced sodium hyaluronate" is abbreviated as
"cinnamate derivative-introduced HA"). The introduction ratio of
the cinnamate derivative was 16%. The introduction ratio of the
cinnamate derivative was calculated based on the amount of
cinnamate by an absorbance measurement method (wavelength: 269 nm)
and the amount of hyaluronate by a carbazole sulfate method.
(1-2) Preparation of Cinnamate Derivative-Introduced HA
Solution
[0081] Saline was added to 86 mg of cinnamate derivative-introduced
HA obtained in the above (1-1) to give a total amount of 15.45 ml,
then the solution was shaken over night with a shaker until
uniformly dissolved. The 0.5% by weight solution of cinnamate
derivative-introduced HA (in drying loss 10%) was obtained.
Likewise, the 0.3% by weight and 0.1% by weight solutions of
cinnamate derivative-introduced HA were obtained.
Example 2
(2-1) Preparation of Cinnamate Derivative-Introduced HA
[0082] A 75 mg/5 mL aqueous solution. of HOSu, a 62 mg/5 mL WFI
solution of EDCI.HCl, and a 92 mg/5 mL WFI solution of 6-aminohexyl
cinnamate hydrochloride (Tokyo Chemical Industry Co., Ltd.) were
added to a solution of sodium hyaluronate (1.0 g, 2.5
mmol/disaccharide unit, weight-average molecular weight 1,500,000;
derived from cock's comb, Seikagaku Corporation) in water for
injection (hereinafter referred to as WFI) (150 mL)/dioxane (75
mL). The mixture was stirred for 4 hours and sodium chloride (1.0
g) was added. Ethanol (500 mL) was added and the resulting
precipitation was filtered off and washed twice successively by 80%
ethanol, ethanol. The solid was dried in vacuo at 40.degree. C. to
afford white solid (1.1 g). The introduction ratio of the cinnamate
derivative was 2.7%.
(2-2). Preparation of Crosslinked Cinnamate Derivative-Introduced
HA
[0083] The above cinnamate derivative-introduced HA (12.5 g) was
dissolved in phosphate buffered saline (concentration of phosphate:
1.5 mM, hereinafter abbreviated as "PBS") to prepare 2.5% solution
of cinnamate derivative-introduced HA (500 mL). The 2.5% solution
of cinnamate derivative-introduced HA was irradiated by 800W high
pressure mercury lamp and performed by a heat treatment in an
autoclave at 121.degree. C. for 7.5 min. to yield crosslinked
cinnamate derivative-introduced HA.
[0084] Further, 1 g of the above crosslinked cinnamate
derivative-introduced HA was dissolved in 11.5 ml of WFI to prepare
the 0.2% by weight of crosslinked, cinnamate derivative-introduced
HA.
Example 3
(3-1) Preparation of Cinnamate Derivative-Introduced HA
[0085] A 172 mg/5 mL aqueous solution of HOSu, a 143 mg/5 mL
aqueous solution of EDCI.HCl, and a 181 mg/5 mL aqueous solution of
3-aminopropyl cinnamate hydrochloride (Tokyo Chemical Industry Co.,
Ltd.) were added to a solution of sodium hyaluronate (1.0 g; 2.5
mmol/disaccharide unit, weight-average molecular weight 900,000;
derived from cock's comb, Seikagaku Corporation) in water (150
mL)/dioxane (75 mL). The mixture was stirred for 3 hours and a 750
mg/10 mL aqueous solution of sodium hydrogen carbonate (Japanese
Pharmacopoeia) was' added. After stirring further 2 h 30 min., the
reaction was quenched with acetic acid (214 mg) and sodium chloride
(1.0 g). Ethanol (300 mL) was added and the resulting precipitation
was filtered off and washed twice successively by 80% ethanol, 95%
ethanol. The solid was dried in vacuo at 40.degree. C. to afford
white solid (1.0 g) as cinnamate derivative-introduced HA. The
introduction ratio of the cinnamate derivative was 10.1%.
(3-2). Preparation of Fluorochrome-Labeled Cinnamate
Derivative-Introduced HA
[0086] A 3.0 mmol/mL aqueous solution of HOSu, a 1.5 mmol/mL
aqueous solution of EDCI.HCl and a 1.5 mmol/mL aqueous solution of
4-aminofluorescein (Tokyo Chemical Industry Co., Ltd.) were added
to a solution of cinnamate derivative-introduced HA obtained in the
above (3-1)(1.00 g, 2.5 mmol/disaccharide unit) in water (150
mL)/dioxane (75 mL). The mixture was stirred one day and a 500
mg/10 mL aqueous solution of sodium hydrogen carbonate (Japanese
Pharmacopoeia) was added. After stirring further 4 h 30 min., the
reaction was quenched with acetic acid (2 mL) and sodium chloride
(6.0 g). Ethanol (500 mL) was added and the resulting precipitation
was filtered off and washed four times by 80% ethanol, twice by
ethanol. The solid was dried in vacuo over night to afford
fluorochrome-labeled solid (782 mg) The introduction ratio of the
fluorescence was 0.60%.
Comparative Example 1
[0087] Preparation of fluorochrome-labeled HA A 3.0 mmol/mL aqueous
solution of HOSu, a 1.5 mmol/mL aqueous solution of EDCI.HCl and a
1.5 mmol/mL, aqueous solution of 4-aminofluorescein (Tokyo Chemical
Industry Co., Ltd.) were added to a solution of sodium hyaluronate
(1.00 g, 2.5 mmol/disaccharide weight-average molecular weight
900,000; derived from cock's comb, Seikagaku Corporation) in water
(150 mL)/dioxane (75 mL). The mixture was stirred one day and a 500
mg/10 mL aqueous solution of sodium hydrogen carbonate (Japanese
Pharmacopoeia) was added. After stirring further 4 h 30 min., the
reaction was quenched with acetic acid (2 mL) and sodium chloride
(6.0 g). Ethanol (500 mL) was added and the resulting precipitation
was filtered off and washed four times by 80% ethanol, twice by
ethanol. The solid was dried in vacuo, over night to afford
fluorochrome-labeled solid (830 mg) The introduction ratio of the
fluorescence was 0.32%.
Example 4
Measurement of Ultraviolet Ray Transmittance of Cinnamate
Derivative-Introduced HA Solution
[0088] The 0.1% by weight aqueous solution of cinnamate
derivative-introduced HA obtained in the above (1-1) was prepared,
and the ultraviolet ray transmittance was measured by an
spectrometer (UV-1600, Shimadzu Corporation).
[0089] A spectrum which indicates the transmittance is shown in
FIG. 1, and the transmittance (%) at various wavelengths is shown
in Table 1. As a result, 100% of the transmittance was shown at the
wavelengths of 340 nm or more, but the transmittance at the
wavelengths of approximately 320 nm or less was 20% or less which
was extremely low, and it was demonstrated that this solution
effectively blocks the transmission of the ultraviolet ray.
[0090] In FIG. 1, scales are shown with 65 nm intervals on a
horizontal axis and with 20% intervals on a vertical axis.
TABLE-US-00001 TABLE 1 .lamda. T (%) 234 19.9 340 106.2 380 101.8
450 98.5
[0091] In the table, .lamda. and T represent the wavelength and the
transmittance (%), respectively.
Example 5
The Effect of the Cinnamate Derivative-Introduced HA on the Healing
of, Rabbit Corneal Epithelium.
[0092] The effect of the cinnamate derivative-introduced HA
prepared in Example 1 on the healing of rabbit corneal epithelium
with surgical removal (The surgical Model).
(5-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0093] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0094] One hour and 4 hours after the corneal epithelia were
peeled, 150 ul of saline as the control substance was administered
in the left eye, and 150 ul of 0.5% by weight cinnamate
derivative-introduced HA solution prepared in the above Example
(1-2) as the subject substance was administered in the right eye.
On one day and 2 days after the peeling, a total of 4 times with 3
hour intervals, and at 3 days after the peeling, with 3 hours
interval, the same administration as above was performed. In the
administration, 1 ml injection syringes were used. Six model
rabbits for the corneal epithelial layer disorder described in the
above 1) were used as administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0095] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 3 hours after the final administration at 3 days
after the peeling. When photographed, a focal length was made
constant to make a magnification of photographs constant.
4) Measurement of Corneal Epithelial Defective Region
[0096] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using an image analyzer. A value obtained by subtracting the area
of the peeled site 3 hours after the final administration at 3 days
after the peeling from the area (peeled area) of the peeled site
just before the administration of the subject substance one hour
after peeling the corneal epithelia was performed was rendered as
"healed area."
(5-2) Study Results
[0097] The results of a healed area percentage in each individual
are shown in FIG. 2, and the results of the healed area percentage
and a healed area percentage ratio in each individual are shown in
Table 2. The healed area percentage and the healed area percentage
ratio were calculated as follows.
Healed area percentage (%)=(Healed area/Peeled area).times.100
Healed area percentage ratio=(Healed area percentage in right
eye/Healed area percentage in left eye).times.100
TABLE-US-00002 TABLE 2 Healed area percentage (%) right eye
(administration of cinnamate left eye Healed area Specimen
derivative- (administration percentage Number introduced HA) of
saline) ratio 1 76.90 66.59 115.47 2 69.44 85.90 80.85 3 75.23
61.71 121.91 4 75.54 56.79 133.01 5 68.41 64.53 106.01 6 83.76
66.92 125.16 Mean 74.88 67.07 113.73 Standard 5.57 9.95 --
deviation
[0098] For each individual of individual numbers 1 to 6, the left
column shows the healed area percentage in the right eye
(administration of cinnamate derivative-introduced HA), and the
right column shows the healed area percentage in the left eye
(administration of saline). In FIG. 2 and Table 2, the apparent
effect to facilitate the healing of the corneal epithelial layer
disorder was observed in 5 of the administered 6 individuals.
Example 6
The Effect of the 0.5% Cinnamate Derivative-Introduced HA on the
Healing of Rabbit Corneal Epithelium.
[0099] The effect of the cinnamate derivative-introduced HA
prepared in Example 1 on the healing of rabbit corneal epithelium
with surgical removal (The surgical Model).
(6-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0100] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketatine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0101] One hour and 4 hours after the corneal epithelia were
peeled, 150 ul of saline as the control substance was administered
in the left eye, and 150 ul of 0.5% by weight cinnamate
derivative-introduced HA solution prepared in the above Example
(1-2) as the subject substance was administered in the right eye.
On one day and 2 days after the peeling a total of 4 times with 3
hour intervals, and at 3 days after the peeling, with 3 hour
intervals, the same administration as above was performed. In the
administration, the 1 ml injection syringes were used. Fourteen
model rabbits for the corneal epithelial layer disorder described
in the above 1) were used as the administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0102] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 3 hours after the second administration at 1 to 3
days after the peeling. When, photographed, the focal length was
made constant to make the magnification of photographs
constant.
4) Measurement of Corneal Epithelial Defective Region
[0103] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value obtained by subtracting the
area of the peeled site 3 hours after the final administration at 3
days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as
"healed area."
(6-2) Study Results
[0104] The results of the healed area in each individual are shown
in FIG. 3, and the results of a healing rate in each individual are
shown in FIG. 4. The healed area and the 20 healing rate were
calculated as follows:
Healed area=Area after peeling*-Area at each time point (after 1 to
3 days)
[0105] * Herein after, "area after peeling" means `just before the
administration of the subject substance one hour after the cornea
was peeled` in calculation of healed area.
[0106] Healing rate=Mean of healed areas at respective time points
(after 1 to 3 days)
[0107] In FIGS. 3 and 4, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.5% by weight cinnamate derivative-introduced HA
solution, compared with the healed area of the corneal epithelia in
the control eyes. And it was also observed that the healing rate
was significantly enhanced in the eyes administered with 0.5% by
weight cinnamate derivative-introduced HA solution.
Example 7
The Effect of the 0.3% Cinnamate Derivative-Introduced HA on the
Healing of Rabbit Corneal Epithelium.
[0108] The effect of the cinnamate derivative-introduced HA
prepared in Example 1 on the Migration of rabbit corneal epithelium
with surgical removal (The surgical Model).
(7-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical 10
Model)
[0109] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylaiine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0110] One hour and 4 hours after peeling the corneal epithelia,
150 ul of saline as the control substance was administered in the
left eye, and 150 4l of 0.3% by weight cinnamate
derivative-introduced HA solution prepared in the above Example
(1-2) as the subject substance was administered in the right eye.
At one day and 2 days after the peeling, a total of 4 times with 3
hour intervals, and at 3 days after the peeling, with 3 hour
intervals, the same administration as above was performed. In the
administration, the 1 ml injection syringes were used. Fourteen
model rabbits for the corneal epithelial layer disorder described
in the above 1) were used as the administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0111] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 3 hours after the second administration at 1 to 3
days after the peeling. When photographed, the focal 5 length was
made constant to make the magnification of photographs
constant.
4) Measurement of Corneal Epithelial Defective Region
[0112] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value obtained by subtracting the
area of the peeled site 3 hours after the final administration at 3
days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as
"healed area."
(7-2) Study Results
[0113] The results of the healed area in each individual are shown
in FIG. 5, and the results, of a healing rate in each individual
are shown in FIG. 6. The healed area and the healing rate were
calculated as follows.
Healed area=Area after peeling-Area at each time point (after 1 to
3 days)
Healing rate=Mean of healed areas at respective time points (after
1 to 3 days)
[0114] In FIGS. 5 and 6, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.3% by weight cinnamate derivative-introduced HA
solution at all time points of days 1 to 3, compared with the
healed area of the corneal epithelia in the control eyes. And it
was also observed that the healing rate was significantly enhanced
in the eyes administered with 0.3% by weight cinnamate
derivative-introduced HA solution.
Example 8
[0115] The Effect of the 0.1% Cinnamate Derivative-Introduced HA on
the Healing of Rabbit Corneal Epithelium (0.1% by Weight Cinnamate
Derivative-Introduced HA Aqueous Solution and 0.1% by Weight HA
Aqueous Solution, 4 Times of Eye Drops per day)
[0116] The effect of the cinnamate derivative-introduced HA
prepared in Example 1 on the healing of rabbit corneal epithelium
with surgical removal (The surgical Model).
(8-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0117] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0118] One hour and 4 hours after peeling the corneal. epithelia,
150 ul of an aqueous solution of 0.1% by. weight HA with a weight
average molecular weight 600,000 to 1,200,000 as the control
substance was administered in the left eye, and 150 ul of 0.1% by
weight cinnamate derivative-introduced HA solution prepared in the
above Example (1-2) as the subject substance was administered in
the right eye. At one day and 2 days after the peeling, a total of
4 times with 3 hour intervals, and at 3 days after the peeling,
with 3 hour intervals, the same administration as above was
performed. In the administration, the 1 ml injection syringes were
used. Eight model rabbits for the corneal epithelial layer disorder
described in the above 1) were used as the administration
subjects.
3) Photographing of Corneal Epithelial Defective Region
[0119] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea,
was peeled and 3 hours after the second administration at 1 to 3
days after the peeling. When photographed, the focal length was
made constant to make the magnification of photographs
constant.
4) Measurement of Corneal Epithelial Defective Region
[0120] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value 15 obtained by subtracting the
area of the peeled site, 3 hours after the final administration at
3 days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as
"healed area."
(8-2) Study Results
[0121] The results of the healed area in each individual are shown
in FIG. 7, and the results of the healed area and the healing rate
in each individual are shown in FIG. 8. The healed area and the
healing rate were calculated as follows.
Healed area=Area after peeling-Area at each time point (after 1 to
3 days)
Healing rate=Mean of healed areas at respective time points (after
1 to 3 days)
[0122] In FIGS. 7 and 8, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.1% by weight cinnamate derivative-introduced HA
solution, compared with the healed area of the corneal epithelia in
the control eyes administered with the 0.1% by weight HA aqueous
solution. And it was also observed that the healing rated was
significantly enhanced in the eyes administered with 0.1% by weight
cinnamate derivative-introduced HA solution.
Example 9
The Effect of the 0.1% Cinnamate Derivative-Introduced HA on the
Migration of Rabbit Corneal Epithelium (0.1% by Weight Cinnamate
Derivative-Introduce a HA Aqueous Solution and 0.1% by Weight HA
Aqueous Solution, One Time of Eye Drops Per Day).
[0123] The effect of the cinnamate derivative-introduced HA
prepared in Example 1 on the Migration of rabbit corneal epithelium
with surgical removal (The surgical Model).
(9-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0124] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0125] One hour after the corneal epithelia were peeled, 150 ul of
the aqueous solution of 0.1% by weight HA with a weight average
molecular weight 600,000 to 1,200,000 as the control substance was
administered in the left eye, and 150 ul of 0.1% by weight
cinnamate derivative-introduced HA solution prepared in the above
Example (1-2) as the subject substance was administered in the
right eye. Furthermore, at one to 3 days after the peeling, once a
day, the same administration as above was performed. In the
administration, the 1 ml injection syringes were used. Eight model
rabbits for the corneal epithelial layer disorder described in the
above 1) were used as the administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0126] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 6 hours after the administration at 1 to 3 days
after the peeling. When photographed, the focal length was made
constant to make the magnification of photographs constant.
4) Measurement of Corneal Epithelial Defective Region
[0127] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value obtained by subtracting the
area of the peeled site 3 hours after the final administration at 3
days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as the
"healed area."
9-2) Results
[0128] The results of the healed area in each individual are shown
in FIG. 9, and the results of the healing rate in each individual
are shown in FIG. 10. The healed area and the healing rate were
calculated as follows.
Healed area=Area after peeling-Area at each time point (after 1 to
3 days)
Healing rate=Mean of healed areas at respective time points (after
1 to 3 days)
[0129] In FIGS. 9 and 10, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.1% by weight cinnamate derivative-introduced HA
solution at all time points of the days 1 to 3, compared with the
healed area of the corneal epithelia in the control eyes
administered with the 0.1% by weight HA aqueous solution. And it
was also observed that the healing rate was significantly enhanced
in the eyes administered with 0.1% by weight cinnamate derivative
introduced HA solution.
Example 10
Staying Property at Rabbit Corneal Epithelial Peeling Site Using
Fluorescence Labeled Cinnamate Derivative-Introduced HA
[0130] The effect of the fluorescence labeled cinnamate
derivative-introduced HA prepared in Example and the fluorescence
labeled HA prepared in Comparative Example 1 on the residual
property of rabbit corneal epithelium with surgical removal (The
surgical Model).
(101-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0131] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0132] One hour after the corneal epithelia were peeled, 150 ul of
the aqueous solution of 0.3% by weight fluorescence labeled HA
prepared in the above Comparative Example 1 as the control
substance was administered in the left eye, and 150 ul of the
aqueous solution of 0.3% by weight fluorescence labeled cinnamate
derivative-introduced HA prepared in the above Example 3 as the
subject substance was administered in the right eye. In the
administration, the 1 ml injection syringes were used. Eight model
rabbits for the corneal epithelial layer disorder described in the
above 1) were used as the administration subjects.
3) Removal of Corneal Epithelia and Production of Frozen Blocks
[0133] Two rabbits were given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine per rabbit,
and eyeballs were removed 30 minutes, one hour, one and a half
hours and 2 hours and 30 minutes after the administration of the
subject substance and the control substance. A pore was opened
between the cornea and sclera in the removed eyeball using a
surgical knife, and only the cornea was taken out using the
microscissors. The removed cornea was placed on a biological sample
slicing plate (supplied from Nisshin EM Corporation, Cat No. 428),
and the portion to be observed was cut out using a single-edged
razor blade stainless steel (GEM(R) STAINLESS STEEL UNCOATED,
Nisshin EM Corporation, Cat No. 429). The cut out portion was
immersed in O.C.T. compound (Tissue-Te) (R)4583, Lot. 1178), then
embedded in a cryostat tray (supplied by Murazumi Co., Ltd., Cat.
No. 31) filled with the O.C.T. compound so that the portion to be
observed was at a bottom, and rapidly frozen using liquid nitrogen
in foam polystyrene to make an unfixed frozen block.
4) Production of Frozen Sections
[0134] Subsequently, the frozen block was removed from the cryostat
tray, and attached on a sample table using the O.C.T. compound. The
sample table and a disposable microtome blade (supplied by Leica
Microsystems Japan, Model 818, Lot. No. 913212) were set in a high
performance frozen microtome for research, and the block was sliced
under the condition of a frozen chamber temperature (CT) at
-20.degree. C. and sample side temperature (OT) at -16.degree. C.
to make sections with a thickness of 5 uM using silane coating
slide glasses (supplied by Muto Pure Chemicals Co., Ltd., Star
Frost Slide Glass, Cat. No. 5116).
5) Methods of Observation and Photographing
[0135] The frozen section was set in an incident-light fluorescence
microscope (Olympus Corporation, BH2-RFC), FA images and
autofluorescent images were observed at IB cube BH2-DMIB,
excitation wavelength: 495 nm, absorption wavelength: 460 nm) and U
cube (BH2-DMU, broad band U excitation, absorption wavelength: 435
nm), respectively. The FA image and the autofluorescent image were
photographed using a cooled high sensitivity CCD camera (Keyence
Corporation, VB-6010) under the condition of exposure time for one
second and ISO sensitivity of 200.
(10-2) Study Results
[0136] Photographs of the sampled cornea were shown in FIGS. 11 and
12. From FIGS. 11 and 12, it was identified by color development of
the fluorescence label that the aqueous solution of 0.3% by weight
fluorescence labeled HA which was the control substance stayed
until 30 minutes after. the administration but did not stay after
one hour. Meanwhile, although the fluorescent color development of
the aqueous solution of 0.3% by weight fluorescence labeled
cinnamate derivative-introduced HA which was the subject substance
was weakened with the elapse of time, the color development was
observed at all time points from 30 minutes to 2 hours and 30
minutes after the administration, thereby the high staying
performance was confirmed.
Example 11
[0137] Effect of the 0.3% Cinnamate Derivative-Introduced HA on
Rabbit Eyes after Exposure to Ultraviolet Light
[0138] The protective effect on the cinnamate derivative introduced
HA prepared in Example 1 on the rabbit with corneal superficial
disorder.
(11-1) Study Procedure
[0139] 1) Anesthesia and Eyelid Opening in Rabbit
[0140] Introduced anesthesia by intravenously injecting 5 mg/kg of
ketimine and 2 mg/kg of xylazine and maintained anesthesia by
inhalation of isoflurane were given to the rabbit. Subsequently,
the eyelid was always opened using an eyelid retractor for
kids.
[0141] 2) Administration of Subject Substance and Control
Substance
[0142] In the condition where the eye was opened, 150 ul of an
aqueous solution of 0.3% by weight HA with a weight average
molecular weight 600,000 to 1,200,000 as the control substance was
administered in the right eye, and 150 1.11 of 0.3% by weight
cinnamate derivative-introduced HA prepared in the above Example
(1-2) as the subject substance was administered in the left eye. In
the administration, the 1 Ml injection syringes were used. One
rabbit described in the above 1) was used as the administration
subject.
[0143] 3) Irradiation of Ultraviolet Rays to Rabbit's Cornea
[0144] Ultraviolet rays were irradiated to both eyes from a
distance of approximately 10 cm apart from the rabbit eyeball using
a 15 kW germicidal lamp. The irradiation was performed for 3
hours.
[0145] 4) Photographing of ultraviolet rays irradiated site
[0146] The eyeball was stained with 0.2% sodium fluorescein under
the continuous anesthesia of the rabbit, and photographed under
violet light. When photographed, the focal length was made constant
to make the magnification of photographs constant.
(11-2) Study Results
[0147] Photographs after the irradiation of the ultraviolet rays
were shown in FIG. 13. From FIG. 13, in the eyeball irradiated with
the ultraviolet rays after the administration of the control
substance, the disordered site stained with 0.2% sodium fluorescein
was apparent.
[0148] Meanwhile, in the eyeball irradiated with the ultraviolet
rays after the administration of the subject substance, the
disordered site stained with 0.2% sodium fluorescein clearly
smaller than that of the control substance, and the corneal
disorder caused by the ultraviolet ray was prevented.
Example 12
Moisturizing Effect Using Removed Cornea
[0149] Using the removed cornea of the rabbit, the moisturizing
performance of the cinnamate derivative-introduced HA prepared in
Example 1 was validated.
(12-1) Methods
1) Removal of Cornea
[0150] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, and the
eyeball was removed. A pore was opened between the cornea and the
sclera in the removed eyeball using a surgical knife, and only the
cornea was taken out using the microscissors.
2) Drying Treatment of Cornea
[0151] The drying treatment was performed by placing the removed
cornea on a paraffin block with a False-tooth stability material
and providing cold air from the distance of approximately 1 m from
the cornea using a dryer for 5 minutes.
3) Administration of Test Substances (Subject Substance, Control
Substance and Negative Control Substance)
[0152] After the completion of the drying treatment, 100 ul of
saline as the negative control substance, the aqueous solution of
0.3% by weight HA with a weight average molecular weight of 600,000
to 1,200,000 as the control substance or the aqueous solution of
0.5% by weight cinnamate derivative-introduced HA prepared in the
above Example (1-2) as the subject substance was administered to
two corneas. In the administration, the 1 ml injection syringes
were used. Three rabbits (6 corneas) described in the above 1) were
used as the administration subjects.
4) Measurement of Water Evaporation Amount
[0153] The water evaporation amount was measured using a water
evaporation amount measurement apparatus (AS-TW2, ASAHIBIOMED)
before the administration of the test substance, after the drying
treatment, after the administration of the test substance and until
40 minutes with 10 minute intervals after the administration of the
test substance.
(12-2) Study Results
[0154] The results of measuring the water evaporation amount were
shown in FIG. 14. From FIG. 14, the water evaporation amount was
slightly higher in the HA aqueous solution which was the control
substance than in the saline which was the negative control whereas
the saline became the value close to 0 after 40 minutes. On the
other hand, the water evaporation amount after the administration
of the subject substance kept the high value even when 40 minutes
passed over, thereby the clear moisturizing performance of the
subject substance was confirmed.
Example 13
(13-1) Methods
1) Removal of Corneal Epithelia
[0155] The rabbit was euthanized, and after removing the eyeball,
an entire corneal layer was removed by incising along the sclera.
The removed cornea was preserved in saline, and the corneal
epithelia was fixed by placing it on the paraffin block and the
False-tooth stability material just before the measurement
(hereinafter described as "the Cornea to be measured").
2) Water Evaporation in Corneal Epithelia
[0156] The cornea to be measured was given cold air by the dryer
from the distance of 30 cm for 5 minutes, and left standing at a
room temperature for one hour.
3) Administration of Subject Substance
[0157] After the water is evaporated, two drops (approximately 100
ul) of saline as the negative control substance, the aqueous
solution of 0.3% by weight HA with a weight average molecular
weight of 600,000 to 1,200,000 as the control substance or the
aqueous solution of 0.5% by weight cinnamate derivative-introduced
HA prepared in the above Example (1-2) as the subject substance was
administered by the 1 ml syringe.
4) Measurement of Water Evaporation Amounts
[0158] The amount perceived as an unperceived evaporation amount
(released water amount per m.sub.2 per hour) was directly measured
as the water evaporation amount from the cornea to be measured
using the water evaporation amount measurement apparatus
(AS-TW2).
(13-2) Study Results
[0159] The results of measuring the water evaporation amount were
shown in FIG. 15. From FIG. 15, the water evaporation amount was
slightly higher in the HA aqueous solution which was the control
substance than in the saline which was the negative control whereas
the saline exhibited the value close to 0 after 40 minutes. On the
other hand, the water evaporation amount of the administered
subject substance kept the high value even when 40 minutes had
passed over, thereby it was confirmed that the subject substance
has a more persistent water retention property on the cornea
compared with saline and the HA aqueous solution.
Example 14
Validation of Healing Effect of Crosslinked Cinnamate
Derivative-Introduced HA
[0160] Using a model hamster for xerostomia, the healing effect of
the crosslinked cinnamate derivative-introduced HA prepared in
Example 2 on the xerostomia was validated.
(14-1) Test Method
1) Production of Model Hamster for Xerostomia
[0161] An inside of the oral cavity of a male Syrian hamster was
exposed by inserting a test tube with a diameter of approximately
10 mm in the oral cavity close to a buccal side and reversing it
under the anesthesia with Nembutal. By giving hot air for
approximately 20 seconds, and subsequently giving cold air for 2
minutes and 40 seconds to the exposed, inside of the oral cavity
using a dryer, the model hamster for the xerostomia was obtained.
The inside of the oral cavity was continuously exposed until the
measurement was completed.
2) Administration of Subject Substance and Control Substance
[0162] Immediately after making the xerostomia model, 100 ul of (A)
PBS, (B) 0.2% by weight HA solution (supplied by Seikagaku
Corporation, weight average molecular weight: 1,500,000) or (C)
0.2% by weight crosslinked cinnamate derivative-introduced HA
solution was administered by applying to the inside of the oral
cavity using a microsyringe.
[0163] Hereinafter, the group administered with (A), the 25 group
administered with (B) and the group administered with (C) are
referred to as PBS group, HA group and crosslinked cinnamate
derivative-introduced HA group, respectively. For the
administration classification (administration group composition),
seven hamsters were used for each of the PBS group, the HA group
and the crosslinked cinnamate derivative-introduced HA group.
3) Calculation of Water Evaporation Amount Ratio
[0164] The water evaporation amount in the inside of the oral
cavity in the xerostomia model hamster was measured using the water
evaporation system (Asahibiomed), and the water evaporation amount
ratio was calculated when the measurement value immediately after
making the xerostomia model hamster was 1. The higher this water
evaporation amount ratio is, the more moisturized condition is
maintained (degree of oral cavity dryness is low). The measurement
was performed immediately after, making the erostomia model
hamster, immediately after the administration, 10 minutes and 20
minutes after the administration.
(14-2) Study Results
[0165] The results of measuring the water evaporation amount are
shown in FIG. 16. In the figure, circled numbers 1, 2, 3 and 4 on
the horizontal axis represent data immediately after making the
xerostomia model hamster, immediately after the administration, 10
minutes and 20 minutes after the administration, respectively. P
represents a significance level. Immediately after the
administration, all of administration groups exhibited the water
evaporation amount ratio of 3.3 to 4.5. The water evaporation
amount ratio 10 minutes after the administration was 0.9 on average
in the PBS group and 2.3 on average in the HA group. On the other
hand, it was 3.2 on average in the crosslinked cinnamate
derivative-introduced HA group, thus the higher water evaporation
amount ratio than the PBS group and the HA group was shown.
Further, the water evaporation amount ratio 20 minutes after the
administration was 0.9 on average in the PBS group and 1.3 on
average in the HA group. On the other hand, it was 3.2 on average
in the crosslinked cinnamate derivative-introduced HA group, thus
the extremely higher water evaporation amount ratio compared with
the PBS group and the HA group was indicated. Furthermore, as is
apparent from FIG. 16, the water evaporation amount ratio in the
crosslinked cinnamate derivative-introduced HA group was very
stable regardless of the elapse of time. This indicated that the
crosslinked cinnamate derivative-introduced HA stayed at-the
administered site for a long period of time and exerted a highly
persistent effect.
[0166] From the above results, GAG into which the hydrophobic group
is introduced via the binding chain including the cinnamate
derivative-introduced HA and the crosslinked cinnamate
derivative-introduced HA has been shown to be suitable for the
application to the mucosa and capable of effectively treating the
disorder in the mucosal epithelial layer by being applied to the
mucosa. It has been also shown that the effect of the treatment is
highly persistent.
[0167] Since no adverse effect due to the administration of the
cinnamate derivative-introduced HA and the crosslinked cinnamate
derivative-introduced HA was observed in any of the above animal
studies, the safety of the agent for applying to mucosa of the
present invention can be sufficiently estimated.
Example 15
(15-1) Preparation of Octylamine-Introduced Sodium Hyaluronate
[0168] A 25.8 mg/2 mL solution (ethano1:0.1MHC1=I:1) of octylamine,
2 mL of 0.1 M solution (ethanol:water=1:1) of DMT-MM (Wako Pure
Chemical Industries, Ltd.) were added to a solution of sodium
hyaluronate (502 mg, 1.25 mmol/disaccharide unit, weight-average
molecular weight 900,000) in water (50 mL)/ethanol (50 mL). The
mixture was stirred over night and a 376 mg/5 mL aqueous solution
of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added.
After stirring further 5 hours, the reaction was quenched with
acetic acid (107 mg) and sodium chloride (522 mg). Ethanol (250 mL)
was added and the resulting precipitation was filtered off and
washed twice successively by 80% ethanol, ethanol. The solid was
dried in vacuo to afford white solid (475 mg). The introduction
ratio of octylamine was 12.6% by HPLC.
(15-2) Preparation of Hexadecylamine-Introduced Sodium
Hyaluronate
[0169] A 30 mg/3 mL solution (ethano1:0.1MHC1=1:1) of octylamine,
1.25 mL of 0.1 M solution (ethanol:water=l:1) of DMT-MM (Wako Pure
Chemical Industries, Ltd.) were added to a solution of sodium
hyaluronate (501 mg, 1.25 mmol/disaccharide unit, weight-average
molecular weight 900,000) in water (50 mL)/ethanol (50 mL). The
mixture was stirred over night and a 381.mg/5 mL aqueous solution
of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added.
After stirring further 5 hours, the reaction was quenched with
acetic acid (107 mg) and sodium chloride (497 mg): Ethanol (250 ML)
was added and the resulting precipitation was filtered off and
washed twice successively by 80% ethanol, ethanol. The solid was
dried in vacuo to afford white solid (497 mg). The introduction
ratio of hexadecylamine was 12% by HPLC.
(15-3) Preparation of Sample Solution
[0170] 64 mg of compound obtained in the above (15-1) was added to
5 mM phosphate buffer saline to give a total amount of 59 ml, then
the solution was shaken over night with a shaker. The 0.1% by
weight solution of compound prepared in the above (15-1) was
obtained.
[0171] Likewise, 0.1% by weight solution of compound prepared in
the above (15-2) was obtained.
Example 16
The Effect of the 0.1% Cinnamate Derivative-Introduced HA on the
Healing of Rabbit Corneal Epithelium.
[0172] The effect of the cinnamate derivative-introduced. HA
prepared in Example 1 on the healing of rabbit corneal epithelium
with surgical removal (The surgical Model).
(16-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0173] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0174] One hour and 4 hours after peeling the corneal epithelia,
150 ul of saline as the control substance was administered in the
left eye, and 150 ul of 0.1% by weight cinnamate derivative
introduced HA solution prepared in the above Example (1-2) as the
subject substance was administered in the right eye. At one day and
2 days after the peeling, a total of 4 times with 3 hour intervals,
and at 3 days after the peeling, with 3 hour intervals, the same
administration as above was performed. In the administration, the 1
ml injection syringes were used. Fourteen model rabbits for the
corneal epithelial layer disorder described in the above 1) were
used as the administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0175] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 3 hours after the second administration at 1 to 3
days after the peeling. When photographed, the focal length was
made constant to make the magnification of photographs
constant.
4) Measurement of Corneal Epithelial Defective Region
[0176] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value obtained by subtracting the
area of the peeled site 3 hours after the final administration at 3
days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as
"healed area."
(16-2) Study Results
[0177] The results of the healed area in each individual are shown
in FIG. 17, and the results of a healing rate in each individual
are shown in FIG. 18. The healed area and the healing rate were
calculated as follows.
Healed area=Area after peeling-Area at each time point (after 1 to
3 days)
Healing rate=Mean of healed areas at respective time points (after
1 to 3 days)
[0178] In FIGS. 17 and 18, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.1% by weight cinnamate derivative-introduced HA
solution at all time points of days 1 to 3, compared with the
healed area of the corneal epithelia in the control eyes. And it
was also observed that the healing rate was significantly enhanced
in the eyes administered with 0.1% by weight cinnamate
derivative-introduced HA solution.
Example 17
The Effect of the 0.1% Octylamine-Introduced HA and
Hexadecylamine-Introduced HA on the Healing of Rabbit Corneal
Epithelium.
[0179] The effect of the octylamine-introduced HA and
hexadecylamine-introduced HA prepared in Example 15 on the healing
of rabbit corneal epithelium with surgical removal (The surgical
Model).
(17-1) Methods
1) Surgical Removal of Corneal Epithelium (The Surgical Model)
[0180] The corneal epithelium of the central region was removed by
using a trephine (8 mm I.D), a 23G needle and microscissors after
anesthesia with an intravenous injection of 5 mg/kg of ketamine and
2 mg/kg of xylazine and topical administration of 0.4% oxbuprocaine
hydrochloride.
2) Topical Administration
[0181] One, hour and 4 hours after peeling the corneal epithelia,
150 ul of saline as the control substance was administered in the
left eye; and 150 ul, of 0.1% octylamine-introduced HA and
hexadecylamine-introduced HA solution prepared in the above Example
(15-2) as the subject substance was administered in the right eye.
At one day and 2 days after the peeling, a total of 4 times with 3
hour intervals, and at 3 days after the peeling, with 3 hour
intervals, the same administration as above was performed. In the
administration, the 1 ml injection syringes were used. Eight model
each rabbits for the corneal epithelial layer disorder described in
the above 1) were used as the administration subjects.
3) Photographing of Corneal Epithelial Defective Region
[0182] The rabbit was given general anesthesia by intravenously
injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine,
subsequently, the corneal epithelial loss site was stained with
0.2% sodium fluorescein dissolved in PBS, and photographed under
ultra-violet light. The photographing was performed just before the
administration of the subject substance one hour after the cornea
was peeled and 3 hours after the second administration at 1 to 3
days after the peeling. When photographed, the focal length was
made constant to make the magnification of photographs
constant.
4) Measurement of Corneal Epithelial Defective Region
[0183] The area of the corneal epithelial defective region stained
with sodium fluorescein was measured on the printed photograph
using the image analyzer. The value obtained by Subtracting the
area of the peeled site 3 hours after the final administration at 3
days after the peeling from the area (peeled area) of the peeled
site just before the administration of the subject substance one
hour after the corneal epithelia were peeled was rendered as
"healed area."
(17-2.) Study Results
[0184] The results of the healed area in each individual are shown
in FIG. 19, and the results of a healing rate in each individual
are shown in FIG. 20. In FIG. 19, C8-L(a: control), C8-R(b),
C16-L(c: control), C16-R(d) represents left eye to which saline was
administrated as a control for C8-R, right eye to which 0.1%
octylamine-introduced HA solution was administrated, left eye to
which saline was administrated as a control for C16-R, right eye to
which 0.1% hexadecylamine-introduced HA solution was administrated,
respectively. In FIG. 20, C8 represents results of the above study
using octylamine, and C16 represents results of the above study
using hexadecylamine. The healed area and the healing rate were
calculated as follows.
Healed area=Area after peeling-Area at each time point (after 1 to
3 days)
Healing rate=Mean of healed areas at respective time points (after
1 to 3 days)
[0185] In FIGS. 19 and 20, it was observed that the healed area of
the corneal epithelia was significantly increased in the eyes
administered with 0.1% octylamine-introduced HA and
hexadecylamine-introduced HA solution at all time points of days 1
to 3, compared with the healed area of the corneal epithelia in the
control eyes. And it was also observed that the healing rate was
significantly enhanced in the eyes administered with 0.1%
octylamine-introduced HA or hexadecylamine-introduced HA
solution.
* * * * *