U.S. patent application number 17/574761 was filed with the patent office on 2022-05-05 for composition including glucosaminoglycan derivative and chemokine receptor activity regulator.
This patent application is currently assigned to SEIKAGAKU CORPORATION. The applicant listed for this patent is SEIKAGAKU CORPORATION. Invention is credited to Yuichi CHIKARAISHI, Yasuhiro GOTO, Takahiro HATANAKA, Tomochika KISUKEDA.
Application Number | 20220133776 17/574761 |
Document ID | / |
Family ID | 1000006081257 |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220133776 |
Kind Code |
A1 |
KISUKEDA; Tomochika ; et
al. |
May 5, 2022 |
COMPOSITION INCLUDING GLUCOSAMINOGLYCAN DERIVATIVE AND CHEMOKINE
RECEPTOR ACTIVITY REGULATOR
Abstract
Provided are a composition comprising a GAG derivative and a
chemokine receptor activity regulator, and a pharmaceutical
composition comprising said composition.
Inventors: |
KISUKEDA; Tomochika; (Tokyo,
JP) ; GOTO; Yasuhiro; (Tokyo, JP) ;
CHIKARAISHI; Yuichi; (Tokyo, JP) ; HATANAKA;
Takahiro; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEIKAGAKU CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
SEIKAGAKU CORPORATION
Tokyo
JP
|
Family ID: |
1000006081257 |
Appl. No.: |
17/574761 |
Filed: |
January 13, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15578034 |
Nov 29, 2017 |
11253540 |
|
|
PCT/JP2016/065897 |
May 30, 2016 |
|
|
|
17574761 |
|
|
|
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61K 47/61 20170801;
A61K 45/06 20130101; A61K 47/50 20170801; A61K 47/36 20130101; A61K
31/726 20130101; A61K 31/17 20130101; A61K 31/728 20130101; A61K
31/5375 20130101; A61K 31/216 20130101; A61P 27/02 20180101; A61K
45/00 20130101 |
International
Class: |
A61K 31/728 20060101
A61K031/728; A61K 31/17 20060101 A61K031/17; A61K 31/5375 20060101
A61K031/5375; A61P 27/02 20060101 A61P027/02; A61K 31/216 20060101
A61K031/216; A61K 45/06 20060101 A61K045/06; A61K 45/00 20060101
A61K045/00; A61K 47/50 20170101 A61K047/50; A61K 47/36 20060101
A61K047/36; A61K 31/726 20060101 A61K031/726 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 2015 |
JP |
2015-110784 |
Claims
1. A composition comprising a glycosaminoglycan derivative and a
chemokine receptor activity regulator.
2. The composition according to claim 1, wherein the
glycosaminoglycan derivative is a hydrophobic group-introduced
glycosaminoglycan.
3. The composition according to claim 1, wherein the
glycosaminoglycan derivative is a crosslinked
glycosaminoglycan.
4. The composition according to claim 1, which comprises a covalent
complex of the glycosaminoglycan derivative and the chemokine
receptor activity regulator.
5. The composition according to claim 1, wherein the
glycosaminoglycan derivative is a derivative of hyaluronic acid or
chondroitin sulfate.
6. The composition according to claim 1, wherein the chemokine
receptor activity regulator is a chemokine receptor antagonist.
7. A pharmaceutical composition comprising the composition of claim
1.
8. The pharmaceutical composition according to claim 7, which is a
drug for treating a posterior eye disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a Divisional of U.S. application Ser. No.
15/578,034, which is a National Stage of PCT/JP2016/065897 filed
May 30, 2016, and claims the benefit of JP App. No. 2015-110784
filed May 29, 2015. The disclosure of each of these documents is
herein incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to a composition comprising a
glycosaminoglycan derivative and a chemokine receptor activity
regulator, and to a pharmaceutical composition.
BACKGROUND ART
[0003] Age-related macular degeneration (hereinafter, abbreviated
as "AMD"), which is a posterior eye disease involving pathological
neovascularization, is broadly divided into exudative type and
atrophic type. Exudative AMD is a disease with essential features
of choroidal neovascularization (hereinafter, abbreviated as "CNV")
and its proliferative change caused by changes in the layers of
retinal pigment epithelial cells-Bruch's membrane-choroid in the
macular area, which disease rapidly proceeds and causes permanent,
severely reduced vision.
[0004] A kind of CC chemokine receptors (hereinafter, abbreviated
as "CCR"), CCR3, is known to be expressed specifically in vascular
endothelial cells in CNV collected from an exudative AMD patient
(see, for example, Nature, 2009, 460: 225-230) and, in a
laser-induced CNV model of mouse as an AMD pathological model,
intravitreal administration of a CCR3 inhibitor suppressed CNV and
has been regarded as useful for the treatment of exudative AMD
(see, for example, U.S. Pat. No. 8,592,482).
[0005] Meanwhile, intravitreal administration of heparan sulfate or
heparin, a sulfated glycosaminoglycan (hereinafter,
glycosaminoglycan is abbreviated as "GAG"), into a laser-induced
CNV model of mouse suppressed CNV and is regarded as useful for the
treatment of exudative AMD (see, for example, WO 2011/122321).
Further, Heebeom Koo, et al. conducted intravitreal administration
of a compound of hyaluronic acid (hereinafter, abbreviated as "HA")
having introduced thereinto 5.beta.-cholanic acid and evaluated the
intraocular distribution of this compound using pathological
specimens (see, for example, Biomaterials, 2012, 33:
3485-3493).
CITATION LIST
Patent Literature
[0006] Patent Literature 1: U.S. Pat. No. 8,592,482 [0007] Patent
Literature 2: WO 2011/122321
Non Patent Literature
[0007] [0008] Non Patent Literature 1: Nature, 2009, 460: 225-230.
[0009] Non Patent Literature 2: Biomaterials, 2012, 33:
3485-3493.
SUMMARY OF INVENTION
Problem to be Solved by Invention
[0010] However, a solution described in U.S. Pat. No. 8,592,482,
which contains a specified amount or more of a solubilizing agent
(such as DMSO) for preparing a CCR3 inhibitor with a concentration
sufficient for exhibiting a pharmaceutical effect, may cause rapid
degeneration or the like of tissues (such as lens) in the vicinity
of the vitreous body. Further, Biomaterials, 2012, 33: 3485-3493
and WO 2011/122321 do not disclose or suggest any chemokine
receptor activity regulator.
[0011] An object of the present invention is to provide a
composition and pharmaceutical composition each exhibits, while
suppressing rapid degeneration of tissues caused by administration
thereof, excellent regulatory activity against chemokine receptor
and excellent medicinal effects on a posterior eye disease and the
like.
Means to Solve the Problem
[0012] Specific means to solve the problems described above is
described below, and the present invention encompasses the
following aspects.
[0013] 1 A composition comprising a GAG derivative and a chemokine
receptor activity regulator.
[0014] 2 The composition according to item 1 above, wherein the GAG
derivative is a hydrophobic group-introduced GAG.
[0015] 3 The composition according to item 1 or 2 above, wherein
the GAG derivative is a crosslinked GAG.
[0016] 4 The composition according to any one of items 1 to 3
above, which comprises a covalent complex of the GAG derivative and
the chemokine receptor activity regulator.
[0017] 5 The composition according to any one of items 1 to 4
above, wherein the GAG derivative is a derivative of HA or
chondroitin sulfate (hereinafter, abbreviated as "CS").
[0018] 6 The composition according to any one of items 1 to 5
above, wherein the chemokine receptor activity regulator is a
chemokine receptor antagonist.
[0019] 7 A pharmaceutical composition comprising the composition of
any one of items 1 to 6 above.
[0020] 8 The pharmaceutical composition according to item 7 above,
which is a drug for treating a posterior eye disease.
[0021] 9 Use of the composition of any one of items 1 to 6 above as
a drug for treating a posterior eye disease.
[0022] 10 A method for treating a posterior eye disease, comprising
administering the composition of any one of items 1 to 6 above into
a vitreous body.
Effects of Invention
[0023] According to the present invention, a composition and
pharmaceutical composition each exhibits, while suppressing rapid
degeneration of tissues caused by administration thereof, excellent
regulatory activity against chemokine receptor and excellent
medicinal effects on a posterior eye disease and the like can be
provided.
BRIEF DESCRIPTION OF DRAWINGS
[0024] FIG. 1 is a graph illustrating CNV inhibitory effects of a
Ki19003-introduced CS derivative of the present embodiment
intravitreally administered to an animal model.
[0025] FIG. 2 is a graph illustrating CNV inhibitory effects of a
Ki19003-introduced CS derivative of the present embodiment
intravitreally administered to an animal model.
[0026] FIG. 3 is a graph illustrating CNV inhibitory effects of a
Ki19003-introduced CS derivative of the present embodiment
intravitreally administered to an animal model.
[0027] FIG. 4 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising SB328437 and a CS
derivative, the composition intravitreally administered to an
animal model.
[0028] FIG. 5 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising SB225002 and an HA
derivative, the composition intravitreally administered to an
animal model.
[0029] FIG. 6 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising GW766994 and an HA
derivative, the composition intravitreally administered to an
animal model.
[0030] FIG. 7 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising Ki19003 and an HA
derivative, the composition intravitreally administered to an
animal model.
[0031] FIG. 8 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising AZD3778 and an HA
derivative, the composition intravitreally administered to an
animal model.
[0032] FIG. 9 is a graph illustrating CNV inhibitory effects of the
composition of the present embodiment comprising SB328437 and an HA
derivative, the composition intravitreally administered to an
animal model.
[0033] FIG. 10 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising GW766994 and a
GAG derivative, the composition intravitreally administered to an
animal model.
[0034] FIG. 11 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising Ki19003 and a
GAG derivative, the composition intravitreally administered to an
animal model.
[0035] FIG. 12 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising GW766994 and a
CS derivative, the composition intravitreally administered to an
animal model.
[0036] FIG. 13 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising RS504393 and
an HA derivative, the composition intravitreally administered to an
animal model.
[0037] FIG. 14 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising PS372424 and
an HA derivative, the composition derivative intravitreally
administered to an animal model.
[0038] FIG. 15 is a graph illustrating CNV inhibitory effects of
the composition of the present embodiment comprising GW766994 and a
CS derivative, the composition intravitreally administered to an
animal model.
[0039] FIG. 16 is a graph in which CNV inhibitory effects of CS
intravitreally administered to an animal model are confirmed.
[0040] FIG. 17 is a graph in which CNV inhibitory effects of CS or
a CS derivative intravitreally administered to an animal model are
confirmed.
[0041] FIG. 18A is an image showing the intraocular conditions
after the intravitreal administration of a Ki19003 solution to an
animal.
[0042] FIG. 18B is an image showing the intraocular conditions
after the intravitreal administration of a SB328437 solution to an
animal.
[0043] FIG. 18C is an image showing the intraocular conditions
after the intravitreal administration of a GW766994 solution to an
animal.
[0044] FIG. 18D is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising Ki19003 and a CS derivative to an
animal.
[0045] FIG. 18E is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising GW766994 and a CS derivative to an
animal.
[0046] FIG. 18F is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising Ki19003 and a CS derivative to an
animal.
[0047] FIG. 18G is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising Ki19003 and a CS derivative to an
animal.
[0048] FIG. 18H is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising SB328437 and an HA derivative to an
animal.
[0049] FIG. 19A is an image showing the intraocular conditions
after the intravitreal administration of an RS504393 solution to an
animal.
[0050] FIG. 19B is an image showing the intraocular conditions
after the intravitreal administration of a PS372424 solution to an
animal.
[0051] FIG. 19C is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising RS504393 and an HA derivative to an
animal.
[0052] FIG. 19D is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising PS372424 and an HA derivative to an
animal.
[0053] FIG. 19E is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising GW766994 and a CS derivative to an
animal.
[0054] FIG. 19F is an image showing the intraocular conditions
after the intravitreal administration of the composition of the
present embodiment comprising GW766994 and a CS derivative to an
animal.
MODES FOR CARRYING OUT THE INVENTION
[0055] In the present specification, the term "step" includes not
only an independent step but also a step of an aspect in which,
even though the step is not distinct from another step, the
intended object of the step is achieved. Further, with respect to a
component in a composition, the content of the component in the
composition means, when a plurality of substances each belonging to
the component present in the composition, the total content of the
plurality of substances, unless otherwise mentioned.
[0056] Hereinbelow, the present invention will be described in
detail, referring to some embodiments of the invention.
[0057] (1) Compositions
[0058] The composition of the present invention comprises a GAG
derivative and a chemokine receptor activity regulator. The
composition of the present invention may contain a GAG derivative
and a chemokine receptor activity regulator as compounds
independent of each other, or in the form of a complex in which the
GAG derivative is complexed with the chemokine receptor activity
regulator by physical interaction or through a chemical bond.
[0059] Examples of the "GAG derivatives" include hydrophobic
group-introduced GAGs each in which a GAG-derived group is
covalently bonded to a hydrophobic group optionally via a spacer
group; crosslinked GAGs each in which GAG is intramolecularly or
intermolecularly crosslinked; compounds each having a GAG-derived
group and a spacer group; and the like.
[0060] Examples of the "hydrophobic group-introduced GAGs" include
a compound in which a hydrophobic group is covalently bonded,
optionally via a spacer group, to a carboxyl group of GAG through
an amide bond or ester bond or a hydroxyl group of GAG through an
ether bond or ester bond. Examples of the "crosslinked GAGs"
include a compound formed by covalently bonding GAGs to each other
via a crosslinking group, the crosslinking group intramolecularly
or intermolecularly bonding carboxyl groups or hydroxyl groups of
the GAG, and a compound formed by crosslinking GAG, the
crosslinking intramolecularly or intermolecularly occurring between
carboxyl groups and hydroxyl groups of the GAGs via no crosslinking
group.
[0061] The GAG for constituting the GAG derivative is an acidic
polysaccharide having a structure composed of repetition of a
disaccharide comprising an amino sugar and uronic acid (or
galactose). Examples of such GAGs include HA, chondroitin, CS,
dermatan sulfate and keratan sulfate. Among these, HA and CS are
preferred. The acidic functional groups of the GAGs, such as a
carboxyl group, may be in a free state not forming a salt or in a
state forming a pharmaceutically acceptable salt.
[0062] Examples of the pharmaceutically acceptable salts include
salts with alkali metal ions, such as sodium salts and potassium
salts, and salts with alkaline earth metal ions, such as magnesium
salts and calcium salts. Among these, from the viewpoint of
compatibility and affinity to a living body, salts with
pharmaceutically acceptable alkali metal ions are preferred, and
sodium salts are more preferred.
[0063] The weight average molecular weight of GAG is not
particularly limited and may be appropriately selected depending on
the purpose or the like. As an example of the weight average
molecular weight of GAG, there can be mentioned 500 Da to
10,000,000 Da, or 40,000 Da to 5,000,000 Da. The weight average
molecular weight of GAG may be measured by a light scattering
method.
[0064] The GAG for constituting the GAG derivative may be produced
by a known method depending on the type thereof. As examples of
such methods, there can be mentioned extraction and purification
from animal-derived raw materials, culture and purification from
GAG-producing bacteria or the like, sugar chain modification and
sugar chain synthesis.
[0065] The "group derived from GAG" in the hydrophobic
group-introduced GAG is a group formed by removing a hydroxyl group
from a carboxyl group of GAG; a group formed by removing a hydrogen
atom from a hydroxyl group or the like of GAG; or the like.
[0066] There is no particular limitation with respect to the
"hydrophobic group" in the hydrophobic group-introduced GAG, and
examples include a group derived from an alicyclic compound, such
as cholic acid, lithocholic acid, deoxycholic acid (which are bile
acids), and cholanic acid (the basic skeleton of each of the bile
acids above); and a group derived from a fatty acid, such as
stearic acid and oleic acid. Preferred is at least one member
selected from the groups enumerated above, more preferred is a
group derived from an alicyclic compound, and still more preferred
is a group derived from cholanic acid, in particular,
5.beta.-cholanic acid. The "group derived from an alicyclic
compound" is a group formed by removing a hydrogen atom or hydroxyl
group from the alicyclic compound, and the "group derived from a
fatty acid" is a group formed by removing a hydroxyl group from a
fatty acid.
[0067] There is no particular limitation with respect to the the
degree of substitution of the hydrophobic groups into GAG, and this
ratio may be appropriately selected depending on the purpose or the
like. As an example of the the degree of substitution, there can be
mentioned 0.1 to 80 mol %.
[0068] In the hydrophobic group-introduced GAG, the group derived
from GAG may be covalently bonded to the hydrophobic group via a
spacer group.
[0069] The "spacer group" is a divalent group which is capable of
linking two groups though a covalent bond, and specific examples
include:
--NH--(CH.sub.2).sub.m--NH--, --C(.dbd.O)--(CH.sub.2).sub.m--NH--,
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)--,
--NH--(CH.sub.2).sub.m--O--,
--NH--CH.sub.2--(OCH.sub.2).sub.m--NH--,
--NH--CH.sub.2--(OCH.sub.2).sub.m--O--,
--C(.dbd.O)--CH.sub.2--(OCH.sub.2).sub.m--NH--,
--C(.dbd.O)--CH.sub.2--(OCH.sub.2).sub.m--(C.dbd.O)--,
--C(.dbd.O)--(CH.sub.2).sub.m--O--,
--C(.dbd.O)--CH.sub.2--(OCH.sub.2).sub.m--O-- and the like. At
least one member selected from these groups is preferred, and a
group represented by the formula:
--NH--(CH.sub.2).sub.m--NH-- (I),
--C(.dbd.O)--(CH.sub.2).sub.m--NH-- (II), or
--NH--(CH.sub.2).sub.m--O-- (III)
is more preferred. In these formulae, each m independently
represents an integer of 2 to 12, preferably 2 to 5, and
particularly preferably 2.
[0070] The spacer group is not limited to a linear group and may
have a substituent, such as an alkyl group, aryl group, hydroxyl
group and halogen atom, on the methylene group contained in the
spacer group. At least a part of the methylene group may be
replaced with an oxygen atom, and aryl group or the like.
[0071] The hydrophobic group-introduced GAG can be produced by a
usual method in which GAG, a compound to form a hydrophobic group
and optionally a compound to form a spacer group are bonded
together using a condensing agent or the like.
[0072] The crosslinked GAG can be formed by, for example,
covalently bonding, with a crosslinking compound having two
crosslinking groups, at least one of carboxyl groups and hydroxyl
groups in GAG to each other via the crosslinking group. Examples of
the crosslinking compounds include a compound having a
photoreactive group, a polymerizable functional group, an amino
group or a thiol group. A crosslinking compound having a
photoreactive group or a polymerizable functional group is
preferred. As examples of methods for crosslinking GAG, there can
be mentioned, the methods belonging to the following four
categories:
(a) crosslinking with an aldehyde crosslinking agent, such as
formaldehyde or glutaraldehyde; (b) self-crosslinking between a
carboxyl group and hydroxyl group in GAG, without using a
crosslinking group; (c) crosslinking with a homo-bifunctional
crosslinking agent (such as a diepoxide compound, divinyl sulfone,
a diamine compound or a dihydrazide compound) or a
hetero-bifunctional crosslinking agent (such as an epihalohydrin);
and (d) crosslinking by reaction of GAG having introduced therein a
functional group (such as a photoreactive group, a polymerizable
functional group, an amino group, a thiol group or a halogen atom)
with GAG having introduced therein a reactive group complementary
to the above functional group or a crosslinking agent having two
reactive groups as mentioned above, although there is no particular
limitation with respect to specific methods belonging to these
categories.
[0073] Among these, preferred is a crosslinked GAG obtained by
crosslinking of (d) above, more preferred are a crosslinked GAG
obtained by photocrosslinking of GAG having introduced therein a
photoreactive group and a crosslinked GAG obtained by crosslinking
GAG having introduced therein a polymerizable functional group with
a crosslinking agent having two thiol groups.
[0074] As an example of the photoreactive group, there can be
mentioned a group derived from cinnamic acid. Such a photoreactive
group may be bonded to the GAG-derived group via a spacer group,
and it is preferred that a group derived from cinnamic acid is
covalently bonded to GAG via a spacer group represented by formula
(I), (II) or (III) above. Examples of the polymerizable functional
groups include a group derived from (meth)acrylic acid. These
polymerizable functional groups may be bonded to the GAG-derived
group via a spacer group, and it is preferred that a group derived
from (meth)acrylic acid is covalently bonded to GAG via a spacer
group represented by formula (I), (II) or (III) above. Examples of
the reactive groups complementary to the polymerizable functional
groups include a thiol group. Examples of the crosslinking agents
having two thiol groups include thiol-PEG-thiol.
[0075] There is no particular limitation with respect to the
content of the crosslinking groups in the crosslinked GAG, and the
content is appropriately selected depending on the purpose or the
like.
[0076] The "compound comprising a GAG-derived group and a spacer
group" is a compound comprising a GAG-derived group having
covalently bonded thereto a spacer group at one end thereof. When
the composition contains this compound, it is preferred that a
group derived from the chemokine receptor activity regulator is
covalently bonded to another end of the spacer group. The
GAG-derived group and the spacer group are as described above, and
the group derived from the chemokine receptor activity regulator is
a group formed by removing a hydrogen atom or hydroxyl group from
the chemokine receptor activity regulator described later.
[0077] There is no particular limitation with respect to the
content of the spacer groups in the compound comprising a
GAG-derived group and a spacer group, and the content is
appropriately selected depending on the purpose or the like.
[0078] The "chemokine receptor activity regulator" is a compound
(drug) having an effect on a chemokine receptor or a chemokine
receptor-binding substance to regulate (inhibit or enhance) signal
transduction via the chemokine receptor. Examples of such compounds
include a chemokine receptor inhibitor, anti-chemokine antibody and
chemokine receptor agonist. Examples of drugs which inhibit signal
transduction via the chemokine receptor include a drug which
competitively or non-competitively inhibits the chemokine receptor
as a chemokine receptor antagonist. Among these, preferred are a
CCR3 antagonist, CXC receptor (CXCR) 2 antagonist, CCR2 antagonist
and the like. Examples of drugs which enhance signal transduction
via the chemokine receptor include a drug having an effect on the
chemokine receptor as a chemokine receptor agonist to increase the
action of the receptor. Among these, preferred are a CXCR3 agonist
and the like.
[0079] Specifically, examples of the CCR3 antagonists include
Ki19003 (see, for example, WO 02/059081), SB328437 (see, for
example, Journal of Biological Chemistry, 2000, 275 (47),
36626-31.), GW766994 (see, for example, WO 03/082292), AZD3778
(see, for example, WO 03/004487) and the like, and examples of the
CXCR2 antagonists include SB225002 (see, for example, Bioorganic
& Medicinal Chemistry, 2009, 17 (23), 8102-8112.). Examples of
the CCR2 antagonists include RS504393 (see, for example, J. Biol.
Chem., 2000, 275 (33)). Examples of the CXCR3 agonists include
PS372424 (see, for example, Biochem. Biophys. Res. Commun., 2006,
349 (1), 221-8). Use of a CCR3 antagonist is particularly
preferred. Some specific examples of the formulae of the chemokine
receptor activity regulators are illustrated below, but the
chemokine receptor activity regulators used in the present
invention are not limited to those compounds.
##STR00001##
[0080] Such a chemokine receptor activity regulator may be in the
form of a salt (for example, a pharmaceutically acceptable salt).
Examples of the salts include a hydrochloride, hydrobromide,
hydroiodide, hydrogen sulfate, hydrogenphosphate, methanesulfonate,
nitrate, hydrogen maleate, acetate, hydrogen citrate, hydrogen
fumarate, hydrogen tartrate, hydrogen oxalate, hydrogen succinate,
benzoate and p-toluenesulfonate.
[0081] The chemokine receptor activity regulator may be contained
in the composition in the form of a complex with the GAG derivative
by physical interaction. A complex comprising a GAG derivative
(such as a hydrophobic group-introduced GAG or crosslinked GAG) and
the chemokine receptor activity regulator exhibits, while
suppressing rapid degeneration of tissues caused by the
administration thereof, excellent regulatory activity against
chemokine receptor and excellent medicinal effects against
posterior eye diseases, such as AMD.
[0082] The chemokine receptor activity regulator and the GAG
derivative may be chemically bonded to each other to form a
complex. Particularly preferred is a covalent complex in which a
group derived from GAG is covalently bonded to a group derived from
the chemokine receptor activity regulator via a spacer group. It is
preferred that the covalent complex is that in which a carboxyl
group in GAG and a carboxyl group in the chemokine receptor
activity regulator are covalently bonded to each other via a spacer
group represented by formula (III) below, and it is particularly
preferred that the covalent complex is that in which a carboxyl
group in GAG and a spacer group represented by formula (III) below
form an amide bond:
--NH--(CH.sub.2).sub.m--O-- (III)
[0083] wherein m is an integer of 2 to 12, preferably 2 to 5, more
preferably 2.
[0084] There is no particular limitation with respect to the type
of the covalent bond between the group derived from the chemokine
receptor activity regulator and the spacer group in the
above-mentioned covalent complex, and examples of the types of the
covalent bond include an ester bond.
[0085] There is no particular limitation with respect to the method
for preparing the covalent complex, the degree of substitution of
the chemokine receptor activity regulator and the like, and they
may be appropriately selected depending on the purpose or the
like.
[0086] By the use of the composition of the present invention,
intravitreal administration of a chemokine receptor activity
regulator can be achieved with no use of a solubilizing agent or,
even in a case in which a solubilizing agent is necessary, with an
extremely small amount of a solubilizing agent, even though the
regulator must be conventionally dissolved in a solubilizing agent
for intravitreal administration, even when the concentration of the
regulator is too low to obtain any medicinal effect by the
administration of the regulator alone.
[0087] There is no particular limitation with respect to the
"solubilizing agent" in the present specification, as long as the
use of the agent is capable of dissolving a poorly water-soluble
chemokine receptor activity regulator. Examples of the agent
include organic solvents such as DMSO, polysorbate 80, macrogol
400, cyclodextrin and the like.
[0088] It is preferred that the composition of the present
invention contains such a solubilizing agent (for example, an
organic solvent) in an amount of 10 wt % or less, more preferably
contains substantially no solubilizing agent.
[0089] Further, it is preferred that administration of the
composition of the present invention into an eye causes no
worsening of intraocular hemorrhage. The presence or absence of
worsening of intraocular hemorrhage can be determined by the method
described later in Examples.
[0090] (2) Method for Producing the Composition
[0091] Examples of embodiments of the compositions of the present
invention include a mixture of the GAG derivative and chemokine
receptor activity regulator and a covalent complex formed by
covalently bonding the GAG derivative and chemokine receptor
activity regulator. There is no particular limitation with respect
to the method for mixing or covalently bonding. For example, as an
example of the method for mixing, there can be mentioned a method
in which a PBS solution of the GAG derivative and the chemokine
receptor activity regulator dissolved in a solvent, such as
ethanol, are stirred for mixing. Further, for example, as an
example of the method for covalently bonding, there can be
mentioned a method in which the chemokine receptor activity
regulator is bonded to a functional group of the GAG derivative
(preferably a GAG derivative comprising a spacer group) through an
amide bond, ester bond or ether bond. Then, if necessary, the
resultant composition may be further subjected to various
treatments, such as dialysis, precipitation, freeze drying and
concentration to dryness.
[0092] (2-1) Method for Producing the Composition Comprising the
Hydrophobic Group-Introduced GAG and Chemokine Receptor Activity
Regulator
[0093] (2-1-1) Method for Producing the Hydrophobic
Group-Introduced GAG
[0094] The hydrophobic group-introduced GAG can be obtained by, for
example, covalently bonding a functional group (for example, a
carboxyl group) in a GAG molecule to a functional group (a carboxyl
group) in a hydrophobic group via a spacer group. Although an
explanation is made below, as an example of a method for producing
the hydrophobic group-introduced GAG, with respect to a case in
which the compound to form the hydrophobic group is
5.beta.-cholanic acid, the method is not limited to that of this
case. The hydrophobic group-introduced GAG in which the GAG-derived
group and a 5.beta.-cholanic acid-derived group are covalently
bonded via a spacer can be produced, for example, by a method
comprising the following steps.
[0095] The production method comprising:
(A) condensing and covalently bonding (bonding through an amide
bond) the carboxyl group in 5.beta.-cholanic acid with an amino
group in a spacer-forming molecule, and (B) condensing and
covalently bonding (bonding through an amide bond) the carboxyl
group in GAG with an amino group in the spacer-forming molecule
[0096] In step (A), the carboxyl group in 5.beta.-cholanic acid is
condensed with and covalently bonded (bonded through an amide bond)
to an amino group in a spacer-forming molecule. In this case, the
amino group in the spacer-forming molecule to be reacted with GAG
may have been protected by a usual method, if necessary.
[0097] In step (B), a carboxyl group in GAG is condensed with and
covalently bonded (bonded through an amide bond) to an amino group
in the spacer-forming molecule.
[0098] There is no limitation with respect to the order of
performing steps (A) and (B), as long as the method for producing
the GAG derivative comprises these steps.
[0099] Examples of the spacer-forming molecules include compounds
represented by the following formula:
H.sub.2N--(CH.sub.2).sub.m--NH.sub.2
wherein m is an integer of 1 to 12, preferably 2 to 5, and more
preferably 2.
[0100] The condensation (esterification, amidation) method used for
the production of the hydrophobic group-introduced GAG may be
appropriately selected from usual methods. As examples of such
methods, there can be mentioned a method using a condensing agent,
such as a water-soluble carbodiimide (for example,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC)),
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
hydrate (DMT-MM) and dicyclohexylcarbodiimide, symmetric acid
anhydride method, mixed acid anhydride methods, active ester
methods and the like. The condensation reaction conditions are
appropriately selected depending on the condensation reaction to be
adopted.
[0101] Examples of solvents used for the condensation reaction
include water, DMSO, methanol, ethanol, propanol, butanol,
acetonitrile, DMF, THF, formamide, and mixtures of these solvents.
Preferred are an ethanol/water mixed solvent and a DMF/formamide
mixed solvent.
[0102] (2-1-2) Method for Mixing the Hydrophobic Group-Introduced
GAG and Chemokine Receptor Activity Regulator
[0103] The intended composition can be obtained by mixing a PBS
solution of the hydrophobic group-introduced GAG and a solution of
the chemokine receptor activity regulator dissolved in, for
example, ethanol, and subjecting the resultant mixture to dialysis
and freeze drying. There is no particular limitation with respect
to the method for the mixing, dialysis and freeze drying, and the
method may be appropriately selected from usual methods.
[0104] (2-2) Method 1 for Producing the Composition Comprising the
Crosslinked GAG and Chemokine Receptor Activity Regulator
[0105] (2-2-1) Method for Producing the Crosslinked GAG
[0106] As an example of the crosslinked GAGs, a photocrosslinkable
GAG can be prepared by, for example, the method described in
Japanese Patent Application Kokai Publication No. 2002-249501.
Specifically, the photocrosslinkable GAG can be obtained by
introducing a photoreactive group (for example, a cinnamic acid
derivative) to a carboxyl group in GAG by a condensation reaction.
Further, the crosslinked GAG can be produced by subjecting the
thus-obtained photocrosslinkable GAG to a photoreaction.
[0107] (2-2-2) Method for Mixing the Crosslinked GAG and Chemokine
Receptor Activity Regulator
[0108] The target composition can be prepared by stirring and
mixing an aqueous solution of the above-obtained crosslinked GAG
with the chemokine receptor activity regulator.
[0109] (2-3) Method 2 for Producing the Composition Comprising the
Crosslinked GAG and Chemokine Receptor Activity Regulator
[0110] (2-3-1) Method for Producing a Precursor of the Crosslinked
GAG
[0111] A precursor of the crosslinked GAG can be prepared by
introducing a methacrylate group to a carboxyl group into GAG via a
spacer group, if necessary, by, for example, the method described
later in Examples. Examples of compounds capable of giving the
crosslinked GAG by a reaction with the thus-obtained precursor of
the crosslinked GAG include, but not limited to,
thiol-PEG-thiol.
[0112] (2-3-2) Method for Producing the Composition
[0113] The desired composition can be obtained by, for example,
mixing the precursor of the above-obtained crosslinked GAG with the
chemokine receptor activity regulator and effecting a crosslinking
reaction using a crosslinking agent, such as thiol-PEG-thiol.
[0114] (2-4) Method 3 for Producing the Covalent Complex of the GAG
Derivative and Chemokine Receptor Activity Regulator
[0115] The GAG derivative having covalently bonded thereto a
chemokine receptor activity regulator can be obtained by covalently
bonding a functional group in GAG (for example, a carboxyl group)
and a functional group in the chemokine receptor activity regulator
(for example, a carboxyl group) to each other via a spacer
group.
[0116] (2-4-1) Method for Producing the GAG Having Covalently
Bonded Thereto the Chemokine Receptor Activity Regulator
[0117] The GAG derivative in which the GAG-derived group is
covalently bonded to a group derived from the chemokine receptor
activity regulator via a spacer can be produced by, for example, a
method comprising the following steps.
[0118] The production method comprising:
(A) condensing a carboxyl group in the chemokine receptor activity
regulator with a hydroxyl group in a spacer-forming molecule to
covalently bond them (bonding them though an ester bond), and (B)
condensing a carboxyl group in GAG with an amino group in the
spacer-forming molecule to covalently bond them (bonding them
though an amide bond).
[0119] In step (A), a carboxyl group in the chemokine receptor
activity regulator is condensed with and covalently bonded to a
hydroxyl group in a spacer-forming molecule. The amino group in the
spacer-forming molecule to be reacted with GAG may have been
protected by a usual method, if necessary.
[0120] In step (B), a carboxyl group in GAG is condensed with and
covalently bonded to an amino group in the spacer-forming
molecule.
[0121] There is no limitation with respect to the order of
performing steps (A) and (B), as long as the method for producing
the GAG derivative comprises these steps.
[0122] Examples of the spacer-forming molecules include compounds
each represented by the following formula:
HO--(CH.sub.2).sub.m--NH.sub.2
wherein m is an integer of 1 to 12, preferably 2 to 5, and more
preferably 2.
[0123] The condensation (esterification and amidation) method can
be appropriately selected from usual methods.
[0124] The structure of the GAG derivative may be selected from a
wide variety of structures. As specific examples of the structures
of the GAG derivatives, there can be mentioned, for example, with
respect to CS derivatives, a structure comprising at least one
occurrence of at least one structural unit represented by chemical
formula (IVa) below and, with respect to HA derivatives, a
structure comprising at least one occurrence of at least one
structural unit represented by chemical formula (Va) below.
##STR00002##
[0125] The group represented by R.sup.12 or R.sup.13 is covalently
bonded to the GAG-derived carbonyl group at the position indicated
by * in the formula.
[0126] As specific examples of the structures of the covalent
complexes of the GAG derivative and chemokine receptor activity
regulator, there can be mentioned, for example, when the derivative
is the CS derivative, a structure comprising at least one
occurrence of at least one structural unit represented by chemical
formula (IVb) below and, when the derivative is the HA derivative,
a structure comprising at least one occurrence of at least one
structural units represented by chemical formula (Vb) below.
##STR00003##
[0127] The group represented by R.sup.22 or R.sup.23 is covalently
bonded to the GAG-derived carbonyl group at the position indicated
by * in the formula.
[0128] (3) Pharmaceutical Composition
[0129] A pharmaceutical composition of the present invention
comprises the composition described above, and may further contain
additional components, such as a pharmaceutically acceptable
excipient, if necessary. Examples of the additional components
include a pharmaceutically acceptable excipient, surfactant,
stabilizer and liquid medium.
[0130] There is no particular limitation with respect to the
applications of the pharmaceutical composition, and it is preferred
that the composition is used, for example, for the treatment of a
posterior eye disease.
[0131] (4) Drug for Treating a Posterior Eye Disease
[0132] A drug for treating a posterior eye disease is a
pharmaceutical composition comprising the aforementioned
composition and used for the treatment of a posterior eye
disease.
[0133] The term "treatment (treating)" used in the present
specification means any treatment directed at a disease, such as
remedy, improvement and control of a progress (prevention of
worsening) of a disease.
[0134] There is no particular limitation with respect to the form
of the drug for treating a posterior eye disease, as long as the
drug is in the form of a pharmaceutical preparation or a medicine
which can be administered to a human eye. It is preferred that the
drug at the time of administration is in a liquid form, such as a
solution, suspension or the like. Such a solution or suspension may
be prepared for administration by dissolving a powder of the
composition when needed.
[0135] The amount of the drug for treating a posterior eye disease
in a liquid form for intravitreal administration is about 10 to
1000 .mu.L.
[0136] As an example of the concentration of the drug for treating
a posterior eye disease in a liquid form, there can be mentioned
0.001 to 5 wt % as the concentration of the chemokine receptor
activity regulator and 0.01 to 10 wt % as the concentration of the
GAG derivative.
[0137] Examples of methods for administering the drug for treating
a posterior eye disease include intravitreal administration,
subconjunctival administration, conjunctival sac administration,
sub-Tenon administration, topical instillation, and administration
to a device placed in an eye. Intravitreal administration is
preferred. Among the intravitreal administrations, intravitreal
injection is most preferred.
[0138] The frequency of administration of the drug for treating a
posterior eye disease may be appropriately fixed depending on
pathologic condition, drug concentration and the like, and the drug
may be administered as required when the pathologic condition has
worsened. However, the drug is not limited to be administered in
these cases.
[0139] The drug for treating a posterior eye disease is to be
administered to treat a posterior eye disease. The "posterior eye
disease" means a disease or any other abnormality occurring at a
posterior segment of an eye, which may develop into a pathological
condition caused by new blood vessels or present
neovascularization. Examples of such diseases and the like include
diabetic retinopathy, diabetic macular edema, retinal artery
occlusion, branch retinal vein occlusion (BRVO), central retinal
vein occlusion (CRVO), retinopathy of prematurity, central serous
chorioretinopathy, central exudative chorioretinopathy, neovascular
maculopathy and AMD (exudative AMD with CNV lesion, and atrophic
AMD that has a risk of CNV development and/or transitions to
exudative AMD). Among these, as indications of the drug of the
present invention for treating a posterior eye disease, preferred
are AMD, diabetic retinopathy and diabetic macular edema, and
especially preferred is exudative AMD.
[0140] As examples of embodiments of the drug for treating a
posterior eye disease, there can be mentioned: a liquid preparation
having a content of the solubilizing agent of not more than 10 wt
%, which causes no worsening of intraocular hemorrhage; a liquid
preparation comprising the crosslinked GAG and chemokine receptor
activity regulator, which causes no worsening of intraocular
hemorrhage; a liquid preparation comprising the crosslinked GAG and
chemokine receptor activity regulator, which has the content of the
solubilizing agent of not more than 10 wt % and causes no worsening
of intraocular hemorrhage; a liquid preparation comprising the
photocrosslinked HA and chemokine receptor antagonist, which causes
no worsening of intraocular hemorrhage; a liquid preparation
comprising the photocrosslinked HA and chemokine receptor
antagonist, which has the content of the solubilizing agent of not
more than 10 wt % and causes no worsening of intraocular
hemorrhage; a liquid preparation comprising the hydrophobic
group-introduced GAG and chemokine receptor activity regulator,
which causes no worsening of intraocular hemorrhage; a liquid
preparation comprising the hydrophobic group-introduced GAG and
chemokine receptor activity regulator, which has the content of the
solubilizing agent of not more than 10 wt % and causes no worsening
of intraocular hemorrhage; a liquid preparation comprising the
hydrophobic group-introduced CS and chemokine receptor antagonist,
which causes no worsening of intraocular hemorrhage; a liquid
preparation comprising the hydrophobic group-introduced CS and
chemokine receptor antagonist, which has the content of the
solubilizing agent of not more than 10 wt % and causes no worsening
of intraocular hemorrhage; a liquid preparation comprising the CS
having introduced thereinto a group derived from cholanic acid and
the CCR3 inhibitor, which causes no worsening of intraocular
hemorrhage; and a liquid preparation comprising the CS having
introduced thereinto a group derived from cholanic acid and the
CCR3 inhibitor, which has the content of the solubilizing agent of
not more than 10 wt % and causes no worsening of intraocular
hemorrhage. The drug of these embodiments for treating a posterior
eye disease may be used as a drug for suppressing progress of
exudative AMD, wherein the drug is injected into the vitreous body
of a human eye.
[0141] (5) Method for Treating Posterior Eye Diseases
[0142] A method for treating a posterior eye disease comprises a
step for administering into an eye the drug for treating a
posterior eye disease. If necessary, the method for treating a
posterior eye disease may further comprise an additional step. The
method for treating a posterior eye disease may be carried out in
accordance with the description in "(4) Posterior eye disease
treating agents" above, and preferred conditions, administration
frequency and the like of the drug are as described above.
EXAMPLES
[0143] Hereinbelow, Examples and Test Examples of the present
invention will be described in greater detail, but they are not
construed as limiting the technical scope of the present invention.
With respect to the substituents on GAG and the chemokine receptor
activity regulator, the contents in the composition were measured
by the methods described later. The molecular weight of GAG is a
weight average molecular weight.
[0144] (1) Preparation of GAG Derivative and Composition
(Example 1) Synthesis of Aminoethyl 5.beta.-Cholanoamide
[0145] Methanol (5 ml) and concentrated hydrochloric acid (0.18 ml)
were added to 5.beta.-cholanic acid (1 g, manufactured by Aldrich),
and the resultant mixture was stirred at 60.degree. C. for 6 hours.
Then, the reaction liquid was cooled to room temperature, and the
precipitated solid was collected by filtration. Ethylenediamine (5
ml, manufactured by Wako Pure Chemical Industries, Ltd.) was added
to the obtained compound, and the resultant mixture was stirred at
130.degree. C. for 5 hours. After the target compound was
identified by LCMS, the mixture was brought back to room
temperature. The obtained solid was collected by filtration, washed
with distilled water and dried to thereby obtain Compound 1 (895
mg).
[0146] ESI-MS; Calcd for C267H3746N2O [M.sup.+H].sup.+, 404; found
404
(Example 2) Synthesis of 5.beta.-Cholanic Acid-Introduced HA
[0147] Formamide (40 ml) was added to HA (average molecular weight:
about 210,000, manufactured by Lifecore Biomedical, LLC, 500 mg),
and the resultant mixture was stirred at 40.degree. C. for 2 hours
while heating to give a solution. To this solution, WSC
(manufactured by Wako Pure Chemical Industries, Ltd., 205 mg) and
N-hydroxysuccinimide (manufactured by WATANABE CHEMICAL INDUSTRIES,
LTD., 123 mg) were added, and the resultant mixture was stirred. A
DMF solution (10 ml) of Compound 1 (96 mg) was added to the
mixture, and further DMF (30 ml) was added. The resultant mixture
was stirred at room temperature for 24 hours. The reaction liquid
was put into a dialysis membrane (Spectra/Por RC Biotech Membrane
MWCO 8-10 kDa, purchased from Funakoshi Co., Ltd.) and dialyzed,
for 3 days in total, against methanol:distilled water=3:1,
methanol:distilled water=1:1 and distilled water in this order. The
dialysate was recovered and a cation-exchange resin (DOWEX.TM. 50
W.times.8, 50-100, 2 g, manufactured by Wako Chemical, Ltd.) was
added. The resultant mixture was stirred for 30 minutes, and the
reaction liquid was filtered and freeze dried to thereby obtain
Compound 2 (590 mg). The degree of substitution of 5.beta.-cholanic
acid was 4.7 mol %.
(Example 3) Synthesis of 5.beta.-Cholanic Acid-Introduced CS
[0148] Reactions were effected in substantially the same manner as
in Example 2 using CS (average molecular weight: about 40,000,
manufactured by SEIKAGAKU CORPORATION, 500 mg), to thereby obtain
Compound 3 (490 mg). The degree of substitution of 5.beta.-cholanic
acid was 10.0 mol %. A drug solution for administration to animals
was prepared by dissolving the freeze dried product in PBS so that
the concentration was 10 mg/ml and filtering the resultant solution
through a 0.22 .mu.m filter.
(Example 4) Preparation of Ki19003-Containing HA Derivative
[0149] Ki19003 (synthesized in accordance with WO 02/059081 A2, 35
mg) was dissolved in ethanol (2 ml). To the resultant solution,
Compound 2 (40 mg) synthesized in Example 2 was added in the form
of a PBS (8 ml, pH 7.4) solution and dissolved therein. The
resultant mixture was stirred for mixing and dialyzed (Spectra/Por
RC Biotech Membrane, MWCO 3.5-5 kDa, manufactured by Spectrum
Laboratories, Inc.) for 7 hours against distilled water. The
dialysate was recovered, filtered through a 0.45 .mu.m filter and
freeze dried to thereby obtain Composition 4 (35 mg). The Ki19003
content was 14.0 wt %. The thus obtained Composition 4 was
dissolved in PBS so that the concentration was 3 mg/ml, and the
resultant solution was filtered through a 0.22 .mu.m filter to
thereby prepare a sample for administration to animals.
(Example 5) Preparation of Ki19003-Containing CS Derivative
[0150] Reactions were effected in substantially the same manner as
in Example 4 using a PBS (8 ml, pH 7.4) solution of Compound 3 (35
mg) synthesized in Example 3, to thereby obtain Composition 5 (33
mg). The Ki19003 content was 22.0 wt %. The thus obtained
Composition 5 was dissolved in PBS so that the concentration was 3
mg/ml, and the resultant solution was filtered through a 0.22 .mu.m
filter to thereby prepare a sample for administration to
animals.
(Example 6) Preparation of GW766994-Containing HA Derivative
[0151] GW766994 (synthesized in accordance with WO 03/082292 A1, 30
mg) was dissolved in ethanol (2 ml). To the resultant solution,
Compound 2 (40 mg) synthesized in Example 2 was added in the form
of a PBS (8 ml, pH 7.4) solution and dispersed therein. The
resultant dispersion was dialyzed (Spectra/Por RC Biotech Membrane,
MWCO 3.5-5 kDa) for 7 hours against distilled water. The dialysate
was recovered, filtered through a 0.45 .mu.m filter and freeze
dried to thereby obtain Composition 6 (35 mg). The GW766994 content
was 23.5 wt %. The thus obtained Composition 6 was dissolved in PBS
so that the concentration was 3 mg/ml, and the resultant solution
was filtered through a 0.22 .mu.m filter to thereby prepare a
sample for administration to animals.
(Example 7) Preparation (1) of GW766994-Containing CS
Derivative
[0152] Reactions were effected in substantially the same manner as
in Example 6 using a PBS (8 ml, pH 7.4) solution of Compound 3 (35
mg) synthesized in Example 3, to thereby obtain Composition 7 (31
mg). The GW766994 content was 27.7 wt %. The thus obtained
Composition 7 was dissolved in PBS so that the concentration was 3
mg/ml, and the resultant solution was filtered through a 0.22 .mu.m
filter to thereby prepare a sample for administration to
animals.
(Example 8) Preparation of GW766994-Containing CS Derivative
(2)
[0153] GW766994 (12 mg) was dissolved in ethanol (2 ml). To the
resultant solution, Compound 3 (60 mg) synthesized in Example 3 was
added in the form of a PBS (8 ml, pH 7.4) solution and stirred for
mixing. The resultant mixture was dialyzed (Slide-A-Lyzer, MWCO 3.5
kDa, manufactured by Thermo Fisher Scientific Inc.) for 7 hours
against distilled water. The dialysate was recovered, filtered
through a 0.22 .mu.m filter and freeze dried to thereby obtain
Composition 8 (40 mg). The GW766994 content was 9.4 wt %. The thus
obtained Composition 8 was dissolved in PBS so that the
concentration was 10 mg/ml to thereby prepare a sample for
administration to animals.
(Example 9) Synthesis of 2-Aminoethyl Esterified Ki19003
[0154] Ki19003 (190 mg) was dissolved in methylene chloride (2 ml),
and dimethylaminopyridine (manufactured by Wako Pure Chemical
Industries, Ltd., 13 mg), WSC (manufactured by Wako Pure Chemical
Industries, Ltd., 206 mg) and N-Boc-ethanolamine (manufactured by
Wako Pure Chemical Industries, Ltd., 174 mg) were added. The
resultant mixture was stirred at room temperature for 3 hours under
nitrogen atmosphere. After the completion of the reaction was
confirmed by LCMS, the reaction liquid was washed with saturated
saline, dehydrated with anhydrous magnesium sulfate, filtered and
concentrated. The resultant residue was purified by silica gel
chromatography (hexane:ethyl acetate=1:2) to thereby obtain a white
solid (81 mg). 4 M HCl/AcOEt (10 ml, manufactured by KOKUSAN
CHEMICAL Co., Ltd.) was added to this solid, and the resultant
mixture was stirred at room temperature for 1 hour. After the
target compound was identified by LCMS, the reaction liquid was
concentrated to thereby obtain Compound 9 (71 mg).
[0155] ESI-MS; Calcd for C27H37C13N4O3 [M.sup.+H].sup.+, 573; found
573
(Example 10) Synthesis of Ki19003-Introduced CS
[0156] Distilled water (4 ml) was added to CS (average molecular
weight: about 40,000, manufactured by SEIKAGAKU CORPORATION, 270
mg), and the resultant mixture was stirred for 30 minutes to give a
solution. To this solution, an ethanol solution (4 ml) of Compound
9 (64 mg) was added, and DMT-MM (manufactured by Wako Pure Chemical
Industries, Ltd., 47 mg) was further added. The resultant mixture
was stirred at room temperature overnight. Sodium chloride (270 mg)
was added to the reaction liquid, and the resultant solution was
added dropwise into ethanol (40 ml). The precipitated white solid
was collected by filtration, washed three times with 90% ethanol in
water, and dried overnight with a vacuum pump to thereby obtain
Compound 10 (281 mg, degree of substitution: 18.0 wt %). The thus
obtained Compound 10 was dissolved in PBS so that the concentration
was 3 mg/ml, and the resultant solution was filtered again through
a 0.22 .mu.m filter to thereby obtain Composition 10, which was
used as a sample for administration to animals.
(Example 11) Synthesis 2 of Ki19003-Introduced CS
[0157] A reaction was effected in substantially the same manner as
in Example 10 using CS (average molecular weight: about 150,000,
manufactured by SEIKAGAKU CORPORATION, 150 mg). Sodium chloride
(150 mg) was added to the reaction liquid, and the resultant
solution was added dropwise into ethanol (40 ml). The precipitated
white solid was collected by filtration, washed three times with
90% ethanol in water, and dried overnight with a vacuum pump to
thereby obtain a white solid. The thus obtained solid was dissolved
in distilled water, and the resultant solution was filtered through
a 0.22 .mu.m filter and freeze dried to thereby obtain Compound 11
(91 mg, degree of substitution: 5.3 wt %). The thus obtained
Compound 11 was dissolved in PBS so that the concentration was 10
mg/m to thereby obtain Composition 11, which was used as a sample
for administration to animals.
(Example 12) Synthesis of 2-Aminoethyl Methacrylate
Hydrochloride
[0158] To a solution of N-Boc-ethanolamine (15.5 g, manufactured by
Tokyo Chemical Industry Co., Ltd.) and diisopropylethylamine (25.2
mL, manufactured by Tokyo Chemical Industry Co., Ltd.) in methylene
chloride (180 mL), methacryloyl chloride (10.5 mL, manufactured by
Tokyo Chemical Industry Co., Ltd.) was added dropwise under argon
atmosphere at -78.degree. C. over 3 minutes, and the resultant
mixture was stirred for 1 hour, and then at room temperature for 14
hours. The reaction liquid was washed with water, extracted with
methylene chloride and dried over sodium sulfate. The solid was
removed by filtration, and the liquid was concentrated to thereby
obtain an oil, which was then purified by silica gel chromatography
(hexane:ethyl acetate=4:1). The resultant yellow solid was washed
with diethyl ether and hexane to give an N-Boc ester (12.3 g) as a
white solid.
[0159] .sup.1H-NMR (CDCl.sub.3) .delta.6.71 (1H, br-s), 5.75 (1H,
s), 5.33-5.33 (1H, m), 4.96) 1H, br-s), 3.40-3.43 (2H, m),
3.32-3.35 (2H, m), 1.96-1.96 (3H, m), 1.44 (9H, s)
[0160] 4 M HCl/Dioxane (120 mL, manufactured by KOKUSAN CHEMICAL
Co., Ltd.) was added to this solid, and the resultant mixture was
stirred at room temperature for 3 hours. The reaction liquid was
concentrated, and the precipitated solid was washed with hexane.
The solid was dried in vacuo at room temperature to afford Compound
12 (8.83 g) as a white solid.
[0161] .sup.1H-NMR (D.sub.2O) .delta.5.73 (1H, s), 5.47 (1H, s),
3.15 (2H, t, J=6.0 Hz), 2.54 (2H, t, J=6.0 Hz), 1.90 (3H, s)
(Example 13) Synthesis of Methacrylate Group-Introduced CS
[0162] CS (average molecular weight: about 40,000, manufactured by
SEIKAGAKU CORPORATION, 4.03 g) was dissolved in deionized water
(120 mL). To the resultant solution, Compound 12 (0.23 g) and
DMT-MM (0.36 g) were sequentially added at room temperature, and
the resultant mixture was stirred for 18 hours.
[0163] Sodium bicarbonate (3.0 g) was added to the reaction liquid,
and the resultant mixture was stirred for 30 minutes and
neutralized with acetic acid to pH 7.0. After 30 minutes of
stirring, sodium chloride (12.0 g) was added, and the resultant
mixture was stirred for 30 minutes. 90% Ethanol (240 mL) was added,
and the resultant mixture was stirred for 30 minutes and the
supernatant was discarded. 90% ethanol (240 mL) was added again,
and the resultant mixture was stirred for 30 minutes and the
supernatant was discarded. This operation was repeated two more
times. The solid was then dialyzed (Cellulose Tube 36/32, MWCO 10
kDa, manufactured by EIDIA Co., Ltd.) against distilled water
overnight and freeze dried to thereby obtain Compound 13 (4.34 g).
The degree of substitution was 9.5 mol %.
(Example 14) Preparation of SB328437-Containing CS Derivative
(1)
[0164] 65 .mu.L of a saline solution (0.2 mg/mL) of thiol-PEG-thiol
(molecular weight: 3400, manufactured by Laysan Bio, Inc.) was
added to Compound 13 (50.0 mg) and SB328437 (2.0 mg, synthesized in
accordance with Journal of Biological Chemistry, 2000, 275 (47),
36626-31). Then, 135 .mu.L of saline was added. After stirring for
1 minute, the resultant mixture was allowed to stand for 48 hours
to thereby obtain a gelled compound. The gelled compound was added
to saline (3 mL) and allowed to stand for 24 hours. Only the gelled
compound was transferred to two sterilized syringes connected to
each other at their tips, and plungers were inserted into the
syringe barrels. The air inside the syringes was removed, and the
two plungers were alternately pressed down 30 times. The gelled
compound was then pushed out to one of the syringes to thereby
obtain Composition 14, which was used as a sample for
administration to animals.
(Example 15) Preparation 2 of SB328437-Containing CS Derivative
(2)
[0165] Substantially the same procedure of synthesis as in Example
14 was repeated, except that the reaction was effected with the
amount of the saline solution (0.2 mg/mL) of thiol-PEG-thiol of 100
.mu.L and the amount of the subsequently added saline of 100 .mu.L
to thereby obtain Composition 15, which was used as a sample for
administration to animals.
(Example 16) Preparation of SB328437-Containing CS Derivative
(3)
[0166] Substantially the same procedure of synthesis as in Example
14 was repeated, except that the reaction was effected with the
amount of the saline solution (0.2 mg/mL) of thiol-PEG-thiol of 200
.mu.L to thereby obtain Composition 16, which was used as a sample
for administration to animals.
(Example 17) Synthesis of Aminoethyl Oleamide
[0167] Oleic acid (500 mg, manufactured by Tokyo Chemical Industry
Co., Ltd.) was dissolved in dimethylformamide (8 ml, manufactured
by Wako Pure Chemical Industries, Ltd.) and, to the resultant
solution, triethylamine (manufactured by Wako Pure Chemical
Industries, Ltd., 0.49 ml), WSC (manufactured by Wako Pure Chemical
Industries, Ltd., 408 mg), HOBT (manufactured by KOKUSAN CHEMICAL
Co., Ltd., 406 mg) and N-Boc-diethylamine (manufactured by Wako
Pure Chemical Industries, Ltd., 312 mg) were added. The resultant
mixture was stirred at room temperature for 3 hours under nitrogen
atmosphere. After the completion of the reaction was confirmed by
LCMS, ethyl acetate was added to the reaction liquid, and the
organic phase was sequentially washed with saturated aqueous
solution of sodium bicarbonate, saturated aqueous solution of
ammonium chloride and saturated saline, dehydrated over anhydrous
magnesium sulfate, filtered and concentrated. The resultant residue
was purified by silica gel chromatography (hexane:ethyl
acetate=1:1) to thereby obtain a white solid (360 mg). Methanol (2
ml, manufactured by Wako Pure Chemical Industries, Ltd.) and 4 M
HCl/AcOEt (10 ml, manufactured by KOKUSAN CHEMICAL Co., Ltd.) were
added to this solid, and the resultant mixture was stirred
overnight at room temperature. After the target compound was
identified by LCMS, the reaction liquid was concentrated to afford
Compound 17 (250 mg).
[0168] ESI-MS; Calcd for C.sub.20H.sub.40N.sub.2O [M+H].sup.+, 325;
found 325
(Example 18) Synthesis of Oleic Acid-Introduced CS
[0169] Distilled water (20 ml) was added to CS (average molecular
weight: about 40,000, manufactured by SEIKAGAKU CORPORATION, 500
mg), and the resultant mixture was stirred for 30 minutes to give a
solution. An ethanol solution (20 ml) of Compound 17 (36 mg) was
added, and DMT-MM (manufactured by Tokuyama Corporation, 55 mg) was
further added. The resultant mixture was stirred overnight at room
temperature. A 1 N aqueous solution of sodium hydroxide (2.5 mL)
was added to the reaction liquid, and the resultant mixture was
stirred for 30 minutes. The whole amount of the reaction liquid was
put into a dialysis membrane (Slide-A-Lyzer G2, MWCO10K, 30 mL,
purchased from Funakoshi Co., Ltd.), and dialyzed, for 2 days in
total, against ethanol:distilled water (1:1) and distilled water in
this order. The dialysate was recovered, a cation-exchange resin
(DOWEX.TM. 50 W.times.8 50-100, 2 g, manufactured by Wako Chemical,
Ltd.) was added and the resultant mixture was stirred for 30
minutes. The reaction liquid was filtered and freeze dried to
thereby obtain Compound 18 (277 mg). The degree of substitution of
5.beta.-oleic acid was 10.3 mol %.
(Example 19) Synthesis of GW766994-Containing Oleic Acid-Introduced
CS
[0170] GW766994 (15 mg) was dissolved in ethanol (2 ml), and the
resultant solution was mixed with a PBS (9 ml, pH 7.4) solution of
Compound 18 (90 mg) synthesized in Example 18. The resultant
solution was dialyzed (Slide-A-Lyzer G2, MWCO 3.5K, 15 mL,
purchased from Funakoshi Co., Ltd.) against distilled water for 7
hours. The dialysate was recovered, filtered through a 0.22 .mu.m
filter and freeze dried to thereby obtain Composition 19 (75 mg).
The GW766994 content was 8.62 wt %. The thus obtained Compound 19
was dissolved in PBS so that the concentration was 10 mg/m, and the
resultant solution was filtered through a 0.22 .mu.m filter to
thereby prepare a sample for administration to animals.
(Example 20) Synthesis of Aminoethyl Lithocholamide
[0171] Lithocholic acid (500 mg, manufactured by Tokyo Chemical
Industry Co., Ltd.) was dissolved in dimethylformamide (8 ml,
manufactured by Wako Pure Chemical Industries, Ltd.), and WSC
(manufactured by Wako Pure Chemical Industries, Ltd., 305 mg), HOBT
(manufactured by KOKUSAN CHEMICAL Co., Ltd., 304 mg) and
N-Boc-diethylamine (manufactured by Wako Pure Chemical Industries,
Ltd., 254 mg) were added. The resultant mixture was stirred at room
temperature for 3 hours under nitrogen atmosphere. After the
completion of the reaction was confirmed by LCMS, ethyl acetate was
added to the reaction liquid. The organic phase was washed
sequentially with saturated aqueous solution of sodium bicarbonate,
saturated aqueous solution of ammonium chloride and saturated
saline, dehydrated over anhydrous magnesium sulfate, filtered and
concentrated. The obtained residue was purified by silica gel
chromatography (hexane:ethyl acetate=1:1) to thereby obtain a white
solid (310 mg). THF (2 ml, manufactured by Wako Pure Chemical
Industries, Ltd.) and 4 M HCl/AcOEt (10 ml, manufactured by KOKUSAN
CHEMICAL Co., Ltd.) were added to this solid, and the resultant
mixture was stirred at 45.degree. C. for 1 hour. After the target
compound was identified by LCMS, the reaction liquid was
concentrated to afford Compound 20 (305 mg).
[0172] ESI-MS; Calcd for C.sub.26H.sub.46N.sub.2O.sub.2
[M+H].sup.+, 419; found 419
(Example 21) Synthesis of Lithocholic Acid-Introduced CS
[0173] Distilled water (18 ml) was added to CS (average molecular
weight: about 40,000, manufactured by SEIKAGAKU CORPORATION, 400
mg), and the resultant mixture was stirred for 30 minutes to give a
solution. An ethanol solution (18 ml) of Compound 20 (36 mg) was
added, and DMT-MM (manufactured by Tokuyama Corporation, 55 mg) was
further added. The resultant mixture was stirred overnight at room
temperature. A 1 N aqueous solution of sodium hydroxide (2.0 mL)
was added to the reaction liquid, and the resultant mixture was
stirred for 30 minutes. The whole amount of the reaction liquid was
put into a dialysis membrane (Slide-A-Lyzer G2, MWCO10K, 30 mL,
purchased from Funakoshi Co., Ltd.), and dialyzed, for 2 days in
total, against ethanol:distilled water (1:1) and distilled water in
this order. The dialysate was recovered, a cation-exchange resin
(DOWEX.TM. 50 W.times.8 50-100, 2 g, manufactured by Wako Chemical,
Ltd.) was added and the resultant mixture was stirred for 30
minutes. The reaction liquid was filtered and freeze dried to
thereby obtain Compound 21 (250 mg). The degree of substitution of
5.beta.-lithocholic acid was 10.0 mol %.
(Example 22) Synthesis of GW766994-Containing Lithocholic
Acid-Introduced CS
[0174] GW766994 (11 mg) was dissolved in ethanol (2 ml), and the
resultant solution was mixed with a PBS (6 ml, pH 7.4) solution of
Compound 21 (66 mg) synthesized in Example 21. The resultant
solution was dialyzed (Slide-A-Lyzer G2, MWCO3.5K, 15 mL, purchased
from Funakoshi Co., Ltd.) against distilled water for 7 hours. The
dialysate was recovered, filtered through a 0.22 .mu.m filter and
freeze dried to thereby obtain Composition 22 (75 mg). The GW766994
content was 10.3 wt %. The thus obtained Composition 22 was
dissolved in PBS so that the concentration was 10 mg/m, and the
resultant solution was filtered through a 0.22 .mu.m filter to
thereby prepare a sample for administration to animals.
[0175] (Method for Measuring the Contents of Substituents on GAG
and Chemokine Receptor Activity Regulator)
[0176] (A) Method for Measuring the Degree of Substitution of
5.beta.-Cholanic Acid
[0177] The degree of substitution of 5f3-cholanic acid in each of
Examples 1 and 2 was measured as follows.
[0178] .sup.1H-NMR spectrum was measured in deuterated
water-deuterated methanol (1:1) mixed solvent, and the ratio was
calculated using the following equation:
Degree of substitution of 5.beta.-cholanic acid (mol %)=(Integrated
value for the peak assigned to the methyl group at position 21 in
5.beta.-cholanic acid)/(Integrated value for the peaks assigned to
N-acetyl groups in GAG)
[0179] (B) Method for Measuring the Degree of Substitution of
Methacrylate Groups
[0180] The degree of substitution of methacrylate groups in Example
13 was measured as follows.
[0181] .sup.1H-NMR spectrum was measured in deuterated water, and
the ratio was calculated using the following equation:
Degree of substitution of methacrylate group (mol %)=(Integrated
value for the peak assigned to methacrylate group)/(Integrated
value for the peaks assigned to N-acetyl groups in GAG)
[0182] (C) Method for Measuring the Content of the Chemokine
Receptor Activity Regulator
[0183] Method for Measuring the Content of Ki19003 in Examples 4
and 5
[0184] A calibration curve was prepared using a spectrophotometer
(UV SPECTROPHOTOMETER manufactured by Shimadzu Corporation,
measurement wavelength 220 nm), and then the measurement was
carried out. The content of the drug was calculated using the
following equation.
Content (wt %)=(Weight of Ki19003 in the composition)/(Weight of
the composition).times.100
[0185] Method for Measuring the Content of GW766994 in Examples 6,
7 and 8
[0186] A calibration curve was prepared by HPLC under the following
conditions, and then the measurement was carried out.
Column: ODS-3, 4.6.times.150 mm, 5 um (manufactured by GL Science)
Flow: 0.8 ml/min
Detect: 225 nm
[0187] Fluent: acetonitrile: 20 mM aqueous solution of ammonium
acetate=5:5 (isocratic)
[0188] The content of the drug was calculated using the following
equation.
Content (wt %)=(Weight of GW766994 in the composition)/(Weight of
the composition).times.100
[0189] Method for Calculating the Content of Ki19003 in Examples 10
and 11
[0190] Compound 10 or 11 (with a known measured weight) was
hydrolyzed by heating at 37.degree. C. for 2 hours in a 2 M aqueous
solution of sodium hydroxide. A calibration curve for Ki19003 was
prepared by HPLC under the following conditions, and then the
measurement of the liberated Ki19003 was carried out.
Column: ODS-3, 4.6.times.150 mm, 5 um (manufactured by GL Science)
Flow: 0.8 ml/min
Detect: 225 nm
[0191] Eluent: acetonitrile: 50 mM aqueous solution of formic
acid=3:7 (isocratic)
[0192] The content was calculated using the following equation.
Content (wt %)=(Weight of Ki19003 liberated from the
compound)/(Weight of the compound).times.100
[0193] (2) Evaluation Methods
(Test Example 1) Assay of CNV Inhibitory Effects Using Composition
10
[0194] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Composition 10 or Ki19003.
[0195] Test Substances
[0196] Ki19003 was dissolved in DMSO to obtain a solution of
Ki19003 (0.6 mg/mL). The following test substances were used in the
assay.
1) Composition 10 (containing 0.54 mg/mL of Ki19003) 2) Ki19003
(0.6 mg/mL)
3) DMSO
[0197] (The sample for administration to animals prepared in
Example 10 was used as Composition 10.)
[0198] Method
[0199] (1) Preparation of Laser-Induced CNV Models and
Administration of the Test Substance
[0200] BN/CrlCrlj rats (male, CHARLES RIVER LABORATORIES JAPAN,
INC.) were used for the preparation of animal models. Under general
anesthesia by intraperitoneal administration of an anesthetic
mixture (saline:Somnopentyl=9:1) (about 2 mL/body), Mydrin-P
ophthalmic solution was topically instilled to cause mydriasis in
both eyes. The retina around the optic disk was irradiated with a
laser beam to induce CNV. Used for the laser beam irradiation were
SCOPISOL solution for eye, Fundus 5.4 mm Laser Lens, a laser
photocoagulation device and a slit lamp illumination system.
Immediately after the laser beam irradiation, 5 .mu.L/eye of the
test substance was administered once into the vitreous bodies of
both eyes. Immediately after the administration, one drop of an
antimicrobial drug (VEGAMOX Ophthalmic Solution 0.5%) was
instilled.
[0201] (2) Preparation of Flat Mounts
[0202] After 10 days from the preparation, the models were
euthanized with CO.sub.2. The eyeballs were extracted and immersed
in 10% neutral buffered solution of formalin (room temperature,
about 60 minutes). Eye cups were prepared, washed with PBS,
dehydrated with methanol and immersed in PBS containing 1% bovine
serum albumin and 0.5% Triton X-100 (room temperature, about 60
minutes). The retina was removed, and 60 .mu.L of 0.5% fluorescein
Griffonia simplicifolia lectin I, FITC-conjugate was added to the
eye cups, and the eye cups were allowed to stand (under
refrigeration, overnight). By this treatment, vascular endothelial
cells of CNV were fluorescently stained with FITC-lectin. Eight
slits were made radially in each of the eye cups to thereby obtain
flat mounts. These flat mounts were washed twice with PBS
containing 0.1% Triton X-100 and embedded on a glass slide with
about 120 .mu.L of Prolong Gold Antifade Reagent.
[0203] (3) Imaging and Area Measurement of CNV
[0204] A fluorescence image of CNV in the flat mount embedded on a
glass slide was taken with a fluorescent microscope. The area of
CNV was measured using an image processing software (Image pro
exp).
[0205] Statistical Analysis
[0206] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0207] Results of Assay
[0208] The results of measurement of the CNV area are given in the
table below and FIG. 1.
[0209] Composition 10 showed significant inhibitory effects of CNV,
as compared to DMSO and Ki19003.
TABLE-US-00001 TABLE 1 mean S.E. n Test results DMSO 34980 1125 16
Ki19003 30752 1371 14 * Composition 10 26811 1206 16 ***, # *: P
< 0.05, ***: P < 0.001 (vs. DMSO), t-test #: P < 0.05 (vs.
Ki19003), t-test
(Test Example 2) Assay of CNV Inhibitory Effects Using Composition
11 (1)
[0210] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Composition 11 or Ki19003.
[0211] Test Substances
[0212] Ki19003 was dissolved in DMSO to obtain a solution of
Ki19003 (0.53 mg/mL). The following test substances were used in
the assay.
1) Composition 11 (containing 0.53 mg/mL of Ki19003) 2) Ki19003
(0.53 mg/mL)
3) DMSO
[0213] (The sample for administration to animals prepared in
Example 11 was used as Composition 11.)
[0214] Method
[0215] Substantially the same procedure as in Method for Test
Example 1 was repeated.
[0216] Statistical Analysis
[0217] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0218] Results of Assay
[0219] The results of measurement of the CNV area are given in the
table below and FIG. 2.
[0220] Composition 11 showed significant inhibitory effects of CNV,
as compared to DMSO and Ki19003.
TABLE-US-00002 TABLE 2 mean S.E. n Test results DMSO 43613 2344 9
Ki19003 40222 1419 10 N.S. Composition 11 35660 1064 10 **, # **: P
< 0.01 vs. DMSO), t-test #: P < 0.05 (vs. Ki19003), t-test
N.S.: Not Significant (vs. DMSO), t-test
[0221] Conclusions
[0222] It was shown from Test Examples 1 and 2 that
Ki19003-introduced CS can be used as a drug for treating a
posterior eye disease, especially AMD, which exhibited medicinal
effects higher than those of Ki19003. It was also shown that CS
could be used as GAG to constitute a GAG derivative.
(Test Example 3) Assay of CNV Inhibitory Effects Using Composition
11 (2)
[0223] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Composition 11 or a formulation
comprising CS and Ki19003.
[0224] Test Substances
[0225] CS (19 mg, the same as that used in Example 11, manufactured
by SEIKAGAKU CORPORATION) and Ki19003 (1 mg) were mixed together
with PBS (2 mL). The resultant mixture was shaken to give a
formulation comprising CS and Ki19003 (hereinbelow "Formulation
1"). The following test substances were used in the assay.
1) Composition 11 (containing 0.53 mg/mL of Ki19003) 2) Formulation
1 (containing 0.5 mg/mL of Ki19003)
3) DMSO
[0226] (The sample for administration to animals prepared in
Example 11 was used as Composition 11.)
[0227] Method
[0228] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0229] Statistical Analysis
[0230] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0231] Results of Assay
[0232] The results of measurement of the CNV area are given in the
table below and FIG. 3.
[0233] Composition 11 showed significant inhibitory effects of CNV,
as compared to DMSO.
TABLE-US-00003 TABLE 3 mean S.E. n Test results DMSO 43613 2344 9
Formulation 1 40385 2438 10 N.S. Composition 11 35660 1064 10 **
**: P < 0.01 (vs. DMSO), t-test N.S.: Not Significant (vs.
DMSO), t-test
[0234] Conclusions
[0235] Ki19003-introduced CS was shown to be able to be used as a
drug for treating a posterior eye disease, especially AMD, which
exhibited medicinal effects higher than those of a formulation
comprising CS and Ki19003. It was also shown that a mere blend of
GAG and a chemokine receptor antagonist is not satisfactory as a
drug for treating a posterior eye disease, and the usefulness of a
covalent complex of the GAG derivative with the chemokine receptor
antagonist was thus confirmed.
(Test Example 4) Assay of CNV Inhibitory Effects Using Compositions
14, 15 and 16
[0236] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of each of Compositions 14, Composition
15, Composition 16 and SB328437.
[0237] Test Substances
[0238] SB328437 was dissolved in DMSO to obtain a solution of
SB328437 (2 mg/mL). The following test substances were used in the
assay.
1) Composition 14 (containing 1.33 mg/mL of SB328437) 2)
Composition 15 (containing 1.67 mg/mL of SB328437) 3) Composition
16 (containing 3.33 mg/mL of SB328437) 4) SB328437 (2 mg/mL) (The
samples for administration to animals prepared in Examples 14, 15
and 16 were used as Compositions 14, 15 and 16, respectively.)
[0239] Method
[0240] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0241] Statistical Analysis
[0242] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0243] Results of Assay
[0244] The results of measurement of the CNV area are given in the
table below and FIG. 4.
[0245] Each of Compositions 14, 15 and 16 showed significant
inhibitory effects of CNV, as compared to SB328437.
TABLE-US-00004 TABLE 4 mean S. E. n Test results SB328437 26040 933
8 Composition 14 22647 628 8 ** Composition 15 20957 1079 8 **
Composition 16 20953 1015 8 ** **: P < 0.01 (vs. SB328437),
t-test
[0246] Conclusions
[0247] The composition comprising SB328437 and the crosslinked CS
was shown to be able to be used as a drug for treating posterior
eye disease, especially AMD, which exhibited medicinal effects
higher than those of SB328437. The crosslinked GAG was shown to be
able to be used as the GAG derivative, and CS was shown to be able
to be used as GAG. Further, SB328437 was shown to be able to be
used as a chemokine receptor antagonist.
(Test Example 5) Assay of CNV Inhibitory Effects Using a
Composition Comprising SB225002 and Crosslinked HA (Gel-One)
[0248] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of each of a composition comprising
SB225002 and Gel-One (photocrosslinked HA: manufactured by
SEIKAGAKU CORPORATION) as well as SB225002.
[0249] Test Substances
[0250] SB225002 (synthesized in accordance with Bioorganic &
Medicinal Chemistry, 2009, 17 (23), 8102-8112) was dissolved in
DMSO to obtain a solution of SB225002 (0.02 mg/mL). SB225002 was
dissolved in DMSO to obtain a solution of SB225002 (0.2 mg/mL), and
0.15 mL of the resultant SB225002 (0.2 mg/mL) solution was mixed
with Gel-One (1.5 mL) and stirred to thereby obtain Composition 31.
SB225002 was dissolved in DMSO to obtain a solution of SB225002 (2
mg/mL), and the resultant 2 mg/mL solution of SB225002 (0.015 mL)
was mixed with Gel-One (1.5 mL) and stirred to thereby obtain
Composition 32. The following test substances were used in the
assay.
1) Composition 31 (containing 0.018 mg/mL of SB225002) 2)
Composition 32 (containing 0.02 mg/mL of SB225002) 3) SB225002
(0.02 mg/me
4) DMSO
[0251] Method
[0252] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0253] Statistical Analysis
[0254] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0255] Results of Assay
[0256] The results of measurement of the CNV area are given in the
table below and FIG. 5.
[0257] Each of Compositions 31 and 32 showed significant inhibitory
effects of CNV, as compared to DMSO and SB225002.
TABLE-US-00005 TABLE 5 mean S. E. n Test results DMSO 42892 2424 10
SB225002 43121 1836 9 N. S. Composition 31 34453 2664 10 *, #
Composition 32 33387 3009 10 *, # *: P < 0.05 (vs. DMSO), t-test
#: P < 0.05 (vs. SB225002), t-test N. S.: Not Significant (vs.
DMSO), t-test
[0258] Conclusions
[0259] The composition comprising SB225002 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of SB225002. The crosslinked GAG was shown to be able to be used as
the GAG derivative, and HA was shown to be able to be used as GAG.
Further, SB225002 was shown to be able to be used as a chemokine
receptor antagonist.
(Test Example 6) Assay of CNV Inhibitory Effects Using a
Composition Comprising GW766994 and Crosslinked HA (Gel-One)
[0260] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of a composition comprising GW766994
and Gel-One (photocrosslinked HA: manufactured by SEIKAGAKU
CORPORATION) as well as GW766994.
[0261] Test Substances
[0262] GW766994 was dissolved in DMSO to obtain a solution of
GW766994 (2 mg/mL). GW766994 was dissolved in DMSO to obtain a
solution of GW766994 (50 mg/mL), and the resultant 50 mg/mL
solution of GW766994 (0.0417 mL) was mixed with Gel-One (1.0 mL)
and stirred to thereby obtain Composition 33. The following test
substances were used in the assay.
1) Composition 33 (containing 2 mg/mL of GW766994) 2) GW766994 (2
mg/mL)
3) DMSO
[0263] Method
[0264] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0265] Statistical Analysis
[0266] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0267] Results of Assay
[0268] The results of measurement of the CNV area are given in the
table below and FIG. 6.
[0269] Composition 33 showed significant inhibitory effects of CNV,
as compared to DMSO and GW766994.
TABLE-US-00006 TABLE 6 mean S. E. n Test results DMSO 42613 1404 9
GW766994 41548 1258 9 N. S. Composition 33 34476 1052 10 ***, ###
***: P < 0.001 (vs. DMSO), t-test ###: P < 0.001 (vs.
GW766994), t-test N. S.: Not Significant (vs. DMSO), t-test
[0270] Conclusions
[0271] The composition comprising GW766994 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of GW766994. Further, GW766994 was shown to be able to be used as a
chemokine receptor antagonist.
(Test Example 7) Assay of CNV Inhibitory Effects Using a
Composition Comprising Ki19003 and Crosslinked HA (Gel-One)
[0272] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of a composition comprising Ki19003 and
Gel-One (photocrosslinked HA: manufactured by SEIKAGAKU
CORPORATION) as well as Ki19003.
[0273] Test Substances
[0274] Ki19003 was dissolved in DMSO to obtain a solution of
Ki19003 (0.5 mg/mL). Ki19003 was dissolved in DMSO to obtain a
solution of Ki19003 (25 mg/mL), and the resultant 25 mg/mL solution
of Ki19003 (0.0204 mL) was mixed with Gel-One (1.0 mL) and stirred
to thereby obtain Composition 34. The following test substances
were used in the assay.
1) Composition 34 (containing 0.5 mg/mL of Ki19003) 2) Ki19003 (0.5
mg/ml)
3) DMSO
[0275] Method
[0276] Substantially the same procedure as in Method for Test
Example 1 was repeated.
[0277] Statistical Analysis
[0278] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0279] Results of Assay
[0280] The results of measurement of the CNV area are given in the
table below and FIG. 7.
[0281] Composition 34 showed significant inhibitory effects of CNV,
as compared to DMSO.
TABLE-US-00007 TABLE 7 mean S. E. n Test results DMSO 40833 1147 10
Ki19003 41393 2614 10 N. S. Composition 34 36423 1483 10 * *: P
< 0.05 (vs. DMSO), t-test N. S. Not Significant (vs. DMSO),
t-test
[0282] Conclusions
[0283] The composition comprising Ki19003 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of Ki19003. Test Examples 1, 2, 3 and 7 showed that a plurality of
types of GAG derivatives (hydrophobic group-introduced GAG or
crosslinked GAG) could be used in combination with Ki19003. This
fact showed that the GAG derivative and chemokine receptor
antagonist which might be used in combination were not always in
one-to-one correspondence.
(Test Example 8) Assay of CNV Inhibitory Effects Using a
Composition Comprising AZD3778 and Crosslinked HA (Gel-One)
[0284] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of a composition comprising AZD3778
(synthesized in accordance with WO 03/004487 A1) and Gel-One
(photocrosslinked HA: manufactured by SEIKAGAKU CORPORATION) as
well as AZD3778.
[0285] Test Substances
[0286] AZD3778 was dissolved in DMSO to obtain a solution of
AZD3778 (0.1 mg/mL). AZD3778 was dissolved in DMSO to obtain a
solution of AZD3778 (5 mg/mL), and the resultant 5 mg/mL solution
of AZD3778 (0.0204 mL) was mixed with Gel-One (1.0 mL) and stirred
to thereby obtain Composition 35. The following test substances
were used in the assay.
1) Composition 35 (containing 0.1 mg/mL of AZD3778) 2) AZD3778 (0.1
mg/ml)
3) DMSO
[0287] Method
[0288] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0289] Statistical Analysis
[0290] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0291] Results of Assay
[0292] The results of measurement of the CNV area are given in the
table below and FIG. 8.
[0293] Composition 35 showed significant inhibitory effects of CNV,
as compared to AZD3778.
TABLE-US-00008 TABLE 8 mean S. E. n Test results DMSO 42579 2016 10
AZD3778 43935 1952 10 N. S. Composition 35 35345 1441 10 **, ## **:
P < 0.01 (vs. DMSO), t-test ##: P < 0.01 (vs. AZD3778),
t-test N. S.: Not Significant (vs. DMSO), t-test
[0294] Conclusions
[0295] The composition comprising AZD3778 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of AZD3778. Further, AZD3778 was shown to be able to be used as a
chemokine receptor antagonist.
(Test Example 9) Assay of CNV Inhibitory Effects Using a
Composition Comprising SB328437 and Crosslinked HA (Gel-One)
[0296] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of a composition comprising SB328437
and Gel-One (photocrosslinked HA: manufactured by SEIKAGAKU
CORPORATION) as well as SB328437.
[0297] Test Substances
[0298] SB328437 was dissolved in DMSO to obtain a solution of
SB328437 (1 mg/mL). SB328437 was dissolved in DMSO to obtain a
solution of SB328437 (50 mg/mL), and the resultant 50 mg/mL
solution of SB328437 (0.02 mL) was mixed with Gel-One (1.0 mL) and
stirred to thereby obtain Composition 36. The following test
substances were used in the assay.
1) Composition 36 (containing 1 mg/mL of SB328437) 2) SB328437 (1
mg/ml)
3) DMSO
[0299] Method
[0300] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0301] Statistical Analysis
[0302] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0303] Results of Assay
[0304] The results of measurement of the CNV area are given in the
table below and FIG. 9.
[0305] Composition 36 showed significant inhibitory effects of CNV,
as compared to SB328437.
TABLE-US-00009 TABLE 9 mean S. E. n Test results DMSO 43155 1548 10
SB328437 38911 988 10 * Composition 36 35012 751 10 ***, ## *: P
< 0.05, *** P < 0.001 (vs. DMSO), t-test ##: P < 0.01 (vs.
SB328437), t-test
[0306] Conclusions
[0307] The composition comprising SB328437 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of SB328437. Further, SB328437 was shown to be able to be used as a
chemokine receptor antagonist. Test Examples 5, 6, 7, 8 and 9
showed that a plurality of types of chemokine receptor antagonists
(GW766994, Ki19003, AZD3778, SB328437 and SB225002) could be used
in combination with the crosslinked GAG. This fact showed that the
GAG derivative and chemokine receptor antagonist which might be
used in combination were not always in one-to-one
correspondence.
(Test Example 10) Assay of CNV Inhibitory Effects Using
Compositions 6 and 7
[0308] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of each of Compositions 6, 7 and
GW766994.
[0309] Test SubstancesGW766994 was dissolved in DMSO to obtain a
solution of GW766994 (1.5 mg/mL). The following test substances
were used in the assay.
1) Composition 6 (containing 0.71 mg/mL of GW766994) 2) Composition
7 (containing 0.83 mg/mL of GW766994) 3) GW766994 (1.5 mg/mL)
4) DMSO
[0310] (The samples for administration to animals prepared in
Examples 6 and 7 were used as Compositions 6 and 7,
respectively.)
[0311] Method
[0312] Substantially the same procedure as in Method for Test
Example 1 was repeated.
[0313] Statistical Analysis
[0314] The areas of CNV in the DMSO group and other groups were
analyzed by Dunnett's test. The significance level was 5% on both
sides. The areas of CNV in the GW766994 group and other
Compositions groups were analyzed by Dunnett's test. The
significance level was 5% on both sides.
[0315] Results of Assay
[0316] The results of measurement of the CNV area are given in the
table below and FIG. 10.
[0317] Each of Compositions 6 and 7 showed significant inhibitory
effects of CNV, as compared to GW766994.
TABLE-US-00010 TABLE 10 mean S. E. n Test results DMSO 32889 3371 8
GW766994 30795 1701 8 N. S. Composition 6 25387 1010 7 *, #
Composition 7 24614 969 8 *, ## *: P < 0.05 (vs. DMSO),
Dunnett's test N. S.: Not Significant (vs. DMSO), Dunnett's test #:
P < 0.05, ##: P < 0.01 (vs. GW766994), Dunnett's test
[0318] Conclusions
[0319] Each of the composition comprising GW766994 and hydrophobic
group-introduced HA and the composition comprising GW766994 and
hydrophobic group-introduced CS was shown to be able to be used as
a drug for treating a posterior eye disease, especially AMD, which
exhibited medicinal effects higher than those of GW766994. Further,
each of HA and CS was shown to be able to be used as GAG to
constitute the hydrophobic group-introduced GAG.
(Test Example 11) Assay of CNV Inhibitory Effects Using
Compositions 4 and 5
[0320] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Compositions 4, 5 and Ki19003.
[0321] Test Substances
[0322] Ki19003 was dissolved in DMSO to obtain a solution of
Ki19003 (0.6 mg/mL).
The following test substances were used in the assay. 1)
Composition 4 (containing 0.42 mg/mL of Ki19003) 2) Composition 5
(containing 0.66 mg/mL of Ki19003) 3) Ki19003 (0.6 mg/mL)
4) DMSO
[0323] (The samples for administration to animals prepared in
Examples 4 and 5 were used as Compositions 4 and 5,
respectively.)
[0324] Method
[0325] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0326] Statistical Analysis
[0327] The areas of CNV in the DMSO group and other groups were
analyzed by Dunnett's test. The significance level was 5% on both
sides.
[0328] Results of Assay
[0329] The results of measurement of the CNV area are given in the
table below and FIG. 11. Each of Compositions 4 and 5 showed
significant inhibitory effects of CNV, as compared to DMSO.
TABLE-US-00011 TABLE 11 mean S. E. n Test results DMSO 34980 1125
16 Ki19003 30752 1371 14 N. S. Composition 4 28766 1226 13 **
Composition 5 27525 1346 15 *** **: P < 0.01, ***: P < 0.001
(vs. DMSO), Dunnett's test N. S.: Not Significant (vs. DMSO),
Dunnett's test
[0330] Conclusions
[0331] Each of the composition comprising Ki19003 and hydrophobic
group-introduced HA and the composition comprising Ki19003 and
hydrophobic group-introduced CS was shown to be able to be used as
a drug for treating a posterior eye disease, especially AMD, which
exhibited medicinal effects higher than those of Ki19003. Further,
each of HA and CS was shown to be able to be used as GAG to
constitute the hydrophobic group-introduced GAG.
(Test Example 12) Assay of CNV Inhibitory Effects and Influence on
Intraocular Hemorrhage Using Composition 8
[0332] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect and influence on intraocular
hemorrhage were examined with respect to intravitreal
administration of Composition 8 or a formulation comprising heparin
and GW766994.
[0333] Test Substances
[0334] GW766994 was dissolved in DMSO to obtain a solution of
GW766994 (10 mg/mL). Heparin (10 mg, manufactured by Aldrich) was
dissolved in PBS (1 mL) to obtain a heparin solution (10 mg/mL).
The heparin solution and 10 mg/mL solution of GW766994 (0.1 mL)
were mixed to thereby obtain Formulation 2 comprising heparin and
GW766994. The following test substances were used in the assay.
1) Composition 8 (containing 0.96 mg/mL of GW766994) 2) Formulation
2 (containing 0.91 mg/mL of GW766994)
3) PBS
[0335] (The sample for administration to animals prepared in
Example 8 was used as Composition 8.)
[0336] Method
(Neovascularization Inhibitory Effects)
[0337] Substantially the same procedure as in Methodfor Test
Example 1 was repeated.
[0338] (Influence on Intraocular Hemorrhage)
[0339] Immediately after the administration of the test substance
and after 10 days from the administration, intraocular hemorrhage
was evaluated by scoring, and the influence on intraocular
hemorrhage was examined based on the presence or absence of
worsening of intraocular hemorrhage.
[0340] (1) Evaluation of Intraocular Hemorrhage by Scoring
Immediately after the Administration of the Test Substance
[0341] Introocular observation was carried out using a slit lamp
and the like, and the intraocular hemorrhage was scored in
accordance with the following criteria of intraocular hemorrhage
for scoring.
Score 0: No hemorrhage was observed in the observed area. Score 1:
The area with observed hemorrhage was 1/4 or less of the observed
area. Score 2: The area with observed hemorrhage was 1/4 or more
and 1/2 or less of the observed area. Score 3: The area with
observed hemorrhage was 1/2 or more of the observed area.
[0342] (2) Evaluation of Intraocular Hemorrhage by Scoring 10 Days
after the Administration
[0343] Immediately after the preparation of the eye cup, the
vitreous humor and retina were observed and hemorrhage in each of
the vitreous humor and retina was scored in accordance with the
following criteria of hemorrhage in the vitreous humor and retina
for scoring. The average value of the scores of hemorrhage in the
vitreous humor and that in the retina was obtained as the score of
intraocular hemorrhage.
Score 0: No hemorrhage was observed in the observed area. Score 1:
The area with observed hemorrhage was 1/4 or less of the observed
area. Score 2: The area with observed hemorrhage was 1/4 or more
and 1/2 or less of the observed area. Score 3: The area with
observed hemorrhage was 1/2 or more of the observed area.
[0344] (3) Calculation of the Presence or Absence of Worsening of
Intraocular Hemorrhage
[0345] The differences in score of intraocular hemorrhage after 10
days from the administration of the test substance relative to that
immediately after the administration and that were calculated and,
based on these differences, the numbers of eyes with (the
difference was positive)/without (the difference was negative or 0)
worsening of hemorrhage were determined.
[0346] Statistical Analysis
[0347] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0348] The presence or absence of worsening of intraocular
hemorrhage in each group was analyzed by 2.times.2 Fisher's exact
test. The significance level was 5% on one side.
[0349] Results of Assay
[0350] The results of measurement of the CNV area are given in
Table 12 below and FIG. 12. Composition 8 showed significant
inhibitory effects of CNV, as compared to PBS.
[0351] The results of examination of the influence on intraocular
hemorrhage are given in Table 13. The number of eyes with worsening
of intraocular hemorrhage for Formulation 2 comprising heparin and
GW766994 was shown to be significantly large, as compared to that
for each of PBS and Composition 8.
TABLE-US-00012 TABLE 12 mean S. E. n Test results PBS 43914 1824 14
Formulation 2 42506 1710 20 N. S. Composition 8 38469 1454 18 * *:
P < 0.05 (vs. PBS), t-test N. S.: Not Significant (vs. PBS),
t-test
TABLE-US-00013 TABLE 13 Worsening of Number Test intraocular
bleeding of eyes Test substances Absent Present examined results
PBS 13 1 14 Formulation 2 12 8 20 *, # Composition 8 16 2 18 N. S.
*: P < 0.05 (vs. PBS) #: P < 0.05 (vs. Composition 8) N. S.:
Not Significant (vs. PBS)
[0352] Conclusions
[0353] The composition comprising GW766994 and hydrophobic
group-introduced CS was shown to be able to be used as a drug for
treating a posterior eye disease, especially AMD, which exhibited
medicinal effects higher than those of Formulation 2 comprising
heparin and GW766994. It was also shown that a mere blend of GAG
and a chemokine receptor antagonist is not satisfactory as a drug
for treating a posterior eye disease, and the usefulness of a
composition comprising the GAG derivative and chemokine receptor
antagonist was thus confirmed.
[0354] In view of occurrence of worsening of intraocular
hemorrhage, Formulation 2 comprising heparin and GW766994 was shown
to be unsuitable for intravitreal administration.
(Test Example 13) Assay of Degeneration of Tissues Caused by
Intravitreal Administration of a Chemokine Receptor Antagonist
Solution Used in Patent Literature 1, which Contains a Solubilizing
Agent (DMSO), as Well as the Composition of the Present
Invention
[0355] Test substances were intravitreally administered once to
rats, and the intraocular conditions were observed.
[0356] Test Substances
[0357] The following test substances were used in the assay.
1) Ki19003 (0.6 mg/mL) 2) SB328437 (1 mg/mL) 3) GW766994 (1.5
mg/mL) 4) Composition 5 (containing 0.66 mg/mL of Ki19003) 5)
Composition 7 (containing 0.83 mg/mL of GW766994) 6) Composition 10
(containing 0.54 mg/mL of Ki19003) 7) Composition 11 (containing
0.53 mg/mL of Ki19003) 8) Composition 36 (containing 1 mg/mL of
SB328437) (The samples for administration to animals prepared in
Examples 5, 7, 10 and 11 and Test Example 9 were used as
Composition 5, 7, 10, 11 and 36, respectively.)
[0358] Solutions of Ki19003, SB328437 and GW766994 were obtained in
substantially the same manner as in Test Examples 1, 9 and 10,
respectively.
[0359] Method
[0360] (1) Administration of Test Substances
[0361] BN/CrlCrlj rats (male, CHARLES RIVER LABORATORIES JAPAN,
INC.) were used as an animal for this assay. Under general
anesthesia by intraperitoneal administration of an anesthetic
mixture (saline:Somnopentyl=9:1) (about 2 mL/body), Mydrin-P
ophthalmic solution was topically instilled to cause mydriasis in
both eyes. Thereafter, 5 .mu.L/eye of the test substance was
administered once into the vitreous bodies of both eyes.
Immediately after the administration, one drop of an antimicrobial
drug (VEGAMOX Ophthalmic Solution 0.5%) was instilled.
[0362] (2) Intraocular Imaging
[0363] Photographs of the conditions of inside of eyes of the rats
were taken with a digital microscope.
[0364] Results of Assay
[0365] The results of the intraocular imaging are shown in FIGS.
18A to 18H.
[0366] As pointed out with an arrow in the figures, each of the
chemokine receptor antagonist solutions (Ki19003 (FIG. 18A),
SB328437 (FIG. 18B) and GW766994 (FIG. 18C)) containing a
solubilizing agent caused degeneration of the lenticular tissue. In
contrast, with respect to Composition 5 (FIG. 18D), Composition 7
(FIG. 18E), Composition 10 (FIG. 18F), Composition 11 (FIG. 18G)
and Composition 36 (FIG. 18H), each of which is a composition
comprising the GAG derivative and chemokine receptor antagonist, no
degeneration of the lenticular tissue was observed.
[0367] Summary
[0368] The composition comprising the GAG derivative and chemokine
receptor antagonist was shown to exhibit excellent antagonistic
activity against a chemokine receptor, as compared to a chemokine
receptor antagonist solution containing a solubilizing agent, while
suppressing occurrence of rapid degeneration of tissues.
(Test Example 14) Assay of CNV Inhibitory Effects Using a
Composition Comprising RS504393 and Crosslinked HA (Gel-One)
[0369] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of each of a composition comprising
RS504393 and Gel-One (photocrosslinked HA: manufactured by
SEIKAGAKU CORPORATION) as well as RS504393.
[0370] Test Substances
[0371] RS504393 (10 mg, manufactured by Abeam) was dissolved in
DMSO to obtain a solution of RS504393 (0.01 mg/mL). RS504393 was
dissolved in DMSO to obtain a solution of RS504393 (1 mg/mL), and
the resultant 1 mg/mL solution of RS504393 (0.01 mL) was mixed with
Gel-One (1.0 mL) and stirred to thereby obtain Composition 37. The
following test substances were used in the assay.
1) Composition 37 (containing 0.01 mg/mL of RS504393) 2) RS504393
(0.01 mg/ml)
3) DMSO
[0372] Method
[0373] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0374] Statistical Analysis
[0375] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0376] Results of Assay
[0377] The results of measurement of the CNV area are given in the
table below and FIG. 13.
[0378] Composition 37 showed significant inhibitory effects of CNV,
as compared to DMSO and RS504393.
TABLE-US-00014 TABLE 14 mean S.E. n Test results DMSO 41672 2812 10
RS504393 44137 2231 10 N.S. Composition 37 34418 1624 10 *, ## *: P
< 0.05 (vs. DMSO), t-test ##: P < 0.01 (vs. RS504393), t-test
N.S.: Not Significant (vs. DMSO), t-test
[0379] Conclusions
[0380] The composition comprising RS504393 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of RS504393. Further, RS504393 was shown to be able to be used as a
chemokine receptor antagonist.
(Test Example 15) Assay of CNV Inhibitory Effects Using a
Composition Comprising PS372424 and Crosslinked HA (Gel-One)
[0381] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of a composition comprising PS372424
and Gel-One (photocrosslinked HA: manufactured by SEIKAGAKU
CORPORATION) as well as PS372424.
[0382] Test Substances
[0383] PS372424 (10 mg, manufactured by Calbiochem) was dissolved
in DMSO to obtain a solution of PS372424 (0.1 mg/mL). PS372424 was
dissolved in DMSO to obtain a solution of PS372424 (1 mg/mL), and
the resultant 1 mg/mL solution of PS372424 (0.1 mL) was mixed with
Gel-One (LO mL) and stirred to thereby obtain Composition 38. The
following test substances were used in the assay.
1) Composition 38 (containing 0.091 mg/mL of PS372424) 2) PS372424
(0.1 mg/ml)
3) DMSO
[0384] Method
[0385] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0386] Statistical Analysis
[0387] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0388] Results of Assay
[0389] The results of measurement of the CNV area are given in the
table below and FIG. 14.
[0390] Composition 38 showed significant inhibitory effects of CNV,
as compared to DMSO and PS372424.
TABLE-US-00015 TABLE 15 mean S.E. n Test results DMSO 42437 1873 9
PS372424 36842 1023 10 * Composition 38 31285 1553 10 ***, ## *: P
< 0.05 (vs. DMSO), t-test ***: P < 0.001 (vs. DMSO), t-test
##: P < 0.01 (vs. PS372424), t-test
[0391] Conclusions
[0392] The composition comprising PS372424 and Gel-One was shown to
be able to be used as a drug for treating a posterior eye disease,
especially AMD, which exhibited medicinal effects higher than those
of PS372424. Further, PS372424 was shown to be able to be used as a
chemokine receptor agonist.
(Test Example 16) Assay of CNV Inhibitory Effects Using Composition
22 (Lithocholic Acid-Introduced CS: Example 22) and Composition 19
(Oleic Acid-Introduced CS: Example 19)
[0393] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Compositions 22 and 19.
[0394] Test Substances
[0395] The following test substances were used in the assay.
1) PBS
[0396] 2) Composition 22 (containing 0.86 mg/mL of GW766994) 3)
Composition 19 (containing 1.03 mg/mL of GW766994) (The samples for
administration to animals prepared in Examples 22 and 19 were used
as Compositions 22 and 19, respectively.)
[0397] Method
[0398] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0399] Statistical Analysis
[0400] The areas of CNV in the PBS group and other groups were
analyzed by a test (t-test) for unpaired two groups. The
significance level was 5% on both sides.
[0401] Results of Assay
[0402] The results of measurement of the CNV area are given in the
table below and FIG. 15. Each of Compositions 22 and 19 showed
significant inhibitory effects of CNV, as compared to PBS.
TABLE-US-00016 TABLE 16 mean S.E. n Test results PBS 46885 1877 10
Composition 22 39428 1423 10 ** Composition 19 39917 1987 10 * *: P
< 0.05 (vs. PBS), t-test **: P < 0.01 (vs. PBS), t-test
[0403] As described in Test Examples 6 and 10, GW766994 dissolved
in DMSO (2 mg/mL and 1.5 mg/mL) did not have inhibitory effects of
CNV, at both concentrations, as compared to DMSO.
[0404] Conclusions
[0405] The composition comprising GW766994 and hydrophobic
group-introduced CS was shown to be able to be used as a drug for
treating a posterior eye disease, especially AMD, which exhibited
medicinal effects higher than those of GW766994. Further, as the
"hydrophobic group" in the hydrophobic group-introduced GAG, a
group derived from an alicyclic compound, such as cholanic acid
(the basic skeleton of bile acids, such as lithocholic acid), and a
group derived from a fatty acid, such as oleic acid, were shown to
be able to be used.
(Test Example 17) Assay of Degeneration of Tissues Caused by
Intravitreal Administration of a Chemokine Receptor Activity
Regulator Solution Used in U.S. Pat. No. 8,592,482, which Contains
a Solubilizing Agent (DMSO), as Well as the Composition of the
Present Invention
[0406] Test substances were intravitreally administered once to
rats, and the intraocular conditions were observed.
[0407] Test Substances
[0408] The following test substances were used in the assay.
1) RS504393 (0.01 mg/mL) 2) PS372424 (0.1 mg/mL) 3) Composition 37
(containing 0.01 mg/mL of RS504393) 4) Composition 38 (containing
0.1 mg/mL of PS372424) 5) Composition 22 (containing 0.86 mg/mL of
GW766994) 6) Composition 19 (containing 1.03 mg/mL of GW766994)
(The samples for administration to animals prepared in Examples 14
(Composition 37 and RS504393) and 15 (Composition 38 and PS372424)
and 22 (Composition 22) and 19 (Composition 19) were used as
Composition 37, RS504393, Composition 38, PS372424, Composition 22
and Composition 19, respectively.)
[0409] Method
[0410] Substantially the same procedure as in Method for Test
Example 13 was repeated.
[0411] Results of Assay
[0412] The results of the intraocular imaging are shown in FIGS.
19A to 19F.
[0413] As pointed out with an arrow in the figures, each of the
chemokine receptor activity regulator solutions (RS504393 (FIG.
19A) and PS372424 (FIG. 19B)) containing a solubilizing agent
caused degeneration of the lenticular tissue. In contrast, with
respect to Composition 37 (FIG. 19C), Composition 38 (FIG. 19D),
Composition 22 (FIG. 19E) and Composition 19 (FIG. 19F), each of
which is a composition comprising the GAG derivative and chemokine
receptor activity regulator, no degeneration of the lenticular
tissue was observed.
[0414] Summary
[0415] The composition comprising the GAG derivative and chemokine
receptor activity regulator was shown to exhibit excellent
regulatory activity against a chemokine receptor, as compared to a
chemokine receptor activity regulator solution containing a
solubilizing agent, while suppressing occurrence of rapid
degeneration of tissues.
(Test Example 18) Assay of CNV Inhibitory Effects Using CS
[0416] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of CS.
[0417] Test Substances
[0418] CS (average molecular weight: about 140,000, manufactured by
SEIKAGAKU CORPORATION) was dissolved in PBS to obtain a CS solution
(20 mg/mL). The following test substances were used in the
assay.
1) CS (20 mg/mL)
2) PBS
[0419] Method
[0420] Substantially the same procedure as in (Method) for Test
Example 1 was repeated.
[0421] Statistical Analysis
[0422] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0423] Results of Assay
[0424] The results of measurement of the CNV area are given in the
table below and FIG. 16.
[0425] CS did not show significant inhibitory effects of CNV, as
compared to PBS.
TABLE-US-00017 TABLE 17 mean S. E. n Test results PBS 29359 1743 10
CS 30462 1865 8 N. S. N. S.: Not Significant (vs. PBS), t-test
[0426] Administration of CS alone did not show significant
medicinal effects as a drug for treating age-related macular
degeneration.
(Test Example 19) Assay of CNV Inhibitory Effects Using Compound
3
[0427] Laser-induced CNV models of rat were prepared, and
neovascularization inhibitory effect was examined with respect to
intravitreal administration of Compound 3
[0428] Test Substances
[0429] CS (average molecular weight: about 40,000, manufactured by
SEIKAGAKU CORPORATION) was dissolved in PBS to obtain a CS solution
(10 mg/mL). The following test substances were used in the
assay.
1) Compound 3
[0430] 2) CS (10 mg/mL)
[0431] Method
[0432] Substantially the same procedure as in (Method) for Test
Example 1 was repeated, except that flat mounts were prepared 4
days after the preparation of models, instead of 10 days after the
preparation of models in Test Example 1.
[0433] Statistical Analysis
[0434] The areas of CNV in each group were analyzed by a test
(t-test) for unpaired two groups. The significance level was 5% on
both sides.
[0435] Results of Assay
[0436] The results of measurement of the CNV area are given in the
table below and FIG. 17.
[0437] Compound 3 did not show significant inhibitory effects of
CNV, as compared to CS.
TABLE-US-00018 TABLE 18 mean S. E. n Test results CS 50236 2371 8
Compound 3 51925 2776 8 N. S. N. S.: Not Significant (vs. CS),
t-test
[0438] Conclusions
[0439] Like the administration of CS alone, administration of the
cholanic acid-introduced CS alone did not show significant
medicinal effects as a drug for treating age-related macular
degeneration.
[0440] The entire disclosure of Japanese Patent Application No.
2015-110784 (filed: May 29, 2015) is incorporated herein by
reference.
[0441] All publications, patent applications and technical
standards mentioned in the present specification are incorporated
herein by reference to the same extent as if each individual
publication, patent application or technical standard was
specifically and individually indicated to be incorporated by
reference.
* * * * *