U.S. patent application number 11/908614 was filed with the patent office on 2009-01-08 for promoter for hard tissue formation.
This patent application is currently assigned to SEIKAGAKU CORPORATION. Invention is credited to Yuji Kaneda, Takashi Takata.
Application Number | 20090012278 11/908614 |
Document ID | / |
Family ID | 36991675 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090012278 |
Kind Code |
A1 |
Takata; Takashi ; et
al. |
January 8, 2009 |
Promoter For Hard Tissue Formation
Abstract
The present invention provides an agent which induces
acceleration of hard tissue formation, acceleration of cell
differentiation and increase in cellular alkaline phosphatase
activity, by directly acting on the cell. Specifically, it provides
a hard tissue formation promoter, a cell differentiation inducer
and a cellular alkaline phosphatase activity reinforcing agent
comprising, as an active ingredient, a glycosaminoglycan or a salt
thereof that keeps sulfate group and has the characteristics of the
following (1) and (2): (1) a basic structure is a disaccharide
repeating structure consisting of a hexuronic acid residue and a
glucosamine residue, (2) one or less of the position among a
2-position hydroxyl group of a hexuronic acid residue, a 6-position
hydroxyl group of a glucosamine residue and a 2-position amino
group of the glucosamine residue in the basic structure of the
aforementioned (1) does not have the sulfate group. In this
connection, it is preferable that the hexuronic acid residue is a
glucuronic acid residue or an iduronic acid residue.
Inventors: |
Takata; Takashi; (Hiroshima,
JP) ; Kaneda; Yuji; (Tokyo, JP) |
Correspondence
Address: |
SUGHRUE-265550
2100 PENNSYLVANIA AVE. NW
WASHINGTON
DC
20037-3213
US
|
Assignee: |
SEIKAGAKU CORPORATION
Tokyo
JP
|
Family ID: |
36991675 |
Appl. No.: |
11/908614 |
Filed: |
March 14, 2006 |
PCT Filed: |
March 14, 2006 |
PCT NO: |
PCT/JP2006/305052 |
371 Date: |
September 14, 2007 |
Current U.S.
Class: |
536/21 ;
536/55.1 |
Current CPC
Class: |
A61P 19/00 20180101;
A61K 31/728 20130101; A61K 31/726 20130101; A61P 43/00 20180101;
A61K 31/727 20130101; A61P 19/10 20180101; A61P 1/02 20180101; A61P
19/08 20180101 |
Class at
Publication: |
536/21 ;
536/55.1 |
International
Class: |
C08B 37/10 20060101
C08B037/10; C08B 37/00 20060101 C08B037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2005 |
JP |
2005-071023 |
Jun 16, 2005 |
JP |
2005-176311 |
Claims
1. A hard tissue formation promoter comprising, as an active
ingredient, a glycosaminoglycan or a salt thereof which has a
sulfate group and characteristics of the following (1) and (2): (1)
a basic structure is a disaccharide repeating structure consisting
of a hexuronic acid residue and a glucosamine residue, (2) one or
less of the position among a 2-position hydroxyl group of the
hexuronic acid residue, a 6-position hydroxyl group of the
glucosamine residue and a 2-position amino group of the glucosamine
residue in the basic structure of the aforementioned (1) does not
have the sulfate group.
2. The hard tissue formation promoter according to claim 1, wherein
the hexuronic acid residue is a glucuronic acid residue or an
iduronic acid residue.
3. The hard tissue formation promoter according to claim 1 or 2,
wherein the glycosaminoglycan which has sulfate group and the
aforementioned characteristics (1) and (2) is 1 or 2 or more
substances selected from a group consisting of heparin, a substance
which does not have the sulfate group on the 2-position hydroxyl
group of the hexuronic acid residue of a heparin, a substance which
does not have the sulfate group on the 6-position hydroxyl group of
the glucosamine residue of the heparin and a substance which does
not have the sulfate group on the 2-position amino group of the
glucosamine residue of the heparin.
4. The hard tissue formation promoter according to claim 1, wherein
the hard tissue is a bone.
5. A cell differentiation promoter comprising, as an active
ingredient, a glycosaminoglycan or a salt thereof which has a
sulfate group and has characteristics of the following (1) and (2):
(1) a basic structure is a disaccharide repeating structure
consisting of a hexuronic acid residue and a glucosamine residue,
(2) one or less of the position among a 2-position hydroxyl group
of the hexuronic acid residue, a 6-position hydroxyl group of the
glucosamine residue and a 2-position amino group of the glucosamine
residue in the basic structure of the aforementioned (1) does not
have the sulfate group.
6. The cell differentiation promoter according to claim 5, wherein
the hexuronic acid residue is a glucuronic acid residue or an
iduronic acid residue.
7. The cell differentiation promoter according to claim 5 or 6,
wherein the glycosaminoglycan which has sulfate group and has the
aforementioned characteristics (1) and (2) is 1 or 2 or more
substances selected from a group consisting of a heparin, a
substance which does not have the sulfate group on the 2-position
hydroxyl group of the hexuronic acid residue of the heparin, a
substance which does not have the sulfate group on the 6-position
hydroxyl group of the glucosamine residue of the heparin and a
substance which does not have the sulfate group on the 2-position
amino group of the glucosamine residue of the heparin.
8. The cell differentiation promoter according to claim 5, wherein
the cell is a bone marrow-derived mesenchymal cell or an
osteoblast.
9. A cellular alkaline phosphatase activity reinforcing agent
comprising, as an active ingredient, a glycosaminoglycan or a salt
thereof which has a sulfate group and the characteristics of the
following (1) and (2): (1) a basic structure is a disaccharide
repeating structure consisting of a hexuronic acid residue and a
glucosamine residue, (2) one or less of the position among a
2-position hydroxyl group of the hexuronic acid residue, a
6-position hydroxyl group of the glucosamine residue and a
2-position amino group of the glucosamine residue in the basic
structure of the aforementioned (1) does not have the sulfate
group.
10. The alkaline phosphatase activity reinforcing agent according
to claim 9, wherein the hexuronic acid residue is a glucuronic acid
residue or an iduronic acid residue.
11. The alkaline phosphatase activity reinforcing agent according
to claim 9 or 10, wherein the glycosaminoglycan which has sulfate
group and the aforementioned characteristics (1) and (2) is 1 or 2
or more substances selected from a group consisting of a heparin, a
substance which does not have the sulfate group on the 2-position
hydroxyl group of the hexuronic acid residue of the heparin, a
substance which does not have the sulfate group on the 6-position
hydroxyl group of the glucosamine residue of the heparin and a
substance which does not have the sulfate group on the 2-position
amino group of the glucosamine residue of the heparin.
12. The alkaline phosphatase activity reinforcing agent according
to claim 9, wherein the cell is a bone marrow-derived mesenchymal
cell or an osteoblast.
13. Use of a glycosaminoglycan or a salt thereof which has a
sulfate group and has characteristics of the following (1) and (2),
for producing a hard tissue formation promoter: (1) a basic
structure is a disaccharide repeating structure consisting of a
hexuronic acid residue and a glucosamine residue, (2) one or less
of the position among a 2-position hydroxyl group of the hexuronic
acid residue, a 6-position hydroxyl group of the glucosamine
residue and a 2-position amino group of the glucosamine residue in
the basic structure of the aforementioned (1) does not have the
sulfate group.
14. Use of a glycosaminoglycan or a salt thereof which has a
sulfate group and has characteristics of the following (1) and (2),
for producing a cell differentiation promoter: (1) a basic
structure is a disaccharide repeating structure consisting of a
hexuronic acid residue and a glucosamine residue, (2) one or less
of the position among a 2-position hydroxyl group of the hexuronic
acid residue, a 6-position hydroxyl group of the glucosamine
residue and a 2-position amino group of the glucosamine residue in
the basic structure of the aforementioned (1) does not have the
sulfate group.
15. Use of a glycosaminoglycan or a salt thereof which has a
sulfate group and has characteristics of the following (1) and (2),
for producing a cellular alkaline phosphatase activity reinforcing
agent: (1) a basic structure is a disaccharide repeating structure
consisting of a hexuronic acid residue and a glucosamine residue,
(2) one or less of the position among a 2-position hydroxyl group
of the hexuronic acid residue, a 6-position hydroxyl group of the
glucosamine residue and a 2-position amino group of the glucosamine
residue in the basic structure of the aforementioned (1) does not
have the sulfate group.
Description
TECHNICAL FIELD
[0001] The present invention relates to a hard tissue formation
promoter comprising glycosaminoglycan or a salt thereof and the
like.
BACKGROUND OF THE INVENTION
[0002] The following abbreviations are used in the application
documents of the present application.
ALP: alkaline phosphatase .alpha.MEM: minimum essential medium
alpha medium BMP-2: bone morphogenetic protein-2 BSP: bone
sialoprotein COLI: type I collagen FBS: fetal bovine serum GAG:
glycosaminoglycan GlcN: glucosamine GlcA: glucuronic acid Hep:
heparin HexA: hexuronic acid HS: heparan sulfate IdoA: iduronic
acid PCR: polymerase chain reaction RT-PCR: reverse transcription
PCR 2DSH: 2-O-desulfated Hep (2-O-desulfated heparin) (a substance
in which a 2-position hydroxyl group of HexA residue in Rep is
desulfated) [0003] 6DSH: 6-O-desulfated Hep (6-O-desulfated
heparin) (a substance in which a 6-position hydroxyl group of GlcN
residue in Hep is desulfated) [0004] NDSH: N-desulfated Rep
(N-desulfated heparin) (a substance in which a 2-position amino
group of GlcN residue in Hep is desulfated)
[0005] Background art of the present invention is described in the
following.
[0006] In Patent Reference 1, an intercellular connection promoter
comprising, as the active ingredient, GAG which has a sulfate and a
disaccharide repeating structure consisting of a HexA residue and a
GlcN residue as the basic structure, wherein said GAG comprises the
HexA residue in which a 2-position hydroxyl group is not
esterificated with sulfuric acid or the GlcN residue in which a
2-position amino group is not sulfaminated is described.
[0007] In Patent Reference 2, an intercellular connection
suppressor comprising, as the active ingredient, GAG which has a
sulfate group and a disaccharide repeating structure consisting of
a HexA residue and a GlcN residue as the basic structure, wherein
said GAG contains the Glc residue in which a 6-position hydroxyl
group is not esterificated with sulfuric acid is described,
[0008] Patent Reference 3 discloses a certain species of desulfated
Rep, and discloses an accelerator of a basic fibroblast growth
factor activity comprising the same as an active ingredient and a
composition which comprises the same and the basic fibroblast
growth factor and accelerates activity of the basic fibroblast
growth factor upon the cell growth.
[0009] Patent Reference 4 discloses a hyaluronic acid fraction
having a bone formation activity and a molecular weight of from 20
kDa to 40 kDa.
Patent Reference 1: JP-A-2003-113090
Patent Reference 2: JP-A-2003-119146
Patent Reference 3: International Publication No. 96/01278
Patent Reference 4: Japanese Patent No. 3333205
[0010] However, there is no disclosure or suggestion that a GAG or
a salt thereof, which has a sulfate group and a disaccharide
repeating structure consisting of a HexA residue and a GlcN residue
as a basic structure, wherein one or less of the position among a
2-position hydroxyl group of the HexA residue, a 6-position
hydroxyl group of the GlcN residue and a 2-position amino group of
the GlcN residue in the basic structure does not have the sulfate
group, by itself has activity of directly accelerating hard tissue
formation, accelerating differentiation of a cell and increasing
ALP activity of the cell.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0011] The object of the present invention is providing an agent
which induces acceleration of hard tissue formation, acceleration
of cell differentiation and increase in ALP activity of a cell, by
directly acting on the cell.
Means for Solving the Problems
[0012] To solve the aforementioned problems, the inventors of the
present invention have conducted intensive studies and found as a
result that a certain species of GAG or a salt thereof induces
acceleration of hard tissue formations acceleration of cell
differentiation and increase in ALP activity of a cell, by directly
acting upon the cell. As a result, the present invention was
accomplished.
[0013] Accordingly, the present invention provides a hard tissue
forming agent comprising, as an active ingredient, a GAG or a salt
thereof that has a sulfate group and the characteristics of the
following (1) and (2) (to be referred to as "forming promoter of
the present invention" hereinafter):
[0014] (1) a basic structure is a disaccharide repeating structure
consisting of a HexA residue and a GlcN residue,
[0015] (2) one or less of the position among a 2-position hydroxyl
group of the HexA residue, a 6-position hydroxyl group of the GlcN
residue and a 2-position amino group of the GlcN residue in the
basic structure of the aforementioned (1) does not have the sulfate
group.
[0016] It is preferable that the HexA residue is a GlcA residue or
an IdoA residue. Also, as the GAG which has the sulfate group and
the aforementioned characteristics, 1 or 2 or more substances
selected from the group consisting of Hep, a substance which does
not have the sulfate group on the 2-position hydroxyl group of HexA
residue of Hep, a substance which does not have the sulfate group
on the 6-position hydroxyl group of GlcN residue of Hep and a
substance which does not have the sulfate group on the 2-position
amino group of GlcN residue of Hep. Additionally, it is desirable
that the hard tissue is a bone.
[0017] Also, the present invention provides a cell differentiation
promoter comprising, as an active ingredient, a GAG or a salt
thereof that has a sulfate group and the characteristics of the
following (1) and (2) (to be referred to as "differentiation
promoter of the present invention" hereinafter):
[0018] (1) a basic structure is a disaccharide repeating structure
consisting of a HexA residue and a GlcN residue,
[0019] (2) one or less of the position among a 2-position hydroxyl
group of the HexA residue, a 6-position hydroxyl group of the GlcN
residue and a 2-position amino group of the GlcN residue in the
basic structure of the aforementioned (1) does not have the sulfate
group.
[0020] It is preferable that the HexA residue is a GlcA residue or
an IdoA residue, Also, as the GAG which has sulfate group and the
aforementioned characteristics, 1 or 2 or more substances selected
from the group consisting of a Hep, a substance which does not have
the sulfate group on a 2-position hydroxyl group of the HexA
residue of the Hep, a substance which does not have the sulfate
group on a 6-position hydroxyl group of the GlcN residue of the Hep
and a substance which does not have the sulfate group on the
2-position amino group of the GlcN residue of the Hep is
specifically preferable. Additionally, it is preferable that the
cell is a bone marrow-derived mesenchymal cell or an
osteoblast.
[0021] Also, the present invention provides a cellular ALP activity
reinforcing agent comprising, as an active ingredient, a GAG or a
salt thereof that has a sulfate group and the characteristics of
the following (1) and (2) (to be referred to as "activity
reinforcing agent of the present invention" hereinafter):
[0022] (1) a basic structure is a disaccharide repeating structure
consisting of a HexA residue and a GlcN residue,
[0023] (2) one or less of the position among a 2-position hydroxyl
group of the HexA residue, a 6-position hydroxyl group of the GlcN
residue and the 2-position amino group of the GlcN residue in the
basic structure of the aforementioned (1) does not have the sulfate
group.
[0024] It is preferable that the HexA residue is a GlcA residue or
an IdoA residue. Also, as the GAG which has the sulfate group and
the aforementioned characteristics, 1 or 2 or more substances
selected from a group consisting of a Hep, a substance which does
not have sulfate group on a 2-position hydroxyl group of the HexA
residue of the Hep, a substance which does not have the sulfate
group on a 6-position hydroxyl group of the GlcN residue of the Hep
and a substance which does not have the sulfate group on a
2-position amino group of the GlcN residue of the Hep is
specifically preferable. Additionally, it is preferable that the
cell is a bone marrow-derived mesenchymal cell or an
osteoblast.
[0025] Hereinafter, the forming promoter of the present invention,
differentiation promoter of the present invention and activity
reinforcing agent of the present invention are called "agent of the
present invention" as a whole.
EFFECT OF THE INVENTION
[0026] The agent of the present invention is markedly useful, since
the GAG which is the active ingredient exerts superior effect by
directly acting on the hard tissue formation, cell differentiation
acceleration or cellular ALP activity increase.
BEST MODE FOR CARRYING OUT THE INVENTION
[0027] The following describes the present invention in detail
based on the best mode for carrying out the present invention.
<1> Active Ingredient of the Agent of the Present
Invention
[0028] The agent of the present invention comprising as an active
ingredient, a GAG or a salt thereof that has sulfate group and the
characteristics of the following (1) and (2):
[0029] (1) a basic structure is a disaccharide repeating structure
consisting of a HexA residue and a GlcN residue,
[0030] (2) one or less of the position among a 2-position hydroxyl
group of the HexA residue, a 6-position hydroxyl group of the GlcN
residue and the 2-position amino group of the GlcN residue in the
basic structure of the aforementioned (1) does not have the sulfate
group.
[0031] In this case, although the HexA residue is not particularly
limited, it is preferable that the Hex A residue is a GlcA residue
or an IdoA residue. Particularly, it is preferable that the GlcA
residue is a D-GlcA residue and it is preferable that the IdoA
residue is a L-IdoA residue. Also, although the GlcN residue is not
particularly limited, it is preferable that the GlcN residue is a
D-GlcN residue.
[0032] Additionally, amino group of the GlcN residue may be
acetylated. In that case, the moiety of the GlcN residue becomes
N-acetylglucosamine (GlcNAc).
[0033] The basic structure of the GAG as the active ingredient of
the agent of the present invention is a disaccharide repeating
structure consisting of such HexA residue and GlcN residue. Namely,
when the HexA residue is regarded as "a", and the GlcN residue as
"b", the GAG as the active ingredient of the agent of the present
invention consists of a basic structure of "a-b-a-b-.cndot. .cndot.
.cndot. .cndot." or "b-a-b-a-.cndot. .cndot. .cndot. .cndot.". In
this connection, the "-" represents glycosidic linkage. When "a" is
GlcA residue, it is preferable that the glycosidic linkage between
"a" and "b" (a-b) is .beta.1.fwdarw.4 linkage, Also, when "a" is
the IdoA residue, it is preferable that the glycosidic linkage
between "a" and "b" (a-b) is .alpha.1.fwdarw.4 linkage.
Additionally, it is preferable that the glycosidic linkage between
"b" and "a" (b-a) is .alpha.1.fwdarw.4 linkage.
[0034] Such a GAG as the active ingredient of the agent of the
present invention comprises a sulfate group. Although the position
which has the sulfate group in the aforementioned basic structure
is not particularly limited, at least 1 position selected from a
2-position hydroxyl group of the HexA residue, a 6-position
hydroxyl group of the GlcN residue and a 2-position amino group of
the GlcN residue is preferable. Examples of the GAG which has the
sulfate group at such a position include Hep and HS, for
example.
[0035] However, in order to use it as the active ingredient of the
agent of the present invention, it is necessary 1 or less of the
positions among the 2-position hydroxyl group of the HexA residue,
the 6-position hydroxyl group of the GlcN residue and the
2-position amino group of the GlcN residue in the aforementioned
basic structure does not have the sulfate group. Accordingly,
examples of the active ingredient of the agent of the present
invention include a Hep (all of the 2-position hydroxyl group of
HexA residue, the 6-position hydroxyl group of the GlcN residue and
the 2-position amino group of the GlcN residue have the sulfate
group), 2DSH, 6DSH, NDSH and the like.
[0036] In this connection, GAG is a polysaccharide (polymer), and
it does not have to say that it is a technical common sense of said
technical field that the complete sameness of the size of molecule,
composition and sequences of the constituting sugars, position of
the sulfate group, the number of the sulfate groups, and the like,
is substantially impossible. Additionally, it is also a technical
common sense of said technical field that, for example, when it is
said that "the 2-position hydroxy group of the HexA residues does
not have the sulfate group" in such a GAG, it does not mean that
"the 2-position hydroxy groups of all of the HexA residues in the
GAG molecule do not have the sulfate group completely", but means
that "it is sufficient when the majority of the 2-position hydroxy
groups of HexA residues in the GAG molecule do not have the sulfate
group, so that a case in which some 2-position hydroxy groups of
HexA residues have the sulfate group is not excluded". Thus, these
descriptions in the application documents of the instant
application are also should be understood in accordance with the
technical common sense in said technical field.
[0037] It is necessary that 1 or less of the positions among the
2-position hydroxyl group of the HexA residue, the 6-position
hydroxyl group of the GlcN residue and the 2-position amino group
of the GlcN residue in the aforementioned basic structure do not
have the sulfate group.
[0038] The origin of GAG as the active ingredient of the agent of
the present invention is not limited too. A natural product may be
used as such; a natural product may further processed by chemical
methods, enzymatic methods or other methods, or a chemically
synthesized substance may be used.
[0039] For example, when a Hep is used as the active ingredient of
the agent of the present invention, a Hep obtained from a natural
product or the like by a conventionally known method may be used as
such, or a Hep which is already on the market as a pharmaceutical
preparation or a reagent may be used. Alternatively, a substance in
which the degree of sulfation of HS is increased to a similar level
of Hep by conventionally known chemical methods, enzymatic methods
or other methods may be used. The term "Hep" in the application
documents of the present application includes not only a pure and
simple Hep but also such a substance which can be recognized as
substantially the same as the Hep.
[0040] Additionally, also in the case where 2DSH, 6DSH or NDSH as
the active ingredient of the agent of the present invention are
used, it can be produced by the method described in the
aforementioned Patent Reference 1 and Patent Reference 2.
[0041] Size (weight average molecular weight) of the GAG which can
be used as the active ingredient of the agent of the present
invention is not particularly limited, as long as it can be
recognized as a "polysaccharide" by those skilled in the art in the
technical field. For example, its weight average molecular weight
is preferably from about 5,000 to 20,000, more preferably from
about 10,000 to 15,000. In this connection, the weight average
molecular weight can be measured in accordance with the method
described in WO 00/06608.
[0042] Also, purity of the GAG which is used as the active
ingredient of the agent of the present invention is not
particularly limited too, and can be appropriately selected
according to the case, object and the like of applying the agent of
the present invention.
[0043] For example, when the agent of the present invention is
applied to objects which require high degree sterility, cleanness
and the like such as a tissue in the living body, a cell cultured
under an aseptic condition and the like, it is preferable to use a
substance which is highly purified and substantially does not
include medically and culturally unacceptable materials such as
endotoxin, microorganisms and the like. Additionally, when the
agent of the present invention is applied to an object which does
not require high degree sterility, cleanness and the like to that
extent, the purification degree may be appropriately reduced in
response to the case and object.
[0044] Also, the "salt of GAG" is not particularly limited too, and
can be appropriately selected according to the case, object and the
like of applying the agent of the present invention. For example,
when it is applied to a tissue in the living body, a medically
acceptable salt may be selected. As the salt of GAG, for example,
an alkali metal salt (sodium salt, lithium salt, potassium salt or
the like), an alkaline earth metal salt, salts with inorganic bases
such as an ammonium salt and the like, or salts with organic bases
and the like such as a diethanolamine salt, a cyclohexylamine salt,
an amino acid salts and the like can be exemplified. A sodium salt
is particularly preferable.
[0045] The agent of the present invention may contain one species
of the aforementioned GAG or a salt thereof alone as the active
ingredient, or may contain two or more species of the
aforementioned GAG or salts thereof as the active ingredient.
[0046] By using such a GAG or a salt thereof, preparation of a
forming promoter of the present invention, a differentiation
promoter of the present invention and an activity reinforcing agent
of the present invention, which has markedly excellent effects is
possible.
<2> Dosage Forms and the Like of the Agent of the Present
Invention
[0047] Dosage forms and the like of the agent of the present
invention are not particularly limited, as long as they contain the
aforementioned GAG or a salt thereof, and the acceleration activity
of hard tissue formation, acceleration activity of cell
differentiation and reinforcing activity of ALP activity of a cell
are exerted by it. They can be appropriately selected according to
the case, object and the like of applying the agent of the present
invention.
[0048] For example, the agent of the present invention may be made
into an agent of a solution state containing the aforementioned GAG
or solid preparations of a salt thereof, or into powders, granules
and the like. Alternatively, for example, when it is provided as a
solution state agent, it may be provided under a frozen state;
provided as the solution as such; or provided as an agent which is
dissolved prior to its use.
[0049] Concentration of the aforementioned GAG or a salt thereof in
the agent of the present invention is not particularly limited too,
and can be appropriately selected according to the case, object and
the like of applying the agent of the present invention.
[0050] Preparation of the Agent of the Present Invention into
Pharmaceutical preparations can be carried out by conventionally
known methods. Additionally, components for removing other agent
and a stabilizer, an emulsifying agent, an adjusting agent of
osmotic pressure, a buffer agent, a tonicity agent, a corrective, a
preservative, a pH adjusting agent, a soothing agent, a coloring
agent, an excipient, a binder, a lubricant, a disintegrating agent
and the like other components can be formulated in preparing the
pharmaceutical preparations, as long as they do not have a bad
influence upon the aforementioned GAG or a salt thereof and also do
not exert influence upon the effect of the present invention,
<3> Use, Using Method and the Like of the Agent of the
Present Invention
[0051] The agent of the present invention can be made into a hard
tissue formation promoter, a cell differentiation promoter or a
cell ALP activity reinforcing agent. When it is made into a hard
tissue formation promoter (formation promoter of the present
invention), it is preferable that the "hard tissue" is a bone. In
this connection, the "acceleration of hard tissue formation"
includes a meaning of accelerating calcification. Also, when it is
made into a cell differentiation promoter (differentiation promoter
of the present invention) or a cell ALP activity reinforcing agent
(activity reinforcing agent of the present invention), it is
preferable that the "cell" is a bone marrow-derived mesenchymal
cell or an osteoblast. Additionally, the agent of the present
invention may be made into a single agent at one of these uses or a
single agent aiming at two or more of these uses.
[0052] Administration and application methods of the agent of the
present invention are not limited, as long as the aforementioned
GAG or a salt thereof which is used as its active ingredient is
under such a condition that it can contact with an object to be
applied such as a living body tissue, cell or the like.
[0053] The agent of the present invention can be used as a
medicament, a reagent and the like aiming, for example, at
accelerating hard tissue formation, accelerating cell
differentiation or reinforcing cell ALP activity. When the agent of
the present invention is used as a medicament, its dose should be
individually set depending on the purpose of the administration
(prevention, maintenance (prevention of deterioration), alleviation
(improvement of symptoms) or treatment), kind of the disease,
symptom, sex, age and body weight of the patient, administration
region, administration method and the like. Although it is not
particularly limited, in general, approximately from 0.03
mg/administration region to 3 mg/administration region per once per
adult can be administered as the aforementioned GAG or a salt
thereof.
[0054] When the agent of the present invention is made into a
medicament, although the animal to which it is administered is not
particularly limited too, a vertebrate is preferable; a mammal is
more preferable; and human is particularly preferable. In this
case, it can be administered for example to the following animals,
according to each use.
[0055] When it is used as a hard tissue formation promoter, it can
be administered to an animal which is under a condition that
acceleration of the formation of a hard tissue is required.
Examples of such a condition include a case of suffering from bone
fracture, bone damage, osteoporosis or the like, a condition of
just after the application of a dental treatment, an orthopedic
treatment, bone prosthesis or the like, and the like.
[0056] When it is used as a cell differentiation promoter, it can
be administered to an animal which is under such a condition that
differentiation of cells is required. Particularly, it is desirable
to administer it to an animal which is under a condition that
differentiation of a bone marrow-derived mesenchymal cell, an
osteoblast or the like is required. Examples of the condition in
which differentiation of such a cell is required are the same as
the case of the aforementioned hard tissue formation promoter.
[0057] When it is used as a cell ALP activity reinforcing agent, it
can be administered to an animal which is under a condition that
increase in ALP activity in cells is required. Examples of such a
condition are the same as the case of the aforementioned hard
tissue formation promoter and cell differentiation promoter.
[0058] When the agent of the present invention is used as a
reagent, although its application object is not particularly
limited too, examples of the object includes a tissue collected
from a living body, a cultured cell and the like.
[0059] Additionally, the aforementioned GAG or a salt thereof as
the active ingredient of the agent of the present invention may be
used as a raw material with the aim of accelerating hard tissue
formation, accelerating cell differentiation or reinforcing ALP
activity in cells, by immobilizing it on the surface of an
appropriate insoluble carrier or mixing it with an appropriate raw
material. As such an insoluble carrier (raw material), those which
have various shapes, such as beads, film, plate, monofilament,
non-woven fabric, sponge, cloth, knitting, staple fiber, tube,
hollow fiber, hydroxyapatite and the like, can be used.
Specifically, it can be used in an implant, bone cement, a bone
prosthetic agent, a root canal filler, a bone plate, an artificial
joint and the like as medical composite materials. Additionally,
the agent of the present invention can also be applied as a raw
material in the field of regeneration medical treatment.
EXAMPLES
[0060] The present invention is specifically explained in detail
based on examples as follows:
<1> Materials
[0061] The materials used in the examples are described below.
1. Cell Strains
[0062] A mouse bone marrow-derived mesenchymal cell strain, ST2
cell strain, and a mouse osteoblast-like cell strain having more
high differentiation degree, MC3T3-E1 cell strain, (both obtained
from RIKEN CELL BANK), were used. In the following, culturing of
these cells was carried out all under a condition of 37.degree. C.
and 5% CO.sub.2, Additionally, antibiotics (100 U/ml in final
concentration of penicillin G, 100 .mu.g/ml in final concentration
of streptomycin and 250 .mu.g/ml in final concentration of
gentamicin) were allowed to coexist in all of the media to be used
for the culturing of these cells.
2. Samples to be tested
[0063] The followings were used as the samples to be tested. All of
the samples provided by Seikagaku Corporation were used. In this
connection, the abbreviations in parentheses mean those shown in
FIG. 1.
[0064] Hyaluronic acid having a weight average molecular weight of
10,000 (hyaluronic acid 10,000)
[0065] Hyaluronic acid having a weight average molecular weight of
60,000 (hyaluronic acid 60,000)
[0066] Hyaluronic acid having a weight average molecular weight of
350,000 (hyaluronic acid 350,000)
[0067] Hyaluronic acid having a weight average molecular weight of
1,000,000 (hyaluronic acid 1,000,000)
[0068] Hyaluronic acid having a weight average molecular weight of
1,500,000 (hyaluronic acid 1,500,000)
[0069] Hyaluronic acid having a weight average molecular weight of
2,500,000 (hyaluronic acid 2,500,000)
[0070] Hep
[0071] 2DSH
[0072] 6DSH
[0073] NDSH
[0074] NSH (a substance in which the 2-position amino group of the
GlcN residue in the Hep is sulfated, and the 2-position hydroxyl
group of the HexA residue and the 6-position hydroxyl group of the
GlcN residue do not have the sulfate group)
[0075] 6SH (6-O-sulfated Rep; 6-O-sulfated heparin; a substance in
which the 6-position hydroxyl group of the GlcN residue is
sulfated, and the 2-position hydroxyl group of the HexA residue and
the 2-position amino group of the GlcN residue do not have the
sulfate group)
[0076] 2SH (2-O-sulfated Hep; 2-O-sulfated heparin; a substance in
which the 2-position hydroxyl group of the HexA residue in the Hep
is sulfated, and the 6-position hydroxyl group of the GlcN residue
and the 2-position amino group of the GlcN residue do not have the
sulfate group)
[0077] DSH (desulfated Hep; desulfated heparin; a substance in
which the Hep was completely desulfated)
[0078] NAH (N-acetyl heparosan)
[0079] CDS (dermatan sulfate)
[0080] 6OSCDS (the 6-position of galactosamine residue in dermatan
sulfate was sulfated)
[0081] All of these sugars can be obtained from Seikagaku
Corporation. As the Hep, an article available from the same company
as a reagent was used. In this connection, the 2DSH, 6DSH and NDSH
were produced using Hep as the material in accordance with the
methods described in JP-A-2003-113090 and JP-A-2003-119146.
[0082] The Hep, 2DSH, 6DSH and NDSH used herein were analyzed by
the "Enzymatic disaccharide analysis by a combination of digestion
with a GAG degrading enzyme and high performance liquid
chromatography" described in WO 00/06608 (Table 1). In the table,
.DELTA.DiHS-0S represents
2-acetamido-2-deoxy-4-O-(4-deoxy-.alpha.-L-threo-hex4-enopyranosyluronic
acid)-D-glucose, .DELTA.DiHS-NS represents
2-deoxy-2-sulfamino-4-O-(4-deoxy-.alpha.-L-threo-hex-4-enopyranosyluronic
acid)-D-glucose, .DELTA.DiHS-6S represents
2-acetamido-2-deoxy-4-O-(4-deoxy-.alpha.-L-threo-hex-4-enopyranosyluronic
acid)-6-O-sulfo-D-glucose, .DELTA.DiHS-US represents
2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-.alpha.-L-threo-hex-4-enopyran-
osyluronic acid)-D-glucose, .DELTA.DiHS-di(6,N)S represents
2-deoxy-2-sulfamino-4-O-(4-deoxy-.alpha.-L-threo-hex-4-enopyranosyluronic
acid)-6-O-sulfo-D-glucose, .DELTA.DiHS-di(U,N)S represents
2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-.alpha.-L-threo-hex-4-enopyran-
osyluronic acid)-D-glucose, .DELTA.DiHS-di(U,6)S represents
2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-.alpha.-L-threo-hex-4-enopyran-
osyluronic acid)-6-O-sulfo-D-glucose and .DELTA.DiHS-tri(U,6,N)S
represents
2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-.alpha.-L-threo-hex-4-enopyran-
osyluronic acid)-6-O-sulfo-D-glucose, respectively.
TABLE-US-00001 TABLE 1 Substance .DELTA.DiHS- name 0S NS 6S US
di(6,N)S di(U,N)S di(U,6)S tri(U,6,N)S Hep 3.9 2.3 3.8 1.8 11.4 6.4
1.5 68.9 2DSH 6.4 12.2 4.6 0 76.8 0 0 0 6DSH 9.3 25.9 0 6.7 4.7
53.4 0 0 NDSH 3.5 0 21.8 7.1 3.1 0 58.9 0
[0083] Additionally, when their weight average molecular weights
were measured by the method described in WO 00/06608, Hep was
14,000, 2DSH was 12,000, 6DSH was 12,500 and NDSH was 11,000.
3. Other Reagent
[0084] As FBS, manufactured by EQUITECH-BIO was used.
<2> Methods and Results
1. Actions on cell differentiation (1)
[0085] Cellular ALP activity was measured as the index of cell
differentiation. Cells were inoculated onto a 24 well culture plate
at a density of 5.times.10.sup.3 cells/well and cultured by adding
50 g/ml in final concentration of each sample to 2% FEBS-containing
.alpha.MEM. On the first week after the cells reached confluent,
ALP activity was measured by the following enzymologic method
(Bessey-Lowry method).
Measuring Method of ALP Activity
[0086] After washing the cultured cells with PBS, the cells were
disrupted and stirred for 40 seconds in 10 mM Tris-HCl buffer (pH
7.4, 500 .mu.l) using an ultra sonic homogenizer (Handy Sonic model
UR-20P; mfd. by Tomy Seiko Co., Ltd.). Next, 25 .mu.l of the sample
liquid was added to 125 .mu.l of an ALP buffer (0.1 M carbonate
buffer pH 9.8, 6.7 mM p-nitrophenyl phosphate, 2 mM MgCl.sub.2) and
incubated at 37.degree. C. for 30 minutes. After terminating the
reaction by adding 125 .mu.L of 0.2 N NaOH, the absorbance at 405
nm was measured by using a microplate reader (Model 550,
manufactured by Bio-Rad Laboratories). The calculated ALP activity
was shown by unit per the number of cells. Additionally, a case in
which the sample to be tested was not added was used as a
control.
[0087] The results in which each sample to be tested was added to
the cell strain MC3T3-E1 are shown in FIG. 1, and the results in
which a sample to be tested (Hep, 2DSH, 6DSH or NDSH) was added to
the cell strain MC3T3-E1 are shown in FIG. 2, and the results in
which a sample to be tested (Hep, 2DSH, 6DSH or NDSH) was added to
the cell strain ST2 are shown in FIG. 3, respectively.
[0088] As shown in FIG. 1 and FIG. 2, each of the Rep, 2DSH, 6DSH
and NDSH increased the ALP activity of the cell strain MC3T3-E1.
Among them, it was shown that Rep is particularly effective for the
increase in ALP activity of the cell strain MC3T3-E1.
[0089] Also, as shown in FIG. 3, each of the Hep, 2D SH, 6DSH and
NDSH increased the ALP activity of the cell strain ST2. Among them,
it was shown that NDSH is particularly effective for the increase
in ALP activity of the cell strain ST2.
[0090] From these results, it was shown that all of the Hep, 2DSH,
6DSH and NDSH increase the ALP activity of the bone marrow-derived
mesenchymal cell and osteoblast. This indicates that having sulfate
groups at two or more positions selected from the group consisting
of the 2-, 6- and N-positions in the Hep structure is necessary for
the increase in ALP activity of these cells.
[0091] It was shown that Rep among them particularly effectively
increases the ALP activity of osteoblast, and NDSH particularly
effectively increases the ALP activity of the bone marrow-derived
mesenchymal cell.
2. Actions on Cell Differentiation (2)
[0092] The ALP activity was measured in the same manner, by
changing the medium of the aforementioned "Actions on cell
differentiation (1)" to a medium which does not contain serum
(serum-free medium). The results of adding each sample to be tested
(Hep, 2DSH, 6DSH or NDSH) to the cell strain MC3T3-E1 are shown in
FIG. 4, and the results of adding each sample to be tested (Hep,
2DSH, 6DSH or NDSH) to the cell strain ST2 are shown in FIG. 5,
respectively. In this connection, the calculated ALP activity was
shown by a numerical value of cells per DNA weight.
[0093] As shown in FIG. 4, all of the Hep, 2DSH, 6DSH and NDSH
increased the ALP activity of the cell strain MC3T3-E1 under the
serum-free condition. It was shown that Hep, 2DSH and NDSH among
them are particularly effective in reinforcing the ALP activity of
the cell strain MC3T3-E1.
[0094] Also as shown in FIG. 5, all of the Hep, 2DSH, 6DSH and NDSH
increased the ALP activity of the cell strain ST2 under the
serum-free condition. It was shown that 2DSH among them is
particularly effective in reinforcing the ALP activity of the cell
strain ST2.
[0095] From these results, it was shown that all of the Hep, 2DSH,
6DSH and NDSR increase the ALP activity of the bone marrow-derived
mesenchymal cell and osteoblast under a serum-free condition. This
indicates that a Hep in two or more positions selected from the
group consisting of the 2-, 6- and N-positions in the Hep structure
which have the sulfate group has the action to increase ALP
activity by directly acting upon these cells without requiring
various factors contained in the serum.
[0096] It was shown that Hep, 2DSH and NDSH among them particularly
effectively increase the ALP activity of osteoblast, and 2DSH
particularly effectively increases the ALP activity of the bone
marrow-derived mesenchymal cell.
3. Actions on. Cell Differentiation (3)
[0097] In the aforementioned "Actions on cell differentiation (1)",
respective cells were recovered and total RNA was extracted on the
first and second weeks after the addition of a sample to be tested,
and expression of mRNA of COLI, ALP, osteocalcin, BSP and BMP-2 was
analyzed by RT-PCR.
[0098] As a result, high induction of ALP mRNA was confirmed in the
cell strain ST2 also by the RT-PCR analysis. In the cell strain
MC3T3-E1, induction of COLI and BSP mRNA was also observed in
addition to that of ALP.
4. Actions on Cell Differentiation (4)
[0099] In order to examine calcification ability of the cells,
alizarin staining (ALZ staining; stained in red indicates
accelerated calcification) was carried out in accordance with the
usual way, in the aforementioned "Actions on cell differentiation
(1)" on the first week after the cells reached confluent. The cells
were washed with PBS, fixed with 10 neutral-buffered formalin and
then stained with 0.01 Alizarin Red S (manufactured by Wako Pure
Chemical Industries).
[0100] As a result, Hep, 2DSH, 6DSH and NDSH accelerated formation
of calcification matter on both of the cell strains ST2 and
MC3T3-E1. Particularly on the cell strain ST2, Hep, 2DSH and NDSH
showed strong acceleration.
5. Actions on Cell Differentiation (5)
[0101] A Wistar rat of 8 weeks of age was subjected to general
anesthesia with ethyl carbamate 20 solution at a dose of 0.5 ml per
100 g body weight, and then the forehead skin and periosteum were
incised. Each sample to be tested (Hep, 2DSH, 6DSH or NDSH; 100
.mu.l for each) diluted to a concentration of 3 mg/ml with PBS was
transplanted under the cranial periosteum. In this case,
hydroxyapatite was used as the carrier.
[0102] Speficially, a block of hydroxyapatite (size: 3 mm in
length.times.2 mm in breadth.times.2 mm in height; adsorbs about
0.1 ml of solution) was soaked for 30 minutes in each sample to be
tested of a concentration of 3 mg/ml. It was used for the
transplantation.
[0103] A substance in which test sample-free PBS was used was used
as the control.
[0104] Each animal was sacrificed on the fourth week and eighth
week after transplantation of the sample to be tested, Then slices
of the transplanted parts were prepared and subjected to
hematoxylin-eosin staining (HE staining). It was observed under an
optical microscope to evaluate degree of the formation of a hard
tissue (bone).
[0105] As a result, only blood vessels and fibrous connective
tissues were observed but formation of bone was not observed in the
control group. On the other hand, formation of bone was observed in
each of the Hep group, 2DSH group, 6DSH group and NDSH group.
Remarkable bone formation was found particularly in the 2DSH
group.
6. Actions on Cell Differentiation (6)
[0106] In the same manner as in the aforementioned "5. Actions on
cell differentiation (5)", slices of the transplanted parts were
prepared by sacrificing each animal on the fourth week and sixth
week after transplantation of the sample to be tested (n=2 for each
group). Ratio of the area of formed bone (hard tissue) to the area
of the hydroxyapatite region observed in the image of said slice
was analyzed by using an image analyzing software (Scion image;
Scion Corporation). The results are shown in FIG. 6. In this
connection, the gray bar in FIG. 6 shows the results on the fourth
week, while the black bar shows the results on the sixth week,
respectively.
[0107] From FIG. 6, it was shown that formation of bone (hard
tissue) was confirmed within a broad range in the Hep group, 2DSH
group, 6DSH group and NDSH group, both on the fourth week and sixth
week, although it was hardly found in the control group. This
effect was remarkable particularly in the 2DSH group and NDSH
group.
7. Actions on Cell Differentiation (7)
[0108] In the same manner as in the aforementioned "5. Actions on
cell differentiation (5)", slices of the transplanted parts were
prepared by sacrificing each animal on the fourth week, eighth week
and twelfth week after transplantation of the sample to be tested
(n=5 for each group). Ratio of the area of bone tissue to the area
of the total tissues observed in the image of the slices of
transplanted region was analyzed using an image analyzing software
(Scion image; Scion Corporation). The results are shown in FIG. 7.
In this connection, the bars of respective weeks in FIG. 7 show the
results in the control group, 2DSH group, 6DSH group, NDSH group
and Hep group, respectively, starting from the left side.
[0109] From FIG. 7, although it was shown that formation of bone
(hard tissue) was confirmed within a broad range in the Hep group,
2DSH group, 6DSH group and NDSH group, all in the fourth week,
eighth week and twelfth week, it was hardly found in the control
group. This effect was remarkable particularly in the 2DSH group,
Hep group and NDSH group. Since these results are consistent with
the results of the "5. Actions on cell differentiation (5)" and "6.
Actions on cell differentiation (6)" which were carried out
separately and independently, it was shown that they are
reproducible.
8. Actions on Cell Growth
[0110] Respective cells were inoculated onto a 24 well culture
plate (manufactured by Falcon) in 5.times.10.sup.3 cells/well
portions. Starting on the first day after the inoculation, each
sample to be tested (Hep, 2DSH, 6DSH or NDSH) was added to the
culture liquid (.alpha.MEM containing 2% FBS) to a final
concentration of 50 .mu.g/ml. On the zero day, first day, third day
and sixth day after addition of the sample to be tested, the number
of cells was counted using a Coulter counter (COULTER ZI,
manufactured by Coulter Electronics Inc.). A case in which the
sample to be tested was not added was used as the control. The
results on the cell strain MC3T3-E1 are shown in FIG. 8, and the
results on the cell strain ST2 are shown in FIG. 9. In FIG. 8 and
FIG. 9, the axis of abscissa shows the number of days after
addition of the sample to be tested, and the axis of ordinate shows
the number of cells.
[0111] As shown in FIG. 8, although all of the samples to be tested
suppressed growth of the cell strain MC3T3-E1, but the growth
suppressing effects of 2DSH, 6DSH and NDSH were small in comparison
with that of Hep. Additionally, as shown in FIG. 9, although 2DSH
accelerated growth of the cell strain ST2, but Hep suppressed it on
the contrary.
[0112] From these results, it was shown that 2DSH exerts the action
to accelerate growth of the bone marrow-derived mesenchymal cell.
Also, it was shown that Hep exerts the action to suppress growth of
the bone marrow-derived mesenchymal cell.
[0113] Additionally it was shown that all of the Hep, 2DSH, 6DSH
and NDSH exert the action to suppress growth of the osteoblast.
[0114] When the above results were synthetically considered, it was
shown that 2DSH and NDSH are particularly effective in increasing
the ALP activity in the bone marrow-derived mesenchymal cell and
osteoblast and in forming a bone (hard tissue).
9. Preparation Examples
[0115] Although preparation examples of the agents of the present
invention are shown in the following, these are only examples and
dosage forms of the agents of the present invention are not limited
thereto.
TABLE-US-00002 (1) Ointments Hep 10 mg Sorbitan monostearate 7 mg
Polyoxyethylene sorbitan monostearate 7 mg Isopropyl palmitate 37
mg Petrolatum 37 mg Liquid paraffin 37 mg Cetanol 50 mg Glycerol 70
mg Magnesium stearate 2 mg
[0116] By adding purified water to the above components, 1 g of a
cream was prepared.
TABLE-US-00003 (2) Tablets 2DSH 100 mg Lactose 670 mg Potato starch
150 mg Crystalline cellulose 60 mg Light anhydrous silicic acid 50
mg
[0117] After mixing the above components, a solution prepared by
dissolving 30 mg of hydroxypropylcellulose in methanol (10% by
weight of hydroxypropylcellulose) was added thereto, and the
resulting mixture was kneaded and granulated. It was made into
granular forms by extruding through a 0.8 mm screen. After drying,
it was mixed with 15 mg of magnesium stearate and then 200 mg by
200 mg of it was subjected to tablet making to obtain tablets.
TABLE-US-00004 (3) Capsules 6DSH 100 mg Lactose 80 mg
[0118] The above components were uniformly mixed and filled in a
hard capsule to obtain capsules.
TABLE-US-00005 (4) Injections NDSH 30 mg
[0119] The above component was dissolved in 2 ml of 5% mannitol
aqueous solution. It was subjected to aseptic filtration and then
put into an ampoule and sealed.
TABLE-US-00006 (5) Injections for dissolution when used (A) 2DSH
(freeze-dried) 30 mg (enclosed in an ampoule) (B) Aseptically
filtered PBS 2 ml (enclosed in an ampoule)
[0120] Using the above (A) and (B) as one set, injections for
dissolution when used were produced. When used, (A) can be used by
dissolving it in (B).
[0121] In this connection, the active ingredients of the agents of
the present invention showed no particular changes when conditions
of the cells and animals were observed every day in the
aforementioned drug effect pharmacological tests. Based on this,
safety of the agents of the present invention can be sufficiently
estimated.
[0122] While the present invention has been describe in detail and
with reference to specific embodiments thereof, it will be apparent
to one skilled in the art that various changes and modifications
can be made therein without departing from the spirit and scope of
the present invention.
[0123] This application is based on a Japanese patent application
filed on Mar. 14, 2005 (Japanese Patent Application No.
2005-071023) and a Japanese patent application filed on Jun. 16,
2005 (Japanese Patent Application No. 2005-1763 11), and the
contents thereof are incorporated by reference. All of the
references cited herein are incorporated as a whole.
INDUSTRIAL APPLICABILITY
[0124] The agents of the present invention can be used as
medicines, reagents and the like with the purpose of accelerating
hard tissue formation, accelerating differentiation of cells and
reinforcing ALP activity of cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0125] FIG. 1 is a graph showing increase in ALP activity in
MC3T3-E1 cell.
[0126] FIG. 2 is a graph showing increase in ALP activity in
MC3T3-E1 cell.
[0127] FIG. 3 is a graph showing increase in ALP activity in ST2
cell.
[0128] FIG. 4 is a graph showing increase in ALP activity in
MC3T3-E1 cell under serum-free condition.
[0129] FIG. 5 is a graph showing increase in ALP activity in ST2
cell under serum-free condition.
[0130] FIG. 6 is a graph showing ratio of the area of a region
where a bone (hard tissue) was formed.
[0131] FIG. 7 is a graph showing ratio of the area of a region
where a bone (hard tissue) was formed.
[0132] FIG. 8 is a graph showing influence upon the growth of
MC3T3-E1 cell.
[0133] FIG. 9 is a graph showing influence upon the growth of ST2
cell.
* * * * *