U.S. patent application number 12/680995 was filed with the patent office on 2010-11-11 for novel chondroitin sulfate having decreased molecular weight and use thereof.
This patent application is currently assigned to Seikagaku Corporation. Invention is credited to Yukio Goto, Kazuhiro Kojima, Hiroshi Maeda.
Application Number | 20100286085 12/680995 |
Document ID | / |
Family ID | 40526190 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286085 |
Kind Code |
A1 |
Kojima; Kazuhiro ; et
al. |
November 11, 2010 |
NOVEL CHONDROITIN SULFATE HAVING DECREASED MOLECULAR WEIGHT AND USE
THEREOF
Abstract
An object of the present invention is to provide a chondroitin
sulfate having a decreased molecular weight which has utilization
as an inhibitor of peritoneal disorder caused by long-term use of a
peritoneal dialysis fluid containing glucose or a polysaccharide
thereof as an osmotic agent, utilization as an osmotic agent in a
peritoneal dialysis fluid, and the like. The chondroitin sulfate
having a decreased molecular weight of the present invention as a
means for achieving the object is characterized by having a weight
average molecular weight of from 1000 to 20000 and containing a
constituent disaccharide unit represented by the following
structural formula in an amount of from 65 o to 100% (molar ratio)
of the total. -[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA
represents a D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated)
Inventors: |
Kojima; Kazuhiro; (Tokyo,
JP) ; Goto; Yukio; (Tokyo, JP) ; Maeda;
Hiroshi; (Tokyo, JP) |
Correspondence
Address: |
KRATZ, QUINTOS & HANSON, LLP
1420 K Street, N.W., 4th Floor
WASHINGTON
DC
20005
US
|
Assignee: |
Seikagaku Corporation
Tokyo
JP
|
Family ID: |
40526190 |
Appl. No.: |
12/680995 |
Filed: |
October 1, 2008 |
PCT Filed: |
October 1, 2008 |
PCT NO: |
PCT/JP2008/067805 |
371 Date: |
June 9, 2010 |
Current U.S.
Class: |
514/54 ;
536/53 |
Current CPC
Class: |
C08L 5/08 20130101; C08K
3/08 20130101; C08K 3/02 20130101; C08J 3/28 20130101; A61K 31/737
20130101; A61P 13/12 20180101; C08K 5/09 20130101; C08L 5/08
20130101; C08L 5/08 20130101; C08L 5/08 20130101; C08K 3/08
20130101; C08K 3/08 20130101; A61K 31/726 20130101; C08K 5/09
20130101; A61M 1/287 20130101; C08J 2305/08 20130101; C08B 37/0069
20130101; C08K 3/02 20130101; A61P 7/08 20180101; C08K 5/09
20130101; C08K 3/02 20130101; A61P 7/02 20180101; A61P 39/06
20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/54 ;
536/53 |
International
Class: |
A61K 31/715 20060101
A61K031/715; C08B 37/08 20060101 C08B037/08; A61P 7/02 20060101
A61P007/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 2007 |
JP |
2007-257945 |
Apr 18, 2008 |
JP |
2008-109492 |
Claims
1. A chondroitin sulfate having a decreased molecular weight,
characterized by having a weight average molecular weight of from
1000 to 20000 and containing a constituent disaccharide unit
represented by the following structural formula in an amount of
from 65% to 100% (molar ratio) of the total.
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated)
2. A peritoneal dialysis fluid, characterized by containing the
chondroitin sulfate having a decreased molecular weight according
to claim 1.
3. A compounding agent for a peritoneal dialysis fluid,
characterized by containing the chondroitin sulfate having a
decreased molecular weight according to claim 1 as an active
ingredient.
4. An inhibitor of peritoneal disorder caused by a peritoneal
dialysis fluid containing glucose and/or a polysaccharide thereof
as an osmotic agent, characterized by containing the chondroitin
sulfate having a decreased molecular weight according to claim 1 as
an active ingredient.
5. An inhibitor of production of an advanced glycation end product
(AGE), characterized by containing the chondroitin sulfate having a
decreased molecular weight according to claim 1 as an active
ingredient.
6. A reactive oxygen scavenger, characterized by containing the
chondroitin sulfate having a decreased molecular weight according
to claim 1 as an active ingredient.
7. An inhibitor of lipid peroxidation, characterized by containing
the chondroitin sulfate having a decreased molecular weight
according to claim 1 as an active ingredient.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel chondroitin sulfate
having a decreased molecular weight and use thereof. More
particularly, the present invention relates to a chondroitin
sulfate having a decreased molecular weight which has utilization
as an inhibitor of peritoneal disorder caused by long-term use of a
peritoneal dialysis fluid containing glucose or a polysaccharide
thereof as an osmotic agent, utilization as an osmotic agent in a
peritoneal dialysis fluid, and the like, and use thereof.
BACKGROUND ART
[0002] Artificial dialysis for patients with end-stage renal
failure is broadly divided into hemodialysis and peritoneal
dialysis. Among these, the peritoneal dialysis is a method in which
a hypertonic dialysis fluid containing glucose or a polysaccharide
thereof as an osmotic agent is injected into the peritoneal cavity
and excess waste products, water, and electrolytes in the body are
removed with the use of the function of the peritoneum as a
semipermeable membrane. The peritoneal dialysis has many advantages
as compared with the hemodialysis in that the need for dietary or
activity restriction is lower, the effect on hemodynamics is lower,
preservation of residual renal function is better, rehabilitation
is easier, etc. However, in recent years, various disorders due to
prolonged peritoneal dialysis, for example, poor water removal or
insufficient removal of waste products accompanying deterioration
of peritoneal function has become a serious problem. The
deterioration of peritoneal function is deeply associated with an
advanced glycation end product (AGE) generated by a reaction
between glucose contained in a peritoneal dialysis fluid as an
osmotic agent and a protein, a reactive oxygen species (ROS)
produced by binding of AGE to a cell having a receptor thereof or
exposure of the cell to high concentration of glucose.
[0003] In order to solve the problems as described above, a search
for a substance that inhibits deterioration of peritoneal function
caused by glucose or a polysaccharide thereof and a search for a
novel osmotic agent alternative to glucose or a polysaccharide
thereof have been performed, and a chondroitin sulfate (CS) which
is a sulfated glycosaminoglycan has already been proposed as a
candidate substance (For example, Patent Document 1 and Non-patent
Document 1).
[0004] Patent Document 1: JP-A-1-151462
[0005] Non-patent Document1:OhnoT., ImadaA., "The usefulness of
sodium chondroitin sulfate as an osmotic agent for peritoneal
dialysis", Toseki kaishi (The Journal of the Japanese Society for
Dialysis Therapy), 30(1): 65, 1997
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
[0006] A chondroitin sulfate is a substance in the body and
therefore has high biocompatibility and little effect on the blood
coagulation system unlike heparin. Accordingly, a chondroitin
sulfate is considered to be promising also from a safety
standpoint. However, although it has been reported that in the
evaluation using an animal model, a chondroitin sulfate has an
action of protecting the peritoneal function (Non-patent Document
1), it has also been reported that a chondroitin sulfate does not
have such a protective action (Suyama K., Kumano K., Sakai T.,
"Agents preventing decrease in ultrafiltration in a rat model with
peritoneal hyperpermeability", Nippon Jinzo Gakkai Shi (The
Japanese Journal of Nephrology), 37(9): 491, 1995). Thus, the
current situation is that the views as to the usefulness of a
chondroitin sulfate are still controversial. In addition, in the
chondroitin sulfate, there are several types based on a difference
in the binding site of a sulfate group such as chondroitin sulfate
A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin
sulfate E, and also there is significant structural diversity.
Therefore, it is considered that functional diversity may arise
from the structural diversity, however, as far as the present
inventors know, there has been no report that a relationship
between the structure and the function of a chondroitin sulfate in
peritoneal dialysis is elucidated.
[0007] Accordingly, an object of the present invention is to
provide a novel chondroitin sulfate useful in peritoneal
dialysis.
Means for Solving the Problems
[0008] In view of the above points, the present inventors conducted
intensive studies, and as a result, they found that a chondroitin
sulfate having a decreased molecular weight having a specific
molecular weight and containing a specific constituent disaccharide
unit in a specific percentage of the total is useful as an
inhibitor of peritoneal disorder caused by long-term use of a
peritoneal dialysis fluid containing glucose or a polysaccharide
thereof as an osmotic agent, useful as an osmotic agent in a
peritoneal dialysis fluid, and the like, based on excellent action
of inhibiting AGE production and action of scavenging reactive
oxygen.
[0009] As described in claim 1, a chondroitin sulfate having a
decreased molecular weight of the present invention completed based
on the above findings is characterized by having a weight average
molecular weight of from 1000 to 20000 and containing a constituent
disaccharide unit represented by the following structural formula
in an amount of from 65% to 100% (molar ratio) of the total.
-[4GlcA.beta.1-3GalNAc(6S).beta.1]-
(wherein GlcA represents a D-glucuronic acid residue; GalNAc
represents an N-acetyl-D-galactosamine residue; .beta.1-3
represents a .beta.1-3 glycosidic linkage; .beta.1-4 represents a
.beta.1-4 glycosidic linkage; and (6S) indicates that position 6 of
the monosaccharide residue is sulfated)
[0010] Further, as described in claim 2, a peritoneal dialysis
fluid of the present invention is characterized by containing the
chondroitin sulfate having a decreased molecular weight according
to claim 1.
[0011] Further, as described in claim 3, a compounding agent for a
peritoneal dialysis fluid of the present invention is characterized
by containing the chondroitin sulfate having a decreased molecular
weight according to claim 1 as an active ingredient.
[0012] Further, as described in claim 4, an inhibitor of peritoneal
disorder caused by a peritoneal dialysis fluid containing glucose
and/or a polysaccharide thereof as an osmotic agent of the present
invention is characterized by containing the chondroitin sulfate
having a decreased molecular weight according to claim 1 as an
active ingredient.
[0013] Further, as described in claim 5, an AGE production
inhibitor of the present invention is characterized by containing
the chondroitin sulfate having a decreased molecular weight
according to claim 1 as an active ingredient.
[0014] Further, as described in claim 6, a reactive oxygen
scavenger of the present invention is characterized by containing
the chondroitin sulfate having a decreased molecular weight
according to claim 1 as an active ingredient.
[0015] Further, as described in claim 7, an inhibitor of lipid
peroxidation of the present invention is characterized by
containing the chondroitin sulfate having a decreased molecular
weight according to claim 1 as an active ingredient.
Effect of the Invention
[0016] According to the present invention, a chondroitin sulfate
having a decreased molecular weight which has utilization as an
inhibitor of peritoneal disorder caused by long-term use of a
peritoneal dialysis fluid containing glucose or a polysaccharide
thereof as an osmotic agent, utilization as an osmotic agent in a
peritoneal dialysis fluid, and the like can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] [FIG. 1] A graph showing an effect of protecting the
peritoneum (action of inhibiting a decrease in water removal rate)
in peritoneal dialysis of a chondroitin sulfate having a decreased
molecular weight of the present invention in Example 3.
[0018] [FIG. 2] A graph showing an effect of protecting the
peritoneum (action of inhibiting lipid peroxidation of the
peritoneum) in peritoneal dialysis of a chondroitin sulfate having
a decreased molecular weight of the present invention in Example
4.
[0019] [FIG. 3] A graph showing an effect of protecting the
peritoneum in peritoneal dialysis of a chondroitin sulfate having a
decreased molecular weight of the present invention in Example 7
(comparison with known AGE production inhibitors).
[0020] [FIG. 4] A graph showing an effect of protecting the
peritoneum in peritoneal dialysis of a chondroitin sulfate having a
decreased molecular weight of the present invention in Example 8
(comparison with known reactive oxygen scavengers).
BEST MODE FOR CARRYING OUT THE INVENTION
[0021] The chondroitin sulfate having a decreased molecular weight
of the present invention, which has utilization as an inhibitor of
peritoneal disorder caused by long-term use of a peritoneal
dialysis fluid containing glucose or a polysaccharide thereof as an
osmotic agent, utilization as an osmotic agent in a peritoneal
dialysis fluid, and the like, is characterized by having a weight
average molecular weight of from 1000 to 20000 and containing a
constituent disaccharide unit represented by the following
structural formula in an amount of from 65 to 100% (molar ratio) of
the total.
-[4GlcA.beta.1-3GalNAc(6S).beta.1]-
(wherein GlcA represents a D-glucuronic acid residue; GalNAc
represents an N-acetyl-D-galactosamine residue; .beta.1-3
represents a .beta.1-3 glycosidic linkage; .beta.1-4 represents a
.beta.1-4 glycosidic linkage; and (6S) indicates that position 6 of
the monosaccharide residue is sulfated)
[0022] The chondroitin sulfate having a decreased molecular weight
of the present invention can be prepared by, for example, using
chondroitin sulfate C (chondroitin 6-sulfate) containing a
constituent disaccharide unit represented by the following
structural formula in an amount of from 65% to 100% (molar ratio)
of the total as a starting material and reducing the molecular
weight of the starting material such that the weight average
molecular weight becomes 1000 to 20000, preferably 5000 to 18000.
As the chondroitin sulfate C to be used as the starting material,
for example, Prionace glauca-derived chondroitin sulfate C (trade
name of Seikagaku Corporation: Chondroitin sulfate C, sodium salt
(shark cartilage), SG) can be used. The reduction of the molecular
weight thereof can be performed by a degradation method using
electron beam irradiation described in JP-A-2004-43645 or the like
as well as the methods such as a chemical degradation method using
hydrochloric acid and an enzymatic degradation method using ovine
testicular hyaluronidase which are known methods of reducing the
molecular weight of a chondroitin sulfate. Incidentally, the
content percentage of the constituent disaccharide unit represented
by the following structural formula in the starting material is not
necessarily 65% or more (molar ratio) of the total and may be less
than 65% (molar ratio) as long as a fraction of 65% or more (molar
ratio) can be obtained by fractional purification. Further, the
chondroitin sulfate having a decreased molecular weight of the
present invention may be a product obtained by chemical synthesis
or extraction from a cultured product or a fermented product of a
biological tissue of shark or the like.
-[4GlcA.beta.1-3GalNAc(6S).beta.1]-
(wherein GlcA represents a D-glucuronic acid residue; GalNAc
represents an N-acetyl-D-galactosamine residue; .beta.1-3
represents a .beta.1-3 glycosidic linkage; .beta.1-4 represents a
.beta.1-4 glycosidic linkage; and (6S) indicates that position 6 of
the monosaccharide residue is sulfated)
[0023] The chondroitin sulfate having a decreased molecular weight
of the present invention has excellent action of inhibiting AGE
production and action of scavenging reactive oxygen. Therefore, by
adding the chondroitin sulfate to a peritoneal dialysis fluid
containing glucose or a polysaccharide thereof as an osmotic agent,
an effect as an inhibitor of peritoneal disorder (e.g., lipid
peroxidation) caused by glucose or a polysaccharide thereof due to
long-term use of such a peritoneal dialysis fluid can be exhibited.
Further, the chondroitin sulfate having a decreased molecular
weight of the present invention can also be added to a peritoneal
dialysis fluid as an osmotic agent in the peritoneal dialysis
fluid.
[0024] In the case where the chondroitin sulfate having a decreased
molecular weight of the present invention is added to a
conventional peritoneal dialysis fluid containing glucose or a
polysaccharide thereof as an osmotic agent, the chondroitin sulfate
may be added at a concentration of from 0.01% (w/v) to 1% (w/v).
Further, in the case where a peritoneal dialysis fluid is prepared
by adding the chondroitin sulfate having a decreased molecular
weight of the present invention as an osmotic agent alternative to
glucose or a polysaccharide thereof, the chondroitin sulfate may be
added at a concentration of from 1% (w/v) to 10% (w/v) to
constitute the peritoneal dialysis fluid together with components
known as constituent components of the peritoneal dialysis fluid
such as sodium, magnesium, calcium, chlorine, and lactic acid.
[0025] Further, the action of inhibiting AGE production of the
chondroitin sulfate having a decreased molecular weight of the
present invention is effective in the treatment of diabetes,
various diabetic complications (for example, diabetic retinopathy,
diabetic nephropathy, diabetic neuropathy, and diabetic vascular
complications), etc., and the action of scavenging reactive oxygen
of it is effective in the treatment of cancer, cataract,
arteriosclerosis, Alzheimer's disease, asthma, etc. Therefore, the
chondroitin sulfate having a decreased molecular weight of the
present invention can also be used as an active ingredient for
treating such a disease. In this case, the chondroitin sulfate
having a decreased molecular weight of the present invention may be
administered orally or parenterally at a dose appropriately
determined based on the degree of symptoms, age, and body weight of
a patient or the like. It can be administered in a known dosage
form.
Examples
[0026] Hereinafter, the present invention will be described in
further detail with reference to Examples, however, the invention
is not limited to the following description. Incidentally, in the
Examples, the molecular weight is indicated in "kDa" (1
kDa=1000).
Example 1
Preparation of Chondroitin Sulfate Having Decreased Molecular
Weight of the Present Invention (1)
[0027] Prionace glauca-derived chondroitin sulfate C (weight
average molecular weight: 30 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate C, sodium salt (shark cartilage),
SG) was used as a starting material and dissolved in an amount of 1
g in 50 mL of PBS (pH 5.3). To this solution, 100,000 U of ovine
testicular hyaluronidase (manufactured by Sigma, type V) was added,
and the enzymatic reaction was allowed to proceed at 37 .degree. C.
A portion of the reaction mixture was taken over time and analyzed
by GPC-HPLC to examine the degree of reduction of the molecular
weight. When a desired molecular weight was obtained, the reaction
mixture was boiled to stop the enzymatic reaction. In the case
where the desired molecular weight was not obtained, ovine
testicular hyaluronidase was further added to the reaction mixture
and the enzymatic reaction was allowed to proceed. When the desired
molecular weight was obtained, the reaction mixture was boiled to
stop the enzymatic reaction. After completion of the reaction,
activated carbon was added to the thus obtained desired molecular
weight fraction, and the reaction was allowed to proceed at
50.degree. C. for 1 hour. Thereafter, the reaction mixture was
filtered, and to the filtrate, sodium acetate trihydrate was added,
and then, ethanol was added thereto to obtain precipitates. The
obtained precipitates were purified by washing with ethanol and
drying. By the above-mentioned method, a chondroitin sulfate having
a decreased molecular weight of the present invention having a
weight average molecular weight of 10 kDa and containing a
constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) in an amount of 74.0% (molar ratio) of the
total was obtained as white powder (the detail of the composition
of the constituent disaccharide is as shown in Table 1).
[0028] Incidentally, the analysis of the composition of the
constituent disaccharide was performed as follows. 100 .mu.L of a
measurement sample (about 200 .mu.g/mL) was taken, 40 .mu.L of 100
mM Tris-HCl buffer (pH 8.0) and Chondroitinase ABC (83 mU,
manufactured by Seikagaku Corporation) were added thereto, and the
total volume was made up to 200 .mu.L. The reaction was allowed to
proceed at 37.degree. C. for 3 hours, and the resulting reaction
mixture was filtered through a 10000 cut-off ultrafilter, and the
filtrate was subjected to HPLC (column: YMC gel PA-120,
manufactured by YMC Co., Ltd.). (The same shall apply to Example
2)
TABLE-US-00001 TABLE 1 Disaccharide composition ratio (%) 0S 6S 4S
SD SB SE CSC 10 kDa 1.0 74.0 14.0 10.1 -- 0.9 0S:
-[4GlcA.beta.1-3GalNAc.beta.1]- 6S:
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- 4S:
-[4GlcA.beta.1-3GalNAc(4S).beta.1]- SD:
-[4GlcA(2S).beta.1-3GalNAc(6S).beta.1]- SB:
-[4GlcA(2S).beta.1-3GalNAc(4S).beta.1]- SE:
-[4GlcA.beta.1-3GalNAc(4S,6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (2S), (4S), and (6S) indicate that position 2, 4, or 6
of the monosaccharide residue is sulfated respectively)
Example 2
Preparation of Chondroitin Sulfate Having Decreased Molecular
Weight of the Present Invention (2)
[0029] Prionace glauca-derived chondroitin sulfate C (weight
average molecular weight: 30 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate C, sodium salt (shark cartilage),
SG) was used as a starting material and subjected to electron beam
irradiation at an irradiation energy of 200 kGy according to the
method described in JP-A-2004-43645, whereby a chondroitin sulfate
having a decreased molecular weight of the present invention having
a weight average molecular weight of 10 kDa and containing a
constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; p1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) in an amount of 73.0% (molar ratio) of the
total was obtained as white powder (the detail of the composition
of the constituent disaccharide is as shown in Table 2).
TABLE-US-00002 TABLE 2 Disaccharide composition ratio (%) 0S 6S 4S
SD SB SE CSC 10 kDa 2.9 73.0 15.3 7.8 -- 1.0 0S:
-[4GlcA.beta.1-3GalNAc.beta.1]- 6S:
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- 4S:
-[4GlcA.beta.1-3GalNAc(4S).beta.1]- SD:
-[4GlcA(2S).beta.1-3GalNAc(6S).beta.1]- SB:
-[4GlcA(2S).beta.1-3GalNAc(4S).beta.1]- SE:
-[4GlcA.beta.1-3GalNAc(4S,6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (2S), (4S), and (6S) indicate that position 2, 4, or 6
of the monosaccharide residue is sulfated respectively)
Example 3
Effect of Protecting Peritoneum in Peritoneal Dialysis of
Chondroitin Sulfate Having Decreased Molecular Weight of the
Present Invention (1)
(Experimental Method)
[0030] To a Wistar male rat at 8 weeks of age, Midpeliq 250 (trade
name, a peritoneal dialysis fluid containing glucose at a
concentration of 2.5% (w/v) manufactured by Terumo Corporation) in
which a test substance was dissolved at a concentration of 0.1%
(w/v) was repetitively administered into the peritoneal cavity once
daily at 15 mL/body for 7 days under ether anesthesia. To a control
group (Control), Midpeliq 250 was administered in the same manner.
On the next day after the final administration, a peritoneal
equilibration test was performed, and the peritoneal function was
evaluated. To be more specific, Midpeliq 250 was injected into the
peritoneal cavity at 60 mL/kg, and 4 hours thereafter, the fluid
remaining in the peritoneal cavity was recovered. The amount of the
recovered fluid was measured, and the ultrafiltration capacity
(water removal rate) of the peritoneum was evaluated.
[0031] Incidentally, as the test substance, the following
substances were used.
[0032] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 300 kGy according to the
method of Example 2 and having a weight average molecular weight of
7 kDa (CSC 7 kDa).
[0033] (b) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 200 kGy according to the
method of Example 2 and having a weight average molecular weight of
10 kDa (CSC 10 kDa).
[0034] (c) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 100 kGy according to the
method of Example 2 and having a weight average molecular weight of
17 kDa (CSC 17 kDa).
[0035] (d) Prionace glauca-derived chondroitin sulfate C (weight
average molecular weight: 30 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate C, sodium salt (shark cartilage),
SG) used as the starting material (CSC 30 kDa).
(Experimental Results)
[0036] The results are shown in FIG. 1. As is apparent from FIG. 1,
a statistically significant effect of protecting the peritoneum was
observed in the chondroitin sulfates having a decreased molecular
weight of the present invention having a weight average molecular
weight of 7 kDa, 10 kDa, and 17 kDa as compared with the control
group, however, the statistically significant effect was not
observed in the shark shoulder cartilage-derived chondroitin
sulfate C having a weight average molecular weight of 30 kDa used
as the starting material (in the drawing, the expression "Normal"
denotes the result in the case where the peritoneal dialysis fluid
was not administered). Further, the statistically significant
effect was not observed also in a chondroitin sulfate having a
decreased molecular weight prepared by using shark fin
cartilage-derived chondroitin sulfate C (weight average molecular
weight: 20 kDa) as a starting material and hydrolyzing it with
hydrochloric acid, having a weight average molecular weight of 10
kDa and containing a constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) in an amount of 59.0% (molar ratio) of the
total (based on another experiment).
Example 4
Effect of Protecting Peritoneum in Peritoneal Dialysis of
Chondroitin Sulfate Having Decreased Molecular Weight of the
Present Invention (2)
(Experimental Method)
[0037] To a Wistar male rat at 8 weeks of age, Midpeliq 250 (trade
name, a peritoneal dialysis fluid containing glucose at a
concentration of 2.5% (w/v) manufactured by Terumo Corporation) in
which a chondroitin sulfate having a decreased molecular weight of
the present invention prepared according to the method of Example 1
and having a weight average molecular weight of 10 kDa (CSC 10 kDa)
was dissolved as a test substance at a concentration of 0.1% (w/v)
was repetitively administered into the peritoneal cavity once daily
at 15 mL/body for 7 days under ether anesthesia. To a control group
(Control), Midpeliq 250 was administered in the same manner. On the
next day after the final administration, the peritoneum (greater
omentum) was excised and the content of lipid peroxide in the
tissue was quantitatively determined by a thiobarbituric acid
method. An effect of protecting the peritoneum of the test
substance was evaluated using an action of inhibiting lipid
peroxidation of the peritoneum as an index.
(Experimental Results)
[0038] The results are shown in FIG. 2. As is apparent from FIG. 2,
a statistically significant effect of protecting the peritoneum was
observed in the chondroitin sulfate having a decreased molecular
weight of the present invention having a weight average molecular
weight of 10 kDa as compared with the control group (in the
drawing, the expression "Normal" denotes the result in the case
where the peritoneal dialysis fluid was not administered).
Example 5
Action of Inhibiting AGE Production of Chondroitin Sulfate Having
Decreased Molecular Weight of the Present Invention
(Experimental Method)
[0039] An experiment was performed according to the method of T.
Kiho et al. (Biosci Biotechnol Biochem, 68: 200, 2004). To be more
specific, a test substance was dissolved in 0.1 M phosphate buffer
(pH 7.4) containing 500 mM glucose and 10 mg/mL bovine serum
albumin at a concentration of 0.1% (w/v), and the resulting
solution was incubated at 37.degree. C. for 4 weeks. After 4 weeks,
a value of fluorescence emitted by AGE was measured at an
excitation light wavelength of 355 nm and a measurement wavelength
of 460 nm, and the amount of produced AGE was determined, and then,
an inhibition rate of AGE production was calculated according to
the following calculation equation.
Inhibition rate of AGE production (%)=[1-(Fluorescence value when
test substance was added)/(Fluorescence value when test substance
was not added)].times.100 [Equation 1]
(Experimental Results)
[0040] The results are shown in Table 3. Incidentally, as the test
substance, the following substances were used. Further, as a
positive control substance, 1 mM aminoguanidine (a known AGE
production inhibitor) was used.
[0041] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared according to the method of
Example 1 and having a weight average molecular weight of 10 kDa
(CSC 10 kDa).
[0042] (b) A chondroitin sulfate having a decreased molecular
weight prepared from whale-derived chondroitin sulfate A (weight
average molecular weight: 30 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate A, sodium salt (whale cartilage),
SG) by the same method, having a weight average molecular weight of
10 kDa and containing a low proportion (20% to 30%) of a
constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (CSA 10 kDa).
[0043] (c) A chondroitin sulfate having a decreased molecular
weight prepared from squid-derived chondroitin sulfate E (weight
average molecular weight: 75 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate E, sodium salt (squid cartilage))
by the same method, having a weight average molecular weight of 10
kDa and containing a low proportion (10% to 20%) of a constituent
disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (CSE 10 kDa).
[0044] (d) A dermatan sulfate having a decreased molecular weight
prepared by subjecting a cockscomb-derived dermatan sulfate (weight
average molecular weight: 40 kDa) to electron beam irradiation at
an irradiation energy of 200 kGy according to the method of Example
2, having a weight average molecular weight of 10 kDa and
containing a low proportion (10% or less) of a constituent
disaccharide unit represented by -[4GlcA.beta.1-3GalNAc(6S)-
(wherein GlcA represents a D-glucuronic acid residue; GalNAc
represents an N-acetyl-D-galactosamine residue;62 1-3 represents a
.beta.1-3 glycosidic linkage; .beta.1-4 represents a .beta.1-4
glycosidic linkage; and (6S) indicates that position 6 of the
monosaccharide residue is sulfated) (DS 10 kDa).
TABLE-US-00003 TABLE 3 Concentration Inhibition rate Test substance
CSA 10 kDa 0.1% (w/v) 20.7% CSC 10 kDa Same as above 54.0% CSE 10
kDa Same as above 8.0% DS 10 kDa Same as above 7.3% Positive
control substance Aminoguanidine 1 mM 52.7%
[0045] As is apparent from Table 3, only the chondroitin sulfate
having a decreased molecular weight of the present invention having
a weight average molecular weight of 10 kDa (CSC 10 kDa) had an
action of inhibiting AGE production comparable to that of
aminoguanidine which is a positive control substance.
Example 6
Action of Scavenging Reactive Oxygen of Chondroitin Sulfate Having
Decreased Molecular Weight of the Present Invention
(Experimental Method)
[0046] An experiment was performed according to the method of Olga
T. et al. (Toxicological Science, 76: 376, 2003). To be more
specific, HL-60 cells differentiated into neutrophil-like cells
were suspended in a culture medium at 2.times.10.sup.6 cells/mL,
and the resulting cell suspension was added to a 96-well plate at
50 .mu.L/well. Then, a test substance was added to the well at 50
.mu.L/well (final concentration: 0.03%, 0.1%, and 0.3% (w/v)).
Further, the culture medium was added to the well as a negative
control at 50 .mu.L/well. L-012 (final concentration: 100 .mu.M,
manufactured by Wako Pure Chemical Industries, Ltd.) which is a
fluorescent substrate for ROS detection and a phorbol ester
(phorbol-12-myristate-13-acetate, manufactured by Sigma, final
concentration: 5 ng/mL) which is an agent for stimulating ROS
production were added to the well at 50 .mu.L/well each, and then,
the plate was incubated at 37.degree. C. for 25 minutes in a 5%
CO.sub.2 incubator. As a background, the plate to which the culture
medium was added in place of the phorbol ester was incubated in the
same manner. After completion of the incubation, a luminescence
intensity (CPS) was measured using a multilabel counter, ARVOSX
1420 (manufactured by PerkinElmer) and an ROS scavenging rate was
calculated according to the following calculation equation.
ROS scavenging rate (%)={1-[(CPS in the case of adding test
substance)-(CPS in the case of background)]/[(CPS in the case of
negative control)-(CPS in the case of background)]}.times.100
[Equation 2]
(Experimental Results)
[0047] The results are shown in Tables 4 and 5. Incidentally, as
the test substance, the following substances were used. Further, as
a positive control substance, 20 nM staurosporine (a known phorbol
ester signal inhibitor) was used.
[Table 4]
[0048] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared according to the method of
Example 1 and having a weight average molecular weight of 1 kDa
(CSC 1 kDa).
[0049] (b) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared according to the method of
Example 1 and having a weight average molecular weight of 10 kDa
(CSC 10 kDa).
[0050] (c) Prionace glauca-derived chondroitin sulfate C (weight
average molecular weight: 30 kDa, trade name of Seikagaku
Corporation: Chondroitin sulfate C, sodium salt (shark cartilage),
SG) used as the starting material (CSC 30 kDa).
[0051] (d) A chondroitin having a decreased molecular weight
prepared by the same method using chondroitin (weight average
molecular weight: 5 kDa, trade name of Seikagaku Corporation:
Chondroitin, sodium salt) as a starting material, having a weight
average molecular weight of 1 kDa and containing no constituent
disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (Ch 1 kDa).
[0052] (e) The chondroitin used as the starting material (Ch 5
kDa).
[0053] (f) A chondroitin sulfate having a decreased molecular
weight prepared by the same method using whale-derived chondroitin
sulfate A (weight average molecular weight: 30 kDa, trade name of
Seikagaku Corporation: Chondroitin sulfate A, sodium salt (whale
cartilage), SG) as a starting material, having a weight average
molecular weight of 1 kDa and containing a low proportion (20% to
30%) of a constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (CSA 1 kDa).
[0054] (g) A chondroitin sulfate having a decreased molecular
weight having a weight average molecular weight of 5 kDa and the
others are the same as in (f) (CSA 5 kDa).
[0055] (h) A chondroitin sulfate having a decreased molecular
weight having a weight average molecular weight of 10 kDa and the
others are the same as in (f) (CSA 10 kDa).
[0056] (i) The whale-derived chondroitin sulfate A used as the
starting material (CSA 30 kDa).
[0057] (j) A chondroitin sulfate having a decreased molecular
weight prepared by the same method using squid-derived chondroitin
sulfate E (weight average molecular weight: 75 kDa, trade name of
Seikagaku Corporation: Chondroitin sulfate E, sodium salt (squid
cartilage)) as a starting material, having a weight average
molecular weight of 5 kDa and containing a low proportion (10% to
20%) of a constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (CSE 5 kDa).
[0058] (k) A chondroitin sulfate having a decreased molecular
weight having a weight average molecular weight of 10 kDa and the
others are the same as in (j) (CSE 10 kDa).
[0059] (l) A chondroitin sulfate having a decreased molecular
weight having a weight average molecular weight of 25 kDa and the
others are the same as in (j) (CSE 25 kDa).
[0060] (m) The squid-derived chondroitin sulfate E used as the
starting material (CSE 75 kDa).
[0061] (n) A dermatan sulfate having a decreased molecular weight
prepared by using a cockscomb-derived dermatan sulfate (weight
average molecular weight: 40 kDa) as a starting material and
subjecting it to electron beam irradiation at an irradiation energy
of 300 kGy according to the method of Example 2, having a weight
average molecular weight of 5 kDa and containing a low proportion
(10% or less) of a constituent disaccharide unit represented by
-[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (DS 5 kDa).
[0062] (o) A dermatan sulfate having a decreased molecular weight
prepared by electron beam irradiation at an irradiation energy of
200 kGy and having a weight average molecular weight of 10 kDa and
the others are the same as in (n) (DS 10 kDa).
[0063] (p) The cockscomb-derived dermatan sulfate used as the
starting material (DS 40 kDa).
[0064] (q) A cockscomb-derived hyaluronic acid (manufactured by
Seikagaku Corporation) having a weight average molecular weight of
900 kDa and containing no constituent disaccharide unit represented
by -[4GlcA.beta.1-3GalNAc(6S).beta.1]- (wherein GlcA represents a
D-glucuronic acid residue; GalNAc represents an
N-acetyl-D-galactosamine residue; .beta.1-3 represents a .beta.1-3
glycosidic linkage; .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (6S) indicates that position 6 of the monosaccharide
residue is sulfated) (HA 900 kDa).
[Table 5]
[0065] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 300 kGy according to the
method of Example 2 and having a weight average molecular weight of
7 kDa (CSC 7 kDa).
[0066] (b) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 200 kGy according to the
method of Example 2 and having a weight average molecular weight of
10 kDa (CSC 10 kDa).
[0067] (c) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 100 kGy according to the
method of Example 2 and having a weight average molecular weight of
17 kDa (CSC 17 kDa).
TABLE-US-00004 TABLE 4 Concentration ROS scavenging rate Test
substance Ch 1 kDa 0.1% (w/v) 11.8% Ch 5 kDa Same as above 13.6%
CSA 1 kDa Same as above 39.7% CSA 5 kDa Same as above 41.0% CSA 10
kDa Same as above 23.9% CSA 30 kDa Same as above 33.4% CSC 1 kDa
Same as above 72.2% CSC 10 kDa Same as above 97.0% CSC 30 kDa Same
as above 5.0% CSE 5 kDa Same as above -9.4% CSE 10 kDa Same as
above 35.3% CSE 25 kDa Same as above 22.4% CSE 75 kDa Same as above
21.9% DS 5 kDa Same as above 39.4% DS 10 kDa Same as above 24.7% DS
40 kDa Same as above 11.6% HA 900 kDa Same as above 11.5% Positive
control substance Staurosporine 20 nM 97.4%
TABLE-US-00005 TABLE 5 Concentration ROS scavenging rate Test
substance 0.03% (w/v) 57.0% CSC 7 kDa 0.1% (w/v) 75.5% 0.3% (w/v)
91.5% 0.03% (w/v) 54.0% CSC 10 kDa 0.1% (w/v) 71.5% 0.3% (w/v)
86.5% 0.03% (w/v) 34.5% CSC 17 kDa 0.1% (w/v) 63.5% 0.3% (w/v)
83.5% Positive control substance Staurosporine 20 nM 98.1%
[0068] As is apparent from Table 4, only the chondroitin sulfates
having a decreased molecular weight of the present invention having
a weight average molecular weight of 1 kDa and 10 kDa (CSC 1 kDa
and CSC 10 kDa) showed excellent ROS scavenging rate, and the ROS
scavenging rate for CSC 10 kDa was comparable to that of
staurosporine which is a positive control substance. Further, as is
apparent from Table 5, the action of scavenging reactive oxygen of
the chondroitin sulfate having a decreased molecular weight of the
present invention is dependent on its concentration.
Example 7
Effect of Protecting Peritoneum in Peritoneal Dialysis of
Chondroitin Sulfate Having Decreased Molecular Weight of the
Present Invention (Comparison with Known AGE Production
Inhibitors)
(Experimental Method)
[0069] To a Wistar male rat at 8 weeks of age, Midpeliq 250 (trade
name, a peritoneal dialysis fluid containing glucose at a
concentration of 2.5% (w/v) manufactured by Terumo Corporation) in
which a test substance was dissolved at a given concentration was
repetitively administered into the peritoneal cavity once daily at
15 mL/body for 7 days under ether anesthesia. To a control group
(Control), Midpeliq 250 was administered in the same manner. On the
next day after the final administration, a peritoneal equilibration
test was performed, and the peritoneal function was evaluated. To
be more specific, Midpeliq 250 was injected into the peritoneal
cavity at 60 mL/kg, and 4 hours thereafter, the fluid remaining in
the peritoneal cavity was recovered. The amount of the recovered
fluid was measured, and the ultrafiltration capacity (water removal
rate) of the peritoneum was evaluated. Further, the concentration
of glucose in the recovered fluid was measured, and the peritoneal
permeability was evaluated.
[0070] Incidentally, as the test substance, the following
substances were used.
[0071] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 200 kGy according to the
method of Example 2 and having a weight average molecular weight of
10 kDa (CSC 10 kDa), concentration: 0.1% (w/v).
[0072] (b) Aminoguanidine which is a known AGE production
inhibitor, concentration: 0.1% (w/v).
[0073] (c) Pyridoxamine which is a known AGE production inhibitor,
concentration: 0.05% (w/v).
(Experimental Results)
[0074] The results are shown in FIG. 3. As is apparent from FIG. 3,
a statistically significant effect of protecting the peritoneum was
observed in the chondroitin sulfate having a decreased molecular
weight of the present invention having a weight average molecular
weight of 10 kDa as compared with the control group, and the effect
was higher than that of aminoguanidine and pyridoxamine both of
which are known AGE production inhibitors (in the drawing, the
expression "Normal" denotes the result in the case where the
peritoneal dialysis fluid was not administered).
Example 8
Effect of Protecting Peritoneum in Peritoneal Dialysis of
Chondroitin Sulfate Having Decreased Molecular Weight of the
Present Invention (Comparison with Known Reactive Oxygen
Scavengers)
(Experimental Method)
[0075] In the same manner as in Example 7, the ultrafiltration
capacity (water removal rate) of the peritoneum and the peritoneal
permeability were evaluated. Incidentally, as the test substance,
the following substances were used.
[0076] (a) A chondroitin sulfate having a decreased molecular
weight of the present invention prepared by electron beam
irradiation at an irradiation energy of 200 kGy according to the
method of Example 2 and having a weight average molecular weight of
10 kDa (CSC 10 kDa), concentration: 0.1% (w/v).
[0077] (b) L-ascorbic acid which is a known reactive oxygen
scavenger, concentration: 0.5 mM.
[0078] (d) Trolox which is a known reactive oxygen scavenger,
concentration: 0.5 mM.
[0079] (d) N-acetyl-L-cysteine which is a known reactive oxygen
scavenger, concentration: 10 mM.
(Experimental Results)
[0080] The results are shown in FIG. 4. As is apparent from FIG. 4,
a statistically significant effect of protecting the peritoneum was
observed in the chondroitin sulfate having a decreased molecular
weight of the present invention having a weight average molecular
weight of 10 kDa as compared with the control group, and the effect
was higher than that of known reactive oxygen scavengers (in the
drawing, the expression "Normal" denotes the result in the case
where the peritoneal dialysis fluid was not administered).
Preparation Example of Peritoneal Dialysis Fluid 1
[0081] A peritoneal dialysis fluid having the following composition
was prepared according to a common procedure.
TABLE-US-00006 Glucose 2.5 (w/v %) Sodium 135.0 (mEq/L) Magnesium
1.5 (mEq/L) Calcium 4.0 (mEq/L) Chlorine 105.5 (mEq/L) Lactic acid
35.0 (mEq/L) Chondroitin sulfate having decreased 0.1 (w/v %)
molecular weight of present invention Osmotic pressure ratio about
(to physiological saline) 1.4 to 1.6 pH 6.3 to 7.3
Preparation Example of Peritoneal Dialysis Fluid 2
[0082] A peritoneal dialysis fluid having the following composition
was prepared according to a common procedure.
TABLE-US-00007 Chondroitin sulfate having decreased 2.5 (w/v %)
molecular weight of present invention Sodium 135.0 (mEq/L)
Magnesium 1.5 (mEq/L) Calcium 4.0 (mEq/L) Chlorine 105.5 (mEq/L)
Lactic acid 35.0 (mEq/L) Osmotic pressure ratio about (to
physiological saline) 1.4 to 1.6 pH 6.3 to 7.3
INDUSTRIAL APPLICABILITY
[0083] The present invention has industrial applicability in that
it can provide a chondroitin sulfate having a decreased molecular
weight which has utilization as an inhibitor of peritoneal disorder
caused by long-term use of a peritoneal dialysis fluid containing
glucose or a polysaccharide thereof as an osmotic agent,
utilization as an osmotic agent in a peritoneal dialysis fluid, and
the like.
* * * * *