JP2000044601A - New galactosaminoglucan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a galactosaminoglucan capable of allowing an effective concentration in the blood to be retained when administered in an organism, and mildly preventing progress of thrombus, or dissolving or promoting the dissolution of the thrombus. SOLUTION: This galactosaminoglucan has the following characteristics: (1) having 40,000-200,000 dalton molecular weight measured by multiangle light scattering method; (2) providing about 16-25% proportion of 2-acetamindo-2- deoxy-3-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-sulfo-D-galactose in unsatured disaccharides analyzed by high-performance liquid chromatography when digested by chondroitinase ABC; (3) having about 7-18 wt.% nondigestible part when chondroitinase B is allowed to act thereon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel galactosaminoglycan which has a sulfate group at a specific position and is hardly digested by chondroitinase B.

[0002]

2. Description of the Related Art Galactosaminoglycan is a general term for glycosaminoglycans containing galactosamine as an amino sugar, and specifically includes dermatan sulfate and chondroitin sulfate (Annu. Rev. Biochee).
m. , 60, 443-475, 1991). Its basic structure is a repeating structure of a disaccharide consisting of N-acetyl-D-galactosamine and D-glucuronic acid or L-iduronic acid, and it is known that galactosaminoglycans have different binding positions of sulfate groups depending on types. (See FIG. 1).

In addition, one of the functions of galactosaminoglycan is as follows.
Finally, the blood anticoagulant activity promoting action is known, and its mechanism of action has been reported in detail (sugar chain engineering,
p348-356, Nobuko Seno et al.).

[0004] Dermatan sulfate, one of the galactosaminoglycans, is prepared by mincing a chicken cop, hydrolyzing with a protease, and performing alcohol precipitation in the presence of sodium acetate. It is reported that it has a higher molecular weight than the following dermatan sulfate (WO95 / 01988). Dermatan sulfate, one of the galactosaminoglycans,
Although it is prepared from various raw materials, it is usually prepared from the small intestine or skin of pigs or cattle. In 1984, N
have reported that two types of dermatan sulfate having different molecular weights and structures from chicken cocks have been prepared (Carbohydrate Research,
131 (1984) 301-314). Furthermore, in addition, it has thrombolytic activity and antithrombin activity,
Dermatan sulfate having a specific oligo sequence (Table 6)
No. 510081).

Dermatan sulfate (hereinafter referred to as “CCDS”) described in WO 95/01988 has an intrinsic viscosity of 0.8.
(100 mL / g) or more, the average molecular weight determined by GPC-HPLC is 38,000 daltons, which is higher than other dermatan sulfates, and the ΔDi-0S content of the constituent disaccharide composition is 2% or more and 7%. % Or less, and the content of heparin (Hep) or heparan sulfate (HS) is extremely low,
It was retained longer in the blood than other dermatan sulfates.
In addition, CCDS has a fibrinolysis-promoting activity in addition to an anticoagulant activity mediated by heparin cofactor II, and has a function of further enhancing fibrinolytic activity when used in combination with tissue plasminogen activator (t-PA). Therefore, it is known that both the anticoagulant and fibrinolytic activities have the effect of enhancing the thrombolytic action.

In recent years, DIC (general intravascular coagulation syndrome)
And heparin, urokinase, t for treating or preventing thrombosis typified by heart disease, myocardial infarction, deep vein thrombosis, etc.
Although a drug such as PA is used, it has problems such as a strong bleeding tendency, an extremely short half-life in blood, and a high price. CCDS was developed to solve these problems, but a drug that has a longer half-life in blood, does not cause severe bleeding, and acts more mildly is expected.

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to maintain an effective blood concentration for a longer period of time when administered to a living body, to mildly prevent the development of a thrombus and to dissolve or promote a thrombus. The purpose of the present invention is to provide a novel galactosaminoglycan having characteristics different from those of conventional galactosaminoglycans.

[0008]

Means for Solving the Problems The present inventors have made intensive studies in view of the above-mentioned circumstances, and as a result, from a specific animal tissue, especially a cockscomb, a novel galactosami having a relatively high molecular weight and characteristic disaccharide composition. The inventors have succeeded in purifying noglycan, and have found that when the novel galactosaminoglycan is administered to a living body, it is retained in the blood for a long time and exhibits a milder thrombolytic activity. Thus, the present invention has been completed.

That is, the present invention relates to: [1] Galactosaminoglycan having the following characteristics: (A) 2-Acetamide-based compound in an unsaturated disaccharide composition analyzed by high performance liquid chromatography when digested with chondroitinase ABC. 2-deoxy-3-O- (4
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 4 to 40%. (B) When chondroitinase B is allowed to act, about 3 to
43% by weight are not digested.

[2] galactosaminoglycan of [1] having the following properties: (A) 2-acetamido-2 in an unsaturated disaccharide composition analyzed by high performance liquid chromatography when digested with chondroitinase ABC -Deoxy-3-O- (4
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 4 to 25%. (B) When chondroitinase B is allowed to act, about 3 to
17% by weight are not digested. (C) The range of the molecular weight measured by the multi-angle light scattering method is 2
Not less than 10,000 daltons and not more than 200,000 daltons.

[3] The molecular weight distribution measured by the multi-angle light scattering method is as follows: 79 to 99% of a molecular weight of 40,000 or less;
The galactosaminoglycan of [2], wherein 1 to 20% of the total is 100% or more and 1% or less,

[4] The galactosaminoglycan of [2], wherein the weight-average molecular weight measured by a multi-angle light scattering method is not less than 28,000 daltons and not more than 43,000 daltons.

[5] Galactosaminoglycan having the following properties: (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3 in an unsaturated disaccharide composition analyzed by high performance liquid chromatography. -O- (4
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 16 to 25%. (B) When chondroitinase B is acted on,
18% by weight is not digested. (C) The molecular weight range measured by the multi-angle light scattering method is 4
Not less than 10,000 daltons and not more than 200,000 daltons.

[6] The molecular weight distribution measured by the multi-angle light scattering method is such that the molecular weight is 40% or less, 5% or less, 40,000 to 100,000 is 90% or more, and 100,000 or more is 5% or less. [5] galactosaminoglycan,

[7] The galactosaminoglycan of [5], wherein the weight-average molecular weight measured by a multi-angle light scattering method is not less than 520,000 daltons and not more than 79,000 daltons.

[8] Galactosaminoglycan having the following properties: (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3 in an unsaturated disaccharide composition analyzed by high performance liquid chromatography. -O- (4
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 26-40%. (B) When chondroitinase B is allowed to act, about 15
~ 43% by weight is not digested. (C) The molecular weight range measured by the multi-angle light scattering method is 7
Not less than 10,000 daltons and not more than 1 million daltons.

[0017]

[9] The molecular weight distribution measured by the multi-angle light scattering method is 40,000 to 100,000, 40 to 70%, 100,000 or more, 30 to 60%, and substantially does not contain a molecule having a molecular weight of 40,000 or less. A galactosaminoglycan according to [6],

[10] The weight average molecular weight measured by the multi-angle light scattering method is 720,000 daltons or more and 11.0
The galactosaminoglycan of [8], which is not more than 10,000 daltons.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Although the method for preparing the galactosaminoglycan of the present invention is not limited, it is usually prepared using tissues or organs such as birds and mammals such as the coron, skin, umbilical cord, and internal organs as raw materials, crushing, extracting, enzymatic decomposition, and organic solvents. Separation and purification can be carried out by appropriately combining methods usually used for separation and purification of glycosaminoglycan, such as precipitation, salting out, salt solution, and various types of chromatography. Preferably, for example, the chicken crown is hydrolyzed with a protease to decompose the protein, and after filtering the hydrolyzed solution,
A galactosaminoglycan fraction is precipitated from the filtrate using an alcohol such as ethanol in the presence of sodium chloride. If necessary, when the content of heparin / heparan sulfate is to be reduced, the collected precipitate is treated with nitrite as described below (Shivery and Conrad method, Biochemistry, 1).
5, 3932-3942 (1976)) or ion-exchange chromatography (WO95 / 09188), or a combination of these two treatments to remove impurities, alcohol precipitation, filtration, and desalting to remove galactosaminoglycan. A preparation (hereinafter referred to as "CCGG") is obtained. still,
The separation and purification of the galactosaminoglycan preparation is not limited to the above-mentioned method, but is performed by extraction according to a known method such as Japanese Patent Publication No. 60-9042, Japanese Patent Publication No. 61-21241, and the like.
After filtration, the filtrate can be precipitated by alcohol in the presence of sodium chloride in the same manner as described above.

Further, the above-mentioned CCGG is fractionated by a separation method based on the difference in molecular weight, and two high molecular fractions, galactosaminoglycan I and galactosaminoglycan II (hereinafter, referred to as “galactosaminoglycan II”).
"CCGGI" and "CCGGII", respectively. This molecular weight fractionation can be performed by a conventional method, for example, ultracentrifugation, gel filtration, molecular exclusion chromatography, ultrafiltration, or a combination of these methods. Among them, a method of performing a combination of ultrafiltration and molecular exclusion chromatography is preferable.

The weight average molecular weight of the galactosaminoglycan of the present invention is determined to be about 28,000 to about 43,000 daltons, preferably about 3.2, by multi-angle light scattering described below.
10,000 daltons to 38,000 daltons, CCGGI is about 5.2
10,000 daltons to about 79,000 daltons, preferably 6.0 to
71,000 daltons, CCGGII is about 720,000 daltons
It is about 110,000 daltons, preferably about 84,000 to about 98,000 daltons. In the gel filtration high-performance liquid chromatography (GPC-HPLC) method described below, CCGG contains about 30,000 to 46,000 daltons, preferably about 35,000 to about 41,000 daltons.
CGGI is about 45,000 daltons to about 69,000 daltons,
Preferably about 53,000 Dalton to 61,000 Dalton, C
CGG II is about 68,000 daltons to about 102,000 daltons, preferably about 79,000 daltons to about 91,000 daltons. The range of the molecular weight of the galactosaminoglycan of the present invention measured by the multi-angle light scattering method (the range of the minimum molecular weight and the maximum molecular weight of the galactosaminoglycan contained in the specific fraction) is 2 to 2 for CCGG. 200,000, CCGG
The preferred range of the molecular weight distribution (percentage) measured by the same method is shown in Table 1, and the preferred range of the molecular weight distribution (percentage) measured by the GPC-HPLC method is shown in Table 1. Are shown in Table 2.

[0022]

[Table 1] Table 1 分子 Molecular weight 40,000 or less 40,000-100,000 100,000 or more 表──────── CCGG 79% to 99% 1% to 20% 1% or less CCGG I 5% or less 90% or more 5% or less CCGGII 0% 40% to 70% 30% to 60% ──── ────────────────

[0023]

[Table 2] Table 2 分子 Molecular weight 20,000 or less 20,000 to 80,000 or more ──────────── ──────── CCGG 5% to 30% 70% to 95% 0% CCGG I 10% or less 75% to 95% 25% or less CCGGII 0% 35% to 60% 40% to 65% ─── ─────────────────

The composition of unsaturated disaccharide when galactosaminoglycan is digested with chondroitinase ABC indicates that the composition of 2-acetamido-2-deoxy-3-O-
The content of (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose (hereinafter referred to as “ΔDi-6S content”) is usually about 4%. % To 40%, especially about 4% for CCGG.
About 25%, preferably about 7% to about 16%, CCGGI
Is about 16% to about 25%, preferably about 16% to 20%,
CCGGII is about 26% to 40%, preferably about 30% to
35%. Here, the ΔDi-6S content is a value obtained by a disaccharide composition analysis by high-performance liquid chromatography, which will be described later, and includes various unsaturated disaccharides resulting from the degradation of galactosaminoglycan with chondroitinase ABC (FIG. 1). Reference) means the content (mol%) of N-acetyl-D-galactosamine which is sulfated at the 6th position.

2-acetamido-2-deoxy-3-O
-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose (hereinafter referred to as “Δ
Di-0S ”) is usually about 4% to about 12%.
In particular, CCGG is about 4% to about 10%, preferably about 4.5% to about 10%, CCGGI is about 5% to about 10%, preferably about 6% to 9%, and CCGGII is about 5%
To about 12%, preferably about 7% to about 10%.

2-acetamido-2-deoxy-3-O
The content of-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose (hereinafter referred to as “ΔDi-4S”) is usually about 52%. % To about 87%, particularly CCGG of about 6%.
3% to about 85%, preferably about 68% to about 84%, CC
GGI is about 55% to about 70%, preferably about 60% to 7%.
0%, CCGGII is about 52% to about 65%, preferably about 52% to about 60%.

2-acetamido-2-deoxy-3-O
-(4-Deoxy-2-O-sulfo-α-L-threo-
Hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose (hereinafter referred to as "ΔDi- _SD ")
Is usually in the range of about 0% to about 5%, especially CC
GG is about 0.5% to about 2.2%, preferably about 0.8%
~ 1.7%.

2-acetamido-2-deoxy-3-O
-(4-Deoxy-2-O-sulfo-α-L-threo-
Hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose (hereinafter referred to as “ΔDi-S _B ”)
Is usually in the range of about 1% to about 10%, especially C
CGG is about 3% to about 8%, preferably about 2% to about 7%,
CCGGI is about 2% to about 5%, preferably about 3% to 5%.
%, CCGGII is about 1.5% to about 4%, preferably about 2%.
% To about 3%.

2-acetamido-2-deoxy-3-O
-(4-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D
- galactose (hereinafter, referred to as "[Delta] Di-S _E") content of is usually about 0 to about 2%.

The names and abbreviations of the various unsaturated disaccharides are Yo
shida, K. et al., Analytical Biochemistry, 177, 32
7-332 (1989).

Further, the content of the fraction that is not digested when chondroitinase B is allowed to act on the galactosaminoglycan of the present invention (hereinafter referred to as “chondroitinase B-resistant fraction”) is usually about 3% by weight. % To about 43% by weight, preferably about 3% to about 23% by weight, and CCGG is about 3% by weight.
To about 17% by weight, preferably about 5% to about 15% by weight, CCGGI about 5% to about 18% by weight, preferably about 7% to about 14% by weight, and CCGGII about 15% to about 15% by weight. 43% by weight, preferably about 20% to about 30% by weight. Here, the content of the chondroitinase B-resistant fraction is a value obtained by analyzing the chondroitinase B digestion pattern described below, and the content (%) of the unreacted chondroitinase B digestion. Means

As described above, the galactosaminoglycan of the present invention is a novel galactosaminoglycan having a relatively high molecular weight and a characteristic disaccharide composition. Among the galactosaminoglycans of the present invention, especially CCGGI and CCGGII
Has a low content of low molecular fraction, and when administered to animals,
Compared with CCDS, which is one of galactosaminoglycans, and known dermatan sulfate derived from organs of animals such as cattle and pigs, it has a longer effective blood concentration and exhibits milder thrombolytic activity. CCGG has a longer blood retention time than known dermatan sulfate derived from organs of animals such as cattle and pigs, and is also useful as a raw material for preparing CCGGI and CCGGII. Galactosaminoglycan of the present invention is a multi-organ failure associated with generalized intravascular coagulation syndrome and multiple thrombus, thrombosis and glomerulonephritis such as deep vein thrombosis,
It also has an excellent effect on inflammation associated with collagen disease, intractable vasculitis, autoimmune disease, multiple sclerosis, and the like.

[0033]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

<1> ccgg, ccggI, and ccg
Preparation of gII To 1,500 kg of cockscomb, 4000 L of water was added, minced and then boiled. After cooling, protease (Pronase, Kaken Pharmaceutical Co., Ltd.) was added and hydrolyzed overnight. In the hydrolysis solution,
After adding 32 L of benzalkonium chloride solution, the mixture was filtered through diatomaceous earth, and the filtered supernatant was discarded to obtain 180 kg of diatomaceous earth. 350 L of water and 42 kg of sodium chloride were added to the diatomaceous earth, followed by filtration. 250 L of ethanol was added to the filtrate, and the obtained precipitate was dried to obtain 1.3 kg of powder.

The above powder was dissolved in 1.3 L of water to prepare a 10% solution, and Shivery and Conrad were prepared.
Heparin / heparan sulfate was removed by performing nitrous acid treatment by the method. That is, the solution in which the powder is dissolved is added to a solution of 0.1.
The mixture was mixed with a 1% nitrous acid aqueous solution, left at room temperature for 10 minutes, and the precipitate was removed by filtration. The filtrate was adjusted to pH 10.5, sodium chloride was added to a final concentration of 1%, and ethanol was further added to a final concentration of 48%.
Add with stirring over ~ 40 minutes. Activated carbon was added to the resulting precipitate, and the mixture was subjected to suction filtration.
The solution was desalted through iaion SA-12A (Mitsubishi Chemical Corporation), and ethanol was added to the filtrate to obtain 105 g of a galactosaminoglycan preparation (ccgg).

The obtained ccgg (105 g) was added in an amount of 0.2 mol.
1 / L sodium chloride solution to a concentration of 0.7%, and subjected to molecular ultrafiltration using a membrane (manufactured by Amicon) having a molecular exclusion limit of 100,000 Da, and 0 times twice the original volume. .
Washed with 2 mol / L sodium chloride solution.

This solution was dissolved in a 0.2 mol / L sodium chloride solution so as to have a concentration of 5%.
Gel filtration using a sodium chloride solution as a solvent (Sepha
cryl S-400, Pharmacia, φ1.0 × 1
20 cm). 210 of each fraction (1 mL / fraction)
The absorbance at nm was measured and the fraction I (ccggI)
(56 mL to 120 mL) and Fraction II (ccggII) (0
５５55 mL). After concentrating each fraction with a rotary evaporator, a hydrophilic membrane filter (0.
22 μm, Millipore) and precipitated with 2 volumes of ethanol. The precipitate was collected, washed first with 90% ethanol, then with 95% ethanol, and finally with 100% ethanol. The washed product was vacuum dried for at least 48 hours.

<2> ccgg, ccggI and ccgg
Physicochemical properties of II (1) Measurement of weight average molecular weight by GPC-HPLC method The weight average molecular weights of ccgg, ccggI and ccggII were determined by the method of Arai et al. (Biochem. Bioch).
em. Biophys. Acta, 1117, 60-7
0,1992). Chondroitin sulfate of known molecular weight (weight average molecular weight 39100, 180
00,8050) and sodium hyaluronate (weight average molecular weight 104000) as standard, gel filtration using high performance liquid chromatography (GPC-HPLC)
Determined by the elution time at The column is TSKge
l G4000PW _XL , G3000PW _XL and G25
00PW _XL (φ7.8X300mm each, Tosoh Corporation)
Was used. The solvent used was a 0.2 mol / L sodium chloride solution, the flow rate was 0.6 ml / min, and the detector used was a differential refractive index detector (RI-8000, Tosoh Corporation).

(2) Measurement of molecular weight by multi-angle light scattering method The molecular weight of ccgg, ccggI and II was measured by multi-angle light scattering method. The differential refractive index detector (Sho
dex RI-71, Showa Denko KK) and a multi-angle light scattering detector (DAWN, Wyatt Technology).
y CORPORATION, and ASTRA software (Wyatt Technology CO)
(RPORATION) and the molecular weight was determined by analysis. Column is SB-806
HQ (φ8 × 300 mm, Showa Denko KK), the solvent used was a 0.1 mol / L sodium nitrate solution, and the flow rate was 1.0 mL / min.

(3) Analysis of chondroitinase B digestion pattern 1% solutions of ccgg, ccggI and ccggII were each 1
Add buffer A (0.001 mol / L calcium acetate, 0.02 mol / L Tris-HCl, pH
7.5) 0.03 U of chondroitinase B in 10 μL
(Seikagaku Corporation) was added and digested at 37 ° C. for 2 hours. The reaction was stopped by heating in a boiling water bath for 1 minute, and 10 μL corresponding to 100 μg of digest was subjected to GPC at 40 ° C.
-Analyzed using HPLC. Column is TSK gel
G4000PW _XL , G3000PW _XL and G250
What was connected with 0PW _XL (each φ7.8 × 300 mm, Tosoh Corporation) was used. The solvent used was a 0.2 mol / L sodium chloride solution, the flow rate was 0.6 ml / min, and the detectors were a differential refractive index detector (RI-8020, Tosoh Corporation) and an ultraviolet-visible detector (UV-8020, A2
30 nm, Tosoh Corporation) was used.

(4) Analysis of Disaccharide Composition Analysis of the positions of the sulfate groups of ccgg, ccggI and II was carried out in accordance with the description of Shinsei Kagaku Jikken Koza 3 Carbohydrate II, p49-62. That is, ccgg, ccggI and II are completely digested into disaccharides by chondroitinase ABC (Seikagaku Corporation), and the resulting disaccharides containing unsaturated bonds (unsaturated disaccharides) are analyzed by GPC-HPLC. analyzed. Further, under these conditions, ΔDi-diS _B and ΔDi
To separate the -dis _E, it was digested with digests further chondro-6-sulfatase (Seikagaku Co.)
ΔDi-diS _B was shifted to ΔDi-0S and analyzed by GPC-HPLC in the same manner. The two results were compared and analyzed to calculate the disaccharide composition ratio. Details are described below.

1) Digestion with chondroitinase ABC Digestion method of ccgg and ccggI In a 1% ccgg solution or 50 μL of a 1% ccggI solution,
Buffer B (0.01 mol / L sodium acetate, 0.0
5 mol / L Tris-HCl, pH 7.5) 10 μ
A solution of 0.5 U of chondroitinase ABC dissolved in L was added and digested at 37 ° C. for 2 hours. The reaction was stopped by heating in a boiling water bath for 1 minute and centrifuged to remove insolubles.

A solution obtained by dissolving 0.5 U of chondroitinase ABC in 10 μL of buffer B was added to the digest, and the digest was digested at 37 ° C. for 6 hours. The reaction was stopped by heating in a boiling water bath for 1 minute and centrifuged to remove insolubles.

Digestion method of ccggII In 50 μL of 0.8% ccggII solution, add 20 μl of buffer B
L was added with 1.0 U of chondroitinase ABC dissolved therein, and digested at 37 ° C. After 3 hours on the way, 2.0 U of chondroitinase ABC was added to 4 μl of distilled water.
L and added. Twenty-four hours after the start of digestion, the mixture was heated in a boiling water bath for one minute to stop the reaction, and centrifuged to remove insolubles.

2) Digestion with chondro-6-sulfatase Buffer C (0.02 mol) per 100 μg equivalent of the digestion with chondroitinase ABC (above and above)
/ L Tris-AcOH, pH 7.0) dissolved in 0.25 U of chondro-6-sulfatase was added to 50 μL, digested at 37 ° C. for 24 hours, and centrifuged to remove insolubles.

3) Analysis by HPLC The digested product of 1) or 2) above, ie, chondroitinase ABC digested solution or digested solution thereof further digested with chondro-6-sulfatase was analyzed by HPLC, respectively. The column is YMC-Pack PA-
120-S5 ion exchange column (φ2.6 × 250m
m, YMC). 6 at a flow rate of 1.5 mL / min
2% 0.8 mol / L sodium hydrogen phosphate in 0 minutes
Flowed with a linear concentration gradient from to 100%. Based on the elution position of the unsaturated chondro-disaccharide kit (Seikagaku Corporation), 232 n
m and the sum of the peak areas identified as unsaturated disaccharides was calculated as 100%, and the disaccharide composition ratio was determined.

(5) Measurement of Intrinsic Viscosity The intrinsic viscosity of ccgg and ccggI was measured in accordance with the thirteenth revised Japanese Pharmacopoeia. An automatic viscosity measuring device (VMC-052, Rigosha Co., Ltd.) was used as the measuring device. The solvent used was a 0.2 mol / L sodium chloride solution, and the same solution was used for measurement of the flow time of an Ubbelohde type viscosity tube. The viscosity was measured at 30 ± 0.1 ° C., and one hundredth of a second of the flow time was rounded off, and the measured value having a difference within 0.1 second for three consecutive times was used for calculating the intrinsic viscosity.

The limiting viscosity is the reduced viscosity (= (t _s / t ₀ -1)
/ C, t _s : flow time of the solution, t ₀ : flow time of the solvent,
(C: sample concentration (% by weight)) was plotted on the ordinate and the concentration was plotted on the abscissa.

(6) Results Table 3 summarizes the results of (1) to (5). The range in parentheses indicates the range, and the abbreviation of the disaccharide composition follows that of FIG. The proportion of the disaccharide moiety was calculated by analyzing the chondroitinase B digestion pattern. Table 3 shows the results.

[0050]

[Table 3]

The molecular weight determined by the multi-angle light scattering method is ccg
gI is about 66,000 daltons, ccggII is about 91,000 daltons, and the average molecular weight determined by GPC-HPLC method is 570,000 daltons for ccggI and ccggII for ccggI.
Has a very high molecular weight of 85,000 daltons.

The chondroitinase B digestion resistant fraction was not present in CCDS, whereas ccgg was 5.3% by weight, ccggI was 13.9% by weight, and ccgg
In II it was 33.9% by weight.

Further, in the unsaturated disaccharide composition, ΔDi-
The 6S content is 12.0% by weight for ccgg and 1 for ccggI.
9.7% by weight, 31.1% by weight for ccggII and CCD
As compared with S, the composition ratio was as high as 3 times or more and 8 times or less.

(7) Analysis of Disaccharide Composition Ratio Range When ccgg, ccggI and ccggII were digested with chondroitinase ABC, the range of unsaturated disaccharide composition analyzed by high performance liquid chromatography was examined. ccg
g of 10 preparations, ΔDi-0S, ΔDi-
6S, ΔDi-4S, ΔDi− _SD , ΔDi−SB _{+ E} , Δ
Table 4 shows the results of measuring the composition ratio of Di-triS in the same manner as in (4) above.

[0055]

[Table 4]

In addition, for the other three lots of the ccgg preparation, ΔDi-0S, ΔDi-6S, ΔDi-4S,
[Delta] Di-S _D, the results of examining the composition of ΔDi-S _{B + E,} and [Delta] Di-Tris shown in Table 5. Note that ΔDi-S
_{B + E} represents the total amount of [Delta] Di-S _B and [Delta] Di-S _E.

[0057]

Table 5 Composition ratio of disaccharide (ccgg) ──────────────────────────────────── Lot .No. ΔDi-0S ΔDi-6S ΔDi-4S ΔDi-S _D ΔDi-S _{B + E} ΔDi-triS total ──────────────────────────────────── RDX045A 6.40 14.27 72.38 1.40 5.55 0.00 100.00 RDX045B 6.16 9.88 76.15 1.36 6.46 0.00 100.00 RDX045C 5.76 7.76 79.46 1.30 5.71 0.00 100.00 ────────────────────────────────────

(8) Analysis of molecular weight distribution GPC-HPL of ccgg, ccggI and ccggII
The molecular weight distribution was examined from the elution pattern of C. GPC-H
Table 6 shows the results of the PLC method. Table 7 shows the results of the multi-angle light scattering method.

[0059]

[Table 6] Table 6 分子 Molecular weight 20,000 or less 20,000 to 40,000 to 40,000 to 60,000 ~ 80,000 or more (%) (%) (%) (%) (%) ──────────────────────────── ccgg 12.9 61.6 23.9 1.7 0.0 ccgg I 1.7 15.9 42.8 26.5 13.2 ccggII 0.0 2.3 14.1 31.6 52.0 ────────────────────────────

[0060]

[Table 7]

<3> ccgg, ccggI and ccgg
Biological activity of II (1) Activated partial thromboplastin time (APTT)
[Measurement method] An APTT reagent containing phospholipids and calcium ion were added to plasma, and the time for forming fibrin was measured for various galactosaminoglycans (ccgg, ccggI).
And the time extended by adding II) was measured.

[0062] Plasma was prepared using SD (sprague-Dawl).
ey) Male rat (6-7 weeks old)
Blood is collected at 1/10 dose of 2% citric acid, and blood is collected at 3000 r.
Obtained by centrifugation at pm for 10 minutes. 100 μL of plasma and c
The final concentration of each galactosaminoglycan was 0, 3, 10, 30, 100, 300, 1, cgg, ccgg I and II.
The solution was diluted to 000 μg / mL, placed in a measurement cup, and kept at 37 ° C. for 1 minute. Thereafter, actin (APTT reagent, international reagent) 1 pre-incubated at 37 ° C
00 μL was added, and the mixture was further kept warm for 2 minutes. Then 37 ° C
100 μL of a 0.02 mol / L calcium chloride solution (international reagent) kept at room temperature, and the time until coagulation occurs from this time is measured by a blood coagulation automatic analyzer (KC-10).
A, Amelung).

[Results] As shown in FIG. 2, the galactosaminoglycan of the present invention prolonged the fibrin formation time in a dose-dependent manner. ccgg, ccggI and II were dermatan sulfate (BIDS, Cel) derived from bovine small intestine used as a reference.
sus Lab Inc. (Sales Cosmo Bio)
The activity was milder than that of. This tendency is for polymers,
The higher the concentration, the more prominent. This suggests that when used for treatment, the increase in activity due to an increase in blood concentration is close to a straight line, and if the concentration is used as an index, it directly leads to the evaluation of the activity, suggesting that it is easy to use in clinical settings. is there.

(2) Comparison of blood duration after administration to rats [Measurement method] SD male rats (Charlesleever) of 6.5 to 7.5 weeks of age were used. Cc for test drug
gg, ccggI and BIDS were used. After the rats were anesthetized with ether, each test drug was administered to the tail vein at a dose of 10 mg / Kg. At 10 and 120 minutes after administration, blood was collected from the inferior vena cava with 1/10 dose of 3.2% citric acid under ether anesthesia, and plasma was centrifuged to obtain plasma. The amount of galactosaminoglycan in plasma was determined by
I, measured according to p175-179. That is, plasma is converted to PD
After desalting with a -10R column (Pharmacia), freeze-drying, digesting the dried product with chondroitinase ABC, ultrafiltration through a membrane with a molecular weight cutoff of 5,000 (manufactured by Millipore), and filtering the obtained filtrate by HPLC analyzed.

[Results] The results are shown in FIG. 3 as a percentage (residual ratio) of the blood volume after a certain period of time with respect to the blood volume immediately after administration of ccgg, ccggI and BIDS. Administration 10
Min, the residual rate after ccgg is 2.2 compared to BIDS.
CcggI was 2.3 times higher. At 120 minutes after administration, 9.3% for ccgg and 1 for ccggI
Although 4.4% remained, BIDS was hardly detected. From the above results, it is considered that the amount of galactosaminoglycan remaining in the blood depends on the molecular weight, and it is expected that the residual rate will be higher with ccggII having a higher molecular weight.

[0066]

According to the present invention described above, the present invention provides a novel galactosaminoglycan that maintains an effective blood concentration for a long time, mildly inhibits thrombus development, and dissolves or promotes thrombolysis. I can do it.

[Brief description of the drawings]

FIG. 1 is a view showing names and structures of unsaturated disaccharide isomers constituting galactosaminoglycan.

FIG. 2. Various galactosaminoglycans (BIDS,
FIG. 4 shows the activated partial thromboplastin time (APTT) activity of ccgg, ccggI and II).

FIG. 3. Galactosaminoglycan (cc) after administration to rats
gg, ccggI) and bovine small intestine dermatan sulfate (BID)
The figure which shows the blood duration of S).

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Akihisa Suda 5-8-1-1403 Kurihama, Yokosuka City, Kanagawa Prefecture (72) Inventor Yoshiyuki Sakura 1-18-15, Teramae, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Mamoru Kyokajima 3-19-7 Minamigai, Higashiyamato-shi, Tokyo F-term (reference) 4C086 AA02 AA03 EA22 EA26 GA17 NA12 NA14 ZA40 ZA54 4C090 AA01 BA70 BB20 BB22 BB24 BB95 BC27 BD37 BD41 DA09 DA23

Claims (10)

[Claims] 1. A galactosaminoglycan having the following properties: (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3-O- (4) in unsaturated disaccharide composition analyzed by high performance liquid chromatography.
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 4 to 40%. (B) When chondroitinase B is allowed to act, about 3 to
43% by weight are not digested. 2. The galactosaminoglycan according to claim 1, which has the following properties. (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3-O- (4) in unsaturated disaccharide composition analyzed by high performance liquid chromatography.
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 4 to 25%. (B) When chondroitinase B is allowed to act, about 3 to
17% by weight are not digested. (C) The range of the molecular weight measured by the multi-angle light scattering method is 2
Not less than 10,000 daltons and not more than 200,000 daltons. 3. The molecular weight distribution measured by a multi-angle light scattering method is as follows: 79 to 99% for 40,000 or less, 1 to 20% for 40,000 to 100,000, and 1% or less for 100,000 or more. The galactosaminoglycan according to claim 2, wherein 4. The galactosaminoglycan according to claim 2, wherein the weight average molecular weight measured by a multi-angle light scattering method is not less than 28,000 daltons and not more than 43,000 daltons. 5. A galactosaminoglycan having the following properties: (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3-O- (4) in unsaturated disaccharide composition analyzed by high performance liquid chromatography.
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 16 to 25%. (B) When chondroitinase B is acted on,
18% by weight is not digested. (C) The molecular weight range measured by the multi-angle light scattering method is 4
Not less than 10,000 daltons and not more than 200,000 daltons. 6. The molecular weight distribution measured by the multi-angle light scattering method is as follows: when the molecular weight is 40,000 or less, 5% or less;
The galactosaminoglycan according to claim 5, wherein the percentage is not less than 5% and not less than 100,000. 7. The galactosaminoglycan according to claim 5, wherein the weight-average molecular weight measured by a multi-angle light scattering method is not less than 520,000 daltons and not more than 79,000 daltons. 8. A galactosaminoglycan having the following properties: (A) When digested with chondroitinase ABC, 2-acetamido-2-deoxy-3-O- (4) in unsaturated disaccharide composition analyzed by high performance liquid chromatography.
-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose is about 26-40%. (B) When chondroitinase B is allowed to act, about 15
~ 43% by weight is not digested. (C) The molecular weight range measured by the multi-angle light scattering method is 7
Not less than 10,000 daltons and not more than 1 million daltons. 9. The molecular weight distribution measured by the multi-angle light scattering method is 40 to 70,000 for 40,000 to 100,000 and 30 for 100,000 or more.
The galactosaminoglycan according to claim 8, wherein the galactosaminoglycan has a molecular weight of up to 60% and does not substantially contain a molecule having a molecular weight of 40,000 or less. 10. The galactosaminoglycan according to claim 8, wherein the weight average molecular weight measured by a multi-angle light scattering method is not less than 720,000 daltons and not more than 110,000 daltons.