JP2000103801A - Fructose-1,6-bisphosphoric acid aldolase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a useful and practical material which inhibits glycolysis system by making the material have a disaccharide-repeating structure as a fundamental structure comprising a gluosamine residue and an uronic acid residue and contain glycosaminoglycan as an effective component having sulfate group in the fundamental structure. SOLUTION: An inhibitor to glycolysis of fructose-1,6-bisphosphoric acid aldolase contains glycosaminoglycan as an effective component. Regarding a sulfate group, a content of sulfated secondary amine of a glycosamine residue is preferably not less than 40 mol% based on the amount of the of glycosamine residue composing a fundamental structure or a content of sulfated secondary hydroxy group of an uronic acid residue is preferably not less than 40 mol% based on the amount of the uronic acid residue. It is preferable that thromboplastin time does not exceed 100 seconds in the existence of 3 μg/ml of glycosaminoglycan. It is also preferable that an anti-coagulation activity of blood is kept at a low level in order to maintain safety to a living thing.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to glycolytic inhibitors, and more particularly, to glycolytic enzymes fructose 1,6-
It relates to inhibitors of bisphosphate aldolase.

[0002]

2. Description of the Related Art Heparin is an antithrombin III (ATII).
I), it is known to have binding properties to lipoprotein lipase, various cell growth factors and cytokines (FEBS
Lett. (1980) 117, 203-206; J. Biol. Chem. (1981) 256,
12893-12898; J. Cell Biol. (1990) 111, 1651-165.
9), Research on structures involved in the above-mentioned binding has attracted attention.

[0003] Based on the above-mentioned research, it has been suggested that, for example, by reducing the ATIII-binding structure in a heparin molecule, the anticoagulant effect inherent to heparin is reduced and the interaction with a bioactive factor is enhanced. For the purpose, chemical modification centering on desulfation of heparin has been performed (J. Carbohydr. Chem. (1993) 12, 507-521; Carbohydr.
Res. (1989) 193, 165-172; Carbohydr. Res. (1976) 46, 87
-95; WO 95/30424).

On the other hand, fructose 1,6-bisphosphate aldolase (EC 4.1.2.13), which is a key enzyme of glycolysis,
It is known that it may also be involved in the abnormal enhancement of glycolysis associated with malaria infection (J. Immunol. (199
0) 144, 1497-1503). Known inhibitors of the above-mentioned fructose 1,6-bisphosphate aldolase include Band3 peptide (B3P) and N-methyl-N-nitrosourea (MNU). It lacks practicality and has not been put to practical use.

[0005]

SUMMARY OF THE INVENTION In order to obtain a therapeutic agent for a disease in which the glycolytic system is enhanced, a useful and practical substance which can inhibit the glycolytic system and has few side effects on the living body has been obtained. Therefore, the development of such a substance has been desired.

[0006]

Means for Solving the Problems In view of the above problems, the present inventors have searched for a substance having an activity of inhibiting fructose 1,6-bisphosphate aldolase. As a result, heparin and modified heparin having a high affinity for the enzyme have been found. Have surprisingly found that they inhibit the activity of the enzyme, and have found that they can be used as fructose 1,6-bisphosphate aldolase inhibitors to complete the present invention. Heparin also has a significant change in its affinity for fructose 1,6-bisphosphate aldolase depending on the binding position of the sulfate group, and glucosamine constituting the basic skeleton, which is a main site to which the sulfate group contained in heparin binds. 6-position hydroxyl group or 2-position amino group of the residue, or uronic acid residue
Glycosamino with reduced anticoagulant activity while having the same fructose 1,6-bisphosphate aldolase inhibitory activity as heparin by regulating the number of sulfate groups in the sulfate group bound to the 2-hydroxyl group The inventors have found that glycans can be obtained and completed the present invention.

[0007] That is, the affinity of heparin or a modified product thereof for fructose 1,6-bisphosphate aldolase is determined by two glucosamine residues in the basic skeleton of heparin.
It is clarified that the sulfate group bonded to the amino group is most involved, and the effect decreases in the order of the sulfate group bonded to the 2-hydroxyl group of the uronic acid residue and the sulfate group bonded to the 6-hydroxyl group of the glucosamine residue. The fact that the modified heparin having more sulfate groups bound to the 2-position amino group of the glucosamine residue has a strong affinity for the above enzyme, and the inhibition of the activity of fructose 1,6-bisphosphate aldolase by heparin or modified heparin is a competitive inhibition. Yes, heparin or modified heparin with higher affinity for the enzyme was found to have a strong inhibitory effect on the enzyme activity.

That is, the present invention relates to a fructose 1,6-bisphosphate aldolase inhibitor. The gist of the present invention is to use a repeating structure of a disaccharide composed of a glucosamine residue and a uronic acid residue as a basic skeleton, and to include a sulfate group in the basic skeleton. The other gist is that the mol% of the glucosamine residue in which the 2-position amino group is sulfated is 4% with respect to the glucosamine residue constituting the basic skeleton.
An inhibitor containing glycosaminoglycan as an active ingredient in an amount of 0% or more, and a further gist is a uronic acid residue in which a 2-position hydroxyl group is sulfated with respect to the amount of uronic acid residues constituting the basic skeleton. An inhibitor comprising a glycosaminoglycan having a mol% of at least 40% as an active ingredient.

[0009] These inhibitors of the present invention include fructose 1,
Has high affinity for 6-bisphosphate aldolase,
At the same time, it exhibits inhibitory activity, and its active ingredient, glycosaminoglycan, has low anticoagulant activity and is therefore highly safe as a pharmaceutical.It is extremely high in comparison with conventional fructose 1,6-bisphosphate aldolase inhibitors. Useful.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to embodiments. The inhibitor of the present invention is an inhibitor of fructose 1,6-bisphosphate aldolase among a series of enzymes involved in glycolysis, and has a repeating structure of a glucosamine residue and a uronic acid residue as a basic skeleton, Glycosaminoglycan having a sulfate group in the basic skeleton is used as the active ingredient. Glycosaminoglycan (hereinafter, referred to as glycosaminoglycan of the present invention) which is an active ingredient of the inhibitor of the present invention is a sugar chain whose basic skeleton is sulfated.
The mol% of the glucosamine residue in which the 2-position amino group is sulfated with respect to the amount of glucosamine residue constituting the basic skeleton, or the uronic acid in which the 2-position hydroxyl group is sulfated with respect to the amount of uronic acid residue which constitutes the basic skeleton. Any of the mole% of residues is 4
It is preferably a glycosaminoglycan of 0% or more.

In order to ensure safety when the inhibitor of the present invention is used as a medicine, it is preferable that glycosaminoglycan as the active ingredient has low anticoagulant activity, Preferably, under the conditions containing the glycosaminoglycan at 3 μg / ml, the activated partial thromboplastin time (hereinafter referred to as “AP
TT ”)).
Preferably, the APTT does not exceed 100 seconds, more preferably it does not exceed 50 seconds. In addition, under the conditions containing the glycosaminoglycan at 1 μg / ml, the thrombin time (hereinafter also referred to as “TT”) described in the examples below.
When measured according to the measurement method of
Preferably it does not exceed 100 seconds, more preferably it does not exceed 50 seconds. The inhibitor of the present invention containing glycosaminoglycan having an anticoagulant activity in a low range as such as an active ingredient has extremely high safety when administered to a living body as a medicine.

By the way, heparin is known to have an effect on the living body such as an anticoagulant activity. This activity is determined by a disaccharide assay using 2-deoxy-2-sulfamino-.
4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as “ΔDiHS-tri (U , 6, N) S ”), which is a sulfate-rich structure detected by antithrombin II in blood.
It has been clarified to be caused by binding to I, and in the disaccharide analysis of sugar chains, the ΔDiHS-tri
If the structure detected as (U, 6, N) S is small, the glycosaminoglycan has low anticoagulant activity, which is a serious side effect on the living body. Therefore, the glycosaminoglycan of the present invention is one of the unsaturated disaccharides in the disaccharide analysis obtained by combining enzymatic digestion and high performance liquid chromatography described in Test Example 1 described below, among which ΔDiHS-tri (U, 6, N) S Is preferably 50% or less, more preferably 30% or less. Particularly preferred are glycosaminoglycans that are below the detection limit in the disaccharide assay.

The glycosaminoglycan of the present invention includes heparin in which the molar percentage of ΔDiHS-tri (U, 6, N) S is more than 50% in the composition of unsaturated disaccharide in the disaccharide analysis described later. Or, from heparan sulfate, a sulfate group bonded to the 6-position hydroxyl group or 2-position amino group of the glucosamine residue constituting the basic skeleton, or a sulfate group ester-bonded to the 2-position hydroxyl group of the uronic acid residue constituting the basic skeleton. Among them, glycosaminoglycan obtained by desulfating one or two sulfate groups is preferred. Among them, particularly, a sulfate group bonded to the 6-position hydroxyl group of the glucosamine residue, a sulfate group bonded to the 2-position amino group, and the uronic acid residue
Glycosaminoglycans obtained by specifically desulfating only one of the sulfate groups of the sulfate group bonded to the 2-position hydroxyl group are preferred, and from the above-mentioned heparin or heparan sulfate, and / or Even glycosaminoglycans obtained by specifically desulfating a group can be used as glycosaminoglycans of the present invention.

For example, the glycosaminoglycan obtained by desulfating only the sulfate group bonded to the hydroxyl group at position 6 (hereinafter referred to as “glycosaminoglycan of the present invention” for convenience) is based on the total amount of glucosamine residues. The amount of a glucosamine residue in which a sulfate group is ester-bonded to the 6-position hydroxyl group is 85% or less, more preferably 60% or less, and further preferably 30% or less, and most preferably, the disaccharide. In the analysis, 10% or less of the unsaturated disaccharide having a glucosamine residue in which the hydroxyl group at the 6-position is sulfated is 10% or less, and among them, glycosaminoglycan which is particularly undetectable is exemplified.

In the glycosaminoglycan of the present invention, in the repeating structure of a disaccharide composed of a glucosamine residue and a uronic acid residue constituting a basic skeleton, a bonding position of a sulfate group to each of the monosaccharides, and The amount can be calculated from the analysis of the composition of the unsaturated disaccharide (disaccharide composition) by combining hydrolysis with glycosaminoglycan degrading enzyme and high performance liquid chromatography.

Here, the “disaccharide composition” refers to an unsaturated disaccharide represented by the following general formula (1) in a disaccharide analysis combining enzyme digestion and high performance liquid chromatography described in Test Example 1 described later. Total amount of sugar [2-acetamido-2-deoxy-4-
O- (4-deoxy-α-L-threo-hex-enopyranosyluronic acid) -D-glucose (hereinafter referred to as ΔDiHS-0S), 2-deoxy-2-sulfamino-4-O- (4 -Deoxy-α-L-threo-h
ex-4-enopyranosyluronic acid) -D-glucose (hereinafter ΔD
iHS-NS), 2-acetamido-2-deoxy-4-O-
(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as ΔDiHS-6S), 2-acetamido-2-deoxy- 4-O- (4-deoxy-2-
O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid)-
D-glucose (hereinafter referred to as ΔDiHS-US), 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L-threo-hex-4
-Enopyranosyluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as ΔDiHS-di (6, N) S), 2-deoxy-2-sulfamino-4-O- (4-deoxy -2-O-sulfo-α-L-threo-h
ex-4-enopyranosyluronic acid) -D-glucose (hereinafter ΔD
iHS-di (U, N) S), 2-acetamido-2-deoxy-4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyrano (Siluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as ΔDiHS-di (U, 6) S), 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O -Sulfo-α-L-threo-hex-4
-Enopyranosyluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as ΔDiHS-tri (U, 6, N) S) 100%
, And corresponds to the mole% of each of the above unsaturated disaccharides, and the numerical value reflects the position and number of sulfate groups of glycosaminoglycan before enzymatic digestion.

For example, the “mol% of the glucosamine residue in which the sulfate group is ester-bonded to the hydroxyl group at position 6” can be calculated from the disaccharide composition described above. That is,
Unsaturated disaccharide (ΔDiHS-6) generated from a basic skeleton containing a glucosamine residue in which a sulfate group is ester-bonded to the 6-hydroxyl group.
S, ΔDiHS-di (6, N) S, ΔDiHS-di (U, 6) S, and ΔDiHS-tr
The sum of the disaccharide compositions of i (U, 6, N) S) is the above-mentioned “mol% of glucosamine residue in which sulfate group is ester-bonded to hydroxyl group at position 6”.
Is equivalent to Similarly, “mol% of uronic acid residue in which the 2-hydroxyl group is sulfated” is an unsaturated disaccharide (U) formed from a basic skeleton containing a uronic acid residue in which a sulfuric acid group is ester-bonded to the 2-hydroxyl group ( ΔDiHS-US, ΔDiHS-di (U, 6) S,
ΔDiHS-di (U, N) S and ΔDiHS-tri (U, 6, N) S) can be calculated as the sum of the respective disaccharide compositions.
Mole percent of glucosamine residues in which the amino group is sulfated "
Is an unsaturated disaccharide (ΔDiHS-N) generated from a basic skeleton containing a glucosamine residue having a sulfate group bound to the 2-amino group.
S, ΔDiHS-di (6, N) S, ΔDiHS-di (U, N) S, and ΔDiHS-tr
It can be calculated as the sum of each disaccharide composition of i (U, 6, N) S).

[0018]

Embedded image

[0019]

[Table 1]

The structures indicated by the above abbreviations may be described as follows. ΔDiHS-0S: ΔHexA1 → 4GlcNAc, ΔDiHS-NS: ΔHexA1 → 4
GlcNS, ΔDiHS-6S: ΔHexA1 → 4GlcNAc (6S), ΔDiHS-U
S: ΔHexA (2S) 1 → 4GlcNAc, ΔDiHS-di (6, N) S: ΔHexA1
→ 4GlcNS (6S), ΔDiHS-di (U, N) S: ΔHexA (2S) 1 → 4GlcN
S, ΔDiHS-di (U, 6) S: ΔHexA (2S) 1 → 4GlcNAc (6S), ΔDi
HS-tri (U, 6, N) S: ΔHexA (2S) 1 → 4GlcNS (6S). In the above formula, ΔHexA is unsaturated hexuronic acid, GlcNAc is N-acetylglucosamine, GlcNS is N-sulfated glucosamine,
The position in parentheses indicates the bonding position of the sulfate group.

Further, the glycosaminoglycan of the present invention comprises:
With respect to the total amount of glucosamine residues constituting the basic skeleton
The mole% of the glucosamine residue in which the 2-position amino group is sulfated is preferably at least 40%, more preferably at least 50%, most preferably at least 60%. In the glycosaminoglycan of the present invention, mol% of the glucosamine residue in which the 2-position amino group is sulfated is, according to the disaccharide analysis, ΔDiHS-NS, ΔDiHS-di (6, N) S, ΔDiHS-d
It can be calculated from the sum of i (U, N) S and ΔDiHS-tri (U, 6, N) S.

Further, in the glycosaminoglycan of the present invention, it is preferable that the mol% of the uronic acid residue in which the 2-position hydroxyl group is sulfated is at least 40% or more based on the total amount of the uronic acid residue constituting the basic skeleton. And more preferably 50% or more. In the glycosaminoglycan of the present invention, the mol% of the uronic acid residue in which the 2-position hydroxyl group is sulfated is
According to the disaccharide analysis, ΔDiHS-US, ΔDiHS-di (U, 6) S, Δ
It can be calculated from the sum of DiHS-di (U, N) S and ΔDiHS-tri (U, 6, N) S.

Furthermore, the glycosaminoglycan of the present invention is characterized in that, in relation to the amount of a glucosamine residue in which a sulfate group is bonded to the 6-position hydroxyl group of the glucosamine residue, the mole of uronic acid residue in which a sulfate group is bonded to the 2-position is used. % And the mole% of the glucosamine residue having a sulfate group bonded to the 6th position [(ΔD
iHS-US, ΔDiHS-di (U, N) S, ΔDiHS-di (U, 6) S, and ΔDi
HS-tri (U, 6, N) S) and (ΔDiHS-6S, ΔDiHS-di (6, N) S,
ΔDiHS-di (U, 6) S, and the sum of mol% of ΔDiHS-tri (U, 6, N) S) is preferably 170 or less, and if it is 100 or less, the antithrombin III binding structure described below is preferred. Most preferred because it is kept low.

Therefore, when the sugar chain of glycosaminoglycan was analyzed by the above-mentioned disaccharide analysis, it was found that 6
It is preferable that the ratio of the sulfuric acid esterification of the hydroxyl group at the 2-position and the hydroxyl group at the 2-position of the uronic acid residue always satisfies the following formula. That is, 0 <A + B ≦ 170 is preferable, and 0 <A + B ≦ 1
00 is most preferred. Where: A = mol% of unsaturated disaccharide having a sulfate group at position 6 of glucosamine residue in disaccharide analysis; B = unsaturated disaccharide having a sulfate group at position 2 of uronic acid residue in disaccharide analysis It shows the value of mol% of sugar, and A and B are positive numbers of 100 or less.

The glycosaminoglycan obtained by desulfating only the sulfate group bonded to the 2-position amino group (hereinafter referred to as "the glycosaminoglycan of the present invention" for convenience) includes, for example, a basic skeleton With respect to the total amount of glucosamine residues, the mole% of the glucosamine residue in which the sulfate group is bonded to the 6-position hydroxyl group is more than 30%, and the mole% of the glucosamine residue in which the 2-position amino group is sulfated is 40%. Less than 30%, preferably less than 30%, and based on the total amount of uronic acid residues constituting the basic skeleton, the mol% of the uronic acid residue in which the 2-position hydroxyl group is sulfated is 40% or more, preferably 50%. The glycosaminoglycans described above are exemplified.

Further, for example, two of the above uronic acid residues
Glycosaminoglycan in which only a sulfate group bonded to a hydroxyl group is desulfated (hereinafter referred to as "the glycosaminoglycan of the present invention" for convenience) is, for example, 6% of the total amount of glucosamine residues constituting the basic skeleton. More than 30% mol% of the amount of glucosamine residue in which a sulfate group is bonded to a hydroxyl group, and more than 40%, preferably less than 50%, of mol% of a glucosamine residue in which a 2-amino group is sulfated, and 40% by mole of uronic acid residues in which the 2-position hydroxyl group is sulfated with respect to the total amount of uronic acid residues constituting the basic skeleton.
Glycosaminoglycans with less than 30%, preferably less than 30%, are exemplified.

As shown in the examples below, fructose 1,6 of each of the glycosaminoglycans (NDSH) of the present invention and glycosaminoglycans (2ODSH) of the present invention described above.
-With respect to bisphosphate aldolase inhibitory activity, the glycosaminoglycan of the present invention showed slightly lower results than heparin and glycosaminoglycan of the present invention, but both glycosaminoglycan of the present invention and high Since it has fructose 1,6-bisphosphate aldolase inhibitory activity, it is useful as an active ingredient of a fructose 1,6-bisphosphate aldolase inhibitor.

The average molecular weight of the glycosaminoglycan of the present invention is not particularly limited, but is usually 3,000 to 30,000 Da, preferably 4,000 to 20,000 Da, and most preferably 5,000 to 18,000 Da. In addition, the glycosaminoglycan of the present invention preferably has affinity for fructose 1,6-bisphosphate aldolase in affinity chromatography using agarose as a carrier, as shown in Example 4 described below. .

The glycosaminoglycan of the present invention can be obtained by a method for sulfating a 2-position amino group of a glucosamine residue or a 2-position hydroxyl group of a uronic acid residue constituting the basic skeleton, and a method for sulphating the glucosamine residue constituting the basic skeleton. Glycosaminoglycans having the basic skeleton, such as heparin or heparan sulfate, have a 6-position sulfate group or a method of desulfating the 2-position sulfate group of the uronic acid residue constituting the basic skeleton, which is appropriately combined, is used. It can be prepared by adjusting the number of sulfate groups. Of the glucosamine residue constituting the above basic skeleton
As a method for sulfating the 2-position amino group, for example, Nagasaw
(a) (Corbohydr. Res. (1989) 193, 165-172). That is, the glycosaminoglycan having the basic skeleton or a salt thereof is pH 9-10
Dissolved in an alkaline solution (eg, sodium carbonate solution, sodium hydroxide solution, potassium hydroxide solution, etc.) and solid trimethylammonium sulfonate or triethylammonium sulfonate at 50-55 ° C. for 6-24 hours.
Add time, additional.

As a technique for sulfate esterification of the 2-position hydroxyl group of the uronic acid residue constituting the basic skeleton, for example, the method of Nagasawa et al. (Corbohydr. Res. (1989) 1)
93, 165-172). That is, the glycosaminoglycan having the basic skeleton is converted into a tributylammonium salt by salt exchange according to a conventional method, and the resulting tributylammonium salt of glycosaminoglycan is completely dissolved in N, N-dimethylformamide. , 5 to 20 molar equivalents / molar amount of free hydroxyl groups at -10 to 0 ° C for 1 hour. As a method for desulfating a sulfate group bonded to the amino group at position 2 of the glucosamine residue constituting the basic skeleton, for example, Inoue & Na
gasawa method (Carbhydr. Res. (1976) 46.87-95) and the like. That is, the lyophilized powder of heparin pyridine salt prepared in the same manner as described above was mixed with dimethyl sulfoxide (DM
SO) After dissolving in an aqueous solution, usually at least 30 ° C, preferably
Heat at 40-60 ° C, then stop the reaction by adding distilled water, and usually adjust the pH to 9-12, preferably 9.5-
It is possible to desulfate the sulfate group bound to the 2-position amino group of the glucosamine residue constituting the basic skeleton by adjusting to 11.5 and dialyzing and freeze-drying.

As a method for desulfating a 6-position sulfate group of a glucosamine residue constituting the basic skeleton, for example, Mats
A modified version of the method of uo et al. (Carbohydr. Res. (1993) 241, 209-215). For example, as a method for desulfating the 6-position hydroxyl group that has been sulfated from heparin, a sodium salt of heparin is applied to a cation exchange resin column such as an Amberlite IR-120 (H ⁺ type) column and the eluate Is adjusted to a pH of 7 to 9, preferably 7.5 to 8.5 by adding an excess of pyridine, and lyophilized to prepare a pyridinium salt of heparin. After dissolving this heparin pyridinium salt in pyridine, N-methyl-N- (trimethylsilyl) trifluoroacetamide (MTSTFA) was added, and 80-1
20 ° C., preferably 90 to 115 ° C. for 30 minutes or more, preferably 2 minutes
Heat with stirring for about an hour. After stopping the reaction by adding water to the reaction mixture to make MTSTFA non-reactive, dialysis was performed against distilled water, and then the dialysis solution was removed.
After adjusting the pH to 9 to 11, preferably 9.5 to 10.5 with NaOH, it is possible to obtain a desired desulfated heparin by subjecting it to dialysis again and freeze-drying the resulting dialysis solution.

2 of uronic acid residues constituting the basic skeleton
As a technique for desulfating a sulfate group, for example, a method obtained by partially modifying the method of Jaseja et al. (Can. J. Chem. (1989) 67, 1449-1456) can be used. That is, the sodium salt of heparin is dissolved in a NaOH solution and lyophilized immediately. After dissolving the obtained lyophilized powder in distilled water, the pH is adjusted to 6 to 8, preferably 6.5 to 7.5 by adding acetic acid. Next, this solution is subjected to a dialysis treatment and a lyophilization treatment.

By combining these various desulfating methods, it is possible to prepare the glycosaminoglycan of the present invention which can be used as an active ingredient of the inhibitor of the present invention exemplified above.

The dosage form and route of administration of the inhibitor containing the glycosaminoglycan of the present invention as an active ingredient into a living body can be appropriately selected according to the nature and severity of the disease to be treated. it can. For example, pharmaceutical compositions (eg, injections, suspensions, emulsions, suppositories, tablets, capsules, etc.) of the inhibitor as such or together with other pharmacologically acceptable carriers, excipients, diluents, etc. As a liquid, plaster, ointment, lotion, pasta, liniment, gel such as a patch), for warm-blooded animals (eg, human, mouse, rat, hamster, rabbit, dog, cat, horse, etc.) In contrast, it can be safely administered parenterally or orally. For example, in the case of using the inhibitor of the present invention as a therapeutic agent for malaria to be applied after malaria infection, for example, for inhibiting glycolysis in blood, parenteral administration is preferable,
Suitable forms for this administration form include injections,
The administration method is preferably, but not limited to, injection or infusion.

The amount and the amount of glycosaminoglycan, which is an active ingredient of the inhibitor of the present invention, are individually determined according to the method of administration of the preparation, the dosage form, the purpose of use, the specific symptoms of the patient, and the weight of the patient. It is a matter to be determined in, but is not particularly limited, generally can be exemplified a blended amount of about 10μg ~ 50mg per formulation, as a dosage about 100μg / kg ~ 100mg / kg per day. Examples can be given. In addition, the frequency of administration of the above-mentioned preparation can be about once a day, and it can be administered 2 to 4 times a day or more. It is also possible to administer continuously by, for example, infusion.

By the way, a normal heparin mouse (male,
LD) by acute toxicity test for female) ₅₀Is 5,
000 mg / kg or more, 2,500 mg / kg or more by subcutaneous or intraperitoneal administration,
It is known to be 1,000 mg / kg or more by intravenous injection.
It is also commonly used as a pharmaceutical anticoagulant
Therefore, its safety, including bleeding activity, has already been confirmed.
ing. On the other hand, glycosaminog used for the inhibitor of the present invention
Lican is used for cultured cells (A31 cells (BALB / C 3T3))
Toxicity is not observed, and the above-mentioned heparin or anticoagulant
Since the modified heparin has reduced solid activity,
Pharmaceutical compositions containing harmful agents have high safety in biological administration
It can be said that.

Further, according to the examples described later, heparin used as a raw material (raw material heparin) shows the strongest inhibitory activity of fructose 1,6-bisphosphate aldolase as compared with its modified form. However, in view of the high safety required for pharmaceuticals and the inhibitory activity of the useful enzyme, glycosaminoglycan as an active ingredient is more effective than the raw material heparin in using the inhibitor of the present invention as a pharmaceutical composition. Rather, modified heparin is suitable.

[0038]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Test Example 1 [Disaccharide analysis by enzyme digestion] Raw material heparin or various modified heparins (these are hereinafter referred to as "heparins")
The analysis of the disaccharide composition, that is, the position of the sulfate group of the constituent sugars was performed as follows. That is, the unsaturated disaccharide (the above structural formula: Chemical Formula 1) produced by enzymatic digestion of heparins was analyzed by high performance liquid chromatography (HPLC) (Shinsei Kagaku Jikken Kozai 3, Carbohydrate II (Tokyo Kagaku Dojin, 1991), pp. 49-62, "Structural analysis combining 2.8 glycosaminoglycan degrading enzyme and HPLC"). The peak area of each unsaturated disaccharide whose structure could be determined was calculated, and the peak area relative to the total area was expressed as a percentage.

(1) Digestion of heparin with heparin-degrading enzyme According to the method described in Neogene Chemistry Laboratory Course 3, Carbohydrate II, pp. 54-59, 1.0 mg of heparin was added to 20 mM sodium acetate containing 2 mM calcium acetate (pH 7.0). 3) Dissolve in 220 μl and add 20 mU of heparitinase, 20 mU of heparitinase I and II to give 3
The reaction was performed at 7 ° C. for 2 hours. (2) Analysis by HPLC 20 μl of the solution after digestion by (1) was analyzed by HPLC (Medicalization, Model 852). Ion exchange column (Dionex, CarboPac PA-1 column φ4.0mm × 250m
m) was used to measure the absorbance at 232 nm. At a flow rate of 1 ml / min, a gradient system using lithium chloride (50 mM → 2.5
M) (Kariya, et al., Comp. Bio).
chem. Physiol., 103B, 473, (1992)).

Test Example 2 [Measurement of Molecular Weight] 10 μl of a 3% solution of modified heparin was analyzed by gel filtration by HPLC. The column is TSKgel- (G4000 + G300
0 + G2500) PW _XL (Tosoh, φ7.8 mm × 30 cm) was eluted at a flow rate of 1.0 ml / min using 0.2 M sodium chloride as an eluent. A differential refractometer (Shimadzu Corporation, AID-2A) was used to detect the modified heparin. The average molecular weight was determined using heparin, whose molecular weight was specified in advance, as a control (Kaneda
et al., Biochem. Biophys. Res. Comm., 220, 108-112.
(1996)). The molecular weight of the raw material heparin was measured using a light scattering method (Nagasawa et al., J. Biochem., 989-993 (197
7)).

Test Example 3 [Measurement of activated partial thromboplastin time (APTT)] Blood was collected from the rat inferior vena cava with 1/10 volume of 3.2% citric acid, and the blood was centrifuged at 1,000 × g for 10 minutes to obtain plasma. . Plasma 100
μl and 100 μl of a heparin solution of various concentrations were put in a measuring cup and kept at 37 ° C. for 1 minute. After that, 37
100 μl of actin (trade name: Yoshitomi Pharmaceutical Co., Ltd.) kept at ° C. was added, and the mixture was further kept warm for 2 minutes. Next, 100 μl of a 0.02 M CaCl ₂ solution kept at 37 ° C. was added, and the time from this time until coagulation occurred was measured by a blood coagulation automatic measuring device (KC-10A: manufactured by Amelung).

Test Example 4 [Thrombin time (TT) measurement] 3.2% from rat inferior vena cava
Blood was collected with 1/10 volume of citrate, and the blood was centrifuged at 1,000 × g for 10 minutes to obtain plasma. 100 μl of plasma and 100 μl of heparin solutions at various concentrations were placed in a measuring cup and kept at 37 ° C. for 1 minute. Thereafter, 100 ml of thrombin (10 U / ml), which had been kept at 37 ° C. in advance for 5 minutes, was added. From this time, the time until coagulation occurred was measured by an automatic blood coagulation analyzer (KC-10A:
Amelung Co.).

The ¹³ C-NMR Measurement of ¹³ C-NMR] Test Example 5, using a GE Co. QE300 type NMR spectrometer, 100% heavy water (D ₂ O) D of the sample per 5% strength was substituted _{A 2} O solution was prepared, and methanol with a chemical shift of 51.66 ppm when TSP (sodium 3-trimethylsilylfluorodioxide) was set to 0 ppm was used as an internal standard.
C., the pulse width was 60 °, and the number of times of integration was 90,000.

[Raw material heparin in the present specification] As the raw material heparin, heparin derived from bovine small intestine (commercial product: manufactured by SPL) is preferable. For example, heparin having the following physical properties can be used. (1) The disaccharide composition calculated from the values measured by the disaccharide analysis method described in Test Example 1 above is as shown in Table 2 below.

[0045]

[Table 2]

(2) The anticoagulant activity is 160 IU / mg. (3) The average molecular weight by the method described in Test Example 2 is 1
It is in the range of 1,000 to 14,000 Da. (4) Measured by the APTT measurement method described in Test Example 3 above
APTT is 200 seconds or more when the raw material heparin is added to the solution at 3 μg / ml. (5) TT measured by the TT measurement method described in Test Example 4 above
However, when the raw material heparin was added to the solution at 1 μg / ml, 600
Seconds or more.

The molar ratio of the glucosamine residue having a sulfate group bonded to the amino group at the 2-position in the raw material heparin to the total amount of glucosamine residues was calculated from the above disaccharide composition value, and was calculated as 87.9% (ΔD
iHS-NS, ΔDiHS-di (6, N) S, ΔDiHS-di (U, N) S, ΔDiHS-t
ri (U, 6, N) S), the molar ratio of the same sugar residue in which the sulfate group is bonded to the 6-position hydroxyl group is calculated from the disaccharide composition value, 82.3%
(ΔDiHS-6S, ΔDiHS-di (6, N) S, ΔDiHS-di (U, 6) S, ΔD
iHS-tri (U, 6, N) S), ΔDiHS-tri (U, 6, N) S amount is 6
It is 4.2%. ¹³ C-NM of standard heparin according to Test Example 5 above
The R spectrum is as shown in FIG. Further, APTT and TT of the raw material heparin were measured according to Test Examples 3 and 4, and the results are shown in Table 3.

[0048]

[Table 3]

Example 1 [Production of modified heparin] <1> Production of 2-O-desulfated heparin 2-O-desulfated heparin (2ODSH) was prepared by the method of Jaseja et al.
n. J. Chem. (1989) 67, 1449-1456) was synthesized by a partially modified method. That is, 200 mg of the sodium salt of the raw material heparin was dissolved in 20 ml of 0.4N NaOH and immediately subjected to freeze-drying treatment. 20 ml of the freeze-dried powder obtained
Dissolved in distilled water, and then add 1N acetic acid to obtain p.
Adjusted to H7. Next, this solution was sequentially subjected to a dialysis treatment and a freeze-drying treatment. This procedure was repeated twice to obtain 165 mg of 2ODSH. ¹³ C-NMR according to Test Example 5 of 2ODSH
The spectrum is as shown in FIG. The disaccharide composition value of 2ODSH and the molecular weight by gel filtration were measured according to Test Examples 1 and 2 above. Table 4 shows the results.

[0050]

[Table 4]

Unsaturated disaccharide having a sulfate group at the 6-position of the glucosamine residue: 61.7% Unsaturated disaccharide having a sulfate group at the 2-position amino group of the glucosamine residue: 91% Sulfuric acid at the 2-position of the uronic acid residue Unsaturated disaccharide having a group: 16.2% Average molecular weight: 10,900 Da Also, according to Test Examples 3 and 4, APTT and TT of 2ODSH were used.
Was measured, and the results are shown in Table 5.

[0052]

[Table 5]

<2> Production of N-desulfated heparin N-desulfated heparin (NDSH) was synthesized according to the method of Inoue & Nagasawa (Carbohydr. Res. (1976) 46, 87-95). That is, 200 mg of the sodium salt of the raw material heparin was applied to a column (1 × 10 cm) packed with Amberlite IR-120 (H ⁺ type) ion exchange resin (manufactured by Organo), and excess pyridine was added to the eluate to adjust the pH to pH 8. The mixture was lyophilized and freeze-dried to prepare heparin pyridinium salt (200 mg). This heparin pyridinium salt (200 mg) was dissolved in 20 ml of a 90% aqueous solution of 90% dimethyl sulfoxide (DMSO), and then heated at 50 ° C. for 5 hours. The reaction was stopped by adding an equal volume of distilled water to the reaction mixture, and adjusted to pH 10 by adding an appropriate amount of 1N NaOH.

Next, the reaction mixture was subjected to a dialysis treatment and a freeze-drying treatment in that order to remove N-desulfated heparin from the reaction mixture.
0 mg was obtained. Since N-desulfated heparin has a free amino group, the amino group is acetylated to N-desulfated,
Used as N-acetylated heparin (NDSH). That is, 160 mg of NDSH was dissolved in 80 ml of 10% methanol (MeOH) aqueous solution containing 50 mM Na ₂ CO ₃ cooled to 4 ° C., and 400 μl of acetic anhydride was added 5 times during 2 hours to 4 ° C. And stirred.
The pH of the reaction mixture was maintained at 7 to 8 by appropriately adding a 10% aqueous MeOH solution containing saturated Na ₂ CO ₃ . After the reaction was terminated by adding an equal volume of distilled water, dialysis and lyophilization were performed.
0 mg of NDSH was prepared. The ¹³ C-NMR spectrum of NDSH in Test Example 5 is as shown in FIG. The disaccharide composition value of NDSH and the molecular weight by gel filtration were measured according to Test Examples 1 and 2 above. Table 6 shows the results.

[0055]

[Table 6]

Unsaturated disaccharide having a sulfate group at the 6-position of the glucosamine residue: 82.5% Unsaturated disaccharide having a sulfate group at the 2-position amino group of the glucosamine residue: 0% Sulfuric acid at the 2-position of the uronic acid residue Unsaturated disaccharide having a group: 70.4% Average molecular weight: 10,500 Da Further, APTT and TT of NDSH were measured according to Test Examples 3 and 4, and the results are shown in Table 7.

[0057]

[Table 7]

<3> Method for producing 2,6-O-desulfated heparin 2,6-O-desulfated heparin (26ODSH) is prepared by preparing a heparin pyridinium salt by the same method as in <2>. After dissolving 200 mg in 20 ml of pyridine, 2 ml of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MTSTFA) was added, and the mixture was heated with stirring at 110 ° C. for 2 hours. After completion of the reaction, the reaction was stopped by adding 20 ml of water to the reaction mixture to make MTSTFA non-reactive, and then dialyzed against distilled water. Next, the inner dialysis solution was adjusted to pH 10 with 1N NaOH, and then subjected to dialysis treatment again, and the resulting inner dialysis solution was subjected to lyophilization treatment to obtain a lyophilized product. This lyophilized product was produced by further desulfating the 2-position of the uronic acid residue by the method for producing 2ODSH described in <1>. The yield was 180 mg. Test example 5
Of the resulting 2,6-O-desulfated heparin ¹³ C-
NMR was measured and the spectrum is shown in FIG.

<4> Method for producing N, 6-O-desulfated heparin N, 6-O-desulfated heparin (N6ODSH) is prepared by preparing a heparin pyridinium salt by the same method as in <2>, and preparing this heparin pyridinium. After dissolving 200 mg of the salt in 20 ml of pyridine, 2 ml of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MTSTFA) was added, and the mixture was heated with stirring at 110 ° C. for 2 hours. After completion of the reaction, the reaction was stopped by adding 20 ml of water to the reaction mixture to make MTSTFA non-reactive, and then dialyzed against distilled water. Then
After adjusting the internal dialysis solution to pH 10 with 1N NaOH, the internal dialysis solution was subjected to dialysis again, and the obtained internal dialysis solution was subjected to lyophilization treatment to obtain a lyophilized product. This lyophilized product was produced by further desulfating the N-position of the glucosamine residue by the NDSH production method described in <2>. The yield was 150 mg. ¹³ C-NM of N, 6-O-desulfated heparin produced by the method of Test Example 5
R was measured and the spectrum is shown in FIG.

<5> Method for producing N, 2-O-desulfated heparin N, 2-O-desulfated heparin (N2ODSH) is obtained by converting the 2ODSH obtained in the above <1> to the NDSH described in <2>. It was produced by further desulfating the N-position of the glucosamine residue by the production method. The yield was 170 mg. According to the method of Test Example 5,
The resulting N, 2-O-desulfated heparin was measured by ¹³ C-NMR, and the spectrum is shown in FIG.

Example 2 Preparation of Immobilized Gel Carrier of Heparin Compounds The method of Sasaki et al. (J. Chromatogr. (1987) 400, 123-132)
Was dissolved in 5 ml of a 2M phosphate buffer (pH 7.2), each containing 200 mg of the raw material heparin and the various modified heparins prepared in Example 1. Next, 5 g of amino agarose gel powder was suspended, 15 mg of NaB (CN) H ₃ was added, and the reaction was allowed to proceed at room temperature for 24 hours with sufficient stirring. After completion of the reaction, the gel containing the coupling product was filtered. In addition, the washing process
Desalting was performed completely by applying 2-3 times. The prepared gel was suspended in 5 ml of a 0.2 M sodium acetate solution, 2.5 ml of acetic anhydride was added, and the mixture was reacted at 0 ° C. for 30 minutes and then at room temperature for 30 minutes. After completion of the reaction, the generated gel was washed with water and completely desalted. Bitter & Muir method (Anal. Biochem. (19
62) By quantifying carboxyl groups according to 4, 330-334), the weight of the heparins immobilized on the agarose gel per unit weight was estimated. Do, gel wet weight 1g
It was confirmed that about 10 mg of each heparin was immobilized.

Example 3 <1> Purification of fructose 1,6-bisphosphate aldolase A 70 mM bovine cerebrum was subjected to 10 mM Tris containing 200 ml of 10% glycerol, 1 mM EDTA and 0.5 mM 2-mercaptoethanol cooled to 4 ° C. -HCl (pH 7.4; GEM-TBS) was added, and the cerebral tissue was cooled by allowing to stand for a while. Then
The cerebral tissue was taken out, cut into pieces using a razor blade that can be cut well, then added twice (V / W) of GEM-TBS cooled again to 4 ° C, and homogenized well in a Worring blender.
Next, this homogenate was further homogenized with a Teflon homogenizer. The homogenate thus prepared was placed in an appropriate amount of a centrifuge tube, and was subjected to 100,000 × g, 20 at 4 ° C.
Centrifugation for 1 min. After completion of the centrifugation, a bovine cerebral extract was obtained in the supernatant. All the crude extracts were further purified by the following method at a temperature of 4 ° C.

First, in the method for preparing a gel carrier described in Example 2, a chondroitin polysulfate-immobilized gel carrier prepared by using chondroitin polysulfate instead of the raw material heparins was prepared, and placed in a column (φ2 × 20 cm). Packed and fully equilibrated with GEM-TBS as described above. After loading the entire amount of the crude extract onto the chondroitin polysulfate-immobilized agarose affinity column, it was washed with 60 ml of GEM-TBS,
A non-adsorbed fraction was obtained in this washing solution. Adsorbed fraction is 3M NaCl
And eluted with GEM-TBS containing.

Next, a column (φ1 × 10 cm) packed with the raw material heparin-immobilized gel carrier prepared in Example 2
Was sufficiently equilibrated with the above GEM-TBS. After loading the non-adsorbed fraction of agarose affinity column chromatography immobilized with chondroitin polysulfate on this column,
Washed with ml of GEM-TBS. 15 ml per specific adsorption fraction
Was eluted using a linear concentration gradient (0 to 1.5 M) of NaCl with two types of eluates of GEM-TBS and 15 ml of GEM-TBS containing 1.5 M NaCl. The detection was performed based on the absorbance at 220 nm, and the elution pattern is shown in FIG. Since a single peak appeared in the linear gradient of NaCl, this fraction was collected and subjected to polyacrylamide gel electrophoresis (SDS-PAG) together with the fraction adsorbed on the column immobilized on chondroitin polysulfate.
When attached to E), as shown in FIG. 8, the molecular weight was about 40 kDa.
The main band was observed. This fraction had fructose 1,6-bisphosphate aldolase activity.

Next, 10 mM Tris-HCl containing 1 mM EDTA (p
Equipped with a MonoQ-HR5 / 5 column equilibrated with H 7.4)
FPLC was loaded with the specific adsorption fraction of the affinity chromatography using the raw material heparin-immobilized gel carrier, and washed with 15 ml of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA. 10m containing 7.5ml of 1mM EDTA per adsorption fraction
M Tris-HCl (pH 7.4) and 7.5 ml of 0.5 M NaCl, 1 mM ED
N with 10 mM Tris-HCl (pH 7.4) containing TA
Elution was performed using a linear concentration gradient of aCl (0-0.5 M). The detection was carried out based on the absorbance at 220 nm, and the elution pattern is shown in FIG.

As a result, one broad peak immediately before the start of the linear concentration gradient of NaCl (Fr. 1) and four sharp peaks in the linear concentration gradient of NaCl (Fr. 2 to Fr. 5)
Since they appeared, they were separated. 5 obtained
F with Superdex200-HR10 / 30 column per seed fraction
When the gel filtration analysis was performed by the PLC and the fractionation was performed at the same time, the purity was tested and the final purification was performed. As a result, a sharp peak was detected in all the fractions at a retention time of about 13 minutes (detection by absorbance at 220 nm) All the fractions were found to be purified to an extremely high purity. Fr.1-F
The yields of r.5 were 0.16 mg, 1.0 mg, 0.4 mg and 0.3 m, respectively.
g, 0.5 mg. These five fractions were prepared in Example 3
As described in <2>, all had fructose 1,6-bisphosphate aldolase activity.

For these five fractions, the method of Osntein [Ann. NY Acad. Sci. (1964) 121, 321-321] or the method of Davis [Ann. NY Acad. Sci.
1, 404-404], and Fr.
Since the mobility increased in the order of 1, Fr.2, Fr.3, Fr.4 and Fr.5, the negative charge was Fr.1 <Fr.2 <Fr.3
<Fr.4 <Fr.5. Therefore, it was suggested that Fr.1 to Fr.5 are isoforms of fructose 1,6-bisphosphate aldolase. In fact, fructose 1,6-bisphosphate aldolase present in the brain is a tetramer of two subunits, A with a high isoelectric point and C with a low isoelectric point, and A ₄ , A ₃ C, A ₂ It has been reported that five isoforms of C ₂ , AC ₃ and C ₄ exist (Methods Enzymol. (1975) 42, 240-249). These results, Fr.1, Fr.2, Fr.3, Fr.4 , and Fr.5 each A _4, A ₃ C, and which corresponds to A ₂ C _2, AC _3, and C ₄ it was thought.

<2> Measurement of activity of fructose 1,6-bisphosphate aldolase
Fructose 1,6-bisphosphate (F-1,6-P2) acts as a substrate, and the phosphorylated monosaccharide is converted to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAL-3-P)
And fructose 1-phosphate (F-1-P)
Also acts as a substrate, and converts the phosphorylated monosaccharide to dihydroxyacetone phosphate (DHAP) and glyceraldehyde (GA
L). The outline of this enzyme activity measurement is F-1,6-P2
Using glycerol 3-phosphate dehydrogenase system in the presence of triosephosphate isomerase with F-1-P as a substrate, the decrease in the absorbance of NADH at 340 nm when the resulting DHAP is reduced to glycerol 3-phosphate. It is to measure.

In detail, the method of Blostein & Rutter (J. Bi
ol. Chem. (1963) 238, 3280-3285). That is, the following five kinds of reagents were prepared. 0.1 M glycylglycine buffer (pH 7.5) 50 mM fructose 1,6-bisphosphate sodium salt solution 0.1 M fructose 1-phosphate dimonocyclohexylammonium salt solution 10 mg / ml glycerol 3-phosphate dehydrogenase / triose phosphate isomerase Complex solution NADH

The reaction mixture was composed of two kinds of substrate monosaccharides having the above concentrations, 4 mg.
5 to 50 μl of fructose 1,6-bisphosphate aldolase (0.005) per 20 ml of 0.1 M glycylglycine buffer (pH 7.5) containing NADH and 50 μl of a glycerol 3-phosphate dehydrogenase / triose phosphate isomerase complex solution. The reaction was started by adding .about.0.02 unit). The amount of decrease in absorbance at 340 nm per unit time was measured at a temperature of 25 ° C. NA at 340nm
DH molecular extinction coefficient so that 6.22 × 10 ⁶ cm ² · mol of when the change in absorbance was decreased to 6.22 from 12.44, fructose 1,6-bisphosphate or 1μm of each 1μmol
This means that the ol fructose 1-phosphate was cleaved. Based on this, various samples (at a final concentration of 4 μg / ml,
It can be calculated whether 1 / min cleavage has occurred.

The unit (activity; unit) of fructose 1,6-bisphosphate aldolase was defined as 1 unit of an enzyme capable of cleaving 1 μmol of a substrate in 1 minute at 25 ° C. in the above reaction system. Specific activity is defined as the number of units per mg of protein. Next, the activity was measured. The fructose 1,6-bisphosphate aldolase activities of the crude extract fraction described in Example 3 <1>, the fraction not adsorbed on the carrier immobilized with chondroitin polysulfate, and the fraction adsorbed specifically on the carrier immobilized on the raw material heparin were measured. However, the total activity and specific activity of the crude extract fraction were 357.9 and 0.07, respectively, and the total activity and specific activity of the non-adsorbed fraction of the chondroitin polysulfate-immobilized carrier were 33.8 and 0.08, respectively. The total activity and specific activity of the fractions were 29.1 and 29.1, respectively.
And 1.5, and the raw material heparin-immobilized carrier-specific adsorption fraction has a specific activity 22 times that of the crude extract fraction (22
Showed twice the degree of purification).

Furthermore, Fr.1 fractionated on a MonoQ column
_{(A 4), Fr.2 (A} 3 C), Fr.3 (A 2 C 2), Fr.4 (AC 3) and Fr.5
The degree of purification of (C ₄ ) is 33 times, 76 times, 205 times, 134 times, respectively.
And 70 times. In addition, these fractions are
The degree of purification after purification with the ex200 column was 39 times, 89 times, 252 times, 171 times, and 83 times, respectively, in the order described above. Table 8 compares the protein amount, total activity, specific activity, recovery rate and degree of purification for each purification step.

[0073]

[Table 8]

Example 4 Measurement of interaction (affinity) between heparin and fructose 1,6-bisphosphate aldolase Heparin and fructose 1,6-bisphosphate
Measurement of the interaction (affinity) of bisphosphate aldolase
The affinity gel carrier prepared in Example 2 was replaced with FP
The sample was packed in an LC column and subjected to affinity chromatography. That, A ₄ aldolase Oyo C ₄ aldolase and mixtures of these hybrid aldolase, 1mM EDTA, 10mM Tris-HCl equilibrated heparins immobilized gel carrier with (pH 7.5) containing 0.5 mM 2-mercaptoethanol Was loaded on the column.

For the adsorbed fraction, 15 ml of 1 mM EDTA, 0.5 mM
10 mM Tris-HCl containing M 2-mercaptoethanol (pH
7.5) and 10 ml of 10 mM Tris-HCl (pH 7.5) containing 15 ml of 1.0 M NaCl, 1 mM EDTA, 0.5 mM 2-mercaptoethanol.
Elution was carried out using a linear gradient of NaCl (0-1.0 M) with the seed solution. The detection was carried out based on the absorbance at 220 nm, and the strength of the interaction (affinity) between heparins and fructose 1,6-bisphosphate aldolase was calculated by measuring the NaCl concentration corresponding to the peak top of the elution peak.

As a result, the fructose 1,6-bisphosphate aldolase-active fraction was eluted at about 0.37 M from the raw material heparin-immobilized gel carrier, while the sulfate bound to any one of the sulfate-binding sites was removed. Modified heparin (2ODSH, NDSH: hereinafter, when these modified products are collectively referred to as mono-desulfated heparin) whose group is specifically desulfated, is from 0.31 to
Eluted at 0.33M. 2OD of monodesulfated heparin
SH eluted at 0.33M, whereas NDSH eluted at 0.31M, so 2ODSH
Was found to have a slightly higher affinity for fructose 1,6-bisphosphate aldolase than NDSH.

Modified heparin (26ODS) in which the sulfate group bound to any two of the sulfate group binding sites is specifically desulfated
H, N6ODSH, N2ODSH (hereinafter referred to as di-desulfated heparin when these modifications are collectively referred to) eluted between 0.30 and 0.27M. Of the heparin derivatives in which the sulfate groups bound to the two sulfate group binding sites were desulfated, 26OD
SH was eluted at 0.30M, while N6ODSH and N2ODSH were eluted at 0.27M, indicating that 26ODSH had a higher fructose 1,6-bisphosphate aldolase affinity than N6ODSH or N2ODSH. Mono-desulfated heparin showed higher affinity for fructose 1,6-bisphosphate aldolase than di-desulfated heparin. Therefore, the N-sulfate group contributed the most to affinity expression, followed by the 2-O-sulfate group, 6-
It was found that the contribution was large in the order of O-sulfate groups (Fig. 1
0).

[0078] A ₄ aldolase purified fructose 1,6-bisphosphate aldolase inhibitory activity Measurement Example 3 Example 5 heparins (A
_{Using 4} isoforms) and C ₄ aldolase (C ₄ isoform), the inhibitory effect of various heparin derivatives on the fructose 1,6-bisphosphate aldolase activity was examined. In the enzyme reaction system described in Example 3, the concentration [S] of fructose 1,6-bisphosphate (F-1,6-P2) serving as a substrate and the concentration of an inhibitor (raw material heparin or six kinds of modified heparins) [I] was variously changed (0.4 to 4 μg / ml) to carry out an enzyme reaction, and the reaction rate (v) was measured. From these results, 1 / [v] is plotted against 1 / [S],
The Ki value in each case was calculated from the value of the [/ S] intercept. Table 9 shows the results. The mode of inhibition of heparins against aldolase was competitive inhibition. Generally, the inhibitor is Ki
A smaller value indicates a stronger effect.

On the other hand, the starting material heparin
It also showed a very strong inhibitory activity against chromium. C_FourEye
Ki value (0.62 μg / ml) for Soform is A_FourIsopho
It was 4.4 times the Ki value (0.14 μg / ml) for the Next
And A_FourIsoform and C _FourAgainst isoforms
When the inhibitory effects of all modified heparins were determined,
Of modified heparin also against aldolase isoforms
It exhibited a characteristic inhibitory effect. A_FourAgainst isoforms
Ki values of 2ODSH and NDSH are 0.40 and 0.5, respectively.
It was 4 μg / ml. Didesulfated heparin is monodesulfated
The inhibitory activity was weaker than that of heparin. A_FourEye
Looking at the sofo, 26ODSH (Ki = 16.60μg / ml) is N6OD
SH (Ki = 76.6μg / ml) or N2ODSH (Ki = 86.2μg / ml)
It had a higher inhibitory activity in comparison.

On the other hand, the inhibitory activity was measured in the same manner as described above using chondroitin sulfates A and C (both manufactured by Seikagaku Corporation) instead of heparins. A chondroitin sulfate A ₄
Ki values for isoforms 547.8μg / ml, Ki values for A ₄ isoform of chondroitin sulfate C is 275.8μ
g / ml. These results suggested that a basic skeleton is required for the expression of the inhibitory activity of fructose 1,6-bisphosphate aldolase activity, and that the binding position of the sulfate group bound to the basic skeleton is involved. At the binding position of the sulfate group, the sulfate group bonded to the 2-position amino group of the glucosamine residue plays the most important role in the expression of the inhibitory activity, and the sulfate group bonded to the 2-position hydroxyl group of the uronic acid residue plays a role in this. Next, it was found that the participation of the sulfate group bonded to the 6-position hydroxyl group of the glucosamine residue was the lowest. Mono-desulfated heparin has stronger fructose 1,6-bisphosphate aldolase inhibitory activity than di-desulfated heparin, and mono-desulfated heparin is more useful as fructose 1,6-bisphosphate aldolase inhibitor It became clear that it was.

[0081]

[Table 9]

[0082]

Industrial Applicability According to the present invention, there is provided a highly practical inhibitor of fructose 1,6-bisphosphate aldolase, which is a key enzyme of glycolysis, and has high safety using the inhibitor as an active ingredient. Pharmaceutical compositions can also be provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing a ¹³ C-NMR spectrum of a raw material heparin.

FIG. 2 shows a ¹³ C-NMR spectrum of 2-O-desulfated heparin.

FIG. 3 ¹³ C-NMR of N-desulfated and N-acetylated heparin
The figure which shows a spectrum.

FIG. 4 shows a ¹³ C-NMR spectrum of 2,6-O-desulfated heparin.

FIG. 5 shows a ¹³ C-NMR spectrum of N, 6-O-desulfated heparin.

FIG. 6 shows a ¹³ C-NMR spectrum of N, 2-O-desulfated heparin.

FIG. 7 is a view showing an elution pattern in affinity column chromatography using a raw material heparin-immobilized gel carrier.

FIG. 8 shows the fraction adsorbed on the gel carrier immobilized with chondroitin polysulfate and the fraction adsorbed on the gel carrier immobilized with raw material heparin
The figure which shows an SDS-PAGE image.

FIG. 9 is a view showing an elution pattern of ion exchange chromatography by FPLC using a MonoQ-HR5 / 5 column.

FIG. 10 is a view showing the affinity between raw material heparin and modified heparin and fructose 1,6-bisphosphate aldolase.

Claims (9)

[Claims] 1. A fructose 1,6-bisphosphate containing a repeating structure of a disaccharide consisting of a glucosamine residue and a uronic acid residue as a basic skeleton and glycosaminoglycan having a sulfate group in the basic skeleton as an active ingredient. Aldolase inhibitors. 2. The method according to claim 1, wherein the mol% of the glucosamine residue in which the 2-position amino group is sulfated is at least 40% based on the amount of glucosamine residue constituting the basic skeleton of glycosaminoglycan. Inhibitors. 3. The method according to claim 1, wherein the mol% of the uronic acid residue in which the 2-position hydroxyl group is sulfated is 40% or more based on the amount of the uronic acid residue constituting the basic skeleton of glycosaminoglycan. Or the inhibitor of 2. 4. The activated partial thromboplastin time (APTT) does not exceed 100 seconds when the glycosaminoglycan is measured in serum in the presence of 3 μg / ml. An inhibitor according to claim 1. 5. The composition of unsaturated disaccharides in a disaccharide analysis obtained by combining glycosaminoglycan hydrolysis with a glycosaminoglycan-degrading enzyme and high-performance liquid chromatography in the composition of unsaturated disaccharides. 2-
Sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo
The inhibitor according to any one of claims 1 to 4, wherein the molar% of -hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose is 50% or less. 6. The composition of an unsaturated disaccharide in a disaccharide analysis obtained by combining glycosaminoglycan hydrolysis with a glycosaminoglycan degrading enzyme and high performance liquid chromatography in the composition of an unsaturated disaccharide, wherein the glycosaminoglycan is 2-deoxy-glycoside. 2-
Sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo
-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose from heparin or heparan sulfate containing more than 50% by mole of glucosamine residue 2 constituting its basic skeleton.
Glycosaminoglycan obtained by desulfating one of a sulfate group bonded to an amino group and a sulfate group ester-linked to a hydroxyl group at position 2 of a uronic acid residue constituting the basic skeleton. The inhibitor according to any one of claims 1 to 5, characterized in that: 7. The composition of unsaturated disaccharide in the disaccharide analysis obtained by combining glycosaminoglycan hydrolysis with glycosaminoglycan-degrading enzyme and high-performance liquid chromatography in the composition of unsaturated disaccharide. 2-
Sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo
-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose 6% of glucosamine residues constituting its basic skeleton from heparin or heparan sulfate containing more than 50% by mole.
Of the sulfate group bonded to the hydroxyl group at the 2-position and the amino group at the 2-position and the sulfate group ester-linked to the hydroxyl group at the 2-position of the uronic acid residue constituting the basic skeleton by desulfating two sulfate groups. The inhibitor according to any one of claims 1 to 5, wherein the inhibitor is glycosaminoglycan. 8. A pharmaceutical composition comprising the inhibitor according to any one of claims 1 to 7. 9. A glycosaminoglycan having a repeating structure of a disaccharide composed of a glucosamine residue and a uronic acid residue as a basic skeleton, and having a sulfate group in the basic skeleton, and agarose having the immobilized glycosaminoglycan as a carrier. A glycosaminoglycan having an affinity for fructose 1,6-bisphosphate aldolase in affinity chromatography.