Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-22
2002-12-10
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C436S094000
Reexamination Certificate
active
06492503
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a glycosaminoglycan having sulfate groups, in which substantially all the sulfate groups bound to the 6-positions of the glucosamine residues constituting the glycosaminoglycan are removed and the removal of other sulfate groups is minimized, and to pharmaceuticals comprising the glycosaminoglycan as an active ingredient, in addition to a method for producing the glycosaminoglycan.
BACKGROUND ART
Heparin is one of glycosaminoglycans having as a backbone structure being composed of a repetitive structure of a disaccharide unit composed of a uronic acid (iduronic acid (IdoA) or glucuronic acid (GlcA)) residue and a glucosamine (GlcN) residue. Heparin is one of glycosaminoglycans in which the hydroxyl group at the 2-position of the uronic acid residue and the hydroxyl group at the 6-position and the amino group at the 2-position of the glucosamine residue each undergo a certain degree of sulfation. Because heparin has an antithorombin III (hereinafter also referred to as “ATIII”) binding site (FEBS Lett. (1980) 117, 203-206), and binds with ATIII to inhibit the action of the thorombin, thereby giving rise to anticoagulative action, heparin has long been extensively used as a pharmaceutical agent such as anticoagulants for improving the results of dialysis treatment and the like. More recently, it has been found that heparin interacts with various physiological active factors. For example, heparin interacts with lipoprotein lipase (J. Biol. Chem. (1981) 256, 12893-12898) and has an affinity for basic fibroblast growth factor (J. Cell Biol. (1990) 111, 1651-1659).
Under such a situation, attention has been focusing on the domain structures within heparin that take part in the binding between heparin and specific cell growth factors or cytokines. Moreover, considerable research is being conducted relating to chemical modifications represented by desulfation of heparin aiming at reducing the anticoagulative action due to the presence of the ATIII binding site to thereby increase the interaction with physiological active factors (J. Carbohydr. Chem. (1993) 12, 507-521; Carbohydr. Res. (1989) 193, 165-172; Carbohydr. Res. (1976) 46, 87-95; WO 95/30424, etc.).
With respect to the aforementioned desulfation of heparin, in recent years the focus has been on removing the sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the heparin (6-desulfation). As desulfation methods, there can be mentioned the method using solvolysis (WO 95/30424) and the method using a silylating reagent (WO 96/01278).
With the former method, along with the removal of the sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the heparin molecule (6-O-sulfate group), the sulfate group bound to the hydroxyl group at the 2-position of the uronic acid residue (2-O-sulfate group) and the sulfate group bound to the amino group at the 2-position of the glucosamine residue (N-sulfate group) are also removed. Thus, in the course of using the former method to remove substantially all of the 6-O-sulfate groups, almost all of the N-sulfate groups of the glucosamine residues and 2-O-sulfate groups of the uronic acid residues are also lost. While the amino group at the 2-position of the glucosamine residue of the heparin thus modified can be resulfated, resulfation of the 2-position of the uronic acid residue without sulfating the 6-position of the glucosamine residue is difficult.
The latter method is superior to the former method in that it enables a more specific removal of the 6-O-sulfate group of the glucosamine residue. However, with the modified heparin thus obtained with the latter method, the effective disaccharide yield as determined by the enzymatic disaccharide analysis method is low, which means that there is still a problem with the method in that the structural identification therefor may not be enough for pharmaceutical applications. Moreover, while the anticoagulative action of the modified heparin is greatly reduced, it is not absent, and it has not been possible to obtain modified heparin in which the anticoagulative activity has been completely eliminated.
Because heparin or fragments thereof have affinities for various physiological active substances and the affinities are closely related to the functions of such substances, intensive studies have been made for search and development of drugs utilizing heparin or modified heparin. However, despite such efforts, they have yet been used effectively only as a blood anticoagulation agent in pharmaceutical applications.
That is, in focusing on using heparin for applications other than as an anticoagulant, it is important to substantially eliminate its anticoagulation and hemorrhagic actions and, with respect to using it as a pharmaceutical substance, it has to enable its “structural identification as a substance”. With respect to these problems, there must be further improvements, and it has been desired to resolve these remaining problems and utilize heparin's affinities for physiologically active substances to provide drugs that are safe and useful.
DISCLOSURE OF THE INVENTION
As a result of assiduous studies aiming at resolution of the above problems, the present inventors succeeded in, by using a specific method to effect desulfation of glycosaminoglycans such as heparin that have sulfate groups, preparation of a novel glycosaminoglycan in which its anticoagulative and hemorrhagic activities were substantially eliminated while its biologically advantageous effects for living bodies such as its affinities for physiologically active substances were maintained, and unidentifiable structures were markedly reduced to the extent that the “structural identification of the substance” can be readily attained, which is important in terms of pharmaceutical applications. Thus, the present invention has been accomplished.
Specifically, it was confirmed that a glycosaminoglycan obtained by subjecting a glycosaminoglycan having sulfate groups to heat treatment at 100° C. or higher in pyridine in the presence of a silylating agent, N-methyl-N-(trimethylsilyl)-trifluoroacetamide (hereinafter also referred to as “MTSTFA”) to remove substantially all of the 6-O-sulfate groups from the glucosamine residues, then evaporating the pyridine from the reaction mixture, adding water and concentrating under reduced pressure, was highly effective in promoting the healing of skin wounds and treating diabetic skin ulcers, and had fructose-1,6-bis-phosphate aldolase inhibitory activity, and that its anticoagulative and hemorrhagic activities had disappeared.
Further, it was confirmed to be possible to readily specify the structure of the glycosaminoglycan prepared by the above method with precision by using an enzymatic disaccharide analysis method utilizing glycosaminoglycan-degrading enzymes, because of the good digestibility of the glycosaminoglycan with glycosaminoglycan-degrading enzymes. This is in contrast to the previous difficulty in identifying structures of modified heparins that had been modified by being subjected to various types of chemical treatment. Using the glycosaminoglycan of which structure can thus be identified as pharmaceuticals makes it possible to provide pharmaceuticals that are highly safe and useful.
The present inventors further found that the glycosaminoglycan had high affinity for fructose-1,6-bis-phosphate aldolase, a key enzyme in the glycolytic pathway, and could be used as a strong inhibitor of that enzyme. Thus, it has become possible to provide a novel fructose-1,6-bis-phosphate aldolase inhibitor.
That is, the present invention provides the followings.
1. A glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, wherein substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the backbone structure is detected as determined by a chemical disaccharide analysis method
Hara Saburo
Hori Yusuke
Kamei Kaeko
Onaya Junichi
Takano Ryo
LandOfFree
Glycosaminoglycan and drug compositions containing the same does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Glycosaminoglycan and drug compositions containing the same, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Glycosaminoglycan and drug compositions containing the same will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2993343