Glycosaminoglycan derivatives and processes for preparing same

Details Glycosaminoglycan derivatives and processes for preparing same Glycosaminoglycan derivatives and processes for preparing same

2000-08-28

2002-12-24

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S001110, C536S004100, C536S021000, C536S055000, C536S055100, C536S055300, C536S123100, C536S124000, C536S127000

Reexamination Certificate

active

06498246

ABSTRACT:

TECHNICAL FIELD
The present invention relates to novel glycosaminoglycan derivatives having a neurite outgrowth promoting activity and a sialidase inhibiting activity, a process for producing them, and a pharmaceutical composition containing the glycosaminoglycan derivative as an active ingredient.
BACKGROUND ART
Neuronal disease is generated by the growth-inhibition of neural cells, by the induction of neural cell necrosis caused by the inability of the cells to be maintained due to reduction of sugar supply or energy metabolism inhibition in the cells as a result of ischemia, or by the inhibition of neurotransmission due to degradation of neurotransmitter. Accordingly, the treatment of this disease has been made with the aim of maintaining growth of neural cells, preventing ischemia of neural cells, or maintaining energy metabolism in the neural cells. That is, it is considered that the fundamental measures for the neuronal disease are to administer neurotrophic factors as substances having a neural cell growth activity (e.g., nerve growth factor: NGF, brain-derived neurotrophic factor: BDNF, ciliary neurotrophic factor: CNTF, fibroblast growth factor: FGF etc.) from the outside or promote synthesis of the neurotrophic factors, to prevent ischemia of neural cells and reduction of energy metabolism in neural cells caused by excess production of sorbitol formed by the activity of aldose reductase, by applying an aldose reductase inhibitor, and to maintain transmission of neural cells by preventing degradation of acetylcholine through the administration of an acetylcholine esterase. However, there are problems, such as antigenicity due to purity of the substance to be administered, side effects caused by the inhibition of enzyme activity, toxicity of the substance per se to be administered, and the like.
Also, it is known that glycosaminoglycan has the activity to accelerate neurite outgrowth promotion(
J. Cell Physiol.,
135: 293-300 (1988)). Furthermore, it is known that heparin and derivatives thereof, periodate oxidation reduction heparin and per-sulfated heparin, have a neurite outgrowth promoting activity and are effective in treating traumatic, ischemic and toxic neuropathy (JP-A-6-157322), but substances having a sufficient activity to inhibit these diseases for applying to medicaments have not been obtained yet.
On the other hand, it is known that sialidase (neuraminidase) inhibitors, such as GS4104 (WO 96/26933), zanamivir (4-guanidino-Neu4Ac2en: WO 91/16320, WO 94/07885), and the like, are effective as antiviral agents, particularly as influenza treating agents.
Since heparin and derivatives thereof are biological substances, they have a low antigenicity, but their use and concentration in using as medicaments are considerably limited due to their high anticoagulation activity. Generally, when heparin is administered to the living body, bleeding activity due to the high anticoagulation activity becomes a serious problem. Accordingly, a new substance having a low anticoagulation activity and a reinforced neuropathy improving effect is expected.
DISCLOSURE OF THE INVENTION
In order to resolve these problems, the present inventors have conducted intensive studies with the aim of obtaining a substance having a low anticoagulation activity for blood and an excellent neuropathy improving effect, and found, as a result of the efforts, that glycosaminoglycan derivatives having a specified structure have a high neurite outgrowth promoting activity, namely a neuropathy improving activity, and have accomplished the present invention by confirming that the anticoagulation activity of this substance is more sharply reduced than heparin. That is, it was found that novel glycosaminoglycan derivatives having a repeating unit structure of two saccharides of hexosamine and hexuronic acid as the backbone structure, in which the bondage between the 2- and 3-position carbon atoms of hexuronic acid as its constituting monosaccharide is partially cleaved and at least a part of the uncleaved hexuronic acid has no sulfate group on its 2-position, has a low anticoagulation activity and an excellent neurite outgrowth promoting activity. A pharmaceutical composition having an excellent effect for improving neuropathy using these properties can be provided. Additionally, based on the fact that a sialidase activity is increased and ganglioside known to have a relation to the maintenance and acceleration of a neurotrophic factor activity on the cell membrane is reduced when cell death and involution of neurite occur, the present inventors have conducted further investigations and found that the novel glycosaminoglycan derivative has a strong sialidase inhibition activity to provide a sialidase inhibitor using this inhibition activity of the glycosaminoglycan derivatives.
The first embodiment of the present invention relates to novel glycosaminoglycan derivatives having the following properties (a), (b) and (c) and comprising at least one structure represented by general formula (1) described in (d) per molecule of a backbone structure formed by a repeating unit structure of hexosamine and hexuronic acid:
(a) mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-&agr;-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as “&Dgr;DiHS-tri(U,6,N)S”) being from 0 to 10%, mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-&agr;-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as “&Dgr;DiHS-di(6,N)S”) being from 95 to 70%, and mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-&agr;-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter also referred to as “&Dgr;DiHS-NS”) being from 5 to 20%, in a disaccharide composition obtained by a disaccharide analysis through a combination of degradation by a glycosaminoglycan degradation enzyme with analysis by high performance liquid chromatography,
(b) activated partial thromboplastin time (APTT) when measured by adding to standard blood plasma at a final concentration of 3 &mgr;g/ml being 50 seconds or less,
(c) weight average molecular weight being from 9,000 to 13,000 Da (dalton), and
(d) general formula (1):
(wherein R
1
represents H or SO
3
H; and R
2
represents COCH
3
or SO
3
H).
The second embodiment of the present invention relates to a process for producing a novel glycosaminoglycan derivative having the properties (a), (b) and (c) and having the specified structure defined in (d), comprising the following steps (i) and (ii) described below; and the third embodiment of the present invention relates to a pharmaceutical composition comprising the glycosaminoglycan derivative as an active ingredient:
(i) a step in which a sulfated glycosaminoglycan having a repeating unit structure of hexosamine and hexuronic acid as a backbone structure is subjected to a cleavage treatment to cleave between only the 2- and 3-position carbon atoms of at least a part of hexuronic acid having no sulfate group on the 2-position in the backbone structure, and
(ii) a step in which the product of the step (i) is subjected to a desulfation treatment by a desulfation method capable of specifically removing the 2-position sulfate group of hexuronic acid to desulfate 90% or more of sulfate groups of the total hexuronic acid having a sulfate group at the 2-position.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
The present invention is described further in detail in the following.
Substance of the Invention
As described above, the substance of the present invention is a novel glycosaminoglycan derivative which has the properties (a), (b) and (c) and also has the specified structure defined in (d).
The term “hexosamine” as used herein means a monosaccharide in which the 2-position of hexose has an amino group, an acetylamino group or a sulfamino group and the 6-position hydroxyl group is optionally sulfated, the term “hexuronic acid” means a monosaccharide in which the 6-position carbon atom of hexose forms a carboxyl group and the 2-position hydroxyl group is optionally sulfated, the term “glycosaminoglycan” means a pol

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