Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Patent
1994-02-23
1996-08-20
Kunz, Gary L.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
514 62, 536 53, 536 54, 536 551, 536 552, 536 553, A61K 31725
Patent
active
055479448
DESCRIPTION:
BRIEF SUMMARY
This is the U.S. national phase of PCT/EP92/01790, filed Aug. 6, 1992.
Dermatan sulphate is a hexosaminoglycan of the heparin family. It is only recently that it has been looked upon as one of the physiological constituents of the vascular wall, and from this standpoint its anti-thrombogenic property has been examined (Desnoyers P. et al.; Pathologie Biologic, June 1989, 759).
Patent applications claiming the extraction process and utilisation as a thrombosis prevention drug have also been filed. European Patent 199,033 described a process of extraction from aorta and myocardium or other vascularised tissues, such process including micronisation of the tissue, dissociation of the proteoglycan with urea, precipitation and fractionation. The product is claimed as a preventive anttthrombotic agent as it possesses activated antifactor ten activity, (AXa), it induces a lowering of the antithrombin III (ATIII) plasma levels and possesses little or no fibrinslyric activity.
EP 238,994 claims a dermarsh sulphate extracted by means of proteolysis, precipitation as a quaternary ammonium salt (cetyl-dimethylethyl ammonium and similar), and purification of the product through formation of a calcium salt in an alkaline medium. The product is characterised as a polysaccharide consisting of the repetitive unit (II) ##STR2##
One subject of this invention consists of a reasonably mild process such that it will preserve the chemical structure of dermatan, in particular preventing or restricting desulphation thereof, and specific process so that it will select a dermatan sulphate characterised by an oligosaccharide sequence including at least eight monosaccharides with a high degree of sulphation. This sequence, responsible for interaction with heparin colactor II (HClI), has the minimum structure shown in (III) ##STR3## in which: G=glucuronic acid; N-acetyl galactosamine-4-sulphate (Gal-NAc-4SO.sub.3.sup.-)-uronic acid-2-sulphate (Ido-2SO.sub.3.sup.-).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the .sup.13 CNMR spectrum of the dermatan sulfate according to formula III;
FIG. 2 is a flow sheet illustrating the process of preparation of the dermatan sulfate according to formula III;
FIG. 3 and FIG. 4 show the .sup.13 CNMR spectrum of fractions A.sub.2 and B.sub.2 obtained by the process of demolition which is intended to isolate only the sequences containing iduronic acid 2-sulfate (Ido-2OSO.sub.3 --).
A hexasaccharide sequence with high affinity for HCII, consisting of three disulphated disaccharides of formula (IV) was identified in dermatan sulphate (Maimone M. M., Tollefsen D. M., J. Biol. Chem. 265, 30, 18263-18271 (1990)); ##STR4##
The subject of the invention is therefore also a dermatan sulphate in whose structure there is a sequence containing a number of disulphated disaccharides in excess of three. It was found surprisingly that sequences consisting of a higher number of disulphated disaccharides (IV), hitherto never described, are contained in dermatan sulphate and are responsible for greater activity, contrary to what was stated by Maimone and Tollefsen, according to whom the hexasaccharide constitutes the sequence with the highest affinity for and activity in respect of HCII. The sequences of the present invention are included among two glucuronic acids having a structure (V). Glucuronic acid is an epimer on carbon C-5 of iduronic acid (VI). ##STR5##
Surprisingly, through minor signals of non-destructive .sup.13 C-NHR spectral analysis it is possible to identify the intact and peculiar sequence responsible for the activity in the dermatan sulphate chain obtained according to the invention (see FIG. 1).
The dermatan sulphate of the present invention has thrombolytic activity, in other words it is capable not only of inhibiting venous thrombus formation but especially of favouring thrombus dissolution at surprisingly low doses, in the region of 0.2-1 mg/kg according to experimental models, and with a long-lasting action. Dermatan sulphate produced according to the invention inhibits thrombin through activati
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Mascellani, et al to be published in the Journal of Carbohydrate Chemistry.
Desnoyers et al. Path. Biol. 1989, 37(6), 759-767.
Maimone et al. J. Biol. Chem. 1990, 265 (30), 18263-18271.
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Bianchini Pietro
Mascellani Giuseppe
Fonda Kathleen Kahler
Kunz Gary L.
Opocrin S.p.A.
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