Oligosaccharide mixtures having antithrombotic activity

Details Oligosaccharide mixtures having antithrombotic activity Oligosaccharide mixtures having antithrombotic activity

1999-12-03

2002-11-26

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S053000, C536S054000

Reexamination Certificate

active

06486137

ABSTRACT:

TECHNICAL FIELD
The invention concerns oligosaccharide mixtures having antithrombotic activity and is particularly concerned with oligosaccharides derived from dermatan sulphate.
BACKGROUND ART
Dermatan sulphate is a linear polysaccharide which has antithrombotic activity and is composed of alternating glucuronic or iduronic acid residues and N-acetylgalac-tosamine residues, these residues being sulphated to a varying extent. The mechanism of its activity in the inhibition of thrombin depends on the dermatan sulphate binding to both heparin cofactor II (HCII, a serine protease inhibitor) and to thrombin (factor IIa) itself.
Numerous studies have been made in the past of dermatan sulphate and its degradation products and these studies have mostly been directed to the identification and isolation of specific fragments having activity of interest. Thus for example J. Biological Chemistry, vol. 261, July 1986, 8854-58 describes fragments containing 12-14 sugar residues which bind to HCII and the same article also describes di-, tetra-, and hexasaccharide fragments without this binding ability. J. Biological Chemistry, vol. 265, October 1990, 18263-71 on the other hand describes hexasaccharides with a high binding affinity to HCII. Oligosaccharides containing 5 or 6 sugar residues and extended forms which bind to HCII are described in WO 91/15217. Thrombosis Research, 66, 1992, 527-33 suggests that the best balance of biological activity, clearance, half-life of disappearance, bioavailability, and other factors is achieved with oligosaccharides having molecular weights ranging from 4-9 kDa. WO 93/05074 describes oligosaccharides with antithrombotic activity which contain 11-13 sugar residues, and WO 93/05075 describes oligosaccharides having 7-12 sugar residues. Blood, vol. 81, April 1993, 1771-77 concludes that the fragment of minimum size for full catalytic activity is a hexadecasaccharide.
The methods used in the earlier investigations to produce the fragments studied generally involve degradation of the dermatan sulphate starting material and isolation of the fragments by affinity chromatography.
A native low molecular weight dermatan sulphate (Desmin) currently under investigation is characterised by a sulphate to carboxylate ratio of 1.06 (Thrombosis Research, vol. 83, no. 1, 103-109).
It will be appreciated from the above that numerous oligosaccharide fragments can be obtained from dermatan sulphate and the relationship between the nature of these oligosaccharides and their activity is complex and has not yet been completely elucidated.
DISCLOSURE OF THE INVENTION
We have now found that a satisfactory level of antithrombotic activity can be found in fractions which have been separated from material with a lower charge density and contain mixtures of oligosaccharides based on specific disaccharide residues.
The oligosaccharides of the invention are different from prior art since they are more negatively charged. They can be prepared by methods which are commercially more competitive compared to those previously used and described in the prior art proposals in the isolation of specific fragments, since they are less expensive to manufacture. These dermatan sulphate fractions show surprisingly higher affinity towards HQII than native dermatan sulphate and can therefore be used to increase the antithrombotic potency of dermatan sulphate based drugs.
The invention thus provides a mixture of linear oligosaccharides containing different numbers of repeating sulphated disaccharide units derived from L-iduronic acid (IdoA) and N-acetyl-D-galactosamine (Gal-NAc) and having the following characteristics:
(a) a molecular weight in the range of 1600-20000 for 90% or more of the mixture,
(b) a sulphur content of 6.0-8.0% by weight,
(c) a sulphate/carboxylate ratio of 1.2-1.6,
(d) a disulphated disaccharide content of 20-60% by weight of the mono-sulphated disaccharide content, and
(e) an antithrombin activity of 20-60 IU/mg.
The mixture is prepared from dermatan sulphate and may thus contain a small proportion of residues derived from D-glucuronic acid, although it is preferably free or essentially free of such residues. Residues of hexosamines other than Gal-NAc may also be present in a small amount, e.g. residues of N-acetyl-D-glucosamine (Glu-NAc) although Gal-NAc is preferably the only hexosamine present.
The Gal-NAc residues are sulphated at the 4-position in most cases and the IdoA residues are sulphated at the 2-position in some instances. Some cases of sulphation at the 6-position of Gal-NAc are also seen. The sul-phate:carboxyl and disulphated:monosulphated disaccharide ratios and the sulphur content can thus vary, mostly depending on the extent of sulphation of the IdoA residues.
Formula 1 shows the sequence of repeating sulphated L-iduronic acid and N-acetyl-D-galactosamine units generally present in the oligosaccharides of the invention.
where R is H or SO
31
3
, and
n is in the range 3-37 for 90% or more of the material and 10-15 for the peak point of the molecular distribution.
The residues are linked from the IdoA 1-position tothe Gal-NAc 3-position and from the Gal-NAc 1-position to the IdoA 4-position. When prepared by the method described below, a proportion of the terminal Gal-NAc residues have a 2,3,4-trihydroxybutyric acid residue (HOCH
2
—CH(O—)—CH(OH)COO
−
) at the 1-position. The counterion is a mono- or divalent cation, mostly sodium but possibly also other ions such as calcium.
MODES OF CARRYING OUT THE INVENTION
The properties characteristic of the oligosaccharides may be measured by the following methods:
(1) molecular weight by gel permeation chromatography (GPC)—HPLC according to the method of Dedem & Nielsen, Pharmeuropa Vol. 3, No. 3, 202-218 (1991),
(2) sulphur content according to Ph. Eur. V.3.5.3,
(3) sulphate/carboxyl ratio according to Ph. Eur. p. 828,
(4) disulphated/monosulphated disaccharide content by the method of Linhardt et al., Anal. Biochem. 181, 288-296 (1989).
(5) HCII-mediated antithrombin activity by a chromogenic assay (Diagnostica Stago, France) in a plasma free system with the 4. International Heparin Standard (code no. 82/502) as standard.
The sulphur content of the mixtures is generally within the range 6.0-8.0% by weight and is preferably 6.5-8.0%. The sulphate/carboxyl ratio can generally range from 1.2-1.6 and is preferably from 1.3-1.6. The content of disulphated disaccharide is usually from 20-60% by weight of the monosulphated disaccharide content and is preferably 30-60% of the latter. The HCII mediated antithrombin activity of the oligosaccharides of the invention is generally in the range 20-60 IU/mg and preferably 30-60 IU/mg.
The oligosaccharide mixture of the invention may be prepared by depolymerisation of dermatan sulphate by periodate oxidation, followed by borohydride reduction and acid hydrolysis and then ion exchange fractionation.
The dermatan sulphate used as the starting material may be derived from animal intestines such as for example porcine mucosa or bovine trachea and have the following characteristics:
Specific optical rotation (4% in water): −50° to −70°
Sulphur: 5.3-6.3% (w/w)
Sulphate/carboxylate ratio: 1.0-1.3
HCII mediated antithrombin activity: 2-10 IU/mg
The main chemical reactions involved in the depolymerisation process are shown in the reaction scheme below and principally follow the route described in Carbohydrate Research 36, 339-348 (1974).
Depolymerisation of dermatan sulphate:
In the first step, treatment of a dermatan sulphate (1) with periodate results in selective oxidation of non-sulphated IdoA residues to produce a ring-opened dialdehyde (2). Reduction of the latter gives the diol (3) which is then cleaved by acid hydrolysis to give two Gal-NAc-terminated fragments, one of which carries a 2,3,4-trihydroxybutyric acid end group. This cleaving occurs at numerous positions along the dermatan sulphate chain, and gives a mixture of fragments of different chain lengths. A selected fraction of these fragments is then obtained by ion exchange fractionation.
The initial oxida

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