Oligosaccharides, their preparation and pharmaceutical...

Details Oligosaccharides, their preparation and pharmaceutical... Oligosaccharides, their preparation and pharmaceutical...

2000-10-20

2003-09-09

Fonda, Kathleen K. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S054000, C536S021000, C536S018700

Reexamination Certificate

active

06617316

ABSTRACT:

The present invention relates to oligosaccharides of formula:
or mixtures thereof, to diastereoisomers thereof, to a process for their preparation and to pharmaceutical compositions containing them.
Disaccharide sulfates containing a 1,6-anhydro structure at the reducing end have been described by H. P. Wessel, J. Carbohydrate Chemistry, 11(8), 1039-1052 (1992); no pharmacological activity is mentioned for these products.
Trisaccharide sulfates comprising a 1,6-anhydro unit have also been described in patent EP 84999 and by Y. Ichikawa et al., Carbohyd. Res, 141, 273-282 (1985) as intermediates for preparing higher oligosaccharides. These trisaccharides have low anti-factor Xa activity.
In formula (I) n is 0 or an integer from 1 to 25; R
1
, R
3
, R
4
and R
5
, which may be identical or different, represent a hydrogen atom or an SO
3
M radical; R
2
and R
6
, which may be identical or different, represent a hydrogen atom or a radical selected from SO
3
M and COCH
3
; and M is sodium, calcium, magnesium or potassium.
These oligosaccharides thus comprise an even number of saccharide units.
In formula (I), R
4
is preferably a hydrogen atom.
Preferably, n is 0 or an integer from 1 to 10; more preferably 0 or an integer from 1 to 6; even more preferably an integer from 1 to 6.
The oligosaccharides of formula (I) can be prepared by the action of an alkali metal or quaternary ammonium hydroxide on oligosaccharides of formula:
in which n is 0 or an integer from 1 to 25; R
1
, R
3
, R
4
and R
5
, which may be identical or different, represent a hydrogen atom or an SO
3
M radical; R
2
and R
6
, which may be identical or different, represent a hydrogen atom or a radical selected from SO
3
M and COCH
3
and M is sodium, calcium, magnesium or potassium, or a mixture thereof.
This reaction is carried out in aqueous medium, at a temperature of from 40 to 80° C., at a pH of from 10 to 13.
As alkali metal hydroxides which can be used, mention may be made of sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide.
As a quaternary ammonium hydroxide which may be used, mention may be made of tetrabutylammonium hydroxide.
The amount of alkali metal or quaternary ammonium hydroxide must be sufficient for the pH of the reaction medium to remain stable throughout the reaction. It is thus necessary to add the alkali metal or quaternary ammonium hydroxide continuously throughout the reaction.
Preferably, the alkali metal or quaternary ammonium hydroxide is in the form of an aqueous 1 to 5% solution.
Preferably, the reaction is carried out at a temperature of from 60 to 70° C.
Advantageously, the reaction pH is from 11 to 12.5.
The reaction is stopped by acidifying the reaction medium, for example by addition of acidic resin such as Amberlite IR120® resin (Fluka).
The oligosaccharides of formula (I) can be eventually purified by gel permeation chromatography with polyacrylamide-agarose type gel such as Ultrogel ACA202 (R) (Biosepra) as described hereinafter for the intermediate oligosaccharides of formula (II).
The oligosaccharides of formula (I) for which n is 0 or 1 can be also eventually purified on an alumina column with a water-ethanol mixture as eluant.
The intermediate oligosaccharides of formula (II) and mixtures thereof can be obtained by chromatographic separation on gel of a mixture of oligosaccharides (III) obtained by enzymatic depolymerization of heparin or basic depolymerization of the benzyl ester of heparin or of a benzyl ester of semi-synthetic heparin.
This chromatography is carried out on columns filled with gel of polyacrylamide-agarose type such as the gel sold under the brand name Ultrogel ACA202® (Biosepra). Preferably, an array of polyacrylamide agarose gel columns is used. The number of columns used is adapted as a function of the volume, of the gel and of the oligosaccharides to be separated. The mixture is eluted with a solution containing a phosphate buffer and sodium chloride. Preferably, the phosphate buffer is a solution containing 0.02 mol/l of NaH
2
PO
4
/Na
2
HPO
4
(pH 7) containing 0.1 mol/l of sodium chloride. The detection of the various fractions is carried out by UV spectrometry (254 nm) and ionic spectrometry (IBF). The fractions thus obtained can then be optionally purified, for example by SAX (strong anion exchange) chromatography according to the methods known to those skilled in the art and in particular according to the methods described by K. G. Rice and R. J. Linhardt, Carbohydrate Research 190, 219-233 (1989), A. Larnkjaer, S. H. Hansen and P. B. Ostergaard, Carbohydrate Research, 266, 37-52 (1995) and in patent WO 90/01501 (Example 2). The fractions are then freeze-dried, after which they are desalified on a column filled with gel such as a column of Sephadex G10® gel (Pharmacia Biochemicals).
When the purification is not carried out by SAX chromatography, the lyophilizates can be optionally purified by simple or fractional precipitation according to the methods known to those skilled in the art and in particular according to the method described in patent FR 2 548 672. In general, the process is performed according to the following procedure:
The lyophilized fraction to be purified is dissolved at 25° C. in about ten volumes of distilled water. On adding methanol or ethanol, the desired oligosaccharide is precipitated, while monitoring its enrichment by HPLC chromatography (high performance liquid chromatography). The addition of methanol or ethanol is determined as a function of the desired yield and purity of the said oligosaccharide. Similarly, this operation can be carried out in several successive steps starting with the initial solution of lyophilizate. For this, more of the insolubilizing agent (methanol or ethanol) is added portionwise and the precipitate obtained after each addition is isolated. The precipitates thus prepared are analyzed by HPLC chromatography. Depending on the desired yield and purity, the suitable-fractions of precipitate are combined.
For the intermediates of formula (II) for which n=0, 1 or 2, it is preferable to start with a mixture (III) obtained by enzymatic depolymerization of heparin.
This depolymerization is carried out by means of heparinase I (EC 4.2.2.7), in a pH 7 phosphate buffer solution, in the presence of sodium chloride and BSA (bovine serum albumin), at a temperature of between 10 and 18° C., and preferably 15° C., for 8 to 10 days, and preferably 9 days. The depolymerization is stopped, for example, by heating the reaction medium at 100° C. for 2 minutes, and the mixture is recovered by lyophilization. It is preferable to use 7 IU of heparinase I per 25 g of heparin. The phosphate buffer solution generally comprises 0.05 mol/l of NaH
2
PO
4
/Na
2
HPO
4
(pH 7) in the presence of 0.1 mol/l of sodium chloride. The BSA concentration is generally 2%.
For the intermediates of formula (II) for which n=0, 1, 2, 3 or 4, it is,preferable to start with a mixture (III) obtained by depolymerizing a benzyl ester of heparin.
The benzyl ester of heparin can be prepared according to the methods described in patents U.S. Pat. No. 5 389 618, EP 40 144 and FR 2 548 672. The degree of esterification will preferably be between 50 and 100%. More preferably, it will be between 70 and 90%.
The depolymerization is carried out in aqueous medium, by means of an alkali metal hydroxide (for example lithium hydroxide, sodium hydroxide, potassium hydroxide or cesium hydroxide) or of a quaternary ammonium hydroxide (for example tetrabutylammonium hydroxide), preferably at a molarity of between 0.1 and 0.2 mol/l, at a temperature of between 40 and 80° C., for 5 to 120 minutes. In one preferred mode, the process is performed for 5 to 15 minutes, at a temperature of between 60 and 70° C., with a 0.15 mol/l sodium hydroxide solution. The depolymerization reaction is stopped by neutralization by addition of an acid such as acetic acid. After addition of 10% by weight per volume of sodium acetate, the oligosaccharide mixture is precipitated by adding methanol, preferably 2 volumes per 1 volume of rea

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