Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2001-03-26
2003-08-19
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S053000, C514S056000, C514S062000, C536S018700, C536S021000, C536S054000, C536S055000, C536S055100, C536S055200, C536S123100
Reexamination Certificate
active
06608042
ABSTRACT:
The present invention relates to pharmaceutical compositions containing as active principle an oligosaccharide of formula:
or to a mixture of these oligosaccharides, to the novel oligosaccharides of formula (I), to mixtures thereof and to methods for their preparation.
In formula (I), n is an integer from 0 to 25, R
1
, R
3
, R
4
, R
5
, R
6
and R
8
, which may be identical or different, represent a hydrogen atom or an SO
3
M radical, R
2
and R
7
, which may be identical or different, represent a hydrogen atom or an SO
3
M or COCH
3
radical, and M is sodium, calcium, magnesium or potassium.
These oligosaccharides thus comprise an even number of saccharides.
In formula (I), R
4
and R
6
are, preferably, hydrogen atoms.
Oligosaccharides of formula (I) for which n is equal to 0, and either R
1
, R
6
and R
8
represent a hydrogen atom, R
7
represents an SO
3
M or COCH
3
radical and M is sodium, or R
1
and R
6
represent a hydrogen atom, R
7
represents a COCH
3
radical, R
8
represents an SO
3
M radical and M is sodium, or R
6
represents a hydrogen atom, R
1
, R
7
and R
8
represent an SO
3
M radical and M is sodium have already been described by G. H. LEE et al., J. Chromat. 212, 65-73 (1981), but no pharmacological property is described for these products.
Oligosaccharides of formula (I) for which n is equal to 0, and either R
6
and R
7
represent hydrogen atoms, R
1
and R
8
represent an SO
3
M radical and M is sodium, or R
1
, R
6
and R
7
represent a hydrogen atom, R
8
represents an SO
3
M radical and M is sodium, are described by M W McLEAN et al., Eur. J. Biochem., 1984, 145, 607, without any indication of pharmacological activity.
The pharmaceutical compositions which are preferred are those containing an oligosaccharide of formula (I) for which:
n is an integer from 0 to 10, and in particular from 0 to 6, and even more particularly from 1 to 6.
R
1
, R
2
, R
3
, R
5
, R
7
and R
8
are identical or different, and represent a hydrogen atom or an SO
3
M radical, and in particular R
1
, R
2
, R
3
, R
5
, R
7
and R
8
are SO
3
M radicals,
M is sodium.
The pharmaceutical compositions which are particularly preferred are those containing an oligosaccharide of formula (I) for which:
n is equal to 0, R
1
, R
7
and R
8
represent an SO
3
M radical, R
6
represents a hydrogen atom and M is sodium,
n is equal to 1, R
1
, R
2
, R
3
, R
5
, R
7
and R
8
represent an SO
3
M radical, R
4
and R
6
represent a hydrogen atom and M is sodium,
n is equal to 2, R
1
, R
2
, R
3
, R
5
, R
7
and R
8
represent an SO
3
M radical, R
4
and R
6
represent a hydrogen atom and M is sodium,
n is equal to 3, R
1
, R
2
, R
3
, R
5
, R
7
and R
8
represent an SO
3
M radical, R
4
and R
6
represent a hydrogen atom and M is sodium,
n is equal to 4, R
1
, R
2
, R
3
, R
5
, R
7
and R
8
represent an SO
3
M radical, R
4
and R
6
represent a hydrogen atom and M is sodium.
The oligosaccharides of formula (I), with the exception of those for which n is equal to 0 and either R
1
, R
6
and R
8
represent a hydrogen atom, R
7
represents an SO
3
M or COCH
3
radical and M is sodium, or R
1
, and R
6
represent a hydrogen atom, R
7
represents a COCH
3
radical, R
8
represents an SO
3
M radical and M is sodium, or R
6
represents a hydrogen, R
1
, R
7
and R
8
represent an SO
3
M radical and M is sodium, or R
6
and R
7
represent hydrogen atoms, R
1
and R
8
represent an SO
3
M radical and M is sodium, or R
1
, R
6
and R
7
represent a hydrogen atom, R
8
represents an SO
3
M radical and M is sodium, are novel and, as such, form part of the invention.
The oligosaccharides of formula (I) can be prepared by reaction of an alkali metal borohydride or a quaternary ammonium borohydride with oligosaccharides of formula:
in which n is an integer from 0 to 25, R
1
, R
3
, R
4
, R
5
, R
6
and R
8
, which may be identical or different, represent a hydrogen atom or an SO
3
M radical, R
2
and R
7
, which may be identical or different, represent a hydrogen atom or an SO
3
M or COCH
3
radical, and M is sodium, calcium, magnesium or potassium.
This reaction is carried out in aqueous medium, at a temperature in the vicinity of 25° C., at a pH between 7 and 10, and preferably between 9 and 10, for the entire duration of the reaction. The pH is maintained by addition of a sodium hydroxide solution at 0.5 mol/l. The reaction is stopped by acidification of the reaction medium, for example by addition of acetic acid until a pH between 4 and 5 is obtained.
As alkali metal borohydrides, mention may be made of lithium borohydride, sodium borohydride and potassium borohydride.
As a quaternary ammonium borohydride, mention may be made of tetrabutylammonium borohydride.
The oligosaccharides of formula (II) can be obtained by gel chromatography separation 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 that sold under the trade mark Ultrogel ACA202
R
(Biosepra). Preferably, an array of polyacrylamide agarose gel columns is used. The number of columns used is adapted as a function of the volume, the gel and the oligosaccharides to be separated. The mixture is eluted with a solution containing a phosphate buffer and sodium chloride. Preferably, the phosphate buffer solution 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. 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 lyophilized, and then desalified on a gel-filled column 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 persons skilled in the art and in particular according to the method described in patent FR 2 548 672. Generally, 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 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.
According to a variant of the present invention, the lyophilized fraction to be purified can be dissolved in 10 to 200 volumes of water containing from 0 to 30% sodium acetate. The percentage of sodium acetate will be preadjusted as a function of the nature of the oligosaccharide to be treated (a function of the size). On adding methanol, the desired oligosaccharide is precipitated while monitoring its enrichment by HPLC chromatography (high performance liquid chromatography). The addition of methanol is determined as a function of the desired yield and purity of said oligosaccharide. Similarly, this operation can be carried out in several su
Mourier Pierre
Perrin Elisabeth
Viskov Christian
Aventis Pharma S.A.
Lewis Patrick
Newman Irving
Wilson James O.
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