CA1180292A - Short chain oligosaccharides possessing biological properties, their preparation and their use as medicaments - Google Patents

Abstract

ABSTRACT

Short chained ligosaccharides of high structural homogeneity constituted essentially by hexasaccharides of the formula :

in which R represents a hydrogen atom or the -SO3 group.
These hexasaccharides have a highly selective activity on certain steps in blood coagulation and are useful as antithrombotic medicaments.

Description

SHORT CHAINED OLIGOSACCHARIDES HAVING BIOLOGICAL PROPERTIES, A PROCESS FOR MAKING THE SAME AND THE USE THEREOF AS DRUGS

The invention relates to short chained oligosaccharides, having biological properties enabling -them particularly -to con-trol only certain stages of blood coagulation.

It also relates to a process for obtaining -the same and to their uses as active principles of drugs.

The inventors have already described oligosaccharides having biological properties of -the type mentioned above.

When depolymerizing heparin by a chemical or an enzymatic route, the inventors have in particular obtained octasaccharides of great value corresponding to the sequence A-B-C-D-E-F-G-H (denoted below by the abreviation A-H).
COO OR OR COO OR rF<
H~ ~O~b~0~V~~~OU `
0503 NHS03 OH NHAc OH NHS03 OS03 ~ IS03 .. . . .
A B C D E F G H
in which R represents a hydrogen atom or a -S03 group.

These octasaccharides are observed to be particularly valuable because of their high specificity with respect to the activated X factor or Xa factor of the blood (measured according to the Yin-Wessler test) whilst their activity on the total coagula-tion (measured according to the USP or APTT -test) is very low.

The references regarding these tests are given in the Examples.

~q~

In pursuing their work in this field, the inventors were interested, particularly, in the production of olisosaccharides with shorter chains than these octasaccharides, bu-t possessing however an advanta-geous ratio of their Yin-~essler titer to their USP or APTT titer.

They have thus been led to observe that by operating under well-determined conditions, it was possible to selectively shorten the active chains of the A-H octasaccharides and to obtain compositions of high homogeneity in active oligosaccharides with short chains.

It is therefore an object of the invention to provide novel oligosaccharides with shorter chains than the oc-tasaccharides, advantageously possessing at least the biological propertires of thes octasaccharides.

It is another object of the invention to provide an easy practice enabling elimination highly selectively of the A-H octasac-charide units not taking part essentially in the biological action concerned of these products. It is still another object to provide means for obtaining an enzyme particularly suited to the practising of this process. It is also another object to provide active princi-ples of drugs and the drugs themselves capable of inhibiting the Xa factor~ when i-t is present in the blood, with a high degree of selectivity whist possessing very low activi-ty on total coagulation, and useful, -through this fact, for antithrombotic treatments without hemorragic risk for the patient.

The oligosaccharide compositions of high homogeneity 3~ according to the invention, are characterised in that they are formed essentially of hexasaccharides possessing the sequence C'DEFGH (deno-ted below by the abreviation C'-H).

.,~

z CO~- 0~ coo- r"~ rOR

o ~ ~~ ~ o H
OH ~HAc OH NHSO3 0503 NHSO3 C' D ,., in which R represen-ts a hydrogen atom or the group S03 .

The hexasaccharides of the invention are characterised by a high affinity for ATIII.

They are also, characterised by NMR spectra comprising, among others, a signal in the region of the carbon a-t the 2 position ofthe N-sulfate-glucosamine residues, which does not appear with heparin. This signal can be attributed to the presence of a substituent on the oxygen atom at the 3 position, and more particularly to a 3-0-sulfate group on an N-sulfate-D-glucosamine residue, (unit F of the diagram).

The hexasaccharides of the invention are, moreo-ver, characterised by Yin-Wéssler titers largely superior to those of heparin. More especially, hexasaccharide compo-sitions of the invention may have Yin-Wessler titers, hi-gher than 2000 u/mg, capable of attaining even about 2500 u/mg. Advantageously, their APTT or USP titer is observed to be particularly low, of -the order of 10, which corres-ponds to co~positions having ratios of their Yin-Wessler titer to their USP or APTT titer as high as 200, even a-bout 250.
Advantageous hexasaccharides are formed from C'-H
hexasaccharides in which at least one or two of the R groups represent a -S03- group.

The invention is also aimed at a process for obtai-~fu~

.

ning the abovementioned hexasaccharide compositions.

According to this process, there are subjected, under pre-determined conditions, A-H octasaccharides1 possibly octasaccharide compositions formed to a major extent by these A H octasac-charides, to the action of an enzyme.

Unexpectedly, it is observed that the A-H octasac-charides, which may be themselves obtained by the action of an enzyme on the heparin chains, are however still degra-dable by the enzymatic route. By operating under predeter-mined condi-tions, it is then possible to eliminate units not taking part essentially in the activity of these pro-ducts, and this with a high specificity, which enables hexasaccharide compositions of great structural homogenei-ty to be obtained.

The process according to the inven-tion is hence characterised in that the abovementioned A-H octasacchari-des, or octasaccharide compositions formed to a large ex-tent from these octasaccharides, having a high ratio of the Yin-Wessler titer to their APTT or USP ti-ter, are contacted with an enzymatic agent under conditions adjusted so as to fragment these octasaccharides specifically in order to remove the A-B units not taking part essentially in the activity concerned, this specificity leading to the produc-tion of mixtures formed practically entirely of active he-xasaccharides.

According to a feature of the invention, these conditions comprise advantageously the application of the enzyme at high concentrations of the order of 0.25 to l mg per mg of octasaccharides treated, preferably of the order of 0.5 mg of enzyme per mg of octasaccharide.

In order to provide hexasaccharide compositions highly homogeneous in C'-~ hexasaccharides, recourse is advantageously had to a treatment enabling the separation from the degradation mixture of a least the major portion of the C'-H active hexasaccharides.
A suitable treatment comprises a fractiona-tion carried out in order to remove the proteins resulting from the enzymatic reaction and the unreacted reagents.
This fractionation may be carried out, for example, by gel permeation according to the molecular weight and~or the ionic density of the products.
In a preferred embodiment of the invention, the A-H octasaccharide is subjected to the action of an enzyme.
As a suitable enzyme for obtaining selective removal of the AB units of the A~H octosaccharide, recourse is advantageously had to heparinase, more specifically a heparinase of bacterial origin.
Such a heparinase is advantageously of the type of heparinases which can be obtained from Flavo-bacterium heparinum bacteria~
For the satisfactory practising of the process of the-invention for obtaining C'-H hexasaccha-rides, recourse is more especially had to a heparinase ~5 such as obtained according to a process including steps, notably of cultivating bacteria of Flavobacterium he-parinum from the extraction of crude heparinase from these bacteria, and purifications~ carried out so as to obtain a purified heparinase, having sufficiently high activity to effect the desired removal of the A-B units with satisfactory yields.
Advantageously, the heparinase applied possesses a heparinasic activity of at least about 90 000 units, preferably higher than 100,000 units, particularly of the order of 110,000 to 140,000 units.

The activity of the enzyme is evaluated, with respect to the increase in the absorption of a heparin titrating at least ~15 iu/mg at 230 nm.
By units, is then meant, in the description and the claims, the amount of enzymes which results in the appearence of an increase of one thousandth of an optical density unit per minute.
As already indicated, 0.25 to 1 mg of enzyme (having an activitv of the order of at least 90 000 units) was employed per mg of octosaccharide preferably about 0.5 mg of enzyme.
Study of the action of the enzyme on the octasaccharide has shown that it was appropriate to work at a temperature above room temperature, particu-larly between 35 and 40C, preferably of the orderof 37~C.
Under these conditions, a total duration of about 24 hours appeared satisfactory.
Advantageously, the operation is carried out in a buf~er medium, preferably of a pH of the order of 6 to 8, in particular close to neutrality.
Taklng into account the moderate stability of the enzyme, it is pre~erable, in order to increase its efficacity, to add it portion by portion, particular-ly at regular intervals during this lapse of time.
It will be noted that, advantageously, the process of the invention uses a starting product, namely the A-H octasaccharide of high quality : homo-geneous, and highly specific.
Under the conditions developed by the inventors, this octasaccharide is hence observed to be degradable and this, selectively, leading to shorter chains preserving the sequence responsible for the activity of these products and hence possessing advan-tageously biological properties at least as great, even greater than those of the octasaccharide.
Preferably, the process of the invention is applied with a heparinase obtained by induction from Flavobacterium heparinum bacteria, extraction of the crude heparinase from the bacteria, frac-tio-na-tion of the crude heparinase extract obtained followed by several purifications of the fractions possessing the desired heparinasic activity.
The preparation of heparinase is effec-ted from a Flavobacterium heparinum culture madeunder the conditions described by Payza et Coll.
(~ Biol. Chem. 1956, 223, p.853-858).
By operating at a temperature close to room temperature, with aeration and average stirring, a culture period of the order o~ 25 to 30 hours appea-red satisfactory.
Bacteria were then recovered from the culture medium, for example, by centrifugation, prefe-rably carried out at low temperature, particularly below 10C, preferably of the order of 4C.
Before extracting the heparinase, it is advantageous to resuspend the bacteria, then after having subjected them to a dispersing operationl -to lyophilise them.
The bacteria were then subjected to a treatment with a view to the extraction of the hepa-rinase. This treatment advantageously comprises grinding them then their recovery for example, by centrifugation.
In order to obtain satisfactory extraction, it was convenient to carry out several ~rindings, for example, firstly in the dry and then in a buffer medium of pH of the order of 6 to 8, advantageously 7 or close to 7.
The crude heparinase extract recovered ~7 by centrifugation was then subjected,for purification purposes, to a fractionation step. To provide a hepari-nase possessing the desired activity and properties, additional operations of purification of the successi-vely obtained fractions advantageously followed.
Study of these fractionation and purification processes has shown that it was preferable to operate at a tempe-rature below room temperature, particularly below 10C, preferably of about ~ 4C.
The fractionation step is advantageously carr;ed out particularly according to an exclusion chro-matographic process by means of DEAE cellulose, in the presence of ammonium sulfate.
In a first batch step, the DEAE cellulose is advantageously used in the proportion of about at least 3 g per g of bacterial cells, preferably of about 5 g.
The DEAE cellulose is advantageously equili-brated previously by means of a buffer of pH of the order of 6 to 8, preferably of about 7.
The ammonium sulfate is utili~ed in the pro-portion of about at least 3 g/l, preferably of about 6 g/l.
The suspension thus formed is filtered and the filtrate is collected, supplemented advantageously with rinsing solutions frorn the DEAE cellulose. In a second step, the previously obtained solution is subjected to an additional fractionation by means of DEAE cellulose, advantageously, preequilibrated by means o the previously used buffer.This s~ep is carried out with advantage by chromatography in a column, the filtrates being percolated at a rate of about 40 to 60 ml/h.
From the effluent, the heparinase was recovered, for example, by precipitation, particularly by means of ammonium sulfa-te.
The heparinase obtained at the conclusion of this fractionation opera-tion which, under -the particular conditions reported above, occurs in the form of a sulfo-ammoniacal precipitate, is t'nen advan-tageously purified by a process including at least one placing in contact with an agarose of -the Sepharose type, more especially tha-t known under the registered trademark (CM SEPHAROSE
CL6B", followed by contacting the collected purified.
fractions with heparinasic activity, with an agarose of the type marketed under the registered trademark "ULTRAGEL ACA54".
The purification step by means of an agarose of the CM Sepharose CL 6 B type is advantageously carried out in a chromatographic column.
It appears desirable to dissolve -the heparinase (which occurs in the form of a sulfoammoniacal precipi-tate), in distilled water, so as to obtain a solution having a conductivity of the order of 6000 ~icromhos and to adjust its pH to about 6.
The agarose applied is advan-tageously equili-brated previously by means of a bu~fer of pH of the order of 5 to 7, preferably close to 6.
After having washed the column preferably by means of the washing buffer, the chroma-tography of the heparinase solution is carried out and the heparinase is recovered by elution by a linear gradient obtained with the buffer used for washing and this same buffer is brought to a higher ionic s-trength.
The fractions possessing the desired heparinasic activity are recovered to collect the heparinase they contain. For example, a precipitation particularly by means of arnmonium sulfate is carried ou-t, followed by j~
~ .

centrifugation.
As already indicated, it is advantageous -to proceed with an additional purification of the heparinase collected by operating once again a placing in contact with agarose, preferably, an agarose such as ULTROGEL ACA
54. This operation is, preferably carried out in a column equilibrated by means oE a buffer of pH of the order of 6 to 8, particularly of the order of 7.
This buffer is advantageously used to develop the column.
In this way fractions with a high heparinasic activity were recovered.
As already described by the inventors, the A-H
octasaccharide employed in the process of the invention can be obtained by contacting the heparin (or heparinic fractions) possessing anticoagulant activity and chains having a molecular weight of the order of abou-t 2000 to 50 000, with an enzymatic agent, preferably a purified heparinase, more especially, of bacterial origin possess-ing an activity of the order of 45 000 units, it beingunderstood -that the assay is carried out with a heparin titrating at least 215 iu/mg. The conditions used for carrying out this step are adjusted so as to obtain a depolymerisation mixture containing octasaccharide chains having an anti-Xa activity (Yin-Wessler) and comprising a sequence responsable for -the specific anti-Xa activity of these products.
The enzyme causes the cleavage of -the heparinic chains between the anomeric carbon of the N-sulfa-te=
glucosamine residue and the following uronic acid unit.
The biologically active octasaccharides are then separared from the depolymerisation mixture by adsorp-tion on antithrombin III (ATIII) fixed to a suppor-t such as agarose, under conditions enabling the oc-tasaccharides having an a~inity for the ATIII to be fixed or retained on the ATIII.
This s-tep is advantageously followed by the elution of the re-tained or adsorbed products in order to recover them and by their fractionation, Eor example by gel filtration in order to isolate them.
These oli~osaccharides appear capable of exerting a powerful antithrombotic activity. By reason of their low and even practically zero anticoagulant ac-tivity, the risks of hemorrhage are advantageously practically eliminated. It has been moreover observed that this type of oligosaccharide does not cause any reactivity of the blood platelets.
The short chain hexasaccharides of the invention 15 are devoid of ~oxicity. Administration of 10 000 u/kg (Yin-Wessler titer) of C'-H does not cause any toxic reaction in the rabbi-t, nor pyrogenic effect in the pyrogenicity test in the rabbit according to the French Pharmacopoes.
Administration to mice of doses as large as 3200 mg/kg has not permi-tted the determination of the ~he invention hence relates to pharmaceutical preparations which include said hexasaccharides with high anti-Xa activity.
It relates more particularly -to pharmaceutical preparations devoid of pyrogenic substances containing an effective amount of active principle in association with pharmaceutical excipients.
It relates also to compositions in which the pharmaceutical vehicle is suitable for administration by the oral route. Suitable administrative forms of the invention for administration by the oral route can be advantageously gastroresistant capsules, pellets or ~1 )2~2 tablets, pills, or again ln the form of liposomes.
O-ther pharmaceutical compositions comprise these oligosaccharides in association with suitable excipients for administration by the rectal route. Corresponding administrative forms are constituted by suppositories.
Otner administrative forms of the invention are constituted by aerosols or pomades.
The invention also relates to injectable, sterile or sterilisable pharmaceutical compositions.
These solutions advan~ageously contain 1000 to 100 000 u tYin-Wessler)/ml of oligosaccharides, preferably from 5000 to 50 000, for example 25 000 u/ml, when these solutions are in-tended for injection subcutaneously.
They can contain, for example, from 500 to 10 000, lS particularly 5000 u/ml of oligosaccharides when they are intended for injection intraveneously or by perfusion.
Advantageously, such pharmaceutical preparations are offered in the form of non~recoverable syringes, ready for use.
The invention also relates to pharmaceutical eompositions eontaining said oligosaeeharides in asso-ciation with another aetive principle, useful in par-ti-cular for prophylaxis and treatment of thrombosis, such as a venotonic agent such as dihydroergotamine, a nico-tinie aeid salt or a thrombolytie agent like urokinase.
The short-chain oligosaccharides of the invention are advantageously in the form of salts of at least one physiologieally aeeeptable metal sueh as sodium and/or ealeium and/or magnesium.
The pharmaeeutical compositions of the invention are partieularly adapted for the control (preventive or curative) of certain steps in the coagula-tion of the blood in man or in the animal, particularly in the case where the patient is subjeeted to risks of hypercoagula-,~

.?;~2 bili-ty resulting from a distur~ance in -the intrinsic phase, for example consequen-t upon a release by the organism of thromboplas-tins, for example, tissular thromboplas-tins, (surgical operations, atheromateous processes, development of tumeurs and disorders of coagulation by bacterial or enzymatic activators, etc).
In order to illustrate the invention, there is indicated, below, an example of the dosage usable in man :
this posology comprises, for example, the administration to the patient of 1000 to 25 000 u (Yin and Wessler) subcutaneously, twice or three times daily, according to the degree of hypercoagulability risk or the thrombotic condition of the patient, or from 1000 to 2S 000 u/24 hours intraveneously, in discontinuous administrations at regular intervals, or continuously by perfusion, or again from 1000 to 25 000 u (three times weekly) intra-muscularly or subcutaneously (these titers are expressed in Yin-Wessler units). These doses may naturally be adjusted for each patient according to the results and blood analyses carried out previously, the na-ture of the disease from which he suffers, and, generally, his state of health.
The 7nvention also relates to the use of the oligosaccharides of the invention and of frac-tions containing them, to the constitution of biological reagents, useful in the laboratory, particularly as comparison elements for -the study of other substances whose anticoagulant ac-tivity it is desired to test, particularly at the level of inhibition of the Xa factor.
It is directed also at the use of the fractions and oligosaccharides in nuclear medecine, as radiophar-maceutical products. The ilogosaccharides and the frac-tions defined above are labelled by tracers selected from among those currently used in this field, and ff~12~2 particularly by means of technetium 99 m.
For this purpose, the -techne-tium 99 m obtained from commercial generators, in the form of unreactive sodium pertechnetate of valency 7 is conver-ted into a reduced technetium of valency 4 which would be -the most reactive form of technetium. This conversion is carried out by means of a reducing system effected from tin salts (s-tannous chloride), iron salts (ferrous sulfate), titanium salts (titanium trichloride) or other salts.
Most of the time, this mere reduc-tion of the technetium is enough to fix the technetium to the mole-cule concerned under given conditions of pH.
It is possible to use the products of the in-vention, which constitutes in a way a support, at doses of the order of 100 to 200 u Yin-Wessler.
For developing these radiopharmaceutical reagents, it is possible to operate in accordance with the method of P.V.

~ ~ ~f~

KULKARNI et al. in The Journal of Nuclear Medicine 21, N 2, p. 177-121.
The so-labelled products are advantageously used in ~n v~vo tests for the detection and diagnosis of 5 extended thromboses and thrombotic conditions.
THE DRAWINGS
In Figures 1 and 2 are shown the elution graphs obtained by recording the OD at 280 nm of re-spectively (1~ an heparinase preparation chromatog-raphied on CM sepharose CL6B wherein portion D corre-sponds to the fractions with a high heparinase activity, and (2) the centrifugation culot recovered from D, chromatographied on ULT~OGEL ACA 54, in Figure 3 is shown the elution diagram of a de-polymerization mixture of octasaccharide A-H chromatog-raphied on a superfine SEPHADEX G-50 column, and in Figure 4 are given the graphs obtained by measuring O.D at 230 nm of eluted fractions obtained by fragment-ing an hexasaccharide fraction with HNO2 (curve a in continuous lines), the content in 2,5-anhydromannose groups of said fragments (curve b with dotted lines), their content in uronic acids (curve c with dashed lines - - - -) and in N-acetylglucosamine groups (curve d with dashed dotted lines -.-.-.-.).

.~

EXAMPLE 1.
Process for producing ac-tive C'-H hexasaccharides by the _ action of heparinase onA-~I oc-tasaccharide.
_ _ _ This process includes the following -three steps 1 to 3 :
1) the preparation of the heparinase ;

2) the action of -the heparinase on the A-H octasaccharide for the purposes of selective degradation, followed by

3) fractionation of the degradation mixture by filtration on gel, and recovery of the fractions containing the desired hexasaccharides.
These steps are carried out as follows :
1) Preparation of the heparinase.
Enzymes derived from the cultivation of Flavobac-terium heparinum obtained by the following procedure wereused :
In an 1~ liter fermenter, of the type marketed under the trademark BIOLAFFITE, the cultiva-tion of Flavo~
bact~erium heparinum ATCC 13125 was carried out, for 26 hours in a culture broth corresponding to the following composition (in grams per liter of distilled water).
Flavobacterium heparinum culture bro-th which has reached the stationary phase : 500 ml Monosodium sodium phospha-te : 2,5 25 Disodium sodium phosphate : 25 Ammonium sulfate : 1 K Cl : 0.1 Sodium heparinate of -ti-ter equal or greater than 150 iu/mg of Codex quality : 3 30 Calcium chloride : 0,01 Ferric chloride : 0.01 Magnesium sulfate : 0.01 Manganese chloride : 0.01 Sodium molybdate : 0,01 The pH of the broth was Einally adjusted to 7.0 with phosphoric acid or soda.
This culture was carried out at a temperature of +24C with aeration and medium s-tirring.
Af-ter 26 hours of cultivation, the medium was cooled to -~ 4C in a time interval of about 2 hours. The bacteria were recovered by centrifugation at 50,000 rpm, on a centrifuge of -the type of the SHARPLESS pneumatic type T 313 A type and this for 2 hours. The centrifugation culot was taken up again in 1 liter cold distilled water, subjected to dispersion by an ULTRA-TURAX turbine at maximum speed for 5 minutes, then freeze-dried. The total duration of this operation was about 36 hours. Under these conditions, 4.1 grams of cells were obtained.
- Extraction of the cells.
... . . . . _ The lyophilised cells, obtained by the preceding step, were vigorously ground dry, in a mortar, in the presence of 2 g of calcined alumine, for 1 hour, 10 ml of buffer 1 was then added (0.1 M sodium acetate buffer, pH 7). The grinding in the mortar o~ the paste then obtained is continued for 30 minutes at + 4C. 450 ml of buffer was then added cold and the whole was allowed to stand with stirring for 1 hour at + 4C. The suspension obtained was centrifuged on a centrifuge of the SORVALL
RC2 B type, at 18,000 rpm at + 4C, for 20 minutes. The centrifugation culots composed of alumina and cell debris were discarded. The supernatant liquor (orange yellow and viscous) was collected, which constituted the crude cell extract, and corresponds to a volume of 470 ml. The rest o~ the manipulations were carried ou-t at + 4C.
- Exclusion chromatography by DEAE cellulose.
With stirring, at t 4C, 2.82 g of ammonium sulfate was added to the previously obtained supernatant liquor, and then 21 g of DEAE cellulose previously ~,~
.~. ,~

1~81~0~g~

equilibrated with buffer 2) (0.1 M sodium acetate buffer, pH 7 containing 6 g/l of ammonium sulfate). The whole was subjected to stirring for 2 hours at ~ 4C wi-th control and adjustment if necessary of the pH 7Ø Then the DEAE cellulose was separated by filtra-tion on a Buchner funnel and it was washed in the cold with buffer 2), until the absence of proteins in the wash solutions.
The whole filtrate and wash solutions (680 ml~ were passed over a column of 400 ml (23 = 5 cm) of DEAE cellulose preequilibrated in buffer 2)~ at + 4C, at the flow rate of 50 ml/h. The column was finally rinsed with buffer 2), until the absence of protein in the rinse solutions. The column effluents and the rinse solutions were combined, which corresponds to a volume of 1100 ml and 715 g of ammonium sulfa-te was added with stirring. The precipitated proteins were collected by centrifugation at 7000 rpm and this for 30 minutes, on acentrifuge of the SORVALL type.
2.5 g of very wet precipitate which constitutes the hepa-rinase was collected. This precipitate can be stored at - 20C for several weeks.
- Chromatography on CM Sepharose CL 6 B.
The previously obtained sulfoammonical precipi-tate was dissolved in cold distilled water used in a sufficient amount -to obtain a final conductivity of 6000 micromhos. The pH of the solution obtained was adjusted to 6.0 by acetic acid or 2 N soda. The final volume was 440 ml. The solution was then percolated at + 4C over a column of 70 ml (15 x 2.6 cm) of CM Sepharose CL 6 B
previously equilibrated with buffer 3) (0.1 M sodium acetate buffer, 0.22 M NaCl, pH 6.0), at a flow rate of 25 ml/h. The column was rinsed by buffer 3) until the absence of proteins in the effluent. The column effluents were discarded.
The heparinase was then eluted at 60 ml/h by means of a linear gradient formed from 600 ml of buffer 3) and 600 ml of buffer 3) adjusted to 0.34 M NaCl.
The proteins emerging from the column were detected by con-tinuous recording of the optical density (O.D.) at 280 nm and the eluate was collected by frac-tions of 5 ml by means of a fraction collec-tor.
In Figure 1, is shown the elution graph ob-tained by recording the O.D. at 280 nm of the column effluent.
The heparinase ac-tivi-ty of each fraction was assayed. The fractions 45 to 48 ~portion D of the graph) with a high heparinase content were grouped. These frac-tions correspond to a volume of 45 ml.
The proteins were precipitated by the addition of 30 g of ammonium sulfate and they were recovered by centrifugation at ~ 4C at 15,000 rpm, for 10 minutes.
- Gel filtration on ULTROGEL ACA 5~.
The centrifugation culot previously obtained was dissolved incold distilled water and a final volume of 5 ml was obtained. This solution was placed at the top of a column (1 m x 26 mm) of ULT~OGEL ACA 54 equili-brated with 0.1 M of sodium acetate buffer, 0.33 M NaCl, pH 7. The column was developed by this same buffer at a flow rate 15 ml per hour. As previously the pro-teins emerging from thecolumn were detected at 280 nm, and the column effluent was collected by fractions of 5 ml. The elution graph is shown in Figure 2. The heparinase acti-vity of each fraction was assayed. The fraction 32 to 37 (portion F of the graph), which contained the heparinase activity, were grouped together, which corresponds to a volume of 60 ml. This solution contains 7 mg of purified heparinase, having an activity of the order to 100 000 to 110 000 units/mg. (There was taken as an enzyme unit, the amount of enzyme which causes the appearance at 231 nm, of one thousandth of an optical density unit per minute when -the heparinase is contac-ted a-t 38C with heparin titrating at least 215 iu/mg 0.065 % in 0.125 M
tris-HCl buffer, pH 7, into which calcium chloride CaC12 is added).
The solution ob-tained is preserved and frozen at - 20C.
2) Degradation of the octasaccharide hy the heparinase.
10 mg of A-H octasaccharide (batch BC IV 135), obtained according to the process described in Pa-tent Application filed by Applicant in CANAD~ on October 6, 1980, No 361 600. having an anti-Xa (~in-Wessler) activity of 2100 u/mg, was dissolved in 10 ml of 0.1 M sodium acetate buffer calcium chloride, pH 7.2.
The solution was incubated at + 37C. The heparin-ase addition was carried out as follows.
At time t = O, 17 ml of the heparinase solutionpreviously obtained (namely 2 mg of heparinase) was added :
at time tl = 8 hours, 17 ml of heparinase solution was again added : at time t2 = 16 hours, there was added finally 8.5 ml of heparinase solution. At time t3 = 24 hours, the evaporation to dryness under vacuum at 40C
of 52.5 ml of solution followed in an appara-tus of -the Rotavapor B~CHI -type.
3) Fractiona-tion of the degradation mixture by filtration on gel.
The mixture obtained on emerging from the degra-dation step was placed at the top of a superfine Sephadex G-50 column (registered tradernark) (200 x 2.5 cm). Elu-tion of the products followed by means of 0.2 M sodium chloride. The products were detected by their absorption at 230 nm. In Figure 3 is shown the elution diagram ob-tained. Three principle fractions are distinguished.
The first is constituted by undegraded starting material (4 mg), the second contains the desired hexasaccharides 2~:

(7 mg) and the -third disaccharides. A less important fraction is also distinguished in the region of te-tra-saccharides between the peaks of -the hexa and of the disaccharides.
The hexa~accharide fractions are collected -toge~
ther, -the salts were removed and they were freeze-dried.
- S-tudy of the structure o~ the hexasaccharides of the fractions collected.
A study of the structure of these :Eractions by colorimetric analysis of the fragments obtained by degra-dation by means of nitrous acid followed by gel filtra-tion, ensued. The degradation, by means of nitrous acid, was carried out according to the method of SHIVELY and CONRAD
described in Biochemistry, vol. 15 N 12, 1976, O. 3932 to 3942.
The action of nitrous acid is manifested by severance of the glycoside bonds between the N-sulfate-glucosamine units and the following uronic acid and converts the sulfate-glucosamine units in-to 2,5-anhydro-mannose residues.
The hexasaccharide was then converted into te-trasaccharide and into disaccharide. These two oligosaccha-rides were separated by filtration on a column of Super-fine G-50 Sephadex (registered trademark) (200 x 0.~ cm), eluted with 0.2 M sodium chloride. In figure 4, are plot-ted the values of the optical density measured a-t 230 nm, of the eluted fractions (curve a in continuous lines) as well as their content of 2.5 anhydromannose groups (curbe b in dotted lines ... ), uronic acids (curve c in dashed lines 30 -----) and N-acetyl-glucosamine (curve d in dashed - dotted lines -.-.-.) (for the determination of this latter yroup, measurements were carried out before and after acid hydrolysis, the difference obtained corresponding to the content of N-acetyl-glucosamine groups).

U~2 It is to be seen on examining Figure 4 (-the arrow in Figure 4 indicates -the elution volume for the original hexasaccharide) that the unsaturated uronic acid units which absorb the light at 230 nm are contained in the tetrasaccharide fraction whilst -the disaccharide fraction is practically entirely devoid of such compounds.
In the same way, it appears tha-t the N-acetyl-glucosamine groups are only pre~ent, as anticipated, in the tetrasaccharide fraction.
The content of uronic acid groups appears twice as high in the tetrasaccharide fraction as in the disaccha-ride fraction.
It is observed, also, that the 2,5-anhydromannose groups are present in equivalent manner in the two frac-tions, which implies that the disaccharides and thetetrasaccharides are obtained in a molar ratio 1/1.
These results show that a cleavage has been ef~ected on the hexasaccharide, giving rise to the disaccharide not bearing a double bond and the tetrasaccharide (cleavage between F and G).
In addition, these results establish that the hexasaccharide fraction is almost exclusively constituted by a single species which carries an N-sulfate-glucosamine unit at its reducing end, and an unsaturated uronic acid unit at its non-reducing end.
Comparison with the starting octasaccharide structure permits the conclusion that two other glucosa-mine units of which one is N-acetylated and the other N-sulfated-3-0-sulfated, and two other uronic acid residues (one glucuronic and one iduronic-2-0-sulfa-ted) complete the hexasaccharide sequence.
- Study of the in vitro and in vlvo biological activity of the hexasaccharide fraction obt ned according to the above-described process~

~,' The an-ti-Xa activity was de-termined by the Yin-Wessler test described by its authors in J. Lab. Clin.
Med. 1976, 81, 298-300.
The overall anticoagulant activity was rneasured by the USP test or the APTT method.
The USP test is described in ~Pharmacopea of the Uni-ted States of America", XIX, pages 229-230 (see also the second supplement USP-NF, page 62, and -the fourth supplement USP, page 90, respectively intitled ~Drug substances~ and "Dosage forms").
The APTT titer is measured by the method of J. CA~N & al. in "Hemos-tase, expansion scientific française", Paris, 1968, pages 133-135.
The in vivo antithrombotic activity was studied by using the method of Wessler & al. described in J. of appl. physiol. 1959, 14, 943-946, using a different thrombogenic stimulant.
- Anti-Xa activity (Yin-Wessler) : 2400 u/mg.
- USP titer or APTT titerless than 10 u/mg.
The activity of this hexasaccharide fraction was studied in vivo in the rabbit according to the Wessler model. The administration of 250 u anti-Xa per kg before administration of 25 u/kg of a thrombogenic complex (concentrated prothrombin complex sold under the name Konyn by Cutter Laboratories, U.S.A.) prevents the formation of a thrombus.

Claims (15)

C L A I M S

1.- A process for the preparation of short chained oligosaccharide compositions, characterized in that octasaccharides corresponding to the sequence ABCDEFGH (denoted below by the abreviation A-H), of the formula :

or octasaccharide compositions formed to a large extent of these octasaccharides, having a high ratio of their Yin-Wessler titer to their APTT or USP titer, are contacted with an enzymatic agent under conditions adjusted so as to fragment these octasaccharides specifically in order to eliminate A-B units, the fragmentation resulting in chains essentially formed of hexasaccharides possessing the sequence denoted below by the abreviation C'-H :

C' D E F G H

in which R represents a hydrogen atom or the -SO3-group and their physiologically acceptable salts.

2.- A process according to claim 1, comprising the use of a heparinase as enzyme.

3.- A process according to claim 2, wherein said heparinase is of bacterial origin and obtainable from Flavobacterium heparinum bacteria.

4.- A process according to claim 2, characterized in that the heparinase applied possesses a heparinasic activity of about 90 000 units (the determination being effected with a heparin titrating at least 215 iu/mg at 230 nm), this enzyme being applied at concentrations of the order of 0.25 to 1 mg per mg of octasaccharides treated, preferably of the order of 0.5 mg of enzymes per mg of octasaccharides.

5.- A process according to claim 4 wherein the heparinic activity is of about 110 000-140 000 units.

6.- A process according to claim 1, characterised in that, to provide hexasaccharide compositions highly homogeneous in C'-H hexasaccharides, the mixture resulting from the enzymatic reaction is subjected to fractionation, for example, by gel permeation according to the molecular weight and/or the ionic density of the product.

7.- A process according to claim 1, characterised in that said fragmentation is carried out at a tempera-ture higher than room temperature, in a buffered medium.

8.- A process according to claim 7, wherein the temperature is between 35 and 40°C and the pH of 6-8.

9.- A process according to claim 1, characterised in that the enzyme is added portion by portion.

10.- A process according to claim 7, wherein a calcium-containing buffered medium is used.

11.- Short chained oligosaccharide compositions as obtained according to claim 1, characterised in that they are formed essentially of hexasaccharides possessing the sequence denoted below by the abreviation C'-H :

in which R represents a hydrogen atom or the -SO3 group and their physiologically acceptable salts.

12.- Compositions according to claim 11 characterised in that said hexasaccharides possess Yin-Wessler titers higher than 2000 u/mg.

13.- Compositions according to claim 11, charac-terised by APTT or USP titers of the order of 10.

14.- Compositions according to claim 11, charac-terised by Yin-Wessler titers and USP or APTT titers in a ratio of the order of 200 or higher.

15.- Compositions according to claim 11, characterised in that they are formed of C'-H
hexasaccharides in which one or two of the R groups represents a -SO3 group.