Oligosaccharides obtainable from heparin including heparinic constituents of molecular weights ranging from 2000 to 50,000. Said fractions have a yin-Wessler titer and a USP titer in a ratio of at least 30. They consist of chains constituted by no more than 8 saccharidic moities. They possess a strong antithrombotic activity and are then useful as antithrombotic drugs.
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1. An oligosaccharide fraction of the heparin chain which oligosaccharide has improved antithrombotic activity in vivo higher than that of heparin (as measured by the yin-Wessler test), which oligosaccharide fraction (1) comprises not more than 8 saccharide units, (2) of which one is an N-sulfate-3-O-sulfate-D-glucosamine unit (3) has anti-Xa activity at least 10 times that of heparin, (4) specific affinity for AT III, (5) a ratio of yin-Wessler titer to USP titer of at least 30 and (6) virtually no anticoagulant activity (as determined by the USP test) and, the biologically acceptable salts of said oligosaccharide.
2. The oligosaccharide of A=unsaturated or saturated uronic acid B=N-sulfate-D-Glucosamine or N-sulfate-6-O-sulfate-D-glucosamine #8#
C=L-iduronic acid or, where present at a chain end, unsaturated uronic acid #10#
D=N-acetyl-D-glucosamine or N-acetyl-6-O-sulfate-D-glucosamine E=D-glucuronic acid F=N-sulfate-3-O-sulfate-D-glucosamine or N-sulfate-3-O-sulfate-6-O-sulfate-D-glucosamine G=2-O-sulfate-L-iduronic acid, and H=N-sulfate-D-glucosamine or N-sulfate-6-O-sulfate-D-glucosamine.
3. The oligosaccharide of
4. The oligosaccharide of
5. The oligosaccharide of
6. The oligosaccharide of
7. The oligosaccharide of
8. The oligosaccharide of
10. The oligosaccharide of
11. The oligosaccharide of
12. A therapeutic antithrombotic composition which has antithrombotic activity higher than that of heparin (as measured by the yin-Wessler test) which composition comprises a therapeutically acceptable carrier and in a therapeutically effective amount, an oligosaccharide of
13. The therapeutic composition of
14. The therapeutic composition of
15. A therapeutic method for controlling thrombosis in a patient which comprises administering to said patient the therapeutic antithrombotic composition of
16. The therapeutic method of
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This application is a continuation-in-part of patent application Ser. No. 091,164, filed Nov. 5, 1979, which application has been abandoned in favor of pending application Ser. No. 204,505 filed Nov. 6, 1980.
The invention relates to oligosaccharidic fractions and to oligosaccharides having biological properties, particularly the ability of more specifically controlling some steps of the blood coagulation.
The invention also relates to processes for obtaining said products and to the use of said products as active principles in drugs.
The invention relates more particularly to oligosaccharides having a highly selective activity against activated factor X or factor Xa of blood i.e. a strong antithrombotic activity, while avoiding the risk of hemorrhage for the patient, as well as to oligosaccharidic fractions containing such oligosaccharides (the term "oligosaccharidic fractions" is used in the specification and the claims to designate a relatively homogeneous mixture of oligosaccharidic fragments or chains having a variable number of saccharidic moieties).
The inventors have been led to investigate the biologically active oligosaccharidic fractions and the oligosaccharides themselves, such as obtained from heparin.
It will be noted that the term heparin is used in the specification and the claims in its broadest sense, in order to designate either a commercial heparin of pharmaceutical grade or a crude heparin such as obtained by extraction from biological material, particularly from mammalian tissue.
It is admitted that heparin is an heterogeneous polysaccharide with respect to the composition of its oligosaccharidic chains as well as to the molecular weight thereof.
It is generally considered that heparin mainly contains 2-O-sulfate-L-iduronic acid and N-sulfate-D glucosamine (6-O-sulfated or not) and to a lesser extent D-glucuronic acid, L-iduronic acid and N-acetyl-D-glucosamine (6-O-sulfated or not) moieties.
It is also known that heparin produces its anticoagulant activity by potentiating the inhibitory effect of antithrombin III (αATIII) which is a plasma protein against the successive enzymatic reactions of the coagulation cascades. As heparin is able to simultaneously depress a large number of the coagulation factors participating in the creation and the unkeeping of different forms of hypercoagulability, its activity does not appear specific but general.
Although this anticoagulant activity turns out to be valuable, the re-equilibration of the coagulation-fibrinolysis system with patients under treatment is delicate, due to the global nature of its action. As a result the administration (in order to prevent hypercoagulation risks, for example, the spectre of post-surgical thrombosis) of too high doses of anticoagulant drug or the insufficient selectivity of that drug can be responsible for serious hemorrhages.
By thoroughly studying various conditions for depolymerizing heparin and the depolymerization mixtures thus obtained, the inventors have been led to notice that under certain conditions, it is possible to obtain valuable antithrombotic oligosaccharides containing mixtures. These oligosaccharides are more satisfactory than heparin with regard to the specificity of their activity. They are more particularly capable of enhancing the specific activity of ATIII with respect to a smaller number of coagulation factors, more especially with respect to factor Xa. More essentially, it has been found that such fractions and oligosaccharides have a low global anticoagulant activity as measured by the USP method and that consequently the ratio of their anti-Xa activity as expressed in Yin-Wessler units and of their USP titer is high, at least 30.
As is well-known, the Yin-Wessler activity is more specifically representative of the capability of the active fractions to potentiate the inhibition of the activated factors Xa of blood by AT III in the corresponding test and the UPS titer is representative of the capability of the active fractions to inhibit the global coagulation of blood or plasma.
The Yin-Wessler titer is measured by the test described by these authors in J. Lab. Clin. Med. 1976,81,298-300, and the U.S.P. titer is measured by the test which is described in the "Pharmacopea of the United States of America", XIX, pp. 229-230 (see also the second supplement U.S.P.--NF, p, 62 and the fourth supplement U.S.P.--NF p. 90 respectively entitled "Drug Substances" and "Dosage Forms".
Unexpectedly, it has thus been noticed that the desired anti-Xa specific activity was found in the short oligosaccharidic chains, i.e. containing no more than 8 saccharidic moieties, which can be isolated from the depolymerization mixtures by resorting to certain purification steps, carried out under specified conditions.
It will be noted that the terms "saccharidic moiety" is used in the specification and the claims to designate monosaccharides contained in the heparinic chains.
Besides, according to an aspect of great interest, the inventors have found that the anti-Xa activity of said fractions and of the oligosaccharides, such as expressed in Yin-Wessler units, was significant of an antithrombotic activity in vivo.
It is then an object of the invention to provide new oligosaccharides and fractions containing them, having a high anti-Xa activity and a remarkable selectivity in the framework of the successive enzymatic reactions which characterize the coagulation processes. It is another object to provide structural features of these oligosaccharides.
It is a further object of the invention to provide a process of obtaining said fractions which is easy to carry out.
It is still another object of the invention to provide active principles of drugs and the drugs per se, particularly capable of inhibiting the factor Xa with a high degree of selectivity while their activity on global coagulation can be maintained at a very low level. Such drugs are advantageously useful for antithrombotic treatment without haemorrhage risks.
Said oligosaccharidic fractions are of the type obtainable by a process which comprises the steps of
contacting heparin (or heparinic fractions) possessing anticoagulant activity and having chains with molecular weights ranging from about 2000 to about 50000, with an agent capable of depolymerizing or fragmenting the heparinic chains, the conditions used for carrying out that step being adjusted so as to obtain a depolymerization mixture which contains oligosaccharidic fragments or chains, constituted by no more than 8 moieties yet having an anti-Xa Yin-Wessler activity and including a sequence consisting of less than 8 moieties, which sequence is responsible, to a large extent, for the specific anti-Xa activity of the products;
treating the depolymerization mixture to separate at least the major part of the above defined oligosaccharidic chains, said treatment advantageously comprising (a) the contact of the depolymerization mixture with ATIII for selecting at least the major part, advantageously practically the totality of the oligosaccharides possessing a sequence having the specific structure necessary to recognize and bind ATIII, (b) the elimination of the unselected products, (c) the recovery of the selected products. Said oligosaccharides can be characterized by the fact they possess a specific structure capable of binding ATIII. They have an anti-Xa activity higher than heparin and a very low global anticoagulant activity.
The products having an ATIII affinity as recovered from the above mentioned process, are submitted to one or several steps in order to selectively separate the above-defined oligosaccharides having short chains.
According to the optical density measurements, UP fraction was fractionated into di-, tetra-, hexa- and octasaccharides (UP2, UP4, UP6 and UP8) and into another product having a higher polymerization degree (UP+8). UP8 and UP+8 do not appear on the elution curve of FIG. 2 as they only represent a very low percentage (3.4 and 1.3% respectively) and are only detected at high sensitivity. FP fraction was separated into four fractions designated FPa,b,c and d, according to the order of the decreasing elution volume. The major part of FP fractions is eluted slightly before UP fraction.
Results concerning each of UP and FP fractions are given in the table hereinafter.
They comprise: the elution volume (ml) of each of said fractions, issued from UP and FP fractions; the % of each of them in the mixture of the UP and FP fractions respectively, the specific rotatory power of each fraction (measured with an electronic polarimeter in aqueous solutions usually 1%); the absorbance at 230 mn (measured in a 0.01% solution in HCl 0.03 M, the results being expressed as optical density of 1% solution); and the anti-Xa activity in human plasma of FP fractions measured according to the test of Yin and Wessler above mentioned.
| TABLE |
| __________________________________________________________________________ |
| Elution |
| Volume |
| % in the |
| [α]D 20 (c = 1, water) |
| Activity anti-Xa |
| Fraction |
| (ml) |
| mixture |
| on the D band of sodium |
| DO.H2O 230 nm |
| μl/mg Yin-Wessler |
| __________________________________________________________________________ |
| UP 2 |
| 52-60 |
| 52 |
| +5 6 -- |
| UP 4 |
| 47-52 |
| 30,5 |
| +23,5 4,8 -- |
| UP 6 |
| 43-47 |
| 13 |
| +37 3,3 -- |
| UP 8 |
| 40-43 |
| 3,4 |
| +41,5 2,5 -- |
| UP + 8 |
| 37-40 |
| 1,3 2 |
| FPa 40-45 |
| 50 |
| +39 3,2 1200(2000 in another te) |
| FPb 35-39 |
| 23 |
| +41 2,5 930 |
| FPc 27-34 |
| 14 |
| -- -- 200 |
| FPd 23-26 |
| 5 -- -- 330 |
| __________________________________________________________________________ |
It appears from the results given in the table that each of the FPa fractions exibit an anticoagulant activity, FPA having the most important biological activity. It will be noted that it represents 50 to 75% of the FP mixture. The FPa fraction was submitted to various treatments in order to elucidate its structure. The following analytical results were obtained.
The FPa fractions recovered after the gel filtration were incubated with HNO2 in an aqueous medium under conditions enabling degradation of the oligosaccharidic chains. The degradation was carried out according to the method of Shiveley and Conrad in Biochemistry 1976, 15, p. 3932-3942.
Under the action of NHO2 the oligosaccharidic chains are fragmented into di- and tetrasaccharides, the break taking place behind the N-sulfated glucosamine moieties said moieties being converted into 2,5 anhydromannose groups.
The di- and tetrasaccharides so obtained are separated by chromatography of Sephadex G50 (200×0.5 cm; NaCl 0.2 M.) The elution diagram is given on FIG. 3.
The following measurements are made on each fraction: the optical density at 230 nm (curve a) the amount of uronic acids (curve b) of 2,5-anhydro-mannose (curve c) and of glucosamine (before and after acid hydrolysis), the radio activity when the analysed products have been tritiated before being degradated by HNO2.
The optical density measurement of these fractions shows that the major part of the unsaturated molecules are disaccharides, 90% or the optical density being in the peak of the disaccharide while 10% are in the peak of the tetrasaccharidic fragment.
It can be considered therefrom that the tetrasaccharidic unit does not contain an uronic acid moiety with a double bond. Such moiety is part of a disaccharide which contains further an N-sulfoglucosamine, this disaccharide GH is located at the non reducing end of the oligosaccharide before its nitrous degradation.
Besides, by dosing 2,5 anhydro-mannose groups in these fractions (curve c in FIG. 3), 33% of 2,5 anhydromannose groups are found in the tetrasaccharidic fragments and 66% in the disaccharidic fragments. By assuming that the oligosaccharidic chains are N-sulfoglucosamine terminated, it can be concluded from said results that FPa contains two disaccharidic chains for one tetrasaccharidic chain (see curve b on FIG. 3). Said results were confirmed by the dosage of the uronic acids (curve b) according to Bitter and Muir in Annal; Biochem. 1962,4 p. 330-334 and of the glucosamine moieties in the degradated products originating from FPA, i.e. said degradated products comprise twice as many disaccharidic molecules as tetrasaccharidic melocules.
By reducing FPa before the nitrous degradation, the reducing ends of the chains are not converted into 2,5-anhydro-mannose during the nitrous degradation. The reduction is carried out with a sodium borohydride buffer at pH 9.5. The reducing ends of the FPa oligosaccharidic chains are then converted into tritiated hexitols. The reduced product is separated from the salts present in the mixture by filtration on Sephadex G 25. It is then submitted to nitrous acid degradation and gel filtration as described above. The dosage of 2,5-anhydro-mannose groups is then carried out and shows a clear decrease of said groups in the disaccharidic fraction while the tetrasaccharidic fractions remain practically unchanged.
By reducing with tritiated borohydride, 70 to 80% of the radioactivity are found in the disaccharides and 20 to 30% in the tetrasaccharides. Furthermore, it was observed that after said treatment, the amount of 2,5-anhydro-mannose decreases far more in the disaccharidic peak than in the tetrasaccharidic peak--such result being in favour of the presence of a disaccharidic fragment at the reducing end (70% of the molecules) and also of a tetrasaccharide (30%).
The FPa fraction was further submitted to a nitrous acid degradation under very mild conditions. It was thus possible to obtain, after gel filtration and affinity chromatography an oligosaccharide fraction containing mainly the two hexasaccharides with ABCDEF and CDEFGH sequences the latest being also part of the invention.
The procedure involved treatment of the said FPa fraction with nitrous acid as described by Cifonelli and King (Carbohydrate Res. 1972, 21,pp. 173-186), except that the reaction was stopped between 1 and 5 mn and preferably 3 mn. The fragments thus obtained were desalted by gel filtration on Sephadex G 25 and submitted to an affinity chromatography in the same conditions as described above.
Two main species, i.e. CDEFGH and ABCDEF were found in the eluted fractions, as shown by the analytical methods described above for study of the FPa fraction. They still had a high anti-Xa activity (Yin and Wessler).
The octasaccharidic fraction was isolated. It presented a YW titer of 2000 IU/mg and an APTT titer of 4 IU/mg. The 13 C NMR spectrum of this product was recorded (see FIG. 5). If confirms that the product is an octasaccharide. It also confirms the assigned structure (ABCDEFGH sequence). The signals observed are respectively characteristic of the:
anomeric carbon in position 1 (90-105 ppm) of the various moieties (A,B,C,D,E,F,G and H) of the structure (the corresponding moiety being mentioned for each peak to enable their identification); on the figure
carbons in position 6 (about 60 and about 70 ppm) (signal C6) and in position 2 (55-60 ppm) (signal C2) of the glucosamine moieties;
CH3 of --NHAc group in D (about 25 ppm). Furthermore the presence of a new resonance signal in the C-2 region of N-sulfo-amino-glucosamine residue is observed (signal x--this signal does not correspond to any resonance signal in NMR spectra obtained under similar conditions with a conventional heparin).
PAC Isolation of an anti-factor Xa active hexasaccharide fractionIn another set of experiments, the mixture obtained after the affinity chromatography step (81 mg), was chromatographied on a column (200×2.5 cm) of Sephadex G-50 superfine gel.
The elution was performed with 0.2 M sodium chloride. The products were detected by U.V absorption at 232 nm (see FIG. 4).
The major part of the product was eluted in the octasaccharidic region, and presented the same properties as the product previously described.
The hexasaccharide fraction was collected and the salts were eliminated. The product thus obtained was freeze-dried. Then yield was 2 mg.
This compound was highly active in the YW test: 510 IU/mg. Its APTT titer was 3IU/mg. Since this hexasaccharidic fraction is obtained after heparinase degradation, it contains the residues A and H characterized in the octasaccharidic fraction. Moreover, since this product presents affinity for ATIII it should contain a CDEF tetrasaccharidic sequence. Thus it can be represented by ABCDEF structure.
However one cannot exclude the presence, in this material, of small amount of a product having CDEFGH structure, where C is an unsaturated hexuronic acid residue (the 2-OH group being sulfated or not).
A study carried out as described for the octasaccharide (i.e. nitrous acid degradation and examination of the fragments) indicates the presence of both ABCDEF and CDEFGH.
PAC Degradation of ABCDEFGH octasaccharide and oligosaccharides obtained1--By using the method described by L. A. Fransson in Carbohydrate Research 62,235-244, 1978 ABCDEFGH octasaccharide is submitted to an oxidation reaction with sodium periodate in phosphate medium, pH 7 at 37°C during about 14 hours. An alkaline hydrolysis is caused by adding NaOH to a pH of 11-12 and the reaction mixture is allowed to stay at room temperature for 30 minutes. It is then neutralized; a trisaccharide which is likely to have a structure FGH is separated by chromatography on Sephadex G 50. The determination of the anti-Xa activity according to the Yin-Wessler test give a value about 100-200 ui/mg depending on the assay.
2--Said octasaccharide is submitted to the action of a purified heparitinase, extracted from Flavobacterium heparinum. The experimental conditions (in particular, pH, temperature, time) correspond to those used in the enzymatic degradation according to example 2. Two tetrasaccharides to which structures EFGH and ABCD can be given are recovered by filtration on SEPHADEX G 50 (carried out as in example 2). By passing the tetrasaccharides through a column of Sepharose ATIII, EFGH is be retained while ABCD is eliminated. EFGH is then recovered by elution with a solution NaCl 1 M. The Yin-Wessler activity, measured in various assays, is about 100-200 ui/mg.
3--Octasaccharide ABCDEFGH is submitted to a mild nitrous degradation. 1 mg of octasaccharide is used per ml of solution--HNO2 is generated in situ by adding NaNO2 N/1000 and Hcl. The pH is adjusted to 3. After 10 mn, at the ambient, the pH is adjusted to 7. The reaction mixture is submitted to a gel filtration followed by an affinity chromatography, using the conditions disclosed in the preceeding examples. By elating with NaCl 1 M or CaCl2 1 M, a product can be recovered having presumably hexasaccharidic structure CDEFGH. Said product is submitted to the action of an iduronidase extracted from human kidney. The enzymatic degradation step is carried out at pH 7 at 37°C
An oligosaccharide to which pentasaccharidic structure DEFGH is attributed is obtained by filtration of the depolymerization mixture on Sephadex G 50. The anti-Xa (Yin-Wessler) activity of this product appears to be over 400 ui/mg.
PAC NMR characterization of the oligosaccharidic fraction obtained according to example 1The 13 C NMR spectrum of this fraction shows the presence of said signal * in the region of C-2 of glucosamine residues (see FIG. 6). This signal can be assigned to a glucosamine unit which is N-sulfated and substituted by a --OSO3- group in position 3.
This glucosamine unit is sulfated or not in position 6.
Furthermore, the integral curve confirms that the product has an average number of moieties of less than 8 and comprises a major part of hexasaccharidic and octasaccharidic species. Starting from beef lung heparin, after controlled degradation, followed by affinity chromatography and gel filtration as described above an octasaccharide having the structure ABCFGHGH can be isolated. This product was highly active in an anti-Xa assay. An hexasaccharide ABCFGH was also extracted in minute amount. Ic was also active in an anti-Xa assay.
The invention extends of course to the oligosaccharides that may be obtained by other depolymerization techniques of heparin or heparinic compounds followed by the subsequent recovery of the oligosaccharides of low molecular weight having anticoagulant activity as measurable by the YIN-WESSLER test.
As an example of another depolymerization technique of heparin or related compounds, one may cite periodic oxidation. One may also cite the technique which consists in producing an α,β elimination reaction by chemical means on heparin or esters of heparin giving similar results, or the process which comprises:
contacting the starting heparin fraction with antithrombin III immobilized on a Sepharose gel to fix the antithrombin-binding components of said heparin fraction,
digesting the so fixed heparin with a bacterial heparinase and
eluting the antithrombin-binding fragments from the gel whereby an eluant containing the above said fragments, all of which may then be subjected to the further steps of the above-defined process.
Petitou, Maurice, Lormeau, Jean Claude, Choay, deceased, Jean
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