GB2254083A - Anticoagulants from e.coli saccharide - Google Patents

Abstract

The present invention relates to anticoagulants prepared from the K5 saccharide of E. coli, which have good activity and which can be mass produced.

Description

ANTICOAGULANTS AND PROCESSES FOR PREPARING SUCH The present invention relates to multimeric compounds useful as anticoagulants, as well as to processes for their production.

Enzymatically modified polysaccharides, consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine units, have been extensively investigated in relation to the biosynthesis of heparin and heparin sulphate (see, for instance "HEPARIN - Chemical and biological properties, clinical applications", D Lane and U Lindahl Editors, published by Edward Arnold, pages 159-190, 1989; and U Lindahl et al., TIBS 11, May 1986, page 221). The modifications involve the N-deacetylation of the glucosamine units, the subsequent N-sulphation of the resulting free amino groups, the C5-epimerisation of the D-glucuronic residues to L-iduronic residues, and the O-sulphation at various positions (primarily at the C-2 of the iduronic acids and the C-6 of the glucosamine units). An additional enzymatic O-sulphation may also affect the OH-groups at the 3 position of the glucosamine residues.

To date, it has only been possible to perform this sequence of enzymatic steps on a microscale basis, and has been performed purely on an experimental basis to mimic what happens in mammalian mast cells during the biosynthesis of heparin and heparin sulphate. The chemical and biological differences between these two substances are illustrated in B Casu et al., Arz Forsch, 33, 135, (1983). The literature also describes methods for the N-deacetylation of N-acetylhexosamine residues present in polysaccharide molecules (L Thunberg et al., Carbohydrate Res., 100, 393, 1982 and Shaklee et al., Biochem J, 217, 187, 1984), as well as procedures for Nand O-sulphation (Levy et al., Proc. Soc. Exp. Biol.

Med., 109, 901, 1962).

EP-A-333243 discloses compounds resulting from extensive sulphation of a certain K-5 saccharide isolated from E. coli strains.

We have prepared compounds from K-5 which have useful anticoagulant/antithrombotic activity, and which can be produced on larger scale than is provided by the art.

Thus, we provide anticoagulants prepared from the K-5 saccharide of E. coli, thereby allowing their mass production. These products also have particularly good activity.

Accordingly, the present invention provides deacetylated K-5 E. coli saccharide, wherein the deacetylation amounts to at least 35% of the acetyl groups of naturally occurring K-5.

The invention further provides the modified K-5 saccharide as defined above, wherein sulphate groups are substituted in all, or substantially all, of the positions on K-5 which have been deacetylated. Such positions include those which would normally be expected to be acetylated, particularly the amine groups of the glucosamine, especially D-glucosamine, residues.

The invention also provides a modified K-5 as defined above, wherein at least some of the glucuronic acid residues are epimerised to the L iduronic acid residues.

The invention also provides modified R-5 as defined, wherein at least some of the free hydroxyl groups, especially those in the 6- position of the glucuronic acid residues and/or, where appropriate, those in the 2position of the iduronic acid residues, are sulphated, preferably to an extent of at least 25%.

The invention further provides a saccharide or derivative thereof, comprising substantially units of glucuronic acid and glucosamine, especially where such units alternate, modified as defined above for K-5.

The present invention further provides any of the modified compounds as defined, wherein at least some of the residues are 3-O-sulphated.

The present invention also provides the use of the compounds of the invention in therapy.

The present invention further provides use of any of the compounds of the invention in the manufacture of a medicament for the treatment or prevention of conditions requiring antithrombotic or anticoagulant activity.

The present invention also provides a process for the preparation of any of the compounds described above, which process comprises one or more of the following steps: a) subjecting the appropriate starting material to an N-deacetylation process; b) sulphating the free NH2 groups produced by a) above; c) epimerising the products of b) such that at least some of the D-glucuronic acid residues are transformed into L-iduronic acid residues; and d) sulphating at least some of the free hydroxy groups in any resulting compound.

Preferably, said process comprises subjecting polysaccharides of different molecular weights (hereinafter referred to as KS-saccharides), extracted from certain E. coli strains, to a sequence of chemical and enzymatic passages, which can schematically be illustrated as follows: a) the KS-saccharides, which consist essentially of an alternate linear sequence of D-glucuronic acid and N-acetyl-D-glucosamine are subjected to a chemical N-deacetylation process; b) the free NH2 groups of the products obtained under a) are sulphated by means of appropriate sulphating agents; c) the products obtained under b) are incubated with a particular enzyme (D-glucuronyl-L-iduronyl-C5-epimerase), extracted from bovine liver, by virtue of which a certain amount of the D-glucuronic acid residues is transformed into L-iduronic acid residues; d) the products obtained under c) are reacted with suitable sulphating agents, by virture of which a certain amount of the hydrogen of free hydroxy groups in the polysaccharide chain is substituted by sulphate groups.

The novel polysaccharides obtained according to this sequence as well as the intermediates of each reaction step can be recovered as free acids or in the form of their salts, such as their mineral alkali salts, including their sodium, potassium, calcium or magnesium salts, from which, in turn, the compounds per se can be prepared by treatment with mineral or organic acids, for example.

The combination of chemical and enzymatic steps of the present invention is new, and has not previously been performed starting from polysaccharides of bacterial origin.

According to step a) above, K-5 saccharides, which generally have molecular weights in the range of from about 2000 to about 100000 Dalton or more, determined by HPLC, may be treated with hydrazine containing hydrazine sulphate, preferably about 10% by weight of hydrazine sulphate, preferably in a sealed tube, for a period of time suitably varying from about 30 minutes to about 6 hours, at a temperature which may be between about 80 and about 110*C, for example.

By this procedure, a certain percentage of the N-acetylated groups of the glucosamine units is removed and, according to step b), the so obtained compounds are are subsequently treated with suitable sulphating agents in order to transform the free amino groups.into sulphamino groups. Suitable sulphating agents may be selected from the complexes of sulphur trioxide and the nitrogen containing organic bases, such as tri-(C1 4alkyl)amine.sulphur trioxide, pyridine.sulphur trioxide and analogues thereof. Other sulphating agents capable of introducing an S03H group onto the desired position also fall within the scope of the invention.

The N-sulphation reaction is preferably performed at a temperature between about 45 and 64 C and, depending on the period of time for which the reaction is performed, N-sulphation is more or less extensive. In general, about 6 hours are sufficient for the majority of the free amino groups to be sulphated.

The so obtained polysaccharides, which generally consist essentially of alternating D-glucuronic acids and D-glucosamine units containing acetylamino and sulphamino groups in various proportions, may then be subjected to an enzymatic treatment, for example, according to step c) above, in order to epimerise a certain proportion of the D-glucuronic acid residues of the polysaccharide chain into L-iduronic acid residues.

The epimerisation is most preferably achieved by means of the enzyme D-glucuronyl-L-iduronyl-C5-epimerase, obtainable from bovine liver following the procedure of H. Prihar et al., (Biochemistry, 19, 495, 1980).

In preferred practice, the polysaccharides obtained under b) are incubated with the enzyme, at room temperature, under conditions apparent to those skilled in the art, for a period of time of from, say, a few hours up to two days. Again, depending on the type of substrate employed and the incubation time, polysaccharides having different degrees of conversion of D-glucuronic acid residues into L-iduronic acid residues can be obtained. Step d) may be performed substantially as described by A. Ogamo et al., (Carbohydrate Res., 193, 165, 1989).

The polysaccharides obtained under c) are advantageously first converted into the corresponding salts of organic nitrogen containing bases such as, for instance, the trimethylamine, triethylamine or tributylamine salts, and are subsequently treated wiz suitable sulphating agents, such as those employed for the N-sulphation of step b). The reaction is preferably carried out in the presence of an anhydrous, inert organic solvent such as, for instance, dimethylformamide, dimethylacetamide, dimethylsulphoxide or mixtures thereof, at a temperature of between about -5 and 10'C, preferably at about OC.

The degree of O-sulphation depends on the employed substrates, as well as the reaction time and conditions.

For the purposes of the present invention, this passage is run for from about 1 to 2 hours, preferably for about 2 hours. An occasional, partial N-desulphation may occur during the course of this reaction. If desired, the end products deriving from step d) can undergo the same N-sulphation procedure described in step b).

The end polysaccharides object of the present invention are recovered according to techniques known in the art, as an example upon dialysis of the reaction mixture and subsequent lyophilisation of the dialysed 13 solution. They can be characterised by 13C-NMR and 1H-NMR spectroscopy, which provides specific fingerprints of glycosaminoglycans, as evidenced by A S Perl.in in Methods of Carbohydrate Chemistry, Vol 7, R L Whistler, J N BeMiller Editors, Academic Press New York, 1976, page 94, and L Ayotte et al., Carb. Res., 145, 267, 1980. Other characterisation techniques such as for instance, HPLC, can advantageously be employed as well.

More specifically, the NMR spectra allow the identification and quantification of the non-sulphated L-iduronic and D-glucuronic acid residues as evidenced by the signals at 5.35 p.p.m. and 4.55 p.p.m. of the spectra reported in Figures 5, 7 through 11 and 16 (see B Casu in "HEPARIN, Chemical and biological properties", published by Edward Arnold, D Lane and U Lindahl Editors, pages 25-49, 1986).

Other minor signals are detectable in the above 13C-NMR spectra associated with end residues, that is, those of the reducing anomeric carbons at 90-95 and 95-98 p. p. m. (table 1 in A S Perlin and B Casu; The Polysaccharides, Vol 1, Academic Press, New York, 1982, page 133) and those of the unsaturated terminal uronic acid residues at 110 p. p. m. (B Casu et al Biochem. J.

187, 599, 1981; B Casu, Nouv. Rev, Fr. Haematol, 26, 211, 1984; J R Linhardt, J. Biol. Chem. 261, 14448, 1986).

The relative percentages of D-glucuronic acids and L-iduronic acids may also be determined by paper chromatography of the disaccharides obtained by deaminative cleavage of the C5-epimerised polysaccharides, according to the procedure described by J Jacobsson et al., Biochem. J., 179, 77, 1979. The relevant chromatograms are shown in Figure 13.

The analyses of the NMR spectra and of the paper chromatograms indicate that the novel polysaccharides of the present invention have a % content of N-sulphated groups varying from about 35 to about 100%, a % content of N-acetylated groups varying from about 0 to about 65%, a % content of L-iduronic acids, calculated over the total uronic acids, comprised between about 10 and about 25%, and a minimal content of 6-O-sulphated groups of about 25%.

It will be apparent to those skilled in the art that those polysaccharides having lower percentages of N-sulphated groups, higher percentages of N-acetylated groups, a per cent content of iduronic acids higher than 25% and a minimal per cent content of 6-O-sulphated groups lower than 25% can also be prepared according to the above processes. Said compounds, as well as the corresponding intermediates in the various reaction steps, fall within the scope of the present invention.

As stated above, these novel polysaccharides display interesting and useful biological properties. In particular they possess remarkable antithrombotic and anticoagulant properties, and the activities of particular compounds of the invention are given in the accompanying Example 8.

The compounds of the invention can be administered one or more times per day in unitary injectable dosages varying from about 30 to about 300 mg.

The present invention particularly provides a product which can be manufactures on an economically viable scale. It concerns all the aspects applicable on an industrial scale, associated with the use of the products, resulting from the invention for human therapeutic applications such as antithrombotic and anticoagulant agents. For this purpose the compounds that are the object of the present invention may be formulated by conventional techniques using suitable excipients and other such ingredients for pharmaceutical compositions suitable for parenteral administration, for example.

Examples of formulations for parenteral administration include sterile solutions contained in ampoules, and may also contain substances to render the solution isotonic with bodily fluids, for example.

The compounds obtained as the intermediates in each of the various steps of the process of the invention are, in general, isolated and characterised, but can also be used as such in the subsequent transformations.

If they are characterised, this is made by 1H-NMR and C-NMR spectroscopy, or any other appropriate means, as illustrated above for the end polysaccharides.

Thus, for instance, the substances prepared as described in step b) may be polysaccharides consisting essentially of alternating D-glucuronic acids and D-glucosamine residues, containing from about 35 to about 100% of N-sulphated groups and from about 0 to about 65% of N-acetylated groups. In the 13C-NMR spectra, they show characteristic signals at 104 p.p.m., typical of the D-glucuronic acids, as well as characteristic signals at 60 p.p.m. and 24 p.p.m., typical of the N-sulphated and the N-acetylated groups (Figures 4, 5 and 7 through 11).

Other minor signals detectable in the above spectra are located at 109 and 103 p.p.m., which are associated with the terminal uronic acid residues (Figures 4, 5 and 7).

The intermediates of the various reaction steps possess antithrombotic and anticoagulant properties, and fall within the scope of the present invention.

In particular, the polysaccharides obtained as under step b) may be further subjected to extensive O-sulphation, performed substantially as that described in above step d) followed, if desired, by a re-N-sulphation carried out as described above. The resulting compounds, surprisingly, possess affinity for antithrombin III.

This class of polysaccharides may be characterised by having alternating D-glucuronic acid and D-glucosamine residues, a per cent content of N-sulphated groups varying from about 35 to about 100, a per cent content of N-acetylated groups varying from about 0 to about 65 and a minimal per cent content of 6-O-sulphated groups of 25.

This is may be shown, again, by the 13C-NMR spectrum of Figure 17 showing characteristic signals at 69 p.p.m., typical of 6-O-sulphated groups of the D-glucosamine residues.

From the above description, it is apparent to the skilled artisan that it is provided by the present invention a method for the preparation of a wide range of polysaccharides in a very simple and convenient way.

It is known that the extractive procedures for obtaining natural polysaccharides are often tedious and expensive and that the various fractionation and de-polymerisation techniques of the purified native substances are not always capable of providing reproducible products.

The drawbacks of relying on crude animal extracts (animal illness, epidemics and so on), are removed, microorganisms a providing a practically unlimited source of starting materials.

The compounds of the invention, optionally additionally N-sulphated, may serve as the starting materials for a subsequent enzymatic reaction, by virtue of which certain hydroxy groups at the 3-position of the D-glucosamine residues are converted into the corresponding O-sulphated groups. Such compounds are also defined above.

This reaction is preferably carried out in the presence of the enzyme 3-O-sulphotransferase, which may be prepared as described in Preparative Example 1, below.

The polysaccharides may then be incubated with the enzyme under conditions known in the art, to allow 3-O-sulphation.

The KS-saccharides employed as the starting materials in the present invention may be prepared by cultivating under aerobic conditions, in a suitable fermentation medium, strains of Escherichia coli (E.

coli). Strains of E. coli which can be employed for the purposes of the present invention are those showing the presence of the K5 capsular polysaccharide antigen, and are available with several different sources such as for instance the American Type Culture Collection, the International Escherichia Centre, Statens Serum Institut of Copenhagen, Denmark, and others.

Other E. coli strains to be employed for the purposes of the present invention are again available with several different strain collections or are of clinical isolation, mainly from pyelonephritis and urinary tract infections. They may be characterised via API SYSTEM 20 g and as strains showing the presence of the K5 capsular polysaccharide antigen according to W Nimmich et al., Z Gesamte Hyg., 35(10), 583, 1989, or D S Dupte et al., Sem. Microbiol. Letters, 14, 75, 1982.

Some of these clinically isolated E. coli strains have been deposited with Deutsche Sammlung von Mikroorganismen und Sellkulturen GmbH, Mascheroder Weg lb, D-3300, Germany, on February 27, 1991, under the provisions of the Budapest Treaty. These strains were assigned the accession numbers DSM 6371, DSM 6372 and DSM 6373.By way of example their characteristics are reported hereinbelow: TEST SUBSTRATE REACTION/ENZYME DSM DSM DSM 6372 6372 6372 ONPG O-nitrophenyl p-galactoside p-galactosidase + + + ADH arginine arginine dehydrolase - - LDC lysine lysine decarboxylase + + + ODC ornithine ornithine decarboxylase - + + CIT sodium citrate citrate utilisation - - H 2 sodium thio sulphate production of H2 URE urea urease - - TDA tryptophan tryptophan deaminase - - IND tryptophan indole production + + + VP sodium pyruvate acetoin production - - GEL Kohn gelatine gelatinase - - GLU glucose fermentation + + + MAN mannitol fermentation oxidation + + + INO inositol fermentation oxidation - - - SOR sorbitol do + + + RHA rhamnose do + + + SAC saccharose do - + + MEL melibiose do + - + AMY amygdaline do - - ARA arabinose do + + + OX filter paper cytochrome oxidase - - NO3 glucose tube NO2 production + + + NO2 glucose tube reduction of NO2 to N2 MOB APIM (microscope) mobility + + + MAC MacConkey culture in + + + OF medium fermentation glucose (API OF) (under oil) + + + OF glucose (API fermentation (air) (OF) + + + + = positive - = negative These E. coli strains showed the presence of the K5 capsular polys accharide antigen, determined as illustrated above. The E. coli strains to be employed in the present invention can be maintained on standard agar (Merck I), on Loeb agar, or on any medium suitable for E. coli.

Preparation of K-5 and cultivation of E. coli is illustrated below in Preparative Example 2.

The following Examples and Preparative Examples are provided only for the purpose of better illustrating the invention but in no way they have to be construed as a limitation of the scope of the invention itself.

Preparative Example 1 Isolation of 3-O-Sulphotransferase The enzyme 3-O-sulphotransferase may be prepared from Furth mast cells tumours extracted from mice available at the Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575 s-751 23 Uppsala, Sweden. Furth mast cells tumours can be developed in normal mice strains, as described by J Furth et al Proc. Soc. Exp. Biol., 95, 824, 1957.

3-O-sulphotransferase may be prepared as follows.

Mastocytoma tumours (about 70g of tissue) were homogenised in 200ml of 0.05 M Tris-1% Triton X-100, pH 714, containing protease inhibitors: 10 pg/ml of pepstatin, 2mM EDTA and lmM of PMSF (Sigma Chemical Co).

The homogenate was gently stirred for 1 hour at 4 C and was then centrifuged at 100000 x g for 1 hour. The supernatant was passed through a glass fibre filter and applied to the following purification protocol: 1) Heparin-Sepharose, 31ml column, which was first washed with buffer A (0. 05 M Tris - 0. 1% Triton X-100, pH 7. 4, lRg/ml of pepstatin, 2mM EDTA, 20% of glycerol containing 0. 15 M NaCl), and was then eluted using a linear gradient of 0. 15 - 1.0 M NaCl in buffer A.

Effluent fractions of 4ml were assayed for O-sulphotransferase activity following substantially a procedure described by Jansson et al., Biochem. J., 149, 49, 1975. O-desulphated heparin was used as a sulphate acceptor. Ten pg of acceptor, 5pCi of 35S PAPS and enzyme protein in a total volume of 100E1 of 50mM HEPES, lOmM MnCl2, 10mM MgCl2, 5mM Caul2, 3.5cm NaF, 1% TRITON X-100 R, pH 7. 4, were incubated at 37 C for 30 minutes.The reactions were terminated by the addition of 400yl of ethanol, containing 1. 3% sodium acetate, along with 0. 4mg of carrier heparin, and the samples were left at -20'C overnight. After centrifugation (13000 rpm for 10 minutes) the supernatants were discarded and the pellets were dissolved in 100E1 of water. The 35S-labeled polysaccharide was separated from residual unincorporated label by centrifugation through Sephadex, as follows. Syringes (5cm x 0. 9cm i. d. ) were packed with Sephadex G-25, superfine grade, equilibrated with 0. 2 M NH4HC03, and were then centrifuged at 2000 rpm for 5 minutes, suspended in conical centrifuge tubes, to eliminate most of the liquid.The samples (100R1) were applied to packed and centrifuged syringes, which were then again centrifuged in the same manner. The labeled polysaccharides were recovered in the effluents collected in the tubes, whereas low molecular weight labeled compounds were retained by the gels. The effluents were analysed by scintillation counting. The active fractions were pooled and dialysed against buffer A containing 0. 15 M NaCl.

2) Blue Sepharose, 4lml column, flow rate 15ml/hour.

Following application of the sample, the column was washed with buffer A containing 0. 15 M NaCl and again eluted with a linear salt gradient from 0. 15 to 1. 0 M NaCl to obtain the last portion of the enzymatic activity. Fractions of Sml were assayed for O-sulphotransferase activity as described above. The active fractions were pooled, concentrated to about 10ml and dialysed against buffer A - 0. 075 M NaCl. The sample was subsequently added with p-mercaptoethanol to 12 mM concentration.

The obtained degree of purification was 150 fold, with an apparent 100% recovery of O-sulphotransferase activity. The sample obtained by the purification through Blue Sepharose contained demonstrable glucosaminyl 6-O-sulphotransferase, iduronosyl 2-O-sulphotransferase and glucosaminyl 3-O-sulphotrans ferase.

Preparative Example 2 Cultivation of E. coli and Production of K-5 Cultivation Media For producing the KS-saccharides, E. coli may be cultivated under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, assimilable sources of nitrogen and inorganic salts. Said culture media may be any one of a number of nutrient media employed in the fermentation art, however certain media are preferred. The preferred carbon sources are glucose, mannose, galactose, peptone and the like. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone, amino acids and the like.Among the inorganic salts which are incorporated in the culture media are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, phosphate, hydrogenphosphate, dihydrogenphosphate, nitrate and the like anions.

Ordinarily, the KS -saccharides producing strains are percolated in a shake flask, then the cultures are put into jar fermenters for the production of substantial quantities of the above saccharides. A typical representative fermentation medium, which can also be employed for the preculture, has the following composition (for 1 litre): K2HPO4 3. 6 g KH2PO4 1.2 g Casamino acid 20 g Sodium citrate dihydrate 0. 5 g Ammonium sulphate 1 g Glucose 4 g MgSO4 0. 15 g The dial ys able part of 100 g of yeast extract in 100 ml of water (dialysis against water, cut-off of the membrane: 15000 D) 1 litre The pH of the medium is 7. 2.

Fermentation a) Pre-culture - One loop of E. coli from a Loeb agar plate is suspended in Sml of Merck Standard 1 medium and incubated for 6 hours, at pH of about 7.2, at 37 C. The mixture was then put into 700ml of the above culture medium and incubated overnight under the same conditions.

b) Fermentation - The fermentation is carried out under aerobic conditions at the effective pH 6. 8 for a period of time varying from about 1 to 10 hours, at 37'C.

A jar fermenter containing 10 litres of the above medium ie: K2HPO4 36 g KH2PO4 12 g Casamino acid 200 g Sodium citrate dihydrate 5 g Ammonium sulphate 10 g Glucose 40 g MgSO4 1. 5 g The di alys able part of 1000g of yeast extract in 1 litre of water (dialysis against water, cut-off of the membrane: 15000 D) 10 1 Extraction of K-5 Extraction - The KS-saccharides are recovered from the fermentation media as under b), according to the procedure described by W Vann et al., Eur. J. Biochem., 116, 359, 1981.

An alternative procedure is as follows. The fermentation broth is centrifuged for about 35 minutes at 5200 rpm. The obtained sediments are suspended in about 800ml of phosphate buffer and the resulting suspension is centrifuged for 15 minutes at 8000 rpm, and the supernatant is removed. The sediment is suspended in 800ml of 50 mM Tris 5mM EDTA buffer pH 7. 3 (prewarmed at 37 C) and the suspension is kept for 30 minutes at 37'C in a water bath. The suspension is then centrifuged at 9000 rpm for 20 minutes. The supernatant is removed and the extraction is repeated three times.

The supernatants of the Tris/EDTA-extractions are combined and added with an aqueous 10% solution of CETAVLON (Trade mark) until no further precipitation is observed and the mixture is kept overnight at room temperature. The mixture is centrifuged at 20'C and 8000 rpm for 10 minutes, the precipitate is dissolved in about 10-20ml of aqueous 1 M NaCl, the solution added with at least 8 volumes of ethanol, centrifuged and the solid, consisting of the crude KS-saccharide, is collected. It is redissolved in about 10-20ml of 1M NaCl and at least 8 volumes of ethanol are added to the solution. The precipitate is collected by centrifugation, dissolved in water and dialysed in a dialysis bag with a cut-off of 3500 Dalton for 24 hours. The dialysed solution is centrifuged for 20 minutes at 9000 rpm at 4'C and the precipitate is discarded.The obtained supernatant, consisting of purified R5-saccharide is freeze-dried.

Lipopolysaccharides are removed by dissolving in water the obtained purified K5-saccharide to a concentration of 2-3% and centrifuging the obtained solution at 45000 rpm for 4 hours at 4 C. The presence of traces of residual lipopolysaccharides does not affect the processing of the starting materials. In order to remove also ribonucleic acid derived from E. coli, the obtained solution is treated for 24 hours with ribonuclease (Sigma) in phosphate buffer containing MgCl2 (l0mM) and then dialysed against deionised water with a dialysis bag having a cut-off of 3500 Dalton for 24 hours and lyophilised. This procedure yields about 0. 8-lg per 10 1. culture.

Typical representative K5 saccharides which were prepared according to the above outlined procedures are as follows.

A) K5-saccharide having an average molecular weight of about 5000 Daltons determined by HPLC. This K5-saccharide shows the 13C-NMR spectrum reported in Figure 1, showing the typical major signals of D-glucuronic acids at 104 p.p.m., and of N-acetyl-D-glucosamine units at 55 and 98 p.p.m., as taught by W Vann et al in Eur. J. Biochem., 116, 359, 1981, and typical minor ones at 110 and 103 p.p.m., ascribable to terminal residues derived from the D-glucuronic acid, as taught by B Casu et al., Biochem.

J. , 197, 599, 1981.

B) KS-saccharide having an average molecular weight of about 50000 Dalton determined by HPLC. This K5-saccharide shows the 13C-NMR spectrum of Figure 2, showing the typical major signals of D-glucuronic acids at 104 p. p. m. and of N-acetyl-D-glucosamine units at 55 and 98 p. p. m.

C) KS-saccharide having an average molecular weight of about 100000 Dalton, determined by HPLC. The 13C-NMR spectrum of this KS-saccharide is reported in Figure 3.

Typical major signals of D-glucuronic acids and N-acetyl-D-glucosamine units are at 104 p. p. m. and at 55 and 98 p. p. m. respectively.

The above determinations confirm that the starting KS-saccharides have the following repeating disaccharide unit:

The 13C- and 1H-NMR spectra of the compounds prepared according to the multi-step process of the present invention, as well as those of the starting RS-saccharides, were recorded from solutions in D2O with a Bruker CXP-300 spectrometer.

The determination via HPLC of the molecular weights of the KS-saccharides was performed by Superose 6 (Trade mark) and Superose 12 (Trade mark) columns equilibrated with 1 M NaCl in 0. 05 M TRIS-tICl pH 8 buffer, and the polysaccharides were assayed with the carbazole method.

Example 1 - Preparation of N-deacetylated-N-sulphated polysaccharides from K5-saccharide A (steps a) and b)) A) In a vial 100mg of KS-saccharide A and 138mg of hydrazine sulphate were dissolved in 1. 38ml of hydrazine. The solution was frozen by pouring the vial into liquid nitrogen, while keeping the solution under nitrogen atmosphere. The vial was then sealed and slowly brought to room temperature, then heated for 5 hours at 96 'C. The vial was then re-frozen with liquid nitrogen, opened, and slowly brought to room temperature. The solution was poured into a round-bottom vessel, washing the vial with 5ml of toluene.

The solution was brought to small volume under reduced pressure and the operation was twice repeated (each with 20ml of toluene), to evaporate off (together with toluene) most of the hydrazine. The residues was then added to 50ml of distilled water. The resulting solution was then brought to neutrality by means of aqueous 37% hydrochloric acid, then dialysed 5 days through a 3500 Dalton cut-off membrane, against sodium chloride solutions and water (2x2 1. 0. 5 M NaCl the first day, 2x2 1. H2O 0 the fourth and fifth day). The solution was then concentrated under reduced pressure, then dissolved in 65ml of distilled water.

The pH of the obtained solution was adjusted to 9 by solid sodium bicarbonate and the temperature raised to 55'C. At this temperature, under continuous stirring, the solution was added with 65ml of the adduct trimethylamine.sulphur trioxide, kept at this temperature for one hour, added with further 65ml of the same adduct, and the whole was reacted for additional 5 hours. The solution was dialysed against aqueous solutions of sodium chloride of decreasing concentrations and water as described above. The dialysed solution was finally freeze-dried and 80mg of a product, having the C-NMR spectrum of Figure 4, were obtained.

The product shows the following characteristic signals: strong signals: 60 p. p. m., N-sulphated groups 62 p. p. m., unsubstituted 6-hydroxyl groups of glucosamine residues 98 p.p.m., glucosamine residues 104 p. p. m., glucuronic acid residues weak signals: 109 and 103 p.p.m., terminal glucuronic acid residues very weak signals: 24 p.p.m., residual N-acetylated groups.

The per cent content of N-acetylated groups, determined by 13C-NMR from the ratio of the area of the signal at 24 p. p. m. to that of the area of the signals of all anomeric (C-1) carbons in the 100-110 p. p. m. region, was about 5. The per cent content of N-sulphated groups was about 95. No signals ascribable to free -NH2 groups were observed.

B) Following the same procedure of the foregoing preparation, starting from 100mg of KS-saccharide A and limiting to 3 hours the reaction with hydrazine sulphate/hydrazine, 77mg of a product were obtained, having a per cent content of N-acetylated groups of about 15, determined by 13C-NMR (Figure 5) as for the product of Ex. 1A, and a per cent content of N-sulphated 13 groups of about 85.The C-NMR spectrum of Figure 5 shows the following characteristic signals: 24 and 55 p.p.m. (strong) : N-acetylated groups 60 p. p. m. (strong) : N-sulphated groups 62 p. p. m. (strong) : unsubstituted 6-hydroxy groups of D-glucosamine residues 98 p. p. m. (strong) : D-glucosamine residues 104 p. p. m. (strong) : D-glucuronic acid residues 103 and 109 p. p. m. (weak) : terminal D-glucuronic acid residues No signals of free -NH2 are present.

C) Following the same procedure for the preparation of the product of Example 1A, starting from 100mg of K5-saccharide A) and limiting to one hour the reaction with hydrazine sulphate/hydrazine, 75mg of a product were obtained, having a percent content of N-acetylated groups at 2. 1 p. p. m. and the total area of the signals of the anomeric hydrogens (between 5 and 6 p.p.m.), and the 13C-NMR spectrum of Figure 7, showing the same characteristic signals of the compound of Example 1B.

The compound has a per cent content of N-sulphated groups of about 70. No signals of free -NH2 groups are present.

Example 2 - Preparation of an N-deacetylated-N-sulphated polysaccharide from RS-saccharide B (steps a) and b)) Starting from 100mg of KS-saccharide B, and following the same procedure described for the preparation of the product of Example 1A, that is, by carrying on the reaction with hydrazine sulphate/hydrazine for 5 hours, 75mg of a product were obtained having a per cent content of N-acetylated groups less than 5, determined by 13C-NMR (Figure 8) as for the product of Ex. lA, and a per cent content of N-sulphated groups higher than 95. The 13C-NMR spectrum of Figure 8 shows the same characteristic signals of the compound of Example 1A. No signals of free NH2 groups are present.

Example 3 - Preparation of N-deacetylated-N-sulhated polysaccharides from K5-saccharide C (steps a) and b)) The following polysaccharides were prepared, starting from 100mg of K5-saccharide C), following the same procedure illustrated for the preparation of Example lA, with different times for the reaction with hydrazine sulphate/hydrazine.

A) Reaction time with hydrazine sulphate/hydrazine: 5 hours. Yields: 85mg of a product having a per cent content of N-acetylated groups of about 2, and a per cent content of N-sulphated groups of about 98, 13 determined as above described by the C-NMR (Figure 9). The spectrum shows the same characteristic signals as the compound of Example 1A.

B) Reaction time with hydrazine sulphate/hydrazine: 2. 5 hours. Yields: 80mg of a product having a percent content of N-acetylated groups of about 23, a per cent content of N-sulphated groups of about 77, determined as above described by the 13C-NMR spectrum of Figure 10.

The spectrum shows the same characteristic signals as the compound of Example 1B.

C) Reaction time with hydrazine sulphate/hydrazine: 80 minutes. Yield 75mg of a product having a per cent content of N-acetylated groups of about 48, a per cent content of N-sulphated groups of about 52, determined as above described by 13C-NMR spectrum of Figure 11. The spectrum shows the same characteristic signals as the compound of Example 1C.

The NMR spectra of the polysaccharides prepared in Examples 1, 2 and 3 clearly indicate that no other modification of the structure of the starting KS-saccharides has occurred.

Example 4 - Preparation of a CS-epimerised-N-sulphated polysaccharides (step c) A) lOmg of the product of Example 1A were incubated with 8mg of a preparation obtained from bovine liver as described by H Prihar (see above) containing the enzyme D-glucuronyl-L-iduronyl-C5-epimerase, in lml of 0. 05 M Hepes, pH 7. 4, containing 50 mM potassium chloride, 15 mM EDTA and 1% of Triton X-100 The mixture was kept at room temperature for 2 days, then the desired epimerised product was isolated by ion-exchange chromatography on DEAE cellulose. 7mg of product were obtained.

The low-field region of the 1?!-NMR spectrum (Figure 12) clearly shows the typical signals at 4. 7 and 4. 95 p. p. m. of the L-iduronic acid residues (Perlin A S in Methods in Carbohydrate Chemistry Vol. VII, R L Whistler, J N BeMiller, Eds., Academic Press, New York, 1976, p 94), the area of which corresponds to 18% of the total area of uronic acids. A similar result was obtained by the paper chromatogram (Figure 13), obtained according to J Jacobsson et al., (see above), from which a per cent content of L-iduronic acids of 18 was determined.

B) 10mg of the product obtained in Example 2 were epimerised following the same procedure described in the preceding Example. Seven mg of producttwere obtained.

The 1H-NMR spectrum (Figure 14) shows the typical signals of the L-iduronic acid residues at 4. 7 and 4. 95 p.p.m., the area of which corresponds to 22% of the total area of uronic acids.

Figure 13 refers to the paper chromatogram, obtained according to J Jacobsson et al. (see above), from which a per cent content of L-iduronic acids of 20 is determined.

C) lOmg of the product obtained in Example 3C were epimerised following the same procedure described in the preceding Example 7. 7mg of product were obtained having the paper chromatogram of Figure 13, from which a per cent content of L-iduronic acids of 19 was determined.

The NMR spectra of the polysaccharides prepared in Examples 4A and 4B clearly indiate that no other structural modification than the C5-epimerisation has occurred. In view of the starting substrate and the epimerisation procedure, no other structural modification has occurred also for the product of Example 4C.

Example 5 - Preparation of O-sulphated-N-sulphated-CSepimerised polysaccharides (step d) A) 100mg of the product prepared in Example 4A were dissolved in 20ml of water and passed through an R + Amberlite IR-120 H column at 4'C. The column was subsequently washed with 20ml of water. The eluates were collected and brought to pH 5. 5 with a 10% solution of tributylamine in ethanol. The excess of tributylamine was extracted three times with 40ml each of dimethyl ether and the aqueous solution containing the tributylamine salt of the product of Example 42. was freeze-dried. 100mg of the obtained product were dissolved in 32ml of anhydrous dimethylformamide, the solution was added with 765mg of the adduct pyridine.sulphur trioxide dissolved in 15ml of anhydrous dimethylformamide and the resulting mixture was kept at 0 C for 1 hour. An equal volume of water was added, the solution was brought to pH 9 with aqueous 4% sodium hydroxide and the whole was dialysed against aqueous solutions of sodium chloride of decreasing concentrations and water, as described in Example 1A.

The dialysed solution was freeze-dried and the obtained product, upon transformation into the corresponding tributylamine salt, was reacted under the same conditions of the present Example. 180mg of a product were obtained, which was finally reacted with the adduct trimethylamine. sulphur trioxide as described in Example 1A 180mg of the title compound were obtained, having the 13C-NMR spectrum of Figure 15, showing characteristic signals at 60 p. p. m. (strong): N-sulphated groups; 62 p. p. m. (strong): unsubstituted 6-hydroxy groups of D-glucosamine residues; 69 p. p. m.

(medium): 6-O-sulphated groups; 98 p. p. m. (strong): glucosamine residues; 99 p. p. m. (medium): non-sulphated L-iduronic acid residues; 104 p. p. m. (strong): D-glucuronic acid residues.

The product has a minimum per cent content of 6-O-sulphated groups of 52, determined from the area of the same signal at 69 p.p.m.

B) Following the same procedure outlined under Example 5A above, and starting from lOOmg of the product of Example 4B, 160mg of a product were obtained having the 13C-NMR spectrum of Figure 16, showing the same characteristic signals as the compound prepared under Example 5A.

The product has a minimum per cent content of 6-O-sulphated groups of 44, determined from the area of the signal at 69 p. p. m.

C) Following the same procedure outlined under Example SA above, and starting from 5mg of the product of Example 4C, 9mg of a product were obtained.

The NMR spectra of the polysaccharides prepared as under Example 5A and 5B. In view of the starting substrate and the sulphation procedure, no other structural modification has occurred also for the product of Example 5C.

Example 6 - Preparation of a non epimerised N,O-sulphated polysaccharide 100mg of the product prepared as under Example 1A were dissolved in 20ml of water and passed through an Amberlite R IR-120 H + column at room temperature.

The column was subsequently washed with 20ml of water.

The eluates were collected and brought to pH 5. 5 with 3ml of a solution of tributylamine (10% in ethanol).

The excess of tributylamine was extracted (three times) with 40ml of ethyl ether, and the solution containing the tributylamine salt of the product of Example 1A was freeze-dried.

180mg of the obtained salt were dissolved in 32ml of anhydrous dimethylformamide. To this solution 765mg of the adduct pyridine.sulphur trioxide, dissolved in 15ml of anhydrous dimethylformamide, were added and the mixture was kept 1 hour at O'C. The reaction mixture was then added with an equal volume of water and the pH brought to 7.0 with aqueous 4% NaOH. The mixture was then dialysed against NaCl solutions of decreasing concentrations as described in Example 1A.

The dialysed solution was freeze-dried, and the obtained product upon transformation into the corresponding tributylamine salt, was reacted under the same conditions of the present Example. 90mg of a product were obtained, which was treated with the adduct trimethylamine.sulphur trioxide under the same conditions reported in Example 1A. A product was obtained, having a minimum per cent content of 6-O-sulphated groups of 35 determined from the area of the signal at 69 p. p. m. in the 13C-NMR spectrum (Figure 17), and having the same characteristic signals as the product of Example 5A.

Example 7 - Enzymatic 3-O-sulphation A reaction mixture of 2ml contained 2mg of the compound of Example 5B, 0. 4ml of a preparation containing the enzymatic activity obtained as described above (corresponding to about 1. 6mg of protein), 1 mM PAPS in 0. 05 M Hepes, 10 mM MnCl2 - 5 mM CaCl2 - 10 mM MgCl2 - 3. 5 FM NaF - (0. 5 - 1%) Triton X-100, pH 7. 4. The reaction mixture was incubated for 2 hours at 37 C, then the reaction was terminated by heating at 100*C for 2 minutes.The denatured proteins were removed by centrifugation and the supernatant passed through a column (0. 8 x 100cm) of Sephadex G-15 equilibrated with 0. 2 M H4NNO3. The excluded material was freeze-dried.

A sample (500 pg) of this material, dissolved in 500 Fl of 50 mM Tris-0.5M Nail, pH 7. 4, was applied to a 3ml column of antithrombin III - Sepharose, equilibrated in the same buffer. The column was then eluted with 50 mM Tris-2 M NaCl, pH 7. 4. The eluate contained 10% of the total material.

Example 8.

Activity of Compounds of the Invention The activity of certain compounds of the invention were evaluated in vitro by means of the following tests: a) Anti-Xa activity, performed as described by A N Teien et al., Thrombosis Res. 8, 413, 1976; b) Heparin Cofactor II activity, performed as described by D Dupoy et al., Thromb. Haem. 60(2), 237, 1988; c) APTT, performed substantially as described by W N Bell et al., Nature, 174, 880, 1954, but diluting the plasma sample 1: 1 with saline; and d) TT, performed substantially as described by C Eika et al., Lance II, 376, 1972, but diluting the plasma sample 1:1 with saline.

The results obtained are summarised in the following table: TABLE 1 IN VITRO COAGULATION PROFILE COMPOUND Anti-Xa HCII APTT TT OF IC50* IC5O* IC200** IC200** EXAMPLE (Eg/ml) (Eg/ml) (Rg/ml) (g/ml) 5A 11 0.4 20 4 5B 56 0. 46 39 13 5C 59. 7 0. 05 3. 55 1. 2 * concentration necessary to inhibit 50% ** concentration necessary to double the coagulation time The results are indicative of the clinical usefulness of the polysaccharides of this invention as antithrombotic and anticoagulant agents.

Example 9 Properties of the Compound of Example 6.

The coagulation profile of the compound of Example 6, determined as above illustrated, is reported in the following Table 2.

TABLE 2 IN VITRO COAGULATION PROFILE COMPOUND Anti-Xa HCII APTT TT OF ICSO* IC50* IC200** IC200** EXAMPLE (Eg/ml) (Rg/ml) (Eg/ml) (Eg/ml) 6 5. 7 0. 26 8 2. 5 * concentration necessary to inhibit 50% ** concentration necessary to double the coagulation time

Claims (23)

1. Claims 1. Deacetylated K-5 E. coli saccharide, wherein the deacetylation amounts to at least 35% of the acetyl groups of naturally occurring K-5.
2. 2. A saccharide according to claim 1, wherein sulphate groups are substituted in all, or substantially all, of the positions on K-5 which have been deacetylated.
3. 3. A saccharide according to claim 2, wherein the positions include those which would normally be expected to be acetylated.
4. 4. A saccharide according to claim 2 or 3, wherein the positions are the amine groups of the glucosamine residues.
5. 5. A saccharide according to any preceding claim, wherein at least some of the glucuronic acid residues are epimerised to L iduronic acid residues.
6. 6. A saccharide according to any preceding claim, wherein at least some of the free hydroxyl groups, are sulphated.
7. 7. A saccharide according to claim 6, wherein the free hydroxyl groups are those in the 6- position of the glucuronic acid residues and/or, where appropriate, those in the 2- position of the iduronic acid residues.
8. 8. A saccharide according to claim 6 or 7, wherein at least 25% of the residues are sulphated.
9. 9. A saccharide or derivative thereof, comprising substantially units of glucuronic acid and glucosamine modified as defined in any preceding claim.
10. 10. A saccharide or derivative thereof, wherein the units of glucuronic acid and glucosamine alternate.
11. 11. A compound according to any preceding claim, wherein at least some of the residues are 3-O-sulphated.
12. 12. The use of a compound according to any preceding claim in therapy.
13. 13. The use of any of a compound according to any preceding claim in the manufacture of a medicament for the treatment or prevention of conditions requiring antithrombotic or anticoagulant activity.
14. 14. A process for the preparation of a compound according to any preceding claim, which process comprises one or more of the following steps: a) subjecting the appropriate starting material to an N-deacetylation process; b) sulphating the free NH2 groups produced by a) above; c) epimerising the products of b) such that at least some of the D-glucuronic acid residues are transformed into L-iduronic acid residues; and d) sulphating at least some of the free hydroxy groups in any resulting compound.
15. 15. Novel polysaccharides consisting of alternate sequences of uronic acids and glucosamine residues, characterised by the fact that they have a per cent content of N-sulphated groups varying from about 35 to about 100, a per cent content of N-acetylated groups varying from about 0 to about 65, a per cent content of L-iduronic acids varying from about 10 to about 25, a minimal per cent content of 6-O-sulphated groups of about 25, the compounds being further characterised in that the remaining uronic acids are essentially D-glucuronic acid residues.
16. 16. A process for preparing the compounds of Claim 15, which comprises, in sequence, the following steps: a) a k5-saccharide, essentially consisting of an alternate linear sequence of D-glucuronic acid and N-acetyl-D-glucosamine residues is treated with a mixture of hydrazine/hydrazine sulphate, for from about 30 minutes to about 6 hours, at a temperature comprised between about 80 and about 110-C; b) the compounds obtained under a) are treated with a sulphating agent selected from the complexes between sulphur trioxide and nitrogen organic containing bases, at a temperature between about 45 and about 65 C, for a period of time up to a maximum of 6 hours; c) the compounds obtained under b), which are polysaccharides essentially consisting of alternating D-glucuronic acid and D-glucosamine residues containing acetylamino and sulphamino groups in various proportions are subjected to the action of the enzyme D-glucuronyl-L-iduronyl-CS epimerase, at about room temperature, for a period of time up.to a maximum of two days; d) the compounds obtained under c) are converted into the corresponding salts of organic nitrogen containing bases and are subsequently treated with a sulphating agent selected from the complexes between sulphur trioxide and the organic nitrogen containing bases, in an inert organic solvent, at a temperature comprised between about -5 and 10'C, for from about 1 to about 2 hours; the process being further characterised in that the compounds obtained under d) may optionally undergo the sulphation procedure of step b).
17. 17. A process as defined in Claim 16, comprising the additional step of submitting the products as defined in Claim 1 to the action of the enzyme 3-O-sulphotrans ferase.
18. 18. KS-saccharides essentially consisting of alternate linear sequences of D-glucuronic acid and N-acetyl-D-glucosamino residues represented by the following formula:

    having an average molecular weight varying from about 2000 to about 100000 Daltons or more, and showing characteristic signals in the 13C-NMR spectrum at 104, 98 and 55 p. p. m.
19. 19. Polysaccharides essentially consisting of alternate sequences of D-glucuronic acid and D-glucosamine units, characterised in that they contain from about 35 to about 100% of N-sulphated groups, from about 0 to about 65% of N-acetyated groups and showing characteristic signals in the C-NMR spectrum at 104, 60 and 24 p. p. m.
20. 20. Polysaccharides essentially consisting of alternate sequences of D-glucuronic acid and D-glucosamine units, characterised in that they contain from about 35 to about 100% of N-sulphated groups, from about 0 to about 65% of N-acetylated groups, a minimal per cent content of 6-O-sulphated groups of about 25, the compound being further characterised in that they have an affinity for antithrombin III.
21. 21. The compounds of Claim 15 whenever prepared by the process of Claim 16 or 17.
22. 22. The compounds of Claim 19, whenever prepared by the steps a) and b) of Claim 16.
23. 23. A compound prepared from at least partially deacetylated K-5, substantially as described herein, especially with reference to the accompanying Examples.