GB2262531A - Antiviral sulphated polysaccharides - Google Patents

Description

2262531 TITLE "AlTIVIRAL SULPHATED POLYSACCHARIDES11 The present invention

relates to sulphated polysaccharides, produced by marine algae belonging to the class of Rhodophyceae, endowed with a broad spectrum antiviral activity against DNA and RNA viruses including human and nurine retroviruses and, particularly, human immunodeficiency virus (HIV), to a process for their extraction and purification and to pharmaceutical compositions containing them.

Marine algae have been screened for antiviral activity, nainly against herpes simplex virus and influenza virus (Deig, E.F. et al. [1974] Antinicrob. Ag. Chemother.

6: 524-5-25; Richards, J.T. et all. [1978] Antimicrob.

Agents Chemother. 14: 24-30; Blunden, G. et al. [1981] Bot. Marina 24: 267-272). Studies of aqueous extracts indicated that the active agents were sulphated polysaccharides which acted by blocking or coating receptor sites necessary for virus adsorption (Ehresmann, D.W. et al. [1977] J. Phycol. 13: 37-40), their antiviral activity being mainly related to the degree of sulphation of the polysaccharides (Ehresmann, D.W. et al. [1979] Hoppe, H.A., Levring, T. and Tanaka, Y. [Eds] Marine Algae in Pharmaceutical Science, W. de Gruyter, Berlin, New York, 293-302).

More recently, the urgent need for an effective chemotherapy for the acquired immunodeficiency syndrome (AIDS) which is caused by HIV, has boosted the search for selective anti-HIV sulphated polysaccharides; for example 5 lambda-carrageenan, dextran sulphate and pentosan polysulphate showed anti-HIV activity.

Agarose-type polysaccharides are produced by marine algae belonging to the class of Rhodophyceae (red algae). Among Rhodophyta, some genera-such as Ceramium, Gelidium. and Gracilaria are the main sources of agars, a family of agarose-type polysaccharides that differ in the level of substitution by methoxyl, sulphate and pyruvate groups (Duckworth, M. and Yaphe, W. [1971] Carbohydr. Res. 16: 189-197).

Agarose-type polysaccharides are composed of alternating A(1->4)D-galactose and a(1->3)L-galactose repeating units with the latter 4-0-linked sugar usually occurring as the 3,6-anhydride form or, sometimes, as L-. galactose-6-sulphate, which is believed to be the biological precursor of the anhydro-L-galactose residue (Araki, C. [1966) Proc. Int. Seaweed Symp. 5: 3). The molecule having the lowest degree of substitution is referred to agarose and has the highest gelling potential of all the agarose-type polysaccharides (Kennedy, J.F., Griffiths, A.J. and Atkins, D.P. [1984] G.O. Phillips, D.S. Wedlock and P.A. Williams [Eds] Gums and Stabilisers for the Food Industry, Pergamon Press, Oxford, Vol.2, 417).

t t, k Agar usually forms soft gels that are mainly used in the preparation of microbiological media or in the food industry whereas agarose, forming brittle gels, has primarily biochemical applications (Mc Lachlan, J. [1985] 5 Plant and Soil 89: 137). Extracts from cell wall of the agarophytes consist of the particularly valuable component, agarose, and of a charged (mainly sulphated) polysaccharidic fraction usually named agaropectin: in the manufacture of agar, the highly sulphated fraction, which is soluble in 10 cold water, is discarded.

The present invention provides sulphated polysaccharides obtainable from marine algae of the class of Rhodophyceae (red algae) and endowed with antiviral activity.

The sulphated polysaccharides object of the present invention (hereinafter designed as ASP) have a common backbone of the agaroid-type, composed of alternating jC(1->4)D-galactose and a(l->3)L-galactose repeating units. In particular, after being purified to homogeneity by anion exchange chromatography by application of an increasing sodium chloride gradient, dialysed exhaustively against dist_illed water, and freeze- dried, they have the following properties: (a) elementary analysis: 20 - 35 % by weight carbon, 3.2 - 5.5 % by weight hydrogen, less than 1% by weight nitrogen and more than 8 % by weight sulphur, when calculated as anhydrous compound; (b) molecular weight of up to 10000 kDa as measured by high performance size exclusion chromatography; (c) soluble in water, in aqueous phosphate buffers at pH 1 to 13 and in aqueous solvents containing up to 20% by volume of a water-soluble alcohol but insoluble in benzene, chloroform, ethyl ether and in aqueous-alcoholic solutions containing more than 80% by volume methyl- or ethyl-alcohol and 1 g/1 of sodium chloride; (d) soluble in water in the presence of barium chloride, but, after being hydrolysed for 3 hrs at 120 C in aqueous 2 M hydrochloric acid, it gives a precipitate of barium sulphate upon addition of barium chloride; (e) more than 90 molar % of the total monosaccharidic units are galactose and 3,6-anhydrogalactose residues which are unsubstituted or substituted; (f) more than 30 molar % of the total monosaccharidic units consist of 40-linked a-L-galactopyranosidic residues which can carry substituents at positions 2, 3 and/or 6; (g) more than 40 molar % of the total monosaccharidic units 20 consist of 3-0-linked P-D-galactopyranosidic residues which can carry substituents at positions 2, 4 and/or 6; (h) more than 40 molar % of the total monosaccharidic units consist of 4- 0-linked a-L-galactopyranosidic residues which can carry substituents at positions 2, 3 and 6, plus 4-0- linked 3,6-anhydro-a-L-galactopyranosidic residues which can carry a substituent at position 2; (i) pyruvate (1-carboxyethylidene) groups, linked as cyclic ketals bridging 0-4 and 0-6 of P-D-galactopyranosidic residues, occur as substituents in less than 10 molar % of the total nonosaccharidic units; (j) the nolar ratio of methyl ether group substituents per 5 monosaccharide unit does not exceed 0.3:1; (k) sulphate hemiester groups can be present as substituents at positions 2, 4 and 6 of the P-D- galactopyranosidic residues, at positions 2, 3 and 6 of the a- Lgalactopyranosidic residues and at position 2 of the 3,6anhydro-a-L- galactopyranosidic residues, and the total degree of sulphation is always greater than 0.6; and (1) the contribution of sulphate hemiester groups at position(s) 2 and 4 to the total degree of sulphation is always greater than 0.3; and pharnaceutically acceptable salts thereof.

The ASP and salts thereof can be provided in pure form. They can be isolated. Preferably the ASP has a D.S. of approximately 0.9 0.1. The galactose and 3,6anhydrogalactose residues (e) may be substituted by from 1 to 3 substituents. The substituents may be selected from sulphate hemiester, nethyl ether, pyruvate (1carboxyethylidene) and sugar groups. The residues (f) to (h) may be substituted by from I to 3 substituents, the substituents again being selected from sulphate hemiester, methyl ether, pyruvate (1-carboxyethylidene) and sugar groups. The sugar groups may be galactose and/or xylose residues.

The agaroid skeleton of ASP may bear therefore some out-of-chain galactose residues (up to 5 molar %) as branch units, and up to 10 molar % of the 4-0-linked a-Lgalactopyranose residues may be replaced by 4-0- linked a-D5 galactopyranose units.

Traces of xylose, which in the purified ASP does not exceed 5 molar %, are frequently detected. If present, xylose occurs as branch units and may be eliminated by oxidation (KI04), followed by reduction (NaBH4) and mild hydrolysis (H+). The presence of xylose is not considered to be a distinctive feature, as the biological properties of ASP remain substantially unaffected in the presence or absence of this component.

ASP behaves as a relatively homogeneous chemical entity but is polydisperse, with a ratio MW/MN usually greater than 2, and MP ranging 100 to 300 kDa. MW denotes weight-average molecular wieght. MN denotes number-average molecular weight. MP denotes molecular weight and peak maximum. MP can therefore denote the molecular weight of the fraction containing the greatest concentration of sulphated polysaccharide, as determined by high performance size exclusion chromatography. Fractions may be assayed for this purpose by the resorcinol-sulphuric acid method.

A polysaccharide of the invention or a pharmaceutically acceptable salt thereof may be prepared by a process comprising: (1) extracting red algae, residues from the manufacture of 1 agars or unpurified agars with an aqueous solvent; (2) subjecting the extract to refining treatment and elution on a column or to fractional precipitation such as to obtain the said polysaccharide; and (3) if desired, converting the polysaccharide thus obtained into a pharmaceutically acceptable salt thereof.

The sulphated polysaccharides of the present invention may therefore be isolated starting directly from red algae able to produce agarose, from residues of the manufacturing of agars (aqueous extracts) or, still in appreciable amounts, from general purpose commercial agars which have not been extensively purified. Preferably, ASP is obtained from algae collected in resting conditions or, however, fron extracts or agars prepared from old algal tissues.

Also preferably, the aqueous solvent employed in step (1) is an aqueous solution of phosphate buffer or NaCl and the refining treatment employed in step (2) is at least one treatment selected from centrifugation, filtration, precipitation with an organic solvent, dialysis, lyophilisation and ultrafiltration.

The present invention provides two different preferred procedures both useful and tailored for the selective extraction and purification of ASP from agarophytes, agars or residues of the manufacturing of agars.

The first one is based on anion exchange chromatography and is applied to aqueous crude extracts: when starting from algae, the seaweed, dried in the air, are ground to a fine powder, washed with acetone, air dried, and extracted at room temperature with 0. 01 M aqueous phosphate buffer pH 6.9, containing 0.01 M EDTA-Na2 and 0.1 M NaCl such as to obtain an aqueous extract that is cleared by filtration through glass wool and centrifugation. Likewise, when starting from residues of the manufacturing of agars, a similar aqueous extract is obtained and cleared. Such aqueous extracts are concentrated "in vacuo" to small volume, cleared by centrifugation, precipitated by 80% (v/v) of methanol, re-dissolved in water, cleared by centrifugation, dialysed against distilled water (cutoff membrane: 10 kDa) and lyophilised, such as to obtain 'aqueous crude extracts'. When starting from agar, this is suspended in aqueous 0.05 M NaCl and poured in a jacketted column thermostatted at 30 OC. Through a peristaltic pump and an external reservoir containing further 0.05 M NaCl solution, the eluent is re-circulated exhaustively. The eluate is concentrated and freeze-dried, such as to obtain an aqueous crude extract from agar. Any one of the aqueous crude extracts described above is dissolved in 0.1 M phosphate buffer pH 7.4 and loaded on a Fractogel TSK DEAE-650M column or equivalent, equilibrated in 0.1 M phosphate buffer pH 7.4. A gradient elution from the starting buffer to 2 M NaCl in 0.1 M phosphate buffer pH 7.4 is carried out at room temperature. Eluted fractions are t - 9 collected and monitored both for total carbohydrate content (phenol- sulfuric acid method) and for in-vitro activity against HSV-1 infected cells. The active fractions, which generally elute in the range of 1-1.5 M HaC1 concentrations are pooled, dialysed or ultrafiltrated (cut-off membrane: 10 kDa), and freeze-dried, such as to obtain purified ASP.

The second procedure of extraction and purification of ASP is based on quaternary ammonium precipitation. Polyanions are precipitable from aqueous solutions by quaternary ammonium detergents. We carried out fractional precipitation of sulphated polysaccharides in a defined range of degree of sulphation (D.S.) using cetyltrinethylaranonium bromide (CTA bromide, CTAB or "Cetavlon"), precipitating and/or dissolving complexes in the presence of different salt (NaCl) concentrations. The starting material nay be any source of 'crude aqueous extract' of agarophytes, of residues of the manufacturing of agars, namely "agaropectins", or of commercial agars, such extract being obtained as described above. When starting from agar, this can be extracted directly with aqueous 0.5 M NaCl at room temzerature with stirring, and then filtered through glass wool. otherwise, any 'crude aqueous extract' is dissolved in aqueous 0.5 M FaCl. Aqueous 6% (w/v) CTA bromide is added slowly to the aqueous solution until a flocculent precipitate of ASP-CTA is formed. The presence of 0.5 M NaCl prevent pectins and low charge density polysulphates (D.S. < 0.3) from precipitating. The - 10 precipitate is collected by centrifugation, dissolved in 4 M NaCl, and cleared by centrifugation. Water is added slowly with good mixing, until the NaCl concentration is lowered to 1 M. The homogeneous precipitate of ASP-CTA which is formed (D.S. > 0.6) is collected by centrifugation and redissolved in 4 M NaCl. Methanol is added with stirring up to 84% (v/v) concentration to precipitate ASP as the sodium salt, leaving all CTA and most.NaCl in the supernatant. The precipitate is collected by filtration, dissolved in water, completely clarified by filtration and precipitated again by 84% MeOH to remove all NaCl. Final washes with 95% MeOH and 100% MeOH and desiccation afford the purified sodium salt of ASP.

These two procedures are preferred, but the object product ASP may be obtained by any other method able to discriminate between polyanions of different charge densities and aimed to collect only galactan sulphate molecules having a D.S. greater than 0.6.

As the object product was shown to be polydisperse over a MW range from 1 to more than 5000 kDa, the present inventors investigated the influence of chain-length on the antiviral activity of ASP. High performance size exclusion chromatography was applied to the repetitive collection of 18 ASP fractions of different molecular weight, which were recovered by dialysis, freeze-dried and tested for antiHSV-1 activity. Results showed that, as expected, activity decreases on decreasing of molecular weight, but, - 11 interestingly, even low M.W. fractions ( < 10 kDa) still possess remarkable activity (HSV-1 CPE IC50 = 0.6 to 0.8 AgInl); see Table II.

Biological activitv The antiviral activity of the above described product ASP has been demonstrated "in vitro" on respiratory syncytial virus, Semliki forest virus, herpes simplex virus type I (HSV-1) and type 2 (HSV-2) and vaccinia. virus, grown in nonolayers of Hep 12 cells; in addition, the antiviral activity has been observed on AO Influenza virus grown in monolayers of BHK21 cells, on Moloney sarcoma virus (MSV) in cultures of nurine balb/3T3 cells and on human immunodeficiency virus (HIV) in cultures of primary human lymphocytes.

Results reported hereinafter refer to biological assays of ASP lot 8682/54, but almost identical results were obtained for every other lot of ASP tested.

Table III shows the antiviral activity of ASP against DNA viruses, evaluated as TC IC50, CPE IC50 and as selectivity index (SI), that is the ratio of cytotoxicity (TC IC50) to activity (CPE IC50). The tests were performed -e (M.W. 8 kDa), a known in comparison to dextran sulphat sulphated polysaccharide endowed with a broad spectrum antiviral activity. Experimental data indicate that ASP is 25 well tolerated in tissue cultures. The TC IC50 on Hrp = 2 cells is 742 4g/ml, whereas CPE IC50 ranges 0.1 to 7.1 Ag/ml. ASP appears to be more active than dextran sulphate on all tested viruses.

A comparison of the antiviral activity of ASP on 5 RNA viruses with that of dextran sulphate is reported in Table IV. These results indicate that ASP performs better than dextran sulphate also against RNA viruses. This is confirmed also by data in Table V, referring to infectious viruses.

Efficacy of ASP, dextran sulphate and pentosan polysulphate against MSV appears to be almost the same (Table VI). ASP also shows remarkable activity against HIV (Table VII).

In preliminary "in vivo" tests, carried out in mice i.p. infected with HSV-1, ASP (25 to 50 mg/kg) was compared to Acyclovir (25 or 50 or 100 mg/kg). The results showed that ASP, administered (as Acyclovir) twice daily for 3 days by i.p. route, was at least as effective as the reference compound. In preliminary toxicity tests of the present substance, in which ASP was administered by i.v. route at a dose of 50 mg/kg to the CD-1 mice (male and female), all of the mice were alive in the fourteen-day observation period after administration. Upon these treatments, ASP displayed no significant activity on peripheral and central nervous system (Irwin's test). No adverse effect was observed when mice were treated with ASP twice daily for 3 days by i.p. route at a dose of 50 mg/kg. ASP is a safe material which is low in its toxicity.

Use ASP or a pharmaceutically acceptable salt thereof is useful in methods of treatment of the human or animal body by therapy. ASP has antiviral activity. ASP or a salt thereof can be used against DNA and RNA viruses, including human and nurine retroviruses, in humans and other mammals, for example against those viruses mentioned above. In view of the activity of ASP or a salt thereof against HIV, ASP or one of its salts may be used in the treatment of AIDS, AIDS Related Complex and related syndromes. ASP or a salt thereof can be used to ameliorate the condition of a patient, such as a human patient, infected with a virus. ASP is a safe material which has a low toxicity. The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, ASP or a pharmaceutically acceptable salt thereof.

The preferred mode of administration of ASP to man and animals as an antiviral drug is by parenteral route, more preferably by slow i.v. infusion. The dosage of ASP according to the present invention is variable depending on whether the subject is man or animal and according to such factors as age, individual differences, condition of the disease, etc., but usually in the case where the subject is man, the dose is 0.05 to 5 mg/kg per day, and this amount of - 14 ASP is given in one to three portions. ASP may also be combined with suitable adjuvants and administered in a dermal pharmaceutical formulation.

In use of ASP as an antiviral drug, it can be in the form of free acid or in the salt form, for example sodium, potassium, calcium, and the like, usually and preferably in the form of sodium salt. ASP can be offered in any desired form of preparation. In the case of injection the composition may-contain such additives as stabilisers, buffering agents, preservative, isotonizing agents, etc., and is offered in the form of unit-dose ampule or in multiple-dose containers. The composition may take the form of aqueous solution, suspension, solution, emulsion in an oleaginous or aqueous vehicle, and the like.

The preparation which comprises ASP as an active drug may be a powder which, when used, is redissolved in a pharmaceutically accettable vehicle such as pyrogen-free sterilised water or saline to a therapeutically effective concentration. 20 ASP may also be used in combination with one or more other known antiviral drugs such as AZT, acyclovir, etc., without lowering the normal efficacy. Such combined use is indeed an effective means for the treatment of the diseases. The following Preparations and Examples illustrate the invention. In the accompanying drawings: Figure 1 shows the results of DEAE chromatography - 15 of AP1 lot 8682/36 (_) and ASP lot 8682/54 Elution occurred at 70 ml/h employing a 0.1 M phosphate buffer, pH 7.4 and a sodium chloride gradient ().

x-axis denotes volume in nl. The left-hand y-axis denotes absorbance at 485 nm. The right-hand y-axis denotes sodium chloride concentration in M. Fractions were assayed by the phenol-sulphuric acid method.

Figure 2 shows the results of high performance size exclusion chromatography of ASP lot 8682/54. The x-axis denote nolecular weight in kDa. The y-axis denotes concentration in weight % of total. Fractions were assayed by the resorcinol-sulphuric acid method.

Figures 3a and 3b show the infrared spectra of ASP lot 8682/54 and of ASP lot 8682/54 as the phenylcarbamoyl derivative respectively. The x-axis in each case denotes wavenumber in cm-1.

Physico-chenical characterisations of ASPs.

Elementary analysis was carried out as usual, with sulphur assayed by the Schoniger method.

Neutral sugar content was determined by a resorcinol - sulphuric acid method (Monsigny, M., Petit, C.

and Roche A.C. (1988] Anal. Biochem. 175: 525-530) and 3,6 anhydrogalactose according to Yaphe and Arsenault (Yaphe, W.

and Arsenault, G.P. [1965] Anal. Biochem. 13: 143-148).

Monosaccharide composition was determined after acid hydrolysis (0.5 M TFA, 3 h, 130 OC) both by paper - 16 chromatography (6:4:3 v/v/v n-butanol:pyridine:water on Filtrak FN 11; detection by aniline hydrogen phthalate, min at 105 C), by reversed- phase HPLC of the dansylderivatives (Mopper, K. and Johnson, L. [1983] J.

Chromatogr. 256: 27-38) and by GC assay of the oximetrimethylsilyl derivatives (Vanlaeke, G., Cuppens, H., Leyssens, L. and Raus, J. [1989) J. of Pharm. & Biomed. Analysis 7: 1641-1649).

Isolation of galactose following acid hydrolysis (0.5 M TFA, 3 h, 130 OC), neutralisation with barium hydroxide and concentration to a syrup, was carried out by paper chromatography using Whatman 54 filter paper with a butan-1-ol: acetic acid: water (4:1:5, upper layer) solvent. The ratio of D- to L-galactose was determined by the method of Schlegel (Schlegel, R.A., Gerbeck, C.M. and Montgomery, R. [1968] Carbohydr. Res. 7: 193-199) using Dgalactose oxidase purchased from Sigma (cat# G-3385).

Methylation analysis was carried out by a modified Hakomori procedure (Harris, P.J., Henry, R.J., Blakeney, A.B. and Stone, B.A. [1984] Carbohydr. Res. 127: 59-73), reductive hydrolysis of the permethylated polysaccharides (Stevenson, T.T. and Furneaux, R.H. (1991] Carbohydr. Res. 210: 277-298), and GLC-MS assay of the partially methylated aldononitrile derivatives (Stortz, C.A., Matulewicz, M.C.

and Cerezo, A.S. [1982) Carbohydr. Res. 111: 31-39).

Infrared spectra of ASPs were recorded as usual, using KBr discs. In addition, better-defined peaks, - 17 particularly in the range 800-850 cm-1, were obtained from IR spectra of permethylated (Harris, P.J., Henry, R.J., Blakeney, A.B. and Stone, B.A. [1984] Carbohydr. Res. 127: 59-73) or perphenylcarbamoylated (Hirase, S. and Watanabe, IK. [1972] Bull. Chem. Soc. Japan 45: 1529-1533) ASPs derivatives.

Proton decoupled 13C-NMR spectra of 50 mg/ml ASPs solutions in D20 at 55 OC were recorded on a Varian VRX-400S instrument at 100.6 MHz with spectral width of 24 KHz and relaxation delay of 1.5 s.

High Performance Size Exclusion Chromatographic (HPSEC) analyses of ASPs were carried out on a HewlettPackard 1090A liquid chromatograph equipped with autosampler and autoinjector. 25 pl of 10 ing//ml solutions of ASPs in the mobile phase were injected onto a Shodex OHpak KB-805 column (300 x 8 mm ID) provided with a Shodex OHpak KB-800P precolumn (50 x 8 =ID) and thermostatted at 50 OC. The nobile phase, a 10: 90 (v/v) mixture of acetonitrile: aqueous 0.01 M phosphate buffer pH 6.9 containing 0.2 M NaCl, was delivered at 1 ml/min, and eluate fractions of known size were collected and assayed for total carbohydrate content by a resorcinolsulphuric acid nicromethod (Monsigny, M., Petit, C. and Roche A.C. [1988] Anal. Biochem. 175: 525-530). A series of pullulans having narrow molecular-weight distributions was used for calibration (Fishman, M.L., Danert, W.C., Phillips, J.G. and Barford, R.A. (19273 Carbohydr. Res. 160: 215-225).

Alkaline treatment of ASP aimed to remove all alkali-labile sulphate was carried out, according to Rees (Rees, D.A. [1961] J. Chem. Soc. 51685171), as follows: 200 mg of ASP were dissolved in 20 ml water, added of 40 mg of sodium borohydride and left to reduce at room temperature overnight. 3 M NaOH (10 ml) was then added together with further 100 mg of NaBH4 and the mixture heated for 4 hrs at 1000C with moderate stirring. The alkaline solution was neutralised using Amberlite IR-200 (H+) resin, dialysed exhaustively against distilled water and the alkali-treated ASP was finally recovered by freeze-drying.

Preparation A: Crude extract from Gracilaria verrucosa collected in February.

Gracilaria verrucosa, belonging to the genus Gracilaria of the family Gracilariceae of the order Gigartinales in the Rhodophyceae, was harvested in February in the Northen Adriatic Sea and carefully hand- sorted before drying in the air.

The seaweed (200 g, dry weight) was ground to a fine powder, washed twice with acetone (2 1 each, with stirring overnight), air dried, and suspended in 3000 ml-of 0.01 M aqueous phosphate buffer pH 6.9, containing 0.01 M EDTA-Na2 and 0.1 M NaCl. The extraction was carried out with adequate stirring at room temperature, overnight; the seaweed residues were removed by filtering through glass wool, the filtrate concentrated "in vacuo" to one fifth of the original volume and further cleared by centrifugation. Precipitation of the pale, yellow-brown supernatant (600 ml) with methanol (2400 ml), re-dissolution in water (500 ml), centrifugation, dialysis against distilled water and lyophilisation afforded 25 g (dry weight) of crude extract designated as E-GVF (yield 12.5% on a dry-weight basis; HSV-1 CPE IC50 = 4 pg/ml).

Preparation B: Crude extract from Gracilaria verrucosa collected in September.

A second sample of Gracilaria verrucosa was harvested in September in the Northen Adriatic Sea and carefully hand- sorted before dry^Lng in the air.

The seaweed (200 g), processed as described in Preparation A, afforded 38 g of crude extract (E-GVS; yield 19% on a dry-weight basis; HSV-1 CPE IC50 = 8 pg/ml).

Pre,paration C: Crude extract from Gracilaria dura.

Gracilaria dura, belonging to the genus Gracilaria of the fanily Gracilariceae of the order Gigartinales in the Rhodophyceae, was harvested in summer in the Northen Adriatic Sea and carefully hand-sorted before drying in the air.

The seaweed (200 g), processed as described in Preparation A, afforded 35 9 of crude extract (E-GD; yield 17.5% on a dry-weight basis; HSV-1 CPE IC50 = 6 pg/ml).

- 20 Preparation D: Crude extract from Gelidium corneum.

Gelidium corneum, belonging to the family Gelidiaceae of the order Gelidiales in the Rhodophyceae, was harvested in summer in the Northen Adriatic Sea and carefully hand-sorted before drying in the air.

The seaweed (200 g), processed as described in Preparation A, afforded 39 g of crude extract (E-GC; yield 19.5% on a dry-weight basis; HSV-1 CPE IC50 = 8 gg/ml).

Preparation E: Acrueous extract from aqar.

Agar (100 g of Bacto-Agar(TM) from Difco Laboratories, Michigan (U.S.A.)) was suspended in 900 ml of aqueous 0.05 M NaCl and poured in a jacketted column (50x5 cm I.D.) thermostatted at 30 C. Through a peristaltic pump and an external reservoir containing further (500 ml) 0.05 M NaCl solution, the eluent was re-circulated at 8 ml/min for 7 hours, when re-circulation was disconnected and fresh 0.05 M NaCl was applied till 1600 ml of eluate were collected. The eluate was concentrated and freezedried, yielding 15.9 g of aqueous extract (AE) (HSV-1 CPE IC50 = 8.6 gg/ml).

Example 1: Purification of AE by DEAE ChrondtograDhy.

Active antiviral sulphated polysaccharide was purified from aqueous extract AE (Preparation E) by DEAE chromatography. For each run, 3 g of AE was dissolved in 300 ml of 0.1 M phosphate buffer pH 7.4 and loaded on a 5x50 cm (1000 ml) Fractogel TSK DEAE-650M column, equilibrated in - 21 0.1 M phosphate buffer pH 7.4. A gradient elution from the starting buffer to 2 M NaCl in 0.1 M phosphate buffer pH 7.4 was carried out at 600 ml/h, at room temperature. Eluted fractions were collected and monitored both for total carbohydrate content (phenol-sulfuric acid method) and for in-vitro activity against HSV-1 infected cells. The active fractions, whichgenerally eluted in the range of 1-1.5 M Nael concentrations were pooled, dialysed or ultrafiltrated, and freeze-dried. Processing of AE (15.9 g) 10 afforded 370 mg of purified product (lot 7987/44; D.S.

0.84; HSV-1 WE ICSO = 0.2 Ag/ml).

Exar,:)le 1: Extraction and nurification of llaqaroid-tvpellsuli3hated polysaccharides havina a D.S.>0.3 by CTAB 15 preciiDitation (.kPI).

454 g of Bacto-Agar(TM) Difco were suspended in 6 1 of aqueous 0.5 M NaCl, containing 2 mM sodium azide, and vigorously stirred for 2 h at room temperature. The aqueous extract (4400 ml) obtained by filtering through glass wool, was added of 37 nl of 5 M NaCl, 24 ml of 2 M sodium sulphate, and 330 nl of a 6 % (w/v) aqueous solution of CTAB, under stirring, while slightly warmed (40 C) for 30 nin, and allowed to aggregate while standing for at least 2 h at room temperature. The presence of 0.5 M NaCl prevented pectins and low charge density sulphated polysaccharides (D.S.. zO.3) from precipitating.

The precipitate was collected by brief centrifugation and dissolved in 640 ml of 4 M NaCl, while stirring at 40 OC overnight. The residual agarose and insoluble impurities were centrifuged off.

Sodium sulphate (28 nl, 2 M), CTAB (93 ml, 6%) and 4780 ml of water were sequentially and slowly added to the clear supernatant (700 ml, from redissolution in 4 M WaCl) with good mixing, lowering the sodium chloride concentration down to 0.5 M, and promoting an homogeneous precipitation.

After warming (40 C, 30 min) with stirring and standing (: 2 h) at room temperature, the precipitate was collected by centrifugation and redissolved in 430 ml of 4 M NaCl (40 'C, with stirring, overnight).

Methanol (2530 ml) was added slowly, with adequate stirring, to the clear solution (470 ml) to precipitate sulphated polysaccharides as their sodium salts, leaving all CTA and most NaCl in the supernatant (3000 ml, 84% MeOH).

Stirring was maintained for at least 2 h, when ion exchange proceeded to completion._ The precipitate was collected by filtration, dissolved in 220 ml of water (tot. vol. 260 ml), completely clarified by filtration (0.45 pm) and precipitated again in 84% MeOH (by addition of 1500 ml MeOH) to remove all NaCl.

Final washes with 95% MeOH (500 ml, twice) and 100% MeOH (400 ml, twice) and drying afforded the purified sodium salts of the sulphated polysaccharides having a D.S.:0.3 in 1.3% yield (6 g, lot 8682/36, or "AP111; D.S. = 0.75; HSV-1 CPE IC50 = 0.2 gg/ml).

Example 3: 'Preparation of 11APS11.

Boo g of "Agar Type All (cat.n.A4550, Sigma Chemical Company, USA) were processed as described in Example 2, affording 4.6 g of sulphated polysaccharides (lot 11APS11; D.S. = 0.68; HSV-1 CPE IC50 = 0.4 Ag/ml).

Example 4: preparation of 11APA11.

g of "Agar-Agar" (cat.n.412361, Carlo Erba, Italy) were finely pulverized and processed as described in Example 2, scaling down all volumes to maintain the same proportion between the amount of starting material and reactans, finally affording 2. 8 g of sulphated polysaccharides (lot 11APA11; D.S. = 0.70; HSV-1 CPE ICSO 0.2 gg/nl).

Example 1-5: Pre)aration of ASP lot 7987/52B and characterisations.

ng of lot AP1 (Example 2) were subjected to DEAE chromatography. The procedure described in Example 1 was scaled down to use a 1.6x19 cm (40 ml) column. The elution profile (solid line in Figure 1) highlighted the presence of two diffent kinds of sulphated polysaccharides in API, having definite and different charge densities: "Peak All (lot 7987/52A; D.S. = 0.38; HSV-1 CPE 1C50 > 20 Ag/inl) and "Peak W' (lot 7987/52B; D.S. = 0.91; HSV-1 CPE - 24 IC50 = 0.2 pg/inl).

It should be noted that: - "Peak All and "Peak B", since eluted as peaks virtually separated to baseline under constant gradient of NaCl, must be regarded as compounds of different kind, and not as parts of a sole range of polysaccharides continuously varying in charge density. - "Peak W' (D.S.=0.91) is highly active against HSV-1 while "Peak All (D.S.=0.38) is inactive.

- Both have similar molar content of galactose (by PC, HPLC and GC), low 3,6-anhydrogalactose (determined according to Yaphe, W. and Arsenault, G. P. [1965] Anal. Biochen. 13: 143-148), a common backbone mainly consisting of alternating P(1->4)D-galactose and a(l->3)L-galactose repeating units (13C-NMR), and similar HPSEC profile ( Mp about 190 kDa - IR spectra of phenylcarbamoylated or methylated derivatives of the two substances showed sufficient resolution in the range 800 to 850 cm-1 and allowed to attribute most, if not all substitution by sulphate hemihester groups in "Peak All to galactose-6-sulphate (820 cm-1), whereas galactose-6-sulphate (820 cm-1) and galactose-2-sulphate (840 cm- 1), possibly plus galactose-4-sulphate (850 cm-1, shoulder), were detected in "Peak B". Their 13C-NMR spectra were in agreement with these observations. - When alkaline treatment was carried out to remove alkalilabile sulphate groups (namely 6-sulphate), "Peak W' gave a - 25 product which retained some anti-HSV-1 activity (CPE ICSO 0.8 gg/ml) and alkali-stable groups (2- and, possibly, 4sulphate; D.S. = 0.47), whereas "Peak All lost all sulphate groups.

Exan;Dle 6: Preparation of ASP lot 8682Z54.

The procedure described in Example 2 was slightly modified: the second precipitation of sulphated polysaccharides with cetyltrimethylammonium bromide (CTAB) was carried out in the presence of 1 M NaCl (instead of 0.5 M NaCl), thus preventing sulphated polysaccharides having a D.S. < 0.6 from precipitating, in order to collect ASP coded "Peak B" only.

Aqueous extraction from agar, first precipitation with CTAB in 0.5 M NaCl, and dissolution in 4 M NaCl were carried out exactly as described in Example 2, starting from 454 g of Bacto-Agar(TM) Difco.

Sodium sulphate (14 mI, 2 M), CTAB (46 nl, 6%) and 2040 ml of water were sequentially and slowly added to the clear supernatant (700 mI, from redissolution in 4 M NaCl) with good mixing, lowering the sodium chloride concentration down to 1 M, and promoting an homogeneous precipitation. After warming (40 'C, 30 min) with stirring and standing 2 h) at room temperature, the precipitate, which centrifuges upward, was collected and re-dissolved in 300 mI of 4 M NaCl (40 C, with stirring, overnight).

Methanol (1685 rl) was added slowly, with adequate - 26 stirring, to the clear solution (315 nl) to precipitate sulphated polysaccharides as their sodium salts, leaving all CTA and most NaCl in the supernatant (2000 nl, 84% MeOH). Stirring was maintained for at least 2 h, when ion exchange 5 proceeded to completion.

The precipitate was collected by filtration, dissolved in 150 ml of water (tot. vol. 180 ml), completely clarified by filtration (0.45 pm) and precipitated again in 84% MeOH (by addition of 1000 ml MeOH) to remove all NaCl.

Final washes with 95% MeOH (350 inl, twice) and 100% MeOH (300 ml) and drying afforded the purified sodium salts of sulphated polysaccharides having a D.S.kO.6 in 1.0% yield (4.4 g, lot 8682/54; D.S. = 0.85; HSV-1 CPE IC50 = 0.2 Ag/M1) - Characterisation exanple of ASP lot 8682/54.

Note: whenever not otherwise specified, contents of substituents or monosaccharide residues are expressed hereinafter as molar percentages.

ASP lot 8682/54 (Example 6) has a D.S. = 0.85, a 3,6-anhydrogalactose content of 10.4% and 8.1% (by weight) of water (Table I). It has been obtained as the sodium salt (Na: 7.5% by weight), freed from excess of sodium chloride (Cl: 5 0.1% by weight) in the last steps of preparation.

Apart from decomposition products of the acidlabile 3,6-anhydrogalactose, only galactose was detected as constituent sugar in the hydrolysates (found: 58% by weight; - 27 calcd: 59% by weight). When galactose was isolated from the hydrolysates, it showed low, positive values of the specific optical rotation, indicating an excess of the Disomer. The presence of the L- isomer was confirmed by the negative values of [a]D neasured after treatment of the isolated sample of galactose with D-galactose oxidase.

ASP lot 8682/54 is polydisperse, as shown by the HPSEC profile (Figure 2), with Mp ranging 150 - 240 kDa.

The infrared spectrum of the product (Figure 3a) exhibited absorbances typical of sulphate esters ( 1420(w), 1370(w) and 1250 cm-1, together with an unresolved 800 to 850 cm-1 band) and 3,6- anhydrogalactose (930 cm-1). Some information about the sites of sulphation was obtained from the IR sPectrun of phenylcarbamoylated ASP lot 8682/54 (Figure 11b), which showed two major bands at 840 cm-1 (2sulphate) and 820 cm-1 (6-sulphate) - the latter reinforced by a shoulder at 810 cm-1 (3,6-anhydrogalactose-2-sulphate) - and a weak band at 850 cm-1 (4sulphate), but no absorption at 860 cm-1 (3-sulphate).

The negative value of the specific optical rotation of ASP lot 8682/54 ([a]D25: -15'; c=1.0, water; constant in the range 20-60OC) is consistent with an agaroid skeleton, namely alternating 3-0-linked P-Dgalactopyranosyl residues and 4-0-linked a-L-galactopyranosyl residues, thus differing from carrageenans which show positive rotations. The low, negative value of the specific rotation of ASP is reasonable, taking into consideration the low proportion of - 28 3,6-anhydrogalactopyranose residues. Furthermore, lack of e vidence of any variation or hysteresis in la]D/temperature profiles argues against the occurrence of double-helix aggregates in aqueous solutions of ASP lot 8682/54 at room 5 temperature.

The agaroid backbone could be also clearly distinguished from that of carrageenans, expecially looking at the anomeric carbons region of the 13C-NMR spectrum. From this spectrum, several chemical characteristics can be deduced: - methoxyl substitution (59.1 ppm) seems absent or, at least, not significant in this lot of ASP, which was confirmed also by 1H-NMR. pyruvate substitution at 0-4 and 0-6 (1-carboxyethylidene groups) positions of some of the P-D-galactopyranose units was inferred from the signals at 25.7 and 176.4 ppm and estimated around 5%. - anomeric carbon signals, though not well resolved, are consistent with a structure constituted by alternating je(1->4)D-galactopyranose and a(l->3)L-galactopyranose repeating units. In particular the assignments are the following: C-1(A-D) (30 to 40%) at 104.4 ppm when a-L: GAL, 2-S-GAL, 6-S-GAL or 2,6-DS-GAL C-1(fl-D) (10 to 20%) at 102.7 ppm when a-L: 3,6-AN C-l(a-L) (15 to 25%) at 101.6 ppm when a-L: GAL or 6-S-GAL C-l(a-L) (25 to 35%) at 99.2 ppm when a-L: 2-S-GAL, 2,6-DS- GAL, 3, 6-AN or 3,6-AN-2-S-GAL where GAL = unsubstituted galactopyranose 2-S-GAL = galactopyranose-2-sulphate 6-S-GAL = galactopyranose-6-sulphate 2,6-DS-GAL = galactopyranose-2,6-disulphate 3,6-AN = 3,6- anhydrogalactopyranose 3,6-AN-2-S-GAL = 3,6-anhydrogalactopyranose-2- sulphate (sulphation at position 3 was neglected on the basis of the 10 IR spectrum).

- the ratio of the respective integration for P- and alinked anomeric carbon atoms is roughly 1:1. - of the total C-6 in a-L and P-D units, about 60% bear a free hydroxyl group (signal at 61.5 ppm).

is Further information about ASP structure came from nthylation analysis, when the following major products e 11 were identified: 2,4,6-tri-0-methq.,;lgalactose, arising from unsubstituted 3-0-linked P-D-galactopyranose 4,6-di-O-r,iethylgalactose, arising from 3-0-linked P-D-galactopyranose-2-sulphate 2,4-di-0- nethylgalactose, arising from 3-0-linked P-D-galactopyranose-6-sulphate - 2,3,6-tri-0-nethylgalactose, arising from unsubstituted 4-0-linked a-L- galactopyranose 3,6-di-o-methylgalactopyranose, arising from 4-0-linked a-Lgalactopyranose-2-sulphate - 3-0-methylgalactopyranose, arising from 4-0linked a-L-galactopyranose-2,6-disulphate 5 together with traces of: - 2, 6-di-0-methylgalactose, arising from 3-0-linked P-D-galactopyranose-4sulphate - 4-0-methylgalactose, possibly arising from 3-0-linked A-Dgalactopyranose-2,6-disulphate - 2,3,4,6-tetra-0-methylgalactose, suggesting the presence of some branching points. Practically all residues based on 3,6-anhydro-a-L- galactose (10%) were degraded during the acid hydrolysis of permethylated ASP. As the ratio of total a-L- to P-Dmethylated residues was estimated around 4: 5, when taking account of 10% of 3,6-anhydro-a-L- residues, we get an approximate I: 1 ratio of 3-0-linked P-D- to 4-0-linked a- L- galactopyranosyl residues in the original, unmethylated polysaccharide.

Taking into consideration all information obtained from structural analyses, we can regard ASP lot 8682/54 as being composed, on average, of 3-0-linked fl-D-galactopyranosyl residues occurring as 1:2:4:3 pyruvylated: 6-sulphate: 2-sulphate unsubstituted alternating with 4-0-linked a-Lgalactopyranosyl residues occurring as 3:2:2:3 2,6-disulphate: 2-sulphate: 3,6-anhydro-2sulphate: unsubstituted.

This picture is approximate as for ratios and also for some minor structural details; for example some extent of 4-sulphate substitution is likely to be present, as inferred from IR spectrum.

DEAE Chro-,iatoqraDh7 of ASP (lot 8682Z54).

mg of lot 8682/54 (Example 6) were subjected to DEAE chromatography. The procedure described in Example 1 was scaled down to use a 1.6x19 cm (40 ml) column. The elution profile (dashed line in Figure 1) revealed the presence of ASP coded "Peak W' only, thus confirming the procedure in Example 3 as a suitable tool tailored for the selective exitraction and purification of ASP.

EYample 7: Purification of ASP from algal crude extracts.

ASP was purified from algal crude extracts by CTAB precipitation, scaling down the procedure described in Example 6 in view of the smaller amount of polysaccharide to process. E-GVF (25 g, Preparation A), E-GVS (38 g, Preparation B), E-GD (35 g, Preparation C) and E-GC (39 g, Preparation D) were separately dissolved each in 500 nl of aqueous 0.5 M NaCl and precipitated by addition of 2 M sodium sulphate (3 ml) and 55 Tal of a 6 % (w/v) aqueous solution of CTAB, under stirring, while slightly warmed (40 OC) for 30 nin, and allowed to aggregate while standing for at least 2 h at room temperature. The precipitates were collected by brief centrifugation and dissolved in 185 ml of 4 M NaCl, while stirring at 40 OC overnight. Insoluble impurities were centrifuged off.

Sodium sulphate (4 ml, 2 M), CTAB (13 ml, 6%) and 583 ml of water were sequentially and slowly added to the clear supernatants (200 ml, from re-dissolution in 4 M NaCl) with good mixing, lowering the sodium chloride concentration down to 1 M, and promoting an homogeneous precipitation.

After warming (40 C, 30 min) with stirring and standing (: 2 h) at room temperature, the precipitates were collected and re-dissolved in 95 nl of 4 X NaCl (40 'C, with stirring, overnight).

Methanol (525 ml) was added slowly, with adequate stirring, to the clear solutions (100 ml) to precipitate sulphated polysaccharides as their sodium salts, leaving all CTA and most NaCl in the supernatants (625 ml, 84% MeOH).

Stirring was maintained for at least 2 h, when ion exchange proceeded to completion.

The precipitates were collected by filtration, dissolved in 50 ml of water (tot. vol. 60 ml), completely clarified by filtration (0.45 gm) and precipitated again in 84% MeOH (by addition of 315 nl MeOH) to remove all NaCl.

Final washes with 95% MeOH (100 ml, twice) and 100% MeOH (100 ml) and drying afforded the purified sodium salts of sulphated polysaccharides having a D.S.:0.6, respectively:

"GVF" (1.24 g; yield 5.0% from E-GVF or 0.62% from - 33 dry alga; D.S. = 0.92; HSV-1 CPE ICSO = 0.2 AgInl) 11GVS11 (1.04 g; yield 2.7% from E-GVS or 0.52% from dry alga; D.S. = 0. 82; HSV-1 CPE IC50 = 0.2 A91m1) 11GWI (0.92 9; yield 2.6% from E-GD or 0.46% from dry alga; D.S. = 0.85; HSV-1 CPE 1C50 = 0.14 gg/ml) "GV' (0.97 g; yield 2.5% from E-GC or 0.49% from dry alga; D.S. = 0.87; HSV-1 CPE ICSO = 0.2 pg/ml).

"In vitro" assay of antiviral activity and cytotoxicity.

The tests were generally performed by resuspending monclayers of' the permissive cells by tryptic treatment, and by seeding the suspension (4 to 10 x 104 cells/ml, in Mininal Essential Medium of Eagle supplemented with 5% Fetal Calf Serun) in 24 or 96 well flat bottom culture plates. Aliquots of aqueous solutions of the product of the present invention were distributed in two-fold serial dilutions, in duplicate, into the wells, together with either uninfected cells (for the cytotoxicity test) or with cells infected with 30 to 100 I.C.50 of virus/well for the antiviral activity test. The virus was added 15 min after incubation of the product with cells.

Cytotoxicity was evaluated as the concentration required to reduce cell growth by 50% (TC ICSO).

The antiviral activity was observed as reduction of cytopathic effect (CPE), as transforming foci in Moloney sarcoma virus infection and by measuring the amount of the HIV core protein p24 (by Elisa) and the total amount of the - 34 HIV synthesized RNA (by the molecular hibridisation technique with nucleic acids). It was determined as the concentration required to reduce virus induced cytopathogenicity by 50% (CPE IC50), as the concentration required to inhibit MSV induced foci transformation by 50% (F IC50) and as the concentration required to reduce by 50% both the HIV p24 and the HIV RNA. Sometimes the 50% reduction of infectious virus production (IV IC50) was also tested. This was determined by the titration of virus contents in cryolysates of the infected treated and untreated cultures.

Table I

Other physico-chemical properties ccmmon to the range of ASPs in the table are:

Na: 6.7 to 7.6 % (by weight) Cl::5 0. 1 % p: < 100 pr-n Galactose: 55 to 59 % (by weight) (GLC-.M-S [ct]D25: -5 to -20' (c 1.0, water) M.W.: 1 to >5000 kDa (Mp: 170 to 260 kDa) (11PSEC) Ykethyl ether groups: 0 to 0.15 iml per nn.-iosaccharide unit Prnn,-ate groups: 0 to 0. 05 wl per nonosaccharide unit Infrared absorption, (EBr): 3200 to 3700 cm-1 2880 to 2950 cm-1 1600 to 1680 cm-1 1420 (weak.), 1370 (weak) and 1250 cm-1 930 cm-1 800 to 850 crLr-1 (13C-NMR) ( 91 91) (OH groups) (CH2 and CH groups) (C)H groups) (sulphate esters) (3,6-anhydrcgalactose) (sulphate esters) Solubility: greater than 50 ng/nl in water insoluble in organic solvents such as benzene, chloroform, ethyl ether# acetone, nethanol ard ethanol.

Table I (continued) Product lot Exanplet IC50(1) (2) Elenentary analysis aC-al(3) (1Ag/ml) D.S. C% H% N% S% B20% (M01 %) 2 0.2 0.75 26.35 4.36 0.37 8.87 10.62 8.5 3 0.4 0.68 28.27 4.38 0.18 8.59 6.51 12.3 4 0.2 0.70 27.92 5.03 0.37 8.69 8.70 9.4 1 0.2 0.84 23.58 3.83 0.29 8.82 13.18 8.1 7987/52B 5 0.2 0.91 24.32 4.15 0.13 9.84 11.24 7.3 6 0.2 0.85 26.14 4.34 0.16 9.91 8.13 10.4 7 0.2 0.92 24.36 3.84 0.12 10.00 11.57 6.0 7 0.2 0.82 24.35 4.11 0.24 8.84 10.57 9.5 7 0.14 0.85 25.46 4.21 0.25 9.62 10.39 9.4 7 0.2 0.87 26.63 4.36 0.56 9.23 9.67 7.7 AP1 APS 1APA 7987/44 8682/54 T7F G,."S GD GC Products of Dwarples 2, 3 and 4 are not ccupletely purified to hamogeneity, neverthel contain ASP as rajor ingredient.

Products of Dumples 1, 5, 6 and 7 cc:nsist of purified ASP and are preferred.

(1) antiviral activity against herpes simplex virus (HSV-1) as 50% reduction (IC50) of cytcpathic effect (CPE) (2) degree of sulphation (the mean number of sulphate hemiester gr per mnosaccharide unit) molar ratio of 3, 6-anhydrogalactose per monosaccl=ide unit Table II "in vitro" activity data of fractions of ASP lot 8682/54 separated by 1UPSEC.

MY. range (kDa) > 4700 1550 - 4700 890 - 1550 690 - 890 550 430 340 260 200 150 105 71 46 690 550 430 340 260 200 150 105 71 28 - 46 16 - 28 8.3 16 4.0 - 8.3 4.0 HSV-1 CPE ICSO (bia-lmi) 0.1 0.1 0.1 0.2 0.2 0.2 0.1 0.2 0.3 0.2 0.2 0.4 0.3 0.6 0.4 0.6 0.8 0.6 Table III lijn-vitroll antiviral activity of sulphated polysaccharides on replication of M viruses ASP Dextran sulphate lot 8682/54 M.W.= 8 kDa (Sigma) TC IC50 CPE IC50 SI TC IC50 WE IC50 SI (A91M1) (M9/M1) (99/M1) (99/M1) 742 0.2 3710 775 4.1 189 742 0.1 7420 775 1.9 408 742 7.1 104 775 52 15 virus cell HSV-1 HEp 2 FOV-2 HEp 2 Vaccinia HEp 2 Table IV "In-vitroll antiviral activity of sulphated polysaccharicks on replication of M1k viruses ASP lot 8682/54 Dextran sulphate M.W.= 8 kDa (Sigma) TC IC50 CPE IC50 SI (M9/M1) (A91M1) 775 0.8 969 775 32 24 580 > 200 < 3 virus cell TC IC50 CPE 1C50 SI (Pg/rlul) (M91M1) Resp.Sync. REp 2 Sw-liki F. flEp 2 Influe=a EdK 742 0.1 7420 742 0.2 3710 430 15.4 28 Table V #gin-vitroll antiviral activity of sulphated polysacx:=ides on infecticius viruses ASP Dextran sulphate lot 8682/54 M.W.= 8 kDa (Sigma) IV IC50 SI IV IC50 SI 0191ml) (99/ml) 0.2 3710 4.8 161 < 0.1 > 7420 1.5 516 0.3 2473 52 15 0.025 29680 0.15 5152 virus HSV-1 HSV-2 Semliki F.

Resp. Sync.

Table VI

Inhibitory effect of sulphated polysaccharides on ESV-irrhiced transfonration of =ine balb/3T3 cells CCUIPCA u-d ASP lot 8682/54 Dextran sulphate Pentosan polysulphate TC ICS0 P 1C50 SI (99/ra) G491R11) 600 0.8 > 750 600 1.0 > 600 600 0.9 > 600 Table VII

IlIn-vitroll anti-HIV activity of ASP lot 8682/54 Evaluated parameters 50% inhibitory c=mntration (pg/ml) EIV p24 0.05 HIV IRNA 1.5

Claims (8)

CIAIMS

1. A sulphated polysaccharide which has a common backbone of the agaroidtype, composed of alternating P(1->4)D-galactose and a(l->3)L-galactose repeating units, and which has the following properties after being purified to homogeneity by anion exchange chromatography by application of an increasing sodium chloride gradient, dialysed exhaustively against distilled water, and freezedried:

(a) elementary analysis: 20 - 35 % by weight carbon, 3.2 5.5 % by weight hydrogen, less than 1% by weight nitrogen and more than 8 % by weight sulphur, when calculated as anhydrous compound; (b) molecular weight of up to 10000 kDa as measured by high performance size exclusion chromatography; (c) soluble in water, in aqueous phosphate buffers at pH 1 to 13 and in aqueous solvents containing up to 20% by volume of a water-soluble alcohol but insoluble in benzene, chloroform, ethyl ether and in aqueous-alcoholic solutions containing more than 80% by volume methyl- or ethyl-alcohol and 1 g/1 of sodium chloride; (d) soluble in water in the presence of barium chloride, but, after being hydrolysed for 3 hrs at 120 C in aqueous 2 M hydrochloric acid, it gives a precipitate of barium sulphate upon addition of barium chloride; (e) more than 90 molar % of the total nonosaccharidic units - 46 are galactose and 3,6-anhydrogalactose residues which are unsubstituted or substituted; (f) more than 30 molar % of the total monosaccharidic units consist of 4-0-linked a-L-galactopyranosidic residues which 5 can carry substituents at positions 2, 3 and/or 6; (g) more than 40 molar % of the totalmonosaccharidic units consist of 3-0- linked P-D-galactopyranosidic residues which can carry substituents at positions 2, 4 and/or 6; (h) more than 40 molar % of the total monosaccharidic units consist of 4-0-linked a-L-galactopyranosidic residues which can carry substituents at positions 2, 3 and 6, plus 4-0linked 3,6-anhydro-a-L- galactopyranosidic residues which can carry a substituent at position 2; (i) pyruvate (1-carboxyethylidene) groups, linked as cyclic ketals bridging 0-4 and 0-6 of A-D-galactopyranosidic residues, occur as substituents in less than 10 molar % of the total monosaccharidic units; (j) the molar ratio of nethyl ether group substituents per monosaccharide unit does not exceed 0.3:1; (k) sulphate hemiester groups can be present as substituents at positions 2, 4 and 6 of the P-D-galactopyranosidic residues, at positions 2, 3 and 6 of the a-Lgalactopyranosidic residues and at position 2 of the 3, 6anhydro-a-L-galactopyranosidic residues, and the total degree of sulphation is always greater than 0.6; and (1) the contribution of sulphate hemiester groups at position(s) 2 and 4 to the total degree of sulphation is - 47 always greater than 0.3; and pharmaceutically acceptable salts thereof.

2. A sulphated polysaccharide according to claim 1 having a degree of sulphation of 0.9 + 0.1 and 5 pharmaceutically acceptable salts thereof.

3. A sulphated polysaccharide according to claim 1 or 2 with a ratio MW:MN greater than 2:1 and MP ranging from 100 to 300 kDa and pharmaceutically acceptable salts thereof.

4. A process for isolating a sulphated polysaccharide according to any one of the preceding claims or a pharmaceutically acceptable salt thereof, which process comprises: (1) extracting red algae, residues from the manufacture of agars or unpurified agars with an aqueous solvent; (2) subjecting the extract to refining treatment and elution on a column or to fractional precipitation such as to obtain the said polysaccharide; and (3) if desired, converting the polysaccharide thus obtained into a pharmaceutically acceptable salt thereof.

5. A process according to claim 4 wherein the aqueous solvent is an aqueous solution of phosphate buffer or RaCl and the refining treatment is at least one treatment selected from centrifugation, filtration, precipitation with an organic solvent, dialysis, lyophilization and ultrafiltration.

6. A pharmaceutical composition comprising a 48 pharmaceutically acceptable carrier or diluent and, as active ingredient, a sulphated polysaccharide or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 3.

7. A sulphated polysaccharide or pharmaceutically acceptable salt thereof according to any one of claims 1 to for use as antiviral agent.

8. A sulphated polysaccharide or pharmaceutically acceptable salt thereof according to claim 7 for use in AIDS 10 treatment