IE904033A1 - Polyelectrolyte complexes for the treatment and prophylaxis¹of virus diseases, novel polyelectrolyte complexes and a¹process for their preparation - Google Patents

Abstract

Polyelectrolyte complexes consisting of a polyacid and of a polybase are suitable for the treatment and prophylaxis of viral diseases.

Description

Polyelectrolyte complexes for the treatment and prophylaxis of virus diseases, novel polyelectrolyte complexes and a process for their preparation The present invention relates to polyelectrolyte complexes for the treatment and prophylaxis of virus diseases, novel polyelectrolyte complexes and a process for their preparation.

Until now, only a few possibilities were known for the treatment of viral diseases in humans and animals. In particular, until now infection with human immunodeficiency virus (HIV), which has been brought into a close causal relationship with the acquired immune deficiency syndrome AIDS, could only be treated to a limited extent.

In recent investigations, it has been shown that in addition to the preparation azidothymidine (AZT) available on the market until now, which is affected by strong adverse effects, sulfated oligo- and polysaccharides also have an inhibitory action against HIV. This is expressed, inter alia, in vivo in an inhibition of the reverse transcriptase of HIV and inhibition of syncytia formation in infected cultured human T lympho25 cytes. Grounds for an activity in humans also exist.

Xylan polysulfate and its sulfated alkyl ester derivatives in particular have a particularly strong action. With respect to these compounds, a particular problem results in that all sulfated oligo- and polysaccharides have a more or less strong anticoagulatory action. This circumstance alone would not necessarily have to be regarded as a severe adverse effect even on long-term therapy. As, however, the biological half lives of these compounds are in the range of a few hours, in the case of intravenous, intramuscular or subcutaneous injection one or more injections would have to be administered daily in each case. As a result of the reduced coagulability, hemorrhages can in this case occur at the injection site, which in the case of frequent treatment of this type can have serious consequences.

As the oral absorbability of sulfated polysaccharides is not satisfactory in some cases, the need for better, in particular orally absorbable, chemotherapeutics for the control of HIV exists.

Surprisingly, it has now been found that polyelectrolyte complexes formed from at least one polyacid and at least one polybase, one of the components having antiviral activity, exhibit a good antiviral activity and, at the same time, are orally well absorbed. These properties of said polyelectrolytes were not to be expected, in particular since the numerous charges present in the polyacids and polybases rather allow a poor oral absorbability to be expected.

The invention accordingly relates to polyelectrolyte complexes formed from at least one polyacid and at least one polybase, for the treatment and prophylaxis of virus diseases. Preferred polyacids according to the invention are partly or completely sulfated, unsubstituted or substituted oligo- or polysaccharides.

Preferred unsubstituted polysaccharides are described, for example, in DE-OS 3,725,554 and EP 0,270,317. A particularly preferred sulfated polysaccharide is xylan polysulfate.

Preferred substituted polysaccharides according to the invention (as described in German Patent Application P 39 21 761) are polysaccharides which can be linear or branched, consisting of the same or different natural or synthetic monomers, which can also contain one substituted or unsubstituted amino group per monomer unit and whose OH group is on average substituted between 5 and 80% by a group of the formula -X-B-Y, where X is an oxy group or a group of the formula I whose carbon atom is bonded to B, B is a non-aromatic hydrocarbon radical having 2 to 30 carbon atoms, in whose alkyl chain up to 3 methylene units can be replaced by oxy groups, in which up to three C-C double bonds can be present and which can be substituted by up to three Cx-C^-alkyl radicals and Y - if X is an oxy group - can be hydrogen, COOR, OSOaR1 in which R is a physiologically tolerable mono- or divalent cation, or a hydrocarbon radical having up to 20 carbon atoms or a mono- or bisether radical having 3 to 10 carbon atoms, R1 is a physiologically tolerable cation or Y - if X is a radical of the formula I - is hydrogen or COOR in which R has the abovementioned meanings and where the OH groups of the abovementioned polysaccharides are substituted between 10 and 95% by a group of the formula OSO3M, where M is a physiologically tolerable cation and between 0 and 40% of the OH groups are unsubstituted.

The invention furthermore relates to the polyelectrolyte complexes, which were still undescribed until now, consisting of xylan sulfate, its lipophilic derivatives or lipophilic derivatives of dextran sulfate and a polybase or consisting of dextran sulfate and polylysine, a lysine ester having an ester group containing 10-20 carbon atoms, chitosan or poly-(2-N,N-dimethylaminoethyl )-D,L-aspartamide.

The degree of sulfation of the sulfated polysaccharides can extend over a wide range. A degree of sulfation of more than 10% is preferred, particularly preferably of more than 50%, in particular between 90 and 100%.

The mean molecular weight of the unsubstituted and substituted polysaccharides should be between 500 and 80,000 D, preferably between 1000 and 40,000 D, in particular between 4000 and 15,000 D.

The polyelectrolyte complexes according to the invention 10 can be formed with the aid of different polybases. Inter alia, proteins can be employed as polybases, such as, for example, hemoglobin. Furthermore, for example, polylysine, lysine alkyl esters, chitosan poly-(2-N,N-dimethylaminoethyl)-D,L-aspartamide, amino acids, polyamino acids, aminated oligo- and polysaccharides (for example aminated dextrans), poly-[a,^-(spermidinyl)-D,Laspartamide], gelatin and derivatives (for example Polygeline*, Behringwerke AG, Marburg), guaternary ammonium compounds (for example Luviquat*, BASF AG, Ludwigshafen or Mirapol A15*, Miranal Chem. Co., Inc. South Brunswick, New Jersey), C12-alkylsternamine, a bis(N-propylamino)polyethylene glycol and aminated polyethylene glycol derivatives are suitable. Furthermore, covalent compounds formed from polymers containing OH groups (for example dextran) and nitrogen crown ethers and their metal complexes can be employed as polybases.

In the case of polylysine, the polybase should preferably have a molecular weight of 1000 to 200,000, particularly preferably of 2000 to 50,000.

An example of a particularly suitable polyelectrolyte complex according to the invention is a complex of xylan polysulfate and hemoglobin.

It is furthermore possible to optimize the polyelectrolyte complexes according to the invention by variation of the substituents of the polyacids and polybases with respect to their absorbability. Thus, for example, it is possible to provide the polyacids with different substituents, such as already explained above. The polybases can also be modified by substitution reactions. Particularly suitable substituents for the polybases are of hydrophobizing nature, such as, for example, alkyl chains, in particular branched or unbranched alkyl chains having 10-20 carbon atoms, such as, for example, lysine octadecyl ester or cetyltrimethylammonium bromide. Other suitable substituents are, for example, steroid radicals, in particular cholesterol bonded via the alcohol group or the cholesteryl esters of succinic acid or analogous steroids.

Moreover, it is possible to prepare made-to-measure polyelectrolyte complexes by the reaction of a polyacid or a mixture of polyacids with a polybase or a mixture of polybases .

The invention furthermore relates to a process for the preparation of polyelectrolyte complexes which comprises initially introducing either a) the polyacid or b) the polybase, preferably in aqueous solution, and adding dropwise a) the polybase or b) the polyacid at a suitable temperature and at a suitable pH, preferably in aqueous solution.

The respective concentration of the polyacid and the polybase in the aqueous solution, the temperature and the pH can be varied within wide ranges; the optimum parameters in each case can be determined empirically for each combination. Representative parameters are given in the exemplary embodiments.

The proportions in which the polyacids and polybases are brought to reaction with one another can extend over a wide range. A weight ratio of 1:10 to 10:1 is preferred, particularly preferably of 1:3 to 3:1, in particular of about 1:1. In many cases, an approximately equimolar ratio of the two components may also be advantageous.

The invention furthermore relates to the use of poly5 electrolyte complexes for the treatment or prophylaxis of virus diseases, and pharmaceuticals containing polyelectrolyte complexes.

For the treatment or for the prophylaxis of diseases which are caused by viruses, the polyelectrolyte com10 plexes according to the invention can be administered in different ways. For example, they can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, as a continuous drop infusion or orally. Oral administration is preferred.

The pharmaceuticals according to the invention are prepared by bringing at least one polyelectrolyte complex, if desired with other additives and/or auxiliaries, into a suitable presentation form. The additives or auxiliaries originate from the group comprising the excipients, preservatives and other customary auxiliaries, for example, for oral presentation forms auxiliaries such as starch, for example potato, corn or wheat starch, cellulose or its derivatives, in particular microcrystalline cellulose, silica, various sugars such as lactose, magnesium carbonate and/or calcium phosphates can be used. Furthermore, it may be advantageous to add auxiliaries which improve the tolerability of the medicament, such as, for example, mucus-forming agents and resins, to the oral presentation forms. For better tolerability, the medicament can also be administered in the form of gastric juice-insoluble capsules. Moreover, it may be advantageous to add a delayed-release agent to the presentation form, if desired in the form of permeable membranes, such as, for example, those based on cellulose or polystyrene, or based on ion exchangers.

The dosage of the polyelectrolyte complexes according to the invention to be used is dependent on various factors such as the presentation form of the medicament and the condition and weight of the patient. However, a daily dose of about 3000 mg of polyelectrolyte complex should only be exceeded for a short time. About 200 to 1500 mg of polyelectrolyte complex are preferred as a daily dose. Moreover, for individual polyelectrolyte complexes it is expedient to test both composition and dosage in experi10 ments.

The polyelectrolyte complexes according to the invention have an action against viruses, in particular even on oral administration. They can be used both for the treatment and for the prophylaxis of virus diseases.

The polyelectrolyte complexes according to the invention have particular importance for the treatment of retroviruses, in particular in the control of various types of HIV.

The present invention will be illustrated in more detail by the following exemplary embodiments.

Examples 1. Preparation of the polyelectrolyte complex formed from xylan polysulfate and poly-L-lysine: A 0.1% aqueous solution of 29.6 mmol of monomer units of poly-L-lysine (mean molecular weight 3500 D) is added to a 0.1% solution of 5 g of xylan polysulfate (manufacturer: BENE Chemie, Munich), in the sodium salt form (also applies to all following mentions of xylan polysulfate) corresponding to 14.8 mmol of monomer units, in water and the mixture is stirred. The precipitate is filtered off through a glass frit, washed with H20 and lyophilized. The product is characterized by elemental analysis and pH determination of an aqueous solution.

Yield: 8.2 g. 2. a) Preparation of poly-(2-Ν,Ν-dimethylaminoethyl)-D,Laspartamide (PDAA) 22.28 g of polysuccinimide (mean molecular weight about 5 4700, determined by means of viscometry) are dissolved in ml of DMF, then 4.59 g of hydroxypyridine and 45.94 ml of 2-dimethylaminoethylamine are added with ice-cooling and stirring. After stirring for 15 h, the mixture is precipitated by pouring into anhydrous acetone, and the precipitate is filtered off through a glass frit and the residue is washed with acetone until neutral. The product is dried over P2O5 in a vacuum desiccator. Characterization is carried out by means of elemental analysis, NMR and IR spectroscopy; the molecular weight is determined by viscometry. Yield: 51 g. b) Preparation of the polyelectrolyte complex formed from xylan polysulfate and poly-(2-N,N-dimethylaminoethyl )-D,L-aspartamide (PDAA) A 0.1% aqueous solution of xylan polysulfate and PDAA in 20 each case are added together in equal amounts (in each case 1 1) .

After further stirring (1 h), the precipitate is filtered off through a glass frit, washed with H2O and lyophilized. Yield: 1.8 g. Characterization by means of elemental analysis and pH determination of an aqueous solution. 3. Preparation of the polyelectrolyte complex formed from xylan polysulfate and chitosan g of xylan polysulfate (14.8 mmol of monomer units) are dissolved in the minimum amount of water necessary and poured through an ion exchanger in the H+ form. A 0.1% aqueous solution of chitosan (Protan) is then added to the acid eluate until it is neutral. The polyelectrolyte complex precipitates during the course of this. After stirring for 1 h, the product is worked up and characterized as described in Example 2. Yield: 8.7 g. 4. Preparation of the polyelectrolyte complex from dextran sulfate and chitosan mmol of monomer units of dextran sulfate (prepared according to German Patent Application P 39 21 769.2) are dissolved in the minimum amount of water necessary and poured through an ion exchanger in the H+ form. A 0.1% aqueous solution of chitosan (manufacturer: Protan & Fagertun, Drammen, Norway) is then added to the acid eluate until it is neutral. The polyelectrolyte complex precipitates during the course of this. After stirring for 1 h, the product is worked up and characterized as described in Example 2. Yield: 8.2 g.

. Preparation of the polyelectrolyte complex formed from partly palmitoyl-substituted xylan polysulfate and poly-L-lysine The partly palmitoyl-substituted xylan polysulfate is 20 prepared according to German Patent Application P 39 21 761.2. A one percent solution of poly-L-lysine hydrobromide, molecular weight 3900 (manufacturer: Sigma Chemie, Munich) in water, adjusted to pH 4 with HCl, is initially added at room temperature. A one percent solution of the xylan polysulfate derivative in water which has also been adjusted to pH 4 with HCl is added dropwise to this with stirring. The polyelectrolyte complex precipitates during the course of this, and the precipitate is separated off by centrifugation and lyophilized. The dry product is washed with water and centrifuged again. The precipitate is then separated off and lyophilized again. The product is characterized as described in Example 2. Yield: about 50% of theory. 6.a) Preparation of L-lysine octadecyl ester 333 mmol of octadecanol (manufacturer: Riedel-de Haen, Seelze) are melted at 100 °C and 164 mmol of L-lysine hydrochloride (manufacturer: Sigma, Munich) are added. 272 mmol of methanesulfonic acid are then added dropwise to the resulting suspension in the course of 10 minutes. The melt is stirred at 115°C for 2 h, then poured into 1 1 of vigorously stirred diethyl ether at -70*C (under N2). After warming to room temperature, the mixture is stirred for 1 hour, and the precipitate is separated off by means of a G4 glass frit and washed several times with diethyl ether. The residue is added with stirring to a mixture of 600 ml of H20 and 100 ml of CH30H. It is stirred at 50°C for 10 minutes and then cooled to room temperature. About 150 ml of 2 M NaOH are added dropwise over the course of 20 minutes until pH 12 is attained. The resulting thick magma is cooled to 10 °C and extracted once with 500 ml of diethyl ether and then twice with 200 ml of diethyl ether each time. The ether extracts are dried over Na2SO4 and cooled to -10°C. HCl gas is passed in until the extracts are saturated. The precipitate is separated off by means of a G2 glass frit, washed several times with diethyl ether and dried in vacuo. Yield: 75 g (95% of theory). Characterization by means of ^H-NMR and thin layer chromatography in CH3OH: 25% ammonia 9:1 on silica gel, Rf = 0.4. b) Preparation of the polyelectrolyte complex from xylan polysulfate and L-lysine octadecyl ester The procedure is analogous to Example 5, the poly-L30 lysine being replaced by L-lysine octadecyl ester.

Activity test Activity of polyelectrolyte complexes of sulfated polysaccharides against retrovirus infections in the mouse.

As no suitable infection model for HIV infection of the human exists in laboratory animals, other retroviruses have to be resorted to for the testing of chemotherapeuticals in infections. In the present case, infection of the mouse with the Friend leukemia virus is selected.

For this purpose, normal laboratory mice (NMRI = Naval Medical Research Institute) are infected by intravenous infection with mouse serum containing Friend leukemia virus. As a symptom of the infection, a distinct enlarge10 ment of spleen and liver develops in control animals within 2 weeks. The treatment is carried out over 10 days, starting 48 hours after infection or over 16 days, starting 4 days before infection. On the 14th experimental day, the animals are sacrificed by dislocation of the cervical vertebra and opened. The spleen is taken out and weighed. The spleen weight of the treated animals is related to that of the untreated infection control as a measurement of the therapeutic activity.

While the spleen weighs about 1% of the body weight or less in uninfected adult laboratory mice (20-24 g body weight), the spleen in infected animals attains about 10% of the body weight at the end of the experiment.

Table 1 shows the results of the activity test in the Friend leukemia virus infection in the mouse on intra25 peritoneal or oral administration of the compounds according to the invention. A group of mice which remain untreated serves as a control.

The effect of treatment is detectable by the reduction in the enlargement of the spleen.

On oral administration of 2000 mg/kg ten times, the compounds investigated prove to be more active than xylan polysulfate 6000, the activity of which is known against HIV in vitro and in the retroviral mouse model.

(Biesert et al. Inhibition of HIV and virus replication by sulphates polyxylan: HOE/BAY 946, a new antiviral compound. AIDS 2, 449 - 457 (1988). Baba et al. Pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-HIV agent in vitro. Antiviral Res. 9, 335 - 343 (1988)).

The reduction in the spleen weight compared to the infected control is statistically significant (p< 0.05 student's T test).

Activity of polyelectrolyte complexes of sulfated polysaccharides against retrovirus infections in the mouse Table 1 Preparation Dosage (mg/kg) relevant spleen n P Control weight 8.66 ± (%) 3.16 (10) 1.00 15 1. Xylan polysulfate 10 x 2000 p.o. 6.36 ± 2.48 (10) 0.04 2. Xylan polysulfate-PDAA polyelectrolyte complex according to Example 2 10 x 2000 p.o. .35 + 1.38 (10) 0.04 3. Xylan polysulfate-polyL-lysine poly25 electrolyte complex according to Example 1 10 x 2000 p.o. .24 ± 1.63 (10) 0.04 Continuation of Table 1 Preparation Dosage (mg/kg) . Xylan polysulfate-chitosan polyelectrolyte complex according to Example 3 16 x 1000 p.o. x 2000 p.o. relevant spleen n weight (%) 6.37 ± 3.23 5.90 ± 1.51 (5) 0.04 (5) 0.005 The result of treatment was detectable by the reduction in the enlargement of the spleen.

On oral administration of 2000 mg/kg ten times, the compounds investigated proved more active than xylan polysulfate 6000, the activity of which is known against HIV in vitro (1/2) and in the retroviral mouse model (1). The reduction in the weight of the spleen compared to the infected control was statistically significant (p< 0.05 student's T test).

Claims (17)

1. Patent claims:

1. A polyelectrolyte complex formed from at least one polyacid and at least one polybase for the treatment and prophylaxis of virus diseases.

2. The polyelectrolyte complex as claimed in claim 1, wherein the polyacid is a partly or completely sulfated, unsubstituted or substituted oligo- or polysaccharide.

3. The polyelectrolyte complex as claimed in claim 1 or 2, wherein the polyacid is an oligo- or polysaccharide whose OH groups are partly etherified or esterified.

4. The polyelectrolyte complex as claimed in one or more of claims 1 to 3, wherein the polybase is a protein.

5. The polyelectrolyte complex as claimed in one or more of claims 1 to 4, wherein the polybase is hydrophobized by a derivatization reaction.

6. The polyelectrolyte complex as claimed in one or more of claims 1 to 5, wherein the polybase is polylysine, a lysine ester, chitosan or poly-(2-N,N-dimethylaminoethyl)-D,L-aspartamide.

7. . A polyelectrolyte complex which consists of xylan sulfate or its lipophilic derivatives and a polybase.

8. A polyelectrolyte complex which consists of dextran sulfate or its lipophilic derivatives and a polybase.

9. A polyelectrolyte complex which consists of dextran sulfate and polylysine, a lysine ester, chitosan or poly(2-N,N-dimethylaminoethyl)-D,L-aspartamide.

10. A process for the preparation of polyelectrolyte complexes as claimed in one or more of claims 1 to 9, which comprises initially introducing either a) the 11.

11. 12.

12. 13.

13.

14. . 14 .

15. 15.

16. 16.

17. 17. polyacid or b) the polybase in aqueous solution and adding dropwise a) the polybase or b) the polyacid at a suitable temperature and at a suitable pH. The use of a polyelectrolyte complex as claimed in one or more of claims 1 to 9 for the treatment or prophylaxis of virus diseases. A pharmaceutical containing a polyelectrolyte complex as claimed in one or more of claims 1 to 9, if desired in addition to customary auxiliaries and/or excipients. A polyelectrolyte complex according to claim 1, substantially as hereinbefore described and exemplified. A process for the preparation of a polyelectrolyte complex according to claim 1, substantially as hereinbefore described and exemplified. A polyelectrolyte complex according to claim 1, whenever prepared by a process claimed in a preceding claim. Use according to claim 11, substantially as hereinbefore described . A pharmaceutical according to claim 12, substantially as hereinbefore described.