Process for obtaining polymers with antiviral activity

Abstract: The present invention consists in a process for obtaining polymers with antiviral activity which is characterised in that in water or in mixtures of water with water-miscible solvents, ruder specific conditions of temperature, time, and pH, molecules carrying carbonyl groups and hydroxyl groups in α position relative to the carbonyl, such as monosaccharides, react with molecules carrying amino and sulfonated groups, such as aliphatic amino-sulfonates; in that after the reaction ends, the polymeric material is purified by conventional processes, such that its molecular weight is within the range from 1,500 to 100,000; in that this polymeric material has antiviral activity and either lacks or presents very low toxicity and anticoagulant activity, and is thus suitable for production of pharmaceutical preparations suitable for use in topical or systemic antiviral therapy in humans or in animals. It is applicable in the pharmaceutical industry.

Description: The present invention concerns a process for obtaining polymers by chemical reaction, under specific temperature, pH, and time conditions, between molecules carrying carbonyl and hydroxyl groups, with the latter in position α relative to the carbonyl, and molecules carrying amino and sulfonated groups, with the resulting polymers having antiviral activity, as a result of which they are suitable for use in the appropriate galenic form for application in human or veterinary medicine. The antiviral activity, in vitro and in vivo in experimental animals, of specific sulfated polysaccharides and other sulfated polymers, such as agar, carrageenans, sulfated dextran, and sulfated polyvinyl are described. It has also been demostrated that the distribution over a carbonated structure of groups sulfated with a specific spatial distribution produces compounds which present antiviral activity; however, to date, the activity of said compounds in vivo in dose and mode of application which can be used in humans has not been demostrated (E. de Clercq, Trends in Pharmacol. Sci., 11, 198, 1990). The fundamental reasons for this lack of activity in vivo in humans could be predicted from the in vivo results obtained in experimental animals. In these, although antiviral activity was manifested undesirable side effects such as increased coagulation time, toxicity, and inadequate bioavailability were also observed. Said effects are fundamentally associated with the anticoagulant properties of oligo/polysaccharides, with the large molecular size of the specimens used, and the presence of toxic contaminants. Because of all this, various lines of investigation have been proposed which may be summarized as a search for sulfated oligomers derived from sulfated polymers which present antiviral activity without manifesting anticoagulant properties and whose bioavailability can be improved based on new galenic techniques. Nevertheless, there is also the possibility that other carrier groups besides sulfate can provide antiviral activity to oligo/polymers when they are added to them in a specific order. Based on this, the study was proposed of which the results are reflected in the present invention by means of which are obtained by chemical reaction, under specific temperature, pH, and time conditions, between molecules carrying amino and sulfonated groups, polymers carrying sulfonated groups which, in addition to having antiviral activity, also present the advantages compared to the polysaccharides and other sulfated polymers that their anticoagulant activity is low or nonexistent, that they have a molecular weight sufficiently low to enable their transfer and absorption by appropriate galenic means, and that they are not toxic, nor are toxic compounds which could remain as contaminants used in obtaining them. U.S.-A-3,660,377 relates to the preparation of a resin having ion exchange capability by a reaction of a reducting sugar such as dextrose with a nitrogen compound selected from the group consisting of ammonia, protein hydrolysates, peptides, glycine, alanine, creptine, phenylalanine, lysine, tyrosine, taurine and mixtures thereof. However, the resins obtained in this document are not water soluble and this non water solubility renders these resins non usable as antiviral products. Furthermore, the compounds disclosed in this document are ion exchange resins and not compounds having antiviral properties. GENERAL DESCRIPTION OF THE PROCESS Step 1 : Preparation of the reaction mixture. In an appropriate container provided with a cover, is prepared the reaction mixture for dissolving the reacting molecules, one carrying carbonyl and hydroxyl groups and which is soluble in water or in water-miscible solvents, for example, a monosaccharide, such as glucose, mannose, galactose, etc., and the other carrying amino and sulfonated groups and which is soluble in water or in water-miscible solvents, for example, an aminosulfonic acid, such as taurine, cysteic acid, etc., in a quantity such that its concentration is between 1 mM and 2 M and the molar ratio between amino groups and carbonyl groups is between 1:10 and 10:1. Said molecules are dissolved in an aqueous medium or a medium made up of mixtures of water-miscible solvent with water, buffered, with the pH adjusted by addition of acid or base such that the pH is between 1.0 and 9.0. Examplary solvents are dimethylsulfoxide, dimethylformamide, ethanol and methanol. Step 2 : Reaction. After the reacting molecules are dissolved in the appropriate medium, one proceeds to the condensation reaction. For this, the reaction mixture is heated to a temperature between 70 and 105 º C, allowing it to react under moderate agitation no longer than the time sufficient for the concentration of the reacting molecule carrying the amino and sulfonated groups to become zero or to remain constant. Step 3 : Purification. The biochemically active polymeric material obtained as a result of the reaction described in the preceding step and which is soluble in the reaction mixture is purified by conventional processes such as gel filtration, ultrafiltration, or dialysis, etc., using appropriate strata or membranes and collecting the fractions whose molecular weight is between 1,500 and 100,000. EXAMPLE 1 Preparation of the reaction mixture: Glucose is dissolved in 1 L of water to a final concentration between 3 and 12 mM in a 2-liter matrass provided with a teflon lid; between 0.1 and 0.3 L of a solution of cysteic acid in water, prepared to a final concentration of 40 to 60 mM and adjusted to a pH between 6.5 and 8.5 by adding, sodium hydroxide is added. This is agitated until the two solutions are completely mixed. Reaction: The reaction mixture is heated to 90 to 100 º C and held at this temperature under gentle agitation for a period of 3 to 5 days. Purification: The solution resulting from the preceding step is concentrated by rotovapor at 40 to 50 º C to 1:5-1:10 of the original volume. It is dialyzed against water in a 3,000- and 10,000-dalton dialysis membrane for 48 hours. It is lyophilized. It is resuspended in water to a final concentration of 10 g L -1 and dialyzed against water in a 3,000- and 10,000-dalton dialysis membrane for 48 hours. It is lyophilized. The product thus obtained is a polymeric material with a molecular weight between 3,000 and 50,000; its elemental composition is C 27 to 47%, H 3 to 7%, S 5 to 11%, N 2 to 6%, and 0 32 to 58%, and it presents the following characteristics with regard to biological activity: Said product is capable, under the conditions described below, of inhibiting the cytopathic effects of the Herpes Simplex 1 virus on He La cells "in vitro" at a concentration in the cellular culture between 10 and 150 µg mL -1 . At said concentrations, said product does not present any toxicity on the He La cells. The conditions for the analysis of the antiviral effect, based on those described in B. Alarcon et al., Antiviral Res., 4, 231, 1984 and M.E. Gonzalez et al., Antimic. Ag. Chemother., 31, 1388, 1987, are: - Cells and virus: The herpes Simplex type 1 virus (KOS) was grown in Vero cells, in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and containing 10,000 IU of penicillin and 50 mg of streptomycin per ml. The concentration of the virus was estimated by plaque test on Vero cells. -Plaque test: The cells were grown in a single layer until confluence in plaques P60 and were incubated with 0.5 ml of 1:10 dilutions in series of the virus in phosphate-buffered saline (PBS) supplemented with 0.5% FCS. After an absorption period of 1 hour at 37 º C, the inoculum was removed and an overlay of 5 ml of DMEM with 0.6% agar and 2% FCS was added. The single layers were incubated at 37 º C in humidified air containing 6% CO 2 for several days until the appearance of cytopathic effects. After these appeared, the overlay was removed, the single layer of cells was precipitated with trichloroacetic acid (TCA) to 5% and the lysis plaques were counted. Estimation of the cytopathic effect: Single layers of HeLa cells grown in DMEM were infected with HSV1 at a low rate of infection (0.0 to 0.4 plaque forming units (PFU)) in the presence of decreasing concentrations of product between 0 and 200 µg/ml. After 48 hours of incubation at 37 º C, the cytopathic effects are studied in the cells, infected or not, by observation under a phase-contrast microscope. Said product, injected intravenously in male SD rats at a dosage of 30 mg kg -1 does not produce changes in coagulation time, conditions under which sodium heparinate totally prevents coagulation. Under said conditions, no exteenal toxic manifestation is observed.

Claims: Sulfonated polymers having antiviral activity obtained by a process comprising the steps of: reacting a reducing sugar (I) with a nitrogen compound (II) in a medium made of water or water-miscible solvent or mixture thereof within a temperature range from 70 to 105 º C and a pH range from 1,0 to 9,0 until the concentration of the compound II remains constant or becomes 0, and purifying the product formed by conventional means characterized in that the reducing sugar (I) is a monosaccharide with an hydroxyl group (-OH) in position α relative to carbonyl group, the nitrogen compound (II) is an aminosulfonic acid, the ratio of the carbonyl groups of the monosaccharide (I) to the amino groups of the nitrogen compound (II) is from 10:1 to 1:10, the concentration of the monosaccharide (I) and the nitrogen compound (II) in the medium is from 1 mM to 2 M, and upon the purifying step, only the products formed with a molecular weight between 1,500 and 100,000 are collected. Sulfonated polymers according to claim 1, characterized in that the monosaccharide (I) is selected from the group consisting of glucose, galactose, mannose, xylose. Sulfonated polymers according to claim 1 or 2, characterized in that the nitrogen compound (II) is selected from the group consisting of taurine, cysteic acid. Products according to anyone of the preceding claims, characterized in that the temperature range is within 90 and 105ºC and must be maintained throughout the duration of the reaction with no variation greater than ± 5 to 10ºC. Products according to anyone of the preceding claims, characterized in that the water-miscible solvents are selected from the group of dimethyl sulfoxide, dimethylformamide, ethanol and methanol. Sulfonated polymers according to anyone of the preceding claims, characterized in that they have the following elemental composition: C:27 to 47% H:3 to 7% S:5 to 11% N:2 to 6%, and 0:32 to 58% and in that their molecular weight is between 3,000 and 50,000. Use of a sulfonated polymer according to anyone of the preceding claims in the manufacture of a medicament for treating viral conditions in human and animals. Use of a compound according to claim 6 in the manufacture of a medicament for inhibiting the cytopathic effects of the Herpes Simplex 1 virus.

Inventors: ALMELA ARMENDARIZ MARIA JESUS, CARRASCO LLAMAS LUIS, GUERRERO BENITO CARMEN URB EL, GUERRERO GOMEZ-PAMO ANTONIO F, LEAL OJEDA JUAN ANTONIO, PIVEL RANIERI JUAN PABLO, ALMELA ARMENDARIZ, MARIA JESUS, CARRASCO LLAMAS, LUIS, GUERRERO BENITO, CARMEN, URB. EL PARQUE - BLOQ. 12, GUERRERO GOMEZ-PAMO, ANTONIO F., LEAL OJEDA, JUAN ANTONIO, PIVEL RANIERI, JUAN PABLO
Applicants: CANTABRIA IND FARMACEUTICA SA, INDUSTRIA FARMACEUTICA CANTABRIA S.A.
Application year: 1991
Main classification: C08B37
IPC codes: C08B37, A61K31, A61P31