Technical area The present invention refers to the use of positive- ly charged polymers (chitosans) for binding to negatively charged polysaccharides on the surface of mammalian cells. Thus the mammalian cells are protected from in- fection by microbes.

Background to the invention The surface of almost all mammalian cells contain sialic acid-and/or heparan sulfate, which are negatively charged high molecular carbohydrates. Initial attachment of many different microbes to host cells involves adsorp- tion of the microbe to these poly-or oligosaccharides.

After binding to the carbohydrate receptors, the microbes are able to infect the cell by invasion through the cell membranes. This mechanism has been proven to be valid for a large number of viruses (the herpes viruses, dengue virus, HIV, papilloma virus...). Bacteria like Helicobacter pylori, E. Coli, some Strepto-and Staphylococci, and parasites, which cause malaria, attach to and infect mammalian cells by a similar mechanism.

Polymeric cations, like chitosan, which is a linear polysaccharide composed of 1, 4-linked P-D-glucosamine and N-acetyl-p-D-glucosamine residues, bind to the negatively charged polymers on the surface of mammalian cells. Thus, the mammalian cell is protected from infection by hinde- ring the binding of the pathogen to the cell surface.

Chitin is the second most abundant organic polymer in nature and is a linear polysaccharide, built up by N- acetyl-ß-D-glucosamine residues. Chitosan is prepared from chitin by partial or full N-deacetylation by alka- line treatment. By exhaustive alkaline treatment chitosan

with more than 90 % D-glucosamine residues could be obtained. At low pH-values these have a large density of positive charges.

The surfaces of microbes are also covered by negatively charged polymeric substances, and a large number of scientific publications and patents describe the use of chitosan and/or chitosan derivatives, in different formulations, for the removal of microbes (e. g. bacteria) from infected tissue, surfaces and solvents.

These techniques have been applied in the food industry (e. g. antimicrobial sheets for preserving food), medical field (e. g. wound dressings) and for purification of contaminated solutions (e. g. water). The property of chitosan that is utilized in the applications described above is it ; ability to immobilize bacteria by binding to their negatively charged surfaces.

In contrast, the present invention describes a tech- nique where the mammalian cells are protected prior to microbial invasion.

Summary of the invention The present invention has for its main objective the provision of a new technique for the prevention of infec- tion by hindering microbial adhesion to cells. This is achieved by treating the surface of the mammalian cells with a positively charged polymer, which is preferably of natural origin. The polymer should be biodegradable in vivo into nontoxic low molecular fragments. The overall positive charge is dependent on the pH-value of the formulation that is used in the treatment, as well as the density of positive charges on the polymer. Natural polysaccharides, such as celluloses and starches, can be substituted with amino functions by methods known per se, and such substituted natural polysaccharides are also useful in this invention. Although meny positively charged carbohydrate polymers are useful in the invention the further illustration of the invention will be made

with reference to chitosan of varying degrees of N- deacetylation. However, this shall not be construed as limiting the scope of the invention.

Another object of the invention is to use an excess of chitosan in the medium. Thus the microbes are immobi- lized by binding of chitosan-to their negatively charged cell surface and the effect of the treatment is enhanced.

The positively charged polymer used in the present medicament is preferably an aminated polymer, e. g. chi- tosan, having a molecular weight from 2 to 100 kD. As mentioned, by regulating the alkaline treatment of chitin, in the production process, it is possible to manufacture chitosan with varying proportions of D- glucosamine-and N-acetyl-D-glucosamine residues. Chitosan is degraded in vivo by enzymes like lysozyme, chitinase, glycosaminidases etc. The degradation products are non- toxic (mono-, di-and/or oligomers containing D-glucos- amine and N-acetyl-D-glucosamine).

The physical and chemical properties of chitosan are affected by the molecular weight, counter ion and the ratio of D-glucosamine and N-acetyl-D-glucosamine resi- dues; this later is described as the degree of N- deacetylation. Also the distribution (random, regular or block) of N-acetyl groups along the chain is important for the physical and biological properties (e. g. enzy- matic degradation, viscosity etc.) of the polymers. It is preferred in the use of the present invention that chitosan has a degree of N-deacetylation of at least about 50% and preferably between about 70% and about 90%, and a molecular weight ranging from 10 kD to 250 kD. The counter ions are preferably either C1-or OAc-.

At pH-values between 4. 5 and 6. 8, chitosans are positively charged and are able to bind to the negatively charged carbohydrates on the surfaces of mammalian cells.

The preferred pH range for a formulation used in the clinic, with optimal effect for protection against micro-

bial infection is 4. 5-6. At pH-values below 4. 5, a formulation is generally harmful to living tissue in the long run. A particularly preferred pH-range is from about 5.5 to about 6. 8.

The concentration of the positively charged polymer in the medicament involved in this invention can vary within broad ranges, although a practical lower limit is about 0. 005% by weight based on the medicament as a whole. A preferred range is 0. 01 to 2% by weight, such as 0.01 to 1% by weight. The upper limit will practically be set by difficulties of obtaining polymer concentrations higher than 2% by weight.

The medicament to be used to prevent infectious disease by microbial adhesion can be presented in diffe- rent physical. forms, for example as powders, ointments, gels, pastes, suspensions, emulsions, solutions, or films. The formulation to be used is of course, adapted to the natures of the disorder to be treated.

By selecting a suitable formulation, the dermis, corneal surface, oronasopharyngeal and other mucousal membranes (i. e. oral cavity, anogenitial region, and gastro intestinal lumen) can be treated in order to inhibit infections caused by a large number of pathogens.

The invention also involves a method of preventing infections of mammalian cells, the treatment being constituted by administering a pharmaceutically effective amount of a medicament containing a positively charged carbohydrate. Such treatment will result in prevention of infection by the mammalian cells being protected against infection through interaction with the positively charged carbohydrate.

The present invention will in the following be further described in non-limiting examples. In these examples percentages and parts refer to weight unless otherwise indicated. The examples refer to the appended drawings, wherein:

Figs. 1 and 2 illustrate chitosan inhibitory activity against HSV-1 and HSV-2, respectively, as a function of chitosan concentration; Fig. 3 shows the effect of the degree of chitosan deacetylation on HSV-1 and HSV-2 infectivity; and Figs. 4 and 5 show the effect-of chitosan on HSV-1 and HSV-2 infectivity, respectively, at varying pH'es.

As is already mentioned microbes bind to negatively charged carbohydrate polymers on the surface of the mammalian cell e. g. to heparan sulfate and/or sialic acid containing polymers. This binding can be hindered by the adherence of a positively charged polymer prior to in- fection by the microbe. In the following Examples this mechanism has been proved and exemplified by in vitro studies with-chitosan derivatives as the positively charged polymer and herpes simplex virus (HSV-1, oro- labial herpes, and HSV-2 genital herpes) as microbes.

However, this mechanism is general and valid for many different bacteria, virus and parasites. The efficacy of the positively charged polymer binding to the negatively charged cell surface is shown in the Examples to be dependent on the pH-value of the medium (Example 6), the concentration of chitosan (Example 3) and the number of positively charged functional groups in the chitosan molecules (Example 5).

Example 1 Preparation of solutions of chitosan with high degree of N-deacetylation The hydrochloride salt of chitosan (5. 87 g) having a degree of N-deacetylation of 84% and a viscosity of 130 mPas. (Protasan Cl 210, Pronova biopolymers, Oslo, Norway) is dissolved in sterile filtered distilled water (144 g) to form a 3% solution of chitosan, stirring was continued until a clear"84"solution was obtained.

Example 2 The preparation of solutions of chitosans with a low degree of N-acetylation This was done as described in Example 1, with the exception that the hydrochloride salt of chitosan having a degree of N-deacetylation of 65% and a viscosity of 142 mPas (Protasan Cl 211, Pronova biopolymers, Oslo, Norway) was used, resulting in a chitosan"65"solution.

Example 3 Effect of chitosan on HSV-1 infectivity. Differential addition of chitosan and virus to the cells.

Abbreviations: PBS: Phosphate-buffered saline GMK: Green Monkey Kidney AH1 cells HSV-1: Herpes simplex type 1 (orolabial herpes) HSV-2: Herpes simplex type 2 (genital herpes) PFU: Plaque forming unit=infections virus particles RT: Room temperature EMEM: Eagle's minimum essential medium GMK-AH1 cells, HSV-1 strain KOS 321 (Holland et al. 1983) and HSV-2 strain 333 were obtained from the Virological laboratory, Guldhedsgatan 10B, Göteborg, Sweden.

Cells were grown at 37°C in 5% C02 in 6-well plates, area 9 cm2/well (TPP, Switzerland) at a concentration of approximately 400 000 cell/cm2. Washing is performed by flowing of the fluid from the side of each well over the cell surfaces, gently rocking for 30 s, and then emptying the well.

Diluent: 1. Cell culture PBS (137 mM NaCl, 2. 7 mM KC1, 8.1 mM Na2HP04, 1. 5 mM KH2PO4 1 mM CaCl2, 0. 5 mM MgCl2, 0. 1 % D-glucose and approx 1. 1 ml of 3M HCl per 500 ml of PBS to achieve pH 5. 5.

Cells: GMK AH1 cells, 3 days old, 6 well plates

Chitosan solution prepared as in example 1 (chitosan 84 solution) Virus: HSV-1, KOS 321, titer :- 8xl07/ml, dilution to obtain appr. 200 PFU/100 ul 100 pu ouf virus + 10 ml of the diluent, the 100 ul of the previous dilution + 10 ml of the diluent.

HSV-2, to obtain appr.

200 PFU/100 1 100 pl of virus + 10 ml of the diluent, then 100 j1 of the previous dilution + 10 ml of the diluent Procedure: Dilute the chitosan 84 solution in 10 ml tubes as follows 1 ml of stock. + 10 ml of the respec- tive pH diluent: dilution 1: 10 2 ml of previous dilution+8 ml of ibid 1: 50 2 ml previous dilution + 8 ml of the re- spective pH diluent: dilution 1: 250 2 ml ibid. + 8 ml of ibid. 1: 1250 2 ml ibid. + 8 ml of ibid. 1: 6250 0 ml + 8 ml of ibid. control Procedure: the chitosan solution added to cells: i 30 min before addition of the virus, then the cells are washed before addition of virus ii 30 min before addition of the virus, then the cells are not washed before addition of virus iii at the time of virus addition iv 30 min after addition of the virus.

1. Wash the cells with 2 ml of the diluent 2. Add 1 ml per well of the chitosan dilution (4 wells per each dilution) (i) and (ii) 3. Incubate for 30 min at RT 4. Wash the wells (i) once with 2 ml of the diluent, add 1 ml of the diluent

5. To the wells (iv) add 1 ml of the diluent 6. To the well (iii) add 1 ml of the respective chitosan dilution and immediately after appr. 200 PFU of HSV-1 or HSV-2 in 100 pl of the diluent. Add the same virus preparations to wells (i), (ii) and (iv) 7. Incubate for 30 min at RT 8. Wash the cells (i), (ii) and (iii) once with 2 ml of the diluent and then with 2 ml of EMEM, add 3 ml of EMEM, add 3 ml of methylcellulose solution (1 % w/v) 9. Wash the cells (iv) once with 2 ml of the diluent, and then add 1 ml of the chitosan dilution 10. Incubate for 30 h at RT 11. Wash the cells (iv) once with 2 ml of the diluent and then once with 2 ml of EMEM, add 3 ml of methyl- cellulose, solution.

After 2-3 days of incubation, the cells were stained with crystal violet and the viral plaques were counted under light microscope (Trybala 1997) The results are described in appended Fig 1 and show that maximum protection from infection is achieved if the chitosan solution is added 30 min before addition of the virus, and is still present (not washed out) on the cell surface when the virus is added.

Results: As is evident from the diagram of appended Fig 1, maximal protective effect against infection is achieved if the chitosan solution is added to the GMK cells 30 min before the addition of the virus ( ). Washing with buffer solution before addition of the virus has little or no effect which indicates that the electrostatic binding of chitosan to the surface of the mammalian cells is strong (-0-). If the virus and chitosan are added to the cells at the same time the chitosan protects from infection, but not as effective as if chitosan had been added before addition of the virus (-cri-). If the virus

has managed to adhere to the heparan sulfate molecules on the GMK-cell surface then subsequent treatment with chitosan has little or no effect ( Example 4 Example 3 is repeated using HSV-2. The results are shown in appended Fig. 2.

Example 5 Effect of the degree of N-deacetvlation of chitosan on HSV-1 and HSV-2 infectivity.

The experiment was performed by the procedure simi- lar to that described in Example 3. The GMK cells was treated with chitosan solutions from stock solutions which was prepared as described in Example 1 and 2, re- spectively. Chitosan was added to the cells 10-15 min before addition of the virus, and was kept on the cell surfaces during 1 h period of viral adsorption. As is evident from the diagram below, a high number of free amino groups are essential for efficient binding and protection of the GMK cells from infection. Chitosan 85 (-N--G-), is more efficient than chitosan 65 (E1 0-). The results are shown in attended Fia. 3.

Example 6 Effect of chitosan 84 on HSV-1 and HSV-2 infectivity at different pH-values The experiments were essentially performed as in Example 5. The cell culture media (PBS) were adjusted to pH 7. 3, 6. 75, 6. 25 and 5. 75 respectively with 3 M HC1.

The results are described in appended Figures 4 and 5, and show what at lower pH-values a larger number of-NH3+ functional groups are present in each chitosan molecule, which in turn results in a better binding to the nega- tively charged heparan sulfate molecules on cell surface of the green monkey kidney cells.