SE523243C3 - Antimicrobial preparation for treatment and prophylaxis - Google Patents

Description

10 15 25 25 35 35 523 243 2 residues are obtained. At low pH values, these have an extensive positive charge.

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 various formulations for removing microbes (eg bacteria) from infected tissue, surfaces and solvents. . This technology has been applied in the food industry (eg antimicrobial foils to protect food), medical field (eg wound compresses) and for purification of contaminated solutions (eg water). The property of chitosan used in the applications described above is its ability to immobilize bacteria by binding to its negatively charged surfaces.

In contrast, the present invention describes a technique in which mammalian cells are protected from microbial invasion.

SUMMARY OF THE INVENTION The main object of the present invention is to provide new technology for preventing infection by preventing microbial adhesion to cells. This is accomplished 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 to non-toxic low molecular weight fragments. The total positive charge depends on the pH of the formulation used in the treatment, as well as the density of the positive charges on the polymer. Natural polysaccharides can be substituted for ridges, such as celluloses and starches, with amino functions in a manner known per se, and such substituted natural polysaccharides are also useful in the present invention. Although many positively charged carbohydrate polymers are useful in the invention, the further illustration of the invention will be made with reference to the chitosan of varying degrees of N-10 15 20 25 30 35 enn nnn nn nn n nn nnnn nnnn 9 nnnnnu nn nnn nnn nn nn nn rv nn nnnnn anno nnno enn: nn nnnon 1 nn nu nnoonvnnnnnnnnonnnnan n: nn nn: nn -øo men 3 avacetylation. However, this should 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. The microbes are thus immobilized by binding to chitosan - to their negatively charged cell surface and the efficiency of the treatment is improved.

The positively charged polymer used in the present drug is preferably one-aminated polymer, for example chitosan, having a molecular weight of from 2 to 100 kD. As mentioned, by regulating the alkaline treatment of chitin in the production process, it is possible to produce chitosan with varying proportions of D-glucosamine and N-acetyl-D-glucosamine residues. Chitosan is degraded in vivo by enzymes such as lysozyme, chitinase, glucosaminidases, etc. The degradation products are non-toxic (mono-, di- and / or oligomers containing D-glucosamine and N-acetyl-D-glucosamine).

The physical and chemical properties of chitosan are affected by the molecular weight, counterion and ratio of D-glucosamine and N-acetyl-D-glucosamine residues; the latter is described as the degree of N-avacetylation. The distribution (arbitrary, regular or block) of N-acetyl groups along the chain is also important for the physical and biological properties (eg enzymatic degradation, viscosity, etc.) of the polymers. It is preferred in the practice of the present invention that chitosan have a degree of N-avacetylation 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 motions are preferably either Cl 'or OAc'.

At pH values between 4.5 and 6.8, chitosans are positively charged and have the ability to bind to the negatively charged carbohydrates on the surface of mammalian cells. The preferred pH range for a preparation used clinically with optimal effect to protect against microbial infection is 4.5-6. At pH values below 4.5, a preparation is generally harmful to living tissue in the long run. An especially preferred pH range is from about 5.5 to about 6.8.

The concentration of the positively charged polymer in the drug of the present invention can vary within wide limits, although a practical lower limit is about 0.005% by weight based on the drug as a whole. A preferred range is 0.01 to 2% by weight, such as 0.01 to 1% by weight. The upper limit is set practically by difficulties in obtaining polymer concentrations higher than 2% by weight.

The medicament for use in preventing infectious disease by microbial adhesion may be provided in various physical forms, for example as powders, ointments, gels, pastes, suspensions, emulsions, solutions or films. The preparation used is, of course, adapted to the nature of the condition being treated.

By selecting an appropriate preparation, the dermis, corneal surface, oronasopharyngeal and other mucosal membranes (ie in the oral cavity, anogenital area and gastrointestinal lumen) can be treated to inhibit infections caused by a large number of pathogens.

The invention also includes a method of preventing infections of mammalian cells, the treatment comprising administering a pharmaceutically effective amount of a drug containing a positively charged carbohydrate. Such treatment results in preventing infection of the mammalian cells by protecting them from infection by interacting with the positively charged carbohydrate.

The present invention will be further described in the following in non-limiting examples. In these examples, percentages refer to and divide the weight unless otherwise stated. The examples refer to the accompanying drawings, in which: Figures 1 and 2 illustrate inhibitory activity of chitosan against HSV-1 and HSV-2, respectively, as a function of chitosan concentration; 10 l5 20 25 30 35 u - -. »A 523 243 ~ - '| Figure 3 shows the effect of the degree of chitosan avacetylation on HSV-1 and HSV-2 infectivity; and Figures 4 and 5 show the effect of chitosan on HSV-1 and HSV-2 infectivity, respectively, at varying pH.

As already mentioned, microbes bind to negatively charged carbohydrate polymers on the surface of the mammalian cell, for example to polymers containing heparan sulphate and / or sialic acid. This binding can be prevented by adhesion of a positively charged polymer prior to microbial infection. In the following examples, this mechanism has been verified and exemplified by in vitro studies with chitosan derivatives as positively charged polymer and herpes simplex and HSV-2 genital lex virus (HSV-1, orolabial herpes, herpes) as microbes. However, this mechanism is general and applies to many different bacteria, viruses and parasites. The effectiveness of the binding of the positively charged polymer to the negatively charged cell surface is shown in the examples to depend on the pH 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 chitosan solutions with a high degree of N-avacetylation The hydrochloride salt of chitosan (5.87 g) with a degree of N-avacetylation of 84% and a viscosity of 130 mPas (Protasan Cl 210, Pronova Biopolymers, Oslo, Norway ) (144 g) to give a 3% solution of chitosan, and stirring is dissolved in sterile filtered distilled water is maintained until a clear "84" solution is obtained.

Example 2 Preparation of solutions of chitosan with low degree of N-acetylation This was carried out as described in Example 1 except that the hydrochloride salt of chitosan with a degree of N-10 10 15 25 - - «. 65% avacetylation and a viscosity of 142 mPas (Protasan Cl 211, Pronova Biopolymers, Oslo, Norway) were used, resulting in a chitosan "65" solution.

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

Abbreviations: PBS: Phosphate buffered saline GMK: Green Monkey Kidney cells AH1 HSV-1: Herpes simplex type 1 (orolabial herpes) HSV-2: Herpes simplex type 2 (genital herpes) PFU: Plaque-forming unit = infection virus particles RT: Room temperature EMEM Minimum Essential Medium GMK-AH1 cells, (Holland et al. 1983) and HSV-2 strain 333 were obtained from the Virology Laboratory, Guldhedsgatan 10B, Gothenburg, Cells were grown at 37 ° C in 5% CO 2 in 6-well plates, HSV-1 strain KOS 321 Sweden. surface 9cnF / well (TPP, Switzerland) at a concentration of about 400,000 cells / cm 2. The washing is performed by assimilating the 9 9 P medium from the side of each well over the cell surfaces, light weighting for 30 sec and then emptying the well.

Diluent: 1. Cell culture PBS (137 mM NaCl, 2.7 KCl, 8.1 mM Na 2 HPO 4, 1.5 mM KH 2 PO 4, 1 mM CaCl 2, 0.5 mM MgCl 2, 0.1% D-glucose and about 1.1 ml 3M HCl per 500 ml PBS) to a pH of 5.5.

Cells: Chitosan solution prepared as in Example 1 (chitosan 84- GMK AH1 ~ cells, 3 days old, 6 well plates. Solution). 10 l5 20 25. - - v nu 523 243 Virus: Hsv-i, Kos 321, cicer; ~ sx1o7 m1, dilution to give about 200 PFU / 100 ul 100 ul virus + 10 ml diluent, 100 ul of previous dilution + 10 ml diluent.

HSV-2, 333, = titer: ~ 3x107 / ml, dilution to give about 200 PFU / 100 ul 100 ul virus + 10 ml diluent, then 100 ul of previous dilution + 10 ml diluent Procedure: Dilute the chitosan-84 solution in 10 ml tubes as follows 1 ml of base solution + 10 ml of respective pH diluent dilution 1:10 2 ml of previous dilution + 8 ml of ibid 1:50 2 ml of previous dilution + 8 ml of respective pH diluent: dilution 1 : 250 2 ml ibid + 8 ml ibid 1: 1250 2 ml ibid + 8 ml ibid 1: 6250 0 ml + 8 ml ibid control Procedure: the chitosan solution was added to cells: for 30 min before adding virus, the cells are then washed before addition of virus ii 30 min before addition of virus, cells are not washed then before addition of virus iii at the time of virus addition iv 30 min after addition of virus 1.Wash the cells with 2 ml 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 room temperature 4.Wash the cells (i) once with 2 ml of dilution add 1 ml diluent 5.To the wells (iv) add 1 ml diluent 10 15 20 25 30 35 u a n. .- S23 243 8 6.For well dilution and immediately after about 200 PFU of HSV-1 or HSV-2 in 100 μl diluent. Put the same virus preparation (i), 7.Incubate for 30 min at room temperature (i), of the diluent and then with 2 ml EMEM, add 3 ml EMEM, w / v) 9.Wash the cells (iv) a batch with 2 ml diluent and (iii) add 1 ml of the respective chitosan compounds to the wells (ii) and (iv) 8. Wash the cells (ii) and (iii) a batch of 2 ml add 3 ml of methylcellulose solution (1% then add 1 ml of chitosan dilution 10. Incubate for 30 hours at room temperature ll. Wash the cells (iv) a batch with 2 ml diluent and then batch with 2 ml EMEM, add 3 ml methylcellulose solution.

After 2-3 days of incubation, the cells are stained with crystal violet and viral plaques are counted under a light microscope (Trybala 1997).

The results are described in the attached Figure 1 and show that maximum protection from infection is achieved if the chitosan solution is added 30 minutes before the addition of the virus and still acts (if not washed off) on the cell surface when the virus is added.

Results As shown in the diagram in Appendix Figure 1, maximum protective effect against infection is achieved if the chitosan solution is added to the GMK cells 30 minutes before the addition of viri- <--->. the virus has little or no effect, indicating that set Wash with buffer solution before adding the electrostatic bond of chitosan to the surface of (-0-). added to the cells at the same time protects the chitosan from the mammalian cells is strong If the virus and chitosan infection but not as effective as if the chitosan was added before the addition of the virus (- -). If the virus has been able to bind to the heparan sulphate molecules on the GMK cell surface, subsequent treatment with chitosan has little or no effect (-I-).

Example 4 Example 3 is repeated using HSV-2. The results are shown in the attached Figure 2.

Example 5 Effect of the degree of N-avacetylation of chitosan on HSV-1 and HSV-2 infectivity The experiment was performed by a procedure similar to that described in Example 3. The GMK cells were treated with chitosan solutions from base solutions prepared as described in Examples 1 and 2, respectively. The chitosan was added to the cells 10-15 minutes before the addition of the virus and kept on the cell surfaces for 1 hour by viral adsorption. As shown in the diagram, a large number of free amino groups are essential for efficient binding and protection of the GMK cells from infection- (~ I- The results are shown in the attached Figure 3. -0-) are more effective than chitosan 65 tion. Kitosan 85 V-E -O-).

Example 6 Effect of chitosan 84 on HSV-1 and HSV-2 infectivity at different pH values The experiments were performed essentially as in Example 5.

(PBS) 6.75, resp. 5.75 with 3 M HCl. The results are described on the attached Cell Culture Media were adjusted to pH 7.3, 6.25 Figures 4 and 5 and show that at lower pH values a larger number of -NH 3 * functional groups are present in each chitosan molecule, which in turn results in a better binding to the negatively charged heparan sulfate molecules on the cell surface of Green Monkeys kidney cells.

Claims (8)

10 15 20 25 u u ø ~ .o n. U. -. n ~ _ - -. -. . - u o v fl Q- no -. n I v: "° ,, 'u -.. - n u - z z: -g _ _.;;; -_- __ _ .. .. ... --- --- 10 PATENÄKRAV Use of a positively charged carbohydrate polymer for the manufacture of a medicament for preventing infection by protecting mammalian cells with such positively charged polymer, characterized in that said positively charged polymer is chitosan, and that said polymer is present in preparations with a pH ranging from about 4.5 to about 6.8. Use according to claim 1, wherein the chitosan has a degree of N-avacetylation of at least about 50%. Use according to claim 1 or 2, wherein said pH range is from about 4.5 to about 6.0. Use according to claim 1 or 2, wherein said pH range is from about 5.5 to about 6.8. Use according to any one of the preceding claims, wherein said medicament is provided in the form of a gel, a film, a powder, a suspension, a paste or an ointment. Use according to any one of the preceding claims, wherein the medicament contains a suitable pharmaceutically acceptable carrier or excipient. Use according to any one of claims 2 to 5, wherein the chitosan has a degree of N-avacetylation of from about 70 to about 90%. Use according to any one of the preceding claims, wherein the polymer is present in the medicament in an amount of at least about 0.005% by weight.