For the treatment of fungal infections, and gastric and duodenal ulcers caused by helicobacter pylori

 

Proposed: for the treatment of fungal infections, and gastric and duodenal ulcers caused by Helicobacter pylori. As such, using the hyaluronate complexes of zinc or cobalt hyaluronate, formerly known as antimicrobial agent suitable for the treatment of ulcers and bedsores. The invention expands the Arsenal of tools specified destination. 14 table.

The invention relates to pharmaceutical compositions with antimicrobial activity, containing associates (complexes) of hyaluronic acid, as well as the way they are received.

In addition, the invention relates to the use of these associates (complexes) of hyaluronic acid for the preparation of pharmaceutical compositions with antimicrobial activity and mode of treatment caused by microbes of clinical manifestations.

So, we discovered that associates (complexes) of hyaluronic acid, that is, the hyaluronates zinc and cobalt have antimicrobial activity and are particularly pronounced antibacterial and antifungal activity against aerobic and anaerobic microorganisms.

Associates deprotonated hyaluronic acid with metal ions 3d of the 4th period of the Periodic table, such as Gyalwa, discussed in the description of the Hungarian patent 203372.

Macromolecule, more than 50 years, known as hyaluronic acid and is usually found in the form of sodium salt, first described Meuag and others (J. Biol. Chem. 107, 629 (1934); J. Biol. Chem. 114, 689 (1939)). Meyer singled hyaluronic acid in acidic conditions. However, the carboxyl groups remain disocyanate at physiological pH; and a polysaccharide called sodium hyaluronate, if the cation contained in the environment - sodium. Structure determination was made Weissman and others (J. Am. Chem. Soc. 76, 1753 (1954)). Determination of the cation environment is not always a simple task, therefore, as suggested by Balasz (The Biology of Hyaluronan 1989, John Wiley and Sons, Ciba Foundation Symposium, p. 143), the common name of the polysaccharide (hyaluronic acid and hyaluronate sodium) is hyaluronan.

Hyaluronic acid in the native state is glycosaminoglycanes high viscosity, containing alternating residues1-4 glucosamine; its molecular weight is in the range from 50,000 to several million. Hyaluronic acid is found in the connective tissues of all mammals: it is found in high concentrations in the skin, in the vitreous body of the eye, synovial fluid, in the man who W ultrapure hyaluronic acid is described, for example, in U.S. patent 4141973 and 4303676 and in the European patent description 0144019.

A number of references linked hyaluronic acid in wound healing. In accordance with'toole and Gross (C. R.'toole es Gross. The increasing interest among matrix of the regenerating newt limb: synthesis and removal of hyalouronate prior to differentiation, Dev. Biol. 25, 55-57 (1971)), hyaluronic acid as the main component of the extracellular fluid is responsible for the migration of various cell types. The above authors found through experimental studies during wound healing in the local concentration of hialuronowy acid increases, whereby stimulated cellular responses required for tissue regeneration. In addition to the foregoing, other experimental results also confirm that the hyaluronic acid regulates accompanying wound healing physiological processes by providing optimal conditions for migration and proliferation of all cells involved in tissue regeneration.

Until recently, hyaluronic acid has been used in therapy as sodium salt, mostly in ophthalmology, dermatology, surgery, articular therapy and in cosmetics. The hyaluronic acid salt formed with alkali, alkaline earth, magnievye absorption of drugs (see Belgian patent 904547). Heavy metal salts of hyaluronic acid, including the salts of silver are used as fungicides, whereas Sol gold is used for the treatment of arthritis (see WO 87/05517). However, the known strong harmful effects of compounds of silver and gold, which have an impact on the immune system, blood-forming organs and the nervous system (M. Shinogi. S. Maeizumi. Effect of preinduction of metallothenein on tissue distribution of silver anthepatic lipid peroxidation, Biol. Pharm Bull. (Japan) 16, 372-374 (1993); S. Masson et al: Rev. Med. Interne (France) 13, 225-232 (1992)).

We found to our surprise that the associates of hyaluronic acid, that is, the hyaluronates zinc and cobalt, suitable for acceleration of epithelialization of the surface of the body deficient epithelium, ulcer treatment hip and ulcers caused by bedsores, have significant antimicrobial and especially antibacterial and antifungal properties; in addition to antibacterial properties, they also show activity against the bacteria Helicobacter pylori, until recently, considered to be responsible for the development of gastro - duodenal ulcers.

Believed to be antibacterial effects associates hyaluronic acid are uncertain, as is assumed known up to now accelerate epithelization of wound healing impacts the AI of hyaluronic acid in the treatment of wounds, but the presence of the antimicrobial effect of hyaluronic acid does not follow from these data.

In accordance with our studies have shown that zinc and cobalt complexes of hyaluronic acid are highly active against aerobic or anaerobic bacteria, such as Staphylacoccus aureus, Streptococus sp. , Pseudomonas aeruginosa. Salmonella sp., E. Coli and Helicobacter pylori.

As Staphylacoccus aureus and Pseudomonas aeruginosa are two types of bacteria that cause eye infections, the fact that hyaluronic acid complexes are effective against both types of microorganisms is very important and the use of compounds of hyaluronic acid in ophthalmology seems particularly promising.

Known intraocular use in ophthalmology of sodium hyaluronate. It is widely used since 1980 during cataract surgery, implantation of artificial lenses for the eye and keratoplasty. When injected into the anterior chamber of the eyeball it inhibits compression during operation and protects sensitive tissues and cells. After surgery sodium hyaluronate is washed out of the eye to prevent what is happening in some cases, increased eye pressure.

Sodium hyaluronate is also used in the treatment when infection is another important area of application of this invention.

Due to their antibacterial effect is particularly useful in ophthalmic therapy is the use of zinc and cobalt complexes of hyaluronic acid; mainly seems promising in this area of zinc hyaluronate. In addition to its lubricating effect of zinc hyaluronate can be used successfully as a topical antibacterial agent in ophthalmology; and, thanks to its antibacterial effect by overcoming moderate inflammatory reactions (R. L. Goa and other Hyaluronic Acid, a review of its Pharmacology and Use, Drugs 47, 536-566 (1994)) and to reduce the risk of infection during surgery can also be used in eye surgery as a substitute of sodium hyaluronate, which is widely used for intraocular use since 1980. The role of zinc as an essential for the eyes of the long trace was found (D. A. Newsome, R. J. Rothman. Zinc uptake in vitro by human retinal pigment epithelium. Invest Ophtalmol. Vis. Sci. 28. 795-799 (1987)).

The zinc hyaluronate has a certain advantage in that the possible reduction in the content of zinc during eye operations can be bypassed. Namely, hyaluronic acid as the polyanion can take the cations in the washing after the operation. It cannot be excluded t can be reduced using zinc hyaluronate.

It is known that bacterial infection of the eye is a serious health issue. Most often the infection causes the Staphylococcus group. Less common infections through Pseudomonas aeruginosa, which is also very dangerous to the eyes. Eye infections such bacteria can result in complete blindness within 24-48 hours. These eye infections often caused by contaminated ophthalmic solution (eye drops, solutions for storing contact lenses or similar solutions). Such contaminated ophthalmic solutions are found in the rooms for consultations, clinics and often at home use. Contaminating microbes are most often belong to the group of Staphylococcus; however, less frequent but more dangerous germs Pseudomonas aeruginosa is also capable of rapid growth in ophthalmic solutions (Remingtons Ophthalmic Preservatives, Chapter 86, 1588). Thus, the use of hyaluronic compounds of zinc and cobalt in ophthalmology can be very important due to their strong activity against both types of the above microorganisms.

The antimicrobial effect of the compounds was confirmed by microbiological research. Solutions of zinc hyaluronate and the hyaluronate sodium were used as reference solutions.

The following example 1 describes the preparation of a 0.5% solution of zinc hyaluronate. Unless otherwise indicated, further interest is always specified as the ratio of weight/volume.

Solutions with a concentration of 0.1% or 0.2% were obtained, respectively, by dilution of 0.5% solution of zinc hyaluronate distilled water quality according to the example.

Example 1 Preparation of 100 ml of 0.5% solution of zinc hyaluronate Characteristics of sodium hyaluronate used to obtain the solution as follows: Molecular weight: 1000000 daltons protein: 0,045% 1% UV absorption: A: 0,085 257 nm 1% And: 0,050
280 nm
With-->
Viscosity:=17,25 DL/g
25oWith
The content of hyaluronic acid: 99,3%
The operations described below are carried out under sterile conditions.

After weighing 0.50 g of sodium hyaluronate in a 100 ml flask add 12,50 ml solution of zinc chloride with a concentration of 0.10 M/l, prepared in bidistilled water (used water for injection, pyrogen-free and sterile). Then the solution is topped up with bidistilled water up to 50 ml of This solution swells throughout the night, then it dissolve when shaken and brought to the desired volume with bidistilled water. After Phil>Example 2
Preparation of 100 ml of 0.5% solution of sodium hyaluronate
0.50 g used in example 1 of sodium hyaluronate dissolved in 100 ml of mediterranei water (used water for injection, pyrogen-free and sterile) as described above to obtain a 0.5% solution of sodium hyaluronate.

Solutions with a concentration of 0.1% or 0.2% were obtained, respectively, by dilution of 0.5% solution with distilled water to the above.

The first series of experiments
0.2% solutions of zinc hyaluronate and sodium (obtained by dilution of 0.5% in accordance with examples 1 and 2) were artificially infected with the same number of microorganisms of different test organisms, and the change in the number of microorganisms was measured in time. The number of microorganisms determined at different points in time sowing method on cups. Assessment produce visually on Saraste (contamination, the number of colonies) and by automated counting of bacteria and fungi. Applicable environment: Soybean-casein agar (Caso-agar/Merck/)
Microorganisms that are registered in the National collection of strains of the National Institute of health (National Strain Collection of the National Institute of Public Health (HNCMB)), were used as test icans,
Aspergillus niger,
The initial number of test organisms:l06/ml
The number of parallel experiments: 3
Applied solutions:
1 - aqueous 0.2% solution of zinc hyaluronate;
2 - aqueous 0.2% solution of sodium hyaluronate.

The results are shown in table 1 and 2.

As you can see from the above results, summarized in tables 1 and 2, a significant difference in the antimicrobial effect appears after 24 hours. 0.2% solution of zinc hyaluronate gives reduction (caption) the most part of the studied test organisms by several orders of magnitude, whereas in the solution of sodium hyaluronate number (titer) test organisms did not change substantially.

The second series of experiments
To prove the antimicrobial action of the zinc hyaluronate further experiment continues with another, but not 0.2% concentration and with different duration time of the study. Table 3 shows the effect of 0.1% solutions of zinc hyaluronate and hyaluronate sodium, respectively, three different test organism.

You can see that in the experiments with zinc hyaluronate number of test organisms Staphylococcus and Salmonella is practically reduced to zero within 48 hours, whereas the results of ASC is their impact was observed for Pseudomonas aeruginosa, for which a significant decrease was detected only from the second day.

The third series of experiments
Due to the importance for ophthalmology Pseudomonas aeruginosa investigate the effect of different concentrations of zinc hyaluronate on Pseudomonas aeruginosa in comparison with solutions of sodium hyaluronate, taken in the same concentrations. The results are presented in tables 4 and 5.

Presented in the tables of results revealed a favorable picture, and it is obvious that the solutions of zinc hyaluronate more effective than solutions of sodium hyaluronate and 0.1% solution of zinc hyaluronate effect already with the same activity, which are 0.2% and 0.5% solutions of zinc hyaluronate. On the basis of this result can be obtained an effective composition for external use.

Also investigated the antibacterial effect of hyaluronate cobalt (II). Example 3 describes the preparation of a solution of cobalt hyaluronate.

Example 3
Obtaining a 0.1% solution of cobalt hyaluronate
After weighing 0.10 g of sodium hyaluronate according to example 1 in 100 ml flask add 2,50 ml of a solution of cobalt chloride with a concentration of 0.10 ml/l, prepared in bidistilled water (used water for injection, aerogen and, then it is dissolved by shaking and finally lead to the desired volume with bidistilled water. After filtration through a membrane filter with pore size 0.45 µm get to 0.10% solution of cobalt hyaluronate.

Fourth series of experiments
Microbiological studies carried out in the same manner as described above except that the bacterium E. Coli is used instead of Salmonella sp. The research results shown in table 6.

From the table 6 results it is clear that the solution of cobalt hyaluronate shows similar solutions of zinc hyaluronate activity on both the investigated test microorganisms, while used to compare solutions of sodium hyaluronate, here are my inactivity with regard to deviations due to the applied measurement methods.

The fifth series of experiments
Antibacterial compounds of zinc hyaluronate can be characterized based on the values of minimum inhibitory concentration (MIC) and bactericidal concentrations (CID) in comparison with the corresponding values for sodium hyaluronate.

The MIC and CID determined using the 0.2% solution. Determine hold a well-known p is sootvetstvuyuschimi the dilutions selected test microorganisms. After incubation under favorable temperature and during the corresponding time series of solutions with different concentrations assessed visually and determine the minimum concentration that is inhibitory to the growth of microorganisms (MIC) or causing the death of microorganisms (CID). The research results shown in table 7.

0.2% solution of sodium hyaluronate has no inhibitory effect on the growth of test organisms even at a concentration of 2000 μg/ml, therefore the value of the MIC could not be determined.

From table 7 should be pretty obvious that the MIC values for zinc hyaluronate point at its far better by several orders of magnitude compared with hyaluronate sodium, microbiological action.

The sixth series of experiments
A promising possibility to use in ophthalmology associates of hyaluronic acid requires good microbiological stability of the compositions, in particular their resistance to infection by various microorganisms. Conducted extensive studies to determine the microbiological stability of solutions of hyaluronates zinc and cobalt in comparison with commonly used in ophthalmology with sodium hyaluronate. Also investigated the stability of R the trial is carried out in accordance with article USP Edition (p. 1478), entitled "Efficacy of antimicrobial preservative funds". The principle of this research is the following. The test substance artificially infect different test organisms and observe changes over time in the number kolonialismus units. Use microbes, registered in the National collection of strains of the National Institute of health (HNCMB). Studies carried out with solutions of zinc hyaluronate and sodium of different concentrations in the presence or in the absence of a preservative agent.

The number kolonialismus units (number of microorganisms) of the samples was determined by seeding on cups. An estimate of the number of bacteria or fungi are using automatic count colonies of bacteria or fungi or visually evaluated respectively according to the number of colonies.

The results are shown in tables 8-12. Each result is the average of three parallel experiments expressed as number of colonies/ml

In accordance with the description in USP XXII and General requirements the substance is stable if the number of bacteria introduced in the artificial infection, reduced by 99% within 14 days; if the test organisms are not the days of the study.

The results show:
1. Microbiological stability of 0.1% solution of zinc hyaluronate satisfies the rules USP XXII due to the fact that 4 of the organism (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans) from 5 microorganisms are not only required lethality, but cannot be detected living microorganisms in a sample taken on the 28th day. The behavior of Aspergillus niger is a common and even demonstrates a reduction of two orders of magnitude.

2. Microbiological stability of 0.2% solution of zinc hyaluronate is the same as the stability of 0.1% solution of zinc hyaluronate, so it meets the requirements of USP XXII.

3. In accordance with the expectations of the stability of 0.5% solution of zinc hyaluronate meets the requirements of USP XXII; in fact, the results show that the stability is improved by increasing the concentration of zinc hyaluronate.

4. Stable properties of a solution containing 0.5% hyaluronate, zinc and 0.1% of potassium sorbate, enough, but only slightly (insignificantly) better when used as a preservative potassium sorbate.

5. 0.5% solution of sodium hyaluronate containing 0.1% of potassium sorbate, also meets the requirements of USP XXII for each test microorganism.

Of these financial p, oborot, sodium hyaluronate shows the presence of a preservative even smaller compared to the zinc hyaluronate stability. The preservative is definitely necessary for a solution of sodium hyaluronate, which is otherwise easily infected.

These data are of great importance due to the fact that preservatives often have allergic effects and their elimination makes use of zinc hyaluronate very successful, especially in ophthalmology.

The seventh series of experiments
In table 13, the characteristic of the microbiological stability of cobalt hyaluronate.

As can be seen from the table, the microbiological stability of 0.1% solution of cobalt hyaluronate in the presence of the bacteria Pseudomonas aeruginosa as well as stability of 0.1% solution of zinc hyaluronate.

You can see that after inoculation is achieved almost immediate effect. After the end of the 72-hour test result of the test for sterility showed that the effect of cobalt hyaluronate on the test bacteria in nature is not Statisticheskii, but it has a bactericidal action.

Getting ophthalmological and dermatological compositions intended for external use, described in sleeper, containing 0.1% of zinc hyaluronate.

Sodium hyaluronate described in example 1 is used for eye drops.

After weighing 0,19 g of sodium hyaluronate with quality "purum pulvis" (pure powder) in a 100 ml flask add to 1.87 ml of a solution of zinc chloride with a concentration of 0.10 M/l and of 27.50 ml of sorbitol solution with a concentration of 1.00 M/l (solutions prepared with bidistilled water). Then bidistilled water to bring the volume to 50 ml of This solution swells throughout the night, then it dissolve when shaken and brought to the desired volume with bidistilled water. The final solution is filtered through a membrane filter (pore size 0.45 µm).

Example 5
Dermatological composition containing zinc hyaluronate.

Obtain 100 g of gel containing 0.2% of zinc hyaluronate.

Characteristics of sodium hyaluronate used to obtain gel:
Molecular weight: 800000 Dalton
Protein: 0,094%
1%
UV-absorption: A: 0,320
257 nm
1%
And: 0,240
280 nm
With-->
Viscosity:=14,5 DL/g
25oWith
The content of hyaluronic acid: 95,2%
After dissolving 0.2 g of sodium hyaluronate, in about 30 ml of water, add 5.0 ml of 0.1 molar solution of 1.5 hours, the solution swells of 10 to 12 hours, and then add 1.0 ml of 20% sodium hydroxide solution.

Pre-prepared solution of zinc hyaluronate is filtered through a filter with pore size of 0.45 μm and poured into the gel under stirring, and then distilled water to bring the volume to 100 ml.

Good results on stability studies on cultures of fungi and mold are presented in tables 8-12 and show that zinc hyaluronate has not only antibacterial, but also significant antifungal activity.

The eighth series of experiments
The effectiveness of the compounds by the bacteria Helicobacter pylori is also important. Based on this effect, it is possible to use compounds to treat or prevent the development of gastric or duodenal ulcers, and for the most part to prevent re-infection after treatment.

To study the efficacy of compounds against bacteria Helicobacter pylori.

These studies are carried out using 1.0 wt/vol % solution of zinc hyaluronate, acting on the strains of Helicobacter pylori cultured from biopsy specimens of the stomach of patients suffering from peptic ulcer disease. 1% solution of De-Nol (colloidal bismuth subcitrate - colloid with the RA (nutrient medium) supplemented with 10% bovine blood. Cup, not containing compounds, hyaluronate, used as a control. Seeded cups stand 3 to 5 days at 37oWith a gas containing 5% oxygen and from 7-8% carbon dioxide. The minimum inhibitory concentration (MIC) is defined as the minimum concentration of a substance, completely inhibiting the reproduction of growing in the control Cup bacteria. The MIC values for zinc hyaluronate and De-Nol, measured respectively on the scanned strain, shown in table 14.

Presented in table 14 the results indicate that the effect in vitro of zinc hyaluronate for Helicobacter pylori compared with the effect of De-Nol used in therapy. This fact should be considered because De-Nol is containing bismuth composition having side effects (problems of toxicity, besides its ingestion is unpleasant for the patient) that can not be ignored, taking into account the fact that such side effects can not be expected when using zinc hyaluronate.


Claims

The use of complex hyaluronate, zinc or cobalt as a means to treat fungal infections, and gastric and duodenal ulcers caused Helicoba

 

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