DK157120B - Process for producing a pharmaceutical preparation having an antibacterial effect - Google Patents

Description

DK 157120 B

The present invention relates to a process for the preparation of a pharmaceutical composition having antibacterial activity and it is intended to provide the possibility of producing such a preparation as in addition to its antibacterial activity in the gastrointestinal tract.

It is already known that livestock, especially young animals, are exposed to serious infections in the digestive tract, which are mainly due to infections 10 of Escherichia coli and various Salmonella species. The known methods for eliminating such infections include: administration of a therapeutic amount of antibiotics, which is associated with a risk of forming antibiotic resistant bacterial strains; and vaccination of the livestock, leading to high costs because vaccines and vaccinations are costly.

It has now surprisingly been found to be possible to avoid the aforementioned disadvantages and to counteract and / or eliminate such bacterial infections using a pharmaceutical composition having an antibacterial effect prepared by the invention by known pharmaceutical carrier puts an antibacterial System containing lactoperoxidase in an amount of at least 1 mg / kg of product, thiocyanate in an amount of at least 15 ppm 25 expressed as sodium thiocyanate and a solid, water-soluble peroxide donor in an amount of at least 21 ppm expressed as sodium percarbonate and selected from alkali metal peroxates, alkaline earth metal peroxides and urea peroxide.

Thiocyanate should be added in an amount which, in the digestive tract, gives a concentration thereof of at least 0.1 mM, preferably 0.2-0.4 mM and it must not exceed the toxic concentration (10 mM). The amount of solid water-soluble peroxide donor is selected in such a way that an equimolar amount is obtained in the digestive tract.

The concentration of formed is at least 0.1 mM

and preferably 0.2-0.4 mM.

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DK 157120 B

The amount of lactoperoxidase depends on that of the enzyme. activity but based on the assumption that 1 mg containing 50 units (U) should have at least 1 mg of enzyme present per day. liters of digestive fluid. One unit of lactoperoxidase is the amount of lactoperoxidase that forms 1 mg of purogallin from purogallol in 20 seconds. at pH 6.0 and 20 ° C.

Thus, in a solution, the concentration of thiocyanate is at least 0.1 mM, preferably 0.2-0.4 mM, the concentration of peroxide donor is such that the concentration of 10 H2 ° 2 is mMf f ^ t ^ wise 0.2-0.4 mM and the concentration of lactoperoxidase should be 1-2 mg / l (50-100 U / l).

The amount of lactoperoxidase in bovine (unpasteurized) or ultra-filtered whey varies naturally. According to the literature, cow's milk contains lactoperoxidase in an amount of approx. 30 mg / l.

In clinical use, the system is usually administered orally or rectally in the form of a pharmaceutical composition according to the invention containing an antibacterial System in combination with a pharmaceutical carrier.

When in this specification and claims, the antibacterial system prepared by the process of the invention is referred to, any peroxide donor and any thiocyanate are screened, whether the compounds are generally or specifically described; unless the context in which such expressions are used, for example in the examples, does not match the broad meaning. The carrier may be a solid, semi-solid or liquid diluent or a capsule. In the pharmaceutical compositions, the active constituents make up between 0.1 and 99% by weight of the composition, suitably between 2 and 50% by weight in preparations for oral administration.

In the preparation of pharmaceutical compositions containing an antibacterial system in the form of a dosage unit for oral administration, the selected ingredients can be admixed with a solid powdered carrier, e.g. with lactose, 3

DK 157120 B

sucrose, sorbitol, mannitol, starch such as potato starch, corn starch or amylopectin, cellulose derivatives or gelatine, and with a lubricant such as magnesium stearate, calcium stearate or polyethylene glycol wax, on which the mixture squeezes into tablets. If coated tablets are desired, the core prepared in the manner described above may be coated with a polymeric polymer which is dissolved or permeable in the intestinal tract. For this coating, a dye can be added to make it easier to distinguish between tablets with different active ingredients or with different amounts of the active ingredients present.

For the preparation of leaf gelatin capsules (bead-shaped closed capsules) consisting of gelatin and, for example, 15 glycerol, or in the preparation of corresponding closed capsules, the active ingredients are mixed with a vegetable oil. Hard gelatin capsules may contain granules of the active ingredients in combination with a solid powdered carrier such as lactose, sucrose, sorbitol, mannitoline, starch (such as potato starch, corn starch or amylopectin), cellulose derivatives or gelatin.

Dosage units for rectal administration may be prepared in the form of suppositories containing the active compound in a mixture with a neutral fat base, or they may be prepared in the form of rectally deliverable gelatin capsules containing the active ingredients in a mixture with a vegetable oil or paraffin oil.

Liquid preparations for oral administration may take the form of a suspension or suspension, for example, a solution containing 2 to 20% by weight of the active substances described above, the remainder consisting of sugar and a mixture of ethanol, water, glycerol and propylene glycol. If desired, such liquid preparations may contain dyes, flavors, saccharin and carboxymethyl cellulose as a thickener. The peroxide donor is hereby present in the form of microencapsulated particles.

Preparation of pharmaceutical tablets for per- 4

DK 157120 B

oral use can be done as follows:

The incoming solids are ground or sieved to a particular particle size. The binder is homogenized and suspended in a certain amount of solvent. The 5 therapeutic substances and necessary auxiliaries are mixed during a continuous and constant mixing with the binder solution, which may consist of a polymer which is soluble or permeable in the intestinal tract, so that the solution is uniformly distributed in the mass 10 without some of the parts are over-moistened. The wetting of the powder mixture with the binder solution causes the particles to be combined into aggregates and the actual granulation process carried out in such a way that the pulp is pressed through a screen in the form of a stainless steel mesh having a mesh size of approx. 1 mm. The pulp is then placed in thin layers on a tray for drying in a drying cabinet. This drying takes place over 10 hours and a month. is carefully standardized since the moisture content of the granulate is of the utmost importance for the following method of forming the tablets. It is possible to use drying in a fluidized bed. In this case, the pulp is not placed on a tray but is poured into a container with a net bottom. After the drying step, the granulate is sieved to obtain the desired particle size. Under certain circumstances, dust must be removed.

To the finished mixture are added disintegrants, lubricants and anti-adhesives. After this blend, the pulp must have its proper composition for the loss lettering step.

30 The cleaned tablet machine is provided with a designated set of pistons, after which proper adjustment of the loss weight and the degree of compression are tested. The weight of the tablet determines the dose size of each tablet and is calculated from the amount of therapeutic agent in the granules. The degree of compression affects the size of the tablet, its strength and its ability to probe into water. In particular, with regard to the latter two characteristics, the choice of com- 5

DK 157120 B

Pressure pressure (0.5-5 t) something of a balancing act. When the correct adjustment is set, the tablet preparation is started and carried out at a rate of 20,000-200,000 tablets per hour. The tablet pressing requires different time and depends on the portion size.

The tablets are preferably coated with a coating.

This means that they are coated with a layer of a polymer which can be dissolved or is permeable to the intestinal tract.

The tablets are usually packaged by means of 10 machines with an electronic counting means. The different types of packaging consist of glass or plastic jars but also boxes, rudders and special dose-adapted packages.

The daily dose of the active substances varies and depends on the route of administration and the bacterial infection, but as a general rule it is 8-400 mg per day. per day Na thiocyanate and 10-500 mg / day Na percarbonate by administration.

Pharmaceutical compositions containing an antibacterial System prepared by the method of the present invention are intended for use in the treatment of bacterial infections of the gastrointestinal tract produced by species, e.g., Shigella, Salmonella, Escherichia coli, Vibero colera, Pseudomonas species (Pseudomonas pyo -cyanea), Staphylococcus species (Staphylococcus albus, Sta-phylococcus aureus), Streptococcus species (Streptococcus viridans, Streptococcus faecalis and 0-Streptococcus) and Proteus.

The process of the invention will be described in more detail below by means of some examples.

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Example 1 Sodium percarbonate granules containing 10% active oxygen 100 g

Sodium thiocyan 40 g 5 Lactoperoxidase (50 u / mg) 2 g

Polyvinylpyrrolidone 10 g

Lactose 50 g

Magnesium stearate 10 g 10 The lactose peroxydase was mixed with lactose and granulated using a solution of polyvinylpyrrolidone.

Sodium percarbonate was mixed with sodium thiocyanate and the lactoperoxidase granulate. Magnesium stearate was added and granular mixture was tabletted.

Tablets with an average weight of 212 mg were obtained and these were coated with a gastric-resistant layer consisting of "Eudragit" ®.

Example 2

Magnesium peroxide 50 g

Sodium thiocyanate 0f8 g

Lactoperoxidase (50 U / mg) 0.04 g Polyvinylpyrrolidone 5 g

Lactose 100 g

Magnesium stearate 10 g

The three active components were each granulated separately for 3Q using polyvinylpyrrolidone as the granulating agent. Lactose and magnesium stearate were added and the mixture was tableted. The tablets won (1000 pcs) with an average weight of approx. 155 mg was coated with a solution of cellulose acetate phthalate which is 55 resistant to gastric juice, in a solvent mixture of equal parts acetone and isopropanol.

. DK 157120B

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Example 3

Urea 50 g

Sodium thiocyanate 20 g

Lactoperoxidase (50.U / mg) 1 g 5 Lactose 100 g

Stearic acid powder 2 g

The urea peroxide is granulated using a solution of "Eudragit" - S. The lactoperoxidase is mixed with lactose and thiocyanate and the mixture is granulated using "Eudragit" ® S. The two portions of granules are combined and mixed with stearic acid powder and the final mixture was tableted. The tablets had an average weight of approx. 175 mg.

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Example 4 In Sodium Percarbonate 100 g

Mannitol 20 g 20 II Sodium thiocyanate 40 g

Mannitol 20 g III Lactoperoxidase 2 g 25 Mannitol 20 g

Granules were prepared from each of mixtures I, II and III using a solution of "Eudragit" - L. The combined granules were mixed with a suitable flavoring such as sugar, cocoa, micro-encapsulated citrus flavor or mixtures thereof.

A dosing means, e.g., a spoon, provides a dose of approx. 200 mg is added to the package for the granular mixture. The packaging is made of moisture-proof material such as a laminated aluminum foil.

DK 157120 B

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Biological experiments A ^ _Sa | menlignin2_with_ known_technology

From Chemical Abstracts Vol. 83, 1975, No. 16231y (ref. Thesis by B. Bjôrck et al. 9 Appl.

5 'Microbiol. 30 (2 :), 199-204, 1975) it is known that products of oxidation of thiocyanate with lactoperoxidase inhibit gram-positive bacteria that form peroxide. These products were bactericidal for such gram-negative bacteria sorti Pseudomonas ssp. And Escherichia coli under the condition that it is exogenously added to glucose oxidase and glucose. The system produced by the method according to the invention is also effective against gram-positive bacteria, see the following.

In a dissertation by Klebanoff et al, "The Peroxi-15 Dase-Thiocyanate-Hydrogen Peroxide Antimicrobial System" in Biochim.Biophys. Acta 117, 63-72, 1966, it has been described that peroxidase (lactoperoxidase and myeloperoxidase), thiocyanate, and are components of an antimicrobial system effective against a number of organisms. may be endogenously formed (presumably by the metabolism of microorganisms), may be added or may be developed by the addition of various systems which generate hydrogen peroxide, e.g., oxidation of ascorbic acid, oxidation of glucose with glucose oxidase, and oxidation of hypoxanthine with xanthino oxidase.

Two attempts have been made to show that the system produced by the process according to the invention has better effect than that of Klebanoff et al. known.

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Fors0g_A

Medium: A 10% solution of skimmed milk powder containing 0.5 mM SCN was added as sodium thiocyanate and 2 µg / ml lactoperoxidase.

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Test strain: Escherichia coli NCTC 9703.

Antibacterial Test: The skim milk solution was incubated in a water bath at 37 ° C and 0.5 mM H 2 O 2 e - * - * - is added 5 magnesium peroxide. Bottles containing only the medium were incubated as a control. 15 minutes after the addition of the peroxide, the test solutions were acidified by the addition of 0.5M HCl to pH 5.0 to simulate the action of gastric acid. After a further 15 minutes, samples of 10 ml of 10 were taken and inoculated with Escherichia coli NCTC 9703 and further incubated at 37 ° C. The number of surviving bacteria was determined immediately after the inoculation and after 2 hours of incubation.

15 results:

Addition Surviving bacteria, cfu / ml 0 hour 2 hours 20 None, control 8.8 x 10 ~ * 8.8 x 10 ^ 0.5 mM H 2 O 2 8.8 x 1 O5 1.4 x 1 6 mM Mg02 8.8 x 105 3.5 x 102 2 ^ It appears from the result that the number of bacteria was doubled in the control sample while it increased by 59% in the sample with the addition of H2 O2 · In the sample to which Mg02 was added the number of bacteria, on the other hand, has dropped to less than half a thousand.

2Q Klebanoff et al. As mentioned above, we also discuss the addition of an H2 O2 generating System such as a glucose and glucose oxidase system.

Fors0cj_B

Attempts have been made to compare such a System with a system in accordance with the method of the present invention.

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DK 157120 B

Medium: A semi-synthetic medium consisting of 1.0 g NH4 Cl, 1.0 g K2 HPO4, 0.2 g MgSO4.7H2O, 10 g FeSO4.7H2O, 10 mg CaCl2, 3.0 g yeast extract (Difco) and 1000 ml water. also containing 0.25 mM SCN added as sodium thiocyanate, 0.5% glucose and 2 µg / ml lactoperoxidase.

Test strain: Escherichia coli NCTC 9703.

Antibacterial test: The experiment was conducted in an anaerobic tent. The medium was seeded with the test strain and an aqueous solution of sodium percarbonate in an amount corresponding to 0.25 mM urea peroxide in an amount corresponding to 0.25 mM H2 O2 or 0.1 U / ml glucose oxidase was added to various samples. Immediately after inoculation and after 2 hours of incubation at 37 ° C, samples were taken and the number of surviving bacteria determined.

Results: 20 Addition Surviving bacteria, cfu / ml 0 hours 2 hours' Έ n

None, control 1.4x10 2.5x10 0.1 U / ml glucose oxidase 1.4x10 ** 1.4x105 0.25mM Ho00 as Z Z g 1 sodium percarbonate 1.4x10 <3.5x10

None, control 1.5 x 10 ** 2.7 x 10 6 0.1 U / ml glucose oxidase 1.5 x 10 ** 1.7 x 10 4 0.25 mM H2 O2 SOm urea peroxide 1.5 x 10 ** 5.2 x 10 35 11

DK 157120 B

B. Independent £ orse) g

4 tubes containing 10 ml of whey with a content of 21 ppm (0.25 mM) NaSCN were incubated at 30 ° C after inoculation of the whey with Pseudomonas fluorescence EF

5 1998. One test tube was a control sample. Sodium xxer carbonate corresponding to 0.1, 0.2 and 0.3 mM H2 O2, respectively, was added to the respective three other test tubes. The amount of bacteria was determined by grafting and after 2 hours of incubation. Lactoperoxidase is present in an amount of 5 10 pg / ml. The result is shown in Table 1: T a b e 1 1. ς Na percarbonate Number of bacteria / ml corresponding to 0 hours 2 hours mM H 2 O 2 6 6

Control (0.0 mM / 2.1x10 2.1x10 0.1mM 2.1x10e l, 8x105 0.2mM 2, lxl06 2.8x103 0.3mM 2.1χ106 2.6x103

It is clear from Table 1 that a significant improvement in the antibacterial effect is obtained when 0.2 mM H 2 O 2 or more is present. But the addition of 0.1 mM H2 O2 already gives a marked bactericidal effect.

Pseudomonas fluorescence EC 1998 was incubated at 30 ° C in two test tubes 1) and 2), with test tube 1) containing 1 g of commercial milk substitute consisting of 15% ultrafiltrated whey powder, 57% whey powder, 8% fat, 16% animal protein and 4% vitamins and minerals in 10 ml of water, the samples containing 50 µg of lactoperoxidase and 27.5 ppm sodium percarbonate being added and the tube 2) containing the same amount of replacement (50 µg lactoperoxidase) and the tube 2) 21 ppm sodium thiocyanate and 27.5 ppm sodium percarbonate.

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The amount of bacteria in the various test tubes is shown in Table 2 below, with results after 0, 2, 4 and 6 hours.

5 T a b e 1 2

Test 0246 hours of reagent aaaa glass 1 4.5x10 ° 3.5x10 5.3x10 ° 6.0x10 ° reagent 6543 glass 2 4.5x10 ° 3.5x103 5.3x10 5.6x10 15 The table shows that the amount of bacteria in glass tubes 2) after 6 hours, only 1/1000 of the amount in test tubes 1) at the same time, in test tubes 1) no or little growth has taken place while bacteria on the other hand are significantly dead 20 in test tube 2).

The effect of the antibacterial system was determined in vitro. Hereby various Escherichia coli strains were incubated at + 37 ° C in an aqueous solution of 5 g of whey powder / 100 ml, 21 ppm NaSCN and sodium percarbonate corresponding to 25 to 0.25 mM C. The number of cells killed by Escherichia coli after 2 hours is shown in Table 3.

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T a b e 1 3

Escherichia staminé% killed coli 5 ___ 0-139 299/66> 99.98 0-149 853/67> 99.98 0-138 355/67> 99.95 10 0-8 915/66> 99.99 0- 147 949/66> 99.99 0-141 220/65> 99.99 15

Several of the strains of Escherichia coli tested above are resistant to antibiotics. The results thus show that the system is very usable. The high applicability is also related to the system acting against 20 gram-positive bacteria.

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Claims (4)

14 DK 157120 B A process for the preparation of a pharmaceutical composition having antibacterial action, characterized in that an antibacterial System containing lacto-peroxidase containing at least 1 mg / kg of product, 10 mg / kg, is added to a pharmaceutical carrier known per se. cyanate in an amount of at least 15 ppm expressed as sodium thiocyanate, and a solid, water-soluble peroxide donor selected in an amount of at least 21 ppm expressed as sodium percarbonate and selected from alkali metal percarbonates, alkaline earth metal peroxides and urea peroxide. 2. A process according to claim 1, characterized in that the solid, water-soluble peroxide donor is sodium carbonate. 3. A process according to claim 2, characterized in that sodium carbonate in granular form is coated with a protective layer which is soluble in the intestinal tract. Process according to claim 3, characterized in that the protective layer is of cellulose acetate-20 phthalate. 25 30 35