FI74614C - A FRUIT PROCESSING OF A PHARMACEUTICAL PRODUCT WITH ANTIBACTERIC EFFECTS. - Google Patents

Description

1 74614

A process for preparing a pharmaceutical preparation having antibacterial activity

Separated from application 770 708 (patent 62617).

This invention relates to a process for preparing a pharmaceutical preparation having antibacterial activity and comprising an antibacterial system consisting of lactoperoxidase, thiocyanate and a peroxide donor.

It is known that livestock, especially young animals, are susceptible to severe gastrointestinal infections, mainly due to infections caused by E. coli and various Salmonel1 times. Thus, previously known methods for eliminating these infections have involved either administering a therapeutic amount of antibiotics at the risk of developing antibiotic-resistant bacterial species, or vaccinating livestock, resulting in high costs because vaccines and vaccinations are relatively expensive. Among other things, another chemotherapeutically active substance that has been used is e.g. trimetoprimsulfa.

Biochimica et Biophysica Acta 117 (1966) 63-72 discloses an antibacterial system based on 1-lactoperoxidase, thiocyanate and a peroxide donor system formed by a glucose / glucose oxyase combination. From Appi. Microbiol. 3 £ (1975) 199-204 it is further known that the above system has an antibacterial effect in milk against Pseudomonas and other gram-negative bacteria. However, the system has been shown to perform poorly in an anaerobic environment.

It has now surprisingly been found possible to avoid these latter disadvantages and to prevent and / or eliminate such bacterial infections with a pharmaceutical preparation prepared by the method of the present invention, characterized in that at least 16 mycoproxidase is added to a pharmaceutical carrier known per se. ppm of thiocyanate calculated as sodium ocyanate and at least 21 ppr of solid water-soluble peroxide donor which is alkali percarbonate, alkaline earth peroxide or urea peroxide, calculated as trifluorocarbonate.

According to a preferred embodiment of the invention, the molar ratio of peroxide donor to thiocyanate is less than 4, preferably 1-2.

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In a non-preferred embodiment, thiocyanate 160-3500 / preferably 160-1750 ppm / kg nutrient calculated as NaSCN, peroxide donor 210-4000 / preferably 210-2000 ppm / kg nutrient calculated as sodium percarbonate and lactoperoxidase 10-200, preferably 10 -100 mg / kg.

The water-soluble peroxide donor is preferably sodium percarbonate coated with a protective layer which is soluble in the intestine and which layer is preferably cellulose acetate phthalate.

The amount of thiocyanate is chosen so that its concentration in the digestive system is at least 0.1, preferably 0.2-0.4 mM, in which case concentrations of 0.5-1.0 mM may be useful in certain cases, but the concentration must not exceed toxic levels. concentrations (10 mM). The amount of peroxide donor is selected to give an equimolar amount of 11 ^ 2, i. so that the concentration of HjOjJ formed in the digestive organs reaches at least 0.1, preferably 0.2-0.4 mM. The concentration of hydrogen peroxide in each case must be less than 4 times the concentration of thiocyanate. The amount of lactoperoxidase depends on the activity of the enzyme, but assuming that 1 mg contains 50 units (U), at least 1 mg of enzyme per liter of gastric fluid must be present.

The concentration of thiocyanate in the solution is thus at least 0.1 mM, preferably 0.2-0.4 mM, the concentration of the peroxide donor is such that the concentration of hydrogen peroxide is 0.1 mM, preferably 0.2-0.4 mM and the concentration of lactoperoxidase the concentration is 1 mg / l (50 U / l). The latter can also vary but must be 1-2 mg / l (50-100 U / l).

One unit of lactoperoxidase is the amount of lactoperoxidase that forms 1 mg of pyrogallin from pyrogallol in 80 seconds at pH 6.0 and 20 ° C.

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The lactoperoxidase is added in purified form and / or in the form of a lactoperoxidase-containing milk product. The amount of lactoperoxidase in bovine whey (unpasteurized) or ultrafiltered whey can, of course, vary. Bovine milk is reported to contain about 30 mg / l of lactoperoxidase, while the amount of lactoperoxidase in human milk is reported to be 1/30 of bovine milk. Lactoperoxidase is also found in other bovine milk products such as skimmed milk powder, which may just as well be used.

Useful thiocyanate salts include sodium, potassium and ammonium salts. The LD50 value of the thiocyanate is 484 mg / kg body weight when administered intravenously to mice and 764 mg / kg body weight when administered orally to rats.

The pharmaceutical preparation to be prepared by the method of the invention is usually administered to pigs, calves and poultry, as well as to fur farm animals such as minks and foxes. Pets such as cats and dogs can also be cared for.

In clinical use, the pharmaceutical preparations prepared by the method of the invention are usually administered orally or rectally.

Thus, in the method of the invention, the antibacterial system in this context comprises any peroxide donor and / or any thiocyanate, whether the compounds are described in general or in particular, provided that the context in which such terms are used, e.g. in the examples, does not correspond to this broad meaning. The carrier may be a solid, a semi-solid, or a liquid diluent or capsule. The amount of active ingredients is usually 0.1 to 99% by weight of the preparation, preferably 2 to 50% by weight in oral preparations.

In preparing pharmaceutical preparations containing the antibacterial system of the invention in the form of oral dosage units, the selected ingredients may be mixed with a solid powdered carrier such as lactose, sucrose, sorbitol, mannitol, starch such as potato starch, corn starch, corn starch, corn starch, corn starch, corn starch with a friction reducing agent such as magnesium stearate, calcium stearate, polyethylene glycol waxes or the like and compressed into tablets. If coated tablets are desired, the core prepared above may be coated with a solution of a polymer that dissolves or penetrates the intestinal tract. A colorant can be added to this coating to more easily separate tablets containing different active compounds or different amounts of active compounds present.

When preparing soft gelatin capsules (pearl-shaped closed capsules) which are gelatin and, for example, glycerin, or when preparing similar closed capsules, the active compound is mixed with vegetable oil. Hard gelatin capsules may contain granules of the active compound together with a solid powdered carrier such as lactose, sucrose, sorbitol, mannitol, cellulose derivatives of starch (such as potato starch, corn starch or amylopectin) or gelatin.

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Dosage units for rectal administration may be prepared in the form of suppositories containing the active ingredient in admixture with a neutral fatty substance or may be prepared in the form of gelatin supercapsules containing the active ingredient in admixture with vegetable oil or paraffin oil.

Oral liquid preparations may be in the form of syrups or suspensions, e.g., solutions containing from about 0.2% to about 20% by weight of the described active ingredients, the remainder being sugar and a mixture of ethanol, water, glycerol and propylene glycol. If desired, such liquid preparations may contain coloring, flavoring, saccharin and carboxymethylcellulose as a thickening agent. The peroxide donor is then present as micro-enclosed particles.

The preparation of oral pharmaceutical tablets is carried out by the following method:

These solids are ground or screened to a specified particle size. The binder is homogenized and suspended in a certain amount of solvent. The therapeutic compound and the necessary excipients are mixed during continuous and uninterrupted mixing of the binder solution, which may be a polymer that dissolves or penetrates the intestinal fluids and wets, so that the solution is evenly distributed in the mass without wetting any part too much. The amount of solvent is usually adjusted so that the mass acquires a consistency resembling wet snow. Wetting of the powder mixture with the binder solution brings the particles lightly together into aggregates and the actual granulation process is carried out by pressing the mass through a sieve which is a mesh made of stainless steel 7 74614 with a mesh size of about 1 mm. The pulp is then placed in thin layers on a substrate to be dried in a drying chamber, drying takes 10 hours and must be carefully standardized, as the moisture content of the granulate is extremely important for the process below and the properties of the tablets. If possible, drying in a fluidized bed can be used. In this case, the pulp is not placed on a base but poured into a tank with a net bottom.

After the drying step, the granules are screened to obtain the desired particle size. Under certain conditions, the powder must be removed.

Disintegrants, friction reducing and adhesion reducing agents are added to the so-called final mixture. After this mixing, the pulp must have the correct composition for the tableting step.

The cleaned tableting machine is equipped with a specified set of punches and molds, after which the appropriate adjustment for the weight and compression ratio of the tablets is 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 tablets, their strength and their ability to disintegrate in water. As for both of the latter properties in particular, the choice of compression pressure (0.5-5 tons) means finding a kind of balance. Once the correct adjustment is set, tablet production begins, which is performed at a rate of 20,000-200,000 tablets per hour. Compression of the tablets requires different times and depends on the size of the batch.

The tablets are preferably coated. This means that they are coated with a layer of a polymer that dissolves or penetrates the intestinal tract.

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The tablets are usually packed on machines equipped with an electronic calculator. Different types of packaging include glass or plastic cans, but also boxes, tubes, and special portion packs.

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

The pharmaceutical preparations containing the antibacterial system prepared by the method of the invention are intended to be used for the treatment of bacterial infections in the gastrointestinal tract caused by e.g. Chigella, Salmonella, E. coli, Vibero Colera, Pseudomonas (Ps. Pyocyanea), Staphylococcus (staph, albaph , aureus), Streptococcus (Strep, viridans, Strep, faecalis, B-Streptococcus), Proteus.

The invention is described in more detail below by means of examples.

Example 1

Sodium percarbonate granules with 10% active oxygen 100 g

Sodium thiocyanate 40 g

Lactoperoxidase (50 U / mg) 2 g

Polyvinylpyrrolidone 10 g

Lactose 50 g

Magnesium stearate 10 g

Lactoperoxidase was mixed with lactose and granulated using a solution of polyvinylpyrrolidone.

Sodium percarbonate was mixed with sodium thiocyanate and lactoperoxidase granules. Magnesium stearate was added, after which the granular mixture was tableted.

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The resulting tablets with an average weight of 212 mg were coated with a layer of Eudragit® resistant to gastric fluids.

Example 2

Magnesium peroxide 50 g

Sodium thiocyanate 0.8 g

Lactoperoxidase (50 U / mg) 0.04 g

Polyvinylpyrrolidone 5 g

Lactose 100 g

Magnesium stearate 2 g

Each of the three active ingredients was granulated as such using polyvinylpyrrolidone as a granulating agent. Lactose and magnesium stearate were added, after which the mixture was tableted. The obtained tablets (1000 pieces) with an average weight of about 155 mg were coated with a solution of cellulose acetate phthalate resistant to gastric fluids in a mixture of acetone and isopropanol (equal parts).

Example 3

Urea peroxide 50 g

Sodium thiocyanate 20 g

Lactoperoxidase (50 U / mg) 1 g

Lactose 100 g

Stearic acid powder 2 g

Urea peroxide was granulated using Eudragit® S solution. Lactoperoxidase was mixed with lactose and thiocyanate and the mixture was granulated using Eudragit®. Both batches of granules were combined and mixed with stearic acid powder and the final mixture was tableted. The average weight of the tablets was about 175 mg.

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Example 4 I Sodium percarbonate 100 g

Mannitol 20 g II Sodium thiocyanate 40 g

Mannitol 20 g III Lactoperoxidase 2 g

Mannitol 20 g

Granules were prepared from each of I, II and III using Eudragit's solution. The combined granules were mixed with a suitable flavoring agent such as sugar, cocoa, microencapsulated citrus flavoring, or a mixture thereof.

A dosing device, for example a spoon, which gives a dose of about 200 mg, was sealed with the package of the granular mixture. The packaging was made of a moisture-tight material such as laminated aluminum foil.

Biological effect

Four test tubes containing 10 ml of whey with 21 ppm (0.25 mM) sodium thiocyanate were incubated at 30 ° C inoculated with Ps. Fluorescence EF 1998. One of the tubes was a control. Sodium percarbonate was added to the other three test tubes in amounts corresponding to 0.1, 0.2 and 0.3 mM H 2 Cl 2. The number of bacteria was determined at inoculation and 2 hours after inoculation. Lactoperoxidase was present at 5 ug / ml. The results are given in Table 1 below.

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table 1

Sodium percarbonate Number of bacteria / ml in an amount corresponding to _, mM H2O2 0 tUnti 2 hours

Control (0.0 mM) 2.1 * 106 2.1-106 0.1 mM 2.1-106 1.8-105 0.2 mM 2.1-106 2.8-103 0.3 mM 2 , 1-106 2.6-103

As can be seen from this, a strong improvement in antibacterial activity was obtained in the presence of 0.2 mM H 2 O 2 or more. However, even the addition of 0.1 mM gives the bacteria a killing effect.

Ps fluorescence EF 1998 was incubated at + 30 ° C in two test tubes 1) and 2), where tube 1) contained 1 g of commercial milk replacer with 15% ultrafiltered whey powder, 57% whey powder, 8% fat, 16% animal protein and 4% vitamins and minerals in 10 ml of water, containing 50 ug of lactoperoxidases and 27.5 ppm of sodium percarbonate were added thereto, and tube 2) contained the same amount of milk replacer (50 ug of lactoperoxidase) and 21 ppm sodium thiocyanate and 27.5 ppm sodium percarbonate were added.

The number of bacteria in the test tubes is given in Table 2 below, which gives the results after 0, 2, 4 and 6 hours.

Table 2 Sample 0 2 46 hours

Test tube 1 4.5-106 3.5-106 5.3-106 6.0 * 106

Test tube 2 4.5-106 3.5-105 5.3-104 5.6'103 12 7 4 61 4

As can be seen from the table, the number of bacteria in tube 2) after 6 hours is only 1/1000 of the number in tube 1) at the same time that no or only slight growth has occurred in tube 1) but instead bacteria have died in tube 2).

The effect of the antibacterial system was determined in a test tube. In this case, various E. coli strains were incubated at + 37 ° C in an aqueous solution of 5 g whey powder / 100 ml, 21 ppm sodium thiocyanate and sodium percarbonate in an amount corresponding to 0.25 mM 1 O 2 · E. coli '. n mortality after 2 hours is given in Table 3 below.

Table 3 E. coli strain Percent death 0-139 299/66> 99.98 0-149 853/67> 99.98 0-138 355/67> 99.95 0-8 915/66> 99.99 0 -147 949/66> 99.99 0-141 220/65> 99.99

Several of the E. coli strains tested above are resistant to antibiotics. The results thus show a high degree of application of the system.

The high degree of application is also evidenced by the fact that the system also has an effect on gram-positive bacteria.

The antibacterial system according to the invention can also be used in human nutrients as well as in pharmaceutical preparations for animals 13 74 61 4 and humans. Both solid and liquid preparations can be used as pharmaceutical preparations, in which case the antibacterial system is usually added to various pharmaceutical carriers, depending on the type and amount used.

In the test against E. coli 9703, a milk replacer for calves containing 45% by weight of skimmed milk powder (40 mg LP / kg), 22.5% by weight of whey powder, 12.5% by weight of soya powder, 15.0% by weight of % animal fat, 4.5% by weight lactose and 0.5% by weight vitamins. The strain was incubated at + 37 ° C in 10% aqueous milk replacer with 5 μg / ml lactoperoxidase. Two different concentrations of sodium thiocyanate and three different concentrations of 1 ^ 02 ^ 3 in the form of sodium percarbonate were used, as shown in Table 4 below.

Table 4

Concentration in mM Number of bacteria / ml SCN-H2 ° 2 0 hours after 2 hours 0.25 0 2.5-106 7.7-107 0.25 0.25 2.5-106 5.3-104 0 , 50 0 1.8-106 1.0-108 0.50 0.50 1.8-106 1.5-104

As shown in Table 4, the antibacterial system gives a potent bactericidal effect at both concentrations tested, while it has been shown that SCN alone does not produce any effect, but bacterial growth continues in the absence of any peroxide donor.

The peroxide donor may also be an alkali metal persulfate which, in the reaction, yields hydrogen peroxide and sulfate.

Claims (7)

14 7461 4 Patent Requirements A process for the preparation of a pharmaceutical preparation having antibacterial activity and comprising an antibacterial system consisting of 1-lactoperoxidase, a thiocyanate and a peroxide donor, characterized in that at least 1 mg / kg of 1-lactoperoxidase is added to a pharmaceutical carrier known per se, as sodium thiocyanate and at least 21 ppm of a solid, water-soluble peroxide scavenger which is an alkali metal carbonate, niacal or peroxide or urea peroxide, calculated as sodium percarbonate. Process according to Claim 1, characterized in that the solid, water-soluble peroxide generator is sodium percarbonate. Process according to Claim 2, characterized in that the sodium percarbonate in granular form is coated with an enteric-protective protective layer. Process according to Claim 3, characterized in that the protective layer is ethyl acetate phthalate. Process according to Claim 1, characterized in that the molar ratio of peroxide donor to thiocyanate: is less than 4: 1, preferably 1 to 2: 1. Process according to Claim 1, characterized in that 160 to 3500 ppm, preferably 160 to 1750 ppm of thiocyanate are used, 210 to 4000 ppm, preferably 210 to 2000 ppm, of peroxide donor are used, and that 10 to 200 ing / kg are used. preferably 10-100 mg / kg 1 actoperoxidase. is 74 61 4 Process according to Claim 1, characterized in that the peroxy di donor is a magnesium umperoxide.