DE2807089A1 - Lipase stabilisation in pharmaceutical preparations - using globular animal proteins - Google Patents

Abstract

Use of 5-50 pts. wt. of an animal globular protein or protein mixt. (excluding the use of 20-100 ptS. wt. sweet whey powder contg. 8-20% whey protein) for stabilising 1 pt. wt. of non-animal lipase in pharmaceutical prepns. is new. New pharmaceutical prepns. contain lipase of non-animal origin and, per pt. wt. lipase, 5-50 pts. wt. of an animal globular protein or protein mixt. (excluding 20-100 pts. wt. sweet whey powder contg. 8-20% whey protein). Lipase gives improved fat digestion and exerts a favourable effect on impaired liver and bile functions. The lipase is stabilised by the protein. It remains fully active and is immediately bioavailable. Its activity commences immediately after mixing with the digestive juices in the stomach, and is not subject to delay due to disintegration or dissolution times.

Description

Use of proteins for stabilization

of lipase of non-animal origin in medicinal preparations (add on patent ......... (application P 26 38 088.1) In the main patent ..... e ... (application P 26 38 088.1) pharmaceutical preparations are proposed that contain 0.5-5 parts by weight (based on 100 g of the preparation) lipase of non-animal origin and 10 - 50 Contains parts by weight of sweet whey powder with a content of 8-20% whey protein. This improves the stability of the lipase.

It has now been found that lipase of non-animal origin in pharmaceutical Preparations in general with animal globular proteins or protein mixtures can be stabilized.

The invention relates to that characterized by the claim Object.

The following are examples of animal globular proteins into consideration: albumins (ov-albumin, lact-albumin, serum-albumin), globulins, prolamins and Gluteline, Globine Protamine, Histone. Such proteins can be used individually or mixtures of these proteins can also be used.

According to the invention, one part by weight of lipase is used to stabilize 5 to 50 parts by weight, preferably 5 to 25 parts by weight, of protein are used. In particular 10-20 parts by weight of protein are used per part by weight of lipase.

Protein preparations can also be used instead of pure proteins whose main component is an animal globular protein or protein mixture ist0 Such preparations are on the market and are obtained according to the usual extraction methods and possible subsequent enrichment of the globular protein from natural Preserve protein sources (compare Ullmann's Encyclopadie der technischen Chemie, Third edition volume 14, pages 409 = 431) o Examples of such preparations are: Whey protein preparations9 plasma protein preparations, egg white protein preparations.

The amount to be used depends on such preparations contain other ingredients, based solely on the amount of globular protein., The preparations according to the invention can also preferably be 0.1 to 5 parts by weight 1 - 2 parts by weight (based on one part by weight of lipase) contain minerals0 Examples of such minerals are: phosphates, citrates, Chlorides, sulfates, carbonates of sodium, potassium, calcium and magnesium in trace amounts also of iron these Salts can be present individually or as a mixture. If they are in a mixture, the content of the metals in the salt mixture is in Percentages by weight, for example, the following: sodium 2-8, preferably 4-5 percent by weight; Potassium 10-30, preferably 15-20 percent by weight; Calcium 2 - 30, preferably 4-15 percent by weight; Magnesium 0.5-3, preferably 0.8-1.5 percent by weight; Iron 0.01-0.1, preferably 0.02-0.08 percent by weight. Inside the mix come, based on one part by weight of magnesium, for example - - 8, preferably 5 - 7 parts by weight of sodium, 2 - 15, preferably 4 - 8 parts by weight of calcium and 15-30, preferably 18-25, parts by weight of potassium.

The content of acids in percent by weight in the salt mixture is for example the following: phosphate anion (P043-10-50, preferably 15-25 percent by weight; Citrate anion 10-30, preferably 15-25 percent by weight; Chloride anion 10 - 20, preferably 12-16 percent by weight; Sulfate anion 2-8, preferably 3-6 percent by weight; Carbonate anion 2 - 15, preferably 5 - 10 percent by weight, based on 1 g of sulfate anion come within the mixture, for example, 2-5, preferably 2.5-3, parts by weight Chloride anion, 3-10, preferably 4-6 parts by weight of phosphate anion, 3-10, preferably 4-6 parts by weight of citrate anion and 0.5-2, preferably 0.8-1.5, carbonate anion.

According to the invention, a lipase of non-animal origin is stabilized. In particular, these are lipase from plants, for example from castor beans or lipase from microorganisms, for example from fungi such as Rilizopus arrhizus, Rhizopus nigricans, Rhizopus oryzae, Rhizopus delemar, Aspergillus species such as Aspergillus niger, Aspergillus oryzae or Welchia perfringens, Mycotorula lipolytica, Candida cylindracea, Geotrichum candidum.

Based on 100 g weight of the preparation, these generally contain 0.5-5 parts by weight, preferably 0.8-2, in particular 0.8-1.5 parts by weight Lipase.

The lipases are obtained in the manner known for this purpose and is given, for example, in Ullmanns Encyklopadie der technischen Chemie, volume 7 (1956), pages 406-411 or in Bulletin de la Société de Chimie Biologique 1966, 48 No. 6, pages 747-770 and 1968, 50 No. 11, pages 2179-2182.

For example, a fungal lipase is made according to Bulletin de la Société de Chimie Biologique 1966, 48 No. 6, page 747 ff.

obtained as follows. After separation of the mycelium from cultures of The filtrate obtained from fungal spores is concentrated in vacuo at 300.degree. C. and centrifuged and from the upper layer, after dilution, the enzyme is precipitated with acetone at 0 ° C and dried in vacuo. For further purification, it is suspended in water, centrifuged, S04 ions precipitated by adding barium chloride, the enzyme in turn with acetone precipitated and the residue thus obtained after dissolving in distilled water applied via a calcium-loaded exchange column XE6IS at pH 4.7 and through a calcium acetate solution at pH 5.7 eluted. After setting, the eluate becomes to pH 6 with dilute ammonia, the enzyme is precipitated with acetone and the resultant Product by suspension in demineralized water again over a Sephadex column G25 chromatographed.

The eluate thus obtained is freeze-dried (-700 C) Lipase obtained as a powder.

A lipase from Rhizopus species, in particular Rhizopus, is preferably used arrhizus used. According to the procedure given above from Bull.Soc. Chim. Biol. 1966, Page 747 ff., For example, a lipase with an activity of 8,800,000 Units / g (determined by means of an olive oil emulsion). This lipase represents is a pure product and is mainly characterized by the fact that it has two Optima-pH one around pH 7 the other around pH 3.5. She behaves in paper electrophoresis, in polyacrylamide gel electrophoresis as also in chromatography on Sephadex columns like a single protein and approximates in their mode of action in particular on triglycerides of pancreatic lipase. (Further Properties see Bull.Soc. Chim. Biol.

1966, 48 No. 6, pages 756 - 766.) An increase in ectivity of this Lipase can be purified by further purification in a Sephadex column G 100 (inX distilled Water), after which a product is obtained with an activity of 11,000,000 Units / g (see Bull. Soc. Chim. Biol.

1968, 50 No. 11, pages 2179-2182).

The stabilizing agent according to the invention causes in particular one Improving the shelf life of lipase when combined and / or mixed with Auxiliaries and additives used in pharmacy, especially those with surface-active properties such as aluminum hydroxide, aluminum hydroxide gel, aluminum oxide (see H.P. Fiedler, Lexicon of auxiliaries for pharmacy, cosmetics and related Areas, 1971, page 43, 44), Aerosil, magnesium carbonate, aluminum salts (aluminum trisilicates, Aluminum phosphates).

The lipase-containing medicaments according to the invention can furthermore contain other enzymes, in particular proteolytically active fungal enzymes and Amylases. Proteases and amylases, such as them, can preferably be used here for example in enzyme concentrates from Aspergillus species, for example from Aspergillus oryzae or Aspergillus parasitiçus are present.

The enzyme concentrate from Aspergillus oryzae, for example, provides one Multi-enzyme complex with a high proportion of acidic proteinase, which is next to it but contains significant amounts of amylase, cellulase and protopectinases; Further Enzymes with some notable activity are peptidases (K. Lehmann, H. Uhlig, Hoppe Seylers Zeitschrift für Physiologische Chemie 2, 99-104 (1969)), neutral and alkaline proteinases, cellobiase, α-glycosidase and hemicellulases, split the planting gum. The main components - acid proteinase, amylase, cellulase and pectinase - achieve their optimum effect in a pH range of 3.5 - 6 and stability.

This is particularly favorable for pharmaceutical use because it is here the full activity of the enzyme complex can already take effect in the stomach0 The acid proteinase in this enzyme concentrate has, for example, an optimum pH of 3 = 5, the neutral proteinase a pH optimum of 6-7 and the alkaline proteinase a pH-Optxmum of 7-10 (compare H. SprechtD Die Proteinasen from Aspergillus oryza, Natriumwissenschaften 44, 37 = 38 (1957)) o proteases and amylases or the Mixed preparations which, in addition to other enzymes, contain high levels of proteases and amylases are common commercial products and are, for example, according to the procedure included9 in Ulmann's Encyclopadie der technischen Chemie9 Volume 7 (1956) page 407-411 are given (the methods given here are part of the disclosure). In particular, Aspergillus oryzae fungal strains are used.

The protease and / or amylase preparations are produced, for example using the surface method by growing molds (Aspergillus oryzae on wheat bran culture media, extraction of the enzymes formed after drying of Substrate with water or buffer solution, precipitation - if necessary after vacuum concentration (at the lowest possible temperatures) - with solvents such as acetone, ethanol, methanol, Isopropanol and so on or with ammonium sulfate, drying and pulverizing the Precipitation product.

After standardization with common inert substances such as sugar, Glucose, milk sugar, starch, diatomaceous earth and so on, are the products in the Trade brought.

However, it is also possible to manufacture them according to the submerged or Tiofkulturner method in the usual manner using, for example, Aspergillus species, In particular, Aspergillus oryzae possible. It is also possible to use the inventive To add other active ingredients to lipase preparations.

The pharmaceuticals are produced in a known manner, with the known and customary pharmaceutical excipients as well as other customary carriers and diluents can be used.

Such substances, for example, come as such carriers and auxiliaries in question, in the following references as auxiliaries for pharmacy, cosmetics and adjacent areas are recommended or indicated: Ullmanns Encyclopädie der Technische Chemie, Volume 4 (1953), pages 1-39; Journal of Pharmaceutical Sciences, Volume 52 (1963), page 918 and ff., H.v. Czetsch-Lindenwald, auxiliary materials for pharmacy and adjacent areas; Pharm. Ind., No. 2, 1961, page 72 and ff .; Dr. H. P. Fiedler, Lexicon of auxiliaries for pharmacy, cosmetics and related areas Cantor KG. Aulendorf i.

Württ. 1971.

Examples of this are gelatin, natural sugars such as cane sugar or milk sugar, lecithin, pectin, starch (e.g. corn starch), alginic acid, Tylose, talc, lycopodium, silica (for example colloidal), cellulose, cellulose derivatives (For example cellulose ethers, in which the cellulose hydroxyl groups are partially with lower saturated aliphatic alcohols and / or lower saturated aliphatic Oxy alcohols are etherified, for example methyloxypropyl cellulose), stearates, magnesium and calcium salts of fatty acids with 12-22 carbon atoms, especially the saturated ones (e.g. stearates), emulsifiers, oils and fats, especially vegetable (for Example peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat ciceim oil, Sunflower seed oil, cod liver oil, mono-, di- and triglycerides of saturated Fatty acids 0 12H2402 to 0 18H3602 and their mixtures, pharmaceutically acceptable monohydric or polyhydric alcohols and polyglycols such as polyethylene glycols and derivatives thereof, esters of aliphatic saturated or unsaturated fatty acids (2 to 22 carbon atoms, in particular 10 to 18 carbon atoms) with monohydric aliphatic alcohols (1 to 20 carbon atoms) or polyhydric alcohols such as glycols, glycerin, diethylene glycol, Pentaerythritol, sorbitol, mannitol and so on, which may also be etherified can, benzyl benzoate, dioxolanes, glycerol formals, glycolfurol, polyglycol ethers with C1-C12 alcohols, dimethylacetamide, lactamides, lactates, ethyl carbonates, silicones (especially medium viscosity dimethylpolysiloxanes) magnesium carbonate and the like.

Known and customary ones can also be used in the production of the preparations Solubilizers or emulsifiers can be used. As a solubilizer and emulsifiers come for example in question: polyvinylpyrrolidone, Sorbitan fatty acid esters such as sorbitan trioleate, lecithin, acacia, tragacanth, polyoxyethylated Sorbitan monooleate, polyoxyethylated fats, polyoxyethylated olotriglycerides, l. inolized olotriglycerides, polyethylene oxide condensation products of fatty alcohols, Alkyl phenols or fatty acids. Polyoxyethylated here means that the relevant Substances contain polyoxyethylene chains, their degree of polymerization in general between 2 - 40 and in particular between 10-20.

Such polyoxyethylated substances can, for example, by reaction of compounds containing hydroxyl groups (for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid residues can be obtained with ethylene oxide (for example 40 moles of ethylene oxide per mole of glyceride).

Examples of oleopotriglycerides are olive oil, peanut oil, castor oil, Sesame oil, cottonseed oil, corn oil (see also Dr. H.P.

Fiedler "Lexicon of auxiliaries for pharmacy, cosmetics and related Areas 1971, pages 191-195).

In addition, the addition of preservatives, stabilizers, Buffer substances, for example calcium hydrogen phosphate, colloidal aluminum hydroxide, Flavor corrections, antioxidants and complexing agents (for example ethylenediaminetetraacetic acid) and the like possible. If necessary, it can also be used with physiologically compatible Acids or buffers can be adjusted to a certain pII range.

The preparations according to the invention are galenically handled according to the usual standard methods. For example become lipase and further enzymes as well as auxiliaries or carriers by stirring or homogenizing (for example by means of colloid mills, ball mills) well mixed (whereby in general at low temperatures e.g. B. 200 C is worked) and in the usual way Compressed tablets.

The lipase-containing tablets according to the invention are taken orally, preferably as chewable tablets.

The dosage of the tablets is, for example, 1 - 3 times a day 2 tablets, whereby the tablets contain the amounts of enzyme and may contain auxiliary substances.

Indications for the preparations according to the invention are, for example: Flatulence, bloating, pain in the abdomen, tenderness, spastic pain Tablets are well tolerated0 In particular, is an excellent influence spastic pain and often persistent pressure pain.

Digestion is usually done mostly by the pancreas Enzymes are formed that break down the food into a form that can be used by the body. The preparation according to the invention, which is fat-splitting due to its lipase content, its Protease content is protein-splitting and its amylase content is starch-degrading replaced hence a deficit in these enzymes.

The fat digestion, which is improved by the lipase, also has an effect a disturbed liver and biliary function favorable.

The enzyme combination is both acidic and alkaline Area of the digestive tract fully effective and thus enables optimal digestion the food.

The preparation according to the invention has the advantage of immediate availability the fully active enzymes. The effect occurs immediately after the stomach Mixing with the chyme without delay due to disintegration or dissolution times.

Example for a tablet: The tablet consists of the following components: Lipase S 60 from Rhizopus arrhizus 20 mg Enzyme concentrate from Aspergillus 100 mg oryzae With silica gel activated dimethyl 263 mg polysiloxane aluminum hydroxide Ge 1 500 mg bialbumin 270 mg sucrose 515 mg sodium saccharin 2.5 mg sorbitol 135 mg highly disperse silica 44 mg talc 60.5 mg vanillin 2.5 mg caramel flavor 7.5 mg 1920.0 mg - The dimethylpolysiloxane is for example in the German Offenlegungsschrift 2 408 290, page 1, last paragraph and page 2. The ones given above 263 mg of activated dimethylpolysiloxane are composed of 20 mg of pure dimethylpolysiloxane and 13 mg of silica gel. - The silica gel used for activation is, for example, that described in German Offenlegungsschrift 2 408 290, page 3 Silicon dioxide.

Under aluminum hydroxide gel powdery oxides, oxide hydrates, Hydroxides and basic salts of aluminum are understood to be not less than 40 ffi Al2O3 contain (safe Ullmanns Encyclopadie der technischen Chemie, third Edition, Volume 4, Page 545/546 and Volume 13, Page 356). In particular, it acts is an aluminum hydroxide gel, which is produced by the precipitation of aluminum salt solutions (for example sulfate solutions) with ammonium carbonate or sodium carbonate and drying of the filter cake is obtained. (Al 203 content not less than 47%, preferably 50 - 60.) The pH of a 4% (weight / volume) suspension in C02-free water, should not be more than 10.0. Such aluminum hydroxide gels are for example under the designation "Tag" in the trade.

The highly dispersed silica is a silica, obtained by hydrolysis of silicon tetrachloride in a hydrogen flame becomes (Aerosil). For example, such a silica has the following parameters: Surface (m2 / g) according to BET; 50-225, preferably 120-225 or 170-225; middle Size of the primary particles in millimicrons: 12-30, preferably 12-16; Bulk weight (normal goods) in g / liter: approx. 60; Tamped volume (normal goods according to DIN 53 194) in mi / 100 g: 1500-2000, preferably 1700-2000; pH value (according to DIN 53 200) in 4 % aqueous dispersion: 3.5-4.3, preferably 3.6-4.3. - the The tablet is produced, for example, as follows: 1. Granules 1) 25.75 kg of sucrose 50.0 kg of dried aluminum hydroxide gel are sieved (approx. 1.2 mm mesh size) and mixed in a suitable mixer (compulsory mixer, z. B.

Diosna mixer).

= Mixture 2) To mixture I / 1, 2693 kg of activated silica gel are added Dimethylpolysiloxane given and mixed intensively.

= Mixture I / 2 3) Granulate ion 25.75 kg sucrose are at +800 C dissolved in 12.0 kg of demineralized water with stirring. = Solution 1 0.25 kg sodium saccharin are dissolved in 0o85 kg of demineralized water while stirring = solution 2 Die Solution 1 is allowed to cool to room temperature before further processing solution 2 is added to solution 1 with stirring.

= Sugar solution The mixture I / 2 is made with the sugar solution moistened and worked through intensively in a suitable mixer (e.g. Diosna).

Then about -0.95 kg of demineralized water are added. The amount of water, like the mixing time, is to be measured in such a way that it is even Moisturized mass is created.

The moist mass is passed through a granulating machine (3 - 4 mm) and dried at 60 - 650 ° C.

The dry, coarse-grained mass is through a sieve device with 1.2 mm mesh size given.

= Granules for the enzyme chewable tablets Relative humidity of the dried Granules: Maximum 10.

II. Production of the pressed enzyme mass for 80,000 tablets 1) 1.05 kg talc 0.05 kg highly dispersed silica 0.25 kg vanillin 0.75 kg caramel flavor 2.10 kg are sieved (mesh size, 5 mm) and then in a suitable Mixer mixed.

= Mixture II / 1 2) 27.0 kg egg albumin 5.0 kg talc 4.35 kg of highly disperse silica 36.35 kg are mixed in a tumble mixer (e.g. Rotex) mixed and passed through an express sieve (mesh size 1.0 mm).

4 = mixture II / 2 3) 2.0 kg fungal lipase from Rhizopus arrhizus 10.0 kg of enzyme concentrate from Aspergillus oryzae 2.10 kg mixture II / 1 14.10 kg combined with approx. 10 kg mixture II / 2 and passed through an express sieve (mesh size 1.0 mm) given.

Another 5 kg of mixture are added through the express sieve used II / 2 given.

= Mixture 11/3 = 29.10. kg 4) 29.10 kg mixture II / 3 21.35 kg mixture II / 2 (= remainder) 50.45 kg are mixed in a suitable mixture (e.g. Diosna).

= Mixture 11/4 5) 13.75 kg of sorbitol are passed through a sieve with 1.2 mm Mesh size given and dried at 600 C for about 5 hours. Relative humidity of dried sorbitol: 15% (- 5%) 6) 50.45 kg mixture II / 4 13.5 kg sorbitol dried 128.05 kg granules for enzyme chewable tablets 192.00 kg mixed in a suitable mixer (e.g.

Turbula; Turbula barrel approx. 370 liters, 1 hour at 10 revolutions / minute).

= Ready-to-press mass Relative humidity of the ready-to-press mass; Maximum 22nd

Bulk volume: 100 g = approx. 140 ml.

III. Pressing process Tablets are made from the ready-to-press mass a weight of 2.4 g using an eccentric or rotary press.

Claims (2)

Use of proteins to stabilize full lipase non-animal Origin in medicinal preparations (addendum to patent ......... (application P 26 3S 088.1) 4 PATENT CLAIMS 1. Use of 5 50 50 parts by weight of an animal globular protein or protein mixture, with the exception of the use from 20 - 100 parts by weight of sweet molecular powder with a content of 8 - 20% molten protein, to stabilize one part by weight of lipase of non-animal origin i.n pharmaceutical preparations. 2. Pharmaceutical preparations containing lipase of non-animal origin contain, characterized in that they contain one part by weight of lipase 5 to 50 Parts by weight of an animal globular protein or protein mixture (except 20 to 100 parts by weight of sweet molar powder with a content of 8 to 20% whey protein) contain.