DE3147727A1 - Wound-healing composition containing collagen from basement membranes - Google Patents

Abstract

The invention relates to a wound-healing composition which contains basement membrane collagen (type IV) and, where appropriate, customary vehicles and ancillary substances. The wound-healing composition according to the invention is able to influence cell regeneration and increase the rate of wound healing.

Description

Wound healing agents containing collagen from basement membranes

The present invention relates to a wound healing agent which contains basement membrane collagen and optionally contains customary carriers and auxiliaries.

So far, one has, for example, containing prostaglandins to promote wound healing Means proposed (see US-A-4061670). In GB-A-1565340 were enzymatic degraded human and animal tissues for wound healing are described. Interstitial According to DE-OS 2462222, collagens are used to heal connective tissue diseases will.

It has now been found that basement membrane collagen becomes a particular leads to rapid wound healing, with the scars healing particularly nicely.

The present invention provides a highly effective new agent provided for the healing of wounds of various types.

The agent is also particularly suitable for healing burn wounds and from sunburn.

It was surprisingly found that there were interstitial collagens there is still a collagen variant in the basement membrane in the connective tissue. The basement membranes have completely different functions and structure compared to connective tissue is different. The basement membranes lie between the parenchymal cells and the Connective tissue or supporting tissue.

Parechymal cells are the epithelial cells of the epidermis and epidermal parts of the urinary, genital, respiratory and gastrointestinal tracts as well as all exocrine nozzles, endothelial cells of the cardiovascular system that mesothelial cells of body cavities and cells of the central and peripheral nervous system, endocrine cells, muscle fibers and fat cells. The basement membranes thus represent the conclusion? of the endothelial or epithelial tissue Connective tissue and at the same time provide a close connection between the various Tissue. They always come only in small amounts, but practically over the whole Body distributed in front. The most important task of the basement membrane is its filtration function; so the membrane occurs in larger quantities where specific filtration tasks are concerned are to be solved.

Such organs are e.g. lungs, kidneys, eyes, placenta etc.

Their main components are collagenous components and glycoproteins.

The isolation of the basement membrane or its components requires completely measures other than those for interstitial connective tissue or interstitial collagen can be applied.

Not only is the membrane present in small quantities, but it is is also insoluble under physiological conditions, i.e. by simple neutral salt extraction, as it is used with interstitial collagens, not to be gained.

One goes to isolate the basement membrane or its components so that by using detergents and other auxiliaries, the membrane extracted from membrane-rich organs. Components are of a relatively high purity of the membrane get when the zëkleinerte organ with denature the agents, such as e.g. urea is treated with simultaneous reduction of disulfide bridges. The membrane can still be brought into solution by proteolytic degradation will. The subsequent separation of the very complex mixture then leads for obtaining the material used according to the invention.

As has now been shown, the basement membrane collagens have very characteristic properties Differences compared to the interstitial collagens. In principle is the same the content of glycine (7/3) and proline and hydroxyproline (1/5). Also the * see footnote page 4 Histidine, valine, methionine and tyrosine levels are similar.

However, the high degree of hydroxylation is characteristic of basement membrane collagen of proline and lysine. The hydroxylysine content is 40-60 residues per 1Ö00 Amino acids are significantly larger than in interstitial collagen, where there are only 4 - 24 residues amounts to. Almost all hydroxylysine residues are also mono- or disaccharides substituted. The proportion of amino acids with non-polar side chains is significant greater; unlike interstitial collagen, basement membrane collagen contains cysteine, in different concentrations depending on the starting tissue.

These differences in structure and composition have a different effect Behavior when used externally on human and animal skin.-The present The collagen used has the ability to stimulate the Basålmeml) ran adjacent endotlial or epithelial cells, which is expressed by e that it comes to a regeneration, accelerated formation of new cells, so that the tissue tissue destroyed by injury or burns again in a very short time is functional.

The agents according to the invention can also be advantageous for aftertreatment used for wounds that have not already healed or surgical scars. Even for skin rashes that can easily leave marks or discolouration of the skin a positive effect can be achieved through increased cell regeneration.

Basement membrane collagen shows even in very low concentrations 0.005% reproducible acceleration of new skin cell formation or regeneration of the destroyed tissue. Tolerance tests have shown that it is also when used from high concentrations, e.g. from 10% solutions, to no undesirable Side effects comes with external application.

* Biology and Chemistry of Basment Membranes, N.A. Kefalides Academic Press, 43, 1978; A. Rauter, Investigation of basement membrane collagen from human Placenta, LMU Munich, 1980.

For use in wound healing agents, basement membrane collagen can be combined with usual Carriers and auxiliary materials and, if necessary, in combination with disinfectants, bactericidal and / or fungicidal substances, to solutions, emulsions, ointments, pastes, Gels, powders or sprays can be processed. For liquid preparations you can as a solvent, water or customary organic solvents, especially alcohol or DMSO can be used. Solubilizers known per se can also be incorporated. Emulsifiers such as ethoxylated fatty alcohols, Fatty acid alkanolamines, para-sin oils, starch or methyl cellulose and preservatives, but also thickeners can be added. As a carrier, for example Cellulose or skin-friendly plastics, such as those used in the manufacture of dressing materials Can be used.

According to the invention, the basement membrane collagen can also be used in pure form (i.e. in up to 100% concentration) e.g. as fibrous or pulverized Freeze-dried material or possibly due to the isolation process mixed with interstitial collagens.

The collagen that can be used according to the invention is, for example, from human Placenta can be isolated by the following method: Example 1 Placental tissue was after Frozen immediately at birth and stored at -200C until use.

After thawing, the placenta was down the umbilical cord and along their circumference cut off the fetal membranes.

The chopping of the organ was followed by the washing out of the blood with 0.4 M sodium acetate solution Finally, it was washed with 0.5 μl of formic acid, and one to establish a suitable pH for pepsin treatment.

10 kg of moist tissue were suspended in 30 liters of 0.5 iW aminic acid and 1g pepsin (0.1%) (from the gastric mucosa of the pig, 2500 Anson U / mg, Boehringer, mannhein) added in solution. after an exposure time of 16-20 hours It was centrifuged at 20 ° C. and the solid residue was discarded. The following work were carried out at 50C in the cold room to avoid denaturing the collagen. The centrifugate was brought to 6% sodium chloride concentration. After centrifugation the residue was in 10 liters o, o3 i trishydroxymethylaminomethane / HCl buffer with pH 7.6 and dissolved with 0.2 M NaCl content. The pH changed and became adjusted with NaOH solution.

The sample was then placed in dialysis tubes against the same buffer (several times Tirwechsel) dialyzed, on the one hand inactivating the pepsin and on the other hand the excess sodium chloride was diluted away. A precipitate of itself Formed during dialysis had to be centrifuged off. The fractional one followed Salt precipitation with 1.8 H NaCl. In order to completely remove type I collagen, this was done Step repeated. The required centrifugations were at 50,000g one Run for hour.

The residue, still contaminated with a little type III collagen solution was / was dissolved in 0.5 M Tris / FtCl, pH 8.0 and the amount of 2-mercaptoethanol added that it was 0.02 molar and incubated for 20 hours at 20 ° C then the sulfhydryl groups were carboxymethylated by adding 0.018 M iodoacetate, Duration 1 hour at 200 G. The sample was filled into dialysis tubes and against Dialyzed 0.5 M formic acid. The dialysis fluid was changed several times, to remove the excess reagents.

Another breakdown of pepsin followed by adding? mg pepsin per 1 g wet weight of the precipitate precipitated with 1.8 M NaCl. After 16-20 hours During the enzymatic treatment, all of the collagen was precipitated with 6% NaCl.

In a subsequent step, the collagen obtained in this way can still be used further cleaned. The residue obtained was redissolved in 0.03M Tris / HCl, pH 7.4, under dialysis and denatured at 450C for 1 hour. Afterward the solution was brought to 3N NaCl and stirred overnight in a refrigerator. Included high molecular weight components precipitated out of the solution and could by centrifugation removed.

The remaining supernatant ran against 0.1 ìl acetic acid or against 0.2 t ammonium hydrogen carbonate solution dialyzed and finally freeze-dried will. In this way, the purest basement membrane collagen was obtained Isolation of individual subcomponents by chromatographic methods, such as Ion exchange chromatography, molecular sieve chromatography, electrophoresis etc., can be further separated.

Depending on the intensity of the pepsin treatments, they vary in size Obtain peptide fragments. Both the native and the denatured collagen material can by conventional methods known in protein chemistry such. B. spec. proteolytic Enzymes, dilute acids, dilute alkalis, cyanogen bromide, etc., can be further split.

According to the invention, of course, the baalm membrane collagen in native as well as denatured form and nevertheless with a high molecular structure as well as in partially split form (peptide mixture, enriched Pure fractions) The pharmacologically active basement membrane collagen can be converted into aqueous and alcoholic solutions, emulsions, ointments and powders and the like are incorporated will. It can also be used in transparent wound closures made of cross-linked polyethylene oxide, how described in DE-OS 1642155, are polymerized.

A particularly practical form of application is with basement membrane collagen Preparations soaked or impregnated wound dressings and plasters. By certain Drying process, e.g. freeze-drying using the "spin-freezing" process pure basement membrane collagen or a conventional carrier and auxiliary material Preparation can be obtained in a cotton-wool-like structure. From these felt-like fleeces pillows of various sizes can then be produced, which are used directly to cover wounds are suitable. In particular, abrasions, e.g. on the back of the hand, which are often very bad heal can be treated beneficially. The wounds stop hurting and dry quickly. The preparations are also excellently suited for hemostasis.

It goes without saying that only sterile preparations are used. The sterilization can be carried out in the conventional way by adding bactericidal, fungicidal and similar means are guaranteed. However, that is particularly preferred Radiation analysis must be applied.

If the compositions according to the invention are used for oral care, e.g. for treatment Used on inflamed gums or bleeding gums, the Agents according to the invention are used in the form of mouthwashes or medical toothpastes.

It can also be used for inflammation of the conjunctiva of the eye of dilute solutions, e.g. in physiological saline solution, of basement membrane collagen good results can be achieved.

The following examples are intended to demonstrate the use according to the invention of the basement membrane collagen obtained according to Example 1 show without to limit the invention in any way: Example 2 Cream with wound healing Effect that is particularly suitable for treating skin burns. It the following components are processed into a cream in a manner known per se: Basement membrane collagen (according to Example 1) 1 g paraffin oil 30 g stearic acid 8 g triethanolamine 1 g perfume (rose oil) 0.05 g polyvinypyrrolidone (PVP) 0.5 g water, dist. ad 100 g The cream was found to be pleasant when applied externally for the treatment of sunburn felt cooling and largely prevented the formation of blisters on the skin. At local Use for skin burns or abrasions turned out to be very nice Scarring and rapid healing.

Example 3 Toothpaste that causes bleeding gums with regular use prevents: basement membrane collagen 0.1 g vitamin C O, O5 g sodium fluoride 0.22 g calcium chloride.gH20 1.8 g carboxymethyl cellulose 1.7 g methyl p-hydroxybenzoate 0.1 g polymethyl methacrylate powder 30 g glycerine 10 g water to 100 g Example 4 mouthwash, suitable for rinsing the mouth for sores in the mouth and throat as well as with a tendency to bleeding gums: Basement membrane collagen 0.3 g vitamin C 0.05 g flavoring substances 0.01 g potassium dihydrogen phosphate 1 g sodium lauryl sulfate 1 g glycerine 3 g ethanol 30 g water ad 100 g Example 5 Lotion used after shaving or to treat skin abrasions becomes: Quaternary polyvinylpyrrolidone copolymer (Gafguat 755) 0.1 g of polystyrene sulfonate sodium salt 0.1 g (Flexan 500) ethanol \ 2.5 g basement membrane collagen 0.1 g water, dist. ad 100 g Example 6 Lotion for soaking wound dressings: Gafquat 755 0.05 g basement membrane collagen 0.05g Carboxyvinyl polymer (Carbopol 940) 0.04 g ethanol 2 g polyethylene glycol 1 g of allantoin 0.1 g of water to 100 g of Example 7 Disinfectant solution with wound healing Effect: wound gasoline 100 g basement membrane collagen 5 g Example 8 wound powder: talc powder 0.5 g clay powder 0.3 g basement membrane collagen (freeze-dried) 0.2 g Example 9 Pure basement membrane collagen produced according to Example 1 is dissolved in 0.1 M acetic acid dissolved with slight warming.

The solution is frozen according to the "spin-freezing" method. Included is rotated aer the freeze drying bottle in an alcohol bath cooled to -20 ° C the liquid Frozen as a thin layer on the wall. To Freeze-drying results in a thin, white, felt-like residue, which in different sized pillows were cut.

After radiation sterilization (UV light), the cushions are used to cover wounds applicable.

Claims (1)

Claims 1. Wound healing agent, characterized in that it Basement membrane collagen (type IV) and, if necessary, the usual carriers and auxiliaries contains. 2. Wound healing agent according to claim 1, characterized in that it Contains bactericidal or fungicidal substances. 3. Wound healing agent according to one of claims 1 to 2, characterized in that that it is used as a carrier cellulosic material or skin-friendly plastic material contains. 4. Wound healing agent according to one of claims 1 to 3, characterized in that that it is made of a felt-like pillow, preferably made of pure basement membrane collagen, consists. 5. Use of basement membrane collagen to treat wounds. 86. Use of a wound healing agent according to one of claims 1 to 5 for oral and dental care.