DE3147727C2 - Wound healing agents containing collagen from basement membranes - Google Patents

Abstract

The invention relates to a wound healing agent which contains basement membrane collagen (type IV) and, if appropriate, conventional carriers and auxiliaries. The wound healing agent according to the invention can influence the formation of new cells and accelerate wound healing.

Description

The present invention relates to a wound healing agent, which contains basement membrane collagen and, if appropriate, customary carriers and auxiliaries

So far one has to promote wound healing z. B. Prostaglandins containing agents proposed (cf. US-A-40 61 670). In GB-A-15 65 340 were enzymatic degraded human and animal tissues for wound healing are described. Interstitial collagens are to be used according to DE-OS 24 62 222 for curing connective tissue diseases.

It has now been found that basaimembrankoiiagen leads to particularly rapid wound healing, with the scars healing particularly nicely.

The present invention provides a highly effective new means of healing wounds of a wide variety Kind provided. The agent is also particularly suitable for the healing of burn wounds and from sunburn.

It was surprisingly found that it was next to interstitial Collagens in connective tissue still have a collagen variant in basement membranes. The basement membranes Compared to connective tissue, they have completely different functions and their structure is also different. The basement membranes lie between parenchymal cells and the connective tissue or supporting tissue. Parenchymal cells are the epithelial cells of the Epidermis and epidermal cells of the urinary, genital, respiratory and gastrointestinal tracts as well as all exocrine glands, endothelial cells of the cardiovascular system, the 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 closure of the endothelial or epithelial tissue Connective tissue and at the same time provide a close connection between the various tissues. she are only ever found in small quantities, but are practically distributed over the whole body. The most important The task of the basement membrane is its filtration function: so the membrane occurs in larger quantities wherever specific filtration tasks have to be solved. Such organs are z. B. lungs, kidneys, eyes, piazenta etc. you The main components are collagenous components and glycoproteins.

The isolation of the basement membrane or its components requires completely different measures than with interstitial connective tissue or interstitial collagen can be applied. The membrane is not only present in small quantities, but it is also under physiological Conditions insoluble, d. H. by simple neutral salt extraction, as is the case with interstitial collagens is applied not to win. One goes with the isolation of the basement membrane (Biology and Chemistry of Basment Membranes, N.A. Kefalides Academic Press, 43, 1978; A. Rauter, Study of Basement Membrane Collagen from human placenta, LMU Munich, 1980) or its components in such a way that the membrane from membrane-rich organs by using detergents and other auxiliaries is extracted. Components of the membrane are retained in a relatively high order when the comminuted Organ with denaturing agents, such as. B. Urea, with simultaneous reduction of disulfide bridges, is treated. It can be even more effective Membrane can be brought into solution by proteolytic degradation. The subsequent separation of the very complex mixture then leads to the recovery of the material used according to the invention.

As has now been shown, the basement membrane coliagens show very characteristic differences the interstitial collagens. The content of glycine (V3) and prclin and hydroxyproline are basically the same (V5). The levels of histidine, valine, methionine and tyrosine are also similar. Characteristic of basement membrane collagen but is the high degree of hydroxylation of proline and lysine. The hydroxylysine content is significant with 40-60 residues per 1000 amino acids larger than in interstitial collagen, where it is only 4-24 residues. Almost all of the hydroxyiysine residues are also substituted with mono- or disaccharides The proportion of amino acids with non-polar side chains is significantly larger; In contrast to interstitial collagen, basement membrane collagen contains cysteine, namely in different concentrations depending on the starting tissue.

These differences in structure and composition cause a different behavior in external Use on human and animal skin

The collagen used here has the ability to stimulate the endothelial or epithelial cells adjoining the basement membrane, which manifests itself in the fact that there is a regeneration, accelerated new formation of skin cells, so that the tissue destroyed by injury or burns again in a very short time is functional. The agents according to the invention can also be used advantageously for the aftertreatment of wounds that have not healed nicely or surgical scars. Even with skin rashes that leave marks or discoloration of the skin, a positive effect can be achieved through the increased formation of new cells.
Even at a very low concentration of 0.005%, basement membrane collagen shows reproducible acceleration of new skin cell formation or regeneration of the destroyed tissue. Tolerance tests have shown that even when using high concentrations, e.g. B. 10% solutions do not have any undesirable side effects when used externally.

For use in wound healing agents, basal • ncmbran collagen with usual carriers and auxiliaries

as well as possibly in combination with disinfectants, bactericidal and / or fungicidal substances, processed into solutions, emulsions, ointments, pastes, gels, powders or sprays. For liquid Preparations can use water or customary organic solvents, especially alcohol, as the solvent or DMSO can be used. Solubilizers known per se can also be incorporated. As an aid can include emulsifiers such as ethoxylated fatty alcohols, fatty acid alkano mines, paraffin oils, starches 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 for the production of Dressing materials find use, are mentioned.

According to the invention, the basement membrane collagen can also be used in pure form (i.e. in a concentration of up to 100%) z. B. a ȣ fibrous or powdered freeze-dried Material or possibly due to the isolation process with interstitial collagens mixed, can be applied.

The collagen usable in the present invention is, for example, from human placenta by the following method isolable:

Manufacturing example

Placental tissue was frozen immediately after birth and to use stored at - 20 ⁰ C.

After thawing, the placenta became the umbilical cord and cut off the fetal membranes along their perimeter. The Zerhacr in the organ the blood was washed out with 0.4 M sodium acetate solution. Last was with 0.5 M formic acid washed to adjust a suitable pH for pepsin treatment.

10 kg of moist tissue were suspended in 30 liters of 0.5 M formic acid and 1 g of pepsin (0.1%) (from the gastric mucosa of the pig, 4C 2500 Anson U / mg, Boehringer, Mannheim) was added in solution. After a period of action of 16-20 hours at 20 ^° C., the mixture was centrifuged and the solid residue was discarded. The following work was carried out at 5 "C in the cold room to avoid denaturation of the collagen. The centrifugate was brought to 6% sodium chloride concentration. After centrifugation, the residue was dissolved in 10 liters of 0.03 M trishydroxymethylaminomethane / HCl buffer
dissolved at pH 7.6 and with 0.2 M NaCl content. The pH changed and was adjusted with NaOH solution. The sample was then dialysed in dialysis tubes against the same buffer (repeated changes), the pepsin being inactivated on the one hand and the excess sodium chloride being dialyzed off on the other. A precipitate that formed during dialysis had to be centrifuged off. The fractional salt precipitation with 1.8 M NaCl followed. This step was repeated to completely remove type 1 collagen. The required centrifugations were carried out at 50,000 g for one hour.

The residue, still contaminated with some type III collagen was dissolved in 0.5 M Tris / HCl, pH 8.0 and the solution was as much 2-mercaptoethanol added that it was 0,02molar and incubated for 20 hours at 20 ⁰ C. The sulfhydryl groups were then carboxymethylated by adding 0.018 M iodoacetate. Duration 1 hour at 20 ^° C. The sample was filled into dialysis tubes and dialyzed against 0.5 M formic acid. The dialysis fluid was changed several times to remove the excess reagents.

This was followed by a further breakdown of pepsin by adding 1 mg of pepsin per 1 g of wet weight of the 1.8 M NaCl precipitated precipitation. After 16-20 hours of the enzymatic treatment with 6% NaCl the entire collagen precipitated

In a subsequent step, the collagen obtained in this way can be purified even further. The received The residue was redissolved in 0.03 M Tris / HCl, pH 7.4, re-dialyzed and denatured at 45 ° C. for 1 hour. The solution was then brought to 3 M NaCl and stirred in the cold room overnight. Included high molecular weight components precipitated out of the solution and could be removed by centrifugation. Of the Remaining supernatant can be used against 0.1 M acetic acid or against 0.2 M ammonium hydrogen carbonate solution dialyzed and finally freeze-dried. In this way the purest basement membrane collagen was obtained, which, for the purpose of isolating individual subcomponents, still by chromatographic methods, such as Ion exchange chromatography, molecular sieve chromatography, electrophoresis, etc., further separated can be.

Depending on the intensity of the pepsin treatments, peptide fragments of different sizes are obtained. As well as The native as well as the denatured collagen material can be prepared by conventional methods known in protein chemistry such as B. spec. proteolytic enzymes, dilute acids, dilute alkalis, cyanogen bromide, etc., further split will.

According to the invention, of course, the basement membrane can be coated in native ais also in denatured form and nevertheless in high molecular structure as can also be used 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 can be incorporated. It can also be used in transparent wound sealants from networked! Polyethylene oxide, as described in DE-OS 16 42 155, are polymerized.

A particularly practical form of application is those soaked with preparations containing basement membrane collagen or impregnated wound dressings and plasters d, v. By certain drying processes, e.g. B. Freeze drying using the "spin-freezing" method, pure basement membrane collagen or a conventional one can be used Preparation containing carriers and auxiliaries in cotton wool Structure can be obtained. Pillows of various sizes can then be made from these felt-like fleeces which are directly suitable for covering wounds. In particular abrasions, e.g. B. on The back of the hand, which often heals very poorly, can be treated advantageously. The wounds stop pain and dry quickly. The preparations are also ideal for stopping bleeding.

It goes without saying that only sterile preparations are used. The sterilization can be done according to conventional Kind can be guaranteed by adding bactericidal, fungicidal and similar agents. Particularly however, radiation sterilization will be preferred.

If the agents according to the invention for oral care, e.g. B. to treat inflamed gums or used for bleeding gums, so be

the agents according to the invention are used in the form of mouthwashes or medical toothpastes

Even with inflammation of the conjunctiva of the eye, the use of dilute solutions, e.g. B. Good results can be achieved in physiological saline solution, with basal membrane collagen.

The following examples are intended to illustrate the use according to the invention of the production example obtained basal membrane collagen show without restricting the invention in any way:

example 1

Cream with wound healing properties, particularly suitable for treating skin burns The following components are processed into a cream in a manner known per se:

Basement membrane collagen
(according to manufacturing example)
Paraffin oil
Stearic acid
Triethanolamine
Fragrance (rose oil)
Polyvinylpyrrolidone (PVP)
Water, dist.

ad

i s

30 g

8g

Ig

0.05 g
0.5 g
100 g

25th

When used externally, the cream was used for treatment felt as pleasantly cooling from sunburn and largely prevented the formation of blisters of the skin. When applied locally to skin burns or abrasions, a very nice one showed up Scarring and rapid healing.

B e i s ρ i e I 2

Toothpaste that, when used regularly, prevents bleeding gums:

0.1g

0.1g 2.5g 0.1g 100 g

Quaternary polyvinylpyrrolidone copolymer (Gafquat 755)

Polystyrene sulfonate sodium salt

(Flexan 500)

Ethanol

Basement membrane collagen

Water, dist

Example 5
Lotion for soaking wound dressings:

Gafquat 755 0.05 g

Basement membrane collagen 0.05 g carboxyvinyl polymer

(Carbopol 940) 0.04 g

Ethanol 2 g

Polyethylene glycol 1 g

Allantoin 0.1 g water to 100 g

Example 6
Disinfectant solution with wound healing properties:

Mineral spirits
Basement membrane collagen

! 0Og 5g

Example 7

Wound powder:

Talc powder
Clay powder

Basement membrane collars
(freeze-dried)

0.5 g OJg

0.2 g

Example 8

Basement membrane collagen Example 3 ad 0.1g Vitamin C 0.05 g Sodium fluoride 0.22 g Calcium chloride · 2H ₂ O 1.8 g Carboxymethyl cellulose 1.7 g Methyl p-hydroxybenzoate 0.1g Polymethyl methacrylate powder 30 g Glycerin 10g water 100 g

Mouthwash, suitable for rinsing the mouth for sores in the mouth and throat as well as for those who tend to Bleeding gums:

Basement membrane collagen ad 0.3 g Vitamin C 0.05 g Flavorings 0.01g Potassium dihydrogen phosphate ig Sodium lauryl sulfate ig Glycerin 3g Ethanol 30 g water 100 g

B e i s ρ i e 1 4

Lotion used after shaving or to treat skin abrasions:

Pure basement membrane collagen produced according to Example 1 is dissolved in 0.1 M acetic acid with gentle heating solved. The solution is made according to the "spin-frcezing" method frozen. While turning the freeze-drying bottle in one to -20 ° C chilled alcohol bath frozen the liquid as a thin layer on the wall. After freeze drying lies a thin, white, felt-like residue, which was cut into different sized pillows.

After radiation sterilization (UV light) the pillows are can be used to cover wounds.

Example 9

It was once collapsed in the "3-pin freezing" process Type IV (isolated as described in Example 1) and in a second the interstitial collagen type I to one Preparation of cotton wool-like structure processed. After one hour of sterilization (UV light), extremely thin type IV and type I pads were placed on open rub bladders placed on both feet and fixed with a commercially available leukoplast. The one treated with Tyρ I. The wound did not heal until day 5, while the other was treated with type IV The bladder had healed on the 3rd day.

Example 10 A commercially available »Vaseline was once Type I

Collagen and the other time type IV collagen mixed in, each with a 5% concentration of collagen was included in the final product. With these remedies, eczema was pruriginoes, which is mainly associated with both forearms occurred. It was applied twice a day by rubbing in one forearm with the type l-containing and the other with the Type I V-containing preparation. With the type IV preparation; you could get an instant itch relief as well Disappearance of the skin irritations can be noticed after 5 to 6 days. When using the type I preparation a slight improvement could only be noticed after 8 to 10 days.

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30th

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Claims (5)

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