DE4028622C2 - Method for producing collagen sponges - Google Patents

Description

The invention relates to a process for the preparation of Collagen sponges, which for transdermal application of pharmaceutical and cosmetic agents, and in particular suitable for use as face masks.

Collagen sponges are known in the art and are Numerous methods for their preparation have been described.

Some of the older publications relate to the production collagen sponges of the uncrosslinked type from soluble Collagen by freeze-drying or by washing out the water by means of organic, water-miscible solvent a foamed collagen mass (see US 26 10 625 and 31 57 524 and DE 26 25 289). You get an easy way this way moldable product with a sponge-like structure. The However, products can not be used for transdermal application of Active ingredients and in particular not used as face masks because they swell and decay immediately upon wetting. you have no strength when wet.  

The production of water-resistant sponges based on collagen by the addition of crosslinking agents (cf., for example, the in DE 27 34 503, 29 43 520 and 32 03 957 described Method).

Thus, in DE-PS 32 03 957 a process for the preparation of waterproof collagen sponges from bovine or porcine collagen described in which the collagen raw materials in the usual way unlocked and released from impurities, then to Purification of a treatment with aqueous alkali and a Subjecting acid treatment at a pH of about 3, the Material obtained mechanically comminuted, to about 2 wt.% Dry mass diluted and the suspension with a Vernet provides the medium. The material thus treated is subsequently ßend, optionally with the addition of textile fibers within from 30 minutes to 4 hours, preferably within 1 1/2 to Deep-frozen for 2 1/2 hours and then according to usual procedures freeze-dried.

Collagen produced by the process of DE-PS 32 03 957 sponges are stable even in aqueous medium, they contain However, residues of the crosslinking agents used, which in sensitive skin can cause irritation.

Since the crosslinking agents are low molecular weight Substances, they can even penetrate the skin and cause unwanted reactions in the body.

Although therefore by the aforementioned method high quality Collagen masses are provided which are in cosmetics and medicine are used and their structure by kontrol crystallized by a generally pharmacological compatible crosslinkers stabilized uniform, has fine-pored matrix, so it has not been successful, a produce crosslinker-free collagen sponge material,  which also with extremely sensitive skin for example as Face mask can be used.

Object of the invention is therefore, without the use of che mixers, emulsifiers or preservatives to provide stable collagen sponges in an aqueous medium, which for the transdermal administration of pharmaceutical and cosmetic active ingredients are suitable.

To solve the problem, the serve according to claims 1 to 8 prepared collagen sponges.

According to the invention succeeded, completely without the use crosslinked chemical additives, stable in aqueous medium To produce collagen sponges that are pure Natural products. You can with pharmaceutical and kosme be loaded with active ingredients of different nature, wherein especially in face masks through an interaction of cosmetic agents with the collagen an intensification can be done. It creates in this way a real, kosme table "drug delivery system".

For carrying out the method according to the invention are first usual high quality raw materials for the production of collagen, such as for example, beef or pork skins or beef chops longing for a customary pretreatment through soft, limp, Fleshing and splitting off the scar layer of either by Salting or by cooling preserved starting material subjected. For salted material, the storage time can be up to to a few months while the chilled material must be processed within a week. Because the kind and Way of conservation the swelling state and the source the pre-treatment of the Preservation be adapted, in particular by means of more thorough washing and liming of salted material.  

According to the invention, the material is then treated with alkalis and treated immediately with acid. This step is basically known in the art and leads to a Purification of the raw material from all non-collagen contaminants fen. A measure of the chemical cleaning is the determination of the Amide nitrogen, which is about 0.3 compared to the initial value should be lowered to 0.5 mmol / g. In the present case should the amide nitrogen after completion of the chemical treatment about 0.20 to 0.40 mmol / g. The degree of cleaning and swelling of the collagen is described as in the prior art (see. For example, DE 32 03 957) determined.

Unlike in the previously used method, however According to the invention significantly intensifies the alkali treatment. The Alkali treatment occurs over a period of 1 to 5, before preferably 2 to 4 weeks, and the concentration of the used sodium hydroxide is 1 to 3 wt.%. To the Comparison should be noted that in the DE 32 03 957 described procedures (see column 6, 1.2) alkaline treatment at a NaOH concentration of less than 0.4% over a period of only 12 to 24 hours was led.

Then the material is thoroughly washed with water, to remove the alkali residues as completely as possible.

The subsequent acid treatment is carried out as in the state of Technique described (see 32 03 957) for about 10 to 20 hours with about 2 to 4% hydrochloric acid. The pH of the medium should be 0.5 to 1.5 and preferably 1.

The alkali treatment according to the invention leads to denaturation part of the collagen, this part being about 3 to 6% by weight, should be based on the total amount of collagen.  

The mass thus treated is then mixed with running water until washed out until the pH of the material is about 2.5 to 3.5, preferably 2.7 to 3.3.

The collagen mass treated according to the invention is after washing evenly acidified and the surface of the material and the individual fibers are sticky. The inventive method differs fundamentally from this measure the previously known methods in which the chemical Treatment should be carried out so gently that Any damage to the fibers is prevented (see, for example DE 32 03 957).

Surprisingly, by the inventive, intensive Alkali treatment of the starting material better adhesion of the Fibers in the finished sponge causes without the flexibility and softness of the sponge is impaired. However, it is necessary to direct the conduct of the procedure so that the Denaturing process only affects the surface of the fibers and the amount of denatured products is about 3 to 6% by weight (see above).

The degree of denaturation is controlled by a Determination of the hexosamine and amino acid pattern, as well as a Determination of the "native" collagen content (see Examples 2 and 3).

The determination of Hexosaminehaltes is a measure of the collagenous connective tissue impurities, such as glycosaminoglycans, Glycoproteins and proteoglycans. These substances are by the alkaline treatment hydrolyzed and washed out. OF INVENTION According to the invention, less than 1 mole of hexosamine is 1000 amino acids present, an indication of the high degree of purity of the obtained Product.  

The amino acid determination serves to detect the Aminsäuremus ters of the product according to the invention. Also in this case show the analyzes (see Example 1) the purity of the product. So corresponds to the found value of 349 glycine residues per 1000 Amino acid residues largely correspond to the theoretical value for pure Collagen (every third amino acid is glycine). The same applies for the hydroxyproline residues as well as for the remaining amino acids, whose content corresponds to that of pure collagen.

The proportion of denatured collagen in the total mass of the Collagen is, as described in Example 2, with trypsin determined. Since the latter hydrolyze only denatured collagen can, with this enzyme, the denatured portion selectively while the total collagen is acidified by acid hydrolysis (cf. Example 2) or with collagenase can be determined. As above indicated, according to the invention, the proportion of denatured Collagen about 3 to 6%, based on the total collagen amount.

Following the partial denaturation according to the invention and washing to a pH of 2 to 4, preferably 2.7 to 3.3, the material is prepared by known methods, such as For example, in DE 32 03 957, pre-shredded, homogenized and defibred and herge an aqueous dispersion provides. The dry weight of the dispersion should be about 1.5 to 3 wt.%, Preferably 2 wt.% Be and the pH with dilute hydrochloric acid to about 2.5 to 3.5, preferably 3 be set.

Should agents containing sponges be prepared, so the former are added to the dispersion. The inventive Collagen sponge has excellent properties Carrier material for active ingredients and is suitable for loading suitable pharmaceutical or cosmetic active ingredients. In In this context, the collagen itself acts like an ion exchangers, by adding numerous electrolytes, metabolites and Medicinal binds.  

According to a particularly preferred embodiment of the invention are added to the dispersion with ATP-loaded liposomes. The Amount of these liposomes can, based on the collagen dry weight in the dispersion about 2 wt.% Be.

The collagen dispersion of the invention, if necessary angerei chert with active ingredients, is then over a period of 0.5 to 4 hours, preferably 1 to 3 hours at a Temperature of -10 to -30 ° C, preferably -25 ° C in the form of Frozen plates. The thickness of the plates can be 1 to 3 cm, preferably 1.5 to 2.0 cm. The pore size is in the essentially controlled by the freezing rate, they can but additionally by adding surface-active substances to be influenced.

The plates may optionally be between -3 to -5 ° C between be stored. During storage, a conditioning ef a beneficial effect on the final product. It doing a slight drop in viscosity, the Reprodu Improved productability of the product. Preferably, the stored frozen plates for at least 24 hours.

After freezing and, if necessary, interim storage reach the plates for freeze-drying.

In the method previously described in the prior art went it is assumed that the following drying process for the Quality of the product is no longer decisive. OF INVENTION However, it has surprisingly been found that the guidance of freeze-drying for the stability of the sponge products in the wet state is of crucial importance. Leaves namely, the freeze-drying at temperatures between 110 and 150 ° C over a long period of time, so apparently finds a cross-linking of the collagen molecules taking place which collagen modified and increased its stability in the wet state.

This networking is very slow and is caused by the  Exposure of elevated temperature to that already substantially achieved dry material under vacuum. According to the invention is allowed Therefore, temperatures of 110 to at least 12 hours 150 ° C at a vacuum of about 0.5 to 3.0 mbar on the im essentially already act dry material, the End point of the actual freeze-drying in a known manner determined by the pressure increase method and thus the beginning of Temperature post-treatment is set.

The crosslinking reaction is unknown in nature, the However, cross-linking effect can with all the usual Bestim be demonstrated. For example, the Resistance of a collagen structure to collagenase a measure for crosslinking, with non-crosslinked material significantly is degraded faster than with non-crosslinked material the case is (see Example 4).

The finished product can be cut to the desired thickness be, with the mechanical properties of the dry Materials are not affected by the heat treatment. Compared to conventionally dried material has invented However, the product prepared according to the invention in the wet state higher strength and its swelling capacity is lower. The Water absorption is determined by immersion in water and subsequent dripping without mechanical action. By the Heat crosslinking during freeze-drying becomes an Ver Reduction of the water absorbency by 20 to 50 wt.% Causes. The stability of the material in the wet state is in one improved to such an extent that it is possible, the active ingredients to massage the material into the skin.

The crosslinking method according to the invention is physiological absolutely harmless and not comparable with procedures which make use of chemical crosslinking agents. Much more it may be a process analogous to that natural aging proceeds.  

The products according to the invention are excellent carriers for cosmetic and pharmaceutical active substances which are known from Collagen matrix can be applied topically or transdermally. The active ingredients may be, for example, in microencapsulated form be present and released by wetting.

The sponges according to the invention can also be unilaterally with a be provided with water-impermeable coating and they have especially as drug-enriched facial masks proven.

Soluble collagens have long been used in cosmetics used to increase skin moisture. By an occlusion effect, this result can be with Face masks each for a limited period of time as well to reach. According to the invention, however, by face masks, which contained ATP-loaded liposomes, an unexpected and extraordinary increase of this effect is achieved by measuring skin moisture and skin smoothness leaves.

The invention will be explained below by way of examples.

example 1 Production of collagen sponges, enriched with ATP-loaded liposomes

Kalkspaltflanken from salted cattle hides were after of leather production conventional methods by soft, liming, Defrosting and splitting the scar layer prepared. Subsequently, the material was washed for about 30 minutes and preserved with solid salt. The amount of salt was about 300 g / kg. Thereafter, the material was in batches of approximately each 20 kg packed in polyethylene bags and at -30 ° C for 3 weeks stored.  

Subsequently, the material was thawed and in flowing Washed thoroughly. Thereafter, the treatment with 1.5% by weight aqueous NaOH solution; the treatment time was 15 days.

The amide nitrogen content of the material, based on dry weight, was 0.28 mmol / g following the alkali treatment.

After thorough washing, the material was made with 4% hydrochloric acid Soured for 20 hours. The ratio of nakedness to fleet was 1: 1.5 and the material had one after acidification pH value of 0.8.

It was then washed with running water until a pH of about 3.0 was reached. The dry weight of the flanks was after this treatment 14%.

The acidified columns were now cut into pieces, with a mincer (perforated disc diameter 5 mm) crushed and subsequently colloidally ground by adding it a total of four times passed over two colloid mills connected in series.

The mass was then diluted to a dry matter content with water of 2.0% and with dilute hydrochloric acid to a pH of 3 set. To the dispersion thus obtained was 2% by weight, based on the dry weight of the collagen, commercial, with ATP loaded liposomes mixed. After thorough mixing and degassing, the dispersion was filled into aluminum molds (Filling height about 2 cm), taking during this process on bubble-free and uniform filling was respected.

The dispersion was then in the brine bath at -25 ° C about Frozen for 90 minutes, the frozen plates were demolded and in the refrigerator at a temperature of -5 ° C 24 hours stored for a long time.  

The 2 cm thick plates were then a freeze skirt tion under a vacuum of about 1.5 mbar with the following supplied temperature program:

temperature Time 160 ° C 13 hours 120 ° C 6 hours 110 ° C 6 hours 90 ° C 6 hours

The dry plates were subsequently made to a thickness of 0.8 mm and subjected to the usual testing. They were perfect for cosmetic use as collagen masks suitable.

Example 2 Determination of the hexosamine and amino acid pattern

Parallel samples of the acidification and washing material, each weighing 10 mg, by weight, in 10 ml of 3 mol / l and 6 mol / l HCl, respectively, were melted at 105 ° C for 15 and 20 hours in sealed bomb tubes hydrolyzed under nitrogen (post-purified N ₂ ). After cooling the bomb tubes in the refrigerator and opening the same, the contents were transferred into 25 ml pointed piston and in a vacuum rotary evaporator (Rotavapor RE 120, Büchi, Switzerland) at 40 ° C in a water jet vacuum brought to dryness. The residue was dissolved in 5 ml H ₂ O and again evaporated to dryness in a water jet vacuum. Subsequently, the residue was dissolved in 5 ml of 0.2 mol / l Na⁺ buffer with a pH of 2.75 consisting of 16.9 g of tri-lithium citrate 4-hydrate, 15 ml of 32% HCl, 1 ml of liquid Phenol, 2 ml Brÿ solution dissolved in 1000 ml distilled water. added.

To determine the glucosamine and galactosamine levels were aliquots of the solution with the buffer mentioned above Diluted 1:10 ratio and 150 .mu.l of in 3 mol / l HCl hydroly sample into the cartridge of an amino acid analyzer (Alphaplus, type 4151, Pharmacia-LKB, Freiburg) and the injected Results evaluated.

The procedure was carried out with the sample hydrolyzed in 6 mol / l HCl repeated, but only 50 ul in a task cartridge were injected.

The double hydrolysis ensured that all amino acids since maximum values for the hexosamines and tyrosine only after hydrolysis in 3 mol / l HCl, maximum values for valine, Isoleucine and leucine, however, only after hydrolysis in 6 mol / l HCl to be obtained.

The results are shown in Table 1.  

Table 1

Example 3 Determination of the proportion of denatured collagen

30 mg, based on dry weight, of the acid-treated and washed material was dissolved in 30 ml of 0.1 mol / l sodium hydrogen Carbonate solution with a pH of 8.2, which 1.5 ml of a 6 mg / ml trypsin solution (lyophilised preparation from bovine Pancreas, Boehringer, Mannheim) were added for 8 hours incubated at about 23 ° C in a shaker water bath. One left then cool the sample to 4 ° C in a cooling chamber and it was then ultracentred at 4 ° C and 32,000 rpm for 30 minutes trifugiert.

After the centrifugation of the centrifuge, the supernatant was under Stir in an ultrafiltration cell through a Diaflo filter PM10, diameter 25 mm, filtered and 1 ml of the filtrate were in 6 mol / l HCl, as described in Example 1, for 20 hours hydrolyzed at 105 ° C. Further processing and analysis of the Hydrolyzate was carried out as described in Example 1, with the Difference that the sample after two times concentration to dryness was taken up in 500 μl buffer and a subset of 150 μl injected into the task cartridge of the amino acid analyzer has been. The determined after performing the amino acid analysis Hydroxyproline value was then expressed as μmol hydroxyproline / g Determined starting substance. It was 38 μmol and represented the trypsin-degradable denatured portion of the total col lagens.

In parallel to the analysis described above, 30 mg of Starting substance, as described in Example 1, in 6 mol / l HCl hydrolyzed and analyzed. The one obtained from this analysis Hydroxyproline value, which represents the total collagen content was 764 μmol / g starting substance (dry weight).  

The results show that about 5% of the collagen denature were.

Example 4 Determination of collagen resistance of the product according to the invention

1 mg (based on dry weight) of a sample of the invention The end product according to the invention was admixed with 100 units of collagenase Add 2.0 ml stock buffer and incubate at 37 ° C. The sample was checked at intervals of 15 minutes and the time and the buffer solution became clear. The time was up a total of 325 minutes.

In parallel, a sample of non-crosslinked, on conven Alternatively, freeze-dried collagen in the same way examined as described above. The collagen solution was in clear after 73 minutes.

Claims (8)

1. A process for the manufacture of collagen sponges, which comprises subjecting the pre-treated in the usual manner a raw material to an alkali treatment and then an acid treatment and it is then washed, crushed, freezing and freeze-dried, characterized in that

• a) the alkali treatment is carried out with a 1 to 3 wt .-% sodium hydroxide solution over a period of 1 to 5 weeks, so that 3 to 6 wt .-% of the surface of the material, based on the total amount of the raw material to macro molecular peptides be dismantled, and
• b) during the freeze-drying on the substantially already dry material temperatures between 110 and 150 ° C at a vacuum between 0.5 and 3.0 mbar act.

2. The method according to claim 1, characterized in that one the crushed material as an aqueous dispersion at -10 to Freeze -30 ° C over a period of 0.5 to 4 hours and then at -3 to -5 ° C for at least 24 hours outsourced. 3. Process according to claims 1 or 2, characterized gekennzeich net, that you can crush the crushed material before freezing added cosmetic or pharmaceutical agents. 4. The method according to claim 3, characterized in that one as active ingredient with ATP-loaded liposomes added. 5. The method according to claim 3, characterized in that one the material by wetting with water releasable, mi Added encapsulated active ingredients.   6. Process according to claims 1 to 5, characterized gekennzeich net, that one loaded with active material after the Freeze-drying on one side with a water-impermeable Coating provides. 7. Use of the method according to claims 1 to 6 prepared collagen sponges for topical or transdermal paint application of active ingredients. 8. Use according to claim 7 as a face mask.