The method of producing drug collagenase

 

The invention relates to biotechnology, can be used in medicine, cosmetology, dermatology, biochemistry and food industries, as well as for research purposes. The method of producing drug collagenase includes homogenization of the original collagen containing raw material, the separation of the extract by centrifugation, chromatographic purification on ion-exchange sorbent and subsequent elution of active enzymes, dialysis of the eluate and freeze drying of the drug. As a starting raw material containing collagen are the larvae of beetles, cojiedo of the genus Dermestes, homogenization is carried out in sodium chloride solution with the addition of sodium azide at a volume ratio of biomass and a solution of 1:2-2,5, while the chromatographic purification leading to DEAE-sepharose, balanced MES buffer with the addition of 0.005 M calcium chloride, and the elution - triswim buffer, with addition of sodium chloride, calcium chloride and ethyl alcohol. The invention provides a high yield of collagenase increases the specific activity of 5-10 times, reduces the duration of the process of obtaining. 3 C.p. f-crystals, 4 Il.

The invention relates to biotechnology, in particular to the field of obtaining highly purified and visokog the AI and food industry, as well as for research purposes.

Currently, of particular interest are enzymes used in “soft” therapeutic treatment enzymatic methods hypertrophic, keloid and hypertrophic keloid scars of various etiologies. Most drugs used for this purpose, do not have therapeutic activity against chronic scarring. Highly relevant, therefore, is the search for enzyme preparations, which are able to degrade the modified collagen old flat scars and scars of various etiologies. Not all known collagenase able to effectively hydrolyze the scar tissue of the person. Because the number of scars such as hypertrophic and keloid have a composite structure, which includes a chemically modified collagen, many well-known collagenase does not have a significant therapeutic actions in their therapy.

The ability of collagenases to destroy the collagen used in medicine for treatment of burns, chronic venous ulcers, scars, osteochondrosis and other currently know quite a lot of enzyme preparations with collagenolytic action. Collagenase invertebrates on the sources of collagenases, used in medicine. This is sometimes related to the limited amount of source material, so the search for new, unconventional sources of collagenases is of great importance.

In invertebrate animals collagenase widespread and, unlike tissue collagenases higher mammals, are used in the digestive process. For example, sea stars Pycnopodia helianthoides were allocated into two proteases with collagenolytic action. One of them is classified as metalloproteinases, and the other with a lower molecular weight, chymotrypsinogen type. Both enzymes were destroyed labeled collagen from the skin of Guinea pigs, but were almost inactive on the substrate clostridiales PZ-Pro-Leu-Gly-Pro-D-Arg. M. E. Alexander, M. N. Dresden, Comp.Biochem.Physiol. 1980, V. 67B, p. 505-509.

The most studied and used for practical purposes collagenase and complex enzyme preparations obtained from representatives of the family of Decapoda.

The drug RNM-101 containing proteinase krill, was used to treat ulcers and Erosi resulting burn 4 n alkali cornea of rabbits. After 28 days. treatment in 45% of cases observed epithelium (without drug - in 33% of cases), even after 7 days. drug treatment of ulcers of the cornea amansii defects sclera of the eye. This is one of the few examples of the application of collagenases arthropods abroad. Sangwan V. S., E. K. Akpek, Voo I., Zhao T., Pinar V., J. Yang, W. Christen, Baltasar, S., Wild, R., Foster C., S //Cornea. 1999. V I 8. P. 707-11.

A method of obtaining collagenase from the head of the division of different types of leeches described in patent WO 87/00860, publ. 1987.02.12.

According to this patent frozen head Department leeches homogenized in distilled water for 10 min at 4C. the Suspension is then centrifuged for 10 min at 4C and collect the supernatant. Sediment resuspended and centrifuged again. Supernatant unite. The enzyme vymalivayut ammonium sulfate to 40% saturation and centrifuged 20 min at 4C. To the supernatant add ammonium sulfate to 80% saturation and centrifuged in the same conditions. Sediment resuspended 50% ammonium sulfate and cialiswhat against distilled water at 4C.

Activity in all fractions determine incubare 100 μl of each fraction in 100 μl of a solution of collagen 24 h After incubation, the solution was placed in ice for 30 min and the supernatant is determined collagenase activity. 2 ml of the sample from the last sludge applied on a column of Sephadex G-75 superfin the further research using the most active fraction. In addition to collagenase authors have identified inhibitors of 400,000, 45000 and 600 daltons.

Collagenase leeches do not give side effects and act directly on one of the main stimulators of aggregated platelets.

In addition, due to the fact that collagenase leeches destroy the collagen only one connection, it gives the opportunity to apply this enzyme together with other active factors in microsurgery for a long stop bleeding or to preserve blood circulation in congestive grafts or flaps of skin and tissue (on the leg).

The disadvantage of this method is the duration and intensity of the method of producing collagenase from this kind of material.

Known purified preparations of collagenases produced by foreign firms Sigma, ICN. These enzyme preparations prepared from the culture fluid of Clostridium hystolyticum, pathogen, is the causative agent of gas gangrene, therefore, require careful treatment in special conditions.

In the process of selection of enzymes produce Department pigments, toxin, bacterial DNA and related proteolytic enzymes.

The selected solution total protein drug is applied to the chromatographic column with hydroxyapatite, and far dorogostoyashie due to the use of Red-12-agarose and multistage, which leads to the reduction of the output of the active enzymes and increase their value. Thus obtained preparations of the road, their application in the food and medical industry is impossible because of the risk of microbial contamination, and the presence of toxin.

The disadvantages of the method include the use of raw materials, which does not contain collagenases, can have a significant therapeutic effect on old scars of various etiologies (PCT/US 94/24272, C 12 N 9/52, 27.10.1994).

Also known is a method of obtaining collagenase from UltraConnect the culture fluid of Streptomyces lavendulae inmi-36-SV shown in the patent of Russian Federation №2075219 (12 N 9/58, 27.03.1996,). According to this patent, the culture fluid is separated from the mycelium by filtration, the filtrate get ultraconnected, which are planted protein by adding ammonium sulfate to 60% saturation. The precipitate was separated by centrifugation, dissolved and cialiswhat against Tris-Hcl buffer and passed through a column of DEAE-cellulose. The active fraction is concentrated and subjected to gelfiltration on Sephadex G-100, unite and lyophilizers. The drug has a higher collagenase active than the drug from Clostridium histolyticum, however, the method has a number of nedostaje, the patent does not specify the final output of the activity, which makes it impossible to assess the effectiveness of the method.

Russia has been actively developed total drugs proteases from hepatopancreas commercial species of crabs. The interest in this object is associated with the disposal of waste kralovstvi. In addition, an important advantage in comparison with collagenase from C. histolyticum bacteria is the causative agent of gas gangrene is a relative harmlessness of drugs collagenases from crab to humans. The preparations obtained by different methods from hepatopancreas, differ significantly in degree of purity and activity, and enzyme composition.

Based on the above enzyme products produced a large number of medical forms in the form of powders, ointments and immobilized on a dressing materials enzymes for the treatment of purulent wounds, ulcers, scars.

Closest to the claimed is a method for the preparation of collagenase from hepatopancreas commercial species of crab Paralithodes Camtschatica, including homogenization him in 0.1 M ammonium acetate containing 0.1-1.0 mm calcium acetate, pH 6.4, separating the supernatant by centrifugation and precipitation of the drug ammonium sulfate 60-70% saturation. The precipitate, which contains the CA2+. This is followed by ion-exchange chromatography on aminirovaniya silicate materials at pH 5,2-6,4, collagenase elute buffer solution containing 1 M Nad in the presence of 15-25% isopropanol, pH 7.0-8.5 in. For additional purification and concentration of the enzyme collagenase vymalivayut of the eluting solution, saturating it with ammonium sulfate to 60%. The layer containing the active collagenase, separated, deleteroute and freeze-dried.

As aminirovaniya silicate materials used aminosilanes, aminouracil, aminoalkyl. The release of collagenase is 50%, the clearance 40 times.

The disadvantages of the method include low output and insufficient purification of the target product. The latter is particularly important when the medical use of the drug. In addition, the use of anion exchange chromatography leads to loss of valuable components, isoelectric point which is higher than 5. Desalting of the preparation of collagenases dialysis leads to the loss of enzymes, because the collagenase during dialysis (2 days) partially hydrolyzes the dialysis bags. Long stay enzymes in distilled water with a pH of 5.6 leads to partial inactivation of a number of proteolytic enzymes. Moreover, the obtained preparations N 9/64, 28.02.94,

The objective of the invention is to provide a method of producing a new form of the drug collagenase, which has a large specific activity relative to the scar tissue of various etiologies.

Another object of the invention is to increase the output of the drug and the reduction of time in the process.

The task is solved by the fact that in the process for the preparation of collagenase, including homogenization of the original collagen containing raw material, the separation of the extract by centrifugation, chromatographic purification on ion-exchange sorbent and subsequent elution of active enzymes, dialysis of the eluate and freeze drying of the drug, as a starting raw material containing collagen are the larvae of beetles, cojiedo of the genus Dermestes, homogenization is carried out in sodium chloride solution with the addition of sodium azide at a volume ratio of biomass and a solution of 1:2-2,5, while the chromatographic purification leading to DEAE-sepharose, balanced MES buffer with the addition of 0.005 M calcium chloride, and the elution - triswim buffer with the addition of sodium chloride, calcium chloride and ethyl alcohol.

Usually, the homogenization is carried out in 3-4% solution of sodium chloride with the addition of 1-5 μm/l of sodium azide at pH 6-7.

Preference is delovogo alcohol, and dialysis - 0.005 M solution of sodium chloride at 2-6C.

The essence of the method consists in the following. As the source of collagenase used beetle larvae-cojiedo of the genus Dermestes capable as a food substrate to use mummified carcasses of large animals. Larvae of cojiedo used at this stage of development, when the activity of the collagenase maximum.

Larvae decapitate micronozzle, from the body of the larvae out of the digestive tube and homogenization is carried out in 3-4% sodium chloride solution with the addition of trace quantities of sodium azide, no more than 1-5 μm/l to destroy the microflora of the solution.

Purification of the supernatant are ion exchange chromatography, sorption collagenase carried out on DEAE-sepharose in the presence of 50 mm MES buffer (pH=6,4), with 0.005 M calcium chloride, and the elution - triswim buffer pH=8-8,5 with 1.5 M sodium chloride and 20% ethyl alcohol containing 0.005 M calcium chloride.

Desalting and concentration of the target drug is carried out by dialysis with subsequent freeze drying.

The total yield of active product is 50-75% depending on the heterogeneity of the raw materials used.

Specific activity number homogenized in 3% sodium chloride solution with the addition of sodium azide in an amount of 3 μm/l, pH 6-7, glass mechanical homogenizer at a volume ratio of biomass and mortar of 1:2 for 15 min at room temperature.

The obtained homogenate was centrifuged at 10,000 rpm Receive 100 ml of total protein drug digestive tract of the larvae cojiedo.

From the obtained solution, 10 ml applied to a column (1,5 × 20 cm) with DEAE-separate, balanced MES-buffer, pH=6,4, with 0.005 M calcium chloride.

The elution of active enzymes spend 0.05 M triswim buffer at pH 8, 1.5 M sodium chloride, 0.005 M calcium chloride and 20% by volume of ethyl alcohol.

The eluate is subjected to dialysis against 0.005 M solution of calcium chloride during the day at +4C, then freeze-dried. The output of the enzyme preparation is 60%. The specific activity of the obtained enzyme preparation 25 mg of scar tissue /day 0.1 mg of the drug.

Example 2.

The study drug and protein digestive tract of the larvae cojiedo on hypertrophic and keloid scars human conduct in vitro by the following method.

For research to take samples of scars three patients:

1 - 2 years hypertrophic scar

2 - 2-year hypertrophic keloid scar

3 is a 2.5 year old keloid is collagenolysis.

Prepare solutions of trypsin (negative control) and clostridial collagenase (positive control) concentration equal and calculated for collagenase in an investigational drug. Determining the concentration of all the proteins produced by the method of Bradford.

Trypsin is used to check intactness scar tissue, clostridial collagenase - reference collagenase activity.

Collagenosis carried out in a closed glass tube on the rocking chair when 36C, 96 hours.

The results of collagenolysis shown on the drawings.

In Fig.1 shows the splitting of a 2-year hypertrophic scar.

Fig 2 shows the splitting of a 2-year hypertrophic keloid scar.

In Fig.3 shows the dynamics of splitting a 2.5 year old keloid scar.

All drawings numbers buxom match:

No. 1 - the buffer for collagenolysis

No. 2 - trypsin

No. 3 - clostridial collagenase

# 4-drug collagenase larvae cojiedo

As can be seen from the drawings, the study drug is more effective clostridial collagenase in all cases: sample 1 and 3 scars completely dissolved in 48 hours.

In Fig.4 shows data comparing the activity of drugs by splitting zastarelo the SSA attachment scar in percent after splitting.

Column 1 control (without enzyme

Column 2 - added clostridiales

Column 3 - preparation of collagenase from larvae of Dermestes

Column 4-drug Morikazu.

Thus, the proposed method allows to obtain collagenase from grubs of cojiedo with access to 75%, increase the specific activity of 5-10 times, to reduce the process time by reducing the total number of stages of preparation and make the method quite economical due to the use of lower-cost sorbent for the chromatographic purification.

Claims

1. The method of producing drug collagenase, including homogenization of the original collagen containing raw material, the separation of the extract by centrifugation, chromatographic purification on ion-exchange sorbent and subsequent elution of active enzymes, dialysis of the eluate and freeze-dried preparation, characterized in that as a starting raw material containing collagen are the larvae of beetles, cojiedo of the genus Dermestes, homogenization is carried out in sodium chloride solution with the addition of sodium azide at a volume ratio of biomass and a solution of 1:2-2,5, while the chromatographic purification leading to DEAE-sepharose, balanced MES - buffer, with dobavliala alcohol.

2. The method of producing drug collagenase under item 1, wherein the homogenization is carried out in 3-4% solution of sodium chloride with the addition of 1-5 μm/l of sodium azide at pH 6-7.

3. The method of producing drug collagenase under item 1, characterized in that the elution of lead buffer solution containing 1.5 M sodium chloride, 0.005 M calcium chloride and 20% by volume of ethyl alcohol.

4. The method of producing drug collagenase under item 1, wherein the dialysis is carried out in a 0.005 M solution of sodium chloride at 2-6C.

 

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SUBSTANCE: raw material obtained out of gray substance homogenate should be supplemented with ionol (2.6-di-tret-butyl-4-methylphenol) at quantity necessary to prepare 0.0001 M final solution. Addition of this substance to raw material enables to keep its high activity being equal to 12.5-16.0 sec and enables to prolong its storage terms at (-40 ± 2) C up to 10 wk before carrying out sublimation and prevent the decrease of activity during sublimation process.

EFFECT: higher efficiency.

1 ex, 2 tbl

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