JPS60500413A - Pharmaceutical composition with high collagenolytic activity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Preparations with high collagenolytic activity and pharmaceutical compositions containing such ceramics thing The present invention provides preparations with collagenolytic activity, more particularly collagen-enriched preparations with collagenolytic activity. Uncontrolled alteration of structure appears. Formulated with a drug vehicle for particular use in the treatment of human or animal diseases ．． The present invention relates to a pharmaceutical composition comprising said preparation.

Multiple species of microorganisms that produce collagenase have been reported in the literature. However, the formulation of the active ingredient content in these preparations is often is difficult. Furthermore, these preparations always contain a certain amount of toxic components, such as neurotoxins. It is not suitable for use in pharmaceutical compositions.

The purpose of the present invention is to apply a sexual coating to wounds where uncontrolled deterioration of Kolaken appears. Laken structure (5 structures) Enzymatic cleaning selective for genea The present invention can be achieved by providing a pharmaceutical composition capable of The objective is further to determine the relative proportions of the main components of the collagen bath-tactile preparation contained in the composition. Manufacture of compositions balanced in predetermined proportions according to the degree of desired therapeutic effect The characteristics of the pharmaceutical decomposition product of the present invention relating to It specifically recognizes the natural amino acid residue] and specifically recognizes the X-glycyl bond. cleavage that can be further inhibited at least partially by myosin It contains an effective amount of collagenase with specific activity.

Preferred collagenases included in the pharmaceutical composition of the present invention include Nolyticus cbemoVy iophagus culture is attended by Naru Collagenase, or microorganisms derived from said bacteria, or collagenases similar to said bacteria. Collagenase can be derived from cultures of microorganisms from which collagenase can be derived. . The main taxonomic characteristics of the bacteria will be described later. Even more generally, Collagenase that can be used in the pharmaceutical composition of the invention includes collagenase, i.e. Enzyme Nomenture Edition IUPAC-IUB EC in (1978).

The so-called Achromobacter species listed as 3.4.24.8 There are collagenases that can react with selective antibodies prepared against "Lachenase".

This collagenase is derived from the chemovar locus recessed strain. . This bean paste stock is Co1 of lN5TITUT PASTEUR as n'l-029. 1ection Nationale de Cu1turede Micro - Deposited at Organismea (C, N, C, M, ).

This collagenase has a specific active icrocatalytic unit compared to other collagenases. (μkat/mq). This can be seen, for example, in Biochirn, B. iophys, Acta 522.218-228 (1978), KEIL-DLOUHA and B, KEIL's paper” 5ubunit 5t structure of Achromobacter Colla ”.

Regarding the chemical characterization of this enzyme and the microorganisms that produce it, see 1. iochimica et Biophysica Acta, 429 (1 976) Vera KEIL-DLOUHA's Chem listed in 239-251 ical characterization and 5 studies of au todigestio of pure collagenase from1 Boriyoj KEIL published in March 26th, 1979. 23rd volume, nr, 2 “Some newly characterized collagen ases from procaryotes and lower eucary otes'' can be fully referenced.

More specifically, the present invention provides collagenase activity based on the total weight of the composition: - emulsions, ointments, powders, or any composition that is not a true liquid in the sense of the common term; In the case of a drug composition, it is a pharmaceutical composition, and 0.05 A ka per pharmaceutical composition in the case of a solution configured for single topical use t/ml It pertains to a pharmaceutical composition containing a drug at least equal to .

The preferred cartilage of the present invention has a hardness of 90.1 to 2, and a good yield of 90.1 to 2. ．． Contains 5 to 1 μkat of collagenase.

Preferred solutions of the invention for topical Pk application are bath liquids - 90.1 to 2, preferably Contains 0.5 to 1 μkat of collagenase.

Preferred compositions of the invention further comprise a neutral grotease on the one hand and an endonuclease on the other hand. Contains enzyme.

Preferably, grotease and endo for 0.350 μkat collagenase. The ratio of grotease to nuclease is 200 casein units (un). it≦9 ea19≦1nolytiques) or less, endonuclea The enzyme has 500 nuclease units. s) Suitable compositions of the present invention preferably contain the following three components: Genase 0.350/! 1 kat or more, -2 for neutral glothiase content 0 to 200 casein units, For one endonuclease, 30 to 500 nucleotides/7-units It is implied by the relative ratio corresponding to .

In a more preferred Mi pseudomorph, the relative ratios of the components to each other are -collagenase to -collagenase, respectively. - more than 0350 μkat for neutral glothiase, 20 to 50 μkat for neutral glothiase. Zein dissolution unit, 30 to 60 nuclease units for one endonuclease corresponds to

The composition of the present invention can be applied to wounds of the above-mentioned nature (eg, burns, ulcers).

- skin (esearres human collagen-based white hard 6 (68 earres) treatment of keloids (resou blanches), e.g. keloids left after surgery It turns out that it is particularly effective in theory. The composition of the present invention pre-decomposes all of the collagen in connective tissue in a reversal of the therapeutic action.

The composition does not attack myofibers, especially myofibers. Collagen dissolution in the composition This is because the sexual compound is inhibited by muscle myosin VC. It is often referred to as "Achromase". In particular, the composition or collagenase This name contains grotease and endonuclease in the above proportions. Use a nickname.

be. In particular, due to this qualitative effect, macrophages, white blood cells, and Achromas It accumulates at the periphery of the action zone of e. This accumulation of blood components leads to tissue regeneration processes. 0, which has an effect on M-effects on process (e.g., regeneration of damaged tissue due to ulcers and burns). Another feature of the action of Achromase on vegetatively altered tissues is that this action It is to stop at the organization that has come. The action of Achromase is to turn dead tissue into a new one. The protein component of OA and chromase that is broken down to the boundary with the tissue and stops at the point where it breaks down Living tissue is not injured by the action of decomposing compounds. im' live tissue This is because there is an inhibitory system inside. In particular, the following three blocking systems, i.e. 1°) non-damaging polypentate collagenase inhibitor present in tissues, 2°) myosin present in muscle tissue, and 3°) present in the blood circulation system Collagenase and grotease inhibitors, especially α-macroglobulin is involved.

What should be pointed out here is that the ``Oki'' Mi product has extremely active collagen bath activity, especially the above-mentioned V. The collagen lytic activity of the order of 111 shown should not be published. If the number of cases is 0, the uninvented collagen bath-degradable preparation to address the anticipated concerns. The selectivity of the product's action did not change even when the amount of collagenase was increased. That is, , the natural collagenase-blocking properties of healthy tissues and the collagen contained in healthy tissues. This is expected since the genease activity regulator properties and nl are fully exerted. Concerns about ``overdosing of enzymes'' were denied.

Neutral grotease, which forms the second active ingredient of Achromase, is a molecular compound. The action of collagenase by further subdividing fragments of the collagen-soluble starting substrate complement. The preparation used in the pharmaceutical composition of the present invention is When oiling hemovar iophagus, the grotease is preferably Contains no harmful ingredients.

Finally, the third fraction of Achromase, consisting primarily of endonucleases, is , helps destroy the DNA that gets released from the cells in the infected process, and thus free D N 0 that functions to destroy pus mainly composed of A Preferably, the grotease and endonuclease bound to collagenase are Polished rice to the same microorganism and cooked it in a microorganism culture medium. It is obtained at the same time as lagenase.

Conditions for adjusting the mutual ratio of these components will be described later.

The present invention further provides that the collagenase is a collagenase hydrolyzed product, in particular a fermentation of collagen. Preferred collagen-soluble compositions formulated with an effective preparation of It depends. 0 These hydrolysates are mainly derived from, for example, calf skin 7N. Average molecular weight less than io, ooo daltons, especially 6,000 to s, ooo dalton base It is composed of collagen fragments made up of gutide molecules. Hydrolyzed equivalent of these furthermore characterizes some of the constituent amino acids, in particular hydroxy-propylene All characteristics are the presence of phosphorus. For example, the characteristic collagen hydrolyzate is The following amino acids (derived from collagen) are divided into the following proportions - Proline: 8.4% -Hydroxyproline: 11.4% These collagen hydrolysis products are produced by dividing denatured or denatured collagenase as appropriate. It can be obtained by any method. 1. The term "hydrolyzed footwear" refers to For example, cerate is not limited to products obtained by chemical hydrolysis of C Also, the corresponding type of hydrolyzate is absorbed.

It is noteworthy that these collagen hydrolysates are present in the wound. is produced by the decomposition of necrotic collagen and is produced by the collagen-soluble composition of the present invention. It has the same properties as the product that is the target of hydrocatalysis.

Adding the collagenase of the present invention to the above-mentioned collagen = -/1゛odd1, = hydrolysis product. It was found that when added in a certain proportion, the same therapeutic activity of the compound of the present invention appears. Did 0 The collagenase composition obtained by adding these collagen hydrolysis products to the collagenase composition of the present invention Another very important advantage is that the uninvented Kolaken soluble composition has an extremely high storage stability. It will only get better.

Should the collagen hydrolyzate be incorporated into the collagenase preparation at the final stage of preparation? In particular, it would be 'Mausoleum I'. %, these collagen hydrolysates are produced in microbial culture media. Collagenase extracted from the solution can be directly cooled and added at the time of extraction. Limitation of extraction solution to remove all undesirable insoluble and soluble fractions contained in the culture medium After external filtration and additional abrasive treatment, A preferred pharmaceutical composition of the present invention is a collagen hydrolyzed product that has not been subjected to final freezing treatment. The product and collagenase are combined into collagenase 1 to 25, preferably 2 to 1. It contains M in a proportion comparable to that of Qm9 of Kolaken hydrolysis. less than the preferred rate It may be expressed as A preferred composition is 1 part collagen per microtar! l 11 to 25N, it goes without saying that the range shown above for the percentage of these - Whether or not rotiase and endonuclease are combined with collagen. It is perfectly valid.

Therefore, the main isobasho of uninvented mulberry H+iam is the accumulation ζ It consists in destroying easy tissue waste. Natural 4 scarring or by e.g. transplantation In order to start the formation of bush scars induced by this process, the above-mentioned destruction (cleaning) of the braid is necessary. I need to drink it.

The invention furthermore particularly relates to the method of farming the active preparations of the invention containing the three aforementioned fractions. In detail, this method is I am using the following. In the method of the present invention, the microorganisms are grown under conditions known per se. cultured to form a secondary structure, especially one with the helical chain characteristics of collagen or collagen. A suitable 71i: inducing factor is released into the culture medium using sufficient molecular weight of the collagen fragment. Separately, collagenase was stimulated by a method known per se and incubated in the culture medium. This incubation *== is continued until after the induction of neutral grotease production, and collagenase and protein The relative ratio of Teinase Koji to Convex Genbun Renkiji # reaches the desired value within each of the above ranges. The culture can then be interrupted and the active preparation can then be recovered from the medium in a manner known per se. Ru. In particular, after removal of the cells, it is sufficient to reach, for example, 60% of the approximate JK of the salt in the solution. Removes ions introduced into ammonium fertilization at a certain concentration. As a variant, the active ingredient may be purified by ultrafiltration.

In the above, the relative ratio of collagenase or grotease (or proteinase) was determined. We proposed that the entire culture period be adjusted in order to achieve the desired results. Bota, 70 te of external origin Collagenase may be complemented by a V-endonuclease, or On the other hand, if it is possible to purify this bath solution, the culture period can be extended and the collagen In order to increase the yield of enzyme, excess 70-teinase and endonuclease were removed. Good too.

This last treatment requires extremely high levels of collagenase molecules and proteinase molecules. It is a good idea to use all the different things. In detail, the molecular weight of collagenase is 30, The molecule of grotease is larger than 000 and is smaller than 30,000, so for example, Both can be easily separated using a suitable ultrafiltration membrane. In particular, culture interruption and bacterial After separation, the resulting medium can be subjected to three types of ultraviolet oxidation sequentially. It is possible. Initial ultrafiltration removes residual bacteria and removes collagenase and grotease. Obtain a clear liquid containing . In the second ultrafiltration, the molecular weight is 30,000 ρ. Proteins, especially collagenase, are separated using a total ultrafiltration membrane. In the third limit offshore passage, 1 Total recovery of grotease using an ultrafiltration membrane that can separate molecular weights greater than 0,000 I respect you. The collagenase and grotease are then mixed again at the desired ratio. be able to.

According to an additional feature of the invention already described, collagenase and optionally protein After optional further purification of the thease and endonuclease, especially straw fermentation. Collagen hydrolysis products are added to the bath solution before recovery by freeze-drying. may be added in advance, preferably in the proportions mentioned above. In other words, the above The bath liquid is supplemented with a Kolaken hydrolyzate product and then processed by lyophilization.

The collagen hydrolyzed into the dissolved collagenase before recovering the collagenase from the bath solution. The addition of degradation products is also a key step in the method aimed at stabilizing the collagenase. It turned out to be Tep. Therefore, this stabilization method is suitable for collagen Collagen in the presence of all of the collagen hydrolyzate in the relative ratio of the yoke to the collagen All features include the step of lyophilizing the enzyme-containing bath solution.

The techniques described below are used to quantify the various components of the composition. These people's The substrates, reactants and calculation methods used in the technique are also described below.

Z, f’ur Ph) rsiol, Chem, 333.149-151 ) substrate The substrate used was 4-phenylazo-benzyl oxane from Soc+≦LJFlulaI. C-carbonyl-L-Pro-Leu-Gly-L-Pro-D-Arg, 0 reactant with HCt (PZ-PLGPR) The reactants used are as follows: A. Buffer: The buffer consists of the following three solutions.

- Bath solution A1: 160mM veronal (5+5-diethyl)kulvitar 2, associate na thorium salt) and 143mM sodium acetate, 3H! D consisting of 0 One bath iA2: IN HCl; - Bath liquid h a: s, s% NaC6: Buffer' (i: To prepare, 200 Mix the solution layer A1 of - and the solution A3 of 80-, and use all of solution A2 to make pl (8.5 Adjust to. Add water to 1t and add CaCl2, 2H2O to final concentration of 4mM. Added.

B. Substrate solution: To prepare this solution, add 10μt of PZ-PLGPR to iooμt of methanol. dissolve in Add buffer to 10-. The substrate solution thus obtained is Weekly songs are stable.

C1 collagenase sample: A solution of 1~ is maintained in a 1~ buffer to obtain a collagenase sample. unknown Using a sample with specific activity, the optimal dilution is C according to the preliminary reading. , the colorimetric value of the optical density at 320 nm is in the range of 0.1 to 1.0 (later out).

D. Citric acid: This is a 0.5% aqueous solution.

E, ethyl acetate.

formulaic method Process with +j@ below.

1. Preheat solution B to 37°C.

2. (accurately diluted) 0.1-collagenase solution and preheated 0.41-nt. Mix with bath solution B and incubate at 37°C for 15 minutes. Add the bath liquid Dt1'' of λ1- and mix.

Add 4.5 td of ethyl acetate and stir vigorously. Next, remove the upper aiso phase. Remove, add anhydrous Na25O and dry.

6. Test according to steps 1 to 5 above using buffer instead of substrate (bath solution B). A control was prepared under the same conditions as Sanflu, and the test sample was heated to 320°C. Optical density is determined by comparison with control 0 A substance that cleaves peptides (PZ Pro-Leu) (manufactured by Fluka) Calculated from the standard curve obtained with compound B).

enzymolog n, 32: Kunitz M, (1947) J , Gen, Physiol, 30.291) Substrate The substrate used was casein for biochemistry (Merck's product No. 2244, the reactants used were: It is as below.

One solution A: This solution is Aa: 0.2M boric acid (IZ4g/10007) and Ab: 0.05M borax (1 9,059/i　000m). To obtain solution A, add 100 rnt of solution. Adjust liquid Aa to 7.6 degrees with bath liquid Ab (approximately 4 g).

-Solution B: 100ffi7 of 19 casein! in a water bath at 010°C. Dissolve the protein cloud obtained by heating until complete dissolution (approximately 15 minutes) can be maintained at 4°C for one week.

-Solution C 5% aqueous solution of trichloroacetic acid.

Groteinase San 1 Pre-quantitate the dilution of the sample containing grotainase into 2-10μq aliquots. Fushin/rnI! Adjust until it is equal to .

Spirit law This f'L is performed in the following procedure.

1. Twice the number of samples in order to correspond to all controls for each Sannor. Prepare a tube (Vidal 11 x 70 plastic tube).

2. Pour 0.5-liter bath solution B into a container and incubate at 137°C for 5 minutes.

& Add 0.5 d of sample to the container for 1 sample. Incubate for 0 minutes. 1. Add solution c6 of 5-.

4. Add 1,5-solution C2 to the 'control' tube and incubate at 037℃ for 5 minutes. Incubate and add 5 ml of sample.

5. All the tubes? Let stand overnight at 4℃0 6. Centrifuge at 8000 rpm for 10 minutes at room temperature. 07. Compare to corresponding control. and read the optical density at 280 (D0280) of the sample. calculation 1. Standard curve Dilute the pure trypsin solution (0-1m9/mc!HC2, 10M) and The final concentrations of trypsin from the strawberries were adjusted to 1, 2, 4, and 6° 8μ, respectively. Next, the above Perform steps 1 to 7.

2. An active unit corresponds to 1 μq of trypsin. The specific activity unit is This corresponds to 91μ of trypsin per η.

■ Quantification of thioxyribonuclease (Kunitz M, (1950) J .

Gen, Phyaol, 33.349-363) Reactant Ni using the following reactants: - (BOEHRINGER product) Cladero's thioxyribonuclea Ze I (2000U/m9) - Thioxyribonucleic acid (a BOEHRINGER product) Configure as below. 100mM Tris, HCl: 4mM M7SO4: 7 &O: 1.8 mM CaCl2, 2 H2O: pH 7.5 with hydrochloric acid adjust.

solution One enzyme standard = 1 ■ thioxyribonuclease I? ! -1rnt buffer Understand.

One substrate: dilute 150 μt in 50 fnl buffer.

Foot type method Follow the steps below.

1.3m7! The absorbance of the substrate solution at 260 nm is adjusted to 0.

Add 2100 μt of standard enzyme and mix.

3. Record all changes in absorbance for 10 minutes.

4. Determine the ratio ΔAzao (change in absorbance at 260 nm) 7 minutes.

5. Reproduce steps 1 to 4 using new substrate solution and unknown enzyme sample Then, determine the value of ΔA26°7.

calculation m9/rd=280 nTQ optical density K (DOtso) X 0.9 ΔA/min x 1000 x 6 LJ, (1951), J. Biol, Chem, 193.265) Reactants The following reactants are used.

ffJ Solution A: NaOH0, 2% Na1CO3 in IM knee bath solution B2 2% Na, potassium tartrate; - Bath liquid C: 1% CuSO4; One solution = 1-solution B + 1-solution C: Dilute to 100- with solution A.

1. ITn9 bovine serum albumin (BSA, manufactured by Sigma) was added to 2- Prepare a reference protein solution in water.

2. Dilute with water and add a series of dilute P containing 10 to 80μ to a final volume of 400μ. ! , prepare a reference solution.

3. Add the solution from 2-, stir, and let stand at room temperature for 10 minutes.

4. Add 200 μt of bath solution Et- and incubate for 10 minutes at 50°C in the dark. do.

& Comparison with control (water was used as a substitute for B5Al solution in step 2) Then read the optical density at 750 nM using standard white i.

quantitative Unknown sample file (i) treated with each dilution K (final amount 400 μt, step 2) do.

calculation 11 L child equivalent of reference bovine serum albumin (BSA) in protein content of unknown sample Indicated by

Vibrio alginolytic chemova, another characteristic of the present invention This will become clear from the following description regarding the culture conditions of R. riophagus. cormorant.

- Figure 1 shows the relative concentrations of the various enzymes whose production was induced during the cultivation of said bacteria over time. It is a graph shown as a function of.

- Figure 2 illustrates the inhibitory properties of myosin on collagenase.

The bacteria were grown in thiosulfate-citrate-bile-shofi (TCBS) medium for 30 min. Incubate overnight at ℃ to form round yellow colonies 20-30% in diameter. Indole production, lysine decarboxylase, gelatinase (thin film test)), Commercially available wetting agent esterase named TWEEN80, tetrathionate reduc tase, nitrate reduction, glucose, maltose, mannitol, sucrose, toreno Acid production from 10-s, mannose, glycerol, use of citrate (Sim mons). This bacterium is affected by the action of aminoglycoside antibiotics, chloramphenin Cole, tetracycline, colistin, rifampicin, sulfonamides, Nalixide acid (1'acide nalixide), this bacterium is tested by the following shows a negative reaction. Methyl red, alkynine dihydrolase, urease, fluoride Phenylalanine deaminase, 0NPG test, &S production (agar iron IGig ler), glucose gas generation, xylose, arabinose, adonitol, Rhamnose, nrubose, sorbitol, dulcit, lactose, inosytose Salicin, Raffinose, Medibiose, α-methylglucoside, Mucat Production of acid from. This bacterium does not utilize Maroney 1f.

Strain l-029 is extremely halophilic and grows optimally in sodium chloride at sD, ia%. It is clearly resistant to 15% sodium chloride. l, ampicillin It is completely resistant to carbenicillin and partially resistant to meji and cephalotene. O Positive reaction in lunitente carboxylase test and cellobiose fermentation test vinegar.

Culture conditions Tris, H (JO,IM and NacZo, 4M and 2mM CaCl2 , p+17.

For example, 25% casamino acids (Difco Labs, + Detroit, Mich,) Using the added medium, stir at 30°C and aerate with heel control (1.4 atm) and culture the cells. Two hours after the start of culture, add the product name ASF to the medium. Commercially available acids (peptide fragments of calf skin collagen: Add at % 1.Proliferation total condition) 1 at 600 nm for samples extracted every hour. The turbidity is measured by the absorbance of the sample.

Collagenase, proteinase and nuclease producing strains are extracellular nucleases. After the addition of ASF, collagenase It produces ze. At this time, the collagenase activity level is 60 nkat/- reach

In the graph of Figure 1, One piece of music is cell proliferation, The one-curve rotation is the activity of collagenase, One curve two is casein bath dissolvable The activity of roteinase, One song Md is nuclease activity 0 as a function of total time. Preferably, when the activity of collagenase has passed a maximum of 1 period, especially in the case shown, 5 Stop the culture after hours. All collagenase is concentrated and recovered from this medium. Furthermore, this Collagenase may be purified. A method that can be used for this purification is KEIL-D LOUHA, V, 1976゜"Chemical character zation and 5tudy of the autodjgestio nof pure collagenase from Achronoba cter 4 Bioehem.

Biopbys, Acta, 429.239-305-h', F, l'L ECROISEY.

V, KEIL-DLOUHA, D, L WOODS, D, PERRI N et B, KEIL.

1975, “Purification, 5tabiljty and 1nhibition of the collagenase from Ac hromobacter $FEBS Letters59, 167-172 ゜ It is mentioned in 扛.

Achromase-type preparations contain 0.312 parts of protein (dry weight) per total product η. quantity). This preparation is defined by the relative activity of “protein dry weight η”. Sexual pregnancy.

- Collagenase: 0.554 /jkat-Norotease double 45 casein bath Decomposition unit - endonuclease: 477 nuclease unit collagen lysis screen Stabilization of activity Collagener to stabilize collagenolytic activity when lyophilizing the entire preparation I use collagen hydrolysis for all people. This stabilizing effect occurs before freeze-drying. The retention rate (%) of the enzyme activity of collagenase preparations after a given amount can be expressed as In a test in which collagen hydrolyzate was added to collagenase in varying proportions, Therefore, it was measured. As a collagen hydrolyzate, it is sold in France. Etablisserrl: nts Rousselot S, A, products ASFi was used. This is a finely powdered Seratin that is soluble in cold water. The results obtained are shown in the table below. Relative ratio of Achromase and ASF used are shown in the left column of the table.

Examination of this table reveals the following central points: Ach without the existence of ASF When ronase bath G is freeze-dried, 50% of collagen bath Iy+ activity is lost. Jump rope.

- Addition of ASF significantly slows down the hostility decline.

A particularly advantageous result is that ASF 2 to 10 ASF5 for 1 part of Achromase in the ratio of parts by weight It has been found that the enzyme activity of the starting bath solution is maintained approximately at full capacity when the ratio is 1.5%.

In addition to maintaining enzyme activity during freeze-drying, freeze-dried collagenase FJItr! Ko Significant storage stability is obtained by addition of lagen hydrolyzate. That is, collagen The total amount of collagenase, including hydrolysis and other products, is 25 grams of collagen at 4℃ after 7 months. It showed a decrease in bath activity. Collagenase ll dobe part p1 to 10 double door part collage Collagenase preparations containing collagen hydrolysates were incubated at the same time and at the same temperature. Deactivation was maintained at 100%.

When the temperature was changed to 4°C and 20°C, the same stabilizing effect after 7 months was obtained by Kolaken digestion. A 30% reduction in activation was observed in the water-splitting collagenase preparation. However, in the tr preparation containing the same allocation as above, 5% There was only a decrease in activity.

Colleague's stabilization is achieved by another method based on collagenase (or Achromase). Preparations of the type, particularly ready-made leg preparations as described below, were also viewed with interest.

biological properties The obtained collagenase preparation not only has the above-mentioned collagenolytic activity but also has the collagenase preparation described above. It has the property of being inhibited by osin; myosin to collagenase The function of a non-competitive blocking factor has been demonstrated.゛ Collagen degradation by collagenase results in digested and solubilized collagen fragments. can be followed by the quantification of hydroxyproline liberated after hydrolysis of .

Hydroxyproline is a collagen l (specially 4h amino acid) and is a reaction mixture during quantitative determination. Other proteins contained in the compound are not involved.

Buffer Tris, HCI 0.02M, NaCl 0.23M, CaC 45,10-3M.

in pH 7.4 5 to 30 mg of muscle fiber collagen, -CaCJ, Q, l+ in 10-' - incubation buffer or - A mixture of 5 μg/g of myosin suspension in the buffer was incubated at 37°C for 1 hour. Incubate.

After incubation, the reaction mixture was centrifuged for 5 min at 12,00 l (0,5 Hydrolyze the supernatant with 0.5 p of H (J12N) at 110°C overnight. Vacuum dry the sample and place in an equal volume of water for hydroxyproline determination. .

The results of the above test using collagenase at a concentration of 0.098 μkat/mA' are This is shown in the graph in Figure 2. In Figure 2, the curve shows collagenase in the absence of myosin. The ratio 1/v (v is the number of moles of collagen digested per hour at 37°C ) as a function of the ratio 1/s (s is the number of moles of collagen used) (LIN Interpretation by WEAVER BURK).

The curve shows the change in the state of digestion of the same preparation in the presence of 5■ myosin. .

Examination of these graphs indicates that the collagenase preparation has activity on collagen. It turns out that sex is very significantly inhibited.

The collimate used in the present invention is completely non-toxic. This means that J, T, LICHTFIELD and F, WL WILCOXON method Can be proven by mortality test.

The preparation of the present invention has a collagenolytic activity of 0.17 μkat per η protein. The p-50 values were as follows.

-Mouse weight 98.58■.

This lethal dose is converted into units of collagenolytic activity of the preparation.

1.46 μkat per kg of mouse It showed lagenlytic activity.

In these tests, pig skin was pre-burned and treated with the collagenase-containing preparation of the invention. I treated it with something. A temperature of 80°C was applied to several places on the pig's back to cause burns. ℃ hot water was applied for a total of 15 minutes (6 wounds were made on the right side of the pig and 6 wounds were made on the left side).

Dead skin was removed from the burnt surface with boiling water and the wound was exposed to air for 48 hours.

Each couple was approximately 3.8 cm in diameter. Ointment containing collagen 2nase and collagener A double test was conducted with a similar ointment that did not contain any enzymes. The latter is a control Ru.

These cartilage sets are shown below: control ointment Monopolyoxyethylated alcohol 132-Hydroxylation to adjust pH7VC sodium.

- applied ointment to the wound on the left side.

Ointment application was repeated 3, 4, 6, and 13 days after burn occurrence, respectively.

The following observations were made 6 and 13 days after the burn injury occurred.

Burn condition after 6 days Depth of burn and thickness of remaining tissue were compared to burns treated with ointment and control composition. It is almost the same as a burn treated with an object.

Six of the burns treated with control ointment showed intact collagen fibers. Although the presence of skin skin was confirmed, burns treated with the collagenase-containing ointment of the present invention In that case, there was no epidermis at all.

In six burns treated with collagenase-containing ointment, inflammatory cells were present in the upper dermis. It was confirmed that there was an intrusion. In controlled burns, only those three phenomena are the same. showed an elephant. The invasion of inflammatory cells is greater than in burns where such invasion does not occur. This also proves that the formation of factory fatigue is progressing.

However, regarding the degree of healing of the epidermis and connective tissue, treated burns and control burns There is no big difference between the two. However, the degree of healing is slightly faster for treated burns.

Burn condition after 13 days In treated animals, the burns were covered with a thin film of degenerated tissue.

New connective tissue had developed all over the burn area, both directly beneath this thin film and in the deeper dermis. . The epidermis had developed in the form of granulation tissue from the periphery of the wound just below the degenerated tissue membrane.

In treated burns, the collagen part of the skin is dissolved by collagenase and the connective tissue is The regeneration of the tissue extends to the surface in the same manner as in the case of a cut. The formed epidermis is the granulation Can migrate to tissue surfaces.

On the other hand, control burns are covered with a deep skin made of dense collagen. This collagen attaches to and covers the tissue. new connective tissue from burns New skin that forms only on the periphery.It is formed only under the epidermal layer. below the shell The epidermis migrates from the periphery of the burn and penetrates the dermis. Therefore, the epidermis is layer 5j - tissue. separated from Therefore, new epidermal sources are found only at the periphery of the wound. these To ensure complete scarring of the tumor, it is necessary to automatically maintain the correct balance with existing tissue. The aim is to achieve the following. Reproducing this balance completely through surgical methods is It is considered extremely difficult.

The preparation of the present invention may have a shape that can be used as an external medicine. example For example, gels, emulsions, especially ointments, creams or solutions, or rIN mist. Available in Noh form (powder aerosol). Collagenase in each formulation depends on each dosage form mentioned above. It is used in combination with appropriate excipients. For topically applied solutions, isotonic media, Preferably, a physiologically acceptable, preferably sterile, pH 7-8.5 buffer is used. Advantageously, the entire collagenase bath solution is prepared using

Solution-based collagenase preparations using an injectable sterile vehicle can be used to treat scars immediately. It may also be injected subcutaneously near the keloid.

Such injectable solutions may also be used for total deep administration. In particular, e.g. Can be administered deeply to destroy pathological build-up of collagen within the body, such as.

Therefore, the pharmaceutical composition of the present invention can be used to prevent uncontrolled deterioration of collagen-rich structures. It can be used for any type of wound.

Examples of wounds that can be treated include burns, ulcers, skin lesions, collagen-based skin lesions, and skin lesions. For example, keloids that occur after surgery, necrosis, especially elephant death due to recumbency or ulcers. be. Pharmaceutical Mi compositions of the invention may also be used for prophylactic treatment. In particular, plasti or other implantation surgery or orthopedic surgery and the present invention in general Collagenase solutions can also be used to treat dental caries. i.e. well known The dental pulp is mainly composed of dense calcareous collagen. In tooth decay, there is a hole in the tooth. However, a hole opens and calcium leaks out. Therefore, calcareous matter is removed. Beniji The collagen frame that is left behind becomes porous and a breeding ground for bacterial infection. stomach.

When the concentrated collagenase solution of the present invention is introduced into the frame, porous collagen is dissolved. A solution is induced, which can be removed by washing with water.

In healthy calcareous collagen, the action of collagenase naturally ceases.

In particular, the collagen solution used is preferably buffered with borate to pH 7-8.5. Preferably, at least 1 μk of collagenase is contained per solution. child and even higher concentrations, the porous collagen dissolves rapidly.

After cleaning and disinfection, the treated tooth is sealed using techniques known to those skilled in the art. left in teeth The small amounts of collagenase that may be present are primarily acidic and recovered in the dental pulp. Gradually inhibited by pH.

It goes without saying that the present invention is not limited to the cases described above. This is clear from the following description. On the contrary, the invention includes all modifications. as a variant Especially for all the different collagenases that can be used as components of drugs. Some utilize the stabilizing properties of collagen hydrolysis products. As a drug ingredient The collagenase that can be used is one that has the above-mentioned selective activity and that can be used at the same time. Collagen exhibits desirable nontoxicity at doses sufficient to exert its full selective collagenolytic activity. It is genase.

Therefore, the present invention particularly applies to collagenase, regardless of the source of collagenase. Regardless of which microbial culture the enzyme was obtained from, all Contains a combination of collagenase and collagen hydrolyzate. These compositions In this case, the two components are mixed in a ratio similar to the above-mentioned preferred ratio range. In particular, some of the following claims may constitute equivalents of the claimed formulations. Ru.

Similarly, these solutions can be used to stabilize collagenases, especially Achromase 5. The method of freeze-drying a liquid in the presence of a collagen hydrolyzate is particularly effective for collagen hydrolysis. In particular, as long as the water splitting products are similarly used within the relative proportions defined above, It has the effect of stabilizing the collagen solution by freeze-drying.

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Claims (1)

[Claims] 1. In particular, the treatment of human or animal diseases in which uncontrolled deterioration of collagen-rich structures appears. containing an effective amount of collagenase together with a drug vehicle for therapeutic use; The collagenase has the peptide sequence X-glycyl-prolyl [wherein X is glycyl] specifically recognizes natural amino acid residues linked to the N-terminal end of sil residues. The sequence is specifically cleaved at the X-glycyl bond and is cleaved by myosin. A pharmaceutical composition characterized in that it is at least partially inhibited. 2. Collagenase is Vibrio alginolyticus chemo Claims characterized in that it is obtained from a culture of var. opha megagus. 1. The composition according to 1. 3. Collagenase is C, N, C, M. Bacteria deposited with accession number I-029 in 3. The composition according to claim 2, characterized in that it is obtained from a strain. 4. Collagenase content is expressed as collagenase activity relative to the total weight of the composition. At least 1 The range of emulsions, ointments, powders, or liquids in the ordinary sense actually includes 0.05 μkat/11 per drug composition for all compositions that are not rare; - 0.05 μkat/per pharmaceutical composition in the case of solutions formulated for topical use; mj! According to any one of claims 1 to 3, the amount is equal to pharmaceutical composition. 5. Emulsion/ointment, powder or non-truly liquid composition 0.1 to 2 preferred or containing 0.5 to 1 μkat/g collagenase, or per solution! l) Contains 0.1 to 2, preferably 0.5 to 1 μk/g of collagenase. A composition according to claim 4, characterized in that: 6. Also contains protease and endonuclease, and has a capacity of 0.350μka The ratio of protease and endonuclease to collagenase in Regarding tease, less than 200 casein units, and regarding endonuclease. Claims characterized in that the number of nuclease units is less than 500 nuclease units. 6. The pharmaceutical composition according to any one of 5 to 5. 7. The above three ingredients - Collagenase is 0.350 μkat or more, - Neutral protease is 20 to 20 0 casein lysis units - endonucleases 30 to 500 nucleases according to claim 6, characterized in that they are included in relative proportions corresponding to the units. Composition of. 8. The said ingredient - Collagenase activity is 0.350 μkat or more, - Neutral protease activity is 20 to 50 casein lysis units, - endonuclease activity of 30 to 60 nuclease units The combination according to claim 7, characterized in that the combinations are included in relative ratios corresponding to each other. A product. 9. Furthermore, Corabuna-41 weight part vehicle force 1 to 25 weight parts, preferably 2 to 10 weight parts The claim is characterized in that it contains a hydrolyzed product of collagen in a proportion of parts of Pharmaceutical composition according to any one of ranges 1 to 8. 10. 1 part by weight of collagenase to 25 parts by weight, preferably 2 to 10 parts by weight containing a collagen hydrolyzate together with the collagenase. Collagenase-based freeze-dried stable composition. 11. The collagenase contained in the composition is according to any one of claims 1 to 3. 11. The composition according to claim 10, wherein the composition is a collagenase as described above. ■ Collagen from an aqueous solution of collagenase without reducing collagenolytic activity. In order to obtain a lyophilized preparation of the lyophilization of said solution in the presence of a lagen hydrolyzate. Kyo 9 method. 13. Collagenase concentration of the collagenase hydrolysis product introduced in advance into the solution The ratio to the degree is 41 parts of collagen = 1xt hydrolyzed product 1 to 25 parts by weight, preferably 2 to 10 parts by weight. The method according to scope 12. 14. A solution used for the treatment of dental caries and other pulp deterioration in humans or animals. The composition according to any one of claims 1 to 10, which has a shape.