Collagenase composition and application thereof

Abstract

The invention provides a collagenase composition. The collagenase composition comprises type I collagenase, type II collagenase, clostridium protease and metalloproteinase. The collagenase composition comprises the following components in parts by mass: more than 75 parts of collagenase, 1-20 parts of metalloprotease and 0.05-3.0 parts of clostridium protease, and the mass ratio of the metalloprotease to the clostridium protease is 2-30. The collagenase composition is high in titer, good in stability and excellent in treatment effect on diseases (especially intervertebral disc protrusion).

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a collagenase composition and its application in treating diseases, in particular to treating intervertebral disc herniation. Background Art

[0002] Lumbar disc herniation (LDH) is common in adults and the elderly. It refers to a clinical syndrome caused by degenerative lesions of the lumbar disc, rupture of the annulus fibrosus, protrusion of the nucleus pulposus, and stimulation or compression of the nerve roots under various factors, especially external forces. Long-term pain seriously affects the quality of life and mental health of patients. It not only causes an economic burden on patients, but also easily causes psychological symptoms such as anxiety and depression in patients. At present, there are many ways to treat LDH, including conservative treatments such as bed rest and oral nonsteroidal anti-inflammatory drugs, traditional Chinese medicine such as massage and bioelectric therapy, minimally invasive treatments such as chemical nucleolysis, corticosteroid injection, and transforaminal endoscopic discectomy, as well as surgical operations. Among them, chemical nucleolysis is widely used due to its advantages of less trauma, less damage to spinal stability, good postoperative effect, and low cost.

[0003] Chemical nucleolysis can be understood as injecting a certain amount of collagenase into the herniated intervertebral disc. The collagenase will directly dissolve the nucleus pulposus tissue. At this time, the nucleus pulposus will denature and shrink, and its volume will decrease, thereby reducing the pressure on the nerve roots, improving the pain symptoms, and ultimately achieving the purpose of eliminating numbness and relieving pain.

[0004] Collagenase (abbreviated as collagenase) is a highly specific proteolytic enzyme that acts on the helical structure of collagen tissue. It has the function of hydrolyzing natural collagen at physiological pH and temperature. Collagenase is widely present in nature and can be isolated from human skin culture fluid and amphibian tissue fluid. Many microorganisms can produce collagenase, such as Pseudomonas, Achromobacter detoxificans, Clostridium and Candida albicans. Summary of the invention

[0005] In order to further improve the efficacy of treating intervertebral disc herniation, the present application combines collagenase with metalloproteinase and clostripain in a certain proportion. The results show that the activity level of specific hydrolysis of collagen substrate is higher and the stability is better, especially in the treatment of intervertebral disc herniation. It has a good efficacy, such as fast onset and better effect. The specific scheme is as follows:

[0006] The first aspect of the present invention provides a collagenase composition, wherein the collagenase composition comprises collagenase.

[0007] The collagenase can hydrolyze collagen or water-soluble denatured collagen.

[0008] Preferably, the collagenase can hydrolyze both water-soluble collagen and water-insoluble collagen.

[0009] The collagen can be natural collagen or non-natural collagen. The non-natural collagen at least includes a triple helix structure.

[0010] The collagenase may be of animal, microbial or plant origin.

[0011] Preferably, the collagenase is produced by a microorganism, such as Clostridium, Bacillus, Achromobacter, Pseudomonas, Klebsiella, Candida and / or Vibrio.

[0012] Preferably, the microorganism may be Hathewaya histolytica, Clostridium perfringens, Clostridium tetani, Bacillus cereus, Bacillus subtilis, Bacillus pumilus, Klebsiella pneumoniae or Candida albicans, etc.

[0013] In a specific embodiment of the present invention, the collagenase is obtained by culturing Clostridium histolytica.

[0014] Preferably, the culture medium formula for culturing Hathewaya histolytica includes, by mass percentage, 0.2-1% yeast extract, 0.2-1% yeast powder, 1%-3% gelatin, and 2%-6% peptone.

[0015] Preferably, the culture temperature is 37°C-42°C, such as 37°C, 38°C, 39°C, 40°C, 41°C or 42°C.

[0016] Preferably, the culture is static fermentation.

[0017] Preferably, the fermentation period of the static fermentation is 40-45 hours.

[0018] Calculated by weight, the collagenase composition comprises:

[0019] Collagenase: at least 75 parts; preferably at least 80 parts, more preferably at least 90 parts;

[0020] Metalloproteinase: 1-20 parts; and,

[0021] Clostridain (Endoproteinase-Arg-C): 0.05-3.0 parts.

[0022] In a specific embodiment of the present invention, the mass fraction of collagenase contained in the collagenase composition is 75-99 parts, preferably 82-99 parts, and further preferably 88-99 parts; for example, 75 parts, 79 parts, 80 parts, 81 parts, 83 parts, 85 parts, 87 parts, 88 parts, 91 parts, 93 parts, 94 parts, 95 parts, 96 parts or 99 parts, etc.

[0023] The collagenase includes type I collagenase and type II collagenase, wherein type I collagenase and type II collagenase can be obtained by extracting and purifying the fermentation liquid of Hathewaya histolytica static fermentation.

[0024] Preferably, the extraction comprises filtering the fermentation broth, precipitating with ammonium sulfate, and then dissolving the precipitate in a buffer solution and desalting under low temperature and nitrogen pressure to obtain a crude product.

[0025] Type Ⅰ collagenase was obtained by anion chromatography, cation chromatography and hydrophobic chromatography.

[0026] Type II collagenase was obtained after anion chromatography, cation chromatography, hydrophobic chromatography and molecular sieve chromatography.

[0027] In some embodiments, type I collagenase is translated from the colG gene and type II collagenase is translated from the colH gene.

[0028] Preferably, the mass ratio of type I collagenase to type II collagenase is 0.8-1.4, preferably 0.8-1.3, and further preferably 0.9-1.2; for example, 0.80, 0.81, 0.82, 0.90, 0.94, 0.95, 1, 1.02, 1.05, 1.07, 1.10, 1.15, 1.16, 1.17, 1.20, 1.21, 1.25, 1.28, 1.29, 1.30, 1.38 or 1.40, etc.

[0029] The mass fraction of type I collagenase in the collagenase composition is 20-55 parts, for example, 20 parts, 25 parts, 28.8 parts, 30 parts, 33 parts, 33.8 parts, 34 parts, 35 parts, 35.90 parts, 38.11 parts, 40 parts, 45 parts, 45.81 parts, 45.65 parts, 46.81 parts, 47.90 parts, 48.96 parts, 49.95 parts, 50 parts, 50.4 parts, 50.45 parts, 51.25 parts, 52.23 parts, 54.32 parts or 55.00 parts, etc., preferably 30-51 parts.

[0030] The mass fraction of type II collagenase in the collagenase composition is 30-60 parts, for example, 30 parts, 33.21 parts, 34.68 parts, 35.00 parts, 37.02 parts, 37.72 parts, 39.02 parts, 39.32 parts, 40 parts, 40.12 parts, 41.5 parts, 42.98 parts, 45.89 parts, 47.99 parts, 48.68 parts, 49.34 parts, 50 parts, 50.50 parts, 55 parts, 56.61 parts, 58.24 parts or 60 parts, etc., preferably 34-46 parts.

[0031] Preferably, the mass percentage of clostripain to collagenase is 0.1%-3%, preferably 0.3%-2%, for example 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.34%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.63%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 0.100%, 0.150%, 0.20%, 0.25%, 0.30%, 0.34%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.63%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.91%, 0.93%, 0.95%, 0.96%, 0.98%, 0.99%, 0.100%, 0.150%, 0.2 %, 0.95%, 1%, 1.10%, 1.20%, 1.23%, 1.30%, 1.34%, 1.40%, 1.50%, 1.52%, 1.60%, 1.70%, 1.80%, 1.89%, 1.90%, 1.96%, 2%, 2.10%, 2.20%, 2.30%, 2.40%, 2.50%, 2.60%, 2.70%, 2.80%, 2.90% or 3%, etc.

[0032] Preferably, the mass fraction of clostripain in the collagenase composition is 0.05-3.0 parts, for example 0.05 parts, 0.10 parts, 0.20 parts, 0.24 parts, 0.30 parts, 0.33 parts, 0.34 parts, 0.40 parts, 0.50 parts, 0.54 parts, 0.60 parts, 0.70 parts, 0.80 parts, 0.90 parts, 1 part, 1.10 parts, 1.20 parts, 1.24 parts, 1.30 parts, 1.40 parts, 1.50 parts, 1.54 parts, 1.60 parts, 1.63 parts, 1.70 parts, 1.80 parts, 1.90 parts, 2 parts, 2.50 parts, 2.6 parts, 2.7 parts, 2.8 parts, 2.9 parts or 3 parts, etc., preferably 0.2-3 parts.

[0033] The metalloprotease can be obtained by fermenting Hathewaya histolytica or by prokaryotic expression.

[0034] Preferably, the mass percentage of metalloproteinase to collagenase is 1%-25%, more preferably 3%-22%, for example 1%, 2%, 2.15%, 3%, 3.80%, 4%, 4.84%, 5%, 6%, 7%, 8%, 8.21%, 9%, 9.04%, 10%, 11%, 12%, 13%, 13.07%, 14%, 14.97%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 21.56%, 22%, 23%, 24% or 25%, etc.

[0035] Preferably, the mass fraction of metalloprotease in the collagenase composition is 1-20 parts, for example, 1 part, 2 parts, 3 parts, 3.36 parts, 3.46 parts, 3.84 parts, 4.00 parts, 4.48 parts, 5.00 parts, 6.00 parts, 6.97 parts, 7 parts, 8 parts, 8.77 parts, 9 parts, 9.9 parts, 10 parts, 10.06 parts, 11 parts, 11.06 parts, 12 parts, 12.48 parts, 13 parts, 13.07 parts, 14 parts, 14.97 parts, 15 parts, 16 parts, 16.1 parts, 17 parts, 17.10 parts, 18 parts, 18.29 parts, 19 parts or 20 parts, etc., preferably 3-17 parts.

[0036] Preferably, the mass ratio of metalloprotease to clostripain is 2-30, preferably 3-27 or 8-27, for example 2, 2.88, 3, 3.61, 4, 5, 5.75, 6, 6.55, 7, 7.66, 8, 8.11, 8.4, 9, 9.34, 10, 11, 11.29, 11.40, 12, 13, 13.24, 14, 15, 16, 16.56, 17, 18, 19, 19.31, 20, 20.48, 21, 22, 22.27, 23, 24, 25, 25.79, 26, 27, 27.16, 28, 28.12, 29, 29.37 or 30, etc.

[0037] Preferably, the collagenase composition comprises, by weight:

[0038] 20-55 parts of type I collagenase, for example, 20 parts, 25 parts, 28.8 parts, 30 parts, 33 parts, 33.8 parts, 34 parts, 35 parts, 35.90 parts, 38.11 parts, 40 parts, 45 parts, 45.81 parts, 45.65 parts, 46.81 parts, 47.90 parts, 48.96 parts, 49.95 parts, 50 parts, 50.4 parts, 50.45 parts, 51.25 parts, 52.23 parts, 54.32 parts or 55 parts, etc.;

[0039] 30-60 parts of type II collagenase, for example 30 parts, 33.21 parts, 34.68 parts, 35 parts, 37.02 parts, 37.72 parts, 39.02 parts, 39.32 parts, 40.00 parts, 40.12 parts, 41.5 parts, 42.98 parts, 45.89 parts, 47.99 parts, 48.68 parts, 49.34 parts, 50.00 parts, 50.50 parts, 55 parts, 56.61 parts, 58.24 parts or 60 parts;

[0040] 1-20 parts of metalloproteinase, for example, 1 part, 2 parts, 3 parts, 3.36 parts, 3.46 parts, 3.84 parts, 4.00 parts, 4.48 parts, 5 parts, 6 parts, 6.97 parts, 7 parts, 8 parts, 8.77 parts, 9 parts, 9.9 parts, 10 parts, 10.06 parts, 11 parts, 11.06 parts, 12 parts, 12.48 parts, 13 parts, 13.07 parts, 14 parts, 14.97 parts, 15 parts, 16 parts, 16.1 parts, 17 parts, 17.10 parts, 18 parts, 18.29 parts, 19 parts or 20 parts, etc.;

[0041] The amount of clostripain in the mixture can be 0.05-3.0 parts, for example, 0.05, 0.10, 0.20, 0.24, 0.30, 0.33, 0.34, 0.40, 0.50, 0.54, 0.60, 0.70, 0.80, 0.90, 1, 1.10, 1.20, 1.24, 1.30, 1.40, 1.50, 1.54, 1.60, 1.63, 1.70, 1.80, 1.90, 2, 2.50, 2.6, 2.7, 2.8, 2.9 or 3 parts, etc.

[0042] Further preferably, the collagenase composition comprises, by weight:

[0043] Type I collagenase 30-51 copies;

[0044] Type II collagenase 34-46 copies;

[0045] 3-17 parts of metalloproteinase;

[0046] 0.2-3.0 parts of clostripain.

[0047] Preferably, the collagenase composition may also include one or more of a pH regulator, an excipient, a stabilizer, a surfactant, a solubilizer, a cosolvent, an antioxidant, a buffer, a suspending agent, an emulsifier, a flavoring agent, a thickener, a wetting agent, a filler, an adhesive, a lubricant, a disintegrant, an antibacterial agent and a preservative.

[0048] Preferably, the collagenase composition can be in any suitable form, such as liquid (such as solution, emulsion or suspension), solid, semi-solid or gas.

[0049] Preferably, the collagenase composition is a medicine, a cosmetic, an animal feed or a food.

[0050] The second aspect of the present invention provides a method for preparing the above collagenase composition, which comprises mixing the components.

[0051] The third aspect of the present invention provides a method for preparing the above collagenase composition, the method comprising performing static fermentation on Clostridium histolytica to obtain a fermentation broth, and extracting the fermentation broth to obtain a crude product.

[0052] The extraction comprises filtering the fermentation liquid, precipitating with ammonium sulfate, and then dissolving the precipitate in a buffer solution and desalting under low temperature and nitrogen pressure.

[0053] In a specific embodiment of the present invention, the crude product is chromatographed on a DEAE cellulose column and / or a Sephadex G-25 column to obtain a collagenase composition.

[0054] The equilibrium buffer for DEAE cellulose column chromatography is HAC-NaAc (preferably pH 5-6), and the equilibrium buffer for Sephadex G-25 column is conductive water (preferably pH 6-7.5).

[0055] In a specific embodiment of the present invention, the crude product is purified to obtain type I collagenase and type II collagenase.

[0056] Preferably, the purification to obtain type I collagenase includes anion chromatography, cation chromatography, and hydrophobic chromatography.

[0057] Preferably, the purification of type II collagenase includes anion chromatography, cation chromatography, hydrophobic chromatography and molecular sieve chromatography.

[0058] The preparation method also includes obtaining the metalloproteinase by prokaryotic expression.

[0059] The amino acid sequence of the metalloprotease is shown in A0A4U9R6P3 (uniprot encoding).

[0060] In a specific embodiment of the present invention, the collagenase composition comprises a mixture of purified type I collagenase and type II collagenase, prokaryotic expressed metalloproteinase and clostripain.

[0061] The fourth aspect of the present invention provides a medicine, which comprises the collagenase composition, and preferably further comprises a pharmaceutically acceptable excipient.

[0062] In a specific embodiment of the present invention, the pharmaceutically acceptable excipients include one or more of pH regulators, excipients, stabilizers, surfactants, solubilizers, cosolvents, antioxidants, buffers, suspending agents, emulsifiers, flavoring agents, thickeners, wetting agents, fillers, binders, lubricants, disintegrants, antibacterial agents and preservatives.

[0063] The drug can be in any suitable dosage form, such as a dosage form for gastrointestinal administration or a dosage form for parenteral administration, such as tablets, powders, emulsions, injections (preferably solution injections), suspensions, capsules, pills, granules, powder injections, lozenges, sprays, aerosols, powder sprays, lotions, ointments, pastes, patches, eye drops, nasal drops, suppositories, effervescent tablets, pills, gels and films.

[0064] The drug may contain a collagenase composition in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).

[0065] The medicine is for human use or veterinary use.

[0066] The fifth aspect of the present invention provides a method for hydrolyzing collagen, which comprises mixing the collagenase composition with collagen.

[0067] Preferably, the hydrolysis temperature is 30-40°C, preferably 35-40°C, for example 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C, etc.

[0068] Preferably, the hydrolysis pH is 6-9, preferably 7-8, such as 6, 6.5, 7, 7.5, 8, 8.5 or 9, etc.

[0069] The sixth aspect of the present invention provides collagen obtained by the method described in the fifth aspect.

[0070] The seventh aspect of the present invention provides an application of the collagenase composition in food, brewing, meat processing, cosmetics or animal feed, or in the preparation of drugs for treating and / or preventing diseases.

[0071] Preferably, the disease comprises tissue fibrosis, intervertebral disc herniation (such as lumbar disc herniation or cervical disc herniation), Dupuytren's contracture, chronic total coronary artery occlusion, diabetes, ulcer (such as bedsore ulcer, arterial ulcer, dermal ulcer, venous ulcer, stasis ulcer), arthritis (such as osteoarthritis or rheumatoid arthritis), burns, wound healing or scar repair.

[0072] The scars may be surgical scars, hypertrophic scars, cosmetic scars, keloids, and the like.

[0073] The eighth aspect of the present invention provides a method for treating and / or preventing a disease, which comprises administering an effective amount of the above-mentioned collagenase composition or drug to a subject in need thereof.

[0074] The administration method can be any suitable administration route, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0075] Beneficial effects:

[0076] 1. The combination of collagenase, metalloproteinase and clostripain in a specific ratio effectively improves the therapeutic effect of intervertebral disc herniation.

[0077] 2. The combination of collagenase, metalloproteinase and clostripain in a specific ratio has higher potency and better stability.

[0078] 3. Metalloproteinases and clostripain, which are often used as impurities in the prior art, are unexpectedly found to have higher potency and better efficacy in treating intervertebral disc herniation when combined with collagenase, which simplifies the impurity removal steps in the prior art when preparing collagenase by fermentation method. DETAILED DESCRIPTION

[0079] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] Unless otherwise defined, all scientific and technical terms used in the application have the same meanings as commonly understood by one of ordinary skill in the art to which the invention relates.

[0081] The "metalloprotease" described in the present invention is a type of protease that can efficiently hydrolyze proteins and polypeptides. Different from the hydrolysis mechanism of other proteases, the metalloprotease has one or more metal ions at its active neutral site.

[0082] The "clostridain (Endoproteinase-Arg-C)" of the present invention is a double-chain protease isolated from Clostridium histolyticum, which has esterase, amidase and protease activities and has a high specificity for the carboxyl peptide bond of arginine.

[0083] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0084] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0085] The term "prevention" as used herein refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in an organism.

[0086] The "effective amount" of the present invention refers to the amount or dosage of the collagenase composition or drug of the present invention that provides the desired treatment or prevention after being administered to a subject or an organ in a single or multiple doses.

[0087] The "subject" described in the present invention can be a human or non-human mammal, or a cell, tissue or organ of a human or non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0088] The disclosures of various publications, patents, and published patent specifications cited in this application are incorporated herein by reference in their entirety.

[0089] Unless otherwise specified, the materials used in the embodiments of the present invention are all commercially available.

[0090] Unless otherwise specified, the parts, percentages or ratios described in the embodiments of the present invention are all based on mass.

[0091] Example 1: Preparation of collagenase composition

[0092] The collagenase composition described in this embodiment is obtained by fermenting Clostridium histolytica using an improved culture medium. The improved culture medium formula: in terms of mass percentage, it is 0.2-1% yeast extract, 0.2-1% yeast powder, 1%-3% gelatin, and 2%-6% meat peptone. Fermentation conditions: at 37°C-42°C, static fermentation, the cycle is 40-45 hours, the fermentation liquid is sterile filtered, and ammonium sulfate is added to precipitate at below 10°C to make the concentration reach 45-55% (weight / volume). The obtained precipitate is dissolved in pH 7-8, Tris-Ca 2+ The buffer was desalted by ultrafiltration membrane under low temperature and nitrogen pressure, and the crude collagenase was obtained by freeze drying. After chromatography on a DEAE cellulose column balanced with HAC-NaAc buffer at pH 5-6, the column liquid was further passed through a Sephadex G-25 column balanced with conductivity water at pH 6.0-7.5 to obtain a collagenase composition, which was structurally identified to contain type I and type II collagenase, metalloproteinase and clostripain.

[0093] Example 2-8: Preparation of collagenase composition

[0094] The collagenase described in this embodiment is obtained by fermenting Clostridium histolytica using an improved culture medium. The improved culture medium formula: in terms of mass percentage, it is 0.2-1% yeast extract, 0.2-1% yeast powder, 1%-3% gelatin, and 2%-6% meat peptone. Fermentation conditions: at 37°C-42°C, static fermentation, the cycle is 40-45 hours, the fermentation liquid is sterile filtered, and ammonium sulfate is added to precipitate at below 10°C to make the concentration reach 45-55% (weight / volume). The obtained precipitate is dissolved in pH 7-8, Tris-Ca 2+ The buffer solution was desalted by ultrafiltration membrane under low temperature and nitrogen pressure, and the crude collagenase was obtained by freeze drying. The crude collagenase was subjected to anion chromatography, cation chromatography, and hydrophobic chromatography to obtain pure type I collagenase (purity>95%), and to anion chromatography, cation chromatography, hydrophobic chromatography, and molecular sieve chromatography to obtain pure type II collagenase (purity>95%).

[0095] Clostripain was purchased from a commercial reagent (Sigma, Lot: BCCD0771).

[0096] A0A4U9R6P3 (uniprot encoding) was used as an example for protein expression and purification of metalloprotease. Plasmid was constructed by gene synthesis, and protein expression and purification were performed using an E. coli expression system to obtain pure metalloprotease (purity> 95%).

[0097] Example 2-8 is pipetted according to the proportions in Table 1, and then mixed and shaken to mix well.

[0098] Table 1

[0099]

[0100] Comparative Examples 1-5: Preparation of Collagenase Compositions

[0101] In comparative examples 1-5, type I collagenase, type II collagenase and metalloproteinase were prepared according to the method of examples 2-8, and clostripain was purchased as a commercial reagent (Sigma, Lot: BCCD0771). The mixture was pipetted according to the proportions in Table 2, and then mixed and shaken.

[0102] Comparative Example 5 was pipetted according to the ratio in Table 2, and a certain mass of human serum albumin (purchased from Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd., batch number 202303035A) was also pipetted to make the total mass fraction of the collagenase composition consistent with that of Example 7.

[0103] Table 2

[0104]

[0105] Effect Example 1: Potency Determination

[0106] Exemplarily, the potency of Examples 2-8 and Comparative Examples 1-5 was determined.

[0107] 1. The method for determining the potency is:

[0108] The collagenase composition obtained in each example acts on collagen at 37°C and pH 7.5. The collagen hydrolyzed per hour is colored with ninhydrin, and leucine is calculated, with 1 micromole of leucine as one activity unit. Each example and comparative example is measured three times in parallel.

[0109] 2. The results are shown in Table 3.

[0110] The potency test results of Examples 2-8 and Comparative Examples 1-5 are shown in Table 3.

[0111] Table 3

[0112]

[0113]

[0114] In the table, "*" represents the P value of the comparison between the potency of the comparative example and the potency of the corresponding example, *P<0.05, **P<0.01.

[0115] The results in Table 3 show that the titers of the collagenase compositions of each ratio in the examples are higher than those in the comparative examples. Further analysis is as follows:

[0116] Comparative Example 1 is compared with Example 7. The total mass fraction of (type I + type II), the mass fraction of metalloproteinase and clostripain are similar, all within the required range, only the type I / type II ratio is not within the range, type I / type II is 0.72, and the average potency of the collagenase composition (1449U / mg) is lower than the average potency of Example 7 (1698U / mg). SPSS25 software was used for T test, and the results showed that there was a significant difference.

[0117] Comparative Example 2 is compared with Example 4. The total mass fraction of (type I + type II), the mass fraction of metalloproteinase and clostripain are similar, all within the required range, only the type I / type II ratio is not within the range, type I / type II is 1.53, and the average potency of the collagenase composition (1543U / mg) is lower than the average potency of Example 4 (2104U / mg). SPSS25 software was used for T test, and the results showed that there was a significant difference.

[0118] Compared with Example 7, the total mass fraction of (Type I + Type II) in Comparative Example 3 and the ratio of Type I / Type II are similar thereto, the metalloproteinase / clostripain is not within the ratio range, the metalloproteinase / clostripain is 1.80, and the average potency of the collagenase composition (1483 U / mg) is lower than the average potency of Example 7 (1698 U / mg). SPSS25 software was used for T test, and the results showed significant differences.

[0119] In Comparative Example 4, the total mass fraction (type I + type II) is 70.95, the collagenase content is low, and the ratio of metalloproteinase / clostripain is high, which is not within the range. The average potency of the collagenase composition is lower than the average potency of the embodiments in which all components are within the range.

[0120] The total mass fraction of (type I + type II) in comparative example 5 is 75.03, and type I / type II is similar to that in example 7, but it does not contain metalloproteinase and clostripain, so the average potency of its collagenase composition (1464U / mg) is lower than the average potency of example 7 (1698U / mg), and the results of T test using SPSS25 software show that there is a significant difference. It can be seen that the presence of metalloproteinase and clostripain has a certain synergistic effect on the activity of collagenase.

[0121] Effect example 2: Animal experiment

[0122] Exemplary selected Examples 3, 4, 5, 7 and Comparative Examples 1-4 were subjected to animal experiments.

[0123] 1. Experimental method: Extraction of rabbit nucleus pulposus and annulus fibrosus in vitro

[0124] (1) Collection method

[0125] First, the rabbit was anesthetized with urethane at a dose of 4 mL / kg. The hair on the back was removed, the skin and subcutaneous fascia on the back of the rabbit were cut open, the back muscle tissue was separated with a scalpel, and the lumbar part of the spine was carefully and completely removed with large scissors; the muscle tissue on the spine was peeled off with small scissors, and the nucleus pulposus and annulus fibrosus were completely removed with hemostatic forceps and a scalpel and placed in saline for later use.

[0126] (2) The removed nucleus pulposus and annulus fibrosus or other tissues are placed in the prepared collagenase composition solution and incubated in a 37° C. water bath, and the weight of the remaining tissue is recorded at a specific time point during the incubation process.

[0127] 2. Test results

[0128] 1) Changes in the weight of the nucleus pulposus and annulus fibrosus over time after the action of different proportions of collagenase compositions

[0129] Collagenase is an enzyme that acts on the helical structure of collagen with high specificity, and can hydrolyze natural collagen under physiological pH and temperature conditions. The nucleus pulposus and annulus fibrosus of lumbar intervertebral disc contain a large amount of collagen fibers, and it can be concluded that collagenase can dissolve the nucleus pulposus and annulus fibrosus. In the nucleus pulposus / annulus fibrosus (STR) model, after incubation of different proportions of collagenase compositions for 4, 8, 12, 24, 48 and 72 hours, the tissue weight has a certain decrease compared with 0 hours, wherein the decrease of Examples 3, 4, 5, 7 is the most significant, and the intensity of action is better than Comparative Examples 1-4. As the different time changes, the weight of each group gradually decreases, and the weight decrease of Examples 3, 4, 5, 7 is the most obvious (P < 0.01) at 72 hours, and the specific results are shown in Table 4.

[0130] Table 4

[0131]

[0132]

[0133] In the table, "*" represents the P value of the comparison between nucleus pulposus / annulus fibrosus (STR) at different time points and 0 hours.

[0134] *P<0.05, **P<0.01.

[0135] It can be seen from Table 4 that, compared with Example 7, in Comparative Example 1, only the Type I / Type II mass ratio is too low, and the ratios of other components are within the required range. The weights of the nucleus pulposus and the annulus fibrosus after the action of the collagenase composition are significantly different from those in Example 7; compared with Example 4, in Comparative Example 2, the Type I / Type II mass ratio is too high, and the ratios of other components are within the required range. The weights of the nucleus pulposus and the annulus fibrosus after the action of the collagenase composition are significantly different from those in Example 4.

[0136] Compared with Example 7, the ratio of metalloproteinase / clostripain in Comparative Example 3 is lower, which is 1.80, and the ratios of other components are within the required range. The weight of the nucleus pulposus and annulus fibrosus after the action of the collagenase composition is significantly different from that in Example 7.

[0137] In Comparative Example 4, the total mass fraction (Type I + Type II) is 70.95, the collagenase content is low, the ratio of metalloproteinase / clostripain is high and is not within the range. Even if the Type I / Type II mass ratio is within the range, the weight of the nucleus pulposus and annulus fibrosus after the action of the collagenase composition is significantly different from that of the embodiments in which all components are within the range.

[0138] Effect Example 3: Stability Experiment

[0139] Exemplary selected Examples 3, 4, 5, 7 and Comparative Examples 3-4 were subjected to stability experiments.

[0140] 1. Test method:

[0141] The collagenase compositions in the examples and comparative examples were placed in a 30°C water bath for incubation, and taken out after 0, 2, 4, 8, 12, and 24 hours for potency testing, and evaluated using the potency retention rate (%), where the potency retention rate (%) is the percentage of the potency at the corresponding time point to the potency at 0 hour.

[0142] 2. Experimental results:

[0143] After incubation of different proportions of collagenase compositions for 0, 2, 4, 8, 12 and 24 hours, the titer of each decreased to a certain extent, and the decreasing trend of comparative examples 3-4 was the most obvious. After incubation for 8 hours, the titer retention rates of Examples 3, 4, 5, and 7 were more than 90%, while comparative examples 3-4 had dropped to less than 80%. After incubation for 24 hours, the titer retention rates of Examples 3, 4, 5, and 7 were more than 50%, while comparative examples 3-4 had dropped to less than 30%. The specific results are shown in Table 5.

[0144] Table 5

[0145]

[0146] As can be seen from Table 5, compared with Example 7, the ratio of metalloproteinase / clostripain in Comparative Example 3 is lower, which is 1.80, and the proportions of other components are within the required range. After incubation in a 30°C water bath for 24 hours, the potency retention rate of the collagenase composition is 25%, which is lower than the potency retention rate of 51% in Example 7, and the stability is poorer than that in Example 7.

[0147] In Comparative Example 4, the total mass fraction (Type I + Type II) is 70.95, the collagenase content is low, the ratio of metalloproteinase / clostripain is high and is not within the range, even if the Type I / Type II mass ratio is within the range, the potency retention rate of the collagenase composition after incubation in a 30°C water bath for 24 hours is 16%, which is lower than the potency retention rate of the embodiment in which all components are within the range, and the stability is relatively poor.

[0148] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0149] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A collagenase composition, characterized in that: Calculated by weight, the collagenase composition comprises: Collagenase: at least 75 parts; Metalloproteinase: 1-20 parts; Clostripain: 0.05-3.0 parts; Wherein, the mass ratio of metalloprotease to clostripain is 2-30.

2. The collagenase composition according to claim 1, characterized in that The collagenase composition contains collagenase in an amount of 75-99 parts by mass, preferably 82-99 parts by mass, and more preferably 88-99 parts by mass.

3. The collagenase composition according to claim 1, characterized in that The collagenase includes type I collagenase and type II collagenase, and the mass ratio of type I collagenase to type II collagenase is 0.8-1.4, preferably 0.8-1.3, and more preferably 0.9-1.

2.

4. The collagenase composition according to claim 1, characterized in that The mass ratio of metalloprotease to clostripain is 8-30.

5. The collagenase composition according to any one of claims 1 to 4, characterized in that: The collagenase composition comprises, by weight: Type Ⅰ collagenase: 20-55 parts; Type II collagenase: 30-60 parts; Metalloproteinase: 1-20 parts; Clostripain: 0.05-3.0 parts.

6. The collagenase composition according to any one of claims 1 to 5, characterized in that: The collagenase composition comprises, by weight: Type I collagenase: 30-51 copies; Type II collagenase: 34-46 copies; Metalloproteinases: 3-17 parts; Clostripain: 0.2-3.0 parts.

7. The collagenase composition according to any one of claims 1 to 6, characterized in that: The collagenase composition is medicine, cosmetics, food or animal feed.

8. A drug, characterized in that The medicine comprises the collagenase composition according to any one of claims 1 to 7, and pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The pharmaceutically acceptable excipients are selected from one or more of pH regulators, excipients, stabilizers, surfactants, solubilizers, cosolvents, antioxidants, buffers, suspending agents, emulsifiers, flavoring agents, thickeners, wetting agents, fillers, adhesives, lubricants, disintegrants, antibacterial agents and preservatives.

10. The drug according to claim 8 or 9, characterized in that The dosage form of the drug is any one of tablets, powders, emulsions, injections (preferably solution-type injections), suspensions, capsules, pills, granules, powder injections, lozenges, sprays, aerosols, powder sprays, lotions, ointments, pastes, patches, eye drops, nasal drops, suppositories, effervescent tablets, pills, gels and films.

11. A method for hydrolyzing collagen, characterized in that: The method comprises mixing the collagenase composition according to any one of claims 1 to 7 with collagen.

12. Use of the collagenase composition according to any one of claims 1 to 7 in food, brewing, meat processing, cosmetics or animal feed, or in the preparation of a drug for treating and / or preventing a disease; Preferably, the disease comprises tissue fibrosis, intervertebral disc herniation (such as lumbar disc herniation or cervical disc herniation), Dupuytren's contracture, chronic total coronary artery occlusion, diabetes, ulcer (such as bedsore ulcer, arterial ulcer, dermal ulcer, venous ulcer, stasis ulcer), arthritis (such as osteoarthritis or rheumatoid arthritis), burns, wound healing or scar repair.

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