A collagenase composition and its application

Abstract

The present invention provides a collagenase composition, which comprises type I collagenase, type II collagenase, clostripain and metalloproteinase. The collagenase composition includes more than 75 parts by mass of collagenase, 1-20 parts by mass of metalloproteinase, and 0.05-3.0 parts by mass of clostripain, and the mass ratio of metalloproteinase to clostripain is 2-30. The collagenase composition has a high titer, good stability, and excellent curative effect in treating diseases (especially lumbar disc herniation).

Description

Technical Field

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

[0002] Lumbar disc herniation (LDH) is common in adults and the elderly. It refers to a clinical syndrome caused by the degeneration of the lumbar intervertebral disc. Under various factors, especially external forces, the annulus fibrosus ruptures and the nucleus pulposus protrudes, irritating or compressing the nerve root. Long-term pain seriously affects the quality of life and mental health of patients, not only causing economic burden to patients, but also easily leading to psychological symptoms such as anxiety and depression. Currently, there are various ways to treat LDH, including conservative treatments such as bed rest and oral non-steroidal anti-inflammatory drugs, traditional Chinese medicine therapies such as massage and bioelectric therapy, minimally invasive treatments such as chemonucleolysis, corticosteroid injection, and arthroscopic surgery, as well as surgical operations. Among them, chemonucleolysis is widely used due to its advantages such as small trauma, little damage to spinal stability, good postoperative effect, and low cost.

[0003] Chemonucleolysis can be understood as injecting a certain amount of collagenase into the herniated intervertebral disc. The collagenase directly dissolves the nucleus pulposus tissue. At this time, the nucleus pulposus degenerates and retracts, with a smaller volume, thereby reducing the compression on the nerve root and improving the pain symptoms, ultimately achieving the purpose of eliminating numbness and alleviating pain.

[0004] Collagenase, also known as collagenase, is a proteolytic enzyme that specifically acts on the helical structure of collagen tissue. Under physiological pH and temperature, it has the function of hydrolyzing natural collagen. Collagenase widely exists in nature and can be isolated from human skin culture fluid and the tissue fluid of amphibians. Many microorganisms can produce collagenase, such as Pseudomonas, Achromobacter insolitus, Clostridium, and Candida albicans. Summary of the Invention

[0005] In order to further improve the curative effect of treating lumbar disc herniation, in this application, collagenase is combined with metalloproteinase and clostripain under certain proportion conditions. The results show that the activity level of specifically hydrolyzing collagen substrates is higher and the stability is better. Especially in treating lumbar disc herniation, it has a good curative effect, such as rapid onset and more excellent effect. The specific scheme is as follows:

[0006] In the first aspect of the present invention, a collagenase composition is provided. The collagenase composition includes collagenase.

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

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

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

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

[0011] Preferably, the collagenase is produced by microorganisms. The microorganisms are, for example, Clostridium, Bacillus, Achromobacter, Pseudomonas, Klebsiella, Candida and / or Vibrio, etc.

[0012] Preferably, the microorganisms can 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 Hathewaya histolytica.

[0014] Preferably, the culture medium formula for culturing Hathewaya histolytica, calculated by mass percentage, includes 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, etc.

[0016] Preferably, the culture is static fermentation.

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

[0018] The collagenase composition described above, by mass parts, 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 parts of collagenase contained in the collagenase composition are 75 - 99 parts, preferably 82 - 99 parts, 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 described above includes type I collagenase and type II collagenase. Among them, type I collagenase and type II collagenase can be obtained by extraction and purification from the fermentation broth of static fermentation of Clostridium histolyticum.

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

[0025] Type I collagenase is obtained through anion chromatography, cation chromatography, and hydrophobic chromatography.

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

[0027] In some specific embodiments, type I collagenase is translated by the colG gene, and type II collagenase is translated by 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, 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, such as 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, such as 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 in the collagenase is 0.1% - 3%, preferably 0.3% - 2%, such as 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%, 0.95%, 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, such as 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 described metalloprotease can be obtained by fermenting Clostridium histolyticum or by prokaryotic expression.

[0034] Preferably, the mass percentage of the metalloprotease in the collagenase is 1% - 25%, more preferably 3% - 22%, such as 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 parts of the metalloprotease in the collagenase composition are 1 - 20 parts, such as 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., and preferably 3 - 17 parts.

[0036] Preferably, the mass ratio of the metalloprotease to the clostripain is 2 - 30, preferably 3 - 27 or 8 - 27, such as 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, by mass parts, the collagenase composition comprises:

[0038] 20 - 55 parts of type I collagenase, such as 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, such as 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, etc.;

[0040] 1 - 20 parts of metalloproteinase, such as 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] 0.05 - 3.0 parts of clostripain, such as 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.

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

[0043] 30 - 51 parts of type I collagenase;

[0044] 34 - 46 parts of type II collagenase;

[0045] 3 - 17 parts of metalloproteinase;

[0046] 0.2 - 3.0 parts of clostripain.

[0047] Preferably, the collagenase composition may further 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 thickening agent, a wetting agent, a filler, a binder, a lubricant, a disintegrant, an antibacterial agent, and a preservative.

[0048] Preferably, the collagenase composition may be in any suitable form, such as a liquid (e.g., a solution, an emulsion, or a suspension), a solid, a semi-solid, or a gas, etc.

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

[0050] In a second aspect of the present invention, a method for preparing the above-mentioned collagenase composition is provided, and the preparation method includes mixing each component to obtain it.

[0051] In a third aspect of the present invention, a method for preparing the above-mentioned collagenase composition is provided, and the preparation method includes performing static fermentation on Clostridium histolyticum to obtain a fermentation broth, and extracting the fermentation broth to obtain a crude product.

[0052] The extraction includes filtering the fermentation broth, ammonium sulfate precipitation, and then dissolving the precipitate in a buffer solution for desalting under low temperature and nitrogen pressure.

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

[0054] Among them, the equilibration buffer for DEAE cellulose column chromatography is HAC-NaAc (pH is preferably 5-6). The equilibration buffer for Sephadex G-25 column is conductivity water (pH is preferably 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 to obtain type II collagenase includes anion chromatography, cation chromatography, hydrophobic chromatography, and molecular sieve chromatography.

[0058] The preparation method further includes obtaining a metalloprotease through prokaryotic expression.

[0059] The amino acid sequence of the metalloprotease is shown as A0A4U9R6P3 (encoded by uniprot).

[0060] In a specific embodiment of the present invention, the collagenase composition is obtained by mixing type I collagenase and type II collagenase obtained by purification, and metalloprotease and clostripain obtained by prokaryotic expression.

[0061] In a fourth aspect of the present invention, there is provided a drug, which comprises the collagenase composition as described above. Preferably, it further comprises pharmaceutically acceptable excipients.

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

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

[0064] The drug can contain 0.01-99.5% by weight (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%) of the collagenase composition.

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

[0066] In a fifth aspect of the present invention, there is provided 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, such as 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] In a sixth aspect of the present invention, there is provided the collagen obtained by the method described in the fifth aspect.

[0070] In a seventh aspect of the present invention, there is provided an application of the collagenase composition in food, brewing, meat processing, cosmetics or animal feed, or an application in the preparation of a drug for treating and / or preventing diseases.

[0071] Preferably, the diseases include tissue fibrosis, intervertebral disc herniation (such as lumbar disc herniation or cervical disc herniation), Dupuytren's contracture, chronic total coronary artery occlusion, diabetes, ulcers (such as pressure sores, arterial ulcers, dermal ulcers, venous ulcers, stasis ulcers), 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, etc.

[0073] In an eighth aspect of the present invention, there is provided a method for treating and / or preventing diseases, the method comprising administering an effective amount of the above collagenase composition or drug to a subject in need thereof.

[0074] The administration mode may be any suitable administration route, such as gastrointestinal administration (such as oral administration) or parenteral administration (such as intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, infusion, intracerebral, intrathecal, transdermal, rectal, etc.) routes.

[0075] Beneficial effects:

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

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

[0078] 3. Metalloproteinase and clostripain, which are often used as impurities in the prior art, unexpectedly show a higher titer and better curative effect on treating intervertebral disc herniation when combined with collagenase, simplifying the impurity removal steps in the prior art for preparing collagenase by fermentation. Detailed implementation manners

[0079] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0080] Unless otherwise defined, all scientific and technical terms used in this application have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0081] As used herein, the "metalloprotease" is a class of proteases that can efficiently hydrolyze proteins and polypeptides. Different from the hydrolysis mechanisms of other proteases, one or more metal ions exist at the neutral site of the activity of metalloproteases.

[0082] As used herein, the "clostridain (Endoproteinase-Arg-C)" is a double-stranded protease isolated from Clostridium histolyticum. It has esterase, amidase, and protease activities and has a high specificity for the carboxypeptide bond of arginine.

[0083] As used herein, "pharmaceutically acceptable" means that it neither significantly stimulates the organism nor inhibits the biological activities and properties of the active substances of the administered product.

[0084] As used herein, "treatment" means slowing down, 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] As used herein, "prevention" means a manner implemented to prevent or delay the occurrence of a disease, disorder, or symptom in an organism.

[0086] As used herein, an "effective amount" means the amount or dose 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] As used herein, a "subject" can be a human or non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cows, sheep, horses, donkeys, foxes, raccoons, minks, camels, dogs, cats, rabbits, rats (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc.

[0088] All 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 examples of the present invention are commercially available.

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

[0091] Example 1: Preparation of the Collagenase Composition

[0092] The collagenase composition in this example is obtained by fermenting Hathewaya histolytica using an improved culture medium. The formula of the improved culture medium: by mass percentage, it is 0.2 - 1% yeast extract, 0.2 - 1% yeast powder, 1% - 3% gelatin, and 2% - 6% peptone. Fermentation conditions: at 37°C - 42°C, static fermentation, with a cycle of 40 - 45 hours. The fermentation broth is aseptically filtered, and ammonium sulfate is added for precipitation at a temperature below 10°C to make the concentration reach 45 - 55% (weight / volume). The obtained precipitate is dissolved in Tris-Ca 2+ buffer, and desalted through an ultrafiltration membrane under low temperature and nitrogen pressurization, and freeze-dried to obtain the crude collagenase. After column chromatography on a DEAE cellulose column equilibrated with HAC-NaAc buffer at pH 5 - 6, the eluate is further passed through a SephadexG-25 column equilibrated with a conductivity level of pH 6.0 - 7.5 to obtain the collagenase composition. Through structural identification, it contains type I and type II collagenases, metalloproteinases, and clostripain.

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

[0094] The collagenase in this example is obtained by fermenting Hathewaya histolytica using an improved culture medium. The formula of the improved culture medium: by mass percentage, it is 0.2 - 1% yeast extract, 0.2 - 1% yeast powder, 1% - 3% gelatin, and 2% - 6% peptone. Fermentation conditions: at 37°C - 42°C, static fermentation, with a cycle of 40 - 45 hours. The fermentation broth is aseptically filtered, and ammonium sulfate is added for precipitation at a temperature below 10°C to make the concentration reach 45 - 55% (weight / volume). The obtained precipitate is dissolved in Tris-Ca 2+ buffer, and desalted through an ultrafiltration membrane under low temperature and nitrogen pressurization, and freeze-dried to obtain the crude collagenase. The crude collagenase is obtained as a pure product of type I collagenase (purity > 95%) after anion chromatography, cation chromatography, and hydrophobic chromatography, and a pure product of type II collagenase (purity > 95%) after anion chromatography, cation chromatography, hydrophobic chromatography, and molecular sieve chromatography.

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

[0096] Taking A0A4U9R6P3 (uniprot code) as an example, the metalloproteinase is prepared by protein expression and purification. The plasmid is constructed by gene synthesis, and the protein expression and purification are carried out using an Escherichia coli expression system to obtain a pure product of the metalloproteinase (purity > 95%).

[0097] Examples 2 - 8 are taken according to the ratios in Table 1, and then mixed and shaken 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 methods of Examples 2-8, and clostripain was purchased as a commercial reagent (Sigma, Lot: BCCD0771). Transfer according to the ratios in Table 2, and then mix well.

[0102] For Comparative Example 5, transfer according to the ratios in Table 2, and at the same time transfer a certain mass of human serum albumin (purchased from Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd., batch number 202303035A) 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 potencies of Examples 2-8 and Comparative Examples 1-5 were determined.

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

[0108] The collagenase compositions obtained in each example were allowed to act on collagen at 37°C and pH 7.5. The collagen hydrolyzed per hour was colored with ninhydrin. Calculated as leucine, each 1 micromole of leucine is one activity unit, and each example and comparative example were measured in parallel three times.

[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 for the comparison of the potency of the comparative example with that of the corresponding example, *P < 0.05, **P < 0.01.

[0115] The results in Table 3 show that the potencies of the collagenase compositions with various ratios in the examples are all higher than those of the comparative examples. Further analysis is as follows:

[0116] Compared with Example 7, in Comparative Example 1, the total mass fraction of (Type I + Type II), the mass fractions of metalloprotease and clostripain are similar, all within the required range, only the ratio of Type I / Type II is not within the range, and the ratio of Type I / Type II is 0.72. The average titer of this collagenase composition (1449 U / mg) is lower than the average titer of Example 7 (1698 U / mg). T-test was performed using SPSS 25 software, and the results showed significant differences.

[0117] Compared with Example 4, in Comparative Example 2, the total mass fraction of (Type I + Type II), the mass fractions of metalloprotease and clostripain are similar, all within the required range, only the ratio of Type I / Type II is not within the range, and the ratio of Type I / Type II is 1.53. The average titer of this collagenase composition (1543 U / mg) is lower than the average titer of Example 4 (2104 U / mg). T-test was performed using SPSS 25 software, and the results showed significant differences.

[0118] Compared with Example 7, in Comparative Example 3, the total mass fraction of (Type I + Type II) and the ratio of Type I / Type II are similar to it, but the ratio of metalloprotease / clostripain is not within the range, and the ratio of metalloprotease / clostripain is 1.80. The average titer of this collagenase composition (1483 U / mg) is lower than the average titer of Example 7 (1698 U / mg). T-test was performed using SPSS 25 software, and the results showed significant differences.

[0119] In Comparative Example 4, the total mass fraction of (Type I + Type II) is 70.95, the collagenase content is low, and the ratio of metalloprotease / clostripain is high and not within the range. The average titer of this collagenase composition is lower than the average titer of the examples where all components are within the range.

[0120] In Comparative Example 5, the total mass fraction of (Type I + Type II) is 75.03, and the ratio of Type I / Type II is similar to that of Example 7, but it does not contain metalloprotease and clostripain. Therefore, the average titer of its collagenase composition (1464 U / mg) is lower than the average titer of Example 7 (1698 U / mg). T-test was performed using SPSS 25 software, and the results showed significant differences. It can be seen that the presence of metalloprotease and clostripain has a certain synergistic effect on the activity of collagenase.

[0121] Effect Example 2: Animal Experiment

[0122] Exemplarily, Examples 3, 4, 5, 7 and Comparative Examples 1-4 were selected for animal experiments.

[0123] 1. Test method: Extraction of rabbit isolated nucleus pulposus and annulus fibrosus

[0124] (1) Sampling 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] In Comparative Example 3, compared with Example 7, the ratio of metalloprotease / clostripain was lower, being 1.80, and the ratios of other components were all within the required ranges. After the action of this collagenase composition, there were significant differences in the weights of the nucleus pulposus and annulus fibrosus compared with those in Example 7.

[0137] In Comparative Example 4, the total mass fraction of (Type I + Type II) was 70.95, the collagenase content was low, the ratio of metalloprotease / clostripain was high and not within the range. Even though the mass ratio of Type I / Type II was within the range, after the action of the collagenase composition, there were significant differences in the weights of the nucleus pulposus and annulus fibrosus compared with those in the examples where all components were within the range.

[0138] Effect Example 3: Stability experiment

[0139] Exemplarily, Examples 3, 4, 5, 7 and Comparative Examples 3 - 4 were selected for the stability experiment.

[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 h respectively for titer detection, and evaluated by the titer retention rate %. The so-called titer retention rate % is the percentage of the titer at the corresponding time point to the titer at 0 h.

[0142] 2. Experimental results:

[0143] After incubation of collagenase compositions with different ratios for 0, 2, 4, 8, 12, and 24 hours, their titers all decreased to a certain extent, and the decreasing trends of Comparative Examples 3 - 4 were the most obvious. After incubation for 8 h, the titer retention rates of Examples 3, 4, 5, 7 were above 90%, while those of Comparative Examples 3 - 4 had decreased to below 80%. After incubation for 24 h, the titer retention rates of Examples 3, 4, 5, 7 could be above 50%, while those of Comparative Examples 3 - 4 had decreased to below 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, in Comparative Example 3, the ratio of metalloprotease / clostripain was lower, being 1.80, and the ratios of other components were all within the required ranges. After the collagenase composition was incubated in a 30 °C water bath for 24 h, its titer retention rate of 25% was lower than the titer retention rate of 51% in Example 7, and its stability was poorer compared with that in Example 7.

[0147] In Comparative Example 4, the total mass fraction of (Type I + Type II) is 70.95, the collagenase content is low, the ratio of metalloprotease / clostripain is high and not within the range. Even if the mass ratio of Type I / Type II is within the range, after the collagenase composition is incubated in a 30 °C water bath for 24 h, its titer retention rate of 16% is lower than that of the examples where all components are within the range, and the stability is relatively poor.

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

[0149] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. Use of a collagenase composition in the preparation of a drug for treating and / or preventing intervertebral disc herniation, 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; The mass ratio of metalloproteinase to clostripain is 2-30, the collagenase includes type I collagenase and type II collagenase, the mass ratio of type I collagenase to type II collagenase is 0.8-1.4, and the amino acid sequence of the metalloproteinase is shown in uniprot encoding A0A4U9R6P3.

2. The use according to claim 1, characterized in that: The collagenase composition contains 75-99 parts by mass of collagenase.

3. The use according to claim 2, characterized in that: The collagenase composition contains 82-99 parts by mass of collagenase.

4. The use according to claim 3, characterized in that: The collagenase composition contains 88-99 parts by mass of collagenase.

5. The use according to claim 1, characterized in that: The mass ratio of type I collagenase to type II collagenase is 0.8-1.

3.

6. The use according to claim 5, characterized in that: The mass ratio of type I collagenase to type II collagenase is 0.9-1.

2.

7. The use according to claim 1, characterized in that: The mass ratio of metalloprotease to clostripain is 8-30.

8. The use according to claim 1, 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.

9. The use according to claim 1, 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.

10. The use according to claim 1, characterized in that: The medicine comprises a collagenase composition and pharmaceutically acceptable auxiliary materials.

11. The use according to claim 10, 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.

12. The use according to claim 10 or 11, characterized in that: The dosage form of the drug is any one of tablets, powders, emulsions, 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.

13. The use according to claim 12, characterized in that: The injection is a solution-type injection.

14. The use according to claim 1, characterized in that: The intervertebral disc herniation includes lumbar disc herniation or cervical disc herniation.

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