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 present 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 more common in adults and the elderly. It refers to a clinical syndrome caused by degenerative lesions of the lumbar intervertebral disc, which, under the influence of various factors, especially external forces, leads to rupture of the annulus fibrosus and protrusion of the nucleus pulposus, causing irritation or compression of the nerve roots. Long-term pain seriously affects the patient's quality of life and mental health, not only causing an economic burden on patients, but also easily causing them to have psychological symptoms such as anxiety and depression. Currently, there are many ways to treat LDH, including conservative treatments such as bed rest and oral non-steroidal 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 such as less trauma, less damage to spinal stability, good postoperative results, 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 degenerate 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 hydrolyzes native 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, such as Pseudomonas, Achromobacter detoxificans, Clostridium, and Candida albicans, can produce collagenase. Summary of the Invention

[0005] In order to further improve the efficacy of treating intervertebral disc herniation, this application combines collagenase with metalloproteinase and clostripain in a certain ratio. The results show that the activity level of specific hydrolysis of collagen substrate is higher and the stability is better. In particular, it has a good efficacy in treating intervertebral disc herniation, such as rapid onset and better effect. The specific plan is as follows:

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

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

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

[0009] The collagen can be natural collagen or non-natural collagen. The non-natural collagen at least comprises a triple helical 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 Clostridium 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 Clostridium histolytica comprises, by weight 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 collagenase composition contains 75-99 parts by mass of collagenase, preferably 82-99 parts, and more 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 broth 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 more 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 the clostripain to the 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. %, 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 Clostridium histolytica or by prokaryotic expression.

[0034] Preferably, the mass percentage of metalloproteinase in 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;

[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 metalloprotease, 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] 0.05-3.0 parts of clostripain, 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.

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

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

[0044] 34-46 copies 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 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-mentioned collagenase composition, which comprises mixing the components.

[0051] In a third aspect, the present invention provides a method for preparing the above-mentioned collagenase composition, comprising subjecting Clostridium histolytica to static fermentation 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 equilibration buffer for DEAE cellulose column chromatography is HAC-NaAc (preferably pH 5-6), and the equilibration buffer for Sephadex G-25 column chromatography 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 further comprises 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 is obtained by mixing purified type I collagenase and type II collagenase, prokaryotically expressed metalloproteinase and clostripain.

[0061] The fourth aspect of the present invention provides a medicine comprising the collagenase composition, and preferably further comprising 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, adhesives, 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 any one of tablets, powders, emulsions, injections (preferably solution-type injections), suspensions, capsules, pills, granules, powder injections, lozenges, sprays, aerosols, powder mists, 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 or veterinary use.

[0066] A 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.

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

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

[0071] Preferably, the disease includes tissue fibrosis, intervertebral disc herniation (e.g., lumbar intervertebral disc herniation or cervical intervertebral disc herniation), Dupuytren's contracture, chronic total occlusion of coronary artery, diabetes, ulcer (e.g., decubitus ulcer, arterial ulcer, dermal ulcer, venous ulcer, stasis ulcer), arthritis (e.g., osteoarthritis or rheumatoid arthritis), burn, wound healing or scar repair.

[0072] The scar can be a surgical scar, a hypertrophic scar, a cosmetic scar, a keloid, etc.

[0073] In an eighth aspect of the present application, a method for treating and / or preventing a disease is provided, which comprises administering to a subject in need thereof an effective amount of the collagenase composition or medicament described above.

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

[0075] Advantages:

[0076] 1. The combination of collagenase, metalloproteinase and clostridial protease in a specific ratio effectively improves the efficacy of intervertebral disc herniation.

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

[0078] 3. Metalloproteinase and clostridial protease, 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, thereby simplifying the impurity removal step in the prior art when preparing collagenase by fermentation. DETAILED DESCRIPTION

[0079] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0080] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as generally understood by those skilled in the art to which the present application relates.

[0081] The "metalloprotease" described in the present invention is a type of protease that can efficiently hydrolyze proteins and polypeptides. Unlike 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)" described in the present invention is a double-chain protease isolated from Clostridium histolyticum. It has esterase, amidase and protease activities and is highly specific for the carboxyl peptide bond of arginine.

[0083] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the 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-associated signs, symptoms, conditions, or disorders.

[0085] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[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 single or multiple doses are administered to a subject or organ.

[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. The non-human mammal can be a wild animal, zoo animal, commercial animal, pet, laboratory 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 (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

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

[0089] Unless otherwise specified, the materials used in the embodiments of the present invention can be obtained commercially.

[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 is: yeast extract 0.2-1%, yeast powder 0.2-1%, gelatin 1%-3%, and peptone 2%-6%, calculated by mass percentage. Fermentation conditions: static fermentation at 37°C-42°C for 40-45 hours. The fermentation broth is sterile filtered and precipitated with ammonium sulfate at a temperature below 10°C to a concentration of 45-55% (weight / volume). The resulting precipitate is dissolved in Tris-CaCl2 solution at pH 7-8. 2+ The buffer was desalted by ultrafiltration under nitrogen pressure at low temperature and freeze-dried to obtain the crude collagenase. The crude collagenase was then chromatographed on a DEAE cellulose column equilibrated with HAC-NaAc buffer at pH 5-6. The effluent was then passed through a Sephadex G-25 column equilibrated with conductivity water at pH 6.0-7.5 to obtain a collagenase composition. Structural identification revealed that the composition contained type I and type II collagenases, metalloproteinases, and clostripain.

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

[0094] The collagenase described in this example is obtained by fermenting Clostridium histolytica using an improved culture medium. The improved culture medium formula is as follows: yeast extract 0.2-1%, yeast powder 0.2-1%, gelatin 1%-3%, and peptone 2%-6%, calculated by weight percentage. Fermentation conditions: static fermentation at 37°C-42°C for 40-45 hours. The fermentation broth is sterile filtered and precipitated with ammonium sulfate at a temperature below 10°C to a concentration of 45-55% (weight / volume). The resulting precipitate is dissolved in Tris-CaCl2 solution at pH 7-8. 2+ The buffer solution was desalted by ultrafiltration under nitrogen pressure at low temperature and freeze-dried to obtain crude collagenase. Purified type I collagenase (purity > 95%) was obtained by anion chromatography, cation chromatography, and hydrophobic chromatography. Purified type II collagenase (purity > 95%) was obtained by anion chromatography, cation chromatography, hydrophobic chromatography, and molecular sieve chromatography.

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

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

[0097] Pipette the mixture in Example 2-8 according to the proportions in Table 1, and then mix and shake 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 mixtures were pipetted according to the ratio in Table 2 and then mixed and shaken.

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

[0108] The collagenase composition obtained in each example was acted on collagen at 37°C and pH 7.5. The amount of collagen hydrolyzed per hour was visualized with ninhydrin, and leucine was calculated as one unit of activity per micromole of leucine. Each example and comparative example was 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 potency of the collagenase compositions of each ratio in the Examples is higher than that in the Comparative Examples. Further analysis is as follows:

[0116] Comparing Comparative Example 1 with Example 7, the total mass fractions of (Type I + Type II), the mass fractions of metalloproteases, and clostripain were similar and all within the required ranges. Only the Type I / Type II ratio was outside the range, with a Type I / Type II ratio of 0.72. The average potency of this collagenase composition (1449 U / mg) was lower than the average potency of Example 7 (1698 U / mg). A T-test using SPSS 25 software showed significant differences.

[0117] Comparing Comparative Example 2 with Example 4, the total mass fractions of (Type I + Type II), the mass fractions of metalloproteases, and clostripain were similar and all within the required ranges. Only the Type I / Type II ratio was outside the range, with a Type I / Type II ratio of 1.53. The average potency of this collagenase composition (1543 U / mg) was lower than the average potency of Example 4 (2104 U / mg). A T-test using SPSS 25 software showed significant differences.

[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 that there was a significant difference.

[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 embodiment in which all components are within the range.

[0120] The total mass fraction of (Type I + Type II) in Comparative Example 5 was 75.03, and the Type I / Type II composition was similar to that in Example 7, but it did not contain metalloproteinases and clostripain. Therefore, the average titer of the collagenase composition (1464 U / mg) was lower than the average titer (1698 U / mg) in Example 7. The T-test using SPSS25 software showed a significant difference. It can be seen that the presence of metalloproteinases and clostripain has a certain synergistic effect on collagenase activity.

[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

[0124] (1) Material collection method

[0125] First, anesthetize the rabbit with urethane at a dose of 4 mL / kg. Remove the dorsal hair, dissect the skin and subcutaneous fascia, and use a scalpel to separate the dorsal muscle tissue. Use large scissors to carefully and completely remove the lumbar spine. Use small scissors to completely dissect the spinal muscle tissue. Use hemostats and a scalpel to completely remove the nucleus pulposus and annulus fibrosus. Place the nucleus pulposus and annulus fibrosus 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. The weight of the remaining tissue is recorded at specific time points during the incubation process.

[0127] 2. Test results

[0128] 1) Changes in the weight of the nucleus pulposus and annulus fibrosus over time after treatment with different ratios 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. Containing a large amount of collagen fibers in the nucleus pulposus and the annulus fibrosus of lumbar intervertebral disc, it can be concluded that collagenase can produce a dissolving effect on the nucleus pulposus and the annulus fibrosus. In the nucleus pulposus / annulus fibrosus (STR) model, after using different proportions of collagenase compositions to hatch for 4, 8, 12, 24, 48 and 72 hours, tissue weight has a certain decline compared with 0 hour, and wherein embodiment 3, 4, 5, 7 declines the most significantly, and action intensity is better than comparative example 1-4. Along with different time changes, each group weight all declines gradually, and when 72 hours, embodiment 3, 4, 5, 7 weight declines the most obvious (P < 0.01), and 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] As can be seen from Table 4, compared with Example 7, in Comparative Example 1, only the Type I / Type II mass ratio is too low, and the proportions 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 proportions 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] Comparative Example 3 has a lower ratio of metalloprotease / clostridial protease of 1.80, and other component ratios are within the required range. The weights of the nucleus pulposus and the annulus fibrosus after the collagenase composition is applied are significantly different from those of Example 7.

[0137] 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 / clostridial protease is high, and is not within the range. Even though the mass ratio of Type I / Type II is within the range, the weights of the nucleus pulposus and the annulus fibrosus after the collagenase composition is applied are significantly different from those of the examples in which each component is within the range.

[0138] Example 3: Stability experiment

[0139] Exemplary Examples 3, 4, 5, 7, and Comparative Examples 3-4 were subjected to a 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. The potency was detected after 0, 2, 4, 8, 12, and 24 hours, respectively, and was evaluated using the potency retention rate %. The potency retention rate % is the percentage of the potency at the corresponding time point to the potency at 0 hours.

[0142] 2. Experimental results:

[0143] After the collagenase compositions with different ratios were incubated for 0, 2, 4, 8, 12, and 24 hours, the potency decreased to some extent, and the decrease trend of Comparative Examples 3-4 was the most obvious. After 8 hours of incubation, the potency retention rates of Examples 3, 4, 5, and 7 were more than 90%, while those of Comparative Examples 3-4 had decreased to less than 80%. After 24 hours of incubation, the potency retention rates of Examples 3, 4, 5, and 7 were more than 50%, while those of Comparative Examples 3-4 had decreased to less than 30%. The specific results are shown in Table 5.

[0144] Table 5

[0145]

[0146] As can be seen from Table 5, Comparative Example 3 has a lower ratio of metalloprotease / clostridial protease of 1.80, and other component ratios are within the required range. After the collagenase composition is incubated in a 30°C water bath for 24 hours, the potency retention rate is 25%, which is lower than the potency retention rate of 51% of Example 7, and the stability is poorer than that of 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 collagenase composition is incubated in a 30°C water bath for 24 hours. The potency retention rate 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, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection 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 appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A collagenase composition for treating and / or preventing intervertebral disc herniation, characterized in that: Calculated by weight, the collagenase composition comprises: collagenase, metalloproteinase and clostripain; 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 metalloproteinase is a non-collagenase metalloproteinase.

2. The collagenase composition according to claim 1, characterized in that The collagenase composition contains at least 75 parts by mass of collagenase.

3. 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.

4. The collagenase composition according to claim 1, characterized in that The mass ratio of type I collagenase to type II collagenase is 0.8-1.3, preferably 0.9-1.

2.

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

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: 20-55 parts; Type II collagenase: 30-60 parts; Metalloproteinase: 1-20 parts; Clostripain: 0.05-3.0 parts.

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

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 medicament 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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