A pharmaceutical composition having joint repairing effect, preparation method thereof and application thereof

Abstract

The present invention relates to a pharmaceutical composition with joint repair efficacy, which is composed of collagen, chitosan and a pharmaceutically acceptable carrier, wherein the weight ratio of collagen to chitosan in the composition is 11:1-1:11, the average molecular weight of collagen is 3kDa-500kDa, and the weight average molecular weight of chitosan is 10000-200000. The pharmaceutical composition of the present invention is used to treat and relieve pain caused by joint cavity stenosis and bone wear, effectively prevent and treat traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long effective period, easy storage, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition with joint repair efficacy, a preparation method thereof, and an application thereof. Background Art

[0002] Osteoarthritis is one of the chronic joint degenerative diseases commonly suffered by the middle-aged and elderly population. Patients with advanced osteoarthritis may experience symptoms such as difficulty squatting and limited flexion and extension, and severe cases may be unable to walk. Oral and intra-articular injection of non-steroidal anti-inflammatory drugs, antipyretic analgesics, hyaluronic acid, sodium hyaluronate, etc. are mainly used to treat osteoarthritis.

[0003] Chitosan is a deacetylated chitin product. It is a linear polysaccharide acid composed of a large portion of D-glucosamine and a small amount of N-acetyl-D-glucosamine connected by β-(1,4) glycosidic bonds. Its structure is similar to cellulose. Glucosamine generated by the hydrolysis of chitosan in vivo is used to treat joint swelling caused by osteoarthritis or rheumatoid arthritis, promote the synthesis of proteoglycans and hyaluronic acid, improve articular cartilage metabolism, enhance the repair ability of articular cartilage damage, protect articular cartilage and promote the formation of synovial fluid, reduce friction and vibration between bone joints, reduce the damage of free radicals to joint tissues, improve joint function and relieve pain, and delay the progression of bone and joint diseases.

[0004] Medical chitosan has physical barrier and lubrication effects and is widely used in joint cavities, but its effect on articular cartilage repair is poor and its water solubility is poor. It cannot be prepared into freeze-dried powder and needs to be stored at low temperatures. Therefore, it is necessary to prepare a pharmaceutical composition with better therapeutic effect and good water solubility to meet clinical needs. Summary of the invention

[0005] The object of the present invention is to provide a pharmaceutical composition with joint repair efficacy, which is composed of collagen, chitosan and a pharmaceutically acceptable carrier, wherein the weight ratio of collagen to chitosan in the composition is 11:1-1:11, the average molecular weight of collagen is 3kDa-500kDa, and the weight average molecular weight of chitosan is 10000-200000.

[0006] According to a preferred technical solution of the present invention, the weight ratio of collagen to chitosan is 1:2.4-1:10, preferably 1:4-1:8.

[0007] According to a preferred technical solution of the present invention, the collagen is selected from any one of bovine collagen and recombinant human collagen or a combination thereof.

[0008] According to a preferred technical solution of the present invention, the composition contains 1-25% collagen by weight, preferably 2-20%.

[0009] According to a preferred technical solution of the present invention, the collagen is selected from any one of type I, type 4, type 6, type 12, or any one of its folding types of α1, α2, and α3.

[0010] According to a preferred technical solution of the present invention, the average molecular weight of type I collagen is 10 kDa-450 kDa, preferably 50 kDa-400 kDa.

[0011] According to a preferred technical solution of the present invention, the type I collagen has a pH of 3.0-9.0, preferably a pH of 5.8-7.5.

[0012] According to a preferred technical solution of the present invention, the purity of the type I collagen is ≥85%, preferably ≥90%.

[0013] In a preferred technical solution of the present invention, the average molecular weight of the recombinant human collagen is 10 kDa-300 kDa, preferably 20 kDa-100 kDa.

[0014] According to a preferred technical solution of the present invention, the recombinant human collagen has a pH of 3.0-9.0, preferably a pH of 5.8-7.5.

[0015] According to a preferred technical solution of the present invention, the purity of the recombinant human collagen is ≥85%, preferably ≥90%.

[0016] According to a preferred technical solution of the present invention, the composition contains 20-60% chitosan by weight, preferably 25-50%.

[0017] According to a preferred technical solution of the present invention, the weight average molecular weight of the chitosan is 11,000-100,000, preferably 12,000-50,000.

[0018] According to a preferred technical solution of the present invention, the viscosity of the chitosan is 5-300 mPa.s, preferably 10-100 mPa.s.

[0019] According to a preferred technical solution of the present invention, the chitoose has a pH of 5.0-8.0, preferably a pH of 5.5-7.0.

[0020] According to a preferred technical solution of the present invention, the purity of the chitoose is ≥80%, preferably ≥90%.

[0021] In a preferred technical solution of the present invention, the pharmaceutically acceptable carrier is selected from any one of a buffer salt, an osmotic pressure regulator, a lyoprotectant, a pH regulator, or a combination thereof.

[0022] According to a preferred technical solution of the present invention, the buffer salt is selected from any one of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, and calcium chloride, or a combination thereof.

[0023] According to a preferred technical solution of the present invention, the osmotic pressure of the composition is 270-350 mOsm / L, 280-310 mOsm / L.

[0024] According to a preferred technical solution of the present invention, the lyoprotectant is selected from any one of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, and ethylene glycol, or a combination thereof.

[0025] According to a preferred technical solution of the present invention, the composition contains 1-25% by weight, preferably 5-20% by weight, of a freeze-drying protective agent.

[0026] According to a preferred technical solution of the present invention, the pH of the composition is 6.0-8.0, preferably 6.8-7.5.

[0027] According to the preferred technical solution of the present invention, the composition contains, by weight percentage, 1-25% collagen, 20-60% chitosan, 15-60% buffer salt and 5-20% sorbitol, the weight ratio of collagen to chitosan in the composition is 11:1-1:11, and the pH value of the composition is 6.0-8.0.

[0028] According to a preferred technical solution of the present invention, the composition optionally contains any one of hyaluronic acid, cell protein extract, chondroitin sulfate, vitamins, casein phosphopeptide, and calcium carbonate, or a combination thereof.

[0029] According to a preferred technical solution of the present invention, the composition is selected from any one of a lyophilized agent, a gel, and a solution.

[0030] Another object of the present invention is to provide a method for preparing a pharmaceutical composition with joint repair efficacy, wherein the composition is composed of collagen, chitosan and a pharmaceutically acceptable carrier, wherein the weight ratio of collagen to chitosan in the composition is 11:1-1:11, the average molecular weight of collagen is 3kDa-500kDa, and the weight average molecular weight of chitosan is 10000-200000. The preparation comprises the following steps: weighing the required amounts of collagen, chitosan and a pharmaceutically acceptable carrier, and mixing them evenly to obtain.

[0031] In a preferred technical solution of the present invention, a pharmaceutically acceptable carrier is dissolved in water under stirring, collagen is added and mixed, and then chitosan is added until dissolved.

[0032] In a preferred technical solution of the present invention, the mixing is selected from any one of stirring, shearing, high-pressure homogenization, and ultrasound, or a combination thereof.

[0033] In the preferred technical solution of the present invention, the stirring conditions are stirring at 100-30000 rpm for 20 min-48 h, preferably stirring at 150-20000 rpm for 20 min-24 h.

[0034] In the preferred technical solution of the present invention, the shearing conditions are shearing at 500-30000 rpm for 10 min-48 h, preferably shearing at 10000-25000 rpm for 20 min-2 h.

[0035] In the preferred technical solution of the present invention, the standing time is 0.5-2.5h, preferably 1-2h.

[0036] In a preferred technical solution of the present invention, the solution is sterilized before standing, and the sterilization is irradiation sterilization, preferably any one of ultraviolet sterilization, infrared sterilization, and gamma ray sterilization.

[0037] In the preferred technical solution of the present invention, the clear solution is allowed to stand and then freeze-dried to obtain the product.

[0038] According to a preferred technical solution of the present invention, the weight ratio of collagen to chitosan is 1:2.4-1:10, preferably 1:4-1:8.

[0039] According to a preferred technical solution of the present invention, the collagen is selected from any one of bovine collagen and recombinant human collagen or a combination thereof.

[0040] According to a preferred technical solution of the present invention, the composition contains 1-25% collagen by weight, preferably 2-20%.

[0041] According to a preferred technical solution of the present invention, the collagen is selected from any one of type I, type 4, type 6, type 12, or any one of its folding types of α1, α2, and α3.

[0042] According to a preferred technical solution of the present invention, the average molecular weight of type I collagen is 10 kDa-450 kDa, preferably 50 kDa-400 kDa.

[0043] According to a preferred technical solution of the present invention, the type I collagen has a pH of 3.0-9.0, preferably a pH of 5.8-7.5.

[0044] According to a preferred technical solution of the present invention, the purity of the type I collagen is ≥85%, preferably ≥90%.

[0045] In a preferred technical solution of the present invention, the average molecular weight of the recombinant human collagen is 10 kDa-300 kDa, preferably 20 kDa-100 kDa.

[0046] According to a preferred technical solution of the present invention, the recombinant human collagen has a pH of 3.0-9.0, preferably a pH of 5.8-7.5.

[0047] According to a preferred technical solution of the present invention, the purity of the recombinant human collagen is ≥85%, preferably ≥90%.

[0048] According to a preferred technical solution of the present invention, the composition contains 20-60% chitosan by weight, preferably 25-50%.

[0049] According to a preferred technical solution of the present invention, the weight average molecular weight of the chitosan is 11,000-100,000, preferably 12,000-50,000.

[0050] According to a preferred technical solution of the present invention, the viscosity of the chitosan is 5-300 mPa.s, preferably 10-100 mPa.s.

[0051] According to a preferred technical solution of the present invention, the chitoose has a pH of 5.0-8.0, preferably a pH of 5.5-7.0.

[0052] According to a preferred technical solution of the present invention, the purity of the chitoose is ≥80%, preferably ≥90%.

[0053] In a preferred technical solution of the present invention, the pharmaceutically acceptable carrier is selected from any one of a buffer salt, an osmotic pressure regulator, a lyoprotectant, a pH regulator, or a combination thereof.

[0054] According to a preferred technical solution of the present invention, the buffer salt is selected from any one of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, and calcium chloride, or a combination thereof.

[0055] According to a preferred technical solution of the present invention, the osmotic pressure of the composition is 270-350 mOsm / L, 280-310 mOsm / L.

[0056] According to a preferred technical solution of the present invention, the lyoprotectant is selected from any one of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, and ethylene glycol, or a combination thereof.

[0057] According to a preferred technical solution of the present invention, the composition contains 1-25% by weight, preferably 5-20% by weight, of a freeze-drying protective agent.

[0058] According to a preferred technical solution of the present invention, the pH of the composition is 6.0-8.0, preferably 6.8-7.5.

[0059] According to the preferred technical solution of the present invention, the composition contains, by weight percentage, 1-25% collagen, 20-60% chitosan, 15-60% buffer salt and 5-20% sorbitol, the weight ratio of collagen to chitosan in the composition is 11:1-1:11, and the pH value of the composition is 6.0-8.0.

[0060] According to a preferred technical solution of the present invention, the composition optionally contains any one of hyaluronic acid, cell protein extract, chondroitin sulfate, vitamins, casein phosphopeptide, and calcium carbonate, or a combination thereof.

[0061] According to a preferred technical solution of the present invention, the composition is selected from any one of a lyophilized agent, a gel, and a solution.

[0062] Another object of the present invention is to provide a pharmaceutical composition having joint repair efficacy for use in preparing any product for joint repair or cartilage repair.

[0063] In a preferred technical solution of the present invention, the product is used to prevent or treat any one of traumatic or degenerative osteoarthritis or its complications.

[0064] In the preferred technical solution of the present invention, the joint repair or cartilage repair is selected from any one of refractory wounds, knee osteoarthritis, synovitis, cartilage damage, bursitis or a combination thereof.

[0065] In the preferred technical scheme of the present invention, the product is used for any one of or a combination of people who need to care for their bones and joints, people with degenerative diseases in the middle-aged and elderly, people with sports injuries, people who need to maintain their health through sports, and people who need to recover after surgery for bone and joint diseases.

[0066] In the preferred technical solution of the present invention, the product is dissolved in water for injection or physiological saline to prepare an injection solution with a concentration of 1-80 mg / ml, preferably 8-30 mg / ml, for injection into the knee joint cavity.

[0067] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0068] Compared with the prior art, the present invention has the following beneficial technical effects:

[0069] 1. The pharmaceutical composition of the present invention scientifically selects the composition and ratio of collagen and chitosan, synergistically exerts the physical barrier and lubricating effects of chitosan, and supplements the collagen in the joint cavity, thereby repairing damaged joints (cartilage), alleviating the pain caused by joint friction, and slowing down the continuous damage to the joints. It is used to treat and relieve the pain caused by joint cavity stenosis and bone wear, and effectively prevents and treats traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long shelf life, and easy storage.

[0070] 2. The preparation method of the pharmaceutical composition of the present invention has the advantages of simple operation, green environmental protection, better cost, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The pharmaceutical composition of the present invention is studied for promoting the proliferation of mouse chondrocytes. DETAILED DESCRIPTION

[0072] The present invention is further described below with reference to the embodiments.

[0073] Type I collagen (from bovine origin) was purchased from Guangzhou Chuanger Biotechnology Co., Ltd.; the average molecular weight of collagen was 300 kDa, the purity was greater than 90%, and the pH was 4.4.

[0074] Recombinant human collagen: purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd. The average molecular weight is 20 kDa, the purity is greater than 90%, and the pH is 5.0.

[0075] Hydroxymethyl chitosan was purchased from Qingdao Haidefeng Biotechnology Co., Ltd. The weight average molecular weight (Mw) of chitosan was 12430, pH 6.9, purity greater than 90%, and viscosity 11 mPa·s.

[0076] Example 1 Preparation of the pharmaceutical composition of the present invention

[0077] The composition of the pharmaceutical composition of the present invention is:

[0078]

[0079]

[0080] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0081] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0082] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 hour to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 hours to obtain a clear solution, and freeze-dry.

[0083] Example 2 Preparation of the pharmaceutical composition of the present invention

[0084] The composition of the pharmaceutical composition of the present invention is:

[0085]

[0086]

[0087] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0088] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0089] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 h to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 h to obtain a clear solution, sterilize it with cobalt 60 gamma rays at a dose of 12 kgy for 8 h, and freeze-dry it.

[0090] Example 3 Preparation of the pharmaceutical composition of the present invention

[0091] The composition of the pharmaceutical composition of the present invention is:

[0092]

[0093]

[0094] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0095] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100mL, stir magnetically at 200rpm for 20min, filter through 0.22μm filter membrane to obtain the mixed solution, and load it into the shearing machine;

[0096] (2) Weigh the required amount of type I collagen, pour it into a shearing machine containing the mixed solution, shear at 22,000 rpm for 20 minutes, let it stand for 1 hour to obtain a clear solution, then add the required amount of chitosan, shear at 22,000 rpm for 20 minutes, let it stand for 1 hour to obtain a clear solution, and freeze-dry.

[0097] Example 4 Preparation of the pharmaceutical composition of the present invention

[0098] The composition of the pharmaceutical composition of the present invention is:

[0099] composition Content (mg) Collagen Type I 300 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0100] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0101] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and get a mixed solution, which is then loaded into a shearing machine;

[0102] (2) Weigh the required amount of type I collagen, pour it into a shearing machine containing the mixed solution, shear at 22,000 rpm for 20 min, let it stand for 1 hour to obtain a clear solution, then add the required amount of chitosan, shear at 22,000 rpm for 20 min, let it stand for 1 hour to obtain a clear solution, sterilize it with cobalt 60 gamma rays at a irradiation dose of 12 kgy for 8 hours, and freeze-dry it.

[0103] Example 5 Preparation of the pharmaceutical composition of the present invention

[0104] The composition of the pharmaceutical composition of the present invention is:

[0105] composition Content (mg) Collagen Type I 500 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0106] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0107] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0108] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 hour to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 hours to obtain a clear solution, and freeze-dry.

[0109] Example 6 Preparation of the pharmaceutical composition of the present invention

[0110] The composition of the pharmaceutical composition of the present invention is:

[0111] composition Content (mg) Collagen Type I 500 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0112] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0113] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100mL, stir magnetically at 200rpm for 20min, filter through 0.22μm filter membrane to obtain the mixed solution, and load it into the shearing machine;

[0114] (2) Weigh the required amount of type I collagen, pour it into a shearing machine containing the mixed solution, shear at 22,000 rpm for 20 minutes, let it stand for 1 hour, and obtain a clear solution. Then add the required amount of chitosan, shear at 22,000 rpm for 20 minutes, let it stand for 1 hour, bottle the clear solution, and freeze-dry.

[0115] Example 7 Preparation of the pharmaceutical composition of the present invention

[0116] The composition of the pharmaceutical composition of the present invention is:

[0117] composition Content (mg) Collagen Type I 150 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0118] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0119] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0120] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 minutes, let it stand for 1 hour to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 minutes, let it stand for 1.6 hours to obtain a clear solution, and freeze-dry.

[0121] Example 8 Preparation of the pharmaceutical composition of the present invention

[0122] The composition of the pharmaceutical composition of the present invention is:

[0123] composition Content (mg) Collagen Type I 150 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0124] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0125] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0126] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 h to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 h to obtain a clear solution, sterilize it with ultraviolet light for 8 h, and freeze-dry it.

[0127] Example 9 Preparation of the pharmaceutical composition of the present invention

[0128] The composition of the pharmaceutical composition of the present invention is:

[0129] composition Content (mg) Recombinant human collagen 300 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0130] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0131] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0132] (2) Weigh the required amount of recombinant human collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 h to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 h to obtain a clear solution, and freeze-dry.

[0133] Example 10 Preparation of the pharmaceutical composition of the present invention

[0134] The composition of the pharmaceutical composition of the present invention is:

[0135] composition Content (mg) Recombinant human collagen 300 Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0136] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0137] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0138] (2) Weigh the required amount of recombinant human collagen, pour it into a wall-breaking machine containing the mixed solution, stir for 30 minutes, let it stand for 1 hour to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir for 30 minutes, let it stand for 1.6 hours to obtain a clear solution, sterilize it with cobalt 60 gamma rays at a dose of 12 kgy for 10 hours, and freeze-dry it.

[0139] Embodiment 11 Preparation of the pharmaceutical composition of the present invention

[0140] The composition of the pharmaceutical composition of the present invention is:

[0141]

[0142]

[0143] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0144] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0145] (2) Weigh the required amount of recombinant human collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 h to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 h to obtain a clear solution, and freeze-dry.

[0146] Example 12 Preparation of the pharmaceutical composition of the present invention

[0147] The composition of the pharmaceutical composition of the present invention is:

[0148]

[0149]

[0150] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0151] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0152] (2) Weigh the required amount of recombinant human collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 200 rpm for 30 min, let it stand for 1 h to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 200 rpm for 30 min, let it stand for 1.6 h to obtain a clear solution, sterilize it with cobalt 60 gamma rays at a dose of 12 kgy for 12 h, and freeze-dry it.

[0153] Embodiment 13 Preparation of the pharmaceutical composition of the present invention

[0154] The composition of the pharmaceutical composition of the present invention is:

[0155]

[0156]

[0157] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0158] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100 mL, stir magnetically at 200 rpm for 20 min, filter through a 0.22 μm filter membrane, and then put the mixed solution into a wall breaking machine;

[0159] (2) Weigh the required amount of type I collagen, pour it into a wall-breaking machine containing the mixed solution, stir at 10,000 rpm for 30 min, let it stand for 1 hour to obtain a clear solution, then weigh the required amount of chitosan, pour it into the wall-breaking machine, stir at 10,000 rpm for 30 min, let it stand for 1.6 hours to obtain a clear solution, and freeze-dry.

[0160] Embodiment 14 Preparation of the pharmaceutical composition of the present invention

[0161] The composition of the pharmaceutical composition of the present invention is:

[0162]

[0163]

[0164] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0165] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4, sorbitol, MgCl 2 , CaCl 2 , add water to 100mL, stir magnetically at 200rpm for 20min, filter through 0.22μm filter membrane to obtain the mixed solution, and load it into the shearing machine;

[0166] (2) Weigh the required amount of type I collagen, pour it into a shearing machine containing the mixed solution, shear at 1000 rpm for 24 hours, let it stand for 1 hour to obtain a clear solution, then add the required amount of chitosan, shear at 1000 rpm for 24 hours, let it stand for 1 hour to obtain a clear solution, and freeze-dry.

[0167] Comparative Example 1

[0168] The composition of the pharmaceutical composition of the present invention is:

[0169] composition Content (mg) Chitosan 1200 NaCl 1600 KCl 40 <![CDATA[Na 2 HP0 4 ]]> 288 <![CDATA[KH 2 AFTER 4 ]]> 48 <![CDATA[MgCl 2 ]]> 5 <![CDATA[CaCl 2 ]]> 5 Sorbitol 268

[0170] The preparation of the pharmaceutical composition of the present invention comprises the following steps:

[0171] (1) Take the required amount of NaCl, KCl, Na 2 HP0 4 , KH 2 PO 4 , sorbitol, MgCl 2 , CaCl 2 , add water to 100mL, stir magnetically at 200rpm for 20min, filter through 0.22μm filter membrane to obtain the mixed solution, and load it into the shearing machine;

[0172] (2) Weigh the required amount of chitosan, pour it into a shearing machine containing the mixed solution, shear at 22,000 rpm for 20 minutes, let it stand for 1 hour to obtain a clear solution, and freeze-dry it.

[0173] Experimental Example 1 Study on the effect of the pharmaceutical composition of the present invention on promoting the proliferation of mouse chondrocytes 1. Experimental reagents

[0174] DMEM basal medium and complete medium, mouse collagenase type IV (Solarbio, catalog number: C8140), PBS buffered saline, CCK-8 solution, 75% alcohol

[0175] 2. Mouse chondrocyte extraction and culture experiment

[0176] (1) 8-week-old C57 mice were killed by cervical dislocation and soaked in a beaker containing 75% alcohol for 5 min;

[0177] (2) Remove the leg bones of the mice, place them in a culture dish containing 75% alcohol for 2 minutes, and then place them in a culture dish containing PBS. Then, use sterilized surgical scissors in a clean bench to separate the hyaline cartilage at the knee joint into a 15 ml centrifuge tube, and rinse the cartilage twice with an appropriate amount of PBS.

[0178] (3) Use sterilized surgical scissors to cut the washed cartilage into small pieces, place them in a six-well plate, add 0.2% mouse type IV collagenase solution (10 mg type IV collagenase dissolved in 5 mL DMEM basal medium) until the cartilage pieces are just immersed, and incubate in a cell culture incubator for 1 hour. During this period, take out the six-well plate every 15 minutes or so and blow it a few times with a pipette to allow the collagenase solution to fully contact the cartilage pieces, which is conducive to digestion into single cells;

[0179] (4) Add about 6 ml of DMEM complete medium to terminate digestion, pipette and filter with a 200-mesh filter, collect the filtrate in a 50 mL centrifuge tube, place in a centrifuge, adjust the speed to 1000 rpm, and centrifuge for 5 min. Discard the supernatant, add 6 ml of DMEM complete medium to resuspend, place in a centrifuge again, adjust the speed to 500 rpm, centrifuge for 5 min, collect the supernatant and culture in a six-well plate at 2 ml / well, place in a cell culture incubator and culture for 24 h, then change the medium;

[0180] (5) Collect the cartilage blocks remaining on the filter and the precipitate centrifuged in the previous step, place them in a six-well plate, add type IV collagenase solution again to immerse the cartilage blocks, and continue to incubate in the incubator for 2 hours. During this period, blow and shake a few times every 15 minutes or so to ensure that the cartilage blocks are fully in contact with the collagenase solution;

[0181] (6) Add 6 ml of DMEM complete medium to terminate digestion, filter again, collect the filtrate in a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 6 ml of DMEM complete medium to resuspend, put it in the centrifuge again, adjust the speed to 500 rpm, centrifuge for 5 min, collect the supernatant and culture it in a six-well plate at 2 ml / well. The remaining precipitate is also resuspended in 2 mL of DMEM complete medium and cultured in a six-well plate, placed in a cell culture flask and cultured for 24 h before changing the medium;

[0182] (7) Observe the cell growth under a microscope. When the cell growth area reaches 80%-90% of the bottom area of ​​the cell culture flask, inoculate the cells in a 96-well plate.

[0183] 3. Experimental study on the proliferation of chondrocytes by the pharmaceutical composition of the present invention

[0184] (1) The chondrocytes grown in the above six-well plate were plated in a 96-well plate, with about 5,000-10,000 cells per well, 100 μl of DMEM complete medium per well, and placed in a cell culture incubator overnight;

[0185] (2) The next day, 90 μl of DMEM complete medium containing different concentrations of the drug composition was added to each well and incubated in an incubator for 48 h. The preparation method was as follows:

[0186] Test groups 1-12: Take the required amount of the lyophilized powder of the pharmaceutical composition prepared in Examples 1-12 respectively, add 2 ml of DMEM complete medium to dissolve, and prepare solutions of different concentrations (Table 1);

[0187] Control group: Take the required amount of the lyophilized powder of the pharmaceutical composition prepared in Comparative Example 1, add 2 ml of DMEM complete medium to dissolve, and prepare a solution (Table 1);

[0188] (3) After incubation, add 10 μl of CCK-8 solution to each well and incubate in the incubator for about 4 h;

[0189] (4) Detect the absorbance at 450 nm using an enzyme reader.

[0190] Cell proliferation rate (%) = absorbance value of test group / absorbance value of blank group × 100%, the results are shown in Figure 1 The pharmaceutical composition of the present invention can achieve the proliferation of chondrocytes.

[0191] Table 1

[0192]

[0193] Test Example 2: Reconstitution test of the pharmaceutical composition of the present invention

[0194] 100 mg of the lyophilized powder of the pharmaceutical composition prepared in Examples 1-14 was taken respectively, dissolved in 3 ml of physiological saline, and allowed to stand for 12 h to obtain a clear solution. No precipitation was observed after being placed at room temperature for 2 weeks.

[0195] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A lyophilized agent having joint repair efficacy, the lyophilized agent comprising collagen, chitosan and a pharmaceutically acceptable carrier, wherein: The collagen is selected from any one or a combination of type I collagen and recombinant human collagen, the average molecular weight of type I collagen is 10kDa-450kDa, the average molecular weight of recombinant human collagen is 10kDa-300kDa, the weight average molecular weight of chitosan is 11000-100000, the weight ratio of collagen:chitosan in the lyophilized agent is 1:2-1:11, the pharmaceutically acceptable carrier is selected from any one or a combination of buffer salt, lyoprotectant, osmotic pressure regulator, pH regulator, the buffer salt is selected from any one or a combination of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, and calcium chloride, the lyoprotectant is selected from any one or a combination of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, and ethylene diene, 2. The lyophilized agent according to claim 1, wherein the collagen contained in the lyophilized agent is 2-20% by weight.

3. The lyophilized agent according to any one of claims 1 to 2, wherein the weight ratio of collagen to chitosan in the lyophilized agent is 1:2.4-1:

10.

4. The lyophilized agent according to claim 3, wherein the weight ratio of collagen to chitosan in the lyophilized agent is 1:2.4-1:

8.

5. The lyophilized agent according to claim 1, wherein the weight ratio of collagen to chitosan in the lyophilized agent is any one of 1:2, 1:2.4, 15.1:45.4, 1:4, 1:8, and 1:

10. The lyophilized agent according to any one of claims 1 to 2, wherein the average molecular weight of the type I collagen is 50 kDa-400 kDa. The lyophilized agent according to claim 3 , wherein the type I collagen has a pH of 3.0-9.

0. The lyophilized preparation according to claim 7 , wherein the type I collagen has a pH of 5.8-7.

5.

9. The lyophilized agent according to claim 3, wherein the purity of type I collagen is ≥90%. 10 . The lyophilized agent according to claim 3 , wherein the average molecular weight of the recombinant human collagen is 20 kDa-100 kDa. The lyophilized preparation according to claim 3, wherein the recombinant human collagen has a pH of 3.0-9.

0. The lyophilized preparation according to claim 11 , wherein the recombinant human collagen has a pH of 5.8-7.

5.

13. The lyophilized agent according to claim 3, wherein the purity of the recombinant human collagen is ≥ 90%. The lyophilized agent according to claim 3 , wherein the weight average molecular weight of the chitosan is 12,000-50,000. The lyophilized agent according to claim 3 , wherein the viscosity of the chitosan is 10-100 mPa.s. The lyophilized agent according to claim 3 , wherein the chitosan has a pH of 5.5-7.

0.

17. The lyophilized agent according to claim 3, wherein the purity of the chitosan is ≥ 90%.

18. The lyophilized agent according to claim 3, wherein the lyophilized agent contains 5-20% of the lyoprotectant by weight.

19. The lyophilized agent according to claim 3, wherein the osmotic pressure of the lyophilized agent is 270-350 mOsm / L.

20. The lyophilized agent according to claim 19, wherein the osmotic pressure of the lyophilized agent is 280-310 mOsm / L. The lyophilized agent according to claim 3 , wherein the pH of the lyophilized agent is 6.0-8.

0. The lyophilized agent according to claim 21 , wherein the pH of the lyophilized agent is 6.8-7.

5.

23. The lyophilized agent according to claim 3, wherein the lyophilized agent contains, by weight percentage, 2-20% collagen, 15-60% buffer salt, and 5-20% sorbitol, the weight ratio of collagen to chitosan in the lyophilized agent is 1:2.4-1:10, and the pH value of the lyophilized agent is 6.0-8.

0.

24. The lyophilized agent according to claim 23, which optionally contains any one of hyaluronic acid, cell protein extract, chondroitin sulfate, vitamins, casein phosphopeptide, calcium carbonate or a combination thereof.

25. The method for preparing the lyophilized agent according to any one of claims 1 to 24, wherein the lyophilized agent consists of collagen, chitosan and a pharmaceutically acceptable carrier, wherein: The collagen is selected from any one of type I collagen and recombinant human collagen or a combination thereof, the average molecular weight of type I collagen is 10kDa-450kDa, the average molecular weight of recombinant human collagen is 10kDa-300kDa, the weight average molecular weight of chitosan is 11000-100000, the weight ratio of collagen to chitosan in the lyophilized agent is 1:2-1:11, the pharmaceutically acceptable carrier is selected from any one of buffer salt, lyoprotectant, osmotic pressure regulator, pH regulator or a combination thereof, the buffer salt is selected from sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Any one of magnesium chloride and calcium chloride or a combination thereof, the lyophilization protective agent is selected from any one of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, and ethylene glycol or a combination thereof, and the preparation of the lyophilized agent comprises the following steps: 1) placing a required amount of a pharmaceutically acceptable carrier in water, stirring at 100-30000 rpm for 20 min-48 h and shearing at 500-30000 rpm for 10 min-48 h, adding collagen, and dissolving it, then adding chitosan, dissolving it, and lyophilizing it to obtain the lyophilized agent.

26. The method according to claim 25, wherein the stirring condition is 150-20000 rpm, 20 min-24 h.

27. The method according to any one of claims 25 to 26, wherein the shearing condition is 10000-25000 rpm, 20 min to 2 h.

28. The method according to any one of claims 25-26, wherein the lyophilized agent is sterilized before lyophilization.

29. The method according to claim 28, wherein the sterilization is selected from any one of ultraviolet sterilization, infrared sterilization, and gamma ray.

30. The method of claim 28, wherein the sterilization is selected from radiation sterilization.

31. Use of the lyophilized agent according to any one of claims 1 to 24 or the lyophilized agent obtained by the method according to any one of claims 25 to 30 in preparing a medicament for preventing and treating any one of traumatic osteoarthritis, degenerative osteoarthritis and cartilage damage.

32. Use of the lyophilized agent according to any one of claims 1 to 24 or the lyophilized agent obtained by the method according to any one of claims 25 to 30 in preparing a medicament for preventing and treating knee osteoarthritis.

33. The use according to any one of claims 31-32, wherein the drug is used for any one of or a combination of people who need to care for their bones and joints, people with degenerative diseases in the middle-aged and elderly, people with sports injuries, people who need to maintain their health through sports, and people who need to recover after surgery for bone and joint diseases.

34. The use according to any one of claims 31-32, wherein the drug is dissolved in either water for injection or physiological saline to prepare an injection solution with a concentration of 8-30 mg / mL for injection into the knee joint cavity.

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