A pharmaceutical composition and its preparation method and use

Abstract

The present invention provides a pharmaceutical composition, a preparation method, and uses thereof. The pharmaceutical composition of the present invention comprises: collagenase, human serum albumin, a sugar alcohol, and a salt; wherein the weight ratio of the human serum albumin to the sugar alcohol is 1:4-100. The lyophilized collagenase preparation prepared using the pharmaceutical composition of the present invention has excellent long-term stability and excellent activity after being formulated into an aqueous solution, with no significant decrease in activity after 72 hours at 2-8°C. It also has good efficacy in treating nodular complications after collagen implantation.

Description

Technical Field

[0001] The present invention belongs to the field of medicine. Specifically, the present invention relates to a pharmaceutical composition and a preparation method and use thereof. Background Art

[0002] Collagen is the primary structural protein in the human body, playing a crucial role in growth and development, wound healing, platelet activation, and angiogenesis. In the skin, collagen is primarily distributed in the dermis, comprising approximately 75-85%. Collagen provides support and volume, while also providing elasticity and stress resistance. It has excellent nutritional benefits, promoting elasticity, and promoting repair and healing.

[0003] Over the past few decades, collagen and collagen stimulators have been widely used as biomedical materials in clinical fields such as trauma, cosmetic surgery, and tissue repair due to their excellent biocompatibility and low antigenicity, and have been widely accepted by patients and clinicians. However, due to the unique physicochemical properties of injectable collagen, its operation technology differs from that of hyaluronic acid, and it has a higher technical threshold and difficulty. Insufficient understanding of the physicochemical properties of collagen and collagen stimulators and insufficient injection experience can lead to many complications, of which nodules are a very common complication.

[0004] Currently, collagenases are used clinically to specifically degrade collagen, thereby treating nodules caused by collagen deposition. Collagenases (abbreviated as collagenases) are highly specific proteases that act on the helical structure of collagen tissue. At physiological pH and temperature, they hydrolyze native collagen. Collagenases can be isolated from human skin culture fluid and amphibian tissue fluid. Many microorganisms, such as Pseudomonas, Achromobacter detoxificans, Clostridium, and Candida albicans, can also produce collagenases.

[0005] Collagenase is relatively sensitive and easily affected by the environment. Normally, collagenase should be stored under refrigerated conditions, usually 2 to 8°C. This helps to extend the shelf life of collagenase and ensures that it remains active before use. In the pharmaceutical process, in order to ensure the stability and activity of collagenase, it is usually freeze-dried for storage. However, the long-term stability of the lyophilized preparation of collagenase prepared by the current method needs to be improved, and once it is prepared into an aqueous solution, the activity is easily reduced. Summary of the Invention

[0006] To address the above issues, the present invention provides a pharmaceutical composition, preparation method, and use thereof. The lyophilized collagenase preparation prepared using the pharmaceutical composition of the present invention exhibits excellent long-term stability and, after being formulated into an aqueous solution, exhibits excellent activity, with no significant decrease in activity after 72 hours at 2-8°C. Furthermore, the composition exhibits excellent efficacy in treating nodular complications following collagen implantation.

[0007] definition:

[0008] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0009] As used herein, the term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0010] This article uses the term "enzyme activity unit (U)" to express enzyme content. Collagenase is activated on collagen at 37°C and pH 7.5. Ninhydrin is used to visualize the amount of collagen hydrolyzed per hour. One micromole of leucine is considered one unit of enzyme activity.

[0011] The above object of the present invention is achieved by providing the following technical solutions:

[0012] In a first aspect, the present invention provides a pharmaceutical composition comprising: collagenase, human serum albumin, sugar alcohol and salt; wherein the weight ratio of the human serum albumin to the sugar alcohol is 1:4-100.

[0013] Preferably, the weight ratio of human serum albumin to sugar alcohol is 1:4-80, preferably 1:4-30, and more preferably 1:8-30.

[0014] Preferably, the weight ratio of human serum albumin to salt is 1: 0.1-100, preferably 1: 0.2-50, and more preferably 1: 0.2-8.

[0015] Preferably, the ratio of collagenase to human serum albumin is 10-10000 U: 1 mg, preferably 50-5000 U: 1 mg, and further preferably 50-3000 U: 1 mg.

[0016] Preferably, the content of collagenase in the pharmaceutical composition is 100-8000 U / mL, preferably 300-5000 U / mL.

[0017] Preferably, the content of human serum albumin in the pharmaceutical composition is 0.05-30 mg / mL, preferably 0.1-20 mg / mL.

[0018] Preferably, the content of sugar alcohol in the pharmaceutical composition is 0.5-150 mg / mL, preferably 1-120 mg / mL.

[0019] Preferably, the content of salt in the pharmaceutical composition is 0.1-80 mg / mL, preferably 0.8-50 mg / mL.

[0020] Preferably, the sugar alcohol is selected from one or more of sorbitol, xylitol, mannitol and maltitol.

[0021] Preferably, the salt is selected from one or more of sodium chloride, calcium chloride, sodium phosphate, sodium monohydrogen phosphate and sodium dihydrogen phosphate.

[0022] According to some embodiments of the invention, the pharmaceutical composition further comprises a solvent.

[0023] Preferably, the ratio of the collagenase to the solvent is 100-8000 U: 1 mL, more preferably 300-5000 U: 1 mL.

[0024] Preferably, the pH value of the pharmaceutical composition is 6.0-7.6, preferably 6.5-7.5.

[0025] Preferably, the solvent is water for injection.

[0026] In a second aspect, the present invention provides a lyophilized preparation prepared from the pharmaceutical composition according to the first aspect of the present invention.

[0027] In a third aspect, the present invention provides a method for preparing the lyophilized preparation according to the second aspect of the present invention, comprising:

[0028] Collagenase, human serum albumin, sugar alcohol and salt are dissolved in a solvent to obtain a mixed solution, and the mixed solution is lyophilized to obtain the lyophilized preparation.

[0029] Preferably, the pH value of the mixed solution is 6.0-7.6, preferably 6.5-7.5.

[0030] Preferably, the solvent is water for injection.

[0031] In a specific embodiment of the present invention, the freeze-drying step is not particularly limited, and a freeze-drying step of collagenase known to those skilled in the art can be used. In a specific embodiment of the present invention, the freeze-drying comprises pre-freezing, primary drying and secondary drying in sequence; preferably, the pre-freezing is carried out under the following conditions: the pre-freezing temperature is -30°C to -55°C; the pre-freezing time is 90-210min; preferably, the primary drying is carried out under the following conditions: the primary drying temperature is -45-0°C; the primary drying time is 20-45h; preferably, the secondary drying is carried out under the following conditions: the secondary drying temperature is 0-30°C; the secondary drying time is 3-10h.

[0032] Preferably, the method further comprises the following step: filtering the mixed solution before freeze-drying.

[0033] In a fourth aspect, the present invention provides a pharmaceutical product comprising the freeze-dried preparation according to the second aspect of the present invention and a dispersion medium.

[0034] Preferably, the pH value of the pharmaceutical product is 6.0-7.6, preferably 6.5-7.5.

[0035] Preferably, the ratio of collagenase to dispersion medium in the pharmaceutical product is 300-5000 U: 0.5-20 mL.

[0036] Preferably, the dispersion medium is selected from one or more of water for injection, glucose aqueous solution, sodium chloride aqueous solution and calcium chloride aqueous solution.

[0037] Further preferably, the concentration of the glucose aqueous solution is 10-150 g / L.

[0038] Further preferably, the concentration of the sodium chloride aqueous solution is 1-15 g / L.

[0039] Further preferably, the concentration of the calcium chloride aqueous solution is 0.1-5 g / L.

[0040] In a fifth aspect, the present invention provides a method for preparing the pharmaceutical product according to the fourth aspect of the present invention, comprising:

[0041] The lyophilized preparation is dissolved in the dispersion medium to obtain the pharmaceutical product.

[0042] In a sixth aspect, the present invention provides a kit comprising the pharmaceutical composition according to the first aspect of the present invention, the lyophilized preparation according to the second aspect of the present invention, or the pharmaceutical product according to the fourth aspect of the present invention.

[0043] In a seventh aspect, the present invention provides use of the pharmaceutical composition according to the first aspect of the present invention, the freeze-dried preparation according to the second aspect of the present invention, or the pharmaceutical product according to the fourth aspect of the present invention in the preparation of a medicament for preventing and / or treating sarcoidosis, tissue fibrosis, intervertebral disc herniation, Dupuytren's contracture, coronary embolism, arthritis, wound healing and / or scar repair.

[0044] Preferably, the sarcoidosis is a complication of nodules after collagen implantation.

[0045] The present invention has at least the following beneficial effects:

[0046] 1. The lyophilized collagenase preparation prepared using the pharmaceutical composition of the present invention has excellent long-term stability and excellent activity after being formulated into an aqueous solution, with no significant decrease in activity after 72 hours at 2-8°C.

[0047] 2. The collagenase freeze-dried preparation prepared by the pharmaceutical composition of the present invention has a good effect in treating nodule complications after collagen implantation.

[0048] 3. The components of the pharmaceutical composition of this invention, when dissolved in a solvent, have a pH between 6.0 and 7.6. Compared to other pH values, this can further enhance the activity and long-term stability of the lyophilized collagenase formulation and improve the efficacy for treating nodular complications following collagen implantation. Furthermore, the human serum albumin employed in this invention has fewer side effects than other serum albumins, such as bovine serum albumin.

[0049] 4. The collagenase freeze-dried preparation prepared using the pharmaceutical composition of the present invention exhibits rapid resolubility in physiological saline and has a low water content, which is beneficial for improving the stability of the freeze-dried preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The specific embodiments of the present invention are described with reference to the following drawings, in which:

[0051] Figure 1 The figure is a trend diagram showing the activity change over time of the lyophilized collagenase preparations of Example 2 and Comparative Examples 2-4 after being dissolved in physiological saline.

[0052] Figure 2 The figure is a trend diagram showing the change in activity of the lyophilized collagenase preparations of Example 5 and Comparative Examples 1, 5, and 6 over time after being dissolved in physiological saline.

[0053] Figure 3 The long-term stability trend diagram of the lyophilized collagenase preparations of Example 2 and Comparative Examples 2-4 is shown.

[0054] Figure 4The long-term stability trend diagram of the lyophilized collagenase preparations of Example 5 and Comparative Examples 1, 5, and 6 is shown.

[0055] Figure 5 Image of collagen gel implanted subcutaneously on the back of a nude mouse before collagenase injection (×20).

[0056] Figure 6 This is an image of the collagen gel 12 hours after the collagenase injection of Example 2 (×20).

[0057] Figure 7 This is an image of the collagen gel 4 days after the collagenase injection of Example 2 (×20).

[0058] Figure 8 This is an image of the collagen gel 12 hours after the collagenase injection of Comparative Example 4 (×20).

[0059] Figure 9 This is an image of the collagen gel of Comparative Example 4 on the 4th day after the collagenase injection (×20). DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0061] The experimental methods used in the following examples and comparative examples are conventional methods unless otherwise specified. The reagents used in the following examples and comparative examples are commercially available products unless otherwise specified.

[0062] The preparation method of collagenase is as follows:

[0063] (1) Clostridium histolytica is fermented in an improved culture medium (the formula of which is: yeast extract 0.2-1%, yeast powder 0.2-1%, gelatin 1%-3%, and meat peptone 2%-6% by weight) at 37°C-42°C for 40-45 hours to obtain a fermentation broth containing low-toxic collagenase;

[0064] (2) The fermentation broth obtained in step (1) was sterile filtered and ammonium sulfate was added to precipitate the collagenase at a concentration of 45-55% (g / mL) at a temperature below 10°C. The precipitate was dissolved in Tris-CaCl2 solution at pH 7-8. 2+The crude collagenase was desalted by ultrafiltration membrane under low temperature and nitrogen pressure, and then freeze-dried to obtain the crude collagenase. It was then chromatographed on a DEAE cellulose column equilibrated with HAC-NaAc buffer at pH 5-6. The column liquid was then passed through a Sephadex G-25 column equilibrated with conductivity water at pH 6.0-7.5 to obtain a solution that was freeze-dried to obtain collagenase.

[0065] Example 1

[0066] Add 100,000 IU of collagenase, 2000 mg of human albumin, 8000 mg of maltitol, and 400 mg of sodium monohydrogen phosphate to water for injection. Stir to dissolve, then dilute to 100 mL (pH 7.3) to obtain a mixed solution. Filter the mixed solution and fill it into 100 vials, 1 mL per vial. After lyophilization, a lyophilized collagenase preparation for injection is obtained. The lyophilization process comprises the following steps:

[0067] (1) Pre-freezing: cool the sample to -45°C within 5 minutes and keep it for 3 hours;

[0068] (2) Primary drying: keep the pre-frozen sample at -30°C for 30 h;

[0069] (3) Secondary drying: Keep the sample after primary drying at 25°C for 5 h.

[0070] Example 2

[0071] Add 120,000 IU of collagenase, 300 mg of human albumin, 2400 mg of mannitol, and 600 mg of sodium chloride to water for injection. Stir to dissolve, then dilute to 100 mL (pH 6.6) to obtain a mixed solution. Filter the mixed solution and transfer it into 100 vials, 1 mL per vial. Lyophilize under the same conditions as in Example 1 to obtain a lyophilized collagenase preparation for injection.

[0072] Example 3

[0073] Add 30,000 units of collagenase, 10 mg of human serum albumin, 100 mg of xylitol, and 80 mg of sodium dihydrogen phosphate to water for injection. Stir to dissolve, then dilute to 100 mL (pH 7.5) to obtain a mixed solution. Filter the mixed solution and transfer it into 100 vials, 1 mL per vial. Lyophilize under the same conditions as in Example 1 to obtain a lyophilized collagenase preparation for injection.

[0074] Example 4

[0075] Add 500,000 IU of collagenase, 250 mg of human albumin, 8000 mg of sorbitol, and 1000 mg of calcium chloride to water for injection. Stir to dissolve, then dilute to 100 mL (pH 7.0) to obtain a mixed solution. Filter the mixed solution and transfer it into 100 vials, 1 mL per vial. Lyophilize under the same conditions as in Example 1 to obtain a lyophilized collagenase preparation for injection.

[0076] Example 5

[0077] Add 100,000 IU of collagenase, 100 mg of human albumin, 8,000 mg of sorbitol, and 5,000 mg of sodium phosphate to water for injection. Stir to dissolve, then dilute to 100 mL (pH 6.5) to obtain a mixed solution. Filter the mixed solution and transfer it into 100 vials, 1 mL per vial. Lyophilize under the same conditions as in Example 1 to obtain a lyophilized collagenase preparation for injection.

[0078] Example 6

[0079] A lyophilized collagenase preparation for injection was prepared according to the method of Example 4, with the only difference from Example 4 being that the amount of calcium chloride used was 2000 mg.

[0080] Example 7

[0081] A lyophilized collagenase preparation for injection was prepared according to the method of Example 4, the only difference from Example 4 being that the amount of calcium chloride used was 3000 mg.

[0082] Example 8

[0083] A lyophilized collagenase preparation for injection was prepared according to the method of Example 2, the only difference from Example 2 being that the amount of mannitol used was 3750 mg.

[0084] Example 9

[0085] A lyophilized collagenase preparation for injection was prepared according to the method of Example 2, the only difference from Example 2 being that the amount of mannitol used was 9000 mg.

[0086] Example 10

[0087] A lyophilized collagenase preparation for injection was prepared according to the method of Example 2, with the only difference from Example 2 being that the amount of mannitol used was 12000 mg.

[0088] Comparative Example 1

[0089] Compared with Example 5, the only difference is that glycine is used instead of sorbitol.

[0090] Comparative Example 2

[0091] Compared with Example 2, the only difference is that no mannitol was added.

[0092] Comparative Example 3

[0093] Compared with Example 2, the only difference is that sucrose is used instead of human serum albumin.

[0094] Comparative Example 4

[0095] Compared with Example 2, the only difference is that the amount of mannitol is 900 mg.

[0096] Comparative Example 5

[0097] Compared with Example 5, the only difference is that the pH of the mixed solution is adjusted to 5.0 with phosphoric acid.

[0098] Comparative Example 6

[0099] Compared with Example 5, the only difference is that the pH of the mixed solution is adjusted to 8.0 with sodium hydroxide.

[0100] Study on the reconstitution time of lyophilized collagenase preparation

[0101] Exemplary studies were conducted on the reconstitution time of the lyophilized formulations of Examples 1-10. After adding 1 ml of normal saline, the dissolution time was recorded. If the formulation did not dissolve naturally within 10 seconds, the formulation was repeatedly shaken until completely dissolved, and the dissolution time was continuously recorded. The results are shown in the table below.

[0102] Table 1 Results of reconstitution time determination of lyophilized collagenase preparations

[0103]

[0104] The shorter the reconstitution time of the collagenase lyophilized preparation in normal saline, the better the solubility of the preparation. A longer reconstitution time may affect the doctor's operating speed and the patient's treatment experience, and also affect the stability of the product. If the reconstitution time of the lyophilized preparation is controlled within 5 minutes, it is generally considered to meet industry standards. As shown in the table above, the collagenase lyophilized preparations prepared in Examples 1 to 10 of the present invention can be quickly reconstituted in normal saline, meeting the standard requirements.

[0105] Study on the Moisture Content of Freeze-dried Collagenase Preparations

[0106] The moisture content of the freeze-dried preparations of Examples 1-10 was exemplarily determined (Chinese Pharmacopoeia 2020 Edition 0832 Moisture Determination Method 2 Drying Method), and the results are shown in the following table.

[0107] Table 2 Determination of moisture content of collagenase freeze-dried preparations

[0108]

[0109] Moisture content is an important indicator for controlling the quality of freeze-dried preparations. When the moisture content is high, it may affect the appearance of the freeze-dried preparation, causing collapse or shrinkage. Lowering the moisture content can reduce this risk and ensure the stability of the freeze-dried preparation.

[0110] If the moisture content of the freeze-dried preparation is controlled below 5% by weight, it is generally considered to meet industry standards. As can be seen from the above table, the freeze-dried collagenase preparations prepared in Examples 1-10 of the present invention all meet the standard requirements.

[0111] Study on the activity of lyophilized collagenase preparation

[0112] The activity of the lyophilized collagenase preparations of Examples 1-5 and Comparative Examples 1-6 was investigated. Each vial of lyophilized collagenase preparation was dissolved in 10 mL of normal saline and stored at 2-8°C. The potency was tested at 0 h, 12 h, 24 h, 48 h, and 72 h. The results are shown in the table below.

[0113] Potency detection method: Collagenase acts on collagen at 37°C and pH 7.5. The collagen hydrolyzed per hour is colored with ninhydrin and calculated as leucine. One micromole of leucine is one activity unit.

[0114] Table 3 Activity test results of collagenase freeze-dried preparation

[0115]

[0116] In Examples 1 to 5, the lyophilized collagenase preparations were dissolved in 10 ml of physiological saline and stored at 2-8° C. for 72 h. The titers of the lyophilized collagenase preparations were all above 90% of the original titer, indicating good solution stability.

[0117] Compared with Example 5, in Comparative Example 1, glycine was used instead of sorbitol. After being dissolved in physiological saline, the potency of the solution gradually decreased and only reached about 46% of the original potency at 72 h, indicating poor solution stability.

[0118] Compared with Example 2, Comparative Example 2 did not add mannitol. After being dissolved in normal saline, the potency of the solution gradually decreased and only reached about 43% of the original potency at 72 hours, indicating poor solution stability.

[0119] Compared with Example 2, Comparative Example 3 uses sucrose instead of human serum albumin. After being dissolved in physiological saline, its potency gradually decreases and only reaches about 38% of the original potency at 72 hours, and the solution stability is poor.

[0120] In Comparative Example 4, compared with Example 2, the weight ratio of human albumin to mannitol was adjusted from 1:8 to 1:3. After being dissolved in physiological saline, its potency gradually decreased, reaching only about 51% of the original potency at 72 h, and the solution stability was poor.

[0121] Compared with Example 5, the pH value of the mixed solution in Comparative Example 5 was adjusted from 6.5 to 5.0. After being dissolved in physiological saline, its potency gradually decreased and only reached about 35% of the original potency at 72 h, indicating poor solution stability.

[0122] In Comparative Example 6, compared with Example 5, the pH value of the mixed solution was adjusted from 6.5 to 8.0. After being dissolved in physiological saline, its potency gradually decreased, reaching only about 41% of the original potency at 72 h, and the solution stability was poor.

[0123] Figure 1 The activity trends of the lyophilized collagenase preparations of Example 2 and Comparative Examples 2-4 after dissolution in physiological saline over time are shown. As can be seen from the figure, the lyophilized collagenase preparation of Example 2 exhibits excellent stability after dissolution in physiological saline. Even after storage at 2-8°C for 72 hours, its activity remains stable, showing no significant decrease. In contrast, the lyophilized collagenase preparations of Comparative Examples 2-4 show a significant decrease in activity under the same storage conditions.

[0124] Figure 2 The activity trends of the lyophilized collagenase preparations of Example 5 and Comparative Examples 1, 5, and 6 after dissolution in physiological saline over time are shown. As shown, the lyophilized collagenase preparation of Example 5 exhibits excellent stability after dissolution in physiological saline. Even after storage at 2-8°C for 72 hours, its activity remains stable, showing no significant decrease. In contrast, the lyophilized collagenase preparations of Comparative Examples 1, 5, and 6 exhibit a significant decrease in activity under the same storage conditions.

[0125] Study on the long-term stability of lyophilized collagenase preparation

[0126] Examples 1-5 and Comparative Examples 1-6 were used to investigate the long-term stability of lyophilized collagenase formulations. The lyophilized collagenase formulations were stored at 2-8°C and tested for potency at 0, 12, 18, and 24 months (using the same testing methods as above). The results are shown in the table below.

[0127] Table 4 Long-term stability test results of collagenase freeze-dried preparation

[0128]

[0129]

[0130] In Examples 1 to 5, the collagenase freeze-dried preparations were stored at 2-8° C. for 24 months, and their potencies were all above 90% of the original potency, indicating good solution stability.

[0131] Comparative Example 1, compared with Example 5, uses glycine instead of sorbitol. When stored at 2-8°C, the potency gradually decreases and reaches only about 63% of the original potency after 24 months, indicating poor stability.

[0132] Compared with Example 2, Comparative Example 2 did not add mannitol. When stored at 2-8°C, the potency gradually decreased and only reached about 57% of the original potency after 24 months, indicating poor stability.

[0133] Comparative Example 3 Compared with Example 2, sucrose was used instead of human serum albumin. When stored at 2-8°C, its potency gradually decreased and only reached about 50% of the original potency after 24 months, and its stability was poor.

[0134] In Comparative Example 4, compared with Example 2, the weight ratio of human albumin to mannitol was adjusted from 1:8 to 1:3. When stored at 2-8°C, its potency gradually decreased, reaching only about 69% of the original potency after 24 months, and its stability was poor.

[0135] Compared with Example 5, the pH value of the mixed solution in Comparative Example 5 was adjusted from 6.5 to 5.0. When stored at 2-8°C, its potency gradually decreased and only reached about 41% of the original potency after 24 months, indicating poor stability.

[0136] In Comparative Example 6, compared with Example 5, the pH value of the mixed solution was adjusted from 6.5 to 8.0. When stored at 2-8°C, its potency gradually decreased, reaching only about 50% of the original potency after 24 months, and its stability was poor.

[0137] Figure 3 The long-term stability of the lyophilized collagenase preparations of Example 2 and Comparative Examples 2-4 is shown. As shown in the figure, the lyophilized collagenase preparation of Example 2 exhibits excellent long-term stability. Even after 24 months of storage at 2-8°C, its activity remains stable, with no significant decrease. In contrast, the lyophilized collagenase preparations of Comparative Examples 2-4 exhibit a significant decrease in activity under the same storage conditions.

[0138] Figure 4 The long-term stability of the lyophilized collagenase preparations of Example 5 and Comparative Examples 1, 5, and 6 is shown. As shown in the figure, the lyophilized collagenase preparation of Example 5 exhibits excellent long-term stability. Even after 24 months of storage at 2-8°C, its activity remains stable, with no significant decrease. In contrast, the lyophilized collagenase preparations of Comparative Examples 1, 5, and 6 exhibit a significant decrease in activity under the same storage conditions.

[0139] Animal experiments

[0140] Exemplarily, the lyophilized collagenase preparations prepared in Examples 2 and 5 and Comparative Examples 1-4 were stored at 2-8° C. for 12 months and then subjected to animal experiments to evaluate the digestion and degradation performance of collagenase on collagen.

[0141] Collagenase injection: The lyophilized collagenase preparation in each vial prepared in Examples 2 and 5 and Comparative Examples 1-4 was dissolved in 8 mL of normal saline to obtain collagenase injection.

[0142] Each nu-BALB / c mouse was subcutaneously injected with collagen gel (Fu Roumei) at 0.05 mL / point on the left and right sides of the back. One week later, 0.05 mL of the above-mentioned collagenase injection was injected in situ at the collagen gel on the left side. The right side was not injected as a control. The changes in collagen volume 24 hours after injection and the time for complete disappearance of collagen and skin ecchymosis were compared and observed. The test results are shown in the table below.

[0143] Table 5 Animal experimental test results

[0144]

[0145] Comparative Example 1 differs from Example 5 in that glycine was used instead of sorbitol. The resulting lyophilized preparation was stored at 2-8°C for 12 months and then subjected to animal testing. After 24 hours, the volume reduction of collagen decreased from 70% in Example 5 to 43%, and the time required for complete disappearance of collagen and skin ecchymosis decreased from 5 days to 9 days compared to Example 5. This indicates that Comparative Example 1 is less effective than Example 5 in treating nodular complications following collagen (stimulator) implantation.

[0146] Comparative Examples 2, 3, and 4 differed from Example 2 in that no mannitol was added, human albumin was replaced with sucrose, and the ratio of human albumin to mannitol was adjusted to 1:3. The resulting lyophilized preparations were stored at 2-8°C for 12 months and then subjected to animal experiments. After 24 hours, the volume reduction of collagen decreased from 80% in Example 2 to 59%, 48%, and 65%, respectively. The time required for complete disappearance of collagen and skin ecchymosis decreased from 4 days to 8 days, 9 days, and 7 days, respectively, compared to Example 2. Therefore, Comparative Examples 2, 3, and 4 were less effective than Example 2 in treating nodular complications following collagen (stimulator) implantation.

[0147] Pathological sections were examined before, 12 hours after, and 4 days after administration of Example 2 and Comparative Example 4. Nude mice were anesthetized, and skin samples were collected from their backs. The skin was fixed with 4% formaldehyde by weight, and 4 μm paraffin sections were prepared for H&E staining.

[0148] The results are as follows: 0.05 mL of the collagenase for injection of Example 2 and Comparative Example 4 can effectively degrade the collagen gel implanted subcutaneously in mice, wherein the degradation effect of Example 2 is better than that of Comparative Example 4. Figures 5 to 9 shown.

[0149] Figure 5 Image of collagen gel implanted subcutaneously on the back of a nude mouse before collagenase injection. Figure 5 As shown, 1 hour before injection, nude mouse dorsal skin was harvested, collagen gel was recovered, and tissue sections were prepared. The image shows homogeneous pink subcutaneous collagen (blue arrows), with a small number of newly formed fibroblasts. Under crossed polarizers, the polarization signal characteristic of type I collagen is barely visible.

[0150] like Figure 6 As shown, 12 hours after the injection of the injectable collagenase of Example 2, the implanted collagen gel was partially degraded, forming an incomplete micelle (green arrow). The capillaries surrounding the implanted collagen gel were congested, and a small amount of neutrophils infiltrated.

[0151] like Figure 7 As shown, on the 4th day after the injection of the injectable collagenase of Example 2, the collagen gel implanted subcutaneously in most nude mice disappeared, the volume of the collagen gel area remaining subcutaneously did not increase, a large cavity appeared in the center of the gel area (as indicated by the arrow in the figure), fibroblasts proliferated around it, and no inflammatory cell infiltration was observed.

[0152] like Figure 8 As shown, 12 hours after the injection of the collagenase for injection in Comparative Example 4, there was no significant change in the mouse dermis and the implanted collagen gel, indicating that the collagenase digestion effect had not yet emerged. Moderate inflammatory cell infiltration was observed around the subcutaneous collagen gel.

[0153] like Figure 9 As shown, on the 4th day after the injection of the injectable collagenase of Comparative Example 4, the subcutaneous collagen gel was slightly concentrated, part of the collagen was dissolved, many wide cracks appeared inside, the subcutaneous tissue was slightly edematous, and a very small amount of neutrophils infiltrated.

[0154] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art who, without departing from the scope of the present invention, may make slight changes or modifications to the above-disclosed technical contents to obtain equivalent or equivalent embodiments falls within the scope of the present invention.

Claims

1. A lyophilized preparation, which is prepared from a pharmaceutical composition comprising: collagenase, human serum albumin, sugar alcohol and salt; wherein: The weight ratio of the human serum albumin to the sugar alcohol is 1:4-100; the sugar alcohol is selected from one or more of sorbitol, xylitol, mannitol and maltitol, and the pH value of the pharmaceutical composition is 6.0-7.

6.

2. The freeze-dried preparation according to claim 1, wherein The weight ratio of human serum albumin to sugar alcohol is 1:4-80.

3. The freeze-dried preparation according to claim 1, wherein The weight ratio of human serum albumin to sugar alcohol is 1:4-30.

4. The freeze-dried preparation according to claim 1, wherein The weight ratio of human serum albumin to sugar alcohol is 1:8-30.

5. The freeze-dried preparation according to claim 1, wherein The weight ratio of the human serum albumin to the salt is 1:0.1-100. The freeze-dried preparation according to claim 1, wherein The weight ratio of the human serum albumin to the salt is 1:0.2-50.

7. The freeze-dried preparation according to claim 1, wherein The weight ratio of human serum albumin to salt is 1:0.2-8.

8. The freeze-dried preparation according to claim 1, wherein The ratio of the collagenase to human serum albumin is 10-10000 U: 1 mg.

9. The freeze-dried preparation according to claim 1, wherein The ratio of the collagenase to human serum albumin is 50-5000 U: 1 mg.

10. The freeze-dried preparation according to claim 1, wherein The ratio of the collagenase to human serum albumin is 50-3000 U: 1 mg.

11. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of collagenase in the pharmaceutical composition is 100-8000 U / mL.

12. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of collagenase in the pharmaceutical composition is 300-5000 U / mL.

13. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of human serum albumin in the pharmaceutical composition is 0.05-30 mg / mL.

14. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of human serum albumin in the pharmaceutical composition is 0.1-20 mg / mL.

15. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of sugar alcohol in the pharmaceutical composition is 0.5-150 mg / mL.

16. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of sugar alcohol in the pharmaceutical composition is 1-120 mg / mL.

17. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of salt in the pharmaceutical composition is 0.1-80 mg / mL.

18. The freeze-dried preparation according to any one of claims 1 to 10, wherein The content of salt in the pharmaceutical composition is 0.8-50 mg / mL.

19. The pharmaceutical composition according to any one of claims 1 to 10, wherein The salt is selected from one or more of sodium chloride, calcium chloride, sodium phosphate, sodium monohydrogen phosphate and sodium dihydrogen phosphate.

20. The freeze-dried preparation according to any one of claims 1 to 10, wherein The pharmaceutical composition further comprises a solvent.

21. The pharmaceutical composition according to claim 20, wherein The ratio of the collagenase to the solvent is 100-8000 U:1 mL.

22. The pharmaceutical composition according to claim 20, wherein The ratio of the collagenase to the solvent is 300-5000 U:mL.

23. The pharmaceutical composition according to claim 20, wherein The pH value of the pharmaceutical composition is 6.5-7.

5.

24. The pharmaceutical composition according to claim 20, wherein The solvent is water for injection.

25. A method for preparing the lyophilized formulation according to any one of claims 1 to 24, comprising: Collagenase, human serum albumin, sugar alcohol and salt are dissolved in a solvent to obtain a mixed solution, and the mixed solution is lyophilized to obtain the lyophilized preparation.

26. The method according to claim 25, wherein The pH value of the mixed solution is 6.0-7.

6.

27. The method according to claim 25, wherein The pH value of the mixed solution is 6.5-7.

5.

28. The method according to claim 25, wherein The solvent is water for injection.

29. The method according to claim 25, wherein The freeze-drying process includes pre-freezing, primary drying and secondary drying in sequence.

30. The method according to claim 29, wherein The pre-freezing is carried out under the following conditions: the pre-freezing temperature is -30°C to -55°C; and the pre-freezing time is 90-210 minutes.

31. The method according to claim 29, wherein The primary drying is carried out under the following conditions: the primary drying temperature is -45-0°C; and the primary drying time is 20-45h.

32. The method of claim 29, wherein: The secondary drying is carried out under the following conditions: the secondary drying temperature is 0-30° C.; and the secondary drying time is 3-10 hours.

33. The method according to any one of claims 25 to 32, wherein The method further comprises the following step: filtering the mixed solution before freeze-drying.

34. A pharmaceutical product comprising the lyophilized formulation according to any one of claims 1 to 24 and a dispersion medium.

35. The pharmaceutical product according to claim 34, wherein The pH of the drug product is 6.0-7.

6.

36. The pharmaceutical product of claim 34, wherein The pH value of the drug product is 6.5-7.

5.

37. The pharmaceutical product of claim 34, wherein The dispersion medium is selected from one or more of water for injection, glucose aqueous solution, sodium chloride aqueous solution and calcium chloride aqueous solution.

38. A kit comprising the lyophilized formulation according to any one of claims 1 to 24 or the pharmaceutical product according to any one of claims 34 to 37.

39. Use of the lyophilized formulation according to any one of claims 1 to 24 or the pharmaceutical product according to any one of claims 34 to 37 in the preparation of a medicament for preventing and / or treating sarcoidosis, tissue fibrosis, intervertebral disc herniation, Dupuytren's contracture, coronary embolism, arthritis, wound healing and / or scar repair.

40. The pharmaceutical product according to claim 39, wherein The sarcoidosis is a complication of nodules after collagen implantation.

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