Chymotrypsin oral lyophilized powder and its preparation method

Abstract

The application discloses a chymotrypsin oral freeze-dried powder, which comprises chymotrypsin, mannitol, sucrose and dextran 20. Preferably, the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 units:(167-222) mg:(167-222) mg:(83-111) mg. The application also provides a preparation method of the chymotrypsin oral freeze-dried powder. The chymotrypsin oral freeze-dried powder has the advantages of sample stability under high-temperature conditions, and the purity of the chymotrypsin sample does not change obviously and the activity recovery is greater than 90% after being placed at 40 DEG C for 3 months.

Description

Technical Field

[0001] This invention belongs to the field of biological agents, specifically relating to an oral lyophilized chymotrypsin powder and its preparation method. Background Technology

[0002] Chymotrypsin, also known as pancreatic chymotrypsin, is a proteolytic enzyme extracted from bovine or porcine pancreas. It acts as an endopeptidase, specifically hydrolyzing carboxyl-terminal aromatic amino acids (tyrosine, tryptophan, leucine) or large hydrophobic side residues such as methionine by cleaving the carboxyl-terminal peptide chains of tyrosine, tryptophan, and phenylalanine. Additionally, chymotrypsin also acts as a lipase, hydrolyzing certain lipids. As a proteolytic enzyme, chymotrypsin can be used to promote the digestion and removal of blood clots, purulent secretions, and necrotic tissue; in ophthalmic surgery to relax ligaments and alleviate traumatic iridocyclitis; and in wounds or local inflammation to reduce local secretions and edema.

[0003] According to statistics from the International Agency for Research on Cancer, there were approximately 1.089 million new cases of gastric cancer worldwide in 2020, ranking fifth among malignant tumors. There were also approximately 769,000 deaths from gastric cancer worldwide in 2020, ranking fourth among malignant tumor deaths. Early-stage gastric cancer is a curable disease, with a 5-year survival rate exceeding 90% after resection, while the 5-year survival rate for late-stage gastric cancer is only 10% to 20%. Therefore, early detection and treatment are crucial for the cure of gastric cancer. Endoscopy is the most commonly used screening method for early-stage gastric cancer. Gastroscopy is a common diagnostic and treatment method for upper gastrointestinal diseases, not only detecting lesions and clarifying their location, morphology, and nature, but also allowing for endoscopic treatment. However, under normal conditions, the upper gastrointestinal tract contains a significant amount of mucus and foam, which greatly affects the endoscopic field of vision and the detection of early lesions. Repeated flushing increases the endoscopic examination time and also increases the patient's discomfort. Streptomycin acts as a mucin-removing agent to eliminate the protein-induced mucus on the surface of the digestive tract mucosa. However, due to the high price of streptoprotein, it has not been widely adopted, and is often replaced by cheaper chymotrypsin and N-acetylcysteine.

[0004] Currently available chymotrypsin preparations are all injectable chymotrypsin, packaged in 2mL vials, requiring a syringe for use. This is inconvenient for use during gastroscopy, as it is not suitable for oral administration, and its light content makes it unsuitable for bagged packaging. This invention proposes a novel oral chymotrypsin preparation that can be weighed and packaged in bags. It offers advantages such as high stability, convenient storage, easier use, long duration of action, effective removal of gastric mucus, and improved visual clarity. It represents a modification and innovation of traditional processes. This preparation has been submitted for approval as a biopharmaceutical. This oral chymotrypsin preparation of the present invention has significant innovative value for the improvement of biopharmaceutical dosage forms. Summary of the Invention

[0005] One objective of this invention is to provide a novel chymotrypsin formulation, wherein the formulation is in the form of an oral lyophilized powder.

[0006] The oral lyophilized powder of the present invention contains chymotrypsin, mannitol, sucrose and dextran 20, wherein the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 units: 167-222 mg: 167-222 mg: 83-111 mg.

[0007] Preferably, the chymotrypsin is a human chymotrypsin.

[0008] More preferably, the chymotrypsin is recombinant human chymotrypsin.

[0009] Preferably, the ratio of chymotrypsin, mannitol, sucrose and dextran 20 in the oral lyophilized powder of chymotrypsin is 4000 units: 200 mg: 200 mg: 100 mg.

[0010] Another object of the present invention is to provide a method for preparing an oral lyophilized chymotrypsin powder, the method comprising the following steps:

[0011] (1) Pretreatment: Take the chymotrypsin stock solution and concentrate it by dialysis until the conductivity of the filtrate is lower than 0.5 mS / cm to obtain chymotrypsin concentrate;

[0012] (2) Preparation of the mixture: Add 5% mannitol, 5% sucrose and 2.5% dextran 20 to the chymotrypsin concentrate according to the volume ratio, and stir to mix well to obtain the mixture;

[0013] (3) Sterilization filtration: The mixture is sterilized by using a filter membrane;

[0014] (4) Freeze-drying: Freeze-dry the filtrate after filtration in step (3) to obtain freeze-dried powder;

[0015] (5) After pulverizing the freeze-dried powder, it is packaged to obtain the oral freeze-dried powder of chymotrypsin.

[0016] Preferably, in step (1), the chymotrypsin stock solution is concentrated and dialyzed using a 5kD membrane and water for injection at a temperature below 25°C.

[0017] Preferably, the content of chymotrypsin in the chymotrypsin concentration dialysis process in step (1) is 1 mg / mL-4 mg / mL, and the activity is 2000 U / mL-9600 U / mL.

[0018] Preferably, the chymotrypsin concentrate in step (1) has a chymotrypsin content greater than 0.6 mg / mL and an activity greater than 1000 U / mL.

[0019] Preferably, the chymotrypsin concentrate in step (1) has a chymotrypsin activity of 1200 U / mL to 4000 U / mL.

[0020] Preferably, the pH value of the mixture in step (2) is 3.0-5.0.

[0021] Preferably, step (2) uses a 0.22μm filter membrane for sterilization filtration.

[0022] Preferably, the content of chymotrypsin in the mixture of step (2) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1100-1300 U / mL.

[0023] Preferably, the content of chymotrypsin in the filtrate of step (3) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1000-1300 U / mL.

[0024] Preferably, step (4) includes:

[0025] (a) Pre-freezing: The sample is placed in the freeze dryer at room temperature, and after the partition is cooled to -45°C, it is pre-frozen for more than 4 hours;

[0026] (b) Single drying: After the condensation temperature drops below -45°C, vacuum is applied to below 10 Pa; the partition is raised to -30°C at a rate of 5°C / 1.5h, and kept at that temperature for more than 48h until the water of crystallization disappears;

[0027] (c) Secondary drying: Raise the temperature of the partition to 20°C at a rate of 5°C / 1.5h and keep it at that temperature for more than 15h; check that there is no change in the vacuum degree, then end the freeze drying and remove the sample from the cabinet.

[0028] Preferably, the packaging specification in step (5) is 0.5g / bag.

[0029] Preferably, the preparation method includes:

[0030] In step (1), chymotrypsin stock solution is used as raw material, with a protein content of 1.0±0.2 mg / mL. It is concentrated and dialyzed using a 5 kDa membrane. The membrane is first treated with 0.5 M NaOH for at least 30 min, and then washed with water for injection. After washing, the chymotrypsin stock solution is concentrated to 1 / 2-1 / 4 of its original volume, and an equal volume of water for injection is added. After mixing, the concentration and dialysis are repeated until the conductivity of the filtrate is lower than 0.5 mS / cm. During the concentration and dialysis process, the protein concentration is 1-4 mg / mL. After dialysis, the protein concentration is adjusted to 0.6-2.0 mg / mL, and the solution pH is 3.0-5.0, preferably 4.0.

[0031] In step (2), the activity and protein content of the chymotrypsin solution after dialysis and concentration in step (1) are detected. Its activity is diluted with water for injection to 1000-1300 U / mL and the protein content is 0.4-0.7 mg / mL. 5% mannitol, 5% sucrose and 2.5% dextran 20 are added according to the final volume ratio and stirred and mixed.

[0032] In step (3), under Class A laminar flow, the preparation solution from step (2) is sterilized by two-stage 0.22μm filters. The resulting sample is a semi-finished product of chymotrypsin with an activity of 1000-1300U / mL and a protein content of 0.4-0.7mg / mL.

[0033] In step (4), the chymotrypsin semi-finished product from step (3) is dispensed and freeze-dried at 1L / tray under laminar flow. The freeze-drying process includes the following steps:

[0034] (a) Pre-freezing: The sample is placed in the freeze dryer at room temperature, and after the partition is cooled to -45°C, it is pre-frozen for more than 4 hours;

[0035] (b) Single drying: After the condensation temperature drops below -45°C, vacuum is applied to below 10 Pa; the partition is raised to -30°C at a rate of 5°C / 1.5h, and kept at that temperature for more than 48h until the water of crystallization disappears;

[0036] (c) Secondary drying: The temperature of the partition is increased to 20°C at a rate of 5°C / 1.5h and kept at that temperature for more than 15h; the vacuum degree is checked and no change is found. The freeze drying is then stopped and the sample is taken out of the freezer to obtain oral lyophilized chymotrypsin powder.

[0037] In step (5), the lyophilized oral chymotrypsin powder from step (4) is pulverized using a pulverizer and packaged in 0.5g / bags. The packaging material is an opaque aluminum-containing composite film, preferably a polyester / aluminum / polyethylene pharmaceutical composite film. The horizontal and vertical seals are confirmed to be straight and properly sealed, thus completing the preparation of the lyophilized oral chymotrypsin powder.

[0038] The lyophilized oral chymotrypsin powder of the present invention can be used for gastroscopy to remove gastric mucus and improve the clarity of the field of vision. The method of administration is to mix one sachet of lyophilized oral chymotrypsin powder with one sachet of 1g sodium bicarbonate, dissolve in warm water, and take within 1 hour. The volume of warm water is preferably 50-100mL. Attached Figure Description

[0039] Figure 1 Observe the clarity of the solution at different pH values ​​during dialysis.

[0040] Figure 2 .Diagram of spray-dried granulation of human chymotrypsin powder.

[0041] Figure 3SDS-PAGE analysis of different formulations of human chymotrypsin oral granules; Figure A shows the exploration of human chymotrypsin oral granule formulations, and Figure B shows the optimization of human chymotrypsin oral granule formulations, where M is the protein standard molecular weight marker, groups 1-6 represent samples with different formulations, and 4℃ / 25℃ / 37℃ represent samples placed at different temperatures of 4℃ / 25℃ / 37℃.

[0042] Figure 4 SDS-PAGE analysis of human chymotrypsin oral granule samples prepared by spray drying under different conditions; where M is the protein standard molecular weight marker, groups 1-6 represent samples prepared by different spray drying processes, 4℃ / 25℃ / 37℃ represent samples placed at different temperatures of 4℃ / 25℃ / 37℃, before spray drying represents samples before spray drying, vacuum represents samples stored in vacuum packaging, and aluminum bag represents samples stored in non-vacuum aluminum bag packaging.

[0043] Figure 5 Human chymotrypsin was freeze-dried into dry powder and granulated; where M is the protein standard molecular weight marker, groups 1-6 represent samples prepared by different granulation processes, 100% represents samples prepared with 100% ethanol, 80% represents samples prepared with 80% ethanol, and pure water represents samples prepared with purified water.

[0044] Figure 6 The stability of human chymotrypsin oral lyophilized powder with different formulations was investigated over 3 months, and the samples were analyzed by SDS-PAGE. M is the protein standard molecular weight marker, groups 1-2 represent lyophilized samples prepared with different formulations, and 4℃ / 25℃ / 37℃ represent samples placed at different temperatures of 4℃ / 25℃ / 37℃.

[0045] Figure 7 Observation of the solution after mixing human chymotrypsin oral lyophilized powder with sodium bicarbonate (30 min).

[0046] Figure 8 Observation on the effect of different doses of human chymotrypsin oral lyophilized powder on mucus removal in beagle dogs during gastroscopy. Detailed Implementation

[0047] The technical solutions of the present invention will be described in detail below with reference to embodiments and accompanying drawings, thereby better illustrating the features and advantages of the present invention. The provided embodiments should be interpreted as illustrative examples of the methods of the present invention, and not as limiting the technical solutions disclosed in the present invention in any way.

[0048] Unless otherwise specified, all reagents and instruments used in the examples are commercially available.

[0049] [Example 1] Selection of dialysis conditions for human chymotrypsin stock solution

[0050] Human chymotrypsin stock solution was concentrated using a 5kD membrane, and an equal volume of purified water was added. Dialysis and concentration were continued until the conductivity was 0.5 mS / cm. 8 mL samples were taken per tube, and the pH was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.1, 9.5, and 10.0, respectively. When the pH was between 6.0 and 7.0, the sample became turbid (slight turbidity began to appear at pH 5.5). When the pH was adjusted back to 3.5, the sample became clear. Adding 3% 10xPBS to the turbid sample at pH 7.0 clarified the sample. After standing for 2 hours (room temperature) and 20 hours (room temperature and 2–8°C), the samples were observed and their activity was detected. The results showed that during dialysis, the conductivity of chymotrypsin dropped to 0.5 mS / cm, and the sample became clear. After adjusting the pH to 6.0–7.0, the sample became turbid. When the pH was adjusted back to 3.5, the sample returned to clarity, and the activity remained unchanged. The turbid sample at pH 7.0 became clear again after adding salt, but the activity decreased to about half, indicating that salt should not be added for reconstitution during dialysis. Chymotrypsin was relatively stable at pH below 5.0. After being placed at room temperature for 20 hours, the activity was still above 800 U / mg, and the closer the pH was to 3.0, the more stable it was. Therefore, the pH of the solution after dialysis was determined to be pH 3.0–5.0, with pH 4.0 being the preferred value.

[0051] Table 1. Activity detection of chymotrypsin stock solution at different pH solutions after dialysis (Unit: U / mL)

[0052]

[0053] [Example 2] Preparation of oral granules of human chymotrypsin by spray drying

[0054] 1. Study on spray-dried granulation of human chymotrypsin powder

[0055] Human chymotrypsin powder was obtained by spray drying a solution containing 10% mannitol and 1000 U / mL. Granulation was performed using anhydrous ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and purified water, respectively. The results showed that after granulation with different concentrations of ethanol and purified water, the higher the ethanol concentration, the better the particle dispersibility and the more uniform the particle size. Figure 2 However, the lower the recovery rate of particles (>80 mesh), and the less significant the effect of different wetting agents on the sample activity, the more determined we are to use 70% ethanol for subsequent particle stability sample granulation.

[0056] Table 2 Recovery rate of qualified particles from 10 mesh to 80 mesh:

[0057] Anhydrous ethanol 2.08 0.29 83.2 90% ethanol 2.20 0.18 88.0 80% ethanol 2.16 0.17 86.4 70% ethanol 2.34 0.13 93.6 Purified water 0.95 0.02 76.0

[0058] Table 3 Activity detection of samples after granulation with different wetting agents

[0059] Pre-granulation control 2.0 2738 100 Anhydrous ethanol 2.0 2591 94.6 90% ethanol 1.9 2498 91.2 80% ethanol 1.9 2526 92.3 70% ethanol 2.0 2680 97.9 Purified water 2.0 2827 103.3

[0060] 2. Exploration of human chymotrypsin spray-drying formulation

[0061] Different dosages of the drug were designed, namely 1g, 0.5g, and 0.2g, corresponding to human chymotrypsin concentrations of 0.2mg / mL, 0.4mg / mL, and 1mg / mL in 10% mannitol, with activities of 400U / mL, 800U / mL, and 2000U / mL, respectively.

[0062] 1g granules: While mixing 80g of sprayed 1g dry powder, add 70% ethanol until the final volume is 25.6mL. Press the powder into granules using a 20-mesh sieve, dry it overnight in an oven at 37℃, and finally dispense it into 1g portions under vacuum / non-vacuum conditions. The stability of the powder is then examined at 2–8℃, 25℃, and 40℃.

[0063] 0.5g granules: While mixing, add 70% ethanol to 40g of sprayed 0.5g dry powder, with a final volume of 12.8mL. Other steps are the same as above.

[0064] 0.2g granules: While mixing, add 70% ethanol to 16g of sprayed 0.2g dry powder, with a final volume of 5.12mL. Other steps are the same as above.

[0065] 0.2g OsrhCT dry powder + 1g sodium bicarbonate powder were combined and granulated. Add 80g of sodium bicarbonate powder to 16g of sprayed 0.2g dry powder, mix well, and add 70% ethanol while mixing, with a final volume of 30.7mL. The rest is the same as above.

[0066] 0.2g OsrhCT granules + 1g sodium bicarbonate powder: While mixing 16g of sprayed 0.2g dry powder, 70% ethanol was added, with a final volume of 5.12mL. The mixture was then granulated using a 20-mesh sieve and dried overnight in an oven at 37℃. Finally, the powder was vacuum / non-vacuum packaged in 0.2g portions, with 1g of sodium bicarbonate powder added to each package. The stability was then examined at 2–8℃, 25℃, and 40℃.

[0067] The results showed that group 3 (0.2g packaging) had the highest relative protein activity, indicating that the 0.2g packaging was superior to the 1g and 0.5g packaging; vacuum or non-vacuum packaging had little impact on the samples. Group 4 (combined granulation) had the lowest specific activity, followed by group 5 (combined packaging), which also showed low specific activity. SDS-PAGE analysis revealed significant degradation in both groups. Figure 3A) This indicates that human chymotrypsin should not be packaged with sodium bicarbonate, as this will affect the human chymotrypsin. Group 3 showed the highest relative specific activity, but compared to the original solution, the specific activity recovery was below 85%, and the specific activity tended to decrease with increasing temperature, suggesting that spray-dried samples may be less stable than freeze-dried samples.

[0068] Table 4. Results of protein activity assay after 2 weeks (unit: U / mg)

[0069]

[0070]

[0071] 3. Optimization of human chymotrypsin spray-drying formulation

[0072] The activity recovery of the 0.2g sample was higher than that of the 1g and 0.5g samples in the early stage. Therefore, the 0.2g sample was used as the main sample to investigate the protective effect of different stabilizer contents on human chymotrypsin particles.

[0073] Group 1 (2% mannitol + 0.5% dextran 20) - 0.25g specification: Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) and add 1900 mL of pH 3.4 purified water, for a total of 2 L. Add 40 g of mannitol and 10 g of dextran 20, mix well, filter through a 0.22 μm filter membrane, and spray dry.

[0074] Group 2 (5% mannitol + 0.5% dextran 20) - 0.22g specification Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) and add 700 mL of pH 3.4 purified water, for a total of 800 mL. Add 40 g of mannitol and 4 g of dextran 20 and mix well. Repeat the above steps.

[0075] Group 3 (5% mannitol + 1.25% dextran 20) - 0.25g specification Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) and add 700 mL of pH 3.4 purified water, for a total of 800 mL. Add 40 g of mannitol and 10 g of dextran 20 and mix well. Repeat the above steps.

[0076] Group 4 (10% mannitol + 0.1% dextran 20) - 0.2g specification Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL), add 300 mL of pH 3.4 purified water, for a total of 400 mL, add 40 g of mannitol and 0.4 g of dextran 20, mix well, and repeat as above;

[0077] Group 5 (10% mannitol + 0.5% dextran 20) - 0.21g specification Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL), add 300 mL of pH 3.4 purified water, for a total of 400 mL, add 40 g of mannitol and 2 g of dextran 20, mix well, and repeat as above;

[0078] Group 6 (10% mannitol + 2.5% dextran 20) - 0.25g specification Take 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) and add 300 mL of pH 3.4 purified water, for a total of 400 mL. Add 40 g of mannitol and 10 g of dextran 20 and mix well. Repeat the above steps.

[0079] The results of the 2-month stability test showed that the higher the temperature, the higher the polymer content, and the purity of the sample at 4℃ did not change significantly. Figure 3 B) The protein specific activity recovery was around 80% (around 90% after 1 month). For samples at 25℃, the protein specific activity recovery was around 70% (around 80% after 1 month); for samples at 40℃, the protein specific activity recovery was 40%–50% (50%–60% after 1 month). Compared with 1 month, the activity showed a significant decreasing trend, with an overall decrease of about 10%, suggesting that spray drying may not be suitable for sample stability.

[0080] Table 5. Results of protein activity assay after 2 months (unit: U / mg)

[0081]

[0082] 4. Optimization of spray drying process for human chymotrypsin

[0083] The spray drying process was optimized using a formulation of 10% mannitol + 0.1% dextran 20 (which has higher activity recovery than other formulations).

[0084] Group 1: Inlet temperature 90℃, outlet temperature 70℃ (uncontrollable, actual temperature 58℃~60℃), atomization frequency 390~400HZ, sample injection flow rate 10rpm (20rpm cannot be sprayed dry, sample adheres to the wall, water droplets are clearly observed), spray drying is performed;

[0085] Group 2: Inlet temperature 100℃, outlet temperature 70℃ (uncontrollable, actual temperature 60℃~62℃), atomization frequency 390~400HZ, sample injection flow rate 12rpm (20rpm cannot be sprayed dry, sample adheres to the wall, water droplets are clearly observed), spray drying is performed;

[0086] Group 3: Inlet temperature 120℃, outlet temperature 80℃ (uncontrollable, actual temperature 64℃~66℃), atomization frequency 390~400HZ, sample injection flow rate 20rpm, spray drying;

[0087] Group 4: Inlet temperature 120℃, outlet temperature 80℃ (uncontrollable, actual temperature 77℃~80℃), atomization frequency 390~400HZ, injection flow rate 10rpm, spray drying.

[0088] Group 5: Inlet air temperature 150℃, outlet air temperature 80℃ (uncontrollable, actual temperature 75℃~80℃), atomization frequency 390~400HZ, sample injection flow rate 40rpm (50rpm cannot be sprayed dry, sample sticks to the wall, water droplets are clearly observed), spray drying is performed.

[0089] Group 6: Inlet temperature 180℃, outlet temperature 80℃ (uncontrollable, actual temperature 80℃~85℃), atomization frequency 390~400HZ, sample injection flow rate 40rpm (50rpm cannot be sprayed dry, sample adheres to the wall, water droplets are clearly observed), spray drying is performed;

[0090] Table 6. Activity test results before and after spraying and after granulation (unit: U / mg)

[0091]

[0092] Optimizing the spray drying process by lowering the outlet temperature to (90℃, 100℃) and changing the spray speed to (120℃, 10rpm / 20rpm), and increasing both the outlet temperature and spray speed to (150℃, 180℃, 40rpm), showed no significant change in sample activity after spraying and granulation. The final one-month stability test results showed no significant difference between vacuum packaging and non-vacuum aluminum bag packaging; higher temperatures resulted in more aggregates. Figure 4 The activity showed a significant decreasing trend. The specific activity recovery of the protein was 83%–93% for samples at 4℃, 70%–80% for samples at 25℃, and 40%–64% for samples at 40℃. This suggests that optimizing the spray drying process did not significantly improve the stability of human chymotrypsin.

[0093] Table 7. Results of protein activity assay after 1 month (unit: U / mg)

[0094]

[0095] Conclusion: The data from this case study show that the human chymotrypsin prepared by spray drying process has poor stability, and the activity of the sample shows a significant decreasing trend with prolonged storage time.

[0096] [Example 3] Feasibility of freeze-drying preparation of human chymotrypsin oral freeze-dried powder

[0097] 1. Study on freeze-dried granulation of human chymotrypsin powder

[0098] Group 1 (2% mannitol + 0.5% dextran 20) - 0.25g specification One portion of freeze-dried powder was moistened with 80% ethanol and then extruded into granules; another portion of freeze-dried powder was moistened with 100% ethanol and then extruded into granules.

[0099] Group 2 (5% mannitol + 0.5% dextran 20) - 0.22g specification : The freeze-dried powder was extruded and granulated using 80% ethanol;

[0100] Group 3 (5% mannitol + 1.25% dextran 20) - 0.25g specification : The freeze-dried powder was extruded and granulated using 80% ethanol;

[0101] Group 4 (10% mannitol + 0.1% dextran 20) - 0.2g specification Freeze-drying failed;

[0102] Group 5 (10% mannitol + 0.5% dextran 20) - 0.21g specification : The freeze-dried powder is moistened with pure water and then extruded into granules;

[0103] Group 6 (10% mannitol + 2.5% dextran 20) - 0.25g specification : The freeze-dried powder is moistened with pure water and then extruded into granules.

[0104] The results showed that the freeze-dried powder was not suitable for granulation. The sample had high viscosity after wetting, making it difficult to mix evenly. During granulation, the particles were prone to agglomeration and exhibited poor dispersibility. Figure 5 A); simultaneously, electrophoretic detection shows the formation of aggregates and degradation bands in the particles ( Figure 5 B) The activity recovery is less than 80%, and the granules do not dissolve when reconstituted after granulation with 100% ethanol. The dosage form cannot be selected as granules and can only be packaged as powders.

[0105] Table 8. Activity detection of human chymotrypsin lyophilized powder after granulation (unit: U / mg)

[0106]

[0107] 2. Formulation exploration and stability study of human chymotrypsin lyophilized powder

[0108] Currently, commercially available injectable human chymotrypsin preparations are packaged in 2mL vials, requiring syringes for use and unsuitable for oral administration. The contents are also relatively light (less than 50mg), making bagged repackaging inconvenient. To optimize the lyophilization formulation, the following formulation was developed: Group 1: 10% mannitol + 2.5% dextran 20; Group 2: 5% mannitol + 5% sucrose + 2.5% dextran 20. The human chymotrypsin protein concentration is 0.5mg / mL. 300mL of each formulation is lyophilized, and then repackaged in 0.5g bags, resulting in 4000U / bag. The lyophilization process is as follows:

[0109]

[0110]

[0111] After lyophilized human chymotrypsin powder was prepared and stored at different temperatures for 3 months, the purity of the remaining samples showed no significant change except for the non-vacuum-packed aluminum bags in Group 1 (25℃ and 40℃) which showed diffuse bands and degradation bands. Figure 6The activity test results showed that the protein specific activity of the group 1 samples decreased significantly with increasing storage temperature, and the protein specific activity recovery of the samples packaged in aluminum bags at 40℃ was only 9%. The specific activity recovery of the samples in group 2 at all temperatures (4℃, 25℃ and 40℃, vacuum and aluminum bag packaging) was above 90%, indicating that the formulation of group 2 was more stable than that of group 1. The formulation was determined to be 5% mannitol + 5% sucrose + 2.5% dextran 20, with a human chymotrypsin protein concentration of 0.5 mg / mL and an activity of 1000 U / mL.

[0112] Table 9. Activity test results of human chymotrypsin lyophilized powder samples after 3 months (unit: U / mg)

[0113]

[0114] Conclusion: The data from this case study show that the human chymotrypsin lyophilized powder prepared by the freeze-drying method of this invention has the advantage of high-temperature stability. After being placed at 40°C for 3 months, the purity of the human chymotrypsin sample did not change significantly, and the activity recovery was greater than 90%.

[0115] [Example 4] Stability Study of Pilot-Scale Production Samples of Human Chymotrypsin Oral Lyophilized Powder

[0116] Take 5L of human chymotrypsin stock solution, concentrate and dialyze it using a 5kD membrane, then add 5% mannitol + 5% sucrose + 2.5% dextran 20 and mix well. Filter the mixture sterilely using a 0.22μm filter to obtain chymotrypsin semi-finished product. Dispense the product into 1L / tray for lyophilization under laminar flow. The lyophilization process includes the following steps:

[0117] (a) Pre-freezing: The sample is placed in the freeze dryer at room temperature, and after the partition is cooled to -45°C, it is pre-frozen for more than 4 hours;

[0118] (b) Single drying: After the condensation temperature drops below -45°C, vacuum is applied to below 10 Pa; the partition is raised to -30°C at a rate of 5°C / 1.5h, and kept at that temperature for more than 48h until the water of crystallization disappears;

[0119] (c) Secondary drying: The temperature of the separator was increased to 20°C at a rate of 5°C / 1.5h and maintained at this temperature for at least 15h. The vacuum level was checked for any change, and the freeze-drying process was terminated. The sample was removed from the freezer to obtain oral lyophilized chymotrypsin powder. After pulverization, the powder was dispensed in 0.5g packets, and its accelerated stability at 25°C was investigated.

[0120] Table 10 Accelerated stability test data of human chymotrypsin lyophilized powder (25±2℃ / RH 60%±5%)

[0121]

[0122]

[0123] Conclusion: The data from this case study show that the human chymotrypsin lyophilized powder prepared by the freeze-drying method of this invention has good batch-to-batch consistency. After 6 months of accelerated storage, the quality indicators of the samples are still qualified and can be used for large-scale sample production.

[0124] [Example 5] In vitro activity detection of human chymotrypsin oral lyophilized powder

[0125] 1. Exploration of the mixing method between human chymotrypsin oral lyophilized powder and sodium bicarbonate.

[0126] (1) Take 100 mL of purified water, add 1 g of sodium bicarbonate and mix well, then add 0.5 g of human chymotrypsin oral lyophilized powder and mix well. The pH is 8.32. Observe the changes in solution and activity.

[0127] (2) First, add 1g of sodium bicarbonate and 0.5g of human chymotrypsin oral lyophilized powder to the conical flask, then add 100mL of purified water and mix well. The pH is measured to be 8.38. Observe the changes in solution and activity.

[0128] (3) Take 100 mL of purified water, add 0.5 g of human chymotrypsin oral lyophilized powder and mix well, then add 1 g of sodium bicarbonate and mix well. The pH is measured to be 8.38, and the change in solution is observed.

[0129] The results showed that the stability of the solution varied significantly depending on the reconstitution method. When sodium bicarbonate and human chymotrypsin oral lyophilized powder were added first, followed by the addition of 100 mL of purified water, the solution remained clear (even overnight). However, regardless of whether sodium bicarbonate was added first to dissolve and mix before or after adding the human chymotrypsin oral lyophilized powder, the solution became cloudy after approximately 30 minutes. Figure 7 It is recommended that the solid be poured out first, and then water be added to dissolve and mix thoroughly before clinical use. Activity testing results showed no significant change in solution activity within 4 hours (recovery greater than 90%).

[0130] Table 11 Changes in the activity of human chymotrypsin oral lyophilized powder solution after being mixed and dissolved with sodium bicarbonate.

[0131] Activity (U / mL) 37.9 37.0 36.1 36.1 36.8 36.3 Recovery rate (%) 100 97.7 95.3 95.1 97.1 95.9

[0132] 2. Detection of in vitro activity changes of human chymotrypsin oral lyophilized powder in a simulated gastric juice environment.

[0133] This experiment references the national standard "Food Safety Testing of Genetically Modified Organisms and Their Products - Test Method for Stability of Exogenous Protein Digestion in Simulated Gastrointestinal Fluid". The experiment used a gastric fluid volume of 50-100 mL, 0.5 g of human chymotrypsin oral lyophilized powder + 1 g sodium bicarbonate solution in a volume of 100 mL, simulating a gastric digestive fluid:oral solution ratio of 1:1 and 1:2. Reaction times were 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, 2 h, 3 h, and 4 h.

[0134] Add 5 mL and 10 mL of simulated gastric digestion fluid (SGF) to two 50 mL centrifuge tubes, respectively, and incubate at 37 °C for 5 min. Add 10 mL of human chymotrypsin oral lyophilized powder + sodium bicarbonate solution to each tube, rapidly shake to mix, and quickly place in a 37 °C water bath. Accurately record the time. At each reaction time point, quickly aspirate the reaction solution for activity testing. Results showed that after mixing simulated gastric digestion fluid with human chymotrypsin oral lyophilized powder + sodium bicarbonate oral solution at different ratios, the solution pH remained between 5.7 and 6.8. Activity testing showed no significant change in activity within 3 h (recovery above 90%).

[0135] Table 12. Activity test results of simulated gastric digestive fluid: oral solution = 1:1.

[0136]

[0137]

[0138] Table 13. Activity test results of simulated gastric digestive fluid: oral solution = 1:2.

[0139] Activity (U / mL) 35.8 36.0 34.5 33.1 32.5 31.1 Recovery rate (%) 100 100.6 96.5 92.6 90.7 87.0

[0140] [Example 6] Detection of the mucus removal effect of human chymotrypsin oral lyophilized powder in Beagle dogs during gastroscopy

[0141] Ten dogs (sex not limited) were randomly assigned to four groups: a control group (n=1), and low, medium, and high dose groups (n=3 per group). Each group received a blank control (sodium bicarbonate solution) and test samples (recombinant human chymotrypsin oral lyophilized powder) at doses of 60, 150, and 750 U / kg, respectively, administered via gavage. The dogs were euthanized seven days later. Before endoscopy, the animals were under general anesthesia. The first endoscopy was performed to check for foam in the stomach, and the images were saved. During endoscopy, 4000 U of recombinant human chymotrypsin oral lyophilized powder and 1 g of sodium bicarbonate were added to 100 mL of drinking water (20–40°C), shaken to dissolve, and then administered orally to the dogs according to their weight. Endoscopic observation was performed 30 minutes later, and the images were saved. During the experiment, clinical observation, adverse events, survival rate, body temperature / weight, blood biochemistry, visual field clarity evaluation, safety evaluation, gross anatomical observation, and histopathological examination were monitored.

[0142] The results showed that, compared with the preoperative and control groups, the low, medium, and high dose groups significantly removed gastric mucus and foam after administration. Figure 8 After 30 minutes, the gastroscopy showed good clarity and no adverse reactions occurred.

[0143] Table 14 Visual field clarity scores before and after drug administration

[0144]

[0145] Note: Evaluation criteria: Excellent image with no mucus or foam adhering to the gastric mucosa, 1 point; Good image with a small amount of mucus and foam adhering to the gastric mucosa that does not affect observation, 2 points; Poor image with mucus and foam adhering to the gastric mucosa, requiring rinsing with water (less than 50 mL), 3 points; Very poor image with a large amount of mucus and foam adhering to the gastric mucosa, requiring rinsing with water (greater than or equal to 50 mL), 4 points.

Claims

1. A lyophilized oral chymotrypsin powder, characterized in that, The oral lyophilized powder of chymotrypsin contains chymotrypsin, mannitol, sucrose and dextran 20, and the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 units: 167-222 mg: 167-222 mg: 83-111 mg, and the chymotrypsin is human chymotrypsin.

2. The oral lyophilized chymotrypsin powder according to claim 1, characterized in that, The chymotrypsin is recombinant human chymotrypsin.

3. The oral lyophilized powder of chymotrypsin according to claim 1, characterized in that, The ratio of chymotrypsin, mannitol, sucrose, and dextran 20 is 4000 units: 200 mg: 200 mg: 100 mg.

4. A method for preparing an oral lyophilized chymotrypsin powder according to any one of claims 1-3, comprising the following steps: (1) Pretreatment: taking chymotrypsin stock solution and dialysis concentrating it until the conductivity of the filtrate is lower than 0.5 mS / cm to obtain chymotrypsin concentrate; (2) Preparation of a mixture: adding 5% mannitol, 5% sucrose and 2.5% dextran 20 to the chymotrypsin concentrate according to the volume ratio, and stirring to obtain the mixture, wherein the pH value of the mixture is 3.0-5.0; (3) Sterilization filtration: using a filter membrane to sterilize the mixture to obtain filtrate; (4) Freeze-drying: freeze-drying the filtrate after filtration in step (3) to obtain lyophilized powder; (5) pulverizing the lyophilized powder and dispensing it to obtain the oral lyophilized chymotrypsin powder.

5. The preparation method according to claim 4, characterized in that, Step (1) The chymotrypsin stock solution is concentrated and dialyzed using a 5kD membrane and / or water for injection at a temperature below 25°C.

6. The preparation method according to claim 4, characterized in that, In step (1), the chymotrypsin concentration dialysis process has a chymotrypsin content of 1 mg / mL-4 mg / mL and an activity of 2000 U / mL-9600 U / mL.

7. The preparation method according to claim 4, characterized in that, The chymotrypsin concentrate in step (1) has a chymotrypsin content greater than 0.6 mg / mL and an activity greater than 1000 U / mL.

8. The preparation method according to claim 7, characterized in that, The chymotrypsin concentrate has a chymotrypsin activity of 1200 U / ml-4000 U / ml.

9. The preparation method according to claim 4, characterized in that, The content of chymotrypsin in the mixture in step (2) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1100-1300 U / mL.

10. The preparation method according to claim 4, characterized in that, The content of chymotrypsin in the filtrate of step (3) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1000-1300 U / mL.

11. The preparation method according to claim 4, characterized in that, Step (4) includes: (a) Pre-freezing: The sample is placed in the freeze dryer at room temperature, and after the partition is cooled to -45°C, it is pre-frozen for more than 4 hours; (b) First drying: After the condensation temperature is lowered to below -45°C, the vacuum is drawn to below 10 Pa; the partition is raised to -30°C at a rate of 5°C / 1.5h and kept at this temperature for more than 48 hours until the water of crystallization disappears; (c) Second drying: the partition is raised to 20°C at a rate of 5°C / 1.5h and kept at this temperature for more than 15 hours; if the vacuum degree is checked and there is no change, the freeze drying is ended and the sample is taken out of the freezer.

12. The preparation method according to claim 4, characterized in that, The packaging specification described in step (5) is 0.5g / bag.

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