A three-layer osmotic pump controlled-release tablet of a pancreatic kallikreinogen and a preparation method thereof

Abstract

The application discloses a pancreatinase three-layer osmotic pump controlled-release tablet and a preparation method thereof, relates to the technical field of pharmaceutical preparations, and discloses that the pancreatinase three-layer osmotic pump controlled-release tablet comprises, from inside to outside, a three-layer tablet core, an isolation coat layer, a semi-permeable membrane coat layer and a light-shielding coat layer; the three-layer tablet core comprises a drug layer, a boosting layer and an auxiliary permeation layer; and the drug layer is subjected to drug stabilization treatment by using trehalose and mannitol. The pancreatinase three-layer osmotic pump controlled-release tablet has the advantages of stable drug release, simple preparation process, effective maintenance of pancreatinase activity, important clinical value and industrial significance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a pancreatic kallikrein triple-layer osmotic pump controlled-release tablet and its preparation method. Background Technology

[0002] Pancreatic kallikreinase, also known as bradykinin or kallikrein-releasing enzyme, is a proteolytic enzyme extracted from porcine pancreas. It consists of 18 amino acids and 4 sugars, with a molecular weight of approximately 26,800. As a classic microcirculation improver, kallikrein catalyzes the conversion of kininogen into kinins, which then act on vascular endothelial cells, promoting the release of vasodilators such as nitric oxide and prostacyclin. This relaxes vascular smooth muscle, dilates blood vessels, and increases blood perfusion to tissues and organs. Simultaneously, this enzyme can activate plasminogen into plasmin, hydrolyzing insoluble fibrin, inhibiting platelet aggregation and adhesion, and reducing blood viscosity, thereby improving microcirculatory disorders.

[0003] Clinically, kallikrein preparations are widely used for various microcirculatory disorders, such as diabetic nephropathy, peripheral neuropathy, retinopathy, fundus disease, and ischemic cerebrovascular disease. They can also be used as adjunctive therapy for hypertension. Common clinical dosage forms are injectable kallikrein and enteric-coated kallikrein tablets. Currently, enteric-coated kallikrein tablets (60 IU or 120 IU) are administered at a dose of 120-240 units three times daily. However, for patients with chronic diseases such as diabetic nephropathy requiring long-term, lifelong medication, this high-frequency three-times-a-day regimen significantly reduces adherence and easily leads to missed doses. Furthermore, after oral administration, this drug primarily exerts its effect temporarily in the intestines, with minimal absorption into the bloodstream. Frequent dosing causes pulsatile fluctuations in local enzyme activity, making it difficult to sustainably and stably improve microcirculation. Repeated pulsatile dosing may also increase the risk of local gastrointestinal irritation (occasionally causing stomach discomfort), while allergic reactions such as rashes and itching are related to individual immune responses and are not dose-dependent. Therefore, the current dosing frequency is insufficient to meet the needs of stable and effective clinical treatment.

[0004] Furthermore, kallikrein, as a proteolytic enzyme, is highly sensitive to changes in heat, humidity, light, and pH. It is relatively stable at pH 4.5–7.8, but unstable to heat, strong acids, strong alkalis, and oxidizing agents. In existing tablet manufacturing processes, wet granulation and tableting are not only complex and lengthy, resulting in significant raw material and excipient losses, but also prone to potency reduction under humid and hot conditions. The blank granulation method is prone to causing the finished product to have inconsistent content uniformity and unstable quality.

[0005] The process disclosed in patent document CN101069742 requires an investment of at least 120% of the raw material cost to ensure the final product's potency. While the enteric-coated tablets disclosed in patent document CN102813637 use stabilizers, they are still prepared using the blank particle method, which cannot effectively solve the problem of content uniformity. Patent document CN104069486A discloses a kallikrein composition using poloxamer and sodium lactate as stabilizers, but the prepared product is a conventional formulation without sustained-release function.

[0006] Osmotic pump controlled-release tablets are currently the most ideal dosage form for oral controlled-release formulations, capable of releasing drugs at a constant rate using zero-order kinetics, maintaining stable blood drug concentrations for 24 hours, thereby reducing dosing frequency, improving efficacy, and reducing toxic side effects. However, applying osmotic pump technology to pancreatic kallikrein faces the following technical challenges:

[0007] (1) Traditional double-layer push-pull permeation pump (containing drug layer + booster layer) can only drill holes on one side of the drug layer. During the production process, it is necessary to accurately identify the interface between the drug layer and the booster layer (industrialization usually uses near-infrared spectroscopy identification, surface optical reflection difference or sheet geometry differentiation, etc.) and use a high-precision laser drilling device. The process is complicated, the equipment requirements are high and the production efficiency is low.

[0008] (2) Kallikrein is sensitive to damp heat. Traditional wet granulation and high-temperature coating processes can easily lead to significant loss of enzyme activity.

[0009] (3) The design of osmotic pumps for oral protein drugs needs to solve the two major contradictions of release control and activity maintenance. There are no reports of successful application of osmotic pump controlled release technology to pancreatic kallikrein.

[0010] Therefore, developing a controlled-release formulation that has stable drug release, simple preparation process, and can effectively maintain the activity of kallikrein has important clinical value and industrial significance. Summary of the Invention

[0011] In view of the problems of complex pancreatic kallikrein process and easy enzyme inactivation in the existing technology, the purpose of this invention is to provide a pancreatic kallikrein three-layer osmotic pump controlled-release tablet and its preparation method. The pancreatic kallikrein three-layer osmotic pump controlled-release tablet has stable drug release, simple preparation process, and can effectively maintain pancreatic kallikrein activity, which has important clinical value and industrial significance.

[0012] This invention is achieved through the following technical solution:

[0013] In a first aspect, this application provides a pancreatic kallikrein three-layer osmotic pump controlled-release tablet, which, from the inside out, comprises a three-layer tablet core, an isolation coating layer, a semi-permeable membrane coating layer, and a light-shielding coating layer; the three-layer tablet core includes a drug layer, a booster layer, and an auxiliary osmotic layer; the drug layer is stabilized using trehalose and mannitol.

[0014] Secondly, this application provides a method for preparing a pancreatic kallikrein triple-layer osmotic pump controlled-release tablet, comprising the following preparation steps:

[0015] (1) Preparation of drug-containing particles

[0016] ① Drug stabilization treatment: The kallikrein raw material and trehalose-mannitol cocrystal (trehalose:mannitol=1:1) were mixed at a ratio of 1:0.8 and ground in a ball mill at low temperature (4℃) for 30 minutes to obtain drug-cocrystal embedding material.

[0017] ② Micronization: The embedded material is placed in an air jet mill with a feed pressure of 0.6–0.8 MPa and a grinding pressure of 0.8–1.0 MPa to obtain micronized powder with a D90 of 5–15 μm.

[0018] ③ Premixing: Take the micronized mixture and an equal amount of low-substituted hydroxypropyl cellulose (L-HPC, degree of substitution ≤7%), and mix them evenly by adding them in equal amounts (3 to 5 times).

[0019] ④ Main mixing: Place the premix with the remaining L-HPC, hydroxypropyl cellulose (HPC SSL), potassium chloride, citric acid, and poloxamer 188 in a three-dimensional motion mixer and mix at 15-25 rpm for 20-30 minutes.

[0020] ⑤ Dry granulation: Roller pressure 50–100 kg / cm², rotation speed 5–15 rpm, screen aperture 0.8–1.5 mm, granulation. Bulk density 0.4–0.6 g / mL, angle of repose ≤35°.

[0021] ⑥ Granulation: Pass through a 20-mesh sieve

[0022] (2) Preparation of booster layer particles

[0023] ① Mixing: Mix hydroxypropyl cellulose (HPC H), potassium chloride, L-HPC, and crosslinked polyvinylpyrrolidone (PVPP) in a mass ratio of 4:2:2:1 until homogeneous, and place in a three-dimensional motion mixer and mix at 15-25 rpm for 20-30 minutes.

[0024] ② Lubrication: Add sodium stearate fumarate (0.5% to 1% of the total weight of the booster layer) and mix for 3 to 5 minutes.

[0025] ③ Dry granulation: Same as step (1)⑤, to obtain the booster layer particles.

[0026] (3) Preparation of auxiliary permeable layer particles

[0027] ① Mixing: Mix potassium chloride, lactose, and L-HPC in a mass ratio of 3:2:1, and add polyethylene glycol 4000 as a porogen, accounting for 0.5% of the total weight of the layer.

[0028] ② Dry granulation: Same as step (1)⑤, to obtain auxiliary permeation layer particles (without any drug).

[0029] (4) Pressing of three-layer core

[0030] A rotary tablet press with three-stage filling capability is used, with a relative humidity ≤25%.

[0031] ① First feeding station: Fill with drug-containing particles, the filling amount accounts for 20% to 35% of the total weight of the tablet core, and pre-compress 0.5 to 1.5 kN.

[0032] ② Second feeding station: Fill with booster layer particles, the filling amount accounts for 30% to 50% of the total weight of the core, and the pre-compression is 0.5 to 1.5 kN.

[0033] ③ Third feeding station: Filling auxiliary permeation layer particles, the filling amount accounts for 20% to 35% of the total weight of the core, and pre-compression is 0.5 to 1.5 kN.

[0034] ④ Final pressure: main pressure 3~6 kN, core hardness 80~150 N, brittleness ≤0.8%, total core weight 450~550mg.

[0035] (5) Isolation gown covering

[0036] The isolation layer is sprayed with HPMC E5 (2%–3% of the tablet core weight) and talc (1:1), 10% ethanol aqueous solution (ethanol:water = 70:30), resulting in a weight gain of 1%–2%.

[0037] (6) Semi-permeable membrane coating

[0038] ① Preparation of coating solution: Mix cellulose acetate (CA-398-10) and chitosan (degree of deacetylation 85%, molecular weight 50kDa) at a mass ratio of 4:1, add PEG 3350 accounting for 25% of the total polymer weight and triethyl citrate accounting for 5%, dissolve in 2% acetic acid aqueous solution: acetone (20:80, mass ratio) to prepare a coating solution with a solid concentration of 6% to 8%.

[0039] ② Crosslinking treatment: Add glutaraldehyde (50% aqueous solution) at 1% of the chitosan mass to the coating solution and stir for 30 minutes.

[0040] ③ Coating: Place the tablet core obtained in step (5) in a high-efficiency coating pan. The inlet air temperature is 40-55℃, the outlet air temperature is 30-40℃, the material temperature is 35-45℃, the atomization pressure is 0.1-0.2 MPa, the spraying speed is 5-15 mL / min, the pan rotation speed is 8-15 rpm, and the coating weight gain is 4%-12% of the tablet core weight.

[0041] ④ Post-treatment: After coating, dry at 40-50℃ for 15-30 minutes.

[0042] (7) Curing and vacuum drying

[0043] The coated sheets were placed in an oven and heated to maturity at 50±2℃ and relative humidity below 20% for 12 hours. Then they were transferred to a vacuum drying oven (vacuum degree ≤0.08 MPa, 40℃) and dried for 6 hours. Finally, they were equilibrated at 25℃ and relative humidity for 24 hours.

[0044] (8) Laser drilling

[0045] A CO2 laser drilling machine is used to drill a small release hole on the circumferential surface of the tablet core, corresponding to the center position of the drug-containing layer. Drilling parameters: laser aperture 0.2–0.3 mm, hole depth penetrating the semi-permeable membrane and the isolation layer (0.3–0.5 mm), positioning accuracy ±0.2 mm. No hole is drilled at the corresponding position of the auxiliary permeation layer.

[0046] (9) Cover with blackout clothing

[0047] A stomach-soluble film coating premix (containing titanium dioxide) was used to prepare a 15%–20% coating solution with purified water. The coating was carried out in a coating pan, and the coating weight gain was controlled to be 2%–4% of the tablet core weight.

[0048] (10) Packaging

[0049] The aluminum-plastic blister pack contains 1-2 packets of silica gel desiccant per blister pack, and is sealed with nitrogen.

[0050] This invention employs a stabilization pretreatment of kallikrein using trehalose-mannitol eutectic low-temperature ball milling and embedding, enabling it to withstand the stresses of subsequent dry granulation, coating, and curing processes. This is a prerequisite for the effectiveness of all subsequent steps. A three-layer functional zone design is adopted: the drug-containing layer utilizes L-HPC, potassium chloride, citric acid, and poloxamer 188 to ensure the formation of a stable, extrudable solution; the propulsion layer provides continuous driving force through the strong expansibility of high-viscosity HPC H and PVPP; and the auxiliary permeation layer increases the total osmotic pressure gradient and improves compressibility through potassium chloride, lactose, and PEG4000 porogen. These three layers achieve a sequential synergistic effect of "water absorption—swelling—propulsion—release." A composite semipermeable membrane of cellulose acetate and cross-linked chitosan was constructed, in which chitosan was cross-linked with glutaraldehyde to form an interpenetrating polymer network. This not only endowed the membrane with excellent mechanical toughness (friability ≤0.8%), but also precisely controlled the water permeation rate through cationic properties and the pore-forming agent PEG3350. Combined with the plasticizer triethyl citrate, the problem of high brittleness and easy formation of microcracks leading to burst release in pure cellulose acetate membranes was completely solved. A complete low-temperature and low-humidity control process, from ball milling at 4℃ and humidity-controlled tableting (RH≤25%) to vacuum drying and nitrogen-filled sealing, formed a complete moisture-proof, heat-proof, and oxygen-proof protection chain. All technical features in the overall technical solution of this invention are interdependent and indispensable: without drug stabilization, all subsequent processes cannot preserve enzyme activity; without the synergistic function of the three layers, the osmotic pump cannot achieve zero-order release; without the cross-linked composite semipermeable membrane, mechanical integrity and release uniformity cannot be guaranteed; and without full-process environmental control, long-term stability is difficult to achieve.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] (1) The present invention adopts “drug-containing layer + booster layer + auxiliary permeation layer (no drug)”. The auxiliary permeation layer can independently adjust the water entry rate to achieve more precise zero-order release and avoid mutual interference between the drugs on both sides. The pancreatic kallikrein three-layer osmotic pump controlled-release tablet has stable drug release, simple preparation process and can effectively maintain pancreatic kallikrein activity, which has important clinical value and industrial significance.

[0053] (2) In this invention, only one small hole (0.2~0.3 mm) is drilled on the circumferential surface corresponding to the drug-containing layer, and the auxiliary permeation layer is poreless. Compared with the dual-hole design, the uncertainty of the drug release path is reduced, the release deviation caused by the blockage or asymmetry of the dual holes is reduced, and the process is more controllable.

[0054] (3) The present invention uses a cellulose acetate-chitosan blend membrane, which is cross-linked with glutaraldehyde. Chitosan has pH responsiveness and bioadhesion, and cross-linking can regulate membrane permeability and improve mechanical strength.

[0055] (4) In this invention, kallikrein is encapsulated in a trehalose-mannitol co-crystal, followed by low-temperature ball milling and micronization. Trehalose protects enzyme activity, while mannitol acts as a pore-forming agent to aid release. The co-crystal form is more stable than simple physical mixing.

[0056] (5) The expansion agent of the present invention is selected from hydroxypropyl cellulose (HPC) and cross-linked polyvinylpyrrolidone (PVPP), and does not contain hydroxypropyl methylcellulose (HPMC) and polyoxyethylene (PEO), thus avoiding the risk of "hypomeltation" and having better compatibility with kallikrein.

[0057] (6) The present invention adds a vacuum drying step after high-temperature aging, which effectively removes the residual acetic acid and acetone in the semipermeable membrane, prevents the long-term effect of the solvent on enzyme activity, and stabilizes the membrane pore size distribution.

[0058] (7) The preparation method of this invention features a complete low-temperature and low-humidity protection chain throughout the entire process, ensuring long-term stability of enzyme activity. From ball milling at 4℃ and tableting at RH≤25%, to vacuum drying and nitrogen-filled packaging, this invention establishes a complete "heat-proof, moisture-proof, and oxygen-proof" process chain. Accelerated stability tests show that the potency of the product of this invention decreases by only 2.1%~3.2% after 6 months.

[0059] (8) This invention addresses the stability problem of pancreatic kallikrein (a biological macromolecule) through a series of measures such as micronization, co-crystallization, and low-temperature operation, thereby expanding the application of osmotic pump technology in protein drugs.

[0060] (9) The single-hole circumferential surface drilling positioning accuracy requirement of the present invention is relatively low (±0.2 mm), and avoids the complicated process of aligning two drug layers with two holes, making it suitable for industrial production.

[0061] (10) The present invention can independently adjust the water permeation rate and drug release rate by using the composition (potassium chloride + lactose ratio) and thickness of the auxiliary permeation layer and the micropore design of the single pore diameter (0.2~0.3 mm).

[0062] (11) All steps of this invention utilize mature pharmaceutical industry equipment (three-dimensional mixer, dry granulator, rotary tablet press, high-efficiency coating pan, laser punching machine, aluminum-plastic blister packaging machine), without the need for special or non-standard equipment. Furthermore, dry granulation avoids the damage to enzymes caused by moisture in wet granulation and eliminates the need for organic solvent granulation, making it environmentally friendly. This technology platform can be extended to the development of oral osmotic pump formulations of other easily inactivated protein peptides (such as insulin, interferon, growth hormone, etc.). Attached Figure Description

[0063] To more clearly illustrate the technical solutions in this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 The graph shows the detection results of the in vitro release rate test of the controlled-release tablets prepared in Example 1 of the present invention;

[0065] Figure 2 The graph shows the detection results of the in vitro release rate test of the controlled-release tablets prepared in Example 2 of the present invention;

[0066] Figure 3 The graph shows the detection results of the in vitro release rate test of the controlled-release tablets prepared in Example 3 of the present invention;

[0067] Figure 4 The graph shows the results of the in vitro release test of the controlled-release tablets prepared in Comparative Example 1.

[0068] Figure 5 The graph shows the results of the in vitro release test of the controlled-release tablets prepared in Comparative Example 2.

[0069] Figure 6 The graph shows the results of the in vitro release test of the controlled-release tablets prepared in Comparative Example 3. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0071] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0072] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described herein, as well as the features of those different embodiments or examples.

[0073] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0074] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0075] Example 1

[0076] This embodiment provides a method for preparing a three-layer osmotic pump controlled-release tablet of kallikrein. The formulation of the three-layer tablet core is shown in Table 1. The tablet core weighs 500 mg per 1000 tablets.

[0077] Table 1

[0078] Kallikrein raw material (potency: 10U / mg) 35.0 - - Trehalose-mannitol eutectic (1:1) 28.0 - - Low-substituted hydroxypropyl cellulose (L-HPC, degree of substitution 6%) 35.0 40.0 20.0 Hydroxypropyl cellulose (HPC SSL) 14.0 - - Hydroxypropyl cellulose (HPC H) - 80.0 - Potassium chloride 14.0 40.0 60.0 Citric acid 7.0 - - Polosham 188 7.0 - - Cross-linked polyvinylpyrrolidone (PVPP) - 20.0 - Sodium stearate - 1.8 - lactose - - 40.0 Polyethylene glycol 4000 - - 0.6 total 140.0 181.8 120.6

[0079] The outer layer of the isolation gown consists of 10.0g of hydroxypropyl methylcellulose (HPMC E5), 10.0g of talc, and an appropriate amount of 10% ethanol aqueous solution.

[0080] Semi-permeable membrane coating layer: cellulose acetate (CA-398-10) 16.0g, chitosan (85% deacetylation, 50kDa) 4.0g, PEG 3350 5.0g, triethyl citrate 1.0g, glutaraldehyde (50% aqueous solution) 0.04g, 2% acetic acid aqueous solution: acetone (20:80) appropriate amount.

[0081] Light-shielding coating layer: 12.0g of gastric-soluble film coating premix (containing titanium dioxide) and appropriate amount of purified water.

[0082] The specific preparation method is as follows:

[0083] S1. Preparation of drug-containing granules: ① Mix kallikrein and trehalose-mannitol cocrystals in a certain proportion and ball mill at 4℃ for 30 minutes; ② Air-jet mill to D90=8μm; ③ Mix with equal amounts of L-HPC in an incremental manner 4 times; ④ Add the remaining L-HPC, HPC SSL, potassium chloride, citric acid, and poloxamer 188, and mix in a three-dimensional mixer at 20rpm for 25 minutes; ⑤ Dry granulation: granulate with a roller pressure of 75kg / cm², a rotation speed of 10rpm, and a 1.0mm sieve to obtain a bulk density of 0.5g / mL and an angle of repose of 32°; ⑥ Granulate through a 20-mesh sieve.

[0084] S2. Preparation of booster layer particles: ① Mix HPC H, potassium chloride, L-HPC, and PVPP at 20 rpm for 25 minutes; ② Add sodium stearate and mix for 4 minutes; ③ Perform dry granulation using the same method.

[0085] S3. Preparation of auxiliary permeation layer particles: ① Mix potassium chloride, lactose, L-HPC, and PEG4000; ② Dry granulation using the same method.

[0086] S4. Compression of the three-layer tablet core: A three-stage rotary tablet press (RH≤25%) is used, with the drug-containing layer filling 35%, the propulsion layer filling 40%, and the auxiliary penetration layer filling 25%. The pre-compression is 1.0kN, the main compression is 4.5kN, resulting in a tablet core hardness of 120N, a friability of 0.5%, and a tablet weight of 500mg.

[0087] S5. Isolation gown covering: Spray with a 70% ethanol solution of HPMC E5 / talc (1:1), resulting in a weight gain of 1.5%.

[0088] S6, Semi-permeable membrane coating: ① Prepare coating solution (7% solid content); ② Add glutaraldehyde for cross-linking for 30 minutes; ③ Coating: air inlet 50℃, material 40℃, atomization pressure 0.15MPa, spraying speed 10mL / min, weight gain 8%; ④ Dry at 50℃ for 20 minutes.

[0089] S7. Curing and vacuum drying: Curing at 50℃ and RH<20% for 12 hours, followed by vacuum drying at 40℃ and ≤0.08MPa for 6 hours, and finally equilibration at 25℃ / RH50% for 24 hours.

[0090] S8. Laser drilling: CO2 laser drilling, drilling one hole in the center of the drug-containing layer of the tablet core, with a diameter of 0.25mm and a depth of 0.4mm.

[0091] S9. Light-blocking coating: Coated with a 15% solids content gastric-soluble coating solution, resulting in a 3% weight gain.

[0092] S10, Packaging: Aluminum-plastic blister pack, with one pack of silica gel desiccant inside, nitrogen-filled and sealed.

[0093] The three-layer osmotic pump controlled-release tablets prepared in this embodiment have a regular appearance and moderate hardness.

[0094] An in vitro release test (pH 6.8 phosphate buffer, paddle method, 50 rpm) was conducted on the three-layer osmotic pump controlled-release tablets prepared using the method of this embodiment. Figure 1 As shown, the test results indicate that:

[0095] The drug releases approximately 19% at 2 hours, 45% at 6 hours, 75% at 12 hours, and over 90% at 24 hours, exhibiting typical zero-order release characteristics (R). 2 =0.9951). Trehalose-mannitol cocrystal effectively protects the activity of kallikrein during granulation and coating processes. The potency of the finished product was determined to be 96.8% of the labeled amount, and the content decreased by only 3.2% after accelerated testing (6 months of storage at 40℃ / RH75%). The cross-linked chitosan semi-permeable membrane ensures stable osmotic pressure drive and mechanical strength, with precise laser perforation location and good release uniformity (RSD<5%).

[0096] Example 2

[0097] This embodiment provides a method for preparing a three-layer osmotic pump controlled-release tablet of kallikrein. The formulation of the three-layer tablet core is shown in Table 2. The tablet core weighs 480 mg per 1000 tablets.

[0098] Table 2

[0099] Kallikrein raw material (potency: 12U / mg) 30.0 - - Trehalose-mannitol eutectic (1:1) 24.0 - - Low-substituted hydroxypropyl cellulose (L-HPC, degree of substitution 5%) 28.0 35.0 18.0 Hydroxypropyl cellulose (HPC SSL) 12.0 - - Hydroxypropyl cellulose (HPC H) - 70.0 - Potassium chloride 12.0 3.0 54.0 Citric acid 6.0 - - Polosham 188 6.0 - - Cross-linked polyvinylpyrrolidone (PVPP) - 17.5 - Sodium stearate - 1.6 - lactose - - 36.0 Polyethylene glycol 4000 - - 0.5 total 118.0 159.1 108.5

[0100] Coatings and accessories (weight gain ratios are the same as in Example 1, with slight adjustments to the specific quantities): isolation gown weight gain 1.8%, semi-permeable membrane weight gain 6%, and light-blocking gown weight gain 2.5%.

[0101] The specific preparation method is basically the same as in Example 1, except that: the filling ratio of the drug-containing layer is adjusted to 30%, the booster layer to 45%, and the auxiliary permeation layer to 25%; the weight gain of the semi-permeable membrane coating is controlled at 6%; and the laser perforation pore size is 0.20 mm.

[0102] This embodiment reduces the thickness of the semipermeable membrane (6% increase in weight) and shrinks the drug release pore size (0.20 mm), while adjusting the three-layer ratio.

[0103] An in vitro release test (pH 6.8 phosphate buffer, paddle method, 50 rpm) was conducted on the kallikrein triple-layer osmotic pump controlled-release tablets prepared using the method of this embodiment. Figure 2 As shown, the test results indicate that:

[0104] Approximately 20% was released after 2 hours, approximately 50% after 6 hours, approximately 82% after 12 hours, and over 95% after 20 hours. The release rate was slightly faster than in Example 1, but the zero-order release characteristic was still evident (R). 2 =0.9895). A thinner semi-permeable membrane increases the water molecule permeation rate, but the release rate is effectively controlled by reducing the pore size. Accelerated stability studies showed that, due to the slightly higher weight gain of the isolation gown (1.8%), and better barrier properties against water and oxygen, the drug potency retention rate was slightly higher than in Example 1 (a 2.8% decrease over 6 months). This example demonstrates that by adjusting key parameters within the scope of the invention, the release curve can be fine-tuned to meet different clinical needs while maintaining excellent stability.

[0105] Example 3

[0106] This embodiment provides a method for preparing a three-layer osmotic pump controlled-release tablet of kallikrein. The formulation of the three-layer tablet core is shown in Table 3. The tablet core weighs 550 mg per 1000 tablets.

[0107] Table 3

[0108] Kallikrein raw material (potency: 8U / mg) 45.0 - - Trehalose-mannitol eutectic (1:1) 36.0 - - Low-substituted hydroxypropyl cellulose (L-HPC, degree of substitution 7%) 45.0 50.0 25.0 Hydroxypropyl cellulose (HPC SSL) 18.0 - - Hydroxypropyl cellulose (HPC H) - 100.0 - Potassium chloride 18.0 50.0 75.0 Citric acid 9.0 - - Polosham 188 9.0 - - Cross-linked polyvinylpyrrolidone (PVPP) - 25.0 - Sodium stearate - 2.3 - lactose - - 50.0 Polyethylene glycol 4000 - - 0.8 total 180.0 227.3 150.8

[0109] Coatings and accessories: Isolation gowns increased in weight by 2.0%, semi-permeable membranes by 12%, and light-blocking gowns by 4%.

[0110] The specific preparation method is basically the same as in Example 1, except that: the filling ratio of the drug-containing layer is adjusted to 33%, the booster layer to 41%, and the auxiliary permeation layer to 26%; the weight gain of the semi-permeable membrane coating is increased to 12%; and the vacuum drying time after curing is extended to 8 hours.

[0111] This embodiment uses the thickest semi-permeable membrane (12% weight gain) and the thickest light-blocking garment (4% weight gain), and extends the vacuum drying time.

[0112] An in vitro release test (pH 6.8 phosphate buffer, paddle method, 50 rpm) was conducted on the kallikrein triple-layer osmotic pump controlled-release tablets prepared using the method of this embodiment. Figure 3 As shown, the test results indicate that:

[0113] Release rates are as follows: only 8% after 2 hours, approximately 30% after 6 hours, approximately 55% after 12 hours, approximately 85% after 24 hours, and over 90% after 36 hours. The release rate is significantly slower than in Examples 1 and 2. The thick film enhances the mechanical strength of the controlled-release tablet (hardness up to 150N), but leads to a prolonged time lag. The light-blocking coating synergistically reduces the risk of photolysis and hygroscopicity with the lactose in the auxiliary permeation layer. This formulation is suitable for scenarios requiring ultra-long-lasting release (e.g., once daily, effectively covering more than 24 hours). Stability testing shows that, after enhanced vacuum drying and a thicker coating, the product's potency decreased by only 2.1% under high temperature and humidity conditions (40°C / 75% RH for 6 months), demonstrating its excellent environmental tolerance.

[0114] Comparative Example 1

[0115] The comparative example is basically the same as Example 1, except that: when the drug-containing particles were prepared, "drug stabilization treatment" was not performed, that is, the kallikrein raw material was directly mixed with excipients such as L-HPC, and it was not pre-encapsulated by low-temperature ball milling with trehalose-mannitol cocrystallization.

[0116] Results Analysis: This comparative study aims to verify the key role of the co-crystal embedding technology. Unstabilized kallikrein was significantly deactivated during dry granulation (involving mechanical stress and localized heat generation), semi-permeable membrane coating (involving thermal stress and organic solvents), and subsequent curing. The final product potency was only 65.3% of the labeled amount. After accelerated testing (3 months at 40℃ / 75% RH), the potency further decreased to 42.8% of the labeled amount, with a significant increase in degradation products. Figure 4 As shown, in vitro release results indicate that although the release curve morphology is similar to that of Example 1, the actual total amount of active substance released is far lower than expected due to severe loss of drug activity, resulting in unreliable efficacy. This comparative example demonstrates that low-temperature encapsulation of trehalose-mannitol cocrystallization is a necessary step in protecting the activity of the kallikrein osmotic pump during preparation; omitting this step will lead to substantial product failure.

[0117] Comparative Example 2

[0118] The comparative example is basically the same as Example 1, except that chitosan and its cross-linking agent glutaraldehyde were not added to the semipermeable membrane coating solution. Only cellulose acetate (CA-398-10), PEG3350, and triethyl citrate were used. Other components and processes were the same.

[0119] Results Analysis: This comparative study investigated the role of cross-linked chitosan in semipermeable membranes. Pure cellulose acetate semipermeable membranes were relatively brittle; during coating and laser perforation processes, a small number of microcracks appeared at the edges of the wafer cores (approximately 5% of the wafers). Figure 5 As shown, in vitro release experiments revealed that these microcracks caused premature drug leakage from non-release pore areas, resulting in a burst release in the initial stage of the release curve (35% release in 2 hours), followed by unstable release rates and large batch-to-batch variability (RSD > 15%). In Example 1, the introduction of chitosan and cross-linking with glutaraldehyde significantly enhanced the toughness and density of the semipermeable membrane, preventing the formation of microcracks. Furthermore, the membrane without cross-linked chitosan exhibited high water permeability and lacked cationic properties, affecting the stable maintenance of osmotic pressure. The results indicate that the cross-linked network structure of chitosan is crucial for obtaining a semipermeable membrane with high mechanical strength and controllable permeability.

[0120] Comparative Example 3

[0121] The comparative example is basically the same as Example 1, except that the laser drilling position is changed to drilling on the circumferential surface corresponding to the booster layer.

[0122] Results Analysis: This comparative study verifies the specificity requirements of the drug release pore location. The drug release principle of osmotic pump controlled-release tablets is as follows: water enters the tablet core through a semi-permeable membrane, the drug-containing layer forms a saturated drug solution, and the propulsion layer absorbs water and expands, generating thrust to push the drug solution out of the release pores. Drilling pores in the propulsion layer causes the expanded propulsion layer material (such as HPC H and PVPP) to be directly squeezed out of the pores, blocking or partially blocking the channels, while the drug in the drug-containing layer cannot obtain an effective discharge path. Figure 6 As shown, in vitro release results indicate that release is extremely slow (<10%) in the first 8 hours, followed by uncontrolled release (over 60% release within 8-12 hours) due to the potential for excessive expansion of the booster layer to rupture the membrane. This tablet completely loses its order-zero controlled-release characteristics. This comparative example demonstrates that the release orifice must be precisely located in the center of the drug-containing layer to ensure that the drug is preferentially expelled from this orifice, thereby achieving the intended controlled-release behavior.

[0123] The detection results of Examples 1-3 and Comparative Examples 1-3 are shown in Table 4.

[0124] Table 4

[0125] Drug-containing layer stabilization treatment (Eutectic embedding) have have none have have Semipermeable membrane composition CA+ crosslinked chitosan CA+ crosslinked chitosan CA+ crosslinked chitosan CA+ crosslinked chitosan Pure CA CA+ crosslinked chitosan Drug release port location drug-containing layer center drug-containing layer center drug-containing layer center drug-containing layer center drug-containing layer center Booster layer Initial potency (labeled amount %) 96.8% 97.2% 95.9% 65.3% 95.5% 96.1% Accelerating 6-month valence retention 93.6% 94.4% 93.8% 42.8% 90.2% 91.5% <![CDATA[Zero-order fitting of release curve R 2 > 0.9951 0.9895 0.9815 0.9821 0.8827 0.6979 Cumulative release over 2 hours (%) 19% 20% 8% 13% 35% 5% 24-hour cumulative release (%) 92% 96% 85% 89% 88% 78% Inter-batch release RSD (%, n=3) 4.2% 4.8% 3.5% 11.3% 16.7% 23.5% Core brittleness (%) 0.5% 0.6% 0.4% 0.7% 1.2% 0.5% Semipermeable membrane microcrack incidence none none none none Approximately 5% none

[0126] The above test results show that:

[0127] Example 1 (Typical Scheme): Achieving optimal balance across various indicators: drug stabilization treatment resulted in a potency retention rate approaching 97%; an 8% increase in the weight of the semi-permeable membrane, combined with a pore size of 0.25 mm, yielded an ideal zero-order release (R... 2 =0.9951); the process parameters are moderate, and the batch-to-batch variation is controlled within 5%. This scheme is suitable for industrial production and can serve as a basic technology platform.

[0128] Example 2 (Fast Release Variant): A "membrane + micropore" compensation mechanism was achieved by reducing the weight gain of the semipermeable membrane (6%) and decreasing the pore size (0.20 mm). The release rate was slightly faster than in Example 1 (96% at 24 h), but the zero-order characteristic was slightly reduced (R0). 2 =0.9895). The reason is that although a thinner membrane increases water permeation flux, the reduction in pore size does not completely linearly compensate for the release rate, resulting in a slightly faster release curve in the early and middle stages. This approach is suitable for scenarios requiring faster onset of action or complete release within 24 hours.

[0129] Example 3 (Ultra-long-acting variant): Utilizing the thickest semi-permeable membrane (12%) and the thickest light-shielding coating (4%), along with extended vacuum drying time, this variant exhibits the flattest release curve (only 85% released at 24 hours, exceeding 95% at 36 hours), but with a prolonged time lag (only 8% released at 2 hours). This formulation is suitable for situations requiring strictly stable blood drug concentrations, or where patient compliance demands "once a day" and a slightly slower initial onset of action is acceptable. Furthermore, the enhanced drying and coating optimizes its environmental tolerance, making it suitable for sale in tropical or high-humidity regions.

[0130] Comparative Example 1 (without drug stabilization treatment): This comparative example reveals the greatest risk to protein and peptide drugs in the preparation of osmotic pumps: inactivation due to the combined effects of mechanical and thermal stress. Roller pressure during dry granulation (50~100 kg / cm²) 2 Local shear forces, along with the hot air (material temperature 35~45℃) and organic solvent (acetone) environment during semi-permeable membrane coating, can disrupt the tertiary structure of kallikrein. The role of trehalose-mannitol co-crystal is not only physical isolation, but more importantly, the formation of a "drug-co-crystal embedding" through low-temperature ball milling. The glass transition properties of trehalose and the hydrogen bond network of mannitol together act as a "molecular cage" for protection. Omitting this step results in an initial potency loss of up to 34.7%, and the degradation rate increases exponentially under accelerated conditions (a further decrease of 22.5% after 3 months). This comparative example demonstrates that all subsequent process optimizations cannot compensate for the lack of initial stabilization treatment, making this step irreplaceable.

[0131] Comparative Example 2 (Chitosan-free cross-linked semi-permeable membrane): Pure cellulose acetate semi-permeable membranes are rigid glassy after drying and are highly brittle. During the coating pan tumbling, laser drilling, and subsequent curing processes, thermal stress and mechanical impacts easily generate microcracks. In this comparative example, approximately 5% of the tablet cores showed microcracks invisible to the naked eye but sufficient to cause drug leakage (confirmed by scanning electron microscopy). This directly led to two consequences: ① Burst release—moisture prematurely enters the cracked area, causing local dissolution of the drug-containing layer and preferential release from the cracks, reaching 35% release in 2 hours (normally 10-18%); ② Large batch-to-batch variability—the random distribution of cracks resulted in highly inconsistent release behavior among each batch of tablet cores (RSD reaching 16.7%). The introduction of cross-linked chitosan (forming a Schiff base cross-linked network through glutaraldehyde) significantly improved the membrane's flexibility and tensile strength. Simultaneously, the cationic properties of chitosan and the hydrophobic framework of cellulose acetate formed an interpenetrating polymer network (IPN), making the membrane more dense. This comparative example demonstrates that the composite semipermeable membrane is not only an osmotic pressure barrier but also the structural framework of the controlled-release tablet, and its mechanical integrity is a prerequisite for zero-order release.

[0132] Comparative Example 3 (Drug release orifice located in the booster layer): This comparative example fundamentally disrupts the working logic of the osmotic pump. The normal working process of the three-layer osmotic pump is: water enters → drug-containing layer forms solution → booster layer expands → drug-containing layer solution is pushed out from the drug release orifice above the drug-containing layer. When the drug release orifice is placed at the corresponding position in the booster layer, the expanded booster layer material (HPC H and PVPP are both highly expandable polymers) will preferentially be squeezed out from the orifice, forming an "embolism" that blocks the channel. At the same time, the drug solution in the drug-containing layer lacks a directional discharge channel, resulting in an abnormally high internal pressure in the tablet core. In vitro release showed: the release was extremely slow (<10%) in the first 8 hours, indicating that the channel was blocked and the drug-containing layer failed to discharge effectively; from 8 to 12 hours, the internal pressure accumulated to exceed the tensile strength of the semipermeable membrane, causing the membrane to rupture at the weak point, resulting in a "burst release" (the release rate jumped from 10% to 65% in 8 to 12 hours). This "slow at the beginning and burst at the end" release mode completely loses the zero-order controlled release advantage of the osmotic pump, and the in vitro-in vivo correlation is extremely poor. This comparative example demonstrates that the precise positioning of the drug release orifice (including the center of the drug layer) is the foundation of osmotic pump technology, and any positional deviation will lead to mechanistic failure.

[0133] Through the above examples, the core of this invention lies in the deep synergy of four key technical features: First, the stabilization pretreatment of kallikrein through low-temperature ball milling and embedding of trehalose-mannitol co-crystallization ensures that it can withstand all process stresses such as subsequent dry granulation, coating, and curing, which is a prerequisite for the effectiveness of all subsequent steps; Second, a three-layer functional partition design is adopted—the drug-containing layer utilizes L-HPC, potassium chloride, citric acid, and poloxamer 188 to ensure that the drug forms a stable solution that can be extruded, and the propulsion layer relies on high-viscosity HPC. The strong expansibility of H and PVPP provides a continuous driving force. The auxiliary permeation layer increases the total osmotic pressure gradient and improves compressibility through potassium chloride, lactose, and PEG4000 pore-forming agent. The three achieve a sequential synergy of "water absorption-swelling-driving-release". Third, a composite semipermeable membrane of cellulose acetate and cross-linked chitosan is constructed. Chitosan is cross-linked with glutaraldehyde to form an interpenetrating polymer network, which not only gives the membrane excellent mechanical toughness (friability ≤0.8%), but also precisely controls the water permeation rate through cationic properties and pore-forming agent PEG3350. Combined with plasticizer triethyl citrate, the problem of high brittleness and easy microcracks leading to sudden release of pure cellulose acetate membrane is completely solved. Finally, the whole process of low temperature and low humidity control from 4℃ ball milling, humidity-controlled tableting (RH≤25%) to vacuum drying and nitrogen-filled sealing forms a complete moisture-proof, heat-proof, and oxygen-proof protection chain. These four characteristics are interdependent and indispensable: without drug stabilization, all subsequent processes cannot preserve enzyme activity; without the synergistic function of the three layers, the osmotic pump cannot achieve zero-order release; without the cross-linked composite semipermeable membrane, mechanical integrity and release uniformity cannot be guaranteed; and without full-process environmental control, long-term stability is difficult to achieve.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A pancreatic kallikrein triple-layer osmotic pump controlled-release tablet, characterized in that, From the inside out, it includes a three-layer tablet core, an isolation coat layer, a semi-permeable membrane coating layer, and a light-shielding coating layer; the three-layer tablet core includes a drug layer, a booster layer, and an auxiliary penetration layer; the drug layer is stabilized using trehalose and mannitol.

2. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 1, characterized in that, The method for preparing the drug layer includes the following steps: Drug stabilization treatment: The kallikrein raw material was mixed with trehalose-mannitol cocrystal and ground at low temperature to obtain drug-cocrystal embedding material; Micronization: The drug-eutectic embedding material is pulverized to obtain micronized powder; Premix: The micro powder and an equal amount of low-substituted hydroxypropyl cellulose are mixed evenly using an equal-volume incremental addition method to obtain a premix; Main mixture: The premix is ​​mixed with low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, potassium chloride, citric acid, and poloxamer to obtain a mixture; Dry granulation: The mixture is granulated and sized to obtain drug-containing granules.

3. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 1, characterized in that, The method for preparing the booster layer includes the following steps: Mixing: Hydroxypropyl cellulose, potassium chloride, low-substituted hydroxypropyl cellulose, and crospovidone are mixed to obtain a mixture; Lubrication: Add sodium stearate fumarate to the mixture, mix evenly, and then perform dry granulation to obtain the booster layer particles.

4. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 3, characterized in that, The amount of sodium stearate added accounts for 0.5% to 1% of the total weight of the booster layer.

5. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 1, characterized in that, The auxiliary permeation layer is prepared by mixing potassium chloride, lactose, and low-substituted hydroxypropyl cellulose evenly, adding polyethylene glycol pore-forming agent, and then performing dry granulation to obtain auxiliary permeation layer particles.

6. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 1, characterized in that, The preparation method of the three-layer core is as follows: first, fill with drug-containing layer particles and pre-press; then fill with booster layer particles and pre-press; then fill with auxiliary permeation layer particles and pre-press; finally, perform final pressing.

7. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 6, characterized in that, The amount of the drug-containing layer particles accounts for 20% to 35% of the total weight of the tablet core; the amount of the propulsion layer particles accounts for 30% to 50% of the total weight of the tablet core; and the amount of the auxiliary penetration layer particles accounts for 20% to 35% of the total weight of the tablet core.

8. The pancreatic kallikrein triple-layer osmotic pump controlled-release tablet according to claim 6, characterized in that, The hardness of the three-layer core is 80N~150N, and the brittleness is ≤0.8%.

9. A method for preparing the kallikrein triple-layer osmotic pump controlled-release tablet according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) The three-layer core is coated with an isolation coat: hydroxypropyl methylcellulose E5 and talc and ethanol aqueous solution are sprayed to coat the isolation coat. (2) Coating with a semi-permeable membrane: Coating the core containing the isolation coat layer in step (1) with a semi-permeable membrane coating solution; (3) Curing and drying the coated core from step (2); (4) Make a small hole for drug release on the circumferential surface of the tablet core, corresponding to the center position of the drug-containing layer; (5) A coating solution prepared with a gastric-soluble film coating premix was used to coat a light-shielding coating layer to obtain a three-layer osmotic pump controlled-release tablet of pancreatic kallikrein.

10. The method for preparing a kallikrein triple-layer osmotic pump controlled-release tablet according to claim 9, characterized in that, The drilling parameters for the drug release orifice are: laser aperture of 0.2mm~0.3mm, orifice depth penetrating the semi-permeable membrane and the isolation layer, and no orifice drilled at the corresponding position of the auxiliary permeation layer.

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