In-vitro dissolution curve determination method of suspension for inhalation

Abstract

The invention provides an in-vitro dissolution curve determination method of a suspension for inhalation, and belongs to the field of analysis and detection of inhalation preparations. The in-vitro dissolution curve determination method comprises the following steps: (1) placing sample collection filter paper above a grade S4 of a new generation medicinal impactor sieve plate; (2) atomizing the suspension for inhalation to obtain atomized aerosol, and enabling the atomized aerosol to circulate in the new-generation medicinal impactor along with airflow; (3) taking out the sample collection filter paper with the active substances, fixing the sample collection filter paper in a filter paper bracket, carrying out dissolution curve determination by using a paddle method, and then taking solutions with the same volume as solutions to be detected at different time points; and (4) respectively measuring the content of the active substances in the solution to be measured at each time point and the content of the residual active substances on the filter paper after the sampling is finished, so as to obtain the accumulated dissolution rate at each time point and draw a dissolution curve, and the method can better simulate the dissolution and absorption process of the active substances in the suspension for inhalation in the lung.

Description

Technical Field

[0001] The present application relates to the field of analysis and detection of inhalation preparations, and in particular, to a method for determining an in vitro dissolution curve of an inhalation suspension. Background Art

[0002] Inhalation preparations are widely used in the treatment of lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). They can directly reach the lesion site and take effect quickly, avoiding the first-pass effect of the liver, improving the bioavailability of the drug, and reducing toxic side effects. Therefore, oral inhalation administration is considered to be the most ideal route of administration for the treatment of lung diseases. However, due to the rich capillaries in the lungs, large alveolar surface area, and small amount of lung fluid, it is very challenging to simulate the dissolution behavior of drugs in the lungs through in vitro dissolution tests, especially for inhaled suspensions whose active ingredients are usually poorly soluble drugs.

[0003] At present, there are many reports on the dissolution methods of inhalation preparations, such as the paddle device method, the Franz diffusion cell method, and the Transwell system method, but these reports mainly focus on dry powder inhalers (DPI), and there are still few studies on the dissolution methods of suspensions for inhalation. Among them, in the collection stage of active substances, there are currently reports on the use of an insert collection cup equipped with a new generation pharmaceutical impactor (NGI, Next Generation Impactor) to collect particles of a certain stage, but this method has low sensitivity, poor reproducibility and high cost; there are also methods to collect particles of a certain stage at multiple doses to improve sensitivity, but this method is very time-consuming and not very operable, and these two collection methods only collect particles of a certain stage for dissolution research, which cannot represent the dissolution process of fine particles (<5 μm) deposited in the lungs to play a role in the body. At the same time, there is also a method of using the whole sample (wider particle size range) to directly conduct dissolution research, but this test method still cannot simulate the dissolution behavior of fine particles in the body and has major defects. Therefore, it is more scientific to collect all fine particles by atomization for in vitro dissolution research. Therefore, it is urgent to optimize the screening and collection methods of inhalation suspension particles in order to develop a simpler and more reproducible in vitro dissolution curve determination method. Summary of the invention

[0004] The purpose of the present application is to provide a method for determining the in vitro dissolution curve of a suspension for inhalation, which method has the advantages of being simple, easy to operate and having good reproducibility, and can better simulate the process of dissolution and absorption of active substances in the suspension for inhalation in the lungs.

[0005] The embodiment of the present application is implemented as follows: The present application embodiment provides a method for determining an in vitro dissolution curve of a suspension for inhalation, comprising the following steps: (1) Place a sample collection filter paper above the S4 level of the sieve plate of the new generation pharmaceutical impactor; (2) Nebulize the inhalation suspension to obtain an aerosol, and allow the aerosol to flow in the new generation pharmaceutical impactor with the airflow; (3) Take out the sample collection filter paper with the active substance collected above the S4 level of the sieve plate, fix the sample collection filter paper in the filter paper holder, transfer the filter paper holder to the dissolution cup filled with the dissolution medium and use the paddle method to measure the dissolution curve, and then take the same volume of solution at different time points as the test solution; (4) After sampling, use high performance liquid chromatography to determine the content of the active substance in the test solution at each time point and the content of the residual active substance on the sample collection filter paper, so as to obtain the cumulative dissolution at each time point and draw a dissolution curve.

[0006] In the above technical scheme, a circular filter paper with a diameter of 43 mm is placed above the S4 stage of the sieve plate of the new generation of pharmaceutical impactor to cover the entire S4 stage of the sieve plate. The inhalation suspension is atomized into an aerosol by the atomization device and then circulates in the impactor with the airflow. The particles of the S1~S3 stages are respectively collected in the collection cups under the corresponding stages, and the particles of the S4 stage and subsequent stages are retained on the circular filter paper. Therefore, a large number of representative particles (i.e., fine particles with a particle size of less than 5 μm that can be deposited deep in the lungs to exert their effects) can be collected through only one collection process, thereby effectively reflecting the dissolution behavior of the active substance in the lungs. Compared with the collection method in the prior art of only collecting particles of a certain level (the amount of collected samples is small, resulting in low test sensitivity and poor reproducibility) or repeatedly collecting particles of a certain level for multiple times (although the sensitivity can be improved, the repeated operation has the problems of cumbersome steps, time-consuming, low efficiency and inconvenient for promotion), the in vitro dissolution curve determination based on the collection method provided in this application has the advantages of simplicity and good reproducibility, and can comprehensively reflect the dissolution and absorption process of fine particles in the inhalation suspension in the lungs. It is of great significance in guiding the development of prescription processes, conducting in vitro bioequivalence evaluation, exempting clinical trials and proving the quality consistency of products before and after changes.

[0007] In some optional embodiments, the suspension for inhalation is selected from at least one of fluticasone propionate suspension for inhalation, budesonide suspension for inhalation and beclomethasone propionate suspension for inhalation.

[0008] In the above technical solutions, there are many types of suspensions for inhalation that can be applied, and more feasible implementation plans can be provided, thereby facilitating the promotion and application of the technical solutions provided in the embodiments of the present application; at the same time, these types of suspensions for inhalation are the most representative suspension products for inhalation on the market.

[0009] In some optional embodiments, the sample collection filter paper is made of glass fiber.

[0010] In the above technical solution, the sample collection filter paper is made of glass fiber, which has both low resistance and high capture efficiency. Under the premise of not hindering the flow of air in the impactor, it can effectively intercept a large number of S4~MOC grade particles in the atomized aerosol and will not be damaged due to excessive infiltration of the atomized aerosol, resulting in particle loss. At the same time, it can also make the particles in the atomized aerosol uniformly and stably loaded on the surface of the filter paper, thereby facilitating the subsequent wetting and dissolution of the particles layer by layer inward in the dissolution medium, thereby making the dissolution process of the particles in the dissolution medium have good reproducibility.

[0011] In some optional embodiments, the pore size of the sample collection filter paper is 0.2 μm to 0.5 μm.

[0012] In the above technical solution, limiting the pore size of the sample collection filter paper within a specific range can effectively reduce the probability of fine particles passing through the filter paper, thereby improving the particle retention rate of the filter paper.

[0013] In some alternative embodiments, the dissolution medium is a PBS buffer solution containing Tween.

[0014] In the above technical solution, adding Tween to the PBS buffer solution can appropriately increase the dissolution rate of fluticasone propionate, so as to more efficiently obtain the entire in vitro dissolution curve.

[0015] In some alternative embodiments, the type of Tween is Tween 20.

[0016] In the above technical solution, the use of a specific type of Tween (i.e., Tween 20) can moderately increase the dissolution rate of fluticasone propionate without causing interference in the measurement process, so as to obtain a more accurate dissolution curve.

[0017] In some optional embodiments, the mass concentration of Tween 20 in the dissolution medium is 0.3% to 0.5%.

[0018] In the above technical solution, the mass percentage of Tween 20 in the dissolution medium is limited to a specific range, which can effectively improve the dissolution rate of fluticasone propionate, so that each test point has a higher detection sensitivity, thereby obtaining better reproducibility.

[0019] In some optional embodiments, the mass concentration of Tween 20 in the dissolution medium is 0.3%.

[0020] In the above technical solution, the mass percentage of Tween 20 in the dissolution medium is limited to 0.3%, which can also make the dissolution curve have better discrimination, help identify the intrinsic quality and differences of the drug, and thus be more suitable for establishing an in vitro dissolution curve model for the inhalation suspension.

[0021] In some optional embodiments, in the step of transferring the filter paper support into a dissolution cup filled with a dissolution medium and measuring the dissolution curve using a paddle method, the volume of the dissolution medium is 500 mL, and the mass of the active substance collected on the filter paper is 113 μg to 137 μg.

[0022] In the above technical scheme, the volume of the dissolution medium and the mass of the active substance collected on the sample collection filter paper are respectively limited within the above ranges, taking into account the characteristics of the small amount of lung fluid in the lungs and the need to improve the test sensitivity. In addition, the selection of a suitable volume of dissolution medium is conducive to better establishing a dissolution curve model, while also satisfying the sink condition of the dissolution curve determination (i.e., greater than 3 times the saturated solubility), and has the advantages of strong operability and more reasonable test scheme design.

[0023] In some optional embodiments, in the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm to 100 rpm.

[0024] In the above technical solution, the rotation speed of the stirring paddle is limited to a specific range, which is beneficial to the effective circulation of the dissolution medium below and above the filter paper holder. While appropriately increasing the dissolution rate, it can also improve the problem of dead volume easily generated at the bottom of the filter paper holder and the dissolution cup, thereby obtaining better reproducibility (i.e., smaller RSD).

[0025] In some optional embodiments, in the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm to 75 rpm.

[0026] In the above technical solution, limiting the rotation speed of the stirring paddle within a specific range can also make the dissolution curve have better discrimination, which can help identify the intrinsic quality and differences of the drug, and is therefore more suitable for establishing an in vitro dissolution curve model for the inhalation suspension.

[0027] Summarize: In the prior art, for the collection of particles in aerosolized aerosols, usually only particles of a certain stage are collected or particles of a certain stage are collected repeatedly. The first collection method collects a small amount of sample, resulting in low test sensitivity and poor reproducibility. Although the second collection method can improve the sensitivity, the repeated operation has the problems of cumbersome steps, time-consuming, low efficiency and inconvenient promotion. In addition, the dissolution test of particles collected by the above two collection methods cannot represent the dissolution process of fine particles that can be deposited in the lungs to exert their effects in the body. Based on this, in this application, a circular filter paper with a diameter of 43 mm is placed above the S4 level of the new generation pharmaceutical impactor sieve plate to cover the entire S4 level of the sieve plate. The suspension for inhalation is atomized into an aerosol by the atomization device and circulates in the impactor with the airflow. The particles of the S1~S3 levels are collected in the collection cups below the corresponding levels, and the particles of the S4 level and subsequent levels are retained on the circular filter paper, so that a large number of representative particles (i.e., fine particles with a particle size of less than 5 μm that can be deposited deep in the lungs to play a role) can be collected through only one collection process, thereby effectively reflecting the dissolution behavior of the active substance in the drug in the lungs. The in vitro dissolution curve determination based on the collection method provided in this application has the advantages of simplicity and good reproducibility, and can comprehensively reflect the dissolution and absorption process of fine particles of different particle sizes in the suspension for inhalation in the lungs, which is of great significance in guiding the development of prescription processes, conducting in vitro bioequivalence evaluations, exempting clinical trials, and proving the quality consistency of products before and after changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 A schematic diagram of the internal structure of a new generation of pharmaceutical impactor provided in an embodiment of the present application; Figure 2 A schematic diagram of the structure of a sieve plate in a new generation of pharmaceutical impactor provided in an embodiment of the present application; Figure 3 A schematic diagram of the structure of a paddle device provided in an embodiment of the present application; Figure 4 A flow chart of a method for determining an in vitro dissolution curve of an inhalation suspension provided in an embodiment of the present application; Figure 5 The liquid chromatogram corresponding to the PBS dissolution medium containing Tween 80 provided in the examples of the present application; Figure 6 The liquid chromatogram corresponding to the PBS dissolution medium containing Tween 20 provided in the examples of the present application; Figure 7 The saturated solubility curve of fluticasone propionate provided in the embodiment of the present application in PBS dissolution medium containing different mass concentrations of Tween 20; Figure 8 The dissolution curve of fluticasone propionate provided in the embodiment of the present application in PBS dissolution medium containing different mass concentrations of Tween 20; Fig. 9 The dissolution curve of fluticasone propionate at different stirring speeds provided in the embodiments of the present application; Fig.10 This is a comparison chart of the in vitro dissolution curves of different batches of the self-developed product and the original product provided in the examples of this application.

[0030] Icons: 10-new generation pharmaceutical impactor; 11-collection cup; 12-sieve plate; 13-S4 level; 14-sample collection filter paper; 20-paddle device; 21-dissolution cup; 22-stirring paddle; 23-filter paper holder. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0032] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to the three situations of “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0033] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a~value b" includes the two end values ​​"a" and "b", and the "unit" in "value a~value b+unit" represents the "unit" of both "value a" and "value b".

[0034] The following is a detailed description of a method for determining the in vitro dissolution curve of an inhalation suspension in an embodiment of the present application.

[0035] The present application embodiment provides a method for determining an in vitro dissolution curve of a suspension for inhalation, comprising the following steps: (1) Place a sample collection filter paper above the S4 level of the sieve plate of the new generation pharmaceutical impactor; (2) Nebulize the inhalation suspension to obtain an aerosol, and allow the aerosol to circulate in the new generation pharmaceutical impactor with the airflow; (3) Take out the sample collection filter paper with the active substance collected above the S4 level of the sieve plate, fix the sample collection filter paper in the filter paper holder, transfer the filter paper holder to the dissolution cup filled with the dissolution medium and use the paddle method to measure the dissolution curve, and then take the same volume of solution at different time points as the test solution; (4) After sampling, use high performance liquid chromatography to determine the content of the active substance in the test solution at each time point and the content of the residual active substance on the sample collection filter paper, so as to obtain the cumulative dissolution at each time point and draw a dissolution curve.

[0036] It should be noted that the new generation pharmaceutical impactor is one of the commonly used translations of next generation impactor known in the art. Next generation impactor is recorded in the 2020 edition of the Pharmacopoeia - Part IV, specifically as Device 3 in the entry catalog (0951-Determination of aerodynamic characteristics of fine particles of inhalation preparations). The original text records that "Device 3 (nextgeneration impactor, NGI) is a cascade impactor with 7 stages and 1 microporous collector (MOC)."

[0037] In the present application, a circular filter paper with a diameter of 43 mm is placed above the S4 stage of the sieve plate of the new generation pharmaceutical impactor to cover the entire S4 stage of the sieve plate. The inhalation suspension is atomized into an aerosol by a nebulizer and then circulates in the impactor with the airflow. The particles of the S1 to S3 stages are respectively collected in the collection cups below the corresponding stages, and the particles of the S4 stage and subsequent stages are retained on the circular filter paper. Therefore, a large number of representative particles (i.e., fine particles with a particle size of less than 5 μm that can be deposited deep in the lungs to exert their effects) can be collected through only one collection process, thereby effectively reflecting the dissolution behavior of the active substance in the drug in the lungs. Compared with the collection method in the prior art of only collecting particles of a certain level (the amount of collected samples is small, resulting in low test sensitivity and poor reproducibility) or repeatedly collecting particles of a certain level for multiple times (although the sensitivity can be improved, the repeated operation has the problems of cumbersome steps, time-consuming, low efficiency and inconvenient for promotion), the in vitro dissolution curve determination based on the collection method provided in this application has the advantages of simplicity and good reproducibility, and can comprehensively reflect the dissolution and absorption process of fine particles in the inhalation suspension in the lungs. It is of great significance in guiding the development of prescription processes, conducting in vitro bioequivalence evaluation, exempting clinical trials and proving the quality consistency of products before and after changes.

[0038] It should be noted that in the embodiments of the present application, particles of multiple stages are collected at one time for dissolution curve measurement. Compared with collecting particles of multiple stages separately and then mixing them for dissolution curve measurement, the former has the advantages of simpler testing steps and better reproducibility. The specific analysis is as follows: the collection process of NGI can make the particles evenly distributed on the surface of the collection cup or filter paper. Adding a mixing step will not make the particles evenly distributed, and the uneven distribution seriously affects the dissolution rate; furthermore, the particles on the collection cup are adsorbed on the surface of the collection cup, and the transfer process and the transfer tool will cause the loss of particles. These problems will affect the reproducibility.

[0039] In addition, in the embodiments of the present application, particles of multiple stages are collected at one time for dissolution curve measurement. Compared with placing multiple collection cups into the dissolution cup simultaneously for dissolution curve measurement, the former has the advantage of better reproducibility. The specific analysis is as follows: the structure of the dissolution cup is provided with a stirring paddle on the top, and a collection plate or a filter paper holder is placed below the stirring paddle, i.e., at the bottom of the dissolution cup. According to the principle of dissolution curve measurement, the different positions of the test substance placed in the dissolution cup have a great influence on the test result, and the space at the bottom of the dissolution cup is limited, and multiple collection plates or filter paper holders cannot be placed in the same plane position. If multiple collection cups are stacked in the dissolution cup for dissolution curve measurement, multiple samples will have different dissolution rates due to their different positions, thereby affecting the reproducibility.

[0040] It should be noted that the number of filter papers is not limited and can be adaptively adjusted according to actual conditions. For example, only one piece can be placed, or two pieces can be placed in an overlapping manner.

[0041] It should be noted that in step (3), the step of taking the same volume of solution at different time points as the test solution includes: taking the same volume of solution from the dissolution cup at different time points and sequentially filtering through primary filtration and secondary filtration, and then using the filtrate as the test solution.

[0042] As an example, the pore size of the filter element for primary filtration is 1 μm, and the pore size of the filter membrane for secondary filtration is 0.45 μm.

[0043] It should be noted that the calculation steps of the cumulative dissolution of the solution to be tested at each time point include: firstly calculating the dissolution of the active substance at each time point by the ratio of the dissolution amount of the active substance at each time point to the total amount of the active substance (total amount of active substance = residual amount of active substance on the filter paper + dissolution amount), and then calculating the cumulative dissolution of the active substance at each time point. Among them, the method for determining the residual amount of active substance on the filter paper is: after the sampling is completed, take out the filter paper support and put it into a beaker, add 20 mL of acetonitrile-water (60:40) mixed solution, ultrasonicate for 5 minutes, then add 30 mL of 0.3% Tween 20 (mass concentration) PBS buffer solution (pH 7.4), mix well, filter, and use high performance liquid chromatography to determine the content of active substance in the filtrate and use the test result as the residual amount of active substance on the filter paper.

[0044] It should be noted that when the aerosol circulates with the airflow in the new generation of pharmaceutical impactors, the active substances in the aerosol exist as particles of different sizes. When the airflow passes through different levels of the sieve plate, the particles of different sizes are collected in the collection cups below the sieve plate, thereby achieving the screening of particles of different sizes. When the airflow passes through the sample collection filter paper above the S4 level, the fine particles in the aerosol will be intercepted by the filter paper, thereby achieving the collection of fine particles in the active substance.

[0045] In order to better understand the technical solution, the structural diagram of the new generation of pharmaceutical impactor and the position of the sample collection filter paper in the sieve plate are used for auxiliary explanation. For details, please refer to Figure 1 and Figure 2 ,in, Figure 1 It is a schematic diagram of the internal structure of the new generation pharmaceutical impactor 10. Figure 2 FIG. 1 is a schematic diagram of the structure of the sieve plate 12 in the new generation pharmaceutical impactor 10. Figure 1 and Figure 2 It can be clearly seen that the sample collection filter paper 14 is located directly above the S4 level 13 of the sieve plate 12 , and the collection cup 11 corresponding to the S4 level 13 is located directly below the S4 level 13 in the sieve plate 12 .

[0046] In addition, in order to better understand the process of paddle dissolution, a structural diagram of a paddle device is used for auxiliary explanation. Figure 3 The paddle device 20 includes a dissolution cup 21, a stirring paddle 22 and a filter paper holder 23, wherein the filter paper holder 23 is located at the bottom of the dissolution cup 21, the sample collection filter paper is fixed in the filter paper holder 23, and the stirring paddle 22 extends into the dissolution cup 21 and is located above the filter paper holder 23.

[0047] It should be noted that the form of the filter paper holder is not limited. For example, the filter paper holder includes two 40-mesh circular screens that are relatively distributed, and the circumferential edges of the two screens are fixed together by a circular ring fixing member, wherein the sample collection filter paper is accommodated in the two screens. It should be noted that in the stage of collecting active substances, the assembly process and parameter control of the new generation of pharmaceutical impactors, freezers, vacuum pumps and atomizers can be carried out according to conventional settings in the field.

[0048] As an example, the collection process of fine particles in the active substance includes the following steps: placing a sample collection filter paper above the S4 level of the sieve plate of the new generation pharmaceutical impactor, and then precooling the new generation pharmaceutical impactor in a freezer, wherein the precooling temperature is 5°C and the precooling time is not less than 90 min; then connecting the precooled new generation pharmaceutical impactor with a vacuum pump and an atomizer, adding an inhalation suspension to the atomizer, and then starting the vacuum pump and the atomizer in turn for atomization, so that the atomized aerosol circulates in the new generation pharmaceutical impactor with the airflow, wherein the atomization time is 5 min and the gas flow rate is 15 L / min; after the atomization is completed, the atomizer and the vacuum pump are turned off in turn; then the sample collection filter paper placed above the S4 level of the sieve plate is removed, and the fine particles in the active substance are collected.

[0049] It should be noted that according to USP1601, the cut-off particle size (Cut-Offdiameter, i.e. the lower limit of the particle size that can be intercepted) of each stage of NGI at a flow rate of 15 L / min is distributed as follows: S1 is 14.10 μm, S2 is 8.61 μm, S3 is 5.39 μm, S4 is 3.30 μm, S5 is 2.08 μm, S6 is 1.36 μm, and S7 is 0.98 μm. That is, by placing a sample collection filter paper above the S4 stage of the sieve plate, particles with a size less than 5.39 μm can be retained on the sample collection filter paper.

[0050] It should be noted that a new generation of pharmaceutical impactors can be purchased commercially.

[0051] As an example, the suspension for inhalation is selected from at least one of fluticasone propionate suspension for inhalation, budesonide suspension for inhalation and beclomethasone propionate suspension for inhalation.

[0052] It should be noted that the fluticasone propionate suspension for inhalation is the commercially available fluticasone propionate suspension for aerosol inhalation (e.g., fluticasone propionate ® , Yiruiping ® Chen Keshu ® ), budesonide suspension for inhalation, that is, commercially available budesonide suspension for inhalation (e.g., Pulmicort Respules ® , sunny and smooth ®), beclomethasone dipropionate suspension for inhalation, that is, commercially available beclomethasone dipropionate suspension for inhalation (e.g., Biolia ® ).

[0053] In this embodiment, there are many types of applicable suspensions for inhalation, and more feasible implementation plans can be provided, thereby facilitating the promotion and application of the technical solutions provided in the embodiments of the present application; at the same time, these types of suspensions for inhalation are highly representative suspension products for inhalation on the market.

[0054] As an example, the material of the sample collection filter paper is glass fiber.

[0055] In this embodiment, the sample collection filter paper is made of glass fiber, which has both low resistance and high capture efficiency. Under the premise of not hindering the flow of air in the impactor, it can effectively intercept a large number of S4~MOC grade particles in the atomized aerosol and will not be damaged due to excessive infiltration of the atomized aerosol, resulting in particle loss. At the same time, it can also make the particles in the atomized aerosol uniformly and stably loaded on the surface of the filter paper, thereby facilitating the subsequent wetting and dissolution of the particles layer by layer inward in the dissolution medium, thereby making the dissolution process of the particles in the dissolution medium have good reproducibility.

[0056] As an example, the pore size of the sample collection filter paper is 0.2 μm to 0.5 μm, such as but not limited to any point value of 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm and 0.5 μm, or a range of values ​​between any two of the pore sizes.

[0057] In this embodiment, the pore size of the sample collection filter paper is limited to a specific range, which can effectively reduce the probability of fine particles passing through the filter paper, thereby improving the particle retention rate of the filter paper.

[0058] It should be noted that, since the solubility of fluticasone propionate in PBS buffer solution is low, in order to increase the dissolution rate of fluticasone propionate and improve the experimental efficiency, a surfactant can be added to the PBS buffer solution.

[0059] As an example, the dissolution medium is a PBS buffer solution containing Tween.

[0060] In this embodiment, adding Tween to the PBS buffer solution can appropriately increase the dissolution rate of fluticasone propionate, so as to more efficiently obtain the entire in vitro dissolution curve.

[0061] As an example, the type of Tween is Tween20.

[0062] In this embodiment, a specific type of Tween (i.e., Tween 20) is used to appropriately increase the dissolution rate of fluticasone propionate without causing interference in the measurement process, so as to obtain a more accurate dissolution curve.

[0063] As an example, in the dissolution medium, the mass concentration of Tween 20 is 0.3% to 0.5%, such as but not limited to any one of 0.3%, 0.4% and 0.5% by mass percentage or a range between any two of them.

[0064] In this embodiment, the mass percentage of Tween in the dissolution medium is limited to a specific range, which can effectively increase the dissolution rate of fluticasone propionate and enable each test point to have a higher detection sensitivity, thereby obtaining better reproducibility.

[0065] As an example, the mass concentration of Tween 20 in the dissolution medium is 0.3%.

[0066] In this embodiment, the mass percentage of Tween 20 in the dissolution medium is limited to 0.3%, which can also make the dissolution curve have better discrimination, help identify the intrinsic quality and differences of the drug, and thus be more suitable for establishing an in vitro dissolution curve model for the suspension for inhalation.

[0067] As an example, the volume of the dissolution medium in the step of transferring the filter paper holder to the dissolution vessel filled with the dissolution medium and performing the dissolution curve measurement using the paddle method is 500 mL and the mass of the active substance is 113 μg ~ 137 μg.

[0068] In this embodiment, the volume of the dissolution medium and the mass of the active substance on the sample collection filter paper are respectively limited within the above ranges, taking into account the characteristics of the small amount of lung fluid in the lungs and the need to improve the test sensitivity. In addition, selecting a suitable volume of dissolution medium is helpful to better establish a dissolution curve model, while also satisfying the sink condition of the dissolution curve determination (i.e., greater than 3 times the saturated solubility), which has the advantages of strong operability and more reasonable test scheme design.

[0069] As an example, in the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm~100 rpm, for example but not limited to any one of 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm and 100 rpm or a range between any two of them.

[0070] In this embodiment, the rotation speed of the stirring paddle is limited to a specific range, which is beneficial to the effective circulation of the dissolution medium below the filter paper holder and above the filter paper holder. While appropriately increasing the dissolution rate, it can also improve the problem of dead volume easily generated at the bottom of the filter paper holder and the dissolution cup, thereby obtaining better reproducibility (i.e., smaller RSD).

[0071] As an example, in the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm~75 rpm, for example but not limited to any one of 50 rpm, 55 rpm, 60 rpm, 65 rpm, 70 rpm and 75 rpm or a range between any two of them.

[0072] In this embodiment, limiting the rotation speed of the stirring paddle within a specific range can also make the dissolution curve have better discrimination, which can help identify the intrinsic quality and differences of the drug, and is more suitable for establishing an in vitro dissolution curve model for the inhalation suspension.

[0073] As an example, in the step of testing the content of active substances in each test solution by high performance liquid chromatography, the chromatographic conditions are as follows: The detection wavelength was 239 nm, the flow rate was 1 mL / min, the injection volume was 100 μL, the column temperature was 40°C, the chromatographic column was Elite, Hypersil BDS-C18, 4.6 mm×150 mm, 3 μm, the mobile phase A was 0.01 mol / L ammonium dihydrogen phosphate solution (pH 3.5)-acetonitrile-methanol (volume ratio of 35:15:50), and the mobile phase B was methanol.

[0074] It should be noted that the processes and steps not specifically described or limited in the in vitro dissolution curve determination method can be set according to routine selection in the art.

[0075] As an example, the flow chart of the method for determining the in vitro dissolution curve of the suspension for inhalation is as follows: Figure 4 shown.

[0076] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0077] Example 1 (1) Two sample collection filter papers (made of glass fiber, with a pore size of 0.3 μm) are placed above the S4 level of the sieve plate of a new generation pharmaceutical impactor (in this example, the commercial NGI device of Copley Company is used).

[0078] (2) Precool the new generation pharmaceutical impactor in a freezer, wherein the precooling temperature is 5°C and the precooling time is not less than 90 min; then connect the precooled new generation pharmaceutical impactor to a vacuum pump and a nebulizer, and add an inhalation suspension (fluticasone propionate nebulized inhalation suspension: Chenkeshu) to the nebulizer. ® , specifications are 2 mL: 0.5 mg), and then the vacuum pump and the nebulizer are started in sequence for nebulization treatment, so that the nebulized aerosol circulates with the airflow in the new generation of pharmaceutical impactor, wherein the nebulization time is 5 min, and the gas flow rate is 15 L / min; after the nebulization is completed, the nebulizer and the vacuum pump are turned off in sequence; then the sample collection filter paper placed above the S4 level of the sieve plate is removed, and the fine particles in the active substance are collected.

[0079] (3) Take out the sample collection filter paper with active substances collected above the S4 level of the sieve plate, fix the sample collection filter paper in the filter paper holder, transfer the filter paper holder to the dissolution cup filled with dissolution medium and use the paddle method to measure the dissolution curve to simulate the in vivo dissolution process, then take 2 mL of the solution at 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min and then pass through the primary filtration and secondary filtration in sequence, where the filter element pore size of the primary filtration is 1 μm and the membrane pore size of the secondary filtration is 0.45 μm, and then the filtrate is used as the test solution.

[0080] a For the selection of surfactants in the dissolution medium, see Figure 5 and Figure 6 ( Figure 5 This is the liquid chromatogram corresponding to the PBS dissolution medium (pH 7.4) containing Tween 80. Figure 6 The liquid chromatogram corresponding to the PBS dissolution medium (pH 7.4) containing Tween 20) is analyzed as follows: Figure 5 In the figure (the upper one is the chromatogram of the test solution, and the lower one is the blank chromatogram of the dissolution medium without fluticasone propionate), it can be seen from the chromatogram results of Tween 80 that the PBS buffer solution (pH 7.4) containing Tween 80 interferes with the peak position of fluticasone propionate, which leads to a decrease in the accuracy of the determination results of fluticasone propionate; Figure 6 In the figure (the upper one is the chromatogram of the test solution, and the lower one is the blank chromatogram of the dissolution medium without fluticasone propionate), it can be seen from the chromatogram of Tween 20 that the PBS buffer solution (pH 7.4) containing Tween 20 has no interference in the peak position of fluticasone propionate, and the test results are more reliable. Therefore, Tween 20 was selected as the surfactant in the PBS buffer solution.

[0081] The liquid chromatography conditions were as follows: detection wavelength of 239 nm, flow rate of 1 mL / min, injection volume of 100 μL, column temperature of 40 °C, chromatographic column of Elite, Hypersil BDS-C18, 4.6 mm×150 mm, 3 μm, mobile phase A of 0.01 mol / L ammonium dihydrogen phosphate solution (pH 3.5)-acetonitrile-methanol (volume ratio of 35:15:50), mobile phase B of methanol, and gradient elution program as shown in Table 1, so as to obtain the cumulative dissolution at each time point and draw the sample dissolution curve.

[0082] Table 1

[0083] b For the selection of dissolution medium volume and Tween 20 concentration in the dissolution medium, please refer to Table 2, Table 3, Table 4, Figure 7 and Figure 8 , the analysis is as follows: Table 2

[0084] See Table 2 and Figure 7 (Wherein, Table 2 shows the saturated solubility of fluticasone propionate in PBS buffer solutions containing different concentrations of Tween 20, Figure 7 (Figure 2 is a curve drawn based on saturated solubility) It can be seen that as the concentration of Tween 20 in the dissolution medium increases, the solubility of fluticasone propionate also increases. It has been found that the maximum mass of active substances that can be collected on the filter paper each time a sample is atomized is 137 μg. Based on the results in Table 2, the volume of medium required to dissolve the active substance can be calculated. The results are shown in Table 3.

[0085] Table 3

[0086] Referring to Table 3 (the volume of dissolution medium and three times the volume of PBS buffer solutions containing different concentrations of Tween 20 to dissolve all active substances), it can be seen that when the concentration of Tween 20 in the PBS buffer solution is 0.3%, the volume of dissolution medium required to dissolve all active substances is 63 mL, and the three times the volume is 190 mL. That is to say, the volume of the dissolution medium must be at least 190 mL to ensure that the dissolution and release process of the drug is not limited by the volume of the dissolution medium, so that the bioavailability of the drug can be evaluated more accurately. Considering operability and rationality, 500 mL is the best choice for the volume of the dissolution medium.

[0087] In addition, see Table 4 and Figure 8(Table 4 is a summary table of the cumulative dissolution and RSD of fluticasone propionate in a dissolution medium (pH 7.4) containing 0.3% Tween and 0.5% Tween. Specifically, the steps for calculating the cumulative dissolution of the active substance at each time point include: firstly calculating the dissolution of the active substance at each time point by the ratio of the dissolved amount of the active substance at each time point to the total amount of the active substance (total amount of the active substance = the residual amount of the active substance on the filter paper + the dissolved amount), and then calculating the cumulative dissolution of the active substance at each time point. The method for determining the residual amount of the active substance on the filter paper is: after the sampling is completed, the filter paper support is taken out and placed in a beaker, 20 mL of acetonitrile-water (60:40) mixed solution is added, ultrasonicated for 5 minutes, and then 30 mL of 0.3% Tween 20 (mass concentration) PBS buffer solution (pH 7.4) is added, mixed evenly, filtered, and the content of the active substance in the filtrate is determined by high performance liquid chromatography and the test result is used as the residual amount of the active substance on the filter paper, n=3, Figure 8 is the cumulative dissolution curve drawn according to Table 4): Table 4

[0088] See Table 4 and Figure 8 It can be seen that as the concentration of Tween 20 in the dissolution medium increases from 0.3% to 0.5%, the dissolution rate is significantly accelerated. Limiting the mass percentage of Tween 20 in the dissolution medium to 0.3% makes the dissolution curve have better discrimination, which can help identify the intrinsic quality and differences of the drug, and is more suitable for establishing an in vitro dissolution curve model for inhalation suspensions. Therefore, the mass percentage of Tween in the dissolution medium is finally limited to 0.3%.

[0089] c For the selection of stirring speed, please refer to Table 5 and Fig. 9 , the analysis is as follows: Table 5

[0090] See Table 5 and Fig. 9 (Table 5 is a summary table of the cumulative dissolution and RSD of the tested solutions at different time points at different rotation speeds, n=3, the dissolution medium is PBS buffer solution (pH 7.4) containing 0.3% Tween 20, Fig. 9According to the cumulative dissolution curve drawn according to Table 5, it can be seen that limiting the rotation speed of the stirring paddle within the range of 50 rpm ~100 rpm is conducive to the effective circulation of the dissolution medium below and above the filter paper holder, and can improve the problem of dead volume easily generated at the bottom of the filter paper holder and the dissolution cup while appropriately increasing the dissolution rate, thereby obtaining better reproducibility (i.e., smaller RSD); further, limiting the rotation speed of the stirring paddle within the range of 50 rpm ~75 rpm can also make the dissolution curve have better discrimination, which is more suitable for establishing an in vitro dissolution curve model for the inhalation suspension, so the stirring speed was finally determined to be 75 rpm.

[0091] In summary, in the step of transferring the filter paper support to the dissolution cup filled with the dissolution medium and measuring the dissolution curve using the paddle method, it was determined that the dissolution medium was a PBS buffer solution (pH 7.4) containing 0.3% Tween 20 by mass concentration, the volume of the dissolution medium was 500 mL, and the speed of the stirring paddle was 75 rpm.

[0092] (4) The content of active substance at each time point and the mass of active substance remaining on the filter paper are tested by high performance liquid chromatography. For the specific chromatographic conditions, please refer to the chromatographic conditions in the surfactant selection part in step (3) to obtain the cumulative dissolution at each time point and draw the sample dissolution curve.

[0093] Test example In vitro dissolution curve test of self-developed products and original products Test method: Different batches of fluticasone propionate inhalation suspension self-developed product (Chen Keshu ® ) and different batches of original fluticasone propionate suspension for inhalation (Yi Rui Ping ® ) were respectively used as test samples and tested according to the test steps of Example 1. The test results were statistically summarized in Table 6 and the corresponding in vitro dissolution curves were drawn according to Table 6 to compare the in vitro dissolution curves of the self-developed products with those of the original products.

[0094] Table 6

[0095] See Table 6 and Fig.10 It can be seen that the dissolution curves of the self-developed product and the original product (reference preparation) are basically the same, and the RSD of the cumulative dissolution at each time point is small, indicating that the method has good reproducibility. At the same time, the specific sample collection method also makes the test method simple and easy.

[0096] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A method for determining the in vitro dissolution curve of an inhalation suspension, characterized in that: The following steps are involved: (1) Place the sample collection filter paper above the S4 level of the frit plate of the new generation pharmaceutical impactor; (2) atomizing the inhalation suspension to obtain an atomized aerosol, and allowing the atomized aerosol to flow along with the airflow in the new generation pharmaceutical impactor; (3) taking out the sample collection filter paper on which the active substance is collected above the S4 level of the sieve plate, fixing the sample collection filter paper in a filter paper holder, transferring the filter paper holder to a dissolution cup filled with a dissolution medium, and performing a dissolution curve measurement using a paddle method, and then taking the same volume of solution at different time points as the test solution; (4) After sampling, the content of the active substance in the test solution and the content of the active substance remaining on the sample collection filter paper at each time point are determined by high performance liquid chromatography to obtain the cumulative dissolution at each time point and draw a dissolution curve.

2. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 1, characterized in that: The inhalation suspension is selected from at least one of fluticasone propionate inhalation suspension, budesonide inhalation suspension and beclomethasone propionate inhalation suspension.

3. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 1 or 2, characterized in that: The sample collection filter paper is made of glass fiber, or / and, and the pore size of the sample collection filter paper is 0.2 μm~0.5 μm.

4. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 2, characterized in that: The suspension for inhalation is the fluticasone propionate suspension for inhalation, and the dissolution medium is a PBS buffer solution containing Tween.

5. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 4, characterized in that: The type of Tween is Tween 20.

6. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 5, characterized in that: In the dissolution medium, the mass concentration of Tween 20 is 0.3% to 0.5%.

7. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 6, characterized in that: In the dissolution medium, the mass concentration of Tween 20 is 0.3%.

8. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 5, characterized in that: In the step of transferring the filter paper support to a dissolution cup filled with a dissolution medium and measuring the dissolution curve using a paddle method, the volume of the dissolution medium is 500 mL, and the mass of the active substance collected on the filter paper is 113 μg to 137 μg.

9. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 5, characterized in that: In the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm to 100 rpm.

10. The method for determining the in vitro dissolution curve of the suspension for inhalation according to claim 9, characterized in that: In the step of measuring the dissolution curve using the paddle method, the rotation speed of the stirring paddle is 50 rpm~75 rpm.

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