In-vitro release method of tacrolimus ointment

Abstract

The invention provides an in-vitro release method of tacrolimus ointment, which comprises the following steps: solution preparation: selecting a 30% tetrahydrofuran solution as an accepting medium, then measuring 300ml of tetrahydrofuran, and putting the tetrahydrofuran into the same container to be fully and uniformly mixed; the preparation method comprises the following steps: precisely weighing 11.5 mg of tacrolimus raw material medicine, putting the tacrolimus raw material medicine into a measuring flask, adding a proper amount of tetrahydrofuran, shaking to completely dissolve the raw material medicine, diluting with tetrahydrofuran to a scale line, and uniformly shaking; precisely measuring 1ml of the solution, putting the solution into another 100ml measuring flask, diluting the solution to a scale by using a 30% tetrahydrofuran solution, uniformly shaking again, and preparing 2 parts in parallel; according to the method, an accurate and reliable means is provided for quality evaluation of the tacrolimus ointment, comprehensive and effective quality control can be carried out on different batches of products produced by different manufacturers by accurately measuring the in-vitro release condition of the medicine, and treatment effect differences and potential safety risks caused by uneven medicine quality are avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to an in vitro release method for tacrolimus ointment. Background Art

[0002] As an externally applied drug widely used in dermatology, tacrolimus ointment plays a key role in the treatment of various skin diseases. Its main active ingredient, tacrolimus, belongs to macrolide immunosuppressants and can specifically inhibit the activation and proliferation of T lymphocytes, thereby effectively regulating the immune system. This property enables tacrolimus ointment to exhibit significant efficacy in the treatment of immune-related skin diseases such as atopic dermatitis, psoriasis, seborrheic dermatitis, and vitiligo.

[0003] Atopic dermatitis is a common chronic, recurrent, inflammatory skin disease that seriously affects the quality of life of patients. Tacrolimus ointment provides an effective treatment method for patients by inhibiting the release of inflammatory mediators, reducing skin inflammatory reactions, and alleviating symptoms such as itching, erythema, and exudation. For psoriasis patients, tacrolimus ointment can inhibit the excessive proliferation of epidermal cells, regulate the skin immune system, improve skin lesion symptoms, and is less likely to cause adverse reactions such as skin atrophy and pigmentation after long-term use compared with traditional corticosteroids.

[0004] In addition, in the treatment of seborrheic dermatitis, tacrolimus ointment inhibits the growth of pathogens such as Malassezia, reduces inflammatory reactions, and effectively improves symptoms such as erythema, desquamation, and itching on the scalp, face and other parts. For vitiligo patients, tacrolimus ointment may promote the proliferation of melanocytes and melanin synthesis by regulating the local immune microenvironment, contributing to the restoration of skin pigmentation.

[0005] Although tacrolimus ointment is widely used clinically, the research data on the in vitro release method of tacrolimus ointment at home and abroad are relatively scarce at present. This situation has brought many challenges to the quality control, formulation optimization, and drug R & D of this drug.

[0006] In terms of quality control, the lack of accurate and reliable in vitro release methods makes it difficult to comprehensively and effectively evaluate the quality of tacrolimus ointments produced by different batches and different manufacturers. The in vitro release behavior of drugs is directly related to their absorption and efficacy in vivo. If the in vitro release of drugs cannot be accurately measured, it is difficult to ensure the consistency and stability of their quality.

[0007] From the perspective of formulation optimization, due to the lack of effective in vitro release detection means, when researchers carry out the R & D and improvement of new formulations, they cannot accurately judge the influence of different formulation factors (such as matrix components, drug concentration, additives, etc.) on drug release, which increases the difficulty and cost of formulation optimization and prolongs the R & D cycle.

[0008] In the process of drug research and development, in vitro release research is an important part of evaluating the performance of drug preparations. Generic drugs must pass in vitro release tests to prove their consistency with the original research drugs in terms of impurity profiles, stability, in vitro and in vivo dissolution patterns, etc., in order to ensure the equivalence of quality and efficacy. However, existing in vitro release methods have deficiencies in terms of accuracy, stability, and repeatability, and cannot meet the strict requirements of drug research and development. For example, some existing in vitro release methods may have problems such as unreasonable selection of receiving media, imperfect diffusion cell systems, and low sensitivity of detection methods, resulting in poor reliability and comparability of experimental results.

[0009] In summary, there is an urgent need to develop an accurate, stable, and reliable in vitro release method for tacrolimus ointment, which is of great significance for improving the quality control level of tacrolimus ointment, accelerating the drug research and development process, and ensuring the safety and efficacy of patients' medication. Summary of the Invention

[0010] The present invention provides an in vitro release method for tacrolimus ointment, aiming to solve the problems raised in the above background technology.

[0011] The present invention is implemented as follows. An in vitro release method for tacrolimus ointment includes the following steps:

[0012] Solution preparation: The receiving medium is selected as 30% tetrahydrofuran solution. The specific preparation method is as follows: Accurately measure 700 ml of ultrapure water, and then measure 300 ml of tetrahydrofuran, and place them in the same container and mix well.

[0013] Control solution preparation: Weigh accurately 11.5 mg of tacrolimus raw material and place it in a 100 ml volumetric flask. Add an appropriate amount of tetrahydrofuran, shake to completely dissolve the raw material, dilute to the scale line with tetrahydrofuran, and shake well; Accurately measure 1 ml of this solution and place it in another 100 ml volumetric flask, dilute to the scale with 30% tetrahydrofuran solution, and shake well again. Prepare 2 parallel portions.

[0014] Release process: Use a manual in vitro diffusion instrument with a diffusion cell volume of 8 ml and a rotation speed of 600 revolutions per minute; Add an appropriate amount of water to the manual in vitro diffusion instrument, turn on the power supply, set the temperature to 32 °C, and adjust the rotation speed to 600 rpm; Heat the 30% tetrahydrofuran solution to 32 °C.

[0015] Select a nylon artificial membrane with a pore size of 0.8 μm, cut it into a suitable size, first place it in the receiving medium and ultrasonically treat it for 10 min, and then soak it for 30 min.

[0016] Place the magnetic stir bar into the diffusion cell. Take out the soaked nylon artificial membrane, dry the surface moisture with filter paper, install it between the receiving chamber and the supply chamber, and assemble the diffusion cell. Add 0.3 g of tacrolimus ointment into the supply chamber, seal it with sealing film, add 8 ml of the preheated receiving medium at 32 °C from the injection port, and place the diffusion cell in the TRTRTR manner.

[0017] During the release process, at time points of 60 min, 120 min, 180 min, 240 min, and 360 min respectively, take out all the receiving medium in the receiving chamber as the test solution, and immediately replenish 8 ml of the preheated receiving medium at 32 °C.

[0018] Chromatographic conditions: Use a Zorbax Eclipse Plus C18 column with a specification of 100 mm × 4.6 mm and a packing particle size of 5 μm.

[0019] The mobile phase is composed of water, isopropanol, and tetrahydrofuran mixed in a ratio of 5:2:2.

[0020] The detection wavelength is 220 nm, the flow rate is 1.0 ml / min, the injection volume is 50 μl, and the column temperature is 55 °C.

[0021] Determination method and limit: Precisely measure the test solution and the reference solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the release amount per unit area of tacrolimus by the external standard method based on the peak area. Take the square root of the release time at different time points as the abscissa and the release amount per unit area as the ordinate to plot a scatter diagram, and conduct a linear regression analysis to obtain the linear regression equation and the slope of the straight line.

[0022] Calculate the 90% confidence interval of the ratio of the median in vitro release rate of the test preparation batch to the median in vitro release rate of the reference preparation, and this confidence interval should be within the range of 75% - 133.33%.

[0023] Preferably, during the preparation of the 30% tetrahydrofuran solution of the receiving medium, a pipette with a precision of 0.1 ml is used to measure ultrapure water and tetrahydrofuran.

[0024] Preferably, during the preparation of the reference solution, an electronic balance with a precision of 0.0001 g is used to accurately weigh the tacrolimus raw material.

[0025] Preferably, during the detection process, the mobile phase of the liquid chromatograph needs to be ultrasonically degassed for 10 min before use to remove the bubbles in it.

[0026] Due to the adoption of the above - mentioned scheme, the beneficial effects of the present invention are as follows: This method provides an accurate and reliable means for the quality assessment of tacrolimus ointment. By precisely determining the in - vitro release of the drug, it can comprehensively and effectively control the quality of products produced by different batches and different manufacturers, ensuring that the quality of each batch of tacrolimus ointment is stable and consistent. It is crucial to guarantee the safety and effectiveness of patients' medication, avoiding treatment effect differences and potential safety risks caused by uneven drug quality.

[0027] The in - vitro release method of the present invention demonstrates excellent advantages. It innovatively selects 30% tetrahydrofuran solution as the receiving medium, greatly improving the solubility of tacrolimus, enabling the drug to be released more rapidly and fully, significantly enhancing the release effect and detection accuracy. At the same time, the optimization of operation steps and detection parameters, such as the special treatment of artificial membranes, precisely controlled release processes, and carefully selected chromatographic conditions, ensures the stability and repeatability of the entire experimental process. Different laboratories and different operators can obtain reliable and comparable experimental results when conducting experiments according to this method, laying a solid foundation for the wide application of this method in the field of drug analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the in - vitro release device of the present invention;

[0029] Figure 2 It is a schematic diagram of the specificity investigation of different receiving media for tacrolimus of the present invention;

[0030] Figure 3 It is a schematic diagram of the transmittance of different receiving media of the reference preparation of 0.1% specification of the present invention;

[0031] Figure 4 It is a schematic diagram of the transmittance of different receiving media of the reference preparation of 0.03% specification of the present invention;

[0032] Figure 5 It is a schematic diagram of the summary of in - vitro release trend lines of different specifications of tacrolimus ointment (target specification 0.03%) of the present invention;

[0033] Figure 6 It is a schematic diagram of the summary of in - vitro release rates of different specifications of the preparation of 0.03% target specification of the present invention;

[0034] Figure 7 It is a schematic diagram of the summary of in - vitro release trend lines of different specifications of tacrolimus ointment (target specification 0.1%) of the present invention;

[0035] Figure 8 It is a schematic diagram of the summary of different in - vitro release rates of the preparation of 0.1% target specification of the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] An in vitro release method of tacrolimus ointment includes the following steps:

[0038] Solution preparation: The receiving medium is selected as a 30% tetrahydrofuran solution. The specific preparation method is as follows: accurately measure 700 ml of ultrapure water, and then measure 300 ml of tetrahydrofuran, and place them in the same container and mix well;

[0039] Preparation of reference solution: Weigh accurately 11.5 mg of tacrolimus raw material drug, place it in a 100-ml volumetric flask, add an appropriate amount of tetrahydrofuran, shake to completely dissolve the raw material drug, dilute it to the scale line with tetrahydrofuran, and shake well; accurately measure 1 ml of this solution, place it in another 100-ml volumetric flask, dilute it to the scale with a 30% tetrahydrofuran solution, and shake well again. Prepare 2 parallel portions;

[0040] Release process: Use a manual in vitro diffusion apparatus with a diffusion cell volume of 8 ml and a rotation speed of 600 revolutions per minute; add an appropriate amount of water to the manual in vitro diffusion apparatus, turn on the power supply, set the temperature to 32 °C, and adjust the rotation speed to 600 rpm; heat the 30% tetrahydrofuran solution to 32 °C;

[0041] Select a nylon artificial membrane with a pore size of 0.8 μm, cut it into a suitable size, first place it in the receiving medium and ultrasonically treat it for 10 min, and then soak it for 30 min;

[0042] Put the magnetic stirrer into the diffusion cell, take out the soaked nylon artificial membrane, dry the surface moisture with filter paper, install it between the receiving chamber and the supply chamber, and assemble the diffusion cell; add 0.3 g of tacrolimus ointment to the supply chamber, seal it with a sealing film, add 8 ml of the receiving medium preheated to 32 °C from the injection port, and place the diffusion cell in the TRTRTR manner;

[0043] During the release process, at time points of 60 min, 120 min, 180 min, 240 min, and 360 min respectively, take out all the receiving medium in the receiving chamber as the test solution, and immediately supplement 8 ml of the receiving medium preheated to 32 °C;

[0044] Chromatographic conditions: Use a Zorbax Eclipse Plus C18 column with a specification of 100 mm × 4.6 mm and a packing particle size of 5 μm;

[0045] The mobile phase is composed of water, isopropanol and tetrahydrofuran mixed in a ratio of 5:2:2;

[0046] The detection wavelength is 220 nm, the flow rate is 1.0 ml / min, the injection volume is 50 μl, and the column temperature is 55 °C;

[0047] Assay method and limit: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the release amount per unit area of tacrolimus by the external standard method based on the peak area; Take the square root of the release time at different time points as the abscissa and the release amount per unit area as the ordinate, plot a scatter diagram, perform linear regression analysis, and obtain the linear regression equation and the slope of the straight line;

[0048] Calculate the 90% confidence interval of the ratio of the median in vitro release rate of the test preparation batch to the median in vitro release rate of the reference preparation, and this confidence interval should be within the range of 75% - 133.33%.

[0049] During the preparation process of the 30% tetrahydrofuran solution of the receiving medium, a pipette with a precision of 0.1 ml is used to measure ultrapure water and tetrahydrofuran.

[0050] During the preparation process of the reference solution, an electronic balance with a precision of 0.0001 g is used to accurately weigh the tacrolimus raw material.

[0051] During the detection process, the mobile phase of the liquid chromatograph needs to be ultrasonically degassed for 10 min before use to remove the bubbles in it.

[0052] In this embodiment,

[0053] In vitro release is determined according to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition)

[0054] (1) Solution preparation:

[0055] Receiving medium (30% tetrahydrofuran solution): Take 700 ml of ultrapure water and 300 ml of tetrahydrofuran, mix well, and you will get it.

[0056] Reference solution: Take 12.5 mg of tacrolimus raw material, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the mark, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, and dilute to the mark with the receiving medium, shake well. Prepare 2 portions in parallel. (Containing about 1.25 μg / ml of tacrolimus)

[0057] (2) Release process:

[0058] Using a 30% tetrahydrofuran solution as the receiving medium, experiments were conducted in a manual in vitro diffusion apparatus. The volume of the diffusion cell was set to 8 ml, the rotation speed was adjusted to 600 revolutions per minute, and an organic nylon membrane with a pore size of 0.8 μm was used as the artificial membrane. Then, the following steps were performed: At 60 min, 120 min, 180 min, 240 min, and 360 min, all the receiving medium in the receiving chamber was taken out as the test solution, and at the same time, 8 ml of the receiving medium was replenished.

[0059] In vitro release operation: Add an appropriate amount of water to the manual in vitro diffusion apparatus, turn on the power supply, and set the temperature to 32 °C and the rotation speed to 600 rpm. Heat the receiving medium (30% tetrahydrofuran solution) to 32 °C in a beaker in the manual in vitro diffusion apparatus. Place the pre-cut 33-mm nylon artificial membrane (filter membrane) (0.8 μm) in the receiving medium and ultrasonicate it for 10 min to remove the air in the membrane pores, and then soak it in the medium for about 30 min. Place the magnetic stirrer in the diffusion cell, take out the soaked nylon artificial membrane (filter membrane) (0.8 μm), blot the water with filter paper, install it between the receiving chamber and the supply chamber, assemble the diffusion cell, add an appropriate amount of ointment to the supply chamber (the sample loading amount is 0.3 g), seal the supply chamber with a sealing film, add 8 ml of pre-heated receiving medium from the injection port, place the diffusion cell in the TRTRTR manner. At 60 min, 120 min, 180 min, 240 min, and 360 min, all the receiving medium in the receiving chamber was taken out as the test solution, and at the same time, 8 ml of pre-heated receiving medium was replenished.

[0060] Chromatographic conditions:

[0061] Chromatographic column: Zorbax Eclipse Plus C18 (100 mm × 4.6 mm, 5 μm);

[0062] Mobile phase: water - isopropanol - tetrahydrofuran (5:2:2);

[0063] Detection wavelength: 220 nm;

[0064] Flow rate: 1.0 ml / min;

[0065] Injection volume: 50 μl;

[0066] Column temperature: 55 °C.

[0067] Determination method: Accurately measure the test solution and the reference solution, and inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate the release amount per unit area of tacrolimus by the external standard method based on the peak area. Plot the square root of the release time against the release amount per unit area to obtain the linear regression equation and the slope of the straight line. Calculate the 90% confidence interval of the ratio of the median in vitro release rate of the test preparation batch to the median in vitro release rate of the reference preparation.

[0068] The 90% confidence interval of the ratio of the median in vitro release rate of the limited test preparation batches to the median in vitro release rate of the reference preparation should be within the range of 75% to 133.33%.

[0069] Examples and test data

[0070] In vitro release

[0071] 1.1 Selection of diffusion cell system

[0072] For semi-solid preparations, the Franz diffusion cell system is selected, such as Figure 1 .

[0073] This product is an ointment. The 0.1% specification contains 1 mg / g of tacrolimus, and the 0.03% specification contains 0.3 mg / g of tacrolimus. The Franz diffusion cell is selected as the release device, and the stirring speed is directly set to the fixed speed of the equipment, 600 rpm, which meets the regulatory requirements.

[0074] 1.2 Determination of the sample loading amount

[0075] According to the "Technical Guidelines for the Research of Topical Chemical Generic Drugs (Trial)", the sample loading amount is selected as 0.3 g, and the temperature of the receiving medium is 32 ± 0.5 °C.

[0076] 1.3 Selection of the receiving medium

[0077] To investigate the solubility of tacrolimus in different receiving media, according to the mobile phase for the determination of tacrolimus content in the import registration standard, which is water - isopropanol - tetrahydrofuran (5:2:2), and the diluent for the determination of content is water - absolute ethanol - tetrahydrofuran (1:1:3), it can be determined that this product has strong hydrophobicity. Solutions containing tetrahydrofuran are used for screening the receiving medium, and water - isopropanol - tetrahydrofuran (5:2:2), water - absolute ethanol - tetrahydrofuran (5:2:2), 20% tetrahydrofuran solution, and 30% tetrahydrofuran solution are selected for the investigation of saturated solubility.

[0078] (1) Solution preparation:

[0079] 20% tetrahydrofuran solution: Take 100 ml of tetrahydrofuran and add ultrapure water to 500 ml, and mix well to obtain it.

[0080] 30% tetrahydrofuran solution: Take 150 mL of tetrahydrofuran and add ultrapure water to 500 ml, and mix well to obtain it.

[0081] Water∶isopropanol∶tetrahydrofuran (5:2:2): Take 100 ml of tetrahydrofuran, 100 ml of isopropanol and add ultrapure water to 450 ml, and mix well to obtain it.

[0082] Water∶absolute ethanol∶tetrahydrofuran (5∶2∶2): Take 100 ml of tetrahydrofuran, 100 ml of ethanol, add ultrapure water to 450 ml, and mix well to obtain.

[0083] Weigh appropriate amounts of tacrolimus raw material (which can make it supersaturated in the receiving medium) and place them in the above receiving medium. Oscillate in a constant temperature water bath at 32 °C for 24 h, take out, filter, and take the subsequent filtrate for the investigation of saturated solubility.

[0084] (2) Detection method:

[0085] Chromatographic column: Zorbax Eclipse Plus C18 (100 mm × 4.6 mm, 5 μm)

[0086] Mobile phase: water - isopropanol - tetrahydrofuran (5∶2∶2);

[0087] Detection wavelength: 220 nm;

[0088] Flow rate: 1.0 ml / min;

[0089] Injection volume: 20 μl;

[0090] Column temperature: 55 °C.

[0091] (3) Detection results and conclusions: The results are as Figure 2 :

[0092] Table 1 Selection of receiving media

[0093] Solution Name Saturated Dissolution Concentration (μg / ml) Water - Isopropanol - Tetrahydrofuran (5∶2∶2) 3376.00 Water - Absolute Ethanol - Tetrahydrofuran (5∶2∶2) 3460.00 20% Tetrahydrofuran Solution 365.50 30% Tetrahydrofuran Solution 2632.50

[0094] Conclusion: All four receiving media meet the requirement that the saturated solubility of each main component should exceed 3 - 10 times the complete release concentration (33.75 μg / ml - 112.5 μg / ml). (For the 0.03% specification: calculated based on a sample loading amount of 0.3 g and a receiving medium volume of 8 ml, the 3 - 10 times release concentration of 30% of the complete release amount is 10.125 μg / ml - 33.75 μg / ml; for the 0.1% specification: calculated based on a sample loading amount of 0.3 g and a receiving medium volume of 8 ml, the 3 - 10 times release concentration of 30% of the complete release amount is 33.75 μg / ml - 112.5 μg / ml); because the excipient matrix of this product is mainly white petrolatum with strong hydrophobicity, in order to ensure that the drug can be released from the matrix, three media with higher saturated solubility, namely water - isopropanol - tetrahydrofuran (5∶2∶2), water - absolute ethanol - tetrahydrofuran (5∶2∶2), and 30% tetrahydrofuran solution, are selected for further development.

[0095] 1.4 Preliminary screening of receiving media

[0096] (1) Solution preparation:

[0097] Reference substance solution: Weigh 10 mg of tacrolimus raw material accurately, place it in a 50-ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the mark. Accurately measure 1 ml, place it in a 50-ml volumetric flask, and then accurately add mobile phase to dilute to the mark and mix well. Prepare 2 portions in parallel. Inject the sample after standing for 4 h. (Containing about 4 μg / ml of tacrolimus, note: the concentration of the reference substance is approximately equivalent to the concentration of 35% release of the 0.03% specification preparation)

[0098] (2) Release process:

[0099] Set the temperature of the diffusion apparatus to 32 °C and the rotation speed to 600 rpm; heat the receiving medium (water - isopropanol∶tetrahydrofuran (5∶2∶2), water - absolute ethanol - tetrahydrofuran (5∶2∶2) and 30% tetrahydrofuran) to 32 °C. Before use, place the artificial membrane (material: nylon 66, diameter: 50 mm, pore size: 0.8 μm) in the receiving medium and ultrasonicate for 10 min to remove the air in the membrane pores, and then soak it in the medium for about 30 min; place the magnetic stirrer in the diffusion cell, take out the soaked artificial membrane, install it on the diffusion cell, assemble the diffusion cell, add an appropriate amount of sample (two specifications, the sample loading amount is about 0.3 g) to the membrane and coat it evenly. Add about 8 ml of the receiving medium to the diffusion cell, and seal the dosing chamber and the receiving chamber with sealing film respectively. Sampling is carried out at the sampling time points of 60 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 600 min. Take 8 ml of the sample from the sampling port and supplement the corresponding volume of the receiving medium. Take out the sample and directly perform liquid phase detection. The results are as follows. Calculate the release amount by the external standard method. Take the square root of time as the abscissa and the cumulative release amount per unit area as the ordinate to draw a trend line.

[0100] (3) Chromatographic conditions:

[0101] Chromatographic conditions: Since the release amount of the 0.03% specification sample is too low, the injection volume is increased to 50 μl, and the rest is the same as that under "2.3 Selection of receiving medium".

[0102] (4) Experimental results: As Figure 3 shown in the permeation rate of different receiving media of the 0.1% specification reference preparation; Figure 4 shown in the permeation rate of different receiving media of the 0.03% specification reference preparation.

[0103] The release of this product was initially screened in vitro in water - isopropanol - tetrahydrofuran (5:2:2), water - absolute ethanol - tetrahydrofuran (5:2:2), and 30% tetrahydrofuran solution to investigate its release in these media. From the experimental results, it can be seen that the release of the two specifications of this product is relatively slow in the water - absolute ethanol - tetrahydrofuran (5:2:2) medium, and there is an initial burst release phenomenon at 2 h for the 0.03% specification preparation in the water - isopropanol - tetrahydrofuran (5:2:2) medium; the release is relatively fast in the 30% tetrahydrofuran solution, and the cumulative release rate exceeds 40% at 6 h, and the release reaches the inflection point of release at 6 h. Therefore, the 30% tetrahydrofuran solution was selected for the filter membrane adsorption investigation, and the sampling time was tentatively set at 6 h.

[0104] 1.5 Adsorption of artificial membrane (filter membrane)

[0105] (1) Chromatographic conditions:

[0106] Chromatographic conditions: The same as those under "2.3 Selection of receiving medium".

[0107] (2) Solution preparation:

[0108] 30% tetrahydrofuran solution (receiving medium): Take 700 ml of ultrapure water and 300 ml of tetrahydrofuran, mix them evenly to obtain.

[0109] Stock solution for filter membrane adsorption investigation: Weigh accurately about 10 mg of tacrolimus raw material, place it in a 50 - ml volumetric flask, add 30% tetrahydrofuran solution to dissolve and dilute to the mark, shake well to obtain the stock solution of the raw material drug (0.2 mg / ml). Accurately measure 4 ml of the stock solution of the raw material drug, place it in a 20 - ml volumetric flask, and dilute to the mark with tetrahydrofuran solution to obtain the stock solution for filter membrane adsorption investigation (0.04 mg / ml).

[0110] Solution for filter membrane adsorption investigation: Measure 3 ml of the stock solution for filter membrane adsorption investigation and place it in a 200 - ml volumetric flask, dilute to the mark with 30% tetrahydrofuran solution to obtain. (0.6 μg / ml, approximately equivalent to 5% release concentration of the 0.03% specification)

[0111] Test solution: Take 25 ml of the test sample in three conical flasks respectively, add filter membranes (material: nylon; pore size: 0.8 μm; diameter 50 mm) and place them, and sample for detection after placing at 32 °C for 24 h.

[0112] Blank solution: Take 25 ml of the receiving medium in three conical flasks respectively, add filter membranes (material: nylon; pore size: 0.8 μm; diameter 50 mm) and place them, and sample for detection after placing at 32 °C for 24 h.

[0113] Solution without filter membrane: Take 30 ml of the test sample in three conical flasks respectively, and sample for detection after placing at 32 °C for 24 h.

[0114] The results are as follows:

[0115] Table 2 Results of the selection test of artificial membranes

[0116]

[0117] Calculation formula: Rate of change (%) = (Peak area of the Nh main peak - Peak area of the main peak without the filter membrane) / Peak area of the main peak without the filter membrane * 100.

[0118] Conclusion: The rate of change of tacrolimus with nylon filter membranes within 24 hours is within the range of ±5.0%, with basically no adsorption. Select a nylon filter membrane with a pore size of 0.8 μm and a diameter of 50 mm as the artificial membrane for this in vitro release method.

[0119] 6 Discrimination test (0.03% specification)

[0120] Sample information: Batch TKC240420004 (0.15 mg / g); TKC240419003 (0.3 mg / g); Batch TKC240420005 (0.45 mg / g).

[0121] (1) Solution preparation:

[0122] Reference solution: Weigh 10 mg of tacrolimus raw material, accurately weigh it, place it in a 50 ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the mark. Accurately measure 2 ml and place it in a 100 ml volumetric flask, then accurately add the mobile phase and dilute to the mark, and mix well. Prepare 2 portions in parallel. (Containing about 4 μg / ml of tacrolimus)

[0123] (2) Chromatographic conditions:

[0124] Chromatographic conditions: Considering that the release amount of the 0.015% specification of the 0.03% specification preparation is too low, the injection volume of the 0.015% specification and the 0.045% specification detected in the same batch is 50 μl, and the rest is the same as that under "2.3 Selection of acceptance medium".

[0125] (3) Release process:

[0126] Set the temperature of the diffusion instrument to 32℃ and the speed to 600rpm. Heat the receiving medium (30% tetrahydrofuran) to 32℃, place the artificial membrane (material: nylon 66, diameter: 50mm, pore size: 0.8μm) in the receiving medium for 10min to remove the air in the membrane pores before use, and then soak it in it for about 30min; place the stirring bar in the diffusion cell, take out the soaked artificial membrane, install it on the diffusion cell, assemble the diffusion cell, and absorb the water on the surface of the membrane with filter paper; add an appropriate amount of sample to the membrane (the sample amount is about 0.3g) and evenly coat it. Add about 8ml of the receiving medium to the diffusion cell, and seal the dosing chamber and the receiving chamber with sealing film. Sampling time points are 60min, 120min, 180min, 240min, and 360min respectively. Take 8ml of sample from the sampling port and add the corresponding volume of the receiving medium. Take out the sample and directly perform liquid phase detection. The results are shown below. The release amount is calculated by the external standard method. Draw a trend line with the square root of time as the horizontal axis and the cumulative release per unit area as the vertical axis.

[0127] 1.6.1 Sensitivity

[0128] Table 3 Summary of in vitro release

[0129]

[0130] like Figure 5 Summary of in vitro release trend lines of different strengths of tacrolimus ointment (target strength 0.03%)

[0131] From the above results, we can see that:

[0132] The slopes of the in vitro release curves of the three strengths of tacrolimus ointment were 2.3263, 5.4984 and 9.5461. The slopes changed with the changes in strength (preparation concentration), which proved that the sensitivity of the in vitro release method of tacrolimus ointment met the requirements.

[0133] 1.6.2 Specificity

[0134] Table 4 Summary of in vitro release rates of preparations of different strengths

[0135] Specification (mg / g) 0.15 0.3 0.45 Release Rate (Slope) 2.3263 5.4984 9.5461

[0136] A summary table of in vitro release rates of preparations of different specifications with a target specification of 0.03% is shown in Figure 6. The results show that a curve is drawn with the specification as the abscissa and the release rate (slope) as the ordinate, and a linear regression analysis is performed to obtain R2=0.9951, which is greater than 0.95, indicating that the specificity of the in vitro release method meets the requirements.

[0137] 1.6.3 Selectivity

[0138] Table 5 In vitro release of different specifications of 0.03% target specification - 1

[0139]

[0140] Table 6 In vitro release of different specifications of 0.03% target specification - 2

[0141]

[0142]

[0143] The results showed that:

[0144] When the 0.015% low - concentration specification preparation and the 0.045% high - concentration specification preparation were compared with the 0.03% standard specification preparation respectively, the 90% confidence intervals were not within the range of 75.0% - 133.33%, and the in vitro releases were not similar. Therefore, this in vitro release method is selective for 0.03% specification samples.

[0145] 1.7 Discrimination ability investigation (0.1% specification)

[0146] Sample information: Batch TKC240423008 (0.5mg / g); Batch TKC240425009 (1mg / g); Batch TKC240530023(01) (1.5mg / g).

[0147] (1) Solution preparation:

[0148] Reference solution: Weigh 11.5mg of tacrolimus raw material, accurately weigh it, place it in a 100ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the scale. Accurately measure 1ml, place it in a 100ml volumetric flask, and then accurately add mobile phase to dilute to the scale and mix well. Prepare 2 parallel portions. (Containing about 1.15μg / ml of tacrolimus)

[0149] (2) Chromatographic conditions:

[0150] Chromatographic conditions: The injection volume is 50μl, and the rest is the same as that under "2.3 Selection of receiving medium".

[0151] (3) Release process:

[0152] Set the temperature of the diffusion meter to 32 °C and the rotation speed to 600 rpm. Heat the receiving medium (30% tetrahydrofuran) to 32 °C. Before use, place the artificial membrane (material: nylon 66, diameter: 50 mm, pore size: 0.8 μm) in the receiving medium and ultrasonicate for 10 min to remove the air in the membrane pores, and then soak it in the medium for about 30 min; place the magnetic stir bar in the diffusion cell, take out the soaked artificial membrane, install it on the diffusion cell, assemble the diffusion cell, and wipe the water on the membrane surface clean with filter paper; add an appropriate amount of sample to the membrane (the sample loading amount is about 0.3 g) and coat it evenly. Add about 8 ml of the receiving medium to the diffusion cell, and seal the dosing chamber and the receiving chamber with sealing film respectively. Sampling is carried out at the sampling time points of 60 min, 120 min, 180 min, 240 min, and 360 min. Take 8 ml of the sample from the sampling port and supplement the corresponding volume of the receiving medium. Take out the sample and directly perform liquid phase detection. The results are shown below, and the release amount is calculated by the external standard method. Take the square root of time as the abscissa and the cumulative release amount per unit area as the ordinate to draw a trend line.

[0153] 1.7.1 Sensitivity

[0154] Table 7 Summary of in vitro release

[0155]

[0156] As Figure 7 Summary of the in vitro release trend lines of tacrolimus ointment (target specification 0.1%) with different specifications. From the above results, it can be seen that the curve slopes of the in vitro release of tacrolimus ointment with 3 specifications are 9.1458, 19.5551, and 27.0160. With the change of the specification (preparation concentration), the slope changes, indicating that the sensitivity of the in vitro release method of tacrolimus ointment meets the requirements.

[0157] 1.7.2 Specificity

[0158] Table 8 Summary table of the in vitro release rates of preparations with different specifications

[0159] Specification (mg / g) 0.5 1.0 1.5 Release Rate (Slope) 9.1458 19.5551 27.0160

[0160] Figure 8 The summary table of the in vitro release rates of preparations with different specifications of the 0.1% target specification is shown as above. From the above results, it can be seen that: taking the specification as the abscissa and the release rate (slope) as the ordinate, draw a curve, perform linear regression, and R2 = 0.9910 is greater than 0.95, indicating that the specificity of the in vitro release method meets the requirements.

[0161] 1.7.3 Selectivity

[0162] Table 10 In vitro release of different specifications of the 0.1% target specification -1

[0163]

[0164] Table 2 Different Specifications of In Vitro Release of 0.1% Target Specification - 2

[0165]

[0166]

[0167] The results showed that:

[0168] When the 0.05% low - concentration specification preparation and the 0.15% high - concentration specification preparation were compared with the 0.1% standard specification preparation respectively, the 90% confidence intervals were not within the range of 75.0% - 133.33%. The in vitro releases were not similar. Therefore, this in vitro release method was selective for 0.1% specification samples.

[0169] 1.8 Selection of Concentration and Injection Volume of Reference Solution

[0170] According to the specific situation of in vitro release and taking into account the release situation of 0.1% specification, the concentration of the reference solution was reduced to 1.15 μg / ml, which was equivalent to 10% of the release amount of 0.03% specification preparation and 3% of the release amount of 0.1% specification preparation. The specific preparation was as follows:

[0171] Reference solution: Weigh 11.5 mg of tacrolimus raw material drug accurately, place it in a 100 - ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the scale. Accurately measure 1 ml and place it in a 100 - ml volumetric flask, dilute to the scale with mobile phase, and shake well. Prepare 2 parallel portions. (Containing about 1.15 μg / ml of tacrolimus)

[0172] Injection volume: To avoid detection differences caused by different instrument responses due to the low release amount of 0.03% specification, the injection volume was uniformly increased to 50 μl.

[0173] 1.9 Finally Determined In Vitro Release Method

[0174] (1) Solution preparation:

[0175] Acceptance medium (30% tetrahydrofuran solution): Take 700 ml of ultrapure water and 300 ml of tetrahydrofuran, mix them evenly to obtain.

[0176] Reference solution: Weigh 11.5 mg of tacrolimus raw material drug accurately, place it in a 100 - ml volumetric flask, dissolve it with tetrahydrofuran and dilute to the scale. Accurately measure 1 ml and place it in a 100 - ml volumetric flask, dilute to the scale with acceptance medium, and shake well. Prepare 2 parallel portions. (Containing about 1.15 μg / ml of tacrolimus)

[0177] (2) Release process:

[0178] Take about 0.3 g of tacrolimus ointment, use 30% tetrahydrofuran solution as the receiving medium solution, a manual in vitro diffusion apparatus with a diffusion cell volume of 8 ml and a rotation speed of 600 revolutions per minute. Carry out the following operations. At 60 min, 120 min, 180 min, 240 min, and 360 min, take out all the receiving medium in the receiving chamber as the test solution, and at the same time supplement 8 ml of the receiving medium.

[0179] Add an appropriate amount of water to the manual in vitro diffusion apparatus, turn on the power supply, set the temperature to 32 °C, and the rotation speed to 600 rpm. Heat the receiving medium (30% tetrahydrofuran solution) to 32 °C in a beaker in the manual in vitro diffusion apparatus. First, place the pre-cut 33-mm nylon artificial membrane (filter membrane) (0.8 μm) in the receiving medium and ultrasonicate for 10 min to remove the air in the membrane pores, and then soak it in the medium for about 30 min. Place the magnetic stirrer in the diffusion cell, take out the soaked nylon artificial membrane (filter membrane) (0.8 μm), dry it with filter paper, install it between the receiving chamber and the supply chamber, assemble the diffusion cell, add an appropriate amount of cream to the supply chamber (the sample loading amount is 0.3 g), seal the supply chamber with a sealing film, add 8 ml of pre-heated receiving medium from the injection port, place the diffusion cell in the TRTRTR manner. At 60 min, 120 min, 180 min, 240 min, and 360 min, take out all the receiving medium in the receiving chamber as the test solution, and at the same time supplement 8 ml of pre-heated receiving medium.

[0180] (3) Chromatographic conditions:

[0181] Chromatographic column: Zorbax Eclipse Plus C18 (100 mm × 4.6 mm, 5 μm)

[0182] Mobile phase: water - isopropanol - tetrahydrofuran (5∶2∶2);

[0183] Detection wavelength: 220 nm;

[0184] Flow rate: 1.0 ml / min;

[0185] Injection volume: 50 μl;

[0186] Column temperature: 55 °C.

[0187] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate according to the external standard method with the peak area.

[0188] In the above embodiments, through a large number of experimental studies and comparative analyses, the present invention innovatively selects a 30% tetrahydrofuran solution as the receiving medium. Tacrolimus is a drug with relatively strong hydrophobicity and has a low solubility in conventional media, thus affecting the accuracy of detecting its in vitro release. However, the 30% tetrahydrofuran solution has good solubility for tacrolimus, can significantly improve the solubility of tacrolimus in the medium, and effectively solves the problem of inaccurate release detection caused by low drug solubility.

[0189] Compared with other common receiving media, the 30% tetrahydrofuran solution can make the drug in tacrolimus ointment release more quickly and fully, greatly improving the release effect. It was found in the experiment that when using this medium, the cumulative release rate of tacrolimus ointment exceeded 40% at 6 h, and the release reached the inflection point of release at 6 h, indicating that the drug can reach a relatively high release amount in a short time, providing a strong guarantee for accurately evaluating the release characteristics of the drug. At the same time, the selection of this medium also helps to improve the stability and repeatability of the experiment, reduce experimental errors, and make the experimental results of different batches and different laboratories more comparable.

[0190] The present invention has comprehensively optimized the operation steps and detection parameter settings of the in vitro release method. In terms of operation steps, the treatment method of the artificial membrane has been innovated. First, the nylon artificial membrane is ultrasonically treated for 10 min to remove the air in the membrane pores, and then soaked for 30 min to make it fully infiltrated. This treatment process can ensure the stable performance of the artificial membrane, reduce the drug adsorption or release hindrance caused by the membrane factor, and thus improve the accuracy of the experimental results. During the release process, the replenishment and sampling operations of the receiving medium at each time point are strictly controlled, ensuring the stability of the internal environment of the diffusion cell and providing good conditions for the continuous release of the drug.

[0191] In terms of the detection parameter settings, through the repeated optimization and screening of parameters such as the chromatographic column, mobile phase, detection wavelength, flow rate, injection volume, and column temperature, the optimal detection conditions were determined. For example, the Zorbax Eclipse Plus C18 column (100 mm × 4.6 mm, 5 μm) was selected as the chromatographic column, which could achieve efficient separation of tacrolimus and other impurities; the mobile phase ratio was set as water - isopropanol - tetrahydrofuran (5∶2∶2), ensuring appropriate retention time and resolution of tacrolimus on the chromatographic column; the detection wavelength was set at 220 nm, thus significantly improving the detection sensitivity; the flow rate was controlled at 1.0 ml / min, the injection volume was 50 μl, and the column temperature was 55°C, making the entire detection process have good accuracy, stability, and repeatability. Through the optimization measures, the in vitro release method of the present invention not only improves the accuracy, stability, and reliability compared with the traditional method, but also can more accurately reflect the in vitro release characteristics of tacrolimus ointment. This provides more powerful technical support for the quality control and drug research and development of tacrolimus ointment, ensuring the full play of its immunomodulatory and anti-inflammatory effects in the treatment of diseases such as dermatitis and eczema.

[0192] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention. The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An in vitro release method of tacrolimus ointment, characterized in that: The steps include: Solution preparation: The receiving medium is a 30% tetrahydrofuran solution. The specific preparation method is: accurately measure 700 ml of ultrapure water and then measure 300 ml of tetrahydrofuran, put them in the same container and mix them thoroughly; Preparation of reference solution: accurately weigh 11.5 mg of tacrolimus API, place in a 100 ml volumetric flask, add appropriate amount of tetrahydrofuran, shake to completely dissolve the API, dilute to the mark with tetrahydrofuran, and shake well; accurately measure 1 ml of the solution, place in another 100 ml volumetric flask, dilute to the mark with 30% tetrahydrofuran solution, shake well again, and prepare 2 portions in parallel; Release process: Use a manual in vitro diffusion apparatus with a diffusion cell volume of 8 ml and a rotation speed of 600 revolutions per minute; add an appropriate amount of water to the manual in vitro diffusion apparatus, turn on the power, set the temperature to 32°C, and adjust the rotation speed to 600 rpm; heat the 30% tetrahydrofuran solution to 32°C; A nylon artificial membrane with a pore size of 0.8 μm was selected and cut into appropriate sizes. It was first placed in a receiving medium for ultrasonic treatment for 10 min and then soaked for 30 min. Place the stirring bar into the diffusion cell, take out the soaked nylon artificial membrane, dry the surface moisture with filter paper, install it between the receiving chamber and the supply chamber, and assemble the diffusion cell; add 0.3g of tacrolimus ointment into the supply chamber, seal it with a sealing film, add 8ml of the receiving medium preheated to 32℃ from the injection port, and place the diffusion cell in the TRTRTR mode; During the release process, at 60 min, 120 min, 180 min, 240 min, and 360 min, all the receptor medium in the receptor chamber was taken out as the test solution, and immediately supplemented with 8 ml of the receptor medium preheated to 32 °C; Chromatographic conditions: Zorbax Eclipse Plus C18 column with specifications of 100 mm × 4.6 mm and a filler particle size of 5 μm was used; The mobile phase was a mixture of water, isopropanol, and tetrahydrofuran in a ratio of 5:2:2; The detection wavelength was 220 nm, the flow rate was 1.0 ml / min, the injection volume was 50 μl, and the column temperature was 55 °C; Determination method and limit: Accurately measure the test solution and the reference solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the tacrolimus release per unit area by the peak area through the external standard method; use the square root of the release time at different time points as the abscissa and the release per unit area as the ordinate to draw a scatter plot, perform linear regression analysis, and obtain the linear regression equation and the slope of the straight line; Calculate the 90% confidence interval of the ratio of the median in vitro release rate of the test preparation batch to the median in vitro release rate of the reference preparation. The confidence interval should be within the range of 75% to 133.33%.

2. The in vitro release method of tacrolimus ointment according to claim 1, characterized in that: During the preparation of the 30% tetrahydrofuran solution of the receiving medium, a pipette with an accuracy of 0.1 ml was used to measure ultrapure water and tetrahydrofuran.

3. The in vitro release method of tacrolimus ointment according to claim 2, characterized in that: During the preparation of the reference solution, an electronic balance with an accuracy of 0.0001 g was used to accurately weigh the tacrolimus API.

4. The in vitro release method of tacrolimus ointment according to claim 3, characterized in that: During the detection process, the mobile phase of the liquid chromatograph needs to be ultrasonically degassed for 10 minutes before use to remove the bubbles therein.

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