Mometasone furoate nasal spray in-vitro dissolution detection sample and pretreatment method and in-vitro dissolution detection method thereof

Abstract

The invention discloses a mometasone furoate nasal spray in-vitro dissolution detection sample as well as a pretreatment method and an in-vitro dissolution detection method thereof, and belongs to the technical field of pharmaceutical analysis. The pretreatment method of the in-vitro dissolution detection sample comprises the following steps: adding a mometasone furoate nasal spray into cellulase for enzymolysis, dispersing a mixture after enzymolysis on a microfiltration membrane, and drying to obtain the in-vitro dissolution detection sample. The invention discloses a mometasone furoate nasal spray in-vitro dissolution detection sample prepared by the pretreatment method, and an in-vitro dissolution detection method adopting the detection sample. Cellulase is added into the mometasone furoate nasal spray for enzymolysis, proper enzymolysis conditions are screened, and API particles are released from a cross-linked structure formed by the auxiliary materials, so that interference of the auxiliary materials on preparation dissolution detection is eliminated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a sample for in vitro dissolution detection of mometasone furoate nasal spray, a pretreatment method thereof and an in vitro dissolution detection method. BACKGROUND

[0002] Mometasone furoate (MF) nasal spray is a drug-device combination product, which has a long-lasting anti-inflammatory effect after nasal administration and can reduce side effects caused by oral administration. The preparation is a suspension in the form of a gel, which has obvious rheological properties. The active pharmaceutical ingredient (API) mometasone furoate and the excipient microcrystalline cellulose (MCC) in the preparation exist in the form of insoluble particles. The rheological properties of the preparation are to ensure that the drug solution does not easily flow out of the nasal cavity after administration. The gel state of the preparation is conducive to the suspension of drug particles in the drug solution, and the preparation is not prone to stratification and drug particle aggregation during storage. In the suspension preparation, weak intermolecular cross-linking is generated between the excipients MCC and sodium carboxymethyl cellulose (CMC-Na) through hydrogen bonds, and the interaction forms a three-dimensional gel structure, which causes water molecules to be fixed in the gel structure, thereby slowing down the release of drug particles in the dissolution medium. The particle size distribution (PSD) of the API in the preparation is a key quality attribute (CQA) for evaluating the nasal spray product and establishing bioequivalence (BE). Since the therapeutic effect and systemic absorption of the delivered drug depend on the dissolution and release of the drug particles deposited in the body, and the dissolution and release rate is directly related to the particle size of the API in the preparation, the dissolution rate of the API in the preparation can be measured to evaluate the differences in the particle size of the API in the reference and self-prepared preparations or different batches of preparations, which is used for generic drug research and development and quality control. At present, in the latest FDA mometasone furoate nasal spray product guidelines, dissolution detection has been included as one of the in vitro equivalence (in vitro BE) detection items for reference and self-prepared preparations.

[0003] In the prior art, there are few documents about the dissolution method of MF API in suspension preparations. Prior art 1 (G. Farias et al., A Systematic Approach in the Development of the Morphologically-Directed Raman Spectroscopy Methodology for Characterizing Nasal Suspension Drug Products, AAPS J. 23 (2021) 73) discloses that the squeezed nasal suspension drug solution is directly subjected to dissolution detection by paddle method, and PBS buffer (pH 7.4) containing 2.0% SDS (w / v) is used as the dissolution medium. The dissolution of the preparations prepared from MF API with different particle sizes was detected. The results show that the particle size (D50 ) and the dissolution rate (t 0.5 ) of the formulation. The correlation coefficient (R 2 ) was 0.89. Prior art 2 (S.S. Bachhav et al., Systematic Evaluation of the Effect of Formulation Variables on In Vitro Performance of Mometasone Furoate Suspension-Metered Dose Inhalers, AAPS J. 24(1) (2021) 9) discloses a dissolution test method for inhaled suspension of MF: the drug particles of the collected specific level part (ex-throat) were subjected to dissolution test, using 0.5% Tween 80 as dissolution medium, and the formulations prepared from MF API of different particle sizes were subjected to dissolution test. The results show that there is no obvious correlation between the particle size (D 50 ) of the API in the formulation and the dissolution rate (mean dissolution time, MDT) of the formulation. Prior art 3 (Elham Amini et al., Sensitivity of Pharmacokinetics to Differences in the Particle Size Distribution for Formulations of Locally Acting Mometasone Furoate Suspension-Based Nasal Sprays, Mol. Pharmaceutics 2023, 20, 5690-5700) discloses a dissolution test method for inhaled suspension of MF: the collected nasal spray liquid was evenly dispersed on the 70mm diameter filter paper with the loading surface facing up and clamped in the net disc, using 0.5% Tween 80 as dissolution medium, and two self-made formulations of different particle sizes and one reference formulation were subjected to dissolution test. The dissolution profile shows (see the original article Figure 1The dissolution method has the ability to distinguish different API particle size prepared formulations, but the dissolution profile is not smooth, the RSD between the dissolution results is large, which does not meet the requirement that the RSD between the results corresponding to the first sampling point of the dissolution curve is not greater than 20%, and the RSD between the results corresponding to the remaining sampling points is not greater than 10% (General Oral Solid Preparation Dissolution Test Technical Guidelines, National Drug Evaluation Center (CDE), 2016). The dissolution methods reported in these literatures prove that the correlation between the dissolution rate of API in untreated formulations and the particle size of API is not ideal and in some cases will cause the RSD between the dissolution results to be large. The applicant found through previous research that it may be because the insoluble excipients in the formulation can significantly affect the dissolution rate of API, so that the release of API from the excipients becomes the rate-limiting step of the dissolution process. Therefore, using a suitable sample pretreatment method to eliminate the influence of insoluble excipients in the formulation on the dissolution of API has become a problem to be solved by those skilled in the art. SUMMARY

[0004] One of the purposes of the present application is to provide a pretreatment method for in vitro dissolution test samples of mometasone furoate nasal spray.

[0005] The second purpose of the present application is to provide a mometasone furoate nasal spray in vitro dissolution test sample prepared by the pretreatment method.

[0006] The third purpose of the present application is to provide a mometasone furoate nasal spray in vitro dissolution test method, which is carried out by using the above-mentioned mometasone furoate nasal spray in vitro dissolution test sample.

[0007] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The pretreatment method for in vitro dissolution test samples of mometasone furoate nasal spray disclosed by the present application, the excipients of the mometasone furoate nasal spray include microcrystalline cellulose and sodium carboxymethyl cellulose; the pretreatment method comprises the following steps: The mometasone furoate nasal spray is added to cellulase for enzymolysis, and the mixture after enzymolysis is dispersed on a microporous filter membrane and dried to obtain a sample for in vitro dissolution test.

[0008] In the prior art, there is only one reference formulation of mometasone furoate nasal spray, which is produced by Merck and has the trade name of Nasonex. The excipient composition and content of the remaining generic drugs, i.e. mometasone furoate nasal spray, need to be consistent with the reference formulation.

[0009] In some embodiments of the present application, 0.3-1 mg of cellulase is added to 100 μL of mometasone furoate nasal spray, preferably 0.3-0.6 mg, and more preferably 0.5 mg.

[0010] In some embodiments of the present application, the enzyme hydrolysis is performed by adding the cellulase buffer solution to the mometasone furoate nasal spray.

[0011] In some embodiments of the present application, the cellulase buffer solution comprises a sodium acetate buffer solution of cellulase. Preferably, the pH value of the cellulase buffer solution is 4.5-6.5, more preferably 4.8.

[0012] The cellulase has good activity at a pH value of 4.5-6.5.

[0013] In some embodiments of the present application, the enzyme hydrolysis temperature is 37-60℃, preferably 50℃. The enzyme hydrolysis time is 6-24 hours, preferably 12 hours.

[0014] In some embodiments of the present application, the microporous filter membrane comprises a glass fiber membrane. Preferably, the pore size of the microporous filter membrane is 1.6μm.

[0015] In some embodiments of the present application, the drying is room temperature drying.

[0016] In some embodiments of the present application, the mometasone furoate nasal spray is moved into a centrifuge tube, the cellulase buffer solution is added for enzyme hydrolysis, the enzyme hydrolysis mixture is cooled to room temperature, centrifuged, the precipitate is dispersed, and all samples in the centrifuge tube are dispersed onto the microporous filter membrane using a pipette gun. Preferably, 100-300μL of the mometasone furoate nasal spray is moved into the centrifuge tube. Preferably, the centrifugation conditions are: 2000-6000g for 10-60 seconds, more preferably 4000g for 30 seconds. Preferably, the precipitate is gently dispersed using a pipette tip to avoid air bubbles, and all samples in the centrifuge tube are dispersed onto the microporous filter membrane using a pipette gun.

[0017] The mometasone furoate nasal spray prepared by the above pretreatment method of the present application is used as the in vitro dissolution test sample.

[0018] The mometasone furoate nasal spray in vitro dissolution test method of the present application uses the above in vitro dissolution test sample for in vitro dissolution test.

[0019] Preferably, the in vitro dissolution test comprises the following method: the in vitro dissolution test sample is placed in a dissolution assembly with the drug facing upwards, the sealing ring of the assembly is fastened, and then placed in a dissolution cup, the paddle disc method is used for dissolution test, and the dissolution curve is drawn. Preferably, the dissolution medium is a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 5.8) containing 0.2% (w / v) Tween 80, at a temperature of 37±0.5℃ and a paddle speed of 75 rpm; Preferably, the sampling time points for the dissolution test are 7.5, 15, 30, 45, 60, 120, 240, 360, 600, 960, and 1440 minutes, with HPLC injection detection. Preferably, the HPLC chromatographic conditions are as follows: ChromCore™ C18 column (5 μm, 4.6 × 150 mm), acetonitrile:water:acetic acid (55:45:0.2, v / v) as mobile phase, detection wavelength of 254 nm; column temperature of 40 ℃, flow rate of 1.5 mL / min, and injection volume of 100 μL.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention is scientifically designed and ingeniously conceived. By adding cellulase to mometasone furoate nasal spray for enzymatic hydrolysis and screening suitable hydrolysis conditions, the API particles are released from the cross-linked structure formed by excipients, thereby eliminating the interference of excipients on the dissolution detection of the formulation. Attached Figure Description

[0021] Appendix Figure 1 This is a comparison graph of dissolution curves for samples treated by different methods in Example 3.

[0022] Appendix Figure 2 This is a comparison graph of dissolution curves for samples with different degrees of enzymatic hydrolysis in Example 4.

[0023] Appendix Figure 3 The images show a comparison of the appearance of mometasone furoate nasal spray samples before and after enzymatic hydrolysis. The left image shows the sample before hydrolysis, and the right image shows the sample after hydrolysis.

[0024] Appendix Figure 4 SEM images of nasal spray samples before and after enzymatic hydrolysis: (A): before enzymatic hydrolysis; (B): after enzymatic hydrolysis.

[0025] Appendix Figure 5 The graph shows the change in apparent viscosity of samples treated with different methods as a function of shear rate in Experiment Example 2. Appendix Figure 6 The graph shows the hysteresis loop results for samples treated with different methods in Experiment Example 2. Among them, the appendix Figure 5 and attached Figure 6The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase.

[0026] The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase. Figure 7 The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase. The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase.

[0027] The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase. Figure 8 The names corresponding to the reference signs in the drawings are as follows: 1: shear rate rising stage of untreated sample; 2: shear rate falling stage of untreated sample; 3: shear rate rising stage of sample without cellulase treatment; 4: shear rate falling stage of sample without cellulase treatment; 5: shear rate rising stage of sample treated with cellulase; 6: shear rate falling stage of sample treated with cellulase. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions will be described below in connection with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0029] Unless otherwise specified, all the raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by the existing methods.

[0030] Glass fiber membrane, diameter 55 mm, pore size 1.6 μm; provided by Whatman Company.

[0031] Stress rheometer, Anton Paar MCR 302, provided by Anton Paar Company; Cellulase is Trichoderma reesei ATCC26921, activity ≥45 U / mg dw. Glucose detection kit, provided by Shanghai Blue Sky Bio-Technology Co., Ltd. Microplate reader, VICTOR Nivo, PerkinElmer Company. The mometasone furoate nasal spray in the embodiments of the present application is Nasona (trade name), 50 μg / press, produced by MSD Belgium BVBA / SPRL, unless otherwise specified.

[0032] Embodiment 1 The present embodiment discloses a pretreatment method for in-vitro dissolution detection sample of the mometasone furoate nasal spray of the present application, which is as follows: Take the finished product of mometasone furoate nasal spray, gently shake the bottle of nasal spray for 10 seconds, and then remove the spray pump from the bottle. Take 200 μL of mometasone furoate nasal spray into a centrifuge tube, and add 100 μL of sodium acetate buffer (0.1 mol / L, pH 4.8) containing 1 mg of cellulase, and mix thoroughly. After enzymolysis at 50°C overnight (about 12 hours), cool the enzymolysis mixture to room temperature, and centrifuge at 4000 g for 30 seconds. Gently disperse the precipitate with a pipette tip to avoid generating air bubbles. Immediately disperse the sample with a total volume of about 300 μL onto a glass fiber membrane with a pipette. Allow the membrane to dry thoroughly at room temperature to obtain the in vitro dissolution test sample of mometasone furoate nasal spray.

[0033] Example 2 This example discloses the investigation test of different enzymolysis reaction conditions.

[0034] The auxiliary material cellulose in mometasone furoate nasal spray is hydrolyzed to generate glucose under the action of cellulase, and thus the glucose content in the reaction system is related to the degree of hydrolysis of cellulose. The content of glucose generated in the enzymolysis reaction is determined by the o-toluidine method, which is used to screen the conditions of enzymolysis reaction.

[0035] The enzymolysis sample preparation method of this example is as follows: take the finished product of mometasone furoate nasal spray, gently shake the bottle of nasal spray for 10 seconds, and then remove the spray pump from the bottle. Take the mometasone furoate nasal spray into a centrifuge tube, and add the sodium acetate buffer of cellulase, mix thoroughly, and enzymolysis. The amount of cellulase, the volume of buffer, the volume of nasal spray, the enzymolysis reaction temperature and duration are shown in Table 1.

[0036] Centrifuge the enzymolysis samples obtained under each reaction condition at 12000 g for 2 minutes. Take 5 μL of supernatant into a centrifuge tube, add 185 μL of glucose detection reagent, and mix thoroughly. After incubation at 95°C for 8 minutes, cool the reaction to 4°C, and centrifuge at 4000 g for 30 seconds. Transfer 180 μL of the mixture in each tube to a 96-well microplate. Measure the optical density at a wavelength of 630 nm with an enzyme marker, and convert each measurement value to the glucose content according to the glucose standard curve (0.25 mg / mL-2.5 mg / mL), and the results are shown in Table 1.

[0037] Table 1. Results of investigation test of different enzymolysis reaction conditions

[0038] *The glucose content is the amount of glucose generated per milligram of Avicel.

[0039] From the above table, it can be seen that when 300 μg of cellulase is added to 100 μL of mometasone furoate nasal spray and enzymolysis is carried out at 50°C for 12 hours or more, the cellulase can be well enzymolyzed.

[0040] Example 3 This example discloses dissolution curve investigation of the enzymolyzed sample and the sample treated by adding CaCl2 solution according to the present application, and comparison with the dissolution curve of the untreated sample.

[0041] The enzymolyzed sample described in this example is an in vitro dissolution test sample of mometasone furoate nasal spray prepared according to the method of Example 1.

[0042] The preparation method of the sample treated by adding CaCl2 solution described in this example is as follows: a packaged product of mometasone furoate nasal spray is taken, the nasal spray bottle is gently shaken for 10 seconds, and then the spray pump is removed from the bottle. 200 μL of mometasone furoate nasal spray is taken by a pipette into a centrifuge tube, 100 μL of sodium acetate buffer (0.1 mol / L, pH 4.8) containing 1.0 mol / L CaCl2 is added, and the mixture is thoroughly mixed. The sample with a total volume of about 300 μL is immediately dispersed onto a glass fiber membrane by a pipette. The membrane is completely dried at room temperature to obtain the sample treated by adding CaCl2 solution.

[0043] The preparation method of the untreated sample is as follows: a packaged product of mometasone furoate nasal spray is taken, the nasal spray bottle is gently shaken for 10 seconds, and then the spray pump is removed from the bottle. 200 μL of mometasone furoate nasal spray is taken by a pipette and dispersed onto a glass fiber membrane. The membrane is completely dried at room temperature to obtain the untreated sample.

[0044] The test sample is placed into a dissolution assembly with the drug facing upward, and the assembly is placed into a dissolution cup after the sealing ring is fastened. The dissolution test is carried out by the paddle method, and the dissolution curve is drawn.

[0045] The dissolution medium is sodium dihydrogen phosphate / sodium hydrogen phosphate buffer (pH 5.8) containing 0.2% (w / v) Tween 80, and the temperature is 37 ± 0.5°C. The paddle rotation speed is 75 rpm. The sampling time points are 7.5, 15, 30, 45, 60, 120, 240, 360, 600, 960 and 1440 minutes, and HPLC is used for sample detection.

[0046] The high performance liquid chromatography conditions are as follows: ChromCore TM C18 chromatographic column (5 μm, 4.6 × 150 mm), acetonitrile: water: acetic acid (55:45:0.2, v / v) as the mobile phase, detection wavelength is 254 nm. The column temperature is 40°C, the flow rate is 1.5 mL / min, and the injection volume is 100 μL.

[0047] The method was linear well in the concentration range of 0.0061 (LOQ) -0.1220 μg / mL, covering the lowest and highest concentration samples in the dissolution process of MF.

[0048] The results are shown in the attached Figure 1 Figures, relative to the untreated sample, both enzymatic treatment and CaCl2 solution treatment can significantly improve the dissolution rate of mometasone furoate in mometasone furoate nasal spray; and the effect of CaCl2 solution treatment is not as good as that of enzymatic treatment, and the RSD of sample dissolution results is large.

[0049] Example 4 In this example, samples with different degrees of enzymatic hydrolysis were used for dissolution test.

[0050] Preparation method of enzymatic sample 9: compared with the mometasone furoate nasal spray in vitro dissolution test sample of example 1, the difference is that the enzymatic conditions are different, and the rest of the conditions are the same. Enzymatic sample 9 was enzymatically hydrolyzed according to enzymatic condition 9 in table 1.

[0051] Similarly, enzymatic sample 12 and enzymatic sample 18 were enzymatically hydrolyzed according to enzymatic conditions 12 and 18 in 1 respectively, and the rest were the same as example 1.

[0052] Enzymatic sample 9, enzymatic sample 12 and enzymatic sample 18 were respectively subjected to dissolution test according to the method of example 3, and the dissolution curve was drawn. The results are shown in the attached Figure 2 Figures.

[0053] The results show that the degree of enzymatic reaction will affect the dissolution rate and the final dissolution amount of mometasone furoate nasal spray API. But when the amount of cellulase added in the enzymatic system reaches a certain limit, the improvement of dissolution rate and final dissolution amount is not obvious.

[0054] Test example 1 This test example investigates the characteristics of mometasone furoate nasal spray samples before and after enzymatic hydrolysis.

[0055] 1. Appearance Enzymatic sample of mometasone furoate nasal spray was prepared by the method of example 1 as follows: mometasone furoate nasal spray finished product, gently shake the nasal spray bottle for 10 seconds, then remove the spray pump from the bottle. Precisely take 200 μL of mometasone furoate nasal spray into a centrifuge tube, add 100 μL of sodium acetate buffer (0.1 mol / L, pH 4.8) containing 1 mg of cellulase, mix well. After enzymatic hydrolysis at 50℃ overnight (about 12 hours), cool the enzymatic mixture to room temperature.

[0056] Compare the appearance of the samples before and after enzymatic hydrolysis, as shown in the attached Figure 3As shown, the mometasone furoate nasal spray after enzymatic digestion showed obvious stratification, while the sample without enzymatic digestion was in a uniform suspension state.

[0057] 2. Scanning electron microscope (SEM) observation The samples before and after enzymatic digestion were observed by scanning electron microscope (SEM), and the results are shown in the following figures. Figure 4 As shown: obvious fine cellulose particles (A) can be seen in the sample without enzymatic digestion, while these fine particles basically disappeared (B) in the sample after enzymatic digestion. Figure 4 Figure 4

[0058] Test Example 2 This test example used three different sample preparation methods to investigate the effect of enzymatic treatment on the rheological properties of the sample.

[0059] (1) Untreated sample: gently shake the nasal spray bottle for 10 seconds, then remove the spray pump from the bottle, and directly measure 2 mL of the liquid.

[0060] (2) Sample without cellulase treatment: gently shake the nasal spray bottle for 10 seconds, then remove the spray pump from the bottle, and transfer 2 mL of the liquid into a centrifuge tube, add 1 mL of sodium acetate buffer (0.1 mol / L, pH 4.8) and mix thoroughly. After incubation at 50°C overnight, cool the mixture to room temperature, and centrifuge at 4000g for 30 seconds. Gently disperse the precipitate with a pipette tip to avoid air bubbles, and measure 2 mL of the liquid.

[0061] (3) Sample treated with cellulase: gently shake the nasal spray bottle for 10 seconds, then remove the spray pump from the bottle, and transfer 2 mL of the liquid into a centrifuge tube, add 1 mL of sodium acetate buffer (0.1 mol / L, pH 4.8) containing 10 mg of cellulase and mix thoroughly. After incubation at 50°C overnight, cool the mixture to room temperature, and centrifuge at 4000g for 30 seconds. Gently disperse the precipitate with a pipette tip to avoid air bubbles, and measure 2 mL of the liquid.

[0062] A controlled stress rheometer was used to measure the rheological properties of the samples. The rheometer was operated at 25°C, using a sandblasted parallel plate with a diameter of 50 mm. 2 mL of the sample was loaded between the parallel plates with a 0.5 mm gap, and the excess sample outside the gap area was removed with a doctor blade. The remaining sample was allowed to equilibrate for 2 minutes, then the shear rate was increased linearly from 1 s -1 to 500 s -1 over 50 seconds, with 10 measurement points. Immediately after, the shear rate was decreased from 500 s -1 to 1 s -1 over 50 seconds, with 10 measurement points. ​​

[0063] The focus of the shear measurement was the effect of enzymatic treatment on the rheological properties such as shear thinning and thixotropy. The results of apparent viscosity as a function of shear rate are shown in Figure 6: Figure 5 As shown in Figure 6, all samples exhibited shear thinning behavior as the shear rate increased, regardless of whether they were subjected to enzymatic treatment. For the two samples that were not subjected to enzymatic treatment (i.e., the untreated sample and the sample without cellulase treatment), the viscosity decreased sharply as the shear rate increased. In contrast, the samples that were subjected to enzymatic treatment exhibited a slight decrease in viscosity at low shear rates, and then remained nearly constant as the shear rate increased, showing behavior similar to a Newtonian fluid.

[0064] The results of the hysteresis loop investigation of the samples are shown in Figure 7: Figure 6 As shown in Figure 7, different hysteresis loops were observed in the samples with different treatments. The area between the ascending and descending curves (the area within the hysteresis loop) was used to evaluate the degree of thixotropy. Compared with the samples that were not subjected to enzymatic treatment, the samples that were subjected to enzymatic treatment did not exhibit thixotropy, and their hysteresis loop areas were negligible.

[0065] Test Example 3 This test example investigated the dissolution profiles of two batches of mometasone furoate nasal spray prepared with different particle sizes of API before and after enzymatic treatment. In this test example, the preparation method of the mometasone furoate nasal spray was carried out according to the literature “A Systematic Approach in the Development of the Morphologically-Directed Raman Spectroscopy Methodology for Characterizing Nasal Suspension Drug Products (G. Farias, J. Shur, R. Price, E. Bielski, B. Newman, AAPS J. 23 (2021) 73, https: / / doi.org / 10.1208 / s12248-021-00605-w)”.

[0066] One batch of API had a particle size D50 of 1.46 um, and the other batch had a D50 of 4.47 um.

[0067] The samples for in vitro dissolution testing of the two batches of mometasone furoate nasal spray after enzymatic treatment were prepared according to the method of Example 1.

[0068] The samples of the two batches of mometasone furoate nasal spray before enzymatic treatment were prepared according to the preparation method of the untreated sample in Example 3.

[0069] Dissolution tests were performed on the above four samples according to the method in Example 3, and dissolution curves were plotted. The results are attached. Figure 7 and attached Figure 8 As shown. (Attached) Figure 7 The similarity factor f2 of the two dissolution curves is 70.4. Figure 8 The similarity factor f2 of the two dissolution curves is 46.5.

[0070] The results showed that, in the dissolution curves of mometasone furoate nasal sprays prepared with APIs of different particle sizes before and after enzymatic hydrolysis, mometasone furoate dissolved slowly in the formulation before enzymatic hydrolysis, and the effect of particle size on the dissolution rate could not be distinguished; mometasone furoate dissolved significantly faster in the formulation after enzymatic hydrolysis, and the dissolution curves of formulations prepared with mometasone furoate of different particle sizes were clearly distinguishable.

[0071] The results of Experiments 1-3 show that the samples before enzymatic hydrolysis behaved as non-Newtonian fluids, while the samples after enzymatic hydrolysis behaved as Newtonian fluids. Enzymatic hydrolysis disrupted the cross-linked structure formed by the excipients in the formulation, causing the formulation to change from a stable suspension state to a layered state. Enzymatic hydrolysis released the API particles from the cross-linked structure formed by the excipients, making the dissolution process of the API particles the rate-limiting step for the release and dissolution of API from the formulation.

[0072] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention used to illustrate the technical solutions of the present invention, and are not intended to limit them, nor are they intended to limit the patent scope of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but which still solve the same technical problem as the present invention, should be included within the protection scope of the present invention; in addition, the direct or indirect application of the technical solutions of the present invention to other related technical fields are similarly included within the patent protection scope of the present invention.

Claims

1. A pretreatment method for samples used in the in vitro dissolution test of mometasone furoate nasal spray, characterized in that, The excipients of the mometasone furoate nasal spray include microcrystalline cellulose and sodium carboxymethyl cellulose; the pretreatment method includes the following steps: Mometasone furoate nasal spray is added to cellulase for enzymatic hydrolysis. The resulting mixture is dispersed on a microporous filter membrane and dried to obtain a test sample that can be used for in vitro dissolution.

2. The pretreatment method according to claim 1, characterized in that, Add 0.3-1 mg of cellulase, preferably 0.3-0.6 mg, and more preferably 0.5 mg, to every 100 μL of mometasone furoate nasal spray.

3. The pretreatment method according to claim 1 or 2, characterized in that, Add cellulase buffer to mometasone furoate nasal spray for enzymatic hydrolysis.

4. The pretreatment method according to claim 3, characterized in that, The cellulase buffer includes a sodium acetate buffer for cellulase. Preferably, the pH of the cellulase buffer is 4.5 to 6.5, more preferably 4.

8.

5. The pretreatment method according to claim 1 or 2, characterized in that, The enzymatic hydrolysis temperature is 37~60℃, preferably 50℃; The enzymatic hydrolysis time is 6-24 hours, preferably 12 hours.

6. The pretreatment method according to claim 1 or 2, characterized in that, The microporous filter membrane includes a glass fiber membrane.

7. The pretreatment method according to claim 1 or 2, characterized in that, The drying process is carried out at room temperature.

8. The pretreatment method according to any one of claims 1 to 7, characterized in that, Transfer mometasone furoate nasal spray into a centrifuge tube, add cellulase buffer for enzymatic hydrolysis, cool the hydrolysate to room temperature, centrifuge, disperse the precipitate, and use a pipette to disperse all the samples in the centrifuge tube onto a microporous filter membrane. Preferably, 100-300 μL of mometasone furoate nasal spray is transferred into a centrifuge tube; Preferably, the centrifugation conditions are: centrifugation at 2000~6000g speed for 10~60 seconds; more preferably, centrifugation at 4000g speed for 30 seconds; Preferably, the precipitate is gently dispersed with a pipette tip to avoid generating air bubbles, and then all the samples in the centrifuge tube are dispersed onto the microporous filter membrane using a pipette.

9. A sample for in vitro dissolution testing of mometasone furoate nasal spray prepared by the pretreatment method according to any one of claims 1-8.

10. A method for detecting the in vitro dissolution of mometasone furoate nasal spray, characterized in that, In vitro dissolution testing was performed using the in vitro dissolution test sample as described in claim 9; The in vitro dissolution test may include the following methods: placing the in vitro dissolution test sample into the dissolution assembly with the drug side facing up, tightening the sealing ring of the assembly, and then placing it into the dissolution cup; using the paddle disc method to perform the dissolution test and plotting the dissolution curve. The preferred dissolution medium is a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution (pH 5.8) containing 0.2% (w / v) Tween 80, at a temperature of 37±0.5℃ and a paddle speed of 75 rpm. The preferred sampling times for the dissolution test are 7.5, 15, 30, 45, 60, 120, 240, 360, 600, 960, and 1440 minutes, with HPLC injection detection. The preferred HPLC chromatographic conditions are: ChromCore™ C18 column (5 μm, 4.6 × 150 mm), mobile phase of acetonitrile:water:acetic acid (55:45:0.2, v / v), detection wavelength of 254 nm, column temperature of 40 ℃, flow rate of 1.5 mL / min, and injection volume of 100 μL.

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