In-vitro osmotic analysis method of lidocaine hydrochloride gel

Abstract

The invention relates to an in-vitro permeability analysis method of lidocaine hydrochloride gel, which relates to the technical field of medicine, and comprises the following steps: placing a transdermal material with a gasket on a receiving pool of a transdermal instrument, taking a lidocaine hydrochloride gel test sample to be detected, smearing the gasket with the lidocaine hydrochloride gel test sample, and operating according to a method set by the transdermal instrument, receiving liquid in the receiving pool is taken at different time for detection; the transdermal material is a pig bladder mucous membrane, the thickness of the transdermal material is 0.25-0.80 mm, and the receiving medium is a PBS (Phosphate Buffer Solution) with the pH value of 7.4. The method can accurately evaluate the rate and the degree of the lidocaine hydrochloride gel reaching the skin action part or the vicinity, is beneficial to evaluation of the transdermal permeation behavior of the medicine, simulation of the transdermal process of the medicine under physiological conditions, control of the quality of the lidocaine hydrochloride gel, evaluation of the preparation performance and improvement of the safety of the product. The method can quickly evaluate the drug release and absorption characteristics, and is low in cost, short in period, green and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an in vitro permeation analysis method of lidocaine hydrochloride gel. Background Art

[0002] Lidocaine is a local anesthetic and antiarrhythmic drug. It is a derivative of cocaine, but lacks the hallucinogenic and addictive properties of cocaine. Lidocaine hydrochloride is a white crystalline powder that is very slightly soluble in water. Its toxicity is comparable to that of procaine, but its local anesthetic effect is stronger and longer-lasting, with good surface penetration. Lidocaine hydrochloride gel is a colorless, viscous liquid with a pH of 6.0-7.0. It is primarily indicated for transurethral examinations and treatments requiring local anesthesia. It can also be used for mucosal anesthesia during thoracoscopy or abdominal surgery, effectively relieving discomfort and pain associated with the examination or surgery. When used as a urethral anesthetic, lidocaine hydrochloride gel takes effect within 5-10 minutes.

[0003] In Vitro Release Test and In Vitro Permeation Test (IVPT) are important contents of in vitro critical quality attribute studies of generic drugs such as ointments, creams, and gels. Among them, IVPT is used to evaluate the rate and extent of drug reaching the site of action on or near the skin, or to evaluate the behavior of drugs penetrating through the skin and entering the body, simulating the transdermal process of drugs under physiological conditions. IVPT can also characterize and evaluate the bioavailability and safe exposure of drugs in generic drugs and reference preparations. When establishing the IVPT method, the acceptance criteria for the skin model should be established, and the receiving medium, method parameters, etc. should be screened and optimized.

[0004] CN114397411A discloses a method for evaluating a leprocaine cream sample, using an initial transdermal test and a repeated transdermal test. The same transdermal material, pigskin, is used for both the initial and repeated transdermal tests. By repeatedly cross-using the pigskin, the in vitro transdermal test of leprocaine cream is performed on the same pigskin using a self-developed sample and a reference preparation, eliminating differences introduced by different pigskins and reducing the impact of these differences on the in vitro transdermal test of leprocaine cream. However, lidocaine hydrochloride gel is administered intraurethrally, and the transdermal materials disclosed in the prior art often use pigskin. Pigskin has significant physiological and anatomical differences from the urethral mucosa, making it unsuitable as a transdermal material for lidocaine hydrochloride. Furthermore, the surface area of ​​the pig urethral mucosa is too small to be suitable for IVPT experiments. Currently, no reports exist on methods for in vitro permeation testing of lidocaine hydrochloride gel.

[0005] Therefore, it is necessary to develop an in vitro permeation analysis method for lidocaine hydrochloride gel that is simple, accurate, and can simulate the actual permeation conditions of the human body. Summary of the Invention

[0006] The object of the present invention is to provide an in vitro permeation analysis method for lidocaine hydrochloride gel. The method is simple and highly accurate, and solves the problem that there is no in vitro permeation analysis method for lidocaine hydrochloride gel and no suitable transdermal material.

[0007] The technical solutions provided by the present invention are as follows:

[0008] The invention provides an in vitro permeation analysis method of lidocaine hydrochloride gel, comprising the following steps: placing a transdermal material with a gasket on a receiving pool of a transdermal instrument, smearing a lidocaine hydrochloride gel sample to be tested on the gasket, operating the transdermal instrument according to a method set, and taking receiving liquid from the receiving pool at different times for detection.

[0009] As an embodiment of the present invention, the transdermal instrument is a vertical diffusion cell.

[0010] As one embodiment of the present invention, the transdermal material is porcine bladder mucosa or a biomimetic membrane, preferably porcine bladder mucosa. Lidocaine hydrochloride gel is administered through the human urethra. Porcine bladder mucosa is the most similar to porcine urethral mucosa in physiological and anatomical structure, so porcine bladder mucosa is selected as the IVPT membrane.

[0011] As an embodiment of the present invention, the thickness of the pig bladder mucosa is 0.25 mm to 0.80 mm.

[0012] As an embodiment of the present invention, the thickness of the pig bladder mucosa is 0.25 mm to 0.5 mm, preferably 0.3 mm to 0.45 mm, and preferably 0.375 mm.

[0013] As an embodiment of the present invention, the receiving pool contains a receiving medium, which is one of pH 4.5 PBS buffer, pH 5.8 PBS buffer, pH 6.4 PBS buffer, pH 6.8 PBS buffer, pH 7.4 PBS buffer, and normal saline, preferably pH 7.4 PBS buffer.

[0014] As an embodiment of the present invention, the temperature of the transdermal instrument is 37°C.

[0015] As an embodiment of the present invention, the rotation speed of the transdermal instrument is 200-750 rpm, preferably 600 rpm.

[0016] As an embodiment of the present invention, the detection is carried out by high performance liquid chromatography, and the chromatographic column of the high performance liquid chromatography is one of a C18 column, a C8 column, and a C4 column, preferably a C18 column, and more preferably a Yuexu UltimatePlus-C18 4.6mm×50mm, 5μm.

[0017] As an embodiment of the present invention, the mobile phase A of the HPLC method is one of formic acid, trifluoroacetic acid, and phosphoric acid aqueous solution, preferably formic acid aqueous solution, more preferably 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0018] As an embodiment of the present invention, the elution procedure of the high performance liquid chromatography is as follows:

[0019] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 81 19 2.0 57 43 2.1 81 19 4.0 81 19

[0020] Compared with the existing technical solutions, the present invention has the following beneficial effects:

[0021] The in vitro permeation method of the present invention uses a vertical diffusion cell, porcine bladder mucosa as the transdermal material, and pH 7.4 PBS buffer as the receiving medium. This method accurately assesses the rate and extent of lidocaine hydrochloride gel reaching or near the skin's site of action, facilitating the evaluation of drug transdermal permeation behavior and simulating the transdermal process of drugs under physiological conditions. This facilitates the control of lidocaine hydrochloride gel's quality, the evaluation of formulation performance, and the improvement of product safety. The present invention can rapidly assess drug release and absorption characteristics, is low-cost, has a short cycle time, and is environmentally friendly. This simple method enables high-precision evaluation of lidocaine hydrochloride gel quality differences through in vitro transdermal evaluation, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the cumulative permeation curve of the drug in the membrane screening test;

[0023] Figure 2 is the cumulative permeation distribution curve of the membrane screening test;

[0024] Figure 3 is the flux distribution curve of the membrane screening test;

[0025] Figure 4 is the cumulative permeation distribution curve of the mucosal thickness screening experiment;

[0026] Figure 5 is the flux distribution curve of the mucosal thickness screening experiment;

[0027] Figure 6 is the cumulative permeation distribution curve of the sensitivity test;

[0028] Figure 7 is the sensitivity test flux distribution curve;

[0029] Figure 8 is the cumulative permeation distribution curve for the selectivity test;

[0030] Figure 9 is the selectivity test flux distribution curve;

[0031] Figure 10 This is the flux distribution curve of three batches of self-made lidocaine hydrochloride gel;

[0032] Figure 11 This is the cumulative permeation distribution curve of three batches of self-developed lidocaine hydrochloride gel;

[0033] Figure 12 is the cumulative penetration distribution curve of the durability test;

[0034] Figure 13 is the flux distribution curve of the durability test;

[0035] Figure 14 is a permeation rate graph of Example 1;

[0036] Figure 15 is a graph of the degree of penetration of Example 1;

[0037] Figure 16 is a permeation rate graph of Example 2;

[0038] Figure 17 is a graph of the degree of penetration of Example 2;

[0039] Figure 18 is a permeation rate graph of Example 3;

[0040] Figure 19 is a graph of the degree of penetration of Example 3;

[0041] Figure 20 is a permeation rate graph of Example 4;

[0042] Figure 21 is a graph of the degree of penetration of Example 4;

[0043] Figure 22 is a permeation rate graph of Comparative Example 1;

[0044] Figure 23 is a graph of the degree of penetration of comparative example 1;

[0045] Figure 24 is a permeation rate graph of Comparative Example 2;

[0046] Figure 25 This is a graph of the penetration degree of comparative example 2. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0048] The raw materials, reagents, materials, and equipment used in the examples can all be purchased through commercial channels; among them, the test sample is 2% lidocaine hydrochloride gel, which is sourced from Nanjing Hanxin Pharmaceutical Technology Co., Ltd.; the reference preparation is 2% lidocaine hydrochloride gel, which is sourced from Aspen Pharma Trading Limited; and the reference substance is lidocaine hydrochloride, which is sourced from the China National Institute for Food and Drug Control.

[0049] Transdermal material processing: pig bladder was obtained, the mucosal layer and smooth muscle layer were carefully separated, the mucosal layer was taken and cut into circular pieces with a diameter of 25 mm for IVPT experiments.

[0050] Membrane thickness measurement: Take the cut circular pig bladder mucosa and measure the mucosal thickness with a thickness gauge. Each piece of mucosa is measured once at three different positions. The thickness is between 0.25mm and 0.80mm.

[0051] After the treated transdermal material is inspected for membrane thickness and passed, a gasket is placed on each piece of porcine bladder mucosa. This gasket is then placed on the receiving reservoir of the transdermal instrument and secured. The receiving reservoir is filled with the receiving medium and maintained at a constant temperature of 37°C. Approximately 300 mg of lidocaine hydrochloride gel is weighed into a 1 mL syringe and the total weight, m1, is accurately determined. The gel is transferred to the gasket and the syringe is weighed back, m2. The weight of the test sample is calculated using the weight loss method (m1 - m2). The transdermal instrument is run according to the configured protocol, and samples are collected at various time points.

[0052] Diffusion cell parameters (IVPT parameters):

[0053] The transdermal instrument in this application is a vertical diffusion cell, using Logan SYSTEM918-12.

[0054] Stirring speed: 600 rpm / min;

[0055] Diffusion cell temperature: 37°C, diffusion cell volume: 12 mL, lubrication volume: 3 mL, filling volume: 2 mL

[0056] Collection volume: 0.8 mL, needle descent: 24.0 mm, rehydration volume: 18.0 mL, sampling hold time: 10 s

[0057] Pre-action time: 30s;

[0058] Sampling time points: 5min, 15min, 30min, 45min, 60min, 75min, 90min, 120min, 150min, 180min, 240min, 300min, 360min. (Note: Each sampling takes 5 minutes to completely remove the medium from the diffusion cell and then refill it with the receiving medium. Therefore, starting from the second sampling point, 5 minutes of sampling time is added to each sampling point. Therefore, the automatic sampling time points set by the instrument are converted to 5min, 20min, 40min, 60min, 80min, 100min, 120min, 155min, 190min, 225min, 290min, 355min, 420min.)

[0059] Chromatographic conditions:

[0060] Mode: HPLC / UPLC

[0061] Chromatographic column: Yuexu Ultimate Plus-C18 4.6mm×50mm, 5μm, Part Number: 00B-4605-E0

[0062] Mobile phase A: 0.1% formic acid in water

[0063] Mobile phase B: acetonitrile

[0064] Detection wavelength: 210nm

[0065] Flow rate: 0.8 mL / min

[0066] Injection volume: 10 μL

[0067] Column temperature: 30°C

[0068] Elution gradient:

[0069] Table 1 Elution gradient

[0070] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 81 19 2.0 57 43 2.1 81 19 4.0 81 19

[0071] Membrane surface temperature:

[0072] This product is intended for urethral administration and is a topical preparation for use on the urethral mucosal surface. The urethra is an internal cavity of the human body with a surface temperature of approximately 37°C. Therefore, 37°C was selected as the membrane surface temperature for in vitro permeation. The stability of lidocaine hydrochloride solutions in a 40°C receiving medium was investigated. Lidocaine hydrochloride solutions in a 40°C receiving medium showed good stability. Therefore, a membrane surface temperature of 37°C was selected.

[0073] Membrane type screening

[0074] Membrane 1 (Bionic Membrane): This product is administered urethrally, and the urethra is too small to allow for permeation testing. The biomimetic membrane is an artificial cellulose-phospholipid biomimetic membrane with a sandwich-like layered structure. Developed by Permeapad (a Logan partner), it mimics the human intestinal and oral mucosa, useful for predicting buccal absorption effectiveness. It can also be used in various testing devices, such as Franz diffusion cells or the Logan Permetro system, to test the osmotic absorption of drug formulations. Therefore, the Permeapad biomimetic membrane was selected as the IVPT membrane.

[0075] Membrane 2 (porcine bladder mucosa): This product is administered through the urethra. The human urethra wall is composed of a mucosal layer, a submucosa layer, and a muscle layer. The submucosa layer is richly supplied with blood and is mainly composed of connective tissue. Lidocaine hydrochloride must pass through the mucosal layer to reach the submucosa layer. The tissue of the bladder wall is divided into three layers: the mucosal layer, the smooth muscle layer, and the adventitia layer (formed by connective tissue). It is closer to the urethral wall in terms of tissue morphology. Since the surface area of ​​the porcine urethral mucosa is too small, it is difficult to use in IVPT experiments. The porcine bladder mucosa is closest to the porcine urethral mucosa in terms of physiological and anatomical tissue structure, so the porcine bladder mucosa was selected as the IVPT membrane. Pretreatment of porcine bladder mucosa: Take the porcine bladder, carefully separate the mucosal layer and the smooth muscle layer, take the mucosal layer, and cut it into circular pieces with a diameter of 25 mm for IVPT experiments.

[0076] Take the circular Permeapad biomimetic membrane and porcine bladder mucosa cut into 25mm diameter, and measure the thickness of the porcine bladder mucosa with a thickness gauge. Each piece of mucosa is measured once at three different positions. The thickness should be between 0.25mm and 0.80mm. The Permeapad biomimetic membrane with a diameter of 25mm and qualified thickness and the porcine bladder mucosa are respectively installed in the diffusion cell so that the inner surface of the membrane faces upward and the outer side of the mucosa is in contact with the receiving medium in the diffusion cell (PBS buffer with pH 7.4 is selected as the receiving medium). Then the surface temperature of the mucosa is balanced to 37℃±1℃, and the transepidermal water loss of the mucosa is measured with a transepidermal water loss meter. The measurement is repeated three times for each piece of mucosa. The drug cumulative permeation curves of the Permeapad biomimetic membrane and porcine bladder mucosa are shown in Figure 1 , the cumulative permeability distribution curve is shown in Figure 2 , the flux distribution curve is shown in Figure 3 .

[0077] Conclusion: The permeation volume and permeation rate of the biomimetic membrane are lower than those of porcine bladder mucosa. The experimental results conducted on this synthetic membrane cannot reflect the rate and extent of drug penetration into and through the skin and may be misleading. Therefore, porcine bladder mucosa was selected as the experimental membrane for IVPT.

[0078] Film thickness screening

[0079] Different thicknesses of pig bladder mucosa have a significant impact on IVPT results, so the thickness of pig bladder mucosa needs to be screened. During screening, the thickness of thin mucosa is 0.10-0.24mm, the thickness of medium mucosa is 0.25-0.8mm, and the thickness of thick mucosa is 0.81-1.2mm. The other parameters of the screening test are consistent with the membrane type screening. The cumulative permeation distribution curves of the three different thicknesses of mucosa are shown in Figure 4 , the flux distribution curve is shown in Figure 5 .

[0080] Conclusion: Thinner porcine bladder mucosa has high drug permeation, but the membrane is easily damaged, leading to experimental failure. Medium-thick porcine bladder mucosa has moderate drug permeation and is less prone to damage, making it an ideal mucosa for experimental use. Excessively thick porcine bladder mucosa has low permeation and cannot reflect the true drug permeation situation. Therefore, porcine bladder mucosa of moderate thickness should be selected for IVPT experiments. The optimal thickness of porcine bladder mucosa is 0.25-0.8 mm.

[0081] IVPT Methodology Validation

[0082] Discrimination test

[0083] Sensitivity results

[0084] The sensitivity of the method was investigated by examining different sample loading amounts (30 mg, 100 mg, and 300 mg). When the sample loading amount was 30 mg, the flux distribution curve was the lowest; when the sample loading amount was 100 mg, the flux distribution curve was in the middle; and when the sample loading amount was 300 mg, the flux distribution curve was the highest. As the sample loading amount increased or decreased, the IVPT method could give higher or lower flux distribution curves and significantly different cumulative permeation curves, so it is believed that the IVPT method is sensitive. The cumulative permeation distribution and flux distribution curves for different sample loading amounts are shown in Figure 2. Figure 6 and Figure 7 , data are shown in Tables 2 to 4.

[0085] Table 2 Sensitivity test cumulative penetration data-1

[0086]

[0087]

[0088] Table 3 Sensitivity test flux data-2

[0089]

[0090] Table 4 Sensitivity test flux data-3

[0091]

[0092] Conclusion: According to Figure 7As can be seen, the flux distribution curve is lowest when the sample load is 30 mg; it is intermediate when the sample load is 100 mg; and it reaches its highest point when the sample load is 300 mg. The flux distribution curve and cumulative permeation curves of this IVPT method show clear differences with increasing or decreasing sample load, thus demonstrating that this IVPT method is sensitive and discriminatory.

[0093] Selective results

[0094] In order to study whether this IVPT method has the ability to distinguish lidocaine hydrochloride topical preparations with different prescriptions, a reference preparation, lidocaine hydrochloride cream, was prepared. The selectivity of the method was investigated by comparing the test product (lidocaine hydrochloride gel) with lidocaine hydrochloride cream. The flux distribution curve and cumulative permeation distribution curve of this product and the preparation with the changed formula (lidocaine hydrochloride cream) were significantly different, so it is believed that the IVPT method has good selectivity. The cumulative permeation distribution and flux distribution curves of lidocaine hydrochloride topical preparations with different prescriptions are shown in Figure 2. Figure 8 and Figure 9 , data are shown in Tables 5 to 9.

[0095] Table 5 Selectivity test cumulative permeation data-1

[0096]

[0097] Table 6 Selectivity test cumulative permeation data-2

[0098]

[0099]

[0100] Table 7 Selectivity test flux data-1

[0101]

[0102] Table 8 Selectivity test flux data-2

[0103]

[0104] Table 9 Summary of selective test data

[0105]

[0106]

[0107] Conclusion: There are significant differences in the cumulative permeation distribution curve and flux distribution curve between the reference preparation (lidocaine hydrochloride cream) and the test preparation (lidocaine hydrochloride gel). Figure 8 and Figure 9Therefore, the IVPT method has good selectivity and discrimination for preparations with different formulas.

[0108] Precision test

[0109] The intra-batch precision of the method was investigated by testing the same batch of lidocaine hydrochloride gel 36 times in parallel. The inter-batch precision of the method was investigated by testing three batches of lidocaine hydrochloride gel 12 times each (12 porcine mucosal samples per batch, for a total of 36 porcine mucosal samples).

[0110] The same batch of lidocaine hydrochloride gel preparations was tested 36 times (a total of 36 porcine mucosal samples). The coefficients of variation for flux (n=36) and cumulative permeation per unit area (n=36) at the first two time points (5 minutes and 15 minutes) ranged from 23.4% to 28.5%, meeting the requirement (no more than 30%). The coefficients of variation for flux (n=36) and cumulative permeation per unit area (n=36) at all subsequent time points ranged from 11.3% to 16.9%, meeting the requirement (no more than 20%).

[0111] Three batches of lidocaine hydrochloride gel preparations were tested 12 times (12 porcine mucosal samples per batch, for a total of 36 porcine mucosal samples). The coefficients of variation for flux (n=36) and cumulative permeation per unit area (n=36) at the first two time points (5 minutes and 15 minutes) ranged from 31.6% to 35.4%, meeting the requirement (no more than 40%). The coefficients of variation for flux (n=36) and cumulative permeation per unit area (n=36) at all subsequent time points ranged from 12.4% to 18.4%, meeting the requirement (no more than 30%).

[0112] The data are summarized in Tables 10 to 14.

[0113] Table 10 Intra-batch precision test data-1

[0114]

[0115]

[0116] Table 10 Intra-batch precision test data-1 (continued from the previous page)

[0117]

[0118] Table 10 Intra-batch precision test data-1 (continued from the previous page)

[0119]

[0120] Table 11 Intra-batch precision test data-2

[0121]

[0122]

[0123] Table 11 Intra-batch precision test data-2 (continued from the previous page)

[0124]

[0125] Table 11 Intra-batch precision test data-2 (continued from the previous page)

[0126]

[0127]

[0128] Table 12 Inter-batch precision test data-1

[0129]

[0130]

[0131] Table 12 Inter-batch precision test data-1 (continued from the previous page)

[0132]

[0133] Table 12 Inter-batch precision test data-1 (continued from the previous page)

[0134]

[0135]

[0136] Table 13 Inter-batch precision test data-2

[0137]

[0138] Table 13 Inter-batch precision test data-2 (continued from the previous page)

[0139]

[0140]

[0141] Table 13 Inter-batch precision test data-2 (continued from the previous page)

[0142]

[0143]

[0144] Table 14 Summary of precision data

[0145]

[0146] Note: The data of intra-batch precision cumulative permeation per unit area (n=36) are shown in Table 10, the data of intra-batch precision flux (drug permeation rate) (n=36) are shown in Table 11, the data of inter-batch precision cumulative permeation per unit area (n=36) are shown in Table 12, and the data of inter-batch precision flux (drug permeation rate) (n=36) are shown in Table 13.

[0147] Conclusion: The intra-batch precision of the method was investigated by testing the same batch of lidocaine hydrochloride gel 36 times in parallel. The inter-batch precision of the method was investigated by testing three batches of lidocaine hydrochloride gel 12 times each.

[0148] The same batch of self-developed preparations was tested 36 times (a total of 36 porcine mucosal samples). The coefficients of variation for the flux (n=36) and cumulative permeation per unit area (n=36) at the first two time points (5 minutes and 15 minutes) ranged from 23.4% to 28.5%, meeting the requirement (no more than 30%). The coefficients of variation for the flux (n=36) and cumulative permeation per unit area (n=36) at all subsequent time points ranged from 11.3% to 16.9%, meeting the requirement (no more than 20%).

[0149] Three batches of the self-developed preparation were tested 12 times (12 porcine mucosal samples per batch, for a total of 36 porcine mucosal samples). The coefficients of variation for the flux (n=36) and cumulative permeation per unit area (n=36) at the first two time points (5 minutes and 15 minutes) ranged from 31.6% to 35.4%, meeting the requirement (no more than 40%). The coefficients of variation for the flux (n=36) and cumulative permeation per unit area (n=36) at all subsequent time points ranged from 12.4% to 18.4%, meeting the requirement (no more than 30%).

[0150] Therefore, the precision of the method is good.

[0151] Penetration curve and range

[0152] During the entire IVPT study period, flux distribution curves and cumulative permeation distribution curves were drawn for three batches of self-made lidocaine hydrochloride gel (each batch of self-made formulation was tested 36 times, and the average of 36 data was taken). The flux distribution and cumulative permeation distribution curves of the three batches of self-made lidocaine hydrochloride gel are shown in Figures 10 and 11 .

[0153] Conclusion: The trends of the cumulative permeation distribution curves are generally consistent. The flux distribution curves can identify the maximum flux and the subsequent flux decline at multiple time points. The permeation curves and ranges meet the requirements.

[0154] Dose consumption

[0155] Throughout the IVPT study period, the recovery of drug permeated into the receiving fluid per diffusion cell unit was characterized as the "total cumulative permeation of drug into the receiving fluid," expressed as a percentage of the labeled amount of drug in the sample. Three batches of the homemade lidocaine hydrochloride gel preparation were tested 12 times (on 12 porcine mucosal samples). The cumulative drug permeation percentages ranged from 84.6% to 87.5%, meeting the requirement (minimum 80%). The cumulative permeation percentage data for the three batches of the homemade lidocaine hydrochloride gel preparation are shown in Table 15.

[0156] Table 15 Cumulative penetration percentage data summary-1

[0157]

[0158] Conclusion: During the entire IVPT study period, three batches of self-developed preparations were tested 12 times (12 porcine mucosal samples per batch), and the average cumulative drug penetration percentage was between 84.6% and 87.5%, which met the requirements (not less than 80%).

[0159] Durability

[0160] After adjusting the stirring rate ±10% and the receiving medium temperature ±1°C, the ratio of the total cumulative drug permeation AMT to that under normal conditions was between 98.0% and 101.4%, and the maximum drug flux J max The ratios to those under normal conditions are 65.2% to 146.0%, which is within the range of 50% to 150%. Therefore, it can be considered that the IVPT method is durable to changes in stirring rate and receiving medium temperature. The cumulative permeation distribution and flux distribution curves of the durability test are shown in Figure 12 and Figure 13 , durability data are shown in Table 16 to Table 17.

[0161] Table 16 Durability data summary

[0162]

[0163]

[0164] Table 17 Durability data summary

[0165]

[0166] Conclusion: After adjusting the stirring rate ±10% and the receiving medium temperature ±1℃, the total cumulative permeation amount of the drug AMT was 98.0% to 101.4% of that under normal conditions, and the maximum drug flux J max The ratios to those under normal conditions ranged from 65.2% to 146.0%, meeting the requirements (within the range of 50% to 150%). Therefore, it can be considered that the IVPT method is robust to changes in stirring rate and receiving medium temperature.

[0167] in conclusion

[0168] This IVPT analytical method validation study demonstrated that the method's discrimination (sensitivity, selectivity), precision (intra-batch precision, inter-batch precision), permeation curve and range, dose consumption, and durability met acceptable standards.

[0169] Example 1

[0170] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa has a thickness of 0.375mm.

[0171] IVRT parameters and chromatographic conditions were as described previously.

[0172] After the membrane thickness inspection is qualified, place a gasket on each piece of pig bladder mucosa, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe back to m2. Use the weight loss method to calculate the weight of the test sample (m1-m2). Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium, and calculate the cumulative drug penetration per unit area.

[0173]

[0174] Q n is the cumulative permeation per unit area at time n (μg / cm 2 );

[0175] C n is the drug concentration in the receptor medium at sampling time n (μg / mL);

[0176] V is the sample volume (12 ml);

[0177] A is the effective diffusion area of ​​the diffusion cell (3.14×(0.75cm) 2 =1.77cm 2 ).

[0178] The drug permeation rate was expressed as flux (J), while the drug permeation extent was expressed as the total cumulative amount of drug permeated into the receiver fluid during the entire study period.

[0179] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 14 ;

[0180] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 15 .

[0181] Example 2

[0182] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa has a thickness of 0.25mm.

[0183] The IVRT parameters included a diffusion cell temperature of 37° C. and a stirring speed of 750 rpm / min. Other IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0184] After the membrane thickness inspection is qualified, place a gasket on each piece of pig bladder mucosa, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe back to m2. Use the weight loss method to calculate the weight of the test sample (m1-m2). Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium, and calculate the cumulative drug penetration per unit area.

[0185] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 16 ;

[0186] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 17 .

[0187] Example 3

[0188] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa has a thickness of 0.80mm.

[0189] The IVRT parameters included a diffusion cell temperature of 37° C. and a stirring speed of 200 rpm / min. Other IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0190] After the membrane thickness inspection is qualified, place a gasket on each piece of pig bladder mucosa, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe back to m2. Use the weight loss method to calculate the weight of the test sample (m1-m2). Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium, and calculate the cumulative drug penetration per unit area.

[0191] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 18 ;

[0192] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 19 .

[0193] Example 4

[0194] Filter membrane processing: Take the cut biomimetic membrane, the thickness of each piece of membrane is 0.45mm.

[0195] The IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0196] After the membrane thickness inspection is qualified, place a gasket on each biomimetic membrane, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe weight m2. Calculate the weight of the test sample (m1-m2) by the weight loss method. Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium and calculate the cumulative drug penetration per unit area.

[0197] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 20 ;

[0198] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 21 .

[0199] Comparative Example 1

[0200] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa is 1mm thick.

[0201] The IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0202] After the membrane thickness inspection is qualified, place a gasket on each piece of pig bladder mucosa, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe back to m2. Use the weight loss method to calculate the weight of the test sample (m1-m2). Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium, and calculate the cumulative drug penetration per unit area.

[0203] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 22 ;

[0204] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 23 .

[0205] Comparative Example 2

[0206] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa has a thickness of 0.375mm.

[0207] The IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0208] After the membrane thickness inspection is qualified, place a gasket on each piece of pig bladder mucosa, place it on the receiving pool of the transdermal instrument, and fix it. Fill the receiving pool with the receiving medium and keep the temperature at 37°C. Weigh 300 mg of lidocaine hydrochloride cream and place it in a 1 mL syringe. Accurately weigh the total weight m1, transfer the gel to the gasket, and weigh the syringe back to m2. Use the weight loss method to calculate the weight of the test sample (m1-m2). Operate according to the set transdermal instrument method and take samples at different time points. Use HPLC to determine the drug concentration of lidocaine hydrochloride in the receiving medium, and calculate the cumulative drug penetration per unit area.

[0209] Plot the drug permeation rate: plot time on the X-axis and flux (drug permeation rate) on the Y-axis. Figure 24 ;

[0210] Plot the drug penetration rate: plot the time as the X-axis and the cumulative penetration rate per unit area as the Y-axis. Figure 25 .

[0211] Comparative Example 3

[0212] Filter membrane processing: Take the cut circular pig bladder mucosa, each piece of mucosa has a thickness of 0.1mm.

[0213] The IVRT parameters included a diffusion cell temperature of 37° C. and a stirring speed of 750 rpm / min. Other IVRT parameters and chromatographic conditions were consistent with those in Example 1.

[0214] After the membrane thickness inspection is passed, place a gasket on each piece of porcine bladder mucosa, place it on the receiving cell of the transdermal instrument, and secure it. Fill the receiving cell with receiving medium and maintain a constant temperature of 37°C. Weigh 300 mg of lidocaine hydrochloride gel (test sample) into a 1 mL syringe and accurately weigh the total weight m1. Transfer the gel to the gasket and weigh the syringe back to m2. Calculate the test sample weight (m1 - m2) using the weight loss method. Run the transdermal instrument according to the set method, and collect samples at different time points. Determine the lidocaine hydrochloride concentration in the receiving medium using HPLC, and calculate the cumulative drug permeation per unit area.

[0215] The mucosa is easily damaged during the test and is not suitable for continuing the test.

Claims

1. An in vitro permeation analysis method for lidocaine hydrochloride gel, characterized in that: The steps include: Place the transdermal material with the gasket on the receiving pool of the transdermal instrument, take the lidocaine hydrochloride gel test sample to be tested and smear it on the gasket, operate according to the method set by the transdermal instrument, and take the receiving liquid in the receiving pool for detection at different times.

2. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 1, characterized in that: The transdermal instrument is a vertical diffusion cell.

3. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 1, characterized in that: The transdermal material is porcine bladder mucosa or a biomimetic membrane, preferably porcine bladder mucosa.

4. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 3, characterized in that: The thickness of the pig bladder mucosa is 0.25 mm to 0.80 mm.

5. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 4, characterized in that: The thickness of the pig bladder mucosa is 0.25 mm to 0.5 mm, preferably 0.3 mm to 0.45 mm, and preferably 0.375 mm.

6. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 1, characterized in that: The receiving pool contains a receiving medium, which is one of pH 4.5 PBS buffer, pH 5.8 PBS buffer, pH 6.4 PBS buffer, pH 6.8 PBS buffer, pH 7.4 PBS buffer, and normal saline, preferably pH 7.4 PBS buffer.

7. The in vitro permeation analysis method of lidocaine hydrochloride gel according to any one of claims 1 to 6, characterized in that: The temperature of the transdermal instrument was 37°C.

8. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 7, characterized in that: The rotation speed of the transdermal instrument is 200-750 rpm, preferably 600 rpm.

9. The in vitro permeation analysis method of lidocaine hydrochloride gel according to any one of claims 1 to 8, characterized in that: The detection is performed by high performance liquid chromatography, and the chromatographic column of the high performance liquid chromatography is one of a C18 column, a C8 column, and a C4 column, preferably a C18 column, and more preferably a Yuexu Ultimate Plus-C18 4.6mm×50mm, 5μm.

10. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 9, characterized in that: The mobile phase A of the high performance liquid chromatography method is one of formic acid, trifluoroacetic acid, and phosphoric acid aqueous solution, preferably formic acid aqueous solution, more preferably 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile.

11. The in vitro permeation analysis method of lidocaine hydrochloride gel according to claim 10, characterized in that: The elution procedure of the HPLC method is as follows: 。

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