In vitro evaluation method of calcitriol soft capsule content and application thereof
By simulating the dissolution and permeation process of calcitriol soft capsules in the gastrointestinal tract using a diffusion cell device and high-performance liquid chromatography, the problem of in vitro evaluation in existing technologies is solved, achieving simple and accurate in vitro evaluation, which is suitable for prescription screening and process optimization.
Patent Information
- Application Number
- CN202610388382.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies lack effective in vitro evaluation methods to simulate the dissolution and permeation process of calcitriol soft capsules in vivo, making it difficult to accurately predict their in vivo behavior and increasing research and development costs and risks.
A diffusion cell device was used to simulate the gastrointestinal environment. Alcohol and acidic media solutions were used in conjunction with a filter membrane. The release curve of calcitriol was determined by high performance liquid chromatography to simulate the dissolution and initial penetration process of the drug in the gastrointestinal tract.
This provides a simple and accurate in vitro evaluation method that can directly reflect the drug release process from the formulation carrier, has good correlation, and is suitable for formulation screening, process optimization, and generic drug consistency evaluation.
Smart Images
Figure CN122108854A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for evaluating the in vitro dissolution and permeation behavior of calcitriol soft capsule contents and its application. Background Technology
[0002] Calcitriol is the most bioactive metabolite of vitamin D3 and is widely used clinically to treat postmenopausal and senile osteoporosis, renal osteodystrophy, hypoparathyroidism, and rickets. Calcitriol soft capsules are a common oral formulation, consisting of a capsule and oily contents. The clinical efficacy of a drug depends not only on its content but also on its dissolution and absorption rates in vivo. For poorly soluble drugs like calcitriol, the rate at which it is released from the oily contents of the soft capsule and permeates the biomembrane is a key factor affecting its bioavailability and efficacy. However, current quality control of calcitriol soft capsules focuses primarily on content determination and routine dissolution testing, lacking in vitro evaluation methods that can effectively simulate its in vivo dissolution-permeation process. This makes it difficult to accurately predict the in vivo behavior of the product through in vitro methods during formulation development, process optimization, and generic drug consistency evaluation, increasing research and development costs and risks.
[0003] Therefore, developing a method that can accurately and reliably evaluate the in vitro release behavior of calcitriol soft capsule contents is of great significance for ensuring drug quality, guiding formulation process development, and conducting effective generic drug consistency evaluation. Summary of the Invention
[0004] This invention aims to overcome the shortcomings of existing technologies and provide an in vitro evaluation method for the contents of calcitriol soft capsules. This method can simulate the dissolution and initial permeation process of drugs in the gastrointestinal tract, is simple to operate, has good reproducibility, and the results correlate well with in vivo behavior.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: An in vitro evaluation method for the contents of calcitriol soft capsules, using a diffusion cell apparatus, includes the following steps: (1) Add the medium solution to the diffusion cell container, making the liquid level of the medium solution flush with the lower surface of the filter membrane in the middle of the diffusion cell. The medium solution is an acidic medium or buffer salt medium containing a certain proportion of alcohol, used to simulate the gastrointestinal fluid environment and increase the solubility of calcitriol.
[0006] (2) Take the contents of the calcitriol soft capsules and place them quantitatively above the filter membrane. The filter membrane is used to simulate a biological barrier.
[0007] (3) The diffusion cell is placed in a constant temperature environment of 32℃-37℃ (preferably 37℃) and stirred at a speed of 300-600 rpm (preferably 400-600 rpm) to simulate gastrointestinal peristalsis.
[0008] (4) At different time points (e.g., 0.5, 1, 2, 3, 4, 6 hours), samples were taken from the bottom of the diffusion cell, and the concentration of calcitriol in the samples was determined by high performance liquid chromatography (HPLC). The cumulative release percentage was then calculated and the release curve was plotted.
[0009] In a preferred embodiment of the present invention, the alcohol in the medium solution is methanol or ethanol, with a volume percentage of 10% to 70%, preferably 50% to 70%. The acidic medium is a hydrochloric acid solution with a pH of 1.0 to 2.0, and the buffer salt medium is an acetate or phosphate buffer solution with a pH of 4.0 to 6.8.
[0010] As a preferred embodiment of the present invention, the filter membrane material is selected from mixed cellulose ester (MCE), polyethersulfone (PES), nylon, polyvinylidene fluoride (PVDF) or polytetrafluoroethylene (PTFE), with a pore size of 0.22μm-0.45μm, preferably PTFE material with a pore size of 0.45μm.
[0011] As a preferred embodiment of the present invention, the sample is determined by high performance liquid chromatography, and the specific method is as follows: Chromatographic conditions Column: Octadecylsilane-bonded silica gel as the packing material (Waters XBridge C18, 4.6 mm × 150 mm, 3.5 μm or equivalent column). Mobile phase: Acetonitrile-water (80:20) Detection wavelength: 265 nm; Column temperature: 45℃; Flow rate: 1.0 ml / min; Injection volume: 100 μl; Injection tray temperature control: 5℃ Preparation of the reference solution: Weigh an appropriate amount of calcitriol reference standard and dilute it with the corresponding diffusion cell reagent to a concentration of 0.004 ug / ml. The test solution is the medium of the diffusion cell taken at different time points.
[0012] Diffusion cell parameters: medium pH 2.0 hydrochloric acid solution (containing 60% methanol), temperature: 37℃, rotation speed: 400 rpm. Biofilm: PTFE, pore size: 0.45 μm.
[0013] Measurement method: Take samples and measure the medium in the lower part of the diffusion cell at 5, 10, 15, 30, 45, 60, 90 and 120 minutes respectively.
[0014] Methodological validation, results are as follows: Table 1. Accuracy Test Results
[0015] Table 2 shows the results of the linear experiment:
[0016] Table 3 Results of the limit of quantitation test:
[0017] Table 4. Results of solution stability tests:
[0018] The method of this invention can be used to evaluate the feasibility of calcitriol soft capsule formulation processes, differentiate between different formulation products, or compare the consistency of release behavior between generic drugs and original drugs.
[0019] The beneficial effects of this invention are as follows: 1. Innovation: This invention establishes for the first time an in vitro diffusion pool evaluation model for the contents (rather than the whole capsule) of calcitriol soft capsules, which more directly reflects the essential process of drug release from the formulation carrier.
[0020] 2. High predictability: This method simulates the two key steps of drug dissolution and transmembrane permeation by setting up a filter membrane and a specific medium (containing alcohols). Its release curve can better distinguish different formulations and may have a higher correlation with in vivo absorption.
[0021] 3. Good practicality: It is easy to operate, uses common instruments and equipment, and the method validation results show that its accuracy, linearity, precision and solution stability all meet the analytical requirements.
[0022] 4. Wide range of applications: It can be used for calcitriol soft capsule formulation screening, process optimization, product quality control, and in vitro release behavior consistency evaluation between generic and original drugs (such as by calculating similarity factor f2), providing key technical support for research and development and production. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the diffusion cell device used in this invention. Detailed Implementation
[0024] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.
[0025] It should be noted that, unless otherwise specified, the present invention does not specifically limit the source of the raw materials used in the following embodiments. Commercially available products or products prepared by conventional preparation methods that are well known to those skilled in the art can be used. Experimental methods that do not specify specific conditions are all conventional methods and conventional conditions well known in the art.
[0026] To test the applicability of the method of the present invention, the following formulations were set up for comparative study: Prescription 1
[0027] Prescription 2
[0028] Prescription 3
[0029] Prescription 4: Determination of the contents of calcitriol soft capsules from the original manufacturer Roche Pharma (Schweiz) Ltd.
[0030] Example 1: Diffusion cell parameters: medium pH 2.0 hydrochloric acid solution (containing 60% methanol), temperature: 37℃, rotation speed 400 rpm. Biofilm: PTFE, pore size 0.45 μm.
[0031] Measurement method: The diffusion cell is prepared according to the attached... Figure 1 Once the sample is in place, add the above-mentioned medium into the bottle 6, install the filter membrane 5 according to the above conditions, and fix the top cover 3 onto the bottle 6 with the horseshoe clamp 4. Take the sample of the above-mentioned prescription, place it at position 2, and start stirring. Start timing, and at 0.5, 1, 2, 3, 4, and 6 hours, use the sampling needle 1 to take samples from the sampling point 8 at the bottom of the diffusion cell through the sampling tube 7, and measure the medium according to the above-mentioned liquid phase method.
[0032] Example 2: Diffusion cell parameters: medium pH 6.8 hydrochloric acid solution (containing 60% methanol), temperature: 37℃, rotation speed 400 rpm. Biofilm: PTFE, pore size 0.45 μm.
[0033] Measurement method: The diffusion cell is prepared according to the attached... Figure 1 With the sample apparatus set up, add the aforementioned medium into bottle 6. Install the filter membrane 5 according to the above conditions. Secure the top cover 3 to bottle 6 using horseshoe clamp 4. Take the sample of the above formula and place it at position 2. Simultaneously start stirring and begin timing. At 0.5, 1, 2, 3, 4, and 6 hours, use sampling needle 1 through sampling tube 7 to collect samples of the medium at sampling point 8 at the bottom of the diffusion cell. Measure the medium using the liquid chromatography method described above.
[0034] Comparison of dissolution rates of different formulations in this method: Under the conditions of Example 1, the dissolution rates of the four formulations were compared with the original formulation, and the f2 value was calculated. An F2 greater than 50 indicates a similar dissolution trend, while a value less than 50 indicates a dissimilar dissolution rate.
[0035] Table 5 Comparison of dissolution trends under the conditions of Example 1
[0036] Results analysis: Of the three formulations, only formulation 1 had an f2 value greater than 50, indicating that formulation 1 is closest to the original formulation and can be used. This also demonstrates that the method of this invention can be used for formulation screening.
[0037] Repeatability testing of the same batch of samples: Under the conditions of Example 1, the same batch of samples from the original drug were tested three times repeatedly, and the f2 value was calculated by comparing it with the original drug formulation. An F2 value greater than 50 indicates a similar dissolution trend, while a value less than 50 indicates a dissimilar dissolution rate.
[0038] Table 6 Repeatability tests under the conditions of Example 1
[0039] Results analysis: The f2 values of the three retests were all greater than 50, indicating that the method of the present invention is stable and has good repeatability.
[0040] Distinguishing ability between different media: Under the conditions of Example 2, the dissolution rates of the four formulations were compared with the original formulation, and the f2 value was calculated. An F2 greater than 50 indicates a similar dissolution trend, while a value less than 50 indicates dissimilar dissolution rates.
[0041] Table 7. Discriminating power under the conditions of Example 1
[0042] Results Analysis: Under different method conditions, the similarity between Formula 1 and the original formula was detected. This indicates that the method of the present invention has the ability to distinguish between different formulas under different conditions. It can effectively distinguish between different formulas and quality differences between different batches.
[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention are still within the scope of the present invention.
Claims
1. An in vitro evaluation method for the contents of calcitriol soft capsules, characterized in that, The method uses a diffusion cell apparatus and includes the following steps: S1 Media preparation: Add the media solution to the diffusion cell container, making the liquid level of the media solution flush with the lower surface of the filter membrane in the middle of the diffusion cell; the media solution is a hydrochloric acid solution containing alcohol, acetate buffer, or phosphate buffer. S2 Sample addition: Take a sample of the contents of the calcitriol soft capsules and place the single-dose contents of the prescription to be tested above the filter membrane; S3 Diffusion Experiment: Stirring was performed at a temperature of 32℃-37℃ using magnetic stirring at a speed of 300-600 rpm; S4 Sample Collection and Measurement: After placing the contents on the filter membrane, start stirring immediately. Start timing from this time point and take samples from the sampling point at the bottom of the diffusion cell at different time points to measure the concentration of calcitriol in the sample and calculate the cumulative release.
2. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 1, characterized in that, In step S1, the alcohol is a small molecule alcohol that is miscible with water, preferably methanol or ethanol; by volume percentage, the alcohol accounts for 10% to 70% of the medium solution.
3. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 2, characterized in that, The alcohols constitute 50% to 70% of the medium solution.
4. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 1, characterized in that, In step S1, the pH value of the hydrochloric acid solution is 1.0-2.0; the pH value of the acetate buffer or phosphate buffer is 4.0-6.
8.
5. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 1, characterized in that, In step S3, the temperature is 37°C and the stirring speed is 400-600 rpm.
6. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 1, characterized in that, The material of the filter membrane is selected from one or a combination of several of mixed cellulose ester (MCE), polyethersulfone (PES), nylon, polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE); the pore size of the filter membrane is 0.22μm-0.45μm.
7. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 6, characterized in that, The filter membrane is made of polytetrafluoroethylene (PTFE) with a pore size of 0.45 μm.
8. The in vitro evaluation method for the contents of calcitriol soft capsules according to claim 1, characterized in that, In step S4, the different time points are selected from 5, 10, 15, 30, 45, 60, 90, 120 minutes or 0.5, 1, 2, 3, 4, 6 hours.
9. The in vitro evaluation method for the contents of calcitriol soft capsules according to any one of claims 1-8, characterized in that, In step S4, the concentration of calcitriol is determined by high performance liquid chromatography. The chromatographic conditions include: a column packed with octadecylsilane-bonded silica gel, acetonitrile-water as the mobile phase, a detection wavelength of 265 nm, and a column temperature of 45 °C.
10. The in vitro evaluation method for the contents of calcitriol soft capsules according to any one of claims 1-9 is used in evaluating the feasibility of the formulation process of calcitriol soft capsules, distinguishing different formulation products, or comparing the consistency of release behavior between generic drugs and original drugs.