A method for determining the in vitro release rate of lemetmovir inclusion complex solution

Abstract

A method for determining the in vitro release rate of letermovir inclusion complex solution includes diluting the letermovir inclusion complex solution with hydroxyethyl starch solution and adding it to a dialysis bag. The dialysis bag is then placed in a release medium, and the mixture is stirred or shaken using a stirring or shaking instrument, heated in a water bath, and subjected to an in vitro release rate test of the letermovir inclusion complex solution. The release medium is collected at different time points, and the concentration of letermovir in the collected release medium is measured to calculate the in vitro release rate of the letermovir inclusion complex solution. The provided method can effectively distinguish the differences in the in vitro release rate of letermovir inclusion complexes from different cyclodextrin formulations and can be used for formulation screening and quality assessment of letermovir.

Description

Technical Field

[0001] This article relates to the field of pharmaceutical technology, and in particular to a method for determining the in vitro release rate of lemetmovir inclusion complex solution. Background Technology

[0002] Letermovir is a novel non-nucleoside cytomegalovirus (CMV) inhibitor (3,4-dihydroquinazoline). It exhibits novel anti-CMV activity by inhibiting the activity of the CMV terminal enzyme complex, thus preventing viral DNA processing and packaging, thereby exerting its antiviral effect. Unlike DNA polymerase inhibitors, letermovir has higher selectivity for CMV and a significantly enhanced potency. It is also the world's first and currently only approved drug for the prevention of CMV infection in allogeneic hematopoietic stem cell transplant recipients. This lays the foundation for a preventative treatment model for CMV, providing physicians with new options for addressing CMV-related issues during hematopoietic stem cell transplantation and improving overall patient outcomes by shifting the focus of viral control upstream.

[0003] Methylbeta-cyclodextrin, hydroxyethylbeta-cyclodextrin, hydroxypropylbeta-cyclodextrin, and sulfobutylbeta-cyclodextrin, as inclusion complexes, are cyclic oligosaccharides with truncated cone shapes that can encapsulate hydrophobic drug molecules of appropriate molecular size and polarity to varying degrees. These inclusion complexes can improve the chemical stability, membrane permeability, and bioavailability of drugs. This technology has been shown to improve the solubility and dissolution rate of a variety of drugs.

[0004] Letermovir has low solubility. Preparing letermovir inclusion complex solutions using methyl beta-cyclodextrin, hydroxyethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutyl beta-cyclodextrin can improve its solubility. Letermovir must be released from the cyclodextrin inclusion complex molecules into the bloodstream before it can exert its therapeutic effect. A method that can effectively differentiate the release rates of different letermovir inclusion complex solutions will be helpful for the development and quality evaluation of letermovir formulations. Summary of the Invention

[0005] Based on the aforementioned technical background and problems, in order to effectively distinguish the differences between lemetmovir inclusion complex solutions prepared by different formulation processes, and to establish a method for determining the in vitro release rate of lemetmovir inclusion complex solutions, thereby achieving the purpose of rapidly characterizing product properties and controlling product quality, this application provides a method for determining the in vitro release rate of lemetmovir inclusion complex solutions.

[0006] In one aspect, this application provides a method for determining the in vitro release rate of lemetmovir inclusion complex solution, comprising the following steps:

[0007] 1) The termovir inclusion complex solution was diluted with hydroxyethyl starch solution and added to a dialysis bag. The dialysis bag was then placed in the release medium and stirred or shaken with a stirring or shaking instrument. The mixture was heated in a water bath to conduct an in vitro release test of the termovir inclusion complex solution.

[0008] 2) At a set time point, the release medium is taken out, the concentration of letemovir in the taken-out release medium is measured, and the in vitro release rate of the letemovir inclusion complex solution is calculated.

[0009] Compared with the prior art, the beneficial effects of this application are as follows: This application creatively tests the release rate of letermovir inclusion complex solution directly by dissolution apparatus or shaker, uses dialysis bags with specific permeability to selectively separate drug molecules and inclusion components, and simultaneously uses specific diluents (containing 3%-8% hydroxyethyl starch solution (W / V)) to adjust the release rate, thereby achieving the goal of rapidly distinguishing the differences between different formulations of letermovir inclusion complex, guiding the development of pharmaceutical products, and controlling product quality.

[0010] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the solutions described in the description and the accompanying drawings. Attached Figure Description

[0011] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0012] Figure 1 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 1 of this application.

[0013] Figure 2 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 2 of this application.

[0014] Figure 3 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 3 of this application.

[0015] Figure 4 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 4 of this application.

[0016] Figure 5 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 5 of this application.

[0017] Figure 6 This is the in vitro release curve of the lemetmovir inclusion complex solution of Example 6 of this application. Detailed Implementation

[0018] General terms and definitions

[0019] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0020] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0021] The term "measurement ratio" refers to the proportion of various substances by weight.

[0022] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0023] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0024] The term "inclusion complex" as used in this article refers to a unique complex formed when a drug molecule is wholly or partially enclosed in the molecular cavity of another substance. Inclusion complexes are a very active area of ​​research in pharmaceutical formulations. In the 1950s, researchers recognized that inclusion complexes could affect the properties of drugs, such as increasing drug solubility and stability, and influencing drug absorption, distribution, and onset of action in vivo.

[0025] The unit "W / V" used in this article refers to g / mL.

[0026] The term "encapsulated component" as used in this article refers to a substance that encapsulates all or part of the structure of a drug molecule within its molecular cavity, such as methyl beta-cyclodextrin, hydroxyethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutyl beta-cyclodextrin described herein.

[0027] The term "dissolution rate" as used in this article refers to the rate at which a drug dissolves from a solid dosage form in a specified solvent. The term "dissolution extent" refers to the rate and degree at which a drug dissolves from a solid dosage form in a specified solvent.

[0028] The term "release rate" used in this article refers to the rate and extent of release of an orally administered drug from a sustained-release, controlled-release, or enteric-coated formulation in a specified solvent. In vitro release rate testing measures the drug release rate of a formulation under simulated in vivo digestive tract conditions (such as temperature, pH of the medium, and stirring rate), and ultimately establishes reasonable in vitro drug release rate standards to monitor the product manufacturing process and control product quality. Combined with in vitro-in vivo correlation studies, release rate can predict the in vivo behavior of a product to some extent.

[0029] As used in this article, the term "release medium" refers to a liquid medium into which the active substance is released. Examples of release media can be water, simulated intestinal fluid, simulated gastric fluid, simulated saliva, or real physiological forms of these fluids, as well as various buffer solutions.

[0030] The term "release curve" used in this article refers to the change in the concentration of the analyte over time.

[0031] The term "molecular weight cutoff" (MWCO) used in this article refers to the retention performance of an ultrafiltration membrane expressed in terms of molecular weight, also known as the cutoff molecular weight, and its unit is Dalton (D). Since directly measuring the pore size of an ultrafiltration membrane is quite difficult, spherical materials with known molecular weights are used for measurement. If the membrane's retention rate of the retained substance is greater than 90%, the membrane's retention performance is expressed using the molecular weight of the retained substance, and this is called the membrane's MWCO.

[0032] To determine the in vitro release rate of letermovir inclusion complex solutions, directly placing the solution in a dialysis bag for release testing resulted in very slow, time-consuming, inefficient, and incomplete release. Conversely, diluting the solution with a release medium before dialysis resulted in too rapid a release rate and lack of discriminative power. This application utilizes a 3%-8% (w / v) hydroxyethyl starch solution to dilute the letermovir inclusion complex, which effectively regulates the release rate, improving testing efficiency and allowing for effective differentiation between different formulations of letermovir inclusion complexes.

[0033] In one aspect, this application provides a method for determining the in vitro release rate of lemetmovir inclusion complex solution, comprising the following steps:

[0034] 1) After diluting the levofloxacin inclusion complex solution with hydroxyethyl starch solution, add it to the dialysis bag, then place the dialysis bag in the release medium, stir or shake with a stirring or shaking instrument, heat in a water bath, and conduct an in vitro release test of the levofloxacin inclusion complex solution.

[0035] 2) Take out the release medium at different time points, measure the concentration of letemovir in the released medium, and calculate the in vitro release rate of the letemovir inclusion complex solution.

[0036] In some embodiments, in step 1), the concentration of the hydroxyethyl starch solution is about 3%-8% (w / v); optionally, the concentration of the hydroxyethyl starch solution is about 6% (w / v). The 6% hydroxyethyl starch solution can both simulate the plasma environment and maintain the release of letemovir from the letemovir inclusion complex at a certain rate.

[0037] In some embodiments, in step 1), the concentration of letemovir in the inclusion complex solution diluted with hydroxyethyl starch solution is about 2-4 mg / mL, and the volume of the diluted inclusion complex solution is about 1-2 mL.

[0038] In some embodiments, in step 1), the release medium is a buffer solution with a pH of about 6-8 and a volume of about 500-1000 mL; optionally, the release medium is a buffer solution with a pH of about 7.4 and a volume of about 500-1000 mL; optionally, the release medium is a phosphate buffer solution with a pH of about 7.4 and a volume of about 500-1000 mL. In some embodiments, in step 1), the volume of the release medium can be adjusted proportionally to the volume of the solution in the dialysis bag or the concentration of letermovir, thereby scaling up or down proportionally.

[0039] In some embodiments, in step 1), the molecular weight cutoff of the dialysis bag is about 1000-5000D, optionally about 1000-3500D. In some embodiments, in step 1), the molecular weight cutoff of the dialysis bag is about 1000D, 1500D, 2000D, 2500D, 3000D, 3500D, 4000D, 4500D, or 5000D.

[0040] In some implementations, in step 1), the dialysis bag has a flattened width of about 12-54 mm, a diameter of about 7.5-34 mm, and a volume of about 0.45-9.3 mL / cm².

[0041] In some implementations, in step (1), the dialysis bag is soaked in purified water for about 10-30 minutes, then rinsed three or more times with purified water, and then rinsed three or more times with the release medium.

[0042] In some embodiments, in step 1), the inclusion compound in the letrovir inclusion complex solution is selected from one or more of methyl beta-cyclodextrin, hydroxyethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutyl beta-cyclodextrin.

[0043] In some embodiments, in step 1), the stirring or agitating instrument is a shaker or dissolution apparatus, and the stirring or agitating rate is approximately 50-200 rpm; optionally, the stirring or agitating rate is approximately 100-150 rpm. In some embodiments, in step 1), the stirring or agitating rate is approximately 50 rpm, 75 rpm, 100 rpm, 125 rpm, 150 rpm, 175 rpm, or 200 rpm. In some embodiments, other types of instruments for dispersing or mixing the solution may also be used.

[0044] In some implementations, in step 1), the water bath heating temperature is about 25°C-50°C, and optionally the water bath heating temperature is about 37°C±5°C.

[0045] In some implementations, in step 2), a small amount of the release medium (e.g., 1 mL) can be taken at set time points (e.g., 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 18 h, 24 h, and 48 h) for determination of letemovir concentration.

[0046] In some embodiments, in step 2), the method for determining the concentration of letemovir in the extracted release medium is selected from one or both of HPLC and UV. In some embodiments, other methods for determining the concentration of the compound may also be used in step 2).

[0047] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0048] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0049] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0050] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0051] The materials used in the embodiments are shown below:

[0052] 1. Medicine

[0053] Letermovir inclusion complex (10% (w / v) of hydroxypropyl beta-cyclodextrin), 15% (w / v) of hydroxypropyl beta-cyclodextrin, 20% (w / v) of hydroxypropyl beta-cyclodextrin, 20% (w / v) of sulfobutyl beta-cyclodextrin, and letermovir reference standard.

[0054] 2. Reagents and materials

[0055] Potassium dihydrogen phosphate (Aladdin), sodium hydroxide (Sinopharm Group), polysorbate 80 (Chengdu Kelong), hydroxyethyl starch (Maclean).

[0056] Dialysis bags (Ruida Henghui).

[0057] 3. Instruments

[0058] Dissolution apparatus (Sotax), constant temperature shaker (Hetian Scientific), high performance liquid chromatograph (Agilent), ultraviolet-visible spectrophotometer (Agilent)

[0059] Example 1

[0060] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0061] (1) Dialysis bag and sample preparation: Take dialysis bags with 8cm molecular weight cutoff of 1000D RC membrane, 3500D RC membrane and 8000D RC membrane (flattened width of 18mm, diameter of 11.5mm and volume of 1.0ml / cm), soak them in purified water for 30min, rinse them three times with purified water and set them aside; Take 2mL of letermovir inclusion complex solution (hydroxypropyl betacyclodextrin content of 15% (W / V) in the sample formulation, and the concentration of letermovir is 20mg / mL) and add it to the dialysis bag after the above preparation, clamp the dialysis bag and set it aside;

[0062] (2) Release conditions: 1000 mL of pH 7.4 phosphate buffer solution was used as the release medium, and the rotation speed was 100 rpm. The dissolution and release rate were determined according to the method of determination of dissolution and release rate (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II).

[0063] (3) Take the dialysis bag containing the letermovir inclusion complex solution described in step (1), fix the dialysis bag on the paddle, and perform in vitro release test according to the method described in step (2). After 0.5, 1, 2, 4, 6, 12, 18, 24, and 48 hours, take 1 mL of the dissolution solution as the test solution. Take an appropriate amount of letermovir reference standard and prepare a solution of a certain concentration as the reference solution. Take the reference solution and the test solution and determine the in vitro release rate of the letermovir inclusion complex solution by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) or ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0401).

[0064] The formula for calculating the release amount of lemetmovir inclusion complex solution is shown below.

[0065]

[0066] In the formula:

[0067] F is the ratio of the concentration of the reference standard to the peak area or absorption coefficient;

[0068] A 供 The peak area or absorption coefficient of the test solution;

[0069] V 介 The volume of the release medium;

[0070] C 包 This represents the concentration of the inclusion complex solution in the dialysis bag;

[0071] V 包 This represents the volume of the inclusion complex solution in the dialysis bag.

[0072] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 1 are shown in Table 1.

[0073] Table 1. Results of in vitro release assay of lemetmovir inclusion complex solution in Example 1

[0074]

[0075] Note: The 8000D RC membrane had already fully released by 12 hours, so no further tests were conducted at subsequent time points.

[0076] Table 1 shows that when using a dialysis bag with an 8000D RC membrane, the release rate of the letermovir inclusion complex solution in phosphate buffer at pH 7.4 reached over 90% after 2 hours, indicating a rapid release rate but poor differentiation between different formulations. The 1000D RC membrane and the 3500D RC membrane showed better differentiation in the release of the letermovir inclusion complex.

[0077] Example 2

[0078] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0079] (1) Dialysis bag and sample preparation: Take an 8cm dialysis bag with a molecular weight cutoff of 3500 DR RC membrane (flattened width 18mm, diameter 11.5mm, volume 1.0ml / cm), soak it in purified water for 30min, rinse it three times with purified water, and then rinse it three times with release medium for later use; take two formulations of letermovir inclusion complex solutions (the hydroxypropyl betacyclodextrin content in the formulation of batch 1 sample is 10% (W / V), and the hydroxypropyl betacyclodextrin content in the formulation of batch 2 sample is 15% (W / V); the concentration of letermovir is 20mg / mL for both). Dilute the above two formulations of samples with pH 7.4 phosphate buffer, 0.6% polysorbate 80 solution (W / V) and 6% hydroxyethyl starch solution (W / V) to prepare inclusion complex solutions containing about 2mg / ml of letermovir. Take 2ml of the diluted letermovir solution and add it to the prepared dialysis bag, and clamp the dialysis bag tightly;

[0080] (2) Release method: 1000 mL of pH 7.4 phosphate buffer solution was used as the release medium, and the rotation speed was 100 rpm. The dissolution and release rate were determined according to the method of determination of dissolution and release rate (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II).

[0081] (3) Take the dialysis bag containing the lemetmovir inclusion complex solution described in step (1), fix the dialysis bag on the paddle, and perform in vitro release test according to the method described in step (2). After 0.5, 1, 2, 4, 6, 8 and 12 hours, take 1 mL of the dissolution solution for measurement. The measurement method is the same as in Example 1. Calculate the in vitro release rate of the lemetmovir inclusion complex solution.

[0082] The formula for calculating the release amount of the lemetmovir inclusion complex solution is the same as in Example 1.

[0083] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 2 are shown in Table 2.

[0084] Table 2. Results of in vitro release assay of lemetmovir inclusion complex solution in Example 2.

[0085]

[0086] Note 1: The release was complete at 4 hours, and no further tests were conducted at subsequent time points.

[0087] Note 2: The formulation of batch 1 sample contained 10% hydroxypropyl beta-cyclodextrin, and the formulation of batch 2 sample contained 15% hydroxypropyl beta-cyclodextrin; the concentration of letermovir was 20 mg / mL in both samples.

[0088] Table 2 shows that after dilution of the termovir inclusion complex solution with a diluent, containing 2 mg / ml of termovir, the release rate is faster than when undiluted. Using pH 7.4 phosphate buffer as a diluent, 80% release is achieved in 2 hours, and near-complete release is achieved in 4 hours; using 0.6% polysorbate 80 solution as a diluent, 80% release is achieved in 1 hour, and near-complete release is achieved in 2 hours; these two diluents show no significant differentiation between different formulations. Using 6% hydroxyethyl starch solution as a diluent, near-complete release is achieved in 8 hours, and a relatively significant differentiation effect is observed between different formulations, making it suitable for formulation screening and quality control.

[0089] Example 3

[0090] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0091] (1) Dialysis bag and sample preparation: Take an 8cm dialysis bag with a molecular weight cutoff of 3500 DR RC membrane (flattened width 18mm, diameter 11.5mm, volume 1.0ml / cm), soak it in purified water for 30min, rinse it three times with purified water, and then rinse it three times with release medium for later use; take an appropriate amount of letermovir inclusion complex solution (the sample formulation contains 15% hydroxypropyl betacyclodextrin (W / V), and the concentration of letermovir is 20mg / mL), and dilute it with 6% hydroxyethyl starch solution (W / V) to prepare inclusion complex solutions containing 10mg / mL, 4mg / mL, and 2mg / mL of letermovir, respectively. Take 2ml of the diluted letermovir inclusion complex solution and add it to the prepared dialysis bag, then clamp the dialysis bag tightly;

[0092] (2) Release conditions: 1000 mL of pH 7.4 phosphate buffer solution was used as the release medium, and the rotation speed was 100 rpm. The dissolution and release rate were determined according to the method of determination of dissolution and release rate (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II).

[0093] (3) Take the dialysis bag containing the lemetmovir inclusion complex solution described in step (1), fix the dialysis bag on the paddle, and perform in vitro release test according to the method described in step (2). After 0.5, 1, 2, 4, 6, 8 and 12 hours, take 1 mL of the dissolution solution for measurement. The measurement method is the same as in Example 1. Calculate the in vitro release rate of the lemetmovir inclusion complex solution.

[0094] The formula for calculating the release amount of the lemetmovir inclusion complex solution is the same as in Example 1.

[0095] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 3 are shown in Table 3.

[0096] Table 3. Results of in vitro release assay of lemetmovir inclusion complex solution in Example 3.

[0097]

[0098] As shown in Table 3, the release rate of the lemetmovir inclusion complex solution (containing 10 mg / mL lemetmovir) diluted with 6% hydroxyethyl starch solution was slow at a concentration of 10 mg / mL and the release was incomplete after 12 hours. The release rates at concentrations of 4 mg / mL and 2 mg / mL were moderate and had a certain degree of distinguishability.

[0099] Example 4

[0100] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0101] (1) Take an 8cm dialysis bag with a molecular weight cutoff of 3500 DR RC membrane (flattened width 45mm, diameter 29mm, volume 6.4ml / cm), soak it in purified water for 30min, rinse it three times with purified water, and then rinse it three times with release medium. Set aside. Take a letermovir inclusion complex solution (the sample formulation contains 15% hydroxypropyl betacyclodextrin (W / V), and the concentration of letermovir is 20mg / mL), and dilute it with 1%, 3%, 6%, and 8% hydroxyethyl starch solutions (W / V) to prepare inclusion complex solutions containing approximately 2mg / ml of letermovir. Add 2ml of the diluted letermovir solution to the treated dialysis bag and clamp the dialysis bag tightly.

[0102] (2) Release conditions: 1000 mL of pH 7.4 phosphate buffer solution was used as the release medium, and the rotation speed was 100 rpm. The dissolution and release rate were determined according to the method of determination of dissolution and release rate (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II).

[0103] (3) Take the dialysis bag containing the lemetmovir inclusion complex solution described in step (1), fix the dialysis bag on the paddle, and perform in vitro release test according to the method described in step (2). After 0.5, 1, 2, 4, 6, 8 and 12 hours, take 1 mL of the dissolution solution for measurement. The measurement method is the same as in Example 1. Calculate the in vitro release rate of the lemetmovir inclusion complex solution.

[0104] The formula for calculating the release amount of the lemetmovir inclusion complex solution is the same as in Example 1.

[0105] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 4 are shown in Table 4.

[0106] Table 4. Results of in vitro release assay of lemetmovir inclusion complex solution in Example 4

[0107]

[0108]

[0109] As shown in Table 4, the release rate of the lemetmovir inclusion complex solution (containing 2 mg / ml lemetmovir) diluted with 1% hydroxyethyl starch solution is relatively slow, and it is not completely released after 12 hours; while the 3%-8% solution reaches more than 90% after 12 hours and is completely released. Therefore, the concentration of hydroxyethyl starch solution used is 3%-8%; more preferably, the concentration of hydroxyethyl starch solution used is 6%.

[0110] Example 5

[0111] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0112] (1) Take an 8cm dialysis bag with a molecular weight cutoff of 3500 DR RC membrane (flattened width 18mm, diameter 11.5mm, volume 1.0ml / cm), soak it in purified water for 30min, rinse it three times with purified water, and then rinse it three times with release medium. Set aside. Take a letermovir inclusion complex solution (the sample formulation contains 15% hydroxypropyl betacyclodextrin and a letermovir concentration of 20mg / mL), and dilute it with a 6% hydroxyethyl starch solution (w / v) to prepare an inclusion complex solution containing approximately 2mg / ml of letermovir. Add 2ml of the diluted letermovir solution to the treated dialysis bag and clamp the dialysis bag tightly.

[0113] (2) Release method: Use 500 mL of pH 7.4 phosphate buffer solution as the release medium, add it to a beaker, and place it in a constant temperature shaker at 37℃±5℃ to maintain a constant temperature; the shaking rate is 200 rpm per minute.

[0114] (3) Take the dialysis bag containing the lemetmovir inclusion complex solution described in step (1), put it into the beaker described in step (2) and shake it. After 0.5, 1, 2, 4, 6, 8 and 12 hours, take 1 mL of the dissolution solution for measurement. The measurement method is the same as in Example 1. Calculate the in vitro release rate of the lemetmovir inclusion complex solution.

[0115] The formula for calculating the release amount of the lemetmovir inclusion complex solution is the same as in Example 1.

[0116] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 5 are shown in Table 5.

[0117] Table 5. Results of in vitro release assay of lemetmovir inclusion complex solution in Example 5.

[0118] Time (hours) Release rate (%) 0 0 0.5 11 1 28 2 46 4 66 6 78 8 87 12 95

[0119] As shown in Table 5, the release curve of the lemetmovir inclusion complex solution (containing 2 mg / ml lemetmovir) diluted with 6% hydroxyethyl starch solution in pH 7.4 phosphate buffer was basically consistent with the release curve obtained by the paddle method of the dissolution and release determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II) under the same diluent and release medium conditions.

[0120] Example 6

[0121] This embodiment describes a method for determining the in vitro release rate of a lemetmovir inclusion complex solution according to the present invention, comprising the following steps:

[0122] (1) Take an 8cm dialysis bag with a molecular weight cutoff of 3500 DR RC membrane (flattened width 45mm, diameter 29mm, volume 6.4ml / cm), soak it in purified water for 30min, rinse it three times with purified water, and then rinse it three times with release medium for later use; take four different formulations of lemetmovir inclusion complex solutions (batch 1 - 10% (W / V) of hydroxypropyl beta-cyclodextrin in the sample formulation; batch 2 - 15% (W / V) of hydroxypropyl beta-cyclodextrin in the sample formulation; batch 3 - 20% (W / V) of hydroxypropyl beta-cyclodextrin in the sample formulation; batch 4 - 20% (W / V) of sulfobutyl beta-cyclodextrin in the sample formulation), and dilute them with 6% hydroxyethyl starch solution to prepare inclusion complex solutions containing 2mg / mL of lemetmovir. Then take 2ml of the diluted lemetmovir solution and add it to the treated dialysis bag, and clamp the dialysis bag tightly;

[0123] (2) Release conditions: 1000 mL of pH 7.4 phosphate buffer solution was used as the release medium, and the rotation speed was 100 rpm. The dissolution and release rate were determined according to the method of determination of dissolution and release rate (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II).

[0124] (3) Take the dialysis bag containing the lemetmovir inclusion complex solution described in step (1), fix the dialysis bag on the paddle, and perform in vitro release test according to the method described in step (2). After 0.5, 1, 2, 4, 6, 8 and 12 hours, take 1 mL of the dissolution solution for measurement. The measurement method is the same as in Example 1. Calculate the in vitro release rate of the lemetmovir inclusion complex solution.

[0125] The formula for calculating the release amount of the lemetmovir inclusion complex solution is the same as in Example 1.

[0126] (4) The results of the in vitro release of the lemetmovir inclusion complex solution in Example 6 are shown in Table 6.

[0127] Table 6. Results of in vitro release of the letermovir inclusion complex solution from Example 6.

[0128]

[0129] Note: Batch 1—The formulation contains 10% (w / v) hydroxypropyl beta-cyclodextrin; Batch 2—The formulation contains 15% (w / v) hydroxypropyl beta-cyclodextrin; Batch 3—The formulation contains 20% (w / v) hydroxypropyl beta-cyclodextrin; Batch 4—The formulation contains 20% (w / v) sulfobutyl beta-cyclodextrin.

[0130] As shown in Table 6, after diluting different formulations of letemovir inclusion complex solutions to 2 mg / ml with 6% hydroxyethyl starch solution and then performing release tests, this release test method can effectively distinguish the differences in formulations.

[0131] By employing the release assay method provided in this application, such as using a suitable dialysis bag (1000-3500D) and diluting it to a suitable concentration (containing 2-4 mg / mL of lemetmovir) with a suitable diluent (6% hydroxyethyl starch solution), the release assay demonstrates good formulation differentiation for lemetmovir inclusion complex solutions and can be used for formulation screening and quality control.

Claims

1. A method for determining the in vitro release rate of a letimovir inclusion complex solution, comprising the following steps: 1) diluting the letimovir inclusion complex solution with a hydroxyethyl starch solution, then adding the diluted solution into a dialysis bag, and placing the dialysis bag into a release medium, and stirring or oscillating, and water bath heating, to perform the in vitro release test of the letimovir inclusion complex solution; wherein the concentration of the hydroxyethyl starch solution is 3%-8%, the concentration of letimovir in the diluted inclusion complex solution is 2-4 mg / mL, the volume of the diluted inclusion complex solution is 1-2 mL, the release medium is a buffer solution with a pH of 6-8, and the molecular weight cut-off of the dialysis bag is 1000-5000 D; 2) taking out the release medium at a set time point, determining the concentration of letimovir in the taken-out release medium, and calculating the in vitro release rate of the letimovir inclusion complex solution.

2. The assay method of claim 1, wherein, In step 1), the concentration of the hydroxyethyl starch solution is 6%.

3. The assay method of claim 1, wherein, In step 1), the release medium is a buffer solution with a pH of 7.

4.

4. The assay method of claim 3, wherein, In step 1), the release medium is a phosphate buffer solution with a pH of 7.

4.

5. The assay method of claim 1, wherein, In step 1), the molecular weight cut-off of the dialysis bag is 1000-3500 D.

6. The assay method of claim 5, wherein, In step 1), the flattened width of the dialysis bag is 12-54 mm, the diameter is 7.5-34 mm, and the volume is 0.45-9.3 mL / cm.

7. The assay method according to any one of claims 1 to 6, wherein, In step 1), the stirring or oscillation is performed using a dissolution apparatus or an oscillation apparatus, and the stirring or oscillation rate is 50-200 rpm.

8. The assay method of claim 7, wherein, In step 1), the stirring or oscillation rate is 100-150 rpm.

9. The assay method according to any one of claims 1 to 6, wherein, In step 1), the water bath heating temperature is 25°C-50°C.

10. The assay method of claim 9, wherein, In step 1), the water bath heating temperature is 37°C±5°C.

11. The assay method according to any one of claims 1 to 6, wherein, In step 2), the method for determining the concentration of letimovir in the taken-out release medium is selected from one or both of HPLC and UV.

12. The assay method according to any one of claims 1-6, wherein, In step 2), the set time point is selected from one or more of 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 18 h, 24 h, and 48 h.

13. The assay method according to any one of claims 1-6, wherein, In step 1), the inclusion component in the letimovir inclusion complex solution is selected from one or more of methyl betadex, hydroxyethyl betadex, hydroxypropyl betadex, and sulfobutyl betadex.

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