Method for measuring encapsulation efficiency of sulfobutyl-beta-cyclodextrin inclusion drug

Abstract

The invention discloses a method for determining the encapsulation efficiency of a sulfobutyl-beta-cyclodextrin inclusion drug, and relates to the field of quality control of sulfobutyl-beta-cyclodextrin inclusion compounds. The method comprises the following steps: (1) adding a solvent 1 into a freeze-dried product of the sulfobutyl-beta-cyclodextrin inclusion drug, extracting, taking supernate or subsequent filtrate, and determining the free drug amount through liquid chromatography; (2) dissolving the freeze-dried product of the sulfobutyl-beta-cyclodextrin inclusion drug in a solvent 2, and determining the total amount of the drug through liquid chromatography; and (3) calculating the encapsulation efficiency of the inclusion drug according to the free drug amount and the total drug amount. The determination method disclosed by the invention is simpler and more convenient, good in repeatability and high in accuracy.

Description

Technical Field

[0001] This invention relates to the field of quality control of sulfobutyl betacyclodextrin inclusion complexes, and specifically to a method for determining the encapsulation efficiency of drugs encapsulated by sulfobutyl betacyclodextrin. Background Technology

[0002] Poorly soluble drugs significantly increase the challenge of new drug development due to their low solubility and absorption rate. In new drug development, poorly soluble candidate drugs of BCS II and BCS IV classes account for as much as 90%. Solving the solubility problem is crucial to improving the bioavailability and efficacy of these drugs. Drug solubilization methods are classified into chemical modification and physical modification according to physicochemical processes. Chemical modification includes salt formation and prodrug design, while physical modification includes micronization, solid dispersion, emulsification, and cyclodextrin inclusion.

[0003] Cyclodextrins are cyclic oligosaccharides derived from starch. The most commonly used natural cyclodextrins are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, composed of 6, 7, and 8 pyranose units, respectively. The opening of the cyclodextrin ring is too small for many drugs, while γ-cyclodextrin, although having a large molecular hole, has high production costs and cannot be mass-produced industrially. Therefore, β-cyclodextrin is one of the most effective inclusion materials known. Natural β-cyclodextrin exhibits strong nephrotoxicity and poor water solubility due to intramolecular hydrogen bonding, limiting its application in drug solubilization. To address the issues of water solubility and nephrotoxicity, scientists have chemically modified the primary and secondary hydroxyl groups (especially the 2, 3, and 6-position hydroxyl groups) of the β-cyclodextrin molecule, forming representative β-cyclodextrin derivatives such as methyl beta-cyclodextrin, carboxymethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutyl beta-cyclodextrin. Among these, sulfobutyl beta-cyclodextrin is the most widely used in marketed drugs.

[0004] To evaluate the inclusion effect of beta-cyclodextrin inclusion complexes, its encapsulation efficiency needs to be determined. Chinese patent CN116474119A describes placing a solid tetraazine dicarboxamide methyl beta-cyclodextrin inclusion complex in a sealed container containing 20 mL of diethyl ether. After vortexing for 10 minutes, the free tetraazine dicarboxamide is washed out. The diethyl ether containing the free tetraazine dicarboxamide is then poured out and allowed to evaporate naturally in a ventilated area until no ether odor remains, yielding the free tetraazine dicarboxamide. The peak area of ​​the obtained free tetraazine dicarboxamide is determined using high-performance liquid chromatography (HPLC), and the content of free tetraazine dicarboxamide is calculated. This content is then compared with the amount of tetraazine dicarboxamide added to calculate the drug's inclusion efficiency.

[0005] For sulfobutylbetacyclodextrin inclusion complexes, the molecular weight difference between the inclusion complex and the free drug molecule is relatively small. Considering that the permeability of the solute also depends on molecular shape, degree of hydration, ionic charge, and polarity, to ensure rapid permeation of the free drug through the membrane, the molecular weight cutoff of the dialysis membrane should be 100 times the allowable molecular weight, and not higher than 50%-80% of the molecular weight to be retained. The molecular weight ratio of the two substances to be separated should be at least 25. Therefore, it is impossible to find an ultrafiltration tube or dialysis bag with a suitable molecular weight cutoff for separation. Gel chromatography is often used to separate substances of different sizes, but when a small amount of sample solution is injected into the chromatograph, the inclusion complex is diluted by a large amount of mobile phase and dissociates, resulting in the final elution of the inclusion complex without drug molecules.

[0006] Therefore, it is of great significance to develop a simple, convenient, reproducible, and accurate method for determining the encapsulation efficiency of drugs encapsulated by sulfobutyl betacyclodextrin. Summary of the Invention

[0007] To address the limitations of existing methods for separating sulfobutyl betacyclodextrin-encapsulated drugs from free drug molecules, this invention utilizes solubility differences (sulfobutyl betacyclodextrin-encapsulated drugs are poorly soluble in certain organic solvents, while trace amounts of free drug molecules can be completely dissolved in these same organic solvents). The sulfobutyl betacyclodextrin-encapsulated drug is extracted with an organic solvent, centrifuged, or filtered. The amount of free drug in the supernatant or filtrate is determined by liquid chromatography. Then, the sulfobutyl betacyclodextrin-encapsulated drug is completely dissolved in another suitable solvent, and the total drug amount is determined. The encapsulation efficiency of the encapsulated drug can then be calculated. This method is simpler, more repeatable, and more accurate.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A method for determining the encapsulation efficiency of a sulfobutyl betacyclodextrin-encapsulated drug includes the following steps: (1) Take the lyophilized product of the drug encapsulated by sulfobutyl betacyclodextrin, add solvent 1, extract the dissolved free drug that is not encapsulated by sulfobutyl betacyclodextrin, take the supernatant or filtrate, and determine the amount of free drug using an appropriate method. (2) Take another lyophilized product containing sulfobutyl betacyclodextrin containing the drug, add solvent 2 to dissolve it completely, and determine the total amount of drug using an appropriate method; (3) Calculate the encapsulation efficiency of the inclusion drug according to the following formula. .

[0009] The encapsulated drug in sulfobutyl betacyclodextrin contains a high amount of encapsulated drug and a low amount of free drug. To accurately determine the encapsulation efficiency, solvent 1 used for extraction is an organic solvent that is poorly soluble in sulfobutyl betacyclodextrin but soluble in trace amounts of free drug. In step (1), solvent 1 is preferably one or a combination of acetonitrile, anhydrous ethanol, isopropanol, acetone, n-propanol, tetrahydrofuran, n-butanol, isobutanol, tert-butanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, polyethylene glycol 400, Tween 80, and dioxane.

[0010] Preferably, in step (1), the mass-to-volume ratio of the sulfobutyl betacyclodextrin-encapsulated drug to solvent 1 is 1:5-15, g:mL; more preferably 1:10, g:mL.

[0011] Preferably, in step (1), the extraction is ultrasonic extraction or shaking extraction, more preferably shaking extraction, and even more preferably, the shaking extraction time is 1-10 min.

[0012] The determination of the total amount of drug in the sulfobutyl betacyclodextrin inclusion complex requires ensuring that the inclusion complex can be completely dissolved. Preferably, in step (2), the solvent 2 is one or a combination of water, formamide, ethylene glycol, dimethyl sulfoxide, and glycerol, and more preferably water.

[0013] This invention applies to lyophilized sulfobutyl betacyclodextrin inclusion complex drugs whose solubility meets the above requirements. The contents of the drug include sulfobutyl betacyclodextrin inclusion complex drugs currently marketed domestically and internationally. For example, the contents include at least one of voriconazole, ziprasidone mesylate, aripiprazole, amiodarone hydrochloride, carfilzomib, posaconazole, melphalan hydrochloride, carbamazepine, derafloxacin, allogeneolone, remdesivir, fosphenytoin sodium, levothyroxine sodium, docetaxel, and fosaspirant.

[0014] Preferably, in step (1) or step (2), the appropriate method for determining the amount of free drug or the total amount of drug is liquid chromatography.

[0015] The beneficial effects of this invention are as follows: By utilizing the solubility difference between sulfobutyl betacyclodextrin-encapsulated drugs and free drug molecules, the separation and analysis of encapsulated and free drugs can be achieved. The method of this invention has good accuracy, repeatability, and robustness, and can effectively evaluate the encapsulation effect of sulfobutyl betacyclodextrin-encapsulated drugs, providing a technical means for product quality evaluation. Detailed Implementation

[0016] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0017] Example 1 The sulfobutyl betacyclodextrin inclusion complex of carfilzomib was investigated as a lyophilized product. The preparation process was as follows: 750 mL of water for injection was added to 250 g of sodium sulfobutyl betacyclodextrin, and the mixture was magnetically stirred to dissolve it. Then, 5 g of carfilzomib was added and stirred until completely dispersed. Water was added to 1000 mL, and the mixture was stirred overnight. The mixture was then filtered, bottled, and lyophilized.

[0018] Sample solution (1): Accurately weigh about 0.5 g of the lyophilized product containing carfilzomisulfate betacyclodextrin and add 5 mL of acetonitrile. Shake and extract for 5 minutes. After filtration, take 1 mL of the filtrate and place it in a 5 mL volumetric flask. Dilute to the mark with 40% acetonitrile aqueous solution and shake well.

[0019] Sample solution (2): Accurately weigh about 0.25 g of the lyophilized product containing carfilzomisulfate betacyclodextrin and place it in a 50 mL volumetric flask. Add 2.5 mL of water to dissolve it, dilute to the mark with 40% acetonitrile aqueous solution, and shake well.

[0020] Reference solution (1): Weigh an appropriate amount of carfilzomib reference standard accurately, add a small amount of acetonitrile to dissolve it, and dilute quantitatively with 40% acetonitrile aqueous solution to prepare a solution containing approximately 4 μg of carfilzomib per ml.

[0021] Reference solution (2): Take an appropriate amount of carfilzomib reference standard, accurately weigh it, add a little acetonitrile to dissolve it, and quantitatively dilute it with 40% acetonitrile aqueous solution to prepare a solution containing about 0.1 mg of carfilzomib per ml.

[0022] Determination method: Accurately measure sample solution (1), sample solution (2), reference solution (1), and reference solution (2), inject them into the liquid chromatograph respectively, and record the chromatogram.

[0023] The chromatographic conditions used are as follows: Chromatographic column: Waters Symmetry C18, 4.6 mm × 150 mm, 3.5 μm; Mobile phase: 0.1 mol / L sodium perchlorate buffer (adjusted to pH 3.1 with 0.1 mol / L phosphoric acid) - acetonitrile (50:50); Flow rate: 1.2 mL / min; Detection wavelength: 220nm; Column temperature: 28℃; Injection volume: 20 μl.

[0024] Calculation formula:

[0025] In the formula: A T2 The peak area of ​​carfilzomi in the chromatogram of sample solution (2); A S2 The peak area of ​​carfilzomi in the chromatogram of the reference solution (2); W S2 The weight of the lyophilized product in sample solution (2) is in mg; C S2 The concentration of carfilzomib in the reference solution (2) is mg / ml; 50 is the dilution factor of sample solution (2).

[0026]

[0027] In the formula: A T1 The peak area of ​​carfilzomi in the chromatogram of sample solution (1); A S1 The peak area of ​​carfilzomi in the chromatogram of the reference solution (1); W S1 The weight of the lyophilized sample in sample solution (1) is in g; C S1 The concentration of carfilzomib in the reference solution (1) is μg / ml; 25 is the dilution factor of sample solution (1).

[0028] Encapsulation rate (%) = 100% - T2 (%) According to the measurements and calculations, the encapsulation efficiency of the drug encapsulated by carfilzomisulfate betacyclodextrin was 99.8%.

[0029] Effect detection: 1. Repeatability test Completely dissolve the test sample solution: Take about 0.25 g of the lyophilized carfilzomib containing the drug, place it in a 50 mL volumetric flask, add 2.5 mL of water to dissolve it, dilute to the mark with 40% acetonitrile aqueous solution, and shake well.

[0030] Free test solution: Take approximately 0.5 g of the lyophilized carfilzomib-encapsulated drug, add 5 mL of acetonitrile, shake to extract for 5 minutes, filter, and take 1 mL of the filtrate. Place it in a 5 mL volumetric flask, dilute to the mark with 40% acetonitrile aqueous solution, and shake well. Prepare 6 parallel solutions. The repeatability test results are shown in Table 1.

[0031] Table 1. Results of Encapsulation Efficiency Repeatability Tests

[0032] The results showed that the range of free carfilzomib concentration in the six test samples was 0.05%, and the encapsulation efficiency was in the range of 99.78%-99.83%, indicating good repeatability of the analytical method.

[0033] 2. Accuracy assessment The accuracy of the method was examined when the proportion of free carfilzomib in the samples was 0.2%, 1%, and 50%.

[0034] Reference stock solution 1: Accurately weigh an appropriate amount of carfilzomib reference standard, add a small amount of acetonitrile to dissolve it, and dilute with acetonitrile to prepare a solution containing approximately 1 mg per 1 mL.

[0035] Reference stock solution 2: Accurately measure 1 mL of "Reference stock solution 1" and place it in a 50 mL volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0036] Reference stock solution 3: Accurately measure 10 mL of "Reference stock solution 2" and place it in a 50 mL volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0037] 0.2% Free Recovery Solution: Accurately weigh approximately 0.5 g of the lyophilized carfilzomib-encapsulated drug, add 5 mL of "Reference Stock Solution 3", shake to extract for 5 minutes, filter, and take 1 mL of the filtrate. Place it in a 5 mL volumetric flask, dilute to the mark with 40% acetonitrile aqueous solution, and shake well. Prepare 3 parallel solutions.

[0038] 1% Free Recovery Solution: Accurately weigh approximately 0.5 g of the lyophilized carfilzomib-encapsulated drug, add 5 mL of "Reference Stock Solution 2", shake to extract for 5 minutes, filter, and take 1 mL of the filtrate. Place it in a 5 mL volumetric flask, dilute to the mark with 40% acetonitrile aqueous solution, and shake well. Prepare 3 parallel solutions.

[0039] 50% Free Recovery Solution: Accurately weigh approximately 0.5 g of the lyophilized carfilzomib-encapsulated drug, add 5 mL of "Reference Stock Solution 1", shake to extract for 5 minutes, filter, and take 1 mL of the filtrate. Place it in a 5 mL volumetric flask, dilute to the mark with 40% acetonitrile aqueous solution, and mix well. Accurately measure another 1 mL, place it in a 50 mL volumetric flask, dilute to the mark with 40% acetonitrile aqueous solution, and mix well. Prepare three parallel solutions.

[0040] Table 2. Encapsulation accuracy test results

[0041] The results showed that the recovery rate of the analytical method was between 91.22% and 113.31% within the range of 0.2%-50% free drug content, indicating good accuracy of the analytical method.

[0042] 3. Durability assessment Test solution: Accurately weigh approximately 0.5 g of the lyophilized carfilzomib-encapsulated drug, and weigh five parallel portions. Add 5 mL of acetonitrile to each portion, and extract by shaking for 1 minute, 3 minutes, 5 minutes, 7 minutes, and 10 minutes respectively. After filtration, take 1 mL of the filtrate and place it in a 5 mL volumetric flask. Dilute to the mark with 40% acetonitrile aqueous solution and shake well. Investigate the differences in the determination results of the free carfilzomib rate in the samples at different shaking times. The results are shown in Table 3.

[0043] Table 3. Results of the durability test for encapsulation efficiency sample treatment methods

[0044] The results showed that after 1-10 minutes of shaking extraction with acetonitrile on the lyophilized carfilzomi cyclodextrin-encapsulated drug, the range of free carfilzomi was 0.08%, indicating that free carfilzomi could be extracted and dissolved in acetonitrile in a short time.

[0045] Example 2 Unlike Example 1, anhydrous ethanol was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.7%. The analytical method showed good accuracy.

[0046] Example 3 Unlike Example 1, isopropanol was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.8%. The analytical method showed good accuracy.

[0047] Example 4 Unlike Example 1, acetone was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.8%. The analytical method showed good accuracy.

[0048] Example 5 Unlike Example 1, the solvent used for free drug extraction was n-propanol, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.7%. The analytical method showed good accuracy.

[0049] Example 6 Unlike Example 1, tetrahydrofuran was used as the solvent for free drug extraction. The encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.2%. The analytical method showed good accuracy.

[0050] Example 7 Unlike Example 1, the solvent used for free drug extraction was n-butanol, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.9%. The analytical method showed good accuracy.

[0051] Example 8 Unlike Example 1, isobutanol was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.8%. The analytical method showed good accuracy.

[0052] Example 9 Unlike Example 1, tert-butanol was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 97.4%. The analytical method showed good accuracy.

[0053] Example 10 Unlike Example 1, the solvent used for free drug extraction was N,N-dimethylformamide, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 97.9%. The analytical method showed good accuracy.

[0054] Example 11 Unlike Example 1, the solvent used for free drug extraction was N,N-dimethylacetamide, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 98.8%. The analytical method showed good accuracy.

[0055] Example 12 Unlike Example 1, the solvent used for free drug extraction was N-methylpyrrolidone, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 98.2%. The analytical method showed good accuracy.

[0056] Example 13 Unlike Example 1, the solvent used for free drug extraction was polyethylene glycol 400, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.3%. The analytical method showed good accuracy.

[0057] Example 14 Unlike Example 1, Tween 80 was used as the solvent for free drug extraction, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.6%. The analytical method showed good accuracy.

[0058] Example 15 Unlike Example 1, the solvent used for free drug extraction was dioxane, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.3%. The analytical method showed good accuracy.

[0059] Example 16 Unlike Example 1, the volume of acetonitrile used to extract the free drug in sample solution (1) was 2.5 ml, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.8%. The analytical method showed good accuracy.

[0060] Example 17 Unlike Example 1, the volume of acetonitrile used to extract the free drug in sample solution (1) was 7.5 ml, and the encapsulation efficiency was determined using the same method, with an encapsulation efficiency of 99.8%. The analytical method showed good accuracy.

[0061] Example 18 Unlike Example 1, the sample solution (1) was extracted with 5 ml of acetonitrile by ultrasonication for 5 minutes. The encapsulation rate was determined using the same method and was 99.4%. The analytical method showed good accuracy.

[0062] Example 19 Unlike Example 1, the drug examined was a lyophilized product containing sulfobutyl betacyclodextrin of amiodarone hydrochloride. The preparation process was as follows: 225 mL of water for injection was taken, 75 g of sodium sulfobutyl betacyclodextrin was added, and the mixture was magnetically stirred to dissolve it. Then, 1 g of amiodarone hydrochloride was added and stirred until completely dispersed. Water was added to 300 mL, and the mixture was stirred overnight. The mixture was then filtered, bottled, and lyophilized.

[0063] Preparation of reference solution for encapsulation efficiency determination: Reference solution (1): Take an appropriate amount of amiodarone hydrochloride reference standard, accurately weigh it, dissolve it in 50% acetonitrile aqueous solution and quantitatively dilute it to prepare a solution containing about 0.5 μg of amiodarone hydrochloride per 1 ml.

[0064] Reference solution (2): Take an appropriate amount of amiodarone hydrochloride reference standard, accurately weigh it, dissolve it in 50% acetonitrile aqueous solution and quantitatively dilute it to prepare a solution containing about 0.075 mg of amiodarone hydrochloride per 1 ml.

[0065] Chromatographic conditions used: Column: Agela Venusil XBP C18, 4.6 mm × 150 mm, 5 μm; Mobile phase: buffer solution (3.0 ml of glacial acetic acid, 800 ml of water, pH adjusted to 4.9 with ammonia solution, then diluted to 1000 ml with water) - methanol - acetonitrile (30:30:40), isocratic elution; Flow rate: 1.0 mL / min; Detection wavelength: 240nm; Column temperature: 30℃; Injection volume: 10 μl.

[0066] The encapsulation efficiency of the drug encapsulated by amiodarone hydrochloride sulfobutyl betacyclodextrin was 99.8%, and the analytical method showed good accuracy.

[0067] Example 20 Unlike Example 1, the drug under investigation was a lyophilized product of sulfobutyl betacyclodextrin containing fosapitant. The preparation process was as follows: 225 mL of water for injection was taken, 75 g of sodium sulfobutyl betacyclodextrin was added, and the mixture was magnetically stirred to dissolve it. Then, 1 g of fosapitant dimethylglucamine was added and stirred until completely dispersed. Water was added to 300 mL, and the mixture was stirred overnight. The mixture was then filtered, bottled, and lyophilized.

[0068] Preparation of reference solution for encapsulation efficiency determination: Reference solution (1): Take an appropriate amount of fosapitan meglumine reference standard, accurately weigh it, dissolve it in 50% acetonitrile aqueous solution and quantitatively dilute it to prepare a solution containing about 0.05 μg of fosapitan meglumine per 1 ml.

[0069] Reference solution (2): Take an appropriate amount of fosapitan meglumine reference standard, accurately weigh it, dissolve it in 50% acetonitrile aqueous solution and quantitatively dilute it to prepare a solution containing about 0.075 mg of fosapitan meglumine per 1 ml.

[0070] Chromatographic conditions used: Column: Phenomenex Luna Phenyl-Hexyl, 4.6 mm × 250 mm, 5 μm; Mobile phase A: 0.1% aqueous phosphoric acid solution; Mobile phase B: Acetonitrile; The gradient elution procedure is shown in Table 4: Table 4.

[0071] Flow rate: 1.0 mL / min; Detection wavelength: 210nm; Column temperature: 25℃; Injection volume: 20 μl.

[0072] According to the measurements and calculations, the encapsulation efficiency of the fosapitan diglucamine sulfobutyl betacyclodextrin-encapsulated drug was 99.9%, and the analytical method showed good accuracy.

[0073] Comparative Example 1 The free drug and the included drug in carfilzomisulfate-betacyclodextrin inclusion complex were separated by ultrafiltration tube centrifugation.

[0074] Table 5. Ultrafiltration Tube Information

[0075] Test solution (1): Take about 3g of carfilzomisulfate betacyclodextrin containing the drug, dissolve it in water and dilute it to 50mL.

[0076] Test solution (2): Take 1 ml of “test solution (1)”, put it in a 20 ml volumetric flask, dilute with water to the mark, and shake well.

[0077] Reference solution: Take an appropriate amount of carfilzomib reference standard, add a small amount of acetonitrile to dissolve it, and dilute with water to prepare a solution containing about 5 μg per 1 mL.

[0078] The test solution (1) was placed in ultrafiltration tubes with molecular weight cutoffs of 3K and 30K, and the test solution (2) was placed in an ultrafiltration tube with a molecular weight cutoff of 1K. After centrifugation and filtration, the concentrations of sulfobutyl betacyclodextrin and carfilzomib in the original solution and filtrate were determined, and the permeability of sulfobutyl betacyclodextrin and carfilzomib was investigated. The reference solution was placed in ultrafiltration tubes with different molecular weight cutoffs. After centrifugation and filtration, the concentration of carfilzomib in the original solution and filtrate was determined, and the permeability of carfilzomib was investigated. The results are summarized as follows: Table 6. Carfilzomib Transmission Rate

[0079] The results showed that although the molecular weight of carfilzomib was less than 1K, the free carfilzomib molecules in the reference solution could not pass through the Omega membranes at 1K, 3K and 30K. This may be because the contact angle between the free lipid-soluble carfilzomib and the hydrophilic Omega membrane is large, and it cannot pass through the pores. When using an ultrafiltration tube with a molecular weight cutoff of 1K, sulbutylated beta-cyclodextrin and carfilzomib are essentially unable to permeate the membrane, making it impossible to separate the encapsulated drug from the free drug. When using an ultrafiltration tube with a molecular weight cutoff of 30K, the encapsulated sulbutylated beta-cyclodextrin can permeate the membrane into the filtrate. At this point, the dynamic equilibrium of cyclodextrin encapsulation in the stock solution is disrupted, shifting towards the formation of inclusion complexes. This allows the free carfilzomib in the stock solution to be encapsulated and then permeate the membrane into the filtrate. Ultimately, almost all of the cyclodextrin and carfilzomib enters the filtrate. When using an ultrafiltration tube with a molecular weight cutoff of 3K, most of the unencapsulated sulbutylated beta-cyclodextrin can enter the filtrate. At this point, the dynamic equilibrium of cyclodextrin encapsulation in the stock solution is also disrupted. The free carfilzomib in the stock solution may be carried into the filtrate by the unencapsulated sulbutylated beta-cyclodextrin, forming a stable encapsulated drug. Therefore, when using ultrafiltration tubes to separate free drugs from encapsulated drugs, it is easy to disrupt the dynamic equilibrium of the encapsulation process, making it impossible to accurately measure the encapsulation rate.

[0080] Comparative Example 2 Unlike Example 1, the sample solution (1) was prepared as follows: Approximately 0.5 g of the lyophilized carfilzomisulfonyl betacyclodextrin containing the drug was accurately weighed, 5 mL of ether was added, and the mixture was shaken for 5 minutes. The extract was then transferred to a 5 mL volumetric flask and allowed to evaporate naturally in a ventilated area until no ether odor remained. The evaporated product was dissolved in 40% acetonitrile aqueous solution and diluted to the mark. The mixture was then shaken well. The remaining steps were the same.

[0081] The accuracy of this method was examined, and the results are as follows: Table 7. Accuracy Test Results of Comparative Example 2

[0082] The results showed that due to the high volatility of ether, there was a loss due to evaporation during the transfer of the extract. In addition, the free drug has a low solubility in ether. These factors combined resulted in a low recovery rate and poor accuracy of the encapsulation efficiency determination.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the encapsulation efficiency of a drug encapsulated by sulfobutyl betacyclodextrin, characterized in that... Includes the following steps: (1) Take the lyophilized product of the drug encapsulated by sulfobutyl betacyclodextrin, add solvent 1, extract the dissolved free drug that is not encapsulated by sulfobutyl betacyclodextrin, take the supernatant or filtrate, and determine the amount of free drug using an appropriate method. (2) Take another lyophilized product containing sulfobutyl betacyclodextrin containing the drug, add solvent 2 to dissolve it completely, and determine the total amount of drug using an appropriate method; (3) Calculate the encapsulation efficiency of the included drug according to the following formula: 。 2. The determination method according to claim 1, characterized in that, The contents of the sulfobutyl betacyclodextrin inclusion complex include at least one of voriconazole, ziprasidone mesylate, aripiprazole, amiodarone hydrochloride, carfilzomib, posaconazole, melphalan hydrochloride, carbamazepine, derafloxacin, allogeneolone, remdesivir, fosphenytoin sodium, levothyroxine sodium, docetaxel, and fosaspirant.

3. The determination method according to claim 1, characterized in that, Solvent 1 is one or a combination of acetonitrile, anhydrous ethanol, isopropanol, acetone, n-propanol, tetrahydrofuran, n-butanol, isobutanol, tert-butanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, polyethylene glycol 400, Tween 80, and dioxane.

4. The determination method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the sulfobutyl betacyclodextrin-encapsulated drug and solvent 1 is 1:5-15, g:mL.

5. The determination method according to claim 4, characterized in that, In step (1), the mass-to-volume ratio of the sulfobutyl betacyclodextrin-encapsulated drug and solvent 1 is 1:10, g:mL.

6. The determination method according to claim 1, characterized in that, In step (1), the extraction is ultrasonic extraction or shaking extraction.

7. The determination method according to claim 6, characterized in that, In step (1), the extraction is a shaking extraction, and more specifically, the shaking extraction time is 1-10 min.

8. The determination method according to claim 1, characterized in that, In step (2), solvent 2 is one or a combination of water, formamide, ethylene glycol, dimethyl sulfoxide, and glycerol.

9. The determination method according to claim 8, characterized in that, In step (2), the solvent 2 is water.

10. The determination method according to claim 1, characterized in that, In step (1) or step (2), the appropriate method for determining the amount of free drug or the total amount of drug is liquid chromatography.

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