A method for determining the content of insulin liposome drug

Abstract

The present invention relates to the field of pharmaceutics, and discloses a method for determining the drug content of insulin liposomes, characterized in that the method comprises (1) membrane breaking: adding a membrane breaking agent composition to the insulin liposomes, mixing, and (2) detecting the insulin drug content by high performance liquid chromatography, wherein the membrane breaking agent composition comprises an acid and an organic solvent, the acid is selected from one or more of TFA, HCl, and H2SO4, and the organic solvent is selected from one or more of methanol, ethanol, and isopropanol. The method for determining the drug content of insulin liposomes of the present invention is convenient, easy to quantitatively analyze, and has no damage to the chromatographic column and the liquid phase system, laying a foundation for the accurate determination of hydrophilic protein polypeptide drug-related preparations.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutics, and in particular to a method for determining the content of insulin liposome drugs. Background Art

[0002] As the first choice drug for the treatment of insulin-dependent diabetes and severe non-insulin-dependent diabetes, insulin generally needs to be injected due to its large molecular weight, poor stability, low bioavailability and other limitations. Patient compliance is low, and long-term frequent injections will produce adverse reactions such as subcutaneous fat atrophy and subcutaneous nodules, causing great pain to patients. Therefore, drug research and development focuses on developing safe and effective non-injection methods to completely or partially replace insulin injections. Due to the characteristics of insulin itself, it is almost not absorbed when directly administered through non-injection routes, and pharmaceutical carriers can be used to present it to improve its non-injection bioavailability. Among pharmaceutical microparticle carriers, liposomes, especially flexible liposomes, have the advantages of enhanced permeability, increased solubility, high encapsulation rate, and low adverse reactions. Moreover, because they contain edge activators, they can penetrate pores much smaller than their own size in a complete form for drug delivery, becoming an effective and efficient carrier in the study of non-injection preparations of insulin.

[0003] Reliable analytical methods are the quality evaluation means for formulation process research. At present, the main method for determining the insulin content in insulin liposomes is to take insulin liposome suspension, break the membrane with sodium cholate solution, extract lipids with chloroform, vortex, mix and centrifuge to take the supernatant for determination (Zhang Li, Qi Xiangrong, Zhang Chunxia. Determination of drug content and encapsulation efficiency of insulin liposomes, Chinese Pharmacist. 2010, (03): 364-365; Na Zhang, Qi N. Ping, Gui H. Huang, et al. Investigation of lectin-modified insulin liposomes as carriers for oral administration, Pharmaceutical Nanotechnology, 2005, 294 (1-2): 247-259). This method is relatively cumbersome and time-consuming for the pretreatment process of insulin liposome samples, and the treatment process involves the mixing of solutions, which leads to certain deviations in the sample determination results. In addition, there are other documents that use insulin liposomes to directly enter the liquid phase for determination without treatment, and use the mobile phase to break the liposomes during the analysis process to determine the insulin content (You Xu, Yiyue Guo, Yuqi Yang, et al. Stabilization of Deformable Nanovesicles Based on Insulin-Phospholipid Complex by Freeze-Drying, Pharmaceutics, 2019, 11(10): 539; Zhang Qiang, Qi Xianrong. HPLC determination of insulin content in insulin liposomes. Chinese Modern Applied Pharmacy, 2001(06): 451-453). In this method, the organic components of the mobile phase cannot completely break down the liposomes with high lipid concentration. Direct sample injection and analysis of liposomes can easily cause blockage of the chromatographic column and liquid phase system for a long time, affecting the service life. Summary of the invention

[0004] The inventors found during the research process that a composition containing a specific acid and a specific organic solvent can be used to break the membrane of insulin liposomes. Based on this, the technical problem solved by the present invention is to provide a method for determining the drug content in insulin liposomes.

[0005] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for determining the drug content in insulin liposomes, characterized in that the method comprises (1) membrane disruption: adding a membrane disruption agent composition to the insulin liposomes and mixing, and (2) detecting the insulin drug content by high performance liquid chromatography, wherein the membrane disruption agent composition comprises an acid and an organic solvent, the acid is selected from one or more of TFA, HCl, and H2SO4, and the organic solvent is selected from one or more of methanol, ethanol, and isopropanol.

[0007] In one embodiment of the present invention, the insulin is selected from at least one of natural insulin, porcine insulin, bovine insulin, recombinant human insulin and various intermediate and long-acting insulins; the lipid material is selected from at least one of natural phospholipids, synthetic phospholipids, cholesterol, bile acid and its salts.

[0008] In one embodiment of the present invention, the insulin liposomes described in the present invention include liposomes prepared directly from insulin and lipids, and / or liposomes prepared from insulin phospholipid complexes and lipids, and the phospholipids in the insulin phospholipid complexes can be selected from at least one of natural phospholipids or synthetic phospholipids.

[0009] Optionally, the liposome is a flexible liposome containing a surfactant.

[0010] Optionally, the insulin liposome is prepared by any one of the following methods:

[0011] (1) Preparation of insulin phospholipid complex

[0012] The insulin phospholipid complex was prepared by the solvent evaporation method in accordance with the patent "An insulin lipid complex and its preparation method and preparation" (application number CN201110196733.3);

[0013] (2) The preparation of insulin phospholipid complex flexible liposomes can be selected from any of the following methods:

[0014] Method (A): dissolving the insulin phospholipid complex and the surfactant in an organic solvent, evaporating the solvent, and drying to obtain a thin film, and then obtaining a hydrated thin film. Specifically, the insulin phospholipid complex and the surfactant prepared in (1) are dissolved in an organic solvent, evaporating the solvent, and drying to obtain a thin film; hydration of the thin film: taking an appropriate amount of aqueous medium, adding it to the dried thin film, hydrating it at a certain temperature for a certain period of time, and preparing a "liposome solution" by homogenization, that is, obtaining the insulin phospholipid complex flexible liposome;

[0015] Method (B): dissolving the insulin phospholipid complex in an organic solvent, evaporating the solvent, drying to obtain a film, and hydrating the film with an aqueous medium containing a surfactant. Specifically, the insulin phospholipid complex prepared in (1) is dissolved in an organic solvent, evaporating the solvent, and drying to obtain a film; film hydration: dissolving a surfactant in an appropriate amount of aqueous medium, adding the film to the dried film, hydrating at a certain temperature for a certain period of time, and homogenizing to obtain a "liposome solution", that is, the insulin phospholipid complex flexible liposome;

[0016] Method (C): dissolving the insulin phospholipid complex and the surfactant in an organic solvent, adding an aqueous medium, homogenizing to form an emulsion, and removing the organic solvent to obtain. Specifically, the insulin phospholipid complex and the surfactant prepared in (1) are dissolved together in an organic solvent, then an aqueous medium is added, homogenizing to form an emulsion, and the organic solvent is removed by vacuum rotary evaporation to obtain a "liposome solution", that is, the insulin phospholipid complex flexible liposome;

[0017] Method (D): dissolving the insulin phospholipid complex in an organic solvent, mixing and homogenizing the mixture with an aqueous medium containing a surfactant to form an emulsion, and removing the organic solvent to obtain the liposome solution, i.e., obtaining the insulin phospholipid complex flexible liposome. Specifically, the insulin phospholipid complex prepared in (1) is dissolved in an organic solvent, and then the surfactant is dissolved in an aqueous medium, and the two are mixed and homogenized to form an emulsion, and the organic solvent is removed by vacuum rotary evaporation to obtain a "liposome solution", i.e., obtaining the insulin phospholipid complex flexible liposome.

[0018] (3) Preparation of insulin common flexible liposomes

[0019] The insulin phospholipid complex in (2) was replaced by insulin to prepare insulin ordinary flexible liposomes.

[0020] (4) Preparation of insulin phospholipid complex liposomes

[0021] The insulin phospholipid complex and the lipid material are dissolved in an organic solvent, the solvent is evaporated, and a thin film is obtained by drying. Specifically, the insulin phospholipid complex and the lipid material prepared in (1) are dissolved in an organic solvent, the solvent is evaporated, and a thin film is obtained; thin film hydration: an appropriate amount of aqueous medium is added to the dried thin film, hydrated at a certain temperature for a certain time, and a "liposome solution" is prepared by homogenization, that is, the insulin phospholipid complex ordinary liposome is obtained.

[0022] (5) Preparation of insulin liposomes

[0023] Dissolve insulin and lipid materials in an organic solvent, evaporate the solvent, and dry to obtain a thin film. Specifically, dissolve insulin and lipid materials in an organic solvent, evaporate the solvent, and dry to obtain a thin film. Hydration of the thin film: take an appropriate amount of aqueous medium, add it to the dried thin film, hydrate it at a certain temperature for a certain period of time, and prepare a "liposome solution" by homogenization, that is, ordinary insulin liposomes.

[0024] In one embodiment of the present invention, the weight content of insulin drug in the insulin liposome is 0.004-2%, and the weight content of lipid is 0.012-50%.

[0025] In one embodiment of the present invention, the volume ratio of the insulin liposome to the membrane-disrupting agent composition is 1:(5-50), preferably 1:(25-50).

[0026] In one embodiment of the present invention, the weight percentage of the organic solvent in the membrane-disrupting agent composition is 75% to 100%, preferably 90% to 100%.

[0027] In one embodiment of the present invention, the acid in the membrane-breaking agent composition is TFA, with a weight percentage of 0.01-0.5%, preferably 0.01-0.1%.

[0028] In one embodiment of the present invention, the acid in the membrane-breaking agent composition is H2SO4, with a weight percentage of 0.01-0.1%.

[0029] In one embodiment of the present invention, the acid in the membrane-breaking agent composition is HCl, with a weight percentage of 0.1-0.5%.

[0030] In one embodiment of the present invention, the organic solvent is methanol.

[0031] In one embodiment of the present invention, the insulin liposome is a flexible liposome.

[0032] The method of the invention is easy to use, effectively breaks the membrane and dissolves the drug, is easy to quantitatively analyze, and has no damage to the chromatographic column and the liquid phase system, thus laying a foundation for the accurate determination of hydrophilic protein polypeptide drugs and related preparations.

[0033] The temperature or content range disclosed in the present invention is expressed in the form of an upper limit and a lower limit, which can be one or more lower limits, or one or more upper limits respectively; a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range, and all ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range; for example, for a specific parameter, a range of 1-40 and 0.6-50 is listed, and it is understood that a range of 0.6-40 and 1-50 is also expected; in addition, if the minimum range values ​​1 and 0.5 are listed, and if the maximum range values ​​50, 40 and 30 are listed, the following ranges are all expected: 1-30, 1-40, 1-50, 0.5-30, 0.5-40, 0.5-50.

[0034] If not otherwise specified, the weight percentages referred to in the present invention are based on the total weight of the composition.

[0035] Beneficial technical effects

[0036] The present invention uses a composition containing acid and organic solvent to perform membrane rupture treatment on insulin liposomes and then conducts liquid phase determination. This method can accurately quantify the drug in the insulin liposomes without damaging the chromatographic column and the liquid phase system. At the same time, it solves the disadvantages of complicated pre-treatment operations and measurement deviations in the existing measurement technology, ensures that the insulin liposomes are effectively ruptured and the content results are accurately determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the chromatogram of the solvent (0.01% TFA-methanol) in the chromatographic detection of the content of insulin flexible liposomes.

[0038] Figure 2 This is the chromatogram of blank flexible liposomes (blank solution) in the chromatographic detection of insulin flexible liposome content.

[0039] Figure 3 This is the chromatogram of insulin (reference solution) in the chromatographic detection of insulin flexible liposome content.

[0040] Figure 4 This is the chromatogram of insulin phospholipid complex flexible liposomes (test solution) in the chromatographic detection of insulin flexible liposome content. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below by means of the accompanying drawings and examples. Through these exemplary descriptions, the features and advantages of the present invention will become clearer and more specific. Any embodiment described as an example here need not be interpreted as being superior or better than other embodiments.

[0042] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Example 1 Preparation of liposomes

[0044] a. Preparation of insulin phospholipid complex: Prepare by solvent evaporation method. Weigh 60 mg of insulin and dissolve it in 6 mL of 0.1% TFA methanol solution; weigh 600 mg of phospholipid and dissolve it in 54 mL of dichloromethane; mix the above two solutions evenly, and evaporate them under reduced pressure and rotary evaporation at 37°C. When the remaining amount of solvent is about the initial volume of methanol, add 1 times the amount of dichloromethane, dry it, and place it in a vacuum drying oven and dry it overnight to obtain it.

[0045] b. Preparation of flexible liposomes of insulin phospholipid complex: weigh 600 mg of phospholipids, 660 mg of phospholipid complex equivalent to 60 mg of insulin, and 400 mg of Tween20, place in a 100 mL round-bottom flask, add 20 mL of dichloromethane to dissolve them completely, and rotary evaporate to form a film in a 37°C water bath, and vacuum dry for 2 hours; weigh 100 mg of sodium cholate, add an appropriate amount of 0.02 mol / L PBS (pH 7.4) to prepare a PBS solution of sodium cholate, add the solution to the lipid film prepared above, and hydrate for 30 minutes. After homogenization, filter through a 0.22 μm filter membrane to obtain.

[0046] Example 2 Membrane-breaking solvent composition

[0047] (1) Selection of organic solvent

[0048] Accurately pipette 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1 into a 50 mL volumetric flask, and add methanol, ethanol, isopropanol, and acetonitrile containing 0.1% TFA respectively, and gently shake while adding to observe the opalescence of the liposome.

[0049] Table 1 Results of membrane disruption treatment with different organic solvents containing 0.1% TFA

[0050]

[0051]

[0052] (2) Choice of acid

[0053] Accurately pipette 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1 into a 50 mL volumetric flask, add 0.1% TFA, HCl, H2SO4, HAC, H3PO4 methanol solution, and observe that the insulin liposome is dissolved in the methanol solution of various acids. The results are as follows:

[0054] Table 2 Membrane rupture treatment results of methanol solution containing different acids

[0055]

[0056] Accurately pipette 1 mL of the insulin phospholipid complex flexible liposomes prepared in Example 1 into a 50 mL volumetric flask, add 0.01%, 0.1% and 0.5% methanol solutions of TFA, HCl and H2SO4 respectively, and observe the rupture of the liposome membrane.

[0057] Table 3 Effect of acid content on membrane rupture treatment

[0058]

[0059] Accurately pipette 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1 into a 50 mL volumetric flask, add 0.01%, 0.1% and 0.5% TFA, HCl, and H2SO4 methanol solutions respectively, and observe the dissolution. Then prepare a stock solution with a concentration of 100 ug / ml, and perform liquid phase analysis at 0h, 3h, 6h, 9h and 12h to evaluate its solution stability. The results are as follows:

[0060] Table 4 Effect of acid content on solution stability after membrane rupture treatment

[0061] TFA HCl <![CDATA[H2SO4]]> 0.01％ 12h stable \ Low response value and poor solution stability 0.1％ 9h stable Low response value and poor solution stability Low response value and poor solution stability 0.5％ Unstable Low response value and poor solution stability \

[0062] (3) Organic solvent concentration

[0063] 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1 was accurately pipetted into a 50 mL volumetric flask, and 0.1% TFA methanol aqueous solution containing 50%, 75% and 90% methanol was added respectively, and the opalescence of the liposome was observed.

[0064] Table 5 Effect of organic solvent concentration on membrane rupture treatment

[0065]

[0066] (4) Acid concentration

[0067] Accurately pipette 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1 into a 50 mL volumetric flask, add 0.01%, 0.1% and 0.5% TFA methanol and 90% methanol aqueous solution, respectively, to prepare a stock solution with a concentration of 100 ug / ml, and perform liquid phase analysis at 0 h, 3 h, 6 h, 9 h and 12 h, respectively, to evaluate the solution stability.

[0068] Table 6 Effect of organic solvent concentration on solution stability after membrane rupture treatment

[0069] Methanol solution 90% methanol in water 0.01％ 12h 12h 0.1％ 9h 9h 0.5％ Poor solution stability Poor solution stability

[0070] Example 3

[0071] On the basis of Example 1, 0.8 mg, 200 mg and 400 mg of insulin were used respectively, and the other prescriptions and processes were not changed to prepare insulin phospholipid complex flexible liposomes, and the obtained complex flexible liposomes had insulin weight percentages of 0.004%, 1% and 2%, respectively; in the preparation of insulin phospholipid complex flexible liposomes, 2.4 mg, 2.5 g and 5 g of phospholipids were used, and the other prescriptions and processes were not changed to prepare insulin phospholipid complex flexible liposomes, and the obtained complex flexible liposomes had phospholipid weight percentages of 0.012%, 25% and 50%, respectively.

[0072] 1 mL of the insulin phospholipid complex flexible liposome prepared in this example was accurately pipetted into 5, 25, and 50 mL volumetric flasks for quantitative analysis, and 0.01%, 0.1%, and 0.5% TFA methanol solutions were added to dilute to the scale, and 1 mL of insulin flexible liposomes was accurately pipetted and diluted to the corresponding volume with PBS, and the corresponding mass of insulin was weighed and dissolved in the corresponding volume of acid-free methanol solution to observe the membrane rupture and dissolution. The results are as follows:

[0073] (1) The volume ratio of insulin phospholipid complex flexible liposome to solvent is 1:5

[0074]

[0075] *Note: The opalescence states in the table are lighter than the opalescence of insulin flexible nanovesicles diluted with PBS, and the precipitates produced are less than the precipitates produced by the corresponding insulin dissolved in methanol solution without acid.

[0076] (2) The volume ratio of insulin phospholipid complex flexible liposome to solvent is 1:25

[0077]

[0078]

[0079] (3) The volume ratio of insulin phospholipid complex flexible liposome to solvent is 1:50

[0080]

[0081] Example 4 Determination of drug content in insulin phospholipid complex flexible liposomes

[0082] Chromatographic conditions

[0083] Chromatographic column: Agilent ZORBAX 300SB-C18 (250×4.6 mm, 5 μm);

[0084] Mobile phase: 0.2 mol / L sulfate buffer (take 28.4 g of anhydrous sodium sulfate, dissolve it in water, add 2.7 mL of phosphoric acid and water to 800 mL, adjust the pH value to 2.3 with ethanolamine, and add water to 1000 mL)-acetonitrile (74-26);

[0085] Column temperature: 40°C; flow rate: 1.00 mL / min; detection wavelength: 214 nm;

[0086] Injection volume: 20.0 μL, solvent: 0.01% TFA-methanol

[0087] Content determination

[0088] Accurately weigh 10 mg of insulin, place it in a 10 mL volumetric flask, dissolve it with 0.01% TFA methanol solution and dilute to the mark, shake well, accurately transfer 0.6 ml to a 10 mL volumetric flask, dilute to the mark with 0.01% TFA-methanol, shake well, and prepare a reference solution with a concentration of 60 μg / mL. Accurately transfer 1 mL of the insulin phospholipid complex flexible liposome prepared in Example 1, place it in a 50 mL volumetric flask, add 0.01% TFA-methanol to the mark, shake well, and use it as the test solution. Prepare a blank flexible liposome solution according to the method of Example 1 without adding insulin, accurately measure 1 mL of blank flexible liposome, place it in a 50 mL volumetric flask, dilute to the mark with 0.01% TFA-methanol, shake well, and use it as a blank solution. Take 0.01% TFA-methanol, reference solution, test solution and blank solution respectively, and inject and analyze under chromatographic conditions: chromatographic column: Agilent ZORBAX300SB-C18 (250×4.6mm, 5μm); mobile phase: 0.2mol / L sulfate buffer (take 28.4g of anhydrous sodium sulfate, dissolve it in water, add 2.7mL of phosphoric acid, 800mL of water, adjust the pH value to 2.3 with ethanolamine, and add water to 1000mL)-acetonitrile (74:26); column temperature: 40℃; flow rate: 1.00mL / min; detection wavelength: 214nm; injection volume: 20.0μL, and record the chromatogram as shown in the figure.

[0089] The results of methodological validation showed that the content of insulin phospholipid complex flexible liposomes was determined using the above method, and at a wavelength of 214nm, neither solvents nor excipients interfered with the determination; in the range of 6 to 180ug / mL, the peak area and concentration had a good linear relationship; the limit of quantification was 1.500ug / mL; samples of three concentrations, low (6ug / mL), medium (60ug / mL), and high (180ug / mL), were injected continuously for 5 times, and the RSD was in the range of 0.05% to 1.84%; the solution had good stability within 12 hours at room temperature; the average recovery rate of samples at three levels of 80%, 100%, and 120% was 98.86%, and the RSD was 1.01% (n=9); the RSD of the repeated determination of the content of 6 samples was 1.11%.

[0090] The present invention has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, the present invention may be subjected to a variety of substitutions and improvements, all of which fall within the scope of protection of the present invention.

Claims

1. A method for determining the content of insulin liposome drug, characterized in that: The method comprises (1) membrane disruption: adding a membrane disruption agent composition to the insulin liposomes, mixing, and (2) High performance liquid chromatography was used to detect the content of insulin. The membrane-breaking agent composition is an acid-containing organic solvent or an acid-containing organic solvent aqueous solution. The acid in the membrane-breaking agent composition is TFA, with a weight percentage of 0.01-0.1%. The organic solvent is selected from one or more of methanol, ethanol, and isopropanol. The organic solvent aqueous solution is a 75% methanol aqueous solution or a 90% methanol aqueous solution. The weight content of the insulin drug in the insulin liposome is 0.004-2%, and the weight content of the phospholipid is 0.012-50%.

2. The method for determining the insulin liposome drug content according to claim 1, wherein: The volume ratio of the insulin liposome to the membrane-breaking agent composition is 1:(5-50).

3. The method for determining the content of insulin liposome drug according to any one of claims 1 to 2, characterized in that: The organic solvent is methanol.

4. The method for determining the content of insulin liposome drug according to any one of claims 1 to 2, characterized in that: The insulin liposome is a flexible liposome.

Images