Liposome drug protective agent and application thereof

Abstract

The invention discloses a lipidosome drug protective agent and application thereof. The lipidosome drug protective agent comprises 10.0-50.0% (m / v) of polyhydric alcohol, 10.0-50.0% (m / v) of saccharides, 10.0-50.0 mM of an antioxidant and an enzyme inhibitor. The solvent is water; wherein the enzyme inhibitor comprises a protease inhibitor and / or a phosphatase inhibitor. The protective agent can be used for biological analysis of lipidosome drugs, and the situation that due to the fact that lipidosome in plasma leaks in the in-vitro storage and transportation process, the content of free drugs is not accurately measured, and the experimental result is affected is avoided. And the requirement of a producer for accurately measuring the concentration of the free medicine in the production process can be met.

Description

Technical Field

[0001] The invention relates to the technical field of preclinical and clinical bioanalysis of drugs, and in particular to a protective agent for liposome drugs and application thereof. Background Art

[0002] Liposomes are widely used as drug carriers for small molecule drugs, proteins, nucleic acids and imaging agents. They have the characteristics of protecting the encapsulated active ingredients from degradation by the physiological environment, prolonging the half-life of drugs, controlling the release of drug molecules, good biocompatibility and safety, etc. They can also selectively deliver the encapsulated active ingredients to the lesion site through passive and / or active targeting, thereby reducing systemic side effects, increasing the maximum tolerated dose and improving the therapeutic effect. Although liposome drugs have good efficacy, they face huge challenges from the formulation of drug preparations to bioanalysis in preclinical and clinical studies. Most of the liposomal drugs exist in the body in the form of loads, and only a small part of the drugs are free outside the liposomes. For samples with very little free drug content (e.g. <1%), leakage of drugs inside the liposomes or liposome contamination during sample storage or extraction will lead to a significant increase in the free drug concentration, thus generating misleading data.

[0003] The stability of the sample storage process is the key to the reliability of the results of the liposome free component determination, especially to avoid freezing and thawing as much as possible during storage and analysis. According to literature reports, the best method is to separate the free drug from the liposome drug immediately after the plasma sample is freshly collected, which requires appropriate equipment and experienced analysts at the sample collection site. If freezing and thawing cannot be avoided for biological samples, cryoprotectants need to be added to the samples to prevent the liposomes from rupturing during the freezing and thawing steps. Therefore, the formulation development of the protective agent is particularly important.

[0004] At the same time, for liposome drugs after in vivo administration, a suitable and reliable pretreatment method to separate free drugs from loaded drugs is crucial. Existing literature reports that pretreatment methods include gel filtration, ultrafiltration, ultracentrifugation and solid phase extraction (SPE). Among them, the matrix removal of gel filtration is not clean, which is prone to matrix effect; although ultracentrifugation and ultrafiltration can measure the concentration of free drugs, it is easy to cause inaccurate measurement due to residues; and solid phase extraction SPE is the most commonly used in vivo sample analysis method, which can measure free and loaded drugs at the same time, with less residues, and can eliminate matrix effects. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing technology can only separate free drugs from liposome drugs immediately after collecting plasma and then store them, and the stability of the drugs is difficult to ensure.

[0006] In order to solve the above technical problems, the present invention provides a protective agent for liposome drugs and its application, which can protect liposomes from leakage during transportation and storage, and then transport the samples to the laboratory for storage for a period of time before testing.

[0007] The present invention solves the above technical problems through the following technical solutions.

[0008] Specifically, the present invention provides a protective agent, which comprises 10.0-50.0% (m / v) of polyol, 10.0~50.0% (m / v) of sugar, 10.0~50.0 mM of antioxidant, and enzyme inhibitor; the solvent is water.

[0009] In some embodiments, the enzyme inhibitor comprises a protease inhibitor and a phosphatase inhibitor.

[0010] In some embodiments, the enzyme inhibitor comprises a protease inhibitor or a phosphatase inhibitor.

[0011] In some embodiments, the enzyme inhibitor is a commercial enzyme inhibitor.

[0012] In some embodiments, the content of the polyol is 20.0-31.25% (m / v).

[0013] In some embodiments, the content of the carbohydrate is 10.0-15.0% (m / v).

[0014] In some embodiments, the antioxidant is present in an amount of 18.75-31.25 mM.

[0015] In some embodiments, the enzyme inhibitor is selected from A32961 from Thermo Scientific™ and 11836170001 from Roche.

[0016] In some embodiments, the enzyme inhibitor is present in an amount of 0.1-1.0 tablet / mL.

[0017] In some embodiments, the content of the enzyme inhibitor is 0.60-1.0 tablet / mL.

[0018] In some specific embodiments, the content of the polyol is 25.0% (m / v).

[0019] In some specific embodiments, the content of the carbohydrate is 12.5% ​​(m / v).

[0020] In some specific embodiments, the content of the antioxidant is 25.0 mM.

[0021] In some embodiments, the carbohydrate is a disaccharide.

[0022] In some embodiments, the polyol is an alcohol compound containing 2 to 6 hydroxyl groups.

[0023] In some embodiments, the antioxidant is selected from one or more of vitamin C, glutathione, PB2, dithioerythritol, THPP and TCEP.

[0024] In some embodiments, the carbohydrate is a disaccharide.

[0025] In some embodiments, the polyol is an alcohol compound containing 2 to 6 hydroxyl groups.

[0026] In some embodiments, the antioxidant is selected from one or more of vitamin C, glutathione, PB2, dithioerythritol, THPP and TCEP.

[0027] In some embodiments, the enzyme inhibitor is A32961 from Thermo Scientific™.

[0028] In some specific embodiments, the disaccharide is selected from one or more of glucose, lactose, sucrose, trehalose and cellobiose.

[0029] In some specific embodiments, the polyol is selected from one or more of ethylene glycol, propylene glycol, glycerol, mannitol and sorbitol.

[0030] In some specific embodiments, the antioxidant is TCEP.

[0031] In some specific embodiments, the sugar is glucose.

[0032] In some specific embodiments, the polyol is glycerol.

[0033] In some specific embodiments, the protective agent comprises 25.0% (m / v) glycerol, 12.5% ​​(m / v) glucose, 25.0 mM TCEP, and 0.8 tablets / mL of enzyme inhibitor;

[0034] Wherein, the enzyme inhibitor is A32961 from Thermo Scientific™.

[0035] The invention also provides a composition, which comprises the liposome medicine and the protective agent.

[0036] In some embodiments, the content of the liposome drug is 0.5 μg / mL to 500 μg / mL.

[0037] The present invention also provides a kit, which comprises the protective agent.

[0038] The present invention also provides a method for preventing leakage of liposome drugs in a sample, the method comprising mixing plasma containing the liposome drugs with the protective agent, wherein the volume ratio of the protective agent to the sample is (1-10):3.

[0039] In some embodiments, the volume ratio of the protective agent to the sample is (2-5):3.

[0040] In some specific embodiments, the volume ratio of the protective agent to the sample is 2:3.

[0041] In some embodiments, the plasma is human plasma or animal plasma.

[0042] In some specific embodiments, the plasma is cynomolgus monkey plasma.

[0043] The present invention also provides a method for separating free drugs in a sample to be tested, the method comprising the following steps:

[0044] The samples to be tested were separated by SPE, and the drug content in the obtained eluate was determined by mass spectrometry.

[0045] In some embodiments, the sample is a plasma sample.

[0046] In some specific embodiments, the sample is human plasma or animal plasma.

[0047] In some specific embodiments, the drug is doxorubicin hydrochloride.

[0048] The present invention also provides the use of the protective agent or the kit in improving the stability of liposome drugs.

[0049] In some specific embodiments, the liposomal drug is a liposomal drug in plasma.

[0050] In some specific embodiments, the liposome drug is doxorubicin hydrochloride liposome.

[0051] In some specific embodiments, the use is for non-diagnostic or non-therapeutic purposes.

[0052] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0053] The reagents and raw materials used in the present invention are commercially available.

[0054] The positive and progressive effects of the present invention are:

[0055] The protective agent provided by the present invention comprises glycerol, sugars, enzyme inhibitors and antioxidants. The protective agent can be used as a protective agent for liposome drug bioanalysis to prevent leakage of liposomes in plasma during in vitro storage and transportation, resulting in inaccurate determination of free drug content and affecting experimental results. It can then be transported to a laboratory for storage for a period of time after sampling and then tested while maintaining the accuracy of the test, which can meet the needs of manufacturers for accurate determination of free drug concentrations during the production process, and also provides an effective means for pharmacokinetic (PK) testing of similar drugs. DETAILED DESCRIPTION

[0056] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used for the present invention, but not limited to the present invention.

[0057] The invention provides a protective agent based on doxorubicin hydrochloride liposomes in plasma and application thereof. The protective agent comprises glycerol, sugars, enzyme inhibitors and antioxidants, and can be used as a protective agent for doxorubicin hydrochloride liposome drug bioanalysis. Solid phase extraction (SPE) is used to separate the doxorubicin hydrochloride liposomes in plasma, and the free doxorubicin hydrochloride content is determined by LC-MS / MS.

[0058] The protective agent comprises glycerol, sugar, enzyme inhibitor and antioxidant, the sugar is glucose, the enzyme inhibitor is protease and phosphatase inhibitor tablets, and the antioxidant is tri(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0059] A glucose concentration of 10.0-15.0% (m / v) is selected as one of the components of the protective agent. The above-defined concentration range can avoid a concentration that is too low to maintain the osmotic pressure of the liposome during freeze-thaw, and a concentration that is too high to cause leakage.

[0060] The dosage of the protease and phosphatase inhibitor tablets is 0.60~1.0 dose / ml. The above-defined range avoids the situation where too low a dosage leads to insufficient enzyme inhibition effect and too high a dosage leads to waste of reagents.

[0061] The concentration of tri(2-carboxyethyl)phosphine hydrochloride (TCEP) is selected to be 18.75~31.25mM. The above-defined range avoids insufficient antioxidant capacity due to a too low concentration and plasma precipitation due to a too high concentration.

[0062] The concentration of glycerol is selected to be 20.0~31.25% (m / v). The above-limited range avoids a concentration that is too low to provide protection and a concentration that is too high to cause liposome rupture due to high viscosity.

[0063] The volume ratio of the protective agent to the plasma is selected to be 2:3, and the plasma is human plasma or preclinical animal plasma, including mouse plasma, rat plasma or cynomolgus monkey plasma.

[0064] The separation method of free doxorubicin hydrochloride adopts SPE separation, comprising the following steps:

[0065] Step 1: Activate the SPE with pure methanol, equilibrate with 5% glucose solution (m / v), and equilibrate again with a plasma glucose mixture (volume ratio of plasma to 5% glucose aqueous solution is 1:3) before loading;

[0066] Step 2: The plasma containing doxorubicin hydrochloride liposomes and the sample to be tested are loaded, and washed three times with a 5% (m / v) glucose aqueous solution, and then washed once with a 5% (v / v) methanol aqueous solution;

[0067] Step 3: using a methanol solution containing 1% formic acid to elute free doxorubicin hydrochloride and collect it;

[0068] Step 4: Determine the eluted free doxorubicin hydrochloride by LC-MS / MS and calculate the content;

[0069] When doxorubicin hydrochloride was detected by LC-MS / MS, the steps were as follows: separation was performed using a UPLC system, the analytical sample was placed in a 4°C autosampler, the column temperature was 40°C, the flow rate was 450 μL / min, and the injection volume was 10 μL; the relevant liquid phase gradient was as follows: 0-0.5min, phase B was maintained at 15%; 0.5-2.5min, phase B changed linearly from 15% to 95%; 2.5-3.5min, phase B was maintained at 95%; 3.5-3.51min, phase B changed linearly from 95% to 15%; 3.51-4.0min, phase B was maintained at 15%; mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid.

[0070] The mass spectrometry conditions used in LC-MS / MS analysis were set as follows:

[0071] TRIPLE QUAD™ 5500 mass spectrometer (SCIEX) was used for mass spectrometry analysis in positive ion mode. The ESI source positive ion conditions were as follows: Collision Gas: 9; source temperature: 300°C; ion Source Gas1 (Gas1): 40; Ion Source Gas2 (Gas2): 40; Curtain gas (CUR): 40; ion Sapary Voltage Floating (ISVF): 5500 V. The MRM mode was used to detect the ion pairs to be measured. The MRM ion pair information is shown in Table 1.

[0072] Table 1 MRM ion pair information

[0073]

[0074] The data were analyzed using Analyst software. The theoretical concentration of doxorubicin hydrochloride in the standard curve was calculated by linear least squares regression using the ratio of the peak area of ​​doxorubicin hydrochloride to the peak area of ​​the isotope internal standard of doxorubicin hydrochloride. The actual concentration of doxorubicin hydrochloride in the sample was calculated using the obtained regression equation, with a weight factor of 1 / x 2 .

[0075] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0076] Example 1

[0077] Main reagents, consumables and instruments

[0078] Human plasma (from Kangya Hospital, Yiyang, Hunan, EDTA-K2 anticoagulant, half plasma from 10 men and half from women)

[0079] HLB plate (Manufacturer: waters, Specification: 30 mg Sorbent per Well, 30μm, 1 / pk, Catalog No.: WAT058951)

[0080] Doxorubicin hydrochloride liposomes (Manufacturer: Shanghai Fudan Zhangjiang Biopharmaceuticals, Specification: 20mg, 10ml)

[0081] Doxorubicin hydrochloride (Manufacturer: MCE, Product No.: HY-15142)

[0082] Doxorubicin hydrochloride isotope internal standard (Manufacturer: Shanghai Zhenzhun Biotechnology Co., Ltd., Catalog No.: IR-15141)

[0083] Glycerin (Manufacturer: Aladdin, Item No.: G116205)

[0084] Glucose (Manufacturer: Aladdin, Product No.: G107850)

[0085] Protease and phosphatase inhibitor tablets (Manufacturer: Thermo Scientific™, Catalog No.: A32961)

[0086] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Manufacturer: Sigma, Catalog No.: 68957)

[0087] Instrument (AB SCIEX TRIPLE QUAD™ 5500 LC / MS)

[0088] Chromatographic column (ACQUITY UPLC® BEH C18, 1.7 µm, 2.1*50 mm, Part No.: 186002350)

[0089] Step 1: Preparation of protective agent

[0090] S1: Weigh 5.0 g of glycerol into a 15 ml centrifuge tube, add 10.0 ml of purified water, mix well, and prepare a concentration of 50%;

[0091] S2: Weigh 35.83 mg of tri(2-carboxyethyl)phosphine hydrochloride (TCEP) into a 5 ml centrifuge tube, add 4 doses of protease and phosphatase inhibitor tablets, weigh 0.625 g of glucose powder and add it into the above 5 ml centrifuge tube, draw 2.5 ml of purified water and add it into the centrifuge tube, and mix by ultrasonication.

[0092] S3: Pipette 2.0 mL of the solutions in S1 and S2 respectively and mix them.

[0093] Step 2: Stability test

[0094] S1: Pipette 10 µL of liposome doxorubicin hydrochloride solution with a doxorubicin hydrochloride concentration of 2 mg / mL, add 990 µL of human plasma, and mix by pipetting 4-5 times;

[0095] S2: Pipette 400µL of protective agent and 600µL of doxorubicin hydrochloride liposome plasma into a 1.5ml centrifuge tube, and mix them by pipetting 4-5 times;

[0096] S3: Store some of the prepared samples in a -70~-90℃ refrigerator for 7 days before testing, freeze-thaw three times in between, with the interval between each freeze-thaw being no less than 24 hours;

[0097] Step 3: Determination of free doxorubicin hydrochloride content

[0098] S1: Prepare doxorubicin hydrochloride solutions with linear concentrations of 5, 10, 50, 100, 500, 1000, 4000, and 5000 ng / mL using blank matrix (volume ratio of protective agent to human plasma is 2:3);

[0099] S2: Add 1 mL of pure methanol to activate the SPE, then add 1 mL of 5% glucose solution (m / v) for equilibrium, and then add 200 µL of a mixture of plasma and 5% glucose (v / v = 1:3) for equilibrium;

[0100] S3: Pipette 50µL of the S3 solution, S2 solution and the standard curve in step 3 into the SPE plate, add 10µL of 10µg / mL doxorubicin hydrochloride isotope internal standard solution, and then add 150µL of 5% glucose water and let it drain naturally;

[0101] S4: Pipette 300 µL of 5% (m / v) glucose aqueous solution for washing, repeat 3 times, and then pipette 300 µL of 5% (v / v) methanol aqueous solution for washing once;

[0102] S5: Pipette 300 µL of methanol solution containing 1% formic acid for elution and collect the eluate, repeat 3 times;

[0103] S6: measuring the solution collected in S5 in step 3 by LC-MS / MS;

[0104] S7: When using LC-MS / MS to detect doxorubicin hydrochloride, the steps are as follows: use the UPLC system for separation, place the analytical sample in a 4°C autosampler, the column temperature is 40°C, the flow rate is 450 μL / min, and the injection volume is 10 μL; the relevant liquid phase gradient is as follows: 0-0.5min, phase B is maintained at 15%; 0.5-2.5min, phase B changes linearly from 15% to 95%; 2.5-3.5min, phase B is maintained at 95%; 3.5-3.51min, phase B changes linearly from 95% to 15%; 3.51-4.0min, phase B is maintained at 15%; mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

[0105] S8: The mass spectrometry conditions used in LC-MS / MS analysis were set as follows:

[0106] TRIPLE QUAD™ 5500 mass spectrometer (SCIEX) was used for mass spectrometry analysis in positive ion mode. The ESI source positive ion conditions were as follows: Collision Gas: 9; source temperature: 300°C; ion Source Gas1 (Gas1): 40; Ion Source Gas2 (Gas2): 40; Curtain gas (CUR): 40; ion Sapary Voltage Floating (ISVF): 5500 V. The MRM mode was used to detect the ion pairs to be measured. The MRM ion pair information is shown in Table 1.

[0107] S9: Analyst software was used to analyze the data. The peak area of ​​doxorubicin hydrochloride was compared with the peak area of ​​the isotope internal standard of doxorubicin hydrochloride to perform a linear least squares regression calculation on the theoretical concentration of doxorubicin hydrochloride in the standard curve. The obtained regression equation was used to calculate the measured concentration of doxorubicin hydrochloride in the sample, with a weight factor of 1 / x 2 .

[0108] The calculation formula is as follows:

[0109]

[0110]

[0111] T0: Mean value of free doxorubicin hydrochloride content in samples at zero hour

[0112] Tx: Free doxorubicin hydrochloride content of stability samples

[0113] Acceptance criteria: The difference should be no greater than 15.0%, and the coefficient of variation (%CV) should be within 15.0%.

[0114] The results are shown in Table 2 , which shows that the difference was no greater than 15%, and the coefficient of variation (CV) was within 15%, indicating that no leakage of doxorubicin hydrochloride liposomes occurred.

[0115] Table 2 Results in Example 1

[0116]

[0117] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0118] Example 2

[0119] The specific implementation of Example 2 is the same as that of Example 1, except that the plasma in step 2 is cynomolgus monkey plasma, which is sourced from Shanghai Inos Biotechnology Co., Ltd., and the EDTA-K2 anticoagulant is used. The plasma of 6 male and female monkeys is mixed in half. The results are shown in Table 3. Table 3 shows that the difference is no more than 15%, and the coefficient of variation (CV) is within 15%, indicating that the doxorubicin hydrochloride liposomes did not leak.

[0120] Table 3 Results in Example 2

[0121]

[0122] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0123] Comparative Example 1

[0124] The specific implementation of Comparative Example 1 is the same as that of Example 1, except that the protective agent in step 1 is purified water. The results are shown in Table 4. Table 4 shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0125] Table 4 Results of Comparative Example 1

[0126]

[0127] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0128] Comparative Example 2

[0129] The specific implementation of Comparative Example 2 is the same as that of Example 1, except that the protective agent formula in step 1 is as follows:

[0130] S1: Weigh 5.0 g of glycerol into a 15 ml centrifuge tube, add 10.0 ml of purified water, mix well, and prepare a concentration of 50%;

[0131] S2: Weigh 0.625 g of glucose powder into a 5 ml centrifuge tube, add 4 protease and phosphatase inhibitor tablets, pipette 2.5 ml of purified water into the centrifuge tube, and mix by ultrasonication.

[0132] S3: 2.0 mL of the solution in S1 and S2 were taken and mixed. The results are shown in Table 5, which shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0133] Table 5 Results of Comparative Example 2

[0134]

[0135] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0136] Comparative Example 3

[0137] The specific implementation of Comparative Example 3 is the same as that of Example 1, except that the protective agent formula in step 1 is as follows:

[0138] S1: Weigh 5.0 g of glycerol into a 15 ml centrifuge tube, add 10.0 ml of purified water, mix well, and prepare a concentration of 50%;

[0139] S2: Weigh 0.625 g of glucose powder into a 5 ml centrifuge tube, pipette 2.5 ml of purified water into the centrifuge tube, and mix by ultrasonication.

[0140] S3: 2.0 mL of the solution in S1 and S2 were drawn and mixed. The results are shown in Table 6, which shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0141] Table 6 Results of Comparative Example 3

[0142]

[0143] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0144] Comparative Example 4

[0145] The specific implementation of Comparative Example 4 is the same as that of Example 1, except that the protective agent formula in step 1 is as follows:

[0146] S1: Weigh 5.0 g of glycerol into a 15 ml centrifuge tube, add 10.0 ml of purified water, mix well, and prepare a concentration of 50%;

[0147] S2: Weigh 35.83 mg of tri(2-carboxyethyl)phosphine hydrochloride (TCEP) into a 5 ml centrifuge tube, add 4 protease and phosphatase inhibitor tablets, pipette 2.5 ml of purified water into the centrifuge tube, and mix by ultrasonication.

[0148] S3: 2.0 mL of the solution in S1 and S2 were drawn and mixed. The results are shown in Table 7, which shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0149] Table 7 Results of Comparative Example 4

[0150]

[0151] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0152] Comparative Example 5

[0153] The specific implementation of Comparative Example 5 is the same as that of Example 1, except that the protective agent formula in step 1 is as follows:

[0154] S1: Weigh 35.83 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) into a 5 ml centrifuge tube, add 4 doses of protease and phosphatase inhibitor tablets, weigh 0.625 g of glucose powder into the above 5 ml centrifuge tube, draw 5.0 ml of purified water into the centrifuge tube, and mix by ultrasonic. The results are shown in Table 8, which shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0155] Table 8 Results in Comparative Example 5

[0156]

[0157] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

[0158] Comparative Example 6

[0159] The specific implementation of Comparative Example 6 is the same as that of Example 1, except that the protective agent formula in step 1 is as follows:

[0160] S1: Weigh 2.5 g of sucrose and add it to the above 5 ml centrifuge tube, draw 5 ml of purified water and add it to the centrifuge tube, mix by ultrasonication, and the results are shown in Table 9. Table 9 shows that the difference is much greater than 15%, indicating that the doxorubicin hydrochloride liposomes leaked.

[0161] Table 9 Results in Comparative Example 6

[0162]

[0163] Note: T0 refers to the sample stored for 0 hours; Tx refers to the sample stored in a -70~-90℃ refrigerator for 7 days.

Claims

1. A protective agent for liposome drugs, characterized in that: The protective agent comprises 10.0-50.0% (m / v) of polyol, 10.0-50.0% (m / v) of sugar, 10.0-50.0 mM of antioxidant, and enzyme inhibitor; the solvent is water; Wherein, the enzyme inhibitor comprises a protease inhibitor and / or a phosphatase inhibitor.

2. The protective agent according to claim 1, characterized in that The content of the polyol is 20.0-31.25% (m / v); and / or, the content of the carbohydrate is 10.0-15.0% (m / v); And / or, the content of the antioxidant is 18.75-31.25 mM; And / or, the enzyme inhibitor is selected from A32961 of Thermo Scientific™ and 11836170001 of Roche; the content of the enzyme inhibitor is 0.1-1.0 tablet / mL, preferably 0.60~1.0 tablet / mL.

3. The protective agent according to claim 1 or 2, characterized in that The content of the polyol is 25.0% (m / v); and / or, the content of the carbohydrate is 12.5% ​​(m / v); and / or, the content of the antioxidant is 25.0 mM; And / or, the enzyme inhibitor is selected from A32961 of Thermo Scientific™ and 11836170001 of Roche; the content of the enzyme inhibitor is 0.1-1.0 tablet / mL, preferably 0.60~1.0 tablet / mL.

4. The protective agent according to any one of claims 1 to 3, characterized in that The sugar is a disaccharide; and / or, the polyol is an alcohol compound containing 2-6 hydroxyl groups; and / or, the antioxidant is selected from one or more of vitamin C, glutathione, PB2, dithioerythritol, THPP and TCEP; and / or, the enzyme inhibitor is A32961 of Thermo Scientific™; Preferably, the disaccharide is selected from one or more of glucose, lactose, sucrose, trehalose and cellobiose; and / or, the polyol is selected from one or more of ethylene glycol, propylene glycol, glycerol, mannitol and sorbitol; and / or, the antioxidant is TCEP; More preferably, the sugar is glucose; and / or the polyol is glycerol.

5. The protective agent according to any one of claims 1 to 4, characterized in that The protective agent comprises 25.0% (m / v) glycerol, 12.5% ​​(m / v) glucose, 25.0 mM TCEP, and 0.8 tablets / mL of enzyme inhibitor; Wherein, the enzyme inhibitor is A32961 from Thermo Scientific™.

6. A composition, characterized in that The composition comprises a liposome drug and a protective agent as described in any one of claims 1 to 5; Preferably, the content of the liposome drug is 0.5 μg / mL to 500 μg / mL.

7. A kit, characterized in that: The kit comprises the protective agent according to any one of claims 1 to 5.

8. A method for preventing leakage of liposome drugs in plasma, characterized in that: The method comprises mixing plasma containing liposome drugs with the protective agent according to any one of claims 1 to 5, wherein the volume ratio of the protective agent to the sample is (1-10):3; preferably (2-5):3; more preferably 2:3; Preferably, the plasma is human plasma or animal plasma, such as cynomolgus monkey plasma.

9. A method for separating free drugs in a sample, characterized in that: The method comprises the following steps: The sample to be tested is subjected to SPE separation, and the drug content in the obtained eluate is determined by mass spectrometry; Preferably, the sample is a plasma sample, preferably human plasma or animal plasma; and / or, the drug is doxorubicin hydrochloride.

10. Use of the protective agent according to any one of claims 1 to 5 or the kit according to claim 7 in improving the stability of liposome drugs; Preferably, the liposome drug is a liposome drug in plasma; and / or, the liposome drug is doxorubicin hydrochloride liposome; and / or, the application is for non-diagnostic or non-therapeutic purposes.

Images