CN106546705A - Lipidosome medicine in-vitro release testing method - Google Patents

Lipidosome medicine in-vitro release testing method Download PDF

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CN106546705A
CN106546705A CN201510600365.2A CN201510600365A CN106546705A CN 106546705 A CN106546705 A CN 106546705A CN 201510600365 A CN201510600365 A CN 201510600365A CN 106546705 A CN106546705 A CN 106546705A
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drug
release
container
example
liposomal
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CN201510600365.2A
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唐海玲
张金平
毛文学
苏勇
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上海复旦张江生物医药股份有限公司
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Abstract

The invention relates to a lipidosome medicine in-vitro release testing method, in particular to a testing method for simulating the medicine release process of lipidosome medicines entering a body in vitro. Lipidosome medicine solution and air are jointly placed into a container, the dynamic action of internal blood circulation on the medicines is simulated by movement such as oscillation or rotation of the container, so that the air in a system is dispersed in movement to form bubbles, in physical collision with lipidosome, a lipid membrane is damaged, and the medicines can be effectively released. All operation parameters include controllable temperature, rotation speed and volume ratio of the lipidosome medicines to the air in the container, release rate is conveniently adjusted, and the medicines can be effectively released within a short time. Free medicines released by ion-exchange resin absorption, and internal sink states can be simulated.

Description

一种脂质体药物体外释放的测试方法 Test Method vitro release of Liposome

技术领域 FIELD

[0001] 本发明涉及一种脂质体药物的测试方法,尤其涉及一种在体外模拟脂质体药物进入体内后的释药过程的测试方法。 [0001] The present invention relates to a method for testing liposomal drug, and more particularly to a method for entering the test process after the release in vitro in vivo liposomal drug.

背景技术 Background technique

[0002] 自20世纪90年代磷脂双层膜发现以来,脂质体作为药物递送载体的研究获得了长足发展。 [0002] Since the phospholipid bilayer membranes found in the 1990s, studies of liposomes as drug delivery carriers gained rapid development. 脂质体是指将药物包封于类脂质双分子层内而形成的微型泡囊体。 It refers to liposomes entrapping the drug in the capsule type micro-bubbles lipid bilayers formed. 磷脂分子在水中将分子亲水头部插入水中,疏水尾部伸向空气,形成具有脂质双层结构的球形脂质体, 粒径通常为25~lOOOnm。 Phospholipid molecules in the water molecule into the water head hydrophilic, hydrophobic tails toward the air, the spherical form liposomes having a lipid bilayer structure, the particle size is generally 25 ~ lOOOnm.

[0003] 脂质体的双层膜结构类似于生物膜,具有良好的生物相容性;经脂质体包裹的药物,具有靶向性、长效性、低毒性以及包封保护良好的优点。 Bilayer membrane structure [0003] Liposomes are similar to biological membranes, has good biocompatibility; liposome-entrapped drug, with a targeted resistance, long-term, low toxicity and the advantage of good protection of the encapsulated . 因此,脂质体被用于多种活性药物的递送,以改善药物进入体内后的血液循环时间,增加在靶部位的蓄积。 Thus, liposomes are used to deliver more active drug, to improve blood circulation time of the drug into the body, to increase the accumulation at the target site. 通过改变粒子的粒径、脂质处方组成以及脂膜表面特征,可以改变脂质体的功能。 By changing the particle diameter, surface characteristics and composition of the lipid formulation lipid membranes, liposomes may be varied functions. 使用不同脂质材料可以制备长循环脂质体、热敏脂质体、pH敏感脂质体、免疫脂质体等各种功能性脂质体,达到特殊治疗目的。 Various functional long circulating liposomes, thermosensitive liposomes, pH sensitive liposomes, immunoliposomes, etc. may be prepared using different lipid materials to achieve specific therapeutic purposes.

[0004] 因此,为了合理设计脂质体药物递送系统,就需要对药物在体内和体外的释放行为进行详细研究,通过体外模拟脂质体药物进入体内后的释药过程进行测试。 [0004] Thus, for the rational design of liposomal drug delivery system, requires a detailed study of the drug release behavior in vitro and in vivo, the release process after entering the body through the liposomal drugs in vitro test.

[0005] 然而,目前仅有的测试方法都有严重不足之处。 [0005] However, currently the only test methods have serious shortcomings. 以阿霉素脂质体为例,FDA指南中的体外释放考察方法仅仅给出了介质pH和温度这两个实验参数,通过大体积缓冲液或含有人血浆的缓冲液模拟体内生理环境。 Doxorubicin liposomes, for example, in the FDA guidelines in vitro release method only gives the investigated medium pH and temperature parameters of these two experiments, by bulky analog buffer or buffer containing physiological environment in vivo human plasma. 但由于阿霉素在脂质体内以硫酸阿霉素的沉淀形态存在,溶解度很低,导致释放速率太慢,难以满足质量控制体系中快速评估的要求。 However, due to the presence of adriamycin A precipitate forms neomycin sulfate in liposomes, low solubility, resulting in the release rate is too slow, it is difficult to meet the requirements of quality control system for rapid assessment.

[0006] Avi Schroeder和Yechezkel Barenholz米用的低频超声法(LFUS)能在短时间(3 分钟)内释放80%的药物,但低频超声会导致脂质膜瞬态孔样缺陷,对脂质体的破坏剧烈, 释放中的脂质体已无制剂概念,无法模拟体内生理环境。 [0006] The release of the low-frequency ultrasound (LFUS) Avi Schroeder and Yechezkel Barenholz energy meter used in a short time (3 minutes) 80% drug, but low frequency ultrasound can cause a transient pore-like defects in the lipid membrane, liposome severe damage, release of liposomal formulation is no longer the concept can not be simulated in vivo physiological environment. Atsuko Hioki和Yoshie Maitani 采用在高温(50°C)条件下,加入牛血清白蛋白(bovine serum albumin(BSA))来加速药物释放;Koji Nakamura和Hiroaki Kasukawa利用在经pH梯度法制备的脂质体中加入不同浓度铵离子的方法评估药物释放行为,并区分加载了药物的脂质体的不同脂质组分。 Atsuko Hioki and Yoshie Maitani employed at a high temperature (50 ° C) conditions, the addition of bovine serum albumin (bovine serum albumin (BSA)) to accelerate drug release; Koji Nakamura and Hiroaki Kasukawa use of liposomes via a pH gradient Preparation of the method of adding different concentrations of ammonium ions assess the drug release behavior, and to distinguish between the different lipid components loaded liposome drug. 上述方法虽然能在较短时间内完成释放,但其释放曲线十分不稳定,重复性差,无法作为质量评价的标准。 Although the above methods can be relatively short period of time to complete release, but release curve is very unstable, poor reproducibility, not as a quality evaluation criteria.

[0007] Jennifer A. Shabbits 和Lawrence D. Mayer 研究了的多层囊泡"沉淀"过量100 倍环境下的大单层囊泡(large unilamellar vesicles,LUVs~100nm)的药物释放速率, 以模拟血细胞和组织中的大膜池。 [0007] Jennifer A. Shabbits and Lawrence D. Mayer studied multilamellar vesicles "precipitation" large unilamellar vesicles, the drug release rate in excess of 100 times the environment (large unilamellar vesicles, LUVs ~ 100nm) to simulate blood cells and the organization of large membrane cell. 该方法虽然能获得脂质体药物在体内滞留的真实属性, 但由于MLVs的多变性,仍难以实现精确地质量评估。 Although this method can be obtained liposomal drug retention in the body of the true nature, but due to the variability MLVs, still difficult to achieve precise quality assessment.

[0008] 综上可以看出,现有的脂质体药物体外释放测试方法难以在药物释放速率与体内体外相关性及可重复性之间获得平衡。 [0008] In summary it can be seen, conventional liposomes in vitro release test method is difficult to achieve a balance between the rate of drug release in vitro and in vivo correlation and repeatability.

发明内容 SUMMARY

[0009] 本发明的目的在于提供一种脂质体药物体外释放的测试方法,其能高度模拟体内药物释放的生理环境、各操作参数可控、能大大提高释药速率、重复性高。 [0009] The object of the present invention is to provide a method for testing release of liposomal drugs in vitro, which simulate in vivo drug release is highly physiological environment, controllable operating parameters, can greatly increase the release rate and high reproducibility.

[0010] 为实现上述目的,本发明采取了如下技术方案: [0010] To achieve the above object, the present invention takes the following technical solutions:

[0011] -种脂质体药物体外释放的测试方法,所述脂质体药物为溶液形态,该方法包含如下步骤: [0011] - Test Method for in vitro release of the species liposomes, the liposome pharmaceutical form is a solution, the method comprising the steps of:

[0012] (1)在预定的温度下,将所述脂质体药物与空气按照预设的体积比装入容器内,并将所述容器封闭; [0012] (1) at a predetermined temperature, the liposomal drug according to a preset air volume charged into the container, and the container closure;

[0013] (2)令所述容器进行在竖直方向上存在来回位移的运动; [0013] (2) the presence of movement causes the container back and forth displacement in a vertical direction;

[0014] (3)在所需时间点取样,并测定所述脂质体药物中的未释放药物浓度或已释放的药物浓度,计算药物的释放速率。 [0014] (3) the time required for the sampling points, and the measured concentration of the drug concentration of the drug is not released or the release of liposomal drug in the calculated rate of drug release.

[0015] 进一步地,所述脂质体药物为脂质体蒽环类药物、脂质体长春新碱或脂质体两性霉素B,优选为脂质体阿霉素。 [0015] Further, the liposome drug is liposome anthracycline, liposomal vincristine or liposomal amphotericin B, preferably is a liposomal doxorubicin.

[0016] 进一步地,所述步骤(2)中的所述运动为绕一非竖直的旋转轴旋转,且所述容器为条状,所述容器以径向垂直所述旋转轴的方式安装。 The motion [0016] Further, the step (2) to rotate about a non-vertical axis of rotation, and the container is a strip, the container perpendicular to the rotation axis radially mounted .

[0017] 更进一步地,所述步骤(2)的旋转运动利用分子杂交仪完成,所述旋转轴为水平, 所述容器为离心管,所述转速为15~50rpm。 [0017] Furthermore, the step (2) using the rotational movement of hybridization instrument is completed, the rotary shaft is horizontal, the container is a centrifuge tube, the speed of 15 ~ 50rpm.

[0018] 进一步地,所述步骤(1)中,所述体积比为1: (0. 1~5);所述预定的温度为30~ 60摄氏度。 [0018] Further, the step (1), the volume ratio of 1: (0.1 to 5); said predetermined temperature is 30 to 60 degrees Celsius.

[0019] 进一步地,所述步骤(1)中,所述脂质体药物与空气按照预设的体积比可定义为所述容器内还存在不少于其容积25%的空气。 [0019] Further, the step (1), the drug with the liposome according to a preset air volume ratio is defined as the volume there is less than 25% of the air in the container.

[0020] 进一步地,在所述步骤(1)中,向所述容器内加入预设量的用于吸附释放出的游离药物的离子交换树脂,所述的预设量不小于将所述脂质体药物内包封的所有药物全部吸附所需的量;所述步骤(3)中测定的是所述脂质体药物中的未释放药物浓度。 A preset amount [0020] Further, in the step (1) was added a predetermined amount to the container for adsorbing ions free drug released exchange resin, the fat is not less than the All the required amount of medicament drug encapsulation within plastids fully adsorbed; assay (3) the step of liposomal drug in the unreleased drug concentration.

[0021] 更进一步地,当所述脂质体药物内包封的药物选自蒽环类药物、长春新碱或两性霉素B ;所述离子交换树脂为阳离子交换树脂。 [0021] Furthermore, when the drug within the liposome encapsulated drug is selected from anthracyclines, vincristine or amphotericin B; the ion exchange resin is a cation exchange resin.

[0022] 与现有方法相比,本发明具有如下优点: [0022] Compared with the conventional method, the present invention has the following advantages:

[0023] (1)通过容器运动模拟体内血液循环对药物的动力作用,能更好地模拟药物进入体内后的释放行为,其释放过程更接近体内释放的真实情况; [0023] (1) the dynamic action of the drug can better simulate the behavior of release of the drug into the body through the simulated blood circulation container motion, which is closer to the real situation of the release process in vivo release;

[0024] (2)所有操作参数,包括温度、转速、容器内脂质体药物与空气的体积比等均为可控,从而能方便地调节释放速率,可在短时间内实现药物的有效释放; [0024] (2) all operating parameters, including temperature, speed, liposomal drugs in the container and air volume ratio are controlled, so that it can easily adjust the release rate of drug release can be achieved effectively in a short time ;

[0025] (3)释放过程可随时暂停,不影响重启后的药物释放,便于取样; [0025] (3) The release process can be suspended at any time, the drug does not affect the release of the restart, to facilitate sampling;

[0026] (3)实验重复性远高于现有技术的各种方法; [0026] (3) is much higher than the experimental reproducibility of the various methods of the prior art;

[0027] (4)若在容器内加入离子交换树脂,则可利用树脂对释放出来的游离药物进行即时吸附,起到模拟体内漏槽状态的效果,并能快速测定残留的未释放药物浓度,大大提高实验效率。 [0027] (4) When the ion exchange resin in the container can be released from the resin using a free drug adsorption instant, the state of play sink mimic in vivo effect, and quickly measuring the residual concentration of the drug is not released, greatly improve the efficiency of the experiment.

[0028] 综上所述,本发明在药物释放速率与体内体外相关性及可重复性之间获得了平衡,是一种能综合满足体外释放实验各方面需求的方法。 [0028] In summary, the present invention between the drug release rate in vitro and in vivo correlation and repeatability of the obtained balance, is a method for in vitro release experiments various needs can be satisfied synthesis.

附图说明 BRIEF DESCRIPTION

[0029] 图1 :实施例1~3中,不同温度下的药物释放曲线; [0029] Figure 1: Examples 1 to 3 embodiment, the drug release profile at different temperatures;

[0030] 图2 :实施例4、2、5中,不同药物体积下的药物释放曲线。 [0030] Figure 2: Example 4.2.5, the drug in different volumes of drug release profile.

[0031] 图3 :实施例6~8中,不同药物体积下的药物释放曲线。 [0031] Figure 3: Example 6-8, drugs with different volumes of drug release profile.

[0032] 图4 :实施例2、9、10中,不同转速下的药物释放曲线。 [0032] FIG. 4: 2,9,10 embodiment, the drug release profile at different speeds.

[0033] 图5 :实施例11~14中,不同阳离子吸附树脂加入量下的药物释放曲线。 [0033] Figure 5: Examples 11 to 14, different cations adsorbed amount of resin in the drug release profile.

[0034] 图6 :本发明重复性试验:不同样品在不同时间点的阿霉素释放速率。 [0034] FIG. 6: Repeatability of the present invention: the release rate of doxorubicin different samples at different time points.

具体实施方式 Detailed ways

[0035] 本发明的基本原理是通过容器运动模拟体内血液循环对药物的动力作用,体系中的空气在运动中分散,形成气泡与脂质体发生物理碰撞,从而破坏脂膜,达到药物的有效释放。 [0035] The basic principle of the invention is that the system air movement in the dynamic effect of the drug is dispersed through the container to simulate the movement of blood circulation in vivo, the effective bubble forming the liposome physical impact, so that disruption of lipid membrane, to achieve a drug freed.

[0036] 在实验温度方面,选择磷脂相变温度52度附近为宜,优选40~50摄氏度。 [0036] In terms of the experimental temperature, the phase transition temperature phospholipids selected near 52 degrees, and preferably 40 to 50 degrees Celsius. 虽然温度越高脂膜的流动性越好,药物释放速率越快,但过高的温度会破坏脂质体本身,也就谈不上释放。 While the fluidity of the lipid membrane temperature is higher the better, the faster the drug release rate, but too high temperatures will destroy the liposome itself, there are no release. 因为该方法下药物的释放速度不仅能通过温度调节,还可通过转速或空气占比来调节,所以除非是专门针对温度梯度进行试验,可以选择30度或更低、60度或更高,否则无需选择过于极端的温度。 Because the rate of drug release is not only at the process temperature by adjusting the rotational speed may also be adjusted by the proportion or air, so unless specifically tested for a temperature gradient, can be selected 30 degrees or less, 60 degrees or more, or no need to select too extreme temperatures.

[0037] 容器的运动必须具备竖直方向上的位移,否则无法使浮在溶液上方的空气与溶液中脂质体发生相对碰撞。 [0037] The movement of the container must have a displacement in the vertical direction, or not so floating in air above the solution in the liposome solution relative collision occurs. 为达到混合效果,最简单的运动方式就是上下震荡或旋转。 To achieve mixed results, the easiest way to fluctuate up and down movement or rotation. 实际操作中,则利用分子杂交仪进行绕水平轴的旋转最为方便,且离心管需以径向垂直旋转轴的方式竖直设置在旋转轴四周。 In practice, the use of molecular hybridization instrument for rotation about a horizontal axis the most convenient manner and in a centrifuge tube for an axis of rotation perpendicular to the radial vertically disposed rotating shaft around. 这样的旋转方式使得空气的分散效果更好,对体内血液循环的模拟更真实。 Such rotation manner that better dispersion effect of the air, the blood circulation simulation more realistic.

[0038] 同时,还可利用离子交换树脂对释放出来的游离药物进行吸附,释放出的游离药物被交换树脂吸附,就能方便地直接测定残留药物浓度。 [0038] Meanwhile, ion-exchange resin may also be released from adsorption of the free drug, free drug is released exchange resin, measuring the residual concentration of the drug can be directly and easily.

[0039] 阳离子树脂可吸附蒽环类药物(柔红霉素、阿霉素、阿柔比星、表阿霉素、伊达比星、戊柔比星、米托蒽醌)、长春新碱、两性霉素B等,其中强酸型阳离子交换树脂具有强酸性的反应基如磺酸基(_S03H),可以广泛吸附所有的阳离子;弱酸型阳离子交换树脂具有较弱的反应基如羧基(-C00H基),仅可交换弱碱中的阳离子如Ca 2+、Mg2+。 [0039] The cationic resin may be adsorbed anthracyclines (daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, valrubicin, mitoxantrone), vincristine , amphotericin B, wherein the strong acid-type strongly acidic cation exchange resin having a sulfonic acid group such as a reactive group (_S03H), all of widely adsorbed cation; weak acid cation exchange resin having carboxyl group such as a weak reaction (-C00H yl), only exchangeable cations of weak bases such as Ca 2 +, Mg2 +. 阴离子交换树脂主要是强碱型阴离子交换树脂,可以和所有阴离子进行交换吸附。 Strong base anion exchange resin is mainly anion exchange resin, adsorption can be exchanged and all anions.

[0040] 若在实验开始前先加入离子交换树脂,则可起到模拟体内漏槽状态的效果;若在实验完成并取样后再加入离子交换树脂,则仅起到吸附游离药物,简化样品检测前处理操作用。 [0040] When the ion exchange resin is added first before the start of the experiment, the body can play a simulation of the effect sink state; if the ion-exchange resin and sampled after completion of the experiment, the free drug adsorbed only functions, simplifying sample detection pretreatment manipulation.

[0041] 以下通过实施例对本发明的具体实施方式做进一步说明。 [0041] The following examples further illustrate specific embodiments of the present invention do. 实施例考察了以下5个参数:离心管类型、温度、转速、药物溶液与空气体积比以及离子交换树脂加入量,对药物的释放速率的影响。 Example 5 The following parameters were investigated: tube type, temperature, speed, air and drug solution volume ratio of the ion exchange resin and the effect of addition amount of the release rate of the drug.

[0042] 实施例1 :5ml离心管-温度37°C、转速25rpm、药物体积2ml-10%树脂 [0042] Example 1: 5ml centrifuge tube - temperature 37 ° C, the rotational speed of 25rpm, the volume of drug 2ml-10% Resin

[0043] (1)、配置脂质体阿霉素溶液: [0043] (1), arranged liposomal doxorubicin solution:

[0044] 将pH 6. 0的磷酸盐缓冲生理盐水(Phosphate buffered saline,PBS)放于超声清洗机中超声20min除气;用移液枪移取脂质体阿霉素1. 25ml至50ml容量瓶中,用上述PBS定容为40倍稀释液,计算出初始药物浓度。 [0044] The pH phosphate buffered saline (Phosphate buffered saline, PBS) 6. 0 is placed in the ultrasonic cleaning machine ultrasonic degassing 20min; pipetting with a pipette liposomal doxorubicin 1. 25ml to 50ml capacity flask, a constant volume of PBS with the above 40-fold dilution, to calculate the initial drug concentration.

[0045] (2)、阳离子交换树脂预处理 [0045] (2) cation exchange resin pretreated

[0046] 将阳离子交换树脂加入到层析柱中,将水自然沥干后,用洗耳球吹打至无连续液体流出。 [0046] The cation exchange resin was added to a chromatography column, the natural water drain, wash the ear to the ball by pipetting without continuous liquid outflow. (3)、体外释放实验 (3), in vitro release experiment

[0047] a、在5ml的EPPEND0RF离心管中加入2ml脂质体阿霉素稀释液和0· 2g(w/v = 10% )阳离子交换树脂; [0047] a, liposomal doxorubicin was added 2ml diluent and 0 · 2g of EPPEND0RF in 5ml centrifuge tube (w / v = 10%) cation exchange resin;

[0048] b、设定分子杂交仪温度为37°C,转速为Orpm,进行预热; [0048] b, hybridization instrument set temperature of 37 ° C, speed of Orpm, preheating;

[0049] c、将离心管以径向垂直于水平旋转轴的方式固定在分子杂交仪的杂交管架上; [0049] c, the centrifuge tube in a radial axis of rotation perpendicular to the horizontal fixed tube holder hybridizer of the hybrid molecule;

[0050] d、设定分子杂交仪温度为37°C,转速为25rpm,开始旋转并计时。 [0050] d, hybridization instrument set temperature 37 ° C, speed of 25rpm, and counting starts rotating.

[0051] e、在2h、4h、6h时间点取样,一支离心管即为一份样品。 [0051] e, at 2h, 4h, 6h sampling time points, a sample that is a centrifugal tube. 用HPLC检测样品上清液中的残留药物浓度。 With a residual drug concentration in the supernatant samples by HPLC.

[0052] f、药物释放速率=(1-残留药物浓度/初始药物浓度)X 100%,实验结果见下表: [0052] f, the drug release rate = (1 - residual drug concentration / initial concentration of the drug) X 100%, the following table results:

[0053] 表1、实施例1的不同时间点阿霉素释放速率 [0053] Table 1, Example 1 at different time points the rate of release of adriamycin embodiment

Figure CN106546705AD00061

[0055] 实施例2 :5ml离心管-温度49°C、转速25rpm、药物体积2ml-10%树脂 [0055] Example 2: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 25rpm, the volume of drug 2ml-10% Resin

[0056] 具体步骤同实施例1,参数设置如标题,温度改变。 [0056] Step 1 In particular, the parameter set such as the title, the temperature changes were the same embodiment. 实验结果见下表: The results in the table below:

[0057] 表2、实施例2的不同时间点阿霉素释放速率 [0057] Table 2, Example 2 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00062

[0059] 实施例3 :5ml离心管-温度52°C、转速25rpm、药物体积2ml-10%树脂 [0059] Example 3: 5ml centrifuge tube - temperature 52 ° C, the rotational speed of 25rpm, the volume of drug 2ml-10% Resin

[0060] 具体步骤同实施例1,参数设置如标题,温度改变。 [0060] Step 1 In particular, the parameter set such as the title, the temperature changes were the same embodiment. 实验结果见下表: The results in the table below:

[0061] 表3、实施例3的不同时间点阿霉素释放速率 [0061] Table 3, Example 3 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00063

[0063] 附图1是根据实施例1~3的结果作图,在其他条件相同(5ml离心管,药物体积2ml,转速25rpm,加入w/v 10%的阳离子吸附树脂)时,不同温度下的药物释放曲线。 [0063] Figure 1 is plotted according to the results of Examples 1 to 3, when the same other conditions (5ml centrifuge tubes, the volume of drug 2ml, speed 25 rpm, was added w / 10% v of a cationic adsorption resin), at different temperatures the drug release profile. 由此可见,温度会影响阿霉素药物的释放速率,温度升高,释放相对变快。 Thus, the temperature will affect the release rate of the drug doxorubicin, the temperature rises, the release is relatively fast.

[0064] 实施例4 :5ml离心管-温度49°C、转速25rpm、药物体积lml-10%树脂 [0064] Example 4: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 25rpm, the volume of drug lml-10% Resin

[0065] 具体步骤同实施例2,参数设置如标题,药物体积改变。 [0065] Specific steps in Example 2, parameters such as title, drug volume change. 实验结果见下表: The results in the table below:

[0066] 表4、实施例4的不同时间点阿霉素释放速率 [0066] Table 4, Example 4 at different time points the rate of release of doxorubicin embodiment

[0067] [0067]

Figure CN106546705AD00071

[0068] 实施例5 :5ml离心管-温度49°C、转速25rpm、药物体积4ml-10%树脂 [0068] Example 5: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 25rpm, the volume of drug 4ml-10% Resin

[0069] 具体步骤同实施例4,参数设置如标题,药物体积改变。 [0069] Specifically Step 4, parameters such as the title, the volume changes were the same drugs embodiment. 实验结果见下表: The results in the table below:

[0070] 表5、实施例5的不同时间点阿霉素释放速率 [0070] Table 5, Example 5 at different time points of the embodiment of doxorubicin release rate

Figure CN106546705AD00072

[0072] 附图2是根据实施例4、2、5的结果作图,在其他条件相同(5ml离心管,温度49°C, 转速25rpm,加入w/v 10%的阳离子吸附树脂)时,不同药物体积(即药物与空气的体积比)下的药物释放曲线。 [0072] Figure 2 is plotted based on the results of Example 4.2.5, the same other conditions (5ml centrifuge tube, a temperature of 49 ° C, speed 25 rpm, was added w / 10% v of a cationic adsorption resin), the different volume of the drug (i.e., a volume ratio of drug to air) under the drug release profile.

[0073] 从图中可以清晰地看出,药液与空气的体积比会明显改变阿霉素的释放行为。 [0073] can be clearly seen from the figure, the volume ratio of liquid to air may significantly alter the behavior of release of doxorubicin. 随着药物体积从lml增加到4ml,阿霉素释放速率显著变慢。 With the increased volume of drug from 4ml lml, doxorubicin significantly slower release rate. 这是因为该测试方法中,破坏脂膜依靠的就是分散到溶液中的空气,因此通过控制空气体积比,能精确操纵释放速率,操作直观简单。 This is because the test method, is to rely on disruption of lipid membrane solution dispersed into the air, by controlling the ratio of the volume of air, the release rate can be precisely manipulated, intuitive and simple operation.

[0074] 实施例6 :5ml离心管-温度47°C、转速25rpm、药物体积1. 8ml-10%树脂 [0074] Example 6: 5ml centrifuge tube - temperature 47 ° C, the rotational speed of 25rpm, the volume of drug 1. 8ml-10% Resin

[0075] 具体步骤同实施例1,参数设置如标题,取样时间点为lh、3h、5h。 [0075] DETAILED same procedure as in Example 1, parameters such as the title, the sampling time points lh, 3h, 5h. 实验结果见下表: The results in the table below:

[0076] 表6、实施例6的不同时间点阿霉素释放速率 [0076] Table 6, Example 6 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00073

[0078] 实施例7 :5ml离心管-温度47°C、转速25rpm、药物体积2. 4ml-10%树脂 [0078] Example 7: 5ml centrifuge tube - temperature 47 ° C, the rotational speed of 25rpm, the volume of drug 2. 4ml-10% Resin

[0079] 具体步骤同实施例6,参数设置如标题,药物体积略加改变。 [0079] DETAILED steps from Example 6, parameters such as title, slightly changes the volume of the drug. 实验结果见下表: The results in the table below:

[0080] 表7、实施例7的不同时间点阿霉素释放速率 [0080] Table 7, Example 7 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00074

[0082] 实施例8 :5ml离心管-温度47°C、转速25rpm、药物体积3ml-10%树脂 [0082] Example 8: 5ml centrifuge tube - temperature 47 ° C, the rotational speed of 25rpm, the volume of drug 3ml-10% Resin

[0083] 具体步骤同实施例6,参数设置如标题,药物体积略加改变。 [0083] DETAILED steps from Example 6, parameters such as title, slightly changes the volume of the drug. 实验结果见下表: The results in the table below:

[0084] 表8、实施例8的不同时间点阿霉素释放速率 [0084] Table 8, in Example 8 at different time points the rate of release of doxorubicin embodiment

[0085] [0085]

Figure CN106546705AD00081

[0086] 附图3是根据实施例6~8作图,在其他条件相同(5ml离心管,温度47°C,转速25rpm,加入w/v 10%的阳离子吸附树脂)时,不同药物体积(即药物与空气的体积比)下的药物释放曲线。 [0086] Figure 3 is a plot of Examples 6 to 8, at the same other conditions (5ml centrifuge tube, a temperature of 47 ° C, speed 25 rpm, was added w / 10% v of a cationic adsorption resin), the volume of different drugs ( i.e., the drug in a volume ratio of drug to air) release profile. 和附图2相比,该组实验中的药物体积变化幅度较小,结果所得的药物释放曲线无明显区别。 Figures 2 and compared the magnitude of the volume change of the drug in the small set of experiments, the results of the drug release profile obtained no significant difference.

[0087] 实施例9 :5ml离心管-温度49°C、转速15rpm、药物体积2ml-10%树脂 [0087] Example 9: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 15rpm, the volume of drug 2ml-10% Resin

[0088] 具体步骤同实施例2,参数设置如标题,转速改变。 [0088] Specific steps in Example 2, parameters such as heading, speed changes. 实验结果见下表: The results in the table below:

[0089] 表9、实施例9的不同时间点阿霉素释放速率 [0089] Table 9, in Example 9 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00082

[0091] 实施例10 :5ml离心管-温度49°C、转速35rpm、药物体积2ml-10%树脂 [0091] Example 10: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 35rpm, the volume of drug 2ml-10% Resin

[0092] 具体步骤同实施例2,参数设置如标题,转速改变。 [0092] Specific steps in Example 2, parameters such as heading, speed changes. 实验结果见下表: The results in the table below:

[0093] 表10、实施例10的不同时间点阿霉素释放速率 [0093] Table 10, Example 10 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00083

[0095] 附图4是根据实施例2、9、10作图,在其他条件相同(5ml离心管,温度49°C,药物体积2ml,加入w/v 10%的阳离子吸附树脂)时,不同转速下的药物释放曲线。 [0095] Figure 4 is an embodiment 2,9,10 plotted in the same other conditions (5ml centrifuge tube, a temperature of 49 ° C, the volume of drug 2ml, was added w / 10% v of a cationic adsorption resin), the different speed in drug release profile. 由此可见, 转速对药物释放速度的影响也很大。 Thus, the impact of speed on the drug release rate is also high. 转速增加直接加速了气泡对脂膜的破坏,药物释放速率显著提升。 Direct damage accelerated speed increases bubbles lipid membrane, the drug release rate significantly.

[0096] 实施例11 :5ml离心管-温度49°C、转速30rpm、药物体积1. 8ml-0%树脂 [0096] Example 11: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 30rpm, the volume of drug resin 1. 8ml-0%

[0097] 具体步骤同实施例1,参数设置如标题,取样时间点为lh、3h、5h。 [0097] DETAILED same procedure as in Example 1, parameters such as the title, the sampling time points lh, 3h, 5h. 但在旋转前不加入阳离子交换树脂,待旋转完成并取样后,才在样品中加入树脂,用以吸附游离药物。 But before the rotation of the cation exchange resin was not added until complete rotation and sampled, the sample was added to the resin to adsorb the free drug. 实验结果见下表: The results in the table below:

[0098] 表11、实施例11的不同时间点阿霉素释放速率 [0098] Table 11, Example 11 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00084

[0100] 实施例12 :5ml离心管-温度49°C、转速30rpm、药物体积1. 8ml-5%树脂 [0100] Example 12: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 30rpm, the volume of drug 1. 8ml-5% Resin

[0101] 具体步骤同实施例11,参数设置如标题,改变加入的阳离子吸附树脂含量。 [0101] Specific steps in Example 11, parameters such as the title, is added to change the content of the cationic resin adsorbent. 实验结果见下表: The results in the table below:

[0102] 表12、实施例12的不同时间点阿霉素释放速率 [0102] Table 12, Example 12 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00091

[0104] 实施例13 :5ml离心管-温度49°C、转速30rpm、药物体积1. 8ml-10%树脂 [0104] Example 13: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 30rpm, the volume of drug 1. 8ml-10% Resin

[0105] 具体步骤同实施例11,参数设置如标题,改变加入的阳离子吸附树脂含量。 [0105] Specific steps in Example 11, parameters such as the title, is added to change the content of the cationic resin adsorbent. 实验结果见下表: The results in the table below:

[0106] 表13、实施例13的不同时间点阿霉素释放速率 [0106] Table 13, Example 13 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00092

[0108] 实施例14 :5ml离心管-温度49°C、转速30rpm、药物体积1. 8ml-20%树脂 [0108] Example 14: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 30rpm, the volume of drug 1. 8ml-20% Resin

[0109] 具体步骤同实施例11,参数设置如标题,改变加入的阳离子吸附树脂含量。 [0109] Specific steps in Example 11, parameters such as the title, is added to change the content of the cationic resin adsorbent. 实验结果见下表: The results in the table below:

[0110] 表14、实施例14的不同时间点阿霉素释放速率 [0110] Table 14, Example 14 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00093

[0112] 附图5是根据实施例11~14作图,在其他条件相同(5ml离心,温度49°C,转速30rpm,药物体积1.8ml)时,加入不同质量的阳离子吸附树脂后的药物释放曲线。 [0112] Figure 5 is a plot embodiments 11 to 14, in the same other conditions (5ml centrifuge, temperature 49 ° C, speed 30 rpm, the volume of drug 1.8ml), the drug release after resin adding different cations adsorbed mass curve. 其中,包括最小量5% (w/v)在内的所有树脂量,都超过能将离心管内所有药物全部吸附的所需量。 Wherein all the resins comprising an amount of a minimum amount of 5% (w / v), including, more than the required amount of drug can all tubes all adsorbed. 从图中可以看出,树脂的加入对药物释放的影响非常小。 As can be seen from the figure, the influence of the resin was added to the drug release is very small.

[0113] 实施例15 :15ml离心管-温度47°C、转速40rpm、药物体积10ml-20%树脂 [0113] Example 15: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 40rpm, drugs resin volume 10ml-20%

[0114] 具体步骤同实施例1,参数设置如标题,更换了离心管型号,取样时间点为lh、3h。 [0114] DETAILED same procedure as in Example 1, parameters such as the title, the replacement tube model, the sampling time points lh, 3h. 实验结果见下表: The results in the table below:

[0115] 表15、实施例15的不同时间点阿霉素释放速率 [0115] Table 15, Example 15 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00094

[0117] 实施例16 :15ml离心管-温度47°C、转速40rpm、药物体积llml-10%树脂 [0117] Example 16: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 40rpm, the volume of drug llml-10% Resin

[0118] 具体步骤同实施例15,参数设置如标题,药物体积略增,树脂加入量减少。 [0118] Specific steps in Example 15, parameters such as title, a slight increase in volume of the drug, reduce the amount of resin. 实验结果见下表: The results in the table below:

[0119] 表16、实施例16的不同时间点阿霉素释放速率 [0119] Table 16, Example 16 at different time points the rate of release of doxorubicin embodiment

[0120] [0120]

Figure CN106546705AD00101

[0121] 实施例17 :15ml离心管-温度47°C、转速30rpm、药物体积10ml-20%树脂 [0121] Example 17: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 30rpm, drugs resin volume 10ml-20%

[0122] 具体步骤同实施例15,参数设置如标题,转速改变。 [0122] Specific steps in Example 15, parameters such as heading, speed changes. 实验结果见下表: The results in the table below:

[0123] 表17、实施例17的不同时间点阿霉素释放速率 [0123] Table 17, Example 17 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00102

[0125] 实施例18 :15ml离心管-温度47°C、转速20rpm、药物体积10ml-10%树脂 [0125] Example 18: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 20rpm, drugs resin volume 10ml-10%

[0126] 具体步骤同实施例15,参数设置如标题,转速改变。 [0126] Specific steps in Example 15, parameters such as heading, speed changes. 实验结果见下表: The results in the table below:

[0127] 表18、实施例18的不同时间点阿霉素释放速率 [0127] Table 18, Example 18 at different time points the rate of release of doxorubicin embodiment

Figure CN106546705AD00103

[0129] 将上述15ml离心管的数据与5ml离心管的数据对比发现,离心管容积增大,有利于药物释放。 [0129] The above data 5ml 15ml centrifuge tube and centrifuge tube comparison, the tube volume increases, beneficial drug release. 在近似的温度和相同的转速下,15ml离心管中的药物与空气体积比大于5ml 离心管,而达到90%以上的药物释放率所需的时间则短于后者。 At approximately the same speed and temperature, 15ml centrifuge tube and the drug greater than the volume of air 5ml centrifuge tube, and the time required to reach 90% or more drug release rate is shorter than the latter. 因此,通过增大离心管型号来提高药物释放速率也是有效的手段之一。 Thus, by increasing the tube model to enhance drug release rate is one of the effective means.

[0130] 实施例19 :5ml离心管-温度49°C、转速25rpm、药物体积2ml-10%树脂 [0130] Example 19: 5ml centrifuge tube - temperature 49 ° C, the rotational speed of 25rpm, the volume of drug 2ml-10% Resin

[0131] 具体步骤同实施例1,参数设置如标题。 [0131] DETAILED same procedure as in Example 1, the parameter set as the title. 取样时间点为lh、2h、4h、6h、8h,每个时间点取6支样品,验证重复性。 Sampling time points lh, 2h, 4h, 6h, 8h, 6 each time point sample was taken to verify repeatability. 实验结果见下表: The results in the table below:

[0132] 表19、实施例19的不同样品在不同时间点的阿霉素释放速率 [0132] Table 19, the release rate of doxorubicin Example 19 different samples at different time points embodiment

Figure CN106546705AD00104

[0134] 附图6是上述各样品的药物释放曲线对比。 [0134] Figure 6 is a sample of each of the comparison drug release profile. 从图中可以看出,本方法的重复性很好,特别是在较长的时间尺度下,药物释放的稳定性很好。 As it can be seen from the figure, a good reproducibility of the method, especially at longer time scales, a good stability of drug release.

[0135] 以下通过多组实施例和对比例,考察不同运动方式对实验效果的影响。 [0135] The following examples and a plurality of sets, comparative effects of different influence on the motion by the experimental results.

[0136] 实施例20 :15ml离心管-温度47°C、转速60rpm、药物体积10ml-2g树脂-竖直装样 [0136] Example 20: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 60rpm, drugs resin volume 10ml-2g - vertical sample loading

[0137] 对比例1 :15ml离心管-温度47°C、转速60rpm、药物体积10ml-2g树脂-水平装样 [0137] Comparative Example 1: 15ml centrifuge tube - temperature 47 ° C, the rotational speed of 60rpm, drugs resin volume 10ml-2g - sample loading level

[0138] 具体实验步骤基本同实施例1,参数设置如标题。 [0138] DETAILED substantially the same experimental procedure as in Example 1, the parameter set as the title. 区别在于,对比例中,安装离心管时采取径向平行于旋转轴的方式。 The difference is that, in Comparative Example, take the form of radially parallel to the rotation axis of the centrifuge tube during installation. 同时,该两组实验均采取了比较大的转速。 Meanwhile, the two sets of experiments are taking a relatively large speed.

[0139] 取样时间点为111、211、311、411、511,每个时间点取2支样品。 [0139] 111,211,311,411,511 sampling time points, two for each time point sample was taken. 实验结果见下表: The results in the table below:

[0140] 表20、实施例20和对比例1在不同时间点的阿霉素释放速率 [0140] Table 20, Example 20 and Comparative Example 1 at the rate of doxorubicin release at different time points

Figure CN106546705AD00111

[0142] 从上表可以看出,转速较大时,采用径向垂直于旋转轴的装样方式,仅lh就达成了较大的释放比例,而采取水平装样的方式则是放很慢。 [0142] As can be seen from the table, the larger the rotation speed, using the sample loading mode to the radial direction perpendicular to the axis of rotation, only lh reached a large proportion of release, to take sample loading level of release is very slow manner . 这是由于围绕水平的旋转轴进行旋转时,如果离心管同样为水平,则空气和溶液二者之间在竖直方向上的相对位移幅度不大,且由于离心力,空气容易靠在管壁内侧,难以形成剧烈搅动。 This is because the rotation shaft is rotated about a horizontal, if the tube is the same level, the magnitude of the relative displacement between both the air and the solution is not in the vertical direction, and due to centrifugal force, the air easily against the inner wall difficult to form vigorous agitation. 因此,采取竖直装样的方式释放速度较快,水平装样则较慢,从实验效率的角度考量,以竖直装样为优。 Thus, taking the vertical sample loading way to release faster and slower sample loading level, from the viewpoint of efficiency considerations experiment, the sample is preferably attached to the vertical.

[0143] 对比例2 :1. 5ml EP管-温度47°C、震荡频率lOOOrpm、药物体积lml-0. 2g树脂-震荡 . [0143] Comparative Example 2:. 1 5ml EP tube - temperature 47 ° C, oscillation frequency lOOOrpm, the volume of drug lml-0 2g resin - shock

[0144] 该对比例2考察震荡运动方式下的药物释放。 [0144] The release of the drug in Comparative Example 2 Investigation oscillation motion.

[0145] 基本的实验步骤同实施例1,参数设置如标题。 [0145] The basic experimental procedure as in Example 1, the parameter set as the title. 区别在于,装样采用1. 5ml EP管, 利用恒温震荡混匀仪进行震荡,震荡频率lOOOrpm。 The difference is that, using the sample loading 1. 5ml EP tube, vortexed using a constant temperature instrument for oscillation, oscillation frequency lOOOrpm. 取样时间点为lh、2h、3h,每个时间点取4支样品,样品离心,取上清,用HPLC检测阿霉素含量。 Sampling time points lh, 2h, 3h, four time points for each sample, samples were centrifuged, the supernatant, doxorubicin content by HPLC analysis. 实验结果见下表: The results in the table below:

[0146] 表21、对比例2中不同时间点的阿霉素释放速率 [0146] Table 21, the release rate of doxorubicin at different time points in Comparative Example 2

Figure CN106546705AD00112

[0148] 从上表可以看出,同一个时间点,不同样品的释放速率差异较大,这表明采取震荡的运动方式难以保证实验的重复性,可控性较低。 [0148] As can be seen from the table, the same time, the release rate of different samples are quite different, indicating that adopt oscillating motion is difficult to ensure reproducibility of the experiment, the controllability is low. 如果降低振荡频率,则又无法在短时间内达到有效释放。 If you reduce the oscillation frequency, it can not achieve an effective release in a short time.

[0149] 对比例3 :50ml肖特瓶-温度47°C、震荡频率150rpm、药物体积50ml-5g树脂 [0149] Comparative Example 3: 50ml Schott bottle - temperature 47 ° C, oscillation frequency of 150rpm, 50ml-5g resin volume of drug

[0150] 该对比例3考察采用另一种振荡器的震荡运动方式下的药物释放。 [0150] Comparative Example 3 Investigation of the use of drugs in another shaking motion oscillator release.

[0151] 基本的实验步骤同实施例1,参数设置如标题。 [0151] The basic experimental procedure as in Example 1, the parameter set as the title. 区别在于,装样采用50ml肖特瓶, 其中加入50ml阿霉素脂质体稀释液。 The difference is that, using the sample loading 50ml Schott bottle, were added 50ml liposomal doxorubicin diluent. 利用恒温摇床振荡器进行震荡,震荡频率150rpm,摇床上同时安装2瓶溶液同时进行。 Constant temperature shaker oscillator oscillation, oscillation frequency 150 rpm, shaker flask 2 was installed at the same time. 取样时间点为lh、2h、3h、4h、5h、6h,每次取样0. 5ml上清,用HPLC检测阿霉素含量。 Sampling time points lh, 2h, 3h, 4h, 5h, 6h, 0. 5ml each sample supernatant, assayed by HPLC Adriamycin. 实验结果见下表: The results in the table below:

[0152] 表22、对比例3中不同时间点的阿霉素释放速率 [0152] Table 22, the release rate of doxorubicin different time points in Comparative Example 3

Figure CN106546705AD00121

[0155] 从上表可以看出,即使是同一台恒温震荡摇床,同时进行实验的两组样品,药物释放行为差异依然很大。 [0155] As can be seen from the table, even if the same constant temperature shock shaker, at the same time two sets of samples of the experiment, the drug release behavior difference is still great. 这可能是因为摇床对样品的固定不够好,开始震动后样品会发生位移,从而影响实验的重复性。 This may be because the shaker is not good enough for a fixed sample, the sample will start to vibrate after displacement, thus affecting the repeatability of experiments.

[0156] 从对比例2和3可以看出,震荡虽然作为一种运动方式,在释放效率方面能够满足药物实验需求,但在实际操作中,采用实验室的常用设备难以对其进行精确控制,从而导致该运动方式的实用性很低,不如旋转方式。 [0156] From Comparative Examples 2 and 3 can be seen that, although the shock as a motion, in terms of the efficiency of the release of drugs to meet the needs of the experiment, but in practice, it is difficult using conventional laboratory apparatus be accurately controlled, Applicability of the resulting motion is low, as the rotational mode.

[0157] 对比例4 :50ml西林瓶-温度47°C、搅拌速率450rpm、药物体积10ml-2g树脂 [0157] Comparative Example 4: 50ml vial - temperature 47 ° C, stirring speed 450rpm, pharmaceutical resin volume 10ml-2g

[0158] 对比例4考察的是恒温磁力搅拌运动方式下的药物释放。 [0158] Comparative Example 4 is examined in drug release under constant magnetic stirring motion.

[0159] 对比例4的基本的实验步骤同实施例1,参数设置如标题。 [0159] substantially the same Comparative Example 4 The experimental procedure 1, parameters such as the title. 区别在于,装样采用50ml西林瓶,其中加入10ml阿霉素脂质体稀释液。 The difference is that, using the sample loading 50ml vials, 10ml was added doxorubicin liposomes diluent. 利用六孔水浴磁力搅拌器同时对6组样品进行搅拌,搅拌速率为450rpm,取样时间点为111、211、311、411、511、611,每次取样0.51111上清, 用HPLC检测阿霉素含量。 Tin magnetic stirrer using a water bath while stirring for 6 set of samples, a stirring rate of 450 rpm, the sampling time points 111,211,311,411,511,611, 0.51111 each sample supernatant, doxorubicin content by HPLC detection . 实验结果见下表: The results in the table below:

[0160] 表23、对比例4中不同时间点的阿霉素释放速率 [0160] Table 23, the release rate of doxorubicin at different time points in Comparative Example 4

Figure CN106546705AD00122

[0162] 从上表可以看出,即使采用同一台搅拌器,在相同条件下同时进行实验的6个样品,其释放速率差异依然很大。 [0162] As can be seen from the table, even with the same agitator for 6 samples while under the same experimental conditions, the difference in release rate still great. 由此可见磁力搅拌的运动方式可控性同样非常低,不适合选择该方式。 Thus magnetic stirring motion controllability is also very low, is not suitable for the selected mode.

[0163] 对比例5 :溶出杯-温度45°C、搅拌速率250rpm、药物体积250ml-20g树脂 [0163] Comparative Example 5: Dissolution cup - temperature 45 ° C, stirring speed 250rpm, pharmaceutical resin volume 250ml-20g

[0164] 对比例5则采用了溶出实验的通用仪器溶出仪,利用搅拌桨进行搅拌。 [0164] Comparative Example 5 using the general dissolution test apparatus dissolution apparatus by stirring the stirring blade.

[0165] 同时将5个溶出杯内分别加入250ml阿霉素脂质体稀释液,设定转速为250rpm(通常溶出实验采取的转速为几十rpm)。 [0165] Also within five cups were added 250ml eluted doxorubicin liposome dilutions are set speed is 250 rpm (typically taken dissolution test speed of several tens rpm). 取样时间点为lh、2h、3h、4h、5h。 Sampling time points lh, 2h, 3h, 4h, 5h. 该对比例5取样后,测试未释放的阿霉素脂质体残留浓度。 Comparative Example 5 After the sampling, testing unreleased residual concentration of liposomal doxorubicin. 实验结果见下表: The results in the table below:

[0166] 表24、对比例5中不同时间点的阿霉素脂质体残留浓度 [0166] Table 24, the residual concentration of the liposomes at different time points doxorubicin Comparative Example 5

Figure CN106546705AD00131

[0168] 从上表可以看出,即使将温度设定为较高的45°C,采取相对猛烈的搅拌速率,依然无法使阿霉素发生释放。 [0168] As can be seen from the table, even when the temperature is set to a higher 45 ° C, take a relatively violent agitation rate, still can not doxorubicin release occurs. 因此,该方法完全无法用于药物释放实验。 Therefore, this method can not be used to complete drug release experiment.

[0169] 本发明的范围不受所述具体实施方案的限制,所述实施方案只作为阐明本发明各个方面的单个例子,本发明范围内还包括功能等同的方法和组分。 [0169] scope of the present invention is not limited by the specific embodiments that the embodiments illustrate only a single example of the various aspects of the present invention, within the scope of the present invention further comprises methods and functionally equivalent components. 实际上,除了本文所述的内容外,本领域技术人员参照上文的描述和附图可以容易地掌握对本发明的多种改进。 In fact, in addition to what is described herein, those skilled in the art with reference to the accompanying drawings and described above can easily grasp various modifications of the present invention. 所述改进也落入所附权利要求书的范围之内。 The improvement also fall within the scope of the appended claims. 上文提及的每篇参考文献皆全文列入本文作为参考。 Each reference mentioned above are incorporated herein by reference in entirety.

Claims (10)

1. 一种脂质体药物体外释放的测试方法,所述脂质体药物为溶液形态,该方法包含如下步骤: (1) 在预定的温度下,将所述脂质体药物与空气按照预设的体积比装入容器内,并将所述容器封闭; (2) 令所述容器进行在竖直方向上存在来回位移的运动; (3) 在所需时间点取样,并测定所述脂质体药物中的未释放药物浓度或已释放的药物浓度,计算药物的释放速率。 Test Method An in vitro release of liposomes, the liposome pharmaceutical form is a solution, the method comprising the steps of: (1) at a predetermined temperature, the liposomal drugs in accordance with the pre-air the volume ratio of charged provided the container and the container closure; (2) the presence of movement causes the container back and forth displacement in a vertical direction; (3) at the desired sampling time point, and determining the lipid drug concentration in the drug substance is not released or the release of the drug concentration, the rate of drug release is calculated.
2. 如权利要求1所述的测试方法,其特征在于所述预定的温度为30~60°C。 The test method as claimed in claim 1, wherein said predetermined temperature is 30 ~ 60 ° C.
3. 如权利要求1所述的测试方法,其特征在于所述脂质体药物与空气的体积比为1: (0· 1 ~5) 〇 Square (0 · 1 to 5): The test method as claimed in claim 1, wherein the volume ratio of the liposomal drug and air is 1
4. 如权利要求1所述的测试方法,其特征在于所述步骤(2)中的所述运动为绕一非竖直的旋转轴旋转,且所述容器为条状,所述容器以径向垂直所述旋转轴的方式安装。 4. The test method according to claim 1, wherein said step of motion (2) to rotate about a non-vertical axis of rotation and said container is a strip, the vessel diameter the installation in the vertical rotary shaft.
5. 如权利要求4所述的测试方法,其特征在于所述旋转运动利用分子杂交仪完成,所述旋转轴为水平,所述容器为离心管,所述转速为15~50rpm。 5. The test method according to claim 4, characterized in that the rotational movement is completed using hybridization instrument, the rotary shaft is horizontal, the container is a centrifuge tube, the speed of 15 ~ 50rpm.
6. 如权利要求1所述的测试方法,其特征在于在所述步骤⑴中,向所述容器内加入预设量的用于吸附释放出的游离药物的离子交换树脂,所述的预设量不小于将所述脂质体药物内包封的所有药物全部吸附所需的量。 The method of testing as claimed in claim 1 according to predefined requirements, characterized in that said step ⑴, ions for adsorbing a predetermined amount of released free drug was added to the container exchange resin, It is not less than the amount of all the drug encapsulated within the liposomal drug needed to fully adsorbed.
7. 如权利要求6所述的测试方法,其特征在于所述离子交换树脂为阳离子交换树脂。 7. The test method according to claim 6, wherein said ion exchange resin is a cation exchange resin.
8. 如权利要求1所述的测试方法,其特征在于所述脂质体药物为脂质体蒽环类药物、 脂质体长春新碱或脂质体两性霉素B。 8. The test method according to claim 1, characterized in that said liposomal drug liposomes anthracycline, liposomal vincristine or liposomal amphotericin B.
9. 如权利要求8所述的测试方法,其特征在于所述蒽环类药物为柔红霉素、阿霉素、阿柔比星、表阿霉素、伊达比星、戊柔比星或米托蒽醌。 9. The test method according to claim 8, wherein the anthracycline agent is daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, valrubicin or mitoxantrone.
10. 如权利要求1所述的测试方法,其特征在于所述脂质体药物为脂质体阿霉素。 10. The test method according to claim 1, characterized in that the liposome agent is liposomal doxorubicin.
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