CN106546706A - External release testing method of liposome medicaments prepared by pH gradient active drug loading method - Google Patents

External release testing method of liposome medicaments prepared by pH gradient active drug loading method Download PDF

Info

Publication number
CN106546706A
CN106546706A CN201510600419.5A CN201510600419A CN106546706A CN 106546706 A CN106546706 A CN 106546706A CN 201510600419 A CN201510600419 A CN 201510600419A CN 106546706 A CN106546706 A CN 106546706A
Authority
CN
China
Prior art keywords
release
drug
container
liposome
vitro
Prior art date
Application number
CN201510600419.5A
Other languages
Chinese (zh)
Inventor
唐海玲
张金平
毛文学
苏勇
Original Assignee
上海复旦张江生物医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海复旦张江生物医药股份有限公司 filed Critical 上海复旦张江生物医药股份有限公司
Priority to CN201510600419.5A priority Critical patent/CN106546706A/en
Publication of CN106546706A publication Critical patent/CN106546706A/en

Links

Abstract

The invention relates to a method for testing liposome medicaments, and especially relates to external release of liposome medicaments prepared by a pH gradient active drug loading method. A vertical displacement motion mode of a container is used for controlling rate of a release promoting agent for releasing compounds and dissolving balance of components in solution and air, indirectly controlling release rate of medicaments, and simulating dynamic action of internal blood circulation for medicaments. Addition, temperature or rotating speed and other parameters of the release promoting agent are controlled, in order to accurately adjust release speed. The method has the advantages of high controllability, operation convenience, and good repeatability. Ion exchange resin also can be added in order to adsorb released free drug, in order to simulate internal sink condition state effects.

Description

pH梯度主动载药法制备的脂质体药物的体外释放测试方法 In vitro drug liposome pH gradient active loading and drug release test method Method

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。 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. 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] JingXia Cui等人在脂质体中加入氯化铵,然后放于52°C恒温孵育以考察阿霉素释放行为。 [0007] JingXia Cui et al liposomes ammonium chloride was added, then placed in a 52 ° C incubation temperature to examine the release behavior of doxorubicin. 其原理:氯化铵溶液在加热过程中会分解产生氨分子,同时在接近磷脂相变52°C的温度下,脂质体膜流动性提高,于是氨分子可以透过脂质膜进入脂质体内部,而后与硫酸阿霉素的阿霉素发生置换反应,置换出的游离阿霉素分子则透过脂膜,扩散到脂质体外部,从而达到阿霉素的释放。 The principle: ammonium chloride solution in the heating process will break down ammonia molecules, while close to the phase transition temperature of the phospholipid 52 ° C, the fluidity is improved liposome membrane, so the ammonia molecules can enter through the lipid membrane lipid inside of the body, followed by substitution reaction with a neomycin sulfate doxorubicin occurs, replacement of free adriamycin molecule through the lipid membrane, diffuse into the outside of the liposome, so as to achieve the release of doxorubicin. 该过程中,原始脂质体内部的pH是偏低的,氨分子进入脂质体内部后会打破pH平衡,从而令药物释放。 In this process, the internal pH of the liposomes is the original low after ammonia molecules break into the inside of the liposome pH balance, so that the drug release. 关于此方法的详细论述可参见文献: ①J Control Release, 2007Apr2;118(2):204-15, Direct comparison of two pegylated liposomal doxorubicin formulations: Is AUC predictive for toxicity and efficacy ; ② Bioanalysis,2011Feb ;3(3):333-44,In vitro and in vivo characterizations of PEGylated liposomal doxorubicin〇 For a detailed discussion of this method can be found in the literature: ①J Control Release, 2007Apr2; 118 (2): 204-15, Direct comparison of two pegylated liposomal doxorubicin formulations: Is AUC predictive for toxicity and efficacy; ② Bioanalysis, 2011Feb; 3 (3 ): 333-44, in vitro and in vivo characterizations of PEGylated liposomal doxorubicin〇

[0008] 此方法的理念来自于固体制剂溶出释放实验中加入促释剂促进药物释放,是公认的用于考察阿霉素脂质体的方法。 [0008] The idea of ​​this method is that in the preparation released from the solid release enhancing agent is added in the experiment to promote the release of the drug, is recognized as a method for inspection of liposomal doxorubicin. 然而在此方法中,阿霉素释放的主要推动力是氯化铵的加入量,如果要达到理想的阿霉素释放量,则需加入大量氯化铵。 However, in this method, the main driver of doxorubicin release of ammonium chloride is added in an amount of, if you want to achieve the ideal of doxorubicin release, you need to add a lot of ammonium chloride. 但同时,氯化铵的化学作用非常迅速,在短时间〇. 5h就能达到60%的释放,3h即达到平台期。 But at the same time, ammonium chloride chemistry very quickly, in a short time square. 5h can reach 60% release, 3h which reached a plateau. 因此,此方法所获得的释放曲线细节特征不明显,无法将阿霉素的主要释放期时间拉长,以增加不同制剂处方之间的区分性,也就无法模拟药物的体内体外释放关系。 Thus, detailed features of the release profile obtained by this method is not obvious, the main release period time can not be stretched doxorubicin, to increase the distinction between the different pharmaceutical formulations, it can not be simulated in vitro drug release in vivo relationship.

发明内容 SUMMARY

[0009] 本发明的目的在于提供一种释药速率可控性高、重复性好的脂质体药物体外释放的测试方法。 [0009] The object of the present invention is to provide a high release rate controllable, reproducible liposomes in vitro drug release test method.

[0010] -种pH梯度主动载药法制备的脂质体药物的体外释放测试方法,所述脂质体药物为溶液形态,溶解有包封了药物的脂质体颗粒,包含如下步骤: [0010] - pH gradient types in vitro drug active loading Method of liposomal drug release test method, the liposome pharmaceutical form is a solution, dissolving the drug encapsulated liposome particles, comprising the steps of:

[0011] (1)在预设定的温度下,将所述脂质体药物与促释剂的混合溶液装入容器内,并将所述容器封闭,所述容器内还存在不超过其容积30%的空气; [0011] (1) at a preset temperature, the solution was mixed with the liposomal drug release enhancing agent is loaded into the container, and the container closure, there is no more than the volume within said container 30% air;

[0012] (2)令所述容器进行在竖直方向上存在来回位移的运动; [0012] (2) the presence of movement causes the container back and forth displacement in a vertical direction;

[0013] (3)在所需时间点取样,并测定所述脂质体药物的未释放药物浓度或已释放药物浓度,计算药物的释放速率。 [0013] (3) the time required for the sampling points, and determining the release of liposomal drug in the drug concentration or not to release the drug concentration, the rate of drug release is calculated.

[0014] 进一步地,所述步骤(1)中预设定的温度为所述脂质体颗粒的磷脂相变温度±20 摄氏度。 [0014] Further, the step (1) in a pre-set temperature of the lipid particles phospholipid phase transition temperature ± 20 ° C.

[0015] 进一步地,所述药物为蒽环类药物、长春新碱或两性霉素B,优选为阿霉素。 [0015] Further, the drug is anthracycline, vincristine or amphotericin B, preferably doxorubicin.

[0016] 进一步地,所述混合溶液中促释剂的浓度为5~100mM。 [0016] Further, the release enhancing agent in the mixed solution at a concentration of 5 ~ 100mM. 所述混合溶液pH值为5. 5-6. 5〇 The pH of the mixed solution is 5-6. 5〇

[0017] 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 [0017] 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 .

[0018] 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。 [0018] 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.

[0019] 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 [0019] 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.

[0020] 更进一步地,所述离子交换树脂为阳离子交换树脂。 [0020] Still further, the ion exchange resin is a cation exchange resin.

[0021] 与现有方法相比,本发明具有如下优点: [0021] Compared with the conventional method, the present invention has the following advantages:

[0022] (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。 [0022] (1) the blood circulation dynamic action of the drug can better simulate the behavior of the released medicament through the container into the body motion simulation, which is closer to the real situation of the release process in vivo release.

[0023] (2)所有操作参数,包括促释剂浓度、温度、转速等均可控,从而能方便地调节释放速率,无论是需要短时间快速释放,还是需要拉长主要释放期时间以增强多个释药曲线之间的区分性,均能方便地实现。 [0023] (2) all operating parameters, including release-enhancing concentration, temperature, speed etc. controllable, so that it can easily adjust the rate of release, either quick release required short time, or need to lengthen the main release period of time to enhance the distinguishing between a plurality of release profile, it can be easily achieved.

[0024] (3)实验重复性远高于现有技术的各种方法,可靠性高。 [0024] (3) is much higher than the experimental reproducibility of the various methods of the prior art, high reliability.

[0025] (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。 [0025] (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.

[0026] 综上所述,本发明是目前唯一能满足药品生产质量评价体系要求的测试方法。 [0026] In summary, the present invention is the only method able to meet pharmaceutical production quality evaluation test system requirements.

附图说明 BRIEF DESCRIPTION

[0027] 图1 :静置、搅拌、旋转三种不同运动方式下的释放行为对比图; [0027] FIG. 1: standing, stirring, rotation release behavior under three different sports comparison chart;

[0028] 图2 :实施例2中每个时间点的6支样品释放速率曲线对比图; [0028] FIG. 2: Sample 6 Comparative release rate profile view of the embodiment 2 of each time point;

[0029] 图3 :温度及PBS缓冲液的pH值对释放速率的影响; [0029] FIG. 3: PBS buffer solution temperature and pH on the release rate value;

[0030] 图4 :实施例12中不同氯化铵浓度下阿霉素的释放速率对比图; [0030] Figure 4: Example of doxorubicin release rate at different concentrations of ammonium chloride Comparative Figure 12;

[0031] 图5 :实施例14中不同处方脂质体的释放速率对比图; [0031] FIG 5: different embodiments of the liposome formulation the release rate comparison in Figure 14;

[0032] 图6 :实施例14中3ml加液量下各组样品的释放速率对比图; [0032] Figure 6: Example 14 was added an amount of 3ml each set of samples at the rate of release of comparison chart;

[0033] 图7 :实施例15中4ml加液量下各组样品的释放速率对比图。 [0033] Figure 7: Example 15 was added in an amount of 4ml release rate of each set of samples under comparison FIG.

具体实施方式 Detailed ways

[0034] 本专利公开的测试方法适用于所有利用pH梯度主动载药法制备的脂质体,如阿霉素脂质体、长春新碱脂质体等。 [0034] Test method of the present patent disclosure for all pH gradient using Method drug active loading of liposomes, liposomes such as doxorubicin, vincristine liposomes.

[0035] 本专利中,促释剂定义是广义上包括了所有能促进脂质体药物释放的物质,其包括表面活性剂,或能在溶液中释放出氨分子的化合物,如硫酸铵、氯化铵、醋酸铵、乙醇胺、 二乙胺、乙二胺等。 [0035] The present patent, release-enhancing agent is defined as including all the material to promote release of liposomal drug in a broad sense, including a surface active agent, or a compound capable of releasing ammonia molecules in solution, such as ammonium sulfate, chloride , ammonium acetate, monoethanolamine, diethylamine, ethylene diamine and the like. 凡是能够在水溶液中分解出能进入脂质体内部并打破保持药物在脂质体内部稳定存在的pH梯度的化合物都可以使用。 Who can decompose the compound in the aqueous solution can be broken up into the interior of the liposome and the drug to maintain the pH gradient inside the liposomes are stable can be used.

[0036] 促释剂的加入量是重要的控制参数之一。 [0036] The added amount of the release enhancing agent is an important control parameter. 以氯化铵为例,其在溶液中的浓度越低, 释放出的氨分子就越少,混合溶液中脂质体药物能接触到的氨分子也越少,则释放速率会降低。 Ammonium chloride, for example, the lower its concentration in the solution, the less release of the ammonia molecule, a mixed solution of liposomal drug molecules have access to the less ammonia, the release rate decreases. 同时,促释剂的加入量也与其种类有关。 Meanwhile, the amount of release-enhancing agent is also related to its kind. 例如,醋酸铵相对于氯化铵属于弱电解质, 因此,若要电离出有效量的氨分子,则醋酸铵的加入量要比氯化铵多很多。 For example, ammonium chloride, ammonium acetate are weakly with respect to electrolyte, and thus, to an effective amount of ionized molecules of ammonia, ammonium acetate is added in an amount of ammonium chloride a lot more than that.

[0037] 脂质体药物的浓度必须根据实验需要设定,属于不可调节参数,药学实验中通常选择接近人体内药物分布的浓度。 [0037] The concentration of the liposomal drug according to the experimental needs to be set, are non-adjustable parameter, typically chosen close to the pharmaceutical experiments concentration distribution of the drug in humans. 在溶液中,脂质体药物与促释剂的比例不存在严格的限制。 In solution, with the ratio of liposomal drug release enhancing agent is not strictly limited exist. 其质量摩尔比范围可达1: (〇. 1~5),甚至更广,这是因为除了促释剂之外,还有温度、 转速等参数同样可起到调节药物释放速率的作用,这些参数的效果可在一定程度上相互抵消。 The mass range up to a molar ratio 1: (1 to 5 billion.), Or even wider, this is because in addition to release enhancing agent, there are temperature, speed and other parameters may likewise play a role in regulating the rate of drug release, these effect parameters can offset each other to some extent. 并且,促释剂加入太少或过量都不会导致实验失败,只是无法获得速率理想的释药曲线,但有时我们也需要考察极端条件下的释药情况,因此促释剂的加入量不存在一个绝对的"允许区间"。 Also, release-enhancing agent is added too little will not lead to excessive or experiment fails, just can not get the desired release rate curve, but sometimes we also need to examine the case under the extreme conditions of release, and therefore the amount of added release enhancing agent is absent an absolute "allowed range."

[0038] 试验温度通常选择在脂质体颗粒的磷脂相变温度附近。 [0038] The test temperature is generally selected from phospholipid liposomes near the phase transition temperature of the particles. 如阿霉素脂质体的磷脂相变温度为52度,则通常在50度上下进行试验。 Phospholipids such as doxorubicin liposome phase transition temperature of 52 degrees, usually tested at 50 degrees up and down. 由于该方法中的容器必须进行旋转,无法像静置法那样采用水浴,所以控温条件不如静置法,温度通常会有〇. 5~1度的波动,但这种程度的小幅度变温对实验结果影响不大。 Since this method must be rotating in the container, not as a standing method, as a water bath, so that temperature conditions are not as good as a standing method, the temperature will generally square. 1 ~ 5 fluctuation degrees, but the small-amplitude degree of temperature change The results had little effect.

[0039] 在对溶液施加运动方面,则利用分子杂交仪进行绕水平轴的旋转最为方便。 [0039] The solution in motion is applied, using the hybridization instrument is rotated about a horizontal axis the most convenient. 离心管最好以径向向心的方式竖直设置在旋转轴四周。 Centrifuge tube is preferably vertically disposed in a radial direction in a manner rotating shaft center of four weeks. 这样的旋转方式可令离心管内的少量空气形成气泡分散到溶液中,不仅能模拟体内血液循环,更有助于促进药物的释放,气泡与脂质体发生物理碰撞会破坏脂膜,导致药物释放。 Such rotation manner to make a small amount of air bubbles in the centrifuge tube is formed into a dispersion solution, not only can make the blood circulation, but also help to promote the release of the drug, the liposome physical bubble collision will destroy lipid membranes, resulting in release of the drug .

[0040] 不过如果离心管内的空气太多,则虽然能加速药物释放,但空气随着旋转而分散的过程是混沌体系,较难控制。 [0040] However, if too much air in the tube, although it can accelerate the drug release, but the air with the rotation of the dispersion process is chaotic system, difficult to control. 在这种情况下,即使温度、转速、空气体积等各参数都保持一致,多次重复试验所获得的释药曲线的重复性仍不及仅保留少量空气,主要利用促释剂进行化学释放所获得的实验结果。 In this case, even when the temperature, rotation speed, air volume, and other parameters are consistent, reproducible release profile of repeated testing and the obtained still retained only a small amount of air, the main use of release-enhancing chemical release agent obtained experimental results. 由于氯化铵分解出的氨分子必须有一个从溶液进入空气、 再从空气返回溶液中的平衡,所以也不能完全去除容器内的空气。 Since the decomposition of ammonium chloride in the ammonia molecule must have a solution from entering the air, and then return to equilibrium in solutions from the air, so it can not completely remove the air in the container. 另一方面,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。 On the other hand, an ion exchange resin may also be utilized to release free drug adsorption, free drug is released exchange resin, measuring the residual concentration of the drug can be directly and easily.

[0041] 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+。 [0041] 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.

[0042] 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。 [0042] 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.

[0043] 以下通过实施例对本发明的具体实施方式做进一步说明。 [0043] The following examples further illustrate specific embodiments of the present invention do.

[0044] (1)考察静置、搅拌、旋转三种不同运动方式下的释放行为 [0044] (1) Investigation standing, stirring, rotation release behavior under three different sports

[0045] 对比例1:静置-52°(:-10%树脂-331111氯化铵 [0045] Comparative Example 1: left -52 ° (: - 10% ammonium chloride resin -331111

[0046] A、配置阿霉素脂质体溶液: [0046] A, arranged liposomal doxorubicin solution:

[0047] 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;用移液枪移取阿霉素脂质体2. 5ml至100ml容量瓶中,用上述PBS定容为40倍稀释液(pH6. 0),计算出初始药物浓度。 [0047] The pH phosphate buffered saline (Phosphate buffered saline, PBS) 6. 0 is placed in an ultrasonic cleaning machine ultrasonic degassing 20min; pipetting with a pipette liposomal doxorubicin 2. 5ml to 100ml capacity flask, a constant volume of PBS with the above 40-fold dilutions (pH6. 0), the initial drug concentration is calculated.

[0048] B、阳离子交换树脂预处理 [0048] B, pretreated cation exchange resin

[0049] 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。 [0049] 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.

[0050] C、体外释放实验 [0050] C, in vitro release experiment

[0051] a、在5ml的EPPEND0RF离心管中加入3ml阿霉素脂质体稀释液; [0051] a, was added 3ml liposomal doxorubicin dilutions of EPPEND0RF 5ml centrifuge tube;

[0052] b、在溶液中加入氯化铵形成混合溶液,使得氯化铵浓度为33mM ; [0052] b, was added a solution of ammonium chloride to form a mixed solution, such that the concentration of ammonium chloride 33mM;

[0053] c、将离心管密闭后进行52°C恒温水浴,然后在3h、6h、8h后分别取样,每个时间点取3支样品取平均值; [0053] c, after the sealed tube for 52 ° C water bath, and then were taken after 3h, 6h, 8h, three time points for each sample averaged;

[0054] d、将离心管取出后立刻放入冰水浴中,待冷却后,分别加入0. 3g(w/v = 10% )阳离子交换树脂,涡旋震荡30s以令游离药物被吸附,取上清lml在4°C下用HPLC检测样品上清液中的残留药物浓度。 [0054] d, the centrifuge tubes after removal immediately placed in an ice-water bath, after cooling, were added 0. 3g (w / v = 10%) cation exchange resin, vortexed 30s in order to make free drug is adsorbed, taken lml supernatant at 4 ° C for a concentration by HPLC detection of drug residues in the sample supernatant.

[0055] e、药物释放速率=(1-残留药物浓度/初始药物浓度)X 100%。 [0055] e, the drug release rate = (1 - residual drug concentration / initial concentration of the drug) X 100%.

[0056] 对比例2:搅拌-52°(:-10%树脂-331111氯化铵 [0056] Comparative Example 2: A mixture of -52 ° (: - 10% ammonium chloride resin -331111

[0057] 步骤A和B同对比例1。 [0057] Steps A and B with Comparative Example 1.

[0058] 步骤C中,取50ml阿霉素脂质体置于250ml茄形瓶中,氯化铵加入量同样为最终浓度33mM,树脂加入量同样为10% (5g)。 [0058] Step C, 50ml of doxorubicin liposomes was placed 250ml eggplant-shaped flask, the same amount of ammonium chloride was added to a final concentration of 33mM, the same amount of resin was 10% (5g). 然后将茄形瓶放入恒温水浴锅中进行52°C水浴, 同时以210rpm进行搅拌,并在3h、6h、8h后分别取样。 The vial was then placed in a thermostatic eggplant-water bath for 52 ° C water bath, while stirring at 210rpm, and were taken after 3h, 6h, 8h.

[0059] 取样时,先停止搅拌,待溶液静置后再用移液枪移取上清lml,立即放入冰水浴中, 在4°C下用HPLC检测样品上清液中的残留药物浓度。 [0059] Samples, before stirring was stopped, and then allowed to stand until the solution with a pipette lml of supernatant was pipetted, immediately placed in an ice water bath, at 4 ° C for a concentration by HPLC detection of drug residues in sample supernatant .

[0060] 药物释放速率=(1-残留药物浓度/初始药物浓度)X 100%。 [0060] Drug release rate = (1 - residual drug concentration / initial concentration of the drug) X 100%.

[0061] 实施例1:旋转-52°(:-10%树脂-331111氯化铵 [0061] Example 1: rotation -52 ° (: - 10% ammonium chloride resin -331111

[0062] 步骤A和B同对比例1、2。 [0062] Steps A and B with Comparative Example 1.

[0063] C、体外释放实验 [0063] C, in vitro release experiment

[0064] a、在5ml的EPPEND0RF离心管中加入5ml阿霉素脂质体稀释液; [0064] a, liposomal doxorubicin was added 5ml dilutions of EPPEND0RF 5ml centrifuge tube;

[0065] b、在溶液中加入氯化铵形成混合溶液,使得氯化铵浓度为33mM ; [0065] b, was added a solution of ammonium chloride to form a mixed solution, such that the concentration of ammonium chloride 33mM;

[0066] c、在混合溶液中加入0· 5g(w/v = 10% )阳离子交换树脂; [0066] c, was added 0 · 5g (w / v = 10%) cation-exchange resin in the mixed solution;

[0067] d、设定分子杂交仪温度为52°C,转速为Orpm,进行预热同时加样;。 [0067] d, hybridization instrument set temperature 52 ° C, speed of Orpm, while preheating loading;.

[0068] e、将离心管封闭后,以径向向心的方式,垂直于旋转轴地固定在分子杂交仪的杂交管架上; [0068] e, after the tube is closed, in a radial manner to the heart, perpendicular to the rotational axis of the tube is fixed in the hybridization of the hybridization instrument holder;

[0069] f、设定分子杂交仪温度为52°C,转速为25rpm,开始旋转并计时。 [0069] f, hybridization instrument set temperature of 52 ° C, speed of 25rpm, and counting starts rotating.

[0070] g、在3h、6h、8h后分别取样,每个时间点取3支样品取平均值; [0070] g, were taken after 3h, 6h, 8h, three time points for each sample averaged;

[0071] h、将离心管取出后立刻放入冰水浴中,在4°C下用HPLC检测样品上清液中的残留药物浓度。 [0071] h, will be removed immediately after the tube placed in an ice-water bath, at 4 ° C for a concentration by HPLC detection of drug residues in the sample supernatant.

[0072] i、药物释放速率=(1-残留药物浓度/初始药物浓度)X 100%。 [0072] i, the drug release rate = (1 - residual drug concentration / initial concentration of the drug) X 100%.

[0073] 表1、对比例1、对比例2、实施例1的阿霉素释放速率对比 [0073] Table 1, Comparative Example 1, Comparative Example 2, the release rate of doxorubicin Example Comparative Example 1

Figure CN106546706AD00071

[0075] 与表1中实验数据对应的附图1为三种不同运动方式下阿霉素脂质体的释放行为对比。 [0075] Table 1 Experiment 1 Comparative data for the corresponding reference liposomal doxorubicin release behavior of three different motion.

[0076] 从机理上分析,三种运动方式的共同点是促使阿霉素释放的主要动力为氯化铵分解产生的氨分子,主要影响因素是氯化铵浓度。 [0076] from the mechanism analysis, three motions common way is to promote the main driver of doxorubicin release of ammonium chloride decomposition of ammonia molecules, the main factor is the concentration of ammonium chloride. 区别则在于,不同运动方式下,氯化铵分解产生的氨分子运动不同。 The difference is that, under different sports, ammonia molecules different motion generated by the decomposition of ammonium chloride. 氨气极易溶于水且密度比空气轻,在搅拌或转动的运动方式下,其扩散至液面上层后会立刻重溶于溶液中。 After ammonia gas highly soluble in water and a density of lighter than air, under stirring or rotating motion, which diffuse to the upper level immediately re-dissolved in the solution.

[0077] 对比例1的静置条件下,氯化铵释放氨气-氨气跨膜进入脂质体内部-阿霉素游离释放,整个过程取决于化学平衡,无外界作用力干扰。 [0077] allowed to stand under the conditions of Comparative Example 1, the release of ammonia chloride - ammonia gas into the interior of the liposome membrane - release of free doxorubicin, depending on the chemical balance of the whole process, no external force disturbance. 其中氨气的释放和跨膜都是非常快速的过程,因此阿霉素的释放也会在短时间内达到峰值,之后释放逐渐放缓。 Where the ammonia release and transmembrane are very quick process, and therefore will release doxorubicin in a short time peaked after the release of a gradual slowdown.

[0078] 对比例2和实施例1的运动条件下,氨气在空气与溶液中的溶解和释放达到动态平衡。 [0078] Comparative Example 2 and the motion of the conditions of Example 1, the ammonia in the air and solution dissolution and release dynamic equilibrium. 由于离心管/茄形瓶中都有空气的存在,氨分子从氯化铵中分解出来,随着搅拌和转动的过程扩散部分扩散到空气中,而后又在搅拌和转动中重新回到溶液中。 Since the centrifuge tube / eggplant-shaped flask of air are present, decomposition from ammonia molecules of ammonium chloride, with stirring and during the rotation of the divergent section into the air while stirring and then the solution is rotated back in . 该动态过程使得体系内氨气浓度保持平衡。 The dynamic process such that the ammonia concentration in the system to maintain a balance.

[0079] 而对比例2与实施例1的区别在于,采用搅拌方式时,茄形瓶是敞口体系,试验温度比较高,部分氨气会从瓶口挥发。 [0079] 2 and Comparative Example 1, the difference is that, when using the agitation, the system is open at eggplant-shaped flask, the test temperature is relatively high, part of the ammonia will evaporate from the bottle. 这导致在氯化铵浓度相同的条件下,两个体系的初始释放浓度相同,但对比例2的后期释放比例低于密闭体系。 This results in the concentration of ammonium chloride at the same conditions, the same concentration of the initial release of the two systems, but the late release of Comparative Example 2 is lower than the proportion of a closed system.

[0080] 三个体系下,采用封闭容器旋转方式的释放速率最为均一,且最终能达到的释放比例最高。 [0080] the three systems, the use of a closed container rotatably most uniform release rates, and the highest percentage of the final release can be achieved.

[0081] 在静置的条件下,如果想降低初期的释放速率,以便对该阶段的释放行为做更细致的观察,则可降低氯化铵浓度。 [0081] under static conditions, if you want to decrease the initial rate of release, in order to make a more detailed observation of the phase behavior of release, the concentration of chloride can be reduced. 但这也会导致最终难以达到理想的释放比例,比如附图2中最终释放比例约为55%,但如果降低氯化铵浓度,可能只能达到40%就进入平台期,根本无法在降低氯化铵加入量(浓度)的条件下,将整个释放上升期拉开,同时达到有效释放总量。 But it can also lead to the release of the final difficult to achieve the desired ratio, such as 2 final figures released ratio is about 55%, but if you reduce the concentration of ammonium chloride, may only reach 40% to enter the plateau, simply can not reduce chlorine ammonium under addition amount (concentration) will pull the entire release period of rise, while achieving the effective release of the total amount.

[0082] 而本专利公开的方法,不仅前后期释放速率均一,且最终释放比例高,还能通过阳离子吸附树脂的加入,最大限度模拟人体内的释放行为。 [0082] The method of the present patent disclosure, only pre-and post uniform rate of release, and a high proportion of the final release, but also by adsorbing a cationic resin is added, to maximize the release behavior of the simulated human body.

[0083] (2)考察本方法的重复性优劣 [0083] (2) investigate the merits of the process repeatability

[0084] 实施例2 :52°C -10%树脂_48mM氯化铵 [0084] Example 2: 52 ° C -10% ammonium chloride resin _48mM

[0085] 步骤A、B、C均同实施例1。 [0085] Step A, B, C were the same as in Example 1. 区别在于混合溶液中的氯化铵最终浓度为48mM。 Except that the final concentration of the mixed chloride solution is 48mM.

[0086] 在时间点为1 h、2h、4h、6h、8h时取样,且每个时间点取6支样品,并求出标准差(SD)。 [0086] at time point 1 h, 2h, 4h, 6h, 8h, sampling, and each sample time points 6 and obtains standard deviation (SD). 试验结果如表2所示。 The test results are shown in Table 2.

[0087] 表2、实施例2中每个时间点的6支样品释放速率 [0087] Table 2, the release rate of the samples in Example 6 in embodiment 2 for each time point

Figure CN106546706AD00081

[0089] 附图2是对表2数据的作图。 [0089] Figure 2 is a plot of the data in Table 2. 从图中可以看出,在同时操作的6组考察组中,阿霉素释放速率非常接近,方法重复性很好。 As can be seen from the figure, in the study group while the group 6 in operation, very close to the rate of release of doxorubicin, repeatability is good.

[0090] (3)考察温度及PBS缓冲液的pH值对释放速率的影响 [0090] (3) pH value influence on the release rate and the temperature of PBS buffer investigated the

[0091] 实施例3 :37°C -10%树脂_33mM 氯化铵-ρΗ5· 5 [0091] Example 3: 37 ° C -10% ammonium resin _33mM -ρΗ5 · 5

[0092] 实施例4 :37°C -10%树脂_33mM 氯化铵-ρΗ6· 0 [0092] Example 4: 37 ° C -10% ammonium chloride resin _33mM -ρΗ6 · 0

[0093] 实施例5 :37°C -10%树脂_33mM 氯化铵-ρΗ6· 5 [0093] Example 5: 37 ° C -10% ammonium resin _33mM -ρΗ6 · 5

[0094] 实施例3、4、5的实验条件如标题所示,其他均与实施例1相同,区别在于温度设定为37°C ;PBS缓冲液的pH值分别调剂至5. 5、6. 0和6. 5 ;取样时间点为4h、8h,每个时间点取1支样品。 [0094] Experimental conditions of Example 3, 4 embodiment as shown in the title are the same as in Example 1 and the other embodiments, except that the temperature was set to 37 ° C; pH value of the PBS buffer to each toner 5. 5,6 2.0 and 6.5; sampling time point of 4h, 8h, each time a sample was taken.

[0095] 实施例6:47°(:-10%树脂-331111氯化铵-?!15.5 [0095] Example 6: 47 ° (: - 10% resin -331111 chloride - 15.5 ?!

[0096] 实施例7:47°(:-10%树脂-331111氯化铵-?册.0 [0096] Example 7: 47 ° (: - 10% ammonium chloride resin -331,111 - 0.01 Volume?

[0097] 实施例8:47°(:-10%树脂-331111氯化铵-?册.5 [0097] Example 8: 47 ° (: - 10% ammonium chloride resin -331,111 - .5 volumes?

[0098] 实施例6、7、8的实验条件如标题所示,其他均与实施例1相同,区别在于温度设定为47°C ;PBS缓冲液的pH值分别调剂至5. 5、6. 0和6. 5 ;取样时间点为4h、8h,每个时间点取1支样品。 [0098] Experimental conditions of Example 6, 7 as shown in title embodiment are the same as in Example 1 and the other embodiments, except that the temperature was set to 47 ° C; pH value of the PBS buffer to each toner 5. 5,6 2.0 and 6.5; sampling time point of 4h, 8h, each time a sample was taken.

[0099] 实施例9 :52°C -10%树脂_33mM 氯化铵-ρΗ5· 5 [0099] Example 9: 52 ° C -10% ammonium resin _33mM -ρΗ5 · 5

[0100] 实施例10 :52°C -10%树脂_33mM 氯化铵-ρΗ6· 0 [0100] Example 10: 52 ° C -10% ammonium chloride resin _33mM -ρΗ6 · 0

[0101] 实施例11 :52°C -10%树脂_33mM 氯化铵-ρΗ6· 5 [0101] Example 11: 52 ° C -10% ammonium resin _33mM -ρΗ6 · 5

[0102] 实施例9、10、11的实验条件如标题所示,其他均与实施例1相同,区别在于温度设定为52°C ;PBS缓冲液的pH值分别调剂至5. 5、6. 0和6. 5 ;取样时间点为4h、8h,每个时间点取1支样品。 [0102] Experimental conditions of Example 9, 10 of the embodiment as shown in the title are the same as in Example 1 and the other embodiments, except that the temperature was set to 52 ° C; pH value of the PBS buffer to each toner 5. 5,6 2.0 and 6.5; sampling time point of 4h, 8h, each time a sample was taken.

[0103] 上述实施例3~11的实验数据如下表3所示,同时附图4是对表3的作图。 [0103] The experimental data above Examples 3 to 11 shown in the following Table 3, while Figure 4 is a plot of Table 3.

[0104] 表3、实施例3~11的阿霉素释放速率对比 [0104] Table 3, doxorubicin release rate for Example 3 - Comparative 11

Figure CN106546706AD00091

[0106] 由附图3可以看出,相同pH条件下,随着温度的升高,阿霉素释放速率大幅增加。 [0106] 3 can be seen from the drawings, the same pH conditions, as the temperature increases, the release rate of doxorubicin significantly increased. 同时,在相同温度条件下,提高PBS缓冲液的pH值会加速阿霉素的释放,主要是因为pH值的升高更有利于氯化铵中氨分子的分解。 Meanwhile, at the same temperature conditions, increase of PBS buffer pH value accelerated release doxorubicin, mainly because of the increase of pH is more conducive to decomposition of ammonium chloride in the ammonia molecule.

[0107] (4)考察氯化铵浓度对释放速率的影响 [0107] (4) The effects of ammonium chloride concentration on the rate of release of

[0108] 实施例12 :52°C -10%树脂 [0108] Example 12: 52 ° C -10% resin

[0109] 实施例12的实验条件如标题所示,其他均与实施例1相同,区别在于分别将混合溶液中氯化铵的浓度调节为0mM、8mM、17mM、33mM、48mM、64mM、77mM,逐一进行试验。 [0109] The experimental conditions of Example 12 are shown in the title are the same as in Example 1 other, respectively, except that the mixed solution of ammonium chloride concentration was adjusted to 0mM, 8mM, 17mM, 33mM, 48mM, 64mM, 77mM, one by one test. 取样时间点为6h,每个时间点取1支样品。 Sampling time point of 6h, each time a sample was taken.

[0110] 实施例12的数据如下表4所示,附图4为表4的数据作图。 Data [0110] Example 12 are shown in Table 4, Figure 4 is the data in Table 4 are plotted.

[0111] 表4、实施例12中不同氯化铵浓度下阿霉素的释放速率对比 [0111] Table 4 Example Comparative release rates of doxorubicin at different ammonium chloride concentrations of 12

Figure CN106546706AD00101

[0114] 由附图4可以看出,随着氯化铵比例的增加,阿霉素释放速率显著增加。 [0114] As can be seen in Figure 4, with the increase of the proportion of ammonium chloride, the release rate of doxorubicin significantly increased. 阿霉素的释放主要是由氯化铵的加入量来决定,因此可通过控制氯化铵的浓度来控制阿霉素的释放速率。 Doxorubicin release is mainly determined by the amount of ammonium chloride was added, thus the release rate can be controlled by controlling the concentration of doxorubicin ammonium chloride.

[0115] (5)考察本测试方法对不同处方的阿霉素脂质体的区分性 [0115] (5) Investigation of this test method doxorubicin liposomes of different prescriptions distinguishability

[0116] 实施例13 :52°C -10%树脂_33mM氯化铵 [0116] Example 13: 52 ° C -10% ammonium chloride resin _33mM

[0117] 实施例13的实验条件如标题所示,均与实施例1相同。 [0117] The experimental conditions of Example 13. As shown in the title are the same as in Example 1. 区别在受试的阿霉素脂质体为3种不同处方的产品: The difference in the test of liposomal doxorubicin into three different prescription products:

[0118] 处方1 :HSPC(卵磷脂)/Chol(胆固醇)/PEG-(55 :37 :5); [0118] Prescription 1: HSPC (lecithin) / Chol (cholesterol) / PEG- (55: 37: 5);

[0119] 处方2 :HSPC/Chol/PEG-(46 :46 :5); [0119] Prescription 2: HSPC / Chol / PEG- (46: 46: 5);

[0120] 处方3 :HSPC/Chol/PEG-(55 :37 :1)。 [0120] Prescription 3: HSPC / Chol / PEG- (55: 37: 1).

[0121] 三组制剂的特性参数及实验数据如下表5所示,附图6为表5的数据作图。 As shown in Table 5 below characteristic parameter and the experimental data [0121] three groups of the formulation, Figure 6 is the data plotted in Table 5.

[0122] 表5、实施例13中不同处方脂质体的特性参数及释放速率对比 [0122] Table 5, the release rate and characteristic parameter embodiment different liposome formulation in Comparative Example 13

Figure CN106546706AD00102

[0124] 脂质体配方中,胆固醇的作用是脂质膜流动性调节剂。 [0124] Liposome formulations, cholesterol is a lipid membrane fluidity modifier. 其不仅可使膜通透性降低, 减少药物渗漏,还可使脂膜维持一定柔韧性,增强脂质体囊泡抗击外部条件变化的能力。 Membrane permeability can not only reduce, decrease drug leakage, but also to maintain a certain flexibility, lipid membrane, liposome vesicles enhanced ability to fight changing external conditions.

[0125] 与处方1相比,处方2增加了胆固醇含量,从而相对增加了疏水空间的面积。 [0125] Compared with Formulation 1, 2 prescriptions increased cholesterol content, thereby increasing the relative hydrophobicity of the area of ​​space. 由于阿霉素为疏水性化合物,疏水空间的增加更利于其从脂膜内部跨膜释放,因此处方2相对于处方1,其释放速率在理论上应有所增加;处方3仅改变PEG的加入量,对体外释放不会产生显著影响。 Because adriamycin is a hydrophobic compound, the hydrophobic space is more conducive to increased release from the inner membrane lipid membranes, therefore the prescription with the prescription 1 2 that the release rate should be increased in theory; 3 changing only the formulation of the PEG was added amount, no significant effect on the in vitro release.

[0126] 由表5可以看出,通过本测试方法得出的不同处方的释药曲线,其变化规律符合理论推断:处方2的释放速率高于处方1,处方3则无明显差异。 [0126] As can be seen from Table 5, the release profile obtained by this test method different prescriptions which variation estimation consistent with theoretical: 2 is higher than the rate of release formulation Formulation 1, 3 prescription no significant difference. 该结果表明该测试方法对不同处方具有良好的区分性。 The test results show that the method has good resistance to distinguish different prescription.

[0127] 实施例14:49°(:-10%树脂-硫酸铵 [0127] Example 14: 49 ° (: - 10% Resin - ammonium sulfate

[0128] 步骤A、B、C均同实施例1。 [0128] Step A, B, C were the same as in Example 1. 区别在于温度设定为49°C,促释剂改为硫酸铵。 Except that the temperature was set to 49 ° C, release-enhancing agent to ammonium sulfate. 在时间点为lh、2h、4h、6h、8h时取样值。 At the time points lh, 2h, 4h, 6h, 8h, sample values.

[0129] 处方4 :HSPC/Chol/PEG-(56 :37 :5),包封率=95. 35%,粒径=10L 23,硫酸铵浓度=250mM ; [0129] Prescription 4: HSPC / Chol / PEG- (56: 37: 5), 35% encapsulation efficiency = 95, particle size = 10L 23, ammonium sulfate concentration = 250mM;.

[0130] 处方5 :HSPC/Chol/PEG-(62 :37 :5),包封率=96. 10%,粒径=97. 63,硫酸铵浓度=250mM ; [0130] Prescription 5: HSPC / Chol / PEG- (62: 37: 5), 10% encapsulation efficiency = 96, particle size = 9,763, ammonium sulfate concentration = 250mM;..

[0131] 处方6:批?0/〇1〇1^^6-(56:37:5),包封率=95.84%,粒径=97.29,硫酸铵浓度=200mM。 [0131] Prescription 6: 0 Batch / 〇1〇1 ^^ 6-? (56: 37: 5), encapsulation efficiency = 95.84%, particle diameter = 97.29, ammonium sulfate concentration = 200mM.

[0132] 试验结果如表6所示。 [0132] The test results are shown in Table 6.

[0133] 表6、实施例14中不同处方脂质体的特性参数及释放速率对比 [0133] Table 6, Examples and the release rate of different parameters liposome formulation Comparative 14

Figure CN106546706AD00111

[0135] 附图5是对表6的做图。 [0135] Figure 5 is a table of FIG. 6 made. 从图中可以看出,即使只是小幅度地改变处方中磷脂和胆固醇比例,本实验依然能观察到其药物释放行为的明显差异。 As it can be seen from the figure, even if only a slight change in prescription proportion phospholipid and cholesterol, the experiment still significant differences observed in drug release behavior.

[0136] 这是因为,硫酸铵浓度的改变会使脂质体内水相中硫酸阿霉素的沉淀形态有所改变,从而影响其释放行为。 [0136] This is because, to change the concentrations of ammonium sulfate precipitate will form liposomes in the aqueous phase of A neomycin sulfate changed, thus affecting its release behavior. 从结果上看,硫酸铵浓度减小,硫酸阿霉素的沉淀结晶态转变为胶晶态,更有利于阿霉素的释放。 Seen from the results, to reduce the concentration of ammonium sulfate, neomycin sulfate A crystalline precipitate colloidal state into crystalline state more conducive to the release of doxorubicin. 因此,本方法对处方存在微小差异的脂质体具有很好地区分性。 Therefore, this method has a very good area for sub-liposome there is a slight difference prescription.

[0137] (7)考察空气比例对释放速率的影响 Effects [0137] (7) Investigation of the rate of release of air ratio

[0138] 实施例15 :5ml管-52°C -10%树脂_48mM氯化铵-3ml脂质体 [0138] Example 15: 5ml tubes -52 ° C -10% liposome -3ml chloride resin _48mM

[0139] 步骤A、B、C均同实施例2。 [0139] Step A, B, C were the same as in Example 2. 区别在于脂质体药物的加液量为3ml。 Except that the amount of liposomal drug dosing was 3ml. 在时间点为2h、 4h、6h时取样,且每个时间点取6支样品测试,并求出SD值。 At the time point of 2h, 4h, 6h when the sampling time points and each sample was tested six, and calculates the value of SD. 试验结果如表7所示。 The test results are shown in Table 7.

[0140] 表7、实施例15中每个时间点的6支样品释放速率 [0140] Table 7, the release rate of 6 samples for each time point in the embodiment of Example 15

Figure CN106546706AD00112

Figure CN106546706AD00121

[0143] 附图6是对表7数据的作图。 [0143] Figure 6 is a plot of the data in Table 7. 从图中可以看出,加液量降低为3ml时,同组操作的6支样品差异较大,SD值也比较大。 As can be seen from the figure, reduced dosing amount 3ml, 6 branched sample set of operations with a large difference, SD value is relatively large. 和相同条件下进行的实施例2相比,重复性明显下降。 And Example 2 under the same conditions as compared to the repetitive decreased.

[0144] 实施例16 :5ml管-52°C -10%树脂_48mM氯化铵-4ml脂质体 [0144] Example 16: 5ml tubes -52 ° C -10% liposome -4ml chloride resin _48mM

[0145] 步骤A、B、C均同实施例15。 [0145] Step A, B, C were the same as in Example 15. 区别在于脂质体药物的加液量为4ml。 Except that the amount of liposomal drug dosing was 4ml. 试验结果如表8所示。 The test results are shown in Table 8.

[0146] 表8、实施例16中每个时间点的6支样品释放速率 [0146] Table 8, the release rate of the sample 6 in Example 16 embodiment each time point

Figure CN106546706AD00122

[0148] 附图7是对表8数据的作图。 [0148] Figure 7 is a plot of the data in Table 8. 从图中可以看出,与实施例15相比,加液量增加至4ml时,重复性明显提升,但与实施例2所获得的结果相比仍有差距。 As can be seen from the figure, compared to Example 15, increases the amount of liquid added to 4ml, reproducibility improved significantly, but the results obtained in Example 2 as compared to still a gap.

[0149] 综上所述,实施例2、15、16的对比验证了容器内空气体积增加,会导致实验重复性下降的结论。 [0149] In summary, Comparative Example 2,15,16 verified embodiment increases the volume of air within the container will lead to decline in the experimental reproducibility of results. 因此,操作时空气体积最好不要超过容器体积的1/3。 Thus, when the operation of the air volume of preferably not more than 1/3 of the volume of the container. 考虑到实验操作的便捷性,在使用离心管和分子杂交仪作为实验器材的情况下,直接按照离心管操作规程所限定的最大容积装药即可,无需在意残余空气的绝对体积。 Considering the ease of operation of the test, in the case of the centrifuge tube and the analyzer as a hybridization experiment equipment, directly in accordance with procedures defined in the centrifuge tube maximum volume charge can, without taking into absolute volume of residual air. 在此条件下,多次试验所获得的释药曲线的重复性足够满足质量评价体系的需求。 Under these conditions, many tests reproducible release profile of the obtained sufficiently meet the needs of quality evaluation system.

[0150] 以上对本发明的具体实施例进行了描述。 [0150] The foregoing specific embodiments of the invention have been described. 需要理解的是,这些实施例仅用于说明可以通过调节各项参数获得期望的测试效果,而非用于指导参数的具体设定。 It is to be understood that these embodiments are illustrative only and may be obtained by adjusting the parameters of the desired test results, rather than for a particular set of parameters guiding. 在理解了促释剂的种类和加入量,以及温度对药物释放速率的影响原则之后,针对任何处方的脂质体和实验目的,均可通过有限次试验获得最佳测试方案,而并不局限于上述特定实施方式,更不局限于只能用于测试阿霉素或其他蒽环类脂质体的药物释放。 After understanding the types and amount of release enhancing agent, and the principle effect of temperature on the rate of drug release for liposomal any prescription and experimental purposes, you can get the best testing program by a limited number of tests, but not limited to to the specific embodiments, but is not limited to only be used to test drugs doxorubicin or other anthracycline liposomes released. 本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质。 Those skilled in the art can make various changes and modifications within the scope of the appended claims, this does not affect the essence of the present invention.

Claims (10)

1. 一种pH梯度主动载药法制备的脂质体药物的体外释放测试方法,所述脂质体药物为溶液形态,溶解有包封了药物的脂质体颗粒包含如下步骤: (1) 在预设定的温度下,将所述脂质体药物与促释剂的混合溶液装入容器内,并将所述容器封闭,所述容器内还存在不超过其容积30%的空气; (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的未释放药物浓度或已释放药物浓度, 计算药物的释放速率。 A pH gradient liposomes active drug carrier prepared in vitro drug release test Method The method, the liposome pharmaceutical form is a solution, dissolving liposome encapsulated drug particles comprising the steps of: (1) at a preset temperature, the solution was mixed with the liposomal drug release enhancing agent is loaded into the container, and the container closure, there is no air of more than 30% of its volume within the container; ( 2) make the presence of the vessel back and forth movement of displacement in the vertical direction; (3) the time required for the sampling points, and determining the release of liposomal drug was not released drug concentration or drug concentration of the drug calculated The release rate.
2. 如权利要求1所述的体外释放测试方法,其特征在于步骤(1)中预设定的温度为所述脂质体颗粒的磷脂相变温度±20摄氏度。 2. The in vitro release testing in claim 1, characterized in that the step (1) in a pre-set temperature of the lipid particles phospholipid phase transition temperature ± 20 ° C.
3. 如权利要求1所述的体外释放测试方法,其特征在于步骤(1)中所述混合溶液中所述脂质体药物与促释剂的质量摩尔比范围为1: (〇. 1~5)。 3. The in vitro release of the test method according to claim 1, wherein the step (1) in the mixed solution with the mass of liposomal drug release enhancing agent is a molar ratio range 1: (1 billion. 5).
4. 如权利要求1所述的体外释放测试方法,其特征在于步骤(1)中所述混合溶液中促释剂的浓度为5~lOOmM。 4. The in vitro according to a method as claimed in claim release test, wherein the step (1) of said release enhancing agent is a mixed solution of a concentration of 5 ~ lOOmM.
5. 如权利要求1所述的体外释放测试方法,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。 5. The in vitro release testing in claim 1, characterized in that said step of motion (2) to rotate about a non-vertical axis of rotation and said container is a strip, the container installation radially perpendicular to the axis of rotation.
6. 如权利要求5所述的体外释放测试方法,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。 5 6. The in vitro release testing according to claim, characterized in that the rotational movement is completed using hybridization instrument, the rotary shaft is horizontal, the container is a centrifuge tube.
7. 如权利要求1所述的体外释放测试方法,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。 7. The in vitro release testing in claim 1, characterized in that said step ⑴, a predetermined amount of ions for adsorption of released free drug was added to the container exchange resin, according to It is not less than a predetermined amount of drug encapsulated within all of the liposomal drug needed to fully adsorbed.
8. 如权利要求7所述的体外释放测试方法,其特征在于所述离子交换树脂为阳离子交换树脂。 8. The in vitro release of the test method according to claim 7, wherein said ion exchange resin is a cation exchange resin.
9. 如权利要求1所述的体外释放测试方法,其特征在于所述药物为蒽环类药物、长春新碱或两性霉素B。 9. The in vitro release testing in claim 1, characterized in that the drug is anthracycline, vincristine or amphotericin B.
10. 如权利要求9所述的体外释放测试方法,其特征在于所述蒽环类药物为阿霉素。 10. The in vitro release of the test method according to claim 9, characterized in that said anthracycline is doxorubicin.
CN201510600419.5A 2015-09-21 2015-09-21 External release testing method of liposome medicaments prepared by pH gradient active drug loading method CN106546706A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510600419.5A CN106546706A (en) 2015-09-21 2015-09-21 External release testing method of liposome medicaments prepared by pH gradient active drug loading method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510600419.5A CN106546706A (en) 2015-09-21 2015-09-21 External release testing method of liposome medicaments prepared by pH gradient active drug loading method

Publications (1)

Publication Number Publication Date
CN106546706A true CN106546706A (en) 2017-03-29

Family

ID=58362407

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510600419.5A CN106546706A (en) 2015-09-21 2015-09-21 External release testing method of liposome medicaments prepared by pH gradient active drug loading method

Country Status (1)

Country Link
CN (1) CN106546706A (en)

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085132A1 (en) * 2000-05-10 2001-11-15 Leiras Oy Drug delivery device, especially for the delivery of levonorgestrel
CN1420762A (en) * 1999-12-23 2003-05-28 莱拉斯股份公司 Devices for delivery of drugs having antiprogestinic properties
CN1767870A (en) * 2003-01-17 2006-05-03 先灵有限责任公司 Otorhinological delivery device
CA2277956C (en) * 1997-01-31 2007-01-23 Depotech Corporation Method for utilizing neutral lipids to modify in vivo release from multivesicular liposomes
CN101264351A (en) * 2008-04-07 2008-09-17 易生科技(北京)有限公司 Composite coating cardiovascular medicaments elution stent and preparation thereof
CN101466384A (en) * 2006-04-27 2009-06-24 休珀努斯药物公司 Osmotic drug delivery system comprising release enhancing agent
US7585465B2 (en) * 2006-06-15 2009-09-08 Logan Instruments Corp. Pharmaceutical product release rate testing device
CN101528206A (en) * 2006-05-11 2009-09-09 株式会社Peptron;大熊制药株式会社 A process of preparing microspheres for sustained release having improved dispersibility and syringeability
CN101536982A (en) * 2009-04-27 2009-09-23 中国人民解放军第二军医大学 Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof
CN101987083A (en) * 2010-07-16 2011-03-23 钟术光 Preparation method for controlled release preparation, especial for zero-order release controlled release preparation
CN102048680A (en) * 2009-11-11 2011-05-11 河北奥星集团药业有限公司 Enteric sustained-release preparation containing zaltoprofen and preparation method thereof
CN102397553A (en) * 2011-11-24 2012-04-04 中国药科大学 Method for improving drug release in carbon nano tube drug delivery system by displacement object
CN103054832A (en) * 2012-12-26 2013-04-24 河南中帅医药科技发展有限公司 Minocycline hydrochloride sustained-release capsule and preparation method thereof
CN103169662A (en) * 2011-12-22 2013-06-26 上海纳米技术及应用国家工程研究中心有限公司 Paclitaxel polymer nanoparticle and preparation method

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2277956C (en) * 1997-01-31 2007-01-23 Depotech Corporation Method for utilizing neutral lipids to modify in vivo release from multivesicular liposomes
CN1420762A (en) * 1999-12-23 2003-05-28 莱拉斯股份公司 Devices for delivery of drugs having antiprogestinic properties
WO2001085132A1 (en) * 2000-05-10 2001-11-15 Leiras Oy Drug delivery device, especially for the delivery of levonorgestrel
CN1767870A (en) * 2003-01-17 2006-05-03 先灵有限责任公司 Otorhinological delivery device
CN101466384A (en) * 2006-04-27 2009-06-24 休珀努斯药物公司 Osmotic drug delivery system comprising release enhancing agent
CN101528206A (en) * 2006-05-11 2009-09-09 株式会社Peptron;大熊制药株式会社 A process of preparing microspheres for sustained release having improved dispersibility and syringeability
US7585465B2 (en) * 2006-06-15 2009-09-08 Logan Instruments Corp. Pharmaceutical product release rate testing device
CN101264351A (en) * 2008-04-07 2008-09-17 易生科技(北京)有限公司 Composite coating cardiovascular medicaments elution stent and preparation thereof
CN101536982A (en) * 2009-04-27 2009-09-23 中国人民解放军第二军医大学 Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof
CN102048680A (en) * 2009-11-11 2011-05-11 河北奥星集团药业有限公司 Enteric sustained-release preparation containing zaltoprofen and preparation method thereof
CN101987083A (en) * 2010-07-16 2011-03-23 钟术光 Preparation method for controlled release preparation, especial for zero-order release controlled release preparation
CN102397553A (en) * 2011-11-24 2012-04-04 中国药科大学 Method for improving drug release in carbon nano tube drug delivery system by displacement object
CN103169662A (en) * 2011-12-22 2013-06-26 上海纳米技术及应用国家工程研究中心有限公司 Paclitaxel polymer nanoparticle and preparation method
CN103054832A (en) * 2012-12-26 2013-04-24 河南中帅医药科技发展有限公司 Minocycline hydrochloride sustained-release capsule and preparation method thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JIANG W等: "In vitro and in vivo characterizations of PEGylated liposomal doxorubicin", 《BIOANALYSIS》 *
JINGXIA CUI等: "Direct comparison of two pegylated liposomal doxorubicin formulations:Is AUC predictive for toxicity and efficacy?", 《JOURNAL OF CONTROLLED RELEASE》 *
张柳等: "不同药物释放系统的体外评价方法研究进展", 《中国医药工业杂志》 *
王维等: "口服缓释制剂体内外相关性评价方法研究进展", 《药物评价研究》 *
许洁等: "环抱素A 脂质体制备及其体外释药方法学考察", 《抗感染药学》 *

Similar Documents

Publication Publication Date Title
Choucair et al. Active loading and tunable release of doxorubicin from block copolymer vesicles
Lindman et al. Systematic investigation of the thermodynamics of HSA adsorption to N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles. Effects of particle size and hydrophobicity
Laouini et al. Preparation, characterization and applications of liposomes: state of the art
Toy et al. The effects of particle size, density and shape on margination of nanoparticles in microcirculation
AU2003245160B2 (en) Method and apparatus for producing liposomes
Jordan et al. Comparative study of chemoembolization loadable beads: in vitro drug release and physical properties of DC bead and hepasphere loaded with doxorubicin and irinotecan
Horger et al. Films of agarose enable rapid formation of giant liposomes in solutions of physiologic ionic strength
US5962016A (en) Multivesicular liposomes utilizing neutral lipids to modify in vivo release
Li et al. Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection
Kandušer et al. Effect of surfactant polyoxyethylene glycol (C12E8) on electroporation of cell line DC3F
KR840002117B1 (en) Process for preparing magnetically-localizable,biodegrodable hipid miscospheres
JP3676976B2 (en) Regulation of drug encapsulation amount to multivesicular liposomes
Kuntsche et al. Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems
Tagami et al. Optimization of a novel and improved thermosensitive liposome formulated with DPPC and a Brij surfactant using a robust in vitro system
US6193998B1 (en) Method for producing liposomes with increased percent of compound encapsulated
Villar et al. Formation of droplet networks that function in aqueous environments
Lin et al. Peg− Lipids and Oligo (ethylene Glycol) surfactants enhance the ultrasonic permeabilizability of liposomes
US8815292B2 (en) Compositions and methods for treating insulin resistance and diabetes mellitus
Troutier et al. An overview of lipid membrane supported by colloidal particles
RU2573409C2 (en) Lipid particles containing nucleic acids and related methods
US20090274730A1 (en) Compositions and methods for treating inflammation
Lee et al. Equilibrium and dynamic interfacial tension measurements at microscopic interfaces using a micropipet technique. 2. Dynamics of phospholipid monolayer formation and equilibrium tensions at the water− air interface
EP1750671B1 (en) Liposomal assembly for therapeutic and/or diagnostic use
CN100563718C (en) Gas-filled microvesicle assembly for contrast imaging
Sun et al. Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection

Legal Events

Date Code Title Description
PB01
SE01