磷脂膜色谱用于评价药物与有序磷脂膜的相互作用磷脂膜色谱用于评价药物与有序磷脂膜的相互作用

目的使用磷脂膜色谱考察药物与有序磷脂膜的相互作用。方法使用磷脂膜色谱测定药物与有序磷脂膜的相互作用强度，表示为药物的膜亲和性参数(lg K_IAM)。使用正辛醇/水系统测定药物的疏水性参数(lg DO/W,7.4)。结果在有机调节剂百分比(φ) 0～30%范围内，容量因子对数值(lg K′_IAM)与φ呈现良好线性关系；对于3种有机调节剂(甲醇、乙醇和乙腈)，lg K′_IAM与φ间曲线截距相同，但斜率有显著差别。从对羟基苯甲酸甲酯到丁酯，每增加一个CH₂对测定的亲脂性参数差值(Δlg K_IAM和Δlg DO/W,7.4)具有相似的影响，但是从对羟基苯甲酸到甲酯的差值却明显偏离这个规律。结论磷脂膜色谱是一个简便、有效和快速的用于评价药物与有序磷脂膜相互作用的工具。     

磷脂膜色谱及其在生物药剂学中的应用

亲脂性参数在解释药物体内吸收、分布和排泄及预测生物活性等方面的重要性早已被人们认识。几种简单有机溶剂 水分配系统的模型都曾被用于评价药物的亲脂性 ,但其中取得较为成功的为正辛醇 水系统[1 ] 。在定量构效关系和药物合理设计上 ,它已成为标准疏水性参数 ,构成了药物特征参数的数据库。近年来 ,研究表明正辛醇 水系统并不能完全模拟所有类型药物在生物膜模型 (液晶态脂质体膜 )上的分配行为 ,导致其不能解释由不同化学结构类型药物组成集合的药动学和药效学特征。研究结果表明其分配系数与稳态下脑 血浓度比值或穿透血脑屏障渗透系数间无明显联系[2 ] 。这主要是因为各向同性、不带电荷中心的正辛醇相并不是呈液晶态生物膜上有序磷脂双分子层的客观模型。为了建立更精确的生物膜模拟系统 ,曾有文献[3]报道合成了磷脂膜界面 (ｉｍｍｏｂｉｌｉｚｅｄａｒｔｉｆｉｃｉａｌｍｅｍ ｂｒａｎｅ,ＩＡＭ) ,并将其作为固定相填料引入高效液相色谱系统 ,组成了磷脂膜色谱 (ＩＡＭｃｈｒｏｍａｔｏｇｒａｐｈｙ)。对结构相似的苯乙胺衍生物 ,磷脂膜色谱与正辛醇 水系统给出相似的亲脂性测量尺度 (ｒ =0 985 ) [4 ] ;但对于由不同化学...     

两性分子与有序磷脂膜的相互作用模式

目的　揭示两性分子与有序磷脂膜的作用模式。方法　分别使用磷脂膜色谱和正辛醇 水系统测定药物的膜亲和性和疏水性参数。结果　两性分子与有序磷脂膜存在吸引性极性附加作用力 ,其测定的膜亲和性参数要明显比由疏水性参数预测的值高。结论　结合两性分子复杂的微观质子平衡 ,不仅其中性、阳性而且两性的微观离子可能通过匹配的构象和能量有利的作用模式而有效的分布到有序的两性磷脂膜中 ,并且后两者的分配产生了与有序磷脂膜的吸引性极性附加作用力     

磷脂膜色谱与正辛醇/水系统亲脂性测量尺度的比较

磷脂膜色谱 (immobilizedartificialmembranechromatography,IAMC)是 2 0世纪 90年代初发展起来的一个高通量筛选药物与磷脂膜相互作用和具有适宜体内药代动力学特征候选药物的工具[1～ 4 ] 。近年来 ,研究结果[5,6 ] 表明 ,由于磷脂膜色谱能较好的模拟细胞膜有序磷脂层的空间环境 ,因此 ,它通常要比正辛醇 水系统更能准确的评价药物与生物膜的相互作用和获得更精确的药物亲脂性参数。因为尚未见磷脂膜色谱和正辛醇 水系统亲脂性测量尺度的系统性比较研究 ,所以本文选择了 2 7个不同化学结构类型药物的集合 ,包括酸性、碱性、中性和两性化合物 ,分别使用正辛醇 水系统和磷脂膜色谱两种生物膜相互作用评价系统对这一系列化合物的亲脂性参数进行考察 ,进而比较这两种不同的生物膜模拟系统。材料与方法药品与试剂　格帕沙星、环丙沙星、左氧氟沙星和OPC 1 72 0 3由日本大冢制药株式会社提供 ;HSR 90 3和NR 762由日本北陆制药株式会社提供 ;奎尼丁、普萘洛尔、阿莫西林、对氨基水杨酸、吡哌酸、利多卡因、普鲁卡因酰胺和正辛醇 (SigmaChemCo,StLouis,...     

从W/O型乳剂制备多层脂质体的理论和实践

脂质体用作药物载体已有多年,然而众所周知多层脂质体(MLV)的药物包裹率很低,这是由于用薄膜法制备时,将水溶液加至类脂膜中,穿透干燥类脂膜使磷脂水化,由于脂相和水相间的表面积小,在形成脂质体膜时,水溶液中的药物不能密切与类脂质接触,因此药物包裹率低。逆相蒸发法(REV)克服了包裹率低的问题,在形成膜时增加类脂和水相之间的表面积,它将水滴乳化在有机相中(W/O乳剂),再形成脂质体。在W/O乳剂     

脂质体(Liposome)作为药物载体的展望

<正> 药物除口服粉剂外,都靠递药系统(Drugdelivery systems)供病人使用,七十年代以来着重研究脂质体作为药物的载体,希望能选择性地达到靶组织,尤其着重于抗癌药物脂质体的研究。一、什么是脂质体?脂质体是由磷脂、胆固醇的疏水性基因交互作用而构成的双层脂质膜,亲水基团位于膜的表面,疏水性基团位于膜的中间,每层脂膜之间有少量水液,一大片双脂膜连接成一个小球,叫做脂质体(Liposomes)或称脂微球(Ve-sicle)。可将大小不规则的大脂质体超声处理,     

正电性多肽copoly(Lys/Tyr)与磷脂膜的相互作用研究

目的 研究正电性多肽copoly(Lys/Tyr)(CPLT)在模拟生物膜上的透过性。方法 ①配制由卵磷脂(EPC)、2-油酰基磷脂酰乙醇胺(DOPE)和大豆磷脂(SBPL)组成的二分子磷脂膜。②Zeta电位测定法(zeta potential method，ZP)测定由上述磷脂组成的脂质体在加入CPLT后的膜表面Zeta电位。③园二色谱法(circular dichroism spectrcvscopy，CD)检测CPLT分子与磷脂膜作用时的构象情况。④电生理学方法(electrophy siology tech—nique，ET)测量CPLT分子在磷脂膜上引起的跨膜电流。⑤荧光光度分析法(fluorescence spectroscopy，FS)检测CPLT分子与磷脂膜的作用过程中的荧光强度变化。⑥共焦点激光扫描显微分析法(confocal laser scanning microscopy，CLSM)研究CPLT在磷脂膜上透过及其相对透过效率。结果①随着CPLT的加入和其浓度的增加，磷脂膜Zeta电位逐渐增加并趋向饱和。②CPLT分子在水相取β-sheet构象，当与磷脂膜结合后，其β-sheet构象的波峰发生红移，但构象基本不变。③CPLT分子能在一定浓度和一定外加电压条件下，透过二分子磷脂膜引起膜电流。④CPLT与磷脂膜的作用可分为三步：第一步，CPLT分子吸附于磷脂膜上；第二步，CPLT分子通过磷脂膜的疏水区；第三步，CPLT分子从二分子膜的内膜解吸，进入膜内水相。⑤CPLT分子在磷脂膜上的透过效率主要决定于磷脂膜的组成，在磷脂膜中存在带负电性的磷脂时可降低CPLT分子在磷脂膜上的透过效率。⑥在最初的CPLT分子与磷脂膜的相互吸附过程中，CPLT分子与磷脂膜间的静电作用力起主要作用。结论copoly(Lys／Tyr)分子能透过二分子磷脂膜，其透过效率主要决定于磷脂膜的组成。在最初的肽一膜吸附过程中，当存在静电作用力时，静电力起主要作用，疏水效应次之。     

灯盏花素脂质体/水分配系数的研究与应用

目的应用脂质体 /水分配系数 ,初步揭示灯盏花素与磷脂分子间相互的作用机理 ,探讨灯盏花素口服吸收机制 ,并且解释一些因素影响灯盏花素脂质体包封率的规律。方法采用平衡透析法测定灯盏花素的脂质体 /水分布系数。结果与灯盏花素的油 /水分配系数随着 pH值的增加而减小相反 ,脂质体 /水分配系数随着pH值的增加而增大 ;磷脂浓度增大 ,脂质体 /水分配系数有增大的趋势。结论药物与磷脂膜的作用除疏水相互作用 (hydrophobicinteraction)外 ,离子间静电、氢键等相互作用占了很大比例 ;初步推测灯盏花素的吸收差可能是因为其吸收有除简单的被动转运外的其它机制 ;实验结果可以用来解释包封率影响因素的一些规律。     

葫芦素E溶解度、正辛醇-水及脂质体-水分配系数测定

目的测定葫芦素E溶解度、正辛醇-水分配系数及脂质体-水分配系数。方法分别采用平衡法、经典摇瓶法及平衡透析法测定葫芦素E溶解度、正辛醇-水分配系数及脂质体-水分配系数。结果葫芦素E在水中溶解度为0.155 mg.L-1,在乙醇、叔丁醇中有潜溶现象;正辛醇-水分配系数为(2.088±0.054);脂质体-水分配系数为0.003~1.819。结论葫芦素E为水不溶性药物;正辛醇-水分配系数及脂质体-水分配系数均中等偏低。     

7-乙基-10-羟基喜树碱长循环脂质体的制备及药动学研究

目的:研究7-乙基-10-羟基喜树碱长循环脂质体(Lip-SN38)的制备方法以及在大鼠体内的药动学.方法:采用两步合成法制备脂质体空间稳定膜材甲氧基聚乙二醇.磷脂酰乙醇胺(mPEG-PE);同时采用薄膜分散法制备Lip-SN38;用阳离子交换树脂微型小柱层析法分离游离药物和脂质体,紫外分光光度法测定包封率;HPLC法测定大鼠血浆中药物浓度.结果:Lip-SN38平均粒径<200 nm,药物包封率>90%;48 h只有<30%的药物体外释放;大鼠尾静脉注射Lip-SN38,剂量为10 mg·kg-1,与SN3.8溶液剂相比,t1/2β增加4.61倍.结论:采用薄膜分散法可制得包封率高、粒径小的脂质体,mPEG-PE修饰磷脂膜可增加Lip-SN38的t1/2β,延长药物在血中的循环时间.     

Lipophilic and electrostatic forces encoded in IAM-HPLC indexes of basic drugs: their role in membrane partition and their relationships with BBB passage data

The membrane phospholipid affinity data, log k(w)(IAM), for 14 basic drugs spanning a wide lipophilicity range were measured by HPLC on two different phospholipid stationary phases, i.e. IAM.PC.MG and IAM.PC.DD2. These data related weakly with log P(N) values, the n-octanol/water partition coefficients of the neutral forms; poorer relationships were found with log D(7.0) values, the n-octanol/water partition coefficients of the mixtures of neutral and ionized forms at pH 7.0. The lack of collinearity confirms that, differently from partition in n-octanol/water, partition in phospholipids encodes not only lipophilic/hydrophobic intermolecular recognition forces but also ionic bonds, due to electrostatic interactions between electrically charged species and phospholipids, according to the "pH-piston hypothesis". This component of interaction was parameterized by Δ log k(w)(IAM) values; they are the differences between the log k(w)(IAM) values experimentally measured and the values expected for neutral isolipophilic compounds. Δ log k(w)(IAM) values of the various analytes changed almost linearly from positive to negative values at increasing lipophilicity. This behavior is consistent with an interaction mechanism with membrane phospholipids including two intermolecular interaction forces: (i) lipophilic/hydrophobic interactions, which decrease on ionization proportionally to the lipophilicity of the neutral forms, and (ii) electrostatic interactions, which increase on ionization and are quite constant for all the analytes at a given ionization degree. Since BBB passage of the considered compounds is supposed to be based on passive mechanisms, we investigated the possible relationships between log BB values, i.e. the logarithms of the ratio between brain and blood concentrations, and three physico-chemical parameters, i.e. (i) log P(N) (lipophilic interaction of the neutral form), (ii) log k(w)(IAM) (global interaction with phospholipids), and (iii) Δ log k(w)(IAM) (electrostatic component of interaction with phospholipids). The results suggest that the electrostatic interactions encoded in log k(w)(IAM) values might act as trapping forces in a phospholipid barrier. Actually, we observed an inverse linear dependence of log BB on Δ log k(w)(IAM) values, but only for the compounds showing positive Δ log k(w)(IAM) values. We conclude that the driving force for BBB passage is the lipophilicity of the neutral forms, log P(N), and not the lipophilicity actually displayed at the experimental pH, log D(7.0). Indeed, the latter does not adequately take into account the role played by protonation in the analyte/membrane interactions because protonation, although hindering membrane passage, can either reduce or enhance partition in phospholipids, depending on analyte lipophilicity.     

Comparison between immobilized artificial membrane (IAM) HPLC data and lipophilicity in n-octanol for quinolone antibacterial agents

The membrane phospholipid affinity of ten quinolone antibacterial agents, including both acidic and zwitterionic compounds, was measured by HPLC on two different immobilized artificial membrane (IAM) stationary phases, namely IAM.PC.MG and IAM.PC.DD2; it is expressed as the logarithm of the retention factor measured with (or extrapolated to) 100% aqueous eluent at pH 7.0, .

Quinolones are a class of highly potent, orally active, broad-spectrum antibacterial agents. For these compounds, lipophilicity values in n-octanol found in the literature, either calculated or measured, are not consistent with each other and are too low to be compatible with their pharmacokinetic properties.

The values obtained in this study showed no relation with any of the lipophilicity values in the literature (clog P(a), clog P(b), MLP, log D^7.4). In contrast, they were collinear with a new lipophilicity scale we had previously obtained by an original ion-pair reversed-phase HPLC method set up to estimate the lipophilicity of the neutral forms, log P^N. Moreover, when comparing the retention of quinolones on IAM to the retention of structurally unrelated neutral compounds, we observed that they interact with phospholipids with the same affinity as neutral isolipophilic compounds.

The use of an eluent at pH 5.5, instead of pH 7.0, increased the retention on IAM not only for acidic, but also for zwitterionic congeners, indicating that phospholipid affinity is enhanced in the experimental conditions that depress the ionization of the acidic function, even when the ionization of the amino function increases simultaneously.

To gain an insight into the mechanism of quinolones/serum-protein interactions, we investigated about possible relationships between quinolones affinity data for serum proteins and IAM data. Quinolone affinity for both HSA and AGP was already demonstrated poorly related to n-octanol lipophilicity values, probably due to the occurrence of electrostatic interactions. Only poor relationships were found between IAM and HSA affinity data, whereas quite good relationships were found with AGP affinity data. However, IAM.PC.DD2 data correlated better than those on IAM.PC.MG with quinolone affinity for both serum-proteins, mainly due to the fact that IAM.PC.MG phase is scarcely discriminative for the compounds with the highest retention values.

The results suggest that IAM retention can produce a lipophilicity scale that, unlike solvent/water partition coefficients, is consistent with the pharmacokinetic behaviour of zwitterionic quinolones.     

Chromatographic Indexes on Immobilized Artificial Membranes for Local Anesthetics: Relationships with Activity Data on Closed Sodium Channels

Purpose. To elucidate the effectiveness of the different parameters for the prediction of biological activity, the n-octanol/buffer partition coefficients and theoretical calculated lipophilicity parameters of thirteen local anesthetic drugs (LAs), including two -blockers, were compared to the affinity values for phospholipids, calculated by a recent technique. Methods. Interactions with phospholipids were measured by high performance liquid chromatography on a stationary phase made up of phospholipids, the so-called 'Immobilized Artificial Membrane' (IAM). Reference lipophilicity parameters were measured by shake-flask method between n-octanol and buffer phases. Results. Interactions with phospholipids were predicted from log P for all compounds except tocainide, which also showed additive polar extra-interactions. Moreover, when the retention on Immobilized Artificial Membrane (IAM) phase was mainly lipophilicity-based, a unique scale included the correlation between log k_w ^IAM and log P values, for both LAs (bases) and the structurally unrelated (nonionizable and acidic) compounds previously studied. IAM interaction values for LAs were predictive of the partition measures on liposome membranes already reported in literature. The half-blocking doses for closed sodium channel, corrected for ionization at pH 7.4, were successfully correlated with the respective IAM values for eleven compounds while procaine and tetracaine, which are ester-linked compounds and have a p-amino group as well, gave more potent results than predicted by phospholipid interactions. Conclusions. The IAM chromatographic parameters were much more effective than reference lipophilicity values in describing partition on model membranes and in predicting pharmacological potency on closed sodium channels.     

Capillary electrochromatography as a new tool to assess drug affinity for membrane phospholipids

This work proposes a new capillary electrochromatography (CEC) method for determination of drug partition in membrane phospholipids. CEC experiments were carried out in a 100 μm (ID) fused-silica capillary, partially packed with a chromatographic phospholipid stationary phase, so-called Immobilized Artificial Membrane, IAM.PC.DD2. The observed retention values were corrected by both the electro-osmotic and electrophoretic mobility values, measured by capillary electrophoresis (CE) experiments, assuming the values of the logarithms of "chromatographic" affinity factors, log k(CEC) as indexes of affinity for phospholipids. Analogously to biochromatography, all the values were determined with a totally aqueous mobile phase, or extrapolated to 100% aqueous buffer. The analytes were 16 structurally unrelated compounds, of basic, neutral, and acidic nature. To evaluate the effectiveness of CEC data to describe partition in phospholipids, log k(CEC) were related to both log P and log k(w)(IAM) values. log P are the lipophilicity values expressed as the logarithms of n-octanol/water partition coefficients and log k(w)(IAM) are the retention data measured by High Performance Liquid Chromatography (HPLC) on an IAM.PC.DD2 column, assumed as the reference values for phospholipid affinity. Phospholipid affinity scale by CEC related to that achieved by HPLC, but only if two different subclasses were considered separately, i.e. protonated and unprotonated analytes; indeed, all the compounds protonated at the experimental pH value (7.0) were retained stronger in CEC than in HPLC. This discrepancy may be due to the use of different buffers in CEC and HPLC since, to avoid the occurrence of a high current, the eluent in CEC experiments was of different composition and lower ionic strength than in HPLC. CEC analyses were faster and required lower amounts of both solvent and stationary phase than HPLC; moreover, with the exception of only three analytes, all analyses were performed with 100% aqueous eluents avoiding time-consuming and tedious extrapolation procedures.     

Micellar electrokinetic chromatography for determination of drug partition in phospholipids

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely L-alpha-dilauroyl phosphatidylcholine (DLPC) or L-alpha-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.     

Lipophilicity parameter from high-performance liquid chromatography on an immobilized artificial membrane column and its relationships to bioactivity of the group of 2,4-dihydroxythiobenzanilides

Several groups of 2,4-dihydroxythiobenzanilide derivatives are extensively studied in our laboratories as potential antifungal or antibacterial agents. Previous papers showed that their biological activities are closely correlated with the lipophilicity determined in HPLC experiments using on octadecylsilyl stationary phase (RP-18). In the present paper we attempted to measure chromatographic indices of 2,4-dihydroxythiobenzanilides, using a silica-based immobilized artificial membrane (IAM) column especially designed to evaluating biological lipophilicity. The theoretical (extrapolated) retention in pure water the log k(w)(IAM) parameter for 23 derivatives was determined during poly-cratic experiments. Statistical analysis exhibited good correlations between the log k(w)(IAM) parameter and the biological data concerning bacteriostatic activity of these compounds. Retention on the IAM phase can be used as predictor of biological membrane permeability and biological lipophilicity of 2,4-dihydroxythiobenzanilides in spite of the acidic properties of this group of compounds.     

Potential of immobilized artificial membrane chromatography for lipophilicity determination of arylpropionic acid non-steroidal anti-inflammatory drugs

Molecular lipophilicity can be expressed by logP or more conveniently by logk, i.e. determined by the traditional shake-flask technique or by liquid chromatography. The logk of 11 arylpropionic non-steroidal anti-inflammatory drugs (NSAIDs) was determined at pH 7.4 of the eluent using two stationary phases i.e. octadecylsilane phase and an immobilized artificial membrane (IAM.PC.MG) packing. The chromatographic retention factors extrapolated to 100% aqueous phase (logk(wODS) and logk(wIAM)) were correlated with n-octanol/water lipophilicity parameters (logP) and with n-octanol/water partition coefficients corrected for ionization at pH 7.4 (logD7.4). In this series of compounds, significant linear correlations (r>0.94) between the chromatographic parameters (logk(wIAM)) and the reference lipophilicity data (logP and logD7.4) were described. Moreover, regression analysis between the lipophilicity parameters and some pharmacokinetic data for the drugs under study were performed. The logk(wIAM) parameter over n-octanol/water partition data seems to provide a good model to obtain lipophilicity parameters of arylpropionic acid NSAIDs for quantitative structure-activity relationships studies.