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,...     

通过喹诺酮抗生素与磷脂膜的相互作用预测肺泡巨噬细胞单层的体外摄取

目的;预测肺泡巨噬细胞单层的体外摄取.方法:以培养的肺泡巨噬细胞单层为体外模型,用磷脂膜色谱,脂质体/水系统评价药物与磷脂膜的相互作用,分别表示为lg k_(1AM),lg D_(L B,7.4),用正辛醇/水系统测定参考的疏水性参数(lg D_(O/B,7.4).结果:lg D_(L B,7.4)(r~2=0.93)比lg D_(O B,7.4)(r~2=0.65)具有与lg k_(1AM)更显著的相关性.lg k_(1AM)和lg D_(L B,7.4)均 比lg D_(O/B,7.4)与细胞内药物的蓄积度有更好的相关性.但对于由5个两性喹酮抗生素和奎尼丁组成的受试集合,三者均与药物进入细胞内的速度具有相近的显著的正相关性.结论:磷脂膜色谱和脂质体/水系统给出相似的亲脂性测量尺度,且均与正辛醇/水系统区别有显著性.与疏水性参数相比,膜亲和性是更有效的肺泡巨噬细胞内药物蓄积和结合的预测参数.     

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...     

格列美脲蛋白结合作用的高效液相色谱-迎头分析法

目的研究格列美脲与人血清白蛋白(human serum albumin,HSA)结合作用。方法采用高效液相色谱-迎头分析法(high performance liquid chromatography-frontal analysis, HPLC-FA)。色谱柱为内表面反相(internal-surface reversed-phase,ISRP)硅胶柱Pinkerton GFF II-S5-80(150 mm×4.6 mm ID, 5 μm)；流动相为 67 mmol·L^-1磷酸盐等渗缓冲液 (pH 7.4, I=0.17 mol·L^-1)；流速0.2 mL·min^-1；检测波长230 nm；进样量900 μL；柱温37 ℃。结果 应用非线性回归参数估算求得HSA分子上两类结合位点结合的格列美脲分子数及相应的结合平衡常数：n₁=1和K₁=5.1 (μmol·L^-1)^-1；n₂=7和K₂=0.017 (μmol·L^-1)^-1。结论HPLC-FA法操作简便，适用于研究格列美脲与HSA的结合作用。     

滇白珠木脂素苷的研究

目的： 研究民间药滇白珠(Gaultheria yunnanensis (Franch.) Rehd.)的化学成分。方法： 用95%乙醇提取,硅胶柱色谱和聚酰胺柱色谱分离纯化,通过各种光谱分析鉴定其结构。结果： 从正丁醇萃取物中分离鉴定了4个木脂素苷类化合物。其结构分别鉴定为(-)-isolariciresinol-2a-O-β-D-xylopyranoside (D₁),(+)-lyoniresinol-2a-O-β-L-arabinopyranoside (D₂),(-)-5′-methoxyisolariciresinol-2a-O-β-D-xylopyranoside (D₃)和(+)-lyoniresinol-2a-O-β-D-glucopyranoside (D₄)。结论： 化合物D2为一新化合物,命名为滇白珠苷A(Gaultheroside A),D₁,D₃,D₄为首次从杜鹃花科植物中分得。     

辣椒素对培养的大鼠三叉神经节神经元瞬时外向钾电流和延迟整流钾电流的影响

目的探讨辣椒素(capsaicin)对大鼠三叉神经节神经元瞬时外向钾电流(I_A)和延迟整流钾电流(I_K)的不同影响。方法采用全细胞膜片钳方法。结果对于辣椒素敏感神经元，辣椒素可选择性、剂量依赖性地抑制其I_A电流，但辣椒素对I_K无明显选择性作用；对于辣椒素不敏感神经元，辣椒素对I_A和I_K均无作用，且对I_A和_IK激活动力学亦无影响。结论辣椒素可选择性抑制对其敏感的三叉神经节神经元I_A，这可能是初次应用辣椒素时其致痛作用的原因之一。     

对《药物吸收速率常数的一种简便计算法》一文的几点看法

本文对药物吸收速率常数(K_α)的简便计算法的可靠性进行了讨论并对应用中的具体措施提出了不同看法。无论是在特征值左右多取几个血样或是用“二次抛物线拟合”都不能肯定地提高计算精度。对于K_α精度要求较高的药代动力学实验研究,该法还有待进一步改进。     

药物脂水分配系数测定的微乳液电动色谱新方法研究

目的建立一种测定药物脂水分配系数的微乳液电动色谱(MEEKC)新方法，以避免测定时必须使用微乳相标记物，以及减小和评价测量误差。方法以系列化合物在MEEKC中的迁移时间t_m值与其脂水分配系数之间的关系，进行非线性拟合得出微乳相的虚拟迁移时间t_me，再建立标准曲线以测定所选取药物的脂水分配系数；从理论上评价测定结果准确度与t_m相关性，并以此评价测定结果的准确度。结果所选取的4种药物脂水分配系数的MEEKC测定值与摇瓶法测定值基本吻合，其平均差值为0.15，其测定准确度与t_m的相关性符合理论模型的分析。结论无微乳相标记物的MEEKC新方法简单、快速、可靠、重现性好，测定脂水分配系数的准确度较高，可用作药物脂水分配系数的测定。     

Donepezil对大鼠海马及新皮层锥体神经元延迟整流样钾电流的影响

目的探讨donepezil对大鼠海马和新皮层锥体神经元具有延迟整流样特性钾电流(I_K)的影响。方法 用膜片钳全细胞记录法。结果Donepezil在微摩尔水平即可抑制I_K,并呈剂量依赖性和电压依赖性,且可使I_K的稳态激活曲线向超级化的方向移动。结论低浓度donepezil即可抑制大鼠海马和新皮层锥体神经元电压激活的I_K,此作用可能与其抑制胆碱酯酶活性协同,参与药物的治疗效应。     

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.     

壳聚糖-聚乳酸及壳聚糖-聚(R)-3-羟基丁酸甲酯共混膜的制备及评价

用流延法制备壳聚糖与聚乳酸或聚(R)-3-羟基丁酸甲酯共混膜。壳聚糖-聚乳酸(1∶1,w/w)和壳聚糖-聚(R)-3-羟基丁酸甲酯(1∶1,w/w)共混膜的抗拉强度为(25.39±1.63)和(23.49±0.43)MPa,吸水率为(32.65±2.41)%和(33.72±3.11)%。该壳聚糖-聚乳酸膜在人工组织液中浸泡45 d后,降解率为(32.26±0.56)%,提示其可诱导新组织再生。     

Ethanol-induced alterations in rat synaptosomal plasma membrane phospholipids. Relationship to changes in the phospholipid methyltransferases

The effects of ethanol ingestion on the lipids of the synaptic plasma membrane (SPM) have been measured and correlated with the time frame for the development of physical dependence. Alterations were observed in three of the phospholipid fractions: phosphatidylcholine (PC) increased, and the phosphatidylethanolamine (PE) and phosphatidylserine (PS) plus phosphatidylinositol (PI) fractions decreased. These alterations occurred after the animals showed signs of dependence. Because PC can be synthesized from PE by the methyltransferase pathway, synaptosomal methyl group incorporation was measured. Rats were fed ethanol for 6 days before an increase was observed in methyl incorporation, a shorter length of time than was necessary to demonstrate physical dependence or phospholipid alterations (10 to 14 days). After ethanol withdrawal, 7 days of control diet feeding were required for methyl group incorporation to return to control values. In vitro ethanol (10-250 mM) additions to the methyltransferase incubations resulted in a slight increase in methyl incorporation. These data suggest that synaptic membrane lipid alterations may be related to ethanol dependence and that changes in the PC/PE ratio may be the result of an increase in the incorporation of methyl groups into synaptosomal phospholipids.     

Effect of nonsteroidal anti-inflammatory drugs on the cellular membrane fluidity

In this work, fluorescence measurements were performed using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to evaluate the effects of the interaction of nonsteroidal anti-inflammatory drugs--NSAIDs (meloxicam, lornoxicam, and nimesulide) with several membrane systems (liposomes with and without cholesterol, mouse splenocytes, mouse macrophages cell line--J774, human leukemia monocyte cell line--THP-1, and human granulocytes and mononuclear cells). DPH fluorescence quenching studies revealed that the NSAIDs studied were able to efficiently quench the probe located in membrane hydrocarbon region. Fluorescence anisotropy measurements were also made to investigate the effects on membrane fluidity resulting from the interaction between the drugs and membrane systems. All the anti-inflammatory drugs studied show an increase in the membrane fluidity in a concentration dependent manner. Results obtained provide an insight into NSAIDs capacity to be inserted in lipid bilayers and alter the lipid dynamics. The induced changes in lipid dynamics may modulate the activity of inflammatory enzymes or may be related with deleterious topical action of NSAIDs on gastric phospholipid fluidity.     

Simultaneous quantification of ten phenolic acids in Lonicerae Japonicae Flos by HPLC-DAD

A reverse-phase HPLC-DAD method was developed for simultaneous quantification of ten phenolic acids (caffeic acid, chlorogenic acid, neochlorogenic acid, 4-O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid methyl ester, 3,4-O-dicaffeoylquinic acid methyl ester, and 4,5-O-dicaffeoylquinic acid methyl ester) in the dried flower buds of Lonicera japonica Thunb. (Lonicerae Japonicae Flos; LJF). An optimal sample preparation method was established as 30-min ultrasonication with 100 times 50% (v/w) ethanol aqueous solution based on the orthogonal test results. The chromatographic separation of the ten phenolic acids was achieved with an AQ-C18 column (4.6 mm×250 mm, 5 µm) and a gradient elution of acetonitrile, methanol and 0.1% formic acid aqueous solution within 55 min. All calibration curves showed good linearity (r²>0.999) within test ranges. The average recoveries were in the range of 98.57%-103.22% with RSD less than 3%. The method developed was accurate, sensitive and reproducible for determination of ten phenolic acids in LJF.     

Relationship between immobilized artificial membrane chromatographic retention and human oral absorption of structurally diverse drugs

Capacity factors are determined for a set of drugs for which human oral absorption (HOA) data are available, using immobilized artificial membrane (IAM) chromatography. The compound set represented acidic, basic, neutral and amphoteric drugs from various structure classes and having low to high human oral absorption. Effect of mobile phase pH on retention was investigated to determine the optimal condition for better correlation with HOA. The retention (capacity factor, k'(IAM) of each drug was measured by reverse phase HPLC using an IAM.PC.DD2 (1 cm x 3 mm i.d., 12 microm) column with an eluent of acetonitrile - 0.01 M phosphate buffer at pH 4.5-7.4. The pH dependent k'(IAM) was in accordance with pH partition theory. Using non-linear regression analysis the obtained log k'(IAM) values were compared with published data on HOA in order to establish correlation. The better correlation with HOA was observed when the highest log k'(IAM) value selected among pH 4.5-7.4 (R(2)=0.8566) for each drug rather than obtained at more traditional pH 7.4 (R(2)=0.7403). Finally, it was confirmed by Cook's D outlier test that there was no influential observation in the model that affect the relationship between IAM capacity factor and HOA. The assay conditions were optimized and validated to make it suitable for routine analysis and for compound characterization in early discovery where permeability may be an issue.     

Effect of toluene on rat synaptosomal phospholipid methylation and membrane fluidity

This study investigated the effects of toluene (1 g/kg, 1 hr, i.p.) on rat synaptosomal phospholipid methylation (PLM), phospholipid composition, and membrane fluidity. Toluene significantly decreased basal PLM (35%) in studies using [3H]methionine [( 3H]Met) as the methyl donor; this was reflected by similar decreases in phosphatidylmonomethylethanolamine (PME) (30%). No effects were observed in either PLM reactions that used [3H]adenosylmethionine [( 3H]AdoMet) as methyl donor, or AdoMet synthetase, suggesting that toluene preferentially affects PLM reactions that derive methyl groups from [3H]Met. Also, toluene decreased synaptosomal phosphatidylethanolamine (PE) (24%), the initial substrate for PLM, and the addition of PE back to PE-depleted synaptosomes restored methyltransferase activity. Agonist-stimulated PLM using norepinephrine (NE) demonstrated that agonist-receptor coupling returned PLM to control values in synaptosomes from toluene-treated rats. NE-stimulated PLM was also blocked by propranolol (PRO), suggesting a role for toluene in receptor-mediated events. Membrane fluidity studies demonstrated that in vivo administration of toluene increased the outer synaptosomal membrane fluidity, whereas in vitro administration of toluene had no effect. Our observations support a positive relationship between increased PLM activity and increased outer, not core, membrane fluidity. These data demonstrate that specific toluene-phospholipid interactions occur in synaptosomes, resulting in altered membrane composition, function and fluidity.