非基于细胞渗透模型在BSCⅡ类药物渗透性研究中的应用

渗透性是肠道吸收和口服生物利用度的决定因素之一,体外渗透性测定是指药物分子从供体室通过不同的屏障进入受体室,从受体室收集样品来测量渗透率。具有高渗透率、低成本、性质稳定、使用方便、可定制化、可减少对缓冲液生物相容性,增加了表面积与体积比等优点的非基于细胞渗透模型根据其屏障类型分为含有(磷酸)脂质的仿生屏障和无脂质的非仿生屏障。仿生无细胞渗透模型包括平行人工膜通透性测定模型(parallel artificial membrane permeability,PAMPA),基于囊泡的渗透测定模型(vesicle-based permeation assay,PVPA)和PermeaPad_?^。非仿生无细胞渗透工具基于透析膜,例如基于聚醚砜材料的中空纤维膜(Hollow Fiber Membrane,HFM)模型。在国外,应用这四种屏障进行不同目的研究正逐渐成为药物吸收研究的热点,但在国内,仅对PMAPM的应用研究较多,缺乏对其他三种屏障的应用研究。笔者总结了渗透性计算方法,检索了近几年对非细胞渗透工具在BSCⅡ类药物渗透性方面的应用,对3种仿生屏障,和重点介绍的无细胞渗透工具中空纤维膜的特点应用进行归纳总结,以期满足难溶性药物配方开发需求和评估优化不同溶出吸收行为的配方。     

非细胞渗透模型在BSCⅡ类药物渗透性研究中的应用进展

渗透性是肠道吸收和口服生物利用度的决定因素之一。非细胞渗透模型是一种体外渗透性测定工具,具有高效率、低成本、性质稳定、使用方便、可定制化等优点。根据屏障类型,非细胞渗透模型可分为含有(磷酸)脂质的仿生屏障和无脂质的非仿生屏障。仿生非细胞渗透模型包括平行人工膜渗透性测定模型、基于囊泡的渗透测定模型和人工纤维素-磷脂仿生膜测定模型。非仿生无细胞渗透模型有基于聚醚砜材料的中空纤维膜模型等。在国外,应用这4种屏障进行不同目的的研究正逐渐成为药物吸收研究的热点,但在国内,仅对平行人工膜渗透性测定模型的应用研究较多,缺乏对其他3种屏障的应用研究。笔者总结了渗透性装置和渗透性计算方法,检索了近几年非细胞渗透模型在BSCⅡ类药物渗透性方面的应用,对3种仿生屏障,和无脂质的中空纤维膜的特点应用进行归纳总结,以期满足难溶性药物配方开发需求。     

Caco—2模型在肽类药物吸收特性研究中的应用

Caco－2模型作为药物离体口服特性筛选模型,已广泛用于药物在小肠处的各种转运机制研究。该模型来源于人体结肠癌细胞,含有多种代谢酶,接近体内吸收的实际情况,可区分不同药物吸收时摄取和跨膜转运过程。本文对Caco－2模型的建立及其近年来在太类及肽类类似物吸收特性研究方面的应用作一综述。     

角膜上皮细胞培养模型及其在眼部药物研究中的应用进展

 眼部药物吸收主要是通过角膜途径进行,而且角膜上皮为眼部用药的主要吸收屏障。由于动物实验存在较多缺陷,且随着细胞培养技术的不断发展,可以采用细胞培养技术建立角膜上皮细胞培养模型,模拟眼部屏障功能。本文综述了已建立的角膜上皮细胞培养模型,主要包括原代角膜上皮细胞培养模型和永生化角膜上皮细胞培养模型,并对角膜上皮细胞培养模型在眼用药物研究方面的进展进行介绍,包括角膜毒性研究、药物角膜渗透性研究及眼用药物吸收机制研究等。     

渗透促进剂对多肽类药物肺部给药促渗作用的机理

目的从肺细胞膜流动性的角度探讨渗透促进剂增加多肽类药物肺部吸收的作用机制。方法以鲑鱼降钙素(sCT)为模型药物,考查多种渗透促进剂对sCT经肺吸收的促进作用。采用电子自旋共振(ESR)技术和荧光偏振技术测定旋转相关时间(τ_C)及荧光偏振度(P),计算渗透促进剂处理前后膜脂流动性和膜蛋白构象的变化,从而考查渗透促进剂增加药物肺部吸收的原因。结果可以显著促进药物吸收的苄泽78、卵磷脂、辛酸钠均引起膜脂质流动性的增加,同时造成蛋白构象不同程度的松散;牛磺胆酸钠对膜蛋白的影响较弱;壳聚糖降低膜脂流动性,其促渗作用主要来自于改变了膜蛋白的三级结构而使细胞间紧密连接张力减小;2-羟丙基-β-环糊精对药物吸收没有明显贡献,同时其对膜通透性基本没有影响。结论考查体外细胞膜流动性的变化为研究渗透促进剂对药物肺部吸收的促进作用提供了重要依据。     

脂质体肺部给药在多肽类药物方面的应用

多肽类药物由于易被蛋白酶降解失活、半衰期短而局限于注射给药。将其通过脂质体的包裹,并经一定的给药装置传输到肺靶向部位或由肺泡吸收进入血液循环,发挥局部或全身疗效,并达到控制释放的目的,已成为研究热点。此文介绍了脂质体肺部给药的特点、方式和在多肽类药物给药中的应用。     

口服药物吸收促进剂应用与开发研究进展

目的 为口服药物吸收促进剂的合理使用及开发提供参考。方法 分析影响口服药物吸收的具体因素,结合国内外研究成果介绍吸收促进剂的常用种类及具体应用,阐述吸收促进剂增加肠道通透性、提高口服药物生物利用度的机制。结果与结论 口服药物生物利用度的影响因素包括消化道物理、化学屏障及药物处方组成等。口服药物吸收促进剂包括表面活性剂、螯合剂、壳聚糖及其衍生物、多肽类、聚酰胺-胺型树枝状大分子、生物黏附材料。表面活性剂为常用吸收促进剂,其一方面可提高药物的表观溶解度,进而提高亲脂性药物口服递送效率,另一方面可减少多种酶对药物的代谢作用,破坏消化道紧密连接、改善细胞膜流动性、促进肠淋巴转运吸收、增强药物与上皮细胞的亲和性,进而增加药物在消化道的渗透效率,从而提高口服生物利用度。目前能真正用于临床含吸收促进剂的药物仍较少,且安全性有待提高。     

肺泡 Ⅱ型上皮细胞与肺部疾病的研究进展

肺泡上皮细胞主要由肺泡Ⅰ型上皮细胞和肺泡Ⅱ型上皮细胞组成,但肺泡Ⅱ型上皮细胞在维持正常肺功能有着更为重要的作用,如维持屏障功能完整、再生功能、分泌功能等;同时其与肺部疾病的发生发展有密切关系.该文对肺泡Ⅱ型上皮细胞的正常生理功能,及其在急性肺损伤、间质性肺疾病、肺部肿瘤、肺结核、慢性阻塞性肺疾病等肺部疾病的研究进展作一综述.     

多肽类药物口服吸收的研究进展

多肽类药物对癌症、自身免疫性疾病具有良好的治疗前景。目前，市售肽类制剂以注射剂为主，频繁的注射给患者造成很大的痛苦。口服作为一种方便、顺应性好的给药途径，日益成为研究焦点；然而肽类药物口服受消化道屏障限制，生物利用度低，半衰期短，制约了它的发展。提高多肽类药物口服生物利用度的研究工作业已展开。本文综述了限制肽类药物口服吸收的主要影响因素及针对性的解决策略。     

肺泡巨噬细胞单层模型在生物药剂学中的应用

目的　 建立体外肺泡巨噬细胞单层模型 ,用于研究药物在巨噬细胞的分布特征和相应机理 ,并可以用来预测药物 (主要为治疗肺部炎症的抗生素 ) ,在体内分布到肺泡巨噬细胞的程度。 方法 　采用支气管肺泡洗净法(broncho alveolarlavage ,BAL)从大鼠肺部分离肺泡巨噬细胞 ,并进行纯化和体外培养。根据巨噬细胞的吸附特征通过洗净法除去混杂的细胞 ,在 95 %air 5 %CO2 ,37℃的细胞孵育箱下 ,进行肺泡巨噬细胞单层的体外培养。在此基础上 ,使用该模型初步考察了环丙沙星在肺泡巨噬细胞的分布。 结果 　环丙沙星能够逆着细胞内外的浓度差向细胞内转运 ,使得细胞内外 (I/E)药物的浓度比值为 11。 结论 　肺泡巨噬细胞单层是有效的研究药物在体内分布到肺泡巨噬细胞的体外细胞模型     

Transport of proteins and peptides across human cultured alveolar A549 cell monolayer

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway of the peptides and proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium. Human lung adenocarcinoma A549 cells formed continuous monolayers when grown on the polycarbonate filters of Transwell plates. The transport of the peptides and proteins having MW of 3400-22,000 Da was studied under different conditions. The results showed that the apparent permeability coefficients (P(app)) of these macromolecules across A549 cell monolayers ranged from 2x10(-6) to 5x10(-6) cm s(-1) and exhibited a good inverse correlation with molecular weight. No concentration, direction, or temperature dependence was observed in the permeation of sCT, INS, and rHAV2. While the P(app) of rhGH in the BA direction (2.25x10(-6) cm s(-1)) was less than that in the AB direction at both concentrations (3.20x10(-6) and 3.29x10(-6) cm s(-1)). The P(app) values of rhGH were concentration and temperature independent in the AB direction. These findings suggest that the hydrophilic peptides and proteins used in this study, sCT, INS, rHAV2, and rhGH, appear to cross the A549 cell monolayers via a paracellular pathway by a passive diffusion mechanism.     

Regional permeability of salmon calcitonin in isolated rat gastrointestinal tracts: Transport mechanism using Caco-2 cell monolayer

The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250-300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 microm in phosphate-buffered saline, pH 7.4, 37 degrees C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-microm pore size, 0.33 cm2 area) in a humidified 37 degrees C incubator (with 5% CO2). Transport experiments were conducted for sCT solutions (50 microm in Dulbecco's modified eagle's medium [DMEM], pH 7.4) from the apical-to-basolateral (A-to-B) direction and B-to-A direction at 37 degrees C and from the A-to-B direction at 4 degrees C. Cell monolayer integrity was monitored by mannitol permeability and transepithelial electrical resistance (TEER) measurements. The permeability coefficients (x 10(-9), cm/sec) for sCT through rat stomach, duodenum, jejunum, ileum, and colon tissues were 0.482 +/- 0.086, 0.718 +/- 0.025, 0.830 +/- 0.053, 1.537 +/- 0.32, and 0.934 +/- 0.15, respectively. The region of maximum sCT permeability is ileum followed by colon, jejunum, duodenum, and stomach. The permeability coefficients (x 10(-6), cm/sec) for sCT through Caco-2 cell monolayer were 8.57 +/- 2.34 (A-to-B, 37 degrees C), 8.01 +/- 1.22 (A-to-B, 4 degrees C), and 6.15 +/- 1.97 (B-to-A, 37 degrees C). The mechanism of its permeation is passive diffusion through the mucosa as determined from the Caco-2 monolayer permeability of sCT.     

Permeability of Peptides and Proteins in Human Cultured Alveolar A549 Cell Monolayer

Purpose. The transport of peptides or proteins across the alveolar cell monolayer was studied in vitro in order to elucidate their transport pathway. Methods. The permeability of 14 peptides or proteins and 6 dextrans with MW 1,000~150,000 was measured in cultured human lung adenocarcinoma A549 cell monolayers at 37°C or 4°C. The stability of the tested peptides and proteins was also evaluated. Results. The permeability coefficients of these macromolecules across the A549 cell monolayer at 37°C ranged from 10 ^–5 to 10 ^–7 (cm/sec), and exhibited a good inverse correlation with molecular weight. All macromolecules were stable throughout the transport experiment, and degradation by proteases was minimal. Permeability at 4°C did not differ from that at 37°C. Clear selectivity for direction of transport was not observed. Conclusions. These results suggested that the tested peptides and proteins appeared to penetrate the A549 cell monolayer via a paracellular route by passive diffusion.     

Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability

Decursin (DE) and decursinol angelate (DA) were isolated from the roots of Angelica gigas (Apiaceae) and purified by HPLC. DE and DA have been reported to exhibit significant neuropharmacological activities, but their intestinal transport and permeability in terms of CNS penetration across the blood-brain barrier (BBB) are unknown. This study was undertaken to evaluate the IN VITRO intestinal and BBB transport of DE and DA using Caco-2 and MDR-MDCK cell monolayer models, respectively. The bidirectional transport of DE and DA across Caco-2 and MDR-MDCK monolayers was examined for 2 hours. Integrity of the monolayer was determined by TEER value and by monitoring the transport of Lucifer yellow (Ly) across the monolayers. Quantitation of DE and DA was performed by HPLC. DE and DA exhibited bidirectional transport with a Papp value in the range of 9.0-12.0x10(-6) cm/sec and 7.2-11.7x10(-6) cm/sec in Caco-2 and MDR-MDCK monolayers, respectively. The TEER values were in the range of 410-440 and 1170-1230 ohm cm2 for Caco-2 and MDR-MDCK monolayers, respectively. Ly measurement, the fluorescent marker of passive paracellular diffusion, resulted in Papp values of 2.5-5.0x10(-6) in Caco-2 and 6.0-8.0x10(-6) cm/sec in MDR-MDCK monolayers, confirming that the monolayer integrity was intact at the end of the experiment. Caco-2:human colonic adenocarcinoma DA:decursinol angelate DE:decursin Ly:Lucifer yellow MDCK:Madin-Darby canine kidney MDR:multidrug resistant Papp:apparent permeability TEER:transepithelial electrical resistance.     

Adsorption of peptides to poly(D,L-lactide-co-glycolide): 1. Effect of physical factors on the adsorption

Four peptides, salmon calcitonin (sCT), the 8–22 amino acid portion of sCT (CT15), an LHRH superagonist, triptorelin (DP) and a somatostatin analogue (RC160) were studied for adsorption to poly(D,L-lactide-co-glycolide) (PLGA) as a function of peptide and polymer concentration. The adsorption of sCT and DP to the polymer showed a primary transient-equilibrium followed by a more rapid and extensive adsorption without reaching a concentration plateau for sCT which occurred earlier with higher peptide concentration. The amount of adsorption for CT15 was much lower than that for sCT and DP and RC160 showed no adsorption. Adsorption isotherms of sCT and DP were fitted to a Langmuir-type of relationship and the monolayer concentration was close to that observed in the kinetic studies or that calculated by the theoretical approach.     

A comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery.

We have previously reported the formation and characterization of poly(ethylene glycol) (PEG)-coated and chitosan (CS)-coated lipid nanoparticles. In the present work our goal was to study the interaction of these surface-modified lipid nanoparticles with Caco-2 cells and to evaluate the potential of these nanostructures as oral delivery systems for salmon calcitonin (sCT). The interaction of rhodamine-loaded nanoparticles with the Caco-2 cell monolayers was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and fluorimetry, respectively. The ability of these nanoparticles to reversibly enhance the transport of hydrophilic macromolecules through the monolayers was investigated by measuring the transepithelial electric resistance and the permeability to Texas Red-dextran. Finally, in vivo studies of the response to sCT-loaded nanoparticles were performed in rats. The results showed that the association of rhodamine-loaded nanoparticles to the Caco-2 cell monolayer was independent of the surface coating of the nanoparticles (CS-coated versus PEG-coated nanoparticles). However, while PEG-coated nanoparticles did not affect the permeability of Caco-2 monolayers, CS-coated nanoparticles produced a dose-dependent reduction in the transepithelial electric resistance, simultaneously to an enhanced dextran transport. The results obtained following oral administration of sCT-loaded CS-coated nanoparticles to rats showed a significant and prolonged reduction in the serum calcium levels as compared to those obtained for control (sCT solution). In contrast, the hypocalcemic response of sCT-loaded PEG-coated nanoparticles was not significantly different of that provided by the control (sCT solution). Therefore, these results indicate that the surface composition of the particles is a key factor in the improvement of the efficiency of oral sCT formulations. Moreover, the encouraging results obtained for CS-coated nanoparticles underline their potential as carriers for peptide delivery.     

Enhancing effect of chemically conjugated deoxycholic acid on permeability of calcitonin in Caco‐2 cells

One approach for enhancing intestinal absorption of therapeutic peptides is chemical conjugation to bile acids. In this study, recombinant salmon calcitonin (sCT) was chemically modified by covalent attachment of deoxycholic acid (DOCA). Three different sCT‐DOCA conjugates, namely, sCT‐mono‐DOCA, sCT‐di‐DOCA, and sCT‐tri‐DOCA, were prepared and characterized for their structural and biological properties. The permeability of these conjugates in the gastrointestinal tract was evaluated using Caco‐2 cell monolayers to determine their potential as an oral dosage form. The conjugates were isolated by reversed‐phase fast protein liquid chromatography (FPLC) and confirmed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Circular dichroism spectra in 50% trifluoroethanol aqueous condition showed the ordered secondary structure of sCT‐DOCA. The biological activities of sCT‐DOCA conjugates were evaluated by cyclic AMP assay using T‐47D cells, and the mean EC₅₀ values of sCT, sCT‐mono‐DOCA, and sCT‐di‐DOCA were 0.034, 0.076, and 0.46 nM, respectively. The transport experiments using the Caco‐2 cell monolayer showed that the permeability of sCT‐DOCA conjugates in buffer was not altered from the native sCT. However, the permeability of sCT‐DOCA conjugates was increased up to 2.5 times that of the native sCT when sCT‐DOCA was formulated in 1% dimethylsulfoxide (DMSO) solution. The conjugated DOCA of sCT‐DOCA significantly enhanced the absorption of sCT‐DOCA in the intestinal membrane when sCT‐DOCA conjugates were completely solubilized by DMSO. In conclusion, this study proposes that therapeutic peptides that have poor absorption profiles could potentially be developed into orally active drugs by conjugation with DOCA in the formulation with appropriate solubilizing agents. Drug Dev. Res. 64:129–135, 2005. © 2005 Wiley‐Liss, Inc.     

Adsorption of peptides to poly(D,L-lactide-co-glycolide): 2. Effect of solution properties on the adsorption

The effect of pH, ionic strength, polarity and temperature on the adsorption of three peptides to poly(D,L-lactideco-glycolide) (PLGA) was evaluated. Maximum adsorption was found near the pI of the peptide for salmon calcitonin (sCT), triptorelin (DP) and a peptide comprising the 8–22 amino acid portion of sCT. For sCT, almost no adsorption was observed at pH < 6 while there was complete depletion at pH 10. Increase in NaCl concentration enhanced the adsorption of sCT and DP. The dependency on solvent ionic strength and polarity suggested that hydrophobic interactions were playing an important role in the adsorption process. The net adsorption of sCT and DP was greater at 22°C than at 4°C or 37°C.     

Evaluation of HPβCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery

For therapeutic peptides, the lung represents an attractive, noninvasive route into the bloodstream. To achieve optimal bioavailability and control their fast rate of absorption, peptides can be protected by coprocessing with polymers such as polyethylene glycol (PEG). Here, we formulated and characterized salmon calcitonin (sCT)-loaded microparticles using linear or branched PEG (L-PEG or B-PEG) and hydroxypropyl-beta-cyclodextrin (HPβCD) for pulmonary administration. Mixtures of sCT, L-PEG or B-PEG and HPβCD were co-spray dried. Based on the particle properties, the best PEG:HPβCD ratio was 1:1 w:w for both PEGs. In the sCT-loaded particles, the L-PEG was more crystalline than B-PEG. Thus, L-PEG-based particles had lower surface free energy and better aerodynamic behavior than B-PEG-based particles. However, B-PEG-based particles provided better protection against chemical degradation of sCT. A decrease in sCT permeability, measured across Calu-3 bronchial epithelial monolayers, occurred when the PEG and HPβCD concentrations were both 1.6 wt %. This was attributed to an increase in buffer viscosity, caused by the two excipients. sCT pharmacokinetic profiles in Wistar rats were evaluated using a 2-compartment model after iv injection or lung insufflation. The maximal sCT plasma concentration was reached within 3 min following nebulization of sCT solution. L-PEG and B-PEG-based microparticles were able to increase T(max) to 20 ± 1 min and 18 ± 8 min, respectively. Furthermore, sCT absolute bioavailability after L-PEG-based microparticle aerosolization at 100 μg/kg was 2.3 times greater than for the nebulized sCT solution.     

Effects of citicholine and dimethylsulfoxide on transepithelial transport of passively diffused drugs in the Caco-2 cell culture model

The objective of this study was to determine, using a Caco-2 cell monolayer model, the extent to which the paracellular and transcellular routes are altered by citicholine (CDP-Ch) and DMSO in the presence of human serum albumin (HSA). The apparent permeability (Papp) of mannitol in the presence of 4% (w/v) HSA was investigated using 0, 0.5, 1.0, 2.5, 5.0, and 10.0% (v/v)) of DMSO. The Papp for mannitol ranged from 0.56 x 10(-6) to 0.89 x 10(-6) cm/s (mean 0.77 x 10(-6)). Increasing the concentration of DMSO does not appear to have an effect on the paracellular transport of mannitol and on the transepithelial resistance (TEER) of the monolayer, (P>0.05). The effect of citicholine (CDP-Ch) was investigated in confluent Caco-2 cell monolayers incubated in the presence of 2, 4, 10, 40, 60, 100 and 200 mM CDP-Ch at 37 degrees C in an atmosphere of 7% CO(2) and 95% relative humidity. Papp of mannitol and diltiazem in the presence of CDP-Ch ranged from 0.53 x 10(-6) to 8.52 x 10(-6) cm/s and from 1.30 x 10(-5) to 2.71 x 10(-5) cm/s, respectively. CDP-Ch may have an effect on the stability of the tight junction complex resulting in an increase in the apparent permeability of mannitol.