Blood-Brain Barrier Permeability of Asiaticoside,Madecassoside and Asiatic Acid in Porcine Brain Endothelial Cell Model

Neurotherapeutic potentials of Centella asiatica and its reputation to boost memory, prevent cognitive deficits and improve brain functions are widely acknowledged. The plant's bioactive compounds, i.e. asiaticoside, madecassoside and asiatic acid were reported to have central nervous system (CNS) actions, particularly in protecting the brain against neurodegenerative disorders. Hence, it is important for these compounds to cross the blood-brain barrier (BBB) to be clinically effective therapeutics. This study aimed to explore the capability of asiaticoside, madecassoside and asiatic acid to cross the BBB using in vitro BBB model from primary porcine brain endothelial cells (PBECs). Our findings showed that asiaticoside, madecassoside and asiatic acid are highly BBB permeable with apparent permeability (P_app) of 70.61 ± 6.60, 53.31 ± 12.55 and 50.94 ± 10.91 × 10^?6 cm/s respectively. No evidence of cytotoxicity and tight junction disruption of the PBECs were observed in the presence of these compounds. Asiatic acid showed cytoprotective effect towards the PBECs against oxidative stress. This study reported for the first time that Centella asiatica compounds demonstrated high capability to cross the BBB, comparable to central nervous system drugs, and therefore warrant further development as therapeutics for the treatment of neurodegenerative diseases.     

Mucus as a barrier to lipophilic drugs

Mucus is a complex hydrogel, comprising glycoproteins, lipids, salts, DNA, enzymes and cellular debris, covering many epithelial surfaces in the human body. Once secreted, mucin forms a barrier to protect the underlying tissues against the extracellular environment. Mucus can therefore adversely affect the absorption or action of drugs administered by the oral, pulmonary, vaginal, nasal or other routes. Solubility and lipophilicity are key factors determining drug absorption, as a drug has to be soluble in the body fluids at the site of absorption and must also possess enough lipophilicity to permeate the biological membrane. Evidence has accumulated over the past 40 years indicating that poorly soluble drugs will interact with mucus glycoprotein. Studies of the permeability of native or purified mucous gels are important when it comes to understanding the relative importance of hindered diffusion versus drug binding in mucous layers. This review highlights the current understanding of the drug–mucin interaction and also examines briefly the interaction of polymers and particles with the mucus matrix. While the concept of mucoadhesion was thought to provide an intensified and prolonged contact to mucosal absorption sites, mucopenetrating properties are nowadays being discussed for (nano)particulate carriers to overcome the mucus as a barrier and enhance drug delivery through mucus.     

Effect of Iontophoretic Patterns on in Vivo Antidiuretic Response to Desmopressin Acetate Administered Transdermally

Abstract

The effects of concentration, amperage and duration on the antidiuretic response induced by iontophoretic delivery of desmopressin acetate (DDAVP) were examined using a diabetes insipidus model in rats. A higher current density brought about a larger and longer antidiuretic response. Prolonged iontophoretic duration caused an overdose. Repeated short iontophoretic treatments with lower current density maintained a constant response with a short lag time and a rapid disappearance of pharmacological response immediately after cessation of the final treatment. This type of iontophoresis substantially reduced the inter-subject variability of response as compared to the response using an intranasal route of administration.     

Biorelevant media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness

In this work we developed and characterized transport media that simulate the composition of micellar phase of intestinal fluids in the fasted and, especially, in the fed state and are appropriate for evaluating intestinal drug permeability characteristics using the Caco-2 model (FaSSIF-TM_Caco and FeSSIF-TM_Caco, respectively). Media composition was based on FaSSIF-V2 and FeSSIF-V2 and recently reported data on total lipid concentrations in the micellar phase of contents of the upper small intestine in the fasted and the fed state and was adapted for cell culture compatibility. Permeation data were evaluated by compartmental kinetic modeling. Permeability coefficients, P, of hydrophilic drugs were not affected by media composition. In contrast, P values of a series of lipophilic compounds measured with FaSSIF-TM_Caco and FeSSIF-TM_Caco, and reflecting transport by diffusion were smaller than those obtained with a purely aqueous reference transport medium, aq-TM_Caco, following the rank order aq-TM_Caco > FaSSIF-TM_Caco > FeSSIF-TM_Caco. The decline of permeability values was stronger as lipophilicity of the compounds increased. Compared with values estimated using aq-TM_Caco, permeability was reduced, depending on the compound, by more than 20- to 100-fold when measured with FeSSIF-TM_Caco whereas compound ranking in regard to the permeability characteristics was also affected. The impact of reduced P value on flux through the mucosa, hence on drug absorption, in combination with the drug amount loaded on colloidal particles needs to be taken into consideration in PBPK modeling especially when the food effect is evaluated.     

Effects of Ethanol on Intestinal Absorption of Drugs.

The effect of chronic alcohol intake on the intestinal absorption of seven compounds belonging to a homologous series (ciprofloxacin derivatives) was evaluated using an in situ rat gut technique that measures the intrinsic absorption rates of the compounds both in control and chronic alcohol-fed rats. For chronic alcohol treatment, the animals were fed a liquid diet containing ethanol (36% of calories), whereas an isocaloric diet was given to the pair-fed control animals. The biophysical absorption model, relating the intestinal absorption rate constants and partition indexes of the tested compounds, was then established either for control or alcohol-fed animals. Differences were analyzed and tentatively interpreted on the basis of general diffusion principles. Results revealed that, in chronic alcohol-fed animals, hydrophilic homologs are absorbed at a significantly faster rate than in control ones, whereas lipophilic homologs do not change their absorption rate relative to controls. Results demonstrate that the bulk polarity of the microvillous lipoidal membrane is enhanced by chronic ethanol intake, whereas basic features of the aqueous boundary layer are not altered. These observations suggest that the physicochemical properties of the compounds are an important factor in explaining the influence of chronic alcohol intake on passive intestinal absorption of xenobiotics. The possible practical implications of our results are discussed from a speculative viewpoint     

Caco-2建立肠黏膜屏障模型及其在通透性研究中的应用

目的：利用 Caco-2细胞建立体外肠黏膜屏障模型,系统评价并初步探讨其在炎症损伤后黏膜通透性改变中的应用。方法体外培养 Caco-2细胞,接种于 Transwell 板上,每日观察细胞形态;自培养第5天起,隔日测细胞膜的跨上皮电阻(transepithelial electrical resistance,TEER);对于 TEER 值达标准的孔测定荧光黄透过率,进行透射电镜的完整性验证。应用培养21 d 的 Caco-2细胞屏障,加入不同浓度(0、50、100、200 nmol /L)的血小板活化因子(platelet-activating factor,PAF)孵育24 h,光镜、电镜观察形态学改变,检测 TEER 和荧光黄透过率,应用间接免疫荧光和 Western blot 观察 ZO-1蛋白的分布及表达。结果Caco-2细胞单层 TEER 从第5～15天逐渐增加,在第15天已经达到600Ω?cm2,平台期保持至第21天;在此期间,细胞形成紧密单层,电镜下细胞呈高分化,细胞间形成紧密连接,绒毛整齐,极性形成;荧光黄透过量极低,体外肠上皮细胞屏障形成。加入 PAF 后,以100 nmol /L 对黏膜屏障通透性影响最大：电镜下见紧密连接结构破坏、断裂,细胞表面微绒毛脱落、稀疏;免疫荧光可见紧密连接的标志蛋白 ZO-1荧光信号减弱,ZO-1环断裂,胞浆内可见阳性染色,提示其向膜下转移。此时,TEER 值下降,荧光黄透过量明显增加,与对照组比较差异有统计学意义(P ＜0.01),与形态学改变规律一致;ZO-1蛋白表达亦降至最低,与对照组相比差异有统计学意义(P ＜0.01)。结论经形态学及细胞通透性验证,培养2～3周的 Caco-2细胞可形成肠屏障模型,用于体外肠黏膜屏障的研究;PAF 影响紧密连接相关蛋白的表达,破坏紧密连接结构,从而影响肠黏膜屏障通透性。     

Kinetic Modeling of Contrast-Enhanced MRI: An Automated Technique for Assessing Inflammation in the Rheumatoid Arthritis Wrist

In recent years, development of rheumatoid arthritis (RA) drug therapy has been more directly targeted to counteract specific mechanisms of inflammation, and it is now believed that early aggressive treatment with disease modifying drugs is important to inhibit future structural joint damage. The development of these new treatments has increased the need for methodologies to assess disease activity in RA and monitor the effectiveness of drug therapy. Unlike X-ray, which shows only structural bone damage, magnetic resonance imaging (MRI) can depict soft tissue damage and synovitis, the primary pathology of RA. Recent studies have also indicated that MRI is sensitive to pathophysiologic changes that may predate radiographic erosions and may predict future joint damage. In this study, we have developed a computer automated analysis technique for MR wrist images that provides an objective measure of RA synovitis. This method applies a two-compartment pharmacokinetic model to every voxel of a dynamic contrast-enhanced MRI (DCE-MRI) dataset and outputs resulting parametric images. The aim of this technique is to not only objectively quantify the severity of rheumatoid synovitis, but to also locally determine where areas of serious disease activity are situated through kinetic modeling of blood-tissue exchange. Preliminary results show good correlation to early enhancement rate, which has previously been shown to be a useful clinical marker of RA activity. However, the use of tracer kinetic modeling methods potentially provides more specific information regarding underlying RA physiology. This approach could provide a useful new tool in RA patient management and could substantially improve RA therapeutic studies by calculating objective biomarkers of the disease state.     

缺氧缺血对体外血脑屏障通透性的影响及其机制的研究

目的：探讨血脑屏障紧密连接（BBB-tight junction，BBB—TJ）蛋白和基质金属蛋白酶-9（MMP-9）在缺氧缺血诱导的体外血脑屏障（BBB）模型通透性增加中的作用及相互关系。方法：ECV304与原代培养的星形胶质细胞共培养建立体外BBB模型。实验分为正常对照组（C组）、缺氧缺血组（H／I组）、BB-1101预处理组（P组）3组。透射电镜分别观察各组BBB-TJ变化；通过吖计数仪测定核素标记牛血清白蛋白（^125I-BSA）通透量检测BBB屏障功能。直接免疫荧光观察细胞骨架蛋白Actin分布的改变，Westemblot检测Actin、Occludin、ZO-1和MMP-9表达量的改变。结果：电镜观察见C组内皮细胞间形成光滑、连续、较高密度的紧密连接，免疫荧光检测见细胞骨架蛋白Actin主要分布在细胞膜及细胞核周边。形成肌动蛋白丝带，轮廓内包含大量柬状排列有序的微丝结构，细胞间连接紧密。缺氧缺血后．H／I组细胞间连接开放，形成裂隙，而胞膜及胞核周边肌动蛋白丝带模糊、部分断裂，并有应力纤维形成，出现细胞间裂隙，BBB对岱I—BSA通透性明显增高，与C组和P组比较，差异有显著性（P〈0．01），同时ZO-1的表达量显著减少，MMP-9的表达显著增多，而Occludin和Actin的表达量无改变。BB-1101预处理后，P组细胞BBB-TJ破坏减少．周边肌动蛋白丝带重新出现，细胞间裂隙较少，岱I-BSA通透量较H／I组明显下降（P〈0．01）．MMP-9的表达较H／I组也明显减少（P〈0.01）。结论：（1）缺氧缺血能促使BBB—TJ的开放进而导致BBB通透性增加；（2）BBB-TJ的开放可能与BBBActin的重组及ZO-1的表达量降低有关；（3）缺氧缺血能增加MMP-9的表达：（4）MMP-9与缺氧缺血时BBBActin的重组及ZO—1的表达减少相关。     

oxLDL对人脐静脉内皮细胞通透性影响及机制研究

目的探讨氧化低密度脂蛋白（ox LDL）对人脐静脉内皮细胞（HUVECs）通透性的影响及其可能机制。方法采用Transwell单层细胞模型系统和油红O染色,观察ox LDL对单层血管内皮细胞通透性的影响以及THP-1巨噬细胞内脂质的蓄积情况;采用蛋白质印迹法及免疫荧光技术检测ox LDL对HUVECs紧密连接occludin蛋白的表达及分布改变。结果 ox LDL增加单层内皮细胞对LDL的通透性和THP-1巨噬细胞脂质的蓄积;ox LDL降低occludin蛋白的表达,同时免疫荧光染色显示,ox LDL处理后,血管内皮细胞间隙增宽,occludin染色减少或脱失,而抑制氧化应激可以减弱ox LDL对内皮细胞的高通透性反应和occludin蛋白表达和形态学的影响。结论 ox LDL可能是通过occludin蛋白表达下降使内皮细胞构改变来介导内皮通透性增加,从而促进内皮下巨噬细胞内的脂质蓄积。