Biopartitioning micellar chromatography: an in vitro technique for predicting human drug absorption

The main oral drug absorption barriers are fluid cell membranes and generally drugs are absorbed by a passive diffusion mechanism. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to mimic the drug partitioning process in biological systems. In this paper the usefulness of BMC for predicting oral drug absorption in humans is demonstrated. A hyperbolic model has been obtained using the retention data of a heterogeneous set of 74 compounds, which shows predictive ability for drugs absorbed by passive diffusion. The model obtained in BMC is compared with those obtained using the well-known systems (Caco-2 and TC-7) that use intestinal epithelium cell lines. The use of BMC is simple, reproducible and can provide key information about the transport properties of new compounds during the drug discovery process.     

Molecular modeling of blood-brain barrier nutrient transporters: in silico basis for evaluation of potential drug delivery to the central nervous system

For drugs that act in the brain, the blood-brain barrier (BBB) is a considerable physical barrier which influences the distribution of drugs to the brain. The BBB is essentially impermeable for hydrophilic and/or charged compounds. Nutrient membrane transporters have an important physiological role in the transport of essential substances across the BBB required for normal brain function. We and others have shown that these transporters may have utility as drug delivery vectors, thereby increasing brain distribution of these compounds via these systems. In this review, we evaluate molecular (in silico) models of BBB transport proteins. Few BBB membrane transporters have been crystallized, but their crystal structures have a possibility for use in homology modeling. Other techniques commonly used are 2D quantitative structure-activity relationships (QSAR), as well as 3D-QSAR techniques including comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA). Each of these models provides valuable information for ascertaining their potential basis for BBB transport and brain drug delivery.     

Microfluidic blood–brain barrier model provides in vivo‐like barrier properties for drug permeability screening

Efficient delivery of therapeutics across the neuroprotective blood–brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High‐fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo‐like barrier properties in a microfluidic BBB model. This BBB‐on‐a‐chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo‐like values of trans‐endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm² on day 3 on chip and were sustained above 2000 Ω · cm² up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC‐dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB‐on‐a‐chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time‐based design of a microfluidic platform will enable integration with other organ modules to simulate multi‐organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184–194. © 2016 Wiley Periodicals, Inc.     

载药纳米粒作为脑靶向给药系统的研究

血脑屏障使大部分的活性药物很难由血液进入脑内发挥作用。载药纳米粒具有脑靶向性,可显著提高药物在脑内浓度,成为药物突破血脑屏障的有效途径。本文综述了近年来载药纳米粒透过血脑屏障的研究进展,并对纳米粒载中药入脑提出展望。     

A Method to Predict Blood-Brain Barrier Permeability of Drug-Like Compounds Using Molecular Dynamics Simulations

The blood-brain barrier (BBB) is formed by specialized tight junctions between endothelial cells that line brain capillaries to create a highly selective barrier between the brain and the rest of the body. A major problem to overcome in drug design is the ability of the compound in question to cross the BBB. Neuroactive drugs are required to cross the BBB to function. Conversely, drugs that target other parts of the body ideally should not cross the BBB to avoid possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds is of great importance. Two gold-standard experimental measures of BBB permeability are logBB (the concentration of drug in the brain divided by concentration in the blood) and logPS (permeability surface-area product). Both methods are time-consuming and expensive, and although logPS is considered the more informative measure, it is lower throughput and more resource intensive. With continual increases in computer power and improvements in molecular simulations, in silico methods may provide viable alternatives. Computational predictions of these two parameters for a sample of 12 small molecule compounds were performed. The potential of mean force for each compound through a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer is determined by molecular dynamics simulations. This system setup is often used as a simple BBB mimetic. Additionally, one-dimensional position-dependent diffusion coefficients are calculated from the molecular dynamics trajectories. The diffusion coefficient is combined with the free energy landscape to calculate the effective permeability (P_eff) for each sample compound. The relative values of these permeabilities are compared to experimentally determined logBB and logPS values. Our computational predictions correlate remarkably well with both logBB (R² = 0.94) and logPS (R² = 0.90). Thus, we have demonstrated that this approach may have the potential to provide reliable, quantitatively predictive BBB permeability, using a relatively quick, inexpensive method.     

A Method to Predict Blood-Brain Barrier Permeability of Drug-Like Compounds Using Molecular Dynamics Simulations

The blood-brain barrier (BBB) is formed by specialized tight junctions between endothelial cells that line brain capillaries to create a highly selective barrier between the brain and the rest of the body. A major problem to overcome in drug design is the ability of the compound in question to cross the BBB. Neuroactive drugs are required to cross the BBB to function. Conversely, drugs that target other parts of the body ideally should not cross the BBB to avoid possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds is of great importance. Two gold-standard experimental measures of BBB permeability are logBB (the concentration of drug in the brain divided by concentration in the blood) and logPS (permeability surface-area product). Both methods are time-consuming and expensive, and although logPS is considered the more informative measure, it is lower throughput and more resource intensive. With continual increases in computer power and improvements in molecular simulations, in silico methods may provide viable alternatives. Computational predictions of these two parameters for a sample of 12 small molecule compounds were performed. The potential of mean force for each compound through a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer is determined by molecular dynamics simulations. This system setup is often used as a simple BBB mimetic. Additionally, one-dimensional position-dependent diffusion coefficients are calculated from the molecular dynamics trajectories. The diffusion coefficient is combined with the free energy landscape to calculate the effective permeability (P_eff) for each sample compound. The relative values of these permeabilities are compared to experimentally determined logBB and logPS values. Our computational predictions correlate remarkably well with both logBB (R² = 0.94) and logPS (R² = 0.90). Thus, we have demonstrated that this approach may have the potential to provide reliable, quantitatively predictive BBB permeability, using a relatively quick, inexpensive method.     

血脑屏障与脑血管疾病的相关研究

血脑屏障（blood brain barrier,BBB）的主要结构包括：脑毛细血管内皮细胞及其间的紧密连接（tight junction,TJ）、基底膜、基 底膜下星型胶质细胞终足。血脑屏障是存在于血液和脑组织之间的一层屏障系统,在许多大脑疾患的病理过程中,BBB 的破坏导 致通透性增高都是不可避免的一个环节。BBB是保证中枢神经系统的正常生理功能的重要屏障系统。目前已有大量关于血脑屏 障通透性在脑血管疾病中的变化研究。本文分别从血脑屏障的结构和功能,药物通过血脑屏障的方法和功能,脑缺血损伤、阿尔 茨海默病、帕金森病和多发性硬化症等不同的脑病变与血脑屏障通透性的变化及中医药应用等方面做一综述。有针对性地对 BBB和大脑疾病进行进一步的研究与探索,将会为临床治疗相关疾病带来新的视角与机遇。     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

Targeting Therapeutics Across the Blood Brain Barrier (BBB), Prerequisite Towards Thrombolytic Therapy for Cerebrovascular Disorders—an Overview and Advancements

Cerebral tissues possess highly selective and dynamic protection known as blood brain barrier (BBB) that regulates brain homeostasis and provides protection against invading pathogens and various chemicals including drug molecules. Such natural protection strictly monitors entry of drug molecules often required for the management of several diseases and disorders including cerebral vascular and neurological disorders. However, in recent times, the ischemic cerebrovascular disease and clinical manifestation of acute arterial thrombosis are the most common causes of mortality and morbidity worldwide. The management of cerebral Ischemia requires immediate infusion of external thrombolytic into systemic circulation and must cross the blood brain barrier. The major challenge with available thrombolytic is their poor affinity towards the blood brain barrier and cerebral tissue subsequently. In the clinical practice, a high dose of thrombolytic often prescribed to deliver drugs across the blood brain barrier which results in drug dependent toxicity leading to damage of neuronal tissues. In recent times, more emphasis was given to utilize blood brain barrier transport mechanism to deliver drugs in neuronal tissue. The blood brain barrier expresses a series of receptor on membrane became an ideal target for selective drug delivery. In this review, the author has given more emphasis molecular biology of receptor on blood brain barrier and their potential as a carrier for drug molecules to cerebral tissues. Further, the use of nanoscale design and real-time monitoring for developed therapeutic to encounter drug dependent toxicity has been reviewed in this study.KEY WORDS: blood brain barrier (BBB), cerebral ischemic disorders, drug delivery, earthworm protease, neurodegenerative disorder, thrombolytic     

Re-engineering biopharmaceuticals for delivery to brain with molecular Trojan horses

Biopharmaceuticals, including recombinant proteins, monoclonal antibody therapeutics, and antisense or RNA interference drugs, cannot be developed as drugs for the brain, because these large molecules do not cross the blood-brain barrier (BBB). Biopharmaceuticals must be re-engineered to cross the BBB, and this is possible with genetically engineered molecular Trojan horses. A molecular Trojan horse is an endogenous peptide, or peptidomimetic monoclonal antibody (mAb), which enters brain from blood via receptor-mediated transport on endogenous BBB transporters. Recombinant neurotrophins, single chain Fv antibodies, or therapeutic enzymes may be re-engineered as IgG fusion proteins. The engineering of IgG-avidin fusion proteins enables the BBB delivery of biotinylated drugs. The IgG fusion proteins are new chemical entities that are dual or triple function molecules that bind multiple receptors. The fusion proteins are able both to enter the brain, by binding an endogenous BBB receptor, and to induce the desired pharmacologic effect in brain, by binding target receptors in the brain behind the BBB. The development of molecular Trojan horses for BBB drug delivery allows the re-engineering of biopharmaceuticals that, owing to the BBB problem, could not otherwise be developed as new drugs for the human brain.     

Blood-brain barrier-on-a-chip for brain disease modeling and drug testing

The blood-brain barrier (BBB) is an interface between cerebral blood and the brain parenchyma. As a gate keeper, BBB regulates passage of nutrients and exogeneous compounds. Owing to this highly selective barrier, many drugs targeting brain diseases are not likely to pass through the BBB. Thus, a large amount of time and cost have been paid for the development of BBB targeted therapeutics. However, many drugs validated in in vitro models and animal models have failed in clinical trials primarily due to the lack of an appropriate BBB model. Human BBB has a unique cellular architecture. Different physiologies between human and animal BBB hinder the prediction of drug responses. Therefore, a more physiologically relevant alternative BBB model needs to be developed. In this review, we summarize major features of human BBB and current BBB models and describe organ-on-chip models for BBB modeling and their applications in neurological complications.     

聚乳酸纳米粒穿透血脑屏障的分析电镜研究

观察以聚乳酸 (D ,L-polylacticacid,PLA)为材料制备、经吐温-80(T-80)表面改性的纳米粒对血脑屏障的穿透效果并探讨其机制 ,分别将FITC-Dextran、叶绿素铜作为PLA纳米粒的示踪标记 ,应用荧光显微镜、透射电镜及分析电镜观察经静脉注射入小鼠体内的PLA纳米粒在脑组织中的分布、穿透血脑屏障的特性。荧光显微镜观察到小鼠脑组织中散在及沿毛细血管壁分布的荧光颗粒 ,透射电镜可观察到小鼠脑毛细血管内皮细胞及周围脑组织中圆形或类圆形的外源性纳米粒 ;进一步采用分析电镜对颗粒处组织进行能谱分析证实其为叶绿素铜标记的PLA纳米粒。证实了T-80修饰的PLA纳米粒具有穿透血脑屏障的特性 ,机制可能是毛细血管内皮细胞的胞吞转运作用 ,同时 ,为研究纳米粒在组织内的定位提供了新的标记方法.     

Refining in vitro neurotoxicity testing--the development of blood-brain barrier models

The purpose of this paper is to review the current state of development of advanced in vitro blood-brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.     

Nanoparticle Mediated P-Glycoprotein Silencing for Improved Drug Delivery across the Blood-Brain Barrier: A siRNA-Chitosan Approach

The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.     

Role of VEGF in an experimental model of cortical micronecrosis

Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.     

Drug permeability prediction using PMF method

Drug permeability determines the oral availability of drugs via cellular membranes. Poor permeability makes a drug unsuitable for further development. The permeability may be estimated as the free energy change that the drug should overcome through crossing membrane. In this paper the drug permeability was simulated using molecular dynamics method and the potential energy profile was calculated with potential of mean force (PMF) method. The membrane was simulated using DPPC bilayer and three drugs with different permeability were tested. PMF studies on these three drugs show that doxorubicin (low permeability) should pass higher free energy barrier from water to DPPC bilayer center while ibuprofen (high permeability) has a lower energy barrier. Our calculation indicates that the simulation model we built is suitable to predict drug permeability.     

Alteration of blood-brain barrier function by methamphetamine and cocaine

The integrity of the blood-brain barrier (BBB) plays an important role in maintaining a safe neural microenvironment in the brain. Loss of BBB integrity has been recognized as a major cause of profound brain alterations. Psychoactive drugs such as methamphetamine (METH) or cocaine are well-known drugs of abuse that can alter the permeability of the BBB via various mechanisms. In addition, the neurotoxicity of METH is well documented, and alterations in BBB function can contribute to this toxicity. A great deal of effort has been devoted to understanding the cellular and molecular mechanisms of the action of these drugs in the central nervous system. However, only a few investigations have focused on the effects of METH and cocaine on BBB function. The aim of this short review is to summarize our present knowledge of this subject.     

Cover Image

The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood–brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three-dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri-culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri-culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p-glycoprotein (p-gp), which is highly responsible for restrictive drug transport. By conducting the calcein-AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri-culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.     

Specific AHNAK expression in brain endothelial cells with barrier properties

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. Because disruption of the BBB may contribute to many brain disorders, they are of considerable interests in the identification of the molecular mechanisms of BBB development and integrity. We here report that the giant protein AHNAK is expressed at the plasma membrane of endothelial cells (ECs) forming specific blood-tissue barriers, but is absent from the endothelium of capillaries characterized by extensive molecular exchanges between blood and extracellular fluid. In the brain, AHNAK is widely distributed in ECs with BBB properties, where it co-localizes with the tight junction protein ZO-1. AHNAK is absent from the permeable brain ECs of the choroid plexus and is down-regulated in permeable angiogenic ECs of brain tumors. In the choroid plexus, AHNAK accumulates at the tight junctions of the choroid epithelial cells that form the blood-cerebrospinal fluid (CSF) barrier. In EC cultures, the regulation of AHNAK expression and its localization corresponds to general criteria of a protein involved in barrier organization. AHNAK is up-regulated by angiopoietin-1 (Ang-1), a morphogenic factor that regulates brain EC permeability. In bovine cerebral ECs co-cultured with glial cells, AHNAK relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties. Our results identify AHNAK as a protein marker of endothelial cells with barrier properties.