In Situ Blood–Brain Barrier Transport of Nanoparticles

PURPOSE: Two novel types of nanoparticles were evaluated as poten tial carriers for drugs across the blood-brain barrier (BBB). METHODS: Nanoparticles were composed of biocompatible materials including emulsifying wax (E. Wax) or Brij 72. Brij 78 and Tween 80 were used as surfactants for E. Wax nanoparticles (E78 NPs) and Brij 72 nanoparticles (E72 NPs), respectively. Both nanoparticle formulations were prepared from warm microemulsion precursors usin melted E. Wax or Brij 72 as the oil phase. Nanoparticles were radio-labeled by entrapment of [3H]cetyl alcohol, and entrapment efficiency and release of radiolabel were evaluated. The transport of E78 and E72 NPs across the BBB was measured by an in situ rat brai perfusion method. RESULTS: Both formulations were successfully radiolabeled by entrapment of [3H]cetyl alcohol; -98% of radiolabel remained associated with nanoparticles at experimental conditions. The transfer rate (Kin) of E78 NPs from perfusion fluid into the brain was 4.1 +/- 0.5 x 10(-3) ml/s/g, and the permeability-surface area product (PA) was 4.3 +/- 0.7 x 10(-3) ml/s/g. The values for Kin and PA for E72 NPs were 5.7 +/- 1.1 x 10(-3) ml/s/g and 6.1 +/- 1.4 x 10(-3) ml/s/g, respectively. CONCLUSIONS: For both nanoparticle types, statistically significant uptake was observed compared to [14C]sucrose, suggesting central nervous system uptake of nanoparticles. The mechanism underlying th nanoparticle brain uptake has yet to be fully understood.     

Effect of Chronic Hypertension on the Blood–Brain Barrier Permeability of Libenzapril

Very little information is available on the permeability of theblood–brain barrier (BBB) to small polar drugs inchronic hypertension. The blood and cerebrospinal fluid (CSF)pharmacokinetics of liben-zapril (LZP), a potentangiotensin converting enzyme inhibitor, were investigated inhypertensive (SH) and normotensive (SD) rats.Following intravenous bolus administration of this hydrophilic drug, theterminal rate constant for elimination (),steady-state volume of distribution ( ), and systemic clearance (CL) were similar in these two animalgroups. Other pharmacokinetic parameters (C_p°,, k _l2, and k ₂₁)were significantly (P < 0.05) greater in thehypertensive group, except for the volume of the central compartment(V_c) and ratio of V_c to , which were smaller in SH rats. The ratio ofarea under the concentration–time curve (AUC) in CSF toblood was about twofold higher in SH rats compared to normotensive rats,showing increased BBB permeability in hypertensive rats. An acute brainuptake study was also performed in SH, SD, and WK rats by intracarotidadministration of ¹⁴C-LZP along with³H₂O as a reference marker. Both LZP and watertransport was found to be significantly higher (about two-to five-fold) in six of the seven different brain regions inSH rats as compared to the normotensive (SD and WK) controls.Because of this simultaneous increase in concentrations of both the drugand the reference marker, BUI values were not affected. Regional brainconcentrations in SH rats were also linearly correlated with the meanarterial pressure (MAP) values, providing further evidence ofthe systemic pressure related increase in BBB permeability.     

Nanoparticle Surface Charges Alter Blood–Brain Barrier Integrity and Permeability

Purpose: The blood–brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability.

Methods: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion.

Results: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations.

Conclusions: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.     

Transport Characteristics of Tramadol in the Blood–Brain Barrier

Tramadol is a centrally acting analgesic whose action is mediated by both agonistic activity at opioid receptors and inhibitory activity on neuronal reuptake of monoamines. The purpose of this study was to characterize the blood–brain barrier (BBB) transport of tramadol by means of microdialysis studies in rat brain and in vitro studies with human immortalized brain capillary endothelial cells (hCMEC/D3). The K_p,uu,brain value of tramadol determined by rat brain microdialysis was greater than unity, indicating that tramadol is actively taken up into the brain across the BBB. Tramadol was transported into hCMEC/D3 cells in a concentration‐dependent manner. The uptake was inhibited by type II cations (pyrilamine, verapamil, etc.), but not by substrates of organic cation transporter OCTs or OCTN2. It was also inhibited by a metabolic inhibitor but was independent of extracellular sodium or membrane potential. The uptake was altered by changes of extracellular pH, and by ammonium chloride‐induced intracellular acidification, suggesting that transport of tramadol is driven by an oppositely directed proton gradient. Thus, our in vitro and in vivo results suggest that tramadol is actively transported, at least in part, from blood to the brain across the BBB by proton‐coupled organic cation antiporter.     

Recent Advances in Blood–Brain Barrier Disruption as a CNS Delivery Strategy

The blood-brain barrier (BBB) is a complex functional barrier composed of endothelial cells, pericytes, astrocytic endfeets and neuronal cells. This highly organized complex express a selective permeability for molecules that bear, amongst other parameters, adequate molecular weight and sufficient liposolubility. Unfortunately, very few therapeutic agents currently available do cross the BBB and enters the CNS. As the BBB limitation is more and more acknowledged, many innovative surgical and pharmacological strategies have been developed to circumvent it. This review focuses particularly on the osmotic opening of the BBB, a well-documented approach intended to breach the BBB. Since its inception by Rapoport in 1972, pre-clinical studies have provided important information on the extent of BBB permeation. Thanks to Neuwelt and colleagues, the osmotic opening of the BBB made its way to the clinic. However, many questions remain as to the detailed physiology of the procedure, and its best application to the clinic. Using different tools, amongst which MRI as a real-time in vivo characterization of the BBB permeability and CNS delivery, we attempt to better define the osmotic BBB permeabilization physiology. These ongoing studies are described, and data related to spatial and temporal distribution of a molecule after osmotic BBB breaching, as well as the window of BBB permeabilization, are discussed. We also summarize recent clinical series highlighting promising results in the application of this procedure to maximize delivery of chemotherapy in the treatment of brain tumor patients.     

In Vitro and in Vivo Transport of Zidovudine (AZT) Across the Blood–Brain Barrier and the Effect of Transport Inhibitors

The transport of the antiviral nucleoside analogue zidovudine (3-azido-3-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 ± 0.44 cm/min/10³; abluminal, 1.63 ± 0.28 cm/min/10³). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 µg/mL. AZT transport was not affected by pre-treatment of the cells with either metabolic inhibitors (DODG and DODG/NaN₃) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood–brain barrier and the blood–CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.     

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P_app of 19.7 ± 1.9 × 10^?6 cm/s and 38.6 ± 4.1 × 10^?6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009     

Characteristics of Substance P Transport Across the Blood–Brain Barrier

Purpose Substance P (SP; NH₃⁺-Arg⁺-Pro-Lys⁺-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) belongs to a group of neurokinins that are widely distributed in the central nervous system and peripheral nervous system. The biological effects mediated by SP in the central nervous system include regulation of affective behavior, emesis, and nociception. Many of these actions are believed to be the result of the binding of SP to the neurokinin-1 (NK-1) receptor and subsequent transport across the blood–brain barrier (BBB). The objective of the study was to investigate the involvement of the NK-1 receptor in the permeation of SP across the BBB. Methods Transport of ³H SP (1–13 nM) was investigated using BBMEC monolayers grown on polycarbonate membranes mounted on a Side-bi-Side™ diffusion apparatus. ³H SP samples were analyzed by scintillation spectrometry. Liquid chromatography-tandem mass spectrometry was used to monitor the transport at higher concentrations (micromolar). Results SP transport across BBMEC monolayers was found to be saturable (K_m = 8.57 ± 1.59 nM, V_max = 0.017 ± 0.005 pmol min⁻¹ mg⁻¹ protein) in the concentration range of 0–13 nM. Significant (p < 0.05) decline in ³H SP permeation was observed in the presence of unlabeled SP and at 4°C, indicating that the transport process is carrier-mediated. High-performance liquid chromatography analysis showed no significant metabolism of ³H SP in either the donor or receiver chambers. ³H SP transport was inhibited by 2–11 SP (p < 0.05) but not by any other fragments, indicating that both the C- and N-terminal regions are essential for molecular recognition by the receptor. Endocytic inhibitors (chloroquine, phenylarsine oxide, monensin, and brefeldin) did not inhibit SP transport, suggesting the involvement of a nonendocytic mechanism in SP permeation. Pro⁹ SP, a high-affinity substrate for the NK-1 major subtype receptor, significantly (p < 0.05) inhibited the transport of SP. However, Sar⁹Met(O₂)¹¹ SP, a high-affinity substrate for the NK-1 minor subtype receptor, septide, and neurokinin A, inhibitors of NK-1 and neurokinin-2 (NK-2) receptors, respectively, did not produce any inhibition of SP transport. Western blot analysis confirmed the presence of the NK-1 receptor in BBMEC monolayers. Conclusions The above results provide functional and molecular evidence for the existence of a carrier-mediated mechanism in the transport of SP across the BBB. The effects of specific inhibitors and the results of Western blot analyses demonstrate the involvement of the NK-1 receptor in the transport of SP across the BBB.     

Characterization of an In Vitro Blood–Brain Barrier Model System for Studying Drug Transport and Metabolism

Bovine brain micro vessel endothelial cells have been isolated and grown in culture to monolayers. These endothelial cell monolayers have been characterized morphologically with electron microscopy, histochemically for brain endothelium enzyme markers, alkaline phosphatase and -glutamyl trans-peptidase, and by immunofluorescence to detect Factor VIII antigen, an exclusive endothelial antigen. Results of these studies indicate that the cells forming the monolayers are of endothelial origin and possess many features of the in vivo brain endothelium responsible for formation of the blood–brain barrier. This in vitro blood–brain barrier model system was used in experiments to determine the permeability of the cultured monolayer to sucrose, leucine, and propranolol. Leucine rapidly moved across the monolayers of this in vitro system and tended to plateau after approximately 10 min. In contrast, the rates of sucrose and propranolol movement were linear during a 1-hr observation period, with the rate of propranolol movement across the monolayer greater than that of sucrose. The ability to detect differences in the permeability of the monolayers to leucine, propranolol, and sucrose with radioactive tracers suggests that this in vitro model system will be an important tool for the investigation of the role of the blood–brain barrier in the delivery of centrally acting drugs and nutrients.     

Blood–Brain Barrier Transport of Therapeutics via Receptor-Mediation

Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting.     

Coexistence of Passive and Proton Antiporter-Mediated Processes in Nicotine Transport at the Mouse Blood–Brain Barrier

Nicotine, the main tobacco alkaloid leading to smoking dependence, rapidly crosses the blood–brain barrier (BBB) to become concentrated in the brain. Recently, it has been shown that nicotine interacts with some organic cation transporters (OCT), but their influence at the BBB has not yet been assessed in vivo. In this study, we characterized the transport of nicotine at the mouse luminal BBB by in situ brain perfusion. Its influx was saturable and followed the Michaelis–Menten kinetics (K_m = 2.60 mM, V_max = 37.60 nmol/s/g at pH 7.40). At its usual micromolar concentrations in the plasma, most (79%) of the net transport of nicotine at the BBB was carrier-mediated, while passive diffusion accounted for 21%. Studies on knockout mice showed that the OCT Oct1–3, P-gp, and Bcrp did not alter [³H]-nicotine transport at the BBB. Neither did inhibiting the transporters Mate1, Octn, or Pmat. The in vivo manipulation of intracellular and/or extracellular pH, the chemical inhibition profile, and the trans-stimulation experiments demonstrated that the nicotine transporter at the BBB shared the properties of the clonidine/proton antiporter. The molecular features of this proton-coupled antiporter have not yet been identified, but it also transports diphenhydramine and tramadol and helps nicotine cross the BBB at a faster rate and to a greater extent. The pharmacological inhibition of this nicotine/proton antiporter could represent a new strategy to reduce nicotine uptake by the brain and thus help curb addiction to smoking.KEY WORDS: blood–brain barrier, nicotine, organic cation, proton antiporter, transporter     

Pharmacokinetic Consequences of Active Drug Efflux at the Blood–Brain Barrier

Purpose The objective of this simulation study was to investigate how the nature, location, and capacity of the efflux processes in relation to the permeability properties influence brain concentrations. Methods Reduced brain concentrations can be due to either influx hindrance, a gatekeeper function in the luminal membrane, which has been suggested for ABCB1 (P-glycoprotein), or efflux enhancement by transporters that pick up molecules on one side of the luminal or abluminal membrane and release them on the other side. Pharmacokinetic models including passive transport, influx hindrance, and efflux enhancement were built using the computer program MATLAB. The simulations were based on experimentally obtained parameters for morphine, morphine-3-glucuronide, morphine-6-glucuronide, and gabapentin. Results The influx hindrance process is the more effective for keeping brain concentrations low. Efflux enhancement decreases the half-life of the drug in the brain, whereas with influx hindrance the half-life is similar to that seen with passive transport. The relationship between the influx and efflux of the drug across the blood–brain barrier determines the steady-state ratio of brain to plasma concentrations of unbound drug, K_p,uu. Conclusions Both poorly and highly permeable drugs can reach the same steady-state ratio, although the time to reach steady state will differ. The volume of distribution of unbound drug in the brain does not influence K_p,uu, but does influence the total brain-to-blood ratio K_p and the time to reach steady state in the brain.     

Contribution of Carrier-Mediated Transport Systems to the Blood–Brain Barrier as a Supporting and Protecting Interface for the Brain; Importance for CNS Drug Discovery and Development

The blood–brain barrier (BBB) forms an interface between the circulating blood and the brain and possesses various carrier-mediated transport systems for small molecules to support and protect CNS function. For example, the blood-to-brain influx transport systems supply nutrients, such as glucose and amino acids. Consequently, xenobiotic drugs recognized by influx transporters are expected to have high permeability across the BBB. On the other hand, efflux transporters, including ATP-binding cassette transporters such as P-glycoprotein located at the luminal membrane of endothelial cells, function as clearance systems for metabolites and neurotoxic compounds produced in the brain. Drugs recognized by these transporters are expected to show low BBB permeability and low distribution to the brain. Despite recent progress, the transport mechanisms at the BBB have not been fully clarified yet, especially in humans. However, an understanding of the human BBB transport system is critical, because species differences mean that it can be difficult to extrapolate data obtained in experimental animals during drug development to humans. Recent progress in methodologies is allowing us to address this issue. Positron emission tomography can be used to evaluate the activity of human BBB transport systems in vivo. Proteomic studies may also provide important insights into human BBB function. Construction of a human BBB transporter atlas would be a most important advance from the viewpoint of CNS drug discovery and drug delivery to the brain.     

Human Insulin Receptor Monoclonal Antibody Undergoes High Affinity Binding to Human Brain Capillaries in Vitro and Rapid Transcytosis Through the Blood–Brain Barrier in Vivo in the Primate

Purpose. The ability of monoclonal antibodies against the human insulin receptor to undergo transcytosis through the blood-brain barrier (BBB) was examined in the present studies. Methods. Two murine monoclonal antibodies (MAb83-7 and MAb83-14) which bind different epitopes within the -subunit of the human insulin receptor were examined using isolated human brain capillaries, frozen sections of primate brain, and in vivo pharmacokinetic studies in anesthetized Rhesus monkeys. Results. Both antibodies strongly illuminated capillary endothelium in immunocytochemical analysis of frozen sections of brain from Rhesus monkey but not squirrel monkey. Both monoclonal antibodies, in the iodinated forms, bound to human brain microvessels, although the binding and endocytosis of MAb83-14 was approximately 10-fold greater than MAb83-7. The active binding of MAb83-14 to the human insulin receptor was paralleled by a very high rate of transport of this antibody through the BBB in vivo in two anesthetized Rhesus monkeys. The BBB permeability-surface area (PS) product in neocortical gray matter was 5.4 ± 0.6 µL/min/g, which is severalfold greater than previous estimates of the PS product for receptor-specific monoclonal antibody transport through the BBB. The brain delivery of MAb83-14 to the Rhesus monkey brain was high and 3.8 ± 0.4% of the injected dose was delivered to 100 g of brain at 3 hours after a single intravenous injection. In contrast, there was no brain uptake of the mouse IgG_2a isotype control antibody. Conclusions. These studies demonstrate an unexpected high degree of transcytosis of a monoclonal antibody through the primate BBB in vivo.     

Electro-Magnetic Nano-Particle Bound Beclin1 siRNA Crosses the Blood–Brain Barrier to Attenuate the Inflammatory Effects of HIV-1 Infection in Vitro

The purpose of this study was to evaluate a novel drug delivery system comprised of ferric-cobalt electro-magnetic nano-material (CoFe2O4@ BaTiO3; MENP) bound to siRNA targeting Beclin1 (MENP-siBeclin1) to cross the blood–brain barrier (BBB) and attenuate the neurotoxic effects of HIV-1 infection in the central nervous system following on-demand release of siRNA using an in vitro primary human BBB model. Beclin1 is a key protein in the regulation of the autophagy pathway and we have recently demonstrated the importance of Beclin1 in regulating viral replication and viral-induced inflammation in HIV-1-infected microglia. The MENP-siBeclin1 nano-formulation did not compromise the physiological function or integrity of the BBB model. Furthermore, the in vitro BBB data revealed that MENP-siBeclin1 could efficiently attenuate viral replication and viral-induced inflammation, likely due to STAT1/ NF-κB signaling pathways. MENP-siBeclin1 also silenced Beclin1 protein expression in HIV-1-infected microglial cells within the model system. In addition, the cytotoxic effects of direct treatment with siBeclin1 and MENP alone or in nano-formulation on primary human neuronal cells showed a minimal amount of cell death. Overall, the data shows that the nano-formulation can silence the BECN1 gene as an effective mechanism to attenuate HIV-1 replication and viral-induced inflammation in the context of the BBB.     

In Vivo Saturation of the Transport of Vinblastine and Colchicine by P-Glycoprotein at the Rat Blood–Brain Barrier

Purpose. To determine concentration-dependent P-gp-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in rats. Methods. The transport of radiolabeled colchicine and vinblastine across the rat BBB was measured with or without PSC833, a well known P-gp inhibitor, and within a wide range of colchicine and vinblastine concentration by an in situ brain perfusion. Thus, the difference of brain transport achieved with or without PSC833 gives the P-gp-mediated efflux component of the compound transported through the rat BBB. Cerebral vascular volume was determined by coperfusion with labeled sucrose in all experiments. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. P-gp limited the uptake of both colchicine and vinblastine, but the compounds differ in that vinblastine inhibited its own transport. Vinblastine transport was well fitted by a Hill equation giving IC₅₀ at 71 M, a Hill coefficient (n) 2, and a maximal efflux velocity J_max of 9 pmol s^–1 g^–1 of brain. Conclusions. P-gp at the rat BBB may carry out both capacity-limited and capacity-unlimited transport, depending on the substrate, with pharmacotoxicologic significance for drug brain disposition and risk of drug-drug interactions.