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.     

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.     

Mechanisms of Dendritic Cell Trafficking Across the Blood–brain Barrier

Although the central nervous system (CNS) is considered to be an immunoprivileged site, it is susceptible to a host of autoimmune as well as neuroinflammatory disorders owing to recruitment of immune cells across the blood–brain barrier into perivascular and parenchymal spaces. Dendritic cells (DCs), which are involved in both primary and secondary immune responses, are the most potent immune cells in terms of antigen uptake and processing as well as presentation to T cells. In light of the emerging importance of DC traficking into the CNS, these cells represent good candidates for targeted immunotherapy against various neuroinflammatory diseases. This review focuses on potential physiological events and receptor interactions between DCs and the microvascular endothelial cells of the brain as they transmigrate into the CNS during degeneration and injury. A clear understanding of the underlying mechanisms involved in DC migration may advance the development of new therapies that manipulate these mechanistic properties via pharmacologic intervention. Furthermore, therapeutic validation should be in concurrence with the molecular imaging techniques that can detect migration of these cells in vivo. Since the use of noninvasive methods to image migration of DCs into CNS has barely been explored, we highlighted potential molecular imaging techniques to achieve this goal. Overall, information provided will bring this important leukocyte population to the forefront as key players in the immune cascade in the light of the emerging contribution of DCs to CNS health and disease.     

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.     

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.     

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.     

Blood–brain Barrier Transport of Non-viral Gene and RNAi Therapeutics

The development of gene- and RNA interference (RNAi)-based therapeutics represents a challenge for the drug delivery field. The global brain distribution of DNA genes, as well as the targeting of specific regions of the brain, is even more complicated because conventional delivery systems, i.e. viruses, have poor diffusion in brain when injected in situ and do not cross the blood–brain barrier (BBB), which is only permeable to lipophilic molecules of less than 400 Da. Recent advances in the “Trojan Horse Liposome” (THL) technology applied to the transvascular non-viral gene therapy of brain disorders presents a promising solution to the DNA/RNAi delivery obstacle. The THL is comprised of immunoliposomes carrying either a gene for protein replacement or small hairpin RNA (shRNA) expression plasmids for RNAi effect, respectively. The THL is engineered with known lipids containing polyethyleneglycol (PEG), which stabilizes its structure in vivo in circulation. The tissue target specificity of THL is given by conjugation of ∼1% of the PEG residues to peptidomimetic monoclonal antibodies (MAb) that bind to specific endogenous receptors (i.e. insulin and transferrin receptors) located on both the BBB and the brain cellular membranes, respectively. These MAbs mediate (a) receptor-mediated transcytosis of the THL complex through the BBB, (b) endocytosis into brain cells and (c) transport to the brain cell nuclear compartment. The present review presents an overview of the THL technology and its current application to gene therapy and RNAi, including experimental models of Parkinson’s disease and brain tumors.     

Physiologically Based Pharmacokinetic Modelling of Drug Penetration Across the Blood–Brain Barrier—Towards a Mechanistic IVIVE-Based Approach

Predicting the penetration of drugs across the human blood–brain barrier (BBB) is a significant challenge during their development. A variety of in vitro systems representing the BBB have been described, but the optimal use of these data in terms of extrapolation to human unbound brain concentration profiles remains to be fully exploited. Physiologically based pharmacokinetic (PBPK) modelling of drug disposition in the central nervous system (CNS) currently consists of fitting preclinical in vivo data to compartmental models in order to estimate the permeability and efflux of drugs across the BBB. The increasingly popular approach of using in vitro–in vivo extrapolation (IVIVE) to generate PBPK model input parameters could provide a more mechanistic basis for the interspecies translation of preclinical models of the CNS. However, a major hurdle exists in verifying these predictions with observed data, since human brain concentrations can’t be directly measured. Therefore a combination of IVIVE-based and empirical modelling approaches based on preclinical data are currently required. In this review, we summarise the existing PBPK models of the CNS in the literature, and we evaluate the current opportunities and limitations of potential IVIVE strategies for PBPK modelling of BBB penetration.     

Specific Binding,Uptake, and Transport of ICAM-1-Targeted Nanocarriers Across Endothelial and Subendothelial Cell Components of the Blood–Brain Barrier

Purpose

The blood–brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models.

Methods

Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures.

Results

ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB.

Conclusions

CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.     

Hydrogen Bonding Potential as a Determinant of the in Vitro and in Situ Blood–Brain Barrier Permeability of Peptides

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood–brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., Adv. Drug Deliv. Rev. 7:365–386, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH₂, AcF₂NH₂, AcF₃NH₂, AcF₂(NMeF)NH₂, AcF(NMeF)₂NH₂, Ac(NMeF)₃NH₂, and Ac(NMeF)₃NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol–buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05). However, correlations were observed between the permeabilities of these peptides and the number of potential hydrogen bonds the peptides can make with water (r = 0.837 in vitro and r = 0.906 in situ; P < 0.05), suggesting that desolvation of the polar bonds in the molecule is a determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane–ethylene glycol (r = 0.981 in vitro and r = 0.940 in situ ; P < 0.05) or the differences in partition coefficients between octanol–buffer and isooctane–buffer (logPC) (r = 0.961 in vitro and r = 0.962 in situ; P < 0.05), both of which are experimental estimates of hydrogen bond or desolvation potential. These results suggest that the permeability of peptides through the BBB is governed by the same physicochemical parameter (hydrogen bonding potential) as their permeability through the intestinal mucosa.     

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.     

Shedding Light on the Blood–Brain Barrier Transport with Two-Photon Microscopy In Vivo

Pharmaceutical Research - Treatment of brain disorders relies on efficient delivery of therapeutics to the brain, which is hindered by the blood–brain barrier (BBB). The work of Prof....     

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     

Potential γ-Hydroxybutyric acid (GHB) Drug Interactions Through Blood–Brain Barrier Transport Inhibition: A Pharmacokinetic Simulation-Based Evaluation

Recreational abuse or overdose of γ-hydroxybutyric acid (GHB) results in dose-dependent central nervous system (CNS) effects including death. As GHB undergoes monocarboxylic acid transporter (MCT)-mediated transport across the blood–brain barrier (BBB), one possible strategy for the management of GHB toxicity/overdose involves inhibition of GHB BBB transport. To test this strategy, interactions between GHB and MCT substrates (salicylic acid or probenecid) were simulated. Competitive, noncompetitive and uncompetitive inhibition mechanisms were incorporated into the GHB–MCT substrate interaction model for inhibitor dosing either pre-, concurrent or post-GHB administration. Simulations suggested that salicylic acid was the better candidate to limit GHB accumulation in the CNS. A time window of effect (> 10% change) was observed for salicylic acid pre- and post-administration, with maximal transport inhibition occurring within 12 hr of pre- and 2 hr of post-administration. Consistent with the prediction that reduced GHB brain concentrations could translate to decreased pharmacodynamic effects, a pilot study in rats showed that the pronounced GHB sedative/hypnotic effects (24.0 ± 6.51 min; n = 4) in the control group (1.58 mmol/kg GHB plus saline) were significantly (p < 0.05) abrogated by salicylic acid (1.25 mmol/kg) coadministration.     

Pharmacokinetic-Pharmacodynamic Modelling of Morphine Transport Across the Blood-Brain Barrier as a Cause of the Antinociceptive Effect Delay in Rats—A Microdialysis Study

Purpose. To quantify the contribution of distributional processes across the blood-brain barrier (BBB) to the delay in antinociceptive effect of morphine in rats. Methods. Unbound morphine concentrations were monitored in venous blood and in brain extracellular fluid (ECF) using microdialysis (MD) and in arterial blood by regular sampling. Retrodialysis by drug was used for in vivo calibration of the MD probes. Morphine was infused (10 or 40 mg/kg) over 10 min intravenously. Nociception, measured by the electrical stimulation vocalisation method, and blood gas status were determined. Results. The half-life of unbound morphine in striatum was 44 min compared to 30 min in venous and arterial blood (p < 0.05). The BBB equilibration of morphine, expressed as the ratio of areas under the curve between striatum and venous blood, was less than unity (0.28 ± 0.09 and 0.22 ± 0.17 for 10 and 40 mg/kg), respectively, indicating active efflux of morphine across the BBB. The concentration-effect relationship exhibited a clear hysterisis with an effect delay half-life of 32 and 5 min based on arterial blood and brain ECF concentrations, respectively. Conclusions. Eighty five percent of the effect delay was caused by morphine transport across the BBB, indicating possible involvement of rate limiting mechanisms at the receptor level or distributional phenomena for the remaining effect delay of 5 min.     

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     

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.