Pieter J. Gaillard

Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes

OBJECTIVE The aim was to establish a flexible, abundantly available, reproducible and functionally characterized in vitro model of the blood-brain barrier (BBB). METHODS In a first step, bovine brain capillaries and newborn rat astrocytes were isolated. Subsequently, a co-culture of primary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, with astrocytes on the bottom of the filter was established. The cell material was characterized on the basis of specific cell-type properties and (functional expression of) specific BBB properties. RESULTS BCEC displayed: (1) characteristic endothelial cell morphology; (2) expression of endothelial cell markers (i.e., CD51, CD62P, CD71 and cadherin 5); (3) marginal F-actin localization; (4) tight junction formation between the cells; (5) expression of gamma-glutamyl-transpeptidase (gamma-GTP); (6) expression of P-glycoprotein (Pgp); (7) functional transendothelial transferrin transport and uptake; (8) restriction of paracellular transport; and (9) high transendothelial electrical resistance (TEER). Astrocytes displayed characteristic astrocyte morphology and expressed glial fibrillary acidic protein (GFAP). Co-culture with astrocytes increased TEER and decreased paracellular transport. In addition, expression of the glucocorticoid receptor (GR) was demonstrated in the endothelial cells of the BBB, while no expression of the mineralocorticoid receptor (MR) was found. CONCLUSIONS A high quality and mass-production in vitro BBB model was established in which experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and pharmacodynamics) and pathophysiological (e.g., disease influence on BBB permeability) objectives can be reproducibly performed.

Drug Targeting to the Brain

The central nervous system (CNS) is a sanctuary site and is protected by various barriers. These regulate brain homeostasis and the transport of endogenous and exogenous compounds by controlling their selective and specific uptake, efflux, and metabolism in the brain. Unfortunately, potential drugs for the treatment of most brain diseases are therefore often not able to cross these barriers. As a result, various drug delivery and targeting strategies are currently being developed to enhance the transport and distribution of drugs into the brain. Here we discuss briefly the biology and physiology of the blood-brain barrier (BBB) and the blood-cerebro-spinal-fluid barrier (BCSFB), and, in more detail, the possibilities for delivering large-molecular-weight drugs by local and global delivery and by viral and receptor-mediated nonviral drug delivery to the (human) brain.

Enhanced brain delivery of liposomal methylprednisolone improved therapeutic efficacy in a model of neuroinflammation

Neuroinflammation contributes to a wide range of disorders of the central nervous system (CNS). Of the available anti-inflammatory drugs, only glucocorticoids have shown central efficacy in CNS-related disorders, such as multiple sclerosis (MS). However, their side effects are dose limiting. To optimally improve the therapeutic window of methylprednisolone, we enhanced its CNS delivery by using pegylated liposomes conjugated to the brain-targeting ligand glutathione. In healthy rats, plasma circulation and brain uptake were significantly increased after encapsulating methylprednisolone in glutathione pegylated (GSH-PEG) liposomes. Furthermore, the efficacy of GSH-PEG liposomal methylprednisolone was investigated in rats with acute experimental autoimmune encephalomyelitis (EAE), an animal model of MS; rats received treatment (10mg/kg; i.v. injection), before disease onset, at disease onset, or at the peak of disease. Free methylprednisolone and non-targeted pegylated (PEG) liposomal methylprednisolone served as control treatments. When treatment was initiated at disease onset, free methylprednisolone showed no effect, while GSH-PEG liposomal methylprednisolone significantly reduced the clinical signs to 42±6.4% of saline control. Moreover, treatment using GSH-PEG liposomes was significantly more effective compared to PEG liposomes. Our findings hold promise for MS treatment and warrant further investigations into this brain delivery system for the treatment of neuroinflammation.

Glutathione PEGylated liposomes: pharmacokinetics and delivery of cargo across the blood-brain barrier in rats.

Abstract Partly due to poor blood–brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated liposomes (G-Technology®) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p < 0.001). In the microdialysis study 4-fold higher (p < 0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.

Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease

Treatment of neurodegenerative disorders such as Alzheimers disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimers disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain.

Enhanced brain drug delivery: safely crossing the blood–brain barrier

The blood–brain barrier presents a significant hurdle in CNS drug development. Blood-to-brain delivery by effectively crossing this barrier allows therapeutics to reach a large area of the brain. Over the past decades several drug delivery technologies have been developed, some more successful than others, which we hold against 10 key development criteria. Adhering to these criteria will allow a more successful development of therapeutics for patients with devastating brain diseases.

Targeted delivery across the blood–brain barrier

The safest and most effective way of targeting drugs to the entire brain is via delivery systems directed at endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective in animal models including primates, but no clinical trials have been performed so far. This review focuses on the well-characterised transferrin and insulin receptor-targeted systems, as well as on the more recently described systems that use the low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor). The possibilities and limitations of these systems are compared and their future for human application is discussed.

Relationship between permeability status of the blood–brain barrier and in vitro permeability coefficient of a drug

OBJECTIVE The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood-brain barrier (BBB) during in vitro drug transport assays is essential for an accurate estimation of the permeability coefficient of a drug. METHODS An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug transport, sodium fluorescein (P(app,FLU), M(w) 376 Da) and FITC-labeled dextran (P(app,FD4), M(w) 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between P(app) and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). RESULTS P(app,FLU) and P(app,FD4) in control experiments varied from 1.6 up to 17.6 (10(-6)cm/s) and 0.3 up to 7. 3 (10(-6)cm/s), respectively; while for individual filters P(app, FLU) was 4 times higher than P(app,FD4) (R(2)=0.97). As long as TEER remained above 131.Omega cm(2) for FLU or 122.Omega cm(2) for FD4 during the transport assay, P(app) remained independent from the basal permeability of the in vitro BBB. Below these TEER values, P(app) increased exponentially. This nonlinear relationship between basal BBB permeability and P(app) was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (P(FLU) and P(FD4)) were estimated to be 2.2+/-0.1 and 0.48+/-0.03 (10(-6)cm/s), respectively. In the experimentally challenged experiments, a reliable indication for P(FLU) and P(FD4) could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. CONCLUSIONS The assessment of basal BBB permeability status during drug transport assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug transport assay are determined.

Astrocytes Increase the Functional Expression of P-Glycoprotein in an In Vitro Model of The Blood-Brain Barrier

AbstractPurpose. To investigate the influence of astrocytes on P-glycoprotein (Pgp) expression and intracellular accumulation of Pgp substrates, separate from their net transcellular transport across the blood-brain barrier (BBB). Methods. An in vitroBBB model was used, comprising of brain capillary endothelial cells (BCEC) monolayers or BCEC co-cultured with astrocytes. Results. BCEC+astrocyte co-cultures seemed to express a higher level of Pgp compared to BCEC monolayers. Inhibition of Pgp results in an increased intracellular accumulation of Pgp substrates in both BCEC monolayers and BCEC+astrocyte co-cultures, and increased the sensitivity for vinblastine mediated disruption of the in vitro BBB (called the vinblastine exclusion assay). BCEC monolayers were more sensitive to vinblastine mediated disruption compared to BCEC+astrocyte co-cultures. In the latter, but not in BCEC monolayers, an inhibitable polar transport of Pgp substrates was only found from the brain to the blood side of the filter. Conclusions. Astrocytes increase the functional expression of Pgp in our in vitroBBB model. These results also illustrate that an important role for Pgp on the BBB is to protect the barrier against intracellular accumulation of cytotoxic BBB disrupting compounds.

Strategies to Improve Drug Delivery Across the Blood-Brain Barrier

The blood-brain barrier (BBB), together with the blood-cerebrospinal-fluid barrier, protects and regulates the homeostasis of the brain. However, these barriers also limit the transport of small-molecule and, particularly, biopharmaceutical drugs such as proteins, genes and interference RNA to the brain, thereby limiting the treatment of many brain diseases. As a result, various drug delivery and targeting strategies are currently being developed to enhance the transport and distribution of drugs into the brain. In this review, we discuss briefly the biology and physiology of the BBB as the most important barrier for drug transport to the brain and, in more detail, the possibilities for delivering large-molecule drugs, particularly genes, by receptor-mediated nonviral drug delivery to the (human) brain. In addition, the systemic and intracellular pharmacokinetics of nonviral gene delivery, together with targeted brain imaging, are reviewed briefly.