Brain Capillary Endothelium Research Articles

Drug transporters are altered in brain, liver and kidney of rats with chronic epilepsy induced by lithium–pilocarpine

Objective: Pharmacoresistance is a common problem hindering treatment of epilepsy. A possible cause for this resistance is the overexpression of efflux drug transporters, e.g. P-glycoprotein (P-gp), which may decrease extracellular antiepileptic drugs levels in brains of intractable epilepsy patients. Another transmembrane drug transporter is the organic anion transporting polypeptide 2 (Oatp2), which is colocalized with P-gp in many polarized tissues. However, a role for Oatp2 in the development of multidrug resistance has not been established. In the present work, we investigated the expression of drug transporters Oatp2 and P-gp in brain, liver and kidney of rats with chronic epilepsy induced by lithium–pilocarpine.Methods: Chronic epilepsy was elicited after status epilepticus (SE) induced by lithium–pilocarpine in adult Wistar rats. Following SE, expression of Oatp2 and P-gp protein was detected by immunohistochemistry and Western blot analysis and messenger RNA (mRNA) using reverse transcription polymerase chain reaction (RT-PCR). Levels of the drug transporters were compared in brain, liver and kidney of chronic epileptic and control rats.Results: Both Oatp2 and P-gp were expressed in the brain, liver and kidney but predominantly in the brain. In the brain, Oatp2 and P-gp immunopositive cells were observed in brain capillary endothelium and choroid plexus epithelium. Compared with control rats, Oatp2 protein expression in brain was significantly decreased, while P-gp was obviously increased in chronic epileptic rats. P-gp mRNA was also significantly higher in brains of chronic epileptic rats, whereas the expression of Oatp2 did not change. Organic anion transporting polypeptide 2 and P-gp did not change at the mRNA or protein level with epilepsy in the liver or kidney.Conclusions: Organic anion transporting polypeptide 2 and P-gp colocalized in tissues that are important in drug absorption, metabolism and excretion. Seizures seem to induce the cerebral changes of P-gp and Oatp2. However, the changes of expression of two transporters exhibit the opposite trend. Organic anion transporting polypeptide 2 might play a different role than P-gp in intractable epilepsy. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the development of intractable epilepsy.

Polychlorinated Biphenyls Disrupt Blood–Brain Barrier Integrity and Promote Brain Metastasis Formation

BackgroundPolychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties in the brain. However, the effects of PCBs on the development of tumor metastases are not fully understood.ObjectiveWe evaluated the hypothesis that exposure to individual PCB congeners can facilitate the development of brain metastases in immunocompetent mice via the disruption of the integrity of the blood–brain barrier (BBB).MethodsC57/Bl6 mice were exposed to individual PCBs by oral gavage, and 48 hr later they were injected with luciferase-labeled K1735 M2 melanoma cells into the internal carotid artery. The development of metastatic nodules was monitored by bioluminescent imaging. In addition, we evaluated the functional permeability of the BBB by measuring permeability of sodium fluorescein across the brain microvessels. Expression and colocalization of tight junction (TJ) proteins were studied by Western blotting and immunofluorescence microscopy.ResultsOral administration of coplanar PCB126, mono-ortho-substituted PCB118, and non-coplanar PCB153 (each at 150 μmol/kg body weight) differentially altered expression of the TJ proteins claudin-5, occludin, and zonula occludens-1 in brain capillaries. These alterations were associated with increased permeability of the BBB. Most importantly, exposure to individual PCB congeners enhanced the rate of formation and progression of brain metastases of luciferase-tagged melanoma cells.ConclusionsOur results show for the first time that exposure to individual PCBs can facilitate the formation of bloodborne metastases via alterations of the integrity of the brain capillary endothelium.

Polycyclic cage structures as carrier molecules for neuroprotective non-steroidal anti-inflammatory drugs

The blood–brain barrier is formed by the brain capillary endothelium and plays the predominant role in controlling the passage of substances between the blood and the brain. Recent studies on polycyclic structures, i.e. pentacyclo[5.4.0.0 2,6.0 3,10.0 5,9]undecane and amantadine, indicated favourable distribution thereof to the brain and it was concluded that these polycyclic structures and their derivatives penetrate the blood–brain barrier readily. A series of novel polycyclic prodrugs incorporating the well known non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid and ibuprofen, were synthesised and screened for blood–brain barrier permeability and antioxidant activity. Increased levels of both NSAIDs were detected in the brain tissue of C57BL/6 mice after administration of the synthesised prodrugs, indicating favourable blood–brain barrier permeation. Results from a lipid peroxidation assay indicated that the ester and amide prodrugs significantly increased the ability of the drugs to attenuate lipid peroxidation. These novel prodrugs thus readily penetrate the blood–brain barrier and exhibit increased antioxidant activity when compared to the free NSAIDs.

Wnt/β-catenin signaling controls development of the blood–brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

ABCC1: a gateway for pharmacological compounds to the ischaemic brain

By preventing access of drugs to the CNS, the blood-brain barrier hampers developments in brain pharmacotherapy. Strong efforts are currently being made to identify drugs that accumulate more efficaciously in ischaemic brain tissue. We identified an ATP-binding cassette (ABC) transporter, ABCC1, which is expressed on the abluminal surface of the brain capillary endothelium and mildly downregulated in response to focal cerebral ischaemia, induced by intraluminal middle cerebral artery occlusion. In biodistribution studies we show that ABCC1 promotes the accumulation of known neuroprotective and neurotoxic compounds in the ischaemic and non-ischaemic brain, ABCC1 deactivation reducing tissue concentrations by up to two orders of magnitude. As such, ABCC1's expression and functionality in the brain differs from the liver, spleen and testis, where ABCC1 is strongly expressed on parenchymal cells, resulting -- in case of liver and testis -- in directed transport from the tissue into the blood. After focal cerebral ischaemia, ABCC1 deactivation abolished the efficacy of both neuroprotective and neurotoxic compounds. Our data indicate that ABCC1 acts as gateway for pharmacological compounds to the stroke brain. We suggest that the tailoring of compounds binding to abluminal but not luminal ABC transporters may facilitate stroke pharmacotherapy.

Effects of acute exposure to aluminum on blood–brain barrier and the protection of zinc

Aluminum and zinc are two important trace elements in an organism. Although several studies have demonstrated their impacts on the intelligence, very little was known about their effects on the integrity of blood–brain barrier (BBB). To study the effects of aluminum and zinc on the permeability of BBB, different doses of aluminum and appropriate zinc were administered to rats. Evans blue was detected in brain to determine the permeability of BBB. The ultrastructure of BBB was observed under the transmission electron microscope. Immunohistochemistry and Western blot method were used to detect the expression of skeleton protein F-actin and tight junction protein occludin in brain capillary endothelium. The data indicated that compared with the control group, Evans blue in brains increased ( P < 0.01), the ultrastructure of BBB changed and the expression of F-actin and occludin decreased ( P < 0.01) in the aluminum-toxic group. Compared with the aluminum-toxic groups, the permeability of BBB to Evans blue decreased ( P < 0.01), the damage of the BBB ultrastructure was attenuated and the expression of F-actin and occludin increased ( P < 0.05) in the aluminum–zinc group. Our present studies suggest that aluminum increases the permeability of BBB by changing its ultrastructure and the expression of occludin and F-actin. Zinc can protect the integrity of BBB in juvenile rats that are exposed to aluminum and inhibit the decrease of tight junction protein occludin and F-actin expression in BBB.

PRECLINICAL STUDY: Disposition of Δ9 tetrahydrocannabinol in CF1 mice deficient in mdr1a P‐glycoprotein

P-glycoprotein (P-gp) plays a major role in drug efflux. All the transported substrates are more or less hydrophobic and amphiphatic in nature. Being lipophilic, Delta(9) tetrahydrocannabinol (THC), the main cannabis component, could be a potential P-gp substrate. The aim of this project was to determine the contribution of the mdr1a gene product to THC disposition. Therefore, oral THC and digoxin (substrate test for P-gp) pharmacokinetics have been investigated in the intestinal epithelium and in the brain capillary endothelium of CF1 mdr1a-/- mice (mice naturally deficient in P-gp). These pharmacokinetics were compared to THC and digoxin oral pharmacokinetics in wild type mice mdr1a+/+ (not P-gp deficient). The application of Bailer's method showed that THC total exposure measured by the area under the plasma concentration time curve was 2.17-fold higher in CF1 mice naturally deficient in P-gp than in wild type mice after oral administration of 25 mg/kg of THC, and 2.4-fold higher after oral administration of 33 microg/kg of digoxin. As a consequence, the oral bioavailability of THC and digoxin was higher in naturally P-gp-deficient mice. We concluded that P-gp limits THC oral uptake and mediates direct drug excretion from the systemic circulation into the intestinal lumen.

Differential expression of the multidrug resistance‐related proteins ABCb1 and ABCc1 between blood‐brain interfaces

Cerebral homeostasis results from the presence of the protective blood-brain and blood-cerebrospinal fluid barriers located respectively at the brain capillary endothelium and the choroid plexus epithelium. ABCb1 (Pgp) and ABCc1 (Mrp1) transporters are two major proteins of neuroprotection whose localization and functional significance at both barriers remain partly unsettled. We conducted a comparative analysis of their relative protein content between the two blood-brain interfaces. Microvessels and choroid plexuses located in the fourth and lateral ventricles were isolated from developing and adult rat brains, and whole homogenates were submitted to quantitative Western blot analysis by using standard curves generated from one of the samples. In adult, choroid plexus-associated Pgp content was less than 0.5% of the level in microvessels, whereas Mrp1 content in microvessels was 4% of that in the fourth ventricle choroid plexus. Pgp but not Mrp1 was enriched in microvessels over parenchyma. In choroid plexuses, Mrp1 displayed a basolateral epithelial localization, and reached its high adult protein level, early during postnatal development. In postnatal as in adult microvessels, Pgp localization appeared luminal. However, by contrast to Mrp1, the level of this transporter increased 4.6-fold between 9-day-old and adult animals. Western blot analysis of human samples confirmed the mirror image of Pgp and Mrp1 expression between the two barriers. We conclude that there are major differences in the mechanisms by which blood-brain interfaces fulfill their neuroprotective functions. The data also highlight the significance of the neuroprotective function of the choroid plexus during brain maturation, when the microvasculature is still developing.

A Unique Carrier for Delivery of Therapeutic Compounds beyond the Blood-Brain Barrier

BackgroundTherapeutic intervention in many neurological diseases is thwarted by the physical obstacle formed by the blood-brain barrier (BBB) that excludes most drugs from entering the brain from the blood. Thus, identifying efficacious modes of drug delivery to the brain remains a “holy grail” in molecular medicine and nanobiotechnology. Brain capillaries, that comprise the BBB, possess an endogenous receptor that ferries an iron-transport protein, termed p97 (melanotransferrin), across the BBB. Here, we explored the hypothesis that therapeutic drugs “piggybacked” as conjugates of p97 can be shuttled across the BBB for treatment of otherwise inoperable brain tumors.ApproachHuman p97 was covalently linked with the chemotherapeutic agents paclitaxel (PTAX) or adriamycin (ADR) and following intravenous injection, measured their penetration into brain tissue and other organs using radiolabeled and fluorescent derivatives of the drugs. In order to establish efficacy of the conjugates, we used nude mouse models to assess p97-drug conjugate activity towards glioma and mammary tumors growing subcutaneously compared to those growing intracranially.Principal FindingsBolus-injected p97-drug conjugates and unconjugated p97 traversed brain capillary endothelium within a few minutes and accumulated to 1–2% of the injected by 24 hours. Brain delivery with p97-drug conjugates was quantitatively 10 fold higher than with free drug controls. Furthermore, both free-ADR and p97-ADR conjugates equally inhibited the subcutaneous growth of gliomas growing outside the brain. Evocatively, only p97-ADR conjugates significantly prolonged the survival of animals bearing intracranial gliomas or mammary tumors when compared to similar cumulated doses of free-ADR.SignificanceThis study provides the initial proof of concept for p97 as a carrier capable of shuttling therapeutic levels of drugs from the blood to the brain for the treatment of neurological disorders, including classes of resident and metastatic brain tumors. It may be prudent, therefore, to consider implementation of this novel delivery platform in various clinical settings for therapeutic intervention in acute and chronic neurological diseases.

Localization of myosin II regulatory light chain in the cerebral vasculature

The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo.

Drug Targeting to the Brain

The goal of brain drug targeting technology is the delivery of therapeutics across the blood-brain barrier (BBB), including the human BBB. This is accomplished by re-engineering pharmaceuticals to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Certain endogenous peptides, such as insulin or transferrin, undergo receptor-mediated transport (RMT) across the BBB in vivo. In addition, peptidomimetic monoclonal antibodies (MAb) may also cross the BBB via RMT on the endogenous transporters. The MAb may be used as a molecular Trojan horse to ferry across the BBB large molecule pharmaceuticals, including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene medicines. Fusion proteins of the molecular Trojan horse and either neurotrophins or single chain Fv antibodies have been genetically engineered. The fusion proteins retain bi-functional properties, and both bind the BBB receptor, to trigger transport into brain, and bind the cognate receptor inside brain to induce the pharmacologic effect. Trojan horse liposome technology enables the brain targeting of non-viral plasmid DNA. Molecular Trojan horses may be formulated with fusion protein technology, avidin-biotin technology, or Trojan horse liposomes to target to brain virtually any large molecule pharmaceutical.

Abcg2/Bcrp1 Mediates the Polarized Transport of Antiretroviral Nucleosides Abacavir and Zidovudine

The bioavailability and targeted distribution of abacavir (ABC) and zidovudine (AZT) to viral reservoirs may be influenced by efflux transporters. The purpose of this study was to characterize the interaction of these nucleoside reverse transcriptase inhibitors with the Abcg2/Bcrp1 transporter, the murine homolog of human breast cancer resistance protein (BCRP), using a Bcrp1-transfected Madin-Darby canine kidney II cell model. Intracellular accumulation of ABC and AZT was significantly reduced by approximately 90% and approximately 70%, respectively, in Bcrp1-transfected cells compared with the wild-type cells. Both ABC and AZT showed significantly increased basolateral-to-apical (B-to-A) and decreased apical-to-basolateral (A-to-B) transport in Bcrp1 cells compared with wild-type directional flux. The efflux ratio (ratio of B-to-A to A-to-B) in Bcrp1-transfected cells was 22 for ABC and 11 for AZT. N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) inhibited this difference in accumulation between the two cell variants with an EC(50) of 1.32 +/- 0.3 microM for ABC and 0.31 +/- 0.1 microM for AZT. Potent and highly cooperative inhibition by Ko143 (3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester) was observed with an EC(50) of 121 +/- 5 nM for ABC and 19.2 +/- 1.5 nM for AZT (Hill coefficient approximately 3-6). Probenecid, an organic anion inhibitor known to influence AZT biodistribution, had no effect on cellular accumulation in the Bcrp1 model. These studies characterize the Bcrp1-mediated transport of ABC and AZT and show that prototypical BCRP inhibitors GF120918 and Ko143 can inhibit the Bcrp1-mediated transport of these important antiretroviral compounds. The functional expression of BCRP at critical barriers, such as the intestinal enterocytes, brain capillary endothelium, and target lymphocytes, could influence the bioavailability and targeted delivery of these drugs to sanctuary sites.

Vasculitis and rheumatologic diseases may play role in the pathogenesis of acute disseminated encephalomyelitis (ADEM)

Acute disseminated encephalomyelitis (ADEM) is defined as a multifocal, monophasic, demyelinating, and inflammatory disease involving the central nervous system. It typically begins within 6 weeks of an antigenic challenge such as infection or immunization. Perivenous inflammation, edema and demyelination are the pathological hallmarks of ADEM. Reactivity of T-cells against myelin components such as myelin basic protein has been found in children with ADEM. The triggers for immune responses in ADEM are not known, but the two most widely accepted hypotheses are molecular mimicry and self-sensitization secondary to CNS infection. Inflammatory cytokines including tumor necrosis factor alpha (TNFalpha), interleukin 2 (IL2) and interferon gamma (INFgamma) are thought to be important in lesion formation in ADEM. Due to the active role of inflammatory cytokines in the pathogenesis of ADEM, any disease contributing to systemic formation of inflammatory cytokines can potentially be an etiologic factor for the initiation of ADEM. In vasculitis and rheumatologic diseases the number of T-cells, T helper type 1 cytokines and other inflammatory cytokines such as TNFalpha increase substantially. We present this hypothesis that in such setting of inflammation, adhesion molecules are up-regulated on the brain capillary endothelium by cytokines and other inflammatory mediators, altering the permeability of the brain blood barrier and so allowing for inflammatory cell migration. The migratory cells attack the basic myelin protein and the final result is the demyelination seen in ADEM. So we propose that vasculitis and rheumatologic diseases may play role in the pathogenesis of ADEM.

Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle

Cationic bovine serum albumin (CBSA) conjugated poly(ethyleneglycol)-poly(lactide) (PEG-PLA) nanoparticle (CBSA-NP), was designed as a novel drug carrier for brain delivery. In this paper, three formulations of CBSA-NP with different surface CBSA density as well as native bovine serum albumin conjugated nanoparticle (BSA-NP) and CBSA unconjugated pegylated nanoparticle (NP), were formulated. Their brain transcytosis across the blood-brain barrier (BBB) coculture and brain delivery in mice were investigated using 6-coumarin as fluorescent probe. By using free CBSA as specific inhibitor, it was evidenced that CBSA-NP crossed the brain capillary endothelium through absorptive mediated transcytosis. The result of transcytosis across the BBB coculture and brain delivery in mice proved that the increase of surface CBSA density of the nanoparticle enhanced the BBB permeability-surface area but decreased blood AUC. The optimized CBSA number conjugated per averaged nanoparticle was 110, with the maleimide-PEG-PLA/methoxy-PEG-PLA weight ratio 1:10, which can acquire the greatest percentage of injected dose per gram brain (%ID/g brain) by 2.3-fold compared with NP. Besides, “accelerated blood clearance phenomenon” was found through evaluating blood clearance profile of CBSA-NP post-injection of single dose or over a period of successive high doses of CBSA-NP. Understanding these issues is important for the future development of CBSA-NP as a brain delivery carrier and for the attenuation of toxicity or immunological responses to the nanodevice following a consequence of nanomedication.

The influence of interleukin-1β on γ-glutamyl transpepidase activity in rat hippocampus

Brain infections as well as peripheral challenges to the immune system lead to an increased production of interleukin-1beta (IL-1beta), a cytokine involved in leukocyte-mediated breakdown of the blood-brain barrier. The effects of IL-1beta have been reported to depend on whether the route of administration is systemic or intracerebral. Using 50-day-old male rats, we compared the effects of IL-1beta on brain gamma-glutamyl transpeptidase (GGT; an enzymatic marker of brain capillary endothelium) at 2, 24 and 96 h after either an intravenous (i.v.) injection of 5 microg IL-1beta or an intracerebroventricular (i.c.v. - lateral ventricle) infusion of 50 ng IL-1beta. When the i.v. route was used, the GGT activity underwent small but significant changes; decreasing in the hippocampus 2 h after the i.v. injection, increasing 24 h later and returning to control levels at 96 h. No significant changes in the hippocampal GGT activity were observed at 2 and 24 h following the i.c.v. infusion. The GGT activity in the hypothalamus remained unchanged regardless of the route of IL-1beta administrations. Similar changes in GGT activity were revealed histochemically. The labeling was found mainly in the capillary bed, the changes being most evident in the hippocampal stratum radiatum and stratum lacunosum-moleculare. A transient increase in GGT activity at 24 h, together with a less sharp delineation of GGT-stained vessels, may reflect IL-1beta induced increased turnover of glutathione and/or oxidative stress, that may in turn, be related to altered permeability of the blood-brain barrier in some neurological and mental disorders, including schizophrenia.

Single domain antibodies as blood–brain barrier delivery vectors

Antibodies against various receptors that undergo transcytosis across brain capillary endothelium including transferrin, insulin growth factor and low-density lipoprotein receptors have been used as vectors to deliver drugs or therapeutic peptides into the brain. In this study, recently discovered llama single domain antibody FC5 that transmigrates across cerebral endothelial cells (CEC) in vitro and blood–brain barrier (BBB) in vivo was engineered to provide ‘linker’ groups for conjugation with large molecules. Horseradish peroxidase (HRP)-tagged IgG was chemically attached to FC5 through an engineered free cysteine (cysFC5) and HRP-IgG-cysFC5 construct was shown to transmigrate across in vitro BBB model. Transmigration of FC5 across CEC was enhanced by pentamerizing FC5. Pentameric FC5 also demonstrated increased binding to microdissected human, mouse and rat brain vessels.

Dynamics of CNS barriers: evolution, differentiation, and modulation.

(1) Three main barrier layers at the interface between blood and tissue protect the central nervous system (CNS): the endothelium of brain capillaries, and the epithelia of the choroid plexus (CP) and the arachnoid. The classical work on these barriers in situ until the 1970s laid the foundations for modern understanding. Techniques for brain endothelial cell isolation and culture pioneered by Ferenc Joó in the 1970s opened up new fields of examination, enabling study of mechanisms at the cellular and molecular level. (2) Astrocytic glial cells are closely associated with the brain endothelial barrier. During evolution the barrier appears to have shifted from the glial to the endothelial layer, in parallel with the increasing importance of the microvasculature and its regulation. Vestiges of the barrier potential of glia remain in the modern mammalian CNS. (3) Evolutionary evidence suggests that the advantage derived from ionic homeostasis around central synapses was the major selective pressure leading to refinement of CNS barrier systems. This is one element of the modern 'multitasking' barrier function. (4) While epithelia are constitutively able to form barriers at appropriate interfaces, the 'default' condition for endothelia is more leaky; inductive influences from associated cells especially astrocytes are important in generating the full blood-brain barrier (BBB) phenotype in brain capillaries. The underlying mechanisms are being elucidated at the molecular and genomics level. (5) The barrier layers of the nervous system can be modulated by a number of receptor-mediated processes, involving several signal transduction pathways, both calcium dependent and independent. Some agents acting as 'inducers' in the long term can act as 'modulators' in the short-term, with some overlap of signaling pathways. Modulating agents may be derived both from the blood and from cells associated with cerebral vessels. Less is known about the modulation of the CP. (6) The challenge for the next era of CNS barrier studies will be to apply new knowledge from proteomics and genomics to understanding the in vivo condition in physiology and pathology.

Modulation of p-Glycoprotein Transport Function at the Blood-Brain Barrier

The central nervous system (CNS) effects of many therapeutic drugs are blunted because of restricted entry into the brain. The basis for this poor permeability is the brain capillary endothelium, which comprises the blood-brain barrier. This tissue exhibits very low paracellular (tight-junctional) permeability and expresses potent, multispecific, drug export pumps. Together, these combine to limit use of pharmacotherapy to treat CNS disorders such as brain cancer and bacterial or viral infections. Of all the xenobiotic efflux pumps highly expressed in brain capillary endothelial cells, p-glycoprotein handles the largest fraction of commonly prescribed drugs and thus is an obvious target for manipulation. Here we review recent studies focused on understanding the mechanisms by which p-glycoprotein activity in the blood-brain barrier can be modulated. These include (i) direct inhibition by specific competitors, (ii) functional modulation, and (iii) transcriptional modulation. Each has the potential to specifically reduce p-glycoprotein function and thus selectively increase brain permeability of p-glycoprotein substrates.

Potential of Transferrin and Transferrin Conjugates of Liposomes in Drug Delivery and Targeting

Targeted drug delivery has gained recognition in modern therapeutics and attempts are being made to explore the potential of cell biology-related bioevents in the development of specific and target-oriented systems. In connection to modern therapeutic systems, most of the emphasis has been laid upon the bioconjugated drug delivery systems. Bioconjugates involve the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The nature of the linking agent between the pharmacologic agent and the delivery-augmenting moiety dictates the degree of successful delivery and its outcome. The component for the bioconjugated drug delivery includes receptors and ligands, where the receptors act as molecular targets or portals whereas ligands with receptors provide selective and specific trafficking towards the targeting site. Recently, a number of bioconjugated systems have been discovered for the site-specific presentation and delivery of various bioactive substances using biorelevant ligands, including antibodies, glycoprotein, viral proteins, and molecules of endogenous origin. In this review, the potential of transferrin (Tf) and Tf conjugates of liposomes in site-specific drug delivery systems are discussed. Tf is an abundant component of serum with the capacity to bind and transport iron, while Tf receptor (TfR), a dimeric transmembrane glycoprotein, is present on the surface of the most proliferating, higher eukaryotic cells. Tf expression is also found in nonproliferating tissues, such as hepatocytes, tissue macrophages, pituitary cells, pancreatic islet cells, and the endothelium of brain capillaries. Tumor cells frequently carry elevated numbers of TfRs compared with corresponding normal cells, and reduced serum levels of Tf are often observed in patients with tumors. In the past, various strategies have been developed, which include coupling of the liposomal surface with Tf by using various linking agents. Low-molecular weight drugs and proteins as well as liposomes can be linked with Tf. The Tf-coupled vesicular system is physicochemically stable in the bioenvironment and is site-specific. The aim of coupling liposomes with Tf is to improve the physical and biochemical stability of liposomes and make them appropriate for targeting specific organs and cells. Tf may be widely applied either as a carrier or targeting ligand in the active targeting of anticancer agents, proteins, and genes to primarily proliferating malignant cells that overexpress TfRs. Tf has been used as a molecular conjugate to deliver DNA to erythroleukemic, lung, and liver cell lines. Tf can also be modified with the positive charge N-acylurea groups to make them suitable for electrostatic binding of DNA, in order to achieve a well defined DNA-binding ligand for receptor-mediated gene transfer. Association of Tf with lipoplexes, in particular the negatively charged ternary complexes, significantly overcomes the inhibitory effect of serum and facilitates efficient transfection in many cell lines, including HeLa, K-562 cells, and lung carcinoma cells Calu-3 and H-292 cells. Tf-lipoplex has demonstrated high efficiency in tumor-targeted gene delivery and long-term therapeutic accuracy in systemic p53 gene therapy for both human head and neck cancer and prostate cancer. Tf and Tf-coupled liposomal drug delivery systems may prove particularly valuable to enable the use of a drug that seems to be ineffective or toxic if delivered systematically. The delivery of drugs to the brain has been particularly challenging because of the presence of the blood-brain barrier, which restricts the passage of most therapeutic agents into the brain. Therefore, active targeting of the brain is crucial for effective treatment of brain diseases. The anti-TfR antibody, such as OX26, when coupled with therapeutic agents, has shown potential in drug and gene delivery to the brain.

The Blood-Brain Barrier: Bottleneck in Brain Drug Development

The blood-brain barrier (BBB) is formed by the brain capillary endothelium and excludes from the brain approximately 100% of large-molecule neurotherapeutics and more than 98% of all small-molecule drugs. Despite the importance of the BBB to the neurotherapeutics mission, the BBB receives insufficient attention in either academic neuroscience or industry programs. The combination of so little effort in developing solutions to the BBB problem, and the minimal BBB transport of the majority of all potential CNS drugs, leads predictably to the present situation in neurotherapeutics, which is that there are few effective treatments for the majority of CNS disorders. This situation can be reversed by an accelerated effort to develop a knowledge base in the fundamental transport properties of the BBB, and the molecular and cellular biology of the brain capillary endothelium. This provides the platform for CNS drug delivery programs, which should be developed in parallel with traditional CNS drug discovery efforts in the molecular neurosciences.