Carrier-mediated transport of the quaternary ammonium neuronal nicotinic receptor antagonist n,n'-dodecylbispicolinium dibromide at the blood-brain barrier

The quaternary ammonium compound N,N'-dodecyl-bispicolinium dibromide (bPiDDB) potently and selectively inhibits nicotinic receptors (nAChRs) mediating nicotine-evoked [(3)H]dopamine release and decreases nicotine self-administration, suggesting that this polar, charged molecule penetrates the blood-brain barrier (BBB). This report focuses on 1) BBB penetration of bPiDDB; 2) the mechanism of permeation; and 3) comparison of bPiDDB to the cations choline and N-octylnicotinium iodide (NONI), both of which are polar, charged molecules that undergo facilitated BBB transport. The BBB permeation of [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB was evaluated using in situ rat brain perfusion methods. Cerebrovascular permeability surface-area product (PS) values for [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB were comparable (1.33 +/- 0.1, 1.64 +/- 0.15, and 1.3 +/- 0.3 ml/s/g, respectively). To ascertain whether penetration was saturable, unlabeled substrate was added to the perfusion fluid. Unlabeled choline (500 microM) reduced the PS of [(3)H]choline to 0.15 +/- 0.06 microl/s/g (p < 0.01). Likewise, unlabeled bPiDDB (500 microM) reduced the PS of [(14)C]bPiDDB to 0.046 +/- 0.005 microl/s/g (p < 0.01), whereas unlabeled NONI reduced the PS for [(3)H]NONI by approximately 50% to 0.73 +/- 0.31 microl/s/g. The PS of [(14)C]bPiDDB was reduced (p < 0.05) in the presence of 500 microM choline, indicating that the BBB choline transporter may be responsible for the transport of bPiDDB into brain. Saturable kinetic parameters for [(14)C]bPiDDB were similar to those for [(3)H]choline. The current results suggest that bPiDDB uses the BBB choline transporter for approximately 90% of its permeation into brain, and they demonstrate the carrier-mediated BBB penetration of a novel bisquaternary ammonium nAChR antagonist.     

Insulin enhancement of opioid peptide transport across the blood-brain barrier and assessment of analgesic effect

Insulin crosses the blood-brain barrier (BBB) via receptor-mediated transcytosis and has been suggested to augment uptake of peripheral substances across the BBB. The delta-opioid receptor-selective peptide D-penicillamine(2,5) (DPDPE), a Met-enkephalin analog, produces analgesia via a central nervous system-derived effect. In vitro (K(cell), microl. min(-1). mg(-1)) and in situ (K(in), microl. min(-1). g(-1)) analyses of DPDPE transport (K(cell) = 0.56 +/- 0. 15; K(in) = 0.28 +/- 0.03) revealed significant (P <.01) increases in DPDPE uptake by the BBB with 10 microM insulin (K(cell) = 1.61 +/- 0.25; K(in) = 0.48 +/- 0.04). In vitro cellular uptake was significantly increased (P <.05) at 1 microM insulin, whereas no significant uptake was observed with CTAP (a somatostatin opioid peptide analog) or sucrose (a paracellular diffusionary marker). No significant change in uptake was seen with DPDPE, CTAP, or sucrose in the presence of holo-transferrin (0-100 microM), indicating that the effect of insulin on DPDPE was not a generalized effect of receptor endocytosis. Insulin did not affect P-glycoprotein efflux, a mechanism that has shown affinity for DPDPE. A similar uptake of DPDPE into the brain (64% increase) was seen with the in situ brain perfusion model. Analgesic assessment revealed a significant decline in DPDPE (i.v.)-induced analgesia with increasing concentrations of insulin (i.v., i.c.v., s.c.) in a dose-dependent manner. Thus, insulin significantly increases DPDPE uptake across the BBB by a specific mechanism. The analgesic effect seen with DPDPE and insulin coadministration was shown to decrease, indicating that insulin reduces the analgesic effect within the central nervous system rather than at the BBB.     

Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity

Doxorubicin delivery to the brain is often restricted because of the poor transport of this therapeutic molecule through the blood-brain barrier (BBB). To overcome this problem, we have recently developed a technology, Pep:trans, based on short natural-derived peptides that are able to cross efficiently the BBB without compromising its integrity. In this study, we have used the in situ mouse brain perfusion method to evaluate the brain uptake of free and vectorized doxorubicin. Doxorubicin was coupled covalently to small peptide vectors: L-SynB1 (18 amino acids), L-SynB3 (10 amino acids), and its enantio form D-SynB3. We first confirmed the very low brain uptake of free radiolabeled doxorubicin, which is most likely due to the efflux activity of the P-glycoprotein at the level of the BBB. Vectorization with either L-SynB1, L-SynB3, or D-SynB3 significantly increased the brain uptake of doxorubicin (about 30-fold). We also investigated the mechanism of transport of vectorized doxorubicin. We show that vectorized doxorubicin uses a saturable transport mechanism to cross the BBB. The effect of poly(L-lysine) and protamine, endocytosis inhibitors, on the transport across the brain was also investigated. Both inhibitors reduced the brain uptake of vectorized doxorubicin in a dose-dependent manner. These studies indicate that the transport of vectorized doxorubicin appears to occur via an adsorptive-mediated endocytosis.     

Transport of histone through the blood-brain barrier

The present studies were designed to determine if the endogenous cationic protein, e.g., histone, is capable of penetrating the blood-brain barrier (BBB) in vivo. Calf thymus histone was iodinated with [125I]iodine and was found to be taken up rapidly by isolated bovine brain capillaries used as an in vitro model system of the BBB via a time- and temperature-dependent mechanism. The binding was saturable and a Scatchard plot of the binding data was linear, yielding a KD = 15.2 +/- 2.8 microM and a maximal binding = 7.7 +/- 1.0 nmol/mg of protein. Other polycations such as protamine or polylysine markedly inhibited uptake of [125I] histone, but cationized albumin demonstrated minimal inhibition and cationized immunoglobulin caused no inhibition of bovine brain capillary uptake of [125I]histone. The in vivo brain VD of [125I] histone reached 159 +/- 70 microliters/g by 10 min of carotid arterial perfusion as compared to the 10-min VD for [3H]albumin, 17 +/- 7 microliter/g. Most of this uptake represented sequestration by the vasculature, but approximately 8% of the total histone taken up by brain was found to be transported unmetabolized (based on trichloroacetic acid precipitability of brain supernatant [( 125I]) into brain interstitium. These studies demonstrate that histone is transported through the BBB in vivo via absorptive-mediated transport. Thus, histone is an endogenous protein that is capable of transport through the BBB and may be a potential vector for pharmaceutical delivery through the BBB.     

Improved blood-brain barrier penetration and enhanced analgesia of an opioid peptide by glycosylation.

Neuropeptide pharmaceuticals have potential for the treatment of neurological disorders, but the blood-brain barrier (BBB) limits entry of peptides to the brain. Several strategies to improve brain delivery are currently under investigation, including glycosylation. In this study we investigated the effect of O-linked glycosylation on Ser(6) of a linear opioid peptide amide Tyr-D-Thr-Gly-Phe-Leu-Ser-NH(2) on metabolic stability, BBB transport, and analgesia. Peptide stability was studied in brain and serum from both rat and mouse by high-performance liquid chromatography. BBB transport properties were investigated by rat in situ perfusion. Tail-flick analgesia studies were performed on male ICR mice, injected i.v. with 100 microg of peptide ligand. Glycosylation of Ser(6) of the peptide led to a significant increase in enzymatic stability in both serum and brain. Glycosylation significantly increased the BBB permeability of the peptide from a value of 1.0 +/- 0.2 microl x min(-1) x g(-1) to 2.2 +/- 0.2 microl x min(-1) x g(-1) (p < 0.05), without significantly altering the initial volume of distribution. Analgesia studies showed that the glycosylated peptide gave a significantly improved analgesia after i.v. administration compared with nonglycosylated peptide. The improved analgesia profile shown by the glycosylated peptide is due in part to an improvement in bioavailability to the central nervous system. The bioavailability is increased by improving stability and transport into the brain.     

Characterization of blood-brain barrier permeability to PYY3-36 in the mouse

Peptide YY3-36 (PYY) has emerged as an important signal in the gut-brain axis, with peripherally administered PYY affecting feeding and brain function. For these effects to be direct, PYY would have to cross the blood-brain barrier (BBB). Here, we determined the permeability of the BBB to PYY radioactively labeled with 131I (I-PYY). Multiple-time regression analysis showed the unidirectional influx rate (Ki) from blood-to-brain for I-PYY to be 0.49 +/- 0.19 microl/g-min, a rate similar to that previously measured for leptin. Influx was not inhibited by 1 microg/mouse of unlabeled PYY, suggesting PYY crosses the BBB by transmembrane diffusion. About 0.176% of the i.v.-injected dose of I-PYY was taken up by brain, an amount similar to that for other peptides important in gut-brain communication. Capillary depletion showed that 69% of I-PYY crossed the BBB to enter the parenchymal space of the brain, and high-performance liquid chromatography demonstrated that the radioactivity in this space represented intact I-PYY. After intracerebroventricular injection, I-PYY crossed from brain to blood by the mechanism of bulk flow. We conclude that PYY crosses in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms. Passage across the BBB provides a mechanism by which blood-borne PYY can affect appetite and brain function.     

In vivo and in vitro evidence of blood-brain barrier transport of a novel cationic arginine-vasopressin fragment 4-9 analog.

The blood-brain barrier (BBB) transport and metabolism of a novel arginine-vasopressin fragment 4-9 [AVP(4-9), isoelectric point; (pI) = 9.2] analog, that is, cationic AVP(4-9) (C-AVP(4-9), PI = 9.8), were examined in vivo and in vitro. At 45 min after an i.v. administration to mice, the cerebrum-to-plasma concentration ratios of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) were 0.103 and 0.330 ml/g cerebrum, respectively, and the BBB permeation clearances were 1.47 x 10(-4) and 3.10 x 10(-4) ml/min/g cerebrum, respectively. In the in vitro study using mouse brain capillary endothelial cells immortalized by SV40 infection (MBEC4), the acid-resistant binding values of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) to MBEC4 at 120 min were 0.93 and 1.95 microliter/mg protein (as the cell/medium ratios), respectively. (35)S-labeled AVP(4-9) showed two-phase saturable acid-resistant binding, and its half-saturation constants (K(D)) were 3.8 nM (high affinity) and 45.7 microM (low affinity). (125)I-labeled C-AVP(4-9) showed single-phase saturable acid-resistant binding, with a K(D) value of 16.4 microM. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a metabolic inhibitor), and AVP(4-9), poly(L-lysine), and protamine (cationic substances), but not by poly(L-glutamic acid) (an anionic peptide) and the V(1) and V(2) vasopressin receptor antagonists. In addition, the conversion of C-AVP(4-9) to AVP(4-9) in the cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP(4-9) is transported through the BBB more effectively than AVP(4-9), via absorptive-mediated endocytosis, and that C-AVP(4-9) is converted to the neuroactive parent peptide, AVP(4-9), in the cerebrum.     

Drug targeting of a peptide radiopharmaceutical through the primate blood-brain barrier in vivo with a monoclonal antibody to the human insulin receptor.

Peptide radiopharmaceuticals are potential imaging agents for brain disorders, should these agents be enabled to undergo transport through the blood-brain barrier (BBB) in vivo. Radiolabeled Abeta1-40 images brain amyloid in tissue sections of Alzheimer's disease autopsy brain, but this peptide radiopharmaceutical cannot be used to image brain amyloid in vivo owing to negligible transport through the BBB. In these studies, 125I-Abeta1-40 was monobiotinylated (bio) and conjugated to a BBB drug delivery and brain targeting system comprised of a complex of the 83-14 monoclonal antibody (mAb) to the human insulin receptor, which is tagged with streptavidin (SA). A marked increase in rhesus monkey brain uptake of the 125I-bio-Abeta1-40 was observed after conjugation to the 8314-SA delivery system at 3 h after intravenous injection. In contrast, no measurable brain uptake of 125I-bio-Abeta1-40 was observed in the absence of a BBB drug delivery system. The peptide radiopharmaceutical was degraded in brain with export of the iodide radioactivity, and by 48 h after intravenous injection, 90% of the radioactivity was cleared from the brain. In conclusion, these studies describe a methodology for BBB drug delivery and brain targeting of peptide radiopharmaceuticals that could be used for imaging amyloid or other brain disorders.     

GHB (gamma-hydroxybutyrate) carrier-mediated transport across the blood-brain barrier

gamma-Hydroxybutyrate (sodium oxybate, GHB) is an approved therapeutic agent for cataplexy with narcolepsy. GHB is widely abused as an anabolic agent, euphoriant, and date rape drug. Recreational abuse or overdose of GHB (or its precursors gamma-butyrolactone or 1,4-butanediol) results in dose-dependent central nervous system (CNS) effects (respiratory depression, unconsciousness, coma, and death) as well as tolerance and withdrawal. An understanding of the CNS transport mechanisms of GHB may provide insight into overdose treatment approaches. The hypothesis that GHB undergoes carrier-mediated transport across the BBB was tested using a rat in situ brain perfusion technique. Various pharmacological agents were used to probe the pharmacological characteristics of the transporter. GHB exhibited carrier-mediated transport across the BBB consistent with a high-capacity, low-affinity transporter; averaged brain region parameters were V(max) = 709 +/- 214 nmol/min/g, K(m) = 11.0 +/- 3.56 mM, and CL(ns) = 0.019 +/- 0.003 cm(3)/min/g. Short-chain monocarboxylic acids (pyruvic, lactic, and beta-hydroxybutyric), medium-chain fatty acids (hexanoic and valproic), and organic anions (probenecid, benzoic, salicylic, and alpha-cyano-4-hydroxycinnamic acid) significantly inhibited GHB influx by 35 to 90%. Dicarboxylic acids (succinic and glutaric) and gamma-aminobutyric acid did not inhibit GHB BBB transport. Mutual inhibition was observed between GHB and benzoic acid, a well known substrate of the monocarboxylate transporter MCT1. These results are suggestive of GHB crossing the BBB via an MCT isoform. These novel findings of GHB BBB transport suggest potential therapeutic approaches in the treatment of GHB overdoses. We are currently conducting "proof-of-concept" studies involving the use of GHB brain transport inhibitors during GHB toxicity.     

Transport of vasopressin fragments across the blood-brain barrier: in vitro studies using monolayer cultures of bovine brain endothelial cells

A well established in vitro blood-brain barrier (BBB) model, consisting of bovine cerebrovascular endothelial monolayers from primary cultures, was used to study the transport profile of vasopressin and its fragments across the BBB and to assess the metabolic properties of the BBB for the behaviorally active vasopressin fragment arginine vasopressin (AVP)1-8 (desglycinamide-AVP). All vasopressin fragments crossed the in vitro BBB to a measurable extent. Endothelial permeabilities were (in 10(-3) cm/min): AVP1-6, 3.0 +/- 0.2; AVP1-7, 4.6 +/- 0.4; AVP1-8, 2.0 +/- 0.5 and AVP1-9, 2.4 +/- 0.4. A significant effect of molecular size on endothelial permeability was seen. Transport rate of AVP1-8, expressed as BBB-clearance, was not affected by luminal concentration change and proved to be symmetrical. These findings suggest that, in the concentration range studied, vasopressin-like peptides can cross the BBB mainly by paracellular transport and that no relevant carrier mediation is involved. AVP1-8 was metabolized slowly (half-life, 6.5 hr) by a 60 cm2 confluent monolayer to AVP1-7, which was not broken down further, suggesting that carboxypeptidases are responsible for AVP1-8 metabolism in the BBB.     

Delivery across the blood-brain barrier of antisense directed against amyloid beta: reversal of learning and memory deficits in mice overexpressing amyloid precursor protein

Amyloid beta protein (Abeta) may play a causal role in Alzheimer's disease. Previous work has shown that the learning and memory deficits that develop with aging in SAMP8 mice, a strain that overproduces Abeta, can be reversed with i.c.v. injections of an Abeta antisense phosphorothiolate oligonucleotide (Olg). Here, we showed that Olg radioactively labeled with (32)P (P-Olg) was transported intact across the blood-brain barrier (BBB) of mice by a saturable system, termed oligonucleotide transport system-1 (OTS-1). Multiple-time regression analysis found a blood-to-brain unidirectional influx rate for P-Olg of 1.4 +/- 0.39 microl/g-min and capillary depletion showed that P-Olg completely crossed the BBB to enter the parenchymal space of the brain. P-Olg was also shown to enter the cerebrospinal fluid. Transport was especially high into the hippocampus, with the percentage of the i.v. dose taken up by each gram of brain (0.865 +/- 0.115%) being about 1/100 of the i.c.v. dose. An i.v. dose of Olg 100 times that of the effective i.c.v. dose reversed the learning and memory deficits of aged SAMP8 mice. These studies show for the first time that phosphorothiolate oligonucleotides can be delivered to the brain in effective doses by intravenous administration.     

Absorptive-mediated endocytosis of a dynorphin-like analgesic peptide, E-2078 into the blood-brain barrier

The binding and internalization of a novel analog of dynorphin-like analgesic basic peptide, [125I]E-2078 (CH3-[125I] Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5), by isolated bovine brain capillaries were investigated. High-performance liquid chromatographic analysis showed that no significant metabolism of [125I] E-2078 occurred during incubation with brain capillaries for 30 min at 37 degrees C. The binding of [125I]E-2078 to brain capillaries increased with time and the steady-state cell-to-medium concentration ratio was 58.5 +/- 2.6 microliters/mg of protein. Approximately one-fourth of the [125I]E-2078 binding was resistant to acid wash, and showed significant dependence on temperature and medium osmolarity. The acid sensitive binding of [125I]E-2078, which presumably represents surface binding, was saturable and the Scatchard plot gave a maximal binding capacity Bmax = 147 +/- 29 pmol/mg of protein, and a half-saturation constant (KD) = 4.62 +/- 0.59 microM. Pretreatment of brain capillaries with phenylarsine oxide, an endocytosis inhibitor, completely suppressed the acid resistant binding of [125I]E-2078, but did not influence the surface binding of [125I]E-2078. The acid resistant binding of [125I] E-2078 was inhibited by poly-L-lysine and protamine, but not inhibited by insulin, transferrin, dynorphin (1-8), beta-neoendorphin, naloxone or poly-L-glutamate. Moreover, in vivo brain extraction of [125I]E-2078 in rats was 368 +/- 55% higher than that of [3H] sucrose and was significantly inhibited by 1 mM of unlabeled E-2078. These results demonstrate that E-2078 is internalized by brain capillaries via absorptive-mediated endocytosis, which is a polycation-sensitive pathway.     

Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo

The brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, expresses high concentrations of transferrin receptor, and recent studies show that an antitransferrin receptor monoclonal antibody may function as a BBB drug transport vector. The present report examines the pharmacokinetics of clearance of radiolabeled antitransferrin receptor monoclonal antibody from the bloodstream in rats in vivo, and also assesses the extent to which brain selectively extracts the antibody from the blood compared to other peripheral organs such as liver, kidney, myocardium, or lung. [125I]Mouse immunoglobulin G2a control antibody was cleared monoexponentially with a half-time of 9.8 +/- 2.3 h. The clearance of the [3H]OX-26 antitransferrin receptor antibody from blood was biexponential with half-times of 2.2 +/- 0.8 min (61 +/- 10% of clearance) and 3.9 +/- 0.2 h (39 +/- 4% of clearance). The OX-26 antibody was rapidly taken up by liver during the first 60 min after injection, but this uptake reached rapid saturation, and hepatic OX-26 content actually declined subsequent to the first hour after injection. In contrast, brain continuously extracted the OX-26 antibody from the bloodstream, and the brain volume of distribution of OX-26 reached a value 18-fold greater than the volume of distribution of the mouse immunoglobulin G2a at 5 h after injection. There was no specific uptake of the OX-26 by myocardium or lung, and minor uptake by kidney was observed that also reached saturation within the first 60 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2) 相似文献   

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis.     

Surface Characteristics of Nanoparticles Determine Their Intracellular Fate in and Processing by Human Blood–Brain Barrier Endothelial Cells In Vitro

A polarized layer of endothelial cells that comprises the blood–brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.     

Evaluation of effect of charge and lipid coating on ability of 60-nm nanoparticles to cross an in vitro model of the blood-brain barrier

A cell culture model of the blood-brain barrier (BBB) consisting of a coculture of bovine brain capillary endothelial cells and rat astrocytes has been used to examine the ability of 60-nm nanoparticles with different physicochemical characteristics to cross the BBB. Neutral, anionic, and cationic nanoparticles were made from crosslinked malto-dextrins derivatized or not (neutral) with phosphates (anionic), quaternary ammoniums (cationic) ligands. Then, these particles were coated or not with a lipid bilayer made of dipalmitoyl phosphatidyl choline and cholesterol. Lipid coating of ionically charged nanoparticles was able to increase BBB crossing 3- or 4-fold compared with uncoated particles, whereas coating of neutral particles did not significantly alter their permeation characteristics across the endothelial cell monolayer. Lipid-coated nanoparticles were nontoxic toward BBB integrity, and crossed the BBB by transcytosis without any degradation. Furthermore, a 27-fold increase in albumin transport was observed when albumin had previously been loaded in the cationic lipid-coated nanoparticles. The influence of red blood cells was studied; a marked inhibition of the transport was observed, probably due to strong interaction between nanoparticles and red blood cells.     

Transport of recombinant CD4 through the rat blood-brain barrier in vivo.

One class of potential acquired immunodeficiency syndrome therapeutics are derivatives of recombinant CD4 (rCD4). Therefore, the present investigations use in vivo techniques to measure the rate at which [3H]rCD4 is transported through the blood-brain barrier (BBB). In addition, the binding of labeled rCD4 to isolated human and bovine brain capillaries is measured. These studies show that [3H]CD4 is removed rapidly from the bloodstream with a half-time of 12.6 +/- 0.9 min. The volume of distribution (Vd) of the protein in brain increases with time and reaches a Vd that is 11.1 +/- 1.1-fold greater than the brain Vd of plasma marker, native rat serum albumin. In addition, [3H]rCD4 is extracted rapidly by the kidney and the ratio of rCD4 Vd to native rat serum albumin Vd in the rat kidney reaches 99 +/- 5 at 60 min after i.v. injection. rCD4 is shown to undergo transcytosis through the BBB using an internal carotid artery perfusion/capillary depletion method coupled with gel filtration fast protein liquid chromatography. In conclusion, these studies report the unexpected finding that rCD4 is transportable through the BBB. rCD4 is a cationic protein and the mechanism of rCD4 transport through the BBB may be analogous to the absorptive-mediated transcytosis of other polycationic proteins.     

Transport of Steroid Hormones through the Rat Blood-Brain Barrier: PRIMARY ROLE OF ALBUMIN-BOUND HORMONE

These studies were undertaken to investigate (a) the permeability properties of the blood-brain barrier (BBB) to the major gonadal and adrenal steroid hormones, and (b) the role of the binding proteins of plasma (albumin and specific globulins) in the regulation of BBB steroid hormone transport.The permeability of the BBB to [(3)H]-labeled progesterone, testosterone, estradiol, corticosterone, aldosterone, and cortisol, was measured relative to [(14)C]butanol, a freely diffusable reference, in the barbiturate anesthetized rat using a tissue sampling-single injection technique. The isotopes were rapidly injected in a 200-mul bolus of Ringer's solution (0.1 g/dl albumin) via the common carotid artery and the percent extraction of unidirectional influx of hormone was determined after a single pass through brain: progesterone, 83+/-4%; testosterone, 85+/-1%; estradiol, 83+/-3%; corticosterone, 39+/-2%; aldosterone, 3.5+/-0.8%; and cortisol, 1.4+/-0.3%. The selective permeability of the BBB was inversely related to the number of hydrogen bonds each steroid formed in aqueous solution and directly related to the respective 1-octanol/Ringer's partition coefficient.When the bolus injection was 67% human serum, >95% of the labeled steroid was bound as determined by equilibrium dialysis. However, the influx of the steroids through the BBB was inhibited by human serum to a much less extent than would be expected if only the free (dialyzable) hormone was transported; progesterone, estradiol, testosterone, and corticosterone transport was inhibited 18, 47, 70, and 85% respectively, or in proportion to the steroid binding to plasma globulins. Rat serum (67%) only inhibited the transport of these four hormones, 0, 13, 12, and 69%, respectively, reflecting the absence of a sex hormone-binding globulin in rat plasma. However, neonatal rat serum (67%) inhibited progesterone, testosterone, and estradiol transport 0, 0, and 91%, respectively, consistent with the presence of an estradiol-binding protein in neonatal rat serum.The binding of steroid hormone to bovine albumin in vitro (as determined by equilibrium dialysis) was compared to albumin binding in vivo (as determined by the single injection technique). The ratio of apparent dissociation constant in vivo, K(D)(app), to the in vitro K(D) was: >200 for progesterone, >200 for testosterone, 120 for estradiol, and 7.7 for corticosterone. Assuming the steady-state condition, the K(D)(app)/K(D) was found to be proportional to the BBB permeability for each steroid.These data demonstrate (a) the selective permeability properties of the BBB to the major steroid hormones is proportional to the tendency of the steroid to partition in a polar lipid phase and is inversely related to the number of hydrogen bond-forming functional groups on the steroid nucleus; (b) the presence of albumin in serum may bind considerable quantities of steroid hormone, but exerts little inhibitory effects on the transport of steroids into brain, whereas globulin-bound hormone does not appear to be transported into brain to a significant extent. Therefore, the hormone fraction in plasma that is available for transport into brain is not restricted to the free (dialyzable) fraction, but includes the larger albumin-bound moiety.     

Pharmacokinetic analysis of the blood-brain barrier transport of 125I-amyloid beta protein 40 in wild-type and Alzheimer's disease transgenic mice (APP,PS1) and its implications for amyloid plaque formation

Amyloid plaques are formed in the extracellular space of Alzheimer's disease (AD) brain due to the accumulation of amyloid beta (Abeta) proteins such as Abeta40. The relationship between Abeta40 pharmacokinetics and its accumulation within and clearance from the brain in both wild-type (WT) and AD transgenic mice (APP,PS1) was studied to understand the mechanism of amyloid plaque formation and the potential use of Abeta40 as a probe to target and detect amyloid plaques. In both WT and APP,PS1 mice, the (125)I-Abeta40 tracer exhibited biexponential disposition in plasma with very short first and second phase half-lives. The (125)I-Abeta40 was significantly metabolized in the liver kidney > spleen. Coadministration of exogenous Abeta40 inhibited the plasma clearance and the uptake of (125)I-Abeta40 at the blood-brain barrier (BBB) in WT animals but did not affect its elimination from the brain. The (125)I-Abeta40 was shown to be metabolized within and effluxed from the brain parenchyma. The rate of efflux from APP,PS1 brain slices was substantially lower compared with WT brain slices. Since the Abeta40 receptor at the BBB can be easily saturated, the blood-to-brain transport of Abeta40 is less likely to be a primary contributor to the amyloid plaque formation in APP,PS1 mice. The decreased elimination of Abeta40 from the brain is most likely responsible for the amyloid plaque formation in the brain of APP,PS1 mice. Furthermore, inadequate targeting of Abeta40 to amyloid plaques, despite its high BBB permeability, is due to the saturability of Abeta40 transporter at the BBB and its metabolism and efflux from the brain.