Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position.

Drug interactions due to efflux transport inhibition at the blood-brain barrier (BBB) have been receiving increasing scrutiny because of the theoretical possibility of adverse central nervous system (CNS) effects identified in preclinical studies. In this review, evidence from pharmacokinetic, pharmacodynamic, imaging, pharmacogenetic, and pharmacovigilance studies, along with drug safety reports, is presented supporting a low probability of modulating transporters at the human BBB by currently marketed drugs.

Fatty acid uptake and incorporation in brain: studies with the perfusion model.

The contributions of individual components of blood to brain [14C]palmitate uptake and incorporation were studied with the in situ brain perfusion technique in the pentobarbital-anesthetized rat. With whole-blood perfusate, brain unacylated [14C]palmitate uptake was linear with time and extrapolated to zero at T = 0 s of perfusion. Tracer accumulated in brain with a blood-to-brain transfer coefficient of 1.8 +/- 0.1 x 10(-4) mL/s/g (whole cerebral hemisphere). Incorporation into brain lipids was rapid such that approximately 40% of tracer in brain at 45 s of perfusion was in cerebral phospholipids and neutral lipids. Similar rates of uptake were obtained during unacylated [14C]palmitate perfusion in whole rat plasma, serum, or artificial saline containing 2-3% albumin, suggesting that albumin has a key role in determining [14C]palmitate uptake in brain. The excellent match in brain uptake rates between whole blood and albumin-containing saline fluid suggests that the perfusion technique will be useful method for quantifying the individual contributions of blood constituents and albumin binding on brain [14C]palmitate uptake.

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique.

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.

Transport of glutamate and other amino acids at the blood-brain barrier.

In most regions of the brain, the uptake of glutamate and other anionic excitatory amino acids from the circulation is limited by the blood-brain barrier (BBB). In most animals, the BBB is formed by the brain vascular endothelium, which contains cells that are joined by multiple bands of tight junctions. These junctions effectively close off diffusion through intercellular pores; as a result, most solutes cross the BBB either by diffusing across the lipoid endothelial cell membranes or by being transported across by specific carriers. Glutamate transport at the BBB has been studied by both in vitro cell uptake assays and in vivo perfusion methods. The results demonstrate that at physiologic plasma concentrations, glutamate flux from plasma into brain is mediated by a high affinity transport system at the BBB. Efflux from brain back into plasma appears to be driven in large part by a sodium-dependent active transport system at the capillary abluminal membrane. Glutamate concentration in brain interstitial fluid is only a fraction of that of plasma and is maintained fairly independently of small fluctuations in plasma concentration. Restricted brain passage is also observed for several excitatory glutamate analogs, including domoic acid and kynurenic acid. In summary, the BBB is one component of a regulatory system that helps maintain brain interstitial fluid glutamate concentration independently of the circulation.

Cerebrovascular Biology and the various neural barriers: challenges and future directions.

DESPITE MAJOR ADVANCES in neuroscience, potential therapeutic options for the treatment of central nervous system diseases often cannot be optimized secondary to the presence of the blood-brain barrier (BBB). During the next decade of inquiry, it is crucial that basic science and clinical research that is focused on overcoming the BBB, to optimize delivery to the central nervous system, be identified and supported as a priority topic. For this reason, the third international Cerebrovascular Biology and Blood-Brain Barrier Conference was convened in March 1998 in Gleneden Beach, OR. This meeting brought together basic science and clinical researchers from around the world to analyze BBB function and to discuss delivery of effective agents to the central nervous system for treatment of brain disease. This report summarizes the information presented at the meeting and the discussions that ensued. The current state of knowledge, obstacles to further understanding the BBB, and research priorities are identified.

Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited.

The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1 -azi-2,2,2-trifluoroethyl)[2-3H]propyl]-1,3-bis(D-mannose-4-ylo xy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for D-[14C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarly, there was a 23+/-4% increase in total GLUT1/mg of microvessel protein and a 52+/-13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.

Blood-brain barrier choline transport in the senescent rat.

Choline is an important membrane phospholipid constituent and a neurotransmitter precursor that is minimally synthesized in brain. The long-term maintenance of brain choline concentration is dependent on uptake from plasma, which occurs via saturable transporter at the blood-brain barrier. Previous studies have suggested that brain choline uptake declined with age. To reevaluate this, brain choline uptake in 3, 12, 24, and 28-month-old Fischer-344 rats was evaluated using the in situ brain perfusion technique. Minimal differences were found with uptake parameters differing by approximately 10% between aged and adult rats for tracer levels while similar trends were observed at higher choline concentrations. Further, estimated Vmax and Km values differed by <30% between the groups. The results suggest that blood-brain barrier choline uptake changes minimally with aging in the rat.

Beta-amyloid induced increase in choline flux across PC12 cell membranes.

Beta-amyloid peptide is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease. It has been shown to be both neurotoxic and neurotrophic in vivo, and its effects have been suggested to be mediated in part by alterations in membrane transport. In the present study, we investigated the effect of beta-amyloid (1-40) on choline transport in cultured PC12 cells. We found that exposure to 46 or 92 microM beta-amyloid (1-40) increased [14C]choline flux in PC12 cells in a concentration-dependent manner, whereas exposure to reverse sequence beta-amyloid (40-1) had no effect. If there is a similar effect in vivo, the increased beta-amyloid dependent permeability to choline could lead to depletion of cellular choline stores and could contribute to the selective vulnerability of cholinergic neurons in Alzheimer's disease.

Facilitated brain uptake of 4-chlorokynurenine and conversion to 7-chlorokynurenic acid.

7-Chlorokynurenic acid (7-Cl-KYNA) and 5,7-dichlorokynurenic acid (5,7-Cl2-KYNA) are of therapeutic interest as potent glycine/N-methyl-D-aspartate NMDA) receptor antagonists, but are excluded from brain by the blood-brain barrier. We examined whether these compounds could be delivered to brain through their respective precursors, L-4-chlorokynurenine (4-Cl-KYN) and L-4,6-dichlorokynurenine (4,6-Cl2-KYN), which are amino acids. 4-Cl-KYN was shown to be rapidly shuttled into the brain by the large neutral amino acid transporter of the blood-brain barrier (K(m) = 105 +/- 14 microM, Vmax = 16.9 +/- 2.3 nmol min-1 g-1) and to be converted intracerebrally to 7-Cl-KYNA. 4,6-Cl2-KYN also expressed affinity for the transporter, but four-fold less than that of 4-Cl-KYN. In summary, the results show that because of their facilitated uptake 4-Cl-KYN and 4,6-Cl2KYN might be useful prodrugs for brain delivery of glycine-NMDA receptor antagonists.

Identification of two molecular species of rat brain phosphatidylcholine that rapidly incorporate and turn over arachidonic acid in vivo.

In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [3H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2-10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [3H] arachidonate (> 87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn-2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn-1 position. However, 10-15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn-2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn-1 position. Analysis demonstrated that tracer was present in both the 16:1-20:4 and 18:2-20:4 PtdCho species at specific activities 10-40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of < 10 min in 16:1-20:4 and 18:2-20:4 PtdCho and 1-3 h in 16:0-20:4, 18:1-20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1-20:4 and 18:2-20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.

Specific activity of brain palmitoyl-CoA pool provides rates of incorporation of palmitate in brain phospholipids in awake rats.

In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2-10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with > 50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6-1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by approximately 60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.

Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat.

Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [3H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1-10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly (t1/2 < 1 min) to steady-state values that averaged < 5% of plasma specific activity. Incorporation was rapid, as > 85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.

Rapid brain uptake of manganese(II) across the blood-brain barrier.

54Mn2+ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of approximately 6 x 10(-5) ml/s/g for brain and approximately 7 x 10(-3) ml/s/g for choroid plexus. Influx from physiologic saline was three- to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of 54Mn2+ in rat plasma was < or = 30%. Purified albumin, transferrin, and alpha 2-macroglobulin were each found to bind 54Mn2+ significantly and to restrict brain 54Mn2+ influx. The results demonstrate that 54Mn2+ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn2+ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism.

Identification of the cationic amino acid transporter (System y+) of the rat blood-brain barrier.

Cationic amino acids are transported from blood into brain by a saturable carrier at the blood-brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis-Menten kinetics with half-saturation constants (Km) of 50-100 microM and maximal transport rates of 22-26 nmol/min/g for L-lysine, L-arginine, and L-ornithine. The kinetic properties matched that of System y+, the sodium-independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38-fold as compared with whole brain. The results indicate that System y+ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.

Drug delivery to brain and the role of carrier-mediated transport.

In summary, the results suggest that carrier-mediated transport can be used to augment the brain delivery of a wide variety of hydrophilic therapeutic drugs. A large number of carriers are now known to be present at the brain capillary endothelium, and in many instances these carriers have been shown to mediate the brain uptake of exogenous drugs. The findings with D,L-NAM demonstrate that brain delivery can be improved through design of selective, high affinity agents. Although NAM was developed for the large neutral amino acid carrier, high affinity drugs could be produced for other systems, as shown by the work of Schein et al. with nitrogen mustard monosaccharides and by the work of Deves and Krupka on choline derivatives. Lastly, the method may allow some selectivity of delivery because of differential expression of transport carriers between tissues and in various disease states.

Facilitated transport of the neurotoxin, beta-N-methylamino-L-alanine, across the blood-brain barrier.

beta-N-Methylamino-L-alanine (BMAA) is a neurotoxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood-brain barrier was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (Vmax) of 1.6 +/- 0.3 x 10(-3) mumol/s/g and a half-saturation constant (Km) of 2.9 +/- 0.7 mM based on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excess L-leucine, but not by L-lysine, L-glutamate, or methylaminoisobutyric acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier. L-BMAA competitively reduced brain influx of L-[14C]leucine, as expected for cross-inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid carrier of the blood-brain barrier and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age.

Rapid high-affinity transport of a chemotherapeutic amino acid across the blood-brain barrier.

The therapeutic efficacy of many anticancer drugs against intracerebral tumors is limited by poor uptake into the central nervous system. One way to enhance brain delivery is to design agents that are transported into the brain by the saturable nutrient carriers of the blood-brain barrier. In this paper, we describe a nitrogen mustard amino acid, DL-2-amino-7-bis[(2-chloroethyl)amino/bd-1,2,3,4-tetrahydro-2-napthoi c acid, that is taken up into brain with high affinity by the large neutral amino acid carrier of the blood-brain barrier. Brain transport of DL-2-amino-7-bis[(2-chloroethyl)aminol-1,2,3,4-tetrahydro-2-naphth oic acid in the rat was found to be rapid (cerebrovascular permeability-surface area product approximately 2 x 10(-2) ml/s/g), saturable and inhibitable by large neutral amino acids. Maximal influx rate (Vmax) and half-saturation (Km) constants equaled 0.26 nmol/min/g and 0.19 microM, respectively, in the parietal cortex. Regional brain uptake of acid exceeded that of the clinical analogue, melphalan, by greater than 20-fold. The results demonstrate that drug modification to produce high-affinity ligands for the cerebrovascular nutrient carriers is a viable means to enhance drug delivery to brain for the treatment of brain tumors and other central nervous system disorders.

Saturable transport of manganese(II) across blood-nerve barrier of rat peripheral nerve.

To determine whether the blood-nerve barrier of the rat peripheral nerve transports manganese(II) (Mn) by a saturable mechanism similar to that found at the blood-brain barrier, we measured the uptake of 54Mn from blood into desheathed sciatic nerve and into cerebral cortex of awake rats at different plasma concentrations of unlabeled Mn using an intravenous infusion technique. The unidirectional influx (Jin) of Mn into sciatic nerve was facilitated and saturable, when steady-state plasma Mn ranged from 4 to 4,312 ng/ml (0.073-78.4 microM), as was the unidirectional influx of Mn into the cerebral cortex. Michaelis-Menten constants (Km and Vmax) and the passive diffusion constant (Kd), determined by nonlinear least squares, were as follows: for the blood-nerve barrier (sciatic nerve) Km = 4.7 microM, Vmax = 0.56 x 10(-3) nmol.s-1.g wet wt-1, and Kd = 6.3 x 10(-6) ml.s-1.g wet wt-1; for the blood-brain barrier (cerebral cortex) Km = 1.0 microM, Vmax = 0.40 x 10(-3) nmol.s-1.g wet wt-1, and Kd = 0.3 x 10(-6) ml.s-1.g wet wt-1. The results demonstrate facilitated concentration-dependent mechanisms of transport of Mn at the blood-nerve and blood-brain barriers.