William M. Pardridge

The blood-brain barrier: Bottleneck in brain drug development

SummaryThe blood-brain barrier (BBB) is formed by the brain capillary endothelium and excludes from the brain ∼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.

Drug transport across the blood–brain barrier

The blood–brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. This property arises from the epithelial-like tight junctions within the brain capillary endothelium. The BBB is anatomically and functionally distinct from the blood–cerebrospinal fluid barrier at the choroid plexus. Certain small molecule drugs may cross the BBB via lipid-mediated free diffusion, providing the drug has a molecular weight <400 Da and forms <8 hydrogen bonds. These chemical properties are lacking in the majority of small molecule drugs, and all large molecule drugs. Nevertheless, drugs can be reengineered for BBB transport, based on the knowledge of the endogenous transport systems within the BBB. Small molecule drugs can be synthesized that access carrier-mediated transport (CMT) systems within the BBB. Large molecule drugs can be reengineered with molecular Trojan horse delivery systems to access receptor-mediated transport (RMT) systems within the BBB. Peptide and antisense radiopharmaceuticals are made brain-penetrating with the combined use of RMT-based delivery systems and avidin–biotin technology. Knowledge on the endogenous CMT and RMT systems expressed at the BBB enable new solutions to the problem of BBB drug transport.

TRANSPORT OF METABOLIC SUBSTRATES THROUGH THE BLOOD-BRAIN BARRIER'

THE ENDOTHELIAL cells of cerebral capillaries, like epithelial cells, possess tight junctions between the plasmalemma of adjacent cells (BRIGHTMAN et al., 1970). As a consequence, the plasma membranes of brain endothelial cells form a continuous membranous barrier between blood and brain interstitium. Similar to other cell membrane systems, the flux of circulating substances through the blood-brain barrier (BBB) occurs, via either (i) lipid mediation, or (ii) carrier mediation (OLDENDORF, 1976). During the past decade with the introduction of newer techniques such as tissue sampling, single-injection methodology (OLDENDORF, 1970), much quantitative information has been obtained regarding the carrier-mediated transport of metabolic substrates through the BBB. Plasma concentrations, transport K,, and V,,, estimates for substrates transported by one of four independent carrier systems (hexose, neutral amino acid, basic amino acid and monocarboxylic acid) have been recently tabulated (PARDRIDGE et ul., 1975; OLDENWRF, 1976). In addition, four other independent carrier systems have recently been identified; these systems mediate the transport into brain of nucleosides, purines (CORNFORD & OLDENDORF, 1975), acidic amino acids (OLDENDORF & SZABO, 1976), and choline (OLDENDORF & BRAUN, 1976). The 8 independent transport systems are listed in Table 1 relative to the V,,, estimate for each system. If it is assumed that each carrier moves through the membrane at a comparable rate, then the Vma,,,provides a measure of the redundancy of the transport system within the BBB. Generally, as barrier transport capacity (V,,,) decreases, the transport affinity increases, as represented by decreasing K , (Table 1). The non-saturable component of BBB transport of metabolic substrates varies over a 30-fold range (Table 1) and probably reflects transport via either very low affinity, high capacity systems, or via free diffusion.

Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins

Abstract: Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor‐mediated or absorptive‐mediated transport through the brain capillary wall, i.e., the blood‐brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood‐borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology‐based technique for comparing the relative rates of transport of several different blood‐borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood‐borne cationized albumin, cationized IgG, and acetylated low‐density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition, 3H‐labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20–30 μl/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the postcapillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood‐borne peptides and proteins across the BBB of anesthetized animals.

CNS Drug Design Based on Principles of Blood‐Brain Barrier Transport

Abstract: Lipid‐soluble small molecules with a molecular mass under a 400–600‐Da threshold are transported readily through the blood‐brain barrier in vivo owing to lipid‐mediated transport. However, other small molecules lacking these particular molecular properties, antisense drugs, and peptide‐based pharmaceuticals generally undergo negligible transport through the blood‐brain barrier in pharmacologically significant amounts. Therefore, if present day CNS drug discovery programs are to avoid termination caused by negligible blood‐brain barrier transport, it is important to merge CNS drug discovery and CNS drug delivery as early as possible in the overall CNS drug development process. Strategies for special formulation that enable drug transport through the blood‐brain barrier arise from knowledge of the molecular and cellular biology of blood‐brain barrier transport processes.

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.

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 Ringers 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/Ringers 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.

Kinetics of competitive inhibition of neutral amino acid transport across the blood-brain barrier.

The transport of tryptophan across the blood‐brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [14C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α‐methyl tyrosine) is studied with a tissue‐sampling, single injection technique in the barbiturate‐anesthetized rat. The equality of the K1 (determined from cross‐inhibition studies) and the Km (determined from auto‐inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent Km values are computed for the essential neutral amino acids from known data on amino acid transport Km and plasma concentrations. The apparent Km values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.

Drug and Gene Delivery to the Brain: The Vascular Route

Brain drug development of either small molecule or large molecule (recombinant proteins, gene medicines) neurotherapeutics has been limited, owing to the restrictive transport properties of the brain microvasculature, which forms the blood-brain barrier (BBB) in vivo. Widespread drug delivery to the brain, while not feasible via craniotomy and intracerebral injection, is possible if the drug is delivered to brain via the transvascular route through the BBB. Novel brain drug delivery and drug targeting strategies can be developed from an understanding of the molecular and cellular biology of the brain microvascular and BBB transport processes.

Human blood-brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels.

The peripheral production of leptin by adipose tissue and its putative effect as a signal of satiety in the central nervous system suggest that leptin gains access to the regions of the brain regulating energy balance by crossing the brain capillary endothelium, which constitutes the blood-brain barrier in vivo. The present experiments characterize the binding and internalization of mouse recombinant leptin in isolated human brain capillaries, an in vitro model of the human blood-brain barrier. Incubation of 125I-leptin with isolated human brain capillaries resulted in temperature-dependent binding: at 37 degrees C, approximately 65% of radiolabeled leptin was bound per milligram of capillary protein. Two-thirds of the bound radioactivity was resistant to removal by acid wash, demonstrating endocytosis of 125I-leptin into capillary cells. At 4 degrees C, binding to isolated capillaries was reduced to approximately 23%/mg of protein, the majority of which was acid wash resistant. Binding of 125I-leptin to brain capillary endothelial plasma membranes was saturable, described by a two-site binding model with a high-affinity dissociation constant of 5.1+/-2.8 nM and maximal binding capacity of 0.34+/-0.16 pmol/mg of membrane protein. Addition of porcine insulin or insulin-like growth factor at a final concentration of 100 nM had a negligible effect on leptin binding. These results provide evidence for a leptin receptor that mediates saturable, specific, temperature-dependent binding and endocytosis of leptin at the human blood-brain barrier.