Jody L. Buciak

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.

Human Insulin Receptor Monoclonal Antibody Undergoes High Affinity Binding to Human Brain Capillaries in Vitro and Rapid Transcytosis Through the Blood–Brain Barrier in Vivo in the Primate

AbstractPurpose. The ability of monoclonal antibodies against the human insulin receptor to undergo transcytosis through the blood-brain barrier (BBB) was examined in the present studies. Methods. Two murine monoclonal antibodies (MAb83-7 and MAb83-14) which bind different epitopes within the α-subunit of the human insulin receptor were examined using isolated human brain capillaries, frozen sections of primate brain, and in vivo pharmacokinetic studies in anesthetized Rhesus monkeys. Results. Both antibodies strongly illuminated capillary endothelium in immunocytochemical analysis of frozen sections of brain from Rhesus monkey but not squirrel monkey. Both monoclonal antibodies, in the iodinated forms, bound to human brain microvessels, although the binding and endocytosis of MAb83-14 was approximately 10-fold greater than MAb83-7. The active binding of MAb83-14 to the human insulin receptor was paralleled by a very high rate of transport of this antibody through the BBB in vivo in two anesthetized Rhesus monkeys. The BBB permeability-surface area (PS) product in neocortical gray matter was 5.4 ± 0.6 µL/min/g, which is severalfold greater than previous estimates of the PS product for receptor-specific monoclonal antibody transport through the BBB. The brain delivery of MAb83-14 to the Rhesus monkey brain was high and 3.8 ± 0.4% of the injected dose was delivered to 100 g of brain at 3 hours after a single intravenous injection. In contrast, there was no brain uptake of the mouse IgG2a isotype control antibody. Conclusions. These studies demonstrate an unexpected high degree of transcytosis of a monoclonal antibody through the primate BBB in vivo.

Transport of Human Recombinant Brain-Derived Neurotrophic Factor (BDNF) Through the Rat Blood−Brain Barrier in Vivo Using Vector-Mediated Peptide Drug Delivery

The blood–brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA–OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA–OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA–OX26 conjugate.

Protamine-mediated transport of albumin into brain and other organs of the rat. Binding and endocytosis of protamine-albumin complex by microvascular endothelium.

High doses of intravenous protamine cause generalized vascular permeability changes in brain and other organs, and concomitant hypoproteinemia. The present investigations test the hypothesis that protamine has a dual action of both binding serum proteins and of undergoing absorptive-mediated transcytosis through microvascular endothelial barriers. Binding of albumin to protamine was demonstrated using equilibrium dialysis, and protamine was shown to selectively augment the uptake of albumin, but not sucrose, in isolated bovine or human brain capillaries. In contrast, the anionic macromolecule, dextran sulfate, resulted in an increased capillary uptake of both albumin and sucrose in vitro. The selective effects of protamine on albumin transport were also documented in vivo using an external organ technique; the intravenous injection of 1.5 mg/kg protamine resulted in a marked and selective influx of albumin into brain, heart, kidney, lung, and liver, and the increased albumin transport exceeded the increased sucrose uptake in some organs by an order of magnitude. The transcytosis of protamine through the cerebral microvascular barrier was documented with an internal carotid artery perfusion technique. In summary, these studies provide evidence for protamine-mediated vectorial transport of albumin through microvascular barriers in brain and other organs.

Measurement of Free Intracellular and Transfer RNA Amino Acid Specific Activity and Protein Synthesis in Rat Brain in vivo

Brain protein synthesis was measured in anesthetized adult, male Sprague–Dawley rats by an in situ internal carotid arterial perfusion technique using [3H]leucine. The specific activity of free intracellular leucine and of tRNA leucine were determined by HPLC separation of phenylisothiocyanate (PITC) derivatives of amino acids. The specific activity of the leucyl-tRNA pool rapidly equilibrated with the free intracellular leucine pool within 2 min. The specific activity of the tRNA and free leucine pools in brain reached equilibrium by 10 min. Plasma amino acid specific activity, however, remained threefold higher than the specific activity of tRNA and free leucine pools. Estimates of protein synthesis were 0.62 ± 0.06 nmol/min/g and were constant between 10 and 30 min of perfusion. The in situ perfusion model for protein synthesis described is a controlled system suited to measurements of protein synthesis in brain that can be applied to the study of brain metabolism under changing physiological conditions.

Drug delivery of antisense oligonucleotides or peptides to tissues in vivo using an avidin–biotin system

AbstractThe pharmacologic efficacy of antisense oligonucleotide- or peptide-based therapeutics in intact organisms is limited by the poor transport of these highly charged molecules across cellular membranes. Since recent studies have shown that avidin, a cationic protein, is taken up by cells via absorptive-mediated endocytosis, the present studies examine whether the avidin-biotin system may be used to develop soluble transport vectors that mediate the organ uptake in vivo of monobiotinylated antisense oligonucleotides or peptides. The model antisense oligonucleotide used is a 21-mer complementary to the bovine GLUT1 glucose transporter mRNA, and the model peptide used is [desamino-Cys1, d-Lys8]lysine vasopressin (DDLVP). The antisense oligonucleotide was bound to avidin with a noncleavable biotin linker using N-hydroxysuccinimidobiotin, and the DDLVP was coupled to the avidin vector with a cleavable biotin linker using sulfosuccinimidyl - 2 - (biotinamido) ethyl -1, 3 - dithiopropionate. Pharmacokineti...

Differential expression of 53- and 45-kDa brain capillary-specific proteins by brain capillary endothelium and choroid plexus in vivo and by brain capillary endothelium in tissue culture.

Tissue-specific gene expression within the brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, may lead to the production of brain capillary-specific proteins (BSPs). BSPs were defined in the present study by immunocytochemistry and Western blotting using a rabbit polyclonal antiserum made against a plasma membrane fraction of isolated bovine brain microvessels. Following absorption of the antiserum with acetone powders of rat liver and rat kidney, the antiserum illuminated only microvessels in brain and did not stain vessels in heart, choroid plexus, urinary bladder, liver, or kidney. Among these organs, the only other structure stained by the antiserum was the basolateral membrane of the bovine choroid plexus epithelium. Western blotting analysis showed that the antiserum reacted with a triplet of brain capillary proteins of 200, 53, and 45 kDa molecular weight. Although the 200-kDa protein was detected on Western immunoblot analysis with bovine choroid plexus, the 53- and 45-kDa proteins were specific to brain microvasculature. Immunocytochemical electron microscopic studies showed that the antiserum stained the plasma membrane of primary cultures of bovine capillary endothelium, but Western blot studies showed preservation of only the 200-kDa protein in primary tissue culture. These studies identify 53- and 45-kDa blood-brain barrier-specific proteins and show that the expression of these proteins is dependent on trophic factors not present in primary tissue culture. The future structural analyses of these proteins may lead to the elucidation of the possible function served in the regulation of BBB transport physiology.

Measurement of amyloid peptide precursor of Alzheimer disease in human blood by double antibody immunoradiometric assay.

The dementia of Alzheimer disease (AD) correlates with the deposition of extracellular amyloid, and this amyloid arises from the abnormal processing of a high molecular weight amyloid peptide precursor (APP), which is a normal cellular protein that is found in both brain and in peripheral tissues in humans. Overproduction of the APP in AD could cause increased concentrations of this protein in either human blood or cerebrospinal fluid (CSF). However, thus far no direct demonstration of soluble APP in human blood has been possible, owing to poor assay sensitivity and interfering plasma proteins. These two problems were eliminated with the present development of an extracting two-site immunoradiometric assay (IRMA). Two rabbit polyclonal antisera were prepared reacting to two different sites (amino acids 161–180 and 597–624) of the APP molecule. The near N-terminal antiserum (anti-APP 161–180) was covalently coupled to a solid phase support and the near C-terminal directed antiserum (anti-APP 597–624) was indirectly labeled using 125labeled near C-terminal synthetic peptide corresponding to amino acids 597–624. The IRMA was validated by partial purification of the APP from human serum and demonstration of the proteins molecular weight (112 kDa) by Western immunoblot procedures. Results of the IRMA showed that the APP is present in human plasma (mean ± SE concentration=32 ± 6 pM, n=25), and there was no significant difference in the APP concentration in 25 controls, 19 patients with AD, and 10 individuals with Down syndrome (DS). Immunoreactive APP was generally not detectable in control or AD CSF volumes as large as 1 ml. In conclusion, these studies provide the first demonstration of the presence of APP in human blood that is immunoreactive with an antiserum directed against the amyloidotic portion (i.e., amino acids 597–624) of the APP molecule. In addition, these studies show that soluble APP is present in human blood, but that its concentration is not increased in AD.