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 microliters/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the post-capillary 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.

These studies demonstrate an unexpected high degree of transcytosis of a monoclonal antibody through the primate BBB in vivo.

IgG molecules are potential neuropharmaceuticals that may be used for therapeutic or diagnostic purposes. However, IgG molecules are excluded from entering brain, owing to a lack of transport of these plasma proteins through the brain capillary wall, or blood-brain barrier (BBB). The Cationization of bovine immunoglobulin (Cohn fraction II) was performed as described for bovine albumin (9, 18). The isoelectric point (pI) of the native and cationized IgG was determined by polyacrylamide slab gel isoelectric focusing (IEF) by methods described previously (19). Samples (10 Al) containing 2 ug of either native or cationized IgG and 2% Nonidet P40 and 2% Pharmalyte 3-10 were applied to the middle of the gel. IEF was performed at 30 W, at a maximum voltage of 3000 V, and was performed for a duration of 4000 V-hr at 10°C. The IEF gels were fixed with 10% trichloroacetic acid (TCA) and 5% sulfosalicylic acid at room temperature for 60 min, washed in 30%o methanol/10%6 acetic acid for 30 min at 37°C and then overnight at room temperature, and the gels were stained with 0.2% Coomassie blue R-250 in 30% methanol/10%o acetic acid at room temperature for 60 min. The gels were destained with 30% methanol/10% acetic acid overnight and then photographed.To 4761The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue 18-amyloid peptide A81-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo.Transport of 125I-labeled A31-40 (125I-A31B-40) Alzheimer disease (AD) is a severe neurodegenerative disorder, and presently there is no premortem diagnostic test for this disease (1). The dementia of AD correlates with the deposition in brain of amyloid (2), which is principally composed of the 42-to 43-amino acid (3-amyloid peptide, A1(3'42/43 (3,4). One possible diagnostic approach to AD is the development of a premortem brain scan that would allow for semiquantitation of the AP3 amyloid burden in human brain. Previous studies have shown that 125I-labeled A(31-40 (125I-A3'-40) binds to preexisting amyloid plaques in frozen sections of AD brain (5). The delivery to brain of radiolabeled A,f1-40 in conjunction with the use of standard neuroimaging modalities such as single photon emission computed tomography (SPECT) may allow for quantitation of AB3 amyloid in AD brain. Therefore, the present studies examine whether 125I-A3l-40 undergoes significant transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, by using both intravenous/pharmacokinetic and internal carotid artery perfusion techniques. These studies then measure the extent to which BBB transport of 125I-AI3-40 is enhanced with the use of a vector-mediated drug delivery system (6). The latter is composed of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody (mAb) to the transferrin receptor (7), which undergoes receptor-mediated transcytosis through the BBB (6); 125I-A31-40 is monobiotinylated (bio), and 125I,bio-Af140 is then conjugated to the SA-OX26 vector. Since conjugation of 125I-Af3'-40 to the vector-mediated delivery system may inhibit binding of A1'-40 to amyloid plaques, the present studies also examine the saturable binding of 125I-A31-40 to amyloid in AD brain after conjugation to the delivery system. Conjugate Synthesis. The OX26 was conjugated to SA by a thioether linkage as described previously (7,8 Abbreviations: Af31-40, 13-amyloid peptide residues 1-40; BBB, bloodbrain barrier; AD, Alzheimer disease; SA, streptavidin; mAb, monoclonal antibody; 0X26, murine mAb to the rat transferrin receptor; NHS, N-hydroxysuccinimide; SPECT, single photon emission computed tomography; bio, biotinylated; VD, brain volume of distribution; PS, permeability-surface area; AUC, area under the plasma concentration curve; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; %ID, percent of the injected dose. MATERIALS AND METHODS

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.