Effect of endotoxin on expression of TNF receptors and transport of TNF-alpha at the blood-brain barrier of the rat.

The transport mechanism mediating brain uptake of tumor necrosis factor (TNF)-alpha has been studied. When (125)I-labeled rat TNF-alpha was used in internal carotid artery perfusions in rats, the cytokine showed transcytosis through the blood-brain barrier in intact form (permeability-surface area product 0.34 +/- 0.13 microl. min(-1). g(-1)). Uptake was inhibited by low nanomolar concentrations of unlabeled rat TNF-alpha. Human TNF-alpha, which does not interact with the p80 TNF receptor in rodents, showed no brain uptake. mRNA expression of both p60 and p80 receptors could be demonstrated in native brain microvessel preparations. These transcripts increased to 149% (p60) and 127% (p80) of control 4 h after a systemic immune stimulation (2 mg/kg bacterial endotoxin ip). Lipopolysaccharide treatment did not alter the rate of brain uptake of TNF-alpha measured between 4 and 24 h later. In conclusion, a receptor-mediated mechanism is responsible for the transcytosis of TNF-alpha. Saturable transport, requiring the p80 receptor, occurs at concentrations encountered under pathophysiological conditions and therefore constitutes a relevant mechanism of communication between the immune system and the brain.

Delivery of peptides and proteins through the blood-brain barrier.

Peptide and protein therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability of these drugs to the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. However, peptides or protein therapeutics may be delivered to the brain with the use of the chimeric peptide strategy for peptide drug delivery. Chimeric peptides are formed when a non-transportable peptide therapeutic is coupled to a BBB drug transport vector. Transport vectors are proteins such as cationized albumin, or the OX26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive-mediated and receptor-mediated transcytosis through the BBB, respectively. In addition to vector development, another important element of the chimeric peptide strategy is the design of strategies for coupling drugs to the vector that give high efficiency coupling and result in the liberation of biologically active peptides following cleavage of the bond linking the therapeutic and the transport vector. The avidin/biotin system has been recently shown to be advantageous in fulfilling these criteria for successful linker strategies. The use of the OX26 monoclonal antibody, the use of the avidin/biotin system as a linker strategy, and the design of a vasoactive intestinal peptide (VIP) analogue that is suitable for monobiotinylation and retention of biologic activity following cleavage, allowed for the recent demonstration of in vivo pharmacologic effects in brain following the systemic administration of relatively low doses (12 microg/kg) of neuropeptide.

The melanocortin melanocyte-stimulating hormone/adrenocorticotropin(4-10) decreases body fat in humans.

The control of body fat is a prominent factor in human health. Animal studies have indicated a homeostatic central nervous system regulation of body fat with particular involvement of the melanocortin receptor pathway. This study provides evidence for a similar role for melanocortins in the long-term control of fat stores in humans. Thirty-six normal weight humans were assigned to one of three experimental groups. After a 4-week baseline, one group was treated with MSH/ACTH(4-10) (MSH/ACTH(4-10)) representing the core sequence of all melanocortins. Another group received desacetyl-alphaMSH, a selective agonist of the brain melanocortin-4 receptor, which shares the 4-10 sequence with MSH/ACTH(4-10). The third group received placebo. Treatments were given intranasally twice daily for 6 weeks, at equimolar doses (MSH/ACTH(4-10), 0.5 mg; desacetyl-alphaMSH, 0.84 mg). Body weight, body composition, and plasma hormone concentrations were measured before and after treatment. MSH/ACTH(4-10) reduced body fat, on the average, by 1.68 kg (P < 0.05) and body weight by 0.79 kg (P < 0.001). Concurrently, plasma leptin levels were decreased by 24% (P < 0.02), and insulin levels were decreased by 20% (P< 0.05) after MSH/ACTH(4-10). Changes after desacetyl-alphaMSH remained nonsignificant. The finding of reduced body adiposity after MSH/ACTH(4-10) confirms and extends to the human the findings of animal models indicating an essential role of the hypothalamic melanocortin system in body weight control.

Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse.

Drug targeting through the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, may be achieved with peptidomimetic monoclonal antibodies that target peptide transcytosis systems on the BBB in vivo. Murine monoclonal antibodies to the rat transferrin receptor, such as the OX26 monoclonal antibody, are targeted through the BBB on the transferrin receptor in the rat. However, the present studies show the OX26 monoclonal antibody is not an effective brain delivery vector in mice. The emergence of transgenic mouse models creates a need for brain drug-targeting vectors for this species. Two rat monoclonal antibodies, 8D3 and RI7-217, to the mouse transferrin receptor were evaluated in the present studies. Both the RI7-217 and the 8D3 antibody had comparable permeability-surface area products at the mouse BBB in vivo. However, owing to a higher plasma area under the concentration curve, the mouse brain uptake of the 8D3 antibody was higher, 3.1 +/- 0.4% of injected dose [(ID)/g] compared with the brain uptake of the RI7 antibody, 1.6 +/- 0.2% ID/g, at 60 min after i.v. injection. Conversely, the mouse brain uptake of the OX26 antibody, which does not recognize the mouse transferrin receptor, was negligible, 0.06 +/- 0.01% ID/g. The RI7-127 antibody was more selective for brain because this antibody was not measureably taken up by liver. The capillary depletion technique demonstrated transcytosis of the RI7-217 antibody through the mouse BBB in vivo. The brain uptake of the 8D3 antibody was saturable, consistent with a receptor-mediated transport process. In conclusion, these studies indicate rat monoclonal antibodies to the mouse transferrin receptor may be used for brain drug-targeting studies in mice such as transgenic mouse models.

Stability of the disulfide bond in an avidin-biotin linked chimeric peptide during in vivo transcytosis through brain endothelial cells.

Drug delivery of potential neuropharmaceuticals with poor intrinsic permeability through the blood-brain barrier (BBB), such as peptides, is facilitated by coupling to a vector that undergoes receptor-mediated transcytosis through the endothelial cells of brain microvessels. When cleavable disulfide linkers are used in the synthesis of such "chimeric peptides", it is crucial that the S-S-bridge is stable during transcytosis. Cleavage within endothelial cells could result in sequestration of the drug moiety instead of passage through the BBB. In the present study the metabolically stable opioid peptide [3 H]DALDA ([3 H]Tyr-DArg-Phe-Lys-NH2 ) was used as a model drug. It was monobiotinylated with the cleavable biotin reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (NHS-SS-biotin) to obtain bio-[3 H]DALDA. The biotinylated peptide was then bound to a vector for brain delivery after intravenous injection in rats, a covalent conjugate of streptavidin and the transferrin receptor monoclonal antibody, OX26. Compared to peptide without vector, brain uptake of bio-[3 H]DALDA after was increased 18-fold to reach 0.12% of the injected dose per g tissue. Transcranial microdialysis was performed for 60 min after an intravenous bolus of chimeric peptide, followed by reverse phase HPLC of dialysate. Stability of the chimeric peptide during transport through the BBB into brain extracellular fluid was concluded from the absence of a peptide peak generated by disulfide cleavage.

Does short-term treatment with modafinil affect blood pressure in patients with obstructive sleep apnea?

OBJECTIVE: To investigate the effects of modafinil, a central nonamphetamine awakening substance, on blood pressure and heart rate in hypersomnolent patients with obstructive sleep apnea. DESIGN: This double-blind, randomized, placebo-controlled crossover trial was performed over 2 days and 3 nights in a single-center study of hospitalized patients from a referred care center. Twenty-six otherwise healthy men (age range, 30 to 60 years) with mild to moderate obstructive sleep apnea were recruited by the outpatient department of the Marburg University Sleep Laboratory. Patients were given 200 mg oral modafinil in the morning and 100 mg at midday. Placebo was given in the same manner in a crossover design. Mean arterial (radial) blood pressure was monitored continuously during nocturnal sleep and during a series of standardized daytime physical and psychologic performance tests. RESULTS: The difference in the main end point between the treatment with modafinil and placebo was 1.17+/-0.83 (mean +/- SE) mm Hg (95% confidence interval: -0.56 to 2.91 mm Hg). The maximal differences in blood pressure values occurred under loaded conditions (systolic blood pressure, ergometry: 5.62+/-1.13 mm Hg; mental stress test: 6.19+/-1.33 mm Hg). CONCLUSION: Short-term administration of modafinil did not elicit a significant response with regard to the main end point. However, cardiovascular effects during mental and physical load were observed. Longterm studies that include subjects with hypertension are necessary to investigate the clinical relevance of the cardiovascular effects of modafinil.

The O-demethylation of the antidementia drug galanthamine is catalysed by cytochrome P450 2D6.

Galanthamine proved effective in symptomatic treatment of senile dementia of Alzheimer's type. The aim of this study was to elucidate the metabolism of galanthamine. Two novel metabolites of galanthamine have been isolated from the urine of eight young men after single doses of 10-15 mg. Some 19.8% of the doses were excreted as O-demethylgalanthamine glucuronide, 5% as N-demethylgalanthamine, 25.1% as galanthamine, and 0.8% as epigalanthamine. After coadministration of quinidine hydrogen sulfate, which inhibits cytochrome P450 2D6 (CYP2D6) selectively, O-demethylgalanthamine glucuronide was highly diminished in urine. In vitro, human liver microsomes metabolized galanthamine to O-demethylgalanthamine with Vmax 5.2 nmol/mg protein/h and Km 187 microM. Ki of quinidine to inhibit O-demethylation was 28 nM. To inhibit cholinesterases, O-demethylgalanthamine was 10-fold more selective for acetylcholinesterase (AChE) versus butyrylcholinesterase (BuChE) than galanthamine. After glucuronidation, O-demethylgalanthamine failed to inhibit AChE and BuChE. N-Demethylgalanthamine inhibited cholinesterases less potently than galanthamine.

No increase in blood-brain barrier permeability after intraperitoneal injection of endotoxin in the rat.

Reactions mediated by the brain are part of the response to intraperitoneal administration of endotoxin, a model of gram-negative bacterial infection. To test the hypothesis that a compromised blood-brain barrier (BBB) may contribute to these reactions, the integrity of the BBB was measured following lipopolysaccharide administration. Rats received intraperitoneal injections of 50 microg/kg or 2 mg/kg of endotoxin. Brain uptake of a macromolecular vascular marker, 3H-labelled rat serum albumin, and of a poorly permeable low molecular weight substance, [14C]sucrose, was then measured with the intravenous bolus injection method. Compared to controls, neither dose of endotoxin affected the BBB permeability for these tracers. This was true when brain uptake was measured 5 min or 2 h after lipopolysaccharide injection. It is concluded that intraperitoneal endotoxin even at a high dose does not acutely disrupt the BBB.

Blood-brain barrier permeability to morphine-6-glucuronide is markedly reduced compared with morphine.

The blood-brain barrier (BBB) permeability to morphine and morphine-6-glucuronide (M6G) is measured under identical conditions using an intravenous injection method in the rat and HPLC separation of morphine from its metabolites. The brain uptake of M6G expressed as %ID/g was 32-fold lower than that of morphine, and the BBB permeability surface area product (PS) of M6G was 57-fold lower as compared with that of morphine. Consistent with these in vivo data, the 1-octanol/buffer partition study showed the liposolubility of M6G was 187-fold lower than that of morphine. The CNS origin of M6G analgesia after peripheral administration was confirmed because the analgesia was completely blocked by naloxone, which crosses BBB, but not by naloxone methiodide, which does not enter brain from blood. In conclusion, the BBB permeability to M6G is markedly reduced as compared with morphine, consistent with the much lower lipid solubility of M6G relative to morphine.

Brain microvascular and astrocyte localization of P-glycoprotein.

The hypothesis that P-glycoprotein plays a functional role at the brain capillary endothelium, which makes up the blood-brain barrier in vivo, is based largely on immunocytochemical studies showing immunoreactive P-glycoprotein localized to either isolated brain microvessels or microvessels within tissue sections. The present studies use the MRK16 monoclonal antibody to human P-glycoprotein to demonstrate that the pattern of immunolocalization of P-glycoprotein in microvessels of human or primate brain is similar to the pattern of immunolocalization of an astrocyte protein, glial fibrillary acidic protein. In contrast, the discontinuous staining pattern of MRK16 is not colocalized with the continuous immunostaining of the brain endothelial GLUT1 glucose transporter. The MRK16 antibody was radiolabeled with [125I]-iodine, and 125I-MRK16 avidly bound isolated human brain capillaries via a saturable mechanism. However, the 125I-MRK16 antibody was not taken up by primate brain capillaries in vivo following intravenous injection. In conclusion, these studies provide evidence that P-glycoprotein does not play a functional role at the luminal membrane of the brain capillary endothelium in vivo, and that a principal site of immunoreactive P-glycoprotein in brain microvasculature is localized to astrocyte foot processes.

Poor permeability of morphine 3-glucuronide and morphine 6-glucuronide through the blood-brain barrier in the rat.

To better understand the in vivo pharmacological effects of morphine 3-glucuronide (M3G, a weak opioid antagonist) and morphine 6-glucuronide (M6G, a potent opioid agonist), the permeability of the blood-brain barrier (BBB) for these metabolites was compared with morphine. Tracers were prepared by enzymatic glucuronidation of [N-methyl-3H]morphine. Brain uptake in rats was measured by the internal carotid perfusion technique and after intravenous bolus injections. In the perfusion experiments morphine showed a permeability-surface area product (PS) of 3.52 +/- 0.61 microliter min-1 g-1. Uptake seemed to be mediated by passive diffusion and was not saturable by 100 microM morphine in the perfusate. The BBB permeability of [3H]M3G and [3H]M6G was too low to be quantified after 5 min of perfusion. Brain uptake of [3H]M3G and [3H]M6G 60 min after i.v. bolus injection reached 0.0060 +/- 0.0003% and 0.0030 +/- 0.0005% injected dose per g, respectively. From these brain concentrations and the corresponding plasma concentration-time curves, BBB PS values of 0.14 +/- 0.02 microliter min-1 g-1 and 0.11 +/- 0.01 microliter min-1 g-1, respectively, were calculated. The ratio of BBB PS values is complementary to the analgesic potencies of morphine and M6G after different routes of administration. The low PS of M6G explains why it is approximately equipotent to morphine after systemic injection, although it is about 2 orders of magnitude more potent than morphine after administration directly into the central nervous system.

In vivo cleavability of a disulfide-based chimeric opioid peptide in rat brain.

Brain delivery of systemically administered neuropeptide drugs may be achieved by the synthesis of chimeric peptides, wherein the peptide is coupled to transport vectors via avidin-biotin technology. The present study focuses on factors that optimize the linkage of drugs to transport vectors. The vector is the OX26 monoclonal antibody to the transferrin receptor, and the model peptide used in these studies is [Lys7]dermorphin (K7DA). The K7DA is monobiotinylated at the epsilon-amino group of the Lys7 residue with either a cleavable linker, e.g., disulfide, using NHS-SS-biotin, or a noncleavable linker, e.g., amide, using NHS-XX-biotin. Disulfide cleavage of the biotinylated derivative yields the desbiotinylated peptide, which is thiolated. Structures of the K7DA analogues were confirmed by secondary ion mass spectrometry. The biotinylated peptides were coupled to a thiol-ether conjugate of the OX26 antibody and either neutral avidin (NLA) or streptavidin. The binding constants (Ki) of the K7DA, the biotinylated K7DA (bio-XX-K7DA), the desbiotinylated K7DA, and the bio-XX-KD7A conjugated to NLA-OX26 were 0.62 +/- 0.14, 1.59 +/- 0.27, 1.24 +/- 0.24, and > 10 nM, respectively, and were determined with a mu-opioid peptide radioreceptor assay. Comparable results were obtained with in vivo tail-flick analgesia testing following intracerebroventricular (icv) injection of opioid chimeric peptides. Reversibility of pharmacologic action of thiolated peptide was demonstrated by icv naloxone administration. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC and internal carotid artery perfusion of labeled opioid chimeric peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary endothelium.

To study transcytosis at the blood-brain barrier (BBB) in vivo, we used the internal carotid artery perfusion technique in rats. Brain uptake of the OX26 anti-transferrin receptor antibody (IgG2a) coupled to 5-nm colloidal gold (OX26-Au) was morphologically examined after infusion or perfusion into the carotid artery for 10 min. The brain tissue was subsequently perfusion-fixed with 2% glutaraldehyde. By light microscopy, silver enhancement of vibratome sections revealed staining of the vascular tree. Electron microscopy showed binding of gold particles at the luminal plasma membrane of brain capillary endothelia, endocytosis in vesicles (50-100 nm), and particles beyond the abluminal plasma membrane. Studies were performed with an IgG2a isotype control, UPC10. Internal carotid artery infusion of [125I]-UPC10 showed no evidence of brain uptake or binding to endothelium. However, microvessel structures were identified after silver enhancement of vibratome sections of brain following internal carotid artery infusion of 5-nm colloidal gold conjugates of UPC10 (UPC10-Au). The morphologically observed binding of UPC10-Au to brain microvessels may be induced by gold conjugation. These studies describe the use of gold conjugates of antibodies to delineate the subcellular pathway involved in transcytosis through the BBB.

Cationization of a monoclonal antibody to the human immunodeficiency virus REV protein enhances cellular uptake but does not impair antigen binding of the antibody.

Replication of the human immunodeficiency virus (HIV) within cells may be blocked by neutralization of viral-specific proteins that are absolutely required for growth of the virus. One such viral-specific protein is REV, and a monoclonal antibody (mAb) against the REV protein is a potential therapeutic for acquired immune deficiency syndrome (AIDS). However, in order to effect 'intracellular immunization', mAbs must be enabled to target the intracellular compartment. One strategy for transcellular drug delivery of mAb-based therapeutics is cationization, and the present studies describe the cationization of a murine mAb specific to the REV protein of HIV-1. The isoelectric point (pI) of the mAb was raised from 6.6 to more than 9.5. There was virtually no difference in binding to wild-type REV protein between the native or cationized anti-REV mAb, based on studies with a solid-phase immunoradiometric assay. The uptake of the [125I] native anti-REV mAb by human peripheral blood lymphocytes (PBLs) was negligible; however, there was a marked increase in both total cell binding and endocytosis by the human PBLs of the [125I] cationized anti-REV mAb. In conclusion, these studies show that an anti-REV mAb may be cationized to markedly increase endocytosis of the antibody and that this cationization reaction does not significantly alter the affinity of the antibody for its target protein. Cationized anti-REV mAbs may allow for intracellular immunization of the virus and are potential therapeutics for the treatment of HIV.

Blood-brain barrier transport of neuropeptides: analysis with a metabolically stable dermorphin analogue.

To avoid the confounding effect of metabolic degradation, the stable mu-opioid peptide agonist [D-Arg2,Lys4]-dermorphin analogue (DALDA) was used to quantitate blood-brain barrier (BBB) permeability by intravenous injection and internal carotid artery perfusion techniques. With intravenous injection, the BBB permeability-surface area products for [3H]DALDA (0.84 +/- 0.13 microliters.min-1.g-1) and [14C]sucrose (0.39 +/- 0.05 microliters.min-1.g-1) correlated with the lipid solubility of the two molecules: the 1-octanol-Ringer partition coefficient for DALDA was approximately 2 log orders greater than that for sucrose. The brain delivery of [3H]DALDA at 30 min after intravenous administration was 0.019 +/- 0.002% of the injected dose per gram, and analgesia was induced with a 5-mg/kg dose administered systemically. In contrast to the result after intravenous injection, the BBB permeability-surface area product for DALDA estimated with the internal carotid artery perfusion technique was manyfold greater. This was due to nonspecific absorption of the peptide into the cerebral microvasculature, which precluded use of the capillary depletion technique to study transcytosis through the BBB after internal carotid artery perfusion. The present studies show that the brain delivery of a metabolically stable peptide, such as DALDA, is comparable to that for sucrose, correlates with lipid solubility, and is mediated by a nonsaturable mechanism, probably free diffusion.

Development and in vitro characterization of a cationized monoclonal antibody against beta A4 protein: a potential probe for Alzheimer's disease.

The blood-brain barrier (BBB) is impermeable to IgG. Therefore, a delivery strategy has to be applied in order to use monoclonal antibodies (mAb) as diagnostic or therapeutic agents in the brain. It has been demonstrated that cationization of IgG allows for the BBB penetration following peripheral administration. A cationized mAb against beta A4-amyloid could be a sensitive and specific diagnostic tool for Alzheimer's disease (AD). The site-protected cationization and radiolabeling with 111In of the specific anti beta-amyloid mAb, AMY33, is described. The binding affinity of the antibody was retained after these procedures (Kd = 3.1 +/- 0.5 nM), as determined by solid-phase immunoradiometric assay and immunocytochemistry on AD brain sections. The in vitro binding by isolated brain capillaries indicated that the cationized antibody may be delivered to the brain in vivo. The ability of the modified antibody to detect cerebral beta-amyloid deposits in vivo can now be evaluated using single photon emission computed tomography (SPECT) and a suitable animal model for cerebral amyloidosis, such as non-human primates or aged canines.

Synthesis and bioactivity of monobiotinylated DALDA: a mu-specific opioid peptide designed for targeted brain delivery.

Delivery through the blood-brain barrier of opioid peptide-based therapeutic agents may be achieved with the use of conjugation of avidin and blood-brain barrier transport vectors. However, this drug delivery strategy requires that 1) the peptide is monobiotinylated and 2) the peptide is biologically active after cleavage of a disulfide linker and peptide release from the avidin-vector conjugate. Whether these criteria may be successfully fulfilled was examined in the present studies. The highly mu receptor-specific dermorphin analog, Tyr-D-Arg-Phe-Lys-NH2 (DALDA), was selectively monobiotinylated at the epsilon-NH2 group of Lys4 with the cleavable biotin linker, sulfosuccinimidyl-2-(biotinamidoethyl) 1,3'-dithioproprionate to obtain biotinylated DALDA (bio-DALDA). The N-terminal alpha-NH2 group of the peptide was protected during biotinylation with the N-9-fluorenylmethoxycarbonyl group. Cleavage of the disulfide bridge yielded the desbiotinylated derivative, desbio-DALDA. The identity of these peptides was verified by secondary ion mass spectrometry. In receptor binding assays with 3H-Tyr-D-Ala-Gly-Phe-(N-Me)-Gly-ol, the Kis of DALDA, bio-DALDA and desbio-DALDA for mu opioid receptors were determined to be 2.3 +/- 0.4, 6.5 +/- 1.1 and 4.0 +/- 0.9 nM, respectively. Binding of bio-DALDA to avidin resulted in a Ki of 14.5 +/- 2.4 nM. The i.c.v. administration of DALDA and desbio-DALDA induced potent and long-lasting analgesia in the rat tail-flick assay. It was found that 1 microgram of DALDA was equipotent to 3 micrograms of desbio-DALDA and 20 micrograms of morphine. The analgesic effect could be blocked by naloxone pretreatment. In conclusion, these studies 1) described methods for the preparation of a biologically active monobiotinylated mu opioid receptor-specific ligand and 2) demonstrated the advantages of using cleavable biotinylation of opioid peptides because the affinity of desbio-DALDA for the receptor approximated the affinity of DALDA and had a 3- to 4-fold higher affinity than did the bio-DALDA-avidin complex.

Pharmacokinetics and saturable blood-brain barrier transport of biotin bound to a conjugate of avidin and a monoclonal antibody to the transferrin receptor.

The delivery of biotinylated therapeutics through the blood-brain barrier (BBB) may be facilitated by the use of avidin-based chimeric peptide conjugates. The latter are formed by conjugating avidin to a BBB drug delivery vector, which is a protein that undergoes receptor-mediated transcytosis through the BBB. The murine OX26 monoclonal antibody to the rat transferrin receptor undergoes receptor-mediated transport through the BBB, and previous studies have shown that a [3H]biotin/avidin-OX26 conjugate is effectively transported through the BBB. However, avidin is a cationic protein, which causes a marked increase in the systemic clearance of avidin-based conjugates from the plasma compartment. The present studies describe attempts to elevate the reduced plasma area under the curve (AUC) of [3H]biotin/avidin-OX26 by preloading or coloading with unconjugated OX26 antibody or unconjugated avidin. Both systemic clearance and BBB transport of avidin-OX26 were equally affected by OX26 preloading or coloading; this had inverse effects on the plasma AUC and the BBB permeability surface area product with no resulting change in the fractional delivery of [3H]biotin to brain. Conversely, avidin coloading preferentially reduced brain clearance of the [3H]biotin/avidin-OX26 conjugate, without substantial alteration in the plasma AUC and greatly reduced the fractional delivery of [3H]biotin to brain. In summary, these studies show that the use of avidin-based vectors results in rapid systemic clearance, which causes a reduction in the delivery of [3H]biotin to brain, despite a comparable BBB permeability coefficient for either the unconjugated OX26 antibody or the avidin-OX26 conjugate.

Enhanced endocytosis and anti-human immunodeficiency virus type 1 activity of anti-rev antibodies after cationization.

Transmembrane transport and endocytosis of antibodies is facilitated by cationization, when the isoelectric point of the antibody is raised to the cationic range. The present studies describe the preparation of affinity-purified polyclonal antibodies directed against a 16-amino acid synthetic peptide corresponding to amino acids 35-50 of the 116-amino acid rev protein of human immunodeficiency virus type 1 (HIV-1). The concentration of cationized anti-rev35-50 antibody that results in 50% binding to the rev epitope, based on results with an immunoradiometric assay, also results in a statistically significant 37% inhibition of HIV-1 replication in human peripheral blood lymphocytes. The cationized antibodies caused no measurable toxicity to the cells, on the basis of [3H]thymidine incorporation. These studies demonstrate that cationization results in enhanced endocytosis of the antibody and enhanced inhibition of HIV-1 replication, consistent with intracellular immunization of the rev protein.