Targeted delivery of protein and gene medicines through the blood-brain barrier.

The development of biologic drugs (recombinant proteins, therapeutic antibodies, peptides, nucleic acid therapeutics) as new treatments of brain disorders has been difficult, and a major reason is the lack of transport through the blood-brain barrier (BBB) of these large molecule pharmaceuticals. Biologic drugs can be re-engineered as brain-penetrating neuropharmaceuticals using BBB molecular Trojan horse technology. Certain peptidomimetic monoclonal antibodies that target endogenous receptors on the BBB, such as the insulin or transferrin receptor, enable the re-engineering of biologic drugs that cross the BBB.

Brain drug targeting and gene technologies.

Brain drug targeting technology is based on the application of four gene technologies that enable the delivery of drugs or genes across the blood-brain barrier (BBB) in vivo. I) Genetic engineering is used to produce humanized monoclonal antibodies that target endogenous BBB transporters and act as vectors for delivery of drugs or genes to the human brain. The conjugate of a neurotherapeutic and a BBB transport vector is called a chimeric peptide. Epidermal growth factor chimeric peptides have been used for neuroimaging of brain cancer. Brain-derived neutrophic factor chimeric peptides have marked neuroprotective effects in brain stroke models. II) Imaging gene expression in the brain in vivo is possible with sequence-specific antisense radiopharmaceuticals, which are conjugated to BBB drug targeting vectors. III) Brain gene targeting technology enables widespread expression of an exogenous gene throughout the central nervous system following an intravenous injection of a non-viral therapeutic gene. IV) A BBB genomics program enables the future discovery of novel transport systems expressed at the BBB. These transporters may be carrier-mediated transport systems, active efflux transporters, or receptor-mediated transcytosis systems. The future discovery of novel BBB transport systems and the application of brain drug targeting technology will enable the delivery to the brain of virtually any neurotherapeutic, including small molecules, large molecules and gene medicines.

Pharmacokinetics and delivery of tat and tat-protein conjugates to tissues in vivo.

The membrane permeation in vivo of therapeutic proteins may be enhanced by conjugation of the protein to cationic import peptides, such as the tat protein of the human immune deficiency virus. The organ uptake, expressed as a percent of injected dose (ID) per gram of tissue, is a function of both membrane permeability and the area under the plasma concentration curve (AUC), which is a function of the plasma pharmacokinetics. The purpose of the present studies was to examine the effect of the tat peptide on the plasma AUC of a model exogenous protein, streptavidin, and to examine the extent to which changes in the plasma AUC influence organ uptake (%ID/g) of the protein. The cationic portion of the tat protein is comprised of a lysine/arginine-rich sequence, designated tat48-58. A biotin analogue of this cationic peptide, tat-biotin, was radioiodinated and injected intravenously into rats with or without conjugation to streptavidin. The unconjugated tat-biotin peptide was nearly instantaneously cleared from plasma by all tissues with a very high systemic clearance of 29 +/- 4 mL/min/kg and a high systemic volume of distribution of 4160 m+/- 450 mL/kg. The plasma clearance of the tat-biotin/streptavidin conjugate, 1.37 +/- 0.01 mL/min/kg, was reduced relative to the clearance of unconjugated tat peptide, but was higher than the plasma clearance of the unconjugated streptavidin, 0.058 +/- 0.005 mL/min/kg. Conjugation of cationic import peptides such as tat48-58 to higher molecular weight proteins results in a marked increase in the rate of removal of the protein from the circulation, which is reflected in the reduced plasma AUC. In summary, tat conjugation of a protein has opposing effects on membrane permeation and the plasma AUC. Therefore, the organ %ID/g is not increased in proportion to the increase in membrane permeation caused by tat conjugation of proteins.

Brain-specific expression of an exogenous gene after i.v. administration.

The treatment of brain diseases with gene therapy requires the gene to be expressed throughout the central nervous system, and this is possible by using gene targeting technology that delivers the gene across the blood-brain barrier after i.v. administration of a nonviral formulation of the gene. The plasmid DNA is targeted to brain with pegylated immunoliposomes (PILs) using a targeting ligand such as a peptidomimetic mAb, which binds to a transporting receptor on the blood-brain barrier. The present studies adapt the PIL gene targeting technology to the mouse by using the rat 8D3 mAb to the mouse transferrin receptor. Tissue-specific expression in brain and peripheral organs of different exogenous genes (beta-galactosidase, luciferase) is examined at 1-3 days after i.v. injection in adult mice of the exogenous gene packaged in the interior of 8D3-PIL. The expression plasmid is driven either by a broadly expressed promoter, simian virus 40, or by a brain-specific promoter taken from the 5' flanking sequence of the human glial fibrillary acidic protein (GFAP) gene. The transgene is expressed in both brain and peripheral tissues when the simian virus 40 promoter is used, but the expression of the exogenous gene is confined to the brain when the transgene is under the influence of the brain-specific GFAP promoter. Confocal microscopy colocalizes immunoreactive bacterial beta-galactosidase with immunoreactive GFAP in brain astrocytes. These studies indicate that tissue-specific gene expression in brain is possible after the i.v. administration of a nonviral vector with the combined use of gene targeting technology and tissue-specific gene promoters.

Receptor-mediated gene targeting to tissues in vivo following intravenous administration of pegylated immunoliposomes.

PURPOSE: Gene therapy has been limited by the immunogenicity of viral vectors, by the inefficiency of cationic liposomes, and by the rapid degradation in vivo following the injection of naked DNA. The present work describes a new approach that enables the non-invasive, non-viral gene therapy of the brain and peripheral organs following an intravenous injection. METHODS: The plasmid DNA encoding beta-galactosidase is packaged in the interior of neutral liposomes, which are stabilized for in vivo use by surface conjugation with polyethyleglycol (PEG). The tips of about 1% of the PEG strands are attached to a targeting monoclonal antibody (MAb), which acts as a "molecular Trojan Horse" to ferry the liposome carrying the gene across the biological barriers of the brain and other organs. The MAb targets the transferrin receptor, which is enriched at both the blood-brain barrier (BBB), and in peripheral tissues, such as liver and spleen. RESULTS: Expression of the exogenous gene in brain, liver, and spleen was demonstrated with beta-galactosidase histochemistry, which showed persistence of gene expression for at least 6 days after a single intravenous injection of the pegylated immunoliposomes. The persistence of the transgene was confirmed by Southern blot analysis. CONCLUSIONS: Widespread expression of an exogenous gene in brain and peripheral tissues is induced with a single intravenous administration of plasmid DNA packaged in the interior of pegylated immunoliposomes. The liposomes are formulated to target specific receptor systems that enable receptor-mediated endocytosis of the complex into cells in vivo. This approach allows for non-invasive, non-viral gene therapy of the brain.

Differential kinetics of transport of 2',3'-dideoxyinosine and adenosine via concentrative Na+ nucleoside transporter CNT2 cloned from rat blood-brain barrier.

The concentrative Na+ nucleoside transporter type 2 (CNT2), cloned from a rat blood-brain barrier cDNA library, yields very high flux ratios for purine nucleosides after expression in frog oocytes. This high activity of the rat CNT2 produced from the blood-brain barrier-derived cDNA, designated clone A-11, enabled a kinetic analysis of 2',3'-dideoxyinosine transport via the rat CNT2. CNT2 transported both adenosine and 2',3'-dideoxyinosine. The 2',3'-dideoxyinosine transport parameters included a Km of 29.2 +/- 8.3 microM, a V(max) of 0.40 +/- 0.11 pmol/oocyte/min, and a constant of nonsaturable transport (KD) of 15.7 +/- 0.6 nl/oocyte/min. The 2',3'-dideoxyinosine Vmax was 27-fold lower than the adenosine Vmax and the 2',3'-dideoxyinosine KD was >15-fold greater than the KD of adenosine transport. Adenosine inhibited both the saturable component of 2',3'-dideoxyinosine transport with a K(I) of 14.8 +/- 1.6 microM, and inhibited the nonsaturable component of 2',3'-dideoxyinosine transport. Both the saturable and nonsaturable components of 2',3'-dideoxyinosine transport were sodium-dependent with a sodium K0.5 of 8.7 +/- 0.9 mM, and a Hill coefficient of 1.00 +/- 0.10. The transport of 2',3'-dideoxyinosine was strongly inhibited by thymidine, whereas thymidine was a weak inhibitor of adenosine transport via rat CNT2. Thymidine was transported by rat CNT2 with a Km = 130 +/- 44 microM and a Vmax = 1.7 +/- 0.5 pmol/oocyte/min. These studies provide evidence for asymmetric transport sites on rat CNT2, where 2',3'-dideoxyinosine and thymidine compete selectively at a low Vmax site on the transporter, whereas adenosine is transported at a high Vmax site.

Cloned blood-brain barrier adenosine transporter is identical to the rat concentrative Na+ nucleoside cotransporter CNT2.

Adenosine transport into brain is regulated by the activity of the adenosine transporter located at the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo. To facilitate cloning of BBB adenosine transporters, poly A+ RNA was purified from isolated rat brain capillaries for production of a rat BBB cDNA library in the pSPORT vector. The cloned RNA (cRNA) generated from in vitro transcription of this library was injected into frog oocytes followed by measurement of [3H]-adenosine uptake. After dilutional cloning, a full-length, 2905-nucleotide adenosine transporter cDNA, designated clone A-11, was isolated. The A-11 clone yielded [3H]-adenosine flux ratios of 400 to 500 after injection of cRNA in oocytes. The adenosine uptake was sodium-dependent and insensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBTI). The Km and Vmax of adenosine transport in the cRNA-injected oocytes were 23.1 +/- 3.7 micromol/L and 10.8 +/- 0.9 pmol/oocyte. min. The K0.5 for sodium was 2.4 +/- 0.1 mmol/L, with a Hill coefficient (n) of 1.06 +/- 0.07. DNA sequence analysis indicated the rat BBB A-11 adenosine cDNA was identical to rat concentrative nucleoside transporter type 2 (CNT2). The adenosine transporter activity of the rat BBB A-11 CNT2 clone is 50-fold more active than previously reported rat CNT2 clones. In summary, these studies describe the expression cloning of CNT2 from a rat BBB library and show that the pattern of sodium dependency and NBTI insensitivity of the cloned CNT2 are identical to patterns of adenosine transport across the BBB in vivo. These results suggest that BBB adenosine transport in vivo is mediated by CNT2, which would make CNT2 one of the few known sodium-dependent cotransporters that mediate substrate transport across the BBB in the blood to brain direction.

Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system.

BACKGROUND AND PURPOSE: Neuroprotection with brain-derived neurotrophic factor (BDNF) requires direct injection into the brain owing to poor transport of the neurotrophin through the blood-brain barrier (BBB) in vivo. The present studies investigate whether BDNF alone or conjugated to a BBB drug targeting system is neuroprotective in focal, reversible brain ischemia after delayed intravenous administration at 60 or 120 minutes after middle cerebral arterial occlusion. METHODS: BDNF was conjugated to the OX26 murine monoclonal antibody to the rat transferrin receptor, which undergoes transport into brain from blood via the BBB transferrin receptor transcytosis system. After a 1-hour occlusion of the middle cerebral artery in nitrous oxide- ventilated animals with normal blood sugar, the brain was reperfused, and either BDNF or the BDNF/OX26 conjugate was administered as a single intravenous injection at a dose of 50 microg per rat. RESULTS: After the intravenous administration of unconjugated BDNF, there was no neuroprotection on the basis of analysis of brain at either 24 hours or 7 days after a 1-hour middle cerebral arterial occlusion. In contrast, there was a 68% and 70% reduction in cortical stroke volume at 24 hours and 7 days, respectively, after intravenous administration of 50 microg per rat of the BDNF conjugate (P<0.01). No effects on subcortical stroke volume were observed. CONCLUSIONS: These studies demonstrate marked neuroprotection in focal, transient brain ischemia with a single, delayed intravenous injection of BDNF if the neurotrophin is conjugated to a BBB drug targeting system. The neuroprotection is long lasting and persists for at least 7 days after a 1-hour middle cerebral artery occlusion.

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.

Mediated efflux of IgG molecules from brain to blood across the blood-brain barrier.

Dextrans and albumin exit brain from blood following intra-cerebral injection by a slow process of convection with a halftime of 10-12 h in the rat. The present studies show that immunoglobulin G (IgG) molecules rapidly efflux from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. The IgG efflux is rapid with a halftime of 48 min in the rat. The efflux of [3H]mouse IgG(2a) from brain to blood is competitively inhibited by intracerebral injection of unlabeled mouse IgG molecules, but is not inhibited by intracerebral injection of comparable doses of unlabeled rat albumin. The IgG efflux system has characteristics of an Fc receptor, as the efflux from brain is competitively inhibited by Fc fragments but is not blocked by F(ab')(2) fragments. Precipitation of brain homogenate by trichloroacetic acid indicates there is no significant metabolism of the IgG molecules during the experimental time period. In conclusion, these studies provide evidence for a BBB Fc receptor that mediates the reverse transcytosis of IgG molecules in the direction of brain to blood.

Rapid transferrin efflux from brain to blood across the blood-brain barrier.

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. Whereas high-molecular-weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5-fold faster than the efflux of holo-transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood-to-brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.

Conjugation of brain-derived neurotrophic factor to a blood-brain barrier drug targeting system enables neuroprotection in regional brain ischemia following intravenous injection of the neurotrophin.

Neurotrophins such as brain-derived neurotrophic factor (BDNF) are potential neuroprotective agents that could be used in the treatment of acute stroke, should these proteins be made transportable through the blood-brain barrier (BBB) in vivo. One approach to the BBB problem is to attach the nontransportable peptide to a brain targeting vector, which is a peptide or peptidomimetic monoclonal antibody (MAb), that is transported into brain from blood via an endogenous BBB transport system. The present studies describe a conjugate of BDNF and the OX26 monoclonal antibody (MAb) to the transferrin receptor. Avidin-biotin technology is used to link the BDNF and the MAb. The surface of the BDNF is conjugated with 2000 Da polyethylene glycol at carboxyl residues to optimize the plasma pharmacokinetics of the neurotrophin. Adult rats subjected to 24 h of permanent middle cerebral artery occlusion (MCAO) were treated intravenously with either unconjugated BDNF, unconjugated MAb, or the BDNF-OX26 conjugate at a dose of 1, 5 and 50 microg/rat of the BDNF. These doses decreased the infarct volume by 6% (not significant), 43% (P<0.01), and 65% (P<0.01), respectively. Significant reduction in stroke volume was still observed if the administration of the BDNF conjugate was delayed for 1-2 h after MCAO, although the pharmacological effect was progressively diminished in proportion to the time delay between MCAO and treatment. In conclusion, these studies demonstrate that large reductions in stroke volume can be achieved with the noninvasive intravenous administration of neurotrophins such as BDNF, providing the peptide is conjugated to a BBB drug targeting system.

Blood-brain barrier genomics.

The blood-brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction-based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IkappaB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.

Antisense imaging of gene expression in the brain in vivo.

Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [(125)I]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film autoradiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [(125)I]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system.

Antisense-mediated down-regulation of the human huntingtin gene.

The present study determines whether the expression of the huntingtin gene might be subject to antisense (AS)-mediated down-regulation. A series of AS oligodeoxynucleotides (ODNs) complementary to the huntingtin transcript [i.e., nucleotide (nt) -25 to 35] were designed and synthesized, and the AS efficacy was investigated by using a combination of in vitro transcription and translation to mimic in vivo conditions. An oligomer directed to nt -1 to 15 (ODN III) markedly reduced the incorporation of [(3)H]leucine into the huntingtin gene product in a dose-dependent manner (ED(50) of approximately 11.5 microM). ODNs that overlap with ODN III on both 5'- and 3'-flanking regions also produced translation arrest of the huntingtin protein; however, the AS-mediated effect of these ODNs represented approximately 50% of the effect of ODN III. In contrast, an ODN directed to nt 19 to 35 had no AS effect. The efficacy of ODN III also was investigated in an inducible, stably transfected PC-12 cell line expressing a truncated huntingtin exon 1 protein. In accordance with the cell free translation studies, ODN III (1-10 microM) markedly decreased the abundance of the huntingtin-green fluorescence fusion protein to 40 to 46% of the control levels. In summary, a series of putative AS candidates were screened for down-regulation of the huntingtin gene, and an ODN molecule directed to the methionine initiation codon was identified with maximum AS effects.

Selective Lutheran glycoprotein gene expression at the blood-brain barrier in normal brain and in human brain tumors.

The Lutheran (LU) glycoprotein was shown to be a specific marker of brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo. A 1.5 kb partial cDNA encoding the bovine LU was isolated from a bovine brain capillary cDNA library. Sequence analysis showed that the bovine and human LU had a 75% and 79% identity in the amino acid and nucleotide sequences, respectively. Northern blot analysis demonstrated a very high level of gene expression of the LU transcript in freshly isolated bovine brain capillaries, but no measurable LU mRNA in whole bovine brain. The high level of LU gene expression was maintained when bovine brain capillary endothelium was grown in tissue culture. Because many BBB specific genes are downregulated in tissue culture and in brain tumors, the expression of the LU mRNA and immunoactive LU protein was investigated in primary and metastatic human brain tumors. Immunocytochemistry of fresh frozen human brain and human brain tumors showed abundant immunostaining of brain capillary endothelium. Northern blot analysis showed the presence of LU transcripts in a panel of primary and metastatic human brain tumors. These studies demonstrated that the LU glycoprotein was a novel new marker of the BBB, and unlike other BBB specific genes, there was a persistent gene expression of the LU glycoprotein both in brain capillary endothelial cells grown in culture and in the endothelium of capillaries perfusing human brain cancer.