Article Published in the Author Account of

William M Pardridge

Molecular Trojan Horses for Blood-Brain Barrier Drug Delivery

Abstract: Molecular Trojan horses are genetically engineered proteins that cross the blood-brain barrier (BBB) via endogenous receptor-mediated transport processes. Molecular Trojan horses provide a brain drug targeting technology that allows for the non-invasive delivery of large molecule therapeutics to the human brain. The development of BBB drug targeting technology is an arcane area of discovery medicine that suffers from chronic under-development.

Molecular Trojan horses are genetically engineered proteins that cross the blood-brain barrier (BBB) via endogenous receptor-mediated transport processes. Molecular Trojan horses provide a brain drug targeting technology that allows for the non-invasive delivery of large molecule therapeutics to the human brain. The development of BBB drug targeting technology is an arcane area of discovery medicine that suffers from chronic under-development. The wisdom of continuation of this under-development of the BBB should be evaluated within the context of the following factors:

  • The brain is the principle source of morbidity. As the elderly population increases 50% by 2020, the triad of Alzheimer’s disease (AD), stroke, and Parkinson’s disease (PD) will have debilitating effects on science and health care delivery. The U.S. costs for health care of AD alone could approximate $0.5 trillion in 2020.
  • Although it is generally regarded that small molecule drugs cross the BBB, in fact, >98% of all small molecule drugs do not cross the BBB (Pardridge, 2001).
  • Essentially 100% of large molecule drugs do not cross the BBB, which include all the products of biotechnology: recombinant proteins, monoclonal antibodies, RNA interference (RNAi) drugs, and gene therapy.
  • No medium or large sized pharmaceutical company in the world today has a BBB drug targeting program.
  • Even if Big Pharma wanted to develop a BBB drug targeting program, there would be few personnel to hire, because <1% of all academic neuroscience programs in the U.S. include a program in BBB drug targeting or even BBB transport biology (Pardridge, 2006).

The insignificant national effort to solve the BBB drug and gene delivery problem contrasts with the massive effort in the molecular neurosciences and brain drug discovery. One can go to a 3-day conference on drug development for Alzheimer’s disease, stroke, or any other brain disorder and the issue of the BBB will not even be mentioned.

New technology for brain drug targeting originates in the fundamental sciences of the molecular and cellular biology of the BBB. The BBB is found in the brain of all vertebrates and is present within the first trimester of human fetal life. The anatomical basis of the BBB is the epithelial-like, high resistance tight junction that cements the endothelial cells together which comprise brain capillaries (Brightman and Reese, 1969). Owing to the presence of these tight junctions, there is no para-cellular pathway for free solute exchange between blood and brain. The tight junctions restrict pinocytosis across the capillary endothelium of the brain. Consequently, there is minimal trans-cellular pathway for solute exchange. Owing to the presence of the BBB, molecules in blood penetrate the brain via only one of two mechanisms: (a) catalyzed transport, or (b) lipid-mediated free diffusion. Regarding the free diffusion pathway, a small molecule crosses the BBB by lipid-mediation, only if (a) the molecule is lipid soluble, and (b) the molecular weight of the drug is <400 Daltons (Pardridge, 2001). The vast majority of small molecule drugs lack these dual molecular characteristics and do not cross the BBB. If affective disorders are excluded, only 1% of all small molecule drugs are active in the brain (Lipinski, 2000).

There are over 100 billion capillaries in the human brain and the surface area of the human BBB is approximately 20 m2. The BBB evolved because the same neurotransmitters used by the central nervous system (CNS) are also used by the peripheral nervous system and are present in the blood stream. Therefore, the BBB evolved to allow for a very tight regulation of the molecular composition of brain interstitial fluid. The price paid for a clean neuronal environment is the great difficulty in developing pharmaceuticals that can penetrate the brain from the blood.

Medicinal chemistry needs to be re-directed away from attempts to increase the lipid solubility of a CNS lead compound, and toward modifications that make the drug a substrate for one of the many BBB CMT endogenous transporters.

Certain small molecules in the blood stream do penetrate the brain via a form of catalyzed transport called carrier-mediated transport (CMT). D-glucose enters brain via CMT on the BBB GLUT1 glucose transporter (Oldendorf, 1971). Large neutral amino acids are needed for brain protein and neurotransmitter synthesis, and these enter the brain via the BBB LAT1 large neutral amino acid transporter. There are scores of specific CMT systems expressed within the BBB that mediate the blood to brain transport of nutrients, vitamins, and hormones. L-dopa is an effective neuropharmaceutical because this water-soluble molecule penetrates brain via CMT on the BBB LAT1 system.

The BBB also expresses specific receptor-mediated transport (RMT) systems, which transport certain large molecule peptides in the blood (Pardridge, 2001). Insulin gains access to brain via RMT on the BBB insulin receptor. Circulating transferrin enters brain via RMT on the BBB transferrin receptor (TfR). The observation that the peptide receptors exist on the BBB, and that these receptors act as transport systems led to the development of the molecular Trojan horse hypothesis. Insulin or transferrin could be used as a molecular Trojan horse for ferrying across the BBB an attached pharmaceutical via the endogenous BBB RMT system.

Alternatively, one could use a peptidomimetic monoclonal antibody (MAb) that binds an exofacial epitope on the BBB receptor such that the binding enables RMT of the MAb across the BBB. The binding site of the MAb is spatially removed from the endogenous ligand binding site, and there is no interference of endogenous transport when the MAb “piggy backs” across the BBB via the endogenous RMT system. Peptidomimetic MAbs have been developed for BBB drug delivery in mice, rats, monkeys, and man (Pardridge, 2001). For brain drug delivery in mice, the rat 8D3 MAb to the mouse TfR is used; this antibody is not active in rats. For drug delivery in rats, the murine OX26 MAb to the rat TfR is used; this antibody is not active in mice. Drug delivery in old world primates such as Rhesus monkey is accomplished with the murine 83-14 MAb to the human insulin receptor (HIR). This HIRMAb cross-reacts with the insulin receptor of old world primates but not new world primates such as squirrel monkey. For brain drug delivery in humans, genetically engineered forms of the HIRMAb have been developed (Pardridge, 2006).

The molecular Trojan horses have been reduced to in vivo CNS pharmacologic practice for a variety of recombinant proteins, peptides, antisense agents, and even non-viral gene therapy, and this work is summarized in Table 1. Peptides and recombinant proteins such as vasoactive intestinal peptide (VIP), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF)-2, and lysosomal enzymes, such as b-galactosidase, are all potential therapeutics for the brain, should these molecules be enabled to cross the BBB. VIP is a potent cerebral vasodilator when applied topically to brain vessels. Perfusion of VIP into the carotid artery results in no increase of cerebral blood flow, because VIP does not cross the BBB. Attachment of the VIP to the molecular Trojan horse results in a 65% increase in hemispheric brain blood flow in conscious rats following intravenous administration of low doses (10-20 mg/kg) of the neuropeptide.

BDNF is a potent neuroprotective agent when injected directly into the brain in either global or focal cerebral ischemia. However, the intravenous administration of BDNF is not neuroprotective because (a) BDNF does not cross the BBB, and (b) the BBB is intact following ischemia in the initial hours where neuroprotection is still possible. The attachment of BDNF to the molecular Trojan horse results in 100% neuroprotection of pyramidal neurons in the CA1 sector of the hippocampus in transient forebrain ischemia following delayed intravenous administration. Intravenous BDNF alone has no neuroprotective effect in global brain ischemia. Attachment of the BDNF, or FGF-2, to the molecular Trojan horse results in a 65-70% reduction in stroke volume in regional brain ischemia following delayed intravenous administration. There is no reduction in stroke volume following intravenous administration of the neurotrophin alone, when the BBB is intact.

Genes encoding lysosomal enzymes have been cloned and recombinant proteins have been produced for the treatment of lysosomal storage disorders. About 65% of all lysosomal storage disorders affect the brain. The lysosomal enzymes do not cross the BBB and enzyme replacement therapy does not treat the brain in lysosomal storage disorders. One of the lysosomal enzymes is β-galactosidase and the bacterial form of β-galactosidase was used as a model system for BBB delivery of lysosomal enzymes. The intravenous injection of β-galactosidase alone in anesthetized mice resulted in no lysosomal enzyme penetrating the brain. However, when the lysosomal enzyme was conjugated to the BBB molecular Trojan horse, lysosomal enzyme penetrated the brain from blood.

There is a need to develop antisense radiopharmaceuticals for in vivo imaging of gene expression. Sequence-specific antisense agents such as peptide nucleic acids (PNA) can be synthesized and radiolabeled with radionucleides such as 111-indium. However, antisense agents such as PNAs, or oligodeoxynucleotides, are polar molecules that do not readily penetrate cell membranes and do not cross the BBB. Accordingly, it is not possible to image gene expression following intravenous administration of sequence-specific antisense radiopharmaceuticals, unless drug targeting technology is used. When the sequence-specific PNA is attached to a molecular Trojan horse, it is possible to image in vivo the up- or down-regulation of brain gene expression in pathologic conditions such as cerebral ischemia or brain cancer. The intravenous administration of the PNA radiopharmaceutical alone does not allow for imaging gene expression in the brain in vivo, because the PNA does not cross the BBB.

A large molecule pharmaceutical, that normally has no activity in the brain because of lack of BBB transport, is transformed into a powerful neuropharmaceutical following attachment to a BBB molecular Trojan horse.

BBB targeting technology has also enabled, for the first time, the global expression of a transgene in the brain following a simple intravenous administration of a non-viral formulation (Zhang et al., 2003). The non-viral plasmid DNA is encapsulated in Trojan horse liposomes (THLs). The THL is comprised of a 100 nm liposome that encapsulates a single plasmid DNA molecule. The surface of the liposome is covered with polyethylene glycol (PEG) and the tips of 1-2% of the PEG strands are conjugated with the molecular Trojan horse. A eukaryotic expression plasmid encoding β-galactosidase was encapsulated in a THL that was targeted to the Rhesus monkey brain with the HIRMAb. Following intravenous administration of low doses of the plasmid DNA (10 mg/kg), the animal was sacrificed at 48 hours and the brain was removed for β-galactosidase histochemistry. The blue monkey brain shown in Figure 1 illustrates the global expression of a transgene throughout the entire primate brain following a single intravenous administration of a non-viral formulation of the gene. The combined use of THL gene targeting technology and organ specific promoters allows the expression of the transgene to be confined to the specific target organ and this organ specific expression of transgenes following THL administration has been demonstrated in mice, rats, and monkeys.

RNA interference, or RNAi, has been heralded as a new technology that will lead to powerful new therapeutics for multiple diseases including brain disorders. However, unless fundamental delivery problems are solved, it is unlikely that RNAi-based therapeutics will widely be used in clinical medicine. There are two types of RNA therapeutics: DNA-based RNAi, wherein a plasmid DNA encodes for short hairpin RNA (shRNA), or RNA-based RNAi, comprised of short interfering RNA (siRNA) duplexes. The first demonstration of RNAi in the brain following intravenous administration of the therapeutic was shown in an experimental brain cancer model. A plasmid DNA encoding for an shRNA directed against a defined sequence of the human epidermal growth factor receptor (EGFR) was encapsulated in THLs. These THLs were administered intravenously on a weekly basis to adult mice with intra-cranial human brain cancer. Weekly RNAi gene therapy using Trojan horse liposomes resulted in a marked decrease in the brain tumor expression of the EGFR protein, and this was associated with a 90% increase in survival time (Zhang et al., 2004).

The translation of BBB molecular Trojan horses to the treatment of human diseases of the brain is now possible, following the genetic engineering of molecular Trojan horses to enable repeat administration in humans (Pardridge, 2006). Approximately 1-2% of the injected dose of the genetically engineered human molecular Trojan horse enters the Rhesus monkey brain. This level of brain uptake is comparable to the brain uptake of small molecules. The BBB molecular Trojan horses can be used in different pharmaceutics technology platforms that utilize either a fusion protein technology or THL technology to deliver to the human brain virtually any large molecule pharmaceutical, including recombinant proteins and enzymes, monoclonal antibodies, non-viral gene medicines, and RNAi therapeutics.

References and Further Readings

Brightman MW, Reese TS. Junctions between intimately apposed cell membranes in the vertebrate brain. Journal of Cell Biology 40:648-677, 1969.

Lipinski CA. Drug-like properties and the causes of poor solubility and poor permeability. Journal of Pharmacological and Toxicological Methods 44:235-249, 2000.

Oldendorf WH. Brain uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. American Journal of Physiology 221:1629-1639, 1971.

Pardridge WM. Brain Drug Targeting: The Future of Brain Drug Development. Cambridge University Press, Cambridge, United Kingdom, pp1-370, 2001.

Pardridge WM. Molecular Trojan horses for blood-brain barrier drug delivery. Current Opinion in Pharmacology 6:494-500, 2006.

Zhang Y, Schlachetzki F, Pardridge WM. Global non-viral gene transfer to the primate brain following intravenous administration. Molecular Therapy 7:11-18, 2003.

Zhang Y, Bryant J, Zhang YF, Charles A, Boado RJ, Pardridge WM. Intravenous RNAi gene therapy targeting the human EGF receptor prolongs survival in intra-cranial brain cancer. 10(11):3667-3677, 2004.

[Discovery Medicine, 6(34):139-143, 2006]

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