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Blood-Brain Barrier Drug Targeting Enables Neuroprotection in Brain Ischemia Following Delayed Intravenous Administration of Neurotrophins

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The blood-brain barrier (BBB) is the rate-limiting step in the translation of neurotrophin neuroscience into clinically effective neurotherapeutics. Since neurotrophins do not cross the BBB, these proteins cannot be used for neuroprotection following intravenous administration, and it is not feasible to administer these molecules by intra-cerebral injection in human stroke. The present studies describe the development of the chimeric peptide brain drug targeting technology and the use of brain-derived neurotrophic factor (BDNF) chimeric peptides in either global or regional brain ischemia. The BDNF chimeric peptide is formed by conjugation of BDNF to a monoclonal antibody (MAb) to the BBB transferrin receptor, and the MAb acts as a molecular “Trojan Horse” to ferry the BDNF across the BBB via transport on the endogenous BBB transferrin receptor. High degrees of neuroprotection in transient forebrain ischemia, permanent middle cerebral artery occlusion, or reversible middle cerebral artery occlusion are achieved with the delayed intravenous administration of BDNF chimeric peptides. In contrast, no neuroprotection is observed following the intravenous administration of unconjugated BDNF, because the neurotrophin does not cross the BBB in vivo.

Blood-Brain Barrier, Neurotrophins, and Neurological Disease

The blood-brain barrier (BBB) is the rate-limiting step in the translation of molecular neurosciences into clinically effective neurotherapeutics (Fig. 1). If neurotherapeutics are developed in the absence of any consideration to BBB transport, then the central nervous system (CNS) drug development pathway invariably leads to program termination.1 Termination arises because essentially 100% of large molecule neuropharmaceuticals do not cross the BBB and >98% of small molecule neuropharmaceuticals do not cross the BBB.2 Despite these facts, the translation of neuroscience into neurotherapeutics invariably takes place in the absence of any consideration of BBB transport properties, and the neurotrophins are a case study of this problem. Not surprisingly, and despite the fact that the neurotrophin genes were cloned more than 10 years ago, there is not a single CNS disease that is currently being treated with neurotrophic factors. Indeed, virtually all clinical trials with neurotrophins have been halted by large pharmaceutical companies.

Figure 1. Neuroscience, blood-brain barrier, and clinically effective neurotherapeutics.

Figure 1

Neuroscience, blood-brain barrier, and clinically effective neurotherapeutics.

Neurotrophin Drug Development

The history of neurotrophin drug development is outlined in Figure 2. There are more than 30 neurotrophic factors that are powerful neuroprotective agents should these molecules be delivered to the target sites within the brain or spinal cord. These target sites all lie behind the BBB. Therefore, it is not possible to develop neurotrophins as clinically effective neurotherapeutics, because (a) these molecules do not cross the BBB, and (b) neurotrophin drug development took place in the absence of any consideration to BBB transport. The pitfalls of developing drugs for the brain that do not cross the BBB are illustrated in the case of amytrophic lateral sclerosis (ALS), which was treated in the 1990s with 3 different neurotrophins, including ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF)-1, and brain-derived neurotrophic factor (BDNF). The CNTF clinical trials were based only on in vitro tissue culture data and some in vivo data in developing animals.3 The IGF1 clinical trials were based on tissue culture data and in vivo data on the sciatic nerve,3 which is outside the CNS and the BBB. An example of a tissue culture finding with nerve growth factor (NGF) is shown in Figure 2, which demonstrates neuronal differentiation in a dorsal root ganglia following exposure to NGF.4 On the basis of such experiments, patients with ALS were treated with CNTF, IGF-1, or BDNF in separate, costly phase III clinical trials. In all 3 trials, the neurotrophic factor was administered by subcutaneous injection, even though the target neurons to be treated in ALS resided behind the BBB or blood-spinal cord barrier. None of these neurotrophic factors crossed the BBB and not surprisingly, all 3 phase III clinical trials failed in the treatment of ALS. Subsequently, neurodegenerative conditions such as Parkinson's disease (PD) were treated with neurotrophic factors such as glial-derived neurotrophic factor (GDNF) by intracerebral ventricular (ICV) infusion.5

Figure 2. There are >30 different neurotrophic factors known to date and many have neuroprotective and restorative effects when injected directly into the brain.

Figure 2

There are >30 different neurotrophic factors known to date and many have neuroprotective and restorative effects when injected directly into the brain. However, these molecules do not cross the BBB. Pre-clinical research and brain drug development (more...)

Limitations of ICV Infusion as a Brain Drug Delivery System

Figure 2 shows an autoradiogram of rat brain obtained 24 hours after the single injection of radiolabeled BDNF into the lateral ventricle.6 The autoradiography demonstrates that the neurotrophic factor does not distribute beyond the ependymal epithelial layer lining the ventricular surface. This study illustrates that the ICV infusion of drug is a poor means of drug delivery to brain parenchyma. ICV infusion of drug allows for drug distribution to the surface of the brain. However, owing to the limitations of diffusion within the brain and to the rapid rate of bulk flow of CSF through the ventricular flow tracks, the majority of drug that is injected into the ventricule is rapidly distributed into the peripheral circulation rather than penetrate into brain parenchyma by diffusion.7 The second phenomenon demonstrated by the autoradiography in Figure 2 is that CSF flow takes place in a unidirectional manner. Molecules injected into the lateral ventricule quickly move to the third ventricle, then to the fourth ventricle, then over the convexities of the brain and are absorbed into the superior sagittal sinus across the arachnoid villae without significant penetration to the contralateral brain. The third property of ICV drug infusion demonstrated by the autoradiography in Figure 2 is that whereas the brain parenchyma is exposed to very little neurotrophic factor, the ependymal surface of the brain is exposed to very high concentrations of neurotrophic factor. This causes significant sub-ependymal gliosis following the ICV infusion of neurotrophic factors in animals.8,9 The ICV infusion of GDNF resulted in such toxicity without efficacy in the treatment of Parkinson's disease that the clinical trials were halted.5

Small Molecules

The administration of neurotrophic factors by either subcutaneous administration or ICV infusion resulted in either no therapeutic effects or enhanced toxicity. Neurotrophin drug development then evolved into small molecules and in this approach, cloned neurotrophic factor receptors are expressed and analyzed by high throughput screening (HTS) methodologies, which involves the screening of hundreds of thousands of small molecule drug candidates. This approach has not led to the discovery of neurotrophic factor small molecule peptidomimetics, because small molecule peptidomimetics tend to be antagonists, not agonists.3 Even if a small molecule neurotrophic factor peptidomimetic agonist was discovered, it is unlikely this molecule would undergo transport across the BBB in pharmacologically significant amounts. While it is often stated that small molecules freely cross the blood-brain barrier, in fact, only a certain type of small molecule crosses the BBB. Small molecules that do cross the BBB have the dual molecular characteristics of (a) molecular weight under a 400–500 Dalton threshold and (b) lipid solubility.2 The kinds of small molecules selected in receptor-based HTS programs have molecular weights above 400–500 Daltons and/or high degrees of hydrogen bonding and low levels of lipid solubility. The end result is program termination of the neurotrophin CNS drug development program, once the small molecule candidate is found to not cross the BBB. The termination of neurotrophin CNS drug development is most unfortunate considering the years of effort that have been devoted to neurotrophin molecular neuroscience and also considering the millions of individuals suffering from chronic neurodegenerative conditions of the brain. The individuals suffering from Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, stroke, and other neurodegenerative conditions could benefit from neurotrophin neuropharmaceuticals. The problem is that the neurotrophin molecule must undergo a molecular reformulation, within the context of a BBB drug-targeting technology, so that the neurotrophin is able to traverse the BBB and distribute into brain parenchyma following intravenous or subcutaneous administration.

This chapter will review noninvasive neurotrophin delivery to the brain using the chimeric peptide BBB drug-targeting technology. The work demonstrates that profound degrees of neuroprotection can be achieved in either global or permanent or reversible regional brain ischemia following delayed intravenous administration of a neurotrophic factor, providing the neurotrophin is conjugated to a BBB drug targeting system.

Chimeric Peptide Technology

BBB Transport

The capillary endothelial cells of the brain of all vertebrates have unique anatomic specializations characterized by (a) high resistance epithelial-like tight junctions that eliminate any paracellular pathway, and (b) a paucity of pinocytosis across the endothelial cell, which eliminates the transcellular pathway of solute movement from blood to brain interstitium.2 The BBB is laid down within the first trimester of human fetal life and restricts the movement of essentially all molecules between blood and brain. There may be molecular movement across the BBB via one of two generalized pathways: (a) free diffusion via lipid solubility and (b) catalyzed transport. Free diffusion occurs for molecules that have a molecular weight under a 400500 Dalton threshold and have high degrees of lipid solubility and low hydrogen bonding. Catalyzed transport occurs via one of several endogenous transport systems within the BBB and this may take place on either carrier-mediated transport (CMT) systems or receptor-mediated transport (RMT) systems within the brain capillary endothelium. The CMT systems transport small molecule nutrients in milliseconds, and include the Glut1 glucose transporter, the LAT1 large neutral amino acid transporter, the MCT1 monocarboxylic acid transporter, and the CNT2 adenosine transporter.1 Some of the RMT systems expressed at the BBB are shown in Figure 3 and include the insulin receptor (IR), the transferrin receptor (TfR), the IGF-2 or IGF-1 receptor, or the leptin receptor.10–13 Despite the presence of an IGF-1 or an IGF-2 receptor at the BBB, there is minimal transport of these molecules into brain from blood following intravenous administration, because the IGFs are strongly bound >99.9% by IGF specific binding proteins in the blood.14

Figure 3. Blood-barrier peptide receptors and chimeric peptide hypothesis.

Figure 3

Blood-barrier peptide receptors and chimeric peptide hypothesis. From.

Chimeric Peptides

Based on the observation that peptide receptors are present on the brain capillary endothelium and that some of these mediate the transcytosis of the circulating peptide through the BBB, the chimeric peptide technology was developed.7 A chimeric peptide is formed when a nontransportable drug is conjugated to a brain drug delivery vector. The latter may be an endogenous peptide, a modified protein, or a peptidomimetic monoclonal antibody (MAb) that undergoes receptor-mediated or absorptive-mediated transcytosis through the BBB in vivo on one of the endogenous transport systems shown in Figure 3. Insulin undergoes RMT through the BBB in vivo on the BBB insulin receptor.15 However, the intravenous administration of a drug/insulin conjugate could lead to hypoglycemia by triggering insulin receptors in peripheral tissues. The conjugation of drugs to transferrin (Tf) is problematical because the Tf/drug conjugate would compete with the very high concentration (25 μM) of endogenous Tf in the blood. These problems are obviated with the use of peptidomimetic MAbs, which bind exofacial epitopes on the BBB receptor that are removed from the binding site of the endogenous ligand. This binding of the MAb to the BBB receptor allows the MAb to “piggyback” across the BBB on the endogenous RMT system. Any drug attached to the peptidomimetic MAb may also undergo transport across the BBB on the endogenous RMT system.16

RMT on the BBB Transferrin Receptor

The outline of the receptor-mediated transcytosis of circulating transferrin through the BBB via the TfR is shown in Figure 4D. Transcytosis of transferrin through the BBB in vivo was demonstrated with both a capillary depletion technique and thaw mount autoradiography.17 The transcytosis of a transferrin receptor peptidomimetic MAb, OX26, was demonstrated with electron microscopy.16 In these experiments, a 5-nm gold conjugate of the OX26 MAb was infused in the carotid artery of anesthetized rats for 10 minutes and the infusion was followed by saline clearing of the cerebral microvasculature and by perfusion fixation with glutaraldehyde. Immunogold silver enhancement at the light microscopic level is shown in Figure 4A and demonstrates localization of the OX26 MAb/gold conjugate within the capillary endothelium of brain. The tissue was processed for electron microscopy and OX26 MAb/gold conjugate was visible on the luminal surface of the brain capillary endothelium, as shown in Figure 4B. Whenever the OX26/gold conjugate was found in the intraendothelial compartment of the capillary endothelium, these molecules were always found clustered in 100 nm endosomal structures, as shown in low magnification in Figure 4B and high magnification in Figure 4C. The exocytosis of the OX26/gold conjugate into the brain interstitium is demonstrated in Figure 4C.

Figure 4. (A) Silver-enhanced vibratome section of rat brain perfused for 10 min with OX26/5-nm gold conjugate in vivo.

Figure 4

(A) Silver-enhanced vibratome section of rat brain perfused for 10 min with OX26/5-nm gold conjugate in vivo. The coronal section is at the level of the frontal cortex. The magnification bar = 10 μm. (B) Electron micrograph of a brain capillary (more...)

Transferrin Transcytosis at the BBB is Bi-directional

The transcytosis of transferrin in the blood to brain direction was initially demonstrated with internal carotid artery perfusion of radiolabeled transferrin.17–18 The receptor-mediated transcytosis of transferrin in the brain to blood direction was demonstrated recently with the brain efflux index (BEI) method.19 These studies showed that both holo-transferrin and apo-transferrin were rapidly exported from brain to blood via reverse transcytosis on the BBB transferrin receptor and that the apotransferrin was transcytosed approximately 2.5-fold faster than the holo-transferrin. There are other examples of macromolecule secretion from brain to blood via reverse transcytosis across the BBB. IgG molecules injected into the brain are rapidly exported to blood via reverse transcytosis on the BBB Fc receptor.20 Whereas the endothelial TfR mediates bidirectional transcytosis of Tf across the BBB, the brain endothelial FcR mediates the unidirectional transcytosis of IgG molecules in the brain to blood direction only.19–20

Species Specific BBB Transport Vectors

The OX26 antibody is a mouse MAb to the rat TfR and this antibody is active only in rats.21 The OX26 MAb has no biologic activity in mice as it does not bind the mouse TfR (Figure 5). Recently, 2 different rat MAbs to the mouse TfR, the 8D3 MAb or the RI7-217 MAb, were demonstrated to undergo receptor-mediated transcytosis across the mouse BBB via the endogenous TfR in this species.21 Brain drug delivery studies in Old World primates such as the Rhesus monkey are possible using the 8314 murine MAb to the human insulin receptor (HIR).22 This MAb is highly active at the human BBB and is also active at the BBB of Old World primates such as the Rhesus monkey. The HIR MAb is not active in New World primates such as Squirrel monkey.22 The murine 83–14 MAb cannot be used for brain drug targeting in humans owing to the high immunogenecity of a mouse protein in humans. However, the HIR MAb was recently genetically engineered to enable human applications.23 As discussed in later sections of this chapter, the chimeric 83–14 MAb retained 100% of the binding activity for the human insulin receptor, and is a highly efficacious brain drug delivery vector in Rhesus monkeys. Second and third generation BBB drug delivery systems in humans will be humanized forms of the 83–14 MAb and MAbs to brain capillary specific proteins (BSP).1

Figure 5. Monoclonal antibodies (MAb) are species-specific with respect to the reactivity to either the transferrin receptor (TfR) or the insulin receptor (IR).

Figure 5

Monoclonal antibodies (MAb) are species-specific with respect to the reactivity to either the transferrin receptor (TfR) or the insulin receptor (IR). BSP = brain capillary-specific protein. From ref.

Avidin-Biotin Technology in Brain Drug Delivery

Subsequent to the discovery of brain drug targeting vectors for a given species, it is necessary to devise suitable technologies for high efficiency coupling or conjugation of the drug to the transport vector.1 These goals are achieved with the use of avidin-biotin technology in brain drug targeting. In this approach, the drug is monobiotinylated in parallel with the production of a vector/avidin or vector/streptavidin (SA) fusion protein. Owing to the extremely high affinity binding of biotin by avidin, there is instantaneous capture of the biotinylated therapeutic by the avidin/vector fusion protein. Given these properties, a “two vial” format for drug administration was developed. In this approach, the BBB vector, fused to the avidin or SA, is prepared in one vial. The monobiotinylated therapeutic is prepared in another vial. Just prior to intravenous injection of the conjugate, the two vials are mixed to allow for rapid formation of the entire drug/MAb conjugate, which is joined through the avidin/biotin linkage, as depicted in Figure 6. Because avidin or SA has 4 biotin binding sites, there would be the formation of high molecular weight aggregates if the drug had degrees of biotinylation higher than a single biotin residue per drug molecule. Therefore, it is essential that the drug be monobiotinylated. Genetically engineered vector/avidin or vector/SA fusion genes and fusion proteins have been produced and purified and the biologic activity of these proteins has been demonstrated with in vivo brain drug targeting studies.24,25

Figure 6. “Two vial” format for administration of drug/vector conjugates using avidin-biotin technology.

Figure 6

“Two vial” format for administration of drug/vector conjugates using avidin-biotin technology. The BBB transport vector vial contains a genetically engineered fusion protein of avidin (AV) and a peptidomimetic monoclonal antibody (MAb). (more...)

Targeting Chimeric Neurotrophic Factors to the Brain

Vasoactive Intestinal Peptide (VIP)

VIP Neurotherapeutics

VIP is the principle endogenous vasodilator in the CNS,26 and VIP is also a neuroprotective agent.27 The topical application of VIP to brain blood vessels results in vasodilatation.28 However, there is no enhancement of cerebral blood flow following the intravenous or intracarotid administration of VIP in multiple species,29–31 because this neuropeptide does not cross the BBB.32 In order to develop a VIP chimeric peptide that would be pharmacologically active in brain following intravenous administration, a series of experiments were performed to engineer a monobiotinylated form of VIP (bioVIP). This VIP chimeric peptide was then used to augment cerebral blood flow (CBF) in conscious rats following intravenous administration.33

Monobiotinylation of VIP Analogue

The biotin residue can be placed on a variety of functional groups including primary amines on lysine residues, carboxyl moieties on glutamate or aspartate residues, or sulfhydryl moieties on cysteine residues. VIP has 3 internal lysine residues and Lys15 can be biotinylated without any loss of biologic activity.34 Therefore, the lysine residues at positions 20 and 21 were converted to arginine residues to prevent biotinylation at these sites (Fig. 7). The amino terminus was acetylated to prevent biotinylation and also to render the peptide resistant to aminopeptidase activity. The Ile at position 26 was converted to alanine to prolong the duration of action and the methionine at position 17 was converted to norleucine to enable iodination of the peptide (Fig. 7). The structure of the VIP analogue was confirmed by fast atom bombardment mass spectrometry.32 The VIPa was biotinylated with NHS-XX-biotin, where NHS = N-hydroxysuccinimide and XX = bisaminohexanoyl. The XX linker is 14 atoms long and is positioned between the amino moiety of Lys15 on the VIP and the biotin group. The VIPa-XX-biotin was bound by a conjugate of streptavidin (SA) and the OX26 MAb to form the chimeric peptide shown in Figure 8A. The biologic activity of the VIP analogue was demonstrated with a mammalian VIP radioreceptor assay.33 Although conjugation of the VIP to the OX26/SA BBB delivery system did diminish the affinity of the neuropeptide for the VIP receptor, there was still retention of biologic activity in the pharmacologically active range following conjugation of the neuropeptide to the MAb vector (Fig. 8B).

Figure 7. Single-letter amino acid sequence of a VIP analogue designed for monobiotinylation.

Figure 7

Single-letter amino acid sequence of a VIP analogue designed for monobiotinylation. The single site for biotinylation is the lysine (K) residue at position 15 and modification of this lysine residue is possible with retention of biologic activity of (more...)

Figure 8. VIP Radioceptor Assay Shows VIP Chimeric Peptide Has High Affinity for the Mammalian VIP Receptor.

Figure 8

VIP Radioceptor Assay Shows VIP Chimeric Peptide Has High Affinity for the Mammalian VIP Receptor. (A) The structure of the VIP chimeric peptide is comprised of three domains. The first domain is the VIP analogue (VIPa) that binds the VIP receptor. The (more...)

Cerebral Blood Flow Enhancement by VIP Chimeric Peptides

Conscious rats with preimplanted indwelling femoral artery and femoral vein catheters were administered 1 of 4 different formulations intravenously: saline, OX26 MAb alone, the monobiotinylated VIP analogue (bioXXVIPa) alone, or the VIP chimeric peptide.33 These drugs were administered to conscious rats by intravenous administration at low systemic doses (20 μg/kg or 5 μg/rat) of the VIP. Administration of the unconjugated bio-XX-VIPa, without conjugation to the OX26/SA delivery system, increased blood flow in salivary gland by 350%, but had no effect on brain blood flow (Fig. 9). Although the VIP analogue did not cross the BBB in vivo, this neuropeptide was freely transported across the porous capillaries perfusing salivary gland. Salivary gland capillaries are richly innervated by VIPergic nerve endings which result in vasodilatation in that exocrine organ.35 In contrast, the administration of the VIP chimeric peptide caused no increase in salivary gland blood flow, but resulted in a 60% increase in CBF in conscious rats following intravenous injection (Fig. 9). The increase in CBF following intravenous administration of the VIP chimeric peptide was observed because the VIP was able to undergo receptor-mediated transcytosis across the BBB on the endogenous transferrin receptor.33 In contrast, conjugation of the VIP neuropeptide to the OX26/SA delivery system actually impeded transport across salivary gland capillaries. The molecular weight of the bio-XX-VIP, approximately 4000 Daltons, was effectively increased to 204,000 Daltons, following conjugation to the 200,000 Dalton OX26/SA drug-targeting system. The size of the VIP chimeric peptide was too large to enable molecular diffusion across the small pore system of capillaries in exocrine glands such as salivary glands. Therefore, the ratio of CBF to salivary gland blood flow (SBF) was increased 10-fold following conjugation of the VIP analogue to the BBB drug-targeting system (Fig. 9). The CBF/SBF ratio is a measure of the therapeutic index of the VIP chimeric peptide. The 10-fold increase in this therapeutic index illustrates the targeting capabilities of the chimeric peptide technology. Conjugation of the neuropeptide to the BBB drug-targeting system not only causes selective uptake into brain, but also causes decreased uptake in peripheral tissues.33

Figure 9. Selective Enhancement of Brain Blood Flow by Intravenous VIP Chimeric Peptide in Conscious Rats.

Figure 9

Selective Enhancement of Brain Blood Flow by Intravenous VIP Chimeric Peptide in Conscious Rats. (Left) Organ blood flow in brain and salivary gland. (Right) Ratio of cerebral blood flow (CBF) to salivary gland blood flow (SBF) in rats administered VIP (more...)

Brain-Derived Neurotrophic Factor (BDNF)

Neurotrophin Surface Charge and Plasma Pharmacokinetics

The members of the NGF-like family of neurotrophins, which includes NGF, BDNF, neurotrophin (NT)-3, and NT-4/5, all have high degrees of structural homology and are highly cationic neuropeptides. An electrostasis model of NGF is shown in Figure 10 and indicates there is a cationic groove comprised of lysine and arginine residues down the center of the molecule with segregation of the anionic charges attached to glutamate and aspartate residues on the periphery of the cationic groove.36 The cationic groove is responsible for neurotrophin interaction with one of the specific trk receptors.37 The cationic surface charge of the NGF-like neurotrophin also causes rapid uptake by liver, which results in rapid systemic clearance of the neurotrophins from blood, which is characterized by a t1/2 < 5 minutes.38 In pharmacokinetic terms, the plasma area under the concentration curve (AUC) is reduced and the NGF-like neurotrophins all have very poor pharmacokinetic profiles. The plasma pharmacokinetics of the neurotrophins was considered only in the late stages of neurotrophin CNS drug development.3 However, the pharmacokinetic profile of a neurotrophin, or any drug, should be considered early in the CNS drug development process. This is because the actual uptake of a drug by brain following intravenous administration, expressed as % of injected dose (ID)/g brain, is a dual function of the BBB permeability-surface area (PS) product, and the plasma AUC,1

Figure 10. Electrostatic charge model of NGF showing the cationic groove as represented by the black coloration in the central part of the molecule.

Figure 10

Electrostatic charge model of NGF showing the cationic groove as represented by the black coloration in the central part of the molecule. This is comprised of the cationic charges of lysine (Lys) and arginine (Arg) residues. Conversely, the anionic charges (more...)

%ID/g = PS × AUC

The BBB PS product is a function of the BBB drug-targeting system, and the plasma AUC is a function of the pharmacokinetic profile. If the drug is rapidly removed from plasma, the AUC is reduced, and the brain uptake (%ID/g) is reduced proportionately. Therefore, in neurotrophin drug development, it is advisable to reformulate the protein to both (a) enable BBB transport, and (b) optimize the plasma AUC. The rapid removal of proteins from blood can be reversed by protein pegylation.

Protein Carboxyl-Directed Pegylation

Polymers of polyethylene glycol (PEG) of either 2000 or 5000 Daltons molecular weight, designated PEG2000 or PEG5000, respectively, can be conjugated to the surface of NGF-like neurotrophins to prevent the rapid uptake of these cationic proteins from blood.39 PEG conjugation is called pegylation. Prior work on protein pegylation involved attachment of the PEG moieties to ϵ-amino groups on internal lysine residues. However, the NGF-like neurotrophins lose biologic activity when the surface lysine residues are modified,40 principally because the lysine and arginine residues comprise the cationic groove that interacts with the specific trk receptors.37 An alternative to amino-directed protein pegylation is carboxyl directed protein pegylation.41 In this approach, PEG hydrazide derivatives are used to attach the PEG polymers to the carboxyl moieties of surface glutamate or aspartate residues, which are segregated from the cationic groove (Fig. 10). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the increased molecular weight of the BDNF following conjugation with either PEG2000 or PEG5000 on surface carboxyl residues (Fig. 11A). The BDNF-PEG2000 had an average molecular weight of 28,000 Daltons and the BDNF-PEG5000 had an average molecular weight of 50,000 Daltons. These observations indicated approximately 57 PEG monomers were attached per BDNF monomer, indicating that approximately half of the surface glutamate or aspartate residues were conjugated. The pharmacokinetic profile of unconjugated BDNF, BDNF-PEG2000, or BDNF-PEG5000 was measured in rats following iodination of the BDNF analogues. The plasma clearance in ml/min/kg of the 3 different forms of BDNF are shown in Figure 11B and indicate the plasma clearance is reduced 73% and 94% by carboxyl-directed pegylation of BDNF by PEG2000 and PEG55000, respectively.41

Figure 11. (Left) Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of BDNF, BDNF-PEG2000, and BDNF-PEG5000.

Figure 11

(Left) Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of BDNF, BDNF-PEG2000, and BDNF-PEG5000. (Right) Plasma pharmacokinetics showing the rate of clearance (Cl) of [125I]-BDNF, [125I]-BDNF-PEG2000, and [125I]-BDNF-PEG5000. From (more...)

BDNF Chimeric Peptides: Combined Use of Carboxyl-Directed Protein Pegylation Technology and Avidin-Biotin Technology

To enable monobiotinylation of BDNF-PEG2000, the neurotrophin was pegylated with both hydrazide-PEG2000 and hydrazide-PEG2000-biotin in a 7:1 molar ratio.42 Following conjugation of the BDNF-PEG-biotin to the OX26/SA and purification by gel filtration chromatography, a molecular analysis of the BDNF chimeric peptide was performed with SDS-PAGE, film autoradiography, and Western blotting, as shown in Figure 12B. The average molecular weight of the BDNF-PEG2000-biotin was 45,000 Daltons based on either Coomassie blue staining or film autoradiography (Figure 12). The incorporation of the biotin residue into the BDNF-PEG2000- biotin was demonstrated by Western blotting, as shown in lane 5 of Figure 12B.

Figure 12. (A) Structure of BDNF chimeric peptide.

Figure 12

(A) Structure of BDNF chimeric peptide. (B) SDS-PAGE, film autoradiography (auto.), and Western blotting of [125I]-BFNF-PEG2000-biotin. (C) Brain uptake of either the [125I]-BDNF or the [125I] BFNF-PEG2000-biotin conjugated to SA-OX26 in anesthetized (more...)

The brain uptake of the BDNF chimeric peptide was measured at 60 minutes following intravenous injection in anesthetized rats and the level of brain uptake was 0.07% of injected dose (ID) per gram brain (Figure 12C). This level of brain uptake is comparable to the brain uptake of morphine, a neuroactive small molecule, following intravenous injection.43 In contrast, the brain uptake of the unconjugated BDNF was negligible, indicating BDNF does not cross the BBB (Figure 12C).

Biologic Activity of BDNF Chimeric Peptides: trkB Autophosphorylation

3T3 cells permanently transfected with the trkB receptor were serum starved, and then exposed to 1–100 ng/ml of either BDNF, BDNF-PEG2000-biotin, BDNFPEG2000- biotinyl/SA-OX26, or SA/OX26 without BDNF, as shown in Figure 13. The cells were then lysed, immunoprecipitated with a trk B antiserum, separated on SDS-PAGE, followed by Western blotting of phosphotyrosine residues using an antiphosphotyrosine antibody.42 The phosphotyrosine Western blots were scanned and the quantitative results are shown in Figure 13. These results show there is complete retention of biologic activity of the BDNF following either pegylation and formation of the BDNF-PEG2000- biotin or conjugation of the BDNF-PEG2000-biotin to SA-OX26, which is a conjugate of streptavidin and the OX26 MAb. In contrast, there is no autophosphorylation of the trkB receptor following exposure of the cells to the SA-OX26 targeting system without BDNF attached (Figure 13).

Figure 13. (Left) 3T3 cells that were permanently transfected with a gene encoding the trkB BDNF receptor are immunstained using an antiserum to trkB, and this shows abundant expression of the trkB in the majority of these cells.

Figure 13

(Left) 3T3 cells that were permanently transfected with a gene encoding the trkB BDNF receptor are immunstained using an antiserum to trkB, and this shows abundant expression of the trkB in the majority of these cells. (Right) The top panel shows the (more...)

BDNF Chimeric Peptides and Transient Forebrain Ischemia

The transient forebrain ischemia (TFI) model44 for evaluating CNS pharmacological effects of intravenously administered neurotrophins is shown in Figure 14. The animal is maintained on a respirator and is anesthetized with 70% nitrous oxide, 30% oxygen and 0.5% halothane.45 Body temperature was maintained with a thermal blanket. The systolic blood pressure was measured with a rat tail amplifier. Prior to induction of ischemia, the halothane component of the anesthesia was discontinued. Blood was collected via the femoral artery catheter and arterial blood gases were measured. Muscle paralysis was induced with intravenous suxamethonium (0.5 mg/kg) and the electroencephalogram (EEG) was recorded via 2 anchored metal screws with an oscillograph and preamplifier. After the blood gas values were stabilized, forebrain ischemia was induced by clamping both common carotid arteries, and by lowering the blood pressure to a level < 50 mm Hg with a single intravenous injection of 5 mg/kg of trimethaphan and by phlebotomy via the femoral artery catheter. Cerebral ischemia was confirmed by isoelectric EEG.45 After 12 minutes of the ischemic insult, the carotid clamps were released and the shed blood was slowly reinfused at a rate of 2 ml/min. The animal was allowed to recover under a heating lamp for 4 hours and was returned to the vivarium. The animals were sacrificed 7 days later and coronal sections were Nissl stained. The pyramidal neurons of the hippocampal CA1, CA3, and CA4 regions were counted with a measuring grid under light microscopy at 100X magnification. Only neurons with a visible nucleolus were scored. In this TFI model, there is a selective loss of the hippocampal CA1 pyramidal neurons at 7 days following the TFI event, as shown in Figure 14.

Figure 14. Transient forebrain ischemia model.

Figure 14

Transient forebrain ischemia model.

The animals subjected to the TFI and isoelectric EEG were treated with either saline, unconjugated BDNF, unconjugated OX26 MAb, or the BDNF/OX26 chimeric peptide. The BDNF chimeric peptide is designated BDNF-PEG2000-biotin/SAOX26.45 The bifunctionality of the BDNF chimeric peptide is shown in Figure 15. Despite the protein pegylation and attachment to the BBB drug-targeting system, the BDNF is still biologically active and binds actively to the neuronal trkB receptor to mediate neuroprotection.42 Moreover, the BDNF chimeric peptide also binds to the transferrin receptor on the BBB, and this enables transport of the BDNF chimeric peptide across the BBB from blood to brain following intravenous administration. The extended bridge comprised of the PEG2000 and the biotin/SA releases any steric hindrance between the 2 components of the chimeric peptide, and this enables retention of the bifunctional nature of the conjugate, and binding to both the neuronal trkB and BBB Tf receptors (Figure 15).

Figure 15. BDNF chimeric peptide binds to both the BBB transferrin receptor (TfR) to mediate uptake into brain from blood, and to the neuronal trkB receptor, to mediate pharmacologic action within the brain.

Figure 15

BDNF chimeric peptide binds to both the BBB transferrin receptor (TfR) to mediate uptake into brain from blood, and to the neuronal trkB receptor, to mediate pharmacologic action within the brain.

During the 10-minute ischemic period, the blood pressure ranged from 22±2 to 27±6 mm Hg in all 4 treatment groups.45 There were no significant differences in the physiologic parameters, including body temperature or blood gases in the 4 groups. Ischemia was confirmed in all rats by demonstration of isoelectric EEG, which is illustrated in Figure 16. The animals were treated intravenously immediately after recovery from the ischemic period. Seven days after the ischemic episode, the animals were sacrificed for Nissl staining and neuron counting of the hippocampal CA1 sector. These data show a 68±10% decrease in the hippocampal CA1 neurons in the rats subjected to TFI (Figure 16) and there was no therapeutic benefit observed with the intravenous administration of either unconjugated BDNF or unconjugated OX26 MAb. In contrast, there was complete normalization of the CA1 pyramidal neuron density following the intravenous administration of the BDNF chimeric peptide (Figure 16). Representative Nissl stains are shown in Figure 16. The neurons were reduced in number and size in the CA1 sector of the hippocampus in the rats subjected to TFI and treated with unconjugated BDNF. These brains appeared no different from the animals treated with the saline control,45 which is expected considering that BDNF does not cross the BBB.41 Moreover, the BBB is not disrupted following a 10-minute transient forebrain ischemia until very late stages when chances for neuroprotection are no longer present. Since the BBB is intact in the period immediately following global ischemia,46 and since BDNF does not cross the BBB,41 neuroprotection in brain would not be expected following intravenous administration of BDNF unless this neurotrophin was conjugated to a BBB drug-targeting system. When this is done, there is complete normalization of the neuron density in the CA1 sector of the hippocampus in the animals treated with the BDNF chimeric peptide (Figure 16).

Figure 16. (Left) Nissl staining at high and low magnification of brain taken from rats subjected to transient forebrain ischemia and treated with either unconjugated BDNF or BDNF conjugate.

Figure 16

(Left) Nissl staining at high and low magnification of brain taken from rats subjected to transient forebrain ischemia and treated with either unconjugated BDNF or BDNF conjugate. (Right) Density of hippocampal CA1 neurons in control rats and four different (more...)

BDNF Chimeric Peptides and Permanent Middle Cerebral Artery Occlusion

The transient forebrain ischemia model is representative of global brain ischemia such as following cardiac arrest. Regional brain ischemia caused by thrombotic or embolic stroke is modeled by the middle cerebral artery occlusion (MCAO) method.47 Both permanent and reversible forms of MCAO have been examined. Initial studies were performed with the permanent MCAO model.48 Adult Sprague-Dawley rats were anesthetized with 70% nitrous oxide, 30% oxygen, and 0.5% halothane, and systolic blood pressure and arterial blood gases were normalized. Body temperature and blood glucose were also measured. The right common carotid artery and the right external carotid artery were exposed and the occipital artery and superior thyroidal artery were electrocoagulated. The right pterygopalatine artery was ligated and the right common carotid artery was clamped and a 40 nylon suture was inserted retrogradely via arteriectomy of the external carotid artery into the internal carotid artery. The suture was slowly advanced until resistance was felt. The external carotid artery was ligated and the common carotid artery clamp was released and the skin incision was sutured. After surgery, rats were allowed to recover spontaneous breathing and were kept for 24 hours in their cages with free access to food and water. Animals were treated intravenously with 1 of 4 different formulations: the BDNF chimeric peptide (BDNFMAb conjugate), the MAb alone, the BDNF alone, or saline.48 The dose of BDNF administered intravenously was 1, 5, or 50 μg/rat. The BDNF chimeric peptide was administered at either 0, 1, or 2 hours following permanent occlusion of the middle cerebral artery. Following sacrifice, the brain was removed, chilled, and 6x2 mm coronal slices were prepared with a brain matrix. Slice 1 is the most rostral and slice 6 is the most caudal section. These sections were stained with 2% 2,3,5 triphenyltetrazolium chloride (TTC). The TTC-stained viable brain tissue dark red while infarcted tissue is unstained. After staining, the sections were fixed in 10% formalin and then scanned on a 1200 dpi Umax scanner. The images were transferred to Adobe Photoshop 5.5 on a G4 Power Macintosh and quantified using NIH image software. The border between the infarcted and noninfarcted tissue was outlined with the image analysis system and the area of infarction was measured by subtracting the area of the noninfarcted ipsilateral hemisphere from that of the contralateral hemisphere for each of the 6 coronal slices. In addition, the area of brain tissue not stained by TTC was computed and the area of brain edema was calculated for each coronal slice. The infarct areas or brain edema areas for each slice were averaged and multiplied by a total thickness of brain (12 mm) to give the infarct and edema volumes. The statistical significance at the p < 0.05 level was determined by analysis of variance (ANOVA) with Bonferroni correction.48

The TTC stains for 16 rats are shown in Figure 17. The scanned images of each coronal section were inverted in Photoshop to generate the images shown in Figure 17. The infarcted area, which is not stained by TTC, appears black in the inverted image, and the healthy brain, which stains red by TTC, appears white in the inverted image. The data show that unconjugated BDNF, unconjugated OX26, and saline, have no effect on the stroke volume in the permanent MCAO model. However, intravenous administration of the BDNF chimeric peptide (BDNF-MAb conjugate) at a dose of 50 μg/rat of BDNF, reduced the infarct volume by 65% and reduced the edema volume by 66%.48

Figure 17. (Left) TTC stains of coronal sections of rat brain following 24-hour permanent MCAO.

Figure 17

(Left) TTC stains of coronal sections of rat brain following 24-hour permanent MCAO. There were four treatment groups. (Right) The infarct area for each of six different coronal slices is shown, and these values were used to compute the volume of the (more...)

A dose-response relationship was examined by reducing the BDNF administered in the form of the conjugate from 50 μg/rat to 5 μg/rat or 1 μg/rat and the infarct volumes of these animals are shown in Figure 18. The intermediate dose of BDNF chimeric peptide, 5 μg/rat, resulted in a 43% reduction in infarct volume in the total hemisphere and a 71% reduction in hemispheric edema volume.48 There was no significant effect on the infarct or edema volume with a very low dose of BDNF chimeric peptide, 1 mg/rat. A time response study was also performed at the higher dose of BDNF chimeric peptide (50 mg/rat). In this study, the BDNF chimeric peptide was delayed and not administered until either 1 or 2 hours after permanent occlusion of the middle cerebral artery. The BDNF chimeric peptide reduced the infarct and the edema volume by 52% and 59%, respectively, following delayed treatment given 1 hour after permanent occlusion of the middle cerebral artery. There was no significant reduction in infarct volume when the administration of the BDNF chimeric peptide was delayed until 2 hours after permanent occlusion of the middle cerebral artery (Fig. 18).

Figure 18. Dose response (Left) and time response (Right) of BDNF chimeric peptides in permanent middle cerebral artery occlusion.

Figure 18

Dose response (Left) and time response (Right) of BDNF chimeric peptides in permanent middle cerebral artery occlusion. From ref.

The permanent MCAO study shows that the BDNF chimeric peptide, but not the native BDNF, enables neuroprotection in regional brain ischemia following delayed noninvasive (intravenous) administration of the neurotrophin. Unconjugated or native BDNF has no neuroprotective effects in brain because (a) BDNF does not cross the BBB41 and (b) the BBB is not disrupted during the treatment period following occlusion of the middle cerebral artery.49 In the absence of hyperglycemia, the BBB is not disrupted for at least 6 hours after occlusion of the middle cerebral artery.49 In contrast, neuronal loss in regional ischemia occurs in the immediate period following occlusion of the artery when there is no BBB disruption.50

The infarct volume is progressively decreased as the dose of BDNF chimeric peptide is increased from 1 to 5 to 50 μg/rat. The brain uptake of the BDNF chimeric peptide in the rat is approximately 0.1% ID/g (Fig. 12). Therefore, the brain concentration of BDNF after the 5–50 μg/rat doses is increased 550 ng/g brain.48 The effect of the 5 mg/rat dose of intravenous BDNF chimeric peptide is comparable to the pharmacologic effects of ICV infusion of 2 μg/day of BDNF administered directly into the lateral ventricle.51 However, in these studies, it was necessary to begin the ICV infusion of the BDNF 24 hours before occlusion of the middle cerebral artery. The BDNF reaches brain parenchyma slowly by diffusion when the drug is administered by ICV infusion (Figure 2). The efficacy of diffusion decreases with the square of the diffusion distance and this accounts for the poor penetration of BDNF into brain parenchyma following ICV infusion (Figure 2). The diffusion distance in the human brain is 1000-fold greater than the diffusion distance in the rat brain, and the ICV infusion of neurotrophic factors has had not beneficial effect in human neuropathology.5 Moreover, it is not feasible to administer neuroprotective agents by craniotomy in human stroke, much less prior to the cerebral insult.

BDNF Chimeric Peptides and Reversible Middle Cerebral Artery Occlusion

The reperfusion associated with reversible brain ischemia is representative of human stroke and reperfusion can aggravate the development of brain edema in focal ischemia and accentuate neuronal loss.52 Therefore, a series of experiments was performed to determine the neuroprotection of intravenous BDNF chimeric peptides in a model using one hour reversible occlusion of the middle cerebral artery.53 In addition, these studies examine the long-term effects of neuroprotection with the BDNF chimeric peptide and infarct volumes are measured at both 24 hours and 7 days after reversible MCAO. In these studies, the middle cerebral artery was occluded for 60 minutes. In these treatment schedules, the BDNF chimeric peptide was administered intravenously in the femoral vein via a 30-gauge needle at 1 hour after middle cerebral artery occlusion or at the beginning of reperfusion. Alternatively, the administration of the BDNF chimeric peptide was delayed 2 hours after arterial occlusion, which is 1 hour after reperfusion. The hemispheric infarct zones were subdivided into cortical and subcortical infarct areas. None of the treatments resulted in a decrease in subcortical infarct volume (Fig. 19). Although unconjugated BDNF had no effect on the cortical infarct volume, the single intravenous injection of 50 μg/rat of the BDNF chimeric peptide administered at 60 minutes after MCAO resulted in a 68% reduction in cortical stroke volume (Fig. 19). If the intravenous administration of the BDNF chimeric peptide was delayed 2 hours after insertion of the catheter, there was a 31% reduction (p < 0.05) in the cortical infarct volume.53

Figure 19. Total (hemispheric), cortical, and subcortical infarct volume at 7 days after a 60 minute MCAO in rats treated with either a single intravenous injection of BDNF or BDNF chimeric peptide at a dose of 50 μg/rat at 1 hour after occlusion of the middle cerebral artery.

Figure 19

Total (hemispheric), cortical, and subcortical infarct volume at 7 days after a 60 minute MCAO in rats treated with either a single intravenous injection of BDNF or BDNF chimeric peptide at a dose of 50 μg/rat at 1 hour after occlusion of the (more...)

In the seven-day study, additional groups of rats were subjected to 1 hour of MCAO and treated with 50 μg/rat of BDNF or BDNF chimeric peptide. The animals were sacrificed 7 days later and the hemispheric cortical and subcortical infarct volumes were measured. Intravenous administration of the unconjugated BDNF caused no decrease in either total hemispheric infarct volume or cortical infarct volume and these results were comparable to the saline-treated animals (Figure 20). Conversely, the total hemispheric infarct volume at 7 days was reduced 53% (p < 0.01) by the BDNF chimeric peptide, compared to the total hemispheric infarct volume following treatment with the unconjugated BDNF.53 The cortical infarct volume was reduced 70% (p < 0.01) with the BDNF chimeric peptide compared to the unconjugated BDNF (Figure 20). The TTC stains of 6 different coronal sections obtained 7 days after treatment with either the unconjugated BDNF or the BDNF chimeric peptide are shown in Figure 20.

Figure 20. (A) TTC stains are shown for 2 different rats for all 6 coronal slabs.

Figure 20

(A) TTC stains are shown for 2 different rats for all 6 coronal slabs. A representative rat from the group treated with the BDNF chimeric peptide (conjugate) and a representative rat from the group treated with unconjugated BDNF are shown. (B) The area (more...)

Neuroprotection with unconjugated BDNF following intravenous administration has been reported in a 2-hour reversible MCAO model and in this study, BDNF was infused over a 3-hour period and the infusion was started 30 minutes after occlusion of the artery.54 There was a 55% reduction of cortical stroke volume following the intravenous infusion of 300 μg/rat of unconjugated BDNF. This dose is 6-fold higher than the dose of BDNF chimeric peptide administered in the studies described in Figures 19–20. Even though very large doses of unconjugated BDNF were administered intravenously, it is unexpected that neuroprotection is achieved with intravenous unconjugated BDNF. This is because the BBB transport of BDNF is negligible when there is no BBB disruption and when the BDNF is not conjugated to a BBB drug delivery system (Fig. 12). Intravenous administration of unconjugated BDNF was neuroprotective in a reversible MCAO model that was performed with chloral hydrate anesthesia that was associated with a significant level of hyperglycemia and a plasma glucose concentration of 220±47 mg %.54 This level of hyperglycemia, in parallel with a 2-hour reversible MCAO, causes vasculopathy and premature disruption of the BBB.55 This level of modest hyperglycemia may accelerate opening of the BBB in focal brain ischemia and enable high doses of unconjugated neurotrophic factor to enter the brain following intravenous administration. In the absence of hyperglycemia and vasculopathy, it is unlikely that unconjugated BDNF is neuroprotective following intravenous administration, because this protein does not cross the BBB.

One study suggests that BDNF can cross the BBB following intravenous administration, because radioactivity can be recovered in brain following the intravenous administration of [125I] BDNF.56 However, this is an artifact arising from peripheral metabolism of this highly cationic neurotrophin that is rapidly removed from blood by peripheral tissues, particularly the liver.38,41 This rapid uptake by peripheral tissues and metabolic degradation is followed by the release of radiolabeled low molecular weight metabolites such as iodotyrosine back to the bloodstream.1 The [125I]-tyrosine may then cross the BBB on the large neutral amino acid transporter and account for the radioactivity in brain following intravenous injection of [125I]-BDNF. This interpretation is supported by prior work using 2 different methodologies. In the first approach, there was no measurable uptake of radioactivity by brain following the intravenous injection of [125I]-BDNF in rats,41 when the peripheral metabolism of the neurotrophic factor was completely suppressed by pegylation of the neurotrophin (Fig. 11B). Second, the brain uptake of radioactivity following the intravenous administration of radiolabeled neuropeptide is suppressed 10-fold when the neuropeptide is labeled with 111-Indium, as opposed to 125-Iodine.57 Peptide degradation products labeled with 111-Indium, which are formed by metabolism in peripheral tissues, are not re-exported back to blood and are not taken up across the BBB in the form of low molecular weight radiolabeled degradation products.57 The interpretation of artifacts in relation to brain uptake of radiolabeled neuropeptides has been recently reviewed.1

In summary, intravenous doses of unconjugated BDNF at a level of 5–50 μg/rat results in no neuroprotection in regional brain ischemia.48,53 In contrast, these doses of BDNF chimeric peptide are highly neuroprotective in regional brain ischemia owing to transport of the BDNF chimeric peptide across the BBB in vivo on the endogenous BBB transferrin receptor (Fig. 15). Doses of BDNF chimeric peptide as low as 5 μg/rat result in substantial neuroprotection following intravenous administration.48 Moreover, the neuroprotection is observed following delayed intravenous administration.48,53 Neuroprotection is possible only during the first 1–3 hours after focal brain ischemia,50 when the BBB is not usually disrupted.49 Therefore, neurotrophin neuropharmaceuticals must be enabled to undergo transport across the BBB following intravenous administration if these agents are to be neuroprotective in regional brain ischemia.

Targeting Gene Therapeutics to the Brain

The chronic neurodegenerative diseases of the brain may also be amenable to brain gene therapy. Genes encoding neurotrophic factors and inserted in viral vectors cause neuroprotection following the intracerebral implantation of the viral vector in brain.58,59 However, the intracerebral implantation of viral vectors may not be feasible for human brain gene therapy for 2 reasons. First, the viral vectors, either adenovirus or herpes simplex virus (HSV), are highly immunogenic and all individuals have a pre-existing immunity to both of these common viruses. A single intracerebral injection of either adenovirus or HSV results in dose-dependent inflammatory reactions in the brain leading to demyelination.60,61 The viral-induced demyelination has been observed in rodent, primate, and human brain. The second limitation is that the intracerebral implantation of a viral vector will only allow for expression of the gene in a very circumscribed treatment volume1 < 1 mm3. In contrast, the areas requiring neuroprotection in a variety of CNS diseases, including AD, PD, or Huntington's disease, have treatment volumes that are log orders greater than 1 mm3. What is needed is a targeting technology for delivering therapeutic genes across the BBB following intravenous administration of a non-viral vector.

Noninvasive, nonviral gene targeting to the brain is possible with the application of the chimeric peptide technology.62 In this approach, a nonviral plasmid carrying a therapeutic or exogenous gene is encapsulated in the interior of neutral 85 nm pegylated immunoliposomes, as depicted in Figure 21A. Packaging the plasmid in the interior of the liposomes protects the exogenous gene from the ubiquitous endonucleases in the body following intravenous administration. The liposome or other nanocontainer is nonimmunogenic and is formed by either natural lipids or other nonimmunogenic polymeric substances. The injection of a plasmid encapsulated in a simple liposome into the bloodstream would result in rapid uptake of the complex by cells lining the reticuloendothelial system. The nanocontainer carrying the exogenous gene may be stabilized in the bloodstream with the use of pegylation technology.62 In this approach, several thousand strands of PEG2000 are attached to the surface of the liposome using lipid-PEG2000 conjugates. However, the injection of a pegylated liposome into the bloodstream would not allow for targeting to the brain. Brain gene targeting is accomplished by tethering targeting MAbs to the tips of approximately 1% of the PEG strands as depicted in Figure 21A. These MAbs trigger transcytosis of the liposome carrying the gene across the BBB and also trigger endocytosis of the therapeutic gene into the target neurons of brain.62 Targeting across the second barrier comprised of the neuronal or glial cell membrane in brain is achieved owing to expression of the TfR on the plasma membrane of brain cells.63

Figure 21. (A) Non-viral plasmid DNA is encapsulated in pegylated immunoliposomes constructed from neutral lipids.

Figure 21

(A) Non-viral plasmid DNA is encapsulated in pegylated immunoliposomes constructed from neutral lipids. There are approximately 3000 strands of polyethylene glycol of 2000 Daltons molecular weight, designated PEG2000, attached to the liposome surface, (more...)

Two different reporter genes were used in initial evaluation of this brain gene targeting technology, luciferase and β-galactosidase.62 The β-galactosidase histochemistry of rat brain obtained 48 hours after a single intravenous injection of the pegylated immunoliposome carrying the gene is shown in Figure 21B. The brain expresses the β-galactosidase gene widely as seen at low magnification (Fig. 21B). No β-galactosidase activity is observed in either control brain or in brain of animals injected with pegylated immunoliposomes wherein the OX26 MAb was replaced with the mouse IgG2A isotype control (Fig. 21C). Pyramidal neurons of the CA1-CA3 sectors of the hippocampus are clearly visualized as are the choroid plexi in both lateral ventricles and in the dorsal horn and the mamillary recess of the third ventricle. The paired supraoptic nuclei of the hypothalamus at the base of the brain are viewed at low magnification. At higher magnification, the microvasculature, the choroid plexus epithelial cells, and the thalamic nuclei showed β-galactosidase gene expression. Gene expression in neurons was observed throughout the brain in a region-specific manner.

These studies demonstrate it is possible to target therapeutic genes widely throughout the brain in a noninvasive way following a simple intravenous injection of a non-viral therapeutic gene packaged within the interior of pegylated immunoliposomes. The development of non-invasive, nonviral gene therapy of the brain requires a molecular formulation derived from the use of advanced drug targeting technology that brings together multiple disciplines including liposome technology, polymer technology, monoclonal antibody targeting technology, genetic engineering, and the molecular biology of therapeutic gene discovery. There are many diseases where it will be useful to have widespread expression of an exogenous gene throughout the brain including inborn errors of metabolism such as lysosomal storage disorders, fragile X syndrome, Rett's syndrome, Canavan's disease, and the inherited epilepsies. Other conditions that would be amenable to noninvasive, nonviral gene therapy of the brain includes cerebral acquired immune deficiency syndrome (AIDS), brain tumors, and neurodegenerative conditions such as Parkinson's disease or Alzheimer's disease. In the case of Parkinson's disease, it would be desirable to augment dopamine production only in the basal ganglia, and not in the cerebral cortex. Region specific expression of an exogenous gene using targeting technology is possible in the future with the use of cellspecific promoters at the 5'-end of the gene and cell-specific mRNA stabilizers at the 3'-end of the gene.62 The persistence of plasmid based gene formulations in brain cells in vivo can also be optimized as described recently.1 The availability of the complete sequence of the human genome and the emerging applications of the genomics technologies will further augment in the future the need for the development of non-viral, non-invasive gene therapy of the brain.

Drug Targeting to the Human Brain

The chimeric peptide technology has been reduced to practice in both rodents and primates and both neurodiagnostic and neurotherapeutic molecules have been delivered across the BBB in these animals.1 The extension of the chimeric peptide technology to the treatment of human brain disorders requires the genetic engineering of the murine antibodies that are used to target drugs and genes via the endogenous transport systems on the BBB. Human/Mouse chimeric MAbs are genetically engineered antibodies wherein approximately 85% of the sequence is of human origin. The murine sequences encoding the variable region of the heavy chain (VH) or the variable region of the light chain (VL) may be spliced into gene fragments containing the constant regions of the heavy chain (HC) and the light chain (LC) of human immunoglobulins. Recently, the murine 83–14 MAb to the HIR was genetically engineered and the human/mouse chimeric HIR MAb was produced.23 CHO cells that have been permanently transfected with a gene encoding the soluble extracellular domain of the HIR64 were used to establish an ELISA.23 These studies demonstrated a complete retention of the affinity of the chimeric MAb for the HIR following the genetic engineering, relative to the affinity of the original murine HIR MAb for the human insulin receptor (Fig. 22A). The genetically engineered chimeric HIR MAb was radiolabeled with [125I] and added to isolated human brain capillaries and there was rapid uptake of the chimeric HIR MAb as shown in Figure 22B. This uptake at the human BBB was suppressed by high concentrations of murine HIR MAb (Fig. 22B). The chimeric HIR MAb was conjugated with diethylenetriamine pentaacetic acid (DTPA) dianhydride and radiolabeled with 111-Indium. The radiolabeled chimeric HIR MAb was injected into anesthetized Rhesus monkeys and brain scans were obtained 2 hours after intravenous injection in the living monkey.23 As shown by the brain scan in Figure 22C, there was avid uptake of the chimeric HIR MAb by the primate brain and a comparable high uptake in the human brain is anticipated based on the studies with human brain capillaries shown in Figure 22B.

Figure 22. (A) ELISA shows equal affinity of the mouse HIR MAb and chimeric HIR MAb.

Figure 22

(A) ELISA shows equal affinity of the mouse HIR MAb and chimeric HIR MAb. The antigen in these studies was affinity purified soluble extracellular domain of the HIR that was produced from CHO cells. Immunocytochemistry was performed with the CHO cells (more...)

In summary, genetically engineered chimeric HIR MAb has been produced and the chimeric antibody has identical reactivity to the human and primate HIR as the original murine antibody. This chimeric HIR MAb may be used in humans for drug targeting to the brain of neurodiagnostic and neurotherapeutic drugs that normally do not cross the BBB. Based on these preclinical studies, it is anticipated that a fusion protein of the chimeric HIRMAb and BDNF would be highly neuroprotective in human stroke and other neurodegenerative disorders following intravenous administration.

Acknowledgments

Daniel Jeong skillfully prepared the manuscript. This work was supported by NIH grant NS-34698.

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Bookshelf ID: NBK6007
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