A unique carrier for delivery of therapeutic compounds beyond the blood-brain barrier.

BACKGROUND: Therapeutic intervention in many neurological diseases is thwarted by the physical obstacle formed by the blood-brain barrier (BBB) that excludes most drugs from entering the brain from the blood. Thus, identifying efficacious modes of drug delivery to the brain remains a "holy grail" in molecular medicine and nanobiotechnology. Brain capillaries, that comprise the BBB, possess an endogenous receptor that ferries an iron-transport protein, termed p97 (melanotransferrin), across the BBB. Here, we explored the hypothesis that therapeutic drugs "piggybacked" as conjugates of p97 can be shuttled across the BBB for treatment of otherwise inoperable brain tumors. APPROACH: Human p97 was covalently linked with the chemotherapeutic agents paclitaxel (PTAX) or adriamycin (ADR) and following intravenous injection, measured their penetration into brain tissue and other organs using radiolabeled and fluorescent derivatives of the drugs. In order to establish efficacy of the conjugates, we used nude mouse models to assess p97-drug conjugate activity towards glioma and mammary tumors growing subcutaneously compared to those growing intracranially. PRINCIPAL FINDINGS: Bolus-injected p97-drug conjugates and unconjugated p97 traversed brain capillary endothelium within a few minutes and accumulated to 1-2% of the injected by 24 hours. Brain delivery with p97-drug conjugates was quantitatively 10 fold higher than with free drug controls. Furthermore, both free-ADR and p97-ADR conjugates equally inhibited the subcutaneous growth of gliomas growing outside the brain. Evocatively, only p97-ADR conjugates significantly prolonged the survival of animals bearing intracranial gliomas or mammary tumors when compared to similar cumulated doses of free-ADR. SIGNIFICANCE: This study provides the initial proof of concept for p97 as a carrier capable of shuttling therapeutic levels of drugs from the blood to the brain for the treatment of neurological disorders, including classes of resident and metastatic brain tumors. It may be prudent, therefore, to consider implementation of this novel delivery platform in various clinical settings for therapeutic intervention in acute and chronic neurological diseases.

Abeta peptide immunization restores blood-brain barrier integrity in Alzheimer disease.

Immunization with amyloid beta (Abeta) peptides or passive immunization with antibodies against Abeta has been reported to reduce plaque burden, neuritic dystrophy, early Tau pathology, microgliosis as well as reversing learning and memory deficits. This has created a central paradox: how does vaccination in peripheral tissues reduce plaque burden in the brain? No single explanation for these phenomena has yet been presented. To reconcile these observations, we demonstrate that the integrity of the blood-brain barrier (BBB), a structural barrier between the brain and the blood, is compromised in Tg2576 Alzheimer disease (AD) model mice. We immunized Tg2576 mice with Abeta before and after the onset of AD-type neuropathology and observed that BBB permeability, amyloid burden, and microgliosis are decreased in immunized mice. It is concluded that the integrity of the BBB is disrupted in AD mice, and after Abeta immunization the immune system clears Abeta from sources in the brain as it would in peripheral organs lacking barriers. Once Abeta is removed, the integrity of the BBB is restored. The data therefore provide an intellectual framework for understanding how the immune system can clear amyloid deposits from AD brains and suggest new strategies for limiting disease progression in amyloidopathies.

Identification of a novel route of iron transcytosis across the mammalian blood-brain barrier.

OBJECTIVE: This study was undertaken to assess the role of p97 (also known as melanotransferrin) in the transfer of iron into the brain, because the passage of most large molecules is limited by the presence of the blood-brain barrier, including that of the serum iron transporter transferrin. METHODS: To study the function of the soluble form of p97, we followed the uptake of radioiodinated and 55Fe loaded p97 and transferrin by the brain during a 24-hour period. RESULTS: We show that the soluble form of p97 has the ability to transcytose across the murine blood-brain barrier, and its transcytosis can be inhibited in a specific manner. We also provide evidence that p97 transports iron into the brain more efficiently than transferrin. CONCLUSIONS: These data support the idea that p97 is an important iron transporter across the blood-brain barrier in normal physiology and possibly in neurodegenerative diseases, such as Alzheimer disease, in which iron homeostasis in the brain becomes disrupted.

Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model.

OBJECTIVE: To establish the generality of cerebrovascular pathology frequently observed with Alzheimer disease, we have assessed blood-brain barrier (BBB) integrity using the Alzheimer disease model Tg2576 mice in which cognitive deficits and neuritic plaque formation develop around 10-12 months of age. METHODS: We assessed BBB integrity using well-established methods involving albumin and Evans blue uptake and introduce the use of a novel perfusion protocol using succinimidyl ester of carboxyfluorescein diacetate. RESULTS: BBB permeability is increased in the cerebral cortex of 10-month-old Tg2576 mice preceding Alzheimer disease pathology presentation. Furthermore, when compared with their nontransgenic littermates, 4-month-old Tg2576 mice exhibit compromised BBB integrity in some areas of the cerebral cortex. An age-related increase in albumin uptake by the brains of Tg2576 mice, compared with nontransgenic mice, was also observed. These findings were supported by quantitative Evans blue analysis (p = 0.07, two-way analysis of variance). CONCLUSION: A breakdown of BBB was evident in young 4- to 10-month-old Tg2576 mice. Compromised barrier function could explain the mechanisms of Abeta entry into the brain observed in experimental Alzheimer disease vaccination models. Such structural changes to the BBB caused by elevated Abeta could play a central role in Alzheimer disease development and might define an early point of intervention for designing effective therapy against the disease.

p97 as a biomarker for Alzheimer disease.

The search is ongoing for a reliable serum biomarker for AD. The level of iron is elevated in the brain of Alzheimer's disease (AD) patients. Our studies have demonstrated that the level of the iron transport protein, p97, is increased in the serum of AD patients but not in various control groups. These results have recently been confirmed by another laboratory who extended our findings by demonstrating that p97 is not elevated in other neurodegenerative diseases. This qualifies p97 as a potentially powerful biomarker specific for AD. Although the relationship between increased level of iron and p97 in the AD brain is not well understood, our research supports the hypothesis that p97 over-expressed by senile plaque associated reactive microglia is exocytosed and appears in blood. The relationship between elevated levels of serum p97 and AD, together with the possible future clinical application of p97 are considered in this report.

Assessing p97 as an Alzheimer's disease serum biomarker.

The search is ongoing for a reliable serum biomarker for AD. The level of iron is elevated in the brain of Alzheimer's disease (AD) patients. Our studies have demonstrated that the level of the iron transport protein, p97, is increased in the serum of AD patients but not in various control groups. These results have recently been confirmed by another laboratory who extended our findings by demonstrating that p97 is not elevated in other neurodegenerative diseases. This qualifies p97 as a potentially powerful biomarker for AD. Although the relationship between increased level of iron and p97 in the AD brain is not well understood, our research supports the hypothesis that p97 over-expressed by senile plaque associated reactive microglia is exocytosed and appears in blood. The relationship between elevated levels of serum p97 and AD, together with the possible future clinical application of p97 are considered in this report.