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

p97-conjugated ADR increased survival times in mice with intracranial tumors.

<p>The percent increase in survival times for mice with free or conjugated ADR compared to controls treated with PBS clearly indicates that ADR conjugation to p97 results in a survival advantage for mice with intracranial tumors.</p

Stability of p97 in the mouse 1 hr. after intravenous injection.

<p>Iodinated p97 protein is found intact in plasma after 1 h post-<i>i.v.</i> injection, but appears to undergo a cleavage event prior to appearance in urine.</p

Mice bearing subcutaneous tumors were treated with p97-ADR conjugates.

<p>a) Tumor growth of subcutaneous C6 glioma mass in mice treated with p97-ADR conjugate SYN002 (4 mg/kg ADR, σ-σ), ADR (4 mg/kg, ν-ν) or PBS (λ-λ). Arrows indicate injection schedule (also refer to Trial 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002469#pone-0002469-t001" target="_blank">Table 1</a>). (n = 9 for each treatment group) b) Activity of LDH and CPK in serum from mice treated with the p97-ADR conjugate SYN002 (4 mg/kg ADR), ADR alone (4 mg/kg) or PBS. Values represent mean values+standard deviation (n = 3).</p