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

Localization of Sodium, Potassium-Adenosine Triphosphatase in the Cerebral Cortex of Developing Rats

Active transport via Na+, K+-ATPase plays an important role in establishing and maintaining proper ionic gradient across all mammalian cell membranes. A strict regulation of Na+, and K+ ion levels is especially important in excitable cells such as neurons and myocytes. Inhibition of the enzyme is associated with several pathological conditions and has serious detrimental effects on cerebral cortical cells. Yet the enzyme appears to be limited in amount and distribution in newborn rats. In this study, Na+, K+- ATPase expression and distribution in early postnatal rats were studied using Western Immunoblotting, cytochemistry and immunocytochemistry. The Western blot study showed that α1, α2, α3, β1 and β2 isoforms of Na+, K+-ATPase were all present at birth. While the levels of a catalytic subunits remained relatively constant, the β subunits increased considerably during the first postnatal month. This might be associated with the additional role of β2 subunit as an adhesion molecule during neuronal development. For the cytochemical study, p-nitrophenylphosphate (p-NPP) was used as a substrate to study the subcellular and cellular distribution of Na+, K+-ATPase. The results suggested that the enzymatic reaction of Na+, K+-ATPase does not become apparent until the rats are 14 days old and its intensity increased in 21 and 28 day old rats. The ultrastructural study exhibited a p-NPP reaction in neurons and neuroglia. Localization of Na+, K+-ATPase in the developing blood-brain barrier (BBB) was also studied using cytochemical and [immunocytochmical] methods. The presence of all α subunits on the luminal surfaces of endothelial cells were detected using immunocytochemistry. The cytochemical methods, using p-NPP as a substrate, exhibited Na+, K+-ATPase on both the luminal and abluminal surfaces of the endothelial cells. More reaction was noted on the abluminal surface of the developing BBB. The newborn rat brain undergo remarkable remodeling during the first month following birth. During this period, some migrating neurons contact target cells while others undergo apoptosis. Neuroglia differentiate and increase in volume and the BBB becomes fully functional during this period. It is likely that cell-cell contact between various cell types is important in retaining the enzyme on the plasma membranes of cerebral cortical cells. The amount of Na+, K+-ATPase appears to increase considerably during the first month of postnatal development while the brain is being remodeled. Young animals might have regulatory mechanisms other than Na+, K+-ATPase to maintain cell volume and ionic balance

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