No abstract

The safest and most effective way of targeting drugs to the entire brain is via delivery systems directed at endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective in animal models including primates, but no clinical trials have been performed so far. This review focuses on the well-characterised transferrin and insulin receptor-targeted systems, as well as on the more recently described systems that use the low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor). The possibilities and limitations of these systems are compared and their future for human application is discussed.

No abstract

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood-brain barrier (BBB) and its concentration in biological media can be easily quantified. Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 microg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37 degree C. This was approximately 3-fold higher compared to binding at 4 degree C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37 degree C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed. In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.