The blood–brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain‐derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep‐2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep‐2 transport and accumulation in brain endothelial cells were unaffected by the P‐glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P‐glycoprotein. However, competition studies show that activated α2‐macroglobulin, a specific ligand for the low‐density lipoprotein receptor‐related protein‐1 (LRP1) and Angiopep‐2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488‐Angiopep‐2 co‐localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep‐2 transport across the BBB is, in part, mediated by LRP1.

By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [125I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.

This report describes the synthesis and preliminary biological characterization of 2 (ANG1007) and 3 (ANG1009), two new chemical entities under development for the treatment of primary and secondary brain cancers. 2 consists of three doxorubicin molecules conjugated to Angiopep-2, a 19-mer peptide that crosses the blood-brain barrier (BBB) by an LRP-1 receptor-mediated transcytosis mechanism. 3 has a similar structure, with the exception that three etoposide moieties are conjugated to Angiopep-2. Both agents killed cancer cell lines in vitro with similar IC(50) values and with apparently similar cytotoxic mechanisms as unconjugated doxorubicin and etoposide. 2 and 3 exhibited dramatically higher BBB influx rate constants than unconjugated doxorubicin and etoposide and pooled within brain parenchymal tissue. Passage through the BBB was similar in Mdr1a (-/-) and wild type mice. These results provide further evidence of the potential of this drug development platform in the isolation of novel therapeutics with increased brain penetration.