Yanick Bertrand

Involvement of the low‐density lipoprotein receptor‐related protein in the transcytosis of the brain delivery vector Angiopep‐2

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.

High transcytosis of melanotransferrin (P97) across the blood–brain barrier

The blood–brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14‐fold higher than that of holo‐transferrin, with no apparent intra‐endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration‐ and conformation‐dependent, suggesting that the transport of P97 is due to receptor‐mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low‐density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Green tea catechins as novel antitumor and antiangiogenic compounds.

The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease. Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans. Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea. Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied. Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance. This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans. The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities. In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.

Transport characteristics of a novel peptide platform for CNS therapeutics

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.

Brain endothelial cells as pharmacological targets in brain tumors

The blood-brain barrier contributes to brain homeostasis by controlling the access of nutrients and toxic substances to the central nervous system (CNS). The acquired brain endothelial cells phenotype results from their sustained interactions with their microenvironment. The endothelial component is involved in the development and progression of most CNS diseases such as brain tumors, Alzheimer’s disease, or stroke, for which efficient treatments remain to be discovered. The endothelium constitutes an attractive therapeutical target, particularly in the case of brain tumors, because of the high level of angiogenesis associated with this disease. Drug development based on targeting differential protein expression in the vasculature associated with normal tissues or with disease states holds great potential. This article highlights some of the growing body of evidence showing molecular differences between the vascular bed phenotype of normal and pathological endothelium, with a particular focus on brain tumor endothelium targets, which may play crucial roles in the development of brain cancers. Finally, an overview is presented of the emerging therapies for brain tumors that take the endothelial component into consideration.

Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease.

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimers disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimers disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimers disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G-Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimers disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimers disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimers disease.

Abstract 4417: Uptake of the new paclitaxel-derivative (ANG1005) by the low-density lipoprotein receptor-related protein1 (LRP1) is related to the aggressive phenotype of glioblastoma cells

The most common primary brain tumour is glioblastoma multiforme (GBM). Despite latest cancer researches to improve the outcome of patients with GBM, this highly aggressive tumour remains one of the most difficult tumors to treat. Angiochem9s Engineered Peptide Compounds (EPiC) provides a non-invasive and flexible platform for small and large molecules to treat brain diseases. Based on these properties, we have created a portfolio of new drug entities composed of siRNA, peptides and mAbs, The most advanced, ANG1005, is a new taxane derivative currently completing two Phase ½ clinical trials for the treatment of primary and secondary brain tumors. This EPiC drug is comprised of one Angiopep-2 peptide conjugated to three molecules of paclitaxel. Recently, Angiopep-2 has been shown to use the low-density lipoprotein related protein 1 (LRP1) has a gateway across the blood-brain barrier. Interestingly, LRP1 seems to be required in many tumours for the aggressive behaviour and is overexpressed in GBM. Here, we show an increase uptake of Angiopep-2 in implanted brain tumour using in vivo imaging when compared to the contralateral normal brain region. In these tumors, ANG1005 could be detected by LC/MS/MS analysis and immunohistochemistry. We next characterized Angiopep-2 and ANG1005 uptake in human U87 glioblastoma cell line. The transfection of U87 cells with siRNA LRP1 decreased the uptake of both Angiopep-2 and ANG1005 indicating that LRP1 is involved in their internalization in glioblastoma cells. Furthermore, we measured an increase in the uptake of both Angiopep-2 and ANG1005 in glioblastoma (U87) cells under three experimental conditions that mimic the aggressive cancer cell microenvironment: acidic pH, serum deprivation and hypoxia. Interestingly, under these conditions LRP1 expression was also increased. The increase of ANG1005 uptake and LRP1 expression indicates that the aggressive cancer microenvironment phenotype favors the entry of this anticancer drug into glioblastoma cells. Overall these results demonstrate that the EPiC drug ANG1005 can be an effective therapeutic strategy to target brain cancer cells and aggressive tumors such as GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4417.