Anthony Regina

Identification and Design of Peptides as a New Drug Delivery System for the Brain

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.

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells.

Abstract: Two membrane glycoproteins acting as energy‐dependent efflux pumps, mdr‐encoded P‐glycoprotein (P‐gp) and the more recently described multidrug resistance‐associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P‐gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood‐brain barrier, but localization of MRP has not been well characterized yet. Using RT‐PCR and immunoblot analysis, we have compared the expression of P‐gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P‐gp‐encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P‐gp, is not predominantly expressed in the blood‐brain barrier endothelial cells and that Mrp1 and the mdr1b P‐gp isoform may be present in other cerebral cells.

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.

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood–brain barrier. As P‐glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P‐glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 µg/mL puromycin for the first 2 days of culture or 3 µg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin‐treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 × 10−6 cm/s) and a high electrical resistance (up to 500 Ω × cm2). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood–brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2.

Paclitaxel is highly efficacious in the treatment of breast, head and neck, non‐small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P‐glycoprotein (P‐gp) at the blood–brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel.

Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes.

Although it has been well established that the drug efflux pump P‐glycoprotein (P‐gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial. The aim of this study was to compare the expression of P‐gp and of multidrug resistance‐associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types. Western blotting analysis revealed higher expression of P‐gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P‐gp and Mrp1 expression was not modified in more differentiated astrocytes obtained when cultured with db‐cAMP for 48 hr. Our functional analysis of P‐gp showed a modest effect of P‐gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (a substrate of P‐gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P‐gp and MRP) by astrocytes. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P‐gp). Taken together, our results provide the first biochemical and functional evidence supporting the expression of P‐gp and Mrp1 in rat cultured astrocytes. J. Neurosci. Res. 60:594–601, 2000

Drug transport to the brain: Key roles for the efflux pump P-glycoprotein in the blood-brain barrier

1. The blood-brain barrier (BBB) contributes to brain homeostastis and fulfills a protective function by controlling the access of solutes and toxic substances to the central nervous system (CNS). The efflux transporter P-glycoprotein (P-gp) is a key element of the molecular machinery that confers special permeability properties to the BBB. 2. P-gp, which was initially recognized for its ability to expel anticancer drugs from multidrug-resistant cancer cells, is strongly expressed in brain capillaries. Its expression in the BBB limits the accumulation of many hydrophobic molecules and potentially toxic substances in the brain. 3. The purpose of this review is to summarize the current state of knowledge about the expression of P-gp, its cellular localization as well as its possible functions in the BBB.

Multidrug resistance in brain tumors: Roles of the blood-brain barrier

Malignant brain tumors and brain metastases present a formidable clinical challenge against which no significant advances have been made over the last decade. Multidrug resistance (MDR) is one of the main factors in the failure of chemotherapy against central nervous system tumors. The MDR1 gene encoding P-glycoprotein (P-gp), a drug efflux pump which plays a significant role in modulating MDR in a wide variety of human cancers, is highly expressed in the blood–brain barrier (BBB). The BBB controls central nervous system exposure to many endogenous and exogenous substances. The exact molecular mechanisms by which the BBB is involved in the resistance of brain tumors to chemotherapy remain to be identified.The purpose of this review is to summarize reports demonstrating that P-gp, one of the most phenotypically important markers of the BBB, is present in primary brain tumors and thus plays a crucial role in their clinical resistance to chemotherapy.

Curcumin inhibits tumor growth and angiogenesis in glioblastoma xenografts

Among the natural products shown to possess chemopreventive and anticancer properties, curcumin is one of the most potent. In the current study, we investigated the effects of this natural product on the growth of human glioma U-87 cells xenografted into athymic mice. We show here that curcumin administration exerted significant anti-tumor effects on subcutaneous and intracerebral gliomas as demonstrated by the slower tumor growth rate and the increase of animal survival time. While investigating the mechanism of its action in vivo, we observed that curcumin decreased the gelatinolytic activities of matrix metalloproteinase-9. Furthermore, treatment with curcumin inhibited glioma-induced angiogenesis as indicated by the decrease of endothelial cell marker from newly formed vessels and by the diminution of the concentration of hemoglobin in curcumin-treated tumors. We also demonstrate, using an in vitro model of blood-brain barrier, that curcumin can cross the blood-brain barrier to a high level. These are the first results showing that curcumin suppresses tumor growth of gliomas in xenograft models. The mechanisms of the anti-tumor effects of curcumin were related, at least partly, to the inhibition of glioma-induced angiogenesis.

New Angiopep-Modified Doxorubicin (ANG1007) and Etoposide (ANG1009) Chemotherapeutics With Increased Brain Penetration

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.