Glioblastoma multiforme, due to its invasive nature, can be considered a disease of the entire brain. Despite recent advances in surgery, radiotherapy and chemotherapy, current treatment regimens have only a marginal impact on patient survival. A crucial challenge faced by cancer researchers is to effectively deliver drugs to invasive glioma cells residing in a sanctuary within the central nervous system. The blood–brain barrier (BBB) restricts delivery of many small and large molecules into the brain. Drug delivery to the brain is further restricted by active efflux transporters present at the BBB, which transport drugs out of the brain back into the blood. Current clinical assessment of drug delivery and hence efficacy is based on the measured drug levels in the bulk tumor mass that is usually removed by surgery. Mounting evidence suggests that the inevitable relapse and lethality of glioblastoma multiforme is due to a failure to effectively treat invasive glioma cells. These invasive cells hide in areas of the brain that are shielded by an intact BBB where they continue to grow and give rise to the recurrent tumor. Effective delivery of chemotherapeutics to the invasive glioma cells is therefore critical, and long-term efficacy will depend upon the ability of a molecularly targeted agent to penetrate an intact and functional BBB throughout the entire brain. This review highlights the various aspects of the BBB, and also the brain–tumor-cell barrier, a barrier due to expression of efflux transporters in tumor cells, that together can significantly influence drug response. It then discusses the special challenge of glioma as a disease of the whole brain, which lends particular emphasis to the need to effectively deliver drugs across the BBB to reach both the central tumor and the invasive glioma cells.

Gefitinib is an orally active inhibitor of the epidermal growth factor receptor approved for use in patients with locally advanced or metastatic non-small cell lung cancer. It has also been evaluated in several clinical trials for treatment of brain tumors such as high-grade glioma. In this study, we investigated the influence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) on distribution of gefitinib to the central nervous system. In vitro studies conducted in MadinDarby canine kidney II cells indicate that both P-gp and BCRP effectively transport gefitinib, limiting its intracellular accumulation. In vivo studies demonstrated that transport of gefitinib across the blood-brain barrier (BBB) is significantly limited. Steady-state brain-to-plasma (B/P) concentration ratios were 70-fold higher in the Mdr1a/b(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) mice (ratio of approximately 7) compared with wild-type mice (ratio of approximately 0.1). The B/P ratio after oral administration increased significantly when gefitinib was coadministered with the dual P-gp and BCRP inhibitor elacridar. We investigated the integrity of tight junctions in the Mdr1a/b(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) mice and found no difference in the brain inulin and sucrose space between the wild-type and Mdr1a/b(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) mice. This suggested that the dramatic enhancement in the brain distribution of gefitinib is not due to a leakier BBB in these mice. These results show that brain distribution of gefitinib is restricted due to active efflux by P-gp and BCRP. This finding is of clinical significance for therapy in brain tumors such as glioma, where concurrent administration of a dual inhibitor such as elacridar can increase delivery and thus enhance efficacy of gefitinib.Malignant gliomas account for approximately 70% of all new cases of malignant primary brain tumors diagnosed in the United States every year. Glioblastoma multiforme (GBM) is the most common type of glioma, accounting for approximately 60 to 70% of malignant gliomas (Wen and Kesari, 2008; CBTRUS, 2008) and claiming 12,000 lives every year (Davis et al., 2001). Epidermal growth factor receptor (EGFR) and its variant EGFRvIII play a critical role in the development of an aggressive phenotype of GBM; EGFR amplification, mutation, and overexpression are associated with poor prognosis and resistance to therapy (Brandes et al., 2008). Several therapeutic strategies targeting EGFR in GBM have been proposed, including the use of monoclonal antibodies against EGFR or EGFRvIII, vaccine therapies, bispecific antibodies, toxin-linked conjugates, and small molecule tyrosine kinase inhibitors (Omuro et al., 2007).

ATP-binding cassette transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) have been shown to work in concert to restrict brain penetration of several tyrosine kinase inhibitors. It has been reported that P-gp is dominant in limiting transport of many dual P-gp/BCRP substrates across the blood-brain barrier (BBB). This study investigated the influence of P-gp and BCRP on the central nervous system (CNS) penetration of sorafenib, a multitargeted tyrosine kinase inhibitor currently being evaluated in clinical trials for glioma. In vitro studies showed that BCRP has a high affinity for sorafenib. Sorafenib inhibited P-gp, but did not seem to be a P-gp substrate in vitro. CNS distribution studies showed that transport of sorafenib to the brain was restricted because of active efflux at the BBB. The brain-to-plasma equilibrium-distribution coefficient

Vemurafenib [N-(3-{[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane-1-sulfonamide (PLX4032)] is a novel small-molecule BRAF inhibitor, recently approved by the Food and Drug Administration for the treatment of patients with metastatic melanoma with a BRAF V600E mutation. The objective of this study was to investigate the role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in the distribution of vemurafenib to the central nervous system. In vitro studies conducted in transfected Madin-Darby canine kidney II cells show that the intracellular accumulation of vemurafenib is significantly restricted because of active efflux by P-gp and BCRP. Bidirectional flux studies indicated greater transport in the basolateral-to-apical direction than the apical-to-basolateral direction because of active efflux by P-gp and BCRP. The selective P-gp and BCRP inhibitors zosuquidar and (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino(1Ј,2Ј:1,6)pyrido(3,4-b)indole-3-propanoic acid-1,1-dimethylethyl ester (Ko143)

ABSTRACT:The objective of this study was to quantitatively examine the protein expression of relevant transporters and other proteins in the brain capillary endothelial cells isolated from wild-type mice and P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. After the isolation of brain capillary endothelial cells, a highly sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was used to determine the quantitative expression of membrane transporters at the blood-brain barrier (BBB) of the various mouse genotypes. Quantitative expression of 29 protein molecules, including 12 ATP-binding cassette transporters, 10 solute carrier transporters, five receptors, and two housekeeping proteins, was examined by quantitative proteomics in the four mouse genotypes. There was no significant difference in the expression of P-gp between the wild-type and Bcrp1(؊/؊) mice. Likewise, Bcrp expression was not significantly different between the wild-type and Mdr1a/b(؊/؊) mice. There was no significant difference in the expression of any of the measured proteins in the brain capillary endothelial cells across the genotypes, except for the lack of expression of the corresponding protein in the mice that had a genetic deletion of P-gp or Bcrp. In conclusion, using a quantitative proteomic approach, we have shown that there are no changes in the expression of several relevant transporters in brain capillary endothelial cells isolated from single and combination knockout mice. These data suggest that the mechanism behind the functional compensation between P-gp and Bcrp at the BBB is not related to compensatory changes in transporter expression.

ABSTRACT:The objective of this study was to determine the bioavailability and disposition of elacridar (GF120918; N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide) in plasma and brain after various routes of administration in the mouse. Elacridar is a potent inhibitor of P-glycoprotein and breast cancer resistance protein and has been used to examine the influence of these efflux transporters on drug distribution to brain. Friend leukemia virus strain B mice were administered 100 mg/kg elacridar either orally or intraperitoneally. The absolute bioavailability of elacridar after oral or intraperitoneal dosing was determined with respect to an intravenous dose of 2.5 mg/kg. At these doses, the absolute bioavailability was 0.22 for oral administration and 0.01 for intraperitoneal administration. The terminal half-life of elacridar was approximately 4 h after intraperitoneal and intravenous administration and nearly 20 h after oral dosing. The brain-to-plasma partition coefficient (Kp,brain) of elacridar increased as plasma exposure increased, suggesting saturation of the efflux transporters at the blood-brain barrier. The Kp,brain after intravenous, intraperitoneal, and oral dosing was 0.82, 0.43, and 4.31, respectively. The low aqueous solubility and high lipophilicity of elacridar result in poor oral absorption, most likely dissolution-rate-limited. These results illustrate the importance of the route of administration and the resultant plasma exposure in achieving effective plasma and brain concentrations of elacridar and can be used as a guide for future studies involving elacridar administration and in developing formulation strategies to overcome the poor absorption.

The study objective was to develop a formulation of elacridar to overcome its dissolution-rate limited bioavailability. Elacridar is a P-gp and BCRP inhibitor that has been used to improve the brain distribution of drugs that are substrates of P-gp and BCRP. The chronic use of elacridar is restricted due to poor solubility leading to poor oral bioavailability. A microemulsion formulation using Cremophor EL, Carbitol and Captex 355 (6:3:1) was developed. The elacridar microemulsion was effective in the inhibition of P-gp and Bcrp in MDCKII-transfected cells. FVBn mice were used to determine the bioavailability of elacridar after a 10 mg/kg dose of elacridar in the microemulsion, intraperitoneally and orally; and the absolute bioavailability was determined to be 1.3 and 0.47, respectively. Co-administration of elacridar microemulsion intraperitoneally with oral erlotinib in FVBn mice improved the erlotinib brain penetration three-fold. The current study shows that a microemulsion formulation of elacridar is effective in improving the bioavailability of elacridar and is an effective inhibitor of P-gp and Bcrp; in-vitro and in-vivo. It offers an alternative to the suspension and allows a decrease in the dose required to achieve a significant inhibitory effect at the blood-brain barrier.

The study objective was to investigate factors that affect the central nervous system (CNS) distribution of elacridar. Elacridar inhibits transport mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and has been used to study the influence of transporters on brain distribution of chemotherapeutics. Adequate distribution of elacridar across the bloodbrain barrier (BBB) and into the brain parenchyma is necessary to target tumor cells in the brain that overexpress transporters and reside behind an intact BBB. We examined the role of P-gp and Bcrp on brain penetration of elacridar using Friend leukemia virus strain B wild-type, Mdr1a/b(2/2), Bcrp1(2/2), and Mdr1a/b(2/2)Bcrp1(2/2) mice. Initially, the mice were administered 2.5 mg/kg of elacridar intravenously, and the plasma and brain concentrations were determined. The brain-to-plasma partition coefficient of elacridar in the wild-type mice was 0.82, as compared with 3.5 in Mdr1a/b(2/2) mice, 6.6 in Bcrp1(2/2) mice, and 15 in Mdr1a/b(2/2)Bcrp1(2/2) mice, indicating that both P-gp and Bcrp limit the brain distribution of elacridar. The four genotypes were then administered increasing doses of elacridar, and the CNS distribution of elacridar was determined. The observed and model predicted maximum brain-to-plasma ratios (E max ) at the highest dose were not significantly different in all genotypes. However, the ED 50 was lower for Mdr1a/b(2/2) mice compared with Bcrp1(2/2) mice. These findings correlate with the relative expression of P-gp and Bcrp at the BBB in these mice and demonstrate the quantitative enhancement in elacridar CNS distribution as a function of its dose. Overall, this study provides useful concepts for future applications of elacridar as an adjuvant therapy to improve targeting of chemotherapeutic agents to tumor cells in the brain parenchyma.