The Roles Of ABCB1 And ABCG2 Transpoters In Targeted Treatment For Brain Tumors

Brain tumors can be devided into two groups:the primary and metastatic tumors. Gliomas are the most common primary brain tumors. Glioblastomas are by far the most common and most malignant type. After standard treatment, the 2-year overall survival of glioblastoma patients is approximately only 25%. Brain metastases occur in a significant percentage of patients with common malignancies, with 5-year cumulative incidence rates of 16% in lung cancer patients,7% of breast cancer patients, and 5% of patients with colon cancer. In diseases such as melanoma, the incidence of brain metastatic disease is reported to be as high as 40%.Despite the dramatic advances in understanding the molecular basis for carcinogenesis and the development of new targeting agents to treat malignancies, a critical challenge that continues to face cancer researchers is overcoming the sanctuary for primary and metastatic disease found within the central nervous system (CNS). This sanctuary is formed by the blood-brain barrier (BBB), a mechanism that protects the brain from exposure to toxins, both endogenous and exogenous. This barrier prevents many of our traditional and new drugs from crossing from the circulation into the brain parenchyma.The BBB is formed by endothelial cells that are closely linked by tight junctions. In contrast to most endothelial cells in the rest of the body, endothelial cells in the BBB lack fenestra and have low endocytic activity. Moreover, the pericytes and astrocytes intimately surrounding the endothelial cells form a secondary lipid layer. Consequently, the BBB is almost impermeable. Entry of essential nutrients (e.g., glucose) is strictly regulated by a range of uptake transporters. Other substances can only enter the brain by passive diffusion across the BBB, and the ability to do so is determined by a series of molecular parameters such as sufficient lipid solubility, molecular weight, degree of ionization, plasma protein binding and tissue binding. Nonetheless, even compounds that have molecular characteristics in favor of passive diffusion demonstrate much lower brain penetration than expected due to the activity of drug efflux transporters. ABC drug transporters expressed at the BBB have well-known roles in the restriction of therapeutic agents into the brain. Of all the efflux transporters present in the BBB, two transporters are mainly responsible for the efflux of anti-cancer agents back into the blood capillaries. These proteins are ABCB1 and ABCG2Thereby, the key factors for anti-brain tummors are exploring new targeting agents and increasing their brain penetration. There are two efficient ways:firstly, exploring or selecting agents showing good membrane penetration and low affinity for ABCB1 and ABCG2; secondly, combination of ABCB1/ABCG2 specific inhibitors and anti-tumor agents.In this study, we first eveluated the affinity of five EZH2 inhibitors for ABCB1 and ABCG2, and their brain penetraion. Then we investigated the ability of ABCB1/ABCG2 inhibitor-elacridar enhancing the efficacy of vemurafenib anti-brain metastases of melanoma.Part I ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-inhibitorsEZH2 up-regulation in glioblastoma (GBM), a group of highly malignant and lethal tumors of the brain, can maintain sternness of tumor cells by inhibiting differentiation, suggesting EZH2 is necessary for glioma progression. Moreover, functional inactivation of EZH2 might attenuate multiple key signals involved in glioma stem cells self-renewal and survival, pinpointing EZH2 as a potential therapeutic target for GBM.We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcbl and/or Abcg2 knockout mice.We found that all EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by its low membrane permeability. Both P-gp and Bcrpl restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcbl;Abcg2 knockout mice. These results are likely due to its poor membrane permeability and question the clinical usefulness of GSK126.Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.Part II Inhibiting ABCB1 and ABCG2 enhances the efficacy of vemurafenib on melanoma brain metastasesThe incidence of melanoma has been steadily increasing worldwide over the past few decades. Brain metastases happens to more than 40% of patients with stage IV melanoma, which is a major cause of death in melanoma cases. Vemurafenib is a specific and potent inhibitor of mutated BRAF. Vemurafenib is a good substrate of ABCB1 and ABCG2, which limits the efficacy of vemurafenib on melanoma especially on brain metastases.Based on this, we performed the present study to evaluate the roles of ABCB1 and ABCG2 in treatment of brain metastases of melanoma with vemurafenib, in hope that these preclinical data would help in further advance of a durable response or less side effects in patients with melanoma brain metastases.We employed WT and Abcbla/1b;Abcg2-/- mice for intracranial injection of melanoma cells. Then we investigated the efficacy of different doses of vemurafenib combined with or without elacridar.The plasma AUCo-24h of Abcbla/1b;Abcg2-/- mice was only 1.4-fold higher than that of WT mice treated with 25 mg/kg vemurafenib, while the brain concentration in Abcb1a/1b;Abcg2-/- mice was 700-fold higher than in WT mice. Albeit elacridar enhanced the brain entry profoundly, the brain concentration in knockout mice dosed with 10 mg/kg vemurafenib was still 24 times higher than in WT mice with elacridar and same dose of vemurafenib. The bioavailability and brain penetration of vemurafenib is restricted by Abcbla/lb and Abcg2, what is more elacridar can enhance them considerably. We employed the subcutaneous tumor models where the penetration of vemurafenib to tumors was not limited by BBB, finding that the tumors were notably and similarly reduced by vemurafenib in the two treated groups even with different doses. With the intracranial melanoma models, the Abcbl and Abcg2 hindered the efficacy of vemurafenib against the brain metastases of melanoma, and elacridar is not powerful enough to reverse the limitation. Interestingly, in the first week, the growth of tumor in knockout mice was arrested by vemurafenib. But after the seventh treatment tumors regrew to the same size as that at the start of treatment, and did not respond to the therapeutics any more. Several methods were tried to explore the mechanism for the acquired resistance in our experiment. Unluckily, no clue was found.The current study showed the ABCB1 and ABCG2 transporters attenuated the initial response of the brain metastases of melanoma to vemurafenib. Coadministration with the ABC inhibitors (elacridar) is a strategy to improve the potency of vemurafenib, although it could not completely reverse the limitation by ABC transporters. Further studies are needed to explore the quickly resistant mechanism in our study.