Improved Brain Penetration and Antitumor Efficacy of Temozolomide by Inhibition of ABCB1 and ABCG2.

The anticancer drug temozolomide is the only drug with proven activity against high-grade gliomas and has therefore become a part of the standard treatment of these tumors. P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) are transport proteins, which are present at the blood-brain barrier and limit the brain uptake of substrate drugs. We have studied the effect of P-gp and BCRP on the pharmacokinetics and pharmacodynamics of temozolomide, making use of a comprehensive set of in vitro transport experiments and in vivo pharmacokinetic and antitumor efficacy experiments using wild-type, Abcg2-/-, Abcb1a/b-/-, and Abcb1a/b;Abcg2-/- mice. We here show that the combined deletion of Abcb1a/b and Abcg2 increases the brain penetration of temozolomide by 1.5-fold compared to wild-type controls (P < .001) without changing the systemic drug exposure. Moreover, the same increase was achieved when temozolomide was given to wild-type mice in combination with the dual P-gp/BCRP inhibitor elacridar (GF120918). The antitumor efficacy of temozolomide against three different intracranial tumor models was significantly enhanced when Abcb1a/b and Abcg2 were genetically deficient or pharmacologically inhibited in recipient mice. These findings call for further clinical testing of temozolomide in combination with elacridar for the treatment of gliomas, as this offers the perspective of further improving the antitumor efficacy of this already active agent.

Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression.

EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.

Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug transporters P-gp and BCRP.

PURPOSE: Erlotinib (Tarceva®, OSI-774) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. As high-grade gliomas frequently show amplification, overexpression and/or mutation of EGFR, this drug has been tested in several clinical trials with glioblastoma patients, but unfortunately, with little success. As erlotinib is a known substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) we have investigated the effect of these ABC-transporters on the brain penetration of erlotinib. STUDY DESIGN: Erlotinib (50 mg/kg) was given by i.p. administration to wild-type (WT), Mdr1ab(-/-) (single P-gp knockout), Bcrp1(-/-) (single Bcrp1 knockout) and Mdr1ab(-/-)Bcrp1(-/-) (compound P-gp and Bcrp1 knockout) mice. Drug levels in plasma and tissues were determined by reversed-phase high-performance liquid chromatography. RESULTS: Relative to Mdr1ab(-/-)Bcrp1(-/-) mice that are deficient for both drug transporters, the area under the concentration time curve in brain tissue (AUC)(brain) of erlotinib decreased significantly by 1.6-fold in Mdr1ab(-/-) mice where Bcrp1 is present (49.6 ± 3.95 versus 31.1 ± 1.7, µg/g*h; P < 0.01). In Bcrp1(-/-) mice, were P-gp is present, a more pronounced 3.8-fold decrease to 13.0 ± 0.70, µg/g*h (P < 0.01) was observed, which is close to the 4.5-fold decrease in the AUC(brain) of erlotinib found in WT mice where both drug transporters are present (11.0 ± 1.35, P < 0.01). The plasma clearance of erlotinib was similar in mice deficient for P-gp and/or Bcrp1 compared with wild-type mice. In all other tissues the differences between the genotypes were negligible. CONCLUSIONS: Both P-gp and Bcrp1 reduce the brain penetration of erlotinib. Although P-gp appears to be the most effective factor limiting the brain penetration of erlotinib, the highest brain accumulation was observed when Bcrp1 was also absent. Strategies to inhibit P-gp/BCRP in patients to improve delivery of (novel molecular-targeted) substrate agents, such as erlotinib, to the brain may be required for treatment of intracranial malignancies.

Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies.

PURPOSE: To develop a transgenic mouse model of glioma that can be conveniently used for testing therapy intervention strategies. High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Typical for high-grade glioma is that glioma cells infiltrate extensively into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. EXPERIMENTAL DESIGN: High-grade gliomas were induced by stereotactic intracranial injection of lentiviral GFAP-Cre or CMV-Cre vectors into compound LoxP-conditional mice, resulting in K-Ras(v12) expression and loss of p16(Ink4a)/p19(Arf) with or without concomitant loss of p53 or Pten. RESULTS: Tumors reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. Especially, CMV-Cre injection into p53;Ink4a/Arf;K-Ras(v12) mice resulted in high-grade glioma with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up was accomplished by noninvasive bioluminescence imaging, and the practical utility for therapy intervention was shown in a study with temozolomide. CONCLUSION: We have developed a realistic high-grade glioma model that can be used with almost the same convenience as traditional xenograft models, thus allowing its implementation at the forefront of preclinical evaluation of new treatments.

High-grade glioma mouse models and their applicability for preclinical testing.

High-grade gliomas (WHO grade III anaplastic astrocytoma and grade IV glioblastoma multiforme) are the most common primary tumors in the central nervous system in adults. Unfortunately, despite great efforts in finding better therapies, high-grade glioma remains among the most devastating and deadliest of all human cancers. During recent years, genetic and molecular alterations that underlie this disease have been identified and advanced our basic knowledge about gliomagenesis. Moreover, understanding the molecular biology has also led to the development of genetically engineered mouse models that resemble many of the features of human gliomas. Ideally, such "patient-like" models should be instrumental for preclinical testing of novel therapeutics, but thus far they have not yet been widely implemented for this purpose. This review will discuss the advantages and shortcomings of the established high-grade glioma mouse models with emphasis on their potential applicability for preclinical testing of novel drugs and treatment regimens.

Tumor microvasculature supports proliferation and expansion of glioma-propagating cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The identification of 'cancer stem cells' (CSC) has shed new light on the potential mechanism of therapy resistance of these tumors. Because these cells appear to be more resistant to conventional treatments, they are thought to drive tumor regrowth after therapy. Therefore, novel therapeutic approaches that target these cells are needed. Tumor cells interact with their microenvironment. It has been reported that close contact between CSCs and tumor microvascular endothelium in GBM is important for CSCs to preserve their undifferentiated state and self-renewal ability. However, our understanding of this interaction is still rudimentary. This is in part due to a lack of suitable in vitro models that accurately represent the in vivo situation. Therefore, we set up a co-culture system consisting of primary brain tumor microvascular endothelial cells (tMVECs) and glioma propagating cells (GPCs) derived from biopsies of GBM patients. We found that tMVECs support the growth of GPCs resulting in higher proliferation rates comparing to GPCs cultured alone. This effect was dependent on direct contact between the 2 cell types. In contrast to GPCs, the FCS-cultured cell line U87 was stimulated by culturing on tMVEC-derived ECM alone, suggesting that both cell types interact different with their microenvironment. Together, these results demonstrate the feasibility and utility of our system to model the interaction of GPCs with their microenvironment. Identification of molecules that mediate this interaction could provide novel targets for directed therapy for GBM.

Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1-/-/Mdr1a/1b-/- (triple-knockout) and wild-type mice.

UNLABELLED: We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATP-binding cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2 (MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral pharmacokinetics of erlotinib hydrochloride in vivo. In vitro cell survival, drug transport, accumulation, and efflux of erlotinib were done using Madin-Darby canine kidney II [MDCKII; wild-type (WT), MDR1, Bcrp1, and MRP2] and LLCPK (WT and MDR1) cells and monolayers as well as the IGROV1 and the derived human BCRP-overexpressing T8 cell lines. In vivo, the pharmacokinetics of erlotinib after p.o. and i.p. administration was studied in Bcrp1/Mdr1a/1b(-/-) (triple-knockout) and WT mice. In vitro, erlotinib was actively transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was observed. In vivo, systemic exposure (P = 0.01) as well as bioavailability of erlotinib after oral administration (5 mg/kg) were statistically significantly increased in Bcrp1/Mdr1a/1b(-/-) knockout mice (60.4%) compared with WT mice (40.0%; P = 0.02). CONCLUSION: Erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib need to be addressed.

P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan.

PURPOSE: The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. EXPERIMENTAL DESIGN: We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(-/-) and Bcrp1(-/-), and compound Mdr1a/b(-/-)Bcrp1(-/-) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. RESULTS: The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(-/-) and Bcrp1(-/-) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(-/-)Bcrp1(-/-) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was approximately 0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice, respectively, resulting in 2.0-fold (P < 0.01), 0.65-fold (P, not significant), and 3.2-fold (P < 0.01), respectively, higher brain-to-plasma AUC ratios. Results using Mrp4(-/-) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp-mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. CONCLUSIONS: Our results using Mdr1a/b(-/-)Bcrp1(-/-) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.

Determination of topotecan in human and mouse plasma and in mouse tissue homogenates by reversed-phase high-performance liquid chromatography.

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay has been developed and validated for quantification of total topotecan in human and mouse plasma and in mouse tissue samples. Isocratic separation was achieved on a Zorbax SB-C(18) column and topotecan was monitored fluorimetrically. Two ranges of calibrations curves were used to determine lower levels of topotecan more accurately. Acceptable accuracy and precision was achieved for all matrices. Topotecan was stable upon repeated freeze-thawing for three cycles or storage for 24 h at ambient temperatures in spiked plasma samples and tissue homogenates, except in heart homogenates. In an additional validation experiment in which (14)C-labeled topotecan was administered to mice, the levels of unchanged topotecan were about 80-90% of the total radioactivity in tissue homogenates collected 10 min after drug administration and virtually similar as in plasma samples. However, results in tissue homogenates obtained 4 h post-drug administration indicated substantial metabolism of topotecan. This assay is suitable for studying the pharmacokinetics and tissue distribution of topotecan in mice. Our results demonstrate the importance of including all tissues of interest for pharmacokinetic studies in the validation procedure.

Blood-brain barrier and chemotherapeutic treatment of brain tumors.

The blood-brain barrier (BBB) is of pivotal importance to maintain homeostasis of the CNS, as it closely regulates the composition of the interstitial fluid in the brain. Unfortunately, malignancies that grow within the CNS may evade chemotherapeutic drugs using the same barrier, making this disease refractory to most chemotherapy regimens. This review will outline the impact of the BBB in brain cancer and discuss the efforts that have been made to enhance the drug exposure of brain tumors. Although this review will focus on the role of the BBB in primary brain cancer (malignant glioma), its impact on brain metastases will also be briefly discussed.