Activation of KATP channels increases anticancer drug delivery to brain tumors and survival

Several anticancer drugs are ineffective against brain tumor and do not impact patient survival because they fail to cross the blood-brain tumor barrier (BTB) effective levels. One such agent temozolomide is commonly used in brain tumor patients, which works better when combined with radiation or other anticancer agents. Likewise, trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2/neu over expressing gliomas may work well when combined with temozolomide. Nonetheless, both drugs do not cross the BTB to significantly impact patient survival. Beforehand we showed that potassium channel agonists when intracarotidly administered increased carboplatin and Her-2 antibody delivery in animal glioma models by triggering formation of brain vascular endothelial transcytotic vesicles. In this study, we investigated whether, intravenously administered, ATP-sensitive potassium channel (KATP) activator (minoxidil sulfate; MS) increases temozolomide and Herceptin delivery to brain tumors to induce antitumor activity and increase survival in nude mice with Glioblastoma multiforme (GBM) cells. The results clearly demonstrate that when given intravenously temozolomide crosses BTB at a relatively low amount while Herceptin failed to cross the BTB. However, MS co-infusion with [14C]-temozolomide or fluorescently labeled-Herceptin resulted in improved and selective drug delivery to brain tumor. We also showed that combination treatment with temozolomide and Herceptin has enhanced anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with GBM when co-infused with MS. Therefore, brain tumor patients may be benefited when anti-neoplastic agent delivery is increased selectively to the brain tumors using KATP channel agonists.

Introduction

Gliomas, accounting for 40% of all primary brain tumors, are the most common primary tumors that arise within the central nervous system in adults. Conventional treatments including chemotherapy, radiation therapy and surgery are often unsuccessful, resulting in limited improvement in overall survival. Standard chemotherapy regimens are not particularly effective due in part to their inability to pass through a compromised blood-brain barrier (BBB), which is often referred to as the blood-brain tumor barrier (BTB). Most anticancer drugs fail to penetrate the BTB at therapeutically effective concentrations thus allowing tumor cells to develop drug resistance, invade and progress to an untreatable high grade tumor. Drug concentration in cerebrospinal fluid (CSF) is widely used to predict unbound drug concentrationin the brain. However, depending on the physicochemical properties of the drug and the time and site of sampling, concentrations can vary considerably. Moreover the blood-CSF barrier lacks the tight endothelial cell junctions observed in BBB and BTB. This suggests that the CSF is not always an accurate surrogate for predicting unbound drug concentration in the brain, but is only a measure of the blood-CSF permeability. Hence, to predict drug delivery to the tumor, quantitative methods like HPLC-MS or quantitative autoradiography need to be used. Previous work from our laboratory has demonstrated that the BTB permeability can be increased by modulating adenosine triphosphate (ATP)-sensitive K+ channels (KATP) (Ningaraj et al., 2003b). This strategy exploits the responsiveness of brain tumor capillary endothelial cells that overexpress these channels to specific activators, like minoxidil sulphate (MS). MS is an active metabolite of Minoxidil that was clinically developed as a therapeutic for hypertension. In our study, we showed that MS selectively activated KATP channels present in the brain tumor and brain tumor capillary endothelial cells (Ningaraj et al., 2003b). It was also established that MS induced drug delivery increase is through the formation of transport vesicles, and not by opening of the endothelial tight junctions.

KATP channels are heteromultimers expressed in cerebral blood vessels that are composed of pore forming (inward-rectifying Kir 6.1 or 6.2) and sulfonylurea receptor subunits (SUR1 or SUR2). They regulate cerebral vascular tone and mediate the relaxation of cerebral vessels to diverse stimuli, including vasomodulators, in normal (Brayden, 2002) and disease states (Kitazono et al., 1995). KATP channels are also involved in secretion and muscle contraction by coupling metabolic activity to membrane potential. Activating mutations in the Kir 6.2 pore-encoding gene, KCNJ11, have been identified in both transient and permanent neonatal diabetes mellitus. These mutations are familial or more often sporadic in nature. Conformations in the ABCC8 gene-encoded SUR1, induced by the interaction of Mg-nucleotides with this regulatory channel subunit dictate KATP channel gating. The SUR1 also serves as a receptor for sulfonylurea drugs like Glibenclamide, which results in ATPindependent KATP channel inhibition. Critical to its role in channel behavior, polymorphisms of ABCC8 gene manifest as disorders of glucose metabolism (Sattiraju et al., 2008). Interestingly in cancer, the role of KATP channels is not yet clearly illustrated. In brain tumors the KATP channels are unregulated (Ningaraj et al., 2003a), possibly due to the hypoxic environment, which also true in ischemic conditions (Kitazono et al., 1995; Ruoslahti, 2002). Furthermore, endotheliumdependent regulation of cerebral blood vessel function is impaired in brain tumors (Cobbs et al., 1995; Morimoto et al., 2002), which might affect tumor capillary permeability in response to vasomodulators.

Temozolomide (8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4 (3H) one) is a second generation imidazotetrazine derivative. Antitumor activity of temozolomide is exerted by its active metabolite MTIC (5-(3-methyltriazen-1-yl) imidazole-4-carboxamide) which methylates the N7 and O6 position of guanines. The cytotoxicity of temozolomide is a result of the failure of the DNA mismatch repair system to find complementary bases for the methylated guanines, leading to the accumulation of nicks in the DNA that ultimately lead to cell cycle arrest and apoptotic cell death. In phase II and III trials temozolomide had improved the 2-year survival rate to 26% from 10% with radiation therapy alone. The effect of temozolomide and radiation seems to correlate with the methylation status of O-6-methylguanineDNA methyltransferase (MGMT). Glioma patients with tumors showing MGMT methylation had better survival advantage from combined treatment of temozolomide and radiation versus radiation alone. Although temozolomide exhibits great antitumor activity its use in the treatment of GBM is limited due to a variety of reasons, including its insufficient delivery across the BTB and its resistance to the drug. This has led to various studies that use a combination therapy approach where temozolomide is combined with either radiation therapy or other anti-cancer agents. For example, Herceptin, a monoclonal antibody that targets HER2-positive breast cancers. Herceptin alone or in combination with other drugs may possibly be used to treat the 15–20% of primary brain tumor patients who have HER2 positive status; provided that it crosses the BTB in therapeutically effective amounts. In this study we studied the combination effect of temozolomide and Herceptin on GBM cells in vitro and in vivo using a murine xenograft model. The combination study was done in conjunction with BTB permeability modulation using KATP channel agonist.