Modulation of KCa channels increases anticancer drug delivery to brain tumors and prolongs survival in xenograft model

Most anticancer drugs fail to impact patient survival since they fail to cross the blood-brain tumor barrier (BTB) at therapeutic levels. For example, Temozolomide (TMZ) exhibits some anti-tumor activity against brain tumors, so does Trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2 neu overexpressing gliomas. Nevertheless, intact BTB and active efflux system may prevent their entry to brain tumors. Previously we have shown that potassium channel agonists increased carboplatin and Her-2 neu antibody delivery in animal glioma models. Here, we studied whether potassium channel agonist increase TMZ and Herceptin delivery across the BTB to elicit anti-tumor activity and increase survival in nude mice with human glial tumor. The KCa channel activity and expression was also evaluated in human glioma tissues. We administered NS-1619, calcium-dependent potassium (KCa) channel agonist, with [14C]-TMZ, and quantified TMZ delivery. The results clearly demonstrate that when given systemically both TMZ and Herceptin do not cross the BTB in significant amounts, however, NS-1619 co-infusion with [14C]-TMZ and Herceptin resulted in enhanced drug delivery to brain-tumor cells. The combination treatment of TMZ and Herceptin also showed improved anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with brain tumor, when co-infused with KCa channel agonists. In conclusion, KCa channel agonists may benefit brain tumor patients by increasing anti-neoplastic agent’s delivery to brain tumors. A clinical outcome of this research is the discovery of a novel drug delivery system that circumvents the BBB/BTB to benefit brain tumor patients.

Background

Patients with highly aggressive brain tumor such as glioblastoma multiforme (GBM) have limited hope of long-term survival even with aggressive treatment regimens of surgery, radiation and/or chemotherapy. GBM cells have an infiltrative nature that causes them to widely disperse within the normal brain tissue, making complete surgical resection impossible. Chemotherapy often fails to effect a cure with GBM due to the efflux of drugs from tumor cells by p-glycoprotein, the resistance of the tumor cells to the drugs, and the failure of the drugs to cross blood-brain barrier (BBB), (referred to as the blood-brain tumor barrier (BTB) when present around the tumor).1 For chemotherapy with TMZ to be more effective against brain tumors it must overcome the twin hurdles of: (a) penetrating the BBB/BTB to achieve effective dosing, and (b) resistance due to the presence of O6 -methylguanineDNA methyltransferase (MGMT).2 To overcome the BBB several strategies have been investigated, such as osmotic disruption, convection-enhanced drug delivery, and intrathecal administration. None of these approaches are yet to provide substantial clinical benefit to brain tumor patients.

Global BBB disruption can result in unwanted toxic effects of anticancer drugs in normal brain. Our investigative approach is to create selective drug delivery to the tumor by noninvasive biochemical modification of the BTB by KCa channel agonist, such as NS-1619,3,4 which has the advantage of increasing the BTB permeability transiently for selective and enhanced anticancer drug delivery only to brain tumor cells.5 Our strategy is to modify systemic drug delivery through cerebral microvessels/capillaries to enable delivering anti-cancer agents to the dispersed pockets of tumor cells that remain after standard therapy. Previously we reported the enhanced delivery of anticancer agents such as carboplatin by means of potassium channel activation through a mechanism involving accelerated formation of pinocytotic vesicles, which can transport drugs across BTB.3,4

TMZ (8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4 (3H) one) is an imidazotetrazine derivative of dacarbazine. It is an inactive prodrug that undergoes rapid nonenzymatic hydrolysis at physiologic pH to the highly reactive metabolite, 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), which is further degraded to an active cytotoxic metabolite 4-Amino-5-imidazole-carboxamide (AIC).6-8 It is postulated that MTIC exerts its anti-tumor activity by alkylating the O6 and N7 positions of guanine in DNA and RNA.9-11 When TMZ’s high Her-2 receptor overexpressing cancers.19,20 Hence, we undertook a strategy of combining TMZ and Trastuzumab to ascertain whether synergistic therapeutic effect would be evident in nude mice with intracranially implanted GBM. We also investigated whether a survival benefit can be achieved in these mice by increasing drug delivery across the BTB. Our primary focus is to evaluate the efficacy of NS-1619 to modify BTB permeability to affect enhanced drug delivery.

Results

Cell cycle distribution. To investigate the effects of NS-1619, TMZ and Trastuzumabinduced A172 cell cycle perturbations, the cell cycle distribution was analyzed using flow cytometric measurements of cellular DNA content made at regular time intervals following treatment. As observed in other glioma cells21,22 A172 cells exhibited an increase in both S and G2 /M phase arrest at 48 and 72 hours after TMZ addition. However, the G2 /M arrest increased to 84% at 96 hours (Fig. 1). BrdU incorporation studies also confirmed that cells were arrested in G2 /M and not S phase (data not shown). In contrast, Trastuzumab induced clearance from plasma is paired with MTIC’s 2.5 minute halflife, the window for TMZ’s clinical activity is extremely narrow. Therefore, in order to achieve therapeutic concentrations of MTIC, TMZ is typically administered in multiple doses up to a period of two years.12 Despite the growing research evidence of TMZ’s efficacy,13 several limiting factors remain, including; crossing the BTB, adverse side effects brought about by the chronic dosing necessary to achieve in vivo therapeutically effective concentrations,11 and the chemotherapeutic resistance manifested by different brain tumor phenotypes and genotypes.14

To measure BBB or BTB permeability, the drug level in the tumor must be quantified by detection and identification of a drug or its metabolites by a quantitative assay such as the HPLCMS-MS method15 or by quantitative autoradiography (QAR).16 Using QAR, we demonstrated that TMZ, when systemically administered, does not cross the BTB in significant amounts, however, NS-1619 co-infusion with [14C]-TMZ resulted in enhanced drug delivery to brain tumor in a glioma xenograft model. Furthermore, molecularly targeted agents such as imatinib (for chronic myelogenous leukemia and gastro intestinal stromal tumors) and Trastuzumab (for Her-2 neu positive breast cancer) have been developed as potential selective inhibitors of critical signaling pathways and are included in treatment algorithms in variety of cancers.17

Clinical trials for GBM patients are ongoing that combine molecular targeted agents with cytotoxic agents.17 Nearly 20% of GBM patients overexpress Her-2 neu, that can be targeted with Trastuzumab.18 Some clinical studies have shown the benefit of using a combination of cytotoxic drug(s) with Trastuzumab against a 77% G0 /G1 phase arrest at 96 hours post treatment (Fig. 1). Furthermore, the addition of TMZ + Trastuzumab resulted in a 62% G2 /M phase arrest at 48 hours (Fig. 1), which continued until 96 hours