Month: September 2019

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Abstract

This review elucidates ongoing research, which show improved delivery of anticancer drugs alone and/ or enclosed in carriers collectively called nanomedicines to cross the BBB/ BTB to kill tumor cells and impact patient survival. We highlighted various advances in understanding the mechanism of BTB function that has an impact on anticancer therapeutics delivery. We discussed latest breakthroughs in developing pharmaceutical strategies, including nanomedicines and delivering them across BTB for brain tumor management and treatment.

Methods

We performed an extensive literature search and highlighted important studies on the regulation of BTB permeability with respect to nanotech-based nanomedicines for targeted treatment of brain tumors. We have reviewed research articles that describe the development of specialized molecules and nanospheres, which carry payload of anticancer agents to brain tumor cells across the BBB/ BTB and avoid drug efflux systems. We highlighted research on the identification and development of targeted anti-cancer drug delivery to brain tumors. In addition, we discussed multimeric molecular therapeutics and nanomedicines that were encapsulated in nanospheres for treatment and monitoring of brain tumors.

Results

In this context, we quoted our research on large conductance calcium-activated potassium channels (BKCa) and ATP-dependent potassium channels (KATP) as portals of enhanced antineoplastic drugs delivery. We showed that several innovative drug delivery agents such as liposomes, polymeric nanoparticles, dendrimers and many such tools can be utilized to improve anticancer drugs and nanomedicines across the BTB to reach brain tumor cells.

Conclusion

This review might interest both academic and drug company scientists involved in drug delivery to brain tumors. We further seek to present evidence that BTB modulators can be clinically developed as combination drug or/ and as stand-alone anticancer drugs. Eventually, it is expected that unrelenting effort from the scientific community in developing novel drug delivery methods should increase the survival rate of brain tumor patients, which is dismally low presently.

INTRODUCTION

The global cases of primary brain tumors were estimated at 256,213 in 2012, and in the USA, it is estimated to be 26,070 in 2017 as per the Central Brain Tumor Registry of the US. It is reasonable to assume that the incidence of secondary (metastatic) brain tumors is 10-fold higher than the primary brain tumors. Available literature indicates that nearly half the number of patients post brain radiation and/or surgical resection develop recurrences in the brain within a year leading to high mortality [1]. Glioma, specifically glioblastoma multiforme (GBM) is a deadly form of brain tumors. The treatment is extremely difficult because cancer cells hide behind BBB. These tumors develop extreme resistance to most treatment modalities. The BBB consisting of cerebral microvessels/ capillaries prevents toxic agents in the circulation and delivery of small and large therapeutic molecules, including nanomedicines and nanospheres. Studies have shown that even a damaged and more permeable BBB can pose serious challenges to drug delivery into the brain for the management of stroke, Alzheimer’s and Parkinson’s diseases [2]. However, even a damaged and more permeable BBB can pose serious challenges to drug delivery into the brain. Hence many methods are employed to get around the BBB and BTB.

Glioma Treatment

Conventional diagnosis and treatment are not effective in reducing glioma patient mortality [3]. In addition, low penetration of anticancer drugs across the BBB/ BTB makes the treatment very difficult. Besides, detecting diffused gliomas using imaging agents on CT and MRI is difficult because the imaging agents do not penetrate the intact BBB at the tumor edges. Hence complete resection of tumor mass is very hard. In order to address this issue, we biochemically modulated BTB to increase permeability to drug and imaging agents, selectively to brain tumors in experimental glioma models.

BBB/ BTB and Drug Delivery

Sufficient quantities of most anti-cancer drugs fail to cross the BTB. The invading glioma cells in the tumor edges are ideal targets for anti-cancer agents due to the presence of unique gene/protein expression pattern [3, 4]. Several promising anticancer drugs are effective against cancers outside the brain but fail against brain tumors in clinical trials, in part due to poor BTB penetration. For instance, Gleevec (Novartis, USA) is less useful against brain cancer due to its poor BTB penetration but has demonstrated efficacy in patients with chronic myelogenous leukemia and gastrointestinal stromal tumor [5]. Similarly, invading edges of brain tumor are not clearly detected by imaging agents as the agents do not penetrate intact BTB easily [5, 6]. Some studies showed that peripheral benzodiazepine receptors (PBRs), which are overexpressed in leading edges of gliomas [7] may be targeted with a PBR ligand linked to contrast enhancing dyes [7, 8] or chemotherapy. Others have used DCE-MRI method to measure the brain vascular permeability [9-10]. In our laboratory, we used KCa and KATP channel openers to increase Magnevist delivery to brain tumor edges as imaged by DCE-MRI [11-13]. In order to develop methods for increasing the delivery of new wave of targeted drug entities referred as nanomedicines to brain tumor, we need to have a precise understanding of the basics of BBB and BTB biology and their permeability regulation.

Popularly known as “neurovascular unit” consisting of endothelial cells (ECs), tight junction proteins (TJp) connecting the ECs, glial, pericytes, and astrocytic foot processes form the BBB. A cartoon (Fig. 1) depicts the key differences in morphology and phenotype, specifically with respect to potassium channel expression on BBB and BTB. Essential nutrients, such as glucose and amino acids get through receptor-mediated endocytosis and cross BBB to maintain vital brain functions. The nutrients and most anticancer drugs (except lipohilic drug entities) that are generally water soluble (hydrophilic) require carrier-mediated transport, receptor-mediated transcytosis and absorptive-mediated transcytosis to enter the brain cells.

Hence drug delivery strategies must involve an understanding of these BBB constituents and their interaction with tumor cells, as well. The BTB around the tumor allows very little while mostly throwing out anticancer drugs by efflux mechanism, including small molecules and therapeutic monoclonal antibodies (MAbs) back to the circulation. Now researchers are working on a variety of carriers such as nanomedicines and nanospheres that might penetrate the BTB. Such nanomedicines armed with targeted drugs are expected to supplement conventional chemotherapy and radiotherapy. The development of nanomedicines for treatment of cancer is defined by their penetration across BTB vasculature that surrounds the tumor. Further nanomedicines’ retention in tumor cells without being expelled by multi-drug resistant P-glycoprotein (Pgp) efflux system (Fig. 2) determines their efficacy. The strategy of targeting tumor blood vessel–specific marker(s) for improving targeted drug delivery has generated great interest in the development of more precise and less toxic anticancer drugs [14, 15]. The real challenge is to improve the bioavailability of cytotoxic agents to neoplasms while minimizing toxicity to normal tissues. More research is required to study how to increase tumor-specific drug delivery and at the same time minimize toxicity to normal tissues. Due to advances in personalized therapy, more targeted drugs like cetuximab (Erbitux®), and therapeutic MAbs like ABX-EGF, EMD 720000, h-R3 and Herceptin, are found to be effective in treating cancers outside the brain. However, they fail to control brain tumors because they fail to cross the BTB in adequate quantity. These targeted anticancer drugs are ineffective to block epidermal growth factor receptors (EGFR), which are often amplified and mutated in human gliomas. Despite evidence of ‘leaky’ tumor centre, the BBB surrounding the proliferating glioma is still impermeable [16, 17]. So low-grade gliomas are insensitive to some chemotherapeutics partly due to incomplete drug delivery across BTB. Therefore, more research is needed to understand the biochemical regulation of the BBB in its normal and abnormal (BTB) states. Then only efforts to deliver therapeutic compounds to brain tumors might yield favourable results.

Background and Aim

The overexpression and alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) that encodes large-conductance calcium-activated voltage-sensitive potassium (BKCa) channels are implicated in the development of human cancers. Dysfunctional angiogenesis in hypoxic tumors is a challenge to intravenous anticancer drug treatments. Hypoxic factors also lead to abnormal vascular functions posing hurdle for anticancer drug delivery to tumors. The aim of this study was to explore the role of BKCa channels in tumor angiogenesis, specifi cally with regard to release of vascular endothelial growth factor (VEGF). Materials and Methods: We subjected the glioma cells under hypoxia and normoxia and studied the expression and activity of BKCa channels in in vitro and in vivo tumor models. Then, we studied the proangiogenic factor, VEGF, in tumors and monitored the neoangiogenic process. Results: We presented in vivo and cell based in vitro experimental evidence on the direct and indirect interactions of BKCa channels with VEGF signaling. There was evidence that under hypoxia, glioma cells overexpressed KCNMA1 and increased VEGF secretion. By inhibiting KCNMA1, we showed that VEGF secretion was signifi cantly reduced, thus potentially controlling angiogenesis, which has implications for vascular permeability and anticancer drug delivery. Moreover, there were differences in alternate splicing of KCNMA1 between normal and malignant cells under hypoxia and normoxia. Conclusion: We conclude that BKCa channels regulate hypoxia-induced angiogenesis. Therefore, serious effort is needed to better understand the molecular mechanisms of BKCa channelopathies triggering angiogenesis and progression of glioma. The modulators of BKCa channels could be viable in new anticancer therapeutics. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University, Atlanta, GA, USA (approved No. A0706007_01) on July 20, 2007.

INTRODUCTION

Cancer is a chronic disease characterized by uncontrolled cell growth.[1] Cancer cells typically go through four stages – initiation, proliferation, invasion, and metastasis. There are over 100 different types of cancer, and each is classifi ed by the type of cells that are initially affected.[2] Cancer cells divide uncontrollably to form lumps or masses of tissue called tumors that can grow and interfere with several bodily functions. Cell signaling involving vascular endothelial growth factor (VEGF) and its VEGF receptor (VEGFR) plays a major role in cancer progression by promoting new blood vessels formation called neoangiogenesis.[3] Disruption of the genes encoding either VEGF or any of the three receptors of the VEGF family results in embryonic lethality because of failure of blood vessel development.[4] VEGFR2 is the main signal transducing VEGFR for angiogenesis and mitogenesis of endothelial cells, which is directly related to cell cancer. VEGFs are combined with VEGFRs to activate the VEGF signaling cascade leading to angiogenesis. As shown in Table 1, specifi c isoforms of VEGFs are combined with specifi c VEGFRs to regulate several critical cell functions and also impact on human health and diseases.[5-8]

VEGF can be detected in both plasma and serum samples of patients, with much higher level in serum. Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors.[9] Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.[10,11] The autocrine VEGF signaling is crucial for tumor initiation and transformation into highly aggressive cancers.[3] The blocking of autocrine VEGF secretion provides a promising strategy to develop new therapeutic approaches.[12] VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis, such as cancer, psoriasis, and rheumatoid arthritis. Direct role of VEGF in tumor growth has been shown using dominant negative VEGFRs to block in vivo proliferation, as well as blocking antibodies to VEGF or to VEGFR2.[13] Interference with VEGF function by targeting the VEGF signaling pathway is a major interest in drug development for blocking angiogenesis in primary and metastatic brain tumors [Figure 1].

The calcium-activated voltage-sensitive potassium (BKCa) channels interact with a variety of proteins both at the plasma membrane and with intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. However, the role of BKCa channels in tumor microenvironment including hypoxia is yet to be explored. Hypoxia promotes vessel growth by upregulating multiple proangiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology.[14] In general, uncontrolled cancer growth and subsequent neoangiogenesis lead to hypoxic tumor microenvironment.[15] VEGF expression increases dramatically in hypoxic conditions due to a number of activated oncogenes that are overexpressed in hypoxia. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis.[16] Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors.[17] The second-generation multitargeted tyrosine kinase inhibitor targets VEGFR, platelet-derived growth factor receptor, and c-kit as key proteins responsible for tumor growth and survival.[18] Pazopanib exhibits good potency against all of the human VEGFRs and closely relate to tyrosine receptor kinases in vitro and demonstrates antitumor activity in several human tumor xenografts. Therefore, VEGFRs are attractive therapeutic targets.[19]

Recent work has shown the central role of K+ channels affect multiple conditions of the tumor microenvironment including hypoxia and adenosine.[20] It has long been known that the interaction of tumor cells with their host microenvironment, including endothelial cells and the extracellular matrix, plays an important role in tumor growth and invasion.[21,22] Hypoxia induces the transcriptional activation of signaling pathways and regulates tumor growth through differential alternative splicing.[23] Nonetheless, very little is known about the effect of hypoxia on the alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) either in tumor cells or in endothelial cells. Understanding the role of hypoxia in KCNMA1 splicing is extremely crucial to study blood–brain tumor barrier (BTB) function and improve drug delivery. Our previous studies[24-30] have revealed that human brain microvascular endothelial cells adjacent to glioma cells overexpress BKCa channels, as opposed to human brain microvascular endothelial cells in normal brain. Our aim is to seek whether the tumor cells (with or without physical contact) overexpress KCNMA1 or its splice variants to increase secretion of VEGF to induce angiogenesis.

Introduction

Brain tumors are the most common type of solid tumor in both children and adults. It is estimated that 78,980 new cases of primary malignant and nonmalignant brain tumor and other central nervous system tumors will be diagnosed in the United States in 2019. This includes an estimated 23,830 primary malignant brain tumors and 55,150 nonmalignant brain tumors.[1] The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is problematic, in terms of both cure rates and long-term quality of life.[2] Gliomas are insidious due to their highly invasive and destructive manifestation, and thus, GBM patients’ median survival is <15 months.[3] The low-grade gliomas often manifest as astrocytomas or anaplastic astrocytomas and sometimes progress dramatically to a highly malignant GBMs. There is a critical need to develop reliable biomarkers for the early detection and successful treatment to attenuate gliomas’ progression into GBM. Gene expression profiling studies have identified many GBM specific genes, involved in epigenetic inactivation, that drive glioma transformation.[4] GBM has distinct gene expression pattern among different histological types and grades of gliomas. [5] Increasing evidence shows that potassium channels are integral part of glioma cell growth and survival.[6]

The importance of voltage-gated potassium channels in tumor biology has aroused great interest in recognition of ion channels as potential targets for tumor therapy.[5‑8] Several potassium channels have been implicated in tumor progression and cell proliferation. Specifically, large-conductance, voltage-sensitive, Ca2+-activated potassium (BKCa) channels are overexpressed in human glioma cells.[7] Calcium-activated potassium channel subunit alpha‑1 (KCNMA1) encodes the α-subunit of the BKCa channels. They play a key role in cellular functions and have recently emerged as regulators of tumorigenesis. These channels respond to changes in intracellular calcium ([Ca2+]i ) and membrane potential, and their expression correlates with increased malignancy in gliomas.[8‑10] Studies have shown that pharmacological inhibition of BKCa channel by iberiotoxin abolished the activation of K+ ion channel currents and attenuated migration of glioma cells.[11‑13] Elucidation of the molecular mechanisms of KCNMA1 regulation is critical for the understanding of a variety of physiological and pathological conditions. The aim of this study was to study the effect of KCNMA1 modulation in glioma cells, thus offering a promising biomarker for early diagnosis and prognostication of high-grade gliomas.

The entire cloned fragment was sequenced using multiple primers to ensure whether each sequence has a good overlap with the adjacent sequence. This sequence was then compared to the KCNMA1 sequence NM 002247.2 (NCBI, Bethesda, MD, USA). Subsequently, the TOPO clones were digested with HindIII and XbaI to release the 3.7 kb fragment, which was gel purified and ligated into the HindIII-XbaI site of pcDNA6-V5/HIS expression vector (Invitrogen). The resulting plasmid pcDNA6/KCNMA1‑expressing KCNMA1 from a T7 promoter containing blasticidin resistance marker was used for selection of stable transfectants

Elacridar (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) is a potent inhibitor of P-glycoprotein (PgP) and breast cancer resistance protein (BCRP). It has been investigated as modulator of efflux transporters, which are shown to be very active in various cancers. The PgP is over expressed on tumor cells and plays a key role cancer resistance to anti-cancer drugs, specifically chemotherapeutics that are thrown out of the tumor cells making them ineffective over time. Researchers are investigating whether inhibiting the PgP may shut down efflux pumps, particularly on cancer cells, thus facilitating entry of drug in cancer cells leading to selective damage to cancer cells rather than normal cells. Elacridar, one such PgP pump inhibitor holds promise as an adjuvant in cancer therapy since clinical trials are underway to test its safety and efficacy in humans. This short review will address very specifically the prospect of using elacridar as adjuvant with anticancer drugs to brain tumors. Since Blood Brain Barrier (BBB) and Brain Tumor Barrier (BTB) pose hurdles to anticancer drug delivery and their reverse transport possibly due to efflux transport proteins on BTB. This review elucidates ongoing research on elacridar delivery across BBB/BTB, and the drug’s safety, efficacy and toxicity. We further seek to present evidence that PgP modulators along with BTB permeabilizing agents such as potassium channel activators can be clinically developed as adjuvants with anticancer drugs.

Introduction

The Central Brain Tumor Registry (USA) has estimated global incidence of primary brain tumors to be about 256,213 in 2012, which increased to 26,070 in 2017. Furthermore, the incidence of secondary (metastatic) brain tumors will be nearly 10-fold higher than the primary brain tumors. Majority of brain tumor patients have very short life expectancy (1 year) even after receiving brain radiation and/ or surgical resection [1]. Glioma, specifically glioblastoma multiforme (GBM) is a deadly form of brain tumor and its management is extremely difficult because of heterogeneity, chemo resistance followed by inadequate delivery of anticancer drugs due to P-glycoprotein (PgP) transport pumps expression on tumor cells as well as in blood brain tumor barrier (BTB). BBB is a physical and biological barrier consisting of endothelial cells (ECs), tight junction proteins (TJp) connecting the ECs, glial, pericytes, and astrocytic foot processes allows essential nutrients, such as glucose and amino acids through receptor mediated endocytosis to maintain vital brain functions. The nutrients and most anticancer drugs (except lipohilic drug entities) that are generally water soluble (hydrophilic) require carrier-mediated transport, receptor-mediated transcytosis and absorptive-mediated transcytosis to enter the brain cells (Figure 1). Studies have shown that BBB obstructs delivery of over 98% of CNS drugs [2]. Hence, various methods are employed to get around the physiological barrier posed by the blood-brain tumor barrier (BTB), including inhibiting PgP proteins.

BBB/BTB and Drug Delivery

Most anti-cancer drugs generally fail to cross the BTB in sufficient quantities. The invading glioma cells in the tumor edges are ideal targets for anti-cancer agents due to the presence of unique gene/protein expression pattern [3]. Several promising anticancer drugs are effective against cancers outside the brain but fail against brain tumors in clinical trials, in part due to poor BTB penetration. For instance, Gleevec (Novartis, USA) is ineffective against brain tumors since it hardly crosses BTB, but has demonstrated efficacy in patients with chronic myelogenous leukemia and gastrointestinal stromal tumor [4]. Similarly, invading edges of brain tumor are not clearly detected by imaging agents as the agents do not penetrate intact BTB easily [5,6]. In order to develop methods for increasing delivery of new wave of targeted drug entities referred as nanomedicines to brain tumor, we need to have precise understanding of the basics of BBB and BTB biology and their permeability and efflux regulation

Glioma treatment

Conventional diagnosis and treatment are not successful in reducing glioma patient mortality. Further, low penetration of anticancer drugs across BTB makes the treatment very difficult. Complete excision of diffused gliomas is nearly impossible partly due to low detection by CT and MRI as the imaging agents do not fully penetrate the intact BBB at the tumor edges. In order to address this issue, we biochemically modulated BTB to increase permeability to drug and imaging agents, selectively to brain tumors in experimental glioma models [5].

Elacridar- Research and Development

Elacridar development as a clinical drug candidate has been well reviewed elsewhere [7], which documents drug transporter families, including PgP and BCAR. There is extensive discussion on ADME, PK-PD and translational aspect in drug discovery and development of these classes of PgP inhibitors.

Many studies have shown that elacridar is a potent inhibitor of PgP and breast cancer resistance proteins (BCRP) [8,9] and have described elacridar as a multidrug resistance-reversal drug that restored sensitivity of multidrug-resistant tumors to doxorubicin. A recent animal study reported brain distribution and bioavailability of elacridar after different routes of administration [10]. It was shown with different routes of elacridar administration that the brain-to-plasma partition coefficient of elacridar increased as plasma exposure increased, suggesting saturation of efflux transporters at BBB. The role of P-gP and BCRP in limiting the distribution of substrate drugs across BBB has been examined using elacridar as a dual inhibitor of both P-gP and BCRP. Drugs such as morphine and amprenavir were shown to be at higher levels in brain after coadministration with Elacridar [11,12]. Furthermore, elacridar increased brain distribution of several tyrosine kinase inhibitors (TKIs) including imatinib, dasatinib, gefitinib, sorafenib, and sunitinib [13-19]. Studies in mice have shown that P-gP and BCRP at the BBB limits brain penetration of sunitinib and its active metabolite, however, oral administration of elacridar improved brain penetration of sunitinib [19,20].

Furthermore, preclinical studies have shown that paclitaxel penetration was improved by coadministration of elacridar or tariquidar in brain tumor [21]. These studies further advances the claim that elacridar can be clinically useful in delivering and retention of anticancer drugs across BTB for better control of brain tumors

Chronic administration of elacridar poses many hurdles, including formulation due to its unfavorable physicochemical properties. Elacridar is extremely lipophilic making it insoluble in water and poorly soluble in most other aqueous solvents [22]. Preclinical studies have shown the variability in plasma and tissue concentrations. Even clinical trials found inter-subject variability after oral dosing [23]. With respect to brain tumors, its availability in brain tumors and its ability to shut down PgP is the key to its development as adjuvant with anticancer drugs. In this regard, elacridar brain penetration in mice was dose-dependent and affected by the P-gP and BCRP at the BBB as shown by positron emission tomographic imaging [24,25]. Therefore, its versatile clinical candidacy as adjuvant in brain tumor treatment is hampered by its unpredictable adsorption and elimination as shown in both preclinical models and clinical applications. Furthermore, careful elucidation of elacridar BTB penetration mechanism and its distribution in brain tumors when coadministered with anticancer drugs, including monoclonal antibodies (MAbs) and nanomedicines is critical.

In addition, co-administration of elacridar with anticancer drugs that are substrates for P-gP and BCRP might improve its BTB penetration for better efficacy. As the brain tumor is heterogeneous with uneven BTB permeability [26] across the tumor spread (metastatic brain tumors), it may throw an uncertain pharmacokinetic challenge with uneven spread of target tumor cells. Nevertheless, safe and efficacious elacridar is what we need at the moment if we need to control brain tumor growth by targeting tumor cells that express BCRP and PgP. Major concern in glioma therapy is tumor resistance to chemotherapy, partly due to their insufficient delivery and lack of retention in tumor cells [27]. Major interest with this strategy will be the efficient use of targeted therapies such as Herceptin, TRKIs and emerging nanomedicines that have shown promise in peripheral cancers while failing in controlling brain tumors due to reasons discussed above. Added advantage of improved delivery and longer retention of anticancer therapies is the potential use of low doses and milder neuro/ peripheral toxicity

Hence, drug delivery strategies must involve understanding of these BBB/BTB constituents and their interaction with tumor cells, as well. The BTB around the tumor allow very little while mostly throwing out anticancer drugs by efflux mechanism, including small molecules and therapeutic monoclonal antibodies (MAbs) back to the circulation. Researchers are working on variety of carriers such as nanomedicines and nanospheres that might penetrate BTB. Such nanomedicines armed with targeted drugs are expected to supplement conventional chemotherapy and radiotherapy. Development of nanomedicines for treatment of cancer is defined by their penetration across BTB vasculature that surrounds the tumor. Further nanomedicines’ retention in tumor cells without being expelled by multi drug resistant P-glycoprotein (PgP) efflux system (Figure 2) determines their efficacy. Recent success in controlling cancer by targeting tumor and tumor blood vessel-specific marker(s) has renewed interest in development of more precise and less toxic anticancer drugs [28]. More research is required to investigate how to increase tumor-specific drug delivery, improve bioavailability of cytotoxic agents to neoplasms, and at the same time minimize toxicity to normal tissues. Due to advances in personalized therapy, more targeted drugs like cetuximab (Erbitux®), and therapeutic MAbs like Herceptin, ABX-EGF, EMD 720000 and h-R3 are shown to be effective in treating cancers outside of brain. However, they fail to control brain tumors because they fail to cross BTB in adequate quantity. These targeted anticancer drugs are ineffective to block epidermal growth factor receptors (EGFR), which are often amplified and mutated in human gliomas. Despite evidence of ‘leaky’ tumor centre, the capillaries surrounding the proliferating glioma as well as the brain tissue surrounding the tumor are nearly as impermeable as the BBB [29]. It is incorrect to assume that the disrupted BBB facilitates drug delivery to gliomas because diffuse tumor-cell invasion is a hallmark of even low-grade gliomas. Hence, understanding the biochemical regulation of the BBB in its normal and abnormal state (BTB) is of great importance as efforts continue to deliver therapeutic compounds to brain tumors.

Introduction

Blood–Brain Barrier Permeability

The blood–brain barrier (BBB) is a cerebrovascular permeability barrier that strictly regulates the entry of variety of substances, including therapeutics and imaging agents into the brain. Unlike blood vessels that circulate blood to other areas of the body, the microvessels that perfuse the brain consist of special endothelial cells and pericytes that lack fenestrations and are sealed by endothelial tight junction proteins (TJp) (Ningaraj et al., 2003; Black and Ningaraj, 2004). These tight endothelium, pericytes, and astrocytic foot processes provide a physical barrier that, together with metabolic barriers, form the BBB (Fig. 17.1). At the blood–brain tumor barrier (BTB) the TJp and astrocytoic foot processes are compromised, leading to a “leaky” BBB. The BBB protects the brain against pathogens and other damaging agents in the circulatory system, including electrolytes that changes the composition of the systemic blood. At the same time the BBB permits entry of certain substances, such as small fat-soluble (lipophilic) molecules that can freely diffuse through the barrier. The BBB also permits entry of essential nutrients, such as glucose and amino acids through receptor-mediated endocytosis to maintain vital brain functions. These nutrients are generally water soluble (hydrophilic), and require more complex mechanisms to cross the BBB, such as carriermediated transport, receptor-mediated transcytosis and absorptive-mediated transcytosis .

While protective under normal circumstances, the BBB prevents the delivery of drugs, other therapeutic molecules and imaging agents to the brain. Furthermore, the BBB blocks delivery of more than 98% of central nervous system (CNS) drugs (Pardridge, 2002). Therefore, the challenge posed by the BBB is compelling, particularly as the population ages and the incidence of neurodegenerative diseases such as stroke, Alzheimer’s disease, and Parkinson’s disease increase in prevalence. The problem is particularly acute for patients with malignant brain tumors, who cannot benefit from anticancer drugs effective in treating tumors elsewhere in the body. A solution to drug delivery across the BBB would be to produce an exponential increase in the number of drugs available for the treatment and prevention of CNS disorders. Regulation of BBB/BTB permeability function may involve endogenous nitric oxide production and a cyclic GMP-dependent mechanism (Liu and Sundqvist, 1997)

Blood–Brain Tumor Barrier Permeability

An ideal strategy for delivery of therapeutic molecules to brain tumors would involve selective opening of only that portion of the BBB that serves the brain tumor. The portion of the BBB that surrounds a brain tumor is known as the BTB. Over the years, many methods have been used to enhance delivery of anticancer drugs and imaging agents to brain tumors (Misra et al., 2003). Some of these efforts have focused on changing the therapeutic molecule or encapusalting the drugs in nanospheres or nanoparticles (Amrawy et al., 2016), rather than altering the barrier. Well considered drug design or drug delivery techniques were aimed at “lipidizing” otherwise poorly-lipid soluble compounds, by either developing lipophilic analogs or packing hydrophilic drugs in liposomes. This approach has been limited by the relative instability of lipophilic analogs in the blood, and the rapid removal of these analogs from the blood as a direct result of their increased lipid solubility. Other strategies have focused on circumventing the barrier, for example by directly injecting drugs into the brain or through the use of implantable drug delivery devices, such as Gliadel. These strategies are highly

Biochemical Modulation of the Blood–Brain Tumor Barrier

Biochemical disruption is based on the finding that the permeability of tumor capillaries is enhanced relative to that of normal brain capillaries by administration of certain vasoactive molecules (Fig. 17.2). BTB function is generally impaired in brain tumors because the endothelium-dependent regulation of cerebral blood vessel function is abnormal (Morimoto et al., 2002), which might affect BTB permeability in response to vasomodulators (Cobbs et al., 1995). Accordingly, biochemical opening with vasoactive agents holds promise for increased anticancer drug delivery selectively to brain tumors across BTB, compared to the toxic Mannitol-induced osmotic disruption. This is due to its nonselectivity and unwanted distribution of anticancer drugs to normal brain. We and others have tested a variety of vasoactive compounds, such as leukotriene (LTC4 ), bradykinin (BK), cGMP, and certain potassium channel agonists to selectively disrupt the BTB without affecting the BBB, for enhanced anticancer drug delivery in experimental brain tumor models (Sugita and Black, 1998; Hashizume and Black, 2002).

Using immunoblot and immunolocalization studies, we established that BKCa channels were overexpressed in rat brain tumor capillary endothelium and tumor cells, and demonstrated the functional presence of channels in isolated rat brain tumor capillary endothelial and tumor cells (Ningaraj et al. 2002). The BKCa channel has been shown to be the convergence point of a BK signaling pathway involving nitric oxide, soluble guanylyl cyclase, and cGMP (Fig. 17.1). While BK has been shown to activate BKCa channels, other known activators of BKCa do not act as vasodilators; for example, 1,3-dihydro [2-hydroxy(trifluoromethyl)phenyl] (trifluoromethyl)-2H-benzimidazolone (NS- 1619) (Holland et al., 1996). Another major class of potassium channels, KATP channels, have also been shown as overexpressed on BTB and brain tumors (Ruoshlati, 2002; Ningaraj et al., 2003). We have developed methods of increasing delivery of anticancer agents to brain tumor using potassium channel activators, which include compounds that indirectly activate potassium channels, such as nitric oxide, nitric oxide donors, and other activators of soluble guanylyl cyclase. These potassium channel activators selectively increase the permeability of the BBB and, in particular the BTB, for small to large-sized molecules, drugs, and imaging agents (Fig. 17.2).

Functional Ion Channels on Blood–Brain Tumor Barrier

Several researchers, including us, have shown the importance of ion channels on BTB permeability regulation and their role in anticancer drug delivery (Ningaraj, 2006; Ningaraj et al., 2002, 2003; Black and Ningaraj, 2004). We recently reviewed the role of BTB-associated ion channels in increasing the BTB permeability for delivering therapeutic, prophylactic, and diagnostic agents to brain tumors (Ningaraj and Khaitan, 2015). We showed that the BTB can be modulated to increase delivery of combination of drugs: trastuzumab with temozolomide in glioma models (Ningaraj et al., 2009). The underlying mechanism is not completely understood, but it involves formation of brain vascular endothelial transcytotic vesicles to facilitate transport of the drugs, as demonstrated by us earlier (Ningaraj et al., 2002, 2003). Recently, we described the role of BKCa and KATP channels in brain tumor cell growth. We also showed that modulators of BKCa and KATP channels may be utilized to enhance the delivery of antineoplastic drugs and imaging agents to glioma cells in brain tumor models (Ningaraj and Khaitan, 2015)

Nanomedicines and Nanoimaging

Despite the challenges faced due to the presence of BBB/BTB, there has been some progress in developing new strategies to treat gliomas. Specifically, gliomas are diffusive with several leading edges, making it difficult to target them with anticancer drugs as well as imaging agents. A recent review article (Juratli et al., 2013) has described the nanomedicine approaches that have been developed and some of the technologies that are being translated to the clinic. It also discusses the hurdles of effective brain tumor treatment and how various nanomedicine techniques that are being explored to overcome BBB using liposomal and polymeric nanoparticles. It is well studied that the BBB becomes compromised both structurally and functionally to transform itself into the BTB, as the primary and or metastatic tumors grow more than 1–2 mm in diameter. Such tumors also have disorganized or distorted, tortuous blood vessels that are typically referred to as the BTB (Fig. 17.1) (Abbott and Friedman, 2012). It is important to note that BTB capillary ultrastructure is markedly different between a GBM and a brain metastasis (Van Tellingen et al., 2015; Hawkins and Davis, 2005). This leads to differential vascular permeability and often requires different strategies to deliver anticancer drugs and imaging agents across the BTB of primary and metastatic brain tumors. Accordingly, BTB permeability should be assessed with advanced methodologies such as dynamic contrastenhanced magnetic resonance imaging (DCE-MRI) to plan for targeted treatment of different types of brain tumors. In this aspect, we have validated a noninvasive method of BTB permeability measurement using DCE-MRI in brain tumor models (Ningaraj and Khaitan, 2013).

Normal epigenetic modifications such as DNA methylation or histone modifications regulate normal cell differentiation while abnormal epigenetic changes could lead to oncogenic transformation of normal cells. Aberrant or altered DNA methylation cause genomic stability, which is often linked to various pathologies including cancer. Specifically, in glioma epigenetic dysfunctions are often shown to drive oncogenic transformation of low grade glioma to high-grade glioblastoma multiforme (GBM). While few epigenetic events are linked to glioma transformation, more transformation events remain to be identified and validated as viable targets for reversing the epigenetic gene silencing. Reversing the epigenetic silencing or resetting these genes hold a promise in preventing or arresting the progression of low grade glioma to GBM. We treated glioma cells with demethylating agent 5-aza (DNA methyltransferase inhibitor) and Trichostatin A (TSA, Histone deacetylase inhibitor) and whole genome expression array was performed to identify genes that were reactivated by treatment. Array results were confirmed by RT-PCR analysis using glioma cells treated with 5-aza and TSA. Genes were filtered by the following criteria: 2-fold reactivated by treatment as determined by microarray; CpG island in their promoter region; reactivated by treatment in majority of the glioma cell lines, as determined by RT-PCR. Methylation specific PCR (MSP) was carried out to determine methylation status of the promoter region using low grade and high-grade glioma cell lines.

Our results indicate that there is a significant difference in the expression of genes between low and high-grade glioma cells, when treated with 5-aza plus TSA. Induction of CLDN6, NDN, OSMR, ADFP, CDCP1, ANGPT2, ZFP42, BTG4 and MYEOV was seen in most of the glioma cells treated with 5-aza and TSA. MSP-DNA methylation analysis revealed that ADFP, CDCP1 and ZFP42 that were uniquely silenced in high-grade glioma cells lines were reactivated after 5-AZA treatment. This result suggests that studying the methylation status of ADFP, CDCP1 and ZFP42 in brain tumor biopsies may indicate the potential aggressive nature of glioma that helps doctors in accurate and diagnosis and clinical treatment decisions .

Introduction

Gliomas are the most common primary tumors that arise within the central nervous system in adults accounting for 78% of malignant brain tumors. In recent years increasing use of genetic analysis in primary tumors resulted in identification of molecular events and pathways involved in the etiology of brain tumors. However, our understanding of molecular basis of most brain tumor cases remains poor. DNA methylation plays an important role in various cellular functions such as transcriptional silencing, X-chromosome inactivation, genomic imprinting, and genomic stability. Aberrant or altered DNA methylation is linked to various pathologies, including cancer [1]. Tumor cells exhibit global hypomethylation of the genome accompanied by region-specific hypermethylation events. Global hypomethylation occurs mainly in the repetitive sequences leading to genomic instability and tumor formation [2]. Aberrant hypermethylation occurs at CpG islands found in the promoter region of genes and is usually associated with the transcriptional silencing of that gene [3]. Another form of epigenetic gene silencing is the covalent modification of histone proteins. These are post-translational modifications that occur at the amino-terminal tail and include acetylation, methylation, phosphorylation, and ubiquitination. These forms of epigenetic modifications are closely linked to each other. Recent studies suggest that DNA methylation might be dependent on histone modifications and acts to preserve the silenced state rather than initiate silencing [3]. Increasing evidence suggests that epigenetic modifications, in addition to genetic changes, play an important role carcinogenesis [4-6]. DNA methylation changes, particularly CpG island hypermethylation are frequent, early, and common events (as common as mutations) in many types of cancers leading to the inactivation of tumor suppressor genes [7] and potentially aiding the transformation of low grade tumors to higher grades.

Several genetic changes have been identified in astrocytic gliomas and glioblastomas involving heterozygous deletion of 19q13, inactivation/deletion of tumor suppressor genes namely p16INK4A [8], p14ARF [9], RB1 [10], PTEN and p53 gene [11] and amplification of EGF receptor gene (EGFR) [12]. Investigations of the role of epigenetics in glioma pathogenesis revealed several of these genes to be epigenetically silenced by promoter CpG island hypermethylation, e.g., cell cycle regulatory proteins RB1 [13], p16INK4A [14-17], myelin related gene EMP3 [1], DNA repair protein MGMT and matrix metalloproteinases inhibitor TIMP3 [2]. Comprehensive whole-genome microarray studies using inhibitors of epigenetic modification identified several genes like CST6 (putative metastatic suppressor), BIK (apoptosis inducer), TSPYL5 (unknown function), BEX1, and BEX2 (uncharacterized function) as putative tumor suppressors that are frequently methylated in primary gliomas [18,19]. Another genome-wide study using restriction landmark genomic scanning identified as many as 1500 CpG islands to be aberrantly methylated in low grade gliomas [16], highlighting a role for DNA methylation in gliomagenesis.

DNA methylation status not only serves as a diagnostic or prognostic marker but is a potential therapeutic target because of the reversible nature of methylation. Since the primary DNA sequence of epigenetically modified genes remains intact, it is possible to reactivate genes using inhibitors of DNA methylation or histone modifications [20,21]. Clinical trials are being carried out using DNA methylation and histone deacetylase inhibitors to reactivate silenced genes in cancers. Some of the DNA methyltransferases inhibitors, 5-azacytidine (Vidaza), and 5-aza-2’-deoxycytidine (Decitabine), have been used with reasonable success in the treatment of hematologic malignancies [22]. Combination of HDAC inhibitors with DNA methyltransferases inhibitors seems to have a synergistic effect in inducing expression of silenced genes [6]. We set out to study whether there are unique hypomethylation events during the genotypic transformation of low to high-grade gliomas. A better understanding of the molecular basis of glioma progression, particularly identifying tumor suppressor genes that trigger glioma transformation to an aggressive high-grade will provide an opportunity to design novel therapeutic strategies to control an otherwise hard-to-treat brain tumor

Gliomas develop genetic traits to rapidly form aggressive phenotypes. Hence, management of gliomas is complicated and difficult. Besides genetic aberrations such as oncogenic copy number variation and mutations, alternative mRNA splicing triggers prooncogenic episodes in many cancers. In gliomas, we found alternative splicing at the KCNMA transcription process. KCNMA1 encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channels. These channels play critical role in glioma invasion and proliferation. We identified a novel KCNMA1 mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high grade gliomas. We found that KCNMA1 alternative pre-mRNA splicing enhanced glioma growth, progression and diffusion. The role of KCNMA1 and its splicing as a critical posttranscriptional regulator of BKCa channel expression is presented in this chapter. Our research implies that high grade gliomas express KCNMA1v and BKCa channel isoform to accelerate growth and transformation to glioblastoma multiforme (GBM). We demonstrated that tumors hardly develop in mice injected with KCNMA1v transfected cell line expressing short-hairpin RNA (shRNA) compared to mice injected with KCNMA1v transected glioma cells. We conclude that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM.

Introduction

KCNMA1-encoded BKCa channels in glioma

Brain tumors are the most common type of solid tumors. In the United States, an estimated 20,000 new primary brain tumor cases are reported [1]. The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is highly complicated due to their highly aggressive phenotypic and genotypic changes [2]. The median survival among GBM patients is only 15 months or less [3]. GBM contains heterogeneous subpopulations of glioma and other mixed supporting cells that are cancerous cells. They have the intrinsic ability that adapt in the brain tumor microenvironment and invade the normal brain. Gene expression profiling studies have identified many genes that have distinct expression patterns among different histological types and grades of gliomas [4]. The response of “normal cells” to malignant transformation involves changes in gene expression and is thought to be regulated by transcription [5]. The potassium ion channels are implicated in the malignant transformation to a higher grade in several cancers [5–7]. For example, we reported that lowgrade gliomas might undergo certain epigenetic changes to develop into a GBM [8].

rain tumors are the most common type of solid tumors. In the United States, an estimated 20,000 new primary brain tumor cases are reported [1]. The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is highly complicated due to their highly aggressive phenotypic and genotypic changes [2]. The median survival among GBM patients is only 15 months or less [3]. GBM contains heterogeneous subpopulations of glioma and other mixed supporting cells that are cancerous cells. They have the intrinsic ability that adapt in the brain tumor microenvironment and invade the normal brain. Gene expression profiling studies have identified many genes that have distinct expression patterns among different histological types and grades of gliomas [4]. The response of “normal cells” to malignant transformation involves changes in gene expression and is thought to be regulated by transcription [5]. The potassium ion channels are implicated in the malignant transformation to a higher grade in several cancers [5–7]. For example, we reported that lowgrade gliomas might undergo certain epigenetic changes to develop into a GBM [8].

The physiological features of BKCa channels also known as maxi K or BK channels are well described [6–9]. These channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of cytosolic calcium (Figure 1). The BKCa channels provide a crucial link between the metabolic and electrical states of cells. The BKCa channel overexpression was observed in biopsies of patients with malignant gliomas compared with nonmalignant human cortical tissues and the level of expression correlated positively with increased malignancy [7]. Studies have shown the importance of BKCa channels in brain tumor biology [5]. Lastly, BKCa currents in glioma cells are more sensitive to intracellular [Ca2+] compared to BKCa channels in healthy glial cells [9, 10].

Diverse role of KCNMA1 in glioma

KCNMA1-encoded BKCa channel plays a pivotal role in cancer cell proliferation. Amplification of KCNMA1 was observed in breast, ovarian, and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3–7%) and gliomas. KCNMA1 amplification was significantly associated with high tumor stage, highgrade, high tumor cell proliferation, and poor prognosis. Due to the large number of protein interactions and activating factors influencing BKCa channel function, intracellular Ca2+, membrane voltage, pH, shear stress, carbon monoxide, phosphorylation states, and steroid hormones, it is generally difficult to predict its direct role in a given tissue. However, in many diseases including cancers, defective regulation and/or expression of BKCa channels have repeatedly been associated with altered cell cycle progression [11], cell proliferation [11], and cell migration [11]. These altered cell functions are implicated in development of malignancy [11].

KCNMA1: STRING analysis

In order to understand the possible interactions of KCNMA1 with other genes and molecules, we used the tool STRING 9.1. It is a database consisting of known and possible protein– protein interactions with a gene of interest. The gene may have a direct (physical) or indirect (functional) association with other molecules. With this tool we can easily identify possible interaction of KCNMA1 with other associated molecules. We can derive detailed information of the protein being investigated as well as its associated molecules, crystal structure of the proteins with its PDB ID, and combined score [confidence score, neighborhood score, fusion score, homology score] on the basis of some parameters like experimental results, text-mining, co expression, databases, and co-occurrence.

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

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.

Nearly 12.5 million new cancer cases are diagnosed worldwide each year. Although new treatments have been developed, most new anticancer drugs that are effective outside the brain have failed in clinical trials against brain tumours, in part due to poor penetration across the blood–brain barrier and the blood–brain tumour barrier. This review will discuss the challenges of drug delivery across the blood–brain barrier/blood–brain tumour barrier to cancer cells, as well as progress made thus far. This will include a biochemical modulation strategy that transiently opens the barrier to increase anticancer drug delivery selectively to brain tumours. It will also briefly discuss a quantitative non-invasive method to measure permeability changes and tumour response to treatment in the human brain

Introduction

Every year in the US, ∼ 20,000 new primary and nearly 200,000 secondary (metastatic) brain tumour cases are reported. Worldwide numbers are more distressing. Even after surgical resection, brain cancer invariably recurs, severely shortening life expectancy [1]. Conventional treatment using radiation and intravenous chemotherapy often prove unsuccessful primarily because the anticancer drugs fail to cross the blood–brain barrier (BBB) in sufficient quantities [2]. Therefore, understanding the biochemical regulation of the BBB in its normal and abnormal states (in and around tumours) is of great importance as efforts continue to deliver therapeutic compounds to brain cancers. The focus is now on targeted cancer therapy by not only supplementing conventional chemotherapy and radiotherapy, but also by preventing toxicity in normal tissues and drug resistance. In particular, successful treatment of brain tumours involves efficient anticancer drug delivery to brain tumours across the blood–brain tumour barrier (BTB). Although the BTB is ‘leaky’ in the tumour centre, the established microvessels (capillaries) feeding the proliferating tumour edge and the brain tissue surrounding the tumour is nearly as impermeable as the BBB [3]. Therefore, the BTB still poses a major obstacle to anticancer drug delivery to tumours. In this article, the challenges involved in and the progress made, especially in the past decade, towards delivering therapeutic drugs selectively to brain tumours will be reviewed.

The cerebral microvessels/capillaries that form the BBB protect the brain from toxic agents in the blood but also pose a significant hindrance to the delivery of small and large therapeutic molecules. Pardridge reported that the BBB blocks delivery of > 98% of CNS drugs [2,4]. The National Institutes of Health (NIH) cited as high priority goals to understand the function of the BBB and BTB, develop novel drug delivery approaches for molecular-targeted therapy, and to further develop methods to non-invasively image the response of brain tumours to treatment. Different strategies have been developed to circumvent the physiological barrier that is posed by the BBB, often based on a conception of the barrier as being controlled by what is called the neurovascular unit. This consists of endothelial cells (ECs), tight junctional proteins connecting the ECs, glia, pericytes and astrocytic foot processes, which interact with neurons (Figure 1). For the most part, research seeks to understand the interaction among the constituents of the BBB and neurons in normal and pathological conditions [5]. By using in vitro and in vivo models, researchers seek to achieve a better understanding of the effects of neurological disorders on the BBB and, thereby, improve our knowledge of BBB biology. The goal is to better comprehend the initiation and progression of neurological disease and to develop approaches to effectively treat brain diseases such as brain tumours. Novel cancer therapies include antiangiogenic agents, immunotherapy, bacterial agents, viral oncolysis, cyclin-dependent kinases and receptor tyrosine kinase inhibitors, antisense agents, gene therapy and combinations of various methods. Amazing clinical success in treating some types of cancer has been achieved using immunotherapy-based anticancer agents such as cytokines, monoclonal antibodies and cancer vaccines. For example, one promising treatment uses antisense oligonucleotides, such as small interfering RNAs, which have been used in various clinical trials for cancer: but only for cancers outside the brain [6]. Despite these promising approaches, the BBB still causes a significant complication to brain cancer treatment. As whole-brain gene microarrays have detected fewer BBB-specific transcripts [7], the focus of work carried out by Ningaraj and others is on cerebrovascular genomics and proteomic research. The ideal approach is to isolate brain capillaries in normal and diseased brain tissue and then to analyse genomic and proteomic differences. Generally, human brain tissue from a temporal lobectomy of a trauma or epilepsy patient is considered to be normal tissue in these studies as it is unethical to obtain normal, healthy human brain tissue [8].