Background
Gliomas comprise the majority of adult primary brain tumors diagnosed annually in the United States [
1‐
4]. Gliomas are classified by the World Health Organization according to their morphologic characteristics into astrocytic, oligodendroglial, and mixed tumors [
4,
5]. High grade gliomas (HGGs; grades III and IV) are the most common primary brain tumors in adults, and their malignant nature ranks them fourth in incidence of cancer death [
2‐
4]. Approximately 15,000 patients die with glioblastomas (GBM, glioblastoma multiforme; a grade IV glioma) in the U.S.A. annually [
1‐
4]. Malignant brain tumors kill approximately 140,000 people worldwide per year [
6]. Standard treatment for GBM, which typically involves surgical resection followed by a combination of radiation and chemotherapy with the standard-of-care (SOC) temozolomide (TMZ), has not substantially improved overall survival (median survival remains 15 to 18 months, five-year survival rates are <10 %) [
7,
8]. Prognosis is even poorer for recurrent disease, with response rates for cytotoxic chemotherapy typically in the range of 5 to 10 %, and 6-month progression-free survival rates of <15 % [
9,
10].
One therapeutic strategy being actively pursued for multiple cancers is targeting angiogenesis, because without the ability to vascularize, a tumor cannot grow in size. Conversely, normal tissue is already vascularized and is not affected by angiogenic inhibition. Angiogenesis is greatly upregulated in HGGs compared to low-grade gliomas (LGGs) [
8]. Angiogenesis is an essential process that provides excess nutrients to developing tumors even at a very early stage [
11]. In fact, assessing angiogenesis is one of the most important criteria for grading tumors in patients [
12]. In addition to cytotoxic chemotherapy, bevacizumab (Avastin®), an anti-VEGF antibody therapeutic, is also used to inhibit angiogenesis as a treatment for recurrent GBM, but it has not been found to significantly improve the clinical outcome [
7,
8].
AG119 (previously referred to as 3⋅HCl [
13]) is a small molecule discovered in our laboratory and found to possess anti-angiogenic (RTK inhibition) and antimicrotubule cytotoxic activity in a single molecule [
13]. It was further found that AG119 possesses antitumor efficacy in two flank xenograft models – human MDA-MB-435 breast (free of melanoma cross-contamination [
14]) and human U251 glioma, and anti-metastatic activity in a mouse orthotopic breast allograft (4 T1) model, with little systemic toxicity [
13]. Due to the strong
in vivo efficacy and potent angiogenic inhibitory activity of AG119 (decreased VEGFR2 (vascular endothelial growth factor receptor 2) and CD31/PECAM-1 immunohistochemical staining), and since we have previously shown that this compound is not a substrate for ATP binding cassette (ABC) transporters [
13], a mechanism behind why many drugs fail to cross the blood–brain barrier [
15], we tested AG119 as a potential anticancer therapy in an orthotopic allograft mouse (GL261) pre-clinical HGG model.
Discussion
It is well known that GL261 is a syngeneic mouse model of HHG in C57BL/6 mice with several morphological, pathological and genetic similarities to human GBM [
26]. It has also been previously reported that the blood-tumor-barrier in GL261 gliomas are quite “leaky” which allows the penetration of various therapeutic compounds [
27], as well as molecular targeting agents which we have reported on [
28]. In this work we demonstrate in the GL261 glioma model that AG119, a small molecule with combined anti-angiogenic and antimicrotubule activity [
13], results in a significant increase in animal percent survival (
p < 0.001), as well as a significant decrease in GL261 HGG tumor growth
in vivo (
p < 0.001), compared to untreated tumors. AG119 was also found to be similar to the standard-of-care (SOC) TMZ, and relatively new anti-VEGF and anti-c-Met antibody targeted therapies. It should be noted that antibody therapies in this study were not optimized, i.e. doses used elicited therapeutic responses, but did not necessarily induce tumor regression [
29]. Also of note, the survival data included anesthesia-related deaths for the AG119 treatment group which may not properly reflect the actual survival times for this treatment group, and should be repeated in future studies. Regardless, this proof-of-concept study does indicate that AG119 has anti-cancer activity in a pre-clinical glioma model.
AG119 was also found to significantly decrease tumor perfusion rates as well as TMZ (both
p < 0.05, when compared to untreated tumors). Perfusion rates decrease in HGG, such as the GL261 model, as a result of the disorganized capillary architecture from vasculature associated with angiogenesis [
22]. This is in keeping with previous findings that AG119 possessed inhibition of VEGFR2 kinase and anti-angiogenic activity in the chicken embryo chorioallantoic membrane (CAM) assay [
13].
These data are encouraging because currently approved active antimicrotubule agents such as the taxanes do not readily cross the blood–brain barrier and are not useful for central nervous system (CNS) tumors, even though gliomas show taxane sensitivity in culture [
30]. We have also previously shown that AG119 is not subject to resistance mechanisms common to other antimicrotubule agents (beta-III tubulin over-expression [
25], P-glycoprotein) in tumors such as gliomas, suggesting that this agent may have a therapeutic advantage over current agents [
13,
31]. This work suggests that AG119 is also not subject to MGMT mediated resistance, as is the case with TMZ. A study to further explore this idea would be a comparison of AG119 sensitivity in parental U251 cells as compared to T98G cells with MGMT knocked out. Thus AG119 may be useful in treating historically resistant gliomas.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RAT oversaw all aspects of the MRI studies conducted, including data acquisition and data analysis, interpreted the data, generation of the MRI data figures, and writing the bulk of the manuscript. MI oversaw all aspects of the cell studies conducted, interpreted the data, provided the AG119 formulation for treated animal studies, provided relevant sections to the manuscript, and edited drafts of the manuscript. AG was responsible for overseeing the synthesis of AG119, proving funds for the project, contributing to the manuscript text, interpretation of the data, and editing drafts of the manuscript. NS was responsible for MRI data acquisition and data analysis, providing information for the methods section of the manuscript, and editing drafts of the manuscript. DS was responsible for animal manipulations, including setting up animals for MRI studies, physiological monitoring, animal treatments, orthoptopic implantation of glioma cells, contributing to the methods sections, and editing drafts of the manuscript. AB was responsible for making the AG119 formulation for animal treatment studies, conducting the cell study, analysis of data, generating the figure for the cell study, and editing drafts of the manuscript. RP was responsible for the synthesis of AG119, and editing drafts of the manuscript. All authors read and approved the final manuscript.
RAT is the Director of the Advanced Magnetic Resonance Center (AMRC) at the Oklahoma Medical Research Foundation (OMRF), with extensive experience (over 26 years with over 100 refereed publications and 2 patents) in the use of MR techniques to assess pathophysiological processes in animal models for cancer (mainly focusing on gliomas), tissue injury, and inflammation. RAT has used and assessed various orthotopic, xenograft and transgenic models for tumor development in the past 16 years, and has incorporated MR imaging and spectroscopy methods to detect morphological, biophysical, functional and metabolic alterations associated with tumor growth, assess therapeutic responses, and develop anti-cancer therapies. RAT is a member of the International Society for Magnetic Resonance in Medicine, and the American Association for Cancer Research. MI is an Assoc. Prof. of Pharmacology/Toxicology in the Dept. of Pharmaceutical Sciences in the College of Pharmacy at the University of Oklahoma Health Sciences Center), and has extensive expertise (over 20 years and over 50 publications) in preclinical small molecule anticancer drug discovery and development (cells, animals (efficacy, PK, TOX)). MI is a member of the Society of Toxicology and the American Association for Cancer Research. AG is a Professor of Medicinal Pharmacy at Duquesne University (DU), and has extensive expertise (over 150 publications and 31 patents) in synthetic medicinal chemistry, computer-assisted drug design, inhibitors of folate metabolizing enzymes, receptor tyrosine kinase inhibitors, anti-mitotic agents, anti-tumor agents, anti-opportunistic infection agents, nucleosides, heterocyclic chemistry and stereochemistry. AG is a member of the American Association of Pharmaceutical Scientists. NS is an Associate Staff Scientist in the AMRC at OMRF with extensive experience in preclinical MRI evaluation of tumor growth, therapeutic efficacy assessments, and molecular-targeted imaging. DS is a Senior Research Assistant in the AMRC at OMRF with extensive experience in orthotopic models for various cancers, morphological evaluation of tumor growth, and tissue necropsies. AB is a PhD graduate student in MI’s research group, and has expertise in preclinical evaluations of cell viability assays, and assessing in vivo tumor growth, associated with anti-cancer agents. RP is a PhD candidate in AG’s research group at DU, and has expertise in the synthesis of anti-cancer agents.