Vascular Targeting Agent Research Articles

3D microtumors in vitro supported by perfused vascular networks.

There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This “organs-on-chips” approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This “tumor-on-a-chip” platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro.

Vascular disruptive agent OXi4503 and anti-angiogenic agent Sunitinib combination treatment prolong survival of mice with CRC liver metastasis.

BackgroundPreclinical research indicate that vascular disrupting agent (VDA) treatment induces extensive tumor death but also a systemic mobilization of bone marrow derived cells including endothelial progenitor cells (EPC) leading to revascularization and renewed growth within the residual tumor. This study investigates if combination of VDA with the anti-angiogenic agent Sunitinib increases the treatment efficacy in a colorectal liver metastases mouse model.MethodsCBA mice with established liver metastases were given a single dose of OXi4503 at day 16 post tumor induction, a daily dose of Sunitinib starting at day 14 or day 16 post tumor induction or a combination of Sunitinib given daily from day 14 or day 16 post tumor induction in combination with a single dose of OXi4503 at day 16. Treatment was terminated at day 21 post tumor induction and its effects were assessed using stereological and immunohistochemical techniques. Long term effects were assessed in a survival study.ResultsCombination with long (7 day) Sunitinib treatment lead to liver toxicity but this was ameliorated in the shorter (5 day) treatment without significantly altering the effects on tumor reduction. Combination treatment resulted in significant reduction of viable tumor, reduction in tumor vasculature, reduction in tumor proliferation, increase in tumor apoptosis and prolonged mouse survival compared to control and single arm treatments. Complete tumor eradication was not achieved. Redistribution of E-cadherin and strong up regulation of ZEB1 and Vimentin were observed in the surviving tumor; indicative of epithelial to mesenchymal transition (EMT), a mechanism that could contribute to tumor resistance.ConclusionsCombination treatment significantly reduces viable tumor and prolongs animal survival. EMT in the surviving tumor may prevent total tumor eradication and could provide novel targets for a more lasting treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2568-7) contains supplementary material, which is available to authorized users.

Abstract 4194: Assessment of novel benzosuberene-based vascular disrupting agents (VDA) on diverse tumor lines

Abstract Introduction: In developing any new drug, non-invasive assays of efficacy provide vital insight into activity with respect to dosing and pharmacodynamics of action. We have previously demonstrated the utility of dynamic bioluminescence imaging (dBLI) to evaluate VDA activity [1,2]. The current study, which evaluated the vascular-disrupting activity upon treatment with the promising novel benzosuberene-based agents KGP18 and KGP265 [structurally inspired by the natural product, combretastatin A-4 (CA4)] led to significantly decreased and delayed light emission indicative of vascular disruption. Methods: Human breast cancer cells (MDA-MB-231), prostate cancer cells (PC3), and lung cancer cells (A549) along with mouse breast tumor 4T1 cells were implanted in male SCID/Balb/c mice. The dose of KGP265 ranged from 5 to 15 mg/kg and the well studied VDA combretastatin A-4P (CA4P; 120 mg/kg) was used as positive control. Dynamic BLI was performed at baseline, 4, 24, 48 and 72 hours after IP administration of each VDA. Fresh D-luciferin was administered at each time point. As a control, additional tumor bearing mice were injected with the same volume of saline. Results: Dose escalation indicated that higher doses produced more effective vascular shutdown, and doses of 3 mg/kg KGP18 and 7.5 mg/kg KGP265 appeared optimal, being effective while causing little observable toxicity with the MBA-MD-231 tumor model. The optimal dose of KGP265 was 5 mg/kg for 4T1, and 10 mg/kg for A549 and PC3. Treatment of the 4T1, PC3 and A549 tumors with the above dose of KGP265 greatly reduced signal output in BLI images. By 4 hours, about 90% of signal was reduced and the signal stayed down for the next 48 hours post-injection. In vivo observations were confirmed by post mortem histology. Treatment with KGP265 led to significantly decreased and delayed light emission indicative of vascular disruption in different tumor types. The BLI signal was reduced by 90% at 4 hours compared to baseline. Light emission remained significantly depressed up to 48 hours and 72 hours, whereas CA4P showed substantial recovery by 24 hours and further recovery by 48 hours. Meanwhile light emission was highly consistent following administration of saline as control with reproducible kinetics and intensities. Conclusion: The results indicate that KGP18 and KGP265 cause significant vascular disruption. KGP265 is a phosphorylated prodrug of KGP18; it has enhanced water solubility, and maintains substantial vascular disrupting efficacy. Compared to CA4P (optimal dose 120 mg/kg), the new VDAs are effective at lower doses and achieve similar imaging results, suggesting enhanced efficacy. We are continuing to explore efficacy and therapeutic response through combination studies. Acknowledgment: CA4P was kindly provided by Dr. David Chaplin of OXiGENE. Supported by NIH R01CA140674

Femoral Head Growth Plate Dysplasia and Fracture in Juvenile Rabbits Induced by Off-target Antiangiogenic Treatment.

Epiphyseal growth plate dysplasia (chondrodysplasia) might be considered as the pathognomonic feature of antiangiogenic treatment in preclinical species as it is reliably and dose-responsively induced in rodents and monkeys with vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor (FGF) receptor inhibitors, matrix metalloproteinase inhibitors, and vascular targeting agents. Here we report epiphyseal growth plate dysplasia in juvenile rabbits treated with an oral spleen tyrosine kinase inhibitor induced by off-target antiangiogenic inhibition of VEGF and FGF family kinase receptors. Epiphyseal growth plate dysplasia resulted in weakening and fracturing of the femoral head physis in 6 of 10 male and 1 of 10 female animals as well as microfracturing and dysplasia of the distal femoral articular cartilage in 1 male animal. Fracture lines ran through the zone of hypertrophic cartilage (as well as adjacent zones), were orientated parallel to the physeal plane, and often involved displacement of the femoral head. We would suggest that the high prevalence of growth plate fracture in the rabbit may represent a potential additional adverse risk to those already established for children treated with antiangiogenic therapy.

Blocking Blood Flow to Solid Tumors by Destabilizing Tubulin: An Approach to Targeting Tumor Growth.

The unique characteristics of the tumor vasculature offer the possibility to selectively target tumor growth and vascularization using tubulin-destabilizing agents. Evidence accumulated with combretastatin A-4 (CA-4) and its prodrug CA-4P support the therapeutic value of compounds sharing this mechanism of action. However, the chemical instability and poor solubility of CA-4 demand alternative compounds that are able to surmount these limitations. This Perspective illustrates the different classes of compounds that behave similar to CA-4, analyzes their binding mode to αβ-tubulin according to recently available structural complexes, and includes described approaches to improve their delivery. In addition, dissecting the mechanism of action of CA-4 and analogues allows a closer insight into the advantages and drawbacks associated with these tubulin-destabilizing agents that behave as vascular disrupting agents (VDAs).

Not flavone-8-acetic acid (FAA) but its murine metabolite 6-OH-FAA exhibits remarkable antivascular activities in vitro

Flavone-8-acetic acid (FAA) has been proved to be a potent vascular-disrupting agent in mice. Unfortunately, FAA did not produce any anticancer activity in clinical trials. Previously, we had reported that FAA is metabolized by mouse microsomes into six metabolites, whereas it was poorly metabolized by human microsomes, with fewer metabolites formed in lesser amounts. Especially, 6-OH-FAA was not formed by human microsomes. In this work, two major available metabolites, 4'-OH-FAA and 6-OH-FAA, were tested and compared with the parent compound FAA for their potential antivascular activities in vitro. The ability of the products to induce morphological changes, disrupt preformed capillaries of EA.hy926 endothelial cells and inhibit tubulin polymerization in vitro was assessed. The action mechanism was determined using the RhoA and Rac1 inhibitors. At 25 µg/ml, 6-OH-FAA induced morphological changes and membrane blebbing, whereas 300 µg/ml of FAA and 4'-OH-FAA slightly changed the morphology without inducing membrane blebbing. At 300 µg/ml, 6-OH-FAA produced morphological changes that were 2.1-6.9-fold greater than that produced by FAA and 4'-OH-FAA, an effect that was consistent with its much greater inhibitory effect on tubulin polymerization compared with FAA and 4'-OH-FAA. 6-OH-FAA significantly disrupted the EA.hy926 cell capillaries. 6-OH-FAA activities were prevented in EA.hy926 cells pretreated with RhoA, but not Rac1, inhibitor. In this short communication we report for the first time that, in vitro, 6-OH-FAA, a mouse-specific FAA metabolite, exhibits significantly stronger antivascular activities compared with FAA and 4'-OH-FAA, which are mediated through the RhoA kinase pathway.

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091.

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)-2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-to-straight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.

Bioluminescence and MR Imaging of the Safety and Efficacy of Vascular Disruption in Gliomas.

The goal of the present study was to examine the safety and efficacy of the vascular disrupting agent (VDA) EPC2407 (Crolibulin™) in experimental glioma models using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Experimental imaging studies were performed in subcutaneous human U87 glioma xenografts and orthotopic murine gliomas established by intracranial implantation of luciferase-transfected glioma cells (GL261-luc). Correlative histopathology and long-term survival analysis was also performed. Treatment with EPC2407 decreased tumor perfusion and increased necrosis and tumor doubling times in subcutaneous U87 xenografts. Dynamic BLI and T1-weighted contrast-enhanced MRI showed reduction in blood flow of intracranial GL261-luc gliomas within a few hours of VDA treatment. T2-weighted MRI did not show any evidence of hemorrhaging or edema in uninvolved brain tissue of EPC2407-treated animals. A significant increase in median survival (p < 0.05) was observed in the orthotopic GL261-luc model following VDA treatment compared to untreated controls. We demonstrate, for the first time, the biological activity of EPC2407 in experimental gliomas. Further investigation into the potential of VDAs in combination with chemoradiation therapy against gliomas is warranted.

Combretastatin A-4 derived 5-(1-methyl-4-phenyl-imidazol-5-yl)indoles with superior cytotoxic and anti-vascular effects on chemoresistant cancer cells and tumors

5-(1-Methyl-4-phenyl-imidazol-5-yl)indoles 5 were prepared and tested as analogs of the natural vascular-disrupting agent combretastatin A-4 (CA-4). The 3-bromo-4,5-dimethoxyphenyl derivative 5c was far more active than CA-4 with low nanomolar IC50 concentrations against multidrug-resistant KB-V1/Vbl cervix and MCF-7/Topo mamma carcinoma cells, and also against CA-4-resistant HT-29 colon carcinoma cells. While not interfering markedly with the polymerization of tubulin in vitro, indole 5c completely disrupted the microtubule cytoskeleton of cancer cells at low concentrations. It also destroyed real blood vessels, both in the chorioallantoic membrane (CAM) of fertilized chicken eggs and within tumor xenografts in mice, without harming embryo or mouse, respectively. Indole 5c was less toxic than CA-4 to endothelial cells, fibroblasts, and cardiomyocytes. In highly vascularized xenograft tumors 5c induced distinct discolorations and histological features typical of vascular-disrupting agents, such as disrupted vessel structures, hemorrhages, and extensive necrosis. In a first preliminary therapy trial, indole 5c retarded the growth of resistant xenograft tumors in mice. © 2016 Elsevier Science Ltd. All rights reserved.

Anti-angiogenic agents in ovarian cancer: past, present, and future

Angiogenesis plays a pivotal role in normal ovarian physiology as well as in the progression of ovarian cancer through ascites formation and metastatic spread. Bevacizumab (Avastin(®), Genentech; South San Francisco, CA, USA), a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, is the most widely studied anti-angiogenesis agent both across tumor types and specifically in epithelial ovarian cancer. In 2005, single-agent bevacizumab at 15 mg/kg (IV) every 3 weeks was first reported to be active in a case of recurrent high-grade serous ovarian cancer after failing 11th line cytotoxic treatment. Since then, many case series, phase II and phase III trials have confirmed these results leading to regulatory approval in most countries including the US Food and Drug Administration in 2014. Guidelines now give clear recommendations as to when and how bevacizumab should be integrated into the ovarian cancer treatment paradigm. Other anti-VEGF agents such as the VEGF receptor (VEGFR) tyrosine kinase inhibitors have not shown increased activity or reduced toxicity relative to bevacizumab. However, anti-angiogenics other than anti-VEGF/VEGFR agents such as those targeting Angiopoietin-1 and -2 are in development as well as novel combinations with vascular disrupting agents (VDAs), PARP inhibitors and immune checkpoint inhibitors. Clearly, the benefits of anti-angiogenic agents such as bevacizumab must be carefully weighed against the cost and associated toxicities. Although almost all patients with ovarian cancer will receive an anti-angiogenic compound, cures are not increased. Predictive biomarkers are an urgent unmet need.

Anti-tumor effects of Semaphorin 4D blockade unravel a novel pro-invasive mechanism of vascular targeting agents in pNETs

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Co-administration of vascular-disrupting agent and doxorubicin by PEG-polypeptide nanoparticles for cancer therapy

Co-administration of vascular-disrupting agent and doxorubicin by PEG-polypeptide nanoparticles for cancer therapy

Tumor resistance to vascular disrupting agents: mechanisms, imaging, and solutions.

The emergence of vascular disrupting agents (VDAs) is a significant advance in the treatment of solid tumors. VDAs induce rapid and selective shutdown of tumor blood flow resulting in massive necrosis. However, a viable marginal tumor rim always remains after VDA treatment and is a major cause of recurrence. In this review, we discuss the mechanisms involved in the resistance of solid tumors to VDAs. Hypoxia, tumor-associated macrophages, and bone marrow-derived circulating endothelial progenitor cells all may contribute to resistance. Resistance can be monitored using magnetic resonance imaging markers. The various solutions proposed to manage tumor resistance to VDAs emphasize combining these agents with other approaches including antiangiogenic agents, chemotherapy, radiotherapy, radioimmunotherapy, and sequential dual-targeting internal radiotherapy.

Preclinical Activity of the Vascular Disrupting Agent OXi4503 against Head and Neck Cancer.

Vascular disrupting agents (VDAs) represent a relatively distinct class of agents that target established blood vessels in tumors. In this study, we examined the preclinical activity of the second-generation VDA OXi4503 against human head and neck squamous cell carcinoma (HNSCC). Studies were performed in subcutaneous and orthotopic FaDu-luc HNSCC xenografts established in immunodeficient mice. In the subcutaneous model, bioluminescence imaging (BLI) along with tumor growth measurements was performed to assess tumor response to therapy. In mice bearing orthotopic tumors, a dual modality imaging approach based on BLI and magnetic resonance imaging (MRI) was utilized. Correlative histologic assessment of tumors was performed to validate imaging data. Dynamic BLI revealed a marked reduction in radiance within a few hours of OXi4503 administration compared to baseline levels. However, this reduction was transient with vascular recovery observed at 24 h post treatment. A single injection of OXi4503 (40 mg/kg) resulted in a significant (p < 0.01) tumor growth inhibition of subcutaneous FaDu-luc xenografts. MRI revealed a significant reduction (p < 0.05) in volume of orthotopic tumors at 10 days post two doses of OXi4503 treatment. Corresponding histologic (H&E) sections of Oxi4503 treated tumors showed extensive areas of necrosis and hemorrhaging compared to untreated controls. To the best of our knowledge, this is the first report, on the activity of Oxi4503 against HNSCC. These results demonstrate the potential of tumor-VDAs in head and neck cancer. Further examination of the antivascular and antitumor activity of Oxi4503 against HNSCC alone and in combination with chemotherapy and radiation is warranted.

Combination of vascular disrupting agents and nanomedicines for solid tumor therapy

Event Abstract Back to Event Combination of vascular disrupting agents and nanomedicines for solid tumor therapy Xuesi Chen1*, Zhaohui Tang1* and Wantong Song1* 1 Changchun Institute of Applied Chemistry, CAS, Key Laboratory of Polymer Ecomaterials, China Introduction: Nanosized anticancer drug delivery systems hold great promise for safe, simple and effective therapies against malignant solid tumors. Due to the enhanced permeability and retention (EPR) effect of solid tumor tissues, nanoparticles can passively accumulate in tumors, and this resulted in more than ten nanocarrier-based drugs been marketed for the treatment of cancers. However, a main barrier for further improving the therapeutic efficiency of nanomedicines lies in the poor permeability of solid tumors. Especially in the tumor central regions, low penetration of nanoparticles results in low contact probability of drugs to reach a majority of tumor cells and limited therapeutic outcomes. Aiming at this problem, in this study, we proposed a novel strategy for enhancing the treatment efficacy of nanomedicines within the central regions of solid tumor by combining nanomedicines with vascular disrupting agents (VDAs). As shown in Figure 1, nanomedicines mainly act on the tumor periphery, while VDAs eradicate tumor cells in the central regions of a solid tumor. Therefore, the combination is expected to eradicate the entire tumor. Materials and Methods: A typical small-molecule VDA, combretastatin A4 disodium phosphate (CA4P), which is in phase I/II clinical trials was applied here. Cis-diamminedichloroplatinum loaded nanoparticles (CDDP-NPs) prepared from CDDP and poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) (PLG-g-mPEG), which was previously reported and optimized by us was applied here as the model nanomedicine. For in vivo studies, Balb/C mice bearing murine colon carcinoma C26 tumors was applied. Results and Discussion: The effect of CA4P, CDDP-NPs and the combination on tumor necrosis after a single intravenous injection was compared by H-E staining. As shown in Figure 2, in contrast to CDDP-NPs or CA4P alone, the combination of CDDP-NPs+CA4P caused necrosis in both the central and peripheral regions, and resulted in a necrosis rate of 92.8±3.0%. In the following in vivo anticancer efficacy test, the combination group exhibited significant tumor growth suppression without significant bodyweight loss. Conclusion: Our work supports the notion that coadministration of nanomedicines plus vascular disrupting agents will serve as a promising strategy for the treatment of solid tumors with high efficacy and low side effects.

Microenvironment-Mediated Modeling of Tumor Response to Vascular-Targeting Drugs.

The tumor-associated microvasculature is one of the key elements of the microenvironment that helps shape, and is shaped by, tumor progression. Given the important role of the vasculature in tumor progression, and the fact that tumor and normal vasculature are physiologically and molecularly distinct, much effort has gone into the development of vascular-targeting drugs that in theory should target tumors without significant risk to normal tissue. In this chapter, a multiscale hybrid mathematical model of tumor-vascular interactions is presented to provide a theoretical basis for assessing tumor response to vascular-targeting drugs. Model performance is calibrated to quantitative clinical data on tumor response to angiogenesis inhibitors (AIs), preclinical data on response to a cytotoxic chemotherapy, and qualitative preclinical data on response to vascular disrupting agents (VDAs). The calibrated model is then used to explore two questions of clinical interest. First, the hypothesis that AIs and VDAs are complementary treatments, rather than redundant, is explored. The model predicts a minimal increase in antitumor activity as a result of adding a VDA to an AI treatment regimen, and in fact at times the combination can exert less antitumor activity than stand-alone AI treatment. Second, the question of identifying an optimal dosing strategy for treating with an AI and a cytotoxic agent is addressed. Using a stochastic optimization scheme, an intermittent schedule for both chemotherapy and AI administration is identified that can eradicate the simulated tumors. We propose that this schedule may have increased clinical antitumor activity compared to currently used treatment protocols.

Solid Tumor Therapy Using a Cannon and Pawn Combination Strategy.

Nanocarrier-based anti-tumor drugs hold great promise for reducing side effects and improving tumor-site drug retention in the treatment of solid tumors. However, therapeutic outcomes are still limited, primarily due to a lack of drug penetration within most tumor tissues. Herein, we propose a strategy using a nanocarrier-based combination of vascular disrupting agents (VDAs) and cytotoxic drugs for solid tumor therapy. Specifically, combretastatin A-4 (CA4) serves as a “cannon” by eradicating tumor cells at a distance from blood vessels; concomitantly, doxorubicin (DOX) serves as a “pawn” by killing tumor cells in close proximity to blood vessels. This “cannon and pawn” combination strategy acts without a need to penetrate every tumor cell and is expected to eliminate all tumor cells in a solid tumor. In a murine C26 colon tumor model, this strategy proved effective in eradicating greater than 94% of tumor cells and efficiently inhibited tumor growth with a weekly injection. In large solid tumor models (C26 and 4T1 tumors with volumes of approximately 250 mm3), this strategy also proved effective for inhibiting tumor growth. These results showing remarkable inhibition of tumor growth provide a valuable therapeutic choice for solid tumor therapy.

Low Z target switching to increase tumor endothelial cell dose enhancement during gold nanoparticle-aided radiation therapy.

Previous studies have introduced gold nanoparticles as vascular-disrupting agents during radiation therapy. Crucial to this concept is the low energy photon content of the therapy radiation beam. The authors introduce a new mode of delivery including a linear accelerator target that can toggle between low Z and high Z targets during beam delivery. In this study, the authors examine the potential increase in tumor blood vessel endothelial cell radiation dose enhancement with the low Z target. The authors use Monte Carlo methods to simulate delivery of three different clinical photon beams: (1) a 6 MV standard (Cu/W) beam, (2) a 6 MV flattening filter free (Cu/W), and (3) a 6 MV (carbon) beam. The photon energy spectra for each scenario are generated for depths in tissue-equivalent material: 2, 10, and 20 cm. The endothelial dose enhancement for each target and depth is calculated using a previously published analytic method. It is found that the carbon target increases the proportion of low energy (<150 keV) photons at 10 cm depth to 28% from 8% for the 6 MV standard (Cu/W) beam. This nearly quadrupling of the low energy photon content incident on a gold nanoparticle results in 7.7 times the endothelial dose enhancement as a 6 MV standard (Cu/W) beam at this depth. Increased surface dose from the low Z target can be mitigated by well-spaced beam arrangements. By using the fast-switching target, one can modulate the photon beam during delivery, producing a customized photon energy spectrum for each specific situation.

Abstract B174: Complementary activity of the vascular disruption agent BNC105 and the hypoxia-activated prodrug evofosfamide (TH-302) in suppressing the growth of preclinical renal and breast solid tumors

Abstract BNC105 is a vascular disruption agent that selectively disrupts tumor blood vessels resulting in hypoxia and necrosis. Evofosfamide (previously known as TH-302, Evo) is a nitroimidazole-triggered prodrug of bromo-isophosphoramide mustard (Br-IPM), an alkylating agent with DNA cross-linking ability. Evo is reduced by intracellular reductases, and under hypoxic conditions selectively releases Br-IPM. Evo has little activity under normoxic conditions, but is highly cytotoxic under hypoxic conditions. Both BNC105 and Evo are currently being evaluated in Phase 1-3 clinical trials. Our studies sought to establish the potential therapeutic combination of BNC105 and Evo. It was reasoned that tumor hypoxia caused by BNC105 will result in increased intratumoral activation of Evo. A greater localized conversion of Evo to Br-IPM would increase the tumor kill achieved. A dose range-finding study was conducted to define the dose level and administration schedule for the BNC105 + Evo combination. The study assessed the safety of administering these two drugs in combination in three different dosing sequences whereby Evo was administered 1hr prior to, immediately following (concurrently) or 1 hr after BNC105. Each of the drugs was administered weekly for 2 weeks. Evo dose levels examined were 50, 75, 100, and 150 mg/kg, and. BNC105 doses investigated were 10 and 16 mg/kg. The combination was well-tolerated at all dose levels evaluated when the drugs were administered concurrently or when Evo was administered 1hr before BNC105. The anti-tumor efficacy of the combination was assessed in the orthotopic syngeneic RENCA renal cancer model and in the MDA-MB-231 breast cancer xenograft model. BNC105 was used at 10 or 16 mg/kg and Evo at 75 mg/kg. RENCA kidney cancer cells were inoculated under the kidney capsule and tumors were allowed to grow for 11 days prior to commencement of dosing (Day 1). BNC105 and Evo were dosed concurrently on Days 1 and 8. Treatment groups included: vehicle control; BNC105; Evo and BNC105 + Evo. The growth of tumors implanted in the kidneys of mice was significantly impeded by the monotherapies and considerably more pronounced when these two drugs were used as a combination treatment. Mice bearing subcutaneous MDA-MB-231 xenografts were treated with BNC105, Evo, or the BNC105 + Evo combination administered concurrently with the same schedule (dosing on Days 1 and 8 of a 21-day cycle). A total of 3 treatment cycles were administered. Based on tumor growth inhibition, there was a clear advantage in treating MDA-MB-231 tumors with the BNC105 + Evo combination compared to either monotherapy. The combination of 16 mg/kg BNC105 + 75 mg/kg Evo substantially inhibited tumor growth as early as the 1st cycle of treatment (Day 1, 8 treatment). This effect continued through the 2nd and 3rd cycles of treatment with the tumor growth curve remaining relatively static. In both models tested the combination of BNC105 with Evo was found to be more efficacious than either therapy alone. Based on these encouraging data the combination is currently being evaluated in additional models including pancreatic cancer and soft tissue sarcoma. Citation Format: Tina C. Lavranos, Daniel Inglis, Donna Beaumont, Annabell F. Leske, Chloe Hawkins, Michaela Scherer, Charles Hart, Jessica Sun, Brian Elenbaas, Gabriel Kremmidiotis. Complementary activity of the vascular disruption agent BNC105 and the hypoxia-activated prodrug evofosfamide (TH-302) in suppressing the growth of preclinical renal and breast solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B174.

Structural interrogation of benzosuberene-based inhibitors of tubulin polymerization

The discovery of 3-methoxy-9-(3′,4′,5′-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (a benzosuberene-based analogue referred to as KGP18) was originally inspired by the natural products colchicine and combretastatin A-4 (CA4). The relative structural simplicity and ease of synthesis of KGP18, coupled with its potent biological activity as an inhibitor of tubulin polymerization and its cytotoxicity (in vitro) against human cancer cell lines, has resulted in studies focused on new analogue design and synthesis. Our goal was to probe the relationship of structure to function in this class of anticancer agents. A series of twenty-two new benzosuberene-based analogues of KGP18 was designed and synthesized. These compounds vary in their methoxylation pattern and separately incorporate trifluoromethyl groups around the pendant aryl ring for the evaluation of the effect of functional group modifications on the fused six-membered aromatic ring. In addition, the 8,9-saturated congener of KGP18 has been synthesized to assess the necessity of unsaturation at the carbon atom bearing the pendant aryl ring. Six of the molecules from this benzosuberene-series of compounds were active (IC50 < 5μM) as inhibitors of tubulin polymerization while four analogues were comparable (IC50 approximately 1μM) in their tubulin inhibitory activity to CA4 and KGP18. The potency of a bis-trifluoromethyl analogue 74 and the unsaturated KGP18 derivative 73 as inhibitors of tubulin assembly along with their moderate cytotoxicity suggested the potential utility of these compounds as vascular disrupting agents (VDAs) to selectively target microvessels feeding tumors. Accordingly, water-soluble and DMSO-soluble phosphate prodrug salts of each were synthesized for preliminary in vivo studies to assess their potential efficacy as VDAs.