Demonstrating Tumor Vascular Disrupting Activity of the Small-Molecule Dihydronaphthalene Tubulin-Binding Agent OXi6196 as a Potential Therapeutic for Cancer Treatment.

The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development.

Chemiluminescent 1,2-Dioxetane Iridium Complexes for Near-Infrared Oxygen Sensing.

Chemiluminescent iridium-based sensors which demonstrate oxygen dependent responses have been developed. The molecular probes, named IrCL-1, IrCL-2 and IrCL-3 consist of oxygen-sensitive iridium complexes attached to a spiroadamantane 1,2 dioxetane and operate via energy transfer from the chemiexcited benzoate to the corresponding iridium(III) complex. Complexing the iridium(III) center with π-extended ligands results in emission in the biologically relevant, near-infrared (NIR) region. All probes demonstrate varying oxygen tolerance, with IrCL-1 being the most oxygen sensitive. These probes have been further utilized for in vitro ratiometric imaging of oxygen, as well as for intraperitoneal, intramuscular and intratumoral imaging in live mice. To our knowledge, these are the first iridium-based chemiluminescent probes that have been employed for in vitro ratiometric oxygen sensing, and for in vivo tumor imaging.

Imaging-Guided Evaluation of the Novel Small-Molecule Benzosuberene Tubulin-Binding Agent KGP265 as a Potential Therapeutic Agent for Cancer Treatment.

The selective disruption of tumor-associated vasculature represents an attractive therapeutic approach. We have undertaken the first in vivo evaluation of KGP265, a water-soluble prodrug of a benzosuberene-based tubulin-binding agent, and found promising vascular-disrupting activity in three distinct tumor types. Dose escalation in orthotopic MDA-MB-231-luc breast tumor xenografts in mice indicated that higher doses produced more effective vascular shutdown, as revealed by dynamic bioluminescence imaging (BLI). In syngeneic orthotopic 4T1-luc breast and RENCA-luc kidney tumors, dynamic BLI and oxygen enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following the administration of KGP265 (7.5 mg/kg). The BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h, indicating vascular disruption, which continued over 24 h. A correlative histology confirmed increased necrosis and hemorrhage. Twice-weekly doses of KGP265 caused significant growth delay in both MDA-MB-231 and 4T1 breast tumors, with no obvious systemic toxicity. A combination with carboplatin produced significantly greater tumor growth delay than carboplatin alone, though significant carboplatin-associated toxicity was observed (whole-body weight loss). KGP265 was found to be effective at low concentrations, generating long-term vascular shutdown and tumor growth delay, thus providing strong rationale for further development, particularly in combination therapies.

A scalable open-source MATLAB toolbox for reconstruction and analysis of multispectral optoacoustic tomography data.

Multispectral photoacoustic tomography enables the resolution of spectral components of a tissue or sample at high spatiotemporal resolution. With the availability of commercial instruments, the acquisition of data using this modality has become consistent and standardized. However, the analysis of such data is often hampered by opaque processing algorithms, which are challenging to verify and validate from a user perspective. Furthermore, such tools are inflexible, often locking users into a restricted set of processing motifs, which may not be able to accommodate the demands of diverse experiments. To address these needs, we have developed a Reconstruction, Analysis, and Filtering Toolbox to support the analysis of photoacoustic imaging data. The toolbox includes several algorithms to improve the overall quantification of photoacoustic imaging, including non-negative constraints and multispectral filters. We demonstrate various use cases, including dynamic imaging challenges and quantification of drug effect, and describe the ability of the toolbox to be parallelized on a high performance computing cluster.

Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor in Live Animals.

Regulation of physiological pH is integral for proper whole body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, composed of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8 and 8.4, making it a viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished using both common luminescence spectroscopy and advanced optical imaging methods. Using an IVIS Spectrum, pH can be measured through tissue with Ratio-pHCL-1, which is shown in vitro and calibrated in sacrificed mouse models. Intraperitoneal injections of Ratio-pHCL-1 into live mice show high photon outputs and consistent increases in the flux ratio when measured at pH 6, 7, and 8.

Oxygen-Enhanced Optoacoustic Tomography Reveals the Effectiveness of Targeting Heme and Oxidative Phosphorylation at Normalizing Tumor Vascular Oxygenation.

Multispectral optoacoustic tomography (MSOT) is an emerging noninvasive imaging modality that can detect real-time dynamic information about the tumor microenvironment in humans and animals. Oxygen enhanced (OE)-MSOT can monitor tumor vasculature and oxygenation during disease development or therapy. Here, we used MSOT and OE-MSOT to examine in mice the response of human non-small cell lung cancer (NSCLC) xenografts to a new class of antitumor drugs, heme-targeting agents heme-sequestering peptide 2 (HSP2) and cyclopamine tartrate (CycT). HSP2 inhibits heme uptake, while CycT inhibits heme synthesis in NSCLC cells, where heme is essential for ATP generation via oxidative phosphorylation. HSP2 and CycT can inhibit ATP generation and thereby suppress NSCLC cell tumorigenic functions. MSOT showed that treatment of NSCLC tumors with HSP2 or CycT reduced total hemoglobin, increased oxygen saturation, and enhanced the amplitude of response to oxygen gas breathing challenge. HSP2 and CycT normalized tumor vasculature and improved tumor oxygenation, where levels of several hypoxia markers in NSCLC tumors were reduced by treatment with HSP2 or CycT. Furthermore, treatment with HSP2 or CycT reduced levels of angiogenic factor VEGFA, its receptor VEGFR1, and vascular marker CD34. Together, our data show that heme-targeting drugs HSP2 and CycT elicit multiple tumor-suppressing functions, such as inhibiting angiogenic function, normalizing tumor vasculature, alleviating tumor hypoxia, and inhibiting oxygen consumption and ATP generation. SIGNIFICANCE: Heme-targeting agents HSP2 and CycT effectively normalize tumor vasculature and alleviate tumor hypoxia, raising the possibility of their combination with chemo-, radio-, and immunotherapies to improve antitumor efficacy.See related commentary by Tomaszewski, p. 3461.

Bioreductively Activatable Prodrug Conjugates of Combretastatin A-1 and Combretastatin A-4 as Anticancer Agents Targeted toward Tumor-Associated Hypoxia.

The natural products combretastatin A-1 (CA1) and combretastatin A-4 (CA4) function as potent inhibitors of tubulin polymerization and as selective vascular disrupting agents (VDAs) in tumors. Bioreductively activatable prodrug conjugates (BAPCs) can enhance selectivity by serving as substrates for reductase enzymes specifically in hypoxic regions of tumors. A series of CA1-BAPCs incorporating nor-methyl, mono-methyl, and gem-dimethyl nitrothiophene triggers were synthesized together with corresponding CA4-BAPCs, previously reported by Davis (Mol. Cancer Ther. 2006, 5 (11), 2886), for comparison. The CA4-gem-dimethylnitrothiophene BAPC 45 proved exemplary in comparison to its nor-methyl 43 and mono-methyl 44 congeners. It was stable in phosphate buffer (pH 7.4, 24 h), was cleaved (25%, 90 min) by NADPH-cytochrome P450 oxidoreductase (POR), was inactive (desirable prodrug attribute) as an inhibitor of tubulin polymerization (IC50 > 20 µM), and demonstrated hypoxia-selective activation in the A549 cell line [hypoxia cytotoxicity ratio (HCR) = 41.5]. The related CA1-gem-dimethylnitrothiophene BAPC 41 was also promising (HCR = 12.5) with complete cleavage (90 min) upon treatment with POR. In a preliminary in vivo dynamic bioluminescence imaging study, BAPC 45 (180 mg/kg, ip) induced a decrease (within 4 h) in light emission in a 4T1 syngeneic mouse breast tumor model, implying activation and vascular disruption.

Structure Guided Design, Synthesis, and Biological Evaluation of Novel Benzosuberene Analogues as Inhibitors of Tubulin Polymerization.

A promising design paradigm for small-molecule inhibitors of tubulin polymerization that bind to the colchicine site draws structural inspiration from the natural products colchicine and combretastatin A-4 (CA4). Our previous studies with benzocycloalkenyl and heteroaromatic ring systems yielded promising inhibitors with dihydronaphthalene and benzosuberene analogues featuring phenolic (KGP03 and KGP18) and aniline (KGP05 and KGP156) congeners emerging as lead agents. These molecules demonstrated dual mechanism of action, functioning both as potent vascular disrupting agents (VDAs) and as highly cytotoxic anticancer agents. A further series of analogues was designed to extend functional group diversity and investigate regioisomeric tolerance. Ten new molecules were effective inhibitors of tubulin polymerization (IC50 < 5 µM) with seven of these exhibiting highly potent activity comparable to CA4, KGP18, and KGP03. For one of the most effective agents, dose-dependent vascular shutdown was demonstrated using dynamic bioluminescence imaging in a human prostate tumor xenograft growing in a rat.

Kinetics-Based Measurement of Hypoxia in Living Cells and Animals Using an Acetoxymethyl Ester Chemiluminescent Probe.

Oxygenation and tissue hypoxia play critical roles in mammalian biology and contribute to aggressive phenotypes in cancerous tumors, driving research to develop accurate and easy-to-implement methods for monitoring hypoxia in living cells and animal models. This study reports the chemiluminescent probe HyCL-4-AM, which contains a nitroaromatic sensing moiety and, importantly, an acetoxymethyl (AM) ester that dramatically improves operation in cells and animals. HyCL-4-AM provides a selective 60 000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM). For cellular operation, the chemiluminescence response kinetics is sharply dependent on oxygen levels, enabling highly significant and reproducible measurement of hypoxia in living cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.

A Chemiluminescent Probe for HNO Quantification and Real-Time Monitoring in Living Cells.

Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real-time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO, HNOCL-1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nm with high selectivity based on the reaction with a phosphine group to form a self-cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics-based approach, which provides real-time quantitative measurements of HNO concentration at the picomolar level. HNOCL-1 can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.

Tumor physiological changes during hypofractionated stereotactic body radiation therapy assessed using multi-parametric magnetic resonance imaging.

Radiation therapy is a primary treatment for non-resectable lung cancer and hypoxia is thought to influence tumor response. Hypoxia is expected to be particularly relevant to the evolving new radiation treatment scheme of hypofractionated stereotactic body radiation therapy (SBRT). As such, we sought to develop non-invasive tools to assess tumor pathophysiology and response to irradiation. We applied blood oxygen level dependent (BOLD) and tissue oxygen level dependent (TOLD) MRI, together with dynamic contrast enhanced (DCE) MRI to explore the longitudinal effects of SBRT on tumor oxygenation and vascular perfusion using A549 human lung cancer xenografts in a subcutaneous rat model. Intra-tumor heterogeneity was seen on multi-parametric maps, especially in BOLD, T2* and DCE. At baseline, most tumors showed a positive BOLD signal response (%ΔSI) and increased T2* in response to oxygen breathing challenge, indicating increased vascular oxygenation. Control tumors showed similar response 24 hours and 1 week later. Twenty-four hours after a single dose of 12 Gy, the irradiated tumors showed a significantly decreased T2* (-2.9±4.2 ms) and further decrease was observed (-4.0±6.0 ms) after 1 week, suggesting impaired vascular oxygenation. DCE revealed tumor heterogeneity, but showed minimal changes following irradiation. Rats were cured of the primary tumors by 3x12 Gy, providing long term survival, though with ultimate metastatic recurrence.

Developing oxygen-enhanced magnetic resonance imaging as a prognostic biomarker of radiation response.

Oxygen-Enhanced Magnetic Resonance Imaging (OE-MRI) techniques were evaluated as potential non-invasive predictive biomarkers of radiation response. Semi quantitative blood-oxygen level dependent (BOLD) and tissue oxygen level dependent (TOLD) contrast, and quantitative responses of relaxation rates (ΔR1 and ΔR2*) to an oxygen breathing challenge during hypofractionated radiotherapy were applied. OE-MRI was performed on subcutaneous Dunning R3327-AT1 rat prostate tumors (n=25) at 4.7 T prior to each irradiation (2F × 15 Gy) to the gross tumor volume. Response to radiation, while inhaling air or oxygen, was assessed by tumor growth delay measured up to four times the initial irradiated tumor volume (VQT). Radiation-induced hypoxia changes were confirmed using a double hypoxia marker assay. Inhaling oxygen during hypofractionated radiotherapy significantly improved radiation response. A correlation was observed between the difference in the 2nd and 1st ΔR1 (ΔΔR1) and VQT for air breathing rats. The TOLD response before the 2nd fraction showed a moderate correlation with VQT for oxygen breathing rats. The correlations indicate useful prognostic factors to predict tumor response to hypofractionation and could readily be applied for patient stratification and personalized radiotherapy treatment planning.

Synthesis and Biological Evaluation of Benzocyclooctene-based and Indene-based Anticancer Agents that Function as Inhibitors of Tubulin Polymerization.

The natural products colchicine and combretastatin A-4 (CA4) have been inspirational for the design and synthesis of structurally related analogues and spin-off compounds as inhibitors of tubulin polymerization. The discovery that a water-soluble phosphate prodrug salt of CA4 (referred to as CA4P) is capable of imparting profound and selective damage to tumor-associated blood vessels paved the way for the development of a new therapeutic approach for cancer treatment utilizing small-molecule inhibitors of tubulin polymerization that also act as vascular disrupting agents (VDAs). Combination of salient structural features associated with colchicine and CA4 led to the design and synthesis of a variety of fused aryl-cycloalkyl and aryl-heterocyclic compounds that function as inhibitors of tubulin polymerization. Prominent among these compounds is a benzosuberene analogue (referred to as KGP18), which demonstrates sub-nM cytotoxicity against human cancer cell lines and functions (when administered as a water-soluble prodrug salt) as a VDA in mouse models. Structure activity relationship considerations led to the evaluation of benzocyclooctyl [6,8 fused] and indene [6,5 fused] ring systems. Four benzocyclooctene and four indene analogues were prepared and evaluated biologically. Three of the benzocyclooctene analogues were active as inhibitors of tubulin polymerization (IC50 < 5 µM), and benzocyclooctene phenol 23 was comparable to KGP18 in terms of potency. The analogous indene-based compound 31 also functioned as an inhibitor of tubulin polymerization (IC50 = 11 µM) with reduced potency. The most potent inhibitor of tubulin polymerization from this group was benzocyclooctene analogue 23, and it was converted to its water-soluble prodrug salt 24 to assess its potential as a VDA. Preliminary in vivo studies, which utilized the MCF7-luc-GFP-mCherry breast tumor in a SCID mouse model, demonstrated that treatment with 24 (120 mg/kg) resulted in significant vascular shutdown, as evidenced by bioluminescence imaging at 4 h post administration, and that the effect continued at both 24 and 48 h. Contemporaneous studies with CA4P, a clinically relevant VDA, were carried out as a positive control.