Synthesis and in vivo biological evaluation of 68Ga labelled carbonic anhydrase IX targeting small molecules for positron emission tomography

Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [68Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing 68Ga for preclinical CA IX imaging are scarce, and this is one of the first effective 68Ga compounds reported for PET imaging of CA IX. © 2016 American Chemical Societ

Effect of S4 on doxorubicin sensitivity in MDA-MB-231 and FaDu cells.

<p>CAIX protein expression is higher during hypoxia in MDA-MB-231 and FaDu cells (<b>A</b>). Quantification of three independent biological repeats shows an almost twofold increase in CAIX expression (normalized to actin expression levels) in both cell lines (<b>B</b>). Cell viability assays of MDA-MB-231 (<b>C</b>) and FaDu cells (<b>D</b>) with increasing concentrations of doxorubicin. Cells were exposed to vehicle (black) or S4 (green) during normoxia (N), or to vehicle (red) or S4 (blue) during hypoxia (H). Results of three independent biological repeats are shown (mean ± SEM).</p

Effect of S4, doxorubicin, or the combination of both, on HT29 –CAIX high and HT29 –CAIX low tumor xenograft growth.

<p>Relative tumor volume (mean ± SEM) of HT29 –CAIX high (<b>A</b>) or HT29 –CAIX low xenografts (<b>B</b>) treated with vehicle (black), S4 (green), vehicle with doxorubicin (red), or S4 with doxorubicin (blue). Linear fits from relative tumor growth were used to estimate the mean time to reach 2 times start volume (T2XSV) of HT29 –CAIX high (<b>C</b>) or HT29 –CAIX low (<b>D</b>) xenografts.</p

Selectively Targeting Tumor Hypoxia with the Hypoxia-Activated Prodrug CP-506

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 mu mol/L (0.1% O-2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies while retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 were significantly correlated with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506