Hyperpolarized (1-13C)Alaninamide Is a Multifunctional In Vivo Sensor of Aminopeptidase N Activity, pH, and CO2.

Spin hyperpolarization enables real-time metabolic imaging of carbon-13-labeled substrates. While hyperpolarized l-(1-13C)alaninamide is a probe of the cell-surface tumor marker aminopeptidase-N (APN, CD13), its activity in vivo has not been described. Scanning the kidneys of rats infused with hyperpolarized alaninamide shows both conversion to [1-13C]alanine and several additional spectral peaks with distinct temporal dynamics. The (1-13C)alaninamide chemical shift is pH-sensitive, with a pKa of 7.9 at 37 °C, and the peaks correspond to at least three different compartments of pH 7.46 ± 0.02 (1), 7.21 ± 0.02 (2), and 6.58 ± 0.05 (3). An additional peak was assigned to the carboxyamino adduct formed by reaction with dissolved CO2. Spectroscopic imaging showed nonuniform distribution, with the low-pH signal more concentrated in the inner medulla. Treatment with the diuretic acetazolamide resulted in significant pH shifts in compartment 1 to 7.38 ± 0.03 (p = 0.0057) and compartment 3 to 6.80 ± 0.05 (p = 0.0019). While the pH of compartment 1 correlates with blood pH, the pH of compartment 3 did not correspond to the pH of urine. In vitro experiments show that alaninamide readily enters blood cells and can detect intracellular pH. While carbamate formation depends on pH and pCO2, the carbamate-to-alaninamide ratio did not correlate with either arterial blood pH or pCO2, suggesting that it may reflect variations in tissue pH and pCO2. This study demonstrates the feasibility of using hyperpolarized sensors to simultaneously image enzyme activity, pCO2, and pH in vivo.

In vitro and in vivo BNCT investigations using a carborane containing sulfonamide targeting CAIX epitopes on malignant pleural mesothelioma and breast cancer cells.

This study aims at merging the therapeutic effects associated to the inhibition of Carbonic Anhydrase IX (CAIX), an essential enzyme overexpressed by cancer cells including mesothelioma and breast cancer, with those ones brought by the application of Boron Neutron Capture Therapy (BNCT). This task was pursued by designing a sulfonamido-functionalised-carborane (CA-SF) that acts simultaneously as CAIX inhibitor and boron delivery agent. The CAIX expression, measured by Western blot analysis, resulted high in both mesothelioma and breast tumours. This finding was exploited for the delivery of a therapeutic dose of boron (> 20 µg/g) to the cancer cells. The synergic cytotoxic effects operated by the enzymatic inhibition and neutron irradiation was evaluated in vitro on ZL34, AB22 and MCF7 cancer cells. Next, an in vivo model was prepared by subcutaneous injection of AB22 cells in Balb/c mice and CA-SF was administered as inclusion complex with a ß-cyclodextrin oligomer. After irradiation with thermal neutrons tumour growth was evaluated for 25 days by MRI. The obtained results appear very promising as the tumour growth was definitively markedly lower in comparison to controls and the CAIX inhibitor alone. This approach appears promising and it call consideration for the design of new therapeutic routes to cure patients affected by this disease.