Molecular imaging of tumor carbonic anhydrase (CA) with a ^99mTc-labeled small molecule inhibitor

Objectives CA IX is upregulated in solid tumors in response to the hypoxic tumor microenvironment and is expressed constitutively in renal cell carcinoma, making it an attractive molecular target for radiopharmaceutical development. MIP-1355, 4-(2-(((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino)ethyl)benzenesulfonamide, a small molecule benzenesulfonamide (BzSA)-based compound selected from a series of CA IX inhibitors incorporating novel tridentate chelates for labeling with the M(CO)₃ core (M = Re or ^99mTc), was evaluated for targeting CA IX.

Methods MIP-1355 was synthesized starting with a BzSA pharmacophore, radiolabeled via ^99mTc(CO)₃ intermediate, and studied in vitro for binding to normoxic CA IX(-) and hypoxic CA IX(+) HeLa, constitutively expressing CA IX/XII(+) SK-RC-52 and CA IX/XII(-) SK-RC-59 cells. Saturation binding and cellular internalization in HeLa cells was examined. Tissue distribution in xenograft mice with and without cold competition was also conducted.

Results ^99mTc-MIP-1355 exhibited high affinity for CA IX on hypoxic HeLa cells (52 nM IC₅₀ by competition binding with the Re analog; 17 nM K_d by saturation binding with the ^99mTc analog). Specificity for carbonic anhydrase binding was demonstrated by competition with acetazolamide in hypoxic HeLa and SK-RC-52 cells and reduced binding in normoxic HeLa and SK-RC-59 cells. ^99mTc-MIP-1355 was not internalized in HeLa cells. In vivo, ^99mTc-MIP-1355 achieved 2.6-3.0% ID/g in CA IX(+) tumors with tumor:blood >30:1 and tumor:muscle >5:1 at 4h. Co-injection with 10 mg/kg acetazolamide reduced tumor uptake by >85%. Uptake in SK-RC-59 xenografts was 0.55% ID/g, further illustrating specificity for carbonic anhydrase.

Conclusions ^99mTc-MIP-1355 binds to CA IX on cancer cells with high affinity. The tumor uptake of ^99mTc-MIP-1355 in vivo was specific to tumor-associated CA IX/XII. Such a targeted probe has the potential to significantly impact diagnosis, staging, treatment selection and therapy of solid tumors