^99mTc-Labeled carbonic anhydrase IX (CA IX) inhibitors for molecular imaging of cancer

Objectives CA IX is upregulated in cancer in response to hypoxia making it an attractive molecular target for radiopharmaceutical development. A series of small molecule benzenesulfonamide (BzSA)-based compounds incorporating novel tridentate chelates for labeling with the M(CO)₃ core (M = Re or ^99mTc), were synthesized and evaluated as inhibitors of CA IX.

Methods CA IX inhibitors were synthesized starting with a BzSA moiety tethered via an ethylene linker to bis-pyridyl amine or functionalized bis-imidazolyl amine chelates. Re compounds were tested at 1-10,000 nM for inhibition of CA IX and CA II via 4-nitrophenylacetate hydrolysis. ^99mTc compounds were studied in vivo in nude mice bearing HeLa xenografts. Specific tumor localization was determined by competition with a 10 mg/kg acetazolamide (AZO) co-injection.

Results BzSA analogs containing novel chelates were prepared: 4-(2-((X, Y)amino)ethyl)benzenesulfonamide, where X = Y = (pyridin-2-ylmethyl) (1), X = (pyridin-2-ylmethyl), Y = carboxymethyl (2), X = Y = (1-(carboxymethyl)-1H-imidazol-2-yl)methyl (3), and X = Y = (1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl (4). High affinity binding to CA IX was observed (IC₅₀= 23-93 nM, 2<1<3<4) with approximately 10-fold selectivity for CA IX over CA II. In HeLa xenograft mice, tumor uptake ranged from 0.23-2.6 %ID/g with 4>3>2>1 at 1 h. Specificity for carbonic anhydrases was confirmed by competition with AZO.

Conclusions A series of BzSA analogs containing novel chelates were shown to bind to CA IX with high affinity and selectivity over CA II. The uptake of ^99mTc analogs in HeLa xenografts was specific to carbonic anhydrases. These novel compounds may be exploited to significantly impact the current paradigm for diagnosis, staging, treatment selection and therapy of solid tumors.

Research Support 1 R43 CA134013-01A