Evaluation of ^99mTc-labeled small molecule inhibitors of carbonic anhydrase (CA) for imaging hypoxia

Objectives Hypoxia is associated with a poor response to radiation therapy and some types of chemotherapies. CA IX is upregulated in response to the hypoxic tumor microenvironment making it an attractive molecular marker for detecting tumor hypoxia. MIP-1490 and MIP-1505, 4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide where X= ethyl or n-butyloxy, respectively, were evaluated for targeting CA IX in hypoxic cells and tumor.

Methods MIP-1490 and MIP-1505 were radiolabeled with ^99mTc(CO)₃ and studied in vitro for binding to normoxic (CA IX-) and hypoxic (CA IX+) HeLa cells, constitutively expressing SK-RC-52 and CA IX- SK-RC-59 cells. Cellular internalization in HeLa cells was examined. Tissue distribution and SPECT imaging in HeLa xenograft mice with and without 10 mg/kg acetazolamide competition was also conducted.

Results Re-MIP-1490 and Re-MIP-1505 bound to CA IX with high affinity on hypoxic HeLa cells (109 and 35 nM IC₅₀ by competition binding, respectively), but did not internalize. Competition with acetazolamide demonstrated CA specific binding in hypoxic or CoCl₂treated HeLa and SK-RC-52 cells while reduced binding was observed in normoxic HeLa and SK-RC-59 cells. In vivo, ^99mTc-MIP-1490 achieved 2.3%ID/g in HeLa tumors with 11:1 tumor:blood and 5:1 tumor:muscle ratios at 1h while ^99mTc-MIP-1505 achieved 3.2%ID/g in HeLa tumors with 12:1 tumor:blood and 16:1 tumor:muscle ratios. Nontarget binding was low for both compounds, but tumor retention was greater with ^99mTc-MIP-1505. SPECT imaging corroborated these results. Co-injection with 10 mg/kg acetazolamide reduced tumor uptake by >85% for both compounds.

Conclusions ^99mTc-MIP-1490 and ^99mTc-MIP-1505 bind to CA IX on hypoxic cells and tumors with high affinity and specificity. Such targeted probes have the potential to significantly impact diagnosis, staging, and treatment selection of solid tumors