(124)I-iodopyridopyrimidinone for PET of Abl kinase-expressing tumors in vivo

Abstract

Because of the recent development of an iodopyridopyrimidinone Abl protein kinase inhibitor (PKI), (124)I-SKI-212230 ((124)I-SKI230), we investigated the feasibility of a PET-based molecular imaging method for the direct visualization of Abl kinase expression and PKI treatment.

Methods: In vitro pharmacokinetic properties, including specific and nonspecific binding of (124)I-SKI230 to its Abl kinase target and interaction with other PKIs, were assessed in cell-free medium and chronic myelogenous leukemia (CML) cells overexpressing BCR-Abl (K562), in comparison with BT-474 cells that are low in Abl expression. In a xenograft tumor model, we assessed the in vivo pharmacokinetics of (124)I-SKI230 using PET and postmortem tissue sampling. We also tested a paradigm of (124)I-SKI230 PET after treatment of the animal with a dose of Abl-specific PKI for the monitoring of the tumor response.

Results: In vitro studies confirmed that SKI230 binds to Abl kinase with nanomolar affinity, that selective uptake occurs in cell lines known to express Abl kinase, that RNAi knock-down supports specificity of cellular uptake due to Abl kinase, and that imatinib, an archetype Abl PKI, completely displaces SKI230. With SKI230, we obtained successful in vivo PET of Abl-expressing human tumors in a nude rat. We were also able to demonstrate evidence of substrate inhibition of in vivo radiotracer uptake in the xenograft tumor after treatment of the animal as a model of PKI treatment monitoring.

Conclusion: These results support the hypothesis that molecular imaging using PET will be useful for the study of in vivo pharmacodynamics of Abl PKI molecular therapy in humans.

FIGURE 1

Pharmacochemical properties of Abl inhibitor for direct imaging. (A) Chemical structure of ¹²⁴I-SKI230. (B) Time course of binding of ¹³¹I-SKI230 to K562 cells (solid line and ■ = on rate; dotted line and ○ = off rate). (C) Saturation binding of SKI230 to K562 cells (inset Scatchard transformation). (D) Displacement binding of ¹³¹I-SKI230 to K562 cells by SKI230 (solid line and ■) and imatinib (dotted line and ○).

FIGURE 2

In vitro characterization of PKI for direct imaging of Abl expression in tumors. (A) Radiotracer accumulation assay. In vitro uptake studies in cell lines K562 (overexpressing BCR-Abl) and BT-474 (devoid of Abl expression). (B) Inhibitory concentrations of 50% for SKI230 in cytotoxicity assay (WST-1 colorimetric assay). (C) Proof of SKI230 binding specificity using Anti-Abl siRNA in radiotracer accumulation assay: wild-type K562, (N1); K562 transfected with nonspecific siRNA control pool (N2); K562 transfected with 100 nM anti-Abl SiRNA (N3); and K562 transfected with 200 nM anti-Abl SiRNA (N4). (D) Western blot analysis of cells treated with radiolabeled compound. 1, Abl antibody Western blot anti–Human-cAbl; 2, protein standards; 3, A-431; 4, BT-474; 5 and 6, K562 (6, control for siRNA experiment); 7, K562 after 100 nM siRNA; and 8, K562 after 200nM siRNA.

FIGURE 3

Direct in vivo imaging of Abl expression in rat xenograft human CML model. (A) Time series of axial PET Advance images of rat, bearing tumor model K562 overexpressing BCR-Abl representing biodistribution of ¹²⁴I-SKI230 in animal. (B) Sagittal projection of accumulation of ¹²⁴I-SKI230 in Abl-expressing CML tumor model in shoulder area 90 min after administration of direct PKI imaging agent. (C) Pharmacokinetic profile of ¹²⁴I-SKI230 biodistribution in organs and tissues quantified using image analysis of radiotracer accumulation based on ROI analysis of dynamic frames. (D) Ex vivo well counts calculated for tissue samples obtained by postmortem tissue collection (at 90 min) confirming SKI230 pharmacokinetic results.

FIGURE 4

Blocking of ¹²⁴I-SKI230 in vivo uptake within engrafted tumor. (A) Transaxial images of representative rat (n = 3) before PKI treatment. (B) Transaxial images of same rat after cold SKI230 administration, demonstrating in vivo displacement of radiotracer by micromolar concentration of systemically administered PKI. (C) ¹²⁴I-SKI230 tumor uptake after blocking with cold SKI230 (open bars) is reduced, compared with pretreatment uptake (solid bars). (D) Imaging of negative-control A-431 tumor model.