Targeted imaging of cancer by fluorocoxib C , a near-infrared cyclooxygenase-2 probe

Cyclooxygenase-2 (COX-2) is a promising target for the imaging of cancer in a range of diagnostic and therapeutic settings. We report a near-infrared COX-2-targeted probe, fluorocoxib C (FC), for visualization of solid tumors by optical imaging. FC exhibits selective and potent COX-2 inhibition in both purified protein and human cancer cell lines. In vivo optical imaging shows selective accumulation of FC in COX-2-overexpressing human tumor xenografts [1483 head and neck squamous cell carcinoma (HNSCC)] implanted in nude mice, while minimal uptake is detectable in COX-2-negative tumor xenografts (HCT116) or 1483 HNSCC xenografts preblocked with the COX-2selective inhibitor celecoxib. Time course imaging studies conducted from 3 h to 7-day post-FC injection revealed a marked reduction in nonspecific fluorescent signals with retention of fluorescence in 1483 HNSCC tumors. Thus, use of FC in a delayed imaging protocol offers an approach to improve imaging signal-to-noise that should improve cancer detection in multiple preclinical and clinical settings. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.20.5.050502]

Targeted imaging of cancer by fluorocoxib C, a near-infrared cyclooxygenase-2 probe Fluorescent contrast agents that emit light in the near-infrared (NIR) region are particularly useful for in vivo imaging targeted to the early detection of cancer. 1 In the last decade, technological advances have led to the development of target-directed NIR probes for imaging or image-guided surgery. 1,2 However, their nonspecific biodistribution and inefficient clearance from nontargeted organs are obstacles to achieving high signal-to-noise ratios in the short-term following administration. 3 One way to address this problem would be through probes that not only absorb and emit at the NIR region, but are also selectively retained at the target, thereby providing time for clearance from nontarget sites. In fact, some reports have demonstrated an advantage of delayed imaging (up to 3 h) of tumors using [ 18 F]fluorodeoxyglucose positron emission tomography (FDG-PET). [4][5][6][7] For example, in the case of FDG-PET imaging of breast cancer, tumor to nontumor signal ratios were significantly higher in 3 h than in 1.5 h images, and lesion detectability increased from 83% at 1.5 h to 93% at 3 h. 7 Similarly, dynamic FDG-PET scans were recorded with improved contrast between tumors and normal tissues at 2 h compared with 1 h postinjection of the tracer. 8 However, the application of this approach in the case of FDG-PET is limited by the short t 1∕2 of the 18 F tracer (109.7 min). 9 This limitation would not apply to an NIRfluorescent probe.
Cyclooxygenase-2 (COX-2) is a promising target for molecular imaging because it is overexpressed in inflammation and many malignancies. 10,11 We recently discovered fluorocoxib A, a 5-carboxy-rhodamine-labeled COX-2 inhibitor, and described its utility in the selective visualization of COX-2 in inflammation and cancer. 12 The success of fluorocoxib A provides the proof-of-principle for COX-2-targeted imaging, but use of this agent has been limited to imaging within only a few hours following administration. Herein, we report the synthesis and evaluation of a long-lived COX-2-targeted NIR probe, fluorocoxib C (FC). FC is a potent and selective COX-2 inhibitor that exhibits COX-2-targeted uptake in human tumor xenografts in both prompt and delayed NIR imaging protocols. Furthermore, we show that delayed imaging using FC results in markedly improved contrast between signals in tumor and nontumor tissues.
The ability of FC to inhibit purified COX-1 or COX-2 was evaluated in a thin layer chromatography assay using 14 C-arachidonic acid. 12 FC displayed selective and potent COX-2 inhibitory activity (COX-2 IC 50 ¼ 0.5 μM; COX-1 IC 50 > 25 μM) while NIR664 was inactive. Inhibition of COX-2 in intact cells by FC was also evaluated using a previously described method. 12 The IC 50 values for inhibition of COX-2 by FC in lipopolysaccharide (LPS)-activated RAW264.7 cells and human 1483 head and neck squamous cell carcinoma (HNSCC) cells were 0.37 and 0.32 μM, respectively.
We first evaluated the prompt optical imaging potential of FC to visualize COX-2 in a preclinical model of cancer. We implanted xenografts in female nude mice by subcutaneously injecting COX-2-expressing 1483 HNSCC cells in the left hip or COX-2-null human colorectal carcinoma (HCT116) cells in the right hip. The tumors were allowed to grow to approximately 700 to 1000 mm 3 . We administered FC or NIR664 at a low-dose (0.2 mg∕kg, retro-orbitally) to the tumor-bearing animals. In some cases, the animals were pretreated with the COX-2-selective inhibitor celecoxib (2 mg∕kg) prior to FC injection. The animals were imaged at 4 h postinjection of fluorophore on a Xenogen IVIS 200 optical imaging system. The imaging showed good uptake of FC into the COX-2-expressing 1483 HNSCC tumors while minimal uptake was documented in the COX-2-negative HCT116 tumors or in 1483 HNSCC tumors pretreated with celecoxib or imaged using NIR664 dye instead of FC [ Figs. 3(a)-3(d)]. To confirm COX-2-dependent uptake of FC, we collected the tumors by dissection and imaged them ex vivo under the Xenogen IVIS 200 optical imaging system. Fig. 3(i). After imaging, we analyzed the tumor tissues using LC-MS/MS and identified the intact parent FC in the 1483 HNSCC tumors (0.24 pmol/g tissue, n ¼ 3).

Figures 3(e)-3(h) show the tumor images with uptake quantification provided in
These prompt imaging experiments established that FC is a valid COX-2-targeting NIR probe. However, at this 4-h time point, substantial fluorescent signals are also detectable in the peritoneal cavity due to biodistribution of the probe in abdominal organs. This led us to investigate whether these nonspecific signals would clear with time such that only the fluorescence signal would be detectable in the tumor. A group of female nude mice bearing 1483 HNSCC xenograft tumors (750 to 1000 mm 3 ) on their left hip was dosed by injection with a low dose of FC (0.2 mg∕kg). The animals were lightly anesthetized with 2% isoflurane and imaged on a Xenogen IVIS 200 optical imaging system at varying times up to 7 days after FC administration. Control animals that did not receive an FC injection showed a noticeable autofluorescence in both sides of the peritoneal cavity but no signal at the tumor [ Fig. 4(a)]. In FC-injected animals, the fluorescence signal was reproducibly detected in the tumor at 3 h and   Figure 4(s) presents the quantification of signals in tumor, liver, kidney, and muscle from uninjected controls, and from 3 h and 7 days FCtreated animals by AMIDE software. Dynamic changes in signals in the nonspecific organs are due to the clearance from the normal tissues.
In conclusion, we report the discovery of FC, a COX-2targeted NIR probe. Uptake of FC is COX-2-dependent as demonstrated by the lack of uptake of nontargeted NIR664 and by the failure of FC uptake in celecoxib-pretreated or COX-2negative tumors. In addition, FC is effective as a delayed fluorescence imaging agent exhibiting the potential for improved lesion detection accuracy over standard imaging through improved signal-to-noise in differentiating tumor from nontumor tissues. Thus, FC represents the first delayed COX-2-targeted fluorescence imaging NIR probe for detection of tissues containing elevated levels of COX-2 in preclinical and clinical settings.