Selective visualization of cyclooxygenase-2 in inflammation and cancer by targeted fluorescent imaging agents

Abstract

Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon.

Figure 1. Labeling of COX-2-expressing cells by compound 2

The experimental protocols are described in Materials and Methods. a, 1483 HNSCC cells treated with 200 nM compound 2 for 30 min. b, 1483 HNSCC cells pretreated with 5 µM celecoxib for 20 min prior to compound 2 treatment. c, Confocal microscopy of 1483 HNSCC cells treated with both mitotrackerGR in blue (mitochondria) and compound 2 in red (perinuclear).

Figure 2. In vivo labeling of COX-2-expression in inflammation by compound 1, 2, or 3

a, C57BL/6 mouse with carrageenan-induced inflammation in the left foot pad. The left mouse was dosed with the negative control molecule 3 (1 mg/kg, i.p.) and the right mouse was dosed with compound 1 (1 mg/kg, i.p.). Both mice were imaged at 3 hr post-injection. b, Fold increase of fluorescence in inflamed vs. contralateral paw of wild-type and COX-2(−/−) mice at 3 hr post-injection of compound 2 (1 mg/kg, i.p.) (n = 6). c, Carrageenan was injected in the rear left footpads of female C57BL/6 mice, followed by dosing compound 1 (1 mg/kg i.p.) 24 hr later. Animals were reinjected with compound 1 at 3, 5, and 7 days post-carrageenan (n = 9). Mice were imaged at 3 hr after compound injection. The plot shows the fold-increase of fluorescence in swollen vs. contralateral foot (n=6).

Figure 3. In vivo labeling of COX-2-expressing xenografts by compound 2

a, Nude mice with HCT116 xenograft (COX-2 negative), or b, 1483 xenograft (COX-2 positive) were dosed (retro-orbital) with 1 mg/kg compound 2 and imaged at 3.5 hrs post-injection. c, Nude mice with 1483 xenografts were pre-dosed with DMSO prior to injection of compound 2 (2 mg/kg, intraperitoneal), or d, pre-dosed with indomethacin (2 mg/kg, intraperitoneal) 24 hrs and 1 hr prior to compound 2 and imaged at 3 hr post-injection (Xenogen IVIS, DsRed filter, 1 s, f2, 1.5 cm depth). The emission observed around the peritoneal cavity in Figures 3c and d is due to residual 2 at the site of injection.

Figure 4. Analysis of fluorescent material in xenografts and several mouse tissues

a, representative HPLC-UV chromatogram (detection = 581 nm) of 1483 tumor extract (4 hr post-administration) revealed a single major fluorescent compound that coeluted with compound 2 (15.3 min). The peak eluting at 13.3 min integrates for less than 5% of the peak at 15.3 min. Non-specific fluorescent peaks were eluted at or near the void volume of the column. Inset – the Q1 mass spectrum of the extracted fluorescent material was identical to that of an authentic standard of 2. b, Time-course and distribution of compound 2 in various mouse tissues in vivo.

Figure 5. In vivo labeling of COX-2-expression in intestinal polyps by compound 2

C57BL/6J-Min/+ mice bearing small intestinal polyps were euthanized at 2 hr after retro-orbital injection of compound 2 (1 mg/kg), and small intestines were washed, opened, and examined by dissecting fluorescence microscopy. a, Section of small intestine with no polyp, 90 ms exposure. b, Single polyp, 90 ms exposure. c, Polyp cluster, 90 ms exposure.