Synthesis and evaluation of ortho-[¹⁸F] fluorocelecoxib for COX-2 cholangiocarcinoma imaging

Figures & data

Figure 1 Structures of the target compound ortho-F-celecoxib 1, the reported celecoxib, fluorolabeled celecoxib 2, 3, and other tagged NSAID analogs.

Abbreviation: NSAID, nonsteroidal anti-inflammatory drug.

Figure 2 (A) Preparation of ortho-F-1. (B) The less reactive CF₃COOF was formed as the major fluorinating reagent.

Figure 3 The product mixtures obtained from the radiofluorination of ortho-[¹⁸F]F-1 were profiled using high-performance liquid chromatography as shown in the UV− (A) and radiochromatograms (B).

Notes: The fraction collected from t_R of 19.97 min of the radiochromatogram was identified as ortho-[¹⁸F]F-1. Elution conditions: isocratic mode, RP-18 column, 70% EtOH (aq.), 3 mL/min flow rate, λ=254 nm. The isolated fraction was further confirmed using an analytic high-performance liquid chromatography system as shown in (C) UV− and (D) radiochromatogram. For clarification, an authentic celecoxib was added to the isolated fraction. The peak at t_R of 7.80 min corresponds to a radiochemical purity of 99.92%. Elution conditions: isocratic mode, RP-18 column, 70% EtOH (aq.), 0.5 mL/min flow rate, λ=254 nm.

Table 1 Binding data obtained from this study and the literature

Classification	Compound	IC50 (µM)		Selectivity index
		COX-1	COX-2
Direct binding assay	[^18F]F-FBPin^Citation32	0.91±0.68	0.33±0.24	2.76
	ortho-[^18F]F-1	0.039	0.024	1.63
	[^3H]celecoxib (Kd)^Citation28,Citation29	>3.4×10^−3	1.9×10^−3	>1.79
		15	0.04	375
Indirect binding assay	Fenbufen^Citation27	3.9	8.1	0.48
	Celecoxib/^14C-arachidonic acid	>4	0.03	>133
	Celecoxib/enzyme-linked immunosorbent assay	3.7	0.06	61.7

Abbreviation: COX, cyclooxygenase.

Figure 4 Plots of the formation of [¹⁸F]F–ligand–COX, ortho-[¹⁸F]F-1, in the presence of various concentrations of the competitor celecoxib (A and B).

Abbreviation: COX, cyclooxygenase.

Figure 5 Tracer uptake of ortho-[¹⁸F]F-1 in the COX-2-overexpressed murine CCA tumor cells and the murine CCA tumor cells as a control.

Abbreviations: CCA, cholangiocarcinoma; COX, cyclooxygenase.

Figure 6 Competitive inhibition of the tracer accumulation (ortho-[¹⁸F]F-1) in the presence of various concentrations of celecoxib (0.05 nM–250 μM).

Notes: In vitro system included COX-2-overexpressed CCA cells (A) and CCA cells (B). IC₅₀=0.5 and 46.3 μM for COX-2-overexpressed CCA and usual CCA cells, respectively.
Abbreviations: CCA, cholangiocarcinoma; COX, cyclooxygenase.

Figure 7 (A) Selected PET images taken of two F18-tagged ligands in the same.

Notes: Data for [¹⁸F]FDG were obtained by scanning 90–120 min after intravenous injection into the tail vein. Data for ortho-[¹⁸F]F-1 were obtained by scanning 30–60 min postinjection. The red circles and white circles indicate the tumor loci and normal liver, respectively. (B) Activity–time curves of ortho-[¹⁸F]F-1 were constructed by counting the ROIs at the liver tumor loci and the adjacent normal region of the liver vs the scanning time over 1 h at 10-min intervals. (C) Dynamic PET studies of ortho-[¹⁸F]F-1 exemplified by coronal slices encompassing the liver of a CCA rat (left) and a normal rat (right) taken at 30–40 min in a 1-h scan. Red arrow bars indicate the ROIs circled for the tumor lesion (left) and the corresponding normal region (right), respectively. Number of rats used for the PET studies include n=4 for CCA rats and n=2 for normal rats.
Abbreviations: CCA, cholangiocarcinoma; PET, positron emission tomography; ROIs, regions of interest.

Table 2 Comparison between the blocking studies of CCA rats and normal rats in the presence of a competitor, celecoxib, at various doses

Pharmacokinetics	CCA rat (n=5)		Normal rat (n=3)
	ortho-[^18F]F-1	ortho-[^18F]F-1+ celecoxib (1–4 mg)	[^18F]F-1	ortho-[^18F]F-1+ celecoxib (4 mg)
T/N ratio	1.38±0.23	1.48±0.20
Uptake (%ID/g)	1.14±0.25	1.18±0.22	0.99±0.13	0.98

Abbreviations: CCA, cholangiocarcinoma; T/N ratio, tumor-to-normal ratio.

Figure 8 A representative comparison between the static PET images of ortho-[¹⁸F] F-1 in a CCA rat (left) and in the same rat using cold celecoxib (2 mg) as a blocker (right).

Notes: The white arrows in a gross picture (middle) and in the two PET images indicate the tumor loci. The stronger signals in the PET images are comparable to the tumor lesions in the gross picture. The representative tumor grew in the upper-right lobe of liver. The blocking study using cold celecoxib did not result in signal reduction.
Abbreviations: CCA, cholangiocarcinoma; PET, positron emission tomography.

Figure 9 The dose–response curve for the CCA rats in the presence of the competitor celecoxib.

Abbreviations: CCA, cholangiocarcinoma; T/N ratio, tumor-to-normal ratio.

Figure 10 Comparison between the tracer uptake of ortho-[¹⁸F]F-1 in the tumor lesion of CCA rats (n=5) and normal liver region of the rats including CCA rats (n=13), P=0.0021, one-tailed Student’s t-test. **The statistical variation is p<0.005.

Abbreviation: CCA, cholangiocarcinoma.

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