2,3-Diaryl substituted indoles as probes for monitoring cyclooxygenase-2 (COX-2) with PET - radiolabeling, in vitro and in vivo studies

Ziel/Aim:

Cyclooxygenase-2 (COX-2) is an enzyme induced during inflammation, but overexpression of COX-2 also has been observed in carcinogenic processes. Non-invasive monitoring and quantitative characterization of functional expression of COX-2 by means of PET would substantially improve understanding of the role of this enzyme during manifestation and progression of inflammatory diseases and cancer.

Methodik/Methods:

Two compounds having a 2,3-diaryl substituted indole scaffold; 3-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-1H-indole 1 and 3-(4-methoxyphenyl)-2-(4-methylsulfonylphenyl)-1H-indole 2, both showing high inhibitory activity for COX-2 (IC₅₀~20nM) served as non-radioactive references. The fluorine-18 radiolabeled tracer [¹⁸F]1 was synthesized by nucleophilic substitution of an appropriate trimethylammonium-substituted aromatic precursor with [¹⁸F]fluoride and subsequent McMurry cyclization. The carbon-11 radiolabeled probe [¹¹C]2 was formed via a methylation reaction of the corresponding desmethyl precursor with [¹¹C]CH₃I. Unstimulated human monocytic leukemia cells (THP-1), phorbol ester stimulated THP-1 macrophages (THP-1M) and human tumor cell lines showing selectively high COX-2 (FaDu, HT-29, A2058) or COX-1 expression (A375) were used to study the overall uptake or cellular association of [¹⁸F]1 and [¹¹C]2 in vitro. The stability of [¹⁸F]1 was determined by metabolite analysis of arterial blood samples in rats. In vivo kinetics and tumor uptake were investigated by dynamic small animal PET studies on HT-29 tumor bearing mice.

Ergebnisse/Results:

[¹⁸F]1 was synthesized in 10% yield (d.c.) in 98% radiochemical purity with a specific activity of 74-91 GBq/µmol. [¹¹C]2 was obtained in 23% yield (d.c.) in 99% radiochemical purity with a specific activity of 79-89 GBq/µmol. The radiotracer cellular uptake in each model used correlated well with the observed COX protein synthesis. Cell models with prominent COX-2 overexpression showed a substantially higher uptake of both [¹⁸F]1 and [¹¹C]2 in the order FaDu>HT29>THP-1M>A2058 when compared to COX-1 overexpressing A375 cells. The lowest cellular uptake was observed in THP-1 showing no or very low baseline expression of both COX-1 and COX-2. Metabolite analysis revealed 75% of original compound [¹⁸F]1 after 60 min. In contrast to the in vitro results no substantial tumor uptake of [¹⁸F]1 on HT-29 tumor bearing mice was detected.

Schlussfolgerungen/Conclusions:

The radiolabeled COX-2 inhibitors [¹⁸F]1 and [¹¹C]2 were synthesized in good radiochemical yield and high purity. Cellular studies demonstrated well correlation of the overall radiotracer uptake with COX expression/protein synthesis rate. In vivo investigation did not show any accumulation of [¹⁸F]1 in COX-2 overexpressing tumors. Further exploration of new fluorine-18 and carbon-11 radiolabeled COX-2 inhibitors is required and currently under the way.