Impact of structural alterations on the radiopharmacological profile of 18F-labeled pyrimidines as cyclooxygenase-2 (COX-2) imaging agents
Introduction
Cyclooxygenases (COXs) are responsible for the conversion of arachidonic acid to prostaglandins to mediate the body's response to inflammatory conditions. The COX enzyme family consists of isoforms COX-1 and COX-2, which are constitutively and inducibly expressed, respectively. Besides COX-1 and COX-2, also a COX-3 isoform has been described in the literature. However, there is still a debate on its individual role as a drug target for inflammation or if it only represents a splice variant of COX-1 [1]. COX-1 and COX-2 have a long history as important drug target for treatment of inflammatory conditions. The development of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, paracetamol and diclofenac predates the discovery of the COX enzyme family [2,3]. Consequently, most NSAIDs are not selective or only minimally selective in inhibiting COX-1 over COX-2 or vice versa. Selective COX-2 inhibitors were developed only after the discovery of the inducible COX-2 isoform in 1991 [4]. The discovery resulted in an important scientific paradigm which postulated that COX-1 is responsible for the maintenance of normal physiological processes, while acute and chronic inflammatory conditions are mediated by COX-2 [5,6]. Recent research over the past years, however, has also analyzed the involvement of COX-1 during acute and chronic inflammation. Inhibition of housekeeping enzyme COX-1 is one of the main reason for observed severe gastrointestinal side-effects associated with long term NSAID use [7].
The discovery of the COX-2 isoform led to the development of numerous COX-2 selective inhibitors, so called “coxibs”, such as celecoxib, rofecoxib, valdecoxib and others. These drugs promised to deliver the same relief from pain and inflammation as NSAIDs, but without gastrointestinal side-effects.
Development of highly selective compounds has made COX-2 also an attractive target and biomarker for molecular imaging applications [8]. Moreover, COX-2 was also found to be involved in the development and progression of a variety of cancers and neurodegenerative diseases [9,10]. As a result, a large number of COX-2 selective radiotracers have been developed to visualize functional expression of COX-2 in cancer, inflammation and other diseases [[11], [12], [13], [14], [15], [16]].
Despite initial promise, the therapeutic and diagnostic value of coxibs as drugs and radiolabeled molecular probes remained highly challenging. The majority of coxibs in clinical use during the early 2000's had to be withdrawn from market following concerns over their cardiac toxicity profile. Although more than two dozen of PET and SPECT radiotracers have been developed and tested over the past decade, none of them have progressed past initial preclinical testing stages [7].
The challenges of developing a COX-2 specific radiotracer are manifold. Many of the PET and SPECT imaging agents that have been reported to date have failed on the basis of insufficient metabolic stability or lack of COX-2 specific binding in vivo. Other studies found the lack of a well characterized animal model that stably expresses COX-2 to be a major obstacle [7]. The slow progress in the field of COX-2 molecular imaging is partially due to the complex nature of the target itself. Both COX enzymes are intracellular targets, membrane bound on the inside of the endoplasmic reticulum, while the active site is located at the end of a long hydrophobic channel the entrance of which is located inside the membrane [17]. Most widely biomarkers for molecular imaging are extracellular or cross membrane-bound targets, while the targeting of intracellular targets remains a formidable challenge [18]. Efforts to image intracellular targets such as COX-2 using small molecules have struggled to find an ideal structural balance for a favorable pharmacokinetic profile of the radiotracers. Molecular candidates for intracellular imaging must be lipophilic enough to readily diffuse through membranes. However, in that case the inevitable non-specific background uptake of these radiotracers in adipose and muscle tissue is expected to lead to unfavorable target-to-background ratios.
The efforts of our research group in this area led to the development and evaluation of radiotracer [18F]1a [19], a radiolabeled COX-2 inhibitor based on a central pyrimidine scaffold. Despite the formidable challenges associated with the development of COX-2 selective radiotracers for in vivo PET imaging, [18F]1a showed favorable and COX-2 selective tumor uptake profile in a preclinical COX-2-expressing HCA-7 colorectal tumor model [19]. Previous and unsuccessful attempts of developing celecoxib-based 18F-labeled radiotracers for imaging COX-2 [16], designed around a central pyrazole scaffold. His further suggests the importance of proper structure selection and underlines the suitability of a pyrimidine scaffold as central motifs for COX-2 selective imaging agents. Although radiotracer [18F]1a displayed promising COX-2 specific tumor uptake in vivo, the radiotracer also exhibited a number of non-specific off-target interactions. [18F]1a showed high uptake in the muscle tissue, which leads to only modest tumor-to-background ratios after >1 h post injection. In addition, radiotracer [18F]1a also showed significant lung retention upon first pass, which leads to delayed distribution pharmacokinetics [19].
In light of these challenges, the delicate balance of pharmacokinetic properties of COX-2 inhibitors is of crucial importance in the search for suitable COX-2 imaging radiotracer.
Here, we report on the in vitro and in vivo evaluation of two structural analogues of previously reported COX-2 radiotracer [18F]1a [19]. The objective of this study was to investigate whether small structural alterations can improve the pharmacokinetic profile of radiotracer [18F]1a to provide an improved COX-2 selective radiotracer. The synthesis of analogues 2a and 3a, as well as their respective 18F-labeled versions ([18F]2a and [18F]3a) has been reported previously [20]. Compound 1a and its structural analogues 2a and 3a display high COX-2 inhibitory potencies in the low nanomolar range while possessing comparable lipophilicity (logP).
Section snippets
General
All regents and solvents were obtained from Sigma-Aldrich, unless otherwise stated and used without further purification. Column chromatography was conducted using Merck silica gel (mesh size 230–400 ASTM). Thin-layer chromatography (TLC) was performed on Merck silica gel F-254 aluminum plates, with visualization under UV light (254 nm). High performance liquid chromatography (HPLC) purifications and analysis were performed using a Phenomenex LUNA® C18 column (100 Å, 250 × 10 mm, 10 mm) on a
Chemistry and radiochemistry
We have previously reported the design and radiosynthesis of three 18F-labeled COX-2 inhibitors based on a pyrimidine scaffold [20] ([18F]1a, [18F]2a, [18F]3a; Fig. 1a). Radiotracer [18F]1a carries a methyl sulfone COX-2 pharmacophore, while radiotracer [18F]2a carries a sulfonamide COX-2 pharmacophore (Fig. 1a - highlighted in blue). Radiotracer [18F]3a contains an ether bridge, while [18F]1a contains a secondary amine linker (Fig. 1a - highlighted in red).
Lipophilicity, experimentally
Discussion
Efforts to develop a radiotracer for in vivo imaging of COX-2 are hampered by a lack of standardized and well-characterized models [6]. Emerging COX-2 radiotracers are studied in a variety of in vitro and in vivo models, generally without comparison to a previously evaluated tracer. The results of these studies are difficult to interpret because it is seldom clear whether the novel radiotracer or the biological model have an innate inadequacy that leads to the relative inability of the
Conclusion
Two highly potent and selective 18F-labeled COX-2 inhibitors were evaluated in a well characterized COX-2 expressing colorectal cancer model and compared to previously reported evaluation of radiotracer [18F]1a. Despite their structural similarities, the hepatobiliary and renal clearance profiles of the radiotracers varied significantly.
We have shown that small modifications such as changing the methylsulfone COX-2 pharmacophore to a sulfonamide group and the change from the amine to the ether
Acknowledgement
The authors would like to thank John Wilson, Dave Clendening and Blake Lazurko from the Edmonton PET Center for radionuclide production and excellent technical support. We thank Gail Hipperson and Dan McGinn from the Vivarium of the Cross Cancer Institute for supporting the animal work. We also gratefully acknowledge the Dianne and Irving Kipnes Foundation, the Canadian Institute of Health Research (CIHR) and the National Science and Engineering Research Council of Canada (NSERC) -
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