Initial Biological Evaluations of [ 18F]KS-7-51 to Image PPAR-? in Tumor Mice Model
- 1. Department of Radiology, University of Pennsylvania, USA
- 2. Department of Radiology, Wake Forest School of Medicine, USA
- 3. Department of Chemistry, University of Illinois, USA
- 4. Department of Radiology, Washington University in St. Louis, USA
Abstract
Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a ligand-activated nuclear receptor transcription factor that plays a vital role in lipid regulation, antitumor, and anti-inflammatory responses. PET imaging of PPAR-γ could provide critical information on tumor pathogenesis and treatment strategies. In this study, we report the radiochemistry and initial biological evaluations of [18F]KS-7-51, a p-fluoroethoxy phenyl derivative in a murine model of prostate cancer (PC3). In vitro cell uptake studies of [18F]KS-7-51 in PC3 cells showed high selectivity and specificity. Biodistribution in PC3-bearing mice demonstrated modest tumor uptake and blockade with KS-7-51 showed specificity. These results demonstrate the utility of [18F]KS-7-51 as a PPAR-γ PET imaging agent.
Citation
Lee H, Damuka N, Katzenellenbogen JA, Bhuddhika Shrimani LP, Xu J, et al. (2020) Initial Biological Evaluations of [18F]KS-7-51 to Image PPAR-γ in Tumor Mice Model. J Radiol Radiat Ther 7(1): 1086.
Keywords
• PET; PPAR-γ; Bio distribution; Cancer; Cell uptake
ABBREVIATIONS
PPAR-γ: Peroxisome Proliferator-Activated Receptor Gamma; PET: Positron Emission Tomography; PC: Prostate Cancer
INTRODUCTION
Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors, and consist of three commonly studied subtypes PPAR-α, PPAR-δ and PPAR-γ [1,2]. PPAR-γ plays a major role in the regulation of lipid metabolism, lipid storage and adipocyte differentiation. PPAR-γ is also associated with different activities in tumorigenicity and plays a vital role in cancer development through its action on cancer stem cells [1-3]. There were some studies reporting the antiangiogenesis and anti-proliferation effects in tumor progression via PPAR-γ-up regulated signaling pathways [4]. PPAR-γ agonists and antagonists have been used for treatment of several cancers, such as glioma, prostate, liver, colorectal, breast and ovarian cancers [5]. The broad range and pleiotropic functions of PPAR-γ makes it an attractive target for developing imaging biomarkers [6]. Radiolabeled PPAR-γ ligands in conjunction with Positron Emission Tomography (PET) offer a sensitive way to quantify PPAR-γ levels and these imaging agents could aid in identifying patients who might show a favorable response to PPAR-γ ligand-based therapeutic applications. Our lab previously reported [18F], [124I], and [76Br]-radiolabeled PPAR-γ agonists and antagonists with high in vitro specificity and in vivo potency [7,8]. The agonist-based radiotracers showed high binding affinities and metabolic stabilities; however, in vivo target tissue uptake was poor. On the other hand, PPAR-γ antagonist radiotracers with high binding affinities and target selectivity suffered from poor in vivo pharmacokinetics [9,10]. GW9662 is considered as a gold standard PPAR-γ antagonist(Figure 1). We previously reported the synthesis and in vitro PPAR-γ selectivity of a p-fluoroethoxy phenyl derivative, KS-7-51 (IC50 = 47.5 ± 7.6 nM Vs. IC50 = 144.6 ± 7.5 nM of GW9662) [11]. Here we report for the first time the radiochemistry, preliminary in vitro, and in vivo evaluations of [18F]KS-7-51 as a potential PET PPAR-γ imaging agent in a murine xenograft model of prostate cancer.
MATERIALS AND METHODS
All commercially available chemicals including standards, reagents and anhydrous solvents were purchased from Sigma-Aldrich, MO, USA. HPLC columns (both semiprep and QC-analytical) were purchased from PJ Cobert Associates Chromatography Supplies, MO, USA. Human PC3 cells were purchased from ATCC cell lines. Non-radioactive standard KS7-51 and its mesylate precursor 1 were synthesized following previously published procedures with slight modifications [12- 14]. The radiochemical synthesis of [18F]KS-7-51 was achieved by substituting the corresponding mesylate group of precursor 1 with [18F]F¯ using K222-K2 CO3 complex in DMSO for 15 min at 100°C as depicted in scheme 1. The total time for the radiochemistry of [18F]KS-7-51, including [18F]F¯ production, azeotropic drying, radiolabeling, purification and formulation was ~65 min. We then conducted in vitro cell uptake assays in PC3, a humanderived prostate cancer cell line following our previously published protocols [9-15]. GW9662 (15 µM) was used as a blocking agent to evaluate specificity of [18F]KS-7-51. The counts per minute (cpm) values of each well were normalized to the amount of radioactivity added to each well and was expressed as percent uptake relative to the control condition. The data was expressed as % total dose (TD)/mg of protein present in each well (Figure 2). With the promising in vitro cell-uptake results, we conducted standard biodistribution studies in BALB/c mice bearing PC3 tumors. Mice were grouped into four groups (n = 4/ group) based on the uptake time i.e., 5, 30, 60 and 120 min postradiotracer injection. Samples of tumor, blood, brain, heart, lung, liver, spleen, pancreas, kidney, muscle and bone were harvested, weighed, and gamma counted with a standard dilution of the injectate. The percentage of the injected dose per gram of tissue (%ID/g) was shown in Figure 3. The complete experimental details including the radiolabeling procedure, in vitro cell uptake assay, and biodistribution studies were described in detail in the ‘Supplementary Material’ section.
RESULTS AND DISCUSSION
The radiochemical purity of [18F]KS-7-51 was >98% and its identity was confirmed by co-elution with non-radioactive KS-7-51. The specific activity was determined to be ~110±11 GBq/ µmol (n>15) and radiochemical yield ~18% (decay corrected to end of synthesis). The radioactive uptake of [18F]KS-7-51 was ~50%, 44% and 48% blocked by GW9662 at 5, 30 and 60 min incubation times respectively showing high in vitro specificity. [18F]KS-7-51 standard biodistribution displayed rapid clearance from blood, liver and kidneys from 5 min to 120 min i.e., blood with %ID/g 4.32 ± 1.62 (5 min) to 0.58 ± 0.02 (120 min), liver %ID/g 11.83 ± 1.53 (5 min) to 4.11 ± 1.02 (120 min), and kidneys %ID/g 16.35 ± 2.92 (5 min) to 6.32 ± 1.32 (120 min). Bone uptake was lowered from %ID/g of 0.63 ± 0.05 (5 min) to 0.21 ± 0.04 (120 min), suggesting no significant metabolic defluorination in vivo [15]. Tumor uptake at 5 and 30 min was 1.87 ± 0.21 and 1.56 ± 0.1 (%ID/g) respectively, while the uptake was significantly lowered at 60 and 120 min with 0.51 ± 0.09 and 0.11 ± 0.09 respectively. To demonstrate specific binding, we also performed blocking experiments in a subset of mice from 30 min group (n=2). The blocking agent was KS-7-51 (10 mg/ kg) administered 30 min prior to [18F]KS-7-51 injection. Tumor uptake in the blocking group was 1.4-fold lower than the baseline, demonstrating specificity of [18F]KS-7-51.
CONCLUSION
[18F]KS-7-51 was synthesized with high radiochemical purity and specific activity. The in vitro cell uptake assay in PC3 cells indicated good selectivity and specificity of [18F]KS-7-51. Initial tumor kinetics of [18F]KS-7-51 in PC3 tumor-bearing mice was modest, which may be due to lower PPAR-γ expression in PC3 tumors. Further experiments including whole-body microPET/ CT imaging in tumor cells with high PPAR-γ over-expression or knockout, in vivo metabolite assays, and quantitative receptor occupancy studies are warranted to completely characterize the radiotracer.
