The synthesis and evaluation of N1-(4-(2-[18F]-fluoroethyl)phenyl)-N8-hydroxyoctanediamide ([18F]-FESAHA), a PET radiotracer designed for the delineation of histone deacetylase expression in cancer

Abstract

Introduction: Given the significant utility of suberoylanilide hydroxamic acid (SAHA) in chemotherapeutic protocols, a PET tracer that mimics the histone deacetylase (HDAC) inhibition of SAHA could be a valuable tool in the diagnosis, treatment planning and treatment monitoring of cancer. Here, we describe the synthesis, characterization and evaluation of N(1)-(4-(2-[(18)F]-fluoroethyl)phenyl)-N(8)-hydroxyoctanediamide ([(18)F]-FESAHA), a PET tracer designed for the delineation of HDAC expression in cancer.

Methods: FESAHA was synthesized and biologically characterized in vivo and in vitro. [(18)F]-FESAHA was then synthesized in high radiochemical purity, and the logP and serum stability of the radiotracer were determined. In vitro cellular uptake experiments and acute biodistribution and small-animal PET studies were performed with [(18)F]-FESAHA in mice bearing LNCaP xenografts.

Results: [(18)F]-FESAHA was synthesized in high radiochemical purity via an innovative one-pot procedure. Enzymatic inhibition assays illustrated that FESAHA is a potent HDAC inhibitor, with IC(50) values from 3 nM to 1.7 μM against the 11 HDAC subtypes. Cell proliferation experiments revealed that the cytostatic properties of FESAHA very closely resemble those of SAHA in both LNCaP cells and PC-3 cells. Acute biodistribution and PET imaging experiments revealed tumor uptake of [(18)F]-FESAHA and substantially higher values in the small intestine, kidneys, liver and bone.

Conclusion: The significant non-tumor background uptake of [(18)F]-FESAHA presents a substantial obstacle to the use of the radiotracer as an HDAC expression imaging agent. The study at hand, however, does present a number of lessons critical to both the synthesis of hydroxamic acid containing PET radiotracers and imaging agents aimed at delineating HDAC expression.

Figure 1

The structures of SAHA, [¹⁸F]-FAHA, and [¹⁸F]-FESAHA

Figure 2

Model of FESAHA bound to the active site of HDLP (based on the crystal structure published by Finnin, et al.)[32, 59]

Figure 3

The synthetic route to FESAHA

Figure 4

The synthetic route to [¹⁸F]-FESAHA

Figure 5

Dose-response curves for the growth inhibitory action of SAHA and FESAHA with LNCaP (top, A) and PC-3 (bottom, B) prostate cancer cells. Cells were treated with compounds for 3 days, and cell proliferation was measured by MTT assays. Absorbance values are normalized to the untreated control and are the mean ± SEM from 6 experiments.

Figure 6

Time-dependent cell association assay of [¹⁸F]-FESAHA and LNCaP and PC-3 prostate cancer cells. Cells were doses with 4 μCi [¹⁸F]-FESAHA in 1 mL media and allowed to incubate at 37 °C for various amounts of time. After incubation, the radioactive media was removed, and the cells were washed twice before being trypsinized and counted for radioactivity.

Figure 7

Representative coronal and sagittal small animal PET images of nude mice bearing LNCaP xenografts in the right shoulder imaged with [¹⁸F]-FESAHA. Images A and B were the result of a 60 minute dynamic scan (t = 30 minutes) following injection of the mouse with ~400 μCi [¹⁸F]-FESAHA. Images C and D were the result of a 5 minute static scan at t =120 minutes. In A and B, the tumor is marked with a white arrow, and the anomalous activity in the lower quartiles of the images are PET artifacts. Images C and D serve to illustrate both the high levels of non-tumor background uptake and the extent of bone activity due to the putative defluorination of the radiotracer. In order to display maximal bone uptake in images C and D, an image projection was chosen that does not include tumor. Maximum and minimum values for the above images are 3.5 and 0.0.