Evaluation of 6-([18F]fluoroacetamido)-1-hexanoicanilide for PET imaging of Histone Deacetylase in the baboon brain

The sequencing of the human genetic code (DNA base pair sequence) has led to the discovery of genes involved with various human diseases [1]. However, for most of the complex common diseases, the genes identified account for only a small effect in the vulnerability of developing them (i.e., addiction, heart disease, diabetes, depression, cancer, Parkinson’s disease). This is likely to reflect the importance of the interactions between environmental and genetic factors in the development of complex human diseases. It is now widely accepted that epigenetics (regulation of gene activity that is not dependent on DNA sequence) is just as critical as DNA to the healthy development of organisms. Epigenetic processes, such as DNA methylation and histone modifications (e.g. acetylation, methylation, phosphorylation and ubiquitination) play a crucial role in gene expression across species [2]. The epigenetic modifications of the histone proteins of chromatin can have significant impact on most major cell functions including DNA repair, cell growth, differentiation and apoptosis. Of these epigenetic processes, the pattern of histone acetylation, regulated by the enzymes histone deacetylases (HDACs) and histone acetyl transferases (HATs), has been most intensively studied [3–5]. Indeed epigenetic research in cancer has led to new therapeutic approaches (i.e histone deacetylase inhibitors) and to biomarkers to predict treatment response [6]. Though current interest in HDAC as therapeutic targets in cancers, neurodegenerative diseases and inflammation is great, much is not known about the mechanisms that underlie these processes and how they are affected by aging, disease, drugs or other environmental exposures. The 18 identified isoforms of HDAC have been broadly divided into two groups, the Zn2+ dependent protease and the NAD+ dependent (class III) [7–9]. The Zn2+ dependent protease group has been further divided into three classes: class I (HDAC -1, -2, -3 and -8), class II (HDAC -4, -5, -6, -7, -9 and -10) and class IV (HDAC -11). The class I and II groups are therapeutic targets for treatment of cancer and other diseases [10–14]. Class I HDACs are localized mainly in the nucleus while class II enzymes are larger in size and shuttle between the nucleus and cytoplasm. Tissue expression of class I and II HDACs is reported to be generally low, with significant variation within tissue types but with little differences in expression pattern between normal and malignant tissue [15]. More recently an atlas of HDACs 1–11 throughout more than 50 regions of the rat brain was reported [16]. In addition to histone proteins endogenous substrates for HDACs include several non-histone proteins such as transcription factors (eg p53, E2F1, GATA1, Re1A, YY1, and hormone receptors), α-tubulin, nuclear import protein importin-α7, signal transduction protein β-catein, DNA repair enzymes Ku70 and WRN and the heat shock protein 90 (hsp90) [17–18]. The deacetylation of these non-histone proteins affects their activity, suggesting that HDACs exert a much broader range of activities within the cell than control of gene transcription [19]. Much effort has been focused on identifying new HDAC inhibitors (HDIs), but few isoform-specific ligands have been identified thus far [20–22]. Studies on several nonspecific HDIs have advanced to clinical trials with one inhibitor, suberanilohydroxamic acid (SAHA) already approved for treatment of cutaneous T-cell lymphoma [23]. In addition to inhibiting class I and II HDACs, SAHA was recently shown to possess no inhibitory activity to a number of kinases and phosphatases but shown to inhibit the zinc dependent metalloproteases neprilysin and ACE (angiotensin-converting enzyme), albeit with lower potency [24]. A greater understanding of the biology and pharmacology of the different HDAC isoforms is needed to better guide preclinical and clinical investigations. In addition to insufficient pharmacological knowledge there are also few tools for accessing the efficacy of these inhibitors in vivo. Imaging the expression/availability/activity of HDAC, using positron emission tomography (PET), may provide a complementary method for determining inhibitor action, monitoring therapeutic efficacy and may also provide insight on the pharmacokinetics (PK) and biodistribution of some of the available HDAC inhibitors. PET imaging may also provide the opportunity to track and quantify the levels of HDAC non-invasively in human tissues as a function of age, disease, drug use or other environmental exposures. Recently 6-([18F]fluoroacetamido)-1-hexanoicanilide ([18F]FAHA) was reported as a potential PET imaging agent and substrate for HDAC with utility for imaging whole body HDAC expression and tumor activity [25]. In PET studies using rats implanted with carcinoma cells, uptake in tumor xenografts was reported to peak at 30 min at 0.43%ID/g [26]. High HDAC activity was observed in tumors as indicated by tumor to muscle ratios of 2.2–2.4 between 30–60 min post-injection. The same group also reported HDAC activity in the rat brain, using [18F]FAHA, as evidenced by a peak uptake of 0.44 % ID/g [27]. In both rodent studies SAHA was shown to inhibit radiotracer uptake. [18F]FAHA is the first reported PET radiotracer with potential for monitoring an epigenetic process in vivo. The present study attempts to assess the feasibility of using [18F]FAHA for imaging HDAC in the baboon brain and to determine its distribution and pharmacokinetics in peripheral organs. While [18F]FAHA appears promising in rodent PET studies, no characterization of its in vitro binding and specificity has been reported. Given its similarity to the acetylated side chain of lysine, we anticipated that FAHA may be a substrate for HDACs. If so, [18F]FAHA in vivo would be cleaved resulting in a labeled metabolite, [18F]fluoroacetate ([18F]FAC), which has the been shown to cross the blood brain barrier (BBB) and have selective uptake in glial cells [28,29]. The metabolism of [18F]FAHA to [18F]FAC in the brain and periphery could complicate interpretation of PET data depending on the rate of decetylation of [18F]FAHA and the uptake and distribution of its metabolite [18F]FAC. In order to determine the contribution of [18F]FAC to [18F]FAHA imaging studies, we performed the following supporting measurements: 1. the rate of peripheral metabolism of [18F]FAHA was assessed by analysis of arterial plasma in baboon studies; 2. the biodistribution of [18F]FAC was determined to access the degree of brain uptake; 3. rodent/microPET studies of [18F]FAHA were carried out to determine the PK in the rodent brain and the chemical form of F-18 in the brain evaluated by ex vivo analysis of brain tissue; 4. in vitro whole blood and plasma incubation studies were used to investigate [18F]FAHA metabolism ex vivo and 5. in vitro distribution and stability studies were also performed in two cell lines to determine if [18F]FAC is formed intracellularly and whether it diffuses out of the cell.