The ghrelin receptor is expressed in

The ghrelin receptor is expressed in a number of human malignancies including prostatic carcinoma cell lines [18,19] and tissues [20], breast carcinoma tumours and cell lines [21], testicular tumours [22], and malignant ovarian cysts and tumours [23]. In addition, it has an elevated level of expression in tissues from patients with atherosclerosis [24] and in biopsies from those with chronic heart failure [25]. The ghrelin receptor thus represents a potential target for molecular imaging of carcinoma and cardiovascular disease. Among the imaging modalities most commonly utilised in the clinic, positron emission tomography (PET) combines a high spatial resolution with high sensitivity [26]. A number of recent publications have thus described the development of PET radiotracers for visualising the GHS-R1a receptor in disease [[27], [28], [29], [30]]. For example, Hou and co-workers synthesised the first picomolar quinazolinone binder of the ghrelin receptor and successfully radiolabelled two nanomolar compounds in high radiochemical purity [30]. In another study on small-molecule derivatives, in vivo imaging in mice with a [11C]radiotracer revealed a higher specific uptake in the pancreas compared to other organs [28]. However, these studies [[27], [28], [29], [30]] and others [[31], [32], [33]] have primarily focused on either small-molecule quinazolinones [27,28,30,31] or ghrelin-derived compounds [29,32,33]. To the best of our knowledge, a growth hormone secretagogue-based PET probe has not been reported to date. We reasoned that modification of peptidic and peptidomimetic GHSs should result in a clinically translatable PET agent with high target specificity, in vivo stability and favourable pharmacokinetic properties. Fluorine-18 was our preferred choice of radioisotope as it is a small innocuous unit that can be easily installed in the absence of a chelator using the [18F]fluorobenzoyl ([18F]FB) prosthetic group. This modification can be initially trialled with 4-[19F]fluorobenzoic VER 155008 ([19F]FBA), in order to determine the optimal location for the 18F-radioisotope without significantly affecting peptide/peptidomimetic binding to the ghrelin receptor. We describe herein the design, synthesis and biological evaluation of 4-fluorobenzoylated derivatives of GHSs with peptidic (GHRP-1, GHRP-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) structures; the determination of their IC50 values for the ghrelin receptor; the identification of the lead peptide [1-Nal4,Lys5(4-FB)]G-7039 with nanomolar IC50 and EC50 values; and the 18F-radiolabelling of its precursor [1-Nal4]G-7039 to furnish the peptidomimetic growth hormone secretagogue [1-Nal4,Lys5(4-[18F]-FB)]G-7039, which could be applied to PET imaging of diseases via targeting of the ghrelin receptor.
Results and discussion
Conclusions In this study, several families of peptidic (GHRP-1, GHRP-2 and GHRP-6) and peptidomimetic (ipamorelin, G-7039, [1-Nal4]G-7039) ghrelin receptor agonists were derivatised through 4-fluorobenzoylation of the lysine side-chain. This led to a range of half-maximal inhibitory concentrations ranging from 69 nM ([1-Nal4,Lys5(4-FB)]G-7039) to 1920 nM ([Inp1,D-2-Nal4,Lys5(4-FB)]ipamorelin). The peptidomimetic [1-Nal4]G-7039 was identified as the most suitable candidate for 18F-radiolabelling, as its 19F-congener ([1-Nal4,Lys5(4-FB)]G-7039, compound 17) had a nanomolar binding affinity (IC50 = 69 nM), high in vitro potency (EC50 = 1.1 nM) and good serum stability (t1/2 = 718 min). This is most likely due to the presence of a core framework of aromatic amino acids that are involved in strong hydrophobic interactions with hydrophobic pockets of the ghrelin receptor. Prosthetic group radiolabelling of [1-Nal4]G-7039 with [18F]SFB delivered the radiolabelled peptidomimetic in an overall average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of >34 GBq/μmol. Further study of this compound would be required to assess its potential for in vivo PET imaging of diseases exhibiting heightened expression of the ghrelin receptor.