Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands.

INTRODUCTION
Primary aldosteronism accounts for 6-15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres.


METHODS
Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites.


RESULTS
[18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%.


CONCLUSIONS
[18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

A C C E P T E D M A N U S C R I P T

Introduction
The most common secondary cause of hypertension is primary aldosteronism (PA), which is reported in approximately 6-15% of all hypertensive patients. By the year 2025, PA is expected to directly impact one in four people with hypertension globally [1][2][3][4][5][6][7]. PA is characterized by an excess secretion of aldosterone, steroid hormone with mineralocorticoid activity produced by the zona glomerulosa of the adrenal cortex where aldosterone synthase (CYP11B2) enzymes play an essential role in aldosterone production [3,8,9]. Aldosterone increases sodium reabsorption and potassium excretion in the renal distal tubules and collecting ducts of nephrons, influencing water retention and blood pressure [3]. Therefore, pathological conditions showing an overproduction of aldosterone frequently lead to hypertension and hypokalaemia resulting in detrimental effects to an individual's cardiovascular system [1]. Over 90% of all PA patients have a sporadic form [10]. The two most common subtypes of sporadic PA are unilateral aldosterone-producing adenomas (UAPA, 40%) or bilateral adrenal hyperplasia (BAH, 60%) [5,11]. Correctly differentiating between the unique features of UAPA and BAH is crucial in identifying an appropriate intervention. UAPA is curable through surgical removal of the diseased adrenal whilst BAH is treated pharmacologically with mineralocorticoid receptor antagonists (e.g. spironolactone) [12]. The most significant obstacle for decision makers is the need to distinguish UAPA from other causes of PA such as BAH or non-functioning adrenal adenoma (incidentalomas) [13]. Invasive bilateral adrenal vein sampling (AVS) is the current gold-standard diagnostic procedure adopted, with cannulation success rates varying from 8% to 95% [10,[14][15][16].
Computed tomography (CT) and magnetic resonance imaging (MRI) have been used for noninvasive lateralisation of aldosterone hypersecretion. These show low diagnostic accuracy due to A C C E P T E D M A N U S C R I P T 5 suboptimal spatial resolution and sensitivity to detect small UAPA (<1 cm) [14,[17][18][19][20]. Positron emission tomography (PET) is considered an accurate and non-invasive alternative to AVS in the management of patients with PA and adrenal adenoma [21].
Metabolite studies of [ 11 C]MTO in humans revealed that unchanged [ 11 C]MTO accounted for 40% and 28% of total blood-borne radioactivity at 20 min and 40 min post injection, with two polar metabolites present in the plasma [30]. Although [ 11 C]MTO has good imaging properties for visualization of UAPA, [21] novel radiotracers with increased adrenal-to-liver ratio (large liver uptake hampers the assessment of the right adrenal) and a longer half life radionuclide (e.g. 18 F) are needed for a more widespread adoption of this diagnostic approach.
An increased adrenal-to-liver PET signal was achieved by adding a halogen atom (e.g. chlorine or bromine) in the para-position of MTO's benzene group such as chlorine ([ 11 [30,36,37]. A para-fluorinated aromatic (R)-MTO derivative (FAMTO, Fig. 1) has comparable affinity for adrenal enzymes (K i = 7.3 nM measured using [ 131 I]IMTO ) as MTO (K i = 4.02 ± 1.87 nM) in rat adrenal membranes [39]. We therefore developed a radiosynthetic strategy to produce a 18 Fradiolabelled analogue of MTO ([ 18 F]FAMTO) in order to evaluate it as a potential radiotracer for aldosterone-producing adenomas.

General Consideration
3 mg, 18 µmol) and tetrakis(pyridine)copper(II) triflate (Cu(OTf) 2 (py) 4 , 3.6 mg, 5 µmol) were added in a vial. DMF was added to achieve a final volume of 500 µL (see Table 2). Radiofluorination was conducted at 118 °C for 20 min, cooled to room temperature, and diluted with H 2 O.  Table 2). Radiofluorination was conducted at 118 °C for 20 min under air, cooled to room temperature, and diluted with H 2 O.

Purification and Analysis of [ 18 F]FAMTO
The crude mixture was purified by semi-preparative HPLC (see Appendix A). The purified [ 18 F]FAMTO was subsequently diluted with 40 mL of PBS, the solution loaded onto a Sep-Pak tC18 Plus Long SPE Cartridge 900 mg, 37-55 µm (cat. no. WAT036800, Waters) and eluted with ethanol (2 mL). Ten fractions (0.2 mL) were collected and radioactivity determined.
Fractions with highest radioactivity were combined and formulated with saline. The formulated solution (4-6 mL of 10% ethanol in saline) containing 1-10 MBq of [ 18 F]FAMTO was used for in vitro and in vivo experiments. Analytical reverse-phase HPLC (see Appendix A) was used to determine the molar activity, radiochemical and chemical purity. Identification of the radioactive products was confirmed by co-elution of added non-radioactive compounds.

Determination of RCY and molar activity of [ 18 F]FAMTO
The entire quenched reaction mixture was used to determine non-isolated and isolated RCY.
All RCYs are reported as decay corrected values [40]. Non-isolated RCYs were determined by integrating the area under the curve in the preparative radio-HPLC chromatogram or iTLC. The molar activities (GBq/µmol) were calculated by dividing the radioactivity of [ 18 F]FAMTO by the amount of the unlabeled FAMTO determined from the peak area in the UV-HPLC chromatograms (λ= 254 nm, common wavelength for identifying metomidate derivative compounds such as etomidate and iodometomidate [25,38], fig. S1). FAMTO concentrations were determined from a UV-absorbance calibration curve (see Appendix A).

Pig Organ Tissues
Pig tissues were purchased from Seralab, UK. Adrenal glands, kidneys, and liver were collected from an healthy animal (female, 9 months old). Tissues were delivered at 4 °C in Dulbecco's media. Once collected, tissues were cut in small and medium pieces. Pieces were snap frozen in an isopentane bath cooled to -30 °C and stored at -80 °C.

Autoradiography
Frozen sections (20 μm) of pig adrenal glands, kidneys, and liver were prepared in a Cryostat and put on superfrost glass slides. At the start of the experiment, the slides warmed to room temperature and preincubated for 10 min in TRIS buffer (

Animal Studies
Biodistribution and metabolite studies were carried out in adult male Sprague-Dawley rats   3A and Table S1). Data are expressed as mean ± standard error (SE) from three independent replicates, unless otherwise indicated. Statistical analysis of %ID/g in the biodistribution study was performed with IBM SPSS Statistics (version 24). Student t-tests were used to determine statistical significance with P < 0.05 considered significant.

Analysis of Radiolabelled Metabolites in Rats
Blood and urine samples were collected at 10, 30, and 60 minutes post injection to measure the amounts of unchanged tracer and radioactive metabolites. Blood was centrifuged at 6000 rpm for 5 min, the supernatant was treated with MeCN (1:1) and centrifuged to precipitate plasma proteins. Plasma supernatant and urine samples were then injected into an HPLC semipreparative column (see Appendix A), and the eluate collected in 1.5 mL test tubes and gamma-counted.

Stability Studies in Plasma
Human blood (1 mL The tubes were centrifuged for 5 min at 4000×g. The supernatant was then injected onto the HPLC (HPLC method 1) and serial 1.5 mL HPLC fractions were collected and gamma-counted.

Plasma Protein Binding
The plasma protein binding of [ 18 F]FAMTO was measured in the plasma of blood samples.
Plasma was prepared from fresh human blood by centrifugation (
Scheme 1 shows the route used to synthesize 3-6 via Mitsunobu reaction that involves the use of both an oxidising agent such as di-tert-butyl azodicarboxylate (DtBad) and a reducing agent such as triphenylphosphine (PPh 3 ) under mild conditions. Yields between 40-60% were obtained.

A C C E P T E D M A N U S C R I P T
14 Two 18 F-labeling strategies were selected to produce [ 18 F]FAMTO, the first via an aryl sulfonium salt (1, Scheme 1) and the second via an aryl boronic precursor (2).
The strategy of labeling sulfonium salts with 18 F fluoride ion has been reported to be a direct, straightforward nucleophilic substitution [41]. Following the optimised procedure developed by  Table 2).
Neumaier and coworkers have reported an efficient protocol to increase the RCY of coppermediated aromatic radiofluorination using a "low amount of base" strategy [43].   [25]. Blocking studies revealed a high specific binding in the liver and kidney, organs known to be rich in cytochrome P450 enzymes.

Ex Vivo Biodistribution, metabolite analysis and in vivo PET Imaging Studies in Rats
Ex vivo biodistribution data ( Fig. 3A and Table S1), performed in healthy Sprague-Dawley rats, was used to assess the adrenal uptake as well as the adrenal-to-organ ratio. [ 18 F]FAMTO showed high liver uptake in the first 10 minutes (%ID/g = 3.61 ± 1.53) followed by a slight decrease at 30 min (%ID/g = 2.16 ± 1.53) and 60 min (%ID/g = 1.68 ± 0.18). Adrenal uptake increased slowly during the first 30 minutes up to %ID/g = 6.2 ± 0.46, followed by a slow decrease up to %ID/g = 4.92 ± 0.70 at 60 minutes. Adrenal-to-liver ratio increased from 0.66 at 10 minutes to 2.8 at 30 minutes postinjection ( Fig. 3A and Table S1).
Rats that received ETO (1 mg/kg) 10 minutes prior to injection of [ 18 F]FAMTO showed a significant decrease in liver uptake (66%, p = 0.005, see Table S1) and a moderate decrease in the adrenal glands (39%, p = 0.03). Pre-treatment with ETO increased the ratio of %ID/g in adrenal to liver by 38% (p = 0.12) leading to an enhanced adrenal-to-liver PET signal compared to the non-pretreatment group. An in vivo PET image of the adrenal uptake of [ 18 F]FAMTO after pre-treatment with ETO (1 mg/kg) is shown in Fig. 3B and S4.
Plasma metabolite analysis revealed 13%, 8% and 2% of unchanged [ 18 F]FAMTO at 10, 30 and 60 minutes post-injection, respectively. Two hydrophilic metabolites at retention times 3 and 21 minutes were observed (Fig. 4). The metabolite eluting at 3 minutes has been identified as the free acid [ 18 F]12 A C C E P T E D M A N U S C R I P T 17 (Fig. S3). Urine metabolite analysis revealed only one metabolite in solution corresponding to [ 18 F]12 ( Fig. S2).

Stability Studies in Human Blood And Plasma Free Fraction
Plasma-derived metabolites can cause a non-specific signal that reduces the specificity and quality

Discussion
The development of the novel 18 F radiotracer targeting adrenal gland enzymes represents a valuable advancement to the field of PA PET imaging. Our PET radiotracer development approach started from the structure-activity relationship analysis of MTO (K i = 4.02 ± 1.87 nM), a potent inhibitor of CYP11B1 and CYP11B2 adrenal gland enzymes, and its derivatives bearing a halogen atom on the benzene ring (e.g. Br, I and F). [ 11 C]MTO is a prototype radiotracer offering a rapid non-invasive procedure localizing aldosterone-producing adenomas [21,44]. Using the aryl sulfonium strategy, a benzylic 1H-imidazole derivative, ETO fragment, has been radiolabelled by Sander et al. in 31% RCY [41]. Applying the same conditions to 1, the production of  Fig. 4, Fig. S2 and S3). In analogy to the carboxylic acid derivative of MTO, we anticipate that compound 12 would also have negligible affinity for adrenal gland enzymes [34].Low metabolic stability of MTO and FETO has been observed in vitro and in vivo [30,37]