Fully automated, robust and reproducible synthesis of 16α-18F-fluoroestradiol (18F-FES), a PET biomarker for the evaluation of an ER-

targeting drug.

1-Introduction: Breast cancer is the most common cancer in women, with 1,658,370 new cancer cases diagnosed in 2015. Almost 70% of breast cancers are estrogen receptor positive. The ER concentration in tumor cells is a significant indicator for the degree of disease. The monitoring of estrogen receptors (ER) engagement is important for both patient prognosis and evaluation of the effectiveness of therapy.1 Positron emission tomography (PET) is a powerful non-invasive tool for in vivo imaging and can detect disease biomarker expression in malignant tissues and monitor therapeutic response. 16a-18F-fluoro-17b-estradiol (18F-FES) is a steroid-based PET tracer with high affinity for ER, validated in clinical studies and used as a dependable tracer for evaluating and managing patients with estrogen-receptor-positive (ER+) breast cancer (fig.1).2

Guillaume BLUET*a, Cathy AUBERTa, Florence GRUSS-LELEUa, Laurent BESRETa, Sébastien D’HEILLY a, Sébastien ROYa, Muhammad OTABASHIb and Laurent BRICHARDb

a) SANOFI R&D, 13 Quai Jules Guesde, 94400 Vitry-sur-Seine, FRANCE b) TRASIS, 90 rue Gilles Magnee, 4430 ANS, BELGIUM

2-Background: In order to evaluate the in vivo effect of different dosing regimens of a selective drug inducing ER degradation, currently in development, we have developed an “in-house” automated synthesis of 18F-FES. The automated production was performed with the TRASIS AllInOne® radiosynthesizer (AIO) shielded in a lead-shielded hot-cell® and remotely controlled by a workstation. This module is equipped with a complete built-in HPLC system with an injection & column selection valve, a radiation & UV detectors. The pump and the eluent selection box are separate from the synthesizer and located below the HotCell® making them accessible at any time even during production. We have used disposable cassette-manifold equipped with three-way valves on which various consumables such as vials, tubes, connectors , spikes and so on can be freely adapted as depicted on figure 2b.

OH

F18

OHFigure 1: 18F-fluoroestradiol

a) Shielded cabinet hotcell® with AIO radiosynthesizer b) Module AIO & disposable cassette built-in HPLC system: pump and the eluent selection box

Figure 2: TRASIS AllInOne® module and HotCell®

3-Synthetic route: The radio-synthesis of 18F-FES 3 consists of three steps starting from 17b-epiestriol-O-cyclic sulfone (MMSE) 1 as follows:3 a) Incorporation of activated [18F] fluoride ion , from K18F into 1 by a nucleophilic substitution, b) hydrolysis of the protected intermediate 2 in acidic media, c) semi-preparative HPLC purification. (scheme 1).

O

SO O

O

MOMO

OH

OH

F18

O

MOMO

F18

SOOOH

18F-(H218O)n in H2

18O

MMSE (ABX GmbH)

K222 / K2CO3

18F-FES

18O(p,n)18F

K 18F 16α

1 23

b) acidic hydrolysis

a) nucleophilic fluorination c) semi-preparative HPLC

4-Results: As shown in the sequence layout (Scheme 2), no-carrier-added [18F]fluoride in [18O]water (10GBq-270 mCi) was transferred to the module and passed through a QMA cartridge. The trapped [18F]fluoride was eluted into sealed reactor (R1) using a solution of kryptofix K222 / K2CO3 (S1) in acetonitrile/water(10%).5 After complete azeotropic drying (8min.), the precursor 1 (0,5mg) in 1,2 mL of acetonitrile was added to the dried [18KF] and the mixture heated at 105°C for 6 min. 1M H2SO4aq (1 mL) was then added to R1 and the mixture was heated at 110°C for 5 min for hydrolysis. After cooling, water (3 mL) was added for dilution and the solution was transferred into the built-in HPLC sytem for purification conducted with a C18 Kromasil® column (250 x 10 mm, 100-5, Akzo Nobel), using water/EtOH (40/60) solution as mobile phase at 3mL/min@254nm. [18F]-FES eluted at 8min (fig.3). The [18F]-FES was isolated in uncorrected RCY of 11% to 25% (16 runs) with good radiochemical purity (>98%) within a short synthesis time, less than 45 min.

nca (18F]/18OH2

QMA S1

R1

1

WATE

R

H2 SO

4

CH3 CN

18OH

2

HPLC loading

graphical method sequence editor

Scheme 1: radiosynthesis of [18F]-fluoroestradiol, [18F]-FES

Scheme 2: Layout ,Hardware and reagent kit

OH

F18

OH

Figure 3: A) Radio-HPLC of [18F]-FES during purification & UV @254nm; B) Analytical radio-HPLC of isolated [18F]-FES co-injected with authentic standard FES

Collected [18F]-FES peak

OH

F18

OH

OH

F

OH

CT and µPET fused images for evaluation of the selective drug upon [18F]-FES uptake in MCF7Y537S tumor bearing mice are shown below (figure 4). Reduced tumor uptake of [18F]-FES has been observed in mice after injection of the selective drug. Data showed [18F]-FES uptake correlated well with immunohistochemical scoring for ER and indicated that [18F]-FES PET might have potential use in the clinic to assist dosage decision in breast cancer patients elligible for the selective drug therapy.4

5-PET imaging studies:

Residual free fluoride 18F

-

Unknow

n impurity

µCT

µPET Fused images

µCT / µPET Vehicle

injection i.v. of [18F]-FES 10 – 12 MBq in 0.9% NaCl / mice,

With selective drug

6-Conclusion: We have described an “in-house” automated, reliable and reproducible radiosynthesis using the TRASIS AllInOne® , AIO synthesizer for the routine production of [18F]-FES for preclinical studies. The easy to use AIO module allowed us to develop efficient automated synthetic and purification procedures to obtain [18F]-FES in good radiochemical yields (up to 25% ndc) within just 45 min. Isolated radioactivities were in the range of 0.45 GBq to 3.9 GBq and were sufficient to support PET imaging studies. Note that the specific activity SA is not a product releasing criterion since SA has no significant effect on tumor uptake of [18F] FES (expressed as standardized uptake value, SUV).6 Figure 4: PET imaging, upon [18F]-FES uptake in MCF7Y537S tumor bearing mice

1) L. Vollenweider-Zeragui, G. Barrelet, Y. Wong, T. Lemarchabnd-Béraud, F. Gomez, Cancer 1986, 1171; 2) a-M. Dixit, J. Shi, L. Wei, G. Afari, S. Bhattacharyya Int. J.Mol.Imaging 2013, 1;b- MMSE precursor and standard reference FES were purchased from ABX (GmbH) ; Technical 18-Fluoride was purchsed from Cyclopharma (Glisy, France) or Petnet solutions (Siemens, Lisses, France).3) a-First manual synthesis of [18F]-FES: D.O. Kiesewetter, M.R. Killbourn, S.W. Landvatter, D.F. Heiman, J.A. Katzenellenbogen, M.J. Welch J. Nucl. Med. 1984, 1212; b-First automated Synthesis of [18F]-FES: J. Römer, F. Füchtner, J. Steinbach, B. Johannsen, Nucl. Med. Biol. 1996, 911; 4) L. Besret, S. Dheilly, SANOFI Oncology R&D/Preclinical R&D/Translational & Exp. Med., Results not published; 5) Classical automated synthesis of [18F]-FES using GE TRACERLab® K.E. Knott, D. Grätz, S. Hübner, S. Jüttler, C. Zankl, M. Müller J. Label. Compd. Radiopharm. 2011, 749; 6) L.M. Peterson, B.F. Kurland, J.M. Link Nucl. Med. Biol. 2011, 969.