General: Unless otherwise noted, all reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. Exendin-4 (exenatide, Byetta®) was obtained from Amylin/Eli Lilly (San Diego, CA, USA). E4C12 (4163 g mol−1; HGEGTFTSDLSCQMEEEAVRLFIEWLKNGGPSSGAPPPS) was obtained from Genscript (Piscataway, NJ, USA). [18F]-Fluoride (n.c.a.) was purchased from PETNET Solutions (Woburn, MA, USA). 3-maleimido-propanoic acid succinimidyl ester 1, tetrazine (Tz) amine 2 and 18F-trans-cyclooctene (18F-TCO) 4 were synthesized as described elsewhere.13, 16, 17, 27 High performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) analyses and HPLC purifications were performed on a Waters LC-MS system (Milford, MA, USA). For LC–ESI-MS analyses, a Waters XTerra® C18 5 μm column was used. For preparative runs, an Atlantis® Prep T3 OBDTM 5 μm column was used. High-resolution ESI mass spectra were obtained on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) in the Department of Chemistry Instrumentation Facility at the Massachusetts Institute of Technology. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian AS-400 (400 MHz) spectrometer. Chemical shifts for protons are reported in parts per million (ppm) and are referenced against the [D6]acetone lock signal (1H, 2.05 ppm). NMR data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, m=multiplet), coupling constants (Hz) and integration.
3-maleimido propanamide-tetrazine (maleimido-Tz) 3: A solution of 3-maleimido-propanoic acid succinimidyl ester 1 (5 mg, 19 μmol, 20 mg mL−1, 250 μL in dimethylformamide (DMF) was added to a solution of Tz amine 2 (3.5 mg, 19 μmol) and Et3N (5.3 μL) in MeCN (1 mL), and the resulting reaction mixture stirred at RT for 1 h. Volatiles were removed in vacuo and the crude product purified using HPLC to give compound 3 as a pink solid (4.4 mg, 13 μmol, 68 %): 1H NMR (400 MHz, [D6]acetone): δ=10.43 (s, 1 H), 8.52 (d, 3JHH=8.3, 2 H), 7.78 (m, 1 H), 8.58 (d, 3JHH=8.2, 2 H), 6.86 (s, 2 H), 4.52 (d, 3JHH=6.0, 2 H), 3.80 (t, 3JHH=7.4, 2 H), 2.59 ppm (t, 3JHH=7.4, 2 H); LC–ESI-MS(+): m/z (%): 339.2 (100) [M+H]+, 677.4 (29) [2M+H]+; LC–ESI-MS(−): m/z (%): 337.1 (29) [M−H]−, 383.1 (100) [M+HCOO]−, 721.3 (27) [2M+HCOO]−; HRMS-ESI: m/z [M−H]+ calcd for [C16H14N6O3Na]+ 361.1020, found 361.1013 [M+Na]+.
E4Tz12 5: A solution of maleimido-Tz 3 (50 μL 10 mm) in DMF was added to a solution of E4C12 4 (3.0 mg, 0.7 μmol) in 1×PBS (1000 μL), and the resulting solution was stirred at RT for 3 h. The reaction mixture was purified using an Amicon® Ultra 3 kDa centrifugal filter (Millipore, Carrigtwohill, Ireland) before being subjected to HPLC purification, yielding compound 5 as a rose-colored solid (0.8 mg, 0.2 μmol, 29 %): LC–ESI-MS(+): m/z (%): 1125.9 (100) [M+4H]4+, 1501.3 (51) [M+3H]3+; LC–ESI-MS(−): m/z (%): 1498.7 (100) [M−3H]3−.
18F-E4Tz12 7: 2-[18F]-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (18F-TCO) was prepared in a similar manner to previously described procedures13 employing a Synthra RN Plus automated synthesizer (Synthra GmbH, Hamburg, Germany) operated by SynthraView software in an average time of 102 min. The synthesizer reagent vials were filled with the following: A2 with MeCN (350 μL), A3 with (E)-2-(cyclooct-4-enyloxy)ethyl 4-methylbenzenesulfonate (2.0 mg, 12.3 μmol) in DMSO (400 μL), A5 with MeCN (150 μL), and B2 with H2O (800 μL). The starting activity well was filled with [18F]-F− (n.c.a.) (2072 MBq, 56±15 mCi) in H218O (500–1000 μL), tetrabutylammonium bicarbonate (TBAB, 250 μL, 75 mm in H2O), and MeCN (200 μL). The [18F]-F−/TBAB solution was transferred to the reaction vessel and dried by azeotropic distillation with MeCN. After drying, TCO-tosylate (2 mg, 15 mM) in DMSO was added and heated to 90 °C for 10 min. After cooling to 30 °C, the mixture was filtered through an Alumina-N cartridge (100 mg, 1 mL, Waters) into reaction vessel 2. The Alumina-N cartridge was washed with MeCN (150 μL) and the combined filtrates were then diluted with H2O (800 μL). This solution was subsequently subjected to preparative HPLC purification (MeCN/H2O, 50:50). 18F-TCO was collected (tR=13.5 min) in 5–6 mL of solvent and isolated by manual C18 solid phase extraction. It was then eluted with DMSO (2×450 μL) to give 10.1±5.9 mCi of 18F-TCO in 46.1±12.2 % (n=4) decay-corrected radiochemical yield (dcRCY) in an average time of 102 min (once drying of [18F]-F− (n.c.a.) had ended). Analytical HPLC demonstrated >94 % radiochemical purity of 18F-TCO.
E4Tz12 5 (5.5 nmol, 1 mM in DMSO) was added to the 18F-TCO 6 in DMSO. After stirring at RT for 20 min, TCO-beads (150 uL suspension of 10 mg mL−1; TCO loading: 13 nmol mg−1) were added to the mixture and stirred for 20 min. The reaction mixture was filtered using an Amicon® Ultra 3 kDa centrifugal filter (Millipore, Carrigtwohill, Ireland) to give 18F-E4Tz12 7 (1.8±0.9 mCi, 46.7±17.3 % (n=4) dcRCY).
18F-E4Tz12 7 (approx. 14 μCi [0.52 MBq]) in DMSO/1×PBS (4:1, 5 μL) was added to octanol (500 μL) and H2O (MilliQ, 500 μL) in a 1.5-mL microcentrifuge tube. The mixture was vortexed for 1 min at RT and centrifuged (15 000 rpm, 5 min). After centrifugation, 100-μL aliquots of both layers were measured using a γ-counter. The experiment was carried out in quintuplicate. This experiment was repeated with octanol/1×PBS (1:1, 1000 μL).
Cell lines: We chose three different insulinoma tumor cell lines (NIT-1, WTRT2, 916-1), to correlate imaging findings and to elucidate how 18F-E4Tz12 behaves in different insulinoma tumor environments. Both WTRT2 and 916-1 were generously provided by Johanna Joyce (Memorial Sloan–Kettering Cancer Center, New York City, USA). NIT-1 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). WTRT2 and 916-1 were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with fetal bovine serum (10 %), L-glutamine, penicillin (100 I.U.), and streptomycin (100 μg mL−1). NIT-1 were cultured in F-12K medium (Kaighn’s Modification of Ham’s F-12 Medium, ATCC, Manassas, VA) supplemented with fetal bovine serum (10 %), sodium bicarbonate (2 %), L-glutamine, penicillin (100 I.U.), and streptomycin (100 μg mL−1). All cell lines were cultured at 37 °C and 5 % CO2.
Western Blot: 916-1, WTRT2, and NIT-1 cells seeded into six-well plates were washed twice with ice-cold 1×PBS and lysed on ice for 10 min with ice-cold RIPA lysis buffer (100 μL) supplemented with a 100-fold dilution of protease inhibitor cocktail for mammalian cells (Sigma–Aldrich). The lysate was centrifuged (10 min, 10 000 rcf) and the supernatant collected. Protein concentrations were determined using bicinchoninic acid (BCA) protein assays (Pierce, Rockford, IL, USA). Cell lysates (10 μg) were subjected to SDS-PAGE, followed by immunoblotting using anti-GLP-1R antibody (#39072, Abcam, Cambridge, UK), goat-anti-rabbit secondary (Jackson ImmunoResearch, West Grove, PA, USA), and detection with chemiluminescence (Picowestern ECL substrate, Pierce). Blots were stripped using Restore Stripping Buffer (Thermo Scientific), labeled with anti-GAPDH antibody (AF 5718, R&D Systems) followed by detection with chemiluminescence.
Mice: Experiments were performed in Nu/Nu mice (from Massachusetts General Hospital, Boston, MA; for tumor implantations and imaging; n=6), C57BL/6 (B6) mice (from The Jackson Laboratory, Bar Harbor, ME; for biodistribution and pharmacokinetics; n=8), or B6.Cg-Tg(Ins1-EGFP)1Hara/J mice (from The Jackson Laboratory, Bar Harbor, ME; for autoradiography/surface reflectance imaging; n=3).18 B6.Cg-Tg(Ins1-EGFP)1Hara/J mice express the enhanced green fluorescent protein (eGFP) in the islets under the control of the mouse insulin 1 promoter (MIP-GFP). For all surgical procedures and imaging experiments, mice were anesthetized with 2.0 % isoflurane in O2 at 2.0 L min−1. For imaging experiments lasting longer than 1 h, the isoflurane flow rate was reduced to ∼1.0 % isoflurane in O2 at 2.0 L min−1. Surgeries were conducted under sterile conditions with a zoom stereomicroscope (Olympus SZ61). All procedures and animal protocols were approved by the Massachusetts General Hospital subcommittee on research animal care.
Whole pancreas islet imaging: B6.Cg-Tg(Ins1-EGFP)1Hara/J (MIP-GFP) mice18 were administered 18F-E4Tz12 7 (92±12 μCi [3.40±0.44 MBq]) via intravenous tail-vein injection, and the GPL-1 receptor-specific probe was allowed to accumulate and clear for 3 h. Mice were then euthanized, their organs perfused using 1×PBS (30 mL) and the pancreata harvested. They were subsequently weighed and placed between two glass cover slides using a 1 mm rubber gasket, maintaining a constant thickness. Initially, fluorescence reflectance was recorded by imaging the entire pancreas on an OV110 epifluorescence imager (Olympus America, Center Valley, PA, USA). The pancreata were then transferred to an autoradiography phosphor imaging plate (SI, Molecular Dynamics) and exposed at −20 °C for 12 h before the plate was analyzed using a Typhoon scanner (GE Healthcare). Image analysis was conducted using ImageJA 1.45 software.
18F-E4Tz12 7 biodistribution studies: C57BL/6 (B6) mice were used for blood half-life determinations. Mice were administered 18F-E4Tz12 7 (68±12 μCi [2.52±0.44 MBq]) by intravenous tail-vein injection. Blood sampling was performed by retro-orbital puncture using tared, heparinized capillary tubes. Samples were subsequently weighed and activity measured using a Wallac Wizard 3“ 1480 Automatic Gamma Counter (PerkinElmer). Blood half-life data were fitted to a biexponential model using Graphpad Prism 4.0c software (GraphPad Software Inc., San Diego, CA), and results were reported as the weighted average of the distribution and clearance phases. For biodistributions, (B6) mice were intravenously injected via tail vein with 18F-E4Tz12 7 (131±18 μCi [4.85±0.67 MBq]). Animals were euthanized at 3 h and their organs perfused using 1×PBS (30 mL). Tissues were subsequently harvested, weighed and their radioactivity counted using a Wallac Wizard 3” 1480 Automatic Gamma Counter. Statistical analysis was performed using Graphpad Prism 4.0c.
MicroPET-CT imaging: Mice were imaged by PET-CT using an Inveon small animal microPET scanner (Siemens Medical Solutions). Mice were injected with 18F-E4Tz12 7 (557±38 μCi [20.61±1.41 MBq]) via tail-vein injection under isoflurane anesthesia (see above). Acquisition for static microPET images started 2 h post injection and acquisition took approximately 30 min. For dynamic microPET imaging, mice were injected approximately 30 s after the start of microPET acquisition, and data was collected for 2 h. The radioactivity concentration for a tissue was determined by measuring within regions of interest (ROIs) for a given tissue with the units of Bq mL−1 min−1. A tissue density of 1 g mL−1 was assumed and ROIs were converted to Bq g−1 min−1 and divided by the injected activity to obtain an imaging ROI-derived % ID g−1. For GLP-1 receptor blocking experiments, unlabeled exenatide (250 μL, 60 μM) was preinjected 45 min prior to injection of 18F-E4Tz12 7. A high-resolution Fourier rebinning algorithm was used, followed by a filtered back-projection algorithm using a ramp filter, to reconstruct 3D images without attenuation correction. The image voxel size was 0.796×0.861×0.861 mm, for a total of 128×128×159 voxels. Peak sensitivity of the Inveon accounts for 11.1 % of positron emission, with a mean resolution of 1.65 mm. The total counts acquired was 600 million per PET scan. Calibration of the PET signal with a cylindrical phantom containing 18F was performed before all scans. CT images were reconstructed using a modified Feldkamp reconstruction algorithm (COBRA) from 360 cone-beam X-ray projections (80 kVp and 500 μA X-ray tube). The isotropic voxel size of the CT images was 60 μm. The reconstruction of data sets, PET-CT fusion, and image analysis were performed using Inveon Research Workplace (IRW) software (Siemens). 3D visualizations were produced using a digital imaging and communications in medicine (DICOM) viewer (OsiriX Foundation, Geneva, Switzerland).
A compartmental model was used to extrapolate results from mouse-imaging studies to humans. The model includes biexponential loss from the plasma compartment (due to redistribution and clearance), and separate compartments for the endocrine and exocrine pancreas. Exchange with the endocrine tissue (islets) was estimated as a function of the vascular surface area-to-volume ratio (measured at 505±146 cm−1 using CD31 stained histology slides),28 and permeability was estimated at 30 μm s−1 (for this sized molecule in the fenestrated capillary bed).21 Exocrine pancreas was modeled in a similar manner, while the exchange parameters were adjusted to fit experimental data. Within the compartments, the imaging agent is able to bind the target, dissociate, internalize, and be degraded and washed out.24 These rate constants were assumed constant between species. For plasma clearance in humans, the rate constants for exchange and clearance from a two-compartmental model were fit to experimental data taken from patients undergoing an intravenous infusion of exenatide19 using a least-squares fitting algorithm in Matlab (Mathworks, Natick, MA, USA). Estimates for humans were obtained by entering the plasma clearance values from human clinical data into the model together with the microscopic transport rates obtained from mouse experiments.