1. PET Imaging Agent Development with Radiometals Eszter Boros, PhD WIN national meeting July 29, 2014

2. Siemens BrainPET prototype scanner installed inside the MAGNETOM TIM Trio MR scanner (left); BrainPET withdrawn from the MR scanner for stand alone MR operation (right) PET at the Martinos Center

3. Positron Emission Tomography (PET) 3 18 F 18 O β+β+ Decay/positron emission Detection e-e- Positron/ electron encounter  (511 keV)  (511 keV) Annihilation event

4. The high molecular sensitivity of PET allows for imaging of picomolar probe concentations Agawal et al., W. J Nucl Med. 2012, 11, 33 -FDG: F-18 labeled fluorodeoxy- glucose maps glucose metabolism of cells.  Powerful tool for the imaging of cancer, inflamation, …

5. PET nuclides

6. PET nuclides –non-metals versus metals -Short half-life (can not be shipped over large distances) -Incorporation of radionuclide into molecule through covalent chemistry: The product experiences only minimal structural change -Covalent bond formation requires organic solvents and high temperatures, generally resulting in < 60% yield -Longer half-lifes allow for shipping of product over longer distances -Incorporation of radionuclide into molecule through coordination chemistry: The product experiences greater structural change -Coordination chemistry is done in water, under milder reaction conditions

7. PET imaging agents with radiometals Targeting vector Ligand Radiometal -Longer half-lifes: Well matched with the pharmacokinetics of peptides and monoclonal antibodies -Mild labeling conditions: Suitable when used in conjunction of sensitive peptides and monoclonal antibodies -Incorporation: Due to the need for a chelator to capture the radiometal, using the tagged approach is most suitable in vivo target Linker

8. 64 Cu – a highly attractive radioisotope 8 64 Ni (p,n)  64 Cu  β + /β - + 64 Zn/ 64 Ni -Cyclotron production with solid targetry option required -Only decays 17.4 % through positron emission -Cu-chemistry is hard… – Cu(II) is promiscuous and labile -Emitted positron is low energy -> high resolution images similar to 18 F are attainable -Half-life of 12.7 h is convenient for peptides, affibodies, antibodies (dependent on PK) -Metallic PET isotope of choice for many currently

9. One does not fit all! Ligand design for biomedical imaging with metals Huang et al, Bioconjugate Chem., 2011, 22 (2), 256–263 Targeting vector Ligand Cu-64

10. Wadas et al. Chem. Rev., 2010, 110, 2858; Dumont et al., J Nucl Med. 2011, 52, 1276-1284 Prior art on poly-aza macrocycles: 64 Cu chelates 10 DOTA macrocycle chelator: Lacks kinetic inertness Cross-bridged macrocycle: Better kinetic inertness, but requires 90C for 1h for labeling and difficult to synthesize Polyamino cage: Rapid labeling under mild conditions but 2+ charge leads to high kidney uptake Pyridine containing macrocycle chelator: Increased kinetic inertness but lack of synthetic flexibility

11. Pycup – the best of all worlds? 11 -Has the cage feature of caged and cross-bridged chelator and the donor atom set comparable to pyridine macrocycle -Charge equilibration possible -Flexible and simplified conjugation chemistry? -Labeling properties?

12. Facile synthesis of pycup derivatives with variable set of donor atoms 12 Brandes, et al., Eur. J. Org. Chem., 1998, 2349 – 2360 E. Boros, et al ; Mol. Pharmaceutics., 2014, 11 (2), 617–629 CHCl 3, Na 2 CO 3, rt, 24 h. Bn-Br(2.5 eq), MeCN, rt, 18 h. tert-Bu-OAc-Br (1.5 eq), 50 °C, 24 h Bn-Br(0.65 eq), MeCN, rt, 18 h. DCM/TFA, rt, 16 h.

13. Radiolabeling and plasma stability of model Cu complexes with varied donor atoms General radiolabeling conditions: Pre-formation of 64 Cu(citrate), 70 °C for 15 minutes at pH 7.4 yields > 96 % radiochemical yield, with no residual 64 Cu(citrate). Rat plasma stability: All derivatives fully intact after 24h in rat plasma at 37 °C

14. Fibrin – useful imaging target Found in wounds and pathologies but not normal tissue Present at high concentrations Atrial thrombus with atrial fibrillation Stroke Coronary Artery thrombosis Pulmonary embolism Deep vein thrombosis Ventricular thrombus post anterior MI Carotid artery thrombosis Cancer Early inflammatory response MS Fibrin specific imaging

15. Proof of principle: Bioconjugation to fibrin-binding peptide for thrombus imaging -Phage display screen provided 3 peptide families with high affinity to the soluble fibrin fragment DD(E) for the non-invasive detection of thrombus -Well-established rat model of carotid artery thrombosis, previously studied with MR probe (EP-2104) and 64 Cu-DOTA conjugates of fbp1, fbp2 and fbp3 Kolodziej et al, Bioconjugate Chem., 2012, 23 (3), 548–556; Overoye-Chan et al, J. Am. Chem. Soc., 2008, 130 (18), 6025–6039; Ciesienski et al, Mol. Pharmaceutics, 2013, 10 (3), pp 1100–1110

16. Bioconjugate for thrombus imaging: Animal model Ciesienski et al, Mol. Pharmaceutics, 2013, 10 (3), pp 1100–1110 Thrombus CCA ECA ICA Incision in neck, isolate common carotid, crush with hemostat clamp for 5 min and reflow. Imaging 1 hr post clot formation. fibrin binding peptide

17. 64 Cu(citrate), 70 °C for 15 min, pH 7.4 HEPES Labeling and imaging of peptide conjugate 17 0.43 0.13 % ID/cc

18. Conclusions -Learn from other successful concepts for Cu-64 ligand design: Incorporation of donor into cross-bridge - Improvement of radiolabeling conditions for quantitative labeling compared to other cross-bridged ligand systems -Decrease of number of donors does not decrease plasma stability -PROVIDING RADIOCHEMISTS WITH NEW TOOLS FOR PET AGENT DEVELOPMENT

19. Acknowledgements The Thrombus Team Tyson RietzRich Looby Nick RotileDr. Howard Chen Dr. Francesco Blasi Helen Day Prof. Peter Caravan