1. l Molecular imaging is a new discipline that helps understanding complex pathological processes by visualizing unique molecular signatures at the cellular, subcellular or gene level. l This technique l Contributes diagnosis of cancer, neurological and cardiovascular diseases l Also contributes to shorten the time for developing new medicine at lower cost l Is expected to have a major economic impact due to earlier and more precise diagnosis l Among imaging modalities, numerous studies have been made with PET. However, complementary development of PET and SPECT is required for widespread applications of this technique. Molecular Imaging

2. 99m 43 Tc l The first artificially produced element with no stable isotopes l A transitional metal belongs to the second transition series l Its chemistry is close to that of rhenium

4. 99m 43 Tc l Is obtained by a generator system utilizing a radiation equilibrium between 99 Mo and 99m Tc Emits a  -ray of 140 keV with a half-life of 6 h, which is suitable to SPECT imaging The most widely applied radionuclide in diagnostic nuclear medicine

5. 90 Mo- 99m Tc Radiation Equilibrium 99 Mo 99m Tc 99 Tc β - -Decay ， T 1/2 = 65.9 h Nuclear Isomer Transition  -ray (140 keV) T 1/2 = 6 h β - -Decay ， T 1/2 = 2.1 x 10 5 y 99 Ru （ Stable ） Radiation Equilibrium

6. 99 Mo - 99m Tc Generator System Separation of 99m Tc from 99 Mo Only 99m Tc is eluted from the column Alumina Column Saline Vacuum Vial 99 MoO 4 2- / 99m TcO 4 -

7. 99m 43 Tc l Is obtained by a generator system utilizing a radiation equilibrium between 99 Mo and 99m Tc Emits a  -ray of 140 keV with a half-life of 6 h, which is suitable to SPECT imaging The most widely applied radionuclide in diagnostic nuclear medicine

8. PET tracer Positron Range The resolution of PET (Positron Emission Tomography) is affected by positron range PET recognizes the site of positron annihilation Advantage of SPECT over PET SPECT (Single Photon Emission Computed Tomography) recognizes the sites of tracer accumulation Impairs resolution of PET SPECT tracer

9. 123 I-IBZM Moue Brain D2 Receptor 99m Tc-MIBI Mouse Heart Fused Images SPECT Images

10. Simultaneous Dual Isotope Imaging of Perfusion and Dopamine D 2 Receptors in Rat Brain 99m Tc-HMPAO 123 I-IBF Images co-registered with MRI MRI SPECT allows simultaneous images of cerebral blood flow and D 2 receptor function

11. SPECT/CT Images （ Murine Thyroid ） Single pinhole Radius of gyration: 25 mm Acquired for 20 min (360 degree) Multi pinhole Radius of gyration: 35 mm Acquired for 10 min (360 degree) 99m TcO 4 - (50-80 μCi) 6 h post-injection

12. l Reticuloendothelial System l 99m T-Sn colloid l Hepatobiliary excretion l 99m Tc-HIDA l Renal Function l 99m Tc-DTPA l Bone Function l 99m Tc-MDP 99m Tc Radiopharmaceuticals in the 70’ At the initial stage of 99m Tc radiopharmaceutical development, it was thought that Tc is a foreign substance and is recognized as such by the body

13. Breakthrough in Tc Chemistry TcO(V) 3+ Tc(I) + TcN(V) 2+ Tc OH 2 CO OC R O Tc OC CO N S S O Tc NH Tc Core Representative Tc Complex + N N O H O O Tc NH HN

14. Chemical Design of 99m Tc-Labeled Compound for Cerebral Blood Flow Measurement Tc O N N N N O H O Rapid conversion to a hydrophilic compound in the brain GSH can easily attack the Tc center  Neutral, compact and lipophilic complex that penetrates intact BBB  No retention in the brain N N O H O O Tc NH HN Structural modification

15. Chemical Design of 99m Tc-Labeled Compound for Cerebral Blood Flow Measurement NH N SS Tc O EtOOC COOEt Rapid hydrolysis of an ester group to generate a hydrophilic compound The properties of the 99m Tc complex (stable, neutral and lipophilic) are masked so that the pharmacokinetics is governed only by the functional groups  Neutral, compact and lipophilic complexes that penetrates intact BBB  No retention in the brain NH S S O Tc N Structural modification

16. 11 C- Cocaine Cocaine Chemical Design of 99m Tc-Labeled Probes （ Dopamine Transporter ） PET Probe SPECT Probe N COOCH 3 F N N S S O Tc O H 3 C Cl N O N S S 123 I- Cocaine 123 I l The chemical structure of 99m Tc-labeled cocaine analogs differs significantly from that of cocaine l These compounds still possess substrate specificity to dopamine transporter of the brain

17. 99m Tc-Labeled Probe for Assessing Fatty Acid Metabolism in the Heart O H O C [ 11 C]palmitic acid O H O 123 I 15-(p-[ 123 I]iodophenyl)pentadecanoic acid ([ 123 I]IPPA) Tc OC CO CO O H O Transported and recognized as a substrate for  -oxidation by the myocardium J. Med. Chem. 50 (3), 543-549, 2007

18. Present Design of 99m Tc-Labeled Probes ： Targeting unit Ligand (10 -4 M) Tc Complexation Monovalent Ligand Tc (10 -7 M) 99m Tc-Labeled Probe (10 -7 M) Tc Divalent Ligand Monovalent Complex Divalent Complex Avidity Conjugate a chelating molecule with a targeting unit (e.g., tropane, peptide) A large excess ligand is used to obtain 99m Tc labeled compound with high radiochemical yields in short reaction times Divalent ligands provide divalent 99m Tc complexes that possess higher avidity to target molecule than monovalent counterpart

19. 99m Tc-Labeled RGD Peptides for Tumor Imaging N SH O HO N HS O OH OHNONH 5 c(RGDfK)c(RGDfK) 5 Divalent RGD Ligand N S N S O OH O O O HNO c(RGDfK) ONH c(RGDfK) 5 5 Tc Divalent 99m Tc-Labeled RGD

20. SPECT Images Tumor (U87MG cells ） Non purified 99m Tc-TMEC-RGD 2 (contained 10 -4 M ligand) HPLC-Purified 99m Tc-TMEC-RGD 2 (No excess ligand)

21. Problem Blood Target (peripheral) Capillary Wall The presence of excess ligand impairs the accumulation of 99m Tc labeled probes in the target Tc ： 99m Tc-labeled probes ： Ligand

22. Dilemma l Excess ligands are used to prepare 99m Tc-labeled probes in order to achieve high radiochemical yields in short reaction times l The presence of excess ligands reduces target accumulation of 99m Tc-labeled probes by competing for the target molecule l Removal of excess ligands from the 99m Tc-labeled probes by HPLC or solid-phase extraction method is possible l HOWEVER, such manipulation impairs the advantages of 99m Tc-labeled probes l simple and sterile preparation l loss of 99m Tc-labeled probes during the purification process (HPLC separation, evaporation and reconstitution) l ANY OTHER APPROACH ?

23. Preparation of 99m Tc-Labeled Probes Kit （ Ligand + SnCl 2 ) 99m TcO 4 - 99m Tc - Labeled Probe

24. Dilemma l Excess ligands are used to prepare 99m Tc-labeled probes in order to achieve high radiochemical yields in short reaction times l The presence of excess ligands reduces target accumulation of 99m Tc-labeled probes by competing for the target molecule l Removal of excess ligands from the 99m Tc-labeled probes by HPLC or solid-phase extraction method is possible l HOWEVER, such manipulation impairs the advantages of 99m Tc-labeled probes l simple and sterile preparation l loss of 99m Tc-labeled probes during the purification process (HPLC separation, evaporation and reconstitution) l ANY OTHER APPROACH ?

25. New Chemical Design of 99m Tc-Labeled Probes l Change the paradigm from “Development of 99m Tc- labeled probes that provide information similar to those by PET or Radioiodinated Compounds” to “Development of radiolabeled probes that can be best achieved by using 99m Tc” that is l 99m Tc-labeled probes utilizing chemical properties of Tc, the properties as a transitional metal

26. New Concept for Designing 99m Tc-Labeled Probes Synthesis of multivalent (divalent or trivalent) 99m Tc- labeled probes from monovalent ligand The target accumulation of 99m Tc-labeled probes would be less impaired by the presence of excess ligands Tc Divalent Complex Tracer amount Trivalent Complex Tracer amount Tc Monovalent Ligand 10 -5 – 10 -4 M Tc

27. 99m Tc Blood Divalent 99m Tc-labeled probes exhibit higher avidity than monovalent ligands to target 99m Tc Rationale behind the Chemical Design Higher target accumulation Target

28. 99m Tc Target 99m Tc Divalent 99m Tc-labeled probes exhibit slower dissociation from target than monovalent ligands Dissociation No dissociation Higher Retention Blood 99m Tc Rationale behind the Chemical Design 99m Tc

29. Validation of the Chemical Design NH SH O HO HN-c(RGDfK) O 5 N SH O HO N HS O OH OHNONH 5 c(RGDfK)c(RGDfK) 5 Monovalent Ligand Divalent Ligand NH S HN S O OH Tc O O O HNO c(RGDfK) ONH c(RGDfK) 5 5 N S N S O OH O O O HNO c(RGDfK) ONH c(RGDfK) 5 5 Divalent 99m Tc Complex Divalent 99m Tc Complex 99m Tc(V)-GH

30. Multivalent 99m Tc-Labeled Probes 99m Tc CoreLigandLinker Targeting Molecule 99m TcO D -Penicillamine Hydroxyamamide Alkyl Ethylene glycol Peptide RGD Peptide Folate Oligo- aspartic acid Antibody Others 99m TcNDithiocarbamate [ 99m Tc(CO) 3 (OH 2 ) 3 ] + Isonitrile

31. Mixed Ligand [ 99m Tc(CO) 3 (OH 2 ) 3 ] + Compound M: Tc/Re R: Targeting Unit Trivalent Compound Divalent Compound with pharmacokinetic modifier (R’)

32. Synthesis of 99m Tc-Labeled RGD Peptide CN-Hx-c(RGDfK) Divalent compound M: 99m Tc/Re

33. SPECT Images 99m Tc-(CN-Hx-RGD) 2 (300 µCi) 2 h post-injection Multipinhole Radius of gyration: 25 mm. Acquired for 20 min (360 degree) Tumor

34. Synthesis of 99m Tc-Labeled RGD Peptide CN-Hx-c(RGDfK) CN-EG 3 -c(RGDfK) Divalent compound Trivalent compound High hepatic accumulation M: 99m Tc/Re

35. Conclusions  The monovalent penicillamine derivatives provided divalent 99m Tc-labeled compounds in high yields  The pharmacokinetics was manipulated by changing linkage structures between penicillamine and c(RGDfK)  The divalent 99m Tc-[Pen-SSG-c(RGDfK)] 2  visualized tumor in mice by SPECT/CT without removing excess ligands  The use of 99m Tc(CO) 3 (OH 2 ) 3 core provided divalent or trivalent 99m Tc-labeled compounds in high yields  The pharmacokinetics was also manipulated by changing linkage structures between CN and c(RGDfK)  The present chemical design of 99m Tc-labeled multivalent compounds would constitute a new strategy to develop molecular probes for SPECT