Molecular imaging is a new discipline that helps understanding complex pathological processes by visualizing unique molecular signatures at the cellular, subcellular or gene level.

This technique Contributes diagnosis of cancer, neurological and cardiovascular

diseases Also contributes to shorten the time for developing new medicine at

lower cost Is expected to have a major economic impact due to earlier and more

precise diagnosis

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

99m43Tc

The first artificially produced element with no stable

isotopes A transitional metal belongs to the second

transition series Its chemistry is close to that of rhenium

99m43Tc

Is obtained by a generator system utilizing a radiation equilibrium between 99Mo and 99mTc

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

90Mo-99mTc Radiation Equilibrium

99Mo

99mTc

99Tc

β--Decay ，T1/2 = 65.9 h

Nuclear Isomer Transition-ray (140 keV)T1/2 = 6 h

β--Decay ，T1/2 = 2.1 x 105 y

99Ru（ Stable ）

Radiation Equilibrium

99Mo - 99mTc Generator System

Separation of 99mTc from 99Mo

Only 99mTc is eluted from the column

Generation of 99mTc from 99Mo

0 1 2 3 4 5Days

Rad

ioac

tivi

ty (

%)

1

100

10

Decay Curve of 99Mo

Max 99mTc

AluminaColumn

Saline Vacuum Vial

99MoO42-/

99mTcO4-

99m43Tc

Is obtained by a generator system utilizing a radiation equilibrium between 99Mo and 99mTc

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

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 PETSPECT (Single Photon Emission

Computed Tomography) recognizes the sites of tracer

accumulation

Impairs resolutionof PET

SPECT tracer

123I-IBZM Moue Brain D2 Receptor

99mTc-MIBI Mouse Heart

mouse brain

salivaryglands

thyroid

striatum

eyes

I123-IBZMD2 receptor study

5mm

Fused Images

SPECT Images

Simultaneous Dual Isotope Imaging of Perfusion and Dopamine D2 Receptors in Rat Brain

99mTc-HMPAO 123I-IBF

Images co-registered with MRI

MRI MRI

SPECT allows simultaneous images of cerebral blood flow and D2 receptor function

SPECT/CT Images （ Murine Thyroid ）

Single pinholeRadius of gyration: 25 mm

Acquired for 20 min (360 degree)

Multi pinholeRadius of gyration: 35 mm

Acquired for 10 min (360 degree)

99mTcO4- (50-80 μCi)

6 h post-injection

Reticuloendothelial System 99mT-Sn colloid

Hepatobiliary excretion 99mTc-HIDA

Renal Function 99mTc-DTPA

Bone Function 99mTc-MDP

99mTc Radiopharmaceuticals in the 70’

At the initial stage of 99mTc radiopharmaceutical development, it was thought that Tc is a foreign substance and is recognized as such by the body

Breakthrough in Tc Chemistry

TcO(V)3+

Tc(I)+

TcN(V) 2+

Tc

OH2

OH2

OH2

CO

OC

OC

R

O

TcOC

COCO

N

S S

O

Tc

NH

Tc Core Representative Tc Complex

Tc

S

S

S

S

N

RR

+

N N

OH

O

O

Tc

NH HN

Chemical Design of 99mTc-Labeled Compound for Cerebral Blood Flow Measurement

Tc

ON N

N N

OH

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

OH

O

O

Tc

NH HN

Structural modification

Chemical Design of 99mTc-Labeled Compound for Cerebral Blood Flow Measurement

NHN

S S

Tc

OEtOOC COOEt

Rapid hydrolysis of an ester group togenerate a hydrophilic compound

The properties of the 99mTc 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

N

COOCH3

H3C

11C-Cocaine

N

COOCH3

H311C

Cocaine

Chemical Design of 99mTc-Labeled Probes（ Dopamine Transporter ）

PET Probe

SPECTProbe

N

COOCH3

F

NN

SS

O

Tc

O

H3C

Cl

N

Tc

O

N

SS

123I-CocaineN

COOCH3

H3C

123I

The chemical structure of 99mTc-labeled cocaine analogs differs significantly from that of cocaine These compounds still possess substrate specificity to dopamine transporter of the brain

99mTc-Labeled Probe for Assessing Fatty Acid Metabolism in the Heart

OH

O

C

[11C]palmitic acid

OH

O

123I

15-(p-[123I]iodophenyl)pentadecanoic acid ([123I]IPPA)

TcOCCO

CO

OH

O

Transported and recognized as a substrate for -oxidation by the myocardium

J. Med. Chem. 50 (3), 543-549, 2007

Present Design of 99mTc-Labeled Probes

： Targeting unit

Ligand (10-4 M)

Tc

Tc

ComplexationMonovalentLigand

Tc(10-7 M) 99mTc-Labeled Probe (10-7

M)

TcDivalentLigand

MonovalentComplex

Divalent Complex

Avidity

Conjugate a chelating molecule with a targeting unit (e.g., tropane, peptide) A large excess ligand is used to obtain 99mTc labeled compound with high radiochemical yields in short reaction times Divalent ligands provide divalent 99mTc complexes that possess higher avidity to target molecule than monovalent counterpart

99mTc-Labeled RGD Peptides for Tumor Imaging

N

SH

O

HON

HS

O

OH

OHN O NH

5

c(RGDfK)c(RGDfK)

5

Divalent RGD Ligand

N

S

N

S

O

OHO

OO

HN O

c(RGDfK)

O NH

c(RGDfK)

5 5

Tc

Divalent 99mTc-Labeled RGD

SPECT Images

Tumor(U87MG cell

s ）

Non purified 99mTc-TMEC-RGD2

(contained 10-4 M ligand)

HPLC-Purified 99mTc-TMEC-RGD2

(No excess ligand)

Problem

BloodTarget

(peripheral)Capillary

Wall

The presence of excess ligand impairs the accumulation of 99mTc labeled probes in the target

Tc Tc

Tc

： 99mTc-labeledprobes

： Ligand

Dilemma Excess ligands are used to prepare 99mTc-labeled probes in

order to achieve high radiochemical yields in short reaction times

The presence of excess ligands reduces target accumulation of 99mTc-labeled probes by competing for the target molecule

Removal of excess ligands from the 99mTc-labeled probes by HPLC or solid-phase extraction method is possible

HOWEVER, such manipulation impairs the advantages of 99mTc-labeled probes simple and sterile preparation loss of 99mTc-labeled probes during the purification process (HPLC

separation, evaporation and reconstitution)

ANY OTHER APPROACH ?

Preparation of 99mTc-Labeled Probes

Kit（ Ligand + SnCl2)

99mTcO4-

99mTc-LabeledProbe

Dilemma Excess ligands are used to prepare 99mTc-labeled probes in

order to achieve high radiochemical yields in short reaction times

The presence of excess ligands reduces target accumulation of 99mTc-labeled probes by competing for the target molecule

Removal of excess ligands from the 99mTc-labeled probes by HPLC or solid-phase extraction method is possible

HOWEVER, such manipulation impairs the advantages of 99mTc-labeled probes simple and sterile preparation loss of 99mTc-labeled probes during the purification process (HPLC

separation, evaporation and reconstitution)

ANY OTHER APPROACH ?

New Chemical Design of 99mTc-Labeled Probes

Change the paradigm from “Development of 99mTc-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 99mTc”

that is 99mTc-labeled probes utilizing chemical properties of

Tc, the properties as a transitional metal

New Concept for Designing 99mTc-Labeled Probes

Synthesis of multivalent (divalent or trivalent) 99mTc- labeled probes from monovalent ligand The target accumulation of 99mTc-labeled probes would be less impaired by the presence of excess ligands

TcDivalent Complex

Tracer amount

Trivalent ComplexTracer amount

Tc

MonovalentLigand

10-5 – 10-4 M

TcTc

99

mTc

Blood

Divalent 99mTc-labeled probes exhibit higher avidity than monovalent ligands to target

99mTc

99m Tc

Rationale behind the Chemical Design

Higher target accumulation

Target

99mTc

99

mTc

99mTc

99mT

c

99mTc99mTc99mTc

Target

99mTc

Divalent 99mTc-labeled probes exhibit slower dissociation from target than monovalent ligands

Dissociation

No dissociation

Higher Retention

Blood

99m Tc

Rationale behind the Chemical Design

99mTc

Validation of the Chemical Design

NH

SH

O

HO HN-c(RGDfK)

O

5

N

SH

O

HON

HS

O

OH

OHN O NH

5

c(RGDfK)c(RGDfK)

5

MonovalentLigand

Divalent Ligand

NH

S

HN

S

O

OHTc

O

OO

HN Oc(RGDfK)

O NHc(RGDfK)

5 5

N

S

N

S

O

OHO

OO

HN Oc(RGDfK)

O NHc(RGDfK)

5 5

Tc

Divalent99mTc Complex

Divalent 99mTc Complex

99mTc(V)-GH

Multivalent 99mTc-Labeled Probes

99mTc Core Ligand LinkerTargeting Molecule

99mTcOD-Penicillamine

Hydroxyamamide

Alkyl

Ethylene glycol

Peptide

RGD Peptide Folate Oligo-aspartic acid Antibody Others

99mTcN Dithiocarbamate

[99mTc(CO)3

(OH2)3]+ Isonitrile

Mixed Ligand [99mTc(CO)3(OH2)3]+ Compound

M: Tc/ReR: Targeting Unit

Trivalent Compound Divalent Compound withpharmacokinetic modifier (R’)

Synthesis of 99mTc-Labeled RGD Peptide

CN-Hx-c(RGDfK)

Divalent compound

M: 99mTc/Re

SPECT Images

99mTc-(CN-Hx-RGD)2 (300 µCi) 2 h post-injection

MultipinholeRadius of gyration: 25 mm.

Acquired for 20 min (360 degree)

Tumor

Synthesis of 99mTc-Labeled RGD Peptide

CN-Hx-c(RGDfK)

CN-EG3-c(RGDfK)

Divalent compound

Trivalent compound

High hepatic accumulationM: 99mTc/Re

Conclusions The monovalent penicillamine derivatives provided divalent

99mTc-labeled compounds in high yields The pharmacokinetics was manipulated by changing linkage

structures between penicillamine and c(RGDfK)

The divalent 99mTc-[Pen-SSG-c(RGDfK)]2

visualized tumor in mice by SPECT/CT without removing excess ligands

The use of 99mTc(CO)3(OH2)3 core provided divalent or trivalent 99mTc-labeled compounds in high yields

The pharmacokinetics was also manipulated by changing linkage structures between CN and c(RGDfK)

The present chemical design of 99mTc-labeled multivalent compounds would constitute a new strategy to develop molecular probes for SPECT