Positron Emission Tomography Imaging of Drug-Induced Tumor

Apoptosis with a Caspase-Triggered Nanoaggregation Probe

2015-06-02 Bin Shen et al, Angew. Chem. Int. Ed. 2013, 52, 10511 –10514

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Uses molecules labeled with positron-emitting iso-

topes.

Carbon-11, fluorine-18, and copper-64.

To monitor biochemical processes in living object.

In real time with excellent sensitivity.

2015-06-02 Bin Shen et al, Angew. Chem. Int. Ed. 2013, 52, 10511 –10514

Positron emission tomography (PET)

How PET works?

Short lived radioactive isotope radioactive isotope

(C-11, Fl-18, O-15 or N-13) attached to glucose, water,

ammonia into the body.

Where a positron emitted from the radioactive sub-

stance collides with an electron in the tissue

the body.

PET detects the gamma rays given off at that site

Different colors or degrees of brightness on a PET image represent different levels of tissue or or-gan function.

How PET works?

Healthy tissue accumulates some of the tagged

glucose, which will show up on the PET images.

Cancerous tissue uses more glucose than nor-mal tissue, and appear brighter than normal tissue on the PET images.

the body.

PET detects the gamma rays given off at that site

Different colors or degrees of brightness on a PET image represent different levels of tissue or organ function.

Why PET?

• Extremely high sensitivity

• Unlimited penetration depth

• Signal quantification and processing

•Radioisotopes: 11C, 13N, 15O, 18F, 124I, 68Ga, 64Cu….

• Applied to pre-clinical and clinical studies

•Safety issues….

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A Positron is an anti-matter electron, it is identical in

mass but has an apposite charge of +1.

Proton (+1 charge) => neutron (0 charge) + positron

(+1 charge) + neutrino (0 charge)

+

anti-neutrino

positron

What is a Positron?

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DETECTOR

DETECTOR

WHAT DO WE WANT TO DETECT IN PET? 2 photons of 511 keV in coincidence, coming in

a straight line from the same annihilation

e-

e+

UNSTABLE NU-CLEUS

EMITS POSITRON

POSITRON AN-NIHILATES WITH ELEC-TRON

TRUE coinci-dence

(APOPTOSIS) THE ROADS TO RUIN

Apoptotic pathways in multicellular or-gansims

BASIC APOPTOTIC MA-CHINERY

DNA fragmentation,

Chromatin condensation,

Membrane blebbing,

Cell shrinkage

Disassembly into apoptotic bodies

EngulfmentInitiator caspases inactivate proteins that protect cells

from apoptosis

Effector caspases are responsible for cellular changes

associated with apoptosis.

DNA damage

Death receptors

Growth factor

withdrawal

TRIGGERP53Bcl-2 familyCytochrome concogenes

REGULATOR

Apaf-1

Caspases

EXECUTIONERAPOPTOSIS

HOW DO CASPASES DISASSEMBLE A CELL? IT SLICES, IT DICES! Selective cleavage of specific proteins

eg bcl-2, or CAD/ICAD

e.g. nuclear lamins

eg. Gelsolin

Activation of caspase-3, committing the cell to pro-

grammed cell death apoptosis.

Activared fluorescent probes developed for imaging cas-

pase-3 activity in cells and living mice.

To develop a PET tracer that is mechanistically similar to

the activatable fluorescent probes in imaging caspase-3

activity with signal amplification.

ROLE OF CAS-PASE 3

Activation, intramolecular cyclization of [18F]C-SNAT (1) and aggregation of cyclized product

Essential bio-maker for early apoptosis Direct visual-

ization active Caspase-3 Monitoring apoptosis

Peptide substrate of Caspase-3: N-Asp-Glu-Val-Asp—X-16

TARGET: CASPASE-3

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PRINCIPLE

• Imaging of caspase-3 activity Monitoring of chemotherapeutic effect

Caspase-3

Self-assembly

Apoptotic cell

[18F]C-SNAT

• 38C13 murine lymphoma xerografts

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REPORTED PET TRACERS FOR APOPTOSIS IMAGING

• [18F]ICMT-11 : High (~nM) binding affinity with activated caspase-3

• [18F]ML-10 : High affinity with phosphatidylserine (PS).

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SYNTHESIS OF THE PROBE

N

SHN

OBocHN

CNOH

OBocHN

N

SH2NCN

1) i-Buchloroformate, NMM

2)

H2N

HS

O

HN O

N CNCF3CO2H

DIPEA, DMF N

SHN

OBocHN

N

S

O

HN O

N CN

1) TFA, DCM

2) N-Boc-D-Cys(Trt)-OH HBTU, HOBt, DIPEA N

SHN

ONH

N

S

O

HN O

N CN

O

BocHN

TrtS

1) TFA, TIS, DCM

2)

N S SEt

N

SHN

ONH

N

S

O

HN O

N CN

O

H2N

S

SEt

1) Ac-DEVD-OH, HBTU, HOBt

2) TFA, DCM

NH

HN

O N

SHN

O

O

N CN

S

N

OHN

S

SEt

ONH

OHN

NH

AcHN

O

O

HO2C

HO2CCO2H

MeO

N CN

5 steps

Active probe (3a)

Control probe (3b)

• No cyano group• No free thiol group

• L-amino acids• Cyano and thiol groups for cyclization

RADIO HPLC CHROMATOGRAPH

Analytical radio-HPLC showing the radioactive signal composition: a)1in saline; b) 1in-cubated with caspase-3 in solution for 1 h; c) extraction from1cellular uptake in apop-totic cell (4 h);d) extraction from1cellular uptake in healthy cell (4 h). No cyclization

C

Purified

Enzyme reactionin solution

Apoptotic cell

Healthy cell

Purified

Apoptotic cell

04/15/2023 Jongho Jeon 21

IN VITRO TEST

• Doxorubicin treatment: 2 mM, 24 h• In vitro uptake for 4 h

HIGH RESOLUTION IMAGING IN APOP-TOTIC CELL

a

04/15/2023 Jongho Jeon 22

HIGH RESOLUTION IMAGING IN APOP-TOTIC CELL

D. Ye et al. Nat. Chem. 2014, 6, 519-526.

• Alexa 488 conjugated probe • Image a and b from apoptotic cell• Image d and e from drug treated tissue

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ANIMAL MODELS

Injection of HeLa cell s.c. on right shoulder of 6-8 weeks old F nu/nu mice.

10-12 d.

Doxorubicin

HeLa cellsHeLa tumor

Intratumoral injec-tions of Doxorubicin

4 days

Doxorubicin4 days (x 3)

i.v. injections of Doxorubicin

[18F]C-SNATPET Scanning

(baseline)

[18F]C-SNATPET Scanning

(treated)

[18F]C-SNATPET Scanning

(treated)

Enzyme level: 2.3 fold increase

Enzyme level: 3.1 fold increase

FLUORESCENT (CY5.5) LABELED PROBE

04/15/2023 25Jongho Jeon

NH

HN

O N

SHN

O

O

N CN

S

N

OHN

SO

NH

OHN

NH

AcHN

OHO

O

HO O

O

O

OH SEt

NHO

Cy5.5

N

SHN

ONH

O O

SNN

HN

O

SN

NHO

Cy5.5

Caspase-3

in treated tumor

Normalize

d

5 min 15 min 30 min 45 min 60 min 90 min

180 min 300 min 480 min 720 min 1440 min

0.010

0.020

0.031

+ DoxSaline

USES

Detect cancer. Determine whether a cancer has spread in the body. Assess the effectiveness of a treatment plan, such as cancer therapy. Determine if a cancer has returned after treatment. Determine blood flow to the heart muscle. Determine the effects of a heart attack, or myocardial infarction, on ar-

eas of the heart. Identify areas of the heart muscle that would benefit from a procedure

such as angioplasty or coronary artery bypass surgery (in combination with a myocardial perfusion scan).

Evaluate brain abnormalities, such as tumors, memory disorders and seizures and other central nervous system disorders.

To map normal human brain and heart function.

Designed and synthesized an 18F-labeled caspase-3

triggered nanoaggregation PET tracer ([18F]C-SNAT)

Its application for imaging caspase-3 activity in dox-

orubicin-treated tumor xenografts.

This activatable PET tracer undergoes intramolecular

cyclization and subsequent aggregation upon caspase-

3 activation.

Conclusion

Achieved enhanced retention in apoptotic tumors.

Applications of this strategy for other enzyme

targets.

Translation of [18F]C-SNAT into clinical studies

are under investigation

Conclusion