Novel PET Probes to Image the Immune System and Cancer From Discovery to Clinical Applications
Caius Radu, M.D.
UCLA
Department of Molecular and Medical Pharmacology
Crump Institute for Molecular Imaging
Reference book for PET pharmaceuticals 2004
>1,600 PET probes have been synthesized, however only very few are used routinely. Why?
Outline
1. Development of [18F]FAC, a new PET probe to image the immune system and cancer
2. Optimization of [18F]FAC 3. Biological significance of PET assays using the FAC probes
4. Potential clinical utility of the FAC probes The immune system is central to health
autoimmunity •
•
•
vaccines
type 1 diabetes
rheumatoid arthritis
multiple sclerosis, etc. transplantation
• immune deficiencies
• lymphoma
cancer immunotherapy
How can we image immune cells?
The Team Owen Witte
Jenny Shu
Mike Phelps
biology
clinical translation
Johannes Czernin
radiochemistry Nagichettiar Satyamurthy Strategy to develop PET probes to image lymphocytes
target identification
gene expression profiling
activated T‐
lymphocytes
non‐
activated T‐
lymphocytes
identify candidate probes
differential screening
evaluate in mouse models & in humans
in vivo evaluation
Identification of target genes & differential screening
microarray identification of DNA salvage pathway genes preferentially expressed in lymphocytes and upregulated upon T cell activation radioactive probe cell uptake assay deoxyuridine
analogs
thymidine
analogs
deoxycytidine
analogs
VS.
activated T‐ non‐
lymphocyte activated T‐
lymphocyte
Identification of candidate probes
FAC
dFdC
extracellular
membrane
SLC29A1
intracellular
dFdC
deoxycytidine kinase (DCK)
P
dFdC‐PO4
Radu CG, Shu CJ, Nair‐Gill E, Shelly SM, Barrio JR, Satyamurthy N, Phelps ME, Witte ON. Nat Med. 2008 Jul;14(7):783‐8
Radiochemical synthesis of [18F]FAC
Dr. Satyamurthy
Synthesis of D-[18F]FAC
O
PhOCO
18
F
OCOPh
OSO2CF3
PhOCO
O
F
18
PhOCO
HBr
OCOPh
PhOCO
NH2
NHSiMe3
N
PhOCO
O
F
18
Br
Me3SiO
+
O
F
18
PhOCO
PhOCO
PhOCO
N
O
N
NH2
HO
+
N
O
N
NH2
O
F
18
HO
N
O
N
PhOCO
NaOCH3
O
F
18
PhOCO
N
HO
N
O
O
18
F
N
HO
NH2
β−isomer
α−isomer
18
D-[ F]FAC
•chemical and radiochemical purities greater than 99% •specific activity greater than 1 Ci/micromol
•>200 runs, ~80‐100 mCi/run 3.5” wide; 8” deep; 11” high
•a new compact unit operation synthesizer technology platform •it accommodates high pressures and temperatures plug‐and‐play reconfiguration
•allows diverse chemistry reaction schemes
•used to synthesize labeled nucleoside analogs, small molecules & biologics
[18F]FAC vs. traditional PET probes
nucleoside analogs PET probes
[18F]FAC
[18F]FLT
[18F]FMAU
[18F]FDG
B
12.5
12
THY
SC
9.5
14
H
%ID/g
%ID/g
%ID/g
%ID/g
GB
GB
GI
BL
BM
BL
2
BL
3
1.4
1.3
BL
BM
Sagittal
Coronal
SP
Sagittal
Coronal
Sagittal
Coronal
Sagittal
Coronal
SP
SC
K
K
K
GI
Transverse
Thy –thymus; SP‐ spleen
Transverse
Transverse
Transverse
[18F]FAC PET/CT imaging of systemic autoimmunity
Normal mice
Mice susceptible to systemic autoimmunity) ‐ untreated
‐ 2 days of DEX treatment B6.Faslpr/J mice carry the Faslpr mutation. Deficient apoptosis of Faslpr lymphocytes leads to lymphadenopathy, arthritis and immune complex‐mediated glomerulonephrosis
Thy –thymus
LN -lymph nodes
BM -bone marrow
DEX‐ Dexamethasone ‐ potent synthetic member of the glucocorticoid class of steroid 1mm Coronal slices anterior to posterior
hormones. It acts as an anti‐inflammatory and immunosuppressant drug. [18F]FAC PET imaging of severe systemic autoimmunity
Thymus
Brachial
lymph node
Axillary lymph
node
Spleen
Cervical
lymph nodes
Brachial
lymph node
•1. Development of [18F]FAC, a new PET probe to image DNA metabolism
•2. Optimization of [18F]FAC •3. Biological significance of PET assays using the FAC probes
•4. Potential clinical utility of the FAC probes [18F]FAC is deaminated in vivo
HLPC after 45 min. incubation of [18F]FAC with human plasma
FAC deamination increases the number of radiolabeled metabolites
[18F]FAC
Ideal [18F]FAC analog to measure DCK activity
[18F]FAU
CDA
extracellular
extracellular
intracellular
intracellular
SLC29A1
CDA
[18F]FAC
DCK 5’‐NT
[18F]FAU
[18F]FAC analog
5’‐NT
[18F]FAC
P
[18F]FAC
P
[18F]FAC
P P P
DCTD [18F]dFdU
DCK
P
[18F]FAC analog P
P
CDA – cytidine deaminase
How do we develop such a probe?
Criteria for DCK‐specific PET probes
The FAC pharmacophore
D-FMAC
L-FFAC
L-FCAC
L-FMAC
D-FBAC
L-FBAC
D-FCAC
D-FFAC
L-FAC
YES
Mouse
studies
1. Good in vitro data?
Criteria: • not a substrate for CDA
• medium or high affinity for DCK
• low affinity for TK2
• transport
YES
RDRC UCLA
YES
IND
Sofie Biosciences
2. Good in vivo data?
3. Good in human data?
4. Further Multi‐site Evaluation
clinical Criteria: • Metabolic
stability in plasma (HPLC)
• Sensitivity: high signals in DCK‐expressing tissues (thymus and bone marrow)
• Specificity: low signals in tissues and tumors that express low levels of DCK
Criteria: • Metabolic
stability in plasma (HPLC)
• Sensitivity & Specificity: Detection of DCK‐
expressing tissues (bone marrow) and of lymphoma lesions;
•Correlate PET signals in lymphoma lesions with DCK activity measured on biopsy samples
trials
FAC analogs that are amenable to fluorination
deaminated
resistant to deamination
-
Affinity for target (DCK)
Km (µM)
Vmax (µM/min)
Vmax/Km
D‐FAC
0.34
0.219
1.55
L‐FAC
0.009
0.628
71.4
L‐FFAC
0.027
0.387
14.25
L‐FCAC
0.608
0.925
1.519
L‐FBAC
6.544
2.607
0.398
L‐FMAC
1.017
0.982
0.965
*TK2 affinity and transport studies in progress
New FAC analogs in mice
[18F]FAC
[18F]L-FAC
[18F]L-FFAC
[18F]L-FCAC
[18F]L-FBAC
[18F]L-FMAC
12
SG
SG
Thy
Thy
Thy
L
B
GI
L
GI
GI
L
GI
K
GI
B
K
BL
BL
BL
sagittal
S
B
coronal
B
GI
transverse
sagittal
S
BL
B
coronal
sagittal
coronal
B
S
B
GI
transverse
GI
transverse
sagittal
BL
coronal
K
GI
transverse
% ID/g
Thy
BL
sagittal
coronal
sagittal
S
K
S
GI
transverse
coronal
GI
3.0
transverse
[18F]L‐FMAC imaging of systemic autoimmunity
autoimmune mouse
wild type B
Thy
13.9
LNs
13.9
13.9
BL
0.8
SP
GI
0.8
% ID/g
GI
% ID/g
% ID/g
SP
0.8
BL
Preliminary evaluation of the FAC probes in humans
[18F]FAC
[18F]L‐FAC
[18F]L‐FMAC
Salivary Glands
Lung
Heart
Liver
left kidney
e. Kidney
Bone Marrow
Bladder
Muscle
healthy volunteers
Purpose: dosimetry - initial estimates based on mouse studies -~3mCi; revised
estimations ~10-15 mCi.
Johannes Czernin and Matthias Benz, UCLA
•1. Development of [18F]FAC, a new PET probe to image DNA metabolism
•2. Optimization of [18F]FAC •3. Biological significance of PET assays using the FAC probes
•4. Potential clinical utility of the FAC probes What is the role of DCK in DNA metabolism? extracellular Glucose
Glucose
Legend:
de novo
salvage
shared
HK Glucose‐6‐P
Fructose‐6‐P
Glutamine
Uridine (U)
(1) carbamylphosphate synthase II
(2) aspartate carbamoyltransferase
(3) dihydroorotase
(4) dihydroorotate dehydrogenase
(mitochondrial inner membrane) U
orotic acid
Ribose 5P
non‐oxidative pathway
(5,6) UMP synthase
phosphoribosyl pyrophosphate (PRPP)
dTTP
dTDP
pyruvate
LDHA, B
TCA cycle
synthesis of mDNA
(TK2 and DGK)
lactate dA, dG, dC, dT
Lactate, H+
mitochondria
GPR65 proton sensor
UMP
CMP
UDP
DNA
pyruvate kinase
low activity
high activity
C
deficient activity in hereditary orotic aciduria
GA3P
pyruvate
Cytidine (C)
asparate, ATP,
HCO3‐
oxidative pathway
PEP
CDA
dUTP
RNA
dUMP
CTP‐S
UTP
CTP
CDP
RR
dCDP
dCMP
TS
dTMP
DCK
dCyd
TK1
(feedback inhibition by dTTP; activated by dCTP)
dCTP
DNA
dAMP, dATP,
dGMP dGTP
dADP,
dGDP
dT, dU
Thymidine (dT), dU
CDA
dC
dA, dG
How do we interpret an [18F]FAC PET scan?
…or which signals are DCK‐specific & which are not?
[18F]FAC microPET/CT
DCK mRNA
Bone/bone‐
marrow (BM)
THY
L?
GI SP
?
BL
BM
BM
Mouse tissues/cells
BM
Lymphocytes (spleen ‐SP)
Thymus
(THY)
The biological function of DCK and the salvage pathway
Fine tuning of intracellular dNTP pools (rather minor role) • vs. Provides dNTPs for DNA replication and repair (major role)
Approach
Generate a genetic mouse model of DCK deficiency
[18F]FAC PET scans of DCK +/+ and −/− mice
DCK +/+
DCK −/−
13
THY
SP
%ID/g
THY
BM
SP
SP
3.0
THY - Thymus, SP - Spleen, BM - Bone Marrow
07/02/2009
[18F]L‐FAC PET scans of DCK +/+ and −/− mice
DCK −/−
10
%ID/g
DCK +/+
1.0
SG – Salivary Gland, L – Liver, GI – Gastrointestinal Tract, BL – Bladder, B – Bone Marrow 06/17/09
Function vs. Structure
DCK +/+
spleen
D-FAC scan
spleen
DCK −/−
•1. Development of [18F]FAC, a new PET probe to image DNA metabolism
•2. Optimization of [18F]FAC •3. Biological significance of PET assays using the FAC probes
•4. Potential clinical utility of the FAC probes Potential applications for the FAC probes Cancer Treatment stratification for nucleoside prodrugs
(gemcitabine, cytarabine, decitabine, etc.)
Cancer surrogate biomarkers for monitoring drugs that interfere with the de novo pathway for DNA synthesis
Examples: Antifolates, RR2 inhibitors/siRNA
Cancer surrogate biomarkers for monitoring drugs/therapies that induce DNA damage
Autoimmunity
Inflammation, bone marrow transplant
[18F]L‐FAC in autoimmune pancreatitis
[18F]L‐FAC
pancreas head
[18F]FDG
pancreas head
Liver
56 y.o. male with chronic pancreatitis confirmed by biopsy [18F]FAC PET in lymphoma
BM
malignant lympadenopathy
Liver
Spleen
Treatment stratification in cancer
FAC
Gemcitabine (dFdC, Gemzar)
Gemcitabine therapy:
•low response rates (~20%) •side effects •low DCK expression is associated with resistance SLC29A1
dFdC, FAC
DCK
P
dFdC‐PO4, FAC‐PO4
Question: is FAC PET useful to predict tumor responses to nucleoside analog pro‐drugs?
[18F]FAC may predict tumor responses to gemcitabine
[18F]FDG microPET/CT
DCK+
DCK-
[18F]FAC microPET/CT
DCK+
Quantification of PET signals
DCKResponses to gemcitabine Laing et al. Noninvasive prediction of tumor responses to gemcitabine using
positron emission tomography PNAS 2009 vol. 106 no. 8 2847-2852.
DCK mRNA
Heterogeneous expression of DCK in human lymphoma cell lines Collaboration with Dr. Sven De Vos, UCLA
Summary
Unique tools to study DCK and the salvage pathway
Chemistry: PET probes
• FAC and analogs
Biology: Genetically engineered mouse models
• DCK deficient mice
Clinical translation
• 3 approved RDRC/IRB protocols >1,600 PET probes have been synthesized, however only few of these probes are used routinely….
……..maybe because each PET assay requires extensive validation & a good understanding of the biology it measures
……..maybe because sometimes the communication between chemists, biologists and clinicians is suboptimal
……..maybe because the logistics and cost of bringing a PET assay to the commercial domain are often overlooked
Reference book for PET pharmaceuticals 2004
Ultimate goal: to shift the paradigm in PET imaging
High throughput RADIOCHEMISTRY
High throughput SCREENING
High throughput IMAGING
• microfluidic
synthesizers
• microfluidic assay for PET probe binding to target cells
• the “PET‐box”, a low cost, easy to use scanner
UCLA &
Radu/Czernin group
Funding from: NIH, Waxman Foundation, Keck Foundation, Dana Foundation
Jason Lee, Wayne Austin, Rachel Laing, Liu Wei, Matthias Benz, Dean Campbell, Hsiang‐I Liao, Gerald Toy, Andrew Tran, Amanda Armijo Conflict of interest: patent application &