Novel PET Probes to Image the Immune System and Cancer - From Discovery to Clinical Applications
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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
• type 1 diabetes
• rheumatoid • immune deficiencies
arthritis • lymphoma
• multiple
sclerosis, etc.
vaccines transplantation cancer immunotherapy
How can we image immune cells?
The Team
Jenny Shu
Owen Witte Mike Phelps
biology
clinical
radiochemistry
translation
Johannes Czernin Nagichettiar
Satyamurthy
Strategy to develop PET probes to image lymphocytes
evaluate in mouse
identify
target identification models & in
candidate probes
humans
gene expression differential screening in vivo evaluation
profiling
activated T‐
lymphocytes
non‐
activated T‐
lymphocytes
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
VS.
thymidine
analogs
activated T‐ non‐
deoxycytidine
analogs
lymphocyte activated T‐
lymphocyte
Identification of candidate probes
dFdC FAC
extracellular
SLC29A1 membrane
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
Dr. Satyamurthy
Radiochemical synthesis of [18F]FAC
Synthesis of D-[18F]FAC
O 18 O
PhOCO F PhOCO 18
F HBr
OCOPh OCOPh
PhOCO OSO2CF3 PhOCO
NH2 O
PhOCO 18
NHSiMe3 F
N
N
O PhOCO
18 Me3SiO N O N
PhOCO F + O N
Br
O
PhOCO 18
PhOCO F N
PhOCO NH2
NH2 O
HO 18
F
N
O N + HO
NaOCH3
O N
O
18
HO F
N
HO NH2
β−isomer α−isomer
18
D-[ F]FAC
3.5” wide; 8” deep; 11” high
•chemical and radiochemical •a new compact unit operation synthesizer
purities greater than 99% technology platform
•specific activity greater than 1 •it accommodates high pressures and
Ci/micromol temperatures plug‐and‐play reconfiguration
•>200 runs, ~80‐100 mCi/run •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 9.5 14
THY H
SC GB
GB
%ID/g
%ID/g
%ID/g
%ID/g
GI
BM BL BL BL
2 1.4 3 1.3
BL
BM
Sagittal Coronal Sagittal Coronal Sagittal Coronal Sagittal Coronal
SP SP
SC
K K K
GI
Transverse Transverse Transverse Transverse
Thy –thymus; SP‐ spleen[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 Thy –thymus
lymphadenopathy, arthritis and immune complex‐mediated glomerulonephrosis LN -lymph nodes
BM -bone marrow
DEX‐ Dexamethasone ‐ potent synthetic member of the glucocorticoid class of steroid
hormones. It acts as an anti‐inflammatory and immunosuppressant drug. 1mm Coronal slices anterior to posterior[18F]FAC PET imaging of severe systemic autoimmunity
Cervical
lymph nodes
Thymus
Brachial Brachial
lymph node lymph node
Axillary lymph
node
Spleen•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
Ideal [18F]FAC analog to
[18F]FAC [18F]FAU measure DCK activity
CDA
extracellular extracellular
SLC29A1
intracellular intracellular
[18F]FAC CDA [18F]FAU [18F]FAC analog
DCK 5’‐NT 5’‐NT DCK
[18F]FAC P DCTD [18F]dFdU P [18F]FAC analog P
[18F]FAC P P
[18F]FAC P P P
How do we develop such a probe?
CDA – cytidine deaminaseCriteria for DCK‐specific PET probes
YES YES YES
The FAC Mouse RDRC IND
pharmacophore studies UCLA Sofie Biosciences
1. Good in 2. Good in vivo 3. Good in human 4. Further
Multi‐site
vitro data? data? data? Evaluation
clinical
L-FFAC L-FCAC
D-FMAC L-FMAC Criteria: Criteria: Criteria: trials
• not a • Metabolic • Metabolic
D-FBAC L-FBAC
D-FCAC
D-FFAC
L-FAC substrate for stability in stability in
CDA plasma (HPLC) plasma (HPLC)
• medium or • Sensitivity: • Sensitivity &
high affinity high signals in Specificity:
for DCK DCK‐expressing Detection of DCK‐
• low affinity tissues (thymus expressing tissues
for TK2 and bone (bone marrow)
• transport marrow) and of lymphoma
• Specificity: lesions;
low signals in •Correlate PET
tissues and signals in
tumors that lymphoma lesions
express low with DCK activity
levels of DCK measured on
biopsy samplesFAC 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 progressNew 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 Thy
L
B GI
L GI L
GI K
GI GI
% ID/g
K
BL B
BL BL
BL B BL
BL
B
sagittal coronal sagittal coronal sagittal coronal sagittal coronal sagittal coronal sagittal coronal
S B S B S B K S K S GI
GI GI GI GI GI 3.0
transverse transverse transverse transverse transverse transverse[18F]L‐FMAC imaging of systemic autoimmunity
wild type autoimmune mouse
B
Thy
13.9 LNs
13.9 13.9
SP
% ID/g
% ID/g
% ID/g
GI SP
GI
0.8 0.8 0.8
BL
BLPreliminary evaluation of the FAC probes in humans
[18F]FAC [18F]L‐FAC [18F]L‐FMAC
Salivary
Glands
Lung
Heart
Liver
e. Kidney left 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
CDA
Glucose Glutamine Uridine (U) Cytidine (C)
Legend:
de novo asparate, ATP,
salvage HCO3‐
(1) carbamylphosphate synthase II
shared (2) aspartate carbamoyltransferase
Glucose (3) dihydroorotase U C
(4) dihydroorotate dehydrogenase deficient activity in hereditary
(mitochondrial inner membrane) orotic aciduria
HK oxidative pathway orotic acid (5,6) UMP
Glucose‐6‐P Ribose 5P synthase
UMP CMP
phosphoribosyl
Fructose‐6‐P non‐oxidative pathway pyrophosphate (PRPP)
GA3P UDP
PEP
DNA dUTP CTP‐S RR
dTTP RNA UTP CTP CDP dCDP dCTP
pyruvate
kinase
low activity
dTDP dUMP
high activity
pyruvate pyruvate
TS dCMP DNA
dAMP, dATP,
LDHA, B
dTMP dGMP dGTP
TCA cycle TK1 DCK
synthesis of mDNA dADP,
lactate (TK2 and DGK)
(feedback dCyd dGDP
inhibition by dTTP;
dA, dG, activated by dCTP)
dC, dT
mitochondria dT, dU
Lactate, H+ GPR65 proton sensor Thymidine (dT), dU CDA dC dA, dGHow 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)
BM
THY
Mouse tissues/cells
L?
GI SP Lymphocytes
? (spleen ‐SP)
Thymus
BL BM (THY)
BMThe 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 THY
%ID/g
SP
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 +/+ DCK −/− 10
%ID/g
1.0
SG – Salivary Gland, L – Liver, GI – Gastrointestinal Tract, BL – Bladder, B – Bone Marrow
06/17/09Function vs. Structure
D-FAC scan
DCK +/+ DCK −/−
spleen spleen•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
Cancer
surrogate biomarkers
Treatment for monitoring drugs
stratification for that interfere with the
nucleoside prodrugs de novo pathway for
(gemcitabine, DNA synthesis
cytarabine, decitabine,
Examples: Antifolates,
etc.)
RR2 inhibitors/siRNA
Cancer
surrogate biomarkers Autoimmunity
for monitoring
drugs/therapies that Inflammation, bone
induce DNA damage marrow transplant[18F]L‐FAC in autoimmune pancreatitis
[18F]L‐FAC [18F]FDG
pancreas head pancreas head
Liver
56 y.o. male with chronic pancreatitis confirmed by biopsy[18F]FAC PET in lymphoma
BM malignant
lympadenopathy
Liver
SpleenTreatment 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 Question: is FAC PET useful to predict tumor
responses to nucleoside analog pro‐drugs?
DCK
P
dFdC‐PO4, FAC‐PO4[18F]FAC may predict tumor responses to gemcitabine
Quantification of PET
signals
[18F]FDG microPET/CT [18F]FAC microPET/CT
DCK- DCK+ DCK-
DCK+
Responses to gemcitabine
Laing et al. Noninvasive prediction of tumor responses to gemcitabine using
positron emission tomography PNAS 2009 vol. 106 no. 8 2847-2852.Heterogeneous expression of DCK in human lymphoma
cell lines
DCK mRNA
Collaboration with Dr. Sven De Vos, UCLASummary
Unique tools to study DCK and the salvage pathway
Biology: Genetically
Chemistry: PET
engineered mouse Clinical translation
probes
models
• FAC and analogs • DCK deficient mice • 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 2004Ultimate goal: to shift the paradigm in PET imaging
High throughput High throughput High throughput
RADIOCHEMISTRY SCREENING IMAGING
• microfluidic • microfluidic assay • the “PET‐box”, a
synthesizers for PET probe low cost, easy to
binding to target use scanner
cells
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 &You can also read
