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1845-1923
High Spatial Resolution
CT, MRI, Optical, & US
MRI Imaging
– High Density, Internalizing Receptors
Transferrin receptor w. Transferrin MION
Optical Imaging
– Physiologic measurements
Volume, flow, Hb O2
– Fluorescent-labeled ligands
Ultrasound Imaging
– Physiologic measurements
– Targeting with a single, site-directed
bubble.
The Advantage of Radionuclides for
Targeted Imaging, especially Low
Density Sites (<20 nM)
If 10 mCi at a specific activity of >1000 <0.2 nM
µCi/nmol: 10 nmol/70 Kg
For Gd MRI:. 10-100 µM
Iron oxide (T2) increases sensitivity.
For iodinated contrast media >100 µM
Optical imaging and Ultrasound ?
George Charles de Hevesy
The first to identify the radioisotope tracer
principle.
In 1923, he used 10.6 hour lead-212 to study the
uptake of solutions in bean plants, noninvasively.
Used small, non-toxic amounts given the
sensitivity of the radioactivity techniques.
The first experiment in animals used Bi-210 to
label and follow the circulation of Bi-containing
antisyphilitic drugs in rabbits.
In a later book with Fritz Paneth, the tracer
method was introduced as the use of
radioelements as indicators.
•The first practical application of a radioisotope was
made by George de Hevesy in 1911.
•He suspected that meals that appeared regularly
might be made from leftovers.
•To confirm these suspicions de Hevesy put a small
amount of radioactive material into the remains of a
meal.
•When the same meal was served, it was radioactive!
History has forgotten the
landlady, but George de
Hevesy went on to win the
Nobel prize in 1943 and
the Atoms for Peace
award in 1959.
Imaging the In Vivo Distribution
of a Gamma Emitting
Radioisotope
Unprecedented Progress
1937 Discovery of the element Tc
1947 Isolation of Tc-99
1958 Technetium Generator
1970 Instant DTPA, HSA, & RBC Kits
1978 Crystal structures of potential Tc
radiopharmaceuticals
2002-present
– Smaller, neutral, more polar inert chelates
– Maximal effective specific activity
– Tc labeled molecules ~300 MW
1934 photo of Livingston and Lawrence
with the 27” cyclotron at LBL
In 1938, Glenn Seaborg and Emilio
Segre discovered Technetium-99m.
Walter Tucker
Powell Richards
Mechanism: TcO4- + stannous ion = reduced Tc
+ chelating agent or particle =
final product
Kit components = stannous salt (reducing agent)
& chelating agent or particle
RS-[123I]IQNB on 5/11/83
Market Analysis and Future Prospects
U.S. sales of diagnostic radiopharmaceuticals reached
$1.53 billion in 2004 and are expected to rise to $3.20
billion by 2010.
– This growth will be based on introduction of new products,
strong demand for cardiology procedures and increased sales
of oncology products, particularly FDG for PET imaging.
Nuclear cardiology sales of $1.06 billion in 2004 will
increase to $1.89 billion by 2010.
FDG sales for oncology as well as cardiology and
neurology will increase from $249 million in 2004 to
$522 million by 2010.
– In addition, new PET radiopharmaceuticals in the pipeline for
specialized applications should add to these sales estimates.
Market Analysis and Future Prospects
U.S. sales of therapeutic radiopharmaceuticals
were still on the threshold in 2005, with total
sales of $57 million.
– Rapid growth is anticipated over the next 5-6 years. By
2012, therapeutic product sales should reach $1.9
billion, with high continuing growth beyond that time.
– This will be based on the introduction of new therapeutic
radiopharmaceuticals for treating lymphoma, colon
cancer, lung cancer, prostate cancer, bone cancer and
other persistent cancers. These agents will be used in
conjunction with traditional therapies, enhancing their
effectiveness, with better specificity and reduced side
effects. Individualized Medicine!
DEVELOPMENT OF
Enzyme/Receptor Targeting in
humans
DATE PET SPECT
1977 [18F]FDG
1983 [11C]N-MeSpip [123I]IQNB
1984* [18F]Cyclofoxy
1985 [11C]Raclopride [99mTc]NGA
1985 [11C]Carfentanil
1985 [11C]Flumazenil
How many radiotracers have changed clinical care?
How many have been used in combination with pharmaceuticals?
The Principle of PET: Coincidence
Detection of Two 511 KeV Gamma Rays is
Used to Determine the Position
PET RADIONUCLIDE
PRODUCTION
T1/2 (min) E+ (kev) Nuclear Reaction
82Rb 1.3 3350 82Sr generator
11C 20 960 14N (p,) w. 6 ppm O2
13N 10 1190 16O (p,)
15O 2.05 1720 14N (d,n) w. 2% O2
18F 109.6 635 20Ne (d,) w. 0.5%F
2
18F 109.6 635 18O (p,n)
76Br 966 3980 75As (,3n)
64Cu 762 571 64Ni (p,n)
124I 5976 2134 124Te (p,n)
FDG:1976 to 2002
Imaging Saturable Sites with
MRI
Relatively High Capacity, Internalizing Sites.
– Mion-Transferrin
Substrates For Enzymes.
– FDG
High Capacity, Non-internalizing Binding Sites.
– A Gd complex of polyDTPA polyneogalactosyl dextran.
Low Capacity, Non-internalizing Binding Sites.
– Gd antibodies targeted to solid tumors.
– Gd labeled antibodies targeted to endothelial sites.
In Vivo MR Imaging: MR image of a mouse
with TfR+ and TfR- flank tumors
(a) The T1-weighted coronal SE image (3.5 min; 0.3x0.3x3 mm3 resolution). TfR- tumors (right
arrowhead) and TfR+ tumors (left arrowhead) have similar signal intensity.
(b) Corresponding T2-weighted gradient echo image showing significant difference (8 min; same
resolution). TfR-mediated cellular accumulation of the superparamagnetic probe decreases signal
intensity as expected using T2- and T2*-weighted imaging pulse sequences on cellular
internalization.
(c) Composite of a T1-weighted spin echo image obtained for anatomic detail with superimposed R2
changes after Tf-MION administration displayed in a color map.
Other Imaging Modalities for desialylated
glycoproteins binding to the high capacity (~500
nM) hepatic binding protein
[Gd]DTPA-galactosyl-
Dextran
Post-injection: 3.8 min
Liver: 66 % Enhancement
How can targeted imaging
accelerate drug discovery?
William C Eckelman PhD
Bethesda MD
Definitions: Molecular Imaging
The term molecular imaging can be broadly defined as
the in vivo characterization and measurement of
biologic processes at the cellular and molecular level.
[Weissleder & Mahmood, Radiology 2001].
MI techniques directly or indirectly monitor and record
the spatiotemporal distribution of molecular or cellular
processes for biochemical, biologic, diagnostic, or
therapeutic applications [Thakur & Lentle, Radiology
2005].
Targeting Proteins
The Magic Bullet
Paul Ehrlich used the English expression “magic
bullet” for the first time in his Harben Lectures.
The German word “Zauberkugel” appears earlier
in his thoughts and publications, based on his
view of “sidechains”, the precursor of our concept
of receptors, and on the desirable property of
drugs that must not harm the host, but attach
the parasitic invader.
Royal Institute of Public Health (London:Lewis, 1908), Experimental Researches on
Specific therapy. On immunity with special references to the relationship between
distribution and action of antigens, 107.
The Magic Bullet
Ehrlich’s first magic bullet was Salvarsan
or arsphenamine, discovered in 1909, which
provided the only cure for syphilis.
Ehrlich also thought of attaching toxins to
antibodies whereby the antibody would
carry the deadly freight to the site of the
invading parasite. His idea lives on in the
development of immunotoxins.
Both the lock and the key are
necessary in Targeted Imaging
99mTcDTPA or GdDTPA in GFR measurements is a key
without a specific target (lock). GFR is a nontargeted
process.
[18F]FP-TZTP is a M2 muscarinic agonist, which is
transported non specifically across the BBB, but binds
specifically to the M2 receptor (the lock).
Doxorubicin in liposomes is not a targeted drug
although the therapeutic effect is increased by
improvement in liver tox profile.
Emil Fischer, 1894
Imaging & “Molecular Targeting”
Interactions between a probe and a protein
target using pre-genomic techniques.
– “Biochemical probes” such as iodide (~50 years),
receptor binding radiotracers and monoclonal
antibodies (~25 years) from autopsy, linkage and
drug efficacy, etc.
Interactions between a probe and a protein
target using post-genomic techniques.
– Molecular biology, proteomics, genomics,
antisense, reporter genes, protein-protein
interactions. More targets (500 2000-3000)
Why is Targeted Imaging becoming
more important in Drug Development?
As the pharmaceutical industry turns to
targeted drugs, targeted imaging is well
positioned to “biomark” the drug potential.
Target identification is dependent upon
clinical research, i.e., “humanomics” should
be the study of choice.
Lindsay MA. Finding new drug targets in the 21st century. DDT 2005: 23/24: 1683.
The Druggable genome
Nucl Horm R
Russ & Lampel. The Druggable genone: an update. DDT 2005: 23/24: 1607.
Measuring Targeting
with Imaging
for targets of differing
density
In Vitro B/F = Bmax/Ki
Imaging requires B/F ratio ~5, Drugs
do not
Measuring the In Vivo Binding Parameters of
[18F]-Fallypride in Monkeys Using a PET Multiple-
Injection Protocol.
Bmax Bmax/KD LRR DA OCC Koff
(nM) (theory) (DVR) AMP min-1
putamen 27 900 22 19% 0.043
0.028
caudate 23 767 24 18% 0.043
0.029
v. 14 467 4 18% 0.030
striatum 0.025
0.026
thalamus 1.8 60 2 24% 0.056
0.035
0.054
amygdala 0.9 30 2 39% 0.042
0.036
0.052
Mukherjee 2005
Measuring occupancy
with Imaging
for a successful
treatment
Specific binding of [18F]Cyclofoxy was lower by 29 to
32% in Methadone Maintained Patients.
Normal
Control
Methadone
Maintained
Patient
Thalamus 32 ± 15
Amygdala 24 ± 30
Kling et al., J Pharm Exp Therap,
Caudate 24 ± 19
295: 1070-1076, 2000 Ant. cingulate cortex 29 ± 20
Measuring Occupancy
with Imaging
for Multi-target drugs
A single target drug with a multi-target
radiotracer.
A multi-target drug with a single target
radiotracer.
M100907 as measured using
[11C]NMSP PET in humans
Measure possible 5-HT2A receptor occupancy by
measuring frontal cortex to cerebellum ratio.
Measure possible D2 receptor occupancy by
measuring striatum to cerebellum ratio.
Is the Occupancy related to plasma
concentration?
Is the Occupancy time course related to plasma
concentration?
[11C]NMSP Binding at
5-HT2A & D2 receptors
Schizophrenia and Antipsychotic
Drugs
M100907 (aka MDL 100907) is a potent
and selective 5-HT2A receptor antagonist,
but does not bind to the D2 receptor.
Therefore, it has the profile of an atypical
antipsychotic agent.
[11C]NMSP can be used to monitor 5-HT2A
and D2 receptor density changes.
J Clin Pharmacol Suppl 1999
Sensitivity/Identifiability
for Drug Changes
Measuring endogenous
transmitter changes
Measuring increased
dopamine.
22.3% (±2.7) in
schizophrenia vs.
15.5% (±1.8) in
controls.
Schizophrenia is
associated with elevated
amphetamine-induced
synaptic dopamine.
Breier A, Su TP, Saunders R, Carson RE, Kolachana BS, de Bartolomeis A,
Weinberger DR, Weisenfeld N, Malhotra AK, Eckelman WC, Pickar D.
Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2569-74.
Measuring
increased Control Physostigmine
acetylcholine.
[18F]FP-TZTP as a
probe for AChE
inhibitors such as
donepezil,
rivastigmine, &
tacrine.
Increased ACh and
15% decrease of
[18F]FP-TZTP in
Ctx.
Carson RE, Kiesewetter DO, Jagoda E, Der MG, Herscovitch P, Eckelman
WC. Muscarinic with [18F]FP-TZTP: control and competition studies.
J Cereb Blood Flow Metab. 1998 Oct;18(10):1130-42.
Individualized
Medicine
Current Individualized Medicine
Metastatic pheochromocytoma (Pheo) can be detected using
[123I]MIBG (or it that is not available [131I]MIBG can be used)
prior to therapy with [131I]MIBG.
The mechanism of localization is based on the neuroendocrine
character of this disease with the the norepinephrine transporter
(NET) being the key biochemical parameter.
Up to 73% of PHEO cells in vitro express somatostatin receptors
so patients with Pheo have been assessed with somatostatin
receptor imaging (with either [123I]Tyr3-octreotide or
[111In]DTPA-octreotide).
Since the presence of NET and SSR appear to be inversely
related and dependent on cell differentiation, imaging both
pathways can be instrumental in choosing therapy.
Current Individualized Medicine
The American College of Radiology has recently set
practice guidelines for [90Y]ibritumomab tiuxetan
(Zevalin) and [131I]tositumomab (Bexxar), which are
approved by the FDA for radioimmunotherapy of
non-Hodgkin’s lymphoma.
Both antibodies are directed against the CD20
antigen, which is found on the surface of normal and
malignant B lymphocytes.
The preliminary imaging studies are to determine
dosimetry or assess biodistribution before the
radiotherapeutic is administered. The package insert
for these two radiotherapeutics has guidelines for
interpreting the imaging study and these guidelines
must be met before the therapy can commence.
Targeted Drugs
Targeted Imaging
Trastuzumab for HER2 (aka ErbB2 & Neu)
– A cell surface glycoprotein with TK activity
– HER2 amplification/over-expression is
predictive for response in breast caner.
– Overexpression became an entry criteria and
higher objective response was related to level of
overexpression.
Imaging study was developed with Ab
fragment to match the Ga-68 half life.
tra STUH zoo mab; Herceptin
Imaging HER2 Receptor in
response to HSp90 Inhibitors
17-allylaminogeldanamycin (17-AAG)
is the first Hsp90 inhibitor to be
tested in a clinical trial.
Induce proteasomal degradation of
HER2 by binding to Hsp90 chaperone
protein.
Label the F(ab’)2 of the anti-HER2
antibody Herceptin with Ga-68.
Smith-Jones et al. Nat Biotech 2004
MicroPET images (coronal) of mice with BT-
474 tumors with Ga-68-DOTAcHF at 3 h before
and 24 h after 17-AAG
Tumor & kidney
Taken from Smith-Jones et al. Imaging the pharmacodynamics of HER2
degradation in response to Hsp90 inhibitors. Nat Biotechnol. 2004;22:701-6
Targeted Drugs
Imatinib is an inhibitor of BCR-ABL TK.
The Philadelphia chromosome and
BCR-ABL have prognostic significance
for chronic myeloid leukemia (CML).
Also, inhibits TK of the oncogene c-KIT
in GIST.
Imatinib-resistant mutants led to BMS
354825.
im MA ta nib; Gleevec
In Vivo Proteomics:
FDG: Before and after Gleevec
Taken from Demetri et al.
Cancer is not a single gene disease,
yet …..
Imatinib (Gleevec)-effective in GIST and
CML. Mutants appeared, but further TK
inhibitors have high affinity for all
mutants.
Trastuzumab (Herceptin)-best in high
expressors of HER2.
Gefitinib (Iressa)-EGFR TK.
– Shrinks tumor, but no change in survival in
NSCLC. Population specific.
– Erlotinib (Tarceva) & Cetuximab (Erbitux)-MAb
Angiogenesis Inhibitors
DDT 2004:9:1042-1044 Golsteyn RM. DDT 2005 10(6):381.
Drivers for Targeted Imaging
Expansion of SPECT/CT complementing the
continued expansion of PET/CT.
Development of the parallel field of small
animal imaging.
The pharmaceutical company’s need to
increase their success rate from 17% for
established targets and 3% for post-
genomic targets.
The FDA’s need to encourage biomarker
development, especially for human use.
Magnitude of the opportunities
Failures in Phase II or Phase III are often due to
newly identified toxicity or absence of targeting.
2000 drugs have failed to target sufficiently and
are accumulating at a rate of 150-200 per year.
Percentage of success
Arth CV CNS Inf Oncol Eye Metab Uro Women ALL
Kola & Landis Nat Rev DD 2004:3:711-716
Efficient “Molecular Targeting”
Discovery & Development
Streamlining drug discovery: finding the
right drug against the right target to treat
the right disease.
For Targeted Imaging probes: finding the
right molecular probe against the right
target to monitor the right disease.
Molecular Probe Design
Develop Molecular Imaging Probes that target a
protein that changes early in the disease.
Develop molecular tracers that are based on a
reductionist concept where the drug-organism
interplay can be reduced to a drug-target interplay.
Collaborate with or join the Pharmaceutical
Industry
Opportunities
University Faculty
– Chemistry dept
– Pharmacology dept
– Radiology dept
Radiopharmaceutical companies
Pharmaceutical companies
– Drug development using imaging
Target-based drug discovery: Is
something wrong?
Physiologic targets
– For example, blood pressure measurements in vivo
using potential drugs, e.g., the ACE inhibitors.
Targeted Drugs
– Gene Targets, e.g., single gene disease
– Mechanistic Targets (receptors, enzymes)
Combitorial chemisty, HTS, rationale drug discovery
Underestimation of the complexity of the physiology
and lack of relevant disease model.
Targeted Imaging could play a major role.
Sams-Dodd, DDT 2005:10:139
Other Imaging Modalities for desialylated
glycoproteins binding to the high capacity (~500 nM)
hepatic binding protein
[Gd]DTPA-galactosyl-
Dextran
Mattrey, Hall
Post-injection: 3.8 min
Vera UCSD
Liver: 66 % Enhancement
Comparison of HER2 status between
primary tumor and disseminated tumor
cells in primary breast cancer patients.
RESULTS: In 46 of 137 (34%) breast cancer patients, CK-
positive cells were detectable in BM. DTC with HER2
positivity were found in 20 (43%) of these patients.
The HER2 expression on DTC was heterogeneous in 7 of 17
(41%) patients.
Concordance rate of HER2 status between primary tumor
and DTC was 62%. In 12 of 20 patients with HER2
negative tumors HER2 positive DTC were detected.
HER2 positive DTC can be detected in patients with HER2
negative primary tumors.
Solomayer EF et al. Breast Cancer Res Treat. 2006 Mar 22; [Epub ahead of print]
HER2-positive circulating tumor cells (CTC)
indicate poor clinical outcome in stage I to III
breast cancer patients.
We detected one to eight CTCs in the peripheral blood of 17 of 35
patients (48.6%) presenting no overt metastasis.
As a positive control, 7 of 7 (100%) patients with metastatic
disease presented positive.
The presence and frequency of HER2-positive CTCs correlated
with a significantly decreased disease-free survival (P < 0.005)
and overall survival (P < 0.05).
Interestingly, in 12 patients with HER2-positive CTCs, the
primary tumor was negative for HER2 as assessed by
immunohistochemical score and fluorescence in situ
hybridization.
This study provides some evidence of a prognostic effect of
HER2-positive CTCs in stage I to III breast cancer.
Wulfing P et al. Clin Cancer Res. 2006 Mar 15;12(6):1715-20.
Predictive Factors for Outcome in a Phase II
Study of Gefitinib in Second-Line Treatment
of Advanced Esophageal Cancer Patients.
Gefitinib has a modest activity in second-line
treatment of advanced esophageal cancer.
However, the patient outcome was significantly
better in female patients and in patients
demonstrating high EGFR expression or SCC
histology.
The selection of esophageal cancer patients for
future studies with EGFR-TKIs based on the
level of EGFR expression in their tumors or SCC
histology should be considered.
Janmaat ML et al. J Clin Oncol. 2006 Apr 1;24(10):1612-9.
What makes a probe a targeted
molecule?
Does the probe bind to the target?
– If there are a limited number of sites, increased
mass should decrease probe binding.
Is the delivery dependent on flow,
permeability, or protein concentration?
– What does flow dependence look like?
– What does permeability dependence look?
Is the probe sensitive to target change?
Epidermal growth factor receptor
inhibitors in cancer treatment.
The epidermal growth factor receptor (EGFR) is a cellular
transmembrane receptor with tyrosine kinase enzymatic activity
which plays a key role in human cancer. EGFR-dependent signaling is
involved in cancer cell proliferation, apoptosis, angiogenesis, invasion
and metastasis.
Cetuximab (Erbitux(R)), a chimeric human-mouse monoclonal
immunoglobin (Ig)G(1) antibody, which blocks ligand binding and
functional activation of the EGFR, is currently registered in the USA,
Switzerland and the European Union for the treatment of advanced,
irinotecan-refractory colorectal cancer. Gefitinib, (Iressa((R))), a small
molecule EGFR-selective inhibitor of tyrosine kinase activity which
blocks EGF autophosphorylation and activation, has been the first
EGFR-targeting drug to be registered in 28 countries worldwide,
including the USA, for the third-line treatment of chemoresistant non-
small cell lung cancer patients.
Ciardiello F. Future Oncol. 2005 Apr;1(2):221-234.
Antiangiogenic cancer therapies get their act together:
current developments and future prospects of growth
factor- and growth factor receptor-targeted approaches
The identification of surrogate markers that can
monitor the activity and efficacy of antiangiogenic
drugs in patients belongs to the most critical
challenges to exploit the full potential of
antiangiogenic therapies. The opportunities and
obstacles in further development of growth factor-
and growth factor receptor-targeted antiangiogenic
approaches for advanced cancer, including
malignant melanoma, will be discussed herein with
particular reference to selected ongoing clinical
trials.
Gille J. Exp Dermatol. 2006 Mar;15(3):175-86.
Triage for Breast Cancer
