Definitions: Molecular Imaging

1
1845-1923
2
High Spatial ResolutionCT, 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.

3
The Advantage of Radionuclides for Targeted
Imaging, especially Low Density Sites (lt20 nM)
4
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.

5

• 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.
6
Imaging the In Vivo Distribution of a Gamma
Emitting Radioisotope
7
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

8
1934 photo of Livingston and Lawrence with the
27 cyclotron at LBL
9
In 1938, Glenn Seaborg and Emilio Segre
discovered Technetium-99m.
10
Walter Tucker Powell Richards
11
Mechanism TcO4- stannous ion reduced Tc
chelating agent or particle final product
Kit components stannous salt (reducing agent)
chelating agent or particle
12
RS-123IIQNB on 5/11/83
13
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.

14
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!

15
DEVELOPMENT OF Enzyme/Receptor Targeting in humans

• DATE PET SPECT
• 1977 18FFDG
• 1983 11CN-MeSpip 123IIQNB
• 1984 18FCyclofoxy
• 1985 11CRaclopride 99mTcNGA
• 1985 11CCarfentanil
• 1985 11CFlumazenil

How many radiotracers have changed clinical
care? How many have been used in combination with
pharmaceuticals?
16
The Principle of PET Coincidence Detection of
Two 511 KeV Gamma Rays is Used to Determine the
Position
17
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. 0.5F2
• 18F 109.6 635 18O (p,n)
• 76Br 966 3980 75As (a,3n)
• 64Cu 762 571 64Ni (p,n)
• 124I 5976 2134 124Te (p,n)

18
FDG1976 to 2002
19
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.

20
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.
21
Other Imaging Modalities for desialylated
glycoproteins binding to the high capacity (500
nM) hepatic binding protein
GdDTPA-galactosyl-Dextran
Post-injection 3.8 min Liver 66 Enhancement
22
How can targeted imaging accelerate drug
discovery?

• William C Eckelman PhD
• Bethesda MD

23
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.

24
Targeting ProteinsThe 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 (LondonLewis,
  1908), Experimental Researches on Specific
  therapy. On immunity with special references to
  the relationship between distribution and action
  of antigens, 107.

25
The Magic Bullet

• Ehrlichs 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.

26
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.
• 18FFP-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
27
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)

28
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.
29
The Druggable genome
Nucl Horm R
Russ Lampel. The Druggable genone an update.
DDT 2005 23/24 1607.
30
Measuring Targeting with Imaging for targets of
differing density

• In Vitro B/F Bmax/Ki
• Imaging requires B/F ratio 5, Drugs do not

31
Measuring the In Vivo Binding Parameters of
18F-Fallypride in Monkeys Using a PET
Multiple-Injection Protocol.
Mukherjee 2005
32
Measuring occupancywith Imaging for a
successful treatment
33
Specific binding of 18FCyclofoxy was lower by
29 to 32 in Methadone Maintained Patients.
Normal Control
Methadone Maintained Patient
Thalamus 32   15 Amygdala 24 
 30 Caudate 24   19 Ant. cingulate cortex
29   20
Kling et al., J Pharm Exp Therap, 295
1070-1076, 2000
34
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.

35
M100907 as measured using 11CNMSP 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?

36
11CNMSP Binding at 5-HT2A D2 receptors
37
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.
• 11CNMSP can be used to monitor 5-HT2A and D2
  receptor density changes.

J Clin Pharmacol Suppl 1999
38
Sensitivity/Identifiabilityfor Drug Changes

• Measuring endogenous transmitter changes

39
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 1894(6)2569-74.
40
Measuring increased acetylcholine. 18FFP-TZTP
as a probe for AChE inhibitors such as donepezil,
rivastigmine, tacrine. Increased ACh and 15
decrease of 18FFP-TZTP in Ctx.
Control
Physostigmine
Carson RE, Kiesewetter DO, Jagoda E, Der MG,
Herscovitch P, Eckelman WC. Muscarinic with
18FFP-TZTP control and competition studies. J
Cereb Blood Flow Metab. 1998 Oct18(10)1130-42.
41
Individualized Medicine
42
Current Individualized Medicine

• Metastatic pheochromocytoma (Pheo) can be
  detected using 123IMIBG (or it that is not
  available 131IMIBG can be used) prior to
  therapy with 131IMIBG.
• 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 123ITyr3-octreotide or
  111InDTPA-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.

43
Current Individualized Medicine

• The American College of Radiology has recently
  set practice guidelines for 90Yibritumomab
  tiuxetan (Zevalin) and 131Itositumomab
  (Bexxar), which are approved by the FDA for
  radioimmunotherapy of non-Hodgkins 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.

44
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
45
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
46
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.
200422701-6
47
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48
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
49
In Vivo ProteomicsFDG Before and after Gleevec
Taken from Demetri et al.
50
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 200491042-1044
Golsteyn RM. DDT 2005 10(6)381.
51
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 companys need to increase
  their success rate from 17 for established
  targets and 3 for post-genomic targets.
• The FDAs need to encourage biomarker
  development, especially for human use.

52
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 20043711-716
53
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.

54
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

55
Opportunities

• University Faculty
• Chemistry dept
• Pharmacology dept
• Radiology dept
• Radiopharmaceutical companies
• Pharmaceutical companies
• Drug development using imaging

56
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57
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 200510139
58
Other Imaging Modalities for desialylated
glycoproteins binding to the high capacity (500
nM) hepatic binding protein
GdDTPA-galactosyl-Dextran
Mattrey, Hall Vera UCSD
Post-injection 3.8 min Liver 66 Enhancement
59
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
60
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 lt 0.005) and overall
  survival (P lt 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
1512(6)1715-20.
61
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
124(10)1612-9.
62
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?

63
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
Apr1(2)221-234.
64
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 Mar15(3)175-86.
65
Triage for Breast Cancer