The Future Trends in Molecular Imaggging

Dr. Juri Gelovani, MD , PhD . Professor of and Neurology

Chairman, Dept. Experimental Diagnostic Director, Center for Advanced Biomedical Imaging Research (CABIR) Molecular And Genetic Imaging of Cancer

El&AEarly & Accura tDite Diagnos is • To follow Screening (blood genomics and proteomics) • Develop Imaging of Biomarkers (tumor localization, sensitivity)

Tumor Phenotyping • Tumor Profiling (receptors & signal transduction, invasiveness, metabolism, proliferation, apoptosis, etc.) • Stroma Profiling (angiogenesis, tissue re-re-organization,organization, etc.)

Imaging for Selection & Monitoring of Therapy • Drug Target Expression & Activity • Dose Optimization • Early Response Evaluation (Rx adjustment) • Shor t-t<Pterm & Long Term Prognosi s • Monitoring Contained/Chronic Disease status • Early Detection of Relapse Image-Guided Biopsy, Surgery and Radiotherapy

Image-Guided Biopsy Image-Guided Radiotherapy Sentinel lymph node mapping in breast carcinoma patients Sentinel lymph node mapping in breast carcinoma patients Imaging of angiogenesis in breast tumor

SPECT imaging after injection of 99mTc-RGD peptide Potential for earlier assessment of response to chemotherapy • Avoid unnecessary cost and toxicity • Switch to alternative treatment sooner Approaches to Optical Imaging of Integrins in Neo-Angiogenesis

SO 3H C(Gf)Cyclo(RGDfK)-CCy5.5 SO 3H Our pipeline: •MMP9 HO3S SO 3H •EGFR N N •PDGFR

Gly • IL11Rα Arg Asp

Lys phe O

RGD + RGD-Cy5.5 Cy5. 5 RGD-C55Cy5.5 1 hr interval

6l/6 nmol/mouse 600 nmol + 6 nmol 30 nmol Structure of 111In-DTPA-K(IRDye800)-c(KRGDf).

Gly Arg Asp O IRdye800 NH

NH HN N H O O

HN O

H N

O

HOOC COOH NNN HOOC COOH COOH Gamma and near-infrared imaging of 111In-DTPA-K(IRDye800)-c(KRGDf) in nude mice bearing s.c. human melanoma.

M21 tumor is ppgositive in integrin αVβ3 expp,ression, while M21-L tumor is negative in αVβ3 expression. Selective accumulation of 111In-DTPA-K(IRDye800)-c(KRGDf) in M21 melanoma tumors that express integrin αVβ3.

3 P=0.0082

2

Non-blocked P=0.54 Blocked 1

0 M21 M21-L

Accumulation of the radiotracer in M21 tumor can be blocked by the parent RGD peptide. On the other hand, preinjection of the parent RGD peptide do not cause significant reduction in the uptake of 111In- DTPA-K(IRDye800)-c(KRGDf) in αvβ3-negative M21 -L tumors. Development of NIR Imaging Probe for Clinical Applications: RGD Conjugates with TS -ICG Dyes

- SO3 SO3

- SO3 - TS-ICG -c(KRGDf) SO3 + N N Et (CH2)5

CONH-c(KRGDf) - - SO3 SO3

- SO3 - TS-ICG-c(KRGDf) SO3 2 + N N

(CH2)5 (CH2)5

CONH-c(KRGDf) CONH-c(KRGDf)

Structures of conjugates of TS-ICG containing one and two molecules of c(KRGDf) peptide. Ex/Em: 765/796 nm NIR Imaging using Monomeric and Dimeric RGD Conj ugates

TS-ICG-(KRGDf)2 Chemical structure of L-PG-NIR813 or D-PG-NIR813. L- and D- PG-NIR813 differ in their stereoisomeric carbon center (*).

Michaelis-Menten constant s (()Km) of the reaction of CB on L-PG-NIR813 with L-PG molecular weight of 17 KD (A) and 56 KD (B). Dose-dependent inhibition of CB-mediated degradation of L-PG-NIR813 by specific CB inhibitor (Inhibitor II).

DdtiDegradation of PG-NIR813 (8.3% loading) by U87 cells culture for 24 hr (1) and 0 hr (2). Culture medium without cells was used as a control (3). In vivo imaging after intravenous injection of L-PG-NIR813 into U87(TGL) nude mice.

NIRF Images were acquired at 24 hr after i.v. injection of PG-NIR813 (50 nmol/mouse, 17 KD, 8.3 % loading). Note the mouse injection with L-PG-NIR813, but not the mouse injected with D-PG-NIR813, exhibits strong NIRF signal. Co-localization of PG-GdDTPA-NIR813 with isosulfan blue (A-D).

Microphotographs of representative resected lymph node confirming the uptake of PG-GdDTPA-NIR813 in the LN (E-H). E: H&E stained tissue section. F: DIC image. G: NIRF image. H: OlOverlay of the DIC and NIRF images. NIRF silignal is pseudocolored green, and DIC pseudocolored red. Original magnification: 50x. Dual MR/optical imaging of the axial and branchial lymph nodes in normal athymic nude mice.

(A) Pre-contrast overlay of white light and NIRF images. (B) Overlay of white light and NIRF images 1 hr after injection of PG-GdDTPA-NIR813 (0.002 mM/L Gd/kg). (C) NIRF image of the same mouse without skin. (D) Resected lymph nodes showed bright signal. E-F: Representative T1-weighted axial MR images at different times. PG- GdDTPA-NIR813 was injected at a dose of 0.02 mmol Gd/kg (E) and 0.002 mmol Gd/kg (F). The arrows indicate SLN. Visualization of cervical lymph nodes after interstitial injection of PG-GdDTPA-NIR813

PG-GdDTPA-NIR813 (0.02 mmol Gd/kg) was injected into the tongue of a normal mouse (A-D) and a mouse with a human DM14 oral squamous carcinoma tumor grown in the tongue (E-H). H&E stained lymph node sections (I-K) indicating the presence of micrometastases (J) and presumably in-transit metastases in lymphatic duct (K). Note the difference in the pattern of lymph node enhancement between normal (A) and metastatic (E) nodes in T1- weighted MRI. Detection of Tumors by Molecular Imaging of Metabolic Shifts Comparative PET Imaging of Prostate Carcinomas with 18FF--FDGFDG and 11CC--AcAc

PET images of prostate, lymph node, and bone metastases obtained using 18F- FDG and 11C-Ac from 73-y-old man with poorly differentiated (Gleason sum 7) adenocarcinoma of prostate. (A) 18F-FDG PET shows low uptake in prostate, with SUV of 2.87. (B and C) 18F-FDG uptake in left iliac lymph node metastatic lesion (B) and right pubic bone metastatic lesion (C). (D–F) 11C-Acetate PET shows high uptake in prostate(D),withSUVof5.45;inleft iliac lymph node metastatic lesion (E); and in right pubic bone metastatic lesion (F). bn = bone; ly = lymph node; pr = prostate. Oyama, et al. J Nucl Med (2002) Metabolism of Fluoro-Acetate

Aconitase

Glycolysis FDG Fatty Acid Synthesis F-Acetate In vitro 3HH--AcetateAcetate and 14CC--FluoroacetateFluoroacetate DualDual--LabelLabel Radiotracer Accumulation Studies in Breast Carcinoma Cell Lines

SKBr3 MB231

6.0 6.0

3H-Acetic Acid 5.0 3H-Acetic Acid 5.0

4.0 14C-F-Acetic Acid 4.0 14C-F-Acetic Acid

3.0 3.0 Linear (3H-Acetic Linear (3H-Acetic

Acid) Ratio C/M Acid) ell/Medium 202.0 2.0 C Linear (14C-F- Linear (14C-F- Acetic Acid) Acetic Acid) 1.0 1.0

0.0 0.0 0 50 100 150 200 0 50 100 150 200

Time Time

SKOV3

6.0

5.0 3H-Acetic Acid

14C-F-Acetic Acid 4.0

3.0 Linear (3H-Acetic

C/M/Ratio Acid) 2.0 Linear (14C-F- 1.0 Acetic Acid) 0.0 0 50 100 150 200 Time 18F-Fluoroacetate in a Multi-Tumor Model PC3 SKBr3

#1

U87 MB435

#2

15 min #3

40 min 60 min 120 min Kinetics of [18F]-FAc derived radioactivity accumulation (tumor/muscle ratio) in different tumors

Accumulation of F-18 fluoroacetate in mice bearing human carcinoma xenografts 10

PC3 SKBr3 U87 MB435 og (Ratios) LL

1 15 40 60 120 Time (min) STAGES OF IMAGING AGENT DEVELOPMENT Dynamic PET/CT Imaging of [18F]-FA

Mouse Macaque Kinetics of Biodistribution of 18F-FAc in Monkeys

1.000%

Blood Liver Kidney 0.100% Lung Bone Muscle %ID/g

0.010%

0.001% Time (min) 0 20 40 60 80 100 120 140 160 180 200 Analysis of Blood Plasma Metabolites

1.000%

0.100% Whole Blood /g

DD F18FAF-18 FA %I F- 0.010%

0.001%

0.060% 0.000% Whole Blood 0.050% 0 50 100 150 200 F-18 FA %ID/g Time (min) 0.040% F-

0.030%

0.020%

0.010%

0.000% 0 50 100 150 200 Time ( (mi in) ) Pharmacological Dose Estimates for [18F]Fluoroacetate

Calculated Specific Activity and Mass. The total mass per dose and specific activity of 18F-fluoroacetate prepared via no-carrier added synthesis were calculated as follows:

Avogadro's Number 6.02E+23 1Ci in Bq 370E+103.70E+10 Lambda (decay constant) 0.000105022

Molecular weight of the compound 78 Isotope T1/2 (sec) 6600 Dose in mCi 10

N of atoms per dose 3.52306E+12 Moles per dose 5.85E-12 Total weight per dose (g) 4.56E-10

Calculated Spp(.Act. (Ci/mol ) 1.71E+09

The fluoroacetate used for imaging may have a mass of 0.456 ng which is about 1/1000 of fluoroacetate that may be present in a cup of tea. Phase I study using PET/CT with 18F18F--FAcetateFAcetate in Tumors that don’t avidly accumulate FDG

• Prostate carconoma, lymph node mets • Breast carcinoma, lobular, lymph node mets • Brain tumors, GBM

30 patients (10 patients per tumor type)

Primary objectives: Pharmacokinetics Radiolabeled metabolites Biodistribution Radiation dosimetry Dose optimization

Secondary objectives: Feasibility of tumor detection as compared to CT and MRI or biopsy TUMOR DETECTION:

NEW IMAGING AGENTS

FOR

STROMAL BIOMARKERS Pancreatic Carcinoma: Problems in Diagnosis & Therapy

• Pancreatic cancer patients seldom exhibit diseasedisease--specificspecific symptoms until late in the course of the disease process

• Despite significant advances in the treatment of many other human tumors, the 55--yearyear survival rate in pancreatic cancer remains <<55%%..

• At present, no imaging modality is sensitive for visualization of early stage pancreatic carcinomas or small metastases in the lymph nodes, peritoneum, and on the surface or within the liver. liver.

• Novel biomarker screening and diagnostic imaging methods for potential earlier detection and localization of pancreatic carcinomas must be developed in conjunction with new image-guided therapies that would eradicate early neoplastic lesions if survival from this disease is to be improved. improved. Current understanding of the molecular changes in the multi-step ppgrogression model of pancreatic adenocarcinoma Molecular Imaging of Stromal Biomarkers: Volume Amplification Approach

1 mm 5 mm

Intraepithelial Neoplasia Peritumoral Tissue Normal Tissue Gene Expression in parenchyma adjacent to infiltrating pancreatic ductal adenocarcinoma and chronic pancreatitis (Fukushima, et al. Mod Pathol 18, 779-787, 2005) Synthesis of [18F]-Lactose Synthesis of a precursor Radiolabeling of [18F]-Lactose

HO OH 1) Ac O, NaOAc 1) NH , MeOH AcO OAc 2 AcO OAc 3 OH AcO OAc O 120 deg, 2h rt, overnight PMP O OTf BzO HO O O BzO 18 O O AcO O O O Bu4N F O HO O AcO OEt 2) PhSH, BF -Et O AcO 2) 4-MeO-PhCH(OMe)2 HO BzO 3 2 AcO BzO OEt OAc 18F OH rt, 5 h OAc SPh p-TsOH, 60 deg, 3h SPh 80¡ãC, 15m 1 23 13 16

OBn nBu2SnO PMP O BSP, TTBP OH OH NaH, BnBr O 3 A MS, -60 deg HO reflux, 3h PMP O O NaOMe O HO O PMBO O O reflux, 2h Tf2O, EtOH O Bu4NI, PMBCl PMBO SPh HO HO OEt reflux 2h 80¡ãC, 5m OH 18F SPh 5 4 17

OBn nBu SnO OBn Co-injection of purified 16 with cold version of 14 PMP O OBn 2 PMBO reflux, 3h O O O Ac2O, 1h HO HO O ADC1 A, ADC1 CHANNEL A (LG\LG000009. D) OEt HO OEt mAU PMBO OEt PMBO PMBO Bu4NI, PMBCl 1600 1400 reflux 2h 8 11.206 6 1200 7 1000

800

600

400 AcO OAc 200 OAc 6.072 9 AcO OAc 0 O AcO 2 4 6 8 10 12 14 16 min SEt OBn DAD1 A, Sig=254,4 Ref=360,100 (LG\LG000009.D) AcO PMBO OBn mAU 1) DDQ, rt, 1h 10.988 OAc O O O BzO -8 O O O AcO OEt OEt -8.5 OAcPMBO 2) BzCl, Py,5h AcO BzO NIS, TfOH OAc -9 10 11 -9.5

-10

-10.5

2 4 6 8 10 12 14 16 min AcO OAc OH AcO OAc BzO OTf H2, 10% Pd/C O O BzO O Tf2O, Py O O AcO OEt O QC Chromatogram of compound 17 BzO OEt rt, 12 h OAc AcO BzO ADC1 A, ADC1 CHANNEL A (LG\LG000009.D) rt, 3h OAc mAU 12 1600 1400 13 11.206

1200

1000

800 OAc 600 AcO 400 Bu NF HO OH 4 200

BzO NaOMe 6.072 O 0 O HO 2 4 6 8 10 12 14 16 min O O O DAD1 A, Sig=254,4 Ref=360,100 (LG\LG000009.D) 80¡ãC, 15m AcO BzO OEt O mAU OAc F Reflux, 20m HO HO OEt 1600 OH F 1400 14 1200

15 1000

800

600

400

200

0 10.988 2 4 6 8 10 12 14 16 min PET imaging of pancreatic carcinoma

Axial Coronal Saggittal

PET imaging of mice bearing orthotopic Tumor Blood MPanc96 human pancreatic cacinoma Muscle xenografts dttddemonstrated high er rettitention of [18F]FDL (100 uCi i.v.) in the tumor- invaded pancreas, which corresponded with high levels of HIP/PAP protein expression in the peritumoral pancreatic tissue, as evidenced by comparative immunohistochemical analysis in situ. The [18F]FDL was eliminated via the renal clearance. PAP immunostain provided by Time-activity curves Dr. Craig Logsdon, MDACC Molecular Imaging of Drug Target Expression-Activity: Selection of Therapy Current Diagnostic – Therapeutic Paradigm

24 - 48 hours PET/CT 2 months

Therapy “A” X

FDG, FLT

Therapy “B” Mechanisms of selectivity of EGFR kinase imaging agent

Inactive Enzyme Active Enzyme

PO4 Cys773 Cys773 Molecular Imaging Agent for Imaging EGFR kinase expression/activity

NonNon--invasiveinvasive molecular imaging of EGFR expressionexpression--activityactivity with 124II--mIPQAmIPQAPET/CT in orthotopic U87 glioma xenografts in mice

uPET/uCT uPET

uCT

U87 glioma Brain Heart (blood) Non-invasive molecular imaging of EGFR expression-activity with 124I-mIPQA PET/CT in intracerebral U87 glioma xenografts in mice Wild-type EGFR + EGFRvIII mutant

U87 glioma U87EGFRvIII glioma Brain Brain Heart (blood) Heart (blood)

Tumor EGFR Tumor EGFR k1 k3 k1 k3 Blood Blood k2 k4 k2 k4 NSCLCs - GL w ith Iressa

3 PC- 14GL H-441GL 2 H 3255GL

-1 H-1975GL TT WS 1

0 -2 -1 0 1 2 3 Iressa μM (log scale)

PC-14GL H-441GL H 3255GL H-1975GL EC50 45.84 46.19 1.598 25.09 Peg6 IPQA 450 H441GL 400 H3255GL HN I 350 PC14GL H 300 H1975GL N N F 250 H441GL with Iressa Medium // 200 H3255GL with Iressa O 150 PC14GL with Iressa N O Cells 100 H1975GL with Iressa 50 0 6 HO 0 25 50 75 100 125 150 Time (min)

In-Vitro Uptake and Washout Study of I-Peg6-IPQA in NSCLCs Uptake Study of IPQA in NSCLCs H441GL H3255GL 20 PC14GL 400 H441 H1975GL H3255 H441GL-washout 15 H3255-washout PC14 PC14GL-washout 300 H1975GL-washout dium H1975 ee 10

200

Cells/M 5

0 100 0 30 60 90 120 150 Time (min) 0 0306090120 Time (min) H441GL %ID/g H3255GL 5.0

%ID/g 5.0

Axial

0

%ID/g 1.0

0 ClCoronal 0 Baseline

%ID/g 5.0

%ID/g 5.0

Axial

0

%ID/g 1.0

0 Coronal 0 Inhibitor: gefitinib, 100mg/kg,i.p. With Inhibitor 3h 1h prior to the radiotracer H441GL %ID/g %ID/g H3255GL 5.0 1.0

%ID/g 1.0

Axial Axial

0

%ID/g 1.0

0 ClCoronal ClCoronal 0 0 Baseline

%ID/g %ID/g 5.0 1.0

%ID/g 1.0

Axial Axial

0

%ID/g 1.0

0 Coronal 0 Coronal 0 Inhibitor: gefitinib, 100mg/kg,i.p. With Inhibitor 3h 24h 1h prior to the radiotracer A Paradigm for Therapy Selection and Monitoring by Imaging (Part II)

After 1 cycle (or @ 2424--72h)72h)

ChemoChemo-- FDG ChemoChemo-- Radiation FDG Radiation FLT FLT EGFR kk--RasRas EGFR kk--RasRas kk--RasRas mutant inhibitor inhibitor PET/CT

Key Imaging Targets: EGFR, DNA synth.

EGFR mutant FDG FDG EGFR Inhibitor EGFR Inhibitor FLT FLT EGFR EGFR Integration of Molecular Imaging, Interventional Radiology, and Tumor Biomarkers

HISTOPATHOLOGY Immunohistochemistry

PET/CT Imaging Tumor Lysate PK/PD Modeling Parametric Image-Image-GuidedGuided Arrays Parametric Imaging Biopsy (or Intraoperative)

microPET/CT Imaging Drug Target

GENOMICS

Radiolabeling Structural Biochemistry

In Silico Modeling Cyclotron Radionuclides

Radiotracer New Precursors HT/HC Screning