ANGIOGENESIS

IMAGING METHODS

성균관의대 핵의학교실이 경한

AREAS OF DISCUSSION

• What is Angiogenesis ?

• Why Angiogenesis Imaging ?

• Current Methods are Available ?

• Prospects of Future Techniques

WHAT IS ANGIOGENESIS ?

ANGIOGENESIS

“The growth of new blood vessels”

An important natural process occurring in the body, both in health and in disease.

 Occurs in healing wounds and for restoring blood flow to tissues after injury.

The healthy body controls angiogenesis through a series of "on" and "off" switches: - “on” switch: angiogenesis growth factors - “off” switch: angiogenesis inhibitors  

Occurs in cancer, DM blindness, macular degen, RA, psoriasis, …

Occurs when ds. cells produce abnormal amounts of angiogenic GFs, overwhelming the effects of natural angiogenesis inhibitors

Feed ds. tissues, destroy normal tissues, and allow tm metastasis

Excessive Angiogenesis

Occurs in coronary artery disease, stroke, and delayed wound healing

When the tissue cannot produce adequate amounts of angiogenic GFs

Lead to to improper circulation and tissue death

Insufficient Angiogenesis

• > 19 known angiogenic growth factors

• > 5 angiogenic GF being tested in humans to heal wounds

• > 30 known natural angiogenesis inhibitors found in the body.

• > 300 angiogenesis inhibitors have been discovered to date.

• > 184 million could benefit from antiangiogenic therapy

• > 314 million would benefit from pro-angiogenesis therapy

• > 6,500 cancer pts have been treated with antiangiogenic therapy

• > 1,000 heart ds. pts received experimental angiogenic therapy

• > $4 billion invested in R&D angiogenesis-based medicines  

Angiogenesis Facts

• Tm interaction with vasculature switch to angiogenic phenotype, enabling tumor progression

Angiogenic Cascade

- Endothelial receptor binding / activation

- Formation of angiogenic mother vessels

- Morphogenesis of mother vessels

- Basement membrane dissolution

- Endothelial cell proliferation

- Endothelial cell migration

- Vascular tube formation

- Arterial-venous differentiation

- Vascular stabilization

Angiogenin Angiopoietin-1 Del-1 Fibroblast growth factors Follistatin Granulocyte colony-stimulating factor (G-CSF) Hepatocyte growth factor /scatter factor Interleukin-8 (IL-8) Leptin Midkine Placental growth factor Platelet-derived endothelial cell GF Platelet-derived growth factor-BB (PDGF-BB) Pleiotrophin (PTN) Proliferin Transforming growth factor-alpha (TGF-alpha) Transforming growth factor-beta (TGF-beta) Tumor necrosis factor-alpha (TNF-alpha) VEGF / vascular permeability factor  

Known Angiogenic Growth Factors

WHY ANGIOGENESIS IMAGING ?

Animal studies do not directly translate to human studies

Host responder characteristics remain poorly understood

Combination therapy may enhance clinical outcome

Conventional oncology trial strategies require modification

Require new standards rather than change in tumor mass for

monitoring thx response

Lessons from Early Clinical Trials

Experts from imaging modalities: US, CT, MRI, PET/Scan

Controversy over the adequacy of present anatomical imaging meas

urements and definition of response criteria

Challenges from growing number of antiangiogenesis clinical trials

Interest in imaging techniques that can provide an early indicator of

effectiveness at a functional or molecular level has increased

Workshop on Angiogenesis Imaging Methodology

Sponsored by the Biomedical Imaging Program of the NCI, 2000

Gastric carcinoma. Erenoglu C, Dig Surg. 2000

Esophageal cancer. Millikan KW, Am Surg. 2000

Node-positive Breast cancer. Viens P, Breast Cancer Res Treat. 1999

Tumour angiogenesis and prognosis. Morgan KG, Histopathology. 1998

Tumor angiogenesis in prognosis. Fox SB, Invest New Drugs. 1997

Breast cancer. Goulding H, Hum Pathol. 1995

Bladder cancer. Bochner BH, J Natl Cancer Inst. 1995

Squamous cell carcinoma. Zatterstrom UK, Head Neck. 1995

Bladder carcinomas. Dickinson AJ, Br J Urol. 1994

Prognostic Value of Tumor Angiogenesis Assessment

PRESENT METHODS

Present Gold Standard

Immunohistochemistry

Anti-CD31

Invasive

Repeated exam difficult

Large variation

Sampling error

Performed with contrast agents to define the intravascular compartment, including blood flow, blood volume, transit time, and capillary permeability.

Functional CT techniques can delineate increases in tissue perfusion

CT Methods

Can identify vascular features at resolution of 50-200 um vessels

Contrast-enhancement using an intravascular agent can generate an index of blood flow, blood volume, or vascularity within tumor

Targeted imaging using ultrasound destruction of microbubbles may provide even further resolution of the tumor vascular tree.

Ultrasound Methods

Can define blood volume and permeability using dynamic enhancement of blood pool contrast agents.

Gadolinium-DTPA can distinguish between normal versus malignant leaky tissues, reflecting the hyperpermeable tumor vasculature.

Contrast uptake also correlates with microvessel density in experimental tumors.

MRI Methods

SPECT METHODS

Gene Therapy with VEGF for Inoperable CAD

JM Isner, The Annals of Thoracic Surgery, 1999

Gene Therapy for Myocardial Angiogenesis. Initial Clinical Results

with Direct Myocardial Injection of phVEGF165 as Sole Therapy

Stress

Rest

Pre Post

JM Isner, Circulation 1998

Catheter-Based Myocardial Gene Transfer for Angiogenesis

JM Isner, Circulation 2001

Evaluates tumor metabolism, as well as blood flow and volume.

H2015, 11CO, and 18FDG, characterizes neoplastic tissue

Antiangiogenic agents should diminish blood flow and decrease tum

or metabolism

Radiolabeled fluoromisonidazole (FMISO) has been used to quantita

te hypoxia in the rat glioma by PET and may provide functional info

rmation about the results of antiangiogenic therapy.

PET METHODS

MD Anderson, SNM 2001

Future Prospects For Nuclear Imaging Methods

Measure vascularity and its change with high SN and SP

The small size of microvessels precludes direct visualization by conventional angiography

Cases often have late-stage disease and a heavy tumor burden with an extensive established vascular supply: imaging must accurately quantify small changes against a potentially large signal background

As antiangiogenic therapy may require lifelong treatment, a non-invasive and costeffective technique would be highly desirable.

Requirements of Angiogenesis Imaging Methods

Effect of anti-angiogenic drugs on parameters measured by nuclear imaging has not been evaluated

Techniques currently being used in ongoing clinical trials of anti-angiogenic drugs be studied in animal models to evaluate the changes induced by anti-angiogenic therapy.

New approaches include integrins, annexin V, hypoxia agents, proliferative indices, and various receptor ligands

Guidelines for Nuclear Imaging Developement

[18F]Galacto-RGDIntegrin v3

The v3 integrin is expressed on newly formed endothelial cells and is thought to anchor the new blood vessel in the tumoral stroma

Extracellular matrix proteins interact to v3 integrin via RGD sequence

Integrin Imaging Strategies

Osteosarcoma (ß3 positive)

 2.58 ± 0.410.13 ± 0.03

 5.47 ± 0.111.97 ± 0.27

 8.67 ± 0.522.21 ± 0.46

 0.80 ± 0.130.28 ± 0.02

 2.88 ± 0.321.68 ± 0.49

 3.43 ± 0.240.72 ± 0.15

 3.77 ± 0.16

Time (min)

Blood 10 120

Liver 10 120

Kidneys 10 120

Muscle 10 120

Tumor 10 120

Lung 10 120

Colon 10 120 1.59 ± 0.46

Melanoma (ß3 positive)

 2.93 ± 0.530.05 ± 0.01

 5.64 ± 2.271.25 ± 0.05

 8.48 ± 0.841.52 ± 0.10

 1.13 ± 0.180.15 ± 0.02

 3.90 ± 1.361.49 ± 0.10

 3.64 ± 0.520.53 ± 0.05

 1.74 ± 0.553.53 ± 0.44

Melanoma (negative)

 4.70a

0.13 ± 0.07 

6.401.79 ± 0.58

 10.82

1.86 ± 0.46 

1.440.18 ± 0.03

 1.88

0.44 ± 0.24 

4.840.71 ± 0.24

 1.68

0.76 ± 0.27

Biodistribution in Melanoma M21() and M21-L() Bearing Mice

Biodistribution in Tumor Bearing Mice

Dose-dependent blockade of uptake by selective pentapeptide

v

[18F]Galacto-RGD PET of Melanoma Bearing Mice

Haubner R, Cancer Res 2001

SYNTHESIS OF 18F-FLUOROPROPYLSQUALAMINE AS ANGIOGENESIS IMAGING AGENT

Squalamine inhibits angiogenesis and solid tumor growth in vivo

N-fluoropropylsqualamine has similar activities as squalamine

F-18 N-fluoropropylsqualamine was synthesized in 4-7% yield

C-Y Shiue*, Univ. Penn and Magainin Pharmaceuticals Inc. SNM meeting, 2001

Effect of anti-angiogenic drugs on parameters measured by nuclear imaging has not been evaluated

Techniques currently being used in ongoing clinical trials of anti-angiogenic drugs be studied in animal models to evaluate the changes induced by anti-angiogenic therapy.

New approaches include integrins, annexin V, hypoxia agents, proliferative indices, and various receptor ligands

Anti-angiogenic drugs themselves be radiolabeled to directly study the pharmacokinetics of the drug.

Guidelines for Nuclear Imaging Developement

Growth factor antagonists - Inhibition of angiogenic factor production

- Anti-growth factor ribozymes

- Soluble growth factor receptors

- MoAb against angiogenic factors

Endothelial signal transduction inhibition - Receptor tyrosine kinase inhibition

- Protein kinase C inhibition

Inhibitors of endothelial cell proliferation - Cell-cycle inhibitors

Therapeutic Targets in Tumor Angiogenesis

Matrix metalloproteinases inhibition - Selective inhibitors of MMP-2, MMP-9

- Non-selective MMP inhibition

Endothelial surface marker targeting - Anti-integrin antibodies or cyclic peptides

Endothelial cell subpopulation inhibitors - Suppression of endothelial progenitor cells

Endothelial cell destruction - Vascular targeting agents

Known Angiogenesis Inhibitors

Antiangiogenic antithrombin III

Cartilage-derived inhibitor (CDI)

CD59 complement fragment

Platelet factor-4 (PF4)

Prolactin 16kD fragment

Proliferin-related protein (PRP)

Fibronectin fragment

Gro-beta

Heparinases

Heparin hexasaccharide fragment

hCG

Interferon alpha/beta/gamma

Interferon inducible protein (IP-10)

Interleukin-12

Angiostatin (plasminogen fragment)

Kringle 5 (plasminogen fragment)

Endostatin (collagen XVIII fragment)

Metalloproteinase inhibitors

2-Methoxyestradiol

Placental ribonuclease inhibitor

Plasminogen activator inhibitor

Retinoids

Tetrahydrocortisol-S

Thrombospondin-1 (TSP-1)

TGF-b

Vasculostatin

Vasostatin (calreticulin fragment)

01000200030004000500060007000

0 50 100 150Time (min)

CP

M/u

g/m

l pro

tein

3 uCi

6 uCi

123I-Taxol for Angiogenesis Imaging

YS Choe, 1999

Targeted Angiogenesis Tumor Vascular Imaging With Radiolabeled Endostatin

131I-labeled endostatin and 99mTc-labeled endostatin

In tumor-bearing rats, Tm/tissue count ratios incr. with time

Tumor %ID/g was 0.2-0.5 for 99mTc and 0.2-1.2 for 131I

Images visualized tumor clearly with radiolabeled endostatin 99mTc-EC-endostatin could assess treatment response

D. J. Yang,. MD Anderson and EntreMed SNM meeting, 2001

ANGIOSTATIN

Angiostatin is a proteolytic fragment of plasminogen A potent inhibitor of angiogenesis and tumor growth

50 kD

123I-Angiostatin Synthesis

0

50

100

150

200

250

300

0 5 10 15 20

Elu

ted

Act

ivit

y (

Ci)

Time (tube number)

KH Lee, 2001

Intravascular Activity of 123I-Angiostatin

Activity in serumActivity in clot

01020304050607080

5 min 10 min 20 min

% to

tal a

ctiv

ity

Blood Clearance

0

20

40

60

80

100

0 40 80 120 160 200

minutes

(%)

KH Lee, 2001

Tumor Uptake in Colon Cancer Bearing Mice

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Blood Heart Lung Liver Spleen Pancr. Tumor muscle Kidney

KH Lee, 2001

Sequential Tumor Uptake Ratio in Image ROIs

0.0

1.0

2.0

3.0

4.0

5.0

6.0

20 30 40 50 60 70 80 90 100 110 120

Tumor/ContralateralTumor/LungTumor/LiverTumor/Heart

Minutes

Cou

nt R

atio

KH Lee, 2001

Rapidly accumulating knowledge of tumor angiogenesis is providing critical insights into new opportunities for imaging

Angiogenesis imaging is critical for optimizing antiang. therapy

Conventional techniques may be adapted to measure blood flow, blood volume, permeability, microvessel density, and tissue metabolism

Future approaches for imaging angiogenesis per se will likely exploit the molecular features of new blood vessel growth.

Novel imaging targets include cell surface integrins, endothelial apoptosis, angiopoietins and other signatures of angiogenesis.

These new modalities will help create a platform for bringing antiangiogenic cancer therapy into standard oncology practice

SUMMARY

FUTURE DIRECTIONS

Many questions remain about the angiogenic process and how it is regulated. And antiangiogenic imaging methods now in development face uncertainties of efficiency

Despite the obstacles, angiogenesis imaging offer the promise of an additional diagnostic modality for our current armamentarium.

Angiogenesis imaging may turn out to have significant benefits because they target easily accessable cells and are unaffected by resistance.

These imaging methods may also be used to evaluate other diseases characterized by abnormal angiogenesis, such as ischemic disease, arthritis, and benign tumors.

Clearly, then, antiangiogenic drugs have exciting potential as therapies for a number of serious conditions-in addition to cancer.

TUMOR TARGETING WITH RADIOLABELED INTEGRIN aVb 3 BINDING RGD PEPTIDES IN A NUDE MOUSE TUMOR MODEL

• 111In and 99mTc-labeled RGD peptide in ovarian cancer bearing BALB/c mice

• Maximal tumor uptake of 7.5 %ID/g of 111In-labeled peptide at 2 hrs pi.

• Tumors were clearly visualized by gamma camera scintigraphy.

• Tumor growth was significantly delayed after injection of 90Y-RGD peptide

• RGD-peptide labeled with either 111In or 99mTc specifically localizes in human tumor xenografts and in various normal tissues in nude mice. Labeled with 90

Y this peptide has potential for peptide receptor radionuclide therapy.

M. Janssen, Dupont Pharmaceuticals

a) Specific inhibitors of angiogenic growth factors

- Angiozyme (Ribozyme Pharmaceuticals)

- Avicine (AVI Biopharma)

- Suramin (NCI)

- rhu MabVEGF (Genentech)

b) Inhibitors of growth factor-receptor binding

- IMC-1C11 (ImClone)

- IM862 (Cytran)

- PI-88 (Progen Industries)

c) Specific tyrosine kinase inhibitors

- PTK787 (Novartis)

- SU5416 (SUGEN)

- SU6668 (SUGEN)

True Angiogenesis Inhibitors

d) Anti-endothelial proliferative agents

- TNP-470 (TAP Pharmaceuticals)

e) Anti-integrin agents

- EMD121974 (Merck KgaA)

- Vitaxin MedImmune

f) Inhibitors of angiogenic factor production

- Octreotide (Novartis)

g) Upregulators of angiogenesis inhibitors

- ImmTher (Endorex)

h) Unknown mechanism

- Angiostatin (EntreMed)

- Endostatin (EntreMed)

Nonselective Antiangiogenic Agents

a) Low-dose cytotoxic chemotherapy drugs - Cyclophosphamide, 5-Fluorouracil - Methotrexate, Vinblastineb) Matrix metalloproteinase inhibitors - BMS275291 (Bristol-Myers Squibb) - Captopril (Bristol-Myers Squibb) - Col-3 (CollaGenex) - Marimastat (British Biotech) - Neovastat (Aeterna Laboratories) - Prinomastat (Agouron Pharmaceuticals) - Solimastat (British Biotech)c) Anti-cytokine agents - Thalidomide (Celgene Corp.) - CC 4047 (Celgene Corp.) - CC 5013 (Celgene Corp.) - CC 7085 (Celgene Corp.) - CDC801 (Celgene Corp.)d) Cox-2 inhibitors - Celecoxib (GD Searle)e) Anti-tubulin agents - Paclitaxel (Angiotech)

Vascular Targeting Agents

a) Anti-tubulin agents - Combretastatin A4 Prodrug (OXiGENE)b) Ion transport inhibitors - Squalamine (againin Pharmaceuticals)c) Receptor-driven inducers of endothelial apoptosis - CM101 (arboMed)

f) Cell locomotion inhibitors - Interferon alfa2a (Hoffman-LaRoche)g) Ion flux inhibitors - Carboxyamidotriazole (NCI)h) Anti-mitochondrial agents - Apra (Cell Therapeutics)i) Nonspecific tyrosine kinase inhibitors - Flavopiridol (NCI) - Genistein (Amino A)j) Copper-lowering agents - D-Penicillamine (NCI) - Tetrathiomolybdate (University of Michigan)k) Cell cycle inhibitors - Ro 317453 (Roche)

• Widely available

• Fully validated

• Highly sensitive to changes in the biochemical process

• Biochemical parameters can be extracted from a single scan

Desirable Characteristics of Radiotracers for Angiogenesis Imaging

True angiogenesis inhibitors

•Halt only vascular sprouting and do not destroy preestablished tumor blood vessels.

•Generally slow tumor growth within several days to a week or more.

•Expected effect is disease stabilization rather than tumor regression

Vascular targeting agents

•Destroy the pre-existing tumor vasculature.

•In animal studies, effect is observable within hours.

•Acute endothelial cell death, thrombosis, and tumor mass hypoxia and necrosis result.

Non-selective antiangiogenic agents

•Antiproliferative or cytotoxic effects on multiple cell types as well as endothelium.

•Dose adjustment, schedule, or delivery mode may produce anti-endothelial effects.

Classification of Angiogenesis Inhibitors

The Angiogenesis Process: How Do New Blood Vessels Grow?

1. Diseased tissue releases angiogenic GFs that diffuse into the nearby tissues

2. The angiogenic GFs bind to specific Rp on the EC of nearby preexisting blood vessels

3. Once GFs bind to their Rps, the ECs become activated. Signals are sent from the cell's surface to the nucleus. The endothelial cell's machinery begins to produce new molecules including enzymes

4. Enzymes dissolve tiny holes in the basement membrane surrounding all existing blood vessels

5. The endothelial cells begin to divide and migrate out through the dissolved holes of the existing vessel towards the diseased tissue or tumor

6. Specialized molecules called adhesion molecules, or integrins serve as grappling hooks to help pull the sprouting new blood vessel sprout forward

7. Matrix metalloproteinases are produced to dissolve the tissue in front of the sprouting vessel tip in order to accommodate it. As the vessel extends, the tissue is remolded around the vessel

8. Sprouting endothelial cells roll up to form a blood vessel tube

9. Individual blood vessel tubes connect to form blood vessel loops that can circulate blood

10.Finally, newly formed blood vessel tubes are stabilized by specialized muscle cells (smooth muscle cells, pericytes) that provide structural support. Blood flow then begins