Post on 26-Dec-2015
AREAS OF DISCUSSION
• What is Angiogenesis ?
• Why Angiogenesis Imaging ?
• Current Methods are Available ?
• Prospects of Future Techniques
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
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 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
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
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