Our department is involved in a number of research projects and studies.
EGFR - Epidermal Growth Factor Receptors
EGFR targeted therapy is already used clinically, however it requires careful monitoring since it is effective in less than one third of the patients. A precise in vivo evaluation of the EGFR presence and density in various tumors is only possible by further preclinical investigations in animal models and clinical studies utilizing PET labeled biomarkers. In this context, we have pioneered this research endeavor.
Epidermal growth factor (EGF) and erb-2 (HER-2) kinase receptors are among growth factor receptor kinases which play a major role in cancer initiation, development and progression. Over-expression of the EGFR has been linked to the malignant transformation of cells, and is correlated with invasiveness and poor prognosis in patients. Thus, the EGFR is an attractive target for the design and development of compounds that can specifically bind to the receptor and inhibit its tyrosine kinase activity and signal transduction pathway in cancer cells.
Myocardial perfusion imaging
Nowadays, the majority of myocardial perfusion imaging (MPI) scans are performed using single photon computed tomography (SPECT) rather than PET in spite of the clear advantage offered by PET such as increased diagnostic accuracy, improved sensitivity, three dimensional dynamic acquisition and absolute quantitation of myocardial blood flow of the coronary arteries. This is partly due to the limited availability of suitable PET tracers.
We hypothesized that carbon-11 and fluorine-18 labeled ammonium salt derivatives have a great potential as PET MPI agent candidates. Their lipophilicity can be easily manipulated via their functional groups to enable passive diffusion into cardiomyocytes; they are positively charged, enhancing their tendency to undergo renal rather than hepatic excretion and increasing their tendency for cardiac accumulation and retention via interactions with the mitochondria which is plentiful in the heart muscle, and their labeling should be simple, straightforward and easily automated. Additionally, the favored carbon-11's half-life (20.4 min) provides adequate time for chemical transformations and a limited option for delivery to surrounding PET centers while keeping the waiting period between consecutive scans to less than two hours, even for equal doses. The development of fluorine-18 labeled analogs will enhance the potential clinical impact of labeled ammonium salts in MPI.
Ten carbon-11 labeled ammonium salt derivatives were designed, synthesized and radiolabeled in our laboratory. Their structure activity relationship and potential as PET MPI agents were evaluated using biodistribution in mice and compared to those of [99mTc]-MIBI, the prevailing SPECT agent in clinical setting. The lead compound was compared with [13N]-NH3 in healthy rats using our new microPET/CT and its potential in diagnosing myocardial ischemia was evaluated in a newly developed ischemic swine model using clinical PET/CT.
So far, the SAR studies of 11C-labeled ammonium salts suggest that both lipophilicity and charge-density affect the performance of these compounds as MPI agents and a very fine-tuned balance between the two should be preserved in order to meet the prerequisites of MPI probes. In addition, the studies that have been conducted indicated that our labeled lead is capable of detecting myocardial ischemia, thus establishing the introduction of radiolabeled ammonium-salt derivatives for myocardial imaging. Currently, we are in the process of completing all of the studies required to bring it to clinical trials and in parallel, we are designing a fluorine-18 analog of our lead compound.
Iressa (ZD 1839 , Gefitinib) is an EGFR reversible inhibitor recently approved by the FDA for treatment of non-small-cell-lung cancer (NSCLC) and prostate cancer, and several other anti-EGFR targeted molecules such as Tarceva (OSI-774 , Erlotinib) and the anti-EGFR antibody Erbitux, are presently undergoing phase 3 clinical trials. Gefitinib binds at the ATP site and inhibits the kinase activity of EGFR. It is used for treatment of NSCLC, and was approved in 2002, but is effective only in a small percentage of patients in whom EGFR possesses activating mutations in the kinase domain. Erlotinib yielded similar results to Gefitinib. In the absence of accurate measurements of EGFR phosphorylation in the human tumor, it is actually not possible to assess whether the poor response to Gefitinib and Erlotinib is indeed due to lack of the specific activating mutations, the absence of a survival function of EGFR, or to insufficient long-term occupancy of the receptor.
Consequently, there has been a growing interest in the use of EGFR-TK inhibitors as labeled biomarkers for molecular imaging of EGFR overexpressing tumors by a nuclear medicine modalities such as Positron Emission Tomography (PET). Such imaging studies will contribute to the understanding of the molecular biology mechanism of cancer. In a pioneered scientific work, and in collaboration with Prof. Levitzki, we have developed more than 20 EGFR inhibitors labeled with either [I-124], [F-18], [C-11] radioisotopes and evaluated the potential role of EGFR as a specific target for cancer imaging and therapy by in vitro and in vivo methods using our own developed two tumor-bearing animal models. The next steps in this study are to perform several Micro-PET experiments with our animal model under different conditions (dose, time frame, blocking experiment with potential new or existing drugs etc.) in order to identify the best tracer, to evaluate drug potency, to correlate treatment response with EGFR occupancy, and to initiate human studies during 2006. Since the results from our research have proven to be successful, we have been contacted by several scientific groups in the U.S. (M.D. Anderson, Washington University), Europe (University of Bologna Medical School) and Australia (Ludwig Institute, Melbourne) for the opportunity to collaborate with us.
C11-Choline PET in Urinary System Tumors
Methyl Choline labeled with 11C (11C-MC) is a promising new agent for tumor imaging using PET. Evidence suggests increased synthesis of membrane phosphatidylcholine in tumor cells that is correlated with high uptake of this radiopharmaceutical into malignant tissue. In contrast to 18F -fluorodeoxyglucose (FDG), its uptake into benign structures such as the normal brain, heart and urinary tract is negligible, resulting in a higher target to background signal ratio in tumors located near those benign structures.
Another advantage is its rapid disappearance from the blood-pool, within minutes, while the plateau of 18F -FDG uptake into tumors may be reached only after hours. Therefore, diagnostic content of 11C-MC images is already stable after 5-15 minutes. Our aim is to evaluate the usefulness of PET imaging with 11C-MC for the diagnosis, staging and therapeutic follow-up of malignant tumors. An automated synthetic route to 11C-MC was developed in our lab including QC procedure. [11C] carbon dioxide is produced in the cyclotron and subsequently iodinated to form [11C] methyl iodide. [Methyl-11C] choline is synthesized by the reaction of [11C] methyl iodide with "neat" dimethylaminoethanol at 120 degrees C for 5 min.
Purification is achieved by evaporation of the reactants followed by passage of the aqueous solution of the product through a cation-exchange resin cartridge. Radiochemical yield is > 98%. Radiochemical purity is > 98%. Chemical purity is > 90% (dimethylaminoethanol is the only possible impurity). Specific radioactivity of the product is > 133 GBq/mumol. The final product is tested for pyrogens by the limulus lysate test. The recommended dose is around 370 Mbq. A quality assurance method based on cation-exchange HPLC was developed. Human study of [11C]Choline-PET was approved by the Helsinki Committee and more than 50 cases of either brain, prostatic and urinary system tumors have been studied up to now.
Dopamine and PET
Neuroendocrine (NE) tumors include gastroenteropancreatic endocrine tumors [carcinoid tumors and islet cell tumors of the pancreas], pheochromocytoma and medullary thyroid carcinoma (MTC) with their associated familial forms, and neuroblastoma tumors. These tumors have been described as APUD-omas, based on their capacity for amine precursor uptake and decarboxylation.
As a result of this characteristic, the amino acid dihydroxyphenylalanine (L-DOPA) is taken up into the cell and is decarboxylated to form dopamine that is subsequently stored in the storage granules. Based on this process, we initiated a study for carcinoid patients with Prof. Benjamin Glazer and Prof. David Gross from the Department of Endocrinology on the use of F18-Fluorodopa (FDOPA), i.e., L-DOPA labeled with F-18, using PET imaging. A semi-automated one-pot radiosynthesis of [F-18] F-DOPA was developed in our facility. This simple and reliable automated procedure has been used routinely in our laboratory for the last two years. [F-18] F-DOPA was produced with a 20% yield, with high chemical and radiochemical purity. Initial results of FDOPA PET imaging in 20 patients have shown improved accuracy compared to FDG PET and other conventional imaging methods (MIBG and Octreoscan).
Vascular Endothelial Growth Factor Receptors (VEGFR) have a major role in the development of pathological angiogenesis and cancer. In order to assess biochemical response to treatment and contribute to anti-VEGFR drug development, advance in in-vivo molecular imaging methodology is mandatory. Angiogenesis, the recruitment of new blood vessels, is a crucial mechanism in a number of physiological and pathological conditions. It is a tightly regulated, multiple-step process, that results in the formation of blood vessels from pre-existing vasculature.
Under normal conditions, angiogenesis occurs during embryonic development, would recovery, and the female menstrual cycle. Pathological angiogenesis occurs in disease conditions such as psoriasis, diabetic retinopathy, rheumatoid arthritis, chronic inflammation and cancer. In the process of angiogenesis, several events are included: proliferation, migration, and invasion of endothelial cells, organization of endothelial cells into functional tubular structures, maturation of vessels, and vessel regression. One of the major pathways for promotion of angiogenesis is the vascular endothelial growth factor (VEGF) family of growth factors and receptors.
Activation of the VEGF/VEGF-receptor (VEGFR) axis triggers multiple signaling networks that result in endothelial cell survival, mitogenesis, migration, differentiation, and vascular permeability.2 Over-expression of both VEGF and VEGFR mRNA has been associated with angiogenesis, tumor progression and poor prognosis in several tumor systems. While over-expression appears in tumor-associated endothelial cells, over-expression or upregulation was not found in the vasculature surrounding of normal tissues. The over-expression of VEGFR2 (KDR) on tumor endothelial cells offers a unique target for specific anti-angiogenetic therapy. While angiogenetic vascularization collapse does not bring about a full recovery, it could help reduce tumor size to around 2 mm (the maximal tumor diameter without induction of angiogenesis), inhibit metastasis and enable more efficient therapy when administered concomitantly with known chemotherapeutic agents.
The current challenge in VEGFR targeted therapy is not only the inhibition of VEGF signaling via VEGFR antagonism, but the evaluation of anti angiogenetic treatment efficacy in the early stages in order to assess different dosing regiments. Tighter control over dosing could lead to personalized treatment and permit guided treatment over different stages of patient management. Effective evaluation of VEGFR occupancy offers a tool for drug discovery and efficacy appraisal. Today, efficacy is evaluated by shrinkage of tumor size, microvessel density (MVD) and total vascular area (TVA), or evaluation of blood flow. Changes in tumor size are measurable by conventional imaging, however the time window post treatment is too long and is measured in months.
Determination of MVD and TVA is achieved by invasive methods and MRI evaluates blood flow and accumulation rather than microvessel integrity. In order to explore the role of VEGFR in cancer, and the potential of VEGFR targeted therapy, there is a pressing need to develop specific and selective molecular imaging modalities that will enable the measurement of several important parameters such as concentration, and occupancy of the VEGFR in vivo with a noninvasive Nuclear Medicine modality. Specific labeled VEGFR2 antagonists could allow for selective, non-invasive quantitative imaging of angiogenetic blood vessels.
Several compounds of the quinoline and quinazoline families will be synthesized and for each one of them, the potency, affinity and selectivity towards the KDR (VEGFR-2) will be determined. A radiolabeling route for the most promising compounds will be developed and their potential as PET imaging agents will be evaluated. The compounds will be characterized in several biological tests including in vitro & in vivo testing. In vitro assays will be performed in a number of cell lines which over-express the VEGFR-2/KDR in comparison with these same cell lines without over-expression.
The in vitro experiments will include potency assays, specific binding assays & selectivity assays versus different Tyrosine kinase receptors including: VEGFR1, PDGFR, EGFR, INSULIN-R, FGFR, c-Kit & others. Following in vitro characterization, tumor cell lines will be injected into nude athymic mice and the PET imaging biomarker candidates will be evaluated in-vivo by biodistribution studies, and specific binding assays including Micro-PET imaging.Patents