1. Development of Radiotracers for Imaging the Proliferative Status of Solid Tumors

Positron Emission Tomography (PET) is an in vivo imaging technique that is capable of measuring disease-associated changes at the molecular level. The use of PET in the field of oncology has largely centered on studies using the metabolic radiotracer, [¹⁸F]FDG, which measures differences in glucose utilization between tumors and the surrounding normal tissue. Although this strategy is very useful in the diagnosis and staging of tumors, [¹⁸F]FDG does not provide information that can be used to identify an appropriate strategy for treating the disease.

A recent strategy for imaging tumors involves the development of radiotracers that measure cellular proliferation and/or the proliferative status of solid tumors. There are a number of reasons why this information is useful in the treatment of cancer patients: 1) rapidly proliferating tumors are generally aggressive and have a high malignant potential that requires aggressive initial treatment; 2) rapidly proliferating tumors typically respond better to cell cycle specific agents (e.g., Ara-C and 5-fluorouracil) and hyperfractionated radiotherapy, while slowly proliferating tumors respond better to cell cycle nonspecific agents (e.g., cyclophosphamide and BCNU) and conventionally fractionated radiotherapy; and, 3) a reduction in the proliferative status of a tumor can be used as an early predictor of the tumor's response to therapy.

There are two different strategies for imaging the proliferative status of solid tumors with PET. The first strategy involves the use of radiolabeled nucleoside analogs which utilize the salvage pathway of DNA synthesis for their uptake. The second strategy involves the use of radiotracers that image the sigma-2 receptor status of solid tumors. The goal of this research program is to develop a receptor-based imaging method to assess the proliferative status of human breast tumors using PET. This application will focus on the use of the sigma-2 (σ2) receptor as a biomarker of tumor proliferation. The choice of this receptor is based on our published data demonstrating that σ2 receptors are expressed in ~10-fold higher density in proliferative mouse mammary adenocarcinoma cells versus the nonproliferative or quiescent cell population under both in vitro (cell culture) and in vivo (tumor xenographs) conditions. Therefore, a σ2 receptor PET radiotracer has the potential to provide information regarding the proliferative status of breast cancer. An in vivo imaging procedure that can provide information about the proliferative status of primary breast tumors would represent a significant improvement over current methods used in making this assessment. Our preliminary data also indicate that ¹¹C- and ¹⁸F-labeled σ2-selective radiotracers developed in our laboratory have a better tumor:nontumor ratio than other agents, including FDG and radiolabeled nucleoside analogs such as [¹⁸F]FLT (a thymidine analog that is not incorporated into DNA) and [¹⁸F]FMAU (a thymidine analog that is incorporated into DNA (Figure 1). We are currently conducting a series of studies comparing the σ2 receptor-based imaging approach with that of [¹⁸F]FLT and [¹⁸F]FMAU. Initial results indicate the σ2 receptor-based imaging approach is superior to both [¹⁸F]FLT and [¹⁸F]FMAU for imaging prostate tumor xenografts. Since σ2 receptors are expressed in many human tumors (e.g., glioma, lung and head and neck tumors), we believe this procedure has the potential to image the proliferative status of a wide variety of solid tumors (Figure 2).

Figure 1. MicroPET/microCT imaging study an 18F-labeled σ2 selective radiotracer developed in our laboratory in a breast cancer xenograft.

	Figure 2. Imaging study using an 18F-labeled σ2 selective radiotracer in a rodent brain tumor model.

2. PET radiotracers for Imaging Cellular Death

Apoptosis or Programmed Cell Death is an important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Apoptosis is initiated by extracellular or intracellular signals in which a complex machinery is activated to start a cascade of events that ultimately leads to the degradation of nuclear DNA and a dismantling of the cell. Apoptosis is a tightly regulated and conserved mode of cell death that does not result in injury to adjacent cells. However, necrosis is a catastrophic, unregulated mode of cell death that is characterized by an invasion of inflammatory cells and injury to adjacent tissue. Abnormal or unregulated apoptosis is believed to occur in a variety of disease states. For example, elevated apoptosis is believed to play a major role in ischemia-reperfusion injury (i.e., myocardial infarction and stroke), neurodegeneration (Parkinson's Disease, Alzheimer's Disease, ALS), sepsis, and diabetic cardiomyopathy. The inability of a cell to undergo apoptosis is believed to play a major role in the resistance of tumor cells toward radiation, chemotherapy, and immunotherapy. Therefore, the development of a noninvasive imaging procedure that can measure apoptosis would be useful in the study of many pathological conditions.

The current method for imaging apoptosis uses radiolabeled analogs of Annexin V. Annexin V is a protein that binds with high affinity to phosphatidyl serine residues that are exposed as part of membrane inversion that occurs during apoptosis. However, since membrane inversion also occurs during necrosis, imaging studies using radiolabeled Annexin V measure both apoptosis and necrosis.

The goal of this research program is to develop an imaging strategy that can discriminate apoptosis from necrosis and vice versa. Our strategy for imaging apoptosis is to develop radiotracers that can measure the level of caspase-3 activity in the cell. Caspase-3 is an "executioner" caspase and is released from an inactive zymogen (procaspase-3) late in the process of apoptosis. A noninvasive imaging procedure that can quantify caspase-3 activity would be useful both in the study of apoptosis in a wide variety of clinical conditions. Since many cancer chemotherapeutics kill tumor cells by inducing apoptosis, a noninvasive imaging procedure that can measure caspase-3 activation would also be useful in monitoring a positive response to chemotherapy in the treatment of cancer.

We have recently developed a number of small molecule inhibitors of caspase-3 that can be labeled with either fluorine-18 or carbon-11. Once a suitable ¹¹C- or ¹⁸F-labeled caspase-3 radiotracer has been identified from biodistribution and microPET imaging studies, a series of comparison studies between a caspase-3 radiotracer and radiolabeled Annexin V will be conducted in order to determine which agent provides an accurate measurement of apoptosis. Initial imaging studies with an 18F-labeled radiotracer for imaging caspase-3 activation in a well-established animal model of apoptosis is shown in Figure 3.

One of the primary functions of caspase-3 in the induction of apoptosis is to degrade the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a chromatin-associated enzyme that detects and repairs single strand breaks in DNA that are induced by a variety of toxic insults. Caspase-3 recognizes a DEVD motif and cleavage at this site separates the DNA binding domain from the catalytic domain. This degradation of PARP-1 is critical for the ensuing DNA fragmentation that is characteristic of apoptosis. Therefore, the immunohistochemical measurement of the two PARP-1 cleavage products is one method for measuring apoptosis.

Figure 3. MicroPET imaging studies with [18F]WC-89 in an animal model of chemically-induced apoptosis in the liver.

Although the degradation of PARP-1 is a key initial step in the induction of apoptosis, the functional activation of this enzyme plays a critical role in necrosis. Exposure of the cell to a variety of toxic stimuli such as ROS/RNS, ionizing radiation, and genotoxic chemicals results in the formation of nicks and breaks in the DNA strand. In response to this DNA damage, PARP-1 becomes activated and catalyzes the formation of poly-ADP on acceptor proteins such as Topoisomerases I and II, DNA polymerase, DNA ligase 2 and histones. Since NAD⁺ is a substrate for the formation of poly-ADP ribosylation reactions, the activation of PARP-1 under genotoxic stimuli results in a depletion of cellular NAD⁺ levels and a disruption in cellular energetics (i.e., glycolysis, -oxidation of fatty acids and the TCA cycle require NAD⁺ and are disrupted following PARP-1 activation). The hyperactivation of PARP-1 to the extent where NAD⁺ levels are depleted results in cellular death via necrosis (Figure 4).

Our studies for imaging cellular death by necrosis have used focused on the use of PARP-1 as the molecular marker of necrosis. We have recently labeled the potent PARP-1 inhibitor, PJ-34, with carbon-11 and evaluated its utility as a radiotracer for imaging necrosis in an animal model of type I diabetes. The results of this study indicate that there is an increase in uptake of [¹¹C]PJ-34 in tissues undergoing necrosis via the hyperactivation of PARP-1. An imaging study on [¹¹C]PJ-34 in an animal model of ischemia-reperfusion injury in the heart is shown in Figure 5. These data suggest that it is possible to measure cellular death via necrosis using radiolabeled inhibitors of PARP-1.

Figure 4. Strategy used for discriminating apoptosis versus necrosis. Caspase activation occurs in apoptosis and not necrosis and is our molecular target for imaging apoptosis. PARP-1 is cleaved in apoptosis and becomes hyperactivated in cells undergoing necrosis. Therefore, PARP-1 is a suitable molecular marker for studying cellular death via necrosis.

	Figure 5. Increased uptake of [11C]PJ-34 in an animal model of ischemia-repfusion injury in the heart.

3. PET Radiotracers for Imaging the D₂ and D₃ Receptors in CNS Disorders.

Over the past fifteen years there has been a tremendous amount of research conducted in the development of PET and SPECT radiotracers for studying dopamine D₂ receptor function in vivo. This research has been largely driven by the availability of a number of antipsychotics possessing a high affinity for dopamine D₂ receptors, and the alteration of D₂ receptor density identified in postmortem brain samples from a variety of CNS disorders including schizophrenia, Parkinson's disease, Alzheimer's Disease, and substance abuse. However, the radiotracers that have been developed to date are not capable of discriminating between the different subtypes of the D₂-class of receptors. For example, [¹¹C]raclopride and [¹²³I]IBZM bind with high affinity to dopamine D₂ and D₃ receptors, and [¹¹C/¹⁸F]N-methylspiperone binds potently to D₂, D₃ and D₄ receptors. Therefore, measurement of "D₂ receptor binding potential" obtained with these radiotracers consists of a composite of D₂, D₃, and, in the case of N-methylspiperone, D₄ receptor density.

A number of recent studies have suggested that D₂ and D₃ receptors are regulated in an opposing manner in a variety of CNS disorders. For example, it has been reported that there is a 45% reduction in D₃ receptors in the ventral striatum and a 15% increase in D₂ receptors the caudate/putamen of postmortem brain samples of Parkinson's Disease. Other studies have shown an increase in D₃ receptors and a decrease in D₂ receptors in brain samples obtained following chronic exposure to cocaine. Therefore, the development of radiotracers having a high affinity for D₃ versus D₂ receptors, and vice versa, would be of tremendous interest to the imaging (PET and SPECT) and neuroscience (autoradiography) research community since it would enable the independent measurement of D₂ and D₃ receptors in a variety of CNS disorders. The goal of this research project is to develop PET and SPECT radiotracers that are selective for dopamine D₃ versus D₂ receptors and vice versa. We have recently reported a number of conformational-flexible benzamide analogs having a high affinity for D₃ versus D₂ and D₄ receptors. Our group has also synthesized a number of indole analogs having a high affinity for dopamine D₂ versus D₃ and D₄ receptors. We have recently initiated a series of microPET imaging studies aimed at imaging D₃ (radiolabeled conformationally-flexible benzamide analogs; Figure 6) and D₂ (radiolabeled indole analogs) in order to measure independently the density of D₂ and D₃ receptors in the CNS. Once suitable D₃- and D₂-labeled radiotracers have been identified, we plan to study the "dysregulation" of D₂ and D₃ receptors that may occur in a variety of CNS disorders.

	Figure 6. Autoradiography (left and center) and microPET imaging studies of dopamine D3 receptors in the CNS using the radiolabeled benzamide analog, WC-10.

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