Michael R. Kilbourn - US grants

The intent of this Proposal is the preparation, evaluation and validation of new radiopharmaceuticals for neurological PET studies, based on the monoamine (dopamine, norepinephrine, and serotonin) reuptake systems. A large number of clinical problems in degenerative neuronal diseases, major psychiatric illnesses, and drug abuse involve the monoamine neurotransmitter systems. The non-invasive, in vivo study of these systems can be best approached using Positron Emission Tomography, but such studies will require the availability of specific, well characterized radiotracers. It is the goal of this project to provide such radiopharmaceuticals. Target molecules for the dopamine, norepinephrine and serotonin reuptake systems have been chosen, and synthetic routes to their radiolabeling with either carbon-11 or fluorine-18 devised. Each new radiochemical will be tested in a wide variety of preclinical tests for pharmacologic specificity and kinetic properties. The goal is the development of radiotracers which are specific for a single binding site, and whose kinetic properties allow precise identification of the parameter of interest (binding to the reuptake site) and its distinction from non-specific processes involved in ligand distribution (blood flow, vascular permeability and non-specific binding). The regional and pharmacological specificity of new radiotracers will be examined in small animals using ex vivo, in vitro, and autoradiographic methodologies. Physiochemical parameters (log P, protein binding) will be determined in vitro. Formation and biodistribution of radiolabeled metabolites in blood and brain will be examined. As the final pre-clinical step, PET studies of radiotracer distribution in primate brain will be completed and the development and evaluation of mathematical models undertaken. Promising radiopharmaceuticals at this point will undergo determination of radiation dosimetry and toxicology and preliminary-human studies completed to allow evaluation of the kinetic model using human data. Overall, this represents a cohesive, structured approach to the development of new radiopharmaceuticals for PET. New quantitative PET techniques for the study of the monoamine systems will permit direct evaluation of disease-related hypotheses in neurology, psychiatry, and drug abuse research.

DESCRIPTION: A number of important neurologic, mental health and drug abuse problems involve dysfunctioning or losses of the monoarninergic systems of the human brain, and in particular the dopaminergic neurons. In vivo imaging of these systems using radiopharmaceuticals and Positron Emission Tomography (PET provides a unique and valuable approach to the study of these diseases in the living human brain. Choices of appropriate radiotracers, alone and in combinations, will be crucial for the success of such in vivo imaging. This Project will examine animal models of such diseases, using a novel approach of dual in vivo radiotracer studies of the vesicular monoamine transporter (VMAT2 and the dopamine neuronal membrane transporter (DAT). The potential in vivo regulation of these binding sites by disease processes and chronic drug treatments will be evaluated. The short term regulation of such transporters b kinase-mediated phosphorylation reactions will be studied. The relationship between the two transporters, proposed here as markers of nerve terminal integrity (VMAT2) and nerve terminal function (DAT), will be examined over tim in models of degeneration and recovery in animal models of Parkinson's disease and drug abuse. Studies will subsequently be done to determine whether measure of numbers of terminals (VMAT2), functional status (DAT), or function on a per terminal basis (DAT/VMAT'2) provides the best sensitivity for measuring in viv the effects of current and new therapeutic approaches to changing the course o these diseases. This Project will provide crucial information on the proper approach to be taken for in vivo tomographic radionuclide of monoaminergic terminal-related disease inception, progression or treatment in a general patient population. Finally, synthetic radiochemistry efforts will be undertaken to extend this approach to measuring the VMAT2 outside the heavily-dopaminergic innervated striaturn, opening up new avenues of research into the role of monoaminergic (particularly, serotonergic and adrenergic) terminals in a wider variety of neurologic, mental health and drug abuse diseases.

The Cyclotron/Radiochemistry Core provides all of the radionuclides, radioactive precursors, and approved PET radiopharmaceuticals for the research and clinical projects of this Program Grant. This includes production of fluorine-18 and carbon-11 precursors for Project 1, and routine deliveries of (11C]flumazenil, [11C]N-methylpiperidylbenzilate, [11C]raclopride, [11C]methoxytetrabenazine and 5-[123I]iodobenzovesamicol for Projects 2-4. PET radionuclide production is done using a dedicated Cyclotron Corp. CS-30 cyclotron, outfitted with 10 cyclotron targets, 8 of which are located on a vertical beam ladder. Primary and secondary production targets are available for all radionuclides. All cyclotron targetry, mechanical, electrical and computer systems have been constructed in house; the Core provides all routine and emergency repairs for all of these systems, as well as for the cyclotron itself. Radiochemical research and routine production of PET radiopharmaceuticals is done in a well-equipped radiochemistry laboratory, which has the capability to deliver clinical doses of carbon-11 radiopharmaceuticals in sequential fashion at less than 1 hour intervals. All syntheses are done remotely using apparatus designed, constructed and maintained by Core staff members. The Core provides established expertise in radiochemistry, radiation safety, quality control, and documentation for regulatory approvals.

Application of PET to the further understanding of the causes and development of neurological diseases will require more radiopharmaceuticals designed for specific aspects of neuronal biochemistry which might be involved. In this Project, new radiopharmaceuticals are proposed for in vivo studies of biochemical processes which may be important in numerous neurological disease, including Parkinson's, Huntington's, and Alzheimer's disease, among others. These new radiopharmaceuticals include markers of the mitochondrial electron transport chain, specifically at complex I, and radiotracers to study the formation of, concentration of or effects of reactive oxygen species such as free radicals. For study of complex I, it is proposed to prepare positron-emitter labeled derivatives of rotenone and related rotenoids. To study free radicals, appropriately labeled spin trapping agents based on the nitrone structure such as alpha-phenyl-t- butyl-nitrone (PBN) are proposed. This Project will undertake the radiochemical synthesis of these proposed new in vivo radiochemicals and their initial animal evaluation to determine distribution, kinetics and metabolism. Such new radiopharmaceuticals, when available for PET applications, could be used to examine numerous recent hypotheses of the underlying biochemical changes occurring in a large number of neurological disorders.

The Cyclotron/Radiochemistry Core provides all of the radionuclides, radioactive precursors, and approved PET radiopharmaceuticals for the research and clinical projects of this Program Grant. This includes production of fluorine-18 and carbon-11 precursors for Project 1, and routine deliveries of d-threo-[11C]methylphenidate, [11C]raclopride, (+) [11C]dihydrotetrabenazine, [15O]water and 5- [123i]iodobenzovesacicol for Projects 2-4. PET radionuclide production is done using a dedicated Cyclotron Corp. CS-30 cyclotron, outfitted with 10 cyclotron targets, 8 of which are located on a vertical beam ladder. Primary and secondary production targets are available for all radionuclides. All cyclotron targetry, mechanical, electrical and computer systems have been constructed in house; the Core provides all routine and emergency repairs for all of these systems, as well as for the cyclotron itself. Radiochemical research and routine production of PET radiopharmaceuticals is done in a well-equipped radiochemistry laboratory, which has the capability to delivery clinical doses of carbon- 11 radiopharmaceuticals in sequential fashion at less than 1 hour intervals. All syntheses are done remotely using apparatus designed, constructed and maintained by Core staff members. The Core provides established expertise in radiochemistry, radiation safety, quality control (including sterility and pyrogen testing), and documentation for regulatory approvals.

DESCRIPTION (provided by applicant): In vivo imaging of the biochemistry of the neuronal membrane dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) are two important methods for non-invasively measuring the losses of dopaminergic function in Parkinson's disease (PD). Both measures may prove useful as biomarkers for clinical trials of new drugs or gene therapy approaches to prevention or reversal of the losses of dopaminergic nerve terminals in Parkinson's and other degenerative diseases of the dopaminergic system. In this Project, the relative sensitivity of these two measures will be directly and simultaneously compared in the acute MPTP, unilateral 6-hydroxydopamine, and chronic rotenone animal models of PD. Losses of in vivo DAT and VMAT2 binding sites will be determined using dual-radiotracer in vivo studies employing tritiated and carbon-11 forms of the specific radioligands d-threo-methylphenidate and dihydrotetrabenazine. Quantitative measures of radioligand binding in rat brain will be determined using a newly developed dual radiotracer infusion to equilibrium protocol. The time-dependent differential losses of in vivo measures of the two transporter binding sites will first be determined for each animal model. Possible alterations of these sites upon growth factor treatment will be determined in control animals. Subsequently, the two different in vivo radioligand measures of DAT and VMAT2 sites will be evaluated as markers of neuroprotection or neurorescue of dopaminergic nerve terminals in the brain, following administration of important new growth factor (e.g., glial-derived neurotrophic factor, GDNF, and erythropoeitin, EPO). These studies will provide crucial information regarding the proper selection of one or both of these in vivo radioligand methods (DAT and/or VMAT2) as optimal in vivo biomarkers to be employed in clinical trials of emerging new drug and gene therapy approaches to the prevention or reversal of the dopaminergic neurodegeneration found in Parkinson's disease.

Core A: Cyclotron/Radiochemistry (MR Kilbourn) This Core will provide the clinical research projects and the Clinical Core with PET radiopharmaceuticals for measuring nigrostriatal dopamine terminals ([11C]DTBZ), serotonin nerve terminals ([11C]DASB), acetylcholine nerve terminals ([11C]PMP), brain fibrillary afi amyloid deposits ([11C]PIB), and myocardial sympathetic nerve terminals ([11C]HED). All of these PET radiotracers are presently in regular production and human research imaging use in our laboratories. The Core will provide high-quality research radiotracers meeting requirements for human administrations, and will track and monitor reagents and components, synthesis results, and quality control metrics in accordance with applicable medical, University, State, and Federal requirements. The Core includes a comprehensive quality assurance program.

DESCRIPTION (provided by applicant): Osteoporosis is estimated to affect over half of adults in the United States over the age of 50, and 1 in 3 women over the age of 80 will suffer a hip fracture; of those who fracture, nearly 15-20% die within 1 year. Overall, the costs associated with osteoporosis in the United States alone were estimated at $19 billion in 2005. This project involves the design, development and evaluation of an new molecular imaging radiotracers for Positron Emission Tomography (PET) studies of osteoporosis and other musculoskeletal diseases (e.g., osteoarthritis, rheumatoid arthritis, osteoporosis) involving potential bone loss. The project will develop radiolabeled forms of known high affinity inhibitors of cathepsin K, a cyteine protease that in highly expressed in osteoclasts and that is responsible for type I collagen degradation and bone resorption. The potential for in vivo imaging of cathepsin K has been demonstrated using optical methods, and this project will extend this concept to a clinically translatable imaging method using radionuclide labeling. This grant will prepare series of radiolabeled inhibitors based on the published cyanopyrimidine and pyrrolopyrimidine scaffolds, using isotopic (carbon-11) substitution or synthesis of fluorinated analogs (for fluorine-18 labeling). The compounds selected as initial targets for carbon-11 labeling all have high affinity for human cathepsin K (< 10 nanomolar), excellent selectivity over cathepsins L and S (100-1000 fold selective) and all are readily labeled using [11C]methyl iodide. New fluorinated analogs where fluoroethyl groups replace methyl groups will be evaluated in vitro for inhibitory action on cathepsin K enzymatic activity, using a fluorescent assay, and potent (<10 nM) inhibitors labeled using analogous [18F]fluoroalkylation reactions. In vivo proof of concept studies to demonstrate osteoclast-dependent localization of radiotracers will be done in rats using focal skeletal injections of RANKL (receptor activator for nuclear facktorB ligand, to induce osteoclastogenesis) or osteoprotogerin (to inhibit osteoclastogenesis). Extent of bone loss will be monitored by microCT. Verification that radioactivity localization is cathepsin-specific will be done in RANKL treated rats and blocking with cold doses of enzyme inhibitor. Successful radiotracers that exhibit high uptake and retention in RANKL-treated animals that is blocked by cold inhibitor, and reduced uptake in osteoprotegrin-treated animals, can then be further evaluated as potential radioligands for human imaging. Detection of increased osteoclastic cellular activity may provide new diagnostic criteria that take osteoclast activity into account in addition to standard measures of structural integrity, and thus provide immediate feedback on the efficacy of a chosen treatment protocol before waiting for further downstream gain or loss of bone to occur. .

DESCRIPTION (provided by applicant): This project aims to develop new radiolabeled substrates for the enzyme monoamine oxidase-B (MAO- B) in the human brain. In the brain, MAO-B is conspicuously expressed in astrocytes, and increased MAO-B enzymatic activity has been used as a biomarker of gliosis in numerous studies of diseases such as refractory epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, and other neurodegenerative conditions. As reactive gliosis is a recognized pathological marker of localized cellular responses to a wide variety of insults to the brain, non-invasive measurement of the extent and pattern of gliosis could have wide applicability to the localization of injured or diseased tissue (e.g., for resection in epilepsy), differentiation of related diseases (Alzheimer's versus frontal-temporal dementia (FTD) or Lewy Body disease), and evaluation of new therapeutic treatments, and thus could significantly assist in the diagnosis and management of many CNS diseases. This project proposes the design, chemical and radiochemical synthesis, and evaluation in vitro and in vivo of new carbon-11 and fluorine-18 labeled substrates for Positron Emission Tomography (PET) imaging of MAO-B enzymatic activity. In an innovative approach to MAO-B imaging, the project will prepare novel radiolabeled derivatives of 4-thiophenyl- and 4-carbamoyl-N-methyl-1,2,3,6-dihydropyridines that are known to be non-toxic MAO-B substrates that form polar reaction products. 4-Thiophenyl-1,2,3,6-dihydropyridines are oxidized to the corresponding non-toxic dihydropyridinium salts;the 4-carbamoyl-1,2,3,6- dihydropyridines are oxidized to the corresponding dihydropyridinium salts that spontaneously hydrolyze to release polar amines. The polar radiolabeled products from MAO-B enzymatic oxidation will accumulate at the site of enzyme action, thus using the concept of metabolic trapping to provide quantifiable PET imaging data. Candidate radiotracers will be evaluated for in vitro reactivity with MAO- B (Km and kcat) and good in vivo brain uptake and retention in rodents using ex vivo dissection techniques. Promising radiotracers will then be evaluated for brain uptake, irreversible trapping, and overall pharmacokinetics in rhesus monkey brain by microPET imaging. The overall goal of the project is to identify appropriate radiolabeled substrates that can be further evaluated and validated for applications in human PET imaging. PUBLIC HEALTH RELEVANCE: Gliosis is a cellular response to tissue injury or degenerative disease in the human brain. This project will develop new radiopharmaceuticals for the non-invasive PET imaging of monoamine oxidase B, an enzyme highly expressed in gliosis. Imaging of gliosis has diagnostic applications in epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and other neurodegenerative diseases.

DESCRIPTION (provided by applicant): This grant application proposes the design, synthesis, and evaluation (in vitro and in vivo) of new PET (positron Emission Tomography) radioligands for in vivo imaging of the ?---aminobutyric acid (GABA) transporter localized in the presynaptic neuronal membrane. Although GABA is one of the predominant inhibitory neurotransmitters in the mammalian brain, and dysfunctioning of the GABA system is evident in such important human diseases as epilepsy, schizophrenia and autism, there are currently no in vivo imaging agents for studies of the presynaptic GABA---ergic innervation in the human brain. The specific aims of this project are the synthesis of a series of diarylalkenyl ether nipecotic acid analogs of the clinically used drug tiagabine (Gabatril(R)) and evaluation of their in vitro binding affinitie and lipophilicity. Candidate compounds with low nanomolar affinities, and appropriate lipophilicity, will be radiolabeled with carbon---11 or fluorine---18 and tested in vivo in rodentsto determine blood---brain---barrier permeability, metabolism, brain pharmacokinetics, and pharmacological specificity for the GABA transporter type 1 (GAT---1). The most promising radiotracers will then be evaluated in the rhesus monkey brain, to determine pharmacokinetics and potential for quantitative measures of GABA---ergic neuron innervation. The goal of the project is to identify candidate radioligands with suitable brain uptake and pharmacokinetics for further development into PET radiopharmaceuticals for non---invasive studies of the GABA neuronal system in human neurological and psychiatric diseases.