Jogesh Mukherjee - US grants

Our long-term goal is to develop positron emission tomographic (PET) imaging methods that will enable measurement of gene therapeutic efficacy in regenerating dopaminergic terminals. The ability to noninvasively monitor the expression of transduced genes is extremely important for planning treatment of neurodegenerative diseases. Significance of this methodology is very high in developing therapeutic strategies for Parkinson's disease (PD). We propose to use two approaches in order to develop a "marker gene" for use in the gene activation imaging assay (GALA). The first approach would use an available receptor-radioligand system, which does not, or only minimally, localizes in the regions of interest. For this purpose, we have identified the serotonin 5HTla receptor gene. The second approach employs a gene preferentially expressed in the periphery and not in the CNS. For this, we have identified the serotonin 5HT2b receptor gone. This wild-type gent and its mutant reccptor (i.e. functionality removed) will be developed as a GAIA tool. We propose to use mF-MPPF as a PET probe for the measuring expression of 5HT la receptor. For the 5HT2b receptor we propose to develop imaging probes based on the reported 5HT2b-selective indolyl-urea and pyrimidine class of compounds. In vitro and ex vivo radioligand imaging (autoradiogmphic) studies of rats following intrastriatal adenoviral and lentiviral marker gene delivery will demonstrate validity of this approach. These imaging studies will be accompanied by simultaneous in vitro measurement of mRNA levels and receptor levels at the site of delivery and correlated with imaging probe concentration. This will allow correlation of the concentration of mRNA, receptors and the imaging probe. These methods wilt then be applied to unilaterally lesioned (6-OHDA) rats using "marker gene and GDNF gene". These rats will also be evaluated by using an assay to measure functionality of the therapeutic GDNF gone and its relationship to the GAlA system by measuring presynaptic dopamine turnover.

[unreadable] DESCRIPTION (provided by applicant): Serotonin 5HT1A receptors have been investigated in a number of CNS disorders, including depression, anxiety, sleep disorders, epilepsy, Parkinson's disease (PD) and Alzheimer's disease (AD). Our particular interest is to apply 5HT1A receptor imaging for the study of mood disorders and AD. Efforts are underway to improve in vivo properties of 5-HT1A agents, 11 C-WAY-100635, 18 F-MPPF and 18 F-FCWAY currently in human use. We have identified and synthesized F-Mefway, which contains a fluorine-18 on a primary carbon 18 to make the compound more stable to defluorination. Mefway has high affinity for 5HT1A receptors and in vitro rodent brain slices exhibited selective binding of F-mefway in hippocampus, cortex and other brain regions, 18 with limited binding in the cerebellum. Preliminary in vitro studies showed serotonin displaced 18 F-mefway from various brain regions with IC50 in the range of 169-243 nM. PET studies in a rhesus monkey showed F- 18 mefway binding to temporal cortex, hippocampus, raphe and other brain regions with ratios of hippocampus to cerebellum = 10. Plasma analysis indicated the presence of approx. 30% of 18 F-mefway and no observed defluorination. The high ratios in specific brain regions such as the hippocampus suggest that F-mefway has 18 potential as a PET imaging agent for 5HT1A receptors in humans. Therefore, our overall goals in this application are to synthesize and characterize pure cis- and trans-isomers of Mefway, study pharmacological characteristics of the isomers and investigate PET imaging characteristics of the 18 F-fluorine radiolabeled analogs in order to identify which of the isomers will be more suitable for human PET studies. In order to extend our findings to human studies, dosimetry of 18F-mefway will be measured in rats and monkeys. Drug- induced serotonin competition studies will be carried out using PET imaging studies in rodents to study serotonin effects. In order to assess the value of F-mefway in AD, two important areas of expertise at UCI 18 have come together in this application to address specific hypotheses using multidisciplinary approaches that include expertise in brain imaging techniques and a research group focusing on animal models of brain aging at the PET Brain Imaging Center and the Institute for Brain Aging and Dementia, respectively. In vitro studies will be carried out on two mice transgenic models of AD, the Tg2576 and 3xTg mice in order to correlate senile plaque and neurofibrillary tangle formation with the loss of serotonin 5HT1A receptors using F-mefway. Due 18 to the high hippocampus to cerebellum ratio (approx 10) and advantages of the fluorine-18 radiolabel, 18 F- mefway may have potential use in the study of AD. [unreadable] Development of imaging methods for understanding serotonin effects in the brain will help understand mood disorders and methods to study alterations of the serotonin receptor abnormailites in transgenic mice models will have implications in diagnosis, treatment planning and therapeutics development of Alzheimer's disease and other disorders of the central nervous system. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Antagonists used for positron emission tomographic (PET) studies of dopamine D2/D3 receptors do not distinguish between the high-affinity (HA) and low-affinity (LA) states of the receptors. We and others have now accomplished imaging only the HA-state of dopamine D2/D3 receptors using aminotetralin-based and apomorphine-based PET radiotracers. These studies confirm that some of the D2/D3 receptors in the striatum of the living rodent and monkey are present in the HA-state. The ability to image and reliably quantitate the amount of receptors present in the HA-state has a number of applications. These include: (a), the study of etiology of disease states such as schizophrenia, manic depression and Parkinson's disease; (b). study of dopamine release since dopamine now competes with a more sensitive PET radiotracer that is bound only (or predominantly) to the HA-state; (c). study of therapeutic drugs that may shift population of affinity states, from HA- to LA-state. Our goal is this application is two-fold: 1. Develop and evaluate agents that are suitable for HA-state imaging of striatal and extrastriatal receptors; and 2. Evaluate HA-state radiotracers that will be able to discriminate between D2 and D3 receptor subtypes. We have optimized 5- hydroxyaminotetralins for imaging dopamine D2 receptors which has resulted in the development of 18F-5- OH-FPPAT as a suitable imaging agent. We propose to optimize radiosynthesis of the more active isomer S-18F-5-OH-FPPAT and carry out in vitro and in vivo binding properties of both R- and S-isomers. The high- affinity agent 11C-PPHT will be studied for extrastriatal HA-states. Both, R- and S-isomers will be evaluated. Quantitative microPET studies to measure amphetamine-induced dopamine release in striatal and extrastriatal regions will be carried out. In order to direct the tetralins to the D3 receptor, the 7-hydroxy analogs of FPPAT and PPHT will be investigated. Specifically we will synthesize R- and S-isomers of 18F-7- OH-FPPAT and 11C-7-OH-PPHT that may have potential as D3 selective agents. Fluorine-18 analog of PPHT, 18F-FPPHT (both 5- and 7-hydroxy) will also be prepared for extrastriatal imaging of HA-states. Pharmacological characterization of these agents using D3 cell lines, brain homogenates, autoradiographic studies, D2 and D3 receptor knock-out mice microPET studies will be carried out. Finally PET studies will be carried out in monkeys with these agents to demonstrate HA-state binding and receptor subtype selectivity. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A reduction of nicotine receptors is reported in Alzheimer's disease (AD) and, conversely, an increase in the number of these receptors is reported in smokers. Imaging these receptors will therefore offer diagnostic capabilities for the study of both AD as well as nicotine dependency. At University of California-Irvine (UCI), we have two major programs: one in AD within the Center for Aging and Dementia and the other a program on studies related to nicotine dependence. The goal of this application is to develop selective alpha4beta2 nicotine acetylcholinergic receptor (a4b2 nAChR) radiopharmaceuticals for use in human PET imaging at the Brain Imaging Center at UCI. This will support collaborative research studies in the above two areas of interest at UCI. This research will also support investigations on other disorders such as Parkinson's disease and schizophrenia. Over the three-year period, our goal is to design and develop quantitative methods for a4b2 nAChR PET imaging studies using an agonist and an antagonist. We have successfully identified structural features in the pyridylether class of compounds that enable development of an antagonist and enhance the binding kinetics of agonists. Our preliminary efforts in developing agents with these properties have been promising. In this application developmental studies of 3 compounds will be carried out. These are putative agonist 2-18F-fluoro-3-[2-(3-(S)-pyrrolinylmethoxy)]pyridine (18F-nifene), and putative antagonists 5-(3 -18F-fluoropropyl)-(3-[2-(S)-pyrrolidinyl)methoxy]pyridine (18F-nifrolidine) and 5-(3 -18F-fluoropropyl)-(3-[2-(3-(S)-pyrrolinylmethoxy)]pyridine (18F-nifrolene). We hypothesize that 18F- nifrolene will have similar, faster kinetics like 18F-nifene. We propose to carry out pharmacological characterization of these compounds, optimize radiosynthesis, evaluate feasibility of quantitative PET studies and conduct test-retest studies. Based on these results radiation dosimetry measures in male and female rats with 18F-nifene as the agonist and either 18F-nifrolidine or 18F-nifrolene as the antagonist will be carried out. Upon complete characterization toxicity data of two agents will be obtained in order to file for an expedited investigational new drug application. Thus the outcome of this 3-year application would be to have an agonist and an antagonist for PET human imaging studies of the a4b2 receptor system. Continuation of this application will then study the role of these receptors in AD and nicotine dependency. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to advance our knowledge by employing imaging techniques using a small animal positron emission tomograph (microPET Focus 120). This instrument has high resolution and high sensitivity and will therefore allow the study of biochemical mechanisms with greater detail in small animals than has been possible before. The Brain Imaging Center (BIC), Center for Functional Onco-Imaging (CFOI), Departments of Pharmacology, Anatomy and Neurobiology, Neurobiology and Behavior, Biological Chemistry, Medicine, Transdisciplinary Tobacco Use Research Center (TTURC), Radiopharmaceutical Laboratories all at University of California-Irvine (UCI) have been involved in using imaging methodology to study normal and abnormal physiological processess. Researchers at UCI have a long tradition of using PET in human studies of various conditions. A large number of researchers at the various centers and Departments in UCI are involved in studying rodent models. The acquisition of a microPET scanner will allow researchers in numerous projects to study in vivo brain function with a resolution of about 1.5 mm or less. For the first time, investigators at UCI will be able to accurately assess functional activity in small regions in the brain noninvasively. Imaging research at UCI is unique in terms of the close association of investigators in basic animal research with clinical research as exemplified in the various programs. Studies carried out on the animal models will greatly enhance our ability to understand and interpret human studies. The new microPET scanner will be housed in Irvine Hall, where the BIC and Center for CFOI are located and houses the human PET scanners, the MC-17 medical cyclotron, radiopharmaceutical laboratories, MR scanners, Optical Imaging Lab and rodent animal facility. The microPET scanner will be under the direction of Dr. Jogesh Mukherjee, and a team from five different departments (Radiology, Medicine, Pharmacology, Neurobiology and Behavior and Biological Chemistry) that will provide administrative, scientific, and technical expertise necessary for the effective operation and maximal utilization of the microPET will be in place.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03) Biomarker Discovery and Validation and specific Challenge Topic 03-DK-101: "Discovery of biomarkers for disease risk, progression or response to therapy in diseases of interest to NINDK". Loss of insulin producing cells in the pancreatic islets, the endocrine component of pancreas, referred as beta cell mass (BCM), leads to an inability to manage blood sugar levels. This results in diabetes mellitus (DM), type 1 (TIDM) or type 2 (T2DM). T1DM, has previously been known as "insulin-dependent diabetes mellitus," (IDDM) or "juvenile diabetes." TIDM is a life-long condition in which the pancreas stops making insulin due to loss of BCM from an autoimmune response. T2DM, previously known as "noninsulin-dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes", is the most common form of diabetes. About 90 to 95 percent of people who have diabetes have T2DM. There are 23.6 million people in the United States, or 8% of the population, who have diabetes. The total prevalence of diabetes increased 13.5% from 2005-2007 according to the American Diabetes Association. Despite rigorous control of blood sugar, the majority of diabetic patients develop serious late-stage complications including retinopathy, nephropathy, neuropathy, microangiopathy and strokes. A long asymptomatic preclinical period characterized by gradual BCM loss precedes clinical T1DM. Methods to predict the development of clinical T1DM currently rely on the detection of multiple autoantibodies to islet- associated proteins combined with HLA genotyping. Improvement of the power and reliability of methods to predict diabetes would raise the possibility for pharmacological intervention during the preclinical phase and the honeymoon period to either slow down or arrest the ongoing destruction of the remaining [unreadable]-cells. It will also allow the monitoring and management of islet transplantation and help the development of immunosuppressive therapies. A non-invasive imaging approach to monitor BCM would enable earlier and better diagnosis/management of both TIDM and T2DM since pancreas is not an ideal organ for biopsy. Several groups have described non- invasive imaging approaches to detect and follow loss of BCM. Various radiotracer methods are currently underway to study differential pancreatic uptake include 6-deoxy-6-125I-iodo-D-glucose, 3H-monosaccharide D- mannoheptulose, 3H-glibenclamide, 2-14C-alloxan, 11C-acetate, 11C-methionine and 18F-FDG. Recently vesicular monoamine transporter-2 in pancreatic beta cells and in sympathetic nerve terminals that innervate islets and exocrine pancreas, was targeted using the specific radioligand 11C-DTBZ. 18F-FDOPA was used to diagnose infants with congenital hyperinsulinism. The ability to diagnose insulin-related disorders is in great need and the various approaches reported thus far have limitations in their ability to detect changes in BCM. Dopamine D2/D3 receptor expression have recently been demonstrated on rodent and human [unreadable]-cells using isolated islets and beta cell lines. These receptors are present in pancreatic islets where they co-localize with insulin producing granules and may serve as a surrogate marker for imaging alterations in BCM. We have used 18F-fallypride, a high affinity D2/D3 PET imaging agent for the study of islet cells and BCM in rodent pancreas with promising results. 18F-Fallypride binds to pancreas sections and isolated islet cells and is competed off by haloperidol, a D2/D3 inhibitor, indicating specific binding. Depleting [unreadable]-cells by treatment with streptozotocin reduced 18F-fallypride binding by 70% and immunostain for insulin confirmed [unreadable]-cell loss. Following IV 18F- fallypride administration, ex-vivo microPET imaging reveals 18F-fallypride in the pancreas. In order to maximize visualization of the pancreas (allowing clearance from adjacent organs) and monitor transplanted islet cells we propose in this application to develop 124I-epidepride (124I-EPID) (t1/2 124I=4.2 days) which will allow imaging over extended periods compared to 18F-fallypride (t1/2 18F=0.076 days). 124I-Epidepride is a new radiotracer suitable for extended imaging of dopamine D2/D3 receptors and may have applications in monitoring of [unreadable]-cell-loss in DM and provide tools to measure responsiveness to new therapies and evaluate efficiency of islet graft survival. PHS 398/2590 (Rev. 09/04) PUBLIC HEALTH RELEVANCE: This is a challenge request for developing a noninvasive PET imaging agent for the study of diabetes. Diabetes Mellitus is a major health problem currently affecting the US. The proposed approach will help in the management of this patient population. This application addresses broad Challenge Area (03) Biomarker Discovery and Validation and specific Challenge Topic 03-DK-101: "Discovery of biomarkers for disease risk, progression or response to therapy in diseases of interest to NINDK"

DESCRIPTION (provided by applicant): Serotonin abnormalities have been related to brain disorders which afflict a large number of people and include depressive disorders which affect over 18 million and Alzheimer's disease (AD) which affects over 5 million of the American population. Serotonin 5HT1A receptors have been implicated in these disorders and are being intensively studied. Our long-term goal is to develop noninvasive imaging methods of positron emission tomography (PET) that will aid in the early diagnosis of these disorders and may therefore be useful in treatment planning. Human studies of 5-HT1A receptor have been underway using 11C-WAY-100635, 18F- MPPF and 18F-FCWAY. Although these imaging agents have provided excellent insights into several human conditions, applications have been limited due to difficulty in production, short half-life, metabolic instability and moderate signal-to-noise ratios. With the support of an NIH R21 grant application, we have developed and evaluated a new imaging agent, 18F-Mefway which contains a fluorine-18 on a primary carbon to make the compound more stable to defluorination. Mefway has high affinity for 5HT1A receptors and in rodent brain imaging studies exhibited selective binding in hippocampus and cortex with limited binding in the cerebellum. PET studies in rhesus monkeys showed 18F-Mefway binding to temporal cortex, hippocampus, raphe and other brain regions with ratios of hippocampus to cerebellum = 10. Plasma analysis indicated the presence of approx. 20-30% of 18F-Mefway and no observed defluorination. The high ratios in specific brain regions such as the hippocampus suggest that 18F-Mefway has good potential as a PET imaging agent for 5HT1A receptors in humans. Our toxicity results of Mefway suggest that a radiotracer injection of 18F-Mefway is suitable for human use. Therefore, our overall goal in this NIH R33 application is to carry out first human studies with 18F- Mefway. Initial human radiation dosimetry studies will be carried out using a PET/CT scanner on 6 subjects. The toxicity data, radiation dosimetry and chemistry standard operating procedures will all be made available through the NIMH/SNIDD Tracer database for investigators at other institutions to use 18F-Mefway for their applications. Brain distribution of 18F-Mefway will be evaluated in normal volunteers in a test-retest paradigm to establish reproducibility and imaging methodology for quantitative analysis. In order to assess the diagnostic value of 18F-Mefway in aging and mild cognitive impairment (MCI) and AD, two important areas of expertise at UCI have come together in this application. Specific hypotheses will be tested using multidisciplinary approaches that include expertise in brain imaging techniques and the Alzheimer's Disease Research Center (ADRC) at the institute for Mental Impairments and Neurological Disorders (MIND). 18F-Mefway will be evaluated in well-characterized groups of control, MCI and AD subjects and results will be correlated with clinical outcome measures of 18F-FDG, MRI and neuropsychological tests. PUBLIC HEALTH RELEVANCE: Development of human imaging methods for serotonin receptors will help understand several brain disorders, including anxiety, depression, epilepsy and Alzheimer's disease. This grant application will specifically have implications in the potential diagnosis, treatment planning and therapeutics development for Alzheimer's disease.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop and evaluate PET imaging methods for measuring activity and distribution (mass) of brown adipose tissue (BAT). This is relevant to the current request for applications: "Human brown adipose tissue: Methods for measurement of mass and activity", RFA-DK-10-002. Type-2 diabetes mellitus, T2DM, previously known as "noninsulin-dependent diabetes mellitus" (NIDDM) or "adult- onset diabetes", is the most common form of diabetes. About 90 to 95 percent of people who have diabetes have T2DM. There are 23.6 million people in the United States, or 8% of the population, who have diabetes. The total prevalence of diabetes increased 13.5% from 2005-2007 according to the American Diabetes Association. BAT has been shown to have thermogenic properties and can reduce white adipose tissue. It may be an important drug target for preventing or treating obesity. Our goal is to develop new imaging methods that will allow measurement of BAT activity and mass. We propose to use the adrenergic system for measuring both activity and distribution (mass) of BAT. The direct involvement of adrenergic system (epinephrine/norepinephrine and [unreadable]3 adrenergic receptor) via the uncoupling protein (UCP) in BAT thermogenesis has been reported. The [unreadable]3 adrenergic receptor has therefore been pursued as a target for therapeutics development for BAT activation. We propose to investigate two approaches towards activation of BAT using the rodent model by using a [unreadable]3-adrenoreceptor agonist, CL 316243 and the norepinephrine transporter (NET) blocker, tomoxetine. Both approaches are expected to stimulate the adrenergic system and increase BAT 18F-FDG in MicroPET/CT imaging. For measuring BAT mass we will develop 11C-CL 316243 as a selective [unreadable]3-adrenergic agonist radiotracer for imaging BAT distribution and mass, and also evaluate the NET radiotracer 18F-MFP3 as a potential radiotracer for measuring BAT distribution and mass. One of our goals is to evaluate BAT activity and mass using 18F-FDG and 11C-CL 316243 in the diet-induced obesity rodent model. Radiation dosimetry studies of 11C-CL 316243 will be carried out on rodents for an exploratory Investigational New Drug (eIND) application for translation to human studies. Development of the adrenergic neurotransmitter receptor imaging methods will strongly complement ongoing imaging approaches for obesity and diabetes.

DESCRIPTION (provided by applicant): Nicotinic a4[unreadable]2 receptors have been implicated in neurodegeneration and are being studied extensively. At University of California-Irvine (UCI), we have several major programs that would gain from imaging nicotinic receptors. These include: 1) Alzheimer's Disease Research Center (ADRC) at the institute for Mental Impairments and Neurological Disorders (MIND);2) Program on studies related to nicotine dependence;3) Program in the early detection of lung cancer, and 4) Neurobiology of learning and memory. During the previous funding period we have successfully completed preclinical evaluation of a new imaging agent, 18F-Nifene which has high affinity for a4[unreadable]2 receptors and requires an imaging time of less than 60 minutes. In animal PET studies selective binding of 18F-Nifene in thalamus, lateral geniculate, cortex and other brain regions was observed with limited binding in the cerebellum, resulting in specific binding ratios of ~3. Plasma analysis indicated the presence of 18F-Nifene and no observed defluorination. The high ratios in specific brain regions and short scan time suggest that 18F-Nifene to be amongst the most suitable agonist that has good potential as a PET imaging agent for a4[unreadable]2 receptors in humans. Our toxicity results of Nifene suggest that a radiotracer injection of 18F-Nifene is suitable for human use. Therefore, one goal in this NIH application is to carry out first human studies with 18F-Nifene. Human radiation dosimetry studies will be carried out using a PET/CT scanner on 6 subjects. Brain distribution of 18F-Nifene will be evaluated in normal volunteers in a test-retest paradigm to establish reproducibility and imaging methodology for quantitative analysis. A second goal of the proposal is to complete the preclinical development of 18F-Nifrolene which is a putative antagonist for this receptor. Animal studies show high binding in receptor-rich brain areas with a scan time of approx. 90 mins, with specific binding ratios ~4. We propose to complete animal imaging, toxicity testing and radiation dosimetry of 18F-Nifrolene during this funding period. The availability of an agonist and antagonist will allow comparative studies of this receptor system in various disorders. The third goal of this application is to evaluate if 18F-Nifene is able to detect changes in the level of the neurotransmitter, acetylcholine in PET studies. This will be of great value to evaluate efficacy of acetylcholinesterase inhibitors used in AD. The overall proposed research in this application will also support investigations in other disorders such as Parkinson's disease and schizophrenia. PUBLIC HEALTH RELEVANCE: Development of human imaging methods for nicotine receptors will help understand several brain disorders, such as Alzheimer's disease, Parkinson's disease, learning and cognition as well as tobacco dependence and lung cancer. This grant application will specifically have implications in the potential diagnosis, treatment planning and therapeutics development for Alzheimer's disease.