Ian S. Zagon, Ph.D. - US grants

Our laboratory has made the initial observations suggesting that endogenous neuropeptides are related to neuro-oncogenicity, and that alterations of endogenous opioid/opioid receptor interactions influence the course of neoplasia (Science 221:671, 1983). During the first 2 years of this grant, we have carefully defined the role of endogenous opioid systems in neural cancer. A major discovery was that endogenous opioids and opioid receptors are present in all types of human and animal tumors, neural and non-neural in origin. Our hypothesis is that endogenous opioids serve to regulate neoplasia through interaction with opioid receptors associated with tumor cells; this may reflect an autocrine mechanism. In this grant proposal, we continue to explore our thesis with a rigorously-defined murine neuroblastoma model. Prototypic opioids related to growth will be identified through drug displacement studies, and structure-function experiments in tissue culture. Binding assays will be used to establish opioid/receptor interaction, and will include assessment of saturability, binding affinity and capacity. Information as to precursors of growth-related opioids will be gained by immunocytochemical procedures. In vitro investigations will be correlated with physiological/pharmacological responsiveness to opioids in mice with transplanted neuroblastoma. The cellular mechanism of neuropeptide regulation will be studied biochemically and structurally, by opioid transport experiments using opioids conjugated to gold and examined by electron microscopy, and with flow cytometry. Finally, the opioid receptor associated with cell growth will be isolated and identified. Receptor characteristics including size and subunit composition, peptide maps, and binding function as studied by reconstitution experiments, will be explored. Receptors will be quantitated by immunodot assays, and receptor distribution assessed by immunocytochemistry and immunoelectron microscopy. Our research efforts will contribute to comprehending the etiology and pathogenesis of neural neoplasia, and will provide strategies for prevention and therapeutic intervention of neuro-oncogenesis. This research is part of a long-range program in cellular and molecular neurobiology which seeks to understand the fundamental principles underlying normal growth and abnormal growth of the nervous system.

This project is designed to explore spectrin and associated cytoskeletal proteins in regard to the structure and function of the developing nervous system. During the first 2 years of this grant we have discovered two subtypes of brain spectrin (240/235) and (240/235E) in the mammalian brain, and localized these isoforms with immunocytochemistry and immunoelectron microscopy. Brain spectrin (240/235) is located primarily in the axons and presynaptic terminals of neurons. Brain spectrin (240/235E) is found in the cell bodies, dendrites, and postsynaptic terminals of neurons, as well as in certain glial cell types. The ontogeny of these spectrin subtypes was explored in immunoautoradiography experiments, and closely examined with immunocytochemical procedures. Each spectrin subtype had a distinct pattern of expression and distribution in the developing nervous system. In this application we describe experiments which are designed to give us a better understanding of brain spectrin subtypes and associated spectrin binding proteins in regard to the development of the mammalian nervous system. The aims of this proposal are: (1) Determine the location and distribution of brain spectrin subtypes in the developing mouse brain using immunoelectron microscopy. (2) Quantitate brain spectrin (240/235E) with quantitative immunodot assay. (3) Define whether there are structural changes in the isoforms of spectrin during development using immunoprecipitation and peptide mapping techniques. (4) Examine whether there are two ankyrin (syndein) subtypes in the mammalian brain utilizing immunoblots and immunohistochemistry. (5) Determine the ontogeny of ankyrin subtypes in the brain with immunoblots, immunodots, immunohistochemistry, and immunoelectron microscopy. (6) Examine the expression of amelin during mammalian brain development employing the techniques outlined in (5). The studies proposed constitute important and novel inquiries into the functional significance of spectrin-like proteins in the binding proteins in neural cells and tissues. This investigation is part of an ongoing program in cellular and molecular neurobiology which seeks to understand the process of normal and abnormal brain development.

Our laboratory has made the initial observations suggesting that endogenous opioid systems are related to nervous system development, and that perturbations of neuro-ontogeny (Science 221:1179-1180, 1983). During the past 8 years of this project the role of endogenous opioid systems in brain development has been carefully defined. We have identified [Met]-enkephalin, derived from preproenkephalin A (PPE), as the native peptide that serves as an opioid growth factor (OGF). OGF regulates the proliferation of both neuronal and glial precursors through inhibitory channels. The source of this peptide appears to be both autocrine (germinative cells) and paracrine (macronneurons). OGF interacts with the nuclear-associated zeta opioid receptor to regulate growth. The binding subunits have been identified and characterized, and polyclonal and monoclonal antibodies generated to these polypeptides. In this grant proposal, we continue to explore the thesis that an endogenous opioid system is important to neurrobiological development. The aims of this proposal are: (1) Determine the embryogenesis of OGF and PPE gene expression in the rat brain by immunocytochemistry, Northern analysis and in situ hybridization. (2) Define the ontogeny of the zeta receptor quantitatively (immunodot assay) and qualitatively (Western blotting and peptide mapping). (3) Ascertain the location of the zeta receptor by immunocytochemistry, immunoelectron microscopy, and in vitro autoradiography. (4) Isolate, purify, and characterize the native zeta receptor, and assess binding function by reconstitution experiments. (5) Clone and sequence the cDNA for the zeta receptor. (6) Examine the OGF and zeta receptor in developing human brain using immunocytochemistry, in vitro autoradiography, Western and ligand blotting, Northern analysis, and in situ hybridization. This research will contribute to comprehending the processes shaping normal brain development, and should be useful in understanding the etiology of developmentally-based neurobiological dysfunction. This research is part of a long-range program in cellular and molecular neurobiology which seeks to define the fundamental principles underlying normal and abnormal brain development.

Our laboratory has made the initial observations suggesting that endogenous neuropeptides are related to neuro-oncogenicity, and that alterations of endogenous opioid/opioid receptor interactions influence the course of neoplasia (Science 221:671, 1983). During the first 2 years of this grant, we have carefully defined the role of endogenous opioid systems in neural cancer. A major discovery was that endogenous opioids and opioid receptors are present in all types of human and animal tumors, neural and non-neural in origin. Our hypothesis is that endogenous opioids serve to regulate neoplasia through interaction with opioid receptors associated with tumor cells; this may reflect an autocrine mechanism. In this grant proposal, we continue to explore our thesis with a rigorously-defined murine neuroblastoma model. Prototypic opioids related to growth will be identified through drug displacement studies, and structure-function experiments in tissue culture. Binding assays will be used to establish opioid/receptor interaction, and will include assessment of saturability, binding affinity and capacity. Information as to precursors of growth-related opioids will be gained by immunocytochemical procedures. In vitro investigations will be correlated with physiological/pharmacological responsiveness to opioids in mice with transplanted neuroblastoma. The cellular mechanism of neuropeptide regulation will be studied biochemically and structurally, by opioid transport experiments using opioids conjugated to gold and examined by electron microscopy, and with flow cytometry. Finally, the opioid receptor associated with cell growth will be isolated and identified. Receptor characteristics including size and subunit composition, peptide maps, and binding function as studied by reconstitution experiments, will be explored. Receptors will be quantitated by immunodot assays, and receptor distribution assessed by immunocytochemistry and immunoelectron microscopy. Our research efforts will contribute to comprehending the etiology and pathogenesis of neural neoplasia, and will provide strategies for prevention and therapeutic intervention of neuro-oncogenesis. This research is part of a long-range program in cellular and molecular neurobiology which seeks to understand the fundamental principles underlying normal growth and abnormal growth of the nervous system.

Our laboratory has made the initial observations suggesting that endogenous opioid peptides are related to nervous system development, and that perturbations of endogenous opioid/opioid receptor interactions markedly influence the course of neuroontogeny (Science 221:1179-1180, 1983). During the first 2 years of this project, the role of endogenous opioid systems in brain development has been carefully defined. We found that endogenous opioids modulate neurobiological maturation, and influence both cell proliferation, and differentiation. A major discovery was that endogenous opioid systems are involved in human brain development. Our hypothesis is that endogenous opioids serve to control brain development through interaction with opioid receptors associated with developing neural cells; this may reflect an autocrine mechanism of growth. In this grant proposal, we continue to explore this thesis using the developing rat cerebella, as a model. The aims of this proposal are: (1) Identify the prototypic opioids related to growth through drug displacement studies and structure-function experiments. The binding site related to growth will be fully characterized, including assessment of kinetics, saturation, and interactions. (2) determine the location and distribution of growth-related opioids by immunoelectron microscopy. (3) Investigate whether developing neural tissues synthesize opioid precursors using in situ hybridization. (4) Isolate and identify the opioid receptor related to neural growth, and characterize the receptor in terms of size, subunit composition, peptide maps, and binding function as studied by reconstitution experiments. Our research efforts will contribute to comprehending normal brain development, and will have impact on understanding the etiology or neurodevelopmental-based dysfunction. Information derived from these studies may be used in designing strategies for intervention in some developmental disorders of the nervous system. This research is part of a long- range program in cellular and molecular neurobiology which seeks to understand the fundamental principles underlying normal and abnormal brain development.

Pancreatic cancer is the 4th leading cause of cancer-related deaths in the U.S. Little is known about the origin, pathogenesis, or treatment of this neoplasia. The opioid growth factor (OGF), [Met5]-enkephalin, is a native inhibitory peptide that is particularly targeted to cell proliferative events. The repressive activity of OGF on cell growth is mediated by the zeta opioid receptor. CCF is a negative growth factor in pancreatic cancer both in vitro and in vivo, Blockade of OGF action by the potent opioid antagonist naltrexone increases pancreatic cancer cell proliferation, indicating the tonic activity of OGF. Both OGF and zeta receptor are in pancreatic tumor cells. This grant hypothesizes that a native opioid peptide inhibits the growth of human pancreatic cancer, and does so through a unique opioid receptor. To test this hypothesis, we propose to: (l) Examine the cellular mechanisms of endogenous opioid action on human pancreatic tumor cell function, including DNA synthesis and the cell cycle. (2) Investigate the autocrine production of the opioid peptide related to growth of human pancreatic cancer. (3) Define the regulatory properties of opioid peptide-receptor interaction with respect to human pancreatic cancer cells. (4) Clone and sequence the zeta opioid receptor in human pancreatic neoplasia, and elucidate the function of this receptor using overexpression studies. (5) Learn about the mechanisms of endogenous opioid activity with regard to the incidence and growth of human pancreatic tumors from xenografts in nude mice. These studies will contribute to comprehending the biology of human cancer, and may provide clues to strategies for the treatment of pancreatic neoplasia. Information derived from the proposed investigation would be the first to identify a natural inhibitory substance and its receptor that serve to regulate pancreatic cancer. This research is part of a long-range program in cellular and molecular oncology which seeks to define the fundamental principles underlying pancreatic cancer in humans.

DESCRIPTION: (Adapted from the applicant's abstract) The three described specific aims and approach to their accomplishment in this proposal are: 1) to determine the importance of opioid-receptor interaction on homeostatic cellular renewal of the diabetic corneal epithelium by studying OGF/receptor expression and the effects of excess OGF or blockade of OGF activity with naltrexone on DNA synthesis in diabetic rat corneal epithelium limbus and conjunctiva; 2) to define the role of an endogenous opioid system related to growth in the re-epithelialization of denuded corneal epithelium in diabetic rats by examining healing and integrity of the repaired epithelium after systemic and topical treatment with naltrexone of rats subjected to corneal abrasions; and 3) to ascertain the influence of an endogenous opioid system related to growth in the repair of corneal wound healing by studying the effect of opioid receptor blockade on healing of wounded epithelium in human corneas in organ culture.

DESCRIPTION: (Applicant's Abstract) The corneal epithelium modulates fluid transport for normal stromal hydration and corneal transparency, and serves as an anatomical and physiological barrier against ocular infection. This epithelium is in a constant state of renewal, and injury to this epithelium requires prompt resurfacing in order to re-establish visual function. Tools to achieve research and clinical applications to corneal diseases at the molecular level offer exciting avenues for advancement, but are stymied by the need for manipulating gene activity. Progress has been made in gene therapy through the development of a particle-mediated gene transfer delivery system (gene gun), a technique shown to be a rapid, highly efficient, and nontoxic method for transfection of genes both in vivo and in vitro. In this grant we propose to establish the gene gun as a valuable research tool in corneal epithelial biology, obtain preliminary information for the efficacy of the gene gun as a device for clinical treatment, and document the importance of the interaction of a native inhibitory peptide, opioid growth factor (OGF) with its receptor (OGFr) at the molecular level as a system regulating the homeostasis and healing of the ocular surface epithelium in humans and animals. The specific aims include: 1. Determine the importance of OGF-OGFr interfacing on maintenance of rat corneal epithelium by examining the consequences of molecular perturbation of OGF receptor gene and protein activity. 2. Define the role of an endogenous opioid system related to wound healing of the rat corneal epithelium following genetic alteration of the OGF receptor gene and protein expression in rat. 3. Ascertain the significance of OGF and OGFr function during wound healing of the human corneal epithelium using organ culture of tissues genetically modified to yield an excess or deficit of OGF receptor. Information derived from these studies will contribute to a breakthrough understanding of gene delivery systems to the corneal surface. Moreover, these investigations will simultaneously acquire invaluable knowledge about the molecular basis by which an endogenous opioid system relates to corneal epithelial homeostasis, and the restoration of corneal integrity following injury. Ultimately, such data can be employed to design molecular strategies to remedy visual dysfunction.

DESCRIPTION (provided by applicant): Corneal erosions/abrasions ranging from superficial defects to full thickness epithelial lesions are found in 50% of diabetic patients, and this condition is termed diabetic keratopathy. Moreover, difficulty with corneal re-epithelialization and persistent epithelial defects/recurrent erosions is associated with the use of donor corneas from diabetic patients, and with corneal epithelial removal during vitrectomy in diabetic individuals. Such corneal epithelial defects can result in infectious corneal ulcers, secondary scarring, and loss of vision. Compelling evidence shows that blockade of opioid-receptor interactions by the opioid antagonist, naltrexone (NTX), restores corneal epithelial wound healing in uncontrolled diabetes. This grant is designed to make the transition from bench to bedside and explores the hypothesis that topical application of NTX will prevent and/or ameliorate corneal epithelial wound healing complications related to Type 1 diabetes. A multi-disciplinary research team consisting of a basic scientist, two ophthalmologists, and a biostatistician have formed a partnership in order to reach the full therapeutic potential of this advance in cell and molecular biology. In the R21 phase (with completion in the R33 phase), this hypothesis is tested in animal models of well-controlled Type 1 diabetes as to the toxicity and efficacy of NTX in the homeostatic (unwounded) and injured corneal epithelium. In order to prepare for FDA requirements, two species of animals are utilized: rat and rabbit. The grant utilizes a well-documented and reliable model of corneal re-epithelialization in the rat and rabbit, induction of Type 1 diabetes by injection of streptozotocin (STZ) (rats) or alloxan (rabbits), and subcutaneous insulin pellets to maintain normoglycemia. The R21 studies the effects of NTX on uninjured (Specific Aim #1) and injured (Specific Aim #2) corneal epithelium of rats, and the uninjured rabbit corneal epithelium (Specific Aim #3). The R33 investigates the safety and efficacy of NTX treatment in corneal abrasions in the diabetic rabbit (Specific Aim #1). If the animal experiments successfully show a non-toxic dose with efficacy, we will perform due diligence testing under the guidelines of the Food and Drug Administration (Specific Aim #2). These data will be used to secure an IND number in order to pursue clinical trials, and as evidence for Institutional Review Board approval to conduct Phase I clinical trials. The proposed use of biotherapy with NTX is an innovative approach whereby the patient's own growth regulatory mechanisms are manipulated to correct complications from diabetes.

Project Summary Diabetes is increasing globally and more than 9% of the US population has diabetes, with an additional 86 million pre-diabetic adult Americans. Type 1 diabetes mellitus (T1D) affects nearly 1.5 million Americans, accounting for almost $18 billion annually in healthcare costs. Diabetic-related complications such as retinopathy and cardiovascular disease receive much public attention, and 3 corneal disorders including keratopathy, dry eye, and surface insensitivity lead to vision loss, elevated healthcare costs, and decreased job productivity. This application focuses on the Opioid Growth Factor (OGF) ? Opioid Growth Factor Receptor (OGFr) regulatory system and blockade by naltrexone (NTX) to prevent ocular surface complications arising from Type 1 diabetes (T1D). Preventive intervention of diabetes-related complications remains an unmet medical need. The underlying hypothesis in this proposal is that the OGF-OGFr axis becomes dysregulated during the development of diabetes leading to over-expression of the inhibitory peptide and/or dysregulation of OGFr thus causing diabetes- associated ocular surface complications. The proposed research will i) Determine the temporal course and magnitude of defects in the OGF-OGFr regulatory pathway during the development of T1D in rats in order to properly initiate preventive therapy, 2) Determine whether topical or systemic administration of NTX prevents or delays the appearance of ocular surface complications. 3) Identify other molecular and protein biomarkers related to diabetes such as delayed corneal wound healing, dry eye, and insensitivity, and determine whether NTX intervention alters their expression and function. This knowledge will advance precision medicine approaches for the prevention and treatment of diabetic complications. This application represents the culmination of several decades of research determining that OGF-OGFr blockade with NTX is effective at treating diabetic ocular surface complications, and for the first time, will determine the ability of such blockade by NTX to prevent diabetic ocular surface complications. There is evidence that diabetes is accompanied by dysregulation in expression of endogenous opioids (i.e., OGF) and their receptors leading to an elevated expression of inhibitory growth factors. For more than 2 decades our laboratory has conducted research to determine the role of this regulatory axis in contributing to diabetic complications in diabetic rat, mouse, and rabbit and demonstrated that topical NTX reverses dry eye to normal tear production, accelerates delayed corneal wound healing, and restores the diabetic complication of decreased corneal sensitivity to normal in these diabetic animals. The proposed research will determine for the first time the ability of OGF-OGFr axis blockade by NTX to prevent these complications. Our research team has clinical and basic science expertise in all aspects of this research and will be able to rapidly translate warranted findings to the clinic.