S. Michael Owens - US grants

The long-term objective of this project is to use immunological approaches to describe the receptor-mediated effects of phencyclidine (PCP). This unique and promising approach involves the construction of the PCP receptor and ligand system with anti-drug and anti-idiotypic antibodies, respectively. This approach in other receptor studies has shown that appropriately modeled anti-drug antibodies can mimic the recognition pattern of the receptor. These anti-drug antibodies can then be used to stimulate formation of antibodies against the binding site of the anti-drug antibody (i.e., anti-idiotypic antibodies). These anti-idiotypic antibodies should contain an internal peptide image of the primary antibody binding site, which should bind to the neuroreceptor. Therefore, the goal of this research is to develop an immunological model for the PCP receptor and ligand system. The appropriate drug and orientation for molecular mimicry of the neuroreceptor will be determined in preliminary studies by production of rabbit antibodies against different orientations of the PCP molecule and against its more potent analogue, TCP (1-[1-(2-thienyl)cyclohexyl]piperidine), coupled to protein. The choice of an appropriate hapten will be based on RIA cross-reactivity studies with PCP and its analogues and cross-reactivity with the recently discovered endogenous peptide ligand for the PCP receptor. Next, mouse monoclonal anti-drug antibodies will be produced, followed by the production of rat monoclonal anti-idiotype antibodies. The resulting anti-idiotope antibodies will be studied as a new typeof ligand in a PCP receptor binding assay. Finally, the antigen binding fragments (Fab and/or Fab prime 2) of the antibodies will be administered in vivo to rats by central routes of administration to determine their behavioral effects and to dogs by both intravenous and intracerebroventricular injection to determine their pharmacokinetic parameters.

The long-term objective of this project is to use immunological approaches to describe the receptor-mediated effects of phencyclidine (PCP). This unique and promising approach involves the construction of the PCP receptor and ligand system with anti-drug and anti-idiotypic antibodies, respectively. This approach in other receptor studies has shown that appropriately modeled anti-drug antibodies can mimic the recognition pattern of the receptor. These anti-drug antibodies can then be used to stimulate formation of antibodies against the binding site of the anti-drug antibody (i.e., anti-idiotypic antibodies). These anti-idiotypic antibodies should contain an internal peptide image of the primary antibody binding site, which should bind to the neuroreceptor. Therefore, the goal of this research is to develop an immunological model for the PCP receptor and ligand system. The appropriate drug and orientation for molecular mimcry of the neuroreceptor will be determined in preliminary studies by production of rabbit antibodies against different orientations of the PCP molecule and against its more potent analogue, TCP (1-[1-(2-thienyl)cyclohexyl]piperidine), coupled to protein. The choice of an appropriate hapten will be based on RIA cross-reactivity studies with PCP and its analogues and cross-reactivity with the recently discovered endogenous peptide ligand for the PCP receptor. Next, mouse monoclonal anti-drug antibodies will be produced, followed by the production of rat monoclonal anti-idiotype antibodies. The anti-idiotope antibodies will be used for inhibition studies in a PCP receptor binding assay and both monoclonal antibodies will be used for immunohistochemical mapping of cross-reacting sites in the central nervous system. Finally, the in vivo behavioral effects after central nervous system routes of administration and pharmacokinetic parameters after i.c.v. and i.v. administration of the monoclonal antibodies fragments will be studied.

biomedical equipment resource; biomedical equipment purchase;

The long-term objectives for the applicant are to better understand the neurolological and metabolic consequences of phencyclidine (PCP) abuse and to develop immunotherapeutic methods for the treatment of drug abuse. The first objective is designed to test the hypothesis that PCP metabolites can lead to long-term, detrimental effects through irreversible binding to critical macromolecules in the central nervous system and at peripheral sites. For these experiments, an animal model will to provide a source of tissues for in vitro studies of the molecular mechanism(s) underlying the vulnerability to PCP metabolite covalent binding. The in vitro experiments will include studies of specific isoenzyme pathways and other potential targets of covalent binding in normal animals and in animals which are genetically deficient in the isoenzyme pathways responsible for generating the electrophillic metabolites. The research plan will include a series of complimentary biochemical, immunological, physical/chemical, tissue culture and neuroreceptor binding studies. When considered together, these molecular and whole animal data should allow us to better predict the neurobiological consequences of PCP abuse. In a second project, high affinity anti-PCP monoclonal antibodies (MAb) will be used as a treatment to block and/or reverse the acute toxicity of PCP. For these experiments, the Fab (antigen binding fragments) of anti-PCP MAb will be used to systematically study the ability of antibodies to affect the pharmacokinetics and acute toxicity of PCP in rats. First, about 613.5 grams of monoclonal anti-PCP Fab will be generated and purified. Then, using toxic doses of PCP, the effects of these anti-PCP Fab on PCP pharmacokinetics will be determined. Finally, with the knowledge gained in these pharmacokinetic studies, rational strategies will be developed to test the ability of these antibody antagonists to block and reverse the effects of toxic doses of PCP. Knowledge gained from these studies could lead to an important treatment for PCP toxicity in humans. During this funding period the applicant will continue to develop as a research scientist in the areas of immunology, experimental therapeutics, pharmaceutical biotechnology and neuroscience. Scientific growth will be aided through collaboration and consultations with other scientist and visits to other research facilities. Specific training will be obtained in the areas of molecular biology, tissue culture and neuroscience.

The long-term objective of this project is to develop short- and long-acting antibody-based medications for the treatment of drug abuse. Medications are badly needed since repeated use of these drugs can lead to toxicity, psychosis, violent behavior and addiction. These new medications could be used in an emergency room setting for rapidly reversing a drug overdose or in a treatment plan for a recovering addict. Although the long-term goal is to learn how to best use antibody-based "pharmacokinetic antagonists" for treating a wide range of drugs, the main focus of this project will be on phencyclidine (PCP). The experiments in this proposal are designed to systematically test the hypothesis that anti-PCP IgG monoclonal antibodies (MAb) and their antigen binding fragments (Fab) could be used to treat the medical problems associated with PCP abuse. The first studies will address the biopharmaceutical and clinical strategies for anti-PCP Fab treatment of PCP overdose in dogs. In the second studies, mouse MAb with a wide range of affinity constants (from about 1-300 nM) will be generated for use as potential long-acting antagonists of repeated PCP use. These antibody medications will then be used in rats to test our hypothesis that careful selection of antibody affinity can lead to an optimal balance between drug association with the antibody (to block drug effects), and drug dissociation from the antibody (to allow regeneration of antibody binding capacity). Combined pharmacokinetic and pharmacodynamic analysis of these experiments will allow us to design better clinical strategies for using antibody-based medications. In the final series of experiments, we will study the rates of partitioning into, and out of, the rodent brain without and with treatment with our antibody-based medications. These data will help to determine the ability of the MAb to redistribute the drug and the rate at which drugs of abuse enter and leave the brain. These integrated studies of pharmacokinetic, behavioral and central nervous system changes before and after antibody-based therapy will serve as a prototypic model that can be applied to treatment of drugs of abuse in humans.

DESCRIPTION (Applicant's Abstract): The long-term objective of this project is to develop antibody-based medications for the treatment of abuse of synthetic stimulants like methamphetamine (METH). A treatment is badly needed since repeated use of these drugs can lead to cardiovascular problems, severe depression, psychosis, and violent behavior. These new medications could be used in a treatment plan for a recovering addict or in an emergency room setting for rapidly reversing a drug overdose. The experiments in this proposal are designed to systematically test the hypothesis that monoclonal antibodies (MAb) and their antigen binding fragments (Fab) could be used to treat a range of medical problems associated with stimulant abuse. Although the eventual goal is to select a MAb that could be used for treating all of the METH-like synthetic stimulants, the main focus of the project will be on METH. Therefore, high affinity MAb against METH will be selected, and 200-400 g of monoclonal anti-METH IgG will be generated in a non-mammalian bioreactor. After production and purification of the IgG and Fab, preclinical testing will begin. To assess the potential use antibodies for treating addiction, a comprehensive series of studies following active and passive immunization (with the monoclonal IgG) will be conducted in a rat model of human "binge" drug use. These studies will include the behavioral evaluation of the effectiveness of immunotherapy after repeated METH doses over an extended period of time. For evaluating the use of anti-METH Fab for treating overdose, an integrated series of pharmacokinetic, behavioral and neurochemical studies after various types of treatments will be conducted. Finally, the pharmacokinetic and behavioral effects of the anti-METH Fab on METH toxicity will be studied in dogs at toxic doses in preparation for scaling up the therapy to humans. These integrated studies of pharmacokinetic, behavioral and neurochemical changes after antibody-based therapy will serve as a prototypic model that can be applied to treating the effects of other drugs of abuse.

DESCRIPTION (Applicant's Abstract): The long-term objective of this project is to develop antibody-based medications for the treatment of abuse of synthetic stimulants like methamphetamine (METH). A treatment is badly needed since repeated use of these drugs can lead to cardiovascular problems, severe depression, psychosis, and violent behavior. These new medications could be used in a treatment plan for a recovering addict or in an emergency room setting for rapidly reversing a drug overdose. The experiments in this proposal are designed to systematically test the hypothesis that monoclonal antibodies (MAb) and their antigen binding fragments (Fab) could be used to treat a range of medical problems associated with stimulant abuse. Although the eventual goal is to select a MAb that could be used for treating all of the METH-like synthetic stimulants, the main focus of the project will be on METH. Therefore, high affinity MAb against METH will be selected, and 200-400 g of monoclonal anti-METH IgG will be generated in a non-mammalian bioreactor. After production and purification of the IgG and Fab, preclinical testing will begin. To assess the potential use antibodies for treating addiction, a comprehensive series of studies following active and passive immunization (with the monoclonal IgG) will be conducted in a rat model of human "binge" drug use. These studies will include the behavioral evaluation of the effectiveness of immunotherapy after repeated METH doses over an extended period of time. For evaluating the use of anti-METH Fab for treating overdose, an integrated series of pharmacokinetic, behavioral and neurochemical studies after various types of treatments will be conducted. Finally, the pharmacokinetic and behavioral effects of the anti-METH Fab on METH toxicity will be studied in dogs at toxic doses in preparation for scaling up the therapy to humans. These integrated studies of pharmacokinetic, behavioral and neurochemical changes after antibody-based therapy will serve as a prototypic model that can be applied to treating the effects of other drugs of abuse.

9977816
OWENS

This award provides funding to support collaborative research between investigators at the University of Arkansas for Medical Sciences, Little Rock and the University of Arkansas, Fayetteville. This is highly venturesome research with the goal of incorporating into plants the genetic capability to produce antibodies to phencyclidine, a substance better known in drug abuse circles as PCP. Efforts will first be made with Arabidopsis thaliana, a plant commonly used in laboratory experimentation. If the capacity to produce and store large amounts of antibody can be introduced into this first plant, then the long-range goal of transferring this technology to an agronomic crop like soybeans will be attempted. This project has the potential to result in both a cheap way of producing large amounts of antibody for use in drug abuse treatment and a possible new highly lucrative crop to revitalize agriculture in Arkansas.

The goal of this transdisciplinary program project is to facilitate the rapid development of new medications for the treatment of medical problems associated with (+) -methamphetamine [(+)METH]abuse. This will be accomplished through a well-integrated and highly focused research program, designed to manufacture and test monoclonal antibody-based therapy for METH abuse and/or addiction. Project 1 (the core unit) will provide strong leadership, vital supply and support services that include large scale monoclonal antibody production, immunochemical and LC/MS/MS analysis for pharmacokinetic studies, and integrated data analysis of the research findings. Project 2 will use rat and monkey models of human drug dependencies to test the effectiveness of the medications in reducing the addictive properties of (+)METH. These studies will include testing of the antibodies in self-administration and drug discrimination paradigms. Project 3 will determine the efficacy, duration of action and safety of the medications in disease models of acute and chronic drug use. Thee animal models will span a range of human use patterns from infrequent usages to dangerous "binge" usage of (+)METH. Measures of locomotor activity and hemodynamic effects, along with changes in the general health status of the animals, will be used to determine the ability of the medications to reduce effects even in the presence of binge use of (+)METH. Project will involve developing the technology for maximizing large scale, low cost production of the antibodies through the development of advanced agrimedicine manufacturing technology. DNA that codes for anti-(+)METH antibody will be introduced into plants enabling the expression and accumulation of pharmaceutical grade monoclonal antibodies on a very large scale and at an acceptable cost. The results of the studies in this multifaceted program project will serve as the initial preclinical testing of these important new medications for the treatment of the devastating consequences of the (+)METH dependence and abuse.