John L. Neumeyer - US grants

health science research support; university;

The objective of this proposal is the continued synthesis and development of anti-convulsant drugs related to 1) those dopamine agonists which can be considered as congeners of apomorphine, 9,8-dihydro-6H-dibenzo[c,g]azonines, and amonotetralin, and 2) aminoisoquinolines, and their evaluation as anticonvulsant drugs in the Anticonvulsant Screening Project (ASP) carried out by the NINCDS. In this research program we shall also investigate 1) the relationship of dopamine agonist response on the basis of the activity on DA sensitive adenylate cyclase, the competition of binding to various 3H-ligands compared with their anticonvulsant activity both from the ASP screen and from audio- or photic-induced seizures in rodents and baboons; 2) the continued study of the chemistry of aminoisoquinoline to make structural changes in this heterocyclic system and to assess the SAR in a systematic approach to structural modification; 3) the study of the acid catalyzed rearrangement of opium alkaloids which would lead to novel aporphines and related structures of interest for biological evaluation; 4) the continued development of sensitive methods for the quantification of N-n-prophylnorapomorphine (NPA), 2,10,11-trihydroxyaporphine (TNPA) and certain pro-drug derivatives of these agents in biological fluids using electron capture gas chromatography in combination with mass spectrometry. In order to facilitate absorption and pharmacokinetic properties, pro-drug derivatives of the most active anticonvulsant compounds will be investigated.

adenosine; ligands; drug design /synthesis /production; xanthines; diuretics; drug screening /evaluation; drug metabolism; asthma; immunopharmacology; ribose; centrally acting drug;

The objectives of Phase II of this proposal are to continue the development and evaluation of molecular probes for D1 and D2 dopamine receptors. Dysfunctions of brain dopamine systems have been implicated in neurological, psychiatric and endocrine disorders and dopaminergic agents are widely prescribed to alleviate their symptoms. Brain dopamine receptors are the molecular targets of the most effective drugs used in the treatment of schizophrenia and Parkinson's disease. The cellular and subcellular distribution and mobility of these receptors remain poorly understood because the available receptor probes do not provide the resolution or detection capabilities required for this vital research. We propose to synthesize fluorescent and biotin probes for dopamine receptors using antagonist drugs as precursors. We will evaluate the affinity and receptor selectivity at D1 and D2 dopamine receptors, brain distribution and cellular distribution in progressive stages. Receptor- selective molecular probes will permit detection of the receptors by fluorescence, light, and electron microscopy and will be useful for mapping cellular and subcellular distribution of dopamine receptors, prior to and after drug treatment, for monitoring receptor mobility, and cell-sorting of dopamine receptor-containing cells. These novel compounds may expedite a clearer understanding of dopamine receptor function in the diseased state and drug-induced adaptive processes. They may furthermore create novel methodologies in psycho- and neuropharmacology research.

The nature of the adenosine receptor will be studied using adenosine agonists and antagonists modified for selectivity and possible affinity labeling. Adenosine plays a key role in physiological processes and currently no selective A1 or A2 antagonists have as yet been developed. Selective agonists and antagonists for adenosine receptors could have clinical importance in the treatment of asthma, as diurectics, respiratory stimulants and as central depressants or stimulants. The following series of adenosine A1 and A2 agonists and antagonists will be synthesized and evaluated: (a) alkyl substituted xanthines; (b) 8-thienyl substituted xanthines (c) 8- cycloalkyl substituted xanthines; (d) 8-phenylxanthine derivatives -functionalized sulfonamides; (e) thio and dithio xanthines; (f) functionalized 2-arylaminoadenosine derivatives; (g) fluorescent probes for adenosine receptors. The biological evaluation will be carried out in collaboration with the Laboratory of Bioorganic Chemistry, NIADDK, and Nova Pharmaceutical Corp. New products generated in Phase II studies will be further evaluated for possible commercial introduction as research tools to the expanding neuroscience community and evaluated as potential therapeutic agents.

A selective binding site for cocaine has been identified in brain membrane preparations from rodents, monkeys and humans. There is a high degree of correspondence between the relative potencies of various cocaine analogs in vivo and their binding affinity for the cocaine site in vitro. Information on the mechanism of action of cocaine may be gained by characterization of this receptor. Studies of this nature are dependent on the availability of a radiolabeled ligand with higher affinity and/or specific activity than tritiated cocaine. In Phase I, three novel cocaine analogs will be prepared in which the C-3 benzoyl group of cocaine is replaced by a p-fluorophenyl group, a modification which has been demonstrated to increase the potency of cocaine derivatives in behavioral studies above that of cocaine itself. The binding parameters of the analogs at the cocaine receptor in monkey brain will be measured. The most promising compound will be evaluated for cocaine-like activity in vivo by conducting behavioral studies in squirrel monkeys. This analog will be tritiated and its binding properties compared with those of tritiated cocaine. It is anticipated that a high affinity radioligand arising from this research will be a commercially valuable product for pharmacological research and drug screening applications. Phase II will involve chemical studies directed toward more potent analogs as well as more extensive biochemical and behavioral characterization in preparation for commercialization of products.

Functional disorders of dopamine (DA) receptor system has been implicated in endocrine, neurological and psychiatric conditions. Detailed pharmacological evaluation and biochemical and behavioral studies of DA receptors have been made possible by the synthesis of high affinity ligands that are selective for each of the receptor subtypes. Affinity labels, drug molecules that bind specifically and irreversibly to a particular biological system, are important tools in enzyme, hormone and neurotransmitter receptor research. Affinity labeling experiments with neurotransmitter receptors have provided a wealth of information regarding the structure and biochemical and physiological function of adrenergic, cholinergic and opioid receptors. In Phase I study, we have completed the synthesis, chemical characterization and radioligand binding assays of bromoacetyl, fumaramide ester and isothiocyanate derivatives of PPHT (D-2 agonist), spiperone (D-2 antagonist) and SKF 83566 (D-1 antagonist). These novel analogs retained the high affinity and selectivity of the parent ligand for the respective receptor system. Preliminary results also indicate that these agents block DA receptors irreversibly. During the Phase II we will continue the synthesis and evaluation of affinity labels for D-1 and D-2 receptors capitalizing on the promising results from Phase I study.

Dysfunction of serotoninergic (5HT) systems in the brain have been implicated in neurological and psychiatric conditions. Drugs that interact with brain 5HT 1A receptors are effective in relieving anxiety disorders. LSD and other hallucinogenic drugs have been shown to produce their psychoactive effects through stimulation of brain 5HT2 receptors. 5HT3 receptor antagonists are effective in controlling nausea during drug therapy. 5HT reuptake blockers are effective in treating depressive psychosis and in controlling appetite. The progress in the study of the role the 5HT systems in brain function has relied considerably on techniques of radiolabelling the 5HT receptors using ligands of high specific radioactivity. However this technique for monitoring and/or visualizing brain 5HT receptors has limitations. Receptor adaptation involves changes in the levels of receptors, receptor mobility, and the interaction with other cellular constituents. These critical properties of receptors are best monitored using fluorescent probes, which are sensitive to the microenvironment of the receptor. In Phase I of the project we propose to synthesize fluorescent probes as a novel approach to monitor 5HT receptor mobility in brain membranes. Fluorescent compounds will be coupled to 5HT receptor agonists and antagonists. We will first test the affinity and selectivity of these compounds for multiple 5ht receptors. Neural membranes at various levels of tissue organization will be exposed to the fluorescent probe, photobleached by laser beam, and the recovery of fluorescence will be monitored by fluorescence microscopy.

DESCRIPTION: (Adapted from Applicant's Abstract) The objective of this proposal is the synthesis and utilization of functionalized molecular probes specific for the imidazoline/guanidinium receptive site (IGRS), a potential important receptor involved in metabolic and cardiovascular regulation. IGRS-selective molecules will be chemically modified to generate 1) a radioiodinated ligand for cell-localization of IGRS and 2) a radioiodinated photoaffinity adduct for identification and structural characterization of the receptor's hormone binding subunit. Novel derivatives will also be synthesized for construction of an affinity matrix for protein purification. Imidazoline and guanidinium compounds such as clonidine and guanabenz elicit a wide variety of responses in both the central nervous system and peripheral tissues. Although many of these cellular effects are mediated by alpha2-adrenergic receptors, both functional and radioligand binding studies indicate that this class of pharmacologically active compounds interact with a cellular protein (IGRS) distinct from the alpha2-adrenergic receptor. Indeed, IGRS does not recognize catecholamines or other known neurotransmitters but does recognize an endogenous clonidine displacing substance purified from brain suggesting the existence of a previously unidentified hormonal/neurotransmitter-receptor system. Detailed analysis of this system and IGRS is limited by the lack of high-specific activity probes specific for the receptor protein.

DESCRIPTION (Adapted from applicant's abstract):Boron Neutron-Capture Therapy (BNCT) is a binary system for delivering intensely ionizing radiation to tumors using a combination of boron-10 labeled molecules and thermal neutrons in order to achieve a tumorcidal effect. Recent evidence has indicated that there are peripheral type benzodiazepine-binding sites (PBS) on viable tumor cells to which compounds can be specifically bound and that these structures do not become attached to both necrotic tumor and normal cerebral cortex. Ligands which are capable of binding selectively to PBS achieve high concentration levels on malignant cells. Such a ligand is the benzodiazepine antagonist PK 11195, a substituted isoquinoline. The initial objective of the Phase I effort will be the synthesis of several boron-containing isoquinolines structurally related to PK 11195 and their evaluation against malignant glioma cells, U251 human glioblastoma. If these studies are successful and such compounds prove useful in targeting glioma cells under in vitro conditions, then their evaluation under in vivo conditions in tumor-bearing animals would be undertaken during Phase II. The Phase I in vitro results will provide the direction for the preparation of other boron-containing isoquinolines as well as the carborane PBS agonists of the R05-4864 type and for their evaluation, ultimately in diseased animals, during Phase II.

Imidazoline and guanidinium compounds such as clonidine and guanabenz elicit a wide variety of responses in both the central nervous system and peripheral tissues. Although many of these cellular effects are mediated by alpha2-adrenergic receptors, both functional and radioligand binding studies indicate that this class of pharmacologically active compounds interact with a cellular protein (IGRS) distinct from the alpha2- adrenergic receptor. Indeed, IGRS does not recognize catecholamines or other known neurotransmitters but does recognize an endogenous clonidine displacing substance purified from brain suggesting the existence of a previously unidentified hormonal/neurotransmitter-receptor system. Detailed analysis of this system and IGRS is limited by the lack of high- specific activity probes selective for the receptor protein. The continued objectives of this proposal are the synthesis and utilization of functionalized molecular probes for the imidazoline/guanidinium receptive site (IGRS), a potentially important receptor involved in metabolic and cardiovascular regulation. IGRS selective molecules developed during the Phase I study will be modified to generate 1) radiolabeled ligands for cell localization of IGRS 2) a radioiodinated photoaffinity adduct for identification of ligand binding subunit 3) suitable ligand for receptor protein purification. Extensive SAR studies of cirazoline-type molecules will be carried out to generate IGRS specific molecules for functional studies.

DESCRIPTION (Adapted from the application): A program of medicinal chemistry and pharmacology is proposed for the synthesis and evaluation of novel dopamine D1 receptor ligands to treat addiction to cocaine and other psychomotor stimulant drugs of abuse. The impetus for this proposal comes from preliminary findings in preclinical studies showing that the D1 partial agonist SKF 83959 a 1- aryl-3-benzazepine may decrease abuse-related effects of cocaine with little side-effect liability. The applicant proposes to synthesize novel derivatives of racemic SKF 83959 that may have increased potency and longer duration of action. These will include enantiomers of SKF 83959; cogeners with protected hydroxy functions on the catechol moiety, and halogenated or alkylated derivatives. The pharmacology of newly synthesized ligands will be established with: 1) in vitro procedures to determine dopamine D1 receptor selectivity in radioligand binding experiments and 2) in vivo procedures to determine potency and duration of action in a primate species and to further evaluate agonist efficacy in behavioral assays of eyeblinking and reversal of catalepsy induced by dopamine D1 receptor blockade, This research is expected to result in one or more dopamine D1 partial agonists with increased duration of action and minimal side effects for further development as candidate anti-cocaine medications. PROPOSED COMMERCIAL APPLICATION: At present, there are no uniformly effective medications for the treatment of cocaine abuse and dependence, and this remains one of our most serious drug problems. The development of such effective medications would have significant commercial application both for development by the pharmaceutical industry and by the government.

Positron emission tomography (PET) is sensitive and specific non-invasive imaging technology that can provide information about the functional status of neurotransmitter system in-vivo. Recent effects have focused on the development of PET-based radiotracers for use in studies of the dopamine transporter (DAT) abundance and pharmacology. Changes in the density and function of DAT have been implicated in neurodegenerative and neuropsychiatric diseases such as Parkinson's disease, major depression, Huntington's chorea, schizophrenia, and attention deficit-hyperactivity disorders (ADHD). Out earlier studies have focused on fluorine-18 labeled tropane derivatives, such as FP-CIT, in which the N-methyl group of the tropane was replaced by a [18F] fluoropropyl group. During the Phase I project, we characterized the cerebral monoamine transporter binding affinity of a series of novel fluoralkyl-containing tropane derivatives which showed higher DAT affinity and selectivity than FP-CIT. These new ligands are attractive candidates for development of 18F-labeled PET radiotracers for clinical imaging DAT in human brain. Our objectives on this Phase II project are to further synthesize and pharmacologically evaluate novel- or O-fluoroalkyl tropane derivatives with a view toward one-step simplified 18F-radiolabeling. The most promising compounds' physiochemical properties (lipophilicity) will be evaluated. A facile and rapid method of synthesis of 18F labeled tropane derivatives will be developed for the most promising compound(s). The lead compound in this series (presently BRL-308) will be evaluated by PET imaging and pharmacokinetics, in non-human primates. PROPOSED COMMERCIAL APPLICATIONS: The fluoropropyl analogue of beta-CIT will be a useful radiopharmaceutical if labeled with radionuclides for tomographic imaging. [123I] FP-CIT is currently being marketed for sale to the Nuclear Medicine community. There is a need for [18F] FP-CIT for PET imaging primarily for the research community. The availability of a kit for easy production of [18F] FP-CIT or related improved ligands will meet this need.

DESCRIPTION (provided by applicant): The goal of this project is to develop a novel agonist radioligand for evaluating the proportion of D2 receptors in the high state. The D2high receptor is thought to be the functional form of the D2 receptor to which endogenous dopamine binds. An agonist radioligand should be more sensitive to endogenous DA levels than antagonist radioligands since, unlike antagonists, agonist radioligands bind preferentially to the receptor in its high affinity state. Thus, agonist radioligands have the potential to elucidate more detailed information on receptor subtype activity as a function of disease as well as DA system activity as a whole. The majority of available radioligands are based on D2 antagonists and, as such, do not specifically target the D2high state. Among the commercially available agonist ligands, none possess high selectivity for D2 over D3. In contrast, fluoroaporphines, which are the focus of this project, are D2 receptor agonists that specifically and selectively target D2high receptors. The objective of this project is, therefore, to develop 3H-labeled fluoroaporphines as radioligands for in vitro binding studies and for autoradiography to aid in the evaluation and study of the role of the D2high receptor function in the brain.

PROJECT SUMMARY The goal of this project is to develop commercial 18F PET radiopharmaceuticals for evaluating the proportion of D2 receptors in the high affinity state in human diseases, including Parkinson?s disease and restless legs syndrome. The D2high receptor is the functional form of the D2 receptor to which endogenous dopamine binds. An agonist radioligand should be more sensitive to endogenous DA levels than antagonist radioligands since, unlike antagonists, agonist radioligands bind preferentially to the receptor in its high affinity state. Thus, agonist radioligands have the potential to elucidate more detailed information on receptor subtype activity as a function of disease as well as DA system activity as a whole. The majority of known radioligands are based on D2 antagonists and, as such, do not specifically target the D2high state. Among the known agonist ligands, none possess high selectivity for D2 over D3, and more importantly, none of the agonist ligands have been successfully developed as 18F PET radioligands. Indeed, the development of an effective 18F-labeled D2 agonist radioligand has proven challenging, and there are no commercially available agonist PET radioligands for measuring D2 function in the brain. In contrast, fluoroaporphines, which are the focus of this project, are fluorinated D2 receptor agonists that specifically and selectively target D2high receptors in vitro and in vivo. Therefore, the objective of this project is to carry out the necessary preclinical evaluations in order to file an investigational new drug application (IND) and develop the fluoroaporphines as commercial 18F PET radiopharmaceuticals to aid in the diagnosis of dopamine-dependent disorders. The D2 agonist 18F PET radioligands will be used for: a) for early diagnosis of PD/LBD and RLS b) as a diagnostic biomarker in clinical trials, to measure the efficacy of medications being developed for PD, RLS, and other disorders involving the dysfunction of the dopaminergic system Such radioligands will be a superior tool for in vivo evaluation of the human brain, for earlier diagnosis of disease, and will foster the development of more targeted and effective therapies for PD, RLS, schizophrenia, addiction, and other neuropsychiatric disorders involving the D2high receptor. Such radioligands will have a widespread and positive impact on public health worldwide. These18F radiopharmaceuticals will have a positive impact on patients? lives by providing a way to get a correct diagnosis earlier and therefore obtain appropriate treatment earlier, and in turn by lowering the overall socio-economical burden to the patients and their families.