James M. Cook, Ph.D. - US grants

Throughout history pathological or excessive anxiety has been clearly designated as undesirable and the understanding of is origin and treatment are a major concern. The benzodiazepines employed to treat anxiety are a group of compounds with wide therapeutic application as anxiolytics, anticonvulsants, hypnotics and muscle relaxants. Although these agents are extremely important in treatment of disease states, their exact mechanism of action remains controversial. Recently it has been reported that 3-ethoxycarbonyl-Beta-carboline(2), 3-methoxycarbonyl-Beta-carboline(1), 3-t-butoxycarbonyl-Beta-carboline(3), and 3-hydroxymethyl-Beta-carboline(4) all potently inhibit [3H] diazepam binding to benzodiazepine receptors and antagonize the anticonvulsant effects of diazepam. More importantly, in contrast to the "pure" benzodiazepine antagonist R015-1788, these Beta-carbolines have been shown, in our laboratories, to possess intrinsic effects of their own, opposite to those of the benzodiazepines, and the effect is different for each compound. For example, (1) was shown to be a convulsant, (2) a proconvulsant (anxiogenic in monkeys), (4) increased sleep latency, reduced total and non-REM sleep in rats without effecting convulsions, while the t-butyl ester(3) was the first Beta-carboline to exert partial agonist/antagonist activity in vivo. In this vein, different series of 3-hydroxymethyl-, 3-alkylketo-, and 3-alkoxycarbonyl-substituted Beta-carbolines will be snythesized and tested in vitro (synaptosomal membranes) and in vivo (mice, rats, monkeys) to determine what structural requiremens are necessary for potent selective, antagonist or agonist activity; the 3-alkylketo compounds are important for they cannot be metabolized to the inactive acid(5). Since the effects reported for Beta-carbolines are different from those reported for R015-1788, alkylating agents based on the structure of Beta-carbolines will be prepared to label the putative antagonist site(s) of benzodiazepine receptors and data compared to that for benzodiazepine irreversible inhibitors irazepine and kenazepine. Knowledge gained from the above experiments will: 1) result in preparation of selective benzodiazepine antagonists and nonbenzodiazepine agonists, 2) determine whether Beta-carbolines bind to the same receptor site(s) as do benzodiazepines, 3) determine whether benzodiazepine receptors are involved in the physiologic control of sleep (3HMC work), and 4) provide a better understanding of the physiological processes related to, or regulated by the benzodiazepine receptor complex. In addition, gram quantities of analogs of 1-4 will be prepared and screened in vivo to separate out the intrinsic effects of Beta-carbolines, and to design agents specific for interaction with one Bz receptor subtype in preference to another.

During the last several years a number of macroline-related indole alkaloids have been isolated from Alstonia macrophylla Wall, 1a-e Alstonia muelleriana Domin, 2a-f and other Alstonia species. As illustrated in Scheme I, the macroline bases consist of both monomeric and bisindale alkaloids, the complex structures of which have not fallen to total synthesis to date. Recently, the stereospecific formation of 1,3-disubstituted tetrahydro Beta-carbolines by the Pictet-Spengler reaction accompanied by complete transfer of chirality from the chiral amino acid ester of tryptophan to position-1 of the tetrahydro Beta-carboline has been developed in our laboratory in the Na-H, Nb-benzyl series, as well as the Na-CH3, NI series. This technology provides an extremely facile method to construct rings A,B,C, and D of the macroline alkaloids in optically active form. Ring E will then be added in stereospecific fashion via an intramolecular (3,3) sigmatropic rearrangement (an ester enolate Claisen rearrangement). It is proposed then to expand and exploit these methods to prepare the optically active macroline alkaloids, macroline 1, alstophylline 6, alstonisine 7, alstonerine 8, suaveoline 9, and to conclude this investigation by the total synthesis of the bisindole alkaloid, macralstonine 4 (C4-3H52N405). The synthesis of 4 is at a very exciting stage for the critical process of bond formation between the monomeric units of macroline 1 (drived from villastonine) and alstophylline (plant derived) has been carried out thus assuring success in this phase of the study. Philosophically, the approach is a general one capable of future extension to other Alstonia alkaloids including alkaloid H and macrosalhin. It has been suggested by LeQuesne that 1, or a suitable equivalent is the biosynthetic precursor for the macroline portion of the Alstonia bases. Intermediates related to 1 can later be labeled and employed to study the biogenesis of this family of alkaloids. Since 4 has been shown to potently lower blood pressure in dogs, the intermediates which occur late in the synthetic sequence and the target alkaloids will be screened for antihypertensive activity in SHR. Furthermore, Beta-carboline intermediates formed early in the route will be screened in vitro (benzodiazepine receptor) and in vivo (male mice, later rats) in regard to their abilities to antagonize the effects of diazepan, decrease sleep time in animals and to reverse the effects of benzodiazepine-alcohol or barbituate induced CNS depression in laboratory rats. In fact, 3-hydroxymethyl Beta-carboline and related Beta-carbolines have been shown to do just this.

Throughout history pathological or excessive anxiety has been clearly designated as undesirable and the understanding of its origin and treatment are a major concern. The benzodiazepines (Bz) employed to treat anxiety are a group of compounds with wide therapeutic applications as anxiolytics, anticonvulsants, hypnotics and muscle relaxants. These agents are now felt to exert their physiological effects via the GABA/Bz/C1 complex. Drug abuse, tolerance, and withdrawal continue to be a problem with benzodazepines. These phenomena also occur in the case of cocaine and morphine. The present work is designed to determine if the biological response mediated by the Bz1 or the Bz2 receptor subtype is responsible for abuse and for withdrawal in the Bz series. Bz1 receptors are proported to mediate the anxiolytic effects at BzR, while Bz2 receptors are reported to elicit the sedative-hypnotic effects. If one of these receptor subtypes is determined to be more important in abuse and/or withdrawal than the other, a better rational for drug design will be at hand. Recently, a series of rigid, planar dihydropyridodiindoles have been synthesized in our laboratory and shown to demonstrate potent affinity (5 nM) for benzodiazepine receptors (BzR) in vitro. It is felt these rigid analogs may conform to one receptor subsite, but will not be able to rotate, conformationally, to bind to a second subsite. Moreover, if either Bz1 or Bz2 selective ligands cue to cocaine or morphine in discriminative stimulus paradigms this would suggest that "downstream" in the biological response there exists a common biological event for these drugs. The protocol here is to first characterize Bz receptors, and then to design ligands specific for either Bz1 or Bz2 sites. Information gained from the above studies will: 1) result in the preparation of selective benzodiazepine antagonists and nonbenzodiazepine agonists, 2) determine whether or not the pyridodiindoles 2 bind to the same inverse agonist receptor site(s) as beta-carbolines, 3) determine the effect of benzodiazepine receptors on anxiety, sleep, convulsions, and memory by providing a better understanding of the physiological processes influenced by the GABA/Bz/C1 complex. In addition, gram quantities of analogs related to 4, 7, 8, 9, and 10 will be prepared and screened in vivo to differentiate the intrinsic effects of beta-carbolines, and to design agents specific for interaction with one Bz receptor subtype in preference to another. These Bz subtype selective ligands can then be employed to determine whether the effects mediated by a Bz receptor subtype are the same effects that other drugs of abuse such as cocaine, alcohol and morphine cue to in discriminative stimulus paradigms.

Throughout history pathological or excessive anxiety has been clearly designated as undesirable and the understanding of its origin and treatment are a major concern. The benzodiazepines (Bz) employed to treat anxiety are a group of compounds with wide therapeutic applications as anxiolytics, anticonvulsants, hypnotics and muscle relaxants 6,7. These agents are now felt to exert their physiological effects via the GABA/Bz/C1 complex 2,45. Recently, a series of rigid, planar dihydropyridodiindoles have been synthesized in our laboratory and shown to demonstrate potent affinity (5 nM) for benzodiazepine receptors (BzR) in vitro 17. The parent compound 2 (X=H) exhibited inverse agonist activity in vivo 17. Moreover, the 2-methoxy analog 4e was a potent proconvulsant, while the 2-chloro analog 4d demonstrated antagonist activity 17 similar to Ro 15-1788. It is felt these rigid analogs may conform to one receptor subsite, but will not be able to rotate, conformationally 21,61, to bind to a second subtype. In addition to enhanced Bz1 or Bz2 selectivity, these rigid planar, active ligands will be employed to model the pharmacophore for BzR. Based on these leads, different series of rigid 4, 7, 10, and semirigid 8, 9 analogs will be synthesized and tested in vitro (synaptosomal membranes) and in vivo (mice, rats, monkeys) to determine what structural requirements are necessary for potent selective (Bz1, or Bz2) inverse agonist, antagonist or agonist activity; the pyridodiinodoles 4 are important for they should possess long half-lives in vivo. Since the effects observed for beta-carbolines 12, 2 and 4e17 are different from those reported for Ro 15-1788 15, irreversible inhibitors based on these beta- carboline analogs will be prepared to label the "proposed" 32,59,60 discrete inverse agonist/antagonist receptor site. Information gained from the above studies will: 1) result in preparation of selective benzodiazepine antagonists and nonbenzodiazepine agonists, 2) determine whether or not beta-carbolines bind to the same receptor sites(s) as do benzodiazepines, 3) determine the effect of benzodiazepine receptors on anxiety 4,9,22,23, sleep, 28 conclusions 27, and memory 29 by providing a better understanding of the physiological processes influenced by the GABA/Bz/C1 complex 2, 45. In addition, gram quantities of analogs related to 4, 7, 8, 9, and 10 will be prepared and screened in vivo to separate out the intrinsic effects of beta-carbolines, and to design agents specific for interaction with one Bz receptor subtype in preference to another.

biomedical equipment resource; biomedical equipment purchase;

The understanding and treatment of pathological anxiety including panic disorders (14) have long been a prime concern in regard to mental health. In a recent study it was reported, people who suffer panic disorder or panic attacks are as likely to contemplate or attempt suicide as patients with other mental disorders such as major depression. From 3 to 10% of the adult population suffer from panic attacks during their lives. The benzodiazepines (BzR) used to treat these diseases have been shown to exhibit a broad spectrum of pharmacologic efficacies including anticonvulsant (27), sedative-hypnotic (27), muscle-relaxant (28), and anxiolytic/-anticonvulsants (13) which are devoid of the myorelaxant- sedative side effects of the benzodiazepines.(13,20,27,28). It is conceivable that these anxioselective anxiolytics might also exhibit decreased abuse potential, as well as reduced symptoms of withdrawal. In this regard, recent results(15,16) from our laboratory are exciting. A chemical and computer assisted analysis of the pharmacophore for agonists at the BzR has been executed(15). Based on this model the 6- propyl ether (6 PBC) (16) has been synthesized and screened in mice. This new agent was found to elicit anxiolytic/anticonvulsant activity, but was completely devoid (16) of the muscle relaxant/ataxic effects which occur with the benzodiazepines. More importantly, 6 PBC (16) completely antagonized the muscle relaxant effects of diazepam while still producing the anxiolytic effect. Outlined in Schemes III-IX are rigid and semi-rigid ligands which will be prepared to define the exact spatial dimensions of the agonist binding domain (see Figures 4-8) at lipophilic regions, as well as the importance of electron density on the ligands at phi 1 and phi 2. These agents will be synthesized and then tested in vitro and in vivo (mice/rats) for their efficacy. The biological data and SAR will be programmed into the E/S-390 (SYBYL) system to further define the pharmacophore for agonists. Since the principle goal is the synthesis of selective anxiolytic/anticonvulsants, the spatial dimensions and electron density required for selective agonist activity will be determined. This would constitute a real step forward in the search for selective anxiolytics. By the very nature of the computer-assisted design of the ligands depicted in Schemes III-IX, many of these bases will exhibit agonist activity; our principal interest lies in those which elicit a selective agonist profile of activity. CoMFA analysis (17,18) will be executed on these latter analogs whenever the SAR/biology is available. Characterization of the BzR at the molecular level is crucial for understanding the biochemical mechanisms which underlie anxiety (29), including panic disorders (14), and convulsions 27, as well as the design of selective agents (agonists) to treat these disease states. It is believed, the successful execution of the above studies will have far reaching effects in medicinal chemistry and neurobiology.

The understanding and treatment of pathological anxiety including panic disorders (14) have long been a prime concern in regard to mental health. In a recent study it was reported, people who suffer panic disorder or panic attacks are as likely to contemplate or attempt suicide as patients with other mental disorders such as major depression. From 3 to 10% of the adult population suffer from panic attacks during their lives. The benzodiazepines (BzR) used to treat these diseases have been shown to exhibit a broad spectrum of pharmacologic efficacies including anticonvulsant (27), sedative-hypnotic (27), muscle-relaxant (28), and anxiolytic/-anticonvulsants (13) which are devoid of the myorelaxant- sedative side effects of the benzodiazepines.(13,20,27,28). It is conceivable that these anxioselective anxiolytics might also exhibit decreased abuse potential, as well as reduced symptoms of withdrawal. In this regard, recent results(15,16) from our laboratory are exciting. A chemical and computer assisted analysis of the pharmacophore for agonists at the BzR has been executed(15). Based on this model the 6- propyl ether (6 PBC) (16) has been synthesized and screened in mice. This new agent was found to elicit anxiolytic/anticonvulsant activity, but was completely devoid (16) of the muscle relaxant/ataxic effects which occur with the benzodiazepines. More importantly, 6 PBC (16) completely antagonized the muscle relaxant effects of diazepam while still producing the anxiolytic effect. Outlined in Schemes III-IX are rigid and semi-rigid ligands which will be prepared to define the exact spatial dimensions of the agonist binding domain (see Figures 4-8) at lipophilic regions, as well as the importance of electron density on the ligands at phi 1 and phi 2. These agents will be synthesized and then tested in vitro and in vivo (mice/rats) for their efficacy. The biological data and SAR will be programmed into the E/S-390 (SYBYL) system to further define the pharmacophore for agonists. Since the principle goal is the synthesis of selective anxiolytic/anticonvulsants, the spatial dimensions and electron density required for selective agonist activity will be determined. This would constitute a real step forward in the search for selective anxiolytics. By the very nature of the computer-assisted design of the ligands depicted in Schemes III-IX, many of these bases will exhibit agonist activity; our principal interest lies in those which elicit a selective agonist profile of activity. CoMFA analysis (17,18) will be executed on these latter analogs whenever the SAR/biology is available. Characterization of the BzR at the molecular level is crucial for understanding the biochemical mechanisms which underlie anxiety (29), including panic disorders (14), and convulsions 27, as well as the design of selective agents (agonists) to treat these disease states. It is believed, the successful execution of the above studies will have far reaching effects in medicinal chemistry and neurobiology.

DESCRIPTION (Adapted from applicant's abstract): The understanding and treatment of pathological anxiety including panic disorder have long been a prime concern in regard to mental health. In a recent study it was reported, people who suffer panic disorder or panic attacks are as likely to contemplate or attempt suicide as patients with other mental disorders such as major depression. From 3-10% of the adult population suffer from panic attacks during their lives. The benzodiazepines (Bz) used to treat these diseases have been shown to exhibit a broad spectrum of pharmacologic efficacies including anticonvulsant, sedative-hypnotic, muscle-relaxant and anxiolytic effects (2-11). Despite the clinical effectiveness of these drugs, there is a need for selective anxiolytic/anticonvulsants (10-12) which are devoid of the myorelaxant/ataxic and sedative side effects of the benzodiazepines. In this regard recent results (14, 20) from our laboratory are exciting. Based on a chemical and computer-assisted analysis of the inclusive pharmacophore for the BzR, the pharmacophoric descriptors for agonist versus inverse antagonist have been defined. More importantly, the 8-acetylenic substituted imidazobenzodiazepines 1a and 2a have been shown to be 40-70 times more selective for Bz5 (alpha5beta2gamma2) receptor subtypes, while betaCCt 5 has been shown to be 20 times more selective for Bz1 (alpha1beta2gamma2) receptor subtypes. These are the most selective ligands ever reported for alpha5 and alpha1 BzR subtypes, respectively, and serve as lead compounds in the search for BzR subtype specific agents. Rigid and semi-rigid (constrained) ligands will be employed to develop agents selective for Bz1 (Schemes 7,8,9 and 12), and Bz5 (Schemes 4,5,6,10 and 11) receptor subtypes. The goal is to develop ligands that are more than 150 times more selective for either Bz1 or Bz5 receptor subtypes in order to determine which biological function(s) is mediated by which subtype. It is felt that Bz subtype selective agents provide one of the best opportunities to develop anxioselective anxiolytic and anticonvulsants devoid of side effects and to understand the complex physiological processes mediated by the GABA/BzR receptor ion channel. Once selective ligands for these sites are developed, Bz2 (alpha2) and Bz3 (alpha3) as well as Bz4 (D1) alpha4 and Bz6 (D1) alpha6 receptor subtypes will be investigated. Characterization of the pharmacology of BzR receptors at the subtype level is crucial for understanding the physiological processes which underlie anxiety, including panic disorder convulsions, and sleep disorders as well as the design of selective agents (agonists) to treat these disease states with decreased abuse potential.

DESCRIPTION (provided by applicant): The understanding and treatment of pathological anxiety have long been a prime concern in regard to mental health. Alterations in GABAA function from controls are known to occur in many anxiety disorders including panic disorder, epilepsy, hypersensitive behavior, phobias, schizophrenia, alcoholism, Anglemans syndrome and Rhetts syndrome, as well as effects which lead to/or complicate drug abuse. The 1,4-benzodiazepines, employed to treat anxiety disorders as well as sleep disorders exhibit anxiolytic, anticonvulsant, muscle relaxant/ataxic, sedative- hypnotic and amnestic effects. Despite the clinical effectiveness of these drugs, there is a need for selective anxiolytics and anticonvulsants which are devoid of the sedative-hypnotic, muscle-relaxant, ataxic and amnestic effects. Recently, with the aid of computer modeling and chemical synthesis, we have developed an orally active anxioselective anxiolytic (1). This ligand exhibits very poor affinity at the a1 receptor subtype of the BzR/GABAergic system and has a near perfect efficacy profile in oocytes [alpha1beta3gamma2 (no efficacy), alpha2beta3gamma2 (full agonist), alpha3beta3gamma2 (50% agonist), alpha5beta3gamma2 (10-15% agonist);no affinity at alpha4/alpha6 BzR/GABAA receptors]. This ligand is orally active as an anxiolytic at 1mg/kg but has no sedative-hypnotic, muscle relaxant, or ataxic effects in rodents even up to 300 mg/kg. It does not generalize to chlordiazepoxide in the discriminative stimulus paradigm, nor has it shown any amnestic effects. It is also active in primates in the conflict paradigm with no signs of muscle relaxation, or ataxia or sedation. The prodrug (2) of this anxiolytic agent exhibits a similar profile in rodents. These two anxiolytics serve as the lead compounds in this study directed toward the synthesis of alpha2 and alpha3 subtype selective ligands. Moreover, these agents should be long-lived, metabolically stable, water soluble, orally active anxiolytic agents for the clinic. In addition, a new series of S-enantiomers of these agents will be prepared. These fit the computer model perfectly, while the R-isomers should be inactive. It is hoped these R-enantiomers may be antagonists of the S-isomers. It is felt 1 and 2 will serve as the lead compounds for the construction of much better, fast acting, orally active anxiolytic agents devoid of the sedative, ataxic, muscle relaxant and amnestic side effects of classical benzodiazepines, as well as exhibit reduced (or no) abuse potential. The lead compounds are illustrated in Figure I, while the target compounds in this phase of the research are depicted in Figure 2 and Schemes 1 -5. In the second part of this research (Schemes 6 and 7) emphasis is on the development of agonists, antagonists and inverse agonists that bind to alpha5beta3gamma2 subtypes with >400 fold subtype selectivity. Synthesis and pharmacological evaluation of these subtype selective agents will permit the assignment of the correct physiological functions to alpha5 subtypes. This is of special importance here in regard to cognition/amnesia and other processes mediated by the hippocampus. A network of 12 collaborators has been established to work with us on receptor binding, efficacy and pharmacology to understand the fundamental basis of/and treat anxiety disorders, as well as memory-impairment including age associated memory deficits. This latter research may also have implications in Alzheimer's disease and schizophrenia.

Addiction to cocaine and other illicit drugs is estimated to cost our society $181 billion which equates to $603 per U.S. citizen. The cost of addiction can be dramatically lowered through the use of treatments;unfortunately, many drugs of abuse, including cocaine, lack a single approved pharmacotherapy. Addiction to psychomotor stimulants, such as cocaine, is marked by a transition in drug consumption from a casual, recreational style of use to a more compulsive, excessive pattern that arises as a result of drug-induced changes in brain functioning. In order to develop effective treatments, it will likely be necessary to identify and target altered brain functioning underlying addiction. Towards this end, drug-induced changes in glutamate release from cystine-glutamate antiporters have been linked to pathological alterations in neural transmission and normalizing cystine-glutamate exchange blocks compulsive drug-seeking in preclinical models. Further, small-scale clinical studies using acetylcysteine to target cystine-glutamate exchange have shown modest efficacy including reduced drug craving and cocaine use. The efficacy of N-acetyl cysteine is limited due to extensive metabolism in the liver and poor passive transport into the brain. As a result, the present proposal seeks to develop novel chemical entities that are more potent and effective in targeting cystine-glutamate exchange in the brain. Aim 1 will involve the design of 32-40 compounds. Aim 2 will utilize in vitro and in vivo screening techniques to determine which compounds are most effective and potent in targeting cystine-glutamate exchange. Specifically, we will use pure glial cortical cultures to determine the capacity of brain cells to utilize the novel ligands to target cystine-glutamate exchange. Next, we will screen the most promising compounds in vivo by assessing the capacity of these ligands to bypass hepatic metabolism, enter into the brain, and target cystine-glutamate antiporters. Aim 3 will determine the potency and efficacy of these novel compounds in blocking cocaineprimed, stress-primed, and cocaine-paired cue primed reinstatement of cocaine-seeking in preclinical models of compulsive drug seeking. Collectively, these experiments have the potential to identify cystine-glutamate antiporters as a novel target in the treatment of addiction and to generate a series of compounds that may ultimately be effective in treating cocaine addiction.

DESCRIPTION (provided by applicant): Neuropathic pain encompasses a range of painful conditions of diverse origins including diabetic neuropathy, post-herpetic neuralgia and nerve injuries after surgery. It includes pain following paraplegia, hypersensitivity to non-painful stimli (allodynia), for example after surgery or during migraine attacks, spontaneous pain, hyperalgesia and diffuse muscle tenderness of myofacial syndromes. Back pain, cancer pain and AIDS associated pain also qualify as neuropathic pain. Currently prescribed drugs for neuropathic pain are often addictive, are not effective for all patients and have various side effects including tolerance, addiction, sedation, liver toxicity. The financial burden from the los of productivity in the US alone numbers in the billions of dollars notwithstanding the misery these patients suffer. Recently, we have discovered a series of nonsedating alpha2/alpha3 BzR/GABA (A) agonists that are active against neuropathic pain as well as anxiety disorders and convulsions. These agents do not develop tolerance and are comprised of a privileged scaffold (imidazobenzodiazepine), the result of which has less chance for toxicity. Because they exhibit little or no efficacy at a1 and a5 subtypes, they will exhibit very little or no abuse potential. This research centers on the modification of these new agents to prolong duration of action in vivo and to provide better subtype selectivity at either a2 or a3 BzR/GABA(A)ergic subtypes. This would preclude the origin of side effects including sedation, ataxia, amnesia, tolerance and abuse potential. In addition, this work will help to establish which GABAerigc subtype in the spinal cord is the nociceptive target of choice. An eventual goal is to replace the addictive opioid analgesics with these safer, non addictive ligands for treatment of all types of neuropathic and inflammatory pain, in human populations. PUBLIC HEALTH RELEVANCE: The design and development of new nonsedating, nonaddictive agents to treat neuropathic pain is under study. These agents are designed to treat diabetic neuropathy, post-herpetic neuralgia, phantom limb pain, pain from nerve injuries after surgery. This includes pain after paraplegia, hypersensitivity to non-painful stimuli (allodynia) and migraine attacks. It is felt these agents will also be effective in back pain, cancer pain and AIDS associated pain without the side effects of morphine (including addiction) or of COX-2 inhibitors (heart problems). There is a significant unmet need for new drugs to treat neuropathic pain. The financial burden from the loss of productivity in the US alone numbers in the billions of dollars, notwithstanding the misery these patients must endure.

DESCRIPTION (provided by applicant): Schizophrenia is a debilitating disorder that affects almost 1% of the world's population. The burden on the caregivers of patients is immense, and the cost of care in the U.S. is >$60 billion/y. Virtually all of the major antipsychotics approved y the FDA act primarily on dopamine and/or serotonin receptor function. Unfortunately, these antipsychotics produce serious side effects and are ineffective in treating negative symptoms and cognitive deficits of schizophrenia. Thus, there is an urgent need to develop treatments to alleviate these symptoms and deficits for schizophrenia patients preferably with novel drug candidates. Our long term goal is to generate compounds that are -subunit selective ligands of the GABAA receptors to determine the biological functions of different -subunits and to develop new therapies for human diseases. The objective here is to generate nonsedating agents for the treatment of negative symptoms and cognitive deficits of schizophrenia with little or no abuse potential (weak or no efficacy at 1 GABAA receptor subunits). Our central hypothesis is that selective modulators of the ¿3,¿5, or ligands with equal efficacy for the ¿2,¿3, and ¿5 subunits (¿2/¿3/¿5) bearing GABAA receptors, which have been shown to attenuate negative symptoms and cognitive deficits of schizophrenia in animal models of this disease, are potentially the first drug candidates to address these symptoms of schizophrenia. The rationale is that these drug candidates will be available for IND filing and clinical trials once the development of current lea compounds described in this application is successfully completed. The following four specific aims are proposed: 1) Determine the activity of selective GABAA receptor modulators in animal models of schizophrenia; 2) Develop highly ¿5 and ¿2/¿3/¿5 selective GABAA receptor modulators; 3) Develop highly 3 selective GABAA receptor modulators; and 4) Determine in vivo activity of highly subtype selective GABAA receptor modulators. Under specific aim one two lead compounds successfully re- versed the increase of tonic DA transmission in MAM rats, which suggests that these compounds would be effective in alleviating DA-mediated psychosis. Additionally, behavioral sensitivity to psychostimulants was reduced, restoring the rhythmicity within HPC-efferent structure, which is expected to be important for the alleviation of cognitive and negative symptoms of schizophrenia. Additionally, these compounds were able to reverse the cognitive symptoms of schizophrenia in the PPI model of PCP treated rats but induced no signs of catalepsy. Under specific aims 2 and 3, synthetic routes to generate the proposed compounds are already establish and under specific aims 4 nonsedating properties of lead compounds has been evaluated in different animal models including rhesus monkeys. The approach is innovative because it focuses on the development and application of chiral and -subtype selective imidazobenzodiazepines (IBZ) as new therapies for schizophrenia. The proposed work is significant because it represents the first step in a continuum of research to develop the first therapies for impaired cognitive function and negative symptoms of schizophrenia patients.

DESCRIPTION (provided by applicant): New therapeutic strategies for asthma are needed to better control disease symptoms and improve quality of life. Recommended first-line drugs (inhaled corticosteroids and ß-adrenergic agonists) do not always control symptoms, disease may become resistant, and side effects can occur. Recently, it was shown that airway smooth muscle (ASM) express ?-amino butyric acid type A receptors (GABAAR) of the ?4/?5 subtype and corresponding subtype selective agonists cause ASM relaxation. Importantly, cells that participate in inflammation (T-lymphocytes and cells of monocyte/macrophage lineage; IC) have also been shown to express functional GABAAR, including the a4 subtype, and reactivity of these cells can be suppressed by GABAAR modulating agents. Despite the growing appreciation of GABAAR signaling in asthma cell types, a strategy that unifies and targets GABAAR responses has not been developed or exploited therapeutically. The long-term goal of our research is to develop safer and more effective asthma drugs by way of our objective to identify GABAAR subunit selective compounds with activity and specificity for affected lung tissues. Our central hypothesis is that ASM and inflammatory cells (IC) express GABAARs with a limited and overlapping subset of ?-subunits that can be targeted with selective agonists; thus providing desired therapeutic activity (suppression of both ASM hyperresponsiveness and inflammation) while avoiding adverse off-target effects. Targeting GABAAR common to both ASM and IC is a compelling asthma strategy because ASM cells can modulate local immune reactivity and inflammatory mediators can influence ASM responsiveness The rationale is that targeting GABAAR in the lung would have drug design advantages because: i) a single drug substance is expected to affect two cell types (ASM and IC) that conspire in asthma pathophysiology; ii) safety is expected to be improved by avoiding corticosteroid use, and; iii) GABAAR agents are prescribed extensively and have a long history of clinical use. We will pursue our central hypothesis by way of three Specific Aims: 1) identify GABAAR subtypes by library screening that have common activity in ASM and IC; 2) establish efficacy of GABAAR ligands in asthma disease models; and, 3) develop selective GABAAR ligands with optimal pharmacological properties. These Aims will yield significant expected outcomes. First, library screening will reveal a detailed relationship between ?-subunit GABAAR selectivity and pharmacological efficacy in the lung. Second, compounds optimized for ?-subunit selectivity will be efficacious in animal asthma models demonstrating pharmacological proof-of-concept. Third, lead compounds will have pharmaceutical properties suitable for safe oral dosage. The positive impacts of these outcomes are an innovative therapeutic strategy for asthma that unifies GABAAR signaling in the lung, expanded knowledge of (and tools to study) lung signaling mechanisms, and ultimately improved patient care from a new drug choice with a fundamentally novel mode of action, improved dosage form, and reduced potential for adverse effects compared to current drugs.