Part 1. Design and Synthesis of Cysteine/Cystine Prodrugs and Bioisosteres Including Symmetrical and Unsymmetrical Disulfides Designed to Increase Cystine Levels in the CNS in Order to Drive the Cystine/Glutamate Antiporter: A Novel Treatment for Schizophrenia and Drug Addiction. Part 2. Design and Synthesis of Subtype Selective Ester Bioisosteres of BZR Ligands for Gabaa/Benzodiazepine Receptors to Enhance Metabolic Stability

Part 1. Schizophrenia is a debilitating disorder that affects almost 1% of the world\u27s population; pharmacotherapy expenditures for this disorder exceed $10 billion dollars even though existing medications exhibit a poor safety/efficacy profile. It is estimated that 75% of patients discontinue drug treatment, in part due to poor safety/efficacy. The current data set demonstrates that cysteine prodrug NAC reverse the behavioral and neurochemical effects of PCP used to model schizophrenia. As a result cysteine prodrugs represent a highly novel approach to treating schizophrenia; indeed, these compounds may ultimately be more effective than existing medications because these drugs target the pathology underlying schizophrenia and reverse behaviors used to model negative symptoms and diminished cognition produced by PCP, which are behaviors and symptoms that are not treated with current first line medications. Specifically, therapeutic endpoints produced by cysteine prodrugs include increasing stimulation of group II metabotropic glutamate receptors and restoring levels of glutathione. The latter effect has the potential to reverse several specific abnormalities that have been observed in schizophrenia including increased oxidative stress, decreased NMDA receptor function, altered gene expression, and abnormal cell proliferation / synaptic connectivity. Throughout this study, multiple series of compounds have been presented and explored, specifically 2 series of cysteine/cystine prodrugs, 2 series of cysteine/cystine bioisosteres and 1 series involving the coupling of two different series of compounds, namely, unsymmetrical disulfides ( mixed dimers). Also in this study, it will be shown through the use of in vivo and in vitro screening methods, diketopiperazine cystine prodrug monomers and dialkylated versions show high promise as novel antipsychotic agents. Furthermore, the diketopiperazine cystine prodrug dimers and dialkylated dimers also have shown promise in becoming novel antipsychotic agents by overcoming the detrimental effects of PCP-induced deficits in sensorimotor gating by restoring pre-pulse inhibition in multiple screenings. Bioisosteres of cysteine and cystine have shown vast improvements over N-Acetylcysteine by competing with C14 uptake and increasing glutamate levels by driving the cystine/glutamate antiporter. It has also been shown that simple modifications to the cysteine/cystine moiety also improve outcomes far greater then N-Acetylcysteine alone. Once the most effective compounds are determined by screening methods, the research strategy benefits by combining the two such compounds as an unsymmetrical disulfide in order to enhance their effects and help eliminate their disadvantages. As an early example to this approach two mixed dimers were synthesized and have shown extremely positive results in screening methods described here. Part 2. A series of 1,4-benzodiazepines and imidazobenzodiazepines including bioisosteric ligands was synthesized in search of subtype selective ligands for GABAA/benzodiazepine receptor subtypes. In this study, it was clear that the improved method for synthesizing benzodiazepines was successful. This is based on the number and quantities of numerous compounds synthesized utilizing the improved method. Although the efficacy of XHe-II-053 (4) was decreased in Phase I because of the metabolism of the C-3 ester to the acid, the bioisostere EMJ-I-026 (5) has been shown to exhibit non-sedating anxiolytic activity in mice as well as a binding/oocyte profile in vitro consistent with a non-sedating anxiolytic. Seven bioisosteric analogues were designed in order to circumvent any potential metabolic liability in humans of the previously described ligand. In fact, the bioisosteric analogues were much more stable in human liver microsomes than XHe-II-053 (4) again indicating these bioisosteres are potential nonsedating anxiolytics as well as useful for treatment of anxiety disorders in human populations. These ligands were also stable on human blood, brain and kidney. Gratifyingly, ligand 5 was clearly an fÑ3 Bz/GABAergic receptor subtype selective ligand at pharmacologically relevant doses (approximately 100 to 200 nM) and, presumably, provides an agent to study physiologically processes mediated by fÑ3 subtypes including anxiety and, in addition, was much more stable on human liver microsomes. In this regard fÑ3 subtype selective ligand, oxadiazole 5 (EMJ-I-026), has been evaluated in the light dark paradigm and clearly is a nonsedating anxiolytic, wherein this ligand was anxiolytic with no sedative properties, in vivo, as compared to diazepam. This study indicated that the ester function in these molecules can be replaced with a metabolically more stable ester bioisostere and still retain anxiolytic activity. The indepth study of these ligands in animal models and other receptor systems are underway by collaborators

Synthesis of Subtype Selective Bz/GABAA Receptor Ligands for the Treatment of Anxiety, Epilepsy and Neuropathic Pain, as Well as Schizophrenia and Asthma

The α2/α3 subtype selective Bz/GABAA receptor positive allosteric modulator HZ-166 (3) has been shown to be a nonsedating anxiolytic with anticonvulsant and antihyperalgesic activity. However, instability in vitro and in vivo has hindered its advancement into clinical trials. A series of ligands based off HZ-166 (3) were synthesized. Many of these ligands were designed to increase metabolic stability, while others were synthesized to study the effects that electronics and sterics have on the efficacy exerted when bound to the GABAA receptor. The α3 subtype selective methyl ester MP-III-024 (19) was shown to have increased resistance to metabolism in in vitro liver microsomal studies and exhibited significant anxiolytic and antihyperalgesic effects in mice without showing signs of sedation. However, pharmacokinetic studies indicated that esters as a functional group may not be suitable for extensive preclinical studies. A series of heterocyclic bioisosteres were synthesized to specifically overcome short half-lives in vivo. The oxadiazole MP-III-080 (34) and oxazole KRM-II-81 (36) underwent pharmacokinetic studies and were both found to exist in plasma and brain samples in high levels. These results indicated that these and related heterocycles would be stable in vivo to undergo extensive preclinical trials. A dozen ligands were assessed in vivo in an anxiolytic marble burying assay and a rotarod assay designed to measure ataxic effects. The results from these studies and other in vitro protocols led to additional studies using KRM-II-81 (36). This oxazole 36 was found to exhibit significant anxiolytic and anticonvulsant properties, including reducing network firing rate frequency in human brain tissue from a patient suffering from resistance epilepsy. In addition, KRM-II-81 (36) was found to be more efficacious than gabapentin to reverse the effects of hyperalgesia in a neuropathic pain model at a lower dose using rats, as well as exhibiting antidepressant-like effects. The α5 GABAA receptor subtype has been linked to the cognitive disorders in such diseases as schizophrenia, bipolar I disorder and major depressive disorder. The enantiomers SH-053-2\u27F-S-CH3 (51) and SH-053-2\u27F-R-CH3 (52) have been shown to be α2/α3/α5- and α5- subtype selective agonists, respectively. Both ligands (S)-51 and (R)-52 have been shown to reduce some positive symptoms of schizophrenia; the S-enantiomer 51 was active in the poly(I:C) model of schizophrenia while the R-enantiomer 52 was active in the MAM-model of schizophrenia. Due to the high rate of comorbidity of schizophrenia with anxiety, epilepsy and depression, the S-enatiomer (51) is shown here to be useful in these instances exhibiting anxiolytic and anticonvulsant properties. In addition, work on analogs of 52 produced MP-III-004 (63), an α5 subtype selective ligand with reduced efficacy at the α1, α2 and α3 subtypes as compared to 52, as well as the very potent α5 positive allosteric modulator MP-III-022 (65). This methyl amide 65 was shown to activate α5 subtypes in vivo in rats at low concentrations, providing a valuable tool to study the α5 GABAA receptor subtype. Recent work has shown SH-053-2\u27F-R-CH3 (52) and MP-III-022 (65) exert antidepressant-like effects in mice, indicating a new use for α5 subtype selective ligands. Moreover, work by Emala et al. has discovered a use for α5 subtype selective ligands outside of the central nervous system. A number of the ligands, especially the α5 selective acid 73, presented herein have been shown to relax precontracted human and guinea pig airway smooth muscle and may provide a novel treatment for those who suffer from asthma

Probing novel compound classes & a new interacting protein for the Mammalian GАВА(_A) receptor

y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain mediating its fast inhibitory action via GABA(_A) receptors. These receptors are implicated in a number of neurological diseases, making GABA(_A) receptor ligands interesting as potential therapeutic agents. The aims of this research project were two-fold: identifying leads for the discovery of new chemical entities that modify GABA(_A) receptor function. The second aim was to increase the understanding of GABAgeric transmission by studying the pharmacological influence of a new interacting protein for the mammalian GABA(_A) receptor, GRIF-1. In the search for novel ligands for GABA(_A) receptor, the pharmacology of three structurally distinct compound classes was investigated. The first class was the NSAID, Mefenamic acid (MFA) and a group of analogues. Results showed that MFA and a series of analogues selectively modulate GABAAR at the agonist binding site, but did not interact with either the picrotoxin or the benzodiazepine sites. Indeed the most significant result of this study was the identification of common active conformers of MFA compound and the differentiation of two analogues based on MFA structure, with an improvement in apparent efficacy. The second compound studied was Octyi-13-Dglucoside, a small molecule congener of a natural fungal metabolite, Caloporoside. These studies demonstrated that Octyi-13-D-glucoside is a positive modulator of GABAA receptor at the channel site demonstrated by its stimulation of specific [(^35)S] TBPS binding. The level of stimulation was similar to that elicited by diazepam and was occluded by GABA. Preliminary structure-activity study showed that the 13-glycosidic linkage and chain length are crucial for the positive modulation of [(^35)S] TBPS binding to the GABAAR by this novel chemical class. The third compound series were essential oils derived from Melissa officinalis and Lavendula angustifolia. These two oils either singly or in combination have been reported to have a significant benefit in the treatment of agitation in dementia. The purpose of this study was to clarify the sedative and calming mechanisms of these two common essential oils by investigating their effects on the GABAAR complex. Melissa and Lavender both singly and in combination inhibit [(^35)S] TBPS binding to the channel site of GABAAR. Melissa oil displayed the higher affinity. Melissa oil alone also showed a stimulatory effect on [(^3)H] muscimol binding. Interestingly, a combination effect on the inhibition of [(^3)H] flunitrazepam binding to the GABAAR has been shown when Lavender and Melissa oils are applied together (50:50), with no effect when applied alone. Neither Melissa nor Lavender oils demonstrated any effect on the binding of [(^3)H] MK-801 to NMDA receptors, or [3H) nicotine to nicotinic acetylcholine receptors. Furthermore, functional studies have demonstrated that both oils (0.01 mg/ml) applied to rat primary cortical neuron cultures, results in a significant reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission. These data suggests that the calming/sedative effects of Melissa are mediated by multiple mechanisms in the CNS; the net effect is depressant on the overall neuronal network. Finally, a pharmacological study was performed on GRIF-1a, a novel GABAA receptor 132 subunit trafficking protein, to gain further insights into the potential role of this novel protein at the inhibitory synapse. In the present work, evidence was provided that GRIF-1a does not increase a1j32y2 receptor complex numbers, but appears importantly to stabilise the GABAAR in a conformation which facilitates binding to both GABA and benzodiazepines. These findings suggest that GRIF-1 protein may be a novel means of modifying the efficacy of synaptic inhibition. In summary, this thesis provides a clear picture about four novel ways for the modulation of the GABAA receptor inhibitory transmission

The Effect Of Gaba A Antagonism On Locomotor Activity And Dopamine Release In The Mouse Caudate Putamen Following Acute Toluene Inhalation: An In-Vivo Microdialysis Study

Toluene is ubiquitous solvent commonly inhaled recreationally. Despite its frequency of misuse, there is little understanding of how toluene acts within the brain. To examine this, this master\u27s thesis examined the impact of acutely inhaled toluene on dopamine (DA) release in the mouse CPu in vivo using microdialysis techniques. Toluene inhalation produced dose-dependent increases in DA levels as well as changes in locomotor activity. These effects were potentiated by pre-treatment with the GABAA antagonist bicuculline via reverse microdialysis delivery. These results suggest that the DA dynamics of toluene abuse are related to toluene\u27s previously explored effects on the GABA system. It is possible that the action of toluene on GABA in the caudate actually inhibits toluene\u27s action on DA. Another theory is that toluene activates multiple GABA receptor sites, and blocking GABAA blocked a primarily inhibitory pathway

Profiling the protein interactions of the novel anticonvulsant (R)-lacosamide

The full spectrum of proteins that interact with the anticonvulsant (R)-lacosamide remain unknown despite the compound's approval in both Europe and the United States for treatment of partial seizures. This dissertation seeks to identify proteins from the mouse brain proteome that bind to (R)-lacosamide by integrating chemical biology approaches coupled with mass spectrometry for drug-target discovery. (R)-Lacosamide probes functionalized with reactive units and chemical reporters are used to selectively label, detect, and capture target proteins from complex mixtures of mouse brain lysate. Several proteins identified were further evaluated using recombinant protein technology, and the adduction site of the DPYSL2 protein was identified using mass spectrometry. Computational studies modeled the interaction of (R)-lacosamide with the known crystal structure of the DPYSL2 protein. One predicted confirmation is consistent with known structure activity relationships of (R)-lacosamide in vivo. Data evaluating labeling of an (R)-lacosamide derivative with a mutant protein lacking the identified adducted residue are presented

Part I, Unified Pharmacophore Protein Models of the Benzodiazepine Receptor Subtypes ; Part II, Subtype

Part I. New models of unified pharmacophore/receptors have been constructed guided by the synthesis of subtype selective compounds in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with comparative models of the α1β2γ2, α2β2γ2, α3β2γ2 and α5β2γ2 GABA(A) receptors has led to an orientation of the pharmacophore model within the benzodiazepine binding site (Bz BS). These results not only are important for the rational design of new selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket. More importantly, the process summarized here may be used as a general template to help scientists develop novel ligands for receptors for which the three dimensional structure has not yet been confirmed by X-ray crystallography or cryo-electron microscopy. Presented here are new models of the α1β2γ2, α2β2γ2, α3β2γ2 and α5β2γ2 GABA(A) receptors which have incorporated homology models built based on the acetylcholine binding protein. These new models will further our ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists to the Bz BS of the GABA(A) receptor. This approach will also serve as a powerful model for structure based approaches carried out using ligand-protein docking methods. Part II. An effective strategy to alleviate memory deficits would be to enhance memory and cognitive processes by augmenting the impact of acetylcholine released from cholinergic neurons of the hippocampus. Using the included volume pharmacophore presented in Part I, a number of a5 selective compounds were synthesized, notably PWZ-029. PWZ-029 was examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests which are indicative of the effects on memory acquisition, locomotor activity, anxiety, and muscle tone. Improvement of task learning was shown at a dose of 5mg/kg in passive avoidance test without effect on anxiety or muscle tone. Moderate negative modulation at GABA(A) receptors containing the α5 subunit using a moderate inverse agonist such as PWZ-029, is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory. Using low temperature NMR and X-ray analysis, it was shown that enhanced selectivity and potent in vitro affinity of α5 selective benzodiazepine dimers was possible with aliphatic linkers of 3 to 5 carbons in length. Although originally proposed to enhance solubility, oxygen-containing linkers caused the dimer to fold back on itself leading to the inability of dimers to enter the binding pocket. In addition, studies of a series of PWZ-029 analogs found that the electrostatic potential near the ligands\u27 terminal substituent correlated with its binding selectivity toward the α5β2γ2 versus α1β2γ2 Bzr/GABA(A) ergic isoform. Investigations further found that compound PWZ-029, which exhibits reasonable binding selectivity toward GABA(A) receptors containing the a5 subunit and possesses a favorable electrophysiological profile, was able to attenuate scopolamine induced contextual memory impairment in mice. This compound appears to be useful (Harris, Delorey et al.) for the treatment of cognitive deficits in rodents as well as primates (Rowlett et al.) and may well be a compound for the treatment of patients with Alzheimers disease

Ethanol alters the GABAergic neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) at local brain sites: significance of local 3α,5α-THP increases in the ventral tegmental area

Neuroactive steroids are endogenous modulators of neuronal activity that modulate central nervous system inhibitory tone and influence motivation and emotional behaviors. The inhibitory neuroactive steroid (3a,5a)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a potent positive modulator of γ-aminobutyric acid type A (GABAA) receptors. In rats, systemic ethanol administration increases 3α,5α-THP in the blood plasma, cerebral cortex, and hippocampus. Ethanol-induced increases of 3α,5α-THP contribute to the behavioral and neurophysiological effects of ethanol in rats and the subjective effects of alcohol in humans. Previous in vivo studies suggest that ethanol-induced increases of 3α,5α-THP are dependent on the adrenal glands, but in vitro studies suggest ethanol produces local synthesis of 3α,5α-THP. Furthermore, limitations in steroid assays have prevented the study of ethanol-induced changes of 3α,5α-THP in many of the brain regions implicated in alcoholism. The first aim of this project used 3α,5α-THP immunohistochemistry (IHC) to determine ethanol effects on cellular levels of 3α,5α-THP across the rat brain, and determine the role of the adrenal glands in these effects. We showed that ethanol produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. We also showed that ethanol can increase or decrease local iv brain levels of 3α,5α-THP independent of adrenal activation in subcortical brain regions, but increases of 3α,5α-THP in the medial prefrontal cortex (mPFC) are dependent on the adrenal glands. Previous studies have shown that systemic 3α,5α-THP can reduce ethanol reinforcement and consumption. However, systemic 3α,5α-THP is metabolized rapidly and can produce sedation, limiting therapeutic value. Therefore, in aim 2 we used gene delivery to drive local neuroactive steroid synthesis in the nucleus accumbens (NAc) or ventral tegmental area (VTA), which are both implicated in ethanol reinforcement and consumption. We showed that adeno-associated viral vector-mediated local increases of 3α,5α-THP in the VTA were associated with a long-term reduction in ethanol reinforcement and consumption. Further investigation showed that 3α,5α-THP is located in tyrosine hydroxylase (TH) positive and negative neurons, but not astrocytes in the VTA. These studies showed that ethanol can produce divergent effects on local brain levels of 3α,5α-THP and increasing local levels of 3α,5α-THP in the VTA is associated with reduced ethanol reinforcement and consumption.Doctor of Philosoph

PRECLINICAL DEVELOPMENT OF PHYTOCANNABINOID- AND ENDOCANNABINOID- BASED PHARMACOTHERAPIES FOR THE TREATMENT OF ETHANOL-INDUCED NEURODEGENERATION

Excessive ethanol consumption, characteristic of alcohol use disorders (AUDs), is associated with widespread neurodegeneration and cognitive and behavioral impairments that may contribute to the chronic and relapsing nature of alcoholism. Therefore, identifying novel targets that can afford neuroprotection will undoubtedly aid current treatment strategies for AUDs. The cannabinoids have been shown to provide neuroprotection in a variety of preclinical models of neurodegeneration; however minimal data is available regarding the use of cannabinoid-based pharmacotherapies for treating ethanol-induced neurodegeneration. Therefore, the current dissertation examined the overarching hypothesis: the cannabinoids are a therapeutic strategy to afford neuroprotection in the context of ethanol-induced neurodegeneration. Importantly, this overarching hypothesis was approached with translational considerations in mind. Specifically, the use of many cannabinoids in the clinic is hindered due to multiple unfavorable pharmacokinetic/pharmacodynamic profiles, including high first pass metabolism and untoward psychoactivity. Therefore, the studies herein were designed to circumvent these PK/PD obstacles. The first set of studies examined whether transdermal delivery of the phytocannabinoid, cannabidiol (CBD), could attenuate binge ethanol induced neurodegeneration. Transdermal CBD afforded neuroprotection in the entorhinal cortex and neuroprotection was similar in magnitude as intraperitoneal administration. The second set of studies found that binge ethanol treatment transiently down-regulated the main CNS cannabinoid receptor, CB1R. Interestingly, these changes were not accompanied by alterations in one of the major endogenous ligands, anandamide (AEA), or other related n-acylethanolamides (NAEs). The latter finding is in contrast to other literature reports demonstrating that endocannabinoid content is substantially elevated in response to a CNS insult. Nevertheless, studies were carried out to determine if administration of the AEA and NAE catabolism inhibitor, URB597, could attenuate binge ethanol induced neurodegeneration. URB597 failed to produce neuroprotection in the entorhinal cortex and dentate gyrus of the hippocampus. However, additional studies found that URB597 failed to elevate AEA in the entorhinal cortex, and in general the biological activity of URB597 was impaired by ethanol exposure. Therefore, with further drug discovery/development efforts, it may be feasible to optimize such treatment strategies. In conclusion, the studies within the current dissertation demonstrated the feasibility of using some cannabinoid-based agents to prevent ethanol-induced neurodegeneration

Synthesis and pharmacological evaluation of primary amino acid derivatives (PAADs): novel neurological agents for the treatment of epilepsy and neuropathic pain

Epilepsy and neuropathic pain (NP) are chronic neurological disorders that result from dysregulations in neuronal function. Currently, there is a lack of adequate therapeutic agents available to treat these disorders and the need remains to develop compounds that possess a novel mechanism of action to address the shortcomings of current medications. Recently, the role of voltage-gated sodium channels (VGSCs) has been implicated in the pathophysiological mechanisms of NP, while their role in epilepsy has been known for some time. The functionalized amino acid (FAA) (R)-lacosamide is an emerging antiepileptic drug (AED) that has been shown to selectively promote VGSCs into the slow inactivated state and has recently been approved by the EMEA and the US FDA under the trademark Vimpat[registered trademark] for the adjuvant treatment of partial-onset seizures in adults. (R)-Lacosamide has also demonstrated clinical efficacy in treating painful diabetic neuropathy, but has yet to gain regulatory approval for this indication. The pharmaceutical industry has made advances in developing peripheral nervous system (PNS)-specific agents that target specific isoforms of VGSCs for the treatment of NP. We combined the concept of PNS-selectivity with our knowledge of FAAs and proposed that primary amino acid derivatives (PAADs) may selectively target PNS receptor sites, thereby avoiding potential CNS side effects that makes adherence to pain therapy difficult. Additionally, we examined the effect of PAADs on CNS function due to the excellent anticonvulsant activity of FAAs. We synthesized and evaluated over 50 PAADs in whole animal models of epilepsy and NP, and developed a structure-activity relationship (SAR) that defined the structural requirements for PAAD activity. The SAR revealed excellent anticonvulsant activity and pain attenuation for a novel class of compounds, the C(2)-hydrocarbon PAADs. Then, we synthesized over 40 additional PAADs to optimize anticonvulsant activity and pain attenuation. From our optimization studies, we discovered two PAADs that displayed superior anticonvulsant activity and may rival the therapeutic capabilities of (R)-lacosamide. Finally, we evaluated the most active PAADs in a series of binding and enzymatic assays but we did not reveal any new binding targets of therapeutic relevance