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Recently, nanocrystal dispersions have been considered as a promising formulation strategy to improve the bioavailability of the deuterated pyrazoloquinolinone ligand DK-I-56-1 (7‑methoxy-2-(4‑methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one). In the current study, the freeze-drying process (formulation and process parameters) was investigated to improve the storage stability of the previously developed formulation. Different combinations of lyoprotectant (sucrose or trehalose) and bulking agent (mannitol) were varied while formulations were freeze-dried under two conditions (primary drying at -10 or -45 °C). The obtained lyophilizates were characterized in terms of particle size, solid state properties and morphology, while the interactions within the samples were analyzed by Fourier transform infrared spectroscopy. In the preliminary study, three formulations were selected based on the high redispersibility index values (around 95%). The temperature of primary drying had no significant effect on particle size, but stability during storage was impaired for samples dried at -10 °C. Samples dried at lower temperature were more homogeneous and remained stable for three months. It was found that the optimal ratio of sucrose or trehalose to mannitol was 3:2 at a total concentration of 10% to achieve the best stability (particle size < 1.0 μm, polydispersity index < 0.250). The amorphous state of lyoprotectants probably provided a high degree of interaction with nanocrystals, while the crystalline mannitol provided an elegant cake structure. Sucrose was superior to trehalose in maintaining particle size during freeze-drying, while trehalose was more effective in keeping particle size within limits during storage. In conclusion, results demonstrated that the appropriate combination of sucrose/trehalose and mannitol together with the appropriate selection of lyophilization process parameters could yield nanocrystals with satisfactory stability., (Copyright © 2023. Published by Elsevier B.V.)

Ethanol has been shown to suppress essential tremor (ET) in patients at low-to-moderate doses, but its mechanism(s) of action remain unknown. One of the ET hypotheses attributes the ET tremorgenesis to the over-activated firing of inferior olivary neurons, causing synchronic rhythmic firings of cerebellar Purkinje cells. Purkinje cells, however, also receive excitatory inputs from granule cells where the α6 subunit-containing GABA A receptors (α6GABA A Rs) are abundantly expressed. Since ethanol is a positive allosteric modulator (PAM) of α6GABA A Rs, such action may mediate its anti-tremor effect. Employing the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of ethanol and α6GABA A R-selective pyrazoloquinolinone PAMs. The burrowing activity, an indicator of well-being in rodents, was measured concurrently. Ethanol significantly and dose-dependently attenuated action tremor at non-sedative doses (0.4-2.4 g/kg, i.p.). Propranolol and α6GABA A R-selective pyrazoloquinolinones also significantly suppressed tremor activity. Neither ethanol nor propranolol, but only pyrazoloquinolinones, restored burrowing activity in harmaline-treated mice. Importantly, intra-cerebellar micro-injection of furosemide (an α6GABA A R antagonist) had a trend of blocking the effect of pyrazoloquinolinone Compound 6 or ethanol on harmaline-induced tremor. In addition, the anti-tremor effects of Compound 6 and ethanol were synergistic. These results suggest that low doses of ethanol and α6GABA A R-selective PAMs can attenuate action tremor, at least partially by modulating cerebellar α6GABA A Rs. Thus, α6GABA A Rs are potential therapeutic targets for ET, and α6GABA A R-selective PAMs may be a potential mono- or add-on therapy., (© 2023. The American Society for Experimental Neurotherapeutics, Inc.)

Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nanoparticles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (<100 nm), and high encapsulation efficacy (>99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

GABA A receptors containing α6 subunits (α6GABA A Rs) in the cerebellum have -been implicated in schizophrenia. It was reported that the GABA synthesizing enzymes were downregulated whereas α6GABA A Rs were upregulated in postmortem cerebellar tissues of patients with schizophrenia and in a rat model induced by chronic phencyclidine (PCP). We have previously demonstrated that pyrazoloquinolinone Compound 6, an α6GABA A R-highly selective positive allosteric modulator (PAM), can rescue the disrupted prepulse inhibition (PPI) induced by methamphetamine (METH), an animal model mimicking the sensorimotor gating deficit based on the hyper-dopaminergic hypothesis of schizophrenia. Here, we demonstrate that not only Compound 6, but also its structural analogues, LAU463 and LAU159, with similarly high α6GABA A R selectivity and their respective deuterated derivatives (DK-I-56-1, DK-I-58-1 and DK-I-59-1) can rescue METH-induced PPI disruption. Besides, Compound 6 and DK-I-56-I can also rescue the PPI disruption induced by acute administration of PCP, an animal model based on the hypo-glutamatergic hypothesis of schizophrenia. Importantly, Compound 6 and DK-I-56-I, at doses not affecting spontaneous locomotor activity, can also rescue impairments of social interaction and novel object recognition in mice induced by chronic PCP treatments. At similar doses, Compound 6 did not induce sedation but significantly suppressed METH-induced hyperlocomotion. Thus, α6GABA A R-selective PAMs can rescue not only disrupted PPI but also hyperlocomotion, social withdrawal, and cognitive impairment, in both METH- and PCP-induced animal models mimicking schizophrenia, suggesting that they are a potential novel therapy for the three core symptoms, i.e. positive symptoms, negative symptoms, and cognitive impairment, of schizophrenia., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

GABAkines, or positive allosteric modulators of γ-aminobutyric acid-A (GABA A ) receptors, are used for the treatment of anxiety, epilepsy, sleep, and other disorders. The search for improved GABAkines, with reduced safety liabilities (e.g., dependence) or side-effect profiles (e.g., sedation) constituted multiple discovery and development campaigns that involved a multitude of strategies over the past century. Due to the general lack of success in the development of new GABAkines, there had been a decades-long draught in bringing new GABAkines to market. Recently, however, there has been a resurgence of efforts to bring GABAkines to patients, the FDA approval of the neuroactive steroid brexanolone for post-partum depression in 2019 being the first. Other neuroactive steroids are in various stages of clinical development (ganaxolone, zuranolone, LYT-300, Sage-324, PRAX 114, and ETX-155). These GABAkines and non-steroid compounds (GRX-917, a TSPO binding site ligand), darigabat (CVL-865), an α2/3/5-preferring GABAkine, SAN711, an α3-preferring GABAkine, and the α2/3-preferring GABAkine, KRM-II-81, bring new therapeutic promise to this highly utilized medicinal target in neurology and psychiatry. Herein, we also discuss possible conditions that have enabled the transition to a new age of GABAkines. We highlight the pharmacology of KRM-II-81 that has the most preclinical data reported. KRM-II-81 is the lead compound in a new series of orally bioavailable imidazodiazepines entering IND-enabling safety studies. KRM-II-81 has a preclinical profile predicting efficacy against pharmacoresistant epilepsies, traumatic brain injury, and neuropathic pain. KRM-II-81 also produces anxiolytic- and antidepressant-like effects in rodent models. Other key features of the pharmacology of this compound are its low sedation rate, lack of tolerance development, and the ability to prevent the development of seizure sensitization., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Positive allosteric modulators (PAMs) of α5GABA A receptors (α5GABAARs) are emerging as potential therapeutics for a range of neuropsychiatric disorders. However, their role in memory processing of healthy animals is not sufficiently examined. We tested the effects of MP-III-022 (1 mg/kg, 2.5 mg/kg and 10 mg/kg), a PAM known to be selective for α5GABAARs and devoid of prominent side-effects, in different behavioral paradigms (Morris water maze, novel object recognition test and social novelty discrimination) and on GABRA5 expression in Wistar rats, 30 min and 24 h after intraperitoneal treatment administration. The lowest dose tested worsened short-term object memory. The same dose, administered two times in a span of 24 h, improved spatial and impaired object and, at a trend level, social memory. The highest dose had a detrimental effect on all types of long-term memory (object memory at a trend level) and short-term spatial memory, but improved short-term object and social memory. Distinct sets of expression changes were detected in both prefrontal cortex and two regions of the hippocampus, but the latter ones could be assessed as more consequential. An increase of GABRA5 mRNA in CA2 occurred in parallel with improvement of object and social, but impairment of spatial memory, while the opposite happened with a trend level change in CA1. Our study demonstrates the variability of the roles of the α5GABAAR based on its level of expression and localization, in dependence on the type and protocol of cognitive tasks, as well as the respective timing of pharmacological modulation and testing., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: To assess GABA A receptor subtypes involved in benzodiazepine tolerance and dependence, we evaluated the ability of subtype-selective and non-selective ligands to substitute for (i.e., produce "cross-tolerance") or precipitate withdrawal during chronic alprazolam treatment., Methods: Four female rhesus monkeys (Macaca mulatta) were implanted with chronic intravenous catheters and administered alprazolam (1.0 mg/kg every 4 h). Following 14+ days of chronic alprazolam, acute administration of selected doses of non-selective and subtype-selective ligands were substituted for, or administered with, alprazolam, followed by quantitative behavioral observations. The ligands included alprazolam and midazolam (positive modulators, non-selective), zolpidem (positive modulator, preferential affinity for α1-containing GABA A receptors), HZ-166 (positive modulator, preferential efficacy at α2- and α3-containing GABA A receptors), and βCCT (antagonist, preferential affinity for α1-containing GABA A receptors)., Results: Acutely, alprazolam and midazolam both induced observable ataxia along with a mild form of sedation referred to as "rest/sleep posture" at a lower dose (0.1 mg/kg, i.v.), whereas at a higher dose (1.0 mg/kg, i.v.), induced deep sedation and observable ataxia. With chronic alprazolam treatment, observable ataxia and deep sedation were reduced significantly, whereas rest/sleep posture was unchanged or emerged. Zolpidem showed a similar pattern of effects, whereas no behaviors engendered by HZ-166 were changed by chronic alprazolam. Administration of βCCT, but not HZ-166, resulted in significant withdrawal signs., Conclusions: These results are consistent with a role for α1-containing GABA A receptor subtypes in tolerance and dependence observed with chronic alprazolam, although other receptors may be involved in the withdrawal syndrome., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Poor water solubility of new chemical entities is considered as one of the main obstacles in drug development, as it usually leads to low bioavailability after administration. To overcome these problems, the selection of the appropriate formulation technology needs to be based on the physicochemical properties of the drug and introduced in the early stages of drug research. One example of the new potential drug substance with poor solubility is DK-I-60-3, deuterated pyrazoloquinolinone, designed for the treatment of various neuropsychiatric disorders. In this research, based on preformulation studies, nanocrystal technology was chosen to improve the oral bioavailability of DK-I-60-3. Nanocrystal dispersions stabilized by sodium lauryl sulfate and polyvinylpyrrolidone were prepared by modified wet media milling technique, with the selection of appropriate process and formulation parameters. The nanoparticles characterization included particle size and zeta potential measurements, differential scanning calorimetry, X-ray powder diffraction, dissolution and solubility study, and in vivo pharmacokinetic experiments. Developed formulations had small uniform particle sizes and were stable for three months. Nanonization caused decreased crystallite size and induced crystal defects formation, as well as a DK-I-60-3 solubility increase. Furthermore, after oral administration of the developed formulations in rats, two to three-fold bioavailability enhancement was observed in plasma and investigated organs, including the brain.

Introduction: Deficits in somatostatin-positive gamma-aminobutyric acid interneurons (SST+ GABA cells) are commonly reported in human studies of mood and anxiety disorder patients. A causal link between SST+ cell dysfunction and symptom-related behaviors has been proposed based on rodent studies showing that chronic stress, a major risk factor for mood and anxiety disorders, induces a low SST+ GABA cellular phenotype across corticolimbic brain regions; that lowering Sst, SST+ cell, or GABA functions induces depressive-/anxiety-like behaviors (a rodent behavioral construct collectively defined as "behavioral emotionality"); and that disinhibiting SST+ cells has antidepressant-like effects. Recent studies found that compounds preferentially potentiating receptors mediating SST+ cell functions, α5-GABAA receptor positive allosteric modulators (α5-PAMs), achieved antidepressant-like effects. Together, the evidence suggests that SST+ cells regulate mood and cognitive functions that are disrupted in mood disorders and that rescuing SST+ cell function via α5-PAM may represent a targeted therapeutic strategy., Methods: We developed a mouse model allowing chemogenetic manipulation of brain-wide SST+ cells and employed behavioral characterization 30 minutes after repeated acute silencing to identify contributions to symptom-related behaviors. We then assessed whether an α5-PAM, GL-II-73, could rescue behavioral deficits., Results: Brain-wide SST+ cell silencing induced features of stress-related illnesses, including elevated neuronal activity and plasma corticosterone levels, increased anxiety- and anhedonia-like behaviors, and impaired short-term memory. GL-II-73 led to antidepressant- and anxiolytic-like improvements among behavioral deficits induced by brain-wide SST+ cell silencing., Conclusion: Our data validate SST+ cells as regulators of mood and cognitive functions and demonstrate that bypassing low SST+ cell function via α5-PAM represents a targeted therapeutic strategy., (© The Author(s) 2021. Published by Oxford University Press on behalf of CINP.)

Tourette syndrome (TS) is a disabling neurodevelopmental disorder characterized by multiple, recurrent tics. The pharmacological treatment of TS is currently based on dopaminergic antagonists; however, these drugs are associated with extrapyramidal symptoms and other serious adverse events. Recent evidence suggests that positive allosteric modulators (PAMs) of GABA A receptors containing α6 subunits (α6 GABA A Rs) oppose the behavioral effects of dopamine. Building on this evidence, in the present study, we tested the efficacy of DK-I-56-1, a highly selective PAM for α6 GABA A Rs, in mouse models of TS exhibiting tic-related responses. DK-I-56-1 significantly reduced tic-like jerks and prepulse inhibition (PPI) deficits in D1CT-7 transgenic mice, a well-documented mouse model of TS. DK-I-56-1 also prevented the exacerbation of spontaneous eyeblink reflex induced by the potent dopamine D 1 receptor agonist SKF 82958, a proxy for tic-like responses. We also showed that both systemic and prefrontal cortical administration of DK-I-56-1 countered the PPI disruption caused by SKF 82958. Although the effects of DK-I-56-1 were akin to those elicited by dopaminergic antagonists, this drug did not elicit extrapyramidal effects, as measured by catalepsy. These results point to α6 GABA A R PAMs as promising TS therapies with a better safety profile than dopaminergic antagonists.

Aging is associated with reduced brain volume, altered neural activity, and neuronal atrophy in cortical-like structures, comprising the frontal cortex and hippocampus, together contributing to cognitive impairments. Therapeutic efforts aimed at reversing these deficits have focused on excitatory or neurotrophic mechanisms, although recent findings show that reduced dendritic inhibition mediated by α5-subunit containing GABA-A receptors (α5-GABAA-Rs) occurs during aging and contributes to cognitive impairment. Here, we aimed to confirm the beneficial effect on working memory of augmenting α5-GABAA-R activity in old mice and tested its potential at reversing age-related neuronal atrophy. We show that GL-II-73, a novel ligand with positive allosteric modulatory activity at α5-GABAA-R (α5-PAM), increases dendritic branching complexity and spine numbers of cortical neurons in vitro. Using old mice, we confirm that α5-PAM reverses age-related working memory deficits and show that chronic treatment (3 months) significantly reverses age-related dendritic shrinkage and spine loss in frontal cortex and hippocampus. A subsequent 1-week treatment cessation (separate cohort) resulted in loss of efficacy on working memory but maintained morphological neurotrophic effects. Together, the results demonstrate the beneficial effect on working memory and neurotrophic efficacy of augmenting α5-GABAA-R function in old mice, suggesting symptomatic and disease-modifying potential in age-related brain disorders., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Background: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells., Objective: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301., Methods: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration., Results: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma., Conclusion: MIDD0301 undergoes no phase I and moderate phase II metabolism., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Migraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) play an important role. This hyperactivity might originate from an underfunctional GABAergic system in TG. To investigate this possibility, we adapted a mouse model of migraine by inducing migraine-like grimaces in male mice via repeated injections of nitroglycerin (NTG, 10 mg/kg, i.p.), once every 2 days, for up to 5 sessions. Migraine-like facial pain scores were measured using the mouse grimace scale. Repeated NTG treatments in mice caused significant increases in migraine-like grimaces that were aborted and prevented by two anti-migraine agents sumatriptan and topiramate, respectively. After 5 sessions of NTG injections, the GABA-synthesizing enzyme, 65-kDa glutamate decarboxylase (GAD65), but not the GABA transporter 1 (GAT1) or the α6 subunit-containing GABA A receptors (α6GABA A Rs), was downregulated in mouse TG tissues. Taking advantage of the unaffected TG α6GABA A R expression in NTG-treated mice, we demonstrated that an α6GABA A R-selective positive allosteric modulator (PAM), DK-I-56-1, exhibited both abortive and prophylactic effects, comparable to those of sumatriptan and topiramate, respectively, in this migraine-mimicking mouse model. The brain-impermeable furosemide significantly prevented the effects of DK-I-56-1, suggesting its peripheral site of action, likely via preventing α6GABA A R modulation in TG. Results suggest that a decreased GABA synthesis caused by the reduced GAD65 expression in TG contributes to the trigeminovascular activation in this repeated NTG-induced migraine-mimicking model and that the unaltered α6GABA A Rs in TG are potential targets for migraine treatment. Thus, α6GABA A R-selective PAMs are potential anti-migraine agents for both abortive and preventive therapies.

We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABA A R) modulator. The therapeutic efficacy of nebulized MIDD0301 in reducing airway resistance was investigated in spontaneous breathing mice using a whole-body plethysmograph and in unconscious mice using a forced oscillation technique. Prophylactic nebulized MIDD0301 reduced subsequent methacholine-induced bronchoconstriction in ovalbumin and house dust mite allergic asthma models and in normal mice. Nebulized MIDD0301 exhibited comparable or better therapeutic potency compared to nebulized albuterol and oral montelukast. Prophylactic nebulized MIDD0301 was also effective in reducing bronchoconstriction, comparable to nebulized albuterol or fluticasone, in a steroid resistant asthma mouse model induced by intratracheal installation of lipopolysaccharide and interferon-gamma. Oral dexamethasone was ineffective in this model. Nebulized MIDD0301 was also effective in reversing bronchospasm when dosed after methacholine challenge comparable to albuterol. Pharmacokinetic studies showed that about 0.06% of nebulized MIDD0301 entered the mouse lung when using a whole body plethysmograph and therapeutic levels were sustained in the lung for at least 25 min. Consistent with previous reports on orally dosed MIDD0301, high doses of nebulized MIDD0301 resulted in minimal brain exposure and thus no observable adverse sensorimotor or respiratory depression effects occurred. In addition, no adverse cardiovascular effects were observed following 100 mg/kg i.p. dosing. These results further demonstrate that charged imidazodiazepine MIDD0301 can selectively target lung GABA A R without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation., Competing Interests: The authors declare the following competing financial interest(s): L.A.A., B.N.M., and D.C.S. are employees of Pantherics. L.A.A., D.C.S., and C.W.E. have an ownership interest in Pantherics, which has licensed the technology reported in this publication. Some of the research was funded by R41HL147658, which was awarded to Pantherics. Pantherics did not finance this research directly. The funders indicated in the Acknowledgment section had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results., (© 2020 American Chemical Society.)

The need for improved medications for the treatment of epilepsy and chronic pain is essential. Epileptic patients typically take multiple antiseizure drugs without complete seizure freedom, and chronic pain is not fully managed with current medications. A positive allosteric modulator (PAM) of α2/3-containing GABA A receptors (5-(8-ethynyl-6-(pyridin-2-yl)-4 H -benzo[ f ]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81 ( 8 ) is a lead compound in a series of imidazodiazepines. We previously reported that KRM-II-81 produces broad-based anticonvulsant and antinociceptive efficacy in rodent models and provides a wider margin over motoric side effects than that of other GABA A receptor PAMs. The present series of experiments was designed to fill key missing gaps in prior preclinical studies assessing whether KRM-II-81 could be further differentiated from nonselective GABA A receptor PAMs using the anticonvulsant diazepam (DZP) as a comparator. In multiple chemical seizure provocation models in mice, KRM-II-81 was either equally or more efficacious than DZP. Most strikingly, KRM-II-81 but not DZP blocked the development of seizure sensitivity to the chemoconvulsants cocaine and pentylenetetrazol in seizure kindling models. These and predecessor data have placed KRM-II-81 into consideration for clinical development requiring the manufacture of kilogram amounts of good manufacturing practice material. We describe here a novel synthetic route amenable to kilogram quantity production. The new biological and chemical data provide key steps forward in the development of KRM-II-81 ( 8 ) as an improved treatment option for patients suffering from epilepsy.

Opiate analgesics are one of the treatment options for severe chronic pain, including late-stage cancer, chronic back pain and other disorders. The recent resurgence in opioid overdose has highlighted the serious need for alternative medicines for pain management. While a role for potentiators of α2/3-containing GABA A receptors in the modulation of pain has been known for several years, advancements in this area required data from selective compounds. KRM-II-81(5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3- yl)oxazole) and analogs selectively potentiate GABA A receptors containing α2/3 subunits and have recently been shown to attenuate pain behaviors in several acute and chronic pain models in rodents. The present study was designed to ascertain whether KRM-II-81 and the structural analog MP-III-80 (3-ethyl-5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)-1,2,4-oxadiazole) would block chemotherapeutic agent paclitaxel-induced pain in male, C57BL/6 mice. Both compounds significantly inhibited pain behaviors evoked by cold and tactile stimulation in paclitaxel-treated mice as did the neuropathic pain drug gabapentin. Subchronic dosing for 22 days with KRM-II-81 and MP-III-80 demonstrated enduring analgesic efficacy without tolerance development, while the effects of gabapentin showed evidence of tolerance development. KRM-II-81 and MP-III-80 also decreased marble-burying behavior in this mouse strain as did the anxiolytic drug chlordiazepoxide. In contrast to KRM-II-81 and MP-III-80, chlordiazepoxide had motor-impairing effects at anxiolytic-like doses. The data add to the literature documenting that these selective potentiators of α2/3-containing GABA A receptors are effective in a host of animal models used to detect novel analgesic drugs. The anxiolytic-like efficacy of these compounds fits well with the comorbidity of anxiety in patients with chronic pain and cancer., (Copyright © 2020. Published by Elsevier Inc.)

DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d 3 -phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), a recently developed deuterated pyrazoloquinolinone, has been recognized as a lead candidate for treatment of various neuropsychiatric disorders. During preclinical investigation of poorly water-soluble compounds such as DK-I-56-1, the application of nanotechnology could be advantageous due to improved safety and possibly increased bioavailability of nanosized formulation. DK-I-56-1 nanosuspensions stabilized by polysorbate 80, alone or in combination with poloxamers 188 i.e. 407 or d-α-tocopheryl polyethylene glycol 1000 succinate, were prepared using a small-scale media milling device. With particle size 208.7-250.6 nm and polydispersity index <0.250, selected nanodiseprsions were stable for three weeks. Pharmacokinetic and biodistribution studies following intraperitoneal administration of three types of formulation in mice indicated high plasma DK-I-56-1 levels after solution (10,228.6 ± 1037.2 ngh/ml) and nanosuspension (6770.4 ± 770.7 ngh/ml) but not suspension administration (966.0 ± 58.1 ngh/ml). However, distribution of DK-I-56-1 after solution was heavily influenced by its composition, and brain availability of nanosuspension was superior to that of solution formulation. In spontaneous locomotor activity test, the expected hyperlocomotor effect was observed after nanosuspension administration, without compromising impact of the vehicle/excipients used. Therefore, nanonization of drug compound assembled with proper selection of stabilizers may seemingly contribute further thorough testing of DK-I-56-1 preclinical efficacy., Competing Interests: Declaration of Competing Interests none, (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report an improved and scalable synthesis of MIDD0301, a positive GABA A receptor modulator that is under development as oral and inhaled treatments for asthma. In contrast to other benzodiazepines in clinical use, MIDD0301 is a chiral compound that has limited brain absorption. The starting material to generate MIDD0301 is 2-amino-5-bromo-2'-fluorobenzophenone, which has a non-basic nitrogen due to electron withdrawing substituents in the ortho and para positions, reducing its reactivity towards activated carboxylic acids. Investigations of peptide coupling reagents on multigram scale resulted in moderate yields due to incomplete conversions. Secondly, basic conditions used for the formation of the seven-membered 1,4-diazepine ring resulted in racemization of the chiral center. We found that neutral conditions comparable to the pKa of the primary amine were sufficient to support the formation of the intramolecular imine but did not enable the simultaneous removal of the protecting group. Both difficulties were overcome with the application of the N -carboxyanhydride of D-alanine. Activated in the presence of acid, this compound reacted with non-basic 2-amino-5-bromo-2'-fluorobenzophenone and formed the 1,4-diazepine upon neutralization with triethylamine. Carefully designed workup procedures and divergent solubility of the synthetic intermediates in solvents and solvent combinations were utilized to eliminate the need for column chromatography. To improve compatibility with large scale reactors, temperature-controlled slow addition of reagents generated the imidazodiazepine at -20 °C. All intermediates were isolated with a purity of >97% and impurities were identified and quantified. After the final hydrolysis step, MIDD0301 was isolated in a 44% overall yield and purity of 98.9% after recrystallization. The enantiomeric excess was greater than 99.0%., Competing Interests: The authors declare the following competing financial interest(s): Drs. Stafford, Cook and Arnold are inventors of patent application WO2018035246A1, Gaba(A) receptor modulators and methods to control airway hyperresponsiveness and inflammation in asthma. Stafford and Arnold have equity interests in Pantherics Incorporated, which has certain intellectual property rights to these patents.

We describe the effects of pH on the structure and bioavailability of MIDD0301, an oral lead compound for asthma. MIDD0301 interacts with peripheral GABA A receptors to reduce lung inflammation and airway smooth muscle constriction. The structure of MIDD0301 combines basic imidazole and carboxylic acid function in the same diazepine scaffold, resulting in high solubility at neutral pH. Furthermore, we demonstrated that MIDD0301 can interconvert between a seven-membered ring structure at neutral pH and an acyclic compound at or below pH 3. Both structures have two stable conformers in solution that can be observed by 1 H NMR at room temperature. Kinetic analysis showed opening and closing of the seven-membered ring of MIDD0301 at gastric and intestinal pH, occurring with different rate constants. However, in vivo studies showed that the interconversion kinetics are fast enough to yield similar MIDD0301 blood and lung concentrations for neutral and acidic formulations. Importantly, acidic and neutral formulations of MIDD0301 exhibit high lung distribution with low concentrations in brain. These findings demonstrate that MIDD0301 interconverts between stable structures at neutral and acidic pH without changes in bioavailability, further supporting its formulation as an oral asthma medication.

Antinociceptive ligand HZ-166 is a GABA A α2/α3 receptor subtype-selective potentiator. It has been shown to exhibit anxiolytic-like effects in rodent and rhesus monkeys, as well as reduced sedative/ataxic liabilities. In order to improve the metabolic stability of HZ-166, the ethyl ester moiety was bioisosterically replaced with 2,4-disubstituted oxazoles and oxazolines. The new analogs of HZ-166 were synthesized, characterized, and evalutated for their biological activity and docked in the human full-length heteromeric α1β3γ2L GABA A receptor subtype CyroEM structure (6HUO). Importantly no sedation nor ataxia was observed on the rotorod for LKG-I-70 ( 6 ) or KPP-III-51 ( 6c ) at 100 and 120 mg/kg, respectively. These was also no loss of righting response for either ligand.

γ-Aminobutyric acid type A (GABA A ) receptors containing the α6 subunit are located in trigeminal ganglia, and their reduction by small interfering RNA increases inflammatory temporomandibular and myofascial pain in rats. We thus hypothesized that enhancing their activity may help in neuropathic syndromes originating from the trigeminal system. Here, we performed a detailed electrophysiological and pharmacokinetic analysis of two recently developed deuterated structurally similar pyrazoloquinolinone compounds. DK-I-56-1 at concentrations below 1 µM enhanced γ-aminobutyric acid (GABA) currents at recombinant rat α6β3γ2, α6β3δ and α6β3 receptors, whereas it was inactive at most GABA A receptor subtypes containing other α subunits. DK-I-87-1 at concentrations below 1 µM was inactive at α6-containing receptors and only weakly modulated other GABA A receptors investigated. Both plasma and brain tissue kinetics of DK-I-56-1 were relatively slow, with half-lives of 6 and 13 hr, respectively, enabling the persistence of estimated free brain concentrations in the range 10-300 nM throughout a 24-hr period. Results obtained in two protocols of chronic constriction injury of the infraorbital nerve in rats dosed intraperitoneally with DK-I-56-1 during 14 days after surgery or with DK-I-56-1 or DK-I-87-1 during 14 days after trigeminal neuropathy were already established, demonstrated that DK-I-56-1 but not DK-I-87-1 significantly reduced the hypersensitivity response to von Frey filaments. SIGNIFICANCE: Neuropathic pain induced by trigeminal nerve damage is poorly controlled by current treatments. DK-I-56-1 that positively modulates α6 GABA A receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain., (© 2019 European Pain Federation - EFIC®.)

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.

Introduction: High frequency neuronal activity in the cerebral cortex can be induced by noxious stimulation during surgery, brain injury or poisoning. In this scenario, it is essential to block cortical hyperactivity to protect the brain against damage, e.g., by using drugs that act as positive allosteric modulators at GABA A receptors. Yet, cortical neurons express multiple, functionally distinct GABA A receptor subtypes. Currently there is a lack of knowledge which GABA A receptor subtypes would be a good pharmacological target to reduce extensive cortical activity. Methods: Spontaneous action potential activity was monitored by performing extracellular recordings from organotypic neocortical slice cultures of wild type and GABA A R-α1(H101R) mutant mice. Phases of high neuronal activity were characterized using peri-event time histograms. Drug effects on within-up state firing rates were quantified via Hedges' g. Results: We quantified the effects of zolpidem, a positive modulator of GABA A receptors harboring α1-subunits, and the experimental benzodiazepine SH-053-2'F-S-CH3, which preferably acts at α2/3/5- but spares α1-subunits. Both agents decreased spontaneous action potential activity but altered the firing patterns in different ways. Zolpidem reduced action potential firing during highly active network states. This action was abolished by flumazenil, suggesting that it was mediated by benzodiazepine-sensitive GABA A receptors. SH-053-2'F-S-CH3 also attenuated neuronal activity, but unlike zolpidem, failed to reduce high frequency firing. To confirm that zolpidem actions were indeed mediated via α1-dependent actions, it was evaluated in slices from wild type and α(H101R) knock-in mice. Inhibition of high frequency action potential firing was observed in slices from wild type but not mutant mice. Conclusion: Our results suggest that during episodes of scarce and high neuronal activity action potential firing of cortical neurons is controlled by different GABA A receptor subtypes. Exaggerated firing of cortical neurons is reduced by positive modulation of α1-, but not α2/3/5-subunit containing GABA A receptors.

Novel treatments against migraine are an urgent medical requirement. The α6 subunit-containing GABA A receptors (α6GABA A Rs) are expressed in trigeminal ganglia (TG), the hub of the trigeminal vascular system (TGVS) that is involved in the pathogenesis of migraine. Here we reveal an unprecedented role of α6GABA A Rs in ameliorating TGVS activation using several pharmacological approaches in an animal model mimicking pathological changes in migraine. TGVS activation was induced by intra-cisternal (i.c.) instillation of capsaicin in Wistar rats. Centrally, i.c. capsaicin activated the trigeminal cervical complex (TCC) measured by the increased number of c-Fos-immunoreactive (c-Fos-ir) TCC neurons. Peripherally, it elevated calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG and depleted CGRP-ir in the dura mater. Pharmacological approaches included a recently identified α6GABA A R-selective positive allosteric modulator (PAM), the pyrazoloquinolinone Compound 6, two α6GABA A R-active PAMs (Ro15-4513 and loreclezole), an α6GABA A R-inactive benzodiazepine (diazepam), an α6GABA A R-selective antagonist (furosemide), and a clinically effective antimigraine agent (topiramate). We examined effects of these compounds on both central and peripheral TGVS responses induced by i.c. capsaicin. Compound 6 (3-10 mg/kg, i.p.) significantly attenuated the TCC neuronal activation and TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin. All these effects of Compound 6 were mimicked by topiramate, Ro15-4513 and loreclezole, but not by diazepam. The brain-impermeable furosemide antagonized the peripheral, but not central, effects of Compound 6. These results suggest that the α6GABA A R in TG is a novel drug target for TGVS activation and that α6GABA A R-selective PAMs have the potential to be developed as a novel pharmacotherapy for migraine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background and Purpose: The pathophysiological role of α 6 -subunit-containing GABA A receptors, which are mainly expressed in cerebellar granule cells, remains unclear. Recently, we demonstrated that hispidulin, a flavonoid isolated from a local herb that remitted a patient's intractable motor tics, attenuated methamphetamine-induced hyperlocomotion in mice as a positive allosteric modulator (PAM) of cerebellar α 6 GABA A receptors. Here, using hispidulin and a selective α 6 GABA A receptor PAM, the pyrazoloquinolinone Compound 6, we revealed an unprecedented role of cerebellar α 6 GABA A receptors in disrupted prepulse inhibition of the startle response (PPI), which reflects sensorimotor gating deficits manifested in several neuropsychiatric disorders., Experimental Approach: PPI disruptions were induced by methamphetamine and NMDA receptor antagonists in mice. Effects of the tested compounds were measured in Xenopus oocytes expressing recombinant α 6 β 3 γ 2S GABA A receptors., Key Results: Hispidulin given i.p. or by bilateral intracerebellar (i.cb.) injection rescued PPI disruptions induced by methamphetamine, ketamine, MK-801 and phencyclidine. Intracerebellar effects of hispidulin were mimicked by Ro15-4513 and loreclezole (two α 6 GABA A receptor PAMs), but not by diazepam (an α 6 GABA A receptor-inactive benzodiazepine) and were antagonized by furosemide (i.cb.), an α 6 GABA A receptor antagonist. Importantly, Compound 6 (i.p.) also rescued methamphetamine-induced PPI disruption, an effect prevented by furosemide (i.cb.). Both hispidulin and Compound 6 potentiated α 6 β 3 γ 2S GABA A receptor-mediated GABA currents., Conclusions and Implications: Positive allosteric modulation of cerebellar α 6 GABA A receptors rescued disrupted PPI by attenuating granule cell activity. α 6 GABA A receptor-selective PAMs are potential medicines for treating sensorimotor gating deficits in neuropsychiatric disorders. A mechanistic hypothesis is based on evidence for cerebellar contributions to cognitive functioning including sensorimotor gating., (© 2018 The British Pharmacological Society.)

Recent reports indicate that α6β2/3γ2 GABA A R selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABA A R selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABA A R α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABA A Rs and were functionally silent at diazepam sensitive α1β2/3γ2 GABA A Rs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABA A R α6β2/3γ2 subtypes.

Background: Conventional antidepressants are thought to produce their impact on clinical symptoms by increasing the central availability of biogenic amine neurotransmitters (the monoamine hypothesis of depression). These drugs continue to be the primary medicines used in major depressive disorder. Although they have biological effects after acute dosing, full antidepressant response generally takes weeks of daily administration. Lack of rapid onset is a large limitation in antidepressant therapy (e.g., suicide, lack of medication compliance, difficulty switching medications)., Methods: The present review of the literature discusses the preclinical and clinical findings on compounds that can produce immediate symptom relief., Results: These compounds include ketamine, scopolamine, and mechanistically-related drugs. Newer additions to the list of potential rapid-acting agents include antagonists of metabotropic (mGlu) 2/3 receptors, negative allosteric modulators of α5-containing GABAA receptors, and psychedelic compounds. An additional benefit of these compounds is that they have demonstrated large effect sizes and, importantly, demonstrated efficacy in patient's refractory to other treatments. A drawback of some of these compounds, to date, is finding ways to expand the duration of clinical efficacy. In addition, for some compounds, the side-effect profile requires management. A primary mechanism by which rapid effects might be produced is through the amplification of excitatory neurotransmission through activation of AMPA receptors. The extracellular efflux of glutamate induced by these drugs has been documented and provides the hypothesized triggering mechanism for AMPA receptor amplification., Conclusion: The preclinical and clinical literature strongly suggests that rapid-acting antidepressants are the current focus of antidepressant drug discovery. Promising clinical findings exist for several compounds including ketamine and other NMDA receptor antagonists, scopolamine, and psilocybin. Two compounds are in late stage clinical development: GLYX-13 (Rapastinel) and eskekamine., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In this study we assessed organophosphorus (OP) pesticide exposure among children living in two Seattle metropolitan area communities by measuring urinary metabolites, and identified possible exposure risk factors through a parental interview. We recruited children in clinic and outpatient waiting rooms. We obtained spot urine samples in the spring and fall of 1998 from 110 children ages 2-5 years, from 96 households. We analyzed urine samples for six dialkylphosphate (DAP) compounds, the common metabolites of the OP pesticides. Through parental interviews we gathered demographic and residential pesticide use data. At least one of the DAP metabolites was measured in 99% of the children, and the two predominant metabolites (DMTP and DETP) were measured in 70-75% of the children. We found no significant differences in DAP concentrations related to season, community, sex, age, family income, or housing type. Median concentrations of dimethyl and diethyl DAPs were 0.11 and 0.04 micromol/L, respectively (all children). Concentrations were significantly higher in children whose parents reported pesticide use in the garden (0.19 vs. 0.09 micromol/L for dimethyl metabolites, p = 0.05; 0.04 vs. 0.03 micromol/L for diethyl metabolites, p = 0.02), but were not different based on reported pet treatment or indoor residential use. Nearly all children in this study had measurable levels of OP pesticide metabolites. Some of this exposure was likely due to diet. Garden pesticide use was associated with elevated metabolite levels. It is unlikely that these exposure levels would cause acute intoxication, but the long-term health effects of such exposures are unknown. We recommend that OP pesticide use be avoided in areas where children are likely to play.

A method has been developed for determination of the xanthine drug, pentoxifylline, and three of its metabolites (a secondary alcohol and two carboxylic acids) in human milk. The method requires pre-extraction with hexane to remove lipids followed by extraction with dichloromethane or dichloromethane-isopropanol (4:1). Absolute extraction recoveries were between 76-90%. Pentoxifylline and its alcohol metabolite (as the trifluoroacetate) and the carboxylic acid metabolites (as ethyl esters) were measured in separate gas chromatographic steps using a nitrogen detector. Determinations of pentoxifylline and its three metabolites were 96-99% accurate and standard deviations of 5-10% were observed for samples at or above the lower practical sensitivity limit (10 ng/ml) for the assay. Pentoxifylline and its metabolites were stable in breast milk for three weeks when stored at -15 degrees C.