I. Design and Synthesis of Α6-GABAAR Subtype-selective Pyrazoloquinolinones with Improved Metabolic Stability and Enhanced Bioavailability. II & III. Process Development of Subtype-Selective Α2/3-GABAAR Imidazodiazepines: HZ-166 and KRM-II-81 and Α5-GABAAR Imidazodiazepines: MIDD0301 and GL-II-73

Abstract-Part 1 Examination of recent reports indicate that α6β2/3γ2 GABAAR selective ligands may be important for the treatment of trigeminal orofacial pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABAAR selective pyrazoloquinolinones, 49 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 GABAAR α6 subtype was retained. The deuterated pyrazoloquinolinone DK-I-56-1 (8b) was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABAARs and were functionally silent at diazepam sensitive α1β2/3γ2 GABAARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which rendered them interesting candidates for treatment of CNS disorders mediated by GABAAR α6β2/3γ2 subtypes. Recently, the GABAAR α+β- binding site was discovered along with the first α6-GABAAR subtype-selective ligand, PZ-II-029 (8a) and later, LAU463 (8n). Subsequent research led to the development of the lead deuterated pyrazoloquinolinones, DK-I-56-1 (8b), DK-I-58-1 (8o) and DK-II-58-1 (13h) which have been shown to exhibit functional selectivity for α6-GABAARs with increased metabolic stability and enhanced bioavailability. The pursuit of further improvements to the bioavailability of these α6-selective ligands led to the exploration of fluorinated substituents (trifluoromethoxy) as an alternative tactic to limit O-demethylation in vivo of the methoxy groups of these lead ligands DK-I-56-1 (8b) and DK-I-58-1 (8o). Herein, the comparative in vivo and in vitro effects of deuteration versus fluorination of these pyrazoloquinolinones is reported. While the strategy of deuteration and fluorination both increased metabolic stability in HLM and MLM, the pharmacokinetic effect on plasma, brain, liver and kidney exposure was dramatically different. Additionally, the fluorinated analogs exhibited a slightly increased cytotoxic effect in vitro on liver and kidney cell lines. Efficacy and functional selectivity for the α6+β3- interface (PQ Site) of α6-GABAARs was reduced for the fluorinated analogs in comparison to the deuterated ligands, as was as the binding affinity at the Bz site (α+γ- interface). In summary, the deuteration strategy proved superior to fluorination of these pyrazoloquinolinones in terms of maintaining α6-selectivity while enhancing the bioavailability and drugability of these α6-GABAAR ligands without detrimental effects. Finally, a more water-soluble phosphoric acid prodrug of DK-I-58-1 (8o) was also synthesized. Part 2 The need for improved medications for the treatment of epilepsy and chronic pain is essential. Epileptic patients typically take multiple antiepileptic drugs without complete seizure freedom. The (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-[1,4]diazepin-3-yl) oxazole (KRM-II-81) is a lead compound in a series of ligands designed as positive allosteric modulators (PAMs) of 2/3-containing GABAA receptors. Ligand KRM-II-81 produced broad-based anticonvulsant efficacy in rodent models used to predict antiepileptic efficacy in patients and provides a wider margin over motoric side effects than that of other GABAA receptor PAMs. The present series of experiments was designed to fill key missing gaps in prior preclinical assessments to assess whether KRM-II-81 could be further differentiated from non-selective GABAA 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 that of DZP. In addition, KRM-II-81 but not DZP blocks the development of seizure sensitivity to the chemical convulsants cocaine and pentylenetetrazol. This antiepileptogenic effect is considered the holy grail of antiepileptic treatment. These and previous data have placed KRM-II-81 into consideration for clinical development requiring the manufacture of kilogram amounts of GMP 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 for patients who continue to suffer from pharmacoresistent epilepsy. Part 3 MIDD0301 ((R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid) is a novel oral drug under development for the treatment of asthma, which targets peripheral α5-GABAARs found in airway smooth muscle (ASM) and reduces lung inflammation. Because of the promising results of MIDD0301 as an asthma therapy devoid of negative side effects, the need for clinical development was of interest. Consequently, the development of a synthetic procedure amenable to the GMP manufacture of kilograms quantities of MIDD0301 was required to improve upon the discovery route. Described herein is an improved and novel synthetic route amenable to the production of kilogram quantities of MIDD0301. Over the last 15 years worldwide life expectancy increased by 5 years jumping from 66 years to 71 years. With progress in science, medicine, and care one tends to live longer. Such extended life expectancy is still associated with age-related changes, including in the brain. The aging brain goes through various changes that can be called morphomolecular senescence. Overall, the brain volume changes, neuronal activity is modified, and plasticity of the cells diminishes, sometimes leading to neuronal atrophy and death. Altogether, these changes contribute to the emergence of cognitive decline where upon an efficient treatment is still not available. Many studies in the context of cognitive decline focused on pathological aging, targeting beta-amyloid in Alzheimer\u27s disease, for example. However, beta-amyloid plaques are also present in healthy adults and treatments targeting plaques have failed to improve cognitive functions. In order to improve the quality of life of aging populations, it is crucial to focus on the development of novel therapies targeting different systems altered during aging, such as the GABAergic system. In previous studies it has been shown that positive allosteric modulators (PAM) acting at α5-containing GABAA receptors improve cognitive performances, and that these α5-GABAA receptors are implicated in dendritic growth of pyramidal neurons. Here, we hypothesized that targeting the α5-GABAA receptors could contribute to the reduction of cognitive decline, directly through activity of the receptors, and indirectly by increasing neuronal morphology. Using primary neuronal culture and chronic treatment in mice, it was demonstrated that an α5-PAM (GL-II-73) increased dendritic length, spine count and spine density in brain regions involved in cognitive processes (prefrontal cortex and hippocampus). The pro-cognitive efficacy of the α5-PAM, GL-II-73 was confirmed and the washout period diminished the precognitive effects without altering the effect on neuronal morphology. Future studies will be needed to investigate what downstream mechanisms are responsible for the neurotrophic effect of the α5-PAM (GL-II-73). These results and previous data have placed GL-II-73 into consideration for clinical development requiring a route to manufacture of kilogram amounts of GMP material. Described herein is a novel synthetic route amenable to kilogram quantity production

Global Projections of Intense Tropical Cyclone Activity for the Late Twenty-First Century from Dynamical Downscaling of CMIP5/RCP4.5 Scenarios

Global projections of intense tropical cyclone activity are derived from the Geophysical Fluid Dynamics Laboratory (GFDL) High Resolution Atmospheric Model (HiRAM; 50-km grid) and the GFDL hurricane model using a two-stage downscaling procedure. First, tropical cyclone genesis is simulated globally using HiRAM. Each storm is then downscaled into the GFDL hurricane model, with horizontal grid spacing near the storm of 6 km, including ocean coupling (e.g., cold wake generation). Simulations are performed using observed sea surface temperatures (SSTs) (1980-2008) for a control run with 20 repeating seasonal cycles and for a late-twenty-first-century projection using an altered SST seasonal cycle obtained from a phase 5 of CMIP (CMIP5)/representative concentration pathway 4.5 (RCP4.5) multimodel ensemble. In general agreement with most previous studies, projections with this framework indicate fewer tropical cyclones globally in a warmer late-twenty-first-century climate, but also an increase in average cyclone intensity, precipitation rates, and the number and occurrence days of very intense category 4 and 5 storms. While these changes are apparent in the globally averaged tropical cyclone statistics, they are not necessarily present in each individual basin. The interbasin variation of changes in most of the tropical cyclone metrics examined is directly correlated to the variation in magnitude of SST increases between the basins. Finally, the framework is shown to be capable of reproducing both the observed global distribution of outer storm size-albeit with a slight high bias-and its interbasin variability. Projected median size is found to remain nearly constant globally, with increases in most basins offset by decreases in the northwest Pacific

Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study

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

Zolpidem Activation of Alpha 1-Containing GABAA Receptors Selectively Inhibits High Frequency Action Potential Firing of Cortical Neurons

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 GABAA receptors. Yet, cortical neurons express multiple, functionally distinct GABAA receptor subtypes. Currently there is a lack of knowledge which GABAA 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 GABAAR-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 GABAA 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 GABAA 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 GABAA receptor subtypes. Exaggerated firing of cortical neurons is reduced by positive modulation of α1-, but not α2/3/5-subunit containing GABAA receptors

A Featureless Infrared Transmission Spectrum for the Super-puff Planet Kepler-79d

Extremely low-density planets ("super-puffs") are a small but intriguing subset of the transiting planet population. With masses in the super-Earth range (1 – 10 M_⊕) and radii akin to those of giant planets (> 4 R_⊕), their large envelopes may have been accreted beyond the water snow line and many appear to be susceptible to catastrophic mass loss. Both the presence of water and the importance of mass loss can be explored using transmission spectroscopy. Here, we present new Hubble space telescope WFC3 spectroscopy and updated Kepler transit depth measurements for the super-puff Kepler-79d. We do not detect any molecular absorption features in the 1.1 − 1.7 μm WFC3 bandpass, and the combined Kepler and WFC3 data are consistent with a flat-line model, indicating the presence of aerosols in the atmosphere. We compare the shape of Kepler-79d's transmission spectrum to predictions from a microphysical haze model that incorporates an outward particle flux due to ongoing mass loss. We find that photochemical hazes offer an attractive explanation for the observed properties of super-puffs like Kepler-79d, as they simultaneously render the near-infrared spectrum featureless and reduce the inferred envelope mass-loss rate by moving the measured radius (optical depth unity surface during transit) to lower pressures. We revisit the broader question of mass-loss rates for super-puffs and find that the age estimates and mass-loss rates for the majority of super-puffs can be reconciled if hazes move the photosphere from the typically assumed pressure of ~10 mbar to ~10 µbar

Transiting Exoplanet Studies and Community Targets for JWST's Early Release Science Program

The James Webb Space Telescope will revolutionize transiting exoplanet atmospheric science due to its capability for continuous, long-duration observations and its larger collecting area, spectral coverage, and spectral resolution compared to existing space-based facilities. However, it is unclear precisely how well JWST will perform and which of its myriad instruments and observing modes will be best suited for transiting exoplanet studies. In this article, we describe a prefatory JWST Early Release Science (ERS) program that focuses on testing specific observing modes to quickly give the community the data and experience it needs to plan more efficient and successful future transiting exoplanet characterization programs. We propose a multi-pronged approach wherein one aspect of the program focuses on observing transits of a single target with all of the recommended observing modes to identify and understand potential systematics, compare transmission spectra at overlapping and neighboring wavelength regions, confirm throughputs, and determine overall performances. In our search for transiting exoplanets that are well suited to achieving these goals, we identify 12 objects (dubbed "community targets") that meet our defined criteria. Currently, the most favorable target is WASP-62b because of its large predicted signal size, relatively bright host star, and location in JWST's continuous viewing zone. Since most of the community targets do not have well-characterized atmospheres, we recommend initiating preparatory observing programs to determine the presence of obscuring clouds/hazes within their atmospheres. Measurable spectroscopic features are needed to establish the optimal resolution and wavelength regions for exoplanet characterization. Other initiatives from our proposed ERS program include testing the instrument brightness limits and performing phase-curve observations.(Abridged)Comment: This is a white paper that originated from an open discussion at the Enabling Transiting Exoplanet Science with JWST workshop held November 16 - 18, 2015 at STScI (http://www.stsci.edu/jwst/science/exoplanets). Accepted for publication in PAS

Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1); process parameters and cryoprotectant selection through stability study

1. INTRODUCTION Nanocrystal dispersions are considered as the universal formulation strategy for brick dust substances. However, the stability of these systems to aggregation represents a big issue. To overcome this, nanocrystal dispersions are usually solidified by freeze-drying (lyophilization). During this process the risk of aggregation is considered to be high, due to ice formation and/or water loss. To prevent the aggregation, For the particle size preservation, therefore, it is necessary to add cryoprotectants/lyoprotectants, among which sugars are most commonly used. To ensure good structure of the cake, bulking agents are often included in formulations, as well [1,2], although in nanocrystalline dispersions the combination of cryoprotectants and bulking agents is not frequent nor much investigated. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), patent protected pyrazoloquinolinone ligand, have been developed recently, and characterized in terms of physicochemical properties and pharmacokinetics after intraperitoneal administration in mice. These formulations were stable for three weeks [3]. Our aim in this study was to improve the stability by freeze-drying, and investigate the influence of different concentrations and physical form of cryoprotectants (sucrose, trehalose) and bulking agent (mannitol) as well as different primary drying conditions on the aggregation prevention. 2. MATERIALS AND METHODS 2.1. Materials DK-I-56-1 was synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin—Milwaukee, USA. The following other materials were used: polysorbate 80, poloxamer 407, sucrose, mannitol (Sigma-Aldrich Laborchemikalien GmbH, Germany) and trehalose (Carl Roth GmbH, Germany). 2.2. Lyophilization Nanocrystal dispersions stabilized by polysorbate 80 and poloxamer 407 were prepared by wet ball milling [3]. After addition of mannitol (M), sucrose (S), or trehalose (T) alone or in combination samples were freeze- dried. Two processes were applied: (1) freezing at -80 °C (3 h), primary drying at -10 °C, 0.340 mbar, secondary drying at 25 °C (24 h) or (2) freezing at -50 °C (3 h), primary drying at -45 °C, 0.2 mbar (21 h), secondary drying at 20 °C (30 h). Samples were stored in crimped vials at 25 °C (lyophilization 1) or 2-8 ºC (lyophilization 2) for three months. 2.3. Physicochemical characterization Particle size (z-ave) was measured by Zetasizer Nano ZS (Malvern Instruments, UK) and Mastersizer (Malvern Mastersizer 2000 Malvern, UK). Redispersibility index (RDI) was calculated as z-ave (before)/z-ave (after) and expressed in percentages. Physical state of samples was determined by differential scanning calorimetry (DSC1; Mettler Toledo, Switzerland),powder X-ray diffraction (Rigaku Smartlab X-ray Diffractometer) and polarized light microscopy (PLM) (Carl Zeiss ApoTome Imager Z1 microscope Zeiss, Germany). 3. RESULTS AND DISCUSSION Right after preparation, nanocrystal dispersions were with submicron particle size around 160 nm, and PDI below 0.2, suggesting narrow size distribution. In the cryoprotectant screening phase, sucrose and/or mannitol were added in different concentrations. It was shown that 10% of the total stabilizer concentration was needed for the particle size preservation: the achieved RDI was above 95%, while cakes with sucrose alone or in combination with mannitol in ratio 1:1 or 3:2 were also with satisfied appearance (Figure 1). Lyophilization was conducted above or below the glass transition temperature of the maximally freeze-concentrated solution (Tg’) (around -39 ºC). When primary drying was performed at -10 °C, no aggregation was noticed right after lyophilization, but particle size increased significantly, lowering down the RDI to < 50%, after one month storage at 25 °C. This was confirmed by laser diffraction. In lyophilization 2, with primary drying at temperature below Tg’, trehalose was also used in the same concentration as sucrose and in combination with mannitol. Interestingly, in this process parameters setup, sucrose or trehalose alone did not prevent aggregation during freeze-drying. Particle size remained almost unchanged in formulation S+M 3+2 (RDI 95%) or slightly higher in T+M 3+2 (RDI 90%), after three months storage, suggesting it was most probably the optimal combination for the stabilization. Physical state analysis revealed that sucrose and mannitol in samples lyophilized by process 1 were in crystalline state, as well as sucrose when used alone in lyophilization 2. Trehalose, on the other hand was amorphous in all samples containing it. Amorphous state of lyoprotectants allows maximal hydrogen bonding due to higher molecule flexibility and availability of hydroxyl groups [3]. Surprisingly, mannitol as a substance with high crystallization tendency was with low crystallinity in lyophilizates. These observations were confirmed by PLM. It is possible that it formed interactions with sucrose or nanocrystal stabilizers [4]. 4. CONCLUSION Results from this study demonstrated freeze- drying as an important technique for the improvement of nanocrystals stability. However, the selection of cryoprotectant and bulking agent ratio beside process parameters (primary drying at -45 ºC) was crucial to get freeze-dried samples with good stability. Sucrose or trehalose in combination with mannitol (ratio 3+2) in total concentration 10% successfully hindered aggregation, thus prolonging the stability to 3 months at 2-8 ºC. 5. REFERENCES 1. Van Eerdenbrugh, B., et al. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. International journal of pharmaceutics, 2008. 364(1): 64-75. 2. Trenkenschuh, E., and Friess, W. Freeze-drying of nanoparticles: How to overcome colloidal instability by formulation and process optimization. European Journal of Pharmaceutics and Biopharmaceutics, 2021.165: 345-360. 3. Mitrović, J.R., et al. Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand DK-I-60-3 by nanonization: A knowledge-based approach. Pharmaceutics, 2021. 13(8): 1188. 4. Kumar, S., et al. Sugars as bulking agents to prevent nano-crystal aggregation during spray or freeze-drying. International journal of pharmaceutics, 2014. 471(1-2): 303-311. ACKNOWLEDGMENT This research was supported by the Science Fund of the Republic of Serbia, grant No. 7749108, project Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma-2 receptors: In vitro/in vivo delineation by nano- and hiPSC-based platforms-NanoCellEmoCog