Development and validation of an LC-MS/MS method for the determination of ARN14988, an acid ceramidase inhibitor, and its application to a pharmacokinetic study in a mouse model.

Despite aggressive treatment approaches, the overall survival of glioblastoma (GBM) patients remained poor with a strong need for more effective chemotherapeutic agents. A previous study has shown that ARN14988 is more cytotoxic to GBM cells compared to US Food and Drug Administration-approved temozolomide. This finding makes ARN14988 a desirable candidate for further pharmacological assessment. Therefore, an efficient analytical method is needed to quantify ARN14988. Herein, we have developed and validated sample preparation and LC-MS/MS triple quadrupole (QQQ) method for quantification of ARN14988 in mouse plasma. In this method, the liquid-liquid extraction of ARN14988 from mouse plasma was performed using 5% ethyl acetate in hexane. The chromatographic separation was achieved using a C18 -column with mobile phases of 10 mm ammonium acetate (pH 5) and 0.1% formic acid in methanol, within a runtime of 10 min. The monitored transitions were m/z 391.20 → m/z 147.00 for ARN14988, and m/z 455.30 → m/z 165.00 for verapamil (internal standard) in positive electrospray ionization. The developed method for ARN14988 showed linearity over the range of 10-5,000 ng/ml (r2 > 0.99). The selectivity, sensitivity, matrix effect, recovery, stability, inter-day and intraday accuracy and precision were determined using four quality control samples. This validated method was successfully applied to the pharmacokinetic study of ARN14988 in mice.

Structural Analogs of the GABAkine KRM-II-81 Are Orally Bioavailable Anticonvulsants without Sedation.

To provide back-up compounds to support the development of the GABAA receptor (GABAAR) potentiator KRM-II-81, three novel analogs were designed: replacing the pyridinyl with 2'-Cl-phenyl (FR-II-60), changing the positions of the N and O atoms in the oxazole ring with addition of an ethyl group (KPP-III-34 and KPP-III-51), or substituting a Br atom for the ethynyl of KRM-II-81 (KPP-III-34). The compounds bound to brain GABAARs. Intraperitoneal administration of FR-II-60 and KPP-III-34 produced anticonvulsant activity in mice [maximal electroshock (MES)-induced seizures or 6 Hz-induced seizures], whereas KPP-III-51 did not. Although all compounds were orally bioavailable, structural changes reduced the plasma and brain (FR-II-60 and KPP-III-51) exposures relative to KRM-II-81. Oral administration of each compound produced dose-dependent increases in the latency for both clonic and tonic seizures and the lethality induced by pentylenetetrazol (PTZ) in mice. Since KPP-III-34 produced the highest brain area under the curve (AUC) exposures, it was selected for further profiling. Oral administration of KPP-III-34 suppressed seizures in corneal-kindled mice, hippocampal paroxysmal discharges in mesial temporal lobe epileptic mice, and PTZ-induced convulsions in rats. Only transient sensorimotor impairment was observed in mice, and doses of KPP-III-34 up to 500 mg/kg did not produce impairment in rats. Molecular docking studies demonstrated that all compounds displayed a reduced propensity for binding to α1His102 compared with the sedating compound alprazolam; the bromine-substituted KPP-III-34 achieved the least interaction. Overall, these findings document the oral bioavailability and anticonvulsant efficacy of three novel analogs of KRM-II-81 with reduced sedative effects. SIGNIFICANCE STATEMENT: A new non-sedating compound, KRM-II-81, with reduced propensity for tolerance is moving into clinical development. Three new analogs were orally bioavailable, produced anticonvulsant effects in rodents, and displayed low sensorimotor impairment. KPP-III-34 demonstrated efficacy in models of pharmacoresistant epilepsy. Docking studies demonstrated a low propensity for compound binding to the α1His102 residue implicated in sedation. Thus, three additional structures have been added to the list of non-sedating imidazodiazepine anticonvulsants that could serve as backups in the clinical development of KRM-II-81.

Comparative anticonvulsant activity of the GABAkine KRM-II-81 and a deuterated analog.

A series of imidazodiazepines has been developed that possess reduced sedative liabilities but retain efficacy in anticonvulsant screening models. The latest of these compounds, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole known as KRM-II-81) is currently awaiting advancement into the clinic. A deuterated structural analog (D5-KRM-II-81) was made as a potential backup compound and studied here in comparison to KRM-II-81. In the present study, both compounds significantly prevented seizures in mice induced by 6 Hz (44 mA) electrical stimulation without significantly altering motoric function on a rotarod after intraperitoneal administration. Both compounds also significantly prevented clonic seizures, tonic seizures, and lethality induced by pentylenetetrazol in mice when given orally. D5-KRM-II-81 had a slightly longer duration of action against clonic and tonic seizures than KRM-II-81. Oral administration of 100 mg/kg of either KRM-II-81 or D5-KRM-II-81 was significantly less disruptive of sensorimotor function in mice than diazepam (5 mg/kg, p.o.). The present report documents that D5-KRM-II-81 represents another in this series of imidazodiazepines with anticonvulsant activity at doses that do not impair sensorimotor function.

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction in vivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

Metabolism, pharmacokinetics, and anticonvulsant activity of a deuterated analog of the α2/3-selective GABAkine KRM-II-81.

The imidazodiazepine, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo [f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a new α2/3-selective GABAkine (gamma aminobutyric acid A receptor potentiator) with anticonvulsant, anxiolytic, and antinociceptive activity in preclinical models. Reducing metabolism was utilized as a means of potentially extending the half-life of KRM-II-81. In vitro and in vivo studies were conducted to evaluate metabolic liabilities. Incubation of KRM-II-81 in hepatocytes revealed sites of potential metabolism on the oxazole and the diazepine rings. These sites were targeted in the design of a deuterated analog (D5-KRM-II-81) that could be evaluated as a potentially longer-acting analog. In contrast to computer predictions, peak plasma concentrations of D5-KRM-II-81 in rats were not significantly greater than those produced by KRM-II-81 after oral administration. Furthermore, brain disposition of KRM-II-81 was higher than that of D5-KRM-II-81. The half-life of the two compounds in either plasma or brain did not statistically differ from one another but the tmax for D5-KRM-II-81 occurred slightly earlier than for KRM-II-81. Non-metabolic considerations might be relevant to the lack of increases in exposure by D5-KRM-II-81. Alternative sites of metabolism on KRM-II-81, not targeted by the current deuteration process, are also possible. Despite its lack of augmented exposure, D5-KRM-II-81, like KRM-II-81, significantly prevented seizures induced by pentylenetetrazol when given orally. The present findings introduce a new orally active anticonvulsant GABAkine, D5-KRM-II-81.

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

The Effects of pH on the Structure and Bioavailability of Imidazobenzodiazepine-3-Carboxylate MIDD0301.

We describe the effects of pH on the structure and bioavailability of MIDD0301, an oral lead compound for asthma. MIDD0301 interacts with peripheral GABAA 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 1H 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.

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor.

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC50 of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with 3H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAAR) of the α1-3ß2-3γ1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABAARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.

A novel GABAA receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.

Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.

An anthrone-based Kv7.2/7.3 channel blocker with improved properties for the investigation of psychiatric and neurodegenerative disorders.

A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC50 = 0.16 µM in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.

The parmodulin NRD-21 is an allosteric inhibitor of PAR1 Gq signaling with improved anti-inflammatory activity and stability.

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.

A high-throughput screening assay for pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5ß3γ2 selective GABAA receptor (GABAAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5ß3γ2 GABAARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAAR ligands.

Novel VDR antagonists based on the GW0742 scaffold.

The vitamin D receptor is a nuclear hormone receptor that regulates cell proliferation, cell differentiation and calcium homeostasis. The receptor is endogenously activated by 1,25-dihydroxyvitamin D3, which induces transcription of VDR targets genes regulated by coactivator binding. VDR antagonists and partial agonists have been developed based on the secosteroid scaffold of vitamin D. Only a few non-secosteroid VDR antagonists are known. Herein, we report the rational design of non-secosteroid VDR antagonists using GW0742 as a scaffold. GW0742 is a PPARδ agonist previously identified by our group as a VDR antagonist. Several modifications including the replacement of the thiazole ring with an oxazole ring led to compound 7b, which inhibited VDR-mediated transcription (IC50 = 660 nM) without activating PPARδ-mediated transcription. However, inhibition of transcription mediated by other nuclear receptors was observed.

Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABAA Receptor Modulators.

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α4ß3γ2 GABAAR selective compound 1 and acidic α5ß3γ2 selective GABAAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABAARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAAR ligands.

Genetic and pharmacological manipulation of glyoxalase 1 regulates voluntary ethanol consumption in mice.

Previous studies have identified an association between the gene glyoxalase 1 (Glo1) and anxiety-like behavior in mice and have shown that the substrate of GLO1, methylglyoxal, is a competitive partial agonist at GABAA receptors. Given the well-established role of GABAA receptors in the behavioral effects of ethanol (EtOH), we investigated the role of Glo1 in voluntary EtOH consumption in mice using the drinking in the dark (DID) paradigm. Transgenic mice overexpressing Glo1 on both FVB/NJ (FVB) or C57BL/6J (B6) backgrounds showed increased voluntary EtOH consumption compared to their wild-type littermates in DID. Furthermore, transgenic Glo1 knockdown mice on a B6 background showed decreased voluntary EtOH consumption in DID. These genetic manipulations of Glo1 had no effect on sucrose, saccharin or water consumption. Finally, we found that a small molecule GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester (pBBG; 6.25, 12.5 mg/kg)) reduced EtOH consumption compared to vehicle treated B6 mice without altering saccharin or water consumption. Sucrose consumption was only reduced by the higher (12.5 mg/kg) dose of pBBG. We did not observe differences in the loss of righting reflex (LORR) or EtOH-induced foot slips on the balance beam in response to acute EtOH administration (LORR: 4 g/kg, Balance Beam: 1.25 g/kg) in B6 or FVB mice overexpressing Glo1, nor in B6 mice treated with pBBG. These data are the first to implicate Glo1 in EtOH-related behaviors and suggest that GLO1 inhibitors may have therapeutic potential for the treatment of alcohol use disorders.

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

Glo1 inhibitors for neuropsychiatric and anti-epileptic drug development.

Many current pharmacological treatments for neuropsychiatric disorders, such as anxiety and depression, are limited by a delayed onset of therapeutic effect, adverse side effects, abuse potential or lack of efficacy in many patients. These off-target effects highlight the need to identify novel mechanisms and targets for treatment. Recently, modulation of Glo1 (glyoxalase I) activity was shown to regulate anxiety-like behaviour and seizure-susceptibility in mice. These effects are likely to be mediated through the regulation of MG (methylglyoxal) by Glo1, as MG acts as a competitive partial agonist at GABA(A) (γ-aminobutyric acid A) receptors. Thus modulation of MG by Glo1 represents a novel target for treatment. In the present article, we evaluate the therapeutic potential of indirectly modulating MG concentrations through Glo1 inhibitors for the treatment of neuropsychiatric disorders.