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GABA mediates inhibitory actions through various GABAA receptor subtypes, including 19 subunits in human GABAAR. Dysregulation of GABAergic neurotransmission is associated with several psychiatric disorders, including depression, anxiety, and schizophrenia. Selective targeting of α2/3 GABAARs can treat mood and anxiety, while α5 GABAA-Rs can treat anxiety, depression, and cognitive performance. GL-II-73 and MP-III-022, α5-positive allosteric modulators have shown promising results in animal models of chronic stress, aging, and cognitive disorders, including MDD, schizophrenia, autism, and Alzheimer’s disease. Described in this article is how small changes in the structure of imidazodiazepine substituents can greatly impact the subtype selectivity of benzodiazepine GABAAR. To investigate alternate and potentially more effective therapeutic compounds, modifications were made to the structure of imidazodiazepine 1 to synthesize different amide analogs. The novel ligands were screened at the NIMH PDSP against a panel of 47 receptors, ion channels, including hERG, and transporters to identify on- and off-target interactions. Any ligands with significant inhibition in primary binding were subjected to secondary binding assays to determine their Ki values. The newly synthesized imidazodiazepines were found to have variable affinities for the benzodiazepine site and negligible or no binding to any off-target profile receptors that could cause other physiological problems.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle 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 GABA(A)R expressed in rat brain, with IC(50) values of 25.1 nM and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted airway smooth muscle 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 three days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by INFγ and 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 GABA(A)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.

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.

[Image: see text] 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.

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.

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 potentiator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. KRM-II-81 is active against many seizure and pain models in rodents, where it exhibits improved pharmacological properties over standard-of-care agents. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption, and distribution, as well as for efficient methods of bulk synthesis, a hydrochloride salt of KRM-II-81 was prepared. KRM-II-81·HCl was produced from the free base with anhydrous hydrochloric acid. The formation of the monohydrochloride salt was confirmed by X-ray crystallography, as well as 1H NMR and 13C NMR analyses. High water solubility and a lower partition coefficient (octanol/water) were exhibited by KRM-II-81·HCl as compared to the free base. Oral administration of either KRM-II-81·HCl or the free base resulted in high concentrations in the brain and plasma of rats. Oral dosing in mice significantly increased the latency to both clonic and tonic convulsions and decreased pentylenetetrazol-induced lethality. The increased water solubility of the HCl salt enables intravenous dosing and the potential for higher concentration formulations compared with the free base without impacting anticonvulsant potency. Thus, KRM-II-81·HCl adds an important new compound to facilitate the development of these imidazodiazepines for clinical evaluation.

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.

Chronic stress is a major risk factor for developing depressive disorders and animal models of stress recapitulate behavioral, cellular and molecular changes that are observed in human depression. Individuals exposed to chronic stress, or patients with MDD experience mood and cognitive dysfunctions. This is in part due to neuronal shrinkage in brain regions involved in several cognitive functions such as the prefrontal cortex (PFC) and the hippocampus (HPC). Also in the context of depression and chronic stress, expression levels and function of the main inhibitory neurotransmitter GABA are reduced. Thus far, drugs targeting this GABA deficit have failed to produce beneficial effects due to broad activity at various GABA receptor subunits, including the α1-subunit, resulting in broad side effects. However, refined and selective activity at the α2/3/5-subunit is hypothesized to exert beneficial effect, devoid of side effects. Here, we show that GL-II-73 and GL-I-54 exert positive allosteric modulation at the α5, and α2/3/5-contianing GABAA receptors respectively, and that they are effective both independently and in combination. Using unpredictable chronic mild stress (UCMS) experiments in male and female C57BL/6 mice (n=12 per group), we showed that acute and chronic administration of a GL-II-73/GL-I-54 racemic mixture (termed “GL-RM”) reduced anxiety-like phenotypes and reversed a working memory deficit in UCMS exposed mice. Brains from animals receiving chronic treatment were collected and stained using a Golgi staining technique. Using stereological approaches, neuronal morphology was reconstructed and dendritic length, spine count and spine density were assessed in pyramidal neurons of the PFC and hippocampus. Chronic GL-RM rescued spine density depletions caused by UCMS at apical and basal dendrites (PFC and CA1). Interestingly, spine densities in both brain regions were correlated to cognitive performance, confirming ameliorative benefits of GL-RM. Together, results support the value of selectively targeting GABAA receptors, excluding the α1-subunit, to overcome chronic stress-induced mood symptoms and cognitive deficits, as well as detriments in neuronal morphology. This study confirm results that were observed in old mice, using a α5-selective positive allosteric modulator, and reinforce the concept that the α2/3/5-containing GABAA receptor are suitable targets for the treatment of stress-induced disorders.

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 administra- tion. 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 statis- tically 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.

[Image: see text] We report the modification of MIDD0301, an imidazodiazepine GABA(A) receptor (GABA(A)R) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABA(A)R binding site of clonazepam, with IC(50) values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α(1)β(3)γ(2) GABA(A)R structural data, suggests binding of the diazepine core between the α1+/γ2– interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

The pharmacological actions exerted by benzodiazepines are dependent on the discrete α protein subunits of the γ-aminobutyric acid type A receptor (GABA

Dysregulation of GABAergic neurotransmission has long been implicated in several psychiatric disorders, including schizophrenia, depression, and anxiety disorders. Alpha 5 subunit-containing GABA

Different subtypes of GABA

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%.

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 GABAA receptors (5-(8-ethynyl-6-(pyridin-2-yl)-4H-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 GABAA 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 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 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.

Analgesic and anti-inflammatory properties mediated by the &kappa, 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, &micro, opioid receptor (MOR), and &delta, 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 &alpha, 1-3&beta, 2-3&gamma, 1-2 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.

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC(50) of 260 nM and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABA(A)R) α(3) subunit, which can assemble with β(1) and γ(2)/δ subunits to form functional GABA(A)Rs. Mouse microglia contained α(2), α(3) and α(5), in addition to β(1–3), γ(1–2) and δ, mRNA, enabling a more diverse array of GABA(A)Rs than human microglia. Benzodiazepines are well-established modulators of GABA(A)R activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABA(A)R antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABA(A)Rs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the μ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of non-sedative drug candidates for inflammatory pain.

GABAkines, or positive allosteric modulators of γ-aminobutyric acid-A (GABA

The aim of the study was to evaluate different biological properties of fruits extracts of Ficus erecta (Thunb.). The concentrated methanolic extracts and Kupchan partitions were screened for thrombolytic activity, whereas streptokinase was used as standard antioxidant property by evaluating free radical scavenging effect and determining total phenolic content and cytotoxic activity by brine shrimp lethality bioassay. The highest thrombolytic activity was exhibited by carbon tetrachloride soluble partition (CTP) of the plant (27.53±0.41%) against the standard streptokinase (65.16±0.064%). The IC50 value of the petroleum ether soluble partition (PEP) was found to be 7.35±0.08 μg/ml, whereas standard ascorbic acid demonstrated IC50 value of 5.80±0.22 μg/ml. The petroleum ether soluble partition (PEP) also exhibited highest phenolic content (43.69±1.4 mg of GAE/100 g of extractives). A correlation was calculated between total phenolic content and free radical scavenging activity of F. erecta which showed positive result having correlation coefficient (R2) of 0.779. Among the LC50 values of the plant material, petroleum ether soluble partition (PEP) exhibited the lowest value of 1.20±0.028 µg/ml as compared to the standard vincristine sulphate (0.45¬±0.003 µg/ml). The present study showed that, the plant F. erecta demonstrated strong cytotoxic and antioxidant property and moderate thrombolytic activity. Key words: Ficus erecta, thrombolytic, antioxidant, free radical scavenging, phenolic content, brine shrimp lethality bioassay, streptokinase, vincristine sulphate, acetyl salicylic acid.

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports of cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines, given the known anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970’s but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZ’s anti-parasitic target is likely distinct from the human receptors that drive sedation. Therefore, we screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, causing extensive vacuole formation beneath the apical membrane. The imidazobenzodiazepine compound series identified has a dramatically lower (∼1 log) affinity for human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects.Author SummaryOver 200 million people are infected with schistosomiasis, yet there are limited therapeutic options available to treat this disease. The benzodiazepine meclonazepam is known to cure both intestinal and urinary schistosomiasis in animal and human studies, but dose-limiting sedation has been a barrier to its development. Little is known about the structure-activity relationship of meclonazepam and other benzodiazepines on schistosomes, or the identity of the parasite receptor for these compounds. However, schistosomes lack obvious homologs to the human GABAARs that cause sedation. This indicates that the parasite target of this drug is distinct from the host receptors that underpin dose-limiting side effects of meclonazepam, and raises the possibility that benzodiazepines with poor GABAAR affinity may still retain anti-parasitic effects. Here, we report an in vitro screen of various benzodiazepines against schistosomes, and the identification of hit compounds that are active against worms yet possess reduced affinity for the human GABAARs that cause sedation.

A computational study of medium effect on solvation free energy, dipole moment, polarizability, hyperpolarizability and different molecular properties like chemical hardness & softness, chemical potential, electronegativity and electrophilicity index of metronidazole have been reported in this paper. Becke, 3-parameter, Lee-Yang-Parr (B3LYP) level of theory with 6-31G (d,p) basis set was applied for gas phase and solution. The effect of solvent polarity on solvation free energy, dipole moment, polarizability, hyperpolarizability and molecular properties were calculated by employing Solvation Model on Density (SMD). The solvation free energies and dipole moment of metronidazole were found to be increased in nonpolar to polar solvents. The dipole moment of metronidazole was higher in different solvent than that of the gas phase. Moreover, from non-polar to polar solvents the chemical potential, electronegativity and electrophilicity index were increased. On the other hand, opposite relation was found in the case of chemical hardness and softness. The results obtained in this study may lead to understand the stability and reactivity of metronidazole and the results will be of assistance to use the title molecule as reaction intermediates and pharmaceuticals.Bangladesh Pharmaceutical Journal 19(1): 9-14, 2016

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 GABAA 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 GABAA 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 GABAA 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.

Aganosma dichotoma (Roth) K. Schum. is an indigenous plant of Bangladesh. Traditional healers use this plant to treat many diseases. In order to systematically explore the medicinal values of this plant, the crude methanol extract of leaves of A. dichotoma were screened for analgesic and antidiarrheal activities in mice model. The peripheral and central analgesic actions were determined by using acetic acid-induced writhing and tail immersion methods. The extract significantly (p < 0.05) attenuated the acetic acid-induced writhing in a dose dependent manner. A noticeable dose-dependent increase (p < 0.05) of latency period was also observed in the tail immersion method. In the castor oil induced anti-diarrheal assay, the extract exhibited significant (p < 0.05) anti-diarrheal effect at a dose of 400 mg/kg body weight.Bangladesh Pharmaceutical Journal 18(1): 15-19, 2015

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports on cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines given the anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970’s but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZ’s anti-parasitic target is distinct from the human receptors that drive sedation. Therefore, we have screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, resulting in extensive vacuole formation beneath the apical membrane. The hit compound series identified has a dramatically lower (~1000×) affinity for the human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects., Author summary Over 200 million people are infected with schistosomiasis, yet there are limited therapeutic options available to treat this disease. The benzodiazepine meclonazepam is known to cure both intestinal and urinary schistosomiasis in animal and human studies, but dose-limiting sedation has been a barrier to its development. Little is known about the structure-activity relationship of meclonazepam and other benzodiazepines on schistosomes, or the identity of the parasite receptor for these compounds. However, schistosomes lack obvious homologs to the human GABAARs that cause sedation. This indicates that the parasite target of this drug is distinct from the host receptors that underpin dose-limiting side effects of meclonazepam and raises the possibility that benzodiazepines with poor GABAAR affinity may still retain anti-parasitic effects. Here, we report an in vitro screen of various benzodiazepines against schistosomes, and the identification of hit compounds that are active against worms yet display reduced affinity for the human GABAAR that causes sedation.

Objectives The objective of the study was to evaluate Allamanda blanchetii extractives for antioxidant, cytotoxic, thrombolytic, membrane stabilizing and antimicrobial activities. Methods The plant extractives were evaluated for their phenolic content and their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Brine shrimp lethality bioassay was conducted to identify cytotoxic potential of the extractives. The test samples were also involved in thrombolytic and membrane stabilizing activity assays to evaluate their abilities to promote clot lysis and to stabilize erythrocyte membrane under hypotonic and heat induced conditions. The extractives were involved in disc diffusion assay to measure their ability to give zones of inhibition in cultured bacterial medium. Results In DPPH free radical scavenging assay, the carbon tetrachloride soluble fraction demonstrated the highest free radical scavenging activity (IC 50 = 40.50 ± 0.32 μg/ml) where as, in brine shrimp lethality bioassay, the hexane soluble fraction revealed the highest cytotoxic activity with LC 50 value of 0.78 ± 0.74 μg/ml. While evaluating the thrombolytic activity of the extractives, the chloroform soluble fraction showed 32.50 ± 0.63% of clot lysis. This fraction at 1.0 mg/ml concentration also inhibited 46.74 ± 0.73% and 41.33 ± 0.59% of haemolysis of RBCs induced by hypotonic solution and heat as compared to 71.90% and 42.12% by acetyl salicylic acid (0.10 mg/ml), respectively. In disc diffusion assay, the extractives of A. blanchetii revealed zone of inhibition ranging from 7.0 to 13.0 mm. Conclusion Further chemical and bioassay guided investigation on violet Allamanda must be conducted for isolation, identification and characterization of the bioactive constituents.