Cell-Free Supernatant of Lactobacillus rhamnosus and Bifidobacterium breve Ameliorates Ischemic Stroke-Generated Neurological Deficits in Rats.

The beneficial effects of probiotics, postbiotics, and paraprobiotics have already been registered in managing ischemic stroke-generated neuroinflammation and gut dysbiosis. Herein, we examined the impact of cell-free supernatant (CFS) obtained from probiotics (Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01) in a rat transient middle cerebral artery occlusion (MCAO) model of focal cerebral injury. Pre-MCAO supplementation of probiotics (2 × 109 CFU/mL) for 21 days or CFS (1 mL/rat) for 7 days protect the MCAO-induced somatosensory and motor impairments recorded at 24 h and 72 h after reperfusion in foot-fault, rotarod, adhesive removal, and vibrissae-evoked forelimb placing tests. We also noted the reduced infarct area and neuronal degradation in the right hemisphere of probiotics- and CFS-recipient MCAO-operated animals. Moreover, MCAO-induced altered concentrations of glial-fibrillary acidic protein, NeuN, zonula occludens-1 (ZO-1), TLR4, IL-1ß, IL-6, and TNF-α, as well as matrix metalloproteinase-9 (MMP9) were reversed in the treatment groups. Probiotics and CFS treatment ameliorated the elevated levels of IL-6, IL-1ß, and MMP9 in the blood plasma of rats. The disrupted microbial phyla, Firmicutes-to-Bacteroides ratio, villi/crypt ratio, and decreased mucin-producing goblet cells, ZO-1, and occludin in the colon of MCAO-operated rats were recovered following probiotics and CFS treatment. NMR characterization of CFS and rat blood plasma revealed the presence of several important bacterial metabolites. These findings suggest that the CFS obtained from Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01 has the propensity to improve MCAO-generated neurological dysfunctions in rats by dampening neuroinflammation and modulating the gut-brain axis modulators.

Development of Benzimidazole-Substituted Spirocyclopropyl Oxindole Derivatives as Cytotoxic Agents: Tubulin Polymerization Inhibition and Apoptosis Inducing Studies.

A series of spirocyclopropyl oxindoles with benzimidazole substitutions was synthesized and tested for their cytotoxicity against selected human cancer cells. Most of the molecules exhibited significant antiproliferative activity with compound 12 p being the most potent. It exhibited significant cytotoxicity against MCF-7 breast cancer cells (IC50 value 3.14±0.50 µM), evidenced by the decrease in viable cells and increased apoptotic features during phase contrast microscopy, such as AO/EB, DAPI and DCFDA staining studies. Compound 12 p also inhibited cell migration in wound healing assay. Anticancer potential of 12 p was proved by the inhibition of tubulin polymerization with IC50 of 5.64±0.15 µM. These results imply the potential of benzimidazole substituted spirocyclopropyl oxindoles, notably 12 p, as cytotoxic agent for the treatment of breast cancer.

Chitosan revokes controlled-cortical impact generated neurological aberrations in circadian disrupted mice via TLR4-NLRP3 axis.

The severity of inevitable neurological deficits and long-term psychiatric disorders in the aftermath of traumatic brain injury is influenced by pre-injury biological factors. Herein, we investigated the therapeutic effect of chitosan lactate on neurological and psychiatric aberrations inflicted by circadian disruption (CD) and controlled-cortical impact (CCI) injury in mice. Firstly, CD was developed in mice by altering sporadic day-night cycles for 2 weeks. Then, CCI surgery was performed using a stereotaxic ImpactOne device. Mice subjected to CCI displayed a significant disruption of motor coordination at 1-, 3- and 5-days post-injury (DPI) in the rotarod test. These animals showed anxiety- and depression-like behaviors in the elevated plus maze and forced-swim test at 14 and 15 DPI, respectively. Notably, mice subjected to CD + CCI exhibited severe cognitive impairment in Y-maze and novel object recognition tasks. The compromised neurological, psychiatric, and cognitive functions were mitigated in chitosan-treated mice (1 and 3 mg/mL). Immunohistochemistry and real-time PCR assay results revealed the magnified responses of prima facie biomarkers like glial-fibrillary acidic protein and ionized calcium-binding adaptor molecule 1 in the pericontusional brain region of the CD + CCI group, indicating aggravated inflammation. We also noted the depleted levels of brain-derived neurotrophic factor and augmented expression of toll-like receptor 4 (TLR4)-leucine-rich-containing family pyrin domain-containing 3 (NLRP3) signaling [apoptosis-associated-speck-like protein (ASC), caspase-1, and interleukin 1-ß] in the pericontusional area of CD + CCI group. CCI-induced changes in the astrocyte-glia and aggravated immune responses were ameliorated in chitosan-treated mice. These results suggest that the neuroprotective effect of chitosan in CCI-induced brain injury may be mediated by inhibition of the TLR4-NLRP3 axis.

Oxyberberine protects middle cerebral artery occlusion triggered cerebral injury through TLR4/NLRP3 pathway in rats.

Cerebral ischemia is a life-threatening health concern that leads to severe neurological complications and fatalities worldwide. Although timely intervention with clot-removing agents curtails serious post-stroke neurological dysfunctions, no effective neuroprotective intervention is available for addressing post-recanalization neuroinflammation. Herein, for the first time we studied the effect of oxyberberine (OBB), a derivative of berberine, on transient middle cerebral artery occlusion (MCAO)-generated neurological consequences in Sprague-Dawley rats. The MCAO-operated rats exhibited significant somatosensory and sensorimotor dysfunctions in adhesive removal, foot fault, paw whisker, and rotarod assays at 1 and 3 days post-surgery. These MCAO-generated neurological deficits were prevented in OBB-treated (50 and 100 mg/kg) rats, and also coincided with a smaller infarct area (in 2,3,5-triphenyl tetrazolium chloride staining) and decreased neuronal death (in cresyl violet staining) in the ipsilateral hemisphere of these animals. The immunostaining of neuronal nuclear protein (NeuN) and glial-fibrillary acidic protein (GFAP) also echoes the neuroprotective nature of OBB. The increased expression of neuroinflammatory and blood-brain barrier tight junction proteins like toll-like receptor 4 (TLR4), TRAF-6, nuclear factor kappa B (NF-κB), pNF-κB, nNOS, ASC, and IKBα in the ipsilateral part of MCAO-operated rats were restored to normal following OBB treatment. We also observed the decline in plasma levels/mRNA transcription of TNF-α, IL-1ß, NLRP3, IL-6, and matrix metalloproteinase-9 and increased expression of occludin and claudin in OBB-treated rats. These outcomes imply that OBB may prevent the MCAO-induced neurological consequences and neuroinflammation by interfering with TLR4 and NLRP3 signaling in rats.

Oxyberberine an oxoderivative of berberine confers neuroprotective effects in controlled-cortical impact mouse model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is known as a silent epidemic that causes many deaths and disabilities worldwide. We examined the response of oxyberberine (OBB) in lipopolysaccharide-stimulated BV2 microglial cells and a controlled-cortical impact (CCI) mouse model of TBI. METHODS: We synthesized OBB from berberine, and also prepared OBB-nanocrystals (OBB-NC). Male C57BL/6 mice were used for CCI surgery, and post-CCI neurobehavioral deficits were assessed from 1 h after injury through 21 days post-injury (dpi). RESULTS: OBB treatment reduced the lipopolysaccharide-triggered elevated levels of reactive oxygen species, nitric oxide, and nuclear factor kappa B (NF-κB) in BV2 microglial cells, indicating a neuroprotective potential. CCI-operated mice exhibited significant neurological deficits on 1, 3, and 5 dpi in neurological severity scoring and rotarod assay. OBB (25 and 50 mg/kg/day) and OBB-NC (3 mg/kg/day) ameliorated these neurological aberrations. Mice subjected to CCI surgery also displayed anxiogenic- and depression-like behaviours, and cognitive impairments in forced-swimming test and elevated-zero maze, and novel object recognition task, respectively. Administration of OBB reduced these long-term neuropsychiatric complications, and also levels of toll-like receptor 4 (TLR4), high-motility group protein 1 (HMGB1), NF-κB, tumour necrosis factor-alpha and interleukin 6 cytokines in the ipsilateral cortex of mice. CONCLUSION: We suggest that the administration of OBB offers neuroprotective effects via inhibition of HMGB1-mediated TLR4/NFκB pathway.

Multistrain probiotic rescinds quinpirole-induced obsessive-compulsive disorder phenotypes by reshaping of microbiota gut-brain axis in rats.

Obsessive-compulsive disorder (OCD) is a disabling mental condition that poses recurring bothersome intrusive thoughts, obsessions, and compulsions. Considering the positive impact of probiotics on neuropsychiatric disorders, herein, we investigated the effect of multistrain probiotic (Bifidobacterium lactis UBBLa-70, Bacillus coagulans Unique IS-2, Lactobacillus rhamnosus UBLR-58, Lactobacillus plantarum UBLP-40, Bifidobacterium infantis UBBI-01, Bifidobacterium breve UBBr-01, and glutamine) in the management of OCD-like phenotype in rats. Rats injected with quinpirole for 5 weeks showed an increased number of marble burying and self-grooming episodes. Quinpirole-injected animals also did less head dipping in the hole board test and avoided exploration of open spaces in the elevated-plus maze. These repetitive, compulsive, self-directed, and anxiety-like phenotypes were abolished after 8-week of multistrain probiotic treatment. The probiotic formulation also prevented the elevated mRNA expression of interleukin-6, tumor-necrosis factor-α, and C-reactive protein in the amygdala and dysregulated levels of 5-hydroxytryptamine, dopamine, and noradrenaline in the frontal cortex of quinpirole-injected rats. The level of brain-derived neurotrophic factor in the frontal cortex remained unaffected across the groups. The altered levels of goblet cells and crypt-to-villi ratio in quinpirole rats were prevented by multistrain probiotic treatment. The results of 16S-rRNA gene-sequencing of gut microbiota from feces contents revealed an elevation in the abundance of Allobaculum and Bifidobacterium species (specifically Bifidobacterium animalis), while the presence of Lactobacillus species (including Lactobacillus reuteri and Lactobacillus vaginalis) exhibited a decline in quinpirole-induced rats. These results imply that modifying the gut-brain axis may be a possible mechanism by which selective multistrain probiotic therapy prevents OCD-like behaviors.

Benzimidazole derivatives as tubulin polymerization inhibitors: Design, synthesis and in vitro cytotoxicity studies.

A new class of benzimidazole derivatives as tubulin polymerization inhibitors has been designed and synthesized in this study. The in vitro anticancer profile of the developed molecules was reconnoitred on selected human cancer cells. The highest cytotoxicity was illustrated by compounds 7n and 7u with IC50 values ranging from 2.55 to 17.89 µM with specificity toward SK-Mel-28 cells. They displayed 5-fold less cytotoxicity towards normal rat kidney epithelial NRK52E cells, which implies that they are not harmful to normal, healthy cells. The cellular staining procedures like AO/EB, DCFDA, and DAPI were applied to comprehend the inherent mechanism of apoptosis which displayed nuclear and morphological alterations. The Annexin V binding and JC-1 studies were executed to evaluate the extent of apoptosis and the decline in mitochondrial transmembrane potential in SK-Mel-28 cell lines. Compound 7n dose-dependently arrested the G2/M phase of the cell cycle and the target-based outcomes proposed tubulin polymerization inhibition by 7n (IC50 of 5.05±0.13 µM). Computational studies were also conducted on the tubulin protein (PDB ID: 3E22) to investigate the stabilized binding interactions of compounds 7n and 7u with tubulin, respectively.

Partial blood replacement ameliorates middle cerebral artery occlusion generated neurological aberrations by intervening TLR4 and NLRP3 cascades in rats.

Acute ischemic stroke is a catastrophic medical condition that causes severe disability and mortality if the sufferer escapes treatment within a stipulated timeframe. While timely intervention with clot-bursting agents like tissue-plasminogen activators abrogates some post-stroke neurologic deficits, no neuroprotective therapy is yet promisingly addresses the post-recanalization neuroinflammation in post-stroke survivors. Herein, we investigated the effect of partial blood replacement therapy (BRT), obtained from healthy and treadmill-trained donor rats, on neurological deficits, and peripheral and central inflammatory cascades using the ischemia-reperfusion animal paradigm. The cerebral ischemia-reperfusion was induced in rats by occlusion of the middle cerebral artery (MCAO) for 90 min, followed by reperfusion. Rats underwent MCAO surgery displayed remarkable sensorimotor and motor deficits in rotarod, foot fault, adhesive removal, and paw whisker tests till 5 days post-surgery. These behavior abnormalities were ameliorated in the BRT-recipient MCAO rats. BRT also reduced the infarct volume and neuronal death in the ipsilateral hemisphere revealed by TTC and cresyl violet staining compared to the MCAO group. Rats received BRT infusion exhibited the reduced expression of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule-1 (Iba-1), and MyD88 on day 5 post-MCAO in immunohistochemistry and immunofluorescent assays. Moreover, elevated levels of toll-like receptor 4 (TLR4) and mRNA expression of IL-1ß, TNF-α, matrix metalloproteinase-9 and NLRP3, and decreased levels of zonula occludens-1 in MCAO rats, were reversed following BRT. These findings suggest that the partial BRT may rescind MCAO-induced neurological dysfunctions and cerebral injury by intervening in the TLR4 and NLRP3 pathways in rats.

Multistrain Probiotics with Fructooligosaccharides Improve Middle Cerebral Artery Occlusion-Driven Neurological Deficits by Revamping Microbiota-Gut-Brain Axis.

Recent burgeoning literature unveils the importance of gut microbiota in the neuropathology of post-stroke brain injury and recovery. Indeed, ingestion of prebiotics/probiotics imparts positive effects on post-stroke brain injury, neuroinflammation, gut dysbiosis, and intestinal integrity. However, information on the disease-specific preference of selective prebiotics/probiotics/synbiotics and their underlying mechanism is yet elusive. Herein, we examined the effect of a new synbiotic formulation containing multistrain probiotics (Lactobacillus reuteri UBLRu-87, Lactobacillus plantarum UBLP-40, Lactobacillus rhamnosus UBLR-58, Lactobacillus salivarius UBLS-22, and Bifidobacterium breve UBBr-01), and prebiotic fructooligosaccharides using a middle cerebral artery occlusion (MCAO) model of cerebral ischemia in female and male rats. Three weeks pre-MCAO administration of synbiotic rescinded the MCAO-induced sensorimotor and motor deficits on day 3 post-stroke in rotarod, foot-fault, adhesive removal, and paw whisker test. We also observed a decrease in infarct volume and neuronal death in the ipsilateral hemisphere of synbiotic-treated MCAO rats. The synbiotic treatment also reversed the elevated levels/mRNA expression of the glial fibrillary acidic protein (GFAP), NeuN, IL-1ß, TNF-α, IL-6, matrix metalloproteinase-9, and caspase-3 and decreased levels of occludin and zonula occludens-1 in MCAO rats. 16S rRNA gene-sequencing data of intestinal contents indicated an increase in genus/species of Prevotella (Prevotella copri), Lactobacillus (Lactobacillus reuteri), Roseburia, Allobaculum, and Faecalibacterium prausnitzii, and decreased abundance of Helicobacter, Desulfovibrio, and Akkermansia (Akkermansia muciniphila) in synbiotic-treated rats compared to the MCAO surgery group. These findings confer the potential benefits of our novel synbiotic preparation for MCAO-induced neurological dysfunctions by reshaping the gut-brain-axis mediators in rats.

Synthesis and biological evaluation of 1-phenyl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-one/thiones as anticancer agents.

Cancer is associated with uncontrolled cell proliferation invading adjoining tissues and organs. Despite the availability of several chemotherapeutic agents, the constant search for newer approaches and drugs is necessitated owing to the ever-growing challenge of resistance. Over the years, DNA has emerged as an important druggable therapeutic drug due to its role in critical cellular processes such as cell division and maintenance. Further, evading apoptosis stands out as a hallmark of cancer. Hence, designing new compounds that would target DNA and induce apoptosis plays an important role in cancer therapy. In the current work, we carried out the synthesis and anticancer evaluation of 1-aryl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-ones/thiones (26 compounds) against selected human cancer cell lines. Among these, compounds 8ae, 8ad, 8cf, 10ad and Kenpaullone have shown good inhibitory properties against HeLa cells (IC50 < 2 µM) with good selectivity over the non-cancerous human embryonic kidney (Hek293T) cells. In cell cycle analysis, the compounds 8ad and 8cf have exhibited G2/M cell cycle arrest in HeLa cells. In addition, the compounds 8ad and 8cf induced apoptosis in a dose-dependent manner in the Annexin-V FITC staining assay. The DAPI staining clearly demonstrated the condensed and fragmented nuclei in 8ad, 8cf, 8ae and Kenpaullone-treated HeLa cells. In addition, these compounds strongly suppressed the healing after 48 h in in vitro cell migration assay. The DNA binding experiments indicated that compounds 8ae, 8cf, and 8ad as well as Kenpaullone interact with double-stranded DNA by binding in grooves which may interrupt the DNA replication and kill fast-growing cells. Molecular docking studies revealed the binding pose of 8ad and Kenpaullone at HT1 binding pocket of double-stranded DNA. Compounds 8ad and 8cf demonstrated moderate topo II inhibition which could be a possible reason for their anticancer properties. Compounds 8ad and 8cf may cause the topo II and DNA covalent complex, which leads to the inhibition of DNA replication and transcription. This eventually increases the DNA damage in cells and promotes cell apoptosis. With the above interesting biological profile, the new 1-aryl-2,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(4H)-one/thione derivatives have emerged as promising leads for the discovery of new anticancer agents.

Synthesis and cytotoxicity evaluation of DNA-interactive ß-carboline indolyl-3-glyoxamide derivatives: Topo-II inhibition and in silico modelling studies.

In a quest for effective cancer targeted drug therapy, a series of new ß-carboline tethered indole-3-glyoxylamide derivatives, conjoining salient pharmacophoric properties with prominent cytotoxicity, were synthesized. The in vitro cytotoxic ability of the compounds was established, and many of the compounds exhibited remarkable cytotoxicity (IC50 < 10 µM) on human cancer cell lines like HCT116, A549, SK-MEL-28, and MCF7. Precisely, compound 12x expressed the best cytotoxic potential against melanoma cancer cell line (SK-MEL-28) with an IC50 value of 4.37 µM. In addition, cytotoxicity evaluation against normal kidney cell line (NRK52E) entrenched the cytospecificity and selectivity index of 12x. The traditional apoptosis assays advised morphological and nuclear alterations such as apoptotic body formation, condensed/horseshoe-shaped/fragmented nuclei, and generation of ROS. The flow cytometric analysis revealed significant early and slight late-stage induction of apoptosis. The target-based physiochemical assays indicated the ability of compound 12x to bind with DNA and inhibition of Topoisomerase II. Moreover, molecular modeling studies affirm the excellent DNA intercalation potential and stabilized interactions of 12x with DNA base pairs. In silico prediction of physicochemical parameters revealed the promising drug-like properties of the synthesized derivatives.

Fecal microbiota transplantation unveils sex-specific differences in a controlled cortical impact injury mouse model.

Introduction: Contusion type of traumatic brain injury (TBI) is a major cause of locomotor disability and mortality worldwide. While post-TBI deleterious consequences are influenced by gender and gut dysbiosis, the sex-specific importance of commensal gut microbiota is underexplored after TBI. In this study, we investigated the impact of controlled cortical impact (CCI) injury on gut microbiota signature in a sex-specific manner in mice. Methods: We depleted the gut microflora of male and female C57BL/6 mice using antibiotic treatment. Thereafter, male mice were colonized by the gut microbiota of female mice and vice versa, employing the fecal microbiota transplantation (FMT) method. CCI surgery was executed using a stereotaxic impactor (Impact One™). For the 16S rRNA gene amplicon study, fecal boli of mice were collected at 3 days post-CCI (dpi). Results and discussion: CCI-operated male and female mice exhibited a significant alteration in the genera of Akkermansia, Alistipes, Bacteroides, Clostridium, Lactobacillus, Prevotella, and Ruminococcus. At the species level, less abundance of Lactobacillus helveticus and Lactobacillus hamsteri was observed in female mice, implicating the importance of sex-specific bacteriotherapy in CCI-induced neurological deficits. FMT from female donor mice to male mice displayed an increase in genera of Alistipes, Lactobacillus, and Ruminococcus and species of Bacteroides acidifaciens and Ruminococcus gnavus. Female FMT-recipient mice from male donors showed an upsurge in the genus Lactobacillus and species of Lactobacillus helveticus, Lactobacillus hamsteri, and Prevotella copri. These results suggest that the post-CCI neurological complications may be influenced by the differential gut microbiota perturbation in male and female mice.

Binary classification model of machine learning detected altered gut integrity in controlled-cortical impact model of traumatic brain injury.

Aim of the study: To examine the effect of controlled-cortical impact (CCI), a preclinical model of traumatic brain injury (TBI), on intestinal integrity using a binary classification model of machine learning (ML).Materials and methods: Adult, male C57BL/6 mice were subjected to CCI surgery using a stereotaxic impactor (Impact One™). The rotarod and hot-plate tests were performed to assess the neurological deficits.Results: Mice underwent CCI displayed a remarkable neurological deficit as noticed by decreased latency to fall and lesser paw withdrawal latency in rotarod and hot plate test, respectively. Animals were sacrificed 3 days post-injury (dpi). The colon sections were stained with hematoxylin and eosin (H&E) to integrate with machinery tool-based algorithms. Several stained colon images were captured to build a dataset for ML model to predict the impact of CCI vs sham procedure. The best results were obtained with VGG16 features with SVM RBF kernel and VGG16 features with stacked fully connected layers on top. We achieved a test accuracy of 84% and predicted the disrupted gut permeability and epithelium wall of colon in CCI group as compared to sham-operated mice.Conclusion: We suggest that ML may become an important tool in the development of preclinical TBI model and discovery of newer therapeutics.

A narrative review on invasive brain stimulation for treatment-resistant depression

While most patients with depression respond to pharmacotherapy and psychotherapy, about one-third will present treatment resistance to these interventions. For patients with treatment-resistant depression (TRD), invasive neurostimulation therapies such as vagus nerve stimulation, deep brain stimulation, and epidural cortical stimulation may be considered. We performed a narrative review of the published literature to identify papers discussing clinical studies with invasive neurostimulation therapies for TRD. After a database search and title and abstract screening, relevant English-language articles were analyzed. Vagus nerve stimulation, approved by the U.S. Food and Drug Administration as a TRD treatment, may take several months to show therapeutic benefits, and the average response rate varies from 15.2-83%. Deep brain stimulation studies have shown encouraging results, including rapid response rates (> 30%), despite conflicting findings from randomized controlled trials. Several brain regions, such as the subcallosal-cingulate gyrus, nucleus accumbens, ventral capsule/ventral striatum, anterior limb of the internal capsule, medial-forebrain bundle, lateral habenula, inferior-thalamic peduncle, and the bed-nucleus of the stria terminalis have been identified as key targets for TRD management. Epidural cortical stimulation, an invasive intervention with few reported cases, showed positive results (40-60% response), although more extensive trials are needed to confirm its potential in patients with TRD.

LL-00066471, a novel positive allosteric modulator of α7 nicotinic acetylcholine receptor ameliorates cognitive and sensorimotor gating deficits in animal models: Discovery and preclinical characterization.

α7 nicotinic acetylcholine receptor (α7 nAChR) is an extensively validated target for several neurological and psychiatric conditions namely, dementia and schizophrenia, owing to its vital roles in cognition and sensorimotor gating. Positive allosteric modulation (PAM) of α7 nAChR represents an innovative approach to amplify endogenous cholinergic signaling in a temporally restricted manner in learning and memory centers of brain. α7 nAChR PAMs are anticipated to side-step burgeoning issues observed with several clinical-stage orthosteric α7 nAChR agonists, related to selectivity, tolerance/tachyphylaxis, thus providing a novel dimension in therapeutic strategy and pharmacology of α7 nAChR ion-channel. Here we describe a novel α7 nAChR PAM, LL-00066471, which potently amplified agonist-induced Ca2+ fluxes in neuronal IMR-32 neuroblastoma cells in a α-bungarotoxin (α-BTX) sensitive manner. LL-00066471 showed excellent oral bioavailability across species (mouse, rat and dog), low clearance and good brain penetration (B/P ratio > 1). In vivo, LL-00066471 robustly attenuated cognitive deficits in both procognitive and antiamnesic paradigms of short-term episodic and recognition memory in novel object recognition task (NORT) and social recognition task (SRT), respectively. Additionally, LL-00066471 mitigated apomorphine-induced sensorimotor gating deficits in acoustic startle reflex (ASR) and enhanced antipsychotic efficacy of olanzapine in conditioned avoidance response (CAR) task. Further, LL-00066471 corrected redox-imbalances and reduced cortico-striatal infarcts in stroke model. These finding together suggest that LL-00066471 has potential to symptomatically alleviate cognitive deficits associated with dementias, attenuate sensorimotor gating deficits in schizophrenia and correct redox-imbalances in cerebrovascular disorders. Therefore, LL-00066471 presents potential for management of cognitive impairments associated with neurological and psychiatric conditions.

Discovery of a Potent and Selective PI3Kδ Inhibitor (S)-2,4-Diamino-6-((1-(7-fluoro-1-(4-fluorophenyl)-4-oxo-3-phenyl-4H-quinolizin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile with Improved Pharmacokinetic Profile and Superior Efficacy in Hematological Cancer Models.

PI3Kδ inhibitors have been approved for B-cell malignancies like CLL, small lymphocytic lymphoma, and so forth. However, currently available PI3Kδ inhibitors are nonoptimal, showing weakness against at least one of the several important properties: potency, isoform selectivity, and/or pharmacokinetic profile. To come up with a PI3Kδ inhibitor that overcomes all these deficiencies, a pharmacophoric expansion strategy was employed. Herein, we describe a systematic transformation of a "three-blade propeller" shaped lead, 2,3-disubstituted quinolizinone 11, through a 1,2-disubstituted quinolizinone 20 to a novel "four-blade propeller" shaped 1,2,3-trisubstituted quinolizinone 34. Compound 34 has excellent potency, isoform selectivity, metabolic stability across species, and exhibited a favorable pharmacokinetic profile. Compound 34 also demonstrated a differentiated efficacy profile in human germinal center B and activated B cell-DLBCL cell lines and xenograft models. Compound 34 qualifies for further evaluation as a candidate for monotherapy or in combination with other targeted agents in DLBCLs and other forms of iNHL.

Intravenous infusion of xenon-containing liposomes generates rapid antidepressant-like effects.

AIM: Similar to ketamine, xenon gas acts as a glutamatergic N-methyl-d-aspartate receptor antagonist, but devoid of propensity to cause untoward effects. Herein, we loaded xenon gas into a liposomal carrier called xenon-containing liposomes (Xe-liposome) for systemic delivery, and investigated its effect as an antidepressant and also analyzed synaptic biomarkers including brain-derived neurotrophic factor (BDNF), protein kinase B (AKT), mammalian target of rapamycin (mTOR), protein kinase C (PKC) and extracellular signal-regulated kinase-1/2 (ERK1/2) in blood and brain. METHODS: Xe-liposomes (15 µl/mg) were prepared by a pressurized freeze-thaw method, and injected via the lateral tail vein (0.6 mL/rat) in male Wistar rats. The uncaging of xenon gas from circulating Xe-liposome was facilitated by continuous ultrasound application externally on the neck over the internal common carotid artery. One-hour after Xe-liposome infusion, animals were assessed for depression-like behaviors using a forced swimming test (FST), and spontaneous locomotor activity. Blood, as well as frontal cortex and hippocampal samples were obtained for immunoblotting and/or enzyme-linked immune sorbent assays. RESULTS: Acute intravenous infusion of Xe-liposome, at 6 mg/kg, showed an increase in swimming time in the FST (p < 0.006), indicating antidepressant-like phenotypes. Higher doses of Xe-liposomes (9 mg/kg) failed to improve swimming duration. This behavioral discrepancy was not associated with locomotion aberrations, as gross activity of rats remained similar for both doses. In biochemical analyses of frontal cortex, protein levels of BDNF increased by 64%, and enhanced phosphorylation of AKT (43%) and mTOR (93%) was observed at the 6 mg/kg dose level of Xe-liposomes, while these biomarkers and phosphorylated PKC and ERK1/2 levels remained unchanged at the higher dose. Moreover, Xe-liposomal treatment did not change the plasma and protein levels of BDNF, and phosphorylated AKT, mTOR, PKC and ERK1/2 hippocampal expressions. CONCLUSION: Xe-liposomes mediate a rapid antidepressant-like effect through activation of AKT/mTOR/BDNF signaling pathway.

Involvement of the central melanocortin system in the effects of caffeine on anxiety-like behavior in mice.

AIMS: To investigate the role of the melanocortin (MC) system in the framework of the central nucleus of the amygdala (CeA) in the differential effects of the adenosine receptor blocker caffeine on anxiety-like behavior, using the social interaction (SI) test. MAIN METHODS: Caffeine was injected intraperitoneally, alone or in combination with alpha-melanocyte stimulating hormone (α-MSH), the MC4 receptor agonist RO27-3225 or the antagonist HS014 via the intra-CeA route. The effects of chronic (21 days) caffeine, given alone or concurrently with α-MSH, or RO27-3225, were investigated. The effects of withdrawal of these treatments on SI time were also evaluated. Furthermore, the acute effects of HS014 were investigated in different sets of caffeine-withdrawn mice. KEY FINDINGS: Acute injection of caffeine, RO27-3225, or α-MSH produced anxiety-like behavior. Prior treatment with α-MSH, or RO27-3225 potentiated the caffeine-induced anxiety-like behavior. Subchronic treatment with HS014 increased the SI time, which was attenuated by caffeine. Chronic administration of caffeine resulted in tolerance to caffeine's anxiogenic effect, while abrupt discontinuation of the treatment produced peak anxiety-like behavior at 72 h post-withdrawal. Concurrent administration of α-MSH, or RO27-3225 with chronic caffeine delayed the development of tolerance and prevented withdrawal-induced anxiety-like behavior. Moreover, acute treatment with HS014 at 72 h post-withdrawal attenuated the anxiety-like behavior. SIGNIFICANCE: α-MSH, possibly via MC4 receptor in the neuroanatomical framework of the CeA, may contribute to the acute, chronic and withdrawal actions of caffeine associated with anxiety-like behavior in the neuroanatomical framework of the CeA.

Involvement of CART in estradiol-induced anorexia.

Since estradiol exercises inhibitory effect on food intake, we wanted to find out if this influence of estradiol is mediated by cocaine- and amphetamine-regulated transcript peptide (CART), a well established anorectic agent in the brain. Ovariectomized (OVX) rats, replaced with estradiol to produce estrous-phase like conditions, showed a significant decrease in food intake as compared with that in OVX controls. Intracerebroventricular (icv) administration of CART (0.5-1 µg/rat) to OVX rats, resulted in a dose-dependent reduction in the food intake. The lower dose (0.25 µg) had no effect, and was considered subeffective. In estradiol replaced OVX rats, CART at subeffective dose, further reduced food intake. However, CART failed to reduce food intake in estradiol replaced OVX rats pretreated with anti-estrogenic agent tamoxifen (3 mg/kg, subcutaneous). Administration of CART antibody (1:500 dilution/rat, i.c.v.) significantly attenuated estradiol-induced anorexia in the OVX rats. While estradiol replacement significantly increased CART-immunoreactivity in the cells/fibers of paraventricular nucleus (PVN) of OVX rats, fibers in the anteroventral periventricular nucleus (AVPV), and cells/fibers in the arcuate nucleus (ARC) showed considerable reduction. These changes were attenuated following concurrent injection of tamoxifen to the estradiol replaced OVX rats. However, CART-immunoreactive cells/fibers in the periventricular area did not respond to any of the treatments. We suggest that estradiol treatment might influence the hypothalamic CART system in a site specific manner. While increased CART activity in the PVN might produce anorexia, reduction of CART in ARC and AVPV might represent a compensatory homeostatic response.

Effect of nicotine on feeding and body weight in rats: involvement of cocaine- and amphetamine-regulated transcript peptide.

While nicotine treatment to rodents causes a transient anorexia and persistent weight loss, withdrawal produces hyperphagia and weight gain. Herein, we test the hypothesis that endogenous anorectic peptide cocaine- and amphetamine-regulated transcript (CART) may be involved in these nicotine triggered physiological disturbances. In acute study, an anorectic effect of intraperitoneal nicotine was significantly potentiated by intracerebroventricular pre-treatment with CART at 2 and 4 h post-injection time-points. In chronic study, following an initial reduction, food intake, but not body weight, was progressively restored to normal. On the other hand, termination of chronic nicotine treatment resulted in significant hyperphagia and weight gain. These effects of nicotine were abolished if the rats were concomitantly treated with CART. An immunohistochemical profile of hypothalamic CART was studied following different nicotine treatment conditions. Acute nicotine treatment caused a significant increase above control in the CART-immunoreactive cells and fibers in the hypothalamic paraventricular (PVN) and fibers in the arcuate (ARC) nuclei. However, chronic nicotine administration had no effect on the CART-immunoreactivity in the PVN and ARC. While nicotine withdrawal reduced the population of CART-immunoreactive cells and fibers in the PVN, the immunoreactivity in the ARC fibers was increased. The results suggest that hypothalamic CART may process the acute, chronic and withdrawal effects of nicotine on feeding and body weight.