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4-(2-Hydroxyethyl) morpholine (HEM) is widely used as a building block of macromolecules in the manufacture of pharmaceuticals and dietary supplements and could remain as an impurity in the finished products. An evaluation of HEM was conducted to identify endpoints that could be used to determine the point-of-departure (POD) for use in assessing the potential risk from exposure to HEM. No oral repeated dose toxicological studies of appropriate duration were found for HEM. Therefore, suitable analogue(s) were identified. Although oral repeated dose studies were available for the analogues, the studies were not of sufficient duration for use in the assignment of a POD for risk evaluation. Accordingly, the Threshold of Toxicological Concern (TTC) approach, which proposes that a de minimis value can be derived to qualitatively assess risk, was considered for HEM. To determine the appropriate TTC approach (genotoxic or non-genotoxic), the genotoxicity of HEM and its analogues were evaluated. The weight of the evidence indicated that HEM, and the appropriate analogues, are not genotoxic. Considering the chemical structure of HEM, the non-genotoxic Cramer class III TTC value of 1.5 μg/kg bw/day was determined to be appropriate for use in safety assessment of HEM as an impurity in products intended for human consumption., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christine Thiffault, Elaine L. Freeman, and Nga L. Tran reports financial support was provided by Exponent Inc. Gary M. Williams reports financial support was provided by New York Medical College, received an honorarium from Exponent, and has no competing financial interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Everolimus, an allosteric mechanistic target of rapamycin (mTOR) inhibitor, recently demonstrated the therapeutic value of mTOR inhibitors for Central Nervous System (CNS) indications driven by hyperactivation of mTOR. A newer, potent brain-penetrant analog of everolimus, referred to as (1) in this manuscript [(S)-3-methyl-4-(7-((R)-3-methylmorpholino)-2- (thiazol-4-yl)-3H-imidazo[4,5-b]pyridin-5-yl)morpholine,(1)] catalytically inhibits mTOR function in the brain and increases the lifespan of mice with neuronal mTOR hyperactivation., Introduction: Early evaluation of the safety of 1 was conducted in cynomolgus monkeys in which oral doses were administered to three animals in a rising-dose fashion (from 2 to 30 mg/kg/day). 1 produced severe toxicity including the evidence of hepatic toxicity, along with non-dose proportional increases in drug exposure. Investigations of cross-species hepatic bioactivation of 1 were conducted to assess whether the formation of reactive drug metabolites was associated with the mechanism of liver toxicity., Methods: 1 contained two morpholine rings known as structural alerts and can potentially form reactive intermediates through oxidative metabolism. Bioactivation of 1 was investigated in rat, human and monkey liver microsomes fortified with trapping agents such as methoxylamine or potassium cyanide., Results: Our results suggest that bioactivation of the morpholine moieties to reactive intermediates may have been involved in the mechanism of liver toxicity observed with 1. Aldehyde intermediates trappable by methoxylamine were identified in rat and monkey liver microsomal studies. In addition, a total of four cyano conjugates arising from the formation of iminium ion intermediates were observed and identified. These findings may potentially explain the observed monkey toxicity. Interestingly, methoxylamine or cyano adducts of 1 were not observed in human liver microsomes., Conclusion: The bioactivation of 1 appears to be species-specific. Circumstantial evidence for the toxicity derived from 1 point to the formation of iminium ion intermediates trappable by cyanide in monkey liver microsomes. The cyano conjugates were only observed in monkey liver microsomes, potentially pointing to cause at least the hepatotoxicity observed in monkeys. In contrast, methoxylamine conjugates were detected in both rat and monkey liver microsomes, with only a trace amount in human liver microsomes. Cyano conjugates were not observed in human liver microsomes, challenging the team on the drugability and progressivity of 1 through drug development. The mechanisms for drug-induced liver toxicity are multifactorial. These results are highly suggestive that the iminium ion may be an important component in the mechanism of liver toxicity 1 observed in the monkey., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Difenoconazole (DIF) and dimethomorph (DIM) are widely used pesticides frequently detected together in environmental samples, so the deleterious effects of combined exposure warrant detailed examination. In this study, the individual and combined effects of DIM and DIF on conventional developmental parameters (hatching rate, deformity rate, lethality) and gene expression were measured in embryonic zebrafish. Both DIF and DIM interfered with normal zebrafish embryo development, and the most sensitive toxicity index for both was 96 h post-fertilization (hpf) deformity rate (BMDL 10 values of 0.30 and 1.10 mg/L, respectively). The combination of DIF and DIM had mainly synergistic deleterious effects on 96 hpf deformity and mortality rates. Transcriptome analysis showed that these compounds markedly downregulated expression of mcm family genes, cdk 1, and cdc 20, thereby potentially disrupting DNA replication and cell cycle progression. Enhanced surveillance for this pesticide combination is recommended as simultaneous environmental exposure may be substantially more harmful than exposure to either compound alone.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely., (© 2021. The Author(s).)

In animals, research in the past two decades has demonstrated the strong involvement of the endocannabinoid system (ECS) in numerous steps of the reproductive process, including ovarian physiology. Reproductive lifespan is closely related to the number of nongrowing ovarian follicles, called ovarian reserve (OR), which is definitively established during foetal life. Thus, OR damage may lead to poor reproductive outcomes and a shortened reproductive lifespan. We investigated whether prenatal ECS modulation had an effect on the OR at different ages in the rat offspring. Four groups of gestating female rats (F0) were exposed to the CB1-/CB2-receptor agonist WIN55212 (0.5 mg/kg), the CB1R inverse agonist SR141716 (3 mg/kg) or Δ9THC (5 mg/kg) and were compared to negative control groups. OR was histologically assessed at different postnatal timepoints (F1 individuals): postnatal day (PND) 6, PND40 and PND90. At PND6, prenatal exposure had no effect on OR. In the young adult group (PND90) exposed during gestation to WIN55212, we observed a CB1R-mediated delayed OR decrease, which was reversed by prenatal CB1R blockade by SR141716. Conversely, after prenatal SR141716 exposure, we observed higher OR counts at PND90. RT-PCR experiments also showed that prenatal ECS modulation perturbed the mRNA levels of ECS enzymes and OR regulation genes. Our findings support the role of the ECS in OR regulation during the foetal life of rats and highlight the need for further studies to elucidate its precise role in OR physiology.

Background and Purpose: Marijuana is the illicit drug most commonly used among pregnant and breastfeeding women. Different studies reported long-term adverse effects induced by in utero exposure to the main component of marijuana, Δ 9 -tetrahydrocannabinol (THC), both in rodents and in humans. However, little is known about any potential sex-dependent effects of marijuana consumption during pregnancy on newborns at early developmental ages., Experimental Approach: We studied the effects of prenatal exposure to the cannabinoid receptor agonist WIN55,212-2 (WIN; 0.5 mg·kg -1 from GD5 to GD20) on the emotional reactivity and cognitive performance of male and female rat offspring from infancy through adolescence and tested the role of mGlu 5 receptor signalling in the observed effects., Key Results: Prenatally WIN-exposed male infant pups emitted less isolation-induced ultrasonic vocalizations compared with male control pups, when separated from the dam and siblings and showed increased locomotor activity while females were spared. These effects were normalized when male pups were treated with the positive allosteric modulator of mGlu 5 receptor CDPPB. When tested at the prepubertal and pubertal periods, WIN-prenatally exposed rats of both sexes did not show any difference in social play behaviour, anxiety and temporal order memory., Conclusions and Implications: We reveal a previously undisclosed sexual divergence in the consequences of fetal cannabinoids on newborns at early developmental ages, which is dependent on mGlu 5 receptor signalling. These results provide new impetus for the urgent need to investigate the functional and behavioural substrates of prenatal cannabinoid exposure in both the male offspring and the female offspring., (© 2019 The British Pharmacological Society.)

Aim: Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache., Methods: Female Sprague Dawley rats received graded intraperitoneal doses of WIN55,212-2 or Δ-9-tetrahydrocannabinol (Δ-9-THC). Antinociception (tail-flick test), catalepsy and hypomotility (open field test) and impairment of motor function (rotarod test) were assessed to establish effective dosing. Rats were then treated twice daily with equianalgesic doses of WIN55,212-2 or Δ-9-THC, or vehicle, for 7 days and cutaneous tactile sensory thresholds were evaluated during and three weeks following drug discontinuation. Rats then received a one-hour period of bright light stress (BLS) on two consecutive days and tactile sensory thresholds were re-assessed., Results: WIN55,212-2 and Δ-9-THC produced antinociception as well as hypomotility, catalepsy and motor impairment. Repeated administration of WIN55,212-2 and Δ-9-THC induced generalized periorbital and hindpaw allodynia that resolved within 3 weeks after discontinuation of drug. Two episodes of BLS produced delayed and long-lasting periorbital and hindpaw allodynia selectively in rats previously treated with WIN55,212-2, and Δ-9-THC., Interpretation: Cannabinoid receptor agonists including Δ-9-THC produce a state of latent sensitization characterized by increased sensitivity to stress, a presumed migraine trigger. Overuse of cannabinoids including cannabis may increase the risk of medication overuse headache in vulnerable individuals.

Background: Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression., Methods: We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting., Results: Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions., Conclusion: These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70., (Copyright © 2019 Elsevier B.V. All rights reserved.)

SPH3127, a newly developed oral nonpeptide direct renin inhibitor with good tolerance and favorable ADME (absorption distribution metabolism excretion) properties in preclinical species, is now being evaluated in phase Ι clinical trial. In this work, the subchronic toxicity of SPH3127 in Sprague-Dawley rats and cynomolgus monkeys has been characterized. Rats and monkeys received SPH3127 orally (30, 300, 900 and 20, 100, 450 mg/kg/day, respectively) on a consecutive daily dosing schedule for 28 days followed by a 28-days recovery period for one third of the animals. The adverse effects of SPH3127 on rats and monkeys mainly included kidney and cardiovascular toxicity, which were consistent with pharmacologic perturbations of physiologic processes associated with the intended molecular targets for this class of renin signaling inhibitors. Moderate liver weight increases accompanied by CYP3A induction were seen in 300 and 900 mg/kg/day rats but not in monkeys or in vitro human hepatocytes. One 450 mg/kg/day monkey died early at day 23 with apparent myelosuppression characterized by atrophy of thymus and spleen, and the relevance to the action of SPH3127 remained unclear. Most of the treatment-induced effects were reversible upon discontinuation of treatment. The no-observed-adverse-effect level (NOAEL) of SPH3127 was determined to be 30 mg/kg/day for Sprague-Dawley rats and 20 mg/kg/day for cynomolgus monkeys based on the kidney and cardiovascular changes found at mid- and high-dose animals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lysosomes generally maintain the weak acidic microenvironment, to ensure highly efficient activity and functions of hydrolytic enzymes and proteins. Aberrations of the lysosomal pH may result in cellular functional changes and influence human physiology, possibly causing serious diseases. Small-molecular fluorescent probes based imaging techniques capable of providing information on target locations are considerably appreciated. Herein, by reducing the size of the typical lysosome targetable group 4-(2-aminoethyl) morpholine, we rationally designed a rhodamine analogue probe Ly-HN2AM with N-Aminomorpholine as the ring-closed switch and the lysosome targetable moiety for visualizing lysosomal pH changes. With the benefit of constructing multi-pentacyclic intramolecular hydrogen bond when binding with the H + , Ly-HN2AM gives a highly sensitive response towards pH values ranging from 4.79 to 6.07, with a remarkable higher pK a 5.35 over the typical 4-(2-aminoethyl) morpholine modified probes. The new probe was successfully applied to visualize pH value changes in lysosome-associated physiological and pathological processes with excellent photostability and low cytotoxicity, indicating the potential applications of lysosome specific bioimaging., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212-2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB 1 R) and cancelled by concomitant administration of adenosine A 2A receptor (A 2A R) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by A 2A R antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212-2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212-2 (300 nM), an effect partially rescued by the A 2A R antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A 2A R or CB 1 R binding in the hippocampus and in the prefrontal cortex. These results, showing that A 2A R antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A 2A Rs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The emergence of synthetic cannabinoids (SCBs) as drugs of abuse in readily available "Spice" smoking blends has exposed users to much more potent cannabinoids than the phytocannabinoids present in Cannabis sativa L. Increasing reports of adverse reactions are emerging in the clinical literature. SCBs may disrupt the endocannabinoid signalling, which has been shown to be crucial within human endometrium remodelling process. Within this study, a telomerase-immortalised human endometrial stromal cell line (St-T1b) and primary human decidual fibroblasts (HdF) were used to determine the impact of SCBs JWH-122, UR-144 and WIN55,212-2 (WIN) on endometrial stromal cells. Our findings indicate that JWH-122 and UR-144 (0.01-25 μM) induce prompt ROS/RNS formation and endoplasmic reticulum (ER) stress without reduction in cell viability. Disturbances in the normal functions of the ER lead to cell stress response, which is after compensated with the increase in reduced/oxidized glutathione ratio (GSH/GSSG). Instead, WIN induces ER stress, mitochondrial dysfunction and apoptotic cell death. The addition of the CB1 antagonist AM281 significantly reduces the effects on cell viability, suggesting that CB1 plays a key role in WIN-induced apoptosis. Collectively, our data suggests that SCBs have dissimilar effects on human endometrial stromal cells and, thus, may impact human reproductive function through distinct mechanisms that are crucial for the understanding of the pathophysiological outcomes from its abuse., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC 50 values of 5 μM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K + -elicited [Ca 2+ ] c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca 2+ entry and the uncoupling of the Ca 2+ -dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

Cannabinoid CB2 receptor agonists are under investigation for clinical use. At the same time, synthetic cannabinoids have been implicated in a number of deaths. One cause of death is thought to be cardiac arrest subsequent to extreme tachycardia. Central mechanisms are thought to play a role in this, with CB1 but not CB2 receptors thought to mediate central effects. However, the direct effects of cannabinoids on the heart are less well understood. We therefore tested the effects of cannabinoids on isolated rat atria to test whether activation of myocardial CB1 and CB2 receptors could contribute to tachycardia. Although we found a moderate effect that can be attributed to CB1 receptors, we did not find any evidence for chronotropic effects by a CB2 receptor activation. Our results indicate that cannabinoid cardiotoxicity may partially involve CB1 receptors in the myocardium, and that CB2 receptor agonists are unlikely to have significant effects on the heart.

Elucidating how cannabinoids affect brain function is instrumental for the development of therapeutic tools aiming to mitigate 'on target' side effects of cannabinoid-based therapies. A single treatment with the cannabinoid receptor agonist, WIN 55,212-2, disrupts recognition memory in mice. Here, we evaluate how prolonged, intermittent (30 days) exposure to WIN 55,212-2 (1 mg/kg) alters recognition memory and impacts on brain metabolism and functional connectivity. We show that chronic, intermittent treatment with WIN 55,212-2 disrupts recognition memory (Novel Object Recognition Test) without affecting locomotion and anxiety-like behaviour (Open Field and Elevated Plus Maze). Through 14 C-2-deoxyglucose functional brain imaging we show that chronic, intermittent WIN 55,212-2 exposure induces hypometabolism in the hippocampal dorsal subiculum and in the mediodorsal nucleus of the thalamus, two brain regions directly involved in recognition memory. In addition, WIN 55,212-2 exposure induces hypometabolism in the habenula with a contrasting hypermetabolism in the globus pallidus. Through the application of the Partial Least Squares Regression (PLSR) algorithm to the brain imaging data, we observed that prolonged WIN 55,212-2 administration alters functional connectivity in brain networks that underlie recognition memory, including that between the hippocampus and prefrontal cortex, the thalamus and prefrontal cortex, and between the hippocampus and the perirhinal cortex. In addition, our results support disturbed lateral habenula and serotonin system functional connectivity following WIN 55,212-2 exposure. Overall, this study provides new insight into the functional mechanisms underlying the impact of chronic cannabinoid exposure on memory and highlights the serotonin system as a particularly vulnerable target., (© 2018 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

During incubation, round whitefish embryos may experience fluctuating or elevated temperatures from natural (e.g., seasonal temperature changes) and/or anthropogenic sources. Anthropogenic sources like once-through cooling discharges from nuclear power plants can also expose embryos to chemicals (e.g., morpholine) and/or radiation. To examine the effects of these potential stressors on embryogenesis, round whitefish were incubated under fluctuating or constant temperatures, with morpholine or 137 Cs gamma rays. We report the percentage of prehatch and posthatch mortality, developmental rate, hatch dynamics, and morphometrics at 4 development stages. Embryos reared at constant temperatures had delayed developmental stage onset and median hatch, higher mortality at constant 8 °C, and lower mortality at ≤5 °C, compared with embryos reared under seasonal temperature regimes. Embryos incubated with ≥500 mg L -1 morpholine (>200× regulatory limits) had advanced hatch, reduced body size, and increased prehatch (100% at 1000 mg L -1 ) and posthatch (≈95% at 500 mg L -1 ) mortality compared with controls. Relative to controls, embryos irradiated with ≥0.16 mGy/d had larger body mass early in development, and all irradiated embryos had decreased posthatch mortality; the lowest dose was >300× discharge limits. Our study suggests that fluctuating or elevated temperatures and high-dose morpholine can alter development rate, hatch dynamics, and growth, and/or increase mortality compared with embryos reared at constant temperatures of ≤5 °C; conversely, low-dose irradiation had transient developmental effects but may benefit early posthatch survival. Environ Toxicol Chem 2018;37:2593-2608. © 2018 SETAC., (© 2018 SETAC.)

Lysosome fluorescent imaging has been widely used in the field of biological staining and diagnostics, which plays a key role in understanding intracellular metabolism and various physiological processes. However, for most currently used small-molecule lysotrackers, the photostability is often unsatisfactory when used for long-term and real-time visualization of lysosomal dynamics. Herein, we reported a new lysosome-targetable photostable fluorescent probe (i.e. MPL-NPA), and results showed that MPL-NAP possesses superior photostability, appreciable tolerance to pH change, low cytotoxicity and high lysosome targeting ability. These findings confirm that MPL-NAP is a well-suited imaging agent for targeting lysosome and enables long-term and real-time monitor of lysosome morphological changes under physiological processes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Selective activation of the M 1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M 1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M 1 , leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M 1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M 1 -expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [ 3 H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M 1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M 1 PAM activity (EC 50 > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M 1 . Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

Background: Buparlisib is an oral pan-class I phosphotidyinositol-3-kinase (PI3K) inhibitor. The present phase I study evaluated the safety, pharmacokinetics, and efficacy of buparlisib with capecitabine in patients with metastatic breast cancer., Patients and Methods: Patients received buparlisib once daily (range, 50 to 100 mg) for 3 weeks with capecitabine twice daily (range, 1000 to 1250 mg/m 2 ) for 2 weeks with a 1-week break. Dose escalation used a traditional "3 + 3" design with standard definitions of dose-limiting toxicity (DLT) and maximum tolerated dose., Results: Of the 25 patients enrolled, 23 were evaluable for DLT and 17 were evaluable for response. The maximum tolerated dose of the combination was buparlisib 100 mg daily and capecitabine 1000 mg/m 2 twice daily. DLTs included grade 3 hyperglycemia and grade 3 confusion. The most common grade 3 toxicities were diarrhea and elevation of aspartate aminotransferase and alanine transaminase. One patient exhibited a complete response to treatment and four had a confirmed partial response. In cohorts 3 and 4, in which the buparlisib dose remained constant but the capecitabine dose was increased, significant increases in the buparlisib plasma concentration were noted., Conclusion: The combination of buparlisib with capecitabine in patients with metastatic breast cancer was generally well-tolerated, with several patients demonstrating prolonged responses. Unexpectedly low rates of PIK3CA mutations (3 of 17) were seen, and only 2 of 7 tumors with subtyping were luminal, making exploration of these putative predictive markers impossible. Further study of the combination is not unreasonable, with expanded pharmacokinetics and sequencing analysis to better elucidate potential drug-drug interactions and more accurate predictive biomarkers of response., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

In previous studies, we detected the photoinitiators 1-hydroxycyclohexyl phenyl ketone (1-HCHPK), methyl 2-benzoylbenzoate (MBB), and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMP) in intravenous injection solutions. In addition, we reported that 1-HCHPK, MBB, and MTMP exhibited cytotoxicity towards normal human peripheral blood mononuclear cells. A previous in vitro study reported that a free-radical photoinitiator introduced covalently bound purine residues into DNA. However, little is known about the in vitro mutagenicity of 1-HCHPK, MBB, and MTMP. In the present in vitro study, we evaluated the mutagenicity of 1-HCHPK, MBB, and MTMP using the Ames test. We found that untreated 1-HCHPK, MBB, and MTMP were not mutagenic in S. typhimurium strain TA97, TA98, TA100, TA102, or TA1535, regardless of the presence/absence of S9 activation. However, ultraviolet (UV) light-irradiated MTMP exhibited mutagenicity in S. typhimurium strain TA97 in the absence of S9 activation. In conclusion, we suggest that exposure to UV-irradiated MTMP, including in intravenous injection solutions, can result in frameshift mutations.

Two iridium(iii) complexes with ligands containing morpholine groups were used to track mitochondrial pH fluctuation during cell apoptosis as their emissive intensities linearly increased with increase in pH values.

Discharges from industrial cooling water systems can include low levels of morpholine (a chemical pH regulator and corrosion inhibitor), as well as transiently higher temperature effluent water which present a potential source of environmental impact to aquatic biota. The effects of environmental levels of morpholine or heat shock (HS) treatment alone and in combination with a challenge high-dose of 137 Cs ionizing radiation were studied using the cytokinesis block micronucleus assay in a rainbow trout cell line (RTG-2). Morpholine treatment of 10 or 100mgL -1 alone produced no significant effects, and no interaction was observed in combination with 7.75Gy radiation. A 9°C magnitude HS treatment alone significantly increased micronuclei formation. A synergistic response was observed when 9°C HS was combined with 7.75Gy radiation, with 15% more cells containing 3 or more micronuclei than the sum of each individual stressor. A synergistic increase in the average number of micronuclei was observed when morpholine and a 9°C HS were co-treated. These results indicate that morpholine at environmentally-relevant levels does not impact micronuclei formation or cell cycle progression however 9°C HS may be of potential concern both alone and in combination with other stressor treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no existing therapeutic approach to delay or stop progression. Genetic, biochemical and pre-clinical studies have provided evidence that leucine-rich-repeat-kinase-2 (LRRK2) kinase is involved in the pathogenesis of PD, and small molecule LRRK2 inhibitors represent a novel potential therapeutic approach. However, potentially adverse target-related effects have been discovered in the lung and kidneys of LRRK2 knock-out (ko) mice and rats. It is unclear if the LRRK2 ko effect in the kidneys and lung is also induced by pharmacological inhibition of the LRRK2 kinase. Here, we show that treatment with the LRRK2 inhibitor PFE-360 in rats induces a morphological kidney phenotype resembling that of the LRRK2 ko rats, whereas no effects were observed in the lung. The PFE-360 treatment induced morphological changes characterised by darkened kidneys and progressive accumulation of hyaline droplets in the renal proximal tubular epithelium. However, no histopathological evidence of renal tubular injury or changes in the blood and urine parameters that would be indicative of kidney toxicity or impaired kidney function were observed after up to 12 weeks of treatment. Morphological changes were detected in the kidney after 2 weeks of treatment and were partially reversible within a 30 day treatment-free period. Our findings suggest that pharmacological LRRK2 inhibition may not have adverse consequences for kidney function., (Copyright © 2018 H. Lundbeck A/S. Published by Elsevier B.V. All rights reserved.)

In our endeavor towards the development of potent molecules for cancer diseases, we have designed and synthesized a series of 2,4,5-trisubstituted imidazole derivatives (B1-B24) and characterized by using various spectroscopic techniques. All these compounds are further evaluated for their in vitro anti-cancer, anti-oxidant activities and molecular docking studies against EGFR, HER2 protein receptors. The in vitro anti-cancer activity analysis reveals that compounds B11 and B16 were found to be effective scaffolds against the tested human cancer cell lines IMR-32, A549 and HeLa. Particularly, B16 and B11 showed effective cytotoxicity against A549 and IMR-32 with IC 50 values of 09.521±0.54μM and 10.294±0.43μM, respectively. Moreover, compounds B17, B18 and B23 showed potent activity towards the anti-oxidant screening with IC 50 values of 5.87±1.73μM, 6.29±1.27μM and 4.95±1.81μM, respectively compared to standard ascorbic acid. Molecular docking was performed against the EGFR, HER2 protein receptors to provide more insight into their mechanism of interaction by comparing with standard EGFR, HER2 inhibitors like Gefitinib (EFGR), Lapatanib (EGFR), Afitinib (HER2) and Canertinib (HER2). Compounds B15, B16, B11 and B10 were exhibiting their minimum binding energies. Out of the aforementioned docked molecules, B15 and B16 showed the best binding energies of -11.15kcalmol -1 , -10.70kcalmol -1 and -10.49kcalmol -1 , -10.12kcalmol -1 against EGFR and HER2 protein receptors, respectively. The molecular docking results are well corroborated with the in vitro anti-cancer activity finding., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme inhibitory, and antimicrobial activities of a new series of 2-(4-substituted phenyl)-1-[2-(morpholin-4-yl)ethyl]-1 H -benzimidazole derivatives, for their possible use as multi-action therapeutic agents. Target compounds ( n = 15) were synthesized under microwave irradiation conditions in two steps, and their structures were elucidated by FT-IR, ¹H-NMR, 13 C-NMR and high resolution mass spectroscopic analyses. Pharmacological screening studies revealed that two of the compounds ( 2b and 2j ) have inhibitory potential on both COX-1 and COX-2 enzymes. In addition, cytotoxic and genotoxic properties of the compounds 2b , 2j and 2m were investigated via the well-known MTT and Ames tests, which revealed that the mentioned compounds are non-cytotoxic and non-genotoxic. As a concise conclusion, two novel compounds were characterized as potential candidates for treatment of frequently encountered inflammatory diseases., Competing Interests: The authors declare no conflict of interest.

A strong relationship between abnormal functions of Aurora kinases and tumorigenesis has been reported for decades. Consequently, Aurora kinases serve as potential targets for anticancer agents. Here, we identified aminobenzothiazole derivatives as novel inhibitors of Aurora B kinase through bioisosteric replacement of the previous inhibitors, aminobenzoxazole derivatives. Most of the urea-linked aminobenzothiazole derivatives showed potent and selective inhibitory activity against Aurora B kinase over Aurora A kinase. Molecular modeling indicated that compound 15g bound well to the active site of Aurora B kinase and formed the essential hydrogen bonds. The potent compounds, 15g and 15k, were selected, and their biological effects were evaluated using HeLa cell lines. It was found that these compounds inhibited the phosphorylation of histone H3 at Ser10 and induced G 2 /M cell cycle arrest. We suggest that the reported compounds have the potential to be further developed as anticancer therapeutics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chemicals used in the prevention of corrosion and biofouling may be released into the environment via industrial cooling water discharges. The authors assessed the impacts of 2 commonly used chemicals, morpholine and sodium hypochlorite, on development in lake whitefish (Coregonus clupeaformis). Embryos were exposed chronically, beginning at fertilization or at the eyed stage. Acute 96-h exposures were also examined at 4 development stages. Chronic morpholine resulted in median lethal concentrations (LC50s) of 219 ± 54 mg/L when exposure began at fertilization and 674 ± 12 mg/L when exposure began at the eyed stage, suggesting that embryos are more sensitive earlier in development. Chronic morpholine exposure advanced hatching by up to 30%, and the early hatching embryos were up to 10% smaller in body length. A decrease in yolk conversion efficiency was also observed in embryos exposed to chronic morpholine concentrations of 1000 mg/L. The majority of effects from morpholine exposure manifested near hatch, possibly reflecting changes in chorion permeability at the end of embryonic development. Sodium hypochlorite only impacted survival with chronic exposure from fertilization, where the total residual chlorine LC50 was 0.52 ± 0.11 mg/L. Acute exposures to both chemicals had minimal effects up to the highest tested concentrations. Overall, the results suggest that the risk during development from exposure to morpholine and sodium hypochlorite is low under normal operating conditions. Environ Toxicol Chem 2017;36:1955-1965. © 2016 SETAC., (© 2016 SETAC.)

A series of novel amidinourea derivatives was synthesized, and the compounds were evaluated as inhibitors of MDA-MB-231 human breast cancer cell proliferation. In addition, a second series of triazine derivatives designed as rigid congeners of the amidinoureas was synthesized, and the compounds were evaluated for their antiproliferative activity. Among the two series, amidinourea 3 d (N-[N-[8-[[N-(morpholine-4-carbonyl)carbamimidoyl]amino]octyl]carbamimidoyl]morpholine-4-carboxamide) emerged as a potent anticancer hit compound with an IC 50 value of 0.76 μm, similar to that of tamoxifen., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of this study was to evaluate the toxicity of the fungicide dimethomorph to soil microbial activity and the earthworm Eisenia fetida. Multiple biomarkers, namely, four soil enzymes (urease, dehydrogenase, invertase, and acid phosphatase), four earthworm biochemical indices (dismutase, catalase, cellulase, and malondialdehyde), and the transcriptional levels of both target genes (dismutase and catalase) were measured at 1, 10, and 100 mg kg -1 after 1, 7, 21, and 28 days. The degradation rate of dimethomorph in soil was also determined, and the results indicated that most parameters did not differ from the controls at 1 and 10 mg kg -1 dimethomorph by the last exposure time (28 d). However, high concentrations (100 mg kg -1 ) of dimethomorph had varying effects on soil enzymatic activity and earthworms. These effects gradually decreased with prolonged exposure times. Positive correlations (R 2  > 0.57) between the target gene expression levels and antioxidant enzyme activities were observed in this study. We also found that earthworms have improved soil microbial activity and accelerated the degradation of dimethomorph. Overall, higher concentrations of dimethomorph might pose an ecological hazard to soil environments in the short term., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tango ® Super is a two-compound fungicide formulation widely employed in grain protection. However, details of Tango ® Super effects on cell cultures have not been fully investigated. In this study, bovine lymphocytes were exposed to a concentration range 0.5; 1.5; 3; 6; and 15 μg mL -1 for 4 h to assess the cytotoxicity and genotoxicity of the fungicide. Our experiments revealed that this fungicide treatment reduced cell viability, decreased cell proliferation and provoked apoptotic cell death. Cell cycle analysis showed predominant accumulation of cells in the G 0 /G 1 phase of the cell cycle. The fungicide was able to induce mitochondrial superoxide production accompanied by elevated levels of carbonylated proteins and changes in the lipid membrane composition. The fungicide did not induce micronuclei production, but stimulated both DNA double-strand breaks and the formation of p53 binding protein, which is accumulated during the DNA repair process at the site of double-strand breaks. Based on the obtained data we suppose that the fungicide-induced DNA damage is the result of oxidative stress, which may contribute to higher occurrence of apoptotic cell death. Because ergosterol biosynthesis-inhibiting fungicides are widely used in agriculture to ensure higher crop yields and may cause health impairment of animals and humans, there is a need for further testing to elucidate their potential genotoxic effects using in vivo and/or in vitro systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phosphodiesterase type 4D (PDE4D) has been indicated as a promising target for treating neurodegenerative pathologies such as Alzheimer's Disease (AD). By preventing cAMP hydrolysis, PDE4 inhibitors (PDE4Is) increase the cAMP response element-binding protein (CREB) phosphorylation, synaptic plasticity and long-term memory formation. Pharmacological and behavioral studies on our hit GEBR-7b demonstrated that selective PDE4DIs could improve memory without causing emesis and sedation. The hit development led to new molecule series, herein reported, characterized by a catechol structure bonded to five member heterocycles. Molecular modeling studies highlighted the pivotal role of a polar alkyl chain in conferring selective enzyme interaction. Compound 8a showed PDE4D3 selective inhibition and was able to increase intracellular cAMP levels in neuronal cells, as well as in the hippocampus of freely moving rats. Furthermore, 8a was able to readily cross the blood-brain barrier and enhanced memory performance in mice without causing any emetic-like behavior. These data support the view that PDE4D is an adequate molecular target to restore memory deficits in different neuropathologies, including AD, and also indicate compound 8a as a promising candidate for further preclinical development., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Potential genotoxic/cytotoxic effects of the epoxiconazole/fenpropimorph-based fungicide were investigated using single cell gel electrophoresis and cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and fluorescence in situ hybridization in cultured bovine lymphocytes. No statistically significant elevations of DNA damage and increases in cytogenetic endpoints were seen. However, evident cytotoxic effect presented as a decrease in mitotic and proliferation indices were recorded after exposure of bovine lymphocytes to the fungicide for 24 and 48 h at concentrations ranging from 3 to 15 µg mL(-1) (P < 0.05, P < 0.01, P < 0.001). Similarly, for 24 h an inhibition in the cytokinesis block proliferation index (CBPI) was obtained after exposure to the fungicide at concentrations ranging from 1.5 to 15 µg mL(-1) (P < 0.01, P < 0.001) in each donor.

Moroxydine hydrochloride (Mor) is known to have multi-antiviral activities against DNA and RNA viruses but very little information exists on its pharmacology. The paper was undertaken to explore the antiviral response and antiapoptotic mechanism of Mor against grass carp reovirus (GCRV) in Ctenopharyngodon idella kidney (CIK) cells. The results showed that exposing GCRV-infected cell to 6.3 μg mL(-1) of Mor for 96 h avoid ca. 50% apoptosis. Meanwhile, Mor had lower cytotoxicity than ribavirin (Rib) as the value of safe concentration was threefold higher than effective concentration and the compound could ensure sufficient into and out of cells within 4 h when tested at the maximal safe concentration. Mor blocked the GCRV-induced cytopathic effects and eliminated nucleocapsids in CIK cells to keep the normal morphological structure. Moreover, the expressions of viral protein genes were significantly inhibited especially the guanylyl transferase and RNA-dependent RNA polymerase related expression. Furthermore, GCRV caused Bcl-2 down-regulation and Bax mitochondrial translocation was prevented by treatment of CIK cells with Mor. The downstream effector, caspase activity was also significantly inhibited in Mor treated cells. The potential mechanism might be that mitochondrial apoptotic signals were not activated by the intervention of Mor for targeting viral gene expression. Taken together, Mor showed high anti-GCRV activity and had been proved as a secure and promising agent in viral controlling in aquaculture industry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study assessed the effect of the buffers 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) on keratinocyte cell viability and microbial growth. It was observed that RPMI buffered with HEPES, supplemented with l-glutamine and sodium bicarbonate, can be used as a more suitable medium to promote co-culture., (Copyright © 2016. Published by Elsevier B.V.)

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Objective: Radiation therapy, one of the major treatments for liver cancer, causes DNA damage and cell death. Since the liver cancer cells have a strong capacity to repair irradiative injury, new medicines to enhance this treatment are urgently required. In this study, we investigated the effect of NU7441, a synthetic small-molecule compound, as a specific inhibitor of DNA-dependent protein kinase (DNA-PK) in radiosensitization of hepatocellular carcinoma HepG2 cells., Methods: Cell Counting Kit-8 (CCK-8) was first used to evaluate the proliferation of HepG2 cells under NU7441 treatment. SDS-PAGE and Western blot were then performed to study the protein expression leading to the DNA damage repair. Further, neutral single cell gel electrophoresis and immunofluorescence assay were carried out to assess DNA repair. Finally, flow cytometry was implemented to examine the changes in cell cycle., Results: NU7441 reduced the CCK-8 counts in the HepG2 culture, further enhanced 60Cox03B3; radiation injury to HepG2 cells, which was manifested by decreasing the DNA-PKcs (S2056) protein expression, increasing x03B3;H2AX foci number, prolonging the tail moment of the comet cells, and inducing cell cycle arrest at G2/M phase., Conclusion: NU7441 inhibited the growth of liver cancer cells, enhanced the radiosensitization of these cancer cells by interfering with the DNA repair and cell cycle checkpoint. These data implicate NU7441 as a potential radiotherapy sensitizer for the treatment of liver cancer., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Although fulminant hepatitis represents a ubiquitous human health problem, there is a lack of effective therapeutic strategies that have few side‑effects and the precise mechanisms underlying fulminant hepatitis are not fully understood. Phosphoinositide 3‑kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in hepatic diseases. Although previous research indicates that PI3K is involved in cardiac diseases, including myocardial infarction, it currently remains unclear whether the inhibition of PI3K is essential for ameliorating the severity of lipopolysaccharide (LPS)‑induced hepatitis. The aim of the present study was to investigate whether pharmacological blockade of PI3K ameliorates the development of LPS‑induced murine acute hepatic injury. A murine model of LPS‑induced acute hepatic injury was used to investigate the therapeutic effect of the pan‑PI3K inhibitor, LY294002 on murine fulminant hepatitis and to investigate potential underlying mechanisms. The current report presents the in vivo role of LY294002 in protecting the mice from fulminant hepatitis. LY294002 was observed to exert significant protective effects on the liver by reducing the activities of alanine aminotransferase and aspartate aminotransferase, as well as by improving the histological architecture of the liver. In LPS‑induced hepatitis, treatment with LY294002 clearly inhibited intrahepatic synthesis of various disease‑relevant proinflammatory cytokines, including tumor necrosis factor‑α, interleukin (IL)‑6, IL‑1β and interferon‑γ. Furthermore, LY294002 was observed to significantly inhibit IκB phosphorylation in LPS‑injured mouse liver samples. Therefore, LY294002 may protect the liver from LPS‑induced injury by inhibition of the IκB‑nuclear factor κ‑light‑chain‑enhancer of activated B cell dependent signaling pathway. Thus, the current report provides evidence that LY294002 exerts potent effects against LPS‑induced hepatic injury, indicating its potential therapeutic value for the treatment of acute hepatitis.

Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol (2-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences., (Published by Elsevier Ltd.)

Unlabelled: Synthetic cannabinoid WIN55,212-2 (WIN) has shown a promise as an anticancer agent but causes psychoactive side-effects. In the present study, nano-micelles of styrene maleic acid (SMA)-conjugated WIN were synthesized to reduce side-effects and increase drug efficacy. SMA-WIN micelles were characterised and their in vitro cytotoxic effect was compared to that of free WIN against triple-negative breast cancer (MDA-MB-231), hormone receptor-positive breast cancer (MCF-7) and castration-resistant prostate cancer (PC3) cell lines. SMA-WIN micelles were synthesised with a ~15% loading, 132.7 nm average diameter, -0.0388 mV charge, and pH-dependent release rate. A dose-dependent inhibition of cell growth was observed in all three cell lines treated with both free and micellar WIN, with both formulations demonstrating equal cytotoxicity., Conclusion: SMA-WIN demonstrated characteristics theorized to improve in vivo drug biodistribution. Potent cytotoxicity was found against breast and prostate cancer cells in vitro, showing promise as a novel treatment against breast and prostate cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Recently, we found that two cyclodidepsipeptides, 3,6-di-(propan-2-yl)-4-methyl-morpholine-2,5-dione (1) and 3-(2-methylpropyl)-6-(propan-2-yl)-4-methyl- morpholine-2,5-dione (2), are excellent inhibitors of xanthine oxidase. In order to obtain more information about the toxicological potential of compounds 1 and 2 on bone cells, the current study was designed to evaluate the effect of these compounds on viability and proliferation of MC3T3-E1 cells. Compound 1 showed neither cytotoxic nor stimulatory effect on cell viability, while compound 2 showed a slight stimulatory effect on cell viability. Both studied compounds showed slight stimulatory effects on proliferation of MC3T3-E1 cells, in a dose dependent manner. Additionally, an in silico toxicological study of compounds 1 and 2 was performed, and the results indicate that they have a good probability of safe biological intake.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas., (© 2014 UICC.)

Despite extensive study, the mechanisms of cell fate choice upon p53 activation remain poorly understood. Using genome-wide shRNA screening, we recently identified the ATM kinase as synthetic lethal with Nutlin-3, an MDM2 inhibitor that leads to non-genotoxic p53 activation. Here, we demonstrate that while this synthetic lethal interaction relies upon components of both the intrinsic and extrinsic apoptotic pathways (e.g., BAX and BID), it is not due to significant ATM effects on the expression of p53 target genes. Instead, loss of ATM activity results in increased mitochondria and reactive oxygen species that drive apoptosis. Finally, we provide evidence that pharmacologic inhibition of ATM blocks autophagy in direct opposition to p53, which activates this process, and that inhibition of autophagy is sufficient to elicit an apoptotic response when combined with Nutlin-3.

Unlabelled: The influence of WIN 55,212-2 mesylate (WIN) on the anticonvulsant activity and acute neurotoxic potential of clobazam (CLB) and lacosamide (LCM) was studied in the maximal electroshock-induced seizure (MES) model and chimney test in mice., Results: indicate that WIN administered intraperitoneally, at doses of 2.5 and 5 mg/kg significantly enhanced the anticonvulsant action of CLB in the MES test by reducing its median effective dose (ED50) from 20.80 mg/kg to 12.05 mg/kg (P<0.05), and 8.22 mg/kg (P<0.001), respectively. In contrast, WIN (1.25 mg/kg) did not significantly potentiate the anticonvulsant activity of CLB against MES-induced seizures. Similarly, WIN at doses of 1.25, 2.5 and 5 mg/kg had no significant impact on the anticonvulsant action of LCM in the MES test. On the other hand, WIN (5 mg/kg) had no impact on the acute neurotoxic effects of CLB and LCM in the chimney test and the median toxic doses (TD50) for CLB and LCM were almost unchanged. Thus, WIN (5 mg/kg) elevated the protective index values for CLB (from 1.41 to 3.07) and LCM (from 3.60 to 4.91). In conclusion, WIN potentiates suppression of tonic-clonic seizures produced by CLB in the mouse MES model, without affecting acute neurotoxic adverse effects of CLB in the chimney test in mice, which is favorable from a preclinical point of view., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Experiments on pregnant rats have demonstrated the absence of damaging effect of Afobazole administered during the antenatal period on organogenesis in fetuses. Afobazole in a dose of 5 mg/kg given to pregnant rats on gestation days 6-16 reduced pre- and post-implantation fetal mortality and improved fertility; 20-day-old embryos had no developmental abnormalities and did not differ from controls by craniocaudal size, body weight, and skeleton ossification. Afobazole in a dose of 100 mg/kg reduced pre- and post-implantation fetal mortality, but had no effect on fertility. No congenital malformations were found in the fetuses, but they were characterized by lower craniocaudal size, body weight, and number of ossification foci in the sternum and spine.