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Copyright of Tropical Medicine & International Health is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aim: To evaluate cortical excitability with transcranial magnetic stimulation (TMS) in children with new-onset epilepsy before and after antiepileptic drugs (AEDs).Method: Fifty-five drug-naïve patients (29 females, 26 males; 3-18y), with new-onset epilepsy were recruited from 1st May 2014 to 31st October 2017 at the Child Neurology Department, Queen Silvia's Children's Hospital, Gothenburg, Sweden. We performed TMS in 48 children (23 females, 25 males; mean [SD] age 10y [3y], range 4-15y) with epilepsy (27 generalized and 21 focal) before and after the introduction of AEDs. We used single- and paired-pulse TMS. We used single-pulse TMS to record resting motor thresholds (RMTs), stimulus-response curves, and cortical silent periods (CSPs). We used paired-pulse TMS to record intracortical inhibition and facilitation at short, long, and intermediate intervals.Results: There were no differences in cortical excitability between children with generalized and focal epilepsy at baseline. After AED treatment, RMTs increased (p=0.001), especially in children receiving sodium valproate (p=0.005). CSPs decreased after sodium valproate was administered (p=0.050). As in previous studies, we noted a negative correlation between RMT and age in our study cohort. Paired-pulse TMS could not be performed in most children because high RMTs made suprathreshold stimulation impossible.Interpretation: Cortical excitability as measured with RMT decreased after the introduction of AEDs. This was seen in children with both generalized and focal epilepsy who were treated with sodium valproate, although it was most prominent in children with generalized epilepsy. We suggest that TMS might be used as a prognostic tool to predict AED efficacy.What This Paper Adds: Resting motor threshold (RMT) correlated negatively with age in children with epilepsy. No differences in cortical excitability were noted between patients with generalized and focal epilepsy. Treatment with antiepileptic drugs decreased cortical excitability as measured with transcranial magnetic stimulation (TMS). Decreased cortical excitability with increased RMT was recorded, especially after sodium valproate treatment. Paired-pulse TMS was difficult to perform because of high RMTs in children. [ABSTRACT FROM AUTHOR]

Copyright of Developmental Medicine & Child Neurology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aim: To assess: (1) the effect on drooling of bilateral submandibular duct ligation as surgical therapy after the administration of submandibular botulinum neurotoxin A (BoNT-A) for excessive drooling and (2) the predictive value of treatment success with BoNT-A on treatment success after bilateral submandibular duct ligation.Method: This was a within-participant retrospective observational study in which 29 children with severe drooling (15 males, 14 females) received BoNT-A treatment at a mean age of 9 years 6 months (SD 2y 5mo), followed by bilateral submandibular duct ligation at a mean age of 10 years 11 months (SD 2y 4mo). Fifteen children were diagnosed with cerebral palsy (CP), with 12 children classified in Gross Motor Function Classification System levels IV and V. The 14 children without CP had non-progressive developmental disorders. The primary drooling severity outcomes were the Visual Analogue Scale (VAS; subjective assessment) and drooling quotient (objective assessment). Measurements were taken before each intervention and again at 8 and 32 weeks.Results: The VAS was significantly lower after bilateral submandibular duct ligation at follow-up compared to BoNT-A treatment (mean difference -33, p≤0.001; 95% confidence interval [CI]=-43.3 to -22.9). The mean drooling quotient did not significantly differ between BoNT-A treatment and bilateral submandibular duct ligation at follow-up (3.3, p=0.457; 95% CI=-4.35 to 9.62) or between 8 and 32 weeks (4.7, p=0.188; 95% CI=-2.31 to 11.65).Interpretation: BoNT-A treatment and bilateral submandibular duct ligation are both effective treatment modalities for drooling. At 32-week follow-up, subjective drooling severity after bilateral submandibular duct ligation was significantly lower compared to previous BoNT-A injections in participants. However, treatment success with BoNT-A is no precursor to achieving success with bilateral submandibular duct ligation.What This Paper Adds: Bilateral submandibular duct ligation is an effective therapy for drooling after treatment with botulinum neurotoxin A (BoNT-A). Treatment success with BoNT-A is not a predictor of successful therapy with bilateral submandibular duct ligation. [ABSTRACT FROM AUTHOR]

Background: The risk of bacterial contamination and the deterioration of platelet (PLT) quality limit the shelf-life of platelet concentrates (PCs). The INTERCEPT pathogen inactivation system reduces the risk of pathogen transmission by inhibiting nucleic acid replication using a combination of a photo-reactive compound and UVA illumination. The goal of this study was to investigate the effects the INTERCEPT system has on the PLT metabolome and metabolic activity.Study Design and Methods: Paired units of buffy coat-derived PCs were generated using a pool and split strategy (n = 8). The paired PCs were either treated with the INTERCEPT system or left untreated. Samples were collected on Days 1, 2, 4, and 7 of storage. Ultra-performance chromatography coupled with time-of-flight mass spectrometry was used to analyze the extra- and intracellular metabolomes. Constraint-based metabolic modeling was then used to predict the metabolic activity of the stored PLTs.Results: A relatively large number of metabolites in the extracellular environment were depleted during the processing steps of the INTERCEPT system, in particular, metabolites with hydrophobic functional groups, including acylcarnitines and lysophosphatidylcholines. In the intracellular environment, alterations in glucose and glycerophospholipid metabolism and decreased levels of 2-hydroxyglutarate were observed following the INTERCEPT treatment. Untargeted metabolomics analysis revealed residual amotosalen dimers present in the treated PCs. Systems-level analysis of PLT metabolism indicated that the INTERCEPT system does not have a significant impact on the PLT energy metabolism and nutrient utilization.Conclusions: The INTERCEPT system significantly alters the metabolome of the stored PCs without significantly influencing PLT energy metabolism. [ABSTRACT FROM AUTHOR]

Background: The exact mechanism and site of action of triptans in aborting migraine attacks remain under debate. We hypothesized that the clinical efficacy of triptans lies in aborting central sensitization and focused on the question of why triptans are headache specific, that is highly effective in migraine and cluster headache and ineffective in extracephalic pain.Methods: Forty healthy participants were enrolled in this double-blinded, randomized, placebo-controlled study. The effect of sumatriptan (n = 20) versus placebo (n = 20) was investigated in a trigeminal (V1) versus an extracephalic dermatome (forearm) using a topical capsaicin sensitization model. Capsaicin-induced primary and secondary hyperalgesia were evaluated using quantitative sensory testing.Results: After capsaicin application, primary hyperalgesia developed in both the sumatriptan and placebo groups in both dermatomes. However, sumatriptan exclusively prevented secondary hyperalgesia in the V1 dermatome but not on the forearm. Placebo exerted no effects on secondary hyperalgesia in both trigeminal and extracephalic dermatomes. Additionally, sumatriptan reduced the flare size exclusively in the V1 dermatome.Conclusions: Our data suggest that sumatriptan reduces central sensitization (secondary hyperalgesia) without modulating peripheral sensitization (primary hyperalgesia) in a human pain model of capsaicin-induced sensitization. Moreover, despite a systemic administration of sumatriptan, the modulatory effects are trigeminal specific, echoing the clinical effect of triptans in aborting headaches, but not extracephalic pain.Significance: Our data suggest that triptans exert their efficacy by suppressing central sensitization. By revealing a dermatome-specific modulation, our study demonstrates a previously unrecognized interaction between the pharmacodynamics of triptans and the trigeminal nociceptive system that provides new insight into how triptans may work in aborting headache attacks. [ABSTRACT FROM AUTHOR]

As the opioid crisis has continued to grow, so has the number of infants exposed to opioids during the prenatal period. A growing concern is that prenatal exposure to opioids may induce persistent neurological changes that increase the propensity for future addictions. Although alcohol represents the most likely addictive substance that the growing population of prenatal opioid exposed will encounter as they mature, no studies to date have examined the effect of prenatal opioid exposure on future sensitivity to alcohol reward. Using a recently developed mouse model of prenatal methadone exposure (PME), we investigated the rewarding properties of alcohol and alcohol consumption in male and female adolescent PME and prenatal saline exposed (PSE) control animals. Conditioned place preference to alcohol was disrupted in PME offspring in a sex-dependent manner with PME males exhibiting resistance to the rewarding properties of alcohol. Repeated injections of alcohol revealed enhanced sensitivity to the locomotor-stimulating effects of alcohol specific to PME females. PME males consumed significantly more alcohol over 4 weeks of alcohol access relative to PSE males and exhibited increased resistance to quinine-adulterated alcohol. Further, a novel machine learning model was developed to employ measured differences in alcohol consumption and drinking microstructure to reliably predict prenatal exposure. These findings indicate that PME alters the sensitivity to alcohol reward in adolescent mice in a sex-specific manner and suggests prenatal opioid exposure may induce persistent effects on reward neurocircuitry that can reprogram offspring behavioural response to alcohol later in life. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide.Experimental Approach: Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry.Key Results: We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential.Conclusion and Implications: These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process. [ABSTRACT FROM AUTHOR]

Background and Purpose: The μ-opioid receptor (μ receptor) is the primary target for opioid analgesics. The 7-transmembrane (TM) and 6TM μ receptor isoforms mediate inhibitory and excitatory cellular effects. Here, we developed compounds selective for 6TM- or 7TM-μ receptors to further our understanding of the pharmacodynamic properties of μ receptors.Experimental Approach: We performed virtual screening of the ZINC Drug Now library of compounds using in silico 7TM- and 6TM-μ receptor structural models and identified potential compounds that are selective for 6TM- and/or 7TM-μ receptors. Subsequently, we characterized the most promising candidate compounds in functional in vitro studies using Be2C neuroblastoma transfected cells, behavioural in vivo pain assays using various knockout mice and in ex vivo electrophysiology studies.Key Results: Our virtual screen identified 30 potential candidate compounds. Subsequent functional in vitro cellular assays shortlisted four compounds (#5, 10, 11 and 25) that demonstrated 6TM- or 7TM-μ receptor-dependent NO release. In in vivo pain assays these compounds also produced dose-dependent hyperalgesic responses. Studies using mice that lack specific opioid receptors further established the μ receptor-dependent nature of identified novel ligands. Ex vivo electrophysiological studies on spontaneous excitatory postsynaptic currents in isolated spinal cord slices also validated the hyperalgesic properties of the most potent 6TM- (#10) and 7TM-μ receptor (#5) ligands.Conclusion and Implications: Our novel compounds represent a new class of ligands for μ receptors and will serve as valuable research tools to facilitate the development of opioids with significant analgesic efficacy and fewer side-effects. [ABSTRACT FROM AUTHOR]

Our previous studies consistently showed that MDMA-induced locomotor hyperactivity is dramatically increased by coadministration of ethanol (EtOH) in rats, indicating possible potentiation of MDMA abuse liability. Thus, we aimed to identify the brain region(s) and neuropharmacological substrates involved in the pharmacodynamics of this potentiation. We first showed that potentiation of locomotor activity by the combination of ip administration of EtOH (1.5 g/kg) and MDMA (6.6 mg/kg) is delay sensitive and maximal when both drugs are injected simultaneously. Then, we used the 2-deoxyglucose quantitative autoradiography technique to assess the impact of EtOH, MDMA, or their combination on local cerebral metabolic rates for glucose (CMRglcs). We showed a specific metabolic activation in the ventral striatum (VS) under MDMA + EtOH versus MDMA or EtOH alone. We next tested if reversible (tetrodotoxin, TTX) or permanent (6-hydrodoxyopamine, 6-OHDA) lesion of the VS could affect locomotor response to MDMA and MDMA + EtOH. Finally, we blocked dopamine D1 or glutamate NMDA receptors in the VS and measured the effects of MDMA and MDMA + EtOH on locomotor activity. We showed that bilateral reversible inactivation (TTX) or permanent lesion (6-OHDA) of the VS prevented the potentiation by EtOH of MDMA-induced locomotor hyperactivity. Likewise, blockade of D1 or NMDA receptors in the VS also reduced the potentiation of MDMA locomotor activity by EtOH. These data indicate that dopamine D1 and glutamate NMDA receptor-driven mechanisms in the VS play a key role in the pharmacodynamics of EtOH-induced potentiation of the locomotor effects of MDMA. [ABSTRACT FROM AUTHOR]

One of the main challenges in treating opioid-use disorders is relapse during abstinence, triggered by re-exposure to drug-associated cues. Previous studies have demonstrated that drug-seeking in rats progressively increases over time during withdrawal (incubation of drug craving). Here, we used male rats and examined neural mechanisms underlying incubation of craving to oxycodone, a commonly abused prescription opioid, and we focused on orbitofrontal cortex (OFC), a brain region previously implicated in incubation of heroin craving. We first used neuronal activity marker Fos and measured neuronal activation in OFC (ventral and lateral OFC) associated with day-1 and day-15 relapse tests. Next, we determined the effect of pharmacological reversible inactivation of OFC on incubated oxycodone seeking on withdrawal day 15. Finally, we determined the effect of reversible inactivation of OFC on nonincubated oxycodone seeking on withdrawal day 1. We found that lever presses during relapse tests were higher on withdrawal day 15 than on withdrawal day 1 (incubation of oxycodone craving). Incubation of oxycodone craving is accompanied with a time-dependent increase of Fos protein expression in both ventral and lateral OFC. Lastly, OFC inactivation decreased oxycodone seeking on withdrawal day 15 but had no effect on withdrawal day 1. Together with the previous heroin study, results here show that OFC plays a critical role in incubation of opioid craving. [ABSTRACT FROM AUTHOR]

Background and Purpose: Efficacy of current antimalarial treatments is declining as a result of increasing antimalarial drug resistance, so new and potent antimalarial drugs are urgently needed. Azithromycin, an azalide antibiotic, was found useful in malaria therapy, but its efficacy in humans is low.Experimental Approach: Four compounds belonging to structurally different azalide classes were tested and their activities compared to azithromycin and chloroquine. in vitro evaluation included testing against sensitive and resistant Plasmodium falciparum, cytotoxicity against HepG2 cells, accumulation and retention in human erythrocytes, antibacterial activity, and mode of action studies (delayed death phenotype and haem polymerization). in vivo assessment enabled determination of pharmacokinetic profiles in mice, rats, dogs, and monkeys and in vivo efficacy in a humanized mouse model.Key Results: Novel fast-acting azalides were highly active in vitro against P. falciparum strains exhibiting various resistance patterns, including chloroquine-resistant strains. Excellent antimalarial activity was confirmed in a P. falciparum murine model by strong inhibition of haemozoin-containing trophozoites and quick clearance of parasites from the blood. Pharmacokinetic analysis revealed that compounds are metabolically stable and have moderate oral bioavailability, long half-lives, low clearance, and substantial exposures, with blood cells as the preferred compartment, especially infected erythrocytes. Fast anti-plasmodial action is achieved by the high accumulation into infected erythrocytes and interference with parasite haem polymerization, a mode of action different from slow-acting azithromycin.Conclusion and Implications: The hybrid derivatives described here represent excellent antimalarial drug candidates with the potential for clinical use in malaria therapy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown.Experimental Approach: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-β1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function.Key Results: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-β1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-β1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-β1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-β1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-β1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin.Conclusion and Implications: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-β1 inhibited osteogenic differentiation of DFCs via a TGF-β1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth. [ABSTRACT FROM AUTHOR]

The lateral septum (LS) is a limbic nucleus interconnected with several brain areas involved in the regulation of mood and reward. Vasopressin (AVP) is a neuropeptide that has been related to the effects of drugs of abuse, but its role in the addictive process is poorly understood. LS expresses a high density of AVP 1A receptors (V1A ). The aim of this work was to examine whether the modulation of LS AVP system affects the behavioral and neurochemical responses to amphetamine (AMPH) in male rats. Our results show that AMPH-induced conditioned place preference (CPP) produces a decrease in LS AVP content. Besides, we demonstrate that the microinjection of AVP in the LS impairs the expression of AMPH-induced CPP and that this effect is mediated by the activation of the V1A receptor in the LS. AVP microinjection in the LS elicited a decrease in neuronal activity in the nucleus accumbens (NAc) in animals subjected to AMPH conditioning. Finally, AVP microinjection in the LS decreased dopamine (DA) release in the NAc. Overall, our data demonstrate that intra-LS AVP diminishes the expression of AMPH conditioning behavior while decreasing neuronal activity and DA release in the NAc. Presumably, the effects of AVP in the LS produce an inhibition of GABAergic projections to the VTA, increasing local inhibitory tone in this nucleus, which in turn reduces the activity of DA projections to NAc. Thus, these results contribute to the knowledge about the role of AVP in LS in regulating the reward circuit and addictive like behaviors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Malaria is one of the deadliest diseases in the world. Novel chemotherapeutic agents are urgently required to combat the widespread Plasmodium resistance to frontline drugs. Here, we report the discovery of a novel benzonaphthyridine antimalarial, methnaridine, which was identified using a structural optimization strategy.Experimental Approach: An integrated pharmacological approach was used to evaluate the antimalarial profile of methnaridine. The pharmacokinetic properties of methnaridine were investigated along with the associated safety profile. Host immune response patterns were also analysed.Key Results: Methnaridine exhibited potent antimalarial activity against P. falciparum (3D7: IC50 = 0.0066 μM; Dd2: IC50 = 0.0056 μM). In P. berghei-infected mice, oral administration effectively suppressed parasitemia (ED50 = 0.52 mg·kg-1 ·day-1 ) and cured the established infection (CD50 = 10.13 mg·kg-1 ·day-1 ). These results are equivalent to or better than those of other antimalarial agents in clinical use. Notably, a four-dose oral regimen at a dosage of 25 mg·kg-1 achieved a complete cure of P. berghei infection in mice. Methnaridine exhibited a rapid parasiticidal profile (PCT99 = 36.0 h) and showed no cross-resistance to chloroquine. Pharmacokinetic studies revealed that methnaridine is readily absorbed, long-lasting and slowly cleared. The safety profile of methnaridine is also satisfactory (maximum tolerated dose = 1,125 mg·kg-1 ). In addition, following methnaridine treatment, infection-induced Th1 immune response was almost fully alleviated in mice.Conclusion and Implications: Methnaridine is an orally bioavailable, fast-acting and long-lasting agent with excellent antimalarial properties. Our study highlights the potential of methnaridine for clinical development as a promising antimalarial candidate. [ABSTRACT FROM AUTHOR]

Background and Purpose: Peptide P4 was described as a dimerization disruptor of trypanothione reductase (TryR), a homodimeric enzyme essential for survival of trypanosomatids. Determination of the true inhibitory constant (Ki ) for P4 was not achieved because reaction rates continuously decreased with time, even when substrate concentration was kept constant. The aim of this study was to find a suitable kinetic model that could allow characterization of the complex pattern of TryR inhibition caused by P4.Experimental Approach: After showing the slow-binding and pseudoirreversible activity of P4 against Leishmania infantum trypanothione reductase (Li-TryR), analysis of the curvatures of the reaction progress curves at different inhibitor concentrations allowed us to define the apparent inhibitory constants (Kiapp ) at five different substrate concentrations. Analysis of the changes in Kiapp values allowed precise definition of the type of inhibition.Key Results: Li-TryR inhibition by P4 requires two sequential steps that involve rapid generation of a reversible enzyme-inhibitor complex followed by a pseudoirreversible slow inactivation of the enzyme. Recovery of enzyme activity after inhibitor dissociation is barely detectable. P4 is a non-competitive pseudoirreversible inhibitor of Li- TryR that displays an overall inhibition constant (Ki* ) smaller than 0.02 μM.Conclusion and Implications: Li-TryRdimer disruption by peptide P4 is a pseudoirreversible time-dependent process which is non-competitive with respect to the oxidized trypanothione (TS2 ) substrate. Therefore, unlike reversible Li-TryR competitive inhibitors, enzyme inhibition by P4 is not affected by the TS2 accumulation observed during oxidant processes such as the oxidative burst in host macrophages. [ABSTRACT FROM AUTHOR]

Background and Purpose: The development of antinociceptive morphine tolerance is a clinically intractable problem. Earlier work has demonstrated the pivotal roles of PDGF and its receptor PDGFRβ in morphine tolerance. Here, we have investigated the role of spinal heat shock protein 27 (HSP27) in morphine tolerance and its relationship with PDGFRβ activation.Experimental Approach: Rats were treated with morphine for 9 days, and its anti-nociceptive effect against thermal pain was evaluated by a tail-flick latency test. Western blot, real-time PCR, immunofluorescent staining, and various antagonists, agonists, and siRNA lentiviral vectors elucidated the roles of HSP27, PDGFRβ, and related signalling pathways in morphine tolerance.Key Results: Chronic morphine administration increased expression and phosphorylation of HSP27 in the spinal cord. Down-regulating HSP27 attenuated the development of morphine tolerance. PDGFRβ antagonism inhibited HSP27 activation and attenuated and reversed morphine tolerance. PDGFRβ induction increased HSP27 expression and activation and partly decreased morphine analgesia. PDGFRβ inhibition reduced Akt and p38 MAPK activity in morphine tolerance. PI3K and p38 inhibitors reversed morphine tolerance and suppressed morphine-induced HSP27 phosphorylation.Conclusion and Implications: This study demonstrated for the first time that spinal HSP27 participates in PDGFRβ-mediated morphine tolerance via the PI3K/Akt and p38 MAPK signalling pathways. These findings suggest a potential clinical strategy for prolonging the antinociceptive effects of opioids during long-term pain control. [ABSTRACT FROM AUTHOR]

The nucleus accumbens (NAc) is a central component of the brain reward system. It has been known that most of the drugs of abuse such as opioids and cannabinoids affect the NAc. Although cannabinoids can modulate different stages of morphine encounter such as the reinstatement of morphine-induced conditioned place preference (CPP), there is no evidence for the NAc neurons' response to prove it. That is why the present study was designed. The procedure was as follows: The rats were entered to CPP by sc 5 mg/kg morphine in three consecutive days. During the extinction period or in the reinstatement phase, icv WIN 55,212-2 (10mM/5 μL dimethyl sulfoxide [DMSO] 10%) or AM251 (0.5mM/5-μL DMSO 10%) was infused in separate groups. Also, the NAc neurons' response to cannabinoid modulation in reinstatement to morphine was investigated by extracellular single unit recording. As a result, the cannabinoid in the reinstatement phase decreased the NAc neuronal activity. The CB1 receptor inhibition during the extinction period increased the NAc firing rate after ip 1 mg/kg morphine. Also, the inhibition of this receptor in the reinstatement phase increased the NAc neurons' firing rate. The inhibitory effect of cannabinoid on the NAc neuronal activity in the reinstatement has indicated the possible potency of cannabinoid to induce reinstatement of morphine-induced CPP alone and in the absence of a priming dose of morphine. Also, the different effects of the CB1 agonist during the extinction period in the reinstatement phase suggest different mechanisms underlying these two parts. [ABSTRACT FROM AUTHOR]

Background and Purpose: Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel.Experimental Approach: Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1.Key Results: Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin ˃ neomycin ˃ kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments.Conclusion and Implications: Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues. [ABSTRACT FROM AUTHOR]

Background and Purpose: Amphetamine (AMPH) use disorder is a serious health concern, but, surprisingly, little is known about the vulnerability to the moderate and compulsive use of this psychostimulant and its underlying mechanisms. Previous research showed that inherited serotonin transporter (SERT) down-regulation increases the motor response to cocaine, as well as moderate (as measured during daily 1-h self-administration sessions) and compulsive (as measured during daily 6-h self-administration sessions) intake of this psychostimulant. Here, we sought to investigate whether these findings generalize to AMPH and the underlying mechanisms in the nucleus accumbens.Experimental Approach: In serotonin transporter knockout (SERT-/- ) and wild-type control (SERT+/+ ) rats, we assessed the locomotor response to acute AMPH and i.v. AMPH self-administration under short access (ShA: 1-h daily sessions) and long access (LgA: 6-h daily sessions) conditions. Twenty-four hours after AMPH self-administration, we analysed the expression of glutamate system components in the nucleus accumbens shell and core.Key Results: We found that SERT-/- animals displayed an increased AMPH-induced locomotor response and increased AMPH self-administration under LgA but not ShA conditions. Further, we observed changes in the vesicular and glial glutamate transporters, NMDA and AMPA receptor subunits, and their respective postsynaptic scaffolding proteins as function of SERT genotype and AMPH exposure (baseline, ShA, and LgA), specifically in the nucleus accumbens shell.Conclusion and Implications: We demonstrate that SERT gene deletion increases the psychomotor and reinforcing effects of AMPH and that the latter is potentially mediated, at least in part, by homeostatic changes in the glutamatergic synapse of the nucleus accumbens shell and/or core. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pharmacotherapy of atrial fibrillation (AF), the most common cardiac arrhythmia, remains unsatisfactory due to low efficacy and safety concerns. New therapeutic strategies target atrial-predominant ion-channels and involve multichannel block (poly)therapy. As AF is characterized by rapid and irregular atrial activations, compounds displaying potent antiarrhythmic effects at fast and minimal effects at slow rates are desirable. We present a novel systems pharmacology framework to quantitatively evaluate synergistic anti-AF effects of combined block of multiple atrial-predominant K+ currents (ultra-rapid delayed rectifier K+ current, IKur , small conductance Ca2+ -activated K+ current, IKCa , K2P 3.1 2-pore-domain K+ current, IK2P ) in AF.Experimental Approach: We constructed experimentally calibrated populations of virtual atrial myocyte models in normal sinus rhythm and AF-remodelled conditions using two distinct, well-established atrial models. Sensitivity analyses on our atrial populations was used to investigate the rate dependence of action potential duration (APD) changes due to blocking IKur , IK2P or IKCa and interactions caused by blocking of these currents in modulating APD. Block was simulated in both single myocytes and one-dimensional tissue strands to confirm insights from the sensitivity analyses and examine anti-arrhythmic effects of multi-atrial-predominant K+ current block in single cells and coupled tissue.Key Results: In both virtual atrial myocytes and tissues, multiple atrial-predominant K+ -current block promoted favourable positive rate-dependent APD prolongation and displayed positive rate-dependent synergy, that is, increasing synergistic antiarrhythmic effects at fast pacing versus slow rates.Conclusion and Implications: Simultaneous block of multiple atrial-predominant K+ currents may be a valuable antiarrhythmic pharmacotherapeutic strategy for AF. [ABSTRACT FROM AUTHOR]

Background and Purpose: Protein tyrosine phosphatase (PTP) 1B (PTP1B) plays a critical role in the regulation of obesity, Type 2 diabetes mellitus and other metabolic diseases. However, drug candidates exhibiting PTP1B selectivity and oral bioavailability are currently lacking. Here, the enzyme inhibitory characteristics and pharmacological benefits of 3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol (BDB) were investigated in vitro and in vivo.Experimental Approach: Surface plasmon resonance (SPR) assay was performed to validate the direct binding of BDB to PTP1B, and Lineweaver-Burk analysis of the enzyme kinetics was used to characterise the inhibition by BDB. Both in vitro enzyme-inhibition assays and SPR experiments were also conducted to study the selectivity exhibited by BDB towards four other PTP-family proteins: TC-PTP, SHP-1, SHP-2, and LAR. C2C12 myotubes were used to evaluate cellular permeability to BDB. Effects of BDB on insulin signalling, hypoglycaemia and hypolipidaemia were investigated in diabetic BKS db mice, after oral gavage. The beneficial effects of BDB on pancreatic islets were examined based on insulin and/or glucagon staining.Key Results: BDB acted as a competitive inhibitor of PTP1B and demonstrated high selectivity for PTP1B among the tested PTP-family proteins. Moreover, BDB was cell-permeable and enhanced insulin signalling in C2C12 myotubes. Lastly, oral administration of BDB produced effective antidiabetic effects in spontaneously diabetic mice and markedly improved islet architecture, which was coupled with an increase in the ratio of β-cells to α-cells.Conclusion and Implications: BDB application offers a potentially practical pharmacological approach for treating Type 2 diabetes mellitus by selectively inhibiting PTP1B. [ABSTRACT FROM AUTHOR]

Background and Purpose: The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) is a multifunctional μ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-d-Phe], would demonstrate multifunctional μ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective μ-opioid receptor agonists, while preventing both drug- and stress-induced reinstatement of morphine-induced CPP.Experimental Approach: The opioid receptor agonist and antagonist activity of cyclo[Pro-Sar-Phe-d-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55°C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-d-Phe] to block morphine- and stress-induced reinstatement of extinguished CPP was determined.Key Results: cyclo[Pro-Sar-Phe-d-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mg·kg-1 i.p., mediated by μ- and κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-d-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-d-Phe] prevented stress- and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner.Conclusions and Implications: These data suggest that cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for both the treatment of pain with reduced sideeffects and preventing both drug- and stress-induced relapse in morphine-abstinent subjects. [ABSTRACT FROM AUTHOR]

Background and Purpose: There is an urgent but unmet need for mitigating radiation-induced intestinal toxicity while radio sensitising tumours for abdominal radiotherapy. We aimed to investigate the effects of metformin on radiation-induced intestinal toxicity and radiosensitivity of colorectal tumours.Experimental Approach: Acute and chronic histological injuries of the intestine from mice were used to assess radioprotection and IEC-6 cell line was used to investigate the mechanisms in vitro. The fractionated abdominal radiation model of HCT116 and HT29 tumour grafts was used to determine the effects on colorectal cancer.Key Results: Metformin alleviated radiation-induced acute and chronic intestinal toxicity by optimising mitophagy which was AMPK-dependent. In addition, our data indicated that metformin increased the radiosensitivity of colorectal tumours with P53 mutation both in vitro and in vivo.Conclusion and Implications: Metformin may be a radiotherapy adjuvant agent for colorectal cancers especially those carrying P53 mutation. Our findings provide a new strategy for further precise clinical trials for metformin on radiotherapy. [ABSTRACT FROM AUTHOR]

Background and Purpose: l-DOPA-induced dyskinesia (LID) is considered a major complication in the treatment of Parkinson's disease (PD). Buspirone (5-HT1A partial agonist) have shown promising results in the treatment of PD and LID, however no 5-HT-based treatment has been approved in PD. The present study was aimed to investigate how the substantia nigra pars reticulata (SNr) is affected by buspirone and whether it is a good target to study 5-HT antidyskinetic treatments.Experimental Approach: Buspirone was studied using in vivo single-unit, electrocorticogram, local field potential recordings along with microdialysis and immunohistochemistry in naïve/sham, 6-hydroxydopamine (6-OHDA)-lesioned or 6-OHDA-lesioned and l-DOPA-treated (6-OHDA/l-DOPA) rats.Key Results: Local buspirone inhibited SNr neuron activity in all groups. However, systemic buspirone reduced burst activity in 6-OHDA-lesioned rats (with or without l-DOPA treatment), whereas 8-OH-DPAT, a full 5-HT1A agonist induced larger inhibitory effects in sham animals. Neither buspirone nor 8-OH-DPAT markedly modified the low-frequency oscillatory activity in the SNr or synchronization within the SNr with the cortex. In addition, local perfusion of buspirone increased GABA and glutamate release in the SNr of naïve and 6-OHDA-lesioned rats but no effect in 6-OHDA/l-DOPA rats. In the 6-OHDA/l-DOPA group, increased 5-HT transporter and decreased 5-HT1A receptor expression was found.Conclusions and Implications: The effects of buspirone in SNr are influenced by dopamine loss and l-DOPA treatment. The present results suggest that the regulation of burst activity of the SNr induced by DA loss may be a good target to test new drugs for the treatment of PD and LID. [ABSTRACT FROM AUTHOR]

Background and Purpose: Anxiety disorder is a common mental health disorder. However, there are few safe and fast-acting anxiolytic drugs available that can treat anxiety disorder. We previously demonstrated that the interaction of neuronal NOS (nNOS) with its carboxy-terminal PDZ ligand (CAPON) is involved in regulating anxiety-related behaviours. Here, we further investigated the anxiolytic effects of nNOS-CAPON disruptors in chronic stress-induced anxiety in animals.Experimental Approach: Mice were intravenously treated with nNOS-CAPON disruptors, ZLc-002 or Tat-CAPON12C, at the last week of chronic mild stress (CMS) exposure. We also infused corticosterone (CORT) into the hippocampus of mice to model anxiety behaviours and also delivered ZLc-002 or Tat-CAPON12C on the last week of chronic CORT treatment via pre-implanted cannula. Anxiety-related behaviours were examined using elevated plus maze, open field, novelty-suppressed feeding and light-dark (LD) tests. The level of nNOS-CAPON interaction was determined by co-immunoprecipitation (CO-IP) and proximity ligation assay (PLA). The neural mechanisms underlying the behavioural effects of nNOS-CAPON uncoupling in anxiety animal models were assessed by western blot, immunofluorescence and Golgi-Cox staining.Key Results: ZLc-002 and Tat-CAPON12C reversed CMS- or CORT-induced anxiety-related behaviours. ZLc-002 and Tat-CAPON12C increased synaptogenesis along with improved dendritic remodelling in CMS mice or CORT-treated cultured neurons. Meanwhile, blocking nNOS-CAPON interaction significantly activated the cAMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) pathway, which is associated with synaptic plasticity.Conclusion and Implications: Collectively, these results provide evidence for the anxiolytic effects of nNOS-CAPON uncouplers and their underlying mechanisms in anxiety disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: While triptans are used to treat migraine, there is evidence that they also reduce inflammation-induced pain at the spinal level. The cellular mechanisms underlying this spinal enhancement are unknown. We examined whether inflammation alters sumatriptan modulation of synaptic transmission in the rat spinal dorsal horn.Experimental Approach: Three to four days following intraplantar injection of complete Freund's adjuvant (CFA) or saline, whole cell recordings of evoked glutamatergic EPSCs were made from lumbar lamina I-II dorsal horn neurons in rat spinal slices KEY RESULTS: In 2- to 3-week-old animals, sumatriptan reduced the amplitude of evoked EPSCs and this was greater in slices from CFA, compared to saline-injected rats. In CFA-injected animals, sumatriptan increased the paired pulse ratio of evoked EPSCs and reduced the rate of spontaneous miniature EPSCs. The 5-HT1B and 5-HT1D agonists CP9 3129 and PNU109291 both inhibited evoked EPSCs in CFA but not saline-injected rats. By contrast, the 5-HT1A agonist R(+)-8-OH-DPAT inhibited evoked EPSCs in saline but not CFA-injected rats. In CFA-injected rats, the sumatriptan-induced inhibition of evoked EPSCs was reduced by the 5-HT1B and 5-HT1D antagonists NAS181 and BRL-15572. Intriguingly, the difference in sumatriptan inhibition between CFA and saline-injected animals was only observed in animals less than 4 weeks old.Conclusion and Implications: These findings indicate that inflammation induces a developmentally regulated 5-HT1B/1D presynaptic inhibition of excitatory transmission into the rat superficial dorsal horn. Thus, triptans could potentially act as spinal analgesic agents for inflammatory pain in the juvenile setting. [ABSTRACT FROM AUTHOR]

Background and Purpose: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.Experimental Approach: We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials.Key Results: Studying μ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (VM ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (Kir 3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced μ receptor activation was also observed at the level of Kir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met-enkephalin) or only moderately ([D-Ala2 , N-Me-Phe4 , Gly5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors.Conclusion and Implications: Our results indicate a strong ligand-dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hispidulin is a flavonoid isolated from Clerodendrum inerme that was found to inhibit intractable motor tics. Previously, we found that hispidulin attenuates hyperlocomotion and the disrupted prepulse inhibition induced by methamphetamine and N-methyl-d-aspartate (NMDA) receptor antagonists, two phenotypes of schizophrenia resembling positive symptoms. Hispidulin can inhibit COMT, a dopamine-metabolizing enzyme in the prefrontal cortex (PFC) that is important for social interaction. Here, we investigated whether hispidulin would affect social withdrawal, one of the negative symptoms of schizophrenia.Experimental Approach: We examined whether acute administration of hispidulin would attenuate social withdrawal in two mice models, juvenile isolated disrupted-in-schizophrenia-1 mutant (mutDISC1) mice and chronic phencyclidine (PCP)-treated naïve mice.Key Results: In chronic PCP-treated mice, hispidulin (10 mg·kg-1 , i.p.) attenuated social withdrawal similar to that observed with dopamine D1 receptor antagonist (SCH-23390, 0.02 mg·kg-1 , i.p.) and was mimicked by the selective COMT inhibitor, OR-486 (10 mg·kg-1 , i.p.). Hispidulin increased extracellular dopamine levels in the PFC of chronic PCP-treated mice. In isolated mutDISC1 mice, hispidulin also reversed social withdrawal. In both models, intra-PFC microinjection of a D1 agonist (SKF-81297: 10 nmol/mouse/bilateral) reversed the impairment of Ser897 phosphorylation at the GluN1 subunit of NMDA receptors, suggesting the association between GluN1 Ser897 -phosphorylation and D1 activation in the PFC exits in both models.Conclusions and Implications: Hispidulin attenuated social withdrawal by activating D1 receptors indirectly through elevated dopamine levels in the PFC by COMT inhibition. This nature of hispidulin suggests that it a potential novel therapeutic candidate for the treatment of negative symptoms in schizophrenia. [ABSTRACT FROM AUTHOR]

Background: Percutaneous coronary intervention (PCI) is effective in treating patients with acute coronary syndrome (ACS) but is associated with some serious complications. Nicorandil is an anti-anginal agent acting to improve microvascular circulation and to increase coronary blood flow. The objective of this article is to evaluate the effects of intracoronary injection followed with continuous intravenous injection of nicorandil on ECG parameters in patients with unstable angina pectoris (UA) undergoing PCI.Methods: A single-center, self-controlled clinical trial was conducted at the Second Hospital of Tianjin Medical University between January 2019 and April 2019. Sixty-three consecutive patients with UA who received coronary angiography and selective PCI were enrolled. ECG was recorded and analyzed before and 24 hr after nicorandil infusion.Results: Patients were divided into three groups: control group (n = 23, aged 63.43 ± 12.55 years), short-term, and prolonged use with nicorandil group (n = 20 and 20, aged 66.45 ± 8.06 years and 65.80 ± 9.49 years, respectively). Clinical characteristics and ECG parameters were similar before PCI among three groups (p > .05). In nicorandil treatment groups, intervals of QTd and Tp-e in patients post-PCI were significantly shorter than that in control and pre-PCI (p < .05).Conclusions: Nicorandil infusion reduces QTd and Tp-e interval in patients with UA. Further studies will be needed to determine whether these electrophysiological changes are associated with a reduction of ventricular arrhythmias and improved outcomes. [ABSTRACT FROM AUTHOR]

Background and Purpose: GPCRs can signal through both G proteins and β-arrestin2. For the μ-opioid receptor, early experimental evidence from a single study suggested that G protein signalling mediates analgesia, whereas β-arrestin2 signalling mediates respiratory depression and constipation. Consequently, for more than a decade, much research effort has been focused on developing biased μ-opioid agonists that preferentially target G protein signalling over β-arrestin signalling, as it was believed that such drugs would be analgesics devoid of respiratory depressant activity. However, the prototypical compounds that have been developed based on this concept have so far failed in clinical and preclinical development.Experimental Approach: The present study was set up to re-examine opioid-induced respiratory depression in β-arrestin2 knockout mice. To this end, a consortium was formed consisting of three different laboratories located in different countries to evaluate independently opioid-induced respiratory depression.Key Results: Our consensus results unequivocally demonstrate that the prototypical μ-opioid agonist morphine (3.75-100 mg·kg-1 s.c. or 3-30 mg·kg-1 i.p.) as well as the potent opioid fentanyl (0.05-0.35 mg·kg-1 s.c.) do indeed induce respiratory depression and constipation in β-arrestin2 knockout mice in a dose-dependent manner indistinguishable from that observed in wild-type mice.Conclusion and Implications: Our findings do not support the original suggestion that β-arrestin2 signalling plays a key role in opioid-induced respiratory depression and call into question the concept of developing G protein-biased μ-opioid receptor agonists as a strategy for the development of safer opioid analgesic drugs. [ABSTRACT FROM AUTHOR]

The guidance cue receptor DCC controls mesocortical dopamine development in adolescence. Repeated exposure to an amphetamine regimen of 4 mg/kg during early adolescence induces, in male mice, downregulation of DCC expression in dopamine neurons by recruiting the Dcc microRNA repressor, microRNA-218 (miR-218). This adolescent amphetamine regimen also disrupts mesocortical dopamine connectivity and behavioral control in adulthood. Whether low doses of amphetamine in adolescence induce similar molecular and developmental effects needs to be established. Here, we quantified plasma amphetamine concentrations in early adolescent mice following a 4 or 0.5 mg/kg dose and found peak levels corresponding to those seen in humans following recreational and therapeutic settings, respectively. In contrast to the high doses, the low amphetamine regimen does not alter Dcc mRNA or miR-218 expression; instead, it upregulates DCC protein levels. Furthermore, high, but not low, drug doses downregulate the expression of the DCC receptor ligand, Netrin-1, in the nucleus accumbens and prefrontal cortex. Exposure to the low-dose regimen did not alter the expanse of mesocortical dopamine axons or their number/density of presynaptic sites in adulthood. Strikingly, adolescent exposure to the low-dose drug regimen does not impair behavioral inhibition in adulthood; instead, it induces an overall increase in performance in a go/no-go task. These results show that developmental consequences of exposure to therapeutic- versus abused-like doses of amphetamine in adolescence have dissimilar molecular signatures and opposite behavioral effects. These findings have important clinical relevance since amphetamines are widely used for therapeutic purposes in youth. [ABSTRACT FROM AUTHOR]

Background and Purpose: Intestinal mucositis refers to mucosal damage caused by cancer treatment, and irinotecan is one of the agents most associated with this condition. Focusing on the development of alternatives to prevent this important adverse effect, we evaluated the activity of the flavonoid luteolin, which has never been tested for this purpose despite its biological potential.Experimental Approach: The effects of luteolin were examined on irinotecan-induced intestinal mucositis in mice. Clinical signs were evaluated. Moreover, histological, oxidative, and inflammatory parameters were analysed, as well as the possible interference of luteolin in the anti-tumour activity of irinotecan.Key Results: Luteolin (30 mg·kg-1 ; p.o. or i.p.) prevented irinotecan-induced intestinal damage by reducing weight loss and diarrhoea score and attenuating the shortening of the duodenum and colon. Histological analysis confirmed that luteolin (p.o.) prevented villous shortening, vacuolization, and apoptosis of cells and preserved mucin production in the duodenum and colon. Moreover, luteolin treatment mitigated irinotecan-induced oxidative stress, by reducing the levels of ROS and LOOH and augmenting endogenous antioxidants, and inflammation by decreasing MPO enzymic activity, TNF, IL-1β, and IL-6 levels and increasing IL-4 and IL-10. Disruption of the tight junctions ZO-1 and occludin was also prevented by luteolin treatment. Importantly, luteolin did not interfere with the anti-tumour activity of irinotecan.Conclusion and Implications: Luteolin prevents intestinal mucositis induced by irinotecan and therefore could be a potential adjunct in anti-tumour therapy to control this adverse effect, increasing treatment adherence and consequently the chances of cancer remission. [ABSTRACT FROM AUTHOR]

Background and Purpose: Breast cancer is the leading cause of death in women worldwide, with resistance to current therapeutic strategies, including tamoxifen, causing major clinical challenges and leading to more aggressive and metastatic disease. To address this, novel strategies that can inhibit the mechanisms responsible for tamoxifen resistance need to be assessed.Experimental Approach: We examined the effect of the novel, clinically-trialled, thiosemicarbazone anti-cancer agent, DpC, and its potential as a combination therapy with the clinically used estrogen receptor (ER) antagonist, tamoxifen, using both tamoxifen-resistant and -sensitive, human breast cancer cells (MDA-MB-453, MDA-MB-231 and MCF-7) in 2D and 3D cell-culture. Synergy was assessed using the Chou-Talalay method. The molecular and anti-proliferative effects of these agents and their combination was examined via Western blot, immunofluorescence and colony formation assays.Key Results: Combinations of tamoxifen with DpC were highly synergistic, leading to potent inhibition of cell proliferation, colony formation, and ER-α transcriptional activity. The combination also more efficiently reduced major molecular drivers of proliferation of tamoxifen-resistant cells, including c-Myc, cyclin D1, and p-AKT, while up-regulating the cell cycle inhibitor, p27, and inhibiting oncogenic phosphorylation of ER-α at Ser167. Assessing these effects using 3D cell culture further confirmed the greater effects of DpC combined with tamoxifen in reducing ER-α expression, and that of the proliferation marker, Ki-67, in both tamoxifen-sensitive and -resistant MCF-7 spheroids.Conclusions and Implications: These studies demonstrate that the synergistic combination of DpC with tamoxifen could be a promising new therapeutic strategy to overcome tamoxifen resistance in ER-positive breast cancer. [ABSTRACT FROM AUTHOR]

Background and Purpose: Blocking the voltage-gated proton channel HV 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few HV 1 channel antagonists have been reported. Here, we have identified a novel HV 1 channel antagonist from scorpion venom and have elucidated its action mechanism.Experimental Approach: HV 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Site-directed mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-HV 1 interaction was modelled by molecular dynamics simulations.Key Results: The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited HV 1 currents. One AGAP mutant has reduced NaV channel activity but intact HV 1 activity (AGAP/W38F). AGAP/W38F inhibited HV 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited HV 1 currents with less pH dependence than Zn2+ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn2+ in HV 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in HV 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn2+ . Experimental data and MD simulations suggested that AGAP/W38F inhibited HV 1 channel using a Zn2+ -like long-range conformational coupling mechanism.Conclusion and Implications: Our results suggest that the Zn2+ binding pocket in HV 1 channel might be a hotspot for modulators and valuable for designing HV 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study HV 1 channel and a lead compound for drug development. [ABSTRACT FROM AUTHOR]

Background and Purpose: The midbrain periaqueductal grey (PAG) plays a central role in modulating pain through a descending pathway that projects indirectly to the spinal cord via the rostroventral medial medulla (RVM). While opioids are potent analgesics that target the PAG, their cellular actions on descending projection neurons are unclear.Experimental Approach: Patch clamp recordings in voltage- and current-clamp mode were made from acutely prepared PAG slices from animals that received retrograde tracer injections into the RVM.Key Results: The μ-agonist DAMGO reduced GABAergic evoked inhibitory postsynaptic currents (IPSCs) in retro-labelled, RVM-projecting neurons to a greater extent than in unlabelled neurons. The κ-opioid agonist U69593 reduced evoked IPSCs to a similar extent in both neuronal groups, while the δ-opioid agonist deltorphin-II was without effect. DAMGO and U69593 both produced a reduction in the rate, but not amplitude of spontaneous miniature IPSCs and asynchronous evoked IPSCs in retro-labelled neurons. DAMGO and U69593 also suppressed glutamatergic EPSCs in retro-labelled and unlabelled neurons. The DAMGO inhibition of evoked EPSCs, however, was less than that for evoked IPSCs in retro-labelled, but not unlabelled neurons. In current clamp, DAMGO produced a depolarizing increase in evoked postsynaptic potentials in retro-labelled neurons, but directly inhibited unlabelled neurons.Conclusion and Implications: These findings suggest that μ-opioids activate the descending analgesic pathway from the midbrain PAG by a combination of presynaptic disinhibition of RVM-projecting neurons and postsynaptic inhibition of presumptive interneurons. [ABSTRACT FROM AUTHOR]

Background and Purpose: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects.Experimental Approach: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico.Key Results: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor.Conclusions and Implications: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway. [ABSTRACT FROM AUTHOR]

Shati/Nat8l is a novel N-acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex (PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self-administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno-associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC-Shati/Nat8l). Interestingly, the METH-induced conditioned place preference (CPP) was attenuated in the mPFC-Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV-GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC-NAc top-down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH-induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH-induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l-mPFC overexpression did not alter METH-induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward-seeking behaviour but not the psychomotor activity of METH. [ABSTRACT FROM AUTHOR]

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder. [ABSTRACT FROM AUTHOR]

Understanding genetic factors that contribute to cannabis use disorder (CUD) is important, but to date, findings have been equivocal. Single-nucleotide polymorphisms (SNPs) in the cannabinoid receptor 1 gene (CNR1; rs1049353 and rs806378) and fatty acid amide hydrolase (FAAH) gene (rs324420) have been implicated in CUD. Their relationship to addiction endophenotypes such as cannabis-related state satiety, the salience of appetitive cues, and craving after acute cannabinoid administration has not been investigated. Forty-eight cannabis users participated in a double-blind, placebo-controlled, four-way crossover experiment where they were administered treatments in a randomized order via vaporization: placebo, Δ9 -tetrahydrocannabinol (THC) (8 mg), THC + cannabidiol (THC + CBD) (8 + 16 mg), and CBD (16 mg). Cannabis-related state satiety, appetitive cue salience (cannabis and food), and cannabis craving were assessed each day. Participants were genotyped for rs1049353, rs806378, and rs324420. Results indicated that CNR1 rs1049353 GG carriers showed increased state satiety after THC/THC + CBD administration in comparison with placebo and reduced the salience of appetitive cues after THC in comparison with CBD administration; A carriers did not vary on either of these measures indicative of a vulnerability to CUD. CNR1 rs806378 CC carriers showed greater salience to appetitive cues in comparison with T carriers, but there was no evidence for changes in state satiety. FAAH rs324420 A carriers showed greater bias to appetitive cues after THC, in comparison with CC carriers. FAAH CC carriers showed reduced bias after THC in comparison with CBD. No SNPs modulated craving. These findings identify candidate neurocognitive mechanisms through which endocannabinoid system genetics may influence vulnerability to CUD. [ABSTRACT FROM AUTHOR]

Background and Purpose: We previously demonstrated that paracetamol has to be metabolised in the brain by fatty acid amide hydrolase enzyme into AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) to activate CB1 receptors and TRPV1 channels, which mediate its analgesic effect. However, the brain mechanisms supporting paracetamol-induced analgesia remain unknown.Experimental Approach: The effects of paracetamol on brain function in Sprague-Dawley rats were determined by functional MRI. Levels of neurotransmitters in the periaqueductal grey (PAG) were measured using in vivo 1 H-NMR and microdialysis. Analgesic effects of paracetamol were assessed by behavioural tests and challenged with different inhibitors, administered systemically or microinjected in the PAG.Key Results: Paracetamol decreased the connectivity of major brain structures involved in pain processing (insula, somatosensory cortex, amygdala, hypothalamus, and the PAG). This effect was particularly prominent in the PAG, where paracetamol, after conversion to AM404, (a) modulated neuronal activity and functional connectivity, (b) promoted GABA and glutamate release, and (c) activated a TRPV1 channel-mGlu5 receptor-PLC-DAGL-CB1 receptor signalling cascade to exert its analgesic effects.Conclusions and Implications: The elucidation of the mechanism of action of paracetamol as an analgesic paves the way for pharmacological innovations to improve the pharmacopoeia of analgesic agents. [ABSTRACT FROM AUTHOR]

Background and Purpose: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs).Experimental Approach: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology.Key Results: Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4β2, α7, and α3β2 nAChRs which it blocked with IC50 values of 1.8, 7, and 12 μM respectively.Conclusions and Implications: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists. [ABSTRACT FROM AUTHOR]

Background: Platelet engraftment following cord blood (CB) transplantation remains a significant hurdle to this day. The uncontrolled growth of ice, a process referred to as ice recrystallization, is one of several mechanisms that lead to cell loss and decreased potency during freezing and thawing. We hypothesized that reducing cell damage induced by ice recrystallization in CB units (CBUs) would reduce losses of stem and progenitor cells and therefore improve engraftment. We previously demonstrated that the ice recrystallization inhibitor (IRI) N-(2-fluorophenyl)-D-gluconamide (IRI 2) increases the postthaw recovery of CB progenitors. Herein, we set out to ascertain whether IRI 2 can enhance platelet and bone marrow engraftment activity of hematopoietic stem cells (HSCs) in cryopreserved CBUs using a serial transplantation model.Study Design and Methods: CBUs were processed following standard volume/red blood cell reduction procedure and portions frozen with dimethyl sulfoxide (DMSO) supplemented or not with IRI 2. Thawed CB samples were serially transplanted into immunodeficient mice.Results: Our results show that supplementation of DMSO with IRI 2 had several beneficial effects. Specifically, higher levels of human platelets were observed in the peripheral blood (p < 0.05; n = 4) upon transplant of CBUs preserved with the IRIs. In addition, human BM chimerism and the number of human CFU progenitors in the bone marrow were superior in IRI 2 recipients compared to DMSO recipients. Moreover, IRI 2 had no negative impact on the multilineage differentiation and self-renewal activities of HSCs.Discussion: Taken together, these results demonstrate that supplementation of a hematopoietic graft with IRI can improve the postthaw engraftment activities of HSCs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets.Experimental Approach: Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kg-1 ) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6-day period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocin-induced diabetic mouse model was used to study the effect of EPO-pretreatment on the endocrine function of the grafts.Key Results: EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPO-pretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPO-pretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation.Conclusion and Implications: These findings demonstrate that, particularly, short-term EPO-pretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events. [ABSTRACT FROM AUTHOR]

Background and Purpose: Gamma oscillations are fast rhythmic fluctuations of neuronal network activity ranging from 30 to 90 Hz that establish a precise temporal background for cognitive processes such as perception, sensory processing, learning, and memory. Alterations of gamma oscillations have been observed in schizophrenia and are suggested to play crucial roles in the generation of positive, negative, and cognitive symptoms of the disease.Experimental Approach: In this study, we investigated the effects of the novel antipsychotic cariprazine, a D3 -preferring dopamine D3 /D2 receptor partial agonist, on cholinergically induced gamma oscillations in rat hippocampal slices from treatment-naïve and MK-801-treated rats, a model of acute first-episode schizophrenia.Key Results: The D3 receptor-preferring agonist pramipexole effectively decreased the power of gamma oscillations, while the D3 receptor antagonist SB-277011 had no effect. In treatment-naïve animals, cariprazine did not modulate strong gamma oscillations but slightly improved the periodicity of non-saturated gamma activity. Cariprazine showed a clear partial agonistic profile at D3 receptors at the network level by potentiating the inhibitory effects when the D3 receptor tone was low and antagonizing the effects when the tone was high. In hippocampal slices of MK-801-treated rats, cariprazine allowed stabilization of the aberrant increase in gamma oscillation power and potentiated resynchronization of the oscillations.Conclusion and Implications: Data from this study indicate that cariprazine stabilizes pathological hippocampal gamma oscillations, presumably by its partial agonistic profile. The results demonstrate in vitro gamma oscillations as predictive biomarkers to study the effects of antipsychotics preclinically at the network level. [ABSTRACT FROM AUTHOR]

Background and Purpose: The worldwide prevalence of herpes simplex virus (HSV) and shortage of efficient therapeutic strategies to counteract it are global concerns. In terms of treatment, the widely utilized anti-HSV drugs such as acyclovir have serious limitations, for example, drug resistance and side effects. Here, we have identified the guanidine-modified (E,E)-4,6-bis(styryl)-pyrimidine (BS-pyrimidine) derivative compound 5d as an inhibitor of HSV and further elucidated the anti-HSV mechanisms of compound 5d both in vitro and in vivo.Experimental Approach: Cytopathic effect inhibition assay, plaque assay, and immunofluorescence assay were used to evaluate the anti-HSV effects of compound 5d in vitro. Membrane fusion assays, immunofluorescence assays, Western blotting assays, and pull-down assays were used to explore the anti-HSV mechanisms of compound 5d. HSV-1-infected mice, combined with haematoxylin-eosin staining and quantitative RT-PCR, were used to study the anti-HSV effects of compound 5d in vivo.Key Results: The guanidine-modified compound 5d rather than the un-modified compound 3a effectively inhibited both HSV-1 and HSV-2 multiplication in different cell lines, more effectively than acyclovir. Compound 5d may block virus binding and post-binding processes such as membrane fusion, by targeting virus gB protein. In addition, compound 5d may also down-regulate the cellular PI3K/Akt signalling pathway to interfere with HSV replication. Treatment with compound 5d also markedly improved survival and decreased viral titres in HSV-infected mice.Conclusions and Implications: Thus, the guanidine-modified BS-pyrimidine derivatives have the potential to be developed into novel anti-HSV agents targeting both virus gB protein and cellular PI3K/Akt signalling pathways. [ABSTRACT FROM AUTHOR]

Background and Purpose: Memantine and marijuana smoking have been found to inhibit tremor in parkinsonian patients, although the observed effects were relatively weak. The tremorolytic effects of combinations of memantine and cannabinoids have not been studied. Here, we have evaluated the anti-tremor activity of memantine, Δ9 -tetrahydrocannabinol (THC) given alone and of their combination. The involvement of some neuroanatomical structures in the effects of the combination was evaluated.Experimental Approach: Haloperidol-induced tremulous jaw movements (TJMs) in rats were used as a model of parkinsonian-like tremor. To evaluate the role of central receptor systems in the drug effects, receptor ligands were administered locally into certain brain areas.Key Results: Memantine and THC alone were without effect, although co-administration of these drugs decreased the number of haloperidol-induced jaw movements. The anti-tremor activity of the combination was antagonized (a) by injections of l-glutamate into the dorsal striatum, entopeduncular nucleus, substantia nigra pars reticulata, globus pallidus, and supratrigeminal and trigeminal motor nuclei but not into the subthalamic and cuneiform nuclei; (b) by injections of CGS 21680 into the ventrolateral striatum; and (c) by injections of bicuculline into the rostral part of the parvicellular reticular nucleus.Conclusions and Implications: Memantine and THC supra-additively inhibit haloperidol-induced TJMs, suggesting that co-administration of these drugs might be a new approach to the treatment of tremor. Our results identified brain areas influencing parkinsonian-like tremor in rats and can help advance the development of novel treatments for repetitive involuntary movements. [ABSTRACT FROM AUTHOR]

Background and Purpose: Damage to the vasculature caused by chronic iron-overload in both humans and animal models, is characterized by endothelial dysfunction and reduced compliance. In vitro, blockade of the angiotensin II AT1 receptors reversed functional vascular changes induced by chronic iron-overload. In this study, the effect of chronic AT1 receptor blockade on aorta stiffening was assessed in iron-overloaded rats.Experimental Approach: Male Wistar rats were treated for 15 days with saline as control group, iron dextran 200 mg·kg-1 ·day-1 , 5 days a week (iron-overload group), losartan (20 mg·kg-1 ·day-1 in drinking water), and iron dextran plus losartan. Mechanical properties of the aorta were assessed in vivo. In vitro, aortic geometry and biochemical composition were assessed with morphometric and histological methods.Key Results: Thoracoabdominal aortic pulse wave velocity (PWV) increased significantly, indicating a decrease in aortic compliance. Co-treatment with losartan prevented changes on PWV, β-index, and elastic modulus in iron-overloaded rats. This iron-related increase in PWV was not related to changes in aortic geometry and wall stress. but to increased elastic modulus/wall stress ratio, suggesting that a change in the composition of the wall was responsible for the stiffness. Losartan treatment also ameliorated the increase in aorta collagen content of the iron-overload group, without affecting circulating iron or vascular deposits.Conclusions and Implications: Losartan prevented the structural and functional indices of aortic stiffness in iron-overloaded rats, implying that inhibition of the renin-angiotensin system would limit the vascular remodelling in chronic iron-overload. [ABSTRACT FROM AUTHOR]

Background and Purpose: FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 receptors (IP3 R) and ryanodine receptors (RyR) to alter Ca2+ signalling in endothelial cells.Experimental Approach: We investigated the effects of FK506 and rapamycin on Ca2+ release via IP3 R and RyR in hundreds of endothelial cells, using the indicator Cal-520, in intact mesenteric arteries from male Sprague-Dawley rats. IP3 Rs were activated by acetylcholine or localised photo-uncaging of IP3 , and RyR by caffeine.Key Results: While FKBPs were present, FKBP modulation with rapamycin did not alter IP3 -evoked Ca2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP3 -evoked Ca2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP3 -evoked Ca2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP3 -evoked Ca2+ release. These findings suggest that IP3 -evoked Ca2+ release is not modulated by FKBP, but by FK506-mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca2+ signalling suggesting that RyR is not functional in native endothelium.Conclusion and Implications: The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca2+ release and altered FKBP binding to IP3 and RyR. [ABSTRACT FROM AUTHOR]

Background and Purpose: Bleeding is one of the most critical adverse effects of antithrombotic drugs, and many efforts have been made to discover novel antiplatelet agents without bleeding complications. Shear stress-induced platelet aggregation (SIPA), where the interaction of von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib constitutes the initial step, is a promising target to overcome bleeding problems, as SIPA occurs only in pathological conditions. Here, we describe SP-8008, a novel modulator of vWF-GP Ib interactions and evaluated its antiplatelet/antithrombotic effects.Experimental Approach: Newly synthesized compounds were screened for antiplatelet effects in vitro, using human platelets exposed to high shear stress. Aggregation, intracellular calcium level, granule secretion, and integrin activation were assessed. Molecular modelling using virtual docking and flow cytometry were used to evaluate effects on vWF-GP Ib interactions. Antithrombotic effects in vivo were determined in rats, using arterial thrombosis and shear stress-specific thrombosis. Transection tail bleeding time was used to evaluate adverse effects.Key Results: SP-8008 was a potent inhibitor of SIPA, with IC50 of 1.44 ± 0.09 μM. SP-8008 effectively and broadly blocked shear stress-induced platelet activation events, without any significant toxicity. Importantly, SP-8008 was highly selective against SIPA, effectively interfering with vWF-GP Ib engagement. Most importantly, SP-8008 exerted significant antithrombotic effects in vivo in both shear stress-specific and arterial thrombosis, without prolonging bleeding time.Conclusions and Implications: Our results demonstrated that SP-8008 can be a novel selective antiplatelet agent with improved safety profile. [ABSTRACT FROM AUTHOR]