Short-Term Acyl-CoA:Cholesterol Acyltransferase Inhibition, Combined with Apoprotein A1 Overexpression, Promotes Atherosclerosis Inflammation Resolution in Mice.

Acyl-CoA:cholesterol acyltransferase (ACAT) mediates cellular cholesterol esterification. In atherosclerotic plaque macrophages, ACAT promotes cholesteryl ester accumulation, resulting in foam cell formation and atherosclerosis progression. Its complete inactivation in mice, however, showed toxic effects because of an excess of free cholesterol (FC) in macrophages, which can cause endoplasmic reticulum stress, cholesterol crystal formation, and inflammasome activation. Our previous studies showed that long-term partial ACAT inhibition, achieved by dietary supplementation with Fujirebio F1394, delays atherosclerosis progression in apoprotein E-deficient (Apoe -/-) mice by reducing plaque foam cell formation without inflammatory or toxic effects. Here, we determined whether short-term partial inhibition of ACAT, in combination with an enhanced systemic FC acceptor capacity, has synergistic benefits. Thus, we crossbred Apoe -/- with human apoprotein A1-transgenic (APOA1 tg/tg) mice, which have elevated cholesterol-effluxing high-density lipoprotein particles, and subjected Apoe -/- and APOA1 tg/tg/Apoe -/- mice to an atherogenic diet to develop advanced plaques. Then mice were either euthanized (baseline) or fed purified standard diet with or without F1394 for 4 more weeks. Plaques of APOA1 tg/tg/Apoe -/- mice fed F1394 showed a 60% reduction of macrophages accompanied by multiple other benefits, such as reduced inflammation and favorable changes in extracellular composition, in comparison with Apoe -/- baseline mice. In addition, there was no accumulation of cholesterol crystals or signs of toxicity. Overall, these results show that short-term partial ACAT inhibition, coupled to increased cholesterol efflux capacity, favorably remodels atherosclerosis lesions, supporting the potential of these combined therapies in the treatment of advanced atherosclerosis. SIGNIFICANCE STATEMENT: Short-term pharmacological inhibition of acyl-CoA:cholesterol acyltransferase-mediated cholesterol esterification, in combination with increased free cholesterol efflux acceptors, has positive effects in mice by 1) reducing the inflammatory state of the plaque macrophages and 2) favoring compositional changes associated with plaque stabilization. These effects occur without toxicity, showing the potential of these combined therapies in the treatment of advanced atherosclerosis.

The arousal effect of hyperbaric oxygen through orexin/hypocretin an upregulation on ketamine/ethanol-induced unconsciousness in male rats.

Approaches that facilitate the recovery from coma would have enormous impacts on patient outcomes and medical economics. Orexin-producing neurons release orexins (also known as hypocretins) energy-dependently to maintain arousal. Hyperbaric oxygen (HBO) could increase ATP levels by preserving mitochondrial function. We investigated, for the first time, the arousal effects of HBO and orexins mechanisms in a rat model of unconsciousness induced by ketamine or ethanol. A total of 120 Sprague-Dawley male rats were used in this study. Unconsciousness was induced either by intraperitoneal injection of ketamine or ethanol. The HBO treatment (100% O2 at 3 ATA) was administered immediately after unconsciousness induction for 1 hr. SB334867, orexin-1 receptor (OX1R) inhibitor, or JNJ10397049, orexin-2 receptor (OX2R) inhibitor was administered 30 min intraperitoneally before unconsciousness induction. Loss of righting reflex test (LORR) and Garcia test were used to evaluate the unconsciousness duration and neurological deficits after recovering from unconsciousness, respectively. Enzyme-linked immunosorbent assay was used to measure brain tissue ATP and orexin A levels. Ketamine or ethanol injection resulted in LORR immediately and neurological deficits 6 hr after unconsciousness induction. HBO treatment significantly reduced the LORR duration, improved Garcia scores and unregulated ATP and orexin A levels in the brain tissue. Administration of OX1R inhibitor or OX2 R inhibitor abolished arousal and neurological benefits of HBO. In conclusion, HBO exerted arousal-promoting effects on unconscious rats induced by ketamine or ethanol. The underlying mechanism was via, at least in part, ATP/orexin A upregulation. HBO may be a practical clinical approach to accelerate unconsciousness recovery in patients.

Isoamericanoic Acid B from Acer tegmentosum as a Potential Phytoestrogen.

Phytoestrogens derived from plants have attracted the attention of the general public and the medical community due to their potentially beneficial role in relieving menopausal symptoms. The deciduous tree Acer tegmentosum Maxim (Aceraceae) has long been utilized in Korean folk medicine to alleviate many physiological disorders, including abscesses, surgical bleeding, and liver diseases. In order to explore structurally and/or biologically new constituents from Korean medicinal plants, a comprehensive phytochemical study was carried out on the bark of A. tegmentosum. One new phenolic compound with a 1,4-benzodioxane scaffold, isoamericanoic acid B (1), as well as with nine known phenolic compounds (2⁻10), were successfully isolated from the aqueous extracts of the bark of A. tegmentosum. A detailed analysis using 1D and 2D NMR spectroscopy, electronic circular dichroism (ECD) spectral data, and LC/MS afforded the unambiguous structural determination of all isolated compounds, including the new compound 1. In addition, compounds 2, 4, 5, and 9 were isolated and identified from the bark of A. tegmentosum for the first time. All isolated compounds were tested for their estrogenic activities using an MCF-7 BUS cell proliferation assay, which revealed that compounds 1, 2, and 10 showed moderate estrogenic activity. To study the mechanism of this estrogenic effect, a docking simulation of compound 1, which showed the best estrogenic activity, was conducted with estrogen receptor (ER) -α and ER-ß, which revealed that it interacts with the key residues of ER-α and ER-ß. In addition, compound 1 had slightly higher affinity for ER-ß than ER-α in the calculated Gibbs free energy for 1:ER-α and 1:ER-ß. Thus, the present experimental evidence demonstrated that active compound 1 from A. tegmentosum could be a promising phytoestrogen for the development of natural estrogen supplements.

Central Orexin A Affects Reproductive Axis by Modulation of Hypothalamic Kisspeptin/Neurokinin B/Dynorphin Secreting Neurons in the Male Wistar Rats.

It is an established fact that orexin plays an important role in regulating the reproductive axis and the secretions of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH). However, its precise cellular and molecular mechanisms are not fully recognized. Accordingly, the aim of the present study is to find out whether the central injection of orexin A (OXA) and its antagonists, SB-334867 (as orexin receptor antagonist 1; OX1RA) and JNJ-10397049 (as orexin receptor antagonist 2; OX2RA), either alone or in combination, can leave any impact on the reproductive axis (either hormonal or behavioral) in the male Wistar rats. Furthermore, in order to see whether OXA signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin (known as KNDy neurons, a neural network which works upstream of GnRH neurons) or not, the relative gene expression of these neuropeptides were measured. Overall, the data from radioimmunoassay revealed that OXA significantly decreases the mean serum level of LH and testosterone and, in a similar vein, its antagonists neutralize this impact. Moreover, data from real-time quantitative PCR indicated that OXA has significantly reduced the hypothalamic expression of Gnrh. In this line, the gene expressions of Kisspeptin and Neurokinin b decreased. However, OXA antagonists neutralize this impact. Also, the expression of Dynorphin gene was upregulated by the following application of the OXA. The results of this study are related to the impact of orexin on the reproductive axis. It is recommended that KNDy neurons as the interneural pathway relay the information of orexin to the GnRH neurons.

Zeylenone inhibits proliferation and promotes apoptosis in ovarian carcinoma cells via Janus kinase 2 / signal transducers and activators of transcription 3 pathways.

AIM: Ovarian cancer is the fifth common cancer in females. The aim of our study was to determine function of Zeylenone on cell viability and apoptosis of ovarian carcinoma SKOV3 cells. METHODS: Cell viability was measured by Cell counting kit-8 (CCK8) assay; Mitochondrial membrane potential (MMP) and apoptosis were detected by flow cytometry. The mRNA and protein levels of related factors were determined by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: Cell viability was decreased by Zeylenone in a dose-dependent manner. Zeylenone with concentrations of 2.5, 5 and 10 µmol/L was used to treat ovarian carcinoma SKOV3 cells for 24 h in the following study. The loss of MMP and apoptosis were both significantly increased by Zeylenone. The mRNA and protein levels of cytochrome c (cyto c) and apoptosis inducing factor (AIF) in cytosol were increased by Zeylenone. The mRNA and protein levels of Caspase-3, Fas, Fasl and Bax were increased; while the expression of Bcl-2 was decreased by Zeylenone. The expression of (Janus family of tyrosine kinase) p-JAK and signal transducer and activator of transcription (p-STAT) was decreased significantly by Zeylenone. CONCLUSION: Zeylenone inhibited cell proliferation and promoted apoptosis in ovarian carcinoma cells. The JAK-STAT pathway was involved in this progress.

The effect of inflammation on sympathetic nerve mediated contractions in rat isolated caudal artery.

Chronic inflammatory process(es) contributes to changes in vascular function in a variety of diseases. Sympathetic nerve-mediated responses in blood vessels play a pivotal role in regular physiological functions. We tested the hypothesis that sympathetic neuro-effector function will be altered as consequence of inflammatory state. Sympathetic nerve-mediated contractions and alpha adrenergic receptor expressions were evaluated in isolated caudal arteries of rats treated with saline and Complete Freund's adjuvant (CFA). While CFA-treated animals had significantly higher plasma levels of tumor necrosis factor-alpha compared to saline, blood pressure remained unchanged. Immunofluorescence revealed increased expression of ionized calcium adapter binding molecule-1 in the adventitia of blood vessels from CFA-treated animals compared to saline. In isolated arteries, electrical field stimulations between 1.25 and 40Hz resulted in frequency-dependent contractions that wasabolished by tetrodotoxin. Neurogenic contractions from CFA groups were significantly greater than saline. While the presence of alpha1-adrenoceptor antagonist (prazosin) significantly inhibited contractions at lower frequencies of stimulation (1.25-5Hz) in isolated arteries of CFA-treated rats compared to controls, alpha2-adrenoceptor antagonist (rauwolscine) had modest effects. Inhibition of neuronal reuptake by cocaine comparably enhanced field-stimulated responses in vessels of experimental and control animals. Immunofluorescence revealed a difference in expression of alpha1- and alpha2-adrenoceptors in the endothelium of blood vessels of CFA compared to saline controls. Collectively, our observations lend support to enhanced neurogenic contractions in blood vessels of inflamed animals possibly attributing to alterations in responsiveness and/or distribution of post-junctional alpha1-adrenoceptors.

Sex differences in hypothalamic-mediated tonic norepinephrine release for thermal hyperalgesia in rats.

Neuropathic pain is treated using serotonin norepinephrine reuptake inhibitors with mixed results. Pain facilitation mediated by α1-adrenoceptors may be involved, but whether norepinephrine (NE) is tonically released is unclear. The aim of this study was to determine whether NE is tonically released from A7 cells following chronic constriction injury (CCI), and if the lateral hypothalamus (LH) plays a role in this release in male and female rats with nociceptive and neuropathic pain types. Neuropathic groups received left CCI while nociceptive groups remained naïve to injury. Fourteen days later, rats were given intrathecal infusion of either the α1-adrenoceptor antagonist WB4101, the α2-adrenoceptor antagonist yohimbine (74 µg), or normal saline for control. Paw withdrawal latency (PWL) from a thermal stimulus was measured. The generalized estimated equation method was used for statistical analysis. Nociceptive rats given WB4101 had a PWL significantly longer than saline control (7.89 ± 0.63 vs. 5.87 ± 0.52 s), while the PWL of neuropathic rats given WB4101 was 13.20 ± 0.52 s compared to 6.78 ± 0.52 s for the saline control rats. Yohimbine had no significant effect. Microinjection of cobalt chloride (CoCl) in the A7 catecholamine cell group to prevent synaptic transmission blocked the effect of WB4101 in all groups, supporting the notion that spinally descending A7 cells tonically release NE that contributes to α1-mediated nociceptive facilitation. Microinjection of CoCl into the left LH blocked the effect of WB4101 in nociceptive and neuropathic male rats, but had no effect in female rats of either pain type, suggesting differential innervation. These findings indicate that tonic release of NE acts at pronociceptive α1-adrenoceptors, that this effect is greater in rats with nerve damage, and that, while NE comes primarily from the A7 cell group, LH innervation of the A7 cell group is different between the sexes.

Antimicrobial Metabolites from a Marine-Derived Actinomycete in Vietnam's East Sea.

Two new compounds, a quinoline alkaloid (1) and a 1,4-dioxane derivative (2), were isolated from culture broth of the marine-derived actinomycete Micromonospora sp. (strain G019) by bioassay-guided fractionation. This actinomycete strain was isolated from sediment, collected at Cát Bà Peninsula, Vietnam. The taxonomic identification was achieved by analysis of 16S rRNA gene sequences. On the basis of morphological and phylogenetic evidence, strain G019 was assigned to the genus Micromonospora. The structures of 1 and 2 were established by spectroscopic data analysis, including one- and two-dimensional NMR, and MS. Compound 1 was found to have antibacterial activity against Escherichia coli (MIC: 48 µg/mL), Salmonella enterica (MIC: 96 µg/mL) and Enterococcus faecalis (MIC: 128 µg/mL), while compound 2 showed inhibitory activity against Enterococcusfaecalis (MIC: 32 µg/mL) and Candida albicans (MIC: 64 µg/mL).

A novel synthetic Piper amide derivative NED-180 inhibits hyperpigmentation by activating the PI3K and ERK pathways and by regulating Ca2+ influx via TRPM1 channels.

Piper amides have a characteristic, unsaturated amide group and exhibit diverse biological activities, including proliferation and differentiation of melanocytes, although the molecular mechanisms underlying its antimelanogenesis effect remain unknown. We screened a selected chemical library of newly synthesized Piper amide derivatives and identified (E)-3-(4-(tert-butyl)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (NED-180) as one of the most potent compounds in suppressing melanogenesis. In murine melan-a melanocytes, NED-180 downregulated the expression of melanogenic regulatory proteins including tyrosinase, Tyrp1, Dct, and MITF. PI3K/Akt-dependent phosphorylation of GSK3ß by NED-180 decreases MITF phosphorylation and inhibits melanogenesis without any effects on cytotoxicity and proliferation. Furthermore, topical application of NED-180 significantly ameliorated UVB-induced skin hyperpigmentation in guinea pigs. Interestingly, data obtained using calcium imaging techniques suggested that NED-180 reduced the TPA-induced activation of TRPM1 (melastatin), which could explain the NED-180-induced inhibition of melanogenesis. All things taken together, NED-180 triggers activation of multiple pathways, such as PI3K and ERK, and inhibits TRPM1/TRPV1, leading to inhibition of melanogenesis.

Pharmacologic Management of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) remains a condition with suboptimal functional outcomes, especially in the young population. Pharmacotherapy has an accepted role in several aspects of the disease and an emerging role in several others. No preventive pharmacologic interventions for SAH currently exist. Antiplatelet medications as well as anticoagulation have been used to prevent thromboembolic events after endovascular coiling. However, the main focus of pharmacologic treatment of SAH is the prevention of delayed cerebral ischemia (DCI). Currently the only evidence-based medical intervention is nimodipine. Other calcium channel blockers have been evaluated without convincing efficacy. Anti-inflammatory drugs such as statins have demonstrated early potential; however, they failed to provide significant evidence for the use in preventing DCI. Similar findings have been reported for magnesium, which showed potential in experimental studies and a phase 2 trial. Clazosentane, a potent endothelin receptor antagonist, did not translate to improve functional outcomes. Various other neuroprotective agents have been used to prevent DCI; however, the results have been, at best inconclusive. The prevention of DCI and improvement in functional outcome remain the goals of pharmacotherapy after the culprit lesion has been treated in aneurysmal SAH. Therefore, further research to elucidate the exact mechanisms by which DCI is propagated is clearly needed. In this article, we review the current pharmacologic approaches that have been evaluated in SAH and highlight the areas in which further research is needed.

3-deoxysilybin exerts anti-inflammatory effects by suppressing NF-κB activation in lipopolysaccharide-stimulated RAW264.7 macrophages.

3-deoxysilybin (3-DS), also known as (-)-isosilandrin A, is a natural flavonoid of Silybum marianum. This study was designed to investigate the anti-inflammatory effect and the underlying molecular mechanisms of 3-DS in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. 3-DS dose-dependently inhibited the production of NO and the expression of iNOS in LPS-stimulated RAW264.7 macrophages. 3-DS also inhibited the production of pro-inflammatory cytokines (MCP-1, TNF-α, IL-6, and IL-1ß) in LPS-stimulated RAW264.7 macrophages. Moreover, 3-DS decreased the NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Furthermore, 3-DS suppressed NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB in LPS-stimulated RAW264.7 macrophages. Taken together, the present study suggests for the first time that 3-DS may exhibit an anti-inflammatory effect through the suppression of NF-κB transcriptional activation in LPS-stimulated RAW264.7 macrophages.

Marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the GABAA receptor.

RATIONALE: In psychopharmacology, researchers have been interested in the hypnotic effects of terrestrial plant polyphenols and their synthetic derivatives. Phlorotannins, a marine plant polyphenol, could have potential as a source of novel hypnotic drugs. OBJECTIVES: The effects of phlorotannins and major phlorotannin constituent eckstolonol on sleep-wake profiles in mice were evaluated in comparison with diazepam, and their hypnotic mechanism was also investigated. METHODS: The effects of phlorotannin preparation (PRT) and eckstolonol orally given on sleep-wake profiles were measured by recording electroencephalograms (EEG) and electromyograms in C57BL/6N mice. Flumazenil, a GABAA-benzodiazepine (BZD) receptor antagonist, was injected 15 min before PRT and eckstolonol to reveal its hypnotic mechanism. RESULTS: PRT administration (>250 mg/kg) produced a significant decrease in sleep latency and an increase in the amount of non-rapid eye movement sleep (NREMS). Eckstolonol significantly decreased sleep latency (>12.5 mg/kg) and increased the amount of NREMS (50 mg/kg). PRT and eckstolonol had no effect on EEG power density of NREMS. The hypnotic effects of PRT or eckstolonol were completely abolished by pretreatment with flumazenil. CONCLUSIONS: We demonstrated that phlorotannins promote NREMS by modulating the BZD site of the GABAA receptor. These results suggest that phlorotannins can be potentially used as an herbal medicine for insomnia and as a promising structure for developing novel sedative-hypnotics.

Flow-through sensor array applied to cytotoxicity assessment in cell cultures for drug-testing purposes.

The viability of cells cultured in microsystems for drug screening purposes is usually tested with a variety of colorimetric/fluorescent methods. In this work we propose an alternative way of assessing cell viability-flow-through sensor array that can be connected in series with cell microbioreactors as compatible detection system. It is shown, that the presented device is capable of cytotoxic effect detection and estimation of cell viability after treatment with 1,4-dioxane and 5-fluorouracil, which proves that it can be used for truly non-invasive, fast, reliable, continuous cell culture monitoring in microscale.

Novel, broad-spectrum anticonvulsants containing a sulfamide group: pharmacological properties of (S)-N-[(6-chloro-2,3-dihydrobenzo[1,4]dioxin-2-yl)methyl]sulfamide (JNJ-26489112).

Broad-spectrum anticonvulsants are of considerable interest as antiepileptic drugs, especially because of their potential for treating refractory patients. Such "neurostabilizers" have also been used to treat other neurological disorders, including migraine, bipolar disorder, and neuropathic pain. We synthesized a series of sulfamide derivatives (4-9, 10a-i, 11a, 11b, 12) and evaluated their anticonvulsant activity. Thus, we identified promising sulfamide 4 (JNJ-26489112) and explored its pharmacological properties. Compound 4 exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures. Mechanistically, 4 inhibited voltage-gated Na(+) channels and N-type Ca(2+) channels and was effective as a K(+) channel opener. The anticonvulsant profile of 4 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. On the basis of its pharmacology and other favorable characteristics, 4 was advanced into human clinical studies.

Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPS-stimulated RAW264.7 macrophages.

Although individual phlorotannins contained in the edible brown algae have been reported to possess strong anti-inflammatory activity, the responsible components of Eisenia bicyclis have yet to be fully studied. Thus, we evaluated their anti-inflammatory activity via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) and tert-butylhydroperoxide (t-BHP)-induced reactive oxygen species (ROS), along with suppression against expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), in RAW 264.7 cells. The anti-inflammatory activity potential of the methanolic extract and its fractions of E. bicyclis was in the order of dichloromethane>methanol>ethyl acetate>n-butanol. The strong anti-inflammatory dichloromethane fraction was further purified to yield fucosterol. From the ethyl acetate fraction, six known phlorotannins were isolated: phloroglucinol, eckol, dieckol, 7-phloroeckol, phlorofucofuroeckol A and dioxinodehydroeckol. We found that these compounds, at non-toxic concentrations, dose-dependently inhibited LPS-induced NO production. Fucosterol also inhibited t-BHP-induced ROS generation and suppressed the expression of iNOS and COX-2. These results indicate that E. bicyclis and its constituents exhibited anti-inflammatory activity which might attribute to inhibition of NO and ROS generation and suppression of the NF-κB pathway and can therefore be considered as a useful therapeutic and preventive approach to various inflammatory and oxidative stress-related diseases.

5alpha,8alpha-epidioxysterols from a Formosan sponge, Axinyssa sp.

One new 5alpha,8alpha-epidioxysterol, 3-acetylaxinysterol (1), along with one known sterol, axinysterol (2), were isolated from a Formosan sponge, Axinyssa sp.. The structures of the compounds were determined by spectroscopic methods and the absolute configuration of 2 was further confirmed by single-crystal X-ray diffraction analysis for the first time. Compound 2 exhibited significant cytotoxicity against K562 and Molt 4 cancer cell lines.

Photo-oxidative stress by ultraviolet-B radiation and antioxidative defense of eckstolonol in human keratinocytes.

Ultraviolet-B (UV-B) irradiation has been known to generate oxidative stress by increasing reactive oxygen species (ROS) in skin cells. Several naturally occurring antioxidant compounds isolated from marine algae are believed to protect against ROS. In this study, we assessed the antioxidative effect of eckstolonol isolated from Ecklonia cava against UV-B-induced ROS in human keratinocytes (HaCaTs). We investigated the effects of photo-oxidative stress by UV-B (50 mJ/cm(2)) and the antioxidative effects of eckstolonol using fluorometry, flow cytometry, microscopy, and cell viability and comet assays. UV-B irradiation decreased cell viability, which was restored in a dose-dependent manner with eckstolonol treatment (0, 5, 50, 100, and 200 µM). Moreover, eckstolonol reduced UV-B-induced ROS, lipid peroxidation, damaged DNA levels, and cell death. These antioxidative effects seem to be due to the enzymatic activities of catalase (CAT) and superoxide dismutase (SOD). Collectively, these results indicate that eckstolonol is capable of protecting keratinocytes from photo-oxidative stress.

Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain.

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Analgesia induced by 2- or 100-Hz electroacupuncture in the rat tail-flick test depends on the activation of different descending pain inhibitory mechanisms.

UNLABELLED: We evaluated the effectiveness of intrathecal antagonists of α1- (WB4101) and α2- (idazoxan) adrenoceptors and serotonergic (methysergide), opioid (naloxone), muscarinic (atropine), GABA(A) (bicuculline) and GABA(B) (phaclofen) receptors in blocking 2- or 100-Hz electroacupuncture (EA)-induced analgesia (EAIA) in the rat tail-flick test. EA was applied bilaterally to the Zusanli and Sanyinjiao acupoints in lightly anesthetized rats. EA increased tail-flick latency, where the effect of 2-Hz EA lasted longer than that produced by 100-Hz EA. The 2-Hz EAIA was inhibited by naloxone or atropine, was less intense and shorter after WB4101 or idazoxan, and was shorter after methysergide, bicuculline, or phaclofen. The 100-Hz EAIA was less intense and shorter after naloxone and atropine, less intense and longer after phaclofen, shorter after methysergide or bicuculline, and remained unchanged after WB4101 or idazoxan. We postulate that the intensity of the effect of 2-Hz EA depends on noradrenergic descending mechanisms and involves spinal opioid and muscarinic mechanisms, whereas the duration of the effect depends on both noradrenergic and serotonergic descending mechanisms, and involves spinal GABAergic modulation. In contrast, the intensity of 100-Hz EAIA involves spinal muscarinic, opioid, and GABA(B) mechanisms, while the duration of the effects depends on spinal serotonergic, muscarinic, opioid, and GABA(A) mechanisms. PERSPECTIVE: The results of this study indicate that 2- and 100-Hz EA induce analgesia in the rat tail-flick test activating different descending mechanisms at the spinal cord level that control the intensity and duration of the effect. The adequate pharmacological manipulation of such mechanisms may improve EA effectiveness for pain management.