Iruplinalkib (WX­0593), a novel ALK/ROS1 inhibitor, overcomes crizotinib resistance in preclinical models for non-small cell lung cancer.

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX­0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models.

Discovery and preclinical evaluations of WX-0593, a novel ALK inhibitor targeting crizotinib-resistant mutations.

ALK gene rearrangements are oncogenic drivers in approximately 5% of NSCLC. Crizotinib, a first generation ALK inhibitor, is widely prescribed for ALK-positive NSCLC in clinic. Resistance to crizotinib and other ALK inhibitors has been problematic. Addressing resistance, here we describe discovery and development of a novel, proprietary spirocyclic diamine-substituted aryl phosphine oxide series of inhibitors, which led to the identification of WX-0593 (16a) as a potent ALK inhibitor. WX-0593 inhibited the activity of both wild type and resistant mutants of ALK in vitro, showed strong antitumor activity in a crizotinib-resistant mouse PDX model. WX-0593 is currently under development in phase II/III clinical trials.

Two Compounds Isolated From Ganglioside GM1 Promote Angiogenesis in Zebrafish.

Ganglioside has been implicated to play important roles in modulating various cell signaling and biological functions. However, the functional analysis of a single ganglioside in a zebrafish model is so far lacking. In this study, we investigated the angiogenic effects of 2 monosialoganglioside compounds isolated from GM1 in zebrafish embryos. First, we showed the tested compounds are adequate safe. Then, we found that these compounds exhibited significant proangiogenic effect through enhancement of endothelial cell proliferation, migration, and differentiation. Furthermore, the 2 compounds were proved to promote angiogenesis through, at least partially, modulating the level of Notch signaling. This study provides the novel insights into the clinical application of the 2 ganglioside compounds and GM1.

Effect of radio-frequency heating on microbial load, flavor, color, and texture profiles of Cordyceps militaris.

BACKGROUND: Cordyceps militaris is a medicine and food dual-purpose mushroom extensively cultivated and consumed in East and Southeast Asia for centuries. However, it has an extremely short shelf life of 3-4 days at room temperature. C. militaris was pasteurized for 10, 20, and 30 min by radio-frequency (RF) at an electrode gap of 20 mm. The effect of RF heating on the microbial load, color, texture, and flavor attributes of C. militaris was evaluated and compared with those sterilized by conventional high-pressure steam. RESULTS: RF heating contributed to good heating uniformity, uniform temperature distribution, and significant decrease in total microbial load. C. militaris heated by RF exhibited unnoticeable total color difference compared with unpasteurized ones, while those sterilized by high-pressure steam presented undesired and unacceptable browning. Insignificant differences in hardness and chewiness were observed after RF heating. Improvements in mushroom-like flavor occurred after 10 and 20 min of RF heating. CONCLUSION: This study suggests that RF heating for pasteurization of edible mushrooms has promising prospects. Evaluation of RF heating on the taste and nutritional characteristics of edible mushrooms is needed in future work. © 2018 Society of Chemical Industry.

The effects of Jiao-Tai-Wan on sleep, inflammation and insulin resistance in obesity-resistant rats with chronic partial sleep deprivation.

BACKGROUND: Jiao-Tai-Wan (JTW), composed of Rhizome Coptidis and Cortex Cinnamomi, is a classical traditional Chinese prescription for treating insomnia. Several in vivo studies have concluded that JTW could exert its therapeutical effect in insomnia rats. However, the specific mechanism is still unclear. The present study aimed to explore the effect of JTW on sleep in obesity-resistant (OR) rats with chronic partial sleep deprivation (PSD) and to clarify its possible mechanism. METHODS: JTW was prepared and the main components contained in the granules were identified by 3D-High Performance Liquid Chromatography (3D-HPLC) assay. The Male Sprague-Dawley (SD) rats underwent 4 h PSD by environmental noise and the treatment with low and high doses of JTW orally for 4 weeks, respectively. Then sleep structure was analyzed by electroencephalographic (EEG). Inflammation markers including high-sensitivity C reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were examined in the rat plasma. Meanwhile, metabolic parameters as body weight increase rate, fasting plasma glucose (FPG), fasting insulin (FINS) levels and insulin resistance index (HOMA-IR) were measured. The expressions of clock gene cryptochromes (Cry1 and Cry2) and inflammation gene nuclear factor-κB (NF-κB) in peripheral blood monocyte cells (PBMC) were also determined. RESULTS: The result showed that the administration of JTW significantly increased total sleep time and total slow wave sleep (SWS) time in OR rats with PSD. Furthermore, the treatment with JTW reversed the increase in the markers of systemic inflammation and insulin resistance caused by sleep loss. These changes were also associated with the up-regulation of Cry1 mRNA and Cry 2 mRNA and the down-regulation of NF-κB mRNA expression in PBMC. CONCLUSIONS: This study suggests that JTW has the beneficial effects of improving sleep, inflammation and insulin sensitivity. The mechanism appears to be related to the modulation of circadian clock and inflammation genes expressions in PBMC.

GM1 Ameliorates Neuronal Injury in Rats after Cerebral Ischemia and Reperfusion: Potential Contribution of Effects on SPTBN1-mediated Signaling.

Monosialoganglioside GM1 (GM1) has long been used as a therapeutic agent for neurological diseases in the clinical treatment of ischemic stroke. However, the mechanism underlying the neuroprotective function of GM1 is still obscure until now. In this study, we investigated the effects of GM1 in ischemia and reperfusion (I/R) brain injury models. Middle cerebral artery occlusion and reperfusion (MCAO/R) rats were treated with GM1 (60 mg·kg-1·d-1, tail vein injection) for 2 weeks. The results showed that GM1 substantially attenuated the MCAO/R-induced neurological dysfunction and inhibited the inflammatory responses and cell apoptosis in ischemic parietal cortex. We further revealed that GM1 inhibited the activation of NFκB/MAPK signaling pathway induced by MCAO/R injury. To explore its underlying mechanism of the neuroprotective effect, transcriptome sequencing was introduced to screen the differentially expressed genes (DEGs). By function enrichment and PPI network analyses, Sptbn1 was identified as a node gene in the network regulated by GM1 treatment. In the MCAO/R model of rats and oxygen-glucose deprivation and reperfusion (OGD/R) model of primary culture of rat cortical neurons, we first found that SPTBN1 was involved in the attenuation of I/R induced neuronal injury after GM1 administration. In SPTBN1-knockdown SH-SY5Y cells, the treatment with GM1 (20 µM) significantly increased SPTBN1 level. Moreover, OGD/R decreased SPTBN1 level in SPTBN1-overexpressed SH-SY5Y cells. These results indicated that GM1 might achieve its potent neuroprotective effects by regulating inflammatory response, cell apoptosis, and cytomembrane and cytoskeleton signals through SPTBN1. Therefore, SPTBN1 may be a potential target for the treatment of ischemic stroke.

A Biomimetic Adhesive Disc for Robotic Adhesion Sliding Inspired by the Net-Winged Midge Larva.

Net-winged midge larvae (Blephariceridae) are known for their remarkable ability to adhere to and crawl on the slippery surfaces of rocks in fast-flowing and turbulent alpine streams, waterfalls, and rivers. This remarkable performance can be attributed to the larvae's powerful ventral suckers. In this article, we first develop a theoretical model of the piston-driven sucker that considers the lubricated state of the contact area. We then implement a piston-driven robotic sucker featuring a V-shaped notch to explore the adhesion-sliding mechanism. Each biomimetic larval sucker has the unique feature of an anterior-facing V-shaped notch on its soft disc rim; it slides along the shear direction while the entire disc surface maintains powerful adhesion on the benthic substrate, just like the biological counterpart. We found that this biomimetic sucker can reversibly transit between "high friction" (4.26 ± 0.34 kPa) and "low friction" (0.41 ± 0.02 kPa) states due to the piston movement, resulting in a frictional enhancement of up to 93.9%. We also elucidate the frictional anisotropy (forward/backward force ratio: 0.81) caused by the V-shaped notch. To demonstrate the robotic application of this adhesion-sliding mechanism, we designed an underwater crawling robot Adhesion Sliding Robot-1 (ASR-1) equipped with two biomimetic ventral suckers. This robot can successfully crawl on a variety of substrates such as curved surfaces, sidewalls, and overhangs and against turbulent water currents with a flow speed of 2.4 m/s. In addition, we implemented a fixed-wing aircraft Adhesion Sliding Robot-2 (ASR-2) featuring midge larva-inspired suckers, enabling transit from rapid water surface gliding to adhesion sliding in an aquatic environment. This adhesion-sliding mechanism inspired by net-winged midge larvae may pave the way for future robots with long-term observation, monitoring, and tracking capabilities in a wide variety of aerial and aquatic environments.

WX-0593 combined with an epithelial growth factor receptor (EGFR) monoclonal antibody in the treatment of xenograft tumors carrying triple EGFR mutations.

Background: To evaluate the safety and therapeutic efficacy of WX-0593, a newly developed potent anaplastic lymphoma kinase (ALK) inhibitor, in combination with an epithelial growth factor receptor (EGFR) monoclonal antibody (QL1203 or Vectibix) for the treatment of xenograft tumors carrying mutant EGFR and osimertinib-resistant mutations (EGFR/T790M/C797S). Methods: The inhibition of tumor cell proliferation by WX-0593 and Vectibix alone or combined was evaluated in four EGFR triple-mutant cell lines: PC9 (EGFR Del19/T790M/C797S), NCI-H1975 (EGFR L858R/T790M/C797S), Ba/F3 (EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S). The in vivo antitumor efficacy of WX-0593 alone or combined with QL1203 or Vectibix was evaluated in xenograft tumor models of BALB/c nude mice developed from H1975 (EGFR-Del19/T790M/C797S) and Ba/F3 (EGFR-L858R/T790M/C797S) cell lines. Mice were randomized into groups and treated with or without WX-0593, QL1203, Vectibix, or their combination. The tumor volume, mouse body weight, and therapeutic side effects were monitored routinely. Blood samples were obtained from all mice at different time points after the last dosage of treatment to evaluate the pharmacokinetic parameters of the drugs. Results: WX-0593 and Vectibix showed a strong synergistic inhibitory effect on the proliferation of two EGFR triple-mutant Ba/F3 cell lines (EGFR L858R/T790M/C797S and Del19/T790M/C797S), but little synergistic inhibitory effect on the proliferation of NCI-H1975 (EGFR L858R/T790M/C797S) and PC9 (EGFR Del19/T790M/C797S). In vivo, WX-0593 (25 mg/kg) showed a modest therapeutic effect when combined with QL1203 or Vectibix, but had no effect on tumor growth as a monotherapy at this dosage. WX-0593 (75 mg/kg) exhibited modest antitumor efficacy that was further enhanced in combination with QL1203 or Vectibix in both tumor models (H1975 and Ba/F3). No significant body weight alteration, any other side effect, or deaths were observed during treatment. Pharmacokinetic analysis showed that the serum level of QL1203 or Vectibix was significantly increased and lasted longer when combined with WX-0593. Conclusions: WX-0593 exhibited a synergetic effect with an EGFR monoclonal antibody on osimertinib-resistant EGFR-mutant non-small cell lung cancer (NSCLC) both in vitro and in vivo. Their combination showed potent antitumor efficacy and an acceptable safety profile, which may be a promising strategy for the treatment of patients with EGFR triple-mutant NSCLC resistant to osimertinib.

Cross-sectional study: Relationship between serum trace elements and hypertension.

BACKGROUND: A balanced intake of trace elements is beneficial for chronic diseases such as hypertension. However, the available information regarding trace elements that may be independently associated with hypertension is limited, and the relationship between this disorder and element ratios also remains unclear. METHODS: A total of 6,754 subjects from rural China were selected, after exclusion of patients who were under 18, had incomplete data or had additional related disorders, by multi-stage simple random and cluster sampling (participation rate: 95.22 %). Subjects were divided into a hypertensive (H) and a control (C) group. Data were collected on blood pressure and 12 serum trace elements were measured by flame atomic absorption spectrometry and inductively coupled plasma-mass spectrometry. Other basic information was collated from questionnaires and biochemical indicators were measured via kits. RESULTS: Differences in serum levels of magnesium (Mg(mg/l): H: 27.43 ± 12.72; C: 26.33 ± 12.16), iron (Fe(mg/l): H: 1.99 ± 1.24; C: 1.84 ± 1.16), copper (Cu(mg/l): H: 1.19 ± 0.37; C: 1.10 ± 0.36), boron (B(µg/l): H: 50.00 ± 25.21; C: 47.57 ± 26.25), selenium (Se(µg/l): H: 125.12 ± 32.81; C: 118.80 ± 29.72) and chromium (Cr(µg/l): H: 8.77 ± 10.12; C: 10.12 ± 10.72) between the hypertensive and control groups were found. There were no differences in serum contents of calcium (Ca(mg/l): H: 112.43 ± 58.25; C: 111.00 ± 59.49), zinc (Zn(mg/l): H: 1.50 ± 1.97; C: 1.44 ± 1.88), arsenic (As(µg/l): H: 4.17 ± 3.94; C: 4.10 ± 4.00), manganese (Mn(µg/l): H: 4.15 ± 4.03; C: 4.07 ± 4.05), cadmium (Cd(µg/l): H: 1.14 ± 1.11; C: 1.18 ± 1.12) or lead (Pb(µg/l): H: 4.22 ± 8.90; C: 4.26 ± 10.25). The serum Cr and Cd concentrations of hypertensive men were lower than that of male controls while Mg, Cu, Ca and Se concentrations in male controls were lower. Further differences were apparent and Fe, B, Se, Mg and Cu all showed higher levels in hypertensive females whereas Cr concentrations were higher in female controls. Serum Zn and B levels showed age-related variations among hypertensive patients and concentrations of serum Cu, Zn, Se and B showed age-related variations among control subjects. For hypertensive patients, the odds ratio (OR) and 95 % confidence interval (CI) for the association of serum Cu, Se and Cr levels with hypertension were Cu: 1.36 (1.12-1.66); Se: 1.03 (1.01-1.05); Cr: 0.89 (0.83-0.96). Moreover, when the participants in the grouping with the highest copper/zinc (Cu/Zn) and magnesium/manganese (Mg/Mn) ratios were compared with the reference group, the OR and 95 % CI for hypertension were 1.22 (1.04-1.44) and 1.20 (1.01-1.42), respectively. CONCLUSIONS: Levels of serum trace elements showed age- and sex-related differences in a group of rural Chinese adults with hypertension and healthy participants. Serum concentrations of Cu, Se and Cr may be independently associated with hypertension. Higher serum ratios of Cu:Zn and Mg:Mn may also be associated with hypertension. Further randomized trials are necessary to elucidate the true relationship between levels of Cu, Se, Cr, Cu:Zn, Mg:Mn and hypertension.

Design, Synthesis, and Evaluation of Tetrahydropyrrolo[1,2-c]pyrimidines as Capsid Assembly Inhibitors for HBV Treatment.

The discovery of novel tetrahydropyrrolo[1,2-c]pyrimidines derivatives from Bay41_4109 as hepatitis B virus (HBV) inhibitors is herein reported. The structure-activity relationship optimization led to one highly efficacious compound 28a (IC50 = 10 nM) with good PK profiles and the favorite L/P ratio. The hydrodynamic injection model in mice clearly demonstrated the efficacy of 28a against HBV replication.

Structure-activity study of quinazoline derivatives leading to the discovery of potent EGFR-T790M inhibitors.

We have developed a series of 6, 7-disubstituted-4-(arylamino) quinazoline derivatives that functioned as irreversible EGFR inhibitors, and these compounds exhibited excellent enzyme inhibition potency. As compared with afatinib, some of them showed significantly enhanced activities towards H1975 cells (EGFR-T790M). Furthermore, the optimized compounds 7q and 8f also demonstrated good pharmacokinetic profiles, oral bioavailability as well as excellent in vivo efficacy in H1975 and HCC827 xenografts at a non-toxic dose. Based on the improved safety and efficacy against EGFR-T790M resistance, 7q and 8f are promising candidates for further studies.

Synthesis and in vivo SAR study of indolin-2-one-based multi-targeted inhibitors as potential anticancer agents.

A series of indolin-2-one analogues were designed and synthesized, and all of them exhibited excellent in vitro potency. The structure and in vivo activity or toxicity relationship (in-vivo SAR) investigation of indolin-2-one structural analogues was carried out. In vivo efficacy studies indicated that 3b significantly suppressed tumor growth in HT-29 and NCI-H460 xenografts without causing significant loss of body weight. Kinase assay showed that compound 3b effectively inhibited the VEGFR-2, VEGFR-3, FLT3, Ret and PDGFR-ß kinases, but had little effect on the VEGFR-1 kinase. Besides, 3b showed higher selectivity for VEGFR-2 compared with PDGFR-ß. On the basis of its selectivity and safety properties, 3b was identified as a drug candidate for the treatment of cancer.

Discovery of a potent dual EGFR/HER-2 inhibitor L-2 (selatinib) for the treatment of cancer.

To develop potent dual EGFR/HER-2 inhibitors with improved druggability, a series of new lapatinib analogs were designed and synthesized. Compared with lapatinib, L-2, L-4 and M-6 were more active against BT-474 or NCI-N87 cells. In vivo efficacy studies indicated that L-2 significantly suppressed tumor growth in NCI-N87 (94.8% inhibition) or SK-OV-3 xenograft (85.7% inhibition) without causing significant loss of body weight. And the inhibition rates of lapatinib in the two xenograft models were 89.7% and 78.8%, respectively. Moreover, further studies revealed that the potent in vivo activities of L-2 may be mainly attributed to its superior aqueous solubility and oral bioavailability. In addition, a high-yielding one-pot procedure was developed for the synthesis of lapatinib and its analogs.

Expression of the human glucokinase gene: important roles of the 5' flanking and intron 1 sequences.

BACKGROUND: Glucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5' pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5' flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5' flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3' to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.

Long-term renal changes in the liver-specific glucokinase knockout mouse: implications for renal disease in maturity-onset diabetes of the young 2.

To investigate the functional and structural renal changes in a long-term liver-specific glucokinase (gck) knockout mouse, a model was developed of maturity-onset diabetes of the young (MODY2). Hemizygous gck knockout mice, gck(w/-) groups, were compared at 6, 10, and 14 months with their age-matched normal littermates, gck(w/w) groups. To examine changes, we compared body weight, fasting blood glucose, serum insulin, and creatinine levels, as well as 24-h urine samples that were collected for urine volume and protein analysis between the 2 groups. Renal tissues were collected and stained with hemotoxylin-eosin and periodic-acid Schiff for light microscopic observation. The expression of renal transforming growth factor ß1 (TGF-ß1) was determined by Western blot. Our results show that fasting blood glucose levels were significantly higher in gck(w/-) mice compared with gck(w/w) mice (P < 0.01) for all age groups. Compared with age-matched gck(w/w) mice, 10-month old gck(w/-) mice have significantly elevated body weights (P < 0.01) and protein contents (P < 0.001). A gradual increase in mesangial matrix and a thickening of the glomerular basement membrane was observed in gck(w/-) mice at 10 and 14 months. The levels of renal TGF-ß1 expression are increasing in both gck(w/-) and gck(w/w) mice. Our results indicate that renal changes occur in the liver-specific gck knockout mouse model of MODY2 and suggest that TGF-ß1 may play a key role in pathogenesis of these renal changes.

Resistin and insulin resistance in hepatocytes: resistin disturbs glycogen metabolism at the protein level.

Resistin has been considered to link obesity with type 2 diabetes. Liver glycogen metabolism plays an essential role in maintaining glucose homeostasis, we investigated the effect of resistin on liver glycogen metabolism and attempted to identify its role in initiating insulin resistance and type 2 diabetes. Primary culture of rat hepatocytes was treated by resistin and insulin. Glycogen content was determined by the anthrone-reagent method. Real-time PCR, Western blot and enzymatic activity assay were used to detect key enzymes and genes involved in glucose metabolism. Hepatocytes exposed to resistin, but only in the presence of insulin, show a decrease in insulin-stimulated glycogen content. Decreased insulin receptor expression and GS activity and elevated GP activity was observed after the treatment of hepatocytes with resistin. No significant changes in the expression of the genes for these proteins were observed. These results strongly suggest that resistin effects glycogen metabolism at the protein level, and resistin is highly associated with insulin resistance and type 2 diabetes and is a candidate for the prevention and treatment of type 2 diabetes. Our results should lead to the development of novel strategies for the treatment of type 2 diabetes.