Celastrol inhibits angiogenesis and the biological processes of MDA-MB-231 cells via the DEGS1/S1P signaling pathway.

Celastrol (Cel) shows potent antitumor activity in various experimental models. This study examined the relationship between Cel's antivascular and antitumor effects and sphingolipids. CCK-8 assay, transwell assay, Matrigel, PCR-array/RT-PCR/western blotting/immunohistochemistry assay, ELISA and HE staining were used to detect cell proliferation, migration and invasion, adhesion and angiogenesis, mRNA and protein expression, S1P production and tumor morphology. The results showed that Cel could inhibit proliferation, migration or invasion, adhesion and angiogenesis of human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 cells by downregulating the expression of degenerative spermatocyte homolog 1 (DEGS1). Transfection experiments showed that downregulation of DEGS1 inhibited the above processes and sphingosine-1-phosphate (S1P) production of HUVECs and MDA-MB-231 cells, while upregulation of DEGS1 had the opposite effects. Coculture experiments showed that HUVECs could promote proliferation, migration and invasion of MDA-MB-231 cells through S1P/sphingosine-1-phosphate receptor (S1PR) signaling pathway, while Cel inhibited these processes in MDA-MB-231 cells induced by HUVECs. Animal experiments showed that Cel could inhibit tumor growth in nude mice. Western blotting, immunohistochemistry and ELISA assay showed that Cel downregulated the expression of DEGS1, CD146, S1PR1-3 and S1P production. These data confirm that DEGS1/S1P signaling pathway may be related to the antivascular and antitumor effects of cel.

Triptolide Inhibits the Biological Processes of HUVECs and HepG2 Cells via the Serine Palmitoyltransferase Long Chain Base Subunit 2/Sphingosine-1-Phosphate Signaling Pathway.

Triptolide (TP) has demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has a high incidence in men, and its incidence is increasing year by year. Studies have shown that angiogenesis plays an important role in the formation of tumors and that angiogenesis is closely related to tumor growth and metastasis. Deregulation of sphingolipids signaling has been associated with several pathological conditions, including cancer. In the present study, we aimed at exploring the potential molecular mechanism of TP's antivascular and antitumor effects in vitro from the perspective of sphinolipids. Human umbilical vein endothelial cells (HUVECs) and HepG2 cells were, respectively, treated with different concentrations of TP and transfected. Then, the effect of HUVECs on HepG2 cells was investigated using a three-dimensional coculture model system. CCK-8 assay was performed for cell proliferation. Cell migration and invasion abilities were assessed using the transwell assay. Cell adhesion and tube formation were detected by Matrigel. RT-PCR and western blotting were used to detect the mRNA and protein expression. The S1P production was measured via ELISA assay. Our results showed that TP inhibited HUVECs and HepG2 cells proliferation, migration, invasion, adhesion, angiogenesis, and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) expression; upregulating SPTLC2 facilitated the proliferation, migration, invasion, adhesion, angiogenesis, and sphingosine-1-phosphate (S1P) production of HUVECs and HepG2 cells, while interfering with SPTLC2 expression inhibited them; HUVECs facilitated the proliferation, migration, invasion, S1P production, S1PR1, and S1PR2 expression of HepG2 cells, while S1PR3 expression was decreased. In conclusion, SPTLC2 may be associated with the antivascular and antitumor effects of TP, and SPTLC2 is expected to become a new marker for tumor therapy. HUVECs can promote the proliferation, migration, and invasion of HepG2 cells, which may be related to the S1P/sphingosine-1-phosphate receptor (S1PR) signaling pathway.

Licorice root extract and magnesium isoglycyrrhizinate protect against triptolide-induced hepatotoxicity via up-regulation of the Nrf2 pathway.

Triptolide, the predominant biologically active component of the Chinese herb Tripterygium wilfordii Hook f., possesses numerous pharmacological activities, including anti-inflammatory, anti-fertility, anti-neoplastic, and immunosuppressive effects. However, toxicity and severe adverse effects, particularly hepatotoxicity, limit the clinical application of triptolide. Licorice root extract contains various bioactive compounds and is potent hepatoprotective. Magnesium isoglycyrrhizinate, a magnesium salt of the 18α-glycyrrhizic acid stereoisomer of glycyrrhizic acid, is used clinically in China to treat chronic viral hepatitis and acute drug-induced liver injury. The aim of this study was to investigate the role of the factor erythroid 2-related factor 2 pathway in the protective effects of LE and MIG against triptolide-induced hepatotoxicity. Hepatotoxicity models were established in L-02 cells and rats using triptolide, and the protective effects of LE and MIG were investigated in vitro and in vivo, respectively. LE and MIG significantly protected against triptolide-induced cytotoxicity. Additionally, triptolide decreased the mRNA and protein levels of Nrf2 and down-regulated Nrf2 target genes, including UGT1A, BSEP, and MRP2, while pretreatment with LE and MIG reversed these effects. Finally, Nrf2-involved antioxidant responses were activated in the presence of LE and MIG.

Tolerability and pharmacokinetics of ranolazine following single and multiple sustained-release doses in Chinese healthy adult volunteers: a randomized, open-label, Latin square design, phase I study.

BACKGROUND AND OBJECTIVES: Ranolazine was approved by the US Food and Drug Administration in January 2006 for the treatment of chronic angina pectoris, and is the first approved agent from a new class of anti-anginal drugs in almost 25 years. The primary objective of this study was to determine the concentration of ranolazine in human plasma using the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method and to compare the pharmacokinetic properties of ranolazine after administration of single and multiple doses of ranolazine in healthy Chinese adult volunteers. METHODS: A randomized, open-label, single- and multiple-dose study design was used in the study. Subjects were randomized to receive a single dose of 500, 1,000, or 1,500 mg of ranolazine. Those who received the single dose continued on to the multiple-dose phase and received 500 mg twice daily for 7 days. In the single-dose phase, blood samples were collected from 0 to 48 h after drug administration. In the multiple-dose phase, samples were obtained before drug administration at 8:00 am and 8:00 pm on days 6 and 7 to determine the minimum steady-state plasma concentration (C(min,ss)) of ranolazine; on day 8, samples were collected from 0 to 48 h after drug administration. All values were expressed as means (standard deviations [SDs]). Adverse events (AEs) were monitored throughout the study via subject interview, vital signs, and blood sampling. RESULTS: The LC-MS/MS method was developed and validated. Twelve Chinese subjects (six men, six women) were enrolled in the single-dose phase of the pharmacokinetic study. The mean (SD) age of the subjects was 24.7 (1.6) years; their mean (SD) weight was 61.3 (6.4) kg, their mean (SD) height was 165.7 (4.5) cm, and their mean (SD) body mass index was 21.6 (6.6) kg/m(2). The main pharmacokinetic parameters [mean (SD)] for ranolazine after administration of a single oral dose of 500, 1,000, and 1,500 mg were as follows: maximum plasma concentration (C(max)) 741.5 (253.0), 1,355.0 (502.0), and 2,328.7 (890.5) ng/mL, respectively; area under the concentration-time curve from time zero to 48 h (AUC(48)) 9,071.9 (3,400.0), 16,573.5 (6,806.2), and 29,324.5 (10,857.2) ng·h/mL; AUC from time zero extrapolated to infinity (AUC(∞)) 9,826.7 (3,152.0), 16,882.4 (6,790.8), and 29,923.5 (10,706.3) ng·h/mL; time to reach C(max) (t(max)) 5.3 (1.4), 4.2 (1.2), and 5.9 (2.8) h; elimination half-life (t(½)) 6.4 (3.3), 6.4 (3.5), and 6.7 (4.3) h. Mean (SD) values for the main pharmacokinetic parameters for ranolazine after administration of multiple doses were as follows: steady-state C(max) (C(max,ss)) 1,732.9 (547.3) ng/mL; C(min,ss) 838.1 (429.8) ng/mL; steady-state AUC at time t (AUC(ss,(t))) 14,655.5 (5,624.2) ng·h/mL; average steady-state plasma drug concentration during multiple-dose administration (C(av,ss)) 1,221.3 (468.7) ng/mL; t(max) 3.46 (1.48) h; t(½) 6.28 (2.48) h. CONCLUSION: In this group of healthy Chinese subjects, AUC and C(max) increased proportionally with the dose, whereas t(½) was independent of the dose. The pharmacokinetic properties of ranolazine were linear after administration of single oral doses of 500 to 1,500 mg. Compared with the pharmacokinetic parameters of the subjects who received a single dose, those who received multiple doses (twice daily) of ranolazine had a larger AUC from time zero to the time of the last measurable concentration (AUC(last)), AUC(∞), C(max), and apparent total body clearance of drug from plasma after oral administration (CL/F), and shorter t(max) (all p < 0.05). Furthermore, some of the main pharmacokinetic parameters of ranolazine may reflect ethnic differences. This dosage was generally well tolerated by all the subjects.

Lack of effect of continuous glycyrrhizin administration on the pharmacokinetics of the P-glycoprotein substrate talinolol in healthy volunteers.

PURPOSE: To investigate the effects of repeated glycyrrhizin ingestion on the oral pharmacokinetics of talinolol, a probe drug for P-glycoprotein (P-gp) activity in humans. METHODS: Fourteen healthy adult male subjects were enrolled in a two-phase randomized crossover-design study. In each phase the volunteers received placebo or compound glycyrrhizin tablets (75 mg glycyrrhizin three times daily) for 6 days. On the seventh day, a single oral dose of 100 mg talinolol was administered, and blood samples were obtained to determine plasma talinolol concentrations, measured in plasma by high-performance liquid chromatography with an ultraviolet detector. Non-compartmental analysis was used to characterize talinolol plasma concentration-time profiles. All pharmacokinetics parameters were calculated using DAS ver. 2.1 software, and statistical analyses were performed with SPSS ver. 13.0 software. Analysis of variance was used to check the difference of the means of the pharmacokinetic parameters between the two treatments at a significance level of 0.05. RESULTS: All treatments were well tolerated during the study period. The geometric mean ± standard deviation of the AUC(0-∞) for talinolol treated by glycyrrhizin and talinolol treated by placebo was 2,218.3 ± 724.3 and 1,988.2 ± 649.2 ng·h/mL, respectively. The 90 % confidence intervals for the ratio of adjusted geometric means (glycyrrhizin:placebo) for AUC(0-∞) and C (max) fell wholly within the interval [80, 125]. Six days of glycyrrhizin treatment resulted in no significant alterations in the pharmacokinetic parameters (AUC(0-∞), AUC(0-24), C (max), t (max), t (½)) for talinolol. CONCLUSIONS: Continuous glycyrrhizin administration had no induction effect on the expression of P-gp in our trial. Further research is needed to study the direct inhibition effect of glycyrrhizin on the function of P-gp with the simultaneous administration of both glycyrrhizin and P-gp substrate.

Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro.

BACKGROUND: Astragalus polysaccharides (APS) are active constituents of Astragalus membranaceus. They have been widely studied, especially with respect to their immunopotentiating properties, their ability to counteract the side effects of chemotherapeutic drugs, and their anticancer properties. However, the mechanism by which APS inhibit cancer and the issue of whether that mechanism involves the reversal of multidrug resistance (MDR) is not completely clear. The present paper describes an investigation of the effects of APS on P-glycoprotein function and expression in H22 hepatoma cell lines resistant to Adriamycin (H22/ADM). METHODS: H22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1 mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR. RESULTS: When used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123 retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed 24 h, 48 h, and 72 h after administration. APS down regulated P-glycoprotein and MDR1 mRNA expression in a concentration-dependent manner within a final range of 0.8-500 mg/L and in a time-dependent manner from 24-72 h. CONCLUSION: APS can enhance the chemosensitivity of H22/ADM cells. This may involve the downregulation of MDR1 mRNA expression, inhibition of P-GP efflux pump function, or both, which would decrease the expression of the MDR1 protein.

Development and validation of a sensitive U-HPLC-MS/MS method with electrospray ionization for quantitation of ranolazine in human plasma: application to a clinical pharmacokinetic study.

A simple, sensitive and high-throughput ultra high-performance liquid chromatography electrospray ionization mass spectrometry (U-HPLC-ESI-MS/MS) method has been developed and validated for the determination of ranolazine in human plasma. Propafenone was employed as the internal standard (I.S.). The analytes were chromatographically separated on a BEH C(18) column (50 mm × 2.1 mm, 1.7 µm) with a mobile phase consisting of acetonitrile and aqueous ammonium acetate solution (0.06% formic acid, 7.5 mmol L(-1) ammonium acetate, 40:60, v/v). Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 428.3→279.3 for ranolazine and m/z 342.4→115.9 for propafenone. The assay was linear over the concentration range 1-3000 ng mL(-1), with correlation coefficients ≥0.997. The intra- and inter-day coefficients of variation were less than 8.9% in terms of relative standard deviation and accuracy ranged from 93.0 to 108.9% at all quality control levels. The validated method was a simple sample preparation procedure and short run-time (<2.0 min) method, which was successfully applied to a phase I pharmacokinetic study of ranolazine in Chinese healthy volunteers.

Simultaneous quantitative determination of paracetamol and its glucuronide conjugate in human plasma and urine by liquid chromatography coupled to electrospray tandem mass spectrometry: application to a clinical pharmacokinetic study.

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of paracetamol (APAP) and its glucuronide conjugate (PG) in human plasma and urine. Plasma samples were precipitated with the mixture of acetonitrile and propylene glycol (90:10, v/v) solution and urine samples were diluted with the mobile phase, which were used to isolate the analytes from biological matrices followed by injection of the extracts onto a C(18) column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI(+)). The method was validated over the concentration range of 10-30,000 ng/mL and 100-6000 ng/mL for APAP in human plasma and urine as well as 10-15,000 ng/mL and 200-60,000 ng/mL for PG in human plasma and urine, respectively. Inter- and intra-run precisions of APAP and PG were less than 15% and the accuracy was within 85-115% for both plasma and urine. The average extraction recoveries were 93.1% and 89.1% for APAP, and 93.7% and 92.3% for PG in human plasma and urine, respectively. The linearity, recovery and stability were validated for APAP and PG in human plasma and urine. The method proved to be simple, robust and time efficient.

Astragalus polysaccharides can regulate cytokine and P-glycoprotein expression in H22 tumor-bearing mice.

AIM: To investigate the adjunct anticancer effect of Astragalus polysaccharides in H22 tumor-bearing mice. METHODS: To establish a solid tumor model, 5.0 × 10(6)/mL H22 hepatoma cells were inoculated subcutaneously into the right armpit region of Kunming mice (6-12 wk old, 18-22 g). When the tumors reached a size of 100 mm(3), the animals were treated as indicated, and the mice were randomly assigned to seven groups (n = 10 each). After ten days of treatment, blood samples were collected from mouse eyes, and serum was harvested by centrifugation. Mice were sacrificed, and the whole body, tumor, spleen and thymus were weighed immediately. The rate of tumor inhibition and organ indexes were calculated. The expression levels of serum cytokines, P-glycoprotein (P-GP) and multidrug resistance (MDR) 1 mRNA in tumor tissues were detected using enzyme-linked immunosorbent assay, Western blotting, and quantitative myeloid-derived suppressor cells reverse transcription-polymerase chain reaction, respectively. RESULTS: The tumor inhibition rates in the treatment groups of Adriamycin (ADM) + Astragalus polysaccharides (APS) (50 mg/kg), ADM + APS (100 mg/kg), and ADM + APS (200 mg/kg) were significantly higher than in the ADM group (72.88% vs 60.36%, P = 0.013; 73.40% vs 60.36%, P = 0.010; 77.57% vs 60.36%, P = 0.001). The spleen indexes of the above groups were also significantly higher than in the ADM group (0.65 ± 0.22 vs 0.39 ± 0.17, P = 0.023; 0.62 ± 0.34 vs 0.39 ± 0.17, P = 0.022; 0.67 ± 0.20 vs 0.39 ± 0.17, P = 0.012), and the thymus indexes of the ADM + APS (100 mg/kg) and ADM + APS (200 mg/kg) groups were significantly higher than in the ADM group (0.20 ± 0.06 vs 0.13 ± 0.04, P = 0.029; 0.47 ± 0.12 vs 0.13 ± 0.04, P = 0.000). APS was found to exert a synergistic anti-tumor effect with ADM and to alleviate the decrease in the sizes of the spleen and thymus induced by AMD. The expression of interleukin-1α (IL-1α), IL-2, IL-6, and tumor necrosis factor-α (TNF-α) was significantly higher in the ADM + APS (50 mg/kg), ADM + APS (100 mg/kg) and ADM + APS (200 mg/kg) groups than in the ADM group; and IL-10 was significantly lower in the above groups than in the ADM group. APS could increase IL-1α, IL-2, IL-6, and TNF-α expression and decrease IL-10 levels. Compared with the ADM group, APS treatment at a dose of 50-200 mg/kg could down-regulate MDR1 mRNA expression in a dose-dependent manner (0.48 ± 0.13 vs 4.26 ± 1.51, P = 0.000; 0.36 ± 0.03 vs 4.26 ± 1.51, P = 0.000; 0.21 ± 0.04 vs 4.26 ± 1.51, P = 0.000). The expression level of P-GP was significantly lower in the ADM + APS (200 mg/kg) group than in the ADM group (137.35 ± 9.20 mg/kg vs 282.19 ± 20.54 mg/kg, P = 0.023). CONCLUSION: APS exerts a synergistic anti-tumor effect with ADM in H22 tumor-bearing mice. This may be related to its ability to enhance the expression of IL-1α, IL-2, IL-6, and TNF-α, decrease IL-10, and down-regulate MDR1 mRNA and P-GP expression levels.

Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography-tandem mass spectrometry.

A rapid and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250mmx4.6mm, 5.0microm, USA) at 25 degrees C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11-5.42microg/g in tissues and 0.18-9.04microg/ml in plasma, for RFP were 0.12-1200microg/g in tissues and 4.0-200microg/ml in plasma, and for LVX were 0.13-26.2microg/g in tissues and 0.09-4.53microg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13microg/g and for those in mouse plasma were 18.1, 20.0 and 21.8ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05microg/g and for those in mouse plasma were 5.5, 6.0 and 6.6ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.