Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo.

In this study, the therapeutic effect and possible mechanism of the total biflavonoid extract of Selaginella doederleinii Hieron (SDTBE) against cervical cancer were originally investigated in vitro and in vivo. First, the inhibition of SDTBE on proliferation of cervical cancer HeLa cells was evaluated, followed by morphological observation with AO/EB staining, Annexin V/PI assay, and autophagic flux monitoring to evaluate the possible effect of SDTBE on cell apoptosis and autophagy. Cell cycle, as well as mitochondrial membrane potential (ΔÑ°m), was detected with flow cytometry. Further, the apoptosis related protein expression and the autophagy related gene LC3 mRNA transcription level were analyzed by Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Finally, the anti-cervical cancer effect of the SDTBE was also validated in vivo in HeLa cells grafts mice. As results, SDTBE inhibited HeLa cells proliferation with the IC50 values of 49.05 ± 6.76 and 44.14 ± 4.75 µg/mL for 48 and 72 h treatment, respectively. The extract caused mitochondrial ΔÑ° loss, induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating Caspase-9 and Caspase-3, promoting cell autophagy and blocking the cell cycle in G0/G1 phase. Furthermore, 100, 200, and 300 mg/kg SDTBE suppressed the growth of HeLa cells xenografts in mice with the mean inhibition rates, 25.3 %, 57.5 % and 62.9 %, respectively, and the change of apoptosis related proteins and microvascular density was confirmed in xenografts by immunohistochemistry analysis. The results show that SDTBE possesses anti-cervical cancer effect, and the mechanism involves in activating Caspase-dependent mitochondrial apoptosis pathway.

Inflammation affects the pharmacokinetics of risperidone: Does the dose need to be adjusted during the acute-phase reaction?

BACKGROUND: Several studies have indicated that the plasma concentration of risperidone increases 3-5-fold during the acute-phase reaction (APR) of inflammation or infection. Psychiatric symptoms are present or deteriorate when the dose is lowered; thus, the complex effects of inflammation on the pharmacokinetics of risperidone need to be examined. METHODS: We established a APR model in rabbits induced by lipopolysaccharide (LPS) and studied the effect of APR on pharmacokinetics, distribution and disposition of risperidone in vivo and in vitro. RESULTS: Following intramuscular administration, the plasma exposures for risperidone and its active metabolite (9-hydroxyrisperidone) were increased approximately 6-fold on day 2 of inflammation. The exposure values did not change between day 2 and 5 of inflammation, nor did the metabolite-to-parent ratio before and during inflammation. Following oral administration, the increase of risperidone exposure was twice as high as that following intramuscular administration during APR. However, the concentration of risperidone and 9-hydroxyrisperidone in brain tissue was similar between the inflammatory and control groups. Moreover, the plasma protein binding (PPB) of risperidone and 9-hydroxyrisperidone associated with inflammation were all increased to >99 %. In addition, risperidone and 9-hydroxyrisperidone were not substrates of the key transporters, OATP1B3, OCT2, OAT3, MATE-1, or MATE-2 K. The expression of progesterone X receptor and P-glycoprotein was inhibited by LPS. CONCLUSION: During APR, reduced expression of P-glycoprotein and increased PPB were responsible for increased exposure in plasma, while maintaining stable concentrations in the brain, and risperidone does not need to be dose-adjusted so as to achieve psychopharmacological outcomes.

Optimization and validation of the liquid chromatography coupled to tandem mass spectrometry method for assessing octreotide release from microspheres during inflammation in rabbit models.

To study the effect of acute-phase reaction (APR) of inflammation on the release of octreotide acetate microsphere (Sandostatin®, SLAR) at a clinical dose, a more sensitive liquid chromatography coupled to tandem mass spectrometry analysis method needs to be developed because of the low plasma concentrations of octreotide. Solid-phase microextraction with an Oasis® HLB µElution plate was adopted for sample preparation. Extraction recovery ranged from 65.7 % to 73.2 %, and the matrix effect was negligible. High sensitivity and an intense chromatographic peak were acquired by optimizing the chromatography and mass spectrometry conditions. The lower limit of quantitation (LLOQ) was 0.01 ng/mL based on 100 µL of plasma, and linearity ranged from 0.01 to 5.0 ng/mL. The coefficients of variations for intraday and interday precision were less than 4.4 %, and the relative error of accuracy was within 5.7 %. The validated method was successfully applied to pharmacokinetics studies of SLAR in a seven-day inflammation model of rabbits, indicating that the APR did not affected the release and pharmacokinetics of the octreotide microspheres.

9-Cyclopropylmethoxy-dihydrotetrabenazine and its stereoisomers as vesicular monoamine transporter-2 inhibitors.

Aim: To separate and evaluate 9-cyclopropylmethoxy-dihydrotetrabenazine (13a) and its stereoisomers for their high affinity for vesicular monoamine transporter-2 (VMAT2). Method: Stereoisomers of 13a were separated and configurations were ascertained by chiral chromatography and crystal diffraction combined with 1H-1H NOESY assay. Possible binding modes of eight stereoisomers and VMAT2 were explored by molecular docking assays. The VMAT2 affinity of the stereoisomers, inhibition in vivo and pharmacokinetics in rats were evaluated. Results: Three stereoisomers were obtained: P1, P2 and P3, and all had similar VMAT2 binding modes. P2 [(2R, 3R, 11bR)-13a] showed the highest potential VMAT2 binding activity (Ki = 0.75 nM), decreased locomotor activity in rats and had an oral absolute bioavailability of 92.0%. Conclusion: P2 has good efficacy and pharmacokinetic properties and warrants further development to treat tardive dyskinesia.

(+)-9-Trifluoroethoxy-α-Dihydrotetrabenazine as a Highly Potent Vesicular Monoamine Transporter 2 Inhibitor for Tardive Dyskinesia.

Valbenazine and deutetrabenazine are the only two therapeutic drugs approved for tardive dyskinesia based on blocking the action of vesicular monoamine transporter 2 (VMAT2). But there exist demethylated inactive metabolism at the nine position for both them resulting in low availability, and CYP2D6 plays a major role in this metabolism resulting in the genetic polymorphism issue. 9-trifluoroethoxy-dihydrotetrabenazine (13e) was identified as a promising lead compound for treating tardive dyskinesia. In this study, we separated 13e via chiral chromatography and acquired R,R,R-13e [(+)-13e] and S,S,S-13e [(-)-13e], and we investigated their VMAT2-inhibitory activity and examined the related pharmacodynamics and pharmacokinetics properties using in vitro and in vivo models (+)-13e displayed high affinity for VMAT2 (Ki = 1.48 nM) and strongly inhibited [3H]DA uptake (IC50 = 6.11 nM) in striatal synaptosomes. Conversely, its enantiomer was inactive. In vivo, (+)-13e decreased locomotion in rats in a dose-dependent manner. The treatment had faster, stronger, and longer-lasting effects than valbenazine at an equivalent dose. Mono-oxidation was the main metabolic pathway in the liver microsomes and in dog plasma after oral administration, and glucuronide conjugation of mono-oxidized and/or demethylated products and direct glucuronide conjugation were also major metabolic pathways in dog plasma. O-detrifluoroethylation of (+)-13e did not occur. Furthermore, CYP3A4 was identified as the primary isoenzyme responsible for mono-oxidation and demethylation metabolism, and CYP2C8 was a secondary isoenzyme (+)-13e displayed high permeability across the Caco-2 cell monolayer, and it was not a P-glycoprotein substrate as demonstrated by its high oral absolute bioavailability (75.9%) in dogs. Thus, our study findings highlighted the potential efficacy and safety of (+)-13e in the treatment of tardive dyskinesia. These results should promote its clinical development.

Ethyl Acetate Extract of Selaginella doederleinii Hieron Induces Cell Autophagic Death and Apoptosis in Colorectal Cancer via PI3K-Akt-mTOR and AMPKα-Signaling Pathways.

Colorectal cancer is one type of cancer with high incidence rate and high mortality worldwide. Thus, developing new chemotherapeutic drugs is important. The Selaginella doederleinii Hieron ethyl acetate (SDEA) extract showed good anti-colon cancer effect in vitro and in vivo, but its mechanism is unclear. This study aimed to further reveal the anti-colon cancer effect of SDEA and its possible mechanism. The effects on cell viability, apoptosis, autophagy, and cell cycle in colorectal cells (HT29 and HCT116) were studied using MTT assay, fluorescence microscopy, transmission electron microscopy, and flow cytometry. The mechanisms were further studied using cell transfection, Western blot, and real-time quantitative polymerase chain reaction assay. The effect of xenotransplantation in vivo was observed using immunohistochemistry. Results showed that SDEA inhibited cell proliferation and induced cell morphological changes, cell cycle arrest, autophagy, and apoptosis. It also induced loss of mitochondrial membrane potential, increased the autophagic flux, raised the ratio of Bax/Bcl-2, activated caspases, and inhibited PI3K-Akt-mTOR signaling pathways. Furthermore, SDEA inhibited the growth of xenograft tumors in a dose-dependent manner. Immunohistochemistry analysis confirmed the alteration of autophagy- and apoptosis-related proteins and immunohistochemical microvascular density in xenografts, which were consistent with the results in vitro. Therefore, SDEA is important for developing candidate drugs against colorectal cancers.

Simultaneous quantification of five biflavonoids in rat plasma by LC-ESI-MS/MS and its application to a comparatively pharmacokinetic study of Selaginella doederleinii Hieron extract in rats.

Selaginella doederleinii Hieron is a widely used as folk Chinese medicine for treatment of different cancers. Our previous investigations have confirmed that the total biflavonoids in ethyl acetate extract from S. doederleinii (SDEA) have favorable anticancer potentials. However, the in vivo process of its bioactive ingredients remains unknown. In this paper, a sensitive and reliable method was developed for simultaneous quantification of main five biflavonoids, including amentoflavone, robustaflavone, 2″,3″-dihydro-3',3″-biapigenin, 3',3″-binaringenin and delicaflavone in the ethyl acetate extract of S. doederleinii (SDEA extract) in rat plasma by high-performance liquid chromatography with electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed using an Ultimate® XB-C18 (100×2.1mm, 3.5µm) with gradient elution of water (0.5% acetic acid) and acetonitrile at 0.2mL/min. All analytes with internal standard (chrysin) were detected using selective reaction monitoring (SRM) in negative ionization mode. The method showed a good linearity over a wide concentration range (r2>0.99). The limits of quantification for the biflavonoids were less than 10ng/mL. The developed method was applied to the comparatively pharmacokinetic study of the five biflavonoids after oral or intravenous administration of SDEA extract in rats. In addition, in silico assessments of permeability and solubility of these biflavonoids were also performed to understand their poor bioavailability. It is the first time to report the in vivo process profiles of the biflavonoids of SDEA extract in rats.

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. KEY MESSAGES: Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.