br Materials and Methods br Paclitaxel Concentration Determi

Materials and Methods
Paclitaxel Concentration Determination in Cell Pellets and Supernatants Calu-6 cells were treated with 100nM TVB-3166 for 24h or 50nM TVB-3664 for 48h. Paclitaxel was added to the cell culture media to a final concentration of 6nM. After the 2h of incubation with paclitaxel, cells and supernatant were collected for analysis. In the control groups, cells were not pre-treated with FASN inhibitors. Treatments were performed in Advanced formulation MEM (Thermo Fisher) supplemented with 1% charcoal-stripped FBS and 1% l-glutamine. All treatments were performed in duplicate. At the end of the incubation, cell culture media were collected into polypropylene tubes; cell pellets were resuspended in 0.5ml of PBS, collected into the polypropylene tubes, and cells were counted. 100μl of media or cell suspension were treated with 200μl of acetonitrile containing the internal standard. Paclitaxel was extracted from cells and media by extensive shaking and further centrifugation. For cell supernatants, 10μl was subjected to LC-MS-MS analysis. The concentration of paclitaxel in cell pellet and supernatant samples was measured by a quantitative LC-MS-MS method using deuterated paclitaxel as an internal standard. The method involved protein precipitation, extraction of paclitaxel and the internal standard from biological matrices, and injection of diluted extract into LC-MS-MS for paclitaxel detection. Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1mg/ml each. Spiking solutions of paclitaxel were prepared by appropriate dilutions with acetonitrile. HPLC or MS grade reagents were used to prepare chromatographic mobile phases. Chromatographic separations were performed by Thermo Scientific Aria LC system consisting of CTC PAL cooling auto-sampler and Agilent binary pumps. A Thermo BetaBasic-18 column (50×2.1mm, 5μM particle size) was used. Mobile phases composed of water, containing 0.1% formic nk1 antagonist (mobile phase A), and acetonitrile with 0.1% of formic acid (mobile phase B) were used. The elution gradient to achieve chromatographic separation was 5–95% B in 2min, 1min at 95% B, and 0.5min equilibrium at 5% B. Flow rate was set at 1ml/min. The eluted analytes were detected using an API Sciex 4000 in positive electrospray mode. A Parker–Ballston LC-MS gas generator supplied nitrogen. The paclitaxel and IS were analyzed by multiple reaction monitoring mode (MRM) with the transitions of 876.6→308.1 for paclitaxel, and 881.6→313.5 for IS. Both paclitaxel and IS were monitored as their respective most abundant adduct ions [M+Na]+ to achieve better sensitivity. Data were acquired by Analyst 1.5 software. The calibration curves were established by plotting peak area ratios of paclitaxel to IS versus nominal concentration. A weighted least-squares linear regression was applied to generate a calibration curve. The calculation of paclitaxel concentrations was performed with Analyst 1.5 software.
Results
Discussion Combined FASN inhibition and taxane treatment demonstrated significantly enhanced anti-tumor efficacy in diverse xenograft tumor models from lung, ovarian, prostate, and pancreatic cancers compared to single agent therapy. This was observed in 6 of 6 xenograft efficacy studies, and in 3 of 6 studies, combination treatment caused tumor regression, which was not observed with single-agent treatment in any of the tumor models. Previous in vitro and in vivo studies with TVB-3166 supported investigating several different tumor types and also suggested a focused study of KRAS mutant non-small-cell lung tumor models (Ventura et al., 2015). TVB-3166 belongs to a series of proprietary FASN inhibitors with high chemical similarity. TVB-2640, the lead molecule in this series of inhibitors, is completing Phase I clinical development for the treatment of solid tumors, where it demonstrated excellent oral bioavailability and pharmacokinetics that translated into sustained target inhibition and highly promising signs of clinical efficacy at well-tolerated doses (Dean et al., 2016). TVB-2640 clinical development is investigating both monotherapy and combination therapy administration with taxane therapies such as paclitaxel. Our current studies investigated two cell-line-derived and one patient-derived lung tumor xenograft models and significant tumor regression was induced by combination treatment in 2 of these 3 models. The results also demonstrated that significantly improved efficacy occurs when FASN inhibition is combined with either paclitaxel or docetaxel, supporting a model that the mechanism of additivity or synergy is linked directly to the FASN and taxane mechanisms of action. A scientific rationale and model for the enhanced activity observed with the drug combination was developed from cell biology studies (Fig. 7). This model incorporates findings from the current studies and from previously reported studies showing that single-agent FASN inhibition with TVB-3166 induces apoptosis in tumor cells by remodeling cell membranes and disrupting localization of lipid raft-associated proteins, inhibiting signaling pathways that include Akt and beta-catenin, and reprogramming gene expression (Ventura et al., 2015).