Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway.

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G1-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

Development and validation of a quantitative LC-MS/MS method for the simultaneous determination of ceftolozane and tazobactam in human plasma and urine.

The purpose of this work was to develop and validate a single sensitive, selective and rapid bioanalytical method to determine ceftolozane and tazobactam concentrations in human plasma and urine and to use this method to analyze samples from a human clinical study. Human plasma and urine samples were prepared by protein precipitation using a solution of acetonitrile, water and formic acid. Following protein precipitation, samples were analyzed by liquid chromatography tandem mass spectrometry. Chromatographic resolution was achieved on a Kinetex PFP column using a gradient elution, a flow rate of 0.4 mL/min, and a total run time of 5 min. Positive electrospray ionization was employed and analytes were quantitated using multi-reaction monitoring mode. Method validation was conducted in accordance with Unites States Food and Drug Administration's regulatory guidelines for bioanalytical method validation. Calibration curves were determined to linear over the range of 0.1 to 40 µg/mL for ceftolozane and 0.05 to 20 µg/mL for tazobactam. The method was determined to be accurate (-6.24 to 12.53 percent relative error), precise (less than 13.28 percent standard deviation) and sensitive in both human plasma and urine. Ceftolozane and tazobactam were determined to be stable across a battery of stability studies including autosampler, benchtop, freeze/thaw and long-term stability. This validated method successfully applied to human clinical samples to determine the concentration versus time profiles of the intravenously administered combination of Zerbaxa (ceftolozane-tazobactam) in burn patients.

Bioanalytical method development and validation of a liquid chromatography-tandem mass spectrometry method for determination of ß-lapachone in human plasma.

The purpose of this work was to develop and validate a rapid, sensitive and robust liquid chromatography tandem mass spectrometric method for the quantification of ß-lapachone in human plasma and to use that method to analyze human clinical samples. Sample preparation for the developed method involved liquid-liquid extraction using ethyl acetate for extraction of ß-lapachone and cryptotanshinone (internal standard) from human plasma. Chromatographic resolution was achieved on a Kinetex C18 column using a gradient elution and a chromatographic flow rate of 0.5 mL/min. The retention times of ß-lapachone and cryptotanshinone were 1.98 and 2.28 min, respectively, and the method had a total run time of 4 min. Bioanalytical method validation was conducted in accordance with the United States Food and Drug Administration regulatory guidelines. The method was validated over 2 calibration ranges in order to support high- and low-dose clinical studies. Calibration curve-1 covered the range of 0.25-50 ng/mL and calibration curve-2 covered the range of 50-2000 ng/mL. The method was determined to be accurate (percent relative errors between -1.07 to 5.36 %), precise (percent relative standard deviations less than 7.4), and sensitive (LLOQ 0.25 ng/mL). ß-lapachone was determined to be stable (% change from time = 0 between -11.6 and 12.6 %) across the autosampler, benchtop, freeze/thaw and long-term (63 days) stability studies. The validated bioanalytical method was employed to determine ß-lapachone concentrations in human plasma samples from a clinical study.

ATI-2173, a Novel Liver-Targeted Non-Chain-Terminating Nucleotide for Hepatitis B Virus Cure Regimens.

ATI-2173 is a novel liver-targeted molecule designed to deliver the 5'-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5'-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development. ATI-2173 was designed by modifying clevudine with a 5'-phosphoramidate to deliver the 5'-monophosphate to the liver. Bypassing the first phosphorylation step of clevudine, the 5'-monophosphate is converted to the active 5'-triphosphate in the liver. ATI-2173 is a selective inhibitor of HBV with an anti-HBV 50% effective concentration (EC50) of 1.31 nM in primary human hepatocytes, with minimal to no toxicity in hepatocytes, skeletal muscle, liver, kidney, bone marrow, and cardiomyocytes. ATI-2173 activity was decreased by viral polymerase mutations associated with entecavir, lamivudine, and adefovir resistance, but not capsid inhibitor resistance mutations. A single oral dose of ATI-2173 demonstrated 82% hepatic extraction, no food effect, and greatly reduced peripheral exposure of clevudine compared with equimolar oral dosing of clevudine. Despite reduced plasma clevudine exposure, liver concentrations of the 5'-triphosphate were equivalent following ATI-2173 versus clevudine administration. By selectively delivering the 5'-monophosphate to the liver, while retaining the unique anti-HBV activity of the 5'-triphosphate, ATI-2173 may provide an improved pharmacokinetic profile for clinical use, reducing systemic exposure of clevudine and potentially eliminating skeletal myopathy.

Steady-State Ceftazidime-Avibactam Serum Concentrations and Dosing Recommendations in a Critically Ill Patient Being Treated for Pseudomonas aeruginosa Pneumonia and Undergoing Continuous Venovenous Hemodiafiltration.

Ceftazidime-avibactam (CAZ-AVI) is a novel intravenous ß-lactam/ß-lactamase inhibitor combination used in the treatment of multidrug-resistant (MDR) gram-negative infections. Although renal dosing recommendations exist for the medication, limited data are available for dosing in patients receiving continuous renal replacement therapy. In this report, we describe a case in which CAZ-AVI 2.5 g was administered as a 2-hour infusion every 8 hours to a 50-year-old critically ill patient with MDR Pseudomonas aeruginosa (CAZ-AVI minimum inhibitory concentration [MIC] 8 µg/ml) pneumonia who was also receiving continuous venovenous hemodiafiltration (CVVHDF). Total serum concentrations of both ceftazidime and avibactam were measured at ~0.5, 2, 4, and 6 hours after completion of the 2-hour infusion of the 11th dose of CAZ-AVI. Ceftazidime pharmacokinetic parameters were as follows: maximum serum concentration (Cmax ) 152.39 µg/ml, half-life 5.17 hours, volume of distribution at steady state (Vdss ) 11.51 L, clearance 1.54 L/hour, and area under the concentration-time curve (AUC) 1295.38 hour•µg/ml. This regimen achieved free ceftazidime serum concentrations more than 4 times the MIC for 100% of the dosing interval. Avibactam pharmacokinetic parameters were as follows: Cmax 35.83 µg/ml, half-life 5.92 hours, Vdss 12.44 L, clearance 1.45 L/hour, and AUC 343.44 hour•µg/ml, which achieved free avibactam concentrations above 1 µg/ml for 100% of the dosing interval. Higher CAZ-AVI dosing is critical in the treatment of pneumonia due to limited ceftazidime penetration into epithelial lining fluid; however, epithelial lining fluid drug concentrations were not collected or measured. Based on this case report and the available evidence, a dose of CAZ-AVI 2.5 g infused over 2 hours every 8 hours appears to be appropriate for critically ill patients who are being treated for pneumonia and are receiving CVVHDF.

The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection.

The New World alphaviruses Venezuelan, Eastern, and Western equine encephalitis viruses (VEEV, EEEV and WEEV, respectively) commonly cause a febrile disease that can progress to meningoencephalitis, resulting in significant morbidity and mortality. To address the need for a therapeutic agent for the treatment of Alphavirus infections, we identified and pursued preclinical characterization of a ribonucleoside analog EIDD-1931 (ß-D-N4-hydroxycytidine, NHC), which has shown broad activity against alphaviruses in vitro and has a very high genetic barrier for development of resistance. To be truly effective as a therapeutic agent for VEEV infection a drug must penetrate the blood brain barrier and arrest virus replication in the brain. High plasma levels of EIDD-1931 are rapidly achieved in mice after oral dosing. Once in the plasma EIDD-1931 is efficiently distributed into organs, including brain, where it is rapidly converted to its active 5'-triphosphate. EIDD-1931 showed a good safety profile in mice after 7-day repeated dosing with up to 1000 mg/kg/day doses. In mouse model studies, EIDD-1931 was 90-100% effective in protecting mice against lethal intranasal infection when therapeutic treatment was started as late as 24 h post-infection, and partial protection was achieved when treatment was delayed for 48 h post-infection. These results support further preclinical development of EIDD-1931 as a potential anti-alphavirus drug.

UHPLC-MS/MS analysis of arachidonic acid and 10 of its major cytochrome P450 metabolites as free acids in rat livers: effects of hepatic ischemia.

The cytochrome P450 metabolites of arachidonic acid (AA) are mostly present in tissues, such as the liver, as bound to phospholipids, with only a small fraction available as free acids. The purpose of this study was to develop and validate a UHPLC-MS/MS method for quantitation of free liver concentrations of AA and four epoxygenated (5,6-, 8,9-, 11,12-, and 14,15-EET), four dihydroxylated (5,6-, 8,9-, 11,12-, and 14,15-DHET), and two ω/(ω-1) hydroxylated (19- and 20-HETEs) metabolites of AA in rat livers using deuterated internal standards. The analytes were rapidly and efficiently (79-92%) recovered from 100mg of fresh liver into methanol. After evaporation, the reconstituted samples were injected either undiluted (for the simultaneous analysis of the metabolites) into a gradient or diluted (for AA analysis) into an isocratic UHPLC system with run times of 5 and 2 min, respectively. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥ 0.98) in the concentration ranges tested for metabolites (0.19-120 ng/g liver) and AA (7.8-500 µg/g liver). The lower limit of quantitation of the assay was between 0.57 and 5.6 pg injected on column for different AA metabolites. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. Additionally, matrix effect was minimal for most analytes. Freeze-thaw of samples drastically increased the free liver concentrations of analytes, presumably due to their release from the membrane storage sites. Therefore, fresh liver samples should be used for analysis. However, the methanolic extracts may be stored at -80°C for at least two weeks without any compromise. The method was successfully used in the measurement of all the analytes in the rats subjected to 60 min of hepatic ischemia (n=6) or sham operation (n=6). Ischemia resulted in significantly higher free concentrations of AA and most of its studied metabolites. The method is precise, accurate, and sensitive for measurement of free liver concentrations of AA and its P450 metabolites in the rat liver.