Ayako Furugen

Determination of ω-6 and ω-3 PUFA metabolites in human urine samples using UPLC/MS/MS.

The ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the precursors of various bioactive lipid mediators including prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acid, isoprostanes, lipoxins, and resolvins (Rvs). These lipid mediators play important roles in various physiological and pathological processes. The quantitative determination of PUFA metabolites seems necessary for disease research and for developing biomarkers. However, there is a paucity of analytical methods for the quantification of ω-6 and ω-3 PUFA metabolites—the specialized pro-resolving mediators (SPMs) present in the human urine. We developed a method for the quantification of ω-6 and ω-3 PUFA metabolites present in human urine using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). The developed method shows good linearity, with a correlation coefficient >0.99 for all of the analytes. The validation results indicate that our method is adequately reliable, accurate, and precise. The method was successfully used to examine urine samples obtained from 43 healthy volunteers. We could identify 20 PUFA metabolites, and this is the first report of the quantitative determination of RvD1, 17(R)-RvD1, 11-dehydro thromboxane B3, RvE2, and 5(S)-HETE in human urine. The urinary 8-iso PGF2α and PGE2 levels were significantly higher in the men smokers than in the men nonsmokers (p < 0.05). In this study, we developed an accurate, precise, and novel analytical method for estimating the ω-6 and ω-3 PUFA metabolites, and this is the first report that the SPMs derived from EPA and DHA are present in human urine.

A rapid and sensitive LC/ESI–MS/MS method for quantitative analysis of docetaxel in human plasma and its application to a pharmacokinetic study

Docetaxel is a taxane family antineoplastic agent widely employed in cancer chemotherapy. We developed a liquid chromatography/tandem mass spectrometry method for the determination of docetaxel in human plasma. Plasma samples were deproteinized by acetonitrile containing internal standard paclitaxel. Chromatographic separation was performed on a TSKgel ODS-100 V 3 μm (50 mm × 2.0 mm i.d.) column using a mobile phase composed of acetonitrile-methanol-water-formic acid (50:5:45:0.1, v/v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. This method covered a linearity range of 5-5000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra-day precision and inter-day precision (R.S.D.) of analysis were less than 6.7%, and the accuracy (R.E.) was within ± 9.0% at the concentrations of 5, 20, 200, and 2000 ng/mL. The total run time was 5.0 min. This method was successfully applied for clinical pharmacokinetic investigation.

AMP-activated protein kinase regulates the expression of monocarboxylate transporter 4 in skeletal muscle

AIMS The aim of this study was to determine the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, on monocarboxylate transporter 4 (MCT4) expression in rat skeletal muscle and a prototypic embryonal rhabdomyosarcoma cell line (RD cells). MAIN METHODS We examined the alteration in Glucose transporter 4 (GLUT4) and MCT4 mRNA levels by quantitative real-time PCR. Alteration in GLUT4 and MCT4 protein levels was examined by Western blotting. KEY FINDINGS In an in vivo study, AICAR increased MCT4 mRNA and protein levels in a fiber-type specific manner. In an in vitro study, AICAR increased MCT4 mRNA and protein levels. Moreover, AICAR-induced MCT4 expression was blocked by Compound C, an AMPK inhibitor. SIGNIFICANCE In this study, we found that AMPK activation induced expression of MCT4 in RD cells and rat skeletal muscle in a fiber-type specific manner. These results indicate the possible involvement of an AMPK-mediated pathway associated with MCT4 expression in skeletal muscle.

Contribution of multidrug resistance-associated proteins (MRPs) to the release of prostanoids from A549 cells

Previous studies indicated that several members of the multidrug resistance-associated protein (MRP) family mediate the transport of prostanoids. However, theimportance of MRPs in the release process of prostanoids has not been fully elucidated. In this study, we investigated the contribution of MRPs, including MRP1, MRP2, and MRP4, to the release process of the prostanoids from human lung adenocarcinoma epithelial A549 cells. The extracellular levels of PGE2, PGF2α, and TXB2 (a metabolite of TXA2) were decreased by treatment with MRP inhibitors (dipyridamole, MK571, and probenecid). The studies using membrane vesicle suggest that the effects of the inhibitors were in part by inhibiting MRP4 function. The effects of knockdown of each MRP (MRP1, MRP2, and MRP4) were also investigated. The extracellular levels of PGE2 and PGF2α were significantly decreased after MRP4 knockdown. Our results suggest that MRPs including MRP4 contribute the release process of prostanoids in A549 cells.

Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry.

Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E(2) (PGE(2)), PGD(2), PGF(2α), 6-keto PGF(1α), and TXB(2), in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C(18) column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL (r(2)>0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng (r(2)>0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.

A full validated hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the quantification of oxaliplatin in human plasma ultrafiltrates.

Oxaliplatin is a platinum agent that is used for treatment of colorectal cancer. A sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the quantification of oxaliplatin was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin as an internal standard and further diluted with acetonitrile. Chromatographic separation of oxaliplatin and the internal standard was achieved with a column modified with phosphorylcholine and an isocratic mobile phase (acetonitrile/water/acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. The lower limit of quantification for oxaliplatin was 25ng/mL. The linearity range of the method was from 25 to 5000ng/mL. The intra-day precision and inter-day precision (RSD) ranged from 0.8 to 6.1%, and the accuracy (RE) was within ±4.5%. The extraction recoveries from human plasma ultrafiltrates were 83.6-91.6%, and ion suppression caused by matrix components was 86.7-88.5% at three different levels, respectively. This method was applied to a clinical pharmacokinetic study of oxaliplatin in a cancer patient. The maximum concentration of colorectal cancer patient administered oxaliplatin was 1650ng/mL.

Regulation of human monocarboxylate transporter 4 in skeletal muscle cells: The role of protein kinase C (PKC)

In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.

Successful Colistin Treatment of Multidrug-Resistant Pseudomonas aeruginosa Infection Using a Rapid Method for Determination of Colistin in Plasma: Usefulness of Therapeutic Drug Monitoring

A 56-year-old woman with systemic lupus erythematosus had bacteremia due to multidrug-resistant Pseudomonas aeruginosa (MDRP). She was initially treated with imipenem-cilastatin, tobramycin, and aztreonam; however, MDRP was still detected intermittently in her plasma. Multidrug-susceptibility tests demonstrated that MDRP was susceptible only to colistin. Therefore, in addition to these antibiotics, the administration of intravenous colistin methanesulfonate, a prodrug formula of colistin, was started at a daily dose of 2.5 mg/kg (as colistin base activity). The initial dose setting was based on the patients renal function (baseline creatinine clearance=32.7 mL/min). After initiating colistin, the patients C-reactive protein levels gradually decreased. Blood cultures showed no evidence of MDRP on days 8, 14, and 22 after colistin initiation. However, the patients renal function went from bad to worse owing to septic shock induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. A few days later, the trough plasma levels of colistin were 7.88 mg/L, which appeared to be higher than expected. After decreasing the colistin dose, the patients renal function gradually improved. On the final day of colistin treatment, the plasma levels decreased to 0.60 mg/L. MDRP could not be detected in blood culture after colistin treatment. Therefore, we successfully treated a case of bloodstream infection due to MDRP by therapeutic drug monitoring (TDM) of colistin. It is suggested that the monitoring of blood colistin levels by liquid chromatography-tandem mass spectrometry can contribute to safer, more effective antimicrobial therapy of MDRP because TDM facilitates quick decisions on dose adjustments.

Effects of proton pump inhibitors and famotidine on elimination of plasma methotrexate: evaluation of drug-drug interactions mediated by organic anion transporter 3

Methotrexate (MTX) is an antifolate agent used in the treatment of numerous types of cancer, and eliminated by active tubular secretion via organic anion transporter 3 (OAT3). Gastric antisecretory drugs, such as proton pump inhibitors (PPIs) and histamine H2 receptor antagonists, are widely used among patients with cancer in clinical practice. The aim of the present study was to analyse the potential drug–drug interactions between MTX and gastric antisecretory drugs in high‐dose MTX (HD‐MTX) therapy. The impact of PPIs on the plasma MTX concentration on 73 cycles of HD‐MTX therapy was analysed retrospectively in 43 patients. Also investigated was the involvement of OAT3 in PPI–MTX drug interaction in an in vitro study using human OAT3 expressing HEK293 cells. In a retrospective study, patients who received a PPI had significantly higher MTX levels at 48 h (0.38 vs. 0.15 μmol l−1, respectively, p = 0.000018) and 72 h (0.13 vs. 0.05 μmol l−1, respectively, p = 0.0002) compared with patients who did not receive a PPI (but received famotidine). Moreover, in vitro experiments demonstrated that PPIs (esomeprazole, lansoprazole, omeprazole and rabeprazole) inhibited hOAT3‐mediated uptake of MTX in a concentration‐dependent manner (IC50 values of 0.40–5.5 μ m), with a rank order of lansoprazole > esomeprazole > rabeprazole > omeprazole. In contrast to PPIs, famotidine showed little inhibitory effect on hOAT3‐mediated MTX uptake. These results demonstrated that co‐administration of PPI, but not famotidine, could result in a pharmacokinetic interaction that increases the plasma MTX levels, at least in part, via hOAT3 inhibition.

Quantification of intracellular and extracellular eicosapentaenoic acid-derived 3-series prostanoids by liquid chromatography/electrospray ionization tandem mass spectrometry

3-Series prostanoids are bioactive lipid mediators synthesized from eicosapentaenoic acid (EPA). Determination of intracellular and extracellular levels of prostanoids is needed to elucidate the mechanism of action, and we therefore developed a method for quantification of intracellular and extracellular levels of 3-series prostanoids (including prostaglandin E3 (PGE3), PGD3, PGF3α, thromboxane B3 (TXB3), and Δ(17)-6-keto PGF1α) by using liquid chromatography/electrospray ionization tandem mass spectrometry. The separation of prostanoids was performed with a CAPCELL PAK C18 MG II column (2.0mm×150mm, 3µm) with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). This method was validated for measurement of both extracellular and intracellular samples with high levels of precision and accuracy. We applied this method to human lung epithelial A549 cells stimulated with calcium ionophore A23187 under the condition of arachidonic acid or EPA treatment and we could measure PGE3 in both intracellular and extracellular samples.