Development of a New Method for Simultaneous Quantitation of Plasma Concentrations of Voriconazole and Voriconazole N-Oxide Using Column-Switching LC-MS/MS and Its Application in Therapeutic Drug Monitoring.

Background: Voriconazole therapy for fungal infections usually continues for several years and is often administered on an outpatient basis. Maintaining the voriconazole plasma concentration in the therapeutic range is highly important for effective therapy; however, it is difficult to obtain sufficient information to assess the voriconazole concentration in outpatients. Therefore, we developed a method to simultaneously measure the plasma concentrations of voriconazole and its major metabolite, voriconazole N-oxide, to obtain rapid results after outpatient blood collection and before medical consultation and to attain a better understanding of adherence and the drug-drug interactions of voriconazole. Methods: Fifty microliters of patient plasma was deproteinized with methanol, injected into the liquid chromatography-tandem mass spectrometry system, and purified using an online column. Separation was achieved on an InertSustain C18 column (2.1 mm id × 50 mm, 2 µm) with a mobile phase of 30:70 (0.1% formic acid in water:methanol) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode. Results: The analysis time was 4 min. The calibration curve was linear, in the range of 0.1 µg/mL to 20 µg/mL for voriconazole and 0.05 µg/mL to 10 µg/mL for voriconazole N-oxide, with a coefficient of determination at R2 > 0.999. Conclusion: There is no need to dilute the patient's plasma even if the concentration of voriconazole is near the upper limit of measurement. Furthermore, the short measurement-time could immediately inform physicians of the patient's voriconazole concentration during ambulatory medical care. Simultaneous measurement of voriconazole and voriconazole N-oxide may also be useful for the immediate adjustment of voriconazole dosage in outpatients and would help us to understand adherence or drug-drug interactions in plasma voriconazole concentrations.

Cyclic AMP Signaling Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Intestinal Epithelial Cells.

To develop a novel in vitro system for predicting intestinal drug absorption using human induced pluripotent stem (iPS) cell-derived intestinal epithelial cells, the cells need to have sufficient drug-metabolizing enzyme and drug transporter activities. We found that cyclic adenosine monophosphate (cAMP) signaling plays an important role in the differentiation of human iPS cells into intestinal epithelial cells. In this study, we aimed to demonstrate the effects of signaling activation in the intestinal differentiation of human iPS cells and the pharmacokinetic characteristics of human iPS cell-derived intestinal epithelial cells. Human iPS cells were differentiated into intestinal stem cells using activin A and fibroblast growth factor 2. Subsequently, the intestinal stem cells were maturated into intestinal epithelial cells by treatment with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) and 3-isobutyl-1-methylxanthine (IBMX), which activate cAMP signaling. The expression levels of intestinal markers and pharmacokinetics-related genes in the differentiated cells were markedly increased by using 8-Br-cAMP and IBMX. In the cells differentiated with the compound we observed cytochrome P450 (CYP) 3A4 inducibility via pregnane X receptor and vitamin D receptor. The metabolic activities of CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, and UDP-glucuronosyltransferase, which are expressed in the human small intestine, were also markedly increased. Furthermore, uptake of glycylsarcosine via peptide transporter 1 was markedly increased. The cells differentiated with the compounds also had drug transporter activities via organic anion transporters and P-glycoprotein. This study is the first to report that the activation of cAMP signaling promotes differentiation of human iPS cell-derived intestinal epithelial cells.

Spiral MRI on a 9.4T Vertical-bore Superconducting Magnet Using Unshielded and Self-shielded Gradient Coils.

Spiral MRI sequences were developed for a 9.4T vertical standard bore (54 mm) superconducting magnet using unshielded and self-shielded gradient coils. Clear spiral images with 64-shot scan were obtained with the self-shielded gradient coil, but severe shading artifacts were observed for the spiral-scan images acquired with the unshielded gradient coil. This shading artifact was successfully corrected with a phase-correction technique using reference scans that we developed based on eddy current field measurements. We therefore concluded that spiral imaging sequences can be installed even for unshielded gradient coils if phase corrections are performed using the reference scans.

Characteristic Analysis of Intestinal Transport in Enterocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

We previously demonstrated that differentiated enterocytes from human induced pluripotent stem (iPS) cells exhibited drug-metabolizing activities and cytochrome P450 CYP3A4 inducibility. The aim of this study was to apply human iPS cell-derived enterocytes in pharmacokinetic studies by investigating the characteristics of drug transport into enterocyte-like cells. Human iPS cells cultured on feeder cells were differentiated into endodermal cells using activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, epidermal growth factor and small-molecule compounds induced the maturation of the intestinal stem cell-like cells. After differentiation, we performed transepithelial electrical resistance (TEER) measurements, immunofluorescence staining, and transport studies. TEER values increased in a time-dependent manner and reached approximately 100 Ω × cm(2) Efflux transport of Hoechst 33342, a substrate of breast cancer resistance protein (BCRP), was observed and inhibited by the BCRP inhibitor Ko143. The uptake of peptide transporter 1 substrate glycylsarcosine was also confirmed and suppressed when the temperature was lowered to 4°C. Using immunofluorescence staining, villin and Na(+)-K(+) ATPase were expressed. These results suggest that human iPS cell-derived enterocytes had loose tight junctions, polarity, as well as uptake and efflux transport functions. In addition, the rank order of apparent membrane permeability coefficient (Papp) values of these test compounds across the enterocyte-like cell membrane corresponded to the fraction absorbance (Fa) values. Therefore, differentiated enterocytes from human iPS cells may provide a useful comprehensive evaluation model of drug transport and metabolism in the small intestine.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-ß promotes differentiation of human induced pluripotent stem cells into enterocytes.

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-ß inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-ß inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-ß inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes.

Echo-Planar Imaging for a 9.4 Tesla Vertical-Bore Superconducting Magnet Using an Unshielded Gradient Coil.

Echo-planar imaging (EPI) sequences were developed for a 9.4 Tesla vertical standard bore (~54 mm) superconducting magnet using an unshielded gradient coil optimized for live mice imaging and a data correction technique with reference scans. Because EPI requires fast switching of intense magnetic field gradients, eddy currents were induced in the surrounding metallic materials, e.g., the room temperature bore, and this produced serious artifacts on the EPI images. We solved the problem using an unshielded gradient coil set of proper size (outer diameter = 39 mm, inner diameter = 32 mm) with time control of the current rise and reference scans. The obtained EPI images of a phantom and a plant sample were almost artifact-free and demonstrated the promise of our approach.

Generation of enterocyte-like cells with pharmacokinetic functions from human induced pluripotent stem cells using small-molecule compounds.

The small intestine plays an important role in all aspects of pharmacokinetics, but there is no system for the comprehensive evaluation of small-intestinal pharmacokinetics, including drug metabolism and absorption. In this study, we aimed to construct an intestinal pharmacokinetics evaluation system and to generate pharmacokinetically functional enterocytes from human induced pluripotent stem cells. Using activin A and fibroblast growth factor 2, we differentiated these stem cells into intestinal stem cell-like cells, and the resulting cells were differentiated into enterocytes in a medium containing epidermal growth factor and small-molecule compounds. The differentiated cells expressed intestinal marker genes and drug transporters. The expression of sucrase-isomaltase, an intestine-specific marker, was markedly increased by small-molecule compounds. The cells exhibited activities of drug-metabolizing enzymes expressed in enterocytes, including CYP1A1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and sulfotransferase. Fluorescence-labeled dipeptide uptake into the cells was observed and was inhibited by ibuprofen, an inhibitor of the intestinal oligopeptide transporter solute carrier 15A1/PEPT1. CYP3A4 mRNA expression level was increased by these compounds and induced by the addition of 1α,25-dihydroxyvitamin D3. CYP3A4/5 activity was also induced by 1α,25-dihydroxyvitamin D3 in cells differentiated in the presence of the compounds. All these results show that we have generated enterocyte-like cells that have pharmacokinetic functions, and we have identified small-molecule compounds that are effective for promoting intestinal differentiation and the gain of pharmacokinetic functions. Our enterocyte-like cells would be useful material for developing a novel evaluation system to predict human intestinal pharmacokinetics.

Effect of milk on the pharmacokinetics of oseltamivir in healthy volunteers.

We previously showed that oseltamivir, a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802, is a substrate of proton-coupled oligopeptide transporter (PEPT1), and its intestinal absorption in rats is markedly inhibited by administration with milk. To investigate the importance of PEPT1 for oseltamivir absorption in humans, and the characteristics of the drug-milk interaction, a crossover clinical study was conducted in healthy volunteers, who received 75 mg of oseltamivir with 400 mL of water or milk. Milk significantly reduced the maximum plasma concentration (C(max) ) and the area under the plasma concentration-time curve from 0 to 2 h (AUC(0-2) ) of both oseltamivir and Ro 64-0802 (oseltamivir, 68.9% and 34.5%; Ro 64-0802, 69.5% and 14.2%, respectively, vs. water), but had no significant effect on the apparent terminal half-life (t(1/2) ) or AUC(0-∞) . Urinary recovery of oseltamivir and Ro 64-0802 was significantly reduced to 77.5% of the control by milk. The early reduction of oseltamivir absorption might be through the PEPT1 inhibition by milk peptides. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the PEPT1 expression and its contribution. This might be the first report suggesting the clinical drug-food interaction via PEPT1.