Exploring Acute Liver Damage: Slimming Health Foods and CYP3A4 Induction.

Background: Patients taking multiple drugs and various health foods often develop acute hepatitis. We hypothesized that the interaction between health foods and drug metabolism was the cause of severe liver injury in these patients. Therefore, we studied changes in the activity of the drug-metabolizing enzyme, cytochrome P450 (CYP), using slimming health food extracts and elucidated the molecular mechanism of liver injury onset through hepatotoxicity evaluation. Methods: For cytotoxicity testing, health food extract samples were added to HepG2 cells derived from hepatic parenchymal cells and culture medium, and cell viability was calculated 48 h after culture. To evaluate CYP3A4 induction, 3-1-10 cells constructed with a reporter linked to CYP3A4 gene were used, and reporter activity was measured 48 h after culture. Results: In the chronological order of the slimming health food intake history of the patient, niacinamide and Gymnema sylvestre extracts strongly inhibited HepG2 cell viability. In contrast, dietary supplements A and Coleus forskohlii extract strongly induced CYP3A4 reporter activity.To confirm CYP3A4 induction in humans, humanized CYP3A/pregnane X receptor (PXR) mice were treated with forskolin. CYP3A4 mRNA expression levels were elevated 3.9 times compared to that of the control group (P < 0.05). Conclusion: Coleus forskohlii extract showed the strongest transcriptional activation of CYP3A4 gene. In a mouse model of human-type drug metabolism, forskolin induced CYP3A4 transcription. Thus, we concluded that CYP3A4 induction by Coleus forskohlii is one of the causes of crucial hepatocellular injury, which is a type of liver injury caused by the active metabolite of acetaminophen produced by CYP3A4.

Mechanism Underlying Conflicting Drug-Drug Interaction Between Aprepitant and Voriconazole via Cytochrome P450 3A4-Mediated Metabolism.

Background: Voriconazole is an antifungal drug for which therapeutic monitoring is recommended to prevent side effects. Temporary administration of the antiemetic drug fosaprepitant remarkably decreases the plasma concentration of voriconazole from the therapeutic range. The ratio of the major metabolite voriconazole N-oxide to voriconazole exceeded that at any other time for a patient who started chemotherapy during voriconazole therapy. We attributed this unpredictable result to cytochrome P450 3A4 induced by aprepitant that was converted from fosaprepitant in vivo. Methods: Concentrations of voriconazole and voriconazole N-oxide were measured using liquid chromatography-mass spectrometry/mass spectrometry in primary human hepatocytes after incubation with aprepitant. Aprepitant suppressed voriconazole N-oxide formation within 24 h, followed by a continuous increase. Levels of drug-metabolizing cytochrome P450 mRNA were measured using real-time PCR in primary human hepatocytes incubated with aprepitant. Results: Cytochrome P450 3A4 and 2C9 mRNA levels increased ~4- and 2-fold, respectively, over time. Cytochrome P450 3A4 induction was confirmed using reporter assays. We also assessed L-755446, a major metabolite of aprepitant that lacks a triazole ring. Both compounds dose-dependently increased reporter activity; however, induction by L-755446 was stronger than that by aprepitant. Conclusion: These results indicate that aprepitant initially inhibited voriconazole metabolism via its triazole ring and increased cytochrome P450 3A4 induction following L-755446 formation. The decrease in plasma voriconazole concentration 7 days after fosaprepitant administration was mainly attributed to cytochrome P450 3A4 induction by L-755446.

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.

In Vivo Proof of Biological Safety and Physiological Effects of Orally Ingested Water Containing H2-Filled Ultrafine Bubbles (UFBs).

Owing to their unique physicochemical properties and diverse biological effects, ultrafine bubbles (UFBs) have recently been expected to be utilized for industrial and biological purposes. Thus, this study investigated the biological safety of UFBs in water for living beings in drinking the water with a view to future use in health sciences. In this study, we used H2-filled UFBs (NanoGAS®) that can hold hydrogen in the aqueous phase for a long time. Mice were randomly assigned to one of three groups: those receiving NanoGAS® water, reverse osmosis water, or natural mineral water, and they ingested it ad libitum for one month or three months. As a result, subchronic drinking of NanoGAS® water does not affect either the common blood biochemical parameters or the health of the organs and mucosal membranes. Our results, for the first time, scientifically demonstrated the biological safety of H2-filled UFBs water for subchronic oral consumption.

[The structure and efforts of the health sectors in municipalities from the first infected case to the second wave of COVID-19].

Objective　To describe the structure and efforts of the health sectors in municipalities to address the COVID-19 pandemic from the first infected case to the second wave.Method　We conducted self-administered postal questionnaires with the department head or an equivalent position of the 1,741 municipal health departments (108 cities or districts with public health centers (PHC) and 1,633 general municipalities) in Japan as of November 1, 2020. The survey period was from November 11, 2020 to January 8, 2021. The respondents were asked to provide the type of local government they were affiliated with, the number of COVID-19 cases in their municipality between January 16 to November 1, 2020, the operational structure of the health sectors after the pandemic began, and efforts made to address it. The analysis tested for the differences in response rates by cities with PHC and general municipalities, and by population size of the general municipalities.Results　A total of 1,270 valid questionnaires (valid response rate 72.9%) were returned from 83 cities with PHC and 1,187 general municipalities. Concerning the operational structure, over 90% of the cities with PHC transferred personnel from other departments to the department of infection control. Over 80% of all municipalities found a way to hold meetings remotely. More than half of the cities with PHC centers had employees working from home. Fewer than 50% of the general municipalities had a business continuity plan (BCP) prepared and in place for an outbreak, such as a novel influenza. Concerning the efforts within the local government, high rates of "secured supplementary budgets" and "monitored and secured infection control equipment" were reported. Concerning the efforts directed toward related organizations, over 70% of the cities with PHC "supported contact tracing at the PHC" and "monitored the stock of infection control equipment and procured equipment to address the shortages at medical institutions, welfare facilities, etc." Meanwhile, approximately 80.5% of general municipalities "corresponded and coordinated with medical institutions concerning the health examinations and services, etc." Concerning the efforts directed toward the public, over 90% of the respondents, regardless of local government type, "wrote articles and disseminated information regarding the infections in public relations (PR) reports or online" and "responded to inquiries from the public." In general municipalities, the larger the population size, the higher the percentage of implementation.Conclusion　Although the municipalities responded to the transmission of the COVID-19, there were some issues. Further preparation for the pandemic is required.

Rapid detection of mutation in isocitrate dehydrogenase 1 and 2 genes using mass spectrometry.

The 2016 World Health Organization classification of tumors of the central nervous system was recently revised. Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and chromosome 1p/19q codeletion are especially important for both the integrated diagnosis and the determination of surgical strategy. To establish a method for intraoperative molecular diagnosis, a simple, rapid method was developed for the measurement of 2-hydroxyglutarate (2-HG), a specific oncometabolite formed in the presence of IDH gene mutation, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). This method requires only 10 min to measure the level of 2-HG from tissue preparation to completion of examination. Using this method, the level of 2-HG was analyzed in 105 patients with diffuse infiltrating glioma, and showed that IDH mutated glioma had significantly higher level of 2-HG compared to IDH wild-type glioma. Receiver operating characteristic curve analysis showed the area under the curve, sensitivity, and specificity were 0.9815, 97.5, and 100%, respectively. In contrast, tumor grade and presence of chromosome 1p/19q codeletion in the IDH mutated glioma could not be predicted from the level of 2-HG. Measurement of 2-HG level using LC/ESI-MS/MS can provide rapid and accurate information of mutation status in the IDH gene.

Population Pharmacodynamic Analysis of Uric Acid-Lowering Effects of Febuxostat Based on Electronic Medical Records in Two Hospitals.

The aim of this study was to develop a population pharmacodynamic (PPD) model to describe uric acid (UA)-lowering effects in patients treated with febuxostat based on electronic medical records in 2 independent hospitals (university and city hospitals). Interhospital differences in the PPD model were also evaluated. We conducted the following 2 approaches to build the PPD models. A PPD model was developed separately using individual hospital data, and structural models and covariates between the two hospitals were compared (approach A). Another PPD model was developed using all available data from both hospitals, and differences between the 2 hospitals were evaluated by performing a covariate analysis on all PPD parameters (approach B). PPD analyses were performed by NONMEM using data from 358 patients. In both approaches, one indirect response model was established. In approach A, 2 diuretics (loops and thiazides) and renal function tests (Scr or BUN) were selected as covariates for the UA baseline level (serum UA levels just before the febuxostat treatment), whereas 2 diuretics and BUN were selected in approach B. A covariate analysis indicated that loops and thiazides increased UA baseline levels by 7%-14% and 6%-11%, respectively. In approach B, "hospital" was identified as a significant covariate for the UA baseline level; the baseline level was 7% higher in the city hospital. A PPD analysis may provide a precise description of the time course of the UA-lowering effects of febuxostat and quantitatively detect an interhospital difference in the UA baseline level.

Development of a Liquid Chromatography-Tandem Mass Spectrometric Method for Quantification of Mycophenolic Acid and Its Glucuronides in Dried Blood Spot Samples.

BACKGROUND: Personalized immunosuppressive therapy, including accurate drug dosing based on the drug blood level, leads to better clinical outcomes, specifically regarding avoidance of drug-induced adverse effects and maintenance of efficacy. Mycophenolic acid (MPA) is used as an immunosuppressant in transplantation of various solid organs. The aim of this study was to develop a method for quantification of MPA and its metabolites, mycophenolic acid 7-O-glucuronide (MPAG) and mycophenolic acid acyl glucuronide, in dried blood spot (DBS) samples, using liquid chromatography/electrospray ionization/tandem mass spectrometry. METHODS: For sample preparation, a microwave-drying approach was used to deactivate enzymes and reduce drying time. Blood volume was calculated in a DBS disk of 3 mm diameter. Concentrations of analytes in plasma from patients receiving mycophenolate mofetil were compared with DBS samples after hematocrit correction. RESULTS: The method yielded good recoveries of all 3 analytes (90.3%-104.2%). Blood volume in the disk was calculated as 3.0 ± 0.2 µL. Linearity over concentration ranges of 0.1-30 mcg/mL MPA, 0.1-200 mcg/mL MPAG, and 0.125-10 mcg/mL mycophenolic acid acyl glucuronide was obtained with r ≥0.999. Intraday and interday variations were less than 14.6%, and accuracy was within ±11.9%. Passing-Bablok analysis showed no significant differences between plasma concentrations and DBS concentrations after hematocrit correction of MPA and MPAG. CONCLUSIONS: We developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry method for analysis of MPA in DBS samples. The method is useful for monitoring the MPA blood level.

Matrix effect-corrected liquid chromatography/tandem mass-spectrometric method for determining acylcarnitines in human urine.

Administration of pivalate-containing antibiotics decreases serum carnitine and increases urinary pivaloylcarnitine, resulting in hypocarnitinemia. Carnitine and acylcarnitines are important biomarkers in the diagnosis of carnitine deficiency, but the relationship between acylcarnitines and drug-induced hypocarnitinemia remains unclear. Quantification of acylcarnitines enables discovery of new biomarkers for prediction and diagnosis of drug-induced hypocarnitinemia. Here we describe a liquid chromatography/tandem mass-spectrometric method for simultaneously quantifying carnitine, 15 acylcarnitines, and cefditoren (the pivoxilated product of an antibiotic prodrug) in human urine. The matrix effect is corrected in 87.8-103% using deuterium-labeled internal standards (2H9-carnitine, 2H3-hexanoylcarnitine, and 2H3-stearoylcarnitine). The surrogate matrix method had an error of <13% in comparison with a standard addition method. Dynamic ranges were 0.1-100µmol/l for acylcarnitines and 0.3-300µg/ml for cefditoren. Both accuracy and precision were <19.7% at the lower limit of quantification and <14.8% for other quality controls. In an example application of this method, urine samples from eight healthy volunteers (five adults and three children) were analyzed, and individual differences were clearly observed.

A simultaneous determination method for 5-fluorouracil and its metabolites in human plasma with linear range adjusted by in-source collision-induced dissociation using hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

We applied a new technique for quantitative linear range shift using in-source collision-induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5-fluorouracil (5-FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). To control adverse effects after administration of 5-FU, it is important to monitor the plasma concentration of 5-FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5-FU and its metabolites in human plasma by LC/ESI-MS/MS coupled with the technique for quantitative linear range shift using in-source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5-FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile-rich eluent after LC separation improved the ESI-MS response of high polar analytes. Based on the validation results, linear range shifts by in-source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.

Monitoring Serum Levels of Sorafenib and Its N-Oxide Is Essential for Long-Term Sorafenib Treatment of Patients with Hepatocellular Carcinoma.

Sorafenib, an oral multi-kinase inhibitor, is the final therapy prior to palliative care for advanced hepatocellular carcinoma (HCC). However, due to its adverse effects, 20% of patients must discontinue sorafenib within 1 month after first administration. To identify ways to predict the adverse effects and administer the drug for longer periods, we explored the relationship between the duration of sorafenib treatment and the pharmacokinetics of sorafenib and its major metabolite, sorafenib N-oxide. Twenty-five subjects enrolled in the study were divided into two groups: patients with dosage reduced or withdrawn due to adverse effects (n = 8), and patients with dosage maintained for 1 month after initial administration (n = 17). We evaluated early sorafenib accumulation as the area under the curve of sorafenib and sorafenib N-oxide concentrations during days 1-7 (AUC(sorafenib) and AUC(N-oxide), respectively). Inter-group comparison revealed that AUC(N-oxide) and AUC ratio (AUC(N-oxide)/AUC(sorafenib)) were significantly higher in the dosage reduction/withdrawal group (P = 0.031 and P = 0.0022, respectively). Receiver operating characteristic analysis indicated that AUC(N-oxide) and AUC ratio were reliable predictors of adverse effects. When patients were classified by cut-off points (AUC(N-oxide:) 2.0 µg ∙ day/mL, AUC ratio: 0.13), progression-free survival was significantly longer in patients with AUC(N-oxide) ≤ 2.0 µg ∙ day/mL (P = 0.0048, log-rank test). In conclusion, we recommend to simultaneously monitor serum levels of sorafenib and its N-oxide during the early stage after the first administration, which enables us to provide safe and long-term therapy for each HCC patient with sorafenib.

Modeling of Cancer Stem Cell State Transitions Predicts Therapeutic Response.

Cancer stem cells (CSCs) possess capacity to both self-renew and generate all cells within a tumor, and are thought to drive tumor recurrence. Targeting the stem cell niche to eradicate CSCs represents an important area of therapeutic development. The complex nature of many interacting elements of the stem cell niche, including both intracellular signals and microenvironmental growth factors and cytokines, creates a challenge in choosing which elements to target, alone or in combination. Stochastic stimulation techniques allow for the careful study of complex systems in biology and medicine and are ideal for the investigation of strategies aimed at CSC eradication. We present a mathematical model of the breast cancer stem cell (BCSC) niche to predict population dynamics during carcinogenesis and in response to treatment. Using data from cell line and mouse xenograft experiments, we estimate rates of interconversion between mesenchymal and epithelial states in BCSCs and find that EMT/MET transitions occur frequently. We examine bulk tumor growth dynamics in response to alterations in the rate of symmetric self-renewal of BCSCs and find that small changes in BCSC behavior can give rise to the Gompertzian growth pattern observed in breast tumors. Finally, we examine stochastic reaction kinetic simulations in which elements of the breast cancer stem cell niche are inhibited individually and in combination. We find that slowing self-renewal and disrupting the positive feedback loop between IL-6, Stat3 activation, and NF-κB signaling by simultaneous inhibition of IL-6 and HER2 is the most effective combination to eliminate both mesenchymal and epithelial populations of BCSCs. Predictions from our model and simulations show excellent agreement with experimental data showing the efficacy of combined HER2 and Il-6 blockade in reducing BCSC populations. Our findings will be directly examined in a planned clinical trial of combined HER2 and IL-6 targeted therapy in HER2-positive breast cancer.

Outcomes of a Long-term Case Review Program during the On-site Training of Pharmacy Students.

  This study sought to determine whether a long-term case review (LTCR) program helped pharmacy students develop their abilities as pharmacists, and how their level of satisfaction changed. LTCRs were comprised of four elements: self-learning, one-on-one bedside training with advising pharmacists, daily group sessions including three members, and weekly plenary sessions (case conferences). This program conducted on-site training in a hospital for 21 fifth-year students. The students were divided into 7 groups. One member of each group was assigned to a ward for bedside training for three weeks, while other member(s) of the central pharmacy provided support through daily group sessions. Each week, students training in the wards delivered case presentations in the case conference. All students, advising pharmacists, and teachers participated in these weekly case conferences. Upon conclusion of the on-site training, a survey was conducted on the program's efficacy. Through information sharing during group discussions, and in case conferences, continuous patient follow up was possible regardless of students' training schedules in wards or in the central pharmacy. After introducing the LTCR, the mean satisfaction level for case conferences (as scored using a 5-point Likert-type scale) increased from 3.4 to 4.3. Students' levels of understanding also improved. Statistically significant increases in students' self-evaluation scores on professional awareness, presentation skills, and logical thinking were also observed. We concluded that the program helped students to gain practical experience, made them more aware of clinical issues, and improved their presentation skills. Through this program, the students gained clinical competency through a deep understanding of the clinical courses of diseases and patient-oriented pharmaceutical care.

Gastrointestinal toxicities of 5-fluorouracil increase the proportion of regulatory T cells in intestinal tract: advantages of alternate-day S-1 administration.

BACKGROUND: 5-Fluorouracil (5-FU), a core anticancer agent for malignancies, induces gastrointestinal (GI) toxicities. Despite recent advances in tumor immunology, it still remains unknown how GI toxicities affect antitumor immunity. S-1 is a tegafur-based oral 5-FU prodrug which has been widely introduced in Japan and other countries. The alternate-day S-1 administration has been proposed to minimize its GI and other toxicities without reducing its anticancer efficacy. METHODS: In this study, two S-1 administration regimens were compared in mice to evaluate their impact of GI toxicities on immunity. In the daily group as a standard administration model, S-1 was administered for 14 days on and 14 days off, and in the alternate-day group as a non-GI toxicity model, S-1 was administered every other day for 28 days. As well as physical findings, regulatory T cells, Th1 cells and other cells in murine lymphoid tissues were analyzed with flow cytometry. RESULTS: Only the daily group exhibited body weight loss and GI toxicities. In the daily group, a proportion of regulatory T cells in the intestinal lymphoid tissue were demonstrated to be six-fold higher than in the control without S-1, and the proportion of Th1 cells showed a decreasing trend. However, the alternate-day group exhibited almost no change in T-cell subsets. CONCLUSION: GI toxicities of 5-FU may have a negative influence on antitumor immunity due to increased proportions of regulatory T cells and decreased proportions of Th1 cells. The alternate-day S-1 administration may be a useful regimen with its minimal influence on T-cell subsets.

Focused metabolomics using liquid chromatography/electrospray ionization tandem mass spectrometry for analysis of urinary conjugated cholesterol metabolites from patients with Niemann-Pick disease type C and 3ß-hydroxysteroid dehydrogenase deficiency.

BACKGROUND: Various conjugated cholesterol metabolites are excreted in urine of the patients with metabolic abnormalities and hepatobiliary diseases. We aimed to examine the usefulness of precursor ion scan and neutral loss scan for the characterization of conjugated cholesterol metabolites in urine. METHODS: A mixture of authentic standards of conjugated cholesterol metabolites was used for investigating the performance of the present method. The urine of patients with Niemann-Pick diseases type C and 3ß-hydroxysteroid dehydrogenase deficiency were analysed by precursor ion scan of m/z 97, 74, and 124. RESULTS: A precursor ion scan of m/z 97 was effective for identifying conjugates with ester sulphates on hydroxyl groups whereas ester sulphates on phenolic alcohols were signalled by a neutral loss scan of 80 Da. Monosaccharide-conjugated cholesterol metabolites were signalled by a precursor ion scan of m/z 113. Although precursor ion scan of m/z 74 and 124 was effective for finding glycine- and taurine-conjugated metabolites, high intensity of product ions (m/z 74 and 124) disturbed measurement of other multiply conjugated metabolites. The urine samples contained many conjugated cholesterol metabolites, and there were several disease-specific intense peaks. We found several unknown intense peaks with three known peaks in urine of the Niemann-Pick type C patient. In the patient with 3ß-hydroxysteroid dehydrogenase deficiency, intense peaks that were tentatively identified as 5-cholenoic acid sulphates and their glycine and taurine conjugates were present. CONCLUSION: The method should lead to the discovery of new urinary biomarkers for these disturbances of cholesterol catabolism and transport.

Usefulness of alternate-day administration of S-1 and leucovorin in a xenograft mouse model of colorectal cancer: a shorter drug-free interval leads to more efficient antitumor effects.

BACKGROUND: A clinical trial of S-1 with leucovorin (S-1/LV) in metastatic colorectal cancer (CRC) patients demonstrated promising efficacy; however, the gastrointestinal toxicities were so severe that it has not been applied in the clinical setting. On the other hand, alternate-day administration of S-1 has been proposed to attenuate the adverse events without reducing its anticancer activity. Our present study was conducted to confirm the feasibility of alternate-day administration of S-1/LV in in vivo xenograft tumor models. METHODS: Mice were treated with S-1/LV in a daily group (2 weeks of administration followed by 2 weeks of withdrawal) or an alternate-day group (administration on alternate days for 4 weeks), then the mice were killed and the xenograft tumors were resected. We compared body weight changes, condition of feces, mucosal injury and myelosuppression and assessed adverse reactions, tumor volume, tumor growth inhibition (TGI) and expression of Ki67, TUNEL, cIAP2 and XIAP to evaluate the antitumor activity and tumor apoptosis. RESULTS: Severe weight loss, diarrhea, mucosal injury and myelosuppression were observed only in the daily group; however, some myelosuppression was also observed in the alternate-day group. The TGI in the alternate-day group was better than in the daily group, possibly resulting from apoptosis due to the suppression of cIAP2 but not XIAP. CONCLUSION: Our findings suggest that alternate-day administration of S-1/LV for CRC treatment can achieve high antitumor activity without severe adverse reactions, and we propose that clinical trials with this regimen should be conducted in CRC patients.

A quantitative HPLC-UV method for determination of serum sorafenib and sorafenib N-oxide and its application in hepatocarcinoma patients.

Sorafenib, an oral multi-kinase inhibitor, has been approved for treatment of advanced renal-cell and hepatocellular carcinoma (HCC). However, 20% of HCC patients taking sorafenib are forced to withdraw due to adverse effects within one month after administration. Orally administered sorafenib is oxidatively metabolized, predominantly by cytochrome P450 3A4 (CYP3A4), in small-intestinal mucosa or liver. We aimed to characterize the CYP3A4-mediated metabolism of sorafenib in HCC patients and explore the contribution of the major metabolite sorafenib N-oxide to adverse effects and therapeutic efficacy. We have therefore developed a method for quantitative determination of sorafenib and its N-oxide in the present study. To optimize the preanalytical procedure, we initially ascertained the solubility of the analytes. Because they are lipophilic, solvents containing more than 40% acetonitrile were required for efficient recovery. The pretreatment procedure that we ultimately developed consists of acetonitrile precipitation, followed by extraction using octadecyl silyl-silica gel to eliminate water-soluble and hydrophilic components of serum. Application of this procedure before HPLC enabled accurate and reproducible quantitation of analytes in a linear range from 0.03 to 30 µg/mL. After characterizing the peaks in the HPLC-ultraviolet chromatogram obtained from a medicated patient by LC-tandem mass spectrometry, we applied this method to HCC patients taking sorafenib, showing large inter-individual differences in the pharmacokinetic profile. In conclusion, our assay system should be useful for follow-up of patients taking sorafenib and for exploring the association between the pharmacokinetics of sorafenib and its N-oxide and the adverse effects or therapeutic efficacy.

Tandem mass spectrometric characterization of bile acids and steroid conjugates based on low-energy collision-induced dissociation.

We examined the characteristics of several bile acids and some steroid conjugates under low-energy-collision-induced dissociation conditions using a triple quadrupole tandem mass spectrometer. According to conjugation types, we observed characteristic product ions and/or neutral losses in the product ion spectra. Amino acid conjugates afforded specific product ions. For example, glycine-conjugated metabolites routinely produced a product ion at m/z 74, and taurine-conjugated metabolites produced product ions at m/z 124, 107, and 80. When a strong peak appeared at m/z 97, the molecule contained a sulfate group. In contrast to amino acid conjugates, carbohydrate conjugates required a combination of product ions and neutral losses for identification. We could discriminate a glucoside from an acyl galactoside according to the presence or absence of a product ion at m/z 161 and a neutral loss of 180 Da. Discrimination among esters, aliphatic ethers, and phenolic ether types of glucuronides was based upon differences in the intensities of a product ion at m/z 175 and a neutral loss of 176 Da. Furthermore, N-acetylglucosamine conjugates showed a characteristic product ion at m/z 202 and a neutral loss of 203 Da, and the appearance of a product ion at m/z 202 revealed the existence of N-acetylglucosamine conjugated to an aliphatic hydroxyl group without a double bond in the immediate vicinity. Together, the data presented here will help to enable the identification of unknown conjugated cholesterol metabolites by using low-energy collision-induced dissociation.

An efficient synthesis of 4α- and 4ß-hydroxy- 7-dehydrocholesterol, biomarkers for patients with and animal models of the Smith-Lemli-Opitz syndrome.

A highly efficient and improved method for the preparation of stereoisomeric 4α- and 4ß-hydroxy-7-dehydrocholesterol has been developed. These oxysterols are atypical precursors of cholesterol found to be present in increased concentrations in brain, liver, and serum of animals treated with AY9944, an inhibitor of 3ß-hydroxysterol-Δ(7)-reductase (Dhcr7). AY9944 -treated rats are considered a model for Smith-Lemli-Opitz syndrome (SLOS). The principal reactions involved were (1) cis-4α,5α-dihydroxylation of the allylic 3ß-acetoxy-Δ(4) intermediate with in situ generated RuO4 and subsequent dehydration with SOCl2, (2) direct 4ß-hydroxylation of cholesterol with selenium dioxide, and (3) regioselective dehydrogenation at C-7/-8 of the resulting 4α- and 4ß-hydroxylated derivatives with 1,3-dibromo-5,5-dimethylhydantoin/azobisisobutyronitrile, followed by tetrabutyl ammonium bromide/tetrabutyl ammonium fluoride. Chemical instability of these 4-hydroxylated 7-dehydrocholesterols when exposed to UV light, heat or in an acidic medium is briefly discussed.

LC/ESI-MS/MS analysis of urinary 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides: new biomarkers for the detection of Niemann-Pick type C disease.

We developed a sensitive, reliable, and accurate LC/ESI-MS/MS method for measurement of 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine amides in urine. This atypical C24 bile acid has been reported previously to be present in the urine of patients with Niemann-Pick Type C (NPC) disease. In the method, targeted analytes are concentrated at the front edge of a trapping column, Shim-pack MAYI-C8, which permits elimination of contaminating molecules in the urinary matrix. The trapped analytes are then eluted, separated on a YMC-Pack Pro C18, and quantified with MS/MS using selected reaction monitoring. The method could detect (as amount injected) 2pg of nonamidated 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid, 2pg of its glycine-amide, and 0.6pg of its taurine-amide, and is linear up to 300pg. The method was then used to measure the three analytes in the urine of NPC patients (N=2), 3ß-hydroxysteroid dehydrogenase deficiency patients (N=2), and healthy volunteers (N=8). Measurable concentrations of all three analytes were present in all subjects. The urinary concentration of the sum of all three analytes was four hundred times greater in the 3month NPC patient and 40times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides in NPC patients suggests that these compounds may be valuable biomarkers for detection of NPC disease.