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.

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.

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.

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.

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.

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.

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.

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.

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells.

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

Tumor necrosis factor-alpha-nuclear factor-kappa B-signaling enhances St2b2 expression during 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia.

The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.

Screening of antibiotics that interact with organic anion-transporting polypeptides 1B1 and 1B3 using fluorescent probes.

Hepatic organic anion transporters OATP1B1 and OATP1B3 are expressed at the sinusoidal membrane of hepatocytes and contribute to the hepatic uptake of a wide variety of clinically used drugs. To identify the antibiotics that interact with the human organic anion transporters OATP1B1 and OATP1B3, we applied a screening system using fluorescent probes. Twenty-six antibiotics with a variety of mechanisms of action were examined. The screening demonstrated that four antibiotics inhibited OATP1B1-mediated transport and 11 antibiotics inhibited OATP1B3-mediated transport in a concentration-dependent manner. Antibiotics that inhibited OATP1B3-mediated transport tended to exhibit higher affinity than those that inhibited OATP1B1-mediated transport. To clarify whether the antibiotics that interacted with OATP1B1 and/or OATP1B3 were substrates for these transporters, an uptake study was performed. Rifampicin and penicillin were transported by both OATP1B1 and OATP1B3. Moreover, OATP1B3 was involved in the transport of ceftriaxone, cefmetazole, cefoperazone, and cefotaxime. Macrolides were not significantly transported by either transporter. In conclusion, the results demonstrated that our system is a useful method for the rapid screening of transporter-antibiotic interaction, and we found novel substrates. Our results indicate that OATP1B1 and/or OATP1B3 contribute to the transport process of some antibiotics, and that drug-drug interactions associated with these transporters could occur after the administration of antibiotics.

Production of 18O-single labeled peptide fragments during trypsin digestion of proteins for quantitative proteomics using nanoLC-ESI-MS/MS.

We investigated the effect of pH and the additive monoethanolamine on (18)O-atom incorporation into the C-terminal carboxy group of peptide fragments during tryptic digestion in (18)O-labeled water. Although amidase activity was sufficient for digestion at pH 6-11, the second (18)O-atom incorporation at the carboxy oxygen site was inhibited at pH 11 or above. The addition of at least 50 mM monoethanolamine into the reaction mixture also inhibited the carboxy oxygen exchange without reduction in amidase activity. Therefore, tryptic digestion for (18)O-single labeling should be performed in 50 mM phosphate buffer (pH 11) containing 50 mM monoethanolamine. The production ratios of (18)O-single labeled peptides were over 85%, and these results were independent of amino acid sequence. We also investigated the linearity of the (18)O-single labeled to unlabeled ratio ((18)O(1)/(18)O(0)). The use of y ions for calculation of the (18)O(1)/(18)O(0) ratio gave a better correlation between the observed and theoretical (18)O(1)/(18)O(0) ratios in the range of 0.1 to 10 than did the use of precursor ions. In the analysis of a pseudobiomarker spiked into human serum, the present (18)O-single labeling method was found to be robust because it was not affected by incomplete LC separation. The present (18)O-single labeling method represents a useful tool for quantitative proteomics using nanoLC-ESI-MS/MS.

Chemical synthesis of (22E)-3alpha,6alpha,7alpha,12alpha-Tetrahydroxy-5beta-chol-22-en-24-oic acid and its N-acylamidated conjugates with glycine or taurine: precursors of the [22,23-(3)H] labelled tracers.

(22E)-3alpha,6alpha,7alpha,12alpha-Tetrahydroxy-5beta-chol-22-en-24-oic acid and its N-acylamidated conjugates with glycine or taurine were synthesized from cholic acid. The key reactions employed are: 1) degradation of the side chain in intermediary C(24) 3alpha,6alpha,7alpha,12alpha-tetrahydroxylated bile acid to the corresponding C(22) 23,24-dinor-aldehyde, followed by Wittig reaction with methyl (triphenylphosphoranylidene)acetate and 2) N-acylamidation of the unconjugated tetrahydroxy-Delta(22)-5beta-cholenoic acid with glycine (or taurine) in the presence of diethylphosphorocyanide and triethylamine as coupling reagents.

Potential corticoid metabolites: chemical synthesis of 3- and 21-monosulfates and their double-conjugates of tetrahydrocorticosteroids in the 5alpha- and 5beta-series.

Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocortisone (THE) in the 5alpha- and 5beta-series. The principal reactions involved are: (1) selective protection of a specific hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of Delta(4)-3-ketosteroids with 10% Pd(OH)(2)/C to yield 3-oxo-5beta-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5alpha-isomers; (3) reduction of the resulting 3-oxo-5beta- and 3-oxo-5alpha-steroids to the corresponding 3alpha-hydroxy-compounds with Zn(BH(4))(2) and K-Selectride((R)), respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocorticosteroid derivatives with sulfur trioxide-triethylamine complex.

Analysis of phosphorylated peptides by double pseudoneutral loss extraction coupled with derivatization using N-(4-bromobenzoyl)aminoethanethiol.

The analysis of post-translational phosphorylation is a crucial step in understanding the mechanisms of many physiological events. Numerous approaches for the development of analytical methods aimed at the detection and quantification of phosphorylated proteins by mass spectrometry have been reported in the literature. In this paper, we report a new strategy for the identification of phosphorylated serine and threonine residues in phosphoproteins. This method consists of selective derivatization of phosphoproteins coupled with double pseudoneutral loss extraction after nanoLC/ESI-MS/MS analysis. First, we designed and synthesized a new derivatization reagent, N-(4-bromobenzoyl)aminoethanethiol, which can selectively react with alpha,beta-unsaturated ketones produced by beta-elimination of a phosphoryl group from phosphorylated serine or threonine residues. The mass spectrum of the derivatized peptide shows a product ion with a characteristic isotopic pattern. After derivatization, fragment ions of peptides with phosphoserine or phosphothreonine have twin peaks with an intensity ratio of approximately 1:1 and a difference of two mass units, while product ions from peptides without phosphoserine or phosphothreonine have normal isotopic patterns. Therefore, the neutral loss of the derivatized residue in the product ion mass spectrum includes two difference losses caused by (79)Br and (81)Br. The extraction of the product scan mass spectrum with double pseudoneutral losses is very effective for identification of the derivatized peptides produced from phosphorylated peptides. The new strategy represents a useful tool for the analysis of phosphorylated serine and threonine residues in phosphorylated proteins.

Role of enzymatic N-hydroxylation and reduction in flutamide metabolite-induced liver toxicity.

Flutamide is used for prostate cancer therapy but occasionally induces severe liver injury. Flutamide is hydrolyzed in the body into 5-amino-2-nitrobenzotrifluoride (FLU-1) and then further oxidized. In our previous study, N-hydroxy FLU-1 (FLU-1 N-OH) was detected in the urine of patients and exhibited cytotoxicity in rat primary hepatocytes. In the present study, we have assessed the roles of FLU-1 N-oxidation and hepatic glutathione (GSH) depletion in liver injury. FLU-1 (200 mg/kg p.o.) was administered to C57BL/6 mice for 5 days together with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (3 mg/kg i.p.) for the first 3 days. Mice were fasted for the last 2 days to deplete hepatic GSH. Administration of FLU-1 alone did not affect serum alanine aminotransferase activities (ALT), whereas coadministration of FLU-1 and TCPOBOP significantly increased ALT in fasted mice but not in nonfasted mice. Microsomal FLU-1 N-hydroxylation was enhanced approximately 5 times by TCPOBOP treatment. Flutamide metabolite-protein adducts were detected in liver microsomes incubated with FLU-1 N-OH, but not with FLU-1 and flutamide, by immunoblotting using antiflutamide antiserum. In the presence of mouse liver cytosol, FLU-1 N-OH was reduced back into FLU-1. This enzymatic reduction required NAD(P)H as a cofactor. The reduction was enhanced by the coexistence of NAD(P)H and GSH, whereas it was markedly inhibited by allopurinol (20 microM). By using purified bovine xanthine oxidase, the reduction was observed in the presence of NAD(P)H. These results suggest that FLU-1 N-OH is involved in flutamide-induced hepatotoxicity and that cytosolic reduction of FLU-1 N-OH plays a major role in protection against flutamide-induced hepatotoxicity.

[Introducing hazard prediction training 'KYT' to undergraduate pharmacy education on patient safety].

To develop students' sensitivity toword medication hazards, we have introduced a behavioral approach, "Kiken-Yochi Training" (KYT) for hazard prediction training to pharmacy education. KYT was originally implemented in the field of occupational health and safety in Japan. Only recently it has been introduced in the medical arena. The process consists of four steps; identification of hazards, assessing risks, planning countermeasure, and making action plan. One facilitator organizes the KYT class (20 students divided into four or five small groups). Watching a photo or illustration of everyday occurrences, each group follows the above four steps to discuss predictable hazards. Concepts are intensively presented in short time with brainstorming. KYT has been used with five classes thus far. Students learned KYT theory and exhibited desired attitudes and behaviors. Students presented many ideas, then formulated their own action plan within about one hour. More than 95% of KYT-naïve students assessed themselves as capable of applying the methodology in various situations. They also assessed themselves as being more aware of potential hazards and new points of view through the KYT process. Pharmacists must work for safer and more effective pharmacotherapy, predicting hazards as side effect or human error and solving the problems on each patient. KYT is a very useful and effective tool for pro-active safety training for the skill and attitude development. Repeating problem-based learning like KYT at intervals through undergraduate education should improve patient safety.