Publications by authors named "Masamitsu Maekawa"

48 Publications

Role of OATP4C1 in Renal Handling of Remdesivir and its Nucleoside Analog GS-441524: The First Approved Drug for Patients with COVID-19.

J Pharm Pharm Sci 2021 ;24:227-236

Department of Pharmacy, Yamagata University Hospital, and Department of Pharmaceutical Science, Faculty of Medicine, Yamagata University, Yamagata, Japan.

Purpose: Remdesivir and its active metabolite are predominantly eliminated via renal route; however, information regarding renal uptake transporters is limited. In the present study, the interaction of remdesivir and its nucleoside analog GS-441524 with OATP4C1 was evaluated to provide the detailed information about its renal handling.

Methods: We used HK-2 cells, a proximal tubular cell line derived from normal kidney, to confirm the transport of remdesivir and GS-441524. To assess the involvement of OATP4C1 in handling remdesivir and GS-441524, the uptake study of remdesivir and GS-441524 was performed by using OATP4C1-overexpressing Madin-Darby canine kidney II (MDCKII) cells. Moreover, we also evaluated the IC50 and Ki value of remdesivir.

Results: The time-dependent remdesivir uptake in HK-2 cells was observed. The results of inhibition study using OATs and OCT2 inhibitors and OATP4C1 knockdown suggested the involvement of renal drug transporter OATP4C1. Remdesivir was taken up by OATP4C1/MDCKII cells. OATP4C1-mediated uptake of remdesivir increased linearly up to 10 min and reached a steady state at 30 min. Remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner with the IC50 and apparent Ki values of 42 ± 7.8 μM and 37 ± 6.9 μM, respectively.

Conclusions: We have provided novel information about renal handling of remdesivir. Furthermore, we evaluated the potential drug interaction via OATP4C1 by calculating the Ki value of remdesivir. OATP4C1 may play a pivotal role in remdesivir therapy for COVID-19, particularly in patients with kidney injury.
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http://dx.doi.org/10.18433/jpps31813DOI Listing
June 2021

Prolonged high-intensity exercise induces fluctuating immune responses to herpes simplex virus infection via glucocorticoids.

J Allergy Clin Immunol 2021 May 7. Epub 2021 May 7.

Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan; Singapore Immunology Network (SIgN) and Skin Research Institute of Singapore (SRIS), Technology and Research (A∗STAR), Biopolis, Singapore. Electronic address:

Background: Epidemiologic studies have yielded conflicting results regarding the influence of a single bout of prolonged high-intensity exercise on viral infection.

Objective: We sought to learn whether prolonged high-intensity exercise either exacerbates or ameliorates herpes simplex virus type 2 (HSV-2) infection according to the interval between virus exposure and exercise.

Methods: Mice were intravaginally infected with HSV-2 and exposed to run on the treadmill.

Results: Prolonged high-intensity exercise 17 hours after infection impaired the clearance of HSV-2, while exercise 8 hours after infection enhanced the clearance of HSV-2. These impaired or enhanced immune responses were related to a transient decrease or increase in the number of blood-circulating plasmacytoid dendritic cells. Exercise-induced glucocorticoids transiently decreased the number of circulating plasmacytoid dendritic cells by facilitating their homing to the bone marrow via the CXCL12-CXCR4 axis, which led to their subsequent increase in the blood.

Conclusion: A single bout of prolonged high-intensity exercise can be either deleterious or beneficial to antiviral immunity.
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http://dx.doi.org/10.1016/j.jaci.2021.04.028DOI Listing
May 2021

Establishment of an analytical method for simultaneous quantitation of CDK4/6 inhibitors, aromatase inhibitors, and an estrogen receptor antagonist in human plasma using LC-ESI-MS/MS.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Mar 22;1173:122655. Epub 2021 Mar 22.

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Electronic address:

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors (palbociclib, abemaciclib, and ribociclib) are used to treat human epithelial growth factor receptor (HER)-2 negative and hormone receptor (HR) positive advanced breast cancer in combination with aromatase inhibitors (letrozole, anastrozole) or an estrogen receptor antagonist (fulvestrant). Administration of these drugs frequently causes severe side effects, such as neutropenia and diarrhea. Therefore, therapeutic drug monitoring (TDM) of CDK4/6 inhibitors, aromatase inhibitors, and the estrogen receptor antagonist is considered important for ensuring the efficacy and safety of these drugs. In this study, we describe a simple, highly sensitive, and specific liquid chromatography/electrospray ionization tandem mass spectrometry method for simultaneous quantitation of the concentrations of palbociclib, abemaciclib, ribociclib, letrozole, anastrozole, and fulvestrant. In addition, we analyzed plasma samples from patients with HER2-negative and HR-positive advanced breast cancer treated with these compounds using this novel method. In our method, the intra-assay relative error (RE) values ranged from -12.8% to 12.9%, the inter-assay RE values ranged from -4.8% to 6.2%, and the coefficient of variation (CV) values for intra- and inter-assay were ≤8.6% and ≤13.3%, respectively. The analytes showed good stability with RE values ranging from -13.5% to 13.6% and CV values <10.4%. Moreover, all the samples from patients were successfully quantified, and were within the range of measurement. This method can be used for TDM of routine anticancer drugs in clinical practice and for pharmacokinetics/pharmacodynamics research in future studies.
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http://dx.doi.org/10.1016/j.jchromb.2021.122655DOI Listing
March 2021

High-throughput liquid chromatography/electrospray ionization-tandem mass spectrometry method using in-source collision-induced dissociation for simultaneous quantification of imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib in human plasma.

Biomed Chromatogr 2021 Mar 27:e5124. Epub 2021 Mar 27.

Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.

Recent studies have shown that therapeutic drug monitoring of tyrosine kinase inhibitors (TKIs) could improve treatment efficacy and safety. A simple analytical method using high-performance LC/electrospray ionization-tandem mass spectrometry has been developed and validated for simultaneous quantification of BCR-ABL and Bruton's TKIs used for chronic leukemia (imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib) in human plasma. Although these structures and physical properties are similar, owing to their different linear ranges, simultaneously determining the plasma levels of these five TKIs by applying optimal MS parameters remains difficult. A quantitative range exceeding 60,000-fold was required, and the linear dynamic ranges of imatinib, bosutinib, and nilotinib were limited because of the presence of a saturated detection signal. In this study, we applied the in-source collision-induced dissociation technique to control the ion amounts in mass spectrometry. This new method allowed rapid determination within 5 min with simple pretreatment. The method was validated according to the US Food and Drug Administration guidelines. Moreover, all samples of patients with chronic leukemia were successfully measured and their values were within the linear range of measurement. Therefore, our high-throughput analytical system is useful to measure the plasma concentrations of imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib in clinical practice.
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http://dx.doi.org/10.1002/bmc.5124DOI Listing
March 2021

Development of a simultaneous analytical method for clozapine and its metabolites in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry with linear range adjusted by in-source collision-induced dissociation.

Biomed Chromatogr 2021 Jul 4;35(7):e5094. Epub 2021 Mar 4.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.

Clozapine (CLZ) is a key drug in treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of CLZ and its metabolites, N-desmethylclozapine and clozapine N-oxide, is required to monitor and manage the risks of side effects. Although quantification methods for TDM have been developed for CLZ and its metabolites, they were not sufficiently accurate for the quantification of CLZ owing to the upper limits of the calibration curves. An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous measurement of CLZ and its metabolites in human plasma. To expand the concentration range of the calibration curves, we used a linear range shift technique using in-source collision-induced dissociation (CID). Using our approach, the linearity and quantitative range were improved compared to those reported by previous studies, and were sufficient for TDM in clinical practice. The intra- and inter-assay accuracy was 84.6%-114.8%, and the intra- and inter-assay precisions were ≤9.1% and ≤9.9%, respectively. Moreover, all samples from patients with treatment-resistant schizophrenia were successfully quantified. Therefore, our novel analytical method using in-source CID had the appropriate performance to measure the plasma concentrations of CLZ and its metabolites for TDM in clinical practice.
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http://dx.doi.org/10.1002/bmc.5094DOI Listing
July 2021

Functional Assessment of 12 Rare Allelic Variants Identified in a Population of 4773 Japanese Individuals.

J Pers Med 2021 Feb 2;11(2). Epub 2021 Feb 2.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These variants were heterologously expressed in 293FT cells, and the kinetic parameters (, , , catalytic efficiency, and ) of ()-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between ()-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype-phenotype associations based on the novel rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.
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http://dx.doi.org/10.3390/jpm11020094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912942PMC
February 2021

Simultaneous analysis of drugs administered to lung-transplanted patients using liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring.

Biomed Chromatogr 2021 Jun 22;35(6):e5067. Epub 2021 Jan 22.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Miyagi, Japan.

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid- H , voriconazole- H , itraconazole- H , and hydroxyitraconazole- H as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.
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http://dx.doi.org/10.1002/bmc.5067DOI Listing
June 2021

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6-Hydroxylation.

Drug Metab Dispos 2021 Mar 31;49(3):212-220. Epub 2020 Dec 31.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences (M.K., E.M.G.R., N.H., M.H.), Tohoku Medical Megabank Organization (E.H., S.S., D.S., S.T., K.K., M.H.), Advanced Research Center for Innovations in Next-Generation Medicine (E.H., A.U., N.H., M.H.), and Laboratory of Clinical Pharmacy, Faculty of Pharmaceutical Sciences (A.A., M.M., N.M.), Tohoku University, Sendai, Japan; Faculty of Pharmacy, Meijo University, Nagoya, Japan (T.N., A.O.); and Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan (M.M., N.M., N.H., M.H.)

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants ( , , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in gene variants in Asian populations, including Japanese individuals, indicating the presence of polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.
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http://dx.doi.org/10.1124/dmd.120.000261DOI Listing
March 2021

Metabolomic Analysis to Elucidate Mechanisms of Sunitinib Resistance in Renal Cell Carcinoma.

Metabolites 2020 Dec 22;11(1). Epub 2020 Dec 22.

Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8574, Japan.

Metabolomics analysis possibly identifies new therapeutic targets in treatment resistance by measuring changes in metabolites accompanying cancer progression. We previously conducted a global metabolomics (G-Met) study of renal cell carcinoma (RCC) and identified metabolites that may be involved in sunitinib resistance in RCC. Here, we aimed to elucidate possible mechanisms of sunitinib resistance in RCC through intracellular metabolites. We established sunitinib-resistant and control RCC cell lines from tumor tissues of RCC cell (786-O)-injected mice. We also quantified characteristic metabolites identified in our G-Met study to compare intracellular metabolism between the two cell lines using liquid chromatography-mass spectrometry. The established sunitinib-resistant RCC cell line demonstrated significantly desuppressed protein kinase B (Akt) and mesenchymal-to-epithelial transition (MET) phosphorylation compared with the control RCC cell line under sunitinib exposure. Among identified metabolites, glutamine, glutamic acid, and α-KG (involved in glutamine uptake into the tricarboxylic acid (TCA) cycle for energy metabolism); fructose 6-phosphate, D-sedoheptulose 7-phosphate, and glucose 1-phosphate (involved in increased glycolysis and its intermediate metabolites); and glutathione and myoinositol (antioxidant effects) were significantly increased in the sunitinib-resistant RCC cell line. Particularly, glutamine transporter (SLC1A5) expression was significantly increased in sunitinib-resistant RCC cells compared with control cells. In this study, we demonstrated energy metabolism with glutamine uptake and glycolysis upregulation, as well as antioxidant activity, was also associated with sunitinib resistance in RCC cells.
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http://dx.doi.org/10.3390/metabo11010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821950PMC
December 2020

Essential oils can cause false-positive results of medium-chain acyl-CoA dehydrogenase deficiency.

Mol Genet Metab Rep 2020 Dec 5;25:100674. Epub 2020 Nov 5.

Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan.

Newborn screening is a public health care program worldwide to prevent patients from critical illness or conditions. Tandem mass spectrometry allows multiplex, inexpensive, and rapid newborn screening. However, mass spectrometry used for newborn screening to date is not able to separate peaks of compounds with similar , which could lead to false-positive results without additional second-tier tests, such as fragmentation. We experienced three neonatal cases with high levels of markers, octanoylcarnitine and octanoylcarnitine/decanoylcarnitine ratio used to pick up possible cases of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. The babies were born consecutively in a maternity hospital. Their second acylcarnitine profiles were normal, and the genetic tests for were negative. Analysis of samples extracted from their first Guthrie cards where blood was not stained also showed peaks equivalent to octanoylcarnitine and decanoylcarnitine, indicating contamination. Environmental surveillance in the maternity ward suggested that essential oils used there might contain the contaminated compound. LC-HRMS/MS and analysis revealed that false-positive results might be due to contamination with the essential oils in Guthrie cards, and causal agents were sphinganine (d17:0) and 2-[2-hydroxyethyl(pentadecyl)amino]ethanol. Thus, health care providers should be cautioned about use of essential oils when collecting blood samples on Guthrie cards. False-positive results can waste costly social resources and cause a physical and psychological burden for children and parents.
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http://dx.doi.org/10.1016/j.ymgmr.2020.100674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653163PMC
December 2020

Biomarker analysis of Niemann-Pick disease type C using chromatography and mass spectrometry.

J Pharm Biomed Anal 2020 Nov 13;191:113622. Epub 2020 Sep 13.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan; Faculty of Pharmaceutical Sciences, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder with progressive degradation of central nervous system. The age of the onset varies from perinatal to adulthood. Patients with NPC are affected in the central nervous system, peripheral nerves, and systemic organs. From these background, it is extremely difficult to discover NPC clinically and diagnose it correctly. The procedure of the conventional laboratory methods are complicated and it takes long time to obtain the result. Because of the importance of early treatments and the shortcomings of conventional diagnostic methods for NPC, remarkable attention has been paid to biomarkers and chemical diagnoses. In the last decade, many NPC biomarkers have been reported. They are classified as cholesterol-related metabolites, sphingolipid metabolites, and novel phospholipid metabolites, respectively. Therefore, these are all lipid metabolites. Various chemical analysis methods have been used for their identification. In addition, chromatography and mass spectrometry are mainly used for their quantification. This review article outlines NPC biomarkers reported in the last decade and their analytical methods.
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http://dx.doi.org/10.1016/j.jpba.2020.113622DOI Listing
November 2020

Development of a Diagnostic Screening Strategy for Niemann-Pick Diseases Based on Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analyses of N-Palmitoyl-O-phosphocholine-serine and Sphingosylphosphorylcholine.

Biol Pharm Bull 2020 Sep 25;43(9):1398-1406. Epub 2020 Jun 25.

Faculty of Pharmaceutical Sciences, Tohoku University.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. H-Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.
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http://dx.doi.org/10.1248/bpb.b20-00400DOI Listing
September 2020

Accurate quantification of urinary metabolites for predictive models manifest clinicopathology of renal cell carcinoma.

Cancer Sci 2020 Jul 21;111(7):2570-2578. Epub 2020 Jun 21.

Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Using surgically resected tissue, we identified characteristic metabolites related to the diagnosis and malignant status of clear cell renal cell carcinoma (ccRCC). Specifically, we quantified these metabolites in urine samples to evaluate their potential as clinically useful noninvasive biomarkers of ccRCC. Between January 2016 and August 2018, we collected urine samples from 87 patients who had pathologically diagnosed ccRCC and from 60 controls who were patients with benign urological conditions. Metabolite concentrations in urine samples were investigated using liquid chromatography-mass spectrometry with an internal standard and adjustment based on urinary creatinine levels. We analyzed the association between metabolite concentration and predictability of diagnosis and of malignant status by multiple logistic regression and receiver operating characteristic (ROC) curves to establish ccRCC predictive models. Of the 47 metabolites identified in our previous study, we quantified 33 metabolites in the urine samples. Multiple logistic regression analysis revealed 5 metabolites (l-glutamic acid, lactate, d-sedoheptulose 7-phosphate, 2-hydroxyglutarate, and myoinositol) for a diagnostic predictive model and 4 metabolites (l-kynurenine, l-glutamine, fructose 6-phosphate, and butyrylcarnitine) for a predictive model for clinical stage III/IV. The sensitivity and specificity of the diagnostic predictive model were 93.1% and 95.0%, respectively, yielding an area under the ROC curve (AUC) of 0.966. The sensitivity and specificity of the predictive model for clinical stage were 88.5% and 75.4%, respectively, with an AUC of 0.837. In conclusion, quantitative analysis of urinary metabolites yielded predictive models for diagnosis and malignant status of ccRCC. Urinary metabolites have the potential to be clinically useful noninvasive biomarkers of ccRCC to improve patient outcomes.
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http://dx.doi.org/10.1111/cas.14440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385347PMC
July 2020

Mechanism of Bile Acid Reabsorption in the Biliopancreatic Limb After Duodenal-Jejunal Bypass in Rats.

Obes Surg 2020 07;30(7):2528-2537

Department of Surgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.

Background: Bile acids (BAs) are important in the metabolic effects of bariatric surgery. Most BAs are reabsorbed in the ileum and recycled back to the liver. We have reported that this enterohepatic circulation was shortened by duodenal-jejunal bypass (DJB), and the biliopancreatic (BP)-limb plays an important role in reabsorption of BAs. However, the mechanism of BA reabsorption in BP-limb remains uncertain. We aimed to investigate the mechanisms of BA reabsorption after DJB, especially focusing on carrier-mediated transport of BAs and the impact of the presence or absence of lipids on BA reabsorption.

Methods: Otsuka-Long-Evans-Tokushima fatty rats or Sprague-Dawley rats were assigned to a control group and DJB group. BA levels in the divided small intestine were quantified with liquid chromatography-mass spectrometry. Labeled BA was injected and perfused with BA transporter inhibitors or mixture of lipids in the isolated BP-limb, and bile was sampled and analyzed.

Results: Conjugated BA levels in the BP-limb were significantly higher than that of the control group. BA absorption tended to decrease by the apical sodium-dependent BA transporter inhibitor and was significantly decreased by the organic anion-transporting peptide (OATP) inhibitor. BA absorption tended to increase in the absence of lipid solutions compared with that in the presence of lipid solutions.

Conclusion: We attributed the increased BA reabsorption in the BP-limb to lack of food in the BP-limb, which contains concentrated BAs and no lipids. OATP played an important role in BA reabsorption in the BP-limb. Therefore, BAs would be reabsorbed in different manners after DJB.
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http://dx.doi.org/10.1007/s11695-020-04506-3DOI Listing
July 2020

Comprehensive and semi-quantitative analysis of carboxyl-containing metabolites related to gut microbiota on chronic kidney disease using 2-picolylamine isotopic labeling LC-MS/MS.

Sci Rep 2019 12 13;9(1):19075. Epub 2019 Dec 13.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.

Carboxyl-containing metabolites, such as bile acids and fatty acids, have many important functions and microbiota is involved in the production of them. In the previous study, we found that the chronic kidney disease (CKD) model mice raised under germ-free conditions provided more severe renal damage than the mice with commensal microbiota. However, the precise influence by the microbiome and carboxyl-containing metabolites to the renal functions is unknown. In this study, we aimed to develop a novel chemical isotope labeling-LC-MS/MS method using the 2-picolylamine and its isotopologue and applied the analysis of effects of microbiome and CKD pathophysiology. The developed semi-quantitative method provided the high accuracy not inferior to the absolute quantification. By comparing of four groups of mice, we found that both microbiota and renal function can alter the composition and level of these metabolites in both plasma and intestine. In particular, the intestinal level of indole-3-acetic acid, short-chain fatty acids and n-3 type of polyunsaturated fatty acid, which play important roles in the endothelial barrier function, were significantly lower in germ-free conditions mice with renal failure. Accordingly, it is suggested these metabolites might have a renoprotective effect on CKD by suppressing epithelial barrier disruption.
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http://dx.doi.org/10.1038/s41598-019-55600-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910927PMC
December 2019

Structural Determination of Lysosphingomyelin-509 and Discovery of Novel Class Lipids from Patients with Niemann-Pick Disease Type C.

Int J Mol Sci 2019 Oct 10;20(20). Epub 2019 Oct 10.

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation-tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as -acylated and -phosphocholine adducted serine. Additionally, we found that a group of metabolites with -acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.
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http://dx.doi.org/10.3390/ijms20205018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829288PMC
October 2019

Investigation of diagnostic performance of five urinary cholesterol metabolites for Niemann-Pick disease type C.

J Lipid Res 2019 12 4;60(12):2074-2081. Epub 2019 Oct 4.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Aoba-ku, Sendai 980-8574, Japan.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by progressive nervous degeneration. Because of the diversity of clinical symptoms and onset age, the diagnosis of this disease is difficult. Therefore, biomarker tests have attracted significant attention for earlier diagnostics. In this study, we developed a simultaneous analysis method for five urinary conjugated cholesterol metabolites, which are potential diagnostic biomarkers for a rapid, convenient, and noninvasive chemical diagnosis, using LC/MS/MS. By the method, their urinary concentrations were quantified and the NPC diagnostic performances were evaluated. The developed LC/MS/MS method showed high accuracy and satisfied all analytical method validation criteria. When the urine of healthy controls and patients with NPC was analyzed, three of five urinary conjugated cholesterol metabolite concentrations corrected by urinary creatinine were significantly higher in the patients with NPC. As a result of receiver operating characteristics analysis, these urinary metabolites might have excellent diagnostic marker performance. 3β-Sulfooxy-7β-hydroxy-5-cholenoic acid showed particularly excellent diagnostic performance with both 100% clinical sensitivity and specificity, suggesting that it is a useful NPC diagnostic marker. The urinary conjugated cholesterol metabolites exhibited high NPC diagnostic marker performance and could be used for NPC diagnosis.
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http://dx.doi.org/10.1194/jlr.M093971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889718PMC
December 2019

An agonistic anti-Toll-like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy.

Immunology 2019 10;158(2):136-149

Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Immune-checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti-Toll-like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell-surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti-TLR4 mAb in T-cell-mediated antitumour immunity. The anti-TLR4 mAb induced the activation of antigen-specific T-cells in adoptive transfer studies. The growth of ovalbumin (OVA)-expressing tumours was significantly suppressed by administration of OVA and the anti-TLR4 mAb in combination, but not individually. The antitumour effect of anti-PD-1 mAb was enhanced in mice administered with OVA plus the anti-TLR4 mAb. The OVA-specific IFN-γ-producing CD8 T-cells were induced by administration of OVA and the anti-TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T-cells. The inflammatory response to the anti-TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF-κB activation and production of TNF-α, IL-6 and IL-1β. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti-TLR4 mAb) plus OVA induced no or less-marked activation of OVA-specific T-cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti-TLR4 mAb induces potent CD8 T-cell-dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti-TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.
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http://dx.doi.org/10.1111/imm.13095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742770PMC
October 2019

Altered bile acid composition and disposition in a mouse model of non-alcoholic steatohepatitis.

Toxicol Appl Pharmacol 2019 09 12;379:114664. Epub 2019 Jul 12.

Graduate School of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; Faculty of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Electronic address:

Non-alcoholic steatohepatitis (NASH) is a progressive inflammatory and fibrotic disease. However, the progression mechanism of NASH is not well understood. Bile acids are endogenous molecules that regulate cholesterol homeostasis, lipid solubilization in the intestinal lumen, and metabolic signaling via several receptors. In this study, we investigated the relationship between bile acid composition and NASH-associated fibrosis using a mouse model fed choline-deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine (CDAHFD). C57BL/6 J mice fed CDAHFD developed NASH and fibrosis within few weeks. With the progress of NASH-associated liver fibrosis, altered bile acid composition was observed in the liver, bile, and peripheral plasma. Decreased mRNA levels of bile acid metabolizing enzymes such as Cyp7a1 and Baat were observed in contrast to increased Sult2a1 level in the liver. Increased mRNA levels of Ostβ and Abcc4 and decreased in mRNA levels of Bsep, Abcc2, Ntcp, and Oatp1b2, suggesting that bile acids efflux from hepatocytes into the peripheral plasma rather than into bile. In conclusion, the changes in bile acid metabolizing enzymes and transporters expression, resulting in increasing the total bile acid concentration in the plasma, signify a protection mechanism by the hepatocyte to reduce hepatotoxicity during disease progression to NASH but may promote liver fibrosis.
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http://dx.doi.org/10.1016/j.taap.2019.114664DOI Listing
September 2019

Diagnostic performance evaluation of sulfate-conjugated cholesterol metabolites as urinary biomarkers of Niemann-Pick disease type C.

Clin Chim Acta 2019 Jul 12;494:58-63. Epub 2019 Mar 12.

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; Faculty of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi, Aoba-Ku, Sendai 980-8574, Japan.

Background: Niemann-Pick disease type C (NPC) is an autosomal recessive inherited disorder with progressive neuronal degeneration. Because conventional diagnostic methods are complicated and invasive, biomarker tests have drawn attention. We aimed to evaluate three urinary conjugated cholesterol metabolites as diagnostic biomarkers for NPC.

Methods: Urine samples from 23 patients with NPC, 28 healthy controls, and 7 patients with inherited metabolic disorders were analyzed. 3β-Sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates in urine were quantified by liquid chromatography-tandem mass spectrometry. The diagnostic performance of the three metabolites and their total concentration was evaluated.

Result: Creatinine-corrected concentrations of three metabolites and their total concentration were all significantly higher in NPC patients (0.0098 < P < .0448). The area under the receiver operating curve for all metabolites exceeded 0.95, the clinical specificity was 92-100%, and the clinical sensitivity was ~95%. In the urine of patients with other inherited metabolic diseases, the concentrations of the metabolites were lower than those in the urine of patients with NPC.

Conclusion: These conjugated cholesterol metabolites in urine can serve as useful diagnostic markers for noninvasive screening of NPC.
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http://dx.doi.org/10.1016/j.cca.2019.03.1610DOI Listing
July 2019

Changes in Enterohepatic Circulation after Duodenal-Jejunal Bypass and Reabsorption of Bile Acids in the Bilio-Pancreatic Limb.

Obes Surg 2019 06;29(6):1901-1910

Department of Surgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.

Background And Aims: Duodenal-jejunal bypass (DJB) shows great effects on weight loss and diabetes improvement. Previously, we reported that the bilio-pancreatic (BP) limb plays an important role in glycemic improvement and in serum bile acid (BA) level increase as reported by Miyachi et al. (Surgery 159(5):1360-71, 2016). This study aimed to investigate the mechanism of BA elevation after DJB and the relationship between these effects and BP-limb length.

Methods: Otsuka Long-Evans Tokushima Fatty rats with diabetes were randomly assigned into four groups: one sham group and three DJB groups. Three DJB groups were defined according to the BP-limb length: 0 cm, 15 cm, and 30 cm. The lengths of the alimentary limb and common channel were set equally in each DJB groups. Body weight, glucose tolerance, and BA levels in the liver, bile juice, portal vein, and intestinal contents were assessed postoperatively. Changes in enterohepatic circulation of BAs were assessed using labeled BA.

Results: BA elevation after DJB was higher with longer BP-limb. In the 30-cm group, the serum total BA level and BA levels in the portal vein, liver, and bile juice were greater than those in other groups. The enterohepatic circulation was shortened in the 15-cm and 30-cm groups.

Conclusions: Shortening of the "enterohepatic circulation" by early reabsorption of BAs in the BP-limb, not by the early influx of bile juice into the ileum, was the main cause of BA elevation after DJB. Thus, glycemic improvement and elevation of BA concentration after DJB depend on the BP-limb length.
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http://dx.doi.org/10.1007/s11695-019-03790-yDOI Listing
June 2019

Value of global metabolomics in association with diagnosis and clinicopathological factors of renal cell carcinoma.

Int J Cancer 2019 07 24;145(2):484-493. Epub 2019 Jan 24.

Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

Renal cell carcinoma (RCC) is a malignant tumor that currently lacks clinically useful biomarkers indicative of early diagnosis or disease status. RCC has commonly been diagnosed based on imaging results. Metabolomics offers a potential technology for discovering biomarkers and therapeutic targets by comprehensive screening of metabolites from patients with various cancers. We aimed to identify metabolites associated with early diagnosis and clinicopathological factors in RCC using global metabolomics (G-Met). Tumor and nontumor tissues were sampled from 20 cases of surgically resected clear cell RCC. G-Met was performed by liquid chromatography mass spectrometry and important metabolites specific to RCC were analyzed by multivariate statistical analysis for cancer diagnostic ability based on area under the curve (AUC) and clinicopathological factors (tumor volume, pathological T stage, Fuhrman grade, presence of coagulation necrosis and distant metastasis). We identified 58 metabolites showing significantly increased levels in tumor tissues, 34 of which showed potential early diagnostic ability (AUC >0.8), but 24 did not discriminate between tumor and nontumor tissues (AUC ≤0.8). We recognized 6 pathways from 9 metabolites with AUC >0.8 and 7 pathways from 10 metabolites with AUC ≤0.8 about malignant status. Clinicopathological factors involving malignant status correlated significantly with metabolites showing AUC ≤0.8 (p = 0.0279). The tricarboxylic acid cycle (TCA) cycle, TCA cycle intermediates, nucleotide sugar pathway and inositol pathway were characteristic pathways for the malignant status of RCC. In conclusion, our study found that metabolites and their pathways allowed discrimination between early diagnosis and malignant status in RCC according to our G-Met protocol.
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http://dx.doi.org/10.1002/ijc.32115DOI Listing
July 2019

Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation.

Biochem Pharmacol 2018 10 8;156:420-430. Epub 2018 Sep 8.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai 980-8575, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8575, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan. Electronic address:

Genetic variations within cytochrome P450 2B6 (CYP2B6) contribute to inter-individual variation in the metabolism of clinically important drugs, including cyclophosphamide, bupropion, methadone and efavirenz (EFZ). In this study, we performed an in vitro analysis of 40 CYP2B6 allelic variant proteins including seven novel variants identified in 1070 Japanese individuals. Wild-type and 39 variant proteins were heterologously expressed in 293FT cells to estimate the kinetic parameters (K, V, and CL) of EFZ 8-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin (7-ETC) O-deethylation activities. The concentrations of CYP2B6 variant holo-enzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild-type and 28 variants showed a peak at 450 nm. The kinetic parameters were measured for the wild-type and 24 variant proteins. The values for the remaining 15 variants could not be determined because the enzymatic activity was not detected at the highest substrate concentration used. Compared to wild-type, six variants showed significantly decreased EFZ 8-hydroxylation CL values, while these values were significantly increased in another six variants, including CYP2B6.6. Although 7-ETC O-deethylation CL values of CYP2B6 variants did not differ significantly from that of CYP2B6.1, the CL ratios obtained for 7-ETC O-deethylation were highly correlated with EFZ 8-hydroxylation. Furthermore, three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2B6 variants. Our findings could provide evidence of the specific metabolic activities of the CYP2B6 proteins encoded by these variant alleles.
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http://dx.doi.org/10.1016/j.bcp.2018.09.010DOI Listing
October 2018

Phenotypic variability of Niemann-Pick disease type C including a case with clinically pure schizophrenia: a case report.

BMC Neurol 2018 Aug 17;18(1):117. Epub 2018 Aug 17.

Department of Neurology, National Center Hospital, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, 187-8551, Kodaira, Tokyo, Japan.

Background: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with severe prognosis. Disease-specific therapy is crucial to prevent disease progression; however, diagnosing NPC is quite difficult because of remarkably variable clinical presentations. The NPC Suspicion Index (NPC-SI) was developed to overcome this problem. Identifying preclinical cases is important for prevention and therapy. Here, we report three newly diagnosed NPC cases, one typical juvenile-onset case and the cases of two sisters with symptoms neurologically/psychiatrically indistinguishable from dystonia and schizophrenia, respectively.

Case Presentation: In Case 1, a 25-year-old man presented with a 14-year history of intellectual disability, clumsiness, spastic ataxia, dysphagia, and frequent falls. Neurological examination revealed vertical supranuclear gaze palsy and involuntary movements. Ultrasonography revealed mild splenomegaly, and filipin staining of skin fibroblasts was positive with a variant staining pattern. NPC1 gene analysis showed compound heterozygous mutations, including c.1421C > T (p.P474L), a known causative mutation, and c.3722 T > C (p.L1241S), a new mutation. In Case 2, a 28-year-old woman, the proband, who had marked splenomegaly in her childhood, survived well, contrary to the expected severe prognosis of infantile NPC. She had minor neuropsychiatric symptoms including auditory hallucinations, nocturnal urination, and sleep paralysis. At the age of 28 years, she presented with a 1-year history of orofacial and oromandibular painful dystonia. The patient's 35-year-old sister (Case 3) was diagnosed with schizophrenia. In both cases, filipin staining of skin fibroblasts was positive with variant staining patterns, as well as elevated levels of urinary bile acids. NPC1 gene analysis showed compound heterozygous mutations including c.3011C > T (p.S1004 L), a known causative mutation, and c.160_161insG (p.D54GfsX4), a new mutation. Their mother, who was under therapy with modafinil for narcolepsy, shared the latter mutation.

Conclusions: Marked clinical variability was observed in our three cases. NPC could masquerade as a pure neuropsychiatric disorder such as dystonia or schizophrenia. Abdominal ultrasonography, history evaluation, and neurological examination were quite important in the diagnostic process.
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http://dx.doi.org/10.1186/s12883-018-1124-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098631PMC
August 2018

Elevation of plasma lysosphingomyelin-509 and urinary bile acid metabolite in Niemann-Pick disease type C-affected individuals.

Mol Genet Metab Rep 2018 Jun 21;15:90-95. Epub 2018 Mar 21.

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Niemann-Pick disease type C (NPC) is a neurovisceral disorder associated with the accumulation of lipids such as cholesterol and sphingolipids. NPC is caused by either or , which encode lysosomal proteins located at membraneous and soluble fractions, respectively. For the past decade, the oxidation products of cholesterol, such as cholestane-3β,5α,6β-triol and 7-ketocholesterol, have been considered selective biomarkers for NPC. However, recent evidence has indicated numerous novel biomarkers for NPC, which raises the possibility that the diagnosis of NPC might be associated with the elevation of multiple lipid biomarkers, rather than a single biomarker. Sphingosylphosphorylcholine (SPC) has been suggested to be one such biomarker for NPC, in which elevated sphingomyelin is a potential precursor. Thus, we first performed a validation study of plasma SPC using LC-MS/MS. The results showed the following plasma concentrations in the NPC-affected and control individuals, respectively: 8.2 ± 2.8 nM (mean ± SD; median, 7.0 nM; max, 11.7 nM; min, 5.1 nM;  = 5) and 3.1 ± 1.4 nM (median, 2.9 nM; max, 4.8 nM; min, 1.5 nM;  = 7). We further extended the study to plasma lysophingomyelin-509 for NPC, a newly reported biomarker with uncharacterized chemical nature. Based on these result with plasma SPC as a surrogate marker, the value of mean of median of plasma lysophingomyelin-509 in NPC-affected individuals elevated at 65.2 (max, 73.2; min, 26.7;  = 5). Furthermore, the efficacy of plasma SPC and lysosphingomyelin-509 as promising biomarkers for this disorder was supported by the finding that the urinary concentration of 3β-sulfooxy-7β--acetylglucosaminyl-5-cholen-24-oic acid, an established biomarker for NPC, was also elevated in the NPC-affected individuals. These results suggest that a novel combination of plasma biomarkers, such as SPC and/or lysophingomyelin-509, and urinary bile acid metabolite could offer a promising platform for the diagnosis of NPC.
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http://dx.doi.org/10.1016/j.ymgmr.2018.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047109PMC
June 2018

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals.

Drug Metab Dispos 2018 08 16;46(8):1083-1090. Epub 2018 May 16.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences (E.H., Yo.N., F.A., Yu.N., N.H., M.H.), and Tohoku Medical Megabank Organization (S.S., J.Y., M.N., M.Y., M.H.), and Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan (M.M., H.Y., N.M., M.H.)

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, mutations, including , , c.2846A>T, and c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these polymorphisms has been identified in the Asian population. Recently, 21 allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue-native PAGE, respectively. Additionally, the values of three kinetic parameters-the Michaelis constant ( ), maximum velocity ( ), and intrinsic clearance ( = )-were determined for the reduction of 5-FU. Eleven variants exhibited significantly decreased intrinsic clearance compared with wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue-native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.
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http://dx.doi.org/10.1124/dmd.118.081737DOI Listing
August 2018

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

Brain Tumor Pathol 2018 Apr 18;35(2):90-96. Epub 2018 Apr 18.

Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan.

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.
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http://dx.doi.org/10.1007/s10014-018-0317-0DOI Listing
April 2018

Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation.

Drug Metab Pharmacokinet 2018 Feb 22;33(1):82-89. Epub 2017 Nov 22.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, 980-8574, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai, 980-8575, Japan. Electronic address:

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.
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http://dx.doi.org/10.1016/j.dmpk.2017.11.004DOI Listing
February 2018

Simultaneous analysis of oral anticancer drugs for renal cell carcinoma in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry.

Biomed Chromatogr 2018 Jun 1;32(6):e4184. Epub 2018 Feb 1.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Miyagi, Japan.

An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry has been developed and validated for simultaneous measurement of four tyrosine kinase inhibitors used for renal cell carcinoma and their metabolites in human plasma. Despite their similar structures, it is difficult to measure plasma levels of these compounds simultaneously using optimal MS parameters for each compound because a quantitative range exceeding 50,000-fold is required. To overcome this problem, we used a linear range shift technique using in-source collision-induced dissociation. Linearity ranges of sorafenib, sorafenib N-oxide, sunitinib, N-desethyl sunitinib, axitinib and pazopanib were 100-10,000, 10-1,000, 1-100, 1-100, 1-100 and 500-50,000 ng/mL, respectively. The intra- and inter-day precision and accuracy were high, and coefficients of variation and relative error were <10.3% and within ±11.8%, respectively. The matrix effects of all analytes ranged from 87.7 to 114.8%. Extraction recoveries and overall recoveries showed small extraction loss (<15.0%) for all analytes. Moreover, all cancer patient samples used in this study were successfully quantified and fell within the linear range of measurement. Therefore, this novel analytical system using in-source collision-induced dissociation has sufficient performance to measure plasma concentrations of these four tyrosine kinase inhibitors and their metabolites for therapeutic drug monitoring.
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http://dx.doi.org/10.1002/bmc.4184DOI Listing
June 2018

Lipid biomarkers for the peroxisomal and lysosomal disorders: their formation, metabolism and measurement.

Biomark Med 2018 Jan 13;12(1):83-95. Epub 2017 Dec 13.

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Lipid biomarkers play important roles in the diagnosis of and monitoring of treatment in peroxisomal disorders and lysosomal storage disorders. Today, a variety of lipids, including very long chain fatty acids, glycolipids, bile acids and the oxidation products of cholesterol, have been considered as biomarkers for these disorders. In this brief review, the authors summarized the recent advances regarding these lipid biomarkers in terms of their formation, metabolism and measurement in these disorders. An understanding of these biomarkers will offer a key to the development of novel diagnoses and help create more effective therapies in the future.
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http://dx.doi.org/10.2217/bmm-2017-0225DOI Listing
January 2018