Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/ß-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

Wide-Targeted Semi-Quantitative Analysis of Acidic Glycosphingolipids in Cell Lines and Urine to Develop Potential Screening Biomarkers for Renal Cell Carcinoma.

Glycosphingolipids (GSLs), mainly located in the cell membrane, play various roles in cancer cell function. GSLs have potential as renal cell carcinoma (RCC) biomarkers; however, their analysis in body fluids is challenging because of the complexity of numerous glycans and ceramides. Therefore, we applied wide-targeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM) based on theoretical mass to perform a comprehensive measurement of GSLs and evaluate their potency as urinary biomarkers. In semi-quantitative lipidomics, 240 SRM transitions were set based on the reported/speculated structures. We verified the feasibility of measuring GSLs in cells and medium and found that disialosyl globopentaosylceramide (DSGb5 (d18:1/16:0)) increased GSL in the ACHN medium. LC-MS/MS analysis of urine samples from clear cell RCC (ccRCC) patients and healthy controls showed a significant increase in the peak intensity of urinary DSGb5 (d18:1/16:0) in the ccRCC group compared with that in the control group. Receiver operating characteristic analysis indicated that urinary DSGb5 could serve as a sensitive and specific marker for RCC screening, with an AUC of 0.89. This study demonstrated the possibility of urinary screening using DSGb5 (d18:1/16:0). In conclusion, urinary DSGb5 (d18:1/16:0) was a potential biomarker for cancer screening, which could contribute to the treatment of RCC patients.

The Significance of Bile in the Biliopancreatic Limb on Metabolic Improvement After Duodenal-Jejunal Bypass.

INTRODUCTION: Duodenal-jejunal bypass (DJB) is an experimental procedure in metabolic surgery that does not have a restrictive component. Changes in bile acid (BA) dynamics and intestinal microbiota are possibly related to metabolic improvement after DJB. Our previous studies involving obese diabetic rats showed the crucial role of the biliopancreatic limb (BPL) in metabolic improvement after DJB caused by BA reabsorption. We established a new DJB procedure to prevent bile from flowing into the BPL and aimed to elucidate the importance of bile in the BPL after DJB. METHODS: Otsuka Long-Evans Tokushima Fatty rats with diabetes were divided into three groups: two DJB groups and a sham group (n = 11). Duodenal-jejunal anastomosis was performed proximal to the papilla of Vater in the DJB group (n = 11). However, the DJB-D group (n = 11) underwent a new procedure with duodenal-jejunal anastomosis distal to the papilla of Vater for preventing bile flow into the BPL. RESULTS: Glucose metabolism improved and weight gain was suppressed in the DJB group, but not in the DJB-D and sham groups. Serum BA level and conjugated BA concentration were elevated in the DJB group. The gut microbiota was altered only in the DJB group; the abundance of Firmicutes and Bacteroidetes decreased and that of Actinobacteria increased. However, the DJB-D group exhibited no apparent change in the gut microbiota, similar to the sham group. CONCLUSION: BAs are essential in the BPL for metabolic improvement after DJB; they can improve the gut microbiota in these processes.

Elucidation and Regulation of Tyrosine Kinase Inhibitor Resistance in Renal Cell Carcinoma Cells from the Perspective of Glutamine Metabolism.

Tyrosine kinase inhibitors (TKIs) play a crucial role in the treatment of advanced renal cell carcinoma (RCC). However, there is a lack of useful biomarkers for assessing treatment efficacy. Through urinary metabolite analysis, we identified the metabolites and pathways involved in TKI resistance and elucidated the mechanism of TKI resistance. To verify the involvement of the identified metabolites obtained from urine metabolite analysis, we established sunitinib-resistant RCC cells and elucidated the antitumor effects of controlling the identified metabolic pathways in sunitinib-resistant RCC cells. Through the analysis of VEGFR signaling, we aimed to explore the mechanisms underlying the antitumor effects of metabolic control. Glutamine metabolism has emerged as a significant pathway in urinary metabolite analyses. In vitro and in vivo studies have revealed the antitumor effects of sunitinib-resistant RCC cells via knockdown of glutamine transporters. Furthermore, this antitumor effect is mediated by the control of VEGFR signaling via PTEN. Our findings highlight the involvement of glutamine metabolism in the prognosis and sunitinib resistance in patients with advanced RCC. Additionally, the regulating glutamine metabolism resulted in antitumor effects through sunitinib re-sensitivity in sunitinib-resistant RCC. Our results are expected to contribute to the more effective utilization of TKIs with further improvements in prognosis through current drug therapies.

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

The Use of an Antioxidant Enables Accurate Evaluation of the Interaction of Curcumin on Organic Anion-Transporting Polypeptides 4C1 by Preventing Auto-Oxidation.

Flavonoids have garnered attention because of their beneficial bioactivities. However, some flavonoids reportedly interact with drugs via transporters and may induce adverse drug reactions. This study investigated the effects of food ingredients on organic anion-transporting polypeptide (OATP) 4C1, which handles uremic toxins and some drugs, to understand the safety profile of food ingredients in renal drug excretion. Twenty-eight food ingredients, including flavonoids, were screened. We used ascorbic acid (AA) to prevent curcumin oxidative degradation in our method. Twelve compounds, including apigenin, daidzein, fisetin, genistein, isorhamnetin, kaempferol, luteolin, morin, quercetin, curcumin, resveratrol, and ellagic acid, altered OATP4C1-mediated transport. Kaempferol and curcumin strongly inhibited OATP4C1, and the Ki values of kaempferol (AA(-)), curcumin (AA(-)), and curcumin (AA(+)) were 25.1, 52.2, and 23.5 µM, respectively. The kinetic analysis revealed that these compounds affected OATP4C1 transport in a competitive manner. Antioxidant supplementation was determined to benefit transporter interaction studies investigating the effects of curcumin because the concentration-dependent curve evidently shifted in the presence of AA. In this study, we elucidated the food-drug interaction via OATP4C1 and indicated the utility of antioxidant usage. Our findings will provide essential information regarding food-drug interactions for both clinical practice and the commercial development of supplements.

Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice.

Fatty acid-binding protein 7 (FABP7) is vital for uptake and trafficking of fatty acids in the nervous system. To investigate the involvement of FABP7 in noise-induced hearing loss (NIHL) pathogenesis, we used Fabp7 knockout (KO) mice generated via CRISPR/Cas9 in the C57BL/6 background. Initial auditory brainstem response (ABR) measurements were conducted at 9 weeks, followed by noise exposure at 10 weeks. Subsequent ABRs were performed 24 h later, with final measurements at 12 weeks. Inner ears were harvested 24 h after noise exposure for RNA sequencing and metabolic analyses. We found no significant differences in initial ABR measurements, but Fabp7 KO mice showed significantly lower thresholds in the final ABR measurements. Hair cell survival was also enhanced in Fabp7 KO mice. RNA sequencing revealed that genes associated with the electron transport chain were upregulated or less impaired in Fabp7 KO mice. Metabolomic analysis revealed various alterations, including decreased glutamate and aspartate in Fabp7 KO mice. In conclusion, FABP7 deficiency mitigates cochlear damage following noise exposure. This protective effect was supported by the changes in gene expression of the electron transport chain, and in several metabolites, including excitotoxic neurotransmitters. Our study highlights the potential therapeutic significance of targeting FABP7 in NIHL.

Global Proteomics for Identifying the Alteration Pathway of Niemann-Pick Disease Type C Using Hepatic Cell Models.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder with progressive neurodegeneration. Although the causative genes were previously identified, NPC has unclear pathophysiological aspects, and patients with NPC present various symptoms and onset ages. However, various novel biomarkers and metabolic alterations have been investigated; at present, few comprehensive proteomic alterations have been reported in relation to NPC. In this study, we aimed to elucidate proteomic alterations in NPC and perform a global proteomics analysis for NPC model cells. First, we developed two NPC cell models by knocking out NPC1 using CRISPR/Cas9 (KO1 and KO2). Second, we performed a label-free (LF) global proteomics analysis. Using the LF approach, more than 300 proteins, defined as differentially expressed proteins (DEPs), changed in the KO1 and/or KO2 cells, while the two models shared 35 DEPs. As a bioinformatics analysis, the construction of a protein-protein interaction (PPI) network and an enrichment analysis showed that common characteristic pathways such as ferroptosis and mitophagy were identified in the two model cells. There are few reports of the involvement of NPC in ferroptosis, and this study presents ferroptosis as an altered pathway in NPC. On the other hand, many other pathways and DEPs were previously suggested to be associated with NPC, supporting the link between the proteome analyzed here and NPC. Therapeutic research based on these results is expected in the future.

Functional Characterization of 29 Cytochrome P450 4F2 Variants Identified in a Population of 8380 Japanese Subjects and Assessment of Arachidonic Acid ω-Hydroxylation.

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [Vmax , substrate concentration producing half of Vmax (S50 ), and intrinsic clearance (CL int ) as Vmax /S50 ] of AA ω-hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω-hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function.

High throughput LC/ESI-MS/MS method for simultaneous analysis of 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib in human plasma.

Many types of oral molecular-targeted anticancer drugs are clinically used in cancer genomic medicine. Combinations of multiple molecular-targeted anticancer drugs are also being investigated, expecting to prolong the survival of patients with cancer. Therapeutic drug monitoring of oral molecular-targeted drugs is important to ensure efficacy and safety. A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been used for simultaneous determination of these drugs in human plasma. However, the sensitivity of mass spectrometers and differences in the therapeutic range of drugs have rendered the development of simultaneous LC/ESI-MS/MS methods difficult. In this study, a simultaneous quantitative method for 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib was developed based on the results of linear range shifts of the calibration curves using four ion abundance adjustment techniques (collision energy defects, in-source collision-induced dissociation, secondary product ion selected reaction monitoring, and isotopologue selected reaction monitoring). The saturation of the detector for the seven analytes was resolved by incorporating optimal ion abundance adjustment techniques. Furthermore, the reproducibility of this method was confirmed in validation tests, and plasma from patients was measured by this method to demonstrate its usefulness in actual clinical practice. This analytical method is expected to make a substantial contribution to the promotion of personalized medicine in the future.

Therapeutic Drug Monitoring of Blood Sirolimus and Tacrolimus Concentrations for Polypharmacy Management in a Lymphangioleiomyomatosis Patient Taking Two Cytochrome P450 3A Inhibitors.

Patients with lymphangioleiomyomatosis (LAM) and lung transplantations are treated with multiple drugs, such as tacrolimus, mycophenolate mofetil, prednisolone, and itraconazole, for long-term suppression of rejection response and prevention of infection. Additional drugs are required when lung transplant recipients develop graft complications. Therefore, managing polypharmacy is critical because of drug-drug interactions caused by various factors, including drug-metabolizing enzymes such as cytochrome P450 3A (CYP3A). The patient was a 48-year-old woman (height 144.9 cm and weight 38.4 kg) who underwent lung transplantation for LAM. Mycophenolate mofetil, tacrolimus (target blood concentration, 4.0-8.0 ng/mL), and prednisolone were administered for immunosuppression, and itraconazole and clarithromycin were administered to manage graft infection. The patient developed unilateral lymphedema, predominantly in the left leg; therefore, sirolimus was initiated with a target blood concentration of 3.0-5.0 ng/mL. In addition to 1.0 mg/day of sirolimus, tacrolimus (0.3 mg/day), itraconazole (100 mg/day), and clarithromycin (800 mg/day) were added. Blood sirolimus concentrations ranged from 18.8 to 36.9 ng/mL on days 6 to 9; thus, treatment with sirolimus was stopped because of over-target blood concentrations. Blood concentrations of sirolimus and tacrolimus were successfully managed without adverse events using therapeutic drug monitoring (TDM) and azole anti-fungal substitution of azithromycin instead of clarithromycin although sirolimus concentration was relatively lower compared to the target range. Thereby, frequent TDM, management of polypharmacy that influences CYP3A activity, and possibly CYP3A genotyping should be appropriately conducted for personalized medicine.

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity.

CYP3A4, which contributes to the metabolism of more than 30% of clinically used drugs, exhibits high variation in its activity; therefore, predicting CYP3A4 activity before drug treatment is vital for determining the optimal dosage for each patient. We aimed to develop and validate an LC-tandem mass spectrometry (LC-MS/MS) method that simultaneously measures the levels of CYP3A4 activity-related predictive biomarkers (6ß-hydroxycortisol (6ß-OHC), cortisol (C), 1ß-hydroxydeoxycholic acid (1ß-OHDCA), and deoxycholic acid (DCA)). Chromatographic separation was achieved using a YMC-Triart C18 column and a gradient flow of the mobile phase comprising deionized water/25% ammonia solution (100 : 0.1, v/v) and methanol/acetonitrile/25% ammonia solution (50 : 50 : 0.1, v/v/v). Selective reaction monitoring in the negative-ion mode was used for MS/MS, and run times of 33 min were used. All analytes showed high linearity in the range of 3-3000 ng/mL. Additionally, their concentrations in urine samples derived from volunteers were analyzed via treatment with deconjugation enzymes, ignoring inter-individual differences in the variation of other enzymatic activities. Our method satisfied the analytical validation criteria under clinical conditions. Moreover, the concentrations of each analyte were quantified within the range of calibration curves for all urine samples. The conjugated forms of each analyte were hydrolyzed to accurately examine CYP3A4 activity. Non-invasive urine sampling employed herein is an effective alternative to invasive plasma sampling. The analytically validated simultaneous quantification method developed in this study can be used to predict CYP3A4 activity in precision medicine and investigate the potential clinical applications of CYP3A4 biomarkers (6ß-OHC/C and 1ß-OHDCA/DCA ratios).

Glutathione depletion results in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

AIMS: Protein disulfide isomerase (PDI) is an essential enzyme involved in oxidative protein folding. PDI is S-nitrosylated in the brains of Alzheimer's disease patients, and S-nitrosylated PDI is considered one of main causes of Alzheimer's disease. However, the mechanisms underlying PDI S-nitrosylation have not yet been elucidated. Because glutathione (GSH) depletion is a pathological feature of Alzheimer's disease, we investigated the effect of GSH depletion on the S-nitrosylation level of PDI. MAIN METHODS: SH-SY5Y cells, which is a human derived neuroblastoma cells, were used in this study. Glutamate and buthionine sulfoximine (BSO) were used as GSH depletors. S-nitrosylated PDI was detected by biotin-switch assay. KEY FINDINGS: GSH depletion by glutamate, a cystine/glutamate antiporter xCT inhibitor, increased S-nitrosylated PDI at C343 in SH-SY5Y cells, and induced IRE1α phosphorylation. BSO, a γ-glutamylcysteine synthetase inhibitor, also increased S-nitrosylated PDI and phosphorylated IRE1α upon GSH depletion. Because S-nitrosylated PDI at C343 is stable in neuro cells, S-nitrosylated PDI by GSH depletion progresses to neurodegeneration by the induction of endoplasmic reticulum stress via phosphorylated IRE1α signaling from the early to late stage. Furthermore, treatment with neohesperidin, but not N-acetylcysteine (NAC), improved PDI S-nitrosylation level in GSH-depleted SH-SY5Y cells because nitrosylated compound of NAC induces PDI S-nitrosylation. SIGNIFICANCE: The results of our study provide a new insight into the mechanisms of neurodegeneration, and may be useful for the development of drugs for Alzheimer's diseases.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Development of a Highly Sensitive and Rapid Liquid Chromatography-Tandem Mass Spectrometric Method Using a Basic Mobile Phase Additive to Determine the Characteristics of the Urinary Metabolites for Niemann-Pick Disease Type C.

As Niemann-Pick disease type C (NPC) is difficult to diagnose owing to its various clinical symptoms; biomarker tests have been developed. Previously, we revealed urinary sulfated cholesterol metabolites as noninvasive biomarkers for NPC. However, LC/tandem mass spectrometry (LC/MS/MS) requires long separation time and large urine volumes. Recently, a basic mobile phase was reported to increase the MS intensity. Thus, we developed a highly sensitive and rapid LC/MS/MS method for analyzing urinary cholesterol metabolites using a basic mobile phase additive. 3ß-Sulfooxy-7ß-N-acetylglucosaminyl-5-cholenic acid, its glycine and taurine conjugates, 3ß-sulfooxy-7ß-hydroxy-5-cholenic acid, and 7-oxo form were measured, with selected reaction monitoring in negative ion mode. Oasis HLB and L-column 3 were used for column-switching LC/MS/MS and urine diluted 10-fold was employed as the sample. After trapping, gradient separation was performed using solutions containing 1% (v/v) ammonium solution. On average, a 16-fold increase in peak areas was observed compared to that obtained at pH 5.5 with the mobile phases. Although the previous method needed 60 min for separation from interference peaks, we succeeded to separate them in 7 min with optimized LC condition. Further, all compounds showed good linearity from 0.3-1000 ng/mL, with satisfactory intra- and inter-day reproducibility. The developed method was applied to the urinalysis of healthy participants and NPC patients. Overall, the concentrations of metabolites correlated with those obtained using the previous method. Therefore, we succeeded to increasing MS intensity and shorten LC running time; and the method is useful for the noninvasive diagnostic screening of patients with NPC.

Bile Acid-Drug Interaction via Organic Anion-Transporting Polypeptide 4C1 Is a Potential Mechanism of Altered Pharmacokinetics of Renally Excreted Drugs.

Patients with liver diseases not only experience the adverse effects of liver-metabolized drugs, but also the unexpected adverse effects of renally excreted drugs. Bile acids alter the expression of renal drug transporters, however, the direct effects of bile acids on drug transport remain unknown. Renal drug transporter organic anion-transporting polypeptide 4C1 (OATP4C1) was reported to be inhibited by chenodeoxycholic acid. Therefore, we predicted that the inhibition of OATP4C1-mediated transport by bile acids might be a potential mechanism for the altered pharmacokinetics of renally excreted drugs. We screened 45 types of bile acids and calculated the IC50, Ki values, and bile acid−drug interaction (BDI) indices of bile acids whose inhibitory effect on OATP4C1 was >50%. From the screening results, lithocholic acid (LCA), glycine-conjugated lithocholic acid (GLCA), and taurine-conjugated lithocholic acid (TLCA) were newly identified as inhibitors of OATP4C1. Since the BDI index of LCA was 0.278, LCA is likely to inhibit OATP4C1-mediated transport in clinical settings. Our findings suggest that dose adjustment of renally excreted drugs may be required in patients with renal failure as well as in patients with hepatic failure. We believe that our findings provide essential information for drug development and safe drug treatment in clinics.

Evaluation of a Capillary Microsampling Device for Analyzing Plasma Lenvatinib Concentration in Patients With Hepatocellular Carcinoma.

BACKGROUND: The anticancer drug, Lenvima (lenvatinib), has severe side effects. Therapeutic drug monitoring helps ensure its efficacy and safety. Regular and optimally timed blood sampling is tough, especially when lenvatinib is self-medicated. Microsampling using the easy to handle Microsampling Wing (MSW) may help circumvent this problem. However, current lenvatinib detection methods are not sensitive enough to detect its concentrations in microsamples (<50-250 µL). Thus, the aim of this study was 2-fold (1) develop an analytic method to estimate plasma lenvatinib concentrations in microsamples and (2) verify whether this method works on micro (5.6 µL) blood plasma samples obtained clinically through MSW from patients with unresectable hepatocellular carcinoma (HCC). METHODS: A simple, highly sensitive, and specific liquid chromatography-electrospray ionization tandem mass spectrometry method was developed. Using this novel protocol, the trough blood plasma concentration of lenvatinib was measured for both blood sampled conventionally and that using MSW. Thirty-five venous whole blood samples were obtained from 11 patients with HCC. Furthermore, the stability of lenvatinib in MSW samples during storage was evaluated. RESULTS: The mean plasma lenvatinib concentration estimates were not significantly different between the MSW and conventional venous blood samples. CV for interday and intraday assays was low. Up to day 5, the lenvatinib concentration in the MSW samples was 85%-115% of the initial day concentration (when stored at 25°C or 4°C). The interference of endogenous matrix components in the human plasma was low. CONCLUSIONS: These results indicate that the novel mass spectrometry protocol accurately measures lenvatinib in human plasma and is reproducible. Thus, MSW could be a useful microsampling device for lenvatinib therapeutic drug monitoring in patients with HCC when used in combination with this novel liquid chromatography-electrospray ionization tandem mass spectrometry detection method.

Importance of Rare DPYD Genetic Polymorphisms for 5-Fluorouracil Therapy in the Japanese Population.

Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. In Caucasians, four DPYD risk variants are recognized to be responsible for interindividual variations in the development of 5-FU toxicity. However, these risk variants have not been identified in Asian populations. Recently, 41 DPYD allelic variants, including 15 novel single nucleotide variants, were identified in 3,554 Japanese individuals by analyzing their whole-genome sequences; however, the effects of these variants on DPD enzymatic activity remain unknown. In the present study, an in vitro analysis was performed on 41 DPD allelic variants and three DPD risk variants to elucidate the changes in enzymatic activity. Wild-type and 44 DPD-variant proteins were heterologously expressed in 293FT cells. DPD expression levels and dimerization of DPD were determined by immunoblotting after SDS-PAGE and blue native PAGE, respectively. The enzymatic activity of DPD was evaluated by quantification of dihydro-5-FU, a metabolite of 5-FU, using high-performance liquid chromatography-tandem mass spectrometry. Moreover, we used 3D simulation modeling to analyze the effect of amino acid substitutions on the conformation of DPD. Among the 41 DPD variants, seven exhibited drastically decreased intrinsic clearance (CL int ) compared to the wild-type protein. Moreover, R353C and G926V exhibited no enzymatic activity, and the band patterns observed in the immunoblots after blue native PAGE indicated that DPD dimerization is required for its enzymatic activity. Our data suggest that these variants may contribute to the significant inter-individual variability observed in the pharmacokinetics and pharmacodynamics of 5-FU. In our study, nine DPD variants exhibited drastically decreased or no enzymatic activity due to dimerization inhibition or conformational changes in each domain. Especially, the rare DPYD variants, although at very low frequencies, may serve as important pharmacogenomic markers associated with the severe 5-FU toxicity in Japanese population.

Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.

Niemann-Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symptoms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC-MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC-MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology observations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.