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.

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.

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.

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.

The prevalence of end-of-life chemotherapy and targeted therapy in Japan, assessed using a health claims database.

PURPOSE: This study aimed to investigate the current status of end-of-life chemotherapy and targeted therapy and explore the aggressiveness of end-of-life care in Japan using the DeSC database, a large administrative claims database. METHODS: We identified fatal cases of at least one cancer-related diagnosis between April 2015 and November 2020. Patients prescribed at least one anticancer drug were analyzed, and chemotherapy regimens were categorized based on the combination of concomitant anticancer drugs prescribed. RESULTS: Among 1,095,713 individuals enrolled in the National Health Insurance database, 7,300 deaths with cancer-related diagnosis were identified. Of these, 4,010 cases were identified in which at least one anticancer drug was prescribed, and 11.6% of 7,300 death had been prescribed anticancer drugs in their last 30 days of life. The most commonly used regimen was S-1 (tegafur, gimeracil, and oteracil potassium combination) monotherapy, followed by nivolumab monotherapy and nab-paclitaxel plus gemcitabine. Immune checkpoint inhibitor monotherapy was more likely prescribed to patients whose last chemotherapy dose was in the last 30 days of life (p = 0.0066, chi-squared test). CONCLUSIONS: This study provides insights into the current status of end-of-life chemotherapy and targeted therapy in Japan, using a large administrative claims database. The results of this study will inform future research on end-of-life chemotherapy and targeted therapy, and help develop strategies to improve the quality of life of patients with advanced cancer.

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.

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.

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.

Predictive model for recurrence of renal cell carcinoma by comparing pre- and postoperative urinary metabolite concentrations.

To improve treatment outcomes in real practice, useful biomarkers are desired when predicting postoperative recurrence for renal cell carcinoma (RCC). We collected data from patients who underwent definitive surgery for RCC and for benign urological tumor at our department between November 2016 and December 2019. We evaluated the differences in pre- and postoperative urinary metabolites with our precise quantitative method and identified predictive factors for RCC recurrence. Additionally, to clarify the significance of metabolites, we measured the intracellular metabolite concentration of three RCC cell lines. Among the 56 patients with RCC, nine had a recurrence (16.0%). When comparing 27 patients with T1a RCC and 10 with benign tumor, a significant difference was observed between pre- and postoperative concentrations among 10 urinary metabolites. In these 10 metabolites, multiple logistic regression analysis identified five metabolites (lactic acid, glycine, 2-hydroxyglutarate, succinic acid, and kynurenic acid) as factors to build our recurrence prediction model. The values of area under the receiver operating characteristic curve, sensitivity, and specificity in this predictive model were 0.894%, 88.9%, and 88.0%, respectively. When stratified into low and high risk groups of recurrence based on this model, we found a significant drop of recurrence-free survival rates among the high risk group. In in vitro studies, intracellular metabolite concentrations of metastatic tumor cell lines were much higher than those of primary tumor cell lines. By using our quantitative evaluation of urinary metabolites, we could predict postoperative recurrence with high sensitivity and specificity. Urinary metabolites could be noninvasive biomarkers to improve patient outcome.

Clinical Importance of Plasma Drug Concentration of Oral Molecular Targeted Drugs for Renal Cell Carcinoma.

PURPOSE: Therapeutic drug monitoring (TDM) is widely used in clinical practice to maximize drug efficacy and minimize toxicities. Currently, it is also practiced in the use of oral molecular targeted drugs. The objective of this study was to assess the clinical importance of measuring the systemic concentration of oral molecular targeted drugs used to treat renal cell carcinoma (RCC). METHODS: The systemic concentrations of the oral molecular targeted drugs sorafenib, sunitinib, axitinib, pazopanib, and everolimus used for RCC were useful for therapeutic interventions, and clinical outcomes were evaluated retrospectively. RESULTS: The interventional use of systemic drug concentration was confirmed in 26 of 87, and their categories are presented. The systemic concentration of sunitinib was useful in dose reduction and/or discontinuation (n = 10), dose escalation (n = 3), and adherence monitoring (n = 2). Nine of the 10 patients whose dose was reduced showed reduced adverse event. Two patients who were intervened in adherence monitor showed improved adherence. For axitinib, dose reduction and/or discontinuation (n = 1) and dose escalation (n = 6) were confirmed. For pazopanib, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, both of them were confirmed to have reduced adverse events. For everolimus, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, a patient with reduced dose recovered from adverse events. Interventions for sorafenib were not identified. CONCLUSIONS: This study demonstrated that systemic concentrations of oral molecular targeted drugs for RCC were considered to be clinically useful for dose adjustment, monitoring of treatment adherence, and the detection of drug interactions. Moreover, this information could be successfully used to guide individualized therapy to maximize the antitumor effects of these drugs.

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

BACKGROUND: Dementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. METHODS: We used SH-SY5Y cells cultured in high-glucose media. RESULTS: S-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. CONCLUSION: Glucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. GENERAL SIGNIFICANCE: The results may be useful for designing curative treatment strategies for dementia.

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

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.

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.

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.

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.

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.

Real-world anticancer medications for reproductive-age women with breast cancer by using a claims database in Japan.

Aim: To describe real-world breast cancer medications among reproductive-age women. Patients & methods: Using data from a Japanese claims database, anticancer prescriptions were classified into seven categories of amenorrhea risk based on fertility preservation guidelines. Results: We identified 2999 women with records of breast cancer and anticancer prescription from 2005 to 2018. The proportions of prescriptions were as follows: high, 4.1-12.9%; intermediate: 6.0-16.3%; low: 0.4-2.3%; very low/no: 0.3-12.2%; unknown: 33.9-45.5%; unlisted combination: 12.2-23.4%; and unlisted drug: 12.5-26.7%. The common drugs in the unknown category were trastuzumab (n = 1527), docetaxel (n = 1014), and paclitaxel (n = 995). For medications unlisted in the guidelines, various drugs and drug combinations were observed. Conclusion: Numerous anticancer drugs are currently being prescribed with insufficient evidence regarding amenorrhea risk.

Lay abstract The ability to have children for breast cancer patients is one of the key issues of cancer survivorship, especially because recent progress in anticancer treatments has enabled patients to achieve longer survival. The fertility preservation guidelines of the American Society of Clinical Oncology (2006) introduce some anticancer treatments that carry potential risks to future fertility. In this study, the anticancer prescriptions of 2999 patients with breast cancer aged between 15 and 49 years were examined. Results showed that several medications are prescribed despite the lack of information on the risk of infertility. This suggests that further research is required to fill the evidence gap, and that decision aid through adequate counseling should be undertaken.

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

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.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase.

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b' domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b' domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.