Novel metabolic biomarker for early detection and diagnosis to the patients with gastric cardia adenocarcinoma.

BACKGROUND: Gastric cardia adenocarcinoma (GCA) is classified as Siewert type II adenocarcinoma at the esophagogastric junction in Western countries. The majority of GCA patients do not exhibit early warning symptoms, leading to over 90% of diagnoses at an advanced stage, resulting in a grim prognosis, with less than a 20% 5-year survival rate. METHOD: Metabolic features of 276 GCA and 588 healthy controls were characterized through a widely-targeted metabolomics by UPLC-MS/MS analysis. This study encompasses a joint pathway analysis utilizing identified metabolites, survival analysis in both early and advanced stages, as well as high and negative and low expression of HER2 immunohistochemistry staining. Machine learning techniques and Cox regression models were employed to construct a diagnostic panel. RESULTS: A total of 25 differential metabolites were consistently identified in both discovery and validation sets based on criteria of p < 0.05, (VIP) ≥ 1, and FC ≥ 2 or FC ≤ 0.5. Early-stage GCA patients exhibited a more favorable prognosis compared to those in advanced stages. HER2 overexpression was associated with a more positive outcome compared to the negative and low expression groups. Metabolite panel demonstrated a robust diagnostic performance with AUC of 0.869 in discovery set and 0.900 in validation set. CONCLUSIONS: A total of 25 common and stable differential metabolites may hold promise as liquid non-invasive indicators for GCA diagnosis. HER2 may function as a tumor suppressor gene in GCA, as its overexpression is associated with improved survival. The downregulation of bile acid metabolism in GCA may offer valuable theoretical insights and innovative approaches for precision-targeted treatments in GCA patients.

Recent advances in enzymatic and chemoenzymatic synthesis of N- and O-glycans.

Glycosylation is one of the most common post-translational modifications of proteins, which plays essential roles in regulating the biological functions of proteins. Efficient and versatile methods for the synthesis of homogeneous and well-defined N- and O-glycans remain an urgent need for biological studies and biomedical applications. Despite their structural complexity, tremendous progress has been made in the synthesis of N- and O-glycans in recent years. This review discusses some recent advances in the enzymatic and chemoenzymatic synthesis of N- and O-glycans.

Preclinical PK investigation of a novel IDO1/TDO dual inhibitor-SHR9146 in mouse plasma and tissues by LC-MS/MS.

Purpose: The aim of the present study was to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of SHR9146, a novel IDO1/TDO dual inhibitor, in mouse plasma and tissues, and to apply it to investigate the preclinical plasma pharmacokinetics and tissue distribution of SHR9146 in mice. Methods: Samples were spiked with deuterated SHR9146-d4 as an internal standard and pretreated by protein-precipitation extraction with methanol. Chromatographic separation was performed on a Venusil ABS C18 column (150 × 4.6 mm, 5 µm) by isocratic elution with 10 mM ammonium acetate buffer containing 0.1% formic acid solution and methanol as mobile phases. MS detection was conducted in positive electrospray ionization with multiple reaction monitoring at m/z 444.1/229.4 for SHR9146 and m/z 448.4/229.2 for the internal standard. Results: The method showed good linearity in the calibration range from 0.05 to 50.0 µg/mL. Precisions (intra- and inter-run) were in the range from 0.5% to 5.1%, and accuracies (RE) were between -3.0% and 4.4% for all the concentration levels. SHR9146 was stable in all the tested bio-samples with recoveries >90%. Pharmacokinetic parameters were obtained by non-compartmental analysis. SHR9146 has a half-life of 0.713 h when IV-injected, with CL 12 mL/min/kg and Vd 0.666 L/kg. After oral dosing from 20 to 80 mg/kg, Cmax (range from 8.751 to 12.893 µg/mL) and AUC0-t (range from 15.606 to 69.971 µg·h/mL) of SHR9146 showed dose proportionality. Other post-oral pharmacokinetic parameters in plasma were as follows (n=6): Tmax 0.79 ± 0.36 h, t1/2 1.586 ± 0.853 h, CL 19.8 ± 0.9 mL/min/kg, Vd 3.427± 1.617 L/kg, and absolute bioavailability of 54.2% ± 12.6% (range from 40.2% to 64.7%). In addition, SHR9146 was found to be absorbed rapidly and distributed widely and mainly in the stomach, adrenal gland, liver, and lung. Conclusion: The method was simple, sensitive, accurate, and specific and was successfully applied for the preclinical pharmacokinetic and tissue distribution study of SHR9146 in mice. The results showed that SHR9146 had dose-independent kinetics in mice via oral administration and was absorbed rapidly and distributed widely. The study provides a good basis for further drug development assessment.

Increases prognostic value of clinical-pathological nomogram in patients with esophageal squamous cell carcinoma.

Background: This study was intended to construct a brand new prognostic nomogram after combine clinical and pathological characteristics to increases prognostic value in patients with esophageal squamous cell carcinoma. Methods: A total of 1,634 patients were included. Subsequently, the tumor tissues of all patients were prepared into tissue microarrays. AIPATHWELL software was employed to explore tissue microarrays and calculate the tumor-stroma ratio. X-tile was adopted to find the optimal cut-off value. Univariate and multivariate Cox analyses were used to screen out remarkable characteristics for constructing the nomogram in the total populations. A novel prognostic nomogram with clinical and pathological characteristics was constructed on the basis of the training cohort (n=1,144). What's more performance was validated in the validation cohort (n=490). Clinical-pathological nomogram were assessed by concordance index, time-dependent receiver operating characteristic, calibration curve and decision curve analysis. Results: The patients can divide into two groups with cut-off value of 69.78 for the tumor-stroma ratio. It is noteworthy that the survival difference was noticeable (P<0.001). A clinical-pathological nomogram was constructed by combining clinical and pathological characteristics to predict the overall survival. In comparison with TNM stage, the concordance index and time-dependent receiver operating characteristic of the clinical-pathological nomogram showed better predictive value (P<0.001). High quality of calibration plots in overall survival was noticed. As demonstrated by the decision curve analysis, the nomogram has better value than the TNM stage. Conclusions: As evidently revealed by the research findings, tumor-stroma ratio is an independent prognostic factor in patients with esophageal squamous cell carcinoma. The clinical-pathological nomogram has an incremental value compared TNM stage in predicting overall survival.

Survival influence of gender on 42,345 patients with gastric cardia adenocarcinoma.

PURPOSE: Some studies indicated that gender is associated with prognostic of cancer, However, currently the prognostic value of gender for gastric cardia adenocarcinoma (GCA) survival is unclear. The aim of our study is to reveal the influence of gender on the prognosis of patients with GCA. PATIENTS AND METHODS: A total of 42,345 cases Chinese GCA patients were enrolled from our previously established GCA and esophageal cancer databases. The clinicopathological characteristics were retrieved from medical records in hospital. The follow-up was performed through letter, telephone or home interview. Among GCA patients, there were 32,544 (76.9%) male patients with the median age 62 years (range 17-97) and 9,801 (23.1%) female patients with the median age 61 years (range 17-95 years). The Chi-square test and Kaplan-Meier method were used to compare the continuous variables and survival. Cox proportional hazards model was used for competing risk analyses, hazard ratios (HRs) and 95% confidence intervals (CIs) were evaluated. RESULTS: Men had shorter GCA-specific survival than women by multivariate analysis (HR 1.114; 95% CI 1.061 to 1.169; P < 0.001). Whether premenopausal, perimenopausal or postmenopausal, the survival of women was better than that of men (premenopausal vs. male, P < 0.001; perimenopausal vs. male, P < 0.001; postmenopausal vs. male, P = 0.035). It was worth noting that in patients with stages I, II, III, and IV, female patients survive longer than male patients (P = 0.049; P = 0.011; P < 0.001; P = 0.044, respectively). CONCLUSION: Gender is an independent prognostic factor for patients with GCA. In comparison with men, women have a significantly better outcome. Smoking and drinking may be protective factors for male GCA patients.

A Redox-Controlled Substrate Engineering Strategy for Site-Specific Enzymatic Fucosylation.

Fucosylation is one of the most common modifications of oligo-N-acetyllactosamine (oligo-LacNAc) glycans. However, none of known fucosyltransferases (FucTs) could install the α1,3-linked fucose to the oligo-LacNAc substrates in a site-specific manner. Here, we report a facile and general redox-controlled substrate engineering strategy for the site-specific α1,3-fucosylation of complex glycans containing multiple LacNAc units. This strategy takes advantage of an operationally simple oxidation enzyme module by using galactose oxidase (GOase) to convert the LacNAc unit into oxidized C6'-aldehyde LacNAc sequence, which is not a good substrate for recombinant α1,3-FucT from Helicobacter pylori strain 26695 (Hpα1,3FucT), enabling the site-specific α1,3-fucosylation at intact LacNAc sites. The general applicability and robustness of this strategy were demonstrated by the synthesis of a variety of structurally well-defined fucosides of linear and branched O- and N-linked glycans.

Chemical profiling, quantitation, and bioactivities of Du-Zhong tea.

Du-Zhong tea is a health beverage produced from Eucommia ulmoides leaves (EUL) as raw materials using traditional tea processing and Chinese herbal pieces processing methods. To evaluate the differences between tender leaves and mature leaves of Du-Zhong tea, UPLC-Q-TOF MS was used to analyze the constituents in EUL collected in April and August. A total of 52 compounds, including iridoids, phenylpropanoids, flavonoids, lignans, and other types of compounds were identified. The contents of nine ingredients in the tender and mature leaves of E. ulmoides were determined by HPLC-DAD analysis. The results show that the average contents of the compounds in tender leaves were significantly higher than those in mature leaves. Lastly, the antioxidant and antipancreatic lipase activities of commercial Du-Zhong tea made by leaves of different maturity were evaluated. Du-Zhong tea with d-grade exhibited relatively higher antioxidant, while C-grade exhibited greater lipase inhibitory activities.

Transthoracic, thoracoabdominal, and transabdominal surgical approaches for gastric cardia adenocarcinomas: a survival evaluation based on a cohort of 7103 patients.

BACKGROUND: This study compared the survival outcomes of different surgical approaches to determine the optimal approach for gastric cardia adenocarcinoma (GCA) and aimed to standardize the surgical treatment guidelines for GCA. METHODS: A total of 7103 patients with GCA were enrolled from our previously established gastric cardia and esophageal carcinoma databases. In our database, when the epicenter of the tumor was at or within 2 cm distally from the esophagogastric junction, the adenocarcinoma was considered to originate from the cardia and was considered a Siewert type 2 cancer. The main criteria for the enrolled patients included treatment with radical surgery, no radio- or chemotherapy before the operation, and detailed clinicopathological information. Follow-up was mainly performed by telephone or through home interviews. According to the medical records, the surgical approaches included transthoracic, thoracoabdominal, and transabdominal approaches. Kaplan-Meier and Cox proportional hazards regression models were applied to correlate the surgical approach with survival in patients with GCA. RESULTS: There were marked differences in age and tumor stage among the patients who underwent the three surgical approaches (P < 0.001). Univariate analysis showed that survival was related to sex, age, tumor stage, and N stage (P < 0.001 for all). Cox regression model analysis revealed that thoracoabdominal approach (P < 0.001) and transabdominal approach (P < 0.001) were significant risk factors for poor survival. GCA patients treated with the transthoracic approach had the best survival (5-year survival rate of 53.7%), and survival varied among the different surgical approaches for different tumor stages. CONCLUSION: Thoracoabdominal approach and transabdominal approach were shown to be poor prognostic factors. Patients with (locally advanced) GCA may benefit from the transthoracic approach. Further prospective randomized clinical trials are necessary.

Serum Metabolomic Profiling Reveals Biomarkers for Early Detection and Prognosis of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common aggressive malignancies worldwide, particularly in northern China. The absence of specific early symptoms and biomarkers leads to late-stage diagnosis, while early diagnosis and risk stratification are crucial for improving overall prognosis. We performed UPLC-MS/MS on 450 ESCC patients and 588 controls consisting of a discovery group and two validation groups to identify biomarkers for early detection and prognosis. Bioinformatics and clinical statistical methods were used for profiling metabolites and evaluating potential biomarkers. A total of 105 differential metabolites were identified as reliable biomarker candidates for ESCC with the same tendency in three cohorts, mainly including amino acids and fatty acyls. A predictive model of 15 metabolites [all-trans-13,14-dihydroretinol, (±)-myristylcarnitine, (2S,3S)-3-methylphenylalanine, 3-(pyrazol-1-yl)-L-alanine, carnitine C10:1, carnitine C10:1 isomer1, carnitine C14-OH, carnitine C16:2-OH, carnitine C9:1, formononetin, hyodeoxycholic acid, indole-3-carboxylic acid, PysoPE 20:3, PysoPE 20:3(2n isomer1), and resolvin E1] was developed by logistic regression after LASSO and random forest analysis. This model held high predictive accuracies on distinguishing ESCC from controls in the discovery and validation groups (accuracies > 89%). In addition, the levels of four downregulated metabolites [hyodeoxycholic acid, (2S,3S)-3-methylphenylalanine, carnitine C9:1, and indole-3-carboxylic acid] were significantly higher in early cancer than advanced cancer. Furthermore, three independent prognostic markers were identified by multivariate Cox regression analyses with and without clinical indicators: a high level of MG(20:4)isomer and low levels of 9,12-octadecadienoic acid and L-isoleucine correlated with an unfavorable prognosis; the risk score based on these three metabolites was able to stratify patients into low or high risk. Moreover, pathway analysis indicated that retinol metabolism and linoleic acid metabolism were prominent perturbed pathways in ESCC. In conclusion, metabolic profiling revealed that perturbed amino acids and lipid metabolism were crucial metabolic signatures of ESCC. Both panels of diagnostic and prognostic markers showed excellent predictive performances. Targeting retinol and linoleic acid metabolism pathways may be new promising mechanism-based therapeutic approaches. Thus, this study would provide novel insights for the early detection and risk stratification for the clinical management of ESCC and potentially improve the outcomes of ESCC.

A simple and fast LC-MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma.

A simple and fast liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C18 column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate-formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d5 and TNF-d7 were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25-500 ng/mlfor TAF and 0.300-15.0 ng/ml for TNF with acceptable accuracy and precision.

Study on enhanced extraction and seasonal variation of secondary metabolites in Eucommia ulmoides leaves using deep eutectic solvents.

It has been considered as a crucial field for the extraction of active ingredients from herbal medicine to use a green and efficient method in the medicinal and food industries. In recent years, deep eutectic solvents (DESs) have been obtaining increase attention in green chemistry area since its sustainability, safety and biodegradability. In this study, an efficient DES composed of choline chloride and L-(+)-ascorbic acid with a molar ratio of 2:1 performed higher efficacy on the extraction of target compounds (including iridoids, phenolic acids and flavonoids) in Eucommia ulmoides leaves than 50% methanol solution. Considering the extraction efficacy and time consumption, microwave-assisted extraction (MAE) was selected and the operational conditions, including power of microwave, liquid/solid ratio and irradiation time were optimized by response surface methodology (RSM). Water was used as anti-solvent to recover ten target analytes from DES with recovery yields of 97.59%, 94.91%, 96.09%, 90.66%, 95.16%, 87.33%, 86.57%, 82.15%, 89.28% and 80.75% for eucommiol (EU), aucubin (AU), geniposidic acid (GA), chlorogenic acid (CA), asperuloside (AP), rutin (RU), kaempferol-3-O-sambubioside (KS), isoquercitrin (IQ), kaempferol-3-O-rutinoside (KR) and astragaline (AS), respectively. By combining the DES-based MAE and quantitative analysis of multi-components by single mark (QAMS) methods, the contents of ten compounds in the leaves of Eucommia ulmoides were determined to clarify the relationship between the accumulation of secondary metabolites and the harvest period. It was found that the contents of main ingredients reached the highest during May to October. The period appears to be the best harvest period for Eucommia ulmoides leaves. This study provides a novel strategy for the harvesting, processing, and quality control of the raw materials from Eucommia ulmoides leaves.

Long-term effect of hospital volume on the postoperative prognosis of 158,618 patients with esophageal squamous cell carcinoma in China.

Background: The impact of hospital volume on the long-term survival of esophageal squamous cell carcinoma (ESCC) has not been well assessed in China, especially for stage I-III stage ESCC. We performed a large sample size study to assess the relationships between hospital volume and the effectiveness of ESCC treatment and the hospital volume value at the lowest risk of all-cause mortality after esophagectomy in China. Aim: To investigate the prognostic value of hospital volume for assessing postoperative long-term survival of ESCC patients in China. Methods: The date of 158,618 patients with ESCC were collected from a database (1973-2020) established by the State Key Laboratory for Esophageal Cancer Prevention and Treatment, the database includes 500,000 patients with detailed clinical information of pathological diagnosis and staging, treatment approaches and survival follow-up for esophageal and gastric cardia cancers. Intergroup comparisons of patient and treatment characteristics were conducted with the X2 test and analysis of variance. The Kaplan-Meier method with the log-rank test was used to draw the survival curves for the variables tested. A Multivariate Cox proportional hazards regression model was used to analyze the independent prognostic factors for overall survival. The relationship between hospital volume and all-cause mortality was assessed using restricted cubic splines from Cox proportional hazards models. The primary outcome was all-cause mortality. Results: In both 1973-1996 and 1997-2020, patients with stage I-III stage ESCC who underwent surgery in high volume hospitals had better survival than those who underwent surgery in low volume hospitals (both P<0.05). And high volume hospital was an independent factor for better prognosis in ESCC patients. The relationship between hospital volume and the risk of all-cause mortality was half-U-shaped, but overall, hospital volume was a protective factor for esophageal cancer patients after surgery (HR<1). The concentration of hospital volume associated with the lowest risk of all-cause mortality was 1027 cases/year in the overall enrolled patients. Conclusion: Hospital volume can be used as an indicator to predict the postoperative survival of ESCC patients. Our results suggest that the centralized management of esophageal cancer surgery is meaningful to improve the survival of ESCC patients in China, but the hospital volume should preferably not be higher than 1027 cases/year. Core tip: Hospital volume is considered to be a prognostic factor for many complex diseases. However, the impact of hospital volume on long-term survival after esophagectomy has not been well evaluated in China. Based on a large sample size of 158,618 ESCC patients in China spanning 47 years (1973-2020), We found that hospital volume can be used as a predictor of postoperative survival in patients with ESCC, and identified hospital volume thresholds with the lowest risk of death from all causes. This may provide an important basis for patients to choose hospitals and have a significant impact on the centralized management of hospital surgery.

The prognostic value of keratin pearls in patients with esophageal squamous cell carcinoma.

Keratin pearls (KP) is an important indicator of the degree of tumor cell differentiation of esophageal squamous cell carcinomas (ESCC). However, the independent prognostic value of KP in ESCC patients remains unclear. The hematoxylin-eosin (H&E) stained tissue microarrays (TMAs) or whole slides of the patients were prepared to identify the existence of KP. Kaplan-Meier (KM) survival analysis as well as univariate and multivariate Cox regression analyses were used to evaluate the prognostic value of KP. A nomogram based on KP and other clinicopathologic characteristics was constructed. The C-index, calibration curve, Receiver Operating Characteristic (ROC) curve, and Decision Curve Analysis (DCA) were used to evaluate the nomogram. The results indicated KP is a protective factor against lymph node metastasis and is closely associated with the differentiation degree in ESCC patients. KM survival analysis showed that the overall survival (OS) of patients with KP was significantly better than for patients without KP. In addition, multivariate Cox regression analysis revealed that KP was an independent predictor of OS. Furthermore, ROC curve demonstrated that KP combined with differentiation degree could more accurately predict the 5-year survival rate than differentiation degree alone. Importantly, the nomogram showed good discrimination and calibration abilities in both training and validation groups, which could more accurately predict the 3-, 5-, and 10-year survival rates of ESCC patients and adds to the predictive value of TNM stage alone. In conclusion, KP is an independent predictor of prognosis in patients with ESCC and provides incremental prognostic value to degree of differentiation.

Focal amplifications are associated with chromothripsis events and diverse prognoses in gastric cardia adenocarcinoma.

The role of focal amplifications and extrachromosomal DNA (ecDNA) is unknown in gastric cardia adenocarcinoma (GCA). Here, we identify frequent focal amplifications and ecDNAs in Chinese GCA patient samples, and find focal amplifications in the GCA cohort are associated with the chromothripsis process and may be induced by accumulated DNA damage due to local dietary habits. We observe diverse correlations between the presence of oncogene focal amplifications and prognosis, where ERBB2 focal amplifications positively correlate with prognosis and EGFR focal amplifications negatively correlate with prognosis. Large-scale ERBB2 immunohistochemistry results from 1668 GCA patients show survival probability of ERBB2 positive patients is lower than that of ERBB2 negative patients when their surviving time is under 2 years, however, the tendency is opposite when their surviving time is longer than 2 years. Our observations indicate that the ERBB2 focal amplifications may represent a good prognostic marker in GCA patients.

Enantioselective determination of ketamine in dog plasma by chiral liquid chromatography-tandem mass spectrometry.

Ketamine is an N-methyl-d-aspartate receptor antagonist that is usually used clinically as a racemic mixture. Its two enantiomers exhibit different pharmacological activities. To determine whether the enantiomers have different pharmacokinetic profiles, a chiral liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of ketamine enantiomers in dog plasma. The enantiomers of ketamine were extracted from 50 µL of plasma by methyl tert-butyl ether. Adequate chromatographic retention and baseline resolution of the enantiomers were achieved within a runtime of 5 min on a chiral column coated with polysaccharide derivatives, using a gradient mobile phase of acetonitrile and 10 mm ammonium bicarbonate aqueous solution. Ketamine enantiomers were detected by mass spectrometry with multiple reaction monitoring mode using the transitions of m/z 238.3 → 125.9 for the analytes and m/z 237.1 → 194.1 for carbamazepine (internal standard). The method was linear over the concentration range from 0.5 to 500 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was 0.5 ng/mL. The intra- and inter-day precision was <7.3% and 8.5% for R- and S-ketamine, respectively. The accuracy was 92.9-110.4% for R-ketamine and 99.8-102.4% for S-ketamine. The method was successfully applied to characterize the stereoselective pharmacokinetic profiles of ketamine in beagle dogs.

Actinorectispora metalli sp. nov., a novel actinomycete isolated from a mine and emended description of the genus Actinorectispora.

A novel actinomycete, designated strain KC 198T, was isolated from rare earth mine. The results of analysis of the 16S rRNA gene sequence indicated that KC 198T was most closely related to Actinorectisporaindica YIM 75728T (98.4 %). Aerial hyphae differentiated into long, straight chains of cylindrical spores. Growth was observed at 10-45 °C (optimum 28 °C), with 0-10 % (w/v) NaCl (optimum, in the absence of NaCl) and at pH 6.0-8.0 (optimum pH 7.0). KC 198T possessed MK-9(H4) as the predominant respiratory quinone and a minor amount of MK-10(H4). Polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Three unidentified lipids were also detected. The main cellular fatty acids were iso-C16 : 0 (30.9 %), iso-C16 : 1H (22.9 %) and iso-C15 : 0 (14.8 %). The genomic DNA G+C content was 66.8 mol%. On the basis of the phenotypic and genotypic characteristics, we propose that strain KC 198T represents a novel species of the genus Actinorectispora. The name Actinorectispora metalli sp. nov. is, therefore, proposed for the novel species with the type strain KC 198T (=CCTCC AA 2015043T=KCTC 39718T). The description of the genus Actinorectispora has also been emended.

Increased Plasma Exposures of Conjugated Metabolites of Morinidazole in Renal Failure Patients: A Critical Role of Uremic Toxins.

Morinidazole is a 5-nitroimidazole drug. Its sulfate conjugate M7 was a sensitive substrate of organic anion transporter 1 (OAT1) and OAT3, whereas N+-glucuronides M8-1 and M8-2 were only OAT3 substrates. In chronic renal failure (CRF) patients, plasma exposures of the three conjugates increased by 15-fold, which were also found in 5/6 nephrectomized (5/6 Nx) rats in this study. Although the transcriptions of Oat1 and Oat3 in 5/6 Nx rat kidneys decreased by 50%, no difference was observed on the three conjugate uptakes between control and 5/6 Nx rat kidney slices. Thus, the highly elevated endogenous uremic toxins in 5/6 Nx rats and humans, namely, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid (HA), and indoxyl sulfate (IS), were considered as influential factors. In rat kidney slices, the uptake of M7, M8-1, and M8-2 was dose dependently reduced by HA and IS, whose plasma concentrations were elevated 5 times in 5/6 Nx rats. In OAT3-overexpressed cells, the three conjugate uptakes were inhibited by CMPF, HA, and IS with IC50 values of 19.2, 87.4, and 222 µM (M7); 8.53, 39.4, and 161 µM (M8-1); and 6.75, 24.1, and 78.3 µM (M8-2), respectively. In OAT1-overexpressed cells, CMPF, HA, and IS showed weak inhibition on M7 uptake with IC50 values of 187, 162, and 200 µM, correspondingly. Results suggest that the reduced mRNA expression of renal transporters in CRF patients may not influence the activities of these transporters. However, accumulated uremic toxins may inhibit the transporters, particularly OAT3, leading to plasma exposure changes of relevant substrates.

Kibdelosporangium metalli sp. nov., isolated from a rare earth mine.

A Gram-stain-positive, oxidase-negative, catalase-positive isolate forming sporangium-like globular bodies, isolated from the rare earth mine of Bayan Obo in China and designated strain KC 266T, was subjected to a comprehensive taxonomic study. Comparative 16S rRNA gene sequence analysis revealed that strain KC 266T represented a novel lineage within the genus Kibdelosporangium and showed highest 16S rRNA gene similarities to Kibdelosporangiumphilippinense (98.5 %), Kibdelosporangiumaridum subsp. largum (98.2 %), Kibdelosporangiumaridum subsp. aridum (98.2 %) and Kibdelosporangiumphytohabitans (98.0 %). The DNA-DNA relatedness between strain KC 266T and the four species of the genus Kibdelosporangium was less than 60 %. The DNA G+C content of strain KC 266T was 67.9 mol%. The quinone system consisted of major amounts of MK-9(H4) and MK-9(H2), minor amounts of MK-8(H2) and traces of MK-10(H4). The diamino acid of the peptidoglycan was meso-diaminopimelic acid. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol, two unknown phospholipids and one unidentified aminophospholipid. The major cellular fatty acids were iso-C16 : 0, C17 : 1 ω6c, iso-C15 : 0 and iso-C14 : 0. Physiological traits as well as unique traits of the polar lipid profile and the fatty acid pattern distinguished strain KC 266T from the most closely related species. All these results indicate that strain KC 266T represents a novel species of the genus Kibdelosporangium, for which the name Kibdelosporangium metalli sp. nov. is proposed. The type strain is KC 266T (=KCTC 39719T=CCTCC AA 2016002T).

Isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP and 10-hydroxy-NBP, across the rat blood-brain barrier.

AIM: To investigate the mechanisms underlying the isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP (3-OH-NBP) and 10-hydroxy-NBP (10-OH-NBP), across the blood brain barrier (BBB). METHODS: After oral administration of NBP (20 mg/kg) to rats, the pharmacokinetics of two major hydroxylated metabolites, 3-OH-NBP and 10-OH-NBP, in plasma and brains were investigated. Plasma and brain protein binding of 3-OH-NBP and 10-OH-NBP was also assessed. To evaluate the influences of major efflux transporters, rats were pretreated with the P-gp inhibitor tariquidar (10 mg/kg, iv) and BCRP inhibitor pantoprazole (40 mg/kg, iv), then received 3-OH-NBP (12 mg/kg, iv) or 10-OH-NBP (3 mg/kg, iv). The metabolic profile of NBP was investigated in rat brain homogenate. RESULTS: After NBP administration, the plasma exposure of 3-OH-NBP was 4.64 times that of 10-OH-NBP, whereas the brain exposure of 3-OH-NBP was only 11.8% of 10-OH-NBP. In the rat plasma, 60%±5.2% of 10-OH-NBP was unbound to proteins versus only 22%±2.3% of 3-OH-NBP being unbound, whereas in the rat brain, free fractions of 3-OH-NBP and 10-OH-NBP were 100%±9.7% and 49.9%±14.1%, respectively. In the rats pretreated with tariquidar and pantoprazole, the unbound partition coefficient Kp,uu of 3-OH-NBP was significantly increased, while that of 10-OH-NBP showed a slight but not statistically significant increase. Incubation of rat brain homogenate with NBP yielded 3-OH-NBP but not 10-OH-NBP. CONCLUSION: The isomer-selective distribution of 10-OH-NBP and 3-OH-NBP across the BBB of rats is mainly attributed to the differences in plasma and brain protein binding and the efflux transport of 3-OH-NBP. The abundant 10-OH-NBP is not generated in rat brains.

Fasiglifam (TAK-875) Inhibits Hepatobiliary Transporters: A Possible Factor Contributing to Fasiglifam-Induced Liver Injury.

Fasiglifam (TAK-875), a selective G-protein-coupled receptor 40 agonist, was developed for the treatment of type 2 diabetes mellitus; however, its development was terminated in phase III clinical trials because of liver safety concerns. Our preliminary study indicated that intravenous administration of 100 mg/kg of TAK-875 increased the serum total bile acid concentration by 3 to 4 times and total bilirubin levels by 1.5 to 2.6 times in rats. In the present study, we examined the inhibitory effects of TAK-875 on hepatobiliary transporters to explore the mechanisms underlying its hepatotoxicity. TAK-875 decreased the biliary excretion index and the in vitro biliary clearance of d8-taurocholic acid in sandwich-cultured rat hepatocytes, suggesting that TAK-875 impaired biliary excretion of bile acids, possibly by inhibiting bile salt export pump (Bsep). TAK-875 inhibited the efflux transporter multidrug resistance-associated protein 2 (Mrp2) in rat hepatocytes using 5 (and 6)-carboxy-2',7'-dichlorofluorescein as a substrate. Inhibition of MRP2 was further confirmed by reduced transport of vinblastine in Madin-Darby canine kidney cells overexpressing MRP2 with IC50 values of 2.41 µM. TAK-875 also inhibited the major bile acid uptake transporter Na(+)/taurocholate cotransporting polypeptide (Ntcp), which transports d8-taurocholic acid into rat hepatocytes, with an IC50 value of 10.9 µM. TAK-875 significantly inhibited atorvastatin uptake in organic anion transporter protein (OATP) 1B1 and OATP1B3 cells with IC50 values of 2.28 and 3.98 µM, respectively. These results indicate that TAK-875 inhibited the efflux transporter MRP2/Mrp2 and uptake transporters Ntcp and OATP/Oatp, which may affect bile acid and bilirubin homeostasis, resulting in hyperbilirubinemia and cholestatic hepatotoxicity.