BACKGROUND AND AIMS:Metabolic dysfunction-associated steatotic liver disease (MASLD), including its more severe form, metabolic dysfunction-associated steatohepatitis (MASH), is increasingly recognized as a critical global health challenge. This study investigates the role of hepatic GPR75 in MASH progression. APPROACH AND RESULTS:Although GPR75 is not abundantly expressed in the liver in healthy individuals, its protein levels significantly increase during MASH. Depletion of Gpr75 in either the whole liver or specifically in hepatocytes protected mice from diet-induced hepatic steatosis, while hepatocyte-specific overexpression of Gpr75 exacerbated diet-induced MASH and liver fibrosis. The deficiency of hepatic Gpr75 activated the GNAI2-cAMP-PKA signaling pathway in the livers of MASLD mice, reducing SREBP-1c maturation and de novo lipogenesis. Mechanistically, VPS35 stabilized GPR75 by recycling it to the hepatocyte membrane, thereby decreasing its degradation during MASH progression. CONCLUSIONS:This study demonstrates that GPR75 serves as a novel regulator of MASLD/MASH by modulating hepatic fatty acid metabolism. These findings suggest that GPR75 suppression may represent a potential therapeutic strategy for MASLD/MASH treatment.

Background:Mycophenolic acid (MPA), administered as mycophenolate mofetil (MMF) or enteric-coated mycophenolate sodium (EC-MPS), is the first-line immunosuppressant for kidney transplant patients. Traditional plasma-based therapeutic drug monitoring (TDM) for MPA fails to accurately reflect intracellular drug exposure at the pharmacological action site. Purpose:This study aimed to establish and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of MPA and its glucuronide metabolite, MPAG, in peripheral blood mononuclear cells (PBMCs) to support cellular pharmacokinetic (PK) assessment. Methods:Chromatographic separation was performed on a BEH C18 column using methanol-water containing 0.1% formic acid and 5 mmol/L ammonium acetate. A total of 139 PBMC samples from 40 kidney transplant recipients (n=19 receiving EC-MPS; n=21 receiving MMF) were analyzed to evaluate the plasma-PBMC PK correlations and gender-related differences. Results:The method exhibited excellent linearity over the concentration range of 0.2-500 ng/mL (R2 > 0.998), along with acceptable precision, accuracy and matrix effect. The analytes remained stable under various experimental conditions. Significant inter-individual variability in intracellular MPA and MPAG concentrations was observed among patients. Spearman correlation analysis demonstrated a weak-to-moderate positive correlation between plasma and intracellular PBMC concentrations of MPA (EC-MPS, Rs=0.396; MMF, Rs=0.480). Additionally, the concentration-time profiles of MPA and MPAG in PBMCs of kidney transplant recipients after administration of EC-MPS or MMF exhibited different characteristics. No significant gender-based differences in plasma and PBMCs were observed. Conclusion:We established and validated an LC-MS/MS method for simultaneous quantification of intracellular MPA and MPAG. The observed weak-to-moderate correlation between plasma and PBMC exposure highlights the inherent limitations of conventional plasma-based TDM for MPA. Collectively, our study provides a feasible approach for monitoring intracellular drug levels and facilitates the optimization of individualized immunosuppressive dosing regimens for renal transplant recipients.

Polycystic ovary syndrome (PCOS) is a metabolic disorder with clinical heterogeneity. PCOS women with non-hyperandrogenemia (NA) might be misdiagnosed due to a lack of diagnostic markers. This study aims to systematically analyze the differences in steroid hormones between PCOS women with hyperandrogenemia (HA) and NA, and to screen classification diagnosis models for PCOS. The serum samples from 54 HA-PCOS, 79 NA-PCOS and 60 control women (Non-PCOS) aged between 18 and 35 were measured by an integrated steroid hormone-targeted quantification assay using LC-MS/MS. The levels of serum androgens, corticosteroids, progestins and estrogens in the steroid hormone biosynthesis pathway were analyzed in PCOS and Non-PCOS women. Eight machine learning methods including Linear Discriminant Analysis (LDA), K-nearest Neighbors (KNN), Boosted Logistic Regression (LogitBoost), Naive Bayes (NB), C5.0 algorithm (C5), Random Forest (RF), Support Vector Machines (SVM), and Neural Network (NNET) were performed, evaluated and selected for classification diagnosis of PCOS. A 10-fold cross-validation on the training set was performed. The whole metabolic flux from cholesterol to downstream steroid hormones increased significantly in PCOS, especially in HA-POCS women. The RF model was chosen for the classification diagnosis of HA-PCOS, NA-PCOS, and Non-PCOS women due to the maximum average accuracy (0.938, p<0.001), AUC (0.989, p<0.001), and kappa (0.906, p<0.001), and the minimum logLoss (0.200, p<0.001). Five steroid hormones including testosterone, androstenedione, total 2-methoxyestradiol, total 4-methoxyestradiol, and free estrone were selected as the decision trees for the simplified RF model. A total of 37 women were included in the validation set. The diagnostic sensitivity for HA-PCOS, NA-PCOS, and Non-PCOS was 100 %, 93.3 % and 91.7 %, respectively. HA-PCOS, NA-PCOS, and Non-PCOS women showed obvious different steroid hormone profiles. The simplified RF model based on two androgens and three estrogens could be effectively applied to the classification diagnosis of PCOS, further reducing the missed diagnosis rate of NA-PCOS.

Purpose:Tigecycline is widely used to treat multidrug-resistant infections. However, the high incidence of coagulopathy poses a significant clinical challenge. This observational study aimed to characterize the metabolomic profiles of critically ill patients receiving tigecycline and to identify potential metabolic traits to predict tigecycline-induced coagulopathy (TIC). Patients and Methods:A total of 53 patients were enrolled and classified into TIC and non-TIC groups. Serum samples were collected at trough (Cmin), mid-dose (C1/2), and peak (Cmax) tigecycline concentrations. LC-MS-based untargeted metabolomics was applied to characterize metabolic profiles across these timepoints and to identify metabolites potentially predictive of TIC. Results:By sequentially applying univariate analysis and multivariate LASSO-penalized Cox proportional hazards regression analysis, we identified 10, 10, and 9 metabolites at the Cmin, C1/2, and Cmax timepoints, respectively, as predictive markers of TIC. Importantly, patients with lower levels of lysophosphatidylcholines (LysoPCs) and lysophosphatidylethanolamines (LysoPEs) are more susceptible to coagulopathy following tigecycline therapy. In particular, receiver operating characteristic curve analysis of LysoPC (18:0), LysoPC (18:3), LysoPE (18:0), and LysoPE (18:4) measured at Cmin demonstrated an area under the curve close to 0.8, providing strong evidence for their potential as robust biomarkers for predicting TIC. Conclusion:Our study indicated that metabolomics could be a valuable tool for predicting the risk of TIC and suggested that LysoPCs and LysoPEs might serve as hypothesis-generating candidates for future studies exploring potential therapeutic interventions.

Background: Tacrolimus (TAC), an important immunosuppressant for liver transplantation, has a narrow therapeutic index and large individual differences in pharmacokinetics. Ascitic fluid is commonly drained after liver transplantation. However, the distribution of TAC in ascitic fluid and the influence of drained ascitic fluid on whole-blood TAC are unclear. Methods: The ascitic fluid samples from twenty liver transplant recipients who were received TAC treatment within 12h after the transplantation surgery were collected for consecutive 24h in different days after the surgery. The distribution of TAC in ascitic fluid were evaluated by using a sensitive UPLC-MS/MS method. Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column (2.1×100mm, 3.5μm). Mass spectrometry was performed in multiple reaction monitoring (MRM) conditions of transitions m/z 821.4→768.5 for TAC. Results: The concentrations of TAC in ascitic fluid samples range from 0.2 to 3.0 ng/mL, accounting for 1.19-31.87% of whole-blood TAC concentrations. A linear mixed model showed a statistically significant positive correlation between the steady-state trough blood concentration of TAC (C0) and the corresponding amount of TAC excreted in the ascitic fluid for 24 consecutive hours, especially after normalization by the daily dose per unit body weight (D/W). Conclusions: These data suggested that the distribution of TAC in ascitic fluid has great individual differences. The whole-blood TAC concentration, D/W and other confounding factors may contribute to the excretion of TAC in ascitic fluid, but the influence of TAC excretion in drained ascitic fluid on the whole-blood TAC concentration is negligible.

Polygonum cuspidatum is a homology of traditional medicine and functional food widely distributed around the world. Our previous study on the hyperlipidemic animal model demonstrated that Polygonum cuspidatum was effective in ameliorating hyperlipidemia, which is characterized by lipid disorders. Herein, the regulatory effect of Polygonum cuspidatum on lipid metabolism needs to be known if its hypolipidemic mechanism is desired to clarify. In this study, an ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry-based lipidomic strategy was first applied to investigate the lipidomic patterns of high-fat diet-induced hyperlipidemic hamsters when treated with Polygonum cuspidatum. The results showed that Polygonum cuspidatum improved the lipidomic profile of hyperlipidemia. A total of 65 differential lipids related to the hypolipidemic effect of Polygonum cuspidatum were screened out and identified, and these differential lipids covered various categories, such as phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, sphingomyelins and so on. Orally administrated Polygonum cuspidatum restored these differential lipids back to normal or nearly normal levels. This study adopted lipidomics to reveal the key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia, which would provide a scientific basis for its clinical application.

Non-alcoholic steatohepatitis (NASH) is emerging as a serious liver disorder characterized by hepatic steatosis and liver inflammation. Nicotinamide adenine dinucleotide (NAD+) and NAD+-dependent deacetylase, SIRT1, play important roles in lipid metabolism in non-alcoholic fatty liver disease (NAFLD). However, their effects on liver inflammation and homeostasis of bile acids (BAs), the extensively proved pathophysiological actors in NASH, have not been fully understood. NASH animal model was induced by a methionine-choline-deficient (MCD) diet in C57BL/6J mice and intraperitoneally injected with NAD+ precursor, an agonist of upstream rate-limiting enzyme NAMPT or downstream SIRT1, or their vehicle solvents. Free fatty acid (FFA) was applied to HepG2 cells to construct the cell model. Induction of NAMPT/NAD+/SIRT1 axis could remarkably alleviate the aggravated inflammation in the liver of NASH mice, accompanied by decreased levels of total BAs throughout the enterohepatic system and a switch of BA synthesis from the classic pathway to the alternative pathway, resulting in less production of pro-inflammatory 12-OH BAs. The expressions of key enzymes including cyp7a1, cyp8b1, cyp27a1 and cyp7b1 in BA synthesis were significantly modulated after NAMPT/NAD+/SIRT1 axis induction in both animal and cell models. The levels of pro-inflammatory cytokines in liver were significantly negatively correlated with the intermediates in NAD+ metabolism, which may also be related to their regulation on BA homeostasis. Our results indicated that induction of NAMPT/NAD+/SIRT1 axis may be a potential therapeutic strategy for NASH or its complications related with BAs.

Background: Anti-folate drug pemetrexed is a vital chemotherapy medication for non-small cell lung cancer (NSCLC). Its response varies widely and often develops resistance to the treatment. Therefore, it is urgent to identify biomarkers and establish models for drug efficacy evaluation and prediction for rational drug use.Methods: A total of 360 subjects were screened and 323 subjects were recruited. Using metabolomics in combination with machine learning methods, we are trying to select potential biomarkers to diagnose NSCLC and evaluate the efficacy of pemetrexed in treating NSCLC. Furtherly, we measured the concentration of eight metabolites in the tryptophan metabolism pathway in the validation set containing 201 subjects using a targeted metabolomics method with UPLC-MS/MS.Results: In the discovery set containing 122 subjects, the metabolic profile of healthy controls (H), newly diagnosed NSCLC patients (ND), patients who responded well to pemetrexed treatment (S) and pemetrexed-resistant patients (R) differed significantly on the PLS-DA scores plot. Pathway analysis showed that glycine, serine and threonine metabolism occurred in every two group comparisons. TCA cycle, pyruvate metabolism and glycerolipid metabolism are the most significantly changed pathways between ND and H group, pyruvate metabolism was the most altered pathway between S and ND group, and tryptophan metabolism was the most changed pathway between S and R group. We found Random forest method had the maximum area under the curve (AUC) and can be easily interpreted. The AUC is 0.981 for diagnosing patients with NSCLC and 0.954 for evaluating pemetrexed efficiency.Conclusion: We compared eight mathematical models to evaluate pemetrexed efficiency for treating NSCLC. The Random forest model established with metabolic markers tryptophan, kynurenine and xanthurenic acidcan accurately diagnose NSCLC and evaluate the response of pemetrexed.

Abstract Introduction: Bruton’s tyrosine kinase (BTK), a member of the Tec family, plays a crucial role in signaling through B-cell receptor (BCR). BTK inhibition blocks BCR signals and prevents B-cell activation and growth. First-generation BTK inhibitors such as ibrutinib covalently binds to a cysteine residue (C481) of BTK. Their most frequent acquired resistance is the development of a serine mutation in the binding site (C481S). Next generation BTK inhibitors such as LOXO-305 and ARQ 531 are being developed to overcome this resistance to first-generation inhibitors. Methods: HMPL-760 was tested in biochemical assays using recombinant human wild type (WT) and C481S mutant BTKs. Its selectivity was carried out using Eurofins Cerep KinaseProfilerTM panel. Cellular activity of HMPL-760 was evaluated in HEK293 cells stably transfected with BTKWT or BTKC481S, and other tumor cell lines, which are either human diffuse large B cell lymphoma (DLBCL) or mantle cell lymphoma (MCL) cell lines. The in vivo antitumor activity and PKPD correlation of HMPL-760 was studied in HBL-1 xenograft mouse models bearing BTKWT or BTKC481S respectively. Results: In biochemical assays, HMPL-760 strongly inhibits BTK kinase activities towards wild-type BTK (BTKWT) and C481S mutant (BTKC481S), and binds to BTK in a reversible way. HMPL-760 demonstrates high selectivity in a panel containing 413 kinases. In cellular assays, HMPL-760 displays strong anti-proliferative activities in B-cell lymphoma cells (TMD-8, OCI-LY10, REC-1, HBL-1 and HBL-1-BTKC481S) harboring either BTKWT or BTKC481S (GI50: 0.0015-0.046 μM). In human whole blood assay, HMPL-760 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 expression in CD19+cells. HMPL-760 shows ≥ 10-fold inhibitory potency than ARQ 531 in both BTKWT and BTKC481S cells, and ~3-fold higher inhibitory potency than that of LOXO-305 in BTKC481S cells. In cellular assay by detecting p-BTK after compound washout, HMPL-760 maintains a longer duration of target inhibition than LOXO-305 in both BTK wild type (HBL-1) and BTK mutant (HBL-1-BTKC481S) cell lines. HMPL-760 displays dose-dependent antitumor efficacy in multiple human B cell lymphoma xenograft models in mice when orally administered at 3~50 mg/kg once daily. Complete tumor regression occurs in most of the tested models at the high dose levels. HMPL-760 shows much stronger antitumor efficacy than LOXO-305 and ARQ 531 at similar dose level, which may be associated with HMPL-760’s higher drug exposures and more sustainable inhibition on BTK phosphorylation in the tumor tissues. Conclusion: HMPL-760 is a reversible, selective, highly potent, BTK inhibitor targeting both BTKWT and BTKC481S. The first-in-human Phase 1 clinical trials of HMPL-760 are under way in patients with r/r B-NHL (NCT05190068, NCT05176691). Citation Format: Linfang Wang, Junqing Liang, Zhihu Gao, Jia Hu, Weigang He, Xianwen Yang, Fangfang Mao, Wei Zhang, Ying Yu, Qihang Zhang, Na Yang, Chun Zhang, Jian Wang, Yu Cai, Xiong Li, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-760 is a highly potent and selective reversible BTK inhibitor, targeting BTK and BTKC481S in B-cell malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4020.

Background: Mutations in isocitrate dehydrogenase (IDH) 1/2 are frequently identified in various cancers, such as AML, cholangiocarcinoma, chondrosarcoma and glioma. Mutant IDHs (mIDHs) cause accumulated 2-HG, leading to blockage of cell differentiation, thereby inducing malignant transformation. Rare cases were identified carrying co-existing mutations in IDH1 and IDH2. mIDH isoform switching, from mutant IDH1 to mutant IDH2 and vice versa, have been reported as a mechanism of acquired resistance to IDH inhibition in AML and cholangiocarcinoma. Thus, simultaneous inhibition on both mIDH1 and mIDH2 may be a promising strategy to overcome resistance and improve clinical efficacy. HMPL-306, a dual inhibitor of mIDH1/mIDH2, developed by HUTCHMED, is being evaluated in clinical trials. Methods: The inhibition of HMPL-306 on IDH enzymes, including mutant and wild type, was determined by fluorescence-based assay. The selectivity of HMPL-306 was evaluated in 322 kinases (SelectScreenTM) and 88 proteins (Cerep). For cellular activities of HMPL-306, 2-HG production and differentiation were detected in cells harboring mIDH. Human tumor xenograft models carrying IDH1 or 2 mutations were established for evaluating mIDH inhibition by detecting 2-HG in plasma and tumor, and anti-tumor efficacies. Results: HMPL-306 inhibited mutant IDH enzyme activities including IDH1R132H, IDH2R140Q and IDH2R172K, while showed weaker inhibition on IDH1/2 wild type enzymes. HMPL-306 had a superior selectivity profile in a kinase panel and a safety panel, while enasidenib, an approved mIDH2 inhibitor, inhibited Adenosine-A3 with IC50 of 12 nM. In cellular assays, HMPL-306 displayed comparable activities to enasidenib and ivosidenib (approved mIDH1 inhibitor) and suppressed 2-HG through inhibition of mIDH1 or mIDH2 at similar level, indicating an equal potency against mIDH1 and 2. Moreover, in both mIDH1/2 cells, HMPL-306 reduced the levels of histone methylation, and promoted hemoglobin γ and Kruppel1 gene expression, which led to differentiation from immature malignant cells to mature normal cells. Oral administration of HMPL-306 remarkably decreased 2-HG level in plasma and tumor tissues in xenograft models carrying mIDH1 or mIDH2 and the inhibition is more potent and durable than either ivosidenib or enasidenib at the same dose. Pharmacokinetics (PK) study in rodents showed high exposures of HMPL-306 in brain and cerebrospinal fluid, a desirable feature for glioma therapy. Combination treatment of HMPL-306 and azacitidine synergized in releasing the differentiation block in mIDH AML cells. HMPL-306 also significantly improved in vivo anti-tumor efficacy of chemotherapy drugs in solid tumor models with mIDH1/2. Conclusion: HMPL-306 is a potent, dual inhibitor of IDH1/2 mutation. The strong activity and favorable PK profiles support further clinical evaluation. Citation Format: Na Yang, Jia Hu, Tingwen Li, Juntao Yu, Dongxia Shi, Min Cheng, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characteristic of HMPL-306, a CNS-penetrable dual inhibitor of mutant IDH1 and IDH2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 543.

Silybin is widely used as a hepatoprotective agent in various liver disease therapies and has been previously identified as a CYP3A inhibitor. However, little is known about the effect of silybin on CYP3A and the regulatory mechanism during high-fat-diet (HFD)-induced liver inflammation. In our study, we found that silybin restored CYP3A expression and activity that were decreased by HFD and conditioned medium (CM) from palmitate-treated Kupffer cells. Moreover, silybin suppressed liver inflammation in HFD-fed mice and inhibited nuclear factor κ-B translocation into the nucleus through elevation of SIRT2 expression and promotion of p65 deacetylation. This effect was confirmed by overexpression of SIRT2, which suppressed p65 nuclear translocation and restored CYP3A transcription affected by CM. The hepatic NAD+ concentration markedly decreased in HFD-fed mice and CM-treated hepatocytes/HepG2 cells but increased after silybin treatment. Supplementing nicotinamide mononucleotide as an NAD+ donor inhibited p65 acetylation, decreased p65 nuclear translocation, and restored cyp3a transcription in both HepG2 cells and mouse hepatocytes. These results suggest that silybin regulates metabolic enzymes during liver inflammation by a mechanism related to the increase in NAD+ and SIRT2 levels. In addition, silybin enhanced the intracellular NAD+ concentration by decreasing poly-ADP ribosyl polymerase-1 expression. In summary, silybin increased NAD+ concentration, promoted SIRT2 expression, and lowered p65 acetylation both in vivo and in vitro, which supported the recovery of CYP3A expression. These findings indicate that the NAD+/SIRT2 pathway plays an important role in CYP3A regulation during nonalcoholic fatty liver disease. SIGNIFICANCE STATEMENT: This research revealed the differential regulation of CYP3A by silybin under physiological and fatty liver pathological conditions. In the treatment of nonalcoholic fatty liver disease, silybin restored, not inhibited, CYP3A expression and activity through the NAD+/ sirtuin 2 pathway in accordance with its anti-inflammatory effect.

Our previous study suggests that berberine (BBR) lowers lipids by modulating bile acids and activating intestinal farnesoid X receptor (FXR). However, to what extent this pathway contributes to the hypoglycemic effect of BBR has not been determined. In this study, the glucose-lowering effects of BBR and its primary metabolites, berberrubine (M1) and demethyleneberberine, in a high-fat diet–induced obese mouse model were studied, and their modulation of the global metabolic profile of mouse livers and systemic bile acids was determined. The results revealed that BBR (150 mg/kg) and M1 (50 mg/kg) decreased mouse serum glucose levels by 23.15% and 48.14%, respectively. Both BBR and M1 markedly modulated the hepatic expression of genes involved in gluconeogenesis and metabolism of amino acids, fatty acids, and purine. BBR showed a stronger modulatory effect on systemic bile acids than its metabolites. Moreover, molecular docking and gene expression analysis in vivo and in vitro suggest that BBR and M1 are FXR agonists. The mRNA levels of gluconeogenesis genes in the liver, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, were significantly decreased by BBR and M1. In summary, BBR and M1 modulate systemic bile acids and activate the intestinal FXR signaling pathway, which reduces hepatic gluconeogenesis by inhibiting the gene expression of gluconeogenesis genes, achieving a hypoglycemic effect. BBR and M1 may function as new, natural, and intestinal-specific FXR agonists with a potential clinical application to treat hyperglycemia and obesity. SIGNIFICANCE STATEMENT This investigation revealed that BBR and its metabolite, berberrubine, significantly lowered blood glucose, mainly through activating intestinal farnesoid X receptor signaling pathway, either directly by themselves or indirectly by modulating the composition of systemic bile acids, thus inhibiting the expression of gluconeogenic genes in the liver and, finally, reducing hepatic gluconeogenesis and lowering blood glucose. The results will help elucidate the mechanism of BBR and provide a reference for mechanism interpretation of other natural products with low bioavailability.

OBJECTIVE:To detect additional variants for newborn carriers of single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis in Changsha area, and explore the variation spectrum of deafness-related genes in this region.METHODS:For 462 newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene, all exons of the genes were subjected to Sanger sequencing. The pathogenicity of the variants was analyzed by database and literature search.RESULTS:For 305 newborns carrying a heterozygous GJB2 variant, 143 (46.49%) were found to carry additional variants, including 29 (9.51%) with c.109G>A likely pathogenic variant, and 1 (6.48%) with c.551G>A pathogenic variant. Among 153 newborns carrying single heterozygous variant of the SLC26A4 gene, 2 (1.31%) were found with a c.281C>T variant, and 1 (0.65%) with a c.1547_1548ins pathogenic variant. Among 4 newborns simultaneously carrying GJB2 and SLC26A4 variants, two were found to carry c.109G>A and c.844T>C variants (clinical significance unknown), respectively.CONCLUSION:For newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis, the detection rate for other variants is quite high. Sanger sequencing can significantly improve the detection rate of high-risk newborns and enrich the variant spectrum of deafness genes.

High-calorie diet, circadian rhythms and metabolic features are intimately linked. However, the mediator(s) between nutritional status, circadian rhythms and metabolism remain largely unknown. This article aims to clarify the key metabolic pathways bridging nutritional status and circadian rhythms based on a combination of metabolomics and molecular biological techniques. A mouse model of high-fat diet-induced obesity was established and serum samples were collected in obese and normal mice at different zeitgeber times. Gas chromatography/mass spectrometry, multivariate/univariate data analyses and metabolic pathway analysis were used to reveal changes in metabolism. Metabolites involved in the metabolism of purines, carbohydrates, fatty acids and amino acids were markedly perturbed in accordance with circadian related variations, among which purine catabolism showed a typical oscillation. What's more, the rhythmicity of purine catabolism dampened in the high-fat diet group. The expressions of clock genes and metabolic enzymes in the liver were measured. The mRNA expression of Xanthine oxidase (Xor) was highly correlated with the rhythmicity of Clock, Rev-erbα and Bmal1, as well as the metabolites involved in purine catabolism. These data showed that a high-fat diet altered the circadian rhythm of metabolic pathways, especially purine catabolism. It had an obvious circadian oscillation and a high-fat diet dampened its circadian rhythmicity. It was suggested that circadian rhythmicity of purine catabolism is related to circadian oscillations of expression of Xor, Uox and corresponding clock genes.

A simple, sensitive and specific hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric (HILIC–MS) method was developed and validated to determine the plasma concentrations of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously in clinical studies. Plasma samples were first acidified and then protein precipitated with acetonitrile. Chromatographic separation was achieved on a HILIC Chrom Matrix HP amide column (5 μm, 3.0 × 100 mm I.D.). The mobile phase consisted of acetonitrile and 5 mM ammonium formate buffer containing 0.1% formic acid. Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r ≥ 0.999) over the established concentration range of 1.0–1000 ng/mL for metformin and 0.1–100 ng/mL for saxagliptin and its active metabolite 5-hydroxy saxagliptin. The extraction recovery for all of the analytes was >92% and the matrix effect ranged from 91.0 to 110.0%. After validation, the method was successfully applied to a bioequivalence study with a single-pill combination (SPC) consisting of 5 mg saxagliptin and 500 mg metformin in 10 healthy Chinese subjects.

Epalrestat is an inhibitor of aldose reductase in the polyol pathway and is used for the management of diabetic neuropathy clinically. Our pilot experiments and accumulated evidences showed that epalrestat inhibited polyol pathway and reduced sorbitol production, and suggested the potential renal protection effects of epalrestat on diabetic nephropathy (DN). To evaluate the protective effect of epalrestat, the db/db mice were used and exposed to epalrestat for 8 weeks, both the physiopathological condition and function of kidney were examined. For the first time, we showed that epalrestat markedly reduced albuminuria and alleviated the podocyte foot process fusion and interstitial fibrosis of db/db mice. Metabolomics was employed, and metabolites in the plasma, renal cortex, and urine were profiled using a gas chromatography-mass spectrometry (GC/MS)-based metabolomic platform. We observed an elevation of sorbitol and fructose, and a decrease of myo-inositol in the renal cortex of db/db mice. Epalrestat reversed the renal accumulation of the polyol pathway metabolites of sorbitol and fructose, and increased myo-inositol level. Moreover, the upregulation of aldose reductase, fibronectin, collagen III, and TGF-β1 in renal cortex of db/db mice was downregulated by epalrestat. The data suggested that epalrestat has protective effects on DN, and the inhibition of aldose reductase and the modulation of polyol pathway in nephritic cells be a potentially therapeutic strategy for DN.

Berberrubine (BRB) has a strong lipid-lowering effect and can be extensively metabolized into berberrubine-9-O- β -D-glucuronide (BRBG) in vivo . Recently, pharmacokinetics studies showed that the production of BRBG was significantly decreased in the urine of mice fed with a high fat diet (HFD), indicating a decreased glucuronidation capacity. Based on the UGT isoform identification, hepatic and renal microsomal incubation, glucuronidation was examined to suggest the metabolism of BRB in liver and kidneys. The results showed that the renal UGT activity for metabolizing BRB markedly decreased, which may be highly related to the decreased expression and activity of renal Ugt1a7c. Surprisingly, in vitro studies revealed neither BRB nor BRBG inhibited the renal UGT activity. By employing an integrated strategy of metabolomics and pharmacokinetics, we identified and confirmed for the first time the inhibitory effect of some potential endogenous molecules on the renal glucuronidation of C57BL/6J mice, such as glutaric acid and linoleic acid. By employing recombinant human UGTs, we found that glutaric acid and linoleic acid efficiently affect the activity of recombinant human UGT1A7, 1A9 and 1A8 at their normal or abnormal physiological levels in vivo . Glutaric acid (2 mM) markedly inhibited the activity of UGT1A7 by 89.4% and UGT1A9 by 32.8%. The inhibition rates reached 99.3% for UGT1A9, 48.3% for UGT1A7, and 46.8% for UGT1A8 with linoleic acid at 200 μ M. It has been suggested that the endogenous molecules have the potential to affect the efficiency of glucuronidation, which might be a key factor contributing to individual differences in drug metabolism. the deviations of the key with significantly elevated The digital labels the fold change of the selected molecules

Apatinib, a highly selective small-molecule inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2), has attracted many attentions due to its anticancer activity in various malignancies containing non-small-cell lung cancer (NSCLC). Our previous preclinical study confirmed the enhanced anti-tumor efficacy of combined treatment between apatinib and docetaxel for NSCLC. However, the effects of docetaxel on pharmacokinetics and tissue distribution of apatinib are not clear. In present study, a reliable HPLC-MS/MS method was established for determination of apatinib. This method had a good linearity in the range of 1-5000 ng/mL, and the recovery and matrix effect were 100.1-103.5%, 77.6-83.5%, respectively. Plasma exposure level of apatinib and the values of C-max. AUC(0-12h). T-1/2, and MRT were not affected by multi-dose of docetaxel. The tissue distributions (kidney, heart, lung, spleen) of apatinib in combined treatment group were lower at 0.25 h but higher at 2 h, and that in intestine and liver were not significantly changed compared with control group. However, pretreatment with docetaxel had no significant effect on AUC(0-4h) of apatinib in tissues in mice. In conclusion, plasma and tissues exposure levels of apatinib were not affected by long-termed treatment with docetaxel, indicating that docetaxel is less likely to increase the side effect of apatinib such as hypertension, hand-foot syndrome and so on.

Berberrubine (BRB) has a strong lipid-lowering effect and can be extensively metabolized into berberrubine-9-O-β-d-glucuronide (BRBG) in vivo. Recently, pharmacokinetics studies showed that the production of BRBG was significantly decreased in the urine of mice fed with a high-fat diet (HFD), indicating a decreased glucuronidation capacity. Based on the UDP-glucuronosyltransferase (UGT) isoform identification, hepatic and renal microsomal incubation, glucuronidation was examined to suggest the metabolism of BRB in liver and kidneys. The results showed that the renal UGT activity for metabolizing BRB markedly decreased, which may be highly related to the decreased expression and activity of renal Ugt1a7c. Surprisingly, in vitro studies revealed neither BRB nor BRBG inhibited the renal UGT activity. By employing an integrated strategy of metabolomics and pharmacokinetics, we identified and confirmed for the first time the inhibitory effect of some potential endogenous molecules on the renal glucuronidation of C57BL/6J mice, such as glutaric acid (GA) and linoleic acid (LA). By employing recombinant human UGTs, we found that GA and LA efficiently affect the activity of recombinant human UGT1A7, 1A9, and 1A8 at their normal or abnormal physiologic levels in vivo. GA (2 mM) markedly inhibited the activity of UGT1A7 by 89.4% and UGT1A9 by 32.8%. The inhibition rates reached 99.3% for UGT1A9, 48.3% for UGT1A7, and 46.8% for UGT1A8 with LA at 200 μM. It has been suggested that the endogenous molecules have the potential to affect the efficiency of glucuronidation, which might be a key factor contributing to individual differences in drug metabolism.

Objective: Salidroside showed potential pharmacological effect on plateau hypoxia and cardiovascular disease like myocardial ischemia. However, pharmacokinetic differences have not been assessed between the pathological model and the normal animals. This study focused on evaluating the pharmacokinetic properties of salidroside in animals with myocardial ischemia. Methods: A reproducible and sensitive method was established and optimized based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine salidroside in rats plasma. The data showed the AUC(0-infinity) and C-max of salidroside proportionally increased along with dose elevation after singly intragastric administration of salidroside at a dose of 20, 50, and 100 mg/kg. Results: Compared to the single dose, the C-max, and AUC(0-8h) of salidroside markedly decreased while CL/F and V/F increased after multiple dosing. However, the C-max, and AUC(0-8h) of ischemic model rats were 0.35 and 0.39 fold lower than those in normal rats after a single dose at 50 mg/kg, with an increased CL/F and V/F. Surprisingly, after a consecutive administration of salidroside for 7 d, the mean C-max, AUC(0-8h) increased 2.89 and 2.61 fold higher than a single dose in model rats, and even 2.28 and 4.03 fold higher than the normal controls after multiple doses. All the above fold values were statistically different (P < 0.01). Conclusion: The particular PK properties of salidroside in ischemic model rats were presented in our study for the first time, suggesting that myocardial ischemia greatly affected pharmacokinetics exposure of the orally administrated salidroside after a single or multiple doses. (C) 2018 Tianjin Press of Chinese Herbal Medicines. Published by Elsevier B.V. All rights reserved.