[Hypoglycemic mechanism of Psoraleae Fructus and its main chemical constituents].

With the rise of incidence, fatality rate, and number of young cases, diabetes mellitus has been one of the seven major diseases threatening human health. Although many antidiabetic drugs(oral or for injection) are available, the majority have serious side effects during the long-term use. Thus, it is of particularly vital to develop new drugs with low risk and definite effect. Psoraleae Fructus, a traditional medicinal widely used in the folk, has hypoglycemic, anti-osteoporosis, antitumor, estrogen-like, and anti-inflammatory effects. Thus, it has great clinical application potential. Chinese medicine and the active ingredients, characterized by multiple targets, multiple pathways, and multiple effects in the treatment of diabetes mellitus, have distinct advantages in clinical application. However, the safety of Chinese medicine remains to be a challenge, and one of keys is to clarifying the mechanism of a single Chinese medicinal and its active ingredients. With the method of literature research, this study summarized and analyzed the hypoglycemic mechanisms of Psoraleae Fructus and its main active ingredients over the last decade: regulating glucose metabolism, improving insulin resistance, and directly acting on pancreatic ß-cells. The result is expected to serve as a reference for further research on the effects of Psoraleae Fructus and its main chemical constituents in lowering blood glucose and preventing diabetes mellitus and the clinical application.

Evaluation of the Effect of Food on the Pharmacokinetics of SHR6390, An Oral CDK4/6 Inhibitor, in Healthy Volunteers.

BACKGROUND AND INTRODUCTION: SHR6390 is a new developed highly effective and selective small-molecule oral CDK4/6 inhibitor. We aimed to evaluate the effect of food on the pharmacokinetics of SHR6390 tablets. METHODS: In an open-label two-way crossover study, 24 healthy Chinese volunteers were randomly divided into Group A and Group B, and 12 volunteers in each group received a single oral dose of a SHR6390 150-mg tablet under fasting and high-fat conditions. Blood samples were collected and determined for pharmacokinetic analyses. A liquid chromatography-tandem mass spectrometry method was developed and validated for determining the SHR6390 concentration. RESULTS: The time to maximum plasma concentration was not significantly affected by a high-fat diet. Compared with the fasting group, maximum plasma concentration, i.e., the area under the concentration-time curve (AUC0-t and AUC0-∞) was altered significantly, as evidenced by an increase of 56.9%, 38.6%, and 37.5% respectively. We identified seven metabolites of SHR6390 from the plasma samples, and we found no sex differences in metabolic pathways. All treatment-emergent adverse events were Grade 1 or 2. CONCLUSIONS: Food intake increased the maximum plasma concentration, AUC0-t, and AUC0-∞ significantly compared with the fasting condition. Meanwhile, single-dose SHR6390 for two treatment cycles is safe. SHR6390 was administered in a fasting status in the pivotal phase III study (NCT03927456) and chosen for the final drug label.

Pharmacokinetic and Safety Comparison of Two Capecitabine Tablets in Patients with Colorectal or Breast Cancer Under Fed Conditions: A Multicenter, Randomized, Open-Label, Three-Period, and Reference-Replicated Crossover Study.

INTRODUCTION: In this study, we assessed the pharmacokinetics (PK), bioequivalence, and safety of 150 mg capecitabine compared to the branded reference formulation in colorectal or breast cancer patients receiving a high-fat diet. METHODS: This was a multicenter, open, random, balanced, three-period, three-sequence and semi-repetitive cross study with 48 subjects. In each study period, the eligible subject received the test or reference formulation, followed by a 1-day washout period. Serial blood samples for pharmacokinetic assessment were collected at predose up to 8 h postdose. The plasma concentrations of capecitabine were analyzed by LC/MS-MS. Pharmacokinetic parameters (non-compartmental model) were assessed with WinNonlin software. The pharmacokinetic parameters assessed were the area under the plasma concentration-time curve from time 0 to the time of last measurable concentration (AUC0-t), the AUC from time zero to infinity (AUC0-∞), the peak plasma concentration of the drug (Cmax), the time needed to reach maximum concentration (Tmax), the elimination half-life (t1/2), and the terminal elimination rate (λz). All were analyzed using an analysis of variance (ANOVA) model after logarithmic transformation of the data. To establish the bioequivalence (BE) for capecitabine, reference-scaled average bioequivalence (RSABE) acceptance criteria and average bioequivalence (ABE) acceptance criteria were used. Safety and tolerability were assessed during the entire study period. RESULTS: Reference scaled maximum plasma concentration (Cmax) was higher than 0.294, permitting use of RSABE. The within-subject SDs of the reference intervention (SWR) for AUC0-t and AUC0-∞ were < 0.294, meeting ABE criteria. The point estimate for the geometric least squares mean (GLSM) ratio for the point estimate of Cmax was 0.962, within the range of 0.80-1.25. The 90% upper confidence boundary for the test/reference of GLSM ratios was 97.84-105.40% for AUC0-t and 97.33-103.51% for AUC0-∞, all of which were within the prespecified limits. The 90% confidence intervals for AUC0-t and AUC0-∞ and 95% upper confidence limit for Cmax indicated bioequivalence. No serious adverse events were found among the subjects. CONCLUSIONS: According to the criteria for bioequivalence, the test formulation was bioequivalent to the reference formulation in terms of the rate and extent of absorption under fed conditions by measurement of total capecitabine and was safe and well tolerated. TRIAL REGISTRATION: NCT04420871.

Pharmacokinetics and Bioequivalence of Cefprozil for Suspension and Granule Formulation in Healthy Chinese Volunteers: Two Single-Dose Crossover Studies.

INTRODUCTION: Cefprozil, an oral second-generation semi-synthetic cephalosporin, possesses a broad spectrum of antimicrobial activity. A granule formulation has been developed to improve medication adherence of the patients. This study was conducted to assess the bioequivalence of the granule formulation to a dry suspension in healthy Chinese volunteers and estimate the pharmacokinetic (PK) profiles of cefprozil. METHODS: An open-label, randomized, single-dose, two-period, two-group, crossover study was conducted in 60 healthy Chinese volunteers under fasted or fed conditions (30 volunteers for each condition) to assess the bioequivalence between two formulations of cefprozil. Blood samples were collected at specified time intervals, and the plasma concentrations of cis- and trans-cefprozil were determined by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. PK and bioavailability parameters were estimated via non-compartmental methods. Adverse events (AEs) were also recorded. RESULTS: Under fasted conditions, the mean Cmax was (3534.70 ± 634.67) ng/ml, Tmax was (0.98 ± 0.25) h, t1/2 was (1.37 ± 0.13) h and AUC0-t was (9302.86 ± 1618.39) ng·h/ml, respectively, after a single dose of 125 mg cefprozil for suspension. Under fed conditions, the mean Cmax was (2438.80 ± 493.78) ng/ml, Tmax was (1.66 ± 0.76) h, t1/2 was (1.36 ± 0.24) h and AUC0-t was (9332.36 ± 1373.61) ng·h/ml, respectively. The PK parameters of the granule formulation of cefprozil were similar to those of the suspension. The 90% CI values of the GMRs of Cmax, AUC0-t and AUC0-∞ under both fasted and fed conditions were within the prespecified bioequivalence range (80.00-125.00%). CONCLUSIONS: According to the criteria for bioequivalence, the test granule formulations of cefprozil and "Cefprozil for Suspension®" were determined to be bioequivalent whether under fasted or fed conditions by measurement of cis-, trans- and total cefprozil. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT04414254.

Holliday junction recognition protein promotes pancreatic cancer growth and metastasis via modulation of the MDM2/p53 signaling.

Holliday junction recognition protein (HJURP) refers to a histone H3 chaperone that has been implicated in different kinds of malignancies. Yet, its character in pancreatic cancer remains unclear. The expression of HJURP was assessed in PDAC tissues by RT-qPCR, immunoblotting, and immunohistochemistry. HJURP-deficient or overexpressed PDAC cell lines were constructed, using shRNA or plasmids with HJURP insert. MTT, sphere formation assay, migration, and invasion assays were performed to evaluate the viability, proliferation, migration, and invasion of PDAC cells. We used xenograft mice models to assess the tumor growth and metastasis in vivo. RNA-seq was applicated in search of the potential downstream target of HJURP in PDAC and subsequent verification were fulfilled via multiple assays, including immunofluorescence. Additionally, chromatin immunoprecipitation and luciferase reporter assay were conducted to explore the potential regulation of MDM2 expression by HJURP through H3K4me2. In this current research, we found that the expression of HJURP in PDAC cells and tissue was significantly higher than those of adjacent normal tissue, and high HJURP expression predicted poor survival. HJURP significantly promoted the viability, sphere formation, migration, and invasion of PDAC cells in vitro, HJURP also facilitated tumor growth and metastasis in vivo. Mechanically, MDM2/p53 axis is critical for HJURP-mediated malignant behaviors in PDAC, and HJURP regulates MDM2 expression through H3K4me2. HJURP could serve as a promising biomarker, and target for PDAC prognosis and treatment.

An UHPLC-MS/MS method for simultaneous quantification of human amyloid beta peptides Aß1-38, Aß1-40 and Aß1-42 in cerebrospinal fluid using micro-elution solid phase extraction.

Amyloid beta (Aß) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer's disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aß propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of human amyloid beta peptides Aß1-38, Aß1-40 and Aß1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aß peptides 15N51- Aß1-38, 15N53- Aß1-40 and 15N55- Aß1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15ng/mL were prepared. A "linear" regression (1/x2 weighting): y=ax+b was used to fit the calibration curves over the concentration range of 0.1-20ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aß1-38, 6.75% for Aß1-40 and 10.74% for Aß1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aß1-38, Aß1-40 and Aß1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24h at 4°C, 4h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of Aß peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human Aß1-38, Aß1-40 and Aß1-42 from CSF.

Effects of Ganoderma lucidum polysaccharides against doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) is a widely used anthracycline derivative anticancer drug, but the use of DOX in clinical applications is limited by its cardiotoxicity. In the current research, we were aiming to assess the effects of Ganoderma lucidum polysaccharides (GLPS) on DOX-induced cardiotoxicity and to illustrate the associated mechanisms. H9c2 rat cardiomyocytes were treated with DOX in the absence or presence of GLPS, and we found GLPS treatment ameliorated DOX-induced H9c2 cell death. Moreover, results of in vivo studies indicated that GLPS significantly decreased the serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and aspartate aminotransferase (AST) and attenuated DOX-induced histological changes of the heart tissues. In addition, we found DOX administration promoted myocardial apoptosis, potentiated oxidative stress, decreased the activities of antioxidant enzymes and increased the production of pro-inflammatory cytokines. However, GLPS pretreatment markedly attenuated all these untoward effects of DOX. Furthermore, GLPS pretreatment was found to inhibit Cul3-mediated K48-linked polyubiquitination of Nrf2 through suppressing Cul3 expression, thereby stabilizing Nrf2 expression in H9c2 cells after DOX treatment, leading to the decreased expression of P53 and p-P65 and increased levels of MDM2 and HO-1, resulted in the attenuated apoptosis, oxidative stress and inflammation induced by DOX.

Evaluation of the in vitro and in vivo metabolic pathway and cytochrome P450 inhibition/induction profile of Huperzine A.

Huperzine A (HupA), one of the reversible and selective acetylcholinesterase inhibitors derived from Chinese herb Huperzia Serrata, possesses affirmative action of ameliorating cognitive dysfunction of Alzheimer's disease. Up to now, the effects of HupA on human cytochrome P450s (CYPs) have not been fully elucidated. The purpose of the present study was to clarify the metabolic pathway of HupA in vitro and in vivo, and to evaluate the CYPs inhibition/induction profile of HupA in vitro. The catalytic activity of CYP enzymes (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4) was measured by the quantification of specific enzyme substrates using validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods. The in vivo metabolic pathway evaluation was performed in an open, single-dose pharmacokinetic study of HupA in fourteen elderly subjects, with urine collecting at certain intervals. In human liver microsomes, HupA (10 ng/mL) was not metabolized within 90 min, and it showed negligible inhibition against these CYP isoforms within 0.2-100 ng/mL. In human liver hepatocytes, the activities of CYP1A2 and CYP3A4 were not significantly altered when incubated at 2 or 20 ng/mL of HupA. After oral administration of 0.1 mg HupA, the total proportion of HupA excreted through urine was relatively high, accounting to 35± 9% at the limited time period of 48 h. These results suggest that HupA is substantially excreted by kidney unchanged rather than metabolized by human liver, and is unlikely to cause clinically relevant drug-drug interaction (DDI) when co-administrated with drugs that are metabolized by CYP isoenzyme system.

Chronopharmacokinetics of Erlotinib and Circadian Rhythms of Related Metabolic Enzymes in Lewis Tumor-Bearing Mice.

OBJECTIVE: The purpose of the current study was to investigate the effect of the dosing time on the pharmacokinetics of erlotinib and the circadian rhythms of the metabolism enzymes in tumor-bearing mice. METHODS: Female C57BL mice were randomly assigned to six groups. Erlotinib was orally administrated to the mice in each group at six different times of day. The plasma concentration of erlotinib was determined through a high-performance liquid-chromatographic assay, and the total mRNA was extracted from liver tissues to determine the expression of the mRNA of the related drug metabolism enzymes by qRT-PCR. RESULTS: The results indicated that AUC0-24 h and MRT0-24 h were the lowest in the 20:00 group (P < 0.01). T max of the 13 HALO (hour after light onset), 17 HALO and 21 HALO groups was higher than that of the 1 HALO and 5 HALO groups (P < 0.01). CL of the light-phase groups was lower than that of the dark-phase groups (P < 0.01). The peak value of C max appeared in the 5 HALO group (P < 0.01). The mRNA levels of Cyp3a11, Cyp3a13 and Cyp1a2 were generally higher during the afternoon and the dark phase. CONCLUSION: Circadian rhythm plays a critical role in the pharmacokinetics of erlotinib in mice, and the mechanisms may be attributed to gene expression rhythms of drug-metabolizing enzymes in liver tissues.

[Suppression of NAMPT expression enhances the sensitivity of K562 cells to imatinib and its relative mechanism].

The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase (NAMPT) expression on imatinib-sensitivity in chronic myelogenous leukemia (CML) cell line K562 and its mechanisms, NAMPT siRNA was synthesized and transfected into K562 cells. PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 µmol/L imatinib. MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib, then half inhibitory concentration (IC(50)) was calculated. Expression of NAMPT at 3rd-48th hour after exposure to 1 µmol/L imatinib was determined by Western blot. To explore the effect of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes, the microarray data from NCBI GEO Data-Sets was analyzed, then the results were confirmed by Western blot. The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors. The results showed that after exposure to 1 µmol/L imatinib for 3 - 48 h, there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA. After exposure to 1 µmol/L of imatinib for 48 h, the survival rate of NAMPT-siRNA interference group was lower than that of negative control group (P < 0.05), indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells. The IC(50) of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group (P < 0.05) after exposure to different concentrations of imatinib for 48 h, the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01 - 0.1 µmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells treated with either NAMPT-siRNA or imatinib, which was confirmed by Western blot. The inhibitory effect was much more significant when both NAMPT-siRNA and imatinib were used. The results of luciferase reporter assay showed that either NAMPT-siRNA or imatinib decreased transcriptional activity of NF-κB. The decreased effect was much more significant when both NAMPT-siRNA and imatinib were used. It is concluded that survival of K562 cells affected by imatinib may not be due to regulation of expression of NAMPT. When expression of NAMPT decreases, the K562 cells are more sensitive to imatinib, this may be related with the decreased transcriptional activity of NF-κB and its downstream effector Bcl-2.