Sensitive and effective method with 96-well plate for determination of levamlodipine in human plasma using LC-MS/MS.

we developed an effective protein precipitation method for determination of levamlodipine in human plasma using LC-MS/MS. Sample extraction was carried out by using liquid-liquid extraction in 96-well plate format. (S)-Amlodipine-d4 was used as internal standard (IS). The chromatographic separation was achieved using Philomen Chiral MX (2) column (3 µm, 2.1 × 100 mm). Mobile phase A was comprised of Acetonitrile (ACN), Mono ethanol amine (MEA) and Iso-Propyl alcohol (IPA) (1000:1:10, v/v/v), Mobile phase B was IPA-ACN (2:1, v/v). The flow rate was 0.4 mL/min. The total run time of each sample was 4.0 min with gradient elution. LC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 409.20 â†’ 238.15 for levamlodipine and 415.25 â†’ 240.20 for (S)-Amlodipine-d4 (the IS). The method was linear from 50 to 10000 pg/mL（R2＝0.9988489）,and the lower limit of quantification (LLOQ) was 50 pg/mL. This method was applied to a bioequivalence study of levamlodipine.

Safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation study in Chinese healthy subjects.

BACKGROUND AND OBJECTIVE: Neutrophil elastase has been identified as a potential therapeutic target for acute lung injury or acute respiratory distress syndrome, and Sivelestat is a selective, reversible and competitive neutrophil elastase inhibitor. This study was designed to investigate the safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat in healthy Chinese subjects. METHODS: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation clinical trial was carried out. Briefly, healthy volunteers in twelve cohorts with 8 per cohort received 1.0-20.2 mg/kg/h Sivelestat or placebo in an intravenous infusion manner for two hours, and healthy volunteers in four cohorts received two hours intravenous infusion of 2.0-5.0 mg/kg/h Sivelestat or placebo with an interval of twelve hours for seven times. The safety and tolerability were evaluated and serial blood samples were collected for pharmacokinetics and neutrophil elastase inhibitory effects analysis at the specified time-point. RESULTS: A total of 128 subjects were enrolled and all participants completed the study except one. Sivelestat exhibited satisfactory safety and tolerability up to 20.2 mg/kg/h in single-dose cohorts and 5.0 mg/kg/h in multiple-dose cohorts. Even so, more attention should be paid to the safety risks when using high doses. The Cmax and AUC of Sivelestat increased in a dose dependent manner, and Tmax was similar for different dose cohorts. In multiple-dose cohorts, the plasma concentrations reached steady state 48 h after first administration and the accumulation of Cmax and AUC was not obvious. Furthermore, the Cmin_ss of 5.0 mg/kg/h dose cohort could meet the needs of clinical treatment. For some reason, the pharmacodynamics data revealed that the inhibitory effect of Sivelestat on neutrophil elastase content in healthy subjects was inconclusive. CONCLUSION: Sivelestat was safe and well tolerated with appropriate pharmacokinetic parameters, which provided support for more diverse dosing regimen in clinical application. CLINICAL TRIAL REGISTRATION: www.chinadrugtrials.org.cn identifier is CTR20210072.

Detection of sivelestat and its metabolite in small volumes of plasma from Chinese ALI/ARDS patients with SIRS via high-throughput UPLC-MS/MS: A pharmacokinetic study.

In this study, we developed a sensitive and efficient analytical approach combining a 96-well plate-based protein precipitation strategy with ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS) in order to assess the pharmacokinetic (PK) properties of sivelestat and its metabolite XW-IMP-A in samples of plasma from ALI/ARDS patients with SIRS. The samples were separated via gradient elution with a C18 column (Phenomenex Kinetex, C18, 2.6 µm, 100 Å, 50 × 2.1 mm) using 0.1 % formic acid aqueous solution (A) and acetonitrile-methanol (1:1, V:V) (B) as a mobile phase at a 0.6 mL/min flow rate. UPLC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 435.1 → 360.0 for sivelestat, m/z 469.0 → 394.0 for sivelestat-IS, m/z 351.0 → 276.0 for XW-IMP-A, and m/z 384.9 → 310.0 for XW-IMP-A-IS. This assay was run for 2.5 min in total, and achieved lowest limit of quantitation values of 2.0 ng/mL and 0.5 ng/mL for sivelestat and XW-IMP-A, respectively, while remaining highly linear from 2-500 ng/mL for sivelestat (r2 ≥ 0.9900) and from 0.5-125 ng/mL for XW-IMP-A (r2 ≥ 0.9900). These validated data were consistent with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) acceptance criteria. In addition, this method was successfully applied to the steady-state PK evaluation of ALI/ARDS patients with SIRS.

Long Non-coding RNA MALAT1 Inhibits Neuron Apoptosis and Neuroinflammation While Stimulates Neurite Outgrowth and Its Correlation With MiR-125b Mediates PTGS2, CDK5 and FOXQ1 in Alzheimer's Disease.

BACKGROUND: This study aimed to investigate the effect of long noncoding ribonucleic acids (RNAs) metastasis-associated lung adenocarcinoma transcript 1 (lnc-MALAT1) on regulating neuron apoptosis, neurite outgrowth and inflammation, and further explore its molecule mechanism in Alzheimer's disease (AD). METHODS: Control overexpression, lnc-MALAT1 overexpression, control shRNA, and lnc-MALAT1 shRNA were transfected into NGF-stimulated PC12 cellular AD model and cellular AD model from primary cerebral cortex neurons of rat embryo, which were established by Aß1-42 insult. Rescue experiments were performed by transferring lnc-MALAT1 overexpression and lnc-MALAT1 overexpression & miR-125b overexpression plasmids. Neuron apoptosis, neurite outgrowth and inflammation were detected by Hoechst-PI/apoptosis marker expressions, and observations were made using microscope and RT-qPCR/Western blot assays. PTGS2, CDK5 and FOXQ1 expressions in rescue experiments were also determined. RESULTS: In two AD models, lnc-MALAT1 overexpression inhibited neuron apoptosis, promoted neurite outgrowth, reduced IL-6 and TNF-α levels, and increased IL-10 level compared to control overexpression, while lnc-MALAT1 knockdown promoted neuron apoptosis, repressed neurite outgrowth, elevated IL-6 and TNF-α levels, but reduced IL-10 level compared to control shRNA. Additionally, lnc- MALAT1 reversely regulated miR-125b expression, while miR-125b did not influence the lnc- MALAT1 expression. Subsequently, rescue experiments revealed that miR-125b induced neuron apoptosis, inhibited neurite outgrowth and promoted inflammation, also increased PTGS2 and CDK5 expressions but decreased FOXQ1 expression in lnc-MALAT1 overexpression treated AD models. CONCLUSION: Lnc-MALAT1 might interact with miR-125b to inhibit neuron apoptosis and inflammation while promote neurite outgrowth in AD.

Effects of cytochrome P450 3A4 and non-genetic factors on initial voriconazole serum trough concentrations in hematological patients with different cytochrome P450 2C19 genotypes.

1. Polymorphisms of cytochrome P450 2C19 (CYP2C19) is an important factor contributing to variability of voriconazole pharmacokinetics. Polymorphisms of CYP3A4, CYP3A5, CYP2C9 and non-genetic factors such as age, gender, body mass index (BMI), transaminase levels, concomitant medications might also affect voriconazole initial steady serum trough concentration (VICmin) in haematological patients, but the effects were not clear. 2. Eighteen single-nucleotide polymorphisms in CYP2C19, CYP3A4, CYP3A5, CYP2C9 were genotyped. Patients were stratified into two groups according to CYP2C19 genotype. Group 1 were patients with CYP2C19*2 or CYP2C19*3, and Group 2 were homozygous extensive metabolizers. The effects were studied in different groups. VICmin was adjusted on daily dose (VICmin/D) for overcoming effect of dose. 3. A total of 106 blood samples from 86 patients were included. In final optimal scaling regression models, polymorphisms of rs4646437 (CYP3A4), age, BMI was identified to be factors of VICmin/D in Group 1 (R2 = .255, p < .001). Only age was confirmed as a factor of VICmin/D in Group 2 (R2 = 0.144, p = .021). 4. Besides polymorphisms of CYP2C19, in individualized medication of voriconazole in haematological patients, polymorphisms of CYP3A4, and non-genetic factors as BMI, age should also be taken into account, especially for individuals with CYP2C19*2 or CYP2C19*3.