Safety, tolerability, pharmacokinetics, and pharmacodynamics of a soluble guanylate cyclase stimulator, HEC95468, in healthy volunteers: a randomized, double-blinded, placebo-controlled phase 1 trial.

Heart failure is the most costly cardiovascular disorder. New treatments are urgently needed. This study aims to evaluate the safety, pharmacokinetics, and pharmacodynamic profile of HEC95468, a soluble guanylate cyclase (sGC) stimulator, in healthy volunteers. Sixty-two, eighteen, and forty-eight participants were enrolled in the single ascending dose (SAD) study, the food effect (FE) study, and the multiple ascending dose (MAD) study, respectively. The study conforms to good clinical practice and the Declaration of Helsinki. Overall, HEC95468 was safe and tolerable; a higher proportion of HEC95468-treated participants reported mild headaches, dizziness, decreased blood pressure, increased heart rate, and gastrointestinal-related treatment-emergent adverse events (TEAEs), similar to the sGC stimulators riociguat and vericiguat. In terms of pharmacokinetic parameters, the maximum observed plasma concentration (Cmax) and the area under the concentration-time curve (AUC0-t) were dose-proportional over the dose range. Moderate accumulation was observed after multiple administrations of HEC95468. Systolic blood pressure (SBP) and diastolic blood pressure decreased, while 3',5'-cyclic guanosine monophosphate (cGMP) concentration in plasma increased and heart rate was induced. Vasoactive hormones (renin, angiotensin II, and norepinephrine) in plasma were compensatorily elevated after oral administration. These data supported further clinical trials of HEC95468 in the treatment of heart failure and pulmonary arterial hypertension. Systematic Review Registration: http://www.chinadrugtrials.org.cn, identifier CTR20210064.

Safety, pharmacokinetics, and pharmacodynamics in healthy Chinese volunteers treated with SC0062, a highly selective endothelin-A receptor antagonist.

This study evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and food effects (FE) of SC0062, a highly active endothelin-A (ETA ) receptor antagonist, in healthy subjects. The primary objectives of this first-in-human phase I study, comprised of single-ascending-dose, multiple-ascending-dose, and FE parts, were to characterize the safety and tolerability of SC0062, and FE. The secondary objectives were to determine the PK behavior of SC0062 and its major active metabolite M18, whereas exploratory objectives focused on PD effects, principally effects on endothelin-1 (ET-1) and total bile acids (TBA). Single doses of 10 to 100 mg and multiple daily doses of 20 and 50 mg for 6 days were well tolerated. SC0062 was rapidly absorbed and plasma exposure of SC0062 and M18 increased disproportionately with dose, achieving steady state by day 3, with accumulation ratios of 1.22 and 1.89 on day 6 for SC0062 and M18, respectively. The geometric mean (geometric standard deviation) terminal elimination half-life (t1/2 ) values of SC0062 and M18 were 7.25 (1.70) h and 13.73 (1.32) h, respectively. Plasma ET-1 concentrations were dose-proportional, whereas plasma TBA concentrations behaved erratically. Following a single 50 mg dose of SC0062 after a high-fat meal, Cmax values for SC0062 and M18 increased by 41% and 32%, respectively, and median Tmax values for SC0062 were 3 h longer than fasting values; exposure was unaffected. These favorable safety, PK, and PD results provide a foundation for further studies of SC0062 in pulmonary arterial hypertension, chronic kidney disease, and other relevant indications.

Safety, immunogenicity, and efficacy of a modified COVID-19 mRNA vaccine, SW-BIC-213, in healthy people aged 18 years and above: a phase 3 double-blinded, randomized, parallel controlled clinical trial in Lao PDR (Laos).

Background: The mRNA vaccine has demonstrated significant effectiveness in protecting against SARS-CoV-2 during the pandemic, including against severe forms of the disease caused by emerging variants. In this study, we examined safety, immunogenicity, and relative efficacy of a heterologous booster of the lipopolyplex (LPP)-based mRNA vaccine (SW-BIC-213) versus a homologous booster of an inactivated vaccine (BBIBP) in Laos. Methods: In this phase 3 clinical trial, which was randomized, parallel controlled and double-blinded, healthy adults aged 18 years and above were recruited from the Southern Savannakhet Provincial Hospital and Champhone District Hospital. The primary outcomes were safety and immunogenicity, with efficacy as an exploratory endpoint. Participants who were fully immunized with a two-dose inactivated vaccine for more than 6 months were assigned equally to either the SW-BIC-213 group (25 µg) or BBIBP group. The primary safety endpoint was to describe the safety profile of all participants in each group up to 6 months post-booster immunization. The primary immunogenic outcome was to demonstrate the superiority of the neutralizing antibody response, in terms of geometric mean titers (GMTs) of SW-BIC-213, compared with BBIBP 28 days after the booster dose. The exploratory efficacy endpoint aimed to assess the relative efficacy of SW-BIC-213 compared to BBIBP against virologically confirmed symptomatic COVID-19 over a 6-month period. The trial was registered with ClinicalTrials.gov (NCT05580159). Findings: Between October 10, 2022, and January 13, 2023, 1200 participants were assigned to SW-BIC-213 group and 1203 participants in the BBIBP group. All adverse reactions observed during the study were tolerable, transient, and resolved spontaneously. Solicited local reactions were the main adverse reactions in both the SW-BIC-213 group (43.8%) and BBIBP group (14.8%) (p < 0.001). Heterologous boosting with SW-BIC-213 induced higher live virus neutralizing antibodies to SARS-CoV-2 wildtype and BA.5 strains with GMTs reaching 750.1 and 192.9 than homologous boosting with BBIBP with GMTs of 131.5 (p < 0.001) and 47.5 (p < 0.001) on day 29. The statistical findings revealed that, following a period of 14-day to 6-month after booster vaccination, the SW-BIC-213 group exhibited a relative vaccine efficacy (VE) of 70.1% (95% CI: 34.2-86.4) against symptomatic COVID-19 when compared to the BBIBP group. Interpretation: A heterologous booster with the COVID-19 mRNA vaccine SW-BIC-213 manifests a favorable safety profile and proves highly immunogenic and efficacious in preventing symptomatic COVID-19 in individuals who have previously received two doses of inactivated vaccine. Funding: Shanghai Strategic Emerging Industries Development Special Fund, Biomedical Technology Support Special Project of Shanghai "Science and Technology Innovation Action Plan", Shanghai Municipal Science and Technology Commission.

A phase I study comparing the pharmacokinetics and safety of HS628 (tocilizumab biosimilar) and reference tocilizumab in healthy male subjects.

This study aimed to evaluate the pharmacokinetic (PK) similarity of the proposed biosimilar HS628 compared with the reference tocilizumab (Actemra®) and also to demonstrate similar safety and immunogenicity profiles in healthy Chinese male subjects. Eighty eligible subjects were randomized into two treatment groups in a 1:1 ratio to receive a single intravenous infusion of HS628 or tocilizumab at 4 mg/kg over 60 min. Blood samples were collected at the scheduled time points for PK and immunogenicity analysis. PK biosimilarity was determined using the standard bioequivalence criteria 80%-125%. A total of 77 subjects received the study drug and completed the study. The main PK parameters were similar for the test and reference groups. The ratio of geometric least-squares means (GMR) and its 90% CIs for AUC0-t , AUC0-∞ , and Cmax between the test group and reference group were 1.06 (1.00-1.12), 1.07 (1.00-1.14), and 1.04 (0.99-1.10), respectively, which were fully within the predefined bioequivalent range of 80%-125%. The incidence of treatment-emergent adverse events (TEAEs) was similar for HS628 and tocilizumab (p > 0.05). The most common TEAEs were decreased fibrinogen, decreased neutrophils, pharyngalgia, oral ulcer, decreased leukocytes, and increased erythrocyte sedimentation rate. The results of the present study provide strong evidence to support the PK similarity and bioequivalence of HS628 and tocilizumab. The safety and immunogenicity profiles of HS628 were also shown to be similar to those of the reference tocilizumab.

Safety and immunogenicity of a modified COVID-19 mRNA vaccine, SW-BIC-213, as a heterologous booster in healthy adults: an open-labeled, two-centered and multi-arm randomised, phase 1 trial.

BACKGROUND: We assessed the safety and immunogenicity of a core-shell structured lipopolyplex (LPP) based COVID-19 mRNA vaccine, SW-BIC-213, as a heterologous booster in healthy adults. METHODS: We conducted an open-labeled, two-centered, and three-arm randomised phase 1 trial. Healthy adults, who had completed a two-dose of inactivated COVID-19 vaccine for more than six months, were enrolled and randomized to receive a booster dose of COVILO (inactivated vaccine) (n = 20) or SW-BIC-213-25µg (n = 20), or SW-BIC-213-45µg (n = 20). The primary study endpoint was adverse events within 30 days post-boosting. The secondary endpoint was the titers of binding antibodies and neutralizing antibodies against the wild-type (WT) of SARS-CoV-2 as well as variants of concern in serum. The exploratory endpoint was the cellular immune responses. This trial was registered with http://www.chictr.org.cn (ChiCTR2200060355). FINDINGS: Between Jun 6 and Jun 22, 2022, 60 participants were enrolled and randomized to receive a booster dose of SW-BIC-213 (25 µg, n = 20, or 45 µg, n = 20) or COVILO (n = 20). The baseline demographic characteristics of the participants at enrollment were similar among the treatment groups. For the primary outcome, injection site pain and fever were more common in the SW-BIC-213 groups (25 µg and 45 µg). Grade 3 fever was reported in 25% (5/20) of participants in the SW-BIC-213-45µg group but was resolved within 48 h after onset. No fatal events or adverse events leading to study discontinuation were observed. For secondary and exploratory outcomes, SW-BIC-213 elicited higher and longer humoral and cellular immune responses than that in the COVILO group. INTERPRETATION: SW-BIC-213, a core-shell structured lipopolyplex (LPP) based mRNA vaccine, was safe, tolerable, and immunogenic as a heterologous booster in healthy Chinese adults. FUNDING: Shanghai Municipal Government, the Science and Technology and Economic Commission of Shanghai Pudong New Area, and mRNA Innovation and Translation Center of Shanghai.

Foam Cells in Atherosclerosis: Novel Insights Into Its Origins, Consequences, and Molecular Mechanisms.

Foam cells play a vital role in the initiation and development of atherosclerosis. This review aims to summarize the novel insights into the origins, consequences, and molecular mechanisms of foam cells in atherosclerotic plaques. Foam cells are originated from monocytes as well as from vascular smooth muscle cells (VSMC), stem/progenitor cells, and endothelium cells. Novel technologies including lineage tracing and single-cell RNA sequencing (scRNA-seq) have revolutionized our understanding of subtypes of monocyte- and VSMC-derived foam cells. By using scRNA-seq, three main clusters including resident-like, inflammatory, and triggering receptor expressed on myeloid cells-2 (Trem2 hi ) are identified as the major subtypes of monocyte-derived foam cells in atherosclerotic plaques. Foam cells undergo diverse pathways of programmed cell death including apoptosis, autophagy, necroptosis, and pyroptosis, contributing to the necrotic cores of atherosclerotic plaques. The formation of foam cells is affected by cholesterol uptake, efflux, and esterification. Novel mechanisms including nuclear receptors, non-coding RNAs, and gut microbiota have been discovered and investigated. Although the heterogeneity of monocytes and the complexity of non-coding RNAs make obstacles for targeting foam cells, further in-depth research and therapeutic exploration are needed for the better management of atherosclerosis.

Development and validation of a ligand-binding assay for quantification of the F(ab')2 antivenom of Daboia russelii siamensis in human serum and its application to a phase I clinical study.

Daboia russelii siamensis accounts for most of snakebite mortalities in China, yet, specific treatment against the venom toxins is absent in clinical practice. The F(ab')2 antivenom of Daboia russelii siamensis is manufactured and approved for the clinical trial in China. To satisfy the need for clinical pharmacokinetic research, this study aimed to develop a ligand binding assay (LBA) for the quantification of F(ab')2 antivenom of Daboia russelii siamensis in human serum. A diverse combination of conditions was optimized based on the fitness of the calibration curve and selectivity. The established LBA undergoes thorough method validation according to the guidelines of regulatory authorities. In the calibration range 1.0-64 µg/mL, the correlation coefficient r2 was from 0.9970 to 1.000, indicating good fitness. Accuracy and precision were within ± 20%. Dilution linearity was observed in the ultra-high quality-control (QC) samples (500 µg/mL). In addition, the assay was free from hook effect, the endogenous interferences and exogenous interferences. The QC samples were stable under different handling and storage conditions. The validated assay was successfully applied to a phase I clinical study of the F(ab')2 antivenom of Daboia russelii siamensis in Chinese healthy volunteers. The peak concentrations exhibited dose-proportionality. In conclusion, this study provides a novel and reliable LBA method for the clinical pharmacokinetic research of F(ab')2 antivenom of Daboia russelii siamensis. It will facilitate further clinical trials in treating the snakebite of Daboia russelii siamensis.

Liquid chromatography-tandem mass spectrometric assay for TPN171 in human plasma.

A simple, rapid and accurate method for quantitative analysis of a highly selective phosphodiesterase-5 inhibitor (PDE5), TPN171 by high performance liquid chromatography and tandem mass spectrometry in human plasma was proposed and validated successfully using D3-TPN171 as internal standards (ISTD). An aliquot of 100 µL of plasma was mixed with internal standard and was precipitated with acetonitrile. Gradient elution was performed on a ACQUITY HSS T3 column (50 × 2.1 mm, 1.8 µm) coupled with a ACQUITY column in-line filter at 40℃, by 5 mM ammonium acetate in water containing 0.1 % formic acid and 0.1 % formic acid in acetonitrile as the mobile phase. The total analytical run time was 3.5 min. The analyte was monitored using multiple reaction monitoring (MRM) scan in positive polarity mode. The ion transition was m/z 442.2→113.2 and 445.2→116.2 for TPN171 and D3-TPN171 respectively. The method was validated for specificity, sensitivity, precision, accuracy, and other analytical parameters. The results found were satisfactory over the linear calibration range of 1-500 ng/mL. Within-day precisions ranged from 1.8 to 7.3 %, and between-day precisions from 2.3 to 4.9 %, accuracies were 95.5-99.8 %.The validated method was successfully applied to determine the plasma concentration after oral administration of 10 mg TPN171 in six healthy volunteers.

Direct analysis in real time-mass spectrometry for rapid quantification of five anti-arrhythmic drugs in human serum: application to therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

Proteomics analysis reveals a potential new target protein for the lipid-lowering effect of Berberine8998.

Berberine8998 is a newly synthesized berberine derivative with better lipid-lowering activity and improved absorption. The objective of this study was to investigate the effects of berberine8998 on serum cholesterol and lipid levels in vivo and to examine the mechanisms involved. Hamsters on high-fat diet (HFD) were administered berberine or berberine8998 (50 mg·kg-1·d-1, ig) for 3 weeks. Berberine8998 administration significantly lowered the total cholesterol, triglycerides and LDL-C levels in HFD hamsters. Bioinformatics revealed that berberine and berberine8998 shared similar metabolic pathways and fatty acid metabolism was the predominant pathway. Western blot validation results showed that peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and long-chain fatty acid-CoA ligase 1 (ACSL1), two proteins involved in fatty acid metabolism, were expressed differently in the berberine8998 group than in the untreated group and the berberine treatment group. Biochemistry results showed that berberine8998 significantly lowered the non-esterified fatty acid (NEFA) levels, which may lead to a reduction in TG levels in the berberine8998 treatment group and the differences observed in proteomics analyses. Pharmacokinetic analysis conducted in rats. After administration of berberine or berberine8998 (50 mg/kg, ig), berberine8998 exhibited a remarkably improved absorption with increasing bioavailability by 6.7 times compared with berberine. These findings suggest that berberine8998 lowers cholesterol and lipid levels via different mechanisms than berberine, and its improved absorption makes it a promising therapeutic candidate for the treatment of hypercholesterolemia and obesity.

A novel small molecule liver X receptor transcriptional regulator, nagilactone B, suppresses atherosclerosis in apoE-deficient mice.

AIMS: Atherosclerosis is the most common cause of cardiovascular diseases, such as myocardial infarction and stroke. We hypothesized that nagilactone B (NLB), a small molecule extracted from the root bark of Podocarpus nagi (Podocarpaceae), suppresses atherosclerosis in an atherosclerotic mouse model. METHODS AND RESULTS: Male apoE-deficient mice on C57BL/6J background received NLB (10 and 30 mg/kg) for 12 weeks. Compared with the model group, NLB treatment (10 and 30 mg/kg) significantly reduced en face lesions of total aorta areas. In RAW264.7 cells, NLB significantly ameliorated cholesterol accumulation in macrophages via enhancing apolipoprotein A-I and HDL-mediated cholesterol efflux. Mechanistically, NLB induced messenger RNA and protein expression of the ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) in RAW264.7 and THP-1 cells. Liver X receptor (LXR) site mutations in the mouse ABCA1 promoter abrogated NLB-mediated luciferase reporter activity. LXRα and LXRß small interfering RNA suppressed NLB-mediated induction of ABCA1 expression. Consistent with in vitro results, NLB induced ABCA1 expression and suppressed macrophage areas in the aortic sinus. Moreover, NLB treatment did not induce the protein expression of LXR in liver. Hepatic and intestinal cholesterol accumulation was significantly alleviated on NLB treatment. Besides, NLB significantly improved plasma lipid profiles in apoE-deficient mice. CONCLUSION: Selective LXR activation in macrophages with NLB induces ABCA1- and ABCG1-mediated cholesterol efflux while exerting minimal effects on lipogenesis and lipid accumulation in liver, resulting in regression of atherosclerosis, and therefore might be a promising strategy for therapeutics.

Betulin attenuates atherosclerosis in apoE-/- mice by up-regulating ABCA1 and ABCG1.

AIM: Betulin is a pentacyclic triterpenoid isolated from the bark of yellow and white birch trees with anti-cancer and anti-malaria activities. In this study we examined the effects of betulin on atherosclerosis in apoE-/- mice and the underlying mechanisms. METHODS: Murine macrophage RAW264.7 cells and human monocyte-derived THP-1 cells were tested. Foam cell formation was detected with Oil Red O staining. Cholesterol efflux was assessed using [3H]-cholesterol efflux assay. The expression of ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) was examined using RT-PCR and Western-blotting. The ABCA1 promoter activity was evaluated using luciferase activity assay. Male apoE-/- mice fed on a high-fat-diet (HFD), and received betulin (20 and 40 mg·kg-1·d-1, ig) for 12 weeks. The macrophage content and ABCA1 expression in the aortic sinuses were evaluated with immunofluorescence staining. The hepatic, intestinal and fecal cholesterol were also analyzed in the mice. RESULTS: In RAW264.7 cells, betulin (0.1-2.5 µg/mL) dose-dependently ameliorated oxLDL-induced cholesterol accumulation and enhanced cholesterol efflux. In both RAW264.7 and THP-1 cells, betulin increased the expression of ABCA1 and ABCG1 via suppressing the transcriptional repressors sterol-responsive element-binding proteins (SREBPs) that bound to E-box motifs in ABCA1 promoter, whereas E-box binding site mutation markedly attenuated betulin-induced ABCA1 promoter activity. In HFD-fed apoE-/- mice, betulin administration significantly reduced lesions in en face aortas and aortic sinuses. Furthermore, betulin administration significantly increased ABCA1 expression and suppressed macrophage positive areas in the aortic sinuses. Moreover, betulin administration improved plasma lipid profiles and enhanced fecal cholesterol excretion in the mice. CONCLUSION: Betulin attenuates atherosclerosis in apoE-/- mice by promoting cholesterol efflux in macrophages.

Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma.

A quantitative method for clopidogrel using online-SPE tandem LC-MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d 4) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24 h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75 mg single dose clopidogrel tablets in 48 healthy male subjects.

Determination of the novel antiarrhythmic drug sulcardine sulfate in human plasma by liquid chromatography tandem mass spectrometry and its application in a clinical pharmacokinetic study.

Sulcardine sulfate (Sul), a novel antiarrhythmic agent, is currently in phase I and phase II clinical trials. To elucidate its clinical pharmacokinetic characteristics, a rapid and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of Sul in human plasma. Plasma samples were precipitated by acetonitrile and isotope-labeled sulcardine was added as internal standard. The analysis was carried out on a Capcell Pak C18 MG III column (100 × 2.0 mm, 5 µm) with 0.1% formic acid in acetonitrile solution and water (17:83, v/v) as mobile phase. The linear range was 5.0-1000 ng/mL for Sul, with a lower limit of quantification of 5.0 ng/mL. The intra- and inter-batch CVs were within ±11.0% and the accuracies were 4.9-107.3%. Our method, for the first time, allows the rapid (only 3.0 min) and accurate quantification of Sul in human plasma. The method has been successfully applied in the pharmacokinetic study of Sul in a clinical trial following oral administration of Sul to healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.

AIM: Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro. METHODS: HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9. RESULTS: Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 µmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 µmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 µmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 µmol/L) and pravastatin (5 µmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone. CONCLUSION: Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

Sodium tanshinone IIA silate inhibits high glucose-induced vascular smooth muscle cell proliferation and migration through activation of AMP-activated protein kinase.

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G0/G1 phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.

Phase I study on the pharmacokinetics and tolerance of ZT-1, a prodrug of huperzine A, for the treatment of Alzheimer's disease.

AIM: Huperzine A isolated from the Chinese herb Huperzia serrata (Thunb) Trev is a novel reversible and selective AChE inhibitor. The aim of this study was to evaluate the pharmacokinetics and tolerance of single and multiple doses of ZT-1, a novel analogue of huperzine A, in healthy Chinese subjects. METHODS: This was a double-blinded, placebo-controlled, randomized, single- and multiple-dose study. For the single-dose study, 9 subjects were randomly divided into 3 groups receiving ZT-1 (0.5, 0.75 or 1 mg, po) according to a Three-way Latin Square Design. For the multiple-dose study, 9 subjects receiving ZT-1 (0.75 mg/d, po) for 8 consecutive days. In the tolerance study, 40 subjects were randomly divided into 5 groups receiving a single dose of ZT-1 (0.5, 0.75, 1, 1.25 or 1.5 mg, po). Plasma and urine concentrations of ZT-1 and Hup A were determined using LC-MS/MS. Pharmacokinetic parameters, including Cmax, AUC0-72 h and AUC0-∞ were calculated. Tolerance assessments were conducted throughout the study. RESULTS: ZT-1 was rapidly absorbed and converted into huperzine A, thus the plasma and urine concentrations of ZT-1 were below the limit of quantification (<0.05 ng/mL). After single-dose administration of ZT-1, the mean tmax of huperzine A was 0.76-0.82 h; the AUC0-72 h and Cmax of huperzine A showed approximately dose-proportional increase over the dose range of 0.5-1 mg. After the multiple-dose administration of ZT-1, a steady-state level of huperzine A was achieved within 2 d. No serious adverse events were observed. CONCLUSION: ZT-1 is a pro-drug that is rapidly absorbed and converted into huperzine A, and ZT-1 is well tolerated in healthy Chinese volunteers.

Liquid chromatography tandem mass spectrometry method for determination of N-acetylcysteine in human plasma using an isotope-labeled internal standard.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine total N-acetylcysteine in human plasma. Mass spectrometric detection was achieved in positive electrospray ionization and multiple reaction monitoring mode. The mass transition pairs of N-acetylcysteine and the isotope-labeled internal standard d3-N-acetylcysteine were 164 → 122 and 167 → 123, respectively. The method was linear over the range of 10-5000 ng/mL in human plasma. The adoption of trichloroacetic acid significantly enhanced the extraction recovery. The blank matrix was screened to minimize the influence of endogenous N-acetylcysteine. After being fully validated, the method was successfully applied to the pharmacokinetic and bioequivalent study of N-acetylcysteine after oral administration of 600 mg tablets to 24 healthy Chinese volunteers.

Development and validation of a liquid chromatography-isotope dilution tandem mass spectrometry for determination of olanzapine in human plasma and its application to bioavailability study.

A simple, reliable and sensitive liquid chromatography-isotope dilution mass spectrometry (LC-ID/MS) was developed and validated for quantification of olanzapine in human plasma. Plasma samples (50 microL) were extracted with tert-butyl methyl ether and isotope-labeled internal standard (olanzapine-D3) was used. The chromatographic separation was performed on XBridge Shield RP 18 (100 mm x 2.1 mm, 3.5 microm, Waters). An isocratic program was used at a flow rate of 0.4 m x min(-1) with mobile phase consisting of acetonitrile and ammonium buffer (pH 8). The protonated ions of analytes were detected in positive ionization by multiple reactions monitoring (MRM) mode. The plasma method, with a lower limit of quantification (LLOQ) of 0.1 ng x mL(-1), demonstrated good linearity over a range of 0.1 - 30 ng x mL(-1) of olanzapine. Specificity, linearity, accuracy, precision, recovery, matrix effect and stability were evaluated during method validation. The validated method was successfully applied to analyzing human plasma samples in bioavailability study.

Development of a liquid chromatography-isotope dilution mass spectrometry method for quantification of fentanyl in human plasma.

Fentanyl is a potent analgesic drug in relieving chronic pain in patients. In this report, we present a simple, reliable and sensitive LC-ID/MS method for the quantification of fentanyl in human plasma. LC-ID/MS analysis was carried out on a triple quadrupole mass spectrometer operated in positive electrospray ionization multiple-reaction-monitoring using the transitions m/z 337.6 --> 187.9 for fentanyl and m/z 342.6 --> 187.9 for the internal standard (D5-fentanyl). The calibration curve covered the range 0.02-10 ng/mL. The intra- and inter-batch precision were less than 6.739 and 3.126% for fentanyl and IS, with accuracy from 94.16 to 102.0%. The lower limit of quantification was identifiable and reproducible at 0.02 ng/mL. The validated method offered increased sensitivity and wide linear concentration range. This method was successfully adopted for the evaluation of bioequivalence of two fentanyl transdermal preparations after single dose administration to 20 Chinese pain-patients.