Development and validation of a UPLC-MS/MS method for the determination of cucurbitacin B in rat plasma and application to a pharmacokinetic study.

Cucurbitacin B (CuB), one of the most abundant forms of cucurbitacins, is a promising natural anticancer drug candidate. Although the anticancer activity of CuB has been well demonstrated, information regarding the pharmacokinetics is limited. A rapid, selective and sensitive UPLC-MS/MS for CuB was developed and validated using hemslecin A (HeA) as internal standard (IS). Plasma samples were pre-treated by liquid-liquid extraction with dichloromethane. Separation was achieved on a reversed-phase C18 column (50 × 4.6 mm, 5 µm) at 35°C using isocratic elution with water-methanol (25:75, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with positive electrospray ionization mode. The calibration curve was linear (r > 0.995) in a concentration range of 0.3-100 ng/mL with a limit of quantification of 0.3 ng/mL. Intra- and inter-day accuracy and precision were validated by percentage relative error and relative standard deviation, respectively, which were both lower than the limit of 15%. This assay was successfully applied to a pharmacokinetic study of CuB in Wistar rats.

LC-ESI-MS/MS analysis and pharmacokinetics of heterophyllin B, a cyclic octapeptide from Pseudostellaria heterophylla in rat plasma.

Heterophyllin B (HB) is a cyclic octapeptide isolated from Pseudostellaria heterophylla. HB is used as the quality control index for evaluating P. heterophylla in the Chinese Pharmacopoeia. A rapid and sensitive LC-ESI-MS/MS method was developed and validated for the analysis of HB in rat plasma. Sample preparation consisted of a solid-phase extraction step for the removal of interference and preconcentration of the target analyte HB and the internal standard N-acetylcysteine before chromatographic analysis by MS/MS detection. The separation of HB and N-acetylcysteine was performed using a Hypersil GOLD™ C18 column and a mixture of methanol-water (60:40, v/v) containing 10 mmol/L ammonium formate and 0.1% formic acid as the mobile phase. The determination step was optimized in the selected reaction monitoring mode for the highly selective and sensitive quantitation of HB in rat plasma. Intra- and inter-assay precision (as relative standard deviation) was ≤9.1%, and accuracy was between 92.6 and 102.7%. The validated method was successfully applied to quantify HB concentrations up to 7 h after tail intravenous injections of 2.08, 4.16 and 8.32 mg/kg HB in rats. The LC-MS/MS method identified the relevant pharmacokinetic parameters of HB and its studied analog.

LC-MS/MS Determination and Pharmacokinetic Study of Pedunculoside in Rat Plasma after Oral Administration of Pedunculoside and Ilex rotunda Extract.

Ilex rotunda is widely used to treat many disorders as a traditional Chinese medicine (TCM) containing 4%-5% pedunculoside (PDC). A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine PDC in rat plasma by using 3ß,19α-dihydroxyurs-12-en-28-oic acid 28-ß-D-glucopyranosyl ester (DEOG) as an internal standard. The analytes were extracted by protein precipitation and eluted on a C18 chromatography column using a mobile phase of methanol-H2O (70:30, v/v) delivered at a flow rate of 0.6 mL/min. Detection was performed using positive ion electrospray ionization in multiple reaction monitoring modes. The assay was linear over the concentration range of 0.60 ng/mL to 200 ng/mL, with a quantification limit of 0.60 ng/mL. Intra-day and inter-day precisions (%RSD) ranged from 2.12 to 9.51 for PDC, whereas the accuracy was within -7.83%~9.40%. The validated method was successfully applied to the pharmacokinetic study of PDC in rat plasma after oral administration of pure PDC and Ilex rotunda extract (IRE). Pharmacokinetic parameters of PDC in IRE, such as Cmax, AUC0-t, AUC0-∞, t1/2z, and CLz/F, statistically differed from those of the pure monomer (p < 0.01). However, Tmax and MRT showed no significant differences between the two groups. Results suggested that other coexisting components in IRE may decrease the absorption of PDC. Compound-compound interactions between PDC and other herbal extract components can alter the pharmacokinetic behavior of PDC. The study will be helpful in providing references for understanding the action mechanism and clinical application of Ilex rotunda.

CD47 Deficiency in Mice Exacerbates Chronic Fatty Diet-Induced Steatohepatitis Through Its Role in Regulating Hepatic Inflammation and Lipid Metabolism.

Inflammation is one of the hallmarks of non-alcoholic steatohepatitis. CD47 is a widely expressed transmembrane protein that signals through inhibitory receptor signal regulatory protein α (SIRPα) to inhibit macrophage activation and phagocytosis. In this study, we sought to investigate the role of CD47 in hepatosteatosis and fibrosis induced by a chronic high-fat diet (HFD), by comparing disease development in wild-type (WT) and CD47KO mice fed HFD for 40 weeks. The HFD induced remarkably more severe hepatic steatosis and fibrosis but less body weight gain and less subcutaneous fat accumulation in CD47KO mice compared to WT mice. Liver tissues from HFD-fed CD47KO mice exhibited enhanced inflammation characterized by increased proinflammatory cytokine production and increased nuclear factor-κB (NF-κB) activation compared to similarly fed WT mice. Although higher expression of apolipoproteins was observed in CD47KO mice compared to WT mice under a low-fat diet (LFD), HFD-fed WT and CD47KO mice showed comparably prominent downregulation of these apolipoprotein genes, suggesting that the marked difference observed in lipid accumulation and hepatosteatosis between these mice cannot be explained by changes in apolipoproteins. Like apolipoproteins, sirtuin 1 (SIRT1) and peroxisome proliferator activated receptor alpha (PPARα), which are involved in regulation of both lipid metabolism and inflammation, were more highly expressed in CD47KO than WT mice under LFD but more severely suppressed in CD47KO than in WT mice under HFD. Taken together, our results indicate that CD47 plays a significant role in the pathogenesis of HFD-induced hepatosteatosis and fibrosis through its role in regulation of inflammation and lipid metabolism.

Loeffler endocarditis as a rare cause of heart failure with preserved ejection fraction: A case report and review of literature.

RATIONALE: Hypereosinophilic syndrome (HES) is a rare disease characterized by hypereosinophilia and its ensuing organ damage. Cardiac involvement is divided into 3 chronological stages: an acute necrotic stage; a thrombus formation stage; and a fibrotic stage. Infiltration of the myocardium by eosinophilic cells followed by endomyocardial fibrosis is known as "Loeffler endocarditis." PATIENT CONCERNS: We report a case of a 60-year-old man diagnosed with left-sided restrictive cardiomyopathy. DIAGNOSIS: The patient experienced heart failure with preserved ejection fraction. The cardiac MRI showed intense, linear, delayed gadolinium enhancement of the endocardium of the lateral wall of the left ventricle, and obliteration of the LV apex. He was ultimately identified as Loeffler endocarditis. INTERVENTION: A bone marrow smear and biopsy revealed the FIP1L1-PDGFRA fusion gene was positive in 82% of segmented nucleated cells. OUTCOME: Our patient responded well to prednisone at 1 mg/kg/d. LESSONS: HES is a rare disease that often afflicts the heart. Cardiac involvement in hypereosinophilia, especially Loeffler endocarditis, carries a poor prognosis and significant mortality. Early detection and treatment of the disease is therefore essential. Further studies are needed to ascertain therapeutic corticosteroid dosages and develop targeted gene therapies, both important steps to ameliorate the effects of Loeffler endocarditis and improve patient outcomes.

Clinical Characteristics and Outcomes in Young Patients With ST-Segment Elevation Myocardial Infarction After Primary Percutaneous Coronary Intervention.

BACKGROUND: There are few published studies of ST-segment elevation myocardial infarction (STEMI) in younger individuals. The differences between these "younger" and "older" individuals may not be fully appreciated by clinicians. The aim of this study was to determine the reasons for the earlier presentation and help to identify strategies for prevention of recurrent myocardial infarction (MI) in younger patients. METHODS: The study population was a cohort of 2,419 consecutive STEMI patients who were treated with primary percutaneous coronary intervention. The median follow-up time of this retrospective study was 2.2 years. RESULTS: The all-cause mortality rates in patients ≤45 years of age at 30 days, 1 and 2 years were 1.7%, 2.0% and 2.2%, respectively. These rates were lower compared with their older matched counterparts whose all-cause mortality rates were 3.3%, 4.2% and 5.5%, respectively (P = 0.010). The incidence of recurrent MI was 4.0% for all age groups combined, 5.4% for younger patients and 3.8% for older patients. The number of stents showed association with recurrent MI in older patients with a first infarction, whereas only composition factor 1 with significantly higher non-high-density lipoprotein and low-density lipoprotein values was significantly associated with recurrent MI in the younger patients. CONCLUSIONS: STEMI patients ≤45 years of age more often had lower rates of all-cause mortality, but the risk of recurrent MI was similar to that of older patients. Regardless of triglyceride level, neither non-high-density lipoprotein nor low-density lipoprotein were independent predictors for recurrent MI during the long-term follow-up in younger patients.

Is Vascular Amyloidosis Intertwined with Arterial Aging, Hypertension and Atherosclerosis?

Vascular amyloidosis (VA) is a component of aging, but both VA and aging move forward together. Although, not all age-related molecules are involved with VA, some molecules are involved in a crosstalk between both of them. However, the cellular mechanism by which, vascular cells are phenotypically shifted to arterial remodeling, is not only involved in aging but also linked to VA. Additionally, patients with hypertension and atherosclerosis are susceptible to VA, while amyloidosis alone may provide fertile soil for the initiation and progression of subsequent hypertension and atherosclerosis. It is known that hypertension, atherosclerosis and amyloidosis can be viewed as accelerated aging. This review summarizes the available experimental and clinical evidence to help the reader to understand the advance and underlying mechanisms for VA involvement in and interaction with aging. Taken together, it is clear that VA, hypertension and atherosclerosis are closely intertwined with arterial aging as equal partners.

A Comparative Pharmacokinetic Study of Myrislignan by UHPLC-MS After Oral Administration of a Monomer and Myristica fragrans Extract to Rats.

An ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) method was developed and validated to quantify myrislignan in rat plasma using podophyllotoxin as an internal standard (IS). The chromatographic separation of myrislignan and IS was performed on a 3.0 µm Hypersil C18 column (50 mm × 4.6 mm) with methanol and water containing 0.1% acetic acid (80:20, v/v) as the mobile phase at a flow rate of 0.3 mL/min. An electrospray ionization was used in the positive selective-ion monitoring mode for the target ions at m/z 397 and m/z 437 for the quantification of myrislignan and IS. The total run time was 3.6 min for each run. The calibration curve was linear over the range of 0.75-300 ng/mL (r> 0.995) with the lower limit of quantitation at 0.75 ng/mL. Intra- and interday precision was below 11.49%, and the mean accuracy ranged from -9.75 to 7.45%. The proposed method was successfully applied to evaluate the pharmacokinetic properties of myrislignan after oral administration of the myrislignan monomer and Myristica fragrans extract in rats. Statistical analyses indicate that the pharmacokinetic properties of myrislignan in rats have significant differences between two groups.

TREM-1 is a positive regulator of TNF-α and IL-8 production in U937 foam cells.

The purpose of our study was to investigate the expression levels of TREM-1 (triggering receptor expressed on myeloid cells-1) in U937 foam cells and determine whether TREM-1 regulates the production of tumor necrosis factor-alpha and interleukin-8 in these cells. Human U937 cells were incubated with phorbol 12-myristate 13-acetate and then oxidized human low-density lipoprotein to induce foam cell formation. Oil red O staining was used to identify the foam cells. The production of IL-8 and TNF-α by U937 foam cells was assayed by enzyme-linked immunosorbent assay. The expression of TREM-1 mRNA in U937 foam cells was detected by reverse transcription-polymerase chain reaction. Moreover, U937 foam cells were transfected by small interfering RNA using Lipofectamine 2000 to knockdown TREM-1. Western blot was performed to assay protein expression of TREM-1 and ELISA was used to examine the effect of TREM-1 knockdown on IL-8 and TNF-α production. PMA and ox-LDL induced U937 cells to form foam cells. The production of TNF-α and IL-8 was found to be significantly elevated in U937 foam cells, concomitant with a significant up-regulation of TREM-1 mRNA. TREM-1 siRNA was able to partially silence the expression of TREM-1 protein and remarkably inhibited TNF-α and IL-8 production in U937 foam cells, suggesting that TREM-1 is a positive regulator of TNF-α and IL-8 production in U937 foam cells. Our finding that TREM-1 controls the production of IL-8 and TNF-α in U937 foam cells defines a potentially critical role of TREM-1 in the pathogenesis of atherosclerosis and implicates TREM-1 as a potential therapeutic target for the disease.