A novel anastrozole intravaginal ring for endometriosis: Pharmacokinetic characteristics and mucosal irritation.

In order to achieve the long-term management of endometriosis, a more economical and environmentally friendly material, ethylene vinyl acetate (EVA), was used to prepare a intravaginal ring with anastrozole (ATZ). This paper has compared the pharmacokinetic parameters with oral tablets (Aida®) in mini pigs and evaluated the uterine targeted effect and mucosal irritation of the ring. A bioassay method was developed and validated for the determination of ATZ in mini pigs. The determination of ATZ was achieved by LC-MS/MS using terfenadine as the internal standard. Separation was achieved on a Kinetex-C18 110A chromatographic column (3 × 30 mm, 2.6 µm; Phenomenex) with a gradient mobile phase consisting of methanol (0.1% formic acid) and water (0.1% formic acid). The method has been proved to be scientific and sensitive through methodological validation and can be easily and quickly applied to the determination of the content of anastrozole in mini pigs. The pharmacokinetic test results show that there was no significant difference in pharmacokinetic parameters between the two formulations. The intravaginal ring has a passive targeting effect on the uterus, and its mucosal irritation is acceptable. The intravaginal ring provides a new method for long-term management of endometriosis.

LC-MS/MS quantification of progesterone in ovariectomized miniature swine and its application for preliminary pharmacokinetic study of a novel vaginal sustained release system.

OBJECTIVE: We here established a simple, fast, robust and sensitive LC-MS/MS method and validated as well for the quantitative analysis of progesterone (PGT) in ovariectomized (OVX) miniature swine plasma. The method was successfully applied to characterize the pharmacokinetics of a progesterone vaginal drug delivery system. METHODS: Megestrol acetate was utilized as an internal standard (IS). The separation and detection of PGT from endogenous interference was performed successfully by liquid chromatography with gradient elution and mass spectrometry equipped with positive ESI source using MRM mode. The EVA intravaginal rings (IVRs) were manufactured by hot-melt extrusion (HME), afterward were administrated vaginally to OVX minipigs to evaluate PK study. RESULTS: The calibration curve for swine plasma samples across the concentration ranged between 0.25 ng/mL and 100 ng/mL. The intra- and inter-assay accuracy and precision were lower than ±5% and 5.88%, respectively. Recoveries of PGT and IS were ranging from 114-119% and 96.5-112%, respectively. In vitro study showed that the EVA IVRs release 18 mg/day of PGT continuously over 7 days, and corresponding mean PGT plasma concentration in OVX minipigs (CAVG) was 4.892 ng/mL. CONCLUSION: All the study produced reliable results for the measurement of PGT concentration in miniature swine plasma and the method was successfully applied to a PK study for PGT vaginal ring in miniature pigs, which may lay the foundation for further research on the progesterone preparations intended for in assisted reproductive technology.

APN-mediated phosphorylation of BCKDK promotes hepatocellular carcinoma metastasis and proliferation via the ERK signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most prevalent human malignancies worldwide and has high morbidity and mortality. Elucidating the molecular mechanisms underlying HCC recurrence and metastasis is critical to identify new therapeutic targets. This study aimed to determine the roles of aminopeptidase N (APN, also known as CD13) in HCC proliferation and metastasis and its underlying mechanisms. We detected APN expression in clinical samples and HCC cell lines using immunohistochemistry, flow cytometry, real-time PCR, and enzyme activity assays. The effects of APN on HCC metastasis and proliferation were verified in both in vitro and in vivo models. RNA-seq, phosphoproteomic, western blot, point mutation, co-immunoprecipitation, and proximity ligation assays were performed to reveal the potential mechanisms. We found that APN was frequently upregulated in HCC tumor tissues and high-metastatic cell lines. Knockout of APN inhibited HCC cell metastasis and proliferation in vitro and in vivo. Functional studies suggested that a loss of APN impedes the ERK signaling pathway in HCC cells. Mechanistically, we found that APN might mediate the phosphorylation at serine 31 of BCKDK (BCKDKS31), promote BCKDK interacting with ERK1/2 and phosphorylating it, thereby activating the ERK signaling pathway in HCC cells. Collectively, our findings indicate that APN mediates the phosphorylation of BCKDKS31 and activates its downstream pathway to promote HCC proliferation and metastasis. Therefore, the APN/BCKDK/ERK axis may serve as a new therapeutic target for HCC therapy, and these findings may be helpful to identify new biomarkers in HCC progression.

Combined effects of age and polymorphisms in Notch3 in the pathogenesis of cerebral infarction disease.

Cerebral infarction disease is a severe hypoxic ischemic tissue necrosis in the brain, often leading to long-term functional disability and residual impairments. The Notch signaling pathway plays key roles in proliferation and survival of the stem/progenitor cells of the central and peripheral nervous systems. Notch3 is an important member of the pathway, but the relationships between the genetic abnormalities and cerebral infarction disease still remain unclear. The aim of this work was to evaluate variations in Notch3 gene for their possible associations with the cerebral infarction disease. We sequenced the Notch3 gene for 260 patients with cerebral infarction disease, 300 normal controls with old ages and 300 normal controls with younger ages, and identified the variations. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 19.0). The Hardy-Weinberg equilibrium test of the population was carried out using the online software OEGE. Six variations, including rs1044116, rs1044009, rs1044006, rs10408676, rs1043996 and rs16980398 within or near the Notch3 gene, were found. The genetic heterozygosity of rs1044116, rs1044009, rs1044006, and rs1043996 was very high, whereas that of rs10408676 and rs16980398 was very low. Statistical analyses showed that rs1044009 and rs1044006 were associated with the risk of cerebral infarction disease in the Chinese Han agedness population. The SNPs rs1044009 and rs1044006 in the Notch3 gene were associated with the risk of cerebral infarction diseases in the Chinese Han agedness population.