LC-MS/MS method for quick detection of vonoprazan fumarate in human plasma: Development, validation and its application to a bioequivalence study.

A liquid chromatography-tandem mass spectrometry method with vonoprazan fumarate-d4 as a stable isotope-labeled internal standard was developed and validated aiming at quantification of vonoprazan fumarate in human plasma for a bioequivalence study. Chromatographic separation was achieved by acetonitrile one-step protein precipitation using a gradient elution of 0.1% formic acid aqueous solution and acetonitrile with a run time of 3.65 min. Detection was carried out on a tandem mass spectrometer in multiple reaction monitoring mode via a positive electrospray ionization interface. The multiple reaction monitoring mode of precursor-product ion transitions for vonoprazan fumarate and vonoprazan fumarate-d4 were m/z 346.0 → 315.1 and 350.0 → 316.0, respectively. The linear range was 0.150-60.000 ng/ml. This method was fully validated with acceptable results in terms of selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, dilution effect, matrix effect, stability, recovery and incurred sample reanalysis. A successful application of this method was realized in the bioequivalence study of vonoprazan fumarate tablet (20 mg) among healthy Chinese volunteers.

Complete genome of Chinese sacbrood virus from Apis cerana and analysis of the 3C-like cysteine protease.

The complete genome sequence of Chinese sacbrood virus (CSBV), isolated from diseased larvae of Apis cerana in Fujian province, China was analyzed. The viral genome consisted of 8,800 nucleotides, encoding 2,848 amino acids. A phylogenetic tree analysis showed the sacbrood virus (SBV) segregated into three distinct groups. The isolates originated from A. c. indica and were the first distinct evolutionary group. The AcSBV-SBM2 isolate, from A. c. cerana, belonged to the second distinct group. The remaining SBV isolates formed the third group. The phylogenetic relationships of SBV isolates suggest that they are derived from similar honeybee species or geographic origins. The 3C-like cysteine protease protein plays an important role in viral replication. The 3C-like cysteine protease protein of CSBV-FZ was predicted to contain a transmembrane domain. The subcellular localization of 3C-like cysteine protease was distributed as discrete punctate inclusions and co-localized with VP1 of CSBV. These results suggest that the non-structural protein 3C-like cysteine protease might be involved in viral replication. Insect cell cultures can further advance our understanding of picorna-like virus replication.

Replication of Chinese sacbrood virus in primary cell cultures of Asian honeybee (Apis cerana).

A primary cell culture system was established for the first time from embryonic tissues of Asian honeybee, Apis cerana, and used to trace the early infection process of Chinese sacbrood virus (CSBV), an iflavirus in the family Iflaviridae. A monolayer of epithelium-like cells of A. cerana, approximately 8-10 µm in diameter, was grown in Kimura's insect medium at 28 °C within 3-4 days of setting up the cultures. Such cultured cells were inoculated with CSBV purified from infected larvae or pupae for 2 h. In electron and confocal micrographs, viral particles accumulated as filamentous or vesicular inclusions in the cytoplasm of infected cultured cells at 36 h post-inoculation (hpi). Real-time quantitative RT-PCR assay showed that the expression levels of four cistrons of CSBV in the cultured cells increased rapidly between 12 and 48 hpi. This newly established primary cell culture derived from A. cerana will be useful for further studies of infection caused by CSBV.

Age-related pharmacokinetics differences were observed between young and elderly populations of a novel PDE5 inhibitor, youkenafil, and its metabolite M459.

PURPOSE: Youkenafil is a novel oral selective PDE5 inhibitor for treating Erectile Dysfunction. This investigation assessed pharmacokinetics (PK), safety, and tolerability of youkenafil and its main metabolite (M459) after taking 100 mg youkenafil hydrochloride tablets in elderly and young subjects. METHODS: This Phase I, single-center, open-label, parallel-group, single-dose study was conducted on 24 individuals (12 elders and 12 youngsters). Each subject received a single oral 100 mg youkenafil hydrochloride tablets. Blood samples were collected before medication and up to 48 h after medication for PK analysis. Safety and tolerability were also assessed, including treatment-emergent adverse events (TEAEs), laboratory tests, 12-lead ECG, vital sign inspections, color vision examinations, and physical examinations. RESULTS: Plasma concentrations of youkenafil and M459 were quantified. PK parameters were determined by non-compartmental analysis. Median Tmax of elderly and young groups were both 0.733 h. However, Cmax, AUC0-t, and AUC0-∞ of youkenafil were separately 16.8 %, 37.2 %, and 37.5 % higher in elders and t1/2 of youkenafil was 2.1 h longer in elders. More great differences were observed for M459. T1/2 values were 4.05 h longer in elders, with Cmax, AUC0-t and AUC0-∞ 73.7 %, 81.1 %, and 81.4 % higher in elders. Two (8.3 %) elderly subjects reported TEAEs (all grade Ⅰ in severity) and both recovered without any treatment. No serious adverse reactions (SAEs) or serious unexpected suspected adverse reactions (SUSARs) occurred in this study. CONCLUSIONS: This was the first PK research of youkenafil and M459 in elderly men. PK parameters differences between youkenafil and M459 were comparable between elderly and young groups. Moreover, safety and tolerability of youkenafil were favorable in both groups.

Adenosine and L-proline can possibly hinder Chinese Sacbrood virus infection in honey bees via immune modulation.

Sacbrood virus (SBV) infects larvae of honey bees, resulting in infected larvae becoming fluid-filled sacs. Our previous studies showed that the extract of herbal medicine, Radix Isatidis, could inhibit Chinese SBV (CSBV) infection in Asian honey bees (Apis cerana). Here, two compounds, adenosine and L-proline, which were previously reported to be associated with immune modulation, were identified in R. Isatidis extract and then selected for an evaluation of their antiviral effect on CSBV infection in A. cerana. Our results revealed that both adenosine and L-proline could significantly mitigate the impact of CSBV on the growth and development of infected larvae and modulate hosts' immune responses by downregulating the expression of immune genes in infected larvae. The results gained from this study suggest that adenosine and L-proline could possibly interfere CSBV infection via immune modulation to avoid exacerbations and nonspecific damage to infected larvae's own tissues.