Development and validation of an LC-MS/MS method for the simultaneous quantification of milbemycin oxime and praziquantel in plasma: application to a pharmacokinetic study in cats.

Introduction: Milbemycin oxime (MBO) and praziquantel (PZQ) have a broad spectrum of biological activity and are commonly used to treat the parasitic infection in the veterinary clinic. In this study, a fast and efficient LC-MS/MS method was established and validated for the simultaneous determination of MBO, PZQ, cis-4-hydroxylated-PZQ (C-4-OH-PZQ) and trans-4-hydroxylated-PZQ (T-4-OH-PZQ) and in cat plasma. Methods: Extraction of analytes and internal standards from cat plasma by acetonitrile protein precipitation, allows rapid processing of large batches of samples. MBO, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, and internal standard (IS) were eluted for 13.5 min on a C18 column with a 0.1% formic acid water/acetonitrile mixture as the mobile phase. Results: Results showed that the method had good precision, accuracy, recovery, and linearity. The linearity range was 2.5-250 ng/mL for MBO, and 10-1000 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ. The intra-day and inter-day precision CV values of the tested components were within 15%. The extraction recoveries of the four components ranged from 98.09% to 107.46%. The analytes in plasma remained stable for 6 h at room temperature, 26 h in the autosampler (4 °C), after freeze-thaw (-20°C) cycles, and 60 days in a -20°C freezer. Method sensitivity sufficed for assessing pharmacokinetic parameters of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in plasma samples with LLOQ of 2.5 ng/mL for MBO and 10 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ. Conclusion: In this study, a selective and sensitive LC-MS/MS method for the simultaneous quantification of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in cat plasma was developed and validated.This method had been successfully applied to evaluate the pharmacokinetics of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ after a single oral administration of 8 mg MBO and 20 mg PZQ in cats.

Enhancement of the oral bioavailability of isopropoxy benzene guanidine though complexation with hydroxypropyl-ß-cyclodextrin.

Isopropoxy benzene guanidine (IBG) is a novel substituted benzene guanidine analogue with antibacterial activity against multidrug-resistant bacteria. However, the bioavailability of IBG is not optimal due to its finite aqueous solubility, thus hampering its potential therapeutic exploitation. In this study, we prepared IBG/hydroxypropyl-ß-CD (IBG/HP-ß-CD) complex, and characterized it by differential scanning calorimetry, Fourier transform infrared spectroscopy, powder X-ray diffraction, and scanning electron microscopy. Physicochemical characterization indicated that the crystal morphology of IBG transformed into an amorphous state, thus forming IBG/HP-ß-CD inclusion complexes. Complexation with HP-ß-CD significantly improve the aqueous solubility, pharmaceutical properties, absorption, and bioavailability of IBG.

Research Progress of Totipotent Stem Cells.

Totipotent stem cells (TSCs), can develop into complete organisms, are used in biological fields such as regenerative medicine, mammalian breeding, and conservation. However, it is difficult to maintain the developmental totipotency and self-renewal capacity of cells cultured from early-stage embryos, which becomes a key factor limiting the research of TSCs. Fortunately, a breakthrough in the study of induced pluripotent stem cells returning to their totipotent state has been made, resulting in the establishment of multiple TSCs and igniting a new wave of stem cell research. Furthermore, the blastocyst-like structures can be generated by the established TSCs, which lays a foundation for synthetic embryos in vitro. In this review, we summarize the totipotent stage of early embryos, the establishment and cultivation of TSCs, and the developmental ability exploration of TSCs to promote further research of TSCs.

First total syntheses of kanjone and its natural analogues.

Kanjone (1), a bioactive furanoflavone and a potent biomolecule, was first isolated from Pongamia pinnata (L.). Herein, we have developed two approaches to synthesize kanjone as well as its natural analogues 6-methoxyisopongaglabol (2) and 6,3'-dimethoxy-[2″,3″:7,8]furanoflavone (3) starting from khellin and 3-hydroxy-4-methoxy-benzaldehyde, respectively.

IL-21-mediated expansion of Vγ9Vδ2 T cells is limited by the Tim-3 pathway.

Vγ9Vδ2 T cells are the main γδ T subset in the peripheral blood and lymphoid organs. Previous studies have shown that Vγ9Vδ2 T cells could expand in the presence of phosphoantigens and IL-2 and exert antitumor functions. However, their potency was limited because sustained proliferation could not be achieved, possibly due to exhaustion caused by prolonged antigenic stimulation. In this study, we examined the proliferative response of Vγ9Vδ2 T cells to IL-21, a cytokine previously shown to promote NK cell and CD8 T cell cytotoxicity. We found that IL-21 could significantly improve the proliferation of phosphoantigen-stimulated Vγ9Vδ2 T cells in a dose-dependent manner. However, in acute myeloid leukemia (AML) patients, the efficacy of IL-21 was significantly reduced. Vγ9Vδ2 T cells from AML patients exhibited lower expression of IL-21R, and required higher levels of IL-21 for expansion. IL-21-treated Vγ9Vδ2 T cells from AML patients presented lower increase in STAT1 phosphorylation than Vγ9Vδ2 T cells from healthy volunteers. Interestingly, AML Vγ9Vδ2 T cells presented significantly higher Tim-3 expression than healthy Vγ9Vδ2 T cells. IL-21 treatment further induced Tim-3 upregulation. Blocking Tim-3 increased the proliferation and the STAT phosphorylation in Vγ9Vδ2 T cells in response to IL-21. Together, these results demonstrated that IL-21 could significantly expand the Vγ9Vδ2 T cells, but its efficacy was limited since it also increased the expression of checkpoint molecule Tim-3.

Impairment of Vα24-Jα18+Vß11+ natural killer T cells in adult acute lymphoblastic leukemia patients.

Type I natural killer T (NKT) cells are attractive candidates for cancer immunotherapy. In this study, we examined the characteristics of type I NKT cells in patients with adult B-cell acute lymphoblastic leukemia (ALL). We first identified type I NKT cells as Vα24-Jα18 and Vß11 double-positive CD3+ lymphocytes. Using this method, we found that the adult B-cell ALL patients presented significantly lower level of type I NKT cells than the age- and sex-matching control subjects. The expression of IL-21 by type I NKT cells was then examined using intracellular flow cytometry, which showed that with α-GalCer stimulation, the adult B-cell ALL patients presented significantly lower level of IL-21+ type I NKT cells than control subjects. By both flow cytometry and ELISA, we found that the vast majority of IL-21-expressing type I NKT cells expressed IL-21R, which was also reduced in adult B-cell ALL patients. Using an in vitro co-culture system, we demonstrated that IL-21R+, but not IL-21R-, type I NKT cells could promote the IFN-γ, granzyme B, and perforin expression by CD8 T cells in an IL-21-dependent fashion. This type I NKT cell-mediated stimulatory effect was reduced in adult B-cell ALL patients than in control subjects. In addition, we observed a positive correlation between the frequency of IL-21R+ type I NKT cells and the frequencies of IFN-γ-, granzyme B-, and perforin-expressing circulating CD8 T cells in adult B-cell ALL patients directly ex vivo. Overall, this study identified an IL-21-related impairment in type I NKT cells from adult B-cell ALL patients.