A validated LC-MS/MS method for determination of six Anti-SARS-CoV-2 drugs in plasma and its application for a pharmacokinetic study in rats.

Antiviral treatment for COVID-19 is considered an effective tool in reducing the rate of severe cases and deaths. As of June 2023, a total of six small molecule antiviral drugs have been conditionally approved for marketing by the National Medical Products Administration (NMPA) within China. In this study, a method of HPLC-MS/MS was established and validated for the determination of six small molecule antiviral drugs in plasma using Lamivudine as an internal standard. The chromatographic separation was performed using gradient elution with an ACE 3 C18-PFP column (3.0 mm × 150 mm, 3 µm), and the mobile phase consisted of deionized water and acetonitrile/water (90:10, v/v), both with 10 mmol/L of ammonium acetate and 0.1 % ammonium hydroxide added. Quantitative analysis of the six small molecule drugs was carried out through selective reaction monitoring based on the positive ion spray ionization mode. The method exhibited excellent precision, accuracy, recovery, and linearity, and it was used to determine the pharmacokinetic characteristics in rats. Our work not only established a bioanalytical method for six small molecule antiviral drugs but also provided scientific references for clinical pharmacokinetic studies.

Nicotine withdrawal induces hyperalgesia via downregulation of descending serotonergic pathway in the nucleus raphe magnus.

Patients deprived of cigarettes exhibit increased pain sensitivity during perioperative periods, yet the underlying neuroanatomical and molecular bases of this hypersensitivity are unclear. The present study showed that both the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were significantly decreased in a rat model of nicotine withdrawal. These rats showed less tryptophan hydroxylase 2 (TPH2) positive neurons and reduced TPH2 expression in the nucleus raphe magnus (NRM), and thus resulted in decreased 5-hydroxytryptamine (5-HT) levels in cerebrospinal fluid. Intrathecal injection of 5-HT or NRM microinjection of TPH-overexpression adeno-associated virus alleviated nicotine withdrawal-induced hyperalgesia, whereas 5-HT receptor pharmacological blockade by methysergide (a 5-HT receptor antagonist) exacerbated hypersensitivity and diminished the difference between the two groups. Together, these data indicate that hyperalgesia after nicotine withdrawal is mediated by declined descending serotonergic pathways in the NRM. This provides a new perspective to improve the postoperative pain management of patients, especially the smokers.

Pharmacokinetics and tissue distribution of Ebracteolatain A, a potential anti-cancer compound, as determined by an optimized ultra-performance liquid chromatography tandem mass spectrometry method.

Ebracteolatain A is a phloroglucinol derivative from the root of Euphorbia ebracteolata Hayata, a Traditional Chinese Medicine also known as Langdu. It has been shown to have good inhibitory effects in breast cancer cells. In this study, a simple, rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to study the pharmacokinetics (PKs) and tissue distribution of Ebracteolatain A in rats. Ebracteolatain A and Magnolol (internal standard) were extracted by the simple protein precipitation extraction technique using methanol as the precipitating solvent. Chromatographic separation was performed using the Agilent Poroshell 120 EC-C18 column with a mobile phase of acetonitrile:0.1% formic acid (70:30, v/v). The protonated analyte was quantitated in negative ionization by MS/MS via multiple reaction monitoring mode. The assay exhibited a linear dynamic range of 2-2000 ng/mL for Ebracteolatain A in biological samples. The lower limit of quantitation was 2 ng/mL. Non-compartmental PK parameters indicated that Ebracteolatain A was well absorbed into the systemic circulation. The absolute bioavailability of Ebracteolatain A was greater when administered by intraperitoneal administration than by oral administration. The tissue distribution study showed that Ebracteolatain A was distributed in the heart, liver, spleen, lung, kidney, brain, stomach, intestine, uterus, ovary, and breast after intravenous injection. The results of this study further our understanding of the in vivo anti-cancer activity of Ebracteolatain A, and shed light on pharmacological strategies that may be useful for the development of novel breast cancer therapeutics.

Influence of In-Gap States on the Formation of Two-Dimensional Election Gas at ABO3/SrTiO3 Interfaces.

We explored in-gap states (IGSs) in perovskite oxide heterojunction films. We report that IGSs in these films play a crucial role in determining the formation and properties of interfacial two-dimensional electron gas (2DEG). We report that electron trapping by IGSs opposes charge transfer from the film to the interface. The IGS in films yielded insulating interfaces with polar discontinuity and explained low interface carrier density of conducting interfaces. An ion trapping model was proposed to explain the physics of the IGSs and some experimental findings, such as the unexpected formation of 2DEG at the initially insulating LaCrO3/SrTiO3 interface and the influence of substitution layers on 2DEG.

A UPLC-MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study.

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid-liquid extraction with ethyl acetate. The UPLC-MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra- and inter-day precision were both <10.2%, and the accuracy ranged from -11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.

[Expression patterns of PPARgamma2 in differentiation of mouse embryonic stem (ES) cells into adipocytes].

To investigate the expression patterns of peroxisome proliferator activated receptor2 (PPARgamma2) gene in the differentiation of mouse embryonic stem (ES) cells into adipocytes, mouse ES cells were transfected with the vector of pPPARgamma2-promoter-luciferase, and PPARgamma2 expressions were analyzed by detecting luciferase activities and by detecting the protein expressions using western blotting. The results showed that the gene PPARgamma2 did not express in undifferentiated mouse ES cells and in embryoid bodies (EBs) within the first 2 days of differentiation induction after EB formation, and began to express from the third day of differentiation induction after EB formation to the finish of the differentiation. The gene's expression in differentiated adipocytes was much stronger than that in differentiating preadipocytes. In Conclusion our results reported for the first time the five-step expression patterns of the gene PPARgamma2 during the whole differentiation procedures from mouse ES cells into adipocytes via preadipocytes, and supported the previous studies that PPARgamma2 is a fat-specific gene that expresses only in developed and developing adipose tissues.

Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA.

Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.

The labeling of 3T3-L1 preadipocyte cells with enhanced green fluorescent protein.

A cell model is desired for adipocyte differentiation investigation and for high-throughput screening of anti-obesity and anti-diabetes molecules from chemical resources due to the world wide epidemic of obesity and diabetes. In order to establish such a cell model, a plasmid of pPPARgamma2-promoter-EGFP was constructed by inserting a 660bp sequence of mouse PPARgamma2 promoter into the Ase I and Kpn I sites of pEGFP-N3 and transferred into 3T3-L1 preadipocyte cells. The cells were induced to differentiate and the expression of PPARgamma2 was detected by the microscopic observation of EGFP and by RT-PCR assays. The results showed that the EGFP gene expression patterns were similar to that of pPPARgamma2's, which indicated that the EGFP gene was transferred into the mouse 3T3-L1 preadipocyte cells, and its expression was under the control of pPPARgamma2 promoter. RT-PCR assays showed that the EGFP expression authentically represented the stable expression of PPARgamma2. In conclusion, a preadipocyte cell line expressing EGFP under the control of the promoter of adipocyte-specific expression gene PPARgamma2 was generated. The cell line provides a powerful approach for the research of adipocyte differentiation and for the high-throughput screening of anti-obesity and anti-diabetes chemicals.

[RNA interference directed by small hairpin RNA expressed in COS-7 cells].

RNA interference is a phenomenon of gene silencing directed by double-stranded RNA. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans, Drosophila melanogaster, etc. For mammalian cells, dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells. Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells. Yet in other kinds of mammalian cells, because of the existence of interferon pathway, long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis. Therefore, dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells. Elbashir et al found that in vitro synthesized small interfering RNA (siRNA) (19-23 nt) could induce potent RNAi as effective as long dsRNA without showing unspecific effect, so that the interferon pathway could be bypassed. It was shown that during RNAi process, long dsRNA was first degraded into 19-23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week. Recently, it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell. The RNAi effect of U6 promoter-driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS-7 cells was checked. And it was found that the U6 promoter-driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS-7 cells. The result established the feasibility of using RNAi technique directed by U6 promoter-driven shRNA to study genes' function in COS-7 cell line.