Simultaneous measurement of COVID-19 treatment drugs (nirmatrelvir and ritonavir) in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

PAXLOVID™ (Co-packaging of Nirmatrelvir with Ritonavir) has been approved for the treatment of Coronavirus Disease 2019 (COVID-19). The goal of the experiment was to create an accurate and straightforward analytical method using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to simultaneously quantify nirmatrelvir and ritonavir in rat plasma, and to investigate the pharmacokinetic profiles of these drugs in rats. After protein precipitation using acetonitrile, nirmatrelvir, ritonavir, and the internal standard (IS) lopinavir were separated using ultra performance liquid chromatography (UPLC). This separation was achieved with a mobile phase composed of acetonitrile and an aqueous solution of 0.1% formic acid, using a reversed-phase column with a binary gradient elution. Using multiple reaction monitoring (MRM) technology, the analytes were detected in the positive electrospray ionization mode. Favorable linearity was observed in the calibration range of 2.0-10000 ng/mL for nirmatrelvir and 1.0-5000 ng/mL for ritonavir, respectively, within plasma samples. The lower limits of quantification (LLOQ) attained were 2.0 ng/mL for nirmatrelvir and 1.0 ng/mL for ritonavir, respectively. Both drugs demonstrated inter-day and intra-day precision below 15%, with accuracies ranging from -7.6% to 13.2%. Analytes were extracted with recoveries higher than 90.7% and without significant matrix effects. Likewise, the stability was found to meet the requirements of the analytical method under different conditions. This UPLC-MS/MS method, characterized by enabling accurate and precise quantification of nirmatrelvir and ritonavir in plasma, was effectively utilized for in vivo pharmacokinetic studies in rats.

Pharmacokinetics of Anti-rheumatic Drugs Methotrexate and Tofacitinib with its Metabolite M9 in Rats by UPLC-MS/MS.

BACKGROUND: Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. METHODS: Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. RESULTS: The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. CONCLUSION: The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

Liposomes co-loaded with ursolic acid and ginsenoside Rg3 in the treatment of hepatocellular carcinoma.

OBJECTIVE: Liposomes co-loaded with ursolic acid and ginsenoside Rg3 (UA+Rg3-LIP) were prepared to study their effects on the proliferation, apoptosis and cell cycle of hepatocellular carcinoma (HCC) cells. METHODS: Liposomes were prepared by reverse evaporation, and then UA+Rg3-LIP were prepared by the pH gradient method, and followed by liposome characterization. Next, the effects of UA+Rg3-LIP on the proliferation, apoptosis and cell cycle of HepG2 cells were investigated by MTT method and flow cytometry at the cell level. RESULTS: The entrapment efficiency of UA in UA+Rg3-LIP was 78.52% and that of Rg3 was 71.68%, as assayed by low-temperature ultracentrifugation. The in vitro release rates of UA+Rg3-LIP and UA+Rg3 detected by the dialysis membrane method were 1-10 h. The release rate of UA+Rg3 was close to 100%; that of UA+Rg3-LIP was decreased after 10 h and approached 100% after 24 h. It was further confirmed by cell experiments that UA+Rg3-LIP could significantly reduce cells viability while at the same time increase their apoptosis rate and raise the proportion of cells in the G0/G1 phase. CONCLUSION: Liposomes co-loaded with ursolic acid and ginsenoside Rg3 could affect cell proliferation, apoptosis and cell cycle, thus slowing down the in vitro drug release ability of HCC.

Establishment and Verification of UPLC-MS/MS Technique for Pharmacokinetic Drug-Drug Interactions of Selinexor with Posaconazole in Rats.

BACKGROUND: A method for the determination of selinexor by UPLC-MS/MS was established to study the effect of posaconazole on the pharmacokinetics of selinexor in rats. METHODS: The experiment rats were divided into group A (0.5% CMC-Na) and group B (posaconazole, 20 mg/kg), 6 rats in each group. 30 minutes after administration of 0.5% CMC-Na or posaconazole, all the rats were given selinexor (8 mg/kg), and plasma samples were collected. The plasma samples underwent acetonitrile protein precipitation, and were separated by UPLC on an Acquity UPLC BEH C18 column with gradient elution. Acetonitrile and 0.1% formic acid were used as the mobile phases. The analyte detection was used a Xevo TQ-S triple quadrupole tandem mass spectrometer and multiple reaction monitoring (MRM) for analyte monitoring. We use acetonitrile for protein precipitation. RESULTS: Selinexor had good linearity (1.0-1000 ng/mL, r2 =0.996 2), and the accuracy and precision, recovery rate and matrix effects(ME) were also met the FDA approval guidelines. Compared with group A, the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group B increased by 60.33%, 48.28% and 48.27%, and Tmax increased by 53.92%, CLz/F reduced by 32.08%. CONCLUSION: This bioanalysis method had been applied to the study of drug interactions in rats. It was found that posaconazole significantly increased the concentration of selinexor in rats. Therefore, when selinexor and posaconazole are combined, we should pay attention to the possible drug-drug interactions to reduce adverse reactions.

PURB is a positive regulator of amino acid-induced milk synthesis in bovine mammary epithelial cells.

Previous studies have implicated that purine-rich element binding protein B (PURB) is a key regulator of gene transcription and cell physiology. Whether PURB plays a regulatory role in milk synthesis in bovine mammary epithelial cells (BMECs) is not known. We observed that Met and Leu increased PURB expression and nuclear localization. Overexpression of PURB led to increased milk protein and fat synthesis as well as mTOR and SREBP-1c expression whereas PURB knockdown had the opposite effects. The PI3K inhibitor wortmannin totally abolished the stimulation of Met and Leu on PURB expression, and we further confirmed that PURB was required for Met and Leu to stimulate mTOR phosphorylation and SREBP-1c expression. We also demonstrated that PURB binds to the promoters of mTOR and SREBP-1c, and these bindings were increased by Met and Leu stimulation. In summary, our data reveal that PURB is required for amino acids to stimulate mTOR and SREBP-1c gene expression, and PURB is a positive regulator of amino acid-induced PI3K-regulated milk protein and fat synthesis in BMECs.

The role of CYP2C9 genetic polymorphism in carvedilol O-desmethylation in vitro.

We aimed at investigating the role of CYP2C9 in carvedilol O-desmethylation and identifying the effect of 35 CYP2C9 allelic variants we found in Chinese Han population on the in vitro metabolism of carvedilol. Recombinant CYP2C9 and CYP2D6 microsomes of the wild type were used to test and verify the enzymes involved in carvedilol O-desmethylation. Recombinant CYP2C9 microsomes of distinguished genotypes were used to characterize the corresponding enzyme activity toward carvedilol. 2-100 µM carvedilol was incubated for 30 min at 37 °C. The products were detected using high-performance liquid chromatography. CYP2C9 plays a certain role in carvedilol metabolism. Compared with wild-type CYP2C9*1, the intrinsic clearance (V max/K m) values of all variants toward carvedilol O-desmethylation were significantly altered. The variants exhibited significantly decreased values (from 30 to 99.8 %) due to increased K m and/or decreased V max values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings complement the database where CYP2C9 polymorphism interacts with biotransformation of exogenous substances like drugs and toxins.

Evaluation of the impact of cantharidin on rat CYP enzymes by using a cocktail of probe drugs.

The aim of this study was to assess the influence of cantharidin on the activities of the drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in rats. The activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were measured using specific probe drugs. After pretreatment for 1week with cantharidin or physiological saline (control group) by intraperitoneal injection, probe drugs phenacetin (5.0mg/kg; CYP1A2 activity), tolbutamide (1.0mg/kg; CYP2C9 activity), omeprazole (10mg/kg; CYP2C19 activity), metoprolol (20mg/kg; CYP2D6 activity) and midazolam (10mg/kg; CYP3A4 activity) were administered to rats by oral administration. The blood was then collected at different times for ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. The data showed that cantharidin exhibits an inhibitory effect on CYP2D6 and CYP3A4 by increasing t1/2, Cmax and AUC(0-∞), and decreasing CL/F compared with those of the control group. In addition, cantharidin has induction effect on CYP2C9 activity. However, no significant changes in CYP1A2 and CYP2C19 activities were observed. In conclusion, the results indicated that cantharidin could inhibit CYP2D6 and CYP3A4, while induce CYP2C9, which may affect the disposition of medicines primarily dependent on these pathways. Our work may be the basis of related herb-drug interactions in the clinic.