Entecavir (ETV) is a superior nucleoside analogue used to treat hepatitis B virus (HBV) infection. Although its advantages over other agents include low viral resistance and the elicitation of a sharp decrease in HBV DNA, adverse effects such as hepatic steatosis, hepatic damage and lactic acidosis have also been reported. Glycyrrhizin has long been used as hepato-protective medicine. The clinical combination of ETV plus glycyrrhizin in China displays better therapeutic effects and lower rates of liver damage. However, there is little evidence explaining the probable synergistic mechanism that exists between these two drugs from a pharmacokinetics view. Here, alterations in the plasma pharmacokinetics, tissue distribution, subcellular distribution, and in vitro and in vivo antiviral activity of ETV after combination with glycyrrhizic acid (GL) were analysed to determine the synergistic mechanisms of these two drugs. Specific efflux transporter membrane vesicles were also used to elucidate their interactions. The primary active GL metabolite, glycyrrhetic acid (GA), did not affect the plasma pharmacokinetics of ETV but promoted its accumulation in hepatocytes, increasing its distribution in the cytoplasm and nucleus and augmenting the antiviral efficiency of ETV. These synergistic actions were primarily due to the inhibitory effect of GA on MRP4 and BCRP, which transport ETV out of hepatocytes. In conclusion, GA interacted with ETV at cellular and subcellular levels in the liver through MRP4 and BCRP inhibition, which enhanced the antiviral activity of ETV. Our results partially explain the synergistic mechanism of ETV and GL from a pharmacokinetics view, providing more data to support the use of these compounds together in clinical HBV treatment.Download high-res image (320KB)Download full-size image

•A LC–MS/MS method was established for simultaneous determination of tenofovir prodrug tenofovir alafenamide (TAF) and its metabolites tenofovir (TFV) and TFV diphosphate (TFV-DP).•Samples were collected by direct lyzing with 50% methanol, followed by further protein precipitation using pure methanol.•Intracellular pharmacokinetics of TFV-DP, TFV and TAF were investigated in HepG2.2.15 cells. Tenofovir was generated quickly along with rapid elimination of TAF, and immediately further phosphorylated into active form TFV-DP largely.Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.Download high-res image (151KB)Download full-size image

•A sensitive LC–MS/MS method without derivatization/ion-pairing agents was established and validated for etimicin (ETM) quantification.•Drug extraction and purification were achieved by adding trichloroacetic acid, followed by using an SPE clean-up procedure.•Pharmacokinetics of ETM in its potential toxicity targeting organs kidney and internal ear, as well as in plasma were monitored for the first time.•ETM showed a considerable long half-life in kidney and internal ear, and exhibited a stronger affinity with kidney than internal ear.Etimicin (ETM), which belongs to the newest generation of aminoglycosides (AGs), has been proven to not only maintain but also strengthen the advantages of former AGs with relatively less toxicity. Now, it is widely applied for the treatment of bacterial infections in the clinic. Nevertheless, nephrotoxicity and ototoxicity are unavoidable issues for AGs, and while ETM is no exception, the seriousness of these issues is different. To explore the reason why ETM exhibits less toxicity and to better direct the optimization and development of new AGs, it is of great necessity and importance to monitor the pharmacokinetic behaviors of ETM in its potential toxicity target organs, the kidney and internal ear, as well as in plasma. Therefore, a novel, sensitive and efficient LC–MS/MS method without derivatization or ion-pairing agents had been developed and validated for quantification of ETM in rat plasma, kidney and internal ear for the first time. This method showed good linearity over the range of 50–2000 ng/mL for rat plasma/internal ear and 100–5000 ng/mL for rat kidney. The precision was less than 4.4% and the accuracy was below 4.8%. Recovery and matrix effects were 71.3%–82.8% and 97.6%–108.5%, respectively. After intravenous administration of a single dose of ETM, plasma drug concentrations fit well with a two-compartmental model, and the AUC0-∞, t1/2α, t1/2β, MRT and CL were 127.96 ± 5.52 μg *h/mL, 0.53 ± 0.03 h, 3.32 ± 1.11 h, 1.01 ± 0.03 h and 234.80 ± 10.05 mL/h/kg, respectively. Particularly, ETM showed a considerably long half-life in kidney and internal ear, up to 155.96 ± 19.95 h and 83.11 ± 26.60 h, respectively, which might contribute greatly to its toxicity.

•LC–MS/MS method for simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 was developed.•Immediate protein precipitation was used instead of DM1 derivatization for sample preparation.•The method was validated and applied to intracellular catabolism investigations of T-DM1.Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100 nM showed good linearity (r2 > 0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1.