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Objective: To study the bi-direction transport behavior of brucine and strychnine in the MDCK-MDR1 cell monolayer model. Methods: MTT method was employed to confirm the safe concentration of brucine and strychnine towards MDCK-MDR1 cells. The effects of transport time, drug concentration, and P-glycoprotein inhibitor verapamil on cumulative absorption concentration (Ccum) and apparent permeability coefficient (Papp) of brucine and strychnine in MDCK-MDR1 monolayer cells were studied. Results: The Papp value of brucine and strychnine was larger than 1 × 10-5 cm/s and the ratio of Papp(BL→AP) vs Papp(AP→BL) was less than 2. Brucine/ strychnine combined with verapamil decreased the ratio of Papp(BL→AP) vs Papp(AP→BL). Conclusion: The absorption of brucine and strychnine in MDCK-MDR1 cell monolayer model was well and the passive transference was its main intestinal absorption mechanism. The P-gp inhibitor verapamil has a significant inhibitory effect on brucine and strychnine absorption. Brucine and strychnine may be a substrate of P-glycoprotein.
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Objective To explore the effect of microemulsion on the transport and mechanism of puerarin in blood brain barrier (BBB) cell model MDCK-MDR1. Methods MTT assay was used to evaluate the cytotoxicity of puerarin microemulsion and solution, and determine the appropriate concentration of administration. The bilateral transport characteristics of puerarin solution-microemulsion was investigated in MDCK-MDR1 monolayer. Immunohistochemical staining was used to study the expression of tight junction proteins, and the changes in cell membrane fluidity was studied by fluorescence bleaching recovery, and the changes of membrane potential was measured by anion probe combined with flow cytometry. The mechanism of the effect of microemulsion on puerarin transport was clarified. Results The MDCK-MDR1 showed no significant toxicity when the mass concentration of puerarin solution ranged from 50 to 300 μg/mL and the microemulsion dilution was over 500 times. The Papp value in absorption direction of puerarin solution on MDCK-MDR1 monolayer was 1.04 × 10-6 cm/s, and the Papp value of excretion direction was 1.05 × 10-6 cm/s. The Papp value of puerarin in microemulsion was significantly increased compared with that in solution (P < 0.05). Microemulsification could reduce the expression of Claudin-1, Occludin, ZO-1, and F-actin in MDCK-MDR1, promote cell membrane flow, and decrease cell membrane potential. Conclusion Microemulsion can promote the bilateral transport of puerarin in the BBB cell model MDCK-MDR1. The mechanism is closely connected with the opening of tight junctions, increasing the cell membrane fluidity, making the cell depolarizing and reducing membrane potential, and increasing the permeation of paracellular.
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OBJECTIVE: To determine the effects of the amphiphilic block polymers, which have the same hydrophilic block with the different hydrophobic block, on the function of P-glycoprotein(P-gp). METHODS: The three different micelles were prepared by film dispersion method. The particle sizes and distributions were measured by dynamic light scattering. Critical micelle concentrations(CMC) were detected by fluorescence probe technique with the pyrene. Rhodamine 123, a specific probe substrate of P-gp, was applied to determine the effects of polymers on the function of P-gp using uptake and efflux method. RESULTS: The particle sizes of mPEG-PCL, mPEG-PDLLA, mPEG-PLGA were (55.9±0.2), (53.7±1.1) and (61.6±0.6)nm. The CMC values were 2.08, 5.42 and 26.4 μg·mL-1. R123 accumulation in Madin-Darby canine kidney/multidrug resistance 1(MDCK-MDR1)cell detected by uptake assay increased to a maximum in the presence of polymers at concentrations of 250 μg·mL-1 for mPEG-PCL, 1~25 μg·mL-1 for mPEG-PDLLA and mPEG-PLGA. In efflux assay, mPEG-PCL, mPEG-PDLLA, mPEG-PLGA decreased the percentage of efflux of R123 at concentrations above the CMC, below/at the CMC or below the CMC respectively, showed the similar RESULTS with uptake assay. CONCLUSION: The mPEG-PCL, mPEG-PDLLA, mPEG-PLGA polymers might have a potential to inhibit the efflux activity of P-gp, which was likely related to the structures of hydrophobic segments, concentrations and existing states of the polymers.
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Objectives: To investigate the effects of bergapten of Angelicae Dahuricae Radix on the transport of vincristine and its possible mechanism. Methods: The apparent permeability coefficient (Papp) for the transport of vincristine through the membrane of MDCK-MDR1 cells was used as an indicator of the effect of bergapten on vincristine transport. Molecular docking was employed to predict the binding force between bergapten and P-glycoprotein (P-gp). The effects of bergapten on P-gp function and P-gp ATPase activity were determined by rhodamine 123 (Rho123) accumulation and activity analysis, respectively. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was used to study the effects of bergapten on membrane fluidity, and Western blotting and quantitative real-time PCR assays were performed to analyze the effect of bergapten on the protein and mRNA expression of P-gp, respectively. These experiments clarified the effects of bergapten on the transport of vincristine and allowed exploration of the possible mechanism underlying the effects of bergapten. Results: The results showed that bergapten could inhibit the transport of vincristine in MDCK-MDR1 cells, and the binding force between bergapten and P-gp was weaker. Bergapten could reduce the accumulation of Rh123 in MDCK-MDR1 cells, increase the membrane fluidity, and upregulate P-gp protein and mRNA expression but it had no effect on P-gp ATPase activity. Conclusions: Overall, we concluded that the possible mechanism through which bergapten inhibits vincristine transport was related to the bergapten-mediated upregulation of P-gp protein and mRNA expression, membrane fluidity or P-gp enzyme activity.
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Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (Papp) and efflux ratio (RE); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-hOATP1B1, HEK293-hOATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1,
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Objective: To study the effects and mechanisms of p-glycoprotein (P-gp) inhibitor and MRP1 inhibitor on the transportation of gastrodin (GAS). Methods: Cell toxicity of GAS was detected by MTT assay, molecular Docking was employed to predicted binding mode and effect ability of GAS with P-gp and MRP1. MDCK-MDR1 cell model was employed to study the influences of Ver, a P-gp inhibitor, and Probenecid, a MRP1 inhibitor, on the transportation of GAS. Results: There was no cell cytotoxicity of GAS between the concentration of 100 to 1000 μg/mL. There was hydrogen-bond and hydrophobic interaction between P-gp and GAS, and hydrogen-bond, hydrophobic interaction and electrostatic-interaction between MRP1 and GAS. LibDock Score indicated that both P-gp-verapamil (Ver) and MRP1-Probenecid were more stable than P-gp-GAS and MRP1-GAS. The Papp of GAS BL→AP was greater than that of AP→BL, Efflux ratio (ER) of GAS was about 1.5, which indicated the efflux pump protein might involve the transportation of GAS. The Papp of GAS was significant increased but the ER of GAS was significant decreased when co-administrated with Ver (P<0.05). The Papp of GAS was slightly increased and the ER of GAS was slightly decreased when co-administrated with Probenecid, while there was no significance. Conclusion: The results indicate that GAS is the substrate of P-gp. However, whether GAS is the substrate of MRP1 needs further research. Both Ver and Probenecid could enhance the transportation of GAS by competitive binding with P-gp and MRP1.
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Objective: To study the mechanisms of absorption and transport of 3-acetyl-11-keto-β-boswellic acid (AKBA) from Boswellia carterif in Caco-2 cell, MDCK-MDR1, and MDCK-Wild cell models. Methods: The Caco-2, MDCK-MDR1, and MDCK-Wild cell monolayer models were used to study the bi-directional transport of AKBA in apical (AP)→basal (BL) or BL→AP; The concentration of AKBA was measured by LC-MS/MS and apparent permeability coefficient (Papp) was calculated. Results: Papp (AP→BL) and Papp (BL→AP) values of AKBA (50 μmol/L) in Caco-2 cell model were 7.9 × 10-7 and 1.5 × 10-7 cm/s, respectively; Papp (AP→BL) and Papp (BL→AP) values of AKBA (50 μmol/L) in MDCK-MDR1 cell model were 2.6 × 10-7 and 0.8 × 10-7 cm/s, respectively; Papp (AP→BL) and Papp (BL→AP) of AKBA (50 μmol/L) in MDCK-Wild cell model was 2.4 × 10-7 and 0.6 × 10-7 cm/s, respectively; The rates of efflux (RE) for AKBA in Caco-2 and MDCK-MDR1 cell monolayers were both smaller than 2. Conclusion: AKBA is not the substrate of P-gp and its absorption rate is low. AKBA is absorbed through the intestinal epithelial cells by active transport absorption and passive diffusion possibly.