Studies On Intestinal Absorption Mechanisms Of 2'-deoxy-2'-?-fluoro-4'-azidocytidine And Its Modulation Effects On Drug Efflux Transporters

Acquiredimmunesyndrome?AIDS?,whichiscausedbyhuman immunodeficiency virus?HIV?infection,is one of the most devastating epidemics.At present,the anti-HIV drugs are mainly divided into 5 classes based on their mechanisms,which are nucleoside/nucleotide reverse transcriptase inhibitors?NRTIs?,non-nucleoside reverse transcriptase inhibitors?NNRTIs?,integrase inhibitors,protease inhibitors and entry inhibitors.2'-Deoxy-2'-?-fluoro-4'-azidocytidine?FNC?,a novel pyrimidine analog,has been developed for AIDS treatment.Previous studies have indicated that FNC exerts strong antiretroviral activity both in vitro and in vivo.FNC can be rapidly absorbed in rats,and the oral bioavailability is high.It has been approved by the CFDA to enter phase?clinical trial.FNC is administered by oral.Therefore,the small intestine is the main site of FNC absorption.There are many drug transporters expressed in the intestinal,which are essential for the absorption of oral medications.However,antiretroviral therapy itself can regulate the expression of several drug transporters involved in drug disposition,thereby leading changes in the pharmacokinetic properties and effectiveness of antiretroviral therapy.Therefore,it is important to clarify the intestinal absorption characteristics of FNC and the underlying mechanisms.The aim of present study is to investigate the mechanism of FNC absorption and transport in the small intestine and the modulation effects of FNC on the drug transporters.The main contents are as follows:1.Everted gut sacs were applied to investigate the absorption of FNC in the small intestine mucosa.Three concentrations of FNC were used in the experiment.The concentration of FNC in the intestinal solution was measured by LC-MS/MS.The results showed that the absorption of FNC was in a concentration-dependent and saturated manner.The cumulative absorption of FNC increased first with the increase of FNC concentration and then was stable.FNC is mainly absorbed via a carrier-mediated process.In addition,there was no significant difference in the absorption of FNC in different segments of the small intestine?p>0.05?.2.Caco-2 cells monolayer model was established and validated.The integrity of Caco-2 cells monolayer was verified by measuring transepithelium electrical resistance,apparent permeability coefficient?Papp?of fluorescein sodium and alkaline phosphatase activity.The results showed that the Caco-2 cell model was in accordance with the requirements of the drug transport experiment and could be used to investigate the intestinal absorption mechanism of the drug.3.Caco-2 cells was used to investigate the uptake mechanisms of FNC.The results showed that:1)The uptake of FNC increased rapidly in the first 1 h and then stabilized.2)FNC uptake was concentration-dependent and saturable.3)At 4?,the uptake of FNC decreased significantly?p<0.05?.4)The uptake of FNC was significantly affected by the pH of the medium and weak acid pH could enhance the absorption of FNC.5)Metabolic inhibitor NaN3,P-gp inhibitor verapamil,MRP2inhibitor probenecid and MK571,and BCRP inhibitor GF120918 significantly increased the uptake of FNC?p<0.05?.It is speculated that the uptake of FNC is mediated by active transport.FNC might be the substrate of P-gp,MRP2 and BCRP.4.Caco-2 cells model was used to investigate the transport mechanisms of FNC.The results showed that:1)The bi-directional transport of FNC was time-dependent.The amount of FNC transported from the basal side?BL?to the apical side?AP?was greater than that of from AP side to BL side.2)Both the absorption and secretion of FNC were concentration-dependent and saturable.3)In the presence of verapamil,the amount of FNC transported from AP side to BL side did not change significantly,but the amount of FNC transported from BL side to AP side was significantly decreased.In the presence of probenecid,the amount of FNC transported in the direction from AP to BL increased significantly and the amount of FNC transported from BL to AP was significantly decreased.In the presence of MK571,the amount of FNC transported from BL side to AP side decreased,while the amount of FNC transported from AP side to BL side did not change significantly.The effect of GF120918 on the absorption and secretion of FNC was similar to that of probenecid,but to a lesser extent.All of verapamil,probenecid,MK571 and GF120918 can reduce Papp_BL-AP/Papp_AP-BL.The transport experiment further indicated that the absorption and secretion of FNC were mediated by the active transport mechanism.FNC is the substrate of P-gp,MRP2 and BCRP.5.The in situ single pass perfused rat intestinal model was established to investigate the absorption characteristics and mechanisms of FNC in the small intestine.Three concentrations of FNC were used in the experiment.The concentration of FNC in the perfusate was measured by LC-MS/MS.The results showed that FNC was well absorbed in the intestine of rats and the absorption has no significant difference in different segments of the small intestine?p>0.05?.FNC is transported in the carrier-mediated manner and the transport protein can be saturated.The absorption rate constant Ka and apparent permeability coefficient Peff were significantly increased after adding verapamil,probenecid and GF120918?p<0.05?,indicating that FNC was the substrate of P-gp,MRP2 and BCRP.The results of in vivo experiments were consistent with those of in vitro experiments.6.The effect of FNC on the expression of MDR1,MRP2 and BCRP mRNA in Caco-2 cells was investigated by real-time fluorescence quantitative PCR?RT-PCR?.The results showed that:1)The expression of MDR1,MRP2 and BCRP mRNA increased with the increase of FNC concentration from 10?M to 100?M.2)The expression of MDR1,MRP2 and BCRP mRNA increased with the incubation time of FNC from 24h to 72 h.The results indicated that FNC regulated the expression of MDR1,MRP2 and BCRP mRNA in a concentration and time-dependent manner.7.The effect of FNC on the expression of MDR1a,MDR1b,MRP2 and BCRP mRNA in rat small intestine was investigated by RT-PCR.The results showed that:1)FNC did not significantly alter the expression of MDR1a,MDR1b,MRP2 and BCRP mRNA in rat duodenum?p>0.05?.2)In rat jejunum,MDR1a,MDR1b and MRP2mRNA expressions were significantly up-regulated,but BCRP mRNA expression did not change significantly.3)In rat ileum,MDR1a,MRP2 and BCRP mRNA expressions were significantly up-regulated,but MDR1b mRNA expression did not change significantly.The results suggested that FNC could regulate the expression of MDR1a,MDR1b,MRP2 and BCRP mRNA in the small intestine of rats,but the regulation was different based on the intestinal segments.8.The effect of FNC on the expression of P-gp,mrp2 and bcrp in Caco-2 cells was investigated by Western blot.The results showed that:1)The expression of P-gp,mrp2 and bcrp increased with the increase of FNC concentration from 10?M to 100?M.2)The expression of P-gp,mrp2 and bcrp increased with the incubation time of FNC from 24h to 72 h.The results indicated that FNC regulated the expression of P-gp,mrp2 and bcrp in a concentration and time-dependent manner.9.Caco-2 cells were treated with different concentrations of FNC?10-100?M?.The regulation of P-gp,MRP2 and BCRP function by FNC was investigated by measuring the fluorescence intensity of Rhodamine 123,CDF and BODIPY FL prazosin using a microplate reader.The results showed that the activity of P-gp,MRP2 and BCRP increased significantly with the increase of FNC concentrations.