Transport Of Levofloxacin In A Model Of Rat Gastric Mucosa In Vitro And The Transporter May Be Involved In

Objective To evaluate the transport of levofloxacin by using a model in vitro by rat gastric mucosa combined with Ussing chamber and the effect of drug concentration, time, direction, p H, P- glycoprotein inhibitors( verapamil, cyclosporin a) and organic cation transporter inhibitors( cimetidine) on the transport of levofloxacin across the gastric mucosa.Method 1 Establish a transport model in vitro of levofloxacin.Restrict the Wistar rats from feed 24 hours.After anesthesia laparotomy, the stomach was rapidly removed from the abdominal cavity.Then,washed the serosal surface several times with NS,T 4℃.Axially cut along the lesser curvature of the stomach, and washed the mucosal surface with Krebs-ringer solution,p H 7.4,T 4 ℃.The undamaged section of the glandular portion of gastric mucosa was separated from the submucosa by microsurgical instruments and then were carefully mounted in Ussing chambers with 0.04 cm 2 of exposed area. And each side of the tissue was bathed with 6ml of Krebs-Ringer solution saturated with CO2 /O2(5%/95%). The chambers were screwed tight and the entire assembly was maintained at 37℃. After an equilibration period of 30 min,the solutions were replaced with fresh Krebs-Ringer solution,and then the experiments were run.2 Study on the transport of levofloxacin across gastric mucosa.(1)Evaluate the effect of the concentration and direction on the transport of levofloxacin across the gastric mucosa.A 1mg·mL–1 stock solution of levofloxacin was made in Krebs-Ringer solution, diluted to the desired final concentrations ranging from 10,25,50, 100,150 μg·m L–1 by Krebs-Ringer solution.For the absorptive transport, donor was the mucosal compartment(M,6m L), whereas for the excretive transport, donor was the serosal compartment(S,6m L). The permeation experiment lasted 160 min and every 20 min 200 ul of receiver solutions was taken for measuring the drug amount that permeated in the M-S and S-M directions by HPLC analysis separately. An equal volume of heated blank buffer was added immediately after each sample was withdrawn.(2)Evaluate the effect of the inhibitor on the transport of levofloxacin across the gastric mucosa. A 1mg·m L–1 stock solution of levofloxacin was made in Krebs-Ringer solution, diluted to the desired final concentrations being 50μg·m L–1 by Krebs-Ringer solution. For the absorptive transport,the donor solution(M,6m L) was joined with levofloxacin(50μg·m L–1) and inhibitor,whereas for the excretive transport, the donor solution(S,6m L) was joined with levofloxacin(50μg·m L–1),and the receiving solution(M,6m L) was joined with inhibitor.The permeation experiment lasted 160 min and every 20 min 200 ul of receiver solutions was taken for measuring the drug amount that permeated in the M-S and S-M directions by HPLC analysis separately.An equal volume of heated blank buffer was added immediately after each sample was withdrawn.(3) Evaluate the effect of p H on the transport of levofloxacin across the gastric mucosa.The donor solution were diluted by Krebs-ringer solution, p H 6.0, 7.4 or 8.0, depending on the experiment.3 Analysis of samples by HPLC methodHPLC method was used for the assay of levofloxacin. The mobile phase was a mixture of acetonitrile-50mmol·L–1 citrate-1mol·L-1ammonium acetate(17:82:1,V/V/V). The effluent was monitored with UV detection at 295 nm at a flow rate of 1.0m L·min–1.Levofloxacin was successfully separated using a Welch C18(4.6mm×250mm,5μm).The Column temperature was 40℃and the injection volume was 20μL.Evaluate the selectivity, linearity, accuracy, precision, stability and sensitivity of the method.Result(1) The method of mucosa exfoliating, Ussing chamber techniques and the HPLC in this study is stable and reliable.(2) Bidirectional accumulation of levofloxacin were increased with time, but between the various concentrations of levofloxacin,the accumulation and apparent permeability coefficient(Papp) were no significant difference(P>0.05);There were significant differences between the accumulation and the Papp of levofloxacin of two directions with various concentrations of donor solutions, separately( P< 0.05).(3)The Papp of levofloxacinat p H 4.0, 7.4, and 8.0 are(0.95± 0.07),(1.60± 0.11),(1.37 ± 0.24)/10-6 cm·s-1, respectively, with50μg·m L–1 donor solutions.The Papp was the greatest at p H 7.4 and Papp(p H4.0) was significantly lower than Papp(p H7.4)(P<0.01).However, there were no significant difference in Papp between p H7.4 and p H 8.0(P>0.05).(4)After addition of Cyclosporin A, the Papp(S-M)/Papp(M-S) of levofloxacin decreased from 2.20 to 1.06(p<0.05).When verapamil was added, the Papp(S-M)/Papp(M-S) of levofloxacin decreased from 2.20 to 1.08(p<0.05); after addition of cimetidine,the Papp(S-M)/Papp(M-S) of levofloxacin decreased from 2.20 to 1.78(P> 0.05).Conclusion(1)Stripping of stomach mucosa of rat is the technical difficulty of the experiment. With method of blistering,we had successfully obtained the required gastric mucosa.(2)There were significant differences between the accumulation and the Papp of levofloxacin of two directions with various concentrations of donor solutions and a p H-dependent manner.The P-gp inhibitors(Cyclosporin A and verapamil) increased the absorption of levofloxacin and decreased the secretion.It shows levofloxacin may be mediated by a carrier protein and P-glycoprotein may be involved in the transport of levofloxacin in this model in vitro.(3)The organic cation inhibitor(Cimetidine) had no effect on the transport of levofloxacin in this model in vitro.This indirectly shows organic cation transporter may not be expressed in normal gastric mucosa of rats or not participate in.