Studies On DP-01 Transport In Vitro And Urinary Excretion In Rats

β-thalassemia is a typical hereditary hemolytic anemia,which has a relatively high morbidity in southern China.Red blood cell transfusions combined with iron chelation are vital treatments for patients with severe β-thalassemia.Up to now,our country hasn’t had proprietary drugs for iron overload,which made it unaffordable for many ordinary families.New drug development is urgent.DP-01 is a candidate iron chelator,while the absorption,transport,metabolism and excretion properties have been studied rarely.Thus in this study,absorption,excretion and transport of DP-01 were studied in vivo and in vitro models to provide guide for theoretical and practical foundations for the next development.1.Bi-directional transport assay of DP-01 across Caco-2 cell monolayer model.Objective:Caco-2 cell monolayer model was used to investigate the intestinal absorption of DP-01.Methods:Caco-2 cell monolayer model was used to investigate the bi-directional transport mechanism of DP-01.The effects of time,drug concentration and pH on the absorption of DP-01 were studied.The determination of DP-01 was performed by HP LC,and apparent permeability coefficient(Papp)and efflux ratio of DP-01 were calculated.Results:An HPLC method for determination of DP-01 in HBSS was developed.At pH 7.4,the Papp of DP-01 at high,medium and low concentrations were 6.40-8.17×10-6 cm/s for AP-to-BL and 8.21～8.40×10-6 cm/s for BL-to-AP(P>0.05),while the efflux ratios were 1.0,1.1,1.3 respectively.DP-01 had a maximum Papp around pH 6.5(P<0.05).Conclusions:DP-01 is mainly absorbed and transported through intestinal epithelial cells by passive diffusion and has high permeability with optimal absorption around pH 6.5(between pH 5.0 and pH 7.4).2.Urinary excretion study of DP-01 in rats after single oral administration.Objective:To develop a HPLC method for determination of DP-01 in rat urine and to study the urinary excretion in rat after signal oral administration of DP-01.Methods:The urine samples after oral administration of DP-01 were compared with blank rat urine sample and hydrolyzed to observe DP-01 and its potential metabolites in rat urine.Urine was collected after signal oral administration of 44.3 mg/kg DP-01 within 84 hours and was quantitatively detected by HPLC.Urinary excretion rate of DP-01 was calculated.Results:DP-01 was mainly excreted unchanged as parent drug in rat urine.A HPLC method for determination of DP-01 in rat urine was developed.The standard curve was linear over the range of 0.5～50 μg/mL,and the lower limit of quantification was 0.5μg/mL(RSD<3.53%).The assay recovery of DP-01 was 96.5-100.8%,while the extraction recoveries of DP-01 and internal standard were 86.3～87.0%and 86.1%respectively.The RSD of intra-and inter-day precision were below 2.1%.And the stability at different store conditions meets the requirements.The urinary excretion rate of DP-01 within 84 hours after signal oral administration of DP-01 was 57.53±9.2%(n=6).Conclusions:DP-01 was excreted mainly in the form of parent drug in rat urine.The urinary excretion rate of DP-01 within 84 hours after signal oral administration of DP-01 was 57.53±9.2%(n=6).3.In vitro interaction of DP-01 with cellular membrane transporters of hOCTs and hOATl.Objective:To study the potential interaction between DP-01 and hOCTs or hOATl transporters in vitro.Methods:The effects of DP-01 on the accumulation of typical substrates of hOCTs and hOATl were evaluated by MDCK-hOCTs and MDCK-hOAT1 cells respectively.The accumulation of DP-01 was also investigated in MDCK-hOCTs/MDCK-hOAT1 cells and mock cells with or without typical inhibitors.Results:In MDCK-hOCT1,MDCK-hOCT2 and MDCK-hOCT3 cells,the accumulation of MPP+ with 100 μmol/L and 300 μmol/L DP-01 were 60.7%,95.3%,67.5%and 41.8%,71.7%,63.3%,respectively.And in MDCK-hOAT1 cells,the accumulation of 6-CF with 100 μmol/L and 300 μmol/L DP-01 were 47.2%and 60.5%,respectively.In MDCK-hOCT1,MDCK-hOCT3 and MDCK-hOATl cells,the uptake of DP-01 were markedly higher than that in mock cells,and were significantly inhibited by typical inhibitors.Moreover,the uptake ratios of DP-01 in MDCK-hOCT1 or MDCK-hOAT1 cells versus mock cells were greater than 2 fold.Whereas the uptake of DP-01 in MDCK-hOCT2 and mock cells did not show significant difference.Conclusions:DP-01 can obviously inhibit hOCTs and hOAT1.And DP-01 is a substrate for both hOCT1 and hOAT1.