Effect Mechanism Of MDR1Gene Polymorphism On The Absorption Of Rupatadine Fumarate

Background：Rupatadine fumarate is an oral antihistamine and platelet-activating factorantagonist indicated for the management of allergic rhinitis and chronic urticaria.P-gp （P-glycoprotein） playing the role as an “efflux pump” which limitsgastrointestinal absorption. The effect of P-gp may mediated the absorption ofrupatadine fumarate in vivo. The MDR1gene encodes the P-gp which singlenucleotide polymorphism （SNP）（C3435T） in exon26was assosiated with the alteredlevels of P-gp. However, the significant interindividual differences of P-gp’sexpression and function will result in different bioavailability of P-gp’s substrates.Therefore, we try to investigate the absorption mechanism of Rupatadine in vitromodel and the study of MDR1polymorphism on the pharmacokinetic of Rupatadinefumarate will be continued.Objective:To study the effect of P-gp on the absorption of Rupatadine fumarate and MDR1polymorphism on the pharmacokinetics of rupatadine fumarate in healthy Chinesevolunteers, in order to provide the basis information for the clinical application bygenotypes.Methods：1. To establish and valuate the characteristics of Caco-2cell monolayer model,for the preparation of experiment model in vitro.2. A significant direction was observed during the transmembrane transport ofRupatadine fumarate, which inferred that rupatadine was mediated by efflux pump,examining the transport characteristics when P-gp inhibitor Verapamil exists or exits,studying excretion phenomenon via the transport on Caco-2cell monolayer model.3. After investigating the mechanism of absorption of rupatadine in vivo.Genotypes of MDR1C3435T for22Chinese healthy subjects were determined by the PCR-RFLP assay.22subjects received a single oral dose of10mg rupatadinefumarate table, the pharmacokinetics of rupatadine fumarate were compared withinthree genotypes （CC, CT, TT）. The determination of Rupatadine Fumarateconcentration in human plasma samples were applied with HPLC-MS/MS methods.4. The main pharmacokinetic parameters of Rupatadine fumarate were cal-culated by statistics software Das2.1, contrasting and analysing the rupatadinefumarate human pharmacokinetic characteristics of MDR1C3435C、MDR1C34-35T、MDR1T3435T by t-value.Results：1、Caco-2cell had formed a tight junctions and integrity with clear border afterbeen cultured on transwell plates. The value of TEER in Caco-2cell monolayer wasover500·cm2after22²5days.There was also a clear polarization on the Caco-2cell monolayers. In the concentration range of5⁴0μM, the uptake of rupatadinefumarate was linearly increased within20minutes, and then leveled off. WhenHank’s PH value was7.4, the uptake reached a maximum intake but there was nosignificance with PH6.5and PH8.0. Consequently, an inerrable Caco-2cellmonolayer model was well constructed for rupatadine’s transport.2、The Apparent permeability cofficient of Rupatadine fumarate transported overCaco-2cell monolayer had a directivity, which indicated the drug was mediated bytransporters, after add specificity inhibitor of P-gp, the Apparent permeabilitycofficient of rupatadine fumarate had taken significant change,and the PDR decrea-ses from5.426to1.339, which referred that the rupatadine fumarate was a substrateof P-gp.3、PCR-RFLP assay was applied for the genotyping of MDR1C3435T variationin22Chinese healthy subjects;10（45.5%） were homozygous wild C3435C;9（40.9%） were the heterozygous C3435T and3（13.6%） were homozygous mutantT3435T.4、There are significantly differences among the pharmacokinetics parameters ofRupatadine fumarate which included C_max, T_max, AUC_0-t, AUC_0-∞, CL, Vd and T1/2. Asfor CC, CT and TT group, C_maxof TT group （12.52±7.49ug/L） was significant lower than that of CC （18.91±7.20ug/L） and CT （14.90±6.04ug/L） group. AUC_0-tandAUC_0-∞were also significant lower in TT group （29.72±24.79ug/L^*h、31.01±25.74ug/L^*h） than which were in CC group （47.46±15.02ug/L^*h、49.88±16.42ug/L^*h）and CT group （49.30±14.22ug/L^*h、51.73±15.64ug/L^*h）, respectively. T_maxof TTgroup （0.58±0.14h） was significant lower than that of CC （0.83±0.29h） and CT（1.11±0.22h） groups. However，CLz of TT group （468.45±264.42L/h） was higherthan that of CC （234.36±130.38L/h） and CT （206.53±50.05L/h） groups, butwithout significant differences.Conclusion:An integrity Caco-2cell monolayer model has been constructed in this st-udy, which is reliable for the transport experiment of rupatadine fumarate. It has demonstrated a significant direction when rupatadine fumarate across the m-embrane and P-gp’s inhibitor （verapamil） could significantly prevent the excret-ion effect of Rupatadine fumarate, which indicated that Rupatadine fumaratemight be the substrate of P-gp. P-gp was encoded by MDR1which polymorphhisms in exon26which may lead a interindividual variation to rupatadine fu-marate’s oral absorption. Compared to CC and TT groups, the absorption of r-upatadine fumarate was lower in TT groups （p<0.01）. Considering the interindividual variability, we should adjust the dose of rupatadine for MDR1TT carrierduring the clinical applications.