Modulation Of Human P-gp By Multiple Doses Of Astragalus Root Extract Granules Using Fexofenadine As A Phenotyping Probe: A Randomised Placebo-controlled Clinical Trial

Objective:1. To investigate the influences of Astragalus root extract (Huang Qi) granules on human P-gp and anticipate the risk of its drug interactions with common coadministered medications;2. To investigate the effects of MDR1 C3435T and SLCO1B1 T521C polymorphism on fexofenadine (FEX) pharmacokinetics and whether pretreatment of Huang Qi granules produces different effects on subgroup of C3435T or T521C genotype.Methods:1. An assay method was established for determining FEX concentrations in human plasma. Plasma samples(200μL) were deproteinized by precipitation with acetonitrile, centrifuged and the supernatant was reconstituted and injected into HPLC. Separation was achieved on Waters Symmetry C18 (4.6mm×250mm,5μm) with a mixture of 0.1mol/L ammonium acetate-acetonitrile (63:37, v/v) as mobile phase. The flow rate was 1mL/min. The fluorescence detector was set at excitation wavelength of 230 nm and emission wavelength of 290 nm. The internal standard method was used to quantify Fex. The assay was validated and applied to pharmacokinetic study. 2. The study had an open-label, randomized, placebo-controlled, two-period crossover design, with 3 wk washout between periods. Fex was used as an in vivo P-gp phenotyping probe.Fourteen volunteers received multiple doses of Huang Qi granules (4 g bid) or placebo (4 g bid) for one week and then received a single oral dose of 120 mg FEX tablet. Plasma samples were taken at different times and analysed by chromatography. Pharmacokinetic parameters were calculated by non-compartmental method and compared in two groups. Bioequivalence evaluation was performed by the software DAS2.1.1.3. C3435T mutation in exon 26 (MDR1 C3435T) and SLCO1B1 T521C mutation were determined in this pharmacokinetic study. Data mining was performed.Results:1. With respect to chromatographic analysis, endogenous chemicals did not interfere with Fex. The calibration curves were linear in the range of 20～1000μg/L (r=0.9993, n=5), with a limit of quantitation of 20μg/L. The within-and between-day RSDs of quality-control samples at high-, medium-and low-concentrations were less than 10%. The average method recovery was 105.9%. The average absolute recovery was 94.6%.2. Pharamcokinetic parameters derived from placebo group were as follows:T1/2(3.75±1.47 h); Cmax (745.11±137.41μg/L); Tmax (2.25±0.47 h); AUC(0-t) (3894.27±923.45μg/L*h); AUC(0-∞) (3993.84±912.97μg/L*h).Pharamcokinetic parameters derived from Huang Qi group were as follows::T1/2(4.00±1.24 h); Cmax(709.44±170.03μg/L); Tmax (2.21±0.51 h); AUC(0-t) (3832.72±1077.60μg/L*h); AUC(0-∞) (3983.53±1019.83μg/L*h). The effects of Hunag Qi granules on FEX pharmcokinetics were insignificant (P>0.05). FEX tablets in two groups were bioequivalent. The average elimination half life was similar with data derived from Liu's study in 20 Chinese volunteers, but it was shorter than documented data (9.6±2.9 h) abroad. With respect to Cmax, Tmax and AUCo-t, no significant differences were obversed(P<0.05).3. MDR1 C3435T phenotyping results of 14 healthy volunteers were as follows: hemozygous CC (wide type, n=5); hemozygous TT(mutation type,n=3); heterozygous CT(mutation type,n=6). OATP1B1 T521C phenotyping results were as follows:TT(n=12); TC (n=2).4. In MDR1 3435 T allele carriers, T1/2 of FEX was longer than that in MDR1 3435 CC carriers (TT, CT, TT+CT> CC, P<0.01). Gene-dose effects on T1/2 in Huang Qi group were not observed (TT, CT, TT+CT> CC→TT, CT, TT+CT=CC, P> 0.05). By comparison of pharmacokinetic parameters in MDR1 3435 CC carriers in either placebo group or Huang Qi group, difference in T1/2 was significant(P=0.02, T1/2(Placebo)< T1/2(Huangqi)).5. There were no gene-dose effects of OATP1B1 521 C on pharmacokinetic parameters of fexofenadine in placebo group. Cmax in OATPIB1 521 C allele carriers exhibited an increase trend with marginal significance (889.50±11.54 vs 679.43±158.09μg/L, P=0.05) compared with that in OATP1B1 521 TT carriers.Conclusion:1. The RP-HPLC assay is stable, simple, rapid, accurate, sensitive and applicable for determining plasma concentrations of FEX in its pharmacokinetic studies;2. One-week pretreatment of Huang Qi granules has no significant effects on human P-gp activity in Chinese healthy subjects. However, effect of Huang Qi granules on P-gp was MDR1 C3435T genotype-dependent. Huang Qi granules may have inhibitory effects on P-gp activity in MDR1 3435 CC carriers. The decrement degree in 3435 CC carriers was greater than that in 3435 T allele carriers.3. The terminal elimination profile of FEX indicates ethnic differences and further investigation is needed.4. One-week pretreatment of Huang Qi granules may have inhibitory effects on OATP1B1 activity in Chinese healthy subjects.