| Objective To investigate the inhibitory effect of cichoric acid(CRA)on human CYP450 enzyme in vitro,study the transport characteristics of CRA in Caco-2 cells and the pharmacokinetics of CRA in rat intestinal vascular perfusion models and rats.Methods(1)CRA was incubated with pooled human liver microsome,and the activity of main seven isozymes was characterized by phenacetin-O-deethylation(CYP1A2),bupropion hydroxylation(CYP2B6),amoxidine deethylation(CYP2C8),diclofenac-4'-hydroxylation(CYP2C9),S-mephenytoin4'-hydroxylation(CYP2C19),dextromethorphan-Odemethylation(CYP2D6),midazolam 1'-hydroxylation(CYP3A4/5),and testosterone-6?-hydroxylation(CYP3A4/5),respectively.And the system was validated by positive inhibitors.LC-MS/MS method was established for determination the metabolites of eight characteristic reactions,and the inhibitory potency of CRA on seven subtypes of CYP450 enzyme in human liver microsomes was evaluated with IC50 values.(2)A method for the determination of CRA in Caco-2 cells was established.The biomechanical transport of CRA on Caco-2 cell monolayers at different concentrations(1 ?M,10 ?M and 100 ?M)was investigated by using P-gp substrate digoxin as a positive control.(3)A rat model of intestinal vascular perfusion was established and the intestinal absorption of CRA was studied by duodenal administration.(4)Rats were randomly divided into four groups: oral administration of CRA low dose(25mg/kg),medium dose(50 mg/kg),high dose(100 mg/kg)and one intravenous injection group(2 mg/kg).The concentrations of CRA and its metabolites CAFA and CA in plasma were determined by LC-MS/MS.The pharmacokinetic parameters were calculated by pharmacokinetic software DAS2.0.Results(1)CRA had no significant inhibitory effect on CYP450(CYP1A2,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6,CYP3A4/5(substrate for midazolam,testosterone)).(2)The apparent permeability(Papp)of CRA of AP to BL were(0.68 ± 0.07)× 10-6 cm/sec,(0.85 ± 0.04)× 10-6 cm/sec and(0.84 ± 0.05)× 10-6 cm/sec at different concentrations(1 ?M,10 ?M and 100 ?M),respectively.The Papp values of BL to AP were(0.70 ± 0.09)× 10-6 cm/sec,(0.83 ± 0.10)× 10-6 cm/sec and(0.85 ± 0.09)× 10-6 cm/sec,respectively.The efflux rate(ER)values were all close to 1.(3)After the duodenal administration,the concentration of CRA in the perfusate increased with the time in the intestinal perfusion model,which was stable at 90 min and the intestinal absorption rate was(12.6 ± 0.57)%.(4)After oral administered CRA displayed as two compartment model in rats and had large Tmax and elimination.Following a single oral of CRA at the three doses,the pharmacokinetic parameters are as follows: AUC(0-?)were 7886.8 ± 208.9 ?g/L*h?35468.8 ± 1737.4 ?g/L*h and 45422.1 ± 398.4 ?g/L*h,respectively;T1/2z were 4.79 ± 1.09 h?5.15 ± 0.9 h and 4.80 ± 0.70 h,respectively;CLz were 3.3 ± 0.1 L/h/kg?1.4 ± 0.07 L/h/kg and 2.2 ± 0.02 L/h/kg,respectively;Vz were 22.5 ± 4.9 L/kg?10.4 ± 1.4 L/kg and 15.2 ± 2.3 L/kg,respectively;Cmax were 1097.7 ± 84.4 ?g/L?3666.0 ± 195.4 ?g/L and 5597.0 ± 325.0 ?g/L,respectively;Tmax were all 4 h.The pharmacokinetic parameters of CRA at a single intravenous dose of 2 mg/kg,the pharmacokinetic parameters are as follows: AUC(0-?)is 51432.7 ± 3650.2 ?g/L*h;T1/2z is 4.5 ± 0.4 h;CLz is 0.04 ± 0.003 L/h/kg;Vz is 0.25 ± 0.034 L/kg.Conclusions CRA has no inhibitory effect on human CYP450 enzyme,which indicated that it does not cause a potential risk of drug interactions with other drugs in relation to liver enzyme inhibition in clinical drug compatibility.CRA has poor absolute bioavailability in rats.There is significant metabolism in rats.Poor transmembrane transport permeability in intestinal cell,and transmembrane transport of CRA may be mainly passive transport. |
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