Transport Properties And Pharmacokinetics Analysis Of Caffeic Acid Phenethyl Ester And Its P-nitro Derivative In Caco-2 Cells And Rats

Caffeic acid phenethyl ester（CAPE）, a flavonoid-like compound isolated from honeybee propolis, has been shown the biological activities including antioxidant,anti-inflammatory, antiviral, anti-carcinogenic, and immunomodulation effects. CAPE has also been reported to ameliorate myocardial ischemia-reperfusion（IR） induced oxidative injury in rats. It was found, however, that CAPE is hydrolyzed rapidly in vitro,and after intravenous administration of 5, 10, 20 mg/kg in rats, CAPE exhibited a very rapid elimination half-life. To improve the in vitro and in vivo stability of CAPE, We designed and synthesized a new CAPE derivate: p-nitro Caffeic Acid Phenethyl Ester（CAPE-NO₂）, possessed a similar effects with CAPE in attenuating IR-induced myocardial injury. In the present study, we aim to understand the pharmacokinetics profiles of CAPE and CAPE-NO₂ in rat and investigate their absorption mechanisms and effects on the P-glycoprotein in Caco-2 cellsThe main pharmacokinetics parameters of CAPE were as follow: t1/2 = 4.2 ± 1.58 h,Tmax = 0.55 ± 0.32 h,Cmax = 447.1 ± 102.3 ng/m L,AUC0-t= 1659.6 ± 152 ng / h·m L,AUC0-∞= 1973.4 ± 202 ng / h?m L,Vz_Fobs = 18.2 ± 7.8 L/h/kg,MRTINF_obs = 4.69± 1.76 h. And the main pharmacokinetics parameters of CAPE-NO₂ were as follows:t1/2 =20.9 ± 6.07 h, Tmax = 1.59 ± 0.6 h, Cmax = 307.4 ± 35.23 ng/m L, AUC0-t = 3239.9 ± 352 ng / h·m L, AUC0-∞= 3658.7 ± 326 ng / h·m L, Vz_Fobs = 40.9 ± 14.5 L/h/kg,MRTINF_obs = 23.6 ± 8.55 h. These data suggested that CAPE-NO₂ had better bioavailability and slower elimination process compared with CAPE.MTT assay was performed to evaluate the cytotoxicity of CAPE and CAPE-NO₂ on Caco-2 cells. cell survival rate was above 90% after treatment of both CAPE and CAPE-NO₂ at the concentration of 5 and 20 μM compared with the control. In terms of cell viability, no significant difference was found between the cells cultured in 0 to 50μM CAPE for 4 h and 48 h and in 0 to 100 μM CAPE-NO₂ for 4 h and 48 h,respectively and the control group. Both CAPE and CAPE-NO₂ possessed good permeability with Papp values above 10-6 cm/s, but CAPE-NO₂ has better permeability with the Papp values 2-4 folds higher than that of CAPE at the concentration of 5, 10 and 20 μM. The efflux ratios of CAPE and CAPE-NO₂ were less than 1.5 and the transport rates of CAPE and CAPE-NO₂ from the AP to BL side across Caco-2 cell monolayer increased dependently with time, indicating that the transport mechanism of these compounds was via passive diffusion. No difference was observed in the efflux ratio of CAPE in the presence or absence of verapamil. However, the efflux ratio of CAPE-NO₂ decreased from 1.17 to 0.72 after co-culture with verapamil. The accumulation of rhodamine 123 was increased by 1.3-1.9 folds in the presence of CAPE and 1.4-2.3 folds in the presence of CAPE-NO₂ after 1 h administration, in concentration-dependent manner over the range of 5-20 μM. In contrast, after administrated with CAPE or CAPE-NO₂ at the concentration of 5, 10, 20 μM for 48 h,CAPE or CAPE-NO₂ decreased the accumulation of rhodamine 123 by about 65% or78% at 20 μM compared with the untreated cells, respectively.Both CAPE and CAPE-NO₂ significantly up-regulated the P-gp expression level in a time- and concentration-dependent way. The cellular P-gp level was increased by about 2.1 and 1.7 folds, respectively, after the cells were pretreated with CAPE and CAPE-NO₂ at 20 μM for 72 h compared with the untreated cells. When the co-incubation period was shortened to 48 h, the P-gp protein level of the cell pretreatedwith CAPE and CAPE-NO₂ at 20 μM was 1.7 and 1.5 folds, respectively, by that of the control.