| Hesperetin are widely present in many plants, especially abundant in sweet orange and lemon which has been reported to show many pharmacological properties including anti-oxidative, anti-inflammatory, anti-viral, and anti-carcinogenic activities. Although hesperetin showed so many beneficial pharmacological properties, low bioavailability of oral administration and short t1/2 limits its further prospect in pharmaceutical application. To maintain plasma concentration of drug, hesperetin should be administrated frequently. Thus, we have synthesized a series of hesperetin derivatives and screened their anti-inflammatory activity in vivo and ex vivo. Among them, 7, 3′-dimethoxy hesperetin was found to have a higher anti-inflammatory activity than its parent compound hesperetin and can effectively induce apoptosis of fibroblast-like synoviocytes in rats with adjuvant arthritis(AA) in vitro. Another compound 8-isopropylaminomethyl hesperetin(IPHP), which is chemically 3′,5,7-trihydroxy-8-isopropylaminomethyl-4′-methoxy flavanone, was found to have a higher anti-inflammatory activity in vitro than its parent, too. IPHP could significantly inhibit the TNF-α and IL-6 which were produced by RAW264.7 cells after stimulating by LPS. This experiment investigated the pharmacokinetic of IPHP in rats together MDCK monolayer models to study the intestinal absorption mechanism. P-glycoprotein(P-gp) and the multidrug resistance-associated protein 2(MRP2) are prominent efflux transporters in the intestinal absorption and excrete toxic compounds into the intestinal lumen, acting as the first protecting barrier for organisms. The expressions of active P-gp and MRP2 in the cultured MDCK cell monolayers has been clarified. So MDCK cell monolayers were used to study whether the intestinal absorption mechanism of IPHP associated with transpoters. Our study amid at providing information and guidance of new drug design and preclinical study. The main contents are summarized as follows:1. The pharmacokinetics study of IPHP in ratsTotally 40 Sprague-Dawley rats equally were divided into 5 gioups in random. Single doses of 25mg/kg IPHP i.v.(n=8/group), or 25mg/kg IPHP, 50mg/kg IPHP, 100mg/kg IPHP, and 100mg/kg hesperetin oral(n=8, via gastric gavage) were administered. After dosing, all blood samples were collected at different time. The collected blood were centrifuged to obtain plasma and the concentration of drug in plasma were determined by HPLC method described as above, then based on the time process of blood drug concentration, the corresponding pharmacokinetic parameters were calculated The peak plasma concentration of IPHP were 1.07±0.24μg/ml and 1.14±0.58μg/ml the 0.5 and 2 h intake at the concentration of 25mg/kg, 2.58±0.94μg/ml and 2.59±0.79μg/ml the 1 and 3 h intake at the concentration of 50mg/kg,4.09±0.91μg/ml and 4.36±1.73 μg/ml the 0.5 and 2 h intake at the concentration of 100mg/kg, respectively. The Tmax(h) were 2.3±0.67,2.40±0.55 and 2.10±0.55, t1/2(h) were 6.37±1.91, 6.45±1.93 and 6.61±1.98, the Cmax(ug/ml) were 1.15±0.32, 2.60±0.70, 4.36±1.18, the AUC(mg/L? h) were 9.34±1.68, 23.80±4.52, 41.63±7.49 for IPHP at 3 different concentrations, respectively. This suggested that IPHP were not metabolized during absorption. The Tmax(h) was 0.35± 0.11,t1/2(h) was 3.07±1.12, the Cmax(ug/ml) was 4.56±1.09, the AUC(mg/L?h) was 50.23±12.22 for hesperetin. Thepharmacokinetic properties of IPHP after oral administration is extraordinary compared to that of hesperetin, it has a longer t1/2 and a higher absolute bioavailablity.2. Transport of IPHP in MDCK cell membrane modelTo study the transport of IPHP in MDCK cell membrane model. The safety concentration of IPHP in MDCK cells was determined by MTT assay. Five different concentrations(7.5, 15, 30, 60, 120μΜ) were investigated by using MDCK cell lines as an intestinal epithelial cell model. The effects of drug concentration, temperature, PH, P-glycoprotein(P-gp) inhibitor verapamil and multidrug resistance protein 2(MRP2) inhibitors MK-571 or probenecid on IPHP transport across MDCK cell lines were all investigated. The drug concentration was determined by HPLC to calculate the apparent permeability coefficient. According to the formula Papp =(d Q/dt)/ A×C0 and ER = Papp(BL-AP) /Papp(AP-BL), the parameters were calculated to show us the results. The transportation of IPHP was related to drug concentration, the Papp(AP-BL)(×10–5) are 1.49±1.20, 2.81±1.20, 3.28±0.45, 3.45±1.30, 3.47±1.27。Papp(BL-AP)(×10–5)(cm?s-1): 2.12±0.728, 3.87±0.364, 4.27±1.16, 4.08±2.68, 4.15±3.66. Efflux rates are:1.41, 1.37, 1.3, 1.18, 1.19. In vitro study indicated that the transport of IPHP was concentration-dependent and temperature-dependent in both AL-to-BL and the reverse direction, p H also played a role in the transport of IPHP. Verapamil, the chemical inhibitor of P-glycoprotein(P-gp) and MRP2 inhibitors, MK-571 or probenecid were added into bidirectional transport to study the function of transporter protein. The results suggested that MRP2 but P-gp may be involved in the transport of IPHP. |
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