| Herbal medicines have been traditionally used for the treatment of various diseases, including cold symptoms, pain, and inflammation for thousands of years. Currently, these medicines are still utilized worldwide as complementary or alternative medical ingredients with the expectation of promoting health and managing chronic or severe diseases such as cancer and HIV infection. Due to the common perception that herbal supplements are safe, they are often co-administered with conventional prescription drugs in clinical trial, raising the risk of herbal-drug interactions. Metabolic enzyme-mediated drug-drug interactions (DDI) have been extensively studied while studies on DDI related to transporters have been relatively limited, despite their importance in drug disposition.P-glycoprotein (P-gp), a member of the ABC transporter superfamily, is encoded by the ABCB1/MDR1gene. It serves as a key factor in conferring the multi-drug resistance (MDR) phenotype to cancer cells in clinical trial. In addition, P-gp is highly expressed in the apical membrane of several pharmacologically important epithelial barriers such as the kidney, liver, intestine, and blood-brain barrier. P-gp could excrete xenobiotics such as cytotoxic compounds into the gastrointestinal tract, bile and urine. Thus the modulation of P-gp can affect the oral bioavailability, biliary or renal clearance, brain uptake of drugs and increase the risk of DDI. This is particularly concerning for drugs with narrow therapeutic indices such as warfarin and digoxin. Small changes in the systemic exposure of these drugs could result in substantial alterations in therapeutic effects and toxic outcomes.Many commonly used traditional Chinese medicines (TCM) have been reported to interact with P-gp. However, the interaction profile is limited between P-gp and bioactive herbal constituents, which can effectively enter human systemic circulation after oral administration. In addition, controversial experimental results are also obtained in different laboratories due to the selection of cell strain, dosage and duration of drug exposure.Due to the high expression of human P-gp and short culture time, MDR1-MDCKII cells transfected with the human MDR1gene have become an attractive model to study the DDI mediated via P-gp.In the present study, MDR1-MDCKII cells were established by the transfection of a retrovirus carrying the human MDR1gene into parent MDCKII cells. The inhibitory effects of50major herbal constituents present in25commonly used TCM on P-gp were investigated in vitro and in vivo. The underlying inhibitory mechanism of herbal constituents was partially assessed using the ATPase activity assay. In addition, molecular docking analysis was performed to elucidate the structure-activity relationships of herbal compounds with P-gp. Due to the overlapping substrate specificities and tissue distribution of cytochrome P4503A4/5(CYP3A4/5) and P-gp, the herbal constituents that exhibited potential inhibition of P-gp were further examined for their effects on CYP3A4/5activity in human liver microsomes. The effects of a single dose of herbal inhibitors on the pharmacokinetics of digoxin were also evaluated in male SD rats. The results were summarized as follows:1. Establishment of MDR1-MDCKII cells with high expression of human P-gp1.1The results of restriction analysis and DNA sequencing showed the P-gp expressive vector pHaMDRwt carrying a human MDR1gene was accurately amplified and enriched in E.coli.1.2Amphotropic virus was produced after the transfection of plasmid pHaMDRwt into PA317cells. Viral titer of up to8.8×10-5pfu/ml was observed in the culture of MDR1-PA317packaging cells.1.3MDR1-MDCKII cells with high expression of human P-gp were established after MDCKII cells were infected with the retrovirus carrying a human MDR1gene and incubated with colchicine (60ng/ml) for two weeks. RT-PCR and western blotting analyses showed the expression of human P-gp was stable and high in MDR1-MDCKII cells. Moreover, the decreased accumulation of Rhol23and enhanced efflux of digoxin were observed in MDR1-MDCKII cells, indicating the high P-gp activity of MDR1-MDCKII cells.2. Effects of50herbal constituents on P-gp and CYP3A4/5in vitro and in vivo: herb-drug interactions mediated via P-gp and CYP3A4/52.1Among50herbal constituents, emodin,18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and20(.S)-ginsenoside F1[20(S)-GF1](100μM or less, depending on their solubility and cytotoxicity) exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2cells. However, despite the structural similarity and/or identical molecular weight, the isomers or analogues of the4herbal constituents (chrysophanol,18a-GA, andrographolide, and ginsenoside Rh1) had no effect on P-gp function.2.2Emodin was the strongest herbal inhibitor of P-gp (IC50=9.42μM) in MDR1-MDCKII cells, followed by180-GA (IC50=21.78μM),20(S)-GF1(IC50=76.08μM) and DAG (IC50=77.80μM).2.3P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of48.61,29.09μM and71.29,38.45nmol/min/mg protein, respectively. However,18β-GA and20(S)-GF1exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration (100μM).2.418β-GA, DAG,20(S)-GF1and Rh1significantly inhibited CYP3A4/5activity (44,41,23and15%inhibition, respectively), while emodin increased the metabolic rate of midazolam by approximately35%compared to the control.2.5Molecular docking analysis (CDOCKER) elucidated the mechanism for structure-activity relationships of herbal constituents with P-gp and CYP3A4. The results showed the isomerism of18-hydrogen atom (18β-GA), the existence of a hydroxyl (emodin) and double bond (DAG), and the position of glycosyl [20(S)-GF1] could be crucial chemical elements for the inhibition of P-gp. Due to these chemical elements, additional strong hydrogen bonds were formed between herbal inhibitors and P-gp, resulting in stronger inhibitory effects of herbal constituents for P-gp. In addition,18β-GA and20(S)-GF1may display their inhibition on CYP3A4by competitively binding with Arg212which was reported to be one of the key residues for the binding of compounds with CYP3A4, while emodin was able to bind more readily with another residue Thr310via a strong hydrogen bond, reducing the competition for this binding orientation/site and enabling activation to occur.2.6Co-administration of digoxin with emodin increased the AUC of digoxin by55%while the Cmax of digoxin was increased by58%when18β-GA was pretreated in SD rats.In conclusion, MDR1-MDCKII cells with high and stable expression of human P-gp were established in the present study, and it could be used as a valuable in vitro tool for the rapid screening of P-gp substrates/inhibitors and investigating the DDI mediated via P-gp. Among50herbal constituents, four constituents, including emodin,18β-GA, DAG and20(S)-GF1exhibited significant inhibition (>50%) on P-gp in vitro and/or in vivo. Moreover, dual inhibition of P-gp and CYP3A4/5activities could occur due to the overlapping substrate specificities between them. Our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans and will provide useful information for the proper use of prescription drugs in combination with herbal medicines, which is particularly important for drugs with narrow therapeutic indices. IMM-H007, an analog of adenosine, is a novel small molecule compound that could significantly improves lipid metabolism disorders in the hyperlipidemia animal models. The pharmacological studies showed that IMM-H007could inhibit cellular lipid accumulation in OLA-treated HepG2cells. Moreover, IMM-H007(2mg/kg per day and above) reduced elevated serum triglyceride, total cholesterol, low density lipoprotein cholesterol and hepatic cholesterol and triglyceride contents in HFD fed hamsters. The mechanism studies indicated that the activation of AMPK in OLA-induced steatosis of HepG2cells was up-regulated by the treatment with IMM-H007. The hepatic cellular AMPK phosphorylation was also up regulated by IMM-H007(6and18mg/kg) treatment in HFD fed hamsters. As a novel lipid metabolism regulator, IMM-H007has a differernt chemical structure, target and metabolic pathway from statins, and could be potentially used for patients who have adverse reactions to statins in clinical trial.Previous studies showed that IMM-H007contained three acetyl groups, which were liable to be hydrolyzed by plasma/serum esterases rather than CYP450s in vitro and in vivo. IMM-H007could be rapidly hydrolyzed to M1, which was believed to be the active form of IMM-H007in vivo, after an oral administration of IMM-H007to rats. A further study indicated that M1was extensively metabolized in HepG2cells, and several metabolites were observed, including deoxidized product of Ml (M344), deribosyl derivative of M1(M228), glucuronide conjugate of M1, glucuronide conjugate of M344, glucuronide conjugate of M228, sulfate conjugate of Ml, phosphate conjugate of M1(MP) and et al. Because MP was an analog of AMP, which was proved to be an effective activator of AMPK, MP was likely the active form of IMM-H007to activate AMPK in HepG2cells.Previous studies showed that the pharmacokinetic characteristics of IMM-H007and its major metabolites were varied between rat plasma and blood. For example, the concentration of MP was fairly low in rat plasma and far higher in rat blood. In the present study, the concentrations of IMM-H007, M1and MP in rat blood were detected after oral and intravenous dosing of IMM-H007to SD rats. Metabolic stabilities of IMM-H007and Ml were assessed in different biological systems such as simulated gastric/intestinal fluid, Tris-HCl buffer, and plasma, blood, liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. The metabolic stability of IMM-H007in the gut microbiota of SD rats was also investigated in this study. The results were summarized as follows:1. Simultaneous quantification of IMM-H007and its metabolites in rat blood using HPLC-MS/MSBase on the guideline of preclinical pharmacokinetic study, the HPLC-MS/MS method for the quantitation of IMM-H007and its metabolites M1and MP in biological sample has been developed, which provided a simple, reliable, sensitive and specific assay for the preclinical pharmacokinetic study of IMM-H007in SD rats. Linear detection responses were obtained for IMM-H007, M1and MP ranging from1～500,2～1000and10～5000ng/ml, respectively. The intra-and inter-day precisions (R.S.D.%) were within15%for all analytes, while the deviation of assay accuracies was within±5.7%. The average recoveries of IMM-H007, M1and MP were80～91.2%,107.4～108.7%and76.4～80.3%, respectively.2. Pharmacokinetic study of IMM-H007in SD rats2.1IMM-H007and its metabolites M1and MP can be detected in rat blood5min after oral administration of IMM-H007(50,150,450and900mg/kg) to SD rats and the blood concentrations were close to the limit of quantitation (LLOQ) at12-36h postdose. The multiple peak concentration-time curves of IMM-H007were observed, and the concentration of parent drug IMM-H007was much lower than its metabolites in both male and female rats.2.2After oral administration of IMM-H007(50,150,450and900mg/kg) to male rats, the average maximum concentration (Cmax) of M1were37.52,61.13,82.69and141.18ng/ml, AUC0-t were160.47,328.71,799.57and1779.31h*μg/L, MRT0-t were3.18,3.93,7.5and11.3h. The Cmax of MP in male rats were88.2,153.46,204.95and309.42ng/ml, AUC0-t were362.71,676.26,2230.14and4536.37h*μg/L, MRT0-t were 2.87,3.65,8.15and12.07h. Both Cmax and AUC0-t of Ml and MP were increased in a dose-dependent manner, but the MRT0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to male rats. The results indicated the saturation of elimination of M1and MP in male rats at high doses of IMM-H007(450and900mg/kg).2.3After oral administration of IMM-H007(50,150,450and900mg/kg) to female rats, the Cmax of M1were37.78,210.72,256.89and325.77ng/ml, AUC0-t were151.87,854.34,1561.42and3334.16h*μg/L, MRT0-t were3.85,4.25,7.62and10.51h. The Cmax of MP in male rats were132.55,920.83,1214.01and1122.64ng/ml, AUC0-t were698.63,4000.82,8376.97and13900.82h*μg/L, MRT0-t were4.08,4.86,8.45and10.3h. The AUC0-t of M1and MP were increased in a dose-dependent manner, but the Cmax of M1and MP were not increased dose-dependently. In addition, the MRTo0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to female rats. The results indicated the saturation of absorption and elimination of M1and MP in female rats at high doses of IMM-H007(450and900mg/kg).2.4Both Cmax and AUCo-t of Ml and MP were much higher in female rats than those in male rats after oral administration of IMM-H007(150,450and900mg/kg), indicating the gender difference between male and female rats.2.5The oral bioavailability of M1was3.04%in male rats and2.12%in female rats after an oral administration of IMM-H007at50mg/kg; the oral bioavailability of MP was5.61%in male rats and6.55%in female rats after an oral administration of IMM-H007at50mg/kg.3. In vitro metabolic stability of IMM-H0073.1The concentrations of IMM-H007and M1did not change significantly after4h incubation with simulated gastric/intestinal fluid and Tris-HCl buffer. The remaing IMM-H007and M1were more than85%when the reaction was terminated with the addition of ice-cold acetonitrile.3.2IMM-H007was unstable when incubated with animal plasma in vitro for2h. IMM-H007could be hydrolyzed to M1at different rate in the plasma of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. M1was stable after2h incubation with animal plasma. Moreover, no MP was detected in the reaction mixture.3.3IMM-H007was unstable when incubated with the liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans in vitro for2h. More than90%IMM-H007was metabolized in the first5min incubation and the reaction was not NADPH-dependent. M1was stable after2h incubation with liver microsomes. Moreover, no MP was detected in the reaction mixture.3.4Both IMM-H007and M1were unstable when incubated with animal blood in vitro for2h. IMM-H007and M1showed different metabolic rates in the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. In addtion, high concentratin of MP was detected in the reaction mixture. The concentration profile of M1demonstrated an upward phase and a downward phase, suggesting onward metabolism to other products, while MP increased constantly until it reached a maximum level. Rates of increase and decrease of products differed by species.3.5IMM-H007could be extensively metabolized into certain metabolites including M444, M402, M1,M344and M228by the gut microbiota of SD rats. Products M444, M402and M1appeared first, followed by M344and M228. Concentration profiles of M444, M402and M1demonstrated an upward phase and a downward phase, while M344and M228increased constantly until they reached the maximum levels. Products M344and M228also appeared when M1was incubated with the gut microbiota of SD rats for8h.In conclusion, the HPLC-MS/MS method for the simultaneous quantitation of IMM-H007and its metabolites M1and MP in rat blood was developed in the present study. In addition, the pharmacokinetics of IMM-H007, M1and MP in rat blood were investigated after oral and intravenous dosing of IMM-H007to SD rats. The results of metabolic stabilities of IMM-H007and M1showed that IMM-H007and M1could be metabolized into several metabolites such as M444, M402, M1, M344, M228and MP by the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys, humans and the gut microbiota of SD rats. Our findings will provide useful information for the further pharmacokinetic study of IMM-H007in other species and give insights to the possible metabolic pathway of IMM-H007in vivo. |
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