| Cytochrome P450(CYP450) is one of the most important phase I metabolizing enzymes in the human body, participating the metabolism of many endogenous and exogenous substances, mainly mediating oxidation and peroxidation reactions. CYP450enzymes are mainly distributed in the liver, mediate a large number of drug metabolism in the human body. CYP1A2,2A6,2C8,2C9,2C19,2D6,2E1, and3A4/5are closely related with drug metabolism. The inhibitors or inducers of drug-metabolizing enzymes may lead to drug-drug interactions (DDI), and trigger severe side effects. According to current statistics, over90%of clinically DDI are caused by alterations of the CYP450enzyme activity.ATP-binding cassettes transporters are one of the largest transmembrane transporter superfamilies. They translocate or efflux various organic molecules including many drugs, with the energy obtained from hydrolysis of ATP. Among these transporters, P-glycoprotein (ABCBl/P-gp/MDR1), multidrug resistance protein (ABCCs/MRPs)1-5, and breast cancer resistant protein (ABCG2/BCRP) are the most important drug transporters. Many clinical reports suggested the significant roles of P-gp, MRPs, and BCRP in drug disposition. In addition, the overexpression of P-gp, MRP1, and BCRP in tumor cells is an important mechanism in the multidrug resistance (MDR), which could lead the failure of chemotherapy.Organic cation transporter OCT3(SLC22A3) is a transporter mediating translocate of various endogenous and exogenous organic cations, belonging to the solute carrier (Solute carrier, SLC) transporter superfamily.OCT3has a wide variety of substrates, including many endogenous compounds such as monoamine neurotransmitters, as well as some clinical drug such as metformin, quinidine, and lamivudine. OCT3is widely distributed in the body, and is involved in many physiological and pathological activities. In recent years, a number of researches indicated the role of OCT3in the function and diseases of central nervous system, such as depression. It is considered that OCT3is a notable target in the antidepressant treatment.Protoberberine alkaloids are widely existed in many plants. Studies reveal that Protoberberine alkaloids have various pharmacological activities. Berberine and tetrahydropalmatine (THP) are widely used in clinical practice for the treatment of diarrhea and analgesia (THP has a chiral carbon atom,(-)-THP is the main analgesic constitute), respectively. Recent studies have demonstrated that berberine produces antidepressant-like effects in animal experiments. In the aspect of metabolism, there is no report about the inhibitory effects of two enantiomers of THP on the activity of CYP450and ABC transporters. As organic cations, the interaction between protoberberine alkaloids and OCT3was also unknown. Thus, this studie investigate the interactions between THP enantiomers and CYP450s as well as ABC transporters, and the interactions between protoberberine alkaloids and OCT3. Moreover, the potential role of THP enantiomers in reversing MDR in chemotherapy, and the antidepressant effects of berberine and jatrorrhizine and the relation with OCT3are also studied.1. Inhibitory effect of tetrahydropalmatine enantiomers on human CYP450sTo investigate the inhibitory effect of THP enantiomers on human CYP450s, an in vitro cocktail approach was established to screen the activity of human CYP1A2,2A6,2C8,2C9,2C19,2D6,2E1and3A4/5. Probes include phenacetin (1A2), coumarin (2A6), taxol (2C8), tolbutamide (2C9), omeprazole (2C19), dextromethorphan (2D6), chlorzoxazone (2E1), testosterone and midazolam (3A4/5). All the probes were monitored with determining the corresponding metabolites of probes by LC-MS/MS, except the coumarin was determined with its reduction. The method was validated in linear range, assay recovery (within100±15%), and precision (RSD<15%), all meet the requirements.The cocktail was then applied in the investigating the inhibitory effects of THP enantiomers on CYP1A2,2A6,2C8,2C9,2C19,2D6,2E1and3A4/5in human liver microsomes. The results showed that, the activity of CYP1A2,2C8,2C9,2C19,2D6,2E1, and3A4/5was inhibited by THP enantiomers, while the metabolism of coumarin (CYP2A6substrate) was not affected. THP enantiomers showed weak inhibition on the activity of CYP1A2,2C8,2C9,2C19,2E1, and3A4/5, and the inhibition on CYP2D6was stereoselective.(-)-THP exhibited a strong inhibition on the metabolism of dextromethorphan (CYP2D6substrate), whereas the inhibitory effect of (+)-THP was weak. The Ki value of (-)-THP inhibition on dextromethorphan is determined as6.42±0.38μM. Since the concentration of (-)-THP in rat liver is high, it is deduced that the co-administration of (-)-THP and CYP2D6substrates may lead to drug-drug interactions.2. The interaction between THP enantiomers and ABC transportersThe accumulation of rhodamine123(R123), calcein, and rosuvastatin in MDCK-MDR1, MDCK-MRP1, and LLC-PK1-BCRP cells respectively, was performed to investigate the inhibitory effect of THP enantiomers on activity of P-gp, MRPl, and BCRP. The results showed that, THP enantiomers are not inhibitors of MRP1or BCRP, but inhibited the activity of P-gp with stereoselective difference. The inhibitory effect of (+)-THP is stronger than that of (-)-THP. The transport of R123across the MDCK-MDR1cell monolayer was further investigated to evaluate the inhibitory effects of THP enantiomers. The measured IC50values:(+)-THP was19.99μM,(-)-THP was48.63μM. Meanwhile, the transport of THP enantiomer across the MDCK-MDR1and MDCK cell monolayer indicated that, THP enantiomers are highly permeable, it could not be deduced that (-)-THP or (+)-THP was transported substrate of P-gp or not. Considering the concentration of THP enantiomers in rat liver is high, and P-gp is intensely expressed in the liver, it is suggested that co-administration of THP enantiomers and P-gp substrate may lead to drug-drug interactions.The results of Western Blot displayed, the protein expression of P-gp in K562/ADR cells was reduced by the treatment of THP enantiomers for48h, but without concentration-dependent. However, THP enantiomers did not affect the mRNA expression of P-gp in the Real-time PCR. It indicated that such regulation may be in the post-transcriptional or translational level.ABC transporters do not only play an important role in DDI, but are also crucial in MDR of tumor cells after chemotherapy clinically. In MDR tumor cells, MCF-7/ADR and K562/ADR, the MTT assay showed that the combination of THP enantiomers and doxorubicin significantly enhanced the cytotoxicity of doxorubicin, decreased the survival rate of tumor cells, in comparison with doxorubicin alone group. Moreover,(+)-THP effect was slightly stronger than (-)-THP, it was consistent with the result of P-gp inhibition. These results revealed that THP enantiomer are inhibitors of P-gp, and are capable to resensitize the MDR tumor cells to anti-cancer agents.3. Establishment of stably hOCT3-transfected cell model and its application in the uptake study of protoberberine alkaloidsSince the protoberberine alkaloids are organic cations, which are most likely interacting with OCT3, the MDCK cell transfected with human organic cation transporter3was generated in the study. The MDCK-hOCT3cell model was evaluated with the uptake of3model substrates:4-Di-1-ASP+, MPP+, and metformin.The uptake of protoberberine alkaloids in MDCK-hOCT3was estimated. The protoberberine alkaloids include berberine, coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine, THP, and corydaline. The uptake of berberine, coptisine, jatrorrhizine, and epiberberine was enhanced in MDCK-hOCT3, compared with mock cells, suggesting they are substrates of hOCT3. The kinetic parameters were further determined. The Vmax values (pmol/mg protein/min) were44.7±1.2,57.5±1.8,16.5±0.38, and4.04±0.16, the Km (μM) values were2.17±0.20,0.351±0.034,0.453±0.042, and0.273±0.032, the Clint (Vmax/Km) were20.6,164,36.4, and14.8for berberine, coptisine, jatrorrhizine, and epiberberine, respectively.4. The inhibitory effect of protoberberine on hOCT3and the antidepressant-Iike activity of berberine and jatrorrhizineThe inhibitory effect of protoberberine on hOCT3-mediated uptake of MPP+was determined in MDCK-hOCT3cells. All the alkaloids inhibited the MPP+uptake in the study, and the inhibitory potency of coptisine is strongest, while (+)-THP is the weakest inhibitor. The IC50values are:berberine,9.87μM, coptisine,2.15μM, jatrorrhizine,4.92μM, epiberberine,2.27μM, berberrubine,4.88μM, palmatine,17.3μM,(-)THP,21.0μM,(+)-THP,28.4μM, and corydaline,21.1μM.It was reported that berberine produced antidepressant-like activity. The relation of berberine, jatrorrhizine (potential metabolite of berberine) and OCT3was evaluated in the study. In MDCK-hOCT3cells, berberine and jatrorrhizine inhibited hOCT3-mediated5-HT/NE uptake, with the IC50values:0.225μM and0.122μM for berberine and jatrorrhizine on5-HT uptake, respectively;0.566μM and0.205μM for berberine and jatrorrhizine on NE uptake, respectively. In mouse brain synaptosomes, the uptake of5-HT and NE was also inhibited by berberine and jatrorrhizine in concentration-dependent manner, and the uptake-2inhibition was major in the total inhibition. In mouse tail suspension test, jatrorrhizine of5,10, and20mg/kg (i.p.) produced antidepressant-like activity. The results are helpful in elucidating the mechanism of the antidepressant-like activity of berberine and extend the pharmacological activity of jatrorrhizine. |
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