The Interaction Of Coptis Alkaloids With UGT And OCTs And The Mechanism Of Hypnotic Action Of Jiaotai Pills Based On OCTs

In vivo pharmacokinetic interactions mediated by metabolic enzymes and transporters are important cause leading to the change of drug efficacy and toxicity.Glucuronic acid conjugation medicated by UGT,an important phase II metabolic pathway,is a clear and detoxify mechanism of endogenous and exogenous compounds.The excessive expression or inhibition of UGT may result in low or high blood drug concentration,which may lead to treatment failure or poisoning.UGT1A1 is an important UGT widely expressed in the body especially in liver,it participates in the in vivo liver bilirubin metabolism,and is closely related to many liver diseases and drug interactions.Organic cation transporters(OCTs)are transporters that transport all kinds of endogenous and exogenous organic cations,it mainly includes three subtypes,OCT1,OCT2 and OCT3.OCTs are widely distributed in the body,and participate in in vivo transfer process of a variety of endogenous substances and clinical drugs,thus may result in interactions,which on the one hand could weaken the drug efficacy or increased toxicity,on the other hand could be auxiliary means to reduce adverse drug reactions.In the process of new drug development,it is very important to evaluate potential drug interactions.Previous researchers always focused their eyes on drug interactions mediated by CYP450,while UGT and OCTs were less noticed.In recent years,along with deeper understanding of UGT and OCTs,the influence of drugs on them has attracted more and more attention.Coptidis Rhizoma,a famous traditional Chinese medicine,has been commonly employed in traditional oriental medicine for two thousand years.It has the efficacy of clear heat drying dampness and purging fire detoxification.Modern pharmacology study proves that Coptidis Rhizoma has effects of anti-tumor,antidiabetics,neuroprotection,antioxidant,antibacterial,antihypertensive and enhance immunity,and so on.Isoquinoline alkaloids are major components of among which the contents of five kinds of berberine type alkaloids,berberine(BER),epiberberine(EPI),coptisine(COP),jateorhizine(JAT)and palmatine(PAL),are relatively higher.In addition,there is a kind of aporphine alkaloid,magnoflorine(MAG).In recent years,studies have shown that metabolic interaction can occur between Coptidis Rhizoma and other drugs.Researches confirmed that this interaction can be mediated by P450 enzyme,while whether UGT enzymes could medicate that interaction is still unknown.In addition,studies have shown that Coptidis alkaloids could interact with OCTs,however,the existing researches mainly restricted to the in vitro cell model,the in vivo data is still not sufficient.Jiao-Tai-Wan(JTW)mainly contains Coptidis Rhizoma and has been used for centuries for the treatment of insomnia in TCM.Recently some scholars found OCTs as non-classic transporters of monoamine neurotransmitters,also play an important role in their transportation.OCTs are important targets for treatment of the central related diseases,such as depression.While current researches about relation of OCTs with insomnia and whether JTW could regulate activities of OCTs have not been reported yet.Therefore,we investigated the interactions between Coptidis alkaloids and UGT enzymes and OCTs,and regulation of JTW on OCTs,which is of great importance in guiding clinical rational drug use of Coptidis Rhizoma,interpreting mechanism of Coptidis Rhizoma formula compatibility and hypnotic effect mechanism of JTW.Part one:Literature ReviewResearch progresses of UGT,OCTs,and drug-drug interactions medicated by them,interactions between Coptidis Rhizoma and UGT and OCTs,as well as pharmacological effects and mechanism of JTW have been surveyed and summarized.Part two:Experiment Studies1 In vitro and in vivo effects of six Coptidis alkaloids on UGTs and UGT1A1 activities of rat and mice liver microsomesIn the present study,the effects of six Coptidis alkaloids on UGTs and UGT1A1 activities of rat and mice liver microsomes were investigated in vitro and in vivo to study the mechanism of metabolic drug-drug intertions between Coptidis Rhizoma with other drugs.In vitro rat and mice liver microsomal incubation systems supplemented with UDPGA were applied,as well as mice liver microsomes after administration of six Coptidis alkaloids.4-Nitrophenol and P-estradiol were selected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC,respectively.For in vitro rat study,BER,EPI,COP and JAT significantly inhibited rat liver microsome UGTs activity,among them EPI showed the strongest inhibition.UGT1A1 activity was inhibited by JAT with IC50 about 227 μmol·L-1,while COP and MAG significantly activated UGT1A1 activity.For in vitro mice study,BER,COP,JAT and PAL significantly inhibited mice liver microsome UGTs activity,and six alkaloids all significantly activated UGT1A1 activity.For in vivo mice study,UGTs activity was significantly activated only in berberine group,while UGT1A1 activity was significantly activated only in jatrorrhizine group.In conclusibn,effects of Coptidis alkaloids on UGT activity showed significant difference between different species,as well as in vitro and in vivo study,which might be one of the factors causing drug-drug interactions of Coptidis Rhizoma with other drugs.2 Interaction between Coptidis alkaloids and OCTs in ratsAfter investigating the interactions between Coptidis alkaloids and UGT,we further assessed the in vivo influences of them on OCTs activities.We chose metformin as a substrate of OCT1 and OCT2,and measured its plasma concentration and pharmacokinetic parameters in rats,which were induced by Coptidis alkaloids and blank control,to evaluate effects of Coptidis alkaloids on OCT1 and OCT2 activities.Subsequently,the expression of OCTs in liver,kidney,heart and brain of rats in each group was detected by western blot.Results showed that expect for EPI(50 mg·kg-1),alkaloids of other groups all significantly increased the initial plasma concentration(Cmax)and area under the plasma concentration-time curve(AUC0～t and AUC0～∞)of metformin in rats,and decreased systemic clearance(CL)and volume of distribution(Vz),while t1/2 was not significantly influenced by them.The order of action strength was BER(100 mg·kg-1)>BER(50 mg·kg-1)>COP(100 mg·kg-1)≈COP(50 mg·kg-1)≈EPI(100 mg·kg-1)>cimetidine(100 mg·kg-1)>EPI(50 mg·kg-1).Western blot results showed that three alkaloids all inhibited OCT2 expression in kidney,and except for EPI(100 mg·kg-1),others all exhibited potent inhibition on OCT3 expression in heart.Results showed that Coptidis alkaloids inhibited activities and expression of OCTs in rats,which might result in drug-drug interaction when they co-administrate with OCTs substrates.3 Correlation of hypnotic effect of JTW with OCTsAfter confirming the inhibition of Coptidis alkaloids on OCTs activities and expression,considering JTW mainly contains Coptidis Rhizoma,and the correlation of OCTs with central nervous system function,we further investigate the correlation of OCTs with hypnotic effect of JTW.We firstly set up the "heart-kidney imbalance" insomnia rat model by PCPA to inverstigate change of OCTs activities in insomnic rats.We examined the pharmacokinetics of metformin in normal and insomnic rats.Results showed that values of CmaX,AUC0～t and AUC0～∞ were significantly decreased in insomnic rats,while values of CL and Vz were significantly increased without chaning t1/2,suggesting that insomnia pathology can induce activities of OCT1 or OCT2.To further evaluate regulation of JTW on OCTs,we investigated the effects of continuous intragastric administration of JTW for 7 days on pharmacokinetics of metformin in normal and insomnic rats.Results showed that in normal rats,JTW significantly increased the AUC0～t,AUC0～∞ and t1/2 of metformin,and significantly decreased CL,while Cmax was potently increased and Vz was potently decreased.In insomnic rats,JTW significantly increased the Cmax,AUC0～t,AUC0～∞ and t1/2 of metformin,and significantly decreased Vz and CL.That means JTW could inhibit liver OCT1 or kidney OCT2 activity,which might be the mechanism of its hypnotic effect.Part three:Summary and discussionBy summarizing,analyzing and discussing all results of the whole study,we draw the following conclusions.First of all,effects of Coptidis alkaloids on UGT activity showed significant difference between different species,as well as in vitro and in vivo study,which might be one of the factors causing drug-drug interactions between Coptidis Rhizoma with other drugs.Second,Coptidis alkaloids inhibited activities and expression of OCTs in rats,which might result in drug-drug interaction when they co-administrate with OCTs substrates.Last of all,insomnia pathology might be correlated with the rise of OCTs activities,JTW could inhibit liver OCT1 or kidney OCT2 activity,which might be mechanism of its hypnotic effect.In a word,OCTs might be important targets for its treatment of insomnia.Evaluation of drug-drug interactions medicated by metabolic enzymes and transporters on the one hand plays a key role on preventing the possible side effects,on the other hand can explain attenuated synergistic mechanism of compatibility of traditional Chinese medicine and finally promote clinical rational drug use and improve effectiveness and safety.The study of inhibition and induction of Coptidis alkaloids on UGT enzymes and OCTs activities could help to early predict clinical potential side effects and guide rational clinical use of Coptidis Rhizoma.And exploring hypnotic effect mechanism of JTW from the angle of OCTs inhibition could provide the experimental basis and guiding significance to clinical rational drug use of JTW.