Study On Effects Of Silymarin On CYP450 Metabolism Enzymes And P-gp Transporter

Pharmacogenetics studies indicate that activity change of drug-metabolizing enzymes and transpoters is the major mechanism for clinically drug-drug interactions,and inherited variation in activities of drug-metabolizing enzymes and transporters results in interindividual differences in drug metabolism,disposition and efficacy.Recently,herbal medicines are more and more widely used by people around the world.The deepgoing study of pharmacogenetics will help us to choose right drug and adjust drug dose to avoid drug side effect and attain more drug efficacy.Cytochrome P450s(CYP450) superfamily expressed widely in organisms are known to play an important role in the biotransformation of many endogenous and exogenous substances.Inhibition or induction of cytochrome P450 isozymes is one of the major causes for clinical drug-drug interactions. CYP3A enzymes are the most abundantly expressed cytochrome P450 enzymes in liver and intestine and play an essential role in the oxidative biotransformation of endogenous compounds and exogenous chemicals, including more than 60%of medically relevant drugs,such as antitumor drugs. CYP3A4 and CYP3A5 are the predominant functional forms of human CYP3A enzyme in adult.The activity of CYP3A influences curative effect of drugs and the patients' reaction to the toxicity of many medicines.In the meantime,it also has a relationship with the risk that the pollution of the environment leads to disease.The high expression of CYP3A can cause the failure of tumor chemotherapy.Effects of drugs on the activity of CYP3A is one of the main causes of drug-drug interaction.Polymorphic CYP3A5 expression in the adult liver and small intestine is strongly correlated with a single nucleotide polymorphism(6986A＞G),which,located in the intron 3 of CYP3A5,could cause alternative splicing and protein truncation resulting in the absence of CYP3A5 protein or seriously decreasing activity of CYP3A.CYP2C9 is a polymorphic enzyme that is responsible for the metabolism of many clinically useful drugs,such as S-warfarin,tolbutamide,phenytoin,glipizide,losartan and numerous non-steroidal anti-inflammatory drugs.Several allelic variants of CYP2C9 have been identified,with the most important mutations being CYP2C9~*2 and CYP2C9~*3.The CYP2C9~*3 variant is well studied and has been reported to show decreased metabolic activity for many drug substrates of the enzyme,so the therapeutic effects of its substrate drugs can be reduced or more adverse reactions should occur.The change of CYP2C9 activity is a significant factor of racial differences and individual differences in clinical medication and drug interactions.P-glycoprotein,an ATP-dependent efflux transporter coded by multidrug resistence gene(MDR1;ABCB1) and widely located at human normal tissues such as liver,kidney,small intestine and brain,plays the key role in drug absorption,distribution and elimination.Also,P-glycoprotein inhibitors are very important for reversement of multiple resistences of antitumor drugs.Silymarin is a purified extract from the seeds of milk thistle,which has been used for over 2,000 years as one of the most popular herbs worldwide.It is reported that a standardized extract of milk thistle contains at least 70-80% silymarin,that consists predominantly of up to 60%of silibinin,but also contains isosilibinin,silycristin,silydianin,and other closely related avonolignans.Silymarin is used mainly for supportive treatment of alcoholic liver disease,chronic hepatitis and cirrhosis.Milk thistle ranks among the top-selling botanical supplements in the United States and is easily available as an over-the-counter herbal remedy in China.Milk thistle has become one of the most commonly used medical remedies all over the world,and its opportunities applied with other drugs are also increasing.Up to date,although a lot of studies on silymarin pharmacological actions have been done.However,there is little report and study on metabolite and metabolic mechanism of silymarin.In addition,the inhibitive and inducing effects of silymarin on other drugs are not very clear.Inhibition or induction of cytochrome P450 isozymes and drug transporters is one of the major causes for clinical drug-drug interactions.To investigate medicines effect on CYP450s activities has important clinical significance,it helps with clinical rational administration and reducing the drug adverse reaction inducing by drug interactions.Research has found that silymarin could obviously inhibit a variety of CYP450 isozymes in mice and human liver microsomes.However, silymarin's effects on cytochrome P450s expression in human cell lines and in vivo have not been clarified.Based on above information,our study was aimed to find effects of herbal medicine silymarin on some important CYP450 enzymes and drug transporters in vitro and in vivo.By setting up cell-based platform in vitro,the effects of silymarin on CYP450 enzymes have been explored at mRNA and protein level. Furthermore,in strictly designed clinical experiments,using midazolam, losartan,and talinolol as the substrate drugs,the potential impact of silymarin on corresponding CYP450 enzymes and drug transporters based on their common genetic polymorphisms have been clarified accociated with drug pharmacokinetics and herb-drug interactions.The present series of studies have found that:1.Silymarin can significantly inhibit the expression of CYP3A4 mRNA after Chang Liver cells were treated for 48 hours,and the inhibition effect is concentration dependent.We can also find a decline of CYP3A4 mRNA level after 24-hour silymarin treatment,which is of statistic significance at concentration of 40μM;Silymarin can also significantly inhibit the expression of CYP2C9 mRNA after Chang Liver cells were treated for both 24 and 48 hours.The inhibition effect is concentration dependent and more significant for 48 hours.Silymarin's effects on CYP2C19,CYP2D6 and CYP2E1 were not observed.Silymarin can significantly inhibit the expression of CYP3A4 and CYP2C9 proteins after Chang Liver cells were treated for both 24 and 48 hours,and the inhibition effect is concentration dependent for 48 hours.2.420 mg daily administration of silymarin for 14 days significantly inhibits the metabolism of midazolam to 1-OH midazolam in healthy volunteers,with no interaction differing in individuals with different CYP3A5 genotypes.3.420 mg daily administration of silymarin for 14 days significantly inhibits the metabolism of losartan to E-3174,with the magnitude of the interaction differing in individuals with different CYP2C9 genotypes. indicating that Silymarin can significantly inhibits CYP2C9 activity in humans;4.420 mg daily administration of silymarin for 14 days increases the oral bioavailability of talinolol,and the increased plasma concentration of talinolol caused by silymarin co-administration may partially due to inhibition of intestinal talinolol efflux through P-gp.However,the interaction is not differing in individuals with different MDR1 genotypes. The present study has provided molecular mechanism for the herb-drug interactions and interindividual variations involving silymarin both in vitro and in vivo,provides experimental and theoretic guidance for combination use of silymarin formulations with sedative and hypnagogue midazolam, hypotensive drug losartan andβ-receptor antagonist talinolol as well as similar drugs.