The Mechanism Of Metabolism And Drug-drug Interactions Of Anti-HIV Active Ingredient Morusin

Background and ObjectivesMorusin is a prenylated flavone isolated from mulberry,the root bark of Morus alba Linn.(Moraceae)and branch bark of Ramulus mori.An increasing number of studies have proved that multiple pharmacological activities are present in morusin,including antitumor,antibacterial,antioxidant,antinociceptive,anticonvulsant,and anti-inflammatory activities.In 2015,morusin was identified as the standard quality control component in SH formula,which was applied for HIV treatment in Thailand as the first natural botanical medicine in the world.After screening of more than 20 active Chinese medicinal herbs for anti-HIV activity,SH formula had been developed(consists of five traditional Chinese herbs)under the principles of "Monarch,Minister,Assistant,and Guide".Its therapeutic efficiency of HIV was 89%,could be long-term used for HIV-infected people,and it was officially approved for sale in Thailand in August 2012.SH formula is the first natural anti-HIV plant drug in China that has obtained a new drug certificate abroad and listed,which creates a pioneer in the treatment of HIV with Chinese herbal medicine.As the main active ingredient in the SH formula,it is speculated that morusin may also have excellent anti-HIV efficacy.There is no relevant report on the anti-viral efficacy of morusin.On the other hand,morusin is 5,2',4'-Trihydroxyflavone.It has many unbound phenolic hydroxyl groups on the molecule and is very likely to be catalyzed by the glucuronyltransferase to produce conjugation reaction which is also the main metabolic pathway of flavones.At present,there is a lack of in-depth research on the metabolic mechanism of morusin.Previous studies have only conducted brief study on the CYP metabolism of morusin,and its glucuronication metabolic pathway has not been reported in the literature.Although LC-MS/MS method has been applied to determine the morusin in the plasma,the pharmacokinetic characteristics of its metabolites in vivo have not been studied.It is of great significance to fully understand the disposition of drug in vivo for elucidating its pharmacological effects and functional mechanism.Therefore,in this study:(1)the first aim of our study was to identify the anti-HIV activity of morusin,confirmed its pharmacological activity.It would provide the necessary research foundation for the druggability study of morusin to be a new therapeutic anti-HIV drug.(2)systematically study of the metabolic mechanism and pharmacokinetic characteristics of morusin,and elucidate its major exposure forms and levels in vivo,which are helpful to predict its efficacy and potential toxic reactions,and are of great importance to the development and clinical application of morusin.In addition,at present,the clinical treatment of HIV is usually a cocktail therapy including multiple drugs,but long-term use may cause serious toxic and side effects and lead to the emergence of a large number of drug-resistant strains.Traditional Chinese medicine treatment of HIV has the characteristics of peaceful and lasting effects,improving immune function,low toxic and side effects,unlikely drug resistance,low price,suitable for national conditions,good patient compliance,and long-term use.Therefore,the combination of traditional Chinese medicine and chemical drugs in the treatment of HIV has become a new treatment trend,with broad prospects for development.As a potential natural anti-HIV product,when morusin is used in combination with other chemical drugs,we have to consider the metabolism-related drug interactions between various drugs because of its extensive metabolism in vivo.This helps to optimize the dose,avoid side effects,and provide important theoretical basis for the safety and effectiveness of clinical use.In this paper,we have conducted in-depth studies on the metabolic characteristics and metabolism-based drug interactions of the morusin using in vivo,in vitro,and in situ experimental models.First,we used TZM-bl cells to study the inhibitory activity of morusin on HIV-1SF162 infection,and used anti-HIV chemical drug tenofovir as a positive control to verify method reliability and compared with the anti-HIV activity of morusin.Then,rat intestinal perfusion model was used to study the absorption and metabolism characteristics of morusin in rat,and collected bile to investigate the excretion.Recombined humanized CYPs,UGTs,and specific chemical inhibitors was incubated with morusin in vitro to confirm the main enzyme isoforms involved in its metabolic reactions.We compared the enzymatic kinetic parameters of morusin in the human liver microsomes,rat liver microsomes,and major metabolic enzyme isoforms to elucidated the metabolic mechanism of morusin.Finally,a rapid and sensitive LC-MS/MS method for the simultaneous detection of the morusin prototype and the main metabolites was established.Using this method,we investigated the pharmacokinetics study of SD rats administered orally with morasin and clarified the main exposure forms and levels of morusin in vivo after oral administration.At the same time,the drug-drug interactions between morusin and anti-HIV chemical drugs were investigated.We studied the changes in plasma drug concentration-time curves and major pharmacokinetic parameters when the two drugs are used in combination,compared to that each drug is administered alone.Methods1.Study on Anti-HIV Efficacy of Morusin in vitroTZM-bl cells were used to determine the inhibitory activity of morusin on HIV-1SF162 infection,and calculate the drug inhibition rate against the virus.Tenofovir,an anti-HIV chemical drug,was used as a positive control.2.Study on Absorption and Metabolism of Morusin in Rat IntestineWe used rat intestinal perfusion model,and collected perfusate and bile samples,analyzed the absorption and metabolism characteristics of morusin in rat intestine.Then we incubated human intestinal microsomes and prepared rat intestinal microsomes with morusin in a glucuronidation metabolism system to study the metabolism characteristics of morusin in intestine.3.Study on the Metabolic Mechanism of Morusin in vitro9 recombined humanized CYPs,12 UGTs,and specific chemical inhibitors was incubated with morusin in vitro,thus to study the main enzyme isoforms mediated morusin metabolism.Then we compared the enzymatic kinetic parameters of morusin in human liver microsomes,rat liver microsomes,and the major metabolic enzyme isoforms,to elucidate the metabolic mechanism of morusin.4.Study on the Pharmacokinetics of MorusinWe used the rat pharmacokinetic model to study the pharmacokinetics of rats after oral administration of morusin(8 mg/kg and 16 mg/kg).A rapid and sensitive LC-MS/MS method was used to detect the content of morusin and its main metabolites in rat plasma,plotted the corresponding drug concentration-time curves,and calculated the main pharmacokinetic parameters using the WinNonlin(?)3.3 non-compartmental model.5.Drug Interaction between Morusin and Anti-HIV drugsWe used the rat pharmacokinetic model,and simultaneously administered two drugs(Morusin and anti-HIV chemical drug)to rat,then measured the concentrations of drug and its metabolites in plasma.Studied the changes in plasma drug concentration-time curves and major pharmacokinetic parameters when the two drugs are used in combination,compared to that each drug is administered alone.Results1.Study on Anti-HIV Drug Efficacy of Morusin in vitroThe morusin had a significant inhibitory activity against HIV-1SF162 infection(IC50 5.27 ?M).Tenofovir,as a positive control group,its IC50 2.64 ?M.2.Identification of Metabolites of MorusinThrough UPLC-MS/MS mass spectrometry analysis combined with UV spectrum peak deviation method,four phase II(M-5-G,MII-2,M-4'-G and M-2'-G)and two phase I metabolites(MI-1 and MI-2)of morusin were identified.3.Absorption and Metabolism of Morusin in Rat IntestineThrough rat intestinal perfusion experiment,duodenum and jejunum were the main absorption and metabolic positions of morusin in rat intestine,the absorption mode was passive diffusion.M-4'-G was the major metabolite.4.Study on the Mechanism of CYP and UGT Metabolism of MorusinThrough specific chemical inhibitors and recombined humanized enzymes assays,the CYP metabolism of morusin was mainly mediated by CYP2C19 and CYP3A,and CYP2B6,2C9,2D6,and 1A2 also have certain functions.However,CYP2C8 and 2E1 hardly participated in the metabolism.And UGT1A1,1A3,1A7,and 2B7 were mainly responsible for the glucuronidation metabolism of morusin.5.Pharmacokinetics of Morusin in ratsThis study established a sensitive and reliable UPLC-MS/MS quantitative method that can simultaneously detect the morasin and four metabolites in rat plasma.After oral administration(8 and 16 mg/kg),morusin was quickly absorbed into the blood from the rat gastrointestinal tract and converted to four metabolites,including 3 mono-glucuronides and 1 hydroxylated metabolite,namely M-5-G,MII-2,M-4'-G and MI-1.The Morusin prototype peaked at approximately 30 to 40 min and was completely eliminated at 48 h.The Cmax and AUCO-t were respectively 299.12 ±119.52 ng/mL and 98.32 ± 21.13 min·?g/mL(16 mg/kg group).In metabolites,the plasma concentration of M-4'-G is much higher than the other three metabolites,and AUCo-t are over 10 times higher than that of other metabolites.The drug concentration-time curves of M-5-G,M-4'-G,and MI-1 were consistent and peaked within 60 min and then gradually eliminated,but MII-2 was quite different,it peaked very late(672~800 min),resulting in its longer elimination time and residence time in vivo and it could still be detected in plasma at 36 h after administration.The pharmacokinetic curves of morusin and its conjugates of two concentration groups both presented double peaks,showing that an enterohepatic recycling might exist.6.Drug-Interaction of Morusin and Anti-BHIV drugsThis chapter examines the effects of respective pharmacokinetic characteristics of morusin and anti-HIV chemical drugs after they were administrated orally together.First,we investigated the effect of atazanavir sulfate on the pharmacokinetics of morusin.The results showed that when morusin was co-administered with atazanavir sulfate,atazanavir could significantly affect the pharmacokinetics of morusin,resulting in a significant increase in Cmax and AUCo-t of morusin,approximately increased to 1.5 times.The Cmax of each metabolite also increased significantly,by approximately 1.4 to 2 times.Subsequently,we examined the effects of morusin on the pharmacokinetics of anti-HIV chemical agents atazanavir sulfate and efavirenz.The results indicated that the effect of morsuin on the pharmacokinetics of atazanavir sulfate were minor.Only the Cmax of atazanavir was found to increase to 1.32 times,and there was no significant change in other pharmacokinetic parameters.However,when morusin was co-administered with efavirenz,significant drug-drug interactions occurred.The Cmax and AUC0-t of efavirenz were increased to 2.22 and 1.62 times,respectively,and MRT0-t was decreased.The differences were statistically significant.Conclusion1.This study firstly investigated the anti-HIV efficacy of morusin itself in vitro and found that morusin has significant inhibitory activity against HIV-1SF162 infection(IC50 = 5.27 ?M)and has no apparent toxic effect on cells.It is suggested that morusin may become a potential alternative monad for anti-HIV natural product and it has good prospect in development and application.2.In this experiment,we used a combination of in vitro,in situ and in vivo experiments to study the metabolic and pharmacokinetic characteristics of morusin,and found that each data had a good correlation.Morusin could be quickly converted into metabolites in vivo after oral administration.In addition to the prototype drug,the plasma contained three mono-glucuronides(M-5-G,M?-2,M-4'-G)and one hydroxylated metabolites(M?-1),of which M-4'-G was the most important metabolite.The CYP metabolism of morusin was mainly mediated by CYP3A4,3A5 and 2C19,while UGT1A1,1A3,1A7 and 2B7 were mainly involved in its glucuronidation metabolism.The comprehensive investigation of metabolic mechanism and pharmacokinetics study may provide new perspective and theoretical basis for the in vivo efficacy study of morusin,and provide new ideas and methods for improving bioavailability.3.By investigating the drug-drug interactions when morusin was co-administered with anti-HIV chemical drugs,we found that when morusin was administered together with atazanavir sulfate,atazanavir sulfate could significantly affect pharmacokinetic behaviors of morusin.The Cmax and AUC0-t of morusin significantly increased to 1.5 times.The Cmax and AUC0-t of each metabolite also increased.However,the effect of morusin on the pharmacokinetics of atazanavir sulfate is minor.Only found the Cmax of atazanavir was increased to 1.32 times.While morusin was used in combination with efavirenz,there was a significant drug-drug interaction.The Cmax and AUC0-tof efavirenz were increased to 2.22 and 1.62 times,respectively.These results showed that when morusin is used clinically as a new anti-HIV natural product in combination with atazanavir sulfate or efavirenz in future,significant drug-drug interactions may occur,thus increase drug blood concentration and enhance efficacy.People should pay attention to adjust the clinical dose,reduce toxicand side effects.