The In Vitro Metabolism Investigation Of Bufadienolides,Active Constituents Of Chansu

Backgrounds: Chansu,the dried skin secretions of the giant toad(containing Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider),is an important traditional Chinese medicine(TCM)and is widely used in China and other Asian countries for treating a number of ailments in the clinic.Bufadienolides are the prominent active constituent of Chansu and it is contained in many famous complicated TCM formulas distributed as over-the counter drugs,namely Liushen Pill,Shexiang Baoxin Pill,and Kyushin.Modern pharmacological studies have clearly shown that BF possesses significantly pharmacological and toxicological effects including cardiotonic,anesthesia,anti-tumor and cardiotoxic effects.Bufadienolides have attracted the attention of scientists worldwide;leading to the increase of its related research and development case.Although bufadienolides display perfectly capable of killing various tumor cells in vitro,however,it is always obtained unsatisfactory results after administration in vivo,indicating the obvious flaw in the druggability of bufadienolides.Multiple information demonstrate that the metabolism of bufadienolides in vivo are fast,which may influent the effectiveness of bufadienolides.At present,there are some reports regarding the metabolism of bufadienolides,but considerable portion of these studies are achieved by using experimental animals.Considerations that the well-known significant difference on the metabolic enzymesbetween human and animal species,the related information obtained using animal model are debatable.Hitherto,the metabolism route,the involved key metabolic enzyme in human and different animal species as well as the potential interaction with the key metabolic enzyme of bufadienolides are still not well studied.Aims: The prominent aims of the present studies are 1)Characterization the major metabolic route,key metabolic enzyme and the kinetic behaviors;making an attempt to analyze the variation on substituent group towards the enzyme selectivity and metabolic rate both quantitatively and qualitatively.2)Combined with theoretical calculation,finding key factors that may influent the interaction between bufadienolides and metabolic enzyme,as well as clarifying the regulation of the bufadienolides metabolism.3)Investigation of the interspecies difference on the metabolism of bufadienolides,including comparison the similarity and difference of the metabolic profiling,involved enzyme and kinetic behavior between human and common experimental animals.4)Elucidation of interaction between bufadienolides and its key metabolic enzymes,including the inhibitory effects of the representative bufadienolides bufalin towards CYP450 as well as the inhibitory effects of potential co-administered drugs towards the metabolism of bufalin.The collective information could provide research foundation and theoretical basis for the development of new bufadienolides agent as well as the rational use of bufadienolides and it contained traditional Chinese medicines.Methods: 1)By means of in vitro incubation with human liver preparation,investigate the Phase I and Phase II metabolic routes of bufadienolides.Identify the metabolite profiling by metabolic profile analyses and metabolite preparation;characterize the key metabolic enzyme by specific enzyme inhibitor study and correlation analysis;analyses the metabolic stability of bufadienolides by kinetic analysis or in vitro metabolic half-life study.2)By means of molecular docking,investigate the key amino-acid residue or structural domain of metabolic enzyme that influent the interaction of bufadienolides and enzyme.3)characterize the inhibitory specific,inhibitory effect(IC50),inhibitory kinetic and related parameters(Ki、KI、Kinact)ofbufalin towards human CYP450 as well as the inhibitory effects of co-administrated drugs towards CYP450-mediated bufalin metabolism.Results:The in vitro metabolism study of bufadienolides1)The in vitro Phase I metabolism study:On the basic results of the previous study of cinobufagin,the in vitro Phase I metabolic route study of bufalin,resinobufagenin,bufotalin,gamabufotalin and arenobufagin were carried out.It found that the above mentioned bufadienolides could be selectively catalyzed by CYP3As(including CYP3A4 and CYP3A5)in human liver microsomes.The major metabolites were1-hydroxyl-and 5-hydroxyl-bufadienolides.However,the metabolic stability,the catalysis preference between CYP3A4 and CYP3A5,as well as the difference on the apparent kinetic parameters were all varied greatly among the bufadienolides.2)The relationship of buafdienolides-CYP3As: It found that the affinity between bufadienolides and CYP3 As was association with the metabolic stability in human liver microsomes as well as the contribution of CYP3A5.In human liver microsomes,the bufadienolides with small Km always exhibited bad metabolic stability.Additionally,in recombinant CYP3 A,the introduction of different substituent group may influence the affinity between bufadienolides and CYP3A4/3A5 to different degree,thereafter influence the contribution of CYP3A4 and CYP3A5 in the Phase I metabolism of bufadienolides.The relationship of bufadienolides-CYP3 As was as follows: hydroxyl group at C11,keto group at C12 could improve the affinity for bufadienolides of CYP3A5,so then accordingly improve the contribution of CYP3A5 in the metabolism;acetyl group at C16,aldehyde group at C19 could reduce the affinity for bufadienolides of CYP3A4,meanwhile improve the contribution of CYP3A5 in the metabolism,so then accordingly improve the contribution of CYP3A5 in the metabolism3)The in vitro Phase II metabolism study: The in vitro Phase II metabolic route study of cinobufagin,bufalin,resibufogenin,bufotalin,telocinobufagin and deacetylcinobufagin were carried out.It found that the above mentioned bufadienolides couldbe selectively catalyzed by SULT2A1 in human liver S9.The major metabolites were bufadienolides-3-O-sulfates.The formation of conjugated metabolites were obey to substrates inhibition model,however,the apparent kinetic parameters including Km,Ksi and Vmax were all varied greatly among the bufadienolides.4)The relationship of buafdienolides-SULT2A1: The results demonstrated that the configuration of 3-hydrxyl group was the key factors influence the sulfation of bufadienolides,the His99 was the key amino-acid residue responsible for the discernment of bufadienolides.The conformation and orientations of 3-hydroxyl of bufadienolides as well as its interaction with His99 decide the progress of conjugated reaction.The relationship of bufadienolides-SULT2A1 was as follows: the hydrophilic substitution(e.g.hydroxyl group at C11,C14,C16)could weaken the affinity for bufadienolides of SULT2A1,while the hydroxyl group at C5 could improve the affinity;aldehyde group at C19 could hinder the conjugated reaction;acetyl group at C16 could reduce the affinity for bufadienolides of SULT2A1.2.The interspecies difference on the in vitro metabolism of bufadienolides1)The interspecies difference on the Phase I metabolism of bufadienolides: The metabolic profile of bufalin(BF)and resibufogenin(RB)in liver microsomes of different animal species was similar with that in human liver microsomes.The CYP3 As were identified as the prominent enzyme for the metabolism reaction.However,the kinetic behavior and apparent kinetic parameters of 5-hydroxylation of BF and RB were varied greatly among the different animals liver microsomes.The kinetic behavior and related parameters of BF 5-hydroxylation in minipig and mouse liver microsomes were similar with that in human liver microsomes,and the kinetic behavior of RB 5-hydroxylation in monkey liver microsomes were similar with that in human liver microsomes.The catalysis efficiency of monkey liver microsomes for BF and RB were significant higher than that of other animal liver microsomes.The in vitro mouse liver microsomes intrinsic clearance for bufalin and resibufogenin were close to the values of human liver microsomes.2)The interspecies difference on the Phase II metabolism of bufadienolides: Thesulfate conjugated metabolic reaction of BF and RB were could not detected in liver S9 of mouse and dog,while the liver S9 of rat,rabbit,guinea pig,minipig and monkey could mediate the sulfation of the two bufadienolides.The SULT2A1 were identified as the prominent enzyme for the metabolism reaction.The catalysis efficiency of rabbit liver S9 for BF and RB were significant higher than that of other animal liver microsomes.The order of in vitro liver microsomes intrinsic clearance for BF and RB were as follows: Rabbit(human)> minipig > guinea pig > monkey > rat.3.The inhibitory effect investigation of bufalin towards CYP3As1)Bufalin(BF)and its 3-oxdiative metabolite 3-keto-bufalin(KBF)were the strong and selective inhibitors of CYP3 As,the IC50 values were 10.4 and 0.22 μM,respectively.The inhibition kinetic investigation demonstrated that the inhibitory effect of BF and KBF obey to mix-inhibition model,the Ki value were 1.90 and 0.20 μM,respectively.2)The inhibitory effect of BF towards CYP3 As was time-dependent,which was associated with the CYP3A4-mediated formation of KBF.It suggested that the inhibition action towards CYP3A4 was suicide inhibition.Furthermore,the inhibitory effect of KBF towards CYP3A4 was improved after 30 min pre-incubation with NADPH generating system,the IC50 value was reduced to 0.036 μM from 0.37 μM.The inactivation of CYP3A4 was time-and concentration-dependent,obeying to first-order kinetic.In human liver microsomes,the inactivation parameters including KI and Kinact of CYP3 As mediated by BF was 21.1 μM and 0.141 min-1;the inactivation parameters including KI and Kinact of CYP3 As mediated by KBF was 0.66μM and 0.064 min-1,respectively.In recombinant human CYP3A4 s,the inactivation parameters including KI and Kinact of CYP3A4 mediated by BF was 1.13 μM and0.097 min-1;while the inactivation parameters including KI and Kinact of CYP3A4 mediated by KBF was 0.25 μM and 0.161 min-1,respectively.3)The inhibition mechanism of KBF towards CYP3A4 was further disclosed by microsomes washing and adding protective agent assay.The results suggested that the irreversible inhibition towards CYP3A4 was due to the formation of active metabolite.The strong inhibitory effect may lead to the loss of the functional activity of CYP3A4 which incur the serious drug-drug interaction,or acute and chronic cardiotoxity,hepatotoxicity as well as other unforeseen severe toxic reactions.Conclusions:1)The CYP-mediated hydroxylation as well as the SULT-mediated sulfation were identified as the prominent phase I and phase II metabolic pathway of bufadienolides in human.The C5 and C1 was the major metabolic sites of all the investigated bufadienolides which mediated by CYP3 As.Additionally,the C3-hydroxylation was the conjugated site of bufadienolides which catalyzed by SULT2A1.The configuration of 3-hydroxyl was the key factor affecting the occurrence of sulfation reaction.2)The substituent group could influence the bufadienolides-metabolic enzyme interactions,thereafter,the kinetic behavior and parameters were varied greatly.It found that the substituent group could significantly influence the affinity of bufadienolides with CYP3 As and SULT2A1.The investigation of the relationship of bufadienolides-phase I metabolism indicated that the C11-hydroxyl and C12-keto could improve the affinity for bufadienolides of CYP3A5,so then accordingly improve the contribution of CYP3A5 in the metabolism;acetyl group at C16,aldehyde group at C19 could reduce the affinity for bufadienolides of CYP3A4,meanwhile improve the contribution of CYP3A5 in the metabolism,so then accordingly improve the contribution of CYP3A5 in the metabolism.The investigation of the relationship of bufadienolides-SULT2A1 was that the hydrophilic substitution(e.g.hydroxyl group at C11,C14,C16)could weaken the affinity for bufadienolides of SULT2A1,while the hydroxyl group at C5 could improve the affinity;aldehyde group at C19 could hinder the conjugated reaction;acetyl group at C16 could reduce the affinity for bufadienolides of SULT2A1.3)The phase I and phase II metabolic profiles of bufalin and resinobufagenin in various liver preparations obtained from different animals were similar with that in human liver preparations,but the sulfation pathway were defective in liver of mouse and pig.The CYP3 As and SULT2A1 were identified as the prominent enzyme for thephase I and phase II metabolism of bufalin and resinobufagenin in various animals liver preparations.However,the catalytic efficiency for bufalin and resinobufagenin were significant different among the interspecies liver preparations.4)Bufalin and its 3-oxdiative metabolite 3-keto-bufalin were the strong selective inhibitor of CYP3 As.It firstly found that 3-keto-bufalin could lead to the irreversible inhibition towards CYP3A4 possibility by the formation of active metabolite.The strong inhibitory effect of KBF may lead to the loss of the functional activity of CYP3A4 which could classify as suicide inhibition.Our results indicated that the inhibitory effect of bufalin and its metabolite toward CYP3A4 not only affect the metabolism of bufaidneolides,but also may disturb the metabolism of other co-administrated drugs incurring severe unforeseen toxic reactions...