Study On The Interaction Between Polyether Antibiotics And Enrofloxacin In Chickens Based On CYP Enzyme

Polyether antibiotics are mainly used to prevent and treat chicken coccidiosis.They have a broad spectrum of resistance to coccidiosis.They can also promote growth,improve feed utilization,and increase breeding efficiency.Therefore,they are widely used in the livestock and poultry breeding industry.It is estimated that 45%of the feed is added with polyether antibiotics.Enrofloxacin（ENR）is a broad-spectrum antibacterial drug that has a good therapeutic effect on poultry intestinal or respiratory infections caused by Escherichia coli,Pasteurella and Mycoplasma.It has good oral absorption and high bioavailability.Thereby,it is widely used in the treatment of bacterial diseases in the livestock and poultry breeding industry.Due to the wide application of polyether antibiotics and ENR in veterinary clinics,they are often used at the same time.CYP450s is the main metabolic enzyme system for the biotransformation of drugs in the body,and it may be induced or inhibited by drugs.Therefore,the combined use of two drugs may affect the activity of metabolic enzymes,then accelerating or slowing down the metabolism of the two in animal.When the metabolism is accelerated,the effect of drugs will be reduced;when the metabolism slowed down,the toxicity of drugs to animals will be increased.Besides,the withdrawal period of a single drug will also be prolonged,resulting in the risk of excessive drug residues in animal edible products,which can do harm to the health of consumers.According to reports,both ENR and salinomycin（SAL）can inhibit the activity of the metabolic enzyme CYP3A,which is the main metabolic enzyme of ENR and SAL.We speculate that when ENR and SAL are used clinically at the same time,it is likely that there will be an interaction due to the inhibition of CYP3A.However,the effect of polyether drugs and ENR on chicken CYP450 enzyme activity and the drug interaction after their combined use have not been reported.Therefore,this study uses monensin（MON）,maduramycin（MAD）,lasalocid（LAS）,SAL and ENR as the research objects,and uses chicken liver microsomes as in vitro metabolism model to study wheather polyether antibiotics and ENR can induce or inhibit the activities of chicken metabolic enzymes.Through the pharmacokinetic study of the combined administration of SAL and ENR in chickens,it is clarified whether the combination of the two drugs will affect each other’s disposal process in chickens.And the research of expression levels of metabolic enzyme genes and proteins in chicken livers will further clarify the molecular mechanisms of the interaction between SAL and ENR.This study will clarify the risks of the combination of SAL and ENR.It has great significance in guiding the rational clinical application and compatibility of polyether antibiotics and ENR.In the meantime,it will also provide a theoretical basis for the interaction of other drugs.1 Establishment of in vitro incubation system and detection method for chicken liver microsomesThe chicken liver microsomes were prepared by differential centrifugation.The protein concentration,reaction time,and substrate concentration were optimized to determine the optimal reaction conditions.The high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）detection method of four substrates and its’metabolites as well as the high performance liquid chromatography（HPLC）detection method of two substrates were established.The results showed that the six probe drugs can be metabolized into target metabolites in the prepared liver microsome system,indicating that the chicken liver microsome system has been successfully constructed.2 The effects of four polyether antibiotics and ENR on the activity of metabolizing enzymes in chicken liver microsomesSAL and LAS can induce the activity of CYP2A6 in chicken liver microsomes when pre-incubation time is 5 minutes,and the induction rates are 17%and 25%,respectively.SAL induced the activity of CYP2E1 when the pre-reaction time is 0 and 5 minutes,the induction rate is 37%and 23%,respectively.When the pre-reaction time is 0 minutes,LAS induced the activity of CYP2E1 at a rate of 64%.MAD induced CYP2D6 and CYP2E1 without pre-incubation,and the induction rates are 12%and 13%,respectively.When the pre-reaction time is 30 minutes,SAL,LAS,MON and MAD can all inhibit the activities of CYP2D6,CYP 2C23a,CYP 2C45,CYP2E1 and CYP3A4 in chicken liver microsomes,which is time-dependent and all of the maximum inhibition rate were exceeding 50%.ENR can inhibit the activity of CYP3A4 in a time-dependent manner.The inhibition rate is 30%when the pre-incubation is 30 min.However,when the pre-reaction time is 0 and 5 min,it can induce the activity of CYP3A4.The induction rates were 38%and 25%,respectively.But ENR had no significant effect on the enzyme activities of CYP2D6,2C23a,2C45 and 2E1.The results show that both ENR and SAL can inhibit their main metabolic enzyme,suggesting that there is a risk of drug interaction when they are administered in combination.3 Study on the effects of SAL and ENR on the pharmacokinetics of chickensCompared with the ENR single administration group,the AUC_0-36h and AUC_0-∞of ENR in the single combined administration group increased by 31%and 29%,respectively.The AUC_0-36h and AUC_0-∞of ENR in SAL 5-day pretreatment group increased by 20%and 19%,respectively.When SAL and ENR are co-administered,SAL will increase the exposure and exposure time of ENR in vivo,because SAL inhibited the expression of CYP3A,the main metabolic enzyme of ENR,considering the in vitro results of the liver microsome incubation test.Compared with the SAL single administration group,the AUC_0-48h,AUC_0-∞,C_max and V_z of SAL in the single combined administration group were reduced by 32%,42%,33%and 38%,respectively;In the meantime,MRT_0-48h and MRT_0-∞increased by 14%and 15%,respectively.AUC_0-48h,AUC_0-∞,MRT_0-48h,MRT_0-∞,C_max and t_1/2 of SAL in the ENR 5 d pretreatment group increased by 87%,87%,118%,141%,64%and 16%,respectively.This indicates that when SAL and ENR are co-administered,ENR will reduce SAL exposure and exposure time in a short period of time,but SAL exposure level will be increased after a longer period of time.Combined with the results of the liver microsome incubation test,this may be due to the time-dependent characteristics of ENR’s inhibitory effect on the expression of SAL’s main metabolic enzyme CYP3A.In a short period of time,ENR can induce the expression of CYP3A,while ENR will inhibit the expression of CYP3A after prolonged exposure.Therefore,the metabolism of SAL become faster firstly and then slowed down.Above all,the co-administration of ENR and SAL will increase the exposure level of ENR in chickens;while a single co-administration will reduce the exposure level of SAL in vivo,and continuous administration for a long time will increase the exposure level of SAL in vivo.It proved that there is interaction between ENR and SAL,and long-term co-administration will significantly increase the exposure level of ENR and SAL in vivo.The results suggest that clinical co-administration will not only increase the toxicity of ENR and SAL to chickens,but may also prolong the drug withdrawal period in edible tissues of chickens,posing a threat to human health.4 The interaction mechanism of SAL and ENRCompared with the ENR alone administration group,the mRNA expression of CYP2D6,CYP1A2,CYP2C23a and CYP2C45 in the SAL 5 d pretreatment group was significantly reduced by 76.92%,97.45%,95.25%and 93.58%,respectively.In the meanwhile,the protein expression of CYP3A4 in the SAL 5 d pretreatment group was not significantly affected.While the differences of metabolic enzyme mRNA or protein is no significant between the single combined administration and the ENR alone administration group.It supported the pharmacokinetic results,and further shows that after ENR and SAL were co-administered,SAL will inhibit the expression of ENR’s secondary metabolic enzyme genes,resulting in a slowdown in metabolism of ENR and an increase exposure of ENR in body.Compared with the SAL single administration group,the mRNA expression of CYP3A4,CYP2D6,CYP1A2 and CYP2C45 in the single combined administration group were significantly increased by 353%,391%,548%and 356%,respectively;and the protein expression of CYP3A4 in the single combined administration group was significantly increased by 49%.In the ENR 5 d pretreatment group,only the mRNA expression of CYP2C45 increased by 155%.There was no significant difference in the mRNA expression of CYP3A4,but the protein expression of CYP3A4 decreased by 30%.This result is consistent with the pharmacokinetic results,revealing that when ENR and SAL are co-administered,exposure of SAL decreased because ENR increased metabolism of SAL by inducing the mRNA and protein expression of CYP3A,and then increasing the activity of CYP3A in the case of a single co-administration.When continuously administrated with ENR,exposure of SAL increased because ENR decreased metabolism of SAL by inhibiting the protein expression of CYP3A,which is the main metabolic enzyme of SAL.In summary,this project established a liver microsomal metabolism system and the detection method for probe substrates.We have also determined the main metabolic enzyme of SAL and ENR in chickens and the effects of polyether antibiotics and ENR on chicken CYP450 metabolic enzyme activities.The exposure of SAL and ENR after their combined administration clarified that the combined use of SAL and ENR will increase the exposure level of each other,suggesting that the two drugs will increase the risk of toxicity and residual excess when clinically co-administered,resulting harm to consumers.In addition,the molecular mechanism based on the interaction of CYP450 metabolic enzymes was further clarified by detecting the expression of CYP450s’mRNA or CYP3As’protein in the liver after combined medication.This study not only provides risk recommendations for the interaction between ENR and SAL and other polyether antibiotics,but also provides references for the rational use of the two drugs in the clinic.