Study On The Effect Of Doxycycline On The Metabolism Of Florfenicol In Goats In Vivo And In Vitro

At present,florfenicol（FF）is widely used in the treatment of goat infectious pleuropneumonia,colibacillosis and other diseases.FF and Doxycycline（DOX）is a common drug combination in veterinary clinic.When FF is combined with DOX,there will be a synergistic effect.When two drugs are used in combination,it is not only the efficacy that needs to be considered,but also the possible interactions between them.The drug-drug interactions are mainly metabolic interactions,the acceleration of metabolism will lead to the decrease of drug efficacy.Slower metabolism can lead to drug accumulation,which can increase drug toxicity or exacerbate residual problems.In addition,according to the monitoring of the China’s Ministry of Agriculture over the years,the phenomenon of excessive single pollutant residue in fresh food has been reduced,while the coexistence of multiple low-residue pollutants has increased significantly.Therefore,it is necessary to study the interaction between FF and DOX from the perspective of metabolism.In this study,liver microsomes and recombinant CYP450 metabolic enzyme system were used to study the metabolism of FF and DOX in goats in vitro models,a residue elimination test was carried out in goats to study the metabolic rules and residue elimination rules of the combination of the two drugs in vivo,and a reasonable withdrawal time for the combination of the two drugs was proposed.The research contents and results are as follows:1 The effect of DOX on the metabolism of FF in liver microsomesIn order to study the effect of DOX on the metabolism of FF in goat liver microsomes,this experiment was divided into two groups:in the single group,only FF was incubated with liver microsomes;in the combined group,FF and DOX were co-incubated with liver microsomes,and the influence of DOX on FF metabolism was determined by comparing the concentration of florfenicol amine（FFA）.The results showed that compared with the single group,the addition of DOX in the combined group would significantly reduce the concentration of FFA,that is,DOX inhibited the metabolism of FF.In order to further explore the relationship between FF metabolism and CYP450 enzyme,the experiment selected six mainly metabolic enzyme CYP2D6,CYP2E1,CYP2A6,CYP1A,CYP2C9 and CYP3A inhibitors:quinidine,4-methylpyrazole,methoxsalin,geraniol,sulfafenpyrazole,ketoconazole and FF were incubated in liver microsomes.Through the determination of the concentration of FFA,the most important metabolic enzyme of FF metabolism were screened.The results showed that the metabolic enzyme CYP3A had the most significant effect on FFA production,that is,the most critical metabolic enzyme for FF in goat liver microsomes was CYP3A.2 The relationship between CYP3A24 and the metabolism of DOX and FFTo further study the role of CYP3A in the metabolism of FF and DOX.In this experiment,by comparing nucleotide and amino acid sequences,it was determined that the goat CYP3A24 gene had the highest homology with the human CYP3A4 gene.Then,the human CYP3A4 was used as a template for homology modeling and the protein model of goat CYP3A24 was obtained.Then,CYP3A24 was docking molecular with FF and DOX,respectively.The docking molecular results showed that the key amino acids affecting the metabolism of FF may be R105,R372 and R440,the key amino acids affecting the metabolism of DOX may be T392 and R440.The plasmid p ET-28a（+）-CYP3A24 was obtained by prokaryotic vector construction technology.Through site-directed mutation technology,amino acids obtained from molecular docking were mutated,then CYP3A24 and mutant proteins were obtained by protein induction expression and protein purification technology.CYP3A24 and each mutant protein were incubated with FF,respectively.The most critical amino acid that CYP3A24 affects FF metabolism were determined by the change of FFA production.The results showed that when the amino acid R105 was mutated to A105,the metabolism of FF was accelerated,while R372 was mutated to A372 and R440 was mutated to A440,the metabolism of FF was slowed down.Among them,R440 had the most significant effect on the metabolism,that is,R440 may be the most critical amino acid affecting the metabolism of FF.To study the relationship between the effect of DOX on FF metabolism and CYP3A24.There were two drug groups incubated with CYP3A24.One group was only FF,the other was FF and DOX.The results showed that when FF was incubated with CYP3A24protein,the addition of DOX could inhibit the metabolism of FF.Since R440 is the common site of DOX and FF docking with CYP3A24,R440 is the most critical amino acid that affects the metabolism of FF.Therefore,this experiment speculates that when DOX is used in combination with FF,DOX will compete with R440 that metabolizes FF in CYP3A24,thereby affecting FF metabolism.3 Establishment of a method for the determination of FF,FFA and DOX in edible tissues of goatsThe extractant of FF and FFA was 2%ammoniated ethyl acetate.After extraction twice,the supernatant was blown dry with nitrogen,dried and reconstituted with 5%acetic acid.The purification method was MCX solid phase extraction,n-hexane fat-free and 0.22μm filter membrane.The extractant of doxycycline hydrochloride（DOX-HCL）was Na₂EDTA-Macilvaine buffer solution,after two oscillating extractions,it was dried with nitrogen,and the precipitate was dissolved in methanol.The purification method was HLB solid phase extraction column,n-hexane defatting and 0.22μm filter membrane.The mobile phase was 0.1%formic acid water and acetonitrile.The gradient elution is as follows:0-1 min,10%acetonitrile;1-5 min,10%-70%acetonitrile;5-6 min,70%acetonitrile;6-6.1 min,70%-10%acetonitrile;6.1-9 min,10%acetonitrile.Electrospray ionizatio is positive and negative ion mode.The correlation coefficients between the concentrations of FF,FFA and DOX-HCl and their peak areas were all greater than 0.99.The detection limit of this method was 5μg/kg,and the quantitation limit was 10μg/kg.At LOQ,1/2 MRL and MRL supplemental concentrations,the recovery of FF,FFA and DOX-HCL in liver,kidney and muscle was 70.62%to 113.55%,with intra-batch coefficient variation less than 11.08%and inter-batch coefficient variation less than9.93%.This method meets the requirements for residue analysis in the guidelines for veterinary drugs.4 Residue elimination test of FF and DOX-HCL in goatsThere were fifty-four healthy multi-generation goats weighed 32.0±4.0 kg.After 7days of feeding,the goats were randomly divided into 3 groups:single group（FF,24animals）,combined group（DOX-HCL+FF,24 animals）and control group（6 animals）.The administration scheme of single group was:FF（20 mg/kg b.w.）,intramuscular injection in the neck,once every 48 h,twice in total.The administration scheme of combined group was:FF（20 mg/kg b.w.）was intramuscular injected in the neck,once every 48 h,twice in total and DOX-HCL（10 mg/kg b.w.）was intramuscularly injected in the neck,every 24 h was administered once for 3 d.At 0.5 d,1 d,3 d,7 d,14 d and 21 d after drug withdrawal,9 animals were killed at each time,including 1 animal in the control group,4 animals in the single group and 4 animals in the combined group.The liver,kidney,muscle and injection muscle were collected and tested on the machine after sample pretreatment.The results were as follows:the kidney was the residual target tissue of DOX-HCL and the liver was the residual target tissue of FF and FFA.The elimination of DOX-HCL was fastest at the injection muscle,and the elimination half-life was 4.81 d,while the elimination was slowest in the kidney and muscle,the elimination half-life was6.08 d.For FF,the elimination of FF was fastest at the injection muscle in both the combined group and the single group,with elimination half-lifes of 2.30 d and 2.27 d;the elimination of FF was slowest at the liver in both the combined group and the single group,with elimination half-lifes of 3.92 d and 3.85 d.For FFA,the elimination of FFA was fastest at the injection muscle in both the combined group and the single group,and the elimination half-lifes were 3.85 d and 3.63 d,respectively.The elimination of FFA was slowest at the liver in both the combined group and the single group,and the elimination half-lifes were 5.87 d and 5.41 d,respectively.WT1.4 software was used to calculate the withdrawal period of three drugs in each edible tissue of goats.The results were as follows:in the combined group,the withdrawal times of FF in goat tissues were muscle（14.73 d）,liver（5.05 d）,kidney（18.68 d）and injection muscle（19.65 d）,respectively;in the single group,the withdrawal time of FF in goat tissues were muscle（15.32 d）,liver（4.97 d）,kidney（17.18 d）and injection muscle（19.21 d）,respectively.The withdrawal time of DOX-HCL in goat tissues were liver（30.54 d）,kidney（27.75 d）,muscle（28.11 d）and injection muscle（38.41 d）,respectively.In summary,when FF is administered at 20 mg/kg b.w.every 48 h and twice in the neck intramuscular injection,the recommended withdrawal time is 20 d.When FF is given by20 mg/kg b.w.,once every 48 h,twice in total,and DOX-HCL（10 mg/kg b.w.）is given once a day for 3 d,the recommended withdraw time is 39 d.