Disposition And Residue Depletion Of Aditoprim In Pig,chicken,carp And Rat

Aditoprim is a recently developed 2,4-diaminopyrimidine derivative that selectively and specifically inhibits the bacterial dihydrofolate reductase,and has a similar spectrum of antimicrobial activity with trimethoprim(TMP).However,clinical investigations in a variety of animal species showed that ADP has a favorable pharmacokinetic behavior than that of TMP indicating a wide range of applications in veterinary practice.Drug metabolism and disposition not only associated to its pharmacological activity or toxicity,but also affects the residual site and elimination rate.In order to reveal the process of biotransformation and evaluate the effectiveness and safety of ADP,our studies synthesized the tritiated-ADP and evaluated its ADME properties in pig,chicken,carp and rat using radiotrace coupled with LC-v.ARC/MS-IT-TOF technique.Subsequently,the standards of ADP and its three major metabolites were prepared.ADP injection was developed and its excretion in pig was carried out.A sensitive and specific high-performance liquid chromatography-ultraviolet(HPLC-UV)method was developed for the simultaneous and quantitative determination of ADP and its three major metabolites in the major tissues and the depletion of ADP and its major metabolites in pigs,chicken,and carp were investigated to propose the residual target tissue and marker residue of ADP.Finally,the food safety standards for ADP in pigs,chicken,and carp was estimated using the assessment methodologies approved by the Joint FAO/WHO Expert Committee on Food Additives(JECFA).This study will contribute to clinical application and residue monitoring of ADP.1.Synthesis procedure and quality standards of tritium-labeled ADP2-bromine-ADP was synthesized with 3,5-dimethoxy-4-amino-benzonitrile as raw material.The synthesis procedure included the reduction,bromine substitution,Swern Oxidation,Knoevenagel reaction and cyclization reaction.Then,2-bromine-ADP was dissolved in methanol-DMF(1:1,v:v),10%Pd/C was added and the mixture reacted with 250 mmHg tritium gas at 60? to give the crude 2-3H-ADP.The 2-3H-ADP was purified and its quality was determined.The resulting 2-3H-ADP with a specific activity 10 mCi/mol,chemical purity 99%,and radiochemical purity 99.2%was stably stored at-20? for six months2.Mass balance and metabolism of aditoprim in pig,chicken,carp and ratsThe excretion were collected at different time intervals from pig(n=4),chicken(n=4),carp(n=4)and rat(n=4)after 3H-ADP were orally administrated at a single dose of 5 mg/kg BW Total radioactivity levels were determined by LSC after digestion.After preparation,samples were analyzed by HPLC-v.ARC/MS-IT-TOF for metabolite profiling.Our results suggesting the maximal excretion occurred during the first day after dosing,and then slowdown in following days.During day one,the cumulative recovery reached up to 72%of the dose in pig,carp and rat and 85%in chicken.At the period of 0-7d,they raised to 90%of the dose all the four species.During the 14 d collection period,the total radioactive recovery in excreta was 95.6± 1.2%,94.2±3.1%,94.2±2.7%and 94.8±7.9%of the administered dose in pigs,chicken,carp,and rats,respectively.For pigs and rats,the urine excretion was the predominant route of elimination and represented about 78%of the dose within 14 d,with the remainder excreted in the feces.A total of 12,11,3,and 6 metabolites were characterized in pigs,chicken,carp,and rats,respectively,involving demethylation of dimethylamino or methoxyl,a-hydroxylation,N-oxidation,or N-glucuronidation.N-demethylation is the major metabolic pathway resulting in the formation of two major metabolites of N-monodesmethyl-ADP and N-didesmethyl-ADP.However,ADP was the predominant component in all samples.Other metabolites declined rapidly over time.At 6h after dose administration,AO,A1,and A2 displayed 42.3,11.4,11.8%of sample radioactivity in pig urine,89.1,9.7,1.1%of sample radioactivity in pig feces;63.2?12.8?6,5%of sample radioactivity in chicken excreta;87.5,9.9,2.5%of sample radioactivity in carp excreta;and 71.2?14.7?2.6%of sample radioactivity in rat urine,79.6?20.5?0.0%of sample radioactivity in rat feces;respectively.3.Tissues distribution and elimination of aditoprim in pig,chicken,carp and ratsPig(n=24)chicken(n=36)carp(n=42)and rat(n=36)were randomly divided into six(seven for carp)equal treatment groups and received a single daily dose of 3H-ADP at 5 mg/kg BW by oral gavage for 7 consecutive days.One group of animals was slaughtered at different time points after the last administration.All the tissues samples were collected and analyzed.Total radioactivity levels in sample were determined by LSC.After preparation,the extract samples of major tissue were analyzed by HPLC-v.ARC/MS-IT-TOF for metabolite profiling.In all four species,ADP-derived radioactivity underwent a wide distribution across the various tissues and fluids,and reached the highest concentration in bile,followed by the kidney,liver,spleen,lungs,adrenal glands and the immune and digestive systems;the lower levels of radioactivity were found in the heart,muscle,fat,blood,swim bladder,skin and hair.The drug concentrations in most tissues rapidly decreased to one-half within 6 h to 1 d postdose,and then decreased slowly.The concentration of radioactivity in all samples from the four species can be detectable at seventh day(14th for carp)of the last dose.After 14d(21 d for carp),the highest radioactivity was detected in liver,with the concentration of 372±25,160±23,103±21 and 185±25 ?g/kg in pig,chicken,carp and rat,respectively.Followed by the kidney;other tissues can detect only a small amount of radioactivity.Liver has the longest elimination t1/2 for pigs,chicken,carp and rats with a t1/2 4.56,3.38,6.69 and 3.96 d respectively and thus was recommended as the residual target tissue.A total of 12(A0-A11),8(A0,A1,A2,A3,A9,A10,A11,A12),3(A0,A1,A2),and 6(A0,A1,A2,A6,A9,A10)metabolites were observed in the major tissue of pigs,chicken,carp and rats,respectively.Most of the radioactive compounds were found in the liver and kidney,only A0,A1 and A2 were detected in muscle and fat.A0,A1,and A2,the major radioactive components,could be consistently detected within 3 d post last dosing.Other minor metabolite depleted rapidly and vanished at 1 d or 3 d.By 7 d or 14 d after dose administration,AO was the only detectable compound.Unchanged parent was the most predominant component in all detected tissues among four species,representing more than 42%of sample radioactivity in all sampling time point.A1 and A2,the major metabolites of ADP,displayed concentration ranging from 18.2?34.1%,21.3?26.8%,12.4?16.8%,19.2?38.5%and 4.0?14.1%,1.6?7.9%,1.6?2.9%,3.1-5.3%in pig,chicken,carp,and rat.respectively.In the liver,AO was eliminated slowest,and showed the similar concentration-time curve to that of total radioactivity.So,parent drug AO was proposed as the residue marker.4.The standards preparation of aditoprim and its major metabolites and quality researchAditoprim was synthesized according to the improved synthetic route.N-monodesmethyl-ADP was sysnthised with 3,5-dimethoxy-4-amino-benzonitrile as raw material,and the synthesis procedure included the nitrile reduction,Knoevenagel reaction and cyclization reaction.N-didesmethyl-ADP was sysnthised with 4-aminobenzonitrile as raw material,and the synthesis procedure included the bromine substitution,methoxylation,nitrile reduction,Knoevenagel reaction and cyclization.3-desmethyl-ADP was sysnthised with aditoprim as raw material,and the anhydrous aluminum chloride as demethylation reagent.All compounds were purified and dried to give the candidate reference substance.The structures of all the compounds were characterized by UV spectra,IR spectra,HNMR,ESI-MS and El.Base on the results of quality research,the content certified value of ADP,N-monodesmethyl-ADP,N-didesmethyl-ADP and 3-desmethyl-ADP were 98.76%,98.48%,98.41%and 98.32%,respectively.These results meet the requirements of the standard substance for determination.5.Development of ADP injection and its excretion experiment in pigADP injection consists of ADP substance,water for injection and lactic acid.The injection was successfully prepared by dissolving 200mg ADP and 200?L lactic acid in 2mL of water for injection.The mixture were filtered,packed and sterilized to produce ADP injection.The quality and stability of ADP injection was investigated in accordance with the "Veterinary Pharmacopoeia of the people's Republic of China"(2010 edition).Results showed that the preparation is in accordance with the quality standard of the pharmacopoeia.In the 6 month accelerated experiment and 12 month long term experimental study,the content of preparation decreased by less than 5%,and the appearance and other indicators showed no significant change.Consequently,the stability of ADP injection was good.A multi-residue HPLC-UV method was developed for the quantitative analysis of ADP(AO)and its major metabolites(N-monomethyl-ADP(A1),N-didesmethyl-ADP(A2)and 3'-demethyl-ADP(A3))in urine and feces.The urine and feces were collected at different time points from pig(n=4)after single injection ADP at dose 5mg/kg BW After preparation,samples were analyzed by HPLC-UV and LC/MS-IT-TOF.Results suggesting the peak excretion 25.5±2.9%and 26.4±4.7%of the dose occurred during 0-6h and 6h-ld after dosing and then slow down.Within 14d,the total cumulative recovery reached up to 66.7±3.5%of the dose.The remaining dose is presumed to be the minor metabolite excreted in urine.The cumulative recovery from urine was significantly higher than that of feces,which accounted for 61.4±4.7%%and 5.3± 1.7%of the administered dose within 14 d,respectively.The results of MS showed that the compounds were identical to the oral administration,and no new compounds were found6.Tissue depletion of aditoprim in pig,chicken and CarpA multi-residue HPLC-UV method was developed for the quantitative analysis of ADP(AO)and its major metabolites A1,A2,A3 in tissues of pig(Liver,kidney,muscle,fat,heart,lung,stomach,intestine),chicken(Liver,kidney,muscle,fat,heart,lung,stomach,intestine)and carp(liver,kidney,muscle,skin,gastrointestinal,bladder).Pig(n=30)chicken(n=36)and carp(n=60)were randomly divided into six equal treatment groups.Pigs received a intramuscular injection of ADP injection at a dose of 5 mg/kg BW for 7 consecutive days;chicken and carp received an dose of ADP through gavage at a dosage of 5 mg/kg b.w.for 7 consecutive days.One group of animals was slaughtered at different time points after the last administration.Tissues samples were collection and analyzed.Results suggested that the higher residue concentrations were detected in the liver,kidney,and lung.AO was the predominant component in all tissues of all these three species,followed by A1 and A2.No A3 can be detectable in all tissues.In all the three species,the liver showed a longer residue half-life(ti/2)than other tissues.In the liver,AO was the slowest eliminated component with a half-life of 3.04,2.54 and 4.53d in pig,chicken and carp.Therefore,the liver was the residual target tissue and ADP was the marker residue in the three food-producing animals.7.Estimate withdrawal times and maximum residue limits of aditoprim in edible tissues of pig,chicken and carpIn this study,the WDT and MRLs were determined in the edible tissues of pig,chicken and carp using the assessment methodologies approved by the Joint FAO/WHO Expert Committee on Food Additives(JECFA)and other international organization(American FDA and European EMEA).According to our national conditions,the MRLs and WDT from the relatively conservative EMEA evaluation procedures were proposed.In accordance with methods of EMEA,the withdrawal time in pig,chicken and carp is 20 d,17 d and 12 d,respectively.The MRLs in liver,kidney,muscle and fat are 50 ?g/kg.In summary,this research improved the synthesis route of ADP and prepared qualified 2-tritium-ADP.The metabolism and disposition characteristics in pig,chicken,carp and rats were scientifically elucidated.The standards preparation of aditoprim and its major metabolites were prepared.ADP injection was developed and its excretion in pig was clearly determined.The residue depletion of ADP in pig,chicken and carp was investigated and the residual target tissue and marker residue were proposed.In additionally,the scientific WDT and MRLs of ADP in the three food producing animals were proposed.All the results improve the safety assessment of ADP and will contribute to its clinical use.