| Zaltoprofen belongs to the class of non-steroidal COX-2 preferential inhibitors anti-inflammatory drugs,have a strong inhibitory effect on both acute and chronic inflammation,low adverse reactions to the gastric and intestinal mucosa,and are widely used in human medicine.The anti-inflammatory and analgesic effects and safety of zaltoprofen have been confirmed in human and animals experiments,and it has the prospect of developing a veterinary anti-inflammatory drugs.The study of drug metabolism and residue elimination in animals is an important scientific basis for evaluating the safety of new veterinary drugs and food safety.However,there are few studies on the metabolism and residue elimination of zaltoprofen,and there is no relevant domestic literature.Radioisotopes enable the labeled compounds to be accurately located in the body,and have the advantages of high specificity,high sensitivity,and easy to detection.They are widely used in the research of drug disposition in the body.In order to reveal the process of biotransformation and evaluate scientifically the safety of zaltoprofen in pigs and rats,this tritium-labeled zaltoprofen first synthesized,using radioactive tracing combined with Liquid Scintillation Counter(LSC),High-Performance Liquid Chromatography tadem on-line isotope detector(HPLC-v.ARC),Liquid Chromatography Combined with Ion Trap/Time of Flight Mass Spectrometry(LC/MS-IT-TOF)technology,the study of the metabolism,excretion,tissue distribution and residue elimination of zaltoprofen in pigs and rats were carried out.The metabolites of zaltoprofen in pigs and rats are identified and the metabolic pathway was inferred,the residue elimination rules of different metabolites in pigs and rats were analyzed,and the residual target tissues and residual markers in pigs were recommended.This study provides a theoretical basis for the rational use of drugs in clinical practice,which in turn provides a scientific basis for the formulation of food safety standards.1 The synthesis and quality standards of tritium-labeled zaltoprofenIn this study,2-(3-carboxymethyl-4-phenylthiophenyl)propionic acid was used as the raw material,it undergoes substitution reaction with bromine to mark the bromine on the benzene ring after esterification,and then undergoes dehydration condensation with polyphosphoric acid to make the ring is closed,then the ester group is hydrolyzed to obtain 4-bromo-zaltoprofen,finally the tritium-bromide exchange were carried out with tritium gas under the catalyst action of palladium carbon(Pd/C)to produce the crude product of 4-~3H-zaltoprofen,which was separated and purified by using preparative liquid chromatograph.After quality standard research,the tritium-labeled zaltoprofen obtained after separation and purification has high chemical purity(≥99%),high radiochemical purity(≥98%)and high specific activity(29.30 Ci/g),its chemical purity and radiochemical purity can still be maintained above 98%when stored at-20℃for 6months,indicating that its stability is good and the quality standards meet the requirements of animal experiments.2 Mass balance and metabolism of zaltoprofen in pigs and ratsDuroc×Large white×Landrace three-way crossbred castrated pigs of 30-days-old(n=4)and Wistar rats of 8-weeks-old(n=6)were given tritium-labeled zaltoprofen at a dose of 5 mg/kg b.w.and the specific activity of 0.15 Ci/g by intramuscular injection.The samples of urine and feces were collected at different time points after administration and until no radioactivity was detected.The samples of urine,feces,blood and bile which were collected at various time periods were digestioned and measured to analyze the mass balance and excretion of zaltoprofen in pigs and rats by LSC;after extraction and purification,the samples were detected by LC-v.ARC and LC/MS-IT-TOF for qualitative and quantitative analysis of its radioactive compounds.The study results showed that,the cumulative drug recovery rate of pigs and rats reached more than 80%after administration of 0 d-1 d;the cumulative drug recovery rate of both animals reached more than 90%after administration of 0 d-7 d.During the collection period of 14 d,the cumulative drug recovery rates of pigs,female rats and male rats were 95.82±0.51%,96.97±7.28%and 94.77±5.76%,respectively.In pigs,about74.80%of the drug is excreted through urine and 21.13%is excreted through feces;however,in female and male rats,about 26.61%and 17.23%of the drug is excreted through urine,79.73%and 68.16%is excreted through feces.The results showd that zaltoprofen is excreted rapidly in pigs and rats,the excretion was concentrated on 0 d-1 d and then the excretion decreased rapidly,but the main excretion pathways are significantly different,rats may mainly excreted through bile,while in pigs may have a process of bile excretion and reabsorption.A total of 8 radioactive compounds were detected in pigs and rats,namely prototype Z0,S-oxide-10-hydroxy zaltoprofen Z1,S-oxide-zaltoprofen Z2,10-hydroxy-zaltoprofen Z3,R-zaltoprofen glucuronide Z4,S-zaltoprofen glucuronide Z5,R-10-hydroxy zaltoprofen glucuronide Z6,and S-10-hydroxy zaltoprofen glucuronide Z7.Z0 and 7metabolites(Z1,Z2,Z3,Z4,Z5,Z6,Z7)were found in pig urine,Z0 and 5 metabolites(Z1,Z2,Z3,Z4,Z6)were found in rat urine;Z0 and 5 metabolites(Z1,Z2,Z3,Z6,Z7)were found in pig feces,Z0 and 3 metabolites(Z1,Z2,Z3)were found in rat feces.Z0and Z3 were detected in the blood of pigs and rats.Only Z0 was detected in pig bile.The number of radioactive compounds were detected in pigs and rats was 8 and 6,of which Z5 and Z7 were only found in pig urine.According to the metabolism of zaltoprofen in pigs and rats,it is speculated that the mainly metabolic characteristics of zaltoprofen are as follow:one is the carbonyl reduction reaction;second is the sulfur oxidation reaction;third is the simultaneous occurrence of sulfur oxidation and the carbonyl reduction reaction;fourth is the glucuronide conjunction of zaltoprofen,fifth is the glucuronide conjunction after the carbonyl reduction.3 Tissues distribution and elimination of zatoprofen in pigs and ratsDuroc×Large white×Landrace three-way crossbred castrated pigs of 30-days-old(n=28)and Wistar rats of 8-weeks-old(n=42)were randomly divided into 7 groups.One group was a blank group,and the other 6 groups were experimental groups.Both pig and rat experimental groups were given tritium-labeled zaltoprofen by intramuscular injection for 7 consecutive days.The dose of pigs and rats were 5 mg/kg b.w.and 10 mg/kg b.w.,respectively.The specific activity was 0.15 Ci/g.A animal of blank group and a group animal of experimental group were killed at different time points after stopping the drug(12 h,3 d,7 d,14 d,21 d and 28 d for pigs,12 h,1 d,3 d,7 d,14 d and 21 d for rats),collect heart,liver,spleen,lungs,kidneys,stomach,large intestine,small intestine,blood and bile etc(plasma and bile samples is mainly used for metabolism test).A small amount of collected tissue samples were added to the digestion solution for water bath digestion.After the digestion was completed,the total radioactivity was measured to obtain the distribution characteristics of zaltoprofen in animal tissues by LSC.At the same time,a part of main tissue samples were extracted and purified with methanol.After extraction and purification,the samples were detected by LC-v.ARC和LC/MS-IT-TOF for qualitative and quantitative analysis of its radioactive compounds.Zaltoprofen and its metabolites are widely distributed in pigs.The concentration of radioactive products in all tissues is high after stopping the drug of 12 h.The drug concentration in bile is the highest,followed by kidney,adrenal gland,liver,muscle and digestion system,the concentration in the heart,spleen,lung,fat,skin and bladder is relatively low.The drug concentration decreased rapidly after stopping the drug of 3 d,bile decreased about 20 times,adrenal gland decreased about 5 times,liver and muscle decreased about 3 times,and most other tissues were about 1/2 of the concentration at 12h.The radioactivity still be detected in all tissues after stopping the drug of 7 d and 14 d.After stopping the drug of 21 d,no radioactivity was detected in the fat and stomach,higher drug concentration in the liver and kidney was 69±3μg/kg and 135±21μg/kg.On the 28 d after the drug was stopped,no radioactivity was detected in tissues.In the main tissues of pigs,three radioactive substances of Z0,Z2 and Z3 were detected.Z0 and Z3can be detected in the liver;Z0,Z2 and Z3 can be detected in the kidneys;Z0 and Z2were detected in the muscle and fat;only Z0 was detected on the 7 d after the drug was stopped.Among the four tissues,Z0 is the main metabolite,accounting for more than90%.The drug concentration in each tissue of pigs decreased rapidly after drug was stopped of 0.5 d-3 d,and the drug elimination was slowly after drug was stopped of 3d-21 d.The tissues liver,spleen,kidney and injected muscle with longer elimination half-life between 5.32 d-6.14 d;followed by heart,psoas muscle,stomach,small intestine,skin,bladder and blood,with elimination half-life of 5.06 d-5.18 d;again for lung,large intestine,adrenal glands and bile,the elimination half-life is between 4.68 d-4.92 d;the fastest tissue elimination is fat,and the elimination half-life is 3.93 d.Among them,in liver and kidney samples,the drug has the highest residual amount,the longest residence time,and the slowest elimination,the half-lives of liver and kidney are 5.50 d and 6.14 d,respectively,the liver and kidney can be used as residual target tissues,the half-life of Z0in the liver and kidney are 5.36 d and 6.05 d,respectively,which is consistent with the total elimination trend,and Z0 is determined as a residual marker in pigs.Zaltoprofen is also widely distributed in rats,with the highest radioactive concentration in the digestive system,followed by liver,kidney and muscle.The concentration in fat,skin and gonads is relatively low,and the concentration in the brain is the lowest.The drug concentration decreased rapidly after stopping the drug of 1 d,the stomach and large intestine decreased about 20 times,the small intestine decreased about40 times,muscle and fat decreased about 10 times.The drug concentration in all tissues after stopping the drug of 3 d was about 1/2 of the concentration at 1 d.High radioactivity can still be detected in all tissues after stopping the drug of 7 d.After stopping the drug of14 d,the higher drug concentration in the liver and kidney was 134±22μg/kg and 215±90μg/kg,and no radioactivity was detected in the rat tissues after stopping the drug of 21 d.In the main tissues of rats,four radioactive compounds of Z0,Z1,Z2 and Z3 were detected.Z0,Z2 and Z3 can be detected in the liver;Z0,Z1,Z2 and Z3 can be detected in the kidneys;Z0 and Z2 were detected in the muscle and fat;only Z0 was detected on the 3 d after the drug was stopped.Among the four tissues,Z0 is the main metabolite,accounting for more than 90%.After stopping the drug of 0.5 d-1 d,the drug concentration decreased rapidly in each tissue of the rats,which was the fast elimination phase.After stopping the drug of 1 d-14 d,the total drug entered the slow elimination phase.The elimination of heart,liver,kidney and muscle is relatively slow,with a half-life between 5.05 d-6.78 d;followed by lung,stomach,brain,gonads and blood,with an elimination half-life between 4.01 d-4.99 d;The elimination of spleen,skin,large intestine and small intestine is relatively fast,with a half-life of about 3.09 d-3.92 d;fat elimination is the fastest,with a half-life of 2.03 d.Among them,the elimination of the drug in the liver and kidney sample is the slowest,and the elimination half-life is 5.87 d and 6.78 d,the half-lifes of Z0 in the liver and kidney are 5.80 d and 6.33 d,respectively.In summary,the tritium-labeled zaltoprofen in compliance with the quality standard was synthesized and obtained for the first time in this study,the metabolism,excretion,tissue distribution and residue elimination of zaltoprofen in pigs and rats were clarified scientifically,the metabolites of zaltoprofen in different samples of pigs and rats were identified and their metabolic pathways were inferred,the residual elimination rules of each metabolite in pigs and rats were analyzed,the residual target tissue and residual marker of zaltoprofen in pigs were recommended.The results of this study provide a theoretical basis for clinical rational use of zaltoprofen the,and more comprehensive information for the formulation of its safety assessment. |
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