The Antibacterial Activity Of Aditoprim And Its Main Metabolites

Aditoprim(ADP),which belongs to 2,4-Diaminopyrimidines,is a dihydrofolate reductase(DHFR)inhibitor that binds to dihydrofolate reductase and inhibits the transformation of dihydrofolic acid to terahydrofolic acid so as to affect the metabolism of folate.Compared with trimethoprim(TMP),another drug in this class,it has longer elimination half-time and higher distribution volumes.And advantages like low toxicity,security and not mutagenic show it has a promising application prospect.However,there are few researches about the pharmacodynamics of ADP at present,so does the pharmacodynamics of ADP's metabolites.This study is to evaluate the pharmacodynamics of ADP and its main metabolites was based on the former research in our lab.The Minimal Inhibitory Concentration(MIC)and Minimal Bactericidal Concentration(MBC)of ADP against many pathogens from swine,poultry,fishes,calves and sheep were determined.MICs50?MIC90?MBC50 and MBC90 were analyzed for the formulation of antibacterial spectrum.Also we determined the in vitro antibacterial activity of ADP's metabolites against Streptococcus from swine and salmonella from poultry.In the end,? clinical study on therapeutic effect of ADP on swine streptococcosis was carried on so as to provide scientific basis for its further promotion application.1 The in vitro antibacterial activity of ADPMinimum inhibitory concentrations(MICs)and Minimal Bactericidal Concentrations(MBCs)of ADP against common pathogens in swine,poultry,fishes,calves and sheep were determined according to the Clinical and Laboratory Standards Insititute(CLSI)guidelines,and TMP acted as control drug.Due to the selected are facultative anaerobic bacteria,and the in vivo body environment was anaerobic or even anoxia,antibacterial effect under anaerobic conditions were also measured and compared with the ordinary conditions.Results showed that ADP was a broad-spectrum antibacterial agent which has a good effect on gram-positive and gram-negative bacteria and its antibacterial activity was equal to TMP.Salmonella and Streptococcus from swine,Escherichia coli and Salmonella from broilers,Escherichia coli,Streptococcus,Mannheimia,Pasteurella from calves,Streptococcus,Mannheimia from sheep,Escherichia coli,Flavobacterim,Acinetobacter baumannii and Yersinia ruckeri from fishes are highly susceptible to ADP.Haemophilus parasuis from swine,Staphylococcus aureus,Aeromonas punctate,Mycobacterium tuberculosis,Streptococcus agalactiae from fishes,Klebsiella from calves and sheep showed moderate susceptibility,whereas Escherichia coli,Actinobacillus pleuropneumonia,Pasteurella,Staphylococcus aureus,Clostridium perfringens from swines,Staphylococcus aureus,Clostridium perfringens from broilers,Staphylococcus aureus from calves are resistant to ADP.The value of MBC/MIC was from 1 to 4 for the ADP against sensitive bacteria,which indicated a good bactericidal activity of ADP.ADP and TMP had equally in vitro activity against Escherichia coli,Salmonella and Streptococcus from swine,Flavobacterim from fishes.Salmonella from broilers,Escherichia coli,Streptococcus,Mannheimia,Pasteurella from calves,Streptococcus,Mannheimia from sheep,Escherichia coli,Aeromonas punctate,Acinetobacter baumannii,Mycobaterium tuberculosis,Streptococcus agalactiae,Staphylococcus aureus and Yersinia ruckeri from fishes were more susceptible to ADP than TMP,whereas the MICs and MBCs of Escherichia coli from calves and broilers were slightly higher for ADP than for TMP.In the respect of a single strain,the MIC and MBC in anaerobic and aerobic conditions were equal or differ by a gradient;from the aspect of more strains,the MIC50,MBC50,MIC90 and MBC90 of ADP against common pathogenic bacteria in anaerobic and aerobic conditions were exactly the same,which illustrates strong antibacterial activity and bactericidal activity of ADP in aerobic conditions.As a result,the drug has no obvious anaerobic selection activity.2 In vitro antibacterial activity of ADP1,ADP2 and ADP3According to the results of the in vitro antibacterial activity of ADP,we choose 44 Streptococcus from swine and 40 Salmonella from broilers to determine the in vitro effect of ADP1,ADP2 and ADP3.Results showed that the MIC and MBC of ADP3 against all Streptococcus from swine and Salmonella from broilers were greater than 128 ug/ml,which demonstrated that ADP3 had no antimicrobial activity.But for the entire,the MIC50,MBC50,MIC90,MBC90 of ADP1,ADP2 for Streptococcus from swine and Salmonella from broilers were equal to those of ADP,of which the MIC50 against Streptococcus from swine was 2 ?g/ml,MBC50 was 4 ?g/ml,MIC90 was 32 ?g/ml,MBC90 was 64 ?g/ml and the MBC50/MIC50,MBC90/MIC90 were 2.Similarly,MIC50 against 40 Salmonella from broilers was 2 ?g/ml,MBC50 was 4 ?g/ml,MIC90 was 32 ?g/ml,MBC90 was 64 ?g/ml and the MBC50/MIC50,MBC90/MIC90 were 2.We can draw a conclusion that the in vitro antibacterial activity of ADP,ADP1 and ADP2 were very similar and all of them had strong antibacterial activity and bactericidal activity.3 Therapeutic effect of ADP on swine streptococcosisSixty piglets weighing 11-13 kg were randomly divided into six groups,and each group consisted of 10.Blank control group was without artificial challenge and administration.In addition,other experimental groups were infected with 1010 colony-forming unit Streptococcus CVCC607.After the swine streptococcosis disease model seccesfully established,drug control groups was intramuscular injected with compound sulfsdiazine soditim in a dose of 30 mg/kg b.w.,While the negative control group did not receive treatment.The remaining three groups were treated with ADP in a dose of 10 mg/kg b.w.,20 mg/kg bw.,and 40 mg/kg b.w.,which were named as low-dose treatment group,middle-dose treatment group and high-dose treatment group,respectively.All the treatment groups were given drug with a once-daily regimen for five consecutive days and then had a 15-day observation period.According to the symptom score system,the mental state,body temperature and clinical symptoms of' every pig were recorded throughout the testing process.Finally,theraputic effect of ADP was judged based on death rate,efficient rate,cure rate and relative growth rate of each group.After the administration,the recovery of animals in different groups was different.The average symptom score of negative control group began to fall on the fifth day after the administration and returned to normal on the fifteenth day with sevaral pigs still appeared depression and cough;average rectal temperature had a substantial drop-off six days after administration while fell below 39.5? on the ninth day.The rectal temperature of pigs in high-dose treatment group returned to normal the second day after administration,while the average clinical symptom score dropped to 2 minutes the third day and were maintained till the end of trial with 0 score.Similarly,mean rectal temperature of middle-dose group returned to normal on the second day,whereas symptom scores were back to normal on the third day.The average body temperature and symptom scores of each pig in low-dose group were back to normal in the third and fourth day.All cured pig had no recurrence during the observation period.Body weight of pigs in each group were tested and compared before and after the trail.The relative weight gain rate(RAW)of compound sulfsdiazine soditim control group was between high-dose treatment group and middle-dose treatment group,whose RAW were 93.0%,96.5%and 91%,respectively and without significant difference each other(P>0.05).In low-dose treatment group,the relative growth gain rate was 74.7%,which had a significant difference when compared with high-dose group and middle-dose group(P<0.05).pigs in negative control group gained an average of only 1.77 kg with RAW of 34.5%,showing a significant difference with high-dose,middle-dose and low-dose group(P<0.05).In order to evaluate the therapeutic effect of ADP on swine streptococcosis we made a comprehensive analysis about coughing,mental state,body temperature and other symptoms of each group.Results showed that there were no deaths in these three ADP treatment group,whereas the survival rate of negative control group was 70%.The high-dose group of ADP owed 100%efficiency rate and 90%cure rate,while both effective rate and cure rate of middle-dose groups were 90%.The cure rate and effective rate of low-dose group were 80%and 70%,respectively.It indicated that all of the three treatment group of ADP could achieve treatment effects.In short,the therapeutic dose of ADP recommended for swine streptococcosis is 20-40 mg/kg b.w.,we suggest 20-40 mg/kg b.w.ADP for treatment,while 10 mg/kg b.w.for prevention or process of consolidation after curing.In conclusion,based on the related guidelines and standards of Clinical and Laboratory Standards Institute(CLSI),"Phase II And Phase III Clinical Efficacy Evaluation Of Antibiotics Guidelines" and "Experimental Clinical Trail Technology Standard" developed by China Ministry Of Agriculture,this study has determined MICs and MBCs of ADP against common pathogens from swine,broliers,fishes,calves and sheep and established antibacterial spectrum of ADP.CVCC607 was selected to evaluate the phase II clinical study of ADP on therapeutic effect on swine streptococcosis,provided reasonable theoretical foundation for clinical application.In a word,ADP has a strong in vivo and in vitro antibacterial activity and will have a promising application prospect in animal production.