Study On The Drugability Of New Candidates Against Drug-resistant Tuberculosis

Objective and Contents In this paper,we studied the preliminary druggability of eight new candidates against drug-resistant tuberculosis,including the following aspects:(1)We established an analysis method to detect the concentration of HY152-C?HY152-D?HY152-E?HY152-J?HY152-K?HY152-L?HY152-M and HY152-P,respectively,in plasma or other biological samples by HPLC-MS/MS.And we performed a validation on the analysis method according to guidance for the validation of quantitative analysis methods for biological samples.(2)We used the established analysis method to carry out a pharmacokinetic study on the eight candidates after single oral administration of one dose at 50 mg/kg and single intravenous administration of one dose at 5 mg/kg.(3)Finally,we got one candidate,which has a good quality of pharmacokinetic,by filtering.Then we investigated the distribution of the candidate into different tissues of rats after oral administration at a single dose at 50 mg/kg,and the accumulation of it into different tissues of rats after multiple oral administration of one dose at 50 mg/kg.(4)The Caco-2 cell monolayer model was established,and we investigated the interactions of the candidate drug with the transporter P-gp by HPLC-MS/MS assay.(5)And in situ single-pass intestine perfusion model was adopted to study the absorption behaviors of the candidate drug in different intestine segments of rats.(6)We conducted an early safety evaluation on the candidate drug at last.Results(1)Method validation is a principal step to provide accurate results of compounds in biological matrix.Therefore,we established a rapid,sensitive and specific HPLC-MS/MS analysis method to detect the concentration of HY152-C,HY152-D,HY152-E,HY152-J,HY152-K,HY152-L,HY152-M and HY152-P in plasma of rats respectively,and the precision and accuracy of the selectivity,linearity and stability were all within the common validation criteria.(2)After intravenous administration of HY152-C,HY152-D,HY152-E,HY152-J,HY152-K,HY152-L,HY152-M and HY152-P,respectively,in dosage of 5 mg/kg in rats,the mean plasma elimination half-lives were 54.1,43.5,91.1,86.9,129,32.6,78.2 and 63.3 minutes,respectively.The mean plasma clearance was 0.009,0.039,0.036,0.014,0.016,0.013,0.018 and 0.011 L/min/kg respectively.The average plasma clearance of HY152-D was 0.709 times the blood flow of the liver in rats(about 0.55 L/min/kg),which indicated to be eliminated fast in vivo.The average plasma clearance of HY152-E and HY152-M was 0.655 times and 0.33 times the blood flow of the liver in rats respectively.Other candidates was lower than 0.30 times the blood flow of the liver in rats,which indicated to be eliminated slowly in vivo.And the mean apparent volume of distribution was 0.67,2.49,4.82,1.79,3.20,0.59,2.04 and 1.00 L/kg respectively.After oral administration of HY152-C,HY152-D,HY152-E,HY152-J,HY152-K,HY152-L,HY152-M and HY152-P,respectively,in dosage of 50 mg/kg in rats,the mean plasma elimination half-lives were 126,105,66.7,171,137,55.1,323 and 186 minutes respectively.The mean plasma maximum concentrations were 16.4,7.6,5.9,6.1,14.8,34.3,2.5 and 0.1 ?g/mL respectively.And the absolute bioavailability of HY152-C,HY152-D,HY152-E,HY152-J,HY152-K,HY152-L,HY152-M and HY152-P was 13.3%,53.0%,32.0%,58.2%,133%,242%,13.3% and 0.97%.Moreover,there was no obvious gender difference of main pharmacokinetic parameters of eight candidates in rats(3)We obtained HY152-J with a good quality of pharmacokinetic after filtering the eight candidates.It could be widely distributed in various tissues of rats after oral administration of HY152-J in a dosage of 50 mg/kg,especially in the kidney,liver,heart,spleen and lung.The peak time of maximum concentration in most tissues was 4 h after administration.The concentrations in most tissues were higher than it in plasma and it did not indicate a tendency to accumulation in tissues after multiple oral administration of HY152-J.(4)The results indicated that we set up Caco-2 cell monolayer model with the properties of good compactness,integrity,and functionality in the transport studies.The apparent permeability coefficients for three different concentrations of candidate drug HY152-J from apical side to basolateral side were in the range of 1.76×10-5-2.61×10-5 cm/s.Moreover,the efflux rations of Papp for different concentrations of HY152-J from apical side to basolateral side and Papp from apical side to basolateral side were between 0.74-1.27.The inhibition tests for the transport of digoxin across Caco-2 cell monolayer model did not show an obvious decrease from basal side to apical side with different HY152-J concentrations(0.625-10 ?mol/L)compared with the negative group.(5)The absorption rate constants(Ka)and apparent permeability coefficients(Papp)of HY152-J in duodenum,jejunum,ileum and colon of rats had no obvious difference(P > 0.05).Compared with the group of low concentration(25 ?g/mL),the Ka and Papp values of HY152-J had conspicuous difference in high concentration(100 ?g/mL)group(P < 0.01)and the Papp values of HY152-J had apparent difference in moderate concentration(50 ?g/mL)group(P < 0.05).Different pH values of perfusate had no significant effect on the Ka and Papp values of HY152-J(P > 0.05).And the P-gp inhibitor had little influence on the intestinal absorption of HY152-J(P > 0.05).(6)The acute toxicity test results showed that the death of mice could be observed in the whole dose groups except for the lowest dose group during the two-week observation period,after oral administration of HY152-J in dosage of 5 g/kg?4 g/kg?3.2 g/kg?2.56 g/kg and 2.05 g/kg respectively in mice.The median lethal dose of male mice was 3.264 g/kg,and the median lethal dose of female mice was 3.416 g/kg.In the chronic toxicity test,compared with normal group,the food-intake had conspicuous difference(P < 0.05)in experimental group after oral administration of HY152-J in dosage of 250 mg/kg for a month.The body weight had no obvious difference about drug toxicity in experimental group comparing with normal group,but it showed a trend to reduce.And the parameters of hemotology in rats had no significant difference comparing with control group.The biochemical index results indicated that there was an apparent increase in ALT,TP,ALB,CHOL,AST,TG and PT(P < 0.05 or P < 0.01).Another notable finding was that a conspicuous increase in organ index of liver could be observed in experimental group.Moreover,histopathological examination indicated that the liver tissue had hepatic steatosis in experimental group.Conclusion We studied the preliminary druggability of eight candidates against drug-resistant tuberculosis from the point of view of pharmacokinetics,and got one candidate with a good quality of pharmacokinetic by selecting.Then we studied the further pharmacokinetics and safety of HY152-J.The results demonstrated that the absolute bioavailability of HY152-J was 58.2%.The peak time of maximum concentration in most tissues was 4 h after administration.And it could be widely distributed in various tissues of rats,especially in the kidney,liver,heart,spleen and lung.In addition,it did not manifest a trend to accumulation in tissues after multiple administrations.It could be inferred that HY152-J is not a substrate and an inhibitor for transport protein P-gp probably.HY152-J could be absorbed in the whole segments of the intestine in rats,and did not have a special absorption region.The absorption of HY152-J did not show an increase with different concentrations(25 ?g/mL-100 ?g/mL).It preliminarily suggested that the absorption of HY152-J complies with active absorption.The median lethal dose of HY152-J was 3.264 g/kg for male mice and 3.416 g/kg for female mice,which evidenced it might be a compound with low toxicity level.We found obvious difference about drug toxicity on biochemical index,organ index,autopsy,and histopathological examination,which suggesting that the toxic target organ of HY152-J may be liver.