| Rat pharmacokinetics investigation of dihydroartemisininDihydroartemisinin has been recommended by the World Health Organization for the treatment of malaria.Recently,the team of the Institute of Chinese Materia Medica intended to further develop dihydroartemisinin for the treatment of pathological pain in the central nervous system.Therefore,we have carried out the rat pharmacokinetic study to support the development of clinical indications of dihydroartemisinin.In the past,some scholars used radioisotope labeling technology to study the pharmacokinetic study of dihydroartemisinin in rats.However,the analysis technology did not distinguish the prototypes and metabolites of dihydroartemisinin.Since the pharmacodynamic activity of dihydroartemisinin mainly comes from the original compounds,the past analytical methods brought certain limitations to the pharmacokinetic study.To investigate the pharmacokinetics of dihydroartemisinin in rats,the UPLC-MS/MS method was developed for the determination of dihydroartemisinin in rat samples,and based on this,we investigated the rat pharmacokinetic characteristics of dihydroartemisinin(including gastrointestinal absorption,tissue distribution,liver and kidney excretion,etc.).Based on UPLC-MS/MS method,we established a sensitive,reliable,and rapid method for the quantitative analysis of dihydroartemisinin prototypes in rat plasma,tissue homogenate,urine,feces and bile after administration,and referred to the guiding principles for non-clinical pharmacokinetic studies of drugs issued by the National Medical Products Administration(NMPA)in 2014,the reliability of the analysis method was verified.All the pharmacokinetic experiment protocols of rats were approved by the Animal Ethics Committee of the Institute of Traditional Chinese Medicine.The pharmacokinetic experiments of rats were carried out according to the approved protocol.The dosage of dihydroartemisinin was referred to the dosage used in the relevant pharmacodynamic studies(the single intravenous dose was 4 mg/kg,the single oral administration of low,medium,and high doses were 4,8,and 32 mg/kg,respectively,and the once-daily dose for oral administration for 7 consecutive days was 8 mg/kg).The pharmacokinetic parameters of dihydroartemisinin were calculated by a non-compartmental model(Phoenix(?)WinNonlin(?)8.2 software;USA).The quantitative analysis method established for dihydroartemisinin had high selectivity,the linear quantitative range was 1-729 ng/mL,the intra-assay and inter-assay accuracy was 87.0%-114%,and the intra-assay and inter-assay precision was 1.07%-12.7%,The stability of the main steps of the simulation analysis method>87.2%,sample dilution reliability was 97.3%-107%,the residual degree was<20%,the relative matrix effect was<15%,the stability of the main steps of the simulation analysis method was>87.2%,the dilution reliability of the sample was 97.3%-107%.After oral administration,dihydroartemisinin was absorbed rapidly(Tmax,0.25-0.45 h),and its oral bioavailability(F)was lower than 30%,which was 3.6%-22.5%in male rats and 6.6%-25.1%in female rats,with great differences among rats.After oral administration,the plasma AUC0-∞ and Cmax of dihydroartemisinin in male rats increased with the increase of dose(4-32 mg/kg),but the linear relationship was uncertain;in female rats,plasma AUC0-∞ and Cmax did not increase with the increase of dose(4-32 mg/kg),which was a nonlinear correlation.The total clearance rate(CLtot,p)of dihydroartemisinin was 9.2-9.7 L/h/kg,which was about 2.8-2.9 times of rat liver blood flow.The steady-state apparent volume of distribution(Vss)was 2.2-2.5 L/kg,which was 3.3-3.8 times the total fluid volume of rats.The elimination half-life was very short(t1/2:0.18 h;shorter than that of oral administration,0.3-0.8 h).After oral administration for 7 consecutive days,there was no significant difference in plasma AUC0-∞ of male rats on day 7 and that on day 1(P>0.05),but it was significantly lower in female rats on day 7 than that on day 1(P<0.01).After oral administration in rats,dihydroartemisinin was distributed in all tissues except the testis and heart of female rats.The concentration of dihydroartemisinin in tissues was generally higher than that in plasma at the corresponding time point(which was consistent with the larger value of Vss),in which the concentration of dihydroartemisinin in ovaries of female rats was higher,but the concentration in the brain was low.The high concentration in the stomach,small intestine,and large intestine were due to the absorption of the compound in the digestive tract tissue after administration,and the difficulty of being washed away during sampling.The change of dihydroartemisinin concentration in tissues was mostly related to the change of plasma concentration.The excretion fraction of dihydroartemisinin in urine was very low(fe-u,0.007%-0.05%),and in bile was also very low(fe-B,0.09%-0.15%),suggesting that dihydroartemisinin was mainly eliminated from the body through metabolism.The metabolism of dihydroartemisinin in vivo will be completed by other members of the research team.Development and application of a bioanalytical assay for comurins of Psoralea corylifolia FructusIt has been reported that the coumarins of Psoralea corylifolia Fructus have estrogen-like,antitumor,anti-inflammatory,antibacterial and antidepressant activities,which are related to liver,kidney,and reproductive toxicity.In the Pharmacopoeia of the People’s Republic of China of 2020 edition,psoralen and isopsoralen are used as the tested components in the quality analysis method of Psoralea corylifolia,and the total content of psoralen and isopsoralen should not be less than 0.7%.To carry out the pharmacokinetic study related to the safe use of Psoralea corylifolia,we established an analytical method for the simultaneous determination of psoralen,isopsoralen,psoralenoside and isopsoralenoside in rat plasma.The optimization of the analytical conditions focused on:sample pretreatment(acetonitrile protein precipitation method),chromatographic separation(C18 reversed-phase chromatographic column,water and acetonitrile as mobile phase),mass spectrometry detection(in ESI positive ion mode,according to ion pair m/z 187.0/131.0,187.1/131.1,384.2/205.2,384.2/205.1 respectively detect psoralen,isopsoralen,psoralenoside,and isopsoralenoside).The reliability of the new analytical method was evaluated about the "Guiding Principles for Non-clinical Pharmacokinetic Research of Drugs" promulgated by NMPA in 2014.The linear quantitative range of the analytical method is 0.5-364.5 ng/mL(psoralen and isopsoralen)and 1-729 ng/mL(psoralenoside and isopsoralenoside).The intra-assay and inter-assay accuracy was 89.9%-104%,and the intra-assay and inter-assay precision was 3.10%-12.2%,and its technical indicators meet the requirements.We found that psoralenoside and isopsoralenoside were the two main components of Psoraleae Fructus extract,with the contents are 45600 and 24000μg/mL,respectively,and the content of psoralen is 1.71%of psoralenoside The content of isopsoralen is 2.40%of the isopsoralenoside.Rats were orally administered Psoraleae Fructus extract(6 g medicinal materials/kg;the component doses of psoralen,isopsoralen,psoralenoside,and isopsoralenoside were 2.35,1.73,137,and 72 mg/kg,respectively).After a single oral administration of psoralen extract,the peak time of psoralenoside and isopsoralenoside(Tmax:1.0-2.7 h)was significantly earlier than that of psoralen and isopsoralen(Tmax:5.5-6.7 h).The plasma AUC0-∞ of psoralen and isopsoralen(443619-582680 and 167314-276903 ng·h/mL,respectively)was higher than that of psoralenoside and isopsoralenoside(5866-13745 ng-h/mL and 12804-28571 ng·h/mL,respectively),suggesting that the metabolism in vivo is an important factor affecting the exposure of component of Psoraleae Fructus extract after oral administration.The plasma AUC0-∞ of psoralen and isopsoralen on the 7th day was significantly lower than that on the 1st day(P<0.05).The AUC0-∞ of psoralenoside and isopsoralenoside in male rats on the 7th day was also significantly lower than that on the 1st day(P<0.05),and t1/2 and MRT were shorter,indicating that the clearance of these two compounds from the body was accelerated,suggesting that the follow-up studies should pay attention to the effect of continuous administration of Psoraleae Fructus extract on its coumarin composition. |
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