Study On Artemisinin Derivatives Pharmacokinetics In Dogs And Metabolism In Vitro

In malaria treatment clinical practice,the high recurrence rate is a biggest drawback after oral administration artemisinin derivatives. In vivo study had found that taking artemisinin derivatives successively for several days, drug concentration decreased more significantly than the 1st day rather than maintained steady state. The time-dependent pharmacokinetics is due to self-induced metabolic. Therefore, taking these drugs lead to high recurrence rate. In the same time, in vivo study also found that the half time was not changed after taking artemisinin derivatives successively for several days. It may be attributed to first pass metabolism affected drug absorption, then clearance rate increased. Intravenous administration can avoid first-pass metabolism, therefore the study add the intravenous group, comparing the pharmacokinetic parameters of different group to study the first-pass metabolism in the role of self-induced metabolism. In addition, in vitro liver microsomes experiments study metabolism about artemisinin, dihydroartemisinin incubation with microsomes those treatment in different ways.1.The pharmacokinetics study of artemisinin in dogsTo establish a LC-MS/MS method for the determination of artemisinin in dog plasma. Artemether was used as the internal standard. The mobile phase consisted of acetonitrile, 10 mmol·L^-1ammonium acetate (containing 0.1% formic acid) solution (85:15, v/v) . The flow rate was set at 0.3 mL·min^-1. An Agilent-C18 column (50×4.6 mm, 1.8μm) was used for separation. Electro spray ionization source (ESI) was used and operated in the positive ion mode. Multiple reaction monitoring (MRM) was applied with the precursor to product ion combinations of m/z 300.2→209.1 (for ART) and m/z 316.1→267.2 (IS, AMT) . The linear calibration curves were obtained in the concentration range of 2 - 500 ng·mL^-1. The lowest limit of quantification was 2 ng·mL^-1.Ten healthy dogs were randomized into two groups. a group of dogs received oral 1000 mg doses of ART once daily for five consecutive days,and another group of dogs received intravenous 60 mg doses of ART once daily for five consecutive days. Each group was samplinged on the 1 and the 5th day respectively. Applied liquid chromatography tandem mass spectrometric (LC-MS/MS) method to measured the content of artemisinin in plasma and calculated the pharmacokinetic parameters. oral group: peak concentration (C_max) and peak time (T_max) was observed, then semi-logarithmic chart could be drawn. Elimination rate constant k can be calculated from several concentrations of elimination phase. Accordance with k value, t_1/2 can be calculated. Using software to calculate AUC_0-t and AUC_0-∞ . After the 1st day oral administration artemisinin, C_max was 29.1 9.9 ng·mL^-1, T_max was 1.9±0.7 h, t_1/2 was 2.6±0.6 h, AUC_0-t was 88.2 26.8 h·ng·mL^-1,AUC0- was 93.7±28.0 h·ng·mL^-1。After the 5th day oral administration artemisinin, drug concentration can not be detected, though the pharmacokinetic parameters can not be calculated. Intravenous group: It has been drawn logarithm of plasma concentrations versus time profile chart , a line could be obtained by the method of graphing. C0 and k could be obtained from linear slope and intercept, then accordance with C0 and k calculated V and t_1/2. k multiply with V was CL. The ratio of C0 and k was AUC_0-t. After single dose of intravenous injection artemisinin, tl/2 was 0.9±0.7 h, CL was 27.8±12.9 L·h^-1 , V was 26.0±10.6 L, AUC_0-t was 3568.9±3902.2 h·ng·mL^-1. After the 5th day intravenous injection administration artemisinin. tl/2 was 0.2±0.04 h, CL was 20.8±9.5 L·h^-1, V was 7.5±4.5L, AUC_0-t was 3363.7±1375.9 h·ng·mL^-1. plasma concentration was significantly lower than the 1st day after the 5th day oral administration. t_1/2, CL, AUC_0-t was no significant difference after multi-dose intravenous. The results showed that after oral administration of the 5th days, plasma concentration was decreased time-dependently, but this phenomenon wasn't found in the intravenous administration induction. It is indicated that the self-induced metabolism of the main process of the first pass metabolism. In addition the drug oral bioavailability is very low in dogs. Oral administration blood concentration is far lower than intravenous administration.2.The pharmacokinetics study of dihydroartemisinin in dogsTo establish a LC-MS/MS method for the determination of dihydroartemisinin in dog plasma. Artemisinin was used as the internal standard. The mobile phase consisted of acetonitrile, 10 mmol·L^-1 ammonium acetate (containing 0.1% formic acid) solution (65:35, v/v) . The flow rate was set at 0.3 mL·min^-1. An Agilent-C18 column (50×4.6 mm, 1.8μm) was used for separation. Electro spray ionization source (ESI) was used and operated in the positive ion mode. Multiple reaction monitoring (MRM) was applied with the precursor to product ion combinations of m/z 267.2→163.1 (DHA) and m/z 300.3→209.2 (IS ART) . The linear calibration curves were obtained in the concentration range of 2 - 500 ng·mL^-1. The lowest limit of quantification was 2 ng·mL^-1.Ten healthy dogs were randomized into two groups. a group of dogs received oral 160 mg doses of DHA once daily for five consecutive days,and another group of dogs received intravenous 60 mg doses of DHA once daily for five consecutive days. Each group was samplinged on the 1 and the 5th day respectively. Applied liquid chromatography tandem mass spectrometric (LC-MS/MS) method to measured the content of dihydroartemisinin in plasma and calculated the pharmacokinetic parameters. oral group: peak concentration (C_max) and peak time (T_max) was observed, then semi-logarithmic chart could be drawn. Elimination rate constant k can be calculated from several concentrations of elimination phase. Accordance with k value, t_1/2 can be calculated. Using software to calculate AUC_0-t and AUC_0-∞ . After the 1st dayoral administration dihydroartemisinin, C_max was 57.6 38.5 ng·mL^-l ,T_max was 2.1±0.7 h., t_1/2 was 2.8±0.8 h, AUC_0-t and AUC_0-∞ was 165.4 77.6 h·ng·mL^-1and 177.8±71.7 h·ng·mL^-1. After the 5th day oral administration dihydroartemisinin, drug concentration can not be detected, though the pharmacokinetic parameters can not be calculated. Intravenous group: It has been drawn logarithm of plasma concentration and time chart , a line could be obtained by the method of graphing. C0 and k could be obtained from linear slope and intercept, then accordance with C0 and k calculated V and t_1/2, k multiply with V was CL. The ratio of C0 and k was AUC_0-t. After single dose of intravenous injection dihydroartemisinin, t_1/2 was 0.4±0.1 h, CL was 114.7±46.3 L·h^-1, V was 61.8±37.9 L, AUC_0-t was 574.8±162.3 h·ng·mL^-1. After the 5th day intravenous injection administration dihydroartemisinin. t_1/2 was 0.5±0.1 h, CL was 101.6±43.6 L·h^-1, V was 61.8±37.9 L, AUC_0-t was 722.7±396.9 h·ng·mL^-1. Plasma concentration was significantly lower than the 1st day after the 5th day oral administration. V, CL, AUC_0-t was no significant difference after multi-dose intravenous. DHA oral bioavailability is very low. The results showed that after oral administration of the 5th days, plasma concentration was decreased time-dependently, but this phenomenon wasn't found in the intravenous administration induction. It is indicated that the self-induced metabolism of the main process of the first pass metabolism. In addition the drug oral bioavailability is very low in dogs. Oral administration blood concentration is far lower than intravenous administration.3 Artemisinin, dihydroartemisinin in rat liver microsomal pharmacokineticsForty rats were divided into eight groups (ART oral multi-dose induction group, DHA oral multi-dose induction group, oral solvent control group, ART multi-dose intravenous injection group, DHA multi-dose intravenous injection group, ART intravenous injection solvent control group, DHA intravenous injection solvent control group, completely blank group. Treatment has been for 5 days). Liver microsomes was manufactured.To establish a LC-MS/MS method for the determination artemisinin, dihydroartemisinin in different microsomal metabolism groups. Drug incubation with rat liver microsomal for a period of time. The metabolic rate could be calculated by the rest concentration of drug. After incubation ART with liver microsomal of completely blank group, oral solvent control group, ART intravenous injection solvent control group, ART oral multi-dose induction group and ART multi-dose intravenous injection group, respectively for 60 minutes. Metabolic rate were 35.3 %, 37.3 %, 39.2 %, 53.2 %, 36.6 %. This showed that the metabolism enzyme could be induced by the 5th day oral administration ART and not by the 5th day intravenous injection administration ART. Rat liver microsomes could be affect by solvent. After incubation DHA with liver microsomal of completely blank group, oral solvent control group, DHA intravenous injection solvent control group, DHA oral multi-dose induction group and DHA multi-dose intravenous injection group, respectively for 60 minutes. Metabolic rate were 44.6 %, 47.1 %, 44.1 %, 51.8 %, 38.7 %. Metabolic rate of ART multi-dose oral group were faster than other control group. Metabolic rate of DHA multi-dose oral group were slightly faster than other control group, but ART, DHA multi-dose injection group did not has similar phenomenon.