In Vitro Stability And Time-dependent Pharmacokinetic Studies Of Dihydroartemisinin

Dihydroartemisinin(DHA) and artesunate(ARS), two derivatives of artemisinin antimalarials, have become the first-line anti-malarials in areas of severe malarias or multidrug resistance. DHA is also the common active metabolite of its methyl ether (artemether), ethyl ether (arteether) and hemisuccinate ester artesunate (artesunate)、 The built-in chemical instability of DHA causes serious bioanalytical problems in hemolytic plasma and blood during the pharmacokinetic and metabolism studies. Morever, monotherapy with artemisinin drugs results in comparatively high recrudescence rate. Drug exposure decreased after multiple oral doses. Autoinduction of CYP-mediated metabolism, has been supposed to be the underlying mechanism、 Decreased plasma concentrations during the repeated dosing have been reported for QHS and artemether. However, the potential auto-induction metabolism of DHA is still controversial. To provide reasonable suggestions for bioanalysis and clinical medication during preclinical and clinical studies of DHA, the time-dependent pharmacokinetics of DHA and its prodrug ARS were investigated in rats. And the stability of DHA in vitro was also evaluated.1. To evaluate the factors that can induce the degradation of DHA during its bioanalysis, the stability of DHA in vitro, including high temperature, high humidity, strong light irradiation, organic solvents, buffered aqueous solutions at different pH, plasma, simulated hemolytic plasma containing inorganic/organic iron (Fe2+, Fe3+, Hb or hemin), and blood was investigated. The degradation process and the epimerization of DHA were monitored using a HPLC-UV and HPLC-HRMS methodology.The results showed that DHA undergoes different degrees of degradation under high temperature, high humidity and strong light irradiation. DHA mainly undergoes epimerization pathways without degradation in organic solvents, and it is predominantly present as alpha epimeric format in plasma samples. The degradation of DHA was pH-and temperature-dependent in buffered aqueous solutions. Reduced form of hemolytic products (i.e., inorganic/organic Fe2+) could accelerate the degradation of DHA, while Fe3+and hemin played a minor role. Two major degradation products of DHA in hemolytic plasma and blood were identified as peroxyhemiacetal, and the initial degradation of DHA in biological fluids was proposed to arise via ring-opening of the cyclic hemiacetal instead of previously presumed peroxide bridge. The stabilizer (H2O2/EDTA-2Na) added to hemolytic plasma and blood could prevent DHA from degradation during a batch analysis, which included sample pretreatment at room temperature, protein precipitation using organic solvents and instrumental analysis.2. The time-dependent pharmacokinetics of DHA (ARS) and its phase I (monohydroxylated DHA) and phase II (α-DHA-G) metabolite in rats were studied after a single and5-day multiple oral doses. A selective and sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) were established for the determination of DHA (artesunate) and its main phase I and phase II metabolite in rat plasma. DHA, ARS, monohydroxylated DHA, a-DHA-G, and internal standard (IS, artemisinin) in rat plasma were detected by HRMS performed in full-scan mode. The method validation showed high accuracy (<7%) and precision (<15%) at the linear dynamic range (10.0-1000.0nM for DHA (ARS) and20.0-2000.0nM for a-DHA-G, respectively).The pharmacokinetic study was designed to get the direct evidence of the potential auto-induction metabolism for DHA. The gender effect on the pharmacokinetic profiles of DHA and its major metabolites (monohydroxylated DHA and a-DHA-G) were also studied. The AUCo-t and CL/F of the parent drug DHA (ARS), its metabolite monohydroxylated DHA and a-DHA-G did not change significantly (P>0.05) after a5-day oral administration of DHA (ARS) to rats. Gender difference was observed for DHA and its metabolites. The exposure level of DHA and its metabolites were much higher in male rats compared with female rats (P<0.05). However, gender difference was not observed for ARS and its metabolites.