| Artemether is a derivative of artemisinin, which is extracted from Artemisia annua L and appraised as "the best hope for malaria treatment" by the World Health Organization. Although chemotherapy drugs are used most extensively, there are still many problems required to be resolved, such as serious side effect and drug resistance. Artemisinin, as a well-known antimalarial, has rapid effect and low toxicity on the treatment of malaria and has been found its new effects like anti-tumor, anti-schistosomiasis and immune suppression. Of all, anti-tumor activity is the most attracting one. Artemisinin may be a promising anti-tumor agent. It is reported that artemether had successfully been used to treat a few cancer cases in clinic.Artemether is a lipophilic and poorly soluble drug. The aim of this study is to develope a novel dosage form of artemether which has some target activity, can be combined with anti-tumor synergist ferrous salt stably, and can be used by intravenous injection.1. The pre-formulation studies of artemetherWe investigated the solubility and stability of artemether by using the analysis system of Waters Acquity UPLC with photodiode array detector and Empower software. Column, BEH C18,1.7μm,2.1 x 50 mm; mobile phase, acetonitrile:water =62:38 (v:v); flow rate,0.4mL-min-1; column temperature,40℃; injection volume,10μL; wavelength,210nm.The results showed that artemether was poorly soluble in water while easily soluble in organic solvents. Artemether had the highest solubility in ethyl oleate among oil phases tested, and in Cremophor EL35 among emulsifiers tested.When the value of pH was 2.0-12.0, effect of pH on the stability of artemether was not significant. Ferrous salts decreased the stability of artemether in water solutions. Thus, when ferrous salts were used with artemether, the mixture could not be stored for too long.2. Preparation of artemether microemulsionsEthyl oleate, GTCC, and injection soybean oil were selected as oil phase, and Cremophor EL35, lecithin, Tween-80, Cremophor RH40, F68 as emulsifiers, with ethanol as auxiliary emulsifier. Pseudo-ternary phase diagrams and single factor study were used to screen out the oil phase and emulsifier. In the single system, the system comprised of Cremophor EL35/alcohol/ethyl oleate/water and the one comprised of Cremorphor RH40/alcohol/ethyl oleate/water could gain large areas of microemulsion region and had good solubility. Mixed emulsifiers were better than single emulsifier. The area of microemulsions region of mixed emulsifiers was bigger than that of a single emulsifier. The biggest area was formed when Cremophor EL35 was used with lecithin. Since lecithin was easy to become gel and Cremophor EL35 had hemolysis side effect, the mass ratio R of Cremophor EL35 to lecithin was determined to be 3:1. On the basis of the phase diagrams research, the pattern and stability of microemulsions were observed and researched. The microemulsions were prepared at room temperature. Artemether had little effect on the area of microemulsions. The optimal formula of artemether microemulsion was confirmed as ethyl oleate:Cremophor EL35:lecithin:alcohol:water=2:3:1:2:5. 3. The evaluation of artemether microemulsionsDrug-loading capability of artemether microemulsions were investigated by using the analysis system of Waters Acquity UPLC with photodiode array detector and Empower software. Column, BEH C18,1.7μm,2.1×50 mm; mobile phase, acetonitrile:water=40:60 (v:v); flow rate,0.4 mL·min-1; column temperature,40℃; injection volume, 10μL; wavelength,210nm.The maximum drug-loading was 137.72 mg-mL-1. The effect of ferrous salts on the stability of artemether microemulsions was investigated by the same method. Comparing with the results of artemether reference solution, microemulsions could significantly improve the stability of artemether by decreasing the degradation rate by almost four times. When the mass ratio of ferrous salt and artemether was over 1:1, the degradation rate increased significantly, which meant that the amount of ferrous salts should not be more than that of artemether.Quality evaluation was conducted. The indexs included appearance, particle size, distribution, drug-loading, and long-term stability. Artemether microemulsions were pale yellow, clear and transparent liquids with Tyndall effect and good liquidity. Particle size and distribution of artemether microemulsions were observed by transmission electron microscope (TEM). Particle size of blank microemulsions was 30-65 nm and the average was 46nm. Particle size of artemether microemulsions was 28-73 nm, and the average was 47 nm.With dilution multiples of 1,10,100, the diluent diluted by physiological saline and water were both clear and had pale blue opalescence. Tyndall effect could be observed and no drug was separated out.The storage conditions of artemether microemulsions were room temperature and being protected from light. Stability test indicated that the artemether microemulsions were stable. 4. Pharmacokinetics and tissue distributionA sensitive and fast UPLC method was successfully developed to study pharmacokinetics and tissue distribution of artemether microemulsions. Plasma concentrations and organization concentrations of artemether were determined by UPLC and analyzed by 3P97.The results showed that artemether microemulsions were rapidly metabolized after intravenous injection. Artemether could be detected in brain, spleen and liver after 2 minutes of injection. It showed that the transfer of artemether was very fast in vivo and could pass brain barrier.10 minutes later, the concentration of artemether decreased by half in plasma and presented the highest concentration in spleen, followed by brain and liver. After 30 minutes passed, artemether in plasma was almost undetectable, with high concentrations in kidney and spleen, followed by lung, brain, liver and heart. Artemether microemulsions could change the distribution of artemether in vivo, with a certain hepatic targeting.5. The activity of anti-tumorHep3B cells were cultured in vitro. CCK8 method was used to test and analyze the inhibition rate. Cisplatin, artemether solution prepared in DMSO solvent, artemether microemulsions, blank microemulsions, artemether microemulsions mixed with different mixture ratio of ferrous salts were cultured with Hep3B cells. Afterwards, the IC50 values in compounds were calculated and analyzed. The results showed that the anti-tumor effect of artemether could be enhanced by artemether microemulsions. And when artemether microemulsions were combined with ferrous salt in the mass ratio of 2:1, the anti-tumor effect were enhanced more.ConclusionThis thesis provided safety evaluation, pharmacokinetics and pharmacodynamics of artemether microemulsions. The stability of artemether microemulsions and its anti-tumor synergistic agent ferrous salts were investigated, which could provide guidance for the anti-tumor study of artemether in the future. |
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