Investigation On2-Methoxyestradiol/Hydroxypropyl-β-Cyclodextrin Injection

2-methoxyestradiol (2-ME) is a metabolite of estrogen, and pharmacological studies have shown efficacy against angiogenesis, inhibition toward tumor cell proliferation and anti-tumor effects.2-ME is a drug with very low solubility in water (about0.2μg·ml-1), low oral absorption efficiency and no regular pattern, so it is crucial to select a suitable dosage form and route of administration to increase the solubility of the drug and improve the safety of clinical medicine in order to achieve better clinical efficacy. Therefore, cyclodextrin HP-β-CD was screened out in this study to be safe and with good solubilization effect toward2-ME, then was used to prepare the2-ME/HP-β-CD injection.First, the design and preparation of2-ME injection were studied, in which HP-β-CD and SBE-β-CD were tested and compared for their effects to enhance the solubility of2-ME. And considering the safety and cost, HP-β-CD was selected as the clathrate agent. The optimized preparation procedures were as follows:50%HP-P-CD aqueous solution was used for packaging2-ME, the ultrasonic power was450W and the inclusion time was5min. Under these conditions, the solubility of2-ME was increased to33.6mmol·l-1, which is10.147mg·ml-1,4.6times more than the solubility in water, thus the clinically promising2-ME/HP-β-CD injection was prepared.To evaluate the safety of the2-ME/HP-β-CD injection, acute toxicity, stimulation and hemolytic experiments were conducted, and it had been observed that the injection of2-ME/HP-β-CD was with no renal toxicity, hemolytic and stimulation, and the safety of this injection was superior to2-ME control solution group.Pharmacokinetics of2-ME/HP-β-CD injection were investigated in mice, indicating that2-ME/HP-β-CD injection was established into a two-compartment model in mice, with the distribution half-life t1/2α and elimination half-life ti/2p (3.08±0.12) min and (10.72±2.31) min, respectively, the AUC0→240min (175.4± 23)μg·ml-1·min, and was found that the in vivo distribution and elimination of2-ME declined rapidly. When2-ME was combined with Verapamil, its AUC and MRT showed an increasing trend, and CL showed a downward trend, indicating that Verapamil had impacts on the in vivo pharmacokinetics of2-ME/HP-β-CD injection.2-ME/HP-β-CD injection was used for intravenous administration in dogs, and pharmacokinetic characteristics was studied. The injection was proved to be two compartment model, with the half-life (t1/2α)(8.77±2.02) min, elimination half-life (t1/2β)(18.36±2.04) min and AUC0→120min (131.86±23)μg·ml-1·min. Comparing to the kinetic parameters in mice, t1/2α and t1/2β were both lengthened to some extent, but there were significant differences in the kinetic parameters between dogs and mice. And as the kinetic parameters of dogs were more similar to human, thus they could be used as references for human clinical researches. And how to maintain the effective blood concentration of2-ME in vivo became the key factor for future studies of2-ME/HP-β-CD injection.In vivo pharmacodynamics of2-ME/HP-β-CD injection were applied for a preliminary study, and BALB/c inbred mice was transplanted breast cancer cells to investigate the inhibition rate, tumor volume change and lung metastases, which proved2-ME/HP-P-CD injection had a certain therapeutic effect toward murine breast cancer with a inhibition rate of20.9%-36.7%, indicating that the in vitro inhibition effect was superior to the in vivo result, and the reasons needed to be further studied.As for vitro experiments, mouse breast cancer4T1, human lung adenocarcinoma cell line SPC-A1and human prostate cancer PC-3cells were used as study objectives, SRB staining assay was applied to examine the tumor cell growth inhibition effect of2-ME and2-ME/HP-β-CD injection, and when combined with verapamil, changes of IC50and the synergy index were used to explore the anticancer effects of2-ME. The injection of2-ME/HP-β-CD and2-ME were confirmed to have the same anti-tumor effect, and the calcium antagonist verapamil was also found to improve the anticancer activity of2-ME and2-ME/HP-β-CD injection, of which the mechanism may be related to the efflux function of P-glycoprotein (P-gp) or other metabolisms needed to be further studied from the aspects of molecular biology.