Pre-clinical Pharmacokinetics Of Docetaxel Nano-injections Based On High-throughput LC-MS/MS Method

Pharmacokinetic processes are broadly defined as absorption,distribution, metabolism and excretion,giving rise to the frequently used acronym-ADME.The pre-clinical studies of pharmacokinetics may be helpful in developing or optimizing dosing regimens in clinical trials and ensure the safety and rationality of clinical practice.These efforts are important to develop safer and more efficient new drug.As an analogue of natural occurring paclitaxel(Taxol),docetaxel(Taxotere) is one of the most promising anticancer agents known today.More and more studies and researches have proven docetaxel's efficiency.Despite of that,patients receiving chemotherapeutic treatment always suffer from life-threatening side effects.Nowadays a number of paclitaxel formulations in development,which range from tocopherol/surfactant emulsions to polyethylene glycol gels,from protein-stabilized nanoparticles to liposomes,claim to minimize the acute toxicity of therapy and are currently undergoing clinical trials.In hence, docetaxel,as an analog of paclitaxel,modifying its formulation vehicle may be an effective way to improve pharmacokinetic behavior or to control its side effects to some extent.A sery of novel docetaxel nano-injections were designed by Shanghai Institute of Materia Medica.To compare with the normal injection of docetaxel in terms of pharmacokinetic characteristics and acute toxicity,it is necessary to investigate these two aspects in animals in early-stage researches.Accordingly,a more rapid and high-throughput LC-MS/MS method employing liquid-liquid extraction based on 96-well format plates was established and fully improved to evaluate accurately the pharmacokinetics of the drug in pre-clinical researches on animal models.1.The establishment of analytical method of docetaxel in bio-samplesA high-throughput analytical method based on liquid chromatography with positive ion electrospray ionization(ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of docetaxel in bio-samples using paclitaxel as the internal standard(I.S.).Most liquid transfer steps were performed by using eight channel pipetting tools.The analyte and internal standard were chromatographed on a Ultimate XB C-8 column(50 min×4.6 mm,5μm) with a mobile phase consisting of methanol-water-formic acid(90:10:0.01,v/v/v) pumped at 0.3 mL/min.The method had a chromatographic total run time of 3 min.A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring(SRM) mode with the precursor-to-product ion transitions m/z 830.0→549.0(docetaxel) and 876.0→591.0(paclitaxel) used for quantitation.The method was sensitive with a lower limit of quantitation(LLOQ) of 1 ng/ml and good linearity in the range 1～500 ng/mL for docetaxel.All the validation data, such as accuracy,precision,and inter-day repeatability,were within the required limits. Finally,the method reported was successfully applied to the pre-clinical pharmacokinetic study of the novel docetaxel nano-injections.2.Pharmacokinetic study of docetaxel nano-injections in beagle dogsThe beagle dogs(both sexes) were randomized into two groups according to the dosage form in self-cross administration.Blood samples(0.5 ml) were collected via lower limb vein into heparinized tubes at different time points.The LC-MS/MS method described above was applied to study pharmacokinetics after intravenous(i.v.) administration of docetaxel at 5 mg/Kg.The differences in plasma concentrations and pharmacokinetic parameters between the test group and control group were compared by paired t-test.The t_1/2,MRT and V_ss of test group were significantly higher than those of control group,while the AUC is lower than that of docetaxel normal injection.At the same time,the statistical results suggest that the novel drug vehicle can control the releasing rate of docetaxel at the very beginning of administration,and blood concentrations can keep a higher level after the time point of 2h compared with the normal injection,which means that docetaxel nano-injection may decrease the acute toxicity and hematotoxicity by releasing drug controlledly,and the blood circulation time of docetaxel is also pronounced increased.3.Pharmacokinetics and tissue distribution of docetaxel nano-injections in ratsSprague-Dawley rats(both sexes) were divided into 2 groups(6 rats/group) and received novel docetaxel nano-injetion and docetaxel normal injection at the dose of 10 mg/Kg intravenously,respectively.Blood samples were withdrawn at different time until 8 h after the administration of the medication.The plasma concentration-time data were analyzed and pharmacokinetic parameters were estimated by non-compartmental analysis.For distribution study,rats were also randomized into 2 groups(50 rats/group) according to the dosage form and treated intravenously with 10 mg/Kg of docetaxel nano-injection and normal injections respectively.Tissues such as heart,liver,spleen,lung, kidney,brain and muscle were removed at 0.5,1,2,4 and 8h after injection(five rats/time point).The results revealed that the t_1/2 and MRT of docetaxel nano-injections were significantly higher than those of docetaxel normal injection,while the AUC is lower than that of control group.At the same time,according to the plasma concentration-time curve, the novel injections could keep longer circulation time in vivo.Further more,the tissue concentrations of docetaxel were higher for liver,spleen,lung and kidney in test group than that of control group,and the nano-injections were shown to quickly distribute to these tissues,suggesting these tissues may be the temporary drug reservoirs which can sustained release docetaxel back to blood.