| BackgroundAllergic reactions and severe systemic toxicity are two major challenges for the clinical application of docetaxel injection(DTX-IN)for treatment of non-small-cell lung cancer(NSCLC).We developed a novel lung-targeted DTX-loaded liposome(DTX-LP),an efficient drug delivery system,with a patented DBaumNC technology to overcome these deficiencies.ObjectiveTo describe the targeting activity,tumor inhibition rate(TIR),survival,pathology,tumor apoptosis and metabolism of DTX after intravenous injection of DTX-LP compared to the DTX injection(DTX-IN)formulation based on the VX2 orthotopic lung cancer rabbit model.Methods1.The establishment of VX2 orthotopic lung cancer rabbit model.The ultra-minimal invasive percutaneous puncture inoculation method was established for a VX2 orthotopic lung cancer rabbit model with fewer technical difficulties,lower mortality of rabbits,a higher success rate and a shorter operation time.The growth,metastasis and apoptosis of tumor was evaluated by CT scans,necropsy,histological examination,flow cytometry and immunohistochemistry.2.Studies on the anti-tumor effect of DTX-LP based on the VX2orthotopic lung cancer rabbit model.The tumor-bearing rabbits were randomly divided into control group,DTX-IN group,DTX-LP low-dose group and DTX-LP high-dose group.Four groups received an intravenous injection of saline,DTX injection(1mg/kg),DTX-LP(0.5 mg/kg)and DTX-LP(1.0 mg/kg)once a week during six weeks beginning at two weeks post-inoculation.The tumor inhibition rate(TIR),survival,pathology,tumor apoptosis,tumor proliferation,tumor angiogenesis,body weight and tumor metastasis were used to evaluate the anti-tumor effect on the VX2 orthotopic lung cancer rabbit model.3.Determination of tissue distribution and pharmacokinetics based on the VX2 orthotopic lung cancer rabbit model.The tumor-bearing rabbits were randomly divided into DTX-IN group and DTX-LP group.Two groups received an intravenous injection of DTX injection(1.0 mg/kg)and DTX-LP(1.0 mg/kg).HPLC method was established for the determination of DTX in heart,liver,spleen,lungs,kidneys,stomach,brain,and tumor at 30min,1.5h,4h,8h and 12h after the last treatment administration.UPLC-MS/MS method was established for the determination of DTX in plasma at 5min,15min,30min,1h,1.5h,2h,4h and 8h.4.Study on metabolism of DTX-LP in vivo based on the VX2orthotopic lung cancer rabbit model.The tumor-bearing rabbits were randomly divided into DTX-IN group and DTX-LP group.Two groups received an intravenous injection of DTX injection(2.0 mg/kg)and DTX-LP(2.0 mg/kg).Feces during the time periods 0-8 h,8-24 h,24-48 h,liver,lungs,and biles were collected to trace the DTX and its metabolites with UPLC-MS/MS.Results1.The VX2 orthotopic lung cancer rabbit model:The ultra-minimal invasive percutaneous puncture inoculation method was established with fewer technical difficulties,lower mortality of rabbits,a higher success rate and a shorter operation time(15min per case).The tumor-bearing rate was100%.The tumor-bearing rabbits showed local solitary tumor with 2-10mm diameters after two weeks post-inoculation.HE staining showed large tumor nests with distinct cell borders and more pleomorphic nuclei.The mean value of survival time was 42.2±10.8d.The double time of tumor was5.19±0.81d.2.The anti-tumor effect:The CT TIRs(%)of DTX-IN and DTX-LP groups after six weekly administrations were 84.39%±1.48%and94.71%±0.75%(P<0.01).The tumor growth was significantly suppressed.The tumor apoptotic rates in four groups(control group,DTX-IN group,DTX-LP low-dose group and DTX-LP high-dose group)from FCM analysis were 29.64%±3.80%,43.23%±2.13%,44.15%±4.76%and68.75%±5.90%(p<0.01 between DTX-IN and DTX-LP high-dose group).The apoptotic tumor index in four groups from TUNEL analysis were7.9%±1.0%,11.7%±1.1%,11.6%±0.7%and 13.9%±0.9%(P<0.01between DTX-IN and DTX-LP high-dose group).The proliferation index in four groups from PCNA analysis were 41.7%±8.2%,21.1%±3.4%,23.1%±2.1%and 15.6%±2.7%(P<0.05 between DTX-IN and DTX-LP high-dose group).In the DTX-LP high-dose group,the CD31-positive blood vessels were smaller than other groups.These results indicated that DTX-LP increased apoptosis of tumor cells and reduced tumor angiogenesis,thereby reducing tumor growth.The average survival days of the four groups were 41.0±11.1,56.0±11.9,60.5±14.0 and 92.3±21.8 days(P<0.05 between DTX-IN and DTX-LP high-dose group).DTX-LP prevented tumor metastasis to the chest wall,prolonged the average survival time,reduced non-targeted organ toxicity and improved the condition of weight loss.3.The tissue distribution and pharmacokinetics:The concentrations at 30min,1.5h,4h,8h and 12h were 76.97±3.99,50.45±5.46,39.87±3.89,39.42±8.52,28.03±4.10?g/g in lung tissue and 57.31±9.30,48.48±4.21,33.80±2.23,32.05±2.37,28.93±2.01?g/g in tumor tissue for the DTX-LP group.The concentrations of DTX in lung and tumor tissues were significantly increased when the DTX was prepared as the lung-targeted liposome formulation.The concentrations at 5min,15min,30min,1h,1.5h,2h,4h and 8h in plasma for the DTX-LP group were lower than DTX injection group.The AUC0?t?t of DTX-LP group is significantly lower than DTX-IN group(P<0.05).4.The metabolism in vivo:DTX and its known metabolites M-1/3,M-2,and M-4 were found in the feces of both in DTX-LP and DTX-IN groups.Spectra from the DTX-LP group samples showed no significant differences in the metabolism pathway or metabolites when compared to samples from the DTX-IN group.According to the iron abundances of M,M-1/3,M-2 and M-4,the metabolites of DTX reached peak concentration levels during the 8-24 h sampling period in the DTX-IN group and during the 24-48 h sampling period in the DTX-LP group.The liposomal delivery of DTX significantly delayed the drug metabolism in rabbit feces compared to the traditional injection formulation.ConclusionIn summary,the pharmacodynamics of DTX-LP is studied based on VX2 orthotopic lung cancer rabbit model.Compared to DTX-IN,DTX-LP increases the tumor apoptosis,enhances the anti-tumor effect of DTX,prolongs the average survival time nearly doubled and reduces the systemic toxicity of DTX.DTX-LP prepared in our paper achieves the expected design purpose of"high efficiency and low toxicity".It is a promising formulation for targeted therapy of lung cancer with a very practical clinical application prospect. |
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