Research And Toxicity Evaluation Of Novel Lung-targeting Docetaxel Liposome

Lung cancer is the leading cause of cancer mortality and morbidity worldwide.Approximately 80–85%of the patients have non-small cell lung cancer（NSCLC）.Chemotherapy treatment is important to prevent metastasis and recurrence.However,major chemotherapy drugs for NSCLC such as taxanes（DTX and paclitaxel）have not gained much clinical use due to low selective biodistribution.To overcome these drawbacks,researchers are focused on development of high efficiency and low toxicity novel drug or delivery system for treatment of NSCLC.Therefore,a novel lung targeting DTX-LP was researched and developed by patented DBaumNC technology in our research group.Was there any new toxic side effect for altered biodistribution of DTX?What would happen for the targeting tissue of lung and the other non-targeting tissues?In this paper,we aimed to study the toxic effects of DTX-LP on lung or other major tissues as reference preparation of DTX-IN,and to clarify occurrence and development of toxic effects,and to explore the safety and efficacy biological evaluation of novel lung targeting DTX-LP.Firstly,preparation and quality control research of DTX-LP.DTX-LP was prepared by DBaumNC technology with specific submicron size and sustained release in vitro.It had favorable physicochemical stability when sealed stored by solid form under 2⁸℃in dark place at least for 12 M and no crystals precipitated was characterized by DSC and XRD.The drug content was 6.66±0.18mg/g.After hydration,the particle size of DTX-LP was 0.72±0.23μm and the zeta potential was-27.5 mV.The encapsulation efficiency was 90.47±1.65%.Pyrogen test,hemolytic and irritation experiments confirmed that DTX-LP was complying with the requirements of injection for intravenous.The research on quality control of DTX-LP built up a good basis for the further toxicological studies.Secondly,research on lung-targeting of DTX-LP.Based on red blood cells as vehicle to carry DTX-LP,we explored the mechanism of lung-targeting.It is probable that DTX-LP reversibly and noncovalently adsorbed on the surface of red blood cells in the blood stream,and then detached likely due to shear or direct RBC-endothelium contact during their passage through the lung tissue microvasculature.Radionuclide labeled radiography and quantitative biodistribution were used to confirm lung targeting of DTX-LP.On the one hand,aggregation of DTX-LP was visual observation in the rabbit lung by SPECT/CT evaluation.On the other hand,the DTX concentration in tissues was determined by HPLC.In detail,the lung AUC_0-t of DTX-LP was approximately 16-fold greater than that of DTX-IN,which demonstrated outstanding lung targeting for DTX.In contrast to lung,the AUC of heart and kidney was much lower（almost0.51-fold and 0.55-fold）than that of DTX-IN.It was notable that AUC _0-t-t values of liver and spleen were equivalent to that of DTX-IN.It indicated that the novel liposomal formulation could alter the biodistribution of DTX compared with the commercial injection DTX-IN.Thirdly,cytotoxicity of DTX-LP in vitro was researched by MTT method.Two kinds of cell lines（A549 lung cancer cells and MDA-MB-231 breast cancer cells）were used to evaluate the cytotoxicity of BLA-LP and DTX-LP.The results showed that BLA-LP had no significant cytotoxicity.The cytotoxicity of DTX-LP was dose-dependent,and stronger than that of DTX-IN.Fourthly,acute toxicity study of DTX-LP.Single dose toxicity study was used to evaluate the safety of lung targeting DTX-LP in mice and rabbits.Maximum feasible dose（MFD）in mice was about 9.9mg/kg.No mortality was found and the side effects included reduced activity（drowsiness,prone）,weight loss,gastrointestinal reactions（reduction of food intake,diarrhea）.It was found that lungs of mice in the DTX-LP group were edema but the organ coefficient had no significant change.Similar toxicity clinical signs were found in the study of rabbits.Hematologic toxicity was dose-dependent.Target toxicity organ included lung tissue,gastrointestinal tract and the blood system in both experimental animals.DTX-LP did not cause more serious side effects and safety in vivo.Last,further toxicity study of DTX-LP.A randomized,controlled trial study was conducted in rabbits to research repeated dose toxicity of DTX-LP,and accompanied with toxicokinetics.The exposure in rabbit plasma（AUC）of DTX was proportionality with the dose of DTX-LP following i.v.administration.Main toxicokinetics parameters such as reduced AUC,C_max and prolonged t_1/2,were significant different from that of DTX-IN.The kinetic characteristics did not change following three repeated dosing.According to the metabolism studies by UPLC-MS/MS,besides DTX and the known metabolite M-2,another three unreported metabolites were found in bile and feces of rabbits,which were named as Mun-1（relative molecular mass 821）and Mun-2（relative molecular mass837）and Mun-3（relative molecular mass,819）.According to toxicity evaluation criteria for rabbits,DTX-LP could reduce the incidence of severity hematologic toxicity（ⅢandⅣgrade）and the histopathological damage of heart and kidneys compared with the same dose of DTX-IN.Toxicity of DTX-LP on hematologic,liver and spleen was reversible.The weekly schedule significantly decreased myelosuppression when cumulative dose was equivalent to that of 3-weekly.In summary,the novel preparation of DTX-LP with well physicochemical stability and compatibility had the outstanding characteristic that it carried the DTX to the lung and decreased the biodistribution of non-targeting tissues such as blood stream,heart and kidneys.It improved the systemic toxicity of DTX-IN.Our study laid the foundation of further comprehensive safety evaluation for the novel preparation.