Topotecan Reversed Lipid Nanoparticle Oral Preparation Research

Topotecan(TPT)is the semisynthetic analogues of camptothecin(CPT)with high solubility in water.TPT has the specific topoisomerase-I inhibitory activity that could lead to serious DNA damage.The post-marketing conventional topotecan hydrochloride capsules nowadays still got drawbacks such as the serious discomfort in gastrointestinal(GI)tract and poor stability in GI tract which leads to diffusion or hydrolyzation,undercutting the bioavailability.In this study,a new preparation,reversed lipid-based nanoparticle of TPT(RLBN-TPT)was developed,for improving the drug stability and the oral bioavailability as well as decreasing the discomfort in GI tract.In this study,transparence,appearance of oil solution and particle size were set as the indexes to practice single factor tests and orthogonal design to screen and optimize the membrane materials and the drug loading of RLBN-TPT.With the preparation technology oflyophilization,reversed lipid-based nanoparticle of TPT(RLBN-TPT)was obtained after dissolved in MCT.Microstructures and Mean particle sizes of RLBN-TPT were characterized by Transmission Electron Microscope(TEM),Optical Microscope(OM)and Dynamic Light Scattering(DLS)respectively.The results showed the average size of reversed micelle nanoparticles was approximately 60 nm with a homogeneous distribution in shapes of spheres or ellipsoid,and after dispersed in simulated intestinal fluid(SIF),lipid vesicles and emulsion droplets were formed in shapes of spheres.High performance liquid chromatographic method was established to determine the concentrations of both forms of topotecan.Release curve of RLBN-TPT in SIF was investigated to study the stability of TPT in GI tract as well as the protective effect of RLBN for topotecan.The CAPCELL PAK C18 MG?cartridge column(250 × 4.6 mm,5?m)was used with a mobile phase consisting of mobile phase ‘A'(pH 6.4 adjusted with glacial acetic acid)and mobile phase ‘B'(100 % acetonitrile)at a flow rate of 1.0 mL·min-1 using a gradient method [0~ 8min(88%A?60%A);8~17(60%A)].The detection wavelength was 360 nm,and the column temperature was maintained at 55°C.Results showed that both lactone and carboxylate forms of topotecan were well separated and determined preciselyby this HPLC method,and the calibration curves were linear within the range of(0.25-5.00)?g/mL.Compared with free topotecan,RLBN significantly improved the stability of TPT in SIF as the percentage of carboxylate form was remarkably lower than the free topotecan(p<0.05),which showed that RLBN can significantly protect the active form of TPT from hydrolysis into inactive form,thus enhancing the stability of drug in Simulated Intestinal Fluid.The HPLC method for the determination of TPT content and related substance of RLBN was established.The method was validated to be accurate,specific,reproducible,and sensitive.The results of stress testing showed that the RLBN-TPT preparations were sensitive to high-humidity and high-temperature,therefore the preparations need to be stored at low temperature(2?-10?)and should not be damped;The results of the accelerated test showed that RLBN-TPT preparations had no obvious changes in content,particle size or cumulative release.LC-MS/MS method was established to determine the plasma concentrations of topotecan in vivo.The method was validated to be accurate and reproducible,and the calibration curves showed good linearity(r2> 0.997)within ranges of 1.0 ~ 200 ng/mL.In vivo pharmacokinetics of RLBN-TPT were compared with Free topotecan(topotecan hydrochloride capsules).Results showed that the Cmax and AUC0-? increased significantly(more than 1.61 and 1.57 times respectively,p<0.05)after oral administration of RLBN-TPT in rats compared with Free TPT,indicating that the bioavailability absorption of topotecan was remarkably enhanced by our preparation;after blocking of the lymphatic transport,significant decrease of drug absorption was found in RLBN-TPT treated group,suggesting that most of drug in RLBN-TPT was transported to intestinal lymph with the help of RLBN.A tumor-bearing model was established in the Male Balb/c mice with HepG2 cells.The results of in vivo antitumor activity studies showed that the tumor inhibitory rate of RLBN-TPT and Free TPT was 44.08% and 16.3% respectively,which was significantly different(p<0.05).The tumors were also tested by the method of TUNEL and H&E staining to further prove the antitumor activity of RLBN-TPT.Results also showed that RLBN-TPT preparation enhanced antitumor activity compared to the free topotecan since tumor cells of its group were dramatically decreased with much more quantity of apoptotic tumor cells.The severity of jejunum after treated with RLBN-TPT and Free TPT in mice was investigated in gastrointestinal damage studies.Results showed that the rats treated with Free TPT had extremely damaged jejunum,which had extensive mucosal necrosis with cellular debris present in the lumen and there was overt bleeding as well as extensive necrosis throughout the jejunum with evidently extensive vascular congestion.However,the mice treated with RLBN-TPT had no extensive histological differences but only a little vascular congestion compared with saline group,which means our preparation had decrease the gastrointestinal damage compared with Free TPT.In conclusion,RLBN-TPT remarkably enhanced the stability of TPT in GI tract,eased the GI degradation,promoted absorption of lymphatic transport and significantly enhanced oral drug bioavailability.Our preparation can be easily manufactured and effectively absorbed by the human body.It is expected to obtain safer and high-tolerated properties for containingbiocompatible natural lipids while without any toxic organic reagents.Meanwhile,the drug can be largely transported through lymphatic system with the help of RLBN,which can be of great significance in the study of targeted cancer therapyrelated to autoimmune diseases.