| Objective: In this paper, cyclosporine A–loaded pH–sensitive nanoparticles (CyA–NP) and freeze–dried CyA–NP (Fd–CyA–NP) were prepared. The high performance liquid chromatography (HPLC) and high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the determination of CyA in biological samples. This work was designed to study absorption, distribution, metabolism and excretion of CyA–NP in vivo.Methods: (1) Preparation of CyA–NP and Fd–CyA–NP, its pharmaceutical characteristics: CyA–NP was prepared with quasi–emulsion solvent diffusion technique, and Fd–CyA–NP was prepared by freeze–drying technique. The particle size, size distribution, encapsulation efficiency and the morphological characteristic were studied individually. (2) HPLC method for determination of CyA in rat blood and tissues, its application to biodistribution: The mice were orally administered with CyA–NP and Neoral at a single dose. The liquid–liquid extraction method was used in biological samples extraction. The internal standard was cyclosporine D (CyD). The HPLC method was established to determine the concentration of CyA in blood and tissues. The tissues distribution and targeting eficiency were evaluated by pharmacokinetic parameters (AUC, AUQ, Cmax, Tmax) and targeting parameters (Te, RTe, TI). (2) HPLC method to determine CyA in dog blood and its application to pharmacokinetic studies: A random two–way crossover study in 6 dogs was adopted. The drug blood concentration was determined by HPLC method after oral administration of Fd–CyA–NP and Neoral. Pharmacokinetic parameters were calculated by 3P97 program. (3) Validated HPLC–MS/MS method to determine CyA in rat feces and urine and its application to in vivo metabolism of CyA–NP: Urine and feces samples were collected after oral administration of CyA–NP. The analytes CyA and its metabolites in rat urine and feces were extracted from waste samples via liquid–liquid extraction. Turboionspray source was used as a detector. It was operated in a positive ion mode with transitions of m/z1225→m/z1112 for CyA and in a selected multiple reactions monitoring (MRM) mode with transitions of m/z1239→m/z1099 for the internal standard (cyclosporine D, CyD). The identification of the metabolites and elucidation of their structure were performed on the basis of their retention times and MS fragmentation behaviors. (5) HPLC method for determination of CyA in rat liver microsomes and its application to enzyme kinetics of CyA–NP in vitro: The rat liver microsomes was prepared by differential centrifugation technique. Microsomal protein concentration was determined by Lowry. The effect of incubation time, enzyme and substrate concentration on drug metabolism was studied, and the enzyme kinetics parameters were calculated by Lineweave–Brurk.Results: (1) The particle size of CyA–NP and Fd–CyA–NP were 43.9±0.8 nm and 52.7±4.6 nm, displayed high encapsulation efficiency about 99%, leading to final CyA loading values as high as (23.9±0.1)% and (22.9±0.2)%, respectively. The nanoparticles looked round and regular under transmission electron microscopy. (2) HPLC method was developed to determine CyA in blood and tissues. The recoveries were all over 72%, and the intra– and inter–day precision values were less than 15%. The concentration–time data of Neoral and CyA–NP were fitted as a two–compartment open model in mice. The relative bioavailability of CyA–NP were 162.5% compared with Neoral. The sequence of CyA–NP levels in various tissues was liver>heart>kidney>spleen>lung, it shows a targeting efficiency in the kidney and heart compared with Neoral, and there is no accumulation in the liver. (3) HPLC method was developed for the determination of CyA in dog blood. The recoveries were all over 94%, the intra– and inter–day precision values were less than 10%. The concentration–time data of Neoral and Fd–CyA–NP were fitted as a two–compartment open model in dog. The AUC of Fd–CyA–NP was higher than that of Neoral (P<0.05), while the CL(s) significantly decreased (P<0.05). The relative bioavailability of Fd–CyA–NP were 135.9% compared with Neoral. (4) HPLC–MS/MS method was developed and validated for the quantification of CyA and the identification of its metabolites in rat urine and feces. Extraction recoveries of CyA and CyD were both over 80%. The intra– and inter–day precision values were less than 8%, and the accuracy was within±15% for the analytes. About 84 h after the oral administration of CyA–NP, the cumulative amounts of CyA were found to be 625.80±289.08μg in feces and 6.48±1.29μg in urine. Totally seven metabolites in rat feces were identified as Demethyl CyA, hydroxy CyA and dihydroxy CyA, while six of these metabolites were detected in rat urine. (5) HPLC method was developed to determine CyA in rat liver microsomes. The recoveries were all over 87%, and the intra– and inter–day precision values were less than 10%. The CLint of CyA products was higher than that from CyA–NP (P<0.05), but there was no statistical significance in Vmax and Km between them (P>0.05). After incubating for 90 min, the metabolic rate of CyA products (1.049±0.028μg·mL–1) was higher than that from CyA–NP (0.874±0.023μg·mL–1) (P<0.05).Conclusion: The HPLC and HPLC–MS/MS method were proven to be sensitive, simple, reproducible, suitable for the rapid determination of CyA and pharmacokinetic studies. CyA–NP was distributed and metabolized extensively in vivo. The bioavailability of CyA was significantly improved. This novel pH–sensitive nanoparticles delivery system had wide perspective and practicability on the clinical application of CyA.
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