| Objective:Baicalin (BA) is one of main effective components of Scutellaria baicalensis Georgi as a medicinal herb. Though BA exhibits a wide range of pharmacological effects, such as detoxification, scavenging superoxide radicals and anti-tumor, etc, its poor solubility and low bioavailability severely limit the clinical application. Therefore, the objective of this study is to develop BA liposome and evaluate pharmaceutic characterization, pharmacokinetics and biodistribution behaviors in animal model to obtain good pharmacokinetic and biodistribution characterization.Methods:1. The solid dispersion technology was used to prepare BA liposomes for the first time. The morphologies of BA-LP were examined by the transmission electron microscopy (TEM). The particle size of t BA-LP was measured by Malvern ZEN3600. The encapsulation coefficency and in vitro release of BA-LP were studied using a dialysis bag method.2. On the basis of the single factor experiments, formulation and preparation technology were optimized with encapsulation efficiency as the index.3. A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of BA in rat plasma and tissues to investigate pharmacokinetic and biodistribution behaviors.4. A HPLC method was developed and validated for the determination of BA in rabbit plasma and tissues to investigate pharmacokinetic and biodistribution behaviors.Results:1. The tween-80 (T-80), BA and citric acid (CA) were determined as main factors by single factor experiments.2. The optimization formulation was composed of T-80 (50mg), BA (96mg) and CA (50mg) based on the orthogonal experiment.3. The transmission electron microscopy revealed spherical globules and good dispersion of BA-LP. The particle size and encapsulation efficiency of BA-LP were 156±5.35 nm and 82.7±0.59%, respectively. In vitro drug release kinetic model of BA-LP was fitted well with the Weibull distribution equation: lnln (1/(1-Q))=0.6091nt-1.230 (R=0.995).4. A HPLC method was successfully established for the determination of BA in rat plasma and tissues. HPLC system consisted of Dionex ultimate 3000 series including pump (LPG-3400SD), UV-vis detector (VWD-3100), auto injector (WPS-3000) and column oven (TCC-3000). Separation was performed on a reverse phase C18 column (Inertsil ODS-SP; 4.6×250 mm,5um particle size, made in Japan) with a guard column (Phenomenex C18,4.0mm×3.0 mm). The elution was isocratic at 1.0 ml/min with a mobile phase consisted of methanol/acetonitrile/0.4%(v/v) aqueous phosphoric acid (7.5:7.5:85) at the detection wavelength of 278 nm. The column temperature was maintained at 35 ℃ and the injection volume was 20μl. The HPLC method has good specificity and no significant interfering peaks at or near the retention time of BA and internal standard were detected from endogenous substances. Good linearity was obtained between 0.0525-5.25μg/ml for plasma and 0.0525-21 μg/g for tissue samples (r>0.999). The intra- and inter-day assay of precision and accuracy for all bio-samples ranged from ranged from 3.78% to 9.83% and from 83.7% to 104.6%, respectively. The extraction recoveries ranged from 79.8% to 95.5%. Samples were stable during the five freeze-thaw cycles at-20℃.5. Carboxymethyl cellulose suspension containing BA (BA-CMC) as a control, the pharmacokinetic parameters of BA-LP were obtained from rat administered by oral gavage at a dose equivalent to 100 mg/kg of BA as follow: plasma drug concentration-time curve (AUC0-t) of BA-CMC and BA-LP was 12.397 and 37.64 mg/L*h, the mean residence time (MRT) was 9.775and 8.358 h, the elimination half-life (t1/2) was 24.833and 10.628 h, apparent volume of distribution (Vz) was 142.088 and 32.446L/kg, clearance rate (CLZ) was 3.965 and 2.116 L/h/kg, the peak concentration (Cmax) was 1.25 and 3.52mg/L. It can be seen from the in vivo distribution at 30min after administration that, BA-CMC group, drug concentration was relatively high in the kidney and lung and low concentrations in the heart and the brain; drug concentration was significantly increased in the liver, spleen, lung in the case of BA-LP group, which were increased by 2.29 times,2.33 times and 1.25 times in the liver, spleen and lung in comparison with BA-CMC.6. A HPLC method was successfully established for the determination of BA in rabbit plasma and tissues. Chromatographic conditions were the same as the above analysis conditions in rats. The HPLC method has good specificity and no significant interfering peaks at or near the retention time of BA and internal standard were detected from endogenous substances. Good linearity was obtained between 0.05-10 μg/ml for plasma and 0.05-300μ.g/g for tissue samples (r>0.999). The intra-and inter-day assay of precision and accuracy for all bio-samples ranged from ranged from 4.37% to 10.84% and from 80.8% to 98.4%, respectively. The extraction recoveries ranged from 73.1%to 94.6%. Samples were stable during the five freeze-thaw cycles at-20℃.7. BA injection (BA-INJ) was chosen as control, the pharmacokinetic param eters of BA-LP obtained from rabbit after intravenous administration at a dose equivalent to 10 mg/kg of BA:AUC(o-t) was 6.915 and 2.373 mg/L*h, MRT (0.t) was 7.162 and 8.073 h, t1/2 was 5.911 and 11.624 h, V2 was 11.754 and 57.599 L/kg, CLZ was 1.378 and 3.434 L/h/kg, Cmax was 1.961 and 0.634 mg/L respectively.At 30min after intravenous administration, BA-INJ groups, drug concentr ations in the kidney tissue> lung> liver> stomach> spleen> heart> brain in turn; for BA-LP groups, the higher drug distribution in the lung tissue was observed, ranged from 5.627±2.833μg/g increased to 15.693±3.834μg/g, followed by liver tissue, which was higher that of BA-INJ group, moreover, drug concentration in other tissues was lower than that of BA-INJ group.At 12h after intravenous administration, the drug was rapidly eliminated for BA-INJ group, and the slow elimination in the case of BA-LP in tissues was observed. Drug concentration in the lung was still the highest in the case of BA-LP and was 4.2 times higher than BA-INJ.Conclusion:1. BA-LP was successfully developed by solid dispersion technology for the first time. The preparation process is simple and feasible, easy to industrialization. The uniform particle size distribution and high encapsulation efficiency of BA-LP were obtained, with slow release characteristics in vitro.2. The oral bioavailability of BA in rats was improved by BA-LP, which change the pharmacokinetic and biodistribution behavior.3. BA-LP significantly altered biodistribution behavior of BA in the rabbits, which shows relatively high drug concentrations in lung, with lung targeting characteristics.4. BA-LP is a promising drug carrier that can increase the oral bioavailability and can deliver drug to lung in an effective way to increase drug concentration in lung tissue and reduce drug distribution in other organ. |
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