| Objective: To provide preliminary experimental data for the application of Anemoside B4 used in clinical practice,study on the feasibility of making Anemoside B4 into nebulizer.Methods:(1)The HPLC analysis for B4 was established and the methodologies were examined.Solubility of B4 in water and different p H buffers were measured and the octanol-water partition coefficient of B4 was tested.The stability of B4 in simulated lung fluid(SLF)was examined.(2)Calu-3cell monolayers were cultured and C971P/C974 P were screened for drug permeation resistance,and the amount of absorption enhancer was further determined by MTT assay.Respimat was selected as device.Different concentrations of Chitosan,CMC-Na and Xanthan gum were formulated,and next generation pharmaceutical impactor(NGI)was used to detect the fine particle fraction(FPF)which used to select the tackifier of the formulation.The ethylparaben was selected as preservative.The amount of B4 in the formulation was determined.(3)According to the Chinese Pharmacopoeia,the FPF of the B4 nebulizer and the content of the drug per spray were examined.The effects of light and temperature on B4 inhaler were examined.(4)The analytical method of B4 in vivo was established and the methodologies were examined.The method specificity and limit in vivo were examined.B4 nebulizer was administered by intratracheal instillation and intravenous injection group was used as a comparison to study drug concentration-time curve in the plasma.The PKSolver V2.0 was used to process the drug concentration in plasma and calculate the pharmacokinetic parameters.B4 inhaler was administered by intratracheal instillation to examine the distribution in the lung.(5)B4 nebulizer was administered by intratracheal instillation several times.The rats were dissected and the lungs were HE stained.The images of HE staining were observed under a microscope to evaluate the irritation.Results:(1)In vitro analysis showed that the B4 concentration ranged from15.625?g/m L to 500?g/m L was in linear with the peak area.The precision RSD wasless than 2%,repetitive RSD was 1.16%,and the recovery was 98%~ 102%.The solubility of B4 in water is 33.78 mg/ml and the octanol/water partition test proves that B4 is a drug with water solubility.B4 was stable within 24 h in SLF.(2)B4 nebulizer formulation were: B4 dosage 10mg/m L;ethylparaben dosage 0.03%(w/v);C974P and Xanthan gum dosage 0.05%(w/v)and 0.03%(w /v)respectively.(3)TSI test showed the deposition rate of up to 71% in secondary flasks.B4 nebulizer the content of drug is between 100?g/spray~120?g/spray.The light and temperature have no significant effect on B4 inhaler.(4)In vivo method showed that concentration of B4 in the plasma and lung tissue in range of 0.5?g/ml to 100.0?g/ml and 0.5?g/ml to 400.0?g/ml respectively was in linear with peak area,the precision and recovery in vivo assays meet the analytical requirements.B4 nebulizer was administered by intratracheal instillation,the drug slowly infiltrates into the blood and prolonged the retention time in the body,while the drug is rapidly eliminated after the intravenous injection.B4 inhaler was administered via intratracheal instillation,a large amount of drug was retained in the lung,and the concentration of the drug in the lung was still 125.5 ?g/g after 24 hours of release.(5)The results of HE staining showed that B4 nebulizer was administered via intratracheal instillation several time that have irritating to the lung.Conclusion: The absorption enhancers and thickener were screened by cytological experiments and particle size distribution experiments,and anemoside B4 nebulizer was prepared.The FPF of the B4 nebulizer complies with pharmacopoeia regulation,and B4 nebulizer is stable to light and temperature.B4 nebulizer was administered by intratracheal instillation,a large amount of drug was accumulated in the lung and drug slowly infiltrates into the blood and prolonged the retention time in the body.B4 nebulizer was repeated administration via intratracheal instillation that has certain irritation to lung tissue. |
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