The Study Of Pharmacokinetics And Tissue Distribution Of Fargesin And Its Preparation

Objective: Our preliminary experiment showed that fargesin, extracted from traditional Chinese Medcine Biond Magnolia, has obvious vasodilation effect on pulmonary artery and was potential to be used for the treatment of pulmonary hypertension. To take a step further, the aim of this study was to develop a new drug candidate for the treatment of pulmonary hypertension which possessed the sustained release effect and lung targeted property.(1) To establish an accurate quantitative determination method to measure the content of fargesin in blood and other biological samples, explore the regularity of the absorption, distribution and metabolism of the fargesin after intragastric administration or tail vein injection and preliminarily evaluate pharmacokinetics and tissue distribution of fargesin in vivo;(2) To prepare the lung targeted microspheres characterized with sustained release effect by optimizing the fabrication technique and establishing the quality evaluation standard;(3) To analyze the pharmacokinetics and tissue distribution of drug-loaded microspheres in vivo, including the targeting ability, sustained release effect and toxicity.Methods: High performance liquid chromatography(HPLC) was adopted in this research to establish a detection method of fargesin in biological samples. DAS 2.0 drug pharmacokinetics software was used to analyze the pharmacokinetic characteristics of fargesin in Sprague Dawley rats after intragastric administration or tail vein injection. The blood concentration-time curves of two different administration routes were drawn. The fargesin microspheres were prepared by the emulsification-solvent evaporation method. The preparation technology was optimized through single factor experiment and orthogonal test. The encapsulation efficiency and drug-loading rate were detected by high performance liquid chromatography, and the characteristics of the microspheres were evaluated by scanning electron microscope and Zeta potential analyzer. Using high performance liquid chromatography, in vivo imaging technique and laser confocal technique, the tissue targeting ability and sustained release effect of the preparation were evaluated. Besides the pharmacokinetics and tissue distribution of the preparation in animals were defined.Results:High performance liquid chromatography was used to analyze the samples and methanol was chosen as the extractant to extract drug from blood and tissue. The mobile phase consisted of methanol and water(58:42 v/v) with a flow rate of 1.00 m L/min. Column temperature was 25°C and sample volume was 20 ?L. With these conditions, a fargesin-contained biological sample detection method was established with good specificity, high accuracy and reasonable linear range. Two kinds of administration methods, intragastric administration(50 mg/kg) and tail vein injection(20 mg/kg), were used for the administration of fargesin to the SD rats. Drugs can be absorbed rapidly after both administration methods. After intragastric administration, the blood concentration reached the first peak at 1 h, then decreased gradually, and reached the maximum plasma concentration at 290 ± 24.49 min with the peak concentration(Cmax) being 464.38 ±152.11 ng/m L and the half-life being 106.39 ± 84.93 min; The peak concentration after tail vein injection appeared at 20 ± 1.28 min with the peak concentration being 2049 ± 723.32 ng/m L and the half-life being 80.75 ± 36.10 min. The plasma concentration of fargesin decreased below the lowest detection limit after 8 h for both this two administration methods. Tissue distribution study suggested that, after these two administration methods, fargesin was detectable in all tissue samples including heart, liver, lung and kidney at the time points of 0.5 h, 2 h, 6 h and 8 h, except that in the kidney at 0.5 h.However, the concentration of fargesin in lung tissue was lower than that in other tissues. In further experiment, fargesin microspheres were prepared using emulsification-solvent evaporation method, and the best preparation was optimized as follows: the ratio of PLGA and drug was 6:1, the concentration of PVA was 2%, the emulsifying time was 40 s, the ratio of emulsifier and oil phase volume was 10:2. The achieved microspheres were honeycomb spherical and the particle size of about 64.2% of the microspheres distributed at the range of 10.17 ± 3.08 ?m. The Zeta potential was-4.49 m V, and the average density was 0.31 ± 0.02 g/cm3. The encapsulation efficiency and drug loading rate were 76.47% and 15.65%, respectively. The release rate of 15 days in PBS buffer(p H=6.8) was 7.62% and the release rate of 72 h was 80% after adding 20% methanol solution in the PBS buffer. The tissue distribution experiment of drug-loaded microsphere showed that the drug can be rapidly enriched in lung tissue at 1 h after intranasal administration or intravenous injection. The average concentration of fargesin in lung tissue was 1050.12 ± 56.57 ng/m L at 30 min after intranasal administration, and reached to 24869.14 ± 36.87 ng/m L at 8 h. Finally, the concentration of fargesin at 24 h was 2009.88 ± 96.53 ng/m L, maintaining at a relatively high concentration level. After intravenous injection, the average concentration of fargesin in lung tissue was 3364.51 ± 49.51 ng/m L at 30 min, reached 23991.06 ± 77.95 ng/m L at 8 h, and reduced to 10660.74 ± 86.57 ng/m L at 24 h. In vivo imaging results suggested that, fargesin was delivered to the lung tissue after both of the two administrations of drug-loaded microspheres, realizing the purpose of targeting therapeutic. Further observation of the lung tissue sections by laser scanning confocal microscopy directly displayed the obvious lung targeted ability and sustained release process of the preparation. The lung tissue sections of two methods of administration were stained with HE, compared with normal lung tissue slice, and no significant inflammatory lesions or changes were observed.Conclusion: This study established a simple, convenient, reliable biological sample detection method of fargesin and systematically studied pharmacokinetics and tissue distribution after oral and intravenous injection. Furthermore, the lung-targeted microspheres were prepared and the quality was evaluated. The pharmacokinetics and targeting properties of the formulation were evaluated by intranasal administration and intravenous injection, indirectly proving that the preparation could avoid the liver first pass elimination, prolong the effect time, and improve the treatment effect through the both kinds of drug delivery methods. The preparation can be further explored as a candidate drug for the treatment of PH.