中国科学院海洋研究所机构知识库

肾病综合征是由多种病因引起的一种肾脏疾病，主要以大量蛋白尿、低蛋白血症、高脂血症及水肿为特性的一组临床症候群。肾病综合征以激素为一线治疗药物，但是长期应用激素类药物可导致免疫紊乱、感染等并发症，患者如未得到有效的治疗，最终会导致终末期肾病。寻找安全有效的肾病综合征治疗药物是新药开发的重要方向。低分子量褐藻多糖硫酸酯（Low molecular weight fucoidan LMWF）是以中药材昆布（褐藻海带）为原料，经提取、纯化得到褐藻多糖硫酸酯（fucoidan），再经氧化降解获得的低分子量硫酸化多糖。本论文主要开展低分子量褐藻多糖硫酸酯（LMWF）治疗肾病综合征的成药性研究，探索其治疗肾病综合征的疗效，并对其药物代谢动力学和药物制剂制备工艺等进行了系统的研究，为进一步研究开发肾病综合征治疗药物提供科学依据。

首先采用阿霉素诱导的肾病综合征的大鼠模型，考察低分子量褐藻多糖硫酸酯（LMWF）对肾病综合征的治疗作用。大鼠尾静脉一次性注射阿霉素（6.0 mg/kg），制备肾病综合征大鼠模型。阿霉素诱导的肾病综合征大鼠出现大量的蛋白尿，血清尿素氮、血肌酐、总胆固醇、TG水平显著的升高。褐藻多糖硫酸酯及LMWF口服给药30天后明显抑制了尿蛋白排出量，降低了BUN、SCr、TG、TC水平。在相同的剂量下（100mg/kg），低分子量褐藻多糖硫酸酯（LMWF）的疗效优于褐藻多糖硫酸酯。两种多糖对肾病综合征的治疗作用与它们的抗氧化活性有关。

其次本文建立了生物样品中岩藻糖的定量分析方法，岩藻糖是褐藻多糖硫酸酯的代表性组成单糖，岩藻糖的血药浓度的变化可以反映其体内吸收和组织分布情况。岩藻糖的浓度在0.025～2 μg/ml范围内，呈良好的线性关系。岩藻糖经水解后的检测限（LOD）为10ng/ml，定量限(LOQ)为25ng/ml。稳定性考察显示14h内血清及组织样品相对稳定。

小鼠60mg/kg 剂量口服给药后，低分子量褐藻多糖硫酸酯（LMWF）的血药峰值Cmax为1.01±0.56 mg/L，达峰时间出现在2.0h；fucoidan的达峰浓度Cmax为0.66±0.32 mg/L，达峰时间Tmax为2.5h。低分子量褐藻多糖硫酸酯（LMWF）相比于褐藻多糖硫酸酯吸收更快，AUC_0-12更大。口服和静脉给药低分子量褐藻多糖硫酸酯（LMWF）（30 mg/kg）后，LMWF的绝对生物利用度为27.9%。小鼠单次口服给药LMWF（30 mg/kg）1h后，LMWF能分布到大部分各组织，在肾中分布最高，最大Cmax为19.93 ± 11.27 μg/g，次之为脑组织（Cmax=5.23± 2.23 μg/g）；心脏分布较少（Cmax=0.59 ± 0.48 μg/g）。

本论文开展了低分子量褐藻多糖硫酸酯制剂研究。通过处方前、制备工艺、质量等研究，确定了低分子量褐藻多糖硫酸酯胶囊的最佳生产工艺。

低分子量褐藻多糖硫酸酯胶囊为硬胶囊剂，规格：0.2g/粒。处方组成为低分子量褐藻多糖硫酸酯、微晶纤维素及二氧化硅，其生产工艺的主要步骤：粉碎过筛、制粒、混合、胶囊填充等。采用沸腾制粒参数法制备颗粒。此生产工艺在车间成功的实现了放大生产，并且连续生产出的胶囊符合质量标准的要求。

通过对低分子量褐藻多糖硫酸酯治疗肾病综合征药效学、药代动力学和制剂工艺研究，证实低分子量褐藻多糖硫酸酯对肾病综合征具有显著疗效，口服吸收具有较好的生物利用度，在肾脏组织浓度高，平均保留时间长，具有开发成为肾病综合征治疗药物的潜力，本文研究为防治肾病综合征的新型多糖药物开发奠定了基础。

Nephrotic syndrome (NS) is a clinical syndrome with a variety of causes, mainly characterized by heavy proteinuria, hypoalbuminemia, and edema. Many clinical and experimental studies have shown that the pathogenesis of nephrotic syndrome is associated with immune dysfunction. Immunosuppressive treatment, including corticosteroids, is the first-line treatment for nephrotic syndrome. However, steroid resistance or steroid dependence is very common and will frequently cause immune dysfunction and complicated infection, then affected patients without effective treatment will in time develop end-stage renal disease. Therefore identification of effective and less toxic therapeutic interventions for nephrotic syndrome remains to be an important issue. In this study, we isolated fucoidan from S. japonica and prepared its depolymerized fragment (low molecular weight fucoidan, LMWF) by oxidant degradation. Low molecular weight fucoidan is a type of fucose-rich sulfated polysaccharides. Besides fucose, LMWF also contains minor galactose, mannose, glucose, rhamnose and xylose, and the weight average molecular weight was 9.5kDa. In order to develop new drug for nephrotic syndrome, we will explore the effect of LMWF on nephrotic syndrome, its pharmacokinetic properties and the preparation progress for LMWF capsule. This study will provide scientific basis for the further research and development of LMWF.

The effect of LMWF and fucoidan on adriamycin induced nephrotic syndrome were investigated in a rats model. Single dose of adriamycin (6.0 mg/kg body weight) was injected via the femoral vein to induce nephrotic syndrome (NS) model. Rats treated by adriamycin had heavy proteinuria and increased BUN, Scr, TG and TC level. LMWF or fucoidan treatment for 30 days could significantly inhibit proteinuria and decrease the elevated BUN, Scr, TG and TC level in a dose dependent manner. At the same dose (100 mg/kg), LMWF had higher renoprotective activity than fucoidan. Their protective effect on nephrotic syndrome was partly related to their antioxidant activity.

Fucose is the main and representative chemical constituent of LMWF and fucoidan. The concentration of fucose, a core component of fucoidan, is quantified to characterize the pharmacokinetic of fucoidan. The development of Quantitative method for LMWF and fucoidan in biological matrix was evaluated. The standard calibration curve of fucose exhibited nice linearity between the fucose standard concentration of 0.025～2 μg/ml. The LOD and LOQ of this measurement were 10 ng/ml and 25 ng/ml respectively.

Both fucoidan and LMWF were absorbed following intragastric administration. Cmax occurred at 2.0h (1.01±0.56 mg/L) for LMWF and 2.5 h (0.66±0.32 mg/L) for fucoidan at the dose of 60 mg/kg. LMWF was absorbed more quickly. The estimated bioavailability of LMWF was 27.9% after a single dose of 30 mg/kg. It can be seen that LMWF was widely distributed into tissues with the highest concentration of 19.93 ± 11.27 μg/g occurring in kidney, the second highest of 5.23± 2.23 μg/g in the brain, and the least 0.59 ± 0.48 μg/g in the heart. LMWF was absorbed in the stomach and intestines, suggesting that it might be primarily absorbed there.

We study the preparation process of low molecular weight fucoidan capsule. The best prescription of low molecular weight fucoidan capsules was established by the research of preformulation, the preparation technique, quality study and stability study. Low molecular weight fucoidan capsule is hard capsule and specification is 0.2g per capsule. The excipients of low molecular weight fucoidan capsule include microcrystalline cellulose and silicon dioxide. The production progress includes crushing, granular preparation, commixture, capsule filling and other steps. Boiling granulation was used to prepare low molecular weight fucoidan capsule. The prescription can be well put into practice and the capsules consistently manufacturing in the factory met the national standards.

The results suggested that low molecular weight fucoidan (LMWF) had excellent protective effects on adriamycin induced nephrotic syndrome, and therefore the potential to be a promising treatment for nephrotic syndrome. Following oral administration, low molecular weight fucoidan (LMWF) was widely distributed in tissues, primarily found in the kidney and remained there for a relatively long time. This research paved the way for the development of fucoidan-based drugs for nephrotic syndrome.