水飞蓟宾纳米晶释药系统的构建与评价
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摘要
难溶性化合物的制剂研究是新药研发的重要难题,是导致研发高失败率、高风险和高投入的关键原因,也是降低药物临床疗效、增加临床不良反应发生率、加重医疗费用的重要因素。纳米晶是近十几年兴起并逐步应用于工业化生产的药物制剂新技术,具有小尺寸效应和量子隧道效应,其首要特征是提高难溶性药物的溶解度和溶出速率,以及由此派生出的其它诸多理化性质和生理特性。
水飞蓟宾是一种具有低毒高效保肝特性的难溶性黄酮木脂素类化合物,是水飞蓟的主要活性成分。现上市剂型为片剂和胶囊剂,Beagle犬口服生物利用度约为17%,因其水溶性差而导致低生物利用度严重影响了临床疗效。本论文将以纳米晶为改善难溶性药物功效的关键技术、以水飞蓟宾为模型药物,进行其纳米晶释药系统的构建与评价。
本课题主要目的是通过活塞-缝隙式高压均质法构建可供口服和注射给药的两种水飞蓟宾纳米晶制剂,建立水飞蓟宾体内外分析方法,考察其药剂学特性和体内药物动力学及组织分布特征,研究其跨膜转运特性,并通过体内外急性肝损伤模型评价水飞蓟宾纳米晶的药效学特征。
本研究的主要内容包括处方前研究、纳米晶工艺筛选与处方研究、冷冻干燥工艺研究与保护剂筛选、HPLC-MS建立生物样品分析方法、Beagle犬口服和静脉滴注纳米晶药动学研究、小鼠尾静脉注射纳米晶组织分布研究、体外Caco-2细胞跨膜转运研究、体外HL7702肝细胞和体内Beagle犬急性肝损伤模型药效学研究等。
采用HPLC-DAD建立了体外样品中水飞蓟宾的分析方法,方法学考察符合要求。处方前研究结果表明,水飞蓟宾的溶解度随溶剂的pH值的递增而增大,pH7.4的PBS中的溶解度为49.7μg·ml-1,正辛醇-水中油水分配系数1gP约为2.8(1gP<5),提示水飞蓟宾低溶解度所导致的较慢的溶出速率可能是致使水飞蓟宾口服生物利用度低的主要原因。
制备工艺与处方筛选结果表明,活塞-缝隙式高压均质法制备水飞蓟宾纳米晶具有良好的重现性和稳定性,适合其纳米晶的制备,纳米晶粒径随均质压力的增大而逐步减小,其1800 bar时粒径约为800 bar时的1/5；随均质次数的增多,纳米晶粒度分布逐步趋于均一,且以最终压力下均质15次为佳。采用L9(34)正交设计法进行纳米晶处方筛选,以不同浓度的磷脂、泊洛沙姆F68.SDS和PVPK30为筛选对象,以Zeta-电位为考察指标,分析结果表明以0.2%磷脂,0.1%泊洛沙姆F68,0.05％SDS,0.05％PVP K30为处方时纳米晶的Zeta-电位最大,经其安全性和稳定性因素考虑,最终确定处方组成为：每100 ml注射用水中含0.5g水飞蓟宾、0.2 g磷脂和0.1 g泊洛沙姆F68。
为进一步提高水飞蓟宾纳米晶的稳定性,对包括甘露醇、海藻糖、p-环糊精、乳糖、葡萄糖及右旋糖酐等在内的多种多元醇和高分子化合物进行真空冷冻干燥保护剂筛选,以冻干品外观形态、纳米晶与保护剂结合形式等为考察因素,并最终确定5%(w/v)甘露醇为最佳保护剂,冻干品外观为完整的圆饼状,表面平滑,质地轻柔,显微观察表明在冻干过程中甘露醇充分的代替了水-OH作用,与纳米晶交替形成有序网状结构,阻止了纳米晶的相互接触和熟化,并起到支架剂作用；冻干工艺考察表明,以-80℃预冻24 h、真空冻干48 h为宜,冻干品含水量低于3%。
为考察水飞蓟宾纳米晶口服和注射给药的可行性,制备粒径不同大小的纳米晶SN-A和SN-B:800 bar下均质15次并按照冻干工艺干燥,得纳米晶SN-A；1800bar下均质15次并按照冻干工艺干燥,得纳米晶SN-B；并分别对SN-A和SN-B进行物相分析和表征。SN-A的粒径及Zeta..电位分别为641.8±14.7 nm(PI-0.375)和-23.1±0.6 mV,SN-B的粒径及Zeta.电位分别为127±1.9 nm(PI-0.292)和-25.5±0.7 mV；TEM观察表明SN-A和SN-B中纳米晶基本呈完整球形外观,粒径与激光粒度分布仪所测结果基本相符,且粒度分布较为均一；AFM扫描表明SN-A与SN-B的形态间存在一定差异,且可见稳定剂在纳米晶表面形成的保护膜；冻干品SEM观察显示,纳米晶粒度分布均匀,SN-B的粒径小于SN-A,两者外观均呈不规则的颗粒状；DSC法和XRPD法分析纳米晶中水飞蓟宾晶型,结果表明,高压均质过程和冷冻干燥操作未引起水飞蓟宾晶态的转化；pH6.0和pH7.4的PBS中的纳米晶溶出速率考察表明,纳米晶可提高水飞蓟宾溶出速率,且SN-B溶出速率大于SN-A; pH6.0的PBS中,纳米晶可将水飞蓟宾的饱和溶解度提高约2.2倍；SN-A和SN-B的比表面积分别为0.435 m2.g-1和0.484 m2·g-1,SN-A孔隙率为0.00401m1·g-1,孔径为17.05±16.0 nm,SN-B孔隙率为0.00431 ml·g’,孔径为17.03±16.4nm；稳定性加速实验考察表明SN-A和SN-B在6个月内具有良好的稳定性。
采用HPLC-MS建立了犬血浆等生物样品中水飞蓟宾含量检测的分析方法,方法学考察符合《化学药物非临床药代动力学研究技术指导原则》要求。Beagle犬口服给药水飞蓟宾原料药或纳米晶(SN-A, SN-B) 20 mg-kg"1后经DAS2.0药动学程序计算,均符合二室模型。纳米晶SN-A和SN-B生物利用度分别为原料药组的2.3倍和2.9倍；SN-A和SN-B的Cmax高于原料药组；原料药组的MRT值最小,SN-A的较长,而SN-B的为最长；原料药组、SN-A和SN-B的tmax值分别为0.56 h、1.03 h和1.08 h。Beagle犬静脉滴注给药水飞蓟宾溶液或纳米晶(SN-A, SN-B) 15 mg·kg-1后经DAS2.0药动学程序计算,溶液组和SN-B组符合三室模型,SN-A组符合二室模型。与溶液组相比,纳米晶可延长tt/2并增大AUC,尽管溶液组Cmax最高,但清除率高,其MRT较短。该实验表明,对于低溶出速率和体内药动学参数欠佳的水难溶性药物-水飞蓟宾,采用纳米晶技术可改善其药动学特征。
为考察纳米晶体内分布特征和粒径对其分布的影响,进行小鼠以20 nig·kg-1剂量分别尾静脉注射水飞蓟宾溶液、SN-A和SN-B后的组织分布研究。靶向指数、选择性指数、靶向效率和相对靶向效率评价指标均证实水飞蓟宾纳米晶具有肝脏被动靶向性；从药物浓度角度考察,纳米晶可降低血浆和心脏中水飞蓟宾浓度,增加肝脏中的浓度；从靶器官药量角度考察,溶液组在肝脏的分布比例介于15%-35%之间,SN-A和SN-B分布比例分别介于46%-63%和31%-67%之间,且在给药后10h内SN-A在肝脏的分布比例稳定在40%-50%之间,提示纳米晶SN-A小鼠尾静脉注射肝脏靶向性优于纳米晶SN-B。
为考察纳米晶对水飞蓟宾口服吸收的影响,进行水飞蓟宾纳米晶SN-A和SN-B在Caco-2细胞中的跨膜转运研究,结果表明,当纳米晶中水飞蓟宾浓度低于125μg·ml-1时无细胞毒性,且药物不影响Caco-2细胞活性与增殖。在pH6.0和pH7.4的Hanks'缓冲液中,以水飞蓟宾原料药和物理混合物为参比对象,在pH6.0环境下,SN-A、SN-B和原料药的Papp分别为5.17±0.77×10-7 cm·s-1、6.43±0.41×10-7cm.s-1和2.47±0.4×10-7cm.s-1；在pH7.4环境下,SN-A、SN-B和原料药的Papp分别为5.73±0.75×10-7 cm·s-1、1.02±0.17×10-6 cm·s-1和2.70±0.44×10-7 cm·s-1。SN-A和SN-B均能提高水飞蓟宾表观渗透系数,提高药物跨膜转运速率,且SN-B的表观渗透系数大于SN-A。
为进行纳米晶体外药效学考察,以浓度为5 mM的H2O2与HL7702细胞孵育24 h,建立体外肝细胞化学性损伤模型；HL7702肝细胞损伤模型预防性给药时,水飞蓟宾溶液组保护作用呈浓度依赖性,且在10μg·ml-1时保护作用最强,SN-A在50μg·ml-1时最强,并且SN-A的保护效应强于溶液组；HL7702肝细胞损伤模型治疗性给药时,溶液组和SN-A的保护作用未及预防性给药时明显。HL7702肝细胞HE染色证实,以10μg·ml-1预防性给药时,SN-A组细胞数目较多,分布规则,胞浆丰满,细胞核形态及核质比接近于正常细胞；溶液组细胞状况介于模型组和SN-A组之间,个别细胞出现碎核。因此,水飞蓟宾可预防性保护肝细胞免受H2O2的损伤,且SN-A的保护效果优于溶液组。
为进行纳米晶体内药效学考察,以CCl4的花生油溶液(1：1,w/w)按照1ml·kg-1剂量皮下多点注射建立Beagle犬急性肝损伤模型,SN-A滴注给药15mg·kg-1、SN-B灌胃给药20mg·kg-1,复方甘草酸单铵为阳性对照,并设模型组和水飞蓟宾溶液对照组,检测AST、ALT、TBIL等肝功指标,测定肝脏SOD和MDA活性,病理切片检查肝组织病理改变,考察水飞蓟宾纳米晶口服和静脉给药的安全性和有效性。经肝功能检测和肝病理切片观察证实,本文成功建立了CCl4致Beagle犬急性肝损伤模型。与水飞蓟宾溶液组相比,纳米晶可更有效的将偏离正常值的AS、ALT、ALP、TBIL、GGT等向正常值调整回归,并增加肝组织中SOD活性,降低MDA产生量,减少肝细胞气球样变和炎性细胞浸润,避免大面积坏死,保护肝细胞核膜完整性,稳定肝细胞。因此,水飞蓟宾纳米晶SN-B口服给药和SN-A静脉注射给药可有效预防CCl4引起的Beagle犬急性肝损伤,且不会产生药源性肝损伤,进一步证实纳米晶可显著提高水飞蓟宾口服生物利用度和静脉给药靶向性,提高水飞蓟宾的保肝药效作用。
本文成功构建了供注射和口服给药的水飞蓟宾纳米晶释药系统,并进行了体内外评价。应用活塞-缝隙式高压均质法制备了供口服和静脉给药的水飞蓟宾纳米晶,并以甘露醇为保护剂进行冷冻干燥提高其稳定性,水飞蓟宾晶型保持不变,粒度分布均匀,溶解度和溶出速率增加,具备良好的制剂学特性。纳米晶可提高水飞蓟宾的口服生物利用度,增加Caco-2细胞跨膜转运系数；延长静脉滴注给药时水飞蓟宾半衰期,并增加肝脏靶向效率。纳米晶可增强水飞蓟宾口服和静脉给药时对Beagle犬急性肝损伤的保护作用。因此,本文研究表明,纳米晶可提高水飞蓟宾口服生物利用度和并实现静脉被动肝脏靶向传输给药,纳米晶技术是改善包括水飞蓟宾在内诸多难溶性药物体内外功效的重要途径。
The development of water insoluble candidate compounds is a tough problem in the new drug researching. This is also the major reasons of lead to high failure probability, high risk and larger import of drugs development. Furthermore, this is also caused low clinic curative effect, increased adverse reaction and aggravated medical cost. The drug nanocrystals nanotechnology, a new pharmaceutical preparation method, which emerged in the recent decades and applied in the industries, has mini-dimension effective and quantum runnel ling effects. The most important features of insoluble compound nanocrystals are improved solubility and enhanced dissolution velocity, as well as derived the other physicochemical properties and physiological nature.
Silybin, a water insoluble flavanolignan compound, which with low toxicant and high performance of protect hepatic function, is the major active constituents of Silybum marianum (L) Genrtn. Currently, the on market dosage forms of silybin are capsule and tablet. Due to the low solubility, the absolute bioavailability of Beagle dog oral administration is only about 20%. Therefore, the poor solubility of silybin may be severity impaired the clinic affectivity.
The major objective of the present research is to develop silybin nanocrystals for oral and intravenous delivery by piston-crack high pressure homogenization approach, establish the silybin determine method by HPLC-DAD and LC-MS for in vitro and in vivo samples analysis, investigate silybin nanocrystals pharmaceutics properties and pharmacokinetics as well as organs distribution characters, and research the Caco-2 cell monolayer transport properties. Furthermore, the pharmacodynamics evaluation of silybin nanocrystals also will be conducted by in vitro and in vivo acute hepatic injury models.
The major contents include preformulation study, technology and formulation screening of preparation silybin nanocrystals by HPH, establishment of HPLC-MS method for biological specimen analysis, pharmacokinetics study of Beagle dog oral administration and intravenous drop of silybin nanocrystals, organs distribution by mice caudal vein intravenous administration, the in vitro transport across Caco-2 cells monolayer evaluation, in vitro HL7702 cell and in vivo Beagle dog hepatic injure model for pharmacodynamics investigation.
The established HPLC-DAD method for in vitro samples analysis was investigated and corresponded to technology requirement. The experiment results of preformulation study stated that the solubility of silybin increased with pH and at pH7.4 is 49.7μg·ml-1. The log of oil/water partition coefficient is 2.8, which hinted that the poor oral bioavailability of silybin was mainly due to the low solubility resulted slow dissolution velocity.
There was a favorable reproducibility and stability in the HPH method for silybin nanocrystals preparation. The higher preparation pressures the smaller nanocrystals particle size. The particle size of nanocrystals of prepared under 1800 bar is one fifth of the nanocrystals of prepared under 800 bar. The more times of homogenization operations the more uniformity of the prepared nanocrystals and 15 times were selected. A L9(34) orthogonal design were conducted for silybin nanocrystals formulation screening. The different concentration of phospholipids, poloxamer 188, SDS and PVP K30 were selected for the formulation screening. And zeta electric potential was the investigate index. The experiment revealed that when the formulation contents 0.2% phospholipids,0.1% poloxamer 188,0.05% SDS and 0.05% PVP K30 the zeta electric potential was the greatest. In consideration of safety and stability, the final silybin nanocrystals formulation contents of 100 ml water,0.5 g silybin,0.2 g phospholipids and 0.1 g poloxamer 188.
The protectants screening of vacuum freeze-drying was carried out between polyalcohol and high molecular compound, such as mannitol, trehalose,β-cyclodextrin, lactose, glucose and dextran. The freeze-dried nanocrystals were evaluated by appearance morphous as well as integrate format. The experiments result demonstrated that 5%(w/v) mannitol is the most suitable protectants. The freeze-dried product shows integrity round cake shape, smoothing surface, light and soft of texture. The microscope observation and evaluation revealed that the mannitol replaced the role of water of -OH and the mannitol alternated with nanocrystals, which formed order reticulate structure. The regularly structure is better for the stability of nanocrystals and prevented the contact between two nanocrystals. Therefore, the freeze-drying technology manifested that pre-freeze condition is -80℃for 24 h, freeze-drying time is 48 h. The water contain of obtained products is lower than 3%.
To investigate the feasibility of silybin nanocrystals for oral and intravenous administration, tow different particle size distributed nanocrystals were prepared using the following operation parameters. As stated formulation, silybin nanocrystals A (SN-A) were homogenized under 800 bar, followed freeze-drying. Silybin nanocrystals B (SN-B) were homogenized under 1800 bar, followed freeze-drying. Thereafter, facies analysis and superficial syndrome of SN-A and SN-B were conducted. Immediately after freeze-dried, the size and zeta potential (ZP) were found to be 641.8±14.7 nm (PI～0.375) and -23.1±0.6 mV for SN-A, and 127±1.9 nm (PI～0.292) and -25.5±0.7 mV for SN-B, respectively. The TEM observation revealed that with the increase of homogenization pressure, in addition to the particle size decreasing, the silybin nanocrystals appeared to be more regularly shaped. The AFM experiments demonstrated that the silybin nanocrystals with a spherical shape revealed values of 718 nm and 226 nm in their diameters, for SN-A and SN-B, respectively. Additionally, the presence of encapsulate films on the surface of the pure particles of drugs could also be observed by AFM. These results indicate that the thin films should be surfactants. Therefore, AFM analysis reinforced the presence of the stabilizer on the surface of the drug particles and this feature is in accordance with the TEM results.
SEM images of the freeze-dried nanocrystals prepared under different homogenization pressure were clearly confirmed that the higher produce pressure the more uniformly nanocrystals in the morphology and the smaller particle size. Even the decreased size cannot be in proportion to the increased pressure. Hence, it was assumed that SN-B had higher uniform and smaller dimension. These results were in accordance with the study of particle size analysis and PI values. Nanocrystals were freeze-dried to obtain the dried silybin powder. TEM images indicate that the silybin powder was aggregated due to the water-removal. The DSC thermograms clearly showed that both of the two freeze-dried silybin nanocrystals displayed conspicuous melting point of silybin at the temperature with comparable physical mixture of drug and surfactants. These denoted that no any possible transformation to an amorphous state during the HPH and freeze-drying operation and this is important to long-term stability. XRPD experiment confirmed the fact that the diffraction pattern was preserved in silybin nanocrystals, which indicates that the crystalline state of silybin appeared to be unaltered following the HPH operation.
In solubility experiment, nanocrystals powder provided a more than two-fold increase in solubility compared to the un-milled silybin or physical mixture. This increase is more pronounced for SN-B that has smaller particle size. Either in SN-A or in SN-B, dissolution velocity enhancement was clearly observed at both tested pH6.0 and pH7.4. Correlating with the crystalline state analyze results, the dramatically enhanced dissolution rate resulted from the increased effective surface area, and the improved solubility due to the decreased particle size, but no due to the consequence of the presence of amorphous fraction. The specific surface area is 0.435 m2·g-1 and 0.484 m·g-1, for SN-A and SN-B, respectively. The porosity and pore diameter, for SN-A is 0.00401 and 17.05±16.0 nm, for SN-B is 0.00431 ml·g-1 and 17.03±16.4 nm, respectively. The accelerated stability test manifested that the SN-A and SN-B has satisfactory stability.
An LC-MS method for determination of silybin in Beagle dog plasma and mice organs was developed for the first time. The technology investigation coincided with the requirement of SFDA guidance principle of chemicals non-clinic pharmacokinetics. For Beagle oral administration (20 mg-kg-1), both nanocrystals delivery systems showed approximately two-fold or three-fold bioavailability improvements in terms rate and extent compared with the un-milled suspension. Based on comparison of the AUC0-∞> values, the un-milled silybin suspensions showed significantly lower silybin exposure than the nanocrystals (SN-A and SN-B). There was no significant difference in AUC0-∞, between SN-A and SN-B. The Cmax was significantly lower in the un-milled silybin suspensions than that of the nanocrystals SN-A or SN-B. The un-milled silybin suspension has the shortest MRT, the SN-A showed longer and the N-B showed the longest. The tmax was significantly shorter for the un-milled suspension than that for the nanocrystals of SN-A and SN-B. No significant statistical difference between the nanocrystals was found for this parameter. For Beagle intravenous drop administration (15 mg·kg-1), in contrast to solution, the nanocrystals formulations had prolonged t1/2 and augmented AUC. No adverse events were observed during the administration. The solution's AUC0-∞was lower than that of the nanocrystals, including SN-A and SN-B. Although the solution has higher Cmax, the lower MRT and higher clearance rate as well as other parameters also lead to a distinct pharmacokinetics profile when compared to the nanocrystals plots. Therefore, these experiments demonstrated that nanocrystals formulations can be used to improve exposure of compounds with low dissolution rate and poor pharmacokinetic characteristics.
To investigate the effect of nanocrystals particle size on the organ distribution, the mice vena caudalis intravenous silybin nanocrystals (20 mg-kg"1) were carried out. The evaluation index included drug targeting index, drug selectivity index, drug targeting efficiency and relative targeting efficiency, which revealed that silybin nanocrystals has liver passive targeting. In considering of silybin concentration in the organs, nanocrystals could decrease the concentration in the plasma and heart; increase the concentration in the liver. Judging from the quantity of silybin in the organs, the liver aggregation of the solution term, SN-A and SN-B is 15%-35%, 46%-63%and 31%-67%, respectively. Furthermore, in 10 h following administration SN-A, the liver aggregation stabilize at 40%-50%. This experiment revealed that the target efficiency of SN-A is better than SN-B.
In the Caco-2 cell cytotoxicity experiments, when the concentration of silybin was below 125μg·ml-1, there were no significant differences between the experimental groups and the control groups. The effects of nanocrystals, SN-A and SN-B, on facilitating silybin transport across the monolayers were much higher than that of the physical mixture formulation. Additionally, the decrease in particle size could also enhance ability of silybin in SN-B system transport across the monolayers as it has a higher ability to enhance Papp. Nanocrystals presented higher Papp values compared with un-milled silybin and physical mixture. Compared with the silybin nanocrystals, the un-milled silybin presented a lower Papp value in pH 6.0 medium. Based on the Caco-2 cell monolayers permeability date, we may tentatively present a conclusion that nanocrystals could promote of poorly soluble drugs transport cross intestine epithelium.
The injuring of HL7702 hepatic cell model was established by incubation with H2O2 for 24 h at concentration of 5 mM. When the silybin prophylactic administrated to cell injury model, the effective presented concentration dependent tendency. The most effectiveness were presented at 10μg·ml-1 and 50μg·ml-1, for silybin solution and SN-A, respectively. The protection efficiency of solution and SN-A was lower than the prophylactic administration experiments, when the silybin remedially administrated to cell injury model. In order to demonstrate the efficiency of SN-A on the protection of HL7702 is better than the solution formulation; the HE staining study was carried out. When the solution and SN-A administrated at 10μg·ml-1, the cells of SN-A group was presented as the endochylema turgor vitalis, the morphology normally, the nucleus and nuclear-cytoplasmic ratio closed to norm cells. Furthermore, the cells condition of solution formulation group was between model group and SN-A group, and presented some cell debris.
Beagle dog acute liver injure model was constructed by subcutaneously multipoint injected of CCl4-peanut oil solution (1:1, w/w). The SN-A intravenous drop and SN-B orally administrated at dose of 15 mg·kg-1 and 20 mg·kg-1, respectively. Compound ammonium glycyrrhizinate was chosen as positive control. The model group and silybin solution control were also set. The hepatic functional parameter, such as AST, ALT, ALP, TBIL and GGT were detected. The enzymes in the liver tissue, include SOD and MDA, were determined. The pathological section were used to observation the patho-changes of liver tissue. Therefore, the safety and efficiency of orally and intravenously administrated silybin nanocrystals were evaluated. Judging from the hepatic functions detection and observation of the patho-changes, the acute liver injurer of Beagle dogs were successfully established. Compared with solution formulations, the nanocrystals, SN-A for intravenous and SN-B for oral administration, could restore the deviate parameters, including AST, ALT, ALP, TBIL and GGT, toward to normal values. Furthermore, the silybin nanocrystals also could increase the SOD activity and decrease the level of MDA. As observed in the histological section, hepatic cell balloon changes and inflammatory cell infiltrate were decreased in the SN-A and SN-B groups, compared with solution group. And large area necrosis also refrained in the nanocrystals. At the same dose, the nanocrystals could effectively protect the liver cells avoid of injure, maintenance the nucleus and membrane integrity, promote the stabilization of liver cells. Silybin nanocrystals, SN-A for intravenous and SN-B for oral delivery, could effectively prevent the CCl4 induced hepatic injury of Beagle dogs. And these demonstrated that nanocrystals could improve the oral bioavailability and target efficiency of intravenous delivery.
In this study, the silybin nanocrystals for oral and intravenous administration,, could be prepared by piston-gap high pressure homogenization method. The freeze-drying, manicol used as protectant, could improve the stability of silybin nanocrystals. In the preparation, the crystals form maintained and particle size distributed uniformity. Dissolution velocity and saturation solubility is dramatically improved. The transmembrane transport ability and oral bioavailability enhanced. The nanocrystals also could implement liver passive target delivery. The nanocrystals improved the effectiveness of silybin for hepatic injury in vitro and in vivo.
水飞蓟宾是一种具有低毒高效保肝特性的难溶性黄酮木脂素类化合物,是水飞蓟的主要活性成分。现上市剂型为片剂和胶囊剂,Beagle犬口服生物利用度约为17%,因其水溶性差而导致低生物利用度严重影响了临床疗效。本论文将以纳米晶为改善难溶性药物功效的关键技术、以水飞蓟宾为模型药物,进行其纳米晶释药系统的构建与评价。
本课题主要目的是通过活塞-缝隙式高压均质法构建可供口服和注射给药的两种水飞蓟宾纳米晶制剂,建立水飞蓟宾体内外分析方法,考察其药剂学特性和体内药物动力学及组织分布特征,研究其跨膜转运特性,并通过体内外急性肝损伤模型评价水飞蓟宾纳米晶的药效学特征。
本研究的主要内容包括处方前研究、纳米晶工艺筛选与处方研究、冷冻干燥工艺研究与保护剂筛选、HPLC-MS建立生物样品分析方法、Beagle犬口服和静脉滴注纳米晶药动学研究、小鼠尾静脉注射纳米晶组织分布研究、体外Caco-2细胞跨膜转运研究、体外HL7702肝细胞和体内Beagle犬急性肝损伤模型药效学研究等。
采用HPLC-DAD建立了体外样品中水飞蓟宾的分析方法,方法学考察符合要求。处方前研究结果表明,水飞蓟宾的溶解度随溶剂的pH值的递增而增大,pH7.4的PBS中的溶解度为49.7μg·ml-1,正辛醇-水中油水分配系数1gP约为2.8(1gP<5),提示水飞蓟宾低溶解度所导致的较慢的溶出速率可能是致使水飞蓟宾口服生物利用度低的主要原因。
制备工艺与处方筛选结果表明,活塞-缝隙式高压均质法制备水飞蓟宾纳米晶具有良好的重现性和稳定性,适合其纳米晶的制备,纳米晶粒径随均质压力的增大而逐步减小,其1800 bar时粒径约为800 bar时的1/5；随均质次数的增多,纳米晶粒度分布逐步趋于均一,且以最终压力下均质15次为佳。采用L9(34)正交设计法进行纳米晶处方筛选,以不同浓度的磷脂、泊洛沙姆F68.SDS和PVPK30为筛选对象,以Zeta-电位为考察指标,分析结果表明以0.2%磷脂,0.1%泊洛沙姆F68,0.05％SDS,0.05％PVP K30为处方时纳米晶的Zeta-电位最大,经其安全性和稳定性因素考虑,最终确定处方组成为：每100 ml注射用水中含0.5g水飞蓟宾、0.2 g磷脂和0.1 g泊洛沙姆F68。
为进一步提高水飞蓟宾纳米晶的稳定性,对包括甘露醇、海藻糖、p-环糊精、乳糖、葡萄糖及右旋糖酐等在内的多种多元醇和高分子化合物进行真空冷冻干燥保护剂筛选,以冻干品外观形态、纳米晶与保护剂结合形式等为考察因素,并最终确定5%(w/v)甘露醇为最佳保护剂,冻干品外观为完整的圆饼状,表面平滑,质地轻柔,显微观察表明在冻干过程中甘露醇充分的代替了水-OH作用,与纳米晶交替形成有序网状结构,阻止了纳米晶的相互接触和熟化,并起到支架剂作用；冻干工艺考察表明,以-80℃预冻24 h、真空冻干48 h为宜,冻干品含水量低于3%。
为考察水飞蓟宾纳米晶口服和注射给药的可行性,制备粒径不同大小的纳米晶SN-A和SN-B:800 bar下均质15次并按照冻干工艺干燥,得纳米晶SN-A；1800bar下均质15次并按照冻干工艺干燥,得纳米晶SN-B；并分别对SN-A和SN-B进行物相分析和表征。SN-A的粒径及Zeta..电位分别为641.8±14.7 nm(PI-0.375)和-23.1±0.6 mV,SN-B的粒径及Zeta.电位分别为127±1.9 nm(PI-0.292)和-25.5±0.7 mV；TEM观察表明SN-A和SN-B中纳米晶基本呈完整球形外观,粒径与激光粒度分布仪所测结果基本相符,且粒度分布较为均一；AFM扫描表明SN-A与SN-B的形态间存在一定差异,且可见稳定剂在纳米晶表面形成的保护膜；冻干品SEM观察显示,纳米晶粒度分布均匀,SN-B的粒径小于SN-A,两者外观均呈不规则的颗粒状；DSC法和XRPD法分析纳米晶中水飞蓟宾晶型,结果表明,高压均质过程和冷冻干燥操作未引起水飞蓟宾晶态的转化；pH6.0和pH7.4的PBS中的纳米晶溶出速率考察表明,纳米晶可提高水飞蓟宾溶出速率,且SN-B溶出速率大于SN-A; pH6.0的PBS中,纳米晶可将水飞蓟宾的饱和溶解度提高约2.2倍；SN-A和SN-B的比表面积分别为0.435 m2.g-1和0.484 m2·g-1,SN-A孔隙率为0.00401m1·g-1,孔径为17.05±16.0 nm,SN-B孔隙率为0.00431 ml·g’,孔径为17.03±16.4nm；稳定性加速实验考察表明SN-A和SN-B在6个月内具有良好的稳定性。
采用HPLC-MS建立了犬血浆等生物样品中水飞蓟宾含量检测的分析方法,方法学考察符合《化学药物非临床药代动力学研究技术指导原则》要求。Beagle犬口服给药水飞蓟宾原料药或纳米晶(SN-A, SN-B) 20 mg-kg"1后经DAS2.0药动学程序计算,均符合二室模型。纳米晶SN-A和SN-B生物利用度分别为原料药组的2.3倍和2.9倍；SN-A和SN-B的Cmax高于原料药组；原料药组的MRT值最小,SN-A的较长,而SN-B的为最长；原料药组、SN-A和SN-B的tmax值分别为0.56 h、1.03 h和1.08 h。Beagle犬静脉滴注给药水飞蓟宾溶液或纳米晶(SN-A, SN-B) 15 mg·kg-1后经DAS2.0药动学程序计算,溶液组和SN-B组符合三室模型,SN-A组符合二室模型。与溶液组相比,纳米晶可延长tt/2并增大AUC,尽管溶液组Cmax最高,但清除率高,其MRT较短。该实验表明,对于低溶出速率和体内药动学参数欠佳的水难溶性药物-水飞蓟宾,采用纳米晶技术可改善其药动学特征。
为考察纳米晶体内分布特征和粒径对其分布的影响,进行小鼠以20 nig·kg-1剂量分别尾静脉注射水飞蓟宾溶液、SN-A和SN-B后的组织分布研究。靶向指数、选择性指数、靶向效率和相对靶向效率评价指标均证实水飞蓟宾纳米晶具有肝脏被动靶向性；从药物浓度角度考察,纳米晶可降低血浆和心脏中水飞蓟宾浓度,增加肝脏中的浓度；从靶器官药量角度考察,溶液组在肝脏的分布比例介于15%-35%之间,SN-A和SN-B分布比例分别介于46%-63%和31%-67%之间,且在给药后10h内SN-A在肝脏的分布比例稳定在40%-50%之间,提示纳米晶SN-A小鼠尾静脉注射肝脏靶向性优于纳米晶SN-B。
为考察纳米晶对水飞蓟宾口服吸收的影响,进行水飞蓟宾纳米晶SN-A和SN-B在Caco-2细胞中的跨膜转运研究,结果表明,当纳米晶中水飞蓟宾浓度低于125μg·ml-1时无细胞毒性,且药物不影响Caco-2细胞活性与增殖。在pH6.0和pH7.4的Hanks'缓冲液中,以水飞蓟宾原料药和物理混合物为参比对象,在pH6.0环境下,SN-A、SN-B和原料药的Papp分别为5.17±0.77×10-7 cm·s-1、6.43±0.41×10-7cm.s-1和2.47±0.4×10-7cm.s-1；在pH7.4环境下,SN-A、SN-B和原料药的Papp分别为5.73±0.75×10-7 cm·s-1、1.02±0.17×10-6 cm·s-1和2.70±0.44×10-7 cm·s-1。SN-A和SN-B均能提高水飞蓟宾表观渗透系数,提高药物跨膜转运速率,且SN-B的表观渗透系数大于SN-A。
为进行纳米晶体外药效学考察,以浓度为5 mM的H2O2与HL7702细胞孵育24 h,建立体外肝细胞化学性损伤模型；HL7702肝细胞损伤模型预防性给药时,水飞蓟宾溶液组保护作用呈浓度依赖性,且在10μg·ml-1时保护作用最强,SN-A在50μg·ml-1时最强,并且SN-A的保护效应强于溶液组；HL7702肝细胞损伤模型治疗性给药时,溶液组和SN-A的保护作用未及预防性给药时明显。HL7702肝细胞HE染色证实,以10μg·ml-1预防性给药时,SN-A组细胞数目较多,分布规则,胞浆丰满,细胞核形态及核质比接近于正常细胞；溶液组细胞状况介于模型组和SN-A组之间,个别细胞出现碎核。因此,水飞蓟宾可预防性保护肝细胞免受H2O2的损伤,且SN-A的保护效果优于溶液组。
为进行纳米晶体内药效学考察,以CCl4的花生油溶液(1：1,w/w)按照1ml·kg-1剂量皮下多点注射建立Beagle犬急性肝损伤模型,SN-A滴注给药15mg·kg-1、SN-B灌胃给药20mg·kg-1,复方甘草酸单铵为阳性对照,并设模型组和水飞蓟宾溶液对照组,检测AST、ALT、TBIL等肝功指标,测定肝脏SOD和MDA活性,病理切片检查肝组织病理改变,考察水飞蓟宾纳米晶口服和静脉给药的安全性和有效性。经肝功能检测和肝病理切片观察证实,本文成功建立了CCl4致Beagle犬急性肝损伤模型。与水飞蓟宾溶液组相比,纳米晶可更有效的将偏离正常值的AS、ALT、ALP、TBIL、GGT等向正常值调整回归,并增加肝组织中SOD活性,降低MDA产生量,减少肝细胞气球样变和炎性细胞浸润,避免大面积坏死,保护肝细胞核膜完整性,稳定肝细胞。因此,水飞蓟宾纳米晶SN-B口服给药和SN-A静脉注射给药可有效预防CCl4引起的Beagle犬急性肝损伤,且不会产生药源性肝损伤,进一步证实纳米晶可显著提高水飞蓟宾口服生物利用度和静脉给药靶向性,提高水飞蓟宾的保肝药效作用。
本文成功构建了供注射和口服给药的水飞蓟宾纳米晶释药系统,并进行了体内外评价。应用活塞-缝隙式高压均质法制备了供口服和静脉给药的水飞蓟宾纳米晶,并以甘露醇为保护剂进行冷冻干燥提高其稳定性,水飞蓟宾晶型保持不变,粒度分布均匀,溶解度和溶出速率增加,具备良好的制剂学特性。纳米晶可提高水飞蓟宾的口服生物利用度,增加Caco-2细胞跨膜转运系数；延长静脉滴注给药时水飞蓟宾半衰期,并增加肝脏靶向效率。纳米晶可增强水飞蓟宾口服和静脉给药时对Beagle犬急性肝损伤的保护作用。因此,本文研究表明,纳米晶可提高水飞蓟宾口服生物利用度和并实现静脉被动肝脏靶向传输给药,纳米晶技术是改善包括水飞蓟宾在内诸多难溶性药物体内外功效的重要途径。
The development of water insoluble candidate compounds is a tough problem in the new drug researching. This is also the major reasons of lead to high failure probability, high risk and larger import of drugs development. Furthermore, this is also caused low clinic curative effect, increased adverse reaction and aggravated medical cost. The drug nanocrystals nanotechnology, a new pharmaceutical preparation method, which emerged in the recent decades and applied in the industries, has mini-dimension effective and quantum runnel ling effects. The most important features of insoluble compound nanocrystals are improved solubility and enhanced dissolution velocity, as well as derived the other physicochemical properties and physiological nature.
Silybin, a water insoluble flavanolignan compound, which with low toxicant and high performance of protect hepatic function, is the major active constituents of Silybum marianum (L) Genrtn. Currently, the on market dosage forms of silybin are capsule and tablet. Due to the low solubility, the absolute bioavailability of Beagle dog oral administration is only about 20%. Therefore, the poor solubility of silybin may be severity impaired the clinic affectivity.
The major objective of the present research is to develop silybin nanocrystals for oral and intravenous delivery by piston-crack high pressure homogenization approach, establish the silybin determine method by HPLC-DAD and LC-MS for in vitro and in vivo samples analysis, investigate silybin nanocrystals pharmaceutics properties and pharmacokinetics as well as organs distribution characters, and research the Caco-2 cell monolayer transport properties. Furthermore, the pharmacodynamics evaluation of silybin nanocrystals also will be conducted by in vitro and in vivo acute hepatic injury models.
The major contents include preformulation study, technology and formulation screening of preparation silybin nanocrystals by HPH, establishment of HPLC-MS method for biological specimen analysis, pharmacokinetics study of Beagle dog oral administration and intravenous drop of silybin nanocrystals, organs distribution by mice caudal vein intravenous administration, the in vitro transport across Caco-2 cells monolayer evaluation, in vitro HL7702 cell and in vivo Beagle dog hepatic injure model for pharmacodynamics investigation.
The established HPLC-DAD method for in vitro samples analysis was investigated and corresponded to technology requirement. The experiment results of preformulation study stated that the solubility of silybin increased with pH and at pH7.4 is 49.7μg·ml-1. The log of oil/water partition coefficient is 2.8, which hinted that the poor oral bioavailability of silybin was mainly due to the low solubility resulted slow dissolution velocity.
There was a favorable reproducibility and stability in the HPH method for silybin nanocrystals preparation. The higher preparation pressures the smaller nanocrystals particle size. The particle size of nanocrystals of prepared under 1800 bar is one fifth of the nanocrystals of prepared under 800 bar. The more times of homogenization operations the more uniformity of the prepared nanocrystals and 15 times were selected. A L9(34) orthogonal design were conducted for silybin nanocrystals formulation screening. The different concentration of phospholipids, poloxamer 188, SDS and PVP K30 were selected for the formulation screening. And zeta electric potential was the investigate index. The experiment revealed that when the formulation contents 0.2% phospholipids,0.1% poloxamer 188,0.05% SDS and 0.05% PVP K30 the zeta electric potential was the greatest. In consideration of safety and stability, the final silybin nanocrystals formulation contents of 100 ml water,0.5 g silybin,0.2 g phospholipids and 0.1 g poloxamer 188.
The protectants screening of vacuum freeze-drying was carried out between polyalcohol and high molecular compound, such as mannitol, trehalose,β-cyclodextrin, lactose, glucose and dextran. The freeze-dried nanocrystals were evaluated by appearance morphous as well as integrate format. The experiments result demonstrated that 5%(w/v) mannitol is the most suitable protectants. The freeze-dried product shows integrity round cake shape, smoothing surface, light and soft of texture. The microscope observation and evaluation revealed that the mannitol replaced the role of water of -OH and the mannitol alternated with nanocrystals, which formed order reticulate structure. The regularly structure is better for the stability of nanocrystals and prevented the contact between two nanocrystals. Therefore, the freeze-drying technology manifested that pre-freeze condition is -80℃for 24 h, freeze-drying time is 48 h. The water contain of obtained products is lower than 3%.
To investigate the feasibility of silybin nanocrystals for oral and intravenous administration, tow different particle size distributed nanocrystals were prepared using the following operation parameters. As stated formulation, silybin nanocrystals A (SN-A) were homogenized under 800 bar, followed freeze-drying. Silybin nanocrystals B (SN-B) were homogenized under 1800 bar, followed freeze-drying. Thereafter, facies analysis and superficial syndrome of SN-A and SN-B were conducted. Immediately after freeze-dried, the size and zeta potential (ZP) were found to be 641.8±14.7 nm (PI～0.375) and -23.1±0.6 mV for SN-A, and 127±1.9 nm (PI～0.292) and -25.5±0.7 mV for SN-B, respectively. The TEM observation revealed that with the increase of homogenization pressure, in addition to the particle size decreasing, the silybin nanocrystals appeared to be more regularly shaped. The AFM experiments demonstrated that the silybin nanocrystals with a spherical shape revealed values of 718 nm and 226 nm in their diameters, for SN-A and SN-B, respectively. Additionally, the presence of encapsulate films on the surface of the pure particles of drugs could also be observed by AFM. These results indicate that the thin films should be surfactants. Therefore, AFM analysis reinforced the presence of the stabilizer on the surface of the drug particles and this feature is in accordance with the TEM results.
SEM images of the freeze-dried nanocrystals prepared under different homogenization pressure were clearly confirmed that the higher produce pressure the more uniformly nanocrystals in the morphology and the smaller particle size. Even the decreased size cannot be in proportion to the increased pressure. Hence, it was assumed that SN-B had higher uniform and smaller dimension. These results were in accordance with the study of particle size analysis and PI values. Nanocrystals were freeze-dried to obtain the dried silybin powder. TEM images indicate that the silybin powder was aggregated due to the water-removal. The DSC thermograms clearly showed that both of the two freeze-dried silybin nanocrystals displayed conspicuous melting point of silybin at the temperature with comparable physical mixture of drug and surfactants. These denoted that no any possible transformation to an amorphous state during the HPH and freeze-drying operation and this is important to long-term stability. XRPD experiment confirmed the fact that the diffraction pattern was preserved in silybin nanocrystals, which indicates that the crystalline state of silybin appeared to be unaltered following the HPH operation.
In solubility experiment, nanocrystals powder provided a more than two-fold increase in solubility compared to the un-milled silybin or physical mixture. This increase is more pronounced for SN-B that has smaller particle size. Either in SN-A or in SN-B, dissolution velocity enhancement was clearly observed at both tested pH6.0 and pH7.4. Correlating with the crystalline state analyze results, the dramatically enhanced dissolution rate resulted from the increased effective surface area, and the improved solubility due to the decreased particle size, but no due to the consequence of the presence of amorphous fraction. The specific surface area is 0.435 m2·g-1 and 0.484 m·g-1, for SN-A and SN-B, respectively. The porosity and pore diameter, for SN-A is 0.00401 and 17.05±16.0 nm, for SN-B is 0.00431 ml·g-1 and 17.03±16.4 nm, respectively. The accelerated stability test manifested that the SN-A and SN-B has satisfactory stability.
An LC-MS method for determination of silybin in Beagle dog plasma and mice organs was developed for the first time. The technology investigation coincided with the requirement of SFDA guidance principle of chemicals non-clinic pharmacokinetics. For Beagle oral administration (20 mg-kg-1), both nanocrystals delivery systems showed approximately two-fold or three-fold bioavailability improvements in terms rate and extent compared with the un-milled suspension. Based on comparison of the AUC0-∞> values, the un-milled silybin suspensions showed significantly lower silybin exposure than the nanocrystals (SN-A and SN-B). There was no significant difference in AUC0-∞, between SN-A and SN-B. The Cmax was significantly lower in the un-milled silybin suspensions than that of the nanocrystals SN-A or SN-B. The un-milled silybin suspension has the shortest MRT, the SN-A showed longer and the N-B showed the longest. The tmax was significantly shorter for the un-milled suspension than that for the nanocrystals of SN-A and SN-B. No significant statistical difference between the nanocrystals was found for this parameter. For Beagle intravenous drop administration (15 mg·kg-1), in contrast to solution, the nanocrystals formulations had prolonged t1/2 and augmented AUC. No adverse events were observed during the administration. The solution's AUC0-∞was lower than that of the nanocrystals, including SN-A and SN-B. Although the solution has higher Cmax, the lower MRT and higher clearance rate as well as other parameters also lead to a distinct pharmacokinetics profile when compared to the nanocrystals plots. Therefore, these experiments demonstrated that nanocrystals formulations can be used to improve exposure of compounds with low dissolution rate and poor pharmacokinetic characteristics.
To investigate the effect of nanocrystals particle size on the organ distribution, the mice vena caudalis intravenous silybin nanocrystals (20 mg-kg"1) were carried out. The evaluation index included drug targeting index, drug selectivity index, drug targeting efficiency and relative targeting efficiency, which revealed that silybin nanocrystals has liver passive targeting. In considering of silybin concentration in the organs, nanocrystals could decrease the concentration in the plasma and heart; increase the concentration in the liver. Judging from the quantity of silybin in the organs, the liver aggregation of the solution term, SN-A and SN-B is 15%-35%, 46%-63%and 31%-67%, respectively. Furthermore, in 10 h following administration SN-A, the liver aggregation stabilize at 40%-50%. This experiment revealed that the target efficiency of SN-A is better than SN-B.
In the Caco-2 cell cytotoxicity experiments, when the concentration of silybin was below 125μg·ml-1, there were no significant differences between the experimental groups and the control groups. The effects of nanocrystals, SN-A and SN-B, on facilitating silybin transport across the monolayers were much higher than that of the physical mixture formulation. Additionally, the decrease in particle size could also enhance ability of silybin in SN-B system transport across the monolayers as it has a higher ability to enhance Papp. Nanocrystals presented higher Papp values compared with un-milled silybin and physical mixture. Compared with the silybin nanocrystals, the un-milled silybin presented a lower Papp value in pH 6.0 medium. Based on the Caco-2 cell monolayers permeability date, we may tentatively present a conclusion that nanocrystals could promote of poorly soluble drugs transport cross intestine epithelium.
The injuring of HL7702 hepatic cell model was established by incubation with H2O2 for 24 h at concentration of 5 mM. When the silybin prophylactic administrated to cell injury model, the effective presented concentration dependent tendency. The most effectiveness were presented at 10μg·ml-1 and 50μg·ml-1, for silybin solution and SN-A, respectively. The protection efficiency of solution and SN-A was lower than the prophylactic administration experiments, when the silybin remedially administrated to cell injury model. In order to demonstrate the efficiency of SN-A on the protection of HL7702 is better than the solution formulation; the HE staining study was carried out. When the solution and SN-A administrated at 10μg·ml-1, the cells of SN-A group was presented as the endochylema turgor vitalis, the morphology normally, the nucleus and nuclear-cytoplasmic ratio closed to norm cells. Furthermore, the cells condition of solution formulation group was between model group and SN-A group, and presented some cell debris.
Beagle dog acute liver injure model was constructed by subcutaneously multipoint injected of CCl4-peanut oil solution (1:1, w/w). The SN-A intravenous drop and SN-B orally administrated at dose of 15 mg·kg-1 and 20 mg·kg-1, respectively. Compound ammonium glycyrrhizinate was chosen as positive control. The model group and silybin solution control were also set. The hepatic functional parameter, such as AST, ALT, ALP, TBIL and GGT were detected. The enzymes in the liver tissue, include SOD and MDA, were determined. The pathological section were used to observation the patho-changes of liver tissue. Therefore, the safety and efficiency of orally and intravenously administrated silybin nanocrystals were evaluated. Judging from the hepatic functions detection and observation of the patho-changes, the acute liver injurer of Beagle dogs were successfully established. Compared with solution formulations, the nanocrystals, SN-A for intravenous and SN-B for oral administration, could restore the deviate parameters, including AST, ALT, ALP, TBIL and GGT, toward to normal values. Furthermore, the silybin nanocrystals also could increase the SOD activity and decrease the level of MDA. As observed in the histological section, hepatic cell balloon changes and inflammatory cell infiltrate were decreased in the SN-A and SN-B groups, compared with solution group. And large area necrosis also refrained in the nanocrystals. At the same dose, the nanocrystals could effectively protect the liver cells avoid of injure, maintenance the nucleus and membrane integrity, promote the stabilization of liver cells. Silybin nanocrystals, SN-A for intravenous and SN-B for oral delivery, could effectively prevent the CCl4 induced hepatic injury of Beagle dogs. And these demonstrated that nanocrystals could improve the oral bioavailability and target efficiency of intravenous delivery.
In this study, the silybin nanocrystals for oral and intravenous administration,, could be prepared by piston-gap high pressure homogenization method. The freeze-drying, manicol used as protectant, could improve the stability of silybin nanocrystals. In the preparation, the crystals form maintained and particle size distributed uniformity. Dissolution velocity and saturation solubility is dramatically improved. The transmembrane transport ability and oral bioavailability enhanced. The nanocrystals also could implement liver passive target delivery. The nanocrystals improved the effectiveness of silybin for hepatic injury in vitro and in vivo.
引文
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