受体介导的肝靶向载药系统研究进展

介绍近几年已报道的有关受体介导的肝靶向载药系统的研究情况。近年来受体介导的肝靶向载药系统的研究取得了一些可喜进展,相关配体-受体可与药物、脂质体、纳米粒、基因、偶联物等相连,提高药物或载体的肝靶向能力。受体介导机制在肝靶向载药系统的研究领域具有广阔的应用前景,尤其是肝脏特异性受体的不断发掘,丰富了肝主动靶向的理论体系,展示了肝脏疾病治疗的美好未来。     

Ultrasound Exposure Enhances the Biological Action of Interferon in the Liver

Although interferon (IFN) α and β are currently recognized as the most effective agents for treating patients with chronic hepatitis B and C, they are well known to cause various adverse effects. To reduce the dose of IFN necessary for treatment, we tried enhancing the effects of IFN in the liver by ultrasound exposure. Percutaneous insonation in mouse liver following IFN- β injection with the ultrasound power level used at clinical diagnosis enhanced the IFN- β -induced increase of 2',5'-oligoadenylate synthetase (2-5AS) levels in the liver. This enhancement of the 2-5AS level was dependent on the duration as well as on the timing of insonation after the IFN- β injection. In contrast, liver insonation did not enhance 2-5AS levels in the lung or spleen, and moreover, it did not alter tissue distribution of injected IFN. Thus, the combination of IFN- β administration and subsequent liver insonation appears to be a promising method for enhancing the antiviral activity of IFN specifically in the liver, enabling a reduction in the dose necessary for treatment.     

Imparting Bone Affinity to Glycoproteins Through the Conjugation of Bisphosphonates

Purpose. To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. Methods. 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Results. The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-, and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. Conclusion. The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.     

肝靶向微粒给药系统的研究进展

目的对近年来微粒给药系统在肝靶向治疗的研究进展做一综述。方法根据国内外文献资料进行整理归纳。结果纳米粒、微球、脂质体及微乳等微粒系统具有被动靶向于肝的趋势,利用肝细胞表面某些受体则可特异性靶向于肝达到主动靶向作用。结论微粒给药系统在肝靶向治疗领域具有重要意义。     

Regional Delivery of Model Compounds and 5-Fluorouracil to the Liver by Their Application to the Liver Surface in Rats: Its Implication for Clinical Use

Purpose The purpose of this study was to examine drug distribution in the liver after drug application to the rat liver surface.Methods Phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextran (MW 4400, FD-4) as model compounds or 5-fluorouracil (5-FU) was applied to the rat liver surface by employing a cylindrical diffusion cell (i.d. 9 mm, 0.64 cm²). Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites: the region under the diffusion cell attachment site (site 1), the applied lobe except for site 1 (site 2), and non-applied lobes (site 3).Results In the case of i.v. administration, there were no differences in PSP concentrations among the three sites of the rat liver, and the concentrations rapidly decreased. On the other hand, the PSP concentration in site 1 after application to the rat liver surface was considerably higher than in site 2 and site 3. In addition, the area under the curve (AUC) value (AUC_site1), calculated from the PSP concentration profile in site 1, was about 10 times larger than that in site 3. A similar trend of regional delivery advantage by liver surface application was observed in the case of the macromolecule model FD-4, with a marked AUC_site1 of about 5 times larger than the other two sites. Moreover, we clarified that the anticancer drug 5-FU preferentially distributed in site 1 after application to the rat liver surface.Conclusion These results demonstrate the possibility of regional delivery of drugs to the liver by application to the liver surface.     

半乳糖衍生物修饰去甲斑蝥酸钠脂质体的主动靶向性研究

制备半乳糖衍生物修饰的去甲斑蝥酸钠脂质体(GNL),探讨并比较其与去甲斑蝥酸钠脂质体(CNL)的肝靶向作用。以半乳糖为起始物,经乙酰化、溴代、缩合、置换后得化合物Galβ1-(CH2-CH2-O)2-C14-H29作为修饰靶向材料,采用逆相蒸发法制备CNL和GNL;RP-HPLC测定小鼠尾静脉注射给药后小鼠肝脏中的药物浓度。实验表明脂质体表面经化合物Galβ1-(CH2-CH2-O)2-C14-H29修饰后,GNL肝脏靶向效率是CNL的1.8倍,是去甲斑蝥酸钠注射液的2.9倍。     

Effective Targeting of Liposomes to Liver and Hepatocytes In Vivo by Incorporation of a Plasmodium Amino Acid Sequence

Purpose Several species of the protozoan Plasmodium effectively target mammalian liver during the initial phase of host invasion. The purpose of this study was to demonstrate that a Plasmodium targeting amino acid sequence can be engineered into therapeutic nanoparticle delivery systems. Methods A 19-amino peptide from the circumsporozoite protein of Plasmodium berghei was prepared containing the conserved region I as well as a consensus heparan sulfate proteoglycan binding sequence. This peptide was attached to the distal end of a lipid–polyethylene glycol bioconjugate. The bioconjugate was incorporated into phosphatidylcholine liposomes containing fluorescently labeled lipids to follow blood clearance and organ distribution in vivo. Results When administered intravenously into mice, the peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver. Fluorescence and electron microscopy demonstrated that the liposomes were accumulated both by nonparenchymal cells and hepatocytes, with the majority of the liposomal material associated with hepatocytes. Accumulation of liposomes in the liver was several hundredfold higher compared to heart, lung, and kidney, and more than 10-fold higher compared to spleen. In liver slice experiments, liposome binding was specific to sites sensitive to heparinase. Conclusions Incorporation of amino acid sequences that recognize glycosaminoglycans is an effective strategy for the development of targeted drug delivery systems. K. J. Longmuir and R. T. Robertson were the primary investigators for this study.     

Population PK-PD Model of Pegylated Interferon Alfa-2a in Healthy Korean Men

Pegylated interferon alfa-2a (PEG-IFN alfa-2a), which was developed to overcome the disadvantages of conventional formulations, is widely prescribed for hepatitis B or C virus infection. It is characterized by pharmacokinetic (PK) and pharmacodynamic (PD) properties much different from those of conventional forms. The present study developed a population PK-PD model of subcutaneous PEG-IFN alfa-2a in a Korean population. For PK, IFN alfa-2a concentrations were described by a 1-compartment model with first-order absorption, preceded by skin-to-depot first-order input. For PD, serum 2’-5’ oligoadenylsynthetase activity was described by an effect compartment model incorporating a tolerance compartment. The baseline serum 2’-5’ oligoadenylsynthetase level was found to have an inverse relationship with sensitivity to tolerance, leading to high tolerance at low baseline. The model revealed that subjects with low baselines experienced time delay, while those with high baselines showed tolerance in their concentration-effect relationships. The developed models matched well with data and simulation results, and the model showed that the optimal dose decreases with the baseline, with no dose effective for a baseline >250 pmol/dL. Our results can serve as a basis for improving dosing regimens of PEG-IFN alfa-2a in adult patients with chronic hepatitis B or C infection.     

重组肝靶向高密度脂蛋白-药物纳米粒的制备及处方优化

高密度脂蛋白(HDL)可逆向转运血浆中胆固醇至肝脏代谢,在肝靶向传递系统方面具有较大的开发潜力和、应用价值。载脂蛋白A-Ⅰ(apoA-Ⅰ)是HDL的主要组成部分。以apoA-Ⅰ为载体,水溶性抗肿瘤药盐酸多柔比星为模型药,采用薄膜分散法或硫酸铵梯度法制备重组高密度脂蛋白-盐酸多柔比星纳米粒,并以平均粒径或包封率为指标进行优化。结果表明,优化后的薄膜分散法所得制品平均粒径为(48.3±16.1)nm,包封率为(20.2±4.2)%。优化后的硫酸铵梯度法所得制品平均粒径为(113.8±10.3)nm,包封率为(83.3±8.5)%,且无溶血性。采用5%蔗糖为冻干保护剂制得的冻干,品于-20℃放置8个月,稳定性较好。     

Targeting Doxorubicin to Epidermal Growth Factor Receptors by Site-Specific Conjugation of C225 to Poly(L-Glutamic Acid) Through a Polyethylene Glycol Spacer

Purpose. Targeted delivery of chemotherapeutic agents through antibody-polymer conjugates has met with limited success. One of the limiting factors is the loss of antibody's binding affinity upon conjugation with polymeric carriers because of lack of control over the number and site of attachment. This study aims to synthesize monovalent polymeric immunoconjugates through site-specific conjugation and to evaluate the in vitro binding activities of the resulting construct. Methods. Antibody C225 against epidermal growth factor receptors was coupled to the terminus of a doxorubicin-bound block copolymer, poly(L-glutamic acid)-co-polyethylene glycol (PG-PEG). Western blot analysis, confocal fluorescent microscopy, and cytotoxicity assay were performed to confirm the specific binding of C225-PEG-PG-Dox to EGFR. Results. C225 was conjugated to PEG-PG-doxorubicin conjugates by reacting sulfhydryl group introduced to C225 with vinylsulfone group introduced at the terminus of PEG-PG block copolymer. Polymeric immunoconjugate C225-PEG-PG-Dox, but not control (i.e., conjugate without antibody), selectively bound to human vulvar squamous carcinoma A431 cells that overexpress epidermal growth factor receptors. Receptor-mediated uptake of C225-PEG-PG-Dox occurred rapidly (within 5 min), whereas nonspecific uptake of PEG-PG-Dox required an extended period of time (24 h) to internalize. Binding of C225-PEG-PG-Dox to A431 cells could be blocked by pretreatment with C225 antibody. C225-PEG-PG-Dox was more potent than free doxrubicin in inhibiting the growth of A431 cells after a 6-h exposure period. Conclusion. Site-specific conjugation of a monoclonal antibody to the terminus of a polymeric carrier enhances receptor-mediated delivery of anticancer agents.     

乙型肝炎导向干扰素在荷瘤鼠体内的分布实验

将2，2，15细胞接种裸鼠，制得抗乙型肝炎病毒（HBV）动物模型。用125I标记乙型肝炎导向干扰素（T－IFN），观察放射标记物（125I－T－IFN）在荷瘤鼠体内的分布情况，以评价T－IFN在体内的抗HBV导向治疗作用。结果显示，125I－T－IFN在各组织浓度顺序依次为：瘤＞心＞肝＞脾＞肾，且瘤组织与其它各组织比较，有明显差异（P＜0．01）。结果表明，在HBV动物模型体内，T－IFN相对浓集于可分泌HBsAg的瘤区。实验结果显示了T-IFN导向治疗HBV感染的可行性。     

Targeting human secretory phospholipase A2 with designed peptide inhibitors for inflammatory therapy

Phospholipase A2 (PLA2) is potentially an important target for anti-inflammatory therapeutics. Here, we described a systematic scheme that integrated protein docking and peptide redocking, molecular dynamics simulation, and binding affinity analysis to rationally design PLA2 inhibitory peptides based on a solved PLA2 crystal structure. The scheme employed protein docking to sample the interaction modes of PLA2 with its natural inhibitor Clara cell protein, from which a number of peptide fragments, including a pentapeptide LLLGS, were cut off and redocked to serve as the lead entities of PLA2 inhibitory peptides. In addition, a systematic mutation energy map that characterized the binding free energy changes ΔG upon mutations of each position of the putative pentapeptide to 20 amino acids was also profiled, which was subsequently used to guide peptide structure optimization. In order to solidify the computational findings, we performed kinetic and inhibition studies of few designed peptides against human secretory PLA2. Consequently, eight peptides were successfully identified to have potent inhibition potency, in which the LLAYK and AVFRS were found to suppress enzymatic activity significantly (K_i?=?0.75?±?0.06 and 4.2?±?0.3?μM, respectively). A further structure examination revealed that the designed peptides can form intensive nonpolar networks of van der Waals contacts and hydrophobic interactions at their complex interfaces with PLA2, conferring considerable stability and affinity for the formed complex systems.     

Design for Cell-Specific Targeting of Proteins Utilizing Sugar-Recognition Mechanism: Effect of Molecular Weight of Proteins on Targeting Efficiency

Hepatic targeting of proteins utilizing the sugar-recognition mechanism was investigated in mice after intravenous injection. Five proteins with different molecular weights, i.e., bovine -globulins (IgG), bovine serum albumin (BSA), recombinant human superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and chicken egg white lysozyme (LZM), were modified with 2-imino-2-methoxyethyl 1-thiogalactoside to obtain galactosylated proteins (Gal-IgG, Gal-BSA, Gal-SOD, Gal-STI, and Gal-LZM). The numbers of galactose residues were 38, 20, 11, 6, and 5 for Gal-IgG, Gal-BSA, Gal-SOD, Gal-STI, and Gal-LZM, respectively. All galactosylated proteins were dose-dependently taken up by the liver and the relative amount accumulated in the liver was decreased with an increase of the administered dose. At low doses (0.05 and 0.1 mg/kg), Gal-IgG, Gal-BSA, and Gal-SOD could be taken up by the liver up to more than 70–80% of dose within 10 min after intravenous injection, but the maximum amounts accumulated in the liver were approximately 40 and 30% of the dose for Gal-STI and Gal-LZM, respectively. Pharmacokinetic analysis revealed that the hepatic uptake clearance (CL_liver) was quite different around the molecular weight of 32 kDa and correlated with the amount delivered to the liver; Gal-IgG, Gal-BSA, and Gal-SOD has a large CL_liver that is close to the hepatic plasma flow rate (85 ml/hr), whereas those of Gal-STI and Gal-LZM were approximately 10 ml/hr at low doses. As for the total amount accumulated in the liver, high glomerular filtration rate of Gal-STI and Gal-LZM was also shown to cause insufficient delivery to the liver apart from being caused by their low CL_liver.     

半乳糖化脂质体-聚阳离子-DNA复合物的肝靶向性研究

目的 研究半乳糖苷修饰的脂质体-聚阳离子-DNA复合物(Gal-LPD)在体外的肝细胞靶向性.方法 合成胆甾五半乳糖苷(Gal-chol),并用薄膜-超声分散法制备空白阳离子脂质体,再与鱼精蛋白-DNA复合物形成Gal-LPD;用激光粒度仪及电位分析仪测定其粒径和电位;以lacZ质粒DNA作为报告基因,用HepG2肝癌细胞和A549肺癌细胞考察LPD的转染效率;用MTT法测定其细胞的毒性.结果 Gal-LPD的粒径为200 nm;Zeta电位随阳离子成分DDAB-DNA比例的不同而在20～55 mV间变化;与未用半乳糖修饰的LPD.相比,Gal-LPD在HepG2细胞中的转染率提高了2.4倍,但在A549细胞中的转染率却有所下降,而且半乳糖能竞争抑制Gal-LPD在HepG2细胞中的转基因效率;Gal-LPD无明显的细胞毒性.结论 Gal-LPD在HepG2细胞中有较高的转基因效率,具有体外的肝细胞靶向性.     

Evidence for Glucuronide Conjugation of p-Nitrophenol in the Caco-2 Cell Model

Pharmaceutical Research -     

紫外分光光度法测定IL-2靶向5-FURP脂质体中IL-2的含量

目的:建立测定白细胞介素-2(IL-2)靶向5-氟尿苷棕榈酸酯前药(5-FURP)脂质体中IL-2含量的方法。方法:在样品溶液中加入考马斯亮蓝G-250溶液染色,以空白脂质体为空白,在595nm波长处照分光光度法测定IL-2的吸光度,并计算含量。结果:IL-2检测浓度的线性范围为1～100μg·mL-1(r=0.9997);平均回收率为99.26%,RSD=0.59%(n=5)。结论:本方法操作简便、快速、检测灵敏、重复性好,可用于该脂质体中IL-2的含量测定。     

大豆糖苷修饰阳离子脂质体的体外肝细胞靶向性

目的研究肝靶向物质大豆糖苷(soybean-derived sterylglucoside,SG)的加入对阳离子脂质体肝细胞靶向性的影响。方法以荧光素钠(FS)为模型药物，采用HepG₂ 2.2.15细胞模型和SD雄性大鼠，检测SG，SG/Brij-35(卞泽-35)和SG/PEG-DSPE(polyethylene glycol-distearoylphosphatidylethanolamine)修饰的阳离子脂质体的物理化学性质，在细胞培养水平和离体肝脏水平考察阳离子脂质体的转染和肝细胞选择性。结果未修饰以及SG，SG/Brij-35和SG/PEG-DSPE修饰的FS阳离子脂质体在中性溶液中的包封率分别为91.74%，88.46%，89.70%和83.12%，粒径分别为124.4，113.7，110.8和93.0 nm，空白脂质体在溶液中表面电荷为正。细胞培养和肝脏灌流结果说明，阳离子脂质体的转染率显著高于中性脂质体，SG单独修饰后的阳离子脂质体的细胞转染率较未修饰有显著提高，SG/Brij-35修饰的阳离子脂质体则表现出肝实质细胞选择性。结论阳离子脂质体可以促进FS进入肝脏细胞，具有较高的肝细胞摄取率，而SG/Brij-35的修饰可以提高脂质体的肝细胞选择性。     

Liver targeting characteristics of galactosyl poly L lysine

以还原胺化法合成了半乳糖多聚－Ｌ－赖氨酸（ＧａｌＰＬＬ）,并首次利用放射性示踪实验测定了ＧａｌＰＬＬ在小鼠体内的肝靶向特征．结果表明,ＧａｌＰＬＬ具有较高的肝靶向性,其在体内的分布形式与肝细胞去唾液酸糖蛋白受体（ＡＳＧＰＲ）的内源性配体去唾液酸胎球蛋白（ＡＳＦ）相似．１２５Ｉ标记的ＧａｌＰＬＬ经静脉注入小鼠体内后,主要被肝脏吸收,在注射后５ｍｉｎ时肝脏的最大吸收为注射总量的３８．９％,比人血清白蛋白高５．７倍．肝脏对ＧａｌＰＬＬ的吸收可特异地被ＡＳＦ及非标记的ＧａｌＰＬＬ抑制,但不能被未半乳糖基化的多聚－Ｌ－赖氨酸抑制,证明ＧａｌＰＬＬ是通过肝细胞ＡＳＧＰＲ介导的内吞作用特异地被肝脏吸收的．由于ＧａｌＰＬＬ不但具有较高的肝靶向性,而且与ＡＳＧＰＲ的天然配体相比在合成和应用方面均具有许多优势,因此有望成为进行药物或基因肝靶向运送的良好载体．