N-(2-羟丙基)甲基丙烯酰胺聚合物-5-氟尿嘧啶接合物的体外释药规律、体内分布及抗肿瘤活性研究

考察本实验室合成的N-(2-羟丙基)甲基丙烯酰胺［N-(2-hydroxypropyl) methacrylamide，HPMA］聚合物-5-氟尿嘧啶(5-flurouracil，5-FU)接合物(P-FU)的体外释药、体内分布及抗肿瘤活性。以小鼠血浆为介质，考察P-FU中5-FU的释放规律；以小鼠H22肝癌实体瘤模型(皮下型)为肿瘤模型，考察接合物在荷瘤小鼠体内的分布情况、药代动力学规律及抑瘤活性。结果表明，37 ℃时P-FU在小鼠血浆中具有一定的稳定性，半衰期(t_1/2)为32.4 h。与5-FU相比，P-FU在荷瘤小鼠体内的循环时间明显延长(血浆中t_1/2为原药的166倍)，在肿瘤中的沉积量(AUC为5-FU的3.3倍)及滞留时间(t_1/2为5-FU的2.3倍)均有明显增加。体内药效学研究表明，P-FU组对荷瘤小鼠的肿瘤生长抑制率(69.09%)显著高于5-FU组(56.49%，P<0.05)，瘤块组织病理学观察结果也显示P-FU组小鼠肿瘤组织中细胞凋亡程度大于5-FU组。HPMA聚合物可被用于为5-FU构建一种新型实体瘤高分子给药系统。     

N-(2-羟丙基)甲基丙烯酰胺聚合物-5-氟尿嘧啶接合物的体外释药规律、体内分布及抗肿瘤活性研究

考察本实验室合成的N-(2-羟丙基)甲基丙烯酰胺[N-(2-hydroxypropyl)methacrylamide,HPMA]聚合物-5-氟尿嘧啶(5-flurouracil,5-FU)接合物(P-FU)的体外释药、体内分布及抗肿瘤活性。以小鼠血浆为介质,考察P-FU中5-FU的释放规律;以小鼠H22肝癌实体瘤模型(皮下型)为肿瘤模型,考察接合物在荷瘤小鼠体内的分布情况、药代动力学规律及抑瘤活性。结果表明,37℃时P-FU在小鼠血浆中具有一定的稳定性,半衰期(t1/2)为32.4 h。与5-FU相比,P-FU在荷瘤小鼠体内的循环时间明显延长(血浆中t1/2为原药的166倍),在肿瘤中的沉积量(AUC为5-FU的3.3倍)及滞留时间(t1/2为5-FU的2.3倍)均有明显增加。体内药效学研究表明,P-FU组对荷瘤小鼠的肿瘤生长抑制率(69.09%)显著高于5-FU组(56.49%,P<0.05),瘤块组织病理学观察结果也显示P-FU组小鼠肿瘤组织中细胞凋亡程度大于5-FU组。HPMA聚合物可被用于为5-FU构建一种新型实体瘤高分子给药系统。     

多聚N-（2-羟丙基）甲基丙烯酰胺-抗肿瘤药物偶合物的研究进展

大分子聚合物-抗肿瘤药物偶合物能利用增强渗透和滞留效应将抗肿瘤药物-偶合物选择性投放于肿瘤组织,从而提高抗肿瘤药物在肿瘤组织的浓度,减少药物的系统毒性。多聚N-（2-羟丙基）甲基丙烯酰胺（HPMA）作为大分子载体具有许多优点,如生物相容性、非免疫原性等,因此多聚HPMA-抗肿瘤药物偶合物成为当前大分子靶向抗肿瘤药物研究的热点之一。目前进入临床的偶合物有6种,还有许多处于临床前研究。本文综述多聚HPMA-抗肿瘤药物偶合物研究的进展。     

N-(4-取代氨基-4-氧代丁酰基)-N-取代甘氨酸的合成

血管紧张素转化酶抑制剂(ACEI)是近年发展的一类新型抗高血压和治疗心力衰竭的有效药物。有关研究工作还在世界范围内广泛进行。 本研究组前曾报道一类具有降压活性的N-取代甘氨酸的合成。作者等根据Cushman等的血管紧张素转化酶(ACE)与底物的作用模型和构效关系,在保持3个主要作用部分(即ACE     

N-乙酰基-L-谷氨酰基-泼尼松龙肾靶向前体药物研究

目的通过研究N-乙酰基-L-谷氨酰基-泼尼松龙的体内分布，考察该前体药物的肾靶向性。方法小鼠iv后，采用高效液相法，在规定时间段测定各组织脏器的泼尼松龙浓度, 并采用大鼠骨密度的测定仪确证前体药物的副作用。结果小鼠给药后15 min，前体药物组肾脏中泼尼松龙浓度为(86±8) μg·g^-1，泼尼松龙组为(57±4) μg·g^-1，60 min后前体药物组肾脏药物浓度为 (67±5) μg·g^-1；泼尼松龙组(42±4) μg·g^-1。大鼠给药30 d后，股骨的骨密度分别为(0.08±0.03) g·cm^-2 （泼尼松龙）和(0.14±0.06) g·cm^-2(前体药物组)。结论前体药物具有肾靶向性, 并能降低致骨质疏松的副作用。     

脑室注射N-甲基-D-门冬氨酸对小鼠学习记忆的影响

采用一次性被动回避性条件反射方法,给小鼠icv N-甲基-D-门冬氨酸(NMDA)10,1和0.1 ng。结果表明:对正常小鼠,在跳台法中NMDA 10 ng能明显增强记忆巩固过程,在避暗法中NMDA 1 ng能显著增强记忆再现过程;在两种方法中,NMDA 1 ng均能显著改善乙醇及亚硝酸钠所致记忆障碍;而NMDA对记忆获得过程无影响。进一步研究表明,NMDA改善记忆的作用可被其受体特异性拮抗剂AP5所拮抗。结合以前报道和电生理研究结果提示,谷氨酸受体NMDA亚型在记忆过程中起着重要的作用,脑室给药作用的环节主要是影响记忆巩固和再现过程。     

血吸虫病化学治疗的研究——ⅩⅩⅩⅦ.β-(5-硝基-4-溴-2-呋喃)-及β-(4,5-二溴-2-呋喃)丙烯酰胺及其酯类的合成

本文报道β-(4,5-二溴-2-呋喃)-及β-(5-硝基-4-溴-2-呋喃)丙烯酰胺及其酯类衍生物26个的合成。动物筛选结果表明;化合物Ⅲ₆,Ⅲ₈和Ⅲ₁₃对感染日本血吸虫小白鼠有明显的治疗作用。化合物Ⅱ₆有较明显的预防作用。     

N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸、脯氨酸和焦谷氨酸的合成

报道11个预期有血管紧张素转化酶抑制活性的N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸(VII_1～9)、脯氨酸(VII₁₀)和焦谷氨酸(VIl₁₁)的合成和鉴定。所有上述化合物以及与VⅡ_1～9相应的叔丁酯(VI_1～9)均未见文献报道。药理初试结果显示，化合物VII₈，VII₉和VI₁₀均有明显降压活性。     

蝙蝠葛中的新生物碱——N-去甲基蝙蝠葛碱

A new phenolic dauricine-type alkaloid together with the know dauricine, were iso-lated from the rhizoma of Menispermum dauricum DC cultivated in Xianning district, Hubei province.Dauricine was obtained as the major alkaloid and was confirmed by comparison with authentic sample.The new alkaloid is an unstable white powder. Based on spectrometric analysis (UV, IR, FAB--MSand ¹HNMR) and N-methylation which offered clauricine dimethiodide(Ⅴ), the structure was elucidat-ed as RR, N-desmethyldauricine(Ⅱ), which was isolated for the first time from nature.     

血吸虫病化学治疗的研究——ⅩⅩⅩⅣ.α-氯-β-(5-硝基-2-呋喃)丙烯酰胺类及其乙烯噁二唑类衍生物的合成

Synthesis of 35 new compounds of α-chloro-β-(5-nitro-2-furyl) acrylamides and 5-[α-chloro-β-(5-nitro-2-furyl ) vinyl]-oxadiazoles by known methods are reported. In preliminary test in mice 10 compounds were found to possess pronounced activity against Schistosomiasis japonica. Among these Ⅰ₁₂, Ⅰ₁₃, Ⅰ₁₄, Ⅰ₂₀, Ⅱ₁ and Ⅱ₈ were shown to be the most effective.     

Biodegradable star HPMA polymer conjugates of doxorubicin for passive tumor targeting

New biodegradable star polymer–doxorubicin (Dox) conjugates designed for passive tumor targeting were investigated and the present study described their synthesis, physico-chemical characterization, drug release and biodegradation. In the conjugates the core formed by poly(amido amine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin attached by hydrazone bonds, which enabled intracellular pH-controlled drug release, or by a GFLG sequence, which was susceptible to enzymatic degradation. The controlled synthesis utilizing semitelechelic copolymer precursors facilitated preparation of biodegradable polymer conjugates in a broad range of molecular weights (110–295 kDa) while still maintaining low polydispersity (～1.7). The polymer grafts were attached to the dendrimers either through stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high molecular weight polymer carrier to products that were able to be excreted from the body by glomerular filtration. Biodegradability tests showed that the rate of degradation was much faster for reductively degradable conjugates (completed within 4 h) than the degradation of conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (within 72 h). This finding was likely due to the difference in steric hindrance for the small molecule glutathione and the enzyme cathepsin B. As for drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin either under mild acidic conditions or in the presence of lysosomal enzyme cathepsin B, both of which modeled the tumor cell microenvironment.     

Development of HPMA copolymer–anticancer conjugates: Clinical experience and lessons learnt

The concept of polymer–drug conjugates was proposed more than 30 years ago, and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate of doxorubicin covalently bound to the polymer backbone by a Gly-Phe-Leu-Gly peptidyl linker (FCE28068) became the first synthetic polymer-based anticancer conjugate to enter clinical trial in 1994. This conjugate arose from rational design attempting to capitalise on passive tumour targeting by the enhanced permeability and retention effect and, at the cellular level, lysosomotropic drug delivery to improve therapeutic index. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA as a new polymer platform. Subsequent Phase I/II trials have investigated an HPMA copolymer-based conjugate containing a doxorubicin and additionally galactose as a targeting moiety to promote liver targeting (FCE28069), and also HPMA copolymer conjugates of paclitaxel (PNU 166945), camptothecin (PNU 166148) and two platinates (AP5280 and AP5346- ProLindac™). The preclinical and clinical observations made in these, and clinical studies with other polymer conjugates, should shape the development of next generation anticancer polymer therapeutics.     

HPMA在骨靶向药物输送方面的应用

本文简要的阐述了N-2-羟丙基-甲基丙烯酰胺聚合物(HPMA)在靶向药物输送方面的特性,并介绍了把N-2-羟丙基-甲基丙烯酰胺聚合物复合物应用在骨靶向方面的部分进展以及它们的体内体外的实验研究现状,以HPMA-D-Asp8-PGE1复合物为例介绍了N-2-羟丙基-甲基丙烯酰胺聚合物复合物的整体设计。     

Peptide-directed HPMA copolymer-doxorubicin conjugates as targeted therapeutics for colorectal cancer

Synthetic oligopeptides have emerged as a promising class of targeting ligands, providing a variety of choices for the construction of conjugates for desired ligand functionality. To explore the potential of short peptides as ligands for targeted delivery of macromolecular therapeutics for colorectal cancer (CRC), fluorescently labelled HPMA copolymers—bearing either G3-C12 or GE11 for targeting galectin-3 and epidermal growth factor receptor (EGFR), respectively—were synthesised and the mechanisms of their internalisation and subcellular fate in CRC cells were studied. The targetability of the G3-C12 bearing copolymers towards galectin-3 was further compared to that of galactose-containing copolymers. The resulting G3-C12-bearing conjugate actively and selectively targets CRC tumour cells over-expressing galectin-3 and exhibits superior targetability to galectin-3 when compared to the galactose-bearing copolymer. GE11 copolymer conjugate binds specifically and efficiently to EGFR over-expressing cells, thus mediating internalisation to a significantly higher extent relative the copolymer conjugated to a scrambled sequence peptide. We further incorporated doxorubicin (DOX) into GE11 bearing copolymer via an acid-labile hydrazone bond. The GE11-DOX copolymer conjugate demonstrated higher cytotoxicity toward EGFR over-expressing cells relative to the control non-targeted DOX conjugate. Altogether, our results show a proof of principle for the selective delivery of DOX to the target CRC cells.     

HPMA-copolymer conjugates targeted to tumor endothelium using synthetic oligopeptides

Synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA)-copolymer–based drug carriers targeted on specific receptors in the membrane of endothelial cells by oligopeptides (GRGDG, cyclo(RGDfK), and PHSCN) are described in this study. The copolymers containing targeting oligopeptides bound to the polymer via dodeca(ethylene glycol) spacer showed a receptor-specific time-dependent uptake with selected endothelial cell lines. The polymers were labeled with a fluorescent dye to enable monitoring of the interaction of the polymer conjugate with cells using fluorescence microscopy. Cellular uptake and apoptosis induction have been studied in vitro using various cell lines (EA.hy926, 3T3, SW620, and EL4). In vivo accumulation of the conjugate specifically targeted with cyclo(RGDfK) within the tumor vasculature was detected using fluorescence intravital microscopy in mice. The conjugate targeted by cyclo(RGDfK) was accumulated preferentially in the periphery of the growing tumor suggesting that the cyclo(RGDfK) peptide targets the polymer conjugate to the site of neoangiogenesis, rather than to the tumor mass.     

Self-association properties of HPMA copolymers containing an amphipathic heptapeptide

Receptor-binding peptides are suitable targeting moieties for macromolecular therapeutics. Binding several targeting peptides to one macromolecule may improve biorecognition due to the multivalency effect. On the other hand, the resulting amphipathic structure of such conjugates may result in the association of side-chains with a concomitant decrease in the accessibility of the side-chain-bound ligands. Using the one-bead one-compound combinatorial method, we have recently identified a heptapeptide (YILIHRN; HP) ligand for the CD21 receptor (Biomacromolecules 7, 3037, 2006). Here, we evaluated the relationship between structure and self-association of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-HP conjugates using fluorescence resonance energy transfer (FRET) to evaluate their conformation in solution. In addition to HP, HPMA copolymers containing side-chains terminating in tryptophan (energy donor) and dansyl (energy acceptor) were synthesized, and solutions were evaluated using an excitation wavelength of 295 nm (ratio of emission intensity 510 nm/370 nm indicated energy transfer efficiency). It was found that higher HP content correlated with higher FRET efficiency, indicating the formation of compact coils. Modification of the HPMA copolymer backbone by the incorporation of acrylic acid (AA) comonomer units resulted in decreased FRET efficiency, presumably due to the expansion of the polymer coils as a result of electrostatic repulsion. The dependence of FRET efficiency on pH was in agreement with the ionization profile of the AA residues.

To determine the effect of HP content on enzymatic drug release kinetics, HPMA copolymer-HP conjugates containing GFLG side-chains terminating with doxorubicin (DOX) were incubated with papain and the release of free DOX monitored. When HP content increased above a particular threshold, the rate of DOX release decreased as a result of self-association of HPMA copolymer-GFLG-DOX-HP conjugates. The FRET data correlated well with hydrodynamic volumes determined by size exclusion chromatography (SEC), with molecular weights determined by light scattering, and with the kinetics of drug release.     

Novel star HPMA-based polymer conjugates for passive targeting to solid tumors

Novel star polymer-doxorubicin conjugates designed for passive tumor targeting have been developed and their potential for treatment of cancer has been investigated. In the present study the synthesis, physico-chemical characterization, drug release, bio-distribution and preliminary data of in vivo efficacy of the conjugates are described. In the water-soluble conjugates the core of a molecule formed by poly(amido amine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds enabling intracellular pH-controlled hydrolytic drug release, or by GFLG sequence susceptible to enzymatic degradation. The controlled synthesis utilizing semitelechelic copolymer precursors facilitated preparation of polymer conjugates in a broad range of molecular weights (1.1–3.0·10⁵ g/mol). In contrast to free drug or linear conjugates the star polymer-Dox conjugates exhibited prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice indicating important role of the EPR effect. The star polymer-Dox conjugates showed significantly higher anti-tumor activity in vivo than Dox·HCl or its linear or graft polymer conjugates, if treated with a single dose 15 or 5?mg Dox eq./kg. Method of tumor initialization (acute or chronic experimental tumor models) significantly influenced effectiveness of the treatment with much lower success in treatment of mice bearing chronic tumors.     

Beyond oncology — Application of HPMA copolymers in non-cancerous diseases

Macromolecular drug conjugates have been developed to improve the efficacy and safety profile of various therapeutic agents for many years. Among them, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates are the most extensively studied delivery platforms for the effective treatment of cancer. In recent years, the applications of HPMA copolymers for the treatment of a broader range of non-cancerous diseases have also been explored. This review highlights the recent developments in the rational design, synthesis, and evaluation of novel HPMA copolymer-drug conjugates for non-cancerous diseases, such as musculoskeletal diseases, infectious diseases and spinal cord injury. The translation potential of these applications is also briefly discussed.     

正电化修饰的HPMA聚合物-阿霉素接合物的制备表征及对肿瘤细胞摄取的影响

目的:制备2种正电化修饰的N-(2-羟丙基)甲基丙烯酰胺(HPMA)聚合物-阿霉素接合物并表征,分别考察2种接合物的正电基团含量对肿瘤细胞摄取的影响。方法:制备侧链带伯胺基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-APMA)和侧链带胍基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-GPMA),对其药剂学性质如正电基团含量,载药量,Zeta电位和分子量进行表征,进一步考察不同正电基团含量的接合物对MCF-7细胞摄取和毒性的影响。结果:通过自由基聚合反应,2种接合物成功合成。其中pHPMA-DOX-APMA伯胺基含量为0.44~1.57 mmol·g^-1,载药量为7.15%~9.25%;pHPMA-DOX-GPMA胍基含量为0.11~0.54 mmol·g^-1,载药量为7.55%~9.07%;相对分子质量分别为33~38 kDa和32~37 kDa。通过BCA法和MTT法研究分别发现在pHPMA-DOX-APMA中的伯胺基团含量为1.570 mmol·g^-1及pHPMA-DOX-GPMA中的胍基含量为0.260 mmol·g^-1时,肿瘤细胞对阿霉素的摄取量显著增加,二者的IC₅₀与pHPMA-DOX相比显著降低(P<0.05)。结论:成功制备了2种正电化修饰的HPMA聚合物-阿霉素接合物;适当的正电化修饰对阿霉素的肿瘤细胞摄取有促进作用。