单克隆抗体—表阿霉素免疫偶合物的制备和体外活性

用双功能试剂己二酰肼制备腙键连接的聚谷氨酸—表阿霉素,通过控制交联条件,所得产物克服了大分子自身交联的缺点,交联率较高。聚谷氨酸的载药量与分子量呈正比,平均每8～11个谷氨酸单体连接1分子表阿霉素。分子量为14300的聚谷氨酸做载体其载药量为1:11,与单抗交联所得的偶合物McAb:PGA:PAR为1:2:22。偶合物较好地保留了抗体活性,体外细胞毒性较游离药物略有下降,但表现出单抗介导的靶细胞选择性杀伤作用。本研究用腙键交联法成功地制备了药/抗比高且体外有效的免疫偶合物,为进一步制备细胞靶向的肿瘤化疗制剂奠定了基础。     

聚氨基酸胶束在肿瘤治疗中的研究进展

聚氨基酸胶束是一类新型的聚合物胶束,具有高效、长效及药量高载等特点。抗肿瘤药物通过化学结合或物理包裹的方式进入胶束内部,具有较高的稳定性。同时,在EPR(Enhanced Permeability and Retention,EPR)效应作用下,聚氨基酸胶束纳米粒可以有效地蓄积于肿瘤组织中达到很好的抗肿瘤效果,并减少药物在其他正常组织中的分布而降低毒副作用。本文综述了聚天冬氨酸、聚谷氨酸和聚赖氨酸三类聚氨基酸胶束作为抗肿瘤药物载体的理化性质、特点及其相关临床研究进展,对各个研究实例进行简要介绍。     

载吉西他滨介孔二氧化硅纳米粒的制备及其抗肿瘤活性评价

目的：制备载吉西他滨(gemcitabine,GemC)的介孔二氧化硅纳米粒(MSN),并对其体内外抗肿瘤活性进行评价。方法：采用聚合法制备了GemC-MSN,采用激光粒度仪测定了纳米粒的粒度分布和电位,并通过透射电镜对纳米粒的形态进行了表征。应用紫外可见分光光度法评价了纳米粒的载药量、包封率及体外释放特性。采用MTT染色法,考察了GemC-MSN对A549细胞的体外细胞毒性。建立了体内肿瘤动物模型,评价纳米粒的体内抗肿瘤活性。结果：纳米粒分布均一,平均粒径为107.29 nm,PDI为0. 167,Zeta电位为0.107mV;药物的载药量和包封率分别为(37.31±1.25)%和(87.37±2.12)%;体外释放结果显示,纳米粒具有一定的缓释作用,96h时释放达到平衡;体内外抗肿瘤试验结果表明,GemC-MSN较游离GemC具有更强的抗肿瘤活性。结论：MSN作为药物的新型载体,具有良好的生物相容性,并能显著提高GemC的载药量,控制药物的缓慢释放,能显著提高GemC的体内外抗肿瘤活性,将为GemC新型给药系统的深入研究提供参考。     

阿霉素-五味子乙素pH敏感聚合物胶束的制备与制剂学性质

目的以p H敏感聚合物聚乙二醇-聚乳酸-聚组氨酸[poly(ethyleneglyco1)-poly(D,L-lactide)-poly(L-histidine),m PEG-PLA-PHis]胶束为载体,联合包载抗肿瘤药物阿霉素与多药耐药逆转剂五味子乙素制备聚合物胶束,并对其制剂学性质进行研究。方法采用薄膜分散法制备阿霉素-五味子乙素p H敏感聚合物胶束,以包封率、载药量和稳定性(载药胶束24 h的包封率和载药量变化)为评价指标,采用单因素试验及Box-Behnken效应面法筛选最优处方;应用透射电子显微镜观察载药胶束的外观形态,动态光散射法测定载药胶束的粒径及zeta电位;透析法考察载药胶束在不同p H条件下的释药行为。结果制备的阿霉素-五味子乙素p H敏感聚合物胶束平均粒径为64.73 nm,zeta电位为-8.7 m V。最优处方中阿霉素包封率为95.3%,载药量为8.7%,五味子乙素包封率为76.1%,载药量为3.4%,载药胶束稳定性较好。体外释放结果表明,所制备的阿霉素-五味子乙素p H敏感聚合物胶束在弱酸性条件下,药物释放速率明显加快。结论采用星点设计-效应面法优化处方与制备工艺,所制备的阿霉素-五味子乙素p H敏感聚合物胶束粒径分布均匀,包封率和载药量良好,具有明显的p H响应行为。     

聚L-乳酸与聚乙二醇-b-聚L-乳酸电纺纤维中布洛芬的释放

目的制备并评价包载有布洛芬的聚L-乳酸和聚乙二醇嵌段聚L-乳酸电纺纤维毡。方法静电纺丝法制备包载布洛芬的纤维毡,扫描电子显微镜观察纤维形态,广角X射线衍射扫描观察纤维表面结晶状态,差示扫描量热评价药物在高分子材料中的结合状态。高效液相色谱测定体外酶降解药物释放结果。结果纤维直径在0.3～1.5μm之间,表面光滑无结晶态物质析出,X射线衍射没有发现布洛芬特征峰出现。差示扫描量热结果显示布洛芬的加入使纤维的玻璃化温度降低。药物释放结果显示相对于纯聚乳酸材料,当载体材料为聚乙二醇嵌段的聚乳酸时,纤维中的布洛芬呈现更快的释放速率,聚乙二醇有利于释放介质中酶对纤维的作用。20%载药量纤维中的药物释放速率大于10%载药量纤维中的药物释放速率,蛋白酶K加快药物的释放速率。结论成功制得布洛芬电纺纤维,药物被较好包裹在纤维内部,聚乳酸电纺纤维中聚乙二醇嵌段对布洛芬释放行为有明显的影响,需要在进一步研究中注意。     

基于碳纳米角的荧光标记载药体系的构建、表征及体外释放

目的考察碳纳米角(CNHox)经不同浓度壳聚糖(CS)修饰后在水和磷酸缓冲溶液(PBS)中的分散性;构建以CNHox为载体装载多烯紫杉醇(DTX)并通过CS连接CdTe量子点荧光探针的载药体系;考察载药体系体外药物释放行为。方法测定经不同浓度CS修饰的CNHox在水中和PBS中的zeta电位,静置观察分散液的聚沉情况;饱和溶液结晶法装载DTX,热重法测定载药量;以EDC、NHS为交联剂共价连接CdTe量子点与CS,修饰到载有DTX的CNHox表面,构建新型荧光标记抗肿瘤载药体系,对该载药体系进行系列表征;考察药物体外溶出情况,高效液相色谱法测定溶出DTX的含量,并绘制体外释放曲线。结果 CS浓度达到2.50mg·mL-1时,CNHox在PBS中的分散性良好;CNHox装载DTX载药量可以达到1.170 mg·mg-1;载药体系成功标记荧光量子点,且在PBS中的分散性良好;体外累积药物释放量为54.8%,释放曲线有明显缓释行为。结论 CS修饰可提高CNHox在盐溶液中的分散性;载药体系具有较高载药量;量子点成功标记载药体系,为该载药体系的体外示踪研究奠定基础;该载药体系的体外释放试验证实具有缓释效果。     

新型顺铂给药系统的制备及其生物活性评价

以生物发酵法获得的γ-聚谷氨酸经酸降解后得到小分子γ-聚谷氨酸(γ-PGA),柠檬酸(CA)通过酯化反应修饰其侧链羧基制备γ-聚谷氨酸-柠檬酸(γ-PGA-CA),再将顺铂(CDDP)与γ-PGA-CA上的羧基相结合制备得载药复合物γ-PGA-CA-CDDP,凝胶色谱法测定分子量,OPDA法测定载药量及有效结合率,透析法结合HPLC研究其对顺铂的缓释效果,MTT法检测复合物的体外抗肿瘤活性,采用新鲜兔血检测载体材料及复合物的溶血性。实验结果表明:成功获得γ-PGA-CA及γ-PGA-CA-CDDP,该复合物相对分子质量约为66k,有效结合率达65%,载药率达16%～20%,48 h时顺铂的累计释放率达到50%,MTT检测显示该复合物对肿瘤细胞具有良好的抑制效果,相对于游离顺铂具有较低的毒性,且无溶血性。因此,γ-PGA-CA可作为药物载体,γ-PGA-CA-CDDP具有潜在的临床应用价值。     

对乙酰氨基酚/MCM-41载药体系的制备及其缓释性能的研究

目的制备对乙酰氨基酚/MCM-41缓释体系,并考察其体外释放行为。方法在碱性和室温条件下制备MCM-41介孔材料;采用浸渍法将解热镇痛药物对乙酰氨基酚组装到MCM-41的孔道中,通过XRD、IR、低温N2吸附对MCM-41介孔材料及药物组装体进行表征;测定组装体的载药量、载药时间以及在人工胃液中的释放行为。结果介孔材料MCM-41作为药物载体具有较短的载药时间(7 h)、较大的载药量(46.65%)、较低的释放速率(6 h仅释放35.6%)。结论通过测定组装体在体外模拟人工胃液和人工肠液中的释放速率,结果表明制得了对乙酰氨基酚/MCM-41缓释释放体系。     

用HPLC法测定大鼠血浆中吉西他滨的浓度

目的：建立HPLC法测定大鼠血浆中吉西他滨的浓度。方法：采用Phenomenex色谱柱,以5-溴尿嘧啶为内标,流动相为乙腈-0.1%三氟乙酸溶液（3∶97）,流速1.0 mL/min,检测波长268 nm。结果：大鼠血浆中吉西他滨浓度在1-200μg/mL范围内线性关系良好,方法回收率〉97%,提取回收率〉72%,日内、日间RSD均〈6%。结论：本法快速、高效、灵敏,适用于吉西他滨的血药浓度测定。     

吉西他滨联合用药治疗乳腺癌的研究进展

吉西他滨属于嘧啶类抗肿瘤药物,在乳腺癌的治疗中,与其他药物联合应用,可以起到很好的治疗各类乳腺癌的效果。本文对吉西他滨联合其他药物治疗乳腺癌的研究进展进行综述。     

The development of orally administrable gemcitabine prodrugs with d-enantiomer amino acids: Enhanced membrane permeability and enzymatic stability

Gemcitabine prodrugs with d- and l-configuration amino acids were synthesized and their chemical stability in buffers, resistance to glycosidic bond metabolism, enzymatic activation, permeability in Caco-2 cells and mouse intestinal membrane, anti-proliferation activity in cancer cell were determined and compared to that of parent drug, gemcitabine. Prodrugs containing d-configuration amino acids were enzymatically more stable than ones with l-configuration amino acids. The activation of all gemcitabine prodrugs was 1.3–17.6-fold faster in cancer cell homogenate than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of amino acid monoester prodrugs containing d-configuration amino acids in cell homogenates was 2.2–10.9-fold slower than one of amino acid monoester prodrugs with l-configuration amino acids. All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent gemcitabine. Gemcitabine prodrugs showed superior the effective permeability in mouse jejunum to gemcitabine. More importantly, the high plasma concentration of d-amino acid gemcitabine prodrugs was observed more than one of l-amino acid gemcitabine prodrugs. In general, the 5′-mono-amino acid monoester gemcitabine prodrugs exhibited higher permeability and uptake than their parent drug, gemcitabine. Cell proliferation assays in AsPC-1 pancreatic ductal cell line indicated that gemcitabine prodrugs were more potent than their parent drug, gemcitabine. The transport and enzymatic profiles of 5′-d-valyl-gemcitabine and 5′-d-phenylalanyl-gemcitabine suggest their potential for increased oral uptake and delayed enzymatic bioconversion as well as enhanced uptake and cytotoxic activity in cancer cells, would facilitate the development of oral dosage form for anti-cancer agents and, hence, improve the quality of life for the cancer patients.     

Low molecular weight proteins as carriers for renal drug targeting. Preparation of drug-protein conjugates and drug-spacer derivatives and their catabolism in renal cortex homogenates and lysosomal lysates.

Low molecular weight proteins (LMWPs) are known to be reabsorbed and catabolized primarily by the proximal tubular cells of the kidneys. As such, LMWPs might serve as drug carriers that release drugs site-specifically in the kidney. We tested this concept in vitro by coupling different drugs to the LMWP lysozyme both directly (amide bond) and via different spacers: oligopeptides (amide bond), (poly-)alpha-hydroxy acids (ester bond), and a pH sensitive cis-aconityl spacer (amide bond). The capability of the kidney to release the parent drug from such drug-spacer derivatives and drug-LMWP conjugates by enzymatic or chemical hydrolysis of the bond was tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Directly coupled conjugates of terminal carboxyl group containing drugs and lysozyme were catabolized to single amino acids, but did not result in release of the parent drug. The amide bond between the drug and the final amino acid (lysine) appeared to be stable in the incubation milieu. Different oligopeptide spacers coupled to the drugs showed similar results: the oligopeptide itself was cleaved but the amide bond between the drug and different single amino acids remained untouched. Only amide bonds of derivatives of carboxylic drugs with peptide structures themselves were cleaved. Some of the directly coupled conjugates of terminal amino drugs and oligopeptides showed clear release of the parent drug whereas others were stable. Terminal amino drugs were rapidly released from an acid-sensitive cis-aconityl spacer. Terminal carboxyl group containing drugs were enzymatically released from their glycolic and lactic ester spacers at different rates. These kinds of drugs were also released as parent drug from LMWP conjugates with ester spacers like L-lactic acid. Increasing spacer length by intercalating a tetra(L-lactic acid) molecular between the drug and the protein further increased the extent and rate of drug release, indicating increased accessibility of the bond to the enzymes. Terminal amino group containing drugs were rapidly generated as parent drug from LMWP conjugates using an acid-sensitive spacer. In addition the conjugates were found to be adequately stable in plasma, considering their rapid clearance from the bloodstream. It is concluded that LMWPs may indeed be of use as carriers for specific renal delivery of drugs, since renal cortex homogenates and lysosomal lysates are able to catabolize the protein and generate the parent drug from drug-LMWP conjugates bearing suitable spacers. The option of enzymatic release is limited by the narrow specificity of the lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of new compounds derived from metronidazole and amino acids and their esters as antiparasitic agents

A number of new compounds derived from metronidazole and amino acids and their esters have been synthesized through a reaction between 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetic acid and a number of amino acid esters in the presence of N,N′carbonyldiimidazole (CDI). Hydrolysis of the esters derivatives with sodium hydroxide (4%) followed by acidification with hydrochloric acid (3?M) afforded the corresponding acids. The newly synthesized compounds were characterized by elemental analysis and by spectroscopic techniques such as ¹H-NMR, ¹³C-NMR, and mass spectrometry. Some of the prepared compounds exhibited lethal activity against pathogenic protozoan parasites.     

Synthesis and in vitro characterization of novel dextran-methylprednisolone conjugates with peptide linkers: effects of linker length on hydrolytic and enzymatic release of methylprednisolone and its peptidyl intermediates

To control the rate of release of methylprednisolone (MP) in lysosomes, new dextran-MP conjugates with peptide linkers were synthesized and characterized. Methylprednisolone succinate (MPS) was attached to dextran 25 kDa using linkers with 1-5 Gly residues. The release characteristics of the conjugates in pH 4.0 and 7.4 buffers, blood, liver lysosomes, and various lysosomal proteinases were determined using a size-exclusion and/or a newly developed reversed-phase HPLC method capable of simultaneous quantitation of MP, MPS, and all five possible MPS-peptidyl intermediates. We synthesized conjugates with >or=90% purity and 6.9-9.5% (w/w) degree of MP substitution. The conjugates were stable at pH 4.0, but released MP and intact MPS-peptidyl intermediates in the pH 7.4 buffer and rat blood, with faster degradation rates for longer linkers. Rat lysosomal fractions degraded the conjugates to MP and all the possible intermediates also at a rate directly proportional to the length of the peptide. Whereas the degradation of the conjugates by cysteine peptidases (papain or cathepsin B) was relatively substantial, no degradation was observed in the presence of aspartic (cathepsin D) or serine (trypsin) proteinases, which do not cleave peptide bonds with Gly. These newly developed dextran conjugates of MP show promise for controlled delivery of MP in lysosomes.     

聚氨基酸胶束作为肿瘤靶向药物载体的研究进展

聚氨基酸作为一种毒副作用低、生物相容性好的高分子材料,被广泛应用于肿瘤以及基因治疗。聚氨基酸链的活性基团丰富,可通过多种反应途径与目的基团连接,从而实现药物的主动靶向性。同时又因为聚合物胶束的粒径为1～100纳米,而肿瘤组织毛细血管壁与正常组织血管壁相比间隙较宽,可以形成“渗透滞留”效应（EPR效应）,使载药纳米粒在肿瘤组织中不断蓄积,进而实现药物在肿瘤中的被动靶向性,本文简要综述了载药聚天冬氨酸、聚谷氨酸以及聚赖氨酸聚合物胶束的理化性质及优势,如肿瘤靶向性、缓释性等,并对近年来聚氨基酸胶束的研究进展进行综述。     

Tumor-targeted and activated bioconjugates for improved camptothecin delivery

Earlier reports from our laboratory described bioconjugates of camptothecin (CPT) for tumor targeting. In the current work, the rate and site of CPT release from the bioconjugates were modulated using increasingly sterically hindered amino acids and cysteine proteinase-sensitive peptide linkers, respectively. Polyethylene glycol served as a spacer/scaffold between CPT and folic acid. The folic acid receptor, overexpressed on many cancer cells, was targeted using folate. The delivery system was tested in vitro for hydrolytic stability, enzyme-mediated cleavage, cytotoxicity and targeting potential. The linkers successfully modulated the hydrolysis rate (around 1--100 h) and potential site (tumor microenvironment) of CPT release. Preliminary molecular modeling approaches were utilized to assess the influence of molecular volume on hydrolysis half-life (i.e. CPT release). There was a clear, but non-linear, relationship between in vitro CPT release and increasing steric hindrance offered by the peptide linker. The efficacy of four conjugates was studied in a syngeneic rat breast cancer model. Histopathological analysis on treated tumors was performed to evaluate disease prognosis. The results demonstrate that programmed bioconjugates may provide superior efficacy and greater control over the rate and site of CPT release, resulting in higher anti-tumor efficacy and lower toxicity.     

Poly(N-acryl amino acids): a new class of biologically active polyanions

Poly(N-acryl amino acids) bearing side groups with a lipophilic character or having charged functional groups (i.e. -NH(2), -COOH, -SH, -OH, and phenols) were synthesized from the radical polymerization of N-acryl amino acid monomers. Monomers were prepared from the reaction of acryloyl chloride and amino acid esters in dry solvents. Polymers of a broad molecular weight ranging from 3 000 to 60 000 Da were obtained. The polymers were optically active, and their structures were confirmed by (1)H NMR and IR spectra and elemental analysis. Hydroxyl-containing polymers were sulfated in high conversion yields by SO(3)/pyridine complex. The newly synthesized linear homopolyanions were tested for heparin-like activities: (i) inhibition of heparanase enzyme, (ii) release of basic fibroblast growth factor (bFGF) from the extracellular matrix (ECM), and (iii) inhibition of smooth muscle cell (SMC) proliferation. Polymers based on tyrosine and leucine were highly active in all three tests (microgram level). Polymers based on phenylalanine, tert-leucine, and proline were active as heparanase inhibitors and FGF release, and polymers of trans-hydroxyproline, glycine, and serine were active only as heparanase inhibitors. The polymer of cis-hydroxyproline was inactive. It was found that a net anionic charge (i.e. carboxylic acid) is essential for biological activity. Thus, methyl ester derivatives of the active polymers, zwitterionic amino acid dependent groups (lysine, histidine), and decarboxylated amino acids (tyramine, ethanolamine) were inactive. The above active polymers did not exhibit anticoagulation activity which is considered the main limitation of heparin and heparinomimetics for clinical use. These synthetic poly(N-acryl amino acids) may have potential use in the inhibition of heparanase-mediated degradation of basement membranes associated with tumor metastasis, inflammation, and autoimmunity.     

Improvement of physicochemical and biopharmaceutical properties of theophylline by poly(ethylene glycol) conjugates

In the present paper two theophylline esters with poly (ethylene glycol) (PEG) and methoxy poly (ethylene glycol) (mPEG) were prepared. Quantitative yields of the pure products were obtained. Unlike the free drug, the drug-polymer conjugates are freely water-soluble at room temperature. In vitro release experiments in aqueous buffer demonstrate that both conjugates are stable in buffer of pH 7.4 and 1.2. In vivo release studies after oral administration of theophylline conjugates demonstrate a good release of parent drug.     

Chemical and enzymatic stability evaluation of lipoamino acid esters of idebenone.

Lipophilic conjugates of idebenone (IDE) with short-chain alkylamino acids were previously synthesized and evaluated in vitro for their antioxidant properties. In this study, their susceptibility to chemical and enzymatic hydrolysis was evaluated. Results indicated that these derivatives release the parent drug quantitatively via enzymatic hydrolysis by serum and liver esterases, with a cleavage rate related to the length of the alkyl side chain. Consequently, the present lipoamino acid conjugates of IDE are prodrugs and their in vivo effects are mediated through the parent compound released in the body.     

Investigation of targeting mechanism of new dextran-peptide-methotrexate conjugates using biodistribution study in matrix-metalloproteinase-overexpressing tumor xenograft model

We conducted a biodistribution study in HT-1080 bearing mice to investigate the drug targeting mechanism and the cause of side effects of the new dextran-peptide-methotrexate conjugates. HT-1080 is a human fibrosarcoma cell line that is known to overexpress matrix-metalloproteinases (MMPs). The experiments compared conjugates carrying MMP sensitive peptide linkers, conjugates carrying MMP insensitive linkers, and free methotrexate. Passive targeting was evidenced by the prolonged plasma circulation and higher tissue accumulations of both types of the conjugates compared to free methotrexate. Independent of the peptide sequence of the linker, the ratio of drug accumulation at the tumor versus drug accumulation at the major site of side effects (small intestine) for either conjugate was increased by the effect of enhance permeation and retention (EPR). The conjugate released a sufficient amount of peptidyl methotrexate to cause inhibition of tumor growth. There was no significant difference in drug accumulation at the tumor site between the MMP-sensitive and the MMP-insensitive conjugates. We concluded that the tumor targeting effect of the dextran-peptide-methotrexate conjugate was dominantly due to passive targeting and EPR. The difference in the systemic side effects observed for conjugates with different linkers could probably be attributed to their varying susceptibility towards enzymes in normal tissues.