反义药物的研究进展

反义药物是通过基因复制、转录、剪接、转运、翻译等水平上调节靶基因的表达 ,干扰遗传信息从核酸向蛋白质的传递 ,阻止疾病相关蛋白的生物合成 ,从而发挥其反义作用。本文就反义药物的最新进展 ,从反义药物的作用机制及临床研究两方面进行综述     

反义药物

反义药物一般指特定基因(如mRNA)为靶点，合成与之特异性互补结合的一段约由20个核苷酸组成的人工合成寡核苷酸即反义核苷酸(antisense oligonucleotide，ASON)。ASON可进行人工大规模合成和化学修饰，具有特异、高效的药理作用，便于进行药物合理设计等特性。根据核酸互补杂交原理特异性抑制特定基因表达的反义技术，可用于研究和开发以疾病相关基因为靶标的反义药物。以基因为靶向的反义药物是抗肿瘤新药研究和开发的重要方向之一j     

反义药物中国专利战略研究

利用中国专利数据库进行了反义药物的专利数据收集和法律状态的信息检索,应用词频分析技术,对于近年来中国内地有关反义药物的专利战略进行了分析和总结,并对于未来反义药物专利的发展趋势进行了初步的预测。     

bcl-2反义肽核酸诱导HL-60 K562细胞凋亡

目的　探讨不同结构的反义药物对HL 60、K562细胞系生物学活性的影响。方法　应用细胞计数、细胞形态观察和流式细胞术观察并比较反义肽核酸和反义寡核苷酸对白血病细胞HL 60、K562生物学活性的影响。结果　 1 0 μmol/L靶向bcl 2mRNA蛋白编码区的反义肽核酸能有效地抑制HL 60、K562细胞的生长、下调bcl 2蛋白的水平及诱导细胞凋亡。 1 0 μmol/L针对bcl 2mRNA同样靶点的反义肽核酸和寡核苷酸作用HL 60和K562细胞 72h ,反义肽核酸仍表现出较强的促细胞凋亡的作用 ,而反义寡核苷酸诱导细胞凋亡的作用则有所下降。结论　反义bcl 2肽核酸能诱导HL 60、K562细胞的凋亡 ,比反义寡核苷酸有更好的反义作用。     

抗肿瘤反义寡核苷酸药物靶标的研究进展

肿瘤是一种多基因相关疾病，反义寡核苷酸药物可以特异性地与靶基因mRNA互补结合，封闭靶基因的表达，在肿瘤的基因治疗中独树一帜。现已发现许多基因在调节细胞增殖和衰老、细胞凋亡、细胞周期、血管生成及信号转导等方面起着重要作用，并证实它们在抗肿瘤反义治疗中可以作为有效的分子靶标。本文综述了近年来发现的抗肿瘤反义寡核苷酸的治疗靶点，并提出了多靶点反义寡核苷酸联合应用治疗肿瘤的新观念。     

反义寡核苷酸类药物的研究进展

反义寡核苷酸类药物通过与靶mRNA的相互作用调节目的基因的表达，它必须具备三个首要条件，即稳定性，选择性和通透靶向性，各种化学修饰包括第二代，第三代寡核苷酸明显增强了反义寡核苷酸的稳定性；靶序列选择方法的改进为其发挥高度选择性提供基础；各种靶向转运技术的应用大大增强了对作用部位的通透性和靶向性，这些方面的研究进展开阔了反义寡核苷酸类药物的应用领域。     

定量构效关系方程对靶向蛋白激酶C-α mRNA的反义药物的活性预测

以往研究对34个针对蛋白激酶C-α mRNA二级结构设计的反义药物（AS-ODNs）进行了定量构效关系（QSAR）分析并得到QSAR方程. 本研究再次设计10个AS-ODNs验证QSAR方程对药物活性预测的可靠性．结果显示其中4个AS-ODNs IC₅₀值明显低于阳性对照ISIS3521（P<0.05）. 8个AS-ODNs与ISIS3521的实测IC₅₀与QSAR方程预测相符,实测与预测IC₅₀之间相关系数为0.76(P<0.05）. 两个AS-ODNs,AP1261(20)和AP0186(20)的实测IC₅₀与预测不符. 结果提 示QSAR方程一定程度上反映了AS ODNs活性与靶结构的关系,对预测反义药物生物活性有益. 但QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计.     

脂质体Geneshuttle20对STAT6反义核酸转染小鼠脾淋巴细胞影响的研究

目的探讨阳离子脂质体Geneshuttle20对STAT6反义核酸在小鼠脾淋巴细胞摄入、分布的影响作用。方法采用阳离子脂质体介导STAT6反义核酸转染小鼠脾淋巴细胞,应用流式细胞仪、荧光显微镜分别观察反义核酸的细胞摄入和胞内分布。结果反义核苷酸与脂质体(W/W)为2∶4时,细胞摄入率可以达到67.7%,较单独加入反义核酸提高转染效率6倍,细胞内平均荧光强度最强,细胞核染色较深。结论Geneshuttle20提高了细胞对反义寡核苷酸的摄取,促使其进入细胞核内发挥作用。     

反义寡核苷酸药理的研究现状

人工合成的反义寡核苷酸抑制相关基因的转录和翻译，对许多涉及基因表达的疾病有一定的治疗作用。对反义寡核苷酸的药动学和药效学，不良反应已有较多的临床前研究，其对肿瘤，病毒感染等的临床试验初步结果，都显示了它的应用前景。     

Changing therapeutic paradigms in CMV retinitis in AIDS

Cytomegalovirus (CMV) retinitis is a common ocular complication of immunosuppression. The management of CMV retinitis has been continuously evolving over the last decade. The mainstay of therapy remains ganciclovir and foscarnet. However, increasing resistance and ongoing toxicities to these agents remain a challenge. Additional frequently utilised agents include cidofovir and fomivirsen. The advent of highly active antiretroviral therapy (HAART) has allowed the restoration of immunocompetency to many patients previously challenged by CMV infection. In some circumstances, HAART has even eliminated the need for ongoing antiviral therapy. This paper reviews the current treatment modalities, including their toxicities and dosing recommendations.     

The potential of clusterin inhibiting antisense oligodeoxynucleotide therapy for prostate cancer

This review summarise the authors’ recent experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy that targets a cytoprotective gene, clusterin, for the treatment of prostate cancer. The acquisition of resistance to a wide variety of proapototic stimuli was initially demonstrated by introducing the clusterin gene into prostate cancer cells. Furthermore, silencing clusterin expression using AS ODN synergistically enhanced the effects of several conventional therapeutic modalities through the effective induction of apoptosis in prostate cancer xenograft models. Based on these outcomes, Phase I clinical trials were conducted using AS clusterin ODN incorporating 2′-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), and the optimal dose of OGX-011 capable of inducing ≤ 90% suppression of clusterin in human prostate cancer tissue was determined. Collectively, these findings suggest the utility of inactivating clusterin function using AS ODN technology as a novel therapeutic strategy for prostate cancer treatment. There have been four kinds of Phase II studies that have begun to further evaluate the efficacy of OGX-011 in patients with prostate, breast and lung cancers.     

Integrated analysis of preclinical data to support the design of the first in man study of LY2181308, a second generation antisense oligonucleotide

AIMS

To predict the concentration and target inhibition profiles of the survivin inhibitor antisense oligonucleotide LY2181308 in humans.

METHODS

An indirect pharmacokinetic/pharmacodynamic (PK/PD) model was built to predict the inhibition of survivin mRNA and protein in humans following LY2181308 dosing. Plasma and tissue PK data from cynomolgus monkeys were analyzed by non-linear mixed effect modelling techniques. Human PK parameters were predicted using allometric scaling. Assumptions about the pharmacodynamic parameters were made based upon the target and tumour growth inhibition data from mouse xenograft models. This enabled the prediction of the clinical PK/PD profiles.

RESULTS

Following a 750 mg dose, LY2181308 tumour concentrations ranging from 18.8 to 54 µg g⁻¹ were predicted to lead to 50 to 90% target inhibition. In humans, LY2181308 tumour concentrations from 13.9 to 52.8 µg g⁻¹ (n = 4, LY2181308 750 mg) were observed associated with a median survivin mRNA and protein inhibition of 20% ± 34 (SD) (n = 9) and 23% ± 63 (SD) (n = 10), respectively. The human PK parameters were adequately estimated: central V_d, 4.09 l (90% CI, 3.6, 4.95), distribution clearances, 2.54 (2.36, 2.71), 0.0608 (0.033, 0.6) and 1.67 (1.07, 2.00) l h⁻¹, peripheral V_ds, 25 900 (19 070, 37 200), 0.936 (0.745, 2.07) and 2.51 (1.01, 2.922) l, mean elimination clearance 23.1 l h⁻¹ (5.6, 33.4) and mean terminal half-life, 32.7 days (range 22–52 days).

CONCLUSION

The model reasonably predicted LY2181308 PK in humans. Overall, the integration of preclinical PK/PD data enabled to appropriately predict dose and dosing regimen of LY2181308 in humans with pharmacologically relevant survivin inhibition achieved at 750 mg.     

反义寡核苷酸类化合物的体内转运研究进展

反义寡聚核苷酸已成功用于对基因表达的特异性调控与抑制。但是,大多数此类化合物是呈负电性的,不能有效跨越细胞膜。到目前为止,已发展了数种提高寡聚核苷酸类化合物细胞内转运的方法。现对提高寡聚核苷酸细胞内转运的载体和方法作一较详尽的归纳。     

反义寡核苷酸药代动力学研究中的定量分析技术

反义寡核苷酸药物的药代动力学研究要求具备相应的生物定量方法。本文就近年来在临床前和临床药代动力学评价中常用的几种定量方法，包括放射性同位素法、毛细管凝胶电泳法、高效液相色谱法、液质联用和基于杂交技术的酶联免疫法，对其各自的特性、应用前景、操作方法及局限性等方面进行了综述。     

反义核酸药物脂质体肝靶向性的研究

目的研究反义核酸药物脂质体给药系统的肝主动靶向性。方法采用薄膜分散法制备脂质体,以花青染料荧光标记脂质体,以荧光分光光度法定量分析;利用肝组织的体内冷冻切片和小鼠体内组织分布研究肝靶向性;检测大鼠肝非实质细胞与肝实质细胞的荧光强度(INPC和IPC)。结果主动靶向脂质体在肝脏中的荧光强度明显强于非主动靶向脂质体,相对摄取率(re)为5.64;非主动靶向脂质体的荧光值比(IPC/INPC)为1.16±0.89,主动靶向脂质体的IPC/INPC为8.24±1.37。结论主动靶向脂质体具有良好的肝实质细胞靶向性和肝靶向性。     

反义寡核苷酸治疗乳腺癌研究进展(英文)

乳腺癌是一类与多基因相关的恶性肿瘤,有些痛基因如HER-2(c-erbB-2,Neu),bcl-2/6cl-xL,蛋白激酶A(PKA),运铁蛋白受体基因(TfR gene)等的过度表达对乳腺癌的预后有明显影响,有证据表明抑制上述过度表达基因能明显改善乳腺痛的治疗效果。近年,反义治疗这种能抑制特定癌基因过度表达的有效手段被应用于乳腺癌的治疗。研究表明,在多数情况下,反义寡核苷酸(ON)能在mRNA或蛋白水平抑制目的基因的表达,有些反义ON在体和离体对乳腺癌细胞系或动物乳腺癌异植肿瘤均显示出令人鼓舞的治疗效果。此外,反义ON与常规化疗药物合用也能产生协同的抗肿瘤作用。反义ON与化疗药物合用可能在不远的将来是治疗乳腺癌的最佳方法之一。     

反义寡核苷酸聚甲基丙烯酸酯亚微粒入胞行为的研究反义寡核苷酸聚甲基丙烯酸酯亚微粒入胞行为的研究

反义寡核苷酸(antisense oligodeoxynucleotides,AS-ODN)是一种能与特定的DNA或RNA特异性结合并阻断基因表达的生物效应分子,具有作用强、特异性高等优点,受到广泛关注[1]。然而由于AS-ODN为亲水性大分子,细胞通透性差,使得它的应用受到极大限制。为了增加AS-ODN在体内的稳定性     

Clinical trials of a new class of therapeutic agents: antisense oligonucleotides

Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.