Effect of benzalkonium chloride on percutaneous absoption of antisense phosphorothioate oligonucleotides

The effect of benzalkonium chloride on skin permeability of partially modified antisense phosphorothioate oligonucleotides (PS-ODN), which are designed as scar formation inhibitor, was investigated using Franz Diffusion Cell. When the concentration ratio of PS-ODN-quarternary ammonium salt complex is more than 1∶100, the apparent partition coefficient (APC) of each complex was increased in the following order; tetraphenyl phosphonium chloride (TPP)<cetyltrimethyl ammonium bromide (CTAB)<benzalkonium chloride (BZ). The permeability of PS-ODN through the rat skin increased in the presence of BZ. The fluxs of PS-ODN with BZ were increased by addition of Pluronic F 68 or Triton X-100 to phosphate buffered saline (PBS), respectively. When the mole ratio of PS-ODN to BZ is 1∶10, the fluxs penetrated of PS-ODN with BZ was greatest. The increase of the permeability in the presence of BZ might be due to the formation of lipophilic ion-pair complex between PS-ODN and BZ. By regulation of mole ratio of PS-ODN to BZ, the development of topical dosage forms using PS-ODN as scar formation inhibitor will be possible with minimal systemic exposure.     

Increased efficacy of antileishmanial antisense phosphorothioate oligonucleotides in Leishmania amazonensis overexpressing ribonuclease H

Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.     

Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides.

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.     

Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides

Phosphorothioate (PS) oligodeoxynucleotides represent the class of antisense drugs most advanced in development and clinical testing. Exploitation of antisense oligonucleotide technology for development of rationally designed therapeutic drugs has presented a unique set of challenges, some of which relate to their pharmacokinetic behavior in vivo. Pharmacokinetic studies of PS oligodeoxynucleotides demonstrate that they are well absorbed from parenteral sites, rapidly distributed broadly to all peripheral tissues, do not cross the blood-brain barrier, and are eliminated primarily by slow metabolism in tissues. In general, the pharmacokinetic properties of this class of compounds appear to be largely driven by chemistry rather than sequence.     

Target validation and functional analyses using antisense oligonucleotides

The human genome project (HGP) has been described as the single most important project in biology and the biomedical sciences to date. In February 2001, the efforts of the HGP resulted in the publication of a 'working draft' of the entire human genome and it is expected that final sequencing and annotation of the genome will be completed by 2003. Researchers are now focusing efforts on the identification of the function of the reported 30,000 human genes. During the past few years, antisense oligomers have been widely used as potent tools for functional genomics and drug target validation. This article describes the emerging and established antisense technologies that will be used to continue the efforts to unlock the function of the human genome and to discover novel drug targets for the treatment of human diseases.     

Curcumin synergistically augments bcr/abl phosphorothioate antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells

AIM: To investigate the growth inhibition effect of the combination of bcr/abl phosphorothioate antisense oligonucleotides (PS-ASODN) and curcumin (cur), and the possible mechanisms of cur on the chronic myelogenous leukemia cell line K562. METHODS: The K562 cell line was used as a P210( bcr/abl )-positive cell model in vitro and was exposed to different concentrations of PS-ASODN (0-20 micromol/L), cur (0-20 micromol/L), or a combination of both. Growth inhibition and apoptosis of K562 cells were assessed by MTT assay and AO/EB fluorescent staining, respectively. The expression levels of P210( bcr/abl ), NF-kappaB and heat shock protein 90 (Hsp90) were assessed by Western blot. RESULTS: Exposure to cur (5-20 micromol/L) and PSASODN (5-20 micromol/L) resulted in a synergistic inhibitory effect on cell growth. Growth inhibition was associated with the inhibition of the proliferation and induction of apoptosis. Western blot analysis showed that the drugs synergistically downregulated the level of P210( bcr/abl ) and NF-kappaB. Cur downregulated Hsp90, whereas no synergism was observed when cur was combined with PS-ASODN. CONCLUSION: PS-ASODN and cur exhibited a synergistic inhibitory effect on the cell growth of K562. The synergistic growth inhibition was mediated through different mechanisms that involved the inhibition of P210( bcr/abl ).     

Percutaneous absorption of antisense phosphorothioate oligonucleotidein vitro

Antisense oligonucleotides seem to provide a promising new tool for the therapy. Choiet al. (1995) reported antisense phosphorothioate oligonucleotides (PS-ODN, 25 mer) complementary to TGF-β mRNA designed for scar formation inhibitor to eliminate scars, which was caused by undesired collagen deposition due to overexpression of TGF-β, in wounded skin. PS-ODN were evaluatedin vitro for skin penetration using normal and tape-stripped damaged rat skin. Thein vitro skin transports were carried out with partially modified PS-ODN (6S) and fully modified PS-ODN (25S). The cumulative amount of PS-ODN (6S) penetrated through normal rat skin was 0.234±0.041 μg/cm² and that of tape-stripped damaged rat skin was 1.077±0.301 μg/cm² over 8 hrs. PS-ODN (25S) can not be found in receptor medium through normal skin due to high molecular weight (Mol.Wt.=8,000) and polyanionic charge. However, the cumulative amount of PS-ODN (25S) penetrated across damaged rat skin in PBS was 0.340±0.296 μg/cm² over 8 hrs. The absense of dermis raised the cumulative amount of PS-ODN (6S) penetrated through rat skin. And the fluxes of PS-ODN (6S) and PS-ODN (25S) at 8hrs across damaged rat skin were 134.63±37.67 ng/cm² h, and 42.50±36.95 ng/cm² h, respectively. While PS-ODN (25S) was stable in 10% heat inactivated fetal bovine serum (FBS) during 24 hrs, PS-ODN (6S) was less stable than PS-ODN (25S), but was markedly stable than unmodified phosphodiester. It is suggested that the cumulative amount of PS-ODN (6S) penetrated through damaged rat skin is larger than that of PS-ODN (25S) since the former is easier to degrade by nuclease than the latter and then is apt to penetrate into skin. Thus, PS-ODN represents a logical candidate for further evaluation due to the potential for delivery into the wounded skin.     

卡维地洛对映体的抗心肌细胞凋亡作用

目的:研究卡维地洛(carvedilol)R(+)和S(-)型对映体对心肌细胞的凋亡保护作用。方法:异丙肾上腺素(ISO)2 mmol.L-1作用12 h以诱导H9C2-1心肌细胞凋亡,Hoechst 33258染色法定性凋亡细胞,AnnexinⅤ-FITC/PI双染标记细胞后以流式细胞仪测定标记率以定量凋亡率。处理组同时加入R(+)或S(-)carvedilol,分2和10μmol.L-1两个浓度组。并分别测定10μmol.L-1R(+)或S(-)carvedilol单一作用的细胞凋亡率。结果:2 mmol.L-1ISO作用12 h后H9C2-1细胞大量凋亡(P<0.01);R(+)或S(-)carvedilol+ISO组细胞凋亡率显著低于ISO组(P<0.05);与低剂量组间比较,R(+)carvedilol+ISO细胞晚期凋亡率和总凋亡率低于S(-)carvedilol+ISO,差异有显著性意义(P<0.05);高剂量S(-)carvedilol组细胞凋亡率略有升高,但显著低于S(-)Carv 10μmol.L-1+ISO组(P<0.05)。结论:ISO可诱导-H9C2-1心肌细胞凋亡,低剂量R(+)或S(-)carvedilol对此均有明显的抑制作用,并且R(+)和S(-)型carvedilol的抗心肌细胞凋亡效应间存在一定的立体选择性差异,R(+)carvedilol显示出更具潜力。     

Livin mRNA的反义核酸诱导MCF-7乳癌细胞凋亡作用

目的：以livin mRNA为靶点设计反义硫代脱氧寡核苷酸(PS—ODNs)，探讨其体外抗肿瘤效应及其机制。方法：设计以livin mRNA为靶点的反义核酸并作用于MCF-7乳癌细胞，用MTT、RT—PCR和流式细胞仪检测等方法对其进行评价研究。结果：在设计的一些反义核酸中，YMZ05能够有效地抑制livin基因的表达，增加caspase-3的活性，诱导MCF-7细胞凋亡，明显抑制其生长，结论：通过抑制livin基因的表达能够抑制MCF-7乳癌细胞生长、诱导其凋亡，livin可能会成为抗肿瘤的新靶点。     

Structural modifications of antisense oligonucleotides

Antisense oligonucleotides are efficient tools for the inhibition of gene expression in a sequence specific way. Natural oligonucleotides are decomposed rapidly in biological systems, which strongly restrict their application. In contrast, artificial oligonucleotides are designed to be more stable against degradation than the target mRNA, which results in a catalytic effect of the drug. Modification of the phosphate linkage has been the first successful strategy for antisense drug developments and Fomivirsene the first antisense drug in therapy. The launch of Fomivirsene has resulted in a revolutionary spin off to antisense research leading to a second generation of antisense oligonucleotides, which are stable against oligonucleotide cleaving enzymes. Among these, oligonucleotides bearing an alkoxy substituent in position 2' were the most successful ones. The third generation of antisense oligonucleotides contains structure elements, which enhance the antisense action. Zwitterionic oligonucleotides show remarkable results, first, because the stability against ribozymes is largely increased, and secondly, because the electrostatic repulsion between the anionic sense and the zwitterionic antisense cords is minimized. Promising new target molecules in antisense research are oligonucleotide chim?res, which enhance the antisense action (chim?res with intercalators, chelators or polyamines) or enable an application as sequence specific detectors (chim?res with biotin, fluorescein or radioligands).     

Cellular uptake of antisense oligonucleotides

Antisense oligonucleotides (AS ONs) selectively bind to the target mRNA and prevent its translation into the corresponding protein. Various tissue culture studies demonstrated that AS ONs enter into cells via the receptor-mediated endocytosis pathway. There are many different types of receptors, and their characteristics and expression vary with cell types. In this review, we will discuss the characteristics of the various receptors that have been isolated in vitro. We will also discuss the uptake and the bioavailability of AS ONs after being administered in vivo.     

Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention

Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-alpha-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage.     

Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides.

Antisense oligonucleotides have been widely used to specifically and selectively downregulate gene expression at the messenger RNA level. Even though oligonucleotides are commonly used in laboratories and clinical trials, they can induce non-specific effects that can lead to misinterpretation of experimentally-derived results. This review summarizes precautions one should take when using oligonucleotides. In addition, the role of one oligonucleotide, G3139, which is targeted to the coding region of bcl-2 messenger RNA, in inhibiting tumor progression in vitro and in clinical trials, is described.     

Setting sights on the treatment of ocular angiogenesis using antisense oligonucleotides

The application of antisense technology to study physiological and disease processes continues to mature. Antisense approaches are among the most direct means to use genomic sequence information. When developing therapeutics, applications range from early target validation in discovery to the therapeutic product. In this review, we describe the application of antisense oligonucleotides (ASOs) to identify genes that are important in controlling angiogenesis. High-throughput assays in vitro have been used to evaluate many gene targets. Genes that appear to be important in angiogenesis are then evaluated further in animal models of ocular angiogenesis. The ability of ASOs to reduce target-gene expression in the appropriate cells in the eye raises the possibility that this class of compounds could be used for target validation in vivo, and also be developed as a novel class of therapeutics in their own right.     

反义寡核苷酸技术在心血管疾病中的研究应用

随着心血管疾病发病的分子机制日益明确，反义寡核苷酸技术针对发病机制中各个不同的靶基因位点，被广泛应用于各种心血管疾病的研究。在高血压、冠心病、心脏移植后并发症、心力衰竭等疾病的发病机制及其治疗性研究中都起到了积极的作用，显示出其作为药物应用于临床的良好前景。     

Oral delivery of antisense oligonucleotides in man

Treatment of systemic disease with phosphorothioate antisense oligonucleotides (PS ASOs) has been accomplished using local or parenteral routes of administration to date. This report describes, for the first time, the effective oral delivery of a second generation oligonucleotide where significant milligram amounts of intact drug are absorbed in human subjects. In this study, a variety of oral solid dosage formulations were evaluated and it was determined that pulsing the delivery of sodium caprate (C10), a well-known permeation enhancer, in a novel manner may provide optimal ASO plasma bioavailability. Further, these dosage forms, containing C10 and ASO, were well tolerated in both fasted and fed volunteers. Oral absorption of the 2'-O-(2-methoxyethyl) modified antisense oligonucleotide (2'-MOE ASO), ISIS 104838, was demonstrated in healthy volunteers with an average 9.5% plasma bioavailability across four formulations tested. The greatest average performance achieved in this study for a single formulation was 12.0% bioavailability within an individual dose and subject range of 1.96-27.5%. The totality of the data suggests that formulations can be devised that allow oral administration of oligonucleotides that maintain systemic concentrations associated with inhibition of targeted human mRNA.     

Novel non-endocytic delivery of antisense oligonucleotides

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.     

Cellular delivery of antisense oligonucleotides.

Antisense oligonucleotides can be successfully employed to inhibit specifically gene expression. However, many oligonucleotide classes are polyanions and cannot passively transit the cell membrane. Thus, the use of naked oligonucleotides for antisense purposes poses some rather stringent challenges, and it is not a trivial task to appropriately interpret the data derived from experiments in which they have been used. Multiple methods have been developed to improve intracellular, and in particular, intranuclear oligonucleotide delivery, and in doing so, to maximize the performance of the antisense technologies that are currently available. This review discusses the use of cationic lipids, protein and peptide delivery agents, and several novel chemical and viral methods that have recently been explored as delivery vehicles, focussing not only on their strengths, but also on their limitations.     

反义核酸药物KS0604在小鼠的组织分布

目的:研究小鼠静脉注射30 mg.kg-1 KS0604后血浆和组织中原形药物浓度-时间变化。方法:建立组织消化结合基于离子交换和反相分配原理的两步固相萃取法,并采用非胶筛分毛细管电泳技术分析小鼠血浆和组织中的KS0604。结果:小鼠血浆和组织中KS0604在标准曲线范围内呈良好的线性关系。小鼠给药后各组织中以全长序列即药物原形为主,给药后4 h内,血浆中的浓度逐渐下降,2 h和4 h仅个别个体可测到。各组织中肾浓度一直最高,在给药后2 h达峰;肝、肺、心、骨骼肌和脂肪中,均在给药后1 h达到最高,4 h内变化不大。而脾在给药后仅个别个体可测到,脑和胰组织中的浓度均低于定量下限。结论:非胶筛分毛细管电泳方法能满足反义核酸药物KS0604小鼠组织分布实验的要求。小鼠给药后各组织中以全长序列即药物原形为主,给药后4 h内,AUC0～t从大到小依次为肾、肝、肺、心、骨骼肌、脂肪和血浆。     

新型c-Myc反义硫代磷酸寡脱氧核苷酸在小鼠体内的药动学

目的 :研究新筛选合成的c Myc基因反义硫代磷酸寡脱氧核苷酸 (antisensephosphorothioateoligodeoxynucleotidestoc Myc ,c MycAS PS ODN )在小鼠体内的药动学、组织分布和排泄情况。方法 :给小鼠按 5 ,10 ,2 0mg·kg- 13个剂量静脉注射3H c MycAS PS ODN后 ,液闪仪测定不同时间点血浆药物浓度 ;按 10mg·kg- 1静脉注射3H c MycAS PS ODN ,测定不同时间组织器官中3H c MycAS PS ODN的含量和其在粪、尿中排泄率 ,药动学统计程序拟合分析。结果 :静脉注射后 3种剂量血浆药物浓度 时间曲线均符合二室分布模型 ,3种剂量下的AUC之间有明显的线性关系 ;多数组织药物分布浓度 2h达高峰 ,肝、脾、肾、肺和骨髓中药物浓度较高 ,2 4h尿、粪总排泄率 82 %。结论 :c MycAS PS ODN在体内分布快 ,除脑、生殖腺等亲脂性组织外 ,其他组织含量高 ;主要通过尿液排泄。