In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide

BACKGROUND: Current vaccination strategies and antiviral drugs only provide limited protection against influenza virus infection. In this study, we investigated the use of a novel antisense oligonucleotide (named IV-AS), which is specific for the 5'-terminal conserved sequence found in all eight viral RNA segments of influenza A virus. METHODS: The activity of IV-AS was monitored both in vitro, in Madin-Darby canine kidney (MDCK) cells, and in vivo using a mouse model. IV-AS was given intranasally to H5N1-infected mice once daily for 6 days starting 6 h after infection. A three-base mismatch of IV-AS was used as a control. RESULTS: IV-AS inhibited influenza virus A induced cytopathic effects in MDCK cells with the 50% effective concentration (EC50) ranging from 2.2 to 4.4 microM. IV-AS was effective against H5N1 virus in preventing death, lessening weight reduction, inhibiting lung consolidation and reducing lung virus titres. Dosages of 40 and 60 mg/kg/day provided 40% and 60% survival rates and prolonged mean survival days in comparison with the infected control group (P<0.05). The lung index in mice treated with IV-AS, at a dose of 20, 40 or 60 mg/kg/day, had been inhibited on day 4 or 6 (P<0.05 or P<0.01); virus titres in lung had declined to 2.42, 1.51 and 1.54 log10 TCID50/g of lung, respectively, whereas the yields in the infected control mice were 6.00 log10 TCID50/g of lung. CONCLUSIONS: Our results suggest that the 5'-terminal conserved region of influenza A virus RNA segments can be targeted using antisense technology; therefore, IV-AS is a potential drug for prophylaxis and control of influenza virus infections.     

干细胞生长因子反义寡核苷酸对肝癌细胞中原癌基因蛋白质c-kit及碱性成纤维细胞生长因子表达的影响

目的探讨干细胞生长因子(SCF)反义寡核苷酸对肝癌HepG2细胞中原癌基因蛋白质c-kit、碱性成纤维细胞生长因子(bFGF)表达的影响及意义。方法以脂质体作为转染载体,转染SCF反义寡核苷酸于HepG2细胞。实验分三组:(1)空白对照组;(2)转染SCF反义寡核苷酸组;(3)转染SCF错义寡核苷酸组。Western blot方法检测转染前后肝癌细胞中SCF、bFGF蛋白的表达,RT-PCR检测转染前后肝癌细胞中c-kit与bFGF mRNA的表达,用流式细胞仪检测转染SCF反义寡核苷酸后HepG2细胞凋亡率。结果与转染错义寡核苷酸组相比,SCF反义寡核苷酸对HepG2细胞中c-kit、bFGF的表达有明显抑制作用,转染SCF反义寡核苷酸可明显增加HepG2细胞凋亡率(P<0.01)。结论 SCF在肝癌细胞凋亡中有重要调节作用,SCF/c-kit有可能作为PI3K/Akt信号通路的上游对肝癌细胞bFGF的表达起重要调控作用。     

内皮缩血管肽反义寡核苷酸促进骨髓移植小鼠造血重建

目的观察内皮缩血管肽(Endostatin)反义寡核苷酸转染骨髓基质细胞后在骨髓移植(BMT)小鼠骨髓造血恢复过程中的作用。方法以脂质体作为转染载体,转染不同剂量的 FITC 标记的 Endostatin 反义寡核苷酸,荧光倒置显微镜观察转染效率,用流式细胞术检测最佳转染条件下转染率,用 RT-PCR 和 Western blot 方法检测最佳转染条件下 Endostatin 反义寡核苷酸对 BMT 后不同时间点小鼠骨髓基质细胞 Endostatin mRNA 及其蛋白质和血管细胞间黏附分子1(VCAM-1)mRNA 及其蛋白表达水平的影响。实验分为4组:①正常组:未经任何处理组;②BMT 组:空白对照组;③BMT+转染Endostatin 反义寡核苷酸组;④BMT+转染 Endostatin 错义寡核苷酸组。结果①在体外成功地将 En-dostatin 反义寡核苷酸导入骨髓基质细胞,转染率达86%;②以 Endostatin 反义寡核苷酸转染骨髓基质细胞后,BMT 后不同时间点骨髓基质细胞 Endostatin mRNA 及其蛋白表达被显著抑制[Endostatin 的灰度值分别为(0.09±0.03)～(1.44±1.19)和(0.02±0.02)～(0.14±0.05)](P<0.01或P<0.05),表明转染成功;③Endostatin 反义寡核苷酸转染有效促进了 BMT 后不同时间骨髓基质细胞 VCAM-1mRNA 及其蛋白表达[VCAM-1的灰度值分别为(1.60±0.92)～(8.05±0.87)和(0.07±0.02)～(0.67±0.09)](P<0.01或P<0.05);④Endostatin 错义寡核苷酸对 BMT 后不同时间骨髓基质细胞 Endostatin 和 VCAM-1的表达基本无影响(P>0.05)。结论 Endostatin 反义寡核苷酸可降低Endostatin 表达,增强 VCAM-1的表达,从而促进骨髓微血管生成,改善基质细胞与造血细胞之间和细胞外基质与造血细胞之间的联系而影响骨髓造血。     

Pharmacokinetics and pharmacodynamics of dilevalol

The pharmacokinetics and pharmacodynamics of dilevalol, the R,R stereoisomer of labetalol, were evaluated in nine subjects. Dilevalol was given as a single 50 mg intravenous dose and as a 400 mg daily oral dose for 7 days. To study the effects of hepatic enzyme inhibition, each subject received dilevalol in the presence of and absence of cimetidine. Cardiac beta-blockade was assessed by use of standardized treadmill tests for 48 hours after oral dilevalol. The three-compartment model analysis showed that systemic clearance (29.8 +/- 5.7 ml/min/kg), volume of distribution (16.6 +/- 4.1 L/kg), and terminal half-life (11.7 +/- 2.7 hours) were not altered by cimetidine. However, there was a 20% increase in the area under the curve (p less than 0.05) and an 11% increase in systemic bioavailability (p less than 0.05) after oral administration. Dilevalol caused significant cardiac beta-blockade for more than 24 hours, but these effects were not altered by cimetidine. The pharmacokinetic changes are consistent with a decrease in first-pass extraction of a high clearance drug.     

Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1

Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase-1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.     

Pharmacokinetics and pharmacodynamics of esmolol administered as an intravenous bolus

Esmolol is a new ultra-short-acting beta-adrenergic receptor blocking agent that may be useful in the treatment of patients with heart disease. We gave esmolol as an intravenous bolus injection (over 30 seconds) to 12 healthy men in a dose-ranging study; each subject received two doses. Our dosing schedule began with 30 mg in the first subject and ended with 100 mg and 150 mg in the final four subjects. We measured blood esmolol concentration, PR interval, QRS duration, QTc interval, cardiac cycle, systolic blood pressure, and diastolic blood pressure. Esmolol doses of 150 mg produced blood esmolol concentrations of 0.868 to 1.47 micrograms/ml. The peak PR interval recorded after esmolol was significantly longer than the control PR interval in four subjects who received 100 and 150 mg doses (192 +/- 7.9 msec vs. 177 +/- 10.6 msec; P = 0.00002). Peak prolongation of the PR interval was recorded 6 to 10 minutes after the bolus, at which time blood esmolol concentrations were negligible. Esmolol did not consistently affect any other pharmacodynamic variable. Giving esmolol as an intravenous bolus injection may be a simple alternative to loading and maintenance infusion in some clinical settings.     

Pharmacokinetics and pharmacodynamics of ertapenem: an overview for clinicians

Ertapenem, a Group 1 carbapenem, is a once-a-day parenteral beta-lactam antibiotic recently licensed in the USA and Europe. Monotherapy with ertapenem dosed as 1 g once a day has been shown to be highly effective in clinical trials for the treatment of complicated infections of skin and skin structures, complicated intra-abdominal infections, community-acquired pneumonia, acute pelvic infections and complicated urinary tract infections. Dosing modifications have not been recommended for adults on the basis of gender, age, weight or liver disease. Presently there are no data regarding the use of ertapenem in children. Dose reductions are indicated for patients with advanced renal insufficiency. Ertapenem is neither a substrate nor an inhibitor of P-glycoprotein or cytochrome P450 enzymes; significant drug interactions between ertapenem and drugs handled by these systems are not expected.     

Pharmacokinetics and pharmacodynamics of atenolol in children

The pharmacokinetics and pharmacodynamics of intravenous atenolol were studied in 10 children during cardiac electrophysiologic studies. The intravenous pharmacokinetic data were best described by a two-compartment model and revealed the following (mean +/- SD): total body clearance, 0.15 +/- 0.06 L/hr/kg; volume of the central compartment 0.33 +/- 0.06 L/kg; volume of distribution at steady state, 0.83 +/- 0.15 L/kg; distributive elimination half-life, 0.29 +/- 0.08 hour; and terminal elimination half-life, 4.56 +/- 1.05 hours. The data suggest that children have a slightly shorter terminal elimination half-life than that of adults. Pharmacodynamic data showed a significantly (p less than 0.01) increased sinus cycle length and an increase in His to ventricle conduction time (p less than 0.05). Further studies are necessary to determine the optimal oral dose and dosing frequency of atenolol and to access the response of children to long-term treatment.     

Inhaled CD86 antisense oligonucleotide suppresses pulmonary inflammation and airway hyper-responsiveness in allergic mice

The B7-family molecule CD86, expressed on the surface of pulmonary and thoracic lymph node antigen-presenting cells, delivers essential costimulatory signals for T-cell activation in response to inhaled allergens. CD86-CD28 signaling is involved in priming allergen-specific T cells, but it is unclear whether these interactions play a role in coordinating memory T-helper 2 cell responses. In the ovalbumin (OVA)-induced mouse model of asthma, administration of CD86-specific antibody before systemic sensitization suppresses inhaled OVA-induced pulmonary inflammation and airway hyper-responsiveness (AHR). In previously OVA-sensitized mice, systemic and intranasal coadministration of CD86 antibody is required to produce these effects. To directly assess the importance of pulmonary CD86 expression in secondary immune responses to inhaled allergens, mice were sensitized and locally challenged with nebulized OVA before treatment with an inhaled aerosolized CD86 antisense oligonucleotide (ASO). CD86 ASO treatment suppressed OVA-induced up-regulation of CD86 protein expression on pulmonary dendritic cells and macrophages as well as on recruited eosinophils. Suppression of CD86 protein expression correlated with decreased methacholine-induced AHR, airway inflammation, and mucus production following rechallenge with inhaled OVA. CD86 ASO treatment reduced BAL eotaxin levels, but it did not reduce CD86 protein on cells in the draining lymph nodes of the lung, and it had no effect on serum IgE levels, suggesting a local and not a systemic effect. These results demonstrate that CD86 expression on pulmonary antigen-presenting cells plays a vital role in regulating pulmonary secondary immune responses and suggest that treatment with an inhaled CD86 ASO may have utility in asthma and other chronic inflammatory lung conditions.     

吸入性激素药代学和药效学

吸入性激素（ICS）是一种局部激素，全身性作用极小，已成为哮喘长期治疗的一线药物。本文重点讨论ICS的药效学和药代动力学。     

miR-155反义寡核苷酸对乳腺癌HS578T细胞增殖和凋亡的影响

目的:探讨微小RNA 155(microRNA-155,miR-155)反义寡核苷酸(antisense oligonucleotide,ASO)对乳腺癌细胞增殖和凋亡的影响.方法:设计合成全硫代磷酸化修饰的miR-155 ASO,通过LipofectamineTM2000(Invitrogen)转染于乳腺癌HS578T细胞.CCK-8法检测乳腺癌细胞转染后的增殖情况,流式细胞仪分析转染率并检测细胞凋亡情况.结果:流式细胞仪检测转染率达70.5%.CCK-8试验结果显示转染miR～155 ASO后.HS578T细胞存活数明显低于空白组和脂质体组.流式细胞仪检测显示转染miR-155ASO后,HS578T细胞凋亡数明显增加.结论:miR-155 ASO可抑制乳腺癌HS578T细胞增殖,并促进其凋亡.提示miR-155可能成为乳腺癌治疗的靶基因.     

Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin? (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.     

Pharmacokinetics and pharmacodynamics of methylprednisolone in obesity.

Methylprednisolone pharmacokinetics and its directly suppressive effects on plasma cortisol, blood histamine (basophils), and circulating helper T cells were evaluated in six obese (at least 35% above ideal body weight) men and six nonobese male volunteers. Methylprednisolone doses of 0.6 mg/kg total body weight were administered as the 21-succinate sodium salt. Absolute clearance (in liters per hour) of methylprednisolone was 40% less in the obese subjects. Total volume of distribution (Vss) of methylprednisolone was unchanged (about 120 L), but when normalized for total body weight, Vss per kilogram was less in obesity. The patterns of cortisol, blood histamine, and helper T cell responses after methylprednisolone administration were similar in both groups, but more profound effects were observed in the obese subjects. Pharmacodynamic models were applied for these immediate effects of methylprednisolone based on the premise that receptor interactions of steroids are followed by rapid suppression of the circadian rhythm of cortisol and recirculation of basophils and helper T cells, which persist until inhibitory concentrations (IC50) of methylprednisolone disappear. Similar IC50 values for the three effects were obtained in both groups, indicating no intrinsic pharmacodynamic differences in sensitivity to these methylprednisolone effects in obesity. However, methylprednisolone should be administered on the basis of ideal body weight, and the dosing interval should be potentially lengthened because of decreased methylprednisolone clearance in obesity.     

Characterization of complexes of an antisense oligonucleotide with protamine and poly-L-lysine salts.

The objectives of this work were to study the interaction of an antisense oligonucleotide (ISIS 2302) with poly-L-lysine (PLL) and protamine salts, to determine the physico-chemical characteristics of the resulting complex systems and to analyze the influence of permeation enhancers (Na-chenodeoxycholate and Na-caprate) on the dissociation of the complexes. Specific conductivity, zeta potential, particle size distribution and dialysis studies of the resulting complex systems were performed. Conductometric titration defined the molar ratios between the ionic species in the complex. Zeta potential data confirmed the conductometric equivalence points and explained the good physical stability of charged complexes when compared to neutral complexes (+/-40 mV for PLL-based complexes and +/-25 mV for protamine sulfate complexes). The particle size was less than 175 nm for most systems. The incorporation of Na-chenodeoxycholate promoted complex dissociation, while Na-caprate gave opposite results. An increase in the ionic strength of the environment had a destabilizing effect and promoted dissociation of the complexes.     

Pharmacokinetics and pharmacodynamics of prednisolone in obese rats

The pharmacokinetics and pharmacodynamic response to prednisolone were examined in dietary-induced obese rats and matched controls. Pharmacokinetic parameters were examined in absolute and weight normalized terms. After an i.v. dose (range, 4.0-6.3 mg/kg) of prednisolone adjusted to achieve similar initial prednisolone plasma concentrations, the time course of glucocorticoid receptors in hepatic cytosol and hepatic tyrosine aminotransferase (TAT) activity were examined. Plasma prednisolone concentrations declined biexponentially with time. Mean (S.D.) for prednisolone plasma clearance normalized for total body mass (TBM) was 2.3 (0.9) liters/hr/kg in normal rats and 2.7 (0.7) liters/hr/kg in obese rats. The volume of distribution at steady-state averaged 0.82 (0.46) liters/kg of TBM in normal rats vs. 1.08 (0.40) liters/kg of TBM in obese rats. Base-line receptor levels for obese rats were 53% higher than control levels. A model to describe simultaneously kinetics and receptor-mediated dynamics was used to analyze the data and obtain estimates for the efficiency of TAT induction. This efficiency parameter in obese rats was 22% of controls, reflecting the innate degree of diminished TAT response. This decreased response in obese animals may indicate a need for joint pharmacokinetic/dynamic considerations in dosing obese individuals with corticosteroids.     

Pharmacokinetics and pharmacodynamics of long-term continuous-infusion doxorubicin

Steady-state plasma levels of doxorubicin and doxorubicinol were analyzed in 32 patients with advanced cancer, each of whom was given doxorubicin by long-term continuous infusion at progressively increasing infusion rates. Patients received doxorubicin for 2 to 50 weeks at rates of 0.2 to 6.1 mg/m2/day. Dose-limiting stomatitis and leukopenia were observed. The mean maximum steady-state doxorubicin concentration was 6.04 ng/ml at a mean maximum infusion rate of 3.92 mg/m2/day. Clearance mechanisms did not appear to be saturated at the durations or infusion rates used in this study. The maximum steady-state doxorubicin level and the ln (initial WBC) were significant correlates of the ln (nadir WBC) (p = 0.002 and 0.02, respectively). A model was constructed according to these two parameters that significantly describes ln (nadir WBC) (p = 0.001). Neither age, infusion rate, nor doxorubicinol level correlated with nadir WBC. Stomatitis did not correlate with any of these parameters. The demonstration of this pharmacodynamic relationship highlights the potential importance of pharmacologic data collection in ongoing attempts to predict the clinical effects of anticancer drugs.     

Phase I trial of ISIS 104838, a 2'-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor-alpha

ISIS 104838 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that binds tumor necrosis factor-alpha (TNF-alpha) mRNA. It carries a 2'-methoxyethyl modification on the five 3' and 5' nucleotide sugars, with 10 central unmodified deoxynucleotides. ISIS 104838 was identified from a 264 ASO screen in phorbol myristate acetate-activated keratinocytes, and the dose response was assessed in lipopolysaccharide (LPS)-activated monocytes. Healthy males received multiple intravenous (i.v.) ISIS 104838 infusions in a placebo-controlled dose escalation trial (0.1-6 mg/kg). Additional volunteers received single or multiple subcutaneous (s.c.) injections. ISIS 104838 suppressed TNF-alpha protein by 85% in stimulated keratinocytes. The IC50 for TNF-alpha mRNA inhibition in stimulated monocytes was <1 microM. For i.v., C(max) occurred at the end of infusion. The effective plasma half-life was 15 to 45 min at 0.1 to 0.5 mg/kg and 1 to 1.8 h for higher doses. The apparent terminal plasma elimination half-life approximated 25 days. Obese subjects had higher plasma levels following equivalent mg/kg doses. For s.c. injections, C(max) occurred at 2 to 4 h and was lower than with equivalent i.v. dosing. Plasma bioavailability compared with i.v. was 82% following a 200 mg/ml s.c. injection. Transient activated partial thromboplastin time prolongation occurred after i.v. infusions and minimally after s.c. injections. Two subjects experienced rash, one a reversible platelet decrease, and mild injection site tenderness was noted. TNF-alpha production by peripheral blood leukocytes, induced ex vivo by LPS, was decreased by ISIS 104838 (p < 0.01). ISIS 104838, a second-generation antisense oligonucleotide, was generally well tolerated intravenously and subcutaneously. The pharmacokinetics support an infrequent dosing interval. Inhibition of TNF-alpha production ex vivo was demonstrated.