Ultrasonic Nebulization of Cationic Lipid-Based Gene Delivery Systems for Airway Administration

Purpose. This study relates to the development of gene therapies for the treatment of lung diseases. It describes for the first time the use of ultrasonic nebulization for administration of plasmid/lipid complexes to the lungs to transfect lung epithelial cells. Methods. Plasmid complexed to cationic liposomes at a specific stoichiometric ratio was nebulized using an ultrasonic nebulizer. We assessed: (i) the stability of plasmid and plasmid/lipid complexes to ultrasonic nebulization, (ii) the in vitro activity of plasmid in previously nebulized plasmid/lipid complex, (iii) the in vivo transgene expression in lungs following intratracheal instillation of nebulized plasmid/lipid formulations compared to un-nebulized complexes, (iv) the emitted dose from an ultrasonic nebulizer using plasmid/lipid complexes of different size, and (v) the transgene expression in lungs following oral inhalation of aerosolized plasmid/lipid complex generated using an ultrasonic nebulizer. Results. Integrity of plasmid formulated with cationic lipids, and colloidal stability of the plasmid/lipid complex were maintained during nebulization. In contrast, plasmid alone formulated in 10% lactose was fragmented during nebulization. The efficiency of transfection of the complex before and after nebulization was comparable. Nebulization produced respirable aerosol particles. Oral exposure of rodents for 10 minutes to aerosol produced from the ultrasonic nebulizer resulted in transgene expression in lungs in vivo. Conclusions. The performance characteristics of the ultrasonic nebulizer with our optimized plasmid/lipid formulations suggests that this device can potentially be used for administering gene medicines to the airways in clinical settings for the treatment of respiratory disorders.     

Nebulization of Fluids of Different Physicochemical Properties with Air-Jet and Ultrasonic Nebulizers

Purpose. Empirical formulae relate the mean size of primary droplets from jet and ultrasonic nebulizers to a fluid's physicochemical properties. Although the size selective filtering effects of baffling and evaporation may modify the secondary aerosol produced, this research sought to evaluate whether viscosity and surface tension of nebulized fluids influenced the aerosol's size and output characteristics. Methods. Fluid systems of different surface tension and viscosity (glycerol and propylene glycol solutions [10–50% (v/v)] and a range of silicone fluids [200/0.65 cs– l00cs]) were nebulized in three jet and two ultrasonic nebulizers. Secondary aerosol characteristics were measured with a Malvern 2600C laser diffraction sizer and the nebulization times, residual volumes and percentage outputs were determined. Results. While the droplet size appeared to be inversely proportional to viscosity for jet nebulizers, it was directly proportional to viscosity for ultrasonic nebulizers. Although fluid systems with lower surface tensions generally produced slightly smaller MMDs, the relationship between surface tension and droplet size was complex. The more viscous fluids required longer nebulization times and were associated with increased residual amounts (lower outputs). The ultrasonic nebulizers did not effectively, and were on occasion unable to, nebulize the more viscous fluids. Conclusions. It follows that there are cut-off values for viscosity and/or surface tension above or below which ultrasonic devices fail to operate. Moreover, jet nebulizers generated an aerosol with an optimum respirable output from median-viscosity fluids.     

Applicability of an Ultrasonic Nebulization System for the Airways Delivery of Beclomethasone Dipropionate in a Murine Model of Asthma

Purpose We have assessed the use of an ultrasonic nebulization system (UNS), composed of ultrasonic nebulizer and diffusion dryer filled with charcoal, for the effective delivery of beclomethasone to the airways in a murine asthma model.Methods Solution of beclomethasone in ethanol was aerosolized using an ultrasonic nebulizer. Passage of the aerosol through a drying column containing charcoal and deionizer produced dry beclomethasone particles. Particles were delivered to BALB/c mice placed in a whole-body exposition chamber 1 h before intranasal challenge with ovalbumine. Efficacy of beclomethasone delivery was evaluated by examining bronchoalveolar lavage fluid (BALF) cytology.Results Effect of three UNS system parameters on aerosol particle size was investigated. The critical parameter affecting the size of dry particles was beclomethasone concentration in aerosolized solution and solution flow rate while power level of ultrasonic nebulizer generator had no effect. Administration of beclomethasone at calculated dose of 150 μg/kg to mice significantly decreased total cell number and relative eosinophil number in BALF.Conclusions The UNS system produces a monodisperse aerosol that can be used for inhalative delivery of poorly water soluble substances to experimental animals. The UNS system minimizes formulation requirements and allows rapid and relatively simple efficacy and toxicity testing in animals.     

沐舒坦雾化吸入后吸痰治疗小儿肺炎的疗效及护理

目的观察沐舒坦雾化吸入后吸痰治疗小儿肺炎的临床疗效。方法将住院肺炎患儿随机分为治疗组和对照组,两组均给予常规治疗,治疗组给沐舒坦雾化吸入,对照组给生理盐水雾化吸入,雾化吸入后均给予吸痰。结果治疗组症状、体征消失天数较对照组明显缩短,经统计学处理两组处理差异有统计学意义（P〈0.05）。结论沐舒坦雾化吸入后吸痰治疗小儿肺炎能够快速有效清除痰液,控制肺部炎症,提高治愈率。     

盐酸氨溴索雾化吸入治疗慢性阻塞性肺疾病患者151例临床分析

目的探讨盐酸氨溴索雾化吸入治疗慢性阻塞性肺疾病的临床效果。方法 151例慢性阻塞性肺患者被随机分为实验组(n=77)和对照组(n=74),对照组给予常规治疗,实验组在常规治疗基础上给予盐酸氨溴索雾化吸入治疗。结果实验组总有效率为98.7%,显效率为92.2%,对照组总有效率为87.8%,显效率为71.6%,实验组疗效显著优于对照组(P<0.05)。结论盐酸氨溴索雾化吸入结合常规础治疗应用于慢性阻塞性肺患者,能显著提高临床效果,改善患者生活质量,值得在临床上应用和推广。     

Generation of Micro-Particles of Proteins for Aerosol Delivery Using High Pressure Modified Carbon Dioxide

Purpose. To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to generate microparticles of proteins suitable for aerosol delivery from aqueous-based solutions. Methods. The ASES technique using high- pressure carbon dioxide modified with ethanol was utilised for the generation of microparticles of proteins (lysozyme, albumin, insulin and recombinant human deoxyribonuclease (rhDNase)) from aqueous solutions. Particle size, morphology, size distributions and powder aerosol performance were examined. The biochemical integrity of the processed proteins was assessed by testing the level of molecular aggregation using size exclusion chromatography and by bioassay technique for lysozyme. Results. Proteins were precipitated as spherical particles ranging in size from 100 to 500 nm. The primary nano-sized particles agglomerated to form micron-sized particles during the precipitation process. The median size of the particles was a function of the operating conditions. In-vitro aerosol performance tests showed that the percent fine particle mass (< 5m) was approximately 65%, 40% and 20% for lysozyme, albumin and insulin, respectively. Negligible loss in the monomer content or biological activity was observed for lysozyme. Insulin exhibited slight aggregation and 93% of the monomer was retained after processing. Albumin was affected by processing and only 50-75% of the monomer was retained compared with 86% in the original material. However, rhDNase was substantially denatured during processing as shown by the significantly reduced monomer content. Conclusions. Micron-sized particles of lysozyme, albumin and insulin with satisfactory inhalation performance were successfully generated from aqueous solutions using the modified ASES technique. The biochemical integrity of the processed proteins was a function of the operating conditions and the nature of the individual protein.     

Protective effect of poly(I): Poly(C) against cephaloridine nephrotoxicity in the rat

The effect of poly(I):poly(C) during cephaloridine nephrotoxicity in the rat was determined by measurement of urinary enzyme excretion. Urinary alkaline phosphatase, lactate dehydrogenase and muramidase levels were lower in rats receiving a combination of poly(I): poly(C) and cephaloridine as compared to the levels of these enzymes in the urines of rats receiving cephaloridine alone. Poly(I): poly(C) alone did not cause an elevation of these enzymes and incubation of the enzymes in urine with poly(I): poly(C) did not inhibit the enzyme activity. It is concluded that poly(I): poly(C) acted to protect kidney cells from damage by cephaloridine.     

Using dextran of different molecular weights to achieve faster freeze-drying and improved storage stability of lactate dehydrogenase

Freeze-drying of protein formulations is frequently used to maintain protein activity during storage. The freeze-drying process usually requires long primary drying times because the highest acceptable drying temperature to obtain acceptable products is dependent on the glass transition temperature of the maximally freeze-concentrated solution (T_g′). On the other hand, retaining protein activity during storage is related to the glass transition temperature (T_g) of the final freeze-dried product. In this study, dextrans with different molecular weight (1 and 40?kDa) and mixtures thereof at the ratio 3:1, 1:1, and 1:3 (w/w) were used as cryo-/lyoprotectant and their impact on the stability of the model protein lactate dehydrogenase (LDH) was investigated at elevated temperatures (40?°C and 60?°C). The dextran formulations were then compared to formulations containing sucrose as cryo-/lyoprotectant. Because of the higher T_g′ values of the dextrans, the primary drying times could be reduced compared to freeze-drying with sucrose. Similarly, the higher T_g and T_g′ of dextrans relative to sucrose led to benefits during storage which was shown through improved protection of LDH activity.     

Effect of Temperature History on the Freeze-Thawing Process and Activity of LDH Formulations

Purpose. The purpose of the study was to investigate the effect of freeze-thawing processes with different temperature histories on thermal transformations and on protein activity of lactate dehydrogenase (LDH) formulations. Polyethylene glycol (PEG 6000) and maltodextrin were used as cryoprotectants. Methods. The thermal characterization was made by oscillating DSC (ODSC). LDH activity assays were performed spectrophotometrically. Results. The crystallization of the solutions and the melting of the frozen samples occurred at fairly constant heat of crystallisation and heat of fusion values and temperatures. The main difference between the two investigated temperature cycles was an exothermic peak at –45°C, which might reflect the transition between the cubic and hexagonal ice structures. When PEG was added to the system an additional endothermic peak appeared at –15°C in the heating program. It was transformed into the shape of a glass transition at the same temperature when the heating rate was increased. The degree of crystallinity of the samples was evaluated as the quota between the c_p component of heat of transformation and the total heat of transformation values. Only minor differences between the two temperature histories and between the samples were observed. The c_p component of the melting endotherm revealed a complex melting process with two overlapping endothermic transformations. The good protein protecting ability of PEG obtained when cooling and heating rate was low, was greatly reduced with increasing rate. The addition of maltodextrin to PEG-containing solutions lowered the activity recovery. Conclusions. The endothermic transformation of a PEG-ice structure at –15°C in the heating process is strongly correlated to the protective ability of PEG 6000 in the freeze-thawing process of LDH. To obtain the highest protein activity after the freeze-thawing process, the formulation shall be transformed by a low cooling and heating rate. The crystallinity of the system melting at about 2°C is independent of temperature history. The c_p component of the melting endotherm, however, shows a complex transformation, where two phases of different crystallinity and stability might be involved.     

Effect of imatinib on the biochemical parameters of the reproductive function in male Swiss albino mice

Background:

Treatment of cancers with cytotoxic agents such as tyrosine kinase inhibiting drugs often, but not always, result in transient to permanent testicular dysfunction. Germ cells are important targets of many chemicals. Most of the drugs are genotoxins and induce irreversible effect on genetic makeup. These mutagenic changes are proportionally related to carcinogenesis. This is alarmingly dangerous in youth and children, since these effects last longer, affecting fertility or forming basis for carcinogenesis. There is paucity of reports on planned studies of imatinib on the testicular function. Hence, the study was planned to assess the effects of imatinib on biochemical markers of testicular functions in male Swiss albino mice.

Materials and Methods:

Male Swiss albino mice were treated with imatinib and sacrificed at the end of first, second, fourth, fifth, seventh, and tenth week after the last exposure to imatinib. The testis were removed, weighed, and processed for biochemical analysis.

Results:

The intratesticular testosterone level was significantly (P<0.001) reduced in treated groups and severe effect was observed on week 4 and 5. The intratesticular lactate dehydrogenase (LDH) level was significantly increased by imatinib in all treated groups up to week 5.

Conclusion:

Imatinib does affect testosterone and LDH level significantly, but this effect is reversible once the drug is withdrawn. This finding may help the clinicians to plan and address the fertility-related issues in young patients of reproductive age who are being treated with imatinib for gastrointestinal tumors and chronic myeloid leukemia.     

Aerodynamical, Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization

Purpose

Despite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine.

Materials and methods

Cetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+®, a mesh nebulizer AeronebPro® and an ultrasonic nebulizer SYST’AM® LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells.

Results

The aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab.

Conclusions

Altogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.     

Section 3 Observations on lactic dehydrogenase activity in blood of patients treated with D-penicillamine for rheumatoid arthritis

Summary

A proportion of patients (11 out of 23) treated for rheumatoid arthritis with D-penicillamine have shown raised plasma levels of lactic dehydrogenase. No explanation of this has been discovered on investigation. It is not considered that these observations indicate a significant toxic effect of D-penicillamine.     

Stabilizing Peptide Fusion for Solving the Stability and Solubility Problems of Therapeutic Proteins

Purpose Protein aggregation is a major stability problem of therapeutic proteins. We investigated whether a novel stabilizing peptide [acidic tail of synuclein (ATS) peptide] could be generally used to make a more stable and soluble form of therapeutic proteins, particularly those having solubility or aggregation problems. Methods We produced ATS fusion proteins by fusing the stabilizing peptide to three representative therapeutic proteins, and then compared the stress-induced aggregation profiles, thermostability, and solubility of them. We also compared the in vivo stability of these ATS fusion proteins by studying their pharmacokinetics in rats. Results The human growth hormone–ATS (hGH–ATS) and granulocyte colony-stimulating factor–ATS (G-CSF–ATS) fusion proteins were fully functional as determined by cell proliferation assay, and the ATS fusion proteins seemed to be very resistant to agitation, freeze/thaw, and heat stresses. The introduction of the ATS peptide significantly increased the storage and thermal stabilities of hGH and G-CSF. The human leptin–ATS fusion protein also seemed to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. Furthermore, the ATS peptide greatly increased the solubility of the fusion proteins. Finally, pharmacokinetic studies in rats revealed that the ATS fusion proteins are also more stable in vivo. Conclusion Our data demonstrate that a more stable and soluble form of therapeutic proteins can be produced by fusing the ATS peptide. E. N. Lee and Y. M. Kim equally contributed to this work.     

Use of Microcalorimetry and Its Correlation with Size Exclusion Chromatography for Rapid Screening of the Physical Stability of Large Pharmaceutical Proteins in Solution

The utility of microcalorimetry as a rapid screening tool for assessing the solution stability of high molecular weight pharmaceutical proteins was evaluated by using model recombinant antibodies, Protein I and Protein II. Changes in the transition midpoint, T_m, were monitored as a function of pH and/or in the presence of excipients, and results were compared with traditional accelerated stability data from samples that were analyzed by size exclusion chromatography (SEC). The data from microcalorimetry were well correlated with those from SEC for predicting both optimal solution pH as well as excipient effects on solution stability. These results indicate that microcalorimetry can be an efficient screening tool useful in identifying optimal pH conditions and excipients to stabilize pharmaceutical proteins in solution formulations.     

大鼠脑外伤后LDH和Caspase-9的表达及意义

目的:探讨大鼠脑外伤后乳酸脱氢酶(LDH)和Caspase-9 的表达及其时序性变化,并探讨脑损伤的分子机制.方法:采用改良的自由落体法,制备成脑损伤模型.采用分光光度法检测指标LDH,Caspase-9 的活性测定.结果:LDH 1h 检测到下降,6h降低到最低,从6h后开始升高,到24h达到高峰,之后又开始下降.Caspase-9 检测到,1h低表达,3h开始升高,12h 达到高峰,1d 时开始显著下降.结论:大鼠脑外伤后可诱发LDH,Caspase-9的表达,随着时间的增加呈现一定的变化趋势.     

心房颤动与C反应蛋白相关性的临床研究

目的研究心房颤动与C反应蛋白之间的相关性。方法选取我院在2011年7月至2013年8月间收治的90例高血压患者的临床资料,其中永久性房颤36例、阵发性房颤27例、正常对照组27例。对3组患者的C反应蛋白进行测定与比较。结果永久性房颤患者的C反应蛋白水平最高,与阵发性房颤组患者相较,对比差异显著,具有统计学意义(P<0.05)。房颤组患者的C反应蛋白水平更高,明显高于正常对照组,具有统计学意义(P<0.05)。结论在心房颤动患者中,其C反应蛋白上升非常明显,这表明在高血压心房颤动中,炎症状态起到了心房颤动得以维持的作用。     

阿奇霉素和头孢他啶治疗难治性肺炎支原体肺炎患儿的临床对照研究

目的 探讨阿奇霉素和头孢他啶对难治性肺炎支原体肺炎患儿的临床疗效及血清乳酸脱氢酶（LDH）的变化影响。方法 2014年1月—2017年1月选择在郑州市妇幼保健院诊治的120例难治性肺炎支原体肺炎患儿作为研究对象,根据随机信封抽签原则分为观察组与对照组各60例,观察组在常规治疗基础上给予阿奇霉素辅助治疗,0.25 g/次,1次/d,对照组在常规治疗基础上给予头孢他啶辅助治疗,10～50 mg/kg加入0.5%氯化钠注射液50～100 mL内,静脉泵入,2次/d。两组都治疗14 d。结果 观察组与对照组患儿总有效率分别为98.3%和86.7%,观察组患儿的总有效率显著高于对照组,差异有统计学意义（P<0.05）。观察组与对照组患儿治疗后的血清LDH水平分别为（143.22±26.29）U/L和（245.10±30.91）U/L,都明显低于治疗前的（445.20±35.10）U/L和（438.10±56.11）U/L（P<0.05）;且治疗后观察组患儿的血清LDH水平也明显低于对照组,差异有统计学意义（P<0.05）。观察组患儿治疗期间的皮疹、胃肠道反应、疼痛、发热等不良反应发生率为11.7%,对照组为10.0%,两组对比无明显差异。结论 相对于头孢他啶,阿奇霉素治疗难治性肺炎支原体肺炎患儿能提高治疗效果,且不会增加不良反应,其作用机制可能与促进血清LDH含量降低有关。     

植物生物反应器生产药用重组蛋白质的研究进展

利用转基因植物作为生物反应器规模化生产药用重组蛋白质是一个新兴的研究领域.本文就转基因植物生物反应器的优点、几种常用的植物表达系统、采用转基因植物生产药用重组蛋白质如疫苗、重组抗体和重组人类防御素等方面的研究进展作一综述.     

Comparison of the endothelial toxicity induced by short-term amiodarone and diazepam exposure in a human umbilical vein endothelial cell line (EVC304)

Context: Venous irritation is the most common side effect of intravenous therapy. Although many in vitro models have been developed to evaluate intravenous drug irritation, these models are not widely accepted.

Objectives: The aim of this paper is to determine whether delayed or immediate cytotoxicity better reflects the in vivo venous irritation ranking.

Materials and methods: We compared the endothelial toxicity induced by high-concentrations of amiodarone and diazepam after short-term exposure (20?min) in a human umbilical vein endothelial cell line (EVC304) by using five in vitro models: lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD), glutathione (GSH), adenosine triphosphate (ATP), and MTT assays.

Results: In the 24-h MTT assay, the IC₅₀ of diazepam and amiodarone was 1.08 and 1.96?mM, respectively. In the 48-h MTT assay, the IC₅₀ of diazepam and amiodarone was 1.114 and 1.128?mM, respectively. In the intracellular LDH and G6PD assays, the EC₅₀ of diazepam was found to be 3.307 and 1.53?mM, while the values of amiodarone were 0.853 and 0.325?mM, respectively. In the intracellular ATP and GSH assays, the EC₅₀ of diazepam was 0.905 and 1.283?mM, while the values of amiodarone were 0.040 and 0.326?mM, respectively.

Conclusion: Both the results of intracellular macromolecule activities and micromolecule concentrations were similar to that observed in in vivo venous irritation studies. However, the delayed cytotoxicity rank from the MTT assay is inconsistent with the in vivo venous irritation rank, suggesting that initial toxicity, but not the delayed toxicity, is related to venous irritation.     

养心复脉饮对心血管系统的作用

目的:研究养心复脉饮对实验性心肌缺血动物的保护作用。方法:应用垂体后叶素等造成动物心肌缺血和其他心功能改变。结果:养心复脉饮对注射垂体后叶素(p it)大鼠血清中磷酸肌酸激酶(CK)和乳酸脱氢酶(LDH)的升高有一定的缓解作用,能明显减少p it大鼠心肌组织丙二醛(MDA)的生成,保护大鼠心肌组织超氧化物歧化酶(SOD)的活性;对氯仿所致小鼠心律失常有明显的保护作用;能明显延长异丙肾上腺素(Iso)小鼠耐缺氧时间。结论:养心复脉饮对心功能有一定的保护作用。