Effect of DA-8159, a selective phosphodiesterase type 5 inhibitor, on electroretinogram and retinal histology in rabbits

DA-8159, a selective inhibitor of phosphodiesterase type 5, was developed as a new drug for erectile dysfunction. The effect of DA-8159 on the electroretinogram (ERG) and the retinal histopathology were evaluated in rabbits. The ERG was performed prior to, and 1 and 5 hr after DA-8159 (5 to 30 mg/kg) administration. The plasma concentration of DA-8159 was determined at each time point, and retinal microscopic examination was also performed. There was no statistically significant ERG change at any dose or at any time. Though the 30 Hz flicker showed a prolongation of the implicit time at 5 hr after the administration of either DA-8159 15 mg or 30 mg/kg (p<0.05), but concurrent amplitude decreases were not statistically significant. At a dose of 5 mg/kg, no test drug was detected in the blood after either 1 or 5 hr. At either 15 mg/kg or 30 mg/kg, there was a dose-dependent increase in the blood concentration after 1 hr of drug administration, which decreased with time. In light and electron microscopic examinations of the retina, there was no remarkable change at any dose. These results suggest DA-8159 has a low risk potential to the retina, but further evaluation on the visual functions in human is needed.     

玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。     

柔红霉素、骆驼蓬总碱对兔视网膜的毒性观察

目的 探讨药物柔红霉素(daunorubicin,DNR)、骆驼蓬总碱(total alkaloid Of Harmaline,TAH)眼内注射防治后发性白内障对视网膜的毒性作用.方法 于兔右眼晶体囊摘除(extracapsular lens extraction,ECLE)术中分别前房内注射0.2mg/mlTAH和0.2mg/ml DNR溶液0.1ml,通过眼底镜检查、视网膜电图及眼组织病理学等研究对象兔眼视网膜的影响.结果 对照眼与15只注入TAH眼眼底无明显的病理变化;TAH眼手术前后视网膜电图b波的振幅和潜时与对照眼无差别.而10只注入DNR眼出现严重的内皮性角膜水肿混浊,前房大量纤维索性渗出特等明显的炎性反应眼底及视网膜电图无法检查.组织病理学检查显示TAH组眼前节结构及视网膜各层结构正常,细胞排列规律;透射电镜检查TAH眼的视网膜感光细胞外节盘膜结构清晰.排列整齐.视网膜细胞排列紊乱,部分明显呈核固缩,表现出明显的毒性反应.结论 TAH眼内注射对兔视网膜的毒性较小,有可能用于后发性白内障的防治研究,而DNR对兔视网膜及其它组织表现出明显毒性,应进一步研究其使用的安全剂量和剂型.     

Nanoparticle drug loading as a design parameter to improve docetaxel pharmacokinetics and efficacy

Nanoparticle (NP) drug loading is one of the key defining characteristics of an NP formulation. However, the effect of NP drug loading on therapeutic efficacy and pharmacokinetics has not been thoroughly evaluated. Herein, we characterized the efficacy, toxicity and pharmacokinetic properties of NP docetaxel formulations that have differential drug loading but are otherwise identical. Particle Replication in Non-wetting Templates (PRINT^®), a soft-lithography fabrication technique, was used to formulate NPs with identical size, shape and surface chemistry, but with variable docetaxel loading. The lower weight loading (9%-NP) of docetaxel was found to have a superior pharmacokinetic profile and enhanced efficacy in a murine cancer model when compared to that of a higher docetaxel loading (20%-NP). The 9%-NP docetaxel increased plasma and tumor docetaxel exposure and reduced liver, spleen and lung exposure when compared to that of 20%-NP docetaxel.     

An assessment of the ocular safety of excipient maleic acid following intravitreal injection in rabbits

Maleic acid was formulated in 0.7% saline and injected intravitreally in rabbits in order to evaluate ocular safety and tolerability. Maleic acid was formulated within a narrow pH range (2-3), administered in a fixed volume (100 μl), and concentrations ranged from 0.00 to 2.00 mg/eye (0.00 to 12.30 mM vitreous). Ocular evaluations were conducted at 2, 4, and 8 days post injection. Ocular irritation responses were observed at doses from 0.50 mg/eye (3.07 mM vitreous) to 2.00 mg/eye (12.30 mM vitreous) and included conjunctival redness and scleral swelling. Chemosis was observed at 2.00 mg/eye (12.30 mM vitreous). Funduscopic evaluations revealed enlarged retinal blood vessels and optic disk swelling at doses ≥1.50 mg/eye (9.22 mM vitreous), retinal folds and retinal discoloration at 2.00 mg/eye (12.30 mM vitreous). Histopathologic evaluations on days 4 and 8 post injection revealed retinal degeneration at doses ≥1.0 mg/eye (6.15 mM vitreous), conjunctival inflammation at doses ≥1.5 mg/eye (9.22 mM vitreous), and retinal pigment epithelial hypertrophy, optic nerve demyelination, anterior chamber fluid, and conjunctival fibrosis at 2.00 mg/eye (12.30 mM vitreous) maleic acid. The data suggest that maleic acid formulations at ≥1.00 mg/eye (6.15 mM vitreous) were not suitable for intraocular indications.     

Ocular disposition and tolerance of ganciclovir-loaded albumin nanoparticles after intravitreal injection in rats

Cytomegalovirus (CMV) infection mainly affects endothelial cells of ocular vessels, optic nerve and the retina, resulting in direct or autoimmune damages, uveoretinitis and disturbed vision. The use of colloidal carriers for the intravitreal delivery of ganciclovir may prolong its residence in the eye, minimizing the opacification observed for macroscopic implants. The aim of this work was to evaluate the ocular toxicity induced by the prolonged presence of ganciclovir-loaded bovine serum albumin nanoparticles after their intravitreal injection. The intraocular disposition of these carriers was also studied by immunochemistry. Two weeks post-injection, a significant amount of nanoparticles remained in the vitreous cavity, mainly in a thin layer overlying the retina and in the area close to the blood aqueoUs barrier. Their prolonged residence in the eve seemed to be well tolerated and the histological evaluation of the retina, mainly the photoreceptor layer, and adjacent tissues revealed the absence of inflammatory reactions or alterations in the tissue architecture (i.e. cellular infiltrations or vascular inflammation). In addition, nanoparticles neither alter the expression and distribution of arrestin and rhodopsin autoantigens nor the mineralocorticoid receptor. In summary, the vision was not affected by autoimmune phenomena or alterations in the behavior of ophthalmic cells due to the intravitreal injection of these nanoparticles.     

Biocompatibility and biodegradation of intravitreal hyaluronan implants in rabbits

To study the biocompatibility and the biodegradation rate in vivo of new intravitreal implants made with three different hyaluronic acid esters: Hyaff7, Hyaff11 and Hyaff11p75 (100% ethyl ester, 100 and 75% benzyl esters, respectively), the plugs were implanted through a sclerotomy at 3.5 mm from the limbus of rabbit eyes. In order to evaluate the in vivo biodegradation the shaft diameter of the plugs was measured by ultrasound biomicroscopy. Slit lamp microscopy, ophthalmoscopy and ERG were performed periodically. The effects of the implants on ocular tissues were also evaluated histologically. All the plugs showed a good biocompatibilitv. Plugs of both the total esters, Hyaff7 and Hyaff11, were found to undergo a slow dissolution process for 60 and 150 days, respectively. The partial benzyl ester, Hyaff11p75, was completely reabsorbed after 15 days. Analysis of variance showed a high correlation between biodegradation rate and the time of resorption (F = 90.5; p < 0.001). The biodegradation rate of each implant is related to the chemical structure of the three types of Hyaff (F = 4.51; p = 0.005). The present data suggest that intravitreal implants based on hyaluronic acid esters represent useful biocompatible and biodegradable devices for a potential drug delivery system in the treatment of posterior segment ocular diseases.     

阿昔洛韦联合小儿肺热咳喘口服液对婴幼儿呼吸道合胞病毒肺炎的疗效分析

目的 研究阿昔洛韦联合小儿肺热咳喘口服液对婴幼儿呼吸道合胞病毒肺炎的疗效.方法 选择2012年10月～ 2015年9月在我院进行诊治的呼吸道合胞病毒肺炎患儿90例,将其随机分为观察组与对照组,每组各45例.观察组采用阿昔洛韦联合小儿肺热咳喘口服液治疗,对照组采用阿昔洛韦治疗,比较两组治疗效果.结果 观察组总有效率为93.33％,明显高于对照组84.44％(P＜0.05);两组患儿的住院时间、咳嗽减轻时间、体温复常时间、哕音消失时间分别进行统计学比较,均有显著性差异(P＜0.05);治疗前两组患儿CD3+、CD4+、CD8+和IL-2水平比较,差异无统计学意义(P＞0.05),同组治疗后CD4+、CD8+和IL-2水平与治疗前相比,差异有统计学意义(P＞0.05),两组治疗后CD3+、CD4+、CD8+和IL-2水平无明显差异(P＞0.05);两组不良反应发生率比较差异无统计学意义(P＞0.05).结论 阿昔洛韦联合小儿肺热咳喘口服液治疗小儿呼吸道合胞病毒肺炎具有显著的临床效果,值得推广应用.     

The movement of self-assembled amphiphilic polymeric nanoparticles in the vitreous and retina after intravitreal injection

The purpose of this study is to determine the correlation between the distribution of nanoparticles in the vitreous and retina and their surface properties after intravitreal injection. For this purpose, we synthesized seven kinds of nanoparticles through self-assembly of amphiphilic polymer conjugates in aqueous condition. They showed similar size but different surface properties. They were labeled with fluorescent dyes for efficient tracking. After intravitreal injection of these nanoparticles into a rodent eye, their time-dependent distribution in the vitreous and retina was determined in stacking tissue images by confocal microscopy. The results demonstrated that the surface property of nanoparticles is a key factor in determining their distribution in the vitreous and retina after intravitreal injection. In addition, immunohistochemistry and TEM images of retina tissues suggested the important mechanism related with Mülller cells for intravitreally administered nanoparticles to overcome the physical barrier of inner limiting membrane and to penetrate into the deeper retinal structures. Therefore, we expect that this study can provide valuable information for biomedical researchers to develop optimized nanoparticles as drug or gene carriers for retinal and optic nerve disorders such as glaucoma, age-related macular degeneration, and diabetic retinopathy.     

Pulmonary Toxicity after a Quick Course of Combinatorial Vincristine, Bleomycin, and Cisplatin Neoadjuvant Chemotherapy in Cervical Cancer

Pulmonary toxicity is one of the most serious adverse effects associated with a quick course of vincristine, bleomycin, and cisplatin neoadjuvant chemotherapy (NAC-VBP). The aim of this study was to evaluate pulmonary toxicity related to a quick course NAC-VBP. A total of consecutive 61 patients, who underwent at most 3 cycles of NAC-VBP every 10 days in the International Federation of Gynecology and Obstetrics (FIGO) stage IB-IIB cervical cancer from 1995 to 2007, were retrospectively analyzed. Of the 61 study subjects, 7 (11.5%) were identified to have pulmonary toxicity and 2 (3.3%) died of pulmonary fibrosis progression despite aggressive treatment and the use of a multidisciplinary approach. No factor predisposing pulmonary toxicity was identified. Initial symptoms were non-specific, but bronchiolitis obliterans organizing pneumonia and interstitial pneumonitis were characteristic findings by high-resolution computed tomography of the chest. The benefit of steroid therapy was uncertain and was associated with steroid-induced diabetes mellitus requiring insulin therapy in two patients. Fatal pulmonary toxicity is a major concern of a quick course NAC-VBP. In conclusion, these patients require special monitoring for bleomycin-induced pulmonary toxicity.     

自发性高血压大鼠苯丙氨酸代谢动力学异常

目的和方法：自发性高血压大鼠（ＳＨＲ）及相应的正常血压对照大鼠（Ｗｉｓｔａｒ Ｋｙｏｔｏ大鼠,ＷＫＹ）静注［＾３Ｈ］苯丙氨酸后的药物动力学及组织摄取。结果：苯丙氨酸在大鼠的药物动力学符合二室开放模型。与ＷＫＹ相比,ＳＨＲ中苯丙氨酸消除半衰期延长,清除率缩小,药时曲线下面积增加,中央室表观分布容积增加,心脏对苯丙氨酸摄取增加。结论：高血压药可能是一种与遗传性氨基酸代谢异常有关的疾病。     

Reduced occurrence of programmed cell death and gliosis in the retinas of juvenile rabbits after shortterm treatment with intravitreous bevacizumab

OBJECTIVE:

Bevacizumab has been widely used as a vascular endothelial growth factor antagonist in the treatment of retinal vasoproliferative disorders in adults and, more recently, in infants with retinopathy of prematurity. Recently, it has been proposed that vascular endothelial growth factor acts as a protective factor for neurons and glial cells, particularly in developing nervous tissue. The purpose of this study was to investigate the effects of bevacizumab on the developing retinas of juvenile rabbits.

METHODS:

Juvenile rabbits received bevacizumab intravitreously in one eye; the other eye acted as an untreated control. Slit-lamp and fundoscopic examinations were performed both prior to and seven days after treatment. At the same time, retina samples were analyzed using immunohistochemistry to detect autophagy and apoptosis as well as proliferation and glial reactivity. Morphometric analyses were performed, and the data were analyzed using the Mann-Whitney U test.

RESULTS:

No clinical abnormalities were observed in either treated or untreated eyes. However, immunohistochemical analyses revealed a reduction in the occurrence of programmed cell death and increases in both proliferation and reactivity in the bevacizumab-treated group compared with the untreated group.

CONCLUSIONS:

Bevacizumab appears to alter programmed cell death patterns and promote gliosis in the developing retinas of rabbits; therefore, it should be used with caution in developing eyes.     

微透析活体采样比较静脉和玻璃体内注射万古霉素在兔玻璃体内的通透性

背景：目前眼部药代动力学研究的人体及动物实验采样方法,均是在体外进行,存在诸多的弊端。 目的：利用微透析活体采样技术,建立眼后节清醒动物药代动力学模型,比较静脉注射和玻璃体内注射万古霉素在兔玻璃体内的通透性。 方法：纳入15只兔,制备眼内炎模型。将微透析探针植入清醒兔眼玻璃体内24 h后,随机分成3组,即静脉注射组、玻璃体内注射组及静脉注射+玻璃体内注射组,分别根据不同的给药方式注射万古霉素。高效液相色谱-紫外检测法连续检测兔眼玻璃体万古霉素的浓度,3p97药代动力学软件拟合药动学参数。 结果与结论：静脉注射组兔眼玻璃体内的药物浓度较低,达不到有效的治疗效果;玻璃体内注射组及静脉注射+玻璃体内注射组兔眼给药后72 h,玻璃体内万古霉素的浓度均高于有效治疗浓度。提示微透析方法联合高效液相色谱-紫外检测法,可以连续、在线、活体检测清醒动物玻璃体内药物浓度;单次静脉注射万古霉素后,玻璃体内不能达到有效治疗剂量。     

美沙拉嗪肠溶片在健康人体的药代动力学及生物等效性研究

目的:研究进口美沙拉嗪(5-ASA)肠溶片在健康人体的药代动力学,并与国产片剂进行生物等效性比较。方法:18名健康男性受试者分别随机交叉口服美沙拉嗪肠溶片试验药或参比药0.5 g,每8小时一次,连续七次。用柱前衍生化—HPLC—荧光法测定给药后不同时间点血浆中5-ASA和其活性代谢产物乙酰化—5-氨基水杨酸(Ac-5-ASA)的浓度,计算药代动力学参数并评价两种制剂的生物等效性。结果:试验药及参比药的原形药(5-ASA)的药代动力学参数分别为:AUC0-t10.25±3.96和9.45±2.94μg.h.ml-1;Cmax2.21±1.49和1.98±1.18μg.ml-1;tmax4.83±2.32和4.47±3.15 h。其代谢产物(Ac-5-ASA)的药代动力学参数分别为:AUC0-t30.29±8.62和29.90±7.45μg.h.ml-1;Cmax3.21±1.02和3.28±1.20μg.ml-1;tmax5.94±2.54和4.31±3.29 h。经统计分析,上述各项参数间差别均无显著性意义(p>0.05)。进口美沙拉嗪肠溶片相对生物利用度为(108.4±27.4%)。结论:美沙拉嗪肠溶片试验药与参比药具有生物等效性。     

LC-MS/MS测定大鼠血浆中黄藤素含量及其药代动力学研究

目的建立大鼠血浆中黄藤素的LC-MS/MS定量方法,并利用此方法考察黄藤素在大鼠体内的药物动力学。方法色谱柱为ZorbaxEclipseXDB-C1(82.1mm×50mm,1.8μm),流动相为0.1%甲酸溶液-乙腈(70:30),采用多级反应模式(MRM)检测;通过大鼠眼底静脉取血,血浆经乙腈沉淀蛋白,取上清液进行LC-MS/MS分析,测定黄藤素血药浓度,通过DAS2.0计算其药动学参数。结果血浆中黄藤素在0.442~44.2ng/ml范围内呈现良好的线性关系,方法学精密度、回收率、稳定性等均符合要求;大鼠体内黄藤素药动学符合二室模型特征,灌胃给予40mg/kg黄藤素后,大鼠血浆中的黄藤素浓度在2.8h达峰值,t1/2为6h,最大血药浓度为19.8ng/ml。结论本研究建立的大鼠体内黄藤素测定方法灵敏度高、专一性好,可用于黄藤素的体内药物动力学研究,为黄藤素应用研究提供了有力支持。     

进行性肌营养不良患者视网膜眼电图表型与临床分型及基因型的关系

目的 研究进行性肌营养不良（Duchenne/Becker muscular dystrophy,DMD/BMD)患者视网膜眼电图（electroretinogram,ERG)表型与临床分型以及基因型的关系。进一步探讨不同基因型的DMD患者抗肌营养不良蛋白（dystrophin)及其同源蛋白在视网膜上的表面爱功能,揭示DMD出现ERG异常的分子机理,方法 用11对引物对22例临床确诊的DMD／BMD患者作三步多重PCR进行基因缺失分析,并行ERG检查,结果 DMD／BMD患者ERG改变与临床分型及病情严重程度无关,与DMD／BMD的基因型有关,基因中央区缺失型的ERG异常率明显高于基因非缺失型,结论 DMD／BMD的ERG改变与DMD基因突变位点有关,可能DP260转录启动子与视网膜电信号的传导关系最密切。     

近红外实时在位检测系统及其在药代动力学中的应用研究

药代动力学研究中迫切需要一种实时在位检测方法。本文提出一种新的基于荧光光谱技术的实时在位检测方法,并搭建了一套用于大鼠体内药代动力学研究的监测系统,得到荧光强度和染料浓度的关系曲线,对大鼠体内荧光染料Cypate(近红外多次甲基菁染料)进行实时检测,并和体外荧光成像系统的结果比较来验证分析系统的可行性。结果表明:(1)Cypate浓度在0.098～25μg/ml范围内线性回归方程为y=73.249x+130.97(R2=0.999 1,P<0.001),高、中、低浓度的RSD分别为1.23%、6.29%和13.48%,检测灵敏度达到0.098μg/ml,特异性好;(2)Cypate的浓度变化与体外成像系统的结果基本一致(r=0.992 5),很好地反映了它在大鼠体内的代谢过程。论文的结论是为药代动力学研究提供了一种新的实时在位检测方法。