PML-RARα融合基因相关蛋白原核表达载体的构建、表达和纯化

目的 构建PML-RAR o[融合基因相关重组表达质粒,在大肠埃希菌中表达可溶性蛋白并进行纯化.方法 利用聚合酶链反应(Polymerase Chain Reaction,PCR)以PML-RAR α全长质粒为模板分别扩增出长度均为1 200 bp的PML和RAR o[序列,并将它们分别插入PET32a(+)质粒中,构建重组表达质粒,化学法转化大肠埃希菌DH5α进行克隆,菌落PCR筛选阳性转化子,双酶切和测序鉴定重组质粒的正确性.正确重组的表达质粒分别命名为PML-Flag-PET32a(+)和Flag-RARα-PET32a(+).将正确重组的表达质粒转化感受态大肠埃希菌BL21(DE3),经异丙基β-D-半乳糖苷(IPTG)诱导表达,利用表达蛋白的组氨酸“标签”(His-tag)进行Ni2+-树脂柱亲和层析纯化,SDS-PAGE和Western blotting鉴定纯化蛋白质.结果 PCR扩增获得目的基因,重组表达质粒经EcoRI/HindⅢ双酶切和测序鉴定证明构建正确.转化感受态BL21(DE3)并经诱导后得到高效表达,SDS-PAGE和Western blotting显示纯化蛋白为目的蛋白.结论 成功构建重组表达质粒,并经诱导表达后可纯化出目的蛋白,为进一步的抗体制备等实验奠定了基础.     

10种中草药对临床常见致病菌体外抗菌作用的实验

目的:研究黄连、黄芩、连翘等10种中草药对临床常见致病菌(大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌)的抗菌作用。方法:制备黄连、黄芩、连翘等10种中草药提取物,采用纸片琼脂扩散法测定抑菌圈大小,并考察提取物琼脂平板稀释后的细菌生长情况。结果:10种中草药的提取物分别对3种受试菌(大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌)呈现不同程度的抑菌作用,其中黄连、黄芩、连翘对金黄色葡萄球菌呈高度敏感,抑菌圈均大于6 mm;连翘对铜绿假单胞菌抑菌圈大于11 mm,大肠埃希菌抑菌圈均等于6 mm。结论:黄连、黄芩、连翘、大黄、黄柏对金黄色葡萄球有不同程度的抗菌作用,连翘对铜绿假单胞菌有抗菌作用,10种中草药对大肠埃希菌的抗菌作用为最差;由于中草药在体内作用机制比体外更为复杂,在体内能否达到与体外相同效果尚待进一步研究。     

胆管癌相关FXYD6基因功能区在大肠埃希菌表达的初步研究

目的对胆管癌相关基因FXYD6基因的功能区进行克隆和表达,以便进一步展开该基因功能研究。方法借助生物信息学方法分析FXYD6基因,设计FXYD6基因信号肽剪切后功能区蛋白体外表达序列,并将其克隆到pBV220表达载体上,继而以大肠埃希菌为表达系统,使功能区蛋白（不含信号肽）获得体外表达。结果该基因主要功能位点位于18～95 aa。PCR扩增出剪切掉信号肽序列的功能区序列（即包含18～95 aa的肽链段）,并成功构建pBV220/FXYD6-ex基因功能区重组表达质粒克隆,其在大肠埃希菌DH5α中表达量最高,表达产物主要以包涵体形式存在。结论获得胆管癌相关FXYD6基因融合蛋白表达产物,为后续研究打下基础。     

Streptomyces sp.4353中3-氨基-5-羟基-苯甲酸（AHBA）合酶基因的克隆及其在大肠埃希菌中的表达

目的 克隆Streptomyces sp.4353中的3-氨基-5-羟基-苯甲酸(AHBA)合酶基因,并将其在大肠埃希菌中表达.方法参与AHBA生物合成的5个基因,即氨基奎尼酸脱水酶基因、氨基奎尼酸/莽草酸脱氢酶基因、AHBA合酶基因、氧化还原酶基因和磷酸酶基因通常连锁,形成一个AHBA生物合成基因簇.本文通过分析、比较已知AHBA生物合成基因簇的基因组织结构特点和序列保守性,设计引物,以Streptomyces sp.4353的总DNA为模板,在已知737bp AHBA合酶基因部分序列的基础上,通过PCR扩增其上、下游DNA序列,克隆至pMD18-T载体上,测序、拼接并进行生物信息学分析.以pET24a(+)为表达载体,对克隆得到的AHBA合酶基因在大肠埃希菌BL21-Codon Plus(DE3)-RIL中进行表达和SDS-PAGE检测.结果从Streptomyces sp.4353中得到了一段2872bp大小的DNA片段(NCBI GenBank接受号EU734184),其中含有AHBA生物合成基因簇中的AHBA合酶基因、氧化还原酶基因和磷酸酶基因(部分);AHBA合酶基因在大肠埃希菌中得到了成功表达,在SDS-PAGE上可见到清晰的、与预计大小(42.7ku)一致的蛋白表达特异条带.结论 Streptomyces sp.4353中的AHBA合酶基因的克隆以及在大肠埃希菌中的表达为进一步研究该基因的功能奠定了基础.本研究克隆得到的2872bp DNA片段还有可能用于AHBA的杂合/异源生物合成,以及安莎类抗生素的组合生物合成.     

四季青水煎液体外抗内毒素、抗菌和体内抗炎作用

目的 研究四季青水煎液的抗内毒素、抗菌和抗炎作用,揭示四季青清热解毒作用的药效学特点。方法 采用体外鲎试剂凝集实验方法检测其抗内毒素作用;采用琼脂二倍稀释法测定四季青水煎液以及与头孢噻肟、左氧氟沙星和庆大霉素联合使用时对25株甲氧西林敏感金黄色葡萄球菌(MSSA)、耐甲氧西林金黄色葡萄球菌(MRSA)、产超广谱β-内酰胺酶(extended-spectrum β-lactamase, ESBLs)大肠埃希菌、非产ESBLs大肠埃希菌和铜绿假单胞菌的最低抑菌浓度(minimum inhibitory concentration, MIC);采用二甲苯致小鼠耳廓炎症肿胀法观察其抗炎作用。结果 四季青水煎液在生药浓度6.25~100mg/mL时具有抗内毒素作用;对受试菌株MSSA、MRSA的MIC范围均为8~33mg/mL,对产ESBLs大肠埃希菌及非产ESBLs大肠埃希菌的MIC范围均为33~>133mg/mL,对铜绿假单胞菌的MIC为33~133mg/mL;与头孢噻肟、左氧氟沙星和庆大霉素联合使用时抗菌活性有相加作用,分级抑菌浓度(fractional inhibitory concentration, FIC)指数范围为0.5相似文献   

临床药学监测在头孢哌酮/舒巴坦治疗呼吸道感染多重耐药革兰阴性菌中的应用

目的探讨临床药学监测在头孢哌酮/舒巴坦治疗呼吸道感染多重耐药革兰阴性菌中的应用。方法收集2016年1~12月期间我院病原学送检标本5880个,所有标本均进行痰液病原菌培养,采用K-B纸片扩散法测定头孢哌酮/舒巴坦对多重耐药革兰阴性菌的抗菌活性,统计分析所有标本病原菌感染及其耐药性情况。结果本次研究中,共检出革兰阴性菌植株1100株(18.71%),其主要以铜绿假单胞菌263株(4.47%)、肺炎克雷伯菌254株(4.32%)、大肠埃希菌189株(3.21%)为主,多重耐药菌186株(3.16%),其主要以大肠埃希菌62株(1.05%)、鲍曼不动杆菌46株(0.78%)、肺炎克雷伯菌254株(0.48%)为主;抗菌活性分析结果显示,头孢哌酮/舒巴坦对多重耐药革兰阴性菌抗菌活性最高的三种细菌分别为大肠埃希菌93.55%、铜绿假单胞菌85.71%、肺炎克雷伯菌82.14%。结论铜绿假单胞菌、肺炎克雷伯菌、大肠埃希菌是本次呼吸道感染主要的革兰阴性菌感染菌,其中大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌是该疾病主要的多重耐药菌,头孢哌酮/舒巴坦对多重耐药菌的抗菌活性存在差异,应针对病原菌合理性使用。     

3种常用藏药的体外抑菌活性研究

目的 测定藏药七十味珍珠丸、八味秦皮丸、仁青常觉的体外抑菌活性。方法 用肉汤稀释法和牛津杯法研究体外抑菌活性,根据最小抑菌浓度(MIC)及抑菌圈大小判断3种藏药的抑菌活性。结果 七十味珍珠丸的抑菌作用由强到弱为金黄色葡萄球菌>枯草芽孢杆菌>铜绿假单胞菌>乙型副伤寒沙门菌>白色念珠菌>大肠埃希氏菌;八味秦皮丸的抑菌作用由强到弱依次为金黄色葡萄球菌>枯草芽孢杆菌>铜绿假单胞菌>乙型副伤寒沙门菌>大肠埃希氏菌>白色念珠菌;仁青常觉的抑菌作用由强到弱依次为金黄色葡萄球菌>枯草芽孢杆菌>铜绿假单胞菌>白色念珠菌>乙型副伤寒沙门菌>大肠埃希氏菌。结论 3种藏药对6种试验菌株的生长均具有不同程度的抑制作用,普遍对金黄色葡萄球菌、枯草芽孢杆菌和铜绿假单胞菌具有较强的抑制作用,其中八味秦皮丸对大肠埃希菌的生长具有显著的抑制作用,仁青常觉对白色念珠菌的生长抑制作用强于七十味珍珠丸和八味秦皮丸。     

五种碳青霉烯类抗生素对大肠埃希菌和铜绿假单胞菌的体外抗菌活性比较

目的 观察五种碳青霉烯类抗生素对大肠埃希菌和铜绿假单胞菌的体外抗菌活性.方法 采用琼脂稀释法检测5种碳青霉烯类抗生素对55株铜绿假单胞菌、49株产ESBLs的大肠埃希菌的最低抑菌浓度(minimalinhibitoryconcentration,MIC).结果 对抗铜绿假单胞菌活性以美洛培南和比阿培南显示最佳,亚胺培南次之,帕尼培南逊于亚胺培南,厄他培南明显逊于其他四种碳青霉烯类抗生素.对产ESBLs的大肠埃希菌厄他培南的活性明显高于其他几种碳青霉烯类抗生素,而其他几种活性基本相当.结论 铜绿假单胞菌对五种碳青霉烯类抗生素耐药性均较高,而五种碳青霉烯类抗生素对产ESBLs的大肠埃希菌均敏感.     

临床药学监测头孢哌酮/舒巴坦治疗呼吸道感染多重耐药革兰阴性菌中的应用价值分析

目的分析临床药学监测头孢哌酮/舒巴坦治疗呼吸道感染多重耐药革兰阴性菌中的应用价值。方法本次研究对象选取来我院接受病原学检查的标本4000个,所有的标本均进行痰液病原菌培养,采取K-B纸片扩散法来测定头孢哌酮/舒巴坦对多药耐药革兰阴性菌的抗菌活性,对所有标本病原菌感染以及耐药的情况加以对比分析。结果在本次研究中,共检测出革兰阴性菌株749株,其中以大肠埃希菌、铜绿假单胞菌以及肺炎克雷伯菌为主,多药耐药菌126株,其中以肺炎克雷伯菌、鲍曼不动杆菌以及大肠埃希菌为主;抗菌活动的结果显示,头孢哌酮/舒巴坦对多重耐药革兰阴性菌抗菌活性最高的三种细菌分别为肺炎克雷伯菌、铜绿假单胞菌以及大肠埃希菌。结论在呼吸道感染中的革兰阴性感染菌主要包括为大肠埃希菌、肺炎克雷伯菌以及铜绿假单胞菌三种,其中铜绿假单胞菌、肺炎克雷伯菌以及大肠埃希菌是导致该病的主要多药耐药菌,头孢哌酮/舒巴坦的抗菌活性存在差异,应该针对病原菌的不同合理地加以使用。     

五种碳青霉烯类抗生素对大肠埃希菌和铜绿假单胞菌的体外抗菌活性比较

目的观察五种碳青霉烯类抗生素对大肠埃希菌和铜绿假单胞菌的体外抗菌活性。方法采用琼脂稀释法检测5种碳青霉烯类抗生素对55株铜绿假单胞菌、49株产ESBLs的大肠埃希菌的最低抑菌浓度（minimalinhibitoryconcentration,MIC）。结果对抗铜绿假单胞菌活性以美洛培南和比阿培南显示最佳,亚胺培南次之,帕尼培南逊于亚胺培南,厄他培南明显逊于其他四种碳青霉烯类抗生素。对产ESBLs的大肠埃希菌厄他培南的活性明显高于其他几种碳青霉烯类抗生素,而其他几种活性基本相当。结论铜绿假单胞菌对五种碳青霉烯类抗生素耐药性均较高,而五种碳青霉烯类抗生素对产ESBLs的大肠埃希菌均敏感。     

Association of ranibizumab (Lucentis®) or bevacizumab (Avastin®) with dexamethasone and triamcinolone acetonide: An in vitro stability assessment

The in vitro stability of monoclonal antibodies used for age-related macular degeneration, ranibizumab and bevacizumab, was investigated. The aggregation profile of the antibodies was compared, alone and after association with dexamethasone sodium phosphate or triamcinolone acetonide. Commercial formulations of ranibizumab and bevacizumab were dialysed into three different buffers. After dialysis, samples were stored at 4 °C, 25 °C and 40 °C during 35 days, alone and in combination with dexamethasone sodium phosphate, triamcinolone acetonide phosphate solution or triamcinolone acetonide suspension. Combined formulations based on both commercial formulations were investigated as well. The aggregation state of the antibodies was measured by multi-angle light scattering (MALS) after separation by asymmetrical flow field-flow fractionation (AFFF) or size-exclusion chromatography (SEC). Ranibizumab results to be more stable than bevacizumab, alone and in combination with dexamethasone sodium phosphate or triamcinolone acetonide. Elevation in concentration, pH and temperature causes a decrease in stability of both antibodies. The association of triamcinolone acetonide phosphate solution with either ranibizumab or bevacizumab is observed to be the least stable combination of all samples tested. Dexamethasone sodium phosphate was shown to have a stabilizing effect on bevacizumab, although this is not the case for its combination with the commercial formulation Avastin®. The results demonstrate that the in vitro association of either ranibizumab or bevacizumab with dexamethasone sodium phosphate or triamcinolone acetonide suspension does not decrease the stability of these antibodies. Although ranibizumab is more stable than bevacizumab in vitro, further research has to point out how this affects their mechanism of action in vivo.     

玻璃体内注射雷珠单抗与曲安奈德治疗视网膜静脉阻塞继发黄斑囊样水肿的疗效比较

目的：比较玻璃体内注射雷珠单抗与曲安奈德治疗视网膜静脉阻塞（ retinal vein occlusion ,RVO）继发黄斑囊样水肿（ cystoidmacular edema ,CME）的疗效及安全性。方法97例RVO继发CME患者按治疗方法分为雷珠单抗组（n＝47）和曲安奈德组（n＝50）。雷珠单抗组给予玻璃体内注射雷珠单抗0．5 mg（0．05 ml）;曲安奈德组给予玻璃体内注射雷珠单抗4．0 mg（0．1 ml）。治疗后随访6个月,比较2组最佳矫正视力（best corrected visual acuity,BC－VA）、黄斑中心凹厚度（ central macular thickness ,CMT）、眼压及并发症发生率。结果2组治疗后各时间点BCVA均较治疗前显著提高（P＜0．05）,但2组间比较差异无统计学意义（P＞0．05）。2组治疗后各时间点CMT均较治疗前显著降低（P＜0．05）,但2组间比较差异无统计学意义（P＞0．05）。雷珠单抗组治疗后各时间点眼压与治疗前比较差异无统计学意义（ P＞0．05）;曲安奈德组治疗后各时间点眼压均较治疗前和雷珠单抗组显著提高（ P＜0．05）。曲安奈德组和雷珠单抗组眼压升高发生率比较,差异有统计学意义（P＜0．05）。结论玻璃体内注射雷珠单抗治疗RVO继发CME,可有效提高BCVA、降低CMT,其效果与曲安奈德相当,且安全性更高。     

Ranibizumab and diabetic macular oedema: after laser therapy

Diabetic retinopathy is sometimes accompanied by macular oedema, leading to a marked decline in visual acuity. The standard treatment, in addition to glycaemic and blood pressure control, is laser photocoagulation, despite its modest efficacy. Ranibizumab (Lucentis, Novartis), a VEGF (vascular endothelial growth factor) inhibitor, was initially authorised for age-related macular degeneration (AMD) in the European Union. It is now also approved for the treatment of visual loss due to macular oedema in diabetic patients. In this setting, clinical evaluation of ranibizumab is mainly based on two double-blind randomised trials comparing ranibizumab + laser photocoagulation versus placebo + laser photo-coagulation in a total of about 1000 patients. Compared with placebo, addition of ranibizumab to laser therapy led to a marked improvement in visual acuity in approximately 15% of patients after 12 months of treatment. The improvement appeared to persist after 24 months of treatment. In a trial that included a group treated with ranibizumab alone, efficacy did not differ from that of the ranibizumab + laser combination. Uncertainties remain concerning the long-term efficacy of ranibizumab and its benefits in patients with poorly controlled diabetes or proliferative retinopathy. The adverse effect profile of ranibizumab in patients with diabetic macular oedema is similar to that reported in patients with AMD, and mainly includes ocular adverse effects such as pain, bleeding and increased intraocular pressure. A risk of systemic adverse effects, particularly cardiovascular disorders, should be kept in mind in case of long-term treatment. Ranibizumab can cause birth defects, even after intravitreal injection during pregnancy. Monthly treatment with ranibizumab is inconvenient, difficult and expensive. In practice, laser therapy remains the standard treatment for diabetic patients with significantly reduced visual acuity due to macular oedema. Ranibizumab, which requires intravitreal injections, should be restricted to second-line use.     

金针菇免疫调节蛋白基因表达及活性初步研究

目的对金针菇免疫调节蛋白(fungal immunomodulatory protein fromflammulina velutipes,FIP-fve)基因进行原核表达及活性测定。方法通过RT-PCR得到FIP-fve,构建pUC19-FIP-fve,和表达质粒pET30-FIP-fve,转化大肠杆菌DE3(BL21),经IPTG诱导,表达产物纯化后进行活性试验。结果该基因在DE3(BL21)中获得了大量表达。表达产物能促进巨噬细胞吞噬功能(P<0.01)、明显增加小鼠血清中IFN-γ的含量(P<0.01)及抑制小鼠移植性S180肉瘤的生长,其抑瘤率分别为31.84%(P<0.05)和36.04%(P<0.05)。结论表达蛋白具有活性,这对于将其开发为免疫调节、抗过敏、抗肿瘤新药具有重要意义。     

Morphologic changes and visual outcomes in resolved central serous chorioretinopathy treated with ranibizumab

Context: Central serous chorioretinopathy (CSC).

Objective: To evaluate the effect of intravitreal ranibizumab injection and the correlation between foveal morphologic changes and visual outcomes in patients with resolved CSC.

Materials and methods: We measured outer nuclear layer (ONL) thickness, outer layer (OL) thickness and evaluated the integrity of the photoreceptor inner-outer segment (IS/OS) junction, the status of the external limiting membrane (ELM) at the central fovea using spectral-domain optical coherence tomography (OCT) in 35 eyes of 35 patients with resolved CSC. The eyes were divided into two groups: The initial medical treatment administered to Group1 (n?=?17) then received intravitreal ranibizumab injections, Group 2 (n?=?18) received medical treatment. Group 3 was composed of normal eyes (n?=?20, as a control). We also investigated a correlation between the ONL thickness and best corrected visual acuity (BCVA).

Results: The mean age was 45.7?±?7.2 (ranged from 27 to 55 years). The mean follow-up period was 14.2 months (minimum 6, maximum 24 months). The mean ONL and OL thickness in Group 1 were significantly thinner than Group 3 (p?r?=?0.681, p?=?0.001). Thirty-tree patients had improvement in BCVA after treatment. Discontinuity of the IS/OS junction was found in 15 eyes (88.2%) in Group 1, in 5 eyes (27.7%) in Group 2 and in no eyes in Group 3.

Discussion: We demonstrated that prolonged serous detachment results in photoreceptor cell loss (apoptosis) and thinning of the ONL. Thinning of the ONL correlates with poorer vision, which has been found by other investigators. Furthermore, vascular endothelial growth factor (VEGF) may be neuroprotective to the photoreceptors which might explain the additional thinning in the patients treated with ranibizumab. This raises the possibility that treatment with VEGF inhibitors may be unfavourable to patients with CSC, even though it speeds recovery and vision does improve.

Conclusion: Intravitreal ranibizumab injection leads to thinning of the ONL and the OL in patients with resolved CSC. The ONL thickness reduction and discontinuity of the IS/OS junction results in poor visual prognosis in resolved CSC eyes.     

Clinical safety of ranibizumab in age-related macular degeneration

Importance of the field: Clinical safety of pharmaceutical products in the elderly is vital because of their increased risk of cardiac and other adverse events.

Areas covered in this review: Search of the Medline database, including articles and abstracts from 1984 to 2009.

What the reader will gain: Knowledge of ocular and systemic risks: The rate of endophthalmitis was 0.05% per injection (MARINA) and <0.1% per injection (ANCHOR), rates confirmed in a retrospective analysis of 14,320 injections. Moderate increases in intraocular pressure were transient, and incidences of intraocular inflammation were rarely serious. Systemic arterial thromboembolic events occurred in 4.6 and 0% of ranibizumab-treated patients and in 3.8 and 0% of sham-treated patients in MARINA (2 years) and PIER (1 year), respectively. In SAILOR, there was a numerically higher rate of cerebrovascular stroke with 0.5 mg ranibizumab compared with 0.3 mg ranibizumab (1.2 vs 0.7%), which was a non-statistically significant trend in patients with a history of stroke.

Take home message: Although further studies to investigate the risk of stroke with ranibizumab therapy are required, repeated intravitreal ranibizumab was well tolerated and not associated with clinically significant safety risks during up to 2 years of treatment.     

Ranibizumab: new drug. Macular degeneration: second-line use due to risks

(1) Due to a lack of any better alternatives, photodynamic therapy is the standard treatment for subfoveal lesions due to neovascular age-related macular degeneration (AMD). It consists of intravenous injection of a photosensitizer, verteporfin, followed by local red laser activation. This treatment, sometimes repeated every 3 months, stabilises the loss of visual acuity for 2 years in about 50% of patients. Adverse effects are generally acceptable. (2) Ranibizumab is an antibody fragment targeting vascular endothelial growth factor (VEGF). VEGF is implicated in the neovascularisation involved in age-related macular degeneration. Ranibizumab is injected into the vitreous in the same way as pegaptanib, the first VEGF antagonist to be approved for an ocular indication. (3) Clinical evaluation of ranibizumab includes 2 placebo-controlled trials (900 patients in total), a trial versus verteporfin (423 patients), and a trial testing ranibizumab in combination with verteporfin (162 patients). More than 90% of patients treated with ranibizumab in these two trials had no tangible loss of vision for one to two years, compared to about 68% of patients treated with verteporfin (statistically significant difference). These trials did not attempt to determine the optimal interval between intravitreal injections. (4) No trials have directly compared ranibizumab with pegaptanib; indirect comparisons suggest that ranibizumab is better than pegaptanib. (5) Intravitreal injection of ranibizumab can have local adverse effects, similar to pegaptanib. These include inflammatory reactions, infections, and elevated intraocular pressure. Arterial thromboses at distant sites, in particular strokes, have been reported with ranibizumab, at a higher frequency with 0.5 mg per infection (about 1%) than with 0.3 mg per injection. (6) When visual acuity continues to deteriorate in patients with age-related macular degeneration despite treatment with verteporfin, ranibizumab provides an effective alternative for patients with no particular risk factors for stroke.     

One-step solid-oil-water emulsion for sustained bioactive ranibizumab release

ABSTRACT

Background: The advent of therapeutic proteins highlights the need for delivery systems that protect and extend the duration of its action. Ranibizumab-VEGF is one such drug used for treating wet AMD. This paper describes a facile method to sustain bioactive ranibizumab release from PLGA-based particles.

Methods: Two emulsion techniques were explored namely: water-in-oil-in-water (WOW) and solid-in-oil-in-water (SOW) emulsion. The bioactivity of ranibizumab was evaluated by comparing its binding capability to VEGF, measured with ELISA to total protein measured by microBCA.

Results: During the emulsion process, contact of ranibizumab with the water-oil interface is the main destabilizing factor and this can be prevented with the use of amphiphilic PVA and solid-state protein in WOW and SOW emulsion respectively. In vitro release of the ranibizumab-loaded particles indicated that a 15-day release could be achieved with SOW particles while the WOW particles generally suffered from a burst release. Released ranibizumab was capable of inhibiting endothelial cell growth indicating its retention of bioactivity. The suppression of burst release from the SOW particles was attributed to the relatively smooth surface morphology of the SOW microparticles.

Conclusions: The use of SOW encapsulation in modulating ranibizumab release while maintaining their bioactivity has been highlighted.     

Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis

Macromolecules such as therapeutic proteins currently serve an important role in the treatment of eye diseases such as wet age-related macular degeneration and diabetic retinopathy. Particularly, bevacizumab and ranibizumab have been shown to be effective in the treatment of these diseases. Iontophoresis can be employed to enhance ocular delivery of these macromolecules, but the lack of information on the properties of these macromolecules has hindered its development. The objectives of the present study were to determine the effective electrophoretic mobilities and charges of bevacizumab, ranibizumab, and model compound polystyrene sulfonate (PSS) using capillary zone electrophoresis. Salicylate, lidocaine, and bovine serum albumin (BSA), which have known electrophoretic mobilities in the literature, were also studied to validate the present technique. The hydrodynamic radii and diffusion coefficients of BSA, bevacizumab, ranibizumab, and PSS were measured by dynamic light scattering. The effective charges were calculated using the Einstein relation between diffusion coefficient and electrophoretic mobility and the Henry equation. The results show that bevacizumab and ranibizumab have low electrophoretic mobilities and are net negatively charged in phosphate buffered saline (PBS) of pH 7.4 and 0.16M ionic strength. PSS has high negative charge but the electrophoretic mobility in PBS is lower than that expected from the polymer structure. The present study demonstrated that capillary electrophoresis could be used to characterize the mobility and charge properties of drug candidates in the development of iontophoretic drug delivery.     

Long-Term Stability of Anti-Vascular Endothelial Growth Factor (a-VEGF) Biologics Under Physiologically Relevant Conditions and Its Impact on the Development of Long-Acting Delivery Systems

The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.