Formulation Stabilization and Disaggregation of Bevacizumab,Ranibizumab and Aflibercept in Dilute Solutions

Purpose

Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies.

Methods

Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer’s formulation, and the new formulation. Analysis was by SE-HPLC and ELISA. Stability, disaggregation and pre-exposure tests were studied.

Results

When Avastin, Lucentis and Eylea are diluted in PBS or manufacturer’s formulation, there is a 40–50% loss of monomer concentration and drug activity. A formulation containing 0.3% NaCl, 7.5% trehalose, 10 mM arginine and 0.04% Tween 80 at a pH of 6.78 stabilized the mAbs and minimized the drug loss. The formulation also disaggregates mAb aggregation while preserving the activity. Degassing the formulation increases recovery.

Conclusions

We developed a novel formulation that significantly stabilizes mAbs under unfavorable conditions such as low concentration or body temperature. The formulation allows for tissue permeation experimentation. The formulation also exhibits a disaggregating effect on mAbs, which can be applied to the manufacture/packaging of mAbs and bioassay reagents.

     

Photokinetic Drug Delivery: Near infrared (NIR) Induced Permeation Enhancement of Bevacizumab,Ranibizumab and Aflibercept through Human Sclera

Purpose

Permeation studies, with near infrared (NIR) light and anti-aggregation antibody formulation, were used to investigate the in vitro permeation of bevacizumab, ranibizumab and aflibercept through human sclera.

Methods

A vertical, spherical Franz cell diffusion apparatus was used for this scleral tissue permeation model. A photokinetic ocular drug delivery (PODD) testing device accommodated the placement of NIR LEDs above the donor chambers. An adjustable LED driver/square wave generator provided electrical energy with a variable pulse rate and pulse width modulation (duty cycle).

Results

Exposure to non-thermal NIR light had no effect on mAbs with regard to monomer concentration or antibody binding potential, as determined by SE-HPLC and ELISA. The optimal LED wavelength was found to be 950 nm. Duty cycle power of 5% vs 20% showed no difference in permeation. When compared to controls, the combination of non-aggregating antibody formulation and NIR illumination provided an average transscleral drug flux enhancement factor of 3X.

Conclusion

Narrow wavelength incoherent (non-laser) light from an NIR LED source is not harmful to mAbs and can be used to enhance drug permeation through scleral tissue. The topical formulation, combined with pulsed NIR light irradiation, significantly improved scleral permeation of three anti-VEGF antibody drugs.

     

Characterization of Human Sclera Barrier Properties for Transscleral Delivery of Bevacizumab and Ranibizumab

The objectives of this study were to (a) investigate transscleral permeation of antivascular endothelial growth factor drugs bevacizumab and ranibizumab and (b) examine the effects of molecular structures of macromolecules upon permeation across human sclera using bevacizumab, ranibizumab, fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA), FITC-labeled ficoll (FITC-ficoll), and FITC-labeled dextrans (FITC-dextrans) in vitro. The hydrodynamic radii of the macromolecules were measured using dynamic light scattering, their partition coefficients to sclera were determined in uptake experiments, and their permeability coefficients and transport lag times across sclera were evaluated in transport experiments of side-by-side diffusion cells. Macromolecules showed relatively low partition coefficients to sclera. The partition coefficient of FITC-BSA was found to be related to its concentration in the equilibration solution, whereas for other macromolecules, no specific concentration dependency was observed. The macromolecules displayed relatively low permeability coefficients and long transport lag times because of their molecular sizes and hindered diffusion. Bevacizumab, ranibizumab, and FITC-BSA exhibited lower transscleral permeability and longer transport lag times than FITC-dextrans and FITC-ficoll of comparable molecular weights possibly because of the flexible structures of the polysaccharides. Thus, the polysaccharides may not be good surrogate permeants to model transscleral transport of therapeutic proteins in transscleral delivery studies. © 2012Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:892–903, 2013     

无保护流体室温燐光法测定药片中的吲哚美辛含量

目的:研究吲哚美辛的无保护流体室温燐光(NP-RTP)性质,建立 NP-RTP 测定吲哚美辛含量的方法。方法:采用 KI为重原子微扰剂、Na_2SO_3为除氧剂,于激发/发射波长286/447 mm处测定,延迟时间0.10 ms,门控时间2.00 ms。结果:吲哚美辛在1.0×10~(-7)～6.0×10~(-6)mol·L~(-1)范围内,燐光强度与浓度线性关系良好,相关系数0.9992,检出限5.2×10~8mol·L~(-1)。用于吲哚美辛的测定,相对标准偏差1.9%～2.4%,与紫外分光光度法比较,相对误差-3.0%～3.2%。结论:本方法灵敏、简便、准确可靠。     

雷珠单抗超说明书用药情况调查分析

目的:调查雷珠单抗临床应用中的超说明书用药情况,为促进雷珠单抗临床合理应用提供参考。方法:随机抽取北京同仁医院2014年1月1日—2014年12月31日应用雷珠单抗的住院医嘱1 201条,从适应证、给药剂量、给药途径、是否出现不良反应等方面进行调查分析。结果:1 201条住院医嘱中,患者的给药剂量为0.05~0.1m L,给药部位为左眼的共623例,右眼为554例,双眼给药的为12例。全部患者均为玻璃体腔注射。超说明书适应证的共1 022例,占85.1%;给药时机不适宜的主要表现为在给药前后28 d内接受眼内手术,共66例,占5.50%;未观察到不良反应发生。结论:药师应从适应证、给药时机方面规范雷珠单抗的超说明书用药管理,促进雷珠单抗的临床合理应用。     

Hydrodynamic, mass transfer, and dissolution effects induced by tablet location during dissolution testing

Tablets undergoing dissolution in the USP Dissolution Testing Apparatus II are often found at locations on the vessel bottom that are off-center with respect to the dissolution vessel and impeller. A previously validated CFD approach and a novel experimental method were used here to examine the effect of tablet location on strain rates and dissolution rates. Dissolution tests were conducted with non-disintegrating tablets (salicylic acid) and disintegrating tablets (Prednisone) immobilized at different locations along the vessel bottom. CFD was used to predict the velocity profiles and strain rates when the tablets were placed at such locations. A CFD-based model was derived to predict the mass transfer coefficient and dissolution curves, which were then compared to the experimental results. Both non-disintegrating and disintegrating off-center tablets experimentally produced higher dissolution rates than centered tablets. The CFD-predicted strain rate distribution along the bottom was highly not uniform and the predicted strain rates correlated well with the experimental mass transfer coefficients. The proposed CFD-based model predicts mass transfer rates that correlate well with the experimental ones. The exact tablet location has a significant impact on the dissolution profile. The proposed model can satisfactorily predict the mass transfer coefficients and dissolution profiles for non-disintegrating tablets.     

Probing bis-ANS Binding Sites of Different Affinity on Aggregated IgG by Steady-State Fluorescence,Time-Resolved Fluorescence and Isothermal Titration Calorimetry

Fluorescent dyes, for example, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS) are extensively used to detect nonnative protein structures in therapeutic protein products, for example, during formulation development by monitoring the greatly enhanced dye fluorescence upon binding to nonnative species. Our aim was to characterize the level of heterogeneity of bis-ANS binding sites in a thermally stressed monoclonal antibody (IgG) formulation by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry (ITC), and to obtain apparent dissociation constants (K_d) by data fitting. Because the methods differ in their underlying measurement principles, they provide different information on binding properties of bis-ANS to thermally stressed IgG. We found very heterogeneous bis-ANS binding sites on thermally stressed IgG, with apparent K_d values ranging from as low as 50 nM (time-resolved fluorescence) to 63 μM (ITC). Steady-state fluorescence and ITC gave insight into an overall binding affinity of a wide population of dye binding sites with micromolar K_d values. Time-resolved fluorescence was particularly sensitive to high-affinity binding sites with nanomolar K_ds. The heterogeneity of the bis-ANS binding sites reflects the complex, heterogeneous nature of the heat-stressed IgG used in this study. To probe such heterogeneity adequately, one should apply complementary analytical methods under various experimental conditions as presented in this paper.     

回顾性分析贝伐珠单抗致高血压等重要不良反应

目的：分析贝伐珠单抗导致高血压等重要药物不良反应（ADR）的发生情况,为其临床合理应用提供参考。方法：回顾性分析本院2021年3月 ~ 2022年3月使用贝伐珠单抗的172例恶性肿瘤患者的临床资料,纳入用药期间发生贝伐珠单抗相关不良反应的患者,分析年龄、不良反应发生时间、用药方案、临床表现、治疗手段和转归情况。结果：发生贝伐珠单抗相关不良反应患者共52例,男女各26例;年龄42 ~ 86岁,其中50 ～ 79岁较多（44例,84.62%）;发生不良反应的时间为1 ~ 22个疗程,93.55%的不良反应发生在用药10个疗程内。高血压、蛋白尿和出血的发生率分别为18.60%、13.95%和3.49%,其中同时出现累及多个系统的不良反应患者10例。ADR中3级以上不良反应18例（29.03%）,包括3 ~ 4级高血压17例,3级消化道出血1例,所有患者预后良好。22例高血压ADR患者继续使用贝伐珠单抗治疗,17例再次出现高血压;20例蛋白尿ADR患者继续使用贝伐珠单抗治疗,10例再次出现蛋白尿。结论：临床使用贝伐珠单抗需关注其特有的不良反应,尤其是50岁以上中老年和用药10个疗程内患者。发生高血压和消化道出血时需及时药物治疗,继续使用贝伐珠单抗需做好全程用药监护,以提高临床用药安全性。     

Adsorption of Beta-Blockers onto Polyisobutylcyanoacrylate Nanoparticles Measured by Depletion and Dielectric Methods

Pharmaceutical Research -     

Comparative Rates of Freeze-Drying for Lactose and Sucrose Solutions as Measured by Photographic Recording,Product Temperature,and Heat Flux Transducer

Sublimation from lactose and sucrose solutions has been monitored by temperature measurement, visual observation, heat flux sensing and manometric measurements. Estimates of energy transfer rates to the subliming mass made from visual observations and heat flux measurements are in broad agreement, demonstrating for the first time that heat flux sensors can be used to monitor the progress of lyophilization in individual vials with low sample volumes. Furthermore, it is shown that under identical lyophilization conditions the initial rate of drying for lactose solutions is low with little water sublimation for up to 150 minutes, which contrasts markedly with the much faster initial rate of drying for sucrose solutions. Measurement of the initial heat flux between shelf and vial indicated a lower flux to a 10% lactose solution than to a 10% sucrose solution.     

噻托溴铵的安全性考量

鉴于UPLIFT研究数据的有力支持,加之mata分析的统计学局限,FDA认为,目前的数据不支持经HandiHaler给药的噻托溴铵与脑卒中、心脏病或死亡风险的增加相关。     

Hardness and Density Distributions of Pharmaceutical Tablets Measured by Terahertz Pulsed Imaging

We present terahertz pulsed imaging (TPI) as a novel tool to quantify the hardness and surface density distribution of pharmaceutical tablets. Good agreement between the surface refractive index (SRI) measured by TPI and the crushing force measured from diametral compression tests was found using a set of tablets that were compacted at various compression forces. We also found a strong correlation between TPI results and tablet bulk density, and how these relate to tablet hardness. Numerical simulations of tablet surface density distribution by finite element analysis exhibit excellent agreement with the TPI measured SRI maps. These results show that TPI has an advantage over traditional diametral compression and is more suitable for nondestructive hardness and density distribution monitoring and control of pharmaceutical manufacturing processes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2179–2186, 2013     

Bevacizumab in non small cell lung cancer: development, current status and issues

Bevacizumab is a monoclonal antibody directed against Vascular Endothelial Growth Factor (VEGF). Evidence about its efficacy in addition to first-line chemotherapy in non-small-cell-lung-cancer (NSCLC) has been produced by two large randomized phase III clinical trials (ECOG 4599 and AVAiL), conducted in a clinically selected population with non-squamous histology and without major risk factors for bleeding. In the ECOG 4599 trial, the addition of bevacizumab (15 mg/kg) to carboplatin plus paclitaxel produced a statistically significant and clinically relevant improvement in overall survival (OS), that was the primary endpoint of the trial (12.3 months vs 10.3 months, HR 0.79; p=0.003). Furthermore, patients receiving bevacizumab showed a significant improvement in progression-free survival (PFS) and in objective response rates. Treatment with bevacizumab was well tolerated by the majority of patients, but was still associated with increased risk of clinically significant bleeding (4.4% vs 0.7%, p0.001). In the AVAiL trial the addition of bevacizumab (at the dose of 7.5 and 15 mg/kg) to cisplatin plus gemcitabine produced a small improvement in PFS, but no differences in OS. Information from retrospective analysis and two large observational studies (SAIL and ARIES) have confirmed the safety profile of first-line bevacizumab with a wide range of chemotherapy partners, but whether its efficacy is comparable when combined with the different regimens is still unknown. The identification of predictive factors of efficacy would be relevant for the optimal use of the drug, but to date we have no conclusive data in this direction.     

Partition Behaviour of Drugs in Microemulsions Measured by Electrokinetic Chromatography

Pharmaceutical Research -     

雷珠单抗的大肠埃希菌胞外分泌表达、纯化及结构表征

抗血管内皮细胞生长因子(VEGF)抗体可抑制新生血管的形成和生长,在年龄相关性黄斑变性眼科疾病的临床治疗中具有重要应用价值,特别是已上市的商品名为雷珠单抗的VEGF抗体Fab片段,在市场上取得巨大成功。本研究探索了雷珠单抗在大肠埃希菌中的胞外分泌表达技术,所表达的产品利用SDS-PAGE、Western Blot、肽谱图全序列分析和纳米差示扫描量热法(Nano DSC)等手段进行了初步的结构表征。结果显示,本研究成功实现了雷珠单抗的大肠埃希菌胞外分泌表达,经过进一步的Capto L亲和色谱获得了有正确氨基酸序列的Fab产品,经质谱分析具有正确的二硫键配对,且具有与标准品相似的热稳定性,为其进一步的工业生产奠定了基础。     

Molecular Motion of Drugs in Hydrocolloids Measured by Electron Paramagnetic Resonance

The effects of a polymer, the Li-salt copolymer of methyl-methacrylic acid, and its methyl ester on the motion of drug molecules in hydrocolloids were studied. The investigation was carried out by means of electron paramagnetic resonance (EPR) using the model nitroxide tempol, and the spin-labeled drugs lidocaine (si-lid) and dexamethasone (sl-dex). Synthesis of sl-dex was performed. Spin-labeled molecules dissolved in hydrocolloids undergo a fast reorientation motion. The decreasing order of rotational correlation times () —sl-dex > si-lid > tempol—suggests that the size and the shape of the molecules strongly affect their motion. The inhibition of motion of larger molecules depends also on their flexibility. The values indicate proportionality of the microviscosity of hydrocolloids to the polymer concentration. Rotational motion is dependent on the local environment conditioned by the free spaces between polymer molecules.