Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz–type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor

Introduction

A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.

Materials and Methods

Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.

Results and Conclusions

Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.     

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

Mitochondrial dysfunctions cause numerous human disorders. A platform technology based on biodegradable polymers for carrying bioactive molecules to the mitochondrial matrix could be of enormous potential benefit in treating mitochondrial diseases. Here we report a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various mitochondria-acting therapeutics by blending a targeted poly(d,l-lactic-co-glycolic acid)-block (PLGA-b)-poly(ethylene glycol) (PEG)-triphenylphosphonium (TPP) polymer (PLGA-b-PEG-TPP) with either nontargeted PLGA-b-PEG-OH or PLGA-COOH. An optimized formulation was identified through in vitro screening of a library of charge- and size-varied NPs, and mitochondrial uptake was studied by qualitative and quantitative investigations of cytosolic and mitochondrial fractions of cells treated with blended NPs composed of PLGA-b-PEG-TPP and a triblock copolymer containing a fluorescent quantum dot, PLGA-b-PEG-QD. The versatility of this platform was demonstrated by studying various mitochondria-acting therapeutics for different applications, including the mitochondria-targeting chemotherapeutics lonidamine and α-tocopheryl succinate for cancer, the mitochondrial antioxidant curcumin for Alzheimer’s disease, and the mitochondrial uncoupler 2,4-dinitrophenol for obesity. These biomolecules were loaded into blended NPs with high loading efficiencies. Considering efficacy, the targeted PLGA-b-PEG-TPP NP provides a remarkable improvement in the drug therapeutic index for cancer, Alzheimer’s disease, and obesity compared with the nontargeted construct or the therapeutics in their free form. This work represents the potential of a single, programmable NP platform for the diagnosis and targeted delivery of therapeutics for mitochondrial dysfunction-related diseases.     

Fragment-guided design of subnanomolar β-lactamase inhibitors active in vivo

Fragment-based design was used to guide derivatization of a lead series of β-lactamase inhibitors that had heretofore resisted optimization for in vivo activity. X-ray structures of fragments overlaid with the lead suggested new, unanticipated functionality and points of attachment. Synthesis of three derivatives improved affinity over 20-fold and improved efficacy in cell culture. Crystal structures were consistent with the fragment-based design, enabling further optimization to a K_i of 50 pM, a 500-fold improvement that required the synthesis of only six derivatives. One of these, compound 5, was tested in mice. Whereas cefotaxime alone failed to cure mice infected with β-lactamase-expressing Escherichia coli, 65% were cleared of infection when treated with a cefotaxime:5 combination. Fragment complexes offer a path around design hurdles, even for advanced molecules; the series described here may provide leads to overcome β-lactamase-based resistance, a key clinical challenge.     

临时冠单体诱导颊黏膜上皮细胞凋亡的动物实验研究

目的:研究4种临时冠桥材料戴用后的单体释出对颊黏膜上皮细胞的凋亡行为的影响.方法:选用4种临时冠桥材料(丙烯酸自凝树脂、丙烯酸热凝树脂、DMG-TEMP树脂、松风SWIFT-TEMP树脂) 对犬的右侧后牙进行临时冠修复,在牙备前、戴冠时、戴冠1周、2周、1个月5个时间点,收集临时冠对应区域的颊黏膜上皮细胞,并应用流式细胞仪检测其凋亡情况.结果:自凝塑料及热凝塑料组的颊黏膜上皮细胞的凋亡率大小排序为戴冠1周>戴冠2周>戴冠1个月>牙备前、戴冠时,差异有统计学意义(P < 0.01).DMG-TEMP树脂及松风SWIFT-TEMP树脂组的颊黏膜上皮细胞凋亡率在戴冠前后均维持较低水平,差异无统计学意义(P >0.05).颊黏膜上皮细胞的凋亡率与自凝塑料及热凝塑料中的残余单体释出量呈正相关(P < 0.01).结论:自凝塑料冠及热凝塑料冠在戴冠早期均有明显的残余单体释出,并加速诱导了颊黏膜上皮细胞的凋亡.     

雷米芬太尼在老年高血压患者腮腺肿瘤手术中的临床应用

目的:探讨雷米芬太尼在老年高血压患者腮腺肿瘤手术中的临床应用价值。方法:选择拟行腮腺肿瘤手术的高血压患者52例,年龄65～82岁,ASAⅡ或Ⅲ级,随机分成雷米芬太尼组(R)和芬太尼组(F),每组26例。2组均采用全凭静脉麻醉,诱导方法一致。术中R组持续泵入雷米芬太尼0.05～0.2μg/kg.min-1,F组间断给予芬太尼,最大用量不超过8.0g/kg。2组均以丙泊酚和顺式阿曲库铵维持麻醉。记录不同时间点血流动力学参数、术后恢复情况及早期不良反应。结果:R组术中血流动力学更平稳,出血量少,术后苏醒时间和拔管时间较F组显著缩短(P<0.01)。2组患者自主呼吸恢复时间、术后躁动、疼痛和恶心呕吐发生率差异无统计学意义。结论:老年高血压患者行腮腺手术时,采用雷米芬太尼麻醉可维持血流动力学稳定,减少术野出血,提高患者苏醒质量,具有较高的安全性和临床实用性。     

牙周优势厌氧菌耐药性的研究进展

牙周厌氧菌是造成牙周炎的主要因素。随着临床治疗中抗生素的滥用,牙周厌氧菌的耐药情况亦愈发普遍。本文就牙周优势厌氧菌及其耐药现状、耐药机制和耐药基因等研究进展作一综述,以期为临床合理使用抗菌药物和新抗菌药物的研发提供依据。