Physicochemical properties of ferumoxytol, a new intravenous iron preparation
新型静脉注射铁制剂氧化铁的理化性质
首次发表： 2009 年 5 月 6 日 https://doi.org/10.1111/j.1365-2362.2009.02130.xCitations： 114
VS Balakrishnan，医学博士，MRCP，肾病科，750 Washington Street， #391， Boston， MA 02111， USA。电话：（617） 636-8524;传真：（617） 636-8329;
e-mail: vbalakrishnan@tuftsmedicalcenter.org
电子邮件： vbalakrishnan@tuftsmedicalcenter.org
Abstract 抽象
Background Intravenous iron is a critical component of anaemia management. However, currently available preparations have been associated with the release of free iron, a promoter of bacterial growth and oxidative stress.
背景 静脉注射铁剂是贫血管理的重要组成部分。然而，目前可用的制剂与游离铁的释放有关，游离铁是细菌生长和氧化应激的促进剂。
Materials and methods We determined the molecular weight, dialysability and capacity for free iron release of ferumoxytol, a semi-synthetic carbohydrate-coated superparamagnetic iron oxide nanoparticle.
材料和方法 我们测定了氧化铁的分子量、可透析性和游离铁释放能力，氧化铁是一种半合成碳水化合物包被的超顺磁性氧化铁纳米颗粒。
Ferumoxytol was compared with three intravenous iron preparations in clinical use: iron dextran (low molecular weight), sodium ferric gluconate and iron sucrose.
将 Ferumoxytol 与临床使用的 3 种静脉注射铁制剂进行比较： 右旋糖酐铁 （低分子量） 、葡萄糖酸铁钠和蔗糖铁。
Intravenous iron preparations were also incubated in rat, and pooled human sera (at concentrations of 600 μM and 42 μg mL−1 respectively) from healthy subjects.
静脉注射铁制剂也在大鼠中孵育，并混合来自健康受试者的人血清 （浓度分别为 600 μM 和 42 μg mL −1 ）。
Results The molecular weight of ferumoxytol was 731 kDa. The relative order of molecular weight was as follows: ferumoxytol > iron dextran > iron sucrose > sodium ferric gluconate. The least ultrafilterable iron was observed with ferumoxytol and the most with ferric gluconate.
结果 氧化铁的分子量为 731 kDa。分子量的相对顺序如下：氧化铁 > 右旋糖酐铁 > 蔗糖铁 > 葡萄糖酸铁钠。使用氧化铁观察到的超滤铁最少，而葡萄糖酸铁观察到的超滤铁最多。
The least dialysable free iron was observed with ferumoxytol and the most with ferric gluconate. Incubation of intravenous iron preparations in rat or pooled human sera demonstrated minimal free iron release with ferumoxytol.
使用氧化铁观察到的游离铁最少，葡萄糖酸铁最多。在大鼠或混合人血清中静脉注射铁制剂的孵育表明，氧化铁释放的游离铁最小。
The order of catalytic iron release as detected by the bleomycin detectable iron assay was as follows: ferumoxytol < iron dextran < iron sucrose < ferric gluconate. A similar trend was observed for the in vivo serum concentration of free iron in rats.
博来霉素检测铁测定法检测到的催化铁释放顺序如下：氧化铁 < 右旋糖酐铁 < 蔗糖铁 < 葡萄糖酸铁。大鼠体内血清游离铁浓度也观察到类似的趋势。
Conclusions In vitro observations from these experiments suggest that ferumoxytol has a favourable profile in terms of tendency to release free iron, in comparison with currently available intravenous iron preparations.
结论 这些实验的体外观察表明，与目前可用的静脉注射铁制剂相比，氧化铁在释放游离铁的趋势方面具有良好的特性。
Introduction 介绍
Intravenous iron therapy is an integral component of the anaemia management protocol in patients with chronic kidney disease (CKD), particularly those on maintenance haemodialysis (HD), and optimizes response to erythropoiesis stimulating agents.
静脉铁剂治疗是慢性肾脏病 （CKD） 患者贫血管理方案的一个组成部分，尤其是接受维持血液透析 （HD） 的患者，并优化了对红细胞生成刺激剂的反应。
The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) guidelines emphasize that most patients undergoing HD will require parenteral iron therapy on a regular basis to achieve target haemoglobin (Hgb) levels [1]. Currently, the intravenous iron agents widely available for clinical use include low molecular weight iron dextran, sodium ferric gluconate and iron sucrose.
美国国家肾脏基金会肾脏疾病结果质量倡议 （NKF-KDOQI） 指南强调，大多数接受 HD 的患者需要定期肠外铁剂治疗以达到目标血红蛋白 （Hgb） 水平 [ 1]。目前，临床上广泛使用的静脉注射铁剂包括低分子量右旋糖酐铁、葡萄糖酸铁钠和蔗糖铁。
The relatively high incidence of anaphylaxis and anaphylactoid reactions has limited the use of high molecular weight iron dextran in recent years.
近年来，过敏反应和过敏样反应的发生率相对较高，限制了高分子量右旋糖酐铁的使用。
Despite the favourable short-term safety profiles of sodium ferric gluconate and iron sucrose, concerns remain regarding the potential long-term consequences of biologically active or catalytic iron release resulting from the use of intravenous iron preparations.
尽管葡萄糖酸铁钠和蔗糖铁具有良好的短期安全性，但仍然存在对使用静脉铁制剂导致的生物活性或催化铁释放的潜在长期后果的担忧。
At doses ranging from 250 to 500 mg, both iron sucrose and sodium ferric gluconate are associated with gastrointestinal symptoms and hypotension, symptoms similar to those that characterize free iron reactions [2,3]. However, in clinical practise, these doses of iron sucrose and sodium ferric gluconate are not routinely used by nephrologists. At lower doses, iron sucrose has been associated with transferrin oversaturation, oxidative stress and enhanced bacterial growth in vitro [4]. Increased carbonyl stress has been associated with sodium ferric gluconate at a dose of 125 mg, as the increase in carbonylated protein concentration correlates with transferrin oversaturation [5].
在250-500mg的剂量范围内，蔗糖铁和葡萄糖酸铁钠都与胃肠道症状和低血压有关，这些症状与游离铁反应的特征相似[2,3]。然而，在临床实践中，这些剂量的蔗糖铁和葡萄糖酸铁钠并不被肾脏科医生常规使用。在较低剂量下，蔗糖铁与转铁蛋白过饱和、氧化应激和体外细菌生长增强有关 [ 4]。125 mg 剂量的葡萄糖酸铁钠与羰基应力增加有关，因为羰基化蛋白浓度的增加与转铁蛋白过饱和相关 [ 5]。
Ferumoxytol is a novel semi-synthetic carbohydrate-coated superparamagnetic iron oxide nanoparticle that is being developed as an intravenous iron replacement therapy.
Ferumoxytol 是一种新型半合成碳水化合物包被的超顺磁性氧化铁纳米颗粒，正在开发为静脉内铁替代疗法。
Spinowitz and colleagues recently showed, in a phase III study involving CKD patients, that two 510 mg doses of intravenous ferumoxytol administered rapidly within 5 ± 3 days were well tolerated and achieved the intended therapeutic effect [6]. This manuscript details in vitro, ex vivo and in vivo studies comparing ferumoxytol with three commercial parenteral iron preparations with respect to catalytic free iron release. The molecular weights and osmolalities of the four iron preparations were also determined.
Spinowitz 及其同事最近在一项涉及 CKD 患者的 III 期研究中表明，在 5 ± 3 天内快速给药两次 510 mg 剂量的静脉注射氧化铁耐受性良好，并达到了预期的治疗效果 [ 6]。本手稿详细介绍了体外、离体和体内研究，比较了氧化铁与三种市售肠外铁制剂在催化游离铁释放方面的表现。还测定了四种铁制剂的分子量和渗透压。
Materials and methods 材料和方法
Molecular weight determination
分子量测定
The molecular weights of ferumoxytol, sodium ferric gluconate, iron sucrose and low molecular weight iron dextran were analysed by gel permeation chromatography (GPC).
凝胶渗透色谱 （GPC） 分析了氧化铁、葡萄糖酸铁钠、蔗糖铁和低分子量葡聚糖铁的分子量。
Each iron supplement was eluted on a tandem column arrangement: first, an Ultrahydrogel 1000 (Waters, Milford, MA, USA) and then, an Ultrahydrogel 250. The tandem columns were calibrated using commercial protein molecular weight standards.
每种补铁剂均采用串联柱洗脱：首先使用 Ultrahydrogel 1000（Waters，Milford，MA，USA），然后使用 Ultrahydrogel 250。使用市售蛋白质分子量标准品校准串联柱。
The protein standards and iron preparations were monitored using a UV-Vis detector set to a wavelength of 280 nm.
使用波长为 280 nm 的紫外-可见光检测器监测蛋白质标准品和铁制剂。
Particle size determination
粒度测定
Transmission electron microscopy (TEM) was performed on a Philips 420 TEM, at 120 kV at TEM Analysis Services (Fort Worth, TX, USA).
透射电子显微镜 （TEM） 在 TEM 分析服务公司（美国德克萨斯州沃思堡）的 Philips 420 TEM 上以 120 kV 进行。
Samples were agitated in an ultrasonic cleaner for approximately 10 min, and then a small amount was pipetted onto a carbon-coated grid and placed into the TEM for imaging.
将样品在超声波清洗机中搅拌约 10 分钟，然后将少量移液到碳涂层网格上并放入 TEM 中进行成像。
Ultrafilterable free iron
超滤游离铁
Samples of each drug were first diluted with distilled water to a volume of 2 mL at a concentration of 2 mg mL−1. Each sample was placed into a 30 K Centricon filter (Millipore, Billerica, MA, USA) and centrifuged at 3000 × g for 30 min.
首先用蒸馏水将每种药物的样品稀释至 2 mL 的体积，浓度为 2 mg mL −1 。将每个样品置于 30 K Centricon 过滤器（Millipore，Billerica，MA，USA）中，并以 3000 × g 离心 30 分钟。
The amount of iron in the filtrate was determined spectrophotometrically after dissolution of the filtrate in hydrochloric acid and complexation with bipyridine (DU 650 spectrophotometer; Beckman Instruments, Fullerton, CA, USA).
滤液溶于盐酸中并与联吡啶络合后，通过分光光度法测定滤液中铁的量（DU 650分光光度计;Beckman Instruments，美国加利福尼亚州富勒顿）。
Osmolality of iron preparations
铁制剂的渗透压
The osmolality of each drug was determined according to United States Pharmacopoeia method using a Fisk OS Osmometer.
根据美国药典方法，使用 Fisk OS 渗透压计测定每种药物的渗透压。
Dialysable free iron 可渗出的游离铁
The amount of free iron that crossed a dialysis membrane during circulation of each of the four intravenous iron preparations through an in vitro haemodialysis circuit was evaluated. The extracorporeal circuit was set up (as shown in Fig. 1) using an ethylene oxide sterilized Hemoflow F50NR capillary dialyzer (Fresenius Polysulfone high-flux dialyzer; Lexington, MA, USA) and Medisystems ReadySet haemodialysis blood tubing set (Medisystems Corporation, Seattle, WA, USA).
评估了四种静脉注射铁制剂通过体外血液透析回路循环期间穿过透析膜的游离铁量。使用环氧乙烷灭菌的 Hemoflow F50NR 毛细管透析器（费森尤斯聚砜高通量透析器;美国马萨诸塞州列克星敦）和 Medisystems ReadySet 血液透析血管套件（Medisystems Corporation，美国华盛顿州西雅图）。
Each iron supplement was diluted in phosphate-buffered saline (PBS) with 4·5% bovine serum albumin (BSA) at approximately 80 mg L−1 and accurately determined. BSA was added because blood is about 4·5% protein by weight, mostly albumin. Before each experiment, the dialysis membrane was rinsed, first with 5 L water at 80 mL min−1, with no backpressure applied, and then with 2·5 L PBS under the same conditions. Filtrate and retentate samples were collected at the end of the PBS filtration to provide background control samples.
将每种铁补充剂用含 4·5% 牛血清白蛋白 （BSA） 的磷酸盐缓冲盐水 （PBS） 稀释，浓度约为 80 mg L −1 ，并准确测定。添加 BSA 是因为血液中蛋白质的重量约为 4·5%，主要是白蛋白。每次实验前，冲洗透析膜，先用 5 L 水冲洗 80 mL −1 ，不施加背压，然后在相同条件下用 2·5 L PBS 冲洗。在 PBS 过滤结束时收集滤液和截留物样品，以提供背景对照样品。
Schematic representation of the extracorporeal circuit used to evaluate the dialysability of intravenous iron preparations. PBS, phosphate-buffered saline; BSA, bovine serum albumin.
用于评估静脉铁制剂可透析性的体外回路的示意图。PBS，磷酸盐缓冲盐水;BSA，牛血清白蛋白。
The schematic representation shown in Fig. 1 delineates the set-up for the extracorporeal circuit. The retentate (80 mg IV iron supplement in 1 L PBS buffer with 4·5% BSA) was continuously re-circulated through the filter. The filtrate was collected for analysis.
图 1 所示的示意图描述了体外回路的设置。截留物（80 mg IV 铁补充剂，溶于 1 L 含 4·5% BSA 的 PBS 缓冲液中）通过过滤器连续再循环。收集滤液进行分析。
The reservoir, filled with fresh PBS buffer, was used to keep the retentate solution at a constant volume of 1 L while the filtration was proceeding.
在过滤过程中，使用装满新鲜 PBS 缓冲液的储液槽将截留物溶液保持在 1 L 的恒定体积。
Adjustments to the flow rate were made during each experiment to maintain a pressure reading of approximately 1·3 psi. A total volume of 5 L was collected as filtrate in 1 L portions in separate 1 L bottles (1 L every 1–2 h).
在每次实验期间调整流速，以保持约 1·3 psi 的压力读数。将总体积为 5 L 的滤液收集为 1 L 分量，分装在单独的 1 L 瓶中（每 1-2 小时 1 L）。
After each 1 L of filtrate was collected, the tubing and filter were cleared of iron oxide solution by pumping the system with 100 mL of fresh PBS buffer solution. Samples were then taken from both filtrate and retentate.
收集每 1 L 滤液后，用 100 mL 新鲜 PBS 缓冲溶液泵送系统，清除管道和过滤器中的氧化铁溶液。然后从滤液和截留物中取样。
The total weight of filtrate and retentate was measured at each sampling point. Samples from the retentate and filtrate reservoirs were removed for atomic absorption spectroscopy for each sample point.
在每个采样点测量滤液和截留物的总重量。从截留物和滤液储层中取出样品，对每个样品点进行原子吸收光谱分析。
Catalytic or free iron release in rat serum
大鼠血清中的催化或游离铁释放
Ex vivo experiments. For the ex vivo experiments (spiking serum with iron complex), the iron was diluted by weight to 0·35 mg iron mL−1 with deionized water, and 50 μL of the diluted iron was added to 0·5 mL normal rat serum and allowed to stand at room temperature for approximately 10 min before assay for free or catalytic iron using the bleomycin detectable iron (BDI) assay.
离体实验。对于离体实验（含铁复合物的加标血清），用去离子水将铁按重量稀释至 0·35 mg 铁 mL −1 ，并将 50 μL 稀释铁加入 0·5 mL 正常大鼠血清中，并在室温下静置约 10 分钟，然后使用博来霉素检测铁 （BDI） 测定法测定游离铁或催化铁。
Rat serum was obtained from Sigma (Cat. no. S7648).
大鼠血清购自 Sigma（货号 S7648）。
根据大鼠静脉注射铁剂 1·4 mg kg −1 ，血体积为体重的 7%，血清含量为 60%，加标大鼠血清中铁的最终浓度选择为约 600 μM 铁：
In vivo experiments. Each of the iron preparations was administered intravenously to rats at a dose of 1·4 mg kg−1 by tail vein injection. The dose of 1·4 mg kg−1 is based on a dose in humans of 100 mg (for a 70 kg individual), which is the typical single dose administered of low molecular weight iron dextran and iron sucrose. A 1 mL blood sample was collected pre-dose and 5 min post injection.
体内实验。每种铁制剂均通过尾静脉注射以 1·4 mg kg −1 的剂量静脉内给予大鼠。1·4 mg kg −1 的剂量基于人体 100 mg（对于 70 kg 个体）的剂量，这是低分子量葡聚糖铁和蔗糖铁的典型单剂量给药。给药前和注射后 5 min 采集 1 mL 血样。
This time point was chosen because most reactions to intravenous iron in human subjects occur immediately after administration. The samples were analysed for catalytic iron using the BDI assay.
选择这个时间点是因为人类受试者对静脉注射铁剂的大多数反应发生在给药后立即发生。使用 BDI 测定法分析样品中的催化铁。
Catalytic or free iron release in human serum
人血清中的催化或游离铁释放
Ex vivo experiments. In this experiment, catalytic iron release of the four iron preparations in human serum was determined. Each of the iron preparations was added to pooled human serum to obtain a concentration of 42 μg mL−1, the theoretical maximum concentration achieved following a 125 mg dose of parenteral iron in a 70 kg individual (1·79 mg kg−1). Additionally, ferumoxytol was also added to pooled serum at concentrations of 84 and 168 μg mL−1, the theoretical maximum concentration, Cmax, achieved following doses of 250 and 500 mg. The samples were incubated at 37 °C for 10 min before analysis for BDI. Experiments represent an n = 5, apart from the higher concentrations (84 and 168 μg mL−1) of ferumoxytol (n = 2).
离体实验。在本实验中，测定了四种铁制剂在人血清中的催化铁释放。将每种铁制剂添加到混合的人血清中以获得 42 μg mL −1 的浓度，这是在 70 kg 个体 （1·79 mg kg −1 ） 中接受 125 mg 肠外铁剂后达到的理论最大浓度。此外，还将氧化铁以 84 和 168 μg mL −1 的浓度添加到混合血清中，在 250 和 500 mg 剂量后达到理论最大浓度 C max 。将样品在 37 °C 下孵育 10 分钟，然后进行 BDI 分析。实验表示 n = 5，除了较高浓度（84 和 168 μg mL −1 ）的氧化铁 （n = 2） 外。
BDI assay BDI 检测
The procedure followed for the BDI assay was based on previous published methods [4,7,8]. Briefly, for each serum sample or standard, the following reagents were combined in polypropylene tubes in the following order: 100 μL 1 M trishydroxymethylaminomethane buffer, 250 μL 1 mg mL−1 deoxyribonucleic acid solution, 25 μL 1·5 units mL−1 bleomycin solution, 25 μL serum blank, serum containing sample or standard, 50 μL 50 mM magnesium chloride solution and 50 μL 8 mM ascorbic acid solution.
BDI 测定遵循的程序基于先前发表的方法 [ 4,7,8]。简而言之，对于每个血清样品或标准品，将以下试剂按以下顺序混合在聚丙烯管中：100 μL 1 M 三羟甲基氨基甲烷缓冲液、250 μL 1 mg mL −1 脱氧核糖核酸溶液、25 μL 1·5 单位 mL −1 博来霉素溶液、25 μL 血清空白、含血清样品或标准品、50 μL 50 mM 氯化镁溶液和 50 μL 8 mM 抗坏血酸溶液。
The Tris buffer, magnesium chloride, ascorbic acid and DNA solutions were stored over Chelex resin overnight before use in the assay. The total reaction volume was 500 μL. Each tube was capped, vortex mixed and incubated for 1 h at 37 °C.
在用于分析之前，将 Tris 缓冲液、氯化镁、抗坏血酸和 DNA 溶液在 Chelex 树脂上储存过夜。总反应体积为 500 μL。将每个管加盖，涡旋混合并在 37 °C 下孵育 1 小时。
A solution of 0·1 M ethylenediamine tetraacetic acid, 50 μL, was then added to each tube and vortex mixed. For the colour development, 250 μL 1% thiobarbituric acid solution was added, followed by 250 μL 25% hydrochloric acid. The tubes were incubated at 80 °C for 20 min.
然后向每个试管中加入 0·1 M 乙二胺四乙酸 50 μL 溶液，涡旋混合。对于显色，加入 250 μL 1% 硫代巴比妥酸溶液，然后加入 250 μL 25% 盐酸。将试管在 80 °C 下孵育 20 分钟。
The tubes were allowed to cool to room temperature, and 1·5 mL 1-butanol was added. The tubes were capped, vortex mixed and centrifuged for 15 min at 2500 × g.
让试管冷却至室温，并加入 1·5 mL 1-丁醇。加盖试管，涡旋混合并以 2500 × g 离心 15 分钟。
The upper layer was carefully transferred to polystyrene disposable semi-micro cuvettes, and the absorbance at 532 nm was measured.
将上层小心地转移到聚苯乙烯一次性半微量比色皿中，并测量 532 nm 处的吸光度。
Results 结果
Molecular weights and osmolalities of intravenous iron preparations
静脉注射铁剂的分子量和渗透压
The gel permeation chromatogram traces show that all the intravenous iron preparations are largely homogeneous (by UV, λ = 280 nm), except for unidentified small low molecular weight peaks appearing for both iron sucrose and ferric gluconate (Fig. 2 and Table 1). The relative order of molecular weights was as follows: ferumoxytol > iron dextran > iron sucrose > ferric gluconate.
凝胶渗透色谱图迹线显示，所有静脉注射铁制剂基本上都是均相的（通过 UV，λ = 280 nm），除了蔗糖铁和葡萄糖酸铁都出现了未鉴定的低分子量小峰（图 2 和表 1）。分子量的相对顺序如下：氧化铁 > 右旋糖酐铁 > 蔗糖铁 > 葡萄糖酸铁。
Molecular weights of intravenous iron preparations as determined by gel permeation chromatogram.
通过凝胶渗透色谱图测定静脉注射铁剂的分子量。
表 1.静脉铁剂制剂的分子量
| Iron preparation 铁剂制备 | Molecular weight vs. protein standards (kDa) 分子量与蛋白质标准品 （kDa） 的关系 |
|---|---|
| Sodium ferric gluconate 葡萄糖酸铁钠 | 200 |
| Iron sucrose 蔗糖铁 | 252 |
| Low molecular weight iron dextran 低分子量葡聚糖铁 |
410 |
| Ferumoxytol Ferumoxytol （铁氧化醇） | 731 |
The osmolalities of iron sucrose, ferric gluconate, iron dextran and ferumoxytol were 1316, 990, 500 and 291 mOsmol kg−1 respectively. Only ferumoxytol was isotonic.
蔗糖铁、葡萄糖酸铁、右旋糖酐铁和氧化铁的渗透压分别为 1316 、 990 、 500 和 291 mOsmol kg −1 。只有氧化铁是等渗的。
TEM results TEM 结果
TEM results for the four iron preparations (un-dialysed) are shown in Fig. 3. Ferumoxytol particles have a diameter of 6·4 ± 0·4 nm (n = 10). The iron dextran, ferric gluconate and iron sucrose TEM analyses are much less defined than for ferumoxytol, so it is difficult to get accurate particle dimensions, though it is apparent from Fig. 3 that the following trend is observed: ferumoxytol > iron dextran > iron sucrose ≥ ferric gluconate. Previous observations on the TEM of iron sucrose and ferric gluconate were of solutions of the iron oxides that were dialysed to remove low molecular mass components [9]. The average core sizes of iron sucrose and ferric gluconate in this report were 3 ± 2 and 2 ± 1 nm respectively.
四种铁制剂（未透析）的 TEM 结果如图 3 所示。Ferumoxytol 颗粒的直径为 6·4 ± 0·4 nm （n = 10）。葡聚糖铁、葡萄糖酸铁和蔗糖铁 TEM 分析的定义远低于氧化铁，因此很难获得准确的颗粒尺寸，尽管从图 3 中可以明显观察到以下趋势：氧化铁>葡聚糖铁>蔗糖铁≥葡萄糖酸铁。以前对蔗糖铁和葡萄糖酸铁的 TEM 的观察是氧化铁的溶液，这些氧化铁被透析以去除低分子量成分 [ 9]。本报告中蔗糖铁和葡萄糖酸铁的平均核心大小分别为 3 ± 2 和 2 ± 1 nm。
Transmission electron microscopy images of ferumoxytol, iron dextran, iron sucrose and sodium ferric gluconate demonstrating the particulate nature of the iron oxide complexes.
氧化铁、葡聚糖铁、蔗糖铁和葡萄糖酸铁钠的透射电子显微镜图像显示了氧化铁络合物的颗粒性质。
Ultrafilterable free iron
超滤游离铁
Table 2 shows data from ultrafiltration of the four iron preparations. The highest proportion of ultrafilterable free iron was observed with ferric gluconate (295 μg mL−1; 2·36%) and the least with ferumoxytol (0·3 μg mL−1; 0·001%).
表 2 显示了四种铁制剂的超滤数据。葡萄糖酸铁的超滤游离铁比例最高 （295 μg mL −1 ;2·36%），而氧化铁的超滤游离铁比例最低 （0·3 μg mL −1 ;0·001%）。
表 2.通过各种静脉注射铁剂的超滤测量的游离铁
| Iron preparation 铁剂制备 | Free iron (μg mL−1) 游离铁 （μg mL −1 ） |
% Free iron % 游离铁 |
|---|---|---|
| Sodium ferric gluconate 葡萄糖酸铁钠 | 295 | 2·36 |
| Iron sucrose 蔗糖铁 | 7·6 | 0·038 |
| Low molecular weight iron dextran 低分子量葡聚糖铁 |
149 | 0·298 |
| Ferumoxytol Ferumoxytol （铁氧化醇） | 0·3 | 0·001 |
Dialysability of iron preparations
铁制剂的可渗透性
Figure 4 shows the cumulative proportion (%) of iron filtered during circulation through the extracorporeal circuit.
图 4 显示了在通过体外回路的循环过程中过滤的铁的累积比例 （%）。
The proportion of iron filtered represents the catalytic or free iron content of each iron preparation, as intact intravenous iron preparations are not dialysable given their relatively large molecular weight relative to pore size of the standard high-flux dialyzer.
过滤的铁的比例代表每种铁制剂的催化或游离铁含量，因为完整的静脉注射铁制剂是不可透析的，因为它们的分子量相对于标准高通量透析器的孔径相对较大。
Cumulative proportion (%) of iron filtered following circulation of intravenous iron preparations (80 mg of elemental iron) dissolved in PBS with 4·5% bovine serum albumin.
静脉注射铁制剂（80 毫克元素铁）溶解在含有 4·5% 牛血清白蛋白的 PBS 中后循环后过滤的铁的累积比例 （%）。
After subtraction of background readings (obtained from circulation of PBS–BSA through the circuit, Control in Fig. 4), the cumulative proportion of iron measured in 5 L of filtrate was minimal for ferumoxytol and iron dextran (< 1·0%). However, approximately 3% and 5% of total iron was filtered for iron sucrose and ferric gluconate respectively.
减去背景读数（从 PBS-BSA 通过回路的循环中获得，图 4 中的对照）后，氧化铁和葡聚糖铁在 5 L 滤液中测得的铁的累积比例最小 （< 1·0%）。然而，分别过滤了大约 3% 和 5% 的总铁中的蔗糖铁和葡萄糖酸铁。
Catalytic or free iron release in rat serum
大鼠血清中的催化或游离铁释放
Ex vivo experiments: analysis of rat serum spiked with intravenous iron preparations. Results of the BDI assay on rat sera, spiked with iron complex ex vivo, are provided in Table 3. Correlation coefficient of the standard curve for the BDI assay was r2 = 0·985. The baseline concentration of catalytic iron in the rat serum was 0·16 μM. The results clearly demonstrate minimal free iron release with ferumoxytol after incubation in rat serum for 10 min.
离体实验：分析加标静脉铁制剂的大鼠血清。表 3 提供了体外加标铁复合物的大鼠血清的 BDI 测定结果。BDI 测定的标准曲线相关系数为 r 2 = 0·985。大鼠血清中催化铁的基线浓度为 0·16 μM。结果清楚地表明，在大鼠血清中孵育 10 分钟后，氧化铁的游离铁释放量最小。
The order of catalytic iron release as detected by the BDI assay was as follows: ferumoxytol < iron dextran < iron sucrose < ferric gluconate.
BDI 测定检测到的催化铁释放顺序如下：氧化铁 < 右旋糖酐铁 < 蔗糖铁 < 葡萄糖酸铁。
表 3.体外各种静脉注射铁剂对催化铁的贡献比较*
| Iron preparation 铁剂制备 | Catalytic iron 催化铁 | ||
|---|---|---|---|
| μM | μg mL−1 微克 mL −1 | % of dose 剂量百分比 | |
| Sodium ferric gluconate 葡萄糖酸铁钠 | 8·12 | 0·45 | 1·4 |
| Iron sucrose 蔗糖铁 | 4·22 | 0·24 | 0·69 |
| Low molecular weight iron dextran 低分子量葡聚糖铁 |
1·10 | 0·06 | 0·19 |
| Ferumoxytol Ferumoxytol （铁氧化醇） | 0·40 | 0·02 | 0·07 |
-
*Catalytic iron was measured by the bleomycin detectable iron (BDI) assay in rat serum to which iron preparations were added. The baseline concentration of catalytic iron in the serum was 0·16 μM.
*通过添加铁制剂的大鼠血清中的博来霉素可检测铁 （BDI） 测定法测量催化铁。血清中催化铁的基线浓度为 0·16 μM。
In vivo experiments: analysis of serum from rats administered iron preparations by tail vein injection. The BDI assay was performed on serum obtained from rats administered iron preparations at a dose of 1·4 mg kg−1. Correlation coefficient of the standard curve for the BDI assay was r2 = 0·997. Table 4 shows the results for the BDI assay in serum pre-injection and 5 min post-injection for each of the iron preparations. Results represent mean ± SEM of three rats used for each iron preparation. Serum concentration of catalytic iron post-injection was lowest for ferumoxytol.
体内实验：通过尾静脉注射分析施用铁制剂的大鼠血清。对以 1·4 mg kg −1 剂量施用铁制剂的大鼠获得的血清进行 BDI 测定。BDI 测定的标准曲线的相关系数为 r 2 = 0·997。表 4 显示了每种铁制剂在血清注射前和注射后 5 分钟内的 BDI 测定结果。结果代表用于每种铁制剂的三只大鼠的平均 ± SEM。注射后催化铁的血清浓度对于氧化铁最低。
The trend was as follows: ferumoxytol < iron dextran < iron sucrose < ferric gluconate.
趋势如下：氧化铁 < 右旋糖酐铁 < 蔗糖铁 < 葡萄糖酸铁。
表 4.体内施用各种静脉铁制剂的催化铁释放的比较*
| Pre-injection 注射前 | 5 min post-injection 注射后 5 分钟 | |||
|---|---|---|---|---|
| μM | μg mL−1 微克 mL −1 | μM | μg mL−1 微克 mL −1 | |
| Sodium ferric gluconate 葡萄糖酸铁钠 | 0·07 ± 0·009 0·07 ± 0·009 | 0·004 | 1·69 ± 0·21 1·69 ± 0·21 | 0·09 |
| Iron sucrose 蔗糖铁 | 0·11 ± 0·01 0·11 ± 0·01 | 0·006 | 0·92 ± 0·41 0·92 ± 0·41 | 0·05 |
| Low molecular weight iron dextran 低分子量葡聚糖铁 |
0·08 ± 0·01 0·08 ± 0·01 | 0·006 | 0·65 ± 0·09 0·65 ± 0·09 | 0·04 |
| Ferumoxytol Ferumoxytol （铁氧化醇） | 0·10 ± 0·03 0·10 ± 0·03 | 0·003 | 0·33 ± 0·05 0·33 ± 0·05 | 0·02 |
-
*Catalytic iron was measured by the bleomycin detectable iron (BDI) assay (mean ± SEM) in serum obtained from rats injected with intravenous iron (1·4 mg kg−1) (n = 3 rats for each iron preparation).
*通过博来霉素可检测铁 （BDI） 测定法（平均 ± SEM）测量静脉注射铁剂 （1·4 mg kg −1 ） 的大鼠血清中的催化铁（每种铁制剂 n = 3 只大鼠）。
Catalytic or free iron release in human serum
人血清中的催化或游离铁释放
Ex vivo experiments: analysis of pooled human serum spiked with iron preparations. Table 5 shows the results of the BDI assay in pooled human serum spiked with the iron preparations at a concentration of 42 μg mL−1 (42–168 μg mL−1 for ferumoxytol). Catalytic or BDI concentrations for the various iron agents ranged from 0·06 to 0·40 μg mL−1, with the lowest concentration noted with ferumoxytol, and the highest with ferric gluconate.
离体实验：分析加标铁制剂的混合人血清。表 5 显示了在加标浓度为 42 μg mL −1 （氧化铁为 42–168 μg mL −1 ）铁制剂的混合人血清中的 BDI 分析结果。各种铁剂的催化或 BDI 浓度范围为 0·06 至 0·40 μg mL −1 ，其中氧化铁浓度最低，葡萄糖酸铁最高。
BDI concentration increased at higher doses of ferumoxytol, but even at the highest dose equivalent (4× the dose), it was still lower than that detected with the 125 mg dose equivalent of ferric gluconate and iron sucrose (0·28 ± 0·10 μg mL−1 vs. 0·41 ± 0·15 μg mL−1 and 0·37 ± 0·10 μg mL−1 respectively).
BDI 浓度在较高剂量的氧化铁下增加，但即使在最高剂量当量（剂量的 4×）下，它仍然低于葡萄糖酸铁和蔗糖铁的 125 mg 剂量当量（分别为 0·28 ± 0·10 μg mL −1 对 0·41 ± 0·15 μg mL −1 和 0·37 ± 0·10 μg mL −1 ）。
表 5.含有氧化铁复合物的人血清中游离铁的离体测定*
| Sodium ferric gluconate 葡萄糖酸铁钠 | Iron sucrose 蔗糖铁 | Low molecular weight iron dextran 低分子量葡聚糖铁 |
Ferumoxytol Ferumoxytol （铁氧化醇） | |||
|---|---|---|---|---|---|---|
| Iron concentration, μg mL−1 铁浓度，μg mL −1 |
42 (n= 5) 42 （n= 5） | 42 (n = 5) 42 （n = 5） | 42 (n = 5) 42 （n = 5） | 42 (n = 5) 42 （n = 5） | 84 (n = 2) 84 （n = 2） | 168 (n = 2) 168 （n = 2） |
| Dose equivalent, mg 剂量当量，mg | 125 | 125 | 125 | 125 | 250 | 500 |
| Mean (±SD) BDI, μg mL−1 平均 （±SD） BDI，μg mL −1 |
0·41 ± 0·15 0·41 ± 0·15 | 0·37 ± 0·10 0·37 ± 0·10 | 0·17 ± 0·05 0·17 ± 0·05 | 0·06 ± 0·03 0·06 ± 0·03 | 0·12 ± 0·05 0·12 ± 0·05 | 0·28 ± 0·10 0·28 ± 0·10 |
| % of dose 剂量百分比 | 0·96 | 0·89 | 0·40 | 0·15 | 0·15 | 0·16 |
-
*Free iron was measured by the bleomycin detectable iron (BDI) concentration in pooled human serum to which iron preparations were added to make an iron complex concentration of 42 μg Fe mL−1 (42–168 μg mL−1 for ferumoxytol).
*游离铁是通过混合人血清中的博来霉素可检测铁 （BDI） 浓度来测量的，其中添加了铁制剂，使铁复合物浓度为 42 μg Fe mL −1 （氧化铁为 42–168 μg mL −1 ）。
Discussion 讨论
The results from the experiments detailed above illustrate that, in contrast to intravenous iron sucrose and sodium ferric gluconate, intravenous ferumoxytol contributes negligible dialysable or ultrafilterable iron as detected by atomic absorption spectrophotometry, and minimal detectable free iron after incubation in rat or human serum or after intravenous administration in rats.
上述实验结果表明，与静脉注射蔗糖铁和葡萄糖酸铁钠相比，静脉注射氧化铁对原子吸收分光光度法检测到的可透析或超滤铁的贡献可以忽略不计，并且在大鼠或人血清中孵育后或在大鼠中静脉内给药后，可检测到的游离铁最少。
Release of bioactive iron from intravenous iron preparations results mainly from intracellular release after clearance of the agent from plasma and uptake by reticuloendothelial cells [10–13]. However, several in vitro studies have shown evidence of iron-induced biological activity, suggesting that release of bioactive or catalytic iron may occur before cellular uptake of the intact parenteral iron agent [14,15]. Van Wyck and colleagues quantified and compared the size of the labile iron fraction among several intravenous iron preparations by examining iron donation to transferrin in vitro [16]. They observed that approximately 2–6% of total iron in these preparations was available for direct donation to transferrin in vitro with the following progression: high molecular weight iron dextran < low molecular weight iron dextran < iron sucrose < ferric gluconate [16]. This fraction may, therefore, contribute to the release of bioactive iron in patients after intravenous iron administration.
静脉注射铁制剂中生物活性铁的释放主要是由于药物从血浆中清除并被网状内皮细胞摄取后的细胞内释放[10–13]。然而，几项体外研究表明铁诱导生物活性的证据，表明生物活性或催化铁的释放可能发生在细胞吸收完整的肠外铁剂之前[14,15]。Van Wyck 及其同事通过检查体外铁剂的供铁，量化并比较了几种静脉注射铁制剂中不稳定铁分数的大小 [ 16]。他们观察到，这些制剂中大约 2-6% 的总铁可在体外直接捐赠给转铁蛋白，其进展如下：高分子量葡聚糖铁 < 低分子量葡聚糖铁 < 蔗糖铁 < 葡萄糖酸铁 [ 16]。因此，该部分可能有助于静脉注射铁剂后患者体内生物活性铁的释放。
Indeed, administration of ferric gluconate at doses ranging from 62·5 to 125 mg during haemodialysis is associated with transferrin saturation exceeding 100% [17].
事实上，在血液透析期间以 62·5 至 125 mg 的剂量施用葡萄糖酸铁与转铁蛋白饱和度超过 100% 相关 [ 17]。
Roob and colleagues observed that after intravenous administration of a 100 mg dose of iron (III) hydroxide sucrose complex in patients with end-stage renal disease during haemodialysis, there was a sharp rise in transferrin saturation which exceeded 100% at 30 min after iron administration [18]. Further, BDI was not detectable before the iron administration, but the level rose significantly after intravenous iron administration.
Roob 及其同事观察到，在血液透析期间，终末期肾病患者静脉注射 100 mg 剂量的氢氧化铁蔗糖复合物后，转铁蛋白饱和度急剧上升，在补铁后 30 分钟超过 100% [ 18]。此外，在补铁前未检测到 BDI，但在静脉补铁后水平显着升高。
At 30 min, there was a strong association between serum BDI concentration and transferrin saturation as well as plasma level of malondialdehyde (a measure of lipid peroxidation) [18]. However, these observations are difficult to interpret given the potential interference of the iron preparation with the assay for serum transferrin-bound iron.
30 分钟时，血清 BDI 浓度与转铁蛋白饱和度以及血浆丙二醛水平（脂质过氧化的量度）之间存在很强的相关性 [ 18]。然而，鉴于铁制剂对血清转铁蛋白结合铁测定的潜在干扰，这些观察结果难以解释。
This interference may account for some or all of the rise in serum iron and transferrin saturation immediately after intravenous iron administration.
这种干扰可能是静脉铁剂给药后血清铁和转铁蛋白饱和度升高的部分或全部原因。
Other investigators have shown that catalytically active or non-transferrin bound iron is detected in the plasma of patients receiving intravenous iron formulations even when transferrin saturation levels are less than 83% [4,15].
其他研究人员表明，即使转铁蛋白饱和度水平低于 83%，在接受静脉铁剂的患者血浆中也能检测到催化活性或非转铁蛋白结合的铁 [ 4,15]。
Intravenous iron agents have a colloidal structure consisting of an iron oxide core surrounded by a carbohydrate shell that stabilizes the iron oxide and slows the release of bioactive iron from the iron oxide core.
静脉注射铁剂具有胶体结构，由氧化铁芯组成，周围环绕着碳水化合物壳，可稳定氧化铁并减缓生物活性铁从氧化铁芯中释放。
As a result of differences in core size and carbohydrate coating, intravenous iron agents differ by overall molecular weight. As MW determinations depend on the method adopted, reported results for a single agent may differ substantially.
由于核心大小和碳水化合物涂层的差异，静脉注射铁剂的总分子量不同。由于 MW 测定取决于所采用的方法，因此单一药物的报告结果可能大不相同。
Thus, direct comparative studies are best suited to assess relative particle sizes of intravenous iron agents.
因此，直接比较研究最适合评估静脉铁剂的相对粒径。
Two such studies have established the relative molecular weights of the currently available intravenous iron preparations, as follows: high molecular weight iron dextran > low molecular weight iron dextran >> iron sucrose > ferric gluconate [19,20].
两项这样的研究已经确定了目前可用的静脉注射铁制剂的相对分子量，如下：高分子量右旋糖酐铁 > 低分子量右旋糖酐铁 >>蔗糖铁 > 葡萄糖酸铁 [ 19,20]。
TEM imaging of the intravenous iron preparations, which detects only the iron oxide core reported here and elsewhere [21], confirms that the relative ordering of the iron oxide core size is ferumoxytol > iron dextran >> iron sucrose > ferric gluconate, establishing the fact that the relative core diameters follow the same sequence as those of the whole molecule [9]. We analysed the molecular weight of ferumoxytol and three commercial intravenous iron preparations by GPC using commercial protein and dextran MW standards.
Regardless of the MW standard used, the relative order of molecular weights remained the same and was as follows: ferumoxytol > iron dextran > iron sucrose > ferric gluconate.
These results have important implications for core surface area available for bioactive iron release.
Overall molecular weight may affect two biological characteristics of intravenous iron agents that are directly relevant to therapeutic use in patients: rate of release of bioactive iron from the ferric hydroxide core and rate of plasma clearance of the agent after intravenous administration [21]. Iron release in vitro appears to be related to total molecular weight in an inverse sequence: the smaller the particle size and molecular weight, the more rapid the release of bioactive iron.
For the same amount of iron, a collection of small spheres would expose a greater total surface area than a collection of larger spheres, because as the radius (r) of a sphere increases, the volume increases by 4/3πr3, but the surface area increases only by 4πr2. In other words, for the same total amount of core iron, surface area available for iron release increases dramatically as core radius decreases [16]. Thus, our observations indicating minimal detectable free iron release from ferumoxytol, in contrast to iron sucrose and ferric gluconate in the ex vivo and in vivo studies detailed in this manuscript, may well be related to the structural characteristics of ferumoxytol.
Release of bioactive or free iron from intravenous iron preparations has implications for both acute as well as chronic adverse effects.
Detailed descriptions of true free iron reactions are available from the early literature on parenteral administration of compounds such as ferric iron [21]. Free-iron-like reactions may account for many of the uncommon but serious adverse drug events reported with the use of IV iron preparations [2,3].
Most patients with advanced chronic kidney disease exist in a microenvironment characterized by infection, oxidative stress and inflammation. Biologically active or free iron may play a role in each of these processes.
Although there is no strong clinical evidence associating intravenous iron with infections, both in vivo and in vitro studies indicate that bioactive iron plays a critical role in microbial growth and leucocyte function. In vitro, bioactive iron in serum from patients administered intravenous iron sucrose enhances growth of selected bacteria [4]. In vivo, transfusional iron overload in patients is associated with microbial infection [22]. Both in patients [23] and in vitro [24], intravenous iron is associated with defects in neutrophil function.
These observations have led to the hypothesis that the release of bioactive iron, arising from the use of intravenous iron preparations currently available, increases the risk of infections in dialysis-dependent patients with chronic kidney disease.
There has been persistent speculation regarding a link between parenteral iron therapy and oxidative stress. Short-term administration of intravenous iron has been shown to induce oxidative stress [25,26], a non-traditional risk factor for atherosclerotic cardiovascular disease.
Rooyakkers and colleagues observed that intravenous infusion of therapeutic doses of ferric saccharate in patients on haemodalysis resulted in increased oxidative stress and acute endothelial dysfunction [27]. Further, chronic administration of intravenous iron preparations with high levels of catalytic free iron may produce pro-oxidant effects that are long lasting.
With higher cumulative doses of such iron preparations administered to haemodialysis patients, there is greater evidence of oxidative stress [28,29]. Indeed, a serum ferritin level in excess of 600 ng mL−1 in dialysis-dependent patients is associated with evidence of elevated lipid peroxidation and reduction in antioxidant defense [28], but this is more likely to be a consequence of co-linearity due to the underlying co-morbidity and inflammation. Furthermore, patients with high levels of inflammatory biomarkers manifest greater evidence of oxidative stress following intravenous iron therapy [28].
The observations above indicate the importance of developing an intravenous iron preparation that leads to negligible free iron release in the extracellular space following intravenous administration in the high-risk population of patients with advanced chronic kidney disease.
Conclusion
Our observations from the ex vivo and in vitro experiments detailed in this manuscript indicate that ferumoxytol, a new isotonic superparamagnetic colloidal iron oxide, results in negligible free iron release as determined by the BDI assay or atomic absorption spectrophotometry in comparison with iron sucrose or ferric gluconate, two widely used intravenous iron preparations.
Further clinical studies are needed to validate these observations.
Acknowledgements
The authors thank the colleagues and laboratory personnel whose contributions allowed the successful completion of the studies detailed in this manuscript.
Address
Division of Nephrology, Tufts Medical Center, Boston, MA, USA (V. S. Balakrishnan, M. Rao, A. T. Kausz); AMAG Pharmaceuticals, Inc., Cambridge, MA, USA (A. T. Kausz, L. Brenner, B. J. G. Pereira, T. B. Frigo, J. M. Lewis).
