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Characterisation


Size


Physicochemical characterisation Structure


Stability In vitro stability


In vitro characterisation Haematology


Distribution Immunotoxicity Figure 1: Steps of NBCD characterisation


modifications (for example, akaganéite, lepidocrocite or ferrihydrite), which have different stability properties.17


The


determination of the morphology of the iron core is therefore an important characteristic of iron carbohydrate NBCDs. The polymorphic form of the core can be examined by X-ray powder diffraction (XRPD) and Mößbauer spectroscopy, a spectroscopic method that uses the recoil free gamma ray absorbance of some elements. If possible, both methods should be combined, because results from these methods are not always overlapping. The grade of the crystallinity is a characteristic, which is also of interest in the structure investigation of NBCDs with inorganic components, as it has an influence on complex degradation. Research techniques from the material sciences such as differential scanning calorimetry (DSC) or XRPD are most suitable for these analyses. Various other methods are available for stability studies. The stability of NBCD products can be tested with regard to temperature and pH, for example, by spectrometric methods such as UV-Vis.6


Impurities


Some impurities can result from the production process of the NBCDs. They may lead to adverse effects, which are not directly related to the


pharmacological effect of the NBCDs (for example, Fe2+


in FeOOH carbohydrate 12


nanoparticles). Impurities can be detected using HPLC methods or ICP-MS. However, some impurities in NBCDs are more challenging to detect, for example, ferrous (divalent) iron. Ferrous iron is considered an impurity in parenteral (ferric) iron preparations. As


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‘free’ ferrous iron, it might generate peroxides, which may be responsible for the development of atherosclerosis18


Few


In vivo characterisation Toxicity


Purity


may oversaturate transferrin binding and generate non-transferrin-bound iron. This is associated with oxidative stress and possible side effects such as myocardial infarction and bacterial infection have observed.18–20


The Immunology Toxicity Clinical effects


determination of labile iron is therefore essential for the characterisation of iron carbohydrate NBCDs. Several tests have been developed to measure the labile iron content. They either mobilise labile iron with chelators such as Ferrozine® EDTA,21 transferrin,22


,6 bleomycin23


polyphenols from white tea,8 it via dialysis.6


and or separate Labile iron tests should . The


ratio of ferric (trivalent) to ferrous iron is therefore an important characteristic for parenteral iron preparations.14


methods to measure this have been established so far, as it is challenging to detect ferrous iron in the presence of ferric iron. Moreover, ferrous iron can be easily oxidised to ferric iron, which makes the task of detecting the content of reduced iron even more difficult. Mößbauer spectroscopy is suitable for ferrous iron determination, but this method is not suitable for trace analysis. For some iron carbohydrates, a cerimetric examination has been reported.8


In vitro methods Various in vitro methods are applicable for NBCD characterisation. If aiming for intravenous administration of the NBCD, a characterisation method should cover issues of haematology, toxicity and immunology such as phagocytosis or cytokine assays. Various cell-based models can be applied for these investigations. These tests cover the standard methods (for example, phagocytosis assay, cytokine detection assays) for characterisation of all parenteral drugs and are widely known. The challenge for NBCD characterisation is developing specific in vitro assays that simulate the relevant in vivo parameters for the specific product. For iron carbohydrates, the amount of labile iron has been identified as such a parameter. The term ‘labile iron’ describes the fraction of iron that is only weakly-bound to the complex and can be easily transferred to transferrin following injection of the iron product. Products containing a high degree of labile iron


estimate the amount of released labile iron after administration as a fraction of the given dose. The EMA proposes its determination by either acid degradation of ferric iron or by measuring the iron transfer to transferrin.14


In vivo methods


As mentioned previously, the in vivo behaviour of NBCDs cannot be predicted by physicochemical and in vitro methods alone. In addition, data from human pharmacokinetic studies cannot reveal the in vivo disposition thoroughly, because blood or plasma concentrations do not deliver sufficient information, for example, on specific tissue distribution.14 Therefore, animal models for tissue/ organ distribution are inevitable. The investigated compartments should be chosen with regard to the potential toxic side effects of the complex, their target tissues and their mode of elimination. In the case of iron, the relevant compartments are plasma, red blood cells, the reticular endothelial system and target tissues including kidney, liver, heart and lungs.14


Besides toxicity and


distribution tests in animals such as rats,10


a promising distribution model for iron complexes has been seen within foetal avians.5,24


Conclusions


An array of methods is available for NBCD characterisation. Due to their heterogeneity, each NBCD requires its own toolbox for specific characterisation. The investigator needs to define the relevant parameters and choose the most appropriate from the multiple methods available. Physicochemical, in vitro, in vivo and clinical studies as well as correlation between in vitro and in vivo parameters are needed for the biopredictive characterisation of this challenging drug class. l


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