Iron-carbohydrate Complexes Research Articles

Nano-scale characterization of iron-carbohydrate complexes by cryogenic scanning transmission electron microscopy: Building the bridge to biorelevant characterization

Iron deficiency and iron deficiency anemia pose significant health challenges worldwide. Iron carbohydrate nanoparticles administered intravenously are a mainstay of treatment to deliver elemental iron safely and effectively. However, despite decades of clinical use, a complete understanding of their physical structure and the significance for their behavior, particularly at the nano-bio interface, is still lacking, underscoring the need to employ more sophisticated characterization methods. Our study used cryogenic Scanning Transmission Electron Microscopy (cryo-STEM) to examine iron carbohydrate nanoparticle morphology. This method builds upon previous research, where direct visualization of the iron cores in these complexes was achieved using cryogenic Transmission Electron Microscopy (cryo-TEM). Our study confirms that the average size of the iron cores within these nanoparticles is approximately 2 nm across all iron-based products studied. Furthermore, our investigation revealed the existence of discernible cluster-like morphologies, not only for ferumoxytol, as previously reported, but also within all the examined iron-carbohydrate products. The application of cryo-STEM for the analyses of product morphologies provides high-contrast and high-resolution images of the nanoparticles, and facilitates the characterization at liquid nitrogen temperature, thereby preserving the structural integrity of these complex samples. The findings from this study offer valuable insights into the physical structure of iron-carbohydrate nanoparticles, a crucial step towards unraveling the intricate relationship between the structure and function of this widely used drug class in treating iron deficiency. Additionally, we developed and utilized the self-supervised machine learning workflow for the image analysis of iron-carbohydrate complexes, which might be further expanded into a useful characterization tool for comparability studies.

Intravenous iron therapy results in rapid and sustained rise in myocardial iron content through a novel pathway.

Intravenous iron therapies contain iron-carbohydrate complexes, designed to ensure iron becomes bioavailable via the intermediary of spleen and liver reticuloendothelial macrophages. How other tissues obtain and handle this iron remains unknown. This study addresses this question in the context of the heart. A prospective observational study was conducted in 12 patients receiving ferric carboxymaltose (FCM) for iron deficiency. Myocardial, spleen, and liver magnetic resonance relaxation times and plasma iron markers were collected longitudinally. To examine the handling of iron taken up by the myocardium, intracellular labile iron pool (LIP) was imaged in FCM-treated mice and cells. In patients, myocardial relaxation time T1 dropped maximally 3 h post-FCM, remaining low 42 days later, while splenic T1 dropped maximally at 14 days, recovering by 42 days. In plasma, non-transferrin-bound iron (NTBI) peaked at 3 h, while ferritin peaked at 14 days. Changes in liver T1 diverged among patients. In mice, myocardial LIP rose 1 h and remained elevated 42 days after FCM. In cardiomyocytes, FCM exposure raised LIP rapidly. This was prevented by inhibitors of NTBI transporters T-type and L-type calcium channels and divalent metal transporter 1. Intravenous iron therapy with FCM delivers iron to the myocardium rapidly through NTBI transporters, independently of reticuloendothelial macrophages. This iron remains labile for weeks, reflecting the myocardium's limited iron storage capacity. These findings challenge current notions of how the heart obtains iron from these therapies and highlight the potential for long-term dosing to cause cumulative iron build-up in the heart.

Iron-carbohydrate complexes treating iron anaemia: Understanding the nano-structure and interactions with proteins through orthogonal characterisation

Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as chronic kidney disease, heart failure and various inflammatory conditions. Even though a plethora of physicochemical characterisation data and clinical studies are available for these products, evidence-based correlation between physicochemical properties of iron-carbohydrate complexes and clinical outcome has not fully been elucidated yet. Studies on other metal oxide NPs suggest that early interactions between NPs and blood upon IV injection are key to understanding how differences in physicochemical characteristics of iron-carbohydrate complexes cause variance in clinical outcomes. We therefore investigated the core-ligand structure of two clinically relevant iron-carbohydrate complexes, iron sucrose (IS) and ferric carboxymaltose (FCM), and their interactions with two structurally different human plasma proteins, human serum albumin (HSA) and fibrinogen, using a combination of cryo-scanning transmission electron microscopy (cryo-STEM), x-ray diffraction (XRD), small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS). Using this orthogonal approach, we defined the nano-structure, individual building blocks and surface morphology for IS and FCM. Importantly, we revealed significant differences in the surface morphology of the iron-carbohydrate complexes. FCM shows a localised carbohydrate shell around its core, in contrast to IS, which is characterised by a diffuse and dynamic layer of carbohydrate ligand surrounding its core. We hypothesised that such differences in carbohydrate morphology determine the interaction between iron-carbohydrate complexes and proteins and therefore investigated the NPs in the presence of HSA and fibrinogen. Intriguingly, IS showed significant interaction with HSA and fibrinogen, forming NP-protein clusters, while FCM only showed significant interaction with fibrinogen. We postulate that these differences could influence bio-response of the two formulations and their clinical outcome. In conclusion, our study provides orthogonal characterisation of two clinically relevant iron-carbohydrate complexes and first hints at their interaction behaviour with proteins in the human bloodstream, setting a prerequisite towards complete understanding of the correlation between physicochemical properties and clinical outcome.

Effect of intravenous iron-carbohydrate complexes in patients with heart failure with reduced ejection fraction and iron deficiency: a meta-analysis of randomized controlled trials

Abstract Background Iron deficiency (ID) has been associated with having the potential to cause heart failure as well as worsening heart failure with reduced ejection fraction (HFrEF). Treatment with a target on ID has been shown to lead to improvement in heart failure patient outcomes. In this study, we performed a meta-analysis of the RCTs to more comprehensively evaluate the effectiveness of intravenous iron-carbohydrate complexes in HF patients with ID. Methods We conducted a systematic literature search on MEDLINE and EMBASE. The search terms included "iron," "iron deficiency," "iron complex," "iron-carbohydrate," "iron sucrose," "heart failure," "HFrEF," and "intravenous." Included studies met the following criteria: (i) a study was published in a peer-reviewed journal; (ii) a study design was a randomized controlled trial; (iii) study enrolled patients with HFrEF and ID, and (iv) a study evaluated intravenous iron-carbohydrate complex. The primary outcome was all-cause mortality during the follow-up. The secondary outcomes were cardiovascular (CV) mortality (deaths due to CV causes), CV admission (hospitalizations due to cardiovascular causes), and HF admission (first hospitalizations due to HF or hospitalizations due to HF worsening). Risk ratios (RRs) with 95% confidence intervals (95%CIs) were calculated with the event/total number in each study and synthesized by a random-effects model. Results We identified 926 articles by the initial database search and additional manual search and retrieved the full text of 18 articles for further review. We then excluded 3 sub-analyses of the included trials to avoid patients’ duplications. Finally, we included 15 RCTs with a total of 3,085 patients (the median or mean age, 62–75 years; female, 25-55%; median follow-up duration, 2–141 weeks). Intravenous iron-carbohydrate complexes were associated with lower risks of all-cause mortality, CV mortality, CV admission, and HF admission, though statistical significance was only observed in HF admission (Figure A-D). CV admission and HF admission provided substantial heterogeneity. Conclusion The results of this meta-analysis showed that intravenous iron-carbohydrate complex therapy in patients with HFrEF and ID was beneficial in lowering the risk of hospitalization due to heart failure. Physicians should be aware of the significance of ID when treating patients with HFrEF.

Abstract 12515: Effect of Intravenous Iron Therapy in Patients With Heart Failure and Iron Deficiency: A Meta-Analysis of Randomized Controlled Trials

Background: Iron deficiency is a frequently observed comorbidity in patients with chronic heart failure (HF). Present guidelines strongly advocate for the routine screening of iron deficiency anemia in all HF patients. Numerous randomized controlled trials (RCTs) have examined the efficacy and safety of intravenous iron-carbohydrate complexes as a supplementation approach for patients with HF with reduced ejection fraction (HFrEF). Nevertheless, there exists a necessity to systematically synthesize the evidence derived from these RCTs through a comprehensive meta-analysis. Methods: A comprehensive search was conducted in major electronic databases to identify relevant RCTs. Studies published from inception to April 2023 were scrutinized. Results: A total of 14 RCTs with 3597 patients were included in the analysis. The pooled analysis demonstrated a significant reduction in the composite outcome of cardiovascular death or hospitalization for worsening HF (OR 0.55, 95% CI= 0.45, 0.67; p < 0.001). Similarly, the composite outcome of all-cause death or cardiovascular hospitalization showed a significant benefit with intravenous iron supplementation (OR 0.42, 95% CI= 0.28, 0.62; p < 0.001). Moreover, the analysis indicated a significant decrease in hospitalizations for HF among patients receiving IV iron (OR 0.49, 95% CI= 0.32, 0.75; p < 0.001). However, no statistically significant difference was observed in all-cause mortality (OR 0.90, 95% CI= 0.74, 1.10; p = 0.31) and cardiovascular mortality (OR 0.85, 95% CI= 0.68, 1.05; p = 0.14) between the two groups. Conclusion: The evidence from this meta-analysis underscores the significance of incorporating IV iron therapy in iron-deficient patients with systolic HF as it demonstrates significant improvements in patient outcomes.

Exploiting Pharma 4.0 Technologies in the Non-Biological Complex Drugs Manufacturing: Innovations and Implications.

The pharmaceutical industry has entered an era of transformation with the emergence of Pharma 4.0, which leverages cutting-edge technologies in manufacturing processes. These hold tremendous potential for enhancing the overall efficiency, safety, and quality of non-biological complex drugs (NBCDs), a category of pharmaceutical products that pose unique challenges due to their intricate composition and complex manufacturing requirements. This review attempts to provide insight into the application of select Pharma 4.0 technologies, namely machine learning, in silico modeling, and 3D printing, in the manufacturing process of NBCDs. Specifically, it reviews the impact of these tools on NBCDs such as liposomes, polymeric micelles, glatiramer acetate, iron carbohydrate complexes, and nanocrystals. It also addresses regulatory challenges associated with the implementation of these technologies and presents potential future perspectives, highlighting the incorporation of digital twins in this field of research as it seems to be a very promising approach, namely for the optimization of NBCDs manufacturing processes.

An Analytical Method for Determination of Total Iron in Pharmaceuticalgrade Intravenous Iron Colloidal Complexes by Redox-Potentiometry.

Iron carbohydrate complexes are colloidal dispersions made up of polynuclear Fe(III)-oxyhydroxide cores surrounded by a carbohydrate shell that stabilizes the complex in iron colloidal formulations. The current study provides an improved method that is precise, accurate, and linear for quantifying total iron in most Iron Carbohydrate Colloid Drug Products. Redox iodometry with a potentiometric determination is used to evaluate total iron in intravenous formulations. The visual indicator approach is more prone to fluctuations at endpoint calculations. Hence, the voltage potential approach is widely accepted as it is more accurate and sensitive. It tracks the actual change in activity that coincides with the equivalence point that is finally considered an endpoint. The principle is based on the idea that ferric iron in formulation reduces to ferrous iron in the presence of the iodide, which oxidizes to iodine. The released iodine is titrated using sodium thiosulfate. The proposed method was precise, with %RSD (relative standard deviation) not more than 1. The method was linear between 80% and 120%, with a linear regression of 0.999. The percent recovery ranged from 98.20 to 99.98 for the concentration ranges of 80-120. The method's robustness was checked by various analysts using different reagent grades. The proposed potentiometric determination method was precise, accurate, linear, and sensitive. The method was successfully validated, and the total iron content determined for commercial batches agrees with the iron claim on the label. Therefore, this method can be adapted widely for total iron content determination in any Intravenous formulation currently available on the market. The proposed method is more accessible at the Quality Control facilities on an industrial scale.

Uncovering the dynamics of cellular responses induced by iron-carbohydrate complexes in human macrophages using quantitative proteomics and phosphoproteomics

Iron-carbohydrate complexes are widely used to treat iron deficiencies. Macrophages play a crucial role in the uptake and fate of these nanomedicines, however, how complexed iron carbohydrates are taken up and metabolized by macrophages is still not fully understood. Using a (phospho-)proteomics approach, we assessed differences in protein expression and phosphorylation in M2 macrophages triggered by iron sucrose (IS). Our results show that IS alters the expression of multiple receptors, indicative of a complex entry mechanism. Besides, IS induced an increase in intracellular ferritin, the loss of M2 polarization, protective mechanisms against ferroptosis, and an autophagic response. These data indicate that macrophages can use IS as a source of iron for its storage and later release, however, the excess of iron can cause oxidative stress, which can be successfully regulated by the cells. When comparing IS with ferric carboxymaltose (FCM) and iron isomaltoside-1000 (IIM), complexes with a higher carbohydrate ligand stability, we observed that FCM and IIM are metabolized at a slower rate, and trigger M2 polarization loss to a lower extent. These results indicate that the surface characteristics of the iron-carbohydrate complexes may influence the cell responses. Our data show that the application of (phospho-)proteomics can lead to a better understanding of metabolic processes, including the uptake, biodegradation and bioavailability of nanomedicines.

Critical nanomaterial attributes of iron-carbohydrate nanoparticles: Leveraging orthogonal methods to resolve the 3-dimensional structure

Intravenous iron-carbohydrate nanomedicines are widely used to treat iron deficiency and iron deficiency anemia across a wide breadth of patient populations. These colloidal solutions of nanoparticles are complex drugs which inherently makes physicochemical characterization more challenging than small molecule drugs. There have been advancements in physicochemical characterization techniques such as dynamic light scattering and zeta potential measurement, that have provided a better understanding of the physical structure of these drug products in vitro. However, establishment and validation of complementary and orthogonal approaches are necessary to better understand the 3-dimensional physical structure of the iron-carbohydrate complexes, particularly with regard to their physical state in the context of the nanoparticle interaction with biological components such as whole blood (i.e. the nano-bio interface).

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes.

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Putting square pegs in round holes: Why traditional pharmacokinetic principles cannot universally be applied to iron-carbohydrate complexes

Intravenous iron-carbohydrate complexes are nanomedicines that are commonly used to treat iron deficiency and iron deficiency anemia of various etiologies. Many challenges remain regarding these complex drugs in the context of fully understanding their pharmacokinetic parameters. Firstly, the measurement of the intact iron nanoparticles versus endogenous iron concentration fundamentally limits the availability of data for computational modeling. Secondly, the models need to include several parameters to describe the iron metabolism which is not completely defined and those identified (e.g. ferritin) exhibit considerable interpatient variability. Additionally, modeling is further complicated by the lack of traditional receptor/enzyme interactions. The known parameters of bioavailability, distribution, metabolism, and excretion for iron-carbohydrate nanomedicines will be reviewed and future challenges that currently prevent the direct application of physiologically-based pharmacokinetic or other computational modeling techniques will be discussed.

Regulatory considerations for generic products of non-biological complex drugs.

The Non-Biological Complex Drug (NBCD) Working Group defines an NBCD as "a medicinal product, not being a biological medicine, where the active substance is not a homo-molecular structure, but consists of different (closely related and often nanoparticulate) structures that cannot be isolated and fully quantitated, characterized and/or described by physicochemical analytical means". There are concerns about the potential clinical differences between the follow-on versions and the originator products and within the individual follow-on versions. In the present study, we compare the regulatory requirements for developing generic products of NBCDs in the European Union (EU) and the United States (US). The NBCDs investigated included nanoparticle albumin-bound paclitaxel (nab-paclitaxel) injections, liposomal injections, glatiramer acetate injections, iron carbohydrate complexes, and sevelamer oral dosage forms. The demonstration of pharmaceutical comparability between the generic products and the reference products through comprehensive characterization is emphasized for all product categories investigated. However, the approval pathways and detailed requirements in terms of non-clinical and clinical aspects may differ. The general guidelines in combination with product-specific guidelines are considered effective in conveying regulatory considerations. While regulatory uncertainties still prevail, it is anticipated that through the pilot program established by the European Medicines Agency (EMA) and the FDA, harmonization of the regulatory requirements will be achieved, thereby facilitating the development of follow-on versions of NBCDs.

The clinical relevance of detectable plasma iron species in iron overload states and subsequent to intravenous iron-carbohydrate administration.

Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

Systematic review and meta-analysis of intravenous iron-carbohydrate complexes in HFrEF patients with iron deficiency.

Iron deficiency (ID) is a common co-morbidity in patients with heart failure (HF). The present meta-analysis evaluates the effect of intravenous (IV) iron-carbohydrate complex supplementation in patients with HF with reduced ejection fraction (HFrEF) and ID/iron deficiency anaemia (IDA). Randomized controlled trials (RCTs) comparing IV iron-carbohydrate complexes with placebo/standard of care in patients with HFrEF with ID/IDA were identified using Embase (from 1957) and PubMed (from 1989) databases through 25 May 2021. Twelve RCTs including 2381 patients were included in this analysis. The majority (90.8%) of patients receiving IV iron-carbohydrate therapy were administered ferric carboxymaltose (FCM); 7.5% received iron sucrose and 1.6% received iron isomaltoside. IV iron-carbohydrate therapy significantly reduced hospitalization for worsening HF [0.53 (0.42-0.65); P<0.0001] and first hospitalization for worsening HF or death [0.75 (0.59-0.95); P=0.016], but did not significantly impact all-cause mortality, compared with control. IV iron-carbohydrate therapy significantly improved functional and exercise capacity compared with the control. There was no significant difference in outcome between IV iron-carbohydrate formulations when similar endpoints were measured. No significant difference in adverse events (AE) was observed between the treatment groups. IV iron-carbohydrate therapy resulted in improvements in a range of clinical outcomes and increased functional and exercise capacity, whereas AEs were not significantly different between IV iron-carbohydrate and placebo/standard of care arms. These findings align with the European Society of Cardiology's 2021 HF guidelines, which recommend the consideration of FCM in symptomatic patients with a left ventricular ejection fraction < 45% and ID.

Tissue biodistribution of intravenous iron-carbohydrate nanomedicines differs between preparations with varying physicochemical characteristics in an anemic rat model

Intravenously administered iron-carbohydrate preparations are a structurally heterogenous class of nanomedicines. Iron biodistribution to target tissues is greatly affected by the physicochemical characteristics of these nanoparticles. Some regulatory agencies have recommended performing studies in animal models for biodistribution characterization and bioequivalence evaluation. In the present work, a systematic comparison of iron exposure, tissue biodistribution and pharmacodynamics of four intravenous iron-carbohydrates in anemic CD rats was conducted. A pilot study was performed to establish the anemic rat model, followed by a control study to evaluate the pharmacokinetics (serum iron, biodistribution) and pharmacodynamics (hematological parameters) in healthy and anemic controls and anemic rats receiving ferric carboxymaltose (FCM). The same parameters were then evaluated in a comparative study in anemic rats receiving FCM, iron sucrose (IS), iron isomaltoside 1000 (IIM), and iron dextran (ID). Despite similar serum iron profiles observed across the investigated nanomedicines, tissue iron biodistribution varied markedly between the individual intravenous iron-carbohydrate complexes. Tissue iron repletion differences were also confirmed by histopathology. These results suggest that employing serum iron profiles as a surrogate for tissue biodistribution may be erroneous. The variability observed in tissue biodistribution may indicate different pharmacodynamic profiles and warrants further study.

Criticality of Surface Characteristics of Intravenous Iron-Carbohydrate Nanoparticle Complexes: Implications for Pharmacokinetics and Pharmacodynamics.

Un-complexed polynuclear ferric oxyhydroxide cannot be administered safely or effectively to patients. When polynuclear iron cores are formed with carbohydrates of various structures, stable complexes with surface carbohydrates driven by multiple interacting sites and forces are formed. These complexes deliver iron in a usable form to the body while avoiding the serious adverse effects of un-complexed forms of iron, such as polynuclear ferric oxyhydroxide. The rate and extent of plasma clearance and tissue biodistribution is variable among the commercially available iron–carbohydrate complexes and is driven principally by the surface characteristics of the complexes which dictate macrophage opsonization. The surface chemistry differences between the iron–carbohydrate complexes results in significant differences in in vivo pharmacokinetic and pharmacodynamic profiles as well as adverse event profiles, demonstrating that the entire iron–carbohydrate complex furnishes the pharmacologic action for these complex products. Currently available physicochemical characterization methods have limitations in biorelevant matrices resulting in challenges in defining critical quality attributes for surface characteristics for this class of complex nanomedicines.

Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways

Intravenous iron-carbohydrate complex preparations (IVIP) are noninterchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of three IVIP we identify a two-pathway model of transient non-transferrin-bound iron (NTBI) generation following single dose administration. Twenty-eight hypoferremic non-anemic patients randomized to 200 mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside- 1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by high-performance liquid chromatography in combination with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, NTBI and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150 hours (h), except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13 μM at 8 h), rapidly increased with Fe-sucrose (0.8 μM at 2 h, 1.25 μM at 4 h), and delayed for Fe-carboxymaltose (0.57 μM at 24 h). NTBI area-under-curve (AUC) were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIP. We propose two mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIP generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Evaluation of Therapeutic Equivalence for the Follow-On Version of Intravenously Administered Non-Biological Complex Drugs.

The interchangeability evaluation for generic drugs formulated as intravenous injections normally only requires assessments of pharmaceutical equivalence (PE) when the medicinal products are simple small-molecule drugs. However, intravenously administered non-biological complex drugs (NBCDs), such as liposomes, microsphere suspension, or fat emulsion, have inherent passive disposition selectivity due to their special formulations, thereby the in vivo drug performances are improved. Because of the complexity in formulation, the in vitro pharmaceutical investigations of follow-on NBCDs are more complicated than those required for generic small-molecule drugs. In addition to qualitative and quantitative sameness of the active and inactive ingredients, it is required to comparatively study the static and kinetic microscopic particle-related physiochemical properties of the follow-on NBCDs versus the reference products. Moreover, for complex formulations that have a significant impact on the biodistribution of the drug compound, an in vivo bioequivalence (BE) study is also important. Since NBCDs that demonstrated bioequivalence through the conventional BE approach have been found inequivalent in efficacy or safety to the reference products, pivotal BE studies for follow-on NBCDs are required to take both encapsulated/total drug and free drug as the analytes to address release kinetics and biodistribution of the active pharmacological ingredient in the body. This manuscript reviews the 26 U.S. FDA published product-specific guidelines for intravenous injections. In general, these NBCDs can be stratified into four groups according to their release kinetics and ability of bio-membrane penetration. Group 1 consists of seven small-molecule, non-complex drugs; group 2 included four NBCDs with either microscale particle size or rapid dissolution property; group 3 include five loosely packed NBCDs (fat emulsions) and one quickly released ophthalmic liposomal drug; and the last group contains four cytotoxic liposomal or protein-bound NBCDs and five iron carbohydrate complexes. The requirements of the corresponding guidelines range from simple proof of PE between the test and thereference products, to a collection of studies that demonstrate the key manufacturing process (e.g. liposome loading), the particle- or vehicle-wise static and kinetic physiological characterizations, the dissolution test, and BEevaluation of both total/encapsulated drug form and free drug form between the follow-on NBCDs and their reference products. Such studies are challenging in implementation. Therefore, a variety of alternative approaches are proposed in this article.

NTBI Is Transiently Generated from Two Compartments during a Single Dose of Intravenous Iron - a Randomized Controlled Clinical Study

Background The prodrug-like intravenous iron-carbohydrate complex preparations (IVIPs) should not be considered interchangeable but with due regard to their different pharmacodynamics (PD) which is determined by the semi-crystalline iron core and carbohydrate shell structures (Bhandari et al, 2018). The pharmacokinetics (PK) of IVIPs influences PD profiles and thus efficacy and safety. Here we examine the complex PK/PD relationships with 3 IVIPs, for the first time identifying a 2-compartment model of transient NTBI generation following a single dose administration. Patients and Methods 28 hypoferremic patients randomized (n=8/arm) to 200mg (iron) of ferric carboxymaltose (FCM), iron sucrose (IS) and iron isomaltoside 1000 (IIM) or placebo (n=4), were followed up for 2 wks on an exploratory PK/PD study. Serum samples (0-312h) were analyzed for total serum iron (Fe), plasma IVIP-iron (IVIP-Fe), transferrin (Tf)-bound iron (TBI) by inductively coupled plasma mass spectrometry, Tf saturation (TSAT) by Tf ELISA and a tripyridyl triazine assay, serum ferritin (SF) by turbidimetry, and non-TBI (NTBI) and hepcidin (hepc) as published (Garbowski et al, Transl Med 2016), (Bansal et al, Rapid Commun Mass Spectrom 2010). A p value &lt;0.05 was statistically significant. The study, sponsored by Vifor Pharma, was conducted according to the Declaration of Helsinki. Results Pts were hypoferremic but non-anemic at baseline: SF 12.4±7.4µg/L, TSAT 18.6±8.5%, hemoglobin 128±8g/L. IVIP-dependent rises in Fe, IVIP-Fe, TSAT, SF, hepc, and NTBI returned to baseline in 2 wks or sooner, except for SF and TSAT. Large differences in the Fe AUCs on study, explainable by large differences in the IVIP-Fe AUCs, were consistent with the known relative differences of plasma half-lives (t1/2) of the IVIPs compared. IVIP-Fe at 4h was highest for IIM and smallest for IS, as was their AUC. With IIM, TSAT reached 80% at 16h, with IS 100% by 4h, and with FCM 98% at 24h; but AUC differences were small. NTBI was low with IIM (0.13µM at 8h), rapidly increased and high with IS (0.8µM at 2h, 1.25µM at 4h), and delayed for FCM (0.58µM at 24h). NTBI appeared after near- (for IIM) or full saturation of Tf but disappeared when TSAT fell &lt;65% (Fig). NTBI AUC for IS and FCM was 7.84- (p&lt;0.01) and 6.78-fold (p=0.04) higher vs. IIM but similar for IS and FCM. Hepc peak time varied, but not AUC or mean levels. SF varied significantly with IVIPs, rising faster with time for IS and FCM, slower for IIM (rate correlated with IVIP t1/2, r=-0.53, p=0.009), with levels highest for FCM (193µg/L at 72h) and IS (152µg/L at 36h) but lowest and latest for IIM (102µg/L at 96h). SF AUC was highest for FCM, suggesting relatively better iron bio-availability, with all IVIPs higher than placebo (Table and Fig). Discussion We propose 2 mechanisms for the observed kinetic NTBI profiles. For IS these have 2 distinct components: a rapid onset and an early peak (2-4h), then decay and a secondary peak/shoulder (16h), disappearing at 36h (Fig). The 1st peak reflects the rapid release of labile iron in plasma directly from circulating IVIPs, occurring &lt;t1/2 of IS and prior to any rise in SF which reports macrophage iron content (direct NTBI). In contrast, since the second peak occurs after &gt;4 t1/2, when &gt;90% of IS entered macrophages (SF half-maximal), the 16h peak likely represents iron re-released from macrophages (indirect NTBI). For IIM with long t1/2, the small NTBI peak at 8h thus represents direct NTBI, while the indirect release at 24h occurs at a rate where Tf does not fully saturate, with NTBI being consequently virtually absent (Fig). FCM first follows the IIM profile with minimal generation of direct NTBI, but displays a starkly different behavior reflecting indirect iron release shown as a strong NTBI peak at 24h, when ~4 plasma t1/2 have passed (Fig). This difference between IIM and FCM is accounted for by the difference in t1/2 of FCM and IIM i.e. by the amount of polymer having been taken up by macrophages (so only 6% of FCM and 30% of IIM still circulates). We conclude that, following IVIP administration, NTBI can arise by 2 mechanisms (direct early release and indirect later release). Different IVIPs generate different NTBI and serum ferritin signatures that are broadly PK-dependent, and may have a bearing both on the iron bio-availability and safety. Longer-term studies with common doses (1g for FCM and IIM) are warranted to link the initial NTBI exposure to subsequent safety and response parameters. Disclosures Garbowski: Vifor Pharma: Consultancy; Imara: Consultancy. Porter:Celgene: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria; Bluebird bio: Consultancy, Honoraria. Burckhardt:Vifor Pharma (previously): Employment. Hider:Vifor Pharma: Research Funding; Renapharma AB: Research Funding.

Iron Oxide Nanoparticle Formulations for Supplementation.

Intravenous (IV) iron is widely used to provide supplementation when oral iron is ineffective or not tolerated. All commercially available intravenous iron formulations are comprised of iron oxyhydroxide cores coated with carbohydrates of varying structure and branch characteristics. The diameter of the iron-carbohydrate complexes ranges from 5-100 nm and meets criteria for nanoparticles. Clinical use of IV iron formulations entered clinical practice beginning of the late 1950s, which preceded the nanomedicine exploration frontier. Thus, these agents were approved without full exploration of labile iron release profiles or comprehensive biodistribution studies. The hypothesis for the pathogenesis of acute oxidative stress induced by intravenous iron formulations is the release of iron from the iron-carbohydrate structure, resulting in transient concentrations of labile plasma iron and induction of the Fenton chemistry and the Haber-Weiss reaction promoting formation of highly reactive free radicals such as the hydroxyl radical. Among available IV iron formulations, products with smaller carbohydrate shells are more labile and more likely to release labile iron directly into the plasma (i.e., before metabolism by the reticuloendothelial system). The proposed biologic targets of labile-iron-induced oxidative stress include nearly all systemic cellular components including endothelial cells, myocardium, liver as well as low density lipoprotein and other plasma proteins. Most studies have relied on plasma pharmacokinetic analyses that require many model assumptions to estimate contribution of the iron-carbohydrate complex to elevations in serum iron indices and hemoglobin. Additionally, the commercially available formulations have not been well studied with regard to optimal dosing regimens, long-term safety and comparative efficacy. The IV iron formulations fall into a class defined by the Food and Drug Administration as "Complex Drugs" and thus present considerable challenges for bioequivalence evaluation.