High doses of ferric derisomaltose and ferric carboxymaltose both increase FGF-23 levels and lead to osteomalacia and bone loss in healthy male mice

doi:10.21203/rs.3.rs-7048209/v1

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High doses of ferric derisomaltose and ferric carboxymaltose both increase FGF-23 levels and lead to osteomalacia and bone loss in healthy male mice

https://doi.org/10.21203/rs.3.rs-7048209/v1

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Ferric carboxymaltose (FCM) and ferric derisomaltose (FDI) are key for treating anemia and iron deficiency. However, FCM has been shown to transiently raise serum fibroblast growth factor (FGF)-23 levels, causing hypophosphatemia and alterations in bone turnover in some patients. To date, detailed effects of FCM and FDI on bone mineralization are still missing.

This study examined FDI and FCM effects on bone mineralization and FGF-23 in healthy mice, avoiding disease confounders. Male 12-week-old C57BL/6J mice received single or weekly FDI, FCM, or placebo injections for 4 weeks.

Repeated FDI and FCM injections affected body weight, blood counts, and caused significant liver iron accumulation and high serum iron. Both reduced most bone parameters by µCT, however, FCM showed falsely high bone density due to iron clusters in the bone marrow. Histology revealed greater bone volume loss with FCM than FDI (-24% FDI, p < 0.05; -36% FCM, p < 0.01), likely from suppressed bone formation. Both iron formulations also led to a prominent increase in osteoid and FGF-23 (intact and C-terminal), raising the i:cFGF-23 ratio. In summary, repeated high doses of FDI and FCM in healthy mice increased i:cFGF-23 ratio and osteoid, while reducing bone formation and volume. Repeated dosing had stronger effects on bone than single dosing.

intravenous iron

osteomalacia

bone mineralization

fibroblast growth factor (FGF)-23

Iron is essential for several physiological processes, including oxygen transport via hemoglobin (Perutz et al. 1998; Ponka 1999) and myoglobin (Ponka 1999; Ordway and Garry 2004), energy production in mitochondria (Rouault and Tong 2008; Sheftel et al. 2012; Lill et al. 2012) and for the activity of enzymes involved in DNA replication and the cell cycle (Cazzola and Skoda 2000; Puig et al. 2017). As such, well-balanced iron levels are indispensable for health, with iron deficiency causing health problems such as anemia, weakness (Lopez et al. 2016) and shortness of breath. Because of its importance in various biochemical processes, the causes of iron deficiency are diverse, ranging from increased physiological demand, such as menstrual blood loss or pregnancy, to inadequate intake due to poverty, malnutrition or diet preference. Iron deficiency can also be a result of genetic diseases like iron-refractory iron-deficiency anemia or other pathologies that disturb the ability of iron absorption, such as bacterial or parasite infections, celiac disease, and chronic inflammatory diseases. To treat iron-deficiency anemia, oral iron therapy is usually sufficient to treat stable patients. However, oral iron supplementation can cause gastrointestinal side effects (Pantopoulos 2024) and worsen the conditions^2–4 in the latter groups of patients, and therefore needs to be replaced by parenteral therapy.

Indeed, different formulations for intravenous administration of iron have been developed to overcome the gastrointestinal adverse effects of the oral therapy. The formulations have been further improved over time to increase the amount of iron administration in each infusion while effectively preventing the toxicity of free labile iron in the circulation. The introduction of ferric carboxymaltose (FCM) made it possible to administer 1,000 mg of iron within a single dose. Ferric derisomaltose (FDI) is the most recent formulation that requires no test dose and can be administered up to 20 mg/kg body weight. Both of them have been shown to be efficacious in resolving iron-deficiency anemia and are better tolerated than oral iron therapy. Recommended by the European Crohn's and Colitis Organization as a first-line treatment, FCM and FDI became the most widely used treatments for iron-deficiency anemia in Europe.

Despite the rapid correction of iron-deficiency anemia, both formulations come with potential side effects. FDI is frequently associated with hypersensitivity reactions, which require immediate medical attention, while FCM has been reported to induce transient hypophosphatemia in a larger number of patients. The induction of hypophosphatemia has been associated with impaired tubular phosphate resorption and low levels of serum 1,25 (OH)₂D₃, mimicking hypophosphatemic conditions caused by excess actions of fibroblast growth factor-23 (FGF-23), such as X-linked hypophosphatemia or tumor-induced osteomalacia (Shimizu et al. 2009). Even though most cases of FCM-induced hypophosphatemia appear to be transient, severe cases of iron-deficiency anemia may require multiple doses of iron to correct red blood cell levels. Thus, it may be possible that repeated doses of intravenous iron could lead to prolonged hypophosphatemia, which may cause osteomalacia, characterized by impaired bone mineralization, pain, and fragility (Zoller et al. 2017; Bartko et al. 2018; Struppe et al. 2023). In fact, case reports revealed bone pain and osteomalacia after prolonged treatment with FCM, often in the context of inflammatory bowel disease, which per se causes bone impairment. Overall, the mechanisms underlying iron-induced FGF-23 levels and hypophosphatemia are not well understood.

Thus, in this study, as a first approach, we administered FDI and FCM parentally to male healthy adult mice to better understand the effects of iron on FGF-23 levels and bone mineralization excluding confounders from other underlying diseases. We used single-dose and multiple dose applications to mimic different clinical scenarios of short-term vs. long-term treatment with iron. Besides the expected iron overload in liver, serum as well as bone marrow, we show that both iron formulations were associated with bone loss due to severely impaired bone formation. Importantly, both iron formulations elevated the ratio of intact to C-terminal FGF23 (i:cFGF23) and the amount of osteoid. As expected, repeated doses of FDI and FCM showed stronger impacts than a single dose application, implying that for bone health, as few iron injections should be used as possible.

Iron application in vivo

Male 12-week-old C57BL/6J mice received 0.5 g/kg body weight iron (either ferric derisomaltose (FDI, Monofer), Pharmacosmos, ferric carboxymaltose (FCM, Ferinject), CSL Vifor, or iron dextran (ID), Sigma, intraperitoneal injections) per week for four weeks according to previous publications (Daba et al. 2013; Robin et al. 2023). In another set of experiments, FDI or FCM were injected once at a dose of 0.5 g/kg body weight and mice were sacrificed 4 weeks later. Mice were fed a standard rodent diet (198 ppm iron) with water ad libitum and were held under a 12 h light/dark cycle and in an air-conditioned room at 23°C. Weight was monitored every week. Mice were euthanized at the age of 16 weeks under deep anesthesia and blood, organs and bones were collected for further analysis. Animal procedures were approved and conducted in compliance with the guidelines of the institutional animal care committee and the Landesdirektion Sachsen (TVV 20/2020).

Blood counts

Blood counts were measured in the peripheral blood of the mice. At sacrifice, blood was collected via heart puncture, diluted with PBS, and analyzed with a Sysmex XN-1000.

Iron measurements

Non-heme iron content in the collected liver and serum was measured using the bathophenanthroline colorimetric method (SFBC) as previously described (Torrance and Bothwell 1968; Rauner et al. 2019). Briefly, 100 mg liver tissue was dried for 3 days at 37°C and afterwards the samples were incubated with 0.01% bathophenanthrolinedisulfonic acid. Values were recorded spectrophotometrically at 535/540 nm. Non-heme iron content is reported as µg iron/g dry tissue weight.

Iron content in histological sections was performed using the Perl’s Prussian Blue staining as previously published (Dogan et al. 2024). Iron-loaded cells were quantified in the femur in an area of 0.24 mm². Iron clusters were identified as larger conglomerates of cells stained with iron and were marked as an area. Iron-covered bone surface was quantified as well using the Osteomeasure software (Osteometrics, USA).

µCT analysis of bone microarchitecture

Bones were measured ex vivo at the end of the experiment. The distal femur and the fourth lumbar vertebra were excised and scanned using a resolution of 10.5 µm with a vivaCT40 (Scanco Medical, Switzerland). For the femora, half the femur was scanned in the scout view of which 100 slices below the growth plate of the distal femur were evaluated for trabecular bone, and 150 slices in the mid-diaphysis were evaluated for cortical bone. For analysis of bone volume, the same slices as for cortical bone were taken, only that the contours were including the bone marrow space instead of the cortical bone. For the vertebral bone, the entire 4th lumbar vertebra was scanned and 100 slices in the middle of the bone were measured. Trabecular and cortical bone parameters were assessed using standard protocols from Scanco Medical. µCT parameters are reported according to international guidelines (Bouxsein et al. 2010; Dempster et al. 2013).

Biomechanical testing

Three-point bending flexural test of the femoral diaphysis was performed to assess bone strength. The femora were stored in 70% ethanol and rehydrated in PBS 24 h prior testing. A Zwick/Roell machine type Z2.5 from Zwick, Germany was used to conduct the mechanical test. Mechanical load was applied to the anterior side of the femoral shaft to measure the maximum load at failure (Fmax, N) and the elastic modulus (Emod, MPa).

Quantitative backscattered electron imaging (qBEI)

Embedded sample blocks were ground and fine-polished and their co-planar surface was sputtered with carbon. Using 20 kV voltage and constant working distance, backscattered-electron images of the vertebral bone were acquired at a magnification of 150x using an electron microscope (Zeiss Crossbeam 340). Images were calibrated based on standards of carbon and aluminum according to previously established protocols, and the conversion of gray values to calcium wt % was performed using a custom written Matlab script. Five mineralization density distribution parameters including the weighted mean calcium-concentration of the bone area (Ca Mean), the peak position of the histogram (Ca Peak), the percentage of highly mineralized bone areas (Ca High), the percentage of lowly mineralized bone areas (Ca Low), and CaWidth (assessed as the full width at half maximum (FWHM) of the histogram curve) as measure for heterogeneity of mineral concentrations were determined as previously described (Roschger et al. 1998, 2008; Milovanovic et al. 2015).

Bone histomorphometry

All mice received two intraperitoneal injections with 20 mg/kg calcein (Sigma) five and two days before sacrifice. For dynamic bone histomorphometry, the third and fourth lumbar vertebrae were fixed in 4% PBS-buffered paraformaldehyde and dehydrated in an ascending ethanol series. Subsequently, bones were embedded in methacrylate and cut into 7 µm sections to assess the fluorescent calcein labels. Sections were analyzed using fluorescence microscopy to determine the mineralized surface/bone surface (MS/BS), the mineral apposition rate (MAR), and the bone formation rate/bone surface (BFR/BS). To assess the osteoid volume (OV), surface (OS) and width (O.Wi), 4 µm methacrylate sections were stained with von Kossa/van Gieson. The Osteomeasure software (Osteometrics, USA) was used to analyze an area of 1.44 mm².

To determine numbers of osteoclasts, the fifth lumbar vertebra was decalcified for one week using Osteosoft (Merck), dehydrated, and embedded into paraffin. Tartrate-resistant acid phosphatase (TRAP) staining was used to identify osteoclasts. Again, an area of 1.44 mm² was analyzed using the Osteomeasure software. Pictures were taken using the CellSens program while fluorescence pictures were taken using the AxioVision 4.8 program.

Serum analysis of bone turnover markers

Serum concentrations of pro-collagen type I amino-terminal propeptide (P1NP), C-terminal telopeptide of type I collagen (CTX), and tartrate resistant acid phosphatase 5b (TRAP5b) were quantified using ELISAs from Immundiagnostik, Bensheim, Germany. Serum levels of Cterminal and intact FGF-23 were also measured with ELISAs from QuidelOrtho, USA.

Statistical analysis

Data are presented as mean ± standard deviation (SD) with presentation of individual data points. One-way ANOVA and pairwise post-hoc tests were used to compare the three groups (FDI vs. FCM vs. PBS); a two-sided Student’s t-tests were used to compare two groups (iron dextran vs. control). Calculations were performed using GraphPad Prism 10 (GraphPad Software Inc, USA). p-values < 0.05 were considered statistically significant.

Mice treated with FDI or FCM become iron overloaded

To investigate how our treatment scheme with FDI or FCM affects the general health of mice, we monitored their body weight weekly and analyzed their blood counts as well as their liver iron content at the end of the experiment. Both, FDI and FCM led to a reduction of body weight towards the end of the experiment (Fig. 1A, FDI: -9%, p < 0.01; FCM: -12%, p < 0.001). Moreover, assessing the liver iron content revealed a heavy iron deposition in the liver with both iron formulations (Fig. 1B). This was accompanied by high serum iron levels and iron-saturated transferrin levels, reaching nearly 100% (Fig. 1C-D). Applying another iron formulation, iron dextran (ID), at the same dose to mice resulted in similar effects, with the mice showing a 5% decrease in body weight (p < 0.05) and a high amount of iron in the liver (Suppl. Figure 1A, B).

Concerning the blood counts, FCM and ID overall showed a similar profile, while FDI showed milder effects (Table 1). FCM and ID significantly reduced the red blood cell count as well as the hematocrit and hemoglobin (Table 1). Mean corpuscular volume was reduced by all three iron treatments (Table 1). All iron sources led to an increase in white blood cells, with FCM and ID showing the largest increase (Table 1). In particular the number of monocytes and neutrophils were increased, while lymphocytes were decreased in number (Table 1). Finally, all treatments led to a reduction in reticulocytes (Table 1). Thus, all iron treatments led to a similar iron overload and inflammatory profile after 4 weeks of treatment.

Table 1
Blood counts in mice treated with multiple doses of iron.
	Control N = 8	FDI N = 8	FCM N = 8	Control N = 8	Iron dextran N = 8
Red blood cells [10⁶/ µl]	9.95 ± 0.75	9.38 ± 0.44	8.78 ± 0.49**	9.59 ± 0.34	8.33 ± 1.11*
Hematocrit [%]	48.7 ± 3.84	44.6 ± 2.72*	41.6 ± 2.58***	48.0 ± 2.00	40.0 ± 5.71**
Hemoglobin [g/dl]	9.08 ± 0.63	8.63 ± 0.56	8.14 ± 0.46**	8.85 ± 0.42	7.69 ± 1.01*
MCV [fl]	48.95 ± 0.61	47.43 ± 0.64***	47.38 ± 1.23**	49.98 ± 0.90	48.28 ± 0.67**
MCH [pg]	0.91 ± 0.025	0.92 ± 0.021	0.93 ± 0.024	0.92 ± 0.02	0.92 ± 0.01
MCHC [g/dl]	18.64 ± 0.43	19.33 ± 0.41**	19.59 ± 0.57**	18.48 ± 0.48	19.13 ± 0.24**
Platelets [10³/ µl]	317.8 ± 29.4	278.0 ± 31.0*	225.6 ± 25.6***	327.0 ± 191.2	648.4 ± 109.4
White blood cells [10³/µl]	10.86 ± 2.39	14.68 ± 3.70*	17.95 ± 3.80***	9.42 ± 4.05	18.23 ± 5.32**
Neutrophils [%]	8.99 ± 1.44	12.38 ± 4.13	14.8 ± 3.80**	5.80 ± 1.77	7.99 ± 2.86
Lymphocytes [%]	87.63 ± 2.18	72.09 ± 14.16*	66.34 ± 6.55***	91.79 ± 2.69	85.21 ± 7.59*
Monocytes [%]	2.86 ± 1.26	15.08 ± 11.97*	18.21 ± 6.67***	2.16 ± 1.73	4.14 ± 1.57*
Reticulocytes [10⁹/L]	232.4 ± 23.97	130.2 ± 37.4***	113.3 ± 75.34***	236.8 ± 48.85	91.76 ± 18.15***
MCV = mean corpuscular volume. MCH = mean corpuscular hemoglobin. MCHC = mean corpuscular hemoglobin concentration. Data represent the mean ± SD. Statistical analysis was conducted using the Student´s t-test for comparisons between Control and Iron dextran, and one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for comparisons among Control, FDI and FCM. p < 0.05; p < 0.01; **p < 0.001.

FDI and FCM show distinct bone and bone marrow characteristics

Next, we investigated the bone microarchitecture of the mice using µCT and tested their bone strength using biomechanical tests (Fig. 2). Except for bone mineral density (BMD) at the distal femur, FDI led to the expected reductions of trabecular bone volume, BMD and tissue mineral density (TMD) at the femur and fourth vertebral body (Fig. 2A-G). Moreover, FDI did not alter the BMD at the femoral cortical bone, but led to a reduction in cortical thickness (Fig. 2H-I). This phenotype resulted in a reduction of bone strength (Fig. 2J). Interestingly, FCM led to different outcomes, showing only a reduction of bone volume at the spine, but no alterations in volume at the femur and even increased (femur) or a trend to an increased (spine) trabecular BMD, which is also reflected by the representative image of the bone microarchitecture (Fig. 2A-B, D, E-F). Importantly, TMD was decreased by FCM treatment at both sites (Fig. 2C, G). Cortical bone was negatively affected by FCM treatment, showing reduced BMD and thickness of the cortex (Fig. 2H-I). ID treatment did not lead to major alterations in trabecular or cortical bone volume, BMD or TMD (data not shown) of the spine or femur (Suppl. Figure 1C-G).

As TMD was decreased in the FCM-treated group, which only takes the mineralized tissue into account, but not BMD, which considers both, the mineralized and soft tissues, we wondered if some of the “density” signal could stem from iron deposition in the bone marrow. To that end, we evaluated the bone marrow compartment of the femoral midshaft using the same BMD threshold as for the trabecular bone analysis of the distal femur. Indeed, mice treated with FCM showed a higher BMD in the bone marrow compartment as compared to PBS or FDI-treated mice (Fig. 3A). Bone volume and TMD, which in general were very low in the bone marrow compartment, was decreased in the FDI group, but not in the FCM group (Fig. 3B-C). Histological analyses of the bones confirmed the µCT data, showing a significant accumulation of iron in the bone marrow of FDI- and FCM-treated mice, however, with distinct forms of deposition (Fig. 3D). While FDI resulted in iron-loaded macrophages scattered throughout the bone marrow, FCM led to a significant accumulation of iron in “iron clusters”, which may result in the increased BMD signal in the µCT (Fig. 3E-F). Both iron treatments increased the amount of iron-covered surface, but FCM showing the largest increase (Fig. 3G). ID resulted in a similar pattern of iron deposition in the bone marrow as FDI (Suppl. Figure 1H).

Taken together, both, FDI and FCM reduced bone TMD, but only FDI also led to reductions in trabecular bone volume and BMD. This may stem from the major accumulation of iron clusters in the bone marrow of FCM-treated mice, which may provide a “false-positive” signal for BMD measurements using µCT.

Bone mineralization is not affected by FDI or FCM

We further analyzed the bone mineral density distribution (BMDD) in the mature trabecular bone region of vertebral bodies of mice treated with PBS, FDI and FCM (Fig. 4A-B), which only showed minor differences between the groups. Mean and peak calcium concentrations were similar between all groups (Fig. 4C-D). CaWidth, (assessed as FWHM), a measure for the heterogeneity of the calcium concentration, showed a trend towards narrower curves in mice treated with FDI (p = 0.08) and a significantly lower value in FCM-treated mice (p < 0.001) (Fig. 4E). CaLow and CaHigh, measures indicating percentage areas with low and high calcium concentrations, respectively, did not show clear differences between the groups. However, in FCM-treated mice, slight trends towards lower bone areas with high and low mineralization indicate a more homogeneous calcium concentration within vertebral bone (Fig. 4F-G).

FDI and FCM significantly reduce bone formation

To address how FDI and FCM affect bone turnover, we performed dynamic bone histomorphometry and analyzed serum bone turnover markers. At histological level, both FDI and FCM led to a reduced bone volume (Fig. 5A), supporting our hypothesis that the µCT picked up false-positive signals from the bone marrow in the FCM group resulting in unaltered bone volume. Both iron formulations led to a drastic reduction in mineralized surface, mineral apposition rate, and the bone formation rate with almost no calcein labels seen in the iron-treated groups (Fig. 5B-C). Accordingly, serum levels of the bone formation marker P1NP were reduced by 25% in both groups (Fig. 5D). The number of osteoclasts was decreased to a similar extent in FDI- and FCM-treated mice, while TRAP serum levels were increased and serum CTX levels showed no difference (Fig. 5E-H). Despite no changes in bone volume, ID treatment also led to an inhibition of bone formation as displayed by the reduced levels of P1NP with no alterations of serum TRAP or CTX levels (Suppl. Figure 1I-K). Taken together, all iron formulations drastically decrease the bone formation rate with smaller effects on osteoclasts.

FDI and FCM result in increased osteoid production and high FGF-23 levels

As the administration of FCM has been associated with an increase in FGF-23 levels and potentially osteomalacia, we analyzed osteoid and FGF-23 levels as well. FDI and FCM led to a marked increase in osteoid width, osteoid volume, and osteoid surface compared to PBS-treated mice (Fig. 6A-C). Large osteoid seams were observed in the iron treated mice, especially in mice treated with FDI (Fig. 6D). Both, FDI and FCM treatment resulted in increased intact and C-terminal serum levels of FGF-23, with FCM leading to higher increases than FDI (Fig. 6E-F). As the intact FGF-23 was stronger up-regulated (6-fold with FDI and 13.5-fold with FCM) than the C-terminal FGF-23 (5.7-fold with FDI and 12.9-fold with FCM), the i:cFGF-23 ratio increased in both FCM- and FDI-treated mice. However, both i:cFGF-23 ratio were elevated to a similar extent (Fig. 6G), potentially explaining the mechanism underlying the increased osteoid formation.

Single injection of FDI and FCM results in milder effects on bone than repeated injections

Even though mouse studies frequently use repeated doses of iron to assess its effects on bone, we wanted to better mimic the clinical application of iron and thus, administered FDI and FCM only once and analyzed the bone outcomes after four weeks. Despite significant iron overload in several tissues (liver, spleen, bone marrow, bone), FDI and FCM treatment did not affect the body weight at the end of the experiment (Suppl. Table 1). Moreover, within the blood, FDI only increased white blood cell counts and reduced the number of reticulocytes (Suppl. Table 1). FCM in contrast already exerted stronger effects on the red blood cell compartment (reduced hematocrit, hemoglobin, MCV, reticulocytes) and elicited a stronger inflammatory response (higher numbers of white blood cells, in particular neutrophils) (Suppl. Table 1).

In the bone, FDI led to non-significant decreases of bone volume, but already increased osteoid surface per bone surface 6-fold as well as intact and C-terminal levels of FGF-23, which however did not lead to an increase in the i:cFGF-23 ratio (Fig. 7A-E). Serum levels of P1NP were not changed, while serum TRAP levels were non-significantly increased by 25% (Fig. 7F-G). In contrast, even after one injection of FCM, bone volume was decreased and osteoid surface increased (3-fold) along with increases in intact and C-terminal FGF-23 (however not resulting in an increased ratio) (Fig. 7H-L). Serum levels of P1NP were significantly reduced, while TRAP levels were increased by 58% four weeks after a single FCM injection (Fig. 7M-N).

Intravenous administration of FDI and FCM are two of the most broadly applied therapeutic interventions for iron-deficiency anemia. Compared to the oral form as well as previous dextran-based formulations, they present outstanding features including higher tolerability, higher stability in the bloodstream and therefore superior ability to correct iron-deficiency anemia. These benefits are owed to their iron-oxyhydroxide structure, which has been shown to be closer to akaganeite rather than magnetite (Blumenstein et al. 2021). As a result, their dissolution rate is lower, rendering them not only more available in the circulation but also present for a longer time. The formulations therefore make it possible to give patients a large amount of iron in a single dose with less toxicity.

Despite the benefits mentioned above, the occurrence of hypophosphatemia has been widely reported after injection of FCM in particular (Wolf et al. 2020a, b; Blumenstein et al. 2021; Boots and Quax 2022; Shahani et al. 2023). Hypophosphatemia is driven by increased serum levels of FGF-23, which is associated with reduced levels of the active form of vitamin D and calcium, followed by increased levels of parathyroid hormone (PTH) (Wolf et al. 2013, 2018, 2020b). Hypophosphatemia caused by administration of FCM was reported not only more frequently than with FDI, but also more severe (Wolf et al. 2020b, c). Even though FCM-induced hypophosphatemia is transient in most cases, case studies have shown that repeated administration of FCM may expose patients to a higher risk of developing osteomalacia, which is associated with bone pain and poor bone strength (Sangrós Sahún et al. 2016; Schaefer et al. 2017; Bartko et al. 2018; Klein et al. 2018; Wolf et al. 2018, 2020c; Fang et al. 2019; Tozzi and Tozzi 2020; Amarnani et al. 2020; Callejas-Moraga et al. 2020; Vilaca et al. 2022; Boots and Quax 2022). The underlying reason of why FCM induces hypophosphatemia more frequently than FDI, however, remains poorly studied.

To investigate the direct effects of FDI and FCM on bone health in more detail, we used healthy male mice as a model to exclude confounders from other underlying diseases that induce anemia and at the same time may affect bone turnover such as inflammatory bowel disease or chronic kidney disease. As expected, we observed accumulation of iron in the liver and serum in mice that received parental iron. Transferrin in the circulation was nearly completely saturated with iron. Repeated dosing of FDI and FCM significantly reduced body weight after the 4-week observation period. In addition to the iron overload, mice developed a hyperchromic microcytic anemia, likely due to exhaustion of erythropoiesis, together with an increased number of white blood cells, which may indicate an inflammatory reaction to this high dose of iron. In another study with a mouse model, iron overload by repeated high dose of iron dextran was also reported to impair hematopoietic system while elevating white blood cell counts (Chai et al. 2015). Importantly, in our study, a single dose of iron also resulted in iron overload without showing an increase in white blood cell numbers, indicating that the repeated dosing led to iron intoxication of the mice, which goes along with inflammation and the formation of ROS.

Besides the systemic iron accumulation, we observed a significant accumulation of iron in bone marrow macrophages followed by negative changes in bone parameters in iron-treated mice. The deleterious effect of iron on bone was even more prominent in those mice that received FCM compared to FDI. Both formulations remarkably reduced cortical thickness at the femoral mid-shaft. Moreover, tissue mineral density at the femur and L4 vertebra was also reduced by iron. Interestingly, loss of trabecular bone volume in the femur and L4 as assessed using µCT was only observed in FDI-treated mice. However, closer analyses using histology revealed that FCM produced a similar extent of trabecular bone loss, but these alterations were disguised in the µCT analyses by the formation of iron clusters in the bone marrow of FCM-treated mice that produced a false-positive signal. Due to these changes in the bone microarchitecture, FDI further reduced flexural strength assessed by the three-point bending test. Interestingly, there was no difference in mice treated with FCM compared to those receiving PBS. This resistance could be attributable to the distinct pattern of FCM deposition in the bone marrow.

The bone loss caused by the parental iron was mainly a result of impaired bone matrix mineralization together with severely reduced bone formation rather than enhanced bone resorption. Even though serum TRAP levels were increased, the number of osteoclasts at the bone surface remained unchanged. As macrophages also express TRAP (Räisänen et al. 2005; How et al. 2014), it is possible that the iron-loaden macrophages in the bone marrow contributed to the elevated serum TRAP levels. These results can be supported by the fact that CTX is normal after iron treatments in our study. In contrast, the persistently high levels of iron, no matter if provided via FDI or FCM, severely reduced the number of osteoblast and impaired their bone-forming properties. Previous studies have shown that osteoblasts are very sensitive to iron-induced ROS formation and ferroptosis. Blocking either iron-induced oxidative stress using N-acetyl cysteine (Tsay et al. 2010) or ferroptosis using ferrostatin-1 (Jiang et al. 2022) rescued bone formation in vitro and in vivo. Thus, our data strongly suggest that to maintain osteoblast function, iron concentrations should be kept to a minimum.

In contrast to studies in humans, in our mouse model, both FDI and FCM induced serum levels of FGF-23. FCM led to a higher increase in intact and C-terminal FGF-23 than FDI, but both resulted in a higher i:cFGF-23 ratio owing to the higher magnitude of iFGF-23 production. It is plausible to believe that the physiological FGF-23 cleaving capacity of the body in order to maintain a normal level of iFGF-23 is challenged by the high dose of these two intravenous iron formulations. Indeed, unlike the effect caused by the repeated doses, single dose of either FDI or FCM elevated both iFGF-23 and cFGF-23 levels but the ratio of i:cFGF-23 was not significant increased, suggesting an absence of iFGF-23 accumulation compared to those with repeated doses. As reported in previous studies with mouse models, this increase of FGF-23 was not associated with alterations in serum phosphate levels or hormones (vitamin D, PTH) due to the enhanced cleavage of FGF-23 (Wang et al. 2008; Sitara et al. 2008; Weidner et al. 2020). How this enhanced cleavage occurs is currently not well understood. FGF-23 was reported to be mainly released by osteocytes. However, in a mouse model of iron deficiency bone marrow sinusoidal endothelial cells were shown to upregulate FGF-23 expression (Li et al. 2023). Similarly, our previous studies on myelodysplastic neoplasms, which are associated with anemia, showed that erythroblasts also can produce high amounts of FGF-23 (Weidner et al. 2020). Therefore, more research is required in order to determine the cellular source of FGF-23 in the iron overload condition caused by intravenous iron.

Regardless of the amount of i:cFGF-23, both formations induced a massive amount of osteoid. This may be the result of iron incorporating into the nascent bone matrix instead of calcium, which blocks further mineralization. Indeed, iron was reported to inhibit crystal growth of hydroxyapatite (Guggenbuhl et al. 2008), the most abundant component in bones. Similar findings were observed in rat and porcine models of osteomalacia induced by aluminum (Sedman et al. 1987; Rodriguez et al. 1990). These alterations support our hypothesis that the overdose of iron led to inflammation in the bone marrow and disrupted mineralization in the bone tissue. Although our qBEI data obtained from a deep layer inside the mineralized bone reflected no changes in calcium distribution, it does not exclude the possibility that iron impaired mineralization by competing with calcium.

Despite careful planning and rigorous experimental design, our study has limitations. First, we studied effects of FCM and FDI on bone of healthy mice instead of using an iron-deficient or anemia model. However, this was a deliberate choice to exclude confounding factors that may further influence the regulation of FGF-23. In addition, we applied doses of iron higher than those used in humans for multiple reasons (considering the higher metabolic rate of mice, the dose is about 2-fold higher). This was done to simulate the long-term accumulation of iron, leading to high but not lethal doses of iron, which served as a proof-of-principle to study the effects on bone homeostasis, mineralization, and FGF-23 levels. The application of high doses also manifested the ability of healthy bodies to handle iron overload. Even though this protocol induced iron toxicity, animals receiving a single dose of FDI or FCM expressed similar, but less pronounced effects, suggesting that also lower doses or iron can induce FGF-23 levels and the formation of excess osteoid.

In summary, our data reveal that high doses of both FDI and FCM negatively affected bone microarchitecture. Reduced bone mineralization and a high accumulation of osteoid associated with high levels of FGF-23 was the main underlying mechanism. Between the two iron formulations, the negative effects on bone were more pronounced in mice treated with FCM, and both formulations showed dose-dependent effects.

Funding: This work was supported by grants from the DFG (FerrOs-FOR 5146) to LCH, UB, and MR.

Conflict of interest

MR reports honoraria for lectures and advisory boards from UCB and Vifor Pharma; LCH reports honoraria for advisory boards from Amgen, Ascendis, Pharmacosmos, and UCB to his institution and himself. All other authors have nothing to disclose.

Competing Interests

Funding:

This work was supported by grants from the DFG (FerrOs-FOR 5146) to LCH, UB, and MR.

Author Contribution

Conceptualization, MR, LH; Data curation, XTLP, VP, MR, MLC, HW, IF, BB, UB; Formal analysis and methodology, XTLP, VP, MR, MLC, HW, IF, BB, UB; Funding acquisition, LCH, UB, MR; Supervision, MR; Writing – original draft, XTLP, MR; Writing – review & editing, all authors.

Acknowledgement

This work was supported by grants from the DFG (FerrOs-FOR 5146 to LCH, UB, and MR).

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Competing interest reported. MR reports honoraria for lectures and advisory boards from UCB and Vifor Pharma; LCH reports honoraria for advisory boards from Amgen, Ascendis, Pharmacosmos, and UCB to his institution and himself. All other authors have nothing to disclose.

Suppl.Tabel1.docx
Supplementaryfigure1.png
Supplementary figure 1. Effects of ID on iron overload, marrow iron, bone microarchitecture and bone turnover. Mice were injected with ID once per week for four weeks (N=8 per group). Dosage is similar to the one used in FDI and FCM experiments. (A) Body weight of the animals was followed over the course of the experiment. (B, H) Iron accumulation was assessed in the liver and the bone marrow via the measurement of liver iron content level and histology, respectively. (C-G) Bone volume over total volume (BV/TV), bone mineral density (BMD) and cortical thickness (Ct.Th) of the femur and vertebra were quantified using µCT. (I-K) Serum levels of P1NP, TRAP and CTX reflected bone remodeling activity after the ID treatment. Individual dots represent individual mice. Mean and SD are indicated as horizontal lines. A two-sided t-test was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001.

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High doses of ferric derisomaltose and ferric carboxymaltose both increase FGF-23 levels and lead to osteomalacia and bone loss in healthy male mice

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