Our previous study reported that erythroferrone (ERFE), a newly identified hormone produced by erythroblasts, responded to recombinant human erythropoietin (rHuEPO) sensitively but its dynamics was complicated by double peaks and circadian rhythm. This study intends to elucidate the underlying mechanisms for the double peaks of ERFE dynamics and further determine whether early ERFE measurements can predict haemoglobin responses to rHuEPO.

Experimental Approach

By using the purified recombinant rat ERFE protein and investigating its deposition in rats, the production of ERFE was deconvoluted. To explore the role of iron in ERFE production, we monitored short-term changes of iron status after injection of rHuEPO or deferiprone. Pharmacokinetic/pharmacodynamic (PK/PD) modelling was used to confirm the mechanisms and examine the predictive ability of ERFE for long-term haemoglobin responses.

Key Results

The rRatERFE protein was successfully purified. The production of ERFE was deconvoluted and showed two independent peaks (2 and 8 h). Transient iron decrease was observed at 4 h after rHuEPO injection and deferiprone induced significant increases of ERFE. Based on this mechanism, the PK/PD model could characterize the complex dynamics of ERFE. In addition, the model predictions further revealed a stronger correlation between ERFE and haemoglobin peak values than that for observed values.

Conclusions and Implications

The complex dynamics of ERFE should be composited by an immediate release and transient iron deficiency-mediated secondary production of ERFE. The early peak values of ERFE, which occur within a few hours, can predict haemoglobin responses several weeks after ESA treatment.

Abbreviations

ERFE: erythroferrone
ESA: erythropoiesis-stimulating agent
PK/PD: pharmacokinetic/pharmacodynamic
rHuEPO: recombinant human erythropoietin
rRatERFE: recombinant rat erythroferrone protein
TfR: transferrin receptor
Tf-Sat: transferrin saturation

What is already known?

ESA administration is guided by haemoglobin levels, but the haemoglobin response is significantly delayed.
Erythroferrone is secreted by erythroblasts following ESA stimulation, exhibiting early response to ESA treatment.

What does this study add?

ERFE dynamics involves immediate release, and secondary production driven by a transient iron decrease.
Early ERFE levels correlate strongly with long-term haemoglobin responses, supported by PK/PD modelling.

What is the clinical significance?

ERFE may revolutionize ESA administration by enabling timely dose adjustments and erythropoietin resistance diagnosis.

1 INTRODUCTION

Erythropoiesis-stimulating agents (ESAs), including recombinant human erythropoietin (rHuEPO, epoetin alfa), darbepoetin alfa and continuous erythropoietin receptor activator (Kiss et al., 2010; Macdougall, 2005), have been widely used to treat anaemic patients with chronic kidney disease (CKD) (Adam et al., 2006; Eschbach, 1989; Winearls et al., 1986). ESAs have revolutionized anaemia management by freeing most patients from blood transfusions and improving their life quality (Adam et al., 2006; Eschbach, 1989; Winearls et al., 1986). However, it requires careful dose titration and personalization for each patient to ensure the benefits outweigh the risks (KDIGO Anaemia Guideline Work Group, 2012). In addition, some observational studies reported that 20%–34% of chronic kidney disease patients showed ESA resistance (Ingrasciotta et al., 2016; Kanbay et al., 2010; Minutolo et al., 2012), which is caused by multiple factors including inflammation, iron deficiency and uraemia (Elliott et al., 2009; Kanbay et al., 2010; Macdougall & Cooper, 2005). For chronic kidney disease patients who have poor responsiveness to ESA, a regular dose titration strategy leads to continuously escalating ESA doses, exposing them to high levels of ESA for a long time before the identification of resistance, and this results in increased cardiovascular events, hospitalization and even mortality (Panichi et al., 2011). Hence, achieving individualized dosing and identifying resistance are critical for the use of ESAs in clinical practice.

Currently, dose adjustments of ESAs and diagnosis of ESA resistance are both based on haemoglobin (HGB) responses (Barbieri et al., 2015; Gaweda et al., 2014; Group, 2012; Hovorka et al., 2004; Martín Guerrero et al., 2003; Martínez-Martínez et al., 2014; McCarthy et al., 2014). There are two major concerns for using as such an index. Firstly, the responses show substantial delays after ESA treatment. It takes a minimum of 4 weeks to observe a significant response before deciding whether to increase or decrease the next dose. The whole titration period for ESAs can extend to several months. Consequently, it takes a considerable amount of time to determine the appropriate dose for an individual. Secondly, responses show oscillations by frequently overshooting or undershooting the target level in patients with chronic kidney disease (Bae et al., 2015; Drüeke & Massy, 2019; Kalantar-Zadeh & Aronoff, 2009), especially under the circumstance that the target range has been further narrowed down to 10–11.5 g·dl⁻¹ according to the latest guideline (McMurray et al., 2012). As a result, it becomes challenging to ascertain the true response to a particular ESA dose, often leading to misleading adjustments in subsequent doses (Singh et al., 2008). Extensive research has focused on identifying earlier biomarkers that can predict responses, such as baseline levels (Pei et al., 2021), reticulocyte haemoglobin content (Singh et al., 2007), hepcidin-20 (Theurl et al., 2014), baseline erythropoietin level (Sanz Ortiz, 2008) and growth/differentiation factor 15 (GDF15) (Tanno et al., 2010). Patients with low baseline, elevated reticulocyte haemoglobin content, or low pre-treatment hepcidin levels were more likely to reach clinically significant improvement in responses. High endogenous erythropoietin levels prior to the initiation of treatment (>100–150 mIU·ml⁻¹) might also predict poor response to ESA or indicate ESA resistance. GDF15 is associated with cellular stress and apoptosis, and high expression of GDF15 could be a biomarker of ineffective erythropoiesis. Currently, there is no direct evidence indicating that any of these indices adequately predict erythropoietic responses or ESA resistance, with the rate of increase remaining the primary diagnostic tool for ESA resistance. Therefore, an early and sensitive biomarker is urgently needed for achieving individualized dosing of ESA and addressing ESA resistance.

A newly identified hormone erythroferrone (ERFE) shows promising potential to be an early biomarker for predicting the erythropoietic response, and it might be further used in dose titration and diagnosis of ESA resistance. Previous reports indicated that an unidentified erythroid regulator existed to facilitate rapid mobilization and absorption of additional iron, thus meeting the large iron demand by synthesis when erythropoiesis is increased (Bothwell et al., 1958; Finch, 1994; Pak et al., 2006; Vokurka et al., 2006). In 2014, ERFE was identified as such an erythroid regulator of iron homeostasis (Kautz et al., 2014; Wang et al., 2017). ERFE is secreted by erythropoietin-stimulated erythroblasts and can act on hepatocytes to suppress hepcidin. By regulating the hepcidin-ferroportin (IREG1) axis, ERFE promotes iron mobilization from storage cells as well as dietary iron absorption to improve iron availability for erythropoiesis (Coffey & Ganz, 2018). ERFE sensitively responds to ESA injection even at a very low dose and blood withdrawal, and strong positive correlations were observed among ERFE levels and ESA doses or concentrations in both healthy subjects and chronic kidney disease patients (Hanudel et al., 2018; Ramirez Cuevas et al., 2020; Robach et al., 2021). It was reported that hepcidin/ERFE ratio could serve as a predictor of responses in anaemic patients undergoing treatment with ferric citrate hydrate. Patients with a low hepcidin/ERFE ratio demonstrated stronger responses (Hara et al., 2019). Additionally, the level of ERFE was a positive marker of anaemia, although the association was not particularly strong (P = 0.0131). Importantly, only baseline ERFE levels were used in this study, and this suggested that the ERFE response may possess more power to predict responses.

Our previous studies supported that ERFE response was a promising biomarker to predict the long-term erythropoietic effects of ESAs, but the results also showed that the dynamics of ERFE is complicated by double peaks (Xu et al., 2022). The peak ERFE concentrations could be the best biomarker to predict the response to ESA treatment, but the complex dynamics of ERFE makes it hard to choose which peak values should be used to predict erythropoietic activity. In addition, the mechanisms underlying the double peaks of ERFE responses to rHuEPO are still unknown and worth in-depth study. In this study, we intend to unravel the mechanisms underlying the complex dynamics of ERFE and further examine the predictive ability of ERFE for erythropoietic responses to ESAs through mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) modelling.

2 METHODS

2.1 Animals

Male Sprague–Dawley rats with weights ranging from 300 to 330 g were supplied by the Laboratory Animal Services Centre at The Chinese University of Hong Kong. Rats in single- and multiple-dose PK/PD studies of rHuEPO were fed a supplemental diet with an extra 1% (w/w) carbonyl iron before rHuEPO treatment. Control rats and all rats in other animal studies were fed standard food without additional iron supplementation. All rats were kept at ambient temperature (25°C) and relative humidity (50%) with a 12/12-h light/dark cycle (three rats per cage) and had free access to food and water. The animals were acclimatized for 1 week in a holding room before any experimental procedures. All studies were conducted after receiving approval from the Animal Ethics Committee of The Chinese University of Hong Kong (Reference Number 20-30-MIS-5 and 18-247-ECS) and are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). Rats were anaesthetised with 5% isoflurane in an induction chamber for 2 min and maintained with 2%–2.5% isoflurane by a nosecone for approximately 3 min to inject substances or collect blood samples (50–100 μl) via tail vein and to stop any bleeding. The euthanasia of rats at the end of the experiments was performed by overdose of carbon dioxide (CO₂).

2.2 Expression and purification of recombinant rat erythroferrone (ERFE) protein

Recombinant rat ERFE protein (rRatERFE) was expressed in human embryonic kidney 293 T (ATCC, Cat# CRL-3216, RRID:CVCL_0063; kindly given by Prof. Billy Ng in CUHK). ERFE gene was synthesized based on the corresponding sequence (ERFE_RAT, Uniprot ID: D4AB34) and fused to a C-terminal FLAG tag. This cDNA was further sequenced and cloned into the mammalian expression vector that confers resistance to ampicillin (pCDNA3.1/Amp). The constructed plasmid was purchased (GENEWIZ, Suzhou, China) and amplified according to the provided information with standard protocols in E.coli using a TIANpure Midi Plasmid extraction Kit (TIANGEN, Beijing, China). Transfection of plasmid in HEK 293T cells was performed with HighGene transfection reagents (ABclonal, MA, USA) according to the manufacturer's protocol. HEK293T cells were seeded in 150 mm TC-treated culture dishes (Corning, AZ, USA) in 20–30 ml complete Dulbecco's modified Eagle's medium (DMEM) that contained 10% FBS and antibiotics (Gibco; Thermo Fisher Scientific, MA, USA) and controlled cell density at 50%–60% before transfection. After 6 h of transfection, the culture medium was replaced by half of fresh and serum-free OPT-MEM (Gibco; Thermo Fisher Scientific, MA, USA). Then, the supernatants were collected every 48 h three times and replaced with serum-free OPT-MEM. The pooled culture medium was further concentrated to 5 ml by ultrafiltration with a 10-kDa cutoff membrane-based filter (Amicon® Ultra-15, Merck, Germany) and purified by anti-FLAG affinity gel according to the manufacturer's protocol (MedChemExpress, Cat# HY-K0217, RRID:AB_3095642). Excess FLAG peptide in elute was removed by desalting columns (Zeba™ Spin Desalting Columns, 7 K MWCO; Sigma, MO, USA). The protein concentration of the final solution was determined by bicinchoninic acid assay (BCA) protein assay (Pierce; Sigma-Aldrich, MO, USA). Western blot analysis was conducted to confirm the purified ERFE-FLAG protein by using an anti-ERFE antibody (AVISCERA BIOSCIENCE, Cat# A00393-03-100, RRID:AB_3095640) and an HRP-conjugated anti-FLAG antibody (ABclonal, Cat# AE024, RRID: AB_2769864) separately. Protein samples were stored at −80°C and thawed only once for further use. All immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018).

2.3 Isolation and culture of primary hepatocytes

Primary rat hepatocytes were isolated from newborn rats by a rapid isolation method without perfusion (Schneider & Potter, 1943). The newborn rats were euthanized by CO₂ overdose followed by decapitation, and then liver tissues were extracted. Livers were minced into pieces (2–3 mm³) and washed three times with Hank's buffer (HBSS). The tissues were digested by 0.05% collagenase (collagenase type II) in HBSS for 30 min at the 37°C incubator. DMEM that contained 20% FBS was added to stop digestion and then filtered tissues through a 70-μm pore nylon cell strainer (SPL Life Sciences, Kyonggi-do, Korea). Hepatocytes were enriched by density gradient centrifugation (25% Percoll, Biochrom GmbH, Berlin, Germany) and washed four times to remove Percoll particles. After gentle resuspension, the isolated hepatocytes were cultured with DMEM containing 20% fetal bovine serum (FBS).

2.4 Quantitative RT-PCR

The isolated rat primary hepatocytes were seeded in 12-wells cell culture plates and exposed to lipopolysaccharide (LPS; 1 ng·ml⁻¹ LPS), 50% concentrated culture medium (CM) from transfected 293T cells, and purified rRatERFE protein (2 μg·ml⁻¹) for 16 h. Then the cells from each well were gathered and total RNA was extracted utilizing TRIzol reagents (RNAiso Plus) as per the instructions provided by the manufacturer. The quantity and purity of the isolated RNA were determined using a nano-drop spectrophotometer, based on a wavelength ratio of A260:A280 (~2.0). A 20 μl reverse-transcription reaction system was employed to create cDNA, utilizing 1 μg of extracted total RNA and 5 μl of 5X PrimeScript RT Master Mix (Takara). Subsequently, the mRNA levels of hepcidin (hamp) were quantified by RT-PCR using TB Green Premix EX Taq (Takara) and corresponding primers. All samples were processed in duplicates on the LightCycler/LightCycler 480 System (Roche, Basel, Switzerland) according to the protocol provided by Takara, in a total volume of 20 μl. The hamp expression was normalized by reference gene actin, with results presented as fold changes as 2^−ΔΔCt (the differences in cycle threshold between the reference and target genes within each group). Primers used in RT-PCR were listed below:

Hamp forward:

5′-CAAGATGGCACTAAGCACTCG--3′.

Hamp reverse:

5′-GCTGGGGTAGGACAGGAATAA--3′.

Actin forward:

5′-GAAATCGTGCGTGACATTAAAGAG--3′.

Actin reverse:

5′-GCGGCAGTGGCCATCTC--3′.

2.5 Deconvolution

The deconvolution/convolution principle is commonly used to evaluate drug release and drug absorption. In our case, the disposition process of ERFE was analysed by conducting a pharmacokinetic (Pk) study of rRatERFE and then the production rates of stimulated ERFE can be determined with the known parameters that describe the elimination of ERFE. The PK analysis and deconvolution were conducted by Phoenix Software (Certara, NJ, USA) (Certara USA, 2019). The details about the deconvolution method are provided in supporting information.

2.6 Experimental design of animal studies

For the pharmacokinetic/pharmacodynamic (PK/PD) studies of rHuEPO to reveal ERFE dynamics, a single-dose study and a multiple-dose study were performed sequentially. RHuEPO (EPOGEN 20,000 units·ml⁻¹; Amgen, CA, USA) was diluted using saline (B Braun Medical, CA, USA) containing 0.25% bovine serum albumin before each injection. In the single-dose study, 36 rats were randomly divided into four groups (each containing nine rats) that received an intravenous injection of either saline, or 100, 450 or 1350 IU·kg⁻¹ of rHuEPO. Drug concentrations and ERFE concentrations were measured based on samples withdrawn at 5, 30 min, and 1, 2, 4, 8, 10, 12, 24, 32 and 48 h after injection. Haematological measurements were monitored three times a week for 3 weeks. In the multiple-dose study, the grouping and dose levels were the same as the single-dose study, while rats received rHuEPO treatment three times a week for 2 weeks. The PK sampling and ERFE concentration monitoring were conducted after the first dose and the last dose at 5 min and 1, 2, 4, 8, 10, 12, 24, 32 and 48 h after injection. Haematological responses were measured three times a week for 2 months. Figure 1a shows the overall study design for the single- and multiple-dose studies. Blood samples were collected into tubes with 2% EDTA as an anticoagulant and then used for erythroid cell counting or centrifuged at 2000 × g for 10 min to get plasma and stored at −20°C for further analysis.

Details are in the caption following the image — **FIGURE 1**
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The erythroferrone (ERFE) and haematological responses after single and multiple doses of recombinant human erythropoietin (rHuEPO) in healthy rats. (a) The overall study design for the single- and multiple-dose PK/PD studies of rHuEPO. (b) ERFE dynamics and long-term erythropoietic effects of rHuEPO after a single dose of intravenous rHuEPO injection. (c) ERFE dynamics and long-term erythropoietic effects of rHuEPO after multiple doses of intravenous rHuEPO injection (thrice a week for 2 weeks). Data are presented as means ± SD (n = 3 for ERFE response; n = 9 for other haematological responses). SD, standard deviation. RET: reticulocytes. RBC: red blood cells. HGB: haemoglobin.

For the study of iron and ERFE interactions, the short-term changes of iron status induced by rHuEPO and the effect of iron on ERFE production were accessed. Twelve rats were randomly divided into a control group and a treatment group (n = 6). The rats in the treatment group received a single dose of 450 IU·kg⁻¹ of rHuEPO intravenously, while control rats received saline. Iron status was accessed by measuring serum iron, transferrin and ferritin concentrations at 0, 1, 2, 4, 8, 10, 12, 16, 20, 24, 28, 32, 36 and 48 h after rHuEPO injection. Hepcidin 25 was also measured at 0, 4, 8, 12, 16, 24, 32 and 48 h post-dose. For further study about the effect of iron decrease on ERFE production, deferiprone was administered to rats to simulate the rHuEPO-induced iron decrease. In this study, six rats were divided into two groups (n = 3) to receive deferiprone (60 mg·kg⁻¹) or saline. Serum iron and ERFE concentrations were measured at 0, 1, 4, 8, 12, 16 and 24 h after deferiprone injection. Blood samples were collected into clot tubes and allowed to be coagulated at room temperature for 30 min, and then centrifuged at 1000 × g for 15 min to get serum and stored as stated above.

For the PK/PD study of rRatERFE, a single dose study with three dose levels (15, 50 and 150 μg·kg⁻¹) was conducted. The dose levels were selected based on the basal level of endogenous ERFE and the detection limit of the ELISA kit for rRatERFE in a half-log escalation factor. Twelve rats were randomly divided into one control group and three treatment groups (n = 3). The purified rRatERFE was diluted using saline before injection. Blood samples were collected at 5, 15 and 30 min, and 1, 2, 4, 8, 12, 24, 32 and 48 h after injection. Haematological changes were measured on days 0, 1, 3, 4, 6, 8, 11, 13 and 15, and hepcidin expression was accessed at 0, 12, 24 and 48 h after treatment. Plasma samples were obtained and stored as stated above.

2.7 Bioassays and haematological measurements

Drug concentrations and PD markers including ERFE, serum iron, transferrin, ferritin and hepcidin were measured mostly by ELISA kits. The concentration of rHuEPO in plasma was quantified by a commercial ELISA kit (DEP00, R&D, Systems Inc., Minneapolis, MN, USA), which shows linearity between 2.5 and 200 mIU·ml⁻¹. All samples were diluted based on previously reported data to make sure the measured values fell in the linear range. ERFE concentrations were measured by a commercial ELISA kit that has a quantification range from 0.156 to 10 ng·ml⁻¹. Serum iron was measured by a chemical colorimetric method by the QuantiChrom™ Iron Assay Kit (DIFE-250; BioAssay Systems, CA, USA), which has a linear detection range from 27 to 1,000 μg·dl⁻¹. Hepcidin 25 was measured by a commercial ELISA kit (ER1504, FineTest, Wuhan, China), which has a detection range between 1.563 and 100 ng·ml⁻¹. Serum transferrin and ferritin concentrations were measured by ELISA kits from Abcam (ab137993 and ab157732, Abcam, Waltham, MA, United States), which have detection range from 0.3 to 3 μg·ml⁻¹ and from 12.5 to 400 ng·ml⁻¹, respectively. The concentrations of rRatERFE in plasma were measured by an anti-FLAG ELISA kit (ab285234; Abcam) with a detection range from 5 to 500 nM, and the concentration unit was converted to ng·ml⁻¹ by a conversion factor (1:1). The calculation of transferrin saturation (Tf-Sat) was derived from iron and transferrin measurements, utilizing the formula below (Petzer et al., 2020):

Tf - Sat (%) = \frac{Fe (μg \cdot {dl}^{- 1})}{Transferrin (mg \cdot {dl}^{- 1})} \times 70.9

Long-term erythropoietic effects were regularly monitored after rHuEPO injection in the single- and multiple-dose studies. Haematological markers including reticulocytes (RET) count, red blood cell (RBC) count and haemoglobin concentrations in peripheral blood were measured by an auto haematology analyser (BC-2800Vet, Mindray Medical International Limited, Shenzhen, China). Reticulocytes will be enumerated by flow cytometry with a thiazole orange stain based on the manufacturer's instructions. The reticulocyte counts will be calculated by multiplying the percentage of RET and the measured red blood cell counts. Blood samples were analysed within 4 h after sampling.

2.8 Mechanism-based PK/PD modelling

Many different PK/PD models have been developed to quantify the pharmacokinetics and erythropoietic effects of rHuEPO (S. Ait-Oudhia et al., 2010a; Krzyzanski et al., 2005; Ramakrishnan et al., 2003; Yan et al., 2012). These models successfully encapsulate the nonlinear disposition of rHuEPO and the differentiation of different stages of erythroid cells. In this present study, we incorporated baseline ERFE and its stimulated responses into the erythropoiesis process. We also included an iron feedback mechanism to capture the complex dynamics of ERFE. The acquired data of rHuEPO concentrations vs. time from both single and multiple dose studies were characterized by employing a two-compartment model, comprised of a central and a tissue compartment. The central compartment facilitates a parallel elimination process that involves a linear first-order elimination rate ( $K_{el}$ ), in conjunction with a nonlinear elimination (Michaelis–Menten kinetics, $V_{\max}$ and $K_{m}$ ). The PD model integrates three interactive physiological processes: erythropoiesis from erythroid progenitors to mature red blood cells, ERFE release and short-term serum iron changes. The erythropoiesis process is characterized by different stages of erythroid lineage cells transitioning from burst-forming unit erythroid cells (BFU-Es; denoted as P1), to colony-forming unit erythroid cells (CFU-Es; denoted as P2), then to early and late stages of erythroblasts (Pro-, Baso-, Poly- and Ortho-EBs; represented by P3), RETs, and finally, red blood cells. The process is represented through a catenary precursor-dependent indirect response model with a series of transit compartments to mimic different stages of erythroid cells. Erythropoietin stimulates erythropoiesis through various mechanisms, and this action is depicted by a depletion effect on burst-forming unit erythroid cells, a retardation effect on the transition of reticulocytes, and a stimulatory influence on mean corpuscular hemoglobin (MCH) synthesis. ERFE is released by erythroblasts, exhibiting a circadian rhythm under baseline conditions without exogenous erythropoietin treatment. Upon exogenous erythropoietin (EPO) stimulation, ERFE shows an immediate release and a subsequent increase regulated by serum iron levels, which undergo a transient decrease due to the high demand for iron. The impact of rHuEPO treatment on the serum iron compartment is portrayed by an indirect response model with a stimulation effect on the elimination rate of serum iron. The complex dynamics of ERFE were captured by an indirect response model detailing the effect of iron decrease on ERFE production, and a surge-based model outlining the immediate release of ERFE from the bone marrow to peripheral blood stimulated by rHuEPO. The details and corresponding differential equations are provided in the supporting information.

3 DATA ANALYSIS AND STATISTICS

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2022). The group size is the number of independent values and the statistical analysis was done using these independent values. Group size for single-dose and multiple-dose of rHuEPO to reveal ERFE dynamics (n = 9) were selected based on previous studies with similar experiments (S. Ait-Oudhia et al., 2010a). Group size in PK/PD studies of rRatERFE was reduced (n = 3) due to the limited production efficiency of rRatERFE in laboratory conditions. This group size is commonly employed in PK/PD studies in rats (Abdallah et al., 2024) because longitudinal data were collected, and continuous measurements can enhance statistical power, thereby reducing the required sample size.

All PK data were first analysed by a non-compartmental approach using Phoenix WinNonlin software. PK/PD modelling was performed in NONMEM (NM7.5; ICON, MD, USA) on the Windows platform with PsN suite (version 4.9.0, [http://psn.sourceforge.net/docs.php]). Data processing, statistical analyses and plotting were performed in R (R Core Team) and GraphPad Prism version 9.0.0 (GraphPad Software, La Jolla, CA, United States). The maximum percentage changes of ERFE and responses were calculated by normalized values by baseline ( $\frac{Value - baseline}{baseline}$ × 100). Statistically significant differences in mean measurements at each time point were assessed using a two-tailed Student's t test for the same dosing regimen and one-way ANOVA between groups followed by a Duncan'spost hoc test. Unless stated otherwise, all results were reported as means $\pm$ SD, and $P <$ 0.05 were considered significant.

3.1 Materials

The normal chow (Rodent Diet 50 IF/6F) and supplemental diet with extra 1% (w/w) carbonyl iron were purchased from Test Diet (St. Louis, MO, USA). Isoflurane (ISO-VET, 250ml) was purchased from AlfaMedic Ltd. (Kong Kong). TIANpure Midi Plasmid extraction Kit was purchased from TIANGEN (Beijing, China). TC-treated culture dishes and culture flask were purchased from Corning (Glendale, AZ, USA). The complete DMEM culture medium, OPT-MEM, Fetal Bovine Serum (FBS) and antibiotics (100X Penicillin-Streptomycin) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 10-kDa cutoff membrane-based filter (Amicon® Ultra-15) was purchased from Merck (Darmstadt, Germany). The 3X FLAG peptide, Zeba™ Spin Desalting Columns, BCA protein assay kits, collagenase type II, lipopolysaccharides (LPS), RIPA cell lysis buffer, ampicillin, bovine serum albumin (BSA) and Ethylenediaminetetraacetic Acid (EDTA)were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hank's buffer (HBSS) was purchased from Life Technologies (Carlsbad, CA, USA). The 70-μm pore nylon cell strainer was purchased from SPL Life Sciences (Kyonggi-do, Korea). Percoll medium was purchased from Biochrom GmbH (Berlin, Germany). The 5X PrimeScript RT Master Mix, TRIzol reagents, TB Green Premix EX Taq (RNAiso Plus; Takara, Shiga, Japan). Rapid SDS-PAGE Gel kit (40% Acr-Bis, 4×Tris/SDS separation gel buffer with pH8.8 and pH8.8, APS power, TEMED) was bought from Biosharp, (Hefei, Anhui, China). Tris–HCl, 10× Tris/glycine/SDS buffer, 10× TBS, Tween 20 were bought from Bio-Rad (Hercules, CA, USA).Deferiprone (DFP) and anti-FLAG affinity gel (Cat# HY-K0217, RRID: AB_3095642) were purchased from MedChemExpress (Shanghai, China). HighGene transfection reagent and HRP-conjugated anti-FLAG antibody (Cat# AE024, RRID: AB_2769864) was purchased from ABclonal (Waltham, MA, USA). The anti-ERFE antibody (Cat# A00393-03-100, RRID: AB_3095640) was purchased from AVISCERA BIOSCIENCE (San Diego, CA, USA). The rat rHuEPO ELISA kit (DEP00) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). The QuantiChromTM Iron Assay Kit (DIFE-250) was purchased from BioAssay Systems (Hayward, CA, USA). Hepcidin 25 ELISA kit (ER1504) was purchased from FineTest (Wuhan, China). The anti-FLAG ELISA kit (ab285234), serum transferrin (ab137993) and ferritin ELISA kits (ab157732) were purchased from Abcam (Waltham, MA, USA).

Details of other materials and suppliers were provided in the specific sections

3.2 Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander, Fabbro, Kelly, et al., 2023a,b,c)

4 RESULTS

4.1 Erythroferrone (ERFE) provided as an early biomarker to recombinant human erythropoietin (rHuEPO), but its dynamics was complicated with double peaks and circadian rhythm.

Figure 1b,c shows the ERFE dynamics and long-term erythropoietic effects of rHuEPO after different dose regimens. In both single- and multiple-dose studies, ERFE responded more sensitively to rHuEPO than haemoglobin. Upon rHuEPO stimulation, ERFE displayed potent responses within a few hours, while took 3–5 days to show significant responses. In addition, ERFE responses became stronger after repeated dosing of rHuEPO. In rats that received a single intravenous injection of rHuEPO at 100, 450 and 1350 IU·kg⁻¹, the maximum percentage increases of ERFE were 64.1%, 139.8% and 329.4% respectively, contrasted with the increases for haemoglobin, which were 5.3%, 11.9% and 19.0% respectively.

We also found that ERFE dynamics was complicated as the ERFE baseline followed a circadian rhythm and ERFE responses to rHuEPO showed double peaks. The ERFE baseline follows a significant circadian rhythm in a 24-h period with a significant percent rhythm estimated to be 89%. ERFE responses to rHuEPO were confirmed to show double peaks at about 2 and 10 h post-dose. However, the mechanisms underlying the double peaks of ERFE responses to rHuEPO are still unknown.

4.2 Protein expression and validation of recombinant rat erythroferrone protein (rRatERFE)

To determine the disposition process of ERFE, we expressed recombinant rat ERFE protein in 293T cells. The results from SDS-PAGE and western blotting demonstrated successful expression of the rRatERFE-FLAG protein in 293T cells and purification of target protein from the collected supernatants using anti-FLAG affinity gel. The SDS-PAGE data exhibited a pronounced protein band with a molecular weight (MW) of approximately 55 kDa (Figure 2a, upper panel), which is consistent with the reported apparent MW of endogenous ERFE (Seldin et al., 2012). The western blot results (anti-FLAG) revealed three protein bands with diverse MWs from 32 to 54 kDa (Figure 2a, lower panel). These bands could signify differentially glycosylated rRatERFE proteins. Furthermore, the anti-ERFE antibody successfully identified the purified rRatERFE from the supernatant, rather than from cell lysis (Figure 2b), providing additional confirmation that the rRatERFE protein was effectively expressed and purified. The bioactivity of rRatERFE was checked by assessing its ability to suppress hepcidin transcription in rat primary hepatocytes. Results showed both the concentrated culture medium and purified rRatERFE protein inhibited hepcidin transcription (Figure 2c).

4.3 The rRatERFE exhibited slight nonlinearity of clearance and could inhibit hepcidin expression in rats

The PK study of rRatERFE was conducted in rats by intravenous administration with three dose levels (15, 50 and 150 μg·kg⁻¹). We here for the first time reported the PK/PD characteristics of rRatERFE. The concentration versus time profiles of rRatERFE are shown in Figure 2d, and Table 1 lists the main PK parameters calculated by NCA methods. The clearance of the rRatERFE exhibits slight nonlinearity, as there is a dose-dependent decrease in the systemic plasma clearance (CL) from 13.19 to 10.62 ml·h⁻¹. The apparent volume of distribution at steady state (V_ss) was out of the normal range of plasma volume of rats, which implies a high affinity of ERFE in tissue. The elimination of rRatERFE showed a clear biphasic process, with the rapid decline stopped at 2 h (initial slope: 0.48–0.77) and then followed by a slower terminal slope (terminal slope: 0.054–0.073). The initial half-lives of three dose levels were similar and showed no significant differences, while the terminal half-lives (t_1/2) increased from 9.52 to 12.91 h with dose escalation.

TABLE 1. Summary of noncompartmental parameters for recombinant rat erythroferrone protein (rRatERFE) pharmacokinetics after single intravenous administration with three dose levels (15, 50 and 150 μg·kg⁻¹).

Parameters (units)	Definition	Dose levels
Parameters (units)	Definition	15 μg·kg⁻¹	50 μg·kg⁻¹	150 μg·kg⁻¹
AUC_0-∞(μg·ml*h⁻¹)	The area under the concentration curve	1140.07 (76.38)	4163.41 (31.37)	14130.43 (323.74)
CL (mL·h⁻¹·kg⁻¹)	Clearance	13.19 (0.87)	12.01 (0.09)	10.619 (0.25)
C_max (μg·ml⁻¹)	Maximum serum concentration	162.38 (5.85)	418.231 (30.09)	2160.83 (114.42)
MRT (h)	Mean residence time	12.66 (0.11)	15.15 (0.22)	16.91 (0.31)
λ_z (h⁻¹)	Terminal slope	0.073(0.007)	0.063 (0.001)	0.054 (0.001)
t_1/2 (h)	Half-life	9.52 (0.089)	11.04 (0.17)	12.91 (0.13)
V_ss (ml·kg⁻¹)	The steady-state volume of distribution	167.18 (12.166)	181.90 (4.08)	179.52 (7.95)

Note: Data are presented as means ± SD.

We also measured haematological changes and hepcidin expression levels after rRatERFE administration in rats. The red blood cell counts and concentrations showed increases after 4 days of the administration, and especially the red blood cell counts showed a dose-dependently increase on Day 8 post-dose (Figure 2e) though the differences were not statistically significant. For hepcidin expression Figure 2f shows rRatERFE administration can suppress hepcidin protein levels within 24 h and there is a significant inhibitory effect in 12 h with a dose-dependent relationship.

4.4 Deconvolution process identified two separate productions of ERFE that responsible for the double peaks in ERFE dynamics

We estimated the kinetic parameters of rRatERFE by fitting the PK data of rRatERFE to a two-compartment model. The mean kinetic parameters were used for further deconvolution process (Table S1). By using two exponential terms and two coefficients as standard kinetic parameters of ERFE, the production rates of stimulated ERFE secretion were calculated by deconvolution and shown in Figure 3a. The production rate of ERFE after recombinant human erythropoietin (rHuEPO) stimulation is separated into two parts that correspond to the double peaks of ERFE responses to rHuEPO. The first production rate occurred at about 2 h and was much higher than the second production rate which occurred at about 8 h. In addition, the first production rate was enhanced after repeated doses while the second production rate showed no significant changes. Those results suggest that additional mechanisms, beyond rHuEPO stimulation, may play a role in the secondary production of ERFE.

4.5 Recombinant human erythropoietin (rHuEPO) induced a transient decrease in iron status

Considering the interaction between ERFE and iron metabolism and iron status is involved in EPO sensitivity (Nai et al., 2015; Pantopoulos, 2015), we assessed the short-term changes of iron status after rHuEPO stimulation (Figure 3b–e). Serum iron levels were significantly decreased after rHuEPO treatment and reached a nadir at 4 hours (control group: 326.5 vs. treatment group: 201.5 μg·ml⁻¹). Ferritin, which indicates the stores of iron, was also measured and showed slight decreases after rHuEPO treatment. Transferrin saturation (Tf-Sat) values were calculated based on the transferrin and serum iron levels, and a significant decrease was observed at 2–4 h post-rHuEPO treatment. The mature isoform of hepcidin (hepcidin 25) decreased after 6 h and remained lower than the control within 48 h of treatment. The rHuEPO-induced decrease of serum iron and Tf-Sat levels reached their nadirs before the second peak of ERFE, which indicates that the iron decrease might act as feedback on ERFE production.

4.6 Acute iron deficiency causes ERFE production

Deferiprone, an iron chelator utilized in the treatment of iron overload, exhibits a high affinity for iron and can effectively chelate iron in the blood. To investigate whether iron decrease alone contributes to ERFE production, deferiprone was employed to induce acute iron decrease that is similar to the effect of rHuEPO on iron status. In rats that received intravenous injections of deferiprone (60 mg·kg⁻¹), serum iron levels significantly decreased at 1 h and returned to baseline at 8 h (Figure 3f). Intriguingly, serum ERFE levels increased from 5 to 20 ng·ml⁻¹ compared to control and showed nearly a tenfold increase compared to baseline at 4 h after deferiprone treatment (Figure 3g). Since rHuEPO-induced serum iron decreased at about 4 h and the transient iron decrease requires 1–4 h to induce ERFE production, the timing of the second peak of ERFE at approximately 10 h after rHuEPO stimulation aligns with the effect of rHuEPO-induced iron decrease on ERFE production. These results indicated that the transient iron decrease should be responsible for the second peak in ERFE dynamics.

4.7 The mechanism-based PK/PD modelling to characterize erythropoietic responses and ERFE responses to rHuEPO

Based on the discovered novel mechanisms for ERFE dynamics, we developed a PK/PD model to describe erythropoiesis, iron metabolism and ERFE to rHuEPO and baseline changes of ERFE. The model structure of the developed PK/PD is shown in Figure 4. The PK/PD model was developed sequentially, and the obtained individual PK parameters of rHuEPO were fixed and used for further fitting of PD responses. Table S2 lists the estimated PK Parameters of rHuEPO and rRatERFE. Table 2 lists the estimated model parameters related to the PD effects of rHuEPO. The estimated baselines of haematological indices are aligned with normal physiological values and values reported in previous studies. The concentration for half the maximum effect of rHuEPO on the immediate release of ERFE (466 mIU·ml⁻¹) is comparable to that on the differentiation of erythroid progenitors (515 mIU·ml⁻¹), which supports that the immediate release of ERFE or the first peak bears a closer relationship to erythropoietic activity. Most parameters are estimated with acceptable precision (CV < 50%), except for the CV of SW is 69.7%. The high CV of SW might be due to the complex model of ERFE since it contains circadian rhythm, interaction with iron and the surge of ERFE.

TABLE 2. Estimated pharmacodynamic parameters with their corresponding RSE of recombinant human erythropoietin (rHuEPO).

Parameters (units)	Definition	Estimate	RSE (%)
S_{max_1}	Maximum stimulation of P1 differentiation to P2 cells by rHuEPO	7.06	16.9
SC_{50_1} (mIU·ml⁻¹)	Concentration of rHuEPO inducing 50% of S_{max_1}	515	35.7
RBC₀₁ (10¹² cells·mL)	The baseline of RBC counts in rats receiving single-dose of rHuEPO	6.96	20.5
RBC₀₂ (10¹² cells·ml⁻¹)	The baseline of RBC counts in rats receiving multiple-dose of rHuEPO	7.57	1.3
T_, (h)	Mean lifespan of RETs	77.7	4.6
T_P1 (h)	Mean lifespan of P1 cells	851	7.5
T_P2 (h)	Mean lifespan of P2 cells	21.5	10.1
IC₅₀ (mIU·ml⁻¹)	Concentration of rHuEPO inducing 50% inhibition for ageing rates of RETs	410	37.1
RET₀ (10¹² cells·ml⁻¹)	The baseline of RET counts	0.325	3.1
MCH₀ (pg per cell)	The baseline of mean corpuscular haemoglobin	19.2	1.5
S_{max_2}	Slope describing the stimulatory effect of rHuEPO on MCH	3.41	4.7
T_H (h)	Mean residence time for MCH in blood	590	14.9
R_m (ng·ml⁻¹)	The mean baseline of ERFE for circadian rhythm	2.38	8.1
R_a (ng·ml⁻¹)	The amplitude of ERFE for circadian rhythm	0.523	28.8
PT (h)	The peak time of ERFE for circadian rhythm	13.5	6.5
K_out (h⁻¹)	First-order elimination constant of ERFE	0.142	28.2
K_{out_fe} (h⁻¹)	First-order elimination constant of serum Fe	0.287	6.1
SC_{50_3} (mIU·ml⁻¹)	Concentration of rHuEPO inducing 50% of S_{max_4}	2,600	7.8
S_{max_4}	Slope describing the stimulatory effect of Fe decrease on ERFE production	3.18	35.2
S_{max_5} * ξ	The convoluted value of S_{max_5} and ξ	802	33.5
SC_{50_5} (mIU·ml⁻¹)	Concentration of rHuEPO inducing 50% of S_{max_5}	466	38.3
TE (h)	The peak time of the surge of ERFE	1.95	42.5
SW	Width of the surge of ERFE	0.03	69.7
ωRBC₀	Interindividual variability for RBC₀	0.282	15.4
ωRET₀	Interindividual variability for RET₀	0.096	23.1
ωR_m	Interindividual variability for R_m	0.202	24.5
δRBC	Proportional error for RBC count	0.037	2.3
δRET1	Proportional error for RET count	0.2	24.6
δRET2	Additional error for RET count	0.094	12.3
δERFE	Proportional error for ERFE concentration	0.265	2.8
δHGB	Proportional error for HGB concentration	0.039	2.2
δFE	Proportional error for Fe concentration	0.136	5.7

Note: RSE, relative standard error; interindividual variability and residual error are expressed as coefficients of variation (%) with RSE on the approximate standard deviation scale (standard error/variance estimate)/2.
Abbreviations: HBG, haemoglobin; RBC, red blood cell; RET reticulocyte.

The final model was further examined by basic goodness-of-fit diagnostic plots as shown in Figures S1 and S2 and visual predictive checks as shown in Figure 5a,b. The population and individual predictions closely represent the observed data, while the observed second peak of ERFE was slightly earlier than the prediction. We have been trying to further improve this without success, which may be indicative of other mechanism(s) for the second peak that was not captured in this model. The conditional weighted residuals displayed no significant trends or distribution patterns with only serval outliers.

4.8 ERFE can predict responses to rHuEPO through a linear regression model

In our previous study, we observed a positive correlation between peak values of ERFE and responses to rHuEPO. However, the observed data cannot accurately represent the true peaks of ERFE responses due to the limitations in sampling intervals and the use of a rotating sampling method in experiments. To further determine whether the early peak values of ERFE can predict long-term responses, we need to clarify the relationship between ERFE and responses after rHuEPO treatment.

Based on the developed PK/PD model, the complete concentration versus time profiles of ERFE and for each individual were predicted, enabling us to quantify the relationship between the real peak values of ERFE and. Our experimental and modelling results suggested that the first peak of ERFE would be more suitable in predicting long-term responses, as it is an immediate release that is triggered by rHuEPO and can reflect the expansion of erythroblast. Therefore, we conducted Pearson correlation analysis using the first peak values of ERFE within 6 h after rHuEPO treatment and the peak values of appeared on about day 15. As expected, we observed a highly significant positive correlation between the predicted peak values of ERFE and responses after rHuEPO treatment (Figure 5d, correlation coefficient R = 0.974), and this correlation is much stronger than that for the observed values (Figure 5c, correlation coefficient R = 0.42). This result implies that the ERFE peak values might be able to predict the maximum responses to rHuEPO. Indeed, a strong positive correlation was also observed between predicted ERFE and the observed peak values (Figure 5e, correlation coefficient R = 0.976). In addition, the model predictions support an accurate linear relationship between ERFE and responses ( $y = 15.56 x + 121.55$ ; r² = 0.96), in which ERFE is the independent variable and can be utilized to predict the maximum responses. Those results demonstrated that early ERFE responses, which occur as early as 2 h after treatment, can predict long-term responses to erythropoiesis-stimulating (ESAs).

5 DISCUSSION

Based on our current research, erythropoietin (EPO) stimulation and iron regulation contribute to the double peaks in erythroferrone |(ERFE) dynamics. The first peak appears to be an immediate release from erythroblasts following rHuEPO stimulation, while the second peak likely results from a feedback response to a transient decrease in iron.

Immediate release is a common trait observed in many hormones in the body, these hormones are swiftly released to manage various physiological functions. For example, in reaction to a sudden surge in blood glucose levels, the pancreas promptly secretes insulin (Malaisse et al., 1979), a hormone that facilitates glucose uptake by cells, thereby decreasing blood glucose levels. The mechanisms driving the immediate release of ERFE could be diverse and may encompass intricate signalling pathways, such as the JAK2-STAT5 signalling pathway. This rapid release of ERFE enables the timely mobilization of iron, which assists the body in recovering from anaemia as a reaction to stress erythropoiesis. The immediate release of ERFE is captured in our model by a surge-based model. In our data, we observed the peak time of the immediate release of ERFE was independent of the rHuEPO dose. This evidence suggests the indirect response model (IDR) is not applicable to describe the rHuEPO-induced immediate release of ERFE, since the response profiles shift as dosage increases (Sharma & Jusko, 1996). Similar to the immediate release of ERFE after rHuEPO treatment, there is an immediate decrease in the serum concentrations after the administration of, a potent GnRH₁ receptor antagonist, cetrorelix (Duijkers et al., 1998). A surge-based model was purposed to capture the response of LH in the PK/PD modelling of cetrorelix, and this model was further modified to describe the effect of budesonide on ACTH and cortisol (Lönnebo et al., 2007; Nagaraja et al., 2003). Therefore, we modified the surge-based model by replacing the surge amplitude parameter with a combination of the stimulation effect of rHuEPO and the erythroblast cell count in the bone marrow. In this way, our model for the first time links the erythroid precursor pool and the immediate release of ERFE.

The second peak of ERFE suggests complex interactions between ERFE and iron homeostasis. The iron changes in the body can be highly dynamic since the iron must be loaded to transferrin for further utilization, while the turnover rate of the transferrin-bound iron complex is as high as 10 times per day (Dautry-Varsat, 1986). Indeed, our data show that the serum iron levels in control rats displayed considerable fluctuations throughout the day. A high dose of exogenous EPO can trigger an acute expansion of erythroblasts in both the bone marrow and spleen (Artuso et al., 2019), thereby consuming substantial amounts of iron. This process is facilitated by the high expression of transferrin receptor 1 (TfR1) on the surfaces of these cells. The demand for iron surpasses the regular supply in circulation, leading to a transient decrease in iron, indicated by low serum iron and Tf-Sat levels. This, in turn, further destabilizes membrane-bound transferrin receptor 2 (TfR2) on erythroid cells. TfR2 in bone marrow might act as a sensor of iron deficiency (Chen et al., 2009; Kawabata et al., 1999; Robb & Wessling-Resnick, 2004). Under regular conditions, TfR2 and erythropoietin receptor interact and localize on the cell surface, regulating the production of red blood cells. However, iron deficiency could deactivate TfR2, and augment erythropoietin receptor associated signalling, which includes ERFE production (Artuso et al., 2018; Forejtnikovà et al., 2010; Ginzburg & Fleming, 2018; Olivari et al., 2021; Wallace et al., 2015). The subsequent amplification of erythropoietin receptor signalling triggers ERFE production, contributing to the secondary production rate in complex ERFE dynamics after rHuEPO stimulation. Our study observed a rapid decrease in iron levels 4 h after the intravenous administration of rHuEPO, and an iron-deficient state induced by the iron chelator deferiprone resulted in an increase of ERFE. This finding offers direct evidence that iron deficiency can stimulate ERFE production and supports the theory that the second peak of ERFE response to rHuEPO is driven by iron feedback regulation through TfR2. This could elucidate why the second peak of ERFE coincides with the circadian ERFE baseline. The circadian rhythm of the ERFE baseline is modelled based on the concept of PD responses with circadian-controlled input rates or loss rates (Ayyar et al., 2019). We assume that its baseline circadian rhythm can be attributed to a time-dependent input rate based on current research that the expression of ERFE is regulated by EPO and EPO is already known to exhibit a circadian rhythm (Sciesielski et al., 2021). The secondary production of ERFE after rHuEPO stimulation is implemented by a stimulation function on the circadian input of ERFE baseline, driven by the magnitude of iron decrease. One limitation of this part of study is that the group size was relatively small (n = 3). However, we are confident in our results as this study involves continuous PD measurements after rHuEPO treatment. In addition, this study was conducted with prior knowledge from the literature that a transient decrease in serum iron was observed after rHuEPO injection in mice (Artuso et al., 2019). Deferiprone was used to evaluate if a decrease in iron could stimulate the production of ERFE. We found that deferiprone effectively lowers serum iron levels, leading us to conduct the experiment with three rats. The marked difference observed between the rats treated with deferiprone and the control group indicated that increasing the number of animals was unnecessary. This dataset was specifically aimed at confirming the mechanism behind the secondary production of ERFE and was not incorporated into the PK/PD modelling.

The expression of rRatERFE and further study in rats not only revealed that ERFE has two distinct release rates post rHuEPO stimulation, but it also, for the first time, reported the PK/PD properties of ERFE. The discovery of two separate ERFE release rates aligns with the mechanism for dual-peak ERFE dynamics as discussed above. In the PK profiles of rRatERFE, the initial slope is much steeper than the terminal slope which indicates that ERFE is promptly diffused from the bloodstream into various body tissues and organs. Considering the role of ERFE in suppressing hepcidin expression in the liver, the highest blood perfusion rates (800−1200 ml·min⁻¹) of the liver ensures the injected rRatERFE protein is rapidly transported to the liver and can bind to its potential receptors (Eipel et al., 2010). To date, specific receptors for ERFE have not been determined, but some evidence suggests that ERFE could interact with bone morphogenetic proteins (BMPs), acting as a natural trap (Arezes et al., 2018; Wang et al., 2020). Here reported PK characteristics imply that the binding between BMPs and ERFE within the Disse space should be fast and have a low dissociation rate, causing a significant portion of circulating ERFE to be retained in the liver. The PD results suggest that rRatERFE might facilitate erythropoiesis by counteracting hepcidin. Our study provides initial evidence that the administration of bioengineered ERFE improves erythropoiesis by increasing red blood cells and concentrations. Given that this is a single-dose study in healthy rats aiming to investigate the pharmacokinetic properties of rRatERFE, it would be worthwhile for future research to explore the effects of recombinant ERFE protein on erythropoiesis with multiple doses in an anaemic condition.

We proposed a mechanism-based PK/PD model that firstly links the effects of rHuEPO on erythropoiesis, ERFE release and production and iron metabolism. Various models have been proposed to represent the erythropoietic effect of rHuEPO (Ait-Oudhia et al., 2010b; Krzyzanski et al., 2005; Ramakrishnan et al., 2003; Sharma et al., 1998; Yan et al., 2012), but our model uniquely integrates the PK of rHuEPO, erythropoiesis from burst-forming unit erythroid cells to mature red blood cells, short-term serum iron changes and the intricate dynamics of ERFE into a mechanism-based model. Particularly, by encompassing the underlying mechanisms of complex ERFE dynamics, the model provides valuable insights such as (1), the circadian pattern of ERFE baseline, which is crucial for either utilizing ERFE as a biomarker or recombinant ERFE protein as a therapeutic drug. This information aids in correcting the measured ERFE values and offers a dosing time reference to prevent interference with endogenous ERFE. (2) The amplitude of the immediate release of ERFE, potentially serving as an index of the erythropoietic ability of an individual. If an individual display no enhancement of the amplitude of immediate release of ERFE, it might suggest the limit of erythropoiesis has been reached and the rHuEPO dose should not be increased, even if the target level is not achieved. (3) The iron changes and the corresponding second peak of ERFE could guide the amount of iron supplements to be administered alongside rHuEPO. (4) The first peak value is more appropriate to be used to predict long-term erythropoietic effects as it is directly related to EPO stimulation and shows comparable sensitivity to rHuEPO compared to erythroid progenitors. Based on the model predictions, a simplified linear regression model could accurately describe the relationship between the ERFE and responses. This method enables straightforward prediction of responses using early ERFE peak values without running a complex PK/PD model, and significantly enhancing the accessibility of ERFE as a clinical biomarker. With the inclusion of additional clinical data, it may be feasible to establish a threshold for ERFE peak values, which is similar to the cut offs of target, in order to facilitate early detection of ESA resistance. Additionally, leveraging prior information and limited sampling after ESA treatment enables the prediction of individual responses through the utilization of PK/PD modelling and Bayesian forecasting employing maximum a posteriori estimation. This approach is frequently employed in therapeutic drug monitoring to forecast future individual PK observations based on historical data and existing population pharmacokinetic information (de Jonge et al., 2005). We recently optimized a simulation method that allows simultaneous dose adaptation according to predefined rules (Xu et al., 2024), this advancement will facilitate the development of a novel algorithm utilizing ERFE levels to guide ESA dosing. Following validation in phase II/III clinical trials, the utilization of ERFE as a biomarker is anticipated to significantly revolutionize anaemia management in clinical practice.

In conclusion, this study elucidated the underlying mechanism for the complex dynamics of ERFE and further demonstrated that ERFE could be used as an early biomarker to predict long-term responses. We revealed a novel mechanism that transient iron deficiency can stimulate ERFE production. The complex dynamics of ERFE is explained as being comprised of an immediate release and a secondary ERFE production mediated by transient iron deficiency. This mechanism was also supported by the developed PK/PD model since the model can adequately describe the ERFE dynamics and the erythropoietic responses to rHuEPO. By using this model, we quantified the relationship between ERFE and responses to rHuEPO and provided an available method to predict maximum responses based on early peak values of ERFE. Although further clinical trials are needed to determine the peak time of ERFE in humans, this method is highly practical as our study demonstrates that the peak time of the immediate release of ERFE is independent of doses and appears as early as 2 h. These results support the hypothesis that early ERFE levels can predict long-term responses to ESAs. In addition, the developed mechanism-based PK/PD model can serve as a key framework for future studies on ERFE.ACKNOWLEDGEMENTSThis work was supported by funding from The Chinese University of Hong Kong, and the postgraduate scholarships of Peng XU was provided by The Chinese University of Hong Kong.

AUTHOR CONTRIBUTIONS

Peng Xu performed the research, analysed the data and wrote the paper. Xiaoyu Yan designed the research study, supervised the overall research and wrote the paper. Raymond S. M. Wong designed the research study.

ACKNOWLEDGEMENTS

This work was supported by the Direct Grant 4054650 and Research Matching Grant Scheme Project 8601261 from The Chinese University of Hong Kong, and the postgraduate scholarships of Peng XU was provided by The Chinese University of Hong Kong. The author extends gratitude to Professor Wojciech Krzyzanski for his insightful advice and valuable discussions regarding the interaction between iron and ERFE, which served as significant inspiration throughout this research.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis, Immunoblotting and Immunochemistry and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.

Open Research

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Supporting Information

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Supplementary Table 1. Calculated kinetic parameters of recombinant rat erythroferrone (rRERFE)

Supplementary Table 2. Estimated pharmacokinetic parameters with their corresponding RSE of recombinant human erythropoietin (rHuEPO) and rRat erythroferrone (ERFE).

Supplementary Figure 1. Basic goodness-of-fit plots of recombinant human erythropoietin (rHuEPO concentrations (A) and rRat erythroferrone (ERFE) (B) in the final model. Upper panels present the observed data (DV) vs. the population predictions (PRED) and individual predictions (IPRED). Lower panels present the conditional weighted residuals (CWRES) vs. TIME (h) and PRED. The red lines are the loess smooth lines. The grey diagonal and horizontal lines are the identity and zero lines.

Supplementary Figure 2. Basic goodness-of-fit plots of PD responses in the final model including RET counts, RBC counts, haemoglobin (HGB) concentrations and erythroferrone (ERFE) concentrations. The top panels present the observed data (DV) vs. the population predictions (PRED). The panels in the first line present the observed data (DV) vs. the individual predictions (IPRED). The panels in the second line present the conditional weighted residuals (CWRES) vs. TIME. The bottom panels present the conditional weighted residual (CWRES) vs. population predictions (PRED). The red lines are the loess smooth lines. The grey diagonal and horizontal lines are the identity and zero lines.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Volume181, Issue16

August 2024

Pages 2833-2850

Early erythroferrone levels can predict the long-term haemoglobin responses to erythropoiesis-stimulating agents

Abstract

Background and Purpose