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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Feb 23;175(7):1054–1065. doi: 10.1111/bph.14143

Sustained plasma hepcidin suppression and iron elevation by Anticalin‐derived hepcidin antagonist in cynomolgus monkey

Andreas M Hohlbaum 1,†, Hendrik Gille 1,†, Stefan Trentmann 1, Maria Kolodziejczyk 1, Barbara Rattenstetter 1, Coby M Laarakkers 2,3, Galina Katzmann 1, Hans Jürgen Christian 1, Nicole Andersen 1, Andrea Allersdorfer 1, Shane A Olwill 1, Bernd Meibohm 4, Laurent P Audoly 1, Dorine W Swinkels 2,3,†, Rachel P L van Swelm 2,3,†,✉
PMCID: PMC5843705  PMID: 29329501

Abstract

Background and Purpose

Anaemia of chronic disease (ACD) has been linked to iron‐restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte‐derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin‐derived, small protein‐based anticalin PRS‐080 hepcidin antagonist with high affinity and selectivity.

Experimental Approach

Anticalin‐ and hepcidin‐specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t 1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS‐080‐PEG30 administration in vivo.

Key Results

PRS‐080 had a hepcidin‐binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS‐080‐PEG30), a t 1/2 of 43 h in cynomolgus monkeys. Dose‐dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS‐080‐PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment‐free interval, all iron parameters returned to pre‐dose values.

Conclusions and Implications

In conclusion, we developed a dose‐dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron‐restricted erythropoiesis that can address the root cause of ACD. PRS‐080‐PEG30 is currently in early clinical development.

Abbreviations

ACD

anaemia of chronic disease

ECL

enhanced chemiluminescence

ESA

erythropoiesis‐stimulating agent

ESRD

end‐stage renal disease

IREG1

ferroportin

NGAL

neutrophil gelatinase‐associated lipocalin

NOAEL

no observed adverse effect level

PD

pharmacodynamic

PEG

maleimide‐methoxy‐polyethylene‐glycol

PK

pharmacokinetic

TIBC

total iron‐binding capacity

TSAT

transferrin saturation

UIBC

unsaturated iron‐binding capacity

Introduction

Anaemia of chronic disease (ACD) develops in patients suffering from infections, inflammatory diseases, cancer and chronic kidney disease, including end‐stage renal disease (ESRD). ACD is also referred to as anaemia of inflammation because it is caused in part by an underlying chronic or acute inflammatory condition (Ganz and Nemeth, 2009; Weiss and Goodnough, 2005). Routine use of erythropoiesis‐stimulating agents (ESAs) has enabled anaemia to be corrected in most patients with ESRD. However, adequate iron stores are essential for achieving maximum benefit from ESAs, and decreased iron stores or decreased availability of iron are the most common reasons for resistance to their effect. Indeed, a major pathophysiological factor in ACD is retention of iron in macrophages, rendering the metal ion unavailable for erythropoiesis. Multiple lines of evidence, both in preclinical models and humans, have revealed that functional iron deficiency in ACD can be attributed to increased concentrations of the main iron regulatory hormone hepcidin (De Falco et al., 2013; Fleming and Sly, 2001; Guo et al., 2013; Kemna et al., 2005; Roy et al., 2007). Hepcidin binding to ferroportin (IREG1), the only known cellular iron exporter, causes its internalization and degradation (Nemeth et al., 2004b) and thereby blocks iron export from macrophages and hepatocytes and iron uptake by duodenal enterocytes (Ganz and Nemeth, 2011). Most ESRD patients receive high‐dose i.v. iron supplementation even though it increases hepcidin expression. Concerns about the long‐term safety of i.v. iron use with respect to oxidative stress, kidney injury and accelerated arteriosclerosis have been raised (Bishu and Agarwal, 2006; Charytan et al., 2015; Macdougall et al., 2016; Slotki and Cabantchik, 2015). In fact, when studied by hepatic MRI, 84% of haemodialysis patients exhibited mild‐to‐severe hepatic iron overload (Rostoker et al., 2012). Therefore, there is a clear unmet need for new treatment modalities directed towards the underlying pathophysiology of ACD.

The hepcidin–IREG1 pathway constitutes an interesting target in this regard (Nemeth, 2010; Ross et al., 2012; Sun et al., 2012). Direct neutralization of hepcidin is attractive as multiple pathways and regulatory circuits converge at the level of hepcidin to control systemic iron homeostasis. In this regard, anti‐hepcidin antibodies have been explored in in vivo models to modulate iron metabolism during anaemia of inflammation (Cooke et al., 2013; Krzyzanski et al., 2016; Sasu et al., 2010). Another way of neutralizing hepcidin directly has been studied using NOX‐H94, an enantiomeric l‐RNA aptamer (also called Spiegelmer), which resulted in iron mobilization in cynomolgus monkeys, healthy human volunteers and healthy volunteers with LPS‐induced systemic inflammation (Boyce et al., 2016; Schwoebel et al., 2013; van Eijk et al., 2014). Other strategies to reduce hepcidin expression with inhibitors of the bone morphogenetic protein‐6/haemojuvelin/mothers against decapentaplegic homologue pathway or inhibitors of the IL‐6/signal transducer and activator of transcription‐3 pathway have been considered as well, since hepcidin expression is controlled by circulating and stored iron and inflammation (Andriopoulos et al., 2009; Boser et al., 2015; Nemeth et al., 2004a; Xia et al., 2008; Crielaard et al., 2017). Lastly, inhibitors of hypoxia‐inducible factor prolyl hydroxylases have been demonstrated to negatively regulate hepcidin expression but were recently shown to exert this effect largely indirectly through their effect on erythropoietin expression (Besarab et al., 2015; Pergola et al., 2016; Provenzano et al., 2016).

Indispensible requisites for effective and safe iron mobilization based on direct hepcidin antagonism are high specificity and tolerability of the hepcidin antagonist. Anticalins are engineered lipocalins and represent a highly promising human, non‐immunoglobulin scaffold. The safety, tolerability and biological activity of a VEGF‐A‐specific anticalin derived from human tear lipocalin was demonstrated in a phase I study (Mross et al., 2013), while the pharmacological activities of other anticalin‐based antagonists directed against c‐MET receptor TK, IL‐4 receptor‐α (cytotoxic T‐lymphocyte‐associated protein‐4) and VEGF‐A were shown in preclinical models (Hohlbaum et al., 2011; Olwill et al., 2013; Schonfeld et al., 2009; Gille et al., 2016). Here, we describe the development of a 21 kDa human neutrophil gelatinase‐associated lipocalin (hNGAL)‐derived anticalin, PRS‐080, engineered to bind human and cynomolgus hepcidin with picomolar affinity and to neutralize its function. Pharmacokinetic/pharmacodynamic (PK/PD) modelling enabled adequate drug candidate modulation to extend the t 1/2 and consequent duration of iron mobilization, which was confirmed in vivo. Importantly, we developed a novel MS‐based assay to measure unbound (free) and, thus, biologically active hepcidin after PRS‐080 administration, in order to effectively quantify target engagement. Indeed, single PRS‐080 administration in cynomolgus showed a dose‐dependent hepcidin suppression and iron mobilization. Moreover, we are the first to show sustained‐free hepcidin suppression and iron mobilization after repeated dosing of a hepcidin antagonist in cynomolgus monkeys. Combined, our data improve our insights of the mutual dynamics of free circulating hepcidin and plasma iron concentrations after administration of a hepcidin antagonist, and vice versa, and show that the biological activity of hepcidin can be effectively and safely antagonized with the PK optimized drug candidate PRS‐080.

Methods

Anticalin selection using phage display and high‐throughput elisa screening

A phage display library of neutrophil gelatinase‐associated lipocalin (NGAL) was used in combination with the Hep‐C‐Bio to select target‐specific lead candidates (Kim et al., 2009). The affinity of the initial lead anticalin for full‐length human hepcidin‐25 was further improved in one cycle of in vitro evolution, by amplifying the central part of its coding region via error‐prone PCR, followed by phage display selection using increased stringency via target limitation and ranking of new variants in a high‐throughput elisa screen basically as described previously (Schonfeld et al., 2009).

Anticalin fermentation, purification and site‐directed PEGylation

The selected hepcidin‐specific anticalin (PRS‐080) was produced by periplasmatic expression in Escherichia (E.) coli or by cytoplasmic expression in E. coli K12 strain BL21 (DE3) when the PRS‐080 variant contained an engineered cysteine at position 87 for site‐directed addition of maleimide‐methoxy‐PEG (PRS‐080‐Cys87) (Schlehuber et al., 2000). The intermediate product PRS‐080‐Cys87 was PEGylated in batch mode, purified by ion exchange chromatography to remove unbound PEG and other process‐related impurities, dialysed and concentrated. Analytical size‐exclusion chromatography and SDS‐PAGE/Western blots were performed for all anticalins to control their purity and quality using standard lab methodology.

Anticalin affinity assessment by surface plasmon resonance

PRS‐080 or control lipocalin (NGAL) was immobilized to a level of 750–1100 resonance units on a CM5 sensor chip using an amine coupling kit (GE Healthcare, Solingen, Germany). Dilutions of different analytes in HBS‐EP+ buffer (GE Healthcare) were applied to the prepared chip surfaces. Data were evaluated with Biacore T200 Evaluation software (V 1.0). The 1:1 binding model and double referencing were used to fit the raw data.

Solution‐binding assay

Meso Scale Discovery (MSD, Rockville, MD, USA) plates were coated with the anti‐human hepcidin mAb 12B9 at 1.5 μg·mL−1. Hep‐C‐Bio, 25 pM, was incubated with variable concentrations of anticalins in assay buffer (PBS, 0.1% Tween 20 and 2% BSA) for 1 h at room temperature and transferred to the antibody‐coated MSD plate to capture unbound (free) Hep‐C‐Bio. Sulfo‐Tag‐labelled streptavidin and the SECTOR Imager 2400 (MSD) were employed for detection as recommended by the manufacturer. The free target concentrations were calculated by using a standard curve of Hep‐C‐Bio and plotted versus the employed anticalin concentrations. Curves were fitted after logarithmic transformation by non‐linear regression with a log inhibitor versus response model (variable slope) in order to determine EC50 values.

Ferroportin internalization assay

A cell‐based IREG1 assay was developed to monitor the effect of the selected hepcidin antagonists on hepcidin activity using a carboxy‐terminally human IREG1 fused with GFP basically as described previously (Nemeth et al., 2004b) and subsequently adapted (Laarakkers et al., 2013). Cells were incubated with PRS‐080 or NGAL and 40 nM of human hepcidin‐25 for 24 h, after which fluorescence was quantified. Stimulation values for wells containing antagonists were normalized with the results from hepcidin‐only treated and untreated cells and plotted versus antagonist concentration. A curve was fitted by non‐linear regression with a four‐parameter logistic equation.

Anticalin‐specific PK/PD model

A PK/PD model for the interaction between PRS‐080 and hepcidin was developed in analogy to that described previously (Xiao et al., 2010). The set of differential equations are described in Supporting Information Figure S1. Binding constants determined by surface plasmon resonance (K on 3.74 106 M−1·s−1 and K off 2.45 10−4 s−1) were used for the interaction between PRS‐080 and cynomolgus hepcidin for all PRS‐080 conjugates, as no potency differences could be detected in a sensitive solution‐binding assay. Since there are no data available on the volume of distribution of hepcidin, the model assumes it to be identical to the central volume of distribution (V c) of PRS‐080. The model was fitted to the experimental total PRS‐080‐PEG30 concentration data obtained from the cynomolgus monkey PK study using non‐linear regression analysis.

Animal studies

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Initial studies investigating PK/PD were performed on non‐naïve, purpose‐bred Macaca fascicularis (cynomolgus monkey; Suzhou Xishan Zhongke Laboratory Animal Co. Ltd, Jiangsu, China) stock animals of approximately 4 years old. The conducting laboratory has an in‐house ethics review procedure, which covers animal welfare within the facility. The activities of the ethical committee, which are not within the scope of the Good Laboratory Practice regulations, were reviewed as part of the Association for Assessment and Accreditation of Laboratory Animal Care International accreditation procedure and the Federation of European Laboratory Animal Science Associations recommendations. The ethical committee ensured that animal use was carefully considered and fully justified and that all possibilities for reduction, refinement and replacement (the 3 Rs) had been evaluated. The study protocols were reviewed and approved after compliance with EU regulations (Directive 2010/63/EC) had been confirmed. After blood sampling had been concluded, animals were returned to stock.

The experiments described in Figures 4 and 5 (cynomolgus monkeys, Bioculture Ltd., Senneville, Mauritius; approximately 4 years old) show data obtained during the formal toxicology programme conducted to facilitate the first human trial with PRS‐080. Both study protocols were reviewed by a designated member of an established animal welfare and ethical review body. The experimental site maintains an establishment licence as issued by the UK competent authority, the Home Office animals in science regulation unit. This licence is issued under the Animals (Scientific Procedures) Act 1986, which implements Directive EU 2010/063 on the protection of animals used for scientific purposes. At the end of these latter studies, animals were killed for the required histopathological assessment. On the day of necropsy, each animal was given an i.v. overdose of sodium pentobarbitone. Immediately subsequent to this, and once death was confirmed, major blood vessels were severed to exsanguinate the animal.

Figure 4.

Figure 4

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Free and total hepcidin‐25 (Hep25), PRS‐080‐PEG30 and iron concentration profiles after single i.v. administration of PRS‐080‐PEG30 in cynomolgus monkeys. (A) Concentration profiles (mean ± SEM) of free PRS‐080‐PEG30 (PRS‐080), plasma iron and free Hep25 after single 10 mg·kg−1 i.v. dose (n = 3). (B) Concentration profiles (mean ± SEM) of total and free PRS‐080‐PEG30 and total Hep25 after single 10 mg·kg−1 i.v. dose (n = 3). (C) Mean duration of free Hep25 suppression (n = 3) and (D) free PRS‐080‐PEG30 concentration profiles for different i.v. doses of PRS‐080‐PEG30 (n = 3).

Figure 5.

Figure 5

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Free and total hepcidin‐25 (Hep25), PRS‐080‐PEG30 and iron concentration profiles after repeated administration in cynomolgus monkeys. Concentration profiles (mean ± SEM) of total and free PRS‐080‐PEG30 (PRS‐080), total and free Hep25 and plasma iron (n = 3; 2 male and 1 female) after five repeated 20 mg·kg−1 s.c. doses administered every other day; ‘#’ means dosing, and ‘*’ means necropsy.

In all studies, animals were randomized to treatment groups and group housed except for short periods of time where experimental procedures dictated otherwise. The animals were housed in cages/pens that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 1989). Animals of the same group and sex were housed in the same pen. The study was not blinded in order to be able to correctly assess compound‐related toxicological observations throughout the study. Water was provided ad libitum via water bottles. Each animal was offered approximately 100 g of primate diet and a 25 g Bonio biscuit (Spillers, Milton Keynes, UK) daily. The animals were provided with a number of items/strategies such as the provision of toys (balls and inert nylon chews), swings and foraging materials for environmental enrichment.

Iron parameters

Iron and unsaturated iron‐binding capacity (UIBC) were measured in plasma samples with the ADVIA 1650 blood biochemistry analyser (Siemens, Erlangen, Germany) using colorimetric assays. Total iron‐binding capacity (TIBC) was calculated using the formula TIBC = iron + UIBC. Transferrin saturation (TSAT) was derived using the formula (100 × iron)∕TIBC.

Total and free PRS‐080‐PEG30

Plasma concentrations of hepcidin‐free PRS‐080‐PEG30 (PRS‐080 coupled to 30 kD PEG) were determined by a sandwich enhanced chemiluminescence (ECL) immunoassay using Hep‐C‐Bio, immobilized via streptavidin as a capturing step and a polyclonal rabbit NGAL‐specific antibody preparation (PL713, Pieris) for detection. The plasma concentrations of total PRS‐080‐PEG30 were determined by a sandwich ECL immunoassay using an affinity‐purified hNGAL‐specific rabbit antibody preparation (PL854, Pieris) as a capturing step and a biotinylated affinity‐purified hNGAL‐specific rabbit antibody preparation (PL1047, Pieris) for detection of the bound PRS‐080.

Total and free hepcidin in the presence of PRS‐080‐PEG30

Free hepcidin concentrations in cynomolgus plasma in the presence of PRS‐080‐PEG30 were measured by weak cation exchange enrichment of hepcidin coupled to time‐of‐flight MS, as described previously for the measurement of full‐length human hepcidin (Kroot et al., 2010; Laarakkers et al., 2013). We used synthetic full‐length rabbit hepcidin‐25 as internal standard as this peptide was shown to have a very low affinity for PRS‐080 (K d = 173 nM, Table 1). For the measurement of total (free and bound) hepcidin, PRS‐080‐PEG30 was released from hepcidin prior to WCX enrichment.

Table 1.

Kinetic parameters and specificity of PRS‐080 to human hepcidin in comparison with hepcidin of other species and additional related or unrelated molecules

Ligand Analyte k a (M−1·s−1) k d (s−1) K d (nM) N
PRS‐080 Hepcidin‐25, human 3.8 (±1.5) · 106 2.0 (±0.7) · 10−4 0.07 (±0.05) 6
Hepcidin‐20, human 5.2 (±0.3) · 106 4.4 (±2.5) · 10−4 0.08 (±0.04) 2
Hepcidin‐25, cynomolgus 1.4 (±2.0) · 107 3.7 (±1.5) · 10−4 0.07 (±0.06) 3
Hepcidin‐25, mouse N/A N/A >1000 3
Hepcidin‐25, rat N/A N/A >500 3
Hepcidin‐25, dog (beagle) 4.0 · 105 1.3 · 10−3 ~3.8 2
Hepcidin‐25, rabbit 3.8 · 104 6.5 · 10−3 ~173 2
Fe‐enterobactin No binding 8
ß‐Defensin No binding 3
VEGF8‐109 No binding 3
HSA No binding 3
NGAL Hepcidin‐25 No binding 6
Fe‐enterobactin 2.1 (±0.7) · 106 8.3 (±1.1) · 10−4 0.42 (±0.14) 3
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Values are means (±SD); N is the number of independent experiments. No binding to the reference channel was detected for all of the investigated targets. All hepcidin analytes exhibited biological activity as verified independently in the cell‐based IREG1 internalization assay (data not shown). HSA, human serum albumin; k a, association rate constant, k d, dissociation rate constant; K d, affinity constant; N/A, not applicable.

Data analysis

The experimental investigations of the PK/PD relationship were conducted in non‐human primates and not in rodents, because PRS‐080 binds to cynomolgus monkey hepcidin but does not appreciably interact with the mouse or rat targets. Ethical considerations limit the use of non‐human primates in research to the absolute minimum required, even for the development of human therapeutics. Consequently, the results presented are of a descriptive nature and not statistically analysed due to a limited sample size. Data were plotted in graphs using GraphPad Prism (v5.03) software. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).

Materials

The anti‐human hepcidin‐specific mAb 12B9 was produced as described in the PCT patent application WO 2009/139822 A1, CDR: SEQ ID# 161‐166. Human, cynomolgus, rat, dog and rabbit hepcidin‐25 as well as C‐terminal biotinylated human hepcidin‐25 (Hep‐C‐Bio) were produced via peptide synthesis and refolding (Bachem AG, Bubendorf, Switzerland, for cynomolgus and PeptaNova GmbH, Sandhausen, Germany, for all others). Fe‐enterobactin (Genaxxon Bioscience, Ulm, Germany), ß‐defensin (Sigma‐Aldrich, Munich, Germany), VEGF8‐109 (Pieris, Freising, Germany) and human serum albumin (Sigma Life Sciences, Munich, Germany) were used as negative controls to demonstrate the binding specificity of PRS‐080 to hepcidin.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

Results

Anticalin PRS‐080 specifically binds hepcidin

Anticalins displaying high affinity and specificity against hepcidin were identified after high‐throughput screening of a NGAL‐based anticalin phage display library. Surface plasmon resonance showed that the anticalin selected, PRS‐080, binds full‐length human hepcidin with a K d = 0.07 ± 0.05 nM (Table 1). Immobilized NGAL bound the bacterial siderophore Fe‐enterobactin with subnanomolar affinity but did not bind hepcidin as expected based on its reported specificity (Goetz et al., 2002). On the other hand, PRS‐080 did not exhibit any measurable affinity towards Fe‐enterobactin. PRS‐080 also binds cynomolgus hepcidin‐25 with comparable affinities to human hepcidin of 0.07 ± 0.06 nM, but no detectable binding could be observed for rat or mouse hepcidin, whereas dog and rabbit hepcidin were bound with nanomolar affinity respectively (Table 1).

Half‐life extension of PRS‐080 does not affect hepcidin binding

PRS‐080 is 21 kDa and has a predicted molecular radius significantly below the size barrier of the glomerular membrane and would be subject to efficient filtration by the kidney (Ohlson et al., 2000). Linear PEG moieties of 20 and 30 kDa and an L2‐branched 40 kDa PEG were individually conjugated to PRS‐080 to increase the hydrodynamic radius and, consequently, its t 1/2. PEGylation did not impact the binding activity for hepcidin as non‐modified PRS‐080 as well as the different conjugates exhibited the same activity (EC50 of 0.05 to 0.06 nM) in a solution‐binding assay that can detect affinity differences in the picomolar range (Supporting Information Table S1).

PRS‐080‐specific, dose‐dependent and complete inhibition of hepcidin‐induced IREG1 degradation could be observed in a cell‐based assay for the non‐modified PRS‐080 and PEGylated variants, whereas NGAL and NGAL‐PEG40 did not have any effect (Figure 1). Furthermore, none of the modifications caused a detectable loss in antagonistic activity compared with the unmodified PRS‐080 hepcidin antagonist consistent with the binding affinity measurements.

Figure 1.

Figure 1

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Inhibition of hepcidin‐induced IREG1 internalization and degradation in vitro. Anticalins (PRS‐080 unmodified, PEG30 or PEG40 conjugated) or lipocalin control (NGAL or PEG40‐conjugated NGAL) were analysed in the cell‐based IREG1 assay. The assay was performed in triplicates and the assay window corresponds to a 1.6‐fold induction (derepression) of GFP fluorescence.

Finally, total drug plasma concentrations profiles as determined from the PK studies in cynomolgus monkeys showed that the terminal t 1/2 of PRS‐080 increased by increasing the MW of the PEG moiety (Table 2).

Table 2.

Key PK parameters of PEGylated versions of PRS‐080 determined after i.v. administration at a dose level of 10 mg·kg−1

C L (mL·h−1·kg) V D (mL·kg−1) AUC0–∞ (h·μg·mL−1) t ½β (h)
Cyno PEG‐20 5.5 135.7 1818 18.8 (±1.1)
PEG‐30 1.3 66.4 8000 43.5 (±6.0)
PEG‐40 0.4 78.5 24 390 167.5 (±10.6)
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PK parameters were derived by non‐compartmental analysis with WinNonLin. AUC0–∞, AUC (from time = 0 to infinity); C L, clearance; V D, volume of distribution.

A novel PK/PD model predicted the duration PRS‐080‐mediated hepcidin suppression in cynomolgus monkey

A cynomolgus PK/PD model for the interaction between PRS‐080 and hepcidin was developed to show the potential extent and the time window of hepcidin neutralization in vivo. The model consists of a two‐compartment PK model for PRS‐080 as free drug and in complex with hepcidin, a turnover model for endogenous hepcidin as previously described and a reversible binding model for the interaction between PRS‐080 and hepcidin (Supporting Information Figure S1) (Xiao et al., 2010). The total cynomolgus plasma concentrations of PRS‐080‐PEG30 concentrations as described in Table 2 were fitted to the PK/PD model. The combined data set of free and total PRS‐080‐PEG30 concentration data could be successfully described by only fitting the turnover rate constant k out,h for hepcidin and the PK parameters for PRS‐080‐PEG30. The second hepcidin turnover rate k in,h was derived from the estimated value for k out,h (9.3 h−1) and the hepcidin baseline value in cynomolgus monkeys (4.3 nM) according to k in,h = Hepcidinbaseline · V c · k out,h.

By keeping the estimated value for k out,h and the measured hepcidin baseline constant, since they are parameters of the biological system rather than the drug compound, the developed modelling approach could also describe the concentration–time profiles for the other two investigated PRS‐080 derivatives (Figure 2A). Single‐dose simulations were performed with the model and model‐derived parameters to explore the time courses of free hepcidin and hepcidin complexed with PRS‐080. The model predicts a suppression of hepcidin below 1 nM for 36, 48 and 48 h for PRS‐080‐PEG20, PRS‐080‐PEG30 and PRS‐080‐PEG40 derivatives, respectively, at the 10 mg·kg−1 dose level. We utilized the same model to simulate a repeated administration of PEG30 and PEG40 modified PRS‐080 conjugates every 48 h. The reduced clearance and thus longer t 1/2 of the PEG40 derivative led to substantial accumulation of the hepcidin–anticalin complex over a 3 week period, whereas a steady state of the complex was reached at about 96 h with the PEG30 derivative (Figure 2B).

Figure 2.

Figure 2

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Prediction of concentration–time profiles of total and free anticalin as well as free hepcidin and hepcidin/anticalin complexes after single and repeated administration in cynomolgus monkeys. (A) Total nM concentrations of anticalin (red squares) were measured in individual cynomolgus monkeys (3 per treatment group) after single i.v. administration of 10 mg·kg−1 of PRS‐080‐PEG20, PRS‐080‐PEG30 or PRS‐080‐PEG40. Concentration–time profiles of total PRS‐080, free PRS‐080 and free hepcidin were predicted using the PK/PD model developed. Hepcidin baseline levels as determined by pre‐dose measurements are indicated by a continuous line. The variable duration of hepcidin suppression below 1 nM is indicated by double arrows and terminal t 1/2 values derived by non‐compartmental PK analysis are indicated for each PRS‐080 conjugate. (B) Concentration–time profiles of hepcidin/anticalin complexes were predicted after repeated administration of 10 mg·kg−1 of PRS‐080‐PEG30 and PRS‐080‐PEG40 every 48 h.

Dose‐dependent iron mobilization in cynomolgus monkeys following single i.v. PRS‐080 administration

The various PRS‐080 formats were tested for their ability to increase plasma iron concentrations following single‐dose administration in cynomolgus monkey. The PRS‐080‐PEG40 format did not further prolong the hyperferremic response compared with PRS‐080‐PEG30 (Figure 3A). In contrast to PRS‐080‐PEG30, NGAL‐PEG40 did not lead to increased plasma iron concentrations, confirming a PRS‐080‐specific effect (Figure 3B). Animals that were given NGAL did show fluctuations in plasma iron concentrations, which are likely explained by diurnal rhythm and variations with food intake (Figure 3B). Further studies with PRS‐080‐PEG30 indicated a linear dose–response relationship between 1 and 20 mg·kg−1 in the cynomolgus monkey. Mobilization of iron was observed first at 1 mg·kg−1, albeit as a relative small and transient plasma iron peak. The AUC for iron mobilization then increased proportionally with the administered dose (Figure 3C).

Figure 3.

Figure 3

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PRS‐080 format and dose‐dependent iron mobilization in healthy cynomolgus monkeys. (A) Concentration–time profiles of total plasma iron measured following a single i.v. dose of 10 mg·kg−1 of PRS‐080 with 20, 30 or 40 kD PEG moiety (PRS‐080‐PEG20, PRS‐080‐PEG30 and PRS‐080‐PEG‐40) in male cynomolgus monkeys, n = 3 per group. Each symbol (square, triangle and circle) represents the measurements in time for each of the three animals respectively. (B) An equimolar dose (12 mg·kg−1 i.v.) of lipocalin (NGAL wt) did not result in an increase in total plasma iron. (C) Dose‐dependency of iron mobilization was also assessed for the PRS‐080‐PEG30 after a single i.v. dose of 0.4, 1.2, 2.4, 4 or 8 mg·kg−1 (n = 3 per dose level), and the AUC of total plasma iron calculated was plotted against the tested dose. At higher‐dose levels, the iron response is extended in duration, whereas 1–3 mg·kg−1 represents the minimal biological effect level in cynomolgus monkeys.

Free hepcidin concentrations correlate with the dose‐dependent increased iron concentrations obtained by single PRS‐080 dosing

Free hepcidin concentrations were measured after single i.v. administration of 1 to 10 mg·kg−1 PRS‐080‐PEG30 and were correlated with the measured iron response and free PRS‐080‐PEG30 concentrations to assess target engagement directly (Figure 4A, data set for 10 mg·kg−1). Free hepcidin declines rapidly from a 7 nM baseline concentration to below 1 nM after administration of PRS‐080‐PEG30 and returns to baseline after ~24 h. At this time point, free PRS‐080‐PEG30 has been reduced from the ~7 μM starting concentration to 250 nM, while elevated plasma iron is maintained for at least 32 h despite the fact that free hepcidin increases transiently above the baseline up to 52 nM at 32 h. Thereafter, plasma iron and free hepcidin return to baseline. Saturation of the administered PRS‐080‐PEG30 dose can also be appreciated by the fact that the measured total hepcidin concentrations of ~2.2 μM equals the total PRS‐080‐PEG30 concentration 24 h after the 4 mg·kg−1 dose (Figure 4B), which coincides with the disappearance of free PRS‐080‐PEG30. Elimination of total hepcidin thereafter is linked to the clearance of PRS‐080‐PEG30. Moreover, these studies showed that the duration of hepcidin suppression below baseline was dependent of the dose of PRS‐080‐PEG30 (Figure 4C) and correlated strongly with the saturation of the active/free form of PRS‐080‐PEG30 (Figure 4D).

Sustained iron mobilization and hepcidin suppression with repeated PRS‐080‐PEG30 dosing

Repeated s.c. dosing of 20 mg·kg−1 PRS‐080‐PEG30 resulted in an apparently stable increase in plasma iron accompanied by a steady free hepcidin concentration (Figure 5). Similar to the single‐dose studies, plasma iron increased to 65 μM and total hepcidin increased to 1600 nM in the 24 h after the first s.c. injection of PRS‐080‐PEG30, whereas free hepcidin remained low. Up to the last s.c. dose, plasma total hepcidin has further increased to 5400 nM, in line with the concentrations of total PRS‐080‐PEG30 (13 000 nM) and free PRS‐080‐PEG30 (6900 nM), whereas the free hepcidin levels remain low. Approximately 24–48 h after the last injection at 192 h, it can be observed that free hepcidin is slightly increasing, similar to what was observed in the single‐dose study, accompanied by a small decline in iron concentration, total and free PRS‐080‐PEG30 levels and total hepcidin level.

In another repeated dose study with PRS‐080‐PEG via i.v. infusion every 48 h for 29 days in cynomolgus monkeys, the no observed adverse effect level (NOAEL) was determined at 120 mg·kg−1, which was the highest dose investigated. In the same study, we assessed the recovery of various iron parameters in time (up to 528 h) after the last PRS‐080‐PEG30 dose at day 29. After the last dosing, plasma iron concentrations and TSAT remained elevated in the PRS‐080‐PEG30‐treated animals for at least 192 h but had returned to the levels observed at baseline and in concurrent controls on post‐dose day 23 (528 h; Figure 6A, B). In concordance, the TIBC and UIBC were lowered in PRS‐080‐PEG30‐treated monkeys compared with controls for 192 h post‐dose but returned to control levels by 528 h post‐dose (Figure 6C, D). Although plasma hepcidin concentrations were not measured in the 120 mg·kg−1 i.v. repeated dose study, the course of the iron parameters suggests an increase in free hepcidin levels after the last PRS‐080‐PEG30 dose within the 528 h post‐dose. Our observations also suggest that any potential temporary increase in free plasma hepcidin after the last PRS‐080‐PEG30 dose as observed in the single‐dose and s.c. repeated dose studies will not lead to plasma iron parameters persistently below nadir levels after drug discontinuation, even at such high toxicologically relevant dose levels.

Figure 6.

Figure 6

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Plasma iron, TSAT, TIBC and UIBC in recovery after repeated i.v. administration in cynomolgus monkeys. To determine the NOAEL and recovery of iron parameters after drug discontinuation, cynomolgus monkeys (n = 10; 5 male and 5 female) were administered 120 mg·kg−1 PRS‐080‐PEG30 via i.v. injection every other day for 28 days. Plasma iron parameters, including (A) plasma iron concentration, (B) TSAT, (C) TIBC and (D) UIBC, were followed up on until 24 h (n = 10) or day 22 (n = 4; 2 male and 2 female) after the last dose. Plasma samples were taken pre‐dosing and at indicated time points after administration. From some animals, no sufficient material was present to determine all parameters; the exact number of samples for analysis per parameter is defined in Supporting Information Table S2. Data are presented as mean ± SEM.

Overall, a dosing interval of 48 h is able to keep the plasma iron concentration high and prevent a negative feedback on plasma iron by free hepcidin. However, subtle fluctuations in iron parameters between dosing may occur.

Discussion

Anticalins are broadly applicable for tight binding of very diverse targets, and their unique cup‐like structure is particularly suited for the engagement of small and compact ligands such as hepcidin. From an extensive library, PRS‐080 was identified as an anticalin with high affinity for hepcidin across different species. Also, the t 1/2‐extended PEGylated PRS‐080 formats showed high hepcidin binding affinity in vitro. Using a PK/PD model with data obtained from PK in vivo studies, we were able to predict suitable PRS‐080 concentrations and dosing intervals to establish optimal elevated systemic iron levels. Target engagement could be verified with a novel MS‐based assay to determine plasma free hepcidin after PRS‐080 administration. Finally, repeated dosing of PRS‐080‐PEG30 in cynomolgus monkey proved successful in maintaining elevated plasma iron levels, without detectable adverse effects on hepcidin regulation.

In a previous study on hepcidin antagonism, it was suggested that hepcidin has a high production rate, due to its small size and fast clearance in order to maintain nanomolar plasma levels (Krzyzanski et al., 2016; Xiao et al., 2010). Based on this suggestion, increased circulating transferrin‐bound iron levels as a result of hepcidin antagonism may, subsequently, lead to a significant hepcidin synthesis by means of counter‐regulation. In turn, this may negatively influence plasma iron levels when the hepcidin antagonism wears off. To address these potential adverse effects of hepcidin counter‐regulation, we increased plasma t 1/2 of the direct hepcidin antagonist PRS‐080 using site‐directed conjugation with PEG moieties of different size and complexity, in order to antagonize newly synthesized hepcidin and ensure prolonged iron mobilization. Indeed, PEGylation resulted in a prolonged iron response after single i.v. PRS‐080 administration in cynomolgus monkeys. Moreover, repeated dosing with 48 h intervals resulted in a sustained iron response, which did not lead to adverse effects on plasma iron parameters after the dosing was discontinued. Using Perls' Prussian Blue special stain, haemosiderin was present within the hepatocytes in the periportal regions of the liver of most treated animals, but there was no clear dose–response relationship. There was no evidence of any cellular injury or inflammatory responses associated with this finding. Moreover, it has to be emphasized that these were doses and frequencies of administration well above those that would be administered in the clinic. Altogether, these studies show the potential of repeated dosing without iron‐related withdrawal effects.

The provided insights in target engagement in our study compared with previous studies on hepcidin antagonism to mobilize reticuloendothelial system iron stores are accomplished by the measurement of free, that is, not antagonist‐bound, hepcidin‐25 by a novel approach using MS. Our data strongly suggest that free hepcidin concentration–time profiles in response to PRS‐080‐PEG30 were independent from total hepcidin profiles. This suggestion is based on three observations. First, we observed a decrease in the free hepcidin concentration, whereas total hepcidin concentration increased due to binding to PRS‐080‐PEG30. Second, we observed a peak in plasma free hepcidin concentration at a later time point than the peak in plasma total hepcidin concentration, which also correlated with the plasma iron concentration. Third, the duration of free hepcidin suppression below baseline increased with higher doses of PRS‐080‐PEG30, despite a stronger accumulation of total hepcidin. Altogether, our observations suggest that the free hepcidin concentration measured in this study is a functional parameter related to free PRS‐080‐PEG30 and iron concentrations in plasma. Moreover, free hepcidin is an essential parameter to effectively study and explain the PKs and PDs of hepcidin antagonism, including the iron response. Furthermore, free hepcidin, in conjunction with plasma iron levels, may be used as a functional parameter to assess the individual patient's response to PRS‐080 treatment and personalize treatment accordingly.

The development of a PK/PD model enabled accurate prediction of the dose and dosing schedule of PRS‐080‐PEG30 in cynomolgus monkey. Indeed, the onset and duration of the experimentally determined iron response mirrored the kinetics of hepcidin suppression predicted in the developed PK/PD model. Robust iron mobilization after single administrations could be observed with PRS‐080‐PEG30 starting at 1 mg·kg−1 in cynomolgus monkey with a dose‐dependent effect duration, while C max of the iron response was capped starting at 3 mg·kg−1 and did not increase at higher doses of up to 120 mg·kg−1. No detectable counter‐regulatory mechanism impaired the iron response upon repeated administration showing that constant inhibition of hepcidin can be achieved if necessary to mobilize iron for an extended period of time through this physiological mechanism to support increased erythropoietic activity.

The NOAEL level of PRS‐080‐PEG30 was determined at 120 mg·kg−1 in cynomolgus monkey, a relatively high‐dose level, which also did not lead to undesirable effects on iron parameters after drug continuation. As such, PRS‐080‐PEG30 is a very promising hepcidin‐antagonist to safely treat iron erythropoiesis of ACD. An important next step is to investigate the hepcidin‐antagonizing potential, increase of erythropoiesis and safety profile of PRS‐080‐PEG30 during (human) ACD, where inflammation and circulating cytokines inhibit erythropoiesis and persistently stimulate hepatic hepcidin synthesis. While inflammation‐induced non‐human primate models of anaemia should be feasible, these are associated with ethical dilemmas and may provide limited additional information. Of note is that rodent models of ACD are not amenable to further evaluate PRS‐080‐PEG30 since based on affinity measurements, PRS‐080‐PEG30 does not bind to rodent hepcidins. In fact, the data presented here, together with safety data of one toxicological species, formed the basis to advance PRS‐080‐PEG30 into clinical development.

To this purpose, the PRS‐080/hepcidin‐specific PK/PD model can be extrapolated to humans by allometric scaling of first‐order rate constants, an approach typically used for scaling of PK/PD models for biotherapeutics (Kagan et al., 2010) and could be further refined by experimental human PK and target engagement data. This may provide a rational basis for the selection of repeat dose levels and dosing regimens in humans, as baseline hepcidin levels and turnover rates are predicted to have a strong impact on the pharmacological response and can vary significantly in different disease populations. PRS‐080‐PEG30 is now advancing towards clinical repeat dosing and may provide a new therapeutic option to alleviate iron retention for the treatment of ACD. The ability to regulate iron in a physiological manner could transform the use of biologics in anaemia‐supportive care as it addresses the root cause of ACD and does necessitate high ESA or high i.v. iron exposure.

Author contributions

A.M.H. and H.G. designed the research, analysed the results, made the figures and drafted the first version of the manuscript; S.T. designed the research and analysed the results; M.K., B.R., C.M.L., G.K. and N.A. performed the research studies and analysed the results; S.A.O. designed the research, analysed the results and wrote the first version of the manuscript; B.M. in collaboration with A.M.H. and H.G. developed the PK/PD model and performed simulations; L.P.A. was involved in designing the research, analysis of results and wrote the first version of the manuscript; and D.W.S. and R.P.L.V.S. designed the research, analysed the results and wrote the subsequent versions of the manuscript. The final version of the manuscript was approved by all authors. All authors take final responsibility for the manuscript.

Conflict of interest

This study does not necessarily reflect the views of the European Commission and in no way anticipates the Commission's future policy in this area. A.M.H., H.G., S.T., M.K., B.R., G.K., H.J.C., N.A., A.A., S.A.O. and L.P.A. are (or were) full‐time employees of Pieris Therapeutics Inc. R.P.L.V.S., C.M.L. and D.W.S. are employees of the Radboudumc that via the http://hepcidinanalysis.com initiative offers high‐quality hepcidin measurements to the scientific, medical and pharmaceutical communities on a fee for service basis.

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 recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1 EC50 values for the PRS‐080 and half‐life extended forms in a solution binding assay.

Table S2 Number of animals per group in determination of plasma iron, TSAT, TIBC and UIBC.

Figure S1 Set of differential equations and a graphical representation that describe the PRS‐080 and hepcidin‐specific PK/PD model. A1f and A2f refer to the amount of free PRS‐080 derivative in the central and peripheral compartment of the PK model respectively. D refers to the dose of anticalin. HP is the amount of free hepcidin in the systemic circulation, and HPA the PRS‐080‐hepcidin complex. k21, k12 and k10 are PK first‐order rate constants, kinh and kout,h are turnover rate constants for hepcidin. k10 serves as a constant for the rate of PRS‐080 elimination from the central compartment and assumed to be equal to the elimination of the PRS‐080‐hepcidin complex. kon and koff are binding constants for the formation and dissociation of the PRS‐080–hepcidin complex. Vc is the volume of distribution of the central compartment for PRS‐080. Ctot denotes the total PRS‐080 concentration, Cf the free PRS‐080 concentration.

Click here for additional data file. (219.1KB, pdf)

Acknowledgements

The authors would like to thank all the technical staff at Pieris Pharmaceuticals for their contributions to this body of work. We thank the http://hepcidinanalysis.com team and in particular Harold Tjalsma and Erwin Wiegerinck for their contribution to the development, validation and interpretation of the free and total hepcidin assays. In addition, thanks to Jakub Jaworski and James Burrows, Queen's University Belfast, for confocal analysis.

This study was funded by Pieris Therapeutics and in part by the European Commission in the context of the FP7 health programme Eurocalin (grant number HEALTH‐F4‐2011‐278408, http://www.eurocalin‐fp7.eu/).

Hohlbaum, A. M. , Gille, H. , Trentmann, S. , Kolodziejczyk, M. , Rattenstetter, B. , Laarakkers, C. M. , Katzmann, G. , Christian, H. J. , Andersen, N. , Allersdorfer, A. , Olwill, S. A. , Meibohm, B. , Audoly, L. P. , Swinkels, D. W. , and van Swelm, R. P. L. (2018) Sustained plasma hepcidin suppression and iron elevation by Anticalin‐derived hepcidin antagonist in cynomolgus monkey. British Journal of Pharmacology, 175: 1054–1065. doi: 10.1111/bph.14143.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1 EC50 values for the PRS‐080 and half‐life extended forms in a solution binding assay.

Table S2 Number of animals per group in determination of plasma iron, TSAT, TIBC and UIBC.

Figure S1 Set of differential equations and a graphical representation that describe the PRS‐080 and hepcidin‐specific PK/PD model. A1f and A2f refer to the amount of free PRS‐080 derivative in the central and peripheral compartment of the PK model respectively. D refers to the dose of anticalin. HP is the amount of free hepcidin in the systemic circulation, and HPA the PRS‐080‐hepcidin complex. k21, k12 and k10 are PK first‐order rate constants, kinh and kout,h are turnover rate constants for hepcidin. k10 serves as a constant for the rate of PRS‐080 elimination from the central compartment and assumed to be equal to the elimination of the PRS‐080‐hepcidin complex. kon and koff are binding constants for the formation and dissociation of the PRS‐080–hepcidin complex. Vc is the volume of distribution of the central compartment for PRS‐080. Ctot denotes the total PRS‐080 concentration, Cf the free PRS‐080 concentration.

Click here for additional data file. (219.1KB, pdf)

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