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. 2018 Apr;175(7):1054-1065.
doi: 10.1111/bph.14143. Epub 2018 Feb 23.

Sustained Plasma Hepcidin Suppression and Iron Elevation by Anticalin-derived Hepcidin Antagonist in Cynomolgus Monkey

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Free PMC article

Sustained Plasma Hepcidin Suppression and Iron Elevation by Anticalin-derived Hepcidin Antagonist in Cynomolgus Monkey

Andreas M Hohlbaum et al. Br J Pharmacol. .
Free PMC article

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 t1/2 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 t1/2 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.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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
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.
Figure 6
Figure 6
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.

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