Overview of preclinical and phase II clinical studies on Pegmolesatide’s long-term erythropoiesis stimulating effect via EPOR-mediated signal transduction

Abstract

Introduction

Anemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO). Pegmolesatide (by Hansoh Pharma) is currently the only marketed long-acting EPO mimetic peptide (EMP) for the treatment of anemia in both dialysis and non-dialysis CKD patients. This paper aimed to explore the long-acting erythropoiesis stimulating molecular mechanism of Pegmolesatide.

Methods

In vitro assays were utilized to assess Pegmolesatide erythropoietin receptor (EPOR) affinity, competitive binding, cell proliferation/survival, apoptosis, cell surface receptor expression, and signal transduction. Pharmacokinetics (PK) and Pharmacodynamics (PD) parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following repeated administration.

Results

In vitro Pegmolesatide demonstrated enhanced binding stability and prolonged residency at EPOR, surpassing erythropoiesis-stimulating agents (ESAs) rHuEPO and Darbepoetin. This sustained EPOR binding facilitated heightened endogenous EPOR expression post-drug withdrawal, maintaining downstream signal transduction pathways (JAK2/STAT5, ERK1/2 MAPK) for erythropoiesis. Pegmolesatide promoted UT-7 cell proliferation & survival and suppressed apoptosis. Following a single 0.08 mg/kg dose of Pegmolesatide in BALB/c mice, reticulocyte count, red blood cells, hemoglobin, and hematocrit persisted at elevated levels 4-6 days after administration. In the two clinical Phase II studies dose-dependent increases in hemoglobin and prolonged response duration were independently observed. Pegmolesatide showed significant PK-PD dual prolongation effects and was well tolerated. Adverse events were mild and manageable, with no reports of severe anaphylaxis.

Discussion

Preclinical and clinical evidence signifies that Pegmolesatide is a unique, potent PEGylated EPO-memetic peptide (EMP) with a prolonged PD efficacy and PK half-life and a good safety-tolerability profile. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play.

Trial registration: Phase IIa clinical study of Pegmolesatide on renal anemia, CTR20140533. Initial Public Notice 14 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. Phase II clinical study of Pegmolesatide on renal anemia, CTR20140539. Initial Public Notice 26 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml.

Introduction

Anemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO) production and impaired release by the damaged kidneys. This condition significantly impacts the long-term survival and quality of life of CKD patients, making its correction a critical clinical objective [1, 2].

EPO is a 30.4 kDa glycoprotein hormone secreted by the kidneys in response to tissue hypoxia. It stimulates the production of red blood cells (RBCs) by regulating erythrocyte production through the activation of the EPO receptor (EPOR). This activation triggers the proliferation and differentiation of erythroid progenitors in the bone marrow, leading to increased reticulocytosis, and ultimately elevating the erythrocyte count and hemoglobin (HGB) concentration in the blood [3,4,5].

Epoetin alfa (Epogen^®) was the first commercially available form of recombinant human erythropoietin (rHuEPO). However, due to its short plasma half-life of 6 to 8 h, rHuEPO must be administered three times per week to effectively raise RBC levels [6]. This frequent dosing regimen can be time-consuming and labor-intensive, often requiring frequent dose adjustments, which may lead to hemoglobin variability [7, 8]. Additionally, long-term rHuEPO treatment can induce the formation of anti-EPO antibodies that neutralize both exogenous and endogenous EPO, resulting in hypo-responsiveness and pure red cell aplasia (PRCA) [9].

To address these issues, various efforts have been made to develop erythropoiesis stimulating agents (ESA) with longer-acting properties or with more convenient administration schedules [10]. EPO derivatives such as darbepoetin and methoxy polyethylene glycol-epoetin beta have been developed through post-translational modifications to rHuEPO to improve plasma half-life. Darbepoetin, for instance, incorporates additional N-linked glycosylation sites, allowing for a dosing schedule of every 1 to 2 weeks in dialysis patients. Methoxy polyethylene glycol-epoetin beta, through PEGylation, extends plasma half-life and dosing intervals [11,12,13,14].

Peginesatide, a long-acting synthetic PEGylated based EPO-mimetic peptide (EMP), was another advancement in this field. Unfortunately, it was found to cause allergenic side effects, including severe anaphylactic reactions in some first-time recipients and as such was withdrawn from the market in 2013 [15].

Pegmolesatide is a novel PEGylated EMP developed by Hansoh Pharmaceutical Group Co., Ltd. Unlike Peginesatide, Pegmolesatide is composed of natural amino acids, reducing the likelihood of generating neutralizing antibodies. As of June 2023, Pegmolesatide has been approved and marketed in mainland China for the treatment of anemia in both dialysis and non-dialysis CKD patients. It is currently the only marketed EPO mimetic peptide. This ESA's ability to sustain erythropoiesis-stimulating activity for up to one month with a single dose and a favorable safety profiles makes it a promising therapeutic option [16]. It remains valuable to consider how Pegmolesatide is able to exert such sustained erythropoiesis stimulating activity upon single dosing up to one month.

We herewith are the first to report on the unique EPOR binding properties and long-acting effects of Pegmolelesatide. We explore its molecular action mechanism in both in vitro and in vivo levels, evaluating its pharmacokinetics (PK) and pharmacodynamics (PD) in BALB/c mice and thru Phase II clinical trials in dialysis and non-dialysis CKD patients with anemia.

Methods

We describe a multifaceted materials and methods approach, comprising of in vitro assays to assess Pegmolesatide EPOR activity in affinity, competitive binding, cell proliferation & survival, apoptosis, cell surface receptor expression, as well as signal transduction. PK and PD parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following single and repeated administration.

In vitro assays

Cell culture and reagents

UT-7 cells and murine BaF3 cells, stable in expressing the EPOR (BaF3-hEPOR), were cultured in RPMI-1640 medium with the addition of 10% FBS, 1% P/S, and 10 ng/mL EPO (Acro, Cat# EPO-H4214). (For supplier details see Supplementary Methods).

Surface plasmon resonance (SPR) for EPOR affinity

Ligand binding analyses were performed using SPR (Biacore™ platform). Protein and peptide binding properties were analyzed using twofold serial dilutions. Affinity data were fitted using Biacore T200 Evaluation software with the “1:1 binding” model. KD = Kd/Ka; residence time = In(2)/kd. (For details, see Supplementary Methods).

SPR competitive binding assay

Pegmolesatide, rHuEPO (ESPO 3000), and EPOR were diluted to 6 nM, 4.8 nM, and 2 nM with running buffer, and mixed in a twofold series to obtain 11 concentrations. Each concentration was incubated with 2 nM EPOR at 4 °C for 18 h, followed by SPR analysis. A sigmoid dose-inhibition curve was plotted to calculate the IC50 value. (For details, see Supplementary Methods).

CellTiter-Glo cell proliferation assay

UT-7 cells were adjusted to 1.2 × 10⁴ cells/mL in complete medium without EPO and cultured in a 96-well plate with various drug concentrations for up to 9 days. Cell viability was assessed using CellTiter-Glo^® 2.0 Cell Viability Assay (Progema, Cat# G9242) and a microplate reader (Bio Tek, H1MFD). (For details, see Supplementary Methods).

Flow cytometry detection of cell apoptosis

UT-7 cells were cultured without EPO and treated with drugs for up to 6 days. Apoptosis was detected using the Annexin V-DY-634 PI Apoptosis Staining/Detection kit (Abcam, Cat# ab214484) and flow cytometry (BECKMAN COULTER, DXFLEX). (For details, see Supplementary Methods).

Flow cytometry detection of EPOR expression on cell surface

UT-7 in logarithmic growth phase was adjusted to a density of 1.2 × 10⁴ cells/mL with complete medium without EPO. UT-7 cells were cultured with drugs for up to 7 days. EPOR expression was analyzed using Human Erythropoietin R PE-conjugated Antibody (R&D, Cat# FAB3072P) and flow cytometry (BECKMAN COULTER, DXFLEX) (For details, see Supplementary Methods).

Western blot analysis

UT-7 or BaF3-hEPOR cells were treated with drugs and lysed. Protein extracts were separated on a 4–20% Criterion™ TGX™ Gel (Bio-Rad, Cat# 5671095) and transferred onto Trans-Blot Turbo Transfer Pack (Bio-Rad, Cat# 1704157). Membranes were probed with specific primary antibodies and visualized using ChemiDoc MP Imaging System (Bio-Rad). (For details, see Supplementary Methods).

Animal models

Single-dose PD study in healthy BALB/c mice

PD study 72 female BALB/c mice were divided into 6 groups and administered different doses of Pegmolesatide, Peptide dimer, or rHuEPO (ESPO 3000). Blood samples were collected at specified intervals and analyzed for hemogram changes.

PK study For the single-dose PK study, BALB/c female mice received injections of Pegmolesatide or Peptide dimer. Blood samples were collected at various time points over 14 days. Serum drug concentration was quantitatively measured using MSD. Pharmacokinetic parameters were analyzed using Phoenix WinNonlin software, including Tmax, Cmax, t1/2, AUC0-t, AUC0-∞, and MRT. The average drug-time curve was plotted based on measured concentrations. This study provides insights into the absorption, distribution, metabolism, and excretion of Pegmolesatide and Peptide dimer in healthy BALB/c mice, essential for understanding their pharmacological profiles and potential clinical applications (For details, see Supplementary Methods).

Phase II clinical trials

Efficacy PK and safety studies in dialysis and non-dialysis CKD patients with anemia

These two multi-centers, single-arm, open-label, dose-finding phase II clinical trials in China strictly adhered to the ethical principles of the Declaration of Helsinki, National Medical Products Administration (NMPA) quality management standards for drug clinical trials, and national China regulatory authority requirements. The aim of these Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, was to evaluate PK-PD characteristics and safety following single and repeated Pegmolesatide administration. (Refer to Fig. 6A for the trial designs). Detailed inclusion criteria, exclusion criteria, dosing regimen, and statistical methods are provided in Supplementary Methods.

Primary efficacy indicators The primary efficacy indicators are the proportion of subjects with HGB values reaching the hemoglobin response target (an increase in HGB relative to baseline of ≥ 1.0 g/dL and HGB ≥ 10.0 g/dL at any time during the trial).

Secondary efficacy indicators Secondary indicators include the mean changes in HGB, and reticulocyte counts relative to baseline and the proportion of subjects responding to the study drug (an increase in HGB relative to baseline of ≥ 1.0 g/dL at any time during the trial).

Blood sampling Blood samples were collected at 10 time points: within 10 min before the first administration, and at 6, 12, 24, 36, 48, 72, 120, 168, and 264 h post-administration. The average drug-time curve was plotted based on the measured blood drug concentrations, and non-compartmental modeling was used to estimate pharmacokinetic parameters, including Tmax, Cmax, t1/2, AUC0-t, AUC0-∞, Vd/F, Kel, MRT, and CL/F.

Safety indicators Safety was assessed by monitoring the incidence and severity of adverse events (AE), as well as changes in body weight, vital signs, clinical examination results, electrocardiograms, and antibody test results.

Statistical analysis

All data were analyzed using GraphPad Prism 10.0 (GraphPad Software Inc., CA, USA) and presented as mean ± standard deviation (SD). Comparisons among multiple groups were conducted using one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. A P-value < 0.05 was considered statistically significant.

Results

Unique EPOR binding properties of Pegmolesatide

Pegmolesatide, a small peptide isolated from a phage display library, has no sequence homology with EPO. It binds to the EPOR receptor with two identical peptide chains simultaneously. PEGylation reduces immunogenicity and prolongs its half-life. The introduction of β-Ala at position 21 minimizes the impact of PEG on binding (Fig. 1A).

Fig. 1

Unique EPOR binding properties of Pegmolesatide. A The sequence and structure of Pegmolesatide. B The EPOR (EC domain, Ig Fc fusion) was immobilized on Biacore chips at 900 RU density. Ligands were then introduced and for the time courses shown, binding was assayed. The peptide dimer refers to the non-PEGylated Pegmolesatide. C Competitive displacement IC₅₀ of Pegmolesatide and ESPO 3000 by EPO. Gradient concentrations of Pegmolesatide and ESPO 3000 were incubated with 2 nM EPOR at 4 ℃ for 18 h; then, each concentration mixture was flowed through the EPO chip. The response value of EPOR binding to EPO was obtained. A nonlinear regression model was used to draw a sigmoid dose-inhibition curve and calculate the IC₅₀ value

To understand Pegmolesatide’s EPOR interactions, Biacore analyses were conducted with an EPOR extracellular domain-IgFc construct. Comparative binding properties of rHuEPO, Darbepoetin, Pegmolesatide, and an unconjugated dimeric peptide (non-PEGylated Pegmolesatide) were analyzed at high and low EPOR densities (Fig. 1B & Supplementary Fig. 1).

SPR results indicate that Pegmolesatide binds EPOR more slowly than other erythropoiesis-stimulating agents but exhibits the longest dissociation time and significantly prolonged residence time (~ 6.8 times longer than rHuEPO YIBIAO), indicating highly stable binding (Table 1 & Supplementary Table 1).

Table 1 Summary ligand-EPOR binding properties including data for association rate constants, dissociation rate constants, equilibrium dissociation constant and EPOR residence time

Gradient concentrations of Pegmolesatide and rHuEPO ESPO 3000 were incubated with 2 nM EPOR at 4 °C for 18 h, then passed through the ligand EPO chip to measure EPOR binding response. Pegmolesatide bound more stably, resulting in less free EPOR and lower response value and IC₅₀ (Fig. 1C). Overall, Pegmolesatide's binding to EPOR is uniquely sustained once ligation is achieved.

Pegmolesatide has a stronger ability to maintain UT-7 cell proliferation than ESPO 3000 at matched biological doses

We assessed the effects of Pegmolesatide on EPOR expression levels by evaluating human erythroid progenitor cell UT-7 growth profiles. First, matched biological dose equivalency was determined for Pegmolesatide and rHuEPO (ESPO 3000) (Fig. 2A). Both Pegmolesatide and ESPO 3000 maintained cell proliferation over 7 consecutive days of treatment. Specifically, the proliferation curves for 1000 ng/mL (20 nM) Pegmolesatide and 4 U/mL ESPO 3000 overlapped; similarly, the curves for 200 ng/mL (4 nM) Pegmolesatide and 0.8 U/mL ESPO 3000 also overlapped. Therefore, in subsequent in vitro experiments, we considered 1000 ng/mL (20 nM) Pegmolesatide and 4 U/mL ESPO 3000 as equivalent biological dose, as well as 200 ng/mL (4 nM) Pegmolesatide and 0.8 U/mL ESPO 3000. Pegmolesatide maintained cell proliferation over 9 consecutive days of treatment, compared to ESPO 3000. Additionally, lower concentrations of Pegmolesatide exhibited a stronger ability to sustain cell proliferation over 8 days compared to the peptide dimer (non-PEGylated Pegmolesatide) (Fig. 2B and Supplementary Fig. 2A).

Fig. 2

Pegmolesatide has a stronger ability to maintain UT-7 cell proliferation than ESPO 3000 at matched biological doses. A and B Effect of drugs on the proliferation of UT-7 cells. UT-7 cells in logarithmic growth phase were adjusted to 1.2 × 10⁴cells/mL with complete medium without EPO. Cells were treated with drugs (Pegmolesatide or ESPO3000 or peptide dimer) for 0, 1, 2, 3, 4, 5, 7, 8, and 9 days. C The effect of drugs on cell survival. After treating cells with drugs for 7 days, centrifuge and discard the supernatant, adjust the cell density to 1.0 × 10⁵cells/mL with complete medium without EPO. Continue to culture the cells for 0, 1, 2, 3, 4, and 7 days. Use Cell Titer-Glo^® 2.0 Cell Viability Assay to detect cell luminescence signals. The magnitude of the signal value is proportional to the number of viable cells. Data are presented as Mean ± SD. N = 3 per time point

After 7 days of treatment with various drugs, cell growth curves were analyzed post-drug withdrawal to assess long-lasting effects. On the first day after withdrawal, all treated groups maintained proliferation, but Pegmolesatide significantly maintained UT-7 cell viability compared to ESPO 3000 and the peptide dimer over time (Fig. 2C).

Pegmolesatide has a superior ability in suppressing UT-7 cell apoptosis compared to ESPO 3000 at a lower matched biological dose

We evaluated Pegmolesatide and other erythropoiesis-stimulating drugs for their ability to inhibit apoptosis in EPO-dependent UT-7 cells. After 24 h in non-serum culture, UT-7 cells were treated with various drug doses. Apoptosis was assessed on days 1, 2, 3, and 6 post-treatments. In the absence of EPO for 24 h, 40% of UT-7 cells underwent apoptosis. Pegmolesatide significantly inhibited apoptosis, though its anti-apoptotic effect at 4 nM and 20 nM did not show dose-dependency. It is possible that higher doses may not lead to further significant effects due to receptor saturation or other pharmacodynamic factors. Compared to 0.8 U/mL ESPO 3000 and 4 nM peptide dimer, 4 nM Pegmolesatide significantly inhibited apoptosis, particularly on day 6, similar to 4 nM Darbepoetin. At 20 nM, Pegmolesatide’s anti-apoptotic effect matched that of 4 U/mL ESPO 3000, 20 nM peptide dimer, and 20 nM Darbepoetin. Overall, Pegmolesatide’s apoptosis inhibition in UT-7 cells was comparable to other drugs within 72 h, but low concentrations of Pegmolesatide maintained this ability after 72 h. (Fig. 3A and 3B).

Fig. 3

Pegmolesatide has a superior ability in suppressing UT-7 cell apoptosis compared to ESPO 3000 at a lower matched biological dose. A and B Inhibitory effect of drugs on apoptosis of UT-7 cells. Cells in logarithmic growth phase were cultured for 24 h without EPO addition. The cell density was adjusted to 5.0 × 10⁵cells/mL using complete culture medium without EPO. Cells were treated with drugs (Pegmolesatide or ESPO3000 or peptide dimer or Darbepoetin) for 0, 1, 2, 3, and 6 days. Data are presented as Mean ± SD. N = 3 per group. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test, ~ versus PBS group, ****P < 0.0001. C The effect of drug withdrawal on apoptosis in UT-7 cells. After treating the cells with drugs for 4 days, centrifuge and discard the supernatant, wash the cells with PBS. Continue to culture the cells for 0, 1, 2, 3, and 4 days. Flow cytometry was used to detect apoptosis. The ordinate represents the proportion of Annexin V-positive cells (the sum of early and late apoptotic cell proportions). Data are presented as Mean ± SD. N = 3 per group. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test, ~ versus EPO group, ***P < 0.001, ****P < 0.0001

Following 4 days of treatment with different drugs and subsequent withdrawal, 20 nM Pegmolesatide significantly reduced apoptosis compared to other drugs (Fig. 3C).

Pegmolesatide enhances cell surface EPOR expression for sustained ESA effects

We investigated the ligand-dependent effects of Pegmolesatide and ESPO 3000 on EPOR expression at matched doses (1000 ng/mL vs. 4 U/mL). UT-7 cells were treated for 7 days, followed by drug withdrawal, and surface EPOR expression was measured. Post-withdrawal, all test drugs increased EPOR expression due to compensatory mechanisms. However, Pegmolesatide-treated cells exhibited significantly higher and more durable EPOR expression compared to those treated with ESPO 3000 and the peptide dimer, as indicated by median fluorescence intensity (Fig. 4A). This enhanced EPOR expression likely contributes to Pegmolesatide’s ability to maintain UT-7 cell viability after drug withdrawal.

Fig. 4

Pegmolesatide enhances cell surface EPOR expression for sustained ESA effects. A Effect of drugs on the expression of EPOR on the surface of UT-7 cells. After treating the cells with drugs for 7 days, centrifuge and discard the supernatant, and adjust the cell density to 3.0 × 10⁵cells/mL with complete medium without EPO. Continue to culture the cells for 0, 1, 2, and 3 days. Cell surface EPOR expression levels were assayed via flow cytometry. Summary data also are shown for replicate analyses (at each day) for median fluorescence intensities. Data are presented as Mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test, ~ versus Pegmolesatide group, *P < 0.05. B Pegmolesatide has a stronger ability to sustain the activation of EPOR downstream signaling. The activation effect of drugs on the downstream signaling of EPOR in Ba/F3-hEPOR cells. BaF3-hEPOR cells in logarithmic growth phase were cultured for 24 h without EPO additives. The cell density was adjusted to 5.0 × 10⁵cells/mL using complete culture medium without EPO. The cells were treated with drugs for 10 min, centrifuged to discard the supernatant, and resuspended in RPMI1640 + 10%FBS + 1%P/S. The cells were cultured for 0 min, 30 min, 1 h, 4 h, 1 day, 2 days, 3 days, and 4 days. Total protein was extracted at each time point. The phosphorylation levels of ERK1/2 and STAT5 were assayed via Western blot

Pegmolesatide binds to EPOR receptors, activating downstream pathways such as STAT5, mediating cell proliferation and differentiation. First, matched biological dose equivalency was determined for Pegmolesatide and rHuEPO (ESPO 3000) in UT-7 and BaF3-hEPOR cell lines (Fig. 2A and Supplementary Fig. 2B). After different drugs were applied to murine BaF3 cells, stable expressing EPOR, for 10 min, the drug was withdrawn, and the changes in downstream signaling pathways were continuously monitored for 4 days. Western blot results showed that the phosphorylation levels of ERK1/2 MAPK and STAT5 peaked 4 h post-withdrawal. Pegmolesatide significantly maintained these phosphorylation levels beyond 4 h, unlike rHuEPO and the peptide dimer, which showed a decline (Fig. 4B & Supplementary Fig. 3). Overall, Pegmolesatide effectively sustains downstream signaling pathway activation, contributing to its prolonged ESA effects.

PK-PD study of Pegmolesatide in healthy mice after single subcutaneous injection

After a single subcutaneous injection of 0.08 and 0.16 mg/kg Pegmolesatide in normal mice, peripheral reticulocytes significantly increased on day 4, and RBC, HGB, and HCT increased significantly on day 6 in a dose-dependent manner. These levels began to decrease on day 8 but remained higher than the Vehicle group until day 14. Pegmolesatide had transient effects on WBC and PLT, with both returning to normal by day 8. In contrast, single doses of the peptide dimer and 10 times the clinical dose of ESPO 3000 showed no efficacy (Fig. 5A & Supplementary Table 2).

Fig. 5

PK-PD study of Pegmolesatide in healthy mice after single subcutaneous injection. A Changes in red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT%) in the serum of mice over time. Seventy-two 7–8-week-old BALB/c female mice were randomly divided into six groups of twelve mice each, and were treated with vehicle, Subcutaneous Injection (SC) injection; 0.08 mg/kg Pegmolesatide, SC injection; 0.16 mg/kg Pegmolesatide, SC injection; 0.08 mg/kg peptide dimer, SC injection; 0.16 mg/kg peptide dimer, SC injection; and 500 IU/kg ESPO3000, iv injection. Whole blood was collected from the orbit on day 4, 6, 8, and 14 after administration. B The concentration of Pegmolesatide in the serum of mice over time. Normal mice were injected with 0.08mpk Pegmolesatide under the skin. Blood samples were collected from the orbital blood of the mice at 0, 0.5, 2, 4, 8, 24, 48, 72, 96, 144, 192, and 336 h after administration. There was a total of 12 time points, with 3 mice at each time point. The average drug-time curve was drawn based on the measured blood drug concentration, and the pharmacokinetic parameters were estimated using a non-compartmental model to obtain the main pharmacokinetic parameters of the drug, in order to fully reflect the characteristics of drug distribution and elimination in mice

The pharmacokinetic results showed a Cmax of 310 ng/mL and an AUC0-∞ of 9197 ng/mL*h for 0.08 mg/kg Pegmolesatide, with a t1/2 of 13.4 h. The peptide dimer's serum concentration was mostly below detection limits, indicating that PEGylation significantly prolongs the half-life (Fig. 5B & Supplementary Table 3).

PK results showed that the serum concentration decreased to 10 ng/mL on day 3 after single administration of 0.08 mg/kg Pegmolesatide, while the EC₅₀ of Pegmolesatide for UT-7 cell proliferation in vitro was 18 ng/mL (data not shown). This indicates that the long-acting effect of Pegmolesatide maintains reticulocyte count, red blood cells, hemoglobin, and hematocrit at high levels for 4-6 days after single administration in mice. In fact, Pegmolesatide is cleared much faster in rodents than in humans (data not shown). Therefore, preclinical data demonstrating a long-acting mechanism will support the clinical use of Pegmolesatide once every 4 weeks.

PK-PD studies in dialysis and non-dialysis CKD patients with anemia

These two Phase II clinical trials included three initial dose groups (0.025 mg/kg, 0.05 mg/kg, and 0.08 mg/kg), with subjects receiving six administrations, each given every four weeks. The first dose was fixed, while subsequent doses could be adjusted to maintain hemoglobin (HGB) levels within 10.0-12.0 g/dL (Fig. 6A).

Fig. 6

PK-PD studies in dialysis and non-dialysis CKD patients with anemia. A A Multi-center, open-label, multi-dose phase II exploratory clinical trial design. Protocol compliant set (PPS): all subjects who complied with the trial protocol, were compliant, and did not use a prohibited medication during the trial. Safety analysis set (SS): all subjects who have used the study drug at least once and have post-dose safety evaluation data. Full Analysis Set (FAS): includes all subjects who have received the study drug at least once and have at least one post-dose pharmacodynamic index measurement. In newly diagnosed CKD dialysis subjects, the efficacy and PK characteristics of Pegmolesatide were investigated at initial doses of 0.025 mg/kg, 0.05 mg/kg, or 0.08 mg/kg. Subjects received subcutaneous injections every 4 weeks, and the dose was adjusted according to hemoglobin levels to achieve a target range of 10.0-12.0 g/dL for 24 weeks of treatment. B Curve of the average change in hemoglobin relative to baseline over time for subjects in each treatment group

Patient compliance and response rates

Dialysis patients: A total of 62 subjects were included in the full analysis set (FAS), and 51 subjects in the per-protocol set (PPS). Among the FAS, 41 subjects achieved the hemoglobin response target, with a compliance rate of 66.1%. The compliance rates for the 0.025 mg/kg, 0.05 mg/kg, and 0.08 mg/kg groups were 70.0%, 63.6%, and 65.0%, respectively. In the PPS, 38 subjects achieved the hemoglobin response target, with a compliance rate of 74.5%, and the rates for the three groups were 76.5%, 73.7%, and 73.3%, respectively.

Overall, 55 subjects in the FAS responded to the study drug, with a response rate of 88.7%. The response rates for the 0.025 mg/kg, 0.05 mg/kg, and 0.08 mg/kg groups were 85.0%, 90.9%, and 90.0%, respectively. In the PPS, 48 subjects responded to the study drug, achieving a response rate of 94.1%, with rates for the three groups being 94.1%, 94.7%, and 93.3% (Supplementary Table 4A).

Non-dialysis patients: A total of 62 subjects were included in the FAS, and 50 subjects in the PPS. Among the FAS, 52 subjects (83.9%) achieved the hemoglobin response target, with compliance rates of 81.0% in both the 0.025 mg/kg and 0.05 mg/kg groups, and 90.0% in the 0.08 mg/kg group. In the PPS, 45 subjects (90.0%) met the target, with compliance rates of 88.2%, 88.2%, and 93.8%, respectively. Additionally, 59 subjects (95.2%) in the FAS and all 50 subjects (100.0%) in the PPS responded to the study drug, with the highest response rate (100.0%) observed in the 0.08 mg/kg group for both sets (Supplementary Table 4B).

Hemoglobin and reticulocyte responses

In the dialysis group, following the first administration, HGB levels in the 0.025 mg/kg dose group decreased slightly at 2- and 4 weeks post-administration. In contrast, HGB levels in the 0.05 mg/kg and 0.08 mg/kg groups increased at 2 weeks, with dose-dependent improvements (average increases of 0.3318 g/dL and 0.648 g/dL, respectively). At 4 weeks, these increases diminished slightly but remained above baseline (average increases of 0.3273 g/dL and 0.3305 g/dL, respectively). From the second administration onwards, with dose adjustments, HGB levels in all dose groups rose significantly compared to baseline. The highest average increases in HGB were 2.0256 g/dL (0.025 mg/kg group), 1.7333 g/dL (0.05 mg/kg group), and 2.0239 g/dL (0.08 mg/kg group) in the FAS. HGB levels reached the target range (10-12 g/dL) around the fifth administration.

In non-dialysis patients, following the first subcutaneous injection of Pegmolesatide, HGB levels increased in all three dose groups by the second week, with the increase proportional to the dose. By week 4, the rise in HGB slightly receded but continued on an upward trend. After the second administration and dose adjustment, the rates of HGB increase in the three groups became similar. Once HGB reached the target range (10-12 g/dL), it remained stable and did not continue to rise. The highest average increases in HGB were 1.8706 g/dL (0.025 mg/kg group), 2.0624 g/dL (0.05 mg/kg group), and 2.7556 g/dL (0.08 mg/kg group) in the FAS. HGB levels reached the target range (10-12 g/dL) around the second administration (Fig. 6B).

In dialysis patients reticulocyte counts increased at 2 weeks post-administration, with dose-dependent changes, and returned to baseline by 4 weeks. In non-dialysis patients, following the first subcutaneous injection of Pegmolesatide, reticulocyte counts increased in all three dose groups by week 2, with higher doses causing greater elevations. By week 4, counts returned to baseline across all groups. After the second dose and subsequent dose adjustments, the rise in reticulocyte counts became similar in all three groups (Supplementary Fig. 4).

Pharmacokinetic analysis

Dialysis patients / Non-dialysis patients The PK studies both included 26 subjects, with (resp. dialysis-/non-dialysis study): 9/9 subjects in the 0.025 mg/kg group, 9/9 subjects in the 0.05 mg/kg group, and 8/8 subjects in the 0.08 mg/kg group. Post-administration, plasma drug concentrations rose with increasing doses. The median T_max values were respectively dialysis-/non-dialysis study as follows: 47.9 h/42.0 h (0.025 mg/kg), 72.0 h/48 h (0.05 mg/kg and 0.08 mg/kg). Geometric mean C_max values were resp. dialysis-/non-dialysis study as follows: 53.3 ng/mL/49.6 ng/mL, 119 ng/mL/127 ng/mL, and 221 ng/mL/175 ng/mL, respectively. Geometric mean T_1/2 values were dialysis-/non-dialysis study as follows 74.2 h/64.5 h, 61.6 h/58.3 h, and 74.9 h/69.7 h, respectively (Fig. 7 and Supplementary Table 5).

Fig. 7

Mean plasma concentration–time curve (mean ± SD) of Pegmolesatide after the first administration. In both studies (A. for non-dialysis patients and B. for dialysis patients), a total of 26 subjects were administered with the first fixed dose. The PK studies showed that the plasma drug concentration increased with increasing dose after the first administration. The blood sampling time points were within 10 min before the first administration and at 6, 12, 24, 36, 48, 72, 120, 168, and 264 h after administration, for a total of 10 blood sampling points

As shown in Table 2, in the dialysis patient group, out of the 62 subjects eligible for the Safety Analysis Subgroup (SAS), 39 (62.9%) reported a total of 114 adverse events (AEs). The incidence of AEs in the 0.025 mg/kg, 0.05 mg/kg, and 0.08 mg/kg dosage groups was 80.0% (16/20), 54.5% (12/22), and 52.6% (10/19), respectively. Note that one subject in the 0.08 mg/kg group was incorrectly assigned a starting dose of 0.06 mg/kg and was thus analyzed separately in the safety analysis. During the study, there were nine instances of serious adverse events (SAEs) reported in eight subjects, representing an incidence of 12.9%.

Table 2 SAEs of dialysis group

Three of these SAEs were deemed possibly related to the study drug: increased liver enzymes, elevated blood pressure, and glaucoma. The remaining SAEs were categorized as possibly unrelated or definitely unrelated, including one case each of congestive heart failure and left femoral neck fracture (possibly unrelated), and two cases of pulmonary infection, and one case each of upper respiratory tract infection and severe anemia (definitely unrelated).

As shown in Table 3, in the non-dialysis group, out of the 62 subjects in the safety population, 45 (72.6%) reported a total of 102 AEs. The incidence of AEs was 71.4% (15/21), 81.0% (17/21), and 65% (13/20) in the 0.025 mg/kg, 0.05 mg/kg, and 0.08 mg/kg dose groups, respectively. Eight secondary serious adverse events occurred in 8 subjects, with an incidence rate of 12.9% (8/62).

Table 3 SAEs of non-dialysis group

These events included one case each of sudden death (probably unrelated), zoster, uremia, incarcerated right hiatal hernia, chronic renal failure, duodenal bulbous ulcer with hemorrhage, acute gastroenteritis, and bilateral pneumonitis (type I respiratory failure), all determined to be unrelated or possibly related to the study.

Overall, in both studies, the AEs were mild and controllable, whilst also similar to those of other marketed ESA. The detailed safety data for Phase II clinical trial involving dialysis and non-dialysis patients is summarized in Supplementary Table 6.

These Phase II clinical trials results demonstrate that Pegmolesatide effectively increases and maintains HGB levels in both dialysis and non-dialysis CKD patients with anemia. The dual prolongation pharmacokinetic and pharmacodynamic profile of Pegmolesatide, evidenced by prolonged HGB responses and reticulocyte counts, support its once-monthly administration. The compliance and response rates and adverse events reporting further validate its favorable efficacy and safety profile in the studied population.

Discussion

This present overview delves into elucidating the long-acting mechanism of Pegmolesatide from the perspective of its EPOR unique binding characteristics. Pegmolesatide is a PEGylated EMP designed and engineered to specifically stimulate EPOR dimer, which regulates erythropoiesis. Pegmolesatide is composed of natural amino acids, reducing the likelihood of generating neutralizing antibodies. Previous studies have demonstrated its advantages, including reduced immunogenicity and an extended duration of action [17]. The peptide component of Pegmolesatide was PEGylated as part of a strategy to enhance PK exposure and PD effect.

There are mainly two subtypes of EPOR, and the stimulation of erythropoiesis is mediated by homodimeric EPOR. Pegmolesatide's symmetrical dimeric structure confers a strong affinity for EPOR (KD = 280 pM), comparable to that of rHuEPO (YIBIAO) and Darbepoetin. Our SPR results reveal that Pegmolesatide exhibits a binding rate constant two times lower than that of an un-PEGylated peptide dimer and rHuEPO (ESPO 3000), indicative of a slower binding process. However, once bound, Pegmolesatide displays the longest dissociation time (Kd decreases by one order of magnitude), resulting in significantly prolonged residence time, demonstrating its high stability (refer to Fig. 1). This phenomenon is further supported by heightened cell surface EPOR expression, as previously observed in studies and illustrated in Fig. 4A [18, 19]. Following drug withdrawal, cells treated with all test compounds exhibit increased surface EPOR expression due to compensatory mechanisms. Notably, cells treated with Pegmolesatide maintain the most persistent expression levels.

The binding of EPO initiates a conformational shift in the extracellular domain of the EPOR, thereby activating a cascade of signal transduction pathways pivotal for erythropoiesis [20]. Subsequently, EPO-EPOR complexes undergo internalization and are directed towards lysosomal degradation facilitated by haematopoietic cell phosphatase, culminating in the reduction of EPOR levels on the cell surface [21]. The ligand may then undergo degradation or be recycled intact back to the cell surface before release [21,22,23]. Extensive research underscores that the interaction between EPO and EPOR significantly amplifies EPOR internalization and breakdown, resulting in a transient period of EPO signaling followed by a rapid decline in cellular EPO sensitivity [21]. In UT-7 cells, the rate of internalization of the rHuEPO-receptor complex was measured at 3.6 per hour, with lysosomal degradation occurring within 15–20 min post-internalization. Consequently, for certain erythropoiesis-stimulating agents, augmenting dosage and frequency may be imperative to sustain downstream effects mediated by EPO-EPOR signaling [21, 23].

Gross and Lodish conducted a comprehensive comparison of receptor binding, internalization, and degradation dynamics between EPO and novel erythropoiesis-stimulating protein (NESP) using hEpoR-expressing cells. Although disparities in receptor binding properties were noted, they surprisingly observed an identical rate of complex internalization. This suggests a potential lack of correlation between EPOR affinity and internalization [23]. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play.

Ligand binding, such as with EPO and Pegmolesatide, induces homodimerization of EPOR chains, activating EPOR and the JAK2/STAT5 and ERK1/2 MAPK pathways [24,25,26]. This signaling cascade promotes differentiation, inhibits apoptosis, and enhances cell proliferation. Pegmolesatide significantly increased phosphorylation levels of JAK2/STAT5 and ERK1/2 MAPK in BaF3-hEPOR cells within 10 min of treatment. The phosphorylation levels peaked 4 h post-drug withdrawal, and Pegmolesatide demonstrated sustained downstream signaling activation longer than other tested drugs (Fig. 4). Compared to previous studies, Pegmolesatide demonstrated superior proliferative, survival, and anti-apoptotic effects in vitro (Figs. 2 and 3) and exhibited long-lasting effects in mice. Despite mouse serum levels decreasing to 10 ng/mL (EC₅₀ of Pegmolesatide for UT-7 cell proliferation in vitro was 18 ng/mL) by day 3 post a single 0.08 mg/kg dose, reticulocyte count, red blood cells, hemoglobin, and hematocrit levels remained highly elevated for 4–6 days. These findings suggest Pegmolesatide’s potential for more sustained therapeutic benefits compared to traditional ESAs, aligning with previous research highlighting the benefits of PEGylation in enhancing drug efficacy and duration [24,25,26].

Pegmolesatide significantly improved anemia in rats with renal insufficiency. 5 out of 6 nephrectomized mice received a single intravenous injection of Pegmolesatide for one week, resulting in peak levels of red blood cells, hemoglobin, and hematocrit, followed by a gradual decline, returning to baseline by the third week (data not shown).

The phase II clinical trials of Pegmolesatide in dialysis and non-dialysis CKD patients with anemia revealed favorable findings regarding its efficacy, PK/PD profiles, and safety. Hemoglobin levels increased and remained elevated above baseline at four weeks. By the fifth administration (18th week of the trial), all groups achieved the target HGB range of 10-12 g/dL. The observed differences in efficacy across various dosage groups may stem from several factors related to renal function, drug clearance, and patient-specific characteristics. First, non-dialysis patients typically reach the target HGB range after the second dose, whereas dialysis patients achieve it only after the fifth dose. This discrepancy is primarily due to differences in renal function and drug clearance. Dialysis patients have more severe kidney dysfunction, leading to faster drug clearance and a more limited duration of drug effects. Consequently, they require more cycles of treatment and dose adjustments to reach the target HGB. In contrast, non-dialysis patients, with better renal function, benefit from endogenous EPO production, enabling quicker and more sustained effects. Second, non-dialysis patients show a greater increase in HGB levels at higher doses (0.08 mg/kg) compared to dialysis patients. This could be due to slower drug clearance in non-dialysis patients, which allows for higher drug concentrations and more effective erythropoiesis. In dialysis patients, the faster drug clearance, compounded by potential bone marrow dysfunction, leads to a less pronounced response despite high doses. Third, in non-dialysis patients, low and medium doses of Pegmolesatide produce a clear dose-dependent effect on HGB levels, likely due to their intact renal function and more responsive bone marrow. However, dialysis patients do not exhibit this dose-dependent response. Factors such as compromised bone marrow function, iron deficiency, chronic inflammation, and the faster clearance of the drug contribute to this lack of a clear dose–response relationship. PK analysis of our studies showed dose-proportional increases in Cmax (Maximum Concentration) and AUC (Area Under the Curve), with a Tmax ranging from 47/42 to 72/48 h and a t1/2 of 61.6/64.5-74.9/69.7 h in dialysis and non-dialysis patients respectively. Patient-specific characteristics, dialysis-related variability, differences in drug absorption & distribution, renal function and drug clearance might contribute to the higher standard deviation in the mean plasma concentration. The dual extension in PK-PD demonstrated its long-lasting effect.

Administration of 0.025, 0.05, 0.08 mg/kg Pegmolesatide doses every four weeks over six cycles resulted in manageable AEs, comparable to other marketed erythropoiesis-stimulating agents (ESAs). A detailed examination of the potential high-risk factors for side effects associated with erythropoiesis-stimulating agents reveals several important considerations. Common side effects of ESAs include elevated blood pressure, hyperkalemia, elevated liver enzymes, and electrocardiogram changes, which are related to the pharmacological actions of these drugs. The differences in side effects between dialysis and non-dialysis patients possibly arise from differences in renal function, drug clearance rates, and the presence of underlying health conditions. Special attention should be given to dosing schedules, drug concentrations, and their overall health status to manage these risks effectively.

In view of this robust and prolonged in vivo activity of Pegmolesatide, usage of this drug for anemia in CKD patients may allow for less frequent dosing, thereby increasing patient convenience. Previous studies have shown that ESAs effectively stimulate erythropoiesis and manage anemia in CKD patients [16, 27, 28]. Pegmolesatide, compares most favorably to these agents. Zhang et al. reported on the Phase III clinical trial, in which 372 patients at 43 dialysis centers in China were randomly assigned (2:1) to receive either Pegmolesatide once every four weeks or epoetin alfa one to three times per week. In the PPS, the mean change in hemoglobin level from baseline to the efficacy evaluation period (calculated as the mean value measured from weeks 17 to 24) was 0.07 g/dL in the Pegmolesatide group and -0.22 g/dL in the epoetin alfa group. The between group difference was 0.29 g/dL (95% confidence interval; 0.11-0.47) confirming non-inferiority of Pegmolesatide to epoetin alfa. AE were common but manageable in both groups, with hypertension being the most frequent treatment-related AE [16]. In summary, the study demonstrated that monthly subcutaneously injection of Pegmolesatide, is safe and effective as conventional epoetin alfa administered one to three times a week in treating anemia in Chinese dialysis patients.

Future preclinical studies will focus on gaining a deeper understanding of the molecular mechanisms underlying the potent pharmacodynamics and favorable pharmacokinetics of Pegmolesatide. Specifically, we plan to investigate the processes of endocytosis, intracellular degradation, and ligand release following Pegmolesatide binding to the erythropoietin receptor. These studies aim to identify key cellular and molecular interactions that contribute to the prolonged activity and enhanced efficacy of Pegmolesatide compared to conventional erythropoiesis-stimulating agents. Understanding these mechanisms will not only provide insights into how Pegmolesatide works at the cellular level, but also help optimize dosing regimens and improve patient outcomes in clinical settings. Additionally, we aim to explore the pharmacodynamic effects of Pegmolesatide in non-CKD anemia models, such as anemia associated with myelodysplastic syndromes, iron deficiency, and cancer, to expand its therapeutic indications. Clinically, future research will focus on validating the pharmacodynamic benefits observed in preclinical models, particularly its long-acting properties and safety profile across diverse patient populations.

Conclusion

Preclinical and clinical studies have demonstrated that Pegmolesatide, a PEGylated EPO-memetic peptide, is a potent stimulator of erythropoiesis with sustained activity and it has been deciphered how it exerts the prolonged anti-anemia effects beyond those explained by its modestly higher-phamacokinetic half-life. Mechanistically, Pegmolesatide exerts the sustained erythropoiesis-stimulating activity in part by promoting EPOR binding stability, maintaining EPOR expression on the cell surface and thus sustaining activation of downstream signaling pathways such as JAK2/STAT5 and ERK1/2 MAPK. This continuous activation inhibits apoptosis and enhances cell proliferation, contributing to the drug's prolonged anti-anemia effects.

These robust findings support Pegmolesatide as a promising therapy for managing both dialysis and non-dialysis CKD patients with anemia, offering prolonged efficacy and a favorable safety profile. It is important to foster future research to provide more insights into long-term safety assessments and broader clinical applications to further establish Pegmolesatide’s role in improving the management of anemia in CKD patients.