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 Tmax 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 Cmax 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 T1/2 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.