PEGylated Recombinant Human Growth Hormone Jintrolong® Exhibits Good Long-Term Safety in Cynomolgus Monkeys and Human Pediatric Growth Hormone Deficiency Patients

Abstract

Jintrolong^® is a long-acting PEGylated recombinant human growth hormone (PEG-rhGH) developed for weekly injection in patients with pediatric growth hormone deficiency (PGHD). Although PEG modification of therapeutic proteins is generally considered safe, concerns persist about the potential for adverse vacuolation in tissues with long-term exposure to PEG-included therapies, particularly in children. We assessed the safety of Jintrolong^® in cynomolgus monkeys with an examination of vacuolation in the brain choroid plexus (CP) and reported long-term clinical safety data obtained from children with PGHD. The toxicity of Jintrolong^® was assessed following the 52-week administration with doses at 0.3, 1, or 3 mg/kg/week. The levels of vacuolation of CP in animals were dose-dependent and at least partially reversible after a 104- or 157-week recovery period. Vacuolation in the CP epithelium did not lead to obvious subcellular structural or cell functional abnormalities. Compared with the clinical dose of 0.2 mg/kg/week Jintrolong^® in PGHD patients, exposure in monkeys under NOAEL 3 mg/kg/week exhibited safety margins greater than 120.5, the predicted minimum dose to induce vacuolation in monkeys is equivalent to 1.29 mg/kg/week in humans, which is 6.45-fold higher than the clinical dose. The safety data acquired in clinical trials for Jintrolong^® were also analyzed, which included phase III (360 patients), phase IV (3,000 patients) of 26-week treatment, and a follow-up study with treatment lasting for 3 years. There was no statistically significant difference in the incidence of adverse reactions between the Jintrolong^® group and the daily rhGH control group (no PEG), and no new adverse effects (AE) were observed in the Jintrolong^® group at the clinical therapeutic dose of 0.2 mg/kg/week.

Keywords: AE; CNS; GHD; MRI; PEG-rhGH; TEM; choroid plexus (CP); vacuolation.

Figure 1

H&E staining and IHC staining of cynomolgus monkeys CP epithelium with 26-week administration of Jintrolong. (A) Excipient control group with 26-week administration. (B) Jintrolong group with 1 mg/kg/week administration for 26 weeks. (C) Jintrolong recovery group with 1 mg/kg/week administration for 26 weeks and following recovery period of 26 weeks. (D) anti-PEG IHC stain of control group. (E) anti-PEG IHC stain of Jintrolong group with 26 weeks of administration. (F) anti-PEG IHC stain of Jintrolong group after recovery. Arrows in (B, C) indicate cytoplasmic vacuoles in CP epithelium. Arrows in (E) indicate representative anti-PEG staining. Black line: under dose 3mg/kg/week; blue line: under dose 1mg/kg/week; red line: under dose 0. 3mg/kg/week; Dotted line: predicted threshold dose to induce vacuolation.

Figure 2

H&E staining of cynomolgus monkeys CP epithelium with 52-week administration of Jintrolong. (A) Excipient control group. (B) Jintrolong group with 0.3 mg/kg/week administration. (C) Jintrolong group with 1 mg/kg/week administration. (D) Jintrolong group with 3 mg/kg/week administration. Arrows in panels (C) and (D) indicate representative cytoplasmic vacuoles.

Figure 3

H&E staining of cynomolgus monkeys CP epithelium with 52-week administration of Jintrolong and after 104-/157- week recovery. (A) Excipient control group. (B) Excipient control group after 104-week recovery. (C) Excipient control group after 157-week recovery. (D–F) 0.3 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. (G–I) 1 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. (J–L) 3 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. Arrows indicate representative cytoplasmic vacuoles.

Figure 4

Anti-PEG IHC staining of cynomolgus monkeys CP epithelium with 52-week administration of Jintrolong and after 104-/157- week recovery. (A) Excipient control group. (B) Excipient control group after 104-week recovery. (C) Excipient control group after 157-week recovery. (D–F) 0.3 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. (G–I) 1 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. (J–L) 3 mg/kg/week Jintrolong group at 52-week, after 104-week recovery, after 157-week recovery, respectively. Arrow in panel (G) indicates anti-PEG staining in vessel. Arrow in panel (J) indicates representative anti-PEG staining in CP epithelium. Arrow in panel (K) indicate weak anti-PEG staining in recovery slides.

Figure 5

Subcellular imaging of cynomolgus monkeys CP epithelium with 52-week administration of Jintrolong and after 104-/157-week recovery. (A) Excipient control group with 52-week administration. (B) Excipient control group after 104-week recovery. (C) Excipient control group after 157-week recovery. (D–F) 0.3 mg/kg/week Jintrolong group at 52-week administration, after 104-week recovery, after 157-week recovery, respectively. (G–I) 1 mg/kg/week Jintrolong group at 52-week administration, after 104-week recovery, after 157-week recovery, respectively. (J–L) 3 mg/kg/week Jintrolong group at 52-week administration, after 104-week recovery, after 157-week recovery, respectively. Arrows in panels (I–L) indicate PEG in vacuoles.

Figure 6

Anti-ki67 IHC staining of cynomolgus monkeys CP epithelium with 52-week administration of Jintrolong. (A) Anti-ki67 staining of excipient control group. (B) Anti-ki67 staining of 0.3 mg/kg/week Jintrolong group. (C) Anti-ki67 staining of 1 mg/kg/week Jintrolong group. (D) Anti-ki67 staining of 3 mg/kg/week Jintrolong group. Arrows in panels (A, C, D) showed representative cells with positive anti-Ki-67 staining. The scale bar is 100 µm.

Figure 7

MRI and 18F-FDG-PET imaging of cynomolgus monkey brains with 52-week administration of Jintrolong and control after 148- week recovery. (A) MRI imaging for axial source of excipient control group. (B) MRI imaging for sagittal source of excipient control group. (C) MRI imaging for axial source of 3 mg/kg/week Jintrolong group. (D) MRI imaging for sagittal source of 3 mg/kg/week Jintrolong group. (E–H) corresponding 18F-FDG-PET imaging. (I–L) corresponding imaging merged by MRI and PET.

Figure 8

Quantification and Model based prediction of vacuolation recovery in the CP of cynomolgus monkeys. (A) The average vacuole size is dose-dependent, the y axis is the average vacuole size by µm². (B) The average vacuole ratio is dose-dependent, the y axis is the average vacuole ratio in total CP epithelium. (C) The vacuolation recovery quantification by average vacuole area multiple average vacuole ratio of cells in CP epithelium. The vacuolation in CP epithelium is completely reversible in middle dose 1 mg/kg/week Jintrolong group and partially reversible in high dose 3 mg/kg/week Jintrolong group. (D) The modeling and simulation data are based on a pharmacokinetic modeling software package NONMEM (the pharmacokinetic modeling method shown in Model Based Prediction of Vacuolation Formation and Recovery in the Cynomolgus Monkeys and in Clinical Pediatric GHD) and the final graphical data analyzed with R programming. It was predicted to be 8.1 years for vacuolation in high dose 3 mg/kg/week Jintrolong^® group to get completely recovered in cynomolgus monkeys. The predicted minimum dose to induce vacuolation in monkey is 0.55 mg/kg/week. Black line: under dose 3mg/kg/week; blue line: under dose 1mg/kg/week; red line: under dose 0. 3mg/kg/week; Dotted line: predicted threshold dose to induce vacuolation.