ASB20123: A novel C-type natriuretic peptide derivative for treatment of growth failure and dwarfism

Abstract

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1-22) and human ghrelin(12-28, E17D). Compared to CNP(1-22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1-22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Fig 1. The amino acid sequences and structures of CNP(1–22), human ghrelin, and ASB20123.

CNP(1–22) is a 22-amino acid (AA) natriuretic peptide. ASB20123 is a 39-AA-designed chimeric natriuretic peptide composed of CNP(1–22) and the 17-AA C-terminal fragment of human ghrelin(12–28). It has a single amino acid exchange as CNP(1–22)ghrelin(12–28, E17D).

Fig 2. Receptor agonist activity of CNP and its derivatives.

(A) NPR-B agonist activity of CNP(1–22), CNP(1–22)/ghrelin(12–28), and ASB20123 was evaluated based on cGMP production in CHO cells stably expressing human NPR-B. (B) NPR-A agonist activity of α-hANP, CNP(1–22), CNP(1–22)/ghrelin(12–28), and ASB20123 was evaluated based on cGMP production in CHO cells stably expressing hNPR-A. (C) GHS-R agonist activity of human ghrelin, CNP(1–22), CNP(1–22)/ghrelin(12–28), and ASB20123 based on the changes in [Ca²⁺]_i in CHO cells stably expressing rat GHS-R1a. Each value represents the mean of duplicate assays.

Fig 3. PK/pharmacodynamic profiles of CNP(1–22) and ASB20123 in rats.

Plasma CNP immunoreactivity-time curves (the upper panels) and cGMP concentrations (the lower panels) after a single iv (20 nmol/kg) or sc (50 nmol/kg) dose of CNP(1–22) (A, B) or ASB20123 (C, D) in anesthetized rats. Each value represents the mean ± SD of 3 rats.

Fig 4. cGMP concentration-time curves in plasma and auricular cartilage after a single sc dose of CNP(1–22) or ASB20123 in mice.

Plasma (A) or auricular cartilage (B) cGMP concentration-time curves after a single sc dose of CNP(1–22) at 1600 nmol/kg or ASB20123 at 200 nmol/kg in mice. Correlation between cGMP concentrations in plasma and in auricular cartilage at each sampling point (C). Each value represents the mean ± SD of 4 mice (A, B) or individual value (C).

Fig 5. Growth curves of female juvenile ICR mice treated with ASB20123 sc during the 8 weeks of the dosing period and the 4 weeks of the washout period.

Body weight (A), body length (B) and tail length (C) data are shown in the upper panels, and the photographs in the lower panel represent the gross appearance of mice at Day 56 (D). Each value represents the mean ± SD of 10 (for the dosing period) or 5 mice (for the washout period). NS: not significant (p > 0.05), *: significant difference (p < 0.05) compared to the control group using Dunnett’s test.

Fig 6

Correlation between plasma CNP immunoreactivity and growth plate thickness of lower limbs [proximal ends of femur (A) and tibia (B), and distal ends of femur (C) and tibia (D)] in rats after sc bolus injections or infusion of ASB20123 at 0, 0.005, 0.015, 0.05, 0.15, and 0.5 mg/kg/day for 7 days. Each value represents the mean ± SD of 4 or 5 rats. The horizontal axis indicates the mean plasma CNP immunoreactivity concentration at 15 min after the 1^st dose for the sc bolus injection groups or mean plasma CNP immunoreactivity on the 2^nd day for the sc infusion groups. The vertical axis indicates the mean percentage of growth plate thickness compared to the respective control groups. Plasma CNP immunoreactivity after sc infusion of ASB20123 at 0.005 mg/kg/day was not detected. NS: not significant (p > 0.05), *: p < 0.05, **: p < 0.01 compared to the control group (0 mg/kg/day) using Dunnett’s test (logarithm conversion).

Fig 7. Increase in body length of male rats receiving sc infusion of ASB20123 at 0.05 and 0.15 mg/kg/day for 12 weeks.

Each value represents the mean ± SD of 4 or 5 rats, **: significant (p < 0.01) compared to the control group using Dunnett’s test. (A) Mean increase in body length after 12 weeks (84 days). (B) Photographs of the exterior appearance on day 84.