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

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

Safety assessment of a novel C-type natriuretic peptide derivative and the mechanism of bone- and cartilage-specific toxicity.

ASB20123, a C-type natriuretic peptide/ghrelin chimeric peptide, was designed as a novel peptide and demonstrated full agonistic activity for natriuretic-peptide receptor B and a significantly longer half-life in plasma compared with the native peptide. We researched the toxicological profile of ASB20123, the correlation between the morphological change of the epiphyseal plate and bone and cartilage toxicity, and biomarkers to detect the toxicity. ASB20123 was systemically administered to male and female rats at daily dose levels of 0.5, 1.5, and 5.0 mg/kg/day for 4 weeks. In this study, toxicity was observed as changes related to bone and cartilage tissues, and no other toxicological changes were observed in all animals. Next, ASB20123 was administered to 12-month-old rats with a little epiphyseal plate. The toxic changes related to bone and cartilage tissues were not observed in any animal with a closed epiphyseal plate, indicating that the toxic changes were triggered by the growth-accelerating effect on the bone and cartilage. Furthermore, we searched for the biomarker related to the bone and cartilage toxicity using rats treated with ASB20123 at doses of 0.005, 0.05, 0.5, and 5.0 mg/kg/day for 4 weeks. A close correlation between necrosis/fibrosis in the epiphysis and metaphysis and thickness of the epiphyseal plate in the femur was confirmed in this study. A decrease in the bone mineral density (BMD) of the femur also was associated with the appearance of bone toxicity. These results indicated that the toxicity of ASB20123 was limited to bone- and cartilage-specific changes, and these changes were triggered by an excessive growth accelerating effect. Furthermore, our data suggested that the thickness of the epiphyseal plate and BMD could be reliable biomarkers to predict bone toxicity

Exogenous C-type natriuretic peptide restores normal growth and prevents early growth plate closure in its deficient rats.

Signaling by C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B, is a pivotal stimulator of endochondral bone growth. We recently developed CNP knockout (KO) rats that exhibit impaired skeletal growth with early growth plate closure. In the current study, we further characterized the phenotype and growth plate morphology in CNP-KO rats, and the effects of exogenous CNP in rats. We used CNP-53, an endogenous form of CNP consisting of 53 amino acids, and administered it for four weeks by continuous subcutaneous infusion at 0.15 or 0.5 mg/kg/day to four-week old CNP-KO and littermate wild type (WT) rats. We demonstrated that CNP-KO rats were useful as a reproducible animal model for skeletal dysplasia, due to their impairment in endochondral bone growth. There was no significant difference in plasma bone-turnover markers between the CNP-KO and WT rats. At eight weeks of age, growth plate closure was observed in the distal end of the tibia and the calcaneus of CNP-KO rats. Continuous subcutaneous infusion of CNP-53 significantly, and in a dose-dependent manner, stimulated skeletal growth in CNP-KO and WT rats, with CNP-KO rats being more sensitive to the treatment. CNP-53 also normalized the length of long bones and the growth plate thickness, and prevented growth plate closure in the CNP-KO rats. Using organ culture experiment of fetal rat tibia, gene set enrichment analysis indicated that CNP might have a negative influence on mitogen activated protein kinase signaling cascades in chondrocyte. Our results indicated that CNP-KO rats might be a valuable animal model for investigating growth plate physiology and the mechanism of growth plate closure, and that CNP-53, or its analog, may have the potential to promote growth and to prevent early growth plate closure in the short stature

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death

<div><p>We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.</p></div

Gross appearance of and growth curves for Δ11 mutant rats.

<p>(A) and (B) Gross appearance of male WT rat and rat with a homozygous Δ11 mutation at two weeks (A) and 14 weeks (B) of age. Black scale bar in each picture denotes 10 mm. (C) Growth curves for Δ11 mutant rats. Upper panels show naso-anal lengths of male (left) and female (right) rats and lower panels exhibit body weight. n = 6, 15, and 10 for male WT, heterozygous Δ11, and homozygous Δ11 rats, respectively; n = 8, 14, and 5 for female WT, heterozygous Δ11, and homozygous Δ11 rats, respectively. *, <i>P</i> < 0.01 vs WT, §, <i>P</i> < 0.01 vs WT, and ¶, <i>P</i> < 0.05 vs WT.</p

Typical examples of H&E-stained specimens of the heart of female WT (left) and CNP KO (homozygous Δ11 mutant, right) rats.

<p>Scale bar shows 1 mm.</p

Histological analysis of the growth plates of CNP KO rats.

<p>(A) Histological pictures of tibial growth plates of three-week-old male WT, homozygous Δ11, and homozygous Δ774 mutant rats. WT rats from Δ774 mutant line were used for this analysis. Upper panels show the results of Alcian blue-H&E staining, while the middle and lower panels show the results of immunohistochemical staining for type II and type X collagens, respectively. (B) and (C) Graphs of the widths of total growth plates (B) and their hypertrophic chondrocyte layers (C). n = 3 in each group and each value is expressed as a ratio (%) of the WT value. *, <i>P</i> < 0.01 vs WT. (D) Histological pictures showing BrdU staining of the tibial growth plates of three-week-old male WT, homozygous Δ11, and homozygous Δ774 mutant rats. (E) Graphs of the number of BrdU positive chondrocytes in the proliferative chondrocyte layers of the growth plates.</p