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. 2009 Mar 12;150(7):3138–3144. doi: 10.1210/en.2008-1676

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Akihiro Yasoda 1, Hidetomo Kitamura 1, Toshihito Fujii 1, Eri Kondo 1, Naoaki Murao 1, Masako Miura 1, Naotetsu Kanamoto 1, Yasato Komatsu 1, Hiroshi Arai 1, Kazuwa Nakao 1
PMCID: PMC2703521  PMID: 19282381

Abstract

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Administration of C-type natriuretic peptide (CNP) rescues short stature and impaired bone growth of mice model of achondroplasia, indicating therapeutic potential of CNP for skeletal dysplasias.

Skeletal dysplasias are a group of genetic disorders characterized by impairment of bone growth. They comprise a diverse group of disorders that, although individually are relatively rare, together affect a large number of individuals and cause significant morbidity and mortality (1). Achondroplasia (Ach) is the most common skeletal dysplasia with a birth prevalence of approximately one of every 10,000 births (2). Recent studies in molecular genetics demonstrated that Ach is caused by constitutive active mutation of fibroblast growth factor receptor 3 (FGFR3), which results in disturbed proliferation and differentiation of growth plate chondrocytes followed by impaired endochondral bone growth (2,3). Current therapy for Ach generally is limited to distraction osteogenesis (4), an orthopedic procedure (5). Although distraction osteogenesis provides some benefit, it is associated with a significant physical burden and time commitment from patients. As a trial for another treatment of Ach, administration of GH was performed (6) but proved to have minimal effect. New therapeutic strategies for Ach are ardently expected at present.

We previously disclosed that the C-type natriuretic peptide (CNP) and its receptor, guanylyl cyclase-B (GC-B) system is the potent stimulatory system for endochondral bone growth. Both CNP and GC-B are expressed in proliferative and pre-hypertrophic chondrocyte layers of growth plate, and mice with targeted overexpression of CNP in cartilage exhibit prominent skeletal overgrowth (7). On the contrary, mice depleted with CNP (8) or GC-B (9) are dwarf due to impaired endochondral bone growth. Furthermore, loss-of-function mutations affecting GC-B are demonstrated to cause one form of autosomal recessive human skeletal dysplasia, acromesomelic dysplasia, type Maroteaux (AMDM) (10,11), indicating that the CNP/GC-B system is crucial for endochondral bone growth in humans as well as in mice.

In our previous report, we demonstrated that cartilage-specific overexpression of a CNP transgene rescues the impaired endochondral bone growth of a mouse model of Ach with targeted expression of constitutive active FGFR3 in cartilage (12) (hereafter called Ach mice) by restoring the decreased matrix production in Ach growth plates through inhibition of FGFR3-mediated MAPK signaling pathway (7). To elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, here we investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component (SAP) promoter or in those treated with a continuous CNP infusion system. Our results indicate that treatment with systemic CNP can be a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Materials and Methods

Mice

Ach mice (FVB background) were created as reported previously (12), whereas the methods used to generate SAP-CNP-Tg mice (C57BL/6J background) will be reported in detail elsewhere (Kake T., H. Kitamura, Y. Adachi, T. Yoshiaki, T. Tachibe, Y. Kawase, K. Jishage, A. Yasoda, M. Mukoyama, and K. Nakao, submitted for publication). Ach mice and SAP-CNP-Tg mice were crossed to generate double-transgenic Ach/SAP-CNP-Tg mice; female F1 progeny were used for the analyses. ICR mice were purchased from Shimizu Experimental Supplies (Kyoto, Japan). Animal care and all experiments were conducted in accordance with the institutional guidelines of Kyoto University Graduate School of Medicine.

Measurement of plasma CNP concentrations

Plasma CNP-22 concentrations were measured using liquid chromatography-mass spectrometry (13). Because the lower limit of detection of liquid chromatography-mass spectrometry was 0.2 ng/ml plasma CNP-22, RIAs for CNP were performed (14) when CNP concentrations were less than 0.2 ng/ml. The cross-reactivity of CNP-53 in the RIA was about 30% on a molar basis.

Administration of CNP to mice

CNP-22 was purchased from the Peptide Institute (Minoh, Japan) and continuously infused into mice via the jugular vein using a mouse continuous infusion system (Instech Laboratories, Plymouth Meeting, PA) equipped with a syringe pump (Harvard Apparatus, Holliston, MA). Female ICR mice or Ach mice (3 wk old) were treated with vehicle or CNP at the indicated doses for 3 or 4 wk.

Skeletal analysis and histology

Skeletal analysis was performed as previously described (15). Briefly, mice were subjected to soft x-ray analysis (30 kVp, 5 mA for 1 min; Softron Type SRO-M5; Softron, Tokyo, Japan), and the lengths of the bones were measured on the soft x-ray film. To evaluate the bone mineral density at the midshaft of the femora, femora of ICR mice at the end of the treatment period were subjected to peripheral quantitative computed tomography using an XCT Research SA instrument (Stratec Medizintechnik GmbH, Pforzheim, Germany), as previously reported (7). For histological analysis, bones were fixed in 10% formalin in 0.01 m PBS (pH 7.4), decalcified in 5% formic acid, and embedded in paraffin. Five-micrometer-thick sections were sliced and stained with safranin-O, hematoxylin, and eosin. Immunohistochemical studies were performed by using rabbit anti-type II collagen antibody (LSL, Tokyo, Japan), rabbit anti-type X collagen antibody (LSL), goat anti-PTH/PTHrP receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-Indian hedgehog antibody (Santa Cruz Biotechnology), or goat anti-Runx2 antibody (Santa Cruz Biotechnology), and the methods will be described in detail elsewhere (Kake T., H. Kitamura, Y. Adachi, T. Yoshiaki, T. Tachibe, Y. Kawase, K. Jishage, A. Yasoda, M. Mukoyama, and K. Nakao, submitted for publication).

Statistical analysis

Data are expressed as means ± sem or sd. The statistical significance of differences between mean values was assessed using Student’s t test.

Results

Rescue of impaired bone growth of Ach mice by blood-borne CNP from a CNP transgene under the control of SAP promoter

To confirm whether or not blood-borne CNP effectively stimulates endochondral bone growth in mice, we developed transgenic mice in which CNP was overexpressed in the liver under the control of human SAP promoter; compared with wild-type mice, these mice showed increased concentrations of plasma CNP-like immunoreactivity (CNP-LI) (Kake T., H. Kitamura, Y. Adachi, T. Yoshiaki, T. Tachibe, Y. Kawase, K. Jishage, A. Yasoda, M. Mukoyama, and K. Nakao, submitted for publication). Two transgenic mouse lines showed phenotypes similar to those of transgenic mice that specifically overproduce CNP in the growth plate; the mouse line with the milder phenotypes was used as the SAP-CNP transgenic mice (SAP-CNP-Tg mouse) in the present study. The plasma CNP-LI concentration was 7.5 pg/ml in SAP-CNP-Tg mice, whereas it was less than 4 pg/ml in wild-type mice. In SAP-CNP-Tg mice, no significant effects were observed for hemodynamic parameters, including systolic blood pressure [104.7 ± 2.0 and 107.2 ± 2.0 (mean ± sd) mm Hg in SAP-CNP-Tg and wild-type mice, respectively], or for blood biochemical parameters, including electrolyte concentrations (Table 1). SAP-CNP-Tg mice exhibited skeletal overgrowth, and at the age of 10 wk, each bone formed through endochondral ossification was longer and its growth plate was wider in SAP-CNP-Tg mice than in their wild-type littermates. Nevertheless, immunohistochemical analyses of tibial growth plates from 10-wk-old mice revealed that the expression patterns and intensities of chondrocyte differentiation markers including type II and X collagens, PTH/PTHrP receptor, Indian hedgehog, and Runx2 are not changed in the SAP-CNP-Tg growth plate compared with those in the wild-type growth plate (Kake T., H. Kitamura, Y. Adachi, T. Yoshiaki, T. Tachibe, Y. Kawase, K. Jishage, A. Yasoda, M. Mukoyama, and K. Nakao, submitted for publication).

Table 1.

Biochemical parameters of SAP-CNP-Tg mice

TP (g/dl) Alb (g/dl) AST (IU/liter) ALT (IU/liter) Al-P (IU/liter) F-Cho (mg/dl) T-Cho (mg/dl) TG (mg/dl) Glu (mg/dl) Ca (mg/dl) BUN (mg/dl) IP (mg/dl) CRE (mg/dl) Na (mEq/liter) K (mEq/liter) Cl (mEq/liter)
SAP-CNP-Tg
 Mean 4.96 2.95 39.08 5.29 482.57a 17.36 65 15.64 242.3 3.15 24.56 7.55 0.31 147.24 5.68 105.31
 sd 0.3 0.17 7.1 1.59 271.92 5.62 12.64 8.82 49.62 1.67 1.66 1.44 0.09 2.06 1.16 2.07
Wild type
 Mean 5.06 2.96 39.71 6.14 210.14 19.71 69.21 20.79 259.5 3.52 22.6 7.32 0.34 146.79 5.25 105.64
 sd 0.28 0.32 7.92 1.83 106.49 3.1 10.18 9.5 33.59 1.57 4.78 1.36 0.02 1.84 1.31 2.45
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For SAP-CNP-Tg and wild-type mice, n = 8 each. TP, Total protein; Alb, albumin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Al-P, alkaline phosphatase; F-Cho, free cholesterol; T-Cho, total cholesterol; TG, triglyceride; Glu, glucose; BUN, blood urea nitrogen; IP, inorganic phosphorus; CRE, creatinine. 

a

P < 0.01, significant difference against wild type (unpaired t test). 

Ach mice crossed with SAP-CNP-Tg mice (double-transgenic Ach/SAP-CNP-Tg mice) showed no marked difference in body length at birth compared with Ach mice, probably because the SAP-CNP transgene was first expressed after birth as previously reported (16). Nevertheless, at the age of 2 wk, Ach/SAP-CNP-Tg mice were longer than their Ach littermates and were similar in length to their wild-type littermates after 6 wk of age (Fig. 1, A and B). Soft x-ray analysis demonstrated that the impaired growth of bones formed via endochondral ossification, such as the humerus, radius, ulna, femur, and tibia, was rescued in Ach/SAP-CNP-Tg mice; indeed, these bones were longer in Ach/SAP-CNP-Tg mice than in wild-type mice (Fig. 1, C and D). As for cranium, the shortness of longitudinal length in Ach mice was not recovered in Ach/SAP-CNP-Tg mice. The width, of which the growth is dependent on membranous ossification, did not differ among the three genotypes (Fig. 1D). Histological analysis revealed that the narrowed growth plate observed in Ach mice was not found in Ach/SAP-CNP-Tg mice (Fig. 2). Chondrocytes in the growth plate, and in particular hypertrophic chondrocytes, were smaller in Ach mice than in wild-type mice, whereas in Ach/SAP-CNP-Tg mice, they were larger than in wild-type mice. These results strongly indicate that CNP produced in the liver was able to affect the chondrocytes in the growth plate.

Figure 1.

Figure 1

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Crossing Ach mice with SAP-CNP-Tg mice rescued the Ach skeletal phenotype. A, Gross appearances of 10-wk-old wild-type (Wt), Ach, and Ach/SAP-CNP-Tg mice; B, growth curves of Wt, Ach, and Ach/SAP-CNP-Tg mice from 2–10 wk after birth (•, Wt mice; ▴, Ach mice; ▵, Ach/SAP-CNP-Tg mice); C, soft x-ray picture of 10-wk-old Wt, Ach, and Ach/SAP-CNP-Tg mice; D, bone lengths of mice at the age of 10 wk measured on soft x-ray films (white bars, Wt mice; black bars, Ach mice; gray bars, Ach/SAP-CNP-Tg mice).

Figure 2.

Figure 2

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Histological analysis of tibial growth plates from 4-month-old wild-type (Wt), Ach, and Ach/SAP-CNP-Tg mice. Samples were stained with safranin-O, hematoxylin, and eosin. Scale bar, 100 μm.

Effects of iv administration of CNP on endochondral bone growth of wild-type mice

Next we examined the effects of systemic CNP administration on bone growth in wild-type mice. The administration of synthetic CNP-22 to 3-wk-old mice via the jugular vein using a continuous infusion system equipped with a syringe pump resulted in a dose-dependent elevation of the plasma CNP-22 concentration. Plasma concentrations of CNP-22 measured using liquid chromatography-mass spectrometry (13) were 5.0 ± 0.3 and 29.3 ± 5.0 (mean ± sd) ng/ml for infusion rates of 0.1 and 1.0 μg/kg mouse body weight per minute, respectively, whereas the concentration was less than 0.2 ng/ml in vehicle-administered mice. Wild-type mice treated with CNP-22 at a dose of 1 μg/kg · min from the age of 3 wk were obviously longer than vehicle-treated mice after 1 wk iv CNP-22 treatment and were significantly elongated after the 4-wk administration period (Fig. 2, A and B). The naso-anal and naso-tail lengths of mice administered 1 μg/kg · min of CNP-22 were 12 and 10% longer, respectively, than those of vehicle-administered mice at the end of the 4-wk administration period (Fig. 2B). The body weights were not changed between the two groups (data not shown). Soft x-ray analysis revealed skeletal overgrowth in the CNP-22-administered mice (Fig. 3, C and D). Bone mineral density at the midshaft of the femur was not substantially different between the two groups [463 ± 30 and 527 ± 81 mg/ml3 (mean ± sd) in groups administered vehicle and CNP-22, respectively; n = 4 for each group]. The thicknesses of the growth plates of the long bones and vertebrae in the CNP-22-administered mice were greater than that in the vehicle-administered mice (Fig. 4A). The thicknesses of the tibial and vertebral growth plates in CNP-22-administered mice were 31 and 32% greater, respectively, than those in the vehicle-administered mice (Fig. 4B). Among the growth plate layers, the hypertrophic chondrocyte layer was markedly thickened in response to CNP-22 (Fig. 4A).

Figure 3.

Figure 3

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Effects of iv administration of synthetic CNP-22 on bone growth. Continuous administration of vehicle or CNP-22 to female ICR mice was performed for 4 wk beginning 3 wk after birth. A, Gross appearances of vehicle-treated (upper panel) or CNP-22-treated at a dose of 5 μg/kg · min (lower panel) ICR mice at the end of the 4-wk administration period beginning at 3 wk of age. B, Growth curves showing the naso-tail length during the administration of vehicle (•, n = 4) or 1 μg/kg · min CNP-22 (○, n = 4–5). *, P < 0.05. C, Soft x-ray examination of mice treated with vehicle (upper panel) or CNP-22 at a dose of 5 μg/kg · min (lower panel). D, Tibial lengths of ICR mice treated with vehicle (white bar) or 1 μg/kg · min CNP (black bar) for 4 wk. n = 4 (vehicle-treated group), and n = 3 (CNP-treated group). *, P < 0.05.

Figure 4.

Figure 4

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Effects of systemic administration of CNP-22 on the growth plates of ICR mice. A, Histological pictures of the tibial (left two panels) and the vertebral (right two panels) growth plates of ICR mice administered vehicle (left panel in each group) or 1 μg/kg · min CNP (right panel in each group) for 4 wk and stained with safranin-O, hematoxylin, and eosin. Areas in the tibial growth plate between the yellow lines (denoted with an H) represent hypertrophic chondrocyte layers. Scale bar, 100 μm. B, Lengths of tibial (left panel) or vertebral (right panel) growth plates of ICR mice treated with vehicle (white bars) or 1 μg/kg · min CNP (black bars) for 4 wk, measured on histological pictures.

We also investigated the effects of sc administration of CNP-22 using the same continuous infusion system; administration of similar doses, however, did not produce significant effects (data not shown).

Rescue of impaired bone growth of Ach mice by systemic administration of CNP

Based on the pilot study of CNP-22 in wild-type mice, CNP-22 was administered iv to 3-wk-old Ach mice. CNP-22 administration resulted in dose-dependent growth in Ach mice (Fig. 5, A and B). At the end of the 3-wk administration period, iv administration of CNP-22 at a dose of 0.1 μg/kg · min rescued 58% of the shortened naso-anal length phenotype of Ach mice, whereas a dose of 1 μg/kg · min resulted in mice that were longer than wild-type controls (Fig. 5, A and B). Soft x-ray analysis revealed promoted skeletal growth of Ach mice administered CNP at the dose of 1 μg/kg · min (Fig. 5C). Radius, ulna, femur, and tibia bones of Ach mice treated with 0.1 μg/kg · min CNP-22 were similar to those of vehicle-treated wild-type mice, whereas CNP-22 administration at a dose of 1 μg/kg · min resulted in longer bones than those from wild-type mice (Fig. 5D). The width of cranium, of which the growth is dependent on membranous ossification, was not changed between all groups (Fig. 5D). In histology, both the proliferative and hypertrophic chondrocyte layers in the tibial and vertebral growth plates were narrow in Ach mice, whereas they were comparable to those of wild-type mice after the administration of CNP at a dose of 1 μg/kg · min for 3 wk (Fig. 6). Hypertrophic chondrocytes were smaller in Ach mice than in wild-type mice, whereas after CNP-22 administration, they were similar in size to the hypertrophic chondrocytes of wild-type mice (Fig. 6A).

Figure 5.

Figure 5

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Rescue of the Ach skeletal phenotype by CNP-22. Continuous iv administration was performed in 3-wk-old female wild-type (Wt) or Ach mice. A, Gross appearances of vehicle-treated Wt (upper panel), Ach (middle panel), or 1 μg/kg · min CNP-22 administered Ach (lower panel) mice at the end of the 4-wk administration period beginning at 3 wk of age. B, The dose-dependent effect of CNP-22 on the growth of Ach mice. Black circles, black triangles, gray triangles, and white triangles represent the naso-anal lengths of Wt mice treated with vehicle, Ach mice treated with vehicle, Ach mice treated with 0.1 μg/kg · min CNP-22, and Ach mice treated with 1 μg/kg · min CNP-22, respectively. The week after the commencement of treatment is shown on the x-axis. C, Soft x-ray analysis of Wt mice treated with vehicle, Ach mice treated with vehicle, and Ach mice treated with 1 μg/kg · min CNP-22 (from top to bottom) at the end of the 4-wk administration period. D, Bone lengths of Wt or Ach mice administered vehicle or CNP-22 for 4 wk. White bars, Wt mice treated with vehicle; black bars, Ach mice treated with vehicle; dark gray bars, Ach mice treated with 0.1 μg/kg · min CNP-22; light gray bars, Ach mice treated with 1 μg/kg · min CNP-22.

Figure 6.

Figure 6

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Effects of systemic administration of CNP-22 on the growth plates of Ach mice. A, Histological pictures of the tibial (upper panel) and vertebral (lower panel) growth plates of mice treated with vehicle or CNP-22 for 4 wk. Samples were stained with safranin-O, hematoxylin, and eosin. From left to right, growth plate of a wild-type (Wt) mouse treated with vehicle, that of an Ach mouse treated with vehicle, and that of an Ach mouse treated with 1 μg/kg · min CNP-22. Scale bar, 50 μm. B, Lengths of tibial (upper panel) and vertebral (lower panel) growth plates of mice treated with vehicle or CNP-22 for 4 wk, measured on histological pictures. White bars, lengths of growth plates of Wt mice treated with vehicle; black bars, those of Ach mice treated with vehicle; gray bars, those of Ach mice treated with 1 μg/kg · min CNP.

Discussion

The present study demonstrates that systemic administration of CNP is a novel therapeutic strategy for skeletal dysplasias including Ach. Previously, we exhibited that the CNP/GC-B system is a potent stimulatory system of endochondral bone growth in the growth plate; CNP and GC-B are expressed mainly in the pre-hypertrophic chondrocyte layer of the growth plate (8), and mice with targeted overexpression of CNP in the growth plate exhibit prominent skeletal overgrowth (7,17), whereas mice depleted with CNP or GC-B exhibit short stature owing to their impaired bone growth (8,9). We started the translational research of the growth-promoting effect of the CNP/GC-B system on bones into skeletal dysplasias, congenital disorders characterized by severe impairment of bone growth. In our previous report, we exhibited that targeted overexpression of CNP in the growth plate of Ach mice could rescue their impaired bone growth, demonstrating that CNP may be an effective treatment for this disorder (7). In the present study, we have investigated whether or not systemic administration of CNP could be a drug therapy for skeletal dysplasias. We exhibited that blood-borne CNP from a CNP transgene specifically expressed in the liver or by continuous iv administration could recover the shortness and the impaired bone growth observed in Ach mice. We also verified the safety of circulating CNP whose plasma concentration affects bone growth; blood pressure, electrolytes, biochemical markers, and metabolic parameters were not significantly changed in SAP-CNP-Tg and wild-type mice. These results demonstrate that systemic administration of CNP is a possible drug therapy for Ach. Because current therapy for Ach generally is limited to distraction osteogenesis (4), an orthopedic procedure (5), and the benefit of distraction osteogenesis is limited, systemic administration of CNP can be a prominent therapeutic strategy for skeletal dysplasias including Ach.

As for the method of systemic administration of CNP, we also investigated the effects of sc administration of CNP-22 using the same continuous infusion system; administration of similar doses, however, did not produce significant effects. This finding could be a result of degradation of CNP-22 by neutral endopeptidase, which reportedly is abundantly expressed in sc tissues of mice (18). Future studies are necessary to evaluate neutral endopeptidase in sc tissues of humans other than mice.

The results showed that a higher plasma CNP-22 concentration was required to rescue the Ach phenotype in the mice with the infusion pump than in the Ach/SAP-CNP-Tg mice. In addition to CNP-22, CNP-53 is an endogenous form of CNP that has a longer biological half-life than CNP-22 (19). The degree of cross-reactivity of CNP-53 in the RIA for CNP is about 30% on a molar basis, indicating that the plasma of the transgenic mice may contain CNP-53 and/or pro-CNP, a precursor of CNP that shows little cross-reactivity in the RIA. Further studies are necessary to elucidate the molecular forms of the CNP-LI proteins secreted from the liver in SAP-CNP-Tg mice. Another reason for the differences between the SAP-CNP-Tg mice and the infusion pump model may be that the increased levels of circulating CNP are present earlier during the development of the transgenic model, i.e. just after birth in Ach/SAP-CNP-Tg mice, compared with 3 wk of age for mice with the CNP infusion pump.

Safety analysis of the systemic administration of CNP-22 showed no change in systolic blood pressure. This is consistent with the result of our previous report demonstrating that systemically administered CNP in humans did not produce significant effects on hemodynamic parameters, including blood pressure (20). In addition, no adverse effects on bone mineral density or blood biochemistry, including electrolyte concentrations, were observed, indicating that chronic CNP treatment is safe. Nevertheless, safety issues with CNP need further study, because only short-term potential toxicity has been examined in the current study.

The clinical significance of CNP and its receptor, GC-B, in endochondral bone growth has been established in humans, because loss-of-function mutations in the human GC-B gene cause AMDM, a form of skeletal dysplasia (10,11); the skeletal phenotypes similar to those of patients suffering from AMDM are also observed in GC-B knockout (9) and GC-B mutant (21,22) mice. Among human skeletal dysplasias, AMDM is likely to be resistant to treatment with CNP. On the other hand, because spontaneous mutations in the mouse CNP gene (23,24) are known to result in phenotypes identical to those observed in CNP knockout mice (8) and CNP mutant mice are rescued by targeted overexpression of CNP in their cartilage (25), patients with loss-of-function mutations in the CNP gene, which have not been reported to date, will likely be very sensitive to CNP administration. In addition, because administration of CNP could successfully stimulate the endochondral bone growth of wild-type mice, CNP would be potentially used for skeletal dysplasias other than achondroplasia or the putative form of skeletal dysplasia caused by loss-of-function mutations in the CNP gene. Recent progress in molecular genetics has identified mutations in various genes as the causes of skeletal dysplasias (1); therefore, in case we try to use CNP for the treatment of one form of skeletal dysplasia caused by mutations in a certain gene, we might better predict the therapeutic effects by investigating the molecular interactions between the gene product and CNP in endochondral ossification.

In conclusion, we have demonstrated the efficacy and safety of iv administration of CNP-22 for impaired endochondral bone growth in Ach mice. These results suggest that systemic administration of CNP or CNP analogs provides a novel therapeutic strategy for human skeletal dysplasias, including Ach.

Acknowledgments

We thank Dr. D.M. Ornitz (Department of Developmental Biology, Washington University Medical School) for Ach mice. We also thank Yoshihiro Ogawa for fruitful discussions and Shinji Yasuno for technical instruction.

Footnotes

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labor, and Welfare of Japan and the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (19591075), a Grant-in-Aid from the Takeda Science Foundation, and a Novo Nordisk Growth and Development Study Award.

Disclosure Summary: T.F., E.K., M.M., N.K., Y.K. and H.A. have nothing to declare. A.Y. receives grant support (2008.12.1∼2011.11.30) from Chugai Pharmaceutical Co., Ltd. H.K. and N.M. are employed by Chugai Pharmaceutical Co., Ltd. K.N. is an inventor of a related U.S. patent (US6743425) and patent applications in Japan (2003-113116 and 2003-104908), Canada (CA 2398030), and Brazil (BR200203172). N.M. is an inventor of patent application PCT/JP2008/051472 (WO2008093762, applied only in Japan).

First Published Online March 12, 2009

Abbreviations: AMDM, Acromesomelic dysplasia, type Maroteaux; Ach, achondroplasia; CNP, C-type natriuretic peptide; CNP-LI, CNP-like immunoreactivity; FGFR3, fibroblast growth factor receptor 3; GC-B, guanylyl cyclase-B; SAP, serum amyloid P component.

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