C-type Natriuretic Peptide Analog Research Articles

Vosoritide treatment for children with hypochondroplasia: a phase 2 trial

Hypochondroplasia is a rare autosomal dominant skeletal dysplasia due to activating variants in FGFR3. It presents with disproportionate short stature with a wide range of clinical severity. There are currently no approved medications to treat short stature in children with hypochondroplasia. Vosoritide is a C-type natriuretic peptide analog that was recently approved for improving growth in children with achondroplasia. We aimed to evaluate the safety and efficacy of vosoritide in children with hypochondroplasia. We conducted a single-arm, phase 2, open-label trial at a single centre in the USA and enrolled 26 children with hypochondroplasia. The trial consists of a 6-month observation period to establish a baseline annualized growth velocity followed by a 12-month intervention period during which vosoritide is administered daily via subcutaneous injection at a dose of 15μg/kg/day. The trial's co-primary endpoints included the incidence of adverse events and the change from baseline in age-sex standardized annualized growth velocity and height standardized deviation score (SDS) after 12 months of treatment. This trial is registered with ClinicalTrials.gov (NCT04219007). Twenty-four participants with a mean age of 5.86 years received vosoritide therapy. The first participant was enrolled on August 4, 2020, and the final participant completed the 18-month trial on September 8, 2023. Vosoritide was well tolerated with no treatment-related serious adverse events. Injection site reactions occurred in 83.3% of participants. No participants discontinued therapy due to an adverse event. Annualized growth velocity increased by 2.26 standard deviations (SD) and height SDS increased by 0.36 SD during the treatment period versus the observation period. Hypochondroplasia specific height SDS increased by 0.38 SD. There was a 1.81cm/year increase in absolute annualized growth velocity. Vosoritide was safe and effective in increasing growth velocity in children with hypochondroplasia. Efficacy was similar to what has been reported in children with achondroplasia. This study was supported by an investigator-initiated grant from BioMarin Pharmaceutical.

Vosoritide therapy in children with achondroplasia aged 3−59 months: a multinational, randomised, double-blind, placebo-controlled, phase 2 trial

Vosoritide is a recombinant C-type natriuretic peptide analogue that increases annualised growth velocity in children with achondroplasia aged 5-18 years. We aimed to assess the safety and efficacy of vosoritide in infants and children younger than 5 years. This double-blind, randomised, placebo-controlled, phase 2 trial was done in 16 hospitals across Australia, Japan, the UK, and the USA. Children younger than 60 months with a clinical diagnosis of achondroplasia confirmed by genetic testing and who had completed a baseline growth study or observation period were enrolled into one of three sequential cohorts based on age at screening: 24-59 months (cohort 1); 6-23 months (cohort 2); and 0-5 months (cohort 3). Each cohort included sentinels who received vosoritide to determine appropriate daily drug dose, with the remainder randomly assigned (1:1) within each age stratum (except in Japan, where participants were randomly assigned within each cohort) to receive daily subcutaneous injections of vosoritide (30·0 μg/kg for infants aged 0-23 months; 15·0 μg/kg for children aged 24-59 months) or placebo for 52 weeks. Participants, caregivers, investigators, and the sponsor were masked to treatment assignment. The first primary outcome was safety and tolerability, assessed in all participants who received at least one study dose. The second primary outcome was change in height Z score at 52 weeks from baseline, analysed in all randomly assigned participants. This trial is registered with EudraCT, 2016-003826-18, and ClinicalTrials.gov, NCT03583697. Between May 13, 2018, and March 1, 2021, 75 participants were recruited (37 [49%] females). 11 were assigned as sentinels, whereas 32 were randomly assigned to receive vosoritide and 32 placebo. Two participants discontinued treatment and the study: one in the vosoritide group (death) and one in the placebo group (withdrawal). Adverse events occurred in all 75 (100%) participants (annual rate 204·5 adverse events per patient in the vosoritide group and 73·6 per patient in the placebo group), most of which were transient injection-site reactions and injection-site erythema. Serious adverse events occurred in three (7%) participants in the vosoritide group (decreased oxygen saturation, respiratory syncytial virus bronchiolitis and sudden infant death syndrome, and pneumonia) and six (19%) participants in the placebo group (petit mal epilepsy, autism, gastroenteritis, vomiting and parainfluenza virus infection, respiratory distress, and skull fracture and otitis media). The least-squares mean difference for change from baseline in height Z score between the vosoritide and placebo groups was 0·25 (95% CI -0·02 to 0·53). Children with achondroplasia aged 3-59 months receiving vosoritide for 52 weeks had a mild adverse event profile and gain in the change in height Z score from baseline. BioMarin Pharmaceutical.

OR18-5 A Prospective Clinical Trial of Vosoritide in Selected Genetic Causes of Short Stature

Abstract Objectives Vosoritide is a C-type natriuretic peptide analog which binds its receptor on chondrocytes, leading to increased chondrocyte proliferation and differentiation via its inhibition of the ERK1/2-MAPK pathway. It was recently approved for increasing linear growth in children with achondroplasia. Our study aims to assess the safety and efficacy of vosoritide in children with genetic mutations in 6 categories: hypochondroplasia, Rasopathies (including Noonan syndrome), aggrecan deficiency, carriers of heterozygous NPR2 mutations, CNP deficiency, and SHOX deficiency. Methods This is a prospective, Phase II study with a goal N of 35 subjects. All subjects must have a documented genetic mutation in one of the 6 categories, be prepubertal between the ages of 3 and 11 for boys and 3 and 10 for girls, and have a height < -2.25 SD. Subjects are followed for a 6-month observation period to establish a baseline annualized growth velocity (AGV) and then receive daily subcutaneous vosoritide (15 mcg/kg/day) for 12 months. The primary outcomes are rate of AEs and change in AGV from baseline. Pharmacokinetic studies and pharmacodynamic studies, using cGMP production as a pharmacodynamic marker, are completed at Day 1 and Months 6 and 12. Results To date, 27 subjects have enrolled in the trial (20 hypochondroplasia, 3 Noonan, 2 NPR2, 2 Aggrecan). Median baseline height is -3.1 SD (IQR -3.6, -2.6). 14 subjects have initiated on vosoritide, and 10 completed 6 months of therapy. Median increase in AGV was 3.9 cm/yr (IQR 2.2, 4.6). The two subjects with NPR2 mutations had increased AGVs of 4.4 and 9.2 cm/yr. The two subjects with Noonan syndrome had an increase in AGV of 7.5 and 3.3 cm/yr. One subject with Noonan syndrome, a 4-year-old male, had previously been treated with growth hormone (GH) for 2 years. His AGV was 6.7 cm/yr while on GH, 4.1 cm/yr during the 6-month observation period, and 11.6 cm/year during the first 6 months of vosoritide treatment. There were no serious adverse events related to vosoritide treatment. One subject had a brief episode of syncope with her first injection which self-resolved, and she continued on therapy with no further incidents. PK parameters were similar to previously published parameters for vosoritide in children with achondroplasia. In preliminary data, vosoritide Cmax and AUC correlated strongly with peak increase in cGMP (r=0.70, p=0.01;r=0.84, p<0.01, respectively) but not with increase in AGV (r=0.35, p=0.32). Conclusions This is the first clinical trial of vosoritide for children with genetic short stature who do not have achondroplasia. Vosoritide treatment may work as a precision therapy to improve growth in multiple genetic conditions which interact with the ERK1/2-MAPK pathway. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Evaluation of FGFR inhibitor ASP5878 as a drug candidate for achondroplasia

Achondroplasia is caused by gain-of-function mutations in FGFR3 gene and leads to short-limb dwarfism. A stabilized analogue of C-type natriuretic peptide (CNP) is known to elongate bone by interacting with FGFR3 signals and thus is a promising drug candidate. However, it needs daily administration by percutaneous injection. FGFR inhibitor compounds are other drug candidates for achondroplasia because they directly fix the mutant protein malfunction. Although FGFR inhibitors elongate the bone of model mice, their adverse effects are not well studied. In this study, we found that a new FGFR inhibitor, ASP5878, which was originally developed as an anti-cancer drug, elongated the bone of achondroplasia model male mice at the dose of 300 μg/kg, which confers an AUC of 275 ng·h/ml in juvenile mice. Although ASP5878 was less effective in bone elongation than a CNP analogue, it is advantageous in that ASP5878 can be administered orally. The AUC at which minimal adverse effects were observed (very slight atrophy of the corneal epithelium) was 459 ng·h/ml in juvenile rats. The positive discrepancy between AUCs that brought efficacy and minimal adverse effect suggests the applicability of ASP5878 to achondroplasia in the clinical setting. We also analyzed effects of ASP5878 in a patient-specific induced pluripotent stem cell (iPSC) model for achondroplasia and found the effects on patient chondrocyte equivalents. Nevertheless, cautious consideration is needed when referring to safety data obtained from its application to adult patients with cancer in clinical tests.

Bone, Growth Plate and Mineral Metabolism

The skeletal research field continues to develop rapidly and has produced several seminal findings in the last year including advances in the treatment of rare skeletal disorders, and an even deeper understanding into the molecular mechanisms that control skeletal development, endochondral bone formation, mineralization of skeletal tissues and skeletal biology. The targeting of the C-type natriuretic peptide (CNP) pathway and options to directly antagonize the overactivity of the FGFR3 pathway in achondroplasia continues to be a subject of high interest and excitement and we here highlight the double-blind, randomized placebo-controlled phase 3 study of a CNP analogue (vosoritide) in children with achondroplasia. We also highlight the identification of a novel gene for autosomal dominant hypophosphatemic rickets, publication of new growth charts for X-linked hypophosphatemia and two large well-designed paediatric vitamin D trials for the prevention of tuberculosis and asthma exacerbation, respectively.

Non-GH Agents and Novel Therapeutics in the Management of Short Stature.

Short stature is one of the most common reasons for referral to pediatric endocrinologists. The vast majority of short children do not have growth hormone (GH) deficiency or another pathologic process that is interfering with normal growth. While GH has been approved in the US for several etiologies of non-GH deficient short stature, its high cost and need for daily injections represent barriers for many families. Alternative agents for the management of short stature include the use of gonadotropin releasing hormone analogs (GnRHas) to delay puberty, and aromatase inhibitors (AIs) in boys to postpone epiphyseal fusion. The results of studies employing GnRHas as either monotherapy or combined with GH are mixed, and there is a dearth of rigorously designed clinical trials that have followed patients to adult height. While AIs have been found to result in modest increases in adult height in some studies, important questions about their long-term safety exist. The C-type natriuretic peptide analog vosoritide is an experimental agent that is emerging as a potential treatment for a few specific conditions including achondroplasia, although its efficacy in attenuating disproportionality is as yet unproven. While each of these therapeutic strategies holds promise, none are currently considered standard of care and several important questions remain. These include the impact of these interventions on quality of life as well as long-term outcomes.

Evaluation of FGFR inhibitor ASP5878 as a drug candidate for achondroplasia

Achondroplasia is caused by gain-of-function mutations in FGFR3 gene and leads to short-limb dwarfism. A stabilized analogue of C-type natriuretic peptide (CNP) is known to elongate bone by interacting with FGFR3 signals and thus is a promising drug candidate. However, it needs daily administration by percutaneous injection. FGFR inhibitor compounds are other drug candidates for achondroplasia because they directly fix the mutant protein malfunction. Although FGFR inhibitors elongate the bone of model mice, their adverse effects are not well studied. In this study, we found that a new FGFR inhibitor, ASP5878, which was originally developed as an anti-cancer drug, elongated the bone of achondroplasia model male mice at the dose of 300 μg/kg, which confers an AUC of 275 ng·h/ml in juvenile mice. Although ASP5878 was less effective in bone elongation than a CNP analogue, it is advantageous in that ASP5878 can be administered orally. The AUC at which minimal adverse effects were observed (very slight atrophy of the corneal epithelium) was 459 ng·h/ml in juvenile rats. The positive discrepancy between AUCs that brought efficacy and minimal adverse effect suggests the applicability of ASP5878 to achondroplasia in the clinical setting. We also analyzed effects of ASP5878 in a patient-specific induced pluripotent stem cell (iPSC) model for achondroplasia and found the effects on patient chondrocyte equivalents. Nevertheless, cautious consideration is needed when referring to safety data obtained from its application to adult patients with cancer in clinical tests.

C-Type natriuretic peptide analogue therapy in children with achondroplasia

C-Type natriuretic peptide analogue therapy in children with achondroplasia

SUN-101 Support for a New Therapeutic Approach of Using a Low-Dose FGFR Tyrosine Kinase Inhibitor (Infigratinib) for Achondroplasia

Background: Achondroplasia (ACH) is the most common non-lethal skeletal dysplasia. Fibroblast growth factor receptor 3 (FGFR3) plays a crucial role in bone elongation, demonstrated by FGFR3 gain-of-function mutations in individuals with ACH and hypochondroplasia. Multiple therapeutic strategies have been considered for ACH, the most advanced is to employ an analog of C-type Natriuretic Peptide (CNP), which antagonizes the MAP kinase (MAPK) pathway. Here, we evaluated a therapeutic strategy that targets all pathways downstream of FGFR3 (e.g., STAT1), not just MAPK. We hypothesized that a very low dose of the oral selective FGFR1-3 tyrosine kinase inhibitor (TKI) infigratinib (BGJ398) would be able to improve defective bone elongation. We also hypothesized that infigratinib would have greater potency at lower concentrations in an ACH cell line than a CNP analog (e.g., vosoritide) given its effects on multiple downstream pathways. Methods: A mouse model (Fgfr3Y367C/+) mimicking ACH was treated with subcutaneous injections of infigratinib daily (0.2 or 0.5 mg/kg/day) or intermittently (1 mg/kg, every 3 days) for a treatment duration of 15 days (PND1-15). In vivo results were compared with vehicle-treated mutant mice. TAN 4-18-chondrocytes, a human chondrocyte line expressing a heterozygous Y373C FGFR3 mutation, were treated with multiple concentrations of infigratinib and vosoritide and MAPK levels were measured. Results: We observed a significant improvement of the upper (humerus +7%, ulna +11%) and lower (femur +11%, tibia +16%) limbs at 0.5 mg/kg/day, and improvement in the foramen magnum. The effect of infigratinib on bone elongation was reduced with a lower dose (0.2 mg/kg), confirming a dose-response relationship. With intermittent injections of infigratinib (1 mg/kg, every 3 days), gain of growth was significant for all the long bones (+7%) and the size of the foramen magnum was increased. Modification of the growth plate structure, displaying better organization, was also seen. In cell line data, compared with FGF18-treated TAN 4-18-chondrocytes, concentrations of 10-6M to 10-10M infigratinib led to statistically significant (p<0.05) improvements. With vosoritide treatment, a concentration of 10-4M led to statistically significant improvements compared with FGF18-treated chondrocytes (p<0.05), although this was not seen at 10-5M. Conclusions: Preclinical ACH mouse model data indicate that low, as well as intermittent, doses of infigratinib promote growth and can improve the foramen magnum. No apparent toxicity of infigratinib was observed, suggesting that TKI therapy has the potential to be a valuable and relevant option for children with ACH. Furthermore, cell line data indicate that infigratinib showed superior potency over a CNP analog, suggesting that inhibition of multiple FGFR3-related pathways vs MAPK inhibition alone may lead to increased efficacy.

Randomized, Double-Masked, Placebo-Controlled Dose Escalation Study of TAK-639 Topical Ophthalmic Solution in Subjects with Ocular Hypertension or Primary Open-Angle Glaucoma.

PurposeTAK-639 is a topical, nine-amino acid, synthetic, C-type natriuretic peptide analog in Phase 1 development for the treatment of ocular hypertension (OHT) and primary open-angle glaucoma (POAG). TAK-639 is postulated to lower intraocular pressure (IOP) through a novel mechanism of action (MOA) that increases trabecular meshwork outflow. We investigated the safety and tolerability of TAK-639 in subjects with OHT or POAG.MethodsThis was a phase 1, multicenter, randomized, double-masked, placebo-controlled, single- and multiple-dose escalation study. Subjects (aged 18–90 years) with OHT or POAG were randomized 5:2 to TAK-639 or placebo. Three dose levels were planned (0.1%, 0.3%, 0.6% TAK-639), each with four dosing regimens (QD, BID, TID, QID). Safety measures included treatment-emergent adverse events (TEAEs) and ophthalmologic examinations. Pharmacokinetics and pharmacodynamics (reduction of IOP) were also evaluated.ResultsIn total, 63 subjects were randomized and received 0.1%, 0.3% and 0.6% TAK-639, as single dose, QD, or BID, and 0.1% and 0.3% TID. The study was terminated before 0.6% TID or QID dosing cohorts were studied; instead, 0.6% BID was repeated in a new cohort. TEAEs were instillation related and of mild-to-moderate intensity. There were no TEAEs leading to premature discontinuation, and no serious TEAEs. The most common treatment-related TEAEs were instillation site pain and transient corneal staining with fluorescein. There were no clinically significant concerns across dose groups for all other safety measures, including drop comfort, best corrected visual acuity, slit-lamp biomicroscopy, and corneal epithelial integrity. Little or no systemic exposure was observed. There was a marginal reduction in IOP in one cohort at the highest dose (0.6%) and regimen (BID) tested, suggesting biological plausibility of targeting the trabecular meshwork through this mechanism.ConclusionTAK-639 was generally well tolerated up to 0.6% BID. Further non-clinical studies will improve understanding of the MOA and the penetration of TAK-639 to the anterior chamber.

Pharmacokinetics and intraocular pressure–lowering activity of TAK-639, a novel C-type natriuretic peptide analog, in rabbit, dog, and monkey

TAK-639 is a topical, 9–amino acid, synthetic, C-type natriuretic peptide analog in development for the treatment of primary open-angle glaucoma and ocular hypertension. This study investigated the impact of TAK-639 on intraocular pressure (IOP), the levels of TAK-639 in aqueous humor, and the pharmacokinetic/pharmacodynamic relationship of TAK-639 following topical ocular administration to normotensive female Dutch belted rabbits, beagle dogs, and cynomolgus monkeys. In the IOP studies, rabbits (n = 6/group) and dogs (n = 8/group) received a single topical ocular dose of TAK-639 0.03%, 0.1%, 0.3%, or 0.6% in the right eye and vehicle in the left eye; monkeys (n = 8/group) received TAK-639 0.1%, 0.3%, 0.6%, 0.9%, or 1.2% in the right eye only. IOP was measured pre dose and at various time points from 0.5 to 24 h post dose for rabbits, and 1–48 h post dose for dogs and monkeys. To assess exposure in aqueous humor, another set of animals received a single ocular dose of TAK-639 0.03%, 0.1%, 0.3%, or 0.6% (rabbits, n = 20/group; dogs, n = 14/group) or TAK-639 0.3%, 0.6%, or 1.2% (monkeys, n = 10/group) in both eyes. Aqueous humor and plasma were collected at the same post dose time points at which IOP was measured. Aqueous humor and plasma TAK-639 concentrations were measured by liquid chromatography–mass spectrometry, and pharmacokinetic parameters were estimated with non-compartmental analysis. Topical ocular administration of TAK-639 resulted in a dose-dependent decrease in IOP, with maximum mean decreases in IOP ranging from −8.90% to −34.4% in the rabbit, from −16.5% to −26.4% in the dog, and from −3.43% to −13.5% in the monkey. The duration of the IOP-lowering effect was 12 h in the rabbit and monkey and 48 h in the dog. TAK-639 exposure in aqueous humor (both maximum concentration and area under the curve) was also dose dependent, with maximum concentration ranging from 0.152 to 93.6 ng/mL (0.03% and 0.6% doses, respectively) in rabbits, 0.490–13.8 ng/mL (0.03% and 0.3% doses, respectively) in dogs, and 1.16–18.1 ng/mL (0.3% and 1.2% doses, respectively) in monkeys. The pharmacokinetic/pharmacodynamic profile, when fitted to an inhibitory sigmoidal model, demonstrated that TAK-639 exposure in aqueous humor correlated well with IOP reduction in these species. The TAK-639 exposure in aqueous humor at half maximal IOP reduction (EC50) was lower in monkey and dog than in rabbit (0.2 and 0.4 vs. 2.0 ng/mL, respectively). In plasma, quantifiable concentrations of TAK-639 were low and detectable predominantly at early time points. In conclusion, in rabbit, dog, and monkey, a single topical ocular drop of TAK-639 had a significant IOP-lowering effect that correlated well with increases in TAK-639 levels in aqueous humor and resulted in minimal systemic exposure of TAK-639.

Effects of TAK-639, a novel topical C-type natriuretic peptide analog, on intraocular pressure and aqueous humor dynamics in mice

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, and individuals with ocular hypertension are at risk to develop POAG. Currently, the only modifiable risk factor for glaucoma progression is lowering of intraocular pressure (IOP). A novel mechanism for lowering IOP involves activation of the type B natriuretic peptide receptor (NPR-B), the naturally occurring agonist of which is C-type natriuretic peptide (CNP). Being a cyclic peptide of 22 amino acids, CNP does not readily penetrate the cornea and its ocular hypotensive effect requires intraocular injection. TAK-639 is a synthetic, cornea-permeable, 9-amino acid CNP analog has been studied for the treatment of ocular hypertension and POAG. We assessed TAK-639 in a receptor binding profile and the effects of TAK-639 on NPR-B-mediated cyclic GMP production in cultured transformed human trabecular meshwork (TM) cells (GTM-3). We also evaluated the effects of topical ocular administration of TAK-639 on mouse IOP and aqueous humor dynamics. Among 89 non-natriuretic peptide receptors, transporters, and channels evaluated, TAK-639 at 10 μM displaced ligand binding by more than 50% to only two receptors: the type 2 angiotensin receptor (IC50 = 8.2 μM) and the cholecystokinin A receptor (IC50 = 25.8 μM). In vitro, TAK-639 selectively activates NPR-B (EC50 = 61 ± 11 nM; GTM-3 cells) relative to NPR-A (EC50 = 2179 ± 670 nM; 293T cells). In vivo, TAK-639 lowered mouse IOP by three mechanisms: increase in aqueous humor outflow facility (C), reduction in the aqueous humor formation rate (Fin), and reduction in episcleral venous pressure (Pe). The maximum mean IOP decreases from baseline were −12.1%, −21.0%, and −36.1% for 0.1%, 0.3%, and 0.6% doses of TAK-639, respectively. Maximum IOP-lowering effect was seen at 2 h, and the duration of action was >6 h. With TAK-639 0.6%, at 2 h post-dose, aqueous outflow facility (C) increased by 155.8%, Fin decreased by 41.0%, the uveoscleral outflow rate (Fu) decreased by 52.6%, and Pe decreased by 31.5% (all p < 0.05). No ocular adverse effects were observed. TAK-639 is an efficacious IOP-lowering agent, with a unique combination of mechanisms of action on both aqueous formation and aqueous outflow facility. Further study of this mechanism of treatment may optimize pharmacologic outcomes and provide disease management in patients with POAG and ocular hypertension.

Foramen magnum stenosis and midface hypoplasia in C-type natriuretic peptide-deficient rats and restoration by the administration of human C-type natriuretic peptide with 53 amino acids.

C-type natriuretic peptide (CNP)-knockout (KO) rats exhibit impaired skeletal growth, with long bones shorter than those in wild-type (WT) rats. This study compared craniofacial morphology in the CNP-KO rat with that in the Spontaneous Dwarf Rat (SDR), a growth hormone (GH)-deficient model. The effects of subcutaneous administration of human CNP with 53 amino acids (CNP-53) from 5 weeks of age for 4 weeks on craniofacial morphology in CNP-KO rats were also investigated. Skulls of CNP-KO rats at 9 weeks of age were longitudinally shorter and the foramen magnum was smaller than WT rats. There were no differences in foramen magnum stenosis and midface hypoplasia between CNP-KO rats at 9 and 33 weeks of age. These morphological features were the same as those observed in CNP-KO mice and activated fibroblast growth factor receptor 3 achondroplasia-phenotype mice. In contrast, SDR did not exhibit foramen magnum stenosis and midface hypoplasia, despite shorter stature than in control rats. After administration of exogenous CNP-53, the longitudinal skull length and foramen magnum size in CNP-KO rats were significantly greater, and full or partial rescue was confirmed. The synchondrosis at the cranial base in CNP-KO rats is closed at 9 weeks, but not at 4 weeks of age. In contrast, synchondrosis closure in CNP-KO rats treated with CNP-53 was incomplete at 9 weeks of age. Administration of exogenous CNP-53 accelerated craniofacial skeletogenesis, leading to improvement in craniofacial morphology. As these findings in CNP-KO rats are similar to those in patients with achondroplasia, treatment with CNP-53 or a CNP analog may be able to restore craniofacial morphology and foramen magnum size as well as short stature.

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.

C-type natriuretic peptide analog treatment of craniosynostosis in a Crouzon syndrome mouse model.

Activating mutations of fibroblast growth factor receptors (FGFRs) are a major cause of skeletal dysplasias, and thus they are potential targets for pharmaceutical intervention. BMN 111, a C-type natriuretic peptide analog, inhibits FGFR signaling at the level of the RAF1 kinase through natriuretic peptide receptor 2 (NPR2) and has been shown to lengthen the long bones and improve skull morphology in the Fgfr3Y367C/+ thanatophoric dysplasia mouse model. Here we report the effects of BMN 111 in treating craniosynostosis and aberrant skull morphology in the Fgfr2cC342Y/+ Crouzon syndrome mouse model. We first demonstrated that NPR2 is expressed in the murine coronal suture and spheno-occipital synchondrosis in the newborn period. We then gave Fgfr2cC342Y/+ and Fgfr2c+/+ (WT) mice once-daily injections of either vehicle or reported therapeutic levels of BMN 111 between post-natal days 3 and 31. Changes in skeletal morphology, including suture patency, skull dimensions, and long bone length, were assessed by micro-computed tomography. Although BMN 111 treatment significantly increased long bone growth in both WT and mutant mice, skull dimensions and suture patency generally were not significantly affected. A small but significant increase in the relative length of the anterior cranial base was observed. Our results indicate that the differential effects of BMN 111 in treating various skeletal dysplasias may depend on the process of bone formation targeted (endochondral or intramembranous), the specific FGFR mutated, and/or the specific signaling pathway changes due to a given mutation.

C-Type Natriuretic Peptide Analog as Therapy for Achondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) is an important regulator of bone formation. Gain-of-function mutations in the FGFR3 gene result in chondrodysplasias which include achondroplasia (ACH), the most common form of dwarfism, in which skull, appendicular and axial skeletons are affected. The skeletal phenotype of patients with ACH showed defective proliferation and differentiation of the chondrocytes in the growth plate cartilage. Both endochondral and membranous ossification processes are disrupted during development. At cellular level, Fgfr3 mutations induce increased phosphorylation of the tyrosine kinase receptor FGFR3, which correlate with an enhanced activation of its downstream signaling pathways. Potential therapeutic strategies have emerged for ACH. Several preclinical studies have been conducted such as the C-type natriuretic peptide (CNP) analog (BMN111), intermittent parathyroid hormone injections, soluble FGFR3 therapy, and meclozine and statin treatments. Among the putative targets to antagonize FGFR3 signaling, CNP (or BMN111) is one of the most promising strategies. BMN111 acts as a key regulator of longitudinal bone growth by downregulating the mitogen-activated protein kinase pathway, which is activated as a result of a FGFR3 gain-of-function mutation. Preclinical studies showed that BMN111 treatment led to a large improvement in skeletal parameters in Fgfr3Y367C/+ mice mimicking ACH. In 2014, a clinical trial (phase 2) of BMN111 in pediatric patients with ACH has started. This first clinical trial marks the first big step towards real treatment for these patients.

Advances in treatment of achondroplasia and osteoarthritis.

Achondroplasia (ACH) is the prototype and most common of the human chondrodysplasias. It results from gain-of-function mutations that exaggerate the signal output of the fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase that negatively regulates growth plate activity and linear bone growth. Several approaches to reduce FGFR3 signaling by blocking receptor activation or inhibiting downstream signals have been proposed. Five show promise in preclinical mouse studies. Two candidate therapies target the extracellular domain of FGFR3. The first is a decoy receptor that competes for activating ligands. The second is a synthetic blocking peptide that prevents ligands from binding and activating FGFR3. Two established drugs, statins and meclozine, improve growth of ACH mice. The strongest candidate therapy employs an analog of C-type natriuretic peptide (CNP), which antagonizes the mitogen-activated-protein (MAP) kinase pathway downstream of the FGFR3 receptor and may also act independently in the growth plate. Only the CNP analog has reached clinical trials. Preliminary results of Phase 2 studies show a substantial increase in growth rate of ACH children after six months of therapy with no serious adverse effects. A challenge for drug therapy in ACH is targeting agents to the avascular growth plate. The application of gene therapy in osteoarthritis offers insights because it faces similar technical obstacles. Major advances in gene therapy include the emergence of recombinant adeno-associated virus as the vector of choice, capsid engineering to target vectors to specific tissues, and development of methods to direct vectors to articular chondrocytes.

Evaluation of the Therapeutic Potential of a CNP Analog in a Fgfr3 Mouse Model Recapitulating Achondroplasia

Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

A novel method for the large-scale production of PG-CNP37, a C-type natriuretic peptide analogue

Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.