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.

Introduction

The C-type natriuretic peptide (CNP) analog is one of the most exciting therapeutic approaches to treat achondroplasia [1]. The binding of CNP to natriuretic-peptide receptor B (NPR-B) inhibits fibroblast growth factor receptor 3 downstream signaling [2], recognized as an important regulator of endochondral bone growth [3]. We recently reported that the exogenous administration of CNP-53 has the potential to stimulate skeletal growth related to short stature, and restore the skull morphology and size of foramen magnum in CNP-KO rats [4, 5]. As a CNP derivative, a clinical trial utilizing BMN-111 is currently proceeding in pediatric patients with achondroplasia [6-8].

We designed ASB20123, a CNP/ghrelin chimeric peptide, as a novel peptide. ASB20123 contains the full-length 22-amino acids of human CNP-22 fused to the 17-amino acids on the C-terminus region of human ghrelin, and the single amino acid is substituted in its ghrelin region. The application of C-terminal part of ghrelin resulted in the higher stability of CNP analogs, compared to that of CNP-22 as the native form; it also improved their bioactivity as stimulators of endochondral bone growth [9-11]. Furthermore, the C-terminal part of the ghrelin includes a BX7B motif, the cluster of basic amino acids might be implicated in the interaction with hyaluronic acid molecules which are the components of extracellular matrix in growth plate [12]. ASB20123, this novel CNP derivative demonstrated full agonistic activity for NPR-B and showed significantly longer half-life in plasma compared with the native forms. A significant and dose-dependent increase in body length was shown in rats after 12 weeks of administration via subcutaneous infusion [13].

CNP is produced in the brain, kidney, bone, blood cells, blood vessels, and heart [14]. NPR-B is expressed in the brain, lung, bone, heart, and ovary. It is also expressed at relatively high levels in fibroblasts and vascular smooth muscle cells [15]. However, the changes that occur after excessive exogenous CNP exposure remain to be clarified. Furthermore, the toxicological profile of the CNP derivative has not been reported previously. In the present study, we evaluated the exhaustive toxicological profile of ASB20123. Furthermore, we researched the relationship between the specific bone and cartilage toxicity and morphological changes of the epiphyseal plate and reliable biomarkers to detect the toxicity.

Materials and methods

Test article

ASB20123 (GLSKGCFGLKLDRIGSMSGLGCVQQRKDSKKPPAKLQPR, C6 and C22 are bound as an intramolecular disulfide bond) was produced by Asubio Pharma Co. Ltd., Japan using a recombinant DNA method in Escherichia coli, purified with high-performance liquid chromatography, and verified by amino acid composition analysis and amino acid sequence analysis [13]. The purity of ASB20123 was 98.4%. Acetate buffer (0.03 mol/L) was added with 10 w/v% sucrose and 1 w/v% benzyl alcohol and used as a vehicle. All chemicals and regents used in the present study were purchased from FUJIFILM Wako Pure Chemical Industries, Ltd., Japan and Otsuka Pharmaceutical Factory, Inc., Japan.

Animals

Sprague-Dawley (SD) rats were purchased from Charles River Laboratories Japan, Inc., Japan and were used for the studies conducted at the Nonclinical Research Center, LSI Medience Corporation, Japan and Asubio Pharma Co., Ltd, Japan. The animals were housed individually in stainless-steel cages in a humidity-, temperature- and ventilation-controlled environment with an automatic 12-h light/dark cycle. They were provided with a standard, pelleted lab chow diet (CRF-1, Oriental Yeast Co., Ltd., Japan) and tap water ad libitum. Rats were acclimatized for at least 1 week prior to the study. Clinical sign was observed at least once a day during the experiment period by trained animal care staff. Throughout all studies, the following criteria were used to exclude an animal from the study and humanely euthanize to prevent undue pain or distress: weight loss (>20%) and moribund condition. No animals were excluded by these criteria, but one animal was found dead on Day 7 of administration period. All animals except for one animal found dead were euthanized and necropsied after blood collection at the end of administration or recovery period. The animals were euthanized by exsanguination from the carotid artery under anesthesia with isoflurane (Mylan Inc., USA). All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of LSI Medience Corporation, Japan and/or Asubio Pharma Co., Ltd., Japan and were approved by the Institutional Animal Care and Use Committee of LSI Medience Corporation, Japan and/or the Committees for Ethics in Animal Experiments of Asubio Pharma Co., Ltd., Japan, respectively.

Study protocol and administration

Toxicity profiling study (study 1)

Twenty male and 20 female rats at 7 weeks of age were randomly divided into 4 groups each with 5 males and 5 females. Dose levels were set at 0.5, 1.5, and 5.0 mg/kg/day. The control animals were treated with the vehicle at the same dosing volume as the test article-treated animals. The dosing volume was set at 1 mL/kg, and the dosing volume for each animal was calculated based on the most recent body weight. Each animal was injected with the dosing formulation subcutaneously once a day for 4 weeks into the back of the animals using a disposable injection needle and syringe. The dose levels were selected based on the result of previous study, in which the rats received at the dose of ASB20123 0.15 mg/kg/day for 12 weeks showed over-growth [13].

Mechanism study (study 2)

Fifteen male and 15 female rats at 12 months of age were randomly divided into 3 groups, each with 5 males and 5 females. Dose levels were set at 0.5 and 5.0 mg/kg/day. Other conditions and procedures were the same as those for study 1.

Biomarker and recovery study (study 3)

Forty male rats at 7 weeks of age were randomly divided into 5 groups with 5 males at the doses of 0, 0.005, 0.05, 0.5, and 5.0 mg/kg/day for biomarker study, and 3 groups with 5 males at the doses of 0, 0.5, and 5.0 mg/kg/day for recovery study withdrawal period for 13 weeks, respectively. Other conditions and procedures were the same as those for study 1.

Observations and examination items

In study 1, clinical observation, measurements of body weight, food and water consumption, ophthalmology, urinalysis, hematology, blood chemistry, measurements of serum alkaline phosphatase (ALP) isozymes and osteocalcin, body length (naso-anal length), bone mineral density (BMD), organ weight, necropsy, and histopathology analyses were conducted. In studies 2 and 3, clinical observation, measurements of body weight, body length, femur bone length, and BMD, necropsy, and histopathology of the femur and tibia were conducted.

Clinical observation

Clinical signs and mortality were observed once or more per day during the administration and recovery period.

Blood chemistry

Blood samples were collected from the posterior vena cava and centrifuged at 1870 × g for 10 minutes to obtain serum samples. The total protein, albumin, A/G ratio, total bilirubin, asparate aminotransferase, alanine aminotransferase, gamma glutamyltranspeptidase, alkaline phosphatase, lactate dehydrogenase, creatine phosphokinase (CPK), total cholesterol, triglycerides, phospholipids, glucose, blood urea nitrogen, creatinine, inorganic phosphorus, and calcium were examined with an auto-analyzer (7170, Hitachi Ltd., Japan), and the sodium, potassium, and chloride were examined with an electrolyte analyzer (EA07, A&T Corporation, Japan).

ALP isozymes and osteocalcin

The remaining serum samples collected for blood chemistry were used for the serum ALP isozyme and osteocalcin measurement. For ALP isozyme measurement, the auto electrophoresis system (Epalyzer 2, Helena Laboratories Co., Ltd., USA) was used. For osteocalcin measurement, an immunoradiometric assay was applied with the Rat Osteocalcin IRMA kit (Immutopics, International, LLC., USA).

Body length (naso-anal length and femur)

At the end of the administration period, naso-anal lengths of each rat were examined with a scale after euthanasia. Femoral lengths were measured with digital calipers after removal at necropsy.

Bone mineral density (BMD)

Bone mineral density (cortical and sponge) of the isolated left femur of each rat was measured using CT scanning (Latheta LCT-200; Hitachi Aloka Medical, Japan).

Histological examination

Whole rat specimens were embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. The thickness of the epiphyseal plate at the proximal end of the femur was measured under a light microscope. It was measured at nine sites for the proximal end of the femur. The average thickness was considered the epiphyseal plate thickness for each rat.

Statistical analysis

Data were analyzed for homogeneity of variance by Bartlett’s test. If the variance was homogeneous, Dunnett’s test was performed for multiple comparisons between the control group and each test article group. If the variance was heterogeneous by Bartlett’s test, Steel’s test was performed for multiple comparisons between the control group and each test article group. A two-tailed test was used as Bartlett’s test, and P values less than 0.05 were considered statistically significant.

Results

Toxicity profile of ASB20123 in study 1

In the clinical observation, abnormal gait appearing as a shuffling gait of the hind limbs was observed in all the test article-treated groups at the dose level of 0.5 mg/kg/day or more at the 3rd week of administration and later. The number of animals exhibiting these symptoms increased dose-dependently. The BMD values of both cortical and trabecular bone in the femur were significantly low in all the test article-treated groups compared with the control group (Fig 1).

Fig 1

BMD of both the cortical and trabecular bone in the femurs of male (A) and female (B) rats treated subcutaneously with ASB20123 for 4 weeks in study 1.

Each value represents the mean ± SD of 5 rats, ** P < 0.01 vs. vehicle-treated group by Dunnett’s multiple comparison test.

The results of histopathological examination are shown in S1 Table, and representative histopathological findings for the proximal femoral bone in rats are shown in Fig 2. The test article-related changes were observed in the bone/cartilage tissues as follows. In the femur, thickening of the epiphyseal plate was observed at the dose levels of 0.5 mg/kg/day or more. This change involved both the proximal and distal portions of the femur, was intense in the proximal portion, and was concomitant with increases in the primary bone and osteoblasts. In the proximal portion, there was necrosis of the epiphysis/metaphysis, fibrosis in the marrow of the head, and ectopic chondrogenesis/osteogenesis. In the tibia, thickening of the epiphyseal plate and an increase in the primary bone were observed in all the test article-treated groups. These changes involved both the proximal and distal portions of the tibia and were intense or highly frequent in the distal portion. There were also increases in osteoblasts in the proximal and distal portions in males, and necrosis of the epiphysis/metaphysis and inflammatory changes in the surrounding tissues in the distal portion.

Fig 2

Representative histopathological findings in the proximal femoral bone in rats in study 1.

(A) Vehicle group (× 40). (B) Vehicle group (× 100). (C) 0.5 mg/kg/day group (× 40). (D) 0.5 mg/kg/day group (× 100). Bidirectional arrows indicate the width of the epiphyseal plate. Arrows indicate the necrosis of cartilage/osseous tissues. Scale bars represent 200 μm.

The changes in body length, CPK, ALP activity, ALP-isozyme fraction, and osteocalcin values are shown in Fig 3. Significantly high values of body length were shown in males at the dose levels of 5.0 mg/kg/day and in females at 0.5 mg/kg/day and more, compared with the vehicle control group. The CPK activity was significantly high or showed a tendency toward higher in all test article treated groups. The ALP activity was significantly high in males at the dose levels of 1.5 and 5.0 mg/kg/day. Although there was no statistically significant difference, the same tendency was observed in males at the dose levels of 0.5 mg/kg/day and in females at 1.5 and 5.0 mg/kg/day. The ALP-isozyme fraction 3 was significantly high in males and females at the dose levels of 5.0 mg/kg/day compared with the vehicle control group, and the same tendency was observed in males administered 0.5 and 1.5 mg/kg/day and females administered 1.5 mg/kg/day. Meanwhile, there were no changes in other items of blood chemistry and the osteocalcin concentrations in any group compared to the control group.

Fig 3

Effects of ASB20123 on the body length (A), CPK activity (B), ALP and ALP-isozyme fraction activity (C), and osteocalcin value (D) in rats treated subcutaneously for 4 weeks in study 1.

Each value represents the mean ± SD of 5 rats, * P < 0.05, ** P < 0.01 vs. vehicle-treated group by Dunnett’s multiple comparison test.

No changes were observed in any group in body weight, food and water consumption, ophthalmology, urinalysis, hematology, necropsy, and organ weight.

Mechanism of the specific bone and cartilage toxicity in study 2

ASB20123 was administered to the 12-month-old rats. One animal was found dead on Day 7 due to the formation of a tumor in the duodenum. The epiphyseal plate closure was observed in all observation sites of the vehicle group of the aged rats, except for the proximal tibia in 2 female rats. The number of rats with the remaining epiphyseal plate in the treatment group was larger than that in the vehicle group. Increases in osteoblasts and primary bone and degeneration/necrosis in the epiphysis and metaphysis were observed in some animals with the epiphyseal plate, but these findings were not observed in animals with a closed epiphyseal plate (Table 1). No test article-related changes were observed in the clinical observation, body weight, femur bone length, and BMD, but abnormal gait was observed in only 1 animal in the clinical observation in the 0.5 mg/kg/day dosage group.

Table 1

Histopathological findings in the femur and tibia in study 2.

	Sex		Male			Female
	Group	Vehicle		ASB20123	Vehicle	ASB20123
Organs / Tissues	Dose (mg/kg/day)	0	0.5	5.0	0	0.5	5.0
Findings*	No. of animals	5	5	4#	5	5	5
Femur (proximal)
Epiphyseal plate closure		5	4	4	5	3	0$
Thickening, epiphyseal plate		-	1 (+)	-	-	2 (+/++)	1$ (++)
Increase, osteoblast and primary bone		-	-	-	-	2 (+)	-
Degeneration/necrosis, epiphysis/metaphysis		-	-	-	-	1 (+)	5 (+/+++)
Femur (distal)
Epiphyseal plate closure		5	5	2	5	2	1
Thickening, epiphyseal plate		-		2 (+++)	-	3 (+/++)	4 (++/+++)
Increase, osteoblast and primary bone		-	-	2 (+)	-	3 (+)	4 (+/++)
Tibia (proximal)
Epiphyseal plate closure		5	0	0	3	0	0
Thickening, epiphyseal plate		-	5 (+)	4 (+)	-	1 (+)	5 (+)
Increase, osteoblast and primary bone		-	3 (+)	4 (+)	-	5 (+)	5 (+)
Tibia (distal)
Epiphyseal plate closure		5	5	5	5	5	5

Grades: -, normal; +, slight; ++, moderate; +++, severe; +/++, slight to moderate; +/+++, slight to severe; ++/+++, moderate to severe. The numbers of animals with histopathological findings are listed. Vehicle: 0.03 mol/L acetic acid buffer solution (pH 4) containing 10 w/v% sucrose and 1 w/v% benzyl alcohol.

*: No test article-related changes were observed in any animal without an epiphyseal plate.

#: One animal was found dead on Day 7 due to the formation of a tumor in the duodenum.

$: The findings of epiphyseal plate were not evaluated in the proximal femoral bone of 4 rats, because the specimen did not have the target tissue.

Searching for the biomarker related to the bone and cartilage toxicity and evaluating for its recovery in study 3

The thickness of the epiphyseal plate, femur bone length, and bone mineral density of the femur were measured in the rats treated with ASB20123 at doses of 0.005, 0.05, 0.5, and 5.0 mg/kg/day for 4 weeks. The epiphyseal plate thickness increased in a dose-dependent manner, and the toxic findings in the epiphysis and metaphysis were observed only in individuals with thickening of more than 200 μm of the epiphyseal plate. A decrease in the BMD in the femur also reflected the appearance of bone toxicity. In contrast, a correlation between the appearance of bone toxicity and body or femur lengths was not observed (Fig 4).

Fig 4

The correlation between bone and cartilage toxicity and several parameters in study 3.

The thickness of the epiphyseal plate of the femur (A), body length (B), and bone length of the femur (C). The BMD of the cortical bone (D) and trabecular bone (E) in the femurs is shown. Bone toxicity observed in each animal is shown in the colored circle, the open circle represents no toxicity, orange indicates slight toxicity, and red indicates severe toxicity. Each bar represents the mean of 5 rats.

The recovery of low BMD value in the femur and bone toxicity observed in the rats treated with ASB20123 for 4 weeks were assessed after 13 weeks withdrawal period. The results of clinical observation, measurements of body weight, body length, femur bone length, BMD, necropsy, and histopathology of the femur and tibia after 4 weeks administration period were similar to those of study 1 (Fig 5A and S2 Table). At the end of the recovery period, necrosis of the epiphysis/metaphysis in the proximal portion of femur and deformation in distal tibia end joint were observed in some individuals (1 or 2 animals/groups) of ASB20123 treated groups (S3 Table). The BMD values of cortical bone were increased during the recovery period from 1112.1 mg/cm3 to 1305.1 mg/cm3 in the control group, from 1079.7 mg/cm3 to 1290.6 mg/cm3 in the 0.5 mg/kg/day group, from 1009.6 mg/cm3 to 1282.4 mg/cm3 in the 5 mg/kg/day group, respectively, it was significantly low in the 5.0 mg/kg/day group. The BMD values of trabecular bone were changed during the recovery period from 456.0 mg/cm3 to 481.1 mg/cm3 in the control group, from 397.7 mg/cm3 to 396.0 mg/cm3 in the 0.5 mg/kg/day group, from 324.6 mg/cm3 to 395.5 mg/cm3 in the 5 mg/kg/day group, respectively, it was significantly low in the 0.5 and 5.0 mg/kg/day group (Fig 5B).

Fig 5

BMD of both the cortical and trabecular bone in the femurs of rats treated subcutaneously with ASB20123 for 4 weeks followed by 13 weeks recovery period in study 3.

(A) At the end of administration period. (B) At the end of recovery period. Each value represents the mean ± SD of 5 rats, ** P < 0.01, * P < 0.05 vs. vehicle-treated group by Dunnett’s multiple comparison test.

Discussion

ASB20123 is a CNP derivative, and this peptide stimulates bone growth through proliferation and differentiation of chondrocytes [13]. In this study, ASB20123 was administered to male and female rats at daily dose levels of 0.5, 1.5, and 5.0 mg/kg/day for 4 weeks to investigate its toxicity. In this study, toxic changes were observed in the bone and cartilage tissues, and no other toxic changes were observed in all animals.

In the histopathological examination, thickening of the epiphyseal plate, which was frequently accompanied by increases in the primary bone and osteoblasts, was observed in the femur and tibia in all the test article-treated groups. Similar cartilage thickening was also detected in the temporomandibular joint and sternum. These findings were characteristic, prominent, and therefore considered to be primary changes based on the pharmacological action of ASB20123. In relation to the above osseous changes, the body length was extended in all the test-article treated groups, and serum ALP-isozyme fraction activity increased, since it is derived from bone and is elevated in the serum as a result of various bone diseases and bone growth. In addition to the above changes due to a pharmacological action of ASB20123, the following toxicity findings were observed in this study. In the proximal portion of the femur, there was necrosis of the epiphysis/metaphysis, fibrosis in the marrow of the head, and ectopic chondrogenesis/osteogenesis in all the test article-treated groups. Necrosis of the trabecula/marrow in the epiphysis of the head and degeneration/necrosis of the peripheral muscle fibers was also sporadically observed. In the tibia, there was necrosis of the epiphysis/metaphysis and inflammatory changes in the surrounding tissues in the distal portion. All these findings associated with the above-mentioned primary changes were probably inflammatory, ischemic, or reactive due to the physical or physiological stimulation following thickening of the epiphyseal plate. These changes were intense or localized in the proximal portion, especially in the head of the femur and in the distal portion of the tibia, suggesting a possibility that the landing-shock on the hind limbs during walking/moving accelerates these bone/cartilage changes. Clinical signs revealed shuffling gait of the hind limbs. This symptom was considered to be caused by the excessive cartilage increase in the distal tibia. The increase of CPK activity might be ascribable to degeneration or necrosis of the muscle with rapid extension of various bones; however, the increase of CPK activity was slight and was judged not to be of serious toxicological significance.

To research the involvement of the epiphyseal plate in the bone-related changes, ASB20123 was administered to the 12-month-old rats with a little epiphyseal plate. The epiphyseal plate closure was observed in all examination sites of the vehicle group, except for in the proximal tibias of 2 female rats. The number of rats with the remaining epiphyseal plate in the test article-treated group was larger than that in the vehicle group. It was suggested that the administration of ASB20123 delayed the epiphyseal plate closure, and this result corresponded to those of our previous reports [4, 5]. An increase in osteoblasts and primary bone and degeneration/necrosis in the epiphysis and metaphysis were not observed in any animal with a closed epiphyseal plate. These results indicated that the toxic changes in the bone and cartilage tissues were triggered by the excessive growth-accelerating effect based on the pharmacological action of ASB20123.

Biomarkers related to bone and cartilage toxicity, thickness of the epiphyseal plate, body length, femur bone length, and BMD of the cortical and trabecular bone in the femur were measured in the rats treated with ASB20123 at the doses of 0.005, 0.05, 0.5, and 5.0 mg/kg/day for 4 weeks. A reliable correlation between necrosis/fibrosis in the epiphysis and metaphysis and thickness of the epiphyseal plate of the femur was confirmed in this study. A decrease in BMD in the cortical bone in the femur was also relevant to the bone and cartilage toxicity. These parameters might be good markers to predict bone- and cartilage-specific toxic changes, because the thickness of the epiphyseal plate can be monitored using radiographic examination, computed tomography, and magnetic resonance imaging in humans [16, 17].

While the decrease of BMD value could be a reliable biomarker to predict bone- and cartilage toxicity, we need to take care of it in the treatment of achondroplasia, because the lower BMD value of achondroplasia population was reported [18]. At the end of the recovery period for 13 weeks, necrosis of the epiphysis/metaphysis in the proximal portion of femur and deformation in distal tibia end joint were still observed in some individuals of ASB20123 treated groups. The incidence of bone toxicity in femur and tibia decreased and considered reversible. Meanwhile, some individuals could not recover; it might be because physical stress exacerbated the lesion through moving freely in the housing environment of stainless-steel cage. The BMD values in the ASB20123 treated groups did not recover completely to the value of control group after 13 weeks recovery period. However, the BMD values in the ASB20123 treated groups increased similarly to those in the control group for 13 weeks. These results indicate that at least a further reduction in BMD could be prevented by discontinuation of treatment.

In this study, we evaluated the toxic profile of ASB20123 the CNP derivative with an extended half-life. As a result, over-dosing of ASB20123 induced excessive growth acceleration through endochondral bone growth, resulting in bone- and cartilage-specific toxicity changes in normal young rats without closed epiphyseal plates. Furthermore, our data suggested that the thickness of the epiphyseal plate and BMD of the cortical bone could be reliable biomarkers to predict bone- and cartilage-specific toxicity. The dosage regimen of CNP derivative would be a key factor for the success as therapeutic drug.

Histopathological findings in rats treated subcutaneously with ASB20123 for 4 weeks in study 1.

(DOC)

Click here for additional data file.

Histopathological findings of femur and tibia in rats treated subcutaneously with ASB20123 for 4 weeks in study 3.

(DOC)

Click here for additional data file.

Histopathological findings of femur and tibia in rats treated subcutaneously with ASB20123 for 4 weeks followed by 13 weeks recovery period in study 3.

(DOC)

Click here for additional data file.

28 Jun 2019

PONE-D-19-14712

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

PLOS ONE

Dear Mr Yotsumoto,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Aug 12 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.  At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines: 1) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) 2) please describe any steps taken to minimize animal suffering and distress, such as by administering analgesics, 3) please include the method of sacrifice and 4) Please describe thecare received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Thank you for your attention to these requests.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Yotsumoto et al provide toxicity data for a novel C-type natriuretic analog that may have utility in treating achondroplasia. Pathological changes observed included necrosis within the metaphysis, fibrosis in the marrow head, and ectopic chondrogenesis/osteogenesis. These pathological events were confined to animals with active growth plates and potentially the result of excessive growth velocity. This is a generally well-written paper that provides new and useful information.

Specific Comments.

1/ Introduction, line 44:- replace “expecting” with “exciting” This line should read “…(CNP) analog is one of the most exciting therapeutic …”.

2/ The reviewer suggest more background information regarding the rational for using a CNP/ghrelin chimeric peptide should be provided eg describe the potential for interaction with hyaluronic acid.

3/ Page 12, line 191:- There is no mention in the results section of any differences in blood chemistry measurements observed between the groups eg triglycerides (mentioned in the methods section). If there were no significant differences, the reviewer suggests line 191 could read “… ophthalmology, blood chemistry, urinalysis …”.

4/ It would be helpful for the reader if the age of the rats was included in the title of Supporting Table 1 or alternatively the title to the table read “Study 1 – histopathology in rats treated subcutaneously with …”.

Reviewer #2: The manuscript titled “Safety assessment of a novel C-type natriuretic peptide derivative and the mechanism of bone and mineral specific toxicity” by Yotsumoto et al describes experiments injecting a new GC-B activator in 12-month old rats and examining effects in bone and cartilage tissue. These data are of potential interest to the field because they suggest that the dosing regimen of the CNP analog may be important for successful clinical results.

Major issues:

Figure 4 of this manuscript shows a very interesting inverse relationship in response to ASB20123 infusion with the drug increasing bone length and decreasing density in both the cortical and trabecular bone. If this is the case, then it may limit the use of the drug because while it increases long bone length, it also results in less bone mineral density, which is unfavorable.

It would be very interesting to see if the BMD increases with time after the initial CNP analog infusion period has ended. In other words, if ASB20123 increases bone length, does it always also decrease bone mineral density or does it return to normal values after the ASB20123 treatment is discontinued. To test this, the investigators should analyze another group of animals 6 weeks after the last ASB20123 injection and determine if BMR is still reduced or whether it increased in the absence of the drug. The question being addressed with this experiment is whether any increase in bone length resulting from ASB20123 infusion also results in permanent reductions in BMD? If the later scenario is observed, then this could limit the utility of ASB20123 in the treatment of achondroplasia if it always results in less dense, and presumably, weaker bone.

Minor issues:

The manuscript would benefit by editing from a native English-speaking person. Trabecular bone is the newer and more common term for “spongy” bone.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Jul 2019

Journal requirement

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response.

According to journal requirement, we have checked our revised manuscript to meet PLOS ONE’s style requirements.

At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: 1) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) 2) please describe any steps taken to minimize animal suffering and distress, such as by administering analgesics, 3) please include the method of sacrifice and 4) Please describe the care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Thank you for your attention to these requests.

Response.

According to journal requirement, we revised the methods section about animal care. Details of animal welfare (e.g., shelter, food, water, environmental enrichment) were mentioned in line 88-92, the method of sacrifice was in line 92-94, and frequency of monitoring was in line 132-133 in the Revised Manuscript with Track Changes, respectively. The steps taken to minimize animal suffering and distress and the criteria used to assess animal health and wellbeing were described in the Guidelines for Animal Experiments of LSI Medience Corporation and/or Asubio Pharma Co., Ltd, and these studies were conducted in accordance with these guidelines.

We are grateful to the Reviewer #1 for the useful comments and suggestions that have helped us to improve our manuscript considerably. As indicated in the following responses, we have taken all your comments and suggestions into account in our revised manuscript. The line numbers appearing in the following responses indicate those of the text in the “Revised Manuscript with Track Changes” file.

Comments by reviewer #1.

Comment 1.

Introduction, line 44:- replace “expecting” with “exciting” This line should read “…(CNP) analog is one of the most exciting therapeutic …”.

Response.

Thank you for your comment. We replaced from “expecting” to “exciting” in the revised version of our manuscript. Please confirm the revised sentence in line 44 in the Revised Manuscript with Track Changes.

Comment 2.

The reviewer suggest more background information regarding the rational for using a CNP/ghrelin chimeric peptide should be provided eg describe the potential for interaction with hyaluronic acid.

Response.

Thank you for your valuable suggestion. We added the following sentence about the background information of ASB20123 in line 55-60 of the Revised Manuscript with Track Changes;

“The application of C-terminal part of ghrelin resulted in the higher stability of CNP analogs, compared to that of CNP-22 as the native form; it also improved their bioactivity as stimulators of endochondral bone growth. Furthermore, the C-terminal part of the ghrelin includes a BX7B motif, the cluster of basic amino acids might be implicated in the interaction with hyaluronic acid molecules which are the components of extracellular matrix in growth plate.”

In addition, we added 4 references in this part and it is shown in line 394-406 in the Revised Manuscript with Track Changes.

Comment 3.

Page 12, line 191

There is no mention in the results section of any differences in blood chemistry measurements observed between the groups eg triglycerides (mentioned in the methods section). If there were no significant differences, the reviewer suggests line 191 could read “… ophthalmology, blood chemistry, urinalysis …”.

Response.

Thank you for pointing out. We missed describing the change of CPK activity. We added in line 196, 199-200 and 201 of the result section, in line 322-324 of the discussion section in the Revised Manuscript with Track Changes.

Comment 4.

It would be helpful for the reader if the age of the rats was included in the title of Supporting Table 1 or alternatively the title to the table read “Study 1 – histopathology in rats treated subcutaneously with …”.

Response.

Thank you for your comment. We added the study No. in the title of all figures and supporting table 1.

We are grateful to the Reviewer #2 for the critical comments and useful suggestions that have helped us to improve our manuscript considerably. As indicated in the following responses, we have taken all your comments and suggestions into account in our revised manuscript. The line numbers appearing in the following responses indicate those of the text in the “Revised Manuscript with Track Changes” file.

Comments by reviewer #2.

Major Comment.

Figure 4 of this manuscript shows a very interesting inverse relationship in response to ASB20123 infusion with the drug increasing bone length and decreasing density in both the cortical and trabecular bone. If this is the case, then it may limit the use of the drug because while it increases long bone length, it also results in less bone mineral density, which is unfavorable.

It would be very interesting to see if the BMD increases with time after the initial CNP analog infusion period has ended. In other words, if ASB20123 increases bone length, does it always also decrease bone mineral density or does it return to normal values after the ASB20123 treatment is discontinued. To test this, the investigators should analyze another group of animals 6 weeks after the last ASB20123 injection and determine if BMD is still reduced or whether it increased in the absence of the drug. The question being addressed with this experiment is whether any increase in bone length resulting from ASB20123 infusion also results in permanent reductions in BMD? If the later scenario is observed, then this could limit the utility of ASB20123 in the treatment of achondroplasia if it always results in less dense, and presumably, weaker bone.

Response.

Thank you for your valuable suggestion. It is very important to clarify the relationship the reduction of BMD and elongation of bone length caused by ASB20123 treatment. It is especially needed to care of the decrease of BMD in the treatment of achondroplasia, because the lower BMD in achondroplasia population was reported (Sims D, et al. Whole-body and segmental analysis of body composition in adult males with achondroplasia using dual X-ray absorptiometry. PLos One, 14(3): e0213806, 2019).

In this study, thickness of epiphyseal plate of femur, BMD and bone related toxicity were observed in the rats treated with ASB20123 at dose of 0.5 mg/kg/day and more for 4-week. Meanwhile, in a previous study, the body length increased significantly after continuous sc infusion at dose of 0.05 mg/kg/day and more for 12-week (Morozumi N, et al. ASB20123: A novel C-type natriuretic peptide derivatives for the treatment of growth failure and dwarfism. PLos One, 14(2): e0212680, 2019). At the same time, the BMD decreased significantly at dose of 0.15 mg/kg/day and more (internal data). We consider that these phenomena were caused mainly by rapid and excessive induction of bone growth in normal juvenile animals.

In addition, we evaluated the recovery of BMD and bone toxicity after 13-week recovery period. These results described in line 256-271 of result section and line 346-358 of discussion section in the revised version of our manuscript and added Fig. 5. The decreased BMD values induced by ASB20123 treatment did not return to the value of control group after 13-week recovery period. However, the BMD values in the ASB20123 treated groups increased similarly to those in the control group for 13 weeks. These results indicate that at least further reduction in BMD could be prevented by discontinuation of treatment.

Considering these results, the dosage regimen of CNP derivative would be a very important key factor for the success as a therapeutic drug.

Minor Comment.

The manuscript would benefit by editing from a native English-speaking person. Trabecular bone is the newer and more common term for “spongy” bone.

Response.

Thank you for pointing out. We replaced from “spongy” to “trabecular” in the revised version of our manuscript. Also, a native English-speaking person in our company have checked our revised manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Aug 2019

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

PONE-D-19-14712R1

Dear Dr. Yotsumoto,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Michael Bader

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

3 Sep 2019

PONE-D-19-14712R1

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

Dear Dr. Yotsumoto:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Michael Bader

Academic Editor

PLOS ONE