A NEW COMBINATION THERAPY FOR THE TREATMENT OF FGFR3- RELATED SKELETAL DISEASE

Abstract

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) and severe short stature, causing achondroplasia and acromesomelic dysplasia type Maroteaux, respectively. In attempt to find a new therapeutic approach for FGFR3-related skeletal disease, the inventors showed that a combination of a NPR2 agonist (e.g. BMN-111) and a phosphatase inhibitor (e.g. LB-100) significantly increases the length of the Fgfr3.sup.Y367C/+ femurs compared to Fgfr3.sup.+/+ femurs and improves the whole growth plate cartilage. The present invention thus relates to the use of a NPR2 agonist (e.g. BMN-111) and a phosphatase inhibitor (e.g. LB-100) for the treatment of FGFR3-related skeletal disease (e.g. achondroplasia).

Claims

1. A method of treatment of FGFR3-related skeletal disease in a patient need thereof comprising administering to the patient a therapeutically effective amount of a combination of a phosphatase inhibitor and an NPR2 agonist.

2. A method of improving bone growth in a patient need thereof comprising in administering to the patient a therapeutically effective amount of a combination of a phosphatase inhibitor and a NPR2 agonist.

3. A method of increasing whole growth plate cartilage in a patient need thereof comprising administering to the patient a therapeutically effective amount of a combination of a phosphatase inhibitor and a NPR2 agonist.

4. The method according to claim 1, wherein the FGFR3-related skeletal disease is selected from the group consisting of thanatophoric dysplasia type I, thanatophoric dysplasia type II, severe achondroplasia with developmental delay and acanthosis nigricans, hypochondroplasia, achondroplasia and FGFR3-related craniosynostosis.

5. The method according to claim 1, wherein the FGFR3-related skeletal diseases is achondroplasia.

6. The method according to claim 1, wherein the phosphatase inhibitor is LB-100.

7. The method according to claim 1, wherein the NPR2 agonist is BMN-111.

8. The method of claim 1, wherein the phosphatase inhibitor and/or the NPR2 agonist are administered as active principles of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

9. The method of claim 4, wherein the FGFR3-related craniosynostosis is Muenke syndrome or Crouzon syndrome with acanthosis nigricans.

Description

FIGURES

[0071] FIG. 1. LB-100 and BMN-111 act synergistically to stimulate growth in fetal femurs from Fgfr3.sup.Y367C/+ mice.(A) Diagram showing sites of action of LB-100 and BMN-111. (B) Representative photographs of fetal femurs from E16.5 day old Fgfr3+/+ (wildtype) and Fgfr3.sup.Y367+ mice, before (D0) and after a 6 day (D6) culture with the indicated treatments. The upper dashed line indicates the groups compared in C-F. (C, D) Measurements of growth in bone length (C) and area (D), showing that in Fgfr3.sup.Y67C/+ bones, BMN-111+LB-100 increases growth more than BMN-111 alone. (E, F) Measurements of growth in bone length (E) and area (F) for Fgfr3+/+ bones, showing that for Fgfr3+/+ bones, BMN-111+LB-100 does not increase growth more than BMN-111 alone. For all experiments, the concentration of BMN-111 was 0.1 μM, and the concentration of LB-100 was 10 μM. Symbols in graphs C-E indicate individual bones (n=4−10). Bars represent mean ±SEM and data were analyzed by unpaired two-tailed t-tests. Asterisks indicate significant differences (p<0.05) between indicated groups; n.s. indicates no significant difference (p>0.05).

[0072] FIG. 2. Dual action of LB-100 and BMN-111 improves chondrocyte differentiation in growth plates of ex-vivo cultured Fgfr3.sup.Y367C/+ femurs. Mean area of individual hypertrophic chondrocytes in proximal growth plates of femurs treated (n=4−6 bones measured for each condition, with 54-137 cells measured for each bone). Data were analyzed by one-way ANOVA followed by the Holm-Sidak multiple comparison test (*p<0.05, **p<0.01, ****p<0.0001).

[0073] FIG. 3. Graphic representation of naso-anal, femur, tibia, ulna and humerus length and % of bone growth of Fgfr3.sup.Y367C/+ mice treated with subcutaneous injection of LB100 (1 mg.kg−1 body weight)+BMN111 (800 ug.kg−1 body weight)

[0074] FIG. 4. Graphic representation of skull and foramen magnum length and foramen magnum area and % of growth of Fgfr3.sup.Y367C/+ mice treated with subcutaneous injection of LB100 (1 mg.kg−1 body weight)+BMN111 (800 ug.kg−1 body weight)

EXAMPLE 1: EX-VIVO

Material & Methods

Mice

[0075] Two mouse lines were used for this study: cGi500 (Thunemann et al., 2013) and Fgfr3.sup.Y367C/+ (Pannier et al., 2009; Lorget et al., 2012). The cGi500 mice were provided by Robert Feil. The use of the cGi500 mice for monitoring cGMP levels in the growth plate has been verified by ELISA measurements (Shuhaibar et al., 2017).

Reagents

[0076] Reagents were obtained from the sources listed in Supplementary Table I. BMN-111 was synthesized by New England Peptide as a custom order with the following sequence: [Cyc(23,39)]H2NPGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC—OH, as described by Lorget et al., 2012. The purity was >95%.

Measurements of cGMP Production in Tibia Growth Plates Using cGi500

[0077] cGMP production in chondrocytes within intact growth plates was measured using tibias dissected from newborn mice (0-1 day old) that globally expressed one or two copies of the the cGi500 FRET sensor, as previously described (Shuhaibar et al., 2017).Tibias were dissected and cultured overnight on Millicell membranes in BGJb medium with 0.1% bovine serum albumin, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. In preparation for imaging, each tibia was slit to remove the tissue overlying the growth plate. Where indicated, the tibia was incubated in LB-100, cantharidin, or control medium, followed by addition of FGF18 (0.5 μg/ml+1 μg/ml heparin) or control medium containing heparin only. The tibia was then placed in a perfusion slide and the growth plate was imaged on the stage of a confocal microscope, as previously described (Shuhaibar et al., 2017).

Rib Chondrocyte Cultures

[0078] Rib cages were dissected from newborn (0-2 day old) mice, and trimmed to remove the skin, spinal cord, and soft tissue around the sternum and ribs. Non-chondrocyte tissue was digested away by incubating the rib cages in 2 mg/ml pronase in PBS for one hour in a shaking water bath at 37° C., and then incubated in 3 mg/ml collagenase D in medium for one hour. After washing, the rib cages were transferred to a dish with fresh collagenase D and incubated for 5-6 hours, with trituration at 2 hours, to release the chondrocytes. The isolated cells were passed through a 40 um nylon cell strainer, resuspended in DMEM/F12 medium with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/m1 of streptomycin, and plated in 35 mm tissue culture dishes, at a cell density corresponding to one newborn mouse per plate. The cells were cultured for 3 days, at which point they were ˜75-90% confluent, then washed with PBS and incubated in serum-free medium for 18 hours. The cells were then incubated in LB-100 (10 μM), or control medium, followed by addition of FGF18 (0.5 ug/ml+1 μg/ml heparin) or control medium containing heparin only.

[0079] At end of the incubation period, dishes were washed in PBS and cells lysed in 250 μl of 1% SDS containing 10 mM sodium fluoride, 1 μM microcystin, and Roche protease inhibitor cocktail. Protein content was determined by a BCA assay. The protein yield per newborn mouse was ˜200-300 μg.

Phostag Gel Electrophoresis, and Western Blotting

[0080] Proteins were separated in a Phos-tag-containing gel, as previously described (Egbert et al., 2014), except that chondrocyte lysates (50 μg protein) were used without immunoprecipitation. Blots were probed with an antibody that was made in guinea pig against the extracellular domain of mouse NPR2 (Ter-Avetisyan et al., 2014). This antibody was a gift from Hannes Schmidt (University of Tubingen), and has been previously validated for western blots (Robinson et al., 2017).

Ex vivo culture of fetal femurs

[0081] Femurs were cultured ex vivo, as described previously (Jonquoy et al., 2012; Lorget et al., 2012). The left femur was cultured in the presence of LB-100 (10 μM), BMN-111 (0.1 μM), or LB-100 (10 μM)+BMN-111 (0.1 μM), and compared with the non-treated right femur. The bone's length was measured on day 0 (D0) and day 6 (D6). Images were captured with an Olympus SZX12 stereo microscope and quantified using cellSens software (Olympus). The results were expressed as the increase in femur length or area (D6-D0) in the presence or absence of LB-100, BMN-111, or LB-100+BMN-111.

Histology

[0082] Fetal femur (E16.5) explants were fixed in 4% paraformaldehyde, decalcified with EDTA (0.4 M), and embedded in paraffin. Serial 5 μm sections were stained with hematoxylin-eosin-safran (HES) reagent, using standard protocols. For immunohistochemical assessment, sections were labeled with the following antibodies and a Dako Envision Kit: anti-COL X (BIOCYC, N.2031501005; 1:50 dilution), anti-phosphorylated ERK1-2 (Thr180/Tyr182) (Cell Signaling Technology, #4370; 1:100 dilution), and anti-S0X9 (polyclonal antibody, Santa Cruz Biotechnology Inc., catalog D0609; dilution 1:75). Images were captured with an Olympus PD70-IX2-UCB microscope and quantified using cellSens software.

[0083] For analysis of the effect of the drug treatments on the area occupied by proliferative chondrocytes, these cells were identified by their round or columnar shape as seen with HES staining, and by the absence of collagen X labeling. We measured the total area occupied by chondrocytes within the whole growth plate and the area occupied by COLX-positive chondrocytes. The area for proliferating chondrocytes was calculated by subtracting the COLX-positive area from the whole growth plate area.

Statistics

[0084] Data were analyzed using Prism 6 or higher (GraphPad Software). To compare more than two groups, we used one-way ANOVA followed by two-tailed t-tests with the Holm-Sidak correction for multiple comparisons, or two-way ANOVA followed by Sidak's multiple comparisons tests. Two groups were compared using either paired or unpaired two-tailed t-tests, as indicated in the figure legends.

Results

LB-100 Counteracts the Inactivation of NPR2 by FGF in Growth Plate Chondrocytes.

[0085] NPR2 activity in chondrocytes of intact growth plates was measured as previously described, using mice expressing a FRET sensor for cGMP, cGi500 (Shuhaibar et al., 2017). Tibias were isolated from newborn mice, and the overlying tissue was excised to expose the growth plate for confocal imaging (Data not shown). When the NPR2 agonist C-type natriuretic peptide (CNP) was perfused across the growth plate, the CFP/YFP emission ratio from cGi500 increased, indicating an increase in cGMP, due to stimulation of the guanylyl cyclase activity of NPR2 (Data not shown). Perfusion of A-type natriuretic peptide (ANP), which activates the NPR1 guanylyl cyclase, or perfusion of a nitric oxide donor (DEA/NO), which activates soluble guanylyl cyclases, did not increase cGMP (Data not shown), showing that among the several mammalian guanylyl cyclases, only NPR2 is active in the chondrocytes of the mouse growth plate. As previously shown, exposure of the growth plate to FGF18 suppressed the cGMP increase in response to CNP perfusion (Data not shown), indicating that FGF receptor activation decreases NPR2 activity.

[0086] Based on previous evidence that a PPP family phosphatase inhibitor, cantharidin (100 μM), inhibits the inactivation of NPR2 in growth plate chondrocytes by FGF (Shuhaibar et al., 2017), we tested a cantharidin derivative, LB-100, for which a phase I clinical trial indicated little toxicity (Chung et al., 2017). LB-100 is a catalytic inhibitor of PPP2 and PPPS, with IC50 values of 0.39±0.013 μM and 1.82±0.093 μM respectively, when tested in vitro with DiFMUP as the substrate (D'Arcy et al., 2019). LB-100 also inhibits PPP1, with an IC50 of 1.80±0.022 μM when tested under these same conditions (Richard Honkanen, personal communication). Based on its similarity to cantharidin, 10 μM LB-100 is also likely to inhibit PPP6, but not PPP4 or PPP7 (Swingle and Honkanen, 2019).

[0087] To investigate if LB-100 counteracts the inactivation of NPR2 by FGF, we used the following protocol:Tibias expressing cGi500 were preincubated with solutions of 1, 5 or 10 μM LB-100 for 60 minutes. FGF was then added and the incubation was continued for an additional 80 minutes. Following these incubations, the tibia was placed in a perfusion slide for confocal imaging, and cGMP production by NPR2 was monitored by measuring the increase in CFP/YFP emission ratio in response to CNP. Incubation with 10 μM LB-100 caused no visible change in chondrocyte morphology as imaged in the live growth plate by confocal microscopy, indicating no obvious toxicity (Data not shown).

[0088] After FGF treatment, the cGMP increase in response to CNP was small (Data not shown). However, when the tibia was preincubated with 5 or 10 μM LB-100 before applying FGF, the CNP-induced cGMP increase was enhanced (Data not shown). 1μM LB-100 had no effect. The CFP/YFP emission ratio attained after CNP perfusion in tibias that had been incubated in 5 or 10 μM LB-100 before the FGF treatment was similar to or greater than that in control tibias without FGF (Data not shown). The CNP-stimulated increases in the CFP/YFP emission ratio from cGi500 under these various conditions, and demonstrates that LB-100 counteracts the inactivation of NPR2 by FGF. LB-100 was more effective than cantharidin, with 5 μM LB-100 resulting in a stimulation equivalent to that seen with 10 μM cantharidin (Data not shown).

LB-100 Counteracts the FGF-induced Dephosphorylation of NPR2 by FGF in Primary Chondrocyte Cultures

[0089] To investigate if LB-100 Counteracts the FGF-induced Dephosphorylation of NPR2, we used Phostag gel electrophoresis (Kinoshita et al., 2009) to analyze the phosphorylation state of NPR2 in isolated chondrocytes from the ribs of newborn mice. After 4 days in culture, the chondrocytes had formed a monolayer with a cobblestone appearance (Data not shown). A 1 hour incubation with 10 μM LB-100 did not cause any visible change in cell morphology (Data not shown). We then compared the phosphorylation state of NPR2 in chondrocytes with and without LB-100 preincubation, and with and without subsequent exposure to FGF. Chondrocyte proteins were separated by Phos-tag gel electrophoresis, which retards migration of phosphorylated proteins, and western blots were probed for NPR2 (Data not shown). Without

[0090] FGF treatment, NPR2 protein from the rib chondrocytes was present in a broad region of the gel. With FGF treatment, the ratio of the signal in the upper vs lower regions decreased (Data not shown), indicating NPR2 dephosphorylation in response to FGF and confirming, with primary chondrocytes, a previous study using a rat chondrosarcoma (RCS) cell line (Robinson et al., 2017). However, if the chondrocytes were preincubated with 10 μM LB-100, the dephosphorylation in response to FGF was only partial, indicating that LB-100 counteracts the FGF-induced dephosphorylation of NPR2 (Data not shown).

In Fgfr3.SUP.Y367C/+ .Femurs, LB-100 Enhances the Stimulation of Bone Growth by the Hydrolysis Resistant NPR2 Agonist BMN-111.

[0091] Previously we showed that the hydrolysis-resistant CNP analog BMN-111 increases bone growth in a mouse model of achondroplasia in which tyrosine 367 is changed to a cysteine (Fgfr3.sup.Y367C/+), resulting in constitutive activation of FGFR3 (Pannier et al., 2009; Lorget et aL, 2012). However, BMN-111 only partially rescued the effect of the FGFR3-activating mutation. Our finding that LB-100 opposes the FGF inhibition of NPR2 activity in chondrocytes suggested that applying LB-100 together with BMN-111 might enhance the stimulation of growth (FIG. 1A). Like CNP, BMN-111 (0.1 μM) stimulated NPR2 activity in growth plate chondrocytes (Data not shown).

[0092] As previously reported (Lorget et al., 2012), 0.1 μM BMN-111 increased the rate of elongation of cultured femurs from embryonic day 16.5 Fgfr3.sup.Y367C/+ mice (FIGS. 1B, 1C, and Data not shown). The mean rate of increase in bone length in the BMN-111-stimulated Fgfr3.sup.Y367C/+ femurs was 1.77 times that in vehicle-treated bones (FIG. 1C). LB-100 alone did not significantly increase the rate of bone elongation, showing a growth rate ratio of 1.04 for LB-100/control (FIGS. 1C). However, when Fgfr3.sup.Y367C/+ femurs were cultured with BMN-111 together with 10 μM LB-100, the mean rate of bone elongation increased to 2.17 times that in untreated bones (FIGS. 1C). Thus, the combination of BMN-111 and LB-100 significantly increased the bone elongation rate to a level —23% higher than that with BMN-111 alone.

[0093] In addition to these measurements of bone length, we also measured the effect of LB-100 and BMN-111 on the rate of increase of the total bone and cartilage area (Data not shown). Based on these area measurements, LB-100 by itself stimulated growth of femurs from Fgfr3.sup.Y367C/+ mice, as did BMN-111 by itself, with a growth rate ratio of 1.40 for LB-100/control, and a growth rate ratio of 1.51 for BMN-111/control (FIGS. 1D). The combination of LB-100 and BMN-111 was even more effective, with a growth rate ratio of 1.93. Thus, the combination of BMN-111 and LB-100 enhanced the rate of increase in area by 27% compared with BMN-111 alone (FIG. 1D).

[0094] With cultured femurs from Fgfr3.sup.+/+ mice, BMN-111 increased the rate of bone growth, but combining BMN-111 with LB-100 did not enhance the growth rate beyond that seen with BMN-111 alone (FIGS. 1E and 1F). This result contrasts with the ability of LB-100 to enhance BMN-111-stimulated bone growth in Fgfr3.sup.Y367C/+ mice. Because Fgfr3.sup.Y367C/+ mice have elevated FGFR3 tyrosine kinase activity due to an activating Fgfr3 mutation (Gibbs and Legeai-Mallet, 2007), their NPR2 would be expected to be less phosphorylated and less active, and LB-100 would be expected to restore their NPR2 phosphorylation and activity towards Fgfr3.sup.+/+ levels, thus increasing bone growth. In contrast, if baseline NPR2 phosphorylation is higher in Fgfr3.sup.+/+ vs Fgfr3.sup.Y367C/+ mice, the growth stimulating effect of LB-100 might be less.

Combined Treatment with LB100 and BMN111 Improves Growth Plate Cartilage Homeostasis.

[0095] Histological analyses of the epiphyseal growth plates of femurs showed that combining BMN-111 and LB-100 treatment modified the cartilage growth homeostasis in both proximal and distal growth plates (Data not shown). Prehypertrophic and hypertrophic chondrocytes produce an extracellular matrix rich in Collagen type X (COLX), allowing us to use COLX immunostaining to label the hypertrophic region, and to visualize and measure individual cells. This labeling revealed a highly beneficial effect of the combined treatment on the size of the cells in the hypertrophic area of Fgfr3.sup.Y367C/+ femurs (FIG. 2). Compared to the Fgfr3+/+ growth plates, the mean cross-sectional areas of the hypertrophic chondrocytes in Fgfr3.sup.Y367C/+ distal and proximal growth plates were reduced by about half (FIG. 2). As previously reported (Lorget et al., 2012), BMN-111 increased the size of the Fgfr3.sup.Y367C/+ hypertrophic chondrocytes, but the cells were smaller than for the wildtype (FIG. 2). However, with the combined treatment of BMN-111 and LB-100, the mean area of the Fgfr3.sup.Y367C/+ hypertrophic cells in the proximal growth plate was 32% greater than with BMN-111 alone, and similar to that of Fgfr3+/+ hypertrophic cells, indicating that the final differentiation of the chondrocytes was restored by the treatment (FIG. 2). Under the conditions used for this analysis, the improvement was only significant in the proximal growth plate (FIG. 2), in which chondrocyte development precedes that in the distal growth plate during the endochondral growth process (Data not shown).We also observed a beneficial effect of the combined treatment on the proliferative region of the growth plate. We measured the area of the proliferative region by subtracting the hypertrophic area, identified by COLX labeling, from the total growth plate area. Based on these measurements, the combined treatment increased the total proliferative growth plate area of the femur by an average of 33% over vehicle, compared to 20% for BMN-111 alone (Data not shown). Thus, the combined treatment increased the proliferative area by 13% compared to BMN-111 alone (Data not shown). In summary, the combined treatment both increased the proliferative growth plate area of the femur and restored chondrocyte terminal differentiation. CNP signaling through NPR2 in the growth plate inhibits the MAP kinase pathway and its extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Ozasa et al., 2005; Krejci et al., 2005). Therefore, we investigated the impact of treatment with LB-100+BMN-111 on the phosphorylation of ERK1/2. In agreement with the constitutive activity of the Y367C Fgfr3 gain-of-function mutation acting to decrease NPR2 activity (Data not shown), we observed by immunostaining a high level of phosphorylated ERK1/2 in the proximal and distal parts of the cartilage compared to controls (Data not shown). The combined LB-100 and BMN-111 treatment decreased the activity of the MAP kinase pathway as demonstrated by the decreased phosphorylation of ERK1/2 in the proximal and distal growth plates of the femurs (Data not shown). These data are in agreement with 1) the role of the MAP kinase pathway as a regulator of chondrocyte differentiation and with 2) our hypothesis that the elevation of cGMP inhibits the MAP kinase pathway thus promoting bone growth. Lastly, we examined the expression of the SOX9, a master transcription factor that is upregulated in Fgfr3 gain-of-function mouse models (Ornitz and Legeai-Mallet, 2017). The activation of ERK1/2 increases SOX9 expression, which functions to suppress chondrocyte terminal differentiation (Lefebvre and Dvir-Ginzberg, 2017; Murakami et al., 2000). Consistent with these findings, we observed that the activation of FGFR3 signaling in the Fgfr3.sup.Y367C/+ femurs increased the expression of SOX9, relative to the Fgfr3+/+ control. SOX9 protein was seen to accumulate visibly in the proliferative and hypertrophic zones of the growth plate cartilage (Data not shown). Treatment of Fgfr3.sup.Y367C/+ femurs with BMN-111+LB-100 reduced SOX9 expression in proximal and distal growth plates, and its expression returned to a more normal level, closer to that of the Fgfr3.sup.+/+ femurs. The fact that SOX9 growth plate expression was decreased confirms the beneficial action of BMN111+LB100 treatment on growth plate cartilage homeostasis.

EXAMPLE 2: IN VIVO

Material & Methods

The Mouse Model

[0096] The Fgfr3.sup.Y367C/+ mouse model has been described previously (Pannier et al 2009). All the mice had a C57BL/6 background. Cartilage and bone analyses were performed on 16-day-old mice. The Fgfr3.sup.Y367C/+ mice were 1-day old upon treatment initiation, and received daily subcutaneous administrations of LB100 (1 mg.kg−1 body weight)+BMN111 (800 ug.kg−1 body weight) or vehicle (3.5 mM HCl, 0.1% DMSO) for 2-weeks. Long bones were measured using a caliper (VWRi819-0013, VWR International).

Combined Drug Treatment

[0097] BMN-111 was synthesized by New England Peptide as a custom order with the following sequence: [Cyc(23,39)]H2NPGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC—OH, as described by Lorget et al., 2012. The purity was >95%.

[0098] LB100 was synthesized by Selleckchem with the following sequence (3-(4-methylpiperazine-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid) as described by Shuhaibar L C et al 2021.

Results

Combined Treatment (LB100+BMN11) Modulates Bone Growth in the Fgfr3 Mouse Model of ACH

[0099] To confirm LB100+BMN-111 in vivo effect on bone growth, we next sought to determine whether or not a combination of BMN-111 and LB-100 could regulate bone growth in the Fgfr3.sup.Y367C/+ murine model of ACH. The Fgfr3.sup.Y367C/+ mice were 1 day old when treatment began and received once-daily subcutaneous administrations of LB-100 (1 mg.kg−1 body weight)+BMN-111 (800 ug.kg−1 body weight) or vehicle for 2-weeks. Significant improvement in dwarfism was noted after only 2 weeks of treatment. The mean naso-anal length was 7.24% (p<0.05)) greater in treated animals than in controls. Likewise, the mean weight was 19.89% (p<0.05) greater in treated animals (FIG. 3). We did not observe any adverse events (e.g. weight loss or death). With regard to long bone growth, the femur and tibia were respectively 5.38% (p<0.01) and 4.82% (p<0.05) longer in treated mice than in controls. Similarly, the humerus and ulna in treated mice were respectively 4.76% (p<0.01) and 7.5% (p<0.01) longer than in non-treated controls (FIG. 3).

[0100] To visualize combined treatment's in vivo on skull and foramen magnum at the skull base, we analyzed KTScanner. We observed that the mean transversal length and longitudinal length of the skull were respectively 3.89% (p<0.05) and 5.30% (p<0.05) greater in treated animals and the foramen magnum area were higher 5.6% (p<0.05) in treated animals (FIG. 4).

[0101] To characterize combined treatment's in vivo mode of action, we analyzed the growth plate. The treatment strongly modified the structural organization of the Fgfr3.sup.Y367C/+ growth plate cartilage and restored the delay of secondary ossification center formation in treated mice (Data not shown). We observed an enlargement of the epiphysis of the proximal part of the femur in treated animals versus untreated, the collagen type X labelling pointed out this change of the epiphysis (Data not shown). A large area of collagen type Xis visible in all animals treated by LB100+BMN-111. As with the ex vivo experiments, and in order to assess combined treatment's in vivo effect on the regulators of chondrocyte differentiation that are perturbed in ACH, we evaluated the expression levels of phosphorylation levels of Erk1-2 (Data not shown). As observed in ex vivo femur cultures, the abnormal in vivo overexpression of phosphorylated Erk1/2 had normalized after combined treatment (Data not shown).

[0102] These in vivo results demonstrate that the combination LB100+BMN-111 can be used to control long bone elongation in FGFR3-related disease.

Conclusion

[0103] Understanding of the mechanisms used by FGFR3 and CNP as important regulators of longitudinal bone growth has allowed the development of an effective therapeutic strategy using a CNP analog (vosoritide, also known as BMN-111) to treat achondroplasia (Savarirayan et al., 2019). The findings described here identify the PPP family phosphatase inhibitor LB-100 as a stimulator of bone growth when used in combination with this CNP analog to stimulate production of cGMP by NPR2. Firstly, using isolated bones incubated with FGF to mimic an achondroplasia-like condition, we show that pretreatment with LB-100 counteracts the decrease in NPR2 guanylyl cyclase activity by FGFR3. Secondly, our results support the hypothesis that FGFR3 acts by dephosphorylating NPR2 in chondrocytes and that LB-100 suppresses the dephosphorylation. Moreover, application of a combination of BMN-111 and LB-100 to bones from the achondroplasia mouse model Fgfr3.sup.Y367C/+ results in growth that exceeds that stimulated by BMN-111 alone, demonstrating the therapeutic potential of this combination treatment for skeletal dysplasias such as achondroplasia.

[0104] Our data also show the benefit of this treatment for growth plate cartilage during bone development in Fgfr3.sup.Y367C/+ mice. During the process of endochondral ossification, chondrocytes actively proliferate in the resting and proliferating chondrocyte zone, and then differentiate to hypertrophic chondrocytes, which lose the capacity to proliferate. The terminally differentiated hypertrophic cells are removed by cell death or transdifferentiate into osteoblasts. It is well known that FGFR3 signaling decreases bone growth by inhibiting both chondrocyte proliferation and differentiation and bone formation. SOX9 is known to be required to permit proliferation and differentiation, which are the regulators (drivers) of bone elongation (Lefebvre and Dvir-Ginzberg, 2017), and it has been proposed that FGFR3 uses ERK1/2 to restrict hypertrophic differentiation (Murakami et al., 2004). Here, we showed that the treatment with BMN-111 and LB-100 reduced the levels of phosphorylated ERK1/2 and SOX9, thus modifying chondrocyte differentiation and allowing bone growth. In addition, we noted an impressive increase in the size of the hypertrophic cells. We concluded that the treatment perfectly restored cartilage homeostasis, and we hypothesize that the elevated cGMP resulting from this treatment could be a key regulator of transdifferentiation of hypertrophic cells into osteoblasts and could control the chondrogenic or osteogenic fate decision.

[0105] The increase in NPR2 phosphorylation by LB-100 is correlated with improved chondrocyte proliferation in Fgfr3.sup.Y367C/+ femurs, consistent with previous results with a mouse model (Npr2-7E) mimicking constitutive phosphorylation of NPR2 (Shuhaibar et al., 2017). LB-100 inhibits multiple PPP family phosphatases (D'Arcy et al., 2019), and thus although our results provide a proof of principle for a possible combination treatment, a more specific phosphatase inhibitor would be more optimal. Determination of the particular phosphatase(s) that dephosphorylate NPR2 in chondrocytes, and development of more specific inhibitors of these phosphatases, could lead to future therapies.

[0106] Recent mouse studies indicate that in addition to increasing prepubertal bone elongation, phosphorylation of NPR2 increases bone density, due to an increase in the number of active osteoblasts at the bone surface (Robinson et al., 2020). Because low bone density is one of the key clinical features of achondroplasia (Matsushita et al., 2016), the combination a CNP analog and a phosphatase inhibitor could also have a beneficial impact on bone density for patients with achondroplasia and related conditions. In addition, this treatment could have potential for treatment of osteoporosis. Lastly, because CNP/NPR2 also plays a key role in regulation of joint homeostasis, inactivation of ERK1/2 due to elevated cGMP could also be beneficial for preventing or minimizing cartilage loss and promoting repair of the damaged articular cartilage in skeletal disorders and osteoarthritis, an extremely common disease of adulthood (Peake et al., 2014). More generally, the combination of natriuretic peptides and phosphatase inhibitors could have therapeutic potential for multiple disorders involving NPR2 and the related guanylyl cyclase NPR1 that also requires phosphorylation for activity (Kuhn, 2016).

[0107] In summary, the combined (LB-100+BMN-111) treatment acts on both chondrocyte proliferation and differentiation, thus promoting better bone growth. In achondroplasia, the homeostasis of the growth plate is disturbed, and proliferation and differentiation are affected by the overactivation of FGFR3. Currently, BMN111 (vosoritide) is being studied in children with ACH, and as demonstrated in preclinical studies, BMN-111 mostly restores the defective differentiation in the growth plate (Lorget et al., 2012). Recently reported phase 2 data demonstrated that vosoritide resulted in a sustained increase in annualized growth velocity for up to 42 months in children 5 to 14 years of age with achondroplasia (Savarirayan et al., 2019). In this study, we described a combination treatment that could increase bone growth rate to a higher level than vosoritide alone, could shorten the required time of treatment, and could be considered as intermittent treatment for patients with achondroplasia.

REFERENCES

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