TYRA-300, an FGFR3-selective inhibitor, promotes bone growth in two FGFR3-driven models of chondrodysplasia

Abstract

Achondroplasia (ACH) and hypochondroplasia (HCH), the two most common types of dwarfism, are each caused by FGFR3 gain-of-function mutations that result in increased FGFR3 signaling, which disrupts chondrogenesis and osteogenesis, resulting in disproportionately shortened long bones. In this study, TYRA-300, a potent and selective FGFR3 inhibitor, was evaluated in 3 genetic contexts: wild-type mice, the Fgfr3Y367C/+ mouse model of ACH, and the Fgfr3N534K/+ mouse model of HCH. In each model, TYRA-300 treatment increased nasoanal length and tibia and femur length. In the two FGFR3-altered models, TYRA-300-induced growth partially restored the disproportionality of long bones. Histologic analysis of the growth plate in Fgfr3Y367C/+ mice revealed that TYRA-300 mechanistically increased both proliferation and differentiation of chondrocytes. Importantly, children with ACH can experience medical complications due to foramen magnum stenosis, and TYRA-300 significantly improved the size and shape of the skull and foramen magnum in Fgfr3Y367C/+ mice. Spinal stenosis is also a frequent complication, and TYRA-300 increased the lumbar vertebrae length and improved the shape of the intervertebral discs in both models. Taken together, these studies demonstrate that the selective FGFR3 inhibitor TYRA-300 led to a significant increase in bone growth in two independent FGFR3-driven preclinical models as well as in wild-type mice.

Keywords: Bone biology; Bone disease; Cell biology; Drug therapy; Mouse models.

Figure 1. TYRA-300 demonstrates dose-dependent increases in growth velocity and long bones in a wild-type mouse model.

(A) Nasoanal length and (B) tail length of female C57BL/6 mice from 3 to 8 weeks of age while receiving daily oral treatment with vehicle (n = 11), 12 mg/kg TYRA-300 (n = 12), or 14 mg/kg TYRA-300 (n = 12) from 4 to 8 weeks of age. Arrows indicate the start of treatment. Significance was assessed using a Mann-Whitney U test at each time point. (C) Tibia and (D) femur lengths measured by calipers on the final day of treatment (8 weeks of age). Significance for TYRA-300–treated groups versus the vehicle-treated group was assessed using a Kruskal-Wallis test. (E) Pharmacokinetic profile for female and male C57BL/6J mice ranging from 1 week old to 12 weeks old after a single subcutaneous dose of TYRA-300 (1.2 mg/kg). The mean AUC_inf (ng × hr/mL) for each age group is shown. For 1- and 2-week-old mice, data points represent n = 6–8 mice per time point. For 3- to 12-week-old mice, data points represent n = 4 mice. The lower limit of quantification was 4 ng/mL for the 1-week-old samples and 8 ng/mL for all other samples. Data in graphs represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 2. TYRA-300 increases long bone length through modulation of the growth plate in the Fgfr3^Y367C/+ mouse model, which mimics the phenotype of achondroplasia.

(A) Representative whole-body radiographs of a vehicle-treated Fgfr3^Y367C/+ mouse on the left versus a TYRA-300–treated Fgfr3^Y367C/+ mouse on the right. Treatment consisted of daily subcutaneous injection of TYRA-300 at 1.2 mg/kg/d for 15 days starting at day 1 after birth. (B) Nasoanal length, (C) tail length, and (D) body weight of vehicle-treated wild-type (Fgfr3^+/+) mice (n = 10), vehicle-treated Fgfr3^Y367C/+ mice (n = 10), and TYRA-300–treated Fgfr3^Y367C/+ mice (n = 8) from 1 to 16 days of age. Significance was assessed using a Mann Whitney U test at each time point. (E) Improvement in tibia, (F) femur, (G) ulna, and (H) humerus length. (I) Representative histological images of H&E-stained distal femurs (original magnification, ×4 [top]; ×10 [bottom]). (J) Quantification of bone volume of the SOC from μCT imaging. (K) Representative histological images of collagen type X (original magnification, ×4 [top], ×10 [middle], and ×20 [bottom]) and (L) PCNA (original magnification, ×20) staining of the distal femurs. (M) Number of HZC cells per region of interest (ROI), quantified from the same ROI within the chondro-osseous junction of the distal part of the right femur of each mouse, as denoted by the black box in K (original magnification, ×20 [950 × 342 pixels]). Scale bar: 200 μm (4× images); 100 μm (10× images); 50 μm (20× images). (N) Bone mineral density (BMD) and (O) bone volume to tissue volume (BV/TV) quantified from μCT imaging of the femurs. PCNA, proliferating cell nuclear antigen; PZC, proliferating zone chondrocytes; HZC, hypertrophic zone chondrocytes; OII, secondary ossification center; BO, bone. Significance for the TYRA-300–treated group versus vehicle-treated group was assessed using a Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The bracket in D denotes that each time point (day 8–16) had a significance value of P < 0.001. Data in graphs represent mean ± SEM.

Figure 3. TYRA-300 improved the size and shape of the axial skeleton in Fgfr3^Y367C/+ mice.

(A) Representative μCT images of the skull. (B) Skull length, (C) width, (D) anteroposterior length, and (E) nasio-occipital length of vehicle-treated wild-type (Fgfr3^+/+) mice (n = 10), vehicle-treated Fgfr3^Y367C/+ mice (n = 10), and TYRA-300–treated (1.2 mg/kg/d s.c.) Fgfr3^Y367C/+ mice (n = 8) from 1 to 16 days of age. (F) Improvement in foramen magnum transverse diameter, (G) sagittal diameter, (H) area, and (I) grade of synchondroses. (J) Representative μCT images of the foramen magnum from each group after treatment. Synchondroses were graded using the following key: I, border of synchondroses were completely separated; II, clear separation of synchondroses with some areas suspicious for bone bridging; III, synchondroses showing bony bridge between 2 borders; IV, completely fused synchondroses with remnants of margin (cartilage); and V, completely fused synchondroses. IOSA, intraoccipital synchondrosis anterior. (K) Length of the L4–L6 lumbar vertebrae, as measured by calipers on the final day of the study. (L) Representative histological images of Alcian blue with Sirius red, Safranin O, collagen type X, and collagen type I staining of the L5 lumbar vertebrae after treatment (original magnification, ×4 [first, second, third, and last row], ×10 [second-to-last row]). The solid arrow line indicates height of nucleus pulpous, and the dashed arrow line indicates width of nucleus pulposus. Scale bar: 200 μm (4× images); 100 μm (10× images). (M) Quantification of the height of the L5 vertebral body from the center point of each Alcian blue/Sirius red image. IVD, intervertebral disc; BO, bone; CEP, cartilage end plate; HY, hypertrophic chondrocyte; OAF, outer annulus fibrosus. Significance was assessed using a Kruskal Wallis test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data in graphs represent mean ± SEM.

Figure 4. TYRA-300 increased bone length and reduced FGFR3 signaling within the growth plate in a Fgfr3^N534K/+ mouse model of hypochondroplasia.

(A–D) Length of (A) tibia, (B) femur, (C) ulna, and (D) humerus in vehicle-treated wild-type (Fgfr3^+/+) mice (n = 12), vehicle-treated Fgfr3^N534K/+ mice (n = 11), and TYRA-300–treated Fgfr3^N534K/+ mice (n = 9) after once daily treatment from 3 to 24 days of age. (E) Representative histological images of H&E (original magnification, ×2 [top], ×10 [bottom]), (F) collagen type X (original magnification, ×10), (G) and pERK1/2 staining of the distal femur (original magnification, ×10). (H) Number of pERK1/2⁺ cells per region of interest (ROI), quantified from the same ROI within the chondro-osseus junction of the right distal femur of each mouse, as designated by the black box in G (1,306 × 587 pixels). Scale bar: 500 μm (2× images); 100 μm (10× images). Col X, collagen type X. Significance was assessed using a Kruskal Wallis test. *P < 0.05, **P < 0.01. Data in graphs represent mean ± SEM.

Figure 5. TYRA-300 increased the size of the foramen magnum and modified the shape of the intervertebral discs in Fgfr3^N534K/+ mice.

(A) Area and (B) sagittal diameter of the foramen magnum of vehicle-treated wild-type (Fgfr3^+/+) mice (n = 12), vehicle-treated Fgfr3^N534K/+ mice (n = 11), and TYRA-300–treated Fgfr3^N534K/+ mice (n = 9) after once daily treatment from 3 to 24 days of age. (C) Improvement in the grade of synchondroses after treatment. (D) Representative μCT images of the foramen magnum from each group. Synchondroses were graded using the following key: I, border of synchondroses were completely separated; II, clear separation of synchondroses with some areas suspicious for bone bridging; III, synchondroses showing bony bridge between 2 borders; IV, completely fused synchondroses with remnants of margin (cartilage); and V, completely fused synchondroses. IOSA, intraoccipital synchondrosis anterior. (E) Representative histological images of staining of the L5 lumbar vertebrae after 21 days of treatment: H&E (original magnification, ×4), collagen type X (original magnification, ×4), and collagen type I (original magnification, ×4). Scale bar: 200 μm. BO, bone; HY, hypertrophic chondrocyte; OAF, outer annulus fibrosus; Col X, collagen type X; Col I, collagen type I. Significance was assessed using a Kruskal Wallis test. *P < 0.05. Data in graphs represent mean ± SEM.