Feasibility of administration of calcitonin gene-related peptide receptor antagonist on attenuation of pain and progression in osteoarthritis

Abstract

Suppressing inflammation and abnormal subchondral bone turnover is essential for reducing osteoarthritis (OA) progression and pain relief. This study focused on calcitonin gene-related peptide (CGRP), which is involved in inflammation and bone metabolism, and investigated whether a CGRP receptor antagonist (rimegepant) could suppress OA progression and relieve pain in two OA models. C57BL/6 mice (10-week-old) underwent surgical destabilization of the medial meniscus, and Rimegepant (1.0 mg/kg/100 μL) or phosphate-buffered saline (100 μL) was administered weekly intraperitoneally after OA surgery and evaluated at 4, 8, and 12 weeks. In the senescence-accelerated mice (SAM)-prone 8 (SAMP8), rimegepant was administered weekly before and after subchondral bone sclerosis and sacrificed at 9 and 23 weeks, respectively. Behavioral assessment and immunohistochemical staining (CGRP) of the dorsal root ganglion (DRG) were conducted to assess pain. In DMM mice, synovitis, cartilage degeneration, and osteosclerosis were significantly suppressed in the rimegepant group. In SAMP8, synovitis, cartilage degeneration, and osteosclerosis were significantly suppressed by rimegepant at 9 weeks; however, not at 23 weeks. Behavioral assessment shows the traveled distance and the number of standings in the rimegepant group were significantly longer and higher. In addition, CGRP expression of the DRG was significantly lower in the rimegepant group at 8 and 12 weeks of DMM and 9 weeks of SAMP8 treatment. No adverse effects were observed in either of the mouse models. Inhibition of CGRP signaling has the potential to be a therapeutic target to prevent OA progression and suppress pain through the attenuation of subchondral bone sclerosis and synovitis.

Figure 1

Histological evaluation of DMM mice at 4, 8, and 12 weeks. Histological evaluation of DMM mice at 4, 8, and 12 weeks. (A) Safranin O staining of knee joint. The bar indicates 500 µm. (B) The OARSI score was obtained by adding the femur and tibia (n = 6 at 4, 8 weeks, n = 12 at 12 weeks for each strain). (C) Micro-CT findings. (D) BV/TV of the subchondral bone epiphysis (n = 6 at 4, 8 weeks, n = 18 at 12 weeks for each strain). (E) HE staining of the knee joint. Bars indicate 100 μm. (F) Synovitis scores of DMM and sham mice at 4 and 12 weeks after surgery (n = 6 for each strain). A comparison of mean values was performed using the Kruskal–Wallis H-test. All data except for Synovitis scores were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 and **P < 0.01 were statistically significant.

Figure 2

Immunohistochemistry analysis on articular cartilage. (A) Immunohistochemistry of Type 10 collagen (Col 10). (B) ADAMTS-5. (C) MMP 13. The bar indicates 100 μm. (D) The ratio of Col 10, MMP13, or ADAMTS-5 positive cells and total cells (n = 6 at 4, 8 weeks, n = 9 at 12 weeks for each strain). All data were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 was statistically significant. ADAMTS-5, a disintegrin and metalloproteinase with thrombospondin motifs 5; MMP13, Matrix metallopeptidase 13.

Figure 3

Effects on bone metabolism in the subchondral bone. (A) Immunohistochemistry of osteocalcin. The bar indicates 500 μm. (B) The number of osteocalcin-positive cells in the subchondral bone's epiphysis (n = 6 at 4, 8, and 12 weeks for each strain). (C) TRAP staining. The bar indicates 100 μm. (D) The number of TRAP-positive cells in the subchondral bone's epiphysis. (E) Immunohistochemistry of CGRP/DAPI in marrow cavity in the subchondral bone. The bar indicates 100 μm. (F) Area of CGRP in the subchondral bone's epiphysis at 4 weeks post-surgery. (G) Immunohistochemistry of CD31/DAPI in marrow cavity in the subchondral bone. Confirmation of nonspecific staining using isotype control. The area surrounded by two white lines is the cartilage layer. All data except for the data of TRAP staining were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 was statistically significant. For TRAP staining data, Wilcoxon t-test with Bonferroni correction was used to compare between 4 and 8 weeks of age or between 4 and 12 weeks of age within each group, and Mann–Whitney U test was used to compare between control and rimegepant group at each time point. *P < 0.05 and **P < 0.01 were statistically significant. NS, not significant. TRAP, tartrate-resistant acid phosphatase; CGRP, calcitonin gene-related peptide.

Figure 4

Assessment of the analgesic effect. (A) Total distance traveled. (n = 6 at 4, 8 weeks, n = 18 at 12 weeks for each strain). (B) The number of standings. (n = 6 at 4, 8 weeks, n = 18 at 12 weeks for each strain). (C) Immunofluorescence staining for FG and CGRP of DRG. The CGRP/FG ratio of DRG in the rimegepant group was significantly lower than that in the control at 8 and 12 weeks post-surgery (n = 4–5 at 8 and 12 weeks for each strain). All data were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 was statistically significant. FG, fluoro-gold; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion.

Figure 5

Histological evaluation in SAMP8 at 9 and 23 weeks. (A) Safranin O staining of the knee joint at 9 weeks. The bar indicates 500 μm. (B) Subchondral bone score (n = 12 for each strain). (C) HE staining. The bar indicates 100 μm. (D) The synovitis score of SAMP8 at 9 weeks (n = 8 for each strain). (E) Safranin O staining of the knee joint at 23 weeks. The bar indicates 500 μm. (F) OARSI score at 23 weeks. (n = 12 for each strain). All data were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 and **P < 0.01 were statistically significant. SAMP8, senescence-accelerated mice-prone 8; NS, not significant.

Figure 6

Bone morphological assessment of SAMP8 using μCT. (A) μ-CT coronal images of the knee joint at 9 weeks. In the control group, bone sclerosis is particularly prominent on the medial tibial plateau, but in the rimegepant group, bone sclerosis is less common. (B) The BV/TV of subchondral bone at 9 weeks and 23 weeks. (n = 12 for each strain. Left and right knees were measured, and the average was calculated as the individual data.). All data were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 was statistically significant. (C) Cross-Sectional observation with 3DCT. BV, bone volume; TV, total bone volume; 3DCT, 3 dimensional computer tomography.

Figure 7

Evaluation of articular cartilage and DRG in SAMP8 at 9 weeks. (A) Immunohistochemistry of ADAMTS-5 and MMP 13. (B) The ratio of MMP13 or ADAMTS-5 positive cells and total cells (n = 6 for each strain). (C) Immunofluorescence staining for FG and CGRP of DRG. CGRP/FG ratio in the rimegepant group was significantly lower than that in the control at 9 weeks (n = 6 for each strain). (D) Immunohistochemistry of CD31/DAPI in marrow cavity in the subchondral bone. Confirmation of nonspecific staining using iso type control. The area surrounded by two white lines is the cartilage layer. The bar indicates 100 μm. All data were compared between control and rimegepant group by Mann–Whitney U test at each time point. *P < 0.05 was statistically significant. ADAMTS-5, a disintegrin and metalloproteinase with thrombospondin motifs 5; MMP13, Matrix metallopeptidase 13; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; FG, fluoro-gold; NS, not significant.

Figure 8

Assessment of the adverse event on bone and liver. (A) μ-CT slices of the sagittal view of the 3rd lumbar vertebra and the axial view of the distal femur in the DMM mice and sham mice at 12 weeks and the SAMP8 model at 23 weeks. (B) HE staining of liver sections. There was no apparent infiltration of inflammatory cells. (C) BMD of the distal femur and L3. In the DMM mice, comparison of mean values was performed using Kruskal Wallis H-test (sham, n = 6; n = 12 for each strain). In the SAMP8, comparison of mean values was performed using Mann–Whitney U test at each time point (n = 12 for each strain). *P < 0.01 was statistically significant. (D) AST and ALT serum level. There were no statistically significant differences in AST and ALT in either the DMM or SAMP8 models. DMM, destabilization of the medial meniscus; SAMP8, senescence-accelerated mice prone 8; BMD, bone mineral density; AST; aspartate aminotransferase; ALT; aminotransferase.