Knee osteoarthritis pain following medial meniscectomy in the nonhuman primate  S. Ogawa, Y. Awaga, M. Takashima, A. Hama, A. Matsuda, H. Takamatsu  Osteoarthritis and Cartilage  Volume 24, Issue 7, Pages 1190-1199 (July 2016) DOI: 10.1016/j.joca.2016.02.006 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Illustration of the study time line and crossover design. A. Knee pressure thresholds and weight bearing measurements were taken before MMx (“Pre, MMx surgery”). NHPs (n = 9) underwent an exercise routine between Day 15 and Day 51, 5 days per week, and periodic measurement of physical parameters and behavioral testing (see Fig. 4 and Supplementary Fig. 1). Sixty-seven days after MMx, macaques were randomized into groups (n = 3/group) following baseline behavioral measurements (“Pre”). The day after randomization, macaques were dosed and tested for five consecutive days. Between Test Cycles, macaques underwent a concurrent washout (9 days in duration) and exercise period (between 4 and 5 days). Testing was completed 33 days after randomization (or 100 days after MMx). B. Treatment test cycles. In each Test Cycle, macaques received either vehicle (1 ml/kg, p.o.), diclofenac (10 mg/kg, p.o.) or aprepitant (10 mg/kg, p.o.). All animals received all of the treatments by the end of the study. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Osteoarthritic pathology of the knee joint following MMx in NHP. Macaques were euthanized 130 days post-MMx and the contralateral and ipsilateral knee joints were coronally sectioned through the medial femoral–tibial joint space. Three μm sections were stained with safranin O and counterstained with hematoxylin and Fast green. A. The contralateral knee joint shows the normal thickness and smooth surface of articular cartilage of the medial femur and tibia. A portion of the medial meniscus (M) is seen. B. The ipsilateral knee joint shows significant reductions of proteoglycan, as shown by diminished safranin O (open arrows). Where present, the surface of the articular cartilage is rough or uneven. Areas of cartilage erosion to the tidemark (black arrows) can be observed and subchondral bone appears to be thickened. Numbered areas (➀,➁,➂) show chondrocytes clusters (“cloning”) within the articular cartilage. C. Higher magnification of numbered areas in B, showing chondrocytes clusters in articular cartilage. Scale bar = 500 μm. C, cartilage; FC, femoral condyle; TP, tibial plateau; M, meniscus; SB, subchondral bone. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of a sham knee surgery on knee pressure thresholds and weight bearing in macaques. Prior to sham surgery (“Pre-”), knee pressure thresholds and weight bearing were measured. Following sham surgery, macaques underwent an exercise regimen similar to that of macaques that underwent MMx and periodic measurement of pressure threshold and weight bearing. A. Pressure thresholds of the ipsilateral knee compared to that of the contralateral knee were converted into a percent (%). The dotted line indicates equal pressure thresholds of the ipsilateral and contralateral knees (100%). Before sham surgery, the pressure threshold of the ipsilateral knee was slightly higher compared to that of the contralateral knee. However, no statistically significant differences in knee pressure were observed over time following sham surgery (P = 0.1546, Dunnett's test). B. The amount of force exerted by the legs was measured. Before sham surgery, weight bearing was about 50% (dotted line). No statistically significant change in weight bearing was observed over time following sham surgery (P = 0.1540, Dunnett's test). Data are expressed as mean ± S.D. n = 3. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Knee pressure threshold and weight bearing in NHP following a MMx. Macaques were tested prior to MMx (“Pre-”). A. Pressure was applied to the knee joint and the threshold was recorded when the animal displayed a pain-related response. The threshold of the ipsilateral (right) knee was compared to that of the contralateral (left) knee and converted into a percent (%). Prior to MMx surgery, knee pressure thresholds were equal (100%; dotted line). Following MMx surgery, a statistically significant decrease in ipsilateral knee pressure threshold was observed over time. B. Amount of force exerted by the legs was measured. Before surgery, animals demonstrate equal weight bearing by both legs (50%; dotted line). Following MMx surgery, a statistically significant decrease in ipsilateral weight bearing was observed over time. Data are expressed as mean ± S.D. n = 9. **P < 0.01 vs Pre-MMx baseline, Dunnett's test. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of drug treatments over time on knee pressure threshold in NHP following a MMx. Pressure was applied to the knee joint and the response threshold was recorded when the animal displayed a pain-related behavior. Threshold of the ipsilateral knee was compared to that of the contralateral knee and converted into a percent (%). Baseline pressure thresholds were measured on Days 0, 14 and 28 and macaques were dosed and tested over the subsequent 5 day period. Data from individual animals are shown from all three Test Cycles (total n = 9). A. Increasing pressure thresholds over time with diclofenac treatment. By contrast, neither aprepitant nor vehicle increased knee pressure thresholds. *P < 0.05, **P < 0.01 vs pre-treatment baseline (“Pre”). B. Significant increases of pressure thresholds following diclofenac treatment compared to that of vehicle. ++P < 0.01 vs vehicle. Data are expressed as mean ± S.D. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effect of drug treatments on weight bearing in NHP following a MMx. Amount of force exerted by the legs was measured. Weight bearing of the ipsilateral limb was compared to the contralateral limb and converted to a percent (%). Baseline weight bearing was measured on Days 0, 14 and 28 and macaques were dosed and tested over the subsequent 5 day period. Data from individual animals are shown from all three Test Cycles (total n = 9). A. Increasing weight bearing over time with diclofenac treatment. By contrast, neither aprepitant nor vehicle increased weight bearing. **P < 0.01 vs pre-treatment baseline (“Pre”). B. Significant increases of weight bearing following diclofenac treatment compared to that of vehicle. +P < 0.05, ++P < 0.01 vs vehicle. Data are expressed as mean ± S.D. Osteoarthritis and Cartilage 2016 24, 1190-1199DOI: (10.1016/j.joca.2016.02.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions