Combining naproxen and a dual amylin and calcitonin receptor agonist improves pain and structural outcomes in the collagen-induced arthritis rat model

Abstract

Background: Pain is a debilitating symptom of rheumatoid arthritis (RA), caused by joint inflammation and cartilage and bone destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain and inflammation in RA, but are not disease-modifying and do not prevent joint destruction when administered alone. KBPs (Key Bioscience peptides) are synthetic peptides based on salmon calcitonin and are expected to inhibit bone resorption and to be chondroprotective. In this study, we investigated if combining a standard of care NSAID (naproxen) with a KBP resulted in improvement in pain scores, as well as disease activity and structural damage in a rat model of RA.

Methods: Collagen-induced arthritis (CIA) was induced in 40 female Lewis rats by immunization with porcine type II collagen; 10 rats were given sham injections. CIA rats were treated with KBP and/or naproxen. Health scores and joint scores were evaluated daily. Mechanical and cold allodynia tests and burrowing tests were used to assess pain-like behaviors. Blood samples were collected for biomarker testing, and paws were collected for histology and microcomputed tomography.

Results: Naproxen monotherapy increased the time until humane endpoints was reached, and improved health score, pain assessments, and trabecular thickness, while KBP monotherapy did not result in improvements. Combination therapy had improved efficacy over naproxen monotherapy; combination therapy resulted in improved health scores, and importantly reduced mechanical and cold allodynia assessment. Furthermore, protection of articular cartilage structure and preservation of bone structure and bone volume were also observed.

Conclusions: This study demonstrates that combining KBP and naproxen may be a relevant therapeutic strategy for RA, resulting in improvements to the overall health, pain, inflammation, and joint structure.

Keywords: Bone; CIA; DACRA; NSAIDs; Pain; Rheumatoid arthritis; Treatment.

Fig. 1

Effects of KBP and naproxen on health and inflammation status. The time until any humane endpoint is presented as a Kaplan-Meier curve (a). Health and inflammation status was assessed using behavioral health scores (b), counting of swollen joints (c), and paw width measurements (d). Data in b–d are presented as the mean ± SEM, n = 10/group, using the last observation carried forward for euthanized rats. Asterisk (*) indicates statistical comparisons to CIA control, and currency sign (¤) indicates comparisons of CIA+Napr and CIA+KBP+Napr. *^/¤P < 0.05; **^/¤¤P < 0.01; ***^/¤¤¤P < 0.001; ****^/¤¤¤¤P < 0.0001

Fig. 2

Analgesic effects of KBP and naproxen. Analgesic effects on mechanical allodynia measured with von Frey filaments and the up-down method (a), on cold hypersensitivity measured with the acetone test (b), and on innate burrowing behavior measured with the burrowing test (c). Data in a are presented as the percent change from baseline, and data in b and c are presented as the mean change from baseline, all ± SEM, n = 10/group, using the last observation carried forward for euthanized rats. Asterisk (*) indicates statistical comparisons to CIA control.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

Effects of KBP and naproxen on bone resorption and type III collagen degradation. Bone resorption was assessed by CTX-I (a) and type III collagen degradation by MMP-9 was assessed using C3M (b). CIA control n = 6; CIA+KBP n = 5; CIA+KBP+Napr n = 10; CIA+Napr n = 10; sham n = 10. Data are presented as the mean fraction of each rat’s baseline measurement ± SEM using the last observation carried forward for euthanized rats. Asterisk (*) indicates statistical comparisons to CIA control.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 4

KBP and naproxen effects on ankle joint structure and immunohistochemical markers. Tibiotarsal joints were stained using the following procedures: SafO + Fast green staining to assess cartilage damage, TRAP and Mayer’s hematoxylin staining for the detection of osteoclasts, and CD68 and Mayer’s hematoxylin staining for the detection of macrophages. The bone marrow was stained using the following procedures: p75 and Mayer’s hematoxylin staining and TRPV1 and Mayer’s hematoxylin staining, both to assess innervation. Representative micrographs are shown for n = 10/group for the SafO staining and n = 4/group for the other stainings. Scale bars indicate 200 μm

Fig. 5

KBP and naproxen effects on bone microstructure. μCT was performed to analyze structural effects of CIA and the treatments. Representative 3D reconstructions (a) with regions of interest (black boxes) and 2D contouring (b, c) indicate the area for three tibiotarsal (d, f) and metatarsal (e, g) analyses. The exact of bone volume to total volume (BV/TV, d, e) and trabecular thickness (Tb.Th, f, g) are reported. The data are presented as the means ± SEM of n = 10/group. Asterisk (*) indicates statistical comparisons to CIA control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001