Pos0838 agomelatine Identified From the Fda-Approved Drug Library Is Therapeutic Against Collagen Induced Arthritis

Abstract

BackgroundTreatment effect of tumor necrosis factor α (TNFα) inhibitors is still low in patients with rheumatoid arthritis (RA), around 50%-70%. Thus more drugs by targeting proliferation of synovial fibroblasts (FLS) and TNFα induced inflammatory cytokine production are needed. Repurposed use of drugs that have been used in clinic is a quick and cost-effective way to find new drugs.ObjectivesThis study aims to screen drugs that could inhibit the proliferation and inflammation induced by TNFα in FLS from FDA approved on market drug library, then to assess the treatment effect of the identified drugs on collagen-induced arthritis (CIA) mouse model.MethodsCCK8 assay was performed to screen the drugs that could inhibit FLS proliferation, followed by qRT-PCR and ELISA to select the drugs that could suppress TNFα induced inflammatory cytokine production. Then, treatment effects of the identified drugs were assessed in CIA mouse model.ResultsThe first and second round drug librabry screening aimed to select out the drugs that could inhibit the proliferation of FLS. Results showed, from 1815 drugs, 372 drugs were indentified at the initial screening (Figure 1A) and 121 drugs were identified from the second screening (Figure 1B). The third round screening was performed to screen the drugs that could inhibit TNFα-induced inflammation, and results showed that a total of 77 drugs could inhibit mRNA expression levels of both IL-6 and IL-8 by over 50%, and 64 drugs could suppress secretion levels of both IL-6 and IL-8 for more than 20% (Figure 1C-F). Then a total of 14 drugs that were not anti-cancer drugs were used to further confirm the inhibitory effect on the TNFα induced inflammation, and results showed only Agomelatine (AOM), Cinacalcet (CCC), Amlodipine (ALM), Simvastatin (SV), and Pindolol (PDL) could suppress mRNA expression and secretion levels of IL-6, IL-8 (Figure 1G-J).As highly proliferated FLS and inflammatory cytokines play a critical role in the pathology of rheumatoid arthritis (RA), we tested the treatment effect of AOM, CCC, ALM, SV, and PDL in the CIA mose model. Results showed ALM (5mg/kg), CCC (10mg/kg), and PDL (10mg/kg) had a trend to reduce the clinical score but did not reach statistical meaning while SV (10mg/kg) did not show any treatment effect. Whereas, AOM (10mg/kg) could effectively alleviate the swelling of the the joint, which was as effective as the positive control drug methotrexate (MTX) (Figure 1K). ALM, AOM, as well as MTX could reduce the pathological score of inflammation and bone erosion at both the knee and the anke while PDL could only reduce the inflammation and relieve bone erosion at the knee(Figure 1L-N).ConclusionAgomelatine indentified from the FDA approved drug library could inhibit FLS proliferation and TNFα induced inflammation, and was therapeutic against CIA mouse model.Figure 1.Agomelatine identified from the FDA-approved drug library is therapeutic against collagen induced arthritis. (A) The first round screening by CCK8 assay. The drugs could inhibit more than 20% absorbance at 450nm, under the red lines, were the target ones, n=372. (B) The second round screening. The drugs could inhibit more than 10% absorbance at 450nm, under the red lines, were the target ones, n=121. (C-F) The third round screening. (C-D) mRNA expression levels of IL-6 and IL-8. (E-F) the secretion levels of IL-6 and IL-8. (G-J) The confirmation of drugs’ inhibitory effect on the inflammation cytokine production. Red asterisk indicated the conparison between TNFα group and DMSO group and blue pound signs suggested the conparison between drugs and TNFα group. (G-H) the mRNA expression levels of IL-6 and IL-8. (I-J) the secretion levels of IL-6 and IL-8. CIA mouse model was established to test the treatment effect of SV (10mg/kg), PDL (10mg/kg), ALM (5mg/kg), CCC (10mg/kg), and AOM (10mg/kg), and MTX (2mg/kg) was set as the positive control. (K) clinical score of joint swelling (L) H&E staining of knee joint (M) H&E staining of ankle joint (N) pathological scoring of H&E staining for joint inflammation and bone erosion.AcknowledgementsThis study is supported by Sichuan Science and Technology Program (2021JDRC0045 and 2021YFS0164), Post-doctoral Research and Development Fund of West China Hospital of Sichuan University (2019HXBH090), and National Natural Science Foundation of China (No. 82201985).Disclosure of InterestsNone Declared.