P142 ROS101, a novel targeted methotrexate prodrug, selectively activated by reactive oxygen species (ROS) shows reduced toxicity in rat CIA model of rheumatoid arthritis
Authors: V Previtali, N Woodworth, MH Hopkins, JH Petersen, I Ahnfelt-Rønne, MH Clausen
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Career situation of first and presenting author</h3> Post-doctoral fellow. <h3>Introduction</h3> The use of methotrexate (MTX) for the treatment of rheumatoid arthritis (RA) is limited by serious adverse effects. Some effects, such as stomatitis and gastric ulcer, can be alleviated by folate supplement. Others are folate-independent, e.g. fibrosis of liver and lung. Moreover, MTX further elevates the micronuclei count, which is generally already increased in RA. In RA, inflamed tissue is characterized by up to 100-fold increased concentrations of reactive oxygen species (ROS, including hydrogen peroxide) compared to healthy tissue. A novel MTX prodrug, ROS101 in development for the treatment of RA, releases MTX at exposure to ROS. This restricts MTX exposure to target tissues with increased ROS levels, e.g. the synovial membrane in RA. <h3>Objectives</h3> To investigate the effect and toxicity of a targeted MTX prodrug in a rat CIA model of rheumatoid arthritis. <h3>Methods</h3> ROS101 was tested in a collagen induced arthritis (CIA) model in dark agouti (DA) rats (10 per group) against vehicle and 0.3 mg MTX i.p./day at equimolar doses. Arthritis was induced by s.c. injection of an emulsion of collagen type II in complete Freund's adjuvant. Treatment was initiated at a mean arthritis severity score of 2/60. Blinded disease evaluation took place 3 times/week from day 10. Bone marrow cells for micronucleus test were obtained from the femur, pictures captured with LARS.4.8 software and analyzed blinded. Individual rat data were included until termination. <h3>Results</h3> Mean and maximum arthritis severity score was reduced in rats in the ROS101 group compared to vehicle (mean score 6.3±3.1 vs. 14.1±3.3, mean±SEM); (max score 12.6±5.3 vs. 32.3±5.3, mean±SEM, p&lt;0.05). All rats in the ROS101 group completed the study until day 32. Reduced arthritis severity in the MTX group after day 22 was accompanied by overt toxicity, causing all rats to be terminated from day 24 to 29. There was no significant difference in body weight between rats in the ROS101 and vehicle groups, whereas body weight in the MTX group decreased significantly from day 22 due to toxicity, including GI effects. Bone marrow micronucleus count was reduced in the ROS101 group in contrast to the MTX group, where micronucleus count was elevated as compared to the vehicle control group. <h3>Conclusions</h3> The CIA study in rats indicates that the MTX prodrug ROS101 may be efficacious for the treatment of RA at an equimolar dose compared to MTX, while avoiding adverse effects known to restrict treatment with MTX. <h3>Acknowledgements</h3> C.A. Hansen, co-founder of ROS Therapeutics. <h3>Disclosure of Interest</h3> V. Previtali: None declared, N. Woodworth: None declared, M. Hopkins: None declared, J. Petersen Shareholder of: ROS Therapeutics, Consultant for: ROS Therapeutics, I. Ahnfelt-Rønne Shareholder of: ROS Therapeutics, Consultant for: ROS Therapeutics, M. Clausen Shareholder of: ROS Therapeutics.