Female lewis rats

Female Lewis rats (150 – 170g; Charles River Laboratories) were house standard shoebox cages, fed Harlan-Tekald diet and water ad libitum and maintained on a 12h light/dark cycle. For induction of arthritis rats were anesthetized with isofluorane and their entire dorsal area shaved. Rats were injected (intradermal) with 100 μl of equal volumes of collagen (Chrondrex, Redmond, WA) and IFA (Sigma Chemicals, St. Louis, MO) emulsion in 10 sites across the dorsal region. At disease onset 11 days following immunization animals were randomized into groups based on their ankle measurements and test article administration was initiated. Rats were dosed orally with vehicle (20% Captisol®), Indomethacin (Sigma Chemicals, St. Louis, MO, 2mg/kg) or CC-509 (15, 25mg/kg QD or 15mg/kg BID) in a dose volume of 5ml/kg. Paw swelling was assessed by caliper measurements on day 11, 12, 13, 14, 15 and 18 post-immunization and % inhibition calculated at day 18 compared to the vehicle control or area under the curve (AUC) calculated from day 11–18. On the last day of the study animals were split into 2 cohorts and bleed twice via tail bleed under isofluorane anesthesia and a third terminal bleed following CO₂ asphyxiation to obtain a time course of plasma compound exposure at 30minutes, 1, 2, 4, 8 and 24h after the last dose of CC-509.

Female Lewis rats with an age of 6–8 weeks were purchased from Charles River (Sulzfeld, Germany). Animals were in the range of 160–180 g when immunized and at least 1 week in the animal facility to get accustomed to the new environment. Rats were housed under standardized, pathogen-free conditions at the local animal facility (Medical Faculty, Ruhr-University Bochum, Bochum, Germany) where food and water were given ad libitum.
For immunization, 10 mg/kg xylazine (Xylavet, CP-Pharma, Burgdorf, Germany) and 50 mg/kg ketamine (CP-Pharma, Burgdorf, Germany) were used intraperitoneally (i.p.) to anesthetize the rats. Two hundred fifty microgram P2_53–78 in phosphate buffer saline (PBS) emulsified in equal volume of CFA containing 1 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, USA) were used to subcutaneously inject into the tail base of the rats. From the day of immunization, animals were weighed and scored daily. To determine a disease score, a tenfold system was used (0 normal; 1 less lively; 2 impaired righting/limb tail; 3 absent righting; 4 ataxic gait, abnormal position; 5 mild paraparesis; 6 moderate paraparesis; 7 severe paraplegia; 8 tetraparesis; 9 moribund; 10 death) (Enders et al. 1998). Animal experiments were approved by the North-Rhine-Westphalia, Germany authorities (Az.: 84-02.04.2015.A420).

Female Lewis rats (177–199 grams, 45 days old) were obtained from Charles River Laboratories, Inc., (Wilmington, MA, USA) and the study was conducted at Bolder BioPATH (Boulder, CO, USA). Animals were housed in a laboratory environment with temperatures ranging between 67–76°F and relative humidity between 30–70%. Automatic timers provided 12 hours of light and 12 hours of dark. Animals were allowed access ad libitum to Harlan Teklad Rodent Chow (Denver, CO, USA) and fresh municipal tap water.

This study was carried out at the University Medical Center Göttingen in strict accordance with recommendations of European and German guidelines for welfare of experimental animals. Animal experiments were approved by the Review Board for the Care of Animal Subjects of the district government of Lower Saxony (LAVES, approval number 13/1197). Female Lewis rats (Charles River Laboratories), aged approximately 3 months, were included in the study. Unless specified otherwise, anesthesia was performed using i.p. injection of ketamine (60 mg/kg) and xylazine (8 mg/kg).

Female Lewis rats, 6–8 weeks old, were purchased from Charles River Laboratories Italia (Calco, Italy) housed at the animal facility of the Foundation IRCCS Neurological Institute Carlo Besta. Rats were housed in groups of three in cages with artificial circadian 12-h light/12-h dark cycle, maintained at air-conditioned room with temperature of 23°C at all time, with free access to a standard stock diet and water provided ad libitum. Procedures involving animals were approved by the Institute Ethical Board and Italian Ministry of Health (1064/2015- PR) and were performed in respect to the Italian Principle of Laboratory Animal Care (D.Lgs 116/92 and D.Lgs 26/2014), in accordance to European Communities Council Directive 86/609/EEC and 2010/63/UE. Animals were sacrificed after deep anesthesia obtained by carbon dioxide; low-grade anesthesia with 2% isoflurane (60:40 N₂O: O₂, flow rate 0.8 l/min) was induced in animals prior to immunizations and treatments.