Male Lewis rats aged 6 weeks (Charles River Japan) were maintained under temperature- controlled environment with a 12 hr light/dark cycle and with access to standard pelleted food (Picolab Rodent Diet 5053, St. Louis, Mo, USA) and water ad libitum. All animal experiments were procedures approved by the Institutional Animal Care and Use Committee of the Hanmi Research Center and performed in accordance with the approved guideline.
Induction of CIA in rats
Lewis rats were injected with 0.6 ml of an emulsion of bovine type II collagen (Chondrex, Redmond, WA, USA) and Incomplete Freund’s Adjuvant (Sigma-Aldrich, St Louis, MO, USA) via intradermal injection at the base of the tail. Seven days later, the 0.3 ml of the same emulsion was immunized in the same manner.
Two independent experiments in CIA rats were performed. In the experiment to determine the therapeutic effects of the HM71224 monotherapy, the rats (n = 7 per group) were orally administered at 0.3, 1, or 3 mg/kg HM71224 once per day for 9 days; in the experiment to determine the combination effects, the rats (n = 5 group) were orally administered HM71224 (1 mg/kg, once daily), MTX (1 mg/kg, twice per week) or HM71224 and MTX for 10 days. In both studies, the treatments were started 6 days after the booster immunization.
Clinical assessment of arthritis
The severity of arthritis was assessed by arthritis score, body weight loss, and paw volume. The arthritis score and body weight were measured three times per week. The arthritis score was determined by the grading of each paw from 0 to 4 (0, normal; 1, mild, but definite redness and swelling of the ankle or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2, moderate redness and swelling of the ankle; 3, redness and swelling of the entire paw including digits; 4, maximally inflamed limb involvement of multiple joints) and was expressed as the sum of the scores of all four paws. The volume of the two hind paws was measured by using a plethysmometer (Ugo Basile, Italy) 9 days after the first administration for the combination effects experiment.
Blood chemistry and hematology
In the experiment to determine the combination effects, ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood and serum were collected on day 9. The complete blood cell counts (CBCs) were obtained by using ADVIA120 automatic blood analyzer (SIEMENS, Germany) and the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine were measured by a Hitachi 7020 automatic chemical analyzer (Hitachi, Japan).
The hind legs of each rat were fixed with 10% neutral-buffered formalin, decalcified with 5% formic acid, and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) and safranin-O to identify bone damage and cartilage damage, respectively. The histopathological score was evaluated microscopically in a blinded manner, and was expressed as the sum of the scores of two legs. The arthritis score was evaluated on a scale of 0 to 4: 0, normal; 1, hyperplasia of the synovial membrane and presence of polymorphonuclear infiltrates; 2, pannus and fibrous tissue formation and focal subchondral bone erosion; 3, articular cartilage destruction and bone erosion; 4, extensive articular cartilage destruction and bone erosion. The bone erosion score was graded from 0 to 4 for severity: 0, normal; 1, focal subchondral erosion; 2, multiple subchondral erosions; 3, multiple subchondral erosions and focal erosion of talus; 4, multiple erosions of the tarsal and metatarsal bones. Synovitis was scored from 0 to 4: 0, normal; 1, mild synovial hypertrophy (< 5 cell layers) with few inflammatory cells; 2, moderate synovial hypertrophy (< 20 cell layers) with the accumulation of inflammatory cells into intrasynovial cysts; 3, pannus and fibrous tissue formation, abscess and interstitial edema; 4, pannus and fibrous tissue formation, abscess, and interstitial edema on both sides of the ankle joint. The cartilage degradation was scored semiquantitatively from 0 to 4: 0, intact; 1, minor depletion (< 10%); 2, moderate depletion (10–50%); 3, high depletion (50–80%); 4, severe depletion (80–100%).
Pharmacokinetics to evaluate drug-drug interactions
In experiment for combination effects, blood was collected in tube containing heparin (1000 IU/mL, Choongwae pharmaceutical, Korea) from the jugular vein at 0, 0.5, 1, 2, 4, 7, and 24 h after the final administration to CIA rats on day 11 and the plasma was obtained by centrifugation at 12,000 rpm for 2 min. Plasma levels of HM71224 and MTX were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS, Agilent™ 1200series, Santa Clara, CA, USA and API 5000™ LC/MS/MS system, Applied Biosystems/MDS SCIEX, Canada). Pharmacokinetic parameters were calculated from the plasma concentration-time data by a non-compartmental method using Phoenix™ WinNonlin® 6.1 (Pharsight, Princeton, NJ, USA).
Except where indicated otherwise, the data were expressed as the mean ± SEM. Differences between the mean values of the groups were compared by a parametric one-way ANOVA test or non-parametric Kruskal-Wallis test using Prism 5.0 software (GraphPad, La Jolla, CA, USA). P values below 0.05 were considered statistically significant. Effective dose levels were also calculated using Prism 5.0 software.