Effect of (3,5,6-Trimethylpyrazin-2-yl)methyl 2-[4-(2-Methylpropyl)-phenyl] propanoate (ITE), a Newly Developed Anti-inflammatory Drug, on Type II Collagen-Induced Arthritis in Mice

Abstract

Type II collagen (CII)-induced arthritis (CIA) in rats 1) and mice 2) has both clinical and histological similarities to human rheumatoid arthritis.1-4) Trentham 1) suggested that an immune reaction to collagen may be involved in the pathogenesis of arthritis. The CIA model has been widely used to evaluated antiarthritic drugs.5-7) ITE (3,5,6-trimethylpyrazin-2-yl)methyl 2-[4-(2-methylpropyl)phenyl]propanoate ( Fig. 1), is a newly developed anti-inflammatory drug.8) In the present study the effect of ITE on CIA in mice and its effect on immune responses to CII in CIA mice were evaluated. MATERIALS AND METHODS AnimalsMale Kun-ming mice weighing 18-22 g were purchased from Shenyang Pharmaceutical University Laboratory Animal Center. Mice were housed in a room under a 12 h light-dark cycle and quarantined for one week before use. The mice were given standard laboratory chow and tap water ad libitum.Drugs ITE synthesized at Hua-tai Institute of Pharmaceutics Science was used. ITE occurs as white powder. Purity of ITE is more than 98.5%. ITE (150, 300, 600 mg/kg) or ibuprofen (IB, 180 mg/kg) was suspended in a 0.5% carboxymethylcellulose (CMC)-saline solution and given orally to mice once a day for 40 d from the day of 1st immunization with CII. Mice in the control group were orally given 0.5% CMC-saline. Type II collagen were purchased from Sigma Co. (St. Louis, U.S.A.). Induction of Arthritis 7)CII was dissolved overnight at 4°C in 0.1 M acetic acid at 2 mg/ml, after which the solution was emulsified in an equal volume of complete Freund's adjuvant (CFA). This emulsion containing 100 mg of CII was injected intradermally into the base of the tail, and the mice were given an intraperitoneal injection of the same emulsion 20 d later.Clinical Assessment 7)The clinical symptoms of arthritis in limbs were evaluated blindly with a visual scoring system every 4 d after day 20. Arthritic lesion of a limb was graded on a scale of 0-4: 0ϭno change, 1ϭswelling and erythema of the digit, 2ϭmild swelling and erythema of the limb, 3ϭgross swelling and erythema of the digit, 4ϭgross deformity and inability to use the limb. The arthritis score of each mouse was the sum of the scores of each of the limbs except the right hind limb, the maximum score being 12. A mouse that showed a score of 1 or more was regarded to be arthritic. The incidence and day of onset of arthritis also were recorded. The right hind paw's volume of each mouse was determined on days 0, 20, 24, 28, 32, 36 and 40 after 1st immunization with CII. Measurement of Anti-CII Antibodies 7)On day 40, blood was collected from the mice, after which the serum level of IgG antibodies to CII was determined by the enzyme-linked immunosorbent assay (ELISA). Briefly a 100 ml sample of serum (1 : 2000) in phosphate-buffered saline (PBS) was added to the wells of a 96 well plate that had been coated with CII overnight at 4°C. The plate was incubated for 2 h at room temperature then, after the washing of the wells with PBS, an addition was made of 100 ml goat antimouse IgG (1 : 40...