Effects of Medium Molecular Weight Heparinyl Phenylalanine on Type I Hypersensitivity

Materials and Methods

Animals. Specific pathogen-free male Wistar rats (8 weeks old), female Wistar rats (5 weeks old), and male ICR mice (3.5 weeks old) were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan), and used for the experiment after a one-week acclimation period. The animals were maintained at 23±2°C (room temperature) and 50±5% relative humidity under an artificial 12-h light-dark cycle (7:00 on-19:00 off). Food and water were given ad libitum during the experimental period. All procedures followed the official regulations for the care and use of laboratory animals approved by the animal experimentation committee of Fuso Pharmaceutical Industries Ltd. (approval number: PDS9611).

Materials. MHR and MHF (mean molecular weight: 8,500-10,000) were synthesized at Fuso Pharmaceutical Industries Ltd. Research and Development Center (Osaka, Japan) (8), and bulk HE (Scientific Protein Laboratories, Waunakee, WI, USA) was used.

Preparation of the anti-ovalbumin rat serum. The anti-ovalbumin (OVA) rat serum was prepared after modification of the method by Stotland LM and Share NN (9). Four days after the operation to remove the spleen from female rats, 0.25 ml of an equal mixture of saline containing 2.0 mg/ml of OVA and 2.0% aluminum hydroxide gel was subcutaneously injected into the limbs. In addition, dead Bordetella pertussis cells (2×10¹⁰ cells/ml saline) were injected intraperitoneally. After five days, as an additional sensitization, 1.0 ml of an equal mixture of saline containing 2.0 mg/ml of OVA and 2.0% aluminum hydroxide gel was subcutaneously injected into the back of rats. Two weeks after the last sensitization, blood was collected from the abdominal aorta and serum was prepared. The PCA antibody titer of this antiserum was 128.

Induction of 48-h homologous PCA in rats and treatment with MHR or MHF. One hundred μl/site of diluted anti-OVA rat serum to antibody titer 16 in saline was intradermally injected into the shaved back of rats. Forty-eight hours later, 10 mg/ml/rat of Evan's blue saline solution containing 1 mg of OVA was injected into the tail vein of rats to induce an allergic reaction. Thirty min later, rats were euthanized by exsanguination and the amount of leaked dye in the back skin was measured using the method of Katayama et al. (10). MHR, MHF (0.3, 3.0, and 30.0 mg/ml/kg, respectively), or saline was injected into the tail vein of rats five min before inducing the allergic reaction.

Administration of MHR, MHF and compound 48/80 in mice. The experiments were carried out according to the method by Tasaka et al. (11). MHR, MHF (10 mg/10 ml/kg), or saline was injected into the tail vein of the mice. Five min later, compound 48/80 (3 mg/10 ml/kg) (Sigma-Aldrich Co., St. Louis, MO, USA) was injected into the tail vein, and the survival time was measured.

Administration of MHR, or MHF and histamine in mice. MHR, MHF (10 mg/10 ml/kg), or saline was injected into the tail vein of mice. Five min later, histamine (Sigma-Aldrich) (400 mg/10 ml/kg) was injected into the tail vein, and 3 h later, survival was evaluated.

Statistical analysis. Data are presented as the mean±S.E., and significance was evaluated by ANOVA followed by the Spjotvoll and Stoline test (corrected Tukey's test) because the number of samples among the groups differed. The differences were assessed at a significance level of 0.05.