It has been well established that myasthenia gravis (MG) is caused by autoimmune responses against nicotinic acetylcholine receptor (AChR) on postsynaptic membrane in neuromuscular junctions.1) Experimental autoimmune myasthenia gravis (EAMG) has been used as an animal model for human MG. This model is prepared by immunizing mice or rats with AChR from Torpedo californica; the animals develop MG-like symptoms, including muscle weakness, with production of autoantibody against AChR.2)The novel immunomodulator (immunosuppressant) FTY720, which was discovered by Fujita et al. 3,4) of our group, is a synthetic structural analogue of myriocin (ISP-I), a metabolite of Isaria cinclairii.5) The efficacy of FTY720 has been well established in preclinical transplantation models, 6) and also recently in renal transplantation in humans. [7][8][9][10] Although the mechanisms of pharmacological action of FTY720 remain to be fully clarified, 11,12) it has been proposed that FTY720 is phosphorylated by sphingosine kinase, and the product, FTY720 monophosphate, suppresses immune response by sequestering lymphocytes from blood and peripheral tissues to the secondary lymphoid tissues.13-16) FTY720 has no inhibitory effect on cytokine production in vitro, in contrast to established immunosuppressants (cyclosporin and tacrolimus hydrate).16) Its mechanism of action is unique and differs from that of the established immunosuppressants. The development of FTY720 was described in our previous report.
17)In this study, we examined the efficacy of FTY720 for preventing the development of EAMG, an animal model of human MG.
MATERIALS AND METHODSAnimals Specific pathogen-free (SPF) C57BL/6 mice (7 weeks of age, females) were purchased from Japan SLC Inc., Shizuoka, Japan. The mice were given g-ray-irradiated food (CRF-1, Oriental Bio Co., Kyoto, Japan) and distilled water for injection (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).FTY720 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720) was kindly provided by Yoshitomi Pharmaceutical Industries, Ltd., Japan.Acetylcholine Receptor from Torpedo californica Electric organ of Torpedo californica was purchased from Aquatic Research Consultants (San Pedro, CA, U.S.A.), and acetylcholine receptor (tAChR) was purified by the method of Froehner and Rafto using cobrotoxin-coupled Sepharose.18) AChR concentration was quantified by the Lowry method.Evaluation of Clinical Symptoms of EAMG Muscle strength was evaluated by the inverted hanging technique as described by Karachunski et al. 19,20) Mice with a holding time of 10 min or more were considered normal, and those with a holding time of less than 10 min were considered as having EAMG.Study Protocol Twelve C57BL/6 mice were immunized intracutaneously on the back with tAChR (5 mg) in the presence of Freund's complete adjuvant at 8, 10 and 11 weeks of age. Six mice (treated group, Nos. 1-6) out of the twelve were orally given FTY720 in water (1.0 mg/kg) three times a week from the day before the first immunization. The other...
