The National Science Foundation's (NSF) Tokyo Office periodically receives and disseminates reports on research developments in Japan that are related to the Foundation's mission. NSF-sponsored researchers currently working in Japan prepare many of these reports. These reports present information for use by NSF program managers and policy makers; they are not statements of NSF policy.
Special Scientific Report #00-14 (November 16, 2000)
Mr. Brian Klein., a graduate student in the Department of Pharmacology and Toxicology at the University of Utah, prepared the following report. Mr. Klein was a participant in the 2000 Summer Institute sponsored in the United States by NSF/NIH/USDA and the Science and Technology Agency and Japan Science and Technology Corporation in Japan. Dr. Kazuo Tsukidate of the Exploratory Safety Assessment Research Division at the Eisai Company in Tsukuba hosted Mr. Klein. Mr. Klein can be reached via email at: b.klein@m.cc.utah.edu.
Multiple Sclerosis (MS) is an autoimmune disease producing inflammation and neurodegeneration in the central nervous system (CNS). Deficits in sensory and motor function and severe fatigue are symptoms of the disease. In the relapsing-remitting form of MS, symptoms lasting days to weeks are followed by recovery. As the disease progresses, it usually evolves into a secondary-progressive disease where relapses are followed by gradual progression of the disease leading to permanent disability.
Current therapies for MS include the use of glucocorticoids to treat active disease and the prophylactic use of synthetic interferon beta (INFb) preparations. Glucocorticoids can reduce symptoms while they are administered and improve the rate of recovery from relapse. However, glucocorticoids have not been shown to slow progression of MS (1,2). Maintenance therapy with glucocorticoids is seldom used due to the numerous adverse effects of long-term glucocorticoid therapy. INFb preparations reduce the frequency of flares by 30 percent in relapsing-remitting MS and may delay progression of the disease (2). The specific mechanisms by which the INFb drugs inhibit MS are not known, but like glucocorticoids, are probably due to their immunosuppressive effects (3). The current therapies for treatment of MS are only partially effective, result in modulation of the immune system and have adverse effects; therefore additional therapeutic options are necessary.
Although the cause of MS is unknown, a rodent animal model for MS called experimental autoimmune encephalomyelitis (EAE) is available for research. The EAE rodent model produces inflammation and demyelination in the CNS with symptoms similar to MS. Macrophages and activated microglia (immune-like cells in the brain) are thought to be involved in the damage leading to demyelination in MS (1). In addition, the neurotransmitter glutamate has been implicated in many neurodegenerative diseases. Oligodendrocytes, the cells that produce myelin, are particularly sensitive to AMPA receptor (a type of glutamate receptor) mediated cell death (4). In MS, disturbances with the glutamate neurotransmitter system have been observed. AMPA receptor antagonists have been demonstrated to be effective in blocking cell death and axon loss in the EAE model without modulatory effects on the immune system (4). Two compounds investigated by Eisai Tsukuba Laboratories referred to as Multiple sclerosis AMPA Receptor Selective antagonist (MARS) 1 and MARS 2, are being pursued for the treatment of MS. Both are orally active and confer protection to oligodendrocytes and neuronal axons in the EAE rodent model for MS.
The objectives of this study were to determine the maximal tolerated dose (MTD) for MARS 1 and MARS 2 for future long-term studies and to characterize any toxicity potential.
Each test compound; MARS 1, MARS 2, and vehicle control (0.5 percent methylcellulose in water) was administered orally once per day to 17 month old beagles (two males and two females per test compound). A dose escalation design was utilized with 0.3 mg/kg given on days 1 and 2, 1.0 mg/kg on days 3 and 4, 3.0 mg/kg on days five and six, and 10 mg/kg on days seven and eight.
Clinical signs, body weight and food consumption were recorded each day of the study. Laboratory measurements including hematology and clinical chemistry were conducted before testing began (pre-test) and after final dose. Toxicokinetic measurements were conducted at pre-dose, 0.5, 1, 2, 4, 8 and 24 hours after dosing on days one, three, five and seven. Postmortem investigation included macroscopic examination and histopathology on the liver, kidneys, heart, lungs, spleen, adrenal glands, testis, epididymis and ovaries.
Administration of MARS 1 at 0.3 mg/kg and 1.0 mg/kg produced no abnormal clinical signs. After the 3.0 mg/kg dosings, abnormal clinical observations included slight ataxia, abnormal gait, recumbency, loose stool and remittance. After the 10.0 mg/kg dosings, abnormal clinical signs were severe ataxia, prostration, loose stool and remittance. Clinical signs after administration of MARS 2 at 0.3 mg/kg and 1.0 mg/kg included ataxia, abnormal gait, recumbency, loose stool and remittance. Administration of MARS 2 at 3.0 mg/kg produced severe ataxia, prostration, loose stool and remittance. The abnormal clinical signs diminished within 90 minutes after dosing for both compounds. The animals receiving MARS 2 were autopsied after the second 3.0 mg/kg dosing on day six due to the severity of the clinical signs. Body weight, food consumption, hematology and clinical chemistry measurements were all normal during the investigation for both MARS 1 and MARS 2. Postmortem macroscopic examination and histopathology revealed no abnormalities associated with either test compound. Toxicokinetic measurements showed peak blood plasma concentrations for MARS 1 and MARS 2 were 100 ng/ml at 0.5 hours after administration. Test compound concentrations in cerebral spinal fluid at autopsy (0.5 hours after final dose) were 1/10 of the plasma maximum concentration for MARS 1 and 1/5 to 1/3 of plasma maximum concentration for MARS 2.
The no-observable-effect-level (NOEL) was 1.0 mg/kg for MARS 1 and below 0.3 mg/kg for MARS 2. The MTD was 3.0 mg/kg and 1.0 mg/kg for MARS 1 and MARS 2 respectively. The only toxicities associated with MARS 1 and MARS 2 during this study were the abnormal clinical signs.
Arnason BGW. Immunologic therapy of multiple sclerosis. Annu. Rev. Med. 50, 291-301, 1999
Riskind P. On the brain. The Harvard Mahoney Neuroscience Institute Letter. (4) Vol. 5. 1996
Munoz-Fernandez MA and Fresno M. “The role of tumor necrosis factor, interleukin 6, interferon-g and inducible nitric oxide synthase in the development and pathology of the nervous system.” Prog. Neurobiol. 56, 307-340, 1998
Smith T, Groom A, Zhu B, Turski L. “Autoimmune encephalomyelitis ameliorated by AMPA antagonists.” Nat. Med. 6, 62-66, 2000