A novel calpain inhibitor for the treatment of acute experimental autoimmune encephalomyelitis

https://doi.org/10.1016/j.jneuroim.2006.08.005Get rights and content

Abstract

Aberrant activation of calpain plays a key role in the pathophysiology of several neurodegenerative disorders. Calpain is increasingly expressed in inflammatory cells in EAE and is significantly elevated in the white matter of patients with multiple sclerosis, thus calpain inhibition could be a target for therapeutic intervention. The experiments reported here employed a myelin oligodendrocyte glycoprotein-induced disease model in C57Bl/6 mice (EAE) and a novel calpain inhibitor, targeted to nervous tissue. CYLA was found to reduce clinical signs of EAE and prevent demyelination and inflammatory infiltration in a dose- and time-dependent manner. Oral administration of the diacetal prodrug was equally effective.

Introduction

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). It is the major disabling disease of young individuals between the ages of 20 and 40 (Bauer et al., 2001, Martin and McFarland, 1995). Despite numerous studies, the etiology of MS remains unknown. Experimental autoimmune encephalomyelitis (EAE), an animal model of MS, has been extensively used to study the pathogenesis of MS (Mendel et al., 1995, Mokhtarian, 1988, Mokhtarian et al., 1984, Mokhtarian et al., 1999, Ofosu-Appiah et al., 1994, Slavin et al., 1998) and its treatment options (Bechtold et al., 2004, Gilgun-Sherki et al., 2003a, Gilgun-Sherki et al., 2003b, Mokhtarian et al., 1996). Currently, the consensus on the pathogenesis of MS is that autoreactive T-cells, activated in the periphery (Mokhtarian et al., 1990, Mokhtarian et al., 1994), cross the blood–brain barrier (BBB) where they re-encounter antigen within the CNS that is presented to them by antigen presenting cells (APC) (Hohlfeld, 1997, Sospedra and Martin, 2005). This process causes secondary influx of additional lymphocytes and blood-born macrophages to the inflammatory site (Compston, 2004, Compston and Coles, 2002, Hohlfeld and Wekerle, 2004). The inflammatory cells produce NO, TNF-alpha and proteases, including calpain, that individually or together contribute to the destruction of myelin and eventually cause injury to the axons (Banati et al., 1993, Benveniste, 1997, Gehrmann et al., 1995).

Calpain has been implicated in myelinolysis (Shields and Banik, 1999, Shields et al., 1997, Shields et al., 1998) and all major myelin proteins are calpain substrates (Banik et al., 1985). Other substrates of calpain are neurofilament proteins (NFP), alpha-spectrin (alpha-fodrin), protein kinase C (PKC) and receptor proteins and enzymes (Bednarski et al., 1995, Ray and Banik, 2003, Saido et al., 1991, Schlaepfer and Zimmerman, 1985). Calpain is significantly elevated in the white matter of MS patients (Shields et al., 1999) and is increasingly expressed in the inflammatory cells in EAE (Shields and Banik, 1998) suggesting its role in the pathogenesis of this demyelinating disease. Increasing evidence suggests that the inhibition of calpain showed less myelin degradation in vitro as compared to the inhibition of other proteases (Deshpande et al., 1995, Smith et al., 1998). Thus, inhibition of the enhanced calpain activity can be a possible target for treatment of these neurodegenerative diseases (Osanai and Nagai, 1984, Ray et al., 2003, Stracher, 1997). Several protease inhibitors have been tested as a potential therapeutic agents in EAE and other animal models of neurodegenerative diseases (Badalamente et al., 1989, Badalamente and Stracher, 2000, Osanai and Nagai, 1984, Smith, 1979, Smith and Amaducci, 1982). A limiting factors for the use of current available protease inhibitors has been their inability to cross the BBB (Osanai and Nagai, 1984, Smith and Amaducci, 1982). Consequently, the development of calpain inhibitors with improved cell membrane and/or BBB permeability has been the focus of intensive research.

In the present study we examined the effect of cysteic–leucyl–argininal (CYLA), a novel calpain inhibitor that is designed to readily cross the BBB in myelin oligodendrocyte glycoprotein (MOG)-induced acute EAE.

Section snippets

Animals, preparation of the peptide antigen and induction of EAE

C57BL/6J female mice (7- to 8-week-old) were purchased from the Jackson Laboratories (Bar Harbor, MD) and housed according to Institutional Animal Care and Use Committee (IACUC) regulations in an Association for Assessment Accreditation of Laboratory Animal Care (AAALAC) certified facility.

MOG 35–55 (NH2–MEVGWYRSPFSRVVHLYRNGK–OH) was synthesized in the Dept. of Biological Chemistry, Biosynthesis and Sequencing Facility at Johns Hopkins University School of Medicine (Baltimore, MD).

EAE was

Evaluation of neurological and body weight changes

EAE mice developed weight loss as the first sign of the disease starting on day 8 post immunization (pi) (Fig. 1A). Early neurological signs such as floppy tail and hind limb weakness appeared on days 11–13 pi (Fig. 1B). Neurological deficits increased in severity in all EAE mice and reached a peak by day 20 pi. Weight loss showed similar pattern. Mice continuously lost weight until day 20 pi corresponding to the progression of the neurological deficits (Fig. 1A and B). The experiment was

Discussion

Mice injected with MOG/CFA exhibit a complex immunopathological cascade that results in activation of macrophages and microglia, and antigen presentation by these cells to effector T cells are important factors in the pathogenesis of EAE (Mokhtarian, 1988). The macrophages and microglia induce damage by the release of proteases known to contribute to myelin degradation, and inhibition of these proteases would suppress the ultimate pathology (Deshpande et al., 1995, Smith et al., 1998).

Acknowledgment

Help from the following people is very much appreciated. Riccardo Bianchi, Barbara Concepcion, Hans VonGizycki, Matt Kelly, Anna Miller, Susanna Popp, William Oxberry, Lynette Sheffield, Mark Stewart, Fasika M. Tedla and Jeremy Weedon. This work was performed in partial fulfillment of the requirements for a PhD degree of Getaw Worku Hassen, at SUNY Downstate at Brooklyn, School of Graduate Studies. This work was partially supported by NIH grant RO1 NS 40157 (FM), Maimonides R&D Fndn and Ceptor

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