Inhibition of the development of chronic experimental autoimmune encephalomyelitis by laquinimod (ABR-215062) in IFN-β k.o. and wild type mice

doi:10.1016/j.jneuroim.2005.11.023

Journal of Neuroimmunology

Volume 173, Issues 1–2, April 2006, Pages 69-78

https://doi.org/10.1016/j.jneuroim.2005.11.023 Get rights and content

Abstract

Laquinimod is a novel oral immunomodulatory substance, which is currently developed for the treatment of multiple sclerosis (MS). The ability of laquinimod to inhibit disease development was investigated in chronic experimental autoimmune encephalomyelitis (chEAE) in IFN-β k.o. mice and wild type mice. Laquinimod was shown to inhibit both disease development and histopathological changes in the CNS. Furthermore, laquinimod was found to be independent of endogenous IFN-β for its effect in chEAE. When laquinimod was combined with exogenous IFN-β, a synergistic disease inhibitory effect was seen. These findings using laquinimod in preclinical disease models for MS emphasize the potential of laquinimod in the future treatment of MS also in patients that do not respond to IFN-β monotherapy. Furthermore, the results indicate that laquinimod may favourably be combined with IFN-β.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) characterized by focal leukocyte infiltration, myelin destruction and axonal loss leading to neurological disability.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of CNS that can be elicited in rodents and represents the major animal model of the human MS. EAE can be induced by either immunization with spinal cord homogenate or purified encephalitogenic antigens and peptides in complete Freund's adjuvant (CFA) (Mendel et al., 1995, Swanborg, 1995, Gijbels et al., 2000, Brunmark et al., 2002). Both the clinical picture and the histopathological characteristics in EAE are similar to MS and the different phases in the development of EAE represent different parts of the human disease.

Laquinimod (Fig. 1), a small molecule (MW: 357D) with high oral bioavailability (unpublished data), has shown favourable pharmacokinetic properties both in mice (Tuvesson et al., 2005) and in man (unpublished data). Laquinimod has shown immunomodulatory activities in preclinical studies (Brunmark et al., 2002, Zou et al., 2002, Jönsson et al., 2004, Yang et al., 2004). Brunmark et al. (2002) showed that laquinimod inhibited disease development in acute experimental autoimmune encephalomyelitis (aEAE) and chronic-relapsing EAE (crEAE) in mice. Laquinimod also inhibited the development of experimental autoimmune neuritis (EAN) in the Lewis rat (Zou et al., 2002). Furthermore laquinimod inhibited disease development and infiltration of inflammatory cells into the CNS in induced aEAE in the Lewis rat (Yang et al., 2004). It was also demonstrated that laquinimod redirected the cytokine production in favour of the Th2/Th3 cytokines IL-4, IL-10 and TGF-β.

The primary objective of treatment with disease modifying therapies in relapsing remitting MS is to reduce the frequency of relapses and slow the progression of neurologic disability. As Glatiramer acetate (GA) and interferon-β (IFN-β), which are approved for treatment of MS (Wiendl and Hohlfeld, 2002) require parenteral administration, an effective oral candidate for treatment of MS would be desirable. Laquinimod (ABR-215062) represents an opportunity for such a novel oral treatment of MS.

A randomized, placebo controlled, double-blind study of three parallel treatment groups of patients with MS (n = 209) has been performed with laquinimod (once daily tablets) treatment (Polman et al., 2005). A statistically significant decrease in disease activity (active lesions) in patients treated with laquinimod (0.3 mg/day) over a 24-week period was demonstrated by using magnetic resonance imaging (MRI) assessments. These findings demonstrate that oral treatment with laquinimod decreases the development of active lesions in MS patients.

The main purpose of this study was to investigate whether laquinimod, for which the mechanism of action is not yet fully elucidated, is dependent on endogenous IFN-β for its inhibition of disease development in EAE. Furthermore the question whether the combination of laquinimod with IFN-β is beneficial as compared to monotherapies was also addressed.

Section snippets

Test compounds and formulations

Laquinimod (ABR-215062) (Fig. 1) was synthesized at Active Biotech Research AB, Lund, Sweden. The compound was dissolved in physiologic saline and administered orally in a volume of 0.2 ml on the day of the immunization to the end of experiment, six days a week. The solutions were dispensed in vials, sufficient for one daily treatment, sealed, stored at 4 °C, and used within one month of preparation. Control mice were similarly injected in a volume of 0.2 ml saline on the day of the

Chronic EAE in IFN-β k.o. mice compared to wild type mice (C57BL/6)

Immunization with the MOG_35–55 peptide in both IFN-β k.o. and their wild type counterparts (C57BL/6 mice) yielded a similar pattern in the onset of disease, but severity of the disease was more pronounced in IFN-β k.o. mice compared to wild type mice (Fig. 2A) (MCS day 9–42; p ≤ 0.0001). This was also seen when body weight gain was compared (body weight change day 42; p ≤ 0.0001) (Fig. 2B).

Inflammatory infiltrate and demyelination in diseased chEAE IFN-β k.o. mice compared with wild type mice (C57BL/6)

A significant increased positive stained area of CD4⁺ (p = 0.029) and MHCII (p = 0.0063) expressing cells was seen

Discussion

This study shows an inhibitory effect of laquinimod on disease development and infiltration of inflammatory cells into the CNS in a chronic EAE model (MOG_35–55 induced chEAE in IFN-β k.o and wild type C57BL/6 mice) resembling the MS disease in man. Results from this study confirm data from earlier studies with laquinimod performed in murine EAE models (Brunmark et al., 2002, Jönsson et al., 2004), in experimental autoimmune neuritis (EAN) in rats (Zou et al., 2002) and in acute EAE in rats (

Acknowledgements

The authors thank Lenore Henriksson, Madeleine Jakobsson-Andén, Helena Karlsson and Ann-Sofi Thornquist for excellent experimental work and Örjan Nordle for statistical analysis. Gunnar Hedlund, Marie Törngren and Tore Nederman are gratefully acknowledged for critical reading of the manuscript.

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Citation Excerpt :
It has been shown to be effective in animal models for other autoimmune conditions. Initial animal studies with laquinimod in relapsing and chronic experimental autoimmune encephalomyelitis (EAE) demonstrated efficacy (Brunmark et al., 2002; Runstrom et al., 2006). When laquinimod was combined with beta-interferon, a synergistic inhibitory effect was seen (Runstrom et al., 2006).
The modern treatment era for multiple sclerosis (MS) began in 1993 with the approval of the first disease-modifying agent. Since then the field has greatly expanded, with 10 therapies currently approved to treat MS. These treatments are effective to reduce relapses and changes on MRI, and slow disability. However, despite these medications some patients continue to have exacerbations, accumulate disability, and develop progressive disease due to partial effectiveness. New molecules with novel mechanisms of action and targets are being explored. Hopefully these agents will yield even greater efficacy without significant safety concerns. As more aggressive therapies are available to treat MS, the goals and expectations of treatment are also likely to change. Some of the emerging therapies, including alemtuzumab, daclizumab, rituximab, ocrelizumab, laquinimod, estriol, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), vitamin D, and stem cell transplantation, will be discussed in this chapter. In the future, therapies with different mechanisms may be combined, but this will need to be evaluated in clinical trials. Neuroprotection and repair definitely warrant further study. The future of MS treatment is very exciting, especially as our armamentarium expands.
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Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6C^hi inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4⁺ T cells and reduced frequency of IFN-γ– and IL-17–producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6C^hi CD115⁺ inflammatory monocytes and CD11b⁺CD11c⁺ dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte–derived DCs and CD4⁺ T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4⁺ T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b⁺ myeloid cells.

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¹: Present address: MicroMorph Histology, Box 724, SE-220 07 Lund, Sweden.

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Inhibition of the development of chronic experimental autoimmune encephalomyelitis by laquinimod (ABR-215062) in IFN-β k.o. and wild type mice

Abstract

Introduction

Section snippets

Test compounds and formulations

Chronic EAE in IFN-β k.o. mice compared to wild type mice (C57BL/6)

Inflammatory infiltrate and demyelination in diseased chEAE IFN-β k.o. mice compared with wild type mice (C57BL/6)

Discussion

Acknowledgements

J. Neuroimmunol.

Curr. Biol.

J. Neuroimmunol.

Clin. Immunol. Immunopathol.

J. Neuroimmunol.

J. Neuroimmunol.

J. Neuroimunol.

Neuropharmacology

Blood–brain barrier permeability and monocyte infiltration in experimental allergic encephalomyelitis. A quantitative MRI study

Brain

Experimental autoimmune encephalomyelitis: an animal model for multiple sclerosis

Neurosci. Res. Commun.