Effects Of Laquinimod Research Articles

Laquinimod Inhibits Microglial Activation, Astrogliosis, BBB Damage, and Infarction and Improves Neurological Damage after Ischemic Stroke.

Glial activation is involved in neuroinflammation and blood-brain barrier (BBB) damage, which plays a key role in ischemic stroke-induced neuronal damage; therefore, regulating glial activation is an important way to inhibit ischemic brain injury. Effects of laquinimod (LAQ) include inhibiting axonal damage and neuroinflammation in multiple neuronal injury diseases. However, whether laquinimod can exert neuroprotective effects after ischemic stroke remains unknown. In this study, we investigated the effect of LAQ on glial activation, BBB damage, and neuronal damage in an ischemic stroke model. Adult ICR mice were used to create a photothrombotic stroke (PT) model. LAQ was administered orally at 30 min after ischemic injury. Neurobehavioral tests, Evans Blue, immunofluorescence, TUNEL, Nissl staining, and western blot were performed to evaluate the neurofunctional outcome. Quantification of immunofluorescence was evaluated by unbiased stereology. LAQ post-treatment significantly reduced infarction and improved forepaw function at 5 days after PT. Interestingly, LAQ treatment significantly promoted anti-inflammatory microglial activation. Moreover, LAQ treatment reduced astrocyte activation, glial scar formation, and BBB breakdown in ischemic brains. Therefore, this study demonstrated that LAQ post-treatment restricted microglial polarization, astrogliosis, and glial scar and improved BBB damage and behavioral function. LAQ may serve as a novel target to develop new therapeutic agents for ischemic stroke.

Laquinimod Protects Against TNF-α-Induced Attachment of Monocytes to Human Aortic Endothelial Cells (HAECs) by Increasing the Expression of KLF2.

IntroductionAs a worldwide health issue, the treatment and prevention of atherosclerosis present an important goal. Increased levels of proinflammatory cytokines such as TNF-α-associated chronic inflammatory response cause endothelial cells to lose their ability to regulate vascular function. Lipid-laden immune cells are recruited to the endothelium where they adhere to the endothelial wall and invade the intimal space, thereby leading to the development of atherosclerotic lesions, fatty plaques, and thickening of the arterial wall. In the present study, for the first time, we investigated the effects of laquinimod, an immunomodulatory agent used for the treatment of multiple sclerosis, on human aortic endothelial in a TNF-α-induced atherosclerotic microenvironment. At present, the mechanism of action of laquinimod is not well defined.MethodsThe effects of laquinimod on the gene expression of IL-6, MCP-1, VCAM-1, E-selectin, and KLF2 were measured by real-time PCR. ELISA assay was used to determine protein secretion and expression. Phosphorylation of ERK5 and the protein level of KLF2 were measured by Western blot analysis. The attachment of monocytes to endothelial cells was assayed by calcein-AM staining and fluorescent microscopy.ResultsOur findings demonstrate that laquinimod reduced the expression of key inflammatory cytokines and chemokines, including IL-6, MCP-1, and HMGB1. We further demonstrate that laquinimod significantly reduced the attachment of monocytes to endothelial cells, which is mediated through reduced expression of the cellular adhesion molecules VCAM-1 and E-selectin. Here, we found that laquinimod could significantly increase the expression of KLF2 through activation of ERK5 signaling. The results of our KLF2 knockdown experiment confirm that the effects of laquinimod observed in vitro are dependent on KLF2 expression.ConclusionTogether, these findings suggest a potential antiatherosclerotic capacity of laquinimod. Further research will elucidate the underlying mechanisms.

Laquinimod, a prototypic quinoline-3-carboxamide and aryl hydrocarbon receptor agonist, utilizes a CD155-mediated natural killer/dendritic cell interaction to suppress CNS autoimmunity

BackgroundQuinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1).MethodsWe explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed.ResultsWe demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing.ConclusionsThis study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance.

Laquinimod protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model

BackgroundThe oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model.MethodsWe induced EAE in C57/BL6 mice via MOG35–55 immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a non-immunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses (Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg laquinimod from day 16 when 60% of the animals had developed clinical signs of EAE.ResultsLaquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered.ConclusionFrom our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

Laquinimod has no effects on brain volume or cellular CNS composition in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease

BackgroundLaquinimod is an anti-inflammatory agent with good central nervous system (CNS) bioavailability, and neuroprotective and myelorestorative properties. A clinical trial in patients with multiple sclerosis demonstrated that laquinimod significantly reduced loss of brain volume. The cellular substrate or molecular events underlying that treatment effect are unknown. In this study, we aimed to explore laquinimod's potential effects on brain volume, animal behavior, cellular numbers and composition of CNS-intrinsic cells and mononuclear cells within the CNS, amyloid beta (Aβ) accumulation and tau phosphorylation in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. MethodsUtilizing a dose response study design, four months old F1 3xTg-AD/C3H mice were treated for 10months between ages 4 and 14months with laquinimod (5, 10, or 25mg/kg), or PBS administered by oral gavage. Brain volumes were measured in a 7 Tesla magnetic resonance imager (MRI) at ages 4 and 14months. Behavioral testing included locomotor and rearing activity and the Morris water maze task. Cell numbers and immunophenotypes were assessed by multiparameter flow cytometry. Aβ deposition and tau phosphorylation were determined by immunohistochemistry. ResultsIn the F1 3xTg-AD/C3H animal model of AD, there was no detectable reduction of brain volume over a period of 10months of treatment, as there was not brain atrophy in any of the placebo or treatment groups. Laquinimod had no detectable effects on most neurobehavioral outcomes. The number or composition of CNS intrinsic cells and mononuclear subsets isolated from the CNS were not altered by laquinimod. ConclusionThis is the first demonstration that there are no age-associated brain volume changes in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. Consequently, laquinimod had no effect on that outcome of this study. Most secondary outcomes on the effects of laquinimod on behavior and the number and composition of CNS-intrinsic cells and mononuclear cells within the CNS were also negative.

Laquinimod dampens hyperactive cytokine production in Huntington's disease patient myeloid cells.

Huntington's disease (HD) is a neurodegenerative condition characterized by pathology in the brain and peripheral tissues. Hyperactivity of the innate immune system, due in part to NFκB pathway dysregulation, is an early and active component of HD. Evidence suggests targeting immune disruption may slow disease progression. Laquinimod is an orally active immunomodulator that down‐regulates proinflammatory cytokine production in peripheral blood mononuclear cells, and in the brain down‐regulates astrocytic and microglial activation by modulating NFκB signalling. Laquinimod had beneficial effects on inflammation, brain atrophy and disease progression in multiple sclerosis (MS) in two phase III clinical trials. This study investigated the effects of laquinimod on hyperactive proinflammatory cytokine release and NFκB signalling in HD patient myeloid cell cultures. Monocytes from manifest (manHD) and pre‐manifest (preHD) HD gene carriers and healthy volunteers (HV) were treated with laquinimod and stimulated with lipopolysaccharide. After 24 h pre‐treatment with 5 μM laquinimod, manHD monocytes released lower levels of IL‐1β, IL‐5, IL‐8, IL‐10, IL‐13 and TNFα in response to stimulation. PreHD monocytes released lower levels of IL‐8, IL‐10 and IL‐13, with no reduction observed in HV monocytes. The effects of laquinimod on dysfunctional NFκB signalling in HD was assessed by inhibitor of kappa B (IκB) degradation kinetics, nuclear translocation of NFκB and interactions between IκB kinase (IKK) and HTT, in HD myeloid cells. No differences were observed between laquinimod‐treated and untreated conditions. These results provide evidence that laquinimod dampens hyper‐reactive cytokine release from manHD and preHD monocytes, with a much reduced effect on HV monocytes. Evidence suggests targeting CNS and peripheral immune disruption may slow Huntington's disease (HD) neurodegenerative processes. The effects of laquinimod, an orally active immunomodulator, on hyperactive cytokine release and dysfunctional NFκB signalling in stimulated myeloid cell cultures from pre‐manifest and manifest HD gene carriers and healthy volunteers were investigated. Laquinimod dampened cytokine release but did not impact NFκB signalling.Read the Editorial Highlight for this article on page 670.

M13 Laquinimod Reduces Hyper-reactive Pro-inflammatory Cytokine Release From Human Primary Huntington's Disease Myeloid Cells

Background Hyper-reactivity of the innate immune response is an early and active component of Huntington’s disease (HD) and is due in part to NFκB pathway dysregulation. There is evidence that the peripheral immune system plays a disease-modifying role in HD and that targeting it may slow disease progression. Laquinimod (LAQ) is an orally active immunomodulator that has beneficial effects in multiple sclerosis and is thought to act via the NFκB pathway. We investigated whether the compound could also reverse the hyper-reactive immune responses observed in HD. Aims Investigate the effects of LAQ on hyper-reactive HD myeloid cells. Methods Primary human monocytes were isolated from blood collected from symptomatic HD patients, premanifest HD gene-carriers (PreHD) and healthy volunteers. Cells were pretreated with 1μM or 5μM LAQ for 2 or 24 hours (h) before being stimulated with LPS for 24h. Cytokine release was measured by ELISA. IκB degradation kinetics, nuclear translocation of NFκB, and interactions between IKK and HTT were assessed in LAQ-treated cells. Results Twenty-four hour pretreatment with 5μM LAQ reduced cytokine release in manifest HD patient monocytes. This included IL-1β, IL-8, TNFα, IL-5, IL-13 and IL-10. Reductions in IL-6, IL-8, IL-10 and IL-13 release by preHD monocytes were also observed using this LAQ dose. When using either 1µM LAQ or 2h pretreatment there was little evidence of a difference between active and control conditions in any of the patient subgroups. We did not observe any significant effect of LAQ on IκB degradation kinetics, NFκB translocation or interactions between IKK and HTT. Conclusions 5μM LAQ applied to HD monocytes for 24h reduces hyper-reactive cytokine production in response to LPS stimulation. The mechanism for this does not appear to be downstream of NFκB activation. We are currently working on elucidating the mechanism of action of LAQ and will go on to determine how this impacts on neuronal survival using neuronal-myeloid cell co-culture models.

Laquinimod Delays and Suppresses Nephritis in Lupus‐Prone Mice and Affects Both Myeloid and Lymphoid Immune Cells

Lupus nephritis depends on autoantibody deposition and activation of multiple immune cell types that promote kidney inflammation, including lymphocytes and monocyte/macrophages. Laquinimod, currently in clinical trials for multiple sclerosis and lupus nephritis, reduces infiltration of inflammatory cells into the spinal cord in experimental autoimmune encephalomyelitis. Activated monocyte/macrophages infiltrate the kidneys during nephritis in systemic lupus erythematosus (SLE). We undertook this study to determine whether using laquinimod to reduce monocyte/macrophage-driven tissue damage as well as to alter lymphocytes in SLE nephritis could have greater therapeutic benefit than current treatments that primarily affect lymphocytes, such as mycophenolate mofetil (MMF). To test laquinimod efficacy, we used the (NZB × NZW)F1 mouse model of SLE, in which disease manifests as nephritis. Preventive and therapeutic studies were performed to determine whether laquinimod could prevent or delay nephritis, as measured by proteinuria, serum creatinine, survival, and renal pathology. Spleen and kidney leukocyte populations and suppression assays were analyzed by flow cytometry. Laquinimod prevented or delayed lupus manifestations at levels equal to or better than MMF. Laquinimod treatment was associated with reduced numbers of monocyte/macrophages, dendritic cells, and lymphocytes, as well as with induction of myeloid-derived suppressor cells in spleens and kidneys. Laquinimod suppressed macrophage-secreted tumor necrosis factor α and induced production of interleukin-10 (IL-10). In addition, laquinimod suppressed interferon-γ and IL-17 production by lymphocytes and down-regulated expression of activation/costimulatory markers on antigen-presenting cells. The effects of laquinimod on myeloid and lymphoid cells may contribute to improvements in (NZB × NZW)F1 mouse survival, proteinuria, and glomerulonephritis. Future development of laquinimod as a therapeutic agent for lupus nephritis is promising.

Effects of Laquinimod on the Human Blood-Brain Barrier (P02.118)

Effects of Laquinimod on the Human Blood-Brain Barrier (P02.118)

Laquinimod Promotes T Cell Immune Modulation in Central Nervous System Autoimmunity Via Type II (M2) Myeloid APC (P02.105)

Objective: We investigated whether T cell immune modulation by laquinimod was mediated through its effects on myeloid antigen presenting cells (APC) or directly on T cells. Background Laquinimod is a novel oral drug that is being evaluated for treatment of RR multiple sclerosis (MS). In the MS model, EAE, laquinimod has been shown to reduce CNS infiltration and demyelination, as well as promote T cell immune modulation. Design/Methods: The effect on the function of myeloid APC was tested by using in vivo laquinimod-treated cells as APC for meylin-specific T cells and by transferring laquinimod treated APC into mice with established EAE. Results: Oral laquinimod treatment reversed established RR-EAE, and was associated with reduced CNS inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c+CD11b+CD4+ dendritic cells and an elevation of CD11bhiGr1hi monocytes. CD11b+ cells from these mice exhibited anti-inflammatory type II (M2) phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, these donor type II monocytes suppressed clinical and histologic signs in recipients with established EAE. We examined whether T cell immune modulation occurred as a direct effect of laquinimod, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Conclusions: Our results show that Laquinimod does not act directly on T cells, but modulates adaptive T cell immune responses via its effect on myeloid APC. Supported by: Support was provided to S.S.Z. by NIH (RO1 AI073737 and NS063008), the NMSS (RG 4124), Teva Pharmaceuticals, and the Maisin Foundation. Disclosure: Dr. Schulze Topphoff has nothing to disclose. Dr. Shetty has received research support from Boehringer Ingelheim. Dr. Varrin-Doyer has nothing to disclose. Dr. Molnarfi has nothing to disclose. Dr. Sagan has received research support from the Guthy Jackson Charitable Foundation. Dr. Sobel has received research support from the NIH. Dr. Nelson has nothing to disclose. Dr. Zamvil has received personal compensation for activities with Biogen Idec, Teva Neuroscience and EMD Serono-Pfizer. Dr. Zamvil has received research support from the National Institutes of Health, National Multiple Sclerosis Society, the Maisin Foundation, the Guthy Jackson Charitable Foundation, Teva Neuroscience, and Boehringer Ingelheim Pharmaceuticals, Inc.

Clinical Efficacy of Laquinimod for the Treatment of Multiple Sclerosis; Pooled Analyses from the ALLEGRO and BRAVO Phase III Trials (S01.007)

Clinical Efficacy of Laquinimod for the Treatment of Multiple Sclerosis; Pooled Analyses from the ALLEGRO and BRAVO Phase III Trials (S01.007)

Laquinimod-Mediated Modulation of B Cells from Multiple Sclerosis Patients: Affecting Direct and Indirect Immune-Regulatory Functions (P02.116)

Objective: The goal of this study was to assess the immunomodulatory in vitro effects of laquinimod on B cells from patients with relapsing-remitting multiple sclerosis (RRMS). Background The benefit of B cell-depleting therapy and other lines of evidence have emphasized a role of B cells in MS. Laquinimod is a novel orally administered drug under development for the treatment of MS, whose mode of action is not fully elucidated. Clinical data so far suggest that laquinimod is safe, well-tolerated and efficacious in reducing MRI measures of disease activity, clinical relapses and slowing the progression of disability. Design/Methods: B cells from naive MS patients were cultured with or without 1uM laquinimod for 24hrs, and analyzed by FACS for known B cell markers. B cells and CD4+ T cells from naive RRMS patients were co-cultured with or without 1uM laquinimod for 72hrs, under stimulation by CD3/CD28 and PMA+ionomycin, and cytokine levels in both B and T cells, and T cell proliferation assessed by FACS. Results: Laquinimod increased the percentage of both CD86+ and IL10+ cells within the CD25+ B cell subset, all markers associated with B cell regulatory functions. The CD25+ B cells demonstrated their regulatory capacity by reducing proliferation of T cells and the percentage of IFNg+ T cells. Laquinimod-treated B cells reduced the expression of IFNg and of IL4 and increased the expression of IL10 and TGFb in T cells, in a B-cell mediated manner. Laquinimod also reduced the expression of IL4, induced IL10 and TGFb expression in B cells in the co-culture, and decreased the percentage of IFNg+ T cells in T cells alone. Conclusions: Laquinimod modulates B cell characteristics and regulatory functions, affecting B cell cytokine and B cell-mediated- T cell cytokine profile, which may be part of the beneficial mode of action of laquinimod in MS patients. Supported by: Financial support from Teva Pharmaceuticals. Disclosure: Dr. Miller has received personal compensation for activities with Teva Pharmaceutical Industries Ltd., Medison Pharma Ltd., Biogen Idec, Merck Serono, Avanir Pharmaceuticals, Novartis, and Bayer Schering Pharma. Dr. Miller has received research support from Teva Pharmaceutical Industries Ltd., Medison Pharma Ltd., Biogen Idec, Merck Serono, and Bayer Schering Pharma. Dr. Nussbaum has nothing to disclose. Dr. Staun-Ram has received research support from Teva Pharmaceuticals. Dr. Snir has nothing to disclose. Dr. Hayardeny Nisimov has nothing to disclose. Dr. Melamed has received research support from Teva Neuroscience. Dr. Toubi has received research support from Teva Pharmaceuticals.

Analysis of Laquinimod Efficiency during Experimental Autoimmune Encephalomyelitis (P02.115)

Objective: The study aims to investigate the cellular mode of action of laquinimod in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Background Laquinimod is an orally administered CNS-active immunomodulator. Phase III data in RRMS patients indicate a positive effects of laquinimod 0.6 mg on clinical disease activity as evidenced by slowing the progression of disability, reducing the rate of MRI-measured brain volume loss and reducing relapse rates, all coupled with a favorable safety and tolerability profile. In EAE, it has been shown that laquinimod acts on autoaggressive T cells by reducing proinflammatory Th1 cytokine production. However, the exact cellular and molecular mechanisms of laquinimod are still incompletely understood. Design/Methods: In this study we set out to test the efficacy of laquinimod in both preventive (application of laquinimod for 14 days starting at the time of immunization) and therapeutic (starting the treatment when first symptoms occurred for 7 days) regimens in the MOG 35-55 -induced murine EAE model. Using intravital two photon microscopy combined with molecular ex vivo -analyses we are currently evaluating the locomotion and function of pathogenic effector T cells during the different phases of EAE as well as their interaction with other immune cells. Results: Our preliminary results show that laquinimod is able to ameliorate EAE in a dose-dependent manner in MOG-induced C57Bl/6 EAE as well as in a more aggressive and fulminant model using transfer of transgenic MOG-specific T cells. This was observed both in the preventive and therapeutic setting. Furthermore, we observed reduced proliferation of MOG-specific T cells and a reduction in the expression of activation markers in the priming phase and later a reduced production of pro-inflammatory cytokines such as IFN-gamma and IL17. Conclusions: In our experimental model used, laquinimod was shown to act early on antigen-specific T cells in the priming/preclinical phase before symptoms start to occur. Supported by: TEVA. Disclosure: Dr. Luhder has received research support from Teva. Dr. Winchenbach has nothing to disclose. Dr. Flugel has received research support from Teva.

Laquinimod Restricts Inflammatory Gene Expression in a Human Model of Reactive Astrogliosis (P02.111)

Objective: Laquinimod is an orally administered CNS-active immunomodulator currently in phase III clinical trials for therapy of relapsing remitting multiple sclerosis (RRMS). Data show that RRMS patients show beneficial effects of laquinimod 0.6mg on clinical disease activity as evidenced by slowing of the progression of disability, reducing the rate of MRI-measured brain volume loss, and reducing relapse rate. The current work examines the mechanisms underlying these effects. Background Laquinimod crosses the blood-brain barrier and enters the CNS, thus in addition to peripheral effects it may act directly on CNS-resident lineages including oligodendrocytes, microglia and astrocytes. Increasing evidence implicates reactive astrocytes as critical regulators of CNS inflammation and repair in MS. Design/Methods: Here, we report that laquinimod profoundly impacts proinflammatory gene expression in a human in vitro model of reactive astrogliosis. Results: Interleukin-1beta (IL-1β) is implicated in lesion pathogenesis in RRMS, and in primary human astrocyte cultures it strongly induced inflammatory factors including cytokines, chemokines and reactive nitrogen species. Importantly, at therapeutic concentrations, laquinimod abrogated IL-1β-induced induction of cytokines including tumor necrosis factor-α (TNFα) and IL-6, and inducible nitric oxide synthase. Laquinimod also differentially regulated IL-1β-induced expression of CXC and CC chemokines, suggesting that it acts as an immunomodulator rather than an immunosuppressant in astrocyte cultures. IL-1β exerts its effects via the transcription factor NF-κB, and suggesting mechanism, laquinimod pretreatment of human astrocytes downregulated NF-κB activation. Conclusions: Collectively, these data reveal laquinimod as a regulator of the proinflammatory phenotype in a human model of reactive astrogliosis, and suggest that it may act centrally on resident CNS cells to restrict pathology in MS lesions. Supported by: A research grant from Teva Pharmaceuticals. Disclosure: Dr. Pham has nothing to disclose. Dr. Zhang has nothing to disclose. Dr. Hayardeny Nisimov has nothing to disclose. Dr. John has received personal compensation for activities with EMD Serono, Teva Neuroscience, and Biogen Idec. Dr. John has received research support from Teva Neuroscience, and Vaccinex Inc.

Laquinimod Add on Effect on Glatiramer Acetate as Well as INFb Treatments (P04.142)

Objective: Studying whether laquinimod treatment added on top of either GA (glatiramer acetate) or IFNb would result in an additive effect on disease severity in MOG-induced EAE. Background Laquinimod is an orally administered CNS-active immunomodulator. Phase III data in RRMS patients indicate clear effects of laquinimod 0.6mg on disease progression and on the accumulation of brain-tissue loss, as measured by MRI, coupled with a favorable safety and tolerability profile. Laquinimod has shown a significant effect in Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. In the EAE model, laquinimod has been shown to significantly abrogate disease severity. It was suggested that Laquinimod exhibits its effect via NFkB signaling inhibition. It was shown to affect CNS residential cells as astrocytes and microglia resulting in reduction of proinflammatory cytokines. Glatiramer acetate (GA), a well-known disease modifying treatment for relapsing RRMS, is an immunomodulator that significantly inhibits disease activity in EAE. GA was shown to bind to various class II major histocompatibility complex (MHC) molecules and to create GA specific Th2 cells. These cells are migrating into the CNS and secreting anti inflammatory cytokines. INFb-which is also in use for RRMS treatment is effective in reducing inflammatory activity in EAE. Laquinimod has shown efficacy, using IFNb knock out mouse model, suggesting that laquinimod efficacy is IFNb independent. Design/Methods: MOG induced EAE C57BL/6 mice were treated with Laquinimod (5 and 25mg/kg) alone or together with suboptimal dose of GA (12.5mg/kg) or IFNb (500,000 and 50,000 IU/mouse). All treatments were started 1 day after immunization. Scoring of EAE clinical signs were carried out daily. Results: Both GA/laquinimod and IFNb/laquinimod adjunct treatment resulted in improved efficacy when compared to each treatment alone. Conclusions: These results suggesting the use of both treatment regimens together as a potential treatment for RRMS patients. Supported by: Teva Pharmaceutical industries. Disclosure: Dr. Hayardeny Nisimov has nothing to disclose. Dr. Birnberg has nothing to disclose. Dr. Raymond has received research support from TEVA Pharmaceutical. Dr. Fine has received research support from Teva Pharmaceutical. Dr. Kaye has received personal compensation for activities with Teva Neuroscience as an employee. Dr. Kaye has received research support from Teva Neuroscience.

Laquinimod Treatment Reduces an Immunogenic Dendritic Cell Population and Protects from Autoimmune CNS Inflammation (P02.117)

Objective: In this study we aimed to investigate whether, and how, laquinimod can prevent the occurrence of relapses in a relapsing-remitting model of MS. Background Laquinimod is an orally administered CNS-active immunomodulator. Phase III data in RRMS patients showed beneficial effects of laquinimod 0.6mg on clinical disease activity as evidenced by slowing the progression of disability, reducing the rate of MRI-measured brain volume loss and reducing relapse rates, all coupled with a favorable safety and tolerability profile. Design/Methods: We used C5BL/6 as well as SJL/J mice to induce EAE with the MOG peptide p35-55 or the PLP peptide p39-151, respectively. Dendritic cells (DCs) were analyzed from either spleen or lymph nodes. T cells were analyzed from spleen, lymph nodes or from the CNS. Results: C57BL/6 mice treated with laquinimod by daily oral gavage, were completely resistant to disease induction. To test whether the disease could also be modulated at a later stage, we induced disease in SJL/J mice, in a protocol that leads to a relapsing-remitting disease. We found that the mice treated with laquinimod were prevented from developing further relapse, in contrast to mice treated with vehicle only. Interestingly, we found that CD4+CD11c+ were almost completely absent in the Laquinimod treated mice, C57BL/6 as well as of the SJL/J mice. In addition, we detected an elevation in the total number of FoxP3+CD4+ regulatory T cells in the laquinimod treated animals. CD11c+CD4+ cells were previously implicated as immunogenic DCs, in contrast to CD11c+CD8+ cells shown to have immunomodulatory properties. Conclusions: We report that treatment of mice with laquinimod has distinct implications for cytokine production and T cell response. We suggest therefore that laquinimod appears to exert its disease-suppressive activity by modulating DC and T cell responses. Supported by: An unrestricted grant from Teva Pharmaceutical Industries (Netanya, Israel). Disclosure: Dr. Waisman has received personal compensation for activities with Teva Neuroscience as a consultant. Dr. Waisman has received research support from Teva Neuroscience. Dr. Masri has nothing to disclose. Dr. Jolivel has nothing to disclose. Dr. Lussi has nothing to disclose. Dr. Siffrin has nothing to disclose. Dr. Zipp has nothing to disclose.

Effect of laquinimod on MRI-monitored disease activity in patients with relapsing-remitting multiple sclerosis: a multicentre, randomised, double-blind, placebo-controlled phase IIb study

A 24-week phase II trial has shown that 0.3 mg of laquinimod given daily to patients with relapsing-remitting multiple sclerosis was well tolerated and reduced the formation of active lesions. We assessed the effect of oral daily 0.3 and 0.6 mg laquinimod on MRI-monitored disease activity in a 36-week double-blind, placebo-controlled phase IIb study. The study was done in 51 centres in nine countries. Inclusion criteria were one or more relapses in the year before entry and at least one gadolinium enhancing (GdE) lesion on screening MRI. Of 720 patients screened, 306 eligible patients were enrolled. Patients, aged 18-50 years, were randomly assigned to placebo (n=102), laquinimod 0.3 mg a day (n=98), or 0.6 mg a day (n=106). Brain MRI scans and clinical assessments were done at week -4, baseline, and monthly from week 12 to week 36. The primary outcome was the cumulative number of GdE lesions at weeks 24, 28, 32, and 36. The principal analysis of the primary endpoint was done on the intention-to-treat cohort. This study is registered with ClinicalTrials.gov, number NCT00349193. Compared with placebo, treatment with laquinimod 0.6 mg per day showed a 40.4% reduction of the baseline adjusted mean cumulative number of GdE lesions per scan on the last four scans (simple means 4.2 [SD 9.2] vs 2.6 [5.3], p=0.0048); treatment with 0.3 mg per day showed no significant effects (3.9 [5.5] vs placebo, p=0.6740). Both doses of laquinimod were well tolerated, with some transient and dose-dependent increases in liver enzymes. A case of Budd-Chiari syndrome-ie, a thrombotic venous outflow obstruction of the liver-occurred after 1 month of exposure in a patient with underlying hypercoagulability who received 0.6 mg laquinimod. Anticoagulant treatment resulted in a decline of liver enzymes to normal without any clinical signs of hepatic decompensation. In patients with relapsing-remitting multiple sclerosis, 0.6 mg per day laquinimod significantly reduced MRI-measured disease activity and was well tolerated.