Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles.

BACKGROUND AND AIMS: We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. APPROACH AND RESULTS: We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6 , and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. CONCLUSIONS: Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.

Laquinimod enhances central nervous system barrier functions.

Laquinimod is currently being tested as a therapeutic drug in multiple sclerosis. However, its exact mechanism of action is still under investigation. Tracking of fluorescently-tagged encephalitogenic T cells during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, revealed that laquinimod significantly reduces the invasion of pathogenic effector T cells into the CNS tissue. T-cell activation, differentiation and amplification within secondary lymphoid organs after immunization with myelin antigen, their migratory capacity and re-activation within the nervous tissue were either only mildly affected or remained unchanged. Instead, laquinimod directly impacted the functionality of the CNS vasculature. The expression of tight junction proteins p120 and ZO-1 in human brain endothelial cells was up-regulated upon laquinimod treatment, resulting in a significant increase in the transendothelial electrical resistance of confluent monolayers of brain endothelial cells. Similarly, expression of the adhesion molecule activated leukocyte cell adhesion molecule (ALCAM) and inflammatory chemokines CCL2 and IP-10 was suppressed, leading to a significant reduction in the migration of memory TH1 and TH17 lymphocytes across the blood brain barrier (BBB). Our data indicate that laquinimod exerts its therapeutic effects by tightening the BBB and limiting parenchymal invasion of effector T cells, thereby reducing CNS damage.

Safety and in vivo immune assessment of escalating doses of oral laquinimod in patients with RRMS.

BACKGROUND: Laquinimod is an oral immunomodulator in clinical development to treat relapsing-remitting multiple sclerosis (RRMS). Laquinimod is in clinical development for the treatment of multiple sclerosis and Huntington Disease (HD). The objective of this study is to assess the safety, tolerability, pharmacokinetics (PK) and cytoimmunologic effects following escalating doses of laquinimod in patients with RRMS. METHODS: One hundred twelve patients were randomly assigned to laquinimod/placebo in a series of separate dose-escalating cohorts starting from a daily oral dose of 0.9 mg/1.2 mg escalating to 2.7 mg, in 0.3 mg increments. RESULTS: Twenty-eight patients received placebo and 84 received laquinimod ranging from 0.9 to 2.7 mg. No deaths occurred. One serious adverse event (SAE) of perichondritis was reported, which was unrelated to laquinimod (0.9 mg). There was no increased incidence of adverse events (AEs) with escalating doses. Laquinimod-treated patients showed more abnormal laboratory levels in liver enzymes, P-amylase, C-reactive protein (CRP), and fibrinogen, but most shifts were clinically non-significant. The exposure of laquinimod was dose proportional and linear in the tested dose range. An immunological substudy showed significant dose-dependent decreases in 6-sulpho LacNAc + dendritic cell (slanDC) frequency following laquinimod compared to placebo. CONCLUSION: Laquinimod doses up to 2.7 mg were safely administered to patients with RRMS. An in vivo effect of laquinimod on the innate immune system was demonstrated. TRIAL REGISTRATION: EudraCT Number: 2009-011234-99 . Registered 23 June 2009.

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-1ß, 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.

Restoration of axon conduction and motor deficits by therapeutic treatment with glatiramer acetate.

Glatiramer acetate (GA; Copaxone) is an approved drug for the treatment of multiple sclerosis (MS). The underlying multifactorial anti-inflammatory, neuroprotective effect of GA is in the induction of reactive T cells that release immunomodulatory cytokines and neurotrophic factors at the injury site. These GA-induced cytokines and growth factors may have a direct effect on axon function. Building on previous findings that suggest a neuroprotective effect of GA, we assessed the therapeutic effects of GA on brain and spinal cord pathology and functional correlates using the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Therapeutic regimens were utilized based on promising prophylactic efficacy. More specifically, C57BL/6 mice were treated with 2 mg/mouse/day GA for 8 days beginning at various time points after EAE post-induction day 15, yielding a thorough, clinically relevant assessment of GA efficacy within the context of severe progressive disease. Therapeutic treatment with GA significantly decreased clinical scores and improved rotorod motor performance in EAE mice. These functional improvements were supported by an increase in myelinated axons and fewer amyloid precursor protein-positive axons in the spinal cords of GA-treated EAE mice. Furthermore, therapeutic GA decreased microglia/macrophage and T cell infiltrates and increased oligodendrocyte numbers in both the spinal cord and corpus callosum of EAE mice. Finally, GA improved callosal axon conduction and nodal protein organization in EAE. Our results demonstrate that therapeutic GA treatment has significant beneficial effects in a chronic mouse model of MS, in which its positive effects on both myelinated and non-myelinated axons results in improved axon function.

Modulation of autoimmune demyelination by laquinimod via induction of brain-derived neurotrophic factor.

Laquinimod is a promising, orally available compound that has been successfully evaluated in placebo-controlled phase II/III studies of relapsing-remitting multiple sclerosis (MS). Studies are ongoing to further define laquinimod's modulatory mechanisms. Analyses in the animal model of experimental autoimmune encephalomyelitis (EAE) demonstrate that laquinimod reduces infiltration of leukocytes into the central nervous system, induces a Th1 to Th2/3 shift, and suppresses Th17 responses. To evaluate the potential neuroprotective capacity of laquinimod via modulation of brain-derived neurotrophic factor (BDNF), we analyzed the expression of BDNF in blood samples from 203 MS patients treated with laquinimod. Furthermore, we investigated the effect of laquinimod in EAE using a conditional BDNF knockout strain lacking BDNF expression in myeloid cells and T cells (LLF mice). Treatment with laquinimod resulted in a significant and persistent increase in BDNF serum levels of MS patients when compared to baseline and placebo-treated patients. LLF mice treated with laquinimod display a more severe EAE disease course in comparison to wild-type mice. Furthermore, laquinimod-treated wild-type monocytes secreted an anti-inflammatory cytokine pattern in comparison to untreated wild-type monocytes and treated LLF monocytes. Adoptive transfer of laquinimod stimulated monocytes into mice with EAE ameliorated the disease course. Consistent with immunomodulatory properties, laquinimod skewed monocytes toward a regulatory phenotype and also acted via modulation of BDNF, which may contribute to neuroprotection in MS patients.

Laquinimod prevents inflammation-induced synaptic alterations occurring in experimental autoimmune encephalomyelitis.

BACKGROUND: There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. OBJECTIVE: Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. METHODS: C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1-30 µM). RESULTS: Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. CONCLUSIONS: Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.

Reduced astrocytic NF-κB activation by laquinimod protects from cuprizone-induced demyelination.

Laquinimod (LAQ) is a new oral immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis (MS). LAQ has well-documented effects on inflammation in the periphery, but little is known about its direct activity within the central nervous system (CNS). To elucidate the impact of LAQ on CNS-intrinsic inflammation, we investigated the effects of LAQ on cuprizone-induced demyelination in mice in vivo and on primary CNS cells in vitro. Demyelination, inflammation, axonal damage and glial pathology were evaluated in LAQ-treated wild type and Rag-1-deficient mice after cuprizone challenge. Using primary cells we tested for effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NF-κB activation in astrocytes and microglia. LAQ prevented cuprizone-induced demyelination, microglial activation, axonal transections, reactive gliosis and oligodendroglial apoptoses in wild type and Rag-1-deficient mice. LAQ significantly decreased pro-inflammatory factors in stimulated astrocytes, but not in microglia. Oligodendroglial survival was not affected by LAQ in vitro. Astrocytic, but not microglial, NF-κB activation was markedly reduced by LAQ as evidenced by NF-κB reporter assay. LAQ also significantly decreased astrocytic NF-κB activation in cuprizone-treated mice. Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NF-κB activation. These effects are CNS-intrinsic and not mediated by peripheral immune cells. Therefore, LAQ downregulation of the astrocytic pro-inflammatory response may be an important mechanism underlying its protective effects on myelin, oligodendrocytes and axons. Modulation of astrocyte activation may be an attractive therapeutic target to prevent tissue damage in MS.

Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis.

BACKGROUND & AIMS: Aramchol is a fatty acid-bile acid conjugate that reduces liver fat content and is being evaluated in a phase III clinical trial for non-alcoholic steatohepatitis (NASH). Aramchol attenuates NASH in mouse models and decreases steatosis by downregulating the fatty acid synthetic enzyme stearoyl CoA desaturase 1 (SCD1) in hepatocytes. Although hepatic stellate cells (HSCs) also store lipids as retinyl esters, the impact of Aramchol in this cell type is unknown. METHODS: We investigated the effects of Aramchol on a human HSC line (LX-2), primary human HSCs (phHSCs), and primary human hepatocytes (phHeps). RESULTS: In LX-2 and phHSCs, 10 µM Aramchol significantly reduced SCD1 mRNA while inducing PPARG (PPARγ) mRNA, with parallel changes in the 2 proteins; ACTA2, COL1A1, ß-PDGFR (bPDGFR) mRNAs were also significantly reduced in LX-2. Secretion of collagen 1 (Col1α1) was inhibited by 10 µM Aramchol. SCD1 knockdown in LX-2 cells phenocopied the effect of Aramchol by reducing fibrogenesis, and addition of Aramchol to these cells did not rescue fibrogenic gene expression. Conversely, in LX-2 overexpressing SCD1, Aramchol no longer suppressed fibrogenic gene expression. The drug also induced genes in LX-2 that promote cholesterol efflux and inhibited ACAT2, which catalyses cholesterol synthesis. In phHeps, Aramchol also reduced SCD1 and increased PPARG mRNA expression. CONCLUSIONS: Aramchol downregulates SCD1 and elevates PPARG in HSCs, reducing COL1A1 and ACTA2 mRNAs and COL1A1 secretion. These data suggest a direct inhibitory effect of Aramchol in HSCs through SCD1 inhibition, as part of a broader impact on both fibrogenic genes as well as mediators of cholesterol homeostasis. These findings illustrate novel mechanisms of Aramchol activity, including potential antifibrotic activity in patients with NASH and fibrosis. LAY SUMMARY: In this study, we have explored the potential activity of Aramchol, a drug currently in clinical trials for fatty liver disease, in blocking fibrosis, or scarring, by hepatic stellate cells, the principal collagen-producing (i.e. fibrogenic) cell type in liver injury. In both isolated human hepatic stellate cells and in a human hepatic stellate cell line, the drug suppresses the key fat-producing enzyme, stearoyl CoA desaturase 1 (SCD1), which leads to reduced expression of genes and proteins associated with hepatic fibrosis, while inducing the protective gene, PPARγ. The drug loses activity when SCD1 is already reduced by gene knockdown, reinforcing the idea that inhibition of SCD1 is a main mode of activity for Aramchol. These findings strengthen the rationale for testing Aramchol in patients with NASH.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation.

BACKGROUND: Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM: To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS: Isolated primary mouse hepatocytes were incubated with 20 µmol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with 13C-uniformly labeled glucose. For the in vivo part of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS: Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACCα/ß(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with 13C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION: Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA ß-oxidation and oxidative phosphorylation.

Author Correction: Laquinimod treatment in the R6/2 mouse model.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Pharmacogenomics strategies to optimize treatments for multiple sclerosis: Insights from clinical research.

Multiple sclerosis (MS) is a chronic, progressive, disabling disorder characterized by immune-mediated demyelination, inflammation, and neurodegenerative tissue damage in the central nervous system (CNS), associated with frequent exacerbations and remissions of neurologic symptoms and eventual permanent neurologic disability. While there are several MS therapies that are successful in reducing MS relapses, none have been effective in treating all patients. The specific response of an individual patient to any one of the MS therapies remains largely unpredictable, and physicians and patients are forced to use a trial and error approach when deciding on treatment regimens. A priori markers to predict the optimal benefit-to-risk profile of an individual MS patient would greatly facilitate the decision-making process, thereby helping the patient receive the most optimal treatment early on in the disease process. Pharmacogenomic methods evaluate how a person's genetic and genomic makeup affects their response to therapeutics. This review focuses on how pharmacogenomics studies are being used to identify biologically relevant differences in MS treatments and provide characterization of the predictive clinical response patterns. As pharmacogenomics research is dependent on the availability of longitudinal clinical research, studies concerning glatiramer acetate and the interferon beta products which have the majority of published long term data to date are described in detail. These studies have provided considerable insight in the prognostic markers associated with MS disease and potential predictive markers of safety and beneficial response.

Laquinimod treatment in the R6/2 mouse model.

The transgenic mouse model R6/2 exhibits Huntington's disease (HD)-like deficits and basic pathophysiological similarities. We also used the pheochromocytoma-12 (PC12)-cell-line-model to investigate the effect of laquinimod on metabolic activity. Laquinimod is an orally administered immunomodulatory substance currently under development for the treatment of multiple sclerosis (MS) and HD. As an essential effect, increased levels of BDNF were observed. Therefore, we investigated the therapeutic efficacy of laquinimod in the R6/2 model, focusing on its neuroprotective capacity. Weight course and survival were not influenced by laquinimod. Neither were any metabolic effects seen in an inducible PC12-cell-line model of HD. As a positive effect, motor functions of R6/2 mice at the age of 12 weeks significantly improved. Preservation of morphologically intact neurons was found after treatment in the striatum, as revealed by NeuN, DARPP-32, and ubiquitin. Biochemical analysis showed a significant increase in the brain-derived neurotrophic factor (BDNF) level in striatal but not in cortical neurons. The number of mutant huntingtin (mhtt) and inducible nitric oxide synthase (iNOS) positive cells was reduced in both the striatum and motor cortex following treatment. These findings suggest that laquinimod could provide a mild effect on motor function and striatal histopathology, but not on survival. Besides influences on the immune system, influence on BDNF-dependent pathways in HD are discussed.

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

BACKGROUND: Laquinimod 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 (Aß) accumulation and tau phosphorylation in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. METHODS: Utilizing 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. Aß deposition and tau phosphorylation were determined by immunohistochemistry. RESULTS: In 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. CONCLUSION: This 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.

Role of Aramchol in steatohepatitis and fibrosis in mice.

Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease (NAFLD) which sets the stage for further liver damage. The mechanism for the progression of NASH involves multiple parallel hits including oxidative stress, mitochondrial dysfunction, inflammation and others. Manipulation of any of these pathways may be an approach to prevent NASH development and progression. Aramchol (arachidyl-amido cholanoic acid) is presently in a phase IIb NASH study. The aim of this study was to investigate Aramchol's mechanism of action and its effect on fibrosis using the methionine- and choline-deficient (MCD) diet model of NASH. We collected liver and serum from mice fed a MCD diet containing 0.1% methionine (0.1MCD) for four weeks, which developed steatohepatitis and fibrosis, as well as mice receiving a control diet; the metabolomes and proteomes were determined. 0.1MCD fed mice were given Aramchol (5mg/kg/day for the last 2 weeks); liver samples were analyzed histologically. Aramchol administration reduced features of steatohepatitis and fibrosis in 0.1MCD fed mice. Aramchol downregulated stearoyl-CoA desaturase 1 (SCD1), a key enzyme involved in triglyceride biosynthesis whose loss enhances fatty acid ß-oxidation. Aramchol increased the flux through the transsulfuration pathway, leading to a rise in glutathione (GSH) and GSH/GSSG ratio, the main cellular antioxidant that maintains intracellular redox status. Comparison of serum metabolomic pattern between 0.1MCD fed mice and NAFLD patients showed a substantial overlap. CONCLUSIONS: Aramchol treatment improved steatohepatitis and fibrosis by 1) decreasing SCD1, and 2) increasing the flux through the transsulfuration pathway maintaining cellular redox homeostasis. We also demonstrated that the 0.1MCD model resembles the metabolic phenotype observed in about 50% of NAFLD patients, which supports the potential use of Aramchol in NASH treatment.

Laquinimod prevents cuprizone-induced demyelination independent of Toll-like receptor signaling.

OBJECTIVE: To test whether Toll-like receptor (TLR) signaling plays a key role for reduced nuclear factor B (NF-κB) activation after laquinimod treatment in the model of cuprizone-induced demyelination, oligodendrocyte apoptosis, inflammation, and axonal damage. METHODS: Ten-week-old C57BL/6J, TLR4(-/-), and MyD88(-/-) mice received 0.25% cuprizone for 6 weeks and were treated daily with 25 mg/kg laquinimod or vehicle. After 6 weeks of demyelination, extent of demyelination, oligodendrocyte density, microglia infiltration, and axonal damage were analyzed in the corpus callosum. Additionally, we analyzed primary mouse astrocytes from C57BL/6J, TLR4(-/-), MyD88(-/-), and TRIF(-/-) mice for alteration in NF-κB signaling. RESULTS: Vehicle-treated controls from C57BL/6J, TLR4(-/-), and MyD88(-/-) mice displayed extensive callosal demyelination as well as microglial activation. In contrast, mice treated with 25 mg/kg laquinimod showed mainly intact callosal myelin. The demyelination score was significantly higher in all untreated mice compared to mice treated with laquinimod. There were significantly fewer APP-positive axonal spheroids, Mac3-positive macrophages/microglia, and less oligodendrocyte apoptosis in the corpus callosum of laquinimod-treated mice in comparison to untreated controls. Stimulated primary mouse astrocytes from laquinimod-treated groups show reduced NF-κB activation compared to vehicle-treated controls. CONCLUSIONS: Our results confirm that laquinimod prevents demyelination in the cuprizone mouse model for multiple sclerosis via downregulation of NF-κB activation. This laquinimod effect, however, does not involve upstream Toll-like receptor signaling.

A New Targeted Model of Experimental Autoimmune Encephalomyelitis in the Common Marmoset.

Multiple sclerosis (MS) is the most common cause for sustained disability in young adults, yet treatment options remain very limited. Although numerous therapeutic approaches have been effective in rodent models of experimental autoimmune encephalomyelitis (EAE), only few proved to be beneficial in patients with MS. Hence, there is a strong need for more predictive animal models. Within the past decade, EAE in the common marmoset evolved as a potent, alternative model for MS, with immunological and pathological features resembling more closely the human disease. However, an often very rapid and severe disease course hampers its implementation for systematic testing of new treatment strategies. We here developed a new focal model of EAE in the common marmoset, induced by myelin oligodendrocyte glycoprotein (MOG) immunization and stereotactic injections of proinflammatory cytokines. At the injection site of cytokines, confluent inflammatory demyelinating lesions developed that strongly resembled human MS lesions. In a proof-of-principle treatment study with the immunomodulatory compound laquinimod, we demonstrate that targeted EAE in marmosets provides a promising and valid tool for preclinical experimental treatment trials in MS research.

Laquinimod rescues striatal, cortical and white matter pathology and results in modest behavioural improvements in the YAC128 model of Huntington disease.

Increasing evidence supports a role for abnormal immune activation and inflammatory responses in Huntington disease (HD). In this study, we evaluated the therapeutic potential of laquinimod (1 and 10 mg/kg), a novel immunomodulatory agent shown to be protective in a number of neuroinflammatory conditions, in the YAC128 mouse model of HD. Treatment with laquinimod for 6 months rescued atrophy in the striatum, in certain cortical regions, and in the corpus callosum of YAC128 HD mice. Diffusion tensor imaging showed that white matter microstructural abnormalities in the posterior corpus callosum were improved following treatment with low dose (1 mg/kg) laquinimod, and were paralleled by reduced levels of interleukin-6 in the periphery of YAC128 HD mice. Functionally, treatment with laquinimod (1 and 10 mg/kg) led to modest improvements in motor function and in depressive-like behaviour. Taken together, these results suggest that laquinimod may improve some features of pathology in HD, and provides support for the role of immune activation in the pathogenesis of HD.

Immune parameters of patients treated with laquinimod, a novel oral therapy for the treatment of multiple sclerosis: results from a double-blind placebo-controlled study.

Laquinimod is a novel orally administered drug for the treatment of relapsing remitting multiple sclerosis (RRMS). In this immunological substudy of the phase III Assessment of Oral Laquinimod in Preventing Progression of MS (ALLEGRO) trial, we performed an ex vivo and in vitro analysis of effects exerted by laquinimod on peripheral blood immune cell populations from RRMS patients with a special focus on monocyte phenotype and function. Approximately 100 patients were enrolled following a standardized protocol. Half of the patients received laquinimod and the other half received placebo. Peripheral blood samples were collected prior to commencement of therapy and after 1, 3, 6, 12, and 24 months of continuous therapy. Main lymphocytic and antigen presenting cell fractions were analyzed in peripheral blood mononuclear cells (PBMCs) ex vivo by flow cytometry. The proliferative response of PBMCs to mitogen or recall antigen was assessed in culture experiments. Untouched monocytes were sorted magnetically and cultured under pro-inflammatory conditions. PBMC analysis showed no significant differences of investigated lymphocytic and antigen presenting cell populations over time within each group, or between the two groups. However, the detailed in vitro analysis of monocytes demonstrated a lower level of CD86 expression on monocytes stimulated with LPS in laquinimod patients beginning from the 1st month of treatment. Upon pro-inflammatory stimulation, monocytes obtained from laquinimod treated patients tended to secrete lower levels of the proinflammatory chemokines CCL2 or CCL5. Taken together, in this prospective study, we demonstrate immune modulation but no immunosuppressive biological activity of laquinimod in a large group of MS patients.