Sarah Al-Izki

Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmune encephalomyelitis

Background: There has been poor translation for the use of immunosuppressive agents from experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), into the treatment of MS. This may be due to the fact that most EAE studies examine prophylactic, pre-treatment regimes that prove to be therapeutically-ineffective in long-established, often progressive, MS. FTY720 (fingolimod/Gilenya) is a sphingosine-1-phosphate receptor modulator. This is a new oral agent that markedly reduces the number of relapses in people with MS, compared with currently licensed injectable agents such as the beta interferons. FTY720 has activity against lymphocytes but may also influence oligodendroglia and could therefore have the potential to influence progressive MS, by promoting remyelination. Methods: The effect of FTY720 was assessed in relapsing-progressive EAE in mice. Results: Early intervention during relapsing EAE could completely inhibit subsequent relapses, inhibited the accumulation of neurodegeneration, and facilitated motor recovery. However, when examined in secondary progressive EAE, that develops after the accumulation of deficit from relapsing disease, long-term treatment with FTY720 failed to slow deterioration when initiated late (4 months) into the disease course. Conclusions: This study indicates that early intervention with immunosuppressive agents may inhibit the generation of the neurodegenerative microenvironment, which is no longer responsive to potent immunosuppression. However, if treatment is initiated too late, progressive, neurological-disease continues unabated. This suggests that immunosuppression is insufficient to control secondary progression in animals, as has been found so far to be the case in MS, and may warrant early intervention with FTY720 for optimal treatment benefit.

Practical guide to the induction of relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse

Biozzi ABH mice develop a reproducible, relapsing-remitting form of experimental autoimmune encephalomyelitis (EAE) that becomes secondary progressive with disease duration. The relapses observed are T-cell dependent and can be inhibited by immune tolerance induction. In contrast the progressive neurodegeneration is T cell-independent and continues despite the re-induction of immune tolerance. Here we present a practical guide to EAE induction in the ABH mouse and approaches used to control relapses such that both autoimmune-independent and autoimmune-dependent mechanisms of neurodegeneration can be explored. Disease-related weight changes are associated with blood-brain barrier dysfunction and clinical disease. A new method for detecting neurodegeneration is described along with new experimental details that will aid in the undertaking of studies in EAE in mice, with particularly emphasis on ABH mice.

Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

Evaluation of the Effects of Sativex (THC BDS: CBD BDS) on Inhibition of Spasticity in a Chronic Relapsing Experimental Allergic Autoimmune Encephalomyelitis: A Model of Multiple Sclerosis

This study investigated the antispasticity potential of Sativex in mice. Chronic relapsing experimental allergic encephalomyelitis was induced in adult ABH mice resulting in hind limb spasticity development. Vehicle, Sativex, and baclofen (as a positive control) were injected intravenously and the “stiffness” of limbs assessed by the resistance force against hind limb flexion. Vehicle alone caused no significant change in spasticity. Baclofen (5 mg/kg) induced approximately a 40% peak reduction in spasticity. Sativex dose dependently reduced spasticity; 5 mg/kg THC + 5 mg/kg CBD induced approximately a 20% peak reduction; 10 mg/kg THC + 10 mg/kg CBD produced approximately a 40% peak reduction in spasticity. Sativex has the potential to reduce spasticity in an experimental mouse model of multiple sclerosis (MS). Baclofen reduced spasticity and served as a positive control. Sativex (10 mg/kg) was just as effective as baclofen, providing supportive evidence for Sativex use in the treatment of spasticity in MS.

Control of spasticity in a multiple sclerosis model using central nervous system-excluded CB1 cannabinoid receptor agonists

The purpose of this study was the generation of central nervous system (CNS)‐excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side‐effect profiling to demonstrate the mechanism of CNS‐exclusion via drug pumps; genome‐wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug‐pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD‐1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1–3 geneticloci. ABCC1 within a cluster showing linkage is a cannabinoid CNS‐drug pump. Global and conditional CB1 receptor‐knockout mice were used as controls. In summary, CNS‐excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.—Pryce, G., Visintin, C., Ramagopalan, S. V., Al‐Izki, S., De Faveri, L. E., Nuamah, R. A., Mein, C. A., Montpetit, A., Hardcastle, A. J., Kooij, G., de Vries, H. E., Amor, S., Thomas, S. A., Ledent, C., Marsicano, G., Lutz, B., Thompson, A. J., Selwood, D. L., Giovannoni, G., Baker, D. Control of spasticity in a multiple sclerosis model using central nervous system‐excluded CB1 cannabinoid receptor agonists. FASEB J. 28, 117–130 (2014). www.fasebj.org

Neurodegeneration progresses despite complete elimination of clinical relapses in a mouse model of multiple sclerosis

BackgoundMultiple Sclerosis has two clinical phases reflecting distinct but inter-related pathological processes: focal inflammation drives the relapse-remitting stage and neurodegeneration represents the principal substrate of secondary progression. In contrast to the increasing number of effective anti-inflammatory disease modifying treatments for relapse-remitting disease, the absence of therapies for progressive disease represents a major unmet clinical need. This raises the unanswered question of whether elimination of clinical relapses will prevent subsequent progression and if so how early in the disease course should treatment be initiated. Experimental autoimmune encephalomyelitis in the Biozzi ABH mouse recapitulates the clinical and pathological features of multiple sclerosis including relapse-remitting episodes with inflammatory mediated demyelination and progressive disability with neurodegeneration. To address the relationship between inflammation and neurodegeneration we used an auto-immune tolerance strategy to eliminate clinical relapses in EAE in a manner analogous to the clinical effect of disease modifying treatments.ResultsBy arresting clinical relapses in EAE at two distinct stages, early and late disease, we demonstrate that halting immune driven demyelination even after the first major clinical event is insufficient to prevent long-term neurodegeneration and associated gliosis. Nonetheless, early intervention is partially neuroprotective, whereas later interventions are not. Furthermore early tolerisation is also associated with increased remyelination.ConclusionsThese findings are consistent with both a partial uncoupling of inflammation and neurodegeneration and that the regenerative response of remyelination is negatively correlated with inflammation. These findings strongly support the need for early combinatorial treatment of immunomodulatory therapies and neuroprotective treatments to prevent long-term neurodegeneration in multiple sclerosis.

Evaluating potential therapies for bladder dysfunction in a mouse model of multiple sclerosis with high-resolution ultrasonography.

Background In multiple sclerosis (MS), demyelinating and neurodegenerative lesions develop throughout the central nervous system, which result in a loss of neurotransmission. As a result, people with MS exhibit a wide range of symptoms including dysfunction of the bladder, which can lead to urinary incontinence or retention. Such signs can develop in animal models of MS. Current assessments of bladder properties in animal models are generally invasive, electrophysiological techniques. Objective The use of a non-invasive, ultrasound system for measuring bladder volume in a mouse model of MS. Methods Chronic relapsing experimental autoimmune encephalomyelitis was induced in mice. The bladder volume was assessed using ultrasonography, during the disease course and following therapy with bethanechol chloride. Results It was demonstrated that volumes obtained ultrasonically positively-correlated (r = 0.960) with the urine volumes obtained by manual expression. It was also shown for the first time that bladder size increased significantly in mice with residual neurological deficit. Indeed, this increase in bladder size showed a strong, positive-correlation (r = 0.951) with the hind limb spasticity. Following treatment with bethanechol chloride, bladder volume significantly decreased in mice with chronic experimental autoimmune encephalomyelitis. Conclusion This study demonstrates a novel outcome measure in experimental MS that allows; repeated, non-invasive, high resolution ultrasonic monitoring of bladder function.

Imidazol-1-ylethylindazole Voltage-Gated Sodium Channel Ligands Are Neuroprotective during Optic Neuritis in a Mouse Model of Multiple Sclerosis

A series of imidazol-1-ylethylindazole sodium channel ligands were developed and optimized for sodium channel inhibition and in vitro neuroprotective activity. The molecules exhibited displacement of a radiolabeled sodium channel ligand and selectivity for blockade of the inactivated state of cloned neuronal Nav channels. Metabolically stable analogue 6 was able to protect retinal ganglion cells during optic neuritis in a mouse model of multiple sclerosis.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β

Objectives: Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes. Methods: Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay. Results: We demonstrate here that fusion proteins of TGF-β, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-β can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease. Conclusions: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti‐metabolite approach to identify drugs that target spasticity.