3-amino-4-(3-hexylphenylamino)-4-oxobutylphosphonic-acid and Disease-Models--Animal

Multiple sclerosis (MS) is an autoimmune-inflammatory neurodegenerative disease that is often accompanied by a debilitating neuropathic pain. Disease-modifying agents slow down the progression of multiple sclerosis and prevent relapses, yet it remains unclear if they yield analgesia. We explored the analgesic potential of fingolimod (FTY720), an agonist and/or functional antagonist at the sphingosine-1-phosphate receptor 1 (S1PR1), because it reduces hyperalgesia in models of peripheral inflammatory and neuropathic pain. We used a myelin oligodendrocyte glycoprotein 35 to 55 (MOG35-55) mouse model of experimental autoimmune encephalomyelitis, modified to avoid frank paralysis, and thus, allow for assessment of withdrawal behaviors to somatosensory stimuli. Daily intraperitoneal fingolimod reduced behavioral signs of central neuropathic pain (mechanical and cold hypersensitivity) in a dose-dependent and reversible manner. Both autoimmune encephalomyelitis and fingolimod changed hyperalgesia before modifying motor function, suggesting that pain-related effects and clinical neurological deficits were modulated independently. Fingolimod also reduced cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord: glutamate-evoked Ca signaling and stimulus-evoked phospho-extracellular signal-related kinase ERK (pERK) expression, as well as upregulation of astrocytes (GFAP) and macrophage/microglia (Iba1) immunoreactivity. The antihyperalgesic effects of fingolimod were prevented or reversed by the S1PR1 antagonist W146 (1 mg/kg daily, i.p.) and could be mimicked by either repeated or single injection of the S1PR1-selective agonist SEW2871. Fingolimod did not change spinal membrane S1PR1 content, arguing against a functional antagonist mechanism. We conclude that fingolimod behaves as an S1PR1 agonist to reduce pain in multiple sclerosis by reversing central sensitization of spinal nociceptive neurons.

Topics: Anilides; Animals; Central Nervous System Sensitization; Disease Models, Animal; eIF-2 Kinase; Female; Fingolimod Hydrochloride; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Motor Activity; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Organophosphonates; Oxadiazoles; Pain Threshold; Peptide Fragments; Receptors, Lysosphingolipid; Sphingosine-1-Phosphate Receptors; Spinal Cord; Spinal Nerve Roots; Subcellular Fractions; Thiophenes

Overexpression of P-glycoprotein (P-gp) in the brain is an important factor leading to drug-resistant epilepsy. Clinical use of P-gp inhibitors is limited by their systemic toxicity. In this study, we tested the hypothesis that FTY720, a sphingosine 1-phosphate (S1P) analogue used for treating multiple sclerosis, modulates the up-regulation of P-gp and improves brain delivery of phenytoin (PHT) through S1P receptor 1 in the hippocampus of a pilocarpine-induced rat model of status epilepticus (SE). We administered vehicle, FTY720 or FTY720+ W146 (an S1P receptor 1 antagonist) to SE rats. Forty-eight hours after SE, we dissected the hippocampus and measured P-gp expression, NF-κB activity and levels of inflammatory mediators (TNF-α and COX-2) by Western blotting and enzyme-linked immunosorbent assay. We also measured hippocampal and plasma concentrations of PHT 30, 60, 90, 120 and 180 min. after an intraperitoneal injection of PHT (50 mg/kg) 48 hr after SE, using microdialysis and high-performance liquid chromatography. FTY720 alleviated the overexpression of hippocampal P-gp in SE rats and reduced NF-κB activity and TNF-α and COX-2 expression, and W146 blocked the effects of FTY720. Furthermore, SE rats that received FTY720 showed significantly greater hippocampal extracellular PHT concentrations than those that received vehicle, and W146 abolished this effect. Our results suggest that FTY720 alleviates seizure-induced overexpression of P-gp by inhibiting S1P receptor 1-mediated inflammation in rat hippocampus and improves PHT delivery to brain. FTY720 shows potential as an adjuvant therapy for drug-resistant epilepsy.

Topics: Anilides; Animals; Anticonvulsants; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclooxygenase 2; Disease Models, Animal; Drug Resistant Epilepsy; Fingolimod Hydrochloride; Hippocampus; Humans; Immunosuppressive Agents; Injections, Intraperitoneal; Lysophospholipids; Male; Organophosphonates; Phenytoin; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Up-Regulation

Sphingosine-1-phosphate (S1P) is an important mediator of inflammation recently shown in in vitro studies to increase the excitability of small-diameter sensory neurons, at least in part, via activation of the S1P(1) receptor subtype. Activation of S1PR(1) has been reported to increase the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide (O(2)(·-)) and nitric oxide synthase (NOS)-derived nitric oxide (NO). This process favors the formation of peroxynitrite (ONOO(-) [PN]), a potent mediator of hyperalgesia associated with peripheral and central sensitization. The aims of our study were to determine whether S1P causes peripheral sensitization and thermal hyperalgesia via S1PR(1) activation and PN formation. Intraplantar injection of S1P in rats led to a time-dependent development of thermal hyperalgesia that was blocked by the S1PR(1) antagonist W146, but not its inactive enantiomer W140. The hyperalgesic effects of S1P were mimicked by intraplantar injection of the well-characterized S1PR(1) agonist SEW2871. The development of S1P-induced hyperalgesia was blocked by apocynin, a NADPH oxidase inhibitor; N(G)-nitro-l-arginine methyl ester, a nonselective NOS inhibitor; and by the potent PN decomposition catalysts (FeTM-4-PyP(5+) and MnTE-2-PyP(5+)). Our findings provide mechanistic insight into the signaling pathways engaged by S1P in the development of hyperalgesia and highlight the contribution of the S1P(1) receptor-to-PN signaling in this process. Sphingosine-1-phosphate (S1P)-induced hyperalgesia is mediated by S1P1 receptor activation and mitigated by inhibition or decomposition of peroxynitrite, providing a target pathway for novel pain management strategies.

Topics: Acetophenones; Anilides; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Hyperalgesia; Lysophospholipids; Male; Metalloporphyrins; NG-Nitroarginine Methyl Ester; Organophosphonates; Oxadiazoles; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Time Factors

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

Topics: Anilides; Animals; Capillary Permeability; Cells, Cultured; CHO Cells; Cricetinae; Disease Models, Animal; Evans Blue; Humans; Lymph Nodes; Lymphocytes; Lysophospholipids; Mice; Mice, Inbred C57BL; Models, Biological; Organophosphonates; Phenotype; Pulmonary Edema; Receptors, Lysosphingolipid; Sphingosine; Stereoisomerism