Sphingosine 1-phosphate Receptor Agonist Research Articles

Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

BackgroundIt is clinically recognized that collateral vessels can form after retinal vein occlusion (RVO) in some cases and these vessels can lead to spontaneous recovery of the pathological condition. In recent years, optical coherence tomography angiography (OCTA) has become a decisive clinical instrument. Unlike previous angiography tests, OCTA enables the non-invasive visualization of fundus vasculature without the need for administration of a contrast agent. However, it remains to be determined if OCTA depicts the ‘true’ histological status as several studies have reported artifacts in OCTA imaging. MethodsWe generated a laser-induced mouse RVO model, and evaluated the subsequent formation of collateral vessels in order to understand the mechanisms by which collateral vessels form using OCTA imaging, as well as molecular and histological assessments. ResultsWe succeeded in visualizing the time course of collateral vessel formation in a mouse RVO model and confirmed the similarity in formation of collateral vessels only within the deep layer of the retina in both human and mouse. We hypothesized that sphingosine 1-phosphate receptor-1 (S1PR1) may play important roles via vascular shear stress linking vein occlusion and collateral vessel formation. Results from OCTA revealed that collateral vessels are increased in response to administration of a S1PR1 agonist in a mouse RVO model. Based on quantitative reverse transcription polymerase chain reaction (qRT-PCR), S1PR1 messenger ribonucleic acid (mRNA) levels in the whole retina peaked 6 h after photocoagulation in this model. Immunohistochemical staining of retinal flat mounts revealed that S1PR1 staining occurred along the laser-occluded blood vessels. ConclusionWe observed the temporal process of collateral vessel formation in a mouse RVO model and identified the relationship between S1PR1 and shear stress as one of the factors in collateral vessel formation in RVO.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

MiR-140 ameliorates neuropathic pain in CCI rats by targeting S1PR1

Objectives Neuropathic pain, with lots of clinical conditions in various diseases, whose physiopathology is implicated in inflammation. MicroRNAs (miRNAs) have largely been shown to exert anti-inflammatory effects against chronic diseases. We then evaluated the effects and regulatory mechanism of miR-140 on neuropathic pain. Methods Rats model with neuropathic pain were established via chronic constriction injury (CCI) and verified by determination of mechanical withdrawal threshold (MWT) and paw withdrawal latency (PWL). The expression level of miR-140 was determined via qRT-PCR (quantitative real-time polymerase chain reaction). Intrathecal injection of miR-140 agomiR was conducted to evaluate the influence of miR-140 on CCI rats via evaluation of MWT, PWL and inflammatory factors secretion. The binding target of miR-140 was predicted and characterized via dual luciferase activity assay. Results Decreased MWT and PWL, as well as increased inflammatory factor secretion, including IL (interleukin)-1β, IL-6 and interferon-γ (IFN-γ), were found in rats under CCI compared with sham rats. MiR-140 was decreased in rats under CCI. Intrathecal injection of miR-140 agomiR increased MWT and PWL, thus attenuating mechanical and thermal hyperalgesia in CCI rats. Moreover, decreased inflammatory factor secretion in rats under CCI injected with miR-140 agomiR, suggesting a negatively regulatory role of miR-140 on neuroinflammation. MiR-140 could bind with Sphingosine-1-phosphate receptor 1 (S1PR1). S1PR1 agonist, SEW2871, could reverse the suppression of miR-140 on neuropathic pain. Conclusions MiR-140 could mollify CCI-stimulated neuropathic pain via targeting S1PR1, suggesting a potential therapeutic target in the treatment of neuropathic pain.

A label-free impedance assay in endothelial cells differentiates the activation and desensitization properties of clinical S1P1 agonists.

Sphingosine-1 phosphate receptor-1 (S1P1 ) activation maintains endothelial barrier integrity, whereas S1P1 desensitization induces peripheral blood lymphopenia. The latter is exploited in the approval and/or late-stage development of receptor-desensitizing agents targeting the S1P1 receptor in multiple sclerosis, such as siponimod, ozanimod, and ponesimod. SAR247799 is a recently described G protein-biased S1P1 agonist that activates S1P1 without desensitization and thus has endothelial-protective properties in patients without reducing lymphocytes. As SAR247799 demonstrated endothelial-protective effects at sub-lymphocyte-reducing doses, the possibility exists that other S1P1 modulators could also exhibit endothelial-protective properties at lower doses. To explore this possibility, we sought to quantitatively compare the biased properties of SAR247799 with the most advanced clinical molecules targeting S1P1 . In this study, we define the β-arrestin pathway component of the impedance profile following S1P1 activation in a human umbilical vein endothelial cell line (HUVEC) and report quantitative indices of the S1P1 activation-to-desensitization ratio of various clinical molecules. In a label-free impedance assay assessing endothelial barrier integrity and disruption, the mean estimates (95% confidence interval) of the activation-to-desensitization ratios of SAR247799, ponesimod, ozanimod, and siponimod were 114 (91.1-143), 7.66 (3.41-17.2), 6.35 (3.21-12.5), and 0.170 (0.0523-0.555), respectively. Thus, we show that SAR247799 is the most G protein-biased S1P1 agonist currently characterized. This rank order of bias among the most clinically advanced S1P1 modulators provides a new perspective on the relative potential of these clinical molecules for improving endothelial function in patients in relation to their lymphocyte-reducing (desensitization) properties.

Abstract P250: Sphingosine-1-phosphate Receptor Agonist And Very Low-density Lipoprotein Regulate Aldosterone Production Via Lipin-1 In Human Adrenocortical Cells

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of both sphingosine-1-phosphate (S1P) and very low-density lipoprotein (VLDL). S1P has been reported to be a novel stimulator of aldosterone secretion and phospholipase D (PLD) activity. VLDL has also been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via PLD. PLD is an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA) which can then be converted to diacylglycerol (DAG) by lipin-1. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s mediation of aldosterone production. We hypothesized that the S1P1 receptor (S1PR1) agonist, SEW2871, (and VLDL) induces steroidogenesis and therefore aldosterone production via lipin-1-mediated metabolism of PA to DAG, with our hypothesis focusing on DAG as the key lipid signal produced by PLD (indirectly). In HAC15 cells, lipin-1 was overexpressed using an adenovirus or inhibited using propranolol followed by treatment with or without SEW2871 (or VLDL) for 24 h. Steroidogenic gene expression and aldosterone levels were monitored by qRT-PCR and radioimmunoassay, respectively. We demonstrated that lipin-1 overexpression (OE) enhanced the SEW2871-stimulated 109-fold increase in CYP11B2 expression by 26% while lipin-1 inhibition decreased the SEW2871-stimulated 56-fold increase in CYP11B2 expression by 74%. While lipin-1 OE had no further effect, propranolol reduced SEW2871-stimulated increases in NR4A1 (2-fold) and NR4A2 (9-fold) mRNA levels by 22% and 52% respectively. The SEW2871-stimulated increase in aldosterone production was inhibited by propranolol (53%), although it was not enhanced by lipin-1 OE. Similar results were obtained with VLDL. Our results are, therefore, suggestive of DAG being the key lipid signal since regulating lipin-1 affects S1PR1 agonist- and VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin-1, or its downstream pathways, to potentially treat obesity-associated hypertension.

S1P1 Contributes to Endotoxin-enhanced B-Cell Functions Involved in Hypersensitivity Pneumonitis.

In a proportion of patients with hypersensitivity pneumonitis, the biological and environmental factors that sustain inflammation are ill defined, resulting in no effective treatment option. Bioaerosols found in occupational settings are complex and often include Toll-like receptor ligands, such as endotoxins. How Toll-like receptor ligands contribute to the persistence of hypersensitivity pneumonitis, however, remains poorly understood. In a previous study, we found that an S1P1 (sphingosine-1-phosphate receptor 1) agonist prevented the reactivation of antigen-driven B-cell responses in the lung. Here, we assessed the impact of endotoxins on B-cell activation in preexisting hypersensitivity pneumonitis and the role of S1P1 in this phenomenon. The impact of endotoxins on pre-established hypersensitivity pneumonitis was studied in vivo. S1P1 levels were tracked on B cells in the course of the disease using S1P1-eGFP knockin mice, and the role of S1P1 on B-cell functions was assessed using pharmacological tools. S1P1 was found on B cells in experimental hypersensitivity pneumonitis. Endotoxin exposure enhanced neutrophil accumulation in the BAL of mice with experimental hypersensitivity pneumonitis. This was associated with enhanced CD69 cell-surface expression on lymphocytes in the BAL. In isolated B cells, endotoxins increased cell-surface levels of costimulatory molecules and CD69, which was prevented by an S1P1 agonist. S1P1 modulators also reduced TNF production by B cells and their capacity to trigger T-cell cooperation ex vivo. An S1P1 ligand directly inhibited endotoxin-induced B-cell activation.

A G protein-biased S1P1 agonist, SAR247799, protects endothelial cells without affecting lymphocyte numbers.

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P1) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P1, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P1-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P1 These findings demonstrate that sustained S1P1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.

Small molecule drugs in the treatment of inflammatory bowel diseases: which one, when and why? - a systematic review.

In the 'treat-to-target' era of inflammatory bowel disease (IBD) management, small molecule drugs (SMDs) represent a promising alternative to biomolecular drugs. Moreover, increasing failure rates of anti-tumor necrosis factor α agents have contributed to the development of new molecules with different mechanisms of action and bioavailability. This review focuses on the positioning of new, orally targeted therapies in the treatment algorithm of both Crohn's disease (CD) and ulcerative colitis (UC), with special consideration to their efficacy and safety. We performed a comprehensive search of PubMed and clinical trial registries to identify randomized controlled trials assessing SMDs in adult patients with moderate-to-severe IBD, irrespective of previous exposure to other biologics. In this review, we included 15 double-blind, placebo-controlled trials that assessed the efficacy and safety of Janus kinase inhibitors, sphingosine-1-phosphate modulators (S1P), SMAD blockers, phosphodiesterase 4 inhibitors and α-4 antagonists. The primary endpoints in UC were achieved for tofacitinib in the phase III OCTAVE study and AJM-300, with a favorable safety profile. S1P receptor agonists, such as etrasimod and ozanimod, demonstrated favorable results in induction studies. For CD, filgotinib and upadacitinib also met the primary outcome criteria. Available data have demonstrated so far that SMDs have an advantageous safety and efficacy profile. However, their use in a clinical setting will eventually require a personalized, mechanism-based therapeutic approach.

T cells are the main population in mouse breast milk and express similar profiles of tight junction proteins as those in mammary alveolar epithelial cells

Immune cells are present in the breast milk of several mammalian species; however, their immunological function and transmigration mechanisms to milk remain unknown. Some researchers hypothesize that milk leukocytes have a mammary gland (MG) origin and transmigrate thorough the paracellular pathway, but mammary alveolar epithelial cells strictly regulate the paracellular movement of milk components during lactation via barrier structures, such as tight junctions (TJs). To investigate this discrepancy, we compared leukocyte populations in mouse MG and milk and explored TJ protein expression profiles in MG leukocytes. The main subsets of milk leukocytes were CD8+ and CD4+ T cells displaying the memory phenotype. The proportions of myeloid, B, and dendritic cells were significantly lower in milk than in the MG. CD8+ T cells expressed genes encoding the TJ proteins claudin-3, -7, -12, and ZO-1 at higher levels when compared with myeloid and B cells in the MG among lactating mice. Alveolar epithelial cells in the MG expressed claudin-3, -4, and -7. Administration of FTY720, an inhibitory agonist of sphingosine 1-phosphate receptor 1 that stabilizes TJ permeability, increased the myeloid cell proportion in milk. Different leukocyte populations in the MG and milk suggest active and selective mechanisms of cell transmigration to milk. Both TJ-forming components in alveolar epithelial cells from the MG and TJ protein expression profiles in leukocytes from the MG appear to regulate milk leukocyte populations. T cells are the main population in mouse breast milk and express similar profiles of TJ proteins as those in mammary alveolar epithelial cells.

Very Low‐Density Lipoprotein and a Sphingosine‐1‐Phosphate Receptor Agonist Regulate Aldosterone Production via Lipin‐1 in Human Adrenocortical Cells

Aldosterone plays an important role in blood pressure homeostasis, and hyperaldosteronism can result in hypertension. Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low‐density lipoprotein (VLDL). VLDL has been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA), a lipid second messenger. In addition, sphingosine‐1‐phosphate (S1P), a bioactive sphingolipid also elevated in obesity, has been reported to be a novel stimulator of aldosterone secretion and PLD activity. The mechanisms underlying the action of these non‐conventional secretagogues of aldosterone remain to be elucidated. Angiotensin II, a classical aldosterone secretagogue, not only activates PLD but also elevates the expression of lipin‐1, an enzyme that converts PA to another lipid second messenger, diacylglycerol (DAG), in human adrenocortical carcinoma (HAC15) cells. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s role in aldosterone production. Herein, we used HAC15 cells to determine whether VLDL and an S1P1 receptor (S1PR1) agonist (SEW2871) stimulate aldosterone production by increasing steroidogenic gene expression via lipin‐1‐mediated metabolism of PA to DAG. We overexpressed lipin‐1 using an adenovirus or inhibited it using propranolol followed by treatment with or without VLDL or SEW2871 for 24 h. The expression of steroidogenic genes and aldosterone secretion were monitored using qRT‐PCR and radioimmunoassay. We demonstrated that lipin‐1 overexpression enhanced the VLDL‐stimulated 55‐fold increase in CYP11B2 expression by 75% while lipin‐1 inhibition decreased the VLDL‐stimulated 21‐fold increase in CYP11B2 expression by 66%. A parallel trend was observed with aldosterone secretion levels: VLDL‐stimulated increase in aldosterone production was enhanced by lipin‐1 overexpression (182%) and was decreased by propranolol (80%). Similar results were obtained with SEW2871. Our results are, therefore, suggestive of DAG being the key lipid signal since lipin‐1 regulates VLDL‐ and S1PR1 agonist‐stimulated expression of steroidogenic genes and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin‐1, or its downstream pathways, to potentially treat obesity‐associated hypertension.Support or Funding InformationThis work was supported in part by the Augusta University Adrenal Center. WBB was also supported by VA Merit Award #BX001357.

In silico Docking Studies of Fingolimod and S1P1 Agonists.

The sphingosine-1-phosphate receptor 1 (S1P1), originally the endothelial differentiation gene 1 receptor (EDG-1), is one of five G protein–coupled receptors (GPCRs) S1P1–5 that bind to and are activated by sphingosine-1-phosphate (S1P). The lipid S1P is an intermediate in sphingolipid homeostasis, and S1P1 is a major medical target for immune system modulation; agonism of the receptor produces a myriad of biological responses, including endothelial cell barrier integrity, chemotaxis, lymphocyte trafficking/targeting, angiogenesis, as well as regulation of the cardiovascular system. Use of in silico docking simulations on the crystal structure of S1P1 allows for pinpointing the residues within the receptor’s active site that actively contribute to the binding of S1P, and point to how these specific interactions can be exploited to design more effective synthetic analogs to specifically target S1P1 in the presence of the closely related receptors S1P2, S1P3, S1P4, and S1P5. We examined the binding properties of the endogenous substrate as well as a selection of synthetic sphingosine-derived S1P1 modulators of S1P1 with in silico docking simulations using the software package Molecular Operating Environment® (MOE®). The modeling studies reveal the relevance of phosphorylation, i.e., the presence of a phosphate or phosphonate moiety within the substrate for successful binding to occur, and indicate which residues are responsible for S1P1 binding of the most prominent sphingosine-1-phosphate receptor (S1PR) modulators, including fingolimod and its structural relatives. Furthermore, trends in steric preferences as for the binding of enantiomers to S1P1 could be observed, facilitating future design of receptor-specific substrates to precisely target the active site of S1P1.

Potential sphingosine-1-phosphate-related therapeutic targets in the treatment of cerebral ischemia reperfusion injury

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that regulates lymphocyte trafficking, glial cell activation, vasoconstriction, endothelial barrier function, and neuronal death pathways in the brain. Research has increasingly implicated S1P in the pathology of cerebral ischemia reperfusion (IR) injury. As a high-affinity agonist of S1P receptor, fingolimod exhibits excellent neuroprotective effects against ischemic challenge both in vivo and in vitro. By summarizing recent progress on how S1P participates in the development of brain IR injury, this review identifies potential therapeutic targets for the treatment of brain IR injury.

Mechanism of sphingosine 1-phosphate clearance from blood.

The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes’ recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.

Fingolimod Rescues Demyelination in a Mouse Model of Krabbe's Disease.

Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination both in vitro and ex vivo models. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. These in vivo findings corroborate initial in vitro studies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease.SIGNIFICANCE STATEMENT This study demonstrates that the administration of the therapy known as fingolimod in a mouse model of Krabbe's disease (namely, the twitcher mouse model) significantly rescues myelin levels. Further, the drug fingolimod also regulates the reactivity of glial cells, astrocytes and microglia, in this mouse model. These protective effects of fingolimod result in an increased life span of twitcher mice.

A Multicentre, Double-Blind, Placebo-Controlled, Parallel-Group Study to Evaluate the Efficacy, Safety, and Tolerability of the S1P Receptor Agonist KRP203 in Patients with Moderately Active Refractory Ulcerative Colitis

Background and Aims: KRP203 is a potent oral agonist of the sphingosine-1-phosphate receptor subtype 1 that induces the sequestration of peripheral lymphocytes, thereby potentially reducing the number of activated lymphocytes circulating to the gastrointestinal tract. Methods: We conducted a multicentre, double-blind, placebo-controlled, parallel-group, proof-of-concept study to evaluate the efficacy, safety, and tolerability of KRP203 in patients with moderately active 5-aminosalicylate-refractory ulcerative colitis (UC). Patients were randomly assigned to receive 1.2 mg KRP203 or placebo daily for 8 weeks. Primary efficacy variable was clinical remission, defined as partial Mayo Score 0–1 and modified Baron Score 0–1 with rectal bleeding subscore 0. Results: KRP203 was safe and well tolerated overall. The most common adverse events (AEs) were gastrointestinal disorders and headache. Importantly, no KRP203-related cardiac AEs were reported. Total peripheral lymphocytes and selectively affected lymphocyte subtypes decreased, causing marked decreases in naive and central memory CD4+ and CD8+ T cells, and also in B cells. Clinical remission occurred in 2/14 (14%) patients under KRP203, compared with 0/8 (0%) under placebo. Conclusions: Overall, KRP203 was safe and well tolerated by patients with UC. Importantly, no cardiac AEs were reported. Although KRP203 did not meet the minimum clinically relevant threshold for efficacy, the results may suggest that KRP203 treatment is superior to placebo. However, in this small study population, the difference was insignificant. Based on these data, studies with an improved design and a larger population should be considered.

Sphingosine 1-phosphate but not Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes.

Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through a family of five G protein-coupled receptors, S1PR1-S1PR5, to regulate cell physiology. The multiple sclerosis drug Fingolimod (FTY720) is a potent S1P receptor agonist that causes peripheral lymphopenia. Recent research has demonstrated direct neuroprotective properties of FTY720 in several neurodegenerative paradigms; however, neuroprotective properties of the native ligand S1P have not been established. We aimed to establish the significance of neurotrophic factor up-regulation by S1P for neuroprotection, comparing S1P with FTY720. S1P induced brain-derived neurotrophic factor(BDNF), leukemia inhibitory factor (LIF), platelet-derived growth factor B (PDGFB), and heparin-binding EGF-like growth factor (HBEGF)gene expression in primary human and murine astrocytes, but not in neurons, and to a much greater extent than FTY720. Accordingly, S1P but not FTY720 protected cultured neurons against excitotoxic cell death in a primary murine neuron-glia coculture model, and a neutralizing antibody to LIF blocked this S1P-mediated neuroprotection. Antagonists of S1PR1and S1PR2both inhibited S1P-mediated neurotrophic geneinduction in human astrocytes, indicating that simultaneous activation of both receptors is required. S1PR2 signaling was transduced through Gα13 and the small GTPase Rho, and was necessary for the up-regulation and activation of the transcription factors FOS and JUN, which regulate LIF, BDNF, andHBEGF transcription. In summary, we show that S1P protects hippocampal neurons against excitotoxic cell death through up-regulation of neurotrophic gene expression, particularly LIF, in astrocytes. This up-regulation requires both S1PR1 and S1PR2 signaling. FTY720 does not activate S1PR2, explaining its relative inefficacy compared to S1P.

Alignment independent 3D-QSAR studies and molecular dynamics simulations for the identification of potent and selective S1P1 receptor agonists

Sphingosine 1-phosphate type 1 (S1P1) receptors are expressed on lymphocytes and regulate immune cells trafficking. Sphingosine 1-phosphate and its analogues cause internalization and degradation of S1P1 receptors, preventing the auto reactivity of immune cells in the target tissues. It has been shown that S1P1 receptor agonists such as fingolimod can be suitable candidates for treatment of autoimmune diseases. The current study aimed to generate GRIND-based 3D-QSAR predictive models for agonistic activities of 2-imino-thiazolidin-4-one derivatives on S1P1 to be used in virtual screening of chemical libraries. The developed model for the S1P1 receptor agonists showed appropriate power of predictivity in internal (r2acc 0.93 and SDEC 0.18) and external (r2 0.75 and MAE (95% data), 0.28) validations. The generated model revealed the importance of variables DRY-N1 and DRY-O in the potency and selectivity of these compounds towards S1P1 receptor. To propose potential chemical entities with S1P1 agonistic activity, PubChem chemicals database was searched and the selected compounds were virtually tested for S1P1 receptor agonistic activity using the generated models, which resulted in four potential compounds with high potency and selectivity towards S1P1 receptor. Moreover, the affinities of the identified compounds towards S1P1 receptor were evaluated using molecular dynamics simulations. The results indicated that the binding energies of the compounds were in the range of −39.31 to −46.18 and −3.20 to −9.75 kcal mol−1, calculated by MM-GBSA and MM-PBSA algorithms, respectively. The findings in the current work may be useful for the identification of potent and selective S1P1 receptor agonists with potential use in diseases such as multiple sclerosis.

Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg−1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance.

ASP1126, a Novel Sphingosine-1-Phosphate–Selective Agonist With a Favorable Safety Profile, Prolongs Allograft Survival in Rats and Nonhuman Primates in Combination With Tacrolimus With a Broad Safety Margin for Bradycardia

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of 5 G protein–coupled receptors (S1P1 to S1P5). Among these, S1P1 is a major regulator of lymphocyte trafficking. Fingolimod, whose active metabolite, fingolimod phosphate, acts as a nonselective S1P-receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating lymphocyte trafficking via downregulation of S1P1 expression on lymphocytes. Here, we describe the pharmacologic profile of a novel S1P1 agonist, ASP1126. ASP1126 preferentially activated S1P1 compared to S1P3 in rat and human guanosine-5'-(γ-thio)-triphosphate (GTPγS) assays. Oral single administration of ASP1126 decreased the number of peripheral lymphocytes and repeated dosing showed a cumulative effect on lymphopenia in both rats and monkeys. ASP1126 prolonged allograft survival in a rat heterotopic heart transplantation model in combination with a subtherapeutic dose of tacrolimus that was independent of drug-drug interactions. In addition, in nonhuman primate (NHP) renal transplantation, pretreatment with ASP1126 reduced not only the number of naive T cells and central memory T cells but also effector memory T cells in the peripheral blood, all of which could contribute to acute graft rejection and prolonged allograft survival in combination with tacrolimus. Further, we confirmed that ASP1126 has a broad ranging safety margin with respect to its effect on lung weight in rats and bradycardia in NHPs, which were the adverse events found in clinical studies of fingolimod. ASP1126 with improved safety profile has the potential to be an adjunct therapy in combination with tacrolimus in clinical transplantation.

RETRACTED ARTICLE: Combination of sphingosine-1-phosphate receptor 1 (S1PR1) agonist and antiviral drug: a potential therapy against pathogenic influenza virus

The pandemic 2009 influenza A H1N1 virus is associated with significant mortality. Targeting S1PR1, which is known to modulate the immune response, provides protection against pathogenic influenza virus. The functional role and molecular mechanism of S1PR1 were analysed by generating inducible endothelial cell-specific S1PR1 knockout mice and assessing the therapeutic efficacy of the selective S1PR1 agonist CYM5442 against acute lung injury (ALI) induced by the 2009 influenza A H1N1 virus. Immune-mediated pulmonary injury is aggravated by the absence of endothelial S1PR1 and alleviated by treatment with CYM-5442, suggesting a protective function of S1PR1 signaling during H1N1 infection. S1PR1 signaling does not affect viral clearance in mice infected with influenza. Mechanistically, the MAPK and NF-kB signaling pathways are involved in the ALI mediated by S1PR1 in infected mice. Combined administration of the S1PR1 agonist CYM-5442 and the antiviral drug oseltamivir provides maximum protection from ALI. Our current study provides insight into the molecular mechanism of S1PR1 mediating the ALI induced by H1N1 infection and indicates that the combination of S1PR1 agonist with antiviral drug could potentially be used as a therapeutic remedy for future H1N1 virus pandemics.