Targeting S1P in Inflammatory Bowel Disease: New Avenues for Modulating Intestinal Leukocyte Migration

Targeting S1P in Inflammatory Bowel Disease: New Avenues for Modulating Intestinal Leukocyte... Abstract Sphingosine 1 phosphate [S1P] is a bioactive lipid mediator involved in the regulation of several cellular processes though the activation of a G protein-coupled receptor family known as the S1P receptors [S1PRs]. Advances in the understanding of the biological activities mediated by S1PRs have sparked great interest in the S1P/S1PRs axes as new therapeutic targets for the modulation of several cellular processes. In particular, the S1P/S1PR1 axis has been identified as key regulator for lymphocyte migration from lymph nodes. The blockade of this axis is emerging as a new therapeutic approach to control the aberrant leukocyte migration into the mucosa in inflammatory bowel disease [IBD]. This review briefly summarises the current evidence coming from clinical studies, and discusses the future prospects of S1P inhibitors for treatment of inflammatory bowel disease. Inflammatory bowel disease, S1P signalling, oral small molecules, lymphocyte circulation modulation 1. Introduction Inflammatory bowel diseases [IBD], which include as major forms ulcerative colitis [UC] and Crohn’s disease [CD], are chronic and relapsing-remitting inflammatory diseases of the gastrointestinal tract. IBD pathogenesis is still poorly understood; however, it is widely accepted that a complex interaction between aberrant inflammatory responses, altered intestinal barrier function, environmental factors and intestinal flora can have a role, in subjects with a genetic predisposition.1 The incidence and prevalence of IBD have increased in the past 50 years, attracting interest from the scientific community to improve the knowledge on these diseases by the identification of new therapeutic targets. However, despite this progress, currently there is no curative therapy. Conventional management of IBD includes glucocorticoids, 5-aminosalicylic acid [for UC], conventional immunomodulators, such as azathioprine, and biologics. The first class of biologics was developed approximately two decades ago, and was represented by anti-tumour necrosis factor [TNF] drugs. Currently licensed anti-TNFs are available for intravenous or subcutaneous administration. However, the efficacy of anti-TNF therapy is often moderate, with potentially serious side effects, is and associated with an increased risk of serious infections. About one-third of patients, defined as primary non-responders, do not respond to induction therapy and another third, called secondary non-responders, despite an initial response show a loss response over time. This secondary loss of response is mainly related to the development of immunogenicity to these protein-based therapies.2 These evidences lead to the need of innovative therapies, with a specific interaction towards new therapeutic targets, to improve the patient’s quality of life and to control the heterogeneous range of symptoms, not adequately treated by the available therapies. In the past decade, an increasing number of newly approved or developing drugs, modulating lymphocyte migration through effects on different mechanisms and acting in a different manner, have shown the potential value of this approach in controlling the aberrant immune response that is co-responsible, with the other above-mentioned factors, for the drastic gut dysfunction observed IBD patients.3 In IBD as in other immune processes, T cells, activated by antigen-presenting cells, play a key role in promoting an immune response. After this activation, circulating T cells enter into lymphoid compartments, where they can differentiate into either tissue resident memory cells or activated T cells. Activated T cells are then released from lymph nodes back into the blood circulation, thus reaching inflamed tissue by extravasation from blood vessels. The process is regulated by adhesion molecules, specific chemokines, and selective binding receptors located in lymph nodes and on appropriate vascular endothelial cells. In the vessels this is a three-step process composed of rolling, regulated by selectins, chemokine-dependent activation and arrest, mediated by integrins. Once lymphocytes have been bound by integrins, they can access the gut through specialised venules, expressing an integrin ligand such as mucosal addressin cell adhesion molecule 1 [MAdCAM-1].4 The potential role of lymphocyte circulation-modulating agents in IBD led to the development of new drugs. A number of drugs blocking specific gut-selective integrin-endothelial ligand interactions have been developed to find a safe and efficacious alternative therapy to anti-TNF-alpha. Vedolizumab is a humanised immunoglobulin [IG] G1 mAb directed against the human lymphocyte integrin α4β7. The α4β7integrin mediates lymphocyte trafficking to gastrointestinal mucosa and gut-associated lymphoid tissue through adhesive interaction with MAdCAM-1, which is expressed on the endothelium of mesenteric lymph nodes and gastrointestinal mucosa.5 Vedolizumab is characterised by high gut selectivity, and it has demonstrated efficacy both for UC and CD treatment.6,7 However, more than 50% of patients could be primary non-responders or could lose response to this drug, both in UC and CD.8,9 Another anti-integrin drug still in development for both indications is etrolizumab. It selectively binds the β7 subunits of the α4β7 and αEβ7 integrins and could, in the future, become an alternative therapy for IBD patients.10,11 The interaction between adhesion molecules and integrins can also be blocked by drugs like anti-MAdCam-1, binding the adhesion molecule at the endothelial level.2 All biologics are administered by either e intravenous infusion or by injection, and are characterised by long half-lives [more than 20 days]. Because of the high rate of non-response or loss of response to marketed drugs, and due to their invasive route of administration, in recent years there has been a growing interest in novel small molecules development. These drugs, orally administered, are characterised by new mechanisms of action. They bind new molecular targets directly located in the lymph node and not at vascular levels. Sphingosine 1-phosphate receptor 1 [S1P1] is a member of these new targets and is involved in the regulation of immune cell circulation. In particular, S1P regulates the re-circulation of naïve and central memory CCR7-positive T cells from the secondary lymphatic organs to the periphery, but does not sequester effector memory CCR7-negative T cells that are responsible for maintaining immune surveillance against pathogens in the periphery, as they do not traffic through lymph nodes.12,13 FTY720, an agonistic activator of S1P receptors also called fingolimod, was shown to reduce lymphocyte egress from secondary lymphoid organs, thus resulting in efficacy in multiple sclerosis patients. This observation led to the identification of S1P1 as a potential target for the treatment of a range of immune diseases. Preclinical studies have revealed an efficient reduction of lymphocyte infiltration in colitic mice treated with FTY720, paving the way for clinical trials in IBD patients.14,15 Here we briefly summarise the S1P pathway and discuss the future role of S1P inhibitors as a novel class of oral small molecule, modulating lymphocyte migration from lymph node to gut in IBD. 2. The S1P pathway Sphingosine-1-phosphate [S1P] is a pleiotropic lipid mediator derived from the metabolism of membrane sphingolipids.16 It is widely recognised that cell membrane constituents, beyond maintaining the structure of the lipid bilayer, act as bioactive metabolites for many cellular processes. Intracellular S1P synthesis consists of sphingosine phosphorylation, catalysed by two sphingosine kinase isoenzymes, SK1 and SK2. Once generated, S1P is translocated by ABC transporters outside the cells, where it exerts biological functions mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed as S1PR1-54,16 [Figure 1]. S1P levels are high in blood and lymph, but very low in tissues and interstitial fluids,17 thus generating a gradient between the blood and tissues. In the lymph node, its heterogeneous concentration distribution has been shown to function as a gradient in directing the trafficking of immune cells such as lymphocytes out of the lymph node and into the circulation.18 Increased levels of S1P have been observed at the site of inflammation, leading to the exacerbation of the inflammatory processes via the recruitment of immune cells and inflammatory mediators.19–21 However, S1P not only promotes inflammation, but it also activates a negative feedback loop on endothelial cells reducing vascular leakage and inhibiting cytokine-induced leukocyte adhesion. Therefore, S1P plays a dual role in inflammatory response.19–25 Figure 1. View largeDownload slide Synthesis and action of sphingosine-1-phosphate. Ceramide, a lipid highly present in cell membranes, is hydrolysed by the ceramidase to generate sphingosine. Once generated, the sphingosine is phosphorylated to form sphingosine-1-phosphate [S1P] by two enzymes, sphingosine kinase 1 and 2. The intracellular levels of S1P are maintained constant by the activity of two enzymes, S1P phosphatase and S1P lyase, that degrade S1P. Whereas the degradation by S1P phosphatase leads to sphingosine and thus is reversible, the action of S1P lyase is irreversible, turning S1P into ethanolamine phosphate and hexadecenal. Intracellular S1P is translocated by ABC transporters outside the cells where it exerts several biological functions, mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed S1P1-5. Figure 1. View largeDownload slide Synthesis and action of sphingosine-1-phosphate. Ceramide, a lipid highly present in cell membranes, is hydrolysed by the ceramidase to generate sphingosine. Once generated, the sphingosine is phosphorylated to form sphingosine-1-phosphate [S1P] by two enzymes, sphingosine kinase 1 and 2. The intracellular levels of S1P are maintained constant by the activity of two enzymes, S1P phosphatase and S1P lyase, that degrade S1P. Whereas the degradation by S1P phosphatase leads to sphingosine and thus is reversible, the action of S1P lyase is irreversible, turning S1P into ethanolamine phosphate and hexadecenal. Intracellular S1P is translocated by ABC transporters outside the cells where it exerts several biological functions, mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed S1P1-5. The wide range of biological functions exerted by S1P signalling reflects the different intracellular pathways triggered by the selective activation of five subtypes of cell-surface heterotrimeric G protein-coupled receptors. They are expressed in a wide variety of tissues, with each subtype exhibiting different cell specificity. Indeed, depending on the specific receptor activated, S1P regulates lymphocyte migration, endothelial permeability, angiogenesis, cellular proliferation, cell migration, cell survival, apoptosis, stress fibre formation, and differentiation signalling.18,26–29 S1P1, S1P2, and S1P3 are ubiquitously expressed, whereas S1P4 and S1P5 have a narrower expression, respectively in the lymphoid and haematopoietic tissues, and in the central nervous system, as well as in some subsets of immune cells.30,31 The regulation of lymphocyte circulation is one of the best-characterised functions of the S1P pathway, mainly mediated by S1P1. S1P1 is the most widely represented S1P receptor; it is mostly expressed on endothelial cells and lymphocytes. S1P-S1P1 interaction on lymphocytes guides their egress from the spleen and lymph nodes into systemic circulation. In 2004, fingolimod [FTY720] [2-amino-2-[2-[4-octyl-phenyl] ethyl]-1,3-propanediol hydrochloride],30 a derivative of myriocin isolated from the fungus Isaria sinclairi, was found to be a structural analogue of S1P, with an antagonist action. Indeed, the oral administration of FTY720 leads to a rapid reduction of peripheral blood lymphocytes, thus modulating T and B cell migration to secondary lymphoid tissues and finally exerting a selective immunosuppressive action. The lack of activity on effector memory CCR7-negative T cells, peripherally sequestered and not circulating through lymph nodes, averts strong immunosuppression.12 This selective immunomodulatory effect of FTY720, after its in vivo phosphorylation [FTY720-phosphate or FTY720-P] by the activity of Sk132,33 or Sk2,34–36 has raised interest in using this drug in several diseases. S1P1 modulators have proven efficacy in neurological disease. The BOLD siponimod study, published in 2013, showed that this S1P1 modulator reduces brain magnetic resonance imaging lesions in the treatment of relapsing-remitting multiple sclerosis.37 This activity was associated with a good safety profile.38 FTY720 [fingolimod] is approved and marketed for the treatment of multiple sclerosis. Several members of this drug class, showing a greater selectivity, have shown promising results in multiple sclerosis [MS].12 Moreover KRP203, similarly to FTY720, ameliorated amyloid-β peptide production, a pathogenic protein causative for Alzheimer disease, in cultured neuronal cells.38 Ponesimod, a new S1P1 modulator also binding S1P3 and S1P5, has demonstrated good efficacy and safety not only in relapsing MS,39 but also38 in patients with moderate to severe chronic plaque psoriasis.40 S1P antagonists showed a good efficacy and safety profile in several immunological diseases. Each drug has a different selectivity for one or more of the five S1P receptors. This aspect is crucial to obtain different pharmacokinetic, pharmacodynamic and safety profiles. 3. S1P1 modulators in IBD 3.1. Pre-clinical study The therapeutic effects of S1P1 modulators in IBD have first been demonstrated in preclinical studies as summarised in Table 1. Table 1. Efficacy of S1P modulators in experimental models of inflammatory bowel disease. Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 IL-10, interleukin 10; DSS, dextran sulphate sodium; TNBS, 2,4,6-trinitrobenzenesulphonic acid. View Large Table 1. Efficacy of S1P modulators in experimental models of inflammatory bowel disease. Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 IL-10, interleukin 10; DSS, dextran sulphate sodium; TNBS, 2,4,6-trinitrobenzenesulphonic acid. View Large In IL-10−/− mice which develop spontaneous chronic colitis similar to that observed in CD patients, the oral administration of FTY720-P [fingolimod] ameliorated severity of colitis, promoting reduction of the CD4+ T lymphocytes in the colonic lamina propria and reduction of IFN-gamma production. Single-dose administration of FTY720 arrested circulating lymphocytes in the secondary lymphoid tissues.32 Similar results were also observed in dextran sulphate sodium-induced and 2,4,6-trinitrobenzenesulphonic acid-induced colitis.41 FTY720 modulated S1P1-expressing lymphocytes in the mesenteric lymph nodes of colitic mice, thus reducing their migration in response to increased S1P gradient.42 The beneficial effects of FTY720 treatment were also observed in the reduction of mucosal levels of TNF-α and IL-12p70, in augmenting of IL-10 and TGF-β, and in the expansion of CD4 + CD25 + regulatory cells [T regs].43,44 Although with lower affinity than S1P1, FTY720 can also activate S1P3, S1P4, and S1P5. It has been postulated that its efficacy is due to S1P1 agonism, whereas its cardiovascular side effects [transient bradycardia and hypertension] are due to S1P3 agonism. In vitro studies showed that among S1P receptors, S1P4 has an inhibitory effect on T cells, suppressing the release of pro-inflammatory cytokines and driving the production of IL-10. Interestingly, S1P4 deficiency affected only partially T cell activities, but it strongly impacted on dendritic cell functions including migration and cytokine production. Additionally, the loss of the receptor led to a shift of the mediated immune response from Th1 to Th2, inhibiting the differentiation toward Th17 lineage.45 Hence, the beneficial effects of FTY720 in the treatment of colitis could be mediated by synergistically inactivation of both S1P1 and S1P4. W-061, another S1P1 modulator with high affinity also for S1P5,46 ameliorated experimental colitis, arresting Th17 and Th1 cells in secondary lymphoid tissues and preventing the changes of intestinal mucosal architecture.46 SEW2871, a newly selective modulator of S1P1 and structurally unrelated to S1P, has been proven to be efficient in IL-10 knockout mice. Similarly to other agonists, SEW2871 suppressed immune cell infiltration, reducing the amount of pro-inflammatory cytokines in the mucosa. However, in addition to these findings, the treatment with SEW2871 also improved epithelial barrier function. An enhancement of tight junction proteins in association with a reduction of epithelial proliferation rate contributed to ameliorating the inflammatory status of IL-10 deficient mice.47 Nevertheless, the mechanisms underlying these effects are not well elucidated. So far there are no studies describing the expression of S1PRs on epithelial cells. In vitro studies using human Caco-2 intestinal epithelial cells have demonstrated that S1P enhances the epithelial barrier integrity. abrogating the TNF-a-dependent disruption.48 Additionally, S1P treatment promoted in a dose-effect manner the expression of E-cadherin expression,49 indicating a role of S1P/S1PRs signalling in controlling intestinal barrier function; but which receptor mediates these effects is still unknown. Chronic inflammation modulates S1P1 expression and tissue S1P levels. Recently Karuppuchamy et al. have suggested that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on dendritic cells migration and vascular barrier function.50 3.2. Clinical study Most clinical studies are evaluating the effects of fingolimod either in MS or in kidney transplantation, but there are no clinical studies registered testing fingolimod in IBD. Data about clinical trials on S1P modulators were obtained by clinicaltrials.gov and pubmed. Currently there are 11 registered clinical trials in IBD with S1P modulators, either ongoing or completed [Table 2]. Table 2. S1P modulator in inflammatory bowel disease. Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 UC, ulcerative colitis; CD, Crohn’s disease; INN, International Nonproprietary Name. View Large Table 2. S1P modulator in inflammatory bowel disease. Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 UC, ulcerative colitis; CD, Crohn’s disease; INN, International Nonproprietary Name. View Large Promising results have been observed with RPC1063 [ozanimod], a selective S1P1 and S1P5 modulator. This drug is currently in development for relapsing multiple sclerosis, ulcerative colitis, and Crohn’s disease. In the TOUCHSTONE phase 2 trial, conducted on 197 adults with moderate-to-severe UC, patients were randomised to receive either ozanimod 0.5 mg or 1 mg or placebo daily for up to 32 weeks. The primary outcome was clinical remission at Week 8. Clinical remission was observed in 16% [p = 0.048], 14% [p = 0,14], and 6% of patients receiving ozanimod 1 mg, 0.5 mg, and placebo, respectively. RPC1063 was significantly more effective at the higher tested dose than either the lower dose or placebo. Secondary endpoints were clinical response and mucosal healing at Week 8. Both clinical response and mucosal healing rates were significantly higher in the ozanimod 1 mg arm [clinical response p = 0.02, mucosal healing p = 0.002]. Clinical remission at Week 32 was 21% in the 1 mg group, 26% in the 0.5 mg group, and 6% in the placebo group [p = 0.01 and p = 0.002]; clinical response at the same time point was 51% in the 1 mg group, 35% in the 0.5 mg group, and 20% in the placebo group [p < 0.001 and p = 0.06]. Finally, mucosal healing was observed in 33% of patients in the 1 mg group vs 32% in the 0.5 mg group vs 12% in the placebo group [p = 0.005 and p = 0.006].51 This study demonstrated the efficacy of ozanimod in UC patients. Moreover, the oral route of administration and the non-protein nature of the drug have potential advantages. The route of administration can in fact be more attractive for patients, and the physicochemical characteristics of the molecule seem to be associated with a negligible formation of anti-drug antibodies, a lower risk of loss of response during treatment.13 Based on these promising results, the maintenance phase of the TOUCHSTONE trial is still ongoing. It is quite possible that 8 weeks may not be enough to demonstrate the maximal response after treatment.52 Preliminary results show that ozanimod keeps its efficacy over time.53A new phase 3 study [TRUE-NORTH] in 900 UC patients has recently been activated, to evaluate efficacy and safety of the drug over 52 weeks.54,55 Preclinical studies using a spontaneous ileitis model have revealed a reduction of mononuclear infiltrate and mucosal thickness in mice treated with RPC1063 compared with vehicle, indicating a potential beneficial activity of this modulator also for CD patients.56 The drug is also under evaluation by a new open-label study, to assess clinical and endoscopic improvement and any changes in mucosal and serum biomarkers in patients with moderate-to-severe active Crohn’s disease receiving oral RPC-1063 as induction therapy.57 The molecular structure of KRP-203 is similar to FTY720. The drug is selective for S1P1. A clinical trial has evaluated the efficacy and safety of KRP-203 in moderately active refractory UC patients, with approximately 72 randomised subjects. Despite the trial termination, no results have been published up to now.58 KRP-203 was firstly developed for immunomodulation in autoimmune diseases and organ transplantation and, similarly to FTY720, provokes lymphopenia by arresting circulating lymphocytes in secondary lymphoid organs. Interestingly, KRP-203 prolonged graft survival and attenuated chronic rejection in rat allograft models, thus suggesting that probably KRP-203 regulates not only T cell trafficking, but also B cell response.59 Likewise, KRP-203 ameliorated clinical and histological severity of experimental colitis, reducing the number of infiltrating immune cells and mucosal pro-inflammatory cytokines, similarly to FTY720.60 KRP-203 selectivity against S1P1 is responsible for an absence of cardiovascular negative signal in telemetered rats, even at high dose levels.61 The positive outcomes associated with these studies have encouraged the development of S1P modulator drugs, more selective, for the treatment of IBD. APD334 [etrasimod], is an oral selective S1P1, S1P4, and S1P5 modulator under evaluation for the treatment of UC patients in two phase 2 studies.62,63 MT1303 [amiselimod] is a selective S1P1 modulator. The phase 2 trials investigating the efficacy and safety of MT1303 in CD patients are completed, but results were not published yet.64,65 Currently the extension phase of the study ongoing.66 The same drug is also under study for psoriasis and multiple sclerosis; the dose finding studies have been completed.67,68 Despite being as potent as fingolimod, MT-1303 seems to be associated with lower rates of adverse events, thus suggesting that the newly developed S1P modulators with improved pharmacokinetics and selectivity may increase patient safety.69,70 The success of negative modulation of S1P1 has led to additional innovative therapeutic strategies based on the S1P pathway for the treatment of immune diseases. Indeed, a highly specific monoclonal antibody directed against S1P, named sphingomab, has been developed. Acting as a molecular sponge, it selectively adsorps and neutralises the extracellular S1P.71 Encouraging data from preclinical studies showed its effectiveness in inhibiting tumour growth of many types of human cancers. This drug has not been studied in experimental models of IBD. S1P is considered a pro-angiogenic factor.72 The neutralisation of increased extracellular levels of S1P could therefore represent a novel approach to prevent the aberrant recruitment of immune cells in the inflamed gut in response to increased levels of S1P. However, it is unclear whether the adsorption of extracellular S1P can also affect the intracellular pool of S1P, with a potential negative influence on other cell processes. The described S1P receptor modulators are shown in Figure 2. Figure.2. View largeDownload slide Modulators of S1P receptors. FTY720, KRP-230, W-061, RPC-1063. and SEW2871, ADP-334. and MT-1303 S1P modulators show affinity to several S1P receptors except S1P2. Each compound can trigger different actions depending on the receptor involved. Only SEW2871 has a high specificity for S1P1. reducing the consequent downstream effects, which in some cases are responsible for the side effects of these compounds as reported for S1P3 after binding with FTY720. The effects mediated by the activation of S1P5 remain still unknown. Figure.2. View largeDownload slide Modulators of S1P receptors. FTY720, KRP-230, W-061, RPC-1063. and SEW2871, ADP-334. and MT-1303 S1P modulators show affinity to several S1P receptors except S1P2. Each compound can trigger different actions depending on the receptor involved. Only SEW2871 has a high specificity for S1P1. reducing the consequent downstream effects, which in some cases are responsible for the side effects of these compounds as reported for S1P3 after binding with FTY720. The effects mediated by the activation of S1P5 remain still unknown. 3.3. Safety Although the immunomodulation mediated by S1P1 specific modulators may be an attractive therapeutic strategy for IBD patients, there is a potential for adverse events related to this therapy. FTY720 has been associated with cardiovascular side effects [transient bradycardia and hypertension],73 at least partially related to S1P3 receptor modulation. The cardiovascular safety profile of amiselimod was more favourable. This molecule showed a high selectivity towards S1P1 and S1P5, a low agonistic activity against S1P4, and no agonistic activity on S1P2 or S1P3 receptors, after 28 days of administration in healthy subjects70 Amiselimod can thus be considered as a new therapeutic agent for autoimmune diseases with a low risk of bradycardia.71 Fingolimod was also associated with other potential adverse events, eg varicella zoster virus encephalitis, abnormal liver function tests, macular oedema, increased risk of fibrosis. These events may not be present with more selective S1P drugs.74 Another potential side effect is related to the regulation of the blood vessel barrier function obtained by S1P1 modulation; the most important aspect resulting from S1P1 is the increased vascular permeability. UC is characterised by increased density of abnormal vessels and, as recently published, by an overexpression of S1P in the colonic mucosa of UC patients. These data confirm the results obtained in experimental colitis. The colonic vascular permeability under basal conditions was increased in mice, due both to the increased vascular density in the inflamed mucosa and to the genetic deletion of S1P1. In conclusion, FTY720 increases bleeding in experimental colitis, demonstrating that S1P1 is critical to maintaining colonic vascular integrity, and may play a role in UC pathogenesis.75 Further clinical trials will better determine if this therapeutic class will demonstrate an overall efficacy, as shown in the TOUCHSTONE trial despite this observation. S1P receptors differ in expression and signalling. Limiting a drug effect to a single receptor subtype 1–5 may provide the therapeutic action, minimising side effects.76 4. Future perspective of S1P-regulating enzymes The essential role of sphingosine-kinase [Sk] 1 and 2 in controlling the synthesis of S1P has raised particular attention on these enzymes as an alternative way to inhibit S1P signalling. The first inhibitors described for Sk1 were DL-threo-dihydrosphingosine [DHS or saphingol] and N,N-Dimethylsphingosine [DMS].77 Both inhibitors have been mainly used in preclinical studies for the treatment of cancer. However, although they effectively inhibited tumour growth in vivo, the use of the inhibitors caused significant hepatotoxicity and haemolysis at higher doses.78 Natural inhibitors of Sk such as B-5354c or F-12509a79 have been discovered. These products may act as potent Sk inhibitors, but the possibility of large-scale production results are unlikely due to their improbable synthesis because of their complex structures. On the other hand, five synthetic non-lipid inhibitors [compounds SkI I–V] have been developed and validated as inducers of cancer cell apoptosis.80 These compounds are commercially available and they have been used in several studies, including murine bone marrow-derived dendritic cells,81 murine cardiac cells,82 human prostatic adenocarcinoma cells,83 and a rat haemorrhagic shock model,84 but there are no evidences of its use in the treatment of colitis. Consistent with the pharmacological effects of Sk1 inhibitors, specific knockdown of Sk1 by small interference RNA triggers apoptosis in multiple types of tumour cells in vitro, including leukaemic,85 melanoma,86 breast,87 glioblastoma,88 and prostate cancer cells.89 Small interference RNA targeted to Sk1 induced activation of effector caspases and cytochrome c release,90 and significantly increased ceramide and sphingosine levels. Furthermore, in human colon cancer cell lines, the knockdown of Sk1 reduced the COX- 2/PGE2 pathway that is known to be implicated in colon carcinogenesis.91 The absence of studies reporting potential effects in the treatment of colitis attenuates its use as a new therapeutic target for IBD. An alternative approach to reduce the S1P gradient responsible of lymphocyte trafficking could be represented by the inhibition of S1P lyase, an important enzyme involved in the S1P metabolism. Indeed, S1P gradient results slowed down after treatment with different S1P lyase inhibitors such as 2-acetyl-4- tetrahydroxybutylimidazole, deoxypyridoxine, or the synthetic compound LX2931.92,93 Importantly, a down-regulation of S1P lyase has been observed in colon cancer cells, which leads to an accumulation of extracellular levels of S1P. Controversially, the specific deletion of S1P lyase on intestinal epithelial cells contributed to the process of tumour transformation by activating STAT3 signalling. STAT3 is a transcription factor that regulates, upon activation by specific cytokines, several cell processes including apoptosis, proliferation, migration, and survival.94,95 STAT3 is activated in epithelial cells of CD patients,96 and genome-wide association studies have identified variants in the STAT3 gene in CD patients.97 Although no association with intestinal permeability was found for STAT3,98 intestinal epithelial cells respond to cytokines that may regulate the epithelial barrier via STAT3. Therefore, the induction of STAT3 activity mediated by the inhibitors of S1P lyase may further impair the intestinal barrier function in these patients, thus raising the suspicion that targeting S1P lyase is not the proper approach to reduce the mucosal levels of S1P. However, further studies are necessary to validate or to definitely exclude the S1P-regulating enzymes as an alternative way to S1P receptor inhibition to block S1P signalling. 5. Conclusions S1P is a bioactive lipid mediator that regulates important cellular functions. Emerging studies have evidenced its active involvement in the intestinal inflammatory processes by modulating activation of T cells, vascular endothelial integrity and, in particular, lymphocyte migration. Although further studies are necessary to completely unveil the molecular mechanisms underlying the S1P activity in the inflamed gut, the neutralisation of S1P signalling, by specific inhibition of its receptors or its regulatory enzymes, shows great promise for the treatment of IBD patients. Indeed, data from clinical studies are encouraging and raise the S1P/S1P1 axis as a new potential therapeutic strategy for the modulation of lymphocyte circulation in IBD. In contrast to anti-integrin antibody inhibition, which arrests the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa, resulting in a strong immunomodulation, S1P1 modulation seems selectively to block the egress of effector T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effector memory T cells [CCR7] into the mucosa [Figure 3]. Figure 3. View largeDownload slide Molecular approaches to modulate lymphocytes circulation in inflammatory bowel disease [IBD]. In contrast to the arrest of lymphocyte trafficking to gastrointestinal mucosa through anti-integrin antibody inhibitiors such as vedolizumab, etrolizumab, and natulizumab, block the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa. This results in a strong immunomodulation. S1P1 modulation seems selectively to block the egress of effectory T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effectory memory T cell [CCR7] into the mucosa, resulting in less immunosuppression for IBD patients. Figure 3. View largeDownload slide Molecular approaches to modulate lymphocytes circulation in inflammatory bowel disease [IBD]. In contrast to the arrest of lymphocyte trafficking to gastrointestinal mucosa through anti-integrin antibody inhibitiors such as vedolizumab, etrolizumab, and natulizumab, block the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa. This results in a strong immunomodulation. S1P1 modulation seems selectively to block the egress of effectory T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effectory memory T cell [CCR7] into the mucosa, resulting in less immunosuppression for IBD patients. In addition, small molecule drugs like S1P modulators have a low molecular weight, no immunogenicity, better pharmacokinetic profile, and are able to diffuse more easily through cell membranes. Moreover the production of S1P modulators is less complex than that of biologic drugs, so the cost could be lower, and also patients tend to prefer the oral drug administration instead of intravenous or subcutaneous administration. So, if the ongoing studies will confirm the efficacy and safety of these new drugs, we may have a novel and easier treatment for IBD patients. 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For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Crohn's and Colitis Oxford University Press

Targeting S1P in Inflammatory Bowel Disease: New Avenues for Modulating Intestinal Leukocyte Migration

Journal of Crohn's and Colitis , Volume 12 (suppl_2) – Aug 1, 2018

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Abstract

Abstract Sphingosine 1 phosphate [S1P] is a bioactive lipid mediator involved in the regulation of several cellular processes though the activation of a G protein-coupled receptor family known as the S1P receptors [S1PRs]. Advances in the understanding of the biological activities mediated by S1PRs have sparked great interest in the S1P/S1PRs axes as new therapeutic targets for the modulation of several cellular processes. In particular, the S1P/S1PR1 axis has been identified as key regulator for lymphocyte migration from lymph nodes. The blockade of this axis is emerging as a new therapeutic approach to control the aberrant leukocyte migration into the mucosa in inflammatory bowel disease [IBD]. This review briefly summarises the current evidence coming from clinical studies, and discusses the future prospects of S1P inhibitors for treatment of inflammatory bowel disease. Inflammatory bowel disease, S1P signalling, oral small molecules, lymphocyte circulation modulation 1. Introduction Inflammatory bowel diseases [IBD], which include as major forms ulcerative colitis [UC] and Crohn’s disease [CD], are chronic and relapsing-remitting inflammatory diseases of the gastrointestinal tract. IBD pathogenesis is still poorly understood; however, it is widely accepted that a complex interaction between aberrant inflammatory responses, altered intestinal barrier function, environmental factors and intestinal flora can have a role, in subjects with a genetic predisposition.1 The incidence and prevalence of IBD have increased in the past 50 years, attracting interest from the scientific community to improve the knowledge on these diseases by the identification of new therapeutic targets. However, despite this progress, currently there is no curative therapy. Conventional management of IBD includes glucocorticoids, 5-aminosalicylic acid [for UC], conventional immunomodulators, such as azathioprine, and biologics. The first class of biologics was developed approximately two decades ago, and was represented by anti-tumour necrosis factor [TNF] drugs. Currently licensed anti-TNFs are available for intravenous or subcutaneous administration. However, the efficacy of anti-TNF therapy is often moderate, with potentially serious side effects, is and associated with an increased risk of serious infections. About one-third of patients, defined as primary non-responders, do not respond to induction therapy and another third, called secondary non-responders, despite an initial response show a loss response over time. This secondary loss of response is mainly related to the development of immunogenicity to these protein-based therapies.2 These evidences lead to the need of innovative therapies, with a specific interaction towards new therapeutic targets, to improve the patient’s quality of life and to control the heterogeneous range of symptoms, not adequately treated by the available therapies. In the past decade, an increasing number of newly approved or developing drugs, modulating lymphocyte migration through effects on different mechanisms and acting in a different manner, have shown the potential value of this approach in controlling the aberrant immune response that is co-responsible, with the other above-mentioned factors, for the drastic gut dysfunction observed IBD patients.3 In IBD as in other immune processes, T cells, activated by antigen-presenting cells, play a key role in promoting an immune response. After this activation, circulating T cells enter into lymphoid compartments, where they can differentiate into either tissue resident memory cells or activated T cells. Activated T cells are then released from lymph nodes back into the blood circulation, thus reaching inflamed tissue by extravasation from blood vessels. The process is regulated by adhesion molecules, specific chemokines, and selective binding receptors located in lymph nodes and on appropriate vascular endothelial cells. In the vessels this is a three-step process composed of rolling, regulated by selectins, chemokine-dependent activation and arrest, mediated by integrins. Once lymphocytes have been bound by integrins, they can access the gut through specialised venules, expressing an integrin ligand such as mucosal addressin cell adhesion molecule 1 [MAdCAM-1].4 The potential role of lymphocyte circulation-modulating agents in IBD led to the development of new drugs. A number of drugs blocking specific gut-selective integrin-endothelial ligand interactions have been developed to find a safe and efficacious alternative therapy to anti-TNF-alpha. Vedolizumab is a humanised immunoglobulin [IG] G1 mAb directed against the human lymphocyte integrin α4β7. The α4β7integrin mediates lymphocyte trafficking to gastrointestinal mucosa and gut-associated lymphoid tissue through adhesive interaction with MAdCAM-1, which is expressed on the endothelium of mesenteric lymph nodes and gastrointestinal mucosa.5 Vedolizumab is characterised by high gut selectivity, and it has demonstrated efficacy both for UC and CD treatment.6,7 However, more than 50% of patients could be primary non-responders or could lose response to this drug, both in UC and CD.8,9 Another anti-integrin drug still in development for both indications is etrolizumab. It selectively binds the β7 subunits of the α4β7 and αEβ7 integrins and could, in the future, become an alternative therapy for IBD patients.10,11 The interaction between adhesion molecules and integrins can also be blocked by drugs like anti-MAdCam-1, binding the adhesion molecule at the endothelial level.2 All biologics are administered by either e intravenous infusion or by injection, and are characterised by long half-lives [more than 20 days]. Because of the high rate of non-response or loss of response to marketed drugs, and due to their invasive route of administration, in recent years there has been a growing interest in novel small molecules development. These drugs, orally administered, are characterised by new mechanisms of action. They bind new molecular targets directly located in the lymph node and not at vascular levels. Sphingosine 1-phosphate receptor 1 [S1P1] is a member of these new targets and is involved in the regulation of immune cell circulation. In particular, S1P regulates the re-circulation of naïve and central memory CCR7-positive T cells from the secondary lymphatic organs to the periphery, but does not sequester effector memory CCR7-negative T cells that are responsible for maintaining immune surveillance against pathogens in the periphery, as they do not traffic through lymph nodes.12,13 FTY720, an agonistic activator of S1P receptors also called fingolimod, was shown to reduce lymphocyte egress from secondary lymphoid organs, thus resulting in efficacy in multiple sclerosis patients. This observation led to the identification of S1P1 as a potential target for the treatment of a range of immune diseases. Preclinical studies have revealed an efficient reduction of lymphocyte infiltration in colitic mice treated with FTY720, paving the way for clinical trials in IBD patients.14,15 Here we briefly summarise the S1P pathway and discuss the future role of S1P inhibitors as a novel class of oral small molecule, modulating lymphocyte migration from lymph node to gut in IBD. 2. The S1P pathway Sphingosine-1-phosphate [S1P] is a pleiotropic lipid mediator derived from the metabolism of membrane sphingolipids.16 It is widely recognised that cell membrane constituents, beyond maintaining the structure of the lipid bilayer, act as bioactive metabolites for many cellular processes. Intracellular S1P synthesis consists of sphingosine phosphorylation, catalysed by two sphingosine kinase isoenzymes, SK1 and SK2. Once generated, S1P is translocated by ABC transporters outside the cells, where it exerts biological functions mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed as S1PR1-54,16 [Figure 1]. S1P levels are high in blood and lymph, but very low in tissues and interstitial fluids,17 thus generating a gradient between the blood and tissues. In the lymph node, its heterogeneous concentration distribution has been shown to function as a gradient in directing the trafficking of immune cells such as lymphocytes out of the lymph node and into the circulation.18 Increased levels of S1P have been observed at the site of inflammation, leading to the exacerbation of the inflammatory processes via the recruitment of immune cells and inflammatory mediators.19–21 However, S1P not only promotes inflammation, but it also activates a negative feedback loop on endothelial cells reducing vascular leakage and inhibiting cytokine-induced leukocyte adhesion. Therefore, S1P plays a dual role in inflammatory response.19–25 Figure 1. View largeDownload slide Synthesis and action of sphingosine-1-phosphate. Ceramide, a lipid highly present in cell membranes, is hydrolysed by the ceramidase to generate sphingosine. Once generated, the sphingosine is phosphorylated to form sphingosine-1-phosphate [S1P] by two enzymes, sphingosine kinase 1 and 2. The intracellular levels of S1P are maintained constant by the activity of two enzymes, S1P phosphatase and S1P lyase, that degrade S1P. Whereas the degradation by S1P phosphatase leads to sphingosine and thus is reversible, the action of S1P lyase is irreversible, turning S1P into ethanolamine phosphate and hexadecenal. Intracellular S1P is translocated by ABC transporters outside the cells where it exerts several biological functions, mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed S1P1-5. Figure 1. View largeDownload slide Synthesis and action of sphingosine-1-phosphate. Ceramide, a lipid highly present in cell membranes, is hydrolysed by the ceramidase to generate sphingosine. Once generated, the sphingosine is phosphorylated to form sphingosine-1-phosphate [S1P] by two enzymes, sphingosine kinase 1 and 2. The intracellular levels of S1P are maintained constant by the activity of two enzymes, S1P phosphatase and S1P lyase, that degrade S1P. Whereas the degradation by S1P phosphatase leads to sphingosine and thus is reversible, the action of S1P lyase is irreversible, turning S1P into ethanolamine phosphate and hexadecenal. Intracellular S1P is translocated by ABC transporters outside the cells where it exerts several biological functions, mostly through the activation of five cell-surface heterotrimeric G protein-coupled receptors termed S1P1-5. The wide range of biological functions exerted by S1P signalling reflects the different intracellular pathways triggered by the selective activation of five subtypes of cell-surface heterotrimeric G protein-coupled receptors. They are expressed in a wide variety of tissues, with each subtype exhibiting different cell specificity. Indeed, depending on the specific receptor activated, S1P regulates lymphocyte migration, endothelial permeability, angiogenesis, cellular proliferation, cell migration, cell survival, apoptosis, stress fibre formation, and differentiation signalling.18,26–29 S1P1, S1P2, and S1P3 are ubiquitously expressed, whereas S1P4 and S1P5 have a narrower expression, respectively in the lymphoid and haematopoietic tissues, and in the central nervous system, as well as in some subsets of immune cells.30,31 The regulation of lymphocyte circulation is one of the best-characterised functions of the S1P pathway, mainly mediated by S1P1. S1P1 is the most widely represented S1P receptor; it is mostly expressed on endothelial cells and lymphocytes. S1P-S1P1 interaction on lymphocytes guides their egress from the spleen and lymph nodes into systemic circulation. In 2004, fingolimod [FTY720] [2-amino-2-[2-[4-octyl-phenyl] ethyl]-1,3-propanediol hydrochloride],30 a derivative of myriocin isolated from the fungus Isaria sinclairi, was found to be a structural analogue of S1P, with an antagonist action. Indeed, the oral administration of FTY720 leads to a rapid reduction of peripheral blood lymphocytes, thus modulating T and B cell migration to secondary lymphoid tissues and finally exerting a selective immunosuppressive action. The lack of activity on effector memory CCR7-negative T cells, peripherally sequestered and not circulating through lymph nodes, averts strong immunosuppression.12 This selective immunomodulatory effect of FTY720, after its in vivo phosphorylation [FTY720-phosphate or FTY720-P] by the activity of Sk132,33 or Sk2,34–36 has raised interest in using this drug in several diseases. S1P1 modulators have proven efficacy in neurological disease. The BOLD siponimod study, published in 2013, showed that this S1P1 modulator reduces brain magnetic resonance imaging lesions in the treatment of relapsing-remitting multiple sclerosis.37 This activity was associated with a good safety profile.38 FTY720 [fingolimod] is approved and marketed for the treatment of multiple sclerosis. Several members of this drug class, showing a greater selectivity, have shown promising results in multiple sclerosis [MS].12 Moreover KRP203, similarly to FTY720, ameliorated amyloid-β peptide production, a pathogenic protein causative for Alzheimer disease, in cultured neuronal cells.38 Ponesimod, a new S1P1 modulator also binding S1P3 and S1P5, has demonstrated good efficacy and safety not only in relapsing MS,39 but also38 in patients with moderate to severe chronic plaque psoriasis.40 S1P antagonists showed a good efficacy and safety profile in several immunological diseases. Each drug has a different selectivity for one or more of the five S1P receptors. This aspect is crucial to obtain different pharmacokinetic, pharmacodynamic and safety profiles. 3. S1P1 modulators in IBD 3.1. Pre-clinical study The therapeutic effects of S1P1 modulators in IBD have first been demonstrated in preclinical studies as summarised in Table 1. Table 1. Efficacy of S1P modulators in experimental models of inflammatory bowel disease. Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 IL-10, interleukin 10; DSS, dextran sulphate sodium; TNBS, 2,4,6-trinitrobenzenesulphonic acid. View Large Table 1. Efficacy of S1P modulators in experimental models of inflammatory bowel disease. Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 Drug Mouse model of colitis S1P activity Outcomes Reference FTY720-P IL-10-/- mice DSS-induced colitis TNBS-induced colitis Oxazolone-induced colitis S1P1 S1P3 S1P4 S1P5 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Expansion of T regulatory cells 32, 41–45 W-061 DSS-induced colitis S1P1 S1P5 Reduction of infiltrating Th17 and Th1 cells 42 SEW2871 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines Improvement of epithelial barrier function 47 RPC-1063 TNBS-induced colitis Adoptive T cell transfer S1P1 S1P5 Reduction of infiltrating immune cells and mucosal thickness 56 KRP-203 IL-10-/- mice S1P1 Reduction of infiltrating immune cells Reduction of pro-inflammatory cytokines 59 IL-10, interleukin 10; DSS, dextran sulphate sodium; TNBS, 2,4,6-trinitrobenzenesulphonic acid. View Large In IL-10−/− mice which develop spontaneous chronic colitis similar to that observed in CD patients, the oral administration of FTY720-P [fingolimod] ameliorated severity of colitis, promoting reduction of the CD4+ T lymphocytes in the colonic lamina propria and reduction of IFN-gamma production. Single-dose administration of FTY720 arrested circulating lymphocytes in the secondary lymphoid tissues.32 Similar results were also observed in dextran sulphate sodium-induced and 2,4,6-trinitrobenzenesulphonic acid-induced colitis.41 FTY720 modulated S1P1-expressing lymphocytes in the mesenteric lymph nodes of colitic mice, thus reducing their migration in response to increased S1P gradient.42 The beneficial effects of FTY720 treatment were also observed in the reduction of mucosal levels of TNF-α and IL-12p70, in augmenting of IL-10 and TGF-β, and in the expansion of CD4 + CD25 + regulatory cells [T regs].43,44 Although with lower affinity than S1P1, FTY720 can also activate S1P3, S1P4, and S1P5. It has been postulated that its efficacy is due to S1P1 agonism, whereas its cardiovascular side effects [transient bradycardia and hypertension] are due to S1P3 agonism. In vitro studies showed that among S1P receptors, S1P4 has an inhibitory effect on T cells, suppressing the release of pro-inflammatory cytokines and driving the production of IL-10. Interestingly, S1P4 deficiency affected only partially T cell activities, but it strongly impacted on dendritic cell functions including migration and cytokine production. Additionally, the loss of the receptor led to a shift of the mediated immune response from Th1 to Th2, inhibiting the differentiation toward Th17 lineage.45 Hence, the beneficial effects of FTY720 in the treatment of colitis could be mediated by synergistically inactivation of both S1P1 and S1P4. W-061, another S1P1 modulator with high affinity also for S1P5,46 ameliorated experimental colitis, arresting Th17 and Th1 cells in secondary lymphoid tissues and preventing the changes of intestinal mucosal architecture.46 SEW2871, a newly selective modulator of S1P1 and structurally unrelated to S1P, has been proven to be efficient in IL-10 knockout mice. Similarly to other agonists, SEW2871 suppressed immune cell infiltration, reducing the amount of pro-inflammatory cytokines in the mucosa. However, in addition to these findings, the treatment with SEW2871 also improved epithelial barrier function. An enhancement of tight junction proteins in association with a reduction of epithelial proliferation rate contributed to ameliorating the inflammatory status of IL-10 deficient mice.47 Nevertheless, the mechanisms underlying these effects are not well elucidated. So far there are no studies describing the expression of S1PRs on epithelial cells. In vitro studies using human Caco-2 intestinal epithelial cells have demonstrated that S1P enhances the epithelial barrier integrity. abrogating the TNF-a-dependent disruption.48 Additionally, S1P treatment promoted in a dose-effect manner the expression of E-cadherin expression,49 indicating a role of S1P/S1PRs signalling in controlling intestinal barrier function; but which receptor mediates these effects is still unknown. Chronic inflammation modulates S1P1 expression and tissue S1P levels. Recently Karuppuchamy et al. have suggested that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on dendritic cells migration and vascular barrier function.50 3.2. Clinical study Most clinical studies are evaluating the effects of fingolimod either in MS or in kidney transplantation, but there are no clinical studies registered testing fingolimod in IBD. Data about clinical trials on S1P modulators were obtained by clinicaltrials.gov and pubmed. Currently there are 11 registered clinical trials in IBD with S1P modulators, either ongoing or completed [Table 2]. Table 2. S1P modulator in inflammatory bowel disease. Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 UC, ulcerative colitis; CD, Crohn’s disease; INN, International Nonproprietary Name. View Large Table 2. S1P modulator in inflammatory bowel disease. Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 Drug INN Developing company Development status S1P activity RPC-1063 Ozanimod Receptos/Celgene UC phase 3 [ongoing] CD phase 2 [ongoing] S1P1-S1P5 KRP-203 Novartis AG/Kyorin Pharmaceutical UC phase 2 [terminated] S1P1-S1P4-S1P5 APD-334 Etrasimod Arena Pharmaceuticals UC phase 2 [ongoing] S1P1-S1P4-S1P5 MT-1303 Amiselimod Mitsubishi Tanebe Pharma CD phase 2 [completed] S1P1-S1P4-S1P5 UC, ulcerative colitis; CD, Crohn’s disease; INN, International Nonproprietary Name. View Large Promising results have been observed with RPC1063 [ozanimod], a selective S1P1 and S1P5 modulator. This drug is currently in development for relapsing multiple sclerosis, ulcerative colitis, and Crohn’s disease. In the TOUCHSTONE phase 2 trial, conducted on 197 adults with moderate-to-severe UC, patients were randomised to receive either ozanimod 0.5 mg or 1 mg or placebo daily for up to 32 weeks. The primary outcome was clinical remission at Week 8. Clinical remission was observed in 16% [p = 0.048], 14% [p = 0,14], and 6% of patients receiving ozanimod 1 mg, 0.5 mg, and placebo, respectively. RPC1063 was significantly more effective at the higher tested dose than either the lower dose or placebo. Secondary endpoints were clinical response and mucosal healing at Week 8. Both clinical response and mucosal healing rates were significantly higher in the ozanimod 1 mg arm [clinical response p = 0.02, mucosal healing p = 0.002]. Clinical remission at Week 32 was 21% in the 1 mg group, 26% in the 0.5 mg group, and 6% in the placebo group [p = 0.01 and p = 0.002]; clinical response at the same time point was 51% in the 1 mg group, 35% in the 0.5 mg group, and 20% in the placebo group [p < 0.001 and p = 0.06]. Finally, mucosal healing was observed in 33% of patients in the 1 mg group vs 32% in the 0.5 mg group vs 12% in the placebo group [p = 0.005 and p = 0.006].51 This study demonstrated the efficacy of ozanimod in UC patients. Moreover, the oral route of administration and the non-protein nature of the drug have potential advantages. The route of administration can in fact be more attractive for patients, and the physicochemical characteristics of the molecule seem to be associated with a negligible formation of anti-drug antibodies, a lower risk of loss of response during treatment.13 Based on these promising results, the maintenance phase of the TOUCHSTONE trial is still ongoing. It is quite possible that 8 weeks may not be enough to demonstrate the maximal response after treatment.52 Preliminary results show that ozanimod keeps its efficacy over time.53A new phase 3 study [TRUE-NORTH] in 900 UC patients has recently been activated, to evaluate efficacy and safety of the drug over 52 weeks.54,55 Preclinical studies using a spontaneous ileitis model have revealed a reduction of mononuclear infiltrate and mucosal thickness in mice treated with RPC1063 compared with vehicle, indicating a potential beneficial activity of this modulator also for CD patients.56 The drug is also under evaluation by a new open-label study, to assess clinical and endoscopic improvement and any changes in mucosal and serum biomarkers in patients with moderate-to-severe active Crohn’s disease receiving oral RPC-1063 as induction therapy.57 The molecular structure of KRP-203 is similar to FTY720. The drug is selective for S1P1. A clinical trial has evaluated the efficacy and safety of KRP-203 in moderately active refractory UC patients, with approximately 72 randomised subjects. Despite the trial termination, no results have been published up to now.58 KRP-203 was firstly developed for immunomodulation in autoimmune diseases and organ transplantation and, similarly to FTY720, provokes lymphopenia by arresting circulating lymphocytes in secondary lymphoid organs. Interestingly, KRP-203 prolonged graft survival and attenuated chronic rejection in rat allograft models, thus suggesting that probably KRP-203 regulates not only T cell trafficking, but also B cell response.59 Likewise, KRP-203 ameliorated clinical and histological severity of experimental colitis, reducing the number of infiltrating immune cells and mucosal pro-inflammatory cytokines, similarly to FTY720.60 KRP-203 selectivity against S1P1 is responsible for an absence of cardiovascular negative signal in telemetered rats, even at high dose levels.61 The positive outcomes associated with these studies have encouraged the development of S1P modulator drugs, more selective, for the treatment of IBD. APD334 [etrasimod], is an oral selective S1P1, S1P4, and S1P5 modulator under evaluation for the treatment of UC patients in two phase 2 studies.62,63 MT1303 [amiselimod] is a selective S1P1 modulator. The phase 2 trials investigating the efficacy and safety of MT1303 in CD patients are completed, but results were not published yet.64,65 Currently the extension phase of the study ongoing.66 The same drug is also under study for psoriasis and multiple sclerosis; the dose finding studies have been completed.67,68 Despite being as potent as fingolimod, MT-1303 seems to be associated with lower rates of adverse events, thus suggesting that the newly developed S1P modulators with improved pharmacokinetics and selectivity may increase patient safety.69,70 The success of negative modulation of S1P1 has led to additional innovative therapeutic strategies based on the S1P pathway for the treatment of immune diseases. Indeed, a highly specific monoclonal antibody directed against S1P, named sphingomab, has been developed. Acting as a molecular sponge, it selectively adsorps and neutralises the extracellular S1P.71 Encouraging data from preclinical studies showed its effectiveness in inhibiting tumour growth of many types of human cancers. This drug has not been studied in experimental models of IBD. S1P is considered a pro-angiogenic factor.72 The neutralisation of increased extracellular levels of S1P could therefore represent a novel approach to prevent the aberrant recruitment of immune cells in the inflamed gut in response to increased levels of S1P. However, it is unclear whether the adsorption of extracellular S1P can also affect the intracellular pool of S1P, with a potential negative influence on other cell processes. The described S1P receptor modulators are shown in Figure 2. Figure.2. View largeDownload slide Modulators of S1P receptors. FTY720, KRP-230, W-061, RPC-1063. and SEW2871, ADP-334. and MT-1303 S1P modulators show affinity to several S1P receptors except S1P2. Each compound can trigger different actions depending on the receptor involved. Only SEW2871 has a high specificity for S1P1. reducing the consequent downstream effects, which in some cases are responsible for the side effects of these compounds as reported for S1P3 after binding with FTY720. The effects mediated by the activation of S1P5 remain still unknown. Figure.2. View largeDownload slide Modulators of S1P receptors. FTY720, KRP-230, W-061, RPC-1063. and SEW2871, ADP-334. and MT-1303 S1P modulators show affinity to several S1P receptors except S1P2. Each compound can trigger different actions depending on the receptor involved. Only SEW2871 has a high specificity for S1P1. reducing the consequent downstream effects, which in some cases are responsible for the side effects of these compounds as reported for S1P3 after binding with FTY720. The effects mediated by the activation of S1P5 remain still unknown. 3.3. Safety Although the immunomodulation mediated by S1P1 specific modulators may be an attractive therapeutic strategy for IBD patients, there is a potential for adverse events related to this therapy. FTY720 has been associated with cardiovascular side effects [transient bradycardia and hypertension],73 at least partially related to S1P3 receptor modulation. The cardiovascular safety profile of amiselimod was more favourable. This molecule showed a high selectivity towards S1P1 and S1P5, a low agonistic activity against S1P4, and no agonistic activity on S1P2 or S1P3 receptors, after 28 days of administration in healthy subjects70 Amiselimod can thus be considered as a new therapeutic agent for autoimmune diseases with a low risk of bradycardia.71 Fingolimod was also associated with other potential adverse events, eg varicella zoster virus encephalitis, abnormal liver function tests, macular oedema, increased risk of fibrosis. These events may not be present with more selective S1P drugs.74 Another potential side effect is related to the regulation of the blood vessel barrier function obtained by S1P1 modulation; the most important aspect resulting from S1P1 is the increased vascular permeability. UC is characterised by increased density of abnormal vessels and, as recently published, by an overexpression of S1P in the colonic mucosa of UC patients. These data confirm the results obtained in experimental colitis. The colonic vascular permeability under basal conditions was increased in mice, due both to the increased vascular density in the inflamed mucosa and to the genetic deletion of S1P1. In conclusion, FTY720 increases bleeding in experimental colitis, demonstrating that S1P1 is critical to maintaining colonic vascular integrity, and may play a role in UC pathogenesis.75 Further clinical trials will better determine if this therapeutic class will demonstrate an overall efficacy, as shown in the TOUCHSTONE trial despite this observation. S1P receptors differ in expression and signalling. Limiting a drug effect to a single receptor subtype 1–5 may provide the therapeutic action, minimising side effects.76 4. Future perspective of S1P-regulating enzymes The essential role of sphingosine-kinase [Sk] 1 and 2 in controlling the synthesis of S1P has raised particular attention on these enzymes as an alternative way to inhibit S1P signalling. The first inhibitors described for Sk1 were DL-threo-dihydrosphingosine [DHS or saphingol] and N,N-Dimethylsphingosine [DMS].77 Both inhibitors have been mainly used in preclinical studies for the treatment of cancer. However, although they effectively inhibited tumour growth in vivo, the use of the inhibitors caused significant hepatotoxicity and haemolysis at higher doses.78 Natural inhibitors of Sk such as B-5354c or F-12509a79 have been discovered. These products may act as potent Sk inhibitors, but the possibility of large-scale production results are unlikely due to their improbable synthesis because of their complex structures. On the other hand, five synthetic non-lipid inhibitors [compounds SkI I–V] have been developed and validated as inducers of cancer cell apoptosis.80 These compounds are commercially available and they have been used in several studies, including murine bone marrow-derived dendritic cells,81 murine cardiac cells,82 human prostatic adenocarcinoma cells,83 and a rat haemorrhagic shock model,84 but there are no evidences of its use in the treatment of colitis. Consistent with the pharmacological effects of Sk1 inhibitors, specific knockdown of Sk1 by small interference RNA triggers apoptosis in multiple types of tumour cells in vitro, including leukaemic,85 melanoma,86 breast,87 glioblastoma,88 and prostate cancer cells.89 Small interference RNA targeted to Sk1 induced activation of effector caspases and cytochrome c release,90 and significantly increased ceramide and sphingosine levels. Furthermore, in human colon cancer cell lines, the knockdown of Sk1 reduced the COX- 2/PGE2 pathway that is known to be implicated in colon carcinogenesis.91 The absence of studies reporting potential effects in the treatment of colitis attenuates its use as a new therapeutic target for IBD. An alternative approach to reduce the S1P gradient responsible of lymphocyte trafficking could be represented by the inhibition of S1P lyase, an important enzyme involved in the S1P metabolism. Indeed, S1P gradient results slowed down after treatment with different S1P lyase inhibitors such as 2-acetyl-4- tetrahydroxybutylimidazole, deoxypyridoxine, or the synthetic compound LX2931.92,93 Importantly, a down-regulation of S1P lyase has been observed in colon cancer cells, which leads to an accumulation of extracellular levels of S1P. Controversially, the specific deletion of S1P lyase on intestinal epithelial cells contributed to the process of tumour transformation by activating STAT3 signalling. STAT3 is a transcription factor that regulates, upon activation by specific cytokines, several cell processes including apoptosis, proliferation, migration, and survival.94,95 STAT3 is activated in epithelial cells of CD patients,96 and genome-wide association studies have identified variants in the STAT3 gene in CD patients.97 Although no association with intestinal permeability was found for STAT3,98 intestinal epithelial cells respond to cytokines that may regulate the epithelial barrier via STAT3. Therefore, the induction of STAT3 activity mediated by the inhibitors of S1P lyase may further impair the intestinal barrier function in these patients, thus raising the suspicion that targeting S1P lyase is not the proper approach to reduce the mucosal levels of S1P. However, further studies are necessary to validate or to definitely exclude the S1P-regulating enzymes as an alternative way to S1P receptor inhibition to block S1P signalling. 5. Conclusions S1P is a bioactive lipid mediator that regulates important cellular functions. Emerging studies have evidenced its active involvement in the intestinal inflammatory processes by modulating activation of T cells, vascular endothelial integrity and, in particular, lymphocyte migration. Although further studies are necessary to completely unveil the molecular mechanisms underlying the S1P activity in the inflamed gut, the neutralisation of S1P signalling, by specific inhibition of its receptors or its regulatory enzymes, shows great promise for the treatment of IBD patients. Indeed, data from clinical studies are encouraging and raise the S1P/S1P1 axis as a new potential therapeutic strategy for the modulation of lymphocyte circulation in IBD. In contrast to anti-integrin antibody inhibition, which arrests the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa, resulting in a strong immunomodulation, S1P1 modulation seems selectively to block the egress of effector T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effector memory T cells [CCR7] into the mucosa [Figure 3]. Figure 3. View largeDownload slide Molecular approaches to modulate lymphocytes circulation in inflammatory bowel disease [IBD]. In contrast to the arrest of lymphocyte trafficking to gastrointestinal mucosa through anti-integrin antibody inhibitiors such as vedolizumab, etrolizumab, and natulizumab, block the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa. This results in a strong immunomodulation. S1P1 modulation seems selectively to block the egress of effectory T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effectory memory T cell [CCR7] into the mucosa, resulting in less immunosuppression for IBD patients. Figure 3. View largeDownload slide Molecular approaches to modulate lymphocytes circulation in inflammatory bowel disease [IBD]. In contrast to the arrest of lymphocyte trafficking to gastrointestinal mucosa through anti-integrin antibody inhibitiors such as vedolizumab, etrolizumab, and natulizumab, block the recruitment of both effector memory T cells [CCR7-] and T cells [CCR7+] to gastrointestinal mucosa. This results in a strong immunomodulation. S1P1 modulation seems selectively to block the egress of effectory T cells [CCR7+] from secondary lymphoid tissue to gastrointestinal mucosa, not affecting the surveillance activities of effectory memory T cell [CCR7] into the mucosa, resulting in less immunosuppression for IBD patients. In addition, small molecule drugs like S1P modulators have a low molecular weight, no immunogenicity, better pharmacokinetic profile, and are able to diffuse more easily through cell membranes. Moreover the production of S1P modulators is less complex than that of biologic drugs, so the cost could be lower, and also patients tend to prefer the oral drug administration instead of intravenous or subcutaneous administration. So, if the ongoing studies will confirm the efficacy and safety of these new drugs, we may have a novel and easier treatment for IBD patients. 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Journal of Crohn's and ColitisOxford University Press

Published: Aug 1, 2018

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