Synthesis, radiosynthesis and biochemical evaluation of fluorinated analogues of sphingosine-1-phosphate receptor 3 specific antagonists using PET.

Sphingosine-1-phosphate and its receptors (S1PRs) are involved in several diseases such as auto immunity, inflammation and cardiovascular disorders. The S1P analogue fingolimod (Gilenya®) is currently in use for the treatment of relapsing multiple sclerosis. S1PRs are also promising targets for clinical molecular imaging in vivo. The organ distribution of individual S1PRs can be potentially achieved by using S1PR subtype-specific (radiolabeled) chemical probes. Here, we report our efforts on synthesis and in vivo potency determination of new ligands for the S1P receptor 3 (S1P3) based on the S1P3 antagonist TY-52156 and in validation of a potential imaging tracer in vivo using Positron Emission Tomography (PET) after 18F-labelling. A p-fluorophenyl derivative exhibited excellent S1P3 antagonist activity in vitro, good serum stability, and medium lipophilicity. In vivo biodistribution experiments using 18F-PET exhibited significant uptake in the myocardium suggesting potential applications in cardiac imaging.

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates bone growth and regeneration. In the present study, a novel regenerative mechanism was connected to S1P signaling within the bone. Activation of its receptor S1PR3 in bone-forming osteoblasts led to secretion of vascular endothelial growth factor a (VEGFa), the most potent vessel-stimulating factor. This stimulated the development of specialized vessels of the bone marrow, the H-type vessels, that supported overall bone regeneration. These findings foster our understanding of regular bone metabolism and suggest that S1P-based drugs may help treat diseases such as age-related osteopenia and posttraumatic bone regeneration, conditions crucially dependent on functional bone microvasculature.

Is myeloid-derived growth factor a ligand of the sphingosine-1-phosphate receptor 2?

Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based ß-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Fine-tuning activation specificity of G-protein-coupled receptors via automated path searching.

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Insight into modulators of sphingosine-1-phosphate receptor and implications for cardiovascular therapeutics.

Cardiovascular disease is the leading cause of death worldwide, and it's of great importance to understand its underlying mechanisms and find new treatments. Sphingosine 1-phosphate (S1P) is an active lipid that exerts its effects through S1P receptors on the cell surface or intracellular signal, and regulates many cellular processes such as cell growth, cell proliferation, cell migration, cell survival, and so on. S1PR modulators are a class of modulators that can interact with S1PR subtypes to activate receptors or block their activity, exerting either agonist or functional antagonist effects. Many studies have shown that S1P plays a protective role in the cardiovascular system and regulates cardiac physiological functions mainly through interaction with cell surface S1P receptors (S1PRs). Therefore, S1PR modulators may play a therapeutic role in cardiovascular diseases. Here, we review five S1PRs and their functions and the progress of S1PR modulators. In addition, we focus on the effects of S1PR modulators on atherosclerosis, myocardial infarction, myocardial ischaemia/reperfusion injury, diabetic cardiovascular diseases, and myocarditis, which may provide valuable insights into potential therapeutic strategies for cardiovascular disease.

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes.

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

Targeting the SPHK1/S1P/S1PR2 axis ameliorates GH-secreted pituitary adenoma progression.

BACKGROUND: Growth hormone-secreted pituitary adenoma (GHPA) is a prominent subtype of pituitary adenoma (PA) associated with progressive somatic disfigurement, various complications, and elevated mortality rates. Existing treatment options have limited efficacy, highlighting the urgent need for novel pharmacological interventions. Previous studies have revealed that sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) signalling have critical roles in the tumour microenvironment, but their role in GHPA remains unclear. METHODS: We performed integrative analyses including bioinformatics analyses, functional studies, and clinical validation to investigate the pathological roles of SPHK1/S1P and evaluated the effectiveness of the S1P receptor 2 (S1PR2) inhibitor JTE-013 in GHPA treatment. RESULTS: SPHK1/S1P signalling is abnormally expressed in patients with GHPA. Knockdown of SPHK1 suppresses S1P-mediated cell proliferation in GH3 Cells. Mechanistically, S1P inhibits apoptosis and autophagy while promoting the secretion of Growth Hormone (GH) by binding to the S1P receptor subtype 2 (S1PR2) in GH3 cells. Moreover, the function of S1PR2 in GH3 cells is mediated by the downstream Akt-Creb pathway. We then identify the S1PR2 as a novel target for therapeutic intervention in GHPA. Systemic administration of the potent and selective S1PR2 antagonist, JTE-013, significantly reduces both tumour size and GH secretion. Importantly, we identify preoperative serum S1P levels as a biomarker predicting poor prognosis in GHPA patients at follow-up. CONCLUSION: Our study shows that blocking SPHK1/S1P/S1PR2 axis can ameliorate the progression of GHPA, providing evidence of a promising therapeutic target for GHPA.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice.

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

The role of SphK/S1P/S1PR signaling pathway in bone metabolism.

There are a large number of people worldwide who suffer from osteoporosis, which imposes a huge economic burden, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine kinase (SphK) is an enzyme that plays a crucial role in the synthesis of sphingosine-1-phosphate (S1P). S1P with paracrine and autocrine activities that act through its cell surface S1P receptors (S1PRs) and intracellular signals. In osteoporosis, S1P is indispensable for both normal and disease conditions. S1P has complicated roles in regulating osteoblast and osteoclast, respectively, and there have been exciting developments in understanding how SphK/S1P/S1PR signaling regulates these processes in response to osteoporosis therapy. Here, we review the proliferation, differentiation, apoptosis, and functions of S1P, specifically detailing the roles of S1P and S1PRs in osteoblasts and osteoclasts. Finally, we focus on the S1P-based therapeutic approaches in bone metabolism, which may provide valuable insights into potential therapeutic strategies for osteoporosis.

Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells.

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

Quantitative Analysis of S1PR1 Expression in the Postmortem Multiple Sclerosis Central Nervous System.

Multiple sclerosis (MS) is an immune-mediated disease that is characterized by demyelination and inflammation in the central nervous system (CNS). Previous studies demonstrated that sphingosine-1-phosphate receptor (S1PR) modulators effectively inhibit S1PR1 in immune cell trafficking and reduce entry of pathogenic cells into the CNS. Studies have also implicated a nonimmune, inflammatory role of S1PR1 within the CNS in MS. In this study, we explored the expression of S1PR1 in the development and progression of demyelinating pathology of MS by quantitative assessment of S1PR1 expression using our S1PR1-specific radioligand, [3H]CS1P1, in the postmortem human CNS tissues including cortex, cerebellum, and spinal cord of MS cases and age- and sex-matched healthy cases. Immunohistochemistry with whole slide scanning for S1PR1 and various myelin proteins was also performed. Autoradiographic analysis using [3H]CS1P1 showed that the expression of S1PR1 was statistically significantly elevated in lesions compared to nonlesion regions in the MS cases, as well as normal healthy controls. The uptake of [3H]CS1P1 in the gray matter and nonlesion white matter did not significantly differ between healthy and MS CNS tissues. Saturation autoradiography analysis showed an increased binding affinity (Kd) of [3H]CS1P1 to S1PR1 in both gray matter and white matter of MS brains compared to healthy brains. Our blocking study using NIBR-0213, a S1PR1 antagonist, indicated [3H]CS1P1 is highly specific to S1PR1. Our findings demonstrated the activation of S1PR1 and an increased uptake of [3H]CS1P1 in the lesions of MS CNS. In summary, our quantitative autoradiography analysis using [3H]CS1P1 on human postmortem tissues shows the feasibility of novel imaging strategies for MS by targeting S1PR1.

Structural determinants of sphingosine-1-phosphate receptor selectivity.

Fingolimod, the prodrug of fingolimod-1-phosphate (F1P), was the first sphingosine-1-phosphate receptor (S1PR) modulator approved for multiple sclerosis. F1P unselectively targets all five S1PR subtypes. While agonism (functional antagonism via receptor internalization) at S1PR1 leads to the desired immune modulatory effects, agonism at S1PR3 is associated with cardiac adverse effects. This motivated the development of S1PR3 -sparing compounds and led to a second generation of S1PR1,5 -selective ligands like siponimod and ozanimod. Our method combines molecular dynamics simulations and three-dimensional pharmacophores (dynophores) and enables the elucidation of S1PR subtype-specific binding site characteristics, visualizing also subtle differences in receptor-ligand interactions. F1P and the endogenous ligand sphingosine-1-phosphate bind to the orthosteric pocket of all S1PRs, but show different binding mode dynamics, uncovering potential starting points for the development of subtype-specific ligands. Our study contributes to the mechanistic understanding of the selectivity profile of approved drugs like ozanimod and siponimod and pharmaceutical tool compounds like CYM5541.

Pharmacological elevation of sphingosine-1-phosphate by S1P lyase inhibition accelerates bone regeneration after post-traumatic osteomyelitis.

Posttraumatic osteomyelitis and the ensuing bone defects are a debilitating complication after open fractures with little therapeutic options. We have recently identified potent osteoanabolic effects of sphingosine-1-phosphate (S1P) signalling and have now tested whether it may beneficially affect bone regeneration after infection. We employed pharmacological S1P lyase inhibition by 4-deoxypyrodoxin (DOP) to raise S1P levels in vivo in an unicortical long bone defect model of posttraumatic osteomyelitis in mice. In a translational approach, human bone specimens of clinical osteomyelitis patients were treated in organ culture in vitro with DOP. Bone regeneration was assessed by µCT, histomorphometry, immunohistology and gene expression analysis. The role of S1P receptors was addressed using S1PR3 deficient mice. Here, we present data that DOP treatment markedly enhanced osteogenesis in posttraumatic osteomyelitis. This was accompanied by greatly improved osteoblastogenesis and enhanced angiogenesis in the callus accompanied by osteoclast-mediated bone remodelling. We also identified the target of increased S1P to be the S1PR3 as S1PR3-/- mice showed no improvement of bone regeneration by DOP. In the human bone explants, bone mass significantly increased along with enhanced osteoblastogenesis and angiogenesis. Our data suggest that enhancement of S1P/S1PR3 signalling may be a promising therapeutic target for bone regeneration in posttraumatic osteomyelitis.

Clinical Pharmacokinetics of Ponesimod, a Selective S1P1 Receptor Modulator, in the Treatment of Multiple Sclerosis.

Ponesimod, a selective, rapidly reversible, and orally active, sphingosine-1 phosphate receptor (S1P) modulator, is indicated for the treatment of relapsing-remitting multiple sclerosis (RRMS). The clinical pharmacokinetics (PK) and pharmacodynamics (PD) of ponesimod was studied in 16 phase I, one phase II, and one phase III clinical studies. Ponesimod population PK was characterized by an open two-compartment disposition model with a terminal half-life of 33 h (accumulation factor of 2- to 2.6-fold), and fast and almost complete oral absorption (absolute oral bioavailability: 84%), reaching peak plasma and blood concentrations within 2-4 h. Ponesimod is highly metabolized, and the parent compound along with its two major (non-clinically active) metabolites are mainly excreted in the feces (recovery: 57.3-79.6%) and to a lesser extent in the urine (recovery: 10.3-18.4%). Additionally, the population PKPD model characterized the ponesimod effects on heart rate: a transient, dose-dependent decrease in heart rate in the first days of dosing, that is mitigated by administering the first doses of ponesimod treatment using a gradual up-titration schedule, before reaching the daily maintenance dose of 20 mg. This selected maintenance dose has been shown to be superior in reducing annualized relapse rate (ARR) when compared with teriflunomide in a pivotal phase III study. Furthermore, a dose-dependent reduction of peripheral lymphocyte counts that is sustained with continued daily oral dosing of ponesimod and is rapidly (4-7 days) reversible upon drug discontinuation has been characterized with an indirect response model.