G-protein-coupled receptors (GPCR) are the largest superfamily of receptors responsible for signaling between cells and tissues, and because they play important physiological roles in homeostasis, they are major drug targets. New technologies have been developed for the identification of new ligands, new GPCR functions, and for drug discovery purposes. In particular, intercellular lipid mediators, such as, lysophosphatidic acid and sphingosine 1-phosphate have attracted much attention for drug discovery and this has resulted in the development of fingolimod (FTY-720) and AM095. The discovery of new intercellular lipid mediators and their GPCRs are discussed from the perspective of drug development. Lipid GPCRs for lysophospholipids, including lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylcholine, free fatty acids, fatty acid derivatives, and other lipid mediators are reviewed.
Drug Discovery* ; Fatty Acids, Nonesterified ; Homeostasis ; Ligands ; Lysophosphatidylcholines ; Lysophospholipids ; Receptors, G-Protein-Coupled ; Sphingosine ; Fingolimod Hydrochloride

OBJECTIVETo investigate the effects of FTY720, a new immunosuppressive agent, on apoptosis and autophagy in multiple myeloma(MM) cell line U266 and to clarify its molecular mechanism.

METHODSU266 cells were treated with 0, 2.5, 5.0, 10.0 and 20.0 µmol/L FTY720 for 24 hours, and the cell viability was assayed by CCK-8 method. Then U266 cells were treated with 20.0 µmol/L FTY720 for 0, 2, 6 and 24 hours, the cell viability was tested. The apoptotic rates induced by different doses and time points of FTY720 were tested by flow cytometry separately. The expression of LC3B was detected by Western blot after U266 cells treated with different doses of FTY720 to see autophagy. U266 cells were treated with FTY720 ± Bafilomycin A1, an inhibitor of autophagy, for 24 hours, then the cell viability and apoptotic rates were tested. Meanwhile the expression of survivin, anti-apoptotic factors, were tested by Western blot.

RESULTSThe cell viability and the apoptotic rates were inhibited significantly by FTY720 (P < 0.05) in time-dependent and dose-dependent manner. The expression of LC3B-II increased significantly in a dose-dependent manner, it indicated that the autophagy was induced by FTY720. Bafilomycin A1 could rescue the cell viability and apoptotic rates in U266 cells treated with FTY720, and it could also rescue the expression of survivin decreased by FTY720.

CONCLUSIONSFTY720 can cause apoptosis and autophagy of U266 cells. The autophagy promote the apoptosis, which maybe due to the degradation of anti-apoptotic factors such as survivin or their upstream factors in lysosomes through autophagy.

Apoptosis ; drug effects ; Autophagy ; drug effects ; Cell Line, Tumor ; Fingolimod Hydrochloride ; Humans ; Multiple Myeloma ; pathology ; Propylene Glycols ; pharmacology ; Sphingosine ; analogs & derivatives ; pharmacology

Here we report a case of pediatric multiple sclerosis treated with interferon beta-1b. Interferon beta is widely used in adult patients with multiple sclerosis (MS). However, its effects and safety in pediatric patients have not been well established. Although supporting data are limited, the use of disease modifying therapies (DMTs) such as interferon beta-1b is recommended early in treatment of children with MS. Reports of interferon beta treatment in pediatric MS patients in Korea are rare. In this report, we describe a Korean girl who was effectively treated with interferon beta-1b for three years. There were no relapses or serious side effects. Therefore, this report provides evidence supporting the use of interferon beta in pediatric MS patients in Korea and other Asian countries. We also reviewed current medical treatment of MS, including some DMTs and second-line treatment options such as natalizumab and cyclophosphamide, and several new oral agents such as fingolimod.
Adult ; Antibodies, Monoclonal, Humanized ; Asian Continental Ancestry Group ; Child ; Cyclophosphamide ; Humans ; Interferon-beta ; Interferons ; Korea ; Multiple Sclerosis ; Propylene Glycols ; Recurrence ; Sphingosine ; Fingolimod Hydrochloride ; Interferon beta-1b ; Natalizumab

Ischemia-reperfusion injury is complicated with multiple injury pathways. If a particular agent is used to restore blood flow and prevent cell death, many damaged neurons may come back to life. However, for stroke victims, there are no effective curative therapeutic approaches available other than thrombolytic treatment. The efficacy of neuroprotective agents are limited by low diffusion, narrow time window, strict dose titration, and lack of confidence at the preclinical level. NXY-059 reflects the fundamental limitation of neuroprotectant. There are recent attempts to overcome these limitations, with use of annexin A2, fingolimod, hydrogen, nitrite, and more. By covering two components, this report reviews what we have recently learned. In addition, it sheds light on some of the newer issues in clinical application.
Annexin A2 ; Benzenesulfonates ; Cell Death ; Diffusion ; Hydrogen ; Light ; Multiple Trauma ; Neurons ; Neuroprotective Agents ; Propylene Glycols ; Reperfusion Injury ; Sphingosine ; Stroke ; Fingolimod Hydrochloride

Sphingosine 1-phosphate (S1P) is an important biologically active lysophospholipid that transmits signals through a family of G-protein-coupled receptors (GPCRs) to regulate the vital functions of several types of immune cells. The S1P GPCRs suppress both generation of specialized functional cytokines, such as IFN-gamma and IL-4, and proliferation of T-cells. Although S1P is chemotactic to T cells, B cells, dendritic cells, and natural killer cells, the major effect of S1P on the immune system is the regulation of lymphocyte recirculation and tissue distribution by S1P and S1P1. Chemotactic response of CD4+CD25+ regulatory T cells to S1P is reduced, but its optimal suppressive activities require S1P. FTY720, a new class of immunomodulator, is rapidly phosphorylated by sphingosine kinase 2 in vivo to form the biologically active phosphorylated-FTY720 (FTY720-P), which closely resembles S1P. The FTY720-P is a true agonist for S1P1, S1P3, S1P4, and S1P5, it affects the tissue distribution and functional activity of T cells, B cells, dendritic cells and regulatory T cells. FTY720 were demonstrated to be a hypotoxic, great effective and reversible immunosuppressive efficacy to prevent allograft rejection and treat some autoimmune diseases. In this article, the synthesis and metabolism of S1P, the expression of S1P GPCRs in immune cells, the effect of S1P on immune cells, the drugs targeted to S1P GPCRs and their clinical implications are reviewed.
Fingolimod Hydrochloride ; Humans ; Immunomodulation ; physiology ; Lysophospholipids ; immunology ; physiology ; Propylene Glycols ; metabolism ; Receptors, G-Protein-Coupled ; immunology ; physiology ; Sphingosine ; analogs & derivatives ; immunology ; metabolism ; physiology

OBJECTIVETo investigate the effect and mechanism of FTY720 on acute graft versus host disease (GVHD) in rat small bowel transplantation (SBTx).

METHODSHeterotopic SBTx was performed using a parent (WF)-into-F1 (WFxACI) rat combination. Recipient rats were divided into experimental group (n=6) and control group (n=6). Rats in the experimental group were administered with FTY720 at 0.5 mg/kg for 14 days. Lymphocyte apoptosis in the liver and the mucosa of intestine and graft was detected by TUNEL and flow cytometry 15 days after transplantation. Recipient survival and lymphocyte apoptosis were compared between the two groups.

RESULTSRecipients in the control group died of GVHD after a mean survival time of (16+/-2.1) days. FTY720-treated recipients had a significantly longer survival (>100 days). After administration of FTY720, the percentage of apoptotic lymphocytes was significantly increased in the graft as compared to that in the control group by flow cytometry. The ratio of apoptotic lymphocyte in the liver and graft was also significantly higher in the experimental group by TUNEL.

CONCLUSIONFTY720 effectively induces the lymphocyte apoptosis, inhibits the lesion of target tissues by GVHD, and prolongs the recipient survival.

Animals ; Apoptosis ; drug effects ; Fingolimod Hydrochloride ; Graft vs Host Disease ; immunology ; prevention & control ; Immunosuppressive Agents ; pharmacology ; Intestine, Small ; transplantation ; Lymphocytes ; cytology ; drug effects ; Male ; Propylene Glycols ; pharmacology ; Rats ; Rats, Inbred WF ; Sphingosine ; analogs & derivatives ; pharmacology ; Transplantation, Heterotopic

Initial discovery on sphingosine 1-phosphate (S1P) as an intracellular second messenger was faced unexpectedly with roles of S1P as a first messenger, which subsequently resulted in cloning of its G protein-coupled receptors, S1P₁₋₅. The molecular identification of S1P receptors opened up a new avenue for pathophysiological research on this lipid mediator. Cellular and molecular in vitro studies and in vivo studies on gene deficient mice have elucidated cellular signaling pathways and the pathophysiological meanings of S1P receptors. Another unexpected finding that fingolimod (FTY720) modulates S1P receptors accelerated drug discovery in this field. Fingolimod was approved as a first-in-class, orally active drug for relapsing multiple sclerosis in 2010, and its applications in other disease conditions are currently under clinical trials. In addition, more selective S1P receptor modulators with better pharmacokinetic profiles and fewer side effects are under development. Some of them are being clinically tested in the contexts of multiple sclerosis and other autoimmune and inflammatory disorders, such as, psoriasis, Crohn’s disease, ulcerative colitis, polymyositis, dermatomyositis, liver failure, renal failure, acute stroke, and transplant rejection. In this review, the authors discuss the state of the art regarding the status of drug discovery efforts targeting S1P receptors and place emphasis on potential clinical applications.
Acute Kidney Injury ; Animals ; Clone Cells ; Cloning, Organism ; Colitis, Ulcerative ; Dermatomyositis ; Drug Discovery* ; Fingolimod Hydrochloride ; Graft Rejection ; In Vitro Techniques ; Liver Failure ; Mice ; Multiple Sclerosis ; Polymyositis ; Psoriasis ; Receptors, Lysosphingolipid* ; Second Messenger Systems ; Sphingosine* ; Stroke

Syl948 is a synthesized selective S1P1 agonist with novel structure. HTRF-IP1 test indicated that Syl948-P, the active form of Syl948 in vitro, has strong activity against S1P1 (EC50: 83 +/- 16 nmol x L(-1)), but its effect on S1P3 was very weak (EC50: 1 026 +/- 90 nmol x L(-1)). In SD rats, oral administration of Syl948 10 mg x kg(-1) significantly decreased the peripheral blood lymphocytes (PBL), with the maximal PBL inhibition rate of 63%, which was as similar as equal dose of fingolimod (FTY720). Oral administration of Syl948 10 mg x kg(-1) had no effect on heart rate of SD rats, which was better than FTY720. Daily oral administration with Syl948 (2 or 4 mg x kg(-1)) significantly prolonged the survival time of the allografts of skin slice on mice. In summary, the above results demonstrated that Syl948 has great selectivity in vitro and good activity in vivo, which indicated its potential use as an anti-rejection drug in skin transplantation.
Animals ; Fingolimod Hydrochloride ; Graft Survival ; drug effects ; Immunosuppressive Agents ; pharmacology ; Lymphocytes ; drug effects ; Mice ; Propylene Glycols ; pharmacology ; Rats ; Receptors, Lysosphingolipid ; agonists ; Skin Transplantation ; Sphingosine ; analogs & derivatives ; pharmacology ; Transplantation, Homologous

OBJECTIVETo explore the operational mechanisms and potential approach to inducing transplantation immune tolerance of FTY720.

METHODSMouse splenocytes were incubated with FTY720, then the DNA was extracted and analyzed using gel electrophoresis. Hearts of inbred BALB/c (H-2(d)) mice were transplanted heterotopically in C57BL/6 (H-2b) mice. Recipients were randomly divided into six groups. Group-1 (n = 6) was the nil-treated control. Groups-2, 3 and 4 were given FTY720 at the dose of 3 mg.kg(-1) by oral gavage once a day with different time courses. Group-2 (n = 14) were administrated from 3 days before transplantation (day-3) to the 11th day after the transplantation (day 11); Group-3 (n = 6) from day 0 to day 14; Group-4 (n = 6) from day-3 to day 0. Group-5 (n = 5) and 6 (n = 5) were treated with Cyclosporine A (10 mg.kg(-1)) and 40-O-(2-hydroxyethyl)-rapamycin (RAD) (3 mg.kg(-1)) respectively by daily gavage from day 3 to day 11. The long survivors (> 100 d) in Group-2 were detected with their IL-4 and IFN-gamma levels and their tolerant state was challenged with second graft: the donor type skin.

RESULTSApoptosis changes of the mouse splenocytes incubated with FTY720 was showed by typical DNA ladders. The median survival time (MST) of Group-1 was 8 d. MST of Group-2 was 55 d and grafts in six mice survived more than 100 d. MST of Group-3 was 16.5 d. Group-4 has a MST of 14 d with one case exceeded 100 d. MST of Group-5 and 6 were 10 d and 13 d respectively. Long survivors of Group-2 can accept donor-type skin graft and the level of IL-4 in their serum is up-regulated while IFN-gamma remained stable.

CONCLUSIONSPretreatment of FTY720 bring about effect on the early events of transplantation immune responses. This effect might be mediated by apoptosis induction in lymphocytes using this drug. We originally designed the regime of FTY720 monotherapy, which started pre-operationally and maintained for a short period of time, and induced stable tolerance the allo-graft in mouse.

Adjuvants, Immunologic ; pharmacology ; Animals ; Apoptosis ; Fingolimod Hydrochloride ; Heart Transplantation ; immunology ; Immune Tolerance ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Myocardium ; pathology ; Propylene Glycols ; pharmacology ; Sphingosine ; analogs & derivatives ; Transplantation Immunology

Sphingosine-1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions in both intracellular and extracellular compartments. It interacts with five G protein-coupled receptors subtypes (S1PR(1-5)) to generate multiple downstream signaling. Activation of S1PR1 has been validated to be involved in the process of immune modulation. Fingolimod (FTY720), the novel S1PR1 agonist, has been approved for the treatment of multiple sclerosis in clinical trials. The study towards discovery of selective S1PR1 agonists has become hot spot for immunological diseases. This article summarized the research progress of S1PR1 agonists, emphasizing their structure types, structure-activity relationship and direction of development.
Animals ; Fingolimod Hydrochloride ; Humans ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; Lysophospholipids ; physiology ; Multiple Sclerosis ; drug therapy ; Propylene Glycols ; pharmacology ; therapeutic use ; Receptors, Lysosphingolipid ; agonists ; classification ; metabolism ; physiology ; Sphingosine ; analogs & derivatives ; pharmacology ; physiology ; therapeutic use ; Structure-Activity Relationship