Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A-D) HeLa cells not expressing (A) or transiently expressing 3xHA-S1PR1-eGFP (B-D) were incubated with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (Carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. HA antibody was used to follow the uptake of S1P1R. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin or HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) Effect of clathrin or AP2 depletion in the uptake of S1PR1 in Hela cells transiently expressing 3xHA-S1P1-eGFP. Cells were depleted of clathrin or of its endocytic adaptor AP2 by transduction with lentiviruses encoding shRNA specific for clathrin heavy chain (CLC) or the μ2 subunit of AP2 (AP2); scrambled shRNA was used as a negative control. The cells were incubated with anti-HA antibody to follow the uptake of S1PR1 upon addition of DMSO only (Carrier) or the ligands (S1P or FTY720P). Transferrin-Alexa Fluor 647 was used to monitor its clathrin-based receptor mediated endocytosis. The representative images correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove surface bound transferrin and anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Transduction, shRNA, Negative Control, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) MEF cells from mice expressing (β-Arrestin1/2+/+) or not (β-Arrestin1/2−/−) β-arrestin1 and β-arrestin2 and stably expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the presence of DMSO only (Carrier) or the ligands S1P or FTY720P. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy. An acid wash step at the end of the experiment was used to remove most of the surface bound anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Stable Transfection, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: HEK 293A cells stably expressing 3xHA-S1PR1-mCherry were incubated or not with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. Data obtained using flow cytometry was from more than 10,000 cells analyzed per condition.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Stable Transfection, Expressing, Incubation, Flow Cytometry

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) HeLa cells transiently expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the absence (− dynasore-OH) or presence (+ dynasore-OH) of the modified more potent cell permeable inhibitor of dynamin, dynasore-OH. Following brief 15 min incubation with dynasore-OH, the media was replaced for 30 min with a new solution only containing DMSO, S1P or FTY720P. Fluorescent transferrin-Alexa Fluor 647 was used to follow the efficiency of the clathrin-based endocytic pathway. Images obtained using spinning disc confocal microscopy correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin and anti-HA antibodies. Scale bar, 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Modification, Confocal Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A – D) Arg1 Yarg Il5 Red5 Il13 Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis and analyzed at the indicated time points. Uninfected mice (Naive) were examined as controls. (A) Expression of KLRG1 and IL-5 by cells gated on CD45 + Lin − cells isolated from peripheral blood (left) or small intestine lamina propria and lung (right). Expression of ST2 and IL-17RB or Arg1 by ILC2s gated as indicated in the top panels. (B and C) Frequencies of KLRG1 + IL-5 (Red5) + ILC2s (B) and percentages of IL-13 reporter–positive ILC2s (C) in peripheral blood. DL, detection limit. (D) Flow cytometry plots of blood (left) or lung (right) ILC2s gated on CD45 + Lin − IL-5 + cells from d5 or d12, highlighting activation as assessed by IL-13 (Sm13) expression and the expression of ST2, Arg1 (Yarg), and IL-17RB. (E) Serum IL-13 levels from WT C57BL/6J mice infected with N. brasiliensis treated with or without FTY720 (FTY) and analyzed at the indicated time points. The x axis represents days after infection. Data displayed as mean ± SEM. u.i., uninfected. Data are from one experiment representative of at least two independent experiments (A, D, and E) or pooled from multiple independent experiments (B and C). *, P < 0.05.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Expressing, Isolation, Flow Cytometry, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A) Arg1 Yarg Il5 Red5 Il13 Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis, and blood (left panels) and lungs (right panels) were analyzed on d5 after infection for IL-5 (Red5) and IL-13 (Sm13) expression in ILCs (gated on CD45 + Lin − ) and CD4 T cells (gated on CD45 + CD4 + ). (B) Mice were infected with N. brasiliensis in the presence or absence of FTY720 (1 mg/kg), and blood (top panels) and lung (bottom panels) ILC2s (gated on CD45 + Lin − GATA3 + ) were examined on d5 or d12 for IL-13 (Sm13) expression and Ki-67 labeling. (C and D) Mice were infected with N. brasiliensis and were i.v. injected with 3 µg of APC-Cy7–labeled CD45.2 antibody on d5 and euthanized 3 min later. (C) Top panels indicate the intravascular (IV) or tissue-resident (Tissue) ILC2s gated on CD45 + Lin − IL5 (Red5) + from the blood, lung, or mLNs (mesLN). Middle and bottom panels show the expression of ST2 and IL-17RB (middle panels) or ST2 and CD69 (bottom panels) from the intravascular or tissue-resident fraction from tissues as indicated. (D) Percentage of tissue and blood (IV) ILC2s in the lung on d5. Data are from one experiment representative of at least two independent experiments. *, P < 0.05; **, P < 0.005. ns, no significant difference.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Expressing, Labeling, Injection

Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s are dependent on tissue extrusion. (A–H) C57BL/6J mice were infected with N. brasiliensis , injected i.p. with saline or FTY720 (1 mg/kg daily from day of infection), and analyzed at the indicated time points. (A) Gating of ILC2s (pregated on live CD45 + Lin − ) from blood or lung. (B) Total number of ILC2s in the blood (left) or lung (right) at d5. (C–E) Flow cytometry plots showing the expression of ST2 and IL-17RB on blood and lung ILC2s from d5 (C) and d12 (E) and quantification of the percentage of IL-17RB + ST2 − ILC2s in the blood and lung on d5 (D). (F) Total number of ILC2s in the blood (left) or lung (right) at d12. Data are representative of two independent experiments with n ≥ 3 individual mice per group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. N.b. , N. brasiliensis ; ns, no significant difference.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Injection, Saline, Flow Cytometry, Expressing

Journal: Journal of Leukocyte Biology

Article Title: Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720

doi: 10.1189/jlb.3vma1114-522rr

Figure Lengend Snippet: Figure 5. RIP1K and HSP90 are involved in neutrophil death induced by FTY720. (A and B) Flow cytometric quantification of Annexin V+PI+, Annexin V+PI2, and HSP27+ neutrophils after preincubation or not with necrostatin-1 (20 mM) or Z-IETD-FMK (20 mM) for 30 min and treatment or not with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01; ***P , 0.001. (C and D) Neutrophil lysates analyzed by Western blotting for caspase-8 cleavage or MLKL phosphorylation after cell death induc- tion by FTY720. Where indicated, Q-VD-OPh (20 mM), necrostatin-1 (20 mM), DPI (20 mM), or NSA (5 mM) was added for 30 min before stimulation. GAPDH analysis was performed as loading control. The results of 1 representative experiment out of 4 are presented. (E) Flow cytometric quantification of HSP27+ neutrophils incubated for 30 min in the presence of geldanamycin (10 mM), DMAG (20 mM), or radicicol (10 mM) before treatment with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01.

Article Snippet: Cells were incubated with FTY720 and FTY720 (S)-phosphate (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or cycloheximide (Sigma, St. Louis, MO, USA) at 3 3 10/ml for 3 h. In some cases, neutrophils were preincubated for 30 min with necrostatin-1 or Z-IETD-FMK (both from Sigma); Q-VD-OPh (R&D Systems, Minneapolis, MN, USA); NSA (Tocris, Bristol, United Kingdom); geldanamycin, 17-DMAG, and radicicol (all from InvivoGen, San Diego, CA, USA); OA (Santa Cruz Biotechnology); or DPI (Sigma).

Techniques: Western Blot, Phospho-proteomics, Control, Incubation

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Mass Spectrometer Settings Used for Metabolite Profiling of FTY720-C2 and FTY720-Mitoxy.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Mass Spectrometry

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Pharmacokinetic Study Design for FTY720-C2 and FTY720-Mitoxy.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Intrinsic Clearance in Liver Microsomes.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Summary of the structural characterization by LC-MS/MS of FTY720-C2 Metabolites Identified in Rat and Human Hepatocytes.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Summary of the structural characterization by LC-MS/MS of FTY720-Mitoxy Metabolites Identified in Rat and Human Hepatocytes.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Using 1 μM of FTY720-C2, we incubated rat (a) and human (b) hepatocytes to obtain the metabolite profile. To simplify presentation, the 0 min incubation time point, data are intentionally not shown. Legend: C2 , FTY720-C2 C2-carboxylic acid; C4 , FTY720-C2 C4-carboxylic acid; C6 , FTY720-C2 C6-carboxylic acid; C8 , FTY720-C2 C8-carboxylic acid; FTY720-C2-OH , hydroxy FTY720-C2.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Incubation

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Mass spectrometry of FTY720-C2 and its metabolites, detected as protonated molecular ions [M+H] + are shown. Our rationale for identifying the structure of the metabolites is demonstrated using colored text and arrows, with blue product ions indicating losses of H 2 O and an N -acetyl group from [M+H] + , red product ions indicating hydroxylation, and green product ions associated with identified carboxylic acid modifications.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Mass Spectrometry

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Using 1 μM of FTY720-Mitoxy, we incubated rat (a) and human (b) hepatocytes to obtain metabolite profiles. To simplify presentation, the 0 min incubation time point data are intentionally not shown. Legend: C4 , FTY720-Mitoxy C4-carboxylic acid; C6 , FTY720-Mitoxy C6-carboxylic acid; C8 , FTY720-Mitoxy C8-carboxylic acid; FTY720-Mitoxy-OH , hydroxy FTY720-Mitoxy.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Incubation

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Mass spectrometry of FTY720-Mitoxy and metabolites detected [M + ] molecular ions. Blue text and arrows indicate losses of H 2 O from [M] + . The ion with a hydroxyl group is shown in red . Ions including a carboxylic acid are shown in green .

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Mass Spectrometry

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Relative Percent Distribution of FTY702-C2 and FTY720-Mitoxy.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Incubation

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: Mean plasma and brain concentrations of FTY720-C2 after IV dosing ( a ) and oral dosing ( b ) (black bar = plasma; white bar = brain); and of FTY720-Mitoxy after IV dosing ( c ) (▲ = plasma; ○ = brain). Units are in ng/mL for plasma and ng/g for brain. Data represent the mean ± SD of two experiments for each time point. Error bars were calculated for all samples and are present on the graphs. However, for samples with little variability error bars do not extend beyond the edges of the symbols so thus, are not apparent.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

doi: 10.1371/journal.pone.0162162

Figure Lengend Snippet: FTY720-C2 increased PP2A activity in adrenal glands at all time points following IV delivery ( a ) and oral delivery ( b ) as compared to untreated control mice. FTY720-Mitoxy increased PP2A activity in the adrenal gland at all time points after IV delivery ( c ). After oral delivery, FTY7220-Mitoxy was not absorbed and adrenal PP2A activity did not increase as compared to untreated controls ( d ). Two mice per time point were evaluated. Data represent mean ± SEM.

Article Snippet: Midazolam, diclofenac, glucose-6-phosphate dehydrogenase, glucose 6 phosphate, magnesium chloride, and testosterone (Sigma-Aldrich, St. Louis, MO); HPLC grade water, methanol, formic acid, and acetonitrile (Fisher Scientific, Pittsburgh, PA); cell free HepatoZYME-SFM (1x) medium (Gibco Life Technology, Grand Island, NY); C17 Sphingosine (Santa Cruz Biotechnology, Dallas, TX); Fingolimod hydrochloride salt (FTY720) (LC laboratories, Woburn, MA); the new compounds FTY720-C2 [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)acetamide] (MW 349.51 g/mol) and FTY720-Mitoxy [ N -(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-3’-triphenylphosphoniumpropanamide] (MW 704.72 g/mol) were synthesized in Dr. Jeffery Arterburn’s laboratory (New Mexico State University, Las Cruces, NM) [ ].

Techniques: Activity Assay

Journal: Immunology and Cell Biology

Article Title: Potential killers exposed: tracking endogenous influenza‐specific CD8 + T cells

doi: 10.1111/imcb.12189

Figure Lengend Snippet: CD25 + CD43 + CD8 + T cells progress to the CD25 − CD43 + phenotype. Mice received 1 mg kg −1 of Fingolimod or PBS i.p. daily, beginning 52 h after i.n. HKx31 infection. Cells were recovered 5, 6 or 7 days after infection and analyzed for D b PA 366 tetramer binding and CD25, CD43, and CD8α expression. (a) Results show the mean ± s.e.m. number of D b PA 244 + CD8 + T cells from MLN, (b) BAL or (c) spleen. ( n = 6 or 7 mice per group from 1 experiment) * P < 0.05, ** P < 0.01 (Mann–Whitney U‐ test). (d) Representative density plots show CD25 versus CD43 expression on gated MLN CD8 + T cells. (e) Results show the mean ± s.e.m. ratio of CD25 + CD43 + to CD25 − CD43 − CD8 + T cells.

Article Snippet: Fingolimod (FTY720, Cayman Chemical, Ann Arbor, MI, USA) was administered i.p. at 1 mg kg −1 (Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Infection, Binding Assay, Expressing, MANN-WHITNEY