FTY720 (S)-Phosphate is an S1PR1 agonist. FTY720 (S)-Phosphate (Tys, 1 µM) does not affect the induction of S1PR1 ubiquitination, but it does sustain the expression of the S1PR1 protein and improve the human pulmonary artery endothelium cells barrier through S1PR1. Additionally, β-arrestin recruitment to S1PR1 is decreased by FTY720 (S)-Phosphate (0.01–50 µM)[1].

In mice suffering from bleomycin-induced acute lung injury (ALI), FTY720 (S)-Phosphate (0.5 mg/kg, ip) reduces lung tissue leukocyte infiltration and maintains S1PR1 expression in the lungs[1].

β-arrestin activation [1]
The Invitrogen Tango™ EDG1--bla U2OS Cell-based Assay was used to determine β-arrestin activation. Briefly, EDG1-bla U2OS cells were grown in FreeStyle™ Expression Medium for 48 h. Then, 1 µM of S1P, Tys, 1R, FTY720, p-FTY720, or 10 µM of SEW was added, and the cells were incubated in a humidified 37°C/5% CO2 incubator for 5 h. Fluorescence substrates were added, and the cells were kept at room temperature in the dark for 2 h. Fluorescence intensity was detected and the blue/green emission ratio for each well was calculated and used as the S1PR1 activation indicator. Experiments were repeated 3 times, and the final ratio was expressed as the mean ± S.E.M.

Bleomycin ALI models [1]
Male C57BL/6 (20–25 g) mice 8–10 weeks old received a single intratracheal dose of bleomycin at 0.6 U/kg (or sterile saline) on Day 0 followed immediately by intraperitoneal injection of Tys (0.5 mg/kg), FTY720 (0.5 mg/kg), or saline. Additional doses of Tys or FTY720 were injected on Days 3 and 6. Bronchoalveolar lavage (BAL) fluid and lungs were then collected on Day 7. BAL fluid was used to detect BAL protein levels, WBC count, and WBC differential count. Lungs were perfused with saline to remove blood for Western blot, tissue albumin, and histopathology evaluation. Peripheral blood was obtained on Day 7 for examination of total cell counts and lymphocytes. Experiments were repeated 3 times. 6–10 mice were used per experimental group.

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To block egress of lymphocytes, 9-week-old female NOD mice were either given 1.5 mg/kg FTY720 in sterile water or equal volume of sterile water by gavage daily for a week. Next the mice were sacrificed and immune cell populations were detected in MLN, PLN, and spleen by flow cytometry.[2]

[1]. FTY720 (s)-phosphonate preserves sphingosine 1-phosphate receptor 1 expression and exhibits superior barrier protection to FTY720 in acute lung injury. Crit Care Med. 2014 Mar;42(3):e189-99.

[2]. Clostridium butyricum CGMCC0313.1 Protects against Autoimmune Diabetes by Modulating Intestinal Immune Homeostasis and Inducing Pancreatic Regulatory T Cells. Front Immunol. 2017 Oct 19;8:1345.

Objective: Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo. Setting: University-affiliated research institute. Subjects: Cultured human pulmonary endothelial cells; C57BL/6 mice. Interventions: Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists. Measurements and main results: FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce β-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation. Conclusion: FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury. [1]

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FTY720 Suppresses the Accumulation of α4β7+ Tregs but Not the Total Tregs in the PLN To confirm if CB0313.1 promoted the migration of gut-primed Tregs to PLN, we administrated NOD mice with FTY720 by gavage, which inhibits T cells circulation and traps them in the lymph node (36). Indeed, FTY720 resulted in a decreased proportion of splenic Tregs (Figure 7A), whereas an increased fraction of Tregs in the MLN compared to the NOD-CB mice (Figure 7B). FTY720 inhibited the migration of α4β7+ Tregs away from MLN (Figure 7C). In the PLN, FTY720 suppressed the accumulation of α4β7+ Tregs (Figure 7E), however, did not significantly change the total proportion of Tregs (Figure 7D), suggesting that the majority of Tregs in the PLN might be induced locally and migrated α4β7+ Tregs represented only a small fraction (Figure S6 in Supplementary Material).[2]

C19H34NO5P

387.45

387.217

C, 58.90; H, 8.85; N, 3.62; O, 20.65; P, 7.99

402616-26-6

(S)-FTY720-phosphonate;1142015-10-8;Fingolimod phosphate;402615-91-2

11452022

White to off-white solid powder

1.2±0.1 g/cm3

584.2±60.0 °C at 760 mmHg

184-186ºC

307.1±32.9 °C

0.0±1.7 mmHg at 25°C

1.541

4.27

4

6

14

26

409

1

OC[C@](CCC1=CC=C(CCCCCCCC)C=C1)(N)COP(O)(O)=O

LRFKWQGGENFBFO-IBGZPJMESA-N

InChI=1S/C19H34NO5P/c1-2-3-4-5-6-7-8-17-9-11-18(12-10-17)13-14-19(20,15-21)16-25-26(22,23)24/h9-12,21H,2-8,13-16,20H2,1H3,(H2,22,23,24)/t19-/m0/s1

[(2S)-2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butyl] dihydrogen phosphate

(S)FTY720P S-FTY720P(S)-FTY720P (S) FTY720P FTY-720 (S)-Phosphate(S)-FTY720 phosphate FTY720 (S)-Phosphate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~1 mg/mL (~2.58 mM)
H2O : < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)