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: Acute inflammatory diseases, such as acute lung injury, are characterized by increased vascular permeability, thus requiring effective treatments to reverse this characteristic. FTY720 is a pharmaceutical analog of a potent barrier-enhancing phospholipid—sphingosine-1-phosphate. Due to certain properties of both FTY720 and sphingosine-1-phosphate that may limit their application in patients with acute lung injury, alternative compounds are needed for treatment. This study aimed to characterize the effects of FTY720(S)-phosphonate (a novel FTY720-phosphate analog) on pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo. Location: University-affiliated research institution. Subjects: Cultured human lung endothelial cells; C57BL/6 mice. Intervention: Endothelial cells were stimulated with a sphingosine-1-phosphate receptor 1 agonist to determine its effect on sphingosine-1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice using bleomycin to evaluate the effect of the sphingosine-1-phosphate receptor 1 agonist. Measurements and main results: FTY720 (S)-phosphonate significantly enhanced the barrier function of human lung endothelial cells in vitro, as measured by transendothelial resistance. This increase in transendothelial resistance was significantly attenuated by reducing sphingosine-1-phosphate receptor 1 (SAPPRP) expression using small interfering RNA. FTY720 (S)-phosphonate maintained SAPPRP protein expression in endothelial cells, unlike SAPPRP, FTY720, or other SAPPRP agonists which reduced SAPPRP protein expression by more than 50%. FTY720 (S)-phosphonate did not induce β-arrestin recruitment, SAPPRP 1 ubiquitination, or proteasome degradation as other agonists did. In normal mice or bleomycin-injured mice, intraperitoneal injection of FTY720 (S)-phosphonate every other day for one week significantly maintained higher levels of pulmonary SAPPRP expression compared to FTY720. FTY720 did not protect mice from bleomycin-induced acute lung injury, while FTY720 (S)-phosphonate significantly reduced lung leakage and inflammation. Conclusion: FTY720 (S)-phosphonate is a promising barrier enhancer that effectively maintains sphingosine-1-phosphate receptor 1 levels and improves the prognosis of a bleomycin-induced acute lung injury model. [1]

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FTY720 inhibited the accumulation of α4β7+ Treg cells but not the accumulation of total Treg cells in the PLN. To verify whether CB0313.1 promoted the migration of gut-initiated Treg cells to the PLN, we administered FTY720 to NOD mice by gavage, which inhibited T cell circulation and retained them in the lymph nodes (36). In fact, FTY720 treatment resulted in a decrease in the proportion of Treg cells in the spleen (Fig. 7A) and an increase in the proportion of Treg cells in the mesenteric lymph nodes (MLN) compared to NOD-CB mice (Fig. 7B). FTY720 inhibited the outward migration of α4β7+ Treg cells from the MLN (Fig. 7C). In the peritoneal lymph nodes (PLN), FTY720 inhibited the accumulation of α4β7+ Treg cells (Fig. 7E), but did not significantly change the total proportion of Treg cells (Fig. 7D), suggesting that most Treg cells in the PLN may be locally induced, while migrating α4β7+ Treg cells account for only a small fraction (see Supplementary Material Fig. S6). [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.)