sphingosine 1-phosphate (S1P)(IC50 = 0.033 nM, in K562 and NK cells); PAK1

Before surviving with NK cells, monocyte-derived immature dendritic cells (iDCs) were exposed to various doses of S1P for variable amounts of time. In these dishes, autologous or allogeneic iDCs that were exposed to 0.2–20 μM S1P for four hours considerably shielded the NK cells. For autologous iDCs and allogeneic iDCs, the IC50 values for S1P were determined to be 160 nM and 34 nM, respectively. Subsequently, fingolimod or SEW2871 at varying doses were able to counteract the inhibitory effect of S1P, with IC50 values of 173 or 15 nM, respectively [1]. It has been observed that fungiolimod causes LPA to be synthesized by preventing lysophospholipase automobility in cells. An important component of axonal development, axonal cAMP concentration, showed a strong correlation with fingolim therapy. Furthermore, fingolimod dramatically lowered the nerve center's LPA levels. Myelin thickness improvements are linked to PF-8380 therapy [2].

Fourteen days after extrusion, fingolimod therapy led to a notable increase in nerves in wild-type C57BL/6 nozzles. On the other hand, T-deficient B-deleted Foxn1-/-nozzles treated with fingolimod demonstrated enhanced nerve regeneration. Though the average rate of nerve growth in Foxn1-/- nozzles treated with Fingolimod and control Foxn1-/- nozzles suggested that T deficit affects nerve regeneration. Possibility of a role, although only Foxn1-/-treated mice significantly outperformed C57BL/6 and fared better in functional examination [2]. Ex vivo radioactive autobinding was used to quantify the amount of 18F-GE180 tracer binding that occurred after animals treated with fingolimod for 28 days. The results showed that the binding potential of 18F-GE180 was significantly reduced (P<0.0001) after treatment with fingolimod.

Detection of cytokines and chemokines release utilizing the ELISArray kits[1]
DCs or NK cells were incubated at a cell concentration of 1 × 106 cells/ml with either media or with 2 μM S1P, 10 nM SEW2871, 10 nM FTY720 or their combinations. DCs were also incubated with 1 μg/ml LPS. After 24 h incubation, the cells were harvested and the cell suspensions were centrifuged at 1,000 × g for 10 min before the supernatants were collected. Detection of the levels of various cytokines and chemokines was carried utilizing the Multi-Analyte ELISArray Kit as described by the manufacturers’ user manual. The kit analyzes the release of IL-1β, IL-4, IL-6, IL-10, IL-12, IL-17A, IFN-γ, TNF-α, TGF-β1, MCP-1, MIP-1α, and MIP-1β. Negative and positive controls supplied by the kits were also included[1].

Immature DCs were left intact or were incubated with 2 μM S1P, 10 nM FTY720 , 10 nM SEW2871 or the combinations of S1P with these drugs for 4 h. As a control 1 μg/ml LPS was used. The cells were washed and incubated in a 96-well plate (v-bottom, 2 × 105 cells per well), washed again and resuspended in PBS buffer containing 0.1% sodium azide. They were labeled with 1 μg/ml FITC-conjugated mouse anti-human CD80, 1 μg/ml FITC-conjugated mouse anti-human CD83, 1 μg/ml FITC-conjugated mouse anti-human CD86, 1 μg/ml FITC-conjugated mouse anti-human HLA-class I, 1 μg/ml FITC-conjugated mouse anti-human HLA-DR, 1 μg/ml FITC-conjugated mouse anti-human HLA-E, or 1 μg/ml FITC-conjugated mouse IgG as a control. The cells were washed twice, and examined in the flow cytometer. Markers were set according to the isotype control FITC-conjugated mouse IgG.[1]
To stain NK cells with antibodies for various NK cell activating receptors, they were either left untreated or incubated with 2 μM S1P for 4 h, washed and stained with 1 μg/ml PE-conjugated mouse anti-human NKp30 (CD337), 1 μg/ml PE-conjugated mouse anti-human NKp44 (CD336), 1 μg/ml PE-conjugated mouse anti-human NKG2D (CD314), or as a control 1 μg/ml PE-conjugated mouse IgG1, for 45 min at 4°C. NK cells were also stained with 1 μg/ml FITC-conjugated anti-killer inhibitory receptor (KIR)/CD158 antibody which recognizes KIR2DL2, KIR2DL3, KIR2DS2 and KIR2DS4, and as a control with FITC-conjugated mouse IgG. The cells were washed twice, and examined in the flow cytometer. Markers were set according to the isotype control PE-conjugated or FITC-conjugated mouse IgG.

Methods:[3]
Chronic focal experimental autoimmune encephalitis (EAE)-like lesions were induced in Lewis rats (n = 24) via stereotactic intrastriatal injection of heat-killed bacillus Calmette-Guérin (BCG) and subsequent activation using an intradermal injection of BCG in complete Freund adjuvant. This process resulted in a delayed-type hypersensitivity (DTH)-like EAE lesion. The extent of neuroinflammation surrounding the lesion was measured using (18)F-GE180 as a PET radioligand. The imaging was performed before and after treatment with fingolimod (0.3 mg/kg/d by mouth, 28 d) or vehicle as a control. In addition to imaging, autoradiography and immunohistochemistry experiments were performed to verify the in vivo results.
Results: [3]
The chronic DTH EAE lesion led to increased ligand binding in the ipsilateral, compared with contralateral, hemisphere when PET imaging was performed with the translocator protein-binding radioligand (18)F-GE180. Treatment with fingolimod led to a highly significant reduction in the binding potential, which could be demonstrated using both in vivo and ex vivo imaging (fingolimod vs. vehicle treatment, P < 0.0001). The area of increased (18)F-GE180 signal mapped closely to the area of activated microglial cells detected by immunohistochemistry.

Absorption, Distribution and Excretion
Fingolimod is absorbed slowly but effectively in the gastrointestinal tract. AUC values vary from patient to patient, and pharmacokinetic studies have shown significant variability in Fingolimod's AUC values. The Tmax of Fingolimod is 12-16 hours, and its bioavailability is 90-93%. Steady-state plasma concentrations of Fingolimod are reached within 1-2 months after a single daily dose. Approximately 81% of orally administered Fingolimod is excreted in the urine as inactive metabolites. Less than 2.5% of the dose is intact Fingolimod and its active metabolites, and these are excreted in the feces. The volume of distribution of Fingolimod is approximately 1200 ± 260 liters. Approximately 86% is distributed in red blood cells (RBCs). According to prescribing information, the plasma clearance of Fingolimod is 6.3 ± 2.3 L/h. Other data indicate a range of 6-8 L/h.

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Metabolism/Metabolites
Sphingosine kinase metabolizes fingolimod to its active metabolite, fingolimod phosphate. Fingolimod metabolism occurs primarily through three pathways: first, phosphorylation of the (S)-enantiomer of fingolimod phosphate (the pharmacologically active phosphate); second, oxidation by cytochrome P450 4F2 (CYP4F2); and third, fatty acid-like metabolism to generate various inactive metabolites. Inactive nonpolar ceramide analogs are also generated during fingolimod metabolism.

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Biological Half-Life
The half-life of fingolimod and its active metabolites is 6-9 days.

Hepatotoxicity
In large randomized controlled trials of fingolimod in patients with multiple sclerosis, 8% to 14% of patients in the fingolimod group experienced serum ALT elevations exceeding three times the upper limit of normal, compared to only 2% to 3% in the placebo group. These enzyme elevations were typically transient, asymptomatic, or without jaundice, and required discontinuation of treatment in less than 1% of cases. No cases of acute hepatitis or clinically significant liver injury were reported in the pre-registration trials of fingolimod. However, following fingolimod's approval and wider use, several cases of clinically significant liver injury attributable to fingolimod have been reported, including acute liver failure requiring urgent liver transplantation. Liver injury typically occurs within days or weeks of treatment initiation, and the pattern of liver enzyme elevation is usually hepatocellular. Furthermore, at least one case of hepatitis B surface antigen (HBsAg) inactive carrier reactivation of the hepatitis B virus has been reported. Therefore, mild to moderate transient elevations of serum enzymes during treatment are not uncommon, and clinically significant liver damage and jaundice due to fingolimod may occur. Probability Score: C (Possible cause of clinically significant liver damage and hepatitis B virus reactivation in susceptible patients). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Although fingolimod and its active metabolites are highly bound in maternal plasma and unlikely to enter breast milk in large quantities, it still poses potential toxicity to breastfed infants. Since there is currently no published experience regarding the use of fingolimod during lactation, expert opinion generally recommends avoiding its use during lactation, especially when breastfeeding newborns or premature infants. However, the manufacturer's label does not recommend contraindication for breastfeeding women. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No relevant published information was found as of the revision date.

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Protein Binding
Fingomod and its active metabolites have a protein binding rate exceeding 99.7%.

[1]. FTY720 and SEW2871 reverse the inhibitory effect of S1P on natural killer cell mediated lysis of K562 tumor cells and dendritic cells but not on cytokine release. Cancer Immunol Immunother. 2010, 59(4), 575-586.

[2]. Fingolimod promotes peripheral nerve regeneration via modulation of lysophospholipid signaling. J Neuroinflammation. 2016 Jun 10;13(1):143.

[3]. In vivo PET imaging demonstrates diminished microglial activation after fingolimod treatment in an animal model of multiple sclerosis. J Nucl Med. 2015 Feb;56(2):305-10.

[4]. Modification of lymphocyte migration to Peyer's patches by inhibition of sphingosine-1-phosphate lyase ameliorates murine colitis. J Gastroenterol Hepatol. 2018 Jan 15.

Pharmacodynamics
In multiple sclerosis, fingolimod binds to sphingosine receptors, thereby reducing associated neuroinflammation. In COVID-19, it may reduce lung inflammation and improve patient clinical outcomes. Fingolimod causes a transient decrease in heart rate and atrioventricular conduction at the beginning of treatment. It has the potential to prolong the QT interval.

C19H33NO2

307.478

307.251

C, 74.22; H, 10.82; N, 4.56; O, 10.41

162359-55-9

Fingolimod-d4;1346747-38-3;Fingolimod-d4 hydrochloride;1346604-90-7;Fingolimod hydrochloride;162359-56-0; 162359-55-9; 402615-91-2 (phosphate); 207113-62-0 (octanoic acid); 1242271-26-6 (palmitamide); 207113-64-2 (hexanoic acid)

107970

White to off-white solid powder

1.0±0.1 g/cm3

479.5±45.0 °C at 760 mmHg

102-107

243.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.531

5.25

3

3

12

22

258

0

KKGQTZUTZRNORY-UHFFFAOYSA-N

InChI=1S/C19H33NO2/c1-2-3-4-5-6-7-8-17-9-11-18(12-10-17)13-14-19(20,15-21)16-22/h9-12,21-22H,2-8,13-16,20H2,1H3

2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propandiol

FTY720 (free base); FTY720; 162359-55-9; 2-Amino-2-(4-octylphenethyl)propane-1,3-diol; 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol; Fingolimod [INN]; 2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol; Fty-720; fingolimodum; Fingolimod HCl; FTY-720; FTY 720; Trade name: Gilenia and Gilenya.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Ethanol : ~7.69 mg/mL (~25.01 mM)
DMSO : ~2 mg/mL (~6.50 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (3.25 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (3.25 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1 mg/mL (3.25 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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