Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application

Abstract

FTY720 (Fingolimod; Gilenya ® ) is an immunemodulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1-and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1-and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Fingolimod targeting protein phosphatase 2A differently affects IL‐33 induced IL‐2 and IFN‐γ production in CD8+ lymphocytes

“…To analyze if FTY720 or FTY720‐P have additional intracellular targets besides the extracellular S1P receptors, we used SphK2 −/− splenocytes. FTY720 is predominantly phosphorylated to FTY720‐P by SphK2, which is prevented in SphK2 −/− splenocytes . As expected, SphK1 but not SphK2 mRNA expression was detectable by qRT‐PCR in SphK2 −/− splenocytes, confirming the complete knockout of the enzyme (Fig.…”

“…These results indicate that the effect of FTY720 is conserved for IFN‐ γ and not dependent on the stimulus. Nonphosphorylated FTY720 will not reach high nanomolar concentrations in the blood of MS patients, but in a previous study we analyzed the subcellular distribution of FTY720 and FTY720‐P in immune cells of the spleen and showed that both substances dramatically accumulate in these splenocytes several 100‐fold . Thus to outline consequences of our data, the inhibition of IFN‐ γ by FTY720 could be beneficial but on the other hand bear additional risks.…”

Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion

“…The stimulatory effect of FTY720 on synaptobrevin was observed when the drug reaches micromolar range. Pertinently, a study of intracellular accumulation of FTY720 demonstrated that micromolar levels are easily achievable 50 . Using LC-MS mass-spectrometry method the authors demonstrated that the lipophilic FTY720 accumulated several hundredfold inside cells reaching micromolar concentrations.…”

Fingolimod (Gilenya®) in multiple sclerosis: bradycardia, atrioventricular blocks, and mild effect on the QTc interval. Something to do with the L‐type calcium channel?

“…Similarly, Yagi et al have demonstrated that in guinea‐pig hearts, concentration of fingolimod was 7.5 times higher than in plasma, while that of fingolimod‐phosphate was six times higher in the heart, compared to plasma. Furthermore, Schröder et al have recently shown in mouse splenocytes that an extracellular concentration of fingolimod‐phosphate of 144 nmol/L corresponds to an intracellular concentration of 49.796 μmol/L, a dramatic 346‐fold elevation. Therefore, our choice to use intrapipette concentrations of fingolimod‐phosphate in the nanomolar range (100 and 500 nmol/L) to evaluate the calcium channel‐blocking effect of the drug in tsA cells appears fairly reasonable.…”