Table 1.
Mass Spectrometer Settings Used for Metabolite Profiling of FTY720-C2 and FTY720-Mitoxy.
Table 2.
Pharmacokinetic Study Design for FTY720-C2 and FTY720-Mitoxy.
Table 3.
Intrinsic Clearance in Liver Microsomes.
Table 4.
Summary of the structural characterization by LC-MS/MS of FTY720-C2 Metabolites Identified in Rat and Human Hepatocytes.
Table 5.
Summary of the structural characterization by LC-MS/MS of FTY720-Mitoxy Metabolites Identified in Rat and Human Hepatocytes.
Fig 1.
Representative LC/MRM Chromatogram of FTY720-C2 Metabolite Profile.
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.
Fig 2.
Proposed Fragmentation Schemes and Mass Spectra for FTY720-C2 and its Metabolites.
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 H2O and an N-acetyl group from [M+H]+, red product ions indicating hydroxylation, and green product ions associated with identified carboxylic acid modifications.
Fig 3.
Representative LC/MRM Chromatogram of FTY720-Mitoxy Metabolite Profile.
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.
Fig 4.
Proposed Fragmentation Scheme and Mass Spectrum for FTY720-Mitoxy and its Metabolites.
Mass spectrometry of FTY720-Mitoxy and metabolites detected [M+] molecular ions. Blue text and arrows indicate losses of H2O from [M]+. The ion with a hydroxyl group is shown in red. Ions including a carboxylic acid are shown in green.
Table 6.
Relative Percent Distribution of FTY702-C2 and FTY720-Mitoxy.
Fig 5.
Plasma and Brain FTY720-C2 and FTY720-Mitoxy concentration profiles.
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.
Fig 6.
PP2A Activity in Mouse Adrenal Gland after Intravenous or Oral Dosing with FTY720-C2 or FTY720-Mitoxy.
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.