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Figure 4 In Vivo Conversion from THI to A6770 Show full caption (A–C) THI was orally administered to rats at 50 mg/kg under a fed condition, with or without pre-administration of antibiotics. At the indicated time points, plasma concentrations of THI (A) or A6770 (B) were measured by LC-MS/MS, and the peripheral lymphocyte number was counted via a hematology system (C). The relative lymphocyte number compared with vehicle treatment was calculated at each point. Data are presented as mean ± SD (n = 3 rats per group).

All the results obtained so far brought to us the expectation that THI might exert its biological effects through the conversion to A6770, which is subsequently phosphorylated into A6770-P and inhibits S1PL in cells. To investigate this possibility, we analyzed the pharmacokinetics of A6770 as a surrogate of A6770-P as well as the pharmacokinetics of THI, in parallel with a transition of the peripheral lymphocyte number after oral administration of THI to rats. As expected, A6770 was definitely detected in plasma and showed a slow appearance with a peak at ≥7 hr after THI administration ( Figure 4 B ). The tendency of the time-dependent transition was consistent with that of the lymphopenic effect, although the peak plasma concentrations of THI were observed approximately 1 hr after dosing, as with previous reports ( Figures 4 A and 4C) (). The detected concentrations of A6770 (<0.32 μg/ml) were extremely low compared with those of THI administered, but it could be speculated that lymphopenia occurs even if the plasma A6770 are at such a low level. This is because 1 mg/kg dosing of A6770 itself resulted in a plasma concentration (≤0.32 μg/ml) similar to that above, while a >30% reduction in lymphocyte number was observed ( Figures 1 B and 1C). In addition, the appearance of A6770 after THI administration was also observed in cynomolgus monkeys ( Figure S4 ). With respect to the marked discrepancy between the plasma THI concentration and the pharmacological response, another group speculated that the building up of the lymphoid S1P concentration following inhibition of the S1PL may be a time-consuming step (). However, our findings raise the novel hypotheses that (1) conversion to A6770 and subsequent phosphorylation into A6770-P are essential for THI to exert its biological effect, and (2) the slow appearance of A6770 in plasma is a major cause of the unsynchronized pharmacokinetic/pharmacodynamic relationship of THI. If these suppositions are true, the question remains as to what prevails in the in vivo conversion route from THI into A6770. Our preceding data suggest that some kind of enterobacterium is likely to be involved in this conversion, because pre-treatment of antibiotics prior to the THI administration canceled both the A6770 appearance in plasma and the lymphopenic effect, without affecting the transition of the plasma concentration of THI ( Figures 4 A–4C). Taking together this finding and the fact that THI causes lymphopenia in rats even via parenteral administrations (), it is speculated that THI undergoes enterohepatic circulation, which could be the reason for the slow appearance of A6770 in plasma. On the other hand, no evidence for the metabolism of THI was obtained in the past study in which [C]THI was orally administered to rats (). We estimate that (1) the slow appearance of A6770, (2) low conversion efficacy from THI to A6770, and (3) the detection method of low sensitivity (thin-layer chromatography [TLC] and autoradiography) could be the reason for this inconsistency, but further verification studies will be needed to fully address the THI-to-A6770 conversion mechanism.