The peripheral blood was examined by the Missouri University Research Animal Diagnostic Laboratory (Columbia, MO) for determination of total blood cell counts and differentials in blood samples. Statistical Analysis. Values are shown as the mean �� S.E. Data were analyzed using a sellectchem standard Student��s t test or one-way analysis of variance, groups were compared by Newman-Keuls test, and significance in all cases was defined at p < 0.05. Results Differential Effects of FTY720 Analogs on Endothelial Cell Barrier Function in Vitro. Novel (R)- and (S)-enantiomers of three FTY720 analogs (1 = phosphonate, 2 = enephosphonate, and 3 = regioisomer) were synthesized as described previously (Lu et al., 2009) (Fig. 1 for the structures of the FTY720 analogs used in this study).

Our initial studies examined the effects of these six compounds on EC barrier integrity as measured by TER, a highly sensitive in vitro measure of permeability. The (R)- and (S)-enantiomers of 1 and 2 are similar to S1P in that they produce rapid and sustained increases in TER (indicative of enhanced EC barrier function), whereas FTY720 itself induced a delayed onset of barrier enhancement as we have reported previously (Dudek et al., 2007) that was slower to rise in TER relative to S1P and the FTY720 analogs [Fig. 2A; note that only (R)-enantiomer TER data are shown. (S)-Enantiomer results are similar and, therefore, not shown for simplicity]. Interestingly, the FTY720 regioisomers 3R and 3S (in which the positions of the amino groups and one of the hydroxymethyl groups are interchanged) were barrier-disruptive at similar concentrations despite being structurally very similar to the parent FTY720 compound (Fig.

1), indicating the sensitivity of this response to minor structural alterations. Although similar to S1P in the rapid induction of increased TER, the barrier-enhancing FTY720 analogs 1R, 1S, and 2R have a greater maximal percentage TER change at 1 ��M compared with both S1P and FTY720 (Fig. 2B). Moreover, when the concentration of these compounds is increased to 10 ��M, analogs 1R, 1S, and 2R exhibit even greater maximal TER elevation, whereas S1P, FTY720, and 2S are now somewhat barrier-disruptive at this dose (Fig. 2C), indicating that the barrier-enhancing effects of analogs 1R, 1S, and 2R are sustained over a wider concentration range than those of either S1P or FTY720.

In fact, dose-response titrations of 1S, 1R, and 2R demonstrate that these analogs retain near Drug_discovery maximal barrier-promoting effects over a range from 1 to 50 ��M, suggesting a potential broader therapeutic index for these compounds compared with S1P or FTY720 (data not shown). The results also highlight the importance of enantiomer-specific effects as the enephosphonate analogs (2R and 2S) have diametrically opposing effects on EC barrier function at higher concentrations (��10 ��M). Fig. 1. Structures of FTY720 analogs.