Comparison of AKP-11 and FTY720 on reversal of lymphopenia in control and EAE animals with different drug treatments followed by withdrawal for 48hrs.

<p>Control and EAE animals were treated with AKP-11 or FTY720 for 14 days and blood lymphocyte subsets were analyzed after 48hrs of discontinue of drug treatment. (A) Total lymphocytes, (B) Total T lymphocytes, (C) Total CD4⁺T lymphocytes, (D) Total CD8⁺T lymphocytes, (E) Total CD62L⁺T lymphocytes populations were reversed to normal levels only in AKP-11 not in FTY720 treated animals. (Total lymphocytes 100% of control 2800 cells/μL, T cells 100% of control 2000 cells/μL, CD4⁺T cells 100% of control 1400 cells/μL and CD8⁺T cells 100% of control 600 cells/μL and homing receptor CD62L⁺T cells 100% of control 1800 cells/μL). Data represents mean ± SEM of three independent experiments (6 animals per group). Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001.</p

AKP-11 and FTY720 treatments attenuate EAE disease in the Lewis rat.

<p>(A-B) EAE was induced in female Lewis rats with guinea pig MBP (25μg/rat). EAE developed rats were divided into 3 groups on day 11 or 12 after immunization and administered vehicle or AKP-11 (3 or 1.3 mg/kg) or FTY720 (1 mg/kg) orally every-day until day 26. Clinical scores: 0, no clinical signs; limp tail or waddling gait with tail tonicity, 1; waddling gait with limp tail (ataxia), 2; ataxia with partial limb paralysis, 2.5; full paralysis of one limb, 3; full paralysis of one limb with partial paralysis of second limb, 3.5; full paralysis of two limbs, 4; moribund stage, 4.5; and death, 5. Data represents mean ± SEM of three independent experiments (6 animals per group). Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001.</p

AKP-11 increases AKT and ERK activation through S1P1 receptor signaling.

<p>(A-C) CHO-S1P1-HA stable cells were stimulated with 100nM AKP-11 or FTY720, or FTY720P at different time points. (5 min, 30 min and 60 min) and were subjected to western analyses of phospho and total AKT and ERK proteins were quantitated. Data represents mean ± SEM of three independent experiments. Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001, NS- not significant.</p

AKP-11 withdrawal increases cell surface S1P1 receptor expression.

<p>(A-B) CHO cells expressing S1P1-HA were pretreated with cycloheximide (15μg/ml) for 30 min to block the synthesis of new S1P1 and then stimulated with 100 nM AKP-11 or FTY720 or FTY720P for 1 hr. The above compounds were washed out and replenished with fresh serum free medium containing 0.5% fatty acid free BSA and cycloheximide and incubated for 2 or 24 hrs. Data represents mean ± SEM of three independent experiments. Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001, NS- not significant.</p

AKP-11 and FTY720 reduce PBL count in rats during EAE.

<p>Control and EAE animals were treated with AKP-11 (1.3mg/kg) or FTY720 (1mg/kg) for 7 days and blood lymphocyte subsets were analyzed by flow cytometry after 24 hrs. of last drug treatment. (A) Total lymphocytes, (B) Total T lymphocytes, (C) Total CD4⁺T lymphocytes, (D) Total CD8⁺T lymphocytes (E) Total CD62L⁺T lymphocytes populations were reduced in AKP-11 and FTY720 treated animals compared to vehicle control animals. (Total lymphocytes 100% of control 2800 cells/μL, T cells 100% of control 2000 cells/μL, CD4⁺T cells 100% of control 1400 cells/μL, CD8⁺T cells 100% of control 600 cells/μL and homing receptor CD62L⁺T cells 100% of control 1800 cells/μL). Data represents mean ± SEM of three independent experiments (6 animals per group). Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001.</p

AKP-11 mediated S1P1 down-regulation is Sphingosine kinase independent and AKP-11 induces less ubiquitinylation.

<p>(A-B) 10μM of SPKII, a dual sphingosine kinase inhibitor treated in CHO-S1P1-HA stable cells for 30 min. After that, the cells were stimulated with 100 nM AKP-11 or FTY720, or FTY720P for 1 hr. Biotinylated cell surface S1P1 HA proteins were immunoblotted and quantitated. (C) CHO-S1P1-HA stable cells were pretreated 20μM MG132 for 2hrs and then stimulated with 1000 nM AKP-11 or FTY720, or FTY720P for 1hr. Cell lysates were immunoprecipitated with HA antibody and ubiquitination of S1P1 was detected by western blotting with ubiquitin antibody. The membrane was re-probed with HA antibody. (D) At the same time input lysates were detected by direct western blot with S1P1 HA antibody. Data represents mean ± SEM of three independent experiments. Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001, NS- not significant.</p

Effect of AKP-11 and FTY720 on lung vascular permeability and heart rate.

<p>(A-B) AKP-11 (1.3mg/kg) and FTY720 (1mg/kg) were orally administered. After 24hrs, Evans blue dye (EBD) was injected through tail vein and after 2hrs the animals were perfused with saline and lungs were photographed. EBD was measured in the lungs after extraction in the dimethylformamide solution. (C-D) Heart rate and blood pressure were measured at 0,1,2,4,6,12,and 24hr post oral administration of vehicle, AKP-11 (1.3mg/kg) and FTY720 (1mg/kg). Data represents mean ± SEM of three independent experiments (6 animals per group). Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001, NS- not significant.</p

AKP-11 and FTY720 treatments reduce PBL count.

<p>Control animals were treated with a single dose of AKP-11 (1.3mg/kg) or FTY720 (1mg/kg) and blood lymphocyte counts were analyzed by flow cytometry after 24 hrs. (A) Total lymphocytes, (B) Total T lymphocytes, (C) Total CD4⁺T lymphocytes, (D) Total CD8⁺T lymphocytes populations were reduced in AKP-11 and FTY720 treated animals compared to vehicle control animals. (Total lymphocytes 100% of control 2800 cells/μL, T cells 100% of control 2000 cells/ ul, CD4⁺T cells 100% of control 1400 cells/ μL, CD8⁺T cells 100% of control 600 cells/μL). Data represents mean ± SEM of three independent experiments (6 animals per group). Statistical significance is indicated as *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001.</p