Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain

Abstract

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, (3)H-verapamil (threefold increase), (3)H-loperamide (fivefold increase), and (3)H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain (14)C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.

Fig. 1.

TNF-α and dPPA act through sphingolipids to reduce luminal P-glycoprotein transport activity in isolated rat brain capillaries. (A) Representative confocal images of brain capillaries after a 60-min incubation with 2 μM NBD-CSA; note the high luminal fluorescence in the control capillary and decreased luminal fluorescence in capillaries exposed to 5 μM PSC833. (Scale bars, 10 μm.) (B) TNF-α acts through SKI, S1PR1, to reduce P-glycoprotein activity. (C) dPPA, a PKCβ1 agonist, acts through SK and S1PR1 to reduce P-glycoprotein transport activity. (D) S1P concentration-dependence of P-glycoprotein–mediated transport. (E) Rapid time course of S1P action. Capillaries were incubated to steady-state (60 min) in medium with 2 μM NBD-CSA. Then 1 μM S1P was added to the medium (time 0 on graph); 30 min later, capillaries were washed and S1P-free medium was added. Each bar represents the mean value for 12–16 capillaries from a single preparation (pooled tissue from five to seven rats); variability is shown as SE bars. Units are arbitrary fluorescence. Statistical comparisons: ***significantly lower than control, P < 0.001.

Fig. 2.

S1PR expression and function in rat brain capillaries. (A) RT-PCR using mRNA isolated from brain capillaries. (Lane 1) 1 kb molecular weight marker; (lane 2) S1PR1; (lane 3) S1PR2; (lane 4) S1PR3; (lane 5) S1PR4; (lane 6) S1PR5. (B) Representative confocal images (magnification, 40×) of capillaries immunostained for S1PR1 and S1PR3. (C) S1PR1 antagonists block the action of S1P on P-glycoprotein transport activity (black bars); an S1PR1 agonist reduces transport activity (gray bars). (D) Sphingosine-induced reduction in P-glycoprotein activity is S1PR1-dependent. (E) Sphingosine reduction in P-glycoprotein activity is SK-dependent. (F) Sphingosine effects on transport depend on a Mrp. Each bar represents the mean value for 8–15 capillaries from a single preparation (pooled tissue from three to six rats); variability is shown as SE bars. Units are arbitrary fluorescence. Statistical comparisons: ***significantly lower than control, P < 0.001. B, basolateral membrane; L, luminal membrane.

Fig. 3.

Fingolimod (FTY720) and its active metabolite, FTY720P, reduce P-glycoprotein transport activity. (A) FTY720 concentration dependence, (B) FTY720P concentration dependence, (C) FTY720 time course of action, (D) FTY720P time course of action. For the time courses, capillaries were loaded to steady state (60 min) in medium with 2 μM NBD-CSA. Then 1 μM FTY720 or FTY720P was added to the medium (time 0 on graph). (E) FTY720 and FTY720P require SK and S1PR1 to reduce P-glycoprotein activity. Each point represents the mean value for 10–15 capillaries from a single preparation (pooled tissue from 5 to 10 rats); variability is shown as SE bars. Units are arbitrary fluorescence. Extended signaling pathway through which TNF-α rapidly reduces basal P-glycoprotein transport activity. Statistical comparisons: ***significantly lower than control, P < 0.001. (F) Agents that activate signaling at various points in the pathway are shown in red. For details on events upstream of S1PR1, see refs. , , and .

Fig. 4.

S1P, FTY720, and FTY720P reduce P-glycoprotein activity (increased brain accumulation of [³H]-verapamil, [³H]-loperamide, and [³H]-paclitaxel) in vivo. (A) Concentration-dependence for S1P-induced increase in brain [³H]-verapamil accumulation. Estimated EC₅₀ from the curve fit is 230 nM. (B) S1P effects on [³H]-verapamil accumulation are blocked by S1PR1 receptor antagonists: VPC is selective for S1PR1 and S1PR3, W146 is selective for the S1PR1. (C) FTY720 and FTY720P reduce P-glycoprotein transport activity in vivo (increased [³H]-verapamil accumulation). (D) FTY720 increases brain accumulation of [³H]-loperamide. (E) FTY720 increases brain accumulation of [³H]-pacitaxel. Results are expressed as the ratio of disintegrations per minute in the brain to disintegrations per minute in the perfusate (R_br μL/g). Each point or bar represents the mean value from four to eight rats; variability is shown as SE bars. Significant differences determined using one-way ANOVA with a Neuman-Keuls post hoc test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; ^##P < 0.01, ^###P < 0.001 vs. S1P (10 μM).