Evaluation of Urea-Based Inhibitors of the Dopamine Transporter Using the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

Abstract

The dopaminergic system is involved in the regulation of immune responses in various homeostatic and disease conditions. For conditions such as Parkinson's disease and multiple sclerosis (MS), pharmacological modulation of dopamine (DA) system activity is thought to have therapeutic relevance, providing the basis for using dopaminergic agents as a treatment of relevant states. In particular, it was proposed that restoration of DA levels may inhibit neuroinflammation. We have recently reported a new class of dopamine transporter (DAT) inhibitors with high selectivity to the DAT over other G-protein coupled receptors tested. Here, we continue their evaluation as monoamine transporter inhibitors. Furthermore, we show that the urea-like DAT inhibitor (compound 5) has statistically significant anti-inflammatory effects and attenuates motor deficits and pain behaviors in the experimental autoimmune encephalomyelitis model mimicking clinical signs of MS. To the best of our knowledge, this is the first study reporting the beneficial effects of DAT inhibitor-based treatment in animals with induced autoimmune encephalomyelitis, and the observed results provide additional support to the model of DA-related neuroinflammation.

Keywords: dopamine; dopamine transporter inhibitor; modafinil; multiple sclerosis; neuroinflammation.

Figure 1.

Representative selected structures of known DAT inhibitors: atypical (1,2), classical (3) ligands, and allosteric modulators (4).

Figure 2.

Inhibition of [3H]-DA uptake by the DAT in the presence of tested compounds. IC₅₀ values were calculated based on at least three independent experiments (n = 4). Tested compounds showed the following inhibitory potency: compound 5 IC₅₀ 17.8 nM (95% CI < 24.8 nM; r² 0.9060); cocaine IC₅₀ 408.9 nM (95% CI: 28.3–598.6 nM; r² 0.9309); nisoxetine IC₅₀ 1.43 μM (95% CI: 0.99–2.1 μM; r² 0.9178); fluoxetine IC₅₀ 16.1 μM (95% CI: 9.8–37.5 μM; r² 0.765); GBR12909 IC₅₀ 22.5 nM (95% CI: 16.1–31.3 nM; r² 0.9284).

Figure 3.

Clinical scores of EAE mice. EAE mice treated with vehicle showed clinical deficits that began with clinical grade 1 from day 13 post-EAE induction and progressed to more severe clinical deficits clinical grades 2–3 from day 13 to day 16 post-EAE induction and clinical grades 3–4 from day 16 to day 23 post-EAE induction (***P < 0.001, F_11,176 = 5.985, one-way ANOVA, vehicle control, n = 12). A 10-day treatment with compound 5 (10 mg/kg, p.o., n = 11), (±)-modafinil (10 mg/kg, p.o., n = 6), or Solu-Medrol (50 mg/kg, p.o., n = 9) significantly reduced clinical deficits in EAE animals (++P < 0.01, +++P < 0.001, two-way ANOVA, Bonferroni post hoc test compared to vehicle control). #, ## P < 0.05–0.01, two-way ANOVA, Bonferroni post hoc test compared Solu-Medrol group to vehicle control.

Figure 4.

Effect of the treatment with DAT inhibitors (compound 5 and (±)-modafinil) or Solu-Medrol on mechanical withdrawal thresholds in EAE mice. The front left paw withdrawal thresholds were measured using an electronic von Frey plantar anesthesiometer in normal control mice (n = 8) and EAE mice treated with vehicle (n = 10), compound 5 (n = 10), Solu-Medrol (n = 9), and modafinil (n = 6), Animals in the EAE-vehicle group, compound 5 group, and modafinil group showed decreased withdraw thresholds (*, ** P < 0.05–0.01, one-way ANOVA). Solu-Medrol-treated animals showed an increased mechanical threshold (+P < 0.05, one-way ANOVA compared to the EAE-vehicle group).

Figure 5.

Effect of the treatment with DAT inhibitors (compound 5 or modafinil) or corticosteroid Solu-Medrol on audible and ultrasonic vocalizations in EAE mice. Audible and ultrasonic vocalizations were recorded in normal control mice (n = 8) and EAE mice treated with vehicle (n = 10), compound 5 (n = 10), Solu-Medrol (n = 9), and modafinil (n = 6) groups on day 24 after EAE induction. Animals in the EAE-vehicle group showed increased audible and ultrasonic vocalizations (*** P < 0.001, one-way ANOVA compared to normal control mice). Solu-Medrol-treated animals showed an increased mechanical threshold (+P < 0.05, one-way ANOVA compared to the EAE-vehicle group). The compound 5 group and modafinil group showed decreased audible and ultrasonic vocalizations (+, ++, +++ P < 0.05–0.001, one-way ANOVA compared to the vehicle-treated group). The Solu-Medrol treated group showed decreased audible vocalizations (+P < 0.05, one-way ANOVA compared to the vehicle-treated group).

Figure 6.

Effect of DAT inhibitor 5 and Solu-Medrol on the GFAP expression in amygdala of EAE mice. (A) Diagram of a coronal brain slice (2.30 mm caudal to bregma) indicates the amygdala (CeA) area shown in the confocal images (B) of GFAP expression in the CeA. C. The number of GFAP cells was significantly increased in EAE mice treated with vehicle (*** P < 0.001 compared to normal, n = 20–22 slices). A 10-day treatment with compound 5 (10 mg/kg, oral dosing) or Solu-Medrol (50 mg/kg, oral dosing) significantly decreased the number of GFAP cells (+++ P < 0.001 compared to EAE-vehicle, n = 15–20 slices). Scale bars, 100 μm.

Figure 7.

Effect of DAT inhibitor 5 and Solu-Medrol on the IL-6 expression in the amygdala in EAE mice. (A) Diagram of a coronal brain slice (2.30 mm caudal to bregma) indicates the amygdala area shown in the confocal image (B) of IL-6-IR expression in the CeA. (C) The number of IL-6-IR cells was significantly increased in EAE mice treated with vehicle (*** P < 0.001 compared to normal, n = 10–12 slices). A 10-day treatment with compound 5 (10 mg/kg, oral dosing) or Solu-Medrol (50 mg/kg, oral dosing) significantly decreased the number of IL-6-IR cells (+++ P < 0.05 compared to the EAE vehicle, n = 9–13). Scale bars, 100 μm.

Figure 8.

Effect of tested compounds on mRNA expression of pro and anti-inflammatory cytokines in EAE mice. Quantitative polymerase chain reaction (qPCR) analyses of inflammatory cytokine expression in CeA (A), spinal cord (B), and lymph node (C) cells isolated from EAE mice. Animals were treated with vehicle, DAT inhibitor 5 (10 mg/kg, p.o.), or Solu-Medrol (50 mg/kg, p.o.). All data represent mean ± SE, and P-values were calculated by ANOVA with Bonferroni post hoc tests, *,**,***P < 0.05, 0.01, and 0.001.