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. 2013 Oct 16;4(10):1382-92.
doi: 10.1021/cn400106n. Epub 2013 Aug 20.

D-512 and D-440 as novel multifunctional dopamine agonists: characterization of neuroprotection properties and evaluation of in vivo efficacy in a Parkinson's disease animal model

Soumava Santra  1 , Liping Xu, Mrudang Shah, Mark Johnson, Aloke Dutta
Affiliations

D-512 and D-440 as novel multifunctional dopamine agonists: characterization of neuroprotection properties and evaluation of in vivo efficacy in a Parkinson's disease animal model

Soumava Santra et al. ACS Chem Neurosci. .

Abstract

In this article, we have demonstrated the in vivo efficacy of D-512 and D-440 in a 6-OHDA-induced unilaterally lesioned rat model experiment, a Parkinson's disease animal model. D-512 is a novel highly potent D2/D3 agonist, and D-440 is a novel highly selective D3 agonist. We evaluated the neuroprotective properties of D-512 and D-440 in the dopaminergic MN9D cells. Cotreatment of these two drugs with 6-OHDA and MPP+ significantly attenuated and reversed 6-OHDA- and MPP+-induced toxicity in a dose-dependent manner in the dopaminergic MN9D cells. The inhibition of caspase 3/7 and lipid peroxidation activities along with the restoration of tyrosine hydroxylase levels by D-512 in 6-OHDA-treated cells may partially explain the mechanism of its neuroprotective property. Furthermore, studies were carried out to elucidate the time-dependent changes in the pERK1/2 and pAkt, two kinases implicated in cell survival and apoptosis, levels upon treatment with 6-OHDA in presence of D-512. The neuroprotective property exhibited by these drugs was independent of their dopamine-agonist activity, which is consistent with our multifunctional drug-development approach that is focused on the generation of disease-modifying symptomatic-treatment agents for Parkinson's disease.

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Figures

Figure 1
Figure 1
Molecular structures of D-512 and D-440.
Figure 2
Figure 2
Effect on turning behavior of D-512, D-440, and ropinirole in 6-OHDA-induced unilaterally lesioned rats studied over 7.5 h. Each point is the mean ± SEM of four rats. All drugs were administered i.p. immediately before counting the rotational activity. (A) For three doses of D-512, one-way ANOVA analysis demonstrated a significant effect among the treatments, F(5, 95) = 13.74 (P < 0.0001). Dunnett’s analysis shows that the effect of three doses (2.5, 5, and 10 μMol/kg) of D-512 are significantly different compared to that of the vehicle (P < 0.01). (B) For two doses of D-440, one-way ANOVA analysis demonstrated a significant effect among the treatments, F(4, 95) = 27.49 (P < 0.0001). Dunnett’s analysis shows that the effect of two doses (5 and 10 μMol/kg) of D-440 are significantly different compared to that of the vehicle (P < 0.01).
Figure 3
Figure 3
Dose-dependent effect of the combination of a pretreatment followed by the cotreatment of D-512, D-440, ropinirole, and N-acetyl cystine (NAC) with 75 μM 6-OHDA on the cell viability of MN9D cells from toxicity caused by 75 μM 6-OHDA. (A, B) Dose-dependent effect of D-440 and D-512 on cell viability. (C) MN9D cells were treated with different concentrations of 6-OHDA (25–125 μM). (D–G) MN9D cells were pretreated with different doses of D-440, D-512, ropinirole, and NAC for 1 h followed by cotreatment with 75 μM 6-OHDA for 24 h. The values shown are the mean ± SD of three independent experiments performed with 4–6 replicates. One-way ANOVA analysis followed by Tukey’s multiple comparison posthoc test was performed. (**p < 0.001 compared to the 6-OHDA group and ##p < 0.001 compared to the control group).
Figure 4
Figure 4
Dose-dependent effect of the combination of a pretreatment followed by the cotreatment of D-512, D-440, and ropinirole with 100 μM MPP+ on the viability of MN9D cells from toxicity caused by 100 μM MPP+. (A) MN9D cells were treated with different concentration of MPP+ (25–125 μM). (B–D) MN9D cells were pretreated with different doses of D-440, D-512, and ropinirole for 1 h followed by cotreatment with 100 μM MPP+ for 24 h. The values shown are the mean ± SD of three independent experiments performed with 4–6 replicates. One-way ANOVA analysis followed by Tukey’s multiple comparison posthoc test was performed. (**p < 0.001 compared to the MPP+ group and ##p < 0.001 compared to the control group).
Figure 5
Figure 5
Morphological changes of MN9D cells treated with control, 75 μM 6-OHDA (24 h), 20 μM D-512 + 75 μM 6-OHDA (1 h pretreatment/24 cotreatment), 100 μM MPP+ (24 h), 20 μM D-512 + 100 μM MPP+ (1 h pretreatment/24 cotreatment), and 20 μM D-512 (24 h) detected by light microscopy at 20× magnification.
Figure 6
Figure 6
Effects of pretreatment of different concentrations of D-512 on the production of thiobarbituric acid reactive substances induced by sodium nitroprusside (SNP, 300 μM). The data from three different experiments are presented as the mean ± SEM. The control data represents the group that was not treated with SNP and is represented as 100% with respect to the other groups. One-way ANOVA analysis followed by Bonferroni’s multiple comparison posthoc test was performed. (##p < 0.001 compared to the control group and **p < 0.001 compared to the SNP + drug group).
Figure 7
Figure 7
Dose-dependent inhibition of caspase 3 activity in 6-OHDA-treated MN9D cells by D-512. The values shown are the mean ± SD of three independent experiments performed with 4–6 replicates. One-way ANOVA analysis followed by Tukey’s multiple comparison posthoc test was performed. (##p < 0.01 control compared to the 6-OHDA alone group and ***p < 0.01 6-OHDA alone group compared to the 6-OHDA + D-512 groups).
Figure 8
Figure 8
Effect of D-512 on 6-OHDA-induced nuclear morphology changes. Apoptotic nuclei were visualized Hoechst 33342 staining. Apoptotic cells are indicated by arrows. The scale bar is 200 μm.
Figure 9
Figure 9
TH protein level was determined by Western blot analysis. MN9D cells were pretreated with 20 μM D-512 for 1 h and co-treated with 75 μM 6-OHDA for 24 h. MN9D cells were also treated with control vehicle, D-512 and 6-OHDA alone. For quantification purpose protein level was normalized with respect to GAPDH protein. The values shown are means ± SDs of three independent experiments performed in triplicate. One way ANOVA analysis followed by Tukey’s Multiple Comparison post hoc test were performed. (**p < 0.05 control compared to the 6-OHDA group. **p < 0.05 6-OHDA compared to the D512 + 6OHDA group).
Figure 10
Figure 10
Determination of the activities of ERK and Akt by Western blot analysis. MN9D cells were pretreated with 10 μM D512 for 1 h followed by cotreatment with 75 μM 6-OHDA separately for 0.25, 2, and 4 h. GAPDH was used as an internal control protein. For quantification purposes, the protein level was normalized with respect to either the total Akt or ERK proteins. The values shown are the mean ± SD of three independent experiments performed in triplicate. One-way ANOVA analysis followed by Tukey’s multiple comparison posthoc test was performed.
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