Figure 1.
Directional transport of Imatinib (A) and INNO-406 (B) across BBMEC monolayers.
The directional transport of two drug analogues Imatinib (A) and INNO-406 (B) were determined in BBMEC. The apical-to-basolateral transport (A→B) (open circles) and basolateral-to-apical (B→A)(closed circles). The data are presented as percent transported as a function of time (means ± SD, n = 3). * Considered statistically significant (p<0.05).
Table 1.
BBMEC Trans-Endothelial Electrical Resistance (TEER).
Table 2.
Directional Apparent Permeability Comparison of Drug Analogues.
Figure 2.
Kinetics of INNO-406 in mouse brain.
Brain levels of INNO-406 (2.5 mg/kg/i.p. or 10 mg/kg/i.p.) in C57 mice 1 h, 4 h, and 24 h after a single injection (A). Brain levels of INNO-406 (2.5 mg/kg/i.p. or 10 mg/kg/i.p. after last injection of MPTP) in C57 mice 1 h, 4 h, and 24 h after single injection in the presence or absence of MPTP (4×20 mg/kg/i.p. at 2 h interval) (B). Brain levels of MPP+ in C57 mice 1 h, 4 h, and 24 h after single injection of MPTP (60 mg/kg/i.p. single injection) in the presence or absence of a single injection of INNO-406 (2.5 mg/kg/i.p. or 10 mg/kg/i.p.) (C).
Figure 3.
The c-Abl inhibitor INNO-406 protects against MPTP-induced loss of DA neurons.
INNO-406 (10 mg/kg/i.p for one week prior to MPTP, 4×20 mg, and one week after MPTP treatment) protects (A–D) substantia nigra DA neurons and (E–H) striatal DA terminals from MPTP-induced toxicity. Pre-treatment of mice with INNO-406 leads to preservation of TH+ positive staining cells within the substantia nigra pars compacta and DA terminals in the striatum compared to MPTP treatment alone. Plots of quantitative stereological data showing the protective effect of INNO-406 in SNpc and striatum has been shown. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). SNpc = Substantia Nigra pars compacta.
Figure 4.
INNO-406 prevents dopaminergic depletion and c-Abl mediated parkin phosphorylation in mice striatum.
(A) INNO-406 prevents MPTP-induced depletion of striatal dopamine and its metabolites DOPAC and HVA in adult male C57BL/J6 mice. Animals received INNO-406 (10 mg/kg, i.p.) as a single daily injection for 7 days before MPTP injection. On day 7 mice were treated with 4×20 mg/kg, i.p. of MPTP at 2 h interval. INNO-406 injection was continued for one more week after the last injection of MPTP. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). (B) Parkin is tyrosine phosphorylated in the striatal lysates of MPTP-treated mice. Animals received INNO-406 (10 mg/kg, i.p.) as a single daily injection for 7 days before MPTP injection. On day 7 mice were treated with 4×20 mg/kg, i.p. of MPTP at 2 h interval. INNO-406 injection was continued for one more week after the last injection of MPTP. Samples were immunoprecipitated with anti-parkin antibody and were immunoblotted for anti-p-tyrosine, anti-parkin or anti-c-Abl. A 10% input samples were immunoblotted for anti-p-c-Abl, anti-parkin, anti-AIMP2, anti-c-Abl and ant-Actin. A pretreatment with INNO-406, a c-Abl kinase inhibitor, blocks oxidative stress-mediated tyrosine phosphorykation of parkin, activation of c-Abl and accumulation of toxic substrate, AIMP2, in the striatum of MPTP-treated mice. The photomicrograph shown is a representative of three repeats from samples pooled from 3 different animals. The optical density quantification data as normalized amounts is presented. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). (C) Cytotoxicity plotted as a percentage of control as measured by LDH in SH-SY5Y cells treated with MPP+ (500 µM). Some samples were incubated with 0.5 or 1.0 µM INNO-406 for 6 h before MPP+ treatment. *p<0.05. Differences among means were analyzed using one-way analysis of variance (ANOVA). All experiments were repeated at least three times and representative examples are presented.