Pre-clinical therapeutic development of a series of metalloporphyrins for Parkinson's disease

Abstract

Reactive oxygen species are a well-defined therapeutic target for Parkinson's disease (PD) and pharmacological agents that catalytically scavenge reactive species are promising neuroprotective strategies for treatment. Metalloporphyrins are synthetic catalytic antioxidants that mimic the body's own antioxidant enzymes i.e. superoxide dismutases and catalase. The goal of this study was to determine if newly designed metalloporphyrins have enhanced pharmacodynamics including oral bioavailability, longer plasma elimination half-lives, penetrate the blood brain barrier, and show promise for PD treatment. Three metalloporphyrins (AEOL 11216, AEOL 11203 and AEOL 11114) were identified in this study as potential candidates for further pre-clinical development. Each of these compounds demonstrated blood brain barrier permeability by the i.p. route and two of three compounds (AEOL 11203 and AEOL 11114) were orally bioavailable. All of these compounds protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity, including dopamine depletion in the striatum, dopaminergic neuronal loss in the substantial nigra, and increased oxidative/nitrative stress indices (glutathione disulfide and 3-nitrotyrosine) in the ventral midbrain of the mice without inhibiting MPTP metabolism. Daily therapeutic dosing of these metalloporphyrins were well tolerated without accumulation of brain manganese levels or behavioral alterations assessed by open field and rotarod tests. The study identified two orally active metalloporphyrins and one injectable metalloporphyrin as clinical candidates for further development in PD.

Keywords: Antioxidants; Dopamine; Drug development; MPTP; Oxidative stress; Pharmacokinetic analysis.

Figure 1. Structures of Metalloporphyrins

Structures of AEOL 11203, AEOL 11215, AEOL 11216, AEOL 11114, AEOL 11209 and AEOL 11207.

Figure 2. AEOL 11209, AEOL 11215 and AEOL 11216 Pharmacokinetic Analysis

The concentration of AEOL 11209 in plasma (A) and brain (B), AEOL 11215 in plasma (C) and AEOL 11216 in plasma (D) and brain (E) of C57BL/6 mice at different times points after a single dose of 15mg/kg administered by i.p or p.o. route. Points represent mean + S.E.M. Each point is the average of 3–4 animals.

Figure 3. AEOL 11203 and AEOL 11114 Pharmacokinetic Analysis

The concentrations of AEOL 11203 in plasma (A) and brain (B), AEOL 11114 in plasma (C) and brain (D) of the C57BL/6 mice at different times points after a single dose (15mg/kg) administered by the i.p. and p.o. route. Points represent mean + S.E.M. Each point is the average of 3–4 animals.

Figure 4. Daily therapeutic dosing of the metalloporphyrins without accumulation of brain manganese levels

Manganese levels in brains of mice at 7 days after injection of vehicle or 15 mg/kg, i.p. daily injections of MnCl₂, AEOL 11114, AEOL 11207 or AEOL 11203. Bars represent mean + S.E.M., *p< 0.01 vs. control. One-way, ANOVA, n=6 mice per group.

Figure 5. Daily therapeutic dosing of the metalloporphyrins without behavioral alterations

Total moving distance (A), moving velocity (B) in 5 minutes measured by open field test and latency (seconds) from acceleration to fall in the rotarod (C) in mice 7 days of vehicle injection or 15 mg/kg, i.p. daily injections of MnCl₂, AEOL 11114, AEOL 11207 or AEOL 11203 i.p. (15mg/kg, daily). Bars represent mean + S.E.M. *p< 0.01 vs. control. One-way, ANOVA, n=6–8 mice per group.

Figure 6. The metalloporphyrins attenuated MPTP-induced dopamine depletion

Dopamine levels in the striata of C57BL/6 mice 3 days after injection of MPTP (15mg/kg×3, s.c., 24h intervals) alone or in the presence of AEOL 11114 (A) or AEOL 11203 (B) 15 mg/kg i.p or p.o. (1 h prior to MPTP and daily thereafter×3). Bars represent mean + S.E.M, n=3–7 mice per group. Statistical analysis: Two-way, ANOVA, Figure (A): interaction [p=0.200], Bonferroni posttests, #p<0.05 MPTP+vehicle versus MPTP+AEOL 11114 i.p and p.o.; (B): interaction [p=0.257], Bonferroni posttests, #p<0.05 MPTP+vehicle versus MPTP+AEOL 11203 i.p.

Figure 7. The metalloporphyrins attenuated MPTP-induced dopaminergic neuronal loss

The number of dopamine neurons in the substantia nigra pars compacta of C57BL/6 mice 7 days after injection of MPTP (15mg/kg×3, s.c., 24h intervals) alone or in the presence of AEOL 11114 (A) and AEOL 11203(B) 15 mg/kg i.p or p.o. (1 h prior to MPTP and daily thereafter×3). Bars represent mean + S.E.M, n=3–6 mice per group. Statistical analysis: Two-way, ANOVA, (A) interaction [*p<0.05], Bonferroni posttests, #p<0.01 MPTP+vehicle versus MPTP+AEOL 11114 i.p. and p.o.; (B): interaction [*p<0.05], Bonferroni posttests, #p<0.01 MPTP+vehicle versus MPTP+AEOL 11203 i.p. and p.o.

Figure 8. The metalloporphyrins attenuated MPTP-induced oxidative/nitrative makers

GSH, GSSG, GSH/GSSG and 3-NT in the ventral midbrain of the C57BL/6 mice 24 hours after injection of MPTP (15mg/kg×3, s.c., daily) alone or in the presence of AEOL 11114 (A) and AEOL 11203 (B) with i.p. or p.o. route (15mg/kg×3, daily). Bars represent mean + S.E.M, n=3–7 mice per group. Statistical analysis: Two-way, ANOVA, (A) interaction: GSH [p=0.061]; GSSG [*p<0.05]; GSH/GSSG [p=0.059]; 3-NT [*p<0.01], Bonferroni posttests, GSH: #p<0.05 MPTP+vehicle versus MPTP+AEOL 11114 i.p., GSSG: #p<0.001 MPTP+vehicle versus MPTP+AEOL 11114 i.p. and p.o.; GSH/GSSG: #p<0.01 MPTP+vehicle versus MPTP+AEOL 11114 i.p, 3-NT: #p<0.001 MPTP+vehicle versus MPTP+AEOL 11114 i.p. and p.o.; (B) interaction: GSH [p=0.053]; GSSG [*p<0.01]; GSH/GSSG [p=0.062]; 3-NT [*p<0.01], Bonferroni posttests, GSH: #p<0.001 MPTP+vehicle versus MPTP+AEOL 11203 i.p., GSSG: #p<0.001 MPTP+vehicle versus MPTP+AEOL 11203 i.p. and p.o.; GSH/GSSG: #p<0.01 MPTP+vehicle versus MPTP+AEOL 11203 i.p, 3-NT: #p<0.001 MPTP+vehicle versus MPTP+AEOL 11203 i.p. and p.o.