Carboxyfullerenes as neuroprotective agents

Abstract

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Figure 1

Structures of carboxyfullerenes showing the paired carboxyl groups on the C₆₀ sphere. The three-dimensional models demonstrate the polar distribution of the carboxyl groups on C₃ and the equatorial distribution on D₃.

Figure 2

Water-soluble carboxylic C₆₀ compounds are effective free radical scavengers. EPR spectra of hydroxyl (^⋅OH) (from 100 μM H₂O₂ with 10 μM Fe²⁺ via the Fenton reaction) and superoxide O_2⨪ (from 25 μM xanthine + 10 mU/ml xanthine oxidase) radicals with 100 mM 5,5-dimethyl-1-pyrolline-N-oxide (DMPO) as the spin-trapping agent: ^⋅OH in the presence of DMPO alone, in the presence of 4 μM C₃, or in the presence of 4 μM D₃, O_2⨪ in DMPO alone, in the presence of 40 μM C₃, or in the presence of 40 μM D₃. The arrow indicates a spurious signal due to an unknown radical in the cavity. The sample was analyzed in a quartz flat cell (60 × 10 × 0.25 mm) in a Bruker 200, X-band EPR spectrometer. Settings were power, 1.6 mW; modulation, 1 G; field modulation, 100 Hz; receiver gain, 3.2 × 10⁵.

Figure 3

The C₃ isomer was a more effective neuroprotective agent than the D₃ isomer. C₃ provided better protection than D₃ against excitotoxic neuronal death induced by exposure to NMDA (200 μM for 10 min, A) or AMPA (8 μM for 24 hr, B). In all figures, the data were normalized to the injury condition (NMDA, AMPA, etc.) without C₃, and were expressed as percent of the injury without C₃. ∗, P < 0.05 vs. untreated injury condition, using ANOVA followed by Student–Newman–Keuls test for multiple comparisons. Values are mean ± SEM, n = 8–16 cultures per condition. ∗, different from excitotoxin alone at P < 0.05, using ANOVA followed by Student–Newman–Keuls test for multiple comparisons. ∗∗, different from C₃.

Figure 4

C₃-reduced neuronal cell death produced by 60 min of combined oxygen-glucose deprivation; n = 8–12 per condition. The data were normalized to oxygen-glucose deprivation without C₃ and were expressed as percent of this injury. ∗, P < 0.05 vs. oxygen-glucose deprivation without C₃, using ANOVA followed by Student–Newman–Keuls test for multiple comparisons.

Figure 5

EPR spectra of spin-labeled lipids (5- or 16-doxyl ketostearic acid) incorporated into mouse brain lipid micelles, with either C₃ or D₃. Both isomers produce a shift in the order parameter (S) of the 5-doxyl group (see Table 1), but C₃ produced a greater change in S. C₃ also altered the lipid (⇓)/aqueous (↓) partition factor, detected by 16-doxyl ketostearic acid, to a greater extent than D₃. Both results suggest that C₃ enters the lipid bilayer to a greater extent than D₃.

Figure 6

C₃ attenuated neuronal death (A) and dihydrorhodamine oxidation (B) induced by serum deprivation. Cell death was determined by manual cell counts of trypan-stained neurons 48 hr after onset of serum deprivation. ∗, P < 0.05 vs. serum deprivation, using ANOVA followed by Student–Newman–Keuls test for multiple comparisons. Values are mean ± SEM, n = 4–8 per condition. Figure is representative of two additional replicates. Confocal images (B) of cortical neurons showing Nomarski images (Upper) of neurons before (CTL) and 8 hr after the onset of serum deprivation (SD). Neurons in the SD condition demonstrate typical apoptotic features, including membrane blebbing and condensation of nuclear contents. (Lower) Concurrent photomicrographs show increased fluorescence due to oxidation of preloaded, nonfluorescent dihydrorhodamine to fluorescent rhodamine 123. Rhodamine fluorescence is quantified with a linear pseudocolor scale corresponding to arbitrary fluorescence intensity units.

Figure 7

C₃-blocked neurodegeneration produced by application Aβ_1–42, (20 μM). Manual cell counts of trypan 48 hr after application of Aβ_1–42 were graphed as mean ± SEM, n = 10–16 per condition.

Figure 8

Survival curves for FALS mice treated with continuous intraperitoneal infusion of saline or 15 mg/kg per day C₃ carboxyfullerene via implanted mini-osmotic pumps for 2 months, starting at age 2 months. Animals treated with carboxyfullerene had increased survival (P = 0.023 by t test). Three surgical deaths occurred during pump implantation, two in the C₃-treated group (after 1 month of treatment, before symptom onset), and one in the control group (with initial pump placement). All procedures conformed to Washington University institutional guidelines for animal welfare.