Carboxyfullerenes as neuroprotective agents

L Dugan

D Turetsky

C Du

D Lobner

Departments of ^{Neurology and}^{Occupational Therapy, and the}^{Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, MO 63110;}^{Department of Chemistry, National Taiwan University, Taipei, Taiwan 106, People’s Republic of China; and}^{Department of Chemistry, Washington University, St. Louis, MO 63130}

^{D.W.C. and T.-S.L. contributed equally to this work.}

Communicated by S. I. Weissman, Washington University, St. Louis, MO

Received 1997 Mar 20; Accepted 1997 Jun 6.

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), α-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 μM, 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 Aβ_1–42 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.

Abstract

Since their discovery in 1985, the pure carbon spheres of C₆₀ (buckminsterfullerene) have generated increasing interest from many different branches of science and engineering, culminating in presentation of the 1996 Nobel Prize in Chemistry to Kroto, Smalley, and Curl for their identification of these unique molecules. Subsequently, investigation into the chemical and physical properties of C₆₀ (and larger fullerenes) has yielded an extensive amount of information about C₆₀, including its avid reactivity with free radicals (1). Buckminsterfullerenes, for example, are capable of adding multiple radicals per molecule; the addition of as many as 34 methyl radicals to a single C₆₀ sphere has been reported, leading Krusic et al. (1) to characterize C₆₀ as a “radical sponge.” However, native C₆₀ is soluble in only a limited number of organic solvents, such as toluene or benzene. We have been interested in the possibility that the potent innate antioxidant properties of C₆₀ could be harnessed for use in biological systems by adding functional groups aimed at enhancing its water solubility.

Glutamate receptor-mediated excitotoxicity has been implicated in the pathogenesis of neuronal loss in the central nervous system in several disease states, including hypoxia-ischemia, epilepsy, and trauma (2–5). Oxygen or nitric oxide radicals are produced as a consequence of glutamate receptor overstimulation (6–10), and free radical scavengers have been shown to attenuate, but not to block, excitotoxic neuronal death (11–15). We recently reported promising neuroprotective effects of antioxidant polyhydroxylated derivatives of C₆₀ on cultured cortical neurons (16). However, further testing revealed considerable synthesis lot-to-lot variability in both water solubility and biological effects, presumably reflecting uncontrolled differences in the number and location of hydroxyl and hemiketal moieties ending up on the C₆₀ shell. To refine this strategy, we have turned to malonic acid derivatives of C₆₀, (C₆₃((COOH)₂)₃), synthesized and purified as two specific regioisomers with C₃ and D₃ symmetry (Fig. (Fig.1)1) and demonstrated that they are effective neuroprotective antioxidants in vitro and in vivo. Although a recent commentary in Science by C. Holden (17) suggested that, “Buckyballs have not lived up to their early promise… . (in applications),” our findings suggest that water-soluble derivatives of C₆₀ may have a novel application as neuroprotective agents.

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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.

Acknowledgments

We thank Scott Schweikart for excellent technical assistance with the FALS mice. This work was supported by National Institutes of Health Grants NS 32636 (L.L.D.), NS 30337 (D.W.C.), and NS 31248 (Teepu Siddique), a grant from Hoffman La Roche (L.L.D., T.-S.L., and D.W.C.), and from the National Science Council of Taiwan (T.-Y.L.).

Acknowledgments

ABBREVIATIONS

NMDA	N-methyl-d-aspartate
AMPA	α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
FALS	familial amyotrophic lateral sclerosis

ABBREVIATIONS

References

1. Krusic P J, Wasserman E, Keizer P N, Morton J R, Preston K F. Science. 1991;254:1183–1185.[PubMed]
2. McGeer E G, McGeer P L. Nature (London) 1976;263:517–524.[PubMed]
3. Rothman S M, Olney J W. Ann Neurol. 1986;19:105–111.[PubMed]
4. Choi D W. Neuron. 1988;1:623–634.[PubMed]
5. McIntosh T, Soares H, Hayes R, Simon R In: Frontiers in Excitatory Amino Acid Research. Cavallo E A, Lehman J, Turski L, editors. New York: Liss; 1988. pp. 653–656. [PubMed][Google Scholar]
6. Garthwaite J, Charles S L, Chess-Williams R. Nature (London) 1988;336:385–388.[PubMed]
7. Dawson V L, Dawson T M, London E D, Bredt D S, Snyder S H. Proc Natl Acad Sci USA. 1991;88:6368–6371.
8. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. Nature (London) 1993;364:535–537.[PubMed]
9. Dugan L L, Sensi S L, Canzoniero L M T, Handran S D, Rothman S M, Lin T T, Goldberg M P, Choi D W. J Neurosci. 1995;15:6377–6388.
10. Reynolds I J, Hastings T G. J Neurosci. 1995;15:3318–3327.
11. Dykens J A, Stern A, Trenkner E. J Neurochem. 1987;49:1222–1228.[PubMed]
12. Chan P H, Chu L, Chen S F, Carlson E J, Epstein C J. Acta Neurochirurgica. 1990;51:245–247.[PubMed]
13. Monyer H, Hartley D M, Choi D W. Neuron. 1990;5:121–126.[PubMed]
14. Yue T-L, Gu J-L, Lysko P G, Cheng H-Y, Barone F C, Feuerstein G. Brain Res. 1992;574:193–197.[PubMed]
15. Chow H S, Lynch J L, Rose K, Choi D W. Brain Res. 1994;639:102–108.[PubMed]
16. Dugan L L, Gabrielsen J K, Yu S P, Lin T S, Choi D W. Neurobiol Dis. 1996;3:129–135.[PubMed]
17. Holden C. Science. 1996;273:1495.[PubMed]
18. Lamparth I, Hirsch A. J Chem Soc Chem Commun. 1994;1994:1727–1728.[PubMed]
19. Hara A, Radin N S. Anal Biochem. 1978;90:420–427.[PubMed]
20. Dugan L L, Bruno V M G, Amagasu S M, Giffard R G. J Neurosci. 1995;15:4545–4555.
21. Koh J Y, Gwag B J, Lobner D, Choi D W. Science. 1995;268:573–575.[PubMed]
22. Hartley D M, Kurth M C, Bjerknes L, Choi D W. J Neurosci. 1993;13:1993–2000.
23. Goldberg M P, Choi D W. J Neurosci. 1993;13:3510–3524.
24. Dugan L L, Creedon D J, Johnson E M, Holtzman D M. Proc Natl Acad Sci USA. 1997;94:4086–4091.
25. Gurney M E, Haifeng P, Chiu A Y, Dal Canto M C, Polchow C Y, Alexander D D, Caliendo J, Hentati A, Kwon Y W, Deng H-X, Chen W, Zhai P, Sufit R L, Siddique T. Science. 1994;264:1772–1775.[PubMed]
26. Basso D M, Beattie M S, Bresnahan J C. J Neurotrauma. 1995;12:1–10.[PubMed]
27. Gwag B J, Koh J K, Chen M M, Dugan L L, Behrens M, Lobner D, Choi D W. NeuroReport. 1995;7:93–96.[PubMed]
28. Choi D W. Curr Opin Neurobiol. 1996;6:667–672.[PubMed]
29. Heron A, Pollard H, Dessi F, Moreau J, Lasbennes F, Ben-Ari Y, Charriaut-Marlangue C. J Neurochem. 1993;61:1973–1976.[PubMed]
30. MacManus J P, Buchan A M, Hill I E, Rasquinha I, Preston E. Neurosci Lett. 1993;164:89–92.[PubMed]
31. Du C, Hu R, Csernansky C A, Hsu C Y, Choi D W. J Cereb Blood Flow Metab. 1996;16:195–201.[PubMed]
32. Hockenberry D M, Oltvai Z N, Yin X M, Milliman C L, Korsmeyer S J. Cell. 1993;75:241–251.[PubMed]
33. Ratan R R, Murphy T H, Baraban J M. J Neurochem. 1994;62:376–379.[PubMed]
34. Greenlund L J, Deckwerth T L, Johnson E M., Jr Neuron. 1995;14:303–315.[PubMed]
35. Koh J Y, Yang L L, Cotman C W. Brain Res. 1990;533:315–320.[PubMed]
36. Wong P C, Pardo C A, Borchelt D R, Lee M K, Copeland N G, Jenkins N A, Sisodia S S, Cleveland D W, Price D L. Neuron. 1995;14:1105–1116.[PubMed]
37. Ripps M E, Huntley G W, Hof P R, Morrison J H, Gordon J W. Proc Natl Acad Sci USA. 1995;92:689–692.
38. Wiedau-Pazos M, Goto J J, Rabizadeh S, Gralla E B, Roe J A, Lee M K, Valentine J S, Bredesen D E. Science. 1996;271:515–518.[PubMed]
39. Gurney M E, Cuttings F B, Zhai P, Doble A, Taylor C P, Andrus P K, Hall E D. Ann Neurol. 1996;39:147–157.[PubMed]