Advertisement

Glyceryl triacetate for Canavan disease: A low-dose trial in infants and evaluation of a higher dose for toxicity in the tremor rat model

  • C. N. Madhavarao
  • P. Arun
  • Y. Anikster
  • S. R. Mog
  • O. Staretz-Chacham
  • J. R. Moffett
  • N. E. Grunberg
  • W. A. Gahl
  • A. M. A. Namboodiri
ORIGINAL ARTICLE

Summary

Canavan disease (CD) is a fatal dysmyelinating genetic disorder associated with aspartoacylase deficiency, resulting in decreased brain acetate levels and reduced myelin lipid synthesis in the developing brain. Here we tested tolerability of a potent acetate precursor, glyceryl triacetate (GTA), at low doses in two infants diagnosed with CD, aged 8 and 13 months. Much higher doses of GTA were evaluated for toxicity in the tremor rat model of CD. GTA was given orally to the infants for up to 4.5 and 6 months, starting at 25 mg/kg twice daily, doubling the dose weekly until a maximum of 250 mg/kg reached. Wild-type and tremor rat pups were given GTA orally twice daily, initially at a dose of 4.2 g/kg from postnatal days 7 through 14, and at 5.8 g/kg from day 15 through 23, and thereafter in food (7.5%) and water (5%). At the end of the trial (~90 to 120 days) sera and tissues from rats were analysed for changes in blood chemistry and histopathology. GTA treatment caused no detectable toxicity and the patients showed no deterioration in clinical status. In the high-dose animal studies, no significant differences in the mean blood chemistry values occurred between treated and untreated groups, and no lesions indicating toxicity were detectable in any of the tissues examined. Lack of GTA toxicity in two CD patients in low-dose trials, as well as in high-dose animal studies, suggests that higher, effective dose studies in human CD patients are warranted.

Keywords

Infant Formula Canavan Disease Magnetic Resonance Spectroscopy Data Human Equivalent Dose Aspartoacylase 

Abbreviations

ASPA

aspartoacylase

ASPAD

ASPA deficiency

CD

Canavan disease

GTA

glyceryl triacetate

MRI

magnetic resonance imaging

IP

intraperitoneally

MRS

magnetic resonance spectroscopy

NAA

N-acetylaspartate

Notes

Acknowledgements

C. N. Madhavarao was supported by the Rosalind Poss Rosen Clinical Research Training Fellowship sponsored by the American Academy of Neurology Foundation, St Paul, MN and Canavan Foundation, New York, NY. The study was supported by Jacob’s Cure Foundation, NTSAD, Boston, Samueli Institute and NIH R56 grant NS039387 for A. M. A. Namboodiri. GTA for the clinical trials was provided gratis by Cognis Oleo Chemicals, Germany. The authors thank the National Bio Resource Project for the Rat, Kyoto University, Kyoto, Japan, for providing breeding pairs of tremor rats.

Supplementary material

10545_2009_1155_MOESM1_ESM.doc (178 kb)
Supplementary Table 1a Biochemical analyses of the blood serum samples of the GTA treated and untreated female tremor and untreated female wild type rats (DOC 178 KB)
10545_2009_1155_MOESM2_ESM.jpg (327 kb)
Supplementary Figure S1

Comparative pictures of normal (a) and homozygous mutant (tremor, b) rat pups at 2 weeks of age. Note the thin coat, ring tail and curled whiskers in the tremor pup compared to the normal features in the wild-type pup (JPG 327 KB).

10545_2009_1155_MOESM3_ESM.jpg (2.6 mb)
Supplementary Figure S2

Stomach sections from the wild-type female (a, b) and male (c, d) rats showing fundic glands (FG), muscularis mucosae (MM), submucosa (SM) and blood vessels (BV). No significant lesion was detected between GTA untreated (a, c) and treated (b, d) wild-type rats and the results were similar in tremor rats (JPG 2.57 MB).

10545_2009_1155_MOESM4_ESM.jpg (3.9 mb)
Supplementary Figure S3

Small intestine (jejunum) sections from the wild-type female (a, b) and male (c, d) rats showing crypts of Lieberkuhn (CL), goblet cells (GC), muscularis mucosae (MM) and lumen (L). No significant lesion was detected between GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 3.86 MB).

10545_2009_1155_MOESM5_ESM.jpg (3.6 mb)
Supplementary Figure S4

Large intestine (colon) sections from the wild-type female (a, b) and male (c, d) rats showing crypts of Lieberkuhn (CL), goblet cells (GC), lumen (L), smooth muscle (SM) and blood vessels (BV). No significant lesion was detected between GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 3.60 MB).

10545_2009_1155_MOESM6_ESM.jpg (2.9 mb)
Supplementary Figure S5

Liver sections from the wild-type female (a, b) and male (c, d) rats showing the lobules of the liver with central vein (CV), sinusoids (Si), hepatocytes (H), plates of liver cells (PL) and portal vein (PV, in Fig. a). No significant lesion was detected between GTA untreated (a, c) and treated (b, d) rats except for diffuse minimal to mild hepatocellular vacuolar change of glycogen-type or lipid type (JPG 2.88 MB).

10545_2009_1155_MOESM7_ESM.jpg (3.5 mb)
Supplementary Figure S6

Kidney cortical labyrinth sections from the wild-type female (a, b) and male (c, d) rats showing glomeruli. The renal capsules (RC) within the Bowman’s space (BS), proximal tubule (PT) and distal tubule (DT) are also shown. No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 3.46 MB).

10545_2009_1155_MOESM8_ESM.jpg (4.6 mb)
Supplementary Figure S7

Spleen sections from the wild-type female (a, b) and male (c, d) rats showing the red pulp (RP) and white pulp (WP) regions. No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 4.57 MB).

10545_2009_1155_MOESM9_ESM.jpg (2.8 mb)
Supplementary Figure S8

Heart sections from the wild-type female (a, b) and male (c, d) rats show the cardiac muscle cell nuclei (N), blood vessels (BV) and cardiac muscle fibers (CMF). No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 2.80 MB).

10545_2009_1155_MOESM10_ESM.jpg (2.9 mb)
Supplementary Figure S9

Lung sections from the wild-type female (a, b) and male (c, d) rats showing the terminal bronchiole (TB), Clara cells (CC) and smooth muscle (SM) of the bronchiole. No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) wild-type rats and tremor rats (JPG 2.90 MB).

10545_2009_1155_MOESM11_ESM.jpg (2.8 mb)
Supplementary Figure S10

Forebrain sections from the wild-type female (a, b) and male (c, d) rats showing the white matter (WM) and gray matter (GM) regions. No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) rats (JPG 2.79 MB).

10545_2009_1155_MOESM12_ESM.jpg (3.2 mb)
Supplementary Figure S11

Cerebellum sections from the wild-type female (a, b) and male (c, d) rats showing the molecular layer (ML), granular layer (GRL) and Purkinje cells (PC). No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) rats (JPG 3.17 MB).

10545_2009_1155_MOESM13_ESM.jpg (3.3 mb)
Supplementary Figure S12

Spinal cord sections from the wild-type female (a, b) and male (c, d) rats showing the neurons (Nu), glial cells (Gl) and blood vessels (BV). No significant lesion was detected among the GTA untreated (a, c) and treated (b, d) rats (JPG 3.34 MB).

10545_2009_1155_MOESM14_ESM.jpg (2.9 mb)
Supplementary Figure S13

Liver sections from the tremor female (a, b) and male (c, d) rats showing the lobules of the liver with central vein (PV), sinusoids (Si), hepatocytes (H) and plates of liver cells (PL). No significant lesion was detected between GTA untreated (a, c) and treated (b, d) rats except for diffuse minimal to mild hepatocellular vacuolar change of glycogen-type or lipid type seen with the treated rats more clearly than the untreated rats (JPG 2.85 MB).

10545_2009_1155_MOESM15_ESM.jpg (2.8 mb)
Supplementary Figure S14

Forebrain sections from the tremor female (a, b) and male (c, d) rats showing the gray matter (GM), white matter (WM) and vacuoles seen in the white matter, typical of the mutant tremor rats. No significant lesion was detected that was attributable to GTA treatment among the GTA untreated (a, c) and treated (b, d) rats (JPG 2.83 MB).

10545_2009_1155_MOESM16_ESM.jpg (3.2 mb)
Supplementary Figure S15

Cerebellum sections from the tremor female (a, b) and male (c, d) rats showing the molecular layer (ML), granular layer (GRL) and Purkinje cells (PC). No significant lesion was detected that was attributable to GTA treatment among the GTA untreated (a, c) and treated (b, d) rats (JPG 3.17 MB).

10545_2009_1155_MOESM17_ESM.jpg (3.1 mb)
Supplementary Figure S16

Spinal cord sections from the tremor female (a, b) and male (c, d) rats showing the neurons (Nu), glial cells (Gl) and blood vessels (BV). Vacuoles are seen as typical of the mutant tremor rats. No significant lesion was detected that was attributable to GTA treatment among the GTA untreated (a, c) and treated (b, d) rats (JPG 3.09 MB).

References

  1. Anonymous (2005) Guidance for industry: Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. US Department of Health and Human Services, FDA, CDER, Rockville, MD, pp 1–30Google Scholar
  2. Bach A, Metais P (1970) [Fats with short and medium chains. Physiological, biochemical, nutritional, and therapeutic aspects]. Ann Nutr Aliment 24:75–144PubMedGoogle Scholar
  3. Bailey JW, Miles JM, Haymond MW (1993) Effect of parenteral administration of short-chain triglycerides on leucine metabolism. Am J Clin Nutr 58:912–916CrossRefPubMedGoogle Scholar
  4. Baslow MH (2003) Brain N-acetylaspartate as a molecular water pump and its role in the etiology of Canavan Disease: a mechanistic explanation. J Mol Neurosci 21:185–190CrossRefPubMedGoogle Scholar
  5. Blüml S (1999) In vivo quantitation of cerebral metabolite concentrations using natural abundance 13C MRS at 1.5 T. J Magn Reson 136:219–225CrossRefPubMedGoogle Scholar
  6. Burri R, Steffen C, Herschkowitz N (1991) N-Acetyl-l-aspartate is a major source of acetyl groups for lipid synthesis during rat brain development. Dev Neurosci 13:403–411CrossRefPubMedGoogle Scholar
  7. Chakraborty G, Mekala P, Yahya D, Wu G, Ledeen RW (2001) Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J Neurochem 78:736–745CrossRefPubMedGoogle Scholar
  8. Chalmers RA, Lawson AM (1982) Organic acids in man: The analytical chemistry, biochemistry and diagnosis of the organic acidurias. Chapman and Hall, New YorkCrossRefGoogle Scholar
  9. D’Adamo AF Jr, Yatsu FM (1966) Acetate metabolism in the nervous system. N-Acetyl-l-aspartic acid and the biosynthesis of brain lipids. J Neurochem 13:961–965CrossRefPubMedGoogle Scholar
  10. D’Adamo AF Jr, Gidez LI, Yatsu FM (1968) Acetyl transport mechanisms. Involvement of N-acetyl aspartic acid in de novo fatty acid biosynthesis in the developing rat brain. Exp Brain Res 5:267–273PubMedGoogle Scholar
  11. Fiume MZ (2003) Final report on the safety assessment of triacetin. Int J Toxicol 22Suppl 2:1–10PubMedGoogle Scholar
  12. Gascon GG, Ozand PT, Mahdi A et al (1990) Infantile CNS spongy degeneration—14 cases: clinical update. Neurology 40:1876–1882CrossRefPubMedGoogle Scholar
  13. Hagenfeldt L, Bollgren I, Venizelos N (1987) N-Acetylaspartic aciduria due to aspartoacylase deficiency—a new aetiology of childhood leukodystrophy. J Inherit Metab Dis 10:135–141CrossRefPubMedGoogle Scholar
  14. Hamaguchi H, Nihei K, Nakamoto N et al (1993) A case of Canavan disease: the first biochemically proven case in a Japanese girl. Brain Dev 15:367–371CrossRefPubMedGoogle Scholar
  15. Hershfield JR, Pattabiraman N, Madhavarao CN, Namboodiri MA (2007) Mutational analysis of aspartoacylase: implications for Canavan disease. Brain Res 1148:1–14CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hoffmann C, Ben-Zeev B, Anikster Y et al (2007) Magnetic resonance imaging and magnetic resonance spectroscopy in isolated sulfite oxidase deficiency. J Child Neurol 22:1214–1221CrossRefPubMedGoogle Scholar
  17. Inoue Y, Kuhara T (2004) Rapid and sensitive screening for and chemical diagnosis of Canavan disease by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 806:33–39CrossRefPubMedGoogle Scholar
  18. Janson CG, McPhee SW, Francis J et al (2006) Natural history of canavan disease revealed by proton magnetic resonance spectroscopy (1H-MRS) and diffusion-weighted MRI. Neuropediatrics 37:209–221CrossRefPubMedGoogle Scholar
  19. Kaul R, Balamurugan K, Gao GP, Matalon R (1994) Canavan disease: Genomic organization and localization of human ASPA to 17p13-ter and conservation of the ASPA gene during evolution. Genomics 21:364–370CrossRefPubMedGoogle Scholar
  20. Kitada K, Akimitsu T, Shigematsu Y et al (2000) Accumulation of N-acetyl-l-aspartate in the brain of the tremor rat, a mutant exhibiting absence-like seizure and spongiform degeneration in the central nervous system. J Neurochem 74:2512–2519CrossRefPubMedGoogle Scholar
  21. Klugmann M, Symes CW, Klaussner BK et al (2003) Identification and distribution of aspartoacylase in the postnatal rat brain. Neuroreport 14:1837–1840CrossRefPubMedGoogle Scholar
  22. Kvittingen EA, Guldal G, Borsting S, Skalpe IO, Stokke O, Jellum E (1986) N-Acetylaspartic aciduria in a child with a progressive cerebral atrophy. Clin Chim Acta 158:217–227CrossRefPubMedGoogle Scholar
  23. Li P, Callery PS, Gan LS, Balani SK (2007) Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction. Drug Metab Dispos 35:1203–1208CrossRefPubMedGoogle Scholar
  24. Lynch JW, Bailey JW (1995) Dietary intake of the short-chain triglyceride triacetin vs. Long-chain triglycerides decreases adipocyte diameter and fat deposition in rats. J Nutr 125:1267–1273PubMedGoogle Scholar
  25. Madhavarao CN, Chinopoulos C, Chandrasekaran K, Namboodiri MA (2003) Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain. J Neurochem 86:824–835CrossRefPubMedGoogle Scholar
  26. Madhavarao CN, Moffett JR, Moore RA, Viola RE, Namboodiri MA, Jacobowitz DM (2004) Immunohistochemical localization of aspartoacylase in the rat central nervous system. J Comp Neurol 472:318–329CrossRefPubMedGoogle Scholar
  27. Madhavarao CN, Arun P, Moffett JR et al (2005) Defective n-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in canavan's disease. Proc Natl Acad Sci U S A 102:5221–5226CrossRefPubMedPubMedCentralGoogle Scholar
  28. Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J (1988) Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am J Med Genet 29:463–471CrossRefPubMedGoogle Scholar
  29. Matalon R, Rady PL, Platt KA et al (2000) Knock-out mouse for canavan disease: a model for gene transfer to the central nervous system. J Gene Med 2:165–175CrossRefPubMedGoogle Scholar
  30. Mathew R, Arun P, Madhavarao CN, Moffett JR, Namboodiri MA (2005) Progress toward acetate supplementation therapy for canavan disease: Glyceryl triacetate administration increases acetate, but not N-acetylaspartate, levels in brain. J Pharmacol Exp Ther 315:297–303CrossRefPubMedGoogle Scholar
  31. Mehta V, Namboodiri MA (1995) N-acetylaspartate as an acetyl source in the nervous system. Brain Res Mol Brain Res 31:151–157CrossRefPubMedGoogle Scholar
  32. Moffett JR, Namboodiri MA (1995) Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain. J Neurocytol 24:409–433CrossRefPubMedGoogle Scholar
  33. Moffett JR, Namboodiri AM (2006) Expression of N-acetylaspartate and N-acetylaspartylglutamate in the nervous system. Adv Exp Med Biol 576:7–26CrossRefPubMedGoogle Scholar
  34. Moffett JR, Ross B, Arun P, Madhavarao CN, Namboodiri AM (2007) N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology. Prog Neurobiol 81:89–131CrossRefPubMedPubMedCentralGoogle Scholar
  35. Moore JK, Perazzo LM, Braun A (1995) Time course of axonal myelination in the human brainstem auditory pathway. Hear Res 87:21–31CrossRefPubMedGoogle Scholar
  36. Tallan HH, Moore S, Stein WH (1956) N-Acetyl-l-aspartic acid in brain. J Biol Chem 219:257–264PubMedGoogle Scholar
  37. Wang J, Leone P, Wu G et al (2009) Myelin lipid abnormalities in the aspartoacylase-deficient tremor rat. Neurochem Res 34:138–148CrossRefPubMedGoogle Scholar
  38. Wittsack HJ, Kugel H, Roth B, Heindel W (1996) Quantitative measurements with localized 1H MR spectroscopy in children with Canavan’s disease. J Magn Reson Imaging 6:889–893CrossRefPubMedGoogle Scholar
  39. Yaron Y, Schwartz T, Mey-Raz N, Amit A, Lessing JB, Malcov M (2005) Preimplantation genetic diagnosis of Canavan disease. Fetal Diagn Ther 20:465–468CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • C. N. Madhavarao
    • 1
  • P. Arun
    • 1
  • Y. Anikster
    • 2
  • S. R. Mog
    • 3
  • O. Staretz-Chacham
    • 4
  • J. R. Moffett
    • 1
  • N. E. Grunberg
    • 5
  • W. A. Gahl
    • 4
  • A. M. A. Namboodiri
    • 1
  1. 1.Department of Anatomy, Physiology and Genetics, Program in Neuroscience and Program in Molecular and Cell BiologyUSUHSBethesdaUSA
  2. 2.Metabolic Disease Unit, Safra Children Hospital, Sheba Medical Center, Tel-Hashomer, and Sackler Medical SchoolTel Aviv UniversityTel AvivIsrael
  3. 3.Division of Comparative PathologyAFRRIBethesdaUSA
  4. 4.Medical Genetics BranchNHGRI, NIHBethesdaUSA
  5. 5.Department of Medical and Clinical Psychology, Program in NeuroscienceUSUHSBethesdaUSA

Personalised recommendations