Orphanet Journal of Rare Diseases (Jun 2022)

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis

  • Emma Hector,
  • Donald Cairns,
  • G. Michael Wall

DOI
https://doi.org/10.1186/s13023-022-02367-w
Journal volume & issue
Vol. 17, no. 1
pp. 1 – 9

Abstract

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Abstract Background Cystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis. Methods Cytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0–72 h in the presence of 25, 50 or 75 μM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 μM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

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