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

Orphanet J Rare Dis. 2022 Jun 16;17(1):231. doi: 10.1186/s13023-022-02367-w.


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 < 0.05).

RESULTS: Neither cysteamine, NACA nor diNACA at 25, 50 or 75 μM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h.

CONCLUSIONS: NACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

PMID:35710564 | DOI:10.1186/s13023-022-02367-w

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