N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) as a novel chelator for Wilson's disease.

Wilson's Disease (WD) is a rare autosomal recessive disorder caused by mutations in the ATP7B gene. These mutations lead to defective copper (Cu) transport and to accumulation of Cu in tissues, primarily in the liver and brain. Current treatment options such as D-penicillamine, trientine, and zinc salts focus on increasing Cu excretion or reducing Cu absorption, but often cause debilitating side effects. N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) is a lipophilic thiol-based compound originally developed for environmental decontamination. It has been shown to chelate toxic metals such as mercury, lead, and cadmium. This study was designed to evaluate the efficacy of NBMI to mitigate Cu overload using both in vitro and in vivo models of WD. HepG2 cells with the ATP7B gene knocked down had increased sensitivity to copper sulfate (CuSO4) compared to wild-type (WT) cells, validating the cell model for WD. Pretreatment with NBMI (2.5-50 μM) improved cell viability, reduced Cu-induced oxidative stress, decreased metallothionein levels, mitigated resulting DNA damage, and reduced overall levels of free intracellular Cu. In an established toxic milk mouse (tx-J) model of WD, 1% dietary NBMI effectively lowered hepatic, cerebral, and renal Cu levels. Treatment with 1% NBMI also improved liver function, as evidenced by reduced ALT levels and normalized hepatocyte morphology. Tx-J mice displayed higher liver-to-body weight ratios compared to WT mice, and treatment with 1% NBMI effectively reduced this ratio. While NBMI did not impact the elevated white blood cell counts and low platelet levels characteristic of tx-J mice, it also did not cause any detrimental effects on red blood cell, hemoglobin, and hematocrit levels. This dose of NBMI also restored homeostasis of other dysregulated essential metal ions in tx-J mice. These findings suggest that dietary administration of NBMI effectively chelates excess free Cu, ameliorates WD symptoms and offers a promising alternative to existing chelators.