DPM-1001 decreased copper levels and ameliorated deficits in a mouse model of Wilson's disease

Abstract

The levels of copper, which is an essential element in living organisms, are under tight homeostatic control. Inactivating mutations in ATP7B, a P-type Cu-ATPase that functions in copper excretion, promote aberrant accumulation of the metal, primarily the in liver and brain. This condition underlies Wilson's disease, a severe autosomal recessive disorder characterized by profound hepatic and neurological deficits. Current treatment regimens rely on the use of broad specificity metal chelators as "decoppering" agents; however, there are side effects that limit their effectiveness. Here, we present the characterization of DPM-1001 {methyl 4-[7-hydroxy-10,13-dimethyl-3-({4-[(pyridin-2-ylmethyl)amino]butyl}amino)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoate} as a potent and highly selective chelator of copper that is orally bioavailable. Treatment of cell models, including fibroblasts derived from Wilson's disease patients, eliminated adverse effects associated with copper accumulation. Furthermore, treatment of the toxic milk mouse model of Wilson's disease with DPM-1001 lowered the levels of copper in the liver and brain, removing excess copper by excretion in the feces while ameliorating symptoms associated with the disease. These data suggest that it may be worthwhile to investigate DPM-1001 further as a new therapeutic agent for the treatment of Wilson's disease, with potential for application in other indications associated with elevated copper, including cancer and neurodegenerative diseases.

Keywords: Wilson's; chelator; copper; toxic milk.

Figure 1.

DPM-1001 was a specific chelator of copper. (A) Chemical structure of DPM-1001. (B) ESI-MS spectra to examine complex formation between DPM-1001 (1 mM) and the indicated metals (8 mM). (C, top left panel) Chemical structure of DPM-1003. (Bottom left panel) Titration of radiolabeled copper (⁶⁴Cu) against DPM-1001 and DPM-1003. (Right panel) ESI-MS spectra to examine complex formation between DPM-1003 (1 mM) and the indicated metals (8 mM). (D) Chemical structure of analog 2. (E) ESI-MS spectra to examine complex formation between analog 2 (1 mM) and the indicated metals (8 mM).

Figure 2.

Treatment with DPM-1001 improved cell viability in the presence of high levels of copper. (A) Representative immunoblot to demonstrate siRNA-induced suppression of ATP7B in HepG2 cells. (B) Survival of control cells with (▴) or without (•) DPM-1001 and ATP7B-KD1 cells with (gray; ▾) and without (▪) DPM-1001 was measured at increasing concentrations of copper, from 0 to 1.5 mM. Cells were preincubated with DPM-1001 at 2 µM for 1 h prior to exposing cells to copper. (C) Survival of control fibroblasts with (▴) and without (▾) DPM-1001 was compared with fibroblasts derived from Wilson's disease patients (GM00032 [left panel], GM00033 [middle panel], and GM05257 [right panel]) with (gray, •) and without (▪) DPM-1001, measured at increasing concentrations of copper, from 0 to 1.5 mM.

Figure 3.

Suppression of copper levels in the toxic milk mouse model of Wilson's disease. (A) Representative sections of liver tissue from the toxic milk mouse model stained with rhodanine following treatment of animals for 2 wk with saline (left panel) or DPM-1001 (right panel). Quantitation is shown at the right. (B–D) Copper levels in the livers (B), brains (C), and kidneys (D) from wild-type (black bars) or toxic milk (gray bars) mice were measured using ICP-MS following treatment with saline, 5 mg/kg DPM-1001 orally every third day, or 5 mg/kg tetrathiomolybdate (TTM) intraperitoneally daily for 2 wk. (E) The levels of copper in the feces of these animals. Data are presented as mean ± SEM. Statistical analysis was performed using ANOVA. (**) P < 0.01; (*) P < 0.1; (ns) not significant.

Figure 4.

Treatment with DPM-1001 ameliorated symptoms in the toxic milk mouse model of Wilson's disease. (A) H&E staining of representative sections of liver tissue from wild-type (top) and toxic milk (bottom; labeled WD for Wilson's disease) mice that were either untreated (left) or treated with DPM-1001 (right). (B,C). Levels of metallothionein in the livers (B) or brains (C) from wild-type (black bars) and toxic milk (gray bars) mice treated with saline, DPM-1001, or tetrathiomolybdate (TTM) were measured. Data are presented as mean ± SEM. Statistical analysis was performed using ANOVA. (**) P < 0.01.