The Specific Copper(II) Chelator TDMQ20 Is Efficient for the Treatment of Wilson's Disease in Mice

Abstract

(1) Background: In patients with Wilson's disease, the deficiency of the copper carrier ATP7B causes the accumulation of copper in the liver, brain and various other organs. Lifelong treatment is therefore mandatory, using copper chelators to increase the excretion of copper and to avoid life-threatening damage. The clinically used reference drug, D-penicillamine, exhibit numerous adverse effects, especially a frequent severe and irreversible neurological worsening, mainly due to its lack of metal selectivity; (2) Methods: A new tetradentate ligand based on an 8-aminoquinoline entity, named TDMQ20, which is highly selective for copper compared with other metal ions, is evaluated in "toxic milk" TX mice as an oral treatment of this Wilson's disease murine model; (3) Results: The concentration of copper in the liver of "toxic milk" TX mice decreased and the fecal excretion of copper increased upon oral treatment with TDMQ20. Both effects are dose-dependent, and more pronounced than those of D-penicillamine; (4) Conclusions: The TDMQ20 copper chelator is more efficient than the reference drug D-penicillamine for the treatment of a Wilson's disease murine model. Pharmacological data obtained with TDMQ20 on the TX mouse model strongly support the selection of this ligand as a drug candidate for this genetic disease.

Keywords: Wilson’s disease; copper chelator; liver; toxic milk mouse.

Figure 1

General formula of TDMQ ligands. For TDMQ20: R = 5,7-dichloro-, n = m = 2.

Figure 2

Dosage of copper in liver (a), feces (b), brain (c), serum (d), kidneys (e) and urine (f) of TX mice (WD) after oral treatment using TDMQ20 at 12.5 mg/kg (TDMQ20-L), 25 mg/kg (TDMQ20-M) or 50 mg/kg (TDMQ20-H). WD mice orally treated with DPA at 200 mg/kg are given for comparison. Control mice are healthy C57BL/6 mice bearing no mutation on ATP7B. Differences with p > 0.05 were considered not significant (ns), * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 6.

Figure 3

HE staining of representative samples of mouse liver. (A) Control group; (B) WD group; (C) TDMQ20-L group; (D) TDMQ20-M group; (E) TDMQ20-H group; (F) DPA group. Bar scales stand for 50 μm. Black, green and light blue arrows stand for anisonucleosis, steatosis and inflammatory cell infiltration, respectively.

Figure 4

Concentration of serum ceruloplasmin labeled with TMT and detected using LC-MS/MS. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Evaluation of the bovine erythrocyte Cu,Zn-SOD activity (named SOD for short) in the presence of DPA or TDMQ20 added at t₀ except otherwise stated. Reduction of a tetrazolium salt by O₂^−• produced by xanthine/xanthine oxidase (X/XO) to its formazan derivative (Absorbance at 450 nm): (b) Fully active Cu,Zn-SOD (black trace); (c) in the presence of DPA at 50 μM (light-blue trace); (c′) in the presence of DPA and Cu²⁺, 1/1, 50 μM, preincubated for 2 h at room temperature (light-blue dashed trace); (d) in the presence of DPA at 100 μM (dark-blue trace); (e) in the presence of DPA at 100 μM added at 10 min (dark-blue dotted trace); (f) in the presence of TDMQ20 at 300 μM (green trace). Reduction of tetrazolium in the absence of Cu,Zn-SOD (red trace, (a)) is given as comparison. The percentage of inhibition of Cu,Zn-SOD is calculated as (y/x) × 100 or (z/x) × 100 for DPA at 100 μM or 50 μM, respectively (traces d and c, respectively).

Figure 6

UV–visible spectrum of bovine erythrocyte Cu,Zn-SOD (10.14 mg/mL in phosphate buffer 100 μM, pH 7.4), either alone (black trace) or in the presence of DPA (0.5, 1.0, or 2.0 mol equivalent, blue, violet and red traces, respectively).

Figure 7

UV–visible spectrum of vitamin B12 alone (20 μM, black trace) or in the presence of DPA (molar ratio = 100/1), in Hepes buffer 50 mM, pH 7.4 (red trace).

Figure 8

UV–visible (λ = 265 nm) kinetic spectra of aerobic ascorbate consumption in the presence of DPA/Cu²⁺, 1.1/1 (light-blue trace), DPA/Cu²⁺, 2.2/1 (dark-blue trace), or TDMQ20/Cu²⁺ 1.1/1 (green trace). Spectra in the presence of no additive (black trace), or CuCl₂ (red trace) are shown as comparison. Spectra were obtained after subtraction of the absorbances at 265 nm of DPA/Cu²⁺, 1.1/1, DPA/Cu²⁺, 2.2/1, or TDMQ20/Cu²⁺ 1.1/1, respectively, from raw data. [Ascorbate] = 100 μM, [Cu²⁺] = 10 μM, [DPA] = 11 or 22 μM, [TDMQ20] = 11 μM.