Letters to the Editor

PENICILLAMINE MAY DETOXIFY COPPER IN WILSON'S DISEASE

Excess copper causes hepatocyte death in hereditary Wilson’s disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents—methanobactins (MBs)—for efficient liver copper depletion in WD rats as well as their safety and effect duration.

Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases.

We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats.

ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

d-Penicillamine, a copper chelator, was the first oral drug introduced for therapy in Wilson disease. This drug is effective to improve the condition of most of the patients. d-Penicillamine is still an accepted and widely used drug for treatment of Wilson disease. Its use is limited by frequent side effects, which necessitate a switch to other treatment modalities.

Initially, the dose should be 250–500 mg/day and should be increased over a few weeks, to the maximal dose of 1000–1500 mg/day (20 mg/kg in children). Supplementation of pyridoxine (vitamin B₆) is needed. The initial lower dose may prevent worsening of neurological symptoms in affected patients. During initial treatment, urinary copper excretion should be 3–8 μmol/24 h. For long-term monitoring, urinary copper excretion measurements after 2 days of d-penicillamine cessation might be considered; the urinary copper excretion in this case should be ≤ 1.6 μmol/24 h. For maintenance therapy, the dose may then be decreased.

Late side effects include nephrotoxicity documented by proteinuria, a lupus-like syndrome marked by hematuria, proteinuria, and positive antinuclear antibody, and Goodpasture’s syndrome. They necessitate immediate cessation of d-penicillamine. Dermatologic toxicities reported include progeric changes in the skin and elastosis perforans serpiginosa.

In mitochondria, copper is a Janus-faced trace element. While it is the essential cofactor of the mitochondrial cytochrome c oxidase, a surplus of copper can be highly detrimental to these organelles. On the one hand, mitochondria are strictly dependent on adequate copper supply for proper respiratory function, and the molecular mechanisms for metalation of the cytochrome c oxidase have been largely characterized. On the other hand, copper overload impairs mitochondria and uncertainties exist concerning the molecular mechanisms for mitochondrial metal uptake, storage and release. The latter issue is of fundamental importance in Wilson disease, a genetic disease characterized by dysfunctional copper excretion from the liver. Prime consequences of the progressive copper accumulation in hepatocytes are increasing mitochondrial biophysical and biochemical deficits. Focusing on this two-sided aspect of mitochondrial copper, we review mitochondrial copper homeostasis but also the impact of excessive mitochondrial copper in Wilson disease.

Wilson disease (WD) is a rare genetic disorder of the copper metabolism leading to systemic copper accumulation, predominantly in the liver. The therapeutic approach in WD patients is the generation of a negative copper balance and the maintenance of copper homeostasis, currently by the use of copper chelators such as D-penicillamine (D-PA). However, in circumstances of delayed diagnosis, poor treatment compliance, or treatment failure, mortality is almost certain without hepatic transplantation. Moreover, even after years of D-PA treatment, high liver copper levels are present in WD patients.

We have recently suggested the use of the bacterial peptide Methanobactin (MB), which has an outstanding binding affinity for copper, as potentially efficient and patient-friendly remedy against copper damage in WD. Here we substantiate these findings considerably, by demonstrating a significant removal of copper from liver samples of WD rats upon short, one week only, MB treatments. Using laser ablation-inductively coupled plasma-mass spectrometry with a spatial resolution down to 4 μm, we demonstrate that only small copper hotspots remain in MB treated animal livers. We further demonstrate in WD rat liver, seven weeks after the stopped MB treatment, a lower liver copper concentration as compared to untreated control animals. Thus, MB highly efficiently depletes liver copper overload with a sustained therapeutic effect.

At present, the copper chelator d-penicillamine (DPA) is the first-line therapy of Wilson’s disease (WD), which is characterized by an excessive copper overload. Lifelong DPA treatments aim to reduce the amount of detrimental excess copper retention in the liver and other organs. Although DPA shows beneficial effect in many patients, it may cause severe adverse effects. Despite several years of copper chelation therapy, discontinuation of DPA therapy can be linked to a rapidly progressing liver failure, indicating a high residual liver copper load. In order to investigate the spatial distribution of remaining copper and additional elements, such as zinc and iron, in rat and human liver samples after DPA treatment, a high resolution (spotsize of 10 μm) laser ablation-inductively coupled plasma-mass spectrometry (LA‐ICP‐MS) imaging method was applied. Untreated LPP^‐/− rats, an established animal model for WD, appeared with a high overall copper concentration and a copper distribution of hotspots distributed over the liver tissue. In contrast, a low (>2-fold decreased) overall copper concentration was detected in liver of DPA treated animals. Importantly, however, copper distribution was highly inhomogeneous with lowest concentrations in direct proximity to blood vessels, as observed using novel zonal analysis. A human liver needle biopsy of a DPA treated WD patient substantiated the finding of an inhomogeneous copper deposition upon chelation therapy. In contrast, comparatively homogenous distributions of zinc and iron were observed. Our study indicates that a high resolution LA‐ICP‐MS analysis of liver samples is excellently suited to follow efficacy of chelator therapy in WD patients.