In Wilson Disease (WD), copper accumulates in the liver and brain causing disease. Bis-choline tetrathiomolybdate (TTM) is a potent copper chelator that may have a lower risk of inducing paradoxical neurological worsening than conventional therapy for neurologic WD. To better understand the mode of action of TTM, we investigated the effects of TTM on copper absorption and biliary excretion. In a double-blind randomized setting, hepatic ⁶⁴Cu activity after orally administered ⁶⁴Cu was examined by PET/CT in 16 healthy volunteers before and after 7 days of TTM treatment (15 mg/d) or placebo. Oral ⁶⁴Cu was administered 1 h after final TTM dose. Changes in hepatic ⁶⁴Cu activity reflected changes in intestinal ⁶⁴Cu uptake. Additionally, in four WD patients, distribution of ⁶⁴Cu in venous blood, liver, gallbladder, kidney, and brain were followed after i.v. ⁶⁴Cu dosing for up to 68 hours before and after seven days of TTM (15 mg/d), using PET/MRI. Increased gallbladder ⁶⁴Cu activity was taken as evidence of increased biliary ⁶⁴Cu excretion. In healthy subjects TTM reduced intestinal ⁶⁴Cu uptake by 82% 15 hours after the oral ⁶⁴Cu dose. In WD patients, gallbladder ⁶⁴Cu activity was negligible before and after TTM, while TTM effectively retained ⁶⁴Cu in the blood, significantly reduced hepatic ⁶⁴Cu activity at all time points and significantly reduced cerebral ⁶⁴Cu activity 2 h after the intravenous ⁶⁴Cu dose. While we did not show an increase in biliary excretion of ⁶⁴Cu following TTM administration, we demonstrated that TTM effectively inhibited most intestinal ⁶⁴Cu uptake and retained ⁶⁴Cu in the blood stream, limiting ⁶⁴Cu exposure to organs like the liver and the brain. Bis-choline tetrathiomolybdate (TTM) is an investigational copper chelator for treatment of Wilson Disease. In animal studies of Wilson Disease models, TTM has been shown to facilitate biliary copper excretion. In the present human study, TTM surprisingly did not facilitate biliary copper excretion but instead reduced intestinal copper uptake to a clinically significant degree. Our study improves the understanding of human copper metabolism and the mechanism of action of TTM. Data is presented from two clinical trials:Absorption study: EudraCT 2020-005832-31Excretion study: EudraCT 2021-000102-25.

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.

Background and Aims: Trientine (TRI) and D-penicillamine (PEN) are used to treat copper overload in Wilson disease. Their main mode of action is thought to be through the facilitation of urinary copper excretion. In a recent study, TRI was noninferior to PEN despite lower 24-hour urinary copper excretion than PEN. We tested whether TRI and/or PEN also inhibit intestinal copper absorption. Approach and Results: Sixteen healthy volunteers were examined with positron emission tomography (PET)/CT 1 and 15 hours after an oral Copper-64 (⁶⁴Cu) dose. They then received 7 days of either PEN or TRI (trientine tetrahydrochloride), after which the ⁶⁴Cu PET/CT scans were repeated. Venous blood samples were also collected. Pretreatment to posttreatment changes of the hepatic ⁶⁴Cu uptake reflect the effect of drugs on intestinal absorption. ⁶⁴Cu activity was normalized to dose and body weight and expressed as the mean standard uptake value. TRI (n=8) reduced hepatic ⁶⁴Cu activity 1 hour after ⁶⁴Cu dose from 6.17 (4.73) to 1.47 (2.97) standard uptake value, p<0.02, and after 15 hours from 14.24 (3.09) to 6.19 (3.43), p<0.02, indicating strong inhibition of intestinal ⁶⁴Cu absorption. PEN (n=8) slightly reduced hepatic standard uptake value at 15 hours, from 16.30 (5.63) to 12.17 (1.44), p<0.04. Conclusions: In this mechanistic study, we show that TRI inhibits intestinal copper absorption, in addition to its cupriuretic effect. In contrast, PEN has modest effects on the intestinal copper absorption. This may explain why TRI and PEN are equally effective although urinary copper excretion is lower with TRI. The study questions whether the same therapeutic targets for 24-hour urinary excretion apply to both drugs.

Background and Aims: Wilson’s disease (WD) is a genetic disease with systemic accumulation of copper that leads to symptoms from the liver and brain. However, the underlying defects in copper transport kinetics are only partly understood. We sought to quantify hepatic copper turnover in patients with WD compared with heterozygote and control subjects using PET with copper‐64 (⁶⁴Cu) as a tracer. Furthermore, we assessed the diagnostic potential of the method. Approach and Results: Nine patients with WD, 5 healthy heterozygote subjects, and 8 healthy controls were injected with an i.v. bolus of ⁶⁴Cu followed by a 90‐min dynamic PET scan of the liver and static whole‐body PET/CT scans after 1.5, 6, and 20 h. Blood ⁶⁴Cu concentrations were measured in parallel. Hepatic copper retention and redistribution were evaluated by standardized uptake values (SUVs). At 90 min, hepatic SUVs were similar in the three groups. In contrast, at 20 h postinjection, the SUV in WD patients (mean ± SEM, 31 ± 4) was higher than in heterozygotes (24 ± 3) and controls (21 ± 4; p < 0.001). An SUV‐ratio of hepatic ⁶⁴Cu concentration at 20 and 1.5 h completely discriminated between WD patients and control groups (p < 0.0001; ANOVA). By Patlak analysis of the initial 90 min of the PET scan, the steady‐state hepatic clearance of ⁶⁴Cu was estimated to be slightly lower in patients with WD than in controls (p = 0.04). Conclusions: ⁶⁴Cu PET imaging enables visualization and quantification of the hepatic copper retention characteristic for WD patients. This method represents a valuable tool for future studies of WD pathophysiology, and may assist the development of therapies, and accurate diagnosis.

Zinc inhibits intestinal copper uptake, an effect utilized for treating Wilson’s disease (WD). We used copper-64 (⁶⁴Cu) PET/CT to examine how much four weeks of treatment with different zinc regimens reduced the hepatic ⁶⁴Cu content after oral ⁶⁴Cu administration and test if alternative regimens were noninferior to the standard regimen of zinc acetate 50 mg × 3 daily. Forty healthy persons were randomized to four different zinc protocols. The WD standard treatment zinc acetate 50 mg × 3 reduced the hepatic ⁶⁴Cu content from 26.9 ± 7.5% to 13.3 ± 5.6% of the administered ⁶⁴Cu. Zinc gluconate 50 mg × 3 was noninferior (P = 0.02) (35.8 ± 9.0% to 17.4 ± 7.5%). Zinc acetate 150 mg × 1 (33.1 ± 9.9% to 17.4 ± 7.5%) and zinc gluconate 150 mg × 1 (28.1 ± 6.7% to 22.0 ± 6.7%) were less effective. These effects were intra- and inter-individually highly variable, and 14% had no effect of any zinc regimen, which may explain disparities in zinc treatment efficacy in WD patients.

In Wilson disease (WD), copper accumulation and increased non-ceruloplasmin-bound copper in plasma lead to liver and brain pathology. To better understand the fate of non-ceruloplasmin-bound copper, we used PET/CT to examine the whole-body distribution of intravenously injected 64-copper (⁶⁴Cu). Eight patients with WD, five heterozygotes, and nine healthy controls were examined by dynamic PET/CT for 90 min and static PET/CT up to 20 h after injection. We measured ⁶⁴Cu activity in blood and tissue and quantified the kinetics by compartmental analysis. Initially, a large fraction of injected ⁶⁴Cu was distributed to extrahepatic tissues, especially skeletal muscle. Thus, across groups, extrahepatic tissues accounted for 45-58% of the injected dose (%ID) after 10 min, and 45-55% after 1 h. Kinetic analysis showed rapid exchange of ⁶⁴Cu between blood and muscle as well as adipose tissue, with ⁶⁴Cu retention in a secondary compartment, possibly mitochondria. This way, muscle and adipose tissue may protect the brain from spikes in non-ceruloplasmin-bound copper. Tiny amounts of cerebral ⁶⁴Cu were detected (0.2%ID after 90 min and 0.3%ID after 6 h), suggesting tight control of cerebral copper in accordance with a cerebral clearance that is 2-3-fold lower than in muscle. Compared to controls, patients with WD accumulated more hepatic copper 6-20 h after injection, and also renal copper at 6 h. Non-ceruloplasmin-bound copper is initially distributed into a number of tissues before being redistributed slowly to the eliminating organ, the liver. Cerebral uptake of copper is extremely slow and likely highly regulated. Our findings provide new insights into the mechanisms of copper control. Maintaining non-ceruloplasmin-bound copper within the normal range is an important treatment goal in WD as this “free” copper is considered toxic to the liver and brain. We found that intravenously injected non-ceruloplasmin-bound copper quickly distributed to a number of tissues, especially skeletal muscle, subcutaneous fat, and the liver, while uptake into the brain was slow. This study offers new insights into the mechanisms of copper control, which may encourage further research into potential new treatment targets. 2016–001975-59.