For decades, the prevailing opinion held by physicians has been that DPA was more potent than trientine-2HCl in the treatment of WD, a perception based on higher levels of UCE in patients previously exposed to DPA [22]. These observations were largely unchallenged because of the difficulties in measuring free copper and fecal copper [27]. However, DPA has a conditional boxed warning label approval in the USA because of potential serious adverse reactions and clinical reports of up to 30% of patients experiencing adverse reactions necessitating discontinuation. In contrast, trientine has a better safety profile and is non-immunogenic. A non-inferiority trial comparing TETA-4HCl with DPA, if confirmed, would provide physicians and patients with an evidence-based alternative to DPA as continuation of first-line treatment, post de-coppering, and prior to the development of adverse events secondary to DPA therapy.

Trientine tetrahydrochloride was approved by the European Medicines Agency in 2017 for the indication of “Wilson disease patients intolerant to d-penicillamine”. For US approval, the sponsor sought approval for a new indication; patients who have been de-coppered, are clinically stable, and tolerant to DPA. The primary intention of this indication was to address an unmet need, providing an alternative to DPA before clinical intolerance was recognized, as some known toxicities of the skin (i.e., elastosis perforans) may develop insidiously. By demonstrating non-inferiority for this population, TETA-4HCl provides an approved alternative therapy.

The CHELATE trial (NCT03539952), a multicenter, randomized, open-label clinical trial, compared TETA-4HCl with DPA in stable patients with WD. The primary hypothesis and endpoint were that the efficacy of TETA-4HCl was non-inferior to penicillamine, as measured using the NCC-Sp assay developed by Orphalan Inc. at 24 weeks post-randomization. The assay was developed and validated [28] at the request of the FDA after limitations of an exchangeable copper assay were identified during the study [28, 29]. The secondary composite efficacy endpoint was NCC and 24-h UCE.

The study enrolled 53 adult subjects (aged 18–75 years) with clinically stable WD, with a Leipzig score ≥4 at diagnosis. The study population was treated with DPA monotherapy for at least 1 year with no dose adjustments in the 6 months prior to screening. In addition, specific inclusion criteria, including laboratory measures of serum NCC levels ≥ 25 and ≤ 150 mcg/L, 24-h UCE between ≥ 100 and ≤ 900 mcg/24 h, and liver function tests (alanine aminotransferase < 2 times the upper limit of normal) defined clinical stability alongside assessments made by an independent adjudication committee of three WD experts who would later be blinded to treatment allocation post-randomization. After 24 weeks, the mean difference in the serum NCC-Sp assay between the penicillamine group and the TETA-4HCl group was −9.1 μg/L (95% CI −24.2, 6.1), with the lower limit of the 95% CI within the defined non-inferiority margin, meeting the primary endpoint of the study (Fig. 3). Urinary copper excretion decreased 50% with TETA-4HCl versus penicillamine (mean difference 237.5 μg/24 h [99% CI 115.6, 359.4]) (Fig. 4).

Mean difference (95% confidence interval) in serum non-ceruloplasmin-bound copper by speciation (NCC-Sp) assay from baseline up to 48-weeks post randomization. DPA d-penicillamine, TETA-4HCL trientine tetrahydrochloride

Primary efficacy measure: serum non-ceruloplasmin-bound copper by speciation at week 24. DPA d-penicillamine, TETA-4HCL trientine tetrahydrochloride

At the end of the extension period (48 weeks), TETA-4HCl remained non-inferior to penicillamine (mean difference NCC −15.5 μg/L [95% CI –34.5, 3.6]). Urinary copper excretion at 48 weeks remained in the expected range for well-treated patients in both study groups, and the mean difference (124.8 μg/24 h [99% CI –37.6, 287.1]) was not significantly different [30].

The CHELATE study is the first prospective head-to-head trial comparing any trientine formulation with DPA. Results demonstrated that TETA-4HCl is non-inferior to DPA as measured by the NCC using a newly developed methodology [28] of copper protein speciation using high-performance liquid chromatography inductively coupled mass spectroscopy assay (NCC-Sp). Trientine tetrahydrochloride was well tolerated and during treatment, more patients achieved the pre-specified composite endpoint of NCC and 24-h UCE within therapeutic target ranges, compared with patients treated with DPA, 50% vs 24%, respectively. Neurological, cognitive, and hepatic assessments confirmed clinical stability over a 48-week period. The independent adjudication committee, blinded to site and treatment allocation, confirmed all enrolled subjects remained clinically stable throughout the 48-week post randomization period.

In summary, in a first of its type of a direct head-to head comparison of two chelators for the treatment of WD, the CHELATE trial demonstrated that TETA-4HCl is non-inferior to DPA. Although trientine-2HCl was first approved in 1985, the breakthrough development program for TETA-4HCl produced the first scored tablet form that could be split, and in contrast to trientine-2HCl, was the first room temperature-stable trientine. Trientine tetrahydrochloride addressed one of the biggest challenges in supply and stability, recognized as important factors affecting patient adherence to therapy. Most trientine-2HCl formulations require storage at 4 °C to mitigate product degradation. In contrast to room temperature stability for TETA-4HCl, trientine-2HCl rapidly converts to a dihydrate form at room temperature, with normal humidity (e.g., relative humidity; 40%). The dihydrate form reduces solubility by 50% compared with the original trientine-2HCl, which may negatively impact the absorption and efficacy of trientine-2HCl in patients [11].

During the early conduct of the phase III CHELATE trial, the NCC exchangeable assay resulted in an underestimation of NCC lacking robust precision and accuracy to be used for assessment of copper load as a primary efficacy endpoint. Recognizing this limitation, Orphalan Inc., in consultation with the FDA, developed an alternative two-step method to determine NCC.

The NCC-Sp assay is a validated, liquid chromatography inductively coupled plasma mass spectroscopy method [28]. Inductively coupled plasma mass spectroscopy is used to quantify total serum copper. The second part utilizes liquid chromatography to separate all the copper-bound proteins; the largest peak is ceruloplasmin-bound copper, followed by albumin and alpha-2 macroglobulin peaks. The area under each peak quantifies the proportion of bound copper to each protein. Non-ceruloplasmin-bound copper by speciation is then calculated by subtracting ceruloplasmin bound copper from total serum copper [28].

The accurate and precise measurement of NCC has the potential to be an important biomarker for WD; assessing copper toxicity and monitoring the efficacy of treatment in WD. Traditional diagnostic assays for NCC have been unreliable, leading to challenges in accurate measurements of copper load. It is estimated that 20% of patients with WD will have a negative estimated NCC because of ceruloplasmin overestimation by an immunoassay [31]. Importantly, the acceptance of the NCC-Sp assay by the FDA as the primary endpoint in the CHELATE trial underscores the reliability, precision, and accuracy of the assay. Importantly, the acceptance of the NCC-Sp assay by the FDA as the primary endpoint in the CHELATE trial underscores the reliability, precision, and accuracy of the assay. In the accompanying editorial to CHELATE trial, Medici and Heffern state, “This method [NCC-Sp] has promise to provide reliable and reproducible quantification due to its direct read-out of copper components excluding those bound to ceruloplasmin” [32]. This tool holds promise for providing clinicians in the future with a reliable assay for assessing treatment response and guiding therapeutic decisions [30].