Copper chelation in patients with hypertrophic cardiomyopathy.

BACKGROUND: Disturbances of copper (Cu) homeostasis can lead to hypertrophic cardiac phenotypes (eg, Wilson's disease). We previously identified abnormal Cu homeostasis in patients with hypertrophic cardiomyopathy (HCM) and, therefore, hypothesised that Cu2+-selective chelation with trientine dihydrochloride may slow or reverse disease progression in HCM. The aim of this study was, therefore to explore the clinical efficacy, safety and tolerability of trientine in HCM. METHODS: In this medicines and healthcare products regulatory agency (MHRA) registered open-label pilot study, we treated 20 HCM patients with trientine for 6 months. Patients underwent a comprehensive assessment schedule including separate cardiac magnetic resonance imaging (CMR) and CMR 31P-spectroscopy at baseline and end of therapy. Predefined end points included changes in left ventricular mass (LVM), markers of LV fibrosis, markers of LV performance and myocardial energetics. Ten matched patients with HCM were studied as controls. RESULTS: Trientine treatment was safe and tolerated. Trientine caused a substantial increase in urinary copper excretion (0.42±0.2 vs 2.02±1.0, p=0.001) without affecting serum copper concentrations. Treatment was associated with significant improvements in total atrial strain and global longitudinal LV strain using both Echo and CMR. LVM decreased significantly in the treatment arm compared with the control group (-4.2 g v 1.8 g, p=0.03). A strong trend towards an absolute decrease in LVM was observed in the treatment group (p=0.06). These changes were associated with a significant change in total myocardial volume driven by a significant reduction in extracellular matrix (ECM) volume (43.83±18.42 mL vs 41.49±16.89 mL, p=0.04) as opposed to pure cellular mass reduction and occurred against a background of significant ECM volume increase in the control group (44.59±16.50 mL vs 47.48±19.30 mL, p=0.02). A non-significant 10% increase in myocardial phosphocreatine/adenosine triphosphate (PCr/ATP) ratio with trientine therapy (1.27±0.44 vs 1.4±0.39) was noted. CONCLUSIONS: Cu2+-selective chelation with trientine in a controlled environment is safe and a potential future therapeutic target. A phase 2b trial is now underway.

Comparison of the Pharmacokinetic Profiles of Trientine Tetrahydrochloride and Trientine Dihydrochloride in Healthy Subjects.

BACKGROUND AND OBJECTIVE: Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism. Chelation of excessive copper is recommended but data on the pharmacokinetics of trientine are limited. The aim of this study was to compare the pharmacokinetics of a new trientine tetrahydrochloride formulation (TETA 4HCl) with those of an established trientine dihydrochloride (TETA 2HCl) salt. METHODS: A randomised single-centre crossover study to evaluate the pharmacokinetics, safety and tolerability of two different oral formulations of trientine (TETA 4HCl tablets vs TETA 2HCl capsules) in 23 healthy adult subjects receiving a single dose equivalent to 600 mg of trientine base was performed. RESULTS: Following oral administration, the median time to reach maximum plasma concentration (Tmax) was 2.00 h (TETA 4HCl) and 3.00 h (TETA 2HCl). The rate (maximum plasma concentration [Cmax]) and extent (area under the plasma concentration-time curve from time zero to infinity [AUC0-∞]) of absorption of the active moiety, trientine, were greater (by approximately 68% and 56%, respectively) for TETA 4HCl than for the TETA 2HCl formulation. The two formulations presented a similar terminal elimination rate (λz) and a similar terminal half-life (t½) for trientine. Differences between TETA 4HCl and TETA 2HCl in the levels of the two main mono- and diacetylated metabolites were less than seen for trientine. For both tested formulations, healthy male volunteers demonstrated higher trientine plasma levels but lower mono- and diacetylated metabolite levels compared with females, with no sex differences in terminal half-life (t½) observed. Single oral doses of both formulations were safe and well tolerated. CONCLUSIONS: Compared with an identical dose of a TETA 2HCl formulation, the TETA 4HCl formulation provided more rapid absorption of trientine and greater systemic exposure in healthy subjects. Clinical Trials Number EudraCT # 2015-002199-25.

[Diagnosis and treatment of Wilson disease in Japan].

Wilson disease is an autosomal recessive disorder based on inborn error of copper metabolism. The copper accumulates in the liver, brain, cornea, kidney, and other organs. This disease should be considered any individual with liver abnormality except infant, any patient older than teenage with neurological (especially for extra pyramidal signs) or neuropsychiatric disorder with or without liver disease and sibling of Wilson disease patient. Typically, a combination of low serum ceruloplasmine levels and high levels of urinary copper contents is sufficient to establish a diagnosis. As other diagnostic tests, measurement of hepatic copper content and ATP7B gene analysis are available. The key strategy of treatment is to reduce the amount of copper in the liver and other tissues by administering both copper-chelating agents, such as D-penicillamine or Trientine, and/or zinc acetate. The author recommend zinc acetate monotherapy for mild to moderate hepatic disorder, Trientine mono therapy for mild to moderate neurologic disorder and combination therapy of Trientine and zinc acetate for sever hepatic or neurologic disorder.

[Wilson's disease presenting as Asperger syndrome].

A 24-year-old man, who had been treated for 3 years as Asperger syndrome in adolescence due to behavioral disturbances, lack of social awareness and inability to socialize, was referred to our hospital shortly after tremors developed. On the basis of clinical features, laboratory findings and the brain MRI, a diagnosis of Wilson's disease (WD) was made. WD was further confirmed by genetic testing (the mutation of ATP7B gene). He was started with trientine hydrochloride 500 mg/day, and after 1 year of follow-up, his psychiatric symptoms have improved. Since psychiatric symptoms may precede the neurological symptoms, the possibility of WD should be always considered in the differential diagnosis of psychiatric disorders in young adults.

Delivery of Copper-chelating Trientine (TETA) to the central nervous system by surface modified liposomes.

The existence of the blood-brain barrier (BBB) complicates the treatment of many central nervous system (CNS) disorders, including the copper storage disease, Wilson's disease. Its CNS symptoms represent a serious problem, since therapeutics for Wilson's disease do not cross the BBB. One strategy to overcome this obstacle is the transfer of drugs across the BBB with colloidal carrier systems like liposomes. The aim of the present study was to encapsulate triethylenetetramine (TETA), a copper chelating agent, into surface modified liposomes and to investigate their permeation across the BBB. Liposomes were modified with cationized bovine serum albumin or penetratin, a cell penetrating peptide. Liposomes were characterized regarding size, PDI, zeta potential and encapsulation efficiency. Size was between 139.4±1.9nm to 171.1±3.5nm with PDI's below 0.2. Zeta potentials of vectorized liposomes were at least 6.9mV higher than those of standard liposomes. Cryo-TEM micrographs displayed liposomal structure, integrity and the similarity of structure and size between loaded, unloaded, vectorized and non- vectorized liposomes. In vivo experiments in rats showed an up to 16-fold higher brain uptake of TETA in vectorized liposomes compared to free TETA or TETA in non-vectorized liposomes, proving successful brain delivery using target seeking surface modifications. Tissue analysis indicated TETA concentrations in the brain being high enough to treat Wilson's disease.

Prospective pilot study of a single daily dosage of trientine for the treatment of Wilson disease.

BACKGROUND: Wilson disease requires lifelong therapy, currently given daily in multiple divided dosages. AIM: To prospectively evaluate once-daily trientine as therapy for Wilson disease. STUDY GROUP: eight patients (seven males) aged 22-71 years with stable Wilson disease treated from 4 to 50 years. Patients were monitored for 3 months then for 12 months on a single daily dose of trientine (15 mg/kg). RESULTS: All patients remained clinically well. ALT and AST fluctuated in some, but none required treatment stoppages or side effects. Liver synthetic function was unchanged. Mean 24-h urine copper and zinc excretions at end of treatment were 313.4 ± 191.7 and 2,214 ± 1,346 µg, respectively. CONCLUSIONS: Once-daily trientine should be explored further for possible maintenance therapy for WD. Single daily dose may improve adherence to therapy. Larger trials and longer-term follow-up will establish the safety and treatment efficacy of this once-daily treatment regimen for WD (registration: NCT01472874).

Chelation therapy in intoxications with mercury, lead and copper.

In the present review we provide an update of the appropriate use of chelating agents in the treatment of intoxications with compounds of mercury, lead and copper. The relatively new chelators meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercapto-propanesulphonate (DMPS) can effectively mobilize deposits of mercury as well as of lead into the urine. These drugs can be administered orally and have relatively low toxicity compared to the classical antidote dimercaptopropanol (BAL). d-Penicillamine has been widely used in copper overload, although 2,3-dimercaptosuccinic acid or tetrathiomolybdate may be more suitable alternatives today. In copper-toxicity, a free radical scavenger might be recommended as adjuvant to the chelator therapy.

Exploratory study of carboplatin plus the copper-lowering agent trientine in patients with advanced malignancies.

PURPOSE: Preclinical data showed that trientine, a copper-lowering agent, re-sensitized cancer cells to carboplatin through enhanced human copper transporter 1 (hCtr1) -mediated platinum uptake. EXPERIMENTAL DESIGN: We studied carboplatin and trientine in patients (n = 55; 45 who had failed platinum) with advanced malignancies (Phase I, modified 3 + 3 design). RESULTS: The most common cancers were head and neck (n = 13), non-small cell lung (n = 10) and epithelial ovarian (n = 8). The median number of prior regimens was four. No dose-limiting toxicity or treatment-related deaths were observed at doses up to carboplatin AUC 6 given with trientine. Eight patients achieved stable disease (SD) ≥ 6 months (six platinum failures) and one patient with platinum-resistant ovarian cancer, partial response (PR) (total SD ≥ 6 months/PR = 9, 16.4 %). The mean nadir serum copper level in the nine patients with SD ≥ 6 months/PR was 0.55 µg/mL (95 % CI, 0.34-0.75) versus 1.22 µg/mL (95 % CI, 1.02-1.42) (p < 0.001) in 38 tested patients with progression. In patients who maintained their ceruloplasmin (major copper-carrying protein) levels at 5-15 mg/dL (n = 9), the median progression-free and overall survivals were 9.2 and 15.2 months versus 1.9 (p = 0.001) and 5.7 months (p = 0.033) in patients who did not (n = 38), respectively. CONCLUSIONS: The combination of a copper-lowering agent with carboplatin was well tolerated and associated with antitumor activity, especially in patients in whom copper and/or ceruloplasmin levels were lowered. Further investigation of this strategy for reversing platinum resistance is warranted.

Co-administration of trientine and flaxseed oil on oxidative stress, serum lipids and heart structure in diabetic rats.

The administration of flaxseed oil or flaxseed oil plus trientine in diabetic rats reduced triglyceride, very low density lipoprotein, and total cholesterol. Furthermore, the combined treatment significantly increased superoxide dismutase activity and attenuated serum Cu2+. The results suggest that the administration of flaxseed oil plus trientine is useful in controlling serum lipid abnormalities, oxidative stress, restoring heart structure, and reducing serum Cu2+ in diabetic rats.

Efficacy and safety of oral chelators in treatment of patients with Wilson disease.

BACKGROUND & AIMS: Wilson disease is a genetic copper storage disorder that causes hepatic and neurologic symptoms. Chelating agents (D-penicillamine, trientine) are used as first-line therapies for symptomatic patients, but there are few data from large cohorts. We assessed the safety of D-penicillamine and trientine therapy and outcomes of patients with Wilson disease. METHODS: We performed a retrospective analysis of data on 380 patients with Wilson disease from tertiary care centers in Germany and Austria, and 25 additional patients from the EUROWILSON registry. Chelator-based treatment regimens were analyzed for their effect on neurologic and hepatic symptoms and for adverse events that led to discontinuation of therapy (Kaplan-Meier estimation; data were collected for a mean of 13.3 y after therapy began). RESULTS: Changes in medication were common, resulting in analysis of 471 chelator monotherapies (326 patients receiving D-penicillamine and 141 receiving trientine). Nine of 326 patients treated with D-penicillamine and 3 of 141 patients given trientine underwent liver transplantation. Adverse events leading to discontinuation of treatment were more frequent among those receiving D-penicillamine than trientine (P = .039). Forty-eight months after therapy, hepatic deterioration was reported in only 4 of 333 patients treated initially with a chelating agent. Hepatic improvements were observed in more than 90%, and neurologic improvements were observed in more than 55%, of therapy-naive patients, and values did not differ significantly between treatments. However, neurologic deterioration was observed less frequently in patients given D-penicillamine first (6 of 295) than those given trientine first (4 of 38; P = .018). CONCLUSIONS: Chelating agents are effective therapies for most patients with Wilson disease; D-penicillamine and trientine produce comparable outcomes, although D-penicillamine had a higher rate of adverse events. Few patients receiving chelation therapy had neurologic deterioration, which occurred more frequently in patients who received trientine.

Comparative evaluation of small-molecule chemosensitizers in reversal of cisplatin resistance in ovarian cancer cells.

Cisplatin-resistance is one of the major challenges in the treatment of epithelial ovarian cancer. Small-molecule chemosensitizers provide a therapeutically feasible approach to overcome cisplatin resistance in ovarian cancer. However, proper selection of chemosensitizer is of prime importance owing to phenotypic differences in cisplatin-resistant ovarian cancers. The resistance reversal activity of chemosensitizers buthionine sulfoximine (BSO), triethylenetetramine (TETA), genistein, rapamycin and colchicine was investigated in various cisplatin-resistant ovarian cancer cells, 2008 C13, CP70 and OVCAR 8 using MTT assays. Cellular accumulation of cisplatin in the presence of chemosensitizers was analyzed by inductively-coupled plasma-mass spectroscopy (ICP-MS). Chemosensitizers exhibited resistance reversal activity in 2008 C13 and CP70 cells in the following order; colchicine> genistein>TETA> rapamycin ≥ BSO (p<0.05), which is in correlation with cellular accumulation of cisplatin. In conclusion, our study demonstrates that resistance reversal activity of chemosensitizers varies with phenotypic behavior of cisplatin-resistant ovarian cancer cells. Data from our study can be utilized to choose a specific chemosensitizer for individualized combination therapy for cisplatin-resistant ovarian cancer.

Functional properties and biodistribution of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) mixtures formed with nucleic acid.

The clinical success of non-viral gene delivery reagents is hampered by their inefficient cellular transgene delivery, which is largely influenced by carrier properties that are currently undefined and misunderstood. In an attempt to further define and understand the requirements for a safe and efficient non-viral gene delivery reagent, research labs often engineer and evaluate many putative products with subtle physiochemical differences in order to delineate requirements for improved in vitro and in vivo success. The synthesis of many putative reagents is often time-intensive, laborious and costly. In a previous manuscript published by our lab, different amounts of poly(triethylenetetramine/cystamine bisacrylamide) (p(TETA/CBA) and its pegylated counterpart, poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) (p(TETA/CBA)-g-PEG) were mixed together to easily identify optimal reagent properties and candidates in vitro, while avoiding the synthesis of many putative candidates for study. This report uses the aforementioned facile approach to evaluate reagent properties of products that were obtained via one-pot synthesis, which improved synthetic ease. As such, synthesis time was reduced from 6days to 3days and had comparable or improved transfection and viability compared to previous works. Moreover, this synthesis resulted in higher molecular weight products than were used in the previous study and allow for lower polymer doses to be used for complexation, which is useful for systemic delivery that is used herein. The physiochemical properties of the formulations derived using these novel reagents was studied prior to investigating their in vivo biodistribution profiles in a murine colon adenocarcinoma model. Interestingly, negatively charged complexes exhibited greater passive tumor accumulation compared to positively charged complexes following their systemic administration. These studies warrant further investigation for the use of negatively charged drug and gene delivery reagents for passive tumor targeting, and they substantiate the use of polycation/PEG-polycation mixtures for facile product evaluation in order to elucidate design and formulation mandates for the clinical success of non-viral gene delivery formulations.

Excess copper chelating therapy for Wilson disease induces anemia and liver dysfunction.

A 37-year-old man was diagnosed with Wilson disease at the age of 14. His first manifestations were neurological. He was treated with trientine for more than 10 years and suffered from anemia and liver dysfunction. Wilson disease is a genetic disorder characterized by accumulation of copper in the body. Excess copper is toxic, but copper is an essential trace element. Copper-binding ceruloplasmin is important for iron metabolism. Excess copper chelating treatment-induced anemia and iron deposition in the liver was suspected. Proper monitoring of copper status is important for the management of Wilson disease.

Pharmacological properties of orally available, amphipathic polyaminocarboxylic acid chelators for actinide decorporation.

Commonly used water-soluble polyaminocarboxylic acid (PACA) chelators, such as EDTA and DTPA, require intravenous or subcutaneous administration due to their poor bioavailability. The bioavailability of PACAs can be improved by the addition of differing lengths of alkyl side chains that alter amphipathic properties. Orally administered amphipathic triethylenetetramine pentaacetic acid (TT) compounds are efficacious for decorporation of plutonium and americium. The synthesis, efficacy, binding affinities, and some initial pharmacokinetics properties of amphipathic TT chelators are reviewed. C-labeled C12TT and C22TT chelators are reasonably well absorbed from the intestine and have a substantial biliary/fecal excretion pathway, unlike DTPA, which is mostly excreted in the urine. Whole body retention times are increased as a function of increasing lipophilicity. Neutron-induced autoradiography studies demonstrate that the oral administration of the chelators can substantially inhibit the redistribution of Pu in skeletal tissues. In summary, amphipathic TT-based chelators have favorable bioavailability, have a significant biliary excretion pathway, have demonstrated efficacy for americium and plutonium, and are thus good candidates for further development. Furthermore, some of the pharmacological properties can be manipulated by changing the lengths of the alkyl side chains and this may have some advantage for decorporation of certain metals and radionuclides.

Evaluation of Cuprimine and Syprine for decorporation of (60)Co and (210)Po.

The acknowledged risk of deliberate release of radionuclides into local environments by terrorist activities has prompted a drive to improve novel materials and methods for removing internally deposited radionuclides. These decorporation treatments will also benefit workers in the nuclear industry, should an exposure occur. Cuprimine and Syprine are oral therapeutics based on the active ingredients D-penicillamine and N,N'-bis-(2-aminoethyl)-1,2-ethanediamine dihydrochloride, respectively. These therapeutic drugs have been used for several decades to treat Wilson's disease, a genetic defect leading to copper overload, by chelation and accelerated excretion of internally deposited copper. Studies were undertaken to evaluate these FDA-approved drugs for the in vivo decorporation of radioactive cobalt (Co) and polonium (Po) using male Wistar-Han rats. In these studies, Co or Po was administered to animals by IV injection, followed by oral gavage doses of either Cuprimine or Syprine. Control animals received the radionuclide alone. For Co studies, animals received a single dose of Cuprimine or Syprine, while for Po studies animals were repeatedly dosed at 24-h intervals for a total of 5 doses. Results show that Syprine significantly increased urinary elimination and skeletal concentrations of Co compared to controls. While Cuprimine had little effect on total excretion of Co, the skeletal, kidney, liver, muscle, and stomach tissues had significantly lower radioactivity compared to control animals. The low overall excretion of Po made it difficult to reliably measure urinary or fecal radioactivity and draw a definitive conclusion on the effect of Cuprimine or Syprine treatment on excretion. However, Cuprimine treatment was effective at reducing spleen levels of Po compared to controls. Similarly, Syprine treatment produced statistically significant reductions of Po in the spleen and skeletal tissues compared to control animals. Based on these promising findings, further studies to evaluate the dose-response pharmacokinetic profiles for decorporation are warranted.

Treatment of Wilson's disease with tetrathiomolybdate: V. Control of free copper by tetrathiomolybdate and a comparison with trientine.

It has become clear that serum "free" copper (the copper not bound to ceruloplasmin in the blood) is the copper causing copper toxicity in Wilson's disease. But up until now, free copper has not been closely followed during initiation of anticopper therapy in neurologically presenting patients. During this period of initial therapy, the future fate of these patients hangs in the balance-if they worsen neurologically as often happens with penicillamine or trientine therapy, many never recover. We hypothesize that free copper levels are a biological marker of clinical outcome in these patients. In this article, we evaluate the control of free copper in 3 studies of initial anticopper treatment in neurologically presenting Wilson's disease patients. The first (study 1) is a 55-patient open-label trial of tetrathiomolybdate, the second (study 2) is a 48-patient double-blind trial comparing tetrathiomolybdate and trientine, and the third (study 3) is a 40-patient double-blind comparison of 2 disease regimens of tetrathiomolybdate. Free copper levels were determined by subtracting ceruloplasmin and tetrathiomolybdate bound copper from total serum copper. Tetrathiomolybdate showed very strong control of free copper levels over the 8 weeks of treatment in the 55-patient open-label study (study 1), reducing it to a mean value of about one fourth, or less, of baseline. In the tetrathiomolybdate/trientine double blind (study 2), tetrathiomolybdate again showed good control of free copper levels over 8 weeks of treatment, which is significantly better than trientine. In the trientine arm of study 2, mean free copper levels actually went up during trientine therapy. The 5 patients who neurologically worsened on trientine therapy over 8 weeks of treatment showed significant spikes in serum free copper levels associated in time with their neurologic worsening. Patients who did not worsen neurologically generally did not show significant spikes in free copper. Tetrathiomolybdate controlled copper less well in the dose regimen study (study 3) than in the previous 2 studies of tetrathiomolybdate treatment, probably because of a change in the way "away from food" tetrathiomolybdate was given.

Therapeutic effects of an oral chelator targeting skeletal tissue damage in experimental postmenopausal osteoporosis in rats.

Earlier studies revealed that age-associated iron accumulation plays an important causal role in osteopenic development after estrogen deficiency. It is believed that an increase in iron content is associated with an increased likelihood of oxidative damage at the point of iron accumulation. However, there is no direct evidence that the iron accumulated in skeletal tissue causes free radical oxidative damage and consequent bone loss. Iron depletion from skeletal tissues of ovariectomized (OVX) rats was carried out with the oral chelator [1-N-docosyl-triethylenetetraminepentaacetic acid (DoTTPA)]. Results suggest the causal role of iron in oxidative damage that may lead to bone loss in the rats. The results also show the therapeutic potential of the bone-targeted chelator to protect against bone loss associated with age-iron accumulation as well as iron overload diseases.

Triethylene tetramine, a novel ligand of G-quadruplex, induces senescence of MCF-7 cells.

Interference with telomerase and telomere maintenance is emerging as an attractive target for antitumor therapies. Ligands stabilizing G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation of telomeres in tumors. Here, we report that long-term treatment with triethylene tetramine (TETA), at 50 or 100 microM, induced marked cellular senescence phenotypes accompanied by increased time of population doubling of MCF-7 cells. Cyclin-dependent kinase inhibitors, including p53 and p21, were also upregulated in TETA-treated MCF-7 cells. TETA is therefore as novel ligand of G-quadruplex and can induce tumor senescence; it is a promising material for tumor treatment.