OF COPPER METABOLISM

FIELD OF THE INVENTION

The present invention provides means and methods for treating disorders of copper metabolism in the brain of a subject comprising administering a therapeutically effective amount of chelators of copper encapsulated into liposomes which are functionalized to cross the blood-brain barrier. These means and methods are particularly suitable for the treatment of neurologic deterioration of subjects suffering from Wilson disease.

BACKGROUND OF THE INVENTION

Because copper is both essential and toxic, its amount, its redox state and its speciation are tightly regulated to maintain the physiological state of the cell. The homeostasis of copper requires regulation of copper uptake, delivery to the copper enzymes, sequestration and excretion. If normal copper homeostasis is impaired as a result, for example, of mutations or dietary imbalances, then both copper deficiency and copper overload impact on human health. Copper deficiency leads to a decrease in the activity of physiologically important enzymes while copper overload produces reactive oxygen species and carbon-centered free radicals which damage cellular components (Rupert Purchase (2013) Science Progress, 96(3), 213-223).

Wilson's disease (WD) or hepatolenticular degeneration is a rare inherited autosomal recessive disorder of copper metabolism characterized by the deposition of copper in the brain, liver, cornea, and other organs. Copper overload in patients with WD is caused by impairment to the biliary route for excretion of dietary copper, leading to an imbalance in copper homeostasis. With a loss of homeostasis, the redox- chemistry of Cu(l)/Cu(ll), which many enzymes and proteins utilize intracellularly, is responsible for copper's cytotoxicity. A combination of neurological, psychiatric and hepatic symptoms, which are a consequence of copper deposition, directly or indirectly, can make the diagnosis of WD challenging. Most symptoms appear in the second and third decades of life. The disease affects between one in 30,000 and one in 100,000 individuals (Rupert Purchase (2013) Science Progress, 96(1 ), 19-32). Young patients, who suffer from mainly internal complications of untreated WD, such as hemolysis, liver and/or renal failure, die within 2 to 7 years. Current guidelines for treating Wilson disease demand a lifelong medical treatment as the copper uptake cannot be controlled by a low copper diet. Independent of the chosen medical regimen, non-adherence or discontinuation of medical therapy in terms of noncompliance is associated with the risk of intractable hepatic or neurologic deterioration. A sequential treatment concept differentiates between the initial (acute) de-coppering therapy and the subsequent maintenance therapy. This concept takes into account that after the initial, more aggressive treatment phase addressing the high copper load, a reduced dose of the chelating drug of choice or less toxic medical approach with zinc might be sufficient to maintain copper homeostasis. Adjustment of the maintenance treatment dose also helps to prevent an overtreatment resulting in a functional copper deficiency.

One major "medical need" in this autosomal recessively inherited copper overload disorder is the disappointing efficacy of the commonly used drugs in neurologic patients. This includes the risk of neurologic deterioration after the initiation of chelation therapy.

Thus, there is a need to provide means and methods that take care of the unsatisfied need in the art as regards the disappointing efficacy of the commonly used drugs in neurologic patients suffering from disorders of copper metabolism in the brain.

The present invention satisfies this need and provides means and methods for treating disorders of copper metabolism in the brain of a subject by the administration of a therapeutically effective amount of chelators of copper encapsulated into liposomes which are functionalized to cross the blood-brain barrier.

Knowing that the presence of copper in the brains of Wilson disease patients is the cause of CNS injury, but this copper has been hard to target directly, since water- soluble medications typically do not cross the blood-brain barrier, the present inventors aimed at a solution. They thus suggest to directly target the CNS with liposomes having encapsulated chelators of copper and which are functionalized to cross the blood-brain barrier. Specifically, the inventors encapsulated triethylenetetramine, a copper chelator, into liposomes with a targeting vector (e.g. cationized cBSA) attached to the liposome surface permitting delivery across the blood-brain barrier into CNS tissue. Such liposomes functionalized to cross the blood-brain barrier were taken up into brain capillary endothelial cells, whereas liposomes without targeting ligand, i.e., non-functionalized liposomes had negligible uptake. Confocal fluorescence microscopy after intravenous administration of functionalized liposomes showed uptake into the brains of rats. These data indicated the feasibility of this strategy.

Accordingly, the present invention provides a method for treating disorders of copper metabolism in the brain of a subject comprising administering a therapeutically effective amount of chelators of copper functionalized to cross the blood-brain barrier.

The subject as referred to herein is a mammal such as a human, dog, cat, horse, cattle, but preferably the subject is a human. Said human is preferably diagnosed to suffer from Wilson disease.

Wilson disease (WD) is an autosomal recessive genetic disorder characterized by the accumulation and/or deposition of copper in the brain, liver, cornea, and other organs; this manifests as neurological or psychiatric symptoms and liver disease. Wilson disease is also called hepatolenticular degeneration. It is treated with medication that reduces copper absorption or removes the excess copper from the body, but occasionally a liver transplant is required. The condition is due to mutations in the Wilson disease protein (ATP7B) gene. A single abnormal copy of the gene is present in 1 in 100 people, who do not develop any symptoms (they are carriers). If a child inherits the gene from both parents, the child may develop Wilson's disease. Symptoms usually appear between the ages of 6 and 20 years, but cases in much older people have been described. Wilson's disease occurs in 1 to 4 per 100,000 people.

The term "treatment" or "treating" or any grammatical form thereof as used herein means in the broadest sense medical procedures or applications that are intended to relieve illness, here the symptoms of Wilson disease. Said term also includes an amelioration of the symptoms of Wilson disease. The term "amelioration" as used herein is synonymous with improvement.

By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered, preferably the effect is a decrease of copper deposits in the brain. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status, and other drugs being administered concurrently. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the adult patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status, and other drugs being administered concurrently. A typical dose can be, for example, in the ranges set forth in the embodiments of the invention and the appended examples; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. It is preferably envisaged that the administration of liposomes as described herein improves neurological symptoms associated with Wilson disease or prevents further worsening.

A chelator of copper is an agent that is capable of chelating copper. "Chelation" involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds, and are called chelants, chelators, chelating agents, or sequestering agents. Preferred copper chelator applied in the means and methods of the present invention comprise penicillamine, trientine, or tetrathiomolybdate.

The present invention makes use of liposomes that are able to cross the blood-brain barrier. This phenomenon is known in the art, since liposomes when they are "functionalized" are able to cross the blood-brain barrier. "Functionalized" means that liposomes are decorated with a targeting entity permitting the liposome to cross the blood-brain barrier.

Whether or not a targeting entity permits a liposome to cross the blood-brain barrier can be tested by in vitro blood-brain barrier (BBB) models such as those described in Eigenmann et al. Fluids and Barriers of the CNS 2013, 10:33 - doi: 10.1 186/2045- 81 18-10-33). Examples of such targeting entities are cationized albumin (cBSA), apolipoprotein E fragment, anti-transferrin receptor antibody, insulin, insulin-like growth factor, leptin or glutathione. These examples of targeting entities are preferred ones in the context of the present invention.

A liposome can be decorated with a targeting entity by embedding the targeting entity into the liposome layer(s), by linkage, e.g., biotin/streptavidin linkage, by covalent ligation, e.g., thiol-maleimide covalent ligation.

The present invention also provides a kit comprising a copper chelator and a targeting entity permitting a liposome to cross the blood-brain barrier.

Furthermore, the present invention provides a kit comprising liposomes having encapsulated a copper chelator, said liposomes are functionalized to cross the blood- brain barrier. The kit of the present invention is preferably a pharmaceutical kit. However, it is also preferred that the kit is a diagnostic kit.