HK1261266A1 - Method and pharmaceutical composition for treatment of neurodegeneration - Google Patents

Description

Methods and pharmaceutical compositions for treating neurodegeneration

Technical Field

The present invention relates to methods and compositions for treating acute or chronic pathological conditions characterized by neurodegeneration.

Background

Neurodegeneration is a term of coverage for a group of pathologies associated with progressive loss of brain tissue structure and/or reduced neuronal function (including death). Examples of such pathologies of the nervous system include parkinson's disease, alzheimer's disease, huntington's disease, age-related brain degeneration, creutzfeldt-jakob disease, and amyotrophic lateral sclerosis, all of which result from neurodegenerative processes. Currently, such neurodegenerative diseases are considered incurable and result in progressive degeneration and/or death of neuronal cells. Additionally, the mechanism of neurodegeneration has been reported to play a key role in the pathophysiology of spinal cord and brain injury with various etiologies, stroke, vascular dementia, and neurotoxicity induced by various agents (Wan et al, 2015; Faden et al, 2016).

Iron, an abundant metallic element in mammals in general and humans in particular, is a vital element that plays a key role in the vital system. In healthy adults, the total amount of iron is 3-4 grams, with some of this iron being associated with the redox and storage of ferryl enzymes, ferritin (including proteins involved in cellular respiration and electron transport). On the other hand, the "unstable" iron pool, in which iron has redox activity, represents a trace amount of iron in transition or transport, which acts as a catalyst for the generation of Reactive Oxygen Species (ROS), including hydroxyl radicals, by fenton's reaction. ROS produce oxidative stress that leads to tissue damage. Many publications link excess tissue (unstable redox activity) iron with ROS-induced tissue damage in a number of pathological phenomena and in different organs and tissues, including the central and peripheral nervous systems.

There is increasing evidence for a relationship between abnormal iron metabolism and/or excessive iron accumulation and the neurodegenerative process in certain brain regions. This connection has been reported for amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, stroke and spinal cord injury, Friedreich's ataxia, brain injury, type of intoxication and some other pathologies (Connor et al, 2001; Rouault.2013). Similar arguments may be stated for aluminum, but to a lesser extent for copper (Kumar and Gill, 2009; Scheiber et al, 2014). Several attempts have been made to apply different iron-chelating compounds in preclinical models to treat these pathologies; however, the main disadvantage of currently available iron chelators is their low ability to penetrate the cell membrane and, more importantly, to cross the blood-brain barrier. In addition, their administration is associated with negative side effects and toxicity.Without excess iron, iron chelators can interfere with the steady state of copper, zinc, and other metal micronutrients. And with Fe³⁺The stability constants of complexes of these iron chelators with other metal ions are significantly smaller than those of complexes of (a).

PrP can fold in a number of structurally different ways, at least one of which can transfer to other prion proteins, forming extremely stable aggregates leading to tissue damage and cell death.prion causes neurodegenerative diseases by extracellular aggregation within the central nervous system forming amyloid plaques that destroy normal brain tissue.this destruction is characterized by "holes" in the tissue due to vacuolization in neurons, with the resulting sponge architecture.according to some reports, redox active iron produced by the Fenton reaction contributes to PrP misfolding (Singh et al 2014; Kim et al 2000.) diseases caused by prion including Creutzfeldt-Jakob disease (CJD) and its subtypes (e.g. iatrogenic D (CJiCJD), variant CJD (CJvCJD) and sporadic CJD (SzsRoth-Creutzfeldt-Jakob disease), the pathological enc disease in the brain-related disease family, the pathological encephalospora protein, Parkinson's disease, the pathological amyloid-related diseases in Alzheimer's disease, Parkinson.

Zinc-desferrioxamine B (Zn-DFO) and gallium-desferrioxamine B (Ga-DFO) are metal complexes that have previously been shown to inhibit iron (and copper) catalysis during ROS formation. Their protective activity can be shown by chelating- "pulling" out and chelating available and redox active iron (which causes harm). In addition to the sequestration of iron by the DFO component of these complexes, the metal component (the relatively inert zinc or gallium ion) is released during the exchange of iron in the complex and further acts as a secondary antioxidant by "pushing" the additional iron ion from its binding site (Chevion, 1988; Chevion, 1991). The steric structure of these complexes is clearly different from that of DFO itself, allowing enhanced penetration of the complex into cells (Chevion, 1991).

As previously shown, treatment with Zn-DFO and/or Ga-DFO produces potent beneficial effects in animal models of human skin and corneal injury, asthma, diabetes, Inflammatory Bowel Disease (IBD), and cataract formation (Siganos et al, 1998; Bibi et al, 2014; US 8,975,294). Furthermore, topical administration of the metal-DFO complex was found to be effective in alleviating the symptoms of exposure to the chemical warfare agent mustard (mustard), more particularly nitrogen mustard (Banin et al, 2003; Morad et al, 2005).

the toxicity of A β oligomers may result from their ability to interact with and disrupt cell membranes, which promotes misfolding of A β plaque formation in cells³⁺) binding to membranes containing GM1 and preventing interaction with A β in cell culture therefore, the process of plaque propagation can be prevented (Williams et al, 2015)³⁺、Gd³⁺、La³⁺And Yb³⁺Similar characteristics, albeit to a lesser extent, were demonstrated. However, the ability of these ions to cross the blood-brain barrier is very limited, especially due to their charge.

Summary of The Invention

In order for a drug to be considered as a candidate for the treatment of neurodegeneration, the drug should first show efficacy providing specific benefits to the damaged brain cells, such as improving the inflammatory response and/or inhibiting the aggregation of amyloid molecules or synuclein. This can be shown even in brain cell culture. However, in order to be considered as a candidate for successful treatment of neurodegeneration, the drug should fulfill the further essential condition that it should be able to effectively penetrate into the brain by crossing e.g. the blood brain barrier or the blood CSF barrier.

It has now been found that metal-DFO complexes such as Zn-DFO complexes according to the invention are able to penetrate into the brain significantly better than DFO alone. In another study, illustrated herein, additional indirect evidence of the permeability of Zn-DFO complexes through the blood-cerebrospinal fluid (CSF) barrier has been obtained, which shows the beneficial effect of Zn-DFO complexes on spinal cord healing from mechanical trauma, as clearly demonstrated by the physiological parameters and structural aspects of the spinal cord. In other words, the studies exemplified herein show that metal-DFO complexes, such as Zn-DFO complexes, are very effective in reducing the rate of, i.e., inhibiting, reducing or ameliorating, neurodegenerative processes, and thus are capable of treating medical conditions associated with or characterized by neurodegenerative processes.

In this respect, it should be emphasized that the iron Fe-DFO complexes resulting from the exchange of zinc of Zn-DFO or gallium of Ga-DFO by iron supported in the tissue, in which iron does not undergo redox cycles, are inert complexes which are completely excreted outside the body.

In one aspect, the present invention therefore relates to a method for preventing, inhibiting, reducing or ameliorating neurodegeneration, and thus more specifically treating a disease, disorder or condition characterized by or associated with neurodegeneration, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one metal-DFO complex (e.g., a Zn-DFO complex) or a combination thereof.

The neurodegeneration prevented, inhibited, reduced, or improved by the methods of the present invention may be associated with: neurodegenerative diseases, disorders or symptoms, such as parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis, huntington's disease, or age-related brain degeneration; a disorder or condition caused by or resulting from injury, e.g., mechanical force, ischemia, toxic agents, or hemorrhage; or a disease, disorder or condition caused by a prion, such as creutzfeldt-jakob disease or a subtype thereof, fatal familial insomnia, kuru, familial spongiform encephalopathy, or multiple system atrophy.

In another aspect, the present invention provides a pharmaceutical composition for preventing, inhibiting, reducing or ameliorating neurodegeneration, and thus more specifically treating a condition characterized by or associated with neurodegeneration, comprising at least one metal-DFO complex (e.g., a Zn-DFO complex), or a combination thereof, and a pharmaceutically acceptable carrier.

In another aspect, the invention relates to metal-DFO complexes (e.g., Zn-DFO complexes) or combinations thereof for use in preventing, inhibiting, reducing, or ameliorating neurodegeneration.

In yet another aspect, the present invention relates to the use of a metal-DFO complex (e.g., a Zn-DFO complex) or a combination thereof in the manufacture of a pharmaceutical composition for preventing, inhibiting, reducing or ameliorating neurodegeneration.

In another aspect, the present invention provides a kit comprising (i) a first pharmaceutical composition comprising DFO or a pharmaceutically acceptable salt thereof; (ii) a second pharmaceutical composition comprising an ion of at least one metal; and (iii) instructions for administering the composition simultaneously or sequentially in any order and over a period of time not exceeding 6 hours, as follows, to obtain a complex of the DFO and the at least one metal in situ, thereby preventing, inhibiting, reducing, or ameliorating neurodegeneration, and thus more specifically treating a condition characterized by or associated with neurodegeneration.

Drawings

FIGS. 1A-1B show recovery-BMS scores for spinal cord injury in mice treated with Zn-DFO (1A); and normalized BMS score (1B) based on the initial value considered as 100%. The values are shown as mean values.

Figure 2 shows white matter as fraction (%) of total area of mice after spinal cord injury treated with Zn-DFO or saline. Values are shown as mean ± standard error.

FIGS. 3A-3C show sections of injured murine spinal cords stained with methylene blue, treated with saline (3A) or Zn-DFO (3B), and untreated (3C). The photographs were taken at magnification x 20.

FIGS. 4A-4B show spinal cords from mice treated with Zn-DFO every 0.01mm after spinal cord injury²The number of axons (4A); and total area of myelin sheath (AU/0.01 mm)²) (4B). Values are shown as mean ± standard error.

FIGS. 5A-5B show sections of H & E stained injured murine spinal cords treated with saline (5A) or Zn-DFO (5B). The photographs were taken at magnification of 60.

Figure 6 shows recovery-body weight of spinal cord injury in mice treated with Zn-DFO. The values are shown as mean values.

Detailed Description

In one aspect, the present invention relates to a method for preventing, inhibiting, reducing or ameliorating neurodegeneration, and thus more specifically treating a disease, disorder or condition characterized by or associated with neurodegeneration, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of at least one, i.e., one, two, three or more metal-DFO complexes.

without being bound by any theory, it is assumed that the protective effect of metal-DFO complexes is caused by several reasons, including inhibition of ROS formation, the ability of metal-DFO complexes to act through a combined "push-pull" mechanism to achieve significant reduction in free radical formation is supported by theoretical considerations and previously reported experimental findings.in the Fenton reaction or metal-mediated Haber-Weiss mechanism, the conversion of low reactive species to highly reactive hydroxyl radicals apparently depends on the availability of trace amounts of redox active ions and labile iron or copper ions, which are essential catalysts in ROS formation (Chevion, 1988; Chevion et al, 1993; Chevion et al, 1998). it is therefore assumed that the complexes, in particular Zn-DFO and Ga-DFO, exert their protective effect through the critical step of interfering with hydroxyl radical formation.

It is speculated that some of the useful roles that DFO plays in inhibiting ROS formation are achieved by its role as a chelator (chelant, chelator, or sequestrant) that is capable of forming soluble complexes, i.e. chelates, with certain metal ions and thus inactivating said metal ions such that they cannot normally react with other elements or ions. Such chelates generally have chemical and biological properties that are significantly different from the chelating agent or metal ion alone. For example, although DFO is a molecule having a noodle-like structure that can penetrate into cells in a small amount, a metal chelate formed from the molecule, such as a zinc-, gallium-or iron-chelate, takes a globular structure that can penetrate into cells. In recent experiments comparing the ability of DFO and Zn-DFO complexes to penetrate cell membranes in tissue culture models (using H9C2 cardiomyocytes), it has been found that the ability of Zn-DFO to penetrate into cells is more than three-fold higher than in DFO alone (data not shown).

The terms "DFO", "desferrioxamine B", "desferrioxamine (desferroxamine)" or "desferroxamine", as used interchangeably hereinRefers to the compound N' - [5- (acetyl-hydroxy-amino) pentyl]-N- [5- [3- (5-aminopentyl-hydroxy-carbamoyl) propionylamino]Pentyl radical]N-hydroxy-succinamide, a bacterial siderophore produced by Streptomyces piloides (generally considered as a safe organism).Iron chelate compounds consisting of six basic units sold in the form of their methanesulfonate salts. When not bound to metals, DFO is a linear (planar) molecule that does not readily penetrate into most cells; however, when the metal is bound, it forms a spherical complex. In addition to iron, DFO also forms a tight complex with zinc. Based on the similarity of ligand chemistry between iron or copper and zinc, it is assumed that the structure of the zinc-DFO complex is also spherical rather than linear. The metal binds to the negatively charged DFO making the molecule less polar. These considerations may explain why DFO complexes more readily penetrate cell membranes and biological barriers and bind more efficiently to redox-active intracellular metals that mediate tissue damage. In this process, two steps provide protection against oxidation: a) redox active iron and copper are removed by their chelation, and b) controlled release of zinc, which itself has antioxidant activity and is required for the full functioning of various metalloenzymes.

The relative stability constants of the DFO complex with Fe (III), Cu (II), Zn (II) and Ga (III) are 10 respectively³¹、10¹⁴、10¹¹And 10²⁸. Based on these thermodynamic properties, complexes of Fe or Cu with high abundance of low molecular weight and redox activity, Zn-DFO complexes exchange Zn with Fe or Cu, and zinc released from the complexes may have additional beneficial antioxidant and/or other effects when permeated into cells. It should be noted that the stability constant of the DFO complex with any lanthanide ion is expected to be less than 10³¹(Orcutt et al, 2010).

The term "subject" as used herein in relation to the methods of the invention refers to any mammal, e.g., a human.

As used herein, the term "therapeutically effective amount" with respect to one or more metal-DFO complexes administered according to the methods of the present invention refers to an amount of the one or more complexes that is sufficient to prevent, inhibit, reduce or ameliorate a neurodegenerative process that occurs in a subject to whom it is administered, when administered within a specified period of time, e.g., days, weeks or months, under a specified regimen. The actual dosage of the one or more metal-DFO complexes administered may be varied so as to obtain an amount of the one or more metal-DFO complexes that is effective to achieve the desired prophylactic/therapeutic response and mode of administration for a particular subject without being toxic to the subject. The selected dosage level depends on a variety of factors including the severity/progression of the disease, the disorder or condition being treated; the particular metal-DFO complex or complexes used, the route of administration and the duration of treatment; (ii) a drug used in combination with one or more metal-DFO complexes, if any, used; and the age, sex, and weight of the subject being treated. In general, it can be postulated that lower doses will be required for prophylactic treatment, whereas higher doses will be required for treatment of subjects already showing said pathological phenotype of neurodegeneration.

In certain embodiments, the metal-DFO complexes administered according to the methods of the invention are each independently a zinc-DFO complex, a gallium-DFO complex, a manganese-DFO complex, an indium-DFO complex, a silver-DFO complex, a cobalt-DFO complex, a gold-DFO complex, or a lanthanide-DFO complex. Specific lanthanides include lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium. According to the present invention, where a combination of metal-DFO complexes is administered, the combination may comprise any quantitative ratio of the metal-DFO complexes. For example, where a combination of two metal-DFO complexes is administered, the combination may comprise the two metal-DFO complexes in the following quantitative ratios: from about 100:1 to about 1:100, for example in the following quantitative ratios: about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1: 100. Similarly, where a combination of three metal-DFO complexes is administered, the combination may comprise the three metal-DFO complexes in the following quantitative ratios, e.g., about 1:1:1, about 1:2:3, about 1:10:50, about 1:20:50, about 1:10:100, or about 1:50: 100.

In specific embodiments, the metal-DFO complex administered according to the methods of the present invention is a Zn-DFO complex, a Ga-DFO complex, a Eu-DFO complex, a Gd-DFO complex, or a combination thereof, i.e., a combination of: Zn-DFO complex and Ga-DFO complex; Zn-DFO complex and Eu-DFO complex; Zn-DFO complex and Gd-DFO complex; Ga-DFO complex and Eu-DFO complex; Ga-DFO complex and Eu-DFO complex; Eu-DFO complex and Gd-DFO complex; Zn-DFO complex, Ga-DFO complex and Eu-DFO complex; Zn-DFO complex, Ga-DFO complex and Gd-DFO complex; Zn-DFO complex, Eu-DFO complex, and Gd-DFO complex; or Ga-DFO complex, Eu-DFO complex and Eu-DFO complex. In more specific such embodiments, a combination of both a Zn-DFO complex and a Ga-DFO complex is administered, e.g., such a combination: wherein the quantitative ratio of Zn-DFO complex to Ga-DFO complex is in the following range: about 100:1 to about 1:100, e.g., about 20:1 to about 1:20, about 10:1 to about 1:10, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1: 1. Some such combinations are those in which the amount of Zn-DFO complex is higher than the amount of Ga-DFO complex, for example the following combinations: wherein the quantitative ratio of the Zn-DFO complex to the Ga-DFO complex is within the following range: about 10:1 to about 2:1, e.g., about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

In certain embodiments, the methods of the invention comprise administering a combination of three or more metal-DFO complexes, for example a combination of a Zn-DFO complex and a Ga-DFO complex as defined above, together with one or more additional metal-DFO complexes (such as, for example, a Eu-DFO complex or a Gd-DFO complex).

According to any one of the embodiments as defined above, the method of the invention is intended to prevent, inhibit, reduce or ameliorate neurodegeneration, and thus actually treat a disease, disorder or condition characterized by or associated with said neurodegeneration. In certain embodiments, the disease, disorder, or condition characterized by or associated with neurodegeneration is a neurodegenerative disease, disorder, or condition, such as, without limitation: parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, or age-related brain degeneration. In other embodiments, the disorder or condition characterized by or associated with neurodegeneration is a disorder or condition caused by or resulting from injury (more particularly spinal cord and brain injury of various etiologies, such as injury caused by mechanical forces, ischemia, toxic agents, or hemorrhage). In further embodiments, the disease, disorder or condition characterized by or associated with the neurodegeneration is a disease, disorder or condition caused by a prion, such as, but not limited to: Creutzfeldt-Jakob disease (CJD) or subtypes thereof (e.g., Iatrogenic CJD (iCJD), variant CJD (vCJD), and sporadic CJD (sCJD)), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, Kuru, familial spongiform encephalopathy, or multiple system atrophy.

In another aspect, the present invention provides a pharmaceutical composition for preventing, inhibiting, reducing or ameliorating neurodegeneration, and thus more specifically treating a disease, disorder or condition characterized by or associated with neurodegeneration, comprising at least one metal-DFO complex (also referred to herein as "one or more active agents") and a pharmaceutically acceptable carrier.

In certain embodiments, the metal-DFO complexes included in the pharmaceutical compositions of the invention are each independently a zinc-DFO complex, a gallium-DFO complex, a manganese-DFO complex, an indium-DFO complex, a silver-DFO complex, a cobalt-DFO complex, a gold-DFO complex, or a lanthanide-DFO complex as defined above (including but not limited to europium-DFO complexes and gadolinium-DFO complexes). According to the present invention, the disclosed pharmaceutical compositions may comprise a combination of more than one, e.g. two or three, metal-DFO complexes as active agents in any quantitative ratio. For example, a composition comprising a combination of two metal-DFO complexes may comprise the two metal-DFO complexes in the following quantitative ratios: from about 100:1 to about 1:100, for example in the following quantitative ratios: about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1: 100. Similarly, where a combination of three metal-DFO complexes is administered, the combination may comprise the three metal-DFO complexes in the following quantitative ratios, e.g., about 1:1:1, about 1:2:3, about 1:10:50, about 1:20:50, about 1:10:100, or about 1:50: 100.

In a specific embodiment, the metal-DFO complex comprised in the pharmaceutical composition of the invention is a Zn-DFO complex, a Ga-DFO complex, a Eu-DFO complex, a Gd-DFO complex, or a combination thereof, i.e. a combination of: Zn-DFO complex and Ga-DFO complex; Zn-DFO complex and Eu-DFO complex; Zn-DFO complex and Gd-DFO complex; Ga-DFO complex and Eu-DFO complex; Ga-DFO complex and Eu-DFO complex; Eu-DFO complex and Gd-DFO complex; Zn-DFO complex, Ga-DFO complex and Eu-DFO complex; Zn-DFO complex, Ga-DFO complex and Gd-DFO complex; Zn-DFO complex, Eu-DFO complex, and Gd-DFO complex; or Ga-DFO complex, Eu-DFO complex and Eu-DFO complex. In more specific such embodiments, the composition comprises a combination of a Zn-DFO complex and a Ga-DFO complex, for example, wherein the quantitative ratio of Zn-DFO complex to Ga-DFO complex is within the following ranges: about 100:1 to about 1:100, e.g., about 20:1 to about 1:20, about 10:1 to about 1:10, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1: 1. Certain such compositions are those in which the amount of Zn-DFO complex is higher than the amount of Ga-DFO complex, for example wherein the quantitative ratio of Zn-DFO complex to Ga-DFO complex in the composition is in the following range: about 10:1 to about 2:1, such as about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

In certain embodiments, the pharmaceutical compositions of the invention comprise a combination of three or more metal-DFO complexes as active agents, for example a combination of a Zn-DFO complex and a Ga-DFO complex as defined above together with one or more further metal-DFO complexes (such as for example a Eu-DFO complex or a Gd-DFO complex).

The pharmaceutical composition of The present invention can be prepared by conventional techniques, for example, as described in Remington: The Science and practice of Pharmacy [ Remington: pharmaceutical science and practice ], 19 th edition, 1995. The composition may be prepared by: for example, uniformly and intimately bringing into association the active agent (i.e., the metal-DFO complex (s)) with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. The active agent may be administered as such, or conjugated to one or more pharmaceutically acceptable groups such as sugars, starches, amino acids, polyethylene glycols (PEGs), polyglyceryl compounds, hydrazines, hydroxylamines, amines, or halides. The compositions may be in liquid, solid or semi-solid form, and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, as well as other inert ingredients and excipients. Each inert ingredient should be pharmaceutically and physiologically acceptable, i.e., both compatible with the other ingredients and not injurious to the subject. In one embodiment, the pharmaceutical composition of the present invention is formulated as nanoparticles or microparticles.

The compositions contemplated herein may be formulated for any suitable route of administration, such as intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, intracerebral, intracerebroventricular, intrapleural, intratracheal, or subcutaneous administration; oral administration; rectal administration; nasal administration; or inhalation. The dosage will depend on the state of the patient and will be determined by the practitioner at different times as deemed appropriate. For example, a physician or veterinarian can start with a dose of one or more active agents used in a pharmaceutical composition at a level below the desired level to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Some routes of administration, such as intraperitoneal or intrarectal, may be used to treat intestinal dysfunction associated with neurodegenerative diseases.

The pharmaceutical compositions of the present invention may be administered, for example, continuously, daily, twice daily, three times daily, or four times daily for different durations, e.g., weeks, months, years, or decades.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable preparations which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limitation, water, ringer's solution, and isotonic sodium chloride solution.

When formulated for administration other than parenteral administration, the pharmaceutical compositions according to the invention may be in a form suitable for oral use, for example as tablets, troches, lozenges or aqueous suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain one or more active agents in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binders, such as starch, gelatin or acacia; and a lubricant. Tablets may be uncoated or coated using known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material may be employed. They may also be coated using the techniques described in U.S. Pat. nos. 4,256,108, 4,166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release.

When formulated for inhalation, the pharmaceutical compositions according to the present invention may be administered using any suitable device known in the art, for example, aerosol-based Metered Dose Inhalers (MDIs), liquid nebulizers, dry powder inhalers (dispensing devices), nebulizers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like.

The pharmaceutical compositions of the present invention may be formulated for controlled release of the active agent. Such compositions may be formulated as controlled release matrices, such as controlled release matrix tablets, in which release of the soluble active agent is controlled by diffusion of the active agent through a gel formed upon swelling of the hydrophilic polymer in contact with dissolved fluids (in vitro) or gastrointestinal fluids (in vivo). Many polymers have been described that are capable of forming such gels, for example derivatives of cellulose, in particular cellulose ethers, such as hydroxypropyl cellulose, hydroxymethyl cellulose, methyl cellulose or methylhydroxypropyl cellulose, and among the different commercial grades of these ethers are those that show higher viscosities. In other configurations, the composition comprises an active agent formulated for controlled release in a microencapsulated dosage form, wherein small droplets of the active agent are surrounded by a coating or film to form particles in the range of a few microns to several millimeters.

Other contemplated formulations are depot systems based on biodegradable polymers, wherein the active agent is slowly released as the polymer degrades. One of the most common types of biodegradable polymers is the hydrolytically unstable polyesters prepared from lactic acid, glycolic acid, or a combination of the two molecules. Polymers prepared from these individual monomers include poly (D, L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D, L-lactide-co-glycolide) (PLG).

The pharmaceutical compositions of the invention as defined in any of the embodiments above may be used to prevent, inhibit, reduce or ameliorate neurodegeneration and, therefore, for the treatment of diseases, disorders or conditions characterized by or associated with such neurodegeneration. The diseases, disorders or conditions treatable by the pharmaceutical compositions of the invention are any disease, disorder or condition characterized by or associated with neurodegeneration, such as neurodegenerative diseases, disorders or conditions; a disorder or condition caused by or resulting from injury, more particularly spinal cord injury or brain injury; or a disease, disorder or condition caused by prions.

The metal-DFO complexes used in accordance with the methods and compositions of the present invention can be prepared using any technique or procedure known in the art (e.g., as described in international publication No. WO 2011021203). The following provides possible procedures for preparing Zn-DFO and Ga-DFO complexes with various Zn-DFO/Ga-DFO stoichiometries. Such complexes with other stoichiometric ratios can be prepared using similar procedures.

A Zn-DFO complex having a stoichiometric ratio of Zn: DFO of 1.0:1.0 can be prepared, for example, by: 10mM DFO solution was mixed with an equal volume of 10mM ZnCl₂The solutions were mixed, titrated to a pH between 5.0 and 7.5, the mixture was heated to 45 ℃ for 30 minutes, and cooled. Alternatively, such complexes may be prepared by: dry 1 Small bottle (500mg, 0.76mmole)By adding 168mg of dry anhydrous zinc acetate (0.76mmole), adding double distilled water until the contents are completely dissolved (about 10ml), heating the solution to 40 ℃ for 45 minutes, and cooling.

A Zn-DFO complex having a stoichiometric ratio of Zn: DFO of 1.25:1.0 can be prepared, for example, by: 10mM DFO solution was mixed with an equal volume of 6mM ZnCl₂The solutions were mixed, titrated to a pH between 5.0 and 7.5, heated to 45 ℃ for 30 minutes, and cooled.

A Zn-DFO complex having a stoichiometric ratio of Zn: DFO of 0.6:1.0 can be prepared, for example, by: 10mM DFO solution was mixed with an equal volume of 12.5mM ZnCl₂The solution was mixed with 10ml of 5.5mM histidine, titrated to a pH between 5.0 and 7.5, heated to 45 ℃ for 30 minutes, and cooled.

A Zn-DFO complex having a stoichiometric ratio of Zn: DFO of 0.2:1.0 can be prepared, for example, by: 50mM DFO solution was mixed with 1/5 volume of 50mM ZnSO₄The solutions were mixed, titrated to a pH between 5.0 and 7.5, heated to 40 ℃ for 45 minutes, and cooled.

Ga-DFO complexes with a Ga: DFO stoichiometric ratio of 1.0:1.0 can be prepared, for example, by: a10 mM DFO solution was mixed with an equal volume of 10mM GaCl at room temperature₃The solutions were mixed, titrated to a pH of about 5.0 (using HCl) and then titrated to a pH between 5.0 and 7.5 (using NaOH). Similar complexes with a Ga: DFO stoichiometric ratio of 0.6:1.0 can be prepared, for example, by: 5mM DFO solution with an equal volume of 3mM GaCl at room temperature₃The solutions were mixed and titrated to a pH between 5.0 and 7.5.

In yet another aspect, the present invention relates to metal-DFO complexes or combinations thereof for use in preventing, inhibiting, reducing or ameliorating neurodegeneration, thereby more specifically treating a disease, disorder or condition characterized by or associated with neurodegeneration.

In yet another aspect, the present invention relates to the use of a metal-DFO complex or a combination thereof for the preparation of a pharmaceutical composition for preventing, inhibiting, reducing or ameliorating neurodegeneration, thereby more specifically treating a disease, disorder or condition characterized by or associated with neurodegeneration.

As previously shown, DFO is capable of extracting metals such as Fe and Zn from human plasma in vitro (soriyaarachhi and Gailer, 2010). It is therefore assumed that under physiological conditions, the simultaneous or sequential administration of DFO and a metal ion, such as Zn-, Ga-, Eu-or Gd-ion, from two separate compositions (provided that the interval between the administration of the two components is determined such that at least the major amount of the first administered component is available (i.e. not secreted) in the circulation when the second component is administered) will result in the in situ formation of the metal-DFO complex.

In another aspect, the present invention therefore relates to a method for preventing, inhibiting, reducing or ameliorating neurodegeneration in a subject in need thereof, thereby treating a disease, disorder or condition characterized by or associated with neurodegeneration, comprising administering to the subject a therapeutically effective amount of DFO or a pharmaceutically acceptable salt thereof and at least one ion of one, two, three or more metals, wherein the DFO or pharmaceutically acceptable salt thereof and the metal ion are administered from two separate compositions, simultaneously or sequentially, in any order and over a time of no more than 6 hours, so as to obtain in situ a complex of the DFO and the at least one metal.

In another aspect, the invention therefore provides a kit comprising (i) a first pharmaceutical composition comprising DFO or a pharmaceutically acceptable salt thereof; (ii) a second pharmaceutical composition comprising an ion of at least one metal; and (iii) instructions for administering the composition simultaneously or sequentially in any order and over a period of time not exceeding 6 hours, as follows, to obtain a complex of the DFO and the at least one metal in situ, thereby preventing, inhibiting, reducing, or ameliorating neurodegeneration, and thereby treating a disease, disorder, or condition characterized by or associated with neurodegeneration.

In certain embodiments, the first pharmaceutical composition contained within a kit of the invention comprises DFO. In other embodiments, the first pharmaceutical composition comprises a DFO salt. Examples of pharmaceutically acceptable salts of DFO include, but are not limited to, mesylate, esylate, tosylate, sulfate, sulfonate, phosphate, carboxylate, maleate, fumarate, tartrate, benzoate, acetate, hydrochloride, and hydrobromide, with mesylate being preferred.

In certain embodiments, the second pharmaceutical composition contained within the kits of the present invention comprises ions of zinc, gallium, indium, silver, cobalt, gold, lanthanides (such as those listed above), or combinations thereof. In certain embodiments, the second pharmaceutical composition comprises ions of more than one metal, for example ions of two or three metals, in any quantitative ratio. For example, a composition comprising two metal ions may comprise the two metals in the following quantitative ratios: from about 100:1 to about 1:100, for example at the following quantitative ratios: about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1: 100.

In a specific embodiment, the second pharmaceutical composition comprised in the kit of the present invention comprises Zn, Ga, Eu, Gd ions or any combination thereof. In more particular such embodiments, the second composition comprises ions of both Zn and Ga, for example, wherein the quantitative ratio of Zn ions to Ga ions is within the following range: about 100:1 to about 1:100, e.g., about 20:1 to about 1:20, about 10:1 to about 1:10, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1: 1. Certain such compositions are those in which the amount of Zn ions is higher than the amount of Ga ions, for example, wherein the quantitative ratio of Zn ions to Ga ions in the composition is in the following range: about 10:1 to about 2:1, e.g., about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

In certain embodiments, the second pharmaceutical composition comprised in the kit of the invention comprises ions of three or more metals, such as ions of Zn, Ga and one or more additional metals (e.g. Eu or Gd).

The metal ion contained in the second pharmaceutical composition may be in the form of a cation (salt) in any possible valence state (depending on the particular metal), or a complex with an organic compound such as aromatic and non-aromatic compounds having a heteroatom-containing moiety, for example, carbonyl compounds, hydroxyl compounds, heterocyclic compounds, alkenes and alkynes (the metal forms a complex having double and triple bonds). Non-limiting examples of ligands (mono-, di-, tridentate-, etc.) that form metal complexes are acetates, gluconates, acetylacetones, stearates, ricinoleates, tris (2-aminoethyl) amine, crown ethers, porphyrins, alkyl phosphates (e.g. dialkyl dithiophosphates) and heterocycles (e.g. terpyridines, pyridinethiones and metallocene compounds).

For example, the zinc ion can be present as a zinc salt (e.g., ZnCl)₂) In the form of, or as complexes such as zinc acetate, zinc stearate, zinc crown ether, zn-porphyrin/crown ether conjugates, zinc protoporphyrin, zinc chlorophyllin and bacteriochlorophyll, monomeric zinc dialkyldithiophosphate, zinc acetylacetonate (trimer; zn₃(AcAct)₆) Zinc terpyridyl (tridentate; [ Zn (Terpy) Cl₂]) Zinc tris (2-aminoethyl) amine, carbonic anhydrase (Zn metalloenzyme), glutamic acid carboxypeptidase II (Zn metalloenzyme), organozinc compounds (such as diethyl zinc (I) and decamethyl di-zinc-metallocene compound (II)), zinc gluconate, zinc pyrithione, and zinc ricinoleate. The gallium ions may be in the form of gallium salts such as GaCl₃Exist in the form of (1).

In certain embodiments, the first and second pharmaceutical compositions contained within the kits of the invention are administered simultaneously. In other embodiments, the compositions are administered sequentially in any order, with two administrations being performed over a period of up to 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

Upon administration of the composition contained in the kit of the invention to a subject in need thereof, at least one metal-DFO complex is obtained in situ that inhibits, reduces or ameliorates neurodegeneration and, thus, treats a disease, disorder or condition characterized by or associated with neurodegeneration in said subject.

The pharmaceutical compositions contained in the kits of the present invention may each be independently formulated for any suitable route of administration, such as intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, intracerebral, intracerebroventricular, intrapleural, intratracheal, or subcutaneous administration; oral administration; rectal administration; nasal administration; or inhalation. It will therefore be appreciated that the two compositions contained within the kit of the invention may be administered using the same or different routes of administration. Some routes of administration, such as intraperitoneal or intrarectal, may be used to treat intestinal dysfunction associated with neurodegenerative diseases.

The kits disclosed herein are particularly advantageous when: where the DFO and metal ion are preferably administered in different dosage forms, for example where one of the components is preferably administered orally and the other component is preferably administered parenterally, or at different dosing intervals; or where titration of one of the components prior to administration is desired by the prescribing physician.

Thus, the kits disclosed herein may comprise each composition in a ready-to-use form, e.g., formulated as a liquid for topical, nasal, or oral administration, or may alternatively comprise one or both of the compositions as a solid composition that can be reconstituted with a solvent to provide a liquid oral dosage form. Where one or both of the compositions are provided in solid form for reconstitution with a solvent, the kit may further comprise a reconstitution solvent and instructions for dissolving the solid composition in the solvent prior to administration. Such solvents should be pharmaceutically acceptable and may be, for example, water, aqueous liquids such as Phosphate Buffered Saline (PBS), non-aqueous liquids, or a combination of aqueous and non-aqueous liquids. Suitable non-aqueous liquids include, but are not limited to, oils, alcohols (e.g., ethanol), glycerol, and glycols (e.g., polyethylene glycol and propylene glycol).

Unless otherwise indicated, all numbers expressing quantities of ingredients and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary by as much as ± 10% depending upon the desired properties sought to be obtained by the present invention.

The invention will now be illustrated by the following non-limiting examples.

Examples of the invention

Study 1 Blood Brain Barrier (BBB) penetration

In this study, the ability of Zn-DFO complexes to penetrate into the brain was examined. Increasing amounts of DFO alone or Zn-DFO complex were injected Intraperitoneally (IP) into rats and the concentration of DFO was measured in their brains to monitor their osmotic capacity.

Specifically, male Sprague-Dawley (SD) rats (300g average body weight, 3 animals per group) were IP injected with DFO (250, 500 or 1000mg/kg body weight). For comparison, a Zn-DFO solution (250mg/kg, 500mg/kg or 1000mg/kg body weight, which corresponds to 883mg, 442mg or 221mg DFO/kg body weight, respectively) in freshly prepared saline was IP injected.

The behavior of the rats was monitored for 90 minutes after injection. Animals were then euthanized using an injection of ketamine-xylazine injection and their hearts, livers, left kidneys and brains were excised and weighed. Brain samples (300mg tissue) were homogenized in 3ml lysis buffer. The tissue homogenates were incubated at 110 ℃ for 5 minutes and centrifuged at 14,500 Relative Centrifugal Force (RCF) for 5 minutes. An equal volume of 40% trichloroacetic acid (TCA) solution was added to form a solution containing 20% TCA, which was vortexed and centrifuged again. The supernatant was transferred to a cuvette and the DFO concentration was measured after addition of 10mM ferric solution (as a weak complex of iron, ferric-nitrilo-tri-acetate, at pH 7.4), scanning in the range λ 380nm-580 nm. Using epsilon according to beer-Lambert law_DFO＝2460M^-1cm^-1Calculating the final concentration; the total amount of DFO per gram of tissue and per whole brain was calculated. The results obtained are summarized in table 1.

Table 1: comparative study of Zn-DFO and DFO on penetration capability in rat brain

^*Results are shown as mean ± standard error.

Below the detection limit of the instrument (<0.005)

Fifteen minutes after 1000mg/kg of Zn-DFO complex was administered, the rats appeared passive and apathy; however, no change in rat behavior was recognized after injection of 500mg/kg or 250mg/kg of Zn-DFO complex. No change in rat behavior was observed for three doses after injection of DFO alone.

At the end of the experiment, the total amount of DFO found in all groups in brain (mg/g) was calculated-3 doses of DFO alone (250mg/kg, 500mg/kg and 1000mg/kg) and 3 doses of Zn-DFO (250mg/kg, 500mg/kg and 1000 mg/kg). The fraction of DFO penetrating into the brain remained almost constant for 3 doses of Zn-DFO, averaging 0.0133%. This value was 0.002% or less for the corresponding dose of DFO alone (due to the limited sensitivity of the instrument). Thus, the permeability of the Zn-DFO complex in the brain is at least 8.6 times higher than that of DFO alone, indicating that the penetration of the Zn-DFO complex into the brain is significantly better than that of DFO alone.

Study 2 spinal cord injury

In this study, the therapeutic effect of metal-DFO complexes on spinal cord injury was tested using a contusion spinal cord injury model in C57BL/6 mice.

The contusion spinal cord injury model was used using an Infinite Horizon spinal cord impactor (Infinite Horizon cord impactor). A 100ms (millisecond) injury was performed on exposed mouse (8 week old males) T12 vertebrae under a force setting of 70 kilodynes, and the mice were anesthetized and fixed with ketamine-xylazine mixture (85: 15). Immediately prior to the challenge, mice were injected with buprenorphine (0.1 mg/ml per kg body weight) as an analgesic.

After contusion, twenty-one mice were divided into three groups (n-7) according to the following treatment (Basso et al, 2006): group 1-control, untreated; group 2-treatment with saline (IP injection 100 μ Ι on days 1,2,3, 4, 6, 8, 10, 12, 14, 17, 20, 23, 27, 31, 35, 39, and 42); group 3-treatment with Zn-DFO complex, 3mg/kg body weight in saline (IP injection 100. mu.l, according to the same protocol as group 2). Both treatment groups (saline and Zn-DFO complex) received the first treatment 10 minutes after the striker-induced trauma.

Recovery after injury from movement of treated or untreated mice was assessed daily on the following 43 days using an open field Barthale Mouse Scale (BMS) score as follows: 0-no ankle movement; 1-mild ankle movements; 2-extensive ankle movements; 3-plantar placement of the paw with or without weight support, or occasional, frequent or continuous instep stepping (dorsal stepping), but no plantar stepping (plantar stepping); 4-occasional footsteps; 5-frequent or continuous footsteps, uncoordinated, or frequent or continuous footsteps, sometimes coordinated, with the paw rotating (R/R) upon initial contact and lift-off; 6-frequent or continuous footsteps, sometimes coordinated, with the paw parallel at initial contact (P/R, P/P), or frequent or continuous footsteps, most coordinated, with the paw rotating at initial contact and lift-off (R/R); 7-frequent or continuous footsteps, mostly coordinated, paws parallel at initial contact and rotated at lift-off (P/R), or frequent or continuous footsteps, mostly coordinated, paws parallel at initial contact and lift-off (P/P), and severe trunk instability; 8-frequent or continuous footsteps, mostly coordinated, paws parallel at initial contact and lift off (P/P), and slight trunk instability, or frequent or continuous footsteps, mostly coordinated, paws parallel at initial contact and lift off (P/P), and normal trunk stability and tail drooping or drooping while lift off; and 9-frequent or continuous footsteps, mostly coordinated, with the paws parallel (P/P) at initial contact and lift off, and normal trunk stability and tail always upward (P-parallel; R-rotation). The basal score at day 0 was taken as 100%, and the values were normalized.

On day 43, animals were euthanized and their spinal cords were excised and stored in 4% Paraformaldehyde (PFA) for histological analysis. Epoxy resin (Epon) blocks were prepared and subjected to histological analysis after staining with methylene blue.

No statistically significant differences were found when comparing the BMS scores recovered after trauma in the untreated group and the saline treated group (fig. 1). Both groups exhibited a limited rise in BMS activity score within the first 13 days, and then both groups reached a plateau of value of 265% (BMS score 4.5) and 278% (BMS score 4.8), respectively, from their initial score (day 0). Mice treated with Zn-DFO showed a faster improvement in the first two weeks, reaching 347% (BMS score 5.1) on day 13. The trend in the Zn-DFO treated group continued over the next two weeks, reaching 389% of its initial score on day 29 (BMS score 5.7/9.0).

Consistent with the results of BMS recovery, histological analysis showed a lack of difference between untreated and saline-treated mice. Both groups exhibited significant loss of white matter from a value of 70% total area fraction (Cohen-ad and Wheeler-kingshot, 2014) to 26.3% ± 2.9% and 25.3% ± 4.2%, respectively (fig. 2), as well as reduced grey matter density. In addition, axonal retraction/retrogradation was observed in the gray matter of these groups. Treatment of fractions with Zn-DFO restored normal morphology and alignment of white and gray matter (white matter area 50.8% ± 3.3%) and its components (fig. 2).

Spinal cord injury had a particularly severe effect on white matter in study animals, every 0.01mm²Gradually decreased to a reported value significantly below 430 ± 52 (Ward et al, 2014) and significantly decreased the amount of structured myelin (fig. 4A-4B).

Treatment with saline limited the effect on the white matter component. In contrast, treatment with Zn-DFO proved significantly more effective (fig. 4-5), restoring axon and axon myelin density.

Body weight measurements showed that animals treated with Zn-DFO gained weight better than untreated or saline treated animals, demonstrating better post-traumatic recovery. However, saline treatment had some beneficial effects when compared to untreated animals (figure 6).

Study 3 model of Experimental autoimmune encephalomyelitis-multiple sclerosis

In this study, the therapeutic effect of metal-DFO complexes on Multiple Sclerosis (MS) was tested using Experimental Autoimmune Encephalomyelitis (EAE) in mice.

By emulsifying 125 μ g myelin oligodendrocyte glycoprotein 35-55 peptide (MOG) in Complete Freund's Adjuvant (CFA) containing 5mg/ml heat-inactivated Mycobacterium tuberculosis_35-55) Subcutaneous (SC) injection into the left lumbar region induced EAE in 8 week old female C57BL/6 mice. Shortly after this and again at 48h, mice were inoculated Intraperitoneally (IP) with 0.5ml pertussis toxin (400 ng). Seven days later, by MOG in CFA_35-55Additional injections of peptide were injected into the right lumbar region to further challenge the mice. Mice were treated daily with Zn-DFO or Ga-DFO (2mg/kg and 6mg/kg) as IP injections.

The severity of the disease was assessed using the scale shown in table 2 (Bittner et al, 2014).

Expected to be exposed to MOG without treatment_35-55The mice will develop disease within 11-13 days after the first injection. The disease worsened within the next 3-4 days, reaching a peak in clinical sign score of 6-7 (see table 2), and within the next two weeks, the pathology of the animals slightly improved, reaching a clinical score of 5-6. Treatment with 2mg/kg Zn-DFO or Ga-DFO is expected to delay the onset of disease to day 14 and reach a peak clinical score of 4-5 on day 17. In the next two weeks, the score will increase to 3-4. The effects exhibited by the two compounds are expected to be similar to each other. Treatment with Ga-DFO 6mg/kg is expected to delay the onset of the disease to day 15 and suppress its peak (day 19) to 3-4. As a result of treatment with Zn-DFO (6mg/kg), disease symptoms will be detected on days 15-16, and the peak clinical score will be less than 3 (day 19). The control group was not expected to express any clinical signs.

TABLE 2 Scale for assessing severity of MS

Study 4 MPTP mouse model for Parkinson's disease

In this study, the therapeutic effect of metal-DFO complexes on Parkinson's disease was tested using the 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) model in mice.

Parkinson's disease was induced for 5 days by four consecutive IP injections of 20mg/kgMPTP every 2 hours in 10-week-old male C57BL/6 mice over 8 hours (Jackson-Lewis and Przedborski, 2007). Animals were treated with Zn-DFO or Ga-DFO (2mg/kg and 6mg/kg) by daily IP injection 30 minutes prior to the first exposure to MPTP. On day 8, animals were euthanized. Immediately after euthanasia, the striatum was dissected for biochemical and histological evaluation of dopaminergic innervation injury.

Exposure to MPTP is expected to manifest as development of a number of nigrostriatal dopamine neuronal pathologies. In exposed and untreated mice, the number of Tyrosine Hydroxylase (TH) -positive neurons in the substantia nigra will be less than 40% of the control group. It is expected that treatment with Zn-DFO or Ga-DFO 2mg/kg will result in a restoration of this value to 65%. Administration of 6mg/kg of either compound is expected to restore the number of TH positive neurons to 85% -90% of the control value.

Additionally, MPTP is expected to reduce brain levels of dopamine to levels 10-12 fold lower than the control. It is expected that treatment with the test complex or combination thereof, administration of 2mg/kg significantly improves brain function and supplements brain dopamine levels, and that administration of 6mg/kg will be significantly better.

Study 5 streptozotocin-induced model of Alzheimer's disease

In this study, the therapeutic effect of metal-DFO complexes on alzheimer's disease was tested using a mouse medium-chain urezotocin (STZ) -induced model.

the disease was induced in 10 week old male C57BL/6 mice the animals were placed in stereotactic frames for mice with nasal and ear bars STZ (3mg/kg) or citrate buffer (control) was injected bilaterally into the lateral ventricle at the coordinates AP-0.5mm, ML + -1.1 mm, DV-2.8mm, in a total volume of 1.5. mu.l to hemisphere relative to the prohalogen, repeated 2 days after the first STZ injection (1.5mg/kg), in order to confirm that the stereotactic coordinates used throughout the study were suitable for STZ injection into the lateral ventricle, FITC-labeled latex microspheres were injected into the lateral ventricle and examined for the presence of fluorescence in the surrounding brain tissue 24 hours after injection, 2mg/kg and 6mg/kg per day by IP injection with Zn-DFO or Ga-DFO, and mice were treated with Eu-DFO as IP injection (4mg/kg) during the 21 days of the experiment, 7 days after 7 days of the experiment and tested for the biochemical observation of amyloid-protein expression per group including rat, beta, rat-synaptophysiologic protein expression, and rat protein expression, etc.

the effect of the Eu-DFO complex is expected to be slightly better than that of the Ga-DFO.

Reference to the literature

Banin,E.；Morad,Y.；Berenshtein,E.；Obolenskt,A.；Yahalom,C.；Goldich,J.；Adibelli,F.M.；Zuniga,G.；DeAnda,M.；Pe’er J.；Chevion,M.Injuryinduced bychemical warfare agents:characterization and treatment of ocular tissuesexposed to nitrogen mustard.Invest Ophthalmol Vis Sci.2003,44(7),2966-2972

Basso,D.M.；Fischer,L.C.；Anderson,A.J.；Jakeman,L.B.；McTigue,D.M.；Popovich,P.G.Basso Mouse Scale for locomotion detects differences in recoveryafter spinal cord injury in five common mouse strains.J Neurotrauma.2006,23(5),635-659

Bibi,H.；Vinokur,V.；Waisman,D.；Elenberg,Y.；Landesberg,A.；Faingersh,A.；Yadid,M.；Brod,V.；Pesin,J.；Berenshtein,E.；Eliashar,R.；Chevion,M.Zn/Ga-DFOiron-chelating complex attenuates the inflammatory process in a mouse modelof asthma.Redox Biol.2014,2,814-819

Bittner,S.；Afzali,A.M.；Wiendl,H.；Meuth,S.G.Myelin oligodendrocyteglycoprotein(MOG35-55)induced experimental autoimmune encephalomyelitis(EAE)in C57BL/6 mice.J Vis Exp.2014,86

Chevion,M.A site-specific mechanism for free radical inducedbiological damage:the essential role of redox-active transition metals.FreeRadio Biol Med.1988,5(1),27-37

Chevion,M.Protection against free radical-induced and transitionmetal-mediated damage:the use of“pull”and“push”mechanisms.Free Radio ResCommun.1991,12-13,691-696

Chevion,M.；Jiang,Y.；Har-El,R.；Berenshtein,E.；Uretzky,G.；Kitrossky,N.Copper and iron are mobilized following myocardial ischemia:possiblepredictive criteria for tissue injury.Proc Natl Acad Sci USA 1993,90(3),1102-1106

Chevion,M.；Berenshtein,E.；Zhu,B-Z.The role of transition metal ionsin free radical-mediated damage.In:Reactive oxygen species in biologicalsystems:an interdisciplinary approach,Colton and Gilbert(Eds.),1998,PlenumPress,New York,pp103-131

Cohen-Adad,J.；Wheeler-Kingshott,C.Quantitative MRI of the SpinalCord,Academic Press,2014,p196

Connor,J.R.；Menzies,S.L.；Burdo,J.R.；Boyer,P.J.Iron and ironmanagement proteins in neurobiology.Pediatr Neurol.2001,25(2),118-129

Faden,A.I.；Wu,J.；Stoica B.A.；Loane,D.J.Progressive inflammation-mediated neurodegeneration after traumatic brain or spinal cord injury.Br JPharmacol.2016 173(4),681-691

Jackson-Lewis,V.；Przedborski,S.Protocol for the MPTP mouse model ofParkinson’s disease.Nat Protoc.2007,2(1),141-151

Kim,N.H.；Park,S.J.；Jin,J.K.；Kwon,M.S.；Choi E.K.；Carp,R.I.；KimY.S.Increased ferric iron content and iron-induced oxidative stress in thebrains of scrapie-infected mice.Brain Res.2000,884(1-2),98-103

Kumar,V.；Gill,K.D.Aluminium neurotoxicity:neurobehavioural andoxidative aspects.Arch Toxicol.2009,83(11),965-978

Mantyh,P.W.；Ghilardi,J.R.；Rogers,S.；DeMaster,E.；Allen,C.J.；Stimson,E.R.,Maggio,J.E.,Aluminum,iron,and zinc ions promote aggregation ofphysiological concentrations of beta-amyloid peptide.J Neurochem.1993,61(3),1171-1174

Morad,Y.；Banin,E.；Averbukh,E.；Berenshtein,E.；Obolensky,A.；Chevion,M.Treatment of ocular tissues exposed to nitrogen mustard:beneficial effectof zinc desferrioxamine combined with steroids.Invest Ophthalmol VisSci.2005,46(5),1640-1646

Orcutt,K.M.；Jones,W.S.；McDonald,A.；Schrock,D.；Wallace,K.J.Alanthanide-based chemosensor for bioavailable Fe³⁺using a fluorescentsiderophore:an assay displacement approach.Sensors 2010,10(2),1326-1337

Ravelli,K.G.；Rosario,B.D.；Camarini,R.；Hernandes,M.S.；Britto,L.R.Intracerebroventricular Streptozotocin as a Model of Alzheimer’s Disease:Neurochemical and Behavioral Characterization in Mice.Neurotox Res.2016

Rouault,T.Iron metabolism in the CNS:implications forneurodegenerative diseases.Nature Reviews Neuroscience 2013,14,551-564

Scheiber,I.F.；Mercer,J.F.；Dringen,R.Metabolism and functions ofcopper in brain.Prog Neurobiol.2014,116,33-57

Siganos,C.S.；Frucht-Pery,J.；Muallem,M.S.；Berenshtein,E.；Naoumidi,I.；Ever-Hadani,P.；Pallikaris I.G.；Siganos D.S.；Chevion,M.Topical use of zincdesferrioxamine for corneal alkali injury in a rabbit model.Cornea 1998,17(2),191-195

Singh,N.；Haidar,S.；Tripathy,A.K.；McElwee,M.K.；Horback,K.；Beserra,A.Iron in neurodegenerative disorders of protein misfolding:a case of priondisorders and Parkinson’s disease.Antioxid Redox Signal 2014 21(3),471-484

Sooriyaarachchi,M.；Gailer,J.Removal of Fe3+and Zn2+from plasmametalloproteins by iron chelating therapeutics depicted with SEC-ICP-AES.Dalton Trans.2010,39(32),7466-7473

Wan,W.；Cao,L.；Kalionis,B.；Xia,S.；Tai,X.Applications of InducedPluripotent Stem Cells in Studying the Neurodegenerative Diseases.Stem CellsInt.2015,2015,382530

Ward,R.E.；Huang,W.；Kostusiak,M.；Pallier,P.N.；Michael-Titus,A.T.；Priestlry,J.V.A characterization of white matter pathology following spinalcord compression injury in the rat.Neuroscience.2014,260,227-239

Williams,T.L.；Urbane,B.；Marshal,K.E.；Vadukul,D.M.；Jenkins,A.T.；Serpell,L.C.Europium as an inhibitor of Amyloid-P(l-42)induced membranepermeation.FEBS Lett.2015,589(21),3228-3236

Claims (24)

1. A method for preventing, inhibiting, reducing or ameliorating neurodegeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one metal-desferrioxamine B complex (metal-DFO complex).

2. The method of claim 1, wherein the metal-DFO complexes are each independently a zinc-, gallium-, indium-, silver-, cobalt-, or gold-DFO complex, or a lanthanide-DFO complex such as a europium-or gadolinium-DFO complex.

3. The method of claim 2, comprising administering a Zn-DFO complex, a Ga-DFO complex, a Eu-DFO complex, a Gd-DFO complex, or a combination thereof.

4. The method of claim 3, comprising administering a combination of a Zn-DFO complex and a Ga-DFO complex, wherein in said combination the quantitative ratio of said Zn-DFO complex to said Ga-DFO complex is in the range of 100:1 to 1: 100.

5. The method of claim 3 or 4, comprising administering a combination of a Zn-DFO complex, a Ga-DFO complex, and at least one additional metal-DFO complex.

6. The method of any one of claims 1-5, wherein the subject has a neurodegenerative disease, disorder, or condition, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, or age-related brain degeneration.

7. The method of any one of claims 1-5, wherein the subject has a disorder or condition resulting from injury, e.g., from mechanical force, ischemia, toxic agents, or hemorrhage.

8. The method of any one of claims 1 to 5, wherein the subject has a prion-caused disease, disorder or condition, such as Creutzfeldt-Jakob disease or a subtype thereof, Giasteman-Sjogren's syndrome, fatal familial insomnia, Kuru, familial spongiform encephalopathy, or multiple system atrophy.

9. A pharmaceutical composition for preventing, inhibiting, reducing or ameliorating neurodegeneration comprising at least one metal-desferrioxamine B complex (metal-DFO complex) and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein the metal-DFO complexes are each independently a zinc-, gallium-, indium-, silver-, cobalt-, or gold-DFO complex, or a lanthanide-DFO complex such as a europium-or gadolinium-DFO complex.

11. The pharmaceutical composition of claim 10, comprising a Zn-DFO complex, a Ga-DFO complex, a Eu-DFO complex, a Gd-DFO complex, or a combination thereof.

12. The pharmaceutical composition of claim 11, comprising a combination of a Zn-DFO complex and a Ga-DFO complex, wherein in said combination the quantitative ratio of said Zn-DFO complex to said Ga-DFO complex is in the range of 100:1 to 1: 100.

13. The pharmaceutical composition of claim 11 or 12, comprising a combination of a Zn-DFO complex, a Ga-DFO complex, and at least one additional metal-DFO complex.

14. The pharmaceutical composition of any one of claims 9 to 13, for use in the treatment of a neurodegenerative disease, disorder or condition, such as parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis, huntington's disease, or age-related brain degeneration.

15. The pharmaceutical composition of any one of claims 9 to 13, for use in treating a disorder or condition resulting from injury, e.g., from mechanical force, ischemia, toxic agents, or hemorrhage.

16. The pharmaceutical composition of any one of claims 9 to 13, for use in treating a disease, disorder or condition caused by a prion, such as creutzfeldt-jakob disease or a subtype thereof, guillain-straussler-scheinker syndrome, fatal familial insomnia, kuru, familial spongiform encephalopathy, or multiple system atrophy.

17. A metal-desferrioxamine B complex (metal-DFO complex) or a combination thereof for use in preventing, inhibiting, reducing or ameliorating neurodegeneration.

18. Use of a metal-desferrioxamine B complex (metal-DFO complex) or a combination thereof in the manufacture of a pharmaceutical composition for the prevention, inhibition, reduction or amelioration of neurodegeneration.

19. A kit, comprising:

(i) a first pharmaceutical composition comprising desferrioxamine B (DFO) or a pharmaceutically acceptable salt thereof;

(ii) a second pharmaceutical composition comprising an ion of at least one metal; and

(iii) instructions for administering the composition simultaneously or sequentially in any order and within a time period of no more than 6 hours to obtain the complex of the DFO and the at least one metal in situ to prevent, inhibit, reduce or ameliorate neurodegeneration are as follows.

20. The kit of claim 19, wherein the second pharmaceutical composition comprises ions of zinc, gallium, indium, silver, cobalt, gold, a lanthanide element such as europium or gadolinium, or a combination thereof.

21. The kit of claim 20, wherein the second pharmaceutical composition comprises ions of Zn, Ga, Eu, Gd, or a combination thereof.

22. The kit of claim 21, wherein said second pharmaceutical composition comprises ions of both Zn and Ga, and the quantitative ratio of the Zn ions to the Ga ions is in the range of 100:1 to 1: 100.

23. The kit of claim 21 or 22, wherein the second pharmaceutical composition comprises ions of Zn, Ga, and at least one additional metal.

24. The kit of any one of claims 19 to 23, wherein the neurodegeneration is associated with: a neurodegenerative disease, disorder or condition, such as parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis, huntington's disease, or age-related brain degeneration; a disorder or condition resulting from injury; or a disease, disorder or condition caused by a prion, such as Creutzfeldt-Jakob disease or a subtype thereof, Gistelman-Straussler syndrome, fatal familial insomnia, Kuru, familial spongiform encephalopathy, or multiple system atrophy.