WO2009056849A1

WO2009056849A1 - Treatment of spinal cord injury

Info

Publication number: WO2009056849A1
Application number: PCT/GB2008/003696
Authority: WO
Inventors: Adina Michael-Titus; Sharon Averill; Von King
Original assignee: Queen Mary & Westfield College
Priority date: 2007-11-02
Filing date: 2008-10-31
Publication date: 2009-05-07
Also published as: GB0721616D0

Abstract

The present invention provides a zinc-chelating agent for use in the treatment or prevention of spinal cord injury.

Description

TREATMENT OF SPINAL CORD INJURY

The present invention relates to the use of zinc-chelating agents, in particular clioquinol, in the treatment of spinal cord injury.

Zinc is a divalent cation essential for the growth and function of the central nervous system (CNS). Zinc-containing neurons, which sequester weakly bound zinc in presynaptic vesicles, are found in the brain and spinal cord. Zinc ions are stored in synaptic vesicles and are released by depolarisation through a calcium-dependent process.

Prolonged depolarisation of neurones, which occurs in the context of brain and spinal cord injury, can lead to cell death. Zinc-linked excitotoxicity was first described in kainic acid-induced epileptic seizures. It was proposed that the observed translocation of zinc between the presynaptic bouton and postsynaptic terminals was contributing to or causing injury. Evidence has since accumulated in support of this theory, in particular in the field of head injury.

The role of zinc in the spinal cord has been less well documented than its effect in the brain. Whilst zinc-containing neurons have been identified in the spinal cord, it is unclear whether they have the same functional role as that postulated at supraspinal level. Evidence accumulated so far suggests that zinc may be involved in sensory and motor transmission at spinal cord level. Recent observations have led authors to suggest that zinc has a protective effect in spinal cord injury (Kalkan et al., 2007, Spinal Cord , 45, 722-730).

Most of the zinc found in the CNS is bound to metalloproteins, which play a central role in zinc dynamics and homeostasis. Metallothioneins are cysteine-rich proteins, which exist as several isoforms (MT-I, MT-II, MT-III and MT-IV) in mammals. MT-I and MT-II are induced by a variety of stimuli, including proinflammatory agents (such as cytokines and free radicals), toxic compounds (including metals such as zinc, copper, cadmium and mercury), drugs (glucocorticoids) and stress, and possibly reflect an endogenous neuroprotective response of the tissue. Astrocytes, the main source of MT-I and MT-II in the brain, may secrete metallothioneins into the extracellular space to protect surrounding neurones.

Clioquinol is an antibiotic and metal chelating agent. Clioquinol was approved by the FDA as an antibiotic and antifungal agent, but was removed from the market over 30 years ago due to adverse side effects including the loss of vitamin B 12. In recent years, there has been renewed interest in clioquinol as a therapeutic agent. For example, WO2006/043153 relates to the use of chelators of zinc and copper ions, including clioquinol, for the treatment of infections caused by blood borne viruses. Zinc chelators generally are also used therapeutically. For example, WO97/09976 relates to the use of pharmaceutically acceptable zinc chelating compounds for the treatment of neurotoxic injury.

Spinal cord injury (SCI) is an important clinical condition. Tissue damage and tissue loss in SCI are due both to the primary and secondary injury. The latter involves excitotoxicity, increased oxidative stress and increased inflammation. Interventions to limit the extent of secondary injury may greatly improve clinical outcome. However, there are currently no treatments for this condition, and therefore the prospects of functional recovery are very limited. There thus exists a need in the art for a method of treating spinal cord injury.

It has now surprisingly been found that clioquinol is effective in the treatment of spinal cord injury.

According to a first aspect of the invention there is therefore provided a zinc- chelating agent for use in the treatment or prevention of spinal cord injury.

The present invention meets a hitherto unmet need for a method of treating spinal cord injury. The invention advantageously provides both methods for treatment and prevention of spinal cord injury. In particular, the present invention can be used to treat the primary injury or to prevent or limit the extent of secondary injury after primary injury has occurred. Treatment with the zinc-chelating agent according to the invention can take the form of a rapid intervention immediately after the initial trauma or other event which causes the primary spinal cord injury, thus preventing or limiting the extent of secondary spinal cord injury. In this aspect of the invention, the zinc- chelating agent can be administered, for example, by a paramedic or other medical professional to a subject immediately after the trauma, for example at the scene of an accident.

The present invention utilises a zinc-chelating agent. Chelation is defined as the process of reversible binding of a ligand to a metal ion, to form a metal complex known as a chelate. A chelating agent is typically an organic compound that is capable of forming coordinate bonds with metal ions through atoms of the organic compound. Accordingly, a zinc-chelating agent is typically an organic compound that is capable of forming coordinate bonds with zinc ions through atoms of the organic compound. The skilled person will appreciate that chelating agents generally have affinity for several metal ions and therefore zinc-chelating agents may also have affinity for other metal ions.

Any suitable zinc-chelating agent can be used in the present invention. Typically, any pharmaceutically acceptable zinc-chelating agent can be used in the present invention. Suitable zinc-chelating agents for use in the invention include compounds of formula (I):

wherein: R₁ can be H, a halogen or -SO₃H;

R₂ can be H, a halogen, a C₁ to C₆ alkyl, a C₆ to C₁₀ aryl, a C₃ to C₁₀ heterocycle, a C₁ to C₆ alkoxy, a nitro or an amide group; R₃ can be - OH or a C₁ to C₆ alkoxy group; or wherein R₂ and R₃ are fused to form a furanic cycle; and phanquinone (4,7-phenanthronine-5,6-dione), ethylenediamine tetraacetic acid (EDTA), disodium-calcium-ethylenediamine tetraacetic acid (CaEDTA), (2S)-2- amino-2-methyl-3-sulfanyl-butanoic acid (D-penicillamine), ethylene glycol tetraacetic acid (EGTA), N,N,N^l,N'-tetrakis(2-pyridyhnethyl)-ethylenediamine (TPEN), diethylenetriamine pentacetic acid (DTPA), 6-methoxy-(8-p- toluenesulfonoamido) quinoline (TSQ), diethyldithiocarbamate (DEDTC), bis(diethylthiocarbamoyi)disulfide (disulfiram), 1,10-phenanthroline, dipicolinate, N- acetyl cysteine, diphenylthiocarbazone (Dithizone), l-[2-(5-carboxyoxazol-2-yl)-6- aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fura-2), dimercaptosuccinic acid (DMSA), dimercapto-1-propanesulfonic acid (DMPS), alphalipoic acid (ALA) and l,2-bis(o-aminophenoxy)-ethane-N,N,N',N'- tetraacetic acid (BAPTA), and derivatives and pharmaceutically-acceptable salts of any thereof. Accordingly, the zinc-chelating agent for use in the invention can be selected from the group consisting of the above-mentioned zinc-chelating agents.

Typically, the halogen is fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Typically, R₁ is H, I, Cl or -SO₃H. Typically, R₂ is H, I or Cl. Typically, R₃ is -OH.

For the purposes of this invention, "C₁ to C₆ alkyl" means a straight chain or branched alkyl radical of 1 to 6 carbon atoms, typically 1 to 4 carbon atoms and most typically

1 to 3 carbon atoms including but not limited to methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, isobutyl, tert-butyl etc. Typically, the C₁ to C₆ alkyl is a methyl. "C₆ to C₁₀ aryl" means an aromatic 6 to 10 membered hydrocarbon containing one ring or being fused to one or more saturated or unsaturated rings including but not limited to phenyl and naphthyl. "C₃ to C₁₀ heterocycle" means an aromatic or non-aromatic 3 to

10 membered carbocyclic group containing one or more heteroatoms selected from sulphur, phosphorous, oxygen and nitrogen and containing one ring or being fused to one or more saturated or unsaturated rings. Typically, the C₃ to C₁₀ heterocycle is a 3 to 7 membered non-aromatic heterocyclic group, which means a heterocyclic group which has no aroniaticity and the number of atoms forming the ring is 4 to 7, including but not limited to azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl etc. Typically, the C₃ to C₁₀ heterocycle is a 5 to 10 membered heteroaryl group, which means an aromatic heterocyclic group in which the number of atoms forming the ring is 5 to 10, including but not limited to pyrrolyl, pyridyl, pyridazinyl etc. "C₁ to C₆ alkoxy" means an oxy group that is bonded to the previously defined alkyl group of 1 to 6 carbon atoms. The alkoxy group is typically an alkoxy group of 1 to 4 carbon atoms and more typically 1 to 3 carbon atoms including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy etc. "Nitro group" means -NO₂. "Amide group" means -RCONH₂, wherein R is typically an alkyl group as defined herein.

Typically, the zinc-chelating agent used in the present invention is a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:

In one embodiment, the compound is a compound of formula (I) and R₁ is H, a halogen or -SO₃H, R₂ is H or a halogen and R₃ is -OH.

In one embodiment, the compound is a compound of formula (I) and R₁ is Cl, R₂ is I and R₃ is -OH. In this embodiment, the compound of formula (I) is clioquinol.

Compounds of formula (I) thus include, for example, 8-hydroxyquinoline:

Compounds of formula (I) also include 8-hydroxyquinoline derivatives such as 8- hydroxyquinolme-5-sulphonic acid (sulphoxine):

SO₃H

Compounds of formula (T) also include halogenated 8-hydroxyquinoline derivatives such as clioquinol (iodochlorhydroxyquin, 5-chloro-7-iodo-8-quinolinol):

Cl

iodoqumol (5,7-diiodo-8-hydroxyquinoline):

and chiniofon (8-hydroxy-7-iodo-5 quinoline sulphonic acid):

SO₃H

Typically, the compound of formula (I) is clioquinol (iodochlorhydroxyquin, 5- chloro-7-iodo-8 -quinolinol) .

Clioquinol chelates zinc as well as various other metal ions such as iron, cobalt, nickel, copper, calcium and magnesium. Clioquinol is hydrophobic and crosses the blood brain barrier. A comprehensive description of clioquinol and methods for its synthesis can be found in Padmanabhan et al Analytical Profiles of Drug Substances vol.18, K. Florey, Ed. (Academic Press, New York, 1989) pages 57-90.

Clioquinol or a pharmaceutically acceptable salt thereof can be used in the present invention. Such salts, as well as the compounds of formula (I) used in the present invention, will have the same zinc-chelating properties as clioquinol. As used herein, all references to "clioquinol" also include pharmaceutically acceptable salts of clioquinol.

Various clioquinol derivatives, and methods of preparing such derivatives, are disclosed in R. Beugelmans et al, Eur. J. Med. Chem. 23 (1988), 539-546.

There is no limitation on the "salt" according to the present invention as long as it forms a salt with the zinc-chelating agent, typically of formula (I), and is pharmaceutically acceptable. Typical examples of salts include hydrohalogenates (for instance, the hydrochloride, hydrobromide, or hydroiodide salt), inorganic acid salts (for instance, the sulphate, nitrate, perchlorate, phosphate, carbonate or bicarbonate salt), organic carboxylic acid salts (for instance, the acetate, maleate, tartrate, fumarate or citrate salt), organic sulfonic acid salts (for instance, the methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate or camphorsulfonate salt), amino acid salts (for instance, the aspartate or glutamate salt), quaternary ammonium salts, alkaline metal salts (for instance, the sodium or potassium salt) and alkaline earth metal salts (for instance, the magnesium or calcium salt). Typically, the "pharmaceutically acceptable salt" of the zinc-chelating agent used in the present invention is a hydrochloride salt, a sulphate salt, a methanesulfonate salt or an acetate salt.

As used herein, an "ester" is typically an alkyl ester, typically a C₁ to C₆ alkyl ester, for example a methyl or ethyl ester.

The zinc-chelating agent is used in the treatment or prevention of spinal cord injury (SCI). The spinal cord is part of the central nervous system (CNS) and is a complex organ containing nerve cells, supporting cells and nerve fibres. The spinal cord is located within the vertebral column and conducts sensory and motor nerve impulses to and from the brain. Spinal cord injury or myelopathy can be defined as damage or trauma to the spinal cord that results in a loss or impaired function causing reduced mobility and/or sensation. Spinal cord injury is commonly caused by trauma, such as automobile accidents, falls, gunshots, diving accidents and war injuries. The spinal cord does not have to be completely severed for there to be a loss of function. In most cases of spinal cord injury the spinal cord remains intact. However, the cellular damage to it results in a loss of function.

Spinal cord injury can be considered as taking two forms. As defined herein, the primary injury is the initial injury, caused for example by an accident or trauma. As defined herein, the secondary injury is damage which develops later, for example in the minutes, hours, days and months following the primary injury.

Secondary injury may occur as a result of compression or spinal instability. Secondary injury can result from, for example, cellular hypoxia, oligaemia and/or oedema due to an injury-induced neurochemical cascade. All of these conditions may be exacerbated by hypotension. Secondary injury can also be due to entry of immune cells, which release free radicals, into the spinal cord. In addition, trauma can cause the release of excess neurotransmitters, leading to excitotoxicity or secondary damage from overexcited nerve cells. Cells may die after spinal cord injury either by necrosis or apoptosis. Axons may also be damaged and nerve cells in the spinal cord below the lesion may die. Zinc has been shown to play an important role in mediating excitotoxicity, oxidative and apoptotic processes and is thus involved in secondary spinal cord injury.

The present invention is used to treat and/or prevent spinal cord injury. Both primary and secondary spinal cord injury can be treated, and secondary spinal cord injury can be prevented. Typically, the present invention is used to treat the consequences of primary injury or to prevent or limit the extent of secondary injury after primary injury has occurred. In particular, treatment with the zinc-chelating agent according to the invention can take the form of a rapid intervention immediately after the initial trauma or other event which causes the primary spinal cord injury, thus preventing or limiting the extent of secondary spinal cord injury. In this aspect of the invention, the zinc-chelating agent can be administered, for example, by a paramedic or other medical professional to a subject immediately after the trauma. This can be done, for example, at the scene of an accident, such as a car accident, allowing rapid intervention and the prevention of secondary spinal cord injury.

The zinc-chelating agent can be administered to the subject by any suitable means. The zinc-chelating agent is typically administered systemically, in particular intraperitoneally (i.p.), intravenously or intramuscularly. In one embodiment, the zinc-chelating agent according to the present invention is administered in a single injection, typically a single intraperitoneal, intravenous or intramuscular injection. In another embodiment, the zinc-chelating agent according to the present invention is administered by means of repeated injections, for example by means of 2, 3, 4, 5 or even 6 injections. Typically, such injections are intraperitoneal, intravenous or intramuscular injections. However, the zinc-chelating agent can also be administered by other enteral or parenteral routes such as by oral, buccal, anal, pulmonary, intraarterial, intra-articular or other appropriate administration routes.

The zinc-chelating agent is administered at a suitable time before or after the spinal cord injury occurs. The time at which the zinc-chelating agent is administered will depend upon the type of spinal cord injury being treated or prevented. For example, for the prevention of secondary spinal cord injury, the zinc-chelating agent in accordance with the invention is administered within a period of time in which it is effective in completely or partially preventing or limiting the extent of secondary injury. The zinc-chelating agent can therefore be administered, for example, between 0 and about 12 hours or between 0 and about 24 hours, up to about 36 hours or about 48 hours and typically between about 1 and about 12 hours, between about 2 and about 10 hours, between about 4 and about 8 hours or between about 5 and about 6 hours after the initial trauma which causes the primary spinal cord injury. The zinc- chelating agent can be administered between 0 and about 1 hour, for example about 30 minutes after the initial trauma which causes the primary spinal cord injury. The administration can be by means of a single injection or repeated injections within this time period. For example, the zinc-chelating agent can be administered by means of repeated injections over the first 24 to 48 hours after the initial trauma which causes the primary spinal cord injury. The zinc-chelating agent can also be administered as an infusion within the post-injury period.

The zinc-chelating agent is typically formulated for use with a pharmaceutically acceptable carrier, vehicle, adjuvant and/or diluent. Suitable carriers, vehicles, adjuvants and/or diluents are well known in the art. Typically, when the zinc- chelating agent is clioquinol the carrier is a mixture of saline, ethanol and Cremophor EL™. Typically, this mixture contains 80% saline, 10% ethanol and 10% Cremophor EL™. Other suitable carriers include carboxymethylcellulose (CMC), methylcellulose, hydroxypropylmethylcellulose (HPMC), phosphate buffered saline (PBS), polyvinyl alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (and other sugars), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). Typically, the zinc-chelating agent is formulated as a liquid formulation, which will generally consist of a suspension or solution of the zinc- chelating agent in a suitable aqueous or non-aqueous liquid carrier or carriers, for example water, ethanol, glycerine, polyethylene glycol or an oil.

The zinc-chelating agent can be administered alone or together with another agent. When the zinc-chelating agent is clioquinol, the clioquinol can be administered alone or together with vitamin B 12.

The zinc-chelating agent for use in the present invention is typically administered to a subject in a therapeutically effective amount. Such an amount is an amount effective to ameliorate, eliminate or prevent one or more symptoms of spinal cord injury, such as the symptoms of primary or secondary spinal cord injury. Such symptoms include, for example, reduced mobility and/or sensation; and cellular hypoxia, oligaemia and/or oedema, hi some embodiments, the treatment of spinal cord injury results in an improved functional outcome in the patient with spinal cord injury. Preferably, the subject to be treated is a human.

The dose of the zinc-chelating agent to be administered may be determined according to various parameters, especially according to the particular zinc-chelating agent used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for a particular patient.

In a second aspect, the invention provides the use of a zinc-chelating agent in the manufacture of a medicament for the treatment or prevention of spinal cord injury.

In a third aspect of the invention, there is provided a method for the treatment or prevention of spinal cord injury comprising administering to a subject in need thereof a therapeutic amount of a zinc-chelating agent. Preferred features for the second and third aspects of the invention are as for the first aspect mutatis mutandis.

In one embodiment, the present invention provides the use of a composition comprising clioquinol and vehicle (80% saline, 10% ethanol and 10% Cremophor EL™) for the treatment of spinal cord injury. Typically, the clioquinol is administered systemically 30 minutes after spinal cord injury.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as limiting on the invention. Reference is made to a number of Figures, in which:

Figure 1 shows the results of autometallographic analysis of spinal cord sections at the lesion site 3 hours (bottom) and 48 hours (top) after spinal cord injury of the rat.

Figure 2 shows the results of autometallographic analysis of spinal cord sections after spinal cord injury in both control animals and those treated with 10 mg/kg clioquinol, at the lesion site (top) and in the dorsal horn of the spinal cord (bottom).

Figure 3a shows the motor score of animals and the degree of impairment caused by spinal cord hemisection injury using the Basso Beattie Bresnahan (BBB) score.

Figure 3b shows the motor score of animals and the degree of impairment caused by compression spinal cord injury using the BBB score (n=7 animals per group).

Figure 4 shows metallothionein expression in control and injured rats using immunostaining. Figure 5a shows metallothionein expression using immunostaining in rats with spinal cord injury receiving vehicle (80% saline, 10% ethanol and 10% Cremophor EL™) alone.

Figure 5b shows metallothionein expression using immunostaining in rats with spinal cord injury receiving clioquinol.

Figure 6 shows the effect of administration of zinc chloride (ZnCl₂) after spinal cord injury, and its negative effects on functional outcome. N=2 animals per group; Group A=ZnCl₂, Group B=saline.

Materials and Methods

In the following Examples, animals received a hemisection spinal cord injury (SCI) or compression SCI, and were given clioquinol (10 mg/kg intraperitoneally) in vehicle (80% saline, 10% ethanol and 10% Cremophor EL™) or vehicle (80% saline, 10% ethanol and 10% Cremophor EL™) alone 30 minutes after injury. Some animals also received ZnCl₂ (20 mg/kg intraperitoneally) or its vehicle (saline) 30 min after SCI. The motor score of all animals was assessed for 7 days following SCI. Animals were then sacrificed and tissue samples were processed using immunocytochemistry.

Example 1 : Zinc accumulates around the lesion site after spinal cord injury

Autometallographic analysis of spinal cord sections at 3 hours and 48 hours after hemisection spinal cord injury of the rats revealed the presence of zinc around the lesion site. The accumulation was marked 3 hours after injury, as shown in Figure 1.

When animals were treated intraperitoneally with 10 mg/kg clioquinol 30 minutes after injury, the presence of zinc was markedly reduced (almost absent) in the spinal cord, as shown in Figure 2. This was seen in both uninjured animals and animals with a spinal cord hemisection. It can be concluded from these results that there is a significant accumulation of zinc around the lesion site after SCI, above the level of zinc seen in uninjured animals, but the administration of clioquinol after SCI leads to a marked decrease in the tissue concentration of zinc in animals with SCI.

Example 2: Effect of clioquinol on functional outcome after spinal cord injury - motor score

The motor score of the animals and the degree of impairment caused by SCI carried out by hemisection or compression was assessed using the Basso Beattie Bresnahan (BBB) score (Basso et al, J Neurotrauma 1995 Feb; 12(1): 1-21). BBB scores were taken daily until the animals were sacrificed, 7 days or 14 days after hemisection or compression injury. The results in hemisection SCI are shown in Figure 3 a.

Animals in both groups demonstrated a significant overall improvement in BBB scores as a function of time after hemisection SCI (p<0.005). Furthermore, animals receiving clioquinol had overall a significantly better functional outcome than those in the vehicle-injected group (p<0.005). The greatest difference in scores was seen at 4 days after injury, where rats in the experimental group scored an average of 6.6 points better than those in the control group (experimental group = 11.3± 1.8, control group = 4.7±2.4). By 7 days post-injury, there was a 4.1 point difference between groups (experimental group = 16.8±1.1, control group = 12.7±2.4). Therefore, the overall improvement in BBB score between day 7 and day 4 was 63% in the control group, and only 32.7% in the experimental group.

hi compression SCI, a more severe model of injury than hemisection SCI, similar observations were made after treatment with clioquinol (experimental group) or vehicle (control group) 30 min after injury (shown in Figure 3b). The animals treated with clioquinol had a much improved functional outcome over 14 days after SCI, compared to the control group. For example, at 7 days after injury the BBB score results were: experimental group = 6.4 ± 1.0, control = 3.0 ±1.3. The difference was still observed at 14 days after compression SCI: experimental group = 9.4 ±1.2, control = 6.3 ± 1.1.

It can be concluded from these results that the administration of clioquinol after SCI leads to a significantly improved neurological outcome in animals with SCI.

Example 3: Metallothionein and the response to spinal cord injury

Control animals

Immunostaining of spinal cord sections from control uninjured rats generally revealed little presence of metallothionein (MT I/II). In both transverse and longitudinal spinal cord sections, MT I/II immunoreactivity appeared to be confined to a few astrocytic- like cells and processes in the outer areas of white matter. In the majority of sections, the grey matter was negative for MT I/II staining. This pattern was consistent throughout the spinal cord, as shown in Figure 4.

Hemisectioned animals

In the spinal cord of hemisectioned rats, there was a significant upregulation of MT I/II in both the animals receiving vehicle and clioquinol, in comparison to uninjured rats.

In the control group, the increase in MT 1711 was evident on the lesioned side. There was a marked increase in MT I/II in both the grey matter (showing dense neurophil staining and astrocytic-like cells) and the white matter (astrocytic-like cells). In some sections, the upregulation of MT I/II appeared to be less significant contralateral to the lesion site, although it was still increased in comparison to the naϊve group, as shown in Figure 5 a.

A similar pattern of immunostaining for MT 1711 was seen in animals injected with clioquinol. However, there appeared to be overall a reduced staining compared to the control group. One animal in particular showed levels of MT I/II almost comparable to naϊve samples, i.e. very close to the limit of detection, as shown in Figure 5b.

Example 4: Administration of zinc chloride (ZnCl?) after spinal cord injury and its functional consequences

Some animals received either ZnCl₂ (20 mg/kg intraperitoneally) or its vehicle (saline) immediately after hemisection SCI, and their motor activity and the degree of impairment caused by SCI was assessed using the BBB score. BBB scores were taken daily until the animals were sacrificed, 7 days after hemisection injury. The results are shown in Figure 6. It was apparent very early after injury that animals which received ZnCl₂ were more impaired than animals receiving the saline vehicle. This difference was maintained until the 7^th day (last day tested), when the 2 groups differed in their score by 3 BBB units.

It can be concluded from the above observations that an excess of zinc in the aftermath of spinal cord injury worsens the outcome. This confirms our hypothesis that zinc chelation leads to an improved outcome, whereas an increase in zinc after injury has the opposite effects.

Claims

1. A zinc-chelating agent for use in the treatment or prevention of spinal cord injury.

2. A zinc-chelating agent for use according to claim 1, wherein the zinc- chelating agent is selected from the group consisting of a compound of formula

(I):

wherein:

R₁ is H, a halogen or -SO₃H;

R₂ is H, a halogen, a C₁ to C₆ alkyl, a C₆ to C₁₀ aryl, a C₃ to C₁₀ heterocycle, a C₁ to C₆ alkoxy, a nitro or an amide group;

R₃ is - OH or a C₁ to C₆ alkoxy group; or wherein R₂ and R₃ are fused to form a furanic cycle; and phanquinone (4,7-phenanthronine-5,6-dione), ethylenediamine tetraacetic acid

(EDTA), disodium-calcium-ethylenediamine tetraacetic acid (CaEDTA), (2S)-2- amino-2-methyl-3-sulfanyl-butanoic acid (D-penicillamine), ethylene glycol tetraacetic acid (EGTA), N,N,N',N'-tetrakis(2-pyridyhnethyl)-ethylenediamine

(TPEN), diethylenetriamine pentacetic acid (DTPA), 6~methoxy-(8-ρ- toluenesulfonoamido) quinoline (TSQ), diethyldithiocarbamate (DEDTC), bis(diethylthiocarbamoyl)disulfide (disulfiram), 1,10-phenanthroline, dipicolinate,

N-acetyl cysteine, diphenylthiocarbazone (Dithizone), l-[2-(5-carboxyoxazol-2- yl)-6-aminobenzofuran-5-oxy]-2-(2^I-amino-5'-methylphenoxy)ethane-N,N,N',N'- tetraacetic acid (Fura-2), dimercaptosuccinic acid (DMSA), dimercapto-1- propanesulfonic acid (DMPS), alphalipoic acid (ALA) and l,2-bis(o- aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and derivatives and pharmaceutically-acceptable salts and esters of any thereof.

3. A zinc-chelating agent for use according to claim 2, wherein the compound of formula (I) is selected from the group consisting of clioquinol (iodochlorhydroxyquin, 5-chloro-7-iodo-8-quinolinol), 8-hydroxyquinoline, 8- hydroxyquinoline-5-sulphonic acid (Sulphoxine), iodoquinol (5,7-diiodo-8- hydroxyquinoline) and chiniofon (8-hydroxy-7-iodo-5 quinoline sulphonic acid).

4. A zinc-chelating agent for use according to claim 3, wherein the zinc- chelating agent is clioquinol.

5. A zinc-chelating agent for use according to any one of the preceding claims, wherein the treatment or prevention of spinal cord injury is treatment of primary spinal cord injury or prevention of secondary spinal cord injury.

6. A zinc-chelating agent for use according to any one of the preceding claims, wherein the zinc-chelating agent is administered intraperitoneally, intravenously or intramuscularly.

7. A zinc-chelating agent for use according to any one of the preceding claims, wherein the zinc-chelating agent is administered in a single injection.

8. Use of a zinc-chelating agent in the manufacture of a medicament for the treatment or prevention of spinal cord injury.

9. Use according to claim 8, wherein the medicament is for the treatment or prevention of spinal cord injury by intraperitoneal, intravenous or intramuscular administration.

10. A method for the treatment or prevention of spinal cord injury comprising administering to a subject in need thereof a therapeutic amount of a zinc-chelating agent.

11. A method according to claim 10, wherein the zinc-chelating agent is administered intraperitoneally, intravenously or intramuscularly.

12. A method according to claim 10 or 11, wherein the zinc-chelating agent is administered in a single injection.