WO2004043483A1

WO2004043483A1 - A method for treating spinal cord injury

Info

Publication number: WO2004043483A1
Application number: PCT/AU2003/001498
Authority: WO
Inventors: Dawei Zang; Surindar Singh Cheema
Original assignee: Monash University
Current assignee: Monash University
Priority date: 2002-11-13
Filing date: 2003-11-13
Publication date: 2004-05-27
Anticipated expiration: 2005-05-13
Also published as: AU2002952658A0

Abstract

The present invention relates generally to a method of treatment and agents useful for same. More particularly, the present invention contemplates a method for treating spinal cord injury (SCI) by promoting locomotor recovery. The method of the present invention is preferably conducted by the administration of leukaemia inhibitory factor (LIF) or a functional homolog, analog or derivative thereof or an agonist of LIF function. The present invention also provides a method for ameliorating the symptoms of trauma or disease to the spinal cord in vertebrate animals and in particular mammals such as humans. The present invention further provides a physiological assessment system generally in the form of an animal model useful in screening for compounds which are capable of treating or ameliorating the symptoms of SCI.

Description

A METHOD FOR TREATING SPINAL CORD INJURY

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

DESCRIPTION OF THE PRIOR ART

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Spinal cord injury (SCI) represents a particularly debilitating condition and frequently results in various levels of paralysis. Substantial efforts have been expended on repairing spinal cords and returning movement to otherwise paralyzed limbs and/or other parts of the body. However, efforts towards finding effective therapeutic interventions for SCI are hampered by the lack of suitable animal models that are fast, reliable and easy to reproduce. To date, only a handful of studies on SCI have been performed on mice (Kuhn and Wrathall, J. Neurotrauma 15: 125-140, 1998; Lang-Lazdunski et al., Stroke 31: 208-213, 2000; Ma et al, Exp. Neurol 169: 239-254, 2001; Seitz et al, J. Neurosci. Res. 67: 337-345, 2002; Seki et al, Neurosurge y 50: 1075-1082, 2002; Tao et al, Neurosci. Lett. 144: 116-120, 1992) despite the numerous advantages in their use. The low body weight of mice makes them ideal when only limited quantities of novel experimental drugs are available. Mice are easy to cage and can easily be put through a battery of locomotor tests. Finally, the availability of transgenic and gene knockout mice should provide unique opportunities for testing the role of specific genes in the inhibition or facilitation of spinal cord repair after injury.

In accordance with the present invention, an animal model for SCI is developed which results in the permanent paralysis of one leg for up to at least 10 months. This model is used to evaluate the effect of Leukaemia Inhibitory Factor (LIF), Minocycline (MIN) and a Vehicle (VEH) solution on locomotor behavior following SCI. LIF is a neuroprotective agent in a variety of neurodegeneration models (Cheema et al, Neuroreport 9: 363-366, 1998; Cheema et al, Journal ofNeuroscience Research 37: 213-218, 1994; Cheema et al, Neuroreport 5: 989-992, 1994; Murphy et al, Prog. Neurobiol. 52: 355-378, 1997; Kurek et al, J. Neurol. Sci. 160: S16-113, 1998). LIF also abrogates oligodendrocyte loss in a rat model of multiple sclerosis (Butzkueven et al, Nat. Med. 8: 613-619, 2002). Furthermore, LIF treatment of adult rats with SCI showed a significant increase in corticospinal axonal re-growth compared to controls with an attendant up-regulation of neurotrophin-3 (Blesch et al, J. Neurosci. 19: 3556-3566, 1999). MIN is a second- generation tetracycline that has been shown to be neuroprotective in a number of neurodegeneration models and is thought to act through several mechanisms including the inhibition of caspases 1 and 3, iNOS, p38 MAPK and activation of microglial cells (Zhu et al, Nature 417: 74-78, 2002). In accordance with the present invention, LIF has been shown to improve locomotive behavior in SCI.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The present invention provides wter alia a rapid and reproducible model of mouse spinal cord injury (SCI) that results in permanent behavioral and neuroanatomical deficits involving a hind limb. This model is used to evaluate the effects of Leukaemia Inhibitory Factor (LIF) and Minocycline (MIN) relative to a control vehicle (VEH). Mice in the VEH and MIN groups with SCI had negligible recovery of locomotor behavior, as measured by four different locomotor tests. In contrast, the LIF group that received LIF by intraperitoneal injections showed a statistically significant improvement in locomotor behavior and mice regained lesion locomotor function. The mouse model shows that LIF is capable of restoring locomotor behavior and is particularly indicated within hours of the trauma or disease leading to SCI.

Accordingly, the present invention contemplates a method of treating SCI or otherwise ameliorating the symptoms of SCI in a vertebrate animal, the method comprising administering to the animal an effective amount of LIF or a functional homolog, analog or derivative thereof or an agonist of LIF for a time and under conditions sufficient for an improvement in locomotor behavior to occur.

The present invention further provides compositions comprising LIF and/or its functional homologs, analogs, derivatives or agonists for use in promoting locomotor behavior in a paralyzed limb or portion of the body following SCI.

The present invention is further directed to the use of LIF or functional homologs, analogs, derivatives or agonists thereof in the manufacture of a medicament for the treatment of paralysis or other form of impaired movement following SCI. The "treatment of paralysis" in this context includes restoring full or partial locomotive behavior.

The subject treated may be any vertebrate animal such as a mammal and in particular a human. SCI may result from physical trauma or from a neurological disease, autoimmune disease or infection by a pathogenic microorganism or virus.

The present invention further provides an animal model of SCI. The animal model comprises an animal having paralysis in at least one limb. Conveniently, the paralysis is caused by SCI following a right hemisection which is extended to include the opposite dorsal and ventral corticospinal tracts.

A list of abbreviations used herein is provided in Table 1.

TABLE 1 Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographic and graphical representation showing coordination and RotaRod analyses of mice with SCI reveal a permanent hind limb paralysis. Panel A shows a normal mouse balancing at the edge of a transparent perpex plate. Panels B and C show the same mouse with SCI ten days (B) and five weeks (C) after SCI. Note the permanent paralysis of the right leg (arrowed). Performance on the RotaRod shows a 94% (D) and 97% (E) reduced capacity after five (D) and ten (E) weeks post lesion. The solid line represents pre-lesion performance at 180 seconds.

Figure 2 is a photographic and graphical representation showing analysis of corticospinal, rubrospinal and bulbospinal systems after SCI. Panels A-C show significant (P<0.0001) reductions in corticospinal neurons retrogradely labelled with Fluorogold in normal (A, D) and SCI mice five (B) and ten (C) weeks after SCI. Panel E shows a significant reduction (P<0.001) in dorsal corticospinal axons below the lesion at Li compared to above the lesion at Tn. Panels F-H show rubrospinal and bulbospinal neurons retrogradely labelled with Fluorogold in the intact (F) and lesioned (G, H) mice. Note the significant (P<0.001) reduction in rubrospinal (G, arrow and I) and bulbospinal (H, arrow and J) neurons. Scale bar, 100 μm.

Figure 3 is a graphical representation showing quantification of locomotor behavior of mice with SCI in the LIF, MIN and VEH groups using four independent tests. Treatment commenced two hours after the induction of SCI. Panel A shows significant (PO.001) improvement on the RotaRod (A) apparatus in the LIF but not MIN and VEH groups. The maximal response in the LIF group is achieved by week three despite continuous treatment for ten weeks. A similar significant (PO.01) improvement in the LIF group was observed in the bar grab (B), bar walk (C) and platform hang (D) tests as indicated by the solid black histograms. Figure 4 is a photographic and graphical representation showing unbiased stereological estimates of myelinated axons above and below the SCI. In panel A, the slashed and horizontal hatches indicate the minimal and maximal extent of the SCI at T₁₂. Myelinated axons were counted in four compartments, namely the lateral, dorsal and ventral white matter (LW, DW & VW) and the dorsal corticospinal tract (dCST). Panels C-E are photomicrographs showing myelinated axons in the LW of an intact (C) mouse and mice from VEH (D) and LIF (E) groups with SCI. There is significantly higher number of myelinated axons in all four compartments only in the LIF group below the SCI at L] compared to above the lesion at T₈ (F-G). * P<0.05~0.01, ** a significant difference (P<0.05) between females and males. Scale bar, 0.5 mm A, B and 50 μm C-E.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the use of an animal model for SCI to ascertain the symptom-ameliorating effects of particular agents LIF. In particular, in accordance with the present invention, it is determined that the administration of LIF promotes restoration of locomotive behavior. It is proposed, therefore, that LIF be used in the manufacture of a medicament to treat vertebrate animals suffering from SCI, and in particular, SCI leading to complete or partial paralysis of limbs or other body parts.

SCI may be induced by physical trauma or may occur following a disease by a pathogenic organism, microorganism or virus. It may also occur following an autoimmune condition.

Accordingly, one aspect of the present invention contemplates a method for treating SCI or the symptoms of SCI in a vertebrate animal, said method comprising administering to said vertebrate animal an effective amount of an agent which promotes or otherwise facilitates restoration of locomotor behavior.

It is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations of components, manufacturing methods, dosage regimens or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to an agent includes a single agent, as well as two or more agents.

In describing and claiming the present invention, the following terminology is used in accordance with the definitions set forth below.

The terms "compound", "active agent", "chemical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound that induces a desired pharmacological and/or physiological effect. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "compound", "active agent", "chemical agent" "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.

Reference to a "compound", "active agent", "chemical agent" "pharmacologically active agent", "medicament", "active" and "drug" includes combinations of two or more actives such as two or more phosphatidylcholines. A "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

For example, a multi-part pharmaceutical pack may have two or more agents separately maintained.

The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent (e.g. agent such as LIF) to provide the desired therapeutic or physiological effect or outcome. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

"Treating" a subject may involve prevention of a condition or other adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the condition.

A "subject" as used herein refers to an animal, preferably a mammal and more preferably human who can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical formulations and methods. A subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.

In a preferred embodiment, the agent of the present invention is generally selected from the list consisting of:-

(1) LIF;

(2) a functional homolog, analog or derivative of LIF;

(3) an agonist of LIF;

(4) a molecule required for LIF gene expression or LIF activity; (5) a target substrate of LIF or its functional homolog, analog, derivative or agonist. The above agents may be used alone or in conjunction with each other such as LIF. Although the preferred agent is LIF, the present invention may be practiced with other molecules such as those contemplated above. The molecules defined in (3) and (4) may also be provided together with LIF or a functional homolog, analog or derivative of LIF. Consequently, an "agent" is not to be construed as being limited to a single molecule or compound and may be a composition of two or more molecules or compounds. In addition, LIF may be administered together with another cytokine, a wound promoting agent, stem cells and/or an agent capable of promoting stem cell differentiation and/or proliferation.

Furthermore, the term "agent" also extends to genetic molecules such as cDNA and genomic DNA molecules and vectors comprising same. Furthermore, where an endogenous gene encodes a product which inhibits expression of a LIF gene (or cDNA), then a genetic agent may comprise an antisense or sense molecule or other molecule which is capable of inducing inhibition at the transcriptional level or at the post-transcriptional level. In other words, gene silencing may be used to reduce the levels of inhibitors of LIF gene expression or of LIF activity.

Reference to a "sense" molecule includes an agent which comprises or which induces RNAi.

A "gene" includes genomic as well as cDNA.

Notwithstanding the range of agents contemplated by the present invention, LIF is the most preferred along with its homologs, analogs and functional derivatives.

Accordingly, another aspect of the present invention provides a method for treating SCI or the symptoms of SCI in a vertebrate animal, said method comprising administering to said vertebrate animal an effective amount of LIF or a functional homolog, analog or derivative thereof which promotes or otherwise facilitates restoration of locomotor behavior. The LIF may be recombinant, synthetic or isolated and naturally occurring. Recombinant LIF is described wter alia in International Patent Application No. PCT/AU88/00093. The LIF may be of any mammalian origin such as of human, murine, porcine or ovine origin. When the LIF is derived from the same species being treated, then the term "homologous LIF" is used. Where the LIF is from a different species to that being treated, then "heterologous LIF" is used.

The present invention extends to the use of homologous and heterologous LIF. Furthermore, LIF from one animal species may be deimmunized (or mammalized) with respect to the mammal being treated. For example, murine LIF can be humanized for use in humans.

The present invention contemplates the treatment and/or prophylaxis of vertebrate animals such as mammals. Reference herein to mammals includes humans, primates, livestock animals (e.g. sheep, horses, cows, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs, hamsters), companion animals (e.g. dogs, cats) and captured wild animals. The treatment of humans, however, is most preferred.

The present invention further extends to the use of a polypeptide or chemical molecule having LIF properties in the manufacture of a medicament for the treatment of SCI.

Such a polypeptide includes LIF. As indicated above, the method and use according to these and other aspects of the present invention may comprise the administration of LIF alone or in combination with one or more other therapeutic agents such as but not limited to one or more other cytokines. The additional agents may be administered simultaneously or sequentially with LIF. Sequential administration means separate administrations within seconds, minutes, hours, days or weeks of LIF and the other agent. LIF and the other agent or agents may be administered in any order.

The present invention further extends to a composition comprising an agent such as LIF for use in the treatment of SCI, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

Preferably, the composition is exclusively used for the treatment of SCI and may be provided with instructions for use.

In this regard, another aspect of the present invention provides a pharmaceutical pack comprising LIF or a homolog, analog or functional derivative in powdered or liquid form, optionally one or more pharmaceutically acceptable carriers and/or diluents which may comprise LIF or be in a separate container and instructions for use, said instructions comprising administering to said vertebrate animal an effective amount of LIF or a homolog, analog or functional derivative thereof which promotes or otherwise facilitates restoration of locomotor behavior.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating expression of a nucleic acid molecule. The vector may, for example, be a viral vector. In this regard, a range of gene therapies are contemplated by the present invention including isolating certain cells, genetically manipulating and returning the cell to the same subject or to a genetically related or similar subject. The present invention further contemplates the administration of "naked" DNA which encodes a LIF polypeptide or an agonist of expression of a LIF gene. LIF may be administered in any number of ways including via intravenous, intraperitoneal, subcutaneous, intrathecal, rectal, intranasal or aerosol administration. Prolonged infusion or sustained release administration is also contemplated. Delivering LIF intraperitoneally or direct to the spinal cord or site of trauma or lesion is particularly preferred. Preparations comprising LIF can be conveniently prepared with reference to Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, U.S.A.

As stated above, the present invention extends to LIF and its homologs, analogs and functional derivatives. A derivative includes a mutant, fragment, part, portion or region of LIF such as a single or multiple amino acid substitution, addition and/or deletion to the LIF amino acid sequence. A derivative also includes hybrid molecules and fusion molecules such as between LIF polypeptides from different species of animals or between polymorphic variants of LIF polypeptides within the one species.

Other derivatives of LIF contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

A "homolog" includes a LIF molecule from a different animal species as well as a structurally and/or functionally related molecule from the same species. A polymorphic variant is regarded herein as a homolog.

LIF "analog" contemplated herein include but are not limited to modifications to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. Analogs may exhibit greater stability, longer serum half-life and enhanced efficacy. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 2.

TABLE 2 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-( -hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine l-carboxy-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane

All derivatives, homologs and analogs of LIF are encompassed by the terms "leukemia inhibitory factor", "LIF", "LIF polypeptide" and a "polypeptide having LIF properties".

The effective amount of LIF contemplated for use in accordance with the subject method in the amount required to ameliorate the symptoms of SCI such as paralysis. Suitable amounts may need to be varied according to the condition and severity of the condition being treated. Multiple doses may be administered or a single bolus may be given. Examples of effective amounts include from about 10 ng/kg body weight to about 10 mg/kg body weight and more particularly from about 0.1 μg/kg body weight to about 5 mg/kg body weight and even more particularly from about 0.5 μg/kg body weight to about 1 mg/kg body weight. Administration may be per hour, per day, per week, or per month.

Generally, recombinant LIF is administered. For the treatment of humans, for example, recombinant human LIF is preferred although the present invention extends to humanized forms of non-human LIF.

The present invention further provides an animal model for SCI. The animal model is useful to screen for agents capable of ameliorating the symptoms of SCI and restore full or partial locomotor behavior. Generally, the animal model involves inducing physical, chemical or genetic trauma to a section or portion of the spinal cord sufficient to induce full or semi-paralysis of at least one limb, digit or portion of a non-human vertebrate animal.

Accordingly, another aspect of the present invention provides an animal model for SCI, said animal model comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal cord to thereby induce full or partial paralysis of a digit, limb or other portion of the body of the vertebrate animal.

The fully or partially paralyzed portion includes an arm, leg, finger, toe or neck -of a vertebrate animal.

The preferred vertebrate animals include mice, rats, rabbits, guinea pigs, hamsters, primates, dogs, cats, pigs, sheep or goats. Mice are particularly convenient due to their rapid ability to breed, small size and easy handling ability.

The animal model comprises, therefore, an intentional derangement or impairment of the spinal cord to facilitate full or partial paralysis of at least one digit, limb or other body portion.

In a particularly preferred embodiment, the animal model is a mouse which has undergone a right hemisection which is extended to include the opposite dorsal and ventral corticospinal tracts.

The animal model of the present invention is useful wter alia in screening for agents which facilitate amelioration of the symptoms of paralysis and promote locomotor behavior.

Accordingly, another aspect of the present invention contemplates a method of treatment of a vertebrate animal suffering SCI, said method comprising administering to said vertebrate animal an effective amount of an agent which when administered to an animal model comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal cord to thereby induce full or partial paralysis of a digit, limb or other portion of the body to a vertebrate animal, locomotor behavior is fully or partially restored.

Still another aspect of the present invention contemplates an agent identified by being able to restore fully or partially locomotor behavior in an animal model, said animal model comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal cord to thereby induce full or partial paralysis of a digit, limb or other portion of the body to a vertebrate animal.

Reference to SCI herein includes any form of physical, chemical or genetic trauma to the spinal cord. A physical trauma includes a tissue insult such as an abrasion, incision, contusion, puncture, compression etc., such as can arise from traumatic contact of a foreign object with any locus of or appurtenant to the head, neck or vertebral column. Other forms of traumatic injury can arise from constriction or compression of CNS tissue by an inappropriate accumulation of fluid (for example, a blockade or dysfunction of normal cerebrospinal fluid or vitreous humor fluid production, turnover, or volume regulation, or a subdural or intracranial hematoma or edema). Similarly, traumatic constriction or compression can arise from the presence of a mass of abnormal tissue, such as a metastatic or primary tumor.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Generation of SCI mouse model

C57BL/6J mice aged six weeks were subject to SCI. Under Ketamine (0.1 mg/gm IP, Parnell Laboratories Australia Pty Ltd) and Ilium Xylazine (0.014 mg/gm IP, Troy Laboratories Pty.Ltd) anaesthesia, the Tj₂ spinal cord segment was exposed. A right hemisection that extended to include the opposite dorsal and ventral corticospinal tracts was made using a pair of iridectomy scissors (Figure 4A). All mice with SCI were able to drink and eat independently and urinate and defecate after recovering from anaesthesia. Mice with SCI show a persistent right hind limb paralysis that is easily seen when they are placed on the edge of a perpex (Figures 1A-C).

In this study, unpaired t-test and one-way analysis of variance (ANOVA) with Tukey's post-hoc-test were performed using Prism software (Version 3.0, GraphPad Software Inc., San Diego, USA). Prior to SCI, the normal mice could balance on the RotaRod for 180 seconds, but five weeks following SCI, they could only manage 10±6 (Mean±SD) seconds, i.e. a reduction of approximately 94% (Figure ID). After ten weeks, the mice could stay on the RotaRod for 6±2 (Mean±SD) seconds, a reduction of approximately 97% (Figure IE). Indeed, one group (n=3) of mice are over 10 months post-lesion and they are normal with the exception of a permanent right hind limb paralysis and inability to stay on the RotaRod for more than 14+12 (Mean±SD) sees.

Tracing with the retrograde tracer Fluorogold (FG) was used to map the status of the corticospinal, rubrospinal and bulbospinal connections (Figure 2). In comparison to mice without SCI, the number of retrogradely labeled neurons associated with the corticospinal, rubrospinal and bulbospinal pathways were severely reduced following SCI (Figures 2A- D, F-J). Furthermore, the number of axons in the dorsal corticospinal tracts showed a consistent loss of axons below the lesion at Li compared to above the lesion at Tn (Figure 2E). These analyses showed a comprehensive loss of major descending tracts following the T12 SCI. EXAMPLE 2 Effects of LIF on SCI mouse models

Thirty mice with SCI were divided into three groups of 10 (five male and five female per group). Mice in each group received thrice weekly intraperitoneal injections of LIF (25 μg/kg) (Azari et al, Brain Res. 922: 144-147, 2001), MIN (10 mg/kg) (Zhu et al, 2002, supra) or VEH (1% w/v albumin in 0.1 M mouse tonicity phosphate-buffered saline). It should be noted that treatments were initiated two hours after the completion of the SCI when the animals had regained consciousness. On the RotaRod test, the LIF group had a significantly (P<0.001) better outcome in terms of recovery of locomotor behaviour compared to the VEH and MIN groups (Figure 3A). The maximal recovery was achieved three weeks post-lesion. Three additional tests of locomotor function namely the bar grab, bar walk and platform hang as described previously were also carried out at week ten (Kuhn and Wrathall, 1998, supra). In all three tests, the outcomes (Figure 3B-D) were significantly (PO.01) better in the LIF compared to the VEH and MIN groups.

EXAMPLE 3 Histological examination

All the mice were killed after the 10-week post-lesion period and their spinal cords fixed and processed for histological estimation of the lesion size using MD2 Microscope Digitizer (Version 3.3J. Minnesota Datametrics Corp. USA). In all the mice with SCI, the right T₁₂ spinal cord was hemisected and, more importantly, in all these animals the contralateral dorsal and ventral corticospinal tracts were also damaged (Figure 4A). The next step was to estimate the number of axons in the spinal cord white matter above and below the lesion. The white matter was for the purposes of counting divided into four compartments, namely, the dorsal, lateral and ventral white matter and the dorsal corticospinal tract (Figure 4B). An unbiased stereological technique was used to estimate the number of myelinated axons (Figure 4C) in these compartments as described previously (Kilpatrick et al, Brain Res. 911: 163-167, 2001). It is evident in histological sections stained with toluidine blue that the number of myelinated axons in the lateral white matter of the LIF group (Figure 4E) was higher compared to the VEH group (Figure 4D). There is a significantly (PO.01) higher number of the corticospinal tract axons below the lesion at Li in the LIF group compared to the MIN and VEH groups (Figure 4F). Counts of myelinated axons below the lesion at Li were also carried out with respect to the gender of the mice. As shown in Figure 4G, there were significantly more myelinated axons both in the male and female SCI mice in the LIF group in all four compartments. However, there was significant difference (P<0.05) between the males and females only in the lateral lesioned white matter.

EXAMPLE 4

Application to treating SCI

In broad terms, two main approaches can be considered in therapeutic intervention that reverse paralysis associated with SCI. The first approach should promote recovery of intact axons that are compromised while the second approach should address the survival, re-growth and synapsis of severed axons. Agents such as LIF may play a vital role in the recovery of function amongst traumatized and severed axons, particularly in relation to oligodendrocytes and restitution of myelin.

In accordance with the present invention, LIF has been shown to have a positive effect on the recovery of locomotor behaviour after SCI. LIF is a multi-functional cytokine with a variety of actions on the developing and adult nervous system (Murphy et al, 1997, supra). The beneficial effect of LIF in relation to SCI may be attributable to any one of its many actions. However, the most likely mode of action of LIF in the SCI model may be via the promotion of oligodendrocyte survival.

An interesting contrast in this study was the complete ineffectiveness of MIN. This tetracycline derivative has multiple actions including the inhibition of caspases 1 and 3, iNOS, p38 MAPK and activation of microglial cells (Zhu et al, 2002, supra). It is unlikely that these processes that usually accompany degeneration are unimportant in SCI. After all, MIN is effective in various model of neurodegeneration including Huntington's disease (Berger, Bmj 321: 70, 2000; Chen et al, Nat. Med. 6: 797-801, 2000), Parkinson's disease (Du et al, Proc. Natl Acad. Sci. USA 98: 14669-14674, 2001; He et al, Brain Res. 909: 187-193, 2001), traumatic brain injury (Sanchez Mejia et al, Neurosurgery 48: 1393- 1401, 2000), excitotoxicity and amyotrophic lateral sclerosis (Zhu et al, 2002, supra; Tikka et al, J. Immunol. 166: 7527-7533, 2001 ; Tikka et al, Brian 125: 722-731, 2002; Nan Den Bosch et al, Neuroreport 13: 1067-1070, 2002). One explanation for the ineffectiveness may be that a suboptimal dose of 10 mg/kg was used; however this dose delayed disease onset and mortality in a transgenic model of amyotrophic lateral sclerosis (Van Den Bosch et al, 2002, supra).

This study also showed that LIF was beneficial despite a two-hour delay before it was administered in this mouse model of SCI. These observations have important clinical implications for the treatment of human SCI. A two-hour time frame is well within the delay that most patients with SCI experience before they are admitted to a hospital. The present invention extends to the administration of LIF after two hours post-SCI. The data in this study clearly demonstrate an effective therapeutic "window" for LIF therapy. It is interesting to note that despite the thrice-weekly administration of LIF over a ten-week span, the maximal recovery peaks at approximately three weeks with little subsequent improvement. Thus, continuous LIF treatment may not be necessary beyond the first three weeks. It is postulated that there is an optimal penetrance of LIF into the damaged spinal cord due to the disrupted blood brain barrier (BBB). It should be noted that systemically administered LIF readily enters the brain and spinal cord by a saturable transport system across the BBB (Pan et al, J. Neuroimmunol 106: 172-180, 2000).

It is proposed that LIF may act by promoting ΝT-3 mediated axonal re-growth or improving myelination by generating new oligdodendrocytes and promoting the survival of existing ones.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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Claims

CLAIMS:

1. A method for treating spinal chord injury (SCI) or symptoms of SCI in a vertebrate animal, said method comprising administering to said vertebrate animal, an effective amount of an agent which promotes or otherwise facilities restoration of locomotive behavior.

2. The method of Claim 1 wherein the agent is Leukemia Inhibitory Factor (LIF).

3. The method of Claim 1 wherein the agent is a functional homolog, analog or derivative of LIF.

4. The method of Claim 1 or 2 wherein the agent is an agonist of LIF or is used in conjunction with LIF.

5. The method of Claim 1 or 2 wherein the agent is a target substrate of LIF or its functional homolog, analog or derivative or agonist or is used in conjunction with same.

6. The method of Claim 2 wherein the LIF is administered together with another cytokine, wound promoting agent, stem cells or an agent capable of promoting stem cell differentiation and/or proliferation.

7. The method of Claim 2 wherein the LIF is recombinant LIF.

8. The method of Claim 7 wherein the LIF is homologous to the species being treated.

9. The method of Claim 7 wherein the LIF is heterologous to the species being treated.

10. The method of Claim 1 wherein the vertebrate animal is a mammal.

11. The method of Claim 10 wherein the mammal is selected for a human, non-human primate, livestock animal, laboratory test animal, companion animal or captured wild animal.

12. The method of Claim 11 wherein the mammal is a human.

13. The method of Claim 11 wherein the mammal is a model of SCI.

14. The method of Claim 1 or 2 wherein the agent is delivered via intravenous, intraperitoneal, subcutaneous, intrathecal, rectal, intranasal or aerosol administration.

15. The method of Claim 1 or 2 wherein the agent is delivered by intraperitoneal administration.

16. The method of Claim 1 or 2 wherein the agent is delivered direct to the spinal chord or site of trauma or lesion.

17. The method of Claim 1 or 2 wherein the agent is administered in an amount of from about 10 ng/kg body weight to about 10 mg/kg body weight.

18. Use of an agent in the manufacture of a medicament for the treatment of SCI or symptoms of SCI in a vertebrate animal.

19. Use of Claim 18 wherein the agent is Leukemia Inhibitory Factor (LIF).

20. Use of Claim 18 wherein the agent is a functional homolog, analog or derivative of LIF.

21. Use of Claim 18 or 19 wherein the agent is an agonist of LIF or is used in conjunction with LIF.

22. Use of Claim 18 or 19 wherein the agent is a target substrate of LIF or its functional homolog, analog or derivative or agonist or is used in conjunction with same.

23. Use of Claim 19 wherein the LIF is administered together with another cytokine, wound promoting agent, stem cells or an agent capable of promoting stem cell differentiation and/or proliferation.

24. Use of Claim 19 wherein the LIF is recombinant LIF.

25. Use of Claim 24 wherein the LIF is homologous to the species being treated.

26. Use of Claim 24 wherein the LIF is heterologous to the species being treated.

27. Use of Claim 18 wherein the vertebrate animal is a mammal.

28. Use of Claim 27 wherein the mammal is selected for a human, non-human primate, livestock animal, laboratory test animal, companion animal or captured wild animal.

29. Use of Claim 28 wherein the mammal is a human.

30. Use of Claim 28 wherein the mammal is a model of SCI.

31. A physiological assessment system for SCI, said physiological assessment system comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal chord to thereby induce full or partial paralysis of a digit, limb or other portion of the body of the vertebrate animal.

32. The physiological assessment system of Claim 32 wherein the vertebrate animal is selected from a non-human primate, mouse, rabbit, guinea pig, hamster, dog, cat, pig, sheep, goat, horse and cow.

33. The physiological assessment of Claim 32 or 33 wherein the animal has undergone a right hemisection which is extended to include the opposite dorsal and ventricle corticospinal tracts.

34. A method of treatment of a vertebrate animal suffering SCI, said method comprising administering to said vertebrate animal an effective amount of an agent which when administered to an animal model comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal cord to thereby induce full or partial paralysis of a digit, limb or other portion of the body to a vertebrate animal, locomotor behavior is fully or partially restored.

35. An agent identified by being able to restore fully or partially locomotor behavior in an animal model, said animal model comprising a live non-human vertebrate animal which has undergone physical, chemical or genetic trauma to the spinal cord to thereby induce full or partial paralysis of a digit, limb or other portion of the body to a vertebrate animal.