NZ537103A

NZ537103A - Metallothionein based neuronal therapeutic and therapeutic methods

Info

Publication number: NZ537103A
Application number: NZ537103A
Authority: NZ
Inventors: Adrian Keith West; Meng Inn Chuah; James Clement Vickers; Roger Steven Chung
Original assignee: Univ Tasmania
Priority date: 2002-06-13
Filing date: 2003-06-13
Publication date: 2007-11-30
Also published as: EP1531905A1; EP1531905A4; AUPS295802A0; WO2003105910A1

Abstract

A method of stimulating neuronal growth or repair in a non human animal comprising exposing a target neuron or neuronal area to a solution of metallothionein isoform MT-IIA. Also described is a therapeutic composition adapted for topic administration to an area of neuronal compromise and comprises metallothionein isoform MT-IIA as an active ingredient.

Description

<div class="application article clearfix" id="description"> WO 03/105910 PCT/AU03/00735 Metallothionein Based Neuronal Therapeutic and Therapeutic Methods Introduction to the Invention 5 This invention relates to the use of metallothionein as an active ingredient in effecting and enhancing recovery of damaged neuronal tissue, particularly following physical trauma and damage thereto. The invention provides a therapeutic incorporating metallothionein and methods of treatment based thereon. 10 Background to the Invention Metallothionein (MT) is a naturally occurring peptide, which is present in most cells of the mammalian body. There are many isoforms in humans, but these resolve into four classes; MT-I and MT-II which are expressed widely, MT-HI which is mainly found in the brain, and MT-IV which is restricted to specific epithelial sites. MTs are 15 intracellular proteins with occasional nuclear localisation, and although there are persistent reports of extracellular detection of MT, the prevailing dogma is fixed that their physiological role is within cells. MTs are metal binding proteins (61-68 amino acids), which normally bind seven zinc ions, although zinc/copper mixtures have been reported. Some isoforms are rapidly 20 induced in response to increases in zinc or copper levels, and also by a large number of hormones and cytokines, including glucocorticoids, interleukin 1 and 6, interferons and so on. Their exact physiological role is unclear. Early suggestions that they act to prevent accumulation of toxic levels of heavy metals are no longer favoured, and if their role is indeed in metal metabolism, it is more likely that they are involved in the 25 intracellular homeostasis of zinc. However, MTs are efficient scavengers of free radicals and are able to protect DNA and other molecules from oxidation, suggesting that their function may be protective. MTs may be considered intracellular stress proteins which respond to a wide variety of stimuli. It is relevant that MT-I/n knockout animals, and those which overexpress MT-I 30 and MT-II are phenotypically normal, except for sensitivity and resistance, respectively, to some chemical and physical stresses. Deficiency of MT-III, the "brain-specific" class ofMT, has been implicated in the pathogenesis of Alzheimer's disease, although this finding has been strongly disputed. MT-IH reduces neuronal survival, and the applicants have shown that, when 35 MT-HI is added to cultured neurons it reduces neurite sprouting. Exogenous MT-HI appears to have an opposing effect to MT-DA and it is expected that comparison of WO 03/105910 PCT/AU03/00735 2 their structures will reveal strategies for designing analogues of both which have specific neurotrophic properties. It has been shown that exposure of rat brain lesions to MT-III causes vacuolisation, consistent with extensive neuronal death. 5 Metallothionein and Heavy Metals There is a large body of literature on the relationship between metallothionein and heavy metals, particularly cadmium. MT was originally isolated as a cadmium-binding protein, and it is known that it acts as the major intracellular sink for this toxic metal. Hence, people exposed to cadmium in the workplace or through contaminated 10 diet will have elevated metallothionein levels, particularly in the kidney. There is no question that MT acts to protect cells against cadmium, however it is not an effective agent, nor is it likely that this is the actual physiological role for the protein: it is likely an adventitious property derived from the chemical similarity between zinc and cadmium. One consequence of this is the numerous studies of the pharmacokinetics of 15 metallothionein bound to heavy metals, following various routes of administration. Whilst cadmiummetallothionein is (not surprisingly) toxic, it is not believed that zinc or copper-metallothioneins will have significant metal-based toxicity at the concentrations employed in the studies below. The applicants have examined the action of metallothionein proteins; including 20 MT-IIA, a major human metallothionein of the MT-I/II class. The studies found that administration of metallothionein to cultured rat neurons increases neuronal survival and enhances the rate of axonal extension. Furthermore, in lesioned rat brains, metallothionein enhances regenerative axonal extension into the lesions and replacement of damaged tissue. Accordingly, the use of metallothionein as an active 25 ingredient in neuronal therapy provides a novel method of stimulating neuronal growth and neuronal survival, a novel class of therapeutic agents and a novel method of treatment for a range of neuronally based disease states. Moreover, metallothionein offers several practical advantages as a therapeutic agent. 30 1. It is a naturally occurring, non-toxic protein 2. It appears possible that intraperitoneally administered metallothionein can enter the CNS compartment, following physical trauma to the brain or spinal cord or breakdown of the blood-brain barrier due to other causes. 3. Metallothionein is not post-translationally modified and hence can be easily 35 produced in bacterial or other expression systems WO 03/105910 PCT/AU03/00735 4. Metallothionein is a small peptide (61 amino acids for MT-IIA, 68 amino acids for human MT-III) and it is very likely that a novel analogue which is amenable to chemical synthesis can be designed. 5 Statement of the Invention In one aspect the invention provides a method of stimulating neuronal growth comprising exposing a target neuron to metallothionein. The target neuron is preferably placed in direct contact with a metallothionein solution. 10 The target neuron may have suffered physical trauma including lesion or other forms of neurodegeneration. The metallothionein may be selected from any one or a combination of known metallothionein classes including MT-I, MT-II, MT-HI and MT-IV and the associated isoforms. 15 Most preferably the metallothionein is selected from MT-II including human MT-IIA. The metallothionein may be a synthetic analogue which combines structural or physical features of any or all known metallothionein isoforms. The metallothionein may be provided in solution at a concentration of up to 20 about 5pg/ml in a neurologically acceptable carrier. Administration of the metallothionein solution may include MT-DA as the sole active ingredient. Alternatively, any one or a combination of the metallothionein classes and isoforms as detailed above may be used. Where combinations of metallothionein are used the different classes and isoforms may be combined in a single 25 dose. Alternatively, the different classes may be administered sequentially. The administration regime may include initial administration of a solution of MT-IIA followed by a subsequent administration of a solution of MT-III. The administration regime may be limited to MT-DA alone as the active ingredient. The method of the invention may be applied to a range of compromised 30 neuronal states including diseased states and injuries. In another aspect the invention provides a method of treatment of any one or a combination of Alzheimers, Parkinsons, Motor Neuron Diseases, head injury, comprising the administration to a patient of a therapeutic including metallothionein as previously described as an active ingredient wherein said therapeutic is applied or 35 administered so as to directly interact with the site of neuronal compromise. WO 03/105910 PCT/AU03/00735 4 In another aspect the invention provides a therapeutic composition comprising metallothionein in any one or a combination of isoforms, or as a synthetic metallothionein comprising features of one or more isoforms, as an active ingredient in a pharmaceutically acceptable carrier wherein said carrier is adapted for topical 5 administration to an area of neuronal compromise. The composition may be adapted for direct topical application to exposed neurons or for administration to non-exposed neurons by indirect routes including intravenous or intraperitoneal administration, which result in accumulation of metallothionein in the compromised region of the brain or other part of the central 10 nervous system. Detailed Description of the Invention Figure 1 shows some effects of human MT-IIA on neuronal survival and neurite elongation of cultured rat cortical neurons. 15 Figure 2 shows by immunocytochemistry the effect of human MT-HA on reactive neurite sprouting following axonal injury. Figure 3 shows by immunocytochemistry the effects of human MT-IIA on reactive neurite sprouting following axonal injury. Figures 4 and 5 show the effect of human MT-HA on lesions in the rat neocortex 20 formed by physical injury. Figure 6 shows the effect of human MT-IH and MT-IIA on neurite formation and initial neurite outgrowth of cultured rat cortical neurons. Figure 7 shows the effect of human MT-in and MT-IIA on the extent and rate of neurite elongation of cultured rat cortical neurons. 25 Figure 8 shows the effect of human MT-DI and MT-HA on the distribution of neurite length of cultured rat cortical neurons. Figure 9A shows immunocytochemistry the effect of human MT-IH and MT-HA on reactive neurite sprouting. Figure 9B shows by immunocytochemistry the effect of human MT-HI and MT-30 nA on reactive axonal growth. Figure 10 shows the quantitative effect of human MT-HI and MT-HA on reactive neurite sprouting and growth. Figure 11 shows by immunohistochemistry the effect of human MT-IIA on axonal sprouting into a lesion site following physical injury in the rat neocortex. 35 Figure 12 shows by immunohistochemistry the effect of human MT-HA on neuronal injury tract repair. WO 03/105910 PCT/AU03/00735 5 The action of MT-IIA (a major human metallothionein of the MT-I/-II class) in two distinct culture models of rat cortical neurons, and in a rat in vh>o model of cortical damage was examined. In culture, it was found that administration of MT-IIA increases neuronal survival, and enhances the rate of axonal extension. In lesioned rat brains, it 5 was found that MT-IIA enhances regenerative axonal growth into the lesion, and replacement of damaged tissue. Metallothionein Action on Cultured Rat Cortical Neurons: Culture Model 1: Rat cortical neurons (El8) were plated at low density in 10 neurobasal medium + B27 supplement, including 150 |ig/ml of a rat brain extract. Recombinant MT-IIA was produced (the major human metallothionein I/H isoform) in E. coli cultures and reconstituted as a zincthionein (7 moles zinc/mole protein). Culture Model 2: Rat cortical neurons (El8) were plated at a higher density in 15 Neurobasal medium + B27 supplement (but without brain extract) and cultured in vitro for 21 days, allowing the formation of neuronal clusters connected by fasciculated axonal bundles. Rat Cortical Injury Model: Focal injuries were performed to the adult rat 20 neocortex by insertion of a 25gauge beveled needle into the Par 1 region of the rat somatosensory cortex to a depth of 1.5mm into the brain. The invention will now be described with reference to a selection of embodiments and examples and Figures 1 to 12. 25 Example 1 Referring in turn to Figures 1 to 7 a series of experiments were conducted using the composition made up of 0.1 to 5p.g/ml of MT-HA in a pharmaceutically and neurologically acceptable carrier. Such a composition when applied topically to a 30 range of in vivo and in vitro neuronal situations clearly demonstrates that MT-HA functions as an active ingredient in enhancing neurite elongation. MT-HA also dramatically increases the extension of processes between clusters following lesions formed by microscapel and ultimately demonstrates the ability of MT-IIA to have a dramatic effect on increasing the rate of recovery from physical injuries. Referring to 35 Figure 1, the bar graph in Figure 1A demonstrates that human MT-IIA promotes neuronal survival in the presence of adult rat brain extract (150ng/ml) after three days. WO 03/105910 PCT/AU03/00735 6 (P < 0.01, ANOVA). Accordingly Zn-MT is not detrimental to the survival of cultured neurons. Referring now to Figure 6E and 6F, under the same conditions human MT-IIA does not increase the initiation of new neurite sprouting over three days, expressed as 5 either the percentage of neurite bearing neurons as shown in Figure 6E or the number of neurites per neurone as shown in Figure 6F. (P > 0.01 ANOVA). This observation has important clinical ramifications as inappropriate sprouting of neurons has been associated with premature neuronal death. Referring now to Figure 7B the bar graph shows MT-HA demonstrating dose 10 dependent promotion of neurite elongation during this period. (P < 0.01, ANOVA). From the above experiments it has been demonstrated that MT-HA is capable of enhancing neurite elongation of cultured rat cortical neurons without increasing the rate of undesirable neurite sprouting. Referring now to Figure 2 a culture of rat cortical neurons was maintained for 15 twenty-one days in order to allow formation of clusters which are interconnected by fasciculated bundles of axons. The axons were cut with a microscapel and a composition as previously described, including recombinant MT-HA, was added. The immunocytochemical results shown in Figures 2A to 2D show that twelve hours after cutting the neuronal bundles with a microscapel there is a marked retraction by 20 transected neurites from the lesion site (which is indicated with a broken line) of up to 100[xm. Whilst in the absence of MT-HA (fig 2A), there are very few neurite extensions, as assessed by NF-M immunoreactive processes (red) extending into the area of retraction (indicated by arrows) in untreated neurons, there are many in the MT-nA treated neurons (fig 2C). Tau and pIH-tubulin immunocytochemical analysis also 25 indicates very few processes extending into the area of retraction (indicated by arrows) in the absence of MT-IIA (fig 2B). However, after twelve hours of incubation with MT-HA at a concentration of lpg/ml, these processes are significantly longer and have extended into the lesion site (fig 2D). Referring now to Figure 3 the experiments detailed in Figure 2 were repeated 30 with a longer exposure period of eighteen hours to recombinant MT-IIA. Eighteen hours after cutting the axonal bundles, the tissue was assessed by immunocytochemical markers and analysis of neurite extension into the lesion. In untreated samples, shown in Figure 3 A, there is minimal neurite growth into lesion site. However, as shown in Figure 3C when a sample is treated with MT-HA at a concentration of 1 ng/ml, the 35 processes have completely traversed the lesion site with the immunoreactive processes shown in red extending from the neurite stumps indicated by arrows and have grown WO 03/105910 PCT/AU03/00735 7 towards the central lesion site indicated by the broken line. From this experimental work it has been demonstrated that MT-IIA promotes growth of TSIF-M immunoreactive processes which are indicated by the arrows across the central lesion site, Tau and (3HI-tubulin immunocytochemical analysis also indicates that a number of processes extend 5 into the area of retraction indicated by arrows in untreated neurons. However, these processes do not cross the transection site shown in Figure 3B. MT-IIA treated samples shown in Figure 3D have been sufficiently promoted such that the growth of processes occurs and extends beyond the transection site indicated by arrows to the opposite stump of the transected neurite bundle. 10 Accordingly, these experiments clearly show that MT-HA dramatically increases the extension of processes, including axons between clusters. This occurs following lesion by microscapel and after eighteen hours of exposure to MT-HA the axonal bundles have bridged the region between the clusters. This effect of MT-HA is a result of a direct topical interaction between the protein, the neurons and the culture 15 medium. Referring now to Figures 4, 5,11 and 12 the action of MT-HA on a rat model of cortical injury was investigated. The rat model of physical damage to the cortex has been previously developed by the inventors and extensively characterised in terms of neuronal damage, orthology, pathology and subsequent recovery. 20 Figures 4, 5,11 and 12 show the extent of the physical injury, and microglial invasion of the cavity. MT-HA administration reduced microglial infiltration and promoted formation of a tissue bridge across the lesion, from the pial surface down. MT-IIA also promoted axonal extension into the lesion site. Very few axonal extensions were seen in the rats treated with vehicle alone (control rats). 25 Figure 4 shows the global location of needle stick injuries as indicated in Panel A. Brain sections underwent immunohistochemistry against SMI-312 (green) and ferritin (red) 4 days post injury. Needle stick injury resulted in a large injury tract, and microglial migration into and surrounding the injury site (B). MT-HA treatment reduced microglial infiltration, and promoted the formation of a tissue bridge enclosing 30 the lesion site from the pial surface down, forming a teardrop like invagination (C). Microglia at the pial surface were small and round, in contrast to the large, amoeboid microglia observed in deeper cortical layers (D, E respectively). MT-HA promoted regenerative axonal growth into the lesion site at both the pial layer (D) and deeper cortical layers (E). In contrast, very few axonal extensions were visualised in control 35 rats, at the pial level (F) or deeper cortical layers (G). Arrowheads indicate the injury tract. WO 03/105910 PCT/AU03/00735 Figure 5 shows immunohistochemical staining against SMI-312 (green) and ferritin (red) 4 days post injury. SMI-312 immunoreactive axonal extensions were often found in close association with small round microglia at the pial surface (A, B). Contrastingly, axonal extensions in deeper cortical layers were often not associated 5 with larger, amoeboid microglia (C, D). Regenerating axons often exhibited a wavy morphology, as if they were constantly changing direction. Occasionally, pyramidal (indicated by arrow, C) and bulb-like (indicated by arrow, D) accumulations were observed along axonal sprouts. The above experiments clearly indicated that the administration of recombinant 10 MT-IIA dramatically increases the rate of recovery from physical injuries. In combination with the tissue culture experiments, this work indicates that the administration of MT-HA following central nervous system injury acts directly on neurons to increase the rate of axonal extension into the lesion. 15 Example 2 Turning now to the effect of human MT-HI as contrasted to MT-HA, a series of experiments were conducted as detailed in Figures 6 to 12. Referring firstly to Figure 6, the effect of MT-III on neurite formation is shown in Figure 6 A in the presence of adult rat brain extract at 150|jg/ml after three days. Figure 6B shows neurite bearing 20 neurons indicated by the arrows. The percentage of neurite bearing neurons is shown in Figure 6C and the number of neurites per neurite-bearing neuron is shown in Figure 6D. From the above it can be seen that MT-HI significantly inhibits neurite outgrowth in both instances at the concentrations tested (p < 0.01, ANOVA). Referring now to Figure 6E human MT-HA had no effect on initial neurite outgrowth over three days. 25 As assessed by both the percentage of neurite bearing neurons or as detailed in Figure 6F, the number of neurites per neurite-bearing neurons. Referring now to Figure 7, it has been shown that human MT-HI prolongs the process of neurite retraction from 0-2 and 2-4 hours after plating as can be seen with reference to Figure 7A. Following this, the rate of neurite elongation is reduced. Referring now to Figure 7B, in contrast to 30 this, application with human MT-IIA significantly increases the rate of neurite elongation. The distribution of neurite lengths three days after MT-HI treatment is shown in Figure 8A, where it is clearly indicated that while MT-HI significantly inhibits neurite growth, a small percentage of neurites were unaffected and grew to lengths comparable 35 to vehicle treated neurites. In contrast to this the distribution of neurite lengths WO 03/105910 PCT/AU03/00735 following MT-HA treatment indicated that a number of neurites grew to lengths greater than that of vehicle treated neurites as can be seen with reference to Figure 8B. Fluorescent double immunocytochemical labelling of cytoskeletal changes both tau shown in red and [3111-tubulin shown in green twelve hours after axonal transection 5 are shown in Figures 9 A Panel A and 9A Panel B. In contrast to this, treatment with MT-HI reduced regenerative neurite sprouting compared to vehicle treated samples as shown in Figures 9A Panel C and 9 A Panel D . The transection site is indicated by a broken line and there is a large area of retraction away from this line. Sprouting neurites are indicated by arrows. The MT-10 HA treatment increased both the number and length of reactive sprouts following injury and this is detailed in Figure 9A Panel E which details the vehicle example and 9A Panel F which indicates the MT-HA example at ljxg/ml (check, please). Referring now to the eighteen hours post axonal transection. This is shown by flourescent double immunocytochemcial labelling of cytoskeletal changes, both tau 15 shown in red and (BIH-tubulin shown in green. The MT-DA can be seen to have promoted reactive axonal growth across the entire transection site shown in Figure 9B Panel A. In contrast such axonal growth is not observed following treatment with either vehicle as shown in Figure 9B Panel B or MT-HI shown in 9B Panel C. Treatment of samples with human MT-HI significantly inhibited both the 20 number and length of reactive sprouts at a twelve hour interval after axonal transection in culture. However, treatment with human MT-HA significantly increased the mean neurite length of reactive sprouts at 12 hours after transection. These findings are detailed in Figures 10A and 10B. 25 Example 3 Further experimental work was done to test the effect of MT-HA and these results are shown in Figures 11 and 12 under immunohistochemical studies for SMI-312 (green axonal marker) and ferritin (red microglial marker). Referring firstly to Figure 11, the results at four days post injury are shown where the needle stick injury 30 resulted in a large injury tract and microglial migration into and surrounding the injury site shown in Figure 11A MT-HA treatment promoted the formation of a tissue bridge enclosing the lesion site from the pial surface down so as to form a tear drop like invagination shown in Figure 1 IB. MT-IIA further promoted axonal sprouting into the lesion site at both the pial layer as shown in Figure 11C and deeper cortical layers 35 shown in Figure 1 ID. In contrast to this, very few axonal sprouts were visualised in 10 10 15 control rats as shown at the pial level in Figure 1 IE and deeper cortical layers as shown in Figure 1 IF. The arrow heads indicate the injury tract. Figure 12 shows details of further experimental work on brain sections at 7 days post injury. In vehicle treated rats, the injury tract was smaller compared to four days post injury shown in Figure 11; although it had not completely closed over, a degree of reactive sprouting is evident in all animals at this time point as shown in Figure 12A. Reactive processes exhibited greater SMI-312 reactivity than uninjured neuronal processes in surrounding neural tissue. Reactive astrocytes also aligned along the borders of the injury tract as shown in Figure 12B. In MT-IIA treated rats, the entire injury tract had closed over, and was demarcated only by a fine line of ferritin immunoreactivity as shown in Figure 12C. Reactive astrocytes also enclose the injury tract, and were found at lower density in adjacent uninjured tissue shown in Figure 12D. In MT-IIA treated animals, numerous reactive axonal processes were observed as shown by the arrows within the injury tract at both deeper cortical levels shown in Figure 12E and pial levels shown in Figure 12F . The above detailed examples demonstrate the clinical application of MT-IIA in promoting nerve cell survival, promoting neuronal regeneration and generally enhancing neurite elongation without causing inappropriate neuronal sprouting. The findings and experimental results support many clinical applications of the invention as detailed below. Disease Indication Role of MTI/II (HA) Alzheimer's disease Buffer metals implicated in development of pathological hallmarks. Promote nerve cell survival. Promote neuronal regeneration. It was demonstrated that MTI/II is upregulated in early stages of the disease (published) Parkinson's disease Promote nerve cell survival. Promote regeneration. Buffer metals implicated in toxicity. Evidence of abnormal metal homeostasis in the brain as well as neurodegeneration. Motor neuron disease Promote nerve cell survival. Evidence of abnormal Promote neuronal regeneration. metal homeostasis in the Buffer metals implicated in brain and spinal cord as toxicity. Reduce oxidative stress well as neurodegeneration. implicated in neuronal degeneration. INTELLECTUAL PROPERTY OFFICE OP N,Z. 1 6 Nov 2006 RECEIVPn WO 03/105910 PCT/AU03/00735 11 Head injury Promote nerve cell survival. Promote neuronal regeneration. It was shown that MT I/n is upregulated at zone of injury. Recombinant protein promotes brain healing and axonal regeneration Spinal cord trauma Promote nerve cell survival. Promote neuronal regeneration. Evidence of delayed neurodegeneration and spinal cavitation following injury. The recombinant protein is potentially capable of promoting neural healing and regeneration. Glaucoma Promote nerve cell survival. Promote neuronal regeneration. Axonal damage followed by neurodegeneration underlies the disease. MTI/II may potentially promote survival of nerve cells and/or appropriate regeneration. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 12 10 15 20 • 25 30 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of stimulating neuronal growth or repair in a non human animal comprising exposing a target neuron or neuronal area to a solution of the metallothionein isoform MT-IIA.

2. A method according to claim 1 wherein said contact is by direct interaction of the target neuron or neuronal site with said solution.

3. A method according to claim 1 or 2 wherein said MT-IIA is naturally occurring human MT-IIA.

4. A method according to claim 1 or 2 wherein said MT-IIA is produced by chemical synthesis or by production in genetically manipulated cells or organisms.

5. A method according to claim 4 wherein said MT-IIA is recombinant human MT-IIA.

6. A method according to any one of claims 1 to 5 wherein said solution has a concentration of up to about 5(iig/ml metallothionein in a neurologically acceptable carrier.

7. A method according to claim 6 wherein said solution has a concentration of about 5|ag/ml metallothionein in solution.

8. A method according to any one of claims 1 to 5 further including exposing said neuron or neuronal area to any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT-IV.

9. A method according to claim 8 wherein said target neuron or neuronal area is exposed simultaneously to a combination of MT-IIA and any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT-IV.

10. A method according to claim 8 wherein said target neuron or neuronal area is exposed sequentially to a combination of MT-IIA followed by any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT- 11. m:\specifications\500000\503000\503056clmmaw.doc INTilLf CTUAL PROPERTY OFFICE OF N.Z. 1 6 NOV 2006 RECEIVED 13

11. A method according to claim 8 wherein said target neuron or neuronal area is exposed sequentially to a combination of any one of metallothionein isoforms selected from MT-I, MT-II, MT-IIA, MT-III and MT-IV. 5

12. A method according to any one of claims 1 to 11 wherein said neuron or neoronal area is located in the brain.

13. A method according to any one of claims 1 to 12 wherein said solution is administered to said neuron or neuronal area by any one or a combination of direct injection, intraperitoneal injection, oral administration or via genetically modified cells including stem cells.

14. A method of treatment of Alzheimer's Disease in a non human animal comprising administration to a patient in need of treatment a therapeutic composition including metallothionein in accordance with the method of any one of claims 1 to 13.

15. A method of treatment of Parkinson's Disease in a non human animal comprising administration to a patient in need of treatment a therapeutic composition including metallothionein in accordance with the method of any one of claims 1 to 13. 20

16. A method of treatment of motor neuron disease in a non human animal comprising administration to a patient in need of treatment a therapeutic composition including metallothionein in accordance with the method of any one of claims 1 to 13.

17. A method of treatment of head injury in a non human animal comprising administration to a patient in need of treatment a therapeutic composition including metallothionein in accordance with the method of any one of claims 1 to 13.

18. A therapeutic composition adapted for topical administration to an area of 30 neuronal compromise said composition characterised by metallothionein isoform MT- IIA as an active ingredient.

19. A composition according to claim 18 wherein said active ingredient is combined with any one or a combination of metallothionein isoforms selected from MT-1, MT-II, 35 MT-III and MT-IV. m:\specifications\500000\503000\503056clnimaw.doc INTELLECTUAL PROPERTY OFFICE OF N.Z. V 6 NOV 2006 RECEIVED 14

20. A composition according to claim 18 or 19 wherein said metallothionein is naturally occurring human MT-IIA.

21. A composition according to any one of claims 18 or 19 wherein said 5 metallothionein is produced by chemical synthesis or by production in genetically manipulated cells or organisms. 10

22. A composition according to claim 21 wherein said metalliothionein is recombinant human MT-IIA.

23. A composition according to any one of claims 18 to 22 further including a neurologically acceptable carrier particularly adapted for a topical administration to an area of neuronal compromise. 15

24. A composition according to claim 23 adapted for direct topical application.

25. A composition according to claim 23 adapted for intraperitoneal or intravenous administration to effect exposure of neurons by a non-topical route. 20

26. A method according to any one of claims 1 to 17 substantially as hereinbefore described with reference to the examples.

27. A composition according to any one of claims 18 to 25 substantially as hereinbefore described with reference to the examples. 25

28. The use of metallothionein isoform MT-IIA for the production of a pharmaceutical composition for the stimulation of neuronal growth

29. The use of metallothionein isoform MT-IIA for the production of a 30 pharmaceutical composition for the stimulation of neuronal growth or repair in the brain.

30. The use of metallothionein isoform MT-IIA for the production of a pharmaceutical composition for the treatment of Alzheimer's Disease. 35 m:\specifications\500000\503000\503056clmmaw.doc INTELLECTUAL PROPERTY OFFICE OF N.Z. 16 NOV 2006 RECEIVED 15

31. The use of metallothionein isoform MT-IIA for the production of a pharmaceutical composition for the treatment of Parkinson's Disease.

32. The use of metallothionein isoform MT-IIA for the production of a 5 pharmaceutical composition for the treatment of motor neurone disease.

33. The use of metallothionein isoform MT-IIA for the production of a pharmaceutical composition for the treatment of head injury. 10

34. The use according to any one of claims 28 to 33 wherein the MT-IIA is naturally occurring human MT-IIA.

35. The use according to any one of claims 33 to 34 wherein said MT-IIA is produced by chemical synthesis or by the production in genetically manipulated cells or 15 organisms.

36. The use according to claim any one of claims 28 to 35 wherein the said MT-IIA is recombinant human MT-IIA. 20

37. The use according to any one of claims 28 to 36 wherein said pharmaceutical composition includes a solution having a concentration of 5p.g/ml metallothionein in a neurologically acceptable carrier.

38. The use according to any one of claims 28 to 37 wherein said solution has a 25 concentration of about 5 jig/ml of metallothionein in solution.

39. The use according to any one of claims 28 to 38 wherein said pharmaceutical composition further includes any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT-IV. 30

40. The use according to any one of claims 28 to 39 wherein said pharmaceutical composition includes a solution adapted for administration to said neuron or neuronal area by any one or a combination of direct injection, intraperitoneal injection, oral administration or via genetically modified cells including stem cells. 35 m:\specifications\500000\503000\503056clmmaw.doc INTELLECTUAL PROPERTY OFFICE OF N.Z. 16 NOV 2006 RECEIVED 16

41. The use according to any one of claims 28 to 40 wherein said pharmaceutical composition is adapted for topical administration to an area of neuronal compromise wherein said composition is characterised by metallothionein isoform MT-IIA as an active ingredient. 5

42. The use according to any one of claims 28 to 41 wherein said active ingredient is combined with any one or a combination of metallothionein isoforms selected from MT-1, MT-II, MT-III and MT-IV. 10

43. Use of metallothionein isoform MT-IIA in the manufacture of a solution for stimulating neuronal growth or repair by a method comprising exposing a target neuron or neuronal area to the said solution.

44. Use according to claim 43 wherein the method comprises direct interaction of 15 the target neuron or neuronal site with said solution.

45. Use according to claim 43 or 44 wherein said MT-IIA is naturally occurring human MT-IIA. 20

46. Use according to claim 43 or 44 wherein said MT-IIA is produced by .chemical synthesis or by production in genetically manipulated cells or organisms.

47. Use according to claim 46 wherein said MT-IIA is recombinant human MT-IIA. 25

48. Use according to any one of claims 43 to 47 wherein said solution has a concentration of up to about 5 |ag/ml metallothionein in a neurologicaily acceptable carrier.

49. Use according to claim 48 wherein said solution has a concentration of about 5 30 (ag/ml metallothionein in solution.

50. Use according to any one of claims 43 to 47 further including the use of any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT-IV in the manufacture of a solution or solutions for stimulating neuronal growth or 35 repair by a method comprising exposing a target neuron or neuronal area to the said solution. INTELLECTUAL PROPERTY OFFICE OF N.Z. m:\specifications\500000\503000\503056clmmaw.doc I 16 NOV 2006 RECEIVED 17

51. Use according to claim 50 wherein the method comprises exposing said target neuron or neuronal area simultaneously to a combination of MT-IIA and any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT- 5 IV.

52. Use according to claim 50 wherein the method comprises exposing said target neuron or neuronal area sequentially to a combination of MT-IIA followed by any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and 10 MT-IV.

53. Use according to claim 50 wherein the method comprises exposing said target neuron or neuronal area sequentially to a combination of any one of metallothionein isoforms selected from MT-I, MT-II, MT-IIA, MT-III and MT-IV. 15

54. Use according to any one of claims 43 to 53 wherein said neuron or neuronal area is located in the brain.

55. Use according to any one of claims 43 to 54 wherein said solutions is in a form 20 suitable for administration to said neuron or neuronal area by any one or a combination of direct injection, intraperitoneal injection, oral administration or via genetically modified cells including stem cells.

56. Use according to any one of claims 43 to 55 wherein the method comprising 25 exposing a target neuron or neuronal area to the said solution is to be used in the treatment of Alzheimer's Disease.

57. Use according to any one of claims 43 to 55 wherein the method comprising exposing a target neuron or neuronal area to the said solution is to be used in the 3 0 treatment of Parkinson's Disease.

58. Use according to any one of claim 43 to 55 wherein the method comprising exposing a target neuron or neuronal area to the said solution is to be used in the treatment of motor neuron disease. 35 m:\specifications\500000\503000\503056clmmaw.doc INTELLECTUAL PROPERTY OFFICE OF NZ 16 NOV 2006 RECEIVED 18

59. Use according to any one of claims 43 to 55 wherein the method comprising exposing a target neuron or neuronal area to the said solution is to be used in the treatment of head injury. 5

60. A therapeutic composition produced in accordance with the use defined in claims 28 to 59, adapted for topical administration to an area of neuronal compromise, said composition being characterised by metallothionein isoform MT-IIA as an active ingredient. 10

61. A composition according to claim 60 wherein said active ingredient is combined with any one or a combination of metallothionein isoforms selected from MT-I, MT-II, MT-III and MT-IV.

62. A composition according to claim 60 or 61 wherein said metallothionein is 15 naturally occurring human MT-IIA.

63. A composition according to any one of claims 60 or 61 wherein said metallothionein is produced by chemical synthesis or by production in genetically manipulated cells or organisms. 20

64. A composition according to claim 63 wherein said metallothionein is recombinant human MT-IIA.

65. A composition according to any one of claims 60 to 64 further including a 25 neurologically acceptable carrier particularly adapted for a topical administration to an area of neuronal compromise.

66. A composition according to claim 65 adapted for direct topical application. 30

67. A composition according to claim 65 adapted for intraperitoneal or intravenous administration to effect exposure of neurons by a non-topical route. Dated this eighth day of November 2006 m:\specifications\500000\503000\503056clmmaw.doc INTELLECTUAL PROPERTY OFFICE OF N.Z. 16 NOV 2006 RECEIVED </div>