CA2926507A1 - Compositions and methods for protection and/or repair of the nervous system - Google Patents

Abstract

The present invention relates to compositions and methods for protecting and/or promoting repair of the nervous system. In particular, the present invention relates to the use of muramyl dipeptide crosslinked to form a microparticle for protecting and/or promoting repair of the nervous system.

Description

Compositions and methods for protection and/or repair of the nervous system Field of the Invention [0001] The present application claims priority from Australian Patent Application No.
2015904055, filed on 6 October 2015.

[0002] The present invention relates to compositions and methods for protecting and/or promoting repair of the nervous system. In particular, the present invention relates to the use of muramyl dipeptide crosslinked to form a microparticle for protecting and/or promoting repair of the nervous system.
Background of the Invention

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0004] The nervous system consists of two main parts, the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS contains the brain and spinal cord, while the PNS consists mainly of nerves, which connect the CNS to every other part of the body.

[0005] At the cellular level, the nervous system contains neurons and glial cells.
Neurons have special structures that allow them to send signals rapidly and precisely to other cells. The signals are in the form of electrochemical waves that travel from the body of the neuron along an axon to other neurons. Glial cells provide structural and metabolic support for neurons. Glial cells include oligodendrocytes, which insulate axons by wrapping around them and forming a myelin sheath, and astrocytes, which provide structural and metabolic support.

[0006] Damage or injury to the nervous system leads to abnormal neurological function, including inability to speak, decreased touch sensation, loss of balance, weakness, mental function problems, visual changes, abnormal reflexes, and walking problems.

[0007] Damage or injury to the nervous system may be the result of a variety of traumas or diseases, including genetic defects (e.g., Huntington's disease and muscular dystrophy), developmental problems (e.g., spina bifida), physical trauma, degeneration (e.g., Parkinson's disease and Alzheimer's disease), loss of blood supply (e.g., stoke), infection (e.g., meningitis), seizures (e.g., epilepsy) and cancer (e.g., brain tumours).
[0100] Damage or injury to the nervous system results in disruption of the precisely controlled microenvironment, resulting in tissue damage and often leading to a lack of significant functional repair and nerve regeneration. When the CNS is injured by trauma or disease, a cascade of secondary damage ensues. Vascular, cellular and chemical responses to the injury include tissue inflammation, reduced blood flow and scar formation. Demyelination occurs on injured axons, slowing the conduction of nerve impulses and stripping axons of protection against further damage.
[0101] There is a need for agents that protect the nervous system from trauma or disease. There is also a need for agents that promote the repair of the nervous system following trauma or disease.

[0008] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
Summary of the Invention

[0009] It has been surprisingly found that muramyl dipeptide crosslinked to form a microparticle (MDP microparticle) enhances motor function following demyelination and/or spinal cord injury, increases the levels of neuroprotective/repair factors and decreases macrophage activation.

[0010] According to another aspect, the present invention provides use of muramyl dipeptide crosslinked to form a microparticle (MDP microparticle) for the manufacture of a medicament for the prophylactic and/or therapeutic treatment of neural injury in a subject.

[0011] According to a further aspect, the present invention provides a composition comprising muramyl dipeptide crosslinked to form a microparticle (MDP
microparticle) for use in the prophylactic and/or therapeutic treatment of neural injury in a subject.

[0012] According to one aspect, the present invention provides a method for the prophylactic and/or therapeutic treatment of neural injury in a subject, comprising administering to the subject a composition comprising murannyl dipeptide crosslinked to form a microparticle (MDP microparticle).

[0013] In one embodiment, the MDP microparticle comprises DNA fragments.

[0014] In another embodiment, the MDP microparticle is isolated from macrophages.

[0015] In another embodiment, the neural injury is spinal cord injury.

[0016] In another embodiment, the neural injury is demyelination.

[0017] In another embodiment, the composition or medicament increases the levels of one or more neuroprotective/repair factors in the subject.

[0018] In another embodiment, the neuroprotective/repair factors are selected from vascular endothelial growth factor (VEGF), insulin growth factor-1 (IGF-1), hepatocyte growth factor (HOE) and erythropoietin (EPO).

[0019] In another embodiment, the composition or medicament reduces macrophage activation.

[0020] In another embodiment, the composition or medicament reduces the number of macrophages expressing MHC class II.

[0021] In another embodiment, the composition or medicament decreases the level of expression of MHC class II on macrophages.

[0022] In another embodiment, the composition or medicament comprises one or more pharmaceutically-acceptable excipients, carriers, vehicles or diluents.

[0023] In another embodiment, the MDP microparticle is not encapsulated in a liposome.

[0024] In another embodiment, the composition or medicament is administered or administrable to the subject parenterally.

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[0025] In another embodiment, the composition or medicament is administered or administrable to the subject intravenously.

[0026] In another embodiment, the composition or medicament is administered or administrable to the subject orally.

[0027] In another embodiment, the composition or medicament is administered or administrable to the subject at a dosage of about lpg to about 100pg of MDP
microparticles.

[0028] In another embodiment, the composition or medicament is administered or administrable to the subject at a dosage of about 100pg to about 1000pg of MDP

microparticles.

[0029] In another embodiment, the composition or medicament is administered to the subject at a dosage of about 100pg to about 700pg of MDP microparticles.

[0030] In another embodiment, the composition or medicament is administered or administrable to the subject at a dosage of about 300pg to about 700pg of MDP
microparticles.

[0031] In another embodiment, the composition or medicament is administered or administrable to the subject at a dosage of about 500pg to about 700pg of MDP
microparticles.

[0032] In another embodiment, the composition or medicament is administered or administrable to the subject at a dosage of about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 pg of MDP microparticles.

[0033] In another embodiment, the composition or medicament is administered or administrable to the subject once a day.

[0034] In another embodiment, the composition or medicament is administered or administrable to the subject once a week.

[0035] In another embodiment, the composition or medicament is administered or administrable to the subject once a fortnight.

[0036] In another embodiment, the composition or medicament is administered or administrable to the subject once a month.

[0037] The appropriate dosage of MDP microparticles, total amount administered and duration of administration can be easily determined by a medical practitioner based on guidance provided herein, the nature and severity of the disease to be treated, and the response by the subject to the treatment. As an example, useful individual dosages may be selected from the range 1pg to 1000pg of MDP microparticles, and may be administered once a day, once a week, once a fortnight or once month depending on the subject's condition, symptoms, tolerance and response to treatment. Doses in a higher range can also be used depending on the requirements, for examples doses in the range of 1000pg to 1500pg of MDP microparticles. Dosages at other frequencies may also be employed. An example of a suitable dosage regimen could be to start with an initial dose of 100 pg followed by escalated doses until appropriate beneficial therapeutic effects are observed in the subject, without significant side-effects. The dosage may be given as single bolus dose or infused overtime, or given in divided doses. The total amount of MDP microparticles administered will depend on subject response and tolerance to treatment. The composition may be administered once a day, once a week, once a fortnight or once a month for a total period that depends on the subject's response.

[0038] MDP microparticle-containing compositions or medicaments may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
The amount of MDP microparticle-containing composition which may be combined with a carrier material to produce a single dose may vary depending upon the subject being treated, and the particular mode of administration.

[0039] The MDP microparticle-containing compositions or medicaments may be administered alone or in combination with pharmaceutically acceptable excipients, carriers, vehicles or diluents, in either single or multiple doses. Suitable pharmaceutical acceptable excipients, carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The compositions formed by combining the MDP microparticle-containing compositions and the pharmaceutically acceptable excipients, carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavourings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as L-arginine, sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrates such as starch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc are often useful for tabletting purposes. Solid composition of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Appropriate materials for this include lactose or milk sugar and high molecular weight polyethylene glycols.
When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavouring agents, colouring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof. The MDP microparticle-containing compositions may also comprise enterically coated dosage forms.

[0040] Suitable formulation protocols and suitable excipients, carriers, vehicles and diluents can be found in standard texts such as Remington: The Science and Practice of Pharmacy, 19th Ed, 1995 (Mack Publishing Co. Pennsylvania, USA), British Pharmacopoeia, 2000, and the like.
Definitions

[0041] In the context of the present invention, the terms "muramyl dipeptide crosslinked to form a microparticle" and "MDP microparticle" refer to a microparticle formed by crosslinked repeats of muramyl dipeptide (MDP), wherein the MDP
repeats are crosslinked to each other. The MDP microparticle may also contain DNA
fragments and/or other agents that stimulate and/or regulate the immune system.

[0042] In the context of the present invention, the words "comprise", "comprising"
and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of "including, but not limited to".

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[0043] In the context of the present invention, the terms "treatment" or "treating"
include preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

[0044] In the context of the present invention, the phrase "therapeutically effective amount" refers to an amount of MDP microparticles that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate or reduce medical symptoms for a period of time. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular composition without necessitating undue experimentation. In certain embodiments, compositions are formulated in a manner such that they will be delivered to a patient in a therapeutically effective amount, as part of a prophylactic or therapeutic treatment. The desired amount of the composition to the administered to a patient will depend on absorption, inactivation and excretion rates of the MDP microparticles, as well as the delivery rate of the MDP
microparticles.

[0045] In the context of the present invention, the phrase "parenteral administration"
as used herein refer to modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0046] In the context of the present invention, the phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0047] In the context of the present invention, the phrase "neural injury"
means injury to the tissue or cells of the nervous system.

[0048] In the context of the present invention, the phrase "nervous system"
encompasses the central nervous system and the peripheral nervous system.
Brief Description of the Figures

[0049] Figure 1: MIS416 treatment enhances motor function recovery in the cuprizone dennyelination model when administered in the recovery/repair phase.

[0050] Figure 2: MIS416 treatment increases the levels of vascular endothelial growth factor (VEGF), Insulin growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and erythropoietin (EPO) in human peripheral blood plasma samples.

[0051] Figure 3: Basso Mouse Scale measure in mice with spinal cord injury (SCI) on day 1, 3 and 7 following injury. MIS416 (3 mg/Kg or 6 mg/Kg) or saline was administered systemically on day 1 post injury. In the saline treated SCI
group there is minimal recovery by day 7 post SCI. In contrast, both MIS416 treated groups show locomotive recovery by day 7, with statistical significance reached in the 6 mg/kg MIS416 treated group compared to saline control.

[0052] Figure 4: Flow cytometric analysis of the spinal cords from mice harvested at day 7 post SCI. Figure 4A shows that macrophage activation is clearly evident in saline treated SCI mice compared to healthy controls, based on the % of total macrophages expressing MHC class II. Both MIS416 treated groups show a clear reduction in the proportion of activated macrophages relative to the saline/SCI treated group.
Figure 4B
shows lower expression levels of MHC class II on MHC II class + macrophages compared to the saline/SCI treated group.
Preferred Embodiment of the Invention

[0053] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0054] The present invention is further described by the following non-limiting examples.
Examples Example 1: Preparation of MIS416

[0055] Prop/on/bacterium acnes was grown to a mid-stationary growth phase and washed to remove contaminants of bacterial culture origin by employing techniques well known to those in the art. Hydrophobic components contained in the cell walls and cytoplasm were sequentially extracted by successive washes with increasing concentrations of ethanol/isopropanol/water (10%:10%:80%, 25%:25%:50% and 40%:40%:20%) at elevated temperatures. The isopropanol was then removed with successive washes with decreasing concentrations (80%, 50%, 40% and 20%) of ethanol at elevated temperatures. The resultant microparticles (MIS416) were then suspended in 6M guanidine-HCI and then washed in water for irrigation and its concentration measured by relating its absorbance at 565 nm to the absorbance of turbidity standards.

[0056] MIS416 contains extensively crosslinked MDP, amino-linked L-alanine-D-isoglutamine dipeptides and bacterial DNA fragments. The MIS416 generated by the present methods can have a broad range of sizes (for example, 0.01 to 30 microns) but the most common size range is from 1 to 7 microns. The preferred size is in the range of 0.5 to 3 microns.

[0057] MIS416 can be isolated from natural sources, as described above, or synthesized using well-known synthetic procedures (see, e.g., Liu et al., Bioorganic and Medicinal Chemistry Letters, 10 (12), 2000, pp. 1361-1363(3); Schwartzman &
Ribi, Prep Biochem. 1980; 10(3): 255-67; Ohya et al. Journal of Bioactive and Compatible Polymers, 1993; 8: 351-364).

[0058] The concentration of MIS416 was adjusted to 0.2 mg/mL in sodium chloride for intravenous administration.

Example 2: Effect of MIS416 on early remyelination

[0059] The effect of MIS416 therapy on early remyelination was examined in the Cuprizone demyelination model. C57/BI6 mice (n=7 or 8 per group) were fed continuously ad libitum a diet of 0.3 A (w/w) cuprizone [oxalic bis-(cyclohexylidenehydrazide); Sigma¨Aldrich] mixed into chow pellets for 6 weeks. After 6 weeks of cuprizone feeding, the mice were fed normal mouse chow to allow spontaneous remyelination to progress (recovery phase) for 2 weeks. To study the effect of MIS416 treatment on the recovery phase, 100 pg/mouse or 200 pg/mouse was administered intravenously on a weekly dosing cycle at weeks 5, 6, 7 and 8 weeks, the first dose overlapping with the last week of cuprizone feeding.

[0060] Cuprizone specifically causes the death oligodendrocytes, which are the myelinating cells within the central nervous system. Their main function is to provide support to axons and also to produce the myelin sheath, which serves to insulate the axons. Loss of oligodendrocytes as a result of cuprizone exposure, leads to progressive loss of motor function.

[0061] The effect of cuprizone demyelination on neurological function was measured weekly, using the rotarod test that measures motor control, coordination and balance.
All rotarod assessments were performed blinded.

[0062] The results show that MIS416 treated animals demonstrated a steady increase in motor function during the recovery phase (week 6 onwards). This is in contrast to untreated animals, where there was little evidence of spontaneous recovery at the early stage of remyelination (Figure 1).
Example 3: Effect of MIS416 on levels of neuroprotective/repair factors

[0063] The effect of MIS416 treatment on the levels of neuroprotective/repair factors was investigated in peripheral blood plasma samples from patients participating in a phase 1b/2a clinical trial for progressive MS. Human trial patient blood plasma was collected at baseline and at 24 hr post MIS46 dosing (500 pg/dose) and stored at -80 C
until analysis for the levels of neuroprotective factors. Levels of vascular endothelial growth factor (VEGF), Insulin growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) were determined using flow cytometry cytokine bead arrays (Antigenix), and erythropoietin (EPO) was quantified using standard ELISA (Cusbio Asia).

[0064] At 24 hr post MIS416 dosing, there were elevated plasma levels of VEGF, EPO, IGF-1 and HGF compared to pre-treatment levels (Figure 2). VEGF has been shown to have neurotrophic and neuroprotective effects on neurons and glial cells (Zachary 2005, Neuro-Signals, 14(5): 207-21; Nicoletti et al 2008, Neuroscience, 151(1): 232-41), as well as promoting angiogenesis in demyelinated lesions (Girolamo et al 2014, Acta Neuropathologica Communications, 2: 84). IGH-1 has been shown to promote the survival of neurons (Heck et al 1999, Journal of Biological Chemistry, 274(14): 9828-35) and its levels are increased in repair responses (Mangolia et al 2014, BioMed Research International, Article ID 736104). HCF has mitogenic, motogenic, morphologic, anti-apoptotic activities and neurotrophic activities, and has been shown to prevent cell death (Kadoyama et al 2011, Current Drug Therapy, 6(3): 197-206).
EPO
decreases the development of pro-inflammatory cytokines and provides trophic support to enable tissue regeneration (Brines & Cerami 2008, Journal of Internal Medicine, 264(5): 405-32). EPO has been shown to enhance nerve recovery after spinal trauma (Celik et al 2002, Proceedings of the National Academy of Sciences of the United States of America, 99(4): 2258-63) and to have a role in neurogenesis and post-stroke recovery (Tsai et al 2006, Journal of Neuroscience, 26(4): 1269-74).
Accordingly, these results demonstrate that administration of MIS416 results in the elevation of neuroprotective/repair factors.
Example 4: MIS416 toxicology studies

[0065] It has been well established that free/soluble MDP has significant toxicity in vivo. Attempts to reduce MDP toxicity have employed procedures to delay release, such as MDP incorporation into liposomes or other related compounds, or modification of terminal groups. Chemical modification has resulted in marked reduction in activity, and designs which change delivery rate have been difficult to control.

[0066] In vivo toxicology studies for MIS416 were performed as summarized in Table 1.

Study ID. Study Title Method Quantity of Outcome G6121 MIS416: MIS416 was administered 10,000, 15,000 Maximum tolerated Acute as single escalating 30,000 and 45,000 dose (MTD) Toxicology doses. Animals were mcg/kg body weight established as Study by IV monitored for toxic signs 15,000 mcg/kg body route in and mortality up to day 15 weight Swiss Albino and subjected to detailed Mice necropsy at terminal sacrifice on day 15 G6122 MIS416: MIS416 was administered 50, 200, 800 and MTD
established as Acute as single escalating 3200 mcg/kg body 3,200 p.g/kg body Toxicology doses. Animals were weight (rabbits) weight Study by IV monitored for toxic signs route in New and mortality up to day 15 Zealand and subjected to detailed White necropsy at terminal Rabbits sacrifice on day 15 G6123 MIS416: MIS416 doses selected 1,000 3,000 and The no-observed-Four Week from acute toxicology 10,000 mcg/kg adverse-effect-level Repeated study were administered body weight (mice) (NOAEL) was Dose twice weekly for 4 weeks. considered to be Toxicology Animals were subjected to 1,000 mg/kg (or a Study by IV detailed necropsy at total weekly dose of Route in terminal sacrifice. 2,000 mcg/kg/week) Swiss Albino Mice G6124 MIS416: MIS416 doses selected 50, 500 and 5,000 The NOAEL was Four Week from acute toxicology mcg/kg body weight considered to be 50 Repeated study were administered mcg/kg injected twice Dose twice weekly for 4 weeks. weekly (100 Toxicology Animals were subjected to mcg/kg/week) Study by IV detailed necropsy at Route in New terminal sacrifice.
Zealand White Rabbits 1370-002 MIS416: A MIS416 was administered 20, 200, 1000 NOAEL in this study 26-Week IV once weekly for 26 mcg/kg body weight could be considered Toxicity weeks. Animals were as being close to 20 Study In subjected to detailed mcg/kg for the Rabbits necropsy at terminal purpose of sacrifice. A one month estimation of human recovery arm comprised 2 safety margins animals/sex/group.
Table 1 Summary of toxicology studies

[0067] The toxicity studies, conducted in mice and rabbits for up to 26 weeks duration, provide adequate safety margins to support long term clinical studies at dosage levels in patients up to 20 mcg/kg/week.

[0068] The toxicity studies show that MI5416 has significantly lower toxicity than free/soluble MDP.
Example 5: Determination of the ability of MIS416 to enhance functional recovery following spinal cord injury

[0069] Mice were anesthetized by IP injection of 50/50 mixture of xylazine (10 mg/kg, Troy laboratories Pty Ltd, Australia) and zoletil (Tiletamine/Zolazepam) (50 mg/kg, Virbac Australia Pty Ltd, Australia). The hair of the dorsal thoracic vertebrae was shaved and bare skin was wiped with saline and sterilized with chlorhexidine and 70%
alcohol. The animal was located in prone position on the fixation plate of a impactor (Precision Systems and Instrumentation (PSI), LLC, USA). Aseptic procedure was maintained during surgery. A vertical incision (-2 cm) was performed over the laminecomy site extending from about thoracic vertebrae T6 to T11 using a type #11 scalpel blade. The fat tissue was located towards the upper vertebrae (around upwards) and was carefully separated on both sides to locate a large vessel (superior vena cava) in that vicinity to prevent potentially fatal bleeding. The paravertebral muscles on both right and left sides of the vertebrate column were dissected with #5 forceps from T10 towards T8. All the muscle flesh in the middle was cut away and the spinous processes of the vertebrae were exposed. T8 and T10 vertebrate were localized as T7, 8 & 9's spinous processes are in caudal direction, T10's is almost in posterior direction, and there is a bigger gap between T10 and T11's spinous processes compared to T10 and T9. T9 lanninectomy was performed by first holding T9's spinous process with Adson forceps and removing the lamina bone on both sides with #2 forceps, then holding T8's spinous process with Adson forceps and removing T9's spinous process.

[0070] After laminectomy, the vertebral column was stabilized by rostral and caudal clamping of T8 and T10's transverse process with Adson microforceps on the impactor's fixation plate. The mice were placed under the impactor and the tip was adjusted by first lowering the tip as close as possible to the spinal cord, then bringing the tip upwards by three counter-clockwise turns of the position sensor. Using the IH
impactor software, a 70 kilodyne-controlled force was applied to the spinal cord by IH-0400 impactor's 1.3 mm diameter probe. Animals were moved away from the impactor, the paravertebral muscles and superficial fascia on both sides of the incision were sutured to cover the injury site, the wound was cleaned with saline, and the skin was clipped. There was no significant difference in delivered force and tissue displacement between experimental groups.

[0071] Mice were given Hartman's solution (25 pL/g) supplemented with gentamycin (1 mg/kg, Troy laboratories Pty limited, Australia) subcutaneous prior to surgery as premedication for possible dehydration during surgery. Animals were visually monitored every 10-15 min after surgery till the reflexes were returned and every 15-30 min till they were fully recovered. Mice were left in recovery room on the heating pad (half on, half off) overnight and moved to the animal holding room the next day. For the first week post surgery mice were weighed and given a dose of subcutaneous gentamycin (1 mg/kg) on daily basis. The bladder was manually expressed twice daily.
Impaired mice were not picked/handled by tail for the duration of the experiment and were supplied with unlimited access to food and water/gel.

[0072] MIS416 at 3 mg/kg or 6 mg/kg was administered systemically via retro-orbital injection, following isoflurane anesthetization at 24 hr following SCI
procedure.

[0073] The Basso Mouse Scale (BMS) for locomotion scoring system, a well-accepted clinical measure of locomotion (Basso et al 2006, Journal of Neurotrauma 23(5):635-59) was used to assess the locomotor recovery post spinal cord injury. To assess the effects of injury, two blinded assessors scored mice independently at days 1,3, and 7 post injury.

[0074] Mice were sacrificed at day 7 post injury. The mice were perfused with PBS
transcardially and the whole spinal cord was isolated and mononuclear cells were extracted for flow cytometry analysis. Briefly, the spinal cord tissue was cut in small pieces and digested with DNAase and Collagenase at 37 C for 45 min. The tissue then was homogenized by passing through 70 pm sieves and mononuclear cells were separated 33% percoll gradient and were collected at the bottom of the tube.
The cells were labelled with the following antibody-fluorophore conjugates (Biolegend, USA):
CD45-BV510, CD115-AF488, CD11b-BV421, CD11c-PE/Dazzle 594, MHC class 11 (IA-IE)-APC/Cy7, CX3CR1-AF647, CCR2-PE, GR1-PerCp/Cy5.5, and acquired using the Fortessa X20 flow cytometer (BD Biosciences, USA). The data was analysed with FlowJo software (Treestar, USA). Peripheral blood-derived macrophages in the spinal cord were distinguished from other peripheral blood-derived myeloid cells and from microglia based on the following phenotype: CD45HICD1Ib+CCR2L0CX3CR1+Gri CD11e. The activation status of these cells was determined by the extent of MHC
class ll co-expression.

[0075] Figure 3 shows the Basso Mouse Scale measure in mice with spinal cord injury (SCI) on day 1, 3 and 7 following injury. MIS416 (3 mg/kg or 6 mg/kg) or saline was administered systemically on day 1 post injury. In the saline treated SCI
group there is minimal recovery by day 7 post SCI. In contrast, both MIS416 treated groups show locomotive recovery by day 7, with statistical significance reached in the 6 mg/kg MIS416 treated group compared to saline control.

[0076] Figure 4 shows the results from flow cytometric analysis of the spinal cords from mice harvested at day 7 post SCI to quantify activated peripheral-blood derived macrophages in response to injury. Macrophage activation in the spinal cord parenchyma is a direct response to the inflamed spinal cord microenvironment.
Activated macrophages are sources of pro-inflammatory cytokines and neurotoxins.
Macrophages that are less inflammatory are more able to support repair pathways.
Macrophage activation is clearly evident in saline treated SCI mice compared to healthy controls, based on the % of total macrophages expressing MHC class ll (Figure 4A).
Both MIS416 treated groups show a clear reduction in the proportion of activated macrophages relative to the saline/SCI treated group. This finding is supported the lower expression level of MHC class II on MHC class II+ macrophages compared to the saline/SCI treated group (Figure 4B)

Claims (16)

- 16 -

1. Use of muramyl dipeptide crosslinked to form a microparticle (MDP
microparticle) for the manufacture of a medicament for the prophylactic and/or therapeutic treatment of neural injury in a subject.

2. The method according to claim 1, wherein the MDP microparticle comprises DNA fragments.

3. The method according to claim 1, wherein the medicament promotes repair of the nervous system following neural injury.

4. The method according to claim 1, wherein the medicament enhances motor function following neural injury.

5. The method according to claim 3 or claim 4, wherein the neural injury is spinal cord injury.

6. The method according to claim 3 or claim 4, wherein the neural injury is demyelination.

7. The method according to any one of claims 1 to 6, wherein the medicament increases the levels of one or more neuroprotective/repair factors in the subject.

8. The method according to claim 7, wherein the neuroprotective/repair factors are selected from vascular endothelial growth factor, insulin growth factor-1, hepatocyte growth factor and erythropoietin.

9. The method according to any one of claims 1 to 8, wherein the medicament reduces macrophage activation.

10. The method according to any one of claims 1 to 9, wherein the medicament further comprises one or more pharmaceutically-acceptable excipients, carriers, vehicles or diluents.

11. The method according to any one of claims 1 to 10, wherein the medicament is administrable to the subject parenterally.

12. The method according to claim 11, wherein the composition is administrable to the subject intravenously.

13. The method according to any one of claims 1 to 10, wherein the composition is administrable to the subject orally.

14. The method according to any one of claims 1 to 13, wherein the composition is administrable to the subject weekly.

15. The method according to any one of claims 1 to 13, wherein the composition is administrable to the subject fortnightly.

16. A composition comprising muramyl dipeptide crosslinked to form a microparticle (MDP microparticle) for use in the prophylactic and/or therapeutic treatment of neural injury in a subject.