WO2020069934A1

WO2020069934A1 - Neuroprotective composition

Info

Publication number: WO2020069934A1
Application number: PCT/EP2019/075826
Authority: WO
Inventors: Robert Fern
Original assignee: Plymouth University
Current assignee: Plymouth University
Priority date: 2018-10-05
Filing date: 2019-09-25
Publication date: 2020-04-09
Anticipated expiration: 2021-04-05

Abstract

Described is a composition for protecting against demyelination and other forms of myelin injury, comprising a combination of an NDMA-type glutamate receptor antagonist and a non-NMDA-type glutamate receptor antagonist.

Description

NEUROPROTECTIVE COMPOSITION

The central nervous system (CNS) is made up of grey matter (GM), where the neuron cell bodies are located and form synapses with other neurons, and white matter (WM), where the long projections (axons) of these neurons run.

The function of WM is the transmission of signals to, from and between neurons and this requires the presence of insulation on many of the long axonal neuronal projection called axons. The insulation is provided by layers of myelin that wrap around the axons, and the myelin is generated by a cell type called the oligodendrocyte.

MR.I imaging reveals white matter myelin damage in many diseases. There are disorders that are effectively caused by“pure” white matter damage such as but not limited to spinal cord injury (SCI), cerebral palsy (CP), vascular dementia (VaD), multiple sclerosis (MS), traumatic brain injury (TBI), ischemic optic neuropathy (ION) and certain kinds of stroke. Damage to myelin in these diseases has serious consequences; for example MS is a product of pure myelin damage.

Other neurological diseases affect grey matter but have an important white matter component, including but not limited to Alzheimer’s, Huntingdon’s and Parkinson’s diseases, depression, schizophrenia and some forms of stroke.

An aspect of the present invention provides a composition for protecting against demyelination and other forms of myelin injury, comprising a combination of an NDMA-type glutamate receptor antagonist and a non-NMDA-type glutamate receptor antagonist.

The concentration of each antagonist may be at a level below a clinically effective uncombined level.

The concentrations of both antagonists may be at least one order of magnitude lower than the clinically effective levels.

The concentration of both antagonists may be at least two orders of magnitude lower than the individually effective levels.

The concentration of each antagonist may be at a level below an in vacuo sub-clinically effective level. In other words, both concentrations are at a level below that which would normally be expected for either drug to be clinically effective when administered individually. For example, the NMDA receptor blocker memantine is typically prescribed at 20 mg/day and the non-NMDA receptor blocker parampanel is typically prescribed at 4-8 mg/day (current UK NICE guidelines). In an embodiment comprising this composition, a 2 mg/day memantine and 0.4-0.8 mg/day parampanel dose may, for example, be prescribed in combination.

I The concentrations of both antagonists may be at least one order of magnitude lower than said in vacuo sub-clinically effective levels.

The concentrations of both antagonists may be at least two or more orders of magnitude lower than said in vacuo sub-clinically effective levels.

Some aspects and embodiments of the present invention are based on a principle of a low-combined dose.

A further aspect relates to the combined use of low concentrations of any NMDA and any non-NMDA antagonist. This is based on data showing that the clinically approved drugs Parampanel (AMPA blocker) + memantine (NMDA blocker) are protective in vitro at very low combined concentrations. The big advantage here is speed of clinical translation due to very low risk.

There is considerable data showing that the AMPA blocker Parampanel and the NMDA blocker memantine work in vitro.

A further aspect relates to the combined use of a selective GluN2C/D NMDA receptor blocker in combination with any non-NMDA blocker, at any concentration range. This is based on data showing that QNZ-46+CP465022 are highly protective at low combined doses in two in vivo models (EAE model of multiple sclerosis and tMCAO model of stroke).

Some aspects and embodiments relate to the use of low concentrations of any NMDA and non-NMDA antagonist in combination at low concentrations.

A further aspect provides the use of the combination of a NDMA-type glutamate receptor antagonist and a non-NMDA glutamate receptor antagonist for protecting against demyelination.

A further aspect provides the use of a combined non-NMDA glutamate receptor and GluN2C/D subunit containing NMDA glutamate receptor antagonist for the prophylaxis and/or treatment of neurological disease.

A further aspect provides the use of a composition comprising an NMDA blocker selected from Table I in combination with an AMPA blocker selected from Table I for the protection of myelin.

Uses described herein may be for the treatment or prophylaxis of neurological disease.

Some aspects and embodiments of the present invention relate to a combined non-NMDA glutamate receptor and a GluN2C/D subunit-containing NMDA glutamate receptor antagonism for protection and treatment of neurological disease, in particular those affecting myelin. The GluN2C/D subunit-containing NMDA glutamate receptor antagonist may comprise QNZ-46 or a functional equivalent thereof. QNZ-46 is a selective, negative allosteric modulator of NMDA receptors that contain a GluN2C/D subunit:

4-[6-Methoxy-2-[(l E)-2-(3-nitrophenyl)ethenyl]-4-oxo-3(4H)quinazolinyl]benzoic acid:

The GluN2C/D subunit-containing NMDA glutamate receptor antagonist may consist of QNZ-46.

The non-NMDA antagonist may comprise an AMPA receptor blocker. The non-NMDA antagonist may comprise CP465022 or a functional equivalent thereof.

CP465022 is a selective non-competitive antagonist of the AMPA non-NMDA receptor.

The non-NMDA antagonist may consist of CP465022. The present invention also provides a neuroprotective composition comprising a selective non-NMDA receptor blocker in combination with a selective GluN2C/D containing receptor blocker. Both blockers may be present at the clinically effective level required when either is delivered individually. Both blockers may be present at concentrations below a clinically effective level required when either is delivered individually/separately.

The present invention also provides a composition for the protection of myelin comprising an NMDA blocker selected from Table I in combination with an AMPA blocker selected from Table I .

Table I

Both blockers may be present at in vacuo sub-clinically effective concentrations. In the case of combined use of a non-NMDA and a NMDA antagonist that is selective for GluN2C/D containing receptor, the antagonists may be present at established clinically effective concentrations.

In broad terms, there are two aspects to the proposal drug use forming part of aspects and embodiments of the present invention: I ) the combined use of low concentrations of any NMDA and any non-NMDA antagonist; 2) the combined use of a selective GluN2C/D NMDA receptor blocker in combination with any non-NMDA blocker, at any concentration range (including the low range).

The present invention also provides the use of a formulation as described herein for the treatment of a neurological disease.

The present invention also provides the use of a formulation as described herein for the treatment of multiple sclerosis.

The present invention also provides the use of a formulation as described herein for the prevention of demyelination.

A further aspect provides a method of treating or preventing myelin injury by administering to a person in need of such treatment an effective amount of a composition as described herein. A further aspect provides use of a composition as described herein in the treatment or prophylaxis of myelin damage.

A further aspect provides use of a composition described herein in the treatment or prophylaxis of disorders or diseases of the nervous system involving myelin pathology including but not limited to: spinal cord injury, cerebral palsy, vascular dementia, multiple sclerosis, traumatic brain injury, ischemic optic neuropathy and stroke.

Other neurological diseases that involve significant myelin damage to which the present invention may be applicable include but are not limited to: Alzheimer’s, Huntingdon’s and Parkinson’s diseases, depression, schizophrenia and disorders affecting the peripheral nervous system (such as diabetic neuropathy), the neurological complications of AIDS, prion diseases, metabolic disorders, genetic disorders and toxicities affecting myelin.

A further aspect relates to use of combined non-NMDA glutamate receptor and GluN2C/D subunit containing NMDA glutamate receptor antagonist for the protection and/or treatment of neurological disease.

A further aspect relates to use of combined AMPA antagonist and GluN2C/D subunit containing NMDA glutamate receptor antagonist for the protection and/or treatment of neurological disease.

Many diseases include myelin damage, and we include herein data from the in vivo tMCAO model of stroke using combined low doses that show protection of areas that are not white matter. The present invention also relates to uses described herein for the protection and/or treatment of non-white matter injury in stroke.

The present invention also provides an ex vivo system for testing the protective effect of drugs against demyelination, comprising the administration of cuprizone in the present of a putative protective drug.

The present invention also provides an ex vivo system for producing selective myelin loss in a sample, for example comprising exposing the sample to cuprizone, LPS or other myelin damaging environment.

This embodiment may form the basis for testing drugs that may be protective against myelin damage in vivo or clinically.

The system may comprise means for visualising myelin or otherwise measuring the functional integrity of myelin.

The present invention also provides a system comprising a means for visualising myelin based upon QNZ- 46 or similar structures. The present invention also provides a system comprising a means for visualising myelin based upon QNZ- 46 or a functionally or a structurally equivalent thereof.

Different aspects and embodiments of the invention may be used separately or together.

Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combination other than those explicitly set out in the claims.

The present invention will now be more particularly described, with reference to the accompanying drawings.

All orientational terms, such as upper, lower, radially and axially, are used in relation to the drawings and should not be interpreted as limiting on the invention or its connection to a closure. Example embodiments are described in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternate forms and should not be construed as limited to the examples set forth herein.

Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail below as examples. There is no intent to limit to the particular forms disclosed and as well as individual embodiments the invention is intended to cover combinations of those embodiments as well. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included. Elements of the example embodiments are consistently denoted by the same reference numerals throughout the drawings and detailed description where appropriate.

The terminology used herein is not intended to limit the scope. The articles“a,”“an,” and“the” are singular in that they have a single referent; however, the use of the singular form in the present document should not preclude the presence of more than one referent. In other words, elements referred to in the singular can number one or more, unless the context clearly indicates otherwise. It will be further understood that the terms“comprises,”“comprising,”“includes,” and/or“including,” when used herein, specify the presence of stated features, items, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, items, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealised or overly formal sense unless expressly so defined herein.

Myelin is formed from the extruded membrane of the oligodendrocyte process and a single oligodendrocyte may extend =<40 inter-nodal myelin segments. An important recent discovery in myelin research is the presence of functional NMDA-type glutamate receptors (GluRs), reported to be expressed at densities comparable to those found at neuronal synapses '^{, 2}. These myelinic NMDA receptors may play a role in the control of myelin development ³ and the uptake of energy substrate ⁴, and we and others have shown that they can be over-stimulated under pathological conditions leading to myelin injury and associated loss of axonal function ⁵' ⁶. Myelin also contains AMPA-type non-NMDA GluRs ⁷, which can also mediate cytotoxic injury ⁸' ⁹. Since AMPA receptors can act to gate NMDA receptor currents, these two receptors may act synergistically in a putative myelinic excitotoxic pathway.

While neuroinflammation remains the leading pathogenic hypothesis in multiple sclerosis research, dysfunction in glutamate regulation has come under increasing scrutiny ¹⁰ and elevated glutamate levels have been reported in the CSF of relapsing multiple sclerosis patients ". While inflammatory cells such as neutrophils, monocytes, macrophages, resident microglia and dendritic cells are all credible sources of abnormal glutamate release in the disorder ^{l 0}, we have recently demonstrated that vesicular glutamate release from the axon cylinder is the principle form of acute pathogenic glutamate release onto myelin, at least under ischemic conditions ⁶.

Multiple sclerosis is a debilitating neuroinflamatory disorder targeting the axon myelin sheath, the insulating, low capacitance layer required for fast action potential propagation.

To test the idea that NMDA receptors are significant in multiple sclerosis demyelination, fixed cryopreserved brain tissue was obtained from the Multiple Sclerosis Society tissue bank at Imperial College London. Normal appearing white matter (NAWM), active lesion (AL) and chronic lesion (CL) tissue from 3 donors was embedded, ultra-sectioned and immune-gold post-stained for the GluN I obligatory NMDA receptor subunit using protocols previously developed for post-embedded immune-electron microscopy (l-TEM) in rodent tissue for this antibody ^{l 2, l 3}, and human tissue using a similar protocol ^l4, in my laboratory. The results (unpublished data) are shown in Figure I and document the elevated expression of the protein in sections from lesion sites, in particular within myelin.

Figure I . GluN I reactivity in multiple sclerosis (NMDA-type GluR expression). A: Low power ultramicrograph of normal appearing white matter (NAWM: used as a control). A: Glial cell soma (“G”) and their nuclei (arrows) are apparent and myelinated axons are randomly oriented (arrowheads). B: At higher power individual axons (“Ax”) contain mitochondria (“m”), microtubules (“mt”) and neurofilaments (“nf”) of normal appearance. Myelin, in general, is compact with occasional structural flaws possibly the result of fixation artifact (arrow heads). C, D: Chronic lesion (CL) tissue exhibits regions of focal myelin decompaction/blebbing. Immuno-gold labeling indicative of GluN I reactivity is present in these regions (D: arrows). E-H: Active lesion (AL) regions contain areas of sever myelin decompaction and splitting (arrow heads) which are often associated with focal points of intense GluN I reactivity (arrows in boxed areas are shown at higher gain). Reactivity can also be seen in non-myelin areas surrounding axons which may be the remnants of glial processes (H“gp”). I: The proportion of myelin occupying the tissue in low- power images, showing the significant myelin loss in lesion sites. J: Immuno-gold particle density in the three regions broken down for whole sections, myelin and non-myelin staining. Note the significant increases in myelin reactivity in CL and AL tissue compared to NAWM. Scale bars as shown.

High profile studies have documented vesicular glutamate release from immature axons ^{l 5 17}, and we have examined vesicular docking in mature myelinated axons via real-time confocal FM4-64 imagining in Thy- l /YFP mice ⁶. No evidence was found for localized vesicular fusion at the axon node of Ranvier or in white matter glial cells, and fusion events occurred largely under the myelin sheath. Extracellular glutamate was measured using biosensor electrodes ⁶' ^{l 8}, reporting evoked [glutamate] rises with a similar time-course to axonal vesicular fusion. Using oxygen-glucose deprivation (OGD) as a pathogenic trigger, large white matter [glutamate] rises were documented which were not significantly affected by block of reverse Na⁺- dependent glutamate uptake, swelling-mediated glutamate release, or cysteine-glutamate antiport. OGD- induced glutamate release was Ca²⁺-dependent as predicted for vesicular release and was sensitive to bafilomycin and rose Bengal, two blockers of vesicular loading. Pathogenic glutamate elevation in white matter is therefore almost entirely dependent upon axonal vesicular release. We also found that glutamate release was central to acute myelin injury. Earlier mixed reports of myelin protection by NMDA receptor blockers were found to result from the slow penetration of drugs into the myelin. Of particular interest, we demonstrated both in vitro and in vivo that myelin damage is strongly mitigated by the drug QNZ-46, a lipid-soluble negative allosteric modulator of NMDA receptors which incorporate GluN2C/D subunits ⁶. We and others have previously documented the selective expression of GluN2C/D containing NMDA receptors in myelin with only sparse expression on the oligodendrocyte somata ^{5 I 2, l 3, l 9}.

Drug lipid solubility was found to be a requirement for effective myelin protection by NMDA blockers, consistent with the expression of GluRs within the myelin sheath ' ². The drug QNZ-46 is a 4-oxo- 3(4H)quinazolinyl derivative containing the trans-stilbene pharmacophore that targets mylein ²⁰ and a quinazolinone backbone which exhibits strong fluorescence ²¹. Quinazolinone derivatives such as QNZ- 46 have a wide range of clinical uses and have a good safety record (see ²²). The drug has a peak emission at 450 nm allowing drug uptake into myelin to be monitored in real time. QNZ-46 loaded into myelin in brain slices and optic nerve from the bath over a 1 20 min time-course and was retained following washout. The drug elevated the resistance of myelin to injury (increasing functional recovery from OGD by ~550 ) even when all the extracellular drug had been removed, a phenomenon we called a“myelin shield” ⁶. Animal studies have shown that NMDA receptor antagonists are highly protective in stroke models but do not translate clinically. For example, a comprehensive trial of early intervention in stroke with the NMDA blocker Mg²⁺ (FAST-MAG trial) recently failed ²³. Other trials have been halted due to on-target side effects following broad-spectrum block of NMDA receptors ²⁴. Quinazolinone such as QNZ-46 are ideally suited to avoid these problem for the following reasons: I ) High lipid-solubility allows rapid myelin access ⁶. 2) Drugability with good brain penetration ⁶. 3) Trans-stilbene pharmacore providing unique myelin trapping ⁶. 4) Negative allosteric mode of action and use-dependent block, predisposing the drug to target pathological over-activation of receptors over normal physiological receptor function ^{20 2S}. 5) Selective for GluN2C/D containing NMDA receptors which are primarily extra-synaptic ^26-28 and are expressed at low levels in grey matter regions than are other NMDA GluR subunits ^{29, 3}°, consistent with limited on-target side effects.

CP465022 (3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-6-fluoro-4(3H)- quinazolinone) is a highly selective and potent non-competitive quinazolinone antagonist of AMPA type non-NMDA GluRs ^{3 I}. Delivered alone, this drug failed to provide protection in a rodent in vivo model of stroke ³². As a close analogue of QNZ-46, CP465022 is likely to share the characteristics of myelin penetration and retention of its fellow quinazolinone. It is a feature of non-NMDA GluRs that they act to gate NMDA GluRs and we here show that both QNZ-46 and CP465022 protect myelin from injury in a model of acute demyelination. The two drugs are shown to provide protection at low concentrations. When applied together at concentrations below those required to provide protection when applied individually, the drugs provide protection when applied in combination. This synergistic effect of the two drugs acting in combination reduces the concentrations required for protection by ~2 orders of magnitude and are in the nM range.

A new assay for testing drugs for protection of myelin injury

For testing the protective effect of drugs against demyelination we have developed an novel ex-vivo cuprizone model (Figures 2-4). This protocol produces a standardized level of myelin damage in mouse brain slices over a 100 min exposure to cuprizone, an agent that is widely used to evoke demyelination in vivo where progression of myelin damage is much slower ^{33, 34}. The advantage of this new approach is it makes practical the generation of dose-response data for drug protection against demyelination. The structural changes seen in myelin at the end of the cuprizone treatment resemble those found in MS patients (Figure 5).

Figure 2. An ex-vivo cuprizone model of demyelination, showing the protocol which involves hemi- secting live mouse brain slices and staining the myelin with a vital dye such as FluoroMyelin Red. The two halves of each section are then exposed to cuprizone for 100 min, with one side simultaneously exposed either to a drug or a vehicle control. The level of myelin retained by the slice is then assessed via an assay such as a confocal fluorescent microscopy.

Figure 3. The advantages of the ex-vivo cuprizone model. Current models for testing drugs against myelin damage (top) require large numbers of animals to be monitored over long periods. The new approach described here generates multiple data from a single animal in a single day, with a higher level of statistical power. Figure 4. Data from the ex-vivo cuprizone model. A: Myelin in the corpus callosum of mouse brain slices is shown stained red. The myelin stain is lower in the slice that has been exposed for 100 min to cuprizone (CPZ) compared to one that has not been exposed (control). The level of myelin staining in 6 pairs of slices is shown to the right showing the significant myelin loss produced by cuprizone exposure. B, C: Similar analysis in this case showing no change in the oligodendrocyte cell bodies and processes that produce the myelin (B) or the astrocyte cells that regulate the extracellular space (C). This demonstrates that the injury is restricted to the myelin.

Figure 5. Ultra-micrograph TEM analysis showing pattern of myelin damage in axons of the corpus callosum fixed after 100 min cuprizone treatment. A: The significant reduction in axon g-ratio following cuprizone treatment over the range of axon diameters, indicative of myelin disruption. B: Representative myelinated axons in the optic nerve and brain slice. Note the similarities to the myelin disruption seen following cuprizone treatment to those in MS patients in Figure I .

This new model is ideal for dose-protection work to identify the concentrations at which drugs protect myelin from injury. The model can be adapted to use other conditions that may be relevant to myelin damage. For example, the bacterial endotoxin lipopolysaccharide (LPS), which activated microglial cells in a fashion similar to the immuno-reaction found in MS and other diseases. We found that 100 min exposure to LPS has a similar effect upon myelin in isolated brain slices to cuprizone (Figure 6).

Figure 6. Ultra-micrograph showing pattern of myelin damage in axons fixed after 100 min LPS treatment.

The new assay shows that combined low-doses of NMDA + AMPA antagonists (both experimental and clinically approved) are protective of myelin injury.

The concentration dependence of the protective effect of GluN2C/D-containing NMDA antagonist QNZ- 46 was investigated using the ex vivo cuprizone assay (Figure 7). A similar set of experiments was conducted for the AMPA antagonist CP465022 (Figure 8). These are highly selective experimental drugs and both are styrl 3(4H)-quinazolinones which we have shown are absorbed into myelin and cross the BBB following oral gavage⁶. Note the concentration-dependent protective effect of either drug when perfused alone. The protective effect of combined treatment with 500 nM QNZ-46 + 10 nM CP465022 is shown in both figures in red and was significantly greater (R<0.001 ) from the degree of myelin protection afforded by these drugs when applied alone at these very low concentrations. These sub-clinical concentrations therefore act synergistically to protect myelin from injury.

Figure 7. The dose-dependence of the protective effect of QNZ-46 against ex vivo cuprizone induced myelin loss assessed via FluoroMyelin Red staining. The stated concentrations of the drug are added 60 min before and continually during the 100 min period of cuprizone treatment. The fold difference in myelin protection is calculated relative to myelin staining in the second hemi-section in absence of the drug. Note

I I the increasing protection with increasing drug concentration. The data point in red is the degree of protection produced by a combined treatment with 500 nM QNZ-46 + 10 nM CP465022.

Figure 8. The dose-dependence of the protective effect of CP465022 against ex vivo cuprizone induced myelin loss assessed via FluoroMyelin Red staining (as in Figure 7). The data point in red is the degree of protection produced by a combined treatment with 500 nM QNZ-46 + 10 nM CP465022.

The protective effect of these drugs was confirmed by recording the compound action potentials (CAP) from the axons of the isolated mouse optic nerve (MON), a central white matter tract (Figure 9). This is a functional assay of white matter. 100 min of cuprizone perfusion significantly reduced the CAP, consistent with the myelin damage described above. This injury was prevented by simultaneous perfusion with I mM QNZ 46 and I mM CP 465022 and these concentrations had no significant protective effect when applied in isolation.

Figure 9. The protective effect of CP465022 and QNZ-46 against ex vivo cuprizone induced loss of the CAP in the isolate MON. A: A combination of I mM QNZ 46 and I mM CP 465022 prevented the significant reduction in CAP amplitude caused by application of I mM cuprizone for 100 minutes (stars = significant CAP loss vs., control). These concentrations were not protective when applied alone. B: The time-course of the mean CAP loss when cuprizone is perfused over the tissue (orange), and the protective effect of combined I mM QNZ 46 and I mM CP 465022 (yellow). C: Representative CAP from these experiments.

The concentration dependence of the protective effect of the clinically approved NMDA antagonist memantine was investigated using the ex vivo cuprizone assay (Figure 10). A similar set of experiments was conducted for the clinically approved AMPA antagonist parampanel (Figure I I ). Note the concentration-dependent protective effect of either drug when perfused alone. The protective effect of combined treatment with 200 nM memantine + 200 nM parampanel is shown in both figures in red and was significantly different (P<0.00 l ) from the degree of myelin protection afforded by these drugs when applied alone at these very low concentrations. These sub-clinical concentrations therefore act synergistically to protect myelin from injury, providing significant protection in combination at 3 orders of magnitude lower concentrations than those required for protection when applied alone.

Figure 10. The dose-dependence of the protective effect of memantine against ex vivo cuprizone induced myelin loss assessed via FluoroMyelin Red staining (as in Figure 7). The data point in red is the degree of protection produced by a combined treatment with 200 nM memantine + 200 nM parampanel.

Figure I I . The dose-dependence of the protective effect of parampanel against ex vivo cuprizone induced myelin loss assessed via FluoroMyelin Red staining (as in Figure 7). The data point in red is the degree of protection produced by a combined treatment with 200 nM memantine + 200 nM parampanel. The protective effect of memantine and parampanel was confirmed by CAP recording (Figure 12). In this assay cuprizone injury was prevented by simultaneous perfusion with I mM memantine and I mM parampanel and these concentrations had no significant protective effect when applied in isolation.

Figure 12. The dose-dependence of the protective effect of memantine and parampanel against ex vivo cuprizone induced loss of the CAP in isolated MON. a. A combination of I mM memantine and I mM parampanel reduced the CAP loss caused by 100 minutes cuprizone exposure. These concentrations had no similar protective effect when applied alone, b. The time-course of the mean CAP loss when cuprizone is perfused over the tissue (orange), and the protective effect of combined I mM memantine and I mM parampanel (yellow). C: Representative CAP from these experiments.

The novel combined low-dose therapy identified using the new in vitro assay translates to established in vivo models.

The data in Figure 7- 12 show that combined AMPA + NMDA receptor antagonists are protective at low combined concentrations, e.g., 10 nM CP465022 + 500 nM QNZ-46. These concentrations were found to be 1 -3 orders of magnitude below those required for protection when the drugs were applied alone and are 2-3 orders of magnitude below the concentrations used to selectively block their respective targets in comparable whole mount CNS preparations ⁶' ^3S. For translation to in vivo studies, a review of the available literature shows that 7.5 mg/Kg CP465022 is required to acutely inhibit the AMPA receptor mediated CA I -schaeffer collateral population spike in mice ³². CP465022 has a half-life of ~4 hours in the circulation ³². A dose of 5 +2 mg/Kg has previously been used in a standard transient middle-cerebral artery occlusion (tMCAO) model of stroke without any protective effect ³². 20 mg/Kg QNZ-46 has been previously used as an effective dose to reduced ischemic injury in vivo with no behavioural effects, and has been modelled to produce a CSF concentration appropriate for block of myelinic GluN2C/D-containing NMDA receptors ⁶. A combined daily dose of I mg/Kg CP465022 + 2 mg/Kg QNZ-46 given via intraperitoneal injection (IP) is therefore ~ one order of magnitude lower than the doses required for each drug in isolation in vivo. It must be recognized that the 4 Hr half-life will result in a much lower circulating concentration for the majority of a 24hr dosing regimen.

The low combined dose was delivered daily (vs., vehicle) in an established in vivo mouse model of demyelination (experimental autoimmune encephalopathy: EAE). Experiments were conducted blind and the drug-treated group had a significantly lower area under the curve (AUC) of neurological injury score and a latter latency to onset, and therefore the treatment showed significant efficacy (Figure 13). The EAE model is widely used for testing of drugs for multiple sclerosis.

Figure 13. Low combined dose treatment (I mg/Kg CP465022 + 2 mg/Kg QNZ-46) significantly protected against functional loss in the EAE model of demyelination in vivo. The treatment was delivered daily from day one. The low combined dose was delivered prior to onset of vascular occlusion in a standard tMCAO model of ischemic stroke (Figure 1 ). Significant protection against the extent of the brain lesion was found, and an improvement in the functional score of the animals. An examination of the ultrastructure of the lesion site using TEM revealed wide-scale protection of myelin in the treated group (Figure 15).

Figure 14. Low combined dose treatment (I mg/Kg CP465022 + 2 mg/Kg QNZ-46) significantly protected against tMCAO injury. Left: Three brain sections are shown vertically arranged, collected form mice subject to tMCAO (60 min) + 24 hr recovery and stained for TCC (the white area indicates the damaged part of the brain). Treatment was delivered 120 min prior to the onset of the tMCAO. The table shows data from individual mice and is collated in the two histograms. These stow the significant reduction in lesion volume (as a % of the brain volume) and the reduced Bederson score, which is indicated of functional improvement.

Figure 1 5. Low combined dose treatment (I mg/Kg CP465022 + 2 mg/Kg QNZ-46) significantly protected against tMCAO injury to myelin. Left: Ultra-micrographs (scales indicated) collected from lesion site of vehicle treated mice. Note the wide-scale cellular injury, including myelin damage and debris (arrows). Right: Comparable images from drug-treated mice showing preserved cellular structures, including myelin (arrows). Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

REFERENCES

1. Micu I, Jiang Q, Coderre E, Ridsdale A, Zhang L, Woulfe J, et al. Nmda receptors mediate calcium accumulation in myelin during chemical ischaemia. Nature. 2006;439:988-992

2. Karadottir R, Cavelier P, Bergersen LH, Attwell D. Nmda receptors are expressed in oligodendrocytes and activated in ischaemia. Nature. 2005;438: l 162- 1 166

3. Lundgaard I, Luzhynskaya A, Stockley JH, Wang Z, Evans KA, Swire M, et al. Neuregulin and bdnf induce a switch to nmda receptor-dependent myelination by oligodendrocytes. PLoS biology.

2013; I I :e 1001743

4. Saab AS, Tzvetavona ID, Trevisiol A, Baltan S, Dibaj P, Kusch K, et al. Oligodendroglial nmda receptors regulate glucose import and axonal energy metabolism. Neuron. 2016;91 : 1 19- 132

5. Salter MG, Fern R. Nmda receptors are expressed in developing oligodendrocyte processes and mediate injury. Nature. 2005;438: l 167- 1 171

6. Doyle S, Hansen DB, Vella J, Bond P, Harper G, Zammit C, et al. Vesicular glutamate release from central axons contributes to myelin damage. Nature communications. 2018;9: 1032

7. Christensen PC, Samadi-Bahrami Z, Pavlov V, Stys PK, Moore GRW. Ionotropic glutamate receptor expression in human white matter. Neuroscience letters. 2016;630: 1 -8

8. Sanchez-Gomez MV, Matute C. Ampa and kainate receptors each mediate excitotoxicity in oligodendroglial cultures. Neurobiology of disease. 1999;6:475-485

9. Ouardouz M, Coderre E, Zamponi GW, Hameed S, Yin X, Trapp BD, et al. Glutamate receptors on myelinated spinal cord axons: li. Ampa and glur5 receptors. Annals of neurology. 2009;65: 160- 166

10. Macrez R, Stys PK, Vivien D, Lipton SA, Docagne F. Mechanisms of glutamate toxicity in multiple sclerosis: Biomarker and therapeutic opportunities. Lancet neurology. 2016; 15: 1089- 1 102 Sarchielli P, Greco L, Floridi A, Floridi A, Gallai V. Excitatory amino acids and multiple sclerosis: Evidence from cerebrospinal fluid. Archive s of neurology. 2003;60: 1082- 1088

12. Alix JJ, Fern R. Glutamate receptor-mediated ischemic injury of premyelinated central axons.

Annals of neurology. 2009;66:682-693

13. Alix JJ, Zammit C, Riddle A, Meshul CK, Back SA, Valentino M, et al. Central axons preparing to myelinate are highly sensitivity to ischemic injury. Annals of neurology. 2012;72:936-951

14. Back SA, Craig A, Kayton RJ, Luo NL, Meshul CK, Allcock N, et al. Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter .Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2007;27:334-347

15. Wake H, Lee PR, Fields RD. Control of local protein synthesis and initial events in myelination by action potentials. Science. 201 I ;333: 1647- 1651

Spitzer S, Volbracht K, Lundgaard I, Karadottir RT. Glutamate signalling: A multifaceted modulator of oligodendrocyte lineage cells in health and disease. Neuropharmacology. 2016

17. Fields RD. A new mechanism of nervous system plasticity: Activity-dependent myelination. Nature reviews. Neuroscience. 2015; 16:756-767 18. Trotman M, Vermehren P, Gibson CL, Fern R. The dichotomy of memantine treatment for ischemic stroke: Dose-dependent protective and detrimental effects. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2015;35:230-239

19. Huria T, Beeraka NM, Al-Ghamdi B, Fern R. Premyelinated central axons express neurotoxic nmda receptors: Relevance to early developing white-matter injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2014

20. Mosley CA, Acker TM, Hansen KB, Mullasseril P, Andersen KT, Le P, et al. Quinazolin-4-one derivatives: A novel class of noncompetitive nr2c/d subunit-selective n-methyl-d-aspartate receptor antagonists. Journal of medicinal chemistry. 2010;53:5476-5490

21. Naleway JJ, Fox CMJ, Robinhold D, Terpetschnig E, Olson NA, Haugland RP. Synthesis and use of new fluorogenic precipitating substrates. Tetrahedron Lett. 1994;35:8569-8572

22. Jafari E, Khajouei MR, Hassanzadeh F, Hakimelahi GH, Khodarahmi GA. Quinazolinone and quinazoline derivatives: Recent structures with potent antimicrobial and cytotoxic activities. Res Pharm Sci. 2016; I 1 : 1 - 14

23. Saver JL, Starkman S, Eckstein M, Stratton SJ, Pratt FD, Hamilton S, et al. Prehospital use of magnesium sulfate as neuroprotection in acute stroke. New Engl J Med. 2015;372:528-536

24. Lipton SA. Failures and successes of nmda receptor antagonists: Molecular basis for the use of open-channel blockers like memantine in the treatment of acute and chronic neurologic insults. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics. 2004; 1 : 101 - 1 10

25. Hamilton NB, Kolodziejczyk K, Kougioumtzidou E, Attwell D. Proton-gated ca(2+)-permeable trp channels damage myelin in conditions mimicking ischaemia. Nature. 2016;529:523-527

26. Brickley SG, Misra C, Mok MH, Mishina M, Cull-Candy SG. Nr2b and nr2d subunits coassemble in cerebellar golgi cells to form a distinct nmda receptor subtype restricted to extrasynaptic sites. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2003;23:4958-4966

27. Momiyama A, Feldmeyer D, Cull-Candy SG. Identification of a native low-conductance nmda channel with reduced sensitivity to mg2+ in rat central neurones. The Journal of physiology. 1996;494 ( Pt 2):479-492

28. Harney SC, Jane DE, Anwyl R. Extrasynaptic nr2d-containing nmdars are recruited to the synapse during Itp of nmdar-epscs. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2008;28: 1 1685- 1 1694

29. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four nmda receptors. Neuron. 1994; 12:529-540

30. Akazawa C, Shigemoto R, Bessho Y, Nakanishi S, Mizuno N. Differential expression of five n- methyl-d-aspartate receptor subunit mrnas in the cerebellum of developing and adult rats. The Journal of comparative neurology. 1994;347: 150- 160 31. Lazzaro JT, Paternain AV, Lerma J, Chenard BL, Ewing FE, Huang J, et al. Functional characterization of cp-465,022, a selective, noncompetitive ampa receptor antagonist. Neuropharmacology. 2002;42: 1 3- 153

32. Menniti FS, Buchan AM, Chenard BL, Critchett DJ, Ganong AH, Guanowsky V, et al. Cp-465,022, a selective noncompetitive ampa receptor antagonist, blocks ampa receptors but is not neuroprotective in vivo. Stroke; a journal of cerebral circulation. 2003;34: 171 - 176

33. Blakemore WF. Observations on oligodendrocyte degeneration, the resolution of status spongiosus and remyelination in cuprizone intoxication in mice. Journal of neurocytology. 1 972; 1 :413-426

34. Yu Q, Hui R, Park J, Huang Y, Kusnecov AW, Dreyfus CF, et al. Strain differences in cuprizone induced demyelination. Cell Biosci. 2017;7:59

35. Garcia-Bereguiain MA, Gonzalez-lslas C, Lindsly C, Butler E, Hill AW, Wenner P. In vivo synaptic scaling is mediated by glua2-lacking ampa receptors in the embryonic spinal cord. Journal of Neuroscience. 2013;33:6791 -6799

Claims

1. A composition for protecting against demyelination, comprising a NDMA-type glutamate receptor antagonist and a non-NMDA glutamate receptor antagonist.

2. A composition as claimed in claim I , in which the concentration of each antagonist is at a level below a clinically effective uncombined level.

3. A composition as claimed in claim 2, in which the concentrations of both antagonists are at least one order of magnitude lower than the clinically effective levels.

4. A composition as claimed in claim 2, in which the concentrations of both antagonists are at least two orders of magnitude lower than the individually effective levels.

5. A composition as claimed in any preceding claim, in which the NMDA antagonist comprises QNZ-46 or a functional equivalent thereof.

6. A composition as claimed in any preceding claim, in which the NMDA antagonist consists of QNZ-46.

7. A composition as claimed in any preceding claim, in which the non-NMDA glutamate receptor antagonist is an AM PA blocker.

8. A composition as claimed in any preceding claim, in which the non-NMDA antagonist comprises CP465022 or a functional equivalent thereof.

9. A composition as claimed in any preceding claim, in which the non-NMDA antagonist consists of CP465022.

10. A neuroprotective composition comprising a selective non-NMDA receptor blocker in combination with a selective GluN2C/D containing receptor blocker.

1 1. A composition as claimed in claim 10, in which both blockers are present at concentrations below a clinically effective level required when either is delivered individually.

12. A combined non-NMDA glutamate receptor and GluN2C/D subunit containing NMDA glutamate receptor antagonist for the protection and/or treatment of neurological disease.

13. An antagonist as claimed in claim 12, in which the disease affects myelin.

14. A composition for the protection of myelin comprising an NMDA blocker selected from Table I in combination with an AMPA blocker selected from Table I .

15. A composition as claimed in claim 14, in which both blockers are present at sub-clinical individually effective concentrations.

16. Use of a composition or antagonist as claimed in any preceding train for the treatment of a neurological disease.

17. Use of a composition or antagonist as claimed in any of claims I to 15 for the treatment of multiple sclerosis.

18. Use of a composition or antagonist as claimed in any of claims I to 15 for the prevention of demyelination.

19. Use of a composition as claimed in any of claims I to 15 in the treatment or prophylaxis of disorders or diseases of the nervous system involving myelin pathology including but not limited to: spinal cord injury, cerebral palsy, vascular dementia, multiple sclerosis, traumatic brain injury, ischemic optic neuropathy and stroke.

20. An ex vivo system for testing the protective effect of drugs against demyelination, comprising the administration of cuprizone in the present of a putative protective drug.

21. An ex vivo system for producing selective myelin loss in a sample, comprising exposing the sample to cuprizone.

22. An ex vivo system for testing the protective effect of drugs against demyelination, comprising the administration of LPS in the present of a putative protective drug.

23. An ex vivo system for producing selective myelin loss in a sample, comprising exposing the sample to LPS.

24. An ex vivo system for testing the protective effect of drugs against demyelination, comprising the administration of a myelin-damaging environment.

25. A system comprising a means for visualising myelin based upon QNZ-46 or similar structures.