Treatment or prevention of central nervous system (CNS) disorder

Abstract

The present invention relates to compounds and compositions for use in the treatment or prevention of central nervous system (CNS) disorder. In particular, the present invention relates to a compound of formula (I): (I) for use in treating or preventing a central nervous system (CNS) disorder, wherein X, Y, R, n, m, Z, A and B are as defined herein.

Description

TREATMENT OR PREVENTION OF CENTRAL NERVOUS SYSTEM (CNS)DISORDERField

[0001] The present invention relates to compounds and compositions for use in the treatment or prevention of central nervous system (CNS) disorder.Background to the invention

[0002] Central nervous system (CNS) disorders, such as multiple sclerosis (MS) are major health problems worldwide. Multiple sclerosis is the most common inflammatory neurological disease in young adults, and in 2016 it is estimated that there were 2,221,188 prevalent cases of multiple sclerosis globally, with and 1,151,478 DALYs (GBD 2016 Multiple Sclerosis Collaborators. Global, regional, and national burden of multiple sclerosis 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019 Mar;18(3):269-285).

[0003] In many chronic neurological disorders - including central nervous system (CNS) disorders such as multiple sclerosis (MS) - chronic active, slowly expanding, smouldering lesions characterized by the accumulation of myeloid cells at the lesion edge are associated with brain atrophy, neurodegeneration, and predict the accumulation of irreversible disability, which in turn drives disease progression (see Preziosa, P. et al. Slowly expanding lesions predict 9-year multiple sclerosis disease progression. Neurol. Neuroimmunol. Neuroinflamm. 9, el 139 (2022)). In these lesions, persistently activated myeloid cells are a continuous source of neurotoxic factors, including tumour necrosis factor (TNF), interleukin- 1 (3 (IL-1 (3), nitric oxide (NO) and reactive oxygen species (ROS), causing remyelination failure and secondary neuronal / axonal damage (see Zrzavy, T. et al. Loss of ‘homeostatic’ microglia and patterns of their activation in active multiple sclerosis. Brain 140, 1900-1913 (2017)).

[0004] In MS-like disease models, axonal injury is followed by a compensatory response, whereby mitochondrial content and activity increases in demyelinated axons to promote neuroprotection (see Licht-Mayer, S. et al. Enhanced axonal response of mitochondria to demyelination offers neuroprotection: implications for multiple sclerosis. Acta Neuropathol. 140, 143-167 (2020)). On the contrary, deficits in neuronal mitochondrial complexes and energy metabolism have been associated with persistent axonal damage, grey-matter atrophy and CNS disease progression (see Campbell, G. & Mahad, D. J.Mitochondrial dysfunction and axon degeneration in progressive multiple sclerosis. FEBS Lett. 592, 1113-1121 (2018)).

[0005] Mitochondrial respiratory complexes and metabolites are also known to control myeloid immune responses (see Peruzzotti-Jametti, L. et al. Macrophage-derived extracellular succinate licenses neural stem cells to suppress chronic neuroinflammation. Cell Stem Cell 22, 355-368 (2018)). It is known in the art that, under inflammatory conditions, elevated intracellular succinate levels in myeloid cells promote a switch from the normal forward electron transport along the respiratory chain to reverse electron transport (RET) through mitochondrial complex I (C)I (see Mills, E. L. et al. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell 167, 457-470 (2016)). This mechanism, which requires a high proton motive force, effectively repurposes mitochondria away from the production of adenosine triphosphate (ATP) towards the generation of superoxide that goes on to form hydrogen peroxide and other ROS, together called mitochondrial ROS (mtROS). Inhibition of succinate dehydrogenase (also known as mitochondrial complex II (CII)) by the reversible inhibitors itaconate or malonate limits RET-induced mtROS production and promotes antiinflammatory effects in myeloid cells in vitro (see Lampropoulou, V. et al. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation. Cell Metab. 24, 158-166 (2016)). However, the role of mitochondrial complexes in perpetuating the activation of microglia in the context of smouldering inflammatory central nervous system (CNS) diseases remains largely unexplored.

[0006] There is a clear unmet clinical need for drugs which treat or prevent CNS injury and neurodegeneration, which may be used for CNS diseases including multiple sclerosis (MS).Summary of the invention

[0007] In view of the above, there remains a need to develop compounds and compositions for use in the treatment and prevention of central nervous system (CNS) disorder, including multiple sclerosis.

[0008] The present invention relates in particular to a compound of formula (I):B ZFormula (I) for use in treating or preventing a central nervous system (CNS) disorder; wherein each X is independently selected from a negative charge, H, or C1-C12 alkyl; wherein Y is selected from H, OH or C1-C12 alkyl; wherein R is O or CH2; wherein n is 0 or 1 ; wherein m is 0 or 1 ; wherein, when no negative charge is present, A is 1 , B is 0 and [Z] is absent; and when at least one negative charge is present, Z is one or more pharmaceutically acceptable cations; and A and B are independently any integer, such that the net charge of the compound is 0.

[0009] The present invention also relates to a composition for use in treating or preventing a central nervous system (CNS) disorder comprising the compound of formula (I) in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.

[0010] The present invention also relates to a method of treating or preventing a central nervous system (CNS) disorder in a subject, the method comprising administering a compound or a composition as defined herein to the subject.

[0011] The present invention also relates to the use of a compound or a composition as defined herein, for the manufacture of a medicament for treating or preventing a central nervous system (CNS) disorder.Brief description of the drawings

[0012] Fig. 1 shows scRNA-seq uniform manifold approximation and projection (UMAP) plot obtained from 22,148 cells coloured by EAE stage (6,205 (control), 3,648(A-EAE), 12,295 (C-EAE)) and fraction of cell types. CAMs, CNS -associated macrophages.

[0013] Fig. 2 shows UMAP plot of clusters and fraction of cells per EAE stage.

[0014] Fig. 3 shows a grouped heat map of the top DEGs for the clusters. The dotted box highlights DAM cluster 4.

[0015] Fig. 4 shows UMAP plots of the unsupervised subcluster analysis of DAM cluster 4 coloured by subcluster (left) and EAE stage (right).

[0016] Fig. 5 shows the top GO terms (by fold enrichment) of DAM cluster 4 subclusters.

[0017] Fig. 6 shows UMAP analysis of DAM cluster 4 subclusters coloured by the mean counts of mitochondrial CI (left) and CII (right).

[0018] Fig. 7 shows quantification of EAE lesions, showing the number of SPP1+ cells expressing the NADH ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4). From left to right, n = 2, 3 and 3 replicates per group. Data are mean ± s.e.m. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test; **P < 0.01. Scale bar, 50 pm.

[0019] Fig. 8 shows expression UMAPs of SPP1, P2RY12, and mitochondrial CI and CII genes in human MAMS from a published study of patients with (MS Schirmer, L. et al. Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature 573, 75-82 (2019)).

[0020] Fig. 9 shows expression UMAPs of SPP1, P2RY12, and mitochondrial CI and CII genes in human MAMS from a published study of patients with (Absinta, M. et al. A lymphocyte-microglia-astrocyte axis in chronic active multiple sclerosis. Nature 597, 709-714 (2021)).

[0021] Fig. 10 shows representative immunofluorescence showing rim-specific expression (dotted lines) of NDUFS4+ and SPP1+ myeloid cells (MHC-II+) in consecutive sections of a chronic active lesion from the secondary progressive MS brain. Scale bar, 60 pm.

[0022] Fig. 11 shows (left) the metabolites significantly altered in A-EAE versus control microglia, n = 5 replicates per group. Statistical analysis was performed using unpaired two-tailed t- tests. Fig. 11 also shows (right) a corresponding correlation analysis of metabolites indicative of A-EAE versus control microglia. FA, fatty acids; P., phosphate.

[0023] Fig. 12 shows (left) the metabolites significantly altered in C-EAE versus A-EAE microglia, n = 5 replicates per group. Statistical analysis was performed using unpairedtwo-tailed t- tests. Fig. 12 also shows (right) corresponding correlation analysis of metabolites indicative of C-EAE versus A-EAE microglia. GSSG, glutathione disulfide.

[0024] Fig. 13 shows selected relevant metabolites, a.u., arbitrary units. n = 5 replicates per group. Statistical analysis was performed using one-way ANOVA with Fisher’s LSD test. The box plots show the median (centre line), quartiles (box limits), minimummaximum values (whiskers).

[0025] Fig. 14 shows genes from the scRNA-seq dataset of Fig. 1 that are involved in itaconate synthesis (Acodl), glycolytic switch (Hifla), DAM phenotype (Apoe), inflammasome (Nlrp3, Ddx3x, Dhx33, Caspl), antioxidant response (Cybb, Txnl, Sodl) and glutathione (Gsr) in the microglial clusters isolated from control, A-EAE and C-EAE mice. The dotted boxes highlight DAMs.

[0026] Fig. 15 shows the levels of mitochondrial proteins (left: representative western blot, values are expressed as fold induction over the control), mitochondrial membrane potential (middle: A | / m; from left to right, n = 10, 4 and 4 replicates per group) and mitochondrial biogenesis (right; mitochondrial / nuclear DNA ratio; from left to right, n = 3, 4 and 4 replicates per group). Statistical analysis was performed using one-way ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P< 0.01, ***P < 0.001.

[0027] Fig. 16 shows mitochondrial CI and CII activity in ex vivo FACS-isolated microglia and infiltrating myeloid cells. OCR, oxygen consumption rate. From left to right, n = 6, 6, 4, 6 and 5 replicates per group. Statistical analysis was performed using oneway ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P< 0.01, ***P < 0.001.

[0028] Fig. 17 shows quantification of fluorescence intensity of the CellROX probe signal using FACS in isolated microglia and infiltrating myeloid cells treated with rotenone. From left to right, n = 16, 16, 4, 4, 4, 4, 4, 4, 4 and 4 replicates per group. Statistical analysis was performed using one-way ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P< 0.01, ***P < 0.001.

[0029] Fig. 18 shows the results from Example 4 and are described in further detail below.

[0030] Fig. 19 shows seahorse metabolic flux analysis of primary microglia derived from wild-type (WT) and Nd6 mice under basal conditions and after stimulation with LPS and IFNy. n = 4 replicates per group. Statistical analysis was performed using one-wayANOVA with Tukey test. Differences in basal respiration, mitochondrial (mt) ATP production and maximal respiration are reported. The violin plots show the median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0031] Fig. 20 shows quantification of mtROS production in LPS + IFNy-stimulated (pro-inflammatory) primary WT and Nd6 microglia after RET induction (RET+). From left to right, n = 18, 18, 17, 18, 18 and 18 replicates per group. Statistical analysis was performed using two-way ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0032] Fig. 21 shows quantification of neuronal neurite length after co-culture with RET+ pro-inflammatory primary WT and Nd6 microglia. From left to right, n = 11, 5, 6, 12, 12, 12 replicates per group. Statistical analysis was performed using two-way ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0033] Fig. 22 shows EAE scores of WT and Nd6 mice up to 30 days after immunization, n = 17 mice per group. Statistical analysis was performed using two-way ANOVA with Bonferroni correction. Data are mean± s.e.m

[0034] Fig. 23 shows scRNA-seq UMAP plots with each cell labelled according to the genotype, obtained from 13,614 cells (7,501 (WT) and 6,113 (Nd6)). Superimposed cluster numbers and the corresponding fraction of cells are shown for control (left) and EAE (right) mice (30 days after immunization).

[0035] Fig. 24 shows selected hMG-like and DAM genes in cluster 0 and 1 DAMs.

[0036] Fig. 25 shows the mitochondrial membrane potential (A | / m) in ex vivo FACS- isolated CD45+CD1 lb+ cells. From left to right, n = 4, 3, 4 and 4 replicates per group. Statistical analysis was performed using one-way ANOVA with Fisher’s LSD test. The violin plots show the median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0037] Fig. 26 shows quantifications of perilesional microglial branching (n = 12 replicates per group) in WT and Nd6 EAE mice. Statistical analysis was performed using two-tailed unpaired t-tests. Scale bars, 30 pm. Data are mean± s.e.m.

[0038] Fig. 27 shows quantifications of IBA1+SPP1+GP91-PHOX+ cells in WT and Nd6 EAE mice (n = 4 replicates per group). Statistical analysis was performed using two- tailed unpaired t-tests. Scale bars, 30 pm. Data are mean ± s.e.m.

[0039] Fig. 28 shows EAE scores of Ndufs4-WT and Ndufs4-KO mice, n = 10 (WT) and 12 (KO) mice per group. Statistical analysis was performed using two-way ANOVA with Bonferroni correction. Data are mean± s.e.m. *P < 0.05,< 0.01, ***P < 0.001, ***P< 0.0001.

[0040] Fig. 29 shows scRNA-seq UMAP plots with each cell labelled according to the genotype, obtained from 10,666 cells (4,180 (Ndufs4 WT) and 6,486 (Ndufs4 KO)). Superimposed cluster numbers and the corresponding fraction of cells are shown for control (left) and EAE (right) mice (50 days after immunization).

[0041] Fig. 30 shows selected hMG-like and DAM genes in cluster 2 and 6 DAMs.

[0042] Fig. 31 shows suspension mass cytometry (CyTOF) analysis of immune cell types at 50 days after immunization obtained from 51,177 cells (23,467 (Ndufs4 WT); 27,710 (Ndufs4 KO)). AA, alternatively activated; pro-inflam., pro-inflammatory.

[0043] Fig. 32 shows quantification of CX3CR1+SPP1+ (left; n = 5 replicates per group) and CASPASE3+IBA1+ (right; n = 4 replicates per group) cells in EAE. Statistical analysis was performed using two-tailed unpaired t-tests. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

[0044] Fig. 33 shows in vivo quantification of perilesional microglial branching (left; n = 12 (WT) and 11 (KO) replicates per group; two-tailed unpaired t-test), GP91-PHOX expression in the EAE spinal cords (middle; n = 5 (WT) and 6 (KO) replicates per group; two-tailed Mann- Whitney U-test) and IBA1+SPP1+GP91-PHOX+ cells (right; n = 4 replicates per group; two-tailed unpaired t-test). Data are mean± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

[0045] Fig. 34 shows quantification of axonal loss (left; n = 5 (WT) and 6 (KO) replicates per group) and axonal degeneration (right; n = 5 replicates per group). Statistical analysis was performed using two-tailed unpaired t-tests. APP, amyloid precursor protein; NHP, neurofilament heavy polypeptide. Data are mean± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

[0046] Fig. 35 shows EAE scores of mice treated with metformin, DMM,DMM + metformin versus saline controls, n = 13 mice per group. Statistical analysis was performed using two-way ANOVA with Bonferroni correction; #P < 0.05 comparing DMM + metformin versus saline. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

[0047] Fig. 36 shows CyTOF analysis of immune cell types at 30 days after immunization obtained from 159,110 cells (32,793 (metformin), 40,864 (DMM), 44,143 (DMM + metformin) and 41,310 (saline)).

[0048] Fig. 37 shows quantification of CX3CR1+SPP1+NDUFS4+ cells, oxidative stress, axonal loss and axonal degeneration in EAE mice, n = 4 replicates per group. Statistical analysis was performed using one-way ANOVA with Tukey test. Data are mean± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.Detailed description of the invention

[0049] A finding underpinning the present invention is the identification of a key mitochondrial mechanism that contributes to the perpetuation of CNS inflammation by sustaining microglial activation and neurotoxic damage. The present inventors have shown that, as CNS inflammation becomes chronic, microglia display lower phosphocreatine and ATP levels that are coupled with transcriptomic changes indicative of a lower antioxidant and inflammasome response. This aligns with previous data suggesting a link between phosphocreatine depletion, low ATP levels and decreased NLRP3 inflammasome activation (Billingham, L. K. et al. Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation. Nat. Immunol. 23, 692-704 (2022)). However, after transitioning from the A-EAE to the C-EAE stage, microglia also show reduced itaconate levels (a known CII inhibitor), higher Hifla transcription, and a progressive alteration of their mitochondrial function.

[0050] Compelling evidence exists that succinate oxidation, coupled with elevated mitochondrial potential, is instrumental for the generation of RET in vitro (Mills, E. L. et al. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell 167, 457-470 (2016)). In the context of acute ischaemiareperfusion injury in vivo, succinate accumulates during ischaemia (when the electrontransport chain is obstructed due to the absence of oxygen) and after reperfusion the oxidation of the succinate pool drives RET (Chouchani, E. T. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515, 431-435 (2014)). The present inventors have determined that heightened succinate oxidation, supported by increased CII activity, in conditions characterized by a high mitochondrial membrane potential and diminished ATP synthesis, propels RET through CI in microglia. Accordingly, it was determined that exposing pro-inflammatory mouse and human microglia in vitro to conditions that boost RET leads to increased oxidative stress and paracrine neurotoxicity that is dependent on CI activity.

[0051] Mechanistically, this process may be prevented in vitro by blocking CI with small molecules or by using Nd6 microglia that have no RET. In an animal disease model in vivo, interfering with the function of the subunits of mitochondrial CI promotes the acquisition of a protective hMG-like phenotype. Moreover, it shifts the transcriptional profile of diverse DAM populations, which together result in the prevention of oxidative stress and neurotoxicity that lead to a protection from irreversible disabilities in mice.Cell-lineage-specific NDUFS4 conditional loss-of-function experiments and in vivo smallmolecule treatments reinforce the rationale for innovative therapeutic strategies designed to reduce mitochondrial CI activity within myeloid cells. This offers a promising route for targeting and resolving long-term CNS inflammation.

[0052] The present invention arises from the surprising finding that malonate compounds and related compounds are particularly effective for the treatment and prevention of central nervous system (CNS) disorder.

[0053] In particular, the invention relates to compounds of formula (I)B ZFormula (I) for use in treating or preventing a central nervous system (CNS) disorder.

[0054] In formula (I), each X is independently selected from a negative charge, H, or Ci- C12 alkyl group. Preferably, each X is independently selected from a negative charge, H or Ci-Cio alkyl. More preferably, each X is independently selected from a negative charge, H or C1-C5 alkyl. Even more preferably, each X is independently selected from H or a methyl group.

[0055] For example, each X may independently be selected from H, methyl, ethyl, n- propyl, iso-propyl, n-butyl, s-butyl, or t-butyl.

[0056] For example, both X groups may be selected from a negative charge, H, or C1-C12 alkyl group. Thus, both X groups may be selected from a negative charge, H or C1-C10 alkyl, or both X groups may be selected from a negative charge, H or C1-C5 alkyl, or both X groups may be selected from H or a methyl group.

[0057] In formula (I), Y is selected from H, OH or C1-C12 alkyl. Preferably, Y is selected from H, OH or C1-C10 alkyl. More preferably, Y is selected from H, OH or C1-C5 alkyl. Even more preferably, Y is selected from H or C1-C4 alkyl, more preferably selected from H or a butyl group. For example, Y may be a hydrogen atom. Alternatively, Y may be OH.

[0058] In formula (I), R is selected from O or CH2. Thus, R may be O. Alternatively, R may be CH2.

[0059] In formula (I), n is 0 or 1. In one embodiment, n is 1. In another embodiment, n is 0.

[0060] In formula (I), m is 0 or 1. In one embodiment, m is 1. In another embodiment, m is 0.

[0061] In one embodiment, no charges are present in the compound of formula (I). In that embodiment, neither X is a negative charge. Each X is independently selected from H, or C1-C12 alkyl group. When the compound of formula (I) is a neutral compound, A is 1, B is 0, and [Z] is absent.

[0062] In one preferred embodiment, the compound of formula (I) has a value of n of 0, a value of m of 0, and neither X group is a negative charge. In this case, the compound is a compound of formula (II):Formula (II) wherein Y is as defined above, and each X’ is independently selected from a negative charge, H, or C1-C12 alkyl group. Preferably, each X’ is independently selected from a negative charge, H or C1-C10 alkyl. More preferably, each X’ is independently selectedfrom a negative charge, H or C1-C5 alkyl. Even more preferably, each X’ is independently selected from H or a methyl group.

[0063] The compound of formula (I) for use in treating or preventing a CNS disorder may be a diester. In this case, the compound is of formula (II), and both X’ groups are independently C1-C12 alkyl groups. For example, both X’ groups may independently be C1-C10 alkyl, or C1-C5 alkyl, or C1-C3 alkyl, or methyl. For example, the compound of formula (II) may be dimethyl malonate:In this embodiment, both X’ groups are methyl, and Y is a H atom.

[0064] In another embodiment, the compound of formula (I) has charged groups and the compound of formula (I) is a salt. For example, one X group may be a negative charge. Alternatively, both X groups may be a negative charge.

[0065] When at least one negative charge is present, Z is one or more pharmaceutically acceptable cations. A and B may independently be any integer (for example, independently 1, 2 or 3), provided that the net charge on the compound of formula (I) is 0. For example, when Z is a +3 cation and the anion is a -2 anion, A is 3 and B is 2, such that the net charge on the salt is 0. In one embodiment, A and B are each independently selected from 1 and 2; for example A is 1 and B is 1 or 2, provided that the net charge of the compound is 0.

[0066] When Z is present, each Z is a pharmaceutically acceptable cation, provided that the net charge of the salt of formula (I) is 0. For example, Z may comprise a cation with a +1 charge, a +2, or a +3 charge. Preferably, Z comprises: a cation selected from Li+, Na+,Zn2+, Al3+and / or NH4+;or a cation selected from Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr2^ Ba2+, Pb2+, Ni2+, Ag+, Sn2+, Cr3+, Zn2+, Al3+and / or NH4+;or a cation selected from Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr^, Ba2+, Pb2+, Ni2+, Sn2+, Cr3+, Zn2+and / or NH4+;or a cation selected from Li+, Na+, K+, Mg2+, Ca2+, Zn2+and / or NF t

[0067] Z may be a single ion, for example a Na+ion, or multiple ions, such as a Na+and a K+ion. Thus, salts with multiple cations (such as sodium potassium malonate) are encompassed by formula (I).

[0068] In a second preferred embodiment, the compound of formula (I) has a value of n of 0, a value of m of 0, and at least one X group is a negative charge. In this case, the compound is a salt of formula (III):Formula (III) wherein A, B, Z, Y and X are as defined above.

[0069] The compound of formula (I) for use in treating or preventing a CNS disorder may be a malonate salt. In this case, the compound is a salt of formula (III), in which Y is a H atom.

[0070] For example, the salt of formula (III) may be disodium malonate, monosodium malonate, dipotassium malonate monopotassium malonate, dilithium malonate, monolithium malonate, calcium malonate, magnesium malonate, ammonium malonate, aluminium malonate or zinc malonate; for example disodium malonate, monosodium malonate, dipotassium malonate monopotassium malonate, dilithium malonate, monolithium malonate, calcium malonate, magnesium malonate, ammonium malonate or zinc malonate.

[0071] The compound for use in treating or preventing a CNS disorder may alternatively be a salt of formula (II), wherein Y is a C1-C5 alkyl, more preferably a butyl group.

[0072] Compounds of the present invention, including malonate compounds, can enter mitochondria by endogenous transport mechanisms, thus allowing the compound to reach the target site in a timely manner. Furthermore, compounds of the present invention have limited toxicity, well established metabolism and have been used as excipients in pharmaceutical development.

[0073] As described above, the present inventors have identified a mitochondrial mechanism that contributes to the perpetuation of CNS inflammation by sustaining microglial activation and neurotoxic damage. As CNS-compartmentalised inflammation becomes chronic (as in the case of progressive MS), microglia display lowerphosphocreatine and ATP levels that are coupled with transcriptomic changes indicative of a lower antioxidant and inflammasome response.

[0074] Compounds of formula (I) have been shown to target microglial cells and to significantly reduce mitochondrial ROS by inhibiting CI. Thus, compounds of formula (I) provide a treatment for diseases and conditions associated with sustained microglial activation, including chronic inflammatory CNS disorders.

[0075] The compound of formula (I) is used in particular to treat or prevent a central nervous system (CNS) disorder i.e. a disorder that is associated with sustained microglial activation. Preferably, the central nervous system disorder is an inflammatory CNS disorder or a neurodegenerative CNS disorder, more preferably an inflammatory and neurodegenerative CNS disorder. Preferably, the CNS disorder is a primary demyelinating disorder of the CNS.

[0076] When the CNS disorder is a demyelinating disorder of the CNS, it is preferably a primary demyelinating disorder of the CNS. The demyelinating CNS disorder may be selected from multiple sclerosis (MS), Neuromyelitis optica spectrum disorders (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), transverse myelitis, and acute disseminated encephalomyelitis (ADEM).

[0077] When the CNS disorder is a neurogenerative disorder, it is preferably a primary neurodegenerative disorder where chronic inflammation has been linked with significantly worse clinical outcomes. The neurodegenerative disorder may be selected from multiple sclerosis (MS), Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia.

[0078] Exemplary conditions that may be treated or prevent by the compound of formula (I) are described in Kouli A et al. Neuroinflammation is linked to dementia risk in Parkinson's disease. Brain. 2024 Mar l;147(3):923-935; Malpetti M et al. Microglial activation in the frontal cortex predicts cognitive decline in frontotemporal dementia.Brain. 2023 Aug 1 ; 146(8):3221 -3231 ; and Nicastro N et al. Gray matter changes related to microglial activation in Alzheimer's disease. Neurobiol Aging. 2020 Oct;94:236-242.

[0079] In a preferred embodiment, the central nervous system disorder is multiple sclerosis (MS). The MS may be any type of MS. For example, the MS may be clinically isolated syndrome (CIS), relapsing-remitting MS, or progressive MS. The MS is typically relapsing-remitting MS, or progressive MS. Typically, the MS is progressive MS, which is preferably selected from secondary progressive MS, or primary progressive MS.

[0080] Alternatively, the compound of formula (I) may be used to treat or prevent a disorder that is associated with sustained microglial activation.

[0081] The compound of formula (I) may be administered by any method in the art. Preferably, the compound of formula (I) is administered orally, topically, subcutaneously, parenterally, intramuscularly, intraperitoneally, intraocularly, intranasally, intra-arterially or intravenously. Most preferably, the compound of formula (I) is administered orally.

[0082] The compound of formula (I) may be administered at a dose in the range of from about 0.1 mg / kg to about 500 mg / kg of body weight. Preferably, the compound of formula (I) is administered at a dose of greater than about 0.2 mg / kg of body weight, preferably greater than about 0.3 mg / kg of body weight, preferably greater than about 0.4 mg / kg of body weight, and preferably greater than about 0.5 mg / kg of body weight. Preferably, the compound of formula (I) is administered at a dose of less than about 450 mg / kg of body weight, preferably less than about 400 mg / kg of body weight, preferably less than about 350 mg / kg of body weight, and preferably less than about 300 mg / kg of body weight.

[0083] The compound of formula (I) may be administered to a patient in combination with one or more additional compounds. Thus, the compound of formula (I) may be administered as part of a combination therapy. The one or more additional compounds may be a compound which inhibits mitochondrial complex I (CI). Alternatively or additionally, the one or more additional compounds may be a compound which inhibits mitochondrial complex II (CII). The one or more additional compounds may be selected from metformin, IACS-010759, intervenolin, ASP4132, phenformin, BAY87-2243, carboxyamidotriazole (CAI), ME344, fenofibrate, lonidamine, a-tocopheryl succinate (a- TOS), VLX600 (6-Methyl-3-{(2E)-2-[l-(2-pyridinyl)ethylidene]hydrazino}-5H- [l,2,4]triazino[5,6-b]indole) and meta-iodobenzylguanidine (mIBG). Preferably, the one or more additional compounds is metformin.

[0084] The compound of formula (I) may be formulated into a composition for use in treating or preventing a central nervous system (CNS) disorder. The composition comprises a compound of formula (I) as defined herein (which may, for example, be a compound of formula (II) or a salt of formula (III)), and one or more pharmaceutically acceptable excipients, carriers or diluents.

[0085] Suitable excipients, carriers and diluents can be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA) andRemington: The Science and Practice of Pharmacy, 2ist Edition (ed. D. B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA).

[0086] Excipients for use in the compositions of the invention include, but are not limited to microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavouring agents, colouring matter or dyes, and, if so desired, emulsifying and / or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

[0087] Pharmaceutical carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, and the like.

[0088] Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials, and mixtures thereof. The compound can be administered to a subject by, for example, subcutaneous implantation of a pellet. The preparation can also be administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation oral administration of a liquid or solid preparation, or by topical application. Administration can also be accomplished by use of a rectal suppository or a urethral suppository.

[0089] Further, as used herein “pharmaceutically acceptable carriers” are well known to those skilled in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.9% saline. Additionally, such pharmaceutically acceptable carriers maybe aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media.

[0090] Pharmaceutically acceptable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

[0091] Pharmaceutically acceptable carriers for controlled or sustained release compositions administrable according to the invention include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[0092] Pharmaceutically acceptable carriers include compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., (1981), pp 367-383). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer- compound abducts less frequently or in lower doses than with the unmodified compound.

[0093] The present invention also relates to a method of treating or preventing a central nervous system (CNS) disorder in a subject, the method comprising administering a compound of formula (I) as defined herein, or a composition as defined herein to the subject. The compound of formula (I) may, for example, be a compound of formula (II) (for example, dimethyl malonate), or a salt of formula (III) (for example, a malonate salt). Preferably, the method comprises administration of said compound of formula (I) to the subject as further defined herein.

[0094] The present invention also relates to the use of a compound of formula (I) as defined herein, or a composition as defined herein for the manufacture of a medicament for treating or preventing a central nervous system (CNS) disorder. The compound of formula (I) may, for example, be a compound of formula (II) (for example, dimethyl malonate), ora salt of formula (III) (for example, a malonate salt). Preferably, said compound of formula (I) is for manufacture of a medicament for preventing a central nervous system (CNS) disorder as further defined herein.

[0095] As used herein, the term “Ci-Cnalkyl” refers to straight chain and branched saturated hydrocarbon groups generally having from 1 to n carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-l-yl, pent-2-yl, pent-3-yl, 3-methylbut-l-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-l-yl, and the like.

[0096] As used herein, the terms “drug”, “drug substance”, “active pharmaceutical ingredient”, and the like, refer to a compound that may be used for treating a subject in need of treatment.

[0097] As used herein, the term “excipient” refers to any substance that may influence the bioavailability of a drug, but is otherwise pharmacologically inactive.

[0098] As used herein, the term “pharmaceutically acceptable” refers to species which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

[0099] As used herein, the term “pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.

[0100] As used herein, the term “subject” as used herein refers to a human or non-human mammal.

[0101] Examples of non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.

[0102] As used herein, the term “body” as used herein refers to the body of a subject as defined above.

[0103] As used herein, the term “therapeutically effective amount” of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect. The therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things.

[0104] As used herein, the term “treating” refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or toreversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition.

[0105] As used herein, the term “treatment” refers to the act of “treating”, as defined above.

[0106] As used herein, the term “preventing” refers to a reduction of the risk of acquiring a given disease or disorder, or a reduction in the severity of symptoms of the given disease or disorder if the disease or disorder is acquired after the preventative measure. Hence, “preventing” refers to the prophylactic treatment of a subject in need thereof. The prophylactic treatment can be accomplished by administering an appropriate dose of a therapeutic agent to a subject having a predisposition to a disorder, or at risk of developing a disorder, even though symptoms of the disorder are absent or minimal, thereby substantially averting onset of the disorder, or substantially reducing the severity of symptoms of the disorder if it is acquired after the preventive measure.

[0107] As used herein, the term “succinate dehydrogenase inhibitor” or “SDHi” refers to a species that inhibit the action of succinate dehydrogenase.

[0108] As used herein the term “comprising” means “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.ExamplesExample 1

[0109] To investigate the molecular mechanisms through which microglia and CNS- infiltrating myeloid cells cooperate to sustain CNS inflammation, ex vivo single-cell RNA- sequencing (scRNA-seq) and liquid chromatography-mass spectrometry (LC-MS)-based analyses were used of Cx3crl-YFPcreERT2R26tdTomatofate-mapping mice, which were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) to induce experimental autoimmune encephalomyelitis (EAE), a model of MS-like disease. RFP+YFP+cells (microglia) and RFP YFP+cells (predominantly consisting of infiltrating myeloid cells) were isolated using fluorescence-activated cell sorting (FACS) from the spinal cord of EAE mice in the acute EAE (A-EAE; 3 days after disease onset) and chronicEAE (C-EAE; 50 days after immunization) disease stages. Non-immunized Cx3crl- YppcreERT2 ^(^tdTomatomjcc were uscc[ashealthy controls.

[0110] scRNA-seq data showed a prevalence of infiltrating myeloid cells in A-EAE mice, while microglia were predominant in control and C-EAE mice (Fig. 1). Unsupervised clustering analysis of the integrated dataset identified 13 cell clusters between control, A- EAE and C-EAE mice (Fig. 2). Clusters 0 and 1 comprised cells with a transcriptional signature reminiscent of homeostatic microglia (hMG-like) and were found mostly in control mice, in which they constituted 91% of all isolated cells. These two clusters differed in the expression of specific homeostatic genes (for example, Siglech, P2ryl2 and Cx3crl, higher in cluster 0) and by the relatively increased expression of AC149090.1, a gene encoding a phospholipid decarboxylase that is involved in lipid metabolism, in cluster 1. This is consistent with evidence supporting differential cellular transcriptional states of microglia (Paolicelli, R. C. et al. Microglia states and nomenclature: a field at its crossroads. Neuron 110, 3458-3483 (2022)). The proportion of hMG-like cells decreased to less than 1% of all isolated cells in A-EAE mice, and subsequently increased to 36% of cells in C-EAE mice, therefore suggesting a partial return to homeostasis in the chronic stage of disease (Fig. 2). This approach also identified several clusters (that is, clusters 3, 4, 5, 7, 8 and 10) of disease-associated microglia (DAMs) that were nearly absent in control mice, increased in A-EAE mice and persisted in C-EAE mice (8%, 46% and 48% of all isolated cells, respectively) (Fig. 2).[OHl] Given the potential pathogenic role of persistent DAM activity in chronic CNS diseases, a clustering analysis of microglia was performed to further understand their activation profile and dynamics over the disease course. The velocity and directionality of RNA expression changes in the different cell and microglial clusters were analyzed. Among all of the DAM clusters, DAM cluster 4 was the only one that consistently increased from control to A-EAE to C-EAE (6%, 23%, 29% of DAMs, respectively), and it showed the lowest probability of transition into other microglial clusters (that is, small RNA velocity), suggestive of a persistent (steady) state in EAE. Transcriptionally, DAM cluster 4 was characterized by the increased expression of the DAM genes Sppl (top differentially expressed gene (DEG)), Cd63, Cst7, Timp2 and Apoe (Fig. 3).

[0112] Additional transcriptomic and subclustering analyses of DAM cluster 4 were performed to identify putative mechanisms driving its persistence in EAE. It was found that DAM cluster 4 was further characterized by DEGs related to Gene Ontology (GO)terms of glycolysis (such as Gapdh zmA Aldoa) and oxidative phosphorylation (for example, Cox4il and Ndufal), while subclustering analysis identified three main subclusters (Fig. 4). DAM subcluster 4.1 was defined by DEGs involved in lipid (Fahp5) and iron metabolism (Fthl and Fltl). DAM subcluster 4.2 was defined by DEGs associated with mitochondrial CI (for example, mt-Ndl and mt-Nd4) and cytochrome b (mt-Cytb). DAM subcluster 4.3 was defined by DEGs involved in myeloid activation (Ccl2 and Ccll2) and mitochondrial metabolism (Tspo). GO-term analysis of DAM subclusters 4.1 and 4.3 revealed enrichment in several pathways involved in myeloid activation and metabolite signalling (Fig. 5). DAM subcluster 4.2 was instead characterized by pathways associated with the electron-transport chain, mitochondrial CI function (NADH to ubiquinone) and energy-coupled proton transport against electrochemical gradient.

[0113] Given the known functional role of CI and CII in electron transport and ROS generation in pro-inflammatory myeloid cells (Mills, E. L. et al. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell 167, 457-470 (2016); Scialo, F., Fernandez- Ayala, D. J. & Sanz, A. Role of mitochondrial reverse electron transport in ROS signaling: potential roles in health and disease. Front. Physiol. 8, 428 (2017)), genes encoding these two mitochondrial complexes were further analysed. No relevant changes were in found in the expression of genes encoding the CII subunits during the different stages of EAE in cluster 4 or its subclusters (Fig. 6). In A- EAE, instead, an increase was observed in genes encoding the CI subunits in DAM cluster 4 and in cluster 2 (predominantly consisting of infiltrating myeloid cells). In C-EAE, the expression of CI subunits further increased in DAM cluster 4 and its subclusters (Fig. 6). Pathological analysis confirmed a 7.6-fold increase in the number of SPP1+DAMs expressing the NADH-ubiquinone oxidoreductase subunit of CI, NDUFS4, in the spinal cord of A-EAE mice versus control mice, followed by a further 2.4-fold increase in SPP1+NDUFS4+DAMs in C-EAE versus A-EAE mice (Fig. 7). This finding was associated with a significant increase in SPP1+RFP+YFP+microglia expressing the marker of oxidative stress GP91-PHOX in C-EAE mice.Example 2

[0114] To investigate the relevance of the findings of Example 1 for human disease, two independent publicly available single-nucleus RNA-seq datasets obtained from people withMS and control individuals post mortem were re-analysed (Schirmer, L. et al. Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature 573, 75-82 (2019); Absinta, M. et al. A lymphocyte-microglia-astrocyte axis in chronic active multiple sclerosis. Nature 597, 709-714 (2021)). In both datasets, a cluster of human microglia activated in progressive MS (MAMS) were identified that displayed a transcriptional profile reminiscent of the mouse DAM cluster 4 identified in the single-cell analysis. Compared with other microglia, MAMS were characterized by the high expression of DAM (for example, SPP1 and APOE) and CI (such as MT-ND1, MT-ND4) genes, but low levels of homeostatic (for example, P2RY12 and SALL1) and antioxidant genes (for example, CYBB and SOD1 (Fig. 8 and Fig. 9). In both datasets, MAMS were almost absent in controls, while most MAMS were found either in chronically active (smouldering) lesions (CALs; 62% of MAMS) or at the edge of CALs (86% of MAMS) in people with MS, in whom they constituted 13% of all microglia. Pathological analysis of the brains of people with progressive MS confirmed the presence of SPP1+NDUFS4+MHC-II+myeloid cells at the CAL edge (Fig. 10).

[0115] Thus, a cluster of persistently activated DAMs with high expression of CI genes and proteins that persists during C-EAE in mice and is found almost exclusively at the edge of CALs in people with progressive MS was identified.Example 3

[0116] To gain further insights into the metabolic features of microglia and infiltrating myeloid cells that sustain chronic CNS inflammation, an LC-MS analysis was performed of the intracellular metabolome of ex vivo isolated myeloid cells. A clear separation was found based on a partial least squares discriminant analysis and a differential abundance of intracellular metabolites based on the cell type and stage of EAE). A-EAE microglia had increased intracellular levels of itaconate, phosphocreatine (an ATP buffer), ascorbate and dehydroascorbate (a ROS scavenger and its oxidized product), as well as glutathione disulfide (which arises from antioxidant reactions) compared with control microglia (Fig. 11). Laser desorption-rapid evaporative ionization mass spectrometry (LD-REIMS) analysis of spinal cord sections confirmed the higher abundance of itaconate and ascorbate within white-matter inflammatory infiltrates in situ. Analysis of the entire LC-MS dataset showed a direct correlation between itaconate levels and ascorbate, as well as dehydroascorbate. C-EAE microglia had instead lower itaconate and significantly loweramounts of glutathione disulfide, phosphocreatine and ATP compared with A-EAE microglia (Fig. 12), which was coupled with significantly increased intracellular levels of creatine and L-citrulline (Figs. 12, 13).

[0117] To correlate these metabolic changes with the expression of relevant genes from the scRNA-seq dataset, the clusters of microglia isolated from control and EAE mice was focused on. The expression of Hifla, which is involved in the switch to aerobic glycolysis in myeloid cells, and the DAM marker Apoe steadily increased in DAM cluster 4 in A- EAE and C-EAE (Fig. 14). Conversely, the expression of aconitate decarboxylase 1 (Acodl), which encodes the enzyme that synthesizes itaconate, was increased in DAM cluster 4 in A-EAE but later decreased in C-EAE. Given the dynamic changes of intracellular phosphocreatine and antioxidants genes related to the inflammasome and the response to ROS were then focused on. It was found that the expression of genes involved in the NLRP3 inflammasome complex (such as Nlrp3 and Dhx33), the antioxidant response (for example, Cybb, Txnl and Sod!) and glutathione synthesis / reduction (Gsr) was increased in DAM cluster 4 during A-EAE, but later decreased in C-EAE (Fig. 14). These findings suggest elevated NO and ROS generation in C-EAE microglia.

[0118] Ex vivo microglia were isolated from control, A-EAE and C-EAE mice for downstream analyses of their mitochondrial function. Mitochondrial complex III, IV and V (CIII, CIV, CV) proteins were reduced in A-EAE and C-EAE, while the CII active subunit SDHA and the CI assembly factor 1 (NDUFAF1) increased in both EAE stages versus control microglia (Fig. 15, left). Although a significant increase in mitochondrial membrane potential starting in A-EAE and persisting in C-EAE microglia (Fig. 15, middle) was found, there was no evidence of increased mitochondrial biogenesis during EAE (Fig. 15, right). Instead, the reanalysis of the scRNA-seq dataset showed that genes positively regulating mitophagy (for example, Ambral, Irgml and Vpsl3d) were reduced in C-EAE versus A-EAE microglia. Accordingly, gene regulatory networks guiding mitophagy were downregulated in DAM cluster 4, while gene regulatory networks guiding CI transcription were upregulated. To connect these metabolic and transcriptional features with the function of mitochondrial complexes, ex vivo metabolic flux analysis were applied at different stages of disease and found that C-EAE microglia had significantly higher levels of CI and CII activity compared with control and A-EAE microglia (Fig. 16).

[0119] On the basis of these integrated data, it is proposed that, after transitioning from the A-EAE to the C-EAE stage, microglia display increased CI-CII activity, reduced ATPlevels and high mitochondrial membrane potential, supporting a repurposing of their mitochondria towards mtROS generation through CI, possibly via RET. Consistent with this, treatment of C-EAE microglia with the CI inhibitor rotenone significantly reduced ROS production to levels observed in control mice (Fig. 17).Example 4

[0120] To further investigate whether mitochondrial CI acts through RET to amplify the oxidative stress seen in persistent smouldering-like inflammatory CNS disease, an in vitro model of RET induction (RET+) was applied to pro-inflammatory mouse microglia. RET was induced in LPS / IENy-stimulated microglial cells through treatment with oligomycin (which blocks mitochondrial CV and increases the mitochondrial proton motive force) in conjunction with succinate (to provide a substrate for oxidation by CII and further sustain a high mitochondrial proton motive force). It was found that the production of mtROS and increased mitochondrial membrane potential of RET+pro-inflammatory microglia were both prevented by rotenone treatment without causing significant cytotoxicity (Fig. 18). These data suggest that CI functions as a hub for mtROS production in pro-inflammatory microglia during RET in vitro.

[0121] To test the pathogenic role of RET+pro-inflammatory rodent microglia, they were co-cultured with SH-SY5Y neuronal cells using a transwell co-culture system that avoided cell-to-cell contacts. No significant change in cell death or neurite length of SH-SY5Y cells that were co-cultured with pro -inflammatory microglia without RET induction (RET ) was observed (Fig. 18). Instead, co-cultures with RET+pro-inflammatory microglia were characterized by a significant increase in CASPASE3 expression and a decrease in neurite length (Fig. 18).

[0122] Blocking CI activity in RET+pro-inflammatory microglia by treatment with rotenone prevented CASPASE3 induction and loss of neurites in co-cultured SH-SY5Y cells (Fig. 18). Conversely, blocking CI activity with the suppressor of site IQ electron leak S1QEL1.1, which inhibits the production of superoxide / hydrogen peroxide without affecting electron movement from COQH2 to NAD, did not significantly rescue SH-SY5Y neurite length versus RET+pro-inflammatory microglia.

[0123] The main findings obtained from mouse microglia were further validated on human induced pluripotent stem (iPS) cell-derived induced microglia (hiMGs) (Fig. 18). RET+pro-inflammatory hiMGs produced significantly more mtROS, had increasedmitochondrial membrane potential and caused increased neurite toxicity in SH-SY5Y cells compared with the controls (Fig. 18). As described for mouse microglia, these effects were all prevented by CI inhibition in hiMGs by treatment with rotenone (Fig. 18).

[0124] Thus, blocking RET in pro-inflammatory rodent and human microglia through CI inhibition protects from excessive mtROS-associated neurotoxicity in vitro.Example 5

[0125] To further establish the role of CI and RET in microglial polarization and function, primary microglia were isolated from Nd6 mice. These mice carry a point mutation in the mitochondrial CI gene Nd6 that blocks RET while preserving normal forward electron transport. Microglial stimulation with LPS and IFNy induced superimposable effects in the expression of genes coding for major pro -inflammatory cytokines in both wild-type (WT) and Nd6 microglia, while stimulated Nd6 microglia displayed significantly higher mitochondrial ATP production (Fig. 19). Nd6 microglia did not increase mtROS production (Fig. 20) or cause significant neurite toxicity in co-culture with of SH-SY5Y cells (Fig. 21) under in vitro conditions that forcing RET in microglia.

[0126] To verify the relevance of these findings in a disease model in vivo, MOG35- 55 EAE was induced in Nd6 and WT mice. Nd6 mice developed a significantly milder EAE throughout the entire disease course compared with WT mice (Fig. 22). Ex vivo scRNA- seq analysis of the entire CNS of WT and Nd6 mice identified 17 cell clusters in nonimmunized control and EAE mice (Fig. 23). In non-immunized control mice, a 1.3-fold increase was found in cluster 4 hMG-like cells in Nd6 versus WT mice (Fig. 23, top). In EAE, Nd6 mice displayed a 14.0-fold and a 4.0-fold increase in hMG-like clusters 3 and 4, respectively (Fig. 23, bottom). Furthermore, Nd6 EAE mice showed substantial changes in DAM phenotypes, the most notable ones being a 23.2-fold reduction of cluster 0 DAM (characterized by the expression of Apoe and Sppl) and a 101-fold increase in cluster 1 DAM (characterized by Fabp5 expression). When comparing the expression of known hMG-like and DAM genes in these two clusters, a reduction in the expression of Trem2 and Apoe was found in Nd6 EAE mice (Fig. 24). The same cluster 0 and cluster 1 DAMs also showed a significant increase in genes associated with mitochondrial ATP- synthesis-coupled electron transport (such as mt-Nd2 and mt-Col), an increased expression of lysosomal genes involved in antigen processing and presentation (for example, Ifi30 and Ctss), but no significant DEGs related to growth-factor activity.

[0127] CD45+Cdl lb+myeloid cells were next isolated from non-immunized control and EAE mice to assess their mitochondrial membrane potential ex vivo (Fig. 25). In nonimmunized control mice, no difference was found in mitochondrial membrane potential between the Nd6 and WT groups. In EAE, myeloid cells isolated from Nd6 mice had a significantly lower mitochondrial membrane potential compared with WT mice. Pathologically, despite not observing significant morphological differences in perilesional microglia in Nd6 and WT EAE mice (Fig. 26), a significant decrease was found in SPP1+IBA1+cells expressing GP91-PHOX in the spinal cords of Nd6 EAE mice (Fig. 27).

[0128] Thus, the lack of RET in Nd6 mice actively regulates microglial responses to neuro inflammation, which results in a reduction in oxidative stress in vivo.Example 6

[0129] To investigate the possibility of therapeutically targeting CI in myeloid cells only during the transition between A-EAE and C-EAE, tamoxifen-inducible transgenic mice were generated that allow for the timed knockout of Ndufs4 in CX3CR1+cells in vivo. MOG35-55 EAE was induced in Cx3cr! -YFPc’eKRI2Ndufs4ilo:'' / ilo:'' mice and administered tamoxifen 1 week after the onset of the EAE clinical signs to obtain Ndufs4- O mice.

[0130] It was found that Ndufs4- O EAE mice had significantly lower disease severity when they reached the C-EAE stage compared with Ndufs4-WT EAE mice (Fig. 28). Ex vivo scRNA-seq of the entire CNS of Ndufs4-^4rT and Ndufs4- O mice revealed 14 cell clusters in non-immunized control and EAE mice (Fig. 29). In non-immunized control mice, Ndufs4- O mice showed a slight decrease (1.1 -fold) in cluster 3 oligodendrocytes and an increase in cluster 0 microglia (2.6-fold), which was characterized by genes involved in cytoskeletal organization such as [3-actin (Actb) and thymosin [3-4 (Tmsb4x) (Fig. 29, top). In EAE, Ndufs4- O mice had a 2.6-fold increase in cluster 0 microglia, an 8.1 -fold increase in cluster 5 microglia (also characterized by cytoskeletal genes, such as tubulin [3-5 chain (Tubb5)) and a 4.9-fold increase in cluster 7 neural progenitor / ependymal cells (Fig. 29, bottom). In EAE, Ndufs4- O mice also showed a 3.6-fold increase of cluster 1 hMG-like cells, which was coupled with a significant reduction in cluster 2 and cluster 6 DAMs expressing Apoe and Cd74 (1.1- and 1.6-fold, respectively). When comparing the expression of known hMG-like and DAM genes in these two latter clusters, it was found that Ndufs4- O EAE mice had reduced DAM gene expression (for example, Cd74, Sppl and Apoe) and increased homeostatic geneexpression (for example, Csflr and Sparc) (Fig. 30). Clusters 2 and 6 showed no significant DEGs related to growth factor activity, but showed a reduction in the expression of genes involved in phagocytosis and antigen processing compared with Ndufs4-W EAE mice, except for Ifi.30.

[0131] Ex vivo mass cytometry analysis of CD45+spinal cord immune cells confirmed a concordant 17% increase in the number of hMG-like cells (Fig. 31) coupled with a lower CII and CI expression in DAMs isolated from Ndufs4- O EAE mice.Pathologically, Ndufs4- O EAE mice showed a significant reduction in CX3CR1+SPP1+cells in the spinal cord (Fig. 32, left) but no significant difference of apoptotic IBA1+cells (Fig. 32, right). Ndufs4- O EAE mice also showed a significant increase in perilesional microglial branching (Fig. 33, left), which was consistent with the differences in genes regulating cytoskeletal organization found in the scRNA-seq dataset. Finally, Ndufs4- O EAE mice showed significantly diminished oxidative stress in the spinal cord (Fig. 33, middle), a reduction in GP91-PHOX expression in SPP1+IBA1+cells (Fig. 33, right) and significantly lower axonal loss and degeneration (Fig. 34).

[0132] Thus, targeting CI activity in myeloid cells in vivo increases hMG-like cells and induces DAM changes that together prevent oxidative stress and associated neurotoxicity.Example 7

[0133] In vitro drug testing was performed in mouse and human microglia to select inhibitors of CI and CII activity for in vivo testing. The CI inhibitors rotenone and metformin, as well as the CII inhibitors dimethyl malonate (DMM) and disodium malonate, were the most effective in reducing mtROS production in vitro. This effect was further enhanced by the combination of selected CI and CII inhibitors.

[0134] In vivo, daily intraperitoneal injections of 4-octyl itaconate (a weak CII inhibitor) or DMM only did not ameliorate MOG35-55 EAE in mice. The combination ofDMM + metformin led to the most significant therapeutic effect on EAE at 30 days after immunization (Fig. 35). Ex vivo mass cytometry analysis of CD45+spinal cord leukocytes revealed a marked effect of DMM + metformin on the frequency of hMG-like (1.4 and 1.3- fold increase, versus saline and DMM, respectively) and DAM (2.5 and 1.6-fold decrease, versus saline and DMM, respectively) (Fig. 36), coupled with a significant reduction in CII and CI expression in DAM clusters. Pathologically, treatment with DMM + metformin led to a significant decrease in CX3CR1+SPP1+NDUFS4+cells, which was coupled withreduced oxidative stress in the spinal cord (Fig. 37), a reduction in GP91-PHOX+expression in IBA1+cells only and a significant protection from axonal loss and degeneration (Fig. 37).

[0135] While specific embodiments of the invention have been described above, it will be appreciated that the invention may be practiced otherwise than as described. The descriptions above are intended to be illustrative, not limiting. Thus it will be apparent to one skilled in the art that modifications may be made to the invention as described without departing from the scope of the claims set out below.

Claims

CLAIMS1. A compound of formula (I):Formula (I) for use in treating or preventing a central nervous system (CNS) disorder; wherein each X is independently selected from a negative charge, H, or C1-C12 alkyl; wherein Y is selected from H, OH or C1-C12 alkyl; wherein R is O or CH2; wherein n is 0 or 1 ; wherein m is 0 or 1 ; wherein, when no negative charge is present, A is 1 , B is 0 and [Z] is absent; and when at least one negative charge is present, Z is one or more pharmaceutically acceptable cations; and A and B are independently any integer, such that the net charge of the compound is 0.

2. The compound for use according to claim 1, wherein n is 0.

3. The compound for use according to claim 1 or claim 2, wherein m is 0,4. The compound for use according to any one of claims 1 to 3, wherein R is O.

5. The compound for use according to any one of claims 1 to 4, wherein each X is independently selected from a negative charge, H, or C1-C5 alkyl, and is preferably selected from H or a methyl group.

6. The compound for use according to any one of claims 1 to 5, wherein Y is selected from H or C1-C5 alkyl, and is preferably selected from H or C1-C4 alkyl, more preferably selected from H or a butyl group.

7. The compound for use according to any one of claims 1 to 6, wherein Y is H.

8. The compound for use according to any one of claims 1 to 7, wherein both X groups are selected from H or C1-C3 alkyl, and are preferably both selected from H or a methyl group.

9. The compound for use according to any one of claims 1 to 8, wherein the compound is dimethyl malonate.

10. The compound for use according to any one of claims 1 to 7, wherein at least one X is a negative charge.11 . The compound for use according to any one of claims 1 to 10, wherein, when present, each Z is independently selected from Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr2-1-, Ba2+, Cu+, Cu2+, Fe2+, Fe3+, Pb2+, Ni2+, Ag+, Sn2+, Cr3+, Zn2+, Al3+and / or NH4+.

12. The compound for use according to any one of claims 1 to 7, 10 or 11, wherein the compound is a malonate salt.

13. The compound for use according to claim 12, wherein the compound is selected from disodium malonate, monosodium malonate, dipotassium malonate monopotassium malonate, dilithium malonate, monolithium malonate, calcium malonate, magnesium malonate, ammonium malonate, aluminium malonate or zinc malonate.

14. The compound for use according to any one of claims 1 to 13, by administering the compound orally, topically, subcutaneously, parenterally, intramuscularly,intraperitoneally, intraocular ly, intranasally, intra-arterially or intravenously.

15. The compound for use according to any one of claims 1 to 14, by administering the compound orally.

16. The compound for use according to any one of claims 1 to 15, wherein the central nervous system disorder is an inflammatory CNS disorder or a neurodegenerative CNS disorder, preferably an inflammatory and neurodegenerative CNS disorder.

17. The compound for use according to any one of claims 1 to 16, wherein the central nervous system disorder is a primary demyelinating disorder of the CNS.

18. The compound for use according to any one of claims 1 to 17, wherein the central nervous system disorder is selected from multiple sclerosis (MS), Neuromyelitis optica spectrum disorders (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), transverse myelitis, acute disseminated encephalomyelitis (ADEM), Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia.

19. The compound for use according to any one of claims 1 to 18, wherein the central nervous system disorder is multiple sclerosis (MS).

20. The compound for use according to claim 19, wherein the MS is relapsingremitting MS or progressive MS, and is preferably progressive MS.

21. The compound for use according to any one of claims 1 to 20, by administering the compound in combination with at least one additional compound which inhibits mitochondrial complex I (CI) and / or mitochondrial complex II (CII).

22. The compound for use according to claim 21, wherein the at least one additional compound which inhibits CI and / or CII is selected from: a second or furthercompound as defined in any one of claims 1 to 13, metformin, IACS-010759, intervenolin, ASP4132, phenformin, BAY87-2243, carboxyamidotriazole (CAI), ME344, fenofibrate, lonidamine, a-tocopheryl succinate (a-TOS), VLX600 (6-Methyl-3 - {(2E)-2-[ 1 -(2-pyridinyl)ethylidene]hydrazino } -5H- [l,2,4]triazino[5,6-b]indole) and meta-iodobenzylguanidine (mIBG).

23. A composition for use in treating or preventing a central nervous system (CNS) disorder comprising the compound for use according to any one of claims 1 to 22 in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.

24. A method of treating or preventing a central nervous system (CNS) disorder in a subject, the method comprising administering a compound as defined in any one of claims 1 to 13, or a composition as defined in claim 23 to the subject.

25. Use of a compound as defined in any one of claims 1 to 13, or a composition as defined in claim 23, for the manufacture of a medicament for treating or preventing a central nervous system (CNS) disorder.

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