Fluorinated glucosamine analogs to reduce injury and promote recovery in neurological disorders

Abstract

Fluorinated glucosamine analogs to reduce injury and promote recovery in neurological disorders.

Description

FLUORINATED GLUCOSAMINE ANALOGS TO REDUCE INJURY AND PROMOTE RECOVERY IN NEUROLOGICAL DISORDERS FIELD

[0001] The present disclosure relates generally to fluorinated glucosamine analogs to reduce injury and promote recovery in neurological disorders.BACKGROUND

[0002] Multiple sclerosis (MS) is a chronic inflammatory disorder characterized by infiltration of leukocytes into the central nervous system (CNS), and activation of CNS-intrinsic microglia, resulting in demyelination and axonal injury1 3. Remyelination is a spontaneous repair response that occurs at early stages of MS but this process is not always efficient46. Remyelination failure contributes to the axonal loss and progression of disability in MS78. The failed repair process could be the consequence of ongoing toxic neuroinflammation and / or presence of inhibitors of oligodendrocyte lineage cells in lesions8-9.

[0003] Various extracellular matrix (ECM) molecules including chondroitin sulfate proteoglycans (CSPGs) contribute to the inhibitory microenvironment of MS lesions10'12. An important subgroup of CSPGs is the lecticans that include 4 members: brevican, neurocan, aggrecan and versican. Versican itself has at least 4 isoforms. Versican-V1 (henceforth called versican) is upregulated in MS lesions13'15and has been described to directly inhibit the differentiation of oligodendrocyte precursor cells (OPCs) into oligodendrocytes and to prevent remyelination15'19. Besides being inhibitory for repair, the elevated CSPGs in MS lesions promote cytotoxic neuroinflammation, such as CSPGs enhancing the generation of highly consequential pro-inflammatory and neurotoxic T helper 17 (Th17) cells in MS and its experimental autoimmune encephalomyelitis (EAE) model15. The in vivo inhibition of CSPG synthesis and deposition in CNS lesions by the use of xylosides or fluorinated glucosamines, such as per-O-acetylated 4-fluoro-N-acetylglucosamine (Ac-4-F-GlcNAc, referred henceforth as fluorosamine) or per-O-acetylated 4,4-difluoro-N-acetylglucosamine (Ac-4,4-diF-GlcNAc, referred henceforth as difluorosamine) enhances remyelination post-injury in MS models1415171920. Thus, lowering CSPG synthesis and deposition in MS can have the dual effect of reducing detrimental Th17 neuroinflammation and also improving remyelination. The possible outcomes in MS would be the decrease of inflammation-promoted relapses, prevention ofworsening of disability that is driven by inflammatory assaults on neuronal axons and oligodendrocytes / myelin, and the increased likelihood of recovery of deficits through remyelination.

[0004] Besides MS, it is possible that CSPG-lowering drugs may also be efficacious in a myriad of other neurological disorders, since the enhanced synthesis and deposition of CSPGs into lesions of the CNS also occurs in traumatic spinal cord and head injuries, neurodegenerative disorders such as Alzheimer’s disease, and stroke21 26. However, different CSPG members may be elevated in each of these disorders, and there are differing pathophysiological processes in these conditions. For instance, where an obvious hematoma does not occur in MS, the primary injury in intracerebral hemorrhage (ICH) is a hematoma (collection of blood) that pools in the CNS parenchyma. Moreover, Th17 cells are not common in ICH27, while this is an important T cell subset present in MS28-29. Thus, it is not obvious that CSPG-lowering drugs would be efficacious across neurological conditions including ICH.

[0005] ICH is a crippling form of stroke characterized by the sudden rupture of cerebral vessels and entry of blood into the brain parenchyma30. ICH constitutes 15 to 20% of all strokes31and is particularly catastrophic with a mortality rate of over 50%32. The pathophysiology of ICH is complex and consists of mechanical disruption, neuroinflammation and demyelination2733. Patients with ICH that survive the stroke recover some neurofunctional deficits several months after31suggesting reorganization or regeneration of neural elements. Indeed, signs of regeneration such as oligodendrogenesis (repopulation of oligodendrocyte precursor cells and oligodendrocytes) are found in autopsied samples of people dying from ICH34. Thus, understanding what promotes or impedes regeneration processes, and overcoming impediments, may enhance recovery from ICH. We recently documented that amongst the lectican CSPGs, neurocan was selectively and profoundly elevated in both murine and human ICH35. We have also reviewed the limited but available literature that ECM members are dysregulated in hemorrhagic (ICH) and ischemic strokes36. Thus, reducing the synthesis and deposition of CSPG members such as neurocan after ICH, and likely also in the more common ischemic type of stroke, appears to be a candidate to reduce inflammation-promoted injury and also improve regenerative processes and functional recovery in stroke.SUMMARY

[0006] In one or more embodiments of the present disclosure, there is provided:

[0007] 1. A compound according to formula 1 , or a pharmaceutically acceptable salt thereof:

[0008] R-i, R2and R3are independently varied between H and an acyl group, and the acyl group is defined as R'CO — or R’XCO — , wherein:

[0009] R' is a substituted or unsubstituted, alkyl or cycloalkyl or heteroalkyl containing up to 20 carbons, optimally between C1-C5, and the heteroalkyl group is defined as an alkyl or cycloalkyl group in which one or more carbon atoms are replaced by heteroatoms such as halogen (F, Cl, Br or I), oxygen (O), nitrogen (N), sulfur (S), or phosphorus (P).

[0010] X is a heteroatom selected from the group consisting of O, N, and S;

[0011] wherein: R4 and R5 are independently varied between H and F, and at least one F is present;

[0012] with the caveat that the general formula 1 does not include the following :

[0013]

[0015] 2. A compound of formula (7, PZ6171), ora pharmaceutically acceptable sat thereof:

[0017] 3. A compound of formula (8), or a pharmaceutically acceptable sat thereof:

[0018] 4. A pharmaceutical composition comprising a compound according toany one of embodiments 1 to 3 and a pharmaceutically acceptable excipient.

[0019] 5. A pharmaceutical composition comprising a compound selected from:

[0020] , and a pharmaceutically acceptable excipient.

[0021] 6. A method of treating multiple sclerosis (MS) or intracerebralhemorrhage (ICH) of stroke in a subject, comprising: administering to the subject in needthereof an effective amount of a compound of any one of embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5.

[0022] 7. The method of embodiment 6, wherein the subject is a human.

[0023] 8. A method of treating a neurological disease or disorder associated withup-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix orchondroitin sulfate proteoglycans, or a tumour associated with up-regulation of anextracellular matrix or chondroitin sulfate proteoglycans, said method comprisingadministering to a subject in need thereof an effective amount of a compound of any oneof embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5.

[0024] 9. The method of embodiment 8, wherein the subject is a human.

[0025] 10. A kit, comprising: a compound of any one of embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5; and a container.

[0026] 11. A commercial package comprising: a compound of any one of embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5; and a container.

[0027] 12. Use of an effective amount of a compound of any one of embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5, for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject, or in the manufacture of a medicament for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject.

[0028] 14. The use of embodiment 12, wherein the subject is a human.

[0029] 15. Use of an effective amount of a compound of any one of embodiments 1 to 3, or a pharmaceutical composition of embodiment 4 or 5, for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or in the manufacture of a medicament for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans.

[0030] 16. The use of embodiment 15, wherein the subject is a human.BRIEF DESCRIPTION OF THE FIGURES

[0031] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0032] Figure 1. Daily difluorosamine treatment for 5 days reduces neurocan and promotes functional recovery in ICH. (A) Chemical structure of difluorosamine (DiF). (B) Experimental paradigm. (C, D) Graphs comparing the latency of rota-rod (C) or forelimb force of grip strength (D) tests between control and DIF mice; each black circle is a different mouse, N= 20. (E) Representative confocal images of perihematomal area (left) and lesion core (right) at day 7 in control and DIF mice stained for DAPI for cell nuclei (blue), neurocan (red), Iba1 (green), GFAP (yellow). The lower left corner inside the dottedlines is the lesion core. Scale bar = 50 pm. (F) Bar graphs comparing the levels of neurocan in perihematomal area, lesion core and contralateral area at day 7 (N= 6). Data are mean ± SEM and analyzed by two-way ANOVA-Tukey’s post hoc test. Western blot analysis (N of 4) of neurocan (G) and quantification (H) comparing the signal ratio of neurocan to p-actin among sham, control, and DIF mice; mean ± SEM, one-way ANOVA-T ukey’s post hoc test; ns: not significant. Significance indicated as **P < 0.01 , ***P < 0.001.

[0033] Figure 2. Difluorosamine reduces neurocan within microglia / macrophages and shifts their functional state towards a regulatory phenotype. (A) Representative confocal images of perihematomal area at day 7 in control (left) and difluorosamine (DIF) (right) mice stained for DAPI for cell nuclei (blue) and Iba1 (green). Scale bar = 50 pm. (B) Quantification comparing the number of Iba1+ cells per mm2of lesion ROI between control and DIF mice. (C) Representative 3D reconstruction images of perihematomal area at day 7 in control (left) and DIF (right) mice using Imaris rendition, and internal accumulation of neurocan within Iba1+ cells. Scale bar = 5 pm. (D) Bar graphs comparing the percentage of Iba1+ cells containing neurocan molecules between control and DIF mice. (E, G, I) Representative confocal images of perihematomal area at day 7 in control (left) and DIF (right) mice stained for the indicated markers. Scale bar = 25 pm or 50 pm. (F, H, J) Quantification showing the percentage of IL-1 p (F), Clec7a (H), Arg1(J) in lesion ROI between control and DIF mice (mean ± SEM of 6 mice). Each dot represents mean of 4 locations per mouse. Unpaired two-tailed Student’s t-test; ns: not significant. *P < 0.05, **P < 0.01

[0034] Figure 3. Difluorosamine improves neurogenesis and oligodendrogenesis over 7 days of ICH. (A, B) Representative confocal images of day 7 perihematomal areas labelled with DAPI (blue), SOX2 (green), Ki67 (red), nestin (yellow) in control (A) and DIF (B) mice. (C, D) Representative confocal images of day 7 perihematomal areas labelled with DAPI (blue), Olig2 (red), PDGFRa (yellow), CC1 (green) in control (C) and DIF (D) mice. The lower left corner inside the dotted lines is the lesion core. Scale bar = 50 pm. (E-G) Bar graphs comparing number of SOX2+ cells (E), SOX2+ Ki67+ (F), the percentage of nestin+ cells in lesion ROI (G). (H-J) Quantifications comparing number of Olig2+ cells (H), OPCs (I) or mature oligodendrocytes (J) per mm2 of lesion ROI between control and DIF mice. Data are presented as the mean ± SEM of 6 mice. Each dot represents mean of 4 locations analyzed per mouse. Unpaired two-tailed Student’s t-test; Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001.

[0035] Figure 4. Difluorosamine reduces the expression of neurocan and improves oligodendrogenesis in ageing mice. (A) Representative confocal images of brain sections from CX3CR1CreER:Ai9 ICH mice at perihematomal area at day 7 stained for DAPI for cell nuclei (blue), Iba1 (green), neurocan (red) in young control (left), old control (middle), and old DIF (right) mice. (B, C) Representative confocal images of CX3CR1CreER:Ai9 ICH mice at day 7 of perihematomal areas labelled with DAPI (blue), Olig2 (red), PDGFRa (green) (B), CC1 (green) (C) in young control (left), old control (middle), and old DIF (right) mice. The lower left corner inside the dotted lines is the lesion core of ICH. Scale bar = 50 pm. Data are presented as mean ± SEM. One-way ANOVA-Tukey’s post hoc test, ns: not significant. Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001.

[0036] Figure 5. Alternate day difluorosamine treatment after ICH promotes functional recovery, reduces neurocan expression and shifts the functional state of microglia / macrophages towards homeostasis over 14 days. (A) Treatment paradigm of difluorosamine in ICH: 14-day experiment. (B, C) Bar graphs comparing the latency of rota-rod test (B) or forelimb force of grip strength test (C) between control and DIF mice. (D, E) Representative confocal images of perihematomal area at day 14 in control (D) and DIF (E) mice stained for DAPI for cell nuclei (blue), neurocan (red), Iba1 (green), GFAP (yellow). Scale bar = 50 pm. (F) Bar graphs comparing neurocan percent area of the lesion region of interest (ROI). (G-l) Quantification showing the percentage of IL-1 p (G), Clec7a (H), Arg1 (I) in lesion ROI between control and DIF mice. Data are presented as the mean ± SEM of 6 mice. Unpaired two-tailed Student’s t-test; Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001.

[0037] Figure 6. Difluorosamine shifts the functional state of microglia / macrophages towards homeostasis over 14 days after ICH. (A-C) Representative confocal images of perihematomal area at day 14 in control (left) and DIF (right) mice stained for DAPI for cell nuclei (blue), Iba1(red) (A, B), IL-1 p (green), Clec7a (green), Iba1 (green) (C), Arg1 (red). The lower left corner inside the dotted lines is the lesion core. Scale bar = 25 pm or 50 pm.

[0038] Figure 7. Difluorosamine improves neurogenesis and oligodendrogenesis over 14 days of ICH. (A, B) Representative confocal images of day 14 perihematomal areas labelled with DAPI (blue), SOX2 (green), Ki67 (red), nestin (yellow) in control (A) and DIF (B) mice. (C, D) Representative confocal images of day 14 perihematomal areas labelled with DAPI (blue), Olig2 (red), PDGFRa (yellow), CC1 (green) in control (C) andDIF (D) mice. The lower left corner inside the dotted lines is the lesion core. Scale bar = 50 pm. (E-G) Bar graphs comparing number of SOX2+ cells (E), SOX2+ Ki67+ (F), the percentage of nestin in lesion ROI (G). (H-J) Quantifications comparing number of Olig2+ cells (H), OPCs (I) or mature oligodendrocytes (J) per mm2 of lesion ROI between control and DIF mice. Data are presented as the mean ± SEM of 6 mice. Each dot represents mean of 4 locations analyzed per mouse. Unpaired two-tailed Student’s t-test; ns: not significant. Significance indicated as *P < 0.05, **P < 0.01, ***P < 0.001.

[0039] Figure 8. MRI shows that difluorosamine alleviates brain tissue loss over 14 days of ICH. (A) A 3D representative of segmented perihematomal area and lesion core. (B, C) Representative segmented lesion core (B) and perihematomal area (blue) (C) of FLASH sequences at day 14 after ICH. Scale bar = 3 mm. (D-G) Representative images of fractional anisotropy (D), mean diffusivity (E), axial diffusivity (F) and radial diffusivity (G) from mice at day 14 after injury. Scale bar = 3 mm. (H-K) Bar graphs comparing data from DTI of the perihematomal area between control and DIF mice: fractional anisotropy (H), mean diffusivity (I), axial diffusivity (J) and radial diffusivity (K). N= 7 in control group and n=6 in DIF group. Data are presented as the mean ± SEM. Unpaired two-tailed Student’s t-test; ns: not significant. Significance indicated as *P < 0.05.

[0040] Figure 9. PZ6171 reduces content of CSPGs produced by astrocytes in culture. Treatment with compounds (50 pM) was done in duplicates and blots were probed using a pan-CSPG antibody.

[0041] Figure 10. PZ6171 administered IP improves functional recovery in ICH-injured mice. The top panel shows the schematic of the experiment while the bottom panel depicts functional capacity as detected by the rotarod test. At 1 and 3 DPI (day post-ICH), mice had deficits (unable to stay on the rotarod) indicating brain injury and this was comparable between the groups prior to initiation of treatment at day 3. With IP treatment at 25 mg / kg PZ6171 or vehicle given on the days indicated in the top panel, there was a significant improvement (functional recovery) in the capacity of mice to stay on the rotating rotarod when given PZ6171. Each dot is an individual mouse. *p<0.05.

[0042] Figure 11. PZ6171 administered intranasally (IN) improves functional recovery in ICH-injured mice. A) In this experiment, IN PZ6171 was initiated at 48 hours after injury, at 100 mg / kg. Drug was given daily from 48 hours to day 8, and every 2 days thereafter. The initial drop in latency (day 1 - 24 hours - after injury) to stay on the rotarod affirms that similar injury had occurred between mice in both groups compared tobaseline pre-ICH level. PZ6171 resulted in enhanced recovery documented at day 14 compared to vehicle mice. B) A similar efficacy was observed with PZ6171 at 50 mg / kg IN dosing. *p<0.05 (2-way ANOVA).

[0043] Figure 12. Intranasal PZ6171 alters microglia / macrophage phenotype in ICH to a less degeneration-associated form. Examples are shown for staining of A) arginase-1 (a regulatory / anti-inflammatory form) and B) Clec7a (a degeneration-associated form) in Iba1+ microglia / macrophages in the core of the ICH lesion.Quantitation in different parts of the ICH lesion for C) arginase-1 and D) Clec7a in vehicle or PZ6171 (100 mg / kg) at day 14 of ICH. Areas of the brain analysed were the perihematomal area (Peri), core of the lesion, and the contralateral (contra) uninvolved hemisphere. *P<0.0001.

[0044] Figure 13. Intraperitoneal PZ6171 (difluorosaminol) reduces EAE disability and neuroinflammation in the spinal cord, correspondent with lowering of CSPG level. A) Schematic of experiment, B) Clinical disability score, with the treatment period shaded, **p<0.01 (2-way ANOVA), C) Spinal cord neuropathology shows higher density of T cells (CD3+) and microglia / macrophages (Iba1+) along with strong CSPG immunoreactivity (represented by chondroitin-4-sulfated glycosaminoglycans CSA), and these were lower in the PZ6171 group. In D, the quantitative differences are displayed; **p<0.01, ***p<0.001. Error bars represent + / - SEM.

[0045] Figure 14. Every other day IP treatment with PZ6171 reduces severity of EAE disability, and this appears to be marginally better, albeit not statistically significant, when compared to PZ6065. Two-way ANOVA, Tukey’s post-hoc test, *p<0.05 when comparing between vehicle and PZ6171. Error bars represent + / - SEM.

[0046] Figure 15. Intranasal (IN) PZ6171 and IP PZ6171 reduce EAE severity. The shaded area in the lower panel refers to the period of daily administration of drug or vehicle (saline) through the IP or intranasal (IN) routes. In the upper panel, the training period before EAE induction refers to the duration when mice were made acquainted with being administered nasal drops.

[0047] Figure 16. Intranasal (IN) PZ6171 and IP PZ6171 reduce levels of CSPGs in the EAE spinal cord, compared to vehicle-treated EAE mice. Mice were killed at day 20 of experiment, as in Figure 15. The corresponding beta-actin blot shows that samples were equally loaded across the groups.

[0048] Figure 17. Examples of other analogs containing a cycloalkyl or heteroalkyl-functionalized acyl group.DETAILED DESCRIPTION

[0049] Here, we disclose the utility of difluorosamine in a mouse model of ICH, associated with reduced accumulation of the neurocan CSPG, with favorable outcomes of a switch of hitherto unknown switch of pro-inflammatory degeneration-associated to regulatory anti-inflammatory microglia / macrophages in lesions, accompanied by improved oligodendrogenesis, better brain tissue integrity as evaluated by MRI, and enhanced functional recovery from a devastating stroke in mice. We also found neurogenesis to occur with difluorosamine treatment in ICH, which was not previously known as an outcome of this drug. Moreover, we describe a new compound, 1,3-Di-O-acetyl-4,4-diF-N-acetyl-D-glucosamine, henceforth called difluorosaminol or PZ6171, that has efficacy in both the EAE model of MS, and the collagenase-induced model of brain ICH, including when administered through the intranasal route. PZ6171 also elicited enhanced functional recovery from ICH when given by the intraperitoneal or intranasal route.

[0050] In some aspects there is described compounds, compositions, methods, and uses, for treating a subject having or suspected of having or at risk of having intracerebral hemorrhage (ICH) stroke.

[0051] Brain injury caused by hemorrhagic stroke is difficult to treat in the clinic and is an important cause of disability.

[0052] Depending on the bleeding site in the brain, hemorrhagic stroke is mainly divided into two types: intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH occurs in the brain, while SAH occurs between the pia mater and the arachnoid.

[0053] In some aspects there is described compounds, compositions, methods, and uses, for treating a subject having or suspected of having or at risk of having an inflammatory disease or disorder.

[0054] As used herein, “inflammatory disease or disorder” may include diseases or disorders associated with inflammation or have an inflammation component.

[0055] In some examples, the inflammatory disease or disorder may include, but is not limited to, a disease or disorder of the central nervous system (CNS), multiple sclerosis (MS), epilepsy, brain ischemia, Alzheimer's disease, experimental autoimmune encephalomyelitis (EAE) and traumatic brain injury, stroke, ALS, Huntington’s disease, Parkinson’s disease, an autoimmune disease or disorder, and / or a cancer.

[0056] In some examples, there is provided a compound according to general formula (1), or a pharmaceutically acceptable salt thereof:R-i, R2and R3are independently varied between H and an acyl group and the acyl group is defined as R'CO — or R’XCO — , wherein:R' is a substituted or unsubstituted, alkyl or cycloalkyl or heteroalkyl containing up to 20 carbons, optimally between C1-C5, and the heteroalkyl group is defined as an alkyl or cycloalkyl group in which one or more carbon atoms are replaced by heteroatoms such as halogen (F, Cl, Br or I), oxygen (O), nitrogen (N), sulfur (S), or phosphorus (P).X is a heteroatom selected from the group consisting of O, N, and S; wherein: R4and R5are independently varied between H and F, and at least one F is present;with the caveat that the general formula 1 does not include the following :

[0057] In one example, there is provided 2. A compound of formula (7), or a pharmaceutically acceptable sat thereof:

[0058] In one example, there is provided a compound of formula (8), or a pharmaceutically acceptable sat thereof:

[0059] In some examples, there is provided a pharmaceutical composition comprising a compound as described herein, and a pharmaceutically acceptable excipient.

[0060] In some examples, there is provided a method of treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject, comprising: administering to the subject in need thereof an effective amount of a compound as described herein, or a pharmaceutical composition as described herein.

[0061] In one example the subject is a human.

[0062] In some examples, there is provided a method of treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, said method comprising administering to a subject in need thereof an effective amount of a compound of as described herein, ora pharmaceutical composition as described herein.

[0063] In some examples, there is provided use of an effective amount of a compound as described herein, or a pharmaceutical composition as described herein, for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject, or in the manufacture of a medicament for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject

[0064] In one example, the subject is a human.

[0065] In one example, there is provided a use of an effective amount of a compound as described herein or a pharmaceutical composition as described herein, for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfateproteoglycans, or in the manufacture of a medicament for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with upregulation of an extracellular matrix or chondroitin sulfate proteoglycans.

[0066] In one example, the subject is a human.

[0067] In some examples, the compounds and compositions described herein may be used in inhibiting cell migration, cell proliferation or cell differentiation. Examples of tissue inflammation include, but are not limited to chronic inflammation and cutaneous inflammation.

[0068] In some example, the compounds and compositions inhibit cell proliferation, cell migration, cell differentiation, and / or production of signaling molecules. In some examples, the cell is a leukocyte, a cancerous cell, or a resident glial cell.

[0069] As used herein, “multiple sclerosis” includes multiple sclerosis or a related disease, and optionally refers to all types and stages of multiple sclerosis, including, but not limited to: benign multiple sclerosis, relapsing remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, chronic progressive multiple sclerosis, transitional / progressive multiple sclerosis, rapidly worsening multiple sclerosis, clinically-definite multiple sclerosis, malignant multiple sclerosis, also known as Marburg's Variant, and acute multiple sclerosis. Optionally, “conditions relating to multiple sclerosis” include, e.g., Devic's disease, also known as Neuromyelitis Optica; myelin oligodendrocyte glycoprotein associated disorder, acute disseminated encephalomyelitis, acute demyelinating optic neuritis, demyelinative transverse myelitis, Miller-Fisher syndrome, encephalomyelradiculoneuropathy, acute demyelinative polyneuropathy, tumefactive multiple sclerosis and Balo's concentric sclerosis.

[0070] As used herein, “progressive” multiple sclerosis refers to forms of the disease which progress towards an ever-worsening disease state over a period of time. Progressive multiple sclerosis includes, for example, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and progressive relapsing multiple sclerosis.

[0071] These subtypes may or may not feature episodic flare-ups of the disease, but are each associated with increased symptoms, such as increased demyelination or pain and reduced capacity for movement, over time.

[0072] The term “Alzheimer's Disease” (AD) generally refers to a mental deterioration in a subject, which clinical manifestations may include, but are not limited to, clinically in progressive memory deficits, confusion, behavioral problems, inability to care for oneself, gradual physical deterioration and, ultimately, death. Alzheimer’s Disease may include preclinical AD, Mild cognitive impairment (MCI), and / or Alzheimer’s dementia.

[0073] The term "cancer" may relate generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.

[0074] The term "malignant" refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).

[0075] The term "metastasize" refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a "metastatic tumor" or a "metastasis." The metastatic tumor contains cells that are like those in the original (primary) tumor.

[0076] The term "benign" or "non-malignant" refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0077] A "cancer cell" refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid cancers and liquid cancers. A "tumor" or "tumor cell" refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.

[0078] The term "relapse" refers to the diagnosis of return, or signs and symptoms of return, of a cancer after a period of improvement or remission.

[0079] The term “subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, cervids, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.

[0080] The term “treatment” or “treat” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.

[0081] "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0082] "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject in the early stage of disease can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent progression.

[0083] In one example the treatment is in vitro treatment. In one example the treatment is in vivo treatment. In one example the treatment is ex vivo treatment.

[0084] In one aspect, there is described a compound selected from:

[0085] In one example, there is provided a compound according to general formula (1), or a pharmaceutically acceptable salt thereof:R-i, R2and R3are independently varied between H and an acyl group, and the acyl group is defined as R'CO — or R’XCO — , wherein:R' is a substituted or unsubstituted, alkyl or cycloalkyl or heteroalkyl containing up to 20 carbons, optimally between C1-C5, and the heteroalkyl group is defined as an alkyl or cycloalkyl group in which one or more carbon atoms are replaced by heteroatoms such as halogen (F, Cl, Br or I), oxygen (O), nitrogen (N), sulfur (S), or phosphorus (P).X is a heteroatom selected from the group consisting of O, N, and S; wherein: R4and R5are independently varied between H and F, and at least one F is present;with the caveat that the general formula 1 does not include the following :

[0086] In one example, there is provided 2. A compound of formula (7), or a pharmaceutically acceptable sat thereof:

[0087] In one example, there is provided a compound of formula (8), or a pharmaceutically acceptable sat thereof:

[0088] As used herein, a "compound" refers to the compound itself, including stereoisomers and tautomers thereof, and its pharmaceutically acceptable salts, solvates, hydrates, complexes, esters, prodrugs and / or salts of prodrugs, unless otherwise specified within the specific text for that compound. Except, when otherwise indicated, e.g. by indication of (R) or (S) configuration at a given location, all stereoisomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. Consequently, compounds described herein may exist in enantiomeric or racemic or diastereomeric forms or as mixtures thereof. The processes for preparation can utilizeracemates or enantiomers as starting materials. When racemic and diastereomeric products are prepared, they can be separated by conventional methods, which for example are chromatographic or fractional crystallization.

[0089] The term “derivative”, “functional derivative” and “physiologically functional derivative” as used herein means an active compound with equivalent or near equivalent physiological functionality to the named active compound when used and / or administered as described herein. As used herein, the term “physiologically functional derivative” includes any pharmaceutically acceptable salts, solvates, esters, prodrugs derivatives, enantiomers, or polymorphs.

[0090] The term "solvate" refers to a complex of variable stoichiometry formed e.g. by a compound of formula (I) and a solvent. The solvent is a pharmaceutically acceptable solvent, such as water, which should not interfere with the biological activity of the solute. Some compounds of the present invention can exist in a tautomeric form which are also intended to be encompassed within the scope of the present invention. "Tautomers" refers to compounds whose structures differ markedly in arrangement of atoms, but which exist in easy and rapid equilibrium. It is to be understood that the compounds of the invention may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be within the scope of the invention, and the naming of the compounds does not exclude any tautomeric form.

[0091] The compounds, salts and prodrugs of the present invention can exist in several tautomeric forms, and such tautomeric forms are included within the scope of the present invention.

[0092] Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present invention includes all tautomers of the present compounds.

[0093] The term "prodrug” used herein refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutical active form in vivo, for example in the subject to which the compound is administered.

[0094] Prodrug and derivatives of niacin, or pharmaceutically acceptable salts or solvates thereof, can be prepared by methods known to those of ordinary skill in the art.

[0095] As used herein, a "subject having intracerebral hemorrhage (ICH) of stroke”, and the like, is a subject known or diagnosed to have an intracerebral hemorrhage (ICH) of stroke. Generally a subject having an intracerebral hemorrhage (ICH) of stroke will havesome objective manifestation of the disease or disorder, such as a sign, symptom, or result of a suitable diagnostic test that indicates the presence of the disease or disorder.

[0096] In some examples, a subject at risk of developing an intracerebral hemorrhage (ICH) of stroke is a subject with a known or suspected predisposition to develop an intracerebral hemorrhage (ICH) of stroke. This may include, but is not limited to a family history to an inflammatory disease or disorder.

[0097] As used herein, a "subject having an inflammatory disease or disorder” is a subject known or diagnosed to have an inflammatory disease or disorder. Generally a subject having an inflammatory disease or disorder will have some objective manifestation of the inflammatory disease or disorder, such as a sign, symptom, or result of a suitable diagnostic test that indicates the presence of the inflammatory disease or disorder.

[0098] In some examples, a subject at risk of developing an inflammatory disease or disorder is a subject with a known or suspected predisposition to develop an inflammatory disease or disorder. This may include, but is not limited to a family history to an inflammatory disease or disorder.

[0099] In some examples, treatment methods comprise administering to a subject a therapeutically effective amount of a compound or composition described herein and optionally consists of a single administration or application, or alternatively comprises a series of administrations or applications.

[0100] In some examples, the compound or pharmaceutical composition is a therapeutically effective amount.

[0101] The term "therapeutically effective amount", as used herein, refers to an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex and / or weight of the subject. The amount of a given compound or composition that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0102] The compounds and / or compositions described herein may be administered either simultaneously (or substantially simultaneously) or sequentially, dependent upon the condition to be treated, and may be administered in combination with other treatment(s). The other treatment(s), may be administered either simultaneously (or substantially simultaneously) or sequentially.

[0103] Administration may be by any suitable means.

[0104] Routes of administration include, but are not limited to, injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, intranasal), oral, inhalation, rectal and transdermal. The pharmaceutical compositions may be given by forms suitable for each administration route. For example, these compositions are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. The injection can be bolus or can be continuous infusion. Depending on the route of administration, a compound or composition described herein can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. A compound or composition described herein can be administered alone, or in conjunction with either another agent as described above or with a pharmaceutically-acceptable carrier, or both. A compound or composition described herein can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, a compound described herein can also be administered in a prodrug form which is converted into its active metabolite, or more active metabolite in vivo.

[0105] Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0106] In some examples, the compound is formulated as a pharmaceutical composition, which is pharmaceutically acceptable.

[0107] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the subject being treated.

[0108] The compound may be formulated with pharmaceutically acceptable carriers, excipients or diluents.

[0109] Pharmaceutically acceptable carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Compositions as described herein may be sterilized by conventional methods and / or lyophilized.

[0110] In one example, treatment comprises administration of a therapeutically effective amount of a compound or a pharmaceutical composition to the central nervous system (CNS) of a subject.

[0111] In one example, treatment provides one or more compound or composition described herein, to the tissues of the CNS by administration directly into the cerebrospinal fluid (CSF).

[0112] In some examples, delivery to the CSF and brain include, but are not limited to, intrathecal (IT), intracerebroventricular (ICV), and intraparenchymal administration. Intrathecal and intracerebroventricular administration may be carried out through the use of surgically implanted pumps that infuse the therapeutic agent into the cerebrospinal fluid. Intraparenchymal delivery may be carried out by the surgical placement of a catheter into the brain.

[0113] As used herein, "delivery to the CSF" and "administration to the CSF" encompass the IT infusion or ICV infusion of one or more compounds or compositions as described herein through the use of an infusion pump. In some embodiments, IT infusion is a suitable means for delivery to the CSF. In other examples, one or more compounds or compositions as described herein is continuously infused into the CSF for the entire course of treatment; such administration is referred to as "continuous infusion" or, in the case of IT infusion, "continuous IT infusion." Also contemplated is continuous intraparenchymal infusion using a pump.

[0114] In some examples, an infusion pump is employed to deliver one or more compounds or compositions as described herein to the CNS. Such infusion pumps and their method of implantation and use are known to the skilled worker. In a specific example, the Medtronic SyncroMed® II pump, is employed. The SyncroMed® II pump is surgically implanted according the procedures set forth by the manufacturer. The pump contains a reservoir for retaining a drug solution, which is pumped at a programmed dose into a catheter that is surgically implanted.

[0115] Method of the invention are conveniently practiced by providing the compounds and / or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0116] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are forillustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0117] EXAMPLES

[0118] Abstract

[0119] The current disclosure relates generally to fluorinated glucosamine analogs and uses thereof, for the treatment of multiple sclerosis (MS) and intracerebral hemorrhage (ICH) of stroke, and other conditions associated with the elevation of the chondroitin sulfate proteoglycans (CSPGs) family of extracellular matrix proteins. It is noted that while different CSPG members are prominently increased in MS (versican) and ICH (neurocan), outcomes of treatment in models of ICH and MS include reduced content of their selective CSPG members in central nervous system (CNS) lesions, better oligodendrogenesis and functional recovery. With treatment, there are lower pro-inflammatory microglia / macrophages in ICH, and fewer T helper 17 cells in MS. In ICH, fluorinated glucosamine analogs additionally promote hematoma resolution and neurogenesis.

[0120] Methods

[0121] Mice

[0122] All animal experiments were performed with ethics approval from the Animal Care Committee at the University of Calgary under regulations of the Canadian Council of Animal Care. For the majority of the ICH experiments, male C57BL / 6 wildtype mice aged 8 to 12 weeks purchased from Charles River (Montreal) were used. For the EAE experiments, C57BL / 6 female mice from Jackson Lab (USA) of 8-12 weeks of age were employed. Mice were housed between 21 °C and 23OOC, in low humidity, with 12h light and 12h dark cycle from 7 am light and starting 7 pm dark, environmental enrichment and free access to food and water.

[0123] Induction of ICH in the brain of mice

[0124] The protocol for ICH induction has been described elsewhere37. In brief, 0.05U of collagenase type VII dissolved in 0.5pl of saline was injected at a rate of O' 1 pl / min over 5 minutes into the right striatum. The needle was maintained at the same spot for additional 5 minutes to prevent reflux. The mice were sutured and then monitored in a thermally controlled environment until recovery. The period of initiation and duration of treatment is described in the Results section.

[0125] ICH brain tissue harvest

[0126] Mice were sacrificed at 7-days or 14-days post-collagenase injection with a lethal dose of ketamine and xylazine. Animals were perfused with a total of 10 ml of phosphate-buffered saline (PBS) and 10 ml of 4% paraformaldehyde (PFA) in PBS via cardiac puncture. The whole brain was collected into 4% PFA in PBS for fixation overnight, and then was transferred into 30% sucrose solution for 72h. The cerebellum was excised, and the remaining brain tissue was frozen in FSC 22 frozen section media (Leica). Brain blocks were cut coronally by a cryostat into 20 pm sections, collected onto microscope slides and stored at -20°C before staining.

[0127] Immunofluorescence staining for ICH brains

[0128] Microscope slides with brain tissues were thawed at room temperature for 30 minutes, then hydrated with PBS for 5 minutes, and permeabilized with 0 1% Triton X-100 in PBS for 5 minutes. Tissue sections were blocked by horse serum blocking solution (0 01 M PBS, 1% bovine serum albumin (BSA), 10% horse serum, 0 1% Triton-X100, 0 1% cold fish gelatin, and 0.05% Tween-20) for 1h at room temperature. Alternatively, for staining using the CC1 antibody in mice, AffiniPure Fab fragment donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch, 715-007-003, 1:50) was added to the blocking buffer. Tissues were incubated with primary antibodies suspended in antibody dilution buffer (0 01 M PBS, 1% BSA, 0 1% Triton-X100, 0 1% cold fish gelatin) overnight at 4°C. Next, slides were washed three times with PBS containing 02% Tween-20 and incubated with fluorophore conjugated secondary antibodies (1:400) and 1 pg / ml of DAPI for 1h. The slides were washed three times and mounted using Fluoromount-G solution (SouthernBiotech).

[0129] Western Blot in ICH experiment

[0130] Lysates were loaded into sodium dodecyl sulphate (SDS) gels (NuPAGE 3-8% Bis-Tris Gel, Invitrogen) and ran with HiMark™ Pre-stained Protein Standard (Invitrogen) at 170V for 1 hour. The proteins were transferred using electroblotting to an 0 2 pm polyvinylidene fluoride membrane (PVDF) (GE Healthcare Life Science). The PVDF membrane was rinsed with tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and blocked with 10% m / v skim powdered milk in TBS for 1h at room temperature. A primary antibody of rabbit anti-mouse neurocan (1 : 1000; Abeam) was added to 3% milk in TBS and incubated overnight at 4°C. The membrane was washed five times (5-min / each) with TBST followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1h at room temperature. The membrane was washed five times (5 min each) with TBST before visualization usingenhanced chemiluminescent (ECL) substrate (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Scientific) and imaged with the BioRad ChemiDoc system. To probe for p-actin, the membranes were washed using Restore™ Western blot stripping buffer (Thermo Scientific) for 30 min before blocking with 5% m / v BSA in TBS for 1h at room temperature. The membrane was then incubated with primary antibody HRP anti-beta actin antibody (Abeam) in 3% m / v BSA in TBS for 1h at room temperature before washing (5x / 5min each). The blots were visualized and imaged using the same methods as above, with quantification using the gel analyzer function in Imaged. The relative amount of protein was normalized to actin.

[0131] Widefield and confocal fluorescence microscopy in ICH experiment

[0132] Overviews of brain sections were imaged with an Olympus VS120 slide scanner using a 1 Ox / o.4NA objective. These were used to locate the lesions. Only the brightness and contrast were adjusted to better display representative images. All samples were then imaged on a Leica TCS SP8 laser scanning confocal microscope using a 25x / o.95 NA water objective. Three-dimensional (3D) z-stacks images (2048 x 2048 x 37 voxels) of the four fluorescent probes were acquired using the 405 nm, 488 nm, 552 nm, and 640 nm lasers sequentially at either 228 x 228 x 567 nm or 114 x 114 x 567 nm voxel size. The two different resolutions were used for quantifying % area of ECM molecules in Regions of Interest (ROIs) and % positive cells with ECM molecules, respectively. Imaging parameters were kept constant for each set of experiments.

[0133] Confocal image analysis of ICH specimens

[0134] Imaged software (NIH) was used to quantify the % ECM molecules in ROIs. For each z-stack image, maximum-intensity projections were created and ROIs were drawn around the perihematomal, lesion center, and contralateral areas according to GFAP or I ba 1 labeling. Image segmentation was achieved using a set intensity threshold for each probe. Negative secondary antibody controls or contralateral controls were used to assess baseline signals and determine thresholds. For each probe, intensity threshold as well as size and circularity filters were kept constant across all samples for each experimental set. Total area and percent area (i.e., % ECM molecules in ROI) were measured.

[0135] Imaris software (Oxford Instruments) was used to determine the percentage of Iba1+ or GFAP+ cells overlapping with ECM molecules. Labelled areas were segmented as surfaces via a set intensity threshold determined as above. Iba1+ or GFAP+ cells were separated using seed points within the Imaris Surface creationworkflow to obtain the total number of cells. For each probe, intensity threshold and surface details were kept constant within each set of experiments. % positive cells with ECM molecules were obtained by dividing the number of positive cells with ECM molecule by the total number of positive cells.

[0136] For better visualization of representative images shown, brightness and contrast were adjusted consistently across all samples and the images were converted to RGB.

[0137] Behavioral tests for ICH mice

[0138] Locomotor functions were evaluated before and 1 , 3, 7, 14 days (the last in a 14-day experiment) after ICH induction using rota-rod test and grip strength test. These tests were previously described37. Mice were transported to the testing room at least 30 minutes prior to training or testing to adjust to the environment before each session.

[0139] MRI imaging of ICH mice

[0140] MRI data were acquired using a 9.4T Bruker BioSpin equipped with a Bruker cryoprobe and operated with ParaVision V.5.1. Mice were anaesthetized initially with 2%-3% isoflurane and a tail vein cannulation were done before imaging. Mice were head-fixed in an animal carrier using tooth and ear bars. Anesthesia was maintained by 1.5% isoflurane. Respiration and body temperature were non-invasively monitored using a small animal monitoring system (Small Animal Instruments). Initially, a shimming procedure was conducted to correct the distortions and optimize the magnetic field homogeneity followed by a scout scan using a Gradient Echo sequence to determine the lesion epicenter and scan range. Subsequently, a T2-weighted FLASH sequence was acquired with 32 slices and 0.25 mm thickness using the following parameters: repetition time=1500 ms, echo time=6.5 ms, acquisition time=25 min, flip angel=90°, averages=2, matrix size=512 x 512; FOV=19.2 mm xi9.2 mm. Diffusion tensor imaging as a common advanced MRI method at the same location was implemented using an Echo Planar Imaging sequence and the following parameters: repetition time=8000 ms, echo time=35.66 ms, slice thickness=0.5 mm, acquisition time=37 min, averages=2, FOV=15 mm xi5 mm, matrix size= 128 x 128; gradient duration=4 ms, diffusion directions=30 and b value=3000 s / mm2.

[0141] Using T2-weighted MRI, each ICH lesion and its penumbra was obtained. Briefly, these anatomical images were converted to a common format (NlfTI-1) used in image processing. These images then underwent brain extraction with the ‘bet4animal’ function implemented in the software FSL (Oxford, UK). The ICH lesions were segmentedin 3-dimention (3D) using a lesion-growing method built in the software ITK-SNAP (Penn Image Computing and Science Laboratory). A 3D Penumbral region was derived by dilating the lesion sphere by 2-pixel sizes followed by subtracting from the original segmentation. All lesion volumes were normalized by the brain volume of the corresponding animal.

[0142] With diffusion tensor imaging, the 30-direction diffusion-weighted volumes were averaged given their optimal brain-skull contrast per animal to reduce inter-volume variability. The resulting average volumes were used to generate individual mouse brain outlines and brain masks with the ‘bet4animal’ function of FSL. The prepared diffusion MRI was then processed using the software DSI-studio that included isotropic resampling assisted by the corresponding brain mask obtained above and eddy current correction, among others, to improve data quality. Finally, quantitative analysis of the images allowed to derive four classical DTI measures: fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity.

[0143] Eventually, the brain extracted T2 MRI scans were aligned with the FA maps as an example of the diffusion tensor imaging measures through a linear coregistration procedure using FSL to ensure anatomical consistency. Average measurements from 3D lesions or penumbra regions were collected from each diffusion tensor imaging measure of each animal, which were subsequently normalized by the values of their contralateral counterpart obtained by flipping the respective 3D ROIs across the midline.

[0144] EAE experiments

[0145] C57BL / 6 mice were induced for EAE using myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 emulsified in complete Freund’s adjuvant supplemented with mycobacterium as described elsewhere15. Mice were scored on a 15-point disability scale38where the individual normal / paresis / paralytic score of the tail and each of the 4 limbs are summed. Intraperitoneal (IP) or intranasal treatment with difluorosamine (PZ6065) or PZ6171 or vehicle (saline) were initiated when mice had obvious signs of disability (that usually starts as tail paresis) (see Results).

[0146] Results

[0147] Daily difluorosamine (PZ6065) treatment ofICH mice reduces lesional neurocan CSPG and elicits functional recovery over 7 days

[0148] We previously reported the substantial elevation of neurocan CSPG, but not versican CSPG, in lesions of murine and human ICH23. This was surprising sinceversican was highly expressed in lesions of MS and its models1415, while neurocan was not elevated14. This informs that the spectrum of CSPGs elevated in ICH and MS differs, and that it remains to be determined whether CSPG-lowering drugs (e.g. PZ6065 or PZ6171) would have efficacy in ICH. To seek to reduce neurocan synthesis and deposition, we initiated daily difluorosamine treatment for 5 days from 2 days post onset of ICH (Fig. 1 A,B). Treatment was given by the intraperitoneal (IP) route. While the initial decline of rotarod and force grip functional activity at days 1 and 3 after ICH (i.e. before treatment was initiated) was comparable between both groups, indicating similar extent of injury, subsequent difluorosamine treatment extended the latency to fall in the rotarod test (Fig. 1C) and enhanced forelimb force of grip strength (Fig. 1D) at day 7. At autopsy, the immunoreactivity for neurocan at lesional regions of interest (ROI) was lowered by difluorosamine (Fig. 1E,F). By corroborative Western blots, the ICH-elevated neurocan was returned to sham injury (i.e. similar experimental procedure except that mice did not have ICH) levels by drug (Fig. 1G,H).

[0149] Figure 1. Daily difluorosamine treatment for 5 days reduces neurocan and promotes functional recovery in ICH (A) Chemical structure of difluorosamine (DiF). (B) Experimental paradigm. (C, D) Graphs comparing the latency of rota-rod (C) or forelimb force of grip strength (D) tests between control and DIF mice; each black circle is a different mouse, N= 20. (E) Representative confocal images of perihematomal area (left) and lesion core (right) at day 7 in control and DIF mice stained for DAPI for cell nuclei (blue), neurocan (red), Iba1 (green), GFAP (yellow). The lower left corner inside the dotted lines is the lesion core. Scale bar = 50 pm. (F) Bar graphs comparing the levels of neurocan in perihematomal area, lesion core and contralateral area at day 7 (N= 6). Data are mean ± SEM and analyzed by two-way ANOVA-Tukey’s post hoc test. Western blot analysis (N of 4) of neurocan (G) and quantification (H) comparing the signal ratio of neurocan to p-actin among sham, control, and DIF mice; mean ± SEM, one-way ANOVA-T ukey’s post hoc test; ns: not significant. Significance indicated as **P < 0.01 , ***P < 0.001.

[0150] In ICH, difluorosamine shifts the functional state of microglia / macrophages towards a regulatory phenotype

[0151] We determined whether difluorosamine affected Iba1 + microglia / macrophages in ICH. Difluorosamine treated mice did not change the density of microglia / macrophages (Fig. 2A,B) but their functional properties were altered. Pro-inflammatory and damage-associated microglia / macrophages have elevated IL-1 p andClec7a while regulatory cells express arginase- 1 (Arg1)39. The results show that difluorosamine treatment reduced IL-1 p and Clec7a in ROI (Fig. 2E-H), whereas the level of Arg1 was elevated (Fig. 2I-J). Collectively, this is a shift towards regulatory microglia / macrophages, which would be potentially less neurotoxic39, upon difluorosamine treatment in ICH. It is currently unknown whether such shift of microglia / macrophages to a regulatory state also occurs in MS following treatment with difluorosamine.

[0152] Figure 2. Difluorosamine reduces neurocan within microglia / macrophages and shifts their functional state towards a regulatory phenotype. (A) Representative confocal images of perihematomal area at day 7 in control (left) and difluorosamine (DIF) (right) mice stained for DAPI for cell nuclei (blue) and Iba1 (green). Scale bar = 50 pm. (B) Quantification comparing the number of Iba1+ cells per mm2 of lesion ROI between control and DIF mice. (C) Representative 3D reconstruction images of perihematomal area at day 7 in control (left) and DIF (right) mice using Imaris rendition, and internal accumulation of neurocan within Iba1+ cells. Scale bar = 5 pm. (D) Bar graphs comparing the percentage of Iba1+ cells containing neurocan molecules between control and DIF mice. (E, G, I) Representative confocal images of perihematomal area at day 7 in control (left) and DIF (right) mice stained for the indicated markers. Scale bar = 25 pm or 50 pm. (F, H, J) Quantification showing the percentage of IL-1 p (F), Clec7a (H), Arg1(J) in lesion ROI between control and DIF mice (mean ± SEM of 6 mice). Each dot represents mean of 4 locations per mouse. Unpaired two-tailed Student’s t-test; ns: not significant. *P < 0.05, **P < 0.01

[0153] Difluorosamine improves neurogenesis and oligodendrogenesis after ICH

[0154] Enhanced neurogenesis and oligodendrogenesis are observed in divergent CNS pathologies344041, although it has not been addressed whether difluorosamine would promote neurogenesis in any conditions. Given a reduced neurocan load and the shift of microglia / macrophages towards a regulatory state, the lesion after ICH in difluorosamine-treated mice could be more conducive for repair. Indeed, the perihematomal area of difluorosamine-treated mice had more SOX2+and nestin+ neuroblasts, many of which were cycling (Ki67+) (Fig. 3A-E). Similarly, there were more Olig2+oligodendrocyte lineage cells, Olig2+PDGFRa+oligodendrocyte precursor cells (OPCs) and Olig2+CC1+mature oligodendrocytes (Fig. 3F-J).

[0155] Collectively, the results highlight neuroreparative processes including neurogenesis and oligodendrogenesis occurring in neurocan-reduced difluorosamine-treated mice with ICH.

[0156] Figure 3. Difluorosamine improves neurogenesis and oligodendrogenesis over 7 days of ICH. (A, B) Representative confocal images of day 7 perihematomal areas labelled with DAPI (blue), SOX2 (green), Ki67 (red), nestin (yellow) in control (A) and DIF (B) mice. (C, D) Representative confocal images of day 7 perihematomal areas labelled with DAPI (blue), Olig2 (red), PDGFRa (yellow), CC1 (green) in control (C) and DIF (D) mice. The lower left corner inside the dotted lines is the lesion core. Scale bar = 50 pm. (E-G) Bar graphs comparing number of SOX2+ cells (E), SOX2+ Ki67+ (F), the percentage of nestin+ cells in lesion ROI (G). (H-J) Quantifications comparing number of Olig2+ cells (H), OPCs (I) or mature oligodendrocytes (J) per mm2 of lesion ROI between control and DIF mice. Data are presented as the mean ± SEM of 6 mice. Each dot represents mean of 4 locations analyzed per mouse. Unpaired two-tailed Student’s t-test; Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001.

[0157] Enhanced oligodendrogenesis also occurs in aging ICH mice after difluorosamine treatment

[0158] The prevalence and worse prognosis of ICH in humans increases with age42. We therefore compared the consequence of ICH in young (6-week-old) versus old (52-week-old) mice with ICH and also addressed whether the older age group would also respond to difluorosamine administered IP.

[0159] Neurocan deposition in the perihematomal area was elevated following injury but the extent was not different between young and aging mice with ICH (young control ICH versus old control ICH) (Fig. 4A,D). The extent of neurocan elevation in the old ICH group was reduced by difluorosamine treatment (Fig. 4A,D). This was accompanied by elevated numbers of Olig2+PDGFRa+ OPCs and Olig2+CC1 + oligodendrocytes in the perihematomal area (Fig. 4B,C,E-G). These results highlight that difluorosamine is also a pro-regenerative drug in aging mice with ICH.

[0160] Figure 4. Difluorosamine reduces the expression of neurocan and improves oligodendrogenesis in ageing mice. (A) Representative confocal images of brain sections from CX3CR1CreER:Ai9 ICH mice at perihematomal area at day 7 stained for DAPI for cell nuclei (blue), Iba1 (green), neurocan (red) in young control (left), old control (middle), and old DIF (right) mice. (B, C) Representative confocal images of CX3CR1CreER:Ai9 ICH mice at day 7 of perihematomal areas labelled with DAPI (blue),Olig2 (red), PDGFRa (green) (B), CC1 (green) (C) in young control (left), old control (middle), and old DIF (right) mice. The lower left corner inside the dotted lines is the lesion core of ICH. Scale bar = 50 pm. Data are presented as mean ± SEM. One-way ANOVA-Tukey’s post hoc test, ns: not significant. Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001.

[0161] A protracted alternate day difluorosamine treatment is efficacious over 14 days of ICH

[0162] Given that the expression of neurocan remained high at day 14 in the perihematomal region of potentially salvageable tissue35, we extended the difluorosamine observations and treatment to day 14. Mice were injected IP once every two days with difluorosamine or saline from day 3 after ICH (Fig. 5A). Behavioral rotarod and grip force tests ensued before injury, and at days 1 , 3, 7, 14 (Fig. 5A). We found that while the initial decline of rotarod and force grip functional activity was comparable between both groups at days 1 and 3 after ICH (indicating similar extent of injury prior to drug initiation), difluorosamine alternate day IP injection initiated from day 3 improved functional recovery that was statistically significant at 14 days (Fig. 5B,C). Brain tissue at day 14 found drug treatment to reduce the level of neurocan immunoreactivity in the perihematomal area (Fig. 5D-F). This was accompanied by difluorosamine-induced switch of microglia / macrophage functional properties from IL-1 [3+ and Clec7a+ pro-inflammatory and damage-associated myeloid cells to Arginase1+ regulatory cells (Fig. 5G-I; Fig. 6). Moreover, there was an elevation of SOX2+Ki67+ and nestin+ neuroblasts in the perihematomal area (Fig. 7A-E). The lesion environment was also more conducive for oligodendrogenesis, with increases of Olig2+ oligodendrocyte lineage cells, Olg2+PDGFRa+ OPCs and olig2+CC1+ oligodendrocytes (Fig. 7F-J).

[0163] Figure 5. Alternate day difluorosamine treatment after ICH promotes functional recovery, reduces neurocan expression and shifts the functional state of microglia / macrophages towards homeostasis over 14 days. (A) Treatment paradigm of difluorosamine in ICH: 14-day experiment. (B, C) Bar graphs comparing the latency of rota-rod test (B) or forelimb force of grip strength test (C) between control and DIF mice. (D, E) Representative confocal images of perihematomal area at day 14 in control (D) and DIF (E) mice stained for DAPI for cell nuclei (blue), neurocan (red), Iba1 (green), GFAP (yellow). Scale bar = 50 pm. (F) Bar graphs comparing neurocan percent area of the lesion region of interest (ROI). (G-l) Quantification showing the percentage of IL-1 p (G), Clec7a (H), Arg1 (I) in lesion ROI between control and DIF mice. Data are presentedas the mean ± SEM of 6 mice. Unpaired two-tailed Student’s t-test; Significance indicated as *P < 0.05, **P < 0.01 , ***P < 0.001.

[0164] Figure 6. Difluorosamine shifts the functional state of microglia / macrophages towards homeostasis over 14 days after ICH. (A-C) Representative confocal images of perihematomal area at day 14 in control (left) and DIF (right) mice stained for DAPI for cell nuclei (blue), Iba1(red) (A, B), IL-1 p (green), Clec7a (green), Iba1 (green) (C), Arg1 (red). The lower left corner inside the dotted lines is the lesion core. Scale bar = 25 pm or 50 pm.

[0165] Figure 7. Difluorosamine improves neurogenesis and oligodendrogenesis over 14 days of ICH. (A, B) Representative confocal images of day 14 perihematomal areas labelled with DAPI (blue), SOX2 (green), Ki67 (red), nestin (yellow) in control (A) and DIF (B) mice. (C, D) Representative confocal images of day 14 perihematomal areas labelled with DAPI (blue), Olig2 (red), PDGFRa (yellow), CC1 (green) in control (C) and DIF (D) mice. The lower left corner inside the dotted lines is the lesion core. Scale bar = 50 pm. (E-G) Bar graphs comparing number of SOX2+ cells (E), SOX2+ Ki67+ (F), the percentage of nestin in lesion ROI (G). (H-J) Quantifications comparing number of Olig2+ cells (H), OPCs (I) or mature oligodendrocytes (J) per mm2 of lesion ROI between control and DIF mice. Data are presented as the mean ± SEM of 6 mice. Each dot represents mean of 4 locations analyzed per mouse. Unpaired two-tailed Student’s t-test; ns: not significant. Significance indicated as *P < 0.05, **P < 0.01, ***P < 0.001.

[0166] Altogether, these results highlight that difluorosamine treatment reduces neurocan content and enhances oligodendrogenesis and neurogenesis over 14 days of ICH.

[0167] The efficacy of difluorosamine in ICH is corroborated in MRI

[0168] Brain MRI imaging was used to analyze ICH lesions and the penumbra regions in response to drug treatment at day 14 both macro- and micro-scopically. Drug was given every other day, IP, from day 3 as described in Figure 5A. Assisted by robust software tools, all 3D lesion-associated ROIs were successfully segmented (Fig. 8A-C). Between treatment groups, there was no significant difference in normalized lesion volume at day 14 (p>0.05).

[0169] Analysis of diffusion tensor imaging (Fig. 8D-G) provided further insight into tissue microscopic characteristics. Specifically, the fractional anisotropy of perihematomal white matter in the striatum of difluorosamine treated mice at day 14 was significantly reduced compared to controls (Fig. 8H). Comparably, the axial diffusivity wassignificantly decreased in the perihematomal white matter of the treatment group at the same timepoint compared to that of the non-treated group (Fig. 8J). Further, there was a non-significant trend in reduction of mean diffusivity and radial diffusivity (Fig. 8I, K) favoring the treatment group towards tissue recovery or myelin repair. These MRI results corroborate the histological and functional recovery data that difluorosamine improves tissue integrity in the perihematomal area over 14 days of ICH.

[0170] Figure 8. MRI shows that difluorosamine alleviates brain tissue loss over 14 days of ICH. (A) A 3D representative of segmented perihematomal area and lesion core. (B, C) Representative segmented lesion core (B) and perihematomal area (blue) (C) of FLASH sequences at day 14 after ICH. Scale bar = 3 mm. (D-G) Representative images of fractional anisotropy (D), mean diffusivity (E), axial diffusivity (F) and radial diffusivity (G) from mice at day 14 after injury. Scale bar = 3 mm. (H-K) Bar graphs comparing data from DTI of the perihematomal area between control and DIF mice: fractional anisotropy (H), mean diffusivity (I), axial diffusivity (J) and radial diffusivity (K). N= 7 in control group and n=6 in DIF group. Data are presented as the mean ± SEM. Unpaired two-tailed Student’s t-test; ns: not significant. Significance indicated as *P < 0.05.

[0171] PZ6171 (difluorosaminol) reduces CSPG content in culture

[0172] Mouse astrocytes in culture produce high levels of CSPGs which are exported out into the culture medium. The latter can then be probed for content of CSPGs by Western blot as described previously20. An inhibitor of CSPG synthesis and export would result in reduced density of CSPG-immunoreactive bands in Western blot. Figure 9 shows an experiment where we identified PZ6171 to be as potent as difluorosamine (PZ6065) in reducing CSPG levels. Not all derivatives of glucosamine reduce CSPG content as we previously reported20, and as noted by PZ12086 or PZ12092 that did not change level compared to controls (no treatment) or that treated with phosphate buffered saline (PBS) and dimethylsulfoxide (DMSO) that are commonly used as vehicle for drugs.

[0173] Figure 9. PZ6171 reduces content of CSPGs produced by astrocytes in culture. Treatment with compounds (50 pM) was done in duplicates and blots were probed using a pan-CSPG antibody.

[0174] Intraperitoneal PZ6171 improves functional recovery in ICH

[0175] Mice were subjected to collagenase-induced ICH37[Li, 2023 #8] and were monitored for functional deficits using the rotarod test, where the capacity and duration of mice to stay on a rotating rod of increasing speed is assessed. After ICH, there was apronounced drop in the capability to stay on the rotarod at days 1 and 3, prior to drug initiation, in both the vehicle (saline) and PZ6171 groups; the equal deficit indicates similar extent of injury in the brain between the 2 groups. IP PZ6171 or saline then ensued as noted in the schematic of Figure 10. At day 14, and consistent with CNS repair although this will be evaluated in brain samples collected, the PZ6171 mice regained the capacity to stay on the rotarod as noted by the statistical difference between the PZ6171 and vehicle groups. Thus, PZ6171 is efficacious in promoting recovery from ICH.

[0176] Figure 10. PZ6171 improves functional recovery in ICH-injured mice. The top panel shows the schematic of the experiment while the bottom panel depicts functional capacity as detected by the rotarod test. At 1 and 3 DPI (day post-ICH), mice had deficits (unable to stay on the rotarod) indicating brain injury and this was comparable between the groups prior to initiation of treatment at day 3. With IP treatment at 25 mg / kg PZ6171 or vehicle given on the days indicated in the top panel, there was a significant improvement (functional recovery) in the capacity of mice to stay on the rotating rotarod when given PZ6171. Each dot is an individual mouse. *p<0.05.

[0177] Intranasal PZ6171 improves functional recovery in ICH

[0178] We addressed whether PZ6171 was effective in recovery from ICH when given by the intranasal route. An increasing literature informs that this route bypasses the blood-brain barrier and has preferential and efficient drug intake into the CNS, thereby potentially increasing potency within the CNS and also reducing the risk of systemic side effects4344. Following collagenase-induced ICH, rotarod test was used to monitor the functional state of mice. PZ6171 (50 or 100 mg / kg) or vehicle was administered intranasally starting from 48 hours after stroke. At 24 hours of ICH, before the first dose of drug was given, a sharp drop in rotarod capability (ability to stay on the rotating rod) was found in all groups, indicating that similar extent of ICH injury had ensued in all animals. In groups given 100 mg / kg (Figure 11A) or 50 mg / kg (Figure 11 B) PZ6171 intranasal, functional improvement was noted at day 14 of injury compared to vehicle control. Thus, PZ6171 is efficacious in promoting recovery from ICH when administered intranasally. Following the behavioral test at day 14, mice were killed and brain sections were analysed for the functional state of microglia / macrophages. Clec7a+ was used as a pro-inflammatory and damage-associated myeloid marker, while Arginase1+ informed on regulatory or anti-inflammatory cells. Figure 12 shows that intranasal PZ6171 (100 mg / kg tested) polarized cells to a regulatory or anti-inflammatory functional state as ascertained by elevation of arginase-1 and reduction of Clec7a.

[0179] Figure 11. PZ6171 administered intranasally improves functional recovery in ICH-injured mice. In panel A, IN PZ6171 (100 mg / kg) was given daily from 48 hours to day 8, and every 2 days thereafter. PZ6171 resulted in enhanced recovery (ability to stay on the rotating rod) documented at day 14 compared to vehicle mice. A similar efficacy was observed with PZ6171 at 50 mg / kg IN dosing (panel B)..

[0180] Figure 12. Intranasal PZ6171 (100 mg / kg tested) alters microglia / macrophage phenotype in ICH to a less degeneration-associated form as determined by elevation of arginase-1 and reduction of Clec7a.

[0181] PZ6171 reduces disease severity in the EAE model of MS

[0182] The potential of PZ6171 to alter disease course in EAE was next tested as shown in the schematic of Figure 13A, as it was not clear that this compound that is different from difluorosamine would have efficacy. Mice were immunized at day 0 with myelin oligodendrocyte glycoprotein (MOG, peptide 35-55) and daily IP treatment of PZ6171 (25 mg / kg) was initiated at day 10 when mice began to acquire signs of EAE (tail impairment). While vehicle (saline)-treated mice became progressively disabled over the next 10 days, to an average score of 8 (equivalent to paralysis of tail and severe paresis of both hind limbs), PZ6171 -treated mice slowly ascended to an average score of 4 (equivalent to paresis of tail, and modest paresis of one hind limb) (Fig. 13B). Mice were then killed to evaluate neuropathology in the spinal cord. Both groups of mice had lesions (hypercellularity and aggregates of immune cells) but the density of immune cells, CSPGs (represented by CSA immunoreactivity), and Iba1+ microglia / macrophages was qualitatively less in the PZ6171 group than in vehicle-treated EAE mice. Figure 13D shows quantitative reduction of CSA and Iba1 (and a trend towards reduced CD3) in the spinal cord of PZ6171 (difluorosaminol)-treated mice.

[0183] Figure 13. Intraperitoneal PZ6171 (difluorosaminol) reduces EAE disability and neuroinflammation in the spinal cord, correspondent with lowering of CSPG level. A) Schematic of experiment, B) Clinical disability score, with the treatment period shaded, **p<0.01 (2-way ANO A), C) Spinal cord neuropathology shows higher density of T cells (CD3+) and microglia / macrophages (Iba1+) along with strong CSPG immunoreactivity (represented by chondroitin-4-sulfated glycosaminoglycans CSA), and these were lower in the PZ6171 group. In D, the quantitative differences are displayed; **p<0.01, ***p<0.001. Error bars represent + / - SEM.

[0184] Next, we compared intraperitoneal PZ6171 against difluorosamine (PZ6065) in EAE; we had previously reported that PZ6065 reduced EAEneuroinflammation and clinical severity, and also promoted remyelination in mice15. In the current experiment, mice were initiated with IP vehicle, PZ6065 or PZ6171 when clinical signs (tail impairment) were detected. Treatment was given IP once every 2 days. Both drugs reduced the accumulation of disability and there was a trend towards a marginally better efficacy of PZ6171 compared to PZ6065 (Fig. 14).

[0185] Figure 14. Every other day IP treatment with PZ6171 reduces severity of EAE disability, and this appears to be marginally better, albeit not statistically significant, when compared to PZ6065. Two-way ANOVA, Tukey’s post-hoc test, *p<0.05 when comparing between vehicle and PZ6171. Error bars represent + / - SEM.

[0186] Next, we tested intranasal administration of PZ6171. In our experiment, we initiated daily intranasal PZ6171 from two days after the onset of clinical signs, and this regimen at 25 mg / kg was as effective as IP PZ6171 in lowering subsequent disability. A higher dose of 50 mg / kg PZ6171 intranasally was even more efficacious (Fig. 15).

[0187] Figure 15. Intranasal (IN) PZ6171 and IP PZ6171 reduce EAE severity. The shaded area in the lower panel refers to the period of daily administration of drug or vehicle (saline) through the IP or intranasal (IN) routes. In the upper panel, the training period before EAE induction refers to the duration when mice were made acquainted with being administered nasal drops.

[0188] PZ6171 lowers content of CSPGs in the spinal cord of EAE mice. Upon termination of the experiment in Figure 15, the spinal cord of mice was harvested and subjected to Western blot for CSPGs, and for beta-actin loading control. The elevation of CSPGs in EAE mice (not shown is the low level of CSPGs in naive control cord) was prevented by IP and IN PZ6171 (Figure 16). Thus, PZ6171 prevented its target protein, CSPGs, from attaining levels seen in EAE disabled mice.

[0189] Figure 16. Intranasal (IN) PZ6171 (50 mg / kg) and IP PZ6171 (25 mg / kg) reduce levels of CSPGs in the EAE spinal cord, compared to vehicle-treated EAE mice. Mice were killed at day 20 of experiment, as in Figure 15. The corresponding beta-actin blot shows that samples were equally loaded across the groups. Here, we used a double amount of PZ6171 intranasally than through IP injection, as there tends to be some fluids being sneezed out by mice during IN administration.

[0190] Discussion

[0191] The microenvironment of ICH and MS lesions is extremely hostile to regenerative processes. For ICH, the accumulation of blood / hematoma, proteases and pro-inflammatory molecules constitutes a toxic environment that injures and destroyssurviving or regenerating elements276263. Moreover, debris such as degraded myelin, or neural fragments that persist such as Nogo-A, are not conducive for survival or maturation of cells such as neuroblasts or OPCs, and they inhibit remyelination. For MS, hematoma is not a pathological finding, and hostile factors within lesions include persistently activated microglia and the accumulation of Th 17 T cells and B lymphocytes6466. Of note, Th 17 and B lymphocytes are not prominent immune cells in ICH, emphasizing different pathophysiology across these conditions. Another impediment after ICH and in MS is the accumulation of non-permissive ECM particularly the family of CSPGs1535[Ghorbani, 2021 #2], We identified neurocan CSPG as the prominent lectican CSPG member to be prominently elevated in murine and human ICH35, while versican-V1 is the major CSPG deposited in MS lesion1415. Regardless of which CSPG member is elevated and deposited in neurological conditions, it is clear that CSPGs are deposited into neural lesions across neurology. Besides being directly inhibitory for oligodendrocyte lineage precursor cells to mature into myelin-forming oligodendrocytes1519, the CSPGs are also pro-inflammatory for many immune cell types15-67. Moreover, deposited CSPGs also inhibit axonal regeneration22242668and neurogenesis41 6970. CSPGs have also been described to stimulate microglia / macrophage production of matrix metalloproteinases14, a family of proteases implicated in ICH injury, so reducing CSPG production would likely lead to less neurotoxic matrix metalloproteinases in lesions. Thus, reducing CSPG synthesis and deposition at sites of CNS injury in ICH and MS theoretically has the dual advantages of overcoming CSPG inhibition of neural repair, and of countering pro-inflammatory responses that can be neurotoxic. However, given the different pathophysiological processes (e.g. Th17 prominence in MS, and hematoma in ICH) and different CSPG members elevated in ICH versus MS, it was not obvious that difluorosamine or PZ6171 would be comparably effective in both conditions until the experiments were performed.

[0192] There are several ways that have been undertaken to overcome CSPG detriments in CNS disorders, including the local application of chondroitinase-ABC to remove the GAG side chains of CSPG2471and the use of peptides that block the interaction of CSPGs with their receptors such as PTP sigma72-73. We have proposed inhibiting CSPG synthesis by using glucosamine analogs20. These compounds block the 4-epimerase enzyme and prevent GAG elongation by impeding the conversion of Uridine-5'-diphosphate-N-acetyl-D-glucosamine to Uridine-5'-diphosphate-N-acetyl-D-galactosamine20-74. While the assembly of GAGs is the primary target, the result oftreatment with fluorosamines is the reduction of the whole CSPG (protein core and GAGs)20.

[0193] The literature of how to overcome CSPGs in ICH is sparse. In murine ICH, blocking PTP sigma by intracellular sigma peptide promoted white matter integrity and functional recovery75. In premature rabbit pups with intraventricular hemorrhage, chondroitinase ABC treatment reduced the expression of neurocan, but did not enhance maturation of oligodendrocytes, myelination, or neurological recovery71. One possible reason is that chondroitinase ABC removes the glycosaminoglycans of CSPGs, but the protein core remains intact and has inhibitory properties.

[0194] In the current work, we now describe PZ6065 (difluorosamine) and PZ6171 (difluorosaminol) to be efficacious in enhancing recovery following ICH, when treatment is initiated 2 or 3 days after the injury. We would propose that the mechanism is through reducing CSPG synthesis and content, with the result that the lesion is less injurious and also more conducive for repair processes such as oligodendrogenesis and remyelination. We note that PZ6171 is also effective when administered through the intranasal route, which potentially will have less impact on the periphery and thus the lessening of potential systemic toxicity.

[0195] In summary, while not wishing to be bound by theory, it is proposed that the reduction of CSPG content by PZ6065 and PZ6171 in models of MS and ICH leads to favorable lesion outcomes that include neurogenesis, oligodendrogenesis, functional recovery, and better microstructural outcomes on MRI. We propose that these compounds are useful medications to improve recovery from neurological conditions.

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[0272] Example 2

[0273] 1) In one example, PZ6171 may cause polarization of microglia / macrophages. PZ6171 may switch microglia / macrophages to a regulatory phenotype from a pro-inflammatory state in both models of ICH and MS, using markers that inform on each functional state. It has been proposed that switching CNS-intrinsic microglia and CNS-infiltrated macrophages from pro-inflammatory to a regulatory state would reduce detrimental processes in the CNS and lead to neurological recovery394546. We anticipate that such a switch will occur with treatment, correspondent with less extent of tissue injury and with higher extent of oligodendrogenesis and neurogenesis

[0274] 2) In one example, difluorosamine or PZ6171 may reduce hematoma size in ICH. Difluorosamine and PZ6171 may reduce hematoma size in ICH over days 3-14, when treatment is initiated from 2 days (48h) after ICH (as in Figures 1 and 10). Reducing hematoma size may lower ongoing injury as well as position the brain in a better capacity for regenerative processes. We anticipate that these CSPG-lowering drugs will reduce hematoma size, since the regulatory microglia / macrophages generated by difluorosamine noted in the ICH model (Fig. 2), and similarly for PZ6171 (Fig. 12), tend to be more phagocytic and can help clear the blood content from the parenchyma of the brain.

[0275] 3) Drugs may be targeted into the CNS. Besides intranasal delivery, difluorosaminol or PZ6171 may be linked to CNS-targeted delivery systems, one example of which is the CAQK (cysteine-alanine-glutamine-lysine) peptide which selectively targets to the CNS upon systemic administration47 due to its recognition of tenascin-C which is preferentially elevated in CNS lesions and not normal CNS parenchyma48. In EAE or ICH, we anticipate that CAQK-linked (or through other CNS-targeted vehicles) difluorosamine or PZ6171, with or without NBD, will localize to higher concentrations inthe brain and spinal cord, and particularly in lesions, and will have less exposure elsewhere in the body. We anticipate that the CNS-targeted drugs will have higher efficacy within the CNS and less side effects throughout the body, should side effects be encountered with the bare drug.

[0276] 4)Difluorosamine and PZ6171 may be combined with an oligodendrocytestimulatory drug to enhance the extent of remyelination and neuroprotection. While lowering CSPG inhibitors within a lesion is conducive for repair processes, CSPG-lowering drugs may be even more efficacious when combined with a drug that directly stimulate oligodendrocyte lineage cells. Several drugs have been reported to stimulate the oligodendrocyte precursor cells (OPCs) directly and to facilitate their maturation into oligodendrocytes95253; one such drug is clemastine, an anti-histamine first reported to promote OPC maturation and remyelination in 201453, and reproduced by many groups54-56including ours57in preclinical models. Clemastine has been tested in a small trial in MS where it marginally but significantly improved optic nerve functions58, lowered serum neurofilament levels59, and have MRI suggestions of some recovery in the corpus callosum60. Interest in clemastine is evident with ongoing trials in the Netherlands (‘RESTORE’)61and a Phase 2a trial of the combination of clemastine and metformin in MS in the UK (NCT05131828). Thus, an example experiment is to combine difluorosamine or PZ6171 with clemastine and determine whether the outcomes of remyelination, neurogenesis and functional recovery is improved, compared to either drugs alone. We anticipate that the combination of clemastine (or other drugs reported to stimulate OPCs) with CSPG-lowering drugs, through stimulating OPCs directly and reducing levels of CSPG inhibitors, will promote repair processes and functional recovery compared to individual drugs.

[0277] 5) Other PZ6171 analogs may be efficacious in models of ICH and MS. We will synthesize and test compounds 1-4 below, which are analogs of PZ6171 that have larger aliphatic or cyclic acyl groups, and compounds 5-8, which are also analogs of PZ6171 but containing a heteroatom-containing functionalized acyl groups (charges or neutral). We anticipate that these compounds will have even better bioavailability and in-vivo activities. These new drugs will first be screened in culture for their capacity to reduce CSPG biosynthesis in cells20, and then be tested in mice in improving outcomes in ICH and MS.

[0278] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments bythose of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0279] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0280] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound according to formula 1 , or a pharmaceutically acceptable salt thereof:R-i, R2and R3are independently varied between H and an acyl group, and the acyl group is defined as R'CO — or R’XCO — , wherein:R' is a substituted or unsubstituted, alkyl or cycloalkyl or heteroalkyl containing up to 20 carbons, optimally between C1-C5, and the heteroalkyl group is defined as an alkyl or cycloalkyl group in which one or more carbon atoms are replaced by heteroatoms such as halogen (F, Cl, Br or I), oxygen (O), nitrogen (N), sulfur (S), or phosphorus (P).X is a heteroatom selected from the group consisting of O, N, and S; wherein: R4and R5are independently varied between H and F, and at least one F is present;with the caveat that the general formula 1 does not include the following :

2. A compound of formula (7, PZ6171), or a pharmaceutically acceptable sat thereof:

3. A compound of formula (8), or a pharmaceutically acceptable sat thereof:

4. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

5. A pharmaceutical composition comprising a compound selected from:, , and a pharmaceutically acceptable excipient.

6. A method of treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject, comprising: administering to the subject in need thereof an effective amount of a compound of any one of claims 1 to 3, or a pharmaceutical composition of claim 4 or 5.

7. The method of claim 6, wherein the subject is a human.

8. A method of treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, said method comprising administering to a subject inneed thereof an effective amount of a compound of any one of claims 1 to 3, or a pharmaceutical composition of claim 4 or 5.

9. The method of claim 8, wherein the subject is a human.

10. A kit, comprising: a compound of any one of claims 1 to 3, ora pharmaceutical composition of claim 4 or 5; and a container.

11. A commercial package comprising: a compound of any one of claims 1 to 3, or a pharmaceutical composition of claim 4 or 5; and a container.

12. Use of an effective amount of a compound of any one of claims 1 to 3, or a pharmaceutical composition of claim 4 or 5, for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject, or in the manufacture of a medicament for treating multiple sclerosis (MS) or intracerebral hemorrhage (ICH) of stroke in a subject.

14. The use of claim 12, wherein the subject is a human.

15. Use of an effective amount of a compound of any one of claims 1 to 3, or a pharmaceutical composition of claim 4 or 5, for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or in the manufacture of a medicament for treating a neurological disease or disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans (CSPGs), an autoimmune disorder associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans, or a tumour associated with up-regulation of an extracellular matrix or chondroitin sulfate proteoglycans.

16. The use of claim 15, wherein the subject is a human.

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