Inhibition of MMP-9 and MMP-12 for the treatment of spinal cord injury or related nerve tissue damage.

Abstract

This invention provides compounds that inhibit matrix metalloproteinases and are useful in the treatment of spinal cord injury (SCI), particularly secondary effects associated with SCI, and related nerve tissue damage. [Solution] A compound or combination of compounds for use in the treatment of SCI or related nerve tissue injury, or for use in the treatment of secondary effects associated with said SCI or related nerve tissue injury, is provided, comprising selectively inhibiting the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after said SCI or related nerve tissue injury.

Description

[Technical Field]

[0001] This invention relates to specific compounds that inhibit matrix metalloproteinases and are useful in the treatment of spinal cord injury (SCI), particularly secondary effects associated with SCI, and related nerve tissue damage. [Background technology]

[0002] SCI (Spine Collision Crusoe) is a serious condition that can lead to significant physical disability and mortality. Globally, the incidence of SCI is 15-40 per million injuries, with 12,000 new cases reported annually in the US and 1,000 in the UK. The most common causes of traumatic SCI are car accidents, falls, sports-related injuries, and interpersonal violence. Clinical outcomes of SCI vary depending on the severity and location of the injury and may include partial or complete loss of sensory and / or motor function below the level of disability. Current treatments for SCI, such as Lyrica (pregabalin), only alleviate the patient's symptoms and do not address the underlying mechanisms that cause neuropathic pain (NP), inflammation, loss of sensory / gait motor function, or blood-spinal barrier (BSCB) disruption. A treatment that could completely restore SCI would address the prevention of further neuronal and glial damage, neutralization of inhibitory molecules, inhibition of glial scarring, replacement of lost neurons, axonal regeneration, and proper synapse formation.

[0003] However, there are no neuroprotective or regenerative therapies explicitly approved by the FDA or EMEA in the field of SCI. Methylprednisolone (Medrol) continues to be used off-label because its drug efficacy is unclear and the high doses required for beneficial effects cause serious adverse side effects. While several promising treatments for SCI are currently in clinical trials, most are in the early stages of testing, and currently, only Lyrica, used to control SCI-induced neuralgia and NP, is an FDA-approved drug.

[0004] Initial mechanical trauma following spinal cord injury (SCI) leads to a cascade of secondary events that exacerbate inflammatory responses around the initial injury site, including blood-spinal barrier (BSCB) disruption, edema, neuronal death, axonal damage, and demyelination.

[0005] Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that target extracellular matrix (ECM) proteins, chemokines, cytokines, and cell surface receptors. To date, 23 MMPs have been identified in humans, of which MMP-2, MMP-9, and MMP-12 are involved in spinal cord injury (SCI) and reliably increase immediately after SCI, with their expression levels directly proportional to the severity of the injury. Excessive MMP activity contributes to BSCB failure, edema, excitotoxicity, mitochondrial apoptosis, inflammation, astrogliosis, NPs, and loss of function. Therefore, since edema correlates with clinical neurological deficits, inhibiting excessive MMP activity after SCI is strategically useful, thereby preventing further damage to the spinal cord's axonal pathways.

[0006] MMP-9 expression peaks within 1 day post-SCI and is detected in glial, macrophage, neutrophil, and vascular elements in SCI 24 hours after injury. Excessive activity of MMP-9 in the acute phase of SCI contributes to BSCB failure, edema, excitotoxicity, leukocyte influx, mitochondrial dysfunction, apoptosis, neuronal demyelination, increased inflammatory response, and astrogliosis. In addition, in rat sciatic nerve ligation mode, MMP-9 overexpression modulates initial neuropathic pain via cleavage of the pro-inflammatory cytokine interleukin-1 (IL-1) and microglial activation.

[0007] MMP-12 is an elastin-degrading protease primarily expressed in macrophages, and its expression increases 189-fold in compression SCI models, peaking 5 days after injury. Post-SCI MMP-12 expression is detrimental and triggers inflammatory responses, acute edema, and secondary injury responses. Therefore, since edema correlates with neurological deficits, preventing edema, a major pathological event after SCI, is strategically useful and would thereby prevent further damage to the axonal pathways within the spinal cord.

[0008] Animal studies have demonstrated that individual gene knockout of MMP-9 and MMP-12 improves functional recovery, reduces BSCB failure, and alleviates neuropathic and inflammatory pain, suggesting that inhibiting MMP-9 or MMP-12 may have beneficial neuroprotective effects.

[0009] The adverse roles of MMP-12 in intracerebral hemorrhage (ICH) and spinal cord injury (SCI) have been reported, and the authors suggest that MMP-12 may be key to the outcome of ICH injury in humans (JEWells, J.Biernaskie, A.Szymanska, PHLarsen, VWYong, D.Corbett, Matrix metalloproteinase (MMP)-12 expression has a negative impact on sensorimotor function following intracerebral hemorrhage in mice. Eur J Neurosci 21,187-196(2005), and JEWells, TKRice, RKNuttall, DREdwards, H.Zekki, S.Rivest, VWYong, An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice. J Neurosci 23,10107-10115(2003)).

[0010] MMP inhibitors, including broad-spectrum inhibitors, such as GM6001 (broad-spectrum MMP inhibitor), Inhibitor I (MMP-9 selective inhibitor), SB-3CT, Lipitor, fluoxetine, and sulforaphane, have been developed and tested in animal models. See the references below.

[0011] Y. Kawasaki, ZZXu, X. Wang, JYPark, ZYZhuang, PHTan, YJGao, K. Roy, G. Corfas, EHLo, RRJi, Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat Med 14, 331-336 (2008).

[0012] H. Kobayashi, S. Chattopadhyay, K. Kato, J. Dolkas, S. Kikuchi, RR Myers, VIShubayev, MMPs initiate Schwann cell-mediated MBP degradation and mechanical nociception after nerve damage. Mol Cell Neurosci 39, 619-627 (2008).

[0013] F. Yu, H. Kamada, K. Niizuma, H. Endo, PHChan, Induction of mmp-9 expression and endothelial injury by oxidative stress after spinal cord injury. J Neurotrauma 25, 184-195 (2008).

[0014] CKWada,JHHolms,MLCurtin,Y.Dai,ASFlorjancic,RBGarland,Y.Guo,HRHeyman,JRStacey,DHSteinman,DHAlbert,JJBouska,INElmore,CLGoodfellow,PAMarcotte,P.Tapang,DWMorgan,MRMichaelides,SKDavidsen,Phenoxyphenyl sulfone N-formylhydroxylamines (retrohydroxamates) as potent, selective, orally bioavailable matrix metalloproteinase inhibitors. J Med Chem 45, 219-232 (2002).

[0015] H. Zhang, M. Chang, CNHansen, DMBasso, LJ Noble-Haeusslein, Role of matrix metalloproteinases and therapeutic benefits of their inhibition in spinal cord injury. Neurotherapeutics 8, 206-220 (2011).

[0016] These inhibitors showed promise in early animal models but have never been tested in human SCI. This is likely due to fears of the potential for adverse musculoskeletal (MSK) side effects of MMP inhibitors, which have been observed under other conditions, particularly due to the length of time (at least 6 weeks) spent using broad-spectrum MMP inhibitors (JTPeterson, Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail Rev 9, 63-79 (2004)).

[0017] U.S. Patent Application No. US2003 / 0139332A1 (Noble et al.) describes inhibitors of MMPs, particularly MMP-9, in SCI and related nervous system injuries.

[0018] Zhang et al. (Neurotherapeutics, Vol. 8, 206-220, April 2011) describe MMPs (particularly MMP-9) and their inhibitors in SCI. Zhang et al. state that MMP-9 promotes wound healing. Therefore, inhibition of MMP-9 does not facilitate wound healing and may lead to scarring. Zhang also states that the use of broad-spectrum MMP inhibitors in more chronically injured spinal cord should proceed with caution.

[0019] Wells et al. (The Journal of Neuroscience, November 5, 2003-23(31), 10107-10115) describe MMP-12 in SCI, its large increase in expression, and the applicability of MMP inhibition in SCI.

[0020] The present disclosure relates to combined selective inhibition of the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 relative to each other after SCI or related neural tissue injury.

[0021] AZD1236 is a potent, reversible and specific inhibitor of human MMP-9 and MMP-12, with 10- to 15-fold selectivity over MMP-2 and MMP-13 and more than 350-fold selectivity over other members of the enzyme family. The IC of AZD1236 50 was measured to be 4.5 nM and 6.1 nM against MMP-9 and MMP-12, respectively. AZD1236 has been clinically used for chronic obstructive pulmonary disease (COPD) by administering 75 mg twice daily to patients in a 6-week randomized controlled trial.

Chemical Formula

[0022] The preparation of AZD1236 is described in WO2006 / 004532 (see, for example, Example 1, page 20). The full chemical name of AZD1236 is (5S)-5-({[4-(2-cyclopropylpyrimidin-5-yl)ethynyl]-3,6-dihydropyridine-1(2H)-yl]sulfonyl}methyl]-5-methylimidazolidine-2,4-dione.

[0023] AZD3342 is another potent inhibitor of human MMP-9 and MMP-12, and the IC measured for MMP-9 and MMP-12 50 are 10 nM and 6 nM (5.9 nM), respectively.

Chemical Formula

[0024] The preparation of AZD3342 is described in WO2002 / 074767 (see, for example, lines 15 to 27 on page 65; lines 23 to 29 on page 120), and the novel crystalline form is described in WO2007 / 106022. The full chemical name of AZD3342 is (5S)-5-[4-(5-chloro-pyridin-2-yloxy)-piperidine-1-sulfonylmethyl]-5-methylimidazolidine-2,4-dione. Summary of the Invention

[0025] This specification demonstrates that certain compounds selectively inhibiting both MMP-9 and MMP-12 are useful in the treatment of SCI or associated nerve tissue injury. In particular, certain compounds selectively inhibiting both matrix metalloproteinases MMP-9 and MMP-12 are shown to prevent SCI-induced edema, suppress inflammatory pain (including neuropathic pain), reduce BSCB failure, reduce scarring, and prevent impairment of sensory and gait function after SCI and associated nerve tissue injury. Certain compounds selectively inhibiting both MMP-9 and MMP-12 also promote axonal regeneration and axonal preservation in the upper and lower parts of the SCI injury site.

[0026] Accordingly, in one embodiment, a compound or combination of compounds is provided for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury (such as traumatic brain injury (TBI)), or for use in the treatment of secondary effects associated with SCI or related nerve tissue injury (such as TBI), wherein the compound or combination of compounds for such use comprises selectively inhibiting the activity or expression of both MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury.

[0027] In further embodiments, compounds or combinations of compounds are provided for use in the treatment of secondary effects associated with SCI or related nerve tissue damage, the compounds or combinations of compounds for use comprising selectively inhibiting the activity or expression of both MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue damage.

[0028] Further embodiments provide compounds or combinations of compounds for use as described herein, where a single selective MMP-9 and MMP-12 inhibitor or a combination of one or more selective MMP-9 inhibitors and one or more distinct selective MMP-12 inhibitors is used.

[0029] In further embodiments, combinations of compounds for use as described herein are provided, in which one or more distinct selective MMP-9 inhibitors and one or more distinct selective MMP-12 inhibitors are used.

[0030] In further embodiments, combinations of compounds for use as described herein are provided, where a combination of one or more distinct selective MMP-9 inhibitors and one or more distinct selective MMP-12 inhibitors (preferably a combination of a single MMP-9 inhibitor and a single MMP-12 inhibitor, i.e., a combination of two compounds) is used.

[0031] In further embodiments, a single compound for use as described herein is provided, wherein a single compound that is both a selective MMP-9 inhibitor and an MMP-12 inhibitor is used.

[0032] In further embodiments, one or more MMP-12 inhibitors have higher activity than one or more MMP-9 inhibitors used. In further embodiments, the MMP-12 inhibitory activity is 10, 100, or 1,000 times higher than the MMP-9 inhibitory activity.

[0033] In further embodiments, one or more MMP-9 inhibitors have higher activity than one or more MMP-12 inhibitors used. In further embodiments, the MMP-9 inhibitory activity is 10, 100, or 1,000 times higher than the MMP-12 inhibitory activity.

[0034] "Selective inhibition of the activity or expression of both MMP-9 and MMP-12" means that the activity or expression of both MMP-9 and MMP-12 is inhibited to a greater extent than the inhibition of other MMPs, particularly MMP-2. While inhibition of both MMP-9 and MMP-12 occurs simultaneously when both are inhibited at the same time, it may be more continuum in nature if the levels of MMP-9 and MMP-12 fluctuate during treatment, and if either MMP-9 or MMP-12 is inhibited earlier, followed by the other significantly later. However, while MMP-9 and MMP-12 levels may fluctuate during treatment, both must always be present to some degree (i.e., there is always at least some degree of simultaneous inhibition of both MMP-9 and MMP-12). Therefore, in the use / treatment described herein, one or more selective inhibitors of both MMP-9 and MMP-12 must always be present simultaneously.

[0035] Thus, compounds or combinations of compounds are provided for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury, or for use in the treatment of secondary effects associated with SCI or related nerve tissue injury, wherein the compounds or combinations of compounds for such use selectively (and simultaneously) inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury, to a greater extent than the inhibition of other MMPs, particularly MMP-2.

[0036] In one embodiment, both the activity and expression of MMP-9 and MMP-12 are selectively inhibited. In a further embodiment, the activity of both MMP-9 and MMP-12 is selectively inhibited.

[0037] In one embodiment, inhibition of MMP-9 and MMP-12 is at least 10 times more selective than inhibition of any other MMP. In another embodiment, inhibition of MMP-9 and MMP-12 is at least 50 or 100 times more selective than inhibition of any other MMP. In a further embodiment, inhibition of MMP-9 and MMP-12 is at least 300 times more selective than inhibition of any other MMP.

[0038] MMP-9 and MMP-12 can be simultaneously inhibited by a single compound having both MMP-9 and MMP-12 inhibitory activity, or by a combination of separate compounds, one of which is a selective MMP-9 inhibitor and the other a selective MMP-12 inhibitor. Administration of the separate compounds may be carried out in a fixed-dose combined composition containing both compounds, or by administering multiple compositions containing the individual compounds separately, sequentially, or simultaneously, on the premise that both compounds are administered in such a way that simultaneous inhibition of both MMP-9 and MMP-12 occurs.

[0039] In one embodiment, "selectively inhibiting the activity or expression of both MMP-9 and MMP-12" also means that the compound or combination of compounds used has substantially equivalent activity against both MMP-9 and MMP-12. For example, IC of AZD1236 50 These values ​​were measured to be 4.5 nM and 6.1 nM for MMP-9 and MMP-12, respectively, for the AZD3342 IC. 50 These were measured to be 10 nM and 6 nM relative to MMP-9 and MMP-12, respectively.

[0040] In one embodiment, a single compound for use as described herein is provided, where a single selective MMP-9 and MMP-12 inhibitory compound is in the range of 1 nM to 50 nM for both MMP-9 and MMP-12; in another embodiment, an IC in the range of 1 nM to 100 nM. 50 It holds.

[0041] In further embodiments, a single compound for use as described herein is provided, wherein a single selective MMP-9 and MMP-12 inhibitory compound has an IC50 greater than 100 nM, or in another embodiment greater than 200 nM, relative to MMP-2. 50 It holds.

[0042] In further embodiments, a single compound for use as described herein is provided, wherein a single selective MMP-9 and MMP-12 inhibitory compound has an IC50 range of 1 nM to 50 nM (in another embodiment, 1 nM to 100 nM) for both MMP-9 and MMP-12. 50 ICs that have and exceed 200 nM compared to MMP-2 50 It holds.

[0043] In further embodiments, the single selective MMP-9 and MMP-12 inhibitory compound for use as described herein is AZD1236 or AZD3342, or a pharmaceutically acceptable salt thereof.

[0044] In further embodiments, the single selective MMP-9 and MMP-12 inhibitory compound for use as described herein is AZD1236 or a pharmaceutically acceptable salt thereof.

[0045] In further embodiments, the MMP-12 inhibitory activity is 10, 100, or 1,000 times higher than the MMP-9 inhibitory activity.

[0046] In further embodiments, the MMP-9 inhibitory activity is 10, 100, or 1,000 times higher than the MMP-12 inhibitory activity.

[0047] In another embodiment, a compound or combination of compounds for use in the treatment of neuropathic pain is provided, wherein the compound or combination of compounds for use comprises selective inhibition of the activity or expression of both MMP-9 (gelatinase B) and metalloelastase MMP-12. Neuropathic pain is pain caused by injury or disease affecting the somatosensory nervous system. In one embodiment, AZD1236, or a pharmaceutically acceptable salt thereof, is provided for use in the treatment of neuropathic pain.

[0048] This disclosure is illustrated and supported by the accompanying drawings. [Brief explanation of the drawing]

[0049] [Figure 1] The levels of MMP-9 and MMP-12, as well as their enzymatic activity, show a sharp increase after DC injury. (A) MMP-9 mRNA levels peak 1 day after injury, while (B) MMP-12 peaks 5 days after injury. (C) MMP-9 protein levels also peak 1 day after injury. (D) MMP-12 protein levels also peak 5 days after injury. (E) MMP-9 activity is high 1 day after injury and peaks 3 days later. RFU = relative fluorescence units. (F) MMP-12 activity peaks 5 days after injury. RFU = relative fluorescence units. Data are represented mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2A] This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. MMP-9 activity is inhibited by oral administration of AZD1236 in both serum and CSF. Data are presented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2B]This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. MMP-12 activity is inhibited by oral administration of AZD1236 in both serum and CSF. Data are presented mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2C] This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. MMP-9 activity is inhibited by intraarachnoid administration of AZD1236 in both serum and CSF. Data are presented mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2D] This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. MMP-12 activity is inhibited by intraarachnoid administration of AZD1236 in both serum and CSF. Data are presented mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2E] This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. The summary table shows the percentage inhibition of MMP-9 and MMP-12 activity by AZD1236 in serum and CSF after oral and subarachnoid administration. n=6 mice / group, two independent experiments, total n=12 mice / group. [Figure 2F] This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. The optimal dose of AZD1236 also significantly inhibits the activity of MMP-9 and MMP-12 in spinal cord homogenate (RFU = relative fluorescence units). Data are presented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 2G]This study demonstrates that AZD1236 significantly inhibits the activity of MMP-9 and MMP-12. In situ zymography shows that in spinal cord sections, high levels of gelatinase enzyme activity (green; arrowhead) after DC injury are suppressed following oral and subarachnoid administration of the optimal dose of AZD1236. Sections are counterstained with GFAP (red) to mark astrocytes in red. # = injury site. Scale bar = 200 μm. [Figure 3A] Spinal cord water content is shown. The mean water content (edema) in the spinal cord increased after DC injury compared to the sham treatment control level, peaked at 3 days, and then decreased. Data are represented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 3B] Spinal cord water content is shown. AZD1236 at 200 mg / kg attenuated the DC injury-induced increase in spinal cord water content 3 days after oral administration immediately following injury. Data are represented mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. **=P=0.01, one-way ANOVA with Dunnett post-hoc test. [Figure 3C] Spinal cord water content is shown. 5 mg / kg of AZD1236 attenuated the increase in spinal cord water content induced by DC injury 3 days after intraarachnoid administration immediately following injury. Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. **=P=0.01, one-way ANOVA with Dunnett post-hoc test. [Figure 3D]Spinal fluid content is shown. Inhibition of both MMP-9 and MMP-12 is necessary to eliminate SCI-induced edema. Note: When MMP408 and inhibitor I were used alone, the above doses were used, but when administered in combination, the dose was halved (i.e., 100 mg / kg = 50 mg / kg MMP408 / 50 mg / kg inhibitor I, 200 mg / kg = 100 mg / kg MMP408 / 100 mg / kg inhibitor I, 300 mg / kg = 150 mg / kg MMP408 / 150 mg / kg inhibitor I). Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury. [Figure 4]This paper compares the ability of MMP inhibitors to suppress spinal cord water content 3 days after DC injury. Posterior column (DC) injury induces and increases water content, which is partially suppressed by different MMP inhibitors. (A) Mean spinal cord water content after treatment with GM6001 (broad-spectrum MMP inhibitor; inhibits MMP-1, MMP-2, MMP-3, MMP-8 and MMP-9). *P=0.05, one-way ANOVA with Dunnett post-hoc test. (B) Mean spinal cord water content after treatment with SB-3CT (selective MMP-2 inhibitor). (C) Mean spinal cord water content after treatment with MMP-9 inhibitor I. *P=0.05, one-way ANOVA with Dunnett post-hoc test. (D) Mean spinal cord water content after treatment with SD2590 (inhibits MMP-2, MMP-3, MMP-8, MMP-9, MMP-13 and MMP-14). *=P=0.05, one-way ANOVA using Dunnett post-hoc test. (E) Mean spinal water content after treatment with ND378 (inhibits MMP-2). (F) Mean spinal water content after treatment with minocycline (no effect on MMP activity). (G) Mean spinal water content after treatment with riluzole (GABA intake inhibitor). *=P=0.05, one-way ANOVA using Dunnett post-hoc test. (H) Mean spinal water content after treatment with glibenclamide (Sur1-regulated NCCa-ATP). *=P=0.05, one-way ANOVA using Dunnett post-hoc test. (I) Mean spinal water content after treatment with melatonin (aquaporin 4). *=P=0.05, one-way ANOVA using Dunnett post-hoc test. (J) When all MMP inhibitors used in this study were compared, AZD1236 was far superior, almost completely attenuating the elevations induced by DC injury and returning these levels to sham treatment control levels. ***=P=0.00012, one-way ANOVA with Dunnett post-hoc test. Data are represented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. *=P=0.05, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury. [Figure 5]Inhibition of MMP-9 and MMP-12 suppresses pro-inflammatory pain markers after DC injury. (A) and (B) Inhibition of MMP-9 and MMP-12 by AZD1236 reduces mRNA levels of the pro-inflammatory pain markers interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), respectively, after oral and subarachnoid administration in a DC injury model. Data are presented mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. ***=P<0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 6] This study demonstrates that AZD1236 suppresses spinal cord hydration, pro-inflammatory pain markers, and MMP activity, and improves pain behavior in a clip compression (CC) model of spinal cord injury (SCI). Oral or intra-arachnoid (it) administration of AZD1236 attenuates (A) injury-induced increase in spinal cord hydration, (B) relative expression of pro-inflammatory pain markers, and (C) MMP-9 and MMP-12 activity. Oral administration of AZD1236 also improves responses to (D) contact allodynia, (E) thermal allodynia, and (F) cold allodynia. Note: AZD1236 significantly reduces pain behavior compared to the currently used neuropathic analgesics pregabalin (30 mg / kg) and gabapentin (100 mg / kg) (concentrations determined and pre-optimized in preliminary CC model experiments before all experiments). Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. *=P=0.04, **=P=0.01; ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury. [Figure 7]This study demonstrates that AZD1236 more effectively reduces pro-inflammatory pain markers after CC injury than other analgesics currently in use. AZD1236 significantly reduced pro-inflammatory pain markers IL-1β, TNF-α, and IL-6 after CC injury compared to pre-optimized pregabalin and gabapentin (whose effects were very weak). Data are presented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. *=P=0.04, **=P=0.01; ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. All drugs were administered by oral force-feeding immediately after injury. [Figure 8] This study demonstrates that oral administration of AZD1236 improves electrophysiological, gait, and sensory outcomes after DC injury. (A) Representative superimposed CAP traces after oral administration of AZD1236. (B) Both CAP amplitude and (C) CAP region show significant improvement after treatment with AZD1236. (D) Both ladder traverse (gait function) and (E) average tape detection / removal time (sensory function) show significant improvement after treatment with AZD1236. #=P<0.00147, linear mixed model; ##=P<0.00114, generalized linear mixed model; ***=P<0.0001, ANOVA. Data are represented mean ± SEM. n=6 mice / group, 3 independent experiments, total n=18 mice / group. [Figure 9] This study demonstrates that intraarachnoid administration of AZD1236 improves electrophysiological outcomes, gait outcomes, and sensory outcomes after DC injury. (A) Representative superimposed CAP traces after intraarachnoid administration of AZD1236. (B) Both CAP amplitude and (C) CAP region show significant improvement after treatment with AZD1236. (D) Both ladder traverse (gait function) and (E) average tape detection / removal time (sensory function) show significant improvement after treatment with AZD1236. #=P<0.0015, linear mixed model; ##=P<0.0011, generalized linear mixed model; ***=P<0.0001, ANOVA. Data are represented mean ± SEM. n=6 mice / group, 3 independent experiments, total n=18 mice / group. [Figure 10]Inhibition of MMP-9 and MMP-12 using AZD1236 promotes DC axonal regeneration and axonal preservation in the upper and lower regions of DC injury in mice. (A) Cholera toxin B (CTB) retrogradely labeled axons that regenerated / sprouted and grew towards the cranial spinal cord at the DC injury site in DC+AZD126-treated animals, whereas no CTB-labeled axons were observed at or beyond the injury site in DC+vehicle-treated controls. Scale bar = 200 μm. (B) A significantly higher proportion of CTB-labeled axons were quantified cranially / caudally at the injury site in DC-AZD1236-treated animals compared to DC+vehicle-treated controls. (C) Immunohistochemical analysis detecting neurofilament (NF) 200+ fibers in cross-sections of the upper (T9) and lower (T7) spinal cord at the injury site in DC+vehicle-treated and DC+AZD1236-treated animals. Scale bar = 200 μm. (D) Quantification of the number of NF200 pixels above and below the injury site, indicating axonal preservation in DC+AZD1236 treated animals compared with DC+vehicle treated controls. Data were expressed as mean ± SEM. n=6 mice / group, 3 independent experiments, total n=18 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury. [Figure 11] This study demonstrates that AZD1236 significantly promotes DC axonal regeneration more effectively than other MMP inhibitors. [Figure 12]This study demonstrates that 24-hour delayed treatment with AZD1236 is as beneficial as immediate treatment. (A) Data showing that AZD1236 significantly suppressed spinal cord hydration due to SCI. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. (B) Expression of pro-inflammatory pain markers is significantly suppressed. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. (C) Activity of MMP-9 and MMP-12 is also suppressed by AZD1236. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. (D) Representative CAP traces treated with spike 2 after delayed treatment with AZD1236. Note: At the end of recording, the CAP traces disappeared due to dorsal hemisectomy of the spinal cord, demonstrating the technical success of the experiment. (E) Significant improvements in CAP amplitude and (F) CAP region were observed after treatment with AZD1236. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. (G) Ladder traversing ability and (H) Tape detection and removal ability improved with AZD1236 treatment. Data are expressed as mean ± SEM. ##=P<0.0012, generalized linear mixed model. #=P<0.0011, linear mixed model. n=6 mice / group, two independent experiments, total n=12 mice / group. [Figure 13] This study demonstrates that a 24-hour delayed treatment with AZD1236 promotes a nearly equivalent rate of axonal regeneration compared to immediate treatment. (A) In DC+ vehicle-treated mice, CTB+ axons stopped at the injury site (#), whereas in animals treated with AZD1236, a significant proportion of CTB+ labeled axons regenerated, penetrating the injury site and entering the cranial spinal cord. C=caudal, R=cranial. Scale bar=200μm. (B) Quantification of the regenerating CTB+ axon rate shows a nearly equivalent rate to that of immediate treatment. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. n=6 mice / group, two independent experiments (total n=12 mice / group). [Figure 14A]Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Inhibition of MMP-9 and MMP-12, as detected by albumin immunoreactivity, suppressed albumin overflow at the injury site (#). AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. Scale bar = 100 μm. [Figure 14B] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification showed suppression of BSCB disruption 3 days after DC injury and treatment with AZD1236 compared to vehicle-treated animals. Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14C] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Laminin immunoreactivity (scar tissue) at the injury site (#) 4 weeks after DC injury and treatment showed significantly suppressed scar tissue in AZD1236-treated animals compared to the vehicle-treated group. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. Scale bar = 100 μm. [Figure 14D] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification of the number of laminin immunoreactive pixels showed a significant decrease in the level of laminin scar tissue at the injury site. Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14E]Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Semaphorin 3A (Sema-3A) immunoreactivity shows that AZD1236 reduces Sema-3A deposition at the injury site (#) when administered immediately after injury or within 24 hours. Scale bar = 100 μm. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14F] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification shows that AZD1236 reduces Sema-3A deposition at the injury site (#) when administered immediately after injury or within 24 hours. Data are expressed as mean ± SEM. AU = arbitrary units. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14G] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. CS-56 immunoreactivity shows that AZD1236 reduces CS-56 deposition at the injury site when administered immediately after or within 24 hours of injury. Scale bar = 100 μm. AZD1236 was administered by oral force-feeding immediately after or 24 hours after injury. [Figure 14H] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification shows that AZD1236 reduces CS-56 deposition at the injury site when administered immediately after injury or within 24 hours. Data are expressed as mean ± SEM. AU = arbitrary units. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14I]Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. CD11b, CD68, and GFAP immunoreactivity are shown in sagittal sections from DC+ vehicle-treated and DC+AZD1236-treated animals. Scale bar = 100 μm. Scale bar = 200 μm in insets i and ii. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14J] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification of the number of CD11b+ immunoreactive pixels showed significantly suppressed levels (immediately and 24 hours later) in DC+AZD1236-treated animals. Data are expressed as mean ± SEM. AU = arbitrary units. n=6 mice / group, two independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14K] Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification of the number of CD68+ immunoreactive pixels showed significantly suppressed levels (immediately and 24 hours later) in DC+AZD1236 treated animals. Data are expressed as mean ± SEM. AU = arbitrary units. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 14L]Oral administration of AZD1236 reduces blood-spinal barrier (BSCB) disruption and scar tissue at the injury site. Quantification of the number of GFAP+ immunoreactive pixels showed significantly suppressed levels (immediately and 24 hours later) in DC+AZD1236 treated animals. Data are expressed as mean ± SEM. AU = arbitrary units. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. AZD1236 was administered by oral force-feeding immediately after injury or 24 hours later. [Figure 15] Common CSF biomarkers in SCI are also shown to be reduced by AZD1236. (A) S100β, (B) NSE, (C) GFAP, (D) pNF-H, and (E) NF-L were all slightly reduced by melatonin but significantly reduced by AZD1236 in comparison. n=12 mice / group. ***=P=0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 16A] This study demonstrates that AZD1236 reduces pro-inflammatory cytokines released from LPS-stimulated microglia in vitro, but does not affect macrophage migration. TNF-α production by primary microglia stimulated by LPS is inhibited by AZD1236. In comparison, other MMP inhibitors, such as GM6001, SD2590, and MMP inhibitor I, only slightly reduce TNF-α levels. n=3 wells / treatment, 3 independent repeats (total n=9 wells / treatment). **=P<0.001; ***=P<0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 16B]This study demonstrates that AZD1236 reduces pro-inflammatory cytokines released from LPS-stimulated microglia in vitro, but does not affect macrophage migration. IL-1β production by primary microglia stimulated by LPS is inhibited by AZD1236. In comparison, other MMP inhibitors such as GM6001, SD2590, and MMP inhibitor I only slightly reduce IL-1β levels. n=3 wells / treatment, 3 independent repeats (total n=9 wells / treatment). **=P<0.001; ***=P<0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 16C] This study demonstrates that AZD1236 reduces pro-inflammatory cytokines released from LPS-stimulated microglia in vitro, but does not affect macrophage migration. IL-6 production by primary microglia stimulated by LPS is inhibited by AZD1236. In comparison, other MMP inhibitors such as GM6001, SD2590, and MMP inhibitor I only slightly reduce IL-6 levels. n=3 wells / treatment, 3 independent repeats (total n=9 wells / treatment). **=P<0.001; ***=P<0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 16D] This study demonstrates that AZD1236 reduces pro-inflammatory cytokines released from LPS-stimulated microglia in vitro, but does not affect macrophage migration. Macrophage migration was unaffected by AZD1236, GM6001, Sd2590, or MMP9 inhibitor I, while positive controls MCP-1 and PMA increased migration indicators in all macrophage populations. n=3 wells / treatment, 3 independent repeats (total n=9 wells / treatment). **=P<0.001; ***=P<0.0001, one-way ANOVA with Dunnett post-hoc test. [Figure 17]This study demonstrates that inhibition of MMP-9 and MMP-12 using AZD3342 is effective in a rat model of DC injury. (A) MMP-9 mRNA levels in rats also peak 1 day after DC injury. (B) MMP-12 mRNA levels in rats also peak 5 days after DC injury. (C) Spinal fluid content (edema) in rats is reduced 3 days after inhibition of MMP-9 and MMP-12. In comparison, melatonin showed only a slight effect. (D) AZD3342 significantly suppresses the activity of MMP-9 and MMP-12. (E) AZD3342 significantly suppresses the expression of pro-inflammatory pain cytokines, while melatonin showed only a slight effect. (F) Representative CAP traces treated with spike 2 6 weeks after treatment with AZD3342 and melatonin. AZD3342 significantly restored the CAP wave after DC injury. (G) AZD3342 improved ladder crossing ability (walking motor function) over 6 weeks. (H) AZD3342 improved tape sensing and removal ability time (sensory function) over 6 weeks compared to vehicle or melatonin-treated rats. Data are expressed as mean ± SEM. n=6 mice / group, 2 independent experiments, total n=12 mice / group. ***P=0.0001, one-way ANOVA with Dunnett post-hoc test. #=P<0.0011, linear mixed model; ##=P<0.0011, generalized linear mixed model. AZD3342 was administered by oral force-feeding immediately after injury. [Figure 18] This study shows that within 4-5 days after the last oral administration of AZD1236, the activity of MMP-9 and MMP-12 at the injury site returns to standard DC injury-inducing levels. (A) MMP-9 activity returns to injury-inducing levels by 7 days, and takes 4 days to return to this level after discontinuation of AZD1236. (B) MMP-12 activity returns to injury-inducing levels by 8 days, and takes 5 days to return to this level after discontinuation of AZD1236. AZD1236 was administered by oral force-feeding immediately after injury. [Figure 19]This shows the activity of MMP-2 after oral and subarachnoid administration of AZD1236. There was no change in MMP-2 activity after oral and subarachnoid administration of AZD1236, indicating that the effect is specific to the inhibition of MMP-9 and MMP-12. Data are presented as mean ± SEM. n=6 mice / group, two independent experiments, total n=12 mice / group. [Figure 20] This shows spinal cord fluid content as a measure of injury-induced edema. Posterior column (DC) injury induces an increase in fluid content, which is completely suppressed by both AZD1236 and AZD3342 (specific inhibitors of MMP-9 and MMP-12). [Figure 21] The water content after aquaporin 4 inhibition is shown. Trifluoperazin (TFP), aquaporin 4 relocalization inhibitors, protein kinase inhibitors (PKAi), and TGN-020 suppress SCI-induced edema. Protein kinase C inhibitors did not affect SCI-induced edema. [Modes for carrying out the invention]

[0050] This specification demonstrates that inhibiting both matrix metalloproteinases MMP-9 and MMP-12 is useful in treating SCI or related nerve tissue damage, or in treating secondary effects associated with SCI or related nerve tissue damage.

[0051] In some embodiments, this can be achieved by using a combination of separate compounds having corresponding MMP-9 and MMP-12 inhibitory activity. In other embodiments, a single compound having both MMP-9 and MMP-12 inhibitory activity may be used.

[0052] AZD1236, a specific inhibitor of MMP-9 and MMP-12, was used, and its effects on edema, BSCB failure, NP, scar formation, and functional and behavioral recovery were confirmed after both oral and intraarachnoid administration. AZD1236 was found to inhibit MMP-9 and MMP-12 in spinal cord tissue, serum, and cerebrospinal fluid, suppress SCI-induced edema, reduce inflammatory pain markers and NP responses (mechanical, thermal, and cold allodynia), improve electrophysiological responses across SCI sites, improve gait and sensory function, reduce BSCB failure and scarring at injury sites, and help protect long-term function. Furthermore, inhibition of MMP-9 and MMP-12 by AZD1236 was found to suppress microglial activation and macrophage infiltration at injury sites, and to reduce axon growth inhibitory molecules derived from scar tissue (e.g., Sema-3A and CS-56). All of these effects contribute to improving the environment for axon regeneration, which in turn promotes the preservation of axons above and below the site of injury.

[0053] The results disclosed herein also demonstrate that AZD3342 suppresses SCI-induced edema, and that a combination of the specific MMP-12 inhibitor MMP408 and the MMP-9 inhibitor Inhibitor I also suppresses SCI-induced edema.

[0054] Since the suppression of either MMP-9 or MMP-12 individually is insufficient, it has been shown herein that the reduction of SCI-induced edema depends on the combined suppression of MMP-9 and MMP-12. Post-SCI clinical outcomes are greatly influenced by spinal cord edema, which, if left untreated, can lead to further injury and death.

[0055] Furthermore, it was found that MMP-12 was expressed at a higher level than MMP-9, and its expression peaked 5 days after SCI. This suggests that suppression of MMP-12, in particular, plays a favorable role in mitigating BSCB failure and promoting better functional recovery after SCI.

[0056] Advantageously, controlling edema using MMP-9 and MMP-12 inhibitors (e.g., AZD1236, AZD3342, or specific MMP-9 and specific MMP-12 inhibitors) reduces swelling with non-surgical methods and therefore also prevents further complications and / or damage from surgery on the site of trauma.

[0057] As shown in the examples, inhibition of both MMP-9 and MMP-12 activity was found to be beneficial in mitigating BSCB failure, thereby suppressing SCI-induced edema and improving functional recovery. Post-SCI outcomes are largely influenced by spinal cord edema. These outcomes are long-lasting and can provide significant feedback to neurosurgeons. In addition, immediate inhibition of BSCB failure likely prevents the influx of inflammatory cells into the spinal cord, inhibits the overflow of molecules such as plasma, and reduces the toxic effects of inflammatory cells, glutamate, and glycine, which can be toxic at high concentrations.

[0058] As shown in the examples, inhibition of MMP9 and 12 by AZD1236 also reduced neutrophil infiltration and mitigated white matter damage after SCI, suggesting that neutrophils are involved in impaired gait recovery. Neutrophils damage brain tissue by producing proteases containing reactive oxygen species and MMPs. Significant suppression of macrophage and microglia activation occurring within and around the injury site was also observed in animals treated with AZD1236. In addition, the examples show that AZD1236 suppresses the release of pro-inflammatory and neuropathic pain-related cytokines from primary microglia activated by LPS in vitro. Furthermore, the examples show that while AZD1236 does not affect macrophage migration in vitro, a decrease in the number of macrophages at the injury site was observed in vivo in animals treated with AZD1236. The decrease in the number of macrophages at the injury site in animals treated with AZD1236 appears to be related to the reduction of BSCB. Overall, all of these effects of AZD1236 contribute to the reduction of overall spinal cord damage, as well as the subsequent decline in sensory and motor function.

[0059] The results disclosed herein demonstrate that labeling albumin, which typically overflows into the spinal cord parenchyma due to BSCB failure, mitigated BSCB damage. Furthermore, AZD1236 promoted the reduction of scarring at the injury site, and simultaneously, astrocyte activation was suppressed as measured by GFAP labeling, suggesting enhanced protection of spinal cord tissue and its normal structure. Both Sema-3A and CSPG are ECM molecules present at the SCI site and are potent axon growth inhibitors. Pharmacological inhibition of Sema-3A and enzymatic degradation of CSPG lead to significant axon regeneration after SCI; therefore, reduction of these molecules by AZD1236 likely results in the observed increase in DC axon regeneration.

[0060] The results of the present specification disclose that inhibition of MMP-9 and MMP-12 by AZD1236 promotes CNS axonal regeneration / sprouting after DC injury, resulting in a significant increase in the number of regenerating axons at the injury site in AZD1236-treated animals, which extend longer distances rostrally. Consistent with this, a significant increase in spare fibers was observed both above and below the injury site, which likely also contributed to the promoted functional recovery observed after AZD1236 treatment. The promoted preservation may be attributable to reduced tissue damage, decreased infiltrating cells, and reduced microglial activity at the injury site observed after AZD1236 treatment. Promotion of axonal regeneration may also be either direct or indirect. For example, immediate activation of both MMP-9 and MMP-12 is involved in BSCB disruption (Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, et al. Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J Neurosci 2000;20(18):7037-42; Noble LJ, Donovan F, Igarashi T, Goussev S, Werb Z. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. J Neurosci 2002;22(17):7526-35), but MMP-9 - / - and MMP-12 - / -Mice showed enhanced behavioral recovery after spinal cord injury, likely due to increased axonal regeneration / formation and reduced BSCB failure (Wells JE, Rice TK, Nuttall RK, Edwards DR, Zekki H, Rivest S, et al. An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice. J Neurosci 2003b;23(31):10107-15). In addition, MMP-9 - / - Mice showed not only a reduction in the complexity of glial scars but also a reduction in the deposition of chondroitin sulfate proteoglycan and NG2 in the subacute phase after SCI (Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH. NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors. J Neurosci 2002;22(7):2792-803; Hsu JY, Bourguignon LY, Adams CM, Peyrollier K, Zhang H, Fandel T, et al. Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord. J Neurosci 2008;28(50):13467-77; Andries L, Van Hove I, Moons L, De Groef L. Matrix Metalloproteinases During Axonal regeneration, a multifactorial role from start to finish (Mol Neurobiol 2017;54(3):2114-25), this is similar to our observations of AZD1236.

[0061] Therefore, MMP-9 and MMP-12 have multiple functions, and experimental evidence suggests that the complex plays a regulatory role in various aspects of axonal regeneration in damaged CNS (Andries L, Van Hove I, Moons L, De Groef L. Matrix Metalloproteinases During Axonal Regeneration, a Multifactorial Role from Start to Finish. Mol Neurobiol 2017;54(3):2114-25). For example, they contribute to initial phagocytosis and glial scar response, while simultaneously promoting the degradation of myelin-derived inhibitors, activation of growth factors, axon guidance, axon elongation, growth cone motion, synapse formation and reinnervation, and final remyelination (Gijbels K, Proost P, Masure S, Carton H, Billiau A, Opdenakker G. Gelatinase B is present in the cerebrospinal fluid during experimental autoimmune encephalomyelitis and cleaves myelin basic protein. J Neurosci Res 1993;36(4):432-40; Proost P, Van Damme J, Opdenakker G. Leukocyte gelatinase B cleavage releases encephalitogens from human myelin basic protein. Biochem Biophys Res Commun 1993;192(3):1175-81; Andries L, Van Hove I, Moons L. De Groef L. Matrix Metalloproteinases During Axonal Regeneration, a Multifactorial Role from Start to Finish. Mol Neurobiol 2017;54(3):2114-25). This specification discloses that attenuating the initial elevation of MMP-9 and MMP-12 after SCI is beneficial.Since the activity of MMP-9 and MMP-12 recovers within 5 days after discontinuation of AZD1236 treatment, it is expected that MMP-9 and MMP-12 can subsequently participate in the normal post-injury healing response (Andries L, Van Hove I, Moons L, De Groef L. Matrix Metalloproteinases During Axonal Regeneration, a Multifactorial Role from Start to Finish. Mol Neurobiol 2017;54(3):2114-25).

[0062] The results disclosed herein demonstrate that AZD1236 suppresses pro-inflammatory pain markers and reduces all characteristics of tactile allodynia, thermal allodynia, cold allodynia, and neuropathic pain (NP). This data supports the practical application of AZD1236 in limiting NP induced by SCI, which is likely due to the reduction of edema. However, since macrophage infiltration, microglia activation, and BSCB barrier disruption correlate with NP, it is possible that suppression of macrophage and microglial activity in the spinal cord also contributes to the suppression of NP (Zhao H, Alam A, Chen Q, M AE, Pal A, Eguchi S, et al. The role of microglia in the pathobiology of neuropathic pain development: what do we know? Br J Anaesth 2017;118(4):504-16; Honjoh K, Nakajima H, Hirai T, Watanabe S, Matsumine A. Relationship of Inflammatory Cytokines From M1-Type Microglia / Macrophages at the Injured Site and Lumbar Enlargement With Neuropathic Pain After Spinal Cord Injury in the CCL21 Knockout(plt) Mouse. Front Cell Neurosci 2019;13: 525; Takeura N, Nakajima H, Watanabe S, Honjoh K, Takahashi A, Matsumine A. Role of macrophages and activated microglia in neuropathic pain associated with chronic progressive spinal cord compression.Sci Rep 2019;9(1): 15656).

[0063] Therefore, all of AZD1236's properties, including not only reduced macrophage and microglia activation and mitigation of BSCB failure, but also, in some cases, neutralization of toxic MMP-9 and MMP-12, likely contribute to limiting infiltrating cells and thus reducing the production of pro-inflammatory cytokines. All of these factors, along with suppressing edema, contribute to the reduction of NP in animals treated with AZD1236. NP is a common secondary complication after SCI, and is estimated to occur in 61% of reported cases. Post-injury NP can manifest at or below the disability level and can cause a decline in quality of life, depression, and sleep disturbances. NP patients are refractory to current drug therapies with Lyrica, the most promising drug in randomized controlled clinical trials (Siddall PJ, McClelland JM, Rutkowski SB, Cousins ​​MJ. A longitudinal study of the prevalence and characteristics of pain in the first 5 years following spinal cord injury. Pain 2003;103(3):249-57; Cardenas DD, Nieshoff EC, Suda K, Goto S, Sanin L, Kaneko T, et al. A randomized trial of pregabalin in patients with neuropathic pain due to spinal cord injury. Neurology 2013;80(6): 533-9).The potential mechanisms of neuropathic pain (NP) development after spinal cord injury (SCI) include peripheral nerve, spinal cord, and brain mechanisms, which are described in detail elsewhere in the literature (Siddall PJ. Management of neuropathic pain following spinal cord injury: now and in the future. Spinal Cord 2009;47(5):352-9; Finnerup NB, Baastrup C. Spinal cord injury pain: mechanisms and management. Curr Pain Headache Rep 2012;16(3):207-16; D'Angelo R, Morreale A, Donadio V, Boriani S, Maraldi N, Plazzi G, et al. Neuropathic pain following spinal cord injury: what we know about mechanisms, assessment and management. Eur Rev Med Pharmacol Sci 2013;17(23):3257-61).

[0064] The results disclosed herein suggest that AZD1236 has several remarkable advantages over other MMP inhibitors whose use is guaranteed in SCI. For example, (1) AZD1236 is superior to other tools and clinical MMP inhibitors in suppressing edema; (2) AZD1236 is superior to other FDA-approved neuropathic pain analgesics (e.g., pregabalin and gabapentin) in suppressing pro-inflammatory pain markers and reducing neuropathic pain behavior; and (3) AZD1236 is nearly equivalent in all parameters measured in our SCI model when administered immediately after or 24 hours after SCI. (4) Short-term (3-day) administration of AZD1236 significantly reduces the likelihood of musculoskeletal side effects observed with long-term use of MMP inhibitors; (5) AZD1236 promotes axonal regeneration and functional recovery; (6) AZD1236 suppresses scar tissue deposition at the site of injury; (6) AZD1236 reduces hematopoietic cell infiltration into the CNS; (7) AZD1236 is unique in that it promotes sensory and gait motor recovery and satisfies many aspects of SCI recovery therapy.

[0065] The results disclosed herein demonstrate that short-term inhibition of MMP-9 and MMP-12 significantly limits the extent of secondary spinal cord injury, and therefore constitute a potential primary pathophysiological approach as a first-line treatment for SCI.

[0066] Furthermore, the results herein disclose that AZD1236 is the first experimental therapy targeting all four aspects of SCI pathophysiology through specific inhibition of MMP-9 and 12. AZD1236 reduces SCI-induced edema, suppresses pro-inflammatory markers of pain, improves animal responsiveness to various NP sources, improves gait and sensory function, and reduces BSCB breakdown and scar tissue formation at the injury site. AZD1236 treatment also results in suppression of macrophage, microglia, and astrocyte activation, promotion of DC axon regeneration through the upper and lower injury sites, and promotion of axon preservation. All of these beneficial effects of AZD1236 treatment contributed to the overall improvement in sensory and gait function observed in the studies disclosed herein.

[0067] In addition, intra-arachnoid administration of AZD1236 required 1 / 40th of the oral dose to produce a similar beneficial effect after SCI. These results suggest that inhibition of MMP-9 and MMP-12 using AZD1236, either orally or intra-arachnoidally, is a promising treatment for SCI.

[0068] The use of lower doses and more target routes compared to oral administration may also limit potential side effects. Advantageously, subarachnoid administration allows the compound(s) to approach steady-state levels in a shorter timeframe than when the compound(s) are administered orally.

[0069] Furthermore, surprisingly, the observed effects are seen after short-term administration, for example, after only the first three days of administration following SCI. In addition, this benefit is also observed when treatment is initiated within 24 hours of SCI. Favorably, this gives healthcare professionals a practical opportunity to treat patients after SCI without compromising any of the benefits associated with treatment. In other words, in certain scenarios, the initiation of treatment for a patient may be delayed in some cases, for example, it may take time for emergency services to arrive at the patient's location after SCI.

[0070] The results disclosed herein are thought to be due to the selective inhibition of MMP-9 and MMP-12, which are increased in expression (along with other MMPs) during SCI. Current symptomatic treatments for SCI (e.g., Lyrica) target NPs but are highly toxic. Other MMP inhibitors (e.g., those used in COPD and cancer treatment) are known to have side effects (e.g., musculoskeletal side effects) and therefore would not be considered attractive candidates. If broad-spectrum MMP inhibitors or MMP-2 inhibitors are used over a long period, for example, for two weeks, the effects of AZD1236 as seen herein would not be obtained.

[0071] Selective inhibition of both MMP-9 and MMP-12 in SCI is unprecedented, and the rapid and significant effects on edema and NP are remarkable. The effect on edema, in particular, helps prevent further damage associated with swelling, thus avoiding further surgical (decompression) interventions. In addition, since cannabinoid receptors are not targeted, addiction issues are also avoided.

[0072] The specific compounds for use described herein that selectively inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 are the compounds described in WO2002 / 074767, WO2007 / 106022 and WO2006 / 004532 or pharmaceutically acceptable salts thereof. This also includes not only tautomers, physical forms, polymorphs, solvates, and hydrates of such compounds, but also optical isomers, diastereomers, and mixtures and racemates of such compounds.

[0073] The specific compounds for use described herein are AZD1236 and AZD3342, or pharmaceutically acceptable salts thereof. Other specific compounds for use described herein are MMP408 (see Li, W., et al. 2009. J. Med. Chem. 52, 1799), a specific MMP-12 inhibitor, and Inhibitor I (see Levin, J.I, et al. 2001. Bioorg. Med. Chem. Lett. 11, 2189), an MMP-9 inhibitor.

[0074] Furthermore, the effects on edema indicate that short-term inhibition of aquaporin channels (AQP4) by compounds (such as TFP, PKAi, and TGN-020) can also rapidly suppress edema in SCIs, producing effects nearly equivalent to those of AZD1236. Therefore, combination therapy is possible, potentially allowing for a significant reduction in the dosage of each drug used. This short-term (1 week) effect of AQP4 inhibitors may help avoid prolonged side effects (such as waterborne illness).

[0075] In the central nervous system (CNS), aquaporin 4 (AQP4), a major water channel protein, regulates spinal cord cytoscopy (BSCB) permeability during spinal cord edema. SCI alters AQP4 expression, and therefore spinal cord water content. AQP4 also regulates astrocyte swelling, which plays a major role in cytotoxic edema after acute SCI. AQP4 null mice showed reduced SCI-induced edema and improved functional recovery during the acute phase after SCI. Pharmacological inhibitors of edema, such as melatonin, and surgical interventions, such as spinal cord resection, also reduced SCI-induced edema by inhibiting AQP4.

[0076] Pharmaceutical composition and dosage In a further embodiment, a pharmaceutical composition is provided comprising a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12, or a pharmaceutically acceptable salt thereof, its hydrate or solvate, together with a pharmaceutically acceptable diluent or carrier.

[0077] The composition may be in a form suitable for oral use (e.g., tablets, lozenges, rigid or flexible capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups, or elixirs), in a form suitable for topical use (e.g., creams, ointments, gels, or aqueous or oily solutions or suspensions), in a form suitable for inhalation administration (e.g., micronized powder or liquid aerosol), in a form suitable for inhalation administration (e.g., micronized powder), or in a form suitable for parenteral or subarachnoid administration (e.g., sterile aqueous or oily solutions for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, or suppositories for rectal administration). In a preferred embodiment, the composition is a tablet.

[0078] The compositions can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Therefore, compositions intended for oral use may contain, for example, one or more colorants, sweeteners, flavorings, and / or preservatives.

[0079] For use in the treatment of spinal cord injury (SCI) or related nerve tissue injury, or in the treatment of secondary effects associated with SCI or related nerve tissue injury, an effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 is sufficient to treat or prevent the conditions referred to herein, slow their progression, and / or alleviate the symptoms associated with those conditions.

[0080] The amount of active ingredient combined with one or more excipients to create a single-dose formulation inevitably varies depending on the individual being treated and the specific route of administration. For example, formulations intended for oral administration to humans generally contain 0.5 mg to 1.0 g of the active ingredient (more preferably 0.5 to 100 mg, e.g., 1 to 30 mg) compounded with an appropriate and convenient amount of excipients, which can vary, for example, from about 5 to about 98% by weight of the total composition.

[0081] The dosage for therapeutic or prophylactic purposes will inevitably vary depending on the nature and severity of the disease, the age and sex of the animal or patient, and the route of administration in accordance with well-known medical principles.

[0082] For such therapeutic or prophylactic purposes, the drug is generally administered in a daily dose ranging from, for example, 0.1 mg / kg to 250 mg / kg (more preferably 0.1 mg / kg to 50 mg / kg) of body weight, divided into doses as needed. Generally, when parenteral or subarachnoid routes are used, lower doses are administered. For example, in the case of intravenous or intraperitoneal administration, doses generally range from, for example, 0.1 mg / kg to 30 mg / kg of body weight are used. Similarly, in the case of inhalation administration, for example, doses range from, for example, 0.05 mg / kg to 25 mg / kg of body weight are used. Oral administration is also sometimes preferred, particularly in tablet form. Typically, a single-dose dosage form contains about 0.5 mg to 0.5 g of the compound of the present invention.

[0083] In one embodiment, the active ingredient is administered once or twice a day.

[0084] In one embodiment, the active ingredient is administered twice a day. In one embodiment, the first daily dose is administered in the morning and the second daily dose is administered in the evening.

[0085] In one embodiment, AZD1236 or a pharmaceutically acceptable salt thereof is administered twice daily.

[0086] In one embodiment, AZD1236 or a pharmaceutically acceptable salt thereof is administered orally at a dose of 50 to 200 mg twice daily, for example, about 100 mg twice daily, or more conveniently, about 75 mg twice daily. In another embodiment, AZD1236 or a pharmaceutically acceptable salt thereof is administered intraarachnoidally at a dose of 1 to 5 mg twice daily, or more conveniently, about 1 to 2.5 mg twice daily, for example, about 1.9 mg twice daily.

[0087] In one embodiment, a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 is administered for up to 3 days, for example, up to 1 or 2 days. Conveniently, a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 is administered for 1, 2, or 3 days, conveniently 3 days. Advantageously, normal MMP-9 and MMP-12 activity was found to recover 4-5 days after the last administration of the compound or combination of compounds, i.e., well before the central nervous system healing phase. While prolonged inhibition of MMP-9 may result in adverse effects due to its role in wound healing, short-term inhibition of MMP-9 and MMP-12 was found to control the initial excessive activation of these enzymes after SCI.

[0088] In one embodiment, the initial dose of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 is administered within up to 24 hours of SCI or associated nerve tissue injury. Advantageously, the beneficial effects resulting from the administration of a compound or combination of compounds that selectively inhibits the activity or expression of both MMP-9 and MMP-12 have been demonstrated to be nearly equivalent whether the compound or combination of compounds is administered immediately after SCI or associated nerve tissue injury, or within 24 hours after injury.

[0089] In one embodiment, the first dose of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 is administered up to 24 hours after SCI or associated nerve tissue injury, and subsequent doses of the compound or combination of compounds are administered thereafter for 1, 2, or 3 days, or conveniently for 3 days. For example, if the first dose is administered 24 hours after injury, the next dose is administered thereafter between 1 and 3 days.

[0090] In one embodiment, the first dose of AZD1236 or a pharmaceutically acceptable salt thereof is administered up to 24 hours after SCI or associated nerve tissue injury, and subsequent doses of the compound or combination of compounds are administered thereafter over 3 days.

[0091] In one embodiment, the first dose of AZD1236 or a pharmaceutically acceptable salt thereof is administered within up to 24 hours of SCI or associated nerve tissue injury, and subsequent doses of the compound or combination of compounds are administered thereafter for 3 days, during which AZD1236 is administered twice daily, for example, orally twice daily.

[0092] In one embodiment, the first dose of AZD1236 or a pharmaceutically acceptable salt thereof is administered up to 24 hours after SCI or associated nerve tissue injury, and subsequent doses of the compound or combination of compounds are administered thereafter for 3 days, during which AZD1236 is administered orally at 50-200 mg twice daily, for example, about 100 mg twice daily, or more conveniently about 75 mg twice daily.

[0093] In one embodiment, the first dose of AZD1236 or a pharmaceutically acceptable salt thereof is administered within up to 24 hours of SCI or associated nerve tissue injury, and subsequent doses of the compound or combination of compounds are administered thereafter for 3 days, during which AZD1236 is administered intra-subarachnoidally at doses of 1 to 5 mg twice daily, preferably 1 to 2.5 mg twice daily, for example, approximately 1.9 mg twice daily.

[0094] In one embodiment, the compound or combination of compounds is administered with food. In another embodiment, the compound or combination of compounds is administered without food.

[0095] Therapeutic use and application In one embodiment, a compound or combination of compounds is provided that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury, or for use in the treatment of secondary effects associated with SCI or related nerve tissue injury. SCI refers to any injury that causes trauma and results in blood-spinal barrier (BSCB) disruption, edema, neuronal death, axonal injury, and demyelination.

[0096] Associated nerve tissue damage refers to any damage that causes trauma and results in nerve tissue breakdown, edema, neuronal death, axonal damage, and demyelination. In one embodiment, associated nerve tissue damage is traumatic brain injury (TBI). TBI is a form of acquired brain injury that occurs when the brain is damaged by accidental trauma. TBI can occur when the head is struck forcefully by an object, or when an object penetrates the skull and enters the brain tissue.

[0097] In another embodiment, a compound or combination of compounds for use in the treatment of secondary effects associated with SCI or related nerve tissue damage is used to treat SCI-induced edema or neuropathic pain (NP).

[0098] Appropriately, the compound or combination of compounds is used to treat neuropathic pain. Appropriately, the compound or combination of compounds suppresses the pro-inflammatory pain markers IL-1β, TNF-α, and / or Il-6 after SCI or related nerve tissue injury. Appropriately, the compound or combination of compounds suppresses the pro-inflammatory pain markers IL-1β, TNF-α, and / or Il-6 after SCI or related nerve tissue injury by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the suppression of pro-inflammatory pain markers is relative to the level in the subject before treatment with the compound or combination of compounds.

[0099] Appropriately, compounds or combinations of compounds are used to treat SCI-induced edema. Appropriately, compounds or combinations of compounds reduce SCI-induced edema by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the edema in the subject prior to treatment with the compound or combination of compounds.

[0100] Other secondary effects associated with SCI or related nerve tissue damage include reduced scarring at the site of BSCB rupture and SCI injury, prolonged protection of function (nervous system), wound healing, prevention of scarring (including in combination with decorin therapy), and / or promotion of axonal regeneration.

[0101] Appropriately, compounds or combinations of compounds are used to treat scarring in the CNS. Appropriately, compounds or combinations of compounds reduce the deposition of scar-related Sema-3A and CS-56 by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compounds are AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the level of scarring in the subject before treatment with the compound or combination of compounds.

[0102] Appropriately, compounds or combinations of compounds are used to suppress the infiltration of hematopoietic cells into the CNS. Appropriately, compounds or combinations of compounds reduce hematopoietic cells such as mononuclear cells, neutrophils, granulocytes, macrophages, and / or natural killer cells by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the infiltration of hematopoietic cells in the subject before treatment with the compound or combination of compounds.

[0103] Other conditions treated by the use of compounds or combinations of compounds that selectively inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 include, but are not limited to, hypertension or hypertonia, Alzheimer's disease, Huntington's disease, epilepsy, cirrhosis, and stroke. In one embodiment, the condition is stroke.

[0104] Alzheimer's disease, SCI, edema, and stroke may be monitored by MRI, and improvement in these conditions results in a decrease in high-signal areas in the brain. Outcomes can also be tracked by neurological assessments of motor and sensory functions, such as measuring reflexes, muscle tone, perception, and improved mental state, which involve monitoring improvements in the nervous system. For example, a 30% improvement, such as 25% to 50% as measured by MRI, would be significant for the patient.

[0105] Further embodiments provide the use of a compound or combination of compounds in the manufacture of a pharmaceutical product for the treatment of spinal cord injury (SCI) or related nerve tissue injury, or for the treatment of secondary effects associated with SCI or related nerve tissue injury, the use of which includes selective inhibition of the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury.

[0106] In further embodiments, methods are provided for a patient in need of treatment for spinal cord injury (SCI) or related nerve tissue injury, or for treatment of secondary effects associated with SCI or related nerve tissue injury, the methods comprising administering to the patient a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury. In one embodiment, the related nerve tissue injury is traumatic brain injury (TBI).

[0107] In further embodiments, a therapeutic kit of parts is provided for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury, or for use in the treatment of secondary effects associated with SCI or related nerve tissue injury, the therapeutic kit of parts is (i) Compounds or combinations of compounds that selectively inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or associated nerve tissue damage, (ii) Includes instructions for instructing the administration of the above-mentioned compound or combination of compounds to patients requiring such treatment.

[0108] The uses and methods described herein may also be used prophylactically, for example, in conjunction with brain or spinal surgery.

[0109] In another embodiment, a compound or combination of compounds is provided for use in the treatment of neuropathic pain that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12.

[0110] In further embodiments, the use of a compound or combination of compounds in the manufacture of a pharmaceutical product for the treatment of neuropathic pain is provided, the use of which includes selective inhibition of the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12.

[0111] In a further embodiment, a method is provided for the treatment of neuropathic pain in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12.

[0112] In a further embodiment, a therapeutic kit of parts is provided for use in the treatment of neuropathic pain, the therapeutic kit of parts is, (i) Compounds or combinations of compounds that selectively inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or associated nerve tissue damage, (ii) Includes instructions for instructing the administration of the above-mentioned compound or combination of compounds to patients requiring such treatment.

[0113] In one embodiment, a method is provided for treating secondary effects associated with SCI or related nerve tissue injury, the method comprising selectively inhibiting the activity or expression of matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after SCI or related nerve tissue injury.

[0114] In one embodiment, a method is provided for treating secondary effects associated with SCI or related nerve tissue damage, the method comprising administering a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 to a patient in need thereof.

[0115] In one embodiment, secondary effects associated with SCI or related nerve tissue damage include SCI-induced edema and / or neuropathic pain (NP).

[0116] Appropriately, a method for treating neuropathic pain is provided, the method comprising administering a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 to a patient in need thereof. Appropriately, the compound or combination of compounds suppresses the pro-inflammatory pain markers IL-1β, TNF-α, and / or Il-6 after SCI or related nerve tissue injury. Appropriately, the compound or combination of compounds suppresses the pro-inflammatory pain markers IL-1β, TNF-α, and / or Il-6 after SCI or related nerve tissue injury by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the suppression of pro-inflammatory pain markers is relative to the level in the subject prior to treatment with the compound or combination of compounds.

[0117] Appropriately, a method for treating SCI-induced edema is provided, which comprises administering a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 to a patient in need thereof. Appropriately, the compound or combination of compounds reduces SCI-induced edema by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the edema in the subject prior to treatment with the compound or combination of compounds.

[0118] Other secondary effects associated with SCI or related nerve tissue damage include reduced scarring at the site of BSCB rupture and SCI injury, prolonged protection of function (nervous system), wound healing, prevention of scarring (including in combination with decorin therapy), and / or promotion of axonal regeneration.

[0119] Appropriately, a method for treating scarring in the CNS is provided, the method comprising administering a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 to a patient in need thereof. Appropriately, the compound or combination of compounds reduces the deposition of scar-related Sema-3A and CS-56 by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the level of scarring in the subject prior to treatment with the compound or combination of compounds.

[0120] Appropriately, a method is provided for suppressing the infiltration of hematopoietic cells into the CNS, the method comprising administering a therapeutically effective amount of a compound or combination of compounds that selectively inhibits the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 to a patient in need thereof. Appropriately, the compound or combination of compounds reduces hematopoietic cells, such as mononuclear cells, neutrophils, granulocytes, macrophages, and / or natural killer cells, by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Conveniently, the compound is AZD1236 or a pharmaceutically acceptable salt thereof. It should be understood that the reduction is relative to the infiltration of hematopoietic cells in the subject prior to treatment with the compound or combination of compounds.

[0121] The uses and methods described herein are particularly useful when treating human patients.

[0122] Combination therapy In certain embodiments, compounds or combinations of compounds intended for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury as defined herein, or in the treatment of secondary effects associated with SCI or related nerve tissue injury, may also be used in addition to conventional surgery and / or other chemotherapy. Other chemotherapy used in combination therapy may include one or more pharmaceutically active agents used in the treatment of SCI or related nerve tissue injury, for example, Lyrica (pregabalin) or methylprednisolone.

[0123] Such combination therapies can be achieved by administering the individual components of the therapy simultaneously, sequentially, or separately. Such combination therapies utilize one or more compounds of the disclosed herein within the dosage range described herein and other pharmaceutically active agents within the approved dosage range.

[0124] Where the term “combination therapy” is used herein, it should be understood to mean administration simultaneously, separately, or sequentially. In one embodiment, “combination therapy” refers to administration simultaneously. In another embodiment, “combination therapy” refers to administration separately. In yet another embodiment, “combination therapy” refers to administration sequentially. When administration is carried out sequentially or separately, delays in the administration of the second component should not result in a loss of the beneficial effect of the combination.

[0125] In a further embodiment, a pharmaceutical composition is provided comprising all the components of a combination therapy along with a pharmaceutically acceptable diluent or carrier.

[0126] In further embodiments, one or more compounds for use as described herein are provided, wherein such use further comprises the administration of an aquaporin 4 inhibitor(s) such as TFP, PKAi, or TGN-020.

[0127] In further embodiments, methods are provided for treating secondary effects associated with SCI or related nerve tissue injury, the methods comprising selectively inhibiting the activity or expression of matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury, wherein the methods further comprise the administration of an aquaporin 4 inhibitor(s) such as TFP, PKAi, or TGN-020.

[0128] In a further embodiment, a pharmaceutical composition is provided comprising all components of a combination therapy including an aquaporin 4 inhibitor(s), along with a pharmaceutically acceptable diluent or carrier.

[0129] Route of administration Compounds or combinations of compounds that selectively inhibit the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12, or one or more pharmaceutical compositions containing one or more of the above compounds, or any combination therapeutic agent, may be administered to the subject by any convenient route of administration (i.e., at the desired site of action), whether systemically, peripherally, locally, or intraarachnoidally.

[0130] Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (e.g., by patches, ointments, etc.); transmucosal (e.g., by patches, ointments, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); lung (e.g., via aerosol, e.g., through the mouth or nose, e.g., by inhalation or inhalation therapy); rectal (e.g., by suppositories or enemas); vaginal (e.g., by vaginal suppositories); parenteral injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, subarachnoid, intrathecal, intraventricular, intrasacral, subcapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, subarachnoid, and intrasternal; e.g., by subcutaneous or intramuscular depot or reservoir implantation.

[0131] In certain embodiments, oral or intraarachnoid administration, particularly intraarachnoid administration, is provided. Such intraarachnoid administration may be used at doses of 1 / 10 to 1 / 50 (e.g., 1 / 20 to 1 / 60), particularly 1 / 40 of the oral dose, to obtain the same beneficial effect after SCI or associated nerve tissue injury. For example, if an oral dose of 200 mg / kg is used, an intraarachnoid dose of 5 mg / kg may be used. Preferably, the compound or combination of compounds is administered intraarachnoidally by an intraarachnoid catheter.

[0132] In further embodiments, the administration may be carried out within a period of up to one or two weeks after SCI or related nerve tissue injury, particularly for a short period, for example, up to the first three days or even just one day.

[0133] In one embodiment, administration is given as a single dose at the start of treatment. In another embodiment, administration is given continuously during treatment, for example, by intravenous infusion. [Examples]

[0134] The following embodiments are provided for illustrative purposes only and do not limit the disclosure.

[0135] animal All animal experiments were authorized by the UK Home Office and ethically approved by the Animal and Ethical Review Board of the University of Birmingham. Experiments were conducted in strict accordance with the UK Animals Scientific Procedures Act, 1986 and the Revised European Directive 1010 / 63 / EU guidelines, and in accordance with the animal use guidelines and recommendations of the Federation of the European Laboratory Animal Science Associations and Animal Research: Reporting of In Vivo Experiments (ARRIVE guidelines).

[0136] Wild-type adult male / female C57BL / 6 mice (in roughly equal proportions) aged 7-9 weeks and weighing 20-30g, and male / female Sprague-Dawley rats (in roughly equal proportions) aged 6-8 weeks and weighing 170-220g (purchased from Charles River, Margate, UK) were maintained in a pathogen-free facility with controlled temperature and humidity under a 12-hour light-dark cycle and fed as needed.

[0137] Research design Generally, all experiments were conducted with n=6 animals / groups and repeated independently 2-3 times (totaling n=12-18 animals / groups). No animals were excluded for any reason during this study. All animals were randomly assigned to different experimental groups, ensuring that each animal cage in each group was identical. All procedures and experimental conditions were blinded to the researchers.

[0138] AZD1236 was administered either orally or by intraarachnoid injection.

[0139] Example 1 Testing of AZD1236 in mouse SCI posterior column (DC) crush and clip compression (CC) models. We selected the DC crush model for spinal cord injury (SCI) because it involves moderate-severity injury traversing the descending corticospinal tract and the ascending sensory gracile fasciculus and cuneiform tract. The axons originate from pyramidal motor neurons in layer V of the contralateral frontal motor neocortex and ipsilateral dorsal root ganglion neurons (DRGN), respectively. The injury response after DC injury in mice has been previously characterized (Z. Ahmed, D. Bansal, K. Tizzard, S. Surey, M. Esmaeili, A. Gonzalez, M. Berry, A. Logan, Decorin blocks scarring and cystic cavitation in acute and induces scar dissolution in chronic spinal cord wounds. Neurobiol Dis 64, 163-176 (2014)), and other literature has shown it to be a suitable model for analyzing regeneration outcomes. However, this model only shows sensitivity to post-injury pain during the first few hours after surgery and cannot be used to evaluate pain behavior (S. Surey, M. Berry, A. Logan, R. Bicknell, Z. Ahmed, Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62-80 (2014)). Therefore, the inventors evaluated pain behavior after treatment with AZD1236 using the clip compression (CC) model of SCI described later.

[0140] in vivo experiments For oral administration of AZD1236, n=6 adult male C57BL6 mice / group (Charles River, Margate, UK) were randomly assigned to one of the following groups: (1) Sham (control; partial laminectomy, but without DC crush injury), (2) DC crush injury + vehicle (partial laminectomy followed by DC crush injury and vehicle injection), (3) DC crush injury + AZD1236 at 100 mg / kg, (4) DC crush injury + AZD1236 at 200 mg / kg, and (5) DC crush injury + AZD1236 at 300 mg / kg. For intra-arachnoid (it) administration of AZD1236, adult male C57BL6 mice (25-30g) (Charles River, Margate, UK) were randomly assigned to one of the following groups: (1) Sham (control; partial laminectomy, but without DC crush injury), (2) DC crush injury + vehicle (partial laminectomy followed by DC crush injury and vehicle injection), (3) DC crush injury + AZD1236 at 2.5 mg / kg, (4) DC crush injury + AZD1236 at 5 mg / kg, and (5) DC crush injury + AZD1236 at 10 mg / kg. For intraarachnoid administration, as previously described, an intra-atlanto-occipital subarachnoid catheter was inserted into the subarachnoid space (FAOladosu, BPCiszek, SCO'Buckley, AGNackley, Novel intrathecal and subcutaneous catheter delivery systems in the mouse. J Neurosci Methods 264, 119-128 (2016)). Briefly, the mouse was placed in a lateral sternal position, a small incision was made in the neck, and the muscles on both sides of the external occipital crest were detached to expose the AO membrane. The membrane was incised, and a 6 cm Alzet mouse intraarachnoid catheter (Alzet, Cupertino, CA, USA) and a polyurethane segment were gently introduced 2.5 cm into the subarachnoid space. The catheter was secured using 4-0 silk sutures (Oasis Medical, IL), and the exposed end of the catheter was sealed with a stainless steel plug and fixed to the upper back. The animals were immediately injected with vehicle or AZD1236, followed by a 10 μl catheter flush with PBS.For the first three days after injury, injections of the drug and vehicle reagent were administered twice daily using a Hamilton microliter syringe (Hamilton Co, USA) over a period of one minute. Where specified, only intact animals that had not undergone surgical procedures were used. All injuries were performed under 5% isoflurane inhalation anesthesia with 1.5 l / min of O2. Analgesia was also administered before and after injury.

[0141] As previously described, DC crush injuries were inflicted on both sides of the T8 vertebral region (S. Surey, M. Berry, A. Logan, R. Bicknell, Z. Ahmed, Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62-80 (2014); MLRead, S. Mir, R. Spice, RJSeabright, EL Suggate, Z. Ahmed, M. Berry, A. Logan, Profiling RNA interference (RNAi)-mediated toxicity in neural cultures for effective short interfering RNA design. J Gene Med 11, 523-534 (2009)). Briefly, graduated watchmaker forceps were inserted 0.5 mm away from the dorsal spinal meninges to a depth of 0.45 mm in mice, and 1 mm away from the dorsal spinal meninges to a depth of 1.0 mm in rats, and the DC was crushed for 3 seconds. AZD1236 or vehicle was administered twice daily by oral force feeding or subarachnoid administration during the first 3 days or 24 hours immediately following the injury, and this treatment was continued twice daily until day 4 (i.e., AZD1236 was administered for 3 days). All experiments were performed with n=6 mice / group and repeated at least 2-3 times independently (total = 12-18 animals / groups / tests).

[0142] After exposure of T6-T9 vertebrae by laminectomy, CC SCI was performed on the T7-T8 vertebral region. As previously described, the aneurysm clip applicator was oriented bilaterally, and an aneurysm clip with a closing force of 24g was applied to the epidural space for 60 seconds (ASRivlin, CHTator, Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 10,38-43 (1978): E. Esposito, I. Pateriti, E. Mazzon, T. Genovese, M. Galuppo, R. Meli, P. Bramanti, S. Cuzzocrea, MK801 attenuates secondary injury in a mouse experimental compression model of spinal cord trauma. BMC Neurosci 12,31 (2011)).

[0143] Bladder was manually emptied twice daily until bladder function recovered. Adult male C57BL6 mice (25-30g) (Charles River, Margate, UK) were randomly assigned to one of the following groups: (1) Sham (control; laminectomy, but without CC SCI); (2) CC SCI + vehicle; (3) CC SCI + AZD1236 200 mg / kg (oral); (4) CC SCI + AZD1236 5 mg / kg (in). All experiments were performed with n=6 mice / group and independently repeated three times (total = 18 animals / groups / tests).

[0144] The efficacy of oral AZD3342 in a DC injury model in adult Sprague-Dawley rats was also evaluated. Animals were divided into six groups: (1) Sham (control); (2) DC crush injury + vehicle; (3) DC crush injury + AZD3342 at 15 mg / kg; (4) DC crush injury + AZD1236 at 75 mg / kg; (5) DC crush injury + AZD3342 at 375 mg / kg; and (6) DC crush injury + melatonin. AZD3342 or vehicle was administered orally twice daily to the animals by blinded experimenters. These experiments were conducted with n=6 rats / group and independently repeated 2-3 times (total n=12-18 rats / group / test).

[0145] As shown in the table below, all tools and clinically appropriate MMP inhibitors were administered and pre-optimized in our injury model.

[0146] [Table 1-1] [Table 1-2]

[0147] Axonal labeling with cholera toxin B To track axons retrogradely, a glass microneedle was used to expose the sciatic nerve in the mid-thigh region, after which 1% cholera toxin B (CTB) (No. 104, List Biologicals Laboratories, Campbell, CA, USA) was injected. The skin was sutured again, and the animals were allowed to recover for one week. After increasing the CO2 concentration, the animals were euthanized and perfused transcardially with 4% paraformaldehyde (TAAB laboratories, Berkshire, UK). Axons labeled with CTB were detected by immunohistochemical testing as described below.

[0148] Quantification of SCI-induced edema As previously described, 3mm on both sides of the injury site in mouse DC models and 5mm on both sides of the injury site in rat DC models were desecrated, and the water content (degree of edema) of the spinal cord was measured 3 days after DC and CC SCI (S. Li, CHTator, Effects of MK801 on evoked potentials, spinal cord blood flow and cord edema in acute spinal cord injury in rats. Spinal Cord 37, 820-832 (1999)). The spinal cord including the injury site was weighed on aluminum foil, dried at 105°C for 24 hours, and weighed again. The percentage of water content was calculated as follows: Water content (%) = [(wet weight - dry weight) / wet weight] × 100%.

[0149] Quantitative RT-PCR (qRT-PCR) The injured area was desecrated +5 mm from both sides (DC and CC SCI, n=6 mice / rat / group, repeated twice independently (total n=12 mice / rat / group)), rapidly frozen in liquid N2, and stored at -80°C until needed. Total RNA was extracted from the spinal cord at an appropriate time after injury, with or without treatment with TRIzol reagent according to the manufacturer's (Invitrogen) instructions. The mRNA levels of MMP-9, MMP-12, IL-1β, TNF-α, and IL-6 were measured using pre-validated mouse primer sequences from complementary DNA prepared from extracted mRNA, and qRT-PCR was performed using a LightCycler PCR machine (Roche, Burgess Hill, UK) (MLRead, S. Mir, R. Spice, RJSeabright, ELSuggate, Z. Ahmed, M. Berry, A. Logan, Profiling RNA interference (RNAi)-mediated toxicity in neural cultures for effective short interfering RNA design. J Gene Med 11, 523-534 (2009)). Mouse primer sequences include MMP-9, catalog number 4331182, Mm0044299_m1; MMP-12, catalog number 4331182, Mm00500554_m1; IL-1β, catalog number 4331182, Mm00434228_m1; TNF-α, catalog number 4331182, Mm00443258_m1; and IL-6, catalog number 4331182, Mm00446190_m1, and rat primers The sequences included MMP-9, catalog number 4331182, Rn00579162_m1; MMP-12, catalog number 4331182, Rn00588640_m1; IL-1β, catalog number 4331182, Rn00580432_m1; TNF-α, catalog number 4331182, Rn01525859_g1; and IL-6, catalog number 4881182, Rn01410330_m1 (all manufactured by ThermoFisher Scientific, Leicestershire, UK).The magnification change was calculated using the ΔΔCt method (MLRead, S. Mir, R. Spice, RJSeabright, ELSuggate, Z. Ahmed, M. Berry, A. Logan, Profiling RNA interference (RNAi)-mediated toxicity in neural cultures for effective short interfering RNA design. J Gene Med 11, 523-534 (2009).

[0150] Detection of MMP-9 and MMP-12 levels in the spinal cord The lesion site was desecrated by +3 mm on both sides (DC and CC SCI) (n=6 mice / rat / group, repeated twice independently, for a total of n=12 mice / rat / group), rapidly frozen in liquid N2, and stored at -80°C until needed. The samples were then homogenized in ice-cold lysis buffer containing a protease inhibitor. MMP-9 (ab253227, Abcam, Cambridge, UK) and MMP-12 (ab213878, Abcam) were detected using ELISA kits obtained from Abcam, following the manufacturer's instructions.

[0151] Detection of MMP-9 and MMP-12 enzyme activity In mice, a 3mm margin on both sides of the injury site (DC and CC SCI), and in rats, a 5mm margin on both sides of the injury site were desecrated (n=6 mice / rat / group, repeated twice independently, totaling n=12 mice / rat / group). These were rapidly frozen in liquid N2 and stored at -80°C until needed. The enzymatic activity of MMP-9 and MMP-12 was measured in 96-well plates using the SensoLyte 520 MMP-9 and MMP-12 fluorescence quantification assay kit, according to the manufacturer's instructions (AnaSpec, Fremont, CA, USA). Fluorescence intensity was measured at Ex / Em = 490 / 520 nm using a Synergy H1 microplate reader (BioTek UK, Swindon, UK).

[0152] In situ zymography and subsequent localization of astrocytes Animals were euthanized with an excess of CO2, perfused transcardially with warm PBS, and unfixed tissue was collected. 3mm and 5mm from both sides of the injury site were desecrated from mice (n=6 mice / rat / group, repeated twice independently, totaling n=12 mice / rat / group), immediately blocked with an optimal cutting temperature compound (OCT; Miles Inc, Elkhart, IL, USA), and stored at -80°C until cryostat sectioning was required. In situ zymography was performed as previously described (Z. Ahmed, RGDent, WE Leadbeater, C. Smith, M. Berry, A. Logan, Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons. Mol Cell Neurosci 28, 64-78 (2005)).

[0153] Briefly, 15 μm thick, unfixed frozen longitudinal sections of spinal cord were prepared in a cryostat and incubated at 25°C for 24 hours in 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.2 mM sodium azide, and 40 ug / ml fluorescein-conjugated DQ® gelatin (Molecular Probes). After proteolysis, highly fluoresceinated gelatin residues were separated and fluoresced. Control sections were incubated either without DQ® gelatin or with 50 μM 1,10-phenanthroline (Sigma, Poole, UK) to inhibit MMP activation. Next, the sections were fixed in 4% paraformaldehyde (TAAB Laboratories), washed with PBS, and incubated with anti-glial fibrillary acidic protein (GFAP) antibody (SAB5700611, 1:400 dilution, GFAP; Sigma) in a humidified chamber at room temperature for 1 hour. Then, the sections were washed with PBS and incubated with a suitable secondary antibody conjugated to Texas Red (Invitrogen). After a final wash with PBS, the sections were mounted in Vectashield containing DAPI (Vector Laboratories, Loughborough, UK) and observed using a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Hertfordshire, UK) equipped with AxioCam HRc and running Axiovision software.

[0154] Immunohistochemical examination After slaughtering the animals by exposure to high concentrations of CO2, the mice were perfused transcardially with 0.1 M phosphate-buffered saline (PBS) containing 4% formaldehyde (TAAB Laboratories, Berkshire, UK). 5 mm from both sides of the injury site was desecrated, fixed with 4% formaldehyde, and then exposed to a stepwise series of cryoprotective sucrose solutions. The spinal cord was blocked with OCT-mounted compound (TAAB Laboratories), and longitudinal sections 15 μm thick were prepared using a cryostat (Brights Instruments, Huntingdon, UK). These sections were then collected on charged glass slides (ThermoFisher Scientific, Loughborough, UK) and stored at -20°C until needed. As previously described, slides were numbered sequentially, and the central section of the injury site was selected for all immunohistochemical analyses (Surey S, Berry M, Logan A, Bicknell R, Ahmed Z. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 2014;275:62-80).

[0155] Three days after injury, the integrity of the spinal cord was assessed using albumin immunoreactivity in the spinal cord as a surrogate marker for BSCB failure. Sections were thawed at room temperature, washed with PBS, and then blocked for endogenous peroxides with H2O2. Next, sections were permeabilized in PBS containing 0.1% Triton X-100 at room temperature for 10 minutes, blocked with PBS containing 4% serum, and incubated overnight with rabbit anti-albumin primary antibody (ab271979; 1:1000 dilution, Abcam). Next, sections were washed with PBS and incubated with HRP-labeled anti-rabbit secondary antibody at room temperature for 1 hour, then washed with PBS and incubated with avidin-biotin complex for 30 minutes. Chromogenication was performed using a 3,3'-diaminobenzidine (DAB) substrate kit, dehydrated via a stepwise series of alcohols, cleared with Histoclear, and mounted with coverslips in Vectormount (all from Vector Labs).

[0156] Fluorescent immunohistochemical testing was performed as previously described (S. Surey, M. Berry, A. Logan, R. Bicknell, Z. Ahmed, Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62-80 (2014)). Briefly, longitudinal spinal cord sections were washed with PBS and incubated in PBS containing 1% (v / v) Triton X-100 (Sigma) to make the cells permeable. The sections were then blocked at room temperature (RT) for 30 minutes using PBS containing 0.05% (w / v) bovine serum albumin (Sigma, Poole, UK) and 0.05% Tween-20 (Sigma), and incubated overnight at 4°C in a humidified chamber with appropriate antibodies. Laminin was detected 4 weeks after DC injury and treatment with rabbit polyclonal anti-laminin primary antibody (ab11575; 1:400 dilution, Abcam). Macrophages were detected using rabbit polyclonal anti-CD68 antibody (ab1525212; 1:500 dilution, Abcam); microglia were detected using rabbit polyclonal antibody against CD11b (ab128797, 1:500 dilution, Abcam); and GFAP was detected using polyclonal anti-GFAP antibody (SAB5700611, 1:400 dilution, Sigma), all of which were detected 10 days after DC injury and treatment. Semaphorin 3A (Sema-3A) and chondroitin sulfate proteoglycan (CSPG) were detected 7 days after injury using monoclonal anti-CS-56 antibody (C8035; 1:200 dilution, Sigma).We selected this time point because several studies had shown that CSPG is observed around the injury site 7 days after injury (e.g., Tang X, Davies JE, Davies SJ. Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J Neurosci Res 2003;71(3):427-44). CTB-labeled axons in mice were detected 6 weeks after DC injury using goat polyclonal anti-CTB antibody (No. 703; 1:1000 dilution, List Biological Labs).

[0157] Next, the sections were washed with PBS and then incubated with Alexa488 and Alex595 conjugated secondary antibodies (Invitrogen; all used at a 1:400 dilution) at room temperature for 1 hour. Then, the sections were washed with PBS and mounted on coverslips using Vectashield mounting medium (containing DAPI) (Vector Laboratories, Peterborough, UK).

[0158] Negative controls were included in each test, but in this case, the primary antibody was omitted, and these slides were used to set the background threshold level before image acquisition. Sections (fluorescent and DAB stained) were observed using AxioCam HRc and Axioplan2 fluorescence microscopes equipped with Axiovision software (all from Zeiss, Hertfordshire, UK).

[0159] Quantification of immunofluorescence All analyses were performed by researchers blinded to the experimental groups. As previously described, relative fluorescence staining intensity was calculated by image analysis (Surey S, Berry M, Logan A, Bicknell R, Ahmed Z. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 2014;275:62-80). Briefly, micrographs taken at 5x magnification using the same standardized exposure settings throughout for each antibody were thresholded, and the average integrated intensity of the pixel / unit area for each antibody in n=12 mice / antibodies was recorded using ImageJ (NIH, USA).

[0160] DC axonal regeneration was quantified from sagittal spinal cord sections of the entire tissue of each mouse (total n=12 mice / group). Using ImageJ software, CTB intensity was quantified at a defined cranial distance from the center of injury, and the variability in tracked efficiency was expressed as a percentage of caudal CTB intensity at the injury site compared to the control.

[0161] Primary microglia culture and treatment Primary mouse microglia derived from the brains of 6-8 week old C57BL / 6 mice were prepared according to a previously described protocol (Moussaud S, Draheim HJ. A new method to isolate microglia from adult mice and culture them for an extended period of time. J Neurosci Methods 2010;187(2):243-53). Briefly, the meninges of the brain were cleaved and finely chopped by enzymatic digestion using 20 units / ml of Papain (all Sigma pipettes). After incubation at 37°C for 90 minutes, the suspension was centrifuged at 200g for 7 minutes, the pellet was resuspended in 0.5 mg / ml of DNase I (Roche, Manheim, Germany), and pulverized using a Pasteur pipette with a small diameter and heat-treated tip. Next, the homogenate was filtered through a 70 μm cell strainer (Beckton Dickinson, Watford, UK) and centrifuged via a Percoll (GE Healthcare, Amersham, UK) gradient. Finally, the cell suspension was resuspended in DMEM / F12 medium (all Invitrogen) supplemented with 10% FBS and 5 ng / ml granulocyte colony and macrophage-stimulating factor (GM-CSF) (No. 415-ML, R&D Systems, Watford, UK), plated into T75 cell culture flasks (Beckton Dickinson) pre-coated with poly-L-lysine, and maintained at 37°C, 5% CO2 for approximately 2 weeks. Once the cells reached confluence, microglia separated, migrated to the air interface, suspended, and proliferated. Next, the supernatant of the flask was collected without prior shaking, and microglia growing in the mixed glial culture substrate were removed and centrifuged at 200 g for 7 minutes. The purity of microglia was measured from each flask by immunocytochemistry against CD11b, confirming 95% purity. Prior to each experiment, microglia were cultured for at least 3 days in DMEM / F12 without GM-CSF.

[0162] Microglia were used in vitro in all experiments using 15-20 day old in vitro (DIV) cells, with 3 × 10⁶ cells. 4 Cells were seeded at a cell / well density onto glass coverslips in 24-well plates. Microglia were exposed to serum-free medium for 24 hours, and then, in preliminary experiments, the cells were exposed to different concentrations of lipopolysaccharide (LPS) to determine the optimal concentration required to maximally activate our primary microglia culture. This was determined to be 10 ng / ml. In the microglia activation assay, cells were exposed to serum-free medium for 24 hours, and then further exposed to LPS for 24 hours with or without different concentrations of AZD1236, GM6001, SD2590, and MMP-9 inhibitor I (all used at 10-500 ng / ml). Next, the cell culture supernatant was collected, centrifuged to remove cell debris, and subjected to ELISA to measure the concentrations of TNF-α, IL-1β, and IL-6. The experiment was performed in 3-well sets and repeated 3 times independently (n=9 wells / treatment).

[0163] ELISA for measuring cytokine levels TNF-α (number MTA00B), IL-1β (number MLB00C), and IL-6 (number M6000B) were measured using commercially available kits from R&D Systems, following the manufacturer's instructions.

[0164] cell culture J774A.1 cells (number TIB-67; ATCC, Middlesex, UK) were cultured in DMEM supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg amphotericin B. The cells were seeded in T75 tissue culture flasks and maintained at 37°C and 5% CO2. RAW264.7 cells (number TIB-71; ATCC) were maintained in DMEM containing 10% FBS.

[0165] Creation of peritoneal macrophages As previously described, commensal peritoneal cells were harvested from adult C57BL / 6 mice (6-8 weeks old) using a 21G needle into the peritoneal cavity and by injecting 10 ml of PBS to collect the cell suspension (Rosas M, Davies LC, Giles PJ, Liao CT, Khafan B, Stone TC, et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 2014;344(6184):645-8). Subsequently, the peritoneal cells were centrifuged at 100 × g for 10 minutes, and the cell pellet was resuspended in DMEM / F12 medium. The cells were incubated at 37°C for 2 hours, and non-adherent cells were removed by gentle washing. The cells were grown in DMEM / F12 medium containing 10% FBS. In the migration assay, cells were grown in RPMI containing 0.02% BSA for 24 hours, and the resting cells were collected and then used in the Transwell migration assay as described below.

[0166] Transwell Migration Assay The migration assay was performed using J774A.1, RAW264.7, and primary mouse peritoneal macrophages in a 6.5 mm Transwell plate (ThermoFisher) with 8 μm well-filled inserts, as previously described (Green et al., 2012). Briefly, the inserts were coated with rat tail type I collagen and 1 × 10⁶ inserts were used. 5Cells (J774A.1, RAW264.7, or primary mouse macrophages) were resuspended in chemotactic buffer (RMPI 1640, containing 0.02% BSA (referred to as RPMI herein)) and added to the upper chamber. They were then incubated with migration media containing or without known chemotactic factors MCP-1 (100 ng / ml) and PMA (100 nM), or AZD1236, GM6001, SD2590, and MMP-9 inhibitor I (all used at 1, 10, 100, and 1000 ng / ml) were added to the lower chamber. The cells were allowed to migrate through the insert membrane at 37°C for 3 hours, after which the insert was washed with PBS. Non-migrating cells remaining on the upper surface were removed with a cotton swab, while migrating cells on the insert were fixed, stained with Diff-Quick (No. 26096, Electron Microscopy Science, Hatfield, UK), and mounted on a glass slide. Migration was visually measured by counting using a light microscope at 40x magnification. The average number of cells in 10 random regions was calculated for each treatment by an experimenter blinded to the treatment conditions. The migration index was calculated by dividing the number of cells that migrated in response to the chemokine by the number of randomly migrating cells (RPMI medium) with a chemotactic reference index > 1.

[0167] Electrophysiological examination As previously described, compound action potentials (CAPs) were recorded 6 weeks after DC injury and treatment. For example, BCHaiins, CYSaab, ACLo, SGWaxman, Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion. SCI. Exp Neurol 188, 365-377 (2004).

[0168] In short, the experimenters were blinded to the treatment of the animals, and the CAP amplitude was calculated between the negative wave after the stimulation artifact and the peak of the next wave. The CAP region was also calculated by rectifying the CAP component (full-wave rectification), and that region was measured. To confirm our records and whether or not CAP was recorded, the dorsal half of the spinal cord was resected between the stimulating electrode and the recording electrode after each experiment.

[0169] Functional testing Functional tests for DC injury and post-treatment were performed as previously described (NDFagoe, CLAttwell, R. Eggers, L. Tuinenbreijer, D. Kouwenhoven, J. Verhaagen, MR Mason, Evaluation of Five Tests for Sensitivity to Functional Deficits following Cervical or Thoracic Dorsal Column Transection in the Rat. PLoS One 11, e0150141 (2016)).

[0170] In short, animals (n=18 / group) were first trained for one week to master traversing a horizontal ladder, and then functional tests were performed. Baseline parameters were established by performing tests 2-3 days before injury. Subsequently, animals were tested 2 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after DC injury and treatment. The experiment involved performing each test in three separate trials, in the same order and at the same time, and with treatment conditions blinded to observers.

[0171] Horizontal Ladder Test: This test assessed the walking motor function of animals and was conducted on a 0.9-meter-long horizontal ladder with a diameter of 15.5 cm and horizontal bars with randomly varied gaps of 3.5-5.0 cm. Animals were evaluated as they crossed the ladder, and the slippage of the left and right hind legs was recorded along with the total number of steps and the average error rate (number of slips / total number of steps).

[0172] Tape Removal (Sensory Function) Test: The tape removal test measures tactile perception of the left hind foot. After holding the animal with both hind legs extended, the time it took the animal to notice and remove a 15 x 15 mm piece of tape (Kip Hochkrepp, Bocholt, Germany) attached to the palm of its left hind foot was recorded, and the average perception time was calculated using these records.

[0173] Assessment of neuropathic pain (NP) Mechanical allodynia was measured in mice by pressing a series of von Frey filaments (0.25g-15g) onto the surface of the hind foot sole, with the treatment conditions blinded to the experimenters. Foot avoidance behavior was recorded as a positive response (F. Nasirinezhad, S. Gajavelli, B. Priddy, S. Jergova, J. Zadina, J. Sagen, Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats. Mol Pain 11,2 (2015)).

[0174] The minimum force required to elicit an escape response in the leg was recorded. Both hind legs were tested, with a 5-minute rest period between tests on the opposite leg.

[0175] Thermal hyperalgesia was tested using an established method for plantar heat sensitivity previously described (S. Surey, M. Berry, A. Logan, R. Bicknell, Z. Ahmed, Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62-80 (2014)). Briefly, animals were acclimatized to a transparent Perspex compartment before post-injury surgery, and treatment data were collected at the same time to ensure consistency. Animals were also acclimatized to the transparent Perspex compartment for 5 minutes before the start of the test. After the mice had settled, an infrared heat source (Harvard Apparatus, Kent, UK) was applied to the plantar surface of the hind paw. Retests were performed on the same paw at 30-second intervals, and reaction times for escape behavior of each hind paw were recorded in five separate tests. A single score for each animal was obtained by averaging the three median scores of the hind paws.

[0176] Cold allodynia was measured by experimenters blinded to the treatment conditions, as the number of foot avoidance behavior responses after applying acetone drops to the plantar surface of the foot (F. Nasirinezhad, S. Gajavelli, B. Priddy, S. Jergova, J. Zadina, J. Sagen, Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats. Mol Pain 11,2 (2015)). The test was repeated five times with a 5-minute interval between each test, and the frequency of responses to acetone was expressed as a percentage of response frequencies ([number of foot avoidance behaviors / number of tests] × 100).

[0177] statistical analysis Statistical significance was calculated from the sample mean using a one-way analysis of variance (ANOVA) with the post-hoc Dunnett method, using SPSS Statistics 19 (IBM, New York, USA). For the horizontal ladder crossing and tape removal tests, data were analyzed using the R package (www.r-project.org) as previously described (Tuxworth RIT, MJ; Anduaga, AM.; Hussien-Ali, A.; Chatzimatthaiou, S.; Longland, J.; Thompson, AM; Almutiri, S.; Alifragis, P.; Kyriacou, CP; Kysela, B.; Ahmed, Z. Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration. Brain Communications 2019;1(1): fcz005).

[0178] In short, in the ladder crossing test, the total time course of injured animals and sham-treated animals was compared using a binomial generalized linear mixed model (GLMM). The binomial GLMM was fitted to R using the lme4 package, which includes the glmer function. Next, p-values ​​were calculated using the parametric bootstrap method. In the tape removal test, a linear mixed model (LMM) was calculated by comparing models in R using the pbkrtest package along with the Kenward-Roger method (NDFagoe, CLAttwell, R. Eggers, L. Tuinenbreijer, D. Kouwenhoven, J. Verhaagen, MR Mason, Evaluation of Five Tests for Sensitivity to Functional Deficits following Cervical or Thoracic Dorsal Column Transection in the Rat. PLoS One 11, e0150141 (2016)).

[0179] In the pain behavior test, data between groups were compared using a two-way ANOVA with repeated measures followed by a Bonferroni post-hoc test.

[0180] All results are expressed as mean ± standard error (SEM). The error bars in the diagram represent the SEM.

[0181] result MMP-9 and MMP-12 show a rapid increase in expression after DC injury. We studied the spatial and temporal expression patterns of MMP-9 and MMP-12 in a mouse model of spinal cord injury (SCI) when the posterior column (DC) was injured bilaterally at the thoracic vertebra (T)8 level (Surey S, Berry M, Logan A, Bicknell R, Ahmed Z. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 2014;275:62-80).

[0182] qRT-PCR was used to confirm the time course of the elevation of MMP-9 and MMP-12 after injury. Consistent with the levels of MMP-9 in human SCI (Casha S, Rice T, Stirling DP, Silva C, Gnanapavan S, Giovannoni G, et al. Cerebrospinal Fluid Biomarkers in Human Spinal Cord Injury from a Phase II Minocycline Trial. J Neurotrauma 2018;35(16):1918-28), mouse MMP-9 mRNA expression also increased 1.9-fold within 1 hour after injury compared to sham-treated mice, peaking at 24 hours and rising to a 4.8-fold increase (Figure 1A). Subsequently, MMP-9 levels decreased, returning to control sham-treated levels 6 days after DC injury (Figure 1A). However, MMP-12 levels did not rise until 3 days after DC injury, peaking at 5 days, at which point they were 11.5 times higher than in sham-treated mice (Figure 1B). MMP-12 levels decreased 6 days after DC injury (Figure 1B). Protein levels of MMP-9 (Figure 1C) and MMP-12 (Figure 1D) reflected their mRNA levels, and the relative activity of these enzymes (Figures 1E and F) correlated with their changes in expression levels over time. These results indicate that both MMP-9 and MMP-12 mRNA, protein levels, and enzyme activity peak within the first 5 days after injury and then decline.

[0183] AZD1236 effectively inhibits the activity of MMP-9 and MMP-12 in the spinal cord, serum, and CSF. The activity levels of MMP-9 and MMP-12 in serum and CSF were measured after DC injury and administration of AZD1236. After oral administration of AZD1236 (200 mg / kg dose), the activity of MMP-9 and MMP-12 was suppressed by 90±2% and 90±3% in serum (Figures 2A and E), and by 74±2% and 69±3% in CSF (Figures 2B and E), respectively. After intra-abdominal administration, the activity of MMP-9 and MMP-12 was suppressed by 71±2% and 71±1% in serum (Figures 2C and E), and by 88±2% and 90±1% in CSF (Figures 2D and E), respectively. These results indicate that the most effective minimum dose of AZD1236 is 200 mg / kg for oral administration and 5 mg / kg for intra-abdominal injection. In the spinal cord, MMP-9 and MMP-12 activity was barely detectable in sham-treated spinal cords (Figure 2F), but DC injury increased MMP-9 and MMP-12 activity by 98±2% and 95±2%, respectively (Figure 2F). After oral and subarachnoid administration of the most effective minimum dose of AZD1236, MMP-9 activity in spinal cord tissue was suppressed by 88±4% and 87±5%, respectively, and MMP-12 activity was suppressed by 85±3% and 86±4%, respectively (Figure 2F).

[0184] Furthermore, in situ zymography of DC-injured spinal cord sections (Ahmed Z, Dent RG, Leadbeater WE, Smith C, Berry M, Logan A. Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons. Mol Cell Neurosci 2005;28:64-78) confirmed that gelatinase activity (green; arrowhead) induced by DC injury in the spinal cord adjacent to the injury site (*) was completely suppressed by orally and subarachnoidally administered AZD1236 (Figure 2G). In summary, these data demonstrate that orally or subarachnoidally administered AZD1236 can significantly suppress the activity of MMP-9 and MMP-12 induced by DC injury.

[0185] Treatment with AZD1236 to reduce water content (edema) caused by DC injury. We investigated whether suppressing the initial increase in MMP-9 and MMP-12 activity reduces water accumulation in the spinal cord caused by DC injury. DC injury significantly increased mean spinal cord water content from 71.0±1.0% to 78.4±1.5%, peaking at 3 days and then decreasing (Figure 3A). Oral administration of clinically appropriate doses of AZD1236 twice daily resulted in a dose-dependent decrease in spinal cord water, reducing it to sham treatment levels at 200 mg / kg and 300 mg / kg (Figure 3B). Intraarachnoid administration of AZD1236 also showed a nearly equivalent dose-dependent decrease in spinal cord water content, but the dose required to achieve the same decrease as oral administration was 1 / 40th of the dose (Figure 3C). Therefore, the increase in spinal cord water content caused by DC injury was reduced to sham treatment levels with AZD1236 at 5.0 and 10.0 mg / kg (Figure 3C). These results indicate that AZD1236 suppresses DC injury-induced edema by both oral and intra-arachnoid administration, and that intra-arachnoid administration requires only 1 / 40th the dose of oral administration.

[0186] We investigated whether the reduction of SCI-induced edema depended on the specific inhibition of MMP-9 or MMP-12. When tool inhibitors (MMP-9 inhibitor I and MMP408) were used alone or in combination, it was shown that combined inhibition of MMP-9 and MMP-12 was necessary to completely halt SCI-induced edema, and that inhibition of MMP-9 or MMP-12 alone was insufficient for therapeutic benefit (Figure 3D). In summary, these results indicate that AZD1236 inhibits SCI-induced edema, and that specific inhibition of both MMP-9 and MMP-12 is necessary to effectively halt SCI-induced edema.

[0187] CNS edema is more effectively eliminated by AZD1236 compared to other MMP inhibitors and is influenced by specific inhibition of both MMP-9 and MMP-12. The inventors compared orally administered AZD1236 with other publicly available clinical and tool-based MMP inhibitors (dosages and routes are shown in Table 1). Tool-based inhibitors included GM6001 (IC50=200pM) (Figure 4A), SB-CT (IC50=400nM) (Figure 4B), MMP-9 inhibitor I (IC50=5nM) (Figure 4C), SD2590 (IC50=0.18nM) (Figure 4D), and ND378 (Figure 4E).

[0188] GM6001 is a broad-spectrum MMP inhibitor also known as Galardin or Ilomastat, with the chemical name (2R)-N 4 -hydroxy-N 1 It is -[(1S)-1-(1H-indole-3-ylmethyl)-2-(methylamino)-2-oxoethyl]-2-(2-methylpropyl)butanediamide. This is commercially available, for example, from Tocris.

[0189] SB-3CT is a selective MMP-2 inhibitor with the chemical name 2-[[(4-phenoxyphenyl)sulfonyl]methyl]thiirane. It is commercially available, for example, from Tocris.

[0190] SD2590 is a potent MMP inhibitor with the chemical name N-hydroxy-1-(2-methoxyethyl)-4-[4-[4-(trifluoromethoxy)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide hydrochloride. It is commercially available, for example, from Tocris.

[0191] Inhibitor I is an MMP-9 inhibitor (see CAS 1177749-58-4), which is commercially available, for example, from Sigma-Aldrich.

[0192] MMP408 is an MMP-12 inhibitor (see CAS1258003-93-8) and is commercially available from Sigma-Aldrich.

[0193] Clinical-grade experimental therapeutic agents reported to inhibit MMPs included minocycline (IC50 = 272 μM) (Figure 4F), riluzole (Figure 4G), glibenclamide (Figure 4H), and melatonin (Figure 4I). Our data showed that melatonin, GM6001, MMP-9 inhibitor I, SD2590, riluzole, and glibenclamide significantly reduced spinal water content compared to DC+ vehicle-treated rats (P = 0.05 to 0.01), but AZD1236 was more effective than any of these compounds (P = 0.0001, AZD1236 vs. melatonin, GM6001, MMP-9 inhibitor I, SD2590, riluzole, and glibenclamide) (Figure 4J).

[0194] AZD1236 reduces pro-inflammatory pain markers and behavioral pain metrics. All of the pro-inflammatory pain markers IL-1β, TNF-α, and IL-6 showed a significant 6-7-fold increase in expression three days after DC injury (Figures 5A and B). However, oral or intra-abdominal administration of AZD1236 at doses of 200 mg / kg and 5 mg / kg reduced the elevation of these pain markers induced by injury, lowering them to sham treatment levels, respectively (Figures 5A and B). These results demonstrate that AZD1236 suppresses pro-inflammatory pain markers after SCI.

[0195] The DC model is a moderate severity model of SCI and can distinguish sensory and gait disturbances, but it is not suitable for detecting the effects of AZD1236 on tactile allodynia, thermal allodynia, and cold allodynia. However, the clip compression (CC) model is for severe injuries and can be used to assess pain in these animals (Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978;10(1):38-43; Esposito E, Paterniti I, Mazzon E, Genovese T, Galuppo M, Meli R, et al. MK801 attenuates secondary injury in a mouse experimental compression model of spinal cord trauma. BMC Neurosci 2011;12:31). Therefore, using the SCI CC model, we investigated whether inhibition of MMP-9 and MMP-12 with AZD1236 attenuates contact allodynia, thermal allodynia, and cold allodynia. First, we confirmed that treatment of CC mice with the same dose of AZD1236 as in the DC injury model, either orally or intraarachnoidally, attenuated spinal cord water content, IL-1β, TNF-α, and IL-6 expression, and MMP-9 / MMP-12 activity in the clip compression model, similar to what was observed in the DC model (Figures 6A-C). The suppression of pro-inflammatory markers, spinal cord water content, and MMP-9 / MMP-12 activity by AZD1236 all significantly improved (to >80% of the sham-treated control) the measured values ​​of mechanical allodynia (Figure 6D), thermal allodynia (Figure 6E), and cold allodynia (Figure 6F). In addition, improvement in mechanical, thermal, and cold allodynia was significantly better in animals treated with AZD1236 than in animals treated with pregabalin and gabapentin, which are currently approved neuropathic pain relievers (Figures 6D-F).These improvements correlate with AZD1236's superior ability to suppress the pro-inflammatory pain markers IL-1β, TNF-α, and IL-6 compared to pregabalin and gabapentin (Figure 7). These results suggest that AZD1236 may also be useful in suppressing SCI-induced neuropathic pain and may even be more effective than currently used analgesics.

[0196] AZD1236 maintains CAP amplitude and improves walking motion and sensory function. To determine whether AZD1236-induced reduction of spinal cord fluid content improves functional outcomes after DC injury, compound action potentials (CAPs) across the entire spinal cord injury site were measured by electrophysiological testing with and without AZD1236 treatment.

[0197] When representative CAP traces of sham controls processed with Spike2 software were superimposed, the DC+vehicle and DC+200 mg / kg AZD1236 groups showed that negative CAP waves were eliminated in the DC+vehicle group compared to the sham-treated controls (Figure 8A). However, oral administration of AZD1236 significantly restored the CAP waves (Figure 8A). At the end of the experiment, unilateral dorsal spinal cord resection stopped CAP tracing in all animals, confirming the technical success of the experiment. The average CAP amplitude was also smaller in the DC+vehicle group, while very large CAP amplitudes were observed at all stimulus intensities after AZD1236 treatment (Figure 8B). Similarly, the CAP area (0.65±0.02mV×ms) in the sham-treated group decreased to 0.06±0.02mV×ms in the DC+vehicle-treated group, but recovered to 80% of the CAP area observed in the sham-treated controls (0.52±0.09mV×ms) (Figure 8C). These improvements in electrophysiological properties across the entire DC injury site resulted in significant improvements in both gait and sensory function (85% better compared to sham-treated animals). For example, the mean error rate in the ladder crossing test remained between 0.15 and 0 over a 6-week period (Figure 8D). After DC injury, the mean error rate increased to 0.59±0.06, and significant impairment (P<0.0012, generalized linear mixed model) persisted over a 6-week period.

[0198] However, treatment with AZD1236 improved the mean error rate at all time points up to 2 weeks after injury, with a significant improvement in animals treated with AZD1236 (P<0.0001, independent sample t-test), and no difference compared to animals treated with sham (Figure 8D). Mean tape detection and removal time also increased by 77.8 ± 5.0 seconds in DC + vehicle treated animals (Figure 8E). Treatment with AZD1236 significantly reduced mean tape detection and removal time, showing a significant improvement by 3 weeks compared to the DC + vehicle treated group (P<0.0001, independent sample t-test), and there was no difference between the animals and the sham treated group (Figure 8E).

[0199] Intra-arachnoid administration of the minimum effective dose of AZD1236 also yielded similarly significant improvements compared to oral administration of AZD1236 in CAP wave, CAP amplitude, CAP area, mean error rate, and mean tape detection and removal time (Figures 9A-E).

[0200] These results indicate that inhibition of MMP-9 and MMP-12 using AZD1236 significantly improved electrophysiological function, gait function, and sensory function. These results also indicate that oral and subarachnoid administration of AZD1236 resulted in similar beneficial improvements in electrophysiological function, gait function, and sensory function after DC injury.

[0201] Inhibition of MMP-9 and MMP-12 by AZD1236 promotes DC axon regeneration and axon preservation in the upper and lower parts of the injured site. Next, we investigated DC axonal regeneration / sprout formation by labeling regenerating fibers at the injury site using the retrograde tracer cholera toxin B (CTB). In DC+vehicle-treated mice, CTB-labeled axons (red) were not observed beyond the injury site (#) (Figure 10A). However, in DC+AZD1236-treated mice, CTB + It was observed that the axon regenerated, penetrating the injury site (#), and extended into the cranial spinal cord (Figure 10A and the inset show high-magnification views of the axon in the cranial spinal cord). CTB + Quantification of labeling intensity showed that the DC+AZD1236 treatment group had a significantly higher proportion of CTB-labeled axons extending at least 1000 μm from the center of the injury compared to the DC+vehicle treatment group (P=0.0001, ANOVA) (Figure 9B).

[0202] NF200 in cross-sectional views of the spinal cord at T9 (upper part of the injury) and T7 (lower part of the injury) +Immunostaining of excess fibers (Figure 10C) and subsequent quantification (Figure 10D) showed that AZD1236 significantly protected axonal fibers in both the upper (T9) and lower (T7) regions of the injury site. These results indicate that oral AZD1236 not only promoted DC axonal regeneration throughout the injury site but also enhanced protection of axons in both the upper and lower regions of the injury site.

[0203] AZD1236 significantly promotes DC axon regeneration more than other MMP inhibitors. Furthermore, CTB regeneration after treatment with AZD1236 + The quantification of DC axon numbers was compared with other MMP inhibitors such as GM6001, SDF2590, and MMP-9 inhibitor I (each MMP inhibitor that was excellent in reducing edema after DC injury). The inventors found that other MMP inhibitors had only a slight effect on axon regeneration, while AZD1236 was far superior in promoting DC axon regeneration after injury (Figure 11).

[0204] Similarly beneficial is the delayed inhibition of MMP-9 and MMP-12 after DC damage. Because establishing accurate clinical diagnosis and treatment regimens for SCI patients can take time, a clinically reasonable 24-hour delay before administering AZD1236 to animals was evaluated. Even with this 24-hour delay, the optimal dose of AZD1236 was as effective as immediate administration in suppressing water content (Figure 12A), pro-inflammatory cytokines (Figure 12B), and MMP-9 and MMP-12 activity (Figure 12C) due to SCI, and further improving dorsal spinal cord potentials (Figure 11D), CAP amplitude (Figure 12E), CAP area (Figure 12F), gait motor function (Figure 12G), and sensory function (Figure 12H). Furthermore, the 24-hour delay treatment with AZD1236 was as effective as immediate administration with the same dose of CTB. + The fibers penetrated the damaged area (#) and facilitated their regeneration and entry into the cranial spinal cord (Figures 13A and B).

[0205] These results demonstrate significant clinical potential for AZD1236 in treating SCI, given that a clinically reasonable time delay in treatment with AZD1236 is as effective as immediate treatment.

[0206] Inhibition of MMP-9 and MMP-12 by AZD1236 (oral administration) reduces BSCB breakdown, scarring, microglia activation, and macrophage infiltration at the site of injury. The blood-spinal cord barrier (BSCB) is functionally equivalent to the blood-brain barrier (BBB) ​​and plays a role in providing a unique microenvironment for the extracellular components of the spinal cord (Bartanusz V, Jezova D, Alajajian B, Digicaylioglu M. The blood-spinal cord barrier: morphology and clinical implications. Ann Neurol 2011;70(2):194-206). Damage to the BSCB occurs after spinal cord injury (SCI) and leads to progressive bleeding resulting in secondary injury mechanisms and permanent neurological deficits (Tran AP, Warren PM, Silver J. The Biology of Regeneration Failure and Success After Spinal Cord Injury. Physiol Rev 2018;98(2):881-917). Three days after injury and treatment (or four days in the 24-hour delayed group), BSCB integrity was assessed using albumin immunoreactivity in the spinal cord as a surrogate marker for BSCB failure.

[0207] Significant albumin immunoreactivity (arrows) was observed throughout the injured area (#) and surrounding spinal cord parenchymal tissue in the section 3 days after injury, indicating that significant BSCB disruption occurred rapidly throughout the spinal cord 3 days after injury in the DC+vehicle treatment group (Figures 14A and B). However, oral administration of AZD1236 significantly suppressed BSCB disruption (arrows) in this area by >75% compared to the DC+vehicle treatment group, both immediately after injury and 24 hours after treatment (P<0.0001, ANOVA), and suppressed it by >73% in the group treated with AZD1236 24 hours later (Figures 14A and 14B), demonstrating that BSCB disruption was prevented.

[0208] Similarly, laminin immunoreactivity defining the extracellular matrix boundaries of scar tissue 4 weeks after DC injury showed broad staining areas (arrows) at the injury site (#) in DC+vehicle-treated animals, indicating the presence of significant scarring (Figure 14C and D). Both groups that received AZD1236 immediately after injury (>80% attenuation, P=0.0001, ANOVA) and those that received a 24-hour delayed treatment (>77% attenuation, P=0.00011, ANOVA) showed only low levels of laminin immunoreactivity (Figure 14C and D).

[0209] These results suggest that when administered immediately after injury or 24 hours later, AZD1236 suppresses both acute BSCB breakdown and injury site scarring.

[0210] AZD1236 treatment also significantly suppressed the immunoreactivity of Sema-3A (Figures 14E and F) (>84% attenuation, P=0.0001, ANOVA) and CS-56 (Figures 14G and H) (>81% attenuation, P<0.0001, ANOVA) at the injury site, both immediately after injury and 7 days (or 8 days in the 24-hour delayed group), indicating that the deposition of scar-related molecules at the injury site was inhibited. Furthermore, AZD1236 treatment, both immediately after injury and 24 hours later, suppressed the immunoreactivity of CD11b (Figures 14I and J; inset i = high-magnification view of the area enclosed by a rectangle showing microglial activation), CD68 (Figures 14I and K; inset ii = high-magnification view of the area enclosed by a rectangle showing macrophage activation / infiltration), and GFAP at the injury site, both immediately after injury and 24 hours later. + (Figures 14I and L) Immunoreactivity was reduced, and all of these were significantly reduced when quantified by image analysis (P=0.0001, ANOVA) (Figures 14J-L).

[0211] These results demonstrate that AZD1236 treatment, whether immediately or with a 24-hour delay, equally and potently suppresses SCI-induced BSCB rupture, scarring at the injury site, macrophage infiltration, and activation of microglia and astrocytes at the injury site.

[0212] Inhibition of MMP-9 and MMP-12 by AZD1236, which removes common CSF biomarkers in SCI. Biomarkers in CSF and serum are increasingly being used to stratify SCI injuries in terms of post-injury severity and potential for recovery. We analyzed some of the most commonly reported post-SCI biomarkers, including S100β, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), phosphorylated neurofilamentous heavy chain (pNF-H), and neurofilamentous light chain (NF-L), and compared their levels after AZD1236 treatment. Levels of S100β, NSE, GFAP, pNF-H, and NF-L all increased significantly 3 days after injury (Figure 15A-E). While melatonin treatment slightly decreased the levels of these biomarkers in CSF, AZD1236 significantly reduced all of these biomarkers to near sham control levels 3 days after injury, either immediately or with a 24-hour delay. These results suggest that AZD1236 also helps suppress common biomarkers of SCI and could be clinically used to monitor SCI progression.

[0213] AZD1236 suppresses LPS-induced cytokine production by microglia but does not affect macrophage migration in vitro. To measure the effects of AZD1236 on microglia activation and macrophage migration, primary adult mouse brain microglia were isolated and subjected to LPS activation with and without AZD1236 treatment. Subsequently, ELISA was performed for TNF-α, IL-1β, and IL-6. 10 ng / ml of LPS stimulated the production of TNF-α, IL-1β, and IL-6 at concentrations of 196±15, 21±4, and 1964±117 pg / ml, respectively (Figures 16A-C). However, increasing the concentration of AZD1236 simultaneously with LPS activation dose-dependently reduced these cytokines, and 100 ng / m³ of AZD1236 returned their concentrations to DMEM / F12 medium control levels (Figures 16A-C). Increasing the concentration of AZD1236 to 500 ng / ml completely suppressed the production of these cytokines, even in the presence of LPS that activates microglia (Figures 16A-C). In contrast, other MMP inhibitors such as GM6001, SD2590, and MMP9 inhibitor I had little effect on suppressing these cytokines (Figures 16A-C).

[0214] AZD1236 and other MMP inhibitors did not affect the migration of primary macrophages or the J744A.1 and RAW264.7 macrophage cell lines, as migration indicators remained at baseline control concentration levels (Figure 16D). On the other hand, both positive controls, MCP-1 and PMA, significantly stimulated chemotaxis in primary peritoneal macrophages and both macrophage cell lines (Figure 16D).

[0215] These results indicate that AZD1236 significantly suppresses microglial activation and therefore the secretion of cytokines such as TNF-α, IL-1β, and IL-6, but does not affect macrophage migration.

[0216] Inhibition of MMP-9 and MMP-12 is also effective in rat models of SCI. We investigated whether suppression of MMP-9 and MMP-12 reduces edema and improves functional recovery in a rat DC injury model that better reproduces the pathophysiology of human SCI (Surey S, Berry M, Logan A, Bicknell R, Ahmed Z. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 2014;275:62-80). For example, similar to humans, after DC injury, rats also form fluid-filled cysts that increase over time, further damaging spinal cord tissue and causing axonal rupture. In addition, these fluid-filled cysts are surrounded by scar tissue, which further hinders axonal regeneration / sprout formation.

[0217] The inventors confirmed that the same mRNA expression profiles for MMP-9 (Figure 17A) and MMP-12 (Figure 17B) were observed in rats after DC injury, compared to mice. Since AZD1236 is inactive against rat MMP-9 / MMP-12, AZD3342, an MMP inhibitor that is active in rats and has nearly equivalent selectivity to AZD1236, was selected (for rat MMP-9 and MMP-12 respectively). 50Using AZD3342 (117nM and 35nM), we investigated whether SCI-induced edema (Figure 17C), MMP-9 and MMP-12 activity (Figure 17D), and pro-inflammatory pain-related cytokines (Figure 17E) could be suppressed in a rat DC injury model. Treatment with AZD3342 significantly improved electrophysiological CAP tracing (Figure 17F), as well as gait function (Figure 17G) and sensory function (Figure 17H), similar to what was observed with AZD1236 in a mouse SCI model. All of these changes in edema, pro-inflammatory pain markers, CAP tracing, gait function, and sensory function were remarkably superior compared to melatonin treatment (used as a positive control). These results indicate that inhibition of MMP-9 and MMP-12 is effective against SCI-induced edema and neuropathic pain, preventing functional loss, even in a rat model of SCI.

[0218] Activity levels of MMP-9 and MMP-12 in a typical injury site within 5 days after discontinuing AZD1236. The time it took for MMP-9 and MMP-12 to return to standard SCI-inducing activity levels at the injury site was investigated after discontinuing oral AZD1236 three days later. The results showed that it took 4 days for MMP-9 and 5 days for MMP-12 to return to the activity levels observed after the DC+ vehicle injury group (Figure 18A and B). These results indicate that standard MMP-9 and MMP-12 activity levels return 4-5 days after the last dose of AZD1236, well before the wound healing phase of the CNS.

[0219] AZD1236 treatment that does not affect MMP-2 activity We investigated whether oral and subarachnoid administration of effective doses of AZD1236 unintentionally affected MMP-2 activity. However, MMP-12 activity assays showed no difference in the increase in MMP-2 activity induced by DC injury after treatment with AZD1236, suggesting that there were no off-target effects on MMP-2 activity by AZD1236 at the doses used (Figure 19).

[0220] Example 2 Spinal fluid content was measured for both AZD1236 and AZD3342 as a measure of injury-induced edema using a technique similar to that described in Example 1 (see Figure 20).

[0221] Example 3 Using a technique similar to that described in Example 1, spinal cord water content was measured as a measure of injury-induced edema for various drugs and aquaporin 4 inhibitors, namely TFP, PKAi, PKCi, and TGN-020 (see Figure 21).

[0222] TFP (trifluoperazine), also known as Stelazine, Eskazinyl, Eskazine, or Jatroneural, is commercially available.

[0223] PKAi are protein kinase A inhibitors, and PKCi are protein kinase C inhibitors.

[0224] TGN-020 is an aquaporin 4 (AQP4) channel blocker with the chemical name N-1,3,4-thiadiazole-2-yl-3-pyridinecarboxamide. It is commercially available, for example, from Tocris.

Claims

1. Compounds or combinations of compounds for use in the treatment of spinal cord injury (SCI) or related nerve tissue injury, or for use in the treatment of secondary effects associated with said SCI or related nerve tissue injury, comprising selectively inhibiting the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after said SCI or related nerve tissue injury.

2. A compound or combination of compounds for use according to claim 1 in the treatment of secondary effects associated with SCI or related nerve tissue damage, comprising selectively inhibiting the activity or expression of both matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue damage.

3. The compound or combination of compounds for use according to claim 2, wherein the secondary effect to be treated is SCI-induced edema or neuropathic pain (NP).

4. The compound or combination of compounds for use according to claim 3, wherein the SCI-induced edema is completely suppressed or suppressed by 25% to 50%, for example, 30%, after treatment.

5. The compound or combination of compounds for use according to claim 1 or 2, wherein the damage to the associated nerve tissue is traumatic brain injury or stroke.

6. A compound or combination of compounds for use according to any one of claims 1 to 5, wherein a single selective MMP-9 and MMP-12 inhibitory compound, or a combination of a selective MMP-9 compound and a separate selective MMP-12 inhibitory compound, is used.

7. A compound for use according to any one of claims 1 to 5, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used.

8. The single selective MMP-9 and MMP-12 inhibitory compound has an IC50 range of 1 nM to 100 nM for both MMP-9 and MMP-12. ５０ A compound for use according to claim 6 or 7, having the properties of:

9. The single selective MMP-9 and MMP-12 inhibitory compound exhibits an IC50+ of 100 nM relative to MMP-2. ５０ A compound for use according to claim 6 or 7, having the properties of:

10. The single selective MMP-9 and MMP-12 inhibitory compound has an IC50 range of 1 nM to 100 nM for both MMP-9 and MMP-12. ５０ , and ICs exceeding 200 nM compared to MMP-2 ５０ A compound for use according to claim 8 or 9, having the properties of:

11. The compound for use according to claim 6, 7, or 8, wherein the single selective MMP-9 and MMP-12 inhibitory compound is AZD1236 or AZD3342, or a pharmaceutically acceptable salt thereof.

12. A compound for use according to claim 6, 7, 8, or 11, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, and the single selective MMP-9 and MMP-12 inhibitory compound is AZD1236 or a pharmaceutically acceptable salt thereof.

13. The compound or combination of compounds for use according to any one of claims 1 to 12, wherein the activity or expression of MMP-9 and MMP-12 is inhibited by administering a single selective MMP-9 and MMP-12 inhibitory compound, or a combination of selective MMP-9 and a separate selective MMP-12 inhibitory compound, for a short period of time, for example, over 1, 2, or 3 days, after injury to SCI or associated nerve tissue.

14. The compound or combination of compounds for use according to any one of claims 1 to 13, wherein the activity or expression of MMP-9 and MMP-12 is inhibited by intraarachnoid administration of a single selective MMP-9 and MMP-12 inhibitor, or a combination of selective MMP-9 and a separate selective MMP-12 inhibitor.

15. The compound or combination of compounds for use according to any one of claims 1 to 14, wherein the activity or expression of MMP-9 and MMP-12 is inhibited by intraarachnoid administration of a single selective MMP-9 and MMP-12 inhibitor, or a combination of a selective MMP-9 and a separate selective MMP-12 inhibitor, at a dose typically of 1 / 40, which is 1 / 20 to 1 / 60 of the dose required for oral administration.

16. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to treat SCI-induced edema.

17. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to treat neuropathic pain (NP).

18. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to mitigate BSCB failure.

19. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to reduce hematopoietic cells in the CNS.

20. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to reduce scarring at the site of SCI injury.

21. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used for the prevention of scarring.

22. A compound for use according to any one of claims 6 to 15, wherein a single selective MMP-9 and MMP-12 inhibitory compound is used, the single selective compound being AZD1236 or a pharmaceutically acceptable salt thereof, and the AZD1236 or a pharmaceutically acceptable salt thereof is used to promote axonal regeneration.

23. The compound for use according to any one of claims 1 to 22, wherein the compound or a pharmaceutically acceptable salt thereof is administered for a short period, for example, over 1, 2, or 3 days, after SCI or associated nerve tissue injury.

24. The compound for use according to claim 23, wherein the AZD1236 or a pharmaceutically acceptable salt thereof is administered orally twice daily at a dose of 50 to 100 mg, for example, about 75 mg twice daily.

25. A method for treating spinal cord injury (SCI) or related nerve tissue injury, or for treating secondary effects associated with said SCI or related nerve tissue injury, comprising selectively inhibiting the activity or expression of matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after said SCI or related nerve tissue injury.

26. A method according to claim 25 for treating secondary effects associated with spinal cord injury (SCI) or related nerve tissue injury, comprising selectively inhibiting the activity or expression of matrix metalloproteinase MMP-9 (gelatinase B) and metalloelastase MMP-12 after such SCI or related nerve tissue injury.

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