Methods for treating spinal cord injury and compositions for use therein

Abstract

Provided is a method of treating spinal cord injury, comprising administering to a subject a compound of Formula (I), (II), or (III), or a pharma- ceutically acceptable salt thereof. JPEG2025505186000009.jpg53170 (wherein X is a halogen) Also provided are compositions for use in a method of treating spinal cord injury comprising administering to a subject in need thereof a composition comprising a compound of Formula (I), (II), or (III) as shown above.

Description

[Technical field]

[0001] The present disclosure relates to methods of treating spinal cord injury and compositions for use in methods of treating spinal cord injury. [Background technology]

[0002] Approximately 27 million individuals worldwide live with a spinal cord injury (SCI), with close to 1 million new cases per year. SCI is therefore a devastating neurological disorder that constitutes a significant portion of the global injury burden, and the number of individuals living with SCI is expected to increase in light of population growth. 1 SCIs are most often caused by a sudden and traumatic impact to the spine, resulting in dislocation or fracture of a vertebra. Following the initial mechanical impact that alters the vascular tissue and blood supply to the area of ​​SCI, secondary injuries can include hemorrhage, ischemia, infiltration of immune cells and release of inflammatory cytokines, and ultimately neuronal death. 2 SCI is generally considered an irreversible neurological injury, and its treatment remains an unmet medical need. Neuroinflammation is a key component of secondary injury mechanisms in SCI 3 Interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) are thought to be crucial mediators of post-traumatic inflammatory responses that can lead to neuronal cell death. 4 Additionally, activation of microglia and astrocytes is a prominent response of neuroinflammation. While astrogliosis is recognized as a deleterious effect of SCI, emerging studies suggest that reactive astrocytes may also play a protective role in SCI. 5 Reactive astrocytes can develop into two types, A1 and A2, respectively. A1 astrocytes have been reported to exert cytotoxic effects on local neurons and oligodendrocytes, whereas A2 astrocytes may promote neuronal outgrowth and survival and contribute to synapse formation and tissue repair. Summary of the Invention

[0003] The present invention provides a novel method of treating spinal cord injury (SCI) by using adenosine analog compounds that are inhibitors of equilibrative nucleoside transporter 1 (ENT1) in the pathogenesis and functional recovery of SCI. In one embodiment of the invention, there is provided a method of treating spinal cord injury, comprising administering to a subject in need thereof a compound of Formula (I), (II), or (III), a pharma- ceutically acceptable salt thereof, or a composition thereof. [ka] (wherein X is a halogen)

[0004] In another aspect of the present invention, a composition for use in a method of treating spinal cord injury comprising administering to a subject in need thereof a composition comprising a compound of formula (I), (II), or (III) as shown above.

[0005] Preferably, the compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 -[(5-halothien-2-yl)methyl]adenosine. More preferably, the compound is selected from the group consisting of N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine, N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6-[(4-chlorothien-2-yl)methyl]adenosine, and N 6 -[(3-chlorothien-2-yl)methyl]adenosine.

[0006] Preferably, the compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 More preferably, the compound is selected from the group consisting of N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromothien-3-yl)methyl]adenosine, N 6 -[(4-bromothien-3-yl)methyl]adenosine, N 6 -[(5-bromothien-3-yl)methyl]adenosine, N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 -[(5-chlorothien-3-yl)methyl]adenosine.

[0007] Preferably, the compound, a pharma- ceutically acceptable salt thereof, or a composition thereof is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical routes. Preferably, the composition further comprises a pharma- ceutically acceptable carrier, excipient, or vehicle. Thus, the present invention provides at least the following advantages: 1. The claimed method can significantly reduce IL-1β, IL-6, and TNF-α mRNA levels, as well as astrocyte and microglia / macrophage activation in the perilesional region of the spinal cord. 2. The claimed methods can reduce neuroinflammation and neuronal damage in SCI, thereby improving functional recovery. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing that J4 was injected intraperitoneally 3 days prior to injury, followed by continuous daily injections for 14 days following spinal cord injury, in accordance with an embodiment of the present invention. [Diagram 2] FIG. 2 illustrates the Basso Mouse Scale (BMS) of J4-treated mice on different days after spinal cord contusion, according to an embodiment of the present invention. [Diagram 3] FIG. 3 shows representative images and quantitative results of footprint analysis of J4-treated mice 14 days post-injury (14 DPI), according to an embodiment of the present invention. [Figure 4] 4 shows Nissl staining of sagittal sections of spinal cords of J4-treated mice at 14 DPI and quantitative results of lesion volume according to an embodiment of the present invention. Scale bar is 500 μm. [Diagram 5] 5 shows representative images of NeuN (green) in the perilesional area of ​​J4-treated mice and the quantitative results thereof, in accordance with an embodiment of the present invention. Scale bar is 50 μm. [Figure 6] FIG. 6 shows protein levels of NeuN at T9-T11 in J4-treated mice 14 DPI and quantitative densitometric analysis of these proteins according to an embodiment of the present invention. [Figure 7] FIG. 7 shows protein levels of c-caspase 3 at T9-T11 in J4-treated mice 14 DPI and quantitative densitometric analysis of these proteins according to an embodiment of the present invention. [Figure 8-1] FIG. 8 shows mRNA levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the perilesional area of ​​J4-treated mice 14 days after injury, according to an embodiment of the present invention. [Figure 8-2]FIG. 8 shows mRNA levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the perilesional area of ​​J4-treated mice 14 days after injury, according to an embodiment of the present invention. [Figure 9] 9 shows representative images of Gfap (green) and Iba-1 (E, red) in the perilesional area of ​​J4-treated mice and their quantitative results according to an embodiment of the present invention. Scale bar is 100 μm. [Figure 10] 10 shows representative images of Iba-1 (red) in the perilesional area of ​​J4-treated mice and the quantitative results thereof, according to an embodiment of the present invention. Scale bar is 100 μm. [Figure 11] FIG. 11 shows immunoblots of C3 and densitometric quantification of the protein in J4-treated mice, according to an embodiment of the present invention. [Figure 12] FIG. 12 shows immunoblots of S100a10 and densitometric quantification of the protein in J4-treated mice, according to an embodiment of the present invention. [Figure 13] FIG. 13 shows immunoblots of TGF-β and densitometric quantification of the protein in J4-treated mice, according to an embodiment of the present invention. [Figure 14] FIG. 14 shows, according to an embodiment of the present invention, that JMF1907 treatment can significantly improve motor function in terms of BMS score (A) and stride length (B) in mice with SCI. [Figure 15] FIG. 15 shows that JMF1907 treatment significantly reduced SCI lesions as examined by Nissl staining and Pdgfβ expression, according to an embodiment of the present invention. [Figure 16] FIG. 16 illustrates that JMF1907 treatment can attenuate neuroinflammation in SCI by reducing the expression of Gfap, according to an embodiment of the present invention. [Figure 17]FIG. 17 illustrates that JMF1907 treatment can attenuate neuroinflammation in SCI by reducing the expression of Iba-1 and CD38, according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In one embodiment, there is provided a method of treating spinal cord injury (SCI), comprising administering to a subject a compound of Formula (I), (II), or (III), a pharma- ceutically acceptable salt thereof, or a composition thereof. [ka] (wherein X is a halogen) Herein, the compound of formula (III) is also referred to as "JMF 1907."

[0010] In another embodiment, the compound is 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 -[(5-halothien-2-yl)methyl]adenosine. Preferably, the compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine (also referred to as "JMF3464" or "J4"), N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine (also called "JMF3818"), N 6 -[(4-chlorothien-2-yl)methyl]adenosine, N 6-[(3-chlorothien-2-yl)methyl]adenosine, or a combination thereof.

[0011] In another embodiment, the compound is 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 -[(5-halothien-3-yl)methyl]adenosine. Preferably, the compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromothien-3-yl)methyl]adenosine, N 6 -[(4-bromothien-3-yl)methyl]adenosine, N 6 -[(5-bromothien-3-yl)methyl]adenosine N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, or N 6 -[(5-chlorothien-3-yl)methyl]adenosine, or a combination thereof. In one embodiment, the compound, a pharma- ceutically acceptable salt thereof, or a composition thereof is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical routes. EXAMPLES

[0012] Materials and Methods animal Induction of spinal cord contusion Mice were anesthetized by intramuscular injection of Zoletil® (50 mg / kg, tiletamine hydrochloride and zolazepam hydrochloride) and Rompun® (12 mg / kg, xylazine hydrochloride) before surgery. After laminectomy at T9-T10 of the spinal column, the spinal cord was exposed and crushed by dropping a 10 g rod with an impact head of 1.2 mm diameter at a height of 6.25 mm above the T10 spinal cord using Impactor model-II spinal cord crush system (RWD68099II) to cause crush injury. In the sham group, the surgical procedure was identical except for the spinal cord crush injury. After injury, the wound was quickly sutured using 4.0 silk thread (Unik Taiwan, New Taipei city, Taiwan). The bladder of the mice was manually and gently massaged once a day to avoid urine retention until reflex control of micturition was restored. Cefazolin (300 mg / kg) was given for three consecutive days after surgery to prevent infection. Mice were sacrificed for analysis 14 days after surgery.

[0013] Pharmacological treatment In one example of treatment of J4, which can be used as an ENT1 inhibitor, male C57BL / 6J mice (8-12 weeks old) were divided into three groups: sham group, SCI+J4 group, and SCI+vehicle group. See Figure 1. In the J4 group, mice were pretreated with 10 mg / kg J4 twice a day for 3 days before injury. After SCI, mice were administered J4 (10 mg / kg) twice a day for 14 consecutive days. In the vehicle group, mice were treated with 5% DMSO instead of J4 following the same protocol as the J4 treatment group. The sham group received the same treatment as the vehicle group. All mice were maintained on a 12-hour light / dark schedule in the Laboratory Animal Center of National Taiwan University (an AAALAC-accredited laboratory animal facility) and were given food and water ad libitum. Procedures involving animals were performed in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

[0014] In another example of treatment with JMF 1907 (i.e., compound of formula (III)), male C57BL / 6J mice (8-12 weeks old) were divided into three groups: JMF1907 group, methylprednisolone (MPSS) group, and vehicle group, in which the mice were administered JMF1907 (10 mg / kg, twice daily for 14 days), methylprednisolone (30 mg / kg, 10 minutes after crush, followed by 2 hours, 4 hours, and 6 hours after crush), or vehicle (0.5% DMSO, twice daily for 14 days).

[0015] Behavioral testing Motor function after SCI was assessed by the Basso Mouse Scale (BMS) open-field locomotor activity rating scale on days 3–14 after surgery. 6 Scoring ranged from 0 for complete paralysis to 9 for normal function. Paw print analysis was assessed 14 days after surgery. The hind limbs of the mice were dipped in red and black dye for the left and right paws, respectively. The mice were then placed on a piece of construction paper surrounded by a cage to encourage straight walking. Stride length was defined as the distance between the midpoints of two consecutive paw prints.

[0016] RT-qPCR analysis Mice were anesthetized by inhalation of isoflurane. Spinal cords were snap frozen in liquid nitrogen and homogenized with a sterile pellet pestle (Thermo Fisher Scientific, MA, USA) using 1 mL of TRIzol™ Reagent (Thermo Fisher Scientific, MA, USA). Total RNA isolation, quality check, cDNA synthesis, and SYBR green-based quantitative real-time PCR assays were performed as previously described. 7The forward / reverse primer sequences for IL-1β, IL-6, TNF-α, and GAPDH are listed in Table 1. The relative expression of target genes normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was calculated by comparative Ct (ΔCt) and expressed as 2-ΔCt. Relative numbers were determined by the following formula: 2-ΔΔCt, where ΔΔCt values ​​were obtained by subtracting ΔCt values ​​of mice with SCI obtained from sham controls.

[0017] [Table 1]

[0018] Immunofluorescence staining After anesthetization with Zoletil® (50 mg / kg) and Rompun® (12 mg / kg), mice were perfused intracardially with ice-cold saline. Spinal cords (T9-T11) were removed and immersed in ice-cold 4% paraformaldehyde. Vertebrae were then placed in cassettes for paraffin embedding. Samples were cut into 4 μm sagittal sections and placed in a water bath at 42° C. Sections were mounted on slides and stored at room temperature until analysis. After deparaffinization, tissue extraction was performed in sodium citrate buffer (pH=6) at 90° C. and spinal cord sections were then washed with phosphate-buffered saline (PBS). Sections were incubated at room temperature in antibody dilution buffer (Roche, Basel, Swiss). Slides were immunostained overnight at 4° C. with the following primary antibodies: chicken anti-neuronal nuclear protein (NeuN) (1:2000, Merck-Millipore, Dramstadt, Germany), rabbit anti-ionized calcium-binding adapter molecule-1 (Iba-1) (1:1000, Abcam, Cambridge, MA, USA), or mouse anti-glial fibrillary acidic protein (Gfap) (1:1000, cell signaling, Danvers, Massachusetts, USA). Corresponding secondary antibodies conjugated with Alexa Fluor 488 or rhodamine (Jackson Immuno Research Laboratories, West Grove, PA, USA) were applied for visualization of immunolabeling. After washing with PBST (phosphate buffered saline with Tween® 20) and PBS, the sections were mounted in mounting gel (DAPI Fluoromount G, Southern Biotech Birmingham, Alabama, USA). Images were acquired by a Zeiss AXIO Imager M1 microscope (Carl Zeiss, Göttingen, Germany). In the perilesional area, the fluorescence intensity of Iba-1 and Gfap was quantified by the mean optical density (mean optical density = integrated optical density (IOD) / area) by ImageJ 1.52 software (National Institutes of Health, Bethesda, Maryland, USA).The number of NeuN-positive cells in three sagittal sections (70 μm apart) of the spinal cord was calculated in bilateral sites of the perilesional area at 20× magnification.

[0019] Nissl staining After deparaffinization, sections were placed in PBS and then in 1% cresyl violet solution. Sections were differentiated in 95% ethyl alcohol and dehydrated in 100% alcohol. After dehydration, sections were processed in xylene and mounted with dibutyl phthalate polystyrene xylene. Areas of traumatic injury were identified by severe tissue destruction or loss of staining. Six Nissl-stained sections spaced apart (100 μm between sections) were selected to estimate proportional lesion size by ImageJ 1.52 software (National Institutes of Health, Bethesda, Maryland, USA).

[0020] Western blot The protein concentration of the samples was determined by Bio-Rad DC Protein Assay Kit (Bio-rad, Hercules, CA, USA). Protein samples (10 μg each) were diluted with loading buffer (200 mM Tris-HCl, 1.43% 2-mercaptoethanol, 0.4% bromophenol blue, and 40% glycerol) and heated at 98°C. Protein samples were then separated on 12% SDS-polyacrylamide gels (for S100a10, c-caspase 3, and GAPDH) or 8% SDS-polyacrylamide gels (for C3, NeuN) in running buffer (0.3% Tris base, 1.88% glycine, and 0.1% SDS) at 90 V for 10 min, followed by separation at 130 V for 60 min. After electrophoresis, the gel was transferred to a nitrocellulose membrane in transfer buffer (0.3% Tris base, 1.88% glycine, and 20% methanol, pH 8.3) at 300 mA for 90 min. Nonspecific binding to the membrane was blocked with BlockPRO™ 1 Min Protein Free Blocking Buffer (Neihu, Taipei City, Taiwan) at room temperature on a shaker at 25 rpm. For detection of C3, NeuN, and GAPDH, membranes were incubated overnight at 4° C. with antibodies against C3 (1:200, Abcam, Cambridge, MA, USA), NeuN (1:1000, Genetex, CA, USA), or GAPDH (1:160000, Biodesign International, Saco, Maine, USA), and for detection of S100a10 and c-caspase 3, membranes were incubated at 4° C. with S100a10 (1:200, Abcam, Cambridge, MA, USA) or c-caspase 3 (1:200, Merck-millipore, Dramstadt, Germany), all diluted in BlockPRO™ 1 Min Protein Free Blocking Buffer.The membrane was washed with TNT buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.2% Tween 20, pH 7.4) and incubated with anti-mouse IgG antibody (1:5000, cell signaling, Danvers, Massachusetts, USA) or anti-rabbit IgG antibody (1:2000, cell signaling, Danvers, Massachusetts, USA) conjugated to horseradish peroxidase (HRP) in TNT buffer at room temperature. Bound antibodies were detected using Chemiluminescence reagent Plus (PerkinElmer Life Sciences, MA, USA) and Bio-rad ChemiDoc™ XRS + system and Image Lab™ software, and images were obtained under appropriate exposure times.

[0021] statistical analysis All statistical analyses were performed by GraphPad Prism v9.1 (GraphPad Software, San Diego, CA, USA). Data of adenosine-related gene alterations in SCI were analyzed by unpaired t-test. Regarding the effect of ENT1 inhibition, all data (i.e., BMS, stride length, RT-qPCR, immunostaining, and Western blotting) were evaluated by one-way ANOVA, except for the data of lesion volume, which was evaluated by unpaired t-test. All data (i.e., BMS, stride length, lesion volume, RT-qPCR analysis, immunostaining, and Western blotting) were evaluated by unpaired t-test. A significance level of 0.05 was considered statistically significant.

[0022] result J4 treatment improves functional recovery after SCI See Figures 1-7, which show that J4 treatment promoted motor function recovery and reduced neuronal cell death in mice with SCI, where data are expressed as the mean ± SEM of 3-7 animals, *p<0.05.

[0023] It has been previously shown that J4, an ENT1 inhibitor, exhibited beneficial effects in the treatment of some neurodegenerative diseases. To investigate whether J4 could also have an effect on SCI, J4 was given to C57BL / 6J mice with SCI, followed by the assessment of motor function and histological changes (see FIG. 1). As shown in FIG. 2, the BMS score of mice with SCI could be improved by J4 treatment. Further tests on the coordination of hindlimb movement showed that J4 treatment improved the stride length of mice with SCI (see FIG. 3). Regarding the lesion, Nissl staining showed that the loss of spinal cord tissue was significantly reduced by J4 treatment 14 days after injury (see FIG. 4).

[0024] These data indicated that motor function recovery and lesions in SCI could be improved by J4 treatment. To further evaluate the preserved neurons in the perilesional area, immunostaining and Western blot for NeuN were performed. As shown in Figure 5, more NeuN-positive cells were identified in the perilesional area of ​​mice treated with J4. Similarly, the protein level of NeuN was higher in the J4-treated group than that in the vehicle group (see Figure 6). Because cleaved caspase-3 (c-caspase 3) is important for cell apoptosis, the protein level of c-caspase 3 was examined. As shown in Figure 7, the level of c-caspase 3 was decreased by J4 treatment for 14 consecutive days after injury. These results indicated that J4 could protect neural cells from apoptosis in SCI.

[0025] J4 treatment reduces inflammatory responses in SCI See Figures 8-10, which show that J4 treatment reduced the inflammatory response in the peri-lesion area of ​​B6 mice with SCI, where data are presented as the mean ± SEM of 3-5 animals, *p<0.05, and # represents the lesion core. Neuroinflammation is important in the progression of SCI, and IL-1β, IL-6, and TNF-α are thought to be important in the post-traumatic inflammatory response. 4 The effect of J4 on the expression of these cytokines in the lesion and perilesional sites of the spinal cord was examined. As shown in parts (A)-(C) of Figure 8, J4 treatment significantly reduced the expression of TNF-α, with a trend towards a reduction in the expression of IL-1β and IL-6 in mice with SCI. In addition to the expression of inflammatory cytokines, astrogliosis and microgliosis are important benchmarks of neuroinflammation. As shown in Figure 9, Gfap-positive astrocytes in the perilesional region are hypertrophied and have thickened branches, indicating morphological changes associated with a reactive state. Compared with that in the vehicle group, the mean fluorescence intensity of Gfap in the J4 group was significantly reduced 14 days after injury. As for microgliosis, activated microglia, like astrocytes, undergo a marked change in cell morphology, changing from a resting state with branched cell morphology to an activated state with amoeboid cell morphology. Ionized calcium-binding adaptor protein-1 (Iba-1), a 17 kDa actin-binding protein, is widely used as an immunohistochemical marker for both microglia and macrophages. As shown in Figure 10, the intensity of Iba-1 positive cells was lower in the J4-treated group compared with the vehicle-treated group. These findings indicated that J4 could reduce inflammatory responses, including the expression of inflammatory cytokines and the activation of astrocytes and microglia / macrophages, in the perilesional region of the spinal cord.

[0026] Modulation of astrocyte phenotype in SCI by J4 treatment See Figures 11-13, which show that J4 treatment modulated the phenotype of astrocytes and microglia / macrophages at T9-T11 in B6 mice with SCI, where data are presented as mean ± SEM of 3-5 animals, *p<0.05.

[0027] Activated astrocytes develop into distinct phenotypes, where A1 astrocytes are considered neurotoxic and A2 astrocytes are restorative. Thus, markers of A1 and A2 astrocytes, respectively, are 8 The expression of C3 and S100a10 was examined. As a result, the protein levels of C3 and S100a10 were significantly decreased and increased, respectively, by J4 treatment (see Figures 11 and 12). These findings indicated that J4 treatment could increase A2 astrocyte expression and decrease A1 astrocyte expression. Since astrocytes can produce transforming growth factor-β (TGF-β), which has been implicated in the induction of axon formation and neurogenesis, 9 Next, the levels of TGF-β were examined. As shown in Figure 13, TGF-β protein was significantly increased by J4 treatment.

[0028] Treatment of SCI in JMF 1907 Similarly, the results showed that treatment with JMF1907 could significantly improve the motor function (in terms of BMS score) (part (A) of FIG. 14) and stride length (part (B) of FIG. 14) of mice with SCI. However, treatment with MPSS did not improve the motor function of mice with SCI. Immunofluorescence analysis and Nissl staining showed that JMF1907 treatment could significantly reduce the lesion area of ​​SCI, consistent with the findings of motor function measurement (FIG. 15). In addition, JMF1907 treatment significantly reduced the levels of Gfap (a marker of reactive astrocytes) (FIG. 16) and Iba-1 and CD68 (both markers of microglia) (FIG. 17) in mice with SCI, indicating the beneficial effect of JMF1907 on reducing neuroinflammation in SCI.

[0029] Consideration Following primary injury to the spinal cord, inflammation plays an important role in the development of secondary injury that can lead to neuronal death. 4Methylprednisolone has been applied to reduce the inflammatory response in SCI, but its use is highly controversial and it is not approved by the US Food and Drug Administration (FDA) for this indication. 10 At present, the treatment of SCI is still considered an unmet medical need. Upon increased extracellular adenosine levels, ENT plays an important role in mediating the cellular uptake of adenosine. The present invention showed that J4, used as a pharmacological inhibitor of ENT1, can alleviate inflammation, reduce lesions, and improve functional recovery in mice with a thoracic spinal cord contusion model. These findings suggest that J4 is a promising candidate for SCI treatment. ENT plays an important role in controlling extracellular levels of adenosine. In the spinal cord, selective inhibition of ENT1 by NBMPR can modulate glutamatergic synaptic transmission via A1R activation. 11 The present invention further demonstrated that inhibition of ENT1 exerts anti-inflammatory effects and improves functional recovery in SCI.

[0030] Neuroinflammation is important in the progression of SCI, and targeting inflammation may provide a means to improve neuronal function and prognosis after SCI. 12 In SCI, neuroinflammation is involved in microgliosis, macrophage infiltration, and astrogliosis in SCI. The expression of Iba-1 (for microglia / macrophages) and Gfap (for astrocytes) was both increased in SCI. Activation of microglia / macrophages and astrocytes can be reduced by pharmacological inhibition. Similar findings have been reported in astrocytes, and Gfap expression is reduced in ENT1 null mice. 13 .

[0031] Regarding the phenotype of activated astrocytes, these results showed that J4 treatment significantly reduced the protein levels of C3 (A1 marker) compared to untreated SCI mice, whereas J4 significantly increased the protein levels of S100a10. A1 astrocytes exhibited neurotoxic effects on oligodendrocytes and neurons in vitro, whereas A2 astrocytes promoted neuronal survival and tissue repair. 14~15 Functional recovery in mice with SCI could be improved by reducing astrocytes with the A1 phenotype. 16 , the increase in A2 astrocytes may provide beneficial effects on reducing the production of proinflammatory cytokines. 17 It has been reported that increasing A2 astrocytes or decreasing A1 astrocytes may be beneficial against injury. 18 Taken together, the modulation of the A1 / A2 phenotype by J4 treatment supports the beneficial effects of J4 in the treatment of SCI.

[0032] conclusion According to the above results, it was shown that mice administered with the compound of the present invention had higher BMS scores and longer stride length compared to controls.Treatment with the compound of the present invention significantly reduced the mRNA levels of IL-1β, IL-6, and TNF-α, as well as the activation of astrocytes and microglia / macrophages in the perilesional area of ​​spinal cord at 14 DPI.Along with the reduction in the size of the lesion volume, more preserved neurons were identified in the perilesional area of ​​J4-treated mice treated with the compound of the present invention.

[0033] In summary, treatment with the compound of the present invention can alleviate neuroinflammation, reduce lesions, and improve motor function recovery in mice with thoracic spinal cord injury.In addition, the compound of the present invention can modulate the A1 / A2 phenotype of activated astrocytes, which can promote nerve regeneration.These results suggest that the compound of the present application is a promising candidate for the treatment of SCI. References JPEG2025505186000005.jpg235168 JPEG2025505186000006.jpg138170

Claims

1. A method of treating spinal cord injury, comprising administering to a subject in need thereof a compound of Formula (I), (II), or (III), a pharma- ceutically acceptable salt thereof, or a composition thereof. 【Chemistry 1】 wherein X is a halogen.

2. The compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 -[(5-halothien-2-yl)methyl]adenosine.

3. The compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine, N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6 -[(4-chlorothien-2-yl)methyl]adenosine, and N 6 -[(3-chlorothien-2-yl)methyl]adenosine.

4. The compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 -[(5-halothien-3-yl)methyl]adenosine.

5. The compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromothien-3-yl)methyl]adenosine, N 6 -[(4-bromothien-3-yl)methyl]adenosine, N 6 -[(5-bromothien-3-yl)methyl]adenosine N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 -[(5-chlorothien-3-yl)methyl]adenosine.

6. 10. The method of claim 1, wherein the compound, a pharma- ceutically acceptable salt thereof, or a composition thereof is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical routes.

7. The method of claim 1 , wherein the composition further comprises a pharma- ceutically acceptable carrier, excipient, or vehicle.

8. 10. The method of claim 1, wherein the treatment of spinal cord injury comprises improving functional recovery after spinal cord injury.

9. 2. The method of claim 1, wherein the treatment of spinal cord injury comprises reducing neuroinflammation in neurons following spinal cord injury.

10. 2. The method of claim 1, wherein the treatment of spinal cord injury comprises modulating astrocyte and microglia / macrophage phenotype in the injured spinal cord.

11. A composition for use in a method of treating spinal cord injury comprising administering to a subject in need thereof a composition comprising a compound of Formula (I), (II), or (III), or a pharma- ceutically acceptable salt thereof. 【Chemistry 2】 wherein X is a halogen.

12. The compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 -[(5-halothien-2-yl)methyl]adenosine.

13. The compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine, N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6 -[(4-chlorothien-2-yl)methyl]adenosine, and N 6 -[(3-chlorothien-2-yl)methyl]adenosine.

14. The compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 -[(5-halothien-3-yl)methyl]adenosine.

15. The compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromothien-3-yl)methyl]adenosine, N 6 -[(4-bromothien-3-yl)methyl]adenosine, N 6 -[(5-bromothien-3-yl)methyl]adenosine N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 -[(5-chlorothien-3-yl)methyl]adenosine.

16. The composition for use according to claim 11, wherein the composition is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.

17. The composition for use according to claim 11, wherein the composition further comprises a pharma- ceutically acceptable carrier, excipient, or vehicle.

18. The composition for use according to claim 11 , wherein the treatment of spinal cord injury comprises improving functional recovery following spinal cord injury.

19. The composition for use according to claim 11 , wherein the treatment of spinal cord injury comprises reducing neuroinflammation following spinal cord injury.

20. The composition for use according to claim 11, wherein the treatment of spinal cord injury comprises modulating the phenotype of astrocytes and microglia / macrophages in the injured spinal cord.

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