JP2011236128A - Central nervous system injury-treating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a central nervous system injury-treating agent which can effectively treat the central nervous system injury.SOLUTION: There is provided the central nervous system injury-treating agent containing a TGF-β1 inhibitor such as an anti-TGF-β1 antibody as an active ingredient. The central nervous system injury-treating agent can effectively treat the central nervous system injury. The treating agent significantly improves a motor function lost by the mechanical contusion of spinal cords. Thereby, it is considered that the treating agent largely contributes to treat the injuries of the central nervous system due to accidents or diseases.

Description

  The present invention relates to a therapeutic agent for central nerve injury.

  Spinal cord injury initiates a series of cellular and biochemical processes that damage both neurons and glia. Cells and axons disappear at the site of injury, and the damage spreads to adjacent areas. Since spinal axons do not regenerate spontaneously, strategies that promote endogenous neuroanatomical repair and functional recovery are needed. It is believed that immune cell activation and inflammatory processes play an important role in sequelae after injury. There are several findings that some inflammatory processes are beneficial because they promote central nervous system repair. It has been reported that anatomical and functional repairs have been improved by transplanting activated macrophages into the spinal cord (Non-patent Document 1). This suggests that some activated inflammatory cells may be beneficial for the damaged central nervous system (CNS). Administration of activated macrophages has been reported to promote axonal growth by cleaning debris from the site of injury and producing various cytokines. Inflammation-induced cytokines and locally released growth factors may contribute to these two effects in CNS inflammation. In contrast, excessively boosting the immune response after CNS injury is detrimental to neurons. Thus, the immune system may play a role in defense and maintenance after injury of the CNS, and the immune response may be beneficial to neurons if well controlled (Non-Patent Document 2).

  TGF-β1 is a multifaceted cytokine that is closely involved in wound repair. TGF-β1 is expressed in several types of CNS pathological models, and its important role in the neurodegenerative process is being recognized (Non-patent Document 3). TGF-β1 enhances the inflammatory response by stimulating the activity of monocytes and lymphocytes. TGF-β1 is upregulated in response to tissue damage in mammals. This isoform is most commonly involved in tissue repair. TGF-β1 induces extracellular matrix deposition by simultaneously stimulating the production of new matrix proteins (collagen, fibronectin and proteoglycan) and is involved in the inflammatory response.

  It is already well established that the ability of regenerated damaged axons is extremely limited in the CNS of mature mammals. In the absence of proper axon regeneration, trauma damage to the adult brain and spinal cord often causes life-long neurological deficits. After CNS injury, various neurite growth inhibitors are present around the injury site. These are believed to be at least partly responsible for the poor axonal regeneration and functional deficits induced by injury. An axon growth inhibitor in myelin is thought to play an important role, but is an extracellular matrix such as chondroitin sulfate proteoglycans (CSPGs) expressed in glial scars that develop around the site of CNS injury The molecule is a significant inhibitor. Administration of TGF-β1 increases glial scars (Non-Patent Documents 4 and 5), and glial scars create physical and chemical barriers to axonal regeneration (Non-Patent Documents 6 and 4) 7)

Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solomon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M (1998) Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med 4: 814-821. Moalem G, Leibowitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M (1999) Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 5: 49-55. Finch CE, Laping NJ, Morgan TE, Nichols NR, Pasinetti GM (1993) TGF-beta 1 is an organizer of responses to neurodegeneration.J Cell Biochem 53: 314322. Laping NJ, Morgan TE, Nichols NR, Rozovsky I, Young-Chan CS, Zarow C, Finch CE (1994) Transforming growth factor-beta 1 induces neuronal and astrocyte genes: tubulin alpha 1, glial fibrillary acidic protein and clusterin. Neuroscience 58 : 563-572. Morgan TE, Laping NJ, Rozovsky I, Oda T, Hogan TH, Finch CE, Pasinetti GM (1995) Clusterin expression by astrocytes is influenced by transforming growth factor beta 1 and heterotypic cell interactions. J Neuroimmunol 58: 101-110. Qiu J, Cai D, Filbin MT (2000) Glial inhibition of nerve regeneration in the mature mammalian CNS. Glia 29: 166-174. Yiu G, He Z (2006) Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7: 617-627. Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats.J Neurotrauma 12: 1-21 Yamashita T, Fujitani M, Yamagishi S, Hata K, Mimura F (2005) Multiple signals regulate axon regeneration through the Nogo receptor complex. Mol Neurobiol 32: 105-111 Tang X, Davies JE, Davies SJ (2003) 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. Neurosci Res 71: 427-444 Pratt BM, McPherson JM (1997) TGF-beta in the central nervous system: potential roles in ischemic injury and neurodegenerative diseases.Cytokine Growth Factor Rev 8: 267-292. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension Nature 389: 990-994. Ren XD, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.EMBO J 18: 578-585. Bareyre FM, Kerschensteiner M, Raineteau O, Mettenleiter TC, Weinmann O, Schwab ME (2004) The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat Neurosci 7: 269-277.

  As described above, a method for promoting the regeneration of damaged central nerves has also been proposed, but the effect is not always sufficient, and there is no practical treatment method for central nerve damage yet.

  Accordingly, an object of the present invention is to provide a therapeutic agent for central nerve injury that can effectively treat damage to the central nerve.

  As a result of intensive studies, the present inventors have found that administration of a TGF-β1 inhibitor to the damaged site of the central nerve promotes axonal elongation at the damaged site and promotes recovery of motor function. The headline and the present invention were completed.

That is, the present invention is as follows.
(1) A therapeutic agent for central nerve injury comprising a TGF-β1 inhibitor as an active ingredient.
(2) The therapeutic agent according to (1) above, wherein the TGF-β1 inhibitor is an anti-TGF-β1 antibody or an antigen-binding fragment thereof.
(3) The therapeutic agent according to (1) or (2) above, wherein the TGF-β1 inhibitor is an anti-TGF-β1 neutralizing antibody.
(4) The therapeutic agent according to any one of (1) to (3), wherein the central nerve is a spinal cord.
(5) A spinal cord injury therapeutic agent comprising an anti-TGF-β1 neutralizing antibody as an active ingredient.

  INDUSTRIAL APPLICABILITY According to the present invention, a therapeutic agent for central nerve injury that can effectively treat central nerve damage has been provided. As specifically described in the following examples, according to the therapeutic agent of the present invention, the motor function lost due to mechanical contusion of the spinal cord is significantly improved. Therefore, the therapeutic agent of the present invention is considered to greatly contribute to the treatment of central nerve damage due to accidents and diseases.

  As described above, the therapeutic agent for central nerve injury of the present invention contains a TGF-β1 inhibitor as an active ingredient. TGF-β is a cytokine known to control cell proliferation / differentiation and promote cell death, and TGF-β1 is one isoform included in the TGF-β family. TGF-β1 itself is well known and well studied.

  The TGF-β1 inhibitor used in the present invention means a substance that inhibits (lost or reduces) the action of TGF-β1, and preferred examples include anti-TGF-β1 antibodies. Anti-TGF-β1 antibodies that inhibit (neutralize) the action of TGF-β1 are widely used for research of TGF-β1 and are commercially available. In the present invention, such commercially available anti-TGF-β1 antibody can be preferably used. The effect of TGF-β1 is known to be exerted by binding to TGF-β1 receptor, and commercially available anti-TGF-β1 antibody inhibits binding between TGF-β1 and TGF-β1 receptor. It has been confirmed that In addition, antibodies that bind to TGF-β1 through an antigen-antibody reaction and inhibit the action thereof are not necessarily antibodies produced using TGF-β1 as an immunogen. It may be an antibody that reacts with an antibody to inhibit its action. However, an antibody having natural TGF-β1 as a corresponding antigen possessed by an animal to be administered is preferable. Therefore, if a treatment target is human, an antibody having human TGF-β1 as a corresponding antigen is preferable.

In general, an antigen-binding fragment of an antibody, that is, a Fab fragment or F (ab ') ₂ fragment containing the variable region of an antibody, often binds to the corresponding antigen of the antibody and exerts the same action as an antibody. Is also known. Therefore, in the present invention, an antigen-binding fragment of an antibody that inhibits the action of TGF-β1 can also be used as an active ingredient. In addition, as an antibody drug, in order to suppress as much as possible the immune response that recognizes the administered antibody drug as a foreign substance, a chimeric antibody in which the constant region is changed to the constant region of the animal (if it is an antibody drug administered to humans, Antibodies with human constant regions), humanized antibodies in which the CDR regions are also changed to human-derived regions, and human antibodies made by immunizing transgenic animals that produce human antibodies are widely used as antibody drugs. Such chimeric antibodies, humanized antibodies and human antibodies can also be preferably used. Techniques for producing these antibodies have already been established in the field of antibody medicine. Furthermore, an antibody produced by genetic engineering may be used. Furthermore, single-chain antibodies such as ScFv are also included in the antigen-binding fragment for interpretation. The antibody may be a polyclonal antibody or a monoclonal antibody.

  The TGF-β1 inhibitor used in the present invention is typically an anti-TGF-β1 antibody or an antigen-binding fragment thereof. TGF-β1 exerts its physiological activity by binding to the TGF-β1 receptor. Therefore, if the binding between TGF-β1 and the TGF-β1 receptor is inhibited, the action of TGF-β1 can be inhibited. In the present invention, the “TGF-β1 inhibitor” includes a substance that inhibits the binding between TGF-β1 and the TGF-β1 receptor. Representative examples include an anti-TGF-β1 receptor antibody that inhibits binding to TGF-β1 and an antigen-binding fragment thereof. Anti-TGF-β1 receptor antibodies that inhibit the binding to TGF-β1 are also commercially available, and such commercially available products can be preferably used.

  Furthermore, a recombinant vector incorporating the gene of the antibody or antigen-binding fragment thereof, which can produce the above-described antibody or antigen-binding fragment thereof in vivo, is also referred to as a “TGF-β1 inhibitor” in the present invention. Is included. Techniques for producing antibodies by genetic engineering have already been widely used, and recombinant vectors incorporating antibody genes can be easily produced. On the other hand, a method of administering a recombinant vector incorporating a gene encoding a desired protein into a living body and expressing the gene in the living body as a result is administered to the living body by gene therapy, particularly a gene. It is widely known in the field of vaccines, and the antibody used in the present invention can be administered by such a gene therapy technique.

  The subject to be treated with the therapeutic agent of the present invention is central nerve damage. The central nerve is preferably the spinal cord. In addition, the damage includes injury caused by diseases such as spinal cord tumors and hernias as well as trauma caused by accidents such as traffic accidents, sports accidents, and falls. The damage is preferably caused by trauma.

  The administration route is preferably local administration to the damaged part. The administration can be performed intermittently by injection, but as described in the following examples, it is preferable to continuously administer to the damaged part using a minipump and a catheter connected thereto. Administration is preferably initiated as soon as possible after injury, and administration is preferably continued for at least 1 week, more preferably at least 2 weeks after injury. However, since the therapeutic effect becomes low after a long period of time after the injury, it is preferable from the viewpoint of economy and patient burden that the administration is about 2 months at most, more preferably about 1 month at most.

  When using an anti-TGF-β1 antibody, the dose can be appropriately set depending on the degree of damage and the patient's condition, etc. For adults, it is usually about 0.01 mg to 2000 mg, preferably about 0.5 mg to 100 mg per day. is there. Moreover, the formulation can use what was formulated as an injection according to the conventional method, for example, the antibody solution etc. which melt | dissolved the antibody in the physiological saline solution can be used. Buffers, pH regulators, osmotic pressure regulators and the like that are commonly used in the pharmaceutical field can be added to injections. If the effects of the present invention are not inhibited, other drugs, for example, bactericides, antibiotics and the like used for the treatment and prevention of trauma can be used in combination.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Materials and Methods Surgical procedures Anesthetized (sodium pentobarbital, 40 mg / kg) female Sprague-Dawley rats (200-250 g) were laminectomy at spinal level T9 / T10 to expose the spinal cord. The spinal cord was crushed using an infinite horizon impactor (IH impactor; Precision System Instrumentation), and a control rabbit IgG immediately after spinal cord injury (SCI) for neutralizing antibody assays in animal models. (13 mice) or an osmotic minipump (200 μL solution, 0.5 μL / h, 14 days delivery, Alzet pump model 2002 (trade name)) filled with anti-TGF-β1 antibody (13 mice) was implanted in each rat. A silicone rubber tube connected to the outlet of the minipump was placed under the back of the animal and placed under the dura mater at the SCI site, and then the muscle and skin layers were sutured. ) Exposed the spinal cord by laminectomy at spinal level T9 / T10, and then sutured the muscle and skin layers.

Immunohistochemistry After deep anesthesia was induced, 4% paraformaldehyde (PFA) solution in 0.1 M phosphate buffer (PB) was perfused in the heart. The spinal cord was excised, postfixed with the above fixative, and cryopreserved overnight in PBS containing 30% sucrose. 10 mm from the damaged site and 10 mm from the caudal side were embedded in Tissue Tek OCT (trade name). Each block was sliced along the cross section (50 μm). Sagittal sections (50 μm) were collected from 10 mm cranial and 10 mm caudal from the injury site. After washing with PBS three times, all sections were blocked with PBS containing 5% bovine serum and 0.1% Triton X-100 (trade name) at room temperature (RT) for 1 hour. The sections were incubated with the primary antibody at 4 ° C. overnight, washed with PBS three times, and incubated with a fluorescein-conjugated secondary antibody (1: 1000, manufactured by Invitrogen) at room temperature for 1 hour. Monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1: 1000, manufactured by Chemicon), monoclonal anti-chondroitin sulfate antibody (1: 200, manufactured by Sigma), anti-type IV collagen antibody (1: 200, manufactured by LSL) ), Anti-Iba1 antibody (1: 500, manufactured by Wako), monoclonal anti-ED1 antibody (1: 100, manufactured by Serotec) and polyclonal anti-serotonin antibody (5HT) (1: 1000, manufactured by Immunostar) are used as primary antibodies. It was.

Prograde labeling of corticospinal tract (CST) In 5 control IgG-treated SCI rats and 5 anti-TGF-β1 antibody-treated SCI rats, descending CST fibers were biotinylated dextranamine (BDA, 10% in saline). , 4.0 μL per cortex, molecular weight 10,000, manufactured by Invitrogen). BDA was injected into the left and right motor cortex (coordinates: 2 mm before Bregma, 2 mm next to Bregma, depth 1.5 mm) under anesthesia 10 weeks after injury. In each injection, 4 μL of BDA was injected over 8 minutes through a glass capillary with an inner diameter of 15 to 20 μm connected to a microliter syringe (manufactured by ITO). Fourteen days after BDA injection, animals were killed by perfusion with PBS followed by 4% paraformaldehyde. The spinal cord was excised, fixed with the above fixative overnight, and cryopreserved overnight in PBS containing 30% sucrose. The spinal cord 40 mm from the injury site, 10 mm from the cranial side and 10 mm from the caudal side was embedded in Tissue Tek OCT. Each block was sliced along the cross section (50 μm). The sections were blocked with PBS containing 0.5% BSA for 1 hour and incubated with Alexa Fluor 488-conjugated streptavidin (trade name, 1: 400, manufactured by Invitrogen) in PBS containing 0.15% BSA for 1 hour.

Quantification The quantification of the immune reaction density of each marker was performed using a microscope. Immunoreactivity in sections 10 mm long (for GFAP, Iba1 and ED1) and 4 mm long (for CSPG and collagen) using Scion Image software (trade name, manufactured by Scion Corporation, Frederick, Maryland, USA) It was measured. The number of pixels in the immunolabeled area was calculated as a percentage of the total area.

  Serotonin positive fibers were measured using a 10 mm cranial section and a 10 mm caudal section from the center of the damaged site. The number of pixels of serotonin positive fibers was calculated as a percentage of the number of pixels in the front corner in the same section.

  The number of pixels of BDA-positive fibers in the 40 mm cranial section from the injury site was counted. The number of pixels of BDA-labeled fibers in gray matter was calculated as a percentage of the number of pixels of the fibers observed in the anterior corner of the posterior column of the same section.

Neurite growth assay Cerebellar granule neurons (cerebellar granule neurons (CGNs)) were excised from postnatal rats (P7-9) by trypsinization (treatment with 0.25% trypsin in PBS at 37 ° C for 15 minutes), and 10% bovine fetus Resuspended in Dulbecco's modified Eagle's medium (DMEM) with formation, ground and washed 3 times with serum-containing DMEM. Neurons were cultured in serum-free DMEM supplemented with B27 and plated on poly-L-lysine (PLL) -coated chamber slides (LabTek II (trade name), manufactured by Nunc, Roskilde, Denmark). Plated neurons were incubated for 24 hours with recombinant human TGF-β1 (10 ng / ml; R & D Systems) or solvent (PBS) as a control. Cells were fixed with 4% paraformaldehyde and stained with monoclonal antibody TuJ1 (1: 1000, manufactured by Covance). The length of the longest neurite in each TuJ1-positive neuron was measured.

Affinity precipitation of GTP-RhoA
It was dissolved in 50 mM Tris, pH 7.5 containing 1% Triton X-100 (trade name), 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl and 10 mM MgCl ₂ and 10 μg / mL leupeptin and aprotinin. The cell lysate was clarified by centrifugation at 13,000 g for 4 minutes at 4 ° C., and the supernatant was incubated with 20 μg of GST-Rho binding domain of Rhotekin beads (trade name) at 4 ° C. for 45 minutes. The beads were washed 3 times with wash buffer (1% Triton X-100 (trade name), 0.1% SDS, 150 mM NaCl and 10 mM MgCl ₂ and 50 mM Tris each containing 10 μg / mL leupeptin and aprotinin, pH 7.5). . RhoA was detected by Western blot using an anti-RhoA antibody (Santa Cruz Biotechnology, Inc.).

Behavioral test The recovery of behavior was evaluated with the Basso-Beattie-Bresnahan (BBB) locomotor rating scale (Non-Patent Document 8) for 12 weeks after injury in the open field. Uninjured sham-operated rats (n = 3) were perfect. Quantification was done blindly.

result
Neutralization of TGF-β1 promotes motor recovery after SCI We examined whether inhibition of endogenous TGF-β1 contributes to functional recovery after SCI. In this example, an anti-TGF-β1 neutralizing antibody (available from R & D Systems) was used. In this example, a rat spinal cord contusion model was used in order to better simulate the clinical state. The spinal cord was injured using an infinite horizontal impactor (trade name IH impactor) at the site of spine level Th9 / T10. Anti-TGF-β1 neutralizing antibody or control IgG was delivered by an osmotic minipump over 2 weeks using a catheter placed in the medullary cavity above the injury site. The animals' ability to exercise was monitored for 12 weeks after injury. Sham-operated rats (FIG. 1) were perfect on the Basso-Beattie-Bresnahan (BBB) motion rating scale, which evaluates hind paw motion. All SCI rats were almost completely paralyzed for 1-7 days after injury. As indicated by the BBB score, these animals gradually showed partial recovery of motor behavior (FIG. 1). Between 5 and 12 weeks after injury, the exercise capacity of rats administered with anti-TGF-β1 antibody tended to be better than that of rats administered with control IgG. After 12 weeks of injury, rats administered with control IgG had an average BBB score of 10.1, whereas rats administered with anti-TGF-β1 antibody had a significantly higher BBB score of 14.4 on average (FIG. 1). . Thus, the data of this example show that administration of anti-TGF-β1 antibody significantly improves motor recovery after spinal cord injury in rats.

Inhibition of TGF-β1 reduces scarring after SCI It is well established that glial scars are an important source of inhibition of axon growth after central nervous system (CNS) injury (9). ). The glial response to injury causes microglia, oligodendrocyte precursors, meningeal cells and astrocytes to collect at the site of injury. Many astrocytes in the damaged area often become hypertrophic, exhibit a reactive phenotype, and become inhibitory extracellular matrix molecules known as CSPGs. Previous studies have reported that the administration of TGF-β1 increases glial scars (Non-patent Documents 4 and 5). Therefore, we quantitatively analyzed the increase of reactive astrocytes in the injured rat spinal cord to determine whether inhibition of TGF-β1 affects the formation of glial scars. To estimate the number of reactive astrocytes, sagittal sections of injured spinal cords obtained from control and anti-TGF-β1 antibody-treated rats were immunostained using anti-GFAP antibody. In control SCI rats, strong immunoreactivity to GFAP was observed in the vicinity of the injury site 2 weeks after SCI, but in SCI rats administered with anti-TGF-β1 antibody, there was a marked decrease in immunoreactivity to GFAP. It was. At 1 week, 2 weeks and 12 weeks after SCI, the increase in astrocytes was estimated by measuring the total area of immunoreactivity against GFAP around the center of the injury site. One week after SCI, there was no significant difference in glial scar formation between anti-TGF-β1 antibody-treated rats and control antibody-treated rats. However, after 2 weeks of SCI, inhibition of TGF-β1 clearly reduced glial scar formation. This difference disappeared 12 weeks after SCI (Figure 2i).

  It has been reported that most CSPGs including versican, brevican and neurocan are up-regulated until 6 months after SCI (Non-Patent Document 10), and these extracellular matrix molecules promote axon growth. It is thought to inhibit. Therefore, next, the expression of CSPGs was examined. Consistent with the increase in reactive astrocytes, anti-TGF-β1 antibody treated rats (Fig. 2j) showed significantly more CSPGs expression 2 weeks after SCI compared to control IgG treated rats (Fig. 2j). Although it was reduced, it was not significantly reduced after 1 week of SCI.

  In addition, collagen formation after SCI was examined. Although there was some tendency for collagen expression to decrease due to anti-TGF-β1 antibody administration (Fig. 2k), between SCI-administered rats and control IgG-administered rats one week after SCI, two weeks There was no significant difference after and after 12 weeks.

TGF-β1 inhibition promotes microglia / macrophage accumulation and activation
TGF-β1 is known to play a role as an anti-inflammatory agent capable of suppressing various functions of activated microglia / macrophages (Non-patent Document 11). Therefore, accumulation of microglia / macrophages after SCI was examined. One week and two weeks after injury, immunohistochemistry for Iba-1 was performed to characterize microglia / macrophages. Numerous Iba-1 positive microglia / macrophages were observed within the injury site after 1 and 2 weeks of SCI. As expected, the number of these Iba-1-positive microglia / macrophages appeared to increase with anti-TGF-β1 antibody administration. To quantitatively analyze the extent of microglia / macrophage accumulation, the area occupied by Iba-1-positive cells around the injury site (10 mm length around the center of the injury site) was measured. As a result, administration of anti-TGF-β1 antibody significantly increased the accumulation of Iba-1-positive cells 2 weeks after SCI (FIG. 3i). Next, anti-ED-1 antibody was used to estimate microglia / macrophage activation. ED-1 is a marker for activated macrophages. In rats treated with anti-TGF-β1 antibody, ED-1 positive microglia / macrophages spread in a wider area around the injury site compared to control IgG-treated rats. Statistical treatment showed that administration of anti-TGF-β1 antibody increased the area occupied by ED-1 positive cells after 2 weeks of SCI (FIG. 3j). Thus, these findings indicate that microglia / macrophage accumulation and activation around the injury site after SCI was enhanced by anti-TGF-β1 antibody administration.

TGF-β1 inhibits neurite outgrowth by a mechanism that is dependent on activation of the RhoA / Rho-kinase pathway The above data showed that inhibition of TGF-β1 promoted post-SCI motor recovery Therefore, it was suggested that TGF-β1 inhibition restores damaged neural circuits. Although the reduction of glial scars and the increase in microglia / macrophage activation / accumulation induced by TGF-β1 inhibition appear to support functional recovery, TGF-β1 also acts as the axis of damaged neurons. It may also play a role in directly inhibiting cord growth. To test the hypothesis that TGF-β1 contributes to inhibition of mammalian CNS neurite growth in vitro, cerebellar granule neurons (CGNs) were used. CGNs obtained from postnatal rats (P7-9) were cultured for 24 hours in the presence or absence of 10 ng / mL recombinant TGF-β1 (rTGF-β1), and the length of neurites was examined. . Neurite growth was significantly inhibited when CGNs were treated with rTGF-β1 (FIG. 4b). In order to utilize the signal transduction mechanism involved in neurite outgrowth inhibition, the effect of TGF-β1 on neurons depends on low molecular weight GTPase RhoA or its downstream effector, Rho-related serine / threonine kinase (Rho-kinase) I investigated whether or not. It was observed that when the neurons were cultured for 24 hours in the presence or absence of 10 μM Y27632 (Non-patent Document 12), a specific inhibitor of Rho-kinase, the inhibitory activity of TGF-β1 was reduced by Y27632. (Figure 4b). In order to directly examine whether RhoA is involved in TGF-β1 signaling, the RhoA binding domain of Rhotekin (Non-patent Document 13), which is an effector protein, was used. This test increased the amount of GTP-RhoA in the cell extract compared to control cells within 10 minutes after the addition of TGF-β1. These results indicate that TGF-β1 inhibits neurite outgrowth by a mechanism that is dependent on RhoA / Rho kinase activation.

Neutralization of TGF-β1 induces the growth and / or preservation of serotonergic axons The results of the neurite outgrowth assay indicate that TGF-β1 may also inhibit axonal growth in vivo Yes. Therefore, we investigated whether inhibition of TGF-β1 promotes axonal growth of damaged neurons. Several descending pathways contribute to the recovery of motor function after SCI. By immunohistochemical analysis, descending serotonergic raphe spinal cord axons in anti-IgG-treated SCI rats were examined. 12 weeks after injury, the anterior horn 10 mm caudal to the injury site of control SCI rats has a 40% higher density of 5-hydroxytryptamine (5-HT) positive fibers compared to the 10 mm cranial slice of the injury site. It decreased (Fig. 4f). In samples obtained from injured rats treated with anti-TGF-β1 antibody, the density of 5-HT positive fibers in the anterior horn at 10 mm cranial to the injury site is approximately the same as the control value in the cranial section (Figure 4f). However, the density of 5-HT positive fibers in the anterior horn 10 mm from the injury site was significantly increased compared to the control value in the caudal section (FIG. 4f). Thus, the administration and anti-TGF-β1 antibody promotes the growth and / or preservation of the raphe spinal cord axons on the caudal side of the injury site. The growth of 5-HT positive fibers may be due to a combination of lateral branch germination in the caudal region and / or long-range axonal growth from cleaved serotonin-containing axons.

  Next, the integrity of the descending corticospinal tract (CST) was examined by injecting biotinylated dextranamine (BDA) into the bilateral sensorimotor cortex. However, no labeled fibers were detected on the caudal side of the center of the injury site in both the SCI rats treated with anti-TGF-β1 antibody and the control IgG-treated rats (data not shown). Next, the distal part of the spinal cord obtained 40 mm from the center of the injury site was examined. This is because side branch germination from CST in this region has been shown to be important for motor recovery (Non-Patent Document 14). In SCI rats, germination of BDA-positive fibers was detected in gray matter, particularly in the gray matter adjacent to the posterior column. Germination of BDA positive fibers was also detected in gray matter in SCI rats treated with anti-TGF-β1 antibody. However, there was no significant difference in the number of BDA positive fibers in gray matter between anti-TGF-β1 antibody-treated SCI rats and control IgG-treated SCI rats (FIG. 4k). Thus, the above data denies that anti-TGF-β1 antibody administration promotes germination or regrowth of damaged CST after SCI.

The figure shows the recovery of the motor ability of rats administered with the therapeutic agent of the present invention after spinal cord injury, compared with rats that did not receive the therapeutic agent of the present invention after spinal cord injury and rats that did not damage the spinal cord. is there. BBB scores were measured at the time points shown in the figure after SCI for rats treated with anti-TGF-β1 antibody (13 mice per group), control IgG administered rats (13 mice per group), and sham-operated rats (3 mice per group). . The average ± standard deviation of each group is shown. The anti-TGF-β1 antibody administration group was statistically different from the control IgG administration group on the day shown in the figure after SCI. *: P <0.05 compared to control group i is a figure which shows the quantitative result of glial scar formation. Using Scion Image Software (trade name), sagittal sections obtained from 5 control anti-IgG-treated rats (open bars) and 6 anti-TGF-β1 antibody-treated rats were analyzed. The area positive for GFAP immunoreactivity was calculated as a percentage of the total area of each section (10 mm length around the center of the damaged site). Data are shown as the mean ± standard deviation of three independent experiments. Significant differences were seen between the two groups after 2 weeks of SCI. *: P <0.05 j as compared with the control group is a graph showing the results of measuring CSPG expression after 1 week and 2 weeks after SCI in the anti-TGF-β1 antibody-administered rat and the control IgG-administered rat. Induction of CSPG expression after SCI was suppressed by administration of anti-TGF-β1 antibody. The area positive for CSPG immunoreactivity was calculated as a percentage of the total area of each section (4 mm length around the center of the damaged site). Data are shown as the mean ± standard deviation of three independent experiments. *: Compared with the control group, P <0.05 k is a graph showing the results of measuring collagen in the contusion spinal cord at 1 week, 2 weeks and 12 weeks after SCI. The area positive for collagen immunoreactivity was calculated as a percentage of the total area of each section (4 mm length around the center of the damaged site). There was no significant difference between the two groups at any time. Data are shown as the mean ± standard deviation of three independent experiments. It is a figure which shows the fixed_quantity | quantitative_assay result of activated microglia / macrophage. Using Scion Image Software (trade name), sagittal shapes obtained from 5 control anti-IgG-treated rats (open bars) and 6 anti-TGF-β1 antibody-treated rats at 1 week and 2 weeks after SCI Sections were analyzed. The area positive for Iba-1 immunoreactivity (i) or ED-1 immunoreactivity (k) was calculated as a percentage of the total area of each section (4 mm length around the center of the damaged site). Data are shown as the mean ± standard deviation of three independent experiments. Two weeks after SCI there was a significant difference between the two groups. *: P <0.05 compared to control group It is a figure which shows that axonal growth is inhibited by TGF-β1. CGNs were conditioned on poly-L-lysine (PLL) coated chamber slides in the presence or absence of 10 μM Y27632 in the presence or absence of TGF-β1 (10 ng / mL). Incubated for 24 hours. b indicates the average length of neurites per neuron. Data are shown as the mean ± standard deviation of three independent experiments. *: P <0.01 compared to the control group (Student's t-test) f indicates the quantification result of 5-HT positive fibers in control IgG-treated rats and anti-TGF-β1 antibody-treated rats 12 weeks after SCI . Serotonergic fibers were measured using 10 mm cranial sections and 10 mm caudal sections from the center of the injury site. There was a significant difference between the two groups 10 mm caudal from the center of the injury site. Open bars: control group, black bars: anti-TGF-β1 antibody administration group. *: P <0.05 k compared to the control group in gray matter in the 40 mg cranial section from the center of the injury site in the control IgG-treated rat and the anti-TGF-β1 antibody-treated rat 12 weeks after SCI. FIG. 6 is a graph showing the ratio between the number of BDA-labeled CST fibers and the number in the same section of normal spinal cord. There was no significant difference between the control IgG administration group and the anti-TGF-β1 antibody administration group.

Claims (5)

  A therapeutic agent for central nerve injury comprising a TGF-β1 inhibitor as an active ingredient.   The therapeutic agent according to claim 1, wherein the TGF-β1 inhibitor is an anti-TGF-β1 antibody or an antigen-binding fragment thereof.   The therapeutic agent according to claim 1 or 2, wherein the TGF-β1 inhibitor is an anti-TGF-β1 neutralizing antibody.   The therapeutic agent according to any one of claims 1 to 3, wherein the central nerve is a spinal cord.   A therapeutic agent for spinal cord injury comprising an anti-TGF-β1 neutralizing antibody as an active ingredient.

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