JP2007008876A - Use of complement inhibitory protein for treating spinal cord injury - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a use of complement inhibitory proteins for treating spinal cord injuries, especially a soluble complement receptor I(sCR1), and to provide a method for ameliorating the prognosis of a patient suffering a spinal cord injury. <P>SOLUTION: By administering a complement inhibitory protein to an individual as soon as possible after an initial spinal cord injury occurs, these secondary effects are treated and/or ameliorated and the locomotor function of an vertebrate that has suffered a spinal cord injury is ameliorated. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the use of complement inhibitory proteins, particularly soluble complement receptor I (sCR1), for treating spinal cord injury.

  It is estimated that 7800 people suffer from spinal cord injury (SCI) each year in the United States, and approximately 250,000-400,000 people currently have some form of spinal cord injury or spinal cord dysfunction (National Spinal Cord Injury Association) . There is currently no cure for spinal cord injury.

  SCI is a condition characterized by nerve tissue contusion that results in a decrease or loss of ability to function properly and transmit nerve impulses. It has been shown that not all nerve damage after an SCI event occurs immediately. Following the initial injury, a series of degenerative processes are initiated that promote tissue damage beyond the original injury site, possibly as part of the immune response to the injury. After mechanical destruction of the initial nerves and nerve fibers at the time of injury, bleeding is usually observed at the site of injury within 30 minutes and can then spread over several hours. Within hours after injury, inflammatory cells, such as neutrophils and macrophages, infiltrate the site and cause further damage to neural tissue, ie cell-mediated damage. These post-traumatic events are called “secondary injuries”.

  After initial trauma, it would be beneficial to prevent further damage to the spinal cord and surrounding tissues after spinal cord injury by treating as soon as possible to prevent the effects of secondary injury.

  Currently, the traditional treatment to reduce or minimize disability resulting from secondary damage is intravenous injection of the glucocorticoid, methylprednisolone (Bracken et al., JAMA, 277 (20): 1597- 1604 (1997)). Unfortunately, long-term administration of glucocorticoids has deleterious systemic side effects, such as increased incidence of sepsis and pneumonia, and the range of treatment periods is limited.

Recently, Stokes et al. Reported an improvement in motor function after injection of liposomes containing dichloromethylene diphosphonic acid (“C1 ₂ MBP”) in Lewis rats with contusional injury to the spinal cord (US Pat. No. 5,932,563). issue). Moreover, a- lipoic acid and / or dihydrolipoic acid (DHL) (U.S. Pat. No. 6,432,434), a _d monoclonal antibodies (U.S. Pat. No. 6,432,404), and monosialoganglioside (GM ₁₎ (US Pat. No. 6,620,793) Progress in improving motor function after spinal cord injury has been reported with the treatments used.

  Neurons and oligodendrocytes may be particularly susceptible to complement-mediated cell death (Gasque et al., 1995, Journal of Immunology, 154 (9): 4726-4733; Agoropoulou et al., 1998, Neuroreport, 9 (5 ): 927-932; Wren et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 9025-9029), this is due to complement activation causing demyelination and neurodegeneration. It suggests that it may exacerbate the disorder of the CNS received. Furthermore, internalization of the cellular receptor of C5a can also induce apoptosis (Farkas et al., 1998, Neuroscience, 86 (3): 903-911; Farkas et al., 1998, J. Physiology, 507 (3): 679-687 ).

  The complement system, which constitutes about 10% of the globulins in normal serum, consists of many different proteins that are important in the immune system's response to foreign antigens. The complement system is activated when its primary component is fragmented and the fragment activates additional complement proteins alone or together with other proteins, causing a proteolytic cascade. Activation of the complement system involves tissues such as increased vascular permeability, phagocytic chemotaxis, inflammatory cell activation, foreign body opsonization, direct cell death, and secondary spinal cord injury Cause damage. Given the continuing need for effective treatment for spinal cord injury, new approaches to address post-traumatic phenomena are desirable.

Summary of the Invention The inventors have shown that the administration of complement inhibitory proteins, particularly soluble complement receptor I, i.e. sCR1, after spinal cord injury inhibits inflammation and the effects of the secondary damage described above associated with spinal cord injury. I found it an unexpectedly effective way to improve Soluble CR1 treatment is effective in improving motor function after traumatic spinal cord injury, as demonstrated herein using a rat spinal cord injury model in vivo.

  Accordingly, in its broadest aspect, the invention relates to a method of treating spinal cord injury in a vertebrate subject comprising administering a complement inhibitory protein after spinal cord injury. This treatment is effective in improving or suppressing the adverse effects of secondary damage at the trauma site and improving motor function. Complement inhibiting proteins are preferably administered as soon as possible after spinal cord injury to prevent the adverse effects of complement activation occurring at the site of injury. In a preferred embodiment, the complement inhibitory protein is soluble complement receptor I (sCR1), ie a truncated non-membrane bound fragment of complement receptor I, which fragment is a native or full-length CR1 protein The ability to inhibit complement activation and / or the ability to bind complement protein C3b or C4b, or (preferably) both C3b and C4b.

  Furthermore, the present invention is directed to a method of ameliorating or suppressing the effects of secondary damage following spinal cord injury in vertebrates, the effects being related to complement activation at the site of injury. Preferably, the treatment comprises administration of complement inhibitory protein as soon as possible after the initial injury. Preferably, the complement inhibiting protein is soluble CR1 comprising at least two short consensus repeats (SCRs) at the N-terminus of CR1, and more preferably full length human comprising, for example, 1-1930 amino acids of mature human CR1 The extracellular domain of CR1, most preferably an sCR1 polypeptide having the amino acid sequence of SEQ ID NO: 3 (TP10; AVANT Immunotherapeutics, Inc., Needham, MA (USA)). Preferably, the complement inhibiting protein is administered with a pharmaceutically acceptable carrier.

  In yet another aspect, the present invention provides a method of improving motor function in a vertebrate suffering from spinal cord injury, such as complement activation that causes further damage to tissues surrounding the spinal cord And administering a complement inhibitory protein after the initial injury to reduce the effects of secondary injury caused by the resulting inflammatory response. In a preferred embodiment, the complement inhibitory protein is a soluble CR1 comprising at least two short consensus repeats (SCRs) at the N-terminus of CR1, more preferably a complete comprising, for example, 1-1930 amino acids of mature human CR1 An extracellular domain of long human CR1, most preferably an sCR1 polypeptide (TP10; AVANT Immunotherapeutics, Inc.) having the amino acid sequence of SEQ ID NO: 3. Preferably, the sCR1 is administered with a pharmaceutically acceptable carrier.

  In another aspect, the present invention provides a pharmaceutical composition suitable for inhibiting secondary injury after spinal cord injury, said composition being the result of complement activation and complement activation following trauma. Complement-inhibiting molecules suitable for inhibiting secondary tissue damage at the site of the resulting injury are included. In a preferred embodiment, the complement inhibitory protein is a soluble CR1 comprising at least two short consensus repeats (SCRs) at the N-terminus of CR1, more preferably, for example, a complete comprising 1 to 1930 amino acids of mature human CR1. An extracellular domain of long human CR1, most preferably an sCR1 polypeptide having the amino acid sequence of SEQ ID NO: 3.

  In yet another aspect, the present invention provides a method of preventing neutrophil activation and neutrophil infiltration as a result of complement activation at the site of spinal cord injury by administration of a complement inhibitory protein. In a preferred embodiment, the complement inhibitory protein is a soluble CR1 comprising at least two short consensus repeats (SCRs) at the N-terminus of CR1, more preferably a complete comprising, for example, 1-1930 amino acids of mature human CR1 An extracellular domain of long human CR1, most preferably an sCR1 polypeptide having the amino acid sequence of SEQ ID NO: 3.

  In a particularly preferred embodiment, it is envisaged that the present invention is suitable for the treatment of humans with spinal cord injury by systemic administration of sCR1 as soon as possible after the injury has occurred.

  The methods of the invention include, for example, CR1, Factor H, C4 binding protein (C4-BP), membrane cofactor protein (MCP), decay accelerating factor (DAF), or fragments thereof that retain complement inhibitory properties, etc. Of any complement inhibiting protein. Alternatively, the complement inhibiting protein can be an antibody specific for complement protein, activated complement protein, or a fragment of a complement protein, and such antibody inhibits complement activation. (Eg, anti-C3, anti-C5b-9, etc.). However, in a preferred embodiment of the invention, the complement inhibiting protein is human CR1, more preferably soluble CR1 (sCR1), and most preferably a CR1 polypeptide or sequence comprising the extracellular domain of mature human CR1 A soluble CR1 polypeptide having the amino acid sequence of No. 3.

Detailed Description of the Invention The present invention is directed to compositions and methods for treating spinal cord injury. In particular, the present invention is directed to compositions and methods that inhibit secondary effects that are known to exacerbate initial damage and are known to be due, at least in part, to complement activation. In particular, the present invention provides a method of treating spinal cord injury comprising administering a complement inhibitory protein as soon as possible after the initial spinal cord injury to ameliorate or suppress the effects of secondary injury resulting from initial trauma. set to target. After an initial traumatic event that causes spinal cord injury (SCI), the inflammatory response at the site of injury causes further damage to the surrounding tissue, thereby exacerbating the effects of the injury and delaying or preventing recovery. Administration of a complement inhibitory protein, such as sCR1, as soon as possible after initial injury according to the present invention inhibits or reduces subsequent complement-mediated inflammatory responses, which in turn reduces the severity and impact of secondary injury. Improve. The treatments described herein can improve motor function over time in subjects with traumatic spinal cord injury.

The methods of the invention can be practiced with any complement inhibitory protein that is effective to block complement activation. Such complement inhibiting proteins include, for example, complement receptor I (CR1), factor H, C4 binding protein (C4-BP), membrane cofactor protein (MCP), decay accelerating factor (DAF), or complement Those fragments that retain complement inhibiting properties, such as the ability to inhibit activation, the ability to bind C3b, the ability to bind C4b, or the ability to bind both C3b and C4b are included. Alternatively, the complement inhibiting protein can be an antibody specific for complement protein, activated complement protein, or a fragment of a complement protein, and such antibody inhibits complement activation. (Eg, anti-C3, anti-C5b-9, etc., collectively referred to as “complement-inhibiting antibodies”). Preferably, the complement inhibiting protein used in the methods described herein is a soluble form (non-membrane bound form) of human CR1. Suitable soluble CR1 polypeptides and preparations are described in detail, for example, in US Pat. No. 5,981,481, US Pat. No. 5,456,909, and US Pat. No. 6,193,979. Special mention is made of a soluble CR1 polypeptide (sCR1-sLe ^x ) that is glycosylated and that exhibits a sialyl Lewis X moiety, as described in US Pat. No. 6,193,979. More preferably, the method of the invention uses a polypeptide comprising the extracellular domain of mature human CR1 (see SEQ ID NO: 2 for full length mature human CR1 sequence). Most preferably, the methods of the invention and the pharmaceutical compositions useful for practicing the invention comprise a soluble human CR1 protein having the amino acid sequence of SEQ ID NO: 3.

  As described more fully below, administration of sCR1 after spinal cord injury in a rat spinal cord injury model resulted in overall recovery, ie motor function, in sCR1-treated rats compared to saline-treated controls. In addition to improving, it has been demonstrated herein to reduce the activity and level of tissue neutrophil infiltration and reduce the level of expression of complement-related proteins at the site of injury. We have thus shown that administration of complement inhibitory proteins after spinal cord injury in vertebrate subjects reduces the effects of secondary damage caused by complement activation after early spinal cord injury. I found

  In certain embodiments, the present invention relates to soluble CR1 polypeptides and their use for the treatment of spinal cord injury. As used herein, a “soluble CR1 polypeptide” or “soluble CR1” or “sCR1” is a full-length CR1 protein that is not expressed on the cell surface like a transmembrane protein, unlike the native CR1 protein. It shall be used to refer to a part. In particular, a CR1 polypeptide that substantially lacks the transmembrane region, or preferably contains all or part of the extracellular portion of CR1, is a soluble CR1 polypeptide. In a preferred embodiment, soluble CR1 polypeptides useful in the present invention are secreted by cells that express them.

  Human C3b / C4b receptor, called complement receptor I (CR1) or CD35, has the presence of erythrocytes, monocytes / macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomeruli. Present on the membrane of podocytes (Fearon DT, 1980, J. Exp. Med., 152: 20, Wilson, JG, et al., 1983, J. Immunol., 131: 684). CR1 specifically binds to C3b, C4b, and iC3b.

  CR1 can also inhibit C3 / C5 convertases of the classical and alternative pathways and can function as a cofactor for the cleavage of C3b and C4b by factor I, so CR1 also functions as a receptor. In addition, it also has a complement regulatory function (Fearon, DT, 1979, Proc. Natl. Acad. Sci. USA, 76: 5867; Iida, KI and Nussenzweig, V., 1981, J. Exp. Med., 153: 1138). In the second pathway of complement activation, the bimolecular complex C3b-Bb is a C3 enzyme (converting enzyme). CR1 can bind to C3b, thereby facilitating the dissociation of the Bb fragment from the complex. In addition, C3b binding to CR1 is susceptible to irreversible proteolytic inactivation by C3b factor I, resulting in the production of inactivated derivatives of C3b (ie, iC3b, C3d and C3dg). . In the classical pathway of complement activation, the C3bC4bC2a complex is a C5 convertase. CR1 binds to C4b and / or C3b, thereby facilitating the dissociation of C2a from the complex. The binding is amenable to irreversible proteolytic inactivation by C4b and / or C3b factor I.

  Several soluble (non-membrane bound) fragments of CR1 have been generated by recombinant DNA methods by deleting the transmembrane and cytoplasmic regions from the expressed DNA (eg, Fearon et al., International Patent Publication). No. WO 89/09220, Oct. 5, 1989; see Fearon et al., International Patent Publication No. WO 91/05047, Apr. 18, 1991). Soluble CR1 fragments are functionally active, i.e., retain the ability to bind to C3b and / or C4b, depending on the natural CR1 region of the CR1 fragment, inhibit complement activation, and I The cofactor activity of the factor is shown. Such constructs inhibit the results of complement activation in vitro, such as neutrophil oxidative burst, complement-mediated hemolysis, and C3a and C5a production. The soluble construct, sCR1 / pBSCR1c, also exhibits in vivo activity in the reverse passive Arthus reaction (Fearon et al., 1989, 1991, supra; Yeh et al., 1991, J. Immunol., 146: 250 (1991)), Inhibition of myocardial inflammation and necrosis after ischemia (Fearon et al., Supra; Weisman et al., 1990, Science, 249: 146-151) and prolonged survival after transplantation (Pruitt et al., 1991, J. Surg. Res., 50: 350; Pruitt et al., 1991, Transplantation, 52: 868 (1991)).

  The complete cDNA coding sequence for human CR1 protein is shown in SEQ ID NO: 1. The amino acid sequence of mature human CR1 is shown in SEQ ID NO: 2.

  Isolation of the full-length CR1 gene, expression and purification of the full-length protein and its active fragments, and proof of activity in full-length proteins and fragments derived from full-length proteins are described in US Pat. Incorporated into the specification.

  Complement inhibiting proteins such as sCR1 useful in the methods of the present invention are advantageously produced in large quantities in host cells, such as bacterial cells, mammalian cells, or plant cells, using recombinant DNA technology that expresses the protein. Generated. For complement inhibitory proteins contemplated herein, mammalian host cells such as Chinese hamster ovary (CHO) cells or African green monkey kidney (COS) cells are preferred. The isolated gene encoding the desired protein can be inserted into an appropriate cloning vector. A number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses. The vector system must be compatible with the host cell used. Such vectors are bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmids or CDM8 plasmids (Seed, B., 1987, Nature, 329: 840-842), or well known thereof. Including, but not limited to, vector derivatives. Recombinant molecules can be introduced into host cells by transformation, transfection, infection, electroporation and the like.

  Recombinant cells that produce the preferred type of sCR1 are available from the American Type Culture Collection, Rockville, MD (accession number CRL 10052). The deposited cell is the Chinese hamster ovary cell line DUX B11 containing plasmid pBSCR1c / pTCSgpt clone 35.6 encoding soluble CR1 having the amino acid sequence of SEQ ID NO: 3. Such sCR1 protein in purified form is produced by AVANT Immunotherapeutics, Inc. (Needham, Mass.) Under the product name TP10.

  After expression in the host cell, soluble CR1 molecules are standard, including chromatographic (eg, ion exchange chromatography, affinity chromatography, and size fractionation column chromatography, high pressure liquid chromatography), centrifugation, and solubility differences. Can be isolated and purified by any convenient method or by any other standard technique for protein purification. Preferred purification methods are described in US Pat. No. 6,316,604, US Pat. No. 5,252,216, and US Pat. No. 5,840,858.

  Soluble CR1 protein is therapeutically useful in the regulation of complement-mediated diseases, ie diseases or conditions characterized by inappropriate or undesirable complement activation. Soluble CR1 protein that can bind to C3b or C4b and / or retain the ability to inhibit the C3 or C5 convertase of the second or classical pathway and / or retain the cofactor activity of factor I Alternatively, the fragment can be used to inhibit complement activation. In the present invention, we use soluble CR1 to ameliorate or inhibit unwanted complement activation after traumatic spinal cord injury, such as post-traumatic inflammatory conditions resulting from complement activation. Proved that it could be.

  In the methods of the invention, to reduce complement activation, to reduce or inhibit inflammation associated with spinal cord injury, and to reduce or inhibit the effects of harmful secondary damage associated with such inflammation To do so, a complement inhibitory protein such as soluble CR1 is preferably administered intravenously to a vertebrate subject suffering from spinal cord injury.

  In the method of treating spinal cord injury according to the present invention, a therapeutically effective amount of a complement inhibiting protein or preparation thereof is administered to a subject in need of such treatment. A preferred subject is a human. The amount administered can be of secondary effects of acute inflammation following inhibition of complement activation or spinal cord injury, such as, for example, reduced neutrophil infiltration into damaged tissue or reduced neutrophil activity. Must be sufficient for inhibition (see Example 3, above). Although determination of a therapeutically effective dose is within the ability of one skilled in the art, as an example, in embodiments of the methods described herein using sCR1 for the treatment of spinal cord injury, an effective dose Will be in the range of 0.01-100 mg / kg, preferably 0.1-10 mg / kg, most preferably 1-10 mg / kg of patient weight. The pharmaceutical composition should be administered as soon as possible after spinal cord injury, and repeated administration is contemplated, eg, to maintain an amount effective to reduce or inhibit complement activation. For example, as demonstrated in the examples below, an effective sCR1 dosing regimen follows a single dose of sCR1 (6 mg / kg) on the day of spinal cord injury, followed by intravenous injections daily during the course of treatment. Consists of similar doses.

  For administration, sCR1 or other therapeutic protein can be prepared in a suitable pharmaceutical composition. Such compositions generally include a therapeutically effective amount of sCR1 or other protein and a pharmaceutically acceptable excipient such as saline, buffered saline, phosphate buffer, dextrose, or sterile water. Or a carrier. The composition may also include certain stabilizers such as sugars including mannose or mannitol.

  Various delivery systems are known and can be used for delivery of CR1 and soluble fragments of CR1, such as, for example, encapsulation in liposomes, microparticles, or microcapsules. Suitable methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, or epidural injection, and oral or pulmonary delivery.

  In a preferred embodiment, the pharmaceutical composition for use in the present invention can be prepared as a pharmaceutical composition for intravenous administration to an individual suffering from spinal cord injury according to conventional methods. In general, compositions for intravenous administration are sterile aqueous buffer solutions. If necessary, the composition may also include a solubilizer and a local anesthetic to relieve pain at the injection site, such as lidocaine. The ingredients are usually provided in unit dosage forms, separately or mixed, as lyophilized powders or water-free concentrates in sealed containers such as ampoules or sachets, and the amount of active substance in active units Is displayed. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  A pharmaceutical pack comprising one or more containers filled with one or more ingredients of the pharmaceutical composition is also contemplated. It is understood that administration of complement inhibitory proteins according to the method of the present invention for the treatment of spinal cord injury can be conveniently administered by emergency personnel on the spot, for example, in the event of a car accident that causes spinal cord injury. It is important that the complement inhibitory protein be administered to individuals with spinal cord injury as soon as possible after injury, preferably immediately or within hours or minutes after the initial spinal cord injury has occurred So it is particularly advantageous.

  The following examples illustrate the method of the present invention. They are provided for purposes of illustration and are not intended to be limiting.

  All data were expressed as mean ± standard deviation (x ± s). Using SPSS 10.0 statistical analysis software, a test for equal variances was first performed between the treatment group and the control group at different time points, followed by a t-test.

Example 1
To test whether complement-inhibiting protein can improve or suppress secondary damage after traumatic events involving the spinal cord and whether it can improve motor function over time (Black) Et al., 1988, Neuro. Surg., 22: 51-60), using a 2 mm diameter concave plastic pad placed at the target site (T ₁₀ segment) and a striking force of 50 g / cm. Sprague-Dawley (SD) rats (n = 80) (Experimental Animal Department of China Medical University) were subjected to spinal cord injury.

  After injury, 80 rats were randomly divided into 10 groups of 8 rats / group. Of the 10 groups, 5 groups (n = 40) consist of rats receiving sCR1 (TP10, AVANT Immunotherapeutics, Inc.) and the remaining 5 groups (n = 40) receive saline injections only. Consists of receiving control rats. Each of the sCR1 group and each of the saline groups were assigned a separate evaluation time point, ie, 12 hours, 1, 3, 7, and 14 days after the initial spinal cord injury, respectively. All rats receiving sCR1 injections (n = 40) received a 6 mg / kg tail vein injection 1 hour after the initial injury, followed by a single daily injection (6 mg / kg) for the duration of the experiment Received. Control rats (n = 40) received a tail vein injection (6 mg / kg) of saline 1 hour after the initial injury, followed by a daily injection (6 mg / kg) for the duration of the experiment.

Evaluation of neural function The recovery of motor function of the lower limbs over time was examined using the inclined plate method. Rats were placed on the same flat plate at 12 hours, 1, 3, 7, and 14 days after injury. The body axis of the rat was aligned along the longitudinal axis of the inclined plate. The vertical angle of the inclined plate was increased by 5 ° and the recovery of movement was evaluated as a function of the maximum inclination angle at which the rat could maintain its position on the plate. Each rat was examined three times and the average angle was recorded. The results of the inclined plate experiment are shown in Table 1.

  As seen in Table 1, there was no significant difference in the angles that rats treated with sCR1 and saline treated (control) 12 hours and 1 day after injury were able to maintain on the tilt plate, but 3 days after treatment, The motor function of rats treated with sCR1 for 7 days and 14 days was significantly improved compared to saline-treated controls. The results in Table 1 demonstrate that administration of sCR1 after spinal cord injury inhibits nerve injury at the site of injury and leads to improvements in recovery of motor function.

Example 2
Histopathological examination Three rats from each time point group were anesthetized at their respective time points (12 hours, 1, 3, 7, and 14 days after injury) and 1 cm spinal cord from the injury site Tissue was collected, fixed and dehydrated. Serial sections 12 μm thick were prepared using a frozen microtome and prepared for hematoxylin and eosin (HE) staining or C3c, C9 or CD59 immunohistochemical staining. Rabbit anti-rat C3c antibody was purchased from Zymed Laboratories (Invitrogen Corp., Carlsbad, CA). Rabbit anti-rat C9 antibody and rabbit anti-CD59 antibody were obtained from Professor Morgan (Wales University, UK). The HE staining kit was purchased from Sigma-Aldrich Biotechnology Center (SABC; Sigma-Aldrich Corp., St. Louis, MO).

Result: HE staining
The results of HE staining were observed with a microscope (200 ×). In the saline control rats, at 12 hours after injury, the gray matter had spotted bleeding and neutrophils began to infiltrate the tissue surrounding the injury site. In the physiological saline group, neurons were swollen one day after injury, showing nuclear asymmetry. Three days after injury, saline rat neurons apparently rounded and expanded, showing nuclear enrichment (suggesting apoptosis) and nuclear fragmentation. Furthermore, there was a large amount of neutrophil infiltration in the tissue derived from the damaged site, and there was large speckled bleeding in gray matter. Seven days after injury, the number of residual neurons decreased, but some neutrophil infiltration was still present in the gray matter. At 14 days after injury, fewer residual neurons and less gray matter neutrophil infiltration were observed. Cavity formation was observed.

  In both sCR1 and control groups, the injured spinal cord deteriorated with time, peaked around day 3 after injury, and tended to stabilize by 14 days after injury. However, in the sCR1-treated group, all symptoms associated with secondary effects at the site of injury, i.e., neuronal cell expansion, degeneration, and neutrophil infiltration, were significantly milder than those in the control group as described above. there were.

Results: The presence of C3c at the site of C3c immunohistochemical staining indicates that complement activation has occurred at that site. The results of C3c immunohistochemical staining demonstrated that C3c expression levels were lower at the site of injury in sCR1-treated rats than saline-treated controls.

Three C3c immunohistochemically stained sections were selected for analysis from each rat in each group. Five high-magnification fields from the front to the back corners of gray matter were selected. The average gray value (AG) of C3c positive reactant was detected using a MetaMorph® automatic color image analyzer (Universal Imaging Corp., Downington, PA). The average gray value is inversely proportional to the C3c immune response intensity. The results of C3c immunohistochemical staining are shown in Table 2.

  At 12 hours post-injury, scattered C3c positive expression was observed in some neuronal cell membranes and cytoplasm in the anterior horn of the spinal cord in both sCR1-treated and saline-treated rats. On day 1 after injury, C3c expression increased in both groups. On day 3 post injury, high levels of C3c positive expression were evident in the remaining neuronal cell membrane, neurotropilem and neuronal cytoplasm. Seven days after injury, C3c expression gradually decreased. On day 14 after injury, some C3c positive neurons were still present in the gray matter and low levels of C3c expression were evident in the neural network.

  In both sCR1 treated and control groups, C3c positive expression in spinal cord injured tissues peaked around day 3 after injury, then began to decrease, and stabilized 2 weeks after injury. However, as seen in Table 2, C3c positive expression levels were significantly lower in sCR1 treated rats at all time points compared to saline treated rats (P <0.01).

Results: C9 and CD59 immunohistochemical staining
Data from C9 and CD59 immunohistochemical staining were obtained using the same method used for detection of C3c expression and observed by light microscopy (200X).

  C3 and C9 are important endogenous components of the complement system. C3 is at the junction of the classical complement activation pathway and the second complement activation pathway and serves as a hub in the complement activation cascade. The presence of C3c, a degradation product of C3 after its inactivation, indicates that complement system activation has progressed to the C3 level. C9, the last molecule that makes up the membrane-damaging complex (MAC), is also the most important complement component for activation of the complement system that attacks and destroys target cells. The anti-C9 antibody used herein was specific for C9 deposited in tissue, which is C9 incorporated into MAC. Therefore, detection of C9 fully reflected the MAC expression status.

  Like C3c, C9 was found to be present in spinal cord injured tissue at each time point after injury in both sCR1 and saline (control) groups, which elicits a complement cascade response in acute spinal cord injury Indicating that it can be activated to the final stage.

The presence of C9 detected in this experiment was similar to that observed in the C3c assay described above. The image analysis results of the C9 detection assay are shown in Table 3.

  As can be seen from the data in Table 3, C9 expression in the sCR1 treated group was significantly lower than that observed in the saline treated group (P <0.01).

  CD59 is a homologous limiting factor in the final stage of complement. It is an important protective complement regulator because its main biological activity is to inhibit the continuation of MAC formation and prevent cell lysis.

  Similar to C3c and C9, CD59 was found to be present in spinal cord injury tissue at each time point after injury in both sCR1 and saline (control) groups, which is a complement cascade response in acute spinal cord injury Indicates that can be induced and activated to the final stage.

  The presence of CD59 detected in this experiment was similar to that observed in the C3c and C9 assays described above. The image analysis results of the CD59 detection assay are shown in Table 4.

  As seen in Table 4, the sCR1 treated groups at 12 hours, 1 day, 3 days and 7 days after injury showed significant differences from the saline treated controls, and sCR1 inhibited complement activation. Showed that. By day 14, the difference in CD59 expression was no longer significant, although it is likely that long-term treatment with sCR1 has a protective effect that reduces the destruction of CD59-expressing cells (eg, neurons, glial cells) in spinal cord injury tissue Would suggest that you have.

  Injured neuronal swelling, degeneration and necrosis were milder in the sCR1 treated group at all time points. In addition, less neutrophil infiltration of damaged tissue was observed in samples taken from the sCR1-treated group.

Example 3
Myeloperoxidase activity Myeloperoxidase (MPO) is an indicator enzyme for neutrophils, the main inflammatory cells involved in acute inflammatory reactions. Myeloperoxidase catalyzes the conversion of hydrogen peroxide (H ₂ O ₂ ) and chloride anions (Cl ⁻ ) to hypochlorous acid (HOCl). Hypochlorous acid is cytotoxic and is produced by neutrophils, destroying invading bacteria and other pathogens. The presence and level of myeloperoxidase at the site of injury is thus a direct indicator of the activity and level of neutrophil infiltration at that site.

  To measure the level of myeloperoxidase activity at the injury site, anesthetize 5 rats from each group at each time point, i.e. 12 hours, 1, 3, 7, and 14 days after injury, and spinal cord A 1 cm tissue sample was taken from the injury site. The tissue was stored in liquid nitrogen and weighed after weighing.

  Prior to homogenization, a tribasic potassium phosphate buffer solution containing 0.5% cetrimonium bromide (CTBA) was added. Each sample was then disrupted by sonication at 4 ° C. for 20 minutes and then centrifuged at 12,500 rpm for 30 minutes. The supernatant was taken and added to 2.9 ml phosphate buffer (pH 7.0) containing 0.5% dianisidine hydrochloride and 0.0005% hydrogen peroxide. Myeloperoxidase levels were measured by spectrophotometry at 460 nm for 2 minutes. One unit of myeloperoxidase activity was defined as the degradation of 1 μmol of hydrogen peroxide per minute at 25 ° C. Myeloperoxidase levels are expressed as units of myeloperoxidase activity per gram of spinal cord tissue.

The results of the myeloperoxidase assay are shown in Table 5.

  The levels of myeloperoxidase in sCR1-treated and saline-treated rats began to increase 12 hours after injury, reached peak levels on day 3 after injury, and then began to decrease. However, as seen in Table 5, the myeloperoxidase level in the sCR1 treated group was consistently lower than the control group at each time point (P <0.01) (see Table 5).

  These results clearly indicate that administration of sCR1 after spinal cord injury reduces the level and amount of neutrophil infiltration at the site of injury, and consequently reduces the effects of inflammation and inflammation-related secondary damage To demonstrate.

  The above example data demonstrate that administration of sCR1 after spinal cord injury in vertebrates inhibits complement activation and inhibits inflammation. Improved recovery of motor function after injury was also observed after treatment with sCR1. These data demonstrate the suitability of the methods disclosed herein for the treatment of spinal cord injury in vertebrates, including humans.

  While numerous embodiments have been described above, it will be appreciated by those skilled in the art that modifications and variations of the described compositions and methods can be made without departing from the scope of the disclosure or the appended claims. Will be understood. The articles and publications cited herein are incorporated by reference.

Claims (18)

  A method of treating spinal cord injury in a vertebrate subject comprising administering a complement inhibitory protein to said vertebrate as soon as possible after said injury. The complement inhibitory protein retains complement receptor I (CR1), factor H, C4 binding protein (C4-BP), membrane cofactor protein (MCP), decay accelerating factor (DAF), and complement inhibitory properties fragment is selected from the group consisting of complement inhibitory antibodies, and sCR1-sLe ^x, a method according to claim 1.   The method of claim 2, wherein the vertebrate is a human.   The method of claim 2, wherein the complement inhibiting protein is a soluble CR1 protein.   5. The method of claim 4, wherein the soluble CR1 is a polypeptide comprising at least two short consensus repeats at the N-terminus of full length human CR1.   6. The method of claim 5, wherein the soluble CR1 is a polypeptide comprising the extracellular domain of mature human CR1.   5. The method of claim 4, wherein the soluble CR1 has the amino acid sequence of SEQ ID NO: 3.   A method for improving motor function of a vertebrate subject undergoing spinal cord injury, comprising administering a complement inhibiting protein to the subject as soon as possible after the injury. The complement inhibitory protein retains complement receptor I (CR1), factor H, C4 binding protein (C4-BP), membrane cofactor protein (MCP), decay accelerating factor (DAF), and complement inhibitory properties fragment is selected from the group consisting of complement inhibitory antibodies, and sCR1-sLe ^x, the method of claim 8.   The method of claim 9, wherein the vertebrate is a human.   10. The method of claim 9, wherein the complement inhibiting protein is a soluble CR1 protein.   12. The method of claim 11, wherein the soluble CR1 is a polypeptide comprising at least two short consensus repeats at the N-terminus of full-length human CR1.   13. The method of claim 12, wherein the soluble CR1 is a polypeptide comprising the extracellular domain of mature human CR1.   12. The method of claim 11, wherein the soluble CR1 has the amino acid sequence of SEQ ID NO: 3.   Use of soluble complement receptor I (sCR1) for the treatment of spinal cord injury.   A pharmaceutical composition for use in the treatment of spinal cord injury comprising a therapeutically effective amount of soluble CR1 protein and a pharmaceutically acceptable excipient or carrier.   The pharmaceutical composition according to claim 16, wherein the soluble CR1 protein is a polypeptide comprising the extracellular domain of mature human CR1.   The pharmaceutical composition according to claim 16, wherein the soluble CR1 protein is a polypeptide having the amino acid sequence of SEQ ID NO: 3.