WO2002067976A1

WO2002067976A1 - Novel ischemia/reperfusion injury inhibitors

Info

Publication number: WO2002067976A1
Application number: PCT/JP2001/007524
Authority: WO
Inventors: Masaki Hirashima; Hiroaki Maeda; Chikateru Nozaki
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 2001-02-28
Filing date: 2001-08-31
Publication date: 2002-09-06

Abstract

Ischemia/reperfusion injury inhibitors which contain as the main component(s) selenoprotein P and/or C-terminal peptide(s) of this protein. These ischemia/reperfusion injury inhibitors are efficaciously usable for diseases caused by ischemia/reperfusion injury such as cerebral infarction, ischemic organ injury, reperfusion injury following organ transplantation or the like, vascular lesion, arteriosclerosis and myocardial infarction.

Description

Specification

Novel ischemia / reperfusion injury inhibitor

Technical field

The present invention relates to the field of ethical pharmaceuticals and to new uses for plasma proteins. More specifically, the present invention relates to a drug for diseases such as vascular disorder, neuropathy, organ / tissue disorder, and motor dysfunction caused by ischemia / reperfusion injury caused during organ transplantation or various shocks. More specifically, the present invention relates to an agent for suppressing ischemia / reperfusion injury, comprising selenoprotein P, which is a kind of plasma protein, and preferably containing the peptide at the C-terminal side of selenoprotein P or the peptide group as an active ingredient. .

Background art

In recent years, diseases such as organ damage due to ischemia-reperfusion injury have been attracting attention.

Ischemia refers to highly localized anemia. The types of ischemia include compression ischemia caused by stenosis or occlusion of the arterial wall due to external compression such as a tumor, obstructive ischemia due to changes in blood vessels or blood vessels themselves, such as thrombosis and arteriosclerosis, and It can be divided into spastic ischemia due to vasospasm such as cerebral anemia and angina, etc., and relative ischemia and absolute ischemia depending on whether the ischemia is due to the force due to stenosis of the vascular lumen or complete occlusion. They are classified into and. Also, ischemia easily occurs in cases other than those caused by the disease state, such as during bleeding prevention and hemostatic treatment during surgery.

Usually, the local changes caused by ischemia depend on the duration of ischemia, the susceptibility of the tissue to ischemia, or the presence or absence of a vascular anastomosis at that site.Generally, oxygen deprivation and nutrients in cells and tissues in the ischemic area Oxygen deficiency results in impairment, through disruption of ATP production, which first impairs function and eventually leads to degeneration, necrosis, or atrophy. Thus, localized ischemic ischemia resulting from circulatory disturbances is an infarction. Infarcts can be broadly divided into anemic and hemorrhagic infarcts. The former are often found in the brain, heart,, kidney, and spleen, and the latter are found in the lungs, intestines, testes, ovaries, and sometimes in the brain. Thus, ischemic conditions that stop blood flow, to varying degrees, adversely affect tissues. In particular, with regard to the brain, cerebral (frontal), cerebellar, vestibular, and spinal ataxia accompany vascular disorders.

Improve ischemic status in cases of early infarction or ischemia during surgery that does not lead to infarction Forces that could avoid infarction would in fact often cause ischemia / reperfusion injury. Ischemic reperfusion injury is a disorder in which reperfusion or reoxygenation associated with resumption of blood flow to an ischemic organ occurs in the cells and tissues of the ischemic organ. The mechanism of post-ischemic injury in the brain, myocardium, lung, liver, kidney, knee, and gastrointestinal tract includes the active oxygen produced mainly by phagocytic cells such as neutrophils and macrophages, and vascular endothelial cells. The generation of free radicals, leukotriene, tropoxane formation, lipid-sugar peroxidation, protein denaturation, inactivation of enzymes, .DNA chain cleavage, nucleobase modification, etc. are one of the causes of tissue damage. It is considered. Simultaneously, damage of endothelial cells causes microcirculation disorder, prolongs ischemia, increases albumin permeability, adherence of neutrophils to endothelial cells, infiltration into tissues, influx of intracellular Ca ions And so on, exacerbating the obstacles. This disorder depends on the time of ischemia, and the longer the time of ischemia, the worse the reperfusion injury.

From these facts, it is necessary to suppress the damage of tissue cells caused by ischemia and the damage caused by reperfusion, in order to improve various disease states associated with ischemia and reduce tissue damage during surgery. It is understood that it is necessary. In other words, in order to reduce the direct damage caused by ischemia, it is necessary to maintain substances that are necessary for maintaining cells, for example, cells in a state where cell death does not easily occur even when oxygen and nutrient concentrations are low. In order to suppress reperfusion injury, it is necessary to improve blood flow through vasodilation, increase antioxidant activity, and control chemical mediators.

Disclosure of the invention

(Technical problems to be solved by the invention)

Currently, in addition to superoxide dismutase ゃ catalase, daltathione peroxidase, and other substances that prevent and control oxidative disorders in many organisms, trimetazidine hydrochloride, a therapeutic agent for ischemic heart disease that is actually used for treatment, is also available. Tozampoxane Synthetic enzyme inhibitor ozadarel sodium, cerebral circulatory metabolism improver difenprodil tartrate, ischemic encephalopathy improver nizophenone fumarate, etc. It cannot be done. In order to ameliorate such various disorders caused by ischemia, substances other than known substances that are currently being developed or used, such as substances that suppress cell death occurring under adverse conditions for cells, Intracellular There is a need to search for substances that increase antioxidant capacity, and the identification of such substances is considered to be of great significance, and new, more effective ischemia / reperfusion injury inhibitors, Particularly in the area of the brain, a drug for improving vascular disorders, neuropathy associated with ischemia / reperfusion injury, and ataxia is in great demand.

The above description (background art and technical problem to be solved by the invention)

"Why do cells die due to ischemia?" By Kunio Tagawa, Kyoritsu Shuppan; "Handbook of stroke" Keiji Sano, IPC; "Today's medical care" CD-ROM version, Medical Shoin;

"Medical Dictionary" CD-ROM version, Nanzan-do; "Latest Medical Dictionary" CD-ROM 2nd edition, referring to the description of Medical and Dental Medicine Publishing.

(How to solve it)

Under the circumstances described above, the inventors of the present application have not previously reported on Serenob Koutin P, which is a protein derived from blood components, and on the C-terminal peptide of the selenoprotein P in a more preferable embodiment. Cell death inhibitory activity was found, and a patent application was filed based on this finding (PCT / JP99 / 063222). The present inventors have conducted intensive studies to provide a new agent for suppressing ischemia / reperfusion injury. The fact that the peptide group can be adequately adapted to humans and other animals as an agent for suppressing ischemia and reperfusion injury, especially as an ataxia-improving agent associated with vascular injury in the brain region, by actual in vivo administration in model animals The present invention was completed based on this finding.

That is, the present invention relates to an agent for suppressing ischemia / reperfusion injury comprising selenoprotein P and / or a C-terminal peptide of the protein or the peptide group as a main component.

In a preferred embodiment of the present invention, the C-terminal peptide of the protein or the peptide group comprises a deletion, substitution or addition of one or several amino acids at the C-terminal 103 amino acid sequence of selenoprotein P. The amino acid sequence, a partial sequence of any one of the amino acid sequences, or a peptide having an amino acid sequence containing the amino acid sequence as a part thereof, or the peptide group.

In a further preferred embodiment of the present invention, the C-terminal peptide of the protein Or the peptide group has the following formula:

, Above): Lys Arg Cys lie Asn Gin Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa Cys Cys His Cys Arg His Leu (location (J number 1) and / or

(I I): Thr Gly Ser Alalie Thr Xaa Gin Cys Lys Glu Asn Leu Pro Ser Leu

Cys Ser Xaa Gin Gly Leu Arg Ala Glu Glu Asn lie (SEQ ID NO: 2)

(Where Xaa represents selenocistine)

An amino acid sequence represented by the following, an amino acid sequence in which one or several amino acids have been deleted, substituted or added, a partial sequence of any one of the above amino acid sequences, or a part thereof. Or a peptide group having the amino acid sequence contained therein.

Selenoprotein P is glutathione peroxidase in 1997

(glutathione-peroxidase) was identified as a selenium-containing protein, and in 1982 it was clarified that selenium was taken up in the form of selenocysteine. Furthermore, in 1991, cloning of the cDNA of selenoprotein P revealed the full-length amino acid sequence, which indicated that the protein could contain up to 10 selenocysteines. (Hill KE and urk RF, Biomed. Environ. Sci., 10, p. 198-208 (1997)).

The present invention relates to this new drug effect of selenoprotein P, and the essential feature of the ischemic / reperfusion injury inhibitor of the present invention is selenoprotein P. The selenoprotein P used here is not particularly limited, and includes any molecular form as long as it has a desired ischemia / reperfusion injury inhibitory activity. That is, various molecular forms such as complete molecular selenoprotein P can be targeted. Among them, a preferred embodiment is a peptide at the C-terminal side of selenoprotein P or the peptide group, and among them, a C-terminal 103 amino acid sequence, and one or several amino acids of the amino acid sequence Particularly preferred are peptides having an amino acid sequence in which is deleted, substituted or added, a partial sequence of any one of the above amino acid sequences, or an amino acid sequence containing the above amino acid sequence as a part thereof, or a group of such peptides. It can be recommended as a mode. Regarding amino acid substitution, especially Cys is replaced with Ser The preferred embodiment is preferred.

In the present invention, the `` peptide group '' refers to a sequence of about 4 to 14 amino acids or one or several amino acids including at least one selenocysteine derived from the amino acid sequence of selenoprotein P. A peptide containing the amino acid sequence that has been deleted, substituted or added, and refers to a collection of peptides having different fine structures due to the presence or absence of sugar chains, differences in charge, diversity of fragmentation, and the like. That is, the selenoprotein P and peptide group of the present invention are derived from the amino acid sequence of selenoprotein P, and if they have a desired ischemia / reperfusion injury inhibitory activity, there are no particular restrictions on the molecular form. These include the complete molecular form of selenoprotein P as well as the C-terminal peptide resulting therefrom. Such a peptide of the present invention can be prepared by a conventional method using a peptide synthesizer, or a chemical compound can be designed using the peptide of the present invention as a lead substance. According to the knowledge obtained during the purification process of the peptide, the peptide or the peptide group was recovered in a molecular weight fraction of 1 OkDa to 30 kDa based on the (a) molecular weight fractionation membrane, and (b) ion As a result of the examination of the binding property to the exchange resin, it was confirmed that it has a structure showing an isoelectric point between pH 7 and pH 8 in blood and a structure showing an isoelectric point above pH 8 (c ) Non-reducing SDS-PAGE shows two bands with a molecular weight of 13 to 14 kDa and two bands of 16 to 17 kDa with sugar chains added to them. d) In SDS-PAGE under reducing conditions, in addition to the above-mentioned band, it has the property of exhibiting bands of 3 to 4 kDa, 7 to 9 kDa and 10 to 12 kDa. It was revealed that the fragmented peptide also had activity.

According to a more detailed analysis, the peptide or peptides are represented by the following formula:

(Izo: Lys Arg Cys lie Asn Gin Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa Cys Cys His Cys Arg His Leu (Distribution (I number 1))

(II): Thr Gly Ser Ala lie Thr Xaa Gin Cys Lys Glu Asn Leu Pro Ser Leu Cys Ser Xaa Gin Gly Leu Arg Ala Glu Glu Asn lie (SEQ ID NO: 2)

(Where Xaa represents selenocistine)

Or a peptide having a partial sequence of the amino acid or Was found to be a peptide group.

The method for producing selenoprotein P or a peptide or peptide group derived from the protein used in the present invention is not particularly limited.For example, a method for separating human blood from human blood, or a method using genetic recombination technology Can be manufactured. Hereinafter, as a preferred embodiment, a method for preparing plasma as a starting material will be outlined. Serenob, which is a main component of the ischemia-reperfusion injury inhibitor used in the present invention, is a peptide, or a group of peptides derived from the protein, which is more heat, denaturing agent and broader than common enzymes. Since it is stable to pH and protease in blood, when purifying and identifying it, in one embodiment, plasma is used as a starting material and various chromatography steps, for example, heparin chromatography, Applicable to on-exchange chromatography, anion exchange chromatography, hydrophobic chromatography, genole filtration chromatography, reversed-phase chromatography, hide-port xyapatite chromatography, affinity chromatography such as antibody columns, etc. In addition to the fractionation method using various carriers, ammonium sulfate Various fractionation methods such as membrane precipitation fractionation, molecular weight membrane fractionation, isoelectric focusing fractionation, and electrophoretic fractionation can be used. By combining these fractionation methods, it is possible to fractionate a desired selenoprotein P or peptide or peptide group. Examples of desirable combinations are shown in Preparation Examples 1 to 3. The outline of the embodiment is, for example, for a peptide or a group of peptides, in order, heparin chromatography, ammonium sulfate precipitation fractionation, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, heparin chromatography, gel These are filtration chromatography, reverse phase chromatography and anion exchange chromatography.

The active fraction obtained by this combination has an estimated purity of 5% or less, for example, when human plasma is used as a starting material, when cell death inhibitory activity is used as an index, 2 X 1 OV lmg protein / m 1 It can be purified in a state showing activity. Since the plasma activity of the starting material is about 20 to 40 / lmg protein / ml, the specific activity increases about 5, 000 to 10, 0,000 times as estimated.

Selenoprotein P or peptide or peptide prepared by the method described above, etc. In order to maintain the activity of the peptide group to the maximum, the selenoprotein P or peptide or peptide group of the present invention should be fresh or when stored at 4 ° C, within about 5 days after storage. preferable. Alternatively, the protein or peptide or peptide group of the present invention can be lyophilized and stored together with a suitable stabilizer, and further, the solution can be frozen and stored.

In the present invention, the ischemia / reperfusion injury inhibitor of the present invention can be prepared by a known method by combining the protein or a peptide or peptide group as an active ingredient with a suitable known excipient. The effective dose of the ischemia / reperfusion injury inhibitor of the present invention varies depending on the age, symptoms and severity of the administration subject, and ultimately varies according to the intention of the doctor. The best mode of administration is a single local (portal) or transarterial large dose (porous) or intravenous drip. In the case of the low molecular weight peptide group, oral or transdermal administration is also possible.

The subject to which the ischemia / reperfusion injury inhibitor of the present invention is administered is not particularly limited as long as it is a patient having a disease caused by ischemia / reperfusion injury, but is not limited to cerebral infarction, ischemic organ injury, and organ. Patients such as reperfusion injury such as transplantation, vascular injury, neuropathy, arteriosclerosis, and myocardial infarction can be considered as targets. When a drug containing selenoprotein P of the present invention or a peptide or peptide group derived therefrom as a main component is used as an agent for suppressing ischemia / reperfusion injury, the drug is administered alone. It can also be used, and co-administration with other therapeutic agents can be expected as an effective means to increase the effect. The ischemia / reperfusion injury inhibitor of the present invention is effective when administered immediately after a patient suffers from ischemic organ injury or before or after a surgical operation for organ transplantation, and is associated with vascular injury. It can be expected to be effective for prophylactic or therapeutic administration for ataxia.

This blood-derived selenoprotein P or the peptide or peptide group used in the present application is a single intravenous dose toxicity test in mice, repeated intravenous dose toxicity test, general pharmacology test, virus inactivation test Its safety has been confirmed by such means.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples, but these do not limit the scope of the present invention in any way. The following preparation examples and actual In Examples, unless otherwise specified, reagents manufactured by Wako Pure Chemical, Takara Shuzo, Toyobo and New England BioLabs were used.

Preparation Example 1

(Preparation of selenoprotein P)

Monoclonal antibodies obtained based on immunization of mice with selenoprotein P (C-terminal 103 amino acids) were immobilized on an immobilization carrier at a ratio of 180 mg Z25 ml (gel) to prepare an antibody column. One liter of plasma was passed through an antibody column equilibrated with phosphorylated saline (PBS), and selenoprotein P was adsorbed on the antibody column. After washing with PBS, the bound substance was eluted with 4 M urea / 2 OmM MES buffer (pH 4.5).

The eluted fraction was passed through heparin sepharose (5 ml) equilibrated with 2 OmM MES buffer (pH 4.5). Next, the heparin column was washed with 2 OmM phosphate buffer (pH 6.5) 2 Om1. Further, it was washed with 2 OmM phosphate buffer (ρ 6.5) 0.5 M NaCl 1 1.5 mM DTT (Dithiothreitol) 3 Oml. 2 OmM phosphate buffer to remove DTT

Washing was performed using (pH 6.5) / 20 ml of 0.5 M NaC1. Thereafter, the adsorbed material was eluted with 30 ml of 2 OmM phosphate buffer (pH 6.5) / l ". OM NaC1.

Elution · The recovered substance was confirmed to be complete molecular selenoprotein P having a molecular weight of about 66 kDa by electrophoresis and Western blotting. The recovery was about 3 mg.

Preparation Example 2

(Preparation of C-terminal peptide derived from selenoprotein P or the peptide group)

Activity tracking in the following preparation steps was based on the following Atsushi method using cell death activity as an index.

(Atsusei method)

D am i cells (Greenberg SM et al., Blood vol. 72, p. 1968-1977) that can be subcultured with serum-free medium SFO 3 (manufactured by Sanko Junyaku) containing 0.05 juM 2ME and 0.1% BSA 1988): 1 X 10 ⁶ ce 1 1 / dish / 3ml) l PMI 1 in lml 640 / D—MEM / F-12: 1: 2: 2 mixed medium (SA medium) (2 ml) was added, followed by culturing for 3 days, and the cells were collected at the time of assay. Cells were washed twice with 50% PBS / SA / 0. 0 3% HSA (SIGMA Co.), 3 1 0 ⁴ with the same medium. After suspending the cell suspension to 6 1 1 / ml, add 20 μl of the obtained cell suspension to the sample only, and add 10 1 to the well for serial dilution. Was dispensed. After adding 2 μl of Atsushi sample to the sample-added well, stirring, serial dilution was carried out for the well containing the 100β1 cell suspension. 3 7 ° and 4 cultured for 5 days in C0 ₂ incubator beta one C was determined.

Assays for the evaluation of Atsushi, from the 4th culture onwards, since live 11 cells with no live | cells are killed and active 1 1 cells continue to survive, It was evaluated whether it was present up to a fold dilution.

The cell death inhibitory activity in plasma indicates heparin binding. Therefore, first, fractionation using a heparin column was performed to collect the heparin-bound fraction in plasma. Using human plasma as a starting material, heparin-binding protein in plasma is adsorbed to a heparin column (Heparin Sepharose: Pharmacia), washed with 0.3 M sodium chloride,

The adsorbed fraction was eluted with 2M sodium chloride. Most of the target cell death inhibitory activity was recovered in the 0.3M sodium chloride wash fraction, but the 2M sodium chloride elution fraction was used for purification of the active substance.

In order to carry out crude fractionation of cell death inhibitory activity bound to heparin, fractionation was performed by ammonium sulfate precipitation. 31.3% w / v (about 2 M) of ammonium sulfate was added to the total amount of the fraction eluted with heparin in 2 M sodium chloride, and the precipitate was collected. The precipitate was dissolved in water and dialyzed against water using a dialysis membrane with a molecular weight of 3,500 cuts. After collecting the dialyzed solution, add 1/50 volume of 1 M Tris-HCl buffer (pH 8.0) to the total volume, and further use 20 mM Tris-HCl buffer (pH 8.0). The solution concentration was adjusted so that the OD280 value was 20-30. The solution was filtered using a filter of 1.0 juni and 0.45 μιη to remove insolubles.

Transfer the filtered protein solution to an anion exchange chromatography carrier (Macro-prep High Q: BioRad; h ^) equilibrated with 20 mM Tris-HCl buffer (pH 8.0). The solution was passed, and anion exchange chromatography was performed. At this time, the non-adsorbed fraction and

Since there was activity in the fraction eluted with 5 OmM sodium chloride, this fraction was collected and mixed. To the active fraction obtained by anion exchange chromatography, 1/50 volume of a solution obtained by mixing 1M citrate buffer (pH 4.0) and 1M citrate in a 6: 4 ratio was added. Then, a protein solution was prepared so as to be 2 OmM citrate buffer (pH about 4.0).

The protein solution described above is passed through a cation exchange chromatography carrier (Macro-prep High S: BioRad) equilibrated with 20 mM citrate buffer (pH 4.0), and then subjected to cation exchange chromatography. Was carried out. After washing with 2 OmM citrate buffer containing 22 OmM sodium chloride (pH 4.0), there was activity in the fraction eluted with 2 OmM citrate buffer containing 55 OmM sodium chloride (pH 4.0) Therefore, this fraction was collected.

1/30 volume of 1 M trisaminomethane solution was added to the total amount of the obtained 550 mM sodium chloride eluted fraction to adjust the pH to about 7.5. To this solution, add 2/3 volume of 3.5M ammonium sulfate solution (add 1/50 volume of 1M Tris-HCl buffer (pH8.5) and adjust ρΗ to about 7.5), and adjust the concentration of ammonium sulfate to 1.4M. The salt concentration was adjusted so that the sodium chloride concentration was 330 mM. Further, in order to remove insoluble substances, the mixture was filtered using a 0.45 // m filtration filter.

Next, the filtered protein was placed on a hydrophobic chromatography carrier (Macro-prep Methyl HIC: BioRad) equilibrated with 2 OmM Tris-HCl buffer (pH 7.5) containing 1.4 ammonium sulfate and 33 OmM sodium chloride. The solution was passed, and hydrophobic chromatography was performed. Since there was activity in the non-adsorbed fraction and the washing fraction in the equilibration buffer (pH 7.5), this fraction was collected. Almost no activity was present in the adsorbed fraction. In order to bind the active fraction to the hydrophobic chromatography carrier, the above-mentioned 3.5 M ammonium sulfate solution having a pH of about 7.5 was added to the active fraction so that the concentration of ammonium sulfate became 2.OM. The sample was passed through a hydrophobic chromatography carrier (Macro-prep Methyl HIC: BioRad) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 2.0 M ammonium sulfate and 240 mM sodium chloride. The components were adsorbed. After washing with equilibration buffer, the adsorbed activity Elution was performed with 20 mM Tris-HCl buffer (pH 8.0). The collected active fraction is dialyzed against water one day and night, and 1/50 volume of 1M citrate buffer (H4.5) is added to ensure that the collected active fraction is adsorbed to the heparin column. PH was adjusted to about 5.0.

Contains 20 mM phosphate buffer (pH 6.5) (buffer A) and 2 M sodium chloride

A 20 mM phosphate buffer (pH 6.2) (buffer B) was prepared, and the active fraction whose pH was adjusted was passed through a heparin column (Hi-Trap Heparin: Pharmacia) equilibrated with buffer A. . Then, after washing the column twice with a solution (0.1 M NaCl) containing 5% of buffer B mixed with buffer A, further mix 20% of buffer B with buffer A The active fraction was eluted with the eluted solution (0.4 MNaCl). The active fraction thus obtained was concentrated to a concentration of about 15 mg / ml using a ^ t condenser (centriprep 3: Amicon). After adding 2% acetic acid to the total amount of the concentrated active fraction, insolubles were removed by a 0.45 / zm filter.

1 ml of the active fraction was passed through a gel filtration chromatography carrier (Superdex 200 pg: Pharmacia) equilibrated with a solution containing 2% acetic acid and 50 OmM sodium chloride, and the gel filtration chromatography was performed. After painting, they were collected.

The above fractions were subjected to C4 reversed-phase HPLC (Wakosil 5C4-200 6 mm x 150 mm: manufactured by Wako) equilibrated with 1% acetonitrile containing 0.1% trifluoroacetic acid and 1% isopropanol. After passing through the column and washing with the equilibration solvent, the active fraction obtained by linear gradient elution of 1% to 40% acetonitrile under the conditions containing 0.1% trifluoroacetic acid and 1% isopropanol was collected. Collected.

In order to further finely fractionate the obtained active fraction, fractionation by ion exchange chromatography one carrier Mini Q (manufactured by Pharmacia) was performed. Linear gradient elution with sodium chloride was performed under the condition of 20 mM ethanolamine (pH 9.15). All fractions had activity, and all fractions reacted with an antibody against the peptide of the present invention. In other words, it was confirmed that the active substance was present in several different structures.

The target active substance at this stage was analyzed by electrophoresis and found to be composed of several bands from 1 OkDa to 30 kDa in the non-reduced state, and 3 to 4 kD in the reduced state. a and one band at 7-9 kDa, two bands at 13-14 kDa, and two bands at 16-17 kDa showed a minimum of six bands. All of these bands were detectable by Western blotting using the aforementioned antibody. In the electrophoresis in the non-reducing state, a protein that reacts with the antibody was confirmed near 28 to 29 kDa, suggesting that the presence of some dimers may exist. Was.

Preparation Example 3

(The heparin-Sepharose-bound fraction of selenoprotein P fragment in plasma using an anti-selenoprotein P antibody-bound carrier column was precipitated with 2 M ammonium sulfate, and the volume of the precipitated fraction was 2 OmM at least 5 times the volume of the precipitated fraction. The precipitate was dissolved using a Tris buffer (pH 8.0), and the selenoprotein P present in this solution was adsorbed to the anti-selenoprotein P antibody-bound carrier conjugate with the anti-selenoprotein p antibody bound to the carrier. After washing with phosphoric acid physiological saline ifcK (PBS), selenoprotein P was eluted with 2 OmM citrate buffer (pH 4.0) containing 4 M urea, and the eluate was further diluted with 20 mM citrate. Absorbed to a cation exchanger (Macroprep High S, BioRad) equilibrated with buffer (pH 4.0) This was eluted with a sodium chloride gradient using sodium chloride and fractions showing cell death inhibitory activity Recovered At this time, it is possible to obtain full-length selenoprotein P. However, this substance has a clearly low cell death inhibitory activity per protein. As a result, it was possible to obtain a selenoprotein P fragment having a strong cell death-suppressing activity per protein, and the fragment obtained here also varies depending on the presence or absence of sugar chains, the presence or absence of intermolecular bonds, and the presence or absence of internal cleavage. This was a mixed fraction containing molecular species of size, and was a group of selenoprotein P fragments showing a size of 10 to 30 kDa by non-reducing electrophoresis.

Example 1

(Effects of selenoprotein P fragment on ischemia / reperfusion injury in cerebral ischemia / reperfusion injury model, Part 1)

Free fatty acids released under ischemic conditions have been considered as one of the factors affecting cranial nerve injury in cerebral infarction. Selenoprotein P is induced by fatty acids In order to examine the effect of selenoprotein P on cerebral infarction, we used 14-week-old gerbils because they can suppress induced cell death (confirmed in NT2). The effect of selenoprotein P fragment on ischemia-reperfusion injury in a cerebral infarction model was examined.

Under general anesthesia by intraperitoneal injection of ketamine hydrochloride (10 O mg Z kg), exposing the jugular vein by midline incision and intravenously administering the selenoprotein P fragment prepared in Preparation Example 2 in an amount of 1 mg Z did. After 30 minutes, the bilateral arterioles were clipped, and after 30 minutes of blockage, reperfusion was performed, and the survival rates after 24 hours were compared.

table 1

The mortality rate of the selenoprotein ノ fragment administration group was clearly four times lower than that of the control saline administration group, suggesting that the rate of recovery from acute cerebral infarction was higher.

Example 2

(Effect of selenoprotein Ρ fragment on ischemia and reperfusion injury in cerebral ischemia-reperfusion injury model, part 2: ataxia amelioration effect) "

The effects of selenoprotein ρ on cerebral ischemia-reperfusion injury and ataxia were evaluated in 12-week-old gerbils to confirm the degree of paralysis. As in Example 1, general anesthesia was performed by intraperitoneal injection of ketamine hydrochloride (100 mg Z kg), the jugular vein was exposed through a midline incision, and 1 mg of the selenoprotein P fragment prepared in Preparation Example 2 was used. Iv dose. After 30 minutes of ischemia and 45 minutes of ischemia, the blood flow was reperfused and evaluated by the degree of paralysis at 6 hours and 24 hours.

The state of paralysis after 6 hours and 24 hours was judged and evaluated by the scores shown in Table 2. The results are shown in Tables 3 and 4. table

ΊΕ ^

刖 There is slight paralysis in the feet, and the movement is dull with the legs bent.

The paralysis of the foot deteriorates slightly and keeps turning to one side ■ · · · 2 The paralysis is bad and falls to one side

In addition, the paralysis worsens and the person cannot move.

5Et 5 Table 3: 30 minute ischemia model

When the state of paralysis after 6 hours and the state of paralysis after 24 hours in the 30-minute ischemia model were evaluated, it was found that the selenoprotein P fragment administration group had clearly better symptoms. In the 45-minute ischemia model, the degree of paralysis after 6 hours was not significantly different, but the degree of paralysis after 24 hours was significantly different.

Example 3 ―

(Improvement of ataxia by administration of selenoprotein P to Klotho mice)

The effect of the selenoprotein P fragment of the present invention was confirmed using Klotho mice for the purpose of improving ataxia associated with vascular disorders. Klotho Mouse is described in Nature, 390: 45-51, 1997, and Yoichi Nabeshima, Graduate School of Medicine, Kyoto University Obtained raw permission from Claire Japan.

Up to 8 weeks of age, 4 chicks of 4-week-old Klotho mice in one group were dissolved in physiological saline with the selenoprotein P fragment (1.5 mg / m 1) obtained in Preparation Example 2 and saline. Water was administered to the peritoneal cavity at a dose of 300 μl each week, and the state change was observed. As a result, raw

No weight gain was observed from the age of 4 weeks in the selenoprotein Ρ fragment administration group, and the root cause was not resolved. Comparing the behaviors, when the selenoprotein fragment administration group placed on the palm of the hand, it was possible to jump off to escape spontaneously, whereas the group administered with the saline diet voluntarily escaped. Could not be taken. This was considered to indicate the effect of selenoprotein Ρ fragment on the improvement of motor impairment.

Example 4

(Effect of selenoprotein Ρ fragment on ischemia ■ reperfusion injury in liver ischemia reperfusion model)

We investigated the effect of selenoprotein 抑制 fragments on ischemia 'reperfusion injury in hepatic ischemia reperfusion model using gerbils. In order to confirm the effect of selenoprotein 事前, pre-administration of selenoprotein Ρ fragment and physiological saline was performed to cause ischemia by blocking portal venous blood flow. Survival rate, blood GOT, GPT, ALP concentration changes, and tissue observation were evaluated.

Gerbils were anesthetized with 10-fold diluted Nembutal (5 O mg / m 1) by intraperitoneal administration of 300 μl, and the inferior vena cava was exposed by abdominal incision, from which selenoprotein prepared in Preparation Example 2 Ρ fragment lmg Z

did. Five minutes later, the portal vein was exposed, and the blood flow was cut off with a clip. At 40 minutes, the gerbils were re-distributed. After abdominal suture, all gerbils were dissected under anesthesia 30 hours later, blood, organs were collected, and liver marker enzymes in the blood were analyzed. Table 5 shows the results.

Table 5

As is clear from the results shown in Table 5, the mortality rate of the physiological saline administration group was 3/4, whereas that of the selenoprotein P fragment administration group was 0/3. ALP In the group administered with saline, the activity was increased about 5 times as compared with the control, whereas the activity was increased in the group treated with selenoprotein P. ALP increases when cell membranes in the liver or intestine are damaged, and the cells that should be damaged were stabilized by selenoprotein P and the damage was reduced.As a result, the selenoprotein P-treated group may have survived. It is suggested.

Claims

The scope of the claims

1. An agent for suppressing ischemia / reperfusion injury comprising selenoprotein P and / or the C-terminal peptide of the protein or the peptide group as a main component.

2. The C-terminal peptide or the peptide group of the protein described above, the C-terminal 103 amino acid sequence of selenoprotein P, the amino acid sequence of which one or several amino acids are deleted, substituted or added, The ischemic / reperfusion injury inhibitor according to claim 1, which has a partial sequence of any one of the amino acid sequences or an amino acid sequence containing the amino acid sequence as a part thereof.

3. The peptide at the C-terminal side of the protein or the peptide group is represented by the following formula:

(I: Lys Arg Cys lie Asn Gin Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu

Leu Ala Pro Arg Ser Xaa Cys Cys His Cys Arg His Leu (Rooster column number 1) and / or

(Where Xaa represents selenocistine)

An amino acid sequence represented by the following, an amino acid sequence in which one or several amino acids have been deleted, substituted or added, a partial sequence of any one of the above amino acid sequences, or a part thereof. 3. The ischemic / reperfusion injury inhibitor according to claim 1 or 2, which is a peptide having an amino acid sequence contained in the peptide or the peptide group.

4. The C-terminal peptide or the peptide group of the protein is recovered in (a) a molecular weight fraction of 10 to 30 kDa based on the molecular weight fractionation membrane, and (b) a study of the binding property to the ion exchange resin. As a result, it has a structure showing an isoelectric point between pH 7 and pH 8 in blood and a structure showing an isoelectric point above pH 8; (c) Non-reducing SDS-PAGE has a molecular weight of 13-14 kDa. It shows two bands and two bands of 1.6 to 17 kDa to which a sugar chain is added, and (d) SDS-PAGE under reducing conditions shows 3 to 4 kD in addition to the above band. The ischemia / reperfusion injury inhibitor according to any one of claims 1 to 3, wherein the agent exhibits bands of a, 7 to 9 kDa and 10 to 12 kDa.

5. Diseases caused by ischemia and reperfusion injury are due to cerebral infarction, ischemic organ tissue damage, reperfusion injury such as organ transplantation, arteriosclerosis, myocardial infarction and ataxia associated with vascular injury. The ischemia / reperfusion injury inhibitor according to any one of claims 1 to 4, which is selected.

6. The ischemia / reperfusion injury inhibitor according to claim ⁵ , wherein the disease caused by ischemia / reperfusion injury is ataxia associated with vascular injury.