JPH09110717A - Therapeutic agent for lupus nephritis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent, comprising a superoxide dismutase having chemically bonded lecithin as an active ingredient and excellent in improving effects on symptoms such as lupus nephritis. SOLUTION: This therapeutic agent for lupus nephritis comprises a superoxide dismutase(SOD) having chemically bonded lecithin [preferably the SOD, represented by the formula (SOD is the superoxide dismutase; X is a residue obtained by removing H in OH at the 2-position of a lysolecithin having the OH at the 2-position of glycerol; (m) is >=2; (n) is >=2] and bonded through chemical cross-linking to the lysolecithin] as an active ingredient. A SOD obtained by chemically modifying cysteine at the 111st position of the SOD (human Cu/ZnSOD), derived from a human and containing copper and zinc at the active center is preferred as the SOD. Concretely, a human Cu/ZnSOD obtained by converting, e.g. the cysteine at the 111st position into S-(2- hydroxyethylthio)cysteine is cited.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the technical field relating to pharmaceutical use of lecithinized superoxide dismutase. More specifically, it belongs to the technical field relating to a therapeutic agent for lupus nephritis containing lecithinized superoxide dismutase as an active ingredient.

[0002]

2. Description of the Related Art Superoxide dismutase (hereinafter, referred to as superoxide dismutase)
SOD is sometimes widely distributed in living organisms such as animals, plants, and microorganisms, and is a reactive oxygen species that is highly reactive among the active oxygen groups (hereinafter sometimes abbreviated as ROS). oxide anion radicals ^- known as enzymes that degrade (hereinafter, simply O ₂ may be abbreviated as). On the drug side, after using antirheumatic drugs,
It is expected that this SOD will be applied to the removal of radicals generated after the use of antithrombotic agents used for myocardial infarction and organ transplantation, and the treatment of various inflammations caused by radicals. Recently, application of this SOD to gastric mucosal injury has also been studied, and its therapeutic effect is expected (Lipid Peroxide Research, Vol. 16, p. 74 (1992)). Generally, when administered intravenously, SOD has a low cell affinity and its half-life in blood is only 4 to 6 minutes, and SOD is rapidly excreted in urine. In order to extend the half-life of SOD in blood, it has been attempted to modify SOD with ficoll, polyethylene glycol, rat albumin, dextran and the like to form a macromolecule.

However, it has been reported that SOD modified with ficoll or polyethylene glycol has a significant decrease in the original enzymatic activity of SOD and still has a low cell affinity. In addition, it has been reported that rat albumin-modified SOD exhibits antigenicity in the human body. Furthermore, SOD modified with dextran is excellent in that the anti-inflammatory action inherent to SOD is enhanced, but it is also reported that antigenicity in the human body is also recognized. recent years,
A substance in which a biologically active protein such as SOD is chemically modified with lecithin (phosphatidylcholine: hereinafter sometimes abbreviated as PC) has been reported. It has been reported that this PC-binding modified bioactive protein has markedly increased cell affinity and is significantly different in biodistribution from conventional non-modified bioactive proteins. Further, it is said that enhancement of pharmacological activity of bioactive proteins, reduction of side effects, and promotion of absorption can be expected.

In addition, a PC SOD (hereinafter referred to as PC-SOD
(Sometimes abbreviated as "abbreviated") has been reported to have an effect of promoting the healing of inflammation caused by burns (Japanese Patent Application Laid-Open No. Hei 3-16).
3100, JP-A-3-170438, U.S. Pat. No. 5,109,118). Furthermore, it is said to be useful as an anti-inflammatory agent without side effects such as antigenicity. JP-A-6-54681 describes the effect of PC-SOD on respiratory resistance by Forssman antiserum. The effect of PC-SOD on ulcerative gastrointestinal disorders has also been reported. Furthermore, PC-SOD for motor neuron diseases such as amyotrophic lateral sclerosis
The effect of is also reported.

[0005]

As described above, further applications of PC-SOD, which can play various roles in vivo, to pharmaceutical applications are expected in the industry. Then, the subject which this invention should solve is establishment of the new medical use of said PC-SOD, and providing this object for medical use.

[0006]

Means for Solving the Problems The present inventor has sought for the applicability of PC-SOD for various medical uses in order to meet the above-mentioned problems. As a result, the present invention has been completed by assuming PC-SOD as an active ingredient of the therapeutic agent for “lupus nephritis”.

[0007] Lupus nephritis is a glomerulonephritis associated with systemic lupus erythematosus and is an immune complex (immune comple
x; hereinafter also referred to as IC) and nephritis associated with activated complement. In the World Health Organization (WHO) classification, lupus nephritis is 1) lupus nephritis in normal glomeruli, 2) lupus nephritis with mesangial proliferative changes,
3) Focal segmental glomerulonephritis, 4) Diffuse proliferative glomerulonephritis, 5) Diffuse membranous glomerulonephritis, 6) End-stage sclerosing glomerulonephritis. , Hematoxylin body, crescent formation, wire / loop lesions, presence / absence of active lesions such as microthrombus, and the form of lupus nephritis is individually identified.

In order to develop this lupus nephritis, R
In- vitro analysis has revealed that the OS is deeply involved in recent years. That is, when IC, activated complement binds to leukocytes, ROS production occurs; R
OS impairs cultured mesangium cells; T cells, macrophages or leukocyte self-produced cytokines increase ROS production; ROS are also produced from glomerular constituent cells such as mesangial cells and endothelial cells; ROS producing cells Intracellular transport system requires intracellular calcium ions through protein kinase C; lipid peroxidation (phosphatidylcholine peroxide; PCOOH) increases in the presence of ROS; contact of ROS with DNA Cleavage of the DNA occurs; and so on.

From this, it is assumed that the symptom of lupus nephritis can be improved by taking measures to effectively eliminate ROS. Therefore, it may be contemplated to improve the symptoms of nephritis by applying the above-mentioned SOD, which is known to have an action of eliminating ROS, to lupus nephritis patients. However, in reality, as described above, SOD has a short blood half-life and a low cell affinity, so that it is skeptical to expect a sufficient pharmacological effect on various symptoms of lupus nephritis. However, when the present inventor examined this point, unfortunately, SOD
It was found that even if it is applied to patients with lupus nephritis, it cannot be an effective therapeutic drug (see Examples below for actual pharmacological effects).

Therefore, the present inventor has conducted extensive studies on the form of SOD effective for improving the symptoms of lupus nephritis,
Various modified SODs were examined for their symptom improving effects on lupus nephritis. As a result, the PC-SOD
Is extremely effective in improving the symptoms of lupus nephritis,
This PC-SOD was found to be extremely useful as an active ingredient of a therapeutic agent for lupus nephritis, and completed the present invention.
That is, the present inventor provides the inventions listed below in each claim.

In a first aspect, there is provided a therapeutic agent for lupus nephritis containing superoxide dismutase chemically bound to lecithin as an active ingredient.

In a second aspect, there is provided a therapeutic agent for lupus nephritis, which contains, as an active ingredient, superoxide dismutase bound to lecithin through chemical crosslinking.

In a third aspect of the present invention, there is provided the therapeutic agent for lupus nephritis according to the second aspect, wherein the superoxide dismutase bound to lecithin through chemical crosslinking is represented by the following general formula (I). SOD- _{[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD represents superoxide dismutase, X is lysolecithin having a hydroxyl group at the 2-position of glycerol, Representing a residue excluding the hydrogen atom of the hydroxyl group at the 2-position, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more).

In a fourth aspect, there is provided the therapeutic agent for lupus nephritis according to the third aspect, wherein the SOD in the general formula (I) is a human-derived SOD.

In claim 5, SOD is human-derived Cu / Zn superoxide dismutase in which SOD is amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine in the general formula (I). A therapeutic agent for lupus nephritis according to claim 3 is provided.

[0016] In the sixth aspect, there is provided the therapeutic agent for lupus nephritis according to any one of the third to fifth aspects, wherein n is an integer of 2 or more and 10 or less in the general formula (I). To do.

[0017] In a seventh aspect, there is provided the therapeutic agent for lupus nephritis according to the sixth aspect, wherein n is 3 in the general formula (I).

In a eighth aspect, there is provided a therapeutic agent for lupus nephritis according to any one of the third to seventh aspects, wherein m is a number of 1 or more and 16 or less in the general formula (I).

A ninth aspect of the present invention provides the therapeutic agent for lupus nephritis according to the eighth aspect, wherein m is 4 in the general formula (I).

[0020]

Embodiments of the present invention will be described below. <1> The therapeutic agent for lupus nephritis of the present invention is a therapeutic agent for lupus nephritis containing, as an active ingredient, superoxide dismutase chemically bound with lecithin, particularly superoxide dismutase bound to lecithin through chemical crosslinking.

In the present specification, "lecithin" means, in addition to ordinary lecithin which means phosphatidylcholine, fatty acid 1 bonded to the 1-position or 2-position of glycerol.
Includes "lysolecithin" from which molecules have been removed.

Further, in the present specification, "superoxide dismutase (SOD)" means active oxygen O in the living body.
₂ ^- as long as capable of exhibiting its inherent function of the degradation of its origin and the like are not particularly limited, and can be widely used SOD with various animals and plants or microorganisms. However, the present invention is an invention for pharmaceutical use, and considering that it is preferable to reduce the antigenicity in vivo as much as possible, depending on the animal species to which the therapeutic agent for lupus nephritis of the present invention is administered, it is appropriate. It is preferable to select the appropriate SOD. For example, when targeting a human for whom the therapeutic agent for lupus nephritis of the present invention is most needed, it is preferable to use human-derived SOD in the present invention in order to reduce the antigenicity in the human body as much as possible. . And among them, human-derived Cu / Zn
SOD (human-derived SOD containing copper and zinc in the active center;
Hereinafter, it may be abbreviated as human Cu / ZnSOD.)
However, the expression level in cells is large, and a production technique by a genetic engineering technique has been established at the time of completion of the present invention, and a large amount can be prepared at the present time, which is particularly preferable.

The human Cu / ZnSOD includes a natural human Cu / ZnSOD produced from human tissue; a human Cu / ZnSOD produced by a genetic engineering method; an amino acid sequence substantially the same as the natural human Cu / ZnSOD. Recombinant human Cu / ZnSOD and the like are available, and any human Cu / ZnSOD can be used as a material for superoxide dismutase (PC-SOD) chemically bound to lecithin used in the present invention.

For reference, this natural human Cu / Zn
The primary structure of the SOD protein is shown in FIG. 1 (SEQ ID NO: 1). Actual human Cu / ZnSOD is a dimer of the protein having the primary structure shown in FIG.

As shown in the amino acid sequence of this protein, the amino acid at position 111 of natural human Cu / ZnSOD is cysteine, but this 111 position was converted to serine by a protein engineering method such as site-directed mutagenesis. Human Cu / ZnSOD (JP-A-62-130684)
Or chemically modified human C of 111 cysteines
u / ZnSOD (Japanese Patent Laid-Open No. 6-199895) has also been reported, and a PC using these human Cu / ZnSOD as a material.
Any of -SOD can be used as an active ingredient of the therapeutic agent for lupus nephritis of the present invention. Of these human Cu / ZnSODs, the cysteine at position 111, which is uniform in charge and molecular weight and has stable SOD activity, is chemically modified. For example, this cysteine is S-
Human C as (2-hydroxyethylthio) cysteine
It is preferable to use u / ZnSOD as a material for the PC-SOD.

In the present specification, human Cu / ZnSOD is obtained by partially converting amino acids by a site-directed mutagenesis method, etc., or a partial amino acid of human Cu / ZnSOD is chemically modified. Including those obtained by
It is simply called human Cu / ZnSOD.

Further, in the present specification, "chemically bound" means that it is covalently bonded, and as long as the covalent bond is present, the presence or absence of the chemical cross-linking described later is It doesn't matter. That is, PC may be directly covalently bonded to SOD, or PC may be covalently bonded to SOD via chemical crosslinking.

The production method of the PC-SOD used in the present invention is not particularly limited as long as it is an SOD chemically bound with lecithin, but usually, a chemical cross-linking agent is bound in advance to the residue of the lecithin. The lecithin derivative is coupled to superoxide dismutase (SOD) via chemical cross-linking formed by its chemical cross-linking agent.
It can be obtained by combining more than one.

The SOD chemically bound to lecithin contained in the therapeutic agent for lupus nephritis of the present invention is not particularly limited, but it is particularly preferable to use the SOD bound to lecithin through chemical crosslinking. .

The lecithin chemically bound to SOD is not particularly limited here, but it is preferable to use PC-SOD chemically bound to lysolecithin.

PC-SOD suitable for this invention
Is expressed by the following equation (I). SOD- _{[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD represents superoxide dismutase, X is lysolecithin having a hydroxyl group at the 2-position of glycerol, Representing the residue except the hydrogen atom of the hydroxyl group at the 2-position, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more)

Here, "2 of glycerol" represented by X
The residue of lysolecithin having a hydroxyl group at the 2-position, excluding the hydrogen atom of the 2-position hydroxyl group, is represented by the following formula (II).

[0033] _{-O-CH (CH 2 OR)} [CH 2 OP (O) (O -) (OCH 2 CH 2 N + (CH 3) 3)] (II) ( wherein, R fatty acid residue ( Is an acyl group))

Particularly, a saturated or unsaturated fatty acid residue having 10 to 28 carbon atoms is preferable as the above R, and more preferably myristoyl group, palmitoyl group, stearoyl group, icosanoyl group, docosanoyl group and other carbon atoms having 14 to 14 carbon atoms.
Among them, there are 22 saturated fatty acid residues, and particularly preferred is a palmitoyl group which is a saturated fatty acid residue having 16 carbon atoms.

Further, in the above formula (I), the residue —C (O) — (CH ₂ ) _n —C (O) — represents a chemical cross-linking residue. The residue of this chemical crosslink is the formula

A linear dicarboxylic acid represented by HO—C (O) — (CH ₂ ) _n— C (O) —OH, an anhydride of this dicarboxylic acid, an ester of this dicarboxylic acid, a halogen of this dicarboxylic acid. And other hydroxyl groups at both ends of the chemical cross-linking agent molecule such as the reactive derivative of this dicarboxylic acid (in the case of the above reactive derivative of dicarboxylic acid, the portion corresponding to the hydroxyl groups at both ends of this dicarboxylic acid) It is a base.

This residue can crosslink SOD and lecithin to form a chemical bond (covalent bond). Therefore, in the present specification, this is generically referred to as "chemical crosslink". The chemical bridge in the above formula (I) is bonded to the lysolecithin residue (II) at one end thereof by an ester bond.

Further, it is considered that the other end of this chemical crosslink is directly bonded to the amino group of SOD by an amide bond or the like. In this chemical cross-linking, the group — (CH ₂ )
_n- is a divalent group formed by removing one hydrogen atom from each of carbon atoms at both ends of a linear alkane, that is, an alkylene group. n is an integer of 2 or more, preferably n is 2 to
It is an integer of 10, particularly preferably 3.

Further, m in the above formula (I) represents the average number of lecithin bound to one SOD molecule through the above-mentioned chemical cross-linking. This m is an integer of 1 or more, and m is preferably 1 to 16, and 4 is particularly preferable.

The binding of the above lecithin derivative to SOD is
Specifically, it can be performed based on, for example, the method described in Japanese Patent Application Laid-Open No. 6-54681, and thereby a PC-SOD can be manufactured. This PC-S
Details of the manufacturing process of the OD will be described in a manufacturing example described later.

The PC-SOD used in the present invention may be a pharmaceutically acceptable salt. The salt that can be used specifically depends on the isoelectric point of PC-SOD used. For example, when the isoelectric point of the PC-SOD molecule is acidic, a pharmaceutically acceptable salt with an alkali can be used. Conversely, when the isoelectric point of the PC-SOD molecule is alkaline, a pharmaceutically acceptable salt with an acid can be used. For example, the isoelectric point of human Cu / ZnSOD obtained by converting the amino acid sequence at position 111 to serine is acidic, and the isoelectric point of PC-SOD obtained by converting this into PC is also acidic. Therefore, this 111-position converted human Cu / Z
As for nSOD, pharmaceutically acceptable salts of salts with alkali, for example, alkali metal salts, alkaline earth metal salts, ammonium salts and the like can be used in the present invention.

The PC-SOD manufactured as described above is
The desired therapeutic agent for lupus nephritis can be obtained by adding it as an active ingredient in the dosage form described below. The therapeutic agent for lupus nephritis of the present invention can be widely applied to lupus nephritis in mammals including human.

For example, in the case of human lupus nephritis, all lupus nephritis according to the above WHO classification, specifically, lupus nephritis in normal glomeruli, lupus nephritis with mesangial proliferative changes, focal segmental glomeruli Nephritis,
The lupus nephritis therapeutic agent of the present invention can be widely applied to diffuse proliferative glomerulonephritis, diffuse membranous glomerulonephritis, end-stage sclerosing glomerulonephritis and the like.

And among these lupus nephritis,
The formation of crescents in renal tissue, which is one of the indicators of lupus nephritis activity, is triggered by the reactive oxygen species released by neutrophils. Nephritis or rapidly progressive nephritis) is a type of lupus nephritis for which administration of the therapeutic agent for lupus nephritis of the present invention is particularly effective.

The above-mentioned disease names are merely examples, and the types of lupus nephritis to which the therapeutic agent for lupus nephritis of the present invention can be applied are not limited to these exemplified diseases. further,
Of course, the therapeutic agent for lupus nephritis of the present invention can be used literally for the purpose of treating lupus nephritis, but not only for pure therapeutic purposes, but also for the prevention, maintenance (prevention of deterioration), and reduction (improvement of symptoms) of lupus nephritis. Etc. can also be administered for the purpose.

<2> In the present invention, any dosage form can be appropriately selected as the dosage form of the therapeutic agent for lupus nephritis of the present invention, depending on the nature and progress of the target disease.
That is, the therapeutic agent for lupus nephritis of the present invention can be orally or parenterally selected and administered according to the administration method such as injection (intramuscular, subcutaneous, intradermal, intravenous injection), oral, inhalation and the like. These drugs can be formulated as appropriate according to these administration methods.

The dosage form that can be selected is not particularly limited, and examples thereof include injections (solutions, suspensions, emulsions, solid preparations for dissolution at the time of use), tablets, capsules, granules, powders, liquids, lipo A wide range of agents, gels, external powders, sprays, inhalants, suppositories, etc. can be selected. In preparing these preparations, it is possible to add components used in the preparation of usual pharmaceuticals such as an excipient, a binder, a lubricant, a coloring agent and a disintegrant.

The compounding amount of PC-SOD which is an active ingredient in the therapeutic agent for lupus nephritis of the present invention and the dosage amount of the therapeutic agent for lupus nephritis of the present invention are the administration method of the preparation, the administration form, the purpose of use and the specific symptoms of the patient It is a matter that should be individually determined according to the weight of the patient, etc., and is not particularly limited.
The clinical dose of C-SOD is about 100 U / day
Kg to 10000 U / Kg [1 U in the present specification]
(Unit) means the cytochrome c method (xanthine-xanthine oxidase-cytochrome c system; J. Biol. Chem., Vol.244, No.22, 6049-6) at pH 7.8 and 30 ° C.
055 (1969)), which represents the amount of PC-SOD enzyme that inhibits the reduction rate of cytochrome c by 50%]. Further, the administration interval of the above preparation may be every other day or every day, and is not particularly limited. Administration is possible even once a day.
It can also be administered in 2 to 4 divided doses per day.

[0049]

EXAMPLES Examples of the present invention will be specifically described below as Production Examples, Test Examples and Formulation Examples. However, these should not limit the technical scope of the present invention.

[Production Example] (1) Human Cu / ZnSOD derivative [human-derived superoxide dismutase in which the amino acid at position 111 of SOD is S- (2-hydroxyethylthio) cysteine]
Production of this human Cu / ZnSOD derivative
It was manufactured by the method described in JP-A-199895.

That is, a known E. coli 545πHR (p
HT351) was cultured, and the obtained cultured cells were disrupted to obtain a crude extract of human Cu / ZnSOD. The crude extract was precipitated with ammonium sulfate, Q-Sepharose FF (Pharmacia)
Column, ammonium sulfate precipitation in order to purify, further 0.2μ
The filtrate filtered through a m filter was lyophilized to give a colorless solid (recombinant human apo SOD). This recombinant human apo SOD was added to a 10 mM triethanolamine buffer (pH 7.
0), bis (2-hydroxyethyl) disulfide was added, and the pH was readjusted to 7.0, followed by stirring at 4 ° C. for 24 hours. NaCl, 2M acetate buffer (pH
5.0) and 0.1 M CuCl ₂ aqueous solution, and added at 4 ° C.
For 13 hours. After this stirring, the pH was adjusted to 7.0 and the precipitate formed by ammonium sulfate precipitation (4 ° C., 100% saturation) was collected. The precipitate was desalted by dialysis with 10 mM triethanolamine buffer (pH 7.0). Q of this desalted fraction
-Pass through a Sepharose FF column and adjust the NaCl concentration to 5 m.
The SOD fraction was collected by gradually increasing the development in the order of M, 10 mM, 20 mM, and 50 mM. Next, this SOD fraction was subjected to ammonium sulfate precipitation (4 ° C., 100% saturation), and the resulting precipitate was collected. The precipitate was used as an aqueous solution, dialyzed against a 0.5 M NaCl aqueous solution, and then purified water, and lyophilized after completion of these dialysis to obtain a human SOD derivative (hereinafter, sometimes simply referred to as SOD). 111 of this human SOD derivative
The amino acid at position became S- (2-hydroxyethylthio) cysteine.

(2) Manufacture of PC-SOD: PC-SOD
Was produced by the method described in JP-A-6-54681. Specifically, it is as follows.

(A) Synthesis of Active Ester of 2- (4-Hydroxycarbonylbutyroyl) lysolecithin Synthesis of 2- (4-Hydroxycarbonylbutyroyl) lysolecithin Chloroform-pyridine of lysolecithin in which the 2-position of glycerol is a hydroxyl group ( 80 ml / 20 ml) suspension, DM
AP (N, N-dimethylaminopyridine) and glutaric anhydride were added, and the mixture was stirred at 60 ° C for 15 hours. Thereafter, the reaction solution was concentrated under reduced pressure, and chloroform: methanol:
The solvent was dissolved by adding a solvent of water = 4: 5: 1, and the solution was passed through an ion exchange column (Dowex 50W-X8, manufactured by Dow Chemical Company) equilibrated with the same solution. The target compound was fractionated by TLC, the solvent was concentrated under reduced pressure, and the residue was purified by a column packed with ODS (octadecylsilane) to obtain the title compound.

Synthesis of Active Ester of 2- (4-Hydroxycarbonylbutyroyl) lysolecithin The carboxylic acid obtained above was dissolved in dichloromethane and cooled at 0 ° C., and N-hydroxysuccinimide and tetrazole were sequentially added thereto. , Then DCC (1,3-
A solution of dicyclohexylcarbodiimide) in dichloromethane was slowly added dropwise, and the mixture was stirred at room temperature for 15 hours. After stirring, the insoluble matter was filtered through Celite to obtain a dichloromethane solution of the active ester compound.

(B) Synthesis of PC-SOD having an average of four lecithin derivatives bound per molecule of SOD: SOD prepared by the above process dissolved in 50 mM borate buffer (pH 8.5) and all amino acids of this SOD. The active ester of 2- (4-hydroxycarbonylbutyroyl) lysolecithin in a 0.8-fold molar amount with respect to the group was reacted by the following method.

Dichloromethane of the above active ester solution was distilled off and dissolved in DMF (N, N-dimethylformamide). This was added to a 50 mM borate buffer (pH 8.5).
, And after filtration of insolubles, dissolve in the same buffer
Was added dropwise to the SOD solution to which 50% of DMF was added. After stirring this at 0 ° C. for 15 hours, the reaction solution was filtered,
The mixture was applied to a gel filtration column using Sephacryl S-300 (manufactured by Pharmacia) as a carrier, and a 50 mM borate buffer (pH
Elution at 8.5), PC-SOD elution fractions were collected and purified by ion exchange chromatography. This was then concentrated by ultrafiltration, and the protein concentration was determined by the Lowry method (Lowry, Oh et al., J. Biol. Chem., 193, 265 (1951)).
)), The residual amino groups of SOD were analyzed by the TNBS method (trinitrobenzene sulfonate, Goodwin, JF, etc., Clin. Che.
m., Vol. 16, p. 24 (1970)).
The number of lecithin derivatives bonded per D1 molecule was determined to be 4.0 on average. The property of this PC-SOD is a blue-green to green transparent aqueous solution with a pH of 7-
It was 8. In addition, when the molecular weight was examined by SDS-polyacrylamide gel electrophoresis, about 1800 per monomer of PC-SOD subunit (PC-SOD is a homodimer of subunit as described above)
It was 0.

[Test Example] PC manufactured by the above manufacturing example
-SOD (concentration 30 mg / ml; specific activity 3.07 × 10 ³ U
(/ Mg protein), a toxicity test and a pharmacological test were performed. PC-SOD was added to a 5% mannitol aqueous solution to give a final concentration of 5
Solution dissolved to give mg / ml (hereinafter PC-SOD
(Sometimes abbreviated as a solution) was used. This PC-S
The OD solution was a blue-green to green transparent aqueous solution and had a pH of 6-8. The osmotic pressure ratio with respect to the physiological saline was about 1.

(1) Acute toxicity test Acute toxicity test using rats PC-SOD solution was administered to each of 5 SD male and female rats by tail vein at 100 mg / Kg. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed. The animals were necropsied on the 15th day, and the major organs were visually observed. As a result, no dead rat was observed, and no change was observed in any of the general condition, body weight, and autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

Acute Toxicity Test Using Monkeys PC-SOD solution was administered to two female cynomolgus monkeys at a dose of 100 mg / Kg through a vein of limbs. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed. As a result, no dead cynomolgus monkeys were observed, and no change was observed in general condition, body weight, or autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

(2) Pharmacological and pharmacological test (a) Lupus nephritis model ((NZB × NZW) F1 mouse) In several% of human lupus nephritis, 50% or more of glomeruli are accompanied by crescent formation, and nephritis rapidly develops. RPGN (rapidly progressive glomerulo nephritis) that progresses into renal failure
) Present an image. This crescent formation means rapid glomerulosclerosis, and is used as an index for measuring the progression rate of nephritis. Moreover, cytokines that increase the reactive oxygen group (ROS) are released from monocytes and macrophages in the infiltrated region of the lesion.

Spontaneous lupus nephritis model (NZB × NZW) F1 mice used in the pharmacological efficacy study of the present invention develop nephritis at 4 weeks of age, resulting in acute progressive glomerulonephritis (RP).
This is a model in which the kidney function is abolished with the image of GN) and the patient died in about 40 weeks. Using the lupus nephritis model mouse, the following pharmacological and pharmacological tests were conducted. The lupus nephritis model ((NZB × NZW) F1 mouse) was obtained from Charles River Japan.

(B) Examination of improvement effect of lupus nephritis by PC-SOD Using the above lupus nephritis model, PC-SOD administration group (hereinafter, also referred to as PC-SOD administration group), PC
SOD (free SOD) administration group (hereinafter, also referred to as free SOD administration group) and PC-SOD
A pharmacological test was conducted in a group to which neither free SOD nor free SOD was administered (hereinafter referred to as non-administration group).

In each group, 6 lupus nephritis model mice were used. In the non-administered group, 200 μl of a 5% aqueous mannitol solution was administered every other day from the tail vein of the lupus nephritis model mouse 4 weeks after birth. Also, free SOD
In the administration group, 200 μl of free SOD was administered every other day so that the weight became 1000 U / Kg body weight. And PC-SO
In the D-administered group, 200 μl of PC-SOD was administered every other day so that the body weight was 1000 U / Kg.

Examination of Effect of PC-SOD Administration on Lupus Nephritis Using PAS-Stained Kidney Section Specimens Time point at which 23 weeks after birth of each lupus nephritis model mouse to be tested after continuing the above administration Then, the kidney was excised from each group of mice, a kidney section sample was prepared, and the section sample was subjected to PAS staining. The stained images are shown in FIGS. 2 to 4.

FIG. 2 is a morphological photograph (40 × magnification) of an organism showing a PAS-stained image in the non-administered group. Second
In the figure, in the whole glomerulus, adhesion with the Bowman's capsule (hemimen formation) was seen to extend all around the glomerulus.
In addition, vitrified glomeruli were also observed. In addition, tubular atrophy was observed and stroma lesions were also prominent.

FIG. 3 shows P in the free SOD administration group.
Fig. 4 is a morphological photograph of an organism showing an AS-stained image (magnification of 40 times), and Fig. 4 is a morphological photograph of the organism showing a PAS-stained image in a PC-SOD administration group (magnification of 40 times).
In FIG. 3, it was observed that the cells in the glomerulus proliferated and, as a result, the glomerulus itself was enlarged. However, the number of glomeruli formed in the crescent and the number of vitrified glomeruli were decreased as compared with FIG.

In FIG. 4, crescent formation, glomerular vitrification, tubular atrophy and interstitial lesions, which are remarkably observed in FIG. 2, and most of the cell proliferation in the glomeruli shown in FIG. 3 are observed. Not observed, but almost normal kidney histology was observed.

In the non-administered group (Fig. 2), the crescent formation was conspicuously observed in the basal membrane at the neutrophil adhesion site accompanying the adhesion of neutrophils (polymorphonuclear leukocytes) to blood vessels. It is known that fibrin released by focal disruptive lesion (FDL) and vascular destruction, as well as proliferation of Bowman's sac epithelium and epithelial cells caused by FDL are involved. The crescent thus formed is involved in the activity of lupus nephritis as described above.

Next, the number of glomeruli and the number of glomeruli forming crescents in each of the above-mentioned sliced specimens were counted, and the ratio of the number of glomeruli forming crescents to the total number of glomeruli was calculated. A comparison was made between the groups (Fig. 5). In addition,
The number of counts was obtained by using section specimens of 6 mice in each group, and 100 glomeruli were examined for each section specimen.

The bar in FIG. 5 shows the standard error. In the results shown in FIG. 5, it was shown that the proportion of crescent formation in the PC-SOD administration group was significantly reduced as compared with the non-administration group and the free SOD administration group (p.
<0.001). The rate of crescent formation in the free SOD administration group was significantly lower than that in the non-administration group (p <0.001).

From the above, it is shown that PC-SOD remarkably suppresses the formation of crescent, which is an index of lupus nephritis activity, and that the administration of PC-SOD suppresses the progress of lupus nephritis. It has been shown.

Examination of the effect of PC-SOD on the active oxygen in the tissue by the cerium-stained sample In order to visualize the generation state of the active oxygen in the tissue of lupus nephritis model, the mice in each group (at 23 weeks after birth) The kidney was removed from the kidney to prepare a kidney section sample, and a known method (Briggs et al., J. Cell Biol. 6 was used for this section sample.
Cerium dyeing based on 7, 566 (1975) was applied. In this staining method, if ROS (hydrogen peroxide (OH ⁻ )) is present in the cells that have taken in cerium chloride (CeCl ₃ ), cerium peroxide (Ce (OH) ₃ OOH)
Formed and electron-dense granular precipitate under electron microscope
It is detected as (hi density granular deposition).

The cerium-stained images are shown in FIGS. 6 to 9. Figures 6 and 7 show cerium-stained images in the non-administered group.
Shown in the figure. Note that FIG. 6 (magnification of 4000 times) is an image inside and around the glomerulus, and FIG. 7 (magnification of 400 times).
(0x) is an image of the renal tubule and its surroundings.

In FIG. 6, a granular precipitate having a high electron density (indicating a site where ROS is present) was observed over the entire image, which indicated the presence of a large amount of ROS.
Especially in the mesangial region of the glomerulus and in the basement membrane, many RO
S was confirmed.

In FIG. 7, the connecting surface of the proximal tubule,
A large amount of electron-dense granules (same as above) was observed in the tubular basement membrane and capillaries. In addition, leukocytes in the capillaries around the proximal tubule release ROS, which causes rolling.
A running image was observed.

As described above, a large amount of ROS was observed in the non-administration group, and white blood cells adhered to the capillary wall and exhibited a typical inflammatory appearance of rolling while releasing ROS. A black image was observed in the cells because ROS was also expressed in leukocytes and monocytes.

Next, cerium-stained images in the PC-SOD administration group are shown in FIGS. 8 and 9. Note that FIG. 8 (magnification of 2500 times) is an image inside and around the glomerulus, and FIG. 9 (magnification of 6000 times) is an image of the renal tubule and its periphery.

In FIG. 8, infiltration of white blood cells (neutrophils) and monocytes was observed in the capillaries, but no image showing the release of granules with high electron density (same as above) was observed. In Fig. 9, the presence of the electron-dense granules (same as above) in the basement membrane was milder than that in Fig. 7. From these results, PC-SOD was shown to suppress ROS, and PC-SOD was shown to suppress the progression of lupus nephritis.

Inducible NO producing enzyme (inducible nitric
P for lupus nephritis using oxide synthase as an index
Examination of effects of C-SOD administration NO (nitoric oxide) is known to exist in the outer medulla, regulate glomerular blood flow, and balance sodium and water. And such N
Inducible NO-producing enzyme for producing O (inducible nit
It is known that ric oxide synthase (hereinafter, also referred to as iNOS) is induced by inflammatory cytokines but not produced by necrotic cells.

In this test, using this iNOS as an index, P
The effect of C-SOD administration on lupus nephritis was examined. That is, kidneys were removed from the mice of each group (at 23 weeks of age), a kidney section sample was prepared, and the kidney section was immunostained with an anti-iNOS antibody to give iN.
The presence or absence of OS was examined. This immunostained image is shown in FIG.

In FIG. 10, the left photograph is the non-administration group, the middle photograph is the free SOD administration group, and the right photograph is P.
Each of the stained images of the C-SOD administration group is shown. In each photograph, the small spherical structure seen throughout the field of view is the cell nucleus. Non-administration group and free SOD
In the administration group, almost no staining with anti-iNOS antibody was observed. This suggests that the cells became necrotic in lupus nephritis, and the iNOS-producing ability that should be originally produced was lost. On the other hand, in the PC-SOD administration group, staining with anti-iNOS antibody was found in the glomerular mesangium and the external medulla. This indicates that iNOS is expressed in glomerular mesangial and external medulla. The expression of iNOS in the PC-SOD administration group suggests that NO is produced,
This suggests that the regulation of glomerular blood flow and the regulation of sodium-water balance are functioning normally. This result suggests that administration of PC-SOD improves the physiological function of the kidney. Also PC-SOD
Suggest that it does not affect the physiological mechanism of the kidney (iNOS expression) such as suppressing the expression.

Examination of the Effect of PC-SOD Administration on Lupus Nephritis Using Manganese SOD (MnSOD) as an Index MnSOD exists in mitochondria in the cytoplasm of eukaryotes. In this test, mice of each group (2 postnatal
The kidney was excised from 3 weeks later), a kidney section sample was prepared, and the kidney section was immunostained with an anti-MnSOD rat antibody to examine the presence or absence of MnSOD. This immunostained image is shown in FIG.

In FIG. 11, the left photograph is the non-administration group, the middle photograph is the free SOD administration group, and the right photograph is P.
Each of the stained images of the C-SOD administration group is shown. In each photograph, the small spherical structure seen throughout the field of view is the cell nucleus. Non-administration group and free SOD
In the administration group, almost no staining with anti-MnSOD antibody was observed. This suggests that the cells became necrotic in lupus nephritis, and the ability to produce MnSOD, which should be produced originally, was lost. In contrast, PC-SO
In the D administration group, strong staining with anti-MnSOD antibody was observed in the glomerulus. This indicates that MnSOD is strongly expressed in glomeruli. M in PC-SOD administration group
The strong expression of nSOD indicates that the physiological removal mechanism of ROS is functioning normally, and MnSO 4
It is suggested that D and PC-SOD removed excess ROS and improved renal physiological function. Also P
C-SOD is the physiological mechanism of ROS removal (MnSOD
It also suggests that it does not affect the expression) such as suppressing the expression.

Pharmacological and pharmacological test for lupus nephritis using mRNA of inflammatory cytokine as an index A protein NF-κB (nuclear factor-κB) that binds to the regulatory region κB of the cytokine of the gene is one of the transcriptional regulatory factors, such as inflammation. It is known to be involved in increased expression of some acute phase proteins in Escherichia coli. NF-κ
B is I-κB (inhibitor-) in the cytoplasm of non-B cells.
Suppressor protein called κB) and complex (NF-κB
-I-κB complex), and inactive NF-
It exists as κB. This NF-κB-I-κB complex is dissociated into NF-κB and I-κB by ROS, and NF-κB is translocated into the cell nucleus to activate NF-κ.
It becomes κB and is thought to activate the transcription of genes for inflammatory cytokines. In addition, after the above dissociation, I-
κB also migrates into the cell nucleus, resulting in uneven distribution of I-κB in the cell nucleus, and the amount of I-κB in the cytoplasm is relatively reduced. In this case, the decreased cytoplasmic I-κB
In order to secure the amount,
In addition to A, I-κB mRNA will also be expressed.

When inflammation occurs in this way,
MRNA and I of inflammatory cytokines in the cytoplasm
-ΚB mRNA is expressed with a strong positive correlation. On the other hand, since lupus nephritis is also a typical type of inflammation, m is a proinflammatory cytokine associated with the onset and progression of lupus nephritis.
The expression levels of RNA and I-κB mRNA are also increased.

Therefore, the presence or absence or the presence or absence of the expression of the inflammatory cytokine is accurately expressed, for example, the presence or absence or the presence or absence of the mRNA of this inflammatory cytokine and the mRNA of I-κB is
It is an excellent indicator of lupus nephritis activity. Therefore, PC-SOD expresses mRNAs such as inflammatory cytokines induced by NF-κB and mR of Iκ-B.
It was examined whether or not the expression of NA was suppressed.

That is, at 23 weeks after birth, kidneys were removed from each group of mice, and the inflammatory cytokine induced by NF-κB [interleukin-6] from these kidneys.
(IL-6), interleukin-2 (IL-2), tumor necrosis factor α (TNFα), interleukin-8 (I
L-8), granulocyte-macrophage stimulating factor (GM-CS)
F)], the complement C ₃ component and the fibronectin B mRN
A and I-κB mRNAs were extracted and each was RT
-PCR method (Frohman MA et al., Proc.Natl.Acad.Sc
iUSA, Vol.85, pp8998-9002 (1988)).

As a control, β-actin mRNA was also examined. Further, in the non-administered group, mRNA was similarly extracted at the 5th and 8th week after birth and detected by the RT-PCR method. Table 1 shows the results. + In this table indicates that each mRNA was detected.

[0089]

[Table 1] (Twenty-three weeks after birth, two mice were used in the non-administration group, and three mice were used in each of the free administration group and the PC-SOD administration group.)

From these results, in the non-administered group, IL-6, I, which is a inflammatory cytokine, at 23 weeks after birth.
MRN of L-2, TNFα, IL-8 and GM-CSF
A was detected (even at 8 weeks of age, IL-6, T
NFα and IL-8 mRNA was detected). In the free SOD administration group, only IL-6 mRNA was detected. However, in the PC-SOD administration group, mRNA of inflammatory cytokine was not detected.

The results show that PC-SOD suppresses the expression of inflammatory cytokine mRNA associated with lupus nephritis, and that PC-SOD can suppress the progress of lupus nephritis.

Further, I-κB mRNA was detected only in the non-administration group, and was observed in the PC-SOD administration group and free S.
It was not found in the OD administration group. From this,
It was shown that PC-SOD suppresses the activation of gene regulatory region of inflammatory cytokine having κB region. This also supports that PC-SOD can suppress the progression of lupus nephritis.

The mRNA of complement component C _{3 and} the mRNA of fibronectin B were detected only in the non-administered group, but not in the free-administered group and the PC-SOD-administered group. As described above, lupus nephritis is a glomerulonephritis associated with systemic lupus erythematosus, and an immune complex (antigen-antibody complex) is formed at the time of onset, which activates the complement system. Therefore, the mRNA of complement component C ₃
Is detected, indicating that the complement system is activated. Fibronectin B is one of the basement membrane components and is known to be significantly involved in the formation of crescents formed with the onset of lupus nephritis. Therefore, mRNA of fibronectin B
Detection of is also an index showing the onset and progression of lupus nephritis.

Therefore, the above-mentioned results concerning the detection of complement component C ₃ mRNA and fibronectin B mRNA indicate that PC-SOD suppresses the expression of these component mRNAs associated with lupus nephritis. -S
It is shown that OD can suppress the progress of lupus nephritis.

[Formulation Example] (1) Injection Preparation PC-SOD (30 mg / m 2) produced in the above Production Example
l) was dissolved in a 5% aqueous solution of mannitol to a final concentration of 5 mg / ml, which was aseptically filtered, dispensed into ampoules in 2 ml portions and sealed to produce injections.

(2) Tablets 100 mg of the freeze-dried product of PC-SOD produced in the above Production Example, 670 mg of lactose, 150 m of potato starch
g, 60 mg of crystalline cellulose and 50 mg of light anhydrous silicic acid were mixed, and a solution of 30 mg of hydroxypropyl cellulose in methanol (10% by weight of hydroxypropyl cellulose) was added and kneaded and granulated. Next, this was extruded through a screen having a diameter of 0.8 mm to obtain granules, and after drying, 15 mg of magnesium stearate was added and compression-molded to produce tablets of 200 mg.

(3) Capsule 100 mg of PC-SOD freeze-dried product produced in the above Production Example, potato starch 150 mg, light anhydrous silicic acid 50 mg, magnesium stearate 10 mg and lactose 7
65 mg was uniformly mixed, 200 mg of the mixture was dispensed and filled into hard capsules to produce capsules.

[0098]

INDUSTRIAL APPLICABILITY The present invention provides a pharmaceutical use invention, that is, a therapeutic agent for lupus nephritis with respect to PC-SOD.

[0099]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 153 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150

[Brief description of the drawings]

FIG. 1 is a drawing showing the primary structure of human Cu / ZnSOD protein.

FIG. 2 is a morphological photograph of an organism showing a PAS-stained image in the non-administration group.

FIG. 3 is a morphological photograph of an organism showing a PAS-stained image in the free SOD administration group.

FIG. 4 is a morphological photograph of an organism showing a PAS-stained image in the PC-SOD administration group.

FIG. 5 is a drawing in which the ratio of the number of glomeruli forming crescents to the total number of glomeruli was calculated and compared among the groups.

FIG. 6 is a morphological photograph of an organism showing a cerium-stained image of the glomerulus and its periphery in the non-administration group.

FIG. 7 is a morphological photograph of an organism showing a cerium-stained image of the tubular tubule and its surroundings in the non-administered group.

FIG. 8 is a morphological photograph of an organism showing a cerium-stained image of the glomerulus and its periphery in the PC-SOD administration group.

FIG. 9 is a morphological photograph of an organism showing a cerium-stained image of the renal tubule and its surroundings in the PC-SOD administration group.

FIG. 10 is a morphological photograph of an organism showing an immunostaining image of a kidney section with an anti-iNOS antibody.

FIG. 11 is a morphological photograph of an organism showing an immunostaining image of a kidney section with an anti-MnSOD rat antibody.

Claims (9)

[Claims] 1. A therapeutic agent for lupus nephritis containing superoxide dismutase chemically bound to lecithin as an active ingredient. 2. A therapeutic agent for lupus nephritis, which contains, as an active ingredient, superoxide dismutase bound to lecithin through chemical cross-linking. 3. A therapeutic agent for lupus nephritis according to claim 2, wherein the superoxide dismutase bound to lecithin through a chemical crosslink is represented by the following general formula (I): SOD- [C (O)-(CH ₂ ) _n -C (O) -X] _m (I) (In the formula, SOD represents superoxide dismutase, and X is lysolecithin having a hydroxyl group at the 2-position of glycerol, and the hydrogen atom of the hydroxyl group at the 2-position is removed Represents a residue, m is the average number of bonds to one molecule of SOD, represents an integer of 1 or more, and n represents an integer of 2 or more). 4. The therapeutic agent for lupus nephritis according to claim 3, wherein SOD in the general formula (I) is of human origin. 5. In the general formula (I), SOD is human-derived Cu / Z, wherein the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine.
The therapeutic agent for lupus nephritis according to claim 3, which is n superoxide dismutase. 6. The therapeutic agent for lupus nephritis according to claim 3, wherein n is an integer of 2 or more and 10 or less in the general formula (I). 7. The therapeutic agent for lupus nephritis according to claim 6, wherein n is 3 in the general formula (I). 8. The therapeutic agent for lupus nephritis according to claim 3, wherein m is a number of 1 or more and 16 or less in the general formula (I). 9. The therapeutic agent for lupus nephritis according to claim 8, wherein m is 4 in the general formula (I).