JPWO2007020922A1 - Therapeutic and preventive agent for brain diseases - Google Patents

Abstract

Novel agents for treating and preventing brain disease in newborns are disclosed. The drug of the present invention contains erythropoietin as an active ingredient and is administered to newborns within 8 weeks of birth. A typical brain disease that can be treated with the therapeutic or prophylactic agent of the present invention is periventricular leukomalacia, which later develops cerebral palsy.

Description

  The present invention relates to the treatment and prevention of brain diseases.

  Brain disease in a newborn is a disease caused by damage to cells or tissues in the brain before, at or just after birth, such as periventricular leukomalacia, which later develops cerebral palsy . Treatment of these diseases is performed by a combination of orthopedic treatment, physical therapy, and occupational therapy, and no drug treatment has been established at present. Therefore, development of an effective treatment method for these diseases is required.

References cited in this specification are as follows. All the contents described in these documents are cited here as a part of this specification. None of these documents is admitted to be prior art to this specification.
Special table 2005-502588

  An object of the present invention is to provide a novel drug for treating and preventing brain diseases in newborns.

  The present inventors have developed a new animal model that mimics periventricular leukomalacia, a neonatal brain disease, and examined the effectiveness of various drugs against brain disease. As a result, erythropoietin is effective. As a result, the present invention was completed.

  The present invention provides a therapeutic or preventive agent for brain diseases comprising erythropoietin as an active ingredient, which is administered to a newborn within 8 weeks of birth. A typical brain disease that can be treated with the therapeutic agent or prophylactic agent of the present invention is periventricular leukomalacia, which later develops cerebral palsy.

FIG. 1 shows improvement of brain damage by EPO administration using a PVL model.

  The therapeutic agent or prophylactic agent of the present invention is administered to a newborn within 8 weeks from birth. The newborn to which the therapeutic agent or prophylactic agent of the present invention is targeted is a newborn within 8 weeks from birth, preferably a newborn within 4 weeks, and more preferably a newborn within 1 week. The newborn in the present invention includes premature babies.

  The therapeutic agent or prophylactic agent of the present invention is not particularly limited as long as it is administered to a newborn within 4 weeks of birth, but is mainly administered to premature infants. In the present invention, the premature infant means a newborn born with a gestational age of less than 33 weeks.

  In the present invention, the brain disease refers to a disease in the brain or cell / tissue damage caused by neurodegeneration, ischemia, hypoxia, inflammation or the like. A typical disease targeted by the therapeutic or prophylactic agent of the present invention is periventricular leukomalasia (PVL), which later develops cerebral palsy. Cerebral palsy is a disease in which paralysis and other neurological disorders occur due to the loss of control of muscles, etc., caused by damage to the brain before, at or after birth. PVL is a disease in which white matter tissue around the ventricle is necrotized. Because there are movement-related nerve fibers in places that are damaged by PVL, movement disorders are likely to occur.

  By administering the therapeutic agent or prophylactic agent of the present invention to a newborn, PVL can be treated or prevented, and as a result, cerebral palsy can be treated or prevented. Therefore, the present invention relates to a therapeutic or prophylactic agent for cerebral palsy or PVL.

  The erythropoietin (EPO) used in the present invention can be any EPO, but is preferably highly purified EPO, more specifically substantially the same as mammalian EPO, particularly human EPO. It has biological activity.

  The EPO used in the present invention may be produced by any method, for example, natural human EPO obtained by purification from human-derived extracts (JP-B-1-38800, etc.), or gene Human EPO, etc. produced by E. coli, yeast, Chinese hamster ovary cells (CHO cells), C127 cells, COS cells, myeloma cells, BHK cells, insect cells, etc., extracted and separated and purified by various methods. Can be used. The EPO used in the present invention is preferably an EPO produced by a genetic engineering technique, and an EPO produced using a mammalian cell (especially a CHO cell) (for example, Japanese Patent Publication No. 1-444317, Kenneth Jacobs). et al., Nature, 313 806-810 (1985), etc.).

  EPO obtained by a genetic recombination method has the same amino acid sequence as naturally-occurring EPO, or has one or more amino acids in the amino acid sequence deleted, substituted, added, etc. Those having the same biological activity as EPO may be used. Amino acid deletion, substitution, addition and the like can be performed by methods known to those skilled in the art. For example, those skilled in the art will recognize site-directed mutagenesis (Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, MJ and Smith, M. (1983) Methods Enzymol. 100, 468- 500; Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, HJ (1987) Methods Enzymol. 154, 350-367; Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766) and the like, and functionally equivalent to EPO by introducing appropriate mutations into the amino acid of EPO. Polypeptide can be prepared. Amino acid mutations can also occur in nature.

  In general, the amino acid residue to be substituted is preferably substituted with another amino acid that preserves the properties of the amino acid side chain. For example, as the properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acids and amide-containing side chains (D, N, E, Q), amino groups having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses represent single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang, A et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

  In the present invention, a fusion protein of EPO and another protein can also be used. In order to prepare a fusion polypeptide, for example, DNA encoding EPO and DNA encoding another protein may be linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Other proteins to be subjected to fusion with the EPO of the present invention are not particularly limited.

  In the present invention, it is also possible to use chemically modified EPO. Examples of chemically modified EPO include, for example, EPO to which a compound such as polyethylene glycol / vitamin B12 or the like or an inorganic or organic compound is bound.

  Furthermore, the EPO of the present invention may be an EPO derivative. In the present invention, the EPO derivative refers to EPO modified with an amino acid in the EPO molecule or EPO modified with a sugar chain in the EPO molecule. The modification of the sugar chain in the EPO molecule includes addition, substitution, deletion and the like of the sugar chain.

  Examples of preferred sugar chain modifications in the present invention include deletion of sialic acid in the EPO molecule. In general, both EPO produced by recombinant animal cells and EPO derived from urine are obtained as an EPO composition containing various EPOs having different sugar chain structures. The number of sialic acids added to EPO molecules in the EPO composition varies depending on individual EPO molecules, but usually 11 to 15 sialic acids are added to one EPO molecule. By removing these sialic acids, it is possible to produce asialized EPO (asialo EPO). The number of sialic acids removed during asialization is not particularly limited, and all sialic acids may be removed, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 sialic acids may be removed. The asialo EPO preferred in the present invention has 10 or less sialic acids added to the EPO molecule, more preferably 5 or less, and particularly preferably 2 or less. The number of sialic acids used in the present invention is the average number of EPO molecules contained in the EPO composition. The average of sialic acid per molecule can be measured by methods known to those skilled in the art (EP 0428267, etc.).

  EPO from which sialic acid has been removed (Asialo EPO) can be prepared by a method known to those skilled in the art, and for example, it can be prepared by treating EPO with an enzyme such as sialidase. Commercially available sialidases can be used (Japanese translations of PCT publication No. 2005-507426, Nobuo Imai et al., Eur. J. Biochem, 194, 457-462 (1990), etc.).

  Examples of the amino acid modification in the EPO molecule include carbamylation, biotinylation, amidineation, acetylation, guanidination, and the like. A preferred amino acid modification in the present invention is carbamylation.

  The amino acid residue to be modified is not particularly limited, and examples thereof include lysine, arginine, glutamic acid, tryptophan and the like. A preferred amino acid modified in the present invention is lysine. Accordingly, as a particularly preferred embodiment of EPO modified with amino acids in the present invention, EPO in which lysine is carbamylated can be mentioned (Marcel L et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science, 2004; 305: 239, Fiordaliso E et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. PNAS, 2005; 102: 2046, etc.). The carbamylation of EPO includes carbamylation by reaction with cyanate ion or the like, alkyl-carbamylation by reaction with alkyl-isocyanate or the like, aryl-carbamylation by reaction with aryl-isocyanate or the like.

  The above-mentioned EPO, recombinant EPO, chemically modified EPO, EPO derivatives, etc. can be highly purified to such an extent that they can be administered to humans and formulated in combination with a pharmaceutically acceptable carrier or excipient. it can. Such purification methods and formulation methods are well known in the art.

  In the preparation of the therapeutic agent or prophylactic agent of the present invention, as necessary, a suspending agent, a solubilizing agent, a stabilizer, a tonicity agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, An excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, and the like can be appropriately added.

  Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate and the like.

  Examples of the solution auxiliary agent include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, tuna gol, castor oil fatty acid ethyl ester and the like.

  Examples of the stabilizer include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfite. It is also possible to add certain amino acids as stabilizers (for example, JP-A-10-182481). Amino acids added as stabilizers include free amino acids, salts such as sodium salts, potassium salts and hydrochlorides thereof. Amino acids can be added alone or in combination of two or more. The amino acid added as a stabilizer is not particularly limited, but preferred amino acids include leucine, tryptophan, serine, glutamic acid, arginine, histidine, and lysine.

  Examples of the isotonic agent include D-mannitol and sorbate.

  Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

  Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol and the like.

  Surfactants include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerin such as glycerin monocaprylate, glycerin monomylate, and glycerin monostearate. Fatty acid ester; polyglycerin fatty acid ester such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate Polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, etc. Xylethylene sorbitan fatty acid ester; polyoxyethylene sorbite fatty acid ester such as polyoxyethylene sorbite tetrastearate and polyoxyethylene sorbite tetraoleate; polyoxyethylene glycerin fatty acid ester such as polyoxyethylene glyceryl monostearate; polyethylene glycol distea Polyethylene glycol fatty acid esters such as rate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxy such as polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether Ethylene polyoxypropylene alkyl ether; polyoxyethylenelenoni Polyoxyethylene alkyl phenyl ethers such as phenyl ether; polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); polyoxyethylene such as polyoxyethylene sorbite beeswax Beeswax derivatives; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; those having HLB 6-18 such as polyoxyethylene fatty acid amides such as polyoxyethylene stearamide; anionic surfactants such as sodium cetyl sulfate, lauryl sulfate Alkyl sulfates having an alkyl group of 10 to 18 carbon atoms such as sodium and sodium oleyl sulfate; average added moles of ethylene oxide such as sodium polyoxyethylene lauryl sulfate Polyoxyethylene alkyl ether sulfates having 2 to 4 and alkyl groups having 10 to 18 carbon atoms; alkyl sulfosuccinates having 8 to 18 carbon atoms in alkyl groups such as sodium lauryl sulfosuccinate; natural Typical examples include surfactants such as lecithin, glycerophospholipid; fingophospholipids such as sphingomyelin; sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms, and the like. One or a combination of two or more of these surfactants can be added to the preparation of the present invention. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60 or 80, with polysorbates 20 and 80 being particularly preferred. Further, polyoxyethylene polyoxypropylene glycol represented by poloxamer (such as Pluronic F-68 (registered trademark)) is also preferable.

  Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, carbon Examples thereof include those having a sulfhydryl group such as thioalkanoic acid having 1 to 7 atoms.

  Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salt, L-ascorbyl palmitate, L-ascorbic acid stearate, sodium bisulfite, Chelating agents such as sodium sulfite, triamyl gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like can be mentioned.

  Furthermore, normally added components such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate; organic salts such as sodium citrate, potassium citrate and sodium acetate May be included.

  When the therapeutic agent or prophylactic agent of the present invention is used as a sustained-release preparation, the sustained-release preparation can be prepared by a known method, for example, a method using a polymer as a base. The polymer used as the base is preferably a polymer that is decomposed in vivo.

  The therapeutic or prophylactic agent of the present invention is preferably administered before the blood brain barrier is fully matured. The therapeutic or prophylactic agent of the present invention is usually administered to newborns within 8 weeks of birth. In order to administer to a newborn within 8 weeks from birth, the therapeutic or prophylactic agent of the present invention may be administered at least once within 8 weeks from birth. The number of administrations may be one time or multiple times. All administrations may be performed within 8 weeks, or only some administrations may be performed within 8 weeks.

  The present inventors have found that the therapeutic or preventive agent of the present invention is effective when administered at a low dose. Therefore, the therapeutic agent or prophylactic agent of the present invention usually has an EPO of 0.001 U to 9999 U / kg / day (0.000005 to 49.995 μg / kg / day), preferably 0.1 U to 1000 U / kg / day (0.0005 to 5 μg / day). kg / day), more preferably 1 U to 100 U / kg / day (0.005 to 0.5 μg / kg / day). The dosage of the therapeutic agent or prophylactic agent of the present invention for each patient is determined by a doctor in consideration of the patient's weight, symptoms, administration route and the like.

  The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2005-236898, which is the application on which the priority of the present application is based, are cited herein as a part of this specification.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Preparation of PVL rat model Wistar rats (SLC Japan) aged 2, 3 or 4 days were used (hereinafter referred to as P2, P3 and P4, respectively). The experiment was conducted with the permission of the Ethics Committee based on the guidelines for animal experiments at Nagoya City University. There were 8-12 sibs per mother. After separation from the mother, a midline incision was made under inhalation anesthesia with isoflurane (introduction 5%, maintenance 2%) on a heated operating table, and the right common carotid artery was exposed by exfoliating the muscle, connective tissue, and vagus nerve And then solidified with a heat coagulator and cut. The closure was sutured with two 6-0 nylon threads. The operation time was performed within 10 minutes. Return to the mother for recovery, heat retention, and breastfeeding, and after 2 hours, immerse in a 37 ° C thermostat adjusted to 6% oxygen with a mixture of oxygen and humidified nitrogen, and heat for 30-120 minutes in a heated container. A low oxygen load was applied. Surviving animals were returned to their mothers after recovery.

  After 48 hours, anesthesia was performed by intraperitoneal injection of Nembutal, perfused transcardially with physiological saline, and then fixed by perfusion with 4% paraformaldehyde. The cervix was cut, and after brain removal, it was fixed for 3-4 hours with the same fixing solution and immersed in 20% sucrose solution. After fixation, it was snap frozen with dry ice and sliced with a sliding microtome with a 40 μm coronal section. The sections were stained with hematoxylin and eosin for histological evaluation with an optical microscope.

Histological evaluation was performed with hematoxylin and eosin staining. Injuries were classified into 4 stages.
No change group is the same as normal control tissue.
In the Slight damage group, white matter such as corpus callosum is normal, but the cortical layer structure is disordered.
Mild damage group is a group with partial loss of white matter and partial destruction of cortical layer structure.
The Severe damage group is a group with extensive loss of white matter and extensive destruction of the total cortical structure.
Mild damage was considered appropriate as a PVL model in rats. The results are shown in the table below.

  At low oxygen load of less than 60 minutes, the mortality rate was low for all of P2, P3, and P4, and it was considered to be resistant to low oxygen load. However, when a low oxygen load of 60 minutes or more was applied, the mortality rate increased.

  The following table shows the classification of P2, P3, and P4 rats according to the degree of damage caused by hematoxylin and eosin staining.

  In P2, almost no brain damage occurred in the surviving animals. In the case of P3, the ratio of Mild damage exceeded 70% at 60 minutes of low oxygen.

  Based on the above, it was considered that a P3 rat was used as a more physiological model, and when hypoxic ischemia was applied at 6% oxygen 60 minutes after occlusion of the right common carotid artery, a state close to PVL could be created.

Improvement of brain damage by EPO administration using PVL model Three-day-old Wistar rats (SLC Japan) were used. The experiment was conducted with the permission of the Ethics Committee based on the guidelines for animal experiments at Nagoya City University. There were 8-12 sibs per mother. After separation from the mother, a midline incision was made under inhalation anesthesia with isoflurane (introduction 5%, maintenance 2%) on a heated operating table, and the right common carotid artery was exposed by exfoliating the muscle, connective tissue, and vagus nerve And then solidified with a heat coagulator and cut. The closure was sutured with two 6-0 nylon threads. The operation time was performed within 10 minutes. Returned to mothers for recovery, heat retention and breastfeeding. Two hours later, erythropoietin (1-30000 U / kg) or carbamylated EPO (CEPO) (50-100 U / kg) was administered intraperitoneally in an amount corresponding to 0.1 ml per 6 g body weight. Carbamylated EPO (CEPO) was prepared by reacting EPO with cyanate ion and carbamylating lysine according to the method described in Jin Zeng (Methods in Enzymology 205: 433-437, 1991). After administration, it was immersed in a 37 ° C. thermostat adjusted to 6% oxygen with a mixture of oxygen and humidified nitrogen, and a low oxygen load for 60 minutes was applied in the heated container. Surviving animals were returned to their mothers after recovery.

  After 48 hours, anesthesia was performed by intraperitoneal injection of Nembutal, perfused transcardially with physiological saline, and then fixed by perfusion with 4% paraformaldehyde. The cervix was cut, and after brain removal, it was fixed for 3-4 hours with the same fixing solution and immersed in 20% sucrose solution. After fixation, it was snap frozen with dry ice and sliced with a sliding microtome with a 40 μm coronal section. Sections were stained with hematoxylin and eosin for histological evaluation by light microscopy, and for immunohistochemical evaluation, O4 (1: 500; MAB345: Chemicon), NG2 (1: 400; MAB5384: Chemicon), MBP (1 : 1000; SMI-94: Sternburg). Injury classification was performed in the same manner as in Example 1. The results are shown in Tables 3 and 4 below and FIG. Table 4 summarizes the effects of EPO administration due to brain damage, with No change-Sligit damage as ameliorated and Mild damage-Severe damage as Constant.

When EPO 50-100 U / kg ip was administered, the mortality rate decreased to 40% or less. Further, when 1 to 10,000 U / kg ip of EPO was administered, the ratio of ameliorated increased to 60% or more, and particularly when 50 to 100 U / kg ip of EPO was administered, the ratio of ameliorated was about 75 to 87.5. Rose to%. From the above, administration of EPO has the effect of improving brain damage caused by hypoxic ischemia, and is considered to have an excellent effect especially when 50 to 100 U / kg ip of EPO is administered.
From the results of administration of 50-100 U / kg ip of carbamylated EPO (CEPO), it is considered that the EPO derivative has the same excellent improvement effect as EPO.

  The present invention is useful for the treatment and prevention of brain diseases.

Claims (7)

A therapeutic or prophylactic agent for brain diseases comprising erythropoietin as an active ingredient, which is administered to a newborn within 8 weeks of birth. The therapeutic or prophylactic agent according to claim 1, wherein the newborn is a premature baby. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the brain disease is cerebral palsy. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the brain disease is periventricular leukomalacia. The therapeutic or prophylactic agent according to any one of claims 1 to 4, wherein erythropoietin is an erythropoietin derivative. The therapeutic or prophylactic agent according to claim 1, wherein the erythropoietin derivative is asialoerythropoietin. The therapeutic or prophylactic agent according to claim 5 or 6, wherein the erythropoietin derivative is carbamylated erythropoietin.

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