JP4831551B2 - Medicine for non-ischemic myocardial injury - Google Patents

Description

  The present invention relates to a medicament for non-ischemic myocardial injury and its use.

  Midkine (hereinafter abbreviated as “MK”) is a basic protein with a molecular weight of 13 kDa and has multifaceted activities such as neurite outgrowth, cell proliferation, cell migration, angiogenesis, induction of inflammation, and fibrinolysis ( Non-patent documents 1 to 4). Involvement of MK in various diseases such as cancer and inflammatory diseases has been reported (Non-Patent Documents 5 and 6).

  The action and effect of MK on myocardial ischemia reperfusion injury was examined (Patent Document 1). As a result, it was shown that MK suppresses the suppression of myocardial damage progression in the acute phase by an anti-apoptotic effect. In the chronic phase after ischemia reperfusion, it was proved that the continuous administration of MK protein promotes angiogenesis and contributes to the improvement of cardiac function. Thus, it was shown that MK is effective for myocardial ischemia reperfusion injury.

International Publication No. 2006/062087 Pamphlet

Journal of Biochemistry 192: 359-371 2002 Journal of Biochemistry 119: 1150-1156 1996 Journal of Clinical Investigation 105: 489-495 2000 Journal of Immunology 167: 3463-3469 Cancer letters 204: 127-143 2004 Oncology 57: 253-257 1999 Clinical Research in Cardiology 96: 469-480 2007 Proceeding of the National Academy of Sciences USA 105 (10): 3915-3920, 2008

  The myocardial injury includes ischemic myocardial injury and non-ischemic myocardial injury such as cardiomyopathy, valvular disease or dilated cardiomyopathy. There is a great gap in the pathogenesis, as well as the pathological conditions and symptoms of ischemic myocardial injury and non-ischemic myocardial injury. In addition, there are many unexplained parts regarding the onset mechanism of non-ischemic myocardial injury, and this is a big foot pad that prevents the establishment of an effective treatment for non-ischemic myocardial injury.

  By the way, in dilated cardiomyopathy, which is one of the non-ischemic myocardial disorders, it is said that the progression of the disorder is suppressed by suppressing autoimmunity by T cells (Non-patent Document 7). On the other hand, it has been reported that when MK is suppressed, activated T cells increase and autoimmune encephalomyelitis is suppressed (Non-patent Document 8). Taken together these two reports, MK suppression is expected to be an effective treatment strategy even in dilated cardiomyopathy.

  There is currently no effective curative treatment for non-ischemic myocardial injury. Therefore, there is no choice but to rely on organ transplantation, but there are many problems to be solved such as chronic donor shortage and excessive burden on patients, and the creation of effective treatments is eagerly desired. Under such circumstances, an object of the present invention is to provide an effective medical means (treatment or prevention means) for non-ischemic myocardial injury.

The present inventors focused on various physiological activities of MK and examined the action of MK using an animal model of non-ischemic myocardial injury. As a result, administration of MK improved the symptoms of non-ischemic myocardial injury. That is, it has been found that enhancing MK rather than suppressing MK is effective in treating non-ischemic myocardial injury. In other words, an unexpected finding that “suppression of MK is an effective treatment strategy even in dilated cardiomyopathy” was brought forward, and the way to establish a treatment for non-ischemic myocardial injury was opened. It was. In addition, as described in the Examples section below, the following findings were obtained: MK is effective for drug-induced dilated cardiomyopathy, and MK for dilated cardiomyopathy caused by arrhythmia. Is effective. The present invention is based on the above results and is as follows.
[1] A medicament for non-ischemic myocardial injury comprising the following (1) or (2) as an active ingredient:
(1) Midkine protein;
(2) A vector carrying a midkine gene.
[2] The medicament according to [1], wherein the midkine protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to the amino acid sequence.
[3] The medicament according to [1], wherein the midkine gene comprises the base sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a base sequence equivalent to the base sequence.
[4] The medicament according to any one of [1] to [3], wherein the non-ischemic cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy or valvular disease.
[5] The medicament according to any one of [1] to [3], wherein the non-ischemic cardiomyopathy is dilated cardiomyopathy.
[6] The medicament according to [5], wherein dilated cardiomyopathy is drug-induced.
[7] Drugs that induce dilated cardiomyopathy are doxorubicin, pharmaceutically acceptable salts of doxorubicin, doxorubicin derivatives (however, those having antitumor activity), pharmaceutically acceptable salts of doxorubicin derivatives The medicament according to [6], which is a drug selected from the group consisting of:
[8] A drug selected from the group consisting of doxorubicin, a pharmaceutically acceptable salt of doxorubicin, a doxorubicin derivative (provided that it has an antitumor action), and a pharmaceutically acceptable salt of a doxorubicin derivative is effective. The medicine according to [7], which is used in combination with a medicine as an ingredient.
[9] The medicament according to [5], wherein dilated cardiomyopathy is caused by arrhythmia.
[10] A method for treating non-ischemic myocardial injury, comprising a step of administering a drug containing the following (1) or (2) as an active ingredient to a patient with non-ischemic myocardial injury:
(1) Midkine protein;
(2) A vector carrying a midkine gene.
[11] A drug selected from the group consisting of doxorubicin, a pharmaceutically acceptable salt of doxorubicin, a doxorubicin derivative (but limited to those having an antitumor action), and a pharmaceutically acceptable salt of a doxorubicin derivative is effective. A method for preventing or treating non-ischemic myocardial injury, comprising the step of administering a drug comprising the following (1) or (2) as an active ingredient to a patient undergoing cancer treatment with a drug as a component:
(1) Midkine protein;
(2) A vector carrying a midkine gene.

Protocol of experiment using drug-induced dilated cardiomyopathy model. Echocardiographic findings in doxorubicin-induced dilated cardiomyopathy model mice. MK protein was intraperitoneally administered with an osmotic pump to doxorubicin-induced dilated cardiomyopathy model mice, and the progress was observed by echocardiography. The upper row shows M mode findings 4 weeks after the start of treatment. There is a clear difference between the findings in the control group and those in the MK treatment group. The lower row shows the measurement results of EF (left ventricular ejection fraction) and FS (functional shortening rate) 4 weeks after the start of treatment. The ratio (mean ± standard deviation) compared with the time of the start of treatment (at the start of MK protein administration; day 0) is shown. * In the graph indicates significance relative to the control group (p <0.05, n = 4). Also, con is the measurement result of the control group, and mk is the measurement result of the treatment group. Echocardiographic findings in doxorubicin-induced dilated cardiomyopathy model mice. The left ventricular diameter during expansion (LVEDd) and the left ventricular diameter during contraction (LVESd) were compared between the MK administration group and the control group. ● is the measurement result of the MK treatment group, ■ ■ is the measurement result of the control group. In the control group, both LVEDd and LVESd increased over time. On the other hand, in the MK treatment group, LVEDd and LVESd are almost constant after the 14th day. The graph which shows the expression state of syndecan 1,3,4 which is a receptor of endogenous MK family protein Pleiotrophin (Pleiotrophin) and MK. Comparison was made between the MK treatment group (4 weeks after the start of treatment) and the control group (mean ± standard error, n = 3). The expression level of the MK treatment group (MK in the graph) was evaluated by the ratio to the expression level of the control group. The graph which shows the expression state of apoptosis related factor Akt1. Comparison was made between the MK treatment group (4 weeks after the start of treatment) and the control group (mean ± standard error, n = 3). The expression level of the MK treatment group (MK in the graph) was evaluated by the ratio to the expression level of the control group. The graph which shows the expression state of myocardial fibrosis related factor TIMP-1,2. Comparison was made between the MK treatment group (4 weeks after the start of treatment) and the control group (mean ± standard error, n = 3). The expression level of the MK treatment group (MK in the graph) was evaluated by the ratio to the expression level of the control group. * Indicates significance relative to the control group (p <0.05, n = 3). Protocol of experiment using rabbit tachycardia model. An MK administration group (MK (+); n = 14), an MK non-administration group (MK (−); n = 17), and a control group (CTL; n = 5) were prepared. Results of experiments using rabbit tachycardia model. The survival rates of the MK administration group (MK (+); n = 14) and the non-MK administration group (MK (−); n = 17) are shown in comparison. Results of experiments using rabbit tachycardia model. Heart morphology (upper left), HE staining results (upper middle), pleural leakage and ascites retention (upper right), heart, lung, liver weight ratio (lower). Results of experiments using rabbit tachycardia model. The upper graph compares echocardiograms, and the lower graph compares ventricular septal wall thickness (IVSWT), left ventricular end-diastolic diameter (LVDd), left ventricular posterior wall thickness (PWT), and left ventricular ejection fraction (EF). Results of experiments using rabbit tachycardia model. The left ventricular free wall and the left / right ventricular junction were stained with Sirius Red. Results of experiments using rabbit tachycardia model. The ratio of TUNNEL positive nuclei was compared between the MK administration group (MK (+)) and the non-MK administration group (MK (-)). Results of experiments using rabbit tachycardia model. The expression state of related molecules was analyzed by Western blot. The ratio of TUNNEL positive nuclei was compared between the MK administration group (MK (+)) and the non-MK administration group (MK (-)). Mechanism of action of MK (assumed).

  The first aspect of the present invention relates to a medicament for non-ischemic myocardial injury. “Non-ischemic myocardial injury” is a term opposite to ischemic myocardial injury and refers to myocardial injury caused by causes other than ischemia. The “medicine for non-ischemic myocardial injury” refers to a drug used for the prevention or treatment of non-ischemic myocardial injury. Representative examples of non-ischemic myocardial injury are dilated cardiomyopathy, hypertrophic cardiomyopathy, and valvular disease. Except as otherwise noted, “non-ischemic myocardial injury” in the present invention is as described above. It is a myocardial injury caused by causes other than ischemia, and is not limited to these three diseases (pathological conditions).

  The medicament of the present invention contains (1) midkine protein or (2) a vector carrying the midkine gene as an active ingredient. In addition, although the pharmaceutical of this invention contains only either (1) and (2) normally, it does not prevent containing these both.

(1) Midkine protein Midkine (MK) is a growth and differentiation factor discovered as a product of a retinoic acid responsive gene and consists of a 13 kDa polypeptide rich in basic amino acids and cysteine (Kadomatsu, K. et al : Biochem. Biophys. Res. Commun., 151: 1312-1318; Tomomura, M. et al .: J. Biol. Chem., 265: 10765-10770, 1990). MK has many physiological activities as described above. The amino acid sequence of MK (GenPept (NCBI), ACCESSION: NP_001012333, DEFINITION: midkine [Homo sapiens]) registered in a public database is shown in SEQ ID NO: 1 in the sequence listing. The midkine protein (hereinafter referred to as “MK protein”), which is an active ingredient of the present invention, is preferably a polypeptide containing the amino acid sequence. A polypeptide containing an amino acid sequence equivalent to the amino acid sequence can also be used as the MK protein. The “equivalent amino acid sequence” here is partially different from the reference amino acid sequence (SEQ ID NO: 1), but this difference is related to the function of the protein (effective action against non-ischemic myocardial injury). An amino acid sequence that has no substantial effect. Therefore, substantial identity is recognized between the reference amino acid sequence (SEQ ID NO: 1) and the equivalent amino acid sequence. In order to determine the presence or absence of substantial identity, for example, using the experimental system (evaluation by an animal model) described in the examples below, two amino acid sequences in terms of action and effect on non-ischemic myocardial injury It can be confirmed that there is no substantial difference between the two. MK has also been identified in mice and rats. The amino acid sequence of mouse MK protein is shown in SEQ ID NO: 5 (GenPept (NCBI), ACCESSION: NP_034914, DEFINITION: midkine [Mus musculus]), and the amino acid sequence of rat MK protein is shown in SEQ ID NO: 9 (GenPept (NCBI) ), ACCESSION: NP_110486, DEFINITION: midkine [Rattus norvegicus]).

  “Different in part of amino acid sequence” typically means deletion or substitution of 1 to several amino acids (upper limit is 3, 5, 7, 10) constituting the amino acid sequence. Alternatively, it means that a mutation (change) has occurred in the amino acid sequence due to addition, insertion, or a combination of 1 to several amino acids (the upper limit is 3, 5, 7, 10). Differences in amino acid sequences here are allowed as long as there is no significant decrease in the above functions. As long as this condition is satisfied, the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions. The term “plurality” as used herein refers to, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, and more preferably a number corresponding to less than about 10%. The number is preferably less than about 5%, and most preferably less than about 1%. That is, the equivalent amino acid sequence is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, even more preferably about 95% or more, and most preferably about 99% or more with the amino acid sequence of SEQ ID NO: 1. Sequence identity.

  It is preferred that the difference between the reference amino acid sequence (SEQ ID NO: 1) and the equivalent amino acid sequence is caused by a conservative amino acid substituent. As used herein, “conservative amino acid substitution” refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.

  By the way, the sequence identity (%) of two amino acid sequences or two nucleic acid sequences (hereinafter, “two sequences” is used as a term including them) can be determined, for example, by the following procedure. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. Sequence identity is a function of the number of identical positions common to the two sequences (ie, sequence identity (%) = number of identical positions / total number of positions × 100), preferably for alignment optimization Take into account the number and size of gaps required.

  Comparison of two sequences and determination of identity can be accomplished using a mathematical algorithm. Specific examples of mathematical algorithms available for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin and Altschul (1993) Proc. Natl. There is an algorithm modified in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search may be performed with score = 100 and wordlength = 12, using the NBLAST program. In order to obtain an amino acid sequence equivalent to a reference amino acid sequence, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. To obtain a gap alignment for comparison, the Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. For details, refer to the NCBI web page. Examples of other mathematical algorithms that can be used for sequence comparison include the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available for example on a GENESTREAM network server (IGH Montpellier, France) or an ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

  The identity of two amino acid sequences, using the Gloss program of the GCG software package, using a Blossom 62 matrix or PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3 Or 4 can be determined. Also, the identity of two nucleic acid sequences can be determined using the GAP program of the GCG software package, with gap weight = 50 and gap length weight = 3.

  MK proteins can be easily prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. Can do. For example, it can be prepared by transforming a suitable host cell (for example, E. coli or yeast) with DNA encoding MK protein and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, various modifications are possible if the MK protein is obtained as a recombinant protein. For example, if a DNA encoding MK protein and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. MK protein can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

  In view of qualitative uniformity and purity, it is preferable to prepare the MK protein by a genetic engineering technique. However, the method for preparing the MK protein is not limited to genetic engineering. For example, MK protein can also be prepared from natural materials by standard techniques (crushing, extraction, purification, etc.).

(2) Vector Holding Midkine Gene In one embodiment of the present invention, a vector holding a midkine gene (hereinafter referred to as “MK gene”) is used as an active ingredient. A “vector” is a carrier for carrying a target gene to a target cell. In the present invention, the MK gene is the target gene. The type of vector is not particularly limited as long as the MK gene can be introduced into the target cell and expressed in the target cell. The “vector” herein includes viral vectors and non-viral vectors. The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors and the like have been developed as virus vectors.

  Non-viral vectors include liposomes, positively charged liposomes (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), HVJ (Hemagglutinating virus of Japan) -Liposome (Dzau, VJ, Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93: 11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita , R., Molecular Med. Today, 5: 298-303, 1999). The vector in the present invention may be constructed as such a non-viral vector.

  In adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, a foreign gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Retroviral vectors are not suitable for gene transfer into non-dividing cells because cell division is required for integration of the viral genome into the host chromosome. On the other hand, lentivirus vectors and adeno-associated virus vectors cause integration of foreign genes into the host chromosome after infection even in non-dividing cells. Therefore, these vectors are effective for stably and long-term expressing foreign genes in non-dividing cells.

  Each viral vector can be prepared according to a previously reported method or using a commercially available dedicated kit. For example, an adenovirus vector can be prepared by the COS-TPC method or full-length DNA introduction method. The COS-TPC method is a homologous recombination that occurs in 293 cells by co-transfecting a recombinant cosmid incorporating the target cDNA or expression cassette and a parent virus DNA-terminal protein complex (DNA-TPC) into 293 cells. (Mayake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I.) (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA introduction method is a method for producing a recombinant adenovirus by subjecting a recombinant cosmid inserted with a target gene to restriction digestion and then transfecting 293 cells (Miho Terashima, Koki Kondo). Hiromi Kanegae, Izumi Saito (2003) Experimental Medicine 21 (7) 931.). The COS-TPC method can be performed using Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.) and Adenovirus genome DNA-TPC (Takara Bio Inc.). The full-length DNA introduction method can be performed using Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.).

  On the other hand, a retroviral vector can be prepared by the following procedure. First, a virus genome (gag, pol, env gene) other than the packaging signal sequence between LTRs (Long Terminal Repeat) existing at both ends of the virus genome is removed, and a target gene is inserted therein. The viral DNA thus constructed is introduced into a packaging cell that constitutively expresses the gag, pol, and env genes. Thereby, only the vector RNA having the packaging signal sequence is incorporated into the viral particle, and a retroviral vector is produced.

  Developed by improving or specificizing fiber protein by modifying adeno vectors (specific infection vectors) and gutted vectors (helper-dependent vectors) that can be expected to improve the expression efficiency of target genes. Has been. The recombinant vector of the present invention may be constructed as such a viral vector.

  The midkine gene inserted into the vector (hereinafter referred to as “MK gene”) is preferably SEQ ID NO: 2 (GenBank (NCBI), ACCESSION: NM_001012334, DEFINITION: Homo sapiens midkine (neurite growth-promoting factor 2) (MDK), transcript variant 1, mRNA), SEQ ID NO: 3 (GenBank (NCBI), ACCESSION: NM_001012333, DEFINITION: Homo sapiens midkine (neurite growth-promoting factor 2) (MDK), transcript variant 2, mRNA), or SEQ ID NO: 4 (GenBank (NCBI), ACCESSION: NM_002391, DEFINITION: Homo sapiens midkine (neurite growth-promoting factor 2) (MDK), transcript variant 3, mRNA). However, DNA having a base sequence equivalent to the base sequence (hereinafter referred to as “equivalent DNA”) can also be used as the MK gene. The “equivalent base sequence” here is partly different from the standard base sequence, but the function of the protein encoded by the difference (effective action against non-ischemic myocardial injury) is substantial. This means a base sequence that is not affected by any significant influence. A specific example of equivalent DNA is DNA that hybridizes under stringent conditions to a base sequence complementary to a reference base sequence (any one of SEQ ID NOs: 2 to 4). The “stringent conditions” here are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). Can be set with reference to. As stringent conditions, for example, hybridization solution (50% formamide, 10 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denaturation Conditions of incubation at about 42 ° C. to about 50 ° C. using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), and then washing at about 65 ° C. to about 70 ° C. using 0.1 × SSC, 0.1% SDS Can be mentioned. More preferable stringent conditions include, for example, 50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

  As another specific example of equivalent DNA, it consists of a base sequence including substitution, deletion, insertion, addition, or inversion of one or more bases with respect to a reference base sequence (any one of SEQ ID NOs: 2 to 4). And a DNA encoding a protein effective against non-ischemic myocardial injury. Base substitution or deletion may occur at a plurality of sites. The term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA It is. Such equivalent DNAs include, for example, restriction enzyme treatment, treatment with exonuclease and DNA ligase, position-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Including mutation, introduction, mutation, and / or inversion using mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Thus, it can obtain by modifying DNA which has a standard base sequence. The equivalent DNA can also be obtained by other methods such as ultraviolet irradiation.

  Still another example of equivalent DNA is DNA in which a base difference as described above is recognized due to a polymorphism represented by SNP (single nucleotide polymorphism).

  The mouse MK gene (Kadomatsu, K. et al., Biochem. Biophy. Res. Commun., 151, 1312 (1988)) and the rat MK gene have been identified. The sequence of the mouse MK gene is shown in SEQ ID NO: 6 (GenBank (NCBI), ACCESSION: NM_010784, DEFINITION: Mus musculus midkine (Mdk), transcript variant 1, mRNA), SEQ ID NO: 7 (GenBank (NCBI), ACCESSION: NM_001012335). , DEFINITION: Mus musculus midkine (Mdk), transcript variant 2, mRNA) and SEQ ID NO: 8 (GenBank (NCBI), ACCESSION: NM_001012336, DEFINITION: Mus musculus midkine (Mdk), transcript variant 3, mRNA) Is shown in SEQ ID NO: 10 (GenBank (NCBI), ACCESSION: NM_030859, DEFINITION: Rattus norvegicus midkine (Mdk), mRNA) in the Sequence Listing.

  The MK gene can be prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, and the like with reference to the sequence information disclosed in this specification or the attached sequence listing. For example, the MK gene can be isolated (and amplified) from a human cDNA library by appropriately using an oligonucleotide probe / primer that can specifically hybridize to the MK gene. As the oligonucleotide probe primer, for example, DNA complementary to the base sequence shown in SEQ ID NO: 2 or a continuous part thereof is used. Oligonucleotide probes and primers can be easily synthesized using a commercially available automated DNA synthesizer. For the method of preparing the library used for preparing the MK gene, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York is helpful.

  An equivalent DNA can be prepared by using a cDNA library derived from a mammalian cell other than a human (eg, monkey, mouse, rat, pig, bovine) instead of the human cDNA library.

  The pharmaceutical preparation of the present invention can be prepared according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  When a vector holding the MK gene is used as an active ingredient, it may be formulated by combining pharmaceutically acceptable media. “Pharmaceutically acceptable medium” refers to a substance that provides advantages or benefits regarding the administration or storage of a vector without substantially affecting the efficacy of the vector (ie, effectiveness against non-ischemic myocardial injury). . Examples of the “pharmaceutically acceptable medium” include deionized water, ultrapure water, physiological saline, phosphate buffered saline (PBS), and 5% dextrose aqueous solution. The composition of the present invention may contain other components such as a suspending agent, a soothing agent, a stabilizer (albumin, Prionex (registered trademark, Penta Farm Japan), etc.), a preservative, and an antiseptic.

  When the vector carrying the MK gene is in the form of a viral vector, it is preferable to use a biocompatible polyol (such as poloxamer 407) in combination. The use of polyols can increase the transduction rate of viral vectors 10 to 100 times (March et al., Human Gene Therapy 6: 41-53, 1995). Therefore, if a polyol is used in combination, the dose of the viral vector can be kept low. In addition, you may decide to use a polyol as one component of the medicine of this invention, or to prepare a polyol (or composition containing it) separately from the medicine of this invention. In the latter case, when the medicament of the present invention is administered, polyol (or a composition containing it) is also administered.

  The dosage form in the case of formulating is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

  The medicament of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (ie, a therapeutically effective amount). The amount of the active ingredient in the medicament of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set within a range of, for example, about 0.1% by weight to about 95% by weight so as to achieve a desired dose.

  The medicament of the present invention is administered to the subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) according to the dosage form. Applied. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed).

  The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the medicament of the present invention is applied to humans.

  The dosage of the medicament of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, when midkine protein is used as an active ingredient, for example, for an adult (body weight of about 60 kg), the dosage is adjusted so that the amount of active ingredient per day is about 0.1 mg to about 500 mg, preferably about 10 mg to about 50 mg. Can be set. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.

  The medicament of the present invention is used for prevention or treatment of non-ischemic myocardial injury. As described above, “non-ischemic cardiomyopathy” is a disease concept including dilated cardiomyopathy, hypertrophic cardiomyopathy and valvular disease. Here, “dilated cardiomyopathy” is cardiomyopathy characterized by a decrease in myocardial contraction of the left ventricle or both left and right ventricles and enlargement of its lumen. Viral infections, genetic predispositions, immune disorders, systemic diseases, metabolic diseases, hypertension, alcohol, drugs, etc. are considered as etiologies. Onset of dilated cardiomyopathy is often observed as a side effect of anthracycline antitumor antibiotics.

  On the other hand, “hypertrophic cardiomyopathy” is cardiomyopathy mainly characterized by left ventricular dilatation due to myocardial hypertrophy. Myocardial hypertrophy is not uniform and strong hypertrophy often occurs locally. Hypertrophic cardiomyopathy is roughly classified into obstructive hypertrophic cardiomyopathy due to thickening of the wall near the aortic valve and non-obstructive hypertrophic cardiomyopathy due to thickening of the apical wall.

  In addition, “valvular disease” is a state in which a heart valve (aortic valve, pulmonary valve, tricuspid valve, mitral valve) has failed and cannot play its original role. Valvular disease is aortic stenosis, aortic regurgitation, pulmonary stenosis, pulmonary regurgitation, tricuspid stenosis, tricuspid regurgitation, tricuspid regurgitation, mitral stenosis and It is roughly divided into mitral regurgitation. There are also congenital and acquired (rheumatic fever, arteriosclerosis, myocardial infarction, degeneration, etc.).

  Considering the experimental results shown in Examples described later, it can be said that the medicament of the present invention exhibits its effectiveness particularly for dilated cardiomyopathy. Thus, in a preferred embodiment, the medicament of the present invention is provided as a medicament for dilated cardiomyopathy.

  Moreover, MK showed a therapeutic effect in an animal model of drug-induced dilated cardiomyopathy (Example 1). In view of this fact, one of the preferred indications for the medicament of the present invention is drug-induced dilated cardiomyopathy. On the other hand, MK showed a therapeutic effect on an animal model of dilated cardiomyopathy caused by arrhythmia (Example 2). In view of this fact, dilated cardiomyopathy due to arrhythmia is also a suitable indication for the medicament of the present invention. Therefore, in a preferred embodiment, the medicament of the present invention is provided as a medicament for drug-induced dilated cardiomyopathy and / or dilated cardiomyopathy caused by arrhythmia. The drug here (ie, drug that induces dilated cardiomyopathy) is typically doxorubicin or a pharmaceutically acceptable salt thereof (eg, hydrochloride) used in the experiments shown in the Examples. However, a drug having the same medicinal effect, that is, a doxorubicin derivative (however, limited to those having an antitumor action) or a pharmaceutically acceptable salt thereof also corresponds to the “drug” herein.

  Here, “doxorubicin” is an anthracycline antitumor antibiotic and is also called adriamycin (common name). A doxorubicin hydrochloride preparation is commercially available under the name Adriacin (trade name, Kyowa Hakko Kogyo Co., Ltd.). Doxorubicin hydrochloride is a malignant lymphoma (reticulosarcoma, lymphosarcoma, Hodgkin's disease), lung cancer, gastrointestinal cancer (gastric cancer, gallbladder / bile duct cancer, pancreatic cancer, liver cancer, colon / rectal cancer, etc.), breast cancer, bladder tumor, osteosarcoma Used to treat. It is also used in combination with other antineoplastic agents and is also applied to breast cancer, endometrial cancer, malignant bone tumor, multiple myeloma, and childhood malignant solid tumor.

  Examples of doxorubicin derivatives are epirubicin, pirarubicin, daunorubicin. All of these compounds have a molecular structure called anthracycline, like doxorubicin. In addition, farmorubicin (registered trademark, Pfizer Inc.), epirubicin hydrochloride (Mylan Pharmaceutical Co., Ltd.) and the like are known as epirubicin hydrochloride preparations. Similarly, pinorbin (trade name, Mercian Corporation, Nippon Kayaku Co., Ltd.) and terarubicin (Meiji Seika Co., Ltd.) are known as pirarubicin hydrochloride preparations, and daunomycin (trade name, Meiji Seika Co., Ltd.) is known as a daunomycin hydrochloride preparation. ing.

  When administering a drug that may induce myocardial injury such as doxorubicin, the pharmaceutical of the present invention is administered in combination to prevent the occurrence of myocardial injury or to reduce the symptoms of myocardial injury. Also good. Thus, the medicament of the present invention can also be used as a countermeasure against the side effects of a specific drug. The following prevention or treatment method is mentioned as a specific example of the said aspect. Namely, a drug selected from the group consisting of doxorubicin, a pharmaceutically acceptable salt of doxorubicin, a doxorubicin derivative (provided that it has an antitumor action) and a pharmaceutically acceptable salt of a doxorubicin derivative as an active ingredient A step of administering the medicament of the present invention (that is, a medicament containing (1) a midkine protein or (2) a vector carrying a midkine gene as an active ingredient) to a patient who receives a cancer treatment with said medicament. And a method for preventing or treating non-ischemic myocardial injury. Of the doxorubicin derivatives, epirubicin and pirarubicin have relatively low cardiotoxicity. However, cardiotoxicity (cause of myocardial injury) is still one of the serious side effects. Therefore, it can be said that the medicament of the present invention is also effective when using a medicament (such as farmorubicin or pinorbin) containing these compounds as an active ingredient.

<Effects of MK on drug-induced dilated cardiomyopathy model>
According to the protocol shown in FIG. 1, the therapeutic effect of MK on non-ischemic myocardial injury was examined. First, doxorubicin-induced dilated myocardium was administered to 7-8 week old mice (C57BL / 6) by intraperitoneal administration of 3 mg / kg of doxorubicin (Sigma) every other day for a total of 6 times (total dose 18 mg / kg). Disease model mice were prepared. One week after the end of doxorubicin administration, this model mouse was supplemented intraperitoneally with an osmotic pump filled with MK protein, and MK administration was started (MK treatment group, n = 4). In 2 weeks, 0.1 mg (100 μL of 1 mg / mL MK protein) was administered. Similarly, physiological saline was administered to the control group (n = 4) with an osmotic pump.

  Two weeks after the start of treatment, the pump was changed and administration of MK protein was continued (0.1 mg of MK protein was administered over 2 weeks). Progress was observed by echocardiography for 4 weeks from the start of treatment. FIG. 2 shows the M mode findings, EF (left ventricular ejection fraction), and FS (functional shortening rate) 4 weeks after the start of treatment. EF and FS are used as indicators of left ventricular contractility. From the M mode findings (upper), significant functional improvement is observed in the MK treatment group. In the MK treatment group, EF and FS also improved (lower row).

  As a result of measuring the left ventricular diameter during expansion (LVEDd) and the left ventricular diameter during contraction (LVESd), the control group showed an increase over time, whereas the MK treatment group showed a clear increase suppression ( FIG. 3).

Next, after killing the MK treatment group and the control group, the myocardial tissue was collected, and the expression state of various molecules and factors was examined.
(1) MK family and receptor The expression state of endogenous MK family protein Pleiotrophin and Syndecan 1, 3 and 4 which are receptors of MK was examined by the following method. MRNA was extracted from the isolated heart 4 weeks after treatment, and RNA expression was measured by quantitative PCR. As a result, the expression of pleiotrophin and syndecans 1, 3, and 4 was clearly enhanced in the MK treatment group ( FIG. 4).

(2) Apoptosis-related factor The expression state of the factor Akt1 involved in apoptosis was examined by the following method. MRNA was extracted from the heart removed 4 weeks after treatment, and RNA expression was measured by quantitative PCR in the same manner as described above. As a result, a clear expression enhancement of Akt1 was observed in the MK treatment group (FIG. 5).

(3) Myocardial fibrosis-related factor The expression state of factors TIMP-1,2 involved in myocardial fibrosis was examined by the following method. MRNA was extracted from the heart isolated 4 weeks after treatment, and RNA expression was measured by quantitative PCR as described above. As a result, the expression of TIMP-1,2 was clearly enhanced in the MK treatment group (Fig. 6).

(4) Summary The results of (1) to (3) show that the expression of the MK receptor is excited by MK administration and the expression of the downstream signal Akt1 is increased. As a result, apoptosis and fibrosis of cardiomyocytes are suppressed, and This suggests that pathological remodeling has been reduced. Increased expression of pleiotrophin, an MK family, seems to have a synergistic effect, but the detailed mechanism is unknown.

<Effect of MK on the tachycardia model>
(1) Creation of rabbit tachycardia model A rabbit tachycardia model was created by the following method (FIG. 7). First, anesthetize and intubate the rabbit, expose the heart after thoracotomy, and sew the tip of the pacemaker lead to the right ventricular epicardium. The pacemaker body is implanted subcutaneously in the back of the rabbit. Wait for recovery and stability after surgery for 10 days, and start tachycardia stimulation with a heart rate of 350 / min (physiological heart rate: average 185 / min) by a programmer. Create myocardial injury by sustaining tachycardia stimulation for 28 days.

(2) Therapeutic effect of MK administration An osmotic pump was implanted subcutaneously at the start of stimulation of tachycardia. In the MK administration group (MK (+); n = 14), 2 mg of MK was administered for 28 days (FIG. 7). Physiological saline was administered to the MK non-administration group (MK (−); n = 17). No treatment was given to the control group (CTL; n = 5). The survival rate after 28 days was MK administration group: 12/14, MK non-administration group: 12/17, and improvement was observed in the MK administration group (FIG. 8). In the isolated heart, marked heart enlargement was observed in the MK non-administered group, but the enlargement was mild in the MK-administered group (upper left in FIG. 9). In addition, the symptoms of dilated cardiomyopathy were milder in the MK administration group than in the MK non-administration group (FIG. 9). Echocardiography showed a marked improvement in the left ventricular contraction rate in the MK administration group compared to the MK non-administration group (Fig. 10, upper panel), and a clear reduction in the left ventricular end-diastolic diameter (LVDd) (The lower part of FIG. 10). Significant improvements were also observed in the other left ventricular function indicators in the MK administration group (FIG. 10, lower panel). As a result of tissue staining, significant suppression of fibrosis was observed in the MK administration group as compared to the MK non-administration group (FIG. 11). Further, the ratio of TUNNEL positive nuclei is also significantly lower in the MK administration group (FIG. 12). As a result of Western blotting, a significant decrease in the Bax / Bcl-2 ratio was observed in the MK administration group as compared to the MK non-administration group (FIG. 13 left). In the MK administration group, increased expression of phosphorylated Akt (p-Akt) and P13 kinase was also observed (FIG. 13 right). The mechanism of action of MK assumed from the above results is shown in FIG.

The following findings are derived from the results of the above two experiments.
(1) Since MK showed a therapeutic effect in an animal model of non-ischemic myocardial injury (Examples 1 and 2), MK is effective for non-ischemic myocardial injury.
(2) Since two animal models (Examples 1 and 2) whose therapeutic effects have been confirmed both reproduce dilated cardiomyopathy, MK is effective for dilated cardiomyopathy.
(3) Since MK showed a therapeutic effect in an animal model of drug-induced dilated cardiomyopathy (Example 1), MK is effective for drug-induced dilated cardiomyopathy. Currently, doxorubicin or its derivatives used for the production of animal models are frequently used for the treatment of malignant lymphoma, lung cancer, digestive organ cancer, breast cancer, bladder tumor, osteosarcoma, etc. “Cardiotoxicity (cause of myocardial injury)” is a problem. The above findings mean that the side effects can be dealt with by the combined use of MK, which is a great gospel for patients.
(4) Since MK showed a therapeutic effect on an animal model of dilated cardiomyopathy caused by arrhythmia (Example 2), MK is effective for dilated cardiomyopathy caused by arrhythmia.

  The medicament of the present invention is effective for treating or preventing non-ischemic myocardial injury. In particular, application to dilated cardiomyopathy caused by drug-induced dilated cardiomyopathy or arrhythmia is expected. In addition, for the purpose of preventing or treating dilated cardiomyopathy induced by an anticancer agent, it is envisaged that the medicament of the present invention is used in combination with the anticancer agent.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (9)

A medicament for non-ischemic myocardial injury comprising the following (1) or (2) as an active ingredient:
(1) Midkine protein;
(2) A vector carrying a midkine gene.   The medicament according to claim 1, wherein the midkine protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to the amino acid sequence.   The medicament according to claim 1, wherein the midkine gene comprises the base sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a base sequence equivalent to the base sequence.   The medicament according to any one of claims 1 to 3, wherein the non-ischemic cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy or valvular disease.   The medicament according to any one of claims 1 to 3, wherein the non-ischemic cardiomyopathy is dilated cardiomyopathy.   The medicament according to claim 5, wherein dilated cardiomyopathy is drug-induced.   The agent that induces dilated cardiomyopathy is composed of doxorubicin, a pharmaceutically acceptable salt of doxorubicin, a doxorubicin derivative (but limited to those having antitumor activity), and a pharmaceutically acceptable salt of doxorubicin derivative The medicament according to claim 6, which is a more selected drug.   An active ingredient is a drug selected from the group consisting of doxorubicin, pharmaceutically acceptable salts of doxorubicin, doxorubicin derivatives (but limited to those having antitumor activity), and pharmaceutically acceptable salts of doxorubicin derivatives. The medicament according to claim 7, which is used in combination with a medicament.   The medicament according to claim 5, wherein dilated cardiomyopathy is caused by arrhythmia.

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