Granulysin and interleukin-6 for infections

FIELD OF THE INVENTION

0001The present invention relates to a therapeutic agent for treating infectious diseases such as tuberculosis.

BACKGROUND OF THE INVENTION

0002In medical care, it goes without saying that a therapeutic agent for infectious diseases is indispensable. At present, a number of therapeutic agents for infections such as antibiotics and synthetic antibacterial agents are provided to be used in medical care.

0003However, currently, an antibacterial agent which is mainly provided as therapeutic agent for infections has in fact an unavoidable problem; appearance of drug resistant bacteria. That is, a new antibacterial agent leads to produce a new drug resistant bacterium, and such a sarcastic state continues until now.

0004For example, tuberculosis occupies the first position of a death rate among single infectious diseases, and its increasing tendency becomes a worldwide problem. Further, a drug resistant bacterium having resistance to almost all antibiotics has been confirmed, and this problem has been obvious.

0005Recently, it has been revealed that granulysin, which is a molecule expressed in Natural Killer cells (NK cell) or Cytotoxic T Lymphocytes (CTL), has direct ability to kill and injure bacteria such as Mycobacterium tuberculosis [<nplcit id="ncit0001" npl-type="s"><text>Stenger, S.et al., Science 282, 121-125 (1998</text></nplcit>)].

0006Granulysin is produced as 15K and, thereafter, processed into 9K in a cytotoxic granule. 15 K and 9K granulysins are respectively incorporated into a macrophage so as to kill and injure bacteria such as Mycobacterium tuberculosis phagocytosed into the macrophage.

0007However, 9K granulysin requires a perforin, which is a molecule derived from the same cytotoxic intragranule, to enter into the macrophage. This is because it is necessary to perforate a target cell by perforin such that 9K granulysin enters into the cell therethrough. Accordingly, 9K granulysin shows cytotoxicity. On the other hand, 15K granulysin does not require perforin capable of perforating the target cell in order to enter into a macrophage, and kills and injures bacteria such as Mycobacterium tuberculosis phagocytosed into the macrophage. Accordingly, 15K granulysin shows no cytotoxicity.

0008The present inventors produced 9K granulysin transgenic mouse and 15K granulysin transgenic mouse, then intravenously administered 5x10⁵ CFU of H37Rv Mycobacterium tuberculosis through tail vein in each mouse and a wild-type control mouse respectively, and after 4 weeks, measured the numbers of Mycobacterium tuberculosis in lungs of each mouse, and confirmed that the number of Mycobacterium tuberculosis in the lung of 15K granulysin transgenic mouse is more reduced than that of 9K granulysin transgenic mouse with a statistically significant difference (p&lt;0.05; Student's t Test). Thus, the present inventors were the first to find that 15K granulysin had significantly stronger killing effect and suppressing effect on Mycobacterium tuberculosis in vivo than 9K granulysin. In addition, the result suggested that 9K Granulysin had very weak suppressing effect on Mycobacterium tuberculosis in vivo.

0009Based on the above result, the present inventors filed an application relating to a therapeutic agent for infections comprising 15K granulysin as an active ingredient, which has no cytotoxicity and has stronger inhibiting effect against Mycobacterium tuberculosis than 9K granulysin, and a therapeutic agent for infections comprising a vector in which a gene encoding 15 K granulysin is incorporated to express 15K granulysin in vivo as an active ingredient. The application has been patented (Japanese Patent No. <patcit id="pcit0001" dnum="JP4149713B"><text>4149713</text></patcit>). However, in order to prepare a stronger therapeutic agent for infections, a therapeutic agent comprising certain substances together with 15K granulysin rather than comprising 15K granulysin itself, which is effective on infections caused by bacteria such as Mycobacterium tuberculosis phagocytosed into the macrophage, is desired.

SUMMARY OF INVENTION

0010The present invention provides a therapeutic agent for infections comprising certain substances together with 15K granulysin rather than only 15K granulysin itself, to which bacteria acquire less resistance, and which has different characteristics from antibiotics and stronger effect on killing bacteria such as Mycobacterium tuberculosis (M. tuberculosis) phagocytosed into the macrophage.

0011One embodiment of the present invention is related to a therapeutic agent for infections comprising as active ingredients: 15K granulysin; and a vector in which a gene encoding 15K granulysin is incorporated to express 15K granulysin in vivo. Another embodiment of the present invention is related to a therapeutic agent for infections comprising as active ingredients: 15K granulysin; and at least one of the following substances (a) to (c). (a) interleukin 6, (b) interleukin 23(c), interleukin 27 Yet another embodiment of the present invention is related to a therapeutic agent for infections comprising as active ingredients: a vector in which a gene encoding 15K granulysin is incorporated to express 15K granulysin in vivo; and at least one of the following substances (a) to (c). (a) interleukin 6, (b) interleukin 23, (c) interleukin 27 Yet another embodiment of the present invention is related to a therapeutic agent for infections comprising the following (a) and (b) as active ingredients: (a) a vector in which a gene encoding 15K granulysin is incorporated to express 15K granulysin in vivo; and(b) vectors in which a gene encoding 65KDA heat-shock protein of M. tuberculosis H37Rv and a gene encoding interleukin 12 are respectively incorporated to express 65KDA heat-shock protein and interleukin 12 in vivo. Yet another embodiment of the present invention is related to the therapeutic agent for infections, wherein the 15K granulysin is a recombinant protein. Yet another embodiment of the present invention is related to the therapeutic agent for infections, wherein causative microorganism of the infections is Mycobacterium tuberculosis.

0012According to one embodiment of the present invention, a therapeutic agent for infections comprising as active ingredients: 15K granulysin; and a vector in which a gene encoding 15K granulysin is incorporated to express 15K granulysin in vivo (Hereinafter, referred to as "15K granulysin in vivo expression vector")is disclosed. The administration of the therapeutic agent shows no cytotoxicity and shows stronger killing effect on bacteria such as M. tuberculosis phagocytosed into the macrophage than that of 15K granulysin itself or of 15K granulysin in vivo expression vector itself.

0013According to another embodiment of the present invention, a therapeutic agent for infections comprising as active ingredients: 15K granulysin; and at least one of interleukin 6 (hereinafter referred to as "IL-6"), interleukin 23 (hereinafter referred to as "IL-23") or interleukin 27 (hereinafter referred to as "IL-27") is disclosed. The administration of the therapeutic agent shows no cytotoxicity and shows stronger killing effect on bacteria such as M. tuberculosis than that of 15K granulysin itself or of at least one of IL-6, IL-23 or IL-27 itself.

0014According to yet another embodiment of the present invention, a therapeutic agent for infections comprising as active ingredients: 15K granulysin in vivo expression vector; and at least one of IL-6, IL-23, or IL-27 is disclosed. The administration of the therapeutic agent shows stronger killing effects on bacteria such as M. tuberculosis than that of 15K granulysin in vivo expression vector itself or of at least one of IL-6, IL-23 or IL-27 itself.

0015According to yet another embodiment of the present invention, a therapeutic agent for infections comprising as active ingredients: 15K granulysin in vivo expression vector; and HVJ-envelope HSP65DNA and IL-12 DNAin vivo expression vectors against M. tuberculosis created by the present inventors (Hereinafter, referred to as "HSP65DNA and IL-12DNA in vivo expression vectors") is disclosed. These HSP65 DNA and IL-12DNA in vivo expression vectors show strong treatment effects on not only mouse family but also Macaca fascicularis family which is considered as the closest model to human in terms of tuberculosis infection. The administration of the therapeutic agent shows stronger killing effects on bacteria such as M. tuberculosis than that of 15K granulysin in vivo expression vector itself or of HSP65 DNA and IL-12 DNA in vivo expression vector itself. In addition, HSP65DNA and IL-12DNA in vivo expression vectors are prepared by incorporating 65KDa heat-shock protein DNA (HSP65DNA) of M. tuberculosis H37Rv and IL-12 DNA into an envelope of Sendai virus (HVJ) , and known to show a strong treatment effect on M. tuberculosis. (<nplcit id="ncit0002" npl-type="s"><text>Okada et al, Jpn.J.Clin.Immunol.,31 (5) 356-368 (2008</text></nplcit>))

0016According to yet another embodiment of the present invention, 15K granulysin recombinant protein is stably provided in great quantities.

0017According to yet another embodiment of the present invention, an agent for infections which is effective to Mycobacterium tuberculosis is provided.

0018The present invention provides a therapeutic agent for infections comprising a combination of 15K granulysin and 15K granulysin in vivo expression vector, a combination of 15K granulysin and at least one interleukin selected from IL-6, IL-23 or IL-27, a combination of 15K granulysin in vivo expression vector and at least one interleukin selected from IL-6, IL-23 or IL-27, or a combination of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors, which has no side effect and shows killing effect on bacteria such as M. tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

0019<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">FIG. 1</figref> is a view showing treatment effects of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector in liver of mice (DBA/1) intraperitoneally-infected with M. tuberculosis.</li><li><figref idref="f0002">FIG.2</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-6 in spleen of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0003">FIG.3</figref> is a view showing treatment effects of 15K granulysin in vivo expression vector and IL-6 in spleen of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0004">FIG.4</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-6 in liver of mice (BALB/c) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0005">FIG.5</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-6 in lung of mice (BALB/c) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0006">FIG.6</figref> is a view showing treatment effects of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector in liver of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0007">FIG.7</figref> is a view showing treatment effects of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector in spleen of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0008">FIG.8</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-23 in spleen of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0009">FIG.9</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-23 in liver of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0010">FIG.10</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-23 in lung of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0011">FIG.11</figref> is a view showing treatment effects of 15K granulysin in vivo expression vector and IL-23 in spleen of mice (DBA/1) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0012">FIG.12</figref> is a view showing treatment effects of 15K granulysin recombinant protein and IL-27 in lung of mice (BALB/c) infected with M. tuberculosis by aerosol.</li><li><figref idref="f0013">FIG.13</figref> is a view showing treatment effects of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors in liver of mice (DBA/1) infected with M. tuberculosis by aerosol.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

0020Embodiments of the present invention will be explained below. 15K granulysin used as an active ingredient of the present therapeutic agent may be obtained by being separated from a living body. However, since 15K granulysin is a trace component in a living body, it is preferable to be used as a recombinant protein obtained by expressing a gene encoding 15K granulysin. Alternatively, it is also a suitable means to produce 15K granulysin in a living body, utilizing 15K granulysin in vivo expression vector in which a gene encoding 15K granulysin is incorporated, as an active ingredient.

(Preparation of 15K Granulysin Recombinant Protein)

0021A sequence of a gene encoding 15K granulysin has been already reported. Based on this, a gene encoding 15K granulysin is effectively prepared to express this gene, which results in preparing a recombinant protein of 15K granulysin.

0022Specifically, the nucleotide chains that are complementary to both ends of a sequence of a gene encoding 15K granulysin are used as amplification primers, and a gene amplification method such as a PCR method is used to prepare a gene amplification product of the gene encoding 15K granulysin. This is incorporated into a suitable gene expression vector, and suitable host such as Escherichia coli, Bacillus subtilis, yeast and insect cell is transformed with such the recombinant vector so as to produce a desired 15K granulysin.

0023It is preferable that, as a gene expression vector used herein, a vector having a promoter and an enhancer at a region upstream of a gene to be expressed, and a transcription termination sequence at a region downstream of the gene is used.

0024In addition, an expression of a 15K granulysin gene is not limited to a direct expression system. For example, a fused protein expression system utilizing a β-galactosidase gene, a glutathione-S-transferase gene or a thioredoxin gene may be used.

0025As a gene expression vector using Escherichia coli as a host, there are exemplified pQE, pGEX, pT7-7, pMAL, pTrxFus, pET, and pNT26CII. As a gene expression vector using Bacillus subtilis as a host, there are exemplified pPL608, pNC3, pSM23, and pKH80.

0026In addition, as a gene expression vector using yeast as a host, there are exemplified pGT5, pDB248X, pART1, pREP1, YEp13, YRp7, and YCp50.

0027In addition, as a gene expression vector using a mammal cell or an insect cell as a host, there are exemplified p91023, pCDM8, pcDL-SRa296, pBCMGSNeo, pSV2dhfr, pSVdhfr, pAc373, pAcYM1, pRc/CMV, pREP4, and pcDNAI.

0028These gene expression vectors may be selected depending on the purpose of expression of 15K granulysin. For example, when 15K granulysin is expressed, it is preferable to select a gene expression vector for which Escherichia coli, Bacillus subtilis or yeast may be selected as a host. When 15K granulysin is expressed to show an assuredly activity even at a small amount, it is preferable to select a gene expression vector for which a mammal cell or an insect cell may be selected as a host.

0029In addition, it is possible to select the existing gene expression vector as described above, but a gene expression vector may be appropriately produced depending on a purpose, and this may be of course used.

0030Transfection of the above-mentioned gene expression vector in which a 15K granulysin gene is incorporated, into a host cell, and a transformation method therewith may be performed by the general method, for example, a calcium chloride method, and an electroporation method are used in the case of using Escherichia coli and Bacillus subtilis as a host cell, and a calcium phosphate method, an electroporation method and a liposome method are used in the case of using a mammal cell and an insect cell as a host.

0031The obtained transformant is cultured according to the conventional methods so as to accumulate a desired 15K granulysin. Medium used in such the culture may be appropriately selected depending on the characteristics of a host. For example, LB medium and TB medium may be appropriately used in the case of using Escherichia coli as a host, and RPMI1640 medium may be appropriately used in the case of using mammal cells as a host.

003215K granulysin may be isolated and purified from the obtained culture according to the conventional method. For example, various treating operations utilizing physical and/or chemical nature of 15K granulysin may be used.

0033Specifically, treatment with a protein precipitating agent, ultrafiltration, gel filtration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography using a specific antibody, and dialysis method can be used alone or by combining these methods.

(Preparation of 15K Granulysin In Vivo Expression Vector)

0034An active ingredient of the present therapeutic agent is a recombinant vector, which is an in vivo expression vector in which a gene encoding 15K granulysin used for expressing the aforementioned recombinant protein is incorporated.

0035Examples of the in vivo expression vector are not limited to, but include an adenovirus vector and a retrovirus vector. For an in vivo expression vector, for example, the aforementioned virus gene is incorporated into a cosmid vector, and a gene capable of expressing 15K granulysin is further incorporated into the cosmid vector and then, this cosmid vector and a parent virus DNA-TP which has been treated with a restriction enzyme are transfected into 293 cells, whereby, homologous recombination occurs in the 293 cells, thus, a desired in vivo expression vector is produced.

0036(Present Therapeutic Agent Comprising 15K granulsin Recombinant Protein and 15K Granulysin In Vivo Expression Vector as Active Ingredient) The combined use of 15K granulsin recombinant protein and 15K granulysin in vivo expression vector shows stronger killing effect on bacteria such as M. tuberculosis phagocytosed into the macrophage than independent use of the above.

003715K granulysin may be formed to a preparation composition by blending an appropriate pharmaceutical preparation thereinto (of course, only 15K glanulysin is possible). As a pharmaceutical preparation carrier, for example, excipients and diluents such as fillers, bulking agents, binders, wetting agents, stabilizers, solubilizers, disintegrating agents and surface active agents which are conventionally used as a pharmaceutical preparation carrier may be freely selected depending on a specific dosage form. A form of a preparation composition is not particularly limited as far as 15K granulysin is effectively used in treatments of infectious diseases. For example, solid agents such as tablets, powders, granules and pills, and also injectable forms such as solutions, suspensions and emulsions may be used. An appropriate carrier to 15K granulysin may be added to produce a dried agent, which is formulated to be liquid upon use.

0038The 15K granulysin in vivo expression vector prepared as described above is isolated and purified, and thereafter administered into a living body, so as to produce 15K granulysin in vivo and exhibit pharmacological effects of 15K granulysin.

0039A dose amount may be appropriately selected depending on an administration method and an administration form of an agent, and symptom of a patient. It is preferable to administer 10,000µg/50kg of the 15K granulysin recombinant protein once per day or by dividing into a few times per day and 6 times at a few dates interval, while it is preferable to administer 2,000µg/50kg of the 15K granulysin in vivo expression vector once per day and 6 times at a few dates interval, but it is not limited thereto.

0040A dosage form of the 15K granulysin in vivo expression vector is generally an injectable form. Also, 15K granulysin in vivo expression vector may be prepared in an injectable form by adding an appropriate carrier thereto. Then, the 15K granulysin recombinant protein and the 15K granulysin in vivo expression vector in the injectable forms may be administered by intravenous, intramuscular, intraosseous, intra-articular, subcutaneous, intracutaneous, or intraperitoneal administration at the same time or alternately at a given interval.

0041(Present Therapeutic Agent Comprising 15K Granulsin Recombinant Protein and at least one Interleukin Selected from IL-6, IL-23 or IL-27 as Active Ingredient) Combined use of the 15K granulysin recombinant protein and at least one interleukin selected from IL-6, IL-23 or IL-27 shows killing effects on bacteria such as M. tuberculosis phagocytosed into the macrophage than independent use of the above materials.

004215K granulysin recombinant protein and at least one interleukin selected from IL-6, IL-23 or IL-27 may be formed in a preparation composition by blending an appropriate pharmaceutical preparation carrier thereinto. As for a pharmaceutical preparation carrier, for example, excipients and diluents such as fillers, bulking agents, binders, wetting agents, stabilizers, solubilizers, disintegrating agents and surface active agents which are conventionally used as a pharmaceutical preparation carrier may be freely selected depending on a specific dosage form. A form of a preparation composition is not particularly limited as far as both the 15K granulysin recombinant protein and the at least one interleukin selected from IL-6, IL-23 or IL-27 are effectively used in treatments of infectious diseases. For example, solid agents such as tablets, powders, granules and pills, and also injectable forms such as solutions, suspensions and emulsions may be used. An appropriate carrier to the 15K granulysin recombinant protein and the at least one interleukin selected from interleukin 6, interleukin 23 or interleukin 27 may be added to produce a dried agent, which is formulated to be liquid upon use.

0043A dose amount of such the therapeutic agent may be appropriately selected depending on an administration method and an administration form of an agent, and symptom of a patient. It is preferable to administer 10,000µg/50kg of 15K granulysin recombinant protein and 2,500µg/50kg in the case of IL-6, 5,000µg/50kg in the case of IL-23, or 2,500µg/50kg in the case of IL-27, which are active ingredients, once per day or by dividing into a few times per day, and 6 times at a few dates to more than 1 week interval, but not limited thereto. The administrations of the two materials may be performed at the same time or alternately at a given interval.

0044Because a combination of 15K granulysin protein and interleukin exhibits strong killing effects on bacteria such as M. tuberculosis phagocytosed into the macrophage, it is possible to reduce the treatment times to 3 times.

0045Such the various forms of pharmaceutical preparations may be administered by an appropriate administration route depending on its form, for example, by intravenous, intramuscular, intraosseous, intra-articular, subcutaneous, intracutaneous, or intraperitoneal administration in the case of an injectable form, or by oral or enteral administration in the case of a solid agent form.

0046(Present Therapeutic Agent Comprising 15K Granulsin In Vivo Expression Vector and at least one Interleukin Selected from IL-6, IL-23 or IL-27 as Active ingredient) Combined use of the 15K granulysin in vivo expression vector and the at least one interleukin selected from interleukin 6, interleukin 23 or interleukin 27 as active ingredients enables to show killing effects on bacteria such as M. tuberculosis phagocytosed into the macrophage than independent use of the above materials.

004715K granulysin in vivo expression vector and the at least one interleukin selected from IL-6, IL-23 or IL-27 are formed to a preparation composition by blending an appropriate pharmaceutical preparation carrier thereinto. A dose amount of such the therapeutic agent may be appropriately decided depending on an administration method and an administration form of an agent, and symptom of a patient. It is preferable to administer 2,000µg/50kg of the 15K granulysin in vivo expression vector once per day and 6 times at a few dates to more than 1 week interval, while it is preferable to administer 2,500µg/50kg in the case of IL-6, 5,000µg/50kg in the case of IL-23, and 2,500µg/50kg in the case of IL-27, once per day or by dividing into a few times per day and 6 times at a few dates to more than 1 week interval, but it is not limited thereto.

0048A dosage form of the 15K granulysin in vivo expression vector is generally an injectable form. Also, the at least one interleukin selected from IL-6, IL-23, or IL-27 may be prepared in an injectable form by adding an appropriate carrier thereto. Then, the both materials in the injectable forms may be administered at the same time or alternately at a given interval.

0049(Present Therapeutic Agent Comprising 15K Granulsin In Vivo Expression Vector and HSP65DNA and IL-12DNA In Vivo Expression Vectors as Active Ingredient) A dosage form of the 15K granulsin in vivo expression vector and an HSP65DNA and IL-12DNA in vivo expression vectors is generally an injectable form, and it may be administered by intravenous, intramuscular, intraosseous, intra-articular, subcutaneous, intracutaneous or intraperitoneal administration. A dose of such the present therapeutic agent may be appropriately selected depending on an administration method and an administration form of an agent, and symptom of a patient, but being not limited.

0050Generally, it is preferable to administer 2,000µg/50kg of 15K granulysin in vivo expression vector and 2,000µg/50kg of the HSP65DNA and IL-12DNA in vivo expression vectors, which are active ingredients, once per day, and 6 times at a few dates interval.

0051Because a combination of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors exhibits strong killing effects on bacteria such as M. tuberculosis phagocytosed into the macrophage, it is possible to reduce the treatment times to 3 times.

[Example]

0052Examples of the present invention will be described below.

0053Improvements of killing effect on bacteria by using (1) 15K granulysin recombinant protein and 15K granulysin in vivo expression vector, (2) 15K granulysin recombinant protein and at least one interleukin selected from IL-6, IL-23 or IL-27, (3) 15K granulysin in vivo expression vector and at least one interleukin selected from IL-6, IL-23 or IL-27, and (4) 15K granulysin in vivo expression vector and HSP65DNA and IL-12 DNA in vivo expression vectors.

0054"Synergetic effect" used herein means effect when a combined use of more than two materials improves killing effect on bacteria compared to an independent use of the materials.

0055G1 to G10 groups used in the examples are shown in TABLE.1.

0056<tables id="tabl0001" num="0001"><table frame="all"><title><TABLE.1></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="12mm" /><colspec colnum="2" colname="col2" colwidth="154mm" /><tbody><row><entry>G1</entry><entry>Control</entry></row><row><entry>G2</entry><entry>15K granulysin recombinant protein and 15K granuysin in vivo expression vector were administered on the same day.</entry></row><row><entry>G3</entry><entry>15K granulysin recombinant protein and Interleukin (IL-6 or IL-23) were administered alternately.</entry></row><row><entry>G4</entry><entry>15K granulysin in vivo expression vector and interleukin (IL-23 or IL-27) were administered alternately.</entry></row><row><entry>G5</entry><entry>Only 15K granulysin recombinant protein was administered in the same day as the administration of 15K granulysin recombinant protein in G2 or G3.</entry></row><row><entry>G6</entry><entry>Only 15K granulysin in vivo expression vector was administered in the same day as the administration of 15K granulysin in vivo expression vector in G2, G4 or G11.</entry></row><row><entry>G7</entry><entry>Only interleukin (IL-6, IL-23 or IL-27) was administered in the same day as the administration of Interleukin in G3 or G4.</entry></row><row><entry>G9</entry><entry>15K granulysin in vivo expression vector was administered in the first half of the experimental period, and IL-6 was administered in the last half of the experimental period.</entry></row><row><entry>G10</entry><entry>Only IL-6 was administered in the same day as the administration of interleukin in G9.</entry></row><row><entry>G11</entry><entry>15K granulysin in vivo expression vector and HSP65DNA and IL-12 DNA in vivo expression vectors were administered alternately.</entry></row><row><entry>G12</entry><entry>Only HSP65DNA and IL-12 DNA in vivo expression vectors were administered in the same day as the administration of HSP65DNA and IL-12 DNA in vivo expression vectors in G11.</entry></row></tbody></tgroup></table></tables>

(Reagent)

005715K granulysin recombinant protein was obtained from R&amp;D System Co (Minneapolis, MN). IL-6 was obtained from Miltenyi Biotec Co. (Auburn, CA). Recombinant mouse IL-23 and recombinant mouse IL-27 were obtained from R&amp;D System Co.

005815K granulysin in vivo expression vector was prepared by inserting 15K granulysin gene into CAG vector, and incorporating the obtained vector into an envelope of Sendai virus (HVJ: Hemagglutinating Virus of Japan). HSP65DNA and IL-12 DNA in vivo expression vectors were prepared by inserting their genes respectively into pcDNA3·1 vectors, and incorporating the obtained vectors into an envelope of Sendai virus (HVJ: Hemagglutinating Virus of Japan). DBA/1 and BALB/c mice were used. These mice are easily infected by M.tuberculosis. For statistical analysis, Student's t Test was used.

0059In the following examples, in the case of an administration of 15K granulysin recombinant protein, 4µg was administered per 1 mouse (20g) per day. In the case of an administration of 15K granulysin in vivo expression vector, 100µg/0.2ml was intramuscularly administered per 1 mouse (20g) per day by 50 µg in an anterior tibial muscle in both sides of inferior limbs. In the case of an administration of IL-6, 1 µg per 1 mouse (20g) per day (>=5x10⁴ International Unit), in the case of an administration of IL-23, 2 µg per 1 mouse (20g) per day (>=5x10⁴ International Unit), and in the case of an administration of IL-27, 1 µg per 1 mouse (20g) per day (>=5x10⁴ International Unit) were intraperitoneally administered by dividing the amounts to three (by 1/3).

0060Bacterium to be used for the infections was M. tuberculosis H37Rv (human type). In the case of an intraperitoneal infection of M.tuberculosis, 1x10⁷ CFU was suspended with 0.2 ml of saline, and this was administered intraperitoneally. In the case of a lung infection (intratracheal infection) of M. tuberculosis, 1x10³ CFU was administered into a respiratory passage of the mouse in an aerosol chamber.

(Experiment 1)

Synergetic Effect of 15K Granulysin Recombinant Protein and 15K Granulysin In Vivo Expression Vector on M. tuberculosis When Infected Intraperitoneally

00618 weeks old DBA/1 mice was intraperitoneally infected with M. tuberculosis. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector (G2), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of only 15K granulysin in vivo expression vector (G6).

0062In G2, 15K granulysin recombinant protein was administered 4 times at intervals, and 15K granulysin in vivo expression vector was administered 7 times at intervals. In G5, only 15K granulysin recombinant protein, and in G6, only 15K granulysin in vivo expression vector, were administered respectively on the same day as G2. Nothing was administered in G1.

0063Then, these mice were dissected after euthanasia to obtain liver tissues including liver cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0064As a result, the number of colonies in G2 was fewer than those in G5 and G6. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform a statistical analysis, and the number of M. tuberculosis in G2 was more reduced than those in G5 and G6 with a statistically significant difference (p&lt;0.05).

0065In addition, the statistical analysis was performed in G1, which is a control, and G2, G5 and G6 in the same way, and the numbers of M. tuberculosis in G2, G5 and G6 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0001">FIG.1</figref>).

0066Accordingly, 15K granulysin recombinant protein and 15K granulysin in vivo expression vector independently exhibit effects in a liver, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 2)

Synergetic Effect of 15K Granulysin Recombinant Protein and IL-6 on M. tuberculosis in DBA/1 mouse

00678 weeks old DBA/1 mice was lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin recombinant protein and IL-6 (G3), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of only IL-6 (G7).

0068In G3, 15K granulysin recombinant protein and IL-6 were alternately administered 6 times each. In G5, only 15K granulysin recombinant protein, and in G7, only IL-6, were administered 6 times respectively. Nothing was administered in G1.

0069Then, these mice were dissected after euthanasia to obtain spleen tissues including spleen cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0070As a result, the number of colonies in G3 was fewer than those in G5 and G7. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G3 was more reduced than those in G5 and G7 with a statistically significant difference (p&lt;0.05).

0071In addition, the statistical analysis was performed in G1, which is a control, and G3, G5 and G7 in the same way, and the numbers of M. tuberculosis in G3, G5 and G7 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0002">FIG.2</figref>).

0072Accordingly, 15K granulysin recombinant protein and IL-6 independently show the effect in a spleen, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis, and show the synergetic effect.

(Experiment3)

Synergetic Effect of 15K Granulysin In Vivo Expression Vector and IL-6 on M. tuberculosis

00738 weeks old DBA/1 mice was lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin in vivo expression vector and IL-6 (G9), a group with an administration of only 15K granulysin in vivo expression vector (G6), and a group with an administration of only IL-6 (G10).

0074In G9, 15K granulysin in vivo expression vector and IL-6 were administered 6 times respectively. 15K granulysin in vivo expression vector was administered in the first half of the experimental period, and IL-6 was administered in the last half of the experimental period. In G6, only 15K granulysin in vivo expression vector, and in G10, only IL-6, were administered 6 times respectively. Nothing was administered in G1.

0075Then, these mice were dissected after euthanasia to obtain spleen tissues including spleen cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0076As a result, the number of colonies in G9 was fewer than those in G6 and G10. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis of G9 was more reduced than those of G6 and G10 with a statistically significant difference (p&lt;0.05).

0077In addition, the statistical analysis was performed in G1, which is a control, and G9, G6 and G10 in the same way, and the numbers of M. tuberculosis in G9, G6 and G10 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0003">FIG.3</figref>).

0078Accordingly, the combined use of 15K granulysin in vivo expression vector and IL-6 significantly improves the killing effect on M. tuberculosis in a spleen compared to the independent use of the above materials, and show the synergetic effect.

(Experiment 4)

Synergetic Effect of 15K Granulysin Recombinant Protein and IL-6 on M. tuberculosis in BALB/c Mouse

0079BALB/c mice, which are commonly used in experiments of tuberculosis infection, were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin recombinant protein and IL-6 (G3), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of only IL-6 (G7).

0080In G3, 15K granulysin recombinant protein and IL-6 were alternately administered 6 times each. In G5, only 15K granulysin recombinant protein, and in G7, only IL-6, were administered 6 times respectively. Nothing was administered in G1.

0081Then, these mice were dissected after euthanasia to obtain liver tissues including liver cells and lung tissues including lung cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0082As a result, the number of colonies in G3 was fewer than those in G5 and G7. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis of G3 was more reduced than those of G5 and G7 with a statistically significant difference (p&lt;0.05).

0083In addition, the statistical analysis was performed in G1, which is a control, and G3, G5 and G7 in the same way, and the numbers of M. tuberculosis in G3, G5 and G7 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0004">FIG.4</figref> (Liver) and <figref idref="f0005">FIG.5</figref> (Lung)).

0084Accordingly, 15K granulysin and IL-6 exhibit effects in liver and lung independently, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 5)

Synergetic Effect of 15K Granulysin Recombinant Protein and 15K Granulysin In Vivo Expression Vector on M. tuberculosis in DBA/1 Mouse When Lung-infected

00858 weeks old DBA/1 mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with administrations of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector (G2), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of only 15K granulysin in vivo expressin vector (G6).

0086In G2, 15K granulysin recombinant protein and 15K granulysin in vivo expression vector were administered 6 times at intervals in the same day. In G5, only 15K granulysin recombinant protein, and in G6, only 15K granulysin in vivo expression vector, were administered 6 times respectively.

0087Then, these mice were dissected after euthanasia to obtain liver tissues including liver cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0088As a result, the number of colonies in G2 was fewer than those in G5 and G6. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G2 was more reduced than those in G5 and G6 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0006">FIG. 6</figref>).

0089Accordingly, the combination of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector in liver significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 6)

Synergetic Effect of 15K Granulysin Recombinant Protein and 15K Granulysin In Vivo Expression Vector on M. tuberculosis When Lung-infected

00908 weeks old DBA/1 mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin recombinant protein and 15K granulysin in vivo expression vector (G2), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of 15K granulysin in vivo expression vector (G6).

0091In G2, 15K granulysin recombinant protein and 15K granulysin in vivo expression vector were alternately administered 6 times each in the same day. In G5, only 15K granulysin recombinant protein, and in G6, only 15K granulysin in vivo expression vector, were administered 6 times respectively. Nothing was administered in G1.

0092Then, these mice were dissected after euthanasia to obtain spleen tissues including spleen cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0093As a result, the number of colonies in G2 was fewer than those in G5 and G6. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G2 was more reduced than those in G5 and G6 with a statistically significant difference (p&lt;0.05).

0094In addition, the statistical analysis was performed in G1, which is a control, and G2, G5 and G6 in the same way, and the number of M. tuberculosis in G2 was more reduced than those in G5 and G6 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0007">FIG.7</figref>).

0095Accordingly, 15K granulysin recombinant protein and 15K granulysin in vivo expression vector exhibit effects in spleen independently, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 7)

Synergetic Effect of 15K Granulysin Recombinant Protein and IL-23 on M. tuberculosis

0096DBA/1 mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin recombinant protein and IL-23 (G3), a group with an administration of only 15K granulysin recombinant protein (G5), and a group with an administration of only IL-23 (G7).

0097In G3, 15K granulysin recombinant protein and IL-23 were alternately administered 6 times each. In G5, only 15K granulysin, and in G7, only IL-23, were administered 6 times respectively. Nothing was administered in G1.

0098Then, these mice were dissected after euthanasia to obtain spleen, liver and lung tissues including spleen, liver and lung cells respectively. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0099As a result, in all of spleen, liver and lung organs, the number of colonies in G3 was fewer than those in G5 and G7. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G3 was more reduced than those in G5 and G7 with a statistically significant difference (p&lt;0.05).

0100In addition, the statistical analysis was performed in G1, which is a control, and G3, G5 and G7 in the same way, and the numbers of M. tuberculosis in G3, G5 and G7 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0008">FIG.8</figref> (spleen), <figref idref="f0009">FIG.9</figref>(liver), and <figref idref="f0010">FIG.10</figref>(lung)).

0101Accordingly, 15K granulysin and IL-23 independently exhibit effects in all of spleen, liver and lung, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 8)

Synergetic Effect of 15K Granulysin in vivo expression vector and IL-23 on M. tuberculosis

0102DBA/1 mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with no administration (G1), a group with administrations of 15K granulysin in vivo expression vector and IL-23 (G4), a group with an administration of only 15K granulysin in vivo expression vector (G6), and a group with an administration of only IL-23 (G7).

0103In G4, 15K granulysin in vivo expression vector and IL-23 were alternately administered 6 times each. In G6, only 15K granulysin in vivo expression vector, and in G7, only IL-23, were administered 6 times respectively. Nothing was administered in G1.

0104Then, these mice were dissected after euthanasia to obtain spleen tissues including spleen cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0105As a result, the number of colonies in G4 was fewer than those in G6 and G7. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G4 was more reduced than those in G6 and G7 with a statistically significant difference (p&lt;0.05).

0106In addition, the statistical analysis was performed in G1, which is a control, and G4, G6 and G7 in the same way, and the numbers of M. tuberculosis in G4, G6 and G7 were more reduced than that in G1 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0011">FIG.11</figref>).

0107Accordingly, 15K granulysin in vivo expression vector and IL-23 exhibit effects in spleen independently, however, the combination of the above enables to significantly improve the killing effect on M. tuberculosis and show the synergetic effect.

(Experiment 9)

Synergetic Effect of 15K Granulysin In Vivo Expression Vector and IL-27 on M. tuberculosis

0108BALB/c mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with administrations of 15K granulysin in vivo expression vector and IL-27 (G4), a group with an administration of only 15K granulysin in vivo expression vector (G6), and a group with an administration of only IL-27 (G7).

0109In G4, 15K granulysin in vivo expression vector and IL-27 were alternately administered 6 times each. In G6, only 15K granulysin in vivo expression vector, and in G7, only IL-27, were administered 6 times respectively.

0110Then, these mice were dissected after euthanasia to obtain lung tissues including lung cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0111As a result, the number of colonies in G4 was fewer than those in G6 and G7. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G4 was more reduced than those in G6 and G7 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0012">FIG.12</figref>).

0112Accordingly, the combination of 15K granulysin in vivo expression vector and IL-27 in lung significantly improves the killing effect on M. tuberculosis and shows the synergetic effect.

(Experiment 10)

Synergetic Effect of 15K granulysin In Vivo Expression Vector and HSP65DNA and IL-12DNA in vivo expression vectors on M. tuberculosis

01138 weeks old DBA/1 mice were lung-infected with M. tuberculosis in an aerosol chamber. The mice were divided into a group with administrations of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors (G11), a group with an administration of only 15K granulysin in vivo expression vector (G6), and a group with an administration of only HSP65DNA and IL-12DNA in vivo expression vectors (G12).

0114In G11, 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors were alternately administered 6 times each. In G6, only 15K granulysin in vivo expression vector, and in G12, only HSP65DNA and IL-12DNA in vivo expression vectors, were administered 6 times respectively.

0115Then, these mice were dissected after euthanasia to obtain liver tissues including liver cells. The obtained tissues were homogenized and cultured on a 7H11 plate agar medium for 14 days. The number of colonies of M. tuberculosis was counted.

0116As a result, the number of colonies in G11 was fewer than those in G6 and G12. The numbers of colonies of 3 mice from each group were put in log₁₀ in order to perform the statistical analysis, and the number of M. tuberculosis in G11 was more reduced than those in G6 and G12 with a statistically significant difference (p&lt;0.05) (See <figref idref="f0013">FIG.13</figref>).

0117Accordingly, the combined use of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vectors in liver significantly improves the killing effect on M. tuberculosis compared to the independent use of the above materials, and shows the synergetic effect.

INDUSTRIAL APPLICABILITY

0118The present invention provides a useful therapeutic agent for infections comprising as an active ingredient: a combination of 15K granulysin and 15K granulysin in vivo expression vector, a combination of 15K granulysin and at least one interleukin selected from IL-6, IL-23 or IL-27, a combination of 15K granulysin in vivo expression vector and at least one interleukin selected from IL-6, IL-23 or IL-27, or a combination of 15K granulysin in vivo expression vector and HSP65DNA and IL-12DNA in vivo expression vector, which has less side effect, and to which bacteria can hardly acquire resistance.