WO2002066069A1

WO2002066069A1 - Remedies for inflammatory/tumor diseases

Info

Publication number: WO2002066069A1
Application number: PCT/JP2002/001445
Authority: WO
Inventors: Akira Matsumori; Jun-Ichi Miyazaki; Atsushi Nakano
Original assignee: Kansai Technology Licensing Organization Co., Ltd.
Priority date: 2001-02-20
Filing date: 2002-02-20
Publication date: 2002-08-29
Also published as: JPWO2002066069A1

Abstract

Remedies for inflammatory/tumor diseases which contain as the active ingredient an expression vector containing an immunosuppressive cytokine gene and/or a soluble cytokine receptor gene in a state allowing expression thereof; and a method of treating inflammatory/tumor diseases which comprises administering a therapeutically efficacious amount of an expression vector containing an immunosuppressive cytokine gene and/or a soluble cytokine receptor gene in a state allowing expression thereof to a patients suffering from an inflammatory/tumor disease.

Description

Specification

Inflammation / neoplastic disease treatment

Technical field

The present invention relates to a therapeutic agent for inflammation / neoplastic disease.

Background art

Heart disease is one of the leading causes of death in developed countries. Among them, acute viral myocarditis is characterized by multifocal inflammation of the heart, and in the chronic phase leads to dilated cardiomyopathy (DCM), ventricular aneurysm, and arrhythmogenic right ventricular dysplasia. Encephalomyocarditis (EMC) virus induced myocarditis in mice, and studies using this experimental model provided important insights into the mechanisms of myocarditis and DCM (Matsumori, 197). In this model, cellular immunity is activated, and enhanced cytokine expression and activation of cytotoxic T cells and macrophages in the myocardium are observed. Therefore, the immune system is considered a therapeutic target.

Direct viral disorders and host immune responses play important roles in the pathogenesis of viral heart disease (Bowles et al., 1998). The immune response plays an important role in fighting viral infections, but at the same time it can have detrimental effects on the host by improperly attacking cardiomyocytes. The balance between such protective and adverse effects may ultimately determine the prognosis of the disease. In addition, increased expression of inflammatory site force-ins in the heart may be involved in reduced myocardial function associated with viral myocarditis (Shioi et al., 1996). Site-in mRNAs containing inulin-leukin (IL)-1, tumor necrosis factor (TNF)-, and interferon (IFN) -γ are induced 3 days after virus inoculation, at which time Cell infiltration is rarely seen (Shioi et al., 1996).

The IL-11 receptor antagonist (IL-lra), a member of the IL-1 family, binds to the IL-11 receptor but does not elicit an intracellular response and is an important endogenous anti-inflammatory. It is a protein (Ai'end et al., 1998; Bresnihan et al., 1998). Viral IL-10 (vIL "10) is thought to be a cellular cytoplasm that is captured by Epstein-Barr virus, and shares many of the anti-inflammatory properties of cellular IL-10. They lack action and therefore can provide excellent immunosuppression, and are promising candidates for the treatment of viral myocarditis. Anti-inflammatory agents utilizing immunosuppressive cytokines are described in JP-A-2000-239182, but it is extremely difficult to administer cytodynamic proteins as proteins. However, there is a disadvantage that the cost is high.

In addition, Japanese Patent Application Laid-Open No. 2000-47115 describes a plasmid into which a cytokine gene has been incorporated. In this publication, inflammation such as myocarditis and the like is described using such a plasmid. No specific method of treatment is described.

An object of the present invention is to provide a therapeutic agent for inflammatory and neoplastic diseases capable of producing an immunosuppressive cytokine in a living body.

BRIEF DESCRIPTION OF THE FIGURES

1A-1D show transgene expression.

Figure 1A is an immunoblot of culture supernatant from BMT-10 cells transfected with pCAGGS-IL-lra showing successful expression of the 17-kDa protein and the correct size of mouse IL-lra. . Each lane shows cells transfected with pCAGGS and cells transfected with pCAGGS-IL-li'a, and recombinant mouse IL-lra. FIG. 1B shows serum levels of IL-lra 5 days after in vivo electroporation. The concentration of IL-lra was 3.5 times higher in EMCV-inoculated mice (n = 5) than in uninfected control mice (n = 4) (p <0.005), and IL-lm gene transfer (IL-lra_EMCV) (n = 5 5) increases serum IL-lra levels 1.9-fold (p <0.01) more than EMCV-inoculated mice (PBS-EMCV).

In FIG. 1C, in the time course, the serum concentration of IL-lra peaks on day 5 (p <0.01 compared to day 0) and then gradually decreases (n = 3 each).

In FIG. ID, the blood concentration on day 5 after the introduction of in vivo electoral poration was IL-lra (l734 pg / ml) and vIL-10 (2294 pg / ml) (n = 5 each).

Figures 2A-2C show the survival curves after EMCV inoculation.

In FIG. 2A, analysis of the survival curve showed that the IL-l treated group (η; η = Γ7) showed significantly higher survival rate than the PBS-treated group (□; n = 29) (p <0.05). .

In FIG. 2B, vIL-10 gene transfer (□; n = 19) also reduced mortality (p <0.01) in mice vaccinated with EMCV compared to PBS treatment (〇; n = 27). In FIG. 2C, empty pCAGGS introduction (C); n = 10 does not change the survival curve compared to PBS treatment (O; n = 10).

Figure 3A shows histological findings. In FIG. 3A, the quantitative pathological score of myocardial invasion shows a significant improvement in both the IL-lra treated group and the vIL-10 treated group (n = 5 each) (IL-lra treated vs. control). , P <0.01; vIL-10 treatment group vs control p <0.01). The lower panel shows representative histological results for each group.

Figure 3B shows histological findings 6 days after EMCV inoculation with soluble c-kit gene transfer. In Figure 3B, the quantitative pathological score for cell invasion was significantly higher in the soluble c-kit treated group (n = 7, 1.4 ± 0.7 mean soil standard deviation) than in the empty plasmid group (n = 9, 0.9 ± 0.4). Significant improvement (p <0.05).

FIG. 4 shows cytokine expression in the heart. In the IL-lra-introduced mouse in Fig. 4A, the expression levels of TNF-α and iNOS in the heart were significantly reduced (n = 5 each). In the vIL-10-introduced mouse of FIG. 4B, the expression levels of IFN-r and iNOS in the heart were significantly reduced. Also decreased in the vIL-10 treatment group (n = 5 each).

Disclosure of the invention

The present invention provides the following therapeutic agents for inflammatory and neoplastic diseases.

(1) A therapeutic agent for inflammation / neoplastic disease, comprising as an active ingredient an expression vector containing an immunosuppressive cytokine gene and / or a soluble cytokine receptor gene in an expressible state.

(2) The therapeutic agent according to (1), wherein the immunosuppressive cytokine and Z or soluble cytokine receptor gene are at least one selected from the group consisting of IL-lra, vIL-10 and soluble c_kit.

(3) The therapeutic agent according to (1), wherein the inflammation and the neoplastic disease is a cardiovascular disease.

(4) Inflammation · The therapeutic agent according to (3), wherein the neoplastic disease is myocarditis.

(5) Inflammation including administering a therapeutically effective amount of an expression vector containing an immunosuppressive cytokine gene and Z or a soluble cytokine receptor gene in a state capable of expressing it to a patient suffering from inflammation or neoplastic disease. Methods for treating neoplastic diseases.

(6) After administration of a therapeutically effective amount of the expression vector, an electric charge is applied across the administration site. The treatment method according to (5), wherein an air pulse is given.

(7) The method according to (5), wherein the immunosuppressive cytokine and / or soluble cytokine receptor gene is at least one selected from the group consisting of IL-lra, vIL'lO and soluble c'kit.

(8) The method according to (5), wherein the inflammation and the neoplastic disease is a cardiovascular disease.

(9) Inflammation · The therapeutic method according to (8), wherein the neoplastic disease is myocarditis. Detailed description of the invention

In the present invention, the immunosuppressive site force-in includes IL-1, IL-lj3, IL-lra, vIL-10, IL-2, 3, 4, 5, 6, 7, 8, 9, 10 , 11,12,13,14,15,16,17,18 inter-phagine, interferon (α, β, r)> TNF-H, TGF-β, GM-CSF, EGF, FGF, RANTES, I-309 / TCA-3, rIP-10, ΜΙΡ-Ι α, MIP-l jS, MIP-2, MCP-1, 2, 3, M-CSF, G-CSF, EPO, TPO, SCF, LIF, Examples include PDGF, KGF, IGF, NGF, BDNF, CDTF, OSM, HGF, and the like. In addition, soluble cytokine receptors are included for the soluble receptors of these cytokins. For example, soluble c-kit is exemplified for SCF, and soluble TNF-α receptor is exemplified for other cytokines such as TNF-Q !. The gene sequences of these immunosuppressive cytokins are known, and it is particularly preferable to use human immunosuppressive cytokine genes.

As an expression vector, for example, pCAGGS can be used, and a promoter Z enhancer sequence, a splicing region, a poly (A) addition site, and the like can be appropriately combined and used. The construction of a cytokine expression vector containing an immunosuppressive cytokine gene in a state capable of expressing it is described in, for example, the following description of the present specification and the description of JP-A-2000-47115. A person skilled in the art can easily perform this. Inflammation's neoplastic diseases include cardiovascular diseases such as myocarditis, cerebral myocarditis, dilated cardiomyopathy, heart failure, coronary artery disease, early coronary atherosclerosis of transplanted hearts, vasculitis, and aortitis, arthritis, back pain, and gout Examples include bronchial asthma, diffuse lung disease, atopy, allergy, hepatitis, flf cirrhosis, nephritis, gastric ulcer, enteritis, encephalomyelitis, multiple sclerosis, autoimmune disease, cancer, and myeloma. In the present invention, the combination of the specific immunosuppressive cytokine and the disease to be treated includes interferon (chronic myelogenous leukemia, multiple myeloma, renal cancer, malignant melanoma, chronic T-cell leukemia, hepatitis), 1-feron / 3 (brain tumors, hepatitis), inferior-feron r (chronic granulomatosis), IL-2 (primary immunodeficiency, malignant melanoma, kidney cancer, liver cancer), IL-6 (platelets) Hypotension), IL-11 (thrombocytopenia), Epo (thrombocytopenia), MCSF (thrombocytopenia, acute leukemia, neutropenia), GCSF (acute leukemia, neutropenia), IL-3 (neutropenia), GMCSF (neutropenia), SCF (neutropenia) power S Examples are shown.

The therapeutic agent for inflammation / neoplastic disease of the present invention is preferably administered by a parenteral route such as injection (intramuscular, subcutaneous, intradermal, intracorporeal, etc.), using any solvent such as physiological saline. be able to. The administration site is preferably in the muscles of the extremities. The therapeutically effective amount is about 0.00001 mg to 1000 mg, preferably about 0.01 mg to 100 mg, more preferably about 0.01 mg to 100 mg per day for an adult, and may be administered once or in several divided doses.

The therapeutic agent for inflammation / neoplastic diseases of the present invention is preferably subjected to electoral poration for applying an electric pulse across the administration site after administration, preferably immediately after administration. The electric pulse is, for example, a DC pulse composed of an arbitrary pulse shape, pulse width, pulse frequency, voltage, and the like. It can be set appropriately according to the injection site, injection amount, etc. of plasmid DNA. In the election port, preferably, electrodes are inserted at a plurality of points around the administration site, a voltage of about 50 to 100 V is applied, the waveform is a rectangular wave, and the pulse width is about 0.1 to about 100 m. Seconds, pulse frequency is 1 to 10 pulses Z seconds, and number of times is 1 to 10 times.

BEST MODE FOR CARRYING OUT THE INVENTION

In the following, an example of treating myocarditis using vIL-10, IL-lra and soluble c-kit (SCF receptor) as immunosuppressive cytodynamics is shown. The therapeutic agent for inflammation / neoplastic disease of the present invention can be obtained.

Methods and results

• Mouse model of myocarditis Four week-old inbredos DBA / 2 mice were inoculated intraperitoneally with lOpfu M mutant of encephalomyocarditis virus (EMCV) diluted in phosphate buffered saline (PBS).

• Plasmid DNA

Mouse IL-lra cDNA was extracted by PCR from total RNA extracted from mouse macrophage cell line J774A.1. Plasmid pCAGGS-IL-lra was constructed by inserting mouse IL-lra cDNA into the Xhol site between the CAG promoter and the 3 'flanking sequence of the egret / 3-glopurin gene on the pCAGGS expression vector. (Niwa et al., 1991). Similarly, plasmid pCAGGS-vIL-10 was constructed by inserting the mouse vIL-10 cDNA into the EcoRI site of the pCAGGS expression vector (Nitta et al., 1998). vIL-10 was derived from pcDSR crBCRF (Suzuki et al., 1995). Similarly, the plasmid pCAGGS-c-kit was constructed similarly by inserting the mouse c-kit cDNA into the EcoRI site of the pCAGGS expression vector. c-kit was derived from pCAGn-c-kit-hFc (Kataoka. Takakura et al., 1997). The plasmid was propagated in Escherichia coli strain DH10B cells, extracted by the alkaline lysis method, and purified by two cycles of ethidium bromide-CsCl equilibrium density gradient centrifugation. The plasmid was further purified by isopropanol precipitation, phenol extraction, phenol Z-cloth form extraction and ethanol precipitation. Plasmid DNA was then dissolved in PBS.

• In vivo electroporation

Mice were anesthetized with sodium pentobarbital. Plasmid DNA (75 g, 1.5 g // l each) was injected into the bilateral tibialis anterior and given electrical pulses as previously described (Aihara et al., 1998). Briefly, a pair of electrode needles was inserted into the muscle at a depth of 5 mni to serve as a DNA injection site. Six pulses were emitted to both muscles by an electric pulse generator (Electro Square Porator T820M; BTX, San Diego, CA) at a rate of 1 pulse / sec, each pulse for a duration of 50 msec.

• SDS-PAGE and Western blotting

pCAGGS-IL-lra and pCAGGS were transfected into BMT-10 cells using Lipofectamine 2000 (GIBCO BRL, Gaithersburg, MD). Two days after transfection, the culture supernatant is removed by one-third volume of SDS sample buffer. (75 mM Tris-HCl, pH 6.8, 6% SDS, 15% glycerol, 15% ME and 0.015% bromophenol), heat at 98 ° C for 5 minutes, and mix with 15% polyacrylamide gel. SDS-PAGE above. Recombinant mouse IL-lm was simultaneously loaded on the gel. After electrophoresis, samples were transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was incubated with goat anti-IL-lra polyclonal antibody (R & D Systems, Minneapolis, MN) for 3 hours at room temperature and washed. The membranes were then incubated for 1 hour at room temperature with anti-goat IgG2a, washed and washed according to the manufacturer's specifications. Autoradiography using chemiluminescence (ECL kit; Amersham Corp., Arlington Heights, IL). Performed photographic processing.

ELISA

For IL-lra ELISA, serum samples obtained from the mouse tail vein were assembled using an ELISA kit (Biosom'ce International, Camarillo, CA) according to the supplier's specifications. Serial dilutions of recombinant mouse IL-lra (R & D system) were used as standards. Serum concentrations of vIL-10 were assayed as follows: 96-well plates were coated overnight at 4 ° C with 22 g / ml rat anti-IL-10 mAb, JES3_9D7 (PliarMingen). Then, the plate was washed with PBS containing 1% BSA at room temperature for 1 hour. After washing with PBS containing 0.05% Tween (PBS / Tween), appropriately diluted samples were added to the wells. Plates were incubated overnight at 4 and washed with PBS / Tween. Biotinylated rat anti-IL-10 mAb (JES3-12G8; 2 gm 1, PharMingen) was added to the wells and plates were incubated with vigorous shaking at room temperature for 3 hours. After washing with PBS / ween, the diluted streptavidin horseradish peroxidase complex was added to the wells. Plates were incubated for 30 minutes at room temperature and washed with PBS / Tween. The substrate (o-phenylenediamine) was added to the wells, and the absorbance at 490 nm was measured with a microplate reader. Using recombinant _v IL-10 and (PharMin _g en) as a standard, the linear range of the ELISA system was 30~2000pg / ml. This ELISA system does not detect mouse IL-10.

• Histological findings

The heart was fixed in 10% formalin, embedded in paraffin, sectioned, and Staining was performed by three-color staining. The heart section specimens were photographed with a CCD camera, and the area of all heart section specimens and the area of the infiltration zone were measured in a blind fashion by NIH Image Software. The extent of cell invasion was calculated by dividing the area of the invasion zone by the area of the whole heart section specimen.

• Real-time PCR

Total RNA was extracted from mouse hearts by guanidine thiocyanate-cesium chloride centrifugation. RNA was reverse transcribed using oligo dT primer and Superscriptll reverse transcriptase (GIBCO BRL, Gaithersburg, MD). Real-time quantitative PCR was performed using the ABI PRISM ™ 7700 System (PE Biosystems, Foster City, Calif.) (Overbergh et al., 1999) to determine mHNA levels of cytokines in the heart. The primer pairs and probes used for these analyzes are as follows.

IL-1 jS forward primer, 5'-CAACCAACAAGTGATATTCTCCATG-3 '

IL-1) 3 back primer, 5 * -GATCCACACTCTCCAGCTGCA-3 '

IL-1 i8 probe, 5'-CTGTGTAATGAAAGACGGCACACCCACC-3 '

TNF-a; forward primer, 5'-CATCTTCTCAAAATTCGAGTGACAA-3 '

TNF-back primer, 5'-TGGGAGTAGACAAGGTACAACCC-3,

TNF-probe, 5'-CACGTCGTAGCAAACCACCAAGTGGA-3 '

IFN-forward primer, 5'-TCAAGTGGCATAGATGTGGAAGAA-3 '

IFN-Avac @ 1 Primer, 5'-TGGCTCTGCAGGATTTTCATG-3 '

IFN-probe, 5'-TCACCATCCTTTTGCCAGTTCCTCCAG-3 '

IL-6 forward primer, 5'-CAGAATTGCCATCGTACAACTCTTTTCTCA-3 '

IL-6 backed primer 1.5'-AAGTGCATCATCGTTGTTCATACA-3

IL-6 probe, 5'-GAGGATACCACTCCCAACAGACC-3 '

iNOS forward primer, 5'-CAGCTGGGCTGTACAAACCTT3 '

iNOS Γ%, liquid primer, 5'-CATTGGAAGTGAAGCGTTTCG-3 'iNOS probe, 5'-CGGGCAGCCTGTGAGACCTTTGA-3'

GAPDH forward primer, 5'-TTCACCACCATGGAGAAGGC-3 ' GAPDH Hack 1D-framer, 5'-GGCATGGACTGTGGTCATGA-3 '

GAPDH probe, 5 'TGCATCCTGCACCACCAACTGCTTAG-3'

These probes were designed to include intron sequences to distinguish appropriate PCR products from amplification products from contaminated genomic DNA. Each was labeled 5 'of the probe.

• Statistical analysis

Survival was analyzed by the Kaplan-Meier method. Statistical comparison was performed by log-rank test and ANOVA. Histological findings, serum levels of cytokines and expression of cardiac cytokines were compared using the Student's t-test.

• Expression of introduced genes

Mouse IL-lra and vIL_10 and c_kit were introduced into pCAGGS expression vector to obtain pCAGGS-IL-lra and pCAGGS'vIL-10 and pCAGGS-c-kit. Immunoblotting of culture supernatants from BM "10 transfected with pCAGGS-IL-lra showed successful expression of the 17 kDa protein, a mouse IL-lra of the correct size ( Figure 1A) Similarly, vIL-10 expression was confirmed by transient transfection of pCAGGS-vIL-10 into BMT-10 cells Similarly, c-kit expression was confirmed by pCAGGS-c Confirmed by transient transfection of -kit into BMT-10 cells EMCV was inoculated to determine whether electroporation of each plasmid resulted in physiologically significant levels of protein. Injected 75 plasmids into the bilateral tibialis anterior muscle of DBA / 2 mice and electrotransferred serum levels of IL-lra or vIL_10 when the level of the introduced gene was predicted to be at its peak. Five days later, it was measured by ELISA (FIG. 1B; Aihara et al., 19). 98) The concentration of IL-lra was 3.5 times higher in EMCV-inoculated mice than in uninfected control mice (p <0.005), and IL-lra gene transfer further reduced serum IL-li'a levels by 1 9-fold increase (p <0.01, compared to EMCV-infected mice) .The serum concentration of IL-lra was After 0, 3, 5, 7 and 14 days after the chilling, the preparation was started. During this time course, the serum concentration of IL-lra peaked on day 5 (p <0.01, compared with day 0), and then gradually decreased (Fig. 1C), which is consistent with the previous report. (Wells, 1993; Vitadello et al., 1994). The vIL-10 concentration 5 days after in vivo electroporation was 2294 pg / ml (FIG. 1D). These results clearly show that in vivo electroporation is superior for delivering cytokines systemically.

• Introduction of IL-lra, vIL_10 and soluble c_kit for myocarditis

By applying the in vivo electrobolition method, the present inventor tried a cytokine gene therapy for experimental myocarditis. 4-week-old DBA / 2 male mice were inoculated with lOpfu EMCV. Less than 20% of these mice survived 14 days after inoculation. Statistical analysis of the results of the IL-lra gene transfer revealed that the treated group showed significantly higher survival (Figure 2A). vIL-10 gene transfer also reduced mortality after EMCV inoculation (FIG. 2B) and resulted in slightly better survival than IL-lra transfer. These data suggested that the two inhibitory cytokines had a beneficial effect. To exclude the possibility of the plasmid itself having an effect, empty pCAGGS plasmid was similarly introduced into infected mice and the viability compared to PBS-injected mice. As shown in FIG. 2C, introduction of empty plasmid did not alter the survival curve. ·

In the case of the soluble c-kit gene transfer, 5 out of 10 animals died in the empty pCAGGS plasmid group, whereas there were no deaths in 10 animals in the soluble c-Mt gene transfer group, and the mortality after EMCV inoculation was significant. (P <0.05).

• Histological findings

In the IL-lra treated group and the vIL-10 treated group, heart section specimens were evaluated histologically on day 5, when inflammation progressed but none of the mice died (Fig. 3A). Quantitative pathological scores for myocardial infiltration were clearly improved in both the IL-lra and vIL_10 treatment groups, suggesting that the improvement in survival was due to alleviation of myocardial damage. did. Transduction of soluble c-Mt gene also reduced myocardial cell infiltration (Fig. 3B). • Cytokine expression in heart

To further clarify the mechanisms underlying the beneficial effects of IL-lra and vIL-10 To this end, the expression levels of inflammatory genes in the heart were measured by real-time quantitative PCR. In IL-Ira transfected mice, expression of TNF-α and iNOS was suppressed to 47% and 83% of the level seen in untreated mice (TNF-α, p <0.001; iNOS, p <0.05; FIG. 4A). On the other hand, in vIL-10-introduced mice, IFN and iNOS were suppressed to 14% and 16%, respectively (IFN-r, p <0.0i; iNOS, p <0.005; FIG. 4B). IL-1 j3 was also reduced in the vIL-10 treated group. Differences in the expression patterns of these inflammatory genes suggest that these two immunomodulatory processes elicit their therapeutic effects by different mechanisms.

At present, infection is thought to be involved in the pathogenesis of many heart diseases. Viral cardiomyositis is of particular concern and has various forms (Matsumori, 1997). In the last few years, proinflammatory cytokines such as cytoin, especially IL-1] 3 and TNF-spin, have been increasingly recognized as important factors in the pathogenesis of myocarditis and cardiomyopathy . In response to various forms of stress, the heart has a proinflammatory site such as IL-1) 3 and TNF-α, chemokines such as monocyte chemoattractant protein-1, and inducible nitric oxide synthesis. It secretes inflammatory enzymes, such as enzymes, and site forces that contain adhesion molecules, such as intracellular adhesion molecule-1. These products are increased in the heart of patients with heart disease, regardless of etiology, as well as in animals with experimental cardiac dysfunction. These products mediate inflammatory and immune responses and are all regulated by NF-KB. Interestingly, expression of the pro-inflammatory sites, IL-1 j8 and TNF-, not only requires activation of NF-κΒ, but also causes its activation. This type of positive regulation loop can enhance and make the local inflammatory response permanent. In this regard, several treatments have been reported that utilize anti-cytokines or immunomodulators to treat viral myocarditis. However, traditional methods of drug delivery have many pitfalls when applied to recombinant proteins: short-lasting efficacy, the need for frequent dosing, and, most importantly, the inadequacy of delivering proteins. And. For example, IL-1 J3 reached a maximum concentration of 150-200 pg / ml during experimental sepsis, and it was 10- 10 to suppress 50% of the IL-1 response in cells expressing the IL-1 receptor. A 0-fold excess of IL-lra is required (Bi'esnihan et al., 1998; Granowitz et al., 1991). In this study, the inventor has overcome these pitfalls by performing gene transfer using in vivo electotomy. In this way, high level Cytokines can be obtained in serum.

IL-lra is a member of the IL-1 gene family and suppresses many effects of IL-1 by competing with the IL-1 receptor both in vitro and in vivo. The agonist effect of IL-1 is regulated, in part, by the interaction of IL-lra and the IL-1 receptor. Administration of recombinant IL-lra improved mortality from TNF administration in mice, and intraperitoneal administration of LPS was more lethal in IL-lra knockout mice than in normal mice (Arend et al., 1998; Evei ' aerdt et al., 1994). On the other hand, mice lacking endogenous IL-lra are less susceptible to Listeria monocytogenes infection than normal mice, suggesting that IL'l plays an important role in resistance to infection by intracellular organisms. To support. These studies establish that the in vivo balance between endogenous IL-1 and IL-lra is important in affecting the host response to infection. IL-lra gene transfer using viral vectors has recently been investigated in experimental animal models of inflammatory diseases such as arthritis and ischemic encephalopathy (Hung et al., 1994; Makarov et al., 1996; Otani et al., 1996). Yan et al., 1997; Yang 6, 1998; Fernandes et al., 1999); The results of these studies suggest a therapeutic role for IL-lra and the important role of IL-1 in the activation of inflammatory cells in vivo. The study of the present invention suggests a novel therapeutic approach for the treatment of myocarditis using IL-lra by gene transfer, where the transduction efficiency was increased by electo-portionation. The present inventors have further analyzed the mechanism by which IL-lra reduces myocardial damage. The IL-1 pathway plays a critical role in regulating myocardial iNOS (Ungureanu-Longrois et al., 1995). TNF-α has a direct cytotoxic and negative inotropic effect on cultured cardiomyocytes and also activates cytotoxic Τ cells, which can cause myocyte damage (Pinsky et al., 1995; Yokoyama et al., 1993; Woodley et al., 1991). It has been suggested that TNF-a plays a key role at the very early stages of the immune response, and that administration of anti-TNF-a mAbs can interfere with the early pathways of acute viral myocarditis (Yamada et al., 1994). In this study, treatment with IL-lra may protect myocardium by suppressing iNOS and TNF-α expression.

IL-lra and vIL-10 were both effective treatments in the mouse model of myocarditis of the present invention, but treatment with in vivo electroporation itself was inoculated with EMCV The survival rate of the mice did not change. IL-10 is considered to be an important regulatory response in the early immune response to viral infection. Mortality and inflammation are increased in IL-10 knockout mice with virus-induced encephalomyelitis (Lin et al., 1998). Recently, the present inventors have shown that treatment with recombinant IL-10 reduces mortality and cytoplasmic force-in expression in a mouse model of myocarditis (Nishio et al., 1999). In the present invention, vIL-10 treatment strongly improved the efficacy of this experimental myocarditis. Cellular IL-10 and vIL-10 share many immunosuppressive properties: they both inhibit INF-α production by activated mouse Thl clones, human peripheral mononuclear cells, and human natural killer cells ( Hsu et al., 1990; Vieira et al., 1991; deWaal Malefyt et al., 1991). According to previous reports, reduced expression of IFN-r was also observed in the vIL-10 treated group. vIL-10 is thought to be a cellular site force captured by the EB virus. However, there are some differences between the effects of IL-10 and vIL-10. Although vIL-10 is less active as a cytokine synthesis inhibitor than IL-10, it lacks some of the immunostimulatory activity of IL-10 on monocytes, lymphocytes and mast cells. Therefore, vIL-10 may provide excellent immunosuppression (Drazan et al., 1996; Debruyne et al., 1998; Henke et al., 2000). These differences may explain the superior effect of vIL-10 in reducing the effects of myocarditis as compared to IL-10. Recently, the structural basis for the different activities of IL-10 and vIL-10 has been elucidated (Ding et al., 2000). The results suggest that a single amino acid is important for the immunostimulatory activity of IL-10. In our model, iNOS expression in the myocardium was also suppressed in vIL-10-treated mice, resulting in direct deactivation of monocyte Z-mace phage or indirect deactivation via inhibition of IFN-r. Suggests. In LPS-stimulated leukocytes, IL-10 is known to induce IL-Ira by mRNA stabilization, while it suppresses the production of IL_1 ^, TNF and IL-8 (Cassatella et al., 1994; Chomarat et al. , 1995). Whether vIL-10 induces irlra is unknown, but mice transfected with vIL-10 show elevated serum concentrations of IL-Ira (874 pg / ml). This additional effect of vIL-10 may explain its higher efficacy compared to IL-lra.

One common effect of cytokine expression is the induction of iNOS. Monoacid nitrogen (NO) is produced during the subacute phase in response to cytokines induced by EMCV. one Sidani Nitrogen is a double-edged sword (Beckman et al., 1990; Beckman, 1991). Nitric oxide is useful as an allergic mechanism of immunological self-protection, while at the same time it plays an important role in killing pathogens. Conversely, the excess of nitric oxide produced by iNOS also has a detrimental effect on myocardial tissue in viral and autoimmune myocarditis. The direct negative inotropic effects of inflammatory cytokines may be mediated by nitric oxide (Balligand et al., 1993; Ungureanu-Longrois et al., 1995). In this study, iNOS expression was suppressed by both IL-lra and vIL-10 administration, suggesting that nitric oxide is a terminal effector of myocardial damage in myocarditis. In conclusion, the present inventors have determined that (1) in vivo electoporation is an effective method for introducing cytokine cytokine genes in vivo, and (2) that the inhibitory cytokines IL-lra and vIL-10 We demonstrated that gene transfer reduced mortality in a mouse model of myocarditis, and (3) that the therapeutic effect was inhibition of immunosuppressive cytokines in the heart. These results strongly support the rationale for developing site-in-force therapy, and cytokine therapy with in vivo electoral poration opens up new therapeutic approaches not only for myocarditis but also for other vascular diseases. It is.

ADVANTAGE OF THE INVENTION According to the therapeutic agent of this invention, it becomes possible to produce | generate and release | release an immunosuppressive cytokine in a living body continuously, and it is not necessary to administer expensive cytokine protein frequently. In addition, by appropriately selecting or combining the types of immunosuppressive cytodynamics involved in the disease, high levels of immunosuppressive cytokines can be obtained in the blood, and one or more inflammatory or neoplastic diseases can be obtained. It can be treated efficiently.

The documents described in this specification are listed below.

References

AIHARA, H., and MIYAZAKI, J. (1998) .Gene transfer into muscle by electroporation in vivo.Nat.Biotechnol. 16,867

AREND, W.P., MALYAK, M., GUTHRIDGE, C.J., and GABAY, C. (1998). Interleukin-1 receptor antagonist: role in Diology. Annu. Rev. Immunol. 16, 24-55.

BALLIGAND, J.-L., UNGUREANU, D "KELLY, R., PIMENTAL, D., MICHEL, I "and SMITH, T. (1993) .Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes floolws exposure to activated macrophage-conditioned medium. J Clin Invest 91, 2314-2319.

BECKMAN. J.S. (1991) .The double-edged role of nitric oxide in brain function and superoxide -me diate d injury.J. Dev.Physiol. 15, 53-59.

BECKMAN, JS, BECKMAN, TW, CHEN, J., MARSHALL, PA, and FREEMAN, BA (1990) .Apparent hydroxyl radical production by perox nitrite * implications for endothelial injury from nitric oxide and superoxide.Proc.Nati.Acad. Sci. USA. 87, 1620-1624.

BOWLES, N.E., and TOWBIN, J.A. (1998). Molecular aspects of myocarditis. Curr. Opin. Cardiol. 13, 179-184.

BRESNIHAN, B., and CUNNANE, G. (1998) .Interleukin-1 receptor antagonist.Emerging therapies for rheumatoid arthritis 24, 615-628.

CASSATELLA, MA, MEDA, L., GASPERINI, S., CALZETTI, F "and BONOEA, S. (1994) .Interleukin 10 (IL-10) upregulates IL-I receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. J. Exp. Med. 179, 1695-1699.

CHOMAEAT, P., VANNIER, E..DECHANET, J., RISSOAN, MC, BANCHEREAU, J., DINARELLO, CA, and MIOSSEC, P. (1995) .Balance of IL-1 receptor antagonist / IL-lb in rheumatoid synovium and its regulation by IL-4 and IL-10. J. Immunol.

DE WAAL MALEFYT, R "HAANEN, J., SPITS, Η ·, RONCAROLO. M G., TE VELDE, A. FIGDOR, C, JOHNSON,, KASTELEIN, R., YSSEL. H., and DE VRIES, JE (1991) .Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via down regulation of class II major histocompatibility complex expression.J. Exp. Med .174, 915-929.

DEBRUYNE, LA, LI, K, CHAN, SY, L., Q., BISHOP, DK, and BROMBERG, JS (1998) .Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses.Gene.Ther. 5, 1079-1087.

DING, Y "QIN, L" KOTENKO. S.V., PESTKA, S., and BROMBERG, J.S. (2000) .A single amino acid determines the immunostimulatory activity of interleukin 10. J. Exp. Med. 191, 213-223.

DRAZAN. KE .. L., W., BULLINGTON, D .. and SHARED, A. (1996) .Viral IL-10 gene therapy inhibits TNF-a and ILrlb, not IL-6, in the newborn endotdxemic mouse. J Pediat. Surg. 31, 411-414.

EVERAERDT, B., BROUCKAERT, P., and FIERS, W. (1994) .Recombinant IL_1 receptor antagonist protects against TNF-induced lethality in mice. J. Immunol. 152, 5041-5049.

FERNANDES, J "TARDIF, G., MARTEL-PELLETIER. J .. LASCAU-COMAN, V., DUPUIS, M., MOLDOVAN, R. SHEPPARD, M., RISHNAN, BR, and PELLETIER, J.-R (1999 In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints' prevention of osteoarthritis progression.Am. J. Pathol. 154, 1159-1169.

GRANOWITZ, EV, SANTOS, A., POUTSIAKA, DD, CANNON, JG, WILMORE, DW "WOLFF, SM, and DINARELLO, CA (1991) .Production of interleukin-1 -receptor antagonist during experimental endotoxaemia.Lancet 338, 1423- 1424.

HENKE, PK, DEBRUNYE, LA. STRIETER, RM, BROMBERG, JS, PRINCE, M., KADELL, AM, SARKAR. M "LONDY, F" and WAKEFIELD, TW (2000) .Viral IL-10 gene transfer decreased inflammation and cell adhesion molecule expression in a rat model of venous thrombosis. J. Immunol. 164, 2131-2141.

HSU, DH, DE WAAL MALEFYT. R, FIORENTINO, DF, DANG, MN, VIEIRA, P., DE VRIES. J., SPITS, H., MOSMANN. TR, and MOORE, KW (1990) .Expression of IL-10 activity by Epstein-Barr virus protein BCRFl.Science 250, 830-832.

HUBER, S.A. (1997) .Autoimmunity in myocarditis: relevance of animal models. Clin. Immunol. Immunopathol. 83, 93-102.

HUBER, S.A., and LODGE, P.A. (1984) .Coxsackievirus B-3 myocarditis in Balb / c mice: Evidence for autoimmunity to myocyte antigens. Am. J. Pathol. 116, 21-29.

HUNG, G 丄., GALEA-LAURI, J "MUELLER, GM, GEORGESCU, HI" LARKIN, LA, SUCHANEK, PD, TINDAL, MH, ROBBINS, PD, and EVANS, CH (1994). Suppression of intra-articular responses to interleukin- 1 by transfer of the interleukin- 1 receptor antagonist gene to synovium. Gene Ther. 1, 64_69.

ISHIYAMA, S., HIROE, M., NISHIKAWA, T "ABE, S" SHIMOJO, T., ITO, H., OZASA, S "YAMAKAWA, K .. MATSUZAKI, M" MOHAMMED, MU, NAKAZAWA, H "KASAJIMA , T "and MARUMO, F. (1997) .Nitric oxide contributes to the progression of myocardial damage in experimental autoimmune myocarditis in rats. Circulation 95, 489-496.

IWASAKI, A "MATSUMORI, A..YAMADA, T" SHIOI, T "WANG, WZ, ONO,, NISHIO, R" OKADA. M "and SASAYAMA, S. (1999) .Pimobendan inhibits the production of proinflammatory cytokines and gene expression of inducible nitric oxide synthase in a murine model of viral myocarditis. J. Am. Coll. Cardiol. 33, 1400-1407.

LIN, M.T., HINTON, D.R., PARRA, B., STOHLMAN, S.A., and VAN DER VEEN, R.C. (1998) .The role of IL-10 in mouse hepatitis virus -induced demyelinating encephalomyelitis.Virology 245, 270-280.

MAKAROV, SS, OLSEN, JC, JOHNSTON, WN, ANDERLE, SK, BROWN, RR, BALDWIN, ASJ, HASKILL, JS, and SCHWAB, JH (1996) .Suppression of experimental arthritis by gene transfer oi interleukin 1 receptor Antagonist cDNA. Proc. Nati. Acad. Sci. USA 93, 402-406.

MATSUMORI, A. (1997) .Molecular and immune mechanisms in the pathogenesis of cardiomyopathy: role of viruses, cytokines, and nitric oxide.Jpn Circ J. 61, 275-291.

MIKAMI, S "KAWASHIMA, S., KANAZAWA, K" HIRATA, K "HOTTA, H., HAYASHI, Y" ITOH. H "and YOKOYAMA, M. (1997) .Lowdose Nome ga-nitro-L-ar inine methyl ester treatment improves survival rate and decreases myocardial injury in a murine model of viral myocarditis induced by coxsackievirus B3. Circ. Res. 81, 504-511.

NISHIO, R., MATSUMORI, A "SHIOI, T" ISHIDA, H., and SASAYAMA, S. (1999) .Treatment of experimental viral myocarditis with interleukin-10. Circulation 100, 1102-1108.

NITTA, Y., TASHIRO, F "TOKUI, M" SHIMADA, A "TAKEI, I" TABAYASHI,, and MIYAZAKI, J. (1998) .Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Hum. Gene. Ther. 9, 1701-1707.

NIWA, H., YAMAMURA,, and MIYAZAKI, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108,

193-200.

OTANI, K., NITA, I., MACAULAY, W "GEORGESCU, HL, ROBBINS, PD, and EVANS, CH (1996) .Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy. J. Immunol. 156, 3558-3562.

OVERBERGH, L., VALCKX. D., WAER, M "and MATHIEU, C. (1999). Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PGR. Cytokine 11, 305-312.

PINSKY, DJ, CAI, B., YANG, X., RODRIGUEZ, C, SCIACCA, RR, and CANNON, PJ (I 995) .The lethal effects of cytokine -induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor b. J. Clin. Invest. 95, 677-685.

SHIOI, T "MATSUMORI, A" and SASAYAMA, S. (1996) .Persistent expression of cytokine in the chronic stage of viral myocarditis in mice.Circulation 94,

2930-7.

SUZUKI, T "TAHARA, H., NARULA, S" MOORE, KW, ROBBINS, PD, and LOTZE, MT (1995) .Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces. local anergy to allogeneic and syngeneic tumors.J. Exp.Med. 182, 477-486.

UNGUREANU-LONGROIS, D "BALLIGAND, J.-L., SIMMONS, WW, OKADA, I" KOBZIK, L "LOWENSTEIN, CJ, KUNKEL, SL, MICHEL. T .. KELLY, RA, and SMITH, TW (1995) Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to b-adrenergic agonists. Circ. Res. 77, 494-502.

VIEIRA, P., DE WAAL-MALEFYT. R., DANG, MN "JOHNSON, KE, KASTELEIN, R., FIORENTINO, DF, DE VRIES, JE, RONCAROLO, MG, MOSMANN, TF, and MOORE, KW (1991) Isolation and expression of human cytokine synthesis inhibitory factor (CSIF / IL-10) cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFl. Proc. Natl. Acad. Sci. USA 88, 1172-1176.

VITADELLO, M., SCHIAFFINO, M.V., PICAED, A., SCARPA, M., and SCHIAFFINO, 5. (1994). Gene transfer in regenerating muscle. Hum. Gene. Ther. 5, 11-18.

WANG, WZ, MATSUMORI, A., YAMADA, T., SHIOI, T "OKADA, I, MATSUI, S., SATO, Y" SUZUKI, H., SHIOTA, K, and SASAYAMA, S. (1997). Beneficial effects of amlodipine in a murine model of congestive heart failure induced by viral myocarditis. Circulation 95, 245-251.

WELLS, DJ (1993) .Improved gene transfer by direct plasmid injection. associated with regeneration in mouse skeletal muscle.FEBS Lett.332, 179-182.

WOODLEY. S 丄., MCMILLAN, M., SHELBY, J., LYNCH, DH, ROBERTS, LK, ENSLEY, RD, and BARRY, WH (1991) Myocyte injury and contraction abnormalities produced by cytotoxic T lymphocytes. Circulation 83, 1410 -1418. YAMADA, T. MATSUMORI, A., and SASAYAMA, S. (1994) .Therapeutic effect of anti-tumor necrosis factor-a antibody on the murine model of viral myocarditis induced by encephalomyocarditis virus. Circulation 89, 846- 851. YANG, GY, LIU, XH, KADOYA, C, ZHAO, YJ "MAO, Y" DAVIDSON, B 丄., And BETZ, AL (1998) .Attenuation of ischemic inflammatory response in mouse brain using an adenoviral vector to induce. overexpression if interleukin-1 receptor antagonist. J. Cereb. Blood. Flow. Metab. 18, 840-847. YANG, GY, ZHAO, YJ, DAVIDSON, B 丄., and BETZ, AL (1997). Overexpression oi interleukin- 1 receptor antagonist in the mouse brain reduce ischemic brain injury.Brain Res. 751, 181-188.

YOKOYAMA. T "VACA, L" ROSSEN, RD, DURANTE, W "HAZARIKA, P., and MANN.DL (1993) .Cellular basis for the negative inotropic effects of tumor necrosis factor-a in the adult mammalian heart. Clin. Invest 92, 2303-2312.

Claims

The scope of the claims

1. A therapeutic agent for inflammation and neoplastic disease, comprising as an active ingredient an expression vector containing an immunosuppressive cytokine gene and / or a soluble cytokine receptor gene in an expressible state.

2. The therapeutic agent according to claim 1, wherein the immunosuppressive cytokine and / or soluble cytokine receptor gene is at least one selected from the group consisting of IL-lra, vIL-10 and soluble c_kit.

3. The therapeutic agent according to claim 1, wherein the inflammation / neoplastic disease is a cardiovascular disease.

4. The therapeutic agent according to claim 3, wherein the inflammation / neoplastic disease is myocarditis.

5. Inflammation / tumor including administering a therapeutically effective amount of an expression vector containing an immunosuppressive cytokine gene and / or a soluble cytokine receptor gene in a state capable of expressing it to a patient suffering from inflammation / neoplastic disease How to treat sexual illness.

6. The treatment method according to claim 5, wherein an electric pulse is applied so as to sandwich the administration site after administration of the therapeutically effective amount of the expression vector.

7. The treatment method according to claim 5, wherein the immunosuppressive cytokine and the Z or soluble cytokine receptor gene are at least one selected from the group consisting of IL-lra, vIL-10 and soluble c_kit.

8. The treatment method according to claim 5, wherein the inflammation / neoplastic disease is a cardiovascular disease.

9. The treatment method according to claim 8, wherein the inflammation / neoplastic disease is myocarditis.