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WO2002066069A1 - Remedes contre l'inflammation/les maladies tumorales - Google Patents

Remedes contre l'inflammation/les maladies tumorales Download PDF

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WO2002066069A1
WO2002066069A1 PCT/JP2002/001445 JP0201445W WO02066069A1 WO 2002066069 A1 WO2002066069 A1 WO 2002066069A1 JP 0201445 W JP0201445 W JP 0201445W WO 02066069 A1 WO02066069 A1 WO 02066069A1
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lra
vil
inflammation
gene
cytokine
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Akira Matsumori
Jun-Ichi Miyazaki
Atsushi Nakano
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Kansai Technology Licensing Organization Co Ltd
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Kansai Technology Licensing Organization Co Ltd
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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a therapeutic agent for inflammation / neoplastic disease.
  • 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.
  • DCM dilated cardiomyopathy
  • EMC Encephalomyocarditis
  • IL-1 inulin-leukin-1
  • TNF tumor necrosis factor
  • IFN interferon
  • 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.
  • Japanese Patent Application Laid-Open No. 2000-47115 describes a plasmid into which a cytokine gene has been incorporated.
  • 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.
  • 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.
  • Figures 2A-2C show the survival curves after EMCV inoculation.
  • Figure 3A shows histological findings.
  • 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.
  • Significant improvement p ⁇ 0.05).
  • FIG. 4 shows cytokine expression in the heart.
  • the present invention provides the following therapeutic agents for inflammatory and neoplastic diseases.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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 ( ⁇ , ⁇ , r)> TNF-H, TGF- ⁇ , GM-CSF, EGF, FGF, RANTES, I-309 / TCA-3, rIP-10, ⁇ - ⁇ ⁇ , 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.
  • soluble cytokine receptors are included for the soluble receptors of these cytokins.
  • soluble c-kit is exemplified for SCF
  • soluble TNF- ⁇ 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.
  • a cytokine 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.
  • 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.
  • 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
  • cardiovascular diseases such as myocarditis, cerebral myocarditis, dilated cardiomyopathy
  • 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.
  • interferon chronic myelogenous leukemia, multiple myeloma, renal cancer, malignant mela
  • 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.
  • 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.
  • pulse frequency is 1 to 10 pulses Z seconds, and number of times is 1 to 10 times.
  • 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.
  • 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.
  • Electric pulse generator Electro Square Porator T820M; BTX, San Diego, CA
  • 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.
  • 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.
  • the substrate o-phenylenediamine
  • the absorbance at 490 nm was measured with a microplate reader.
  • the linear range of the ELISA system was 30 ⁇ 2000pg / ml. This ELISA system does not detect mouse IL-10.
  • 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.
  • IL-1 jS forward primer 5'-CAACCAACAAGTGATATTCTCCATG-3 '
  • IL-1 i8 probe 5'-CTGTGTAATGAAAGACGGCACACCCACC-3 '
  • TNF-back primer 5'-TGGGAGTAGACAAGGTACAACCC-3
  • TNF-probe 5'-CACGTCGTAGCAAACCACCAAGTGGA-3 '
  • IFN-forward primer 5'-TCAAGTGGCATAGATGTGGAAGAA-3 '
  • IFN-probe 5'-TCACCATCCTTTTGCCAGTTCCTCCAG-3 '
  • IL-6 forward primer 5'-CAGAATTGCCATCGTACAACTCTTTTCTCA-3 '
  • iNOS forward primer 5'-CAGCTGGGCTGTACAAACCTT3 '
  • iNOS ⁇ % liquid primer, 5'-CATTGGAAGTGAAGCGTTTCG-3 'iNOS probe, 5'-CGGGCAGCCTGTGAGACCTTTGA-3'
  • GAPDH forward primer 5'-TTCACCACCATGGAGAAGGC-3 ' GAPDH
  • 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.
  • 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)
  • vIL-10 expression was confirmed by transient transfection of pCAGGS-vIL-10 into BMT-10 cells
  • 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.
  • 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).
  • 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. ⁇
  • cytokinines such as cytoin, especially IL-1] 3 and TNF-spin
  • proinflammatory cytokines such as cytoin, especially IL-1] 3 and TNF-spin
  • cytoin especially IL-1
  • TNF-spin proinflammatory cytokines
  • the heart has a proinflammatory site such as IL-1) 3 and TNF- ⁇ , 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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- ⁇ has a direct cytotoxic and negative inotropic effect on cultured cardiomyocytes and also activates cytotoxic ⁇ 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- ⁇ 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).
  • 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- ⁇ 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • cytokine expression is the induction of iNOS.
  • Monoacid nitrogen (NO) is produced during the subacute phase in response to cytokines induced by EMCV.
  • 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.
  • the excess of nitric oxide produced by iNOS also has a detrimental effect on myocardial tissue in viral and autoimmune myocarditis.
  • nitric oxide The direct negative inotropic effects of inflammatory cytokines may be mediated by nitric oxide (Balligand et al., 1993; Ungureanu-Longrois et al., 1995).
  • 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 vivo electoporation is an effective method for introducing cytokine cytokine genes in vivo
  • 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.
  • the therapeutic agent of this invention it becomes possible to produce
  • 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.
  • BECKMAN JS
  • BECKMAN TW
  • CHEN J.
  • MARSHALL PA
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  • Viral IL-10 gene transfer decreased inflammation and cell adhesion molecule expression in a rat model of venous thrombosis. J. Immunol. 164, 2131-2141.
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Abstract

La présente invention concerne des remèdes, destinés au traitement de maladies inflammatoires/tumorales, qui contiennent en tant que principe actif, un vecteur d'expression contenant un gène de cytokine immunosuppresseur et/ou un gène récepteur de cytokine soluble sous une forme permettant l'expression dudit vecteur ; et une méthode de traitement des maladies inflammatoires/tumorales qui consiste à administrer une quantité efficace d'un point de vue thérapeutique d'un vecteur d'expression contenant un gène de cytokine immunosuppresseur et/ou un gène récepteur de cytokine soluble sous une forme permettant l'expression dudit vecteur chez des patients souffrant d'une maladie inflammatoire/tumorale.
PCT/JP2002/001445 2001-02-20 2002-02-20 Remedes contre l'inflammation/les maladies tumorales Ceased WO2002066069A1 (fr)

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Cited By (2)

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JP2007131548A (ja) * 2005-11-08 2007-05-31 Hirosaki Univ プロテオグリカンの新規医薬用途
CN114177295A (zh) * 2021-12-16 2022-03-15 上海市第五人民医院 白细胞介素1受体拮抗剂治疗非酒精性脂肪性肝病的应用

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CN111514237B (zh) * 2020-05-29 2021-09-07 中国人民解放军陆军第八十二集团军医院 一种治疗病毒性心肌炎的中药组合物及其制备方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007131548A (ja) * 2005-11-08 2007-05-31 Hirosaki Univ プロテオグリカンの新規医薬用途
CN114177295A (zh) * 2021-12-16 2022-03-15 上海市第五人民医院 白细胞介素1受体拮抗剂治疗非酒精性脂肪性肝病的应用
CN114177295B (zh) * 2021-12-16 2023-09-19 上海市第五人民医院 白细胞介素1受体拮抗剂治疗非酒精性脂肪性肝病的应用

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