JP2006298780A - Conjugate of hgf gene and macroaggregated albumin-polyethyleneimine (maa-pei) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a conjugate composed of a DNA encoding HGF and bonded to MAA-PEI and provide an injection for the treatment of pulmonary diseases containing the conjugate. <P>SOLUTION: The conjugate is produced by bonding a DNA encoding HGF with a macroaggregated albumin-polyethyleneimine (MAA-PEI). The injection for the treatment of pulmonary diseases contains the conjugate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  In the present invention, DNA encoding hepatocyte growth factor (hereinafter abbreviated as HGF) and coarsely aggregated albumin-polyethyleneimine (hereinafter, coarsely aggregated albumin-polyethyleneimine is also referred to as MAA-PEI) are bound. And a pulmonary disease treatment injection containing the same.

  Pulmonary injury diseases include, for example, pneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, etc., particularly injuries caused by fibrosis of the lungs (eg pneumoconiosis, etc.), patients with reduced immunity and elderly people Aspiration pneumonia and the like may have a poor prognosis, and establishment of a therapeutic agent is strongly desired.

  When the lung is injured, HGF is newly produced by the lung itself, HGF promotes DNA synthesis in the lung epithelial cells via the lung HGF receptor, and cell proliferation in the lung tissue is increased. It is known to promote injury repair. Therefore, prophylactic or therapeutic agents for lung diseases containing HGF or HGF gene have been proposed. (See Patent Document 1). However, it is reported that most of the administered HGF is taken up by the liver and is hardly detected in the lung, or even if detected, it is a small amount of 10% or less of the administered HGF (see Non-Patent Document 1). .) Therefore, in order to use the lung as a target organ for HGF, it is necessary to administer a large amount of HGF.

  In addition, adenoviral vectors have been mainly used as experimental vectors for experimental trials of HGF gene therapy for pulmonary fibrosis. However, adenovirus vectors are known to cause various inflammations, and there are many problems to be solved for clinical application to pulmonary fibrosis, which is a chronic inflammatory disease. It is.

Formulations using polyethyleneimine and genetic macromolecules (eg, DNA, RNA, catalytically active nucleic acids, etc.) resulted in high levels of lung transfection and were shown to be stable during nebulization ( (See Patent Document 2). The preparation is capable of treating cystic fibrosis, asthma, lung cancer, esophageal cancer, colon cancer, leukemia, breast cancer, sarcoma, melanoma, etc. by being administered to the lung by oral inhalation and nasal inhalation. Is described.
However, no description on HGF is found in any of the above documents.
Japanese Patent Laid-Open No. 06-172207 JP-T-2002-541125 MOTHIRO KATO and 4 others, The Journal of Pharmacology and Experimental Therapies, 1999, Vol. 290, Vol. 373-379

  An object of this invention is to provide the composite_body | complex which DNA which codes HGF, and MAA-PEI couple | bonded, and the injection for treatment of the lung disease containing them.

  The present inventors note that coarsely aggregated albumin cannot be passed through the lung capillaries, but is temporarily trapped and can selectively accumulate in the lung as a transient microembolus. And a complex formed by binding HGF-encoding DNA and MAA-PEI selectively accumulates in the lung like coarse aggregated albumin, and expresses DNA at the accumulation site to produce HGF. It was. The inventors have further studied and completed the present invention.

That is, the present invention
(1) a complex formed by binding DNA encoding HGF and coarsely aggregated albumin-polyethyleneimine (MAA-PEI);
(2) The complex according to (1), wherein the DNA is any of the following:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1 in the sequence listing; or (b) DNA comprising a base sequence complementary to the DNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing and stringent conditions DNA comprising a nucleotide sequence that hybridizes underneath and encodes a protein having HGF activity,
(3) The complex according to (1) above, wherein the DNA is any of the following:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing; or (b) DNA comprising a base sequence complementary to the DNA having the base sequence set forth in SEQ ID NO: 2 in the sequence listing and stringent conditions DNA comprising a nucleotide sequence that hybridizes underneath and encodes a protein having HGF activity,
(4) The complex according to any one of (1) to (3), wherein DNA is inserted into an expression vector,
(5) The complex according to (4) above, wherein the expression vector is a plasmid,
(6) The composite according to any one of (1) to (5) above, wherein the particle diameter of the composite is 10 μm or more and less than 100 μm,
(7) An injection for treating lung disease, comprising the complex according to (6),
(8) The pulmonary disease is at least one disease selected from pulmonary fibrosis, interstitial pneumonia, emphysema, pulmonary hypertension, pneumonia, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis and swallowing pneumonia The pulmonary disease treatment injection according to (7),
About.

  The complex of the invention is administered intravenously and then thoroughly mixed with the blood flow in the right heart and sent to the pulmonary capillaries. The complex cannot be passed through the capillaries of the lungs, but is temporarily trapped and becomes a transient microemboli, and can selectively accumulate in the lungs. In the accumulation site, DNA combined with MAA-PEI or DNA inserted into an expression plasmid can be expressed and produce HGF, so that HGF can be efficiently used in the lung. That is, only 10% or less could be distributed in the lung even when HGF was injected conventionally, but when the injection containing the complex of the present invention was administered intravenously or the like, the complex of the present invention was selectively applied to the lung. Therefore, HGF can be distributed locally and in high concentration in the lung.

  In addition, since the complex of the present invention selectively accumulates in the lung and produces HGF protein at the accumulation site, the dose of the HGF expression vector can be reduced to about 1/10 compared with the conventional one. Furthermore, since the complex of the present invention selectively accumulates in the lung, the influence on other organs by administration of the complex of the present invention can be suppressed to a minimum.

  The present invention is a complex of DNA encoding HGF and coarsely aggregated albumin-polyethyleneimine (MAA-PEI).

The DNA encoding HGF used in the present invention refers to DNA capable of expressing HGF.
HGF (SEQ ID NO: 3 in the sequence listing) was originally discovered as a growth promoting factor for hepatocytes, and its gene was cloned in 1989 (Miyazawa,
K. et al, BBRC. 163, 967-973 (1989); Nakamura. T, et al, Nature 342, 440-443 (1989)). Examples of the DNA used in the present invention include, for example, Japanese Patent No. 2777678, Biochem. Biophys. Res. Commun. 1989, 163, p. 967, Biochem. Biophys. Res. Commun. 1990, 172, p. Examples include those obtained by incorporating the HGF DNA described in 321 etc. into an appropriate expression vector (non-viral vector, viral vector). Here, as the base sequence of DNA encoding HGF, those registered in databases such as Genebank (registration number D90334) can be preferably used in addition to those described in the above-mentioned document. Therefore, HGF DNA can be cloned by using an appropriate DNA portion as a PCR primer based on these sequence information and performing, for example, RT-PCR reaction on mRNA such as liver. These cloning methods include, for example, a molecular cloning method (Molecular Cloning, A laboratory Manual, Third Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001, and the like, Molecular Cloning and Third Edition). Accordingly, it can be easily performed by those skilled in the art.
The expression vector used in the complex of the present invention is preferably a non-infectious or non-inflammatory vector. By using a non-infectious or non-inflammatory vector, there is no fear of infection or inflammation due to a foreign virus, which is a concern in vectors derived from viruses such as adenovirus.

  Furthermore, the DNA used in the present invention is not limited to the above. For example, a protein (such as a 5-amino acid-deficient HGF (SEQ ID NO: 4 in the sequence listing)) whose expressed protein has a biological function equivalent to HGF ( For example, DNA encoding homologues (homologues, mutants and derivatives, etc.) is included. The DNA encoding the homologue, variant or derivative is one or more (preferably several) to the DNA that hybridizes with the DNA under stringent conditions or the amino acid sequence of the protein encoded by the DNA. DNA encoding a protein having an action as HGF among DNA encoding a protein consisting of an amino acid sequence in which amino acids are substituted, deleted and / or added.

  The DNA can be easily obtained by, for example, a normal hybridization method or a PCR method, and specifically can be performed with reference to the molecular cloning method or the like.

  In addition, in this specification, DNA is used in a meaning including not only double-stranded DNA but also single-stranded DNA such as sense strand and antisense strand constituting the DNA and tissue-derived RNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, double-stranded DNA containing human genomic DNA and single-stranded DNA containing DNA (positive strand) and single-stranded DNA having a sequence complementary to the positive strand. (Complementary strand) and any of these fragments are included.

  Specifically, as DNA encoding HGF, for example, (a) DNA having a base sequence represented by SEQ ID NO: 1 or 2, or (b) having a base sequence represented by SEQ ID NO: 1 or 2 Preferred examples include DNA that hybridizes with DNA under stringent conditions and that encodes a protein having substantially the same quality of activity as HGF. The DNA hybridized with the DNA having the base sequence represented by SEQ ID NO: 1 or 2 is, for example, a colony hybridization method, plaque hybridization method or Southern blot hybridization method using the above DNA as a probe. Specifically, it means DNA obtained by using, specifically, at about 65 ° C. in the presence of about 0.7 to 1.0 M sodium chloride using a filter on which colony or plaque-derived DNA is immobilized. After hybridization, an SSC solution having a concentration of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate) is used and about 65 ° C. After washing the filter under the conditions of autoradiography Means DNA that can be identified.

  Specifically, the DNA that hybridizes with the DNA having the base sequence represented by SEQ ID NO: 1 or 2 is about 70% or more, preferably about 80%, with the base sequence represented by SEQ ID NO: 1 or 2. As mentioned above, DNA having a base sequence having a homology of about 90% or more, most preferably about 95% or more is preferable. Hybridization can be performed according to a known method such as the molecular cloning method described above. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

  Moreover, it can also be cloned by chemical synthesis using a conventionally known method from the known nucleotide sequence information of HGF. Examples of the chemical synthesis method include a method of chemically synthesizing with a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin Elmer Co., Ltd.) using the phosphoramidite method.

The substitution of the DNA base sequence is performed using PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the gapped duplex method, the Kunkel method, etc. It can carry out according to the well-known method of these, or the method according to them. The cloned DNA encoding the HGF of the present invention can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  The DNA encoding HGF used in the present invention may be modified in order to increase its stability in cells, and to reduce its toxicity if it is toxic. Such modifications include, for example, J. Org. Kawakami et al. Pharm Tech Japan, Vol. 8, p247 (1992); Vol. 8, p395 (1992); T.A. Crooke et al. eds. , Antisense Research and Applications, CRC Press (1993), etc. are used. Further, the DNA or the like encoding HGF used in the present invention may be added with a substance other than the base. Examples of such other substances include sugars; acids or bases; polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton; or increase interaction with cell membranes or increase nucleic acid uptake. Hydrophobic substances such as caking lipids (for example, phospholipids, cholesterol, etc.) can be mentioned. Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). The other substance can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. The DNA or the like of the present invention may have its ends chemically modified. Examples of the terminal modifying group include a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid, and a group for preventing degradation by nucleases such as exonuclease and RNase. Examples of such a capping group include a hydroxyl-protecting group known in the art derived from glycols such as polyethylene glycol and tetraethylene glycol.

The DNA encoding HGF used in the present invention may be inserted into an expression vector. As the expression vector, an expression vector capable of expressing HGF is preferable.
An expression vector into which DNA has been inserted can be constructed, for example, by linking a DNA fragment having a base sequence encoding HGF to an appropriate expression vector. The expression vector can be constructed according to a known technique [Molecular Cloning, Cold Spring Harbor Laboratory, A laboratory manual (1989)].

  The expression vector is preferably a plasmid, for example, a plasmid derived from E. coli (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), or yeast Plasmid (eg, pSH19, pSH15), pGL3 (Promega), pCAGGS [Gene, 108, 193-200 (1991)], pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stragene), pcDL- SRα296 [Molecular and Cellular Biology; 8, 466-472 (1988)), pEF-BOS [Nucleic Acid Research; 18, 5322 (1990)], Special Table 1 -511009 plasmid etc. publication can be mentioned.

  In order to enhance the expression of HGF at the administration site, the expression vector preferably contains a promoter, an enhancer and the like.

  Coarse aggregated albumin (MAA) used in the present invention has a refining property, and after being retained in the lung for a necessary time, it is finely excreted and used for lung scintigraphy and the like. Etc. can be preferably used. MAA is preferably a mammal, particularly human serum albumin. MAA can be prepared by the method of Colombetti et al. (Internetl J Nuclear Med Biol. 2: 180-184 (1975)).

Polyethyleneimine (PEI), which binds coarse aggregated albumin, is a polymer with a high cation density. PEI can be produced by polymerizing ethyleneimine in the presence of an acid catalyst. Examples of the production method include a method of polymerizing ethyleneimine in the presence of a polymetallooxoacid salt or 2,6-dipivaloyloxybenzoic acid (Japanese Patent Laid-Open No. 11-158271).
PEI having a molecular weight of about 20000 to 70000 is preferred.

  The polymer PEI having a high cation density binds to MAA, so that the conjugate (MAA-PEI) can bind to a negatively charged molecule. MAA-PEI can be prepared by the method described in US Pat. No. 6,821,955. Since DNA is usually negatively charged, it can be bound to MAA-PEI.

Also in the present invention, a complex formed by binding HGF-encoding DNA and MAA-PEI (hereinafter referred to as the complex of the present invention) has a positively charged PEI surface and DNA encoding HGF. Alternatively, since the vector is negatively charged, it is bound by ionic bond.
The complex of the present invention is prepared, for example, according to the method described in US Pat. No. 6,821,955, for example, DNA encoding HGF or an expression vector containing the DNA and MAA-PEI, for example, physiological saline or PBS. And then slowly incubating the solution at about 0-40 ° C., preferably at room temperature, for about 10-60 minutes, preferably 15-30 minutes.
The content of DNA encoding HGF in the complex of the present invention is not particularly limited, but is usually about 0.001 to 0.5 (w / w%), preferably about 0.005 to 0.2 (w / W%), about 0.01 to 0.16 (w / w%).

The preparation containing the complex of the present invention can take various preparation forms, but an injection is preferable. An injection can be prepared by a conventional method. For example, it can be prepared by suspending the complex of the present invention in an appropriate base and then filling it in a sterile container. The base is not particularly limited as long as it is a base usually used for injections, and is a salt solution of distilled water, sodium chloride, or a mixture of sodium chloride and other inorganic salts, mannitol, lactose, dextran, glucose. Etc., an amino acid solution such as glycine and arginine, an organic acid solution or a mixed solution of a salt solution and a glucose solution, and the like. In addition, according to conventional methods, these bases are supplemented with osmotic pressure adjusting agents, pH adjusting agents, vegetable oils such as sesame oil and soybean oil, or auxiliary agents such as surfactants such as lecithin or nonionic surfactants. An injection may be prepared as a suspension or dispersion.
A stabilizer can be added to the injection. Examples of the stabilizer include albumin, globulin, gelatin sorbitol, ethylene glycol, and propylene glycol. Furthermore, the injection of the present invention may contain additives necessary for formulation, for example, a reducing agent such as stannous chloride, an antioxidant such as sodium edetate, a soothing agent such as benzyl alcohol, and the like. .
These injections can be made into preparations for use at the time of use by operations such as powdering and freeze-drying.

  The content of the complex of the present invention in the injection is preferably about 10 to 1 million pieces / mL, preferably about 300 to 600,000 pieces / mL.

  The particle size of the complex of the present invention in the injection must be a particle size that can be used as an injection, and preferably does not contain particles of 100 μm or more. The particle size is about 10 or more and less than 100 μm, preferably about 10 to 60 μm. However, the particle diameter is not particularly limited when used in a dosage form other than an injection.

  An injection containing the complex of the present invention can be administered by several administration routes, for example, intravenous, arterial, subcutaneous, intramuscular, etc., with intravenous administration being most preferred. The dose is appropriately adjusted depending on the patient's symptoms, age, weight, etc. Usually, the complex of the present invention is about 10 to 1 million per day, preferably about 30 to 60, per patient (60 kg). 10,000 pieces / day. The administration is preferably performed at intervals of about 1 to 5 days, preferably about 3 to 4 days. When the complex of the present invention is precipitated in a solution, it is preferable to administer the precipitate after stirring.

  The administered complex of the present invention selectively accumulates in the lung, and DNA is expressed at the accumulation site to produce HGF. Therefore, mammals including humans (for example, cows, horses, pigs, Sheep, dogs, cats, etc.) can be used for the treatment of pulmonary diseases for which HGF is effective. Examples of the pulmonary disease include pulmonary fibrosis, interstitial pneumonia, emphysema, pulmonary hypertension, pneumonia, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis or swallowing pneumonia pulmonary fibrosis. Among these lung diseases, it is particularly effective for treating pulmonary fibrosis.

  Examples are given below for illustrating the present invention in more detail, but the present invention is not limited thereto. In the examples, PBS represents phosphate buffered saline, and BLM represents bleomycin. The data shows the mean value ± standard deviation. In order to statistically compare the data of two different groups, Mann-Whitney U test was used, and a risk rate of 5% or less was determined to be significant.

[Preparation of MAA-PEI-HGF expression plasmid]
1. Preparation of HGF expression plasmid Full-length cDNA (2.2 kb) of human HGF was inserted into the Xho I site of pCAGGS (Gene 108: 193-200, 1991) to express the human HGF gene under the control of the chicken β-actin promoter. A plasmid (hereinafter also referred to as HGF / pCAGGS) was prepared.

2. Preparation of MAA-PEI 0.25 mg (0.8 μmol) in a solution (pH 8.5) in which 50 mg (0.72 μmol) of human serum albumin (hereinafter abbreviated as HSA) was mixed with 1 mL of 0.1 M sodium bicarbonate. ) DMSO solution of N-succinimidyl 3- (pyridyldithio) propionate (Pierce, Rockford, IL) was added and reacted (hereinafter also referred to as HSA solution). A solution in which 18 mg (0.72 μmol) of PEI (molecular weight 25,000; Aldrich, Milwaukee, WI) was dissolved in 1 mL of purified water was adjusted to pH 8.5 with 1 M hydrochloric acid (hereinafter also referred to as PEI solution). To the PEI solution, 10 μL of a DMSO solution of 0.25 mg (0.8 μmol) N-succinimidyl 3- (pyridyldithio) propionate was added and stirred for several hours. HSA and PEI solutions were added separately to a NAP-10 column (Pharmacia, Piscataway, NJ) equilibrated with PBS. To the PEI eluate, 15 mg of Redactacryl (Calbiochem, San Diego, CA) was added and reduction treatment was performed for several hours. Next, the PEI eluate was added to the HSA eluate with slow stirring so that the molar ratio of PEI to HSA was 1: 2. Stirring was continued overnight, the solution was adjusted to 5 mL with PBS, and the pH was adjusted to 5.5-6.0 with 1M hydrochloric acid. The prepared solution was agglomerated at 85 ° C. with vigorous stirring. Aggregated MAA-PEI was gently centrifuged and the precipitate (MAA-PEI) was resuspended in PBS and washed twice by centrifugation. The washed MAA-PEI was resuspended in PBS to a final volume of 4 mL.

3. Preparation of MAA-PEI-HGF / pCAGGS The MAA-PEI solution prepared in 2 above was diluted and suspended to 10% by mass with physiological saline. While slowly stirring the diluted suspension solution (175 μL), 25 μL of HGF / pCAGGS (200 ng / μL PBS) prepared in 1 above was added dropwise. The MAA-PEI suspension solution to which the plasmid was added was incubated at room temperature for 20 minutes to obtain MAA-PEI-HGF / pCAGGS.

[Reference example]
Preparation of adenoviral vector In order to compare the pro-inflammatory effect of human HGF expression plasmid with that of adenoviral vector, non-proliferating adenoviral vector not incorporating foreign insertion gene (hereinafter also referred to as AdCMV-Null) Prepared.)

[Test example]
1. Confirmation test of MAA intrapulmonary distribution
60 μL of ^99m Tc-MAA (Lungscinchi Tc-99m, Nippon Mediphysics Co., Ltd.) was injected into the vein from the tail vein of the mouse and imaged over time with a gamma camera equipped with a pinhole collimator.

The results are shown in FIG. A gamma camera image showed that ^99m Tc-MAA was accumulated in the lung region of the mouse (FIG. 1A). Moreover, the radiation dose by ^99m Tc in each organ 1 hour and 6 hours after ^99m Tc-MAA administration is shown in FIG. 1B. Even after 6 hours of ^99m Tc-MAA, the radiation dose of ^99m Tc in the lung was high.

2. Transfection ability in the lung with MAA-PEI-HGF / pCAGGS (1) Relationship between plasmid dosage and HGF expression level HGF / pCAGGS (0, 0.5, 1, 5 μg) and MAA-PEI-HGF / pCAGGS ( 0, 0.5, 1, 5 μg) were injected into each vein from the tail vein of mice (5 mice in each group), and 2 hours later, lung HGF was measured with human HGF ELISA kit (R & D) to produce HGF. The amount.

  The results are shown in FIG. The amount of HGF produced in the lung was highest in the 1 μg administration group in the MAA-PEI-HGF / pCAGGS administration group (black column), and significantly higher (p <0.05) than in the HGF / pCAGGS administration group. . Even when 5 μg was administered, the HGF expression level was higher when MAA-PEI-HGF / pCAGGS was administered than in the HGF / pCAGGS administration group.

(2) Transfection ability HGF / pCAGGS (10 μg) and MAA-PEI-HGF / pCAGGS (1 μg) were injected into each vein from the tail vein of mice (5 mice in each group), and the amount of HGF expression in the lungs over time Was measured with a human HGF ELISA kit (R & D). In addition, MAA-PEI-HGF / pCAGGS (1 μg) was injected intravenously from the tail vein of mice (5 mice in each group), and 3 days and 6 days later, MAA-PEI-HGF / pCAGGS (1 μg) was intravenously injected. The amount of HGF expression in the lung was measured in the same manner over time.

  The results are shown in FIG. The expression of HGF in the mouse lung after administration of HGF / pCAGGS (10 μg) gradually increased from 12 hours to 24 hours, reached the highest level in 2 days, and disappeared in 5 days (FIG. 3; − □ −). In contrast, the duration of HGF expression in the MAA-PEI-HGF / pCAGGS (1 μg) administration group persisted for 14 days or longer (FIG. 3; − ■ −). In the group to which MAA-PEI-HGF / pCAGGS (1 μg) was further administered after 3 days and 6 days, the expression level of HGF in the lung increased until 9 days after the administration and then decreased (FIG. 3; solid triangles).

3. Organ migration test after administration of MAA-PEI-HGF / pCAGGS HGF / pCAGGS (10 μg) and MAA-PEI-HGF / pCAGGS (1 μg) were injected into each vein from mice (5 mice in each group), Three days later, blood was collected from the axillary artery under ether anesthesia, and the lungs, liver, spleen, kidney and heart were removed, and the HGF expression levels in serum and each organ were measured with a human HGF ELISA kit (R & D). .

  The results are shown in FIG. The expression level of HGF in the liver of the MAA-PEI-HGF / pCAGGS administration group was significantly lower than that of the HGF / pCAGGS administration group. In addition, almost no expression of HGF was observed in the spleen, kidney and heart of the MAA-PEI-HGF / pCAGGS administration group. In the HGF / pCAGGS administration group and MAA-PEI-HGF / pCAGGS administration group, no expression of HGF was observed in the serum.

4). Plasmid Intravascular Safety Confirmation Test Adenoviral vector (AdCMV-Null) 1 × 10 ¹⁰ particles, HGF / pCAGGS (10 μg) or MAA-PEI-HGF / pCAGGS (1 μg) from mice (7 mice in each group) from the tail vein After intravenous injection, serum concentrations of inflammatory cytokines [TNF-α (tumor necrosis factor) and interleukin-6 (IL-6)] after 5 hours were measured by ELISA kit (R & D). As a control, PBS was similarly administered intravenously.
The results are shown in FIG. Serum TNF-α and IL-6 were markedly elevated 5 hours after adenoviral vector administration. In contrast, neither serum TNF-α nor IL-6 in the HGF / pCAGGS or MAA-PEI-HGF / pCAGGS administration group showed any effect and statistical difference with PBS administration. This result shows the safety of MAA-PEI-HGF / pCAGGS in blood vessels.

5. Anti-inflammatory effect and anti-fibrotic effect by administration of HGF expression plasmid (1) Treatment of animals C57BL / 6 mice (female, 17-20 g) were anesthetized with ketamine hydrochloride (Sankyo Co., Ltd.) and xylazine hydrochloride (Bayer Co., Ltd.). 90 μg of bleomycin hydrochloride (Nippon Kayaku Co., Ltd.) was administered intranasally. HGF / pCAGGS (10 μg / 200 μL) or MAA-PEI-HGF / pCAGGS (1 μg / 200 μL) was prepared in physiological saline and intravenously administered from the tail vein on the first, fourth and seventh days after bleomycin administration. Mice were bled from the axillary artery under ether anesthesia and killed by blood removal. PBS or pCAGGS was administered as a control.
(2) Anti-inflammatory effect The lung was excised 7 days after bleomycin administration, and inflammatory cytokines (TNF-α and IL-6) in lung tissue were measured by ELISA kit (R & D).
The results are shown in FIG. TNF-α and IL-6 in the lung tissue of the MAA-PEI-HGF / pCAGGS administration group on day 7 after bleomycin administration were significantly lower than those in the PBS administration group.
(3) Antifibrotic effect On the 21st day after administration of bleomycin, the lung was removed and the amount of hydroxyproline in the lung tissue was measured. The measurement of the amount of hydroxyproline was as follows. That is, the formalin-fixed right lung was immersed in acetone for 6 hours and then degreased with ether. Fully dried lungs were ground and hydrolyzed for 16 hours at 115 ° C. with 6N hydrochloric acid. Hydroxyproline content was measured by high performance liquid chromatography. In addition, a pathological section was prepared according to a conventional method, and histological observation was performed.
The results are shown in FIG. In the MAA-PEI-HGF / pCAGGS administration group, suppression of hydroxyproline production by bleomycin was observed.
Moreover, also in the histopathological observation, fibrous lesions due to bleomycin were decreased in the MAA-PEI-HGF / pCAGGS administration group.
(4) Effect on apoptosis The lungs were excised on the 3rd and 7th days after bleomycin administration, and paraffin-embedded sections were prepared according to a conventional method. Ten sections of the sections were randomly selected at a magnification of 1000 times per slide, and apoptotic cells were detected with an apoptosis-detection kit (Takara Bio Inc.). The 10 selected fields were regarded as 1 unit, and the number of apoptotic cells per unit (apoptotic cell / lung unit area) was measured.
The results are shown in FIG. Apoptotic cells increased by bleomycin were significantly reduced in the MAA-PEI-HGF / pCAGGS administration group.

(5) Effect on HGF receptor The effect on HGF receptor was carried out by detecting phosphorylated c-Met present in the lung using Western blot.
That is, the lungs 3 and 7 days after administration of bleomycin were removed, and the lungs were refluxed with cooled PBS containing protease inhibitors and frozen in liquid nitrogen. 0.1 g of this lung was dissolved in 1 mL of a lysate [containing protease inhibitor: 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 25 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100 1 mM Na ₃ VO ₄ (sodium orthovanadate (V)), 50 mM NaF, 1 mM PMSF (phenylmethylsulfonyl fluoride), protease inhibitor cocktail (Sigma) 5 μL / mL]. Then, it stirred at 4 degreeC and 0.5 to 1 hour with the rotator, and centrifuged (15,000 rpm, 30 minutes). The soluble fraction was collected, and protein G-Sepharose (Amersham) was added to the soluble fraction and incubated. The protein G sepharose was removed by centrifugation, and an anti-Met antibody (Santacruz B-2) was added to the resulting supernatant for immunoprecipitation. The immunoprecipitate was reduced with dithiothreitol, electrophoresed by SDS-PAGE using 5% by mass polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane by electrotransfer. Phosphorylated Met transferred to the PVDF membrane was detected using a phosphorylation-recognizing antibody (UBI 7-211; Cosmo Bio Inc.).

The results are shown in FIG. It was found that the MAA-PEI-HGF / pCAGGS administration group induced tyrosine phosphorylation of c-Met.
This suggests that the complex of the present invention produces HGF in the lung and promotes HGF-dependent cell differentiation by triggering tyrosine phosphorylation of the HGF receptor c-Met in the lung. To do.

[Formulation Example 1]
The composite prepared in Example 1 3 mg
Stannous chloride 0.1mg
Benzyl alcohol 10.4mg
Sodium acetate 7.2mg
Propylene glycol 83 mg
Water for injection Appropriate amount The above is mixed according to a conventional method to make the total amount 1 mL. Place in a vial and store frozen for use.

  The complex of the present invention and the injection containing the complex can be safely used for the treatment of lung diseases.

FIG. 1 is a graph showing organ distribution after intravenous administration of ^99m Tc-MAA to mice. In the figure, A shows a gamma camera image, and B shows the radiation dose of ^99m Tc in each organ. (1) shows the radiation dose of ^99m Tc in each organ after 1 hour, and ( ^square) shows the radiation dose of ^99m Tc in each organ after 6 hours. FIG. 2 is a graph showing the relationship between plasmid dosage and HGF expression level. In the figure, □ indicates the HGF / pCAGGS administration group, and ■ indicates the MAA-PEI-HGF / pCAGGS administration group. FIG. 3 is a diagram showing the transfection ability of pCAGGS and MAA-PEI-HGF / pCAGGS. In the figure, □ indicates the HGF / pCAGGS administration group, ■ indicates the MAA-PEI-HGF / pCAGGS administration group, and the black triangle indicates the MAA-PEI-HGF / pCAGGS 3 administration group. The arrow indicates the time of administration of MAA-PEI-HGF / pCAGGS in the MAA-PEI-HGF / pCAGGS 3 times administration group. FIG. 4 shows a diagram in which the expression level of HGF in each organ after MAA-PEI-HGF / pCAGGS administration was tested. In the figure, □ indicates the HGF / pCAGGS administration group, and ■ indicates the MAA-PEI-HGF / pCAGGS administration group. * Indicates a significant difference, p <0.05. FIG. 5 shows inflammatory cytokines after plasmid administration. In the figure, ▪ indicates TNF-α, □ indicates IL-6, * indicates a significant difference, and p <0.05. FIG. 6 shows the effect on bleomycin-induced lung inflammation. In the figure, ▪ indicates TNF-α, □ indicates IL-6, * indicates a significant difference, and p <0.05. FIG. 7 shows the effect on bleomycin-induced pulmonary fibrosis. In the figure, the dot column represents the group administered with HGF expression plasmid alone (HGF / pCAGGS 10 μg), and the hatched column represents the group administered MAA-PEI with HGF expression plasmid (MAA-PEI-HGF / pCAGGS 1 μg). * Indicates a significant difference, p <0.05. FIG. 8 shows the effect on bleomycin-induced lung apoptotic cells. * Indicates a significant difference, p <0.05. FIG. 9 shows the results of Western blotting of phosphorylated c-Met in the lungs 3 and 7 days after bleomycin administration.

Claims (8)

  A complex formed by binding DNA encoding hepatocyte growth factor (HGF) and coarsely aggregated albumin-polyethyleneimine (MAA-PEI). The complex according to claim 1, wherein the DNA is any of the following:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1 in the sequence listing; or (b) DNA comprising a base sequence complementary to the DNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing and stringent conditions DNA consisting of a nucleotide sequence that hybridizes underneath and encodes a protein having hepatocyte growth factor (HGF) activity. The complex according to claim 1, wherein the DNA is any of the following:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing; or (b) DNA comprising a base sequence complementary to the DNA having the base sequence set forth in SEQ ID NO: 2 in the sequence listing and stringent conditions DNA comprising a nucleotide sequence that hybridizes underneath and encodes a protein having HGF activity.   The complex according to any one of claims 1 to 3, wherein DNA is inserted into an expression vector.   The complex according to claim 4, wherein the expression vector is a plasmid.   The composite according to any one of claims 1 to 5, wherein the particle diameter of the composite is 10 µm or more and less than 100 µm.   An injectable preparation for treating pulmonary diseases, comprising the complex according to claim 6. The lung disease is at least one disease selected from pulmonary fibrosis, interstitial pneumonia, emphysema, pulmonary hypertension, pneumonia, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis and swallowing pneumonia Item 8. An injection for treatment of pulmonary disease according to Item 7.