KR101479549B1 - Composition for preventing or treating inflammatory respiratory diseases comprising dexamethasone-conjugated polyethylenimine/MIF expression-inhibitory nucleic acid complex - Google Patents

Abstract

The present invention relates to novel use of a dexamethasone-conjugated polyethylenimine/MIF expression inhibitory nucleic acid complex. A dexamethasone-PEI conjugate efficiently transfers an MIF expression inhibitory nucleic acid into a cell. The transferred MIF expression inhibitory nucleic acid effectively inhibits expression of MIF and Muc5ac genes. Therefore, an inflammatory reaction induced by a particulate material or the like and excessive secretion of mucus can be prevented. The dexamethasone-PEI/nucleic acid complex includes: i) the conjugate of dexamethasone and polyethlyenimine (PEI); and the nucleic acid capable of inhibiting expression of the macrophage migration inhibitory factor (MIF) binding to the PEI by electrostatic attraction.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating an inflammatory respiratory disease including a dexamethasone-linked polyethyleneimine / MIF expression-suppressing nucleic acid complex, and a composition for preventing or treating inflammatory respiratory diseases comprising dexamethasone-conjugated polyethylenimine / MIF expression-

The present invention relates to a new use of dexamethasone-conjugated polyethyleneimine / MIF expression-suppressing nucleic acid complexes.

Epidemiological studies have shown an association between airborne particulate matter (PM) inhalation and human respiratory and cardiovascular disease (Dockery DW, et al. N Engl J Med 1993; 329: 1753-9; Gauderman WJ , et al., Lancet 2007; 369: 571-7; Bell, et al., Annu Rev Public Health 2004; 25: 247-80).

PM has been reported to be associated with a variety of respiratory diseases. The World Health Organization (WHO) estimated that about 30-40% of asthma and 20-30% of respiratory diseases were associated with air pollution (Schäfer T et al. Allergy 1997; 52: 14-22). However, the mechanism of aggravation of asthma and other respiratory diseases due to inhalation of airborne particles has not yet been clarified.

Macrophage migration inhibitory factor (MIF), on the other hand, is a protein known as T cell-derived lymphokine that inhibits macrophage migration during a delayed-type hypersensitivity response (Bloom BR , Science 1966; 153: 80-2; David JR Proc Natl Acad Sci USA 1966; 56: 72-7). Currently, MIF is known to have various effects such as T cell activation, IgE synthesis, cell growth and apoptosis and tumor neovascularization (Santos LL, et al. Clin Exp Immunol . 2008; 152: 372-80; Flores-Romo Immunology 1989; 67: 547-9; Takahashi A, et al., Microbiol Immunol 1999; 43: 61-7; Nishihira J, et al., Ann NY Acad Sci 203; 995: 171-82).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a gene therapy agent capable of effectively inhibiting airway inflammation, in particular inflammation induced by PM inhalation. As a result, it is possible to inhibit PM-induced inflammation reaction and mucus hypersecretion by inhibiting the expression of nucleic acid targeting a macrophage migration inhibiting factor, and it is also possible to inhibit the dexamethasone-polyethyleneimine (PEI) (synergistic therapy effect) in the case of using it as a carrier, and completed the present invention.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory respiratory diseases.

It is another object of the present invention to provide a pharmaceutical composition for inhibiting intraluminal mucus hypersecretion.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for the prophylaxis or treatment of inflammatory respiratory diseases comprising the following complex:

(i) a conjugate of dexamethasone and polyethyleneimine (PEI); And (ii) a dexamethasone-PEI / nucleic acid complex comprising an expression-suppressing nucleic acid for a macrophage migration inhibitory factor (MIF) gene bound electrostatically to said polyethyleneimine,

The expression-inhibiting nucleic acid for the MIF gene is siRNA, shRNA, miRNA, antisense oligonucleotide or nucleic acid aptamer.

According to another aspect of the invention, the present invention provides a pharmaceutical composition for inhibiting mucus hypersecretion in an airway or respiratory tract comprising said complex.

The present inventors have sought to develop a gene therapy agent capable of effectively inhibiting airway inflammation, in particular inflammation induced by PM inhalation. As a result, it is possible to inhibit PM-induced inflammation reaction and mucus hypersecretion by inhibiting the expression of nucleic acid targeting a macrophage migration inhibiting factor, and it is also possible to inhibit the dexamethasone-polyethyleneimine (PEI) It was confirmed that the treatment effect could be synergistic.

As shown in the following examples, when a conjugate of dexamethasone and PEI ("DEXA-PEI") was used as a transporter, MIF siRNA was effectively transfected into cells and the expression of MIF was markedly suppressed (see C in FIG. 2) In animal experiments, the inflammatory response was markedly inhibited by administration of DEXA-PEI / MIF siRNA (see FIGS. 5 and 6), and Muc5ac Expression was also effectively down-regulated (see FIG. 8). These results show that the use of the dexamethasone-PEI / nucleic acid complex of the present invention can provide the preventive and therapeutic effect of inflammatory respiratory diseases and the inhibitory effect on mucus hypersecretion in airways.

According to one embodiment of the present invention, the dexamethasone-PEI / nucleic acid complex has an average particle size of 200-500 nm. In one particular example, the complex has an average particle size of 300-500 nm.

According to one embodiment of the invention, the complex exhibits a zeta potential in the range of 1 to 50 mV. As described above, the complex of the present invention exhibits positive zeta potential and therefore can be efficiently endocytosed into cells. Accordingly, the expression-inhibiting nucleic acid for the MIF gene bound to PEI can be efficiently delivered to a target site.

According to one embodiment of the present invention, the N / P ratio (number of nitrogen atoms of PEI / number of phosphate atoms of nucleic acid) in the complex is 1-30: 1. In one particular example, the N / P ratio is 2-20: 1, in another specific example 2-8: 1, and in another specific example 2-6: 1.

In the present invention, the expression-suppressing nucleic acid for the MIF gene is siRNA, shRNA, miRNA, antisense oligonucleotide or nucleic acid aptamer.

As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method.

In one specific example, the siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to an MIF mRNA sequence) and an antisense strand (a sequence complementary to a MIF mRNA sequence) are positioned on opposite sides to form a double strand . According to another specific example, a siRNA molecule of the invention may have a single stranded structure with self-complementary sense and antisense strands. Examples of the sense strand of the siRNA molecule include strands of the first and third sequences of the sequence listing, and examples of the antisense strand include strands of the second and fourth sequences of the sequence listing.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of MIF gene by RNAi effect. The sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.

SiRNA molecules of the present invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, wherein the siRNA molecule formed by the expression of the nucleotide sequence is a molecule The hairpin structure is formed by hybridization, and the stem-and-loop structure as a whole is formed. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The siRNA of the present invention may be in a chemically modified form to prevent rapid degradation by in vivo nucleic acid degrading enzymes. Methods of chemical modification of siRNAs to make them stable and resistant so that they are not easily degraded are well known to those skilled in the art. The most commonly used method for chemical modification of siRNAs is the modification of boranophosphate or phosphorothioate.

As used herein, the term "shRNA (short hairpin RNA)" is a single-stranded RNA having a length of 45 to 70 nucleotides and is an oligosaccharide linking 3-10 base linkers between a sense of the target gene siRNA sequence and a complementary nonsense When DNA is synthesized and cloned into a plasmid vector or shRNA is inserted into a retrovirus, lentivirus and adenovirus, a shRNA with a looped hairpin structure is produced and converted into an siRNA by a dicer in the cell To show the RNAi effect.

As used herein, the term "miRNA (microRNA)" is a single stranded RNA molecule of 21-25 nucleotides that is a regulatory element that controls gene expression in eukaryotes through inhibition in the step of disrupting or detoxifying the target mRNA. These miRNAs are made up of two stages of processing. The first miRNA transcript is made in the nucleus by the RNase III type enzyme called Drosha, which is made into a stem-loop structure of about 70-90 bases, that is, pre-miRNA, which is then transferred to the cytoplasm and transferred to an enzyme called Dicer To produce a mature miRNA of 21-25 bases. These miRNAs complementarily bind to the target mRNA and act as post-transcriptional gene suppressors, leading to translation inhibition and mRNA destabilization. miRNAs are involved in a variety of physiological phenomena and diseases.

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and refers to a complementary sequence in mRNA, It acts to inhibit translation. The antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the MIF gene and capable of binding to the MIF gene mRNA and can be used for translation of the MIF mRNA, translocation into the cytoplasm, maturation or any other overall biological function Lt; RTI ID = 0.0 > activity. ≪ / RTI > The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, and more preferably 40 bases in 10.

As used herein, the term "nucleic acid aptamer " refers to a nucleic acid that has an antagonistic (i.e., inhibitory) effect on a target by binding to a target by the ability to adopt a particular three dimensional conformal form. The inhibition of a target by an aptamer can be achieved by covalently attaching to the target by reacting with the target in a manner that alters the functional activity of the target or target by catalytically changing the target by binding of the target By promoting the reaction between the target and other molecules. An aptamer can be composed of a mixture of multiple ribonucleotide units, deoxyribonucleotide units or two types of nucleotide residues. The aptamer may further comprise one or more modified base, sugar or phosphate backbone units.

According to one embodiment of the present invention, the inflammatory respiratory disease or intraluminal mucus hypersecretion is induced by inhalation of particulate matter.

As used herein, the term "particulate matter" refers to particulate pollutants in the solid or liquid state resulting from mechanical processes such as crushing and sorting of materials, combustion, synthesis, and the like. When air pollutants are classified according to their characteristics, they are in contrast to gaseous pollutants. Particle size has a great influence on the nature of the substance. When it is present in the air, it causes mental retardation, and some fine dust particles enter the lungs and respiratory system of the human body and cause catastrophic damage.

According to an embodiment of the present invention, the inflammatory respiratory disease is selected from asthma, COPD, acute lung injury, empyema, lung abscess, pneumonia, pulmonary tuberculosis, cough, sputum, bronchitis, Tonsillitis, sinusitis, rhinitis, obstructive bronchitis and laryngitis.

Examples of the asthma include bronchial asthma, atopic asthma, atopic bronchial IgE mediated asthma, non-atopic asthma, allergic asthma and non-allergic asthma. Examples of the bronchial asthma include acute bronchitis, chronic bronchitis, And catarrhal bronchitis.

According to one embodiment of the present invention, the intraluminal mucus hypersecretion can cause airway inflammation.

According to one embodiment of the present invention, the complex may inhibit the inflammatory reaction by inhibiting the expression of the muc5ac gene as well as the MIF gene when administered in a subject.

According to one embodiment of the present invention, the complex can inhibit the secretion of intraluminal mucus by inhibiting the expression of muc5ac gene upon administration to an individual.

According to one embodiment of the present invention, the pharmaceutical composition of the present invention can be administered parenterally, and in the case of parenteral administration, intranasal administration, intravenous injection, subcutaneous injection, muscle injection, Lt; / RTI >

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

According to one embodiment of the present invention, the daily dose or the daily dose of the pharmaceutical composition of the present invention is 0.000001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.

The features and advantages of the present invention are summarized as follows:

(i) The present invention relates to a novel use of a dexamethasone-PEI / MIF expression-suppressing nucleic acid complex, that is, a prophylactic and therapeutic use of inflammatory respiratory diseases and a use for suppressing intraluminal mucus hypersecretion.

(ii) The present invention is based on the finding that, when a dexamethasone-PEI conjugate is used as a carrier of a MIF expression-inhibiting nucleic acid, a synergistic effect can be achieved in the above uses.

(iii) The dexamethasone-PEI conjugate efficiently transfers the MIF-expressing-suppressing nucleic acid into the cell, effectively suppresses the expression of MIF and Muc5ac gene by the transferred MIF-expressing nucleic acid, Can be suppressed.

Figure 1 shows the gel retardation and zeta potential results of the DEXA-PEI / MIF siRNA complex. (A) MIF siRNA was mixed with each polymer at different ratios. (B) Zeta potential of PEI / MIF siRNA complex (♦) and DEXA-PEI / MIF siRNA complex (◇). (C) Particle size of PEI / MIF siRNA complex (♦) and DEXA-PEI / MIF siRNA complex (◇).
Figure 2 is a semi-quantitative RT-PCR analysis of MIF mRNA expression. (A) MIF mRNA expression was increased in SiO ₂ - treated cells. (B) Effect of electroporated MIF siRNA on MIF mRNA expression in SiO ₂ -treated cells. (C) Effect of DEXA-PEI / MIF siRNA on MIF mRNA expression in SiO ₂ -treated cells.
Figure 3 shows the distribution of cy3-labeled MIF siRNA in bronchial lavage cells (X1000 magnification, scale bar 5 [mu] m, each group n = 7). An overlay image of cy3-siRNA (red) and nuclei (DAPI, blue). (A) Intratracheal administration of no-endotoxin water to sham mice. (B) Intravenous administration of SiO ₂ (4 mg / kg per 100 μl of no-endotoxin water). (C) SiO ₂ - the distribution of siRNA in the cy3-MIF treated mice. (D) SiO ₂ - cy3 in treated mice (red) -DEXA-PEI- joint distribution of scrambled siRNA. (E) Distribution of cy3-DEXA-PEI-conjugated MIF siRNA. (F) To evaluate the transfection efficiency of MIF siRNA in BAL cells, cy3 ratio for DAPI staining was analyzed (*: p <0.05).
Figure 4 shows the transfection distribution of cy3-DEXA-PEI-conjugated siRNA in mouse airways (X400 magnification, scale bar 20 [mu] m, each group n = 7). (A) Intratracheal administration of no-endotoxin water to sham mice. (B) Intravenous administration of SiO ₂ (4 mg / kg per 100 μl of no-endotoxin water). (C) SiO ₂ - the distribution of siRNA in the cy3-MIF treated mice. (D) SiO ₂ - cy3 in treated mice (red) -DEXA-PEI- joint distribution of scrambled siRNA. (E) Distribution of cy3-DEXA-PEI-conjugated MIF siRNA. (F) Non-analysis of cy3 and DAPI staining in airway epithelial cells (*: p <0.05).
Figure 5 shows the effect of DEXA-PEI-conjugated MIF siRNA on the number of PM-induced airway inflammatory cells in bronchoalveolar lavage fluid (BALF). (A) Total number of BAL cells after inhalation of SiO ₂ and siRNA. Number of lymphocytes, neutrophils and macrophages in BAL cells after inhalation of (BD) SiO ₂ and siRNA (*: p <0.05; **: p <0.01).
6 is a SiO ₂ - 72 hours of the induced mouse lung tissue shows the lung histology examination result (X400 magnification, scale bar 20 ㎛). (A) The lung flakes were periodic acid-schiff stain. (B) Reduction of the immunopathological response in DEXA-PEI-conjugated MIF siRNA-treated mice. To determine glycogen, chorioiodic sheath histopathology of lung was analyzed (*: p < 0.05).
Figure 7 shows RT-PCR analysis of Muc5ac mRNA expression. (A) Expression of Muc5ac mRNA in SiO ₂ -treated cells. (B) Knockdown analysis of Muc5ac mRNA in SiO ₂ -treated cells using MIF siRNA (*: p <0.05).
Figure 8 shows RT-PCR mutation analysis of MIF and Muc5ac mRNA expression in all experimental mouse groups. (A) Changes in MIF and Muc5ac mRNA by RT-PCR. (B) Expression of MIF mRNA in all experimental animal groups. Only DEXA-PEI infusion reduced MIF mRNA levels and DEXA-PEI-conjugated MIF siRNAs further reduced MIF expression. (C) Analysis of Muc5ac mRNA in all experimental animal groups. To determine the degree of inflammation, Muc5ac mRNA was analyzed by RT-PCR. As with the MIF mRNA levels, only DEXA-PEI infusion reduced Muc5ac mRNA levels, and DEXA-PEI-conjugated MIF siRNAs further reduced Muc5ac expression (*: p <0.05).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

1. Cell culture and particle treatment

Human bronchial epithelial cell line Beas-2b (ATCC, Manassas, VA, US) from the 5% CO ₂ incubator at a temperature of 37 ℃ RPMI-1640 medium (Invitrogen, CA, US), 10% heat-inactivated fetal calf serum and Penicillin (100 units / ml) once every 3 days. For the experimental treatment, the Beas-2b cells 37 ℃, T-75 cell culture flasks (2 x 10 ⁵ cells / ㎖) 10% fetal calf a RPMI-1640 medium containing serum and penicillin in 5% CO ₂ conditions Lt; / RTI &gt; Prior to the treatment of the particles, they were heated in an oven for 8 hours at a temperature of 180 ° C to remove particulate matter (SiO _2, mean diameter = 5-21 nm) endotoxin. The next day after inoculation, the cells were treated with silicon dioxide (SiO ₂ ) particles at various concentrations (0, 0.4, 4, 40 μg / ml) for 8 hours and incubated in a humidified CO ₂ incubator at 37 ° C. for 8 hours .

2. Electroporation

Electroporation was performed using Neon Transfection Systems (Invitrogen) according to the manufacturer's instructions. Briefly, 1 X 10 ⁶ cells were suspended in 100 μl of electroporation R buffer containing 200 pM siRNA and electroporated twice at 1.290 V for 20 ms using a 100-μl electroporation tip.

3. DEXA-PEI synthesis and siRNA conjugation

PEI (PEI2k, 2 kDa) was dissolved in 1.8 ml of anhydrous DMSO containing 2-fold molar excess of the Traut's reagent and dexamethasone-21-mesylate. The reaction was allowed to proceed at room temperature for 4 hours, and the reaction was terminated by the addition of cold ethyl acetate. The precipitate was dissolved in water and dialyzed with pure water for one day using a dialysis membrane (molecular weight cut-off = 1000). The mixture was lyophilized to give DEXA-PEI (46% yield). It was confirmed from the ¹ H-NMR data (300 MHz; Korea Basic Science Institute, Seoul, Korea) that 1 mole of dexamethasone was bonded per 1 mole of PEI.

The optimal PEI / siRNA ratio was determined by gel retardation assay. Briefly, 20 μg of siRNA was dissolved in 10 μl of RNase free water and the siRNA solution was diluted in 10 μl of PBS (pH 7.4) with varying amounts of DEXA-PEI and a weight ratio of 1 to 16 Mixed and then vortexed for a short time. After incubation at room temperature for 15 min, complex formation was confirmed by gel retardation analysis on 8% polyacrylamide gel electrophoresis in Tris-borate-EDTA (TBE) buffer.

4. Gel retardation and dynamic light scattering

The DEXA-PEI / MIF siRNA complexes were prepared at various weight ratios (2: 1, 4: 1, 8: 1, 10: 1, 20: 1) in Hepes-buffered saline (20 mM Hepes, 150 mM NaCl, pH 7.4) Respectively. After incubation at room temperature for 30 minutes, the complexes were electrophoresed on a 2% agarose gel containing GelRed (KOMA BIOTECH, Seoul, Korea; 0.5 μg / ml gel). After electrophoresis, the gel was analyzed on a UV illuminator to confirm the position of the complex. The hydrodynamic diameter of the composites at various weight ratios was analyzed with a Zetasizer Nano ZS system (Malvern Instruments, UK). After 30 min incubation, the complex solution was diluted to 2 ml final volume before measurement. The size of the complexes was measured and analyzed using Dispersion Technology software 4.30. The refractive index ( n = 1.33) and viscosity (0.89) of ultrapure water were measured at room temperature.

5. siRNA and its chemical formula

The human MIF siRNA sequence (targeting open reading frame positions 369-389) is as follows (Genolution Pharmaceuticals, Seoul):

Sense: 5'-GCCCGGACAGGGUCUACAU-3 '(SEQ ID No. 1)

Antisense: 5'-AUGUAGACCCUGUCUCGGGC-3 '(SEQ ID No. 2).

The mouse MIF siRNA sequence (targeting open reading frame positions 414-436) is as follows (Integrated DNA Technologies, San Diego, CA, US):

Sense: 5'-GUUCCACCUUCGCUUGAGUCUCUG-3 '(SEQ ID No. 3)

Antisense: 5'-GCCAAGGUGGAAGCGAACUCAGGACCG -3 '(SEQ ID No. 4).

scrambled siRNA (Intergrated DNA Technologies, San Diego, Calif., USA) was used as a negative control. All siRNAs were fluorescently labeled with Cy ^TM 3 using a Label IT ^ㄾ miRNA labeling kit (Mirus, Madison, WI, US).

6. Animal and particle treatment

Six-week old female BALB / c mice (weight 20 ± 1 g) were raised in plastic wool with high-efficiency particulate air (HEPA) filters under conditions of 28 ± 1 ° C., 50 ± 10% humidity and 12 hours of light period. Prior to particle inhalation, the SiO ₂ particles were heated at 180 ° C for 8 hours. Mice were dosed with SiO ₂ (4 mg / kg) in 100 μl of no-endotoxin water via intratracheal inhalation using a 24G IV catheter.

After inhalation, DEXA-PEI-conjugated siRNA was administered in the same manner as inhalation of the particles at an inoculum dose of 2.5 μg and 10 μg, respectively. As a negative control, DEXA-PEI-scrambled siRNA was administered at a dose of 10 [mu] g. Mice were sacrificed after 72 hours. All treatments and experimental procedures for laboratory animals were carried out in accordance with the Animal Experiment Regulations of Hanyang University and all protocols were approved by IACUC of Hanyang University.

7. Preparation and morphological analysis of lung tissue

After 72 hours of siRNA and PM injection, the mice were anesthetized with pentobarbital sodium (65 mg / kg, intraperitoneally), injected with 1 ml of saline and subjected to BAL (bronchoalveolar lavage) 4 times and the trachea Respectively. Cell counts were determined using a hemocytometer and differential cell counts were performed on slides with cytospin and Diff-Quik staining (Scientific Products, Gibbstown, NJ, US). Immediately after BAL administration, RNA was extracted or fixed with 4% paraformaldehyde and snap-frozen for paraffin embedding. For measurement of goblet cells, tissues were periodic acid-Schiff staining (PAS). Morphometrical analysis was performed under an optical microscope at x400 magnification. The PAS-positive epithelial cells and total epithelial cell counts were measured at four preset locations (12, 3, 6, and 9 o'clock; 12 o) using the ImageJ software (National Institute of Health, Bethesda, MD, 'clock is part of the membrane) with 250 μm basement membrane length. Briefly, the dyed portion was changed to a 50x digitized image. Images representing points of interest were divided into constituent colors using ImageJ software and the threshold was manually adjusted until only the pixels representing positive staining were visible. Automated pixel analysis was used to calculate the pixel area (positive staining area). The pixel length of the basement membrane was determined separately (circumference). Pixel length vs. pixel length. After calibration of the actual length (micrometer), the positive staining area was divided into its circumference. Both the numerator and the denominator are multiplied by 1 mm and the calculated value is expressed in terms of millimeter airway length per millimeter basal membrane per millimeter. The results were expressed as percentage of goblet cells in epithelial cells.

8. Reverse transcription PCR and experimental group

Total RNA was isolated using guanidinium thiocyanate-phenol-chloroform extraction method. DNase I (10,000 U / ml; Stratagene, La Jolla, CA, US) -treated RNA was resuspended in 0.5 mM dNTP, 2.5 mM MgCl ₂ , 5 mM DTT, 1 μl random hexamers (50 ng / 200 units / μl; Life Technologies, Grand Island, NY, US) for 50 min at 42 ° C and thermally inactivated at 70 ° C for 15 min. The cDNA was dispensed into tubes containing primer pairs for human and mouse beta-actin, MIF and Muc5ac genes. The primer sequences are as follows:

&Lt; Human [beta] -actin &

Forward: 5'-GCA CTC TTC CAG CCT TCC C-3 '(Sequence Listing 5 sequence)

Reverse: 5'-TCA CCT TCA CCG TTC CAG TTT TT-3 '(515 bp) (SEQ ID NO: 6)

<Human MIF>

Forward: 5'-CCA TCA TGC CGA TGT TC-3 '(SEQ ID No. 7)

Reverse: 5'-CGA AGG TGG AGT TGT TC-3 '(348 bp) (SEQ ID No. 8)

<Human Muc5ac>

Forward: 5'-GGA GGT GCC CAC TTC TA AC-3 '(SEQ ID NO: 9)

Reverse: 5'-CTT CAG GCA GGT CTC GCT G-3 '(153 bp) (SEQ ID NO: 10)

&Lt; Mouse beta-actin &

Forward: 5'-TAT CGC TGC GCT GGT CGT CG-3 '(SEQ ID NO: 11)

Reverse: 5'-CAC AGC CTG GAT GGC TAC GTA CA-3 '(406 bp) (SEQ ID NO: 12)

<Mouse MIF>

Forward: 5'-GTT TCT GTC GGA GCT CAC-3 '(SEQ ID NO: 13)

Reverse direction; 5'-AGC GAA GGT GGA ACC GTT CCA-3 '(292 bp) (SEQ ID NO: 14)

<Mouse muc5ac>

Square: 5'-CTG TGA CAT TAT CCC ATA AGC CC-3 '(Sequence Listing 15 sequence)

Reverse: 5'-ACC GAT CCC GCC CAG TGA CA-3 '(306 bp) (Sequence Listing 16 sequence).

The amplification of MIF and Muc5ac was performed at 27 and 28 cycles (1 cycle: denaturation at 94 ° C. for 30 seconds, 45 at 45 ° C. and 58.1 ° C.) with initial denaturation (94 ° C., 5 min) Second annealing, extension at 72 캜 for 45 seconds).

9. Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, US). The differences between the experimental groups were compared by the Kruskal-Wallis test. If differences were found to be significant, Mann-Whitney U-test and Tukey's post hoc test were applied to compare the differences between the two samples. When the p value was 0.05, the difference was judged to be significant.

Experiment result

1. Complex formation assay

To confirm complex formation between DEXA-PEI and MIF siRNA, gel retardation analysis was performed. DEXA-PEI / MIF siRNA complexes were prepared at various weight ratios (2: 1, 4: 1, 8: 1, 10: 1, 20: 1) and analyzed on 2% (w / v) agarose gels. Figure 1 A shows that the retention efficiency of the complex was the highest at 4: 1 ratio (DEXA-PEI / MIF siRNA).

The zeta potential of the DEXA-PEI / MIF siRNA complexes prepared with different N / P ratios is shown in Fig. The PEI / MIF siRNA complex showed a negative charge even with an N / P ratio of 4. On the other hand, the DEXA-PEI / MIF siRNA complex showed a positive charge from the N / P ratio of 2, and the charge of the DEXA-PEI / MIF siRNA complex increased to 20 N / P ratio.

After formation of the complex, the particle size was measured. In the N / P ratio of 2, the diameter of the PEI / MIF siRNA complex was 833.53 ± 52.68 nm. The particle size of the PEI / MIF siRNA complex decreased with increasing N / P ratio. The DEXA-PEI / MIF siRNA complex had an average diameter of 355.2 ± 17.33 nm at an N / P ratio of 4. On the other hand, PEI / MIF siRNA had an average diameter of 622.4 ± 34.95 nm at the same N / P ratio (FIG. 1C). These contributions contribute to an increase in transfection efficiency of DEXA-PEI / MIF siRNA and would be suitable for efficient endocytosis by Beas-2b.

In the subsequent experiments, DEXA-PEI / MIF siRNA complexes were used at a weight ratio of 4: 1.

2. Knockdown of MIF

PM (SiO ₂ ) treatment increased the expression of MIF in airway epithelial cell line Beas-2b (FIG. 2A). SiRNA was introduced into the cells by electroporation as a positive control. MIF siRNA successfully down-expressed PM-induced MIF expression in a dose-dependent manner (Fig. 2B). Even when DEXA-PEI was used as a carrier of siRNA, the expression of PM-induced MIF was effectively reduced (FIG. 2C). However, naked siRNA and DEXA-PEI / scrambled siRNA did not show any effect on PM-induced MIF expression.

3. Delivery of siRNA using DEXA-PEI

BALB / c mice were intratracheally administered PM and treated with cy3-labeled DEXA-PEI / MIF siRNA complex. After 72 hours of culture, BAL cells and cell suspension prepared from lung lobes were analyzed under fluorescence microscope.

As shown in FIG. 3, fluorescent signals were observed in BAL cells (mainly alveolar macrophages) of the DEXA-PEI-conjugated siRNA-treated group (D and E in FIG. 3). Fluorescence was not observed in the control, SiO ₂ alone, or BAL cells in the naked MIF siRNA treatment group. DAPI-stained nuclei were counted and red fluorescence was released 3 days after transfection of DEXA-PEI-conjugated siRNA in approximately 25% of the total BAL cells. However, no red fluorescence was observed in BAL cells derived from naked siRNA treatment or SiO ₂ -treated mouse (Fig. 3F).

DEXA-PEI delivered cy3-labeled MIF siRNA to mouse lung tissue. Regardless of whether it was MIF or scrambled, siRNA alone was not efficiently delivered to mouse tissue (Fig. 4C). However, DEXA-PEI-conjugated siRNAs were easily transfected into mouse tissue. Histological analysis of lung tissue using fluorescence microscopy revealed that the cells showed red fluorescence in about 21% of the total cells of lung tissue (Fig. 4F). Red fluorescence was particularly prominent in epithelial cells lined with bronchioles (Fig. 4, D and E).

Transfection of MIF siRNA with carrier molecule (DEXA-PEI) down-expressed the expression of MIF induced by PM treatment in a dose-dependent manner (Fig. 8A).

4. Reduction of inflammation

Inhalation of the SiO ₂ particles induced infiltration of white blood cells circulating in the airways (FIG. 5A). The amounts of leukocytes (FIG. 5B) and neutrophils (FIG. 5C) were increased 3-fold and 15-fold after the inhalation of SiO _2, respectively. The number of infiltrating macrophages also increased 1.4-fold in SiO ₂ -treated mice (FIG. 5D). Airway inflammation after PM treatment was efficiently down-regulated by DEXA-PEI-conjugated MIF siRNA. Treatment of MIF siRNA and DEXA-PEI inhibited leukocyte infiltration and neutrophil infiltration by 39% and 62%, respectively, at 10 μg dose compared to SiO ₂ -treated samples. However, there was no significant change in macrophage count (Fig. 5D). Scrambled siRNA and naked siRNA conjugated with DEXA-PEI also inhibited infiltration of leukocytes (15% and 2% each) and neutrophils (15% and 1% of each), although the inhibition rate was lower than DEXA-PEI / MIF siRNA. There was no significant change in MIF siRNA without carrier molecule treatment.

Histological analysis revealed the potential role of MIF in airway inflammation. As shown in FIG. 6A, the inhalation of SiO ₂ increased airway inflammation, mucous hypersecretion, and remnant cell hyperplasia. Inflammatory cells were observed in the airways of all flaps. The number of inflammatory cells was significantly reduced in the DEXA-PEI-conjugated MIF siRNA treated tissues (Fig. 6A). However, anti-inflammatory effects were not observed in tissues treated with DEXA-PEI-conjugated scrambled siRNA or siRNA (without carrier). In addition, treatment of DEXA-PEI / MIF siRNA significantly reduced the percentage of PAS positive cells in the organs (Fig. 6B).

5. Association between MIF and Muc5ac gene expression

The Muc5ac protein is the major mucin protein in the airways (Cha MH, et al. Mol Cell Proteomics 2007; 6: 56-63). Up-regulation of Muc5ac in epithelial cells by PM treatment has been reported (Ahn MH, et al. Respir Res 2005; 6: 34-42). As shown in Fig. 7 A, the process of the SiO ₂ particles in Beas-2b cell line was also increased as well as the expression of MIF, Muc5ac expression of the gene. When MIF siRNA was treated with Beas-2b cells using DEXA-PEI or electroporation, a decrease in Muc5ac expression was observed with MIF (Fig. 7B). Similar results were obtained from the mouse model. Increased expression of Muc5ac by SiO ₂ treatment dropped to basal level (Figure 8).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-University Cooperation Foundation Hanyang University <120> Composition for preventing or treating inflammatory respiratory          dodecanethasone-conjugated polyethylenimine / MIF          expression-inhibitory nucleic acid complex <130> PN130330 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> Human MIF siRNA (sense) <400> 1 gcccggacag ggucuacau 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> Human MIF siRNA (antisense) <400> 2 auguagaccc uguccgggc 19 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> Mouse MIF siRNA (sense) <400> 3 guuccaccuu cgcuugaguc cug 23 <210> 4 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> mouse MIF siRNA (antisense) <400> 4 gccaaggugg aagcgaacuc aggaccg 27 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human beta-actin <400> 5 gcactcttcc agccttccc 19 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human beta-actin <400> 6 tcaccttcac cgttccagtt ttt 23 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human MIF <400> 7 ccatcatgcc gatgttc 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human MIF <400> 8 cgaaggtgga gttgttc 17 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human Muc5ac <400> 9 ggaggtgccc acttctaac 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human Muc5ac <400> 10 cttcaggcag gtctcgctg 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for maze beta-actin <400> 11 tatcgctgcg ctggtcgtcg 20 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for maze beta-actin <400> 12 cacagcctgg atggctacgt aca 23 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mause MIF <400> 13 gtttctgtcg gagctcac 18 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mause MIF <400> 14 agcgaaggtg gaaccgttcc a 21 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mause muc5ac <400> 15 ctgtgacatt atcccataag ccc 23 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mause muc5ac <400> 16 accgatcccg cccagtgaca 20

Claims (10)

A pharmaceutical composition for the prophylaxis or treatment of inflammatory respiratory diseases, including the following complexes:
(i) a conjugate of dexamethasone and polyethyleneimine (PEI); And (ii) a dexamethasone-PEI / nucleic acid complex comprising an expression-suppressing nucleic acid for a macrophage migration inhibitory factor (MIF) gene bound electrostatically to said polyethyleneimine,
The expression-inhibiting nucleic acid for the MIF gene is siRNA, shRNA or an antisense oligonucleotide.
2. The pharmaceutical composition of claim 1, wherein the dexamethasone-PEI / nucleic acid complex has an average particle size of 200-500 nm.
2. The pharmaceutical composition of claim 1, wherein the dexamethasone-PEI / nucleic acid complex exhibits a zeta potential in the range of 1 to 50 mV.
2. The pharmaceutical composition according to claim 1, wherein the N / P ratio (number of nitrogen atoms of PEI / number of phosphate atoms of nucleic acid) in the dexamethasone-PEI / nucleic acid complex is 1-30: 1.
The pharmaceutical composition according to claim 1, wherein the inflammatory respiratory disease is induced by inhalation of particulate matter.
The method of claim 1, wherein the inflammatory respiratory disease is selected from asthma, COPD, acute lung injury, empyema, lung abscess, pneumonia, pulmonary tuberculosis, cough, sputum, bronchitis, sore throat, tonsillitis, , Rhinitis, bronchiolitis obliterans, and laryngitis.
The pharmaceutical composition according to claim 1, wherein the complex inhibits the inflammatory reaction by inhibiting the expression of the Muc5ac (mucin 5AC) gene as well as the MIF gene upon administration to a subject.
A pharmaceutical composition for inhibiting mucus hypersecretion in an airway or respiratory tract comprising the following complex:
(i) a conjugate of dexamethasone and polyethyleneimine (PEI); And (ii) a dexamethasone-PEI / nucleic acid complex comprising an expression-suppressing nucleic acid for a macrophage migration inhibitory factor (MIF) gene bound electrostatically to said polyethyleneimine,
The expression-inhibiting nucleic acid for the MIF gene is siRNA, shRNA or an antisense oligonucleotide.
9. The pharmaceutical composition according to claim 8, wherein the intraluminal mucus hypersecretion is induced by inhalation of particulate matter.
9. The pharmaceutical composition according to claim 8, wherein the complex inhibits expression of Muc5ac gene upon administration to a subject to inhibit secretion of intraluminal mucus.

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