This is a continuation of Application Ser. No. 09/660,522, filed Sep. 12, 2000 (now abandoned), which is a continuation of Application Ser. No. 09/029,497 filed Jun. 9, 1998 (now U.S. Pat. No. 6,248,722, issued Jun. 19, 2001); which is a 371 National Stage Application of PCT/JP96/02359 filed Aug. 22, 1996, the disclosures of which are incorporated herein by reference.
TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS
The present invention relates to a medicament for use in gene therapy and the like. More particularly, the present invention relates to a medicament comprising a hepatocyte growth factor (HGF) gene as well as a liposome containing the HGF gene.
PRIOR ART
HGF is a physiologically active peptide that exhibits diverse pharmacological activities. The pharmacological activities of HGF are described in, e.g., JIKKEN-IGAKU (Experimental Medicine), Vol. 10, No. 3 (extra issue), 330-339 (1992). In view of its pharmacological activities, HGF is expected to be useful as: a drug for the treatment of cirrhosis of the liver or of renal diseases; epithelial cell growth accelerators; anticancer agents; agents for the prevention of side effects in cancer therapy; agents for the treatment of lung disorders, gastrointestinal damages or cranial nerve disorders; agents for the prevention of side effects in immunosuppression; collagen degradation accelerators; agents for the treatment of cartilage disorders, arterial diseases, pulmonary fibrosis, hepatic diseases, blood coagulopathy, plasma hypoproteinosis or wounds; agents for the improvement of nervous disorders; hematopoietic stem cell potentiators; and hair growth promoters (Japanese Patent KOKAI (Laid-Open) Nos. 4-18028 and 4-49246, EP 492614, Japanese Patent KOKAI (Laid-Open) No. 6-25010, WO 93/8821, Japanese Patent KOKAI (Laid-Open) Nos. 6-172207, 7-89869 and 640934, WO 94/2165, Japanese Patent KOKAI (Laid-Open) Nos. 6-40935, 6-56692 and 741429, WO 93/3061, Japanese Patent KOKAI (Laid-Open) No. 5-213721, etc.).
As to gene therapy, extensive studies and investigations recently have been made all over the world for treatment of adenosine deaminase deficiency, AIDS, cancer, pustulous fibrosis or hemophilia, etc.
However, gene therapy using HGF genes is unknown yet. It is even unclear if such gene therapy will be effective.
Problems to be Solved by the Invention
HGF is known to be one of the drugs that have a short half life in blood. As a result, persistent topical administration for HGF would be desirable.
In view of the diverse pharmacological activities of HGF, HGF is expected to be developed as a drug having extensive applications to various diseases. On the other hand, when HGF is systemically administered, side effects might be caused due to the diverse pharmacological activities of HGF. In addition, when HGF itself is intravenously administered, HGF has the drawback that a considerable amount of HGF is retained in the liver, resulting in reduction of the amount of HGF that reachs the target organ.
Means for Solving the Problems
The present invention has been made to solve the foregoing problems. In summary, the present invention relates to:
-
- (1) a medicament comprising a HGF gene;
- (2) a liposome containing the HGF gene;
- (3) a liposome according to (2), which is a membrane fusion liposome fused to Sendai virus;
- (4) a medicament comprising the liposome according to (2) or (3);
- (5) a medicament according to (1) or (4), for use in the treatment of arterial disorders; and,
- (6) a medicament according to (1) or (4), for use in the treatment of cartilage injuries.
In
BEST MODE FOR CARRYING OUT THE INVENTION
The “HGF gene” employed in the present invention means a gene capable of expressing HGF. Thus, so long as the polypeptide expressed has substantially the same effect as that of HGF, the HGF gene may have a partial deletion, substitution or insertion of the nucleotide sequence, or may have other nucleotide sequences ligated therewith at the 5′-terminus and/or 3′-terminus thereof. Typical examples of such HGF genes include HGF genes as described in Nature, 342, 440 (1989), Japanese Patent KOKAI (Laid-Open) No. 5-111383, Biohem. Biophys. Res. Commun., 163, 967 (1989), etc. These genes may be used in the present invention.
The HGF gene is incorporated into an appropriate vector and the HGF gene-bearing vector is provided for use. For example, the HGF gene may be used in the form of a viral vector having the HGF gene as described hereinafter, or in the form of an appropriate expression vector having the HGF gene.
The “pharmaceutical composition” used in the present invention means a medicament for the treatment or prevention of human diseases, which is attributed to the pharmacological activities of HGF. For example, exemplified are medicaments for the treatment or prevention of the diseases given hereinabove.
According to the present invention, the HGF gene is introduced into cells wherein HGF is expressed in those cells to exhibit the pharmacological actions. Thus, the medicament of the present invention is effectively applicable to the diseases for which HGF itself is effective.
Where the HGF gene is introduced into, e.g., cells, the growth of vascular endothelial cells is accelerated, while undesired growth of vascular smooth muscle cells is not accelerated, as demonstrated in the Examples hereinafter. Moreover, as demonstrated in the Examples hereinafter, where the HGF gene is introduced into the heart in in vivo animal tests using rats, angiogenesis is observed. Therefore, the HGF gene is effective for the treatment and prevention of arterial disorders, in particular, various diseases caused by a disturbance which mainly involves abnormal proliferation of vascular smooth muscle cells (e.g., restenosis after percutaneous transluminal coronary angioplasty (PTCA), arteriosclerosis, insufficiency of peripheral circulation, etc.), and for the treatment and prevention of diseases such as myocardial infarction, myocardia, peripheral angiostenosis, cardiac insufficiency, etc. HGF itself is also useful for the treatment and prevention of the diseases as described above, since HGF promotes the proliferation of vascular endothelial cells but does not promote the growth of vascular smooth muscle cells. The pharmacological effects of the HGF gene are attributed to those of HGF itself.
As demonstrated in the Examples hereinafter, introduction of the HGF gene into the joint results in promoting repair of articular cartilage cells to thereby promote the proliferation of proteoglycan-synthesizing cells. Therefore, the HGF gene is effective for the prevention and treatment of various cartilage injuries such as osteogenetic abnormality, arthritis deformans, discopathy deformans, fracture repair and restoration insufficiency, trauma caused by sports, key puncher's disease, etc. HGF itself is useful for the treatment and prevention of the diseases described above, since HGF promotes repair and growth of cartilage cells. The effects of the HGF gene are based on those of HGF itself.
A “liposome” is a closed vesicle of lipid bilayer encapsulating an aqueous compartment. It is known that the lipid bilayer membrane structure is extremely similar to biological membranes. To prepare the liposomes of the present invention, phospholipids are employed. Typical examples of phospholipids are phosphatidylcholines such as lecithin, lysolecithin, etc.; acidic phospholipids such as phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylic acid, etc.; or phospholipids obtained by replacing an acyl group(s) of these acidic phospholipids with lauroyl, myristoyl, oleoyl, etc.; and sphingo-phospholipids such as phosphatidylethanolamine, sphingomyelin, etc. Neutral lipids such as cholesterol may also be added to these phospholipids. The liposomes may be prepared, in a conventional manner, from naturally occurring materials such as lipids in normal cell membranes. The liposomes containing the HGF gene of the present invention may be prepared, for example, by suspending a thin layer of purified phospholipids in a solution containing the HGF gene and then treating the suspension in a conventional manner, such as by ultrasonication.
The liposomes containing the HGF gene of the present invention may be appropriately fused to viruses, etc. to form membrane fusion liposomes. In this case, it is preferred to inactivate viruses, e.g., through ultraviolet irradiation, etc. A particularly preferred example of the membrane fusion liposome is a membrane fusion liposome fused with Sendai virus (hemagglutinating virus of Japan: HVJ). The membrane fusion liposome may be produced by the methods described in NIKKEI Science, April, 1994, pages 32-38; J. Biol. Chem., 266 (6), 3361-3364 (1991), etc. In more detail, the HVJ-fused liposome (HVJ-liposome) may be prepared, e.g., by mixing purified HVJ inactivated by ultraviolet irradiation, etc. with a liposome suspension containing the HGF gene vector, gently agitating the mixture and then removing unbound HVJ by sucrose density gradient centrifugation. The liposomes may be bound to substances having an affinity to target cells, thereby enhancing the efficiency of gene introduction into the target cells. Examples of substances having an affinity for target cells include ligands such as an antibody, a receptor, etc.
For introduction of the HGF gene into cells, conventional methods are employed, which are roughly classified into introduction via viral vectors and other strategies (NIKKEI Science, April, 1994, pages 20-45; GEKKAN YAKUJI, 36 (1), 23-48 (1994) and references cited therein). Both types of methods can be used for preparation of the medicament of the present invention.
The former type of method using viral vectors comprises the step of incorporating the HGF gene into, e.g., a retrovirus, an adenovirus, an adeno-related virus, a herpes virus, a vaccinia virus, a poliovirus, a sindbis virus or other RNA virus. Of these viruses, a retrovirus, an adenovirus and an adeno-related virus are particularly preferably employed for introduction.
Examples of the other methods include the liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method. Of these methods, particularly preferred is the liposome method.
For practical use of the HGF gene as a medicament, it is advantageous to introduce the HGF gene directly into the body (in vivo method). Alternatively, certain cells are collected from the human, the HGF gene is then introduced into the cells outside the body, and cells having the HGF gene introduced therein are returned to the body (ex vivo method). These methods are described in NIKKEI Science, April, 1994, pages 20-45; GEKKAN-YAKUJI, 36 (1), 23-48 (1994) and references cited therein. Any of these methods is suitable, depending upon the disease to be treated, target organs, etc. and may be applied to the medicament compositions of the present invention.
The in vivo method is less costly, less laborious and therefore more convenient than the ex vivo method, but the latter method provides a higher efficiency of introduction of the HGF gene into cells.
Where the medicament of the present invention is administered by the in vivo method, the medicament may be administered through any route appropriate for diseases to be treated, such as via target organs, etc. The medicament may be administered intravenously, intraarterially, subcutaneously, intramuscularly, etc., or directly to the objective organ of the disease, e.g., kidney, liver, lung, brain, nerve, etc. Direct administration to the objective site can treat the target organ selectively. For example, in gene therapy using a gene for restenosis after PTCA, the composition may be administered intraarterially (JIKKEN-IGAKU, 12 (extra issue 15), 1298-1933 (1994). Preferably, the medicament of the present invention is applied at the tip of a balloon used for PTCA and the tip is rubbed against a blood vessel, whereby the medicament may be introduced directly into vascular endothelial cells and vascular smooth muscle cells.
Where the ex vivo method as described above is used to introduce the HGF gene, human cells (e.g., lymphocytes or hematopoietic stem cells) are harvested in a conventional manner, and the harvested cells are sensitized with the medicament of the present invention for gene introduction. Thereafter the HGF-producing cells are inserted back into the human.
Where the medicament is administered by the in vivo method, the medicament may take various preparation forms, including the form of a liquid preparation. In general, the medicament may be preferably prepared as an injection comprising the HGF gene as an active ingredient. If necessary and desired, conventional carriers may be added to the composition. The injection may be prepared in a conventional manner, e.g., by dissolving the HGF gene in an appropriate solvent (e.g., sterilized water, a buffered solution, a physiological saline solution, etc.), filtering the solution through a filter, etc. for sterilization, filling up a sterile container with the solution. The medicament may be prepared using the HGF gene-containing viral vector, instead of the HGF gene itself. Where the liposomes containing the HGF gene embedded therein (or HVJ-liposomes) are employed, the medicament may be in the form of liposome preparations such as a suspension, a frozen preparation, a centrifugally concentrated frozen preparation, etc.
The content of the HGF gene in the medicament may be appropriately varied depending upon diseases to be treated, target organs, patients' ages or body weights, etc. However, it is appropriate to administer in a dose of 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg when calculated as the HGF gene. The dose may be divided into several days or a few months.
EXAMPLES
Hereinafter the present invention will be described in more detail with reference to the examples but is not deemed to be limited thereto. Materials and methods used in the following examples are outlined below.
Materials and Methods
(1) HGF Expression Vector
The HGF expression vector was prepared by inserting human HGF cDNA (2.2 kb, Biochem. Biophys. Res. Commun., 172, 321-327 (1990); Japanese Patent KOKAI (Laid-Open) No. 5-111383) between the EcoRI and NotI sites of a pUC-SRα expression vector (FEBS, 333, 61-66 (1993)). In this plasmid vector, transcription of HGF cDNA is regulated by the SRa promoter (Nature, 342, 440-443 (1989)).
(2) Cell Culture
Rat coronary endothelial cells were isolated from enzymatically digested heart of 8-week old Sprague-Dawley (SD) rats by density gradient centrifugation (Transplantation, 57, 1653-1660 (1994)). Rat aortic vascular smooth muscle cells (VSMCs) were obtained from 12 week old SD rats by enzymatic treatment (J. Clin. Invest., 93, 355-360 (1994)). These cells were maintained in DMEM medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μ/ml). The cells were incubated at 37° C. in a humidified 95% air-5% CO2 atmosphere. The culture medium was routinely changed at 2 day-intervals. Both immunopathological and morphological observation revealed that these cells were endothelial cells and smooth muscle cells, respectively.
Human aortic endothelial cells (five passages) and human VSMCs (five passages) were obtained from Kurabo Co. The endothelial cells were incubated in a manner similar to the above method in MCDB131 medium supplemented with 5% fetal calf serum, epidermal growth factor (10 ng/ml), basic fibroblast growth factor (2 ng/ml) and dexamethasone (1 μM).
Endothelial cells in the stationary state were prepared according to the method described in J. Clin. Invest., 86, 1690-1697 (1990), ibid., 94, 824-829 (1994).
(3) Transfection of the HGF Gene into HVJ-Liposomes in vitro
Endothelial cells or VSMCs were inoculated for sensitization on a 6-well plate in a cell count of 106 and proliferated to reach 80% confluence. The cells were washed 3 times with a balanced salt solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6; hereinafter abbreviated as “BSS”) supplemented with 2 mM calcium chloride. To the cells was added 1 ml of a solution of the HVJ-liposome-DNA (containing 2.5 mg of lipids and 10 mg of the embedded DNA) obtained in Example 1 hereinafter or 1 ml of a solution of the HVJ-liposome-cont obtained in Comparative Example 1 hereinafter. The resulting mixture was incubated at 4° C. for 5 minutes and at 37° C. for a further 30 minutes. The cells were washed and maintained in a fresh medium containing 10% bovine serum in a CO2 incubator.
(4) Assay for the HGF Concentrations in Endothelial Cells and VSMCs
The concentration of HGF produced from the sensitized endothelial cells and VSMCs was assayed by ELISA. That is, rat or human endothelial cells or VSMCs were inoculated on a 6-well plate (made by Corning) in a cell density of 5×104 cells/cm2, followed by incubation for 24 hours. The medium was replenished 24 hours after the sensitization, and incubation was continued for a further 48 hours. To investigate if HGF was released, the sensitized cells (48 hours after sensitization) were washed and added to 1 ml of a serum-free medium containing 5×10−7 M insulin, 5 μg/ml transferrin and 0.2 mM ascorbate. After 24 hours, the culture media was collected, centrifuged at 600 g for 10 minutes and then stored at −20° C.
The HGF concentration in the media was determined by an enzyme immunoassay using an anti-rat HGF antibody or an anti-human HGF antibody (Exp. Cell Res., 210, 326-335 (1994); Jpn. J. Cancer Res., 83, 1262-1266 (1992)). A rabbit anti-rat or an anti-human HGF IgG was coated onto a 96-well plate (made by Corning) at 4° C. for 15 hours. After blocking with 3% bovine serum albumin-containing PBS (phosphate buffered saline), the culture medium was added to each well, and incubation was performed at 25° C. for 2 hours. After washing each well 3 times with PBS containing 0.025% Tween (PBS-Tween), a biotinated rabbit anti-rat HGF IgG or an anti-human HGF IgG was added to each well followed by incubation at 25° C. for 2 hours. After washing with PBS-Tween, each well was incubated together with horse radish peroxidase-bound streptoavidin-biotin complex (PBS-Tween solution). The enzymatic reaction was initiated by adding thereto a substrate solution (containing 2.5 mM o-phenylenediamine, 100 mM sodium phosphate, 50 mM citrate and 0.015% hydrogen peroxide). The reaction was terminated by adding 1 M sulfuric acid to the system. Absorbance was measured at 490 nm. The anti-human HGF antibody is reactive only with human HGF, and not with rat HGF. The anti-rat HGF antibody is reactive solely with rat HGF, and not with human HGF.
(5) HGF
The human and rat recombinant HGFs employed were purified from the culture solution of CHO cells or C-127 cells transfected with an expression plasmid bearing human or rat HGF cDNA (Cell, 77, 261-271 (1994); J. Clin. Invest., 93, 355-360 (1994)).
(6) Statistical Analysis
All runs were repeated at least 3 times. Data measured are shown by mean±standard error. Statistical analysis of the measured data was made according to Duncan's test.
(7) Hematoxylin-Eosin (HE) Staining and Azan Staining
Ten days after the gene introduction, the rats having the HGF gene introduced therein were sacrificed by perfusion with heparinized physiological saline. Fixation was then made overnight with a 4% paraformaldehyde PBS solution. After fixation, the tissue was embedded in paraffin. Slides were prepared and stained with HE and Azan in a conventional manner. The slides were examined on a microscope to count the number of microvessels.
Example 1 Preparation of HVJ-Liposomes Containing the HGF Expression Vector
Phosphatidylserine, phosphatidylcholine and cholesterol were mixed with tetrahydrofuran in a weight ratio of 1:4.8:2. By distilling tetrahydrofuran off through a rotary evaporator, the lipid mixture (10 mg) was precipitated onto the container wall. After 96 μg of high mobility group (HMG) 1 nuclear protein purified from bovine thymus was mixed with a BBS (200 μl) solution of plasmid DNA (300 μg) at 20° C. for an hour, the mixture was added to the lipid mixture obtained above. The resulting liposome-DNA-HMG 1 complex suspension was mixed with a vortex, ultrasonicated for 3 seconds and then agitated for 30 minutes.
The purified HVJ (strain Z) was inactivated by UV irradiation (110 erg/mm2 sec) for 3 minutes immediately before use. BSS was added to and mixed with the liposome suspension (0.5 ml, containing 10 mg of the lipids) obtained above and HVJ (20,000 hemagglutinating units) to make the total volume 4 ml. The mixture was incubated at 4° C. for 10 minutes and gently agitated at 37° C. for a further 30 minutes. The unreacted HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. That is, the upper layers in the sucrose density gradient were collected to give the HVJ-liposomes containing the HGF expression vector (containing 10 μg/ml of the HGF expression vector). The HVJ-liposomes containing the HGF expression vector is hereinafter often referred to as HVJ-liposome-DNA.
Example 2 Administration of the HVJ-Liposome Containing the HGF Expression Vector to Rats
The HVJ-liposomes containing the HGF expression vector were prepared by the method described in the above Example, using 64 μg of HMG 1 nuclear protein and 200 ìg of plasmid DNA. BSS was added to and mixed with the liposome suspension (0.5 ml, containing 10 mg of the lipids) and HVJ (35,000 hemagglutinating units) to make the whole volume 2 ml.
SD rats (weighing 400-500 g; purchased from Japan Charles River) were anesthetized with intraperitoneal administration of sodium pentobarbital (0.1 ml/100 mg), warmed and breathing was maintained with an automated breather. The rats were subjected to thoracotomy on the left side. The HVJ-liposome-DNA or HVJ-liposome-cont (20 μl) was carefully injected directly through the cardiac apex using a 30 G syringe.
Comparative Example 1
Preparation of HVJ-Liposomes Containing No HGF Expression Vector
A vector bearing no HGF gene was treated in the same manner as described in Example 1 to prepare the HVJ-liposomes containing no HGF expression vector. The HGF expression vector-free HVJ-liposomes are hereinafter referred to as HVJ-liposome-cont.
Comparative Example 2 Preparation of HVJ-Liposomes Containing Human TGF-β Expression Vector
HVJ-liposomes containing human TGF-β expression vector were prepared in a manner similar to that described in Example 1 except for using a human TGF-β expression vector.
The HVJ-liposomes containing human TGF-expression vector are hereinafter referred to as HVJ-liposome-DNA (TGF-β).
Test Example 1 Expression of HGF in Rat Coronary Endothelial Cells Sensitized with HVJ-Liposome-DNA
HVJ-liposome-DNA (concentration of the HGF expression vector in liposomes: 10 μg/ml) was sensitized to rat coronary endothelial cells (cell count: 106). HGF production was determined by ELISA. For control, a similar test was conducted using HVJ-liposome-cont. HGF production was also determined on the non-sensitized rat coronary endothelial cells (no-treatment group). The results are shown in
As shown in
Cell counting in the groups tested revealed that the HGF expression group showed significantly high cell counts.
Test Example 2 Effects of the Sensitized HGF Expression Vector on Proliferation of Endothelial Cells
Human endothelial cells were sensitized with HVJ-liposome-cont. The sensitized cells were incubated in the presence or absence of exogenously added recombinant human HGF (1, 10 and 100 ng/ml) and the cell growth rate (%) was determined. The results are shown in
The (line) graph shown in
The endothelial cells sensitized with HVJ-liposome-DNA (concentration: 10 μg/ml) were similarly incubated, the increase in the number of cells was determined, and the cell growth rate (%) was calculated. For a control, endothelial cells sensitized with HVJ-liposome-cont were also incubated, the increase in the number of cells was determined, and the cell growth rate (%) was calculated. The results are shown in
As is noted from the bar shown in
The aforesaid endothelial cells sensitized with HVJ-liposome-DNA were incubated in the presence or absence of a rabbit anti-human HGF antibody. The increase in the number of cells was determined, and the cell growth rate was calculated. For a control, the endothelial cells sensitized with HVJ-liposome-cont were incubated, and the increase in the number of cells was determined in a similar manner in order to calculate the cell growth rate. The rabbit anti-human HGF antibody (10 μg/ml) was purified by the method described in Jpn. J. Cancer Res., 83, 1262-1266 (1992). This antibody is capable of neutralizing the biological activity of 10 ng/ml at a concentration of 10 μg/ml. The anti-human HGF antibody reacts only with human HGF and not with rat HGF, whereas the anti-rat HGF antibody reacts only with rat HGF and not with human HGF. Normal rabbit serum IgG (10 μg/ml) was used for the control.
The results are shown in
Test Example 3 Effects of the Supernatant of the Incubated HVJ-Liposome-DNA-Sensitized Rat VSMCs on Rat Coronary Endothelial Cells
The supernatant from the incubated HVJ-liposome-DNA-sensitized rat VSMCs was added to the rat coronary endothelial cell culture system (cell count: 105) during the stationary phase. After incubation for 3 days, the increase in the number of endothelial cells was determined. For a control, the supernatant of the incubated HVJ-liposome-cont-sensitized rat VSMCs was added to the rat coronary endothelial cells in a similar manner to that described above, and the increase in the number of endothelial cells was determined as described above. The results are shown in
As shown in
The concentration of HGF in the culture supernatant of the rat VSMCs sensitized with HVJ-liposome-DNA or HVJ-liposome-cont as described above was assayed by ELISA using an anti-human HGF antibody and an anti-rat HGF antibody. The HGF concentration in the culture supernatant of non-sensitized VSMCs was also assayed (no-treatment group).
The results obtained using the anti-human HGF antibody and the anti-rat HGF antibody are shown in
As shown in
As observed in
Test Example 4 Effects of the Supernatant from the Incubated HVJ-Liposome-DNA-Sensitized Rat Coronary Endothelial Cells on rat Coronary Endothelial Cells
The supernatant of the incubated HVJ-liposome-DNA-sensitized rat coronary endothelial cells was added to the rat coronary endothelial cell culture system (cell count: 105) during the stationary phase. After incubation for 3 days, the increase in the number of endothelial cells was examined. For the control, the endothelial cells were incubated in a similar manner, using culture supernatant from the HVJ-liposome-cont-sensitized rat coronary endothelial cells, and the increase in the number of endothelial cells was determined. The results are shown in
As shown in
Next, anti-HGF antibody was added to the culture supernatant of the HVJ-liposome-DNA-sensitized rat coronary endothelial cells. The increase in the number of endothelial cells was determined as described above. The results are shown in
As shown in
Test Example 5 Effects of HVJ-Liposome-DNA-Sensitized Human VSMCs on Human Endothelial Cells
Human VSMCs were inoculated on a cell culture insert (manufactured by Coaster, pore diameter of 0.45 μm), and then grown in DMEM medium supplemented with 10% bovine serum. On the other hand, human endothelial cells were inoculated on a 6-well plate and maintained in DMEM medium supplemented with 10% bovine serum. When the VSMCs reached 80% confluence, the VSMCs were incubated at 4° C. for 5 minutes and at 37° C. for 30 minutes together with HVJ-liposome-DNA (DNA content in the liposomes: 10 μg) or with HVJ-liposome-cont. After sensitization, the insert containing the sensitized VSMCs was added to each well containing human endothelial cells in the stationary phase. VSMCs and the endothelial cells were co-incubated for 3 days in DMEM medium supplemented with 0.5% bovine serum. Thereafter the cell count was determined with a WST-cell counter kit (manufactured by Wako Co.). The results are shown in
The results shown in
Test Example 6 Effects of the HVJ-Liposome-DNA-Sensitized Rat VSMCs on Rat Coronary Endothelial Cells
The HVJ-liposome-DNA-sensitized rat VSMCs (cell count: 106) were co-incubated for 3 days with rat coronary endothelial cells (cell count: 105) in the stationary phase. Thereafter, the increase in the number of endothelial cells was determined. For the control, endothelial cells were co-incubated in a similar manner using the HVJ-liposome-cont-sensitized rat VSMCs, and the increase in the number of endothelial cells was determined. The results are shown in
As shown in
Test Example 7 Growth of Rat VSMCs Sensitized with HVJ-Liposome-DNA
Rat VSMCs sensitized with HVJ-liposome-DNA and rat VSMCs sensitized with HVJ-liposome-cont were incubated, respectively, to compare the increase in the cell counts therebetween. Sensitization with HVJ-liposome-DNA did not affect cell growth at all. The results reveal that HGF has no cell growth promoting effect on VSMCs.
Test Example 8 Induction of Angiogenesis in Rat Heart Muscle Directly Injected with HVJ-Liposome-DNA
Rat heart muscle directly injected with HVJ-liposome-DNA, rat heart muscle directly injected with HVJ-liposome-cont and rat no-treatment heart muscle were stained with HE and Azan, respectively, and examined on a microscope to count the number of microvessels. The results are shown in
As is seen from
Test Example 9 Repair of Articular Cartilage by Directly Introducing HVJ-Liposome-DNA into the Joint
Ten week-old Fischer's rats were injured at the femoral intercondylaris through the subcartilage using Kirschner's wires of 1.8 mm in diameter. One week after the operation, the HVJ-liposome-DNA (100 μl/knee) prepared in Example 1 was introduced directly into the joint. For the control, the HVJ-liposome-cont prepared in Comparative Example 1 and HVJ-liposome-DNA (TGF-β) prepared in Comparative Example 2 were administered directly into the joint in the same amount. Rats were sacrificed 1, 3 and 4 weeks after introduction of these genes, etc. and the histology of the repaired sites was observed.
As shown in
As shown in
INDUSTRIAL APPLICABILITY
The medicament of the present invention provides persistent therapeutic effects, as compared to HGF itself. Moreover, the medicament of the present invention may be topically applied to the target organs so that the effects can be selectively exhibited, resulting in minimizing the side effects of HGF.
