Patent 6,936,594

By introducing hepatocyte growth factor (HGF) gene and/or vascular endothelial growth factor (VEGF) gene into the subarachnoid space in humans, cerebrovascular disorders such as cerebrovascular obstruction, cerebral infarction, cerebral thrombosis, cerebral embolism, stroke, cerebral bleeding, moyamoya disease, cerebrovascular dementia, and Alzheimer's dementia can be effectively treated or prevented.

DISCLOSURE OF THE INVENTION

The present invention relates to novel gene therapy agents for treating or preventing cerebrovascular disorders, and novel methods for administration of said gene therapy agents. More preferably, the present invention relates to novel agents for treating and preventing cerebrovascular disorders comprising hepatocyte growth factor (HGF) gene and/or vascular endothelial growth factor (VEGF) gene as an active ingredient, or novel administration methods comprising administering said therapeutic or preventive agents to the subarachnoid space.

The present inventors investigated in vivo whether the introduction of HGF gene and VEGF gene can induce angiogenesis on the surface of an ischemic brain. As a result, we have revealed that: (a) after the transfection of HGF gene or VEGF gene, these proteins are detected in the brain over a prolonged period of time, (b) therapy with HGF gene or VEGF gene transfection can induce angiogenesis on the surface of an ischemic brain, (c) the transfection of HGF gene or VEGF gene is effective In treating reduced blood flow in the brain caused by vascular obstruction, and (d) the therapy is also useful when performed before obstruction. Furthermore, we have also demonstrated that the introduction of these genes can be more effectively attained by a novel method of administration i.e., introduction into subarachnoid space.

In addition, the present inventors have found that delayed neuronal death due to ischemia in the hippocampus CA-1 region can be suppressed by the introduction of HGF gene.

Based on the foregoing findings, the present invention was completed.

Thus, the present invention provides the inventions described in the following (1) to (23).

(1) A therapeutic and preventive agent for cerebrovascular disorders comprising HGF gene and/or VEGF gene as an active ingredient;

(2) The therapeutic or preventive agent in the above (1) wherein cerebrovascular disorders are cerebrovascular obstruction, cerebral infarction, cerebral thrombosis, cerebral embolism, stroke, cerebral bleeding, moyamoya disease, cerebrovascular dementia, Alzheimer's dementia, and sequelae of cerebral bleeding or cerebral infarction;

(3) A therapeutic or preventive agent for reduced blood flow in the brain comprising HGF gene and/or VEGF gene as an active ingredient;

(4) A promoting agent for angiogenesis in the brain comprising HGF gene and/or VEGF gene as an active ingredient;

(5) A suppressing agent for neuronal death in the brain comprising HGF gene as an active ingredient;

(6) The suppressing agent of the above (5) wherein neuronal death in the brain is delayed neuronal death caused by cerebral ischemia;

(7) A suppressing agent for apoptosis of nerve cells in the brain comprising HGF gene as an active ingredient;

(8) The agent in any of the above (1)-(7) which comprises HGF gene and/or VEGF gene as an active ingredient and which is to be used in combination with HGF protein and/or VEGF protein;

(9) The agent of the above (8) which comprises HGF gene as an active ingredient and which is to be used in combination with HGF protein;

(10) The agent in any of the above (1)-(9) wherein HGF gene and/or VEGF gene are in the form of HVJ-liposome;

(11) The agent in any of the above (1)-(10) to be administered into the subarachnoid space;

(12) A method of producing the agent in any of the above (1)-(11) comprising blending HGF gene and/or VEGF gene with a pharmaceutically acceptable solvent;

(13) A therapeutic or preventive method for cerebrovascular disorders comprising introducing HGF gene and/or VEGF gene into humans;

(14) A therapeutic or preventive method for reduced blood flow comprising introducing HGF gene and/or VEGF gene into humans;

(15) A method of promoting cerebral angiogenesis comprising introducing HGF gene and/or VEGF gene into humans;

(16) A method of suppressing neuronal death in the brain comprising introducing HGF gene into humans;

(17) A method of suppressing apoptosis of nerve cells in the brain comprising introducing HGF gene into humans;

(18) The method in any of the above (13)-(17) comprising administering HGF gene and/or VEGF gene into the subarachnoid space in humans;

(19) The method in any of the above (13)-(18) comprising administering HGF protein and/or VEGF protein together with the introduction of HGF gene and/or VEGF gene;

(20) The method in the above (19) comprising administering HGF protein together with the introduction of HGF gene;

(21) Use of HGF gene and/or VEGF gene in the manufacture of a therapeutic or preventive agent for cerebrovascular disorders;

(22) Use of HGF gene and/or VEGF gene in the manufacture of a therapeutic or preventive agent for reduced blood flow in the brain;

(23) Use of HGF gene and/or VEGF gene in the manufacture of a promoting agent for angiogenesis in the brain;

(24) Use of HGF gene in the manufacture of a suppressing agent for neuronal death in the brain; and

(25) Use of HGF gene in the manufacture of a suppressing agent for apoptosis of nerve cells in the brain.

BEST MODE FOR CARRYING OUT THE INVENTION

As used herein "HGF gene" means a gene that can express HGF (HGF protein). Specifically, there can be mentioned one in which cDNA of HGF as described in Nature 342:440 (1989); Patent Publication No., 2777678; Biochem. Biophys. Res. Commun. 163:967 (1989); and Biochem. Biophys. Res. Commun. 172:321 (1990) was integrated into a suitable expression vector (nonviral vector, viral vector) as described below. The base sequence of cDNA encoding HGF has been described in the above literature and also been registered at databases such as Genbank. Thus, based on such sequence information, a suitable DNA portion is used as a PCR primer; for example, by performing a RT-PCR reaction on mRNA derived from the liver or leukocytes, cDNA of HGF can be cloned. Such cloning can easily be performed by a person skilled in the art according to a basic textbook such as Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989).

Furthermore, the HGF gene of the present invention is not limited to those described above, but any gene may be used as the HGF gene of the present invention as long as the protein expressed by said gene can act in virtually the same manner as HGF. Thus, from among 1) DNA that hybridizes to said cDNA under a stringent condition and 2) DNA encoding a protein comprising an amino acid sequence in which one or a plurality of (preferably several) amino acids have been substituted in, deleted from, and/or added to the amino acid sequence of the protein encoded by said cDNA and the like, those that encode a protein having an action as HGF are encompassed in the category of HGF gene of the present invention. DNA in the above 1) and 2) may be easily obtained by site-directed mutagenesis, a PCR method, or a standard hybridization method and the like, and specifically they may be performed with reference to a basic textbook such as the above Molecular Cloning etc.

As used herein "VEGF gene" means a gene that can express VEGF (VEGF protein). Thus, there can be illustrated one integrated into a suitable expression vector (nonviral vector, viral vector) as described below. By selective splicing of VEGF gene at transcription in humans, the presence of 4 subtypes (VEGF121, VEGF165, VEGF189, VEGF206) have been reported (Science 219:983 (1983); J. Clin. Invest. 84:1470 (1989); Biochem. Biophys. Res. Commun. 161:851 (1989)). According to the present invention, any of these VEGF genes can be used, but from the viewpoint of being biologically most potent, VEGF165 gene is most preferred. Furthermore, as in the case of the above HGF, modified versions of these VEGF genes are encompassed in the category of the VEGF gene of the present invention as long as they encode a protein having an activity as VEGF.

Said VEGF gene, as in the case of HGF gene, may be easily cloned by a person skilled in the art based on sequences as previously described (for example, Science 246:1306 (1989)) and sequence information registered in databases, and their modification can also be easily performed.

According to the present invention, it was demonstrated for the first time that cerebrovascular disorders can be treated or prevented with HGF gene or VEGF gene. Thus, the present invention revealed, for the first time, that (a) after the transfection of HGF gene or VEGF gene, these proteins are detected in the brain over a prolonged period of time, (b) by treatment using HGF gene or VEGF gene transfection, angiogenesis can be induced on the surface of an ischemic brain, (c) the transfection of HGF gene or VEGF gene is effective in treating reduced blood flow in the brain caused by obstruction in the blood vessels, and (d) this treatment method is also effective when performed before obstruction. Thus, HGF gene and VEGF gene may be effectively used as a therapeutic or preventive agent for various cerebrovascular disorders such as disorders resulting from cerebral ischemia, disorders associated with reduced blood flow in the brain, disorders for which improvement is expected by promoting angiogenesis in the brain, and the like.

Specifically they are effectively used as therapeutic or preventive agents (hereinafter, the therapeutic or preventive agents of the present invention are simply designated as gene therapy agents) for cerebrovascular obstruction, cerebral infarction, cerebral thrombosis, cerebral embolism, stroke (including subarachnoid bleeding, transient cerebral ischemia, cerebral atheroscrelosis), cerebral bleeding, moyamoya disease, cerebrovascular dementia, Alzheimer's dementia, sequelae of cerebral bleeding or cerebral infarction, and the like.

Furthermore, the present inventors have found that delayed neuronal death due to ischemia in the hippocampus CA-1 region is suppressed by the introduction of HGF gene, that is. HGF gene has an effect of suppressing neuronal death in the brain. We have also demonstrated that this effect is based on the c-Met-mediated apoptosis-suppressing effect of nerve cells.

The hippocampus CA-1 region as used herein is a region that is densely populated with nerves and a region that is susceptible to neuronal death by cerebral ischemia. Such HGF gene has been found to be able to treat and prevent cerebrovascular disorders based on the both of the angiogenic effect (suppression of reduced blood flow) and the nerve cell protective effect.

Since HGF gene has c-Met-mediated nerve cell protecting effect as described above, it can be effectively used as a therapeutic or preventive agent for neurodegenerative diseases such as Alzheimer's disease, Alzheimer's senile dementia, amyotrophic lateral sclerosis, or Parkinson's disease.

In accordance with the present invention, HGF gene and VEGF gene may be used alone or in combination with each other. They can also be used in combination with the gene of other vascular endothelial growth factors. Furthermore, HGF gene and/or VEGF gene may be used in combination with HGF protein and/or VEGF protein. Preferred are a combination of HGF gene and HGF protein or of VEGF gene and VEGF protein, more preferably of HGF gene and HGF protein. See Example 4 below for details.

HGF protein as used herein may be obtained by any method as long as it has been purified to the extent it may be usable as a pharmaceutical drug. Commercially available products (for example Toyoboseki k. k., Code No. HGF-101, etc.) may also be used. cDNA of HGF obtained by cloning mentioned above is inserted into any suitable expression vector, which is introduced into a host cell to obtain a transformant, from the culture supernatant of which transformant may be obtained recombinant HGF protein of interest (see, for example, Nature 342:440 (1989): Patent Publication No. 2777678). VEGF protein can also be obtained in a similar manner.

Then, a method of gene introduction, form of introduction, amount to be introduced and the like for use in gene therapy of the present invention are explained.

When a gene therapy agent comprising the above gene as an active ingredient is to be administered to patients, the dosage regimens are roughly divided into two: a case in which a nonviral vector is used, and a case in which a viral vector is used. The methods of preparation and administration thereof are explained in detail in experimental manuals (Separate volume of Experimental Medicine, Basic Technology in gene therapy, Yodosha (1996); Separate volume of Experimental Medicine, Experimental Methods in Gene Introduction and Expression Analysis, Yodosha (1997); Handbook for Development and Research of Gene Therapy, edited by Japan Society of Gene Therapy, NTS (1999)). This will be explained in specific terms below.

A. When a Nonviral Vector is Used

Using a recombinant expression vector in which a gene of interest has been integrated into a commonly used gene expression vector may be used to introduce the gene of interest into cells or tissue by the following method etc.

As a method of gene introduction into cells, there can be mentioned the lipofection method, the calcium phosphate co-precipitation method, the DEAE-dextran method, direct DNA introduction methods using micro glass tubes, and the like.

As a method of gene introduction into the tissue, a recombinant expression vector may be incorporated into the cell by subjecting any of a method of gene introduction with internal type liposome, a method of gene introduction with electrostatic type liposome, the HVJ-liposome method, the improved HVJ-liposome method (HVJ-AVE liposome method), the receptor-mediated gene introduction method, a method of introducing DNA molecules together with carriers (metal particles) by a particle gun, a method of directly introducing naked-DNA, a method of introduction with positively-charged polymers and the like.

Among them, HVJ-liposome is a fusion product prepared by enclosing DNA into liposome made of lipid bilayer, which was fused to inactivated Sendai virus (Hemagglutinating virus of Japan: HVJ). The HVJ-liposome method is characterized by a very high fusing activity with the cell membrane compared to the conventional liposome method, and is a preferred mode of introduction. For the method of preparing HVJ-liposome, see, for details, the literature (Separate volume of Experimental Medicine, Basic Technology in gene therapy, Yodosha (1996); Experimental Methods in Gene Introduction and Expression Analysis, Yodosha (1997); J. Clin. Invest. 93:1458-1464 (1994); Am. J. Physiol. 271:R1212-1220 (1996)) and the like, and experimental examples described below for details. As HVJ, the Z strain (available from ATCC) is preferred, but other HVJ strains (for example, ATCC VR-907 and ATCC VR-105) may also be used.

Furthermore, the method of directly introducing naked-DNA is the most simple method among the methods described above, and in this regard a preferred method of introduction.

Expression vectors as used herein may be any expression vectors as long as they permit the expression in vivo of the gene of interest, and include, for example, expression vectors such as pCAGGS (Gene 108:193-200 (1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stratagene) and the like.

B. When a Viral Vector is Used

Representative methods use, as viral vectors such as recombinant adenovirus, retrovirus and the like. More specifically, the gene of interest can be introduced into DNA virus or RNA virus such as detoxified retrovirus, adenovirus. adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, human immunodeficiency virus (HIV) and the like, which is then infected to the cell to introduce the gene into the cell.

Among the above viral vectors, the efficiency of infection is known to be the highest with adenovirus than with other viral vectors. In this regard, it is preferred to use an adenovirus vector system.

As methods of introducing a gene therapy agent into a patient, there are an in vivo method that permits direct introduction of the gene therapy agent into the body, and an ex vivo method in which certain cells are removed from a human and a gene therapy agent is introduced into said cells, which are then returned into the body (Nikkei Science, April 1994 issue pp. 20-24; Monthly Yakuji, 36(1):23-48 (1994); Supplement to Experimental Medicine 12(15) (1994); Handbook for Development and Research of Gene Therapy, edited by Japan Society of Gene Therapy, NTS (1999)). According to the present invention, the in vivo method is preferred.

Sites for administration to patients are selected depending on the disease, disease state and the like to be treated. For example, in addition to making a hole directly into the cranium and introducing the gene therethrough, there is administration to the lateral ventricle or administration to the subarachnoid space. Among them, administration to the subarachnoid space is a novel and efficient method of administration that was disclosed in the present invention. The administration to the subarachnoid space is desired when it is intended to treat the disease based on the original purpose. i.e. when reduced blood flow in the brain is treated by angiogenesis and/or by suppressing neuronal death in the brain.

Dosage forms may take various forms according to various administration regimens described above (for example, liquids). When, for example, an injection containing the gene as an active ingredient is to be used, said injection may be prepared according to a standard method. For example, after dissolving in a suitable solvent (a buffer such as PBS, physiological saline, sterile water, etc.), it is filter-sterilized with filter as needed, and then filled into sterilized containers. Commonly used carriers etc. may be added to the injection. In liposomes such as HVJ-liposome, they may take the form of suspensions, frozen formulations, centrifugation-concentrated frozen formulations and the like.

In order to facilitate delivery of the gene into the periphery of a lesion site, a sustained release preparation (minipellet formulation, etc.) may be prepared and implanted near the affected region, or it can be administered to the affected area continuously and gradually using an osmotic pump etc.

The content of DNA in the formulation may be controlled as appropriate depending on the disease to be treated, age and weight of the patient, etc., and usually it is in the range of 0.0001-100 mg, preferably 0.001-10 mg, as the DNA of the present invention, which is preferably given every few days to every few months.

Claim 1 of 5 Claims

1. A therapeutic or preventive method for cerebrovascular disorders comprising introducing into a human subject a polynucleotide encoding hepatocyte growth factor (HGF) and/or a polynucleotide encoding vascular endothelial growth factor (VEGF) in the form of hemagglutinating virus of Japan (HVJ)-liposomes by direct injection into the subarachnoid space of said subject thereby treating or preventing said cerebrovascular disorders.

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