The present invention relates to a therapeutic preventive agent that includes an angiogenic factor gene (such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hypoxia inducible factor (HIF)) as its active ingredient, and the administration of such an agent into the targeted skin diseases-affected area.

DISCLOSURE OF THE INVENTION

An objective of the present invention relates to a therapeutic or preventive agent for skin diseases that uses an angiogenic factor gene, and the use of these pharmaceutical agents.

The present inventors considered that HGF, which is an angiogenic factor, might promote epithelial repair and angiogenesis during wound healing. The present inventors investigated (i) whether, following gene transfer, human HGF mRNA and protein might distribute and deposit within full-thickness of wounds, (ii) whether the genetically transferred protein might be biologically active, and (iii) whether the transferred protein might have a biological effect on pathological conditions (for example, mitogen activity involving several cells within full-thickness of wounds, as well as re-epithelization in granulation tissues, angiogenesis, and deposition of the extracellular matrix, etc.).

Changes in wound tissues were also investigated to determine whether they related to TGF-.beta.1 secretion. Measurements were made of the wound area, the concentration of human and rat HGF protein in wound tissue after HGF gene transfer, and the expression of the mRNA of other constitutive factors thought to be involved in wound healing such as TGF-.beta.1, collagen type I (Col.alpha.2 (I)), collagen type III (Col.alpha.1 (III)) desmin, and vascular smooth muscle .alpha.-actin (.alpha.-sm-actin) A semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used for these measurements. Morphogenic changes in the wound were investigated by in situ hybridization and immunohistochemical methods.

With these results, the present inventors demonstrated that direct administration of an angiogenic factor gene to a skin diseases-affected area is extremely effective. Specifically, it was found that in skin wounds, administration of an angiogenic factor gene yields effective results.

Because therapy with an angiogenic factor gene is non-invasive, the gene can be administered any number of times depending on the condition of the disease.

Specifically, the subject matter of this invention is as follows: (1) a therapeutic or preventive agent for skin diseases comprising an angiogenic factor gene as the active ingredient; (2) the therapeutic or preventive agent according to (1) wherein the angiogenic factor gene is an HGF gene, VEGF gene, FGF gene, or HIF gene; (3) the therapeutic or preventive agent according to (1), wherein the skin diseases is a wound, alopecia (baldness), skin ulcer, decubitus ulcer (bedsore), scar (keloid), atopic dermatitis, or skin damage following a skin graft including autotransplantation and crosstransplantation; (4) the therapeutic or preventive agent according to (1) or (2), wherein said therapeutic or preventive agent is in the form of a tablet, pill, sugar-coated agent, capsule, liquid preparation, gel, ointment, syrup, slurry, or suspension; (5) the agent according to any one of (1) to (3), wherein said agent is used for transferring a gene into a cell by employing liposome entrapment, electrostatic liposomes, HVJ-liposomes, improved HVJ-liposome, viral envelope vectors, receptor-mediated gene transfer, transfer of DNA into a cell using a particle gun (gene gun) direct introduction of naked-DNA, DNA transfer into a cell by ultrasonication, electroporation, or introduction using a positively charged polymer; (6) a method for treating or preventing skin diseases, wherein the method comprises introduction of an angiogenic factor gene into a mammal; and (7) use of an angiogenic factor gene for producing a therapeutic or preventive agent for skin diseases.

The term "angiogenic factor gene" used in the present invention refers to a gene that can express an angiogenic growth factor. Herein, the term "angiogenic factor" refers to a growth factor that has not only been shown to stimulate in vivo neovascularization and angiogenesis (initiated along with activation of endothelial cells of the parent blood vessel), but has also been shown to be mitogenic for endothelial cells in vitro. Examples of the factor include HGF, VEGF, FGF, and HIF described hereinafter.

In the present invention, the term "HGF gene," as employed herein, refers to a gene that can express HGF (HGF protein). Specifically, the gene includes HGF cDNA (such as that described in Nature, 342, 440 (1989), Japanese Patent Publication No. 2577091, Biochem. Biophys. Res. Commun., 163, 967 (1989), Biochem. Biophys. Res. Commun., 172: 321 (1990)) where incorporated into appropriate expression vectors (e.g. non-viral vectors, viral vectors), such as those mentioned below. The nucleotide sequence of the cDNA encoding HGF is described in the aforementioned literature. The sequence is also registered in databases such as Genbank. Thus, by using DNA segments appropriate to the DNA sequence as PCR primers, HGF cDNA can be cloned in an RT-PCR reaction, using, for example, mRNA derived from liver or leukocytes. This cloning can be readily performed by one skilled in the art by referring to texts such as Molecular Cloning Second Edition, Cold Spring Harbor Laboratory Press (1989).

The term "VEGF gene," as employed herein refers to a gene that can express VEGF (VEGF protein). Specifically, such a gene is exemplified by VEGF cDNA incorporated into appropriate expression vectors (e.g. non-viral vectors, viral vectors) such as those mentioned below. Due to selective splicing during transcription, there are four subtypes of the VEGF gene in humans (VEGF121, VEGF165, VEGF189, and VEGF206) (Science, 219, 983 (1983); J. Clin. Invest., 84, 1470 (1989); Biochem. Biophys. Res. Commun., 161, 851 (1989)). Any of these VEGF genes can be used in the present invention. However, the VEGF165 gene is preferred as its biological activity is the strongest of the VEGF genes. The VEGF gene can also be readily cloned by one skilled in the art, based on the sequences described in the literature (Science, 246, 1306 (1989)) and the sequence information registered in databases. Modification of the VEGF gene can also be easily carried out.

The terms "FGF gene" and "HIF gene" as employed herein refer to genes that can express FGF and HIF respectively. Such genes are exemplified by genes incorporated into appropriate expression vectors (e.g. non-viral vectors, viral vectors) such as those mentioned below. Such genes can also be readily cloned by one skilled in the art, based on the sequences described in known literature and sequence information registered in databases. Modifications of these genes can also be easily carried out.

The angiogenic factor gene of the present invention is not limited to those mentioned above. So long as the protein expressed by the gene is effective as an angiogenic factor, the gene can be used as the angiogenic factor gene of the present invention. More specifically, the angiogenic factor gene of the present invention encompasses: 1) DNA that hybridizes under stringent conditions to the aforementioned cDNA; 2) DNA encoding a protein with the amino acid sequence of the protein encoded by the aforementioned cDNA, wherein one or more (preferably several) amino acids are substituted, deleted, and/or added; and such, so long as the DNA encodes a protein which is effective as the angiogenic factor of this invention. The DNA described above in 1) and 2) can be readily obtained, for example, by employing site-directed mutagenesis, PCR (Current Protocols in Molecular Biology edit., Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4), conventional hybridization (Current Protocols in Molecular Biology edit., Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4), etc.

Specifically, those skilled in the art can isolate DNA that hybridizes with a DNA described above by using the above-mentioned angiogenic factor gene or part thereof as a probe, or by using as a primer an oligonucleotide which specifically hybridizes with the angiogenic factor. Typical stringent hybridization conditions for isolating DNA encoding a protein functionally equivalent to the angiogenic factor are those of "1.times.SSC, 37.degree. C." or the like; more stringently, those of "0.5.times.SSC, 0.1% SDS, 42.degree. C." or the like; much more stringently, those of "0.1.times.SSC, 0.1% SDS, 65.degree. C." or the like. As the hybridization conditions become more stringent, DNA more homologous to the probe sequence can be isolated. However, the above combinations of SSC, SDS, and temperature are only examples, and those skilled in the art can achieve stringencies equivalent to the above by appropriately combining these or other conditions that determine hybridization stringency (probe concentration, probe length, time of reaction, etc.).

When compared to proteins of known angiogenic factor, proteins encoded by genes isolated using hybridization or PCR typically demonstrate high homology at the amino acid level. The term "high homology" means sequence homology of at least 50% or more, preferably 70% or more, more preferably 90% or more (for example, 95% or more). The identity of amino acid and nucleotide sequences can be determined using the BLAST algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Based on this algorithm, programs such as BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When nucleotide sequences are analyzed by BLAST-based BLASTN, the parameters are set, for example, as follows: score=100; and wordlength=12. Alternatively, when amino acid sequences are analyzed by BLAST-based BLASTX, the parameters are set, for example, as follows: score50; and wordlength=3. When BLAST and the Gapped BLAST program are used for the analysis, default parameters are used in each program. The specific techniques used in these analysis methods are already known (see, for example, the National Center for Biotechnology Information web site).

The following describes methods, forms, and amounts of gene transfer when gene therapy is employed as per the present invention.

When a gene therapy agent with the HGF gene as its active ingredient is administered to a patient, the form of administration can be classified into two groups: that using a non-viral vector, and that using a viral vector. Methods for the preparation and administration of these vectors are described in detail in experiment manuals (Jikken Igaku (Experimental Medicine) Supplementary Volume, "Idenshichiryo no Kisogijyutsu (Fundamental Techniques for Gene Therapy)", Yodosha, 1996; Jikken Igaku (Experimental Medicine) Supplementary Volume, "Idenshidonyu & Hatsugenkaiseki Jikkenho (Experimental Methods for Gene Transfer & Expression Analysis)", Yodosha, 1997; "Idenshi-chiryo Kaihatsu Kenkyu Handbook (Handbook of Gene Therapy Research and Development)", Nihon Idenshichiryo Gakkai (The Japan Society of Gene Therapy) Edition, NTS, 1999). Detailed explanations are given below.

A. Use of Non-Viral Vectors

A recombinant vector (where the target gene has been inserted into a conventional gene expression vector) can be used to insert a target gene into cells and tissues as per the methods below.

Examples of methods for gene transfer into cells include: lipofection, calcium phosphate co-precipitation, the use of DEAE-dextran, direct infusion of DNA using a glass capillary tube, electroporation, etc.

Methods for gene transfer into tissues include the use of: internal type liposomes, electrostatic type liposomes, HVJ (hemagglutinating virus of Japan)-liposomes, improved type HVJ-liposomes (HVJ-AVE liposomes), viral envelope vectors, receptor-mediated transfer, gene guns (the use of a particle gun to import a carrier such as metal particles along with DNA)., direct introduction of naked-DNA, positively charged polymers, ultrasonic irradiation, etc.

The aforementioned HVJ-liposome is constructed by incorporating DNA into a liposome formed by a lipid bilayer, then fusing this liposome with an inactivated Sendai virus (hemagglutinating virus of Japan: HVJ). The use of HVJ-liposomes is characterized by extremely high cell membrane fusion compared to conventional liposome methods, and is the preferred form of introduction. Methods of preparing HVJ-liposomes have been described in detail (Experimental Medicine Supplementary Volume, "Idenshichiryo no Kisogijyutsu (Fundamental Techniques of Gene Therapy)", Yodosha, 1996; Experimental Medicine Supplementary Volume, "Idenshidonyu & Hatsugenkaiseki Jikkenho (Experimental Methods for Gene Transfer & Expression Analysis)", Yodosha (1997); J. Clin. Invest. 93: 1458-1464(1994); Am. J. Physiol. 271: R1212-1220(1996), etc.). Use of the HVJ-liposome in transfection also includes, for example, the methods described in Molecular Medicine 30: 1440-1448(1993); Experimental Medicine, 12: 1822-1826(1994); Protein, Nucleic Acid, and Enzyme, 42, 1806-1813(1997); and preferably includes the method described in Circulation 92(Suppl. II): 479-482(1995).

The Z strain (available from ATCC) is the preferred HVJ strain, however in essence, other HVJ strains (for example, ATCC VR-907, ATCC VR-105, etc.) may be used.

Herein, the term "viral envelope vector" refers to a vector that incorporates a foreign gene into a viral envelope. Viral envelope vectors are gene transfer vectors in which the viral genome has been inactivated. Since viral proteins are not produced, the vector is safe and its cytotoxicity and antigenicity are low. By incorporating a gene into such a viral envelope vector (e.g. one that uses an inactivated virus), a highly efficient gene transfer vector that is safe for use with cultured cells and biological tissues can be prepared. Viral envelope vectors can be prepared, for example, using the method described in WO 01/57204 (PCT/JP01/00782). Examples of viruses used to prepare gene transfer vectors include both wild-type viruses and recombinant viruses, and such examples include Retroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxyiridae, Herpesviridae, Baculoviridae, and Hepadnaviridae. A viral envelope vector using HVJ is particularly suitable. Furthermore, a gene transfer vector can be prepared using a recombinant Sendai virus, as described by Hasan, M. K. et al. (Journal of General Virology, 78, 2813-2820 (1997)) or Yonemitsu, Y. et al. (Nature Biotechnology 18, 970-973 (2000)).

Direct transfer of naked-DNA is the most convenient of the methods mentioned above, and is thus the preferred method of introduction.

With respect to the present invention, any expression vector can be used so long as it can express the desired gene in vivo, and includes, for example, pCAGGS (Gene, 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, or pZeoSV (Invitrogen, Stratagene).

B. Use of Viral Vectors

Viral vectors such as recombinant adenoviruses and retroviruses are typically used. More specifically, a desired gene is introduced into a DNA or RNA virus, such as an avirulent retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, or immunodeficiency virus (HIV). The recombinant virus is then infected into the cell, thus introducing the desired gene.

Of the viral vectors mentioned above, the infection efficiency of adenoviruses is known to be much higher than other viral vectors. Thus, the use of the adenovirus vector system is preferred.

Methods for introducing an agent of the present invention during gene therapy include: (i) in vivo introduction of a gene therapy agent directly into the body; and (ii) ex vivo introduction of a gene therapy agent into a cell harvested from the body, followed by reintroduction of the modified cell into the body (Nikkei Science, April 1994, 20-45; Gekkann Yakuji 36 (1), 23-48, 1994; Jikken Igaku (Experimental Medicine) Supplementary Volume, 12 (15), 1994; "Idenshi-chiryo Kaihatsu Kenkyu Handbook (Handbook of Gene Therapy Research and Development)", Nihon Idenshichiryo Gakkai eds. (The Japan Society of Gene Therapy) Edition, NTS, 1999). The in vivo method is preferred in the present invention.

Various formulations (for example, liquid preparations) suitable for each of the above-mentioned methods of administration may be adopted as the form of the preparation. For example, an injection containing a gene as the active ingredient can be prepared by conventional methods which might include dissolving a gene in an appropriate solvent (e.g. a buffer solution, such as PBS, physiological saline, and sterilized water); sterilizing by filtration as necessary, and then loading into a sterile container. Conventional carriers and such like may be added to the injection as required. Alternatively, liposomes such as the HVJ-liposome can be prepared as suspensions, frozen agents, or centrifugally concentrated frozen agents.

For skin diseases, a therapeutic or preventive agent of this invention may be locally administered to the affected area of the skin, preferably in the form of an ointment. This ointment is an entirely homogenous semi-solid external agent with a firmness appropriate for easy application to the skin. Such an ointment normally includes fats, fatty oils, lanoline, Vaseline, paraffin, wax, hard ointments, resins, plastics, glycols, higher alcohols, glycerol, water or emulsifier and a suspending agent. Using these ingredients as a base, a decoy compound can be evenly mixed. Depending on the base, the mixture may be in the form of an oleaginous ointment, an emulsified ointment, or a water-soluble ointment oleaginous ointments use bases such as plant and animal oils and fats, wax, Vaseline and liquid paraffin. Emulsified ointments are comprised of an oleaginous substance and water, emulsified with an emulsifier. They may take either an oil-in-water form (O/W) or a water-in-oil-form (W/O). The oil-in-water form (O/W) may be a hydrophilic ointment. The water-in-oil form (W/O) initially lacks an aqueous phase and may include hydrophilic Vaseline and purified lanoline, or it may contain a water-absorption ointment (including an aqueous phase) and hydrated lanoline. A water-soluble ointment may contain a completely water-soluble Macrogol base as its main ingredient.

A pharmaceutically acceptable and preferable carrier is Vaseline containing 5% stearyl alcohol, or Vaseline alone, or Vaseline containing liquid paraffin. Such carriers enable pharmaceutical compositions to be prescribed in forms appropriate for patient consumption, such as tablets, pills, sugar-coated agents, capsules, liquid preparations, gels, ointments, syrups, slurries, and suspensions.

Alternatively, when locally administered into cells in an affected area or a tissue of interest, a therapeutic or preventive agent of this invention may contain a synthetic or natural hydrophilic polymer as the carrier. Examples of such polymers include hydroxypropyl cellulose and polyethylene glycol. A gene or vector of the present invention is mixed with a hydrophilic polymer in an appropriate solvent. The solvent is then removed by methods such as air-drying, and the remainder is then shaped into a desired form (for example, a sheet) and applied to the target site. Formulations containing such hydrophilic polymers keep well as they have a low water-content. At the time of use, they absorb water, becoming gels that also store well. In the case of sheets, the firmness can be adjusted by mixing a polyhydric alcohol with a hydrophilic polymer similar to those above, such as cellulose, starch and its derivatives, or synthetic polymeric compounds. Hydrophilic sheets thus formed can be used as the above-mentioned sheets.

Genes selected from angiogenic factor genes such as those used in the present invention (e.g. HGF, VEGF, FGF, HIF, etc.) may be used in multiple combinations or alone. Furthermore, factors other than the angiogenic factors mentioned above, and which are known to have an angiogenic effect, may also be used in combination or alone. For example, factors such as EGF have been reported to have an angiogenic effect, and such genes can be used. Furthermore, growth factors such as EGF have been reported to repair a variety of tissue cell injuries, and such genes may also be used.

Skin diseases according to the present invention includes wounds, alopecia (baldness), skin ulcers, decubitus ulcers (bedsores) scars (keloids), atopic dermatitis, and skin damage following skin grafts such as autotransplantation and crosstransplantation. Preventive agent, according to the present invention, refers to a pharmaceutical agent which prevents the onset (or incidence) of the above-mentioned diseases, or a pharmaceutical agent which reduces symptoms caused by the above-mentioned diseases, or a pharmaceutical agent which accelerates amelioration of these symptoms. These preventive agents are also included in the present invention.

Herein, "alopecia" refers to the phenomenon of thinning hair, where the hair cycle becomes extremely short, such that even thick hair falls out mid-growth, and as a result, the hairs that do grow are soft, fine and short. The expression "skin ulcers" means damage to deep tissues, reaching to the dermis or to the hypodermal tissue. Skin ulcers are categorized into ischemic ulcers, congestive ulcers, diabetic ulcers, decubitus ulcers, radiation ulcers instillation leakages, etc. "Decubitus ulcers" refers to a pathological condition where necrosis occurs by occlusion of a tissue's peripheral blood vessels due to continuous compression experienced at the contact surface of the body. Decubitus ulcers are intractable ulcers having a dry necrotic mass with a clear border, which form at sites of long-term compression, such as the back of the head, the back, and the hips of bedridden people. "Scars (keloids)" occur after skin damage and means hypertrophy of the connective tissue in which the wound surface produces a flat protrusion and sometimes forms claw-like projections. Some scars are proliferative, and continue to expand to the surrounding region, and beyond the original wound site. Factors that cause an external wound to form a keloid include systemic factors (such as genetic factors, age, and hormonal factors), and local factors (such as susceptibility to scars depending on the part of the body). Scars are categorized into hyperplastic scars, keloid scars, true keloids, etc.

Administration sites and methods for gene therapy agents of the present invention are selected such that they are appropriate to the disease and symptoms to be treated. The preferred administration method is parenteral administration. Furthermore, the preferred administration site is at the site of skin diseases. Herein, the term "site of skin diseases" refers to a site including the skin diseases-affected area and its surrounding region.

Specifically, administration to the skin diseases site can be carried out intravascularly, intramuscularly, and such, as well as by administration to surface layers with ointments and such Therefore, at the sites of wounds, baldness, decubitus ulcers (bedsores), keloids, atopic dermatitis, and skin grafts such as autotransplantation and crosstransplantation, angiogenesis in the affected area is enhanced, and blood flow is improved by intravascular and intramuscular administration using a syringe or catheter, or by surface application using an ointment or such. In this way, the function of the affected area can be recovered and normalized.

Application of an HGF gene of the present invention by active gene transfer allows treatment of wounds, baldness, skin ulcers, decubitus ulcers (bedsores), scars (keloids), atopic dermatitis, and skin damage following skin grafts such as autotransplantation and crosstransplantation, and enables functional recovery in patients for whom conventional therapeutic methods are not an appropriate option. A therapeutic or preventive agent of the present invention contains an angiogenic factor gene in an amount sufficient to accomplish the objectives intended by the pharmaceutical agent, i.e. it contains an angiogenesis gene in a "therapeutically effective amount" or a "pharmacologically effective amount". A "therapeutically effective amount" or "pharmacologically effective amount" is an amount of pharmaceutical agent required to produce the intended pharmacological results, and is the amount required to relieve the symptoms of the patient to be treated. Assays useful in confirming the effective dose for a particular application include methods for measuring the degree of recovery from target diseases. The amount that should actually be administered varies depending on the individual being treated, and is preferably an amount optimized to achieve the desired effects without marked side effects.

Therapeutically effective amounts, pharmacologically effective amounts, and toxicity can be determined by cell culture assays or optionally, by using appropriate animal models. Such animal models can be used to determine the desired concentration range and administration route for the pharmaceutical agent. Based on these animal models, one skilled in the art can determine the effective dose in a human. The dose ratio of therapeutic effect to toxic effect is called the therapeutic index, and this can be expressed as the ratio ED50:LD50. Pharmaceutical compositions with a large therapeutic index are preferred. An appropriate dose is selected according to the dosage form, the patient's sensitivity, age and other conditions, and the type and severity of the disease. Although the dose of a therapeutic agent of the present invention differs depending on the condition of the patient, the adult dose of an HGF gene is in the range of approximately 1 .mu.g to approximately 50 mg, preferably in the range of approximately 10 .mu.g to approximately 5 mg, and more preferably from the range of approximately 50 .mu.g to approximately 5 mg.

A therapeutic agent of the present invention is preferably administered once every few days or few weeks, where the frequency of administration is selected such that it is appropriate to the patient's symptoms. A characteristic of the therapeutic agent of the present invention is that, due to its non-invasive administration, it can be administered any number of times depending on the symptoms.

With regards to the present invention, there are no restrictions regarding the animal into which the angiogenic factor gene can be transferred, however mammals are preferred. Examples of mammals include, without limitation, humans, and non-human mammals such as monkeys, mice, rats, pigs, cows and sheep.
 

Claim 1 of 7 Claims

1. A method for accelerating the initial stage of skin wound healing, comprising direct administration of a plasmid or viral vector encoding a full length hepatocyte growth factor (HGF) to the wound, wherein the HGF is expressed, thereby accelerating the initial stage of skin wound healing.

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