The present invention relates to novel pharmaceutical compositions for angiogenic therapy. More specifically, the present invention relates to hovel pharmaceutical compositions for angiogenic therapy that contain, as the active ingredients a gene encoding angiogenesis factor and a prostacyclin synthase gene for angiogenic therapy, etc.
Development of new blood vessels and angiogenesis are initiated along with activation of endothelial cells of parental blood vessels. Growth factors that have been shown, in addition to the stimulation of such angiogenesis in vivo, to function mitogenically toward endothelial cells in vitro are termed "angiogenesis factor (angiogenesis growth factor)".
The first therapeutic application of angiogenesis factor was reported by
The strategy wherein angiogenesis factors are used for treating angiopathy (as mentioned above) is referred to as "angiogenic therapy." Recently, extremely active research on angiogenic therapy is in progress for ischemic diseases and arterial diseases using genes of above-mentioned angiogenesis factors.
For example, the present inventors have elucidated the effectiveness of HGF genes against arteriosclerosis obliterans (ASO) (
Furthermore, the effectiveness of the VEGF gene on swine myocardial ischemia model (
Regarding the bFGF gene, it has been reported that the number of blood vessels increase due to intramuscular introduction of the bFGF gene into a mdx mouse, a model for muscular dystrophy (
Prostacyclin (prostaglandin I2; PGI2), a kind of prostaglandin, is an unstable lipid mediator having a half-life of 5 to 10 minutes (
As mentioned above, substances, such as PGI2, that have vasodilating effect and platelet aggregation inhibitory effect are known to be effective against various types of angiopathies. However, these substances have never been used in combination in the aforementioned angiogenic therapy with the HGF gene, and it has not been determined as to what kind of effects can be expected by such combination.
Furthermore, angiogenesis factors, such as HGF, VEGF, bFGF, and EGF, are known to enhance the expression of ets-1 (erythroblastosis virus oncogene homolog 1), a transcription regulatory factor, and activate various types of factors involved in angiogenesis via the ets-1 (
The objective of the present invention is to provide novel pharmaceutical compositions for angiogenic therapy. The object of the present invention is to provide novel pharmaceutical compositions for angiogenic therapy that contain, as the active ingredient, a gene encoding an angiogenesis factor and a prostacyclin synthase gene for angiogenic therapy, etc.
The present inventors examined the effect of the combined use of a gene of the PGI2-synthesizing enzyme (PGI2 synthase, hereinafter referred to as "PGIS") in angiogenic therapy along with the HGF gene. No drug indicating a satisfying effect by the combination in general angiogenic therapy using a gene of an angiogenesis factor has been found so far. Furthermore, effects of combined application with other genes have not been elucidated so far.
As a result of examination using a mouse hind limb ischemia ASO model, it has been revealed that the combined application of HGF gene or VEGF gene with PGIS gene show an unexpectedly remarkable improvement in hind limb blood flow, compared to the use of each of these genes alone. Furthermore, for the first time, the PGIS gene was found to reinforce the angiogenic effect of the HGF gene or VEGF gene, and to express an angiogenic effect even used alone.
According to the above-mentioned result, it was revealed that combined application of substances, such as PGI2, or substances producing them (such as the PGIS gene), having vasodilating effect or platelet aggregation inhibitory effect is extremely effective in angiogenic therapy wherein a gene of an angiogenesis factor is used.
The present invention was accomplished based on the above-mentioned findings.
More specifically, the subjects of the present invention are:
The phrase "gene encoding an angiogenesis factor," as employed herein for angiogenic therapy refers to a gene that encodes a protein or polypeptide that can induce the formation of new blood vessels or parts thereof. The genes are HGF gene and VEGF gene; the HGF gene being more preferable. The genetic sequences of these genes are registered in public.databases and by utilizing these databases, one skilled in the art can readily clone the above-mentioned genes.
Hereinafter, the invention is explained using HGF gene and VEGF gene as the example.
In the present invention, the term "HGF gene," as employed herein refers to a gene that encodes HGF (HGF protein). In addition, an HGF gene incorporated into an expression plasmid to be expressed may also be simply referred to as "HGF gene." Specifically, the gene includes cDNAs of HGF, such as those described in
The HGF gene of the present invention is not limited to those mentioned above. So long as the protein expressed from the gene substantially has the same angiogenic effect as HGF, the gene can be used as the HGF gene of the present invention. More specifically, the HGF gene of the present invention encompasses: 1) DNAs that hybridize under stringent conditions to the aforementioned cDNA; 2) DNAs encoding a protein consisting of 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 they encode a protein with angiogenic effect. The above DNAs of 1) and 2) can be readily obtained, for example, by site-directed mutagenesis method, PCR method, conventional hybridization methods, etc. Specifically, these methods can be performed by referring to the aforementioned reference, such as
The term "VEGF gene," as employed herein refers to a gene encoding VEGF protein. A VEGF gene incorporated into an expression plasmid to be expressed may also be simply referred to as the "VEGF gene." Specifically, such a gene is exemplified by a cDNA of a VEGF incorporated into an appropriate expression vector (non-virus vector, virus vector) such as those mentioned below. Regarding the VEGF genes in humans, the existence of four kinds of subtypes (VEGF121, VEGF165, VEGF189, and VEGF206) due to selective splicing during transcription have been reported (
Similar to the HGF gene, the VEGF gene can also be readily cloned by one skilled in the art based on the sequence described in the literature (for example,
Whether the above-mentioned HGF gene, VEGF gene, or genes encoding the modified forms of them possess angiogenic effect can be investigated, for example, via in vitro measuring the proliferative effect on vascular endothelial cells that is described in
The above-mentioned genes encoding angiogenesis factors can be used alone or in combination in the angiogenic therapy of the present invention.
According to the Example mentioned below, for the first time it was revealed that the combined use of prostacyclin synthase gene (PGIS gene) in angiogenic therapy with HGF gene yields an unexpectedly remarkable effect. More specifically, it was demonstrated for the first time that a synergistic effect exceeding the sum of the effect of HGF gene alone and PGIS gene alone is achieved by the combination.
Herein, PGI2 synthesized by PGIS has vasodilating effect, vascular permeability enhancing effect, and platelet aggregation inhibitory effect as mentioned above. Therefore, the reason for the aforementioned synergistic effect may be that the combined use of the HGF gene and PGIS gene provided an environment wherein the HGF can readily function at the ischemic site; i.e., an environment wherein angiogenesis by the HGF occurs easily through the effects, such as vasodilating effect and platelet aggregation inhibitory effect, possessed by PGI2. As a result, this caused the aforementioned effect beyond expectation.
The phrase "substances having vasodilating effect," as employed herein includes all of the known substances having vasodilating effect (commercially available vasodilating agents, etc.), and may be any substance including, such as genes, proteins, and low molecular weight compounds. Specifically, the following substances can be presented as the examples.
The above term "substances that produce substances having vasodilating effect and/or platelet aggregation inhibitory effect" refers to substances that synthesize, produce, or induce the aforementioned substances having vasodilating effect and/or platelet aggregation inhibitory effect. Specifically, they indicate substances that synthesize, produce, or induce the aforementioned substances that increase prostaglandin. These substances are the PGIS gene.
The genetic sequences of all of these genes are registered in public databases, and those skilled in the art can readily clone the genes using these registered genes.
An example wherein the PGIS gene is used is explained below.
Herein, the term "PGIS gene" refers to a gene that encodes a PGIS protein. The PGIS gene incorporated into an expression plasmid so as to be expressed may also be simply referred to as the "PGIS gene". Specifically, a cDNA of PGIS described in
As with the HGF gene and VEGF gene, the PGIS gene can also be cloned readily by one skilled in the art based on the sequence described in the aforementioned literature, or on the sequence information registered in the database. The PGIS gene can also be modified easily. Whether a protein encoded by the gene has the desired PGIS activity can be measured, for example, by enzyme immunoassay using 6-keto Prostaglandin F1 a enzyme immunoassay kit (Cayman, catalogue No. #515211), or by a method for detecting metabolites of the prostacyclin synthase by thin layer chromatography (TLC). Alternatively, the effect of enhancement of angiogenic effect due to an angiogenesis factor can be measured by measuring the effect of its combined use with an angiogenesis factor on a mouse hind limb ischemia model described below in the Example.
Below appear the method for introduction, form of introduction, and amount of introduction of the pharmaceutical compositions for angiogenic therapy according to the present invention.
To use a gene encoding an angiogenesis factor and a gene, such as the aforementioned PGIS gene, in combination, i.e., a combined application of two or more genes, both genes need to take the form of an agent for gene therapy. Representative combinations include the combination of the HGF gene and PGIS gene, or the VEGF gene and PGIS gene.
The form of administration to administer the agent for gene therapy to a patient can be classified into two groups, one using a non-virus vector, and the other using a virus vector. The method of preparation and administration thereof are described in detail in experiment manuals (
Using a recombinant expression vector, a conventional gene expression vector introduced with an object gene, the object gene can be introduced into cells and tissues by the following method.
Examples of methods of gene transfection into cells include: calcium phosphate co-precipitation method, method of direct infusion of DNA using a glass capillary tube, etc.
Examples of gene transfection into tissues include: the method of gene transfection by internal type liposome, method of gene transfection by electrostatic type liposome, HVJ (hemagglutinating virus of Japan)-liposome method, improved type HVJ-liposome method (HVJ-AVE liposome method), receptor-mediated method of gene transfection, method of importing a carrier (metal particles) along with a DNA molecule into cells by particle gun, method of direct introduction of naked-DNA, method for introduction by a positively charged polymer, and so on. The recombinant expression vector can be introduced into a cell using any of these methods. Among these methods, the method of direct introduction of naked-DNA is most convenient, and thus is a preferred method for introduction from that perspective. Alternatively, due to its extremely high fusion activity with the cell membrane compared to conventional liposome methods, the HVJ-liposome method is a preferred form for transfection. Although the Z strain. (obtained from ATCC) is preferred as the HVJ, fundamentally, other HVJ strains (for example ATCC VR-907 , ATCC VR-105, and such) can be also used.
Any expression vector can be used in the present invention so long as it can express the desired gene in vivo, and includes, for example, pCAGGS (
The two or more genes mentioned above can be transfected into the body simultaneously as a mixture of two or more recombinant expression vectors, which were prepared by incorporating the genes into discrete expression vectors, or separately with a time interval. Alternatively, a single expression vector wherein the two or more genes are incorporated into one expression vector, can be also introduced. Furthermore, with the aforementioned liposomal preparations, transfection can be carried out by enclosing two or more recombinant expression vectors into one liposome, or by enclosing each recombinant expression vector into separate liposomes.
Examples of virus vectors include recombinant adenovirus, retrovirus, etc. More specifically, a gene is introduced into a cell by introducing a desired gene into a DNA virus or RNA virus, such as avirulent retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, and immunodeficiency virus (HIV); the recombinant virus is infected into the cell.
Among the aforementioned virus vectors, the infection efficiency of adenoviruses is known to be much higher than other virus vectors. Thus, from this perspective, the use of the adenovirus vector system is preferred.
Similar to the above-mentioned non-virus vector, these adenovirus vectors mentioned above can be introduced simultaneously as a mixture, or separately with a time interval by preparing recombinant expression vectors introduced with respective two or more genes. Alternatively, a single recombinant expression vector wherein two or more genes are incorporated into one expression vector can be introduced.
Furthermore, two or more genes can be introduced into the living body using both of the aforementioned techniques using non-virus vector and virus vector.
Methods for introducing the agent of the present invention for gene therapy include: (i) the in vivo method that introduces the agent for gene therapy directly into the body; and (ii) the ex vivo method that harvests a certain type of cell from the body, introduces the agent for gene therapy into the cell outside the body, and then returns the modified cell to the body (
When administering by the in vivo method, administration is carried out via an appropriate administration route depending on the disease to be treated, target organ, and so on. For example, the administration can be intravenous, intra-arterial, subcutaneous, intradermal, intramuscular, etc., or via direct local administration to the lesion itself.
Various formulations (for example, liquid preparations, etc.) suitable for each of the above-mentioned forms of administration may be adopted as the form of the preparation. For example, to prepare an injection containing a gene as the active ingredient, the injection can be prepared by conventional methods, for example, by dissolving in an appropriate solvent (buffer solution, such as PBS, physiological saline, sterilized water, etc.); sterilizing by filtration through a filter as necessary, and then loading into a sterile container. A conventional carrier and such may be added as required to the injection. Alternatively, a liposome, such as HVJ-liposome, can be in the form of liposome preparations, such as suspension, frozen agent, or centrifugally concentrated frozen agent.
Furthermore, to facilitate the presence of the genes around the diseased site, a controlled release preparation (miniature pellet preparation, etc.) can be prepared and implanted near the affected area. Alternatively, continuous and gradual administration to the affected area using an osmotic pump is available.
The aforementioned two or more recombinant expression vectors may take different formulations, or it may be a formulation of a mixed combined agent.
The amount of the genes contained in the preparation can be adjusted appropriately depending on the disease to be treated, age and weight of the patient, etc.; however, generally it is preferred that 0.0001 to 100 mg, or preferably 0.001 to 10 mg of each gene is administered once every few days or every few months.
When a gene encoding an angiogenesis factor and a low molecular weight compound, protein, peptide, etc. are used in combination, the gene encoding the angiogenesis factor should be in the form of the aforementioned agent for gene therapy. On the other hand, low molecular weight compounds and such are administered orally or parenterally in a conventional form of a pharmaceutical composition. Representative combinations include the combination of the HGF gene and PGI2 derivative, the VEGF gene and PGI2 derivative, and so on.
Explanation on pharmaceutical compositions containing the aforementioned low molecular weight compound, protein, and so on as the active ingredient are described in the following.
The administration method, dose, etc. of the aforementioned low molecular weight compounds or proteins that are already commercially available as vasodilating agents or platelet aggregation inhibitory agents (antiplatelet agents) can be set according to the statement of virtues. However, in general, examples of the form of administration and method of administration are the following.
For oral administration, it can be administered in an administration form conventionally used in the art. For parenteral administration, it can be administered in administration forms such as local administration agent (transdermal agent, etc.), rectal administration agent, injection, and nasal agent.
Examples of oral agents or rectal administration agents include capsules, tablets, pills, powders, drops, suppositories, liquid preparations, etc. Examples of injections include sterile solutions, suspensions, emulsions, and such; and specifically, water, water-propylene glycol solution, buffering solution, 0.4% physiological saline, and such can be presented as examples. Local administration agents include, for example, cream, ointment, lotion, transdermal agents, and such.
The above-mentioned dosage forms are formulated with pharmaceutically acceptable fillers and additives by methods conventionally performed in the art. Pharmaceutically acceptable fillers and additives include carriers, binders, perfume, buffers, thickeners, coloring agents, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, pH regulating agents, tonicity regulating agents, lubricants, and such. Pharmaceutically acceptable carriers include, for example, magnesium carbonate, lactose, pectin, starch, methyl cellulose, and such.
Such pharmaceutical compositions can be administered via an appropriate administration route depending on the disease to be treated, target organ, and such. For example, the administration can be intravenous, intra-arterial, subcutaneous, intradermal, intramuscular, etc., or direct local administration to the lesion itself. Furthermore, oral administration and administration as a suppository are also possible.
The dose and frequency of administrations vary depending on the symptom, age, weight of the patient, administration form, and such; but it is normally within the range of approximately 0.0001 to approximately 500 mg, preferably within the range of approximately 0.001 to approximately 100 mg for adults per day, which is administered at a time or divided for several administrations.
Pharmaceutical compositions that contain the above-mentioned low molecular weight compounds and proteins as the active ingredients can be administered simultaneously with the agent for gene therapy containing a gene encoding an angiogenesis factor, or they can be administered separately with a time interval.
The pharmaceutical compositions for angiogenic therapy of the present invention that have been described so far can be applied to all diseases that require angiogenic therapy. Specifically, ischemic disease or arterial disease can be exemplified as such diseases. More specifically, examples of heart diseases include ischemic heart disease, myocardial infarction, acute myocardial infarction, myocardosis, angina pectoris, unstable angina, coronary arteriosclerosis, heart failure, and such; and examples of ischemic diseases of the extremities include arteriosclerosis obliterans (ASO), Berger's disease, vascular injury, arterial embolism, arterial thrombosis, arterial occlusion of the organ, aneurysm, and such. Other examples are cerebrovascular diseases. Specifically, examples of cerebrovascular diseases include cerebrovascular occlusion, cerebral infarction, cerebral thrombosis, cerebral embolism, stroke, cerebral hemorrhage, moyamoya disease, cerebrovascular dementia, Alzheimer type dementia, sequela of cerebral hemorrhage, and sequela of cerebral infarction. Among these diseases, the pharmaceutical compositions of the present invention are effectively used particularly against arteriosclerosis obliterans.
Furthermore, the present invention also provides an agent for potentiating the angiogenic effect due to a gene encoding an angiogenesis factor which contains, as the active ingredient, at least one substance selected from the group consisting of substances having vasodilating effect and/or platelet aggregation inhibitory effect, and substances producing them. As mentioned above, the aforementioned substance that is the active ingredient of the pharmaceutical composition for angiogenic therapy of the present invention has the effect of enhancing the angiogenic effect due to a gene encoding an angiogenesis factor. Therefore, as mentioned above, it can be used as one of the components of a pharmaceutical composition for angiogenic therapy, or alternatively, it can be used alone as a potentiating agent to increase the angiogenic effect due to a gene encoding an angiogenesis factor. The potentiating agent of the present invention is used effectively in cases where the effect of the gene encoding the angiogenesis factor is insufficient. The potentiating agent of the present invention may comprise only one component (substance), or pulural components (substances) in combination.
Specifically, the active ingredient of the potentiating agents of the present invention is the aforementioned PGIS gene. The angiogenesis factor is HGF or VEGF as mentioned above.
The administration method, administration form, accommodated disease, and such of the potentiating agent of the present invention are the same as those of the aforementioned pharmaceutical compositions for angiogenic therapy.
Herein below, the present invention will be described using Examples, however, it is not to be construed as being limited thereto.
The cDNA of human HGF (described in Unexamined Published Japanese Patent Application No. (JP-A) Hei 5-111383) was cloned by a conventional method, and was inserted into an expression plasmid pcDNA3. 1 (+) (Invitrogen) containing a cytomegalovirus (CMV) promoter to be used as human HGF gene.
The cDNA of human PGIS (
C57BL/6J mouse (8 weeks old, male) was used. The mouse was anesthetized by intraperitoneal injection of 200 µl of 10-fold diluted Nembutal, and further by ether inhalation when supplementation was necessary. Then, the arteriovenous of the left hind limb was tied up to produce a mouse hind limb ischemia ASO model. Ten days later, the blood flow in both hind limbs was evaluated using Laser Doppler Imager (LDI, Moor Instruments Ltd., MLDI5070), and the left-right ratio was calculated. After the evaluation, 500 µg each of the genes of above (1) was administered into the left hind limb muscle in the form of naked plasmids. Four groups were set up: i. e. , a control group without administration; a group given the HGF gene alone; a group given the PGIS gene alone; and a group given a combined application of the HGF gene and PGIS gene. 2 weeks and 4 weeks after the administration of the genes, the blood flow was evaluated using LDI, and the ratio was calculated. Furthermore, 4 weeks later, the left hind limb muscle was extirpated, and after preparing a frozen section, the capillary density in the muscle was measured by alkaline phosphatase staining. Significant difference test was performed by the Fisher's PLSD method.
The changes in the ratio of the left-right hind limb blood flow with time was measured by LDI, and is shown in
The capillary densities in the muscles 4 weeks after the gene administration are shown in
An expression plasmid carrying the human HGF gene, which is the same as that of Example 1, was used. The cDNA of human ets-1 (GenBank Acc. No. J04101,
Sprauge Dawley rats (12 weeks old, male) were used. The femoral artery from one side was extirpated to produce a rat hind limb ischemia ASO model. One week later, 100 µg each of the genes was administered into the left hind limb muscle using the HVJ-liposome method. Four groups were set up: a control group wherein the vector was administered alone; a group given the HGF gene alone; a group given the ets-1 gene alone; and a group wherein the HGF gene and ets-1 gene were used in combination. Using Laser Doppler Imager (LDI) before gene administration and 4 weeks after gene administration, the blood flow in both hind limbs was evaluated, and the proportion of increase in the left-right blood flow ratio was calculated. Furthermore, the left hind limb muscle was extirpated, and after preparing a frozen section, the capillary density in the muscle was measured by alkaline phosphatase staining. To investigate the influence of the gene administration on the expression of endogenous HGF, intramuscular rat HGF concentration in the ischemic limb was measured using ELISA kit (Institute of Immunology).
By the independent administration of the ets-1 gene, the ets-1 binding activity in the muscular tissue increased. Furthermore, by the administration of the ets-1 gene, the proportion of increase of hind limb blood flow ratio measured using LDI rose (
In the group wherein the ets-1 gene and HGF gene was administered in combination, the proportion of increase of LDI blood flow ratio rose remarkably compared to the groups given ets-1 gene alone or HGF gene alone (
According to the measurement of the intramuscular endogenous HGF concentrations in rat ischemic limbs, the rat HGF concentration was higher in the group wherein the HGF gene and ets-1 gene was used in combination compared to the group given HGF gene alone (
The cDNA of human VEGF165 (gift from Prof. Yonemitsu at Kyushu University Department of Surgery II) was cloned by a conventional method, and was inserted into the EcoRI site of expression plasmid pCAGGS (
The cDNA of human PGIS (
The intramuscular capillary density 4 weeks after the gene administration is shown in
The present invention provides a novel and highly effective pharmaceutical composition for angiogenic therapy which contains as the active ingredients at least one substance selected from the group consisting of substances having vasodilating effect and/or platelet aggregation inhibitory effect, and substances producing them; and a gene encoding an angiogenesis factor. Furthermore, due to the present invention it was newly discovered that genes, such as prostacyclin synthase gene which were not known to be useable for angiogenic therapy can be applied to angiogenic therapy. Finally, pharmaceutical compositions for angiogenic therapy containing these genes as the active ingredients were provided.
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.