Drug composition for angiogenesis therapy

Abstract

Drug compositions of angiogenesis therapy contain gene coding for human prostacyclin synthase (hPGIS) synthesizing prostaglandin I.sub.2 with activities of vasodialation and/or anti-platelet aggregation; drug compositions contain adeno-associated virus (AAV) inserted with gene for angiogenesis factors. The administration of the drug compositions into the aimed treatment region results in transfer of AAV type 1-hPGIS to skeletal muscles and induces a notable expression of human PGIS gene in skeletal muscles. The PGI.sub.2 is produced by mediation of the gene expression in the muscle cells, secreted, induces vessel-protective, neovascularization and anti-platelet aggregation actions, which lead to an improvement in vascular ischemia.

Claims

1. A drug composition of angiogenesis therapy used for treatment and prevention of peripheral arterial disease comprising; an active component, an adeno-associated virus (AAA) inserted with a PGIS gene producing a prostaglandin I2 (PGI2) which induces one or more of the activities selected from the group consisting of vasodialation, anti-platelet aggregation and angiogenesis.

2. The drug composition of angiogenesis therapy according to claim 1, wherein the adeno-associated virus (AAV) is further inserted with a gene coding for an angiogenesis factor.

3. A drug composition of angiogenesis therapy used for treatment and prevention of peripheral arterial disease comprising; an active component, a first adeno-associated virus (AAV) inserted with a PGIS gene producing a prostaglandin I2 (PGI2) which induces one or more of the activities selected from the group consisting of vasodialation, anti-platelet aggregation and angiogenesis, and a second adeno-associated virus (AAV) inserted with a gene coding for an angiogenesis factor.

4. The drug composition of angiogenesis therapy according to any of claim 1, wherein the adeno-associated virus (AAV) is of the type selected from the group consisting of type 1, 2, 5 and 8.

5. The drug composition of angiogenesis therapy according to claim 1, wherein the adeno-associated virus (AAV) is of the type selected from the group consisting of type 1 and 2.

6. The drug composition of angiogenesis therapy according to claim 2, wherein the gene coding for an angiogenesis factor is a gene coding for a material selected from the group consisting of a protein, a peptide and a part thereof capable of inducing angiogenesis.

7. The drug composition of angiogenesis therapy according claim 1, wherein the gene coding for an angiogenesis factor is a vascular endotherial growth factor (VEGF).

8. The drug composition of angiogenesis therapy according to claim 1, further comprising a pharmaceutically permissible carrier containing the adeno-associated virus (AVV).

Description

BRIEF DESCRIPTIN OF THE FIGURES

[0056] FIG. 1 shows subcutaneous blood perfusion by the laser Doppler method of ischemic limbs treated with adeno-associtaed virus-mediated PGIS gene (AAV-PGIS group), saline (control group), adeno-associtaed virus-mediated EGFP gene (AAV-EGFP (enhanced green fluorescence protein) group) and the corresponding macroscopic images.

[0057] FIG. 2 shows qualitative analyses of perfusion rates of ischemic left limbs treated with adeno-associtaed virus-mediated PGIS gene (AAV-PGIS), saline (control), adeno-associtaed virus-mediated EGFP gene (AAV-EGFP) in comparison with the respective control non-ischemic right limbs (n=12)

[0058] FIG. 3 indicates the improvement in limb necrosis rate of AAV-PGIS group mice in comparison with those of control and AAV-EGFP group mice.

[0059] FIGS. 4A-4E show quantitative RT-PCR analysis of the expression of human PGIS, mouse PGIS, mouse VEGF, and a mouse receptor for VEGF, FLK-1 relative to mouse glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mouse in mouse left thigh muscles received an injection of AAV-PGIS, control (saline) or AAV-EGFP. Expression of hPGIS (A), mGAPDH (B), mVEGF (C), and mFLK-1(D) in left limb skeletal muscles were examined by quantitative RT-PCR (n=5).

[0060] FIG. 5 shows the western blot analysis of the expression of human PGIS (hPGIS) in ischemic muscles received an injection of control (saline), AAV-EGFP or AAV-PGIS.

[0061] FIGS. 6A-6C show immunostaining images of human PGIS (FIG. 6A) and mouse von Willebrand factor (vWF) (FIG. 6B), and relative capillary vessel area in hind limb muscles received an injection of control (saline), AAV-EGFP or AAV-PGIS. (FIG. 6A) Human PGIS expressed in hind limb muscles received an injection of control (saline), AAV-EGFP or AAV-PGIS was immunostained by anti-human PGIS antibody. (FIG. 6B) Mouse vWF as a neovascularization marker expressed in hind limb muscles in the three groups was immunostained by Anti-vWF antibody. (FIG. 6C) vWF positive area of the immages in FIG. 6B was quantitatively analyzed.

DETAILED DESCRIPTION

[0062] Below the present invention is concretely explained with a practice example, but the present invention is not restricted by this practice example.

Construction of Plasmids and AAV Vectors

[0063] The expression vector for human PGIS was constructed as described previously. In brief, the blunted HindIII/BamHI fragment of the full-length human PGIS cDNA was ligated into the blunted XhoI site of the pUC-CAGGS expression plasmid. To verify that the pUC/PGIS construct encoded a biologically active PGIS protein, pUC/PGIS was transfected into NIH3T3 cells, and conversion of [.sup.14C]-PGH.sub.2 to 6-keto-[.sup.14C]-PGF1 was measured. pUC-CAGGS vector lacking the insert served as the control vector. Human PGIS genes were then inserted into AAV-CAG plasmids, and AAV-hPGIS vectors were constructed. AAV-EGFP (enhanced green fluorescent protein) vector for control experiments was also prepared as described previously.

Murine Model of Hind Limb Ischemia

[0064] Eight-week-old male BALB/c nude mice (Japan CLEA) were anesthetized with diethyl ether, and the skin was incised over the femoral artery in the mid-portion of the left hind limb. The femoral artery was then gently isolated, and the proximal portion of the artery was ligated with 7-0 silk ligatures. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of the Keio University School of Medicine, and they conformed to the NIH Guide for the Care and Use of Laboratory Animals.

Experimental Protocols

[0065] Mice were divided into 3 groups (control, AAV-EGFP and AAV-hPGIS). The AAV-hPGIS group was administered AAV type 1-hPGIS (1.010.sup.11) into left thigh muscle. The control group was administered the vehicle (0.9% saline) into the left thigh muscle. As negative control, the AAV-EGFP group was administered AAV type 1-EGFP (1.010.sup.11) into same site. After 1 week of gene transfer, the left femoral artery of the mice was ligated to produce hind limb ischemia model. After 2 week of ligation, the incidence of limb necrosis was evaluated and blood perfusion rate of the mice was measured using Laser Doppler perfusion imaging (LDPI) system. The left thigh muscles were excised and utilitied for further analysis.

[0066] To examine the continuity of the expression of human PGIS mRNA , each 4 to 5 mice in the groups of hPGIS, control, and AAV-EGFP were administered AAV type 1-hPGIS (1.010.sup.11, v.g./body), into left thigh muscle of C57/BALB6 mice (8 week-old)

Laser Doppler Blood Perfusion Analysis

[0067] The three groups (AAV-hPGIS, CONTROL and AAV-EGFP) of mice are subjected to ischemia in left hind limbs, and the blood perfusion rate in the ischemic left (L) and normal right (R) hind limb was measured with a Laser Doppler perfusion imaging (LDPI) system (Moor LDI, Moor Instruments, USA). The measurement was carried out by taking LDPI of perfusion state of ischemic hind limbs in each group. The results are shown in FIG. 1.

[0068] In FIG. 1 optical (Macro) and the corresponding Laser Doppler (LDP) perfusion images obtained by measurement with LDPI system.

[0069] In FIG. 1 the images obtained by LDPI method are represented in color. The index for color images representing perfusion state shown below. The index from left to right changes from dark blue to red. Low or nil blood perfusion was displayed as dark blue, and the highest perfusion intervals were displayed as red up to white (maximum).

[0070] FIG. 2 shows quantitative analysis of the blood perfusion rate of the ischemic limb in groups of AAV-PGIS, Control and AAV-EGFP, compared with the non-ischemic control limb (n=12). Representative Laser Doppler perfusion images in each group. (B)

[0071] In FIG. 2 data are meanSD. *P<0.05; ns, not significant.

Quantitative RT-PCR

[0072] RNA extraction and quantitative RT-PCR were performed as described previously. Quantitative RT-PCR was performed with TaqMan probes (Applied Biosystems): human PGIS (Hs00168766_m1), mouse PGIS (Mm00447271_m1), mouse VEGF (Mm01281449_m1), FLK-1 (Mm01222419_m1), FLT-1 (Mm00438971_m1), and Rodent GAPDH. The mRNA levels were normalized by comparison to GAPDH mRNA. In FIG. 4 data are meanSD. *P<0.05; ns, not significant.

Western Blot Analysis

[0073] Membrane fractions of skeletal muscle were prepared by homogenization of thigh muscle in ice-cold buffer as described Immunodetection was performed on membrane extracts with an antibody to human PGIS. The results are shown in FIG. 5.

Histological Analysis

[0074] Samples were fixed with 10% formalin, embedded with paraffin, and cut into 4-m sections. The sections were stained with hematoxylin and eosin. In addition, thigh muscle sections were stained with anti-human PGIS antibody. The results are shown in FIG. 6, which clearly indicates that the remarkable expression of hPGIS mRNA in group of AAV-hPGIS mice with administration of AAV-hPGIS contributed to angiogenesis.

[0075] In FIG. 6 (A) illustrates microscopic images showing expression of hPGIS in left hind limb muscles in groups of AAV-hPGIS, Control and AAV-EGFP. The each muscle in (A) was stained by immnohistochemical methods. (B) Typical examples of microscopic images of von Willebrand factor (vWF)-stained left hind limb muscles mouse. (C) The results of quantitative analysis of microvessels in left hind limb muscles subjected to immunofluorecence staining of vWF. In FIG. 6C data are meanSD. *P<0.05; ns, not significant.

Density of Microvessels

[0076] Animals were sacrificed under anesthesia, and perfusion fixation was performed with 4% paraformaldehyde. The thigh muscles were excised and embedded with OTC compound. Frozen sections (7 m) were cut from the tissue specimens Immunofluorescent staining for endothelial cells was carried out with anti-von Willebrand factor (vWF) antibody (DAKO). Nuclei were stained with TOTO-3 (Molecular Probes). The density of microvessels was calculated from the number of vWF-positive vessels. All confocal microscopy was carried out on a LSM 510 META (Carl Zeiss, Jena, Germany). The capillary density was the ratio between the total area of capillary vessels and the total skeletal muscle area, each measured by Image J software. The data for each mouse were calculated from 30 serial sections.

Statistical Analysis

[0077] All data are presented as the meanSD. Statistical significance was evaluated using the unpaired Student t test. Comparisons among more than three groups were performed using analysis of variance. P<0.05 was considered significant.

Experimental Results

[0078] Duration of hPGIS Expression After Administration of AAV Type 1-hPGIS Vector

[0079] By the present inventors etc. were examined the duration of expression efficiency by AAV type 1-hPGIS vector using 8-weeks control mice (C57/BL/6). Mice were administered AAV type 1-hPGIS vector into the left thigh muscle. For analysis the left thigh muscle from mice treated with before and after 2, 4, 8, 12 weeks vector administration. The results, as analyzed by quantitative RT-PCR, indicates a strong human PGIS mRNA expression from 2 weeks after gene transfer. The strong expression was maintained even after 12 weeks. The results are shown in Table 1.

[0080] Table 1 Change in expression of human PGIS after administration of AAV1-human PGIS vector (1.010.sup.11 (v.g./body)) into left thigh muscle

TABLE-US-00001 TABLE 1 Amount of Number of expressed human Term of administration treated mouse PGIS mRNA*.sup.1 Before administration 8 .sup.0.003 0.005*.sup.2 2 weeks after administration 4 1220 4880 of AAV-human PGIS 4 weeks after administration 5 6310 9470 of AAV-human PGIS 8 weeks after administration 5 4648 2640 of AAV-human PGIS 12 weeks after administration 5 3370 1980 of AAV-human PGIS *.sup.1Relative amount of hPGIS mRNA to mouse GAPDH mRNA. *.sup.2Mean value SD. *.sup.3 mice were administered AAV1-human PGIS vector (1.0 10.sup.11 (v.g./body))

Improvement of Hind Limb Ischemia

[0081] To treat limb ischemia model mice, the present inventor et al administered AAV type 1-hPGIS into left thigh muscle and examined the degree of improvement in the ischemia two weeks after the limb-ischemia operation. In examination the seriousness of limb ischemia was divided into three groups. As shown in FIG. 3, foot or finger necrosis was observed in 63% and 69% mice in groups of Control and AAV-EGFP treated with AAV type 1-EGFP, respectively. But the rate of necrosis in AAV-hPGIS group mice received an administration of AAV type 1-hPGIS, was significantly lowered to 19% and improved. Furthermore, frequency of tertiary foot necrosis in AAV-hPGIS was lowest in the three groups described above. These results indicate the administration of AAV type 1-hPGIS in skeletal muscles is effective in limb ischemia.

Laser Doppler Blood Perfusion Analysis

[0082] Using laser Doppler blood perfusion (LDP) the blood flow in limb muscle tissues of AAV-hPGIS, Control, and AAV-EGFP group mice was analyzed percutaneously. In the LDP images shown in FIG. 1, the blood perfusion of left thigh muscle tissues in the lesion side of mice was lowered in Control and AAV-EGFP groups compared with AAV-hPGIS group. Additionally quantitative analysis (blood perfusion rate) by LDP method indicates 73, 54 or 118% of perfusion rates in the left lesion side of mice as referred to those in respective right healthy sides in the three groups of Control, AAV-EGFP and AAV-hPGIS (see FIG. 2). It is likely from the remark of LDP images that the administration of AAV-hPGIS improves the blood perfusion.

Analysis of mRNA in Ischemic Muscles

[0083] Expression of mRNA in ischemic limb tissues was analyzed using quantitative RT-PCR. Compared with Control and AAV-EGFP groups, a remarkably significant expression of human PGIS gene was observed in AAV-hPGIS group (see FIG. 4A) and the expression of intrinsic mouse PGIS gene was not significantly different among the three group mice. Furthermore, expression of murine mRNA for VEGF, FLK-1 and FLT-1 was analyzed to study neovascularization in the ischemic limb tissues. Consequently, it was significant that expression of VEGF mRNA in AAV-PGIS group was twice compared with that in the other two groups (See FIG. 4C). Although expression of mRNA for FLK-1 and FLT-1 in AAV-PGIS group has a increasing tendency compared that in the other two groups, the difference was not statistically significant among them (see FIG. 4D and 4E). Hereinbefore, strong expression of hPGIS was confirmed in skeletal muscles. Additionally, expression of VEGF mRNA increased in AAV-PGIS group.

Western Blot Analysis of Ischemic Muscles

[0084] Furthermore, gene expression at protein level was examined using western blot analysis. Western blots confirmed hPGIS protein in AAV-hPGIS administration group (see FIG. 5).

Expression of hPGIS in Skeletal Muscles

[0085] Moreover, histological examination of the skeletal muscle of the region administered AAV-PGIS was carried out. Imunostaining with anti-human PGIS antibody confirmed expression of hPGIS gene/protein in muscle cells in AAV-hPGIS administration group (see FIG. 6), and none of immunological changes such as infiltration of inflammatory cells was observed among the three groups administered AAV-PGIS, Control and AAV-EPDGF.

Angiogenesis in Skeletal Muscles

[0086] Additionally to examine improvement at the level of ischemic muscle cells, skeletal muscles were subjected to immunostaining with anti-vWF antibody. In confocal microscopy the increase in vEF-positive vessels was observed in AAV-hPGIS group compared with Control and AAV-EGFP groups, suggesting an increase in capillary density (see FIGS. 6A and 6B). Thus, the density of vVW-positive microvessels was quantitatively analyzed using Image J software.

[0087] The capillary density was expressed as the ratio between the total area of capillary vessels and the total skeletal muscle area. It was statistically significant that the density of vVW-positive microvessels in AAV-hPGIS group was 6.5 folds higher than that in Control and AAV-EGFP groups (see FIG. 6C). Elastica van Gieson (EGV) stain of skeletal muscles was also carried out, but any significant change was not observed among the above three groups.

[0088] As described above, neovascularization at the capillary level occurred in the ischemic skeletal muscles administered AAV-PGIS, suggesting reduction of ischemic injury of skeletal muscles.

[0089] It is shown from the facts described above that improvement of hind limb ischemia is obtained by transfer of human PGIS gene in ischemic skeletal muscles using AAV type 1 vector. Although many serotypes are known as AVV, AAV type 1 is known to have an ability of the strongest and long-term expression in skeletal muscles. It is reported by the present inventor et al that AAV type 1-human PGIS (AAV-hPGIS) shows a long-term strong gene transfer efficiency in the in vitro and in vivo control experiments. Additionally gene expression of AAV type 1 is limited in the local site of administration, and it is not found in remote organs. This characteristic of the expression can reduce the side effect of the gene therapy in remote organs, and is useful in the treatment of patients suffering from serious limb ischemia, which are complicated with arteriosclerosis-induced diseases in other organs.

[0090] As it is clear from previously described experimental results, the rate of necrosis in AAV-hPGIS group mice received an administration of AAV type 1-hPGIS, was significantly lowered and frequency of tertiary foot necrosis was lowest in AAV-hPGIS. Furthermore, in quantitative analysis by LDP the perfusion rate in AAV-hPGIS group caused a statistically significant increase compared with those in the two groups without administration of AAV-hPGIS. LDP image suggests that the development of collateral vessels contribute to the improvement of blood perfusion in ischemic limbs, because disruption of blood perfusion in femoral arteries by ligation, namely the blockage of femoral arteries was confirmed in all groups.

[0091] Subsequently, gene expression was examined with skeletal muscles, the administration site of AAV-hPGIS. The expression of PGIS gene was analyzed by quantitative RT-PCR. A remarkable gene expression of external human PGIS was observed with skeletal muscles in AAV-hPGIS group, and was ten thousand-folds compared with those of the control groups. An enhanced two-fold gene expression of intrinsic VEGF was also observed with skeletal muscles in AAV-hPGIS group, suggesting induction of VEGF by expression of external human PGIS. Using immunostaining of hPGIS a remarkable expression of human PGIS protein was observed in skeletal cells mediated AAV-hPGIS. In addition, any inflammatory changes and tumor formation of muscles was not found by the AAV viral administration. The findings prove the safety of AAV-PGIS, and coincide with the fact that AAV has the lowest immunogenicity among virus vectors.

[0092] Furthermore, by the present inventors skeletal muscles was subjected to immnostaining with anti-vWF antibody in order to examine whether neovascularization occurs or not. In AAV-PGIS group neovascularization was confirmed at the capillary level. Incidentally EVG-staining for elastic fibers in skeletal muscles was carried out and significant changes were not observed in all treated groups, suggesting that the neovascularization at capillary level occurs in skeletal muscles administered AAV-hPGIS.

[0093] It is demonstrated from results mentioned above that transfer of AAV-hPGIS into skeletal muscles improves limb ischemia by a remarkable expression of human PGIS in skeletal muscle cells, leading to production in the muscle cells and secretion of PGI.sub.2 meditated by the expression, which induces activities of vessel protection, induction of neovascularization etc. And it is noteworthy that the effects mentioned above were achieved by single administration. Previous clinical and basic research reports reveal that the strong continuous gene expression could not be maintained due to plasmids, non-viral vectors etc. without a property of long-term expression. Thus, with use of these vectors the multiple-time administration is neccesary to keep the gene expression. The highly safe virus vectors such as the AAV vector achieving a long-term and strong expression can be ideal for clinical applications