EP1037910A2 - Entretien de l'integrite de muscles lisses a l'aide de proteines morphogeniques - Google Patents

Entretien de l'integrite de muscles lisses a l'aide de proteines morphogeniques

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Publication number
EP1037910A2
EP1037910A2 EP98961838A EP98961838A EP1037910A2 EP 1037910 A2 EP1037910 A2 EP 1037910A2 EP 98961838 A EP98961838 A EP 98961838A EP 98961838 A EP98961838 A EP 98961838A EP 1037910 A2 EP1037910 A2 EP 1037910A2
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EP
European Patent Office
Prior art keywords
bmp
gdf
vector
moφhogen
smooth muscle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP98961838A
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German (de)
English (en)
Inventor
Takashi Fourth Dept. of Internal Med. NAKAOKA
Kohei Dept. of Biochem. The Cancer Inst. MIYAZONO
Kuber T. Sampath
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Curis Inc
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Creative Biomolecules Inc
Curis Inc
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Publication of EP1037910A2 publication Critical patent/EP1037910A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to the maintenance of vascular integrity using morphogenic proteins or nucleic acids.
  • vascular smooth muscle Following trauma, characteristic changes occur in vascular smooth muscle. These changes may result in cell death, increased collagen synthesis (and a concomitant reduction in cellular elasticity), inflammatory responses, and a general cellular hypertrophy. Stimuli of cellular trauma include exposure to toxic agents, certain diseases, mechanical stress, and the like.
  • Vascular trauma may result in the synthesis of extracellular matrix proteins, such as collagen. This, coupled with an inflammatory response and cellular proliferation, may result in conditions, such as atherosclerosis, in which the blood vessel intima thickens and loses its elasticity. A resulting loss of blood flow may underlie clinical manifestations of cardiovascular disease.
  • Atherosclerosis is a common form of cardiovascular disease. Typically, atherosclerotic conditions lead to insufficient blood supply to critical organs, resulting in heart attack, stroke, and kidney failure. Additionally, atherosclerosis is a complicating factor in hypertension and diabetes. Vascular smooth muscle cells typically become abnormally proliferative in atherosclerosis. Smooth muscle cell proliferation may reduce blood flow and make vessels susceptible to clotting.
  • Restenosis the recurrence of artery stricture, is an induced form of atherosclerosis.
  • Recent evidence supports a unifying hypothesis of vascular injury in which coronary artery restenosis, coronary vein graft, and cardiac allograft atherosclerosis, represent an accelerated form of the same pathogenic process. Ip, J.H., et al., J. Am. Coll. Cardiol, 75:1667-1687 (1990); Muller, D.W.M., et al, J. Am. Coll. Cardiol, 79:418-432 (1992). Restenosis results from a complex series of fibroproliferative responses to vascular injury involving potent growth- regulatory molecules.
  • TGF- ⁇ 1 is known to regulate vascular smooth muscle cell proliferation m vitro Majack, et al , J Cell Biol , 105 465-471 (1987), Battegay, et al , Cell, 63 515-524 (1990) Whether TGF- ⁇ 1 stimulates or inhibits growth is, however, dependent on many factors, such as the conditions of the cell culture, the presence of other growth-regulatory molecules, the sequence of their addition, and the concentration of TGF- ⁇ l
  • compositions and methods for maintaining smooth muscle integ ⁇ ty More specifically, there is a need in the art for compositions and methods that are capable of reducing the intimal thickening of vascular smooth muscle cells caused by smooth muscle cell proliferation, extracellular mat ⁇ x protein synthesis, and - j - inflammatory responses Furthermore, there exists a need in the art for treatment methods that can also maintain the smooth muscle cell phenotype and thereby preserve the cellular elasticity. The present invention addresses these needs.
  • the invention provides compositions and methods for maintaining vascular integrity, especially vascular smooth muscle integrity.
  • the invention provides compositions and methods for inhibition of smooth muscle cell proliferation.
  • the invention provides compositions and methods for counteracting various cellular responses to trauma.
  • compositions and methods of the invention inhibit collagen synthesis and maintain cellular elasticity of smooth muscle cells, inhibit cellular inflammatory response, protect cells against cytotoxic injury, and maintain the normal balance of Type I and Type III collagen produced by these cells, thereby maintaining cell phenotype.
  • the invention relates to localized delivery of a protein or nucleic acid composition that inhibits smooth muscle cell proliferation and other responses to vascular trauma.
  • a protein or nucleic acid composition preferably comprises a mo ⁇ hogenic protein, or nucleic acid encoding a morphogenic protein, in an acceptable vehicle.
  • the vehicle is a vector that causes expression of a morphogen in mammalian cells.
  • the mo ⁇ hogen preferably has an amino acid sequence with at least 70% sequence homology with the C-terminal 102-106 amino acids, including the conserved seven cysteine domain, of human OP-1, corresponding to residues 326-431 or 330-431 of SEQ ID NO: 1.
  • the mo ⁇ hogenic protein may, therefore, comprise OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-9, BMP- 10, BMP-11, BMP- 12, BMP-15, BMP- 16, DPP, Vgl, Vgr, 6A protein, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, and conservative amino acid variants thereof.
  • the morphogenic protein is OP-1 or BMP-2.
  • an expression vector for delivery of mo ⁇ hogen-encoding nucleic acid may comprise a viral vector, most preferably an adenoviral vector.
  • the vector preferably contains a nucleic acid encoding a mo ⁇ hogen protein in operative association with a promoter, such as the chicken ⁇ -actin promoter.
  • the vector further comprises an enhancer element, such as a CMV-LE enhancer element, in association with the promoter.
  • nucleic acids operatively encoding a morphogen of the invention may be delivered in liposomal or other lipid formulations which enhance delivery to target cells.
  • a morphogen-encoding nucleic acid may be introduced as "naked DNA" (i.e., DNA not associated with proteins or with a vector).
  • Naked DNA may be introduced via intravascular, intraperitoneal, intradermal or transdermal injection, by inhalation, or by other means known in the art.
  • Naked DNA may comprise a linear or circular, single or double-stranded nucleic acid molecule.
  • naked DNA may comprise a plasmid or viral genome, or may be a linear expression sequence excised from a plasmid or viral genome, or amplified in vitro.
  • Naked DNA may be administered substantially free from association with transfection-facilitating proteins, viral particles, liposomal formulations, charged lipids and calcium phosphate precipitating agents as is known in the art (see, e.g., U.S. Pat. No. 5,580,859, incorporated by reference herein). Naked DNA typically expresses its gene product transiently, and does not integrate into the host genome. Morphogen-encoding naked DNA may, therefore, be used to provide protective effects normally associated with mo ⁇ hogens. In the context of the present invention, such DNA provides protective effects against intima thickening, smooth muscle cell proliferation, and inflammatory imbalances caused by damage to blood vessels. Mo ⁇ hogen-encoding naked DNA, in addition to being injected prophylactically, may be administered topically to the vasculature ⁇ e.g., by application on a balloon catheter as described herein) at a site of injury.
  • Methods of the invention provide for the administration of a mo ⁇ hogen-encoding nucleic acid for treatment or prevention of vascular occlusion.
  • Administration of a nucleic acid encoding a mo ⁇ hogen or administration of a mo ⁇ hogen itself is useful in treating vascular proliferative disorders, such as restenosis and atherosclerosis, by, for example, inhibiting smooth muscle cell proliferation.
  • Methods of the invention are also useful to preserve the integrity of vascular tissue, by, for example, protecting cells from cytotoxicity, reducing the occurrence of inflammation of vascular tissue epithelium, and maintaining the balance of extracellular matrix proteins, such as, for example, Type 1 and/or Type III collagen. These effects maintain vascular integrity and cell phenotype.
  • the invention provides for the treatment of smooth muscle cell proliferative disorders and other disorders caused by vascular trauma
  • the invention provides an effective treatment for vascular proliferative disorders, such as atherosclerosis and restenosis, and for prevention of the loss of smooth muscle integrity caused by decreased cellular elasticity and inflammatory responses, through the administration of a morphogenic protein or a nucleic acid encoding a mo ⁇ hogenic protein
  • Figure 1 A shows 3 H-thymidine incorporation into the DNA of rat aortic smooth muscle cells treated with either BMP-2 or TGF- ⁇ 1
  • the closed circles indicate cells that were treated with BMP-2
  • the open triangles indicate cells that were treated with TGF ⁇ -1
  • the open circles indicate cells that were left untreated.
  • Figure IB shows 3 H-thymidine incorporation into the DNA of rat aortic smooth muscle cells treated with either BMP -2 or TGF- ⁇ 1 at various concentrations
  • the closed circles indicate cells that were treated with BMP-2, and the closed triangles indicate cells treated with TGF- ⁇ 1.
  • Figure 1C shows 3 H-thymidine inco ⁇ oration into the DNA of rat aortic smooth muscle cells treated with either BMP-2 at 0.3 pM or TGF- ⁇ 1 at 100 pM
  • Figure ID shows the number of cells grown in plates treated with either BMP-2 or TGF- ⁇ 1.
  • the closed circles indicate cells treated with BMP-2, the open triangles indicate cells treated with TGF- ⁇ 1 , and the open circles indicated cells that were untreated.
  • Figure IE shows the percentage of 3 H-thymidine inco ⁇ oration into the DNA of rat aortic smooth muscle cells that are either untreated or treated with OP-1 at concentrations of 0.1 ng ml, 1 ng/ml, 10 ng/ml or 40 ng/ml
  • Figure IF shows the amount of collagen synthesis by rat aortic smooth muscle cells treated with either BMP-2 or TGF- ⁇ 1
  • the closed circles indicate cells treated with BMP-2
  • the open circles indicate cells treated with TGF- ⁇ 1.
  • Figure 2 is an illustration of the DNA of an adenoviral vector containing a DNA insert encoding BMP-2
  • Figure 3 is an illustration of the DNA of an adenoviral vector containing a DNA insert encoding ⁇ -galactosidase
  • Figure 4A shows a Western blot analysis of media from cells infected with an adenovirus containing a DNA insert encoding BMP-2
  • Lane 1 shows BMP-2
  • lane 2 shows cell culture media from cells infected with an adenoviral vector containing a DNA insert for ⁇ -galactosidase
  • lane 3 shows cell culture media from cells infected with an adenoviral vector contaimng a DNA insert for BMP-2
  • Figure 4B shows ⁇ -thymidme incorporation into the DNA of rat aortic smooth muscle cells treated with an adenovirus contaimng a DNA insert encoding either BMP-2 or ⁇ - galactosidase
  • the closed circles indicate treatment with the adenovirus contaimng a DNA insert encoding BMP-2
  • the open circles indicate treatment with the adenovirus containing a DNA insert encoding ⁇ -galactosidase
  • Figure 5 A shows a cross-section of an Elastica von Gieson-stained rat carotid artery after undergoing balloon injury
  • Figure 5B shows a cross-section of an Elastica von Gieson-stamed rat carotid artery after undergoing balloon injury and subsequent treatment with an adenovirus contaimng a DNA insert encoding BMP-2
  • Figure 5C shows a cross-section of an Elastica von Gieson-stained rat carotid artery after undergoing balloon injury and subsequent treatment with an adenovirus contaimng a DNA insert encoding ⁇ -galactosidase
  • Figure 6A shows the intimal cross-sectional areas of rat carotid arte ⁇ es after undergoing balloon injury and either no treatment or treatment with an adenovirus contaimng a DNA insert encoding either BMP-2 or ⁇ -galactosidase
  • Group 1 of the graph represents non-treated control
  • group 2 represents treatment with the adenoviral vector contaimng the DNA insert encoding BMP-2
  • group 3 represents treatment with the adenoviral vector containing the DNA insert encoding ⁇ -galactosidase.
  • Figure 6B shows the medial cross-sectional areas of rat carotid arteries after undergoing balloon injury and either no treatment or treatment with an adenovirus containing a DNA insert encoding either BMP-2 or ⁇ -galactosidase.
  • Group 1 of the graph represents non-treated control
  • group 2 represents treatment with the adenoviral vector containing the DNA insert encoding BMP-2
  • group 3 represents treatment with the adenoviral vector containing the DNA insert encoding ⁇ -galactosidase.
  • Figure 6C shows the intimal medial ratio of cross-sectional areas of rat carotid arteries after undergoing balloon injury and either no treatment or treatment with an adenovirus containing a DNA insert encoding either BMP-2 or ⁇ -galactosidase.
  • Group 1 of the graph represents non-treated control
  • group 2 represents treatment with the adenoviral vector containing the DNA insert encoding BMP-2
  • group 3 represents treatment with the adenoviral vector containing the DNA insert encoding ⁇ -galactosidase.
  • Figure 7 A shows the amount of ⁇ -actin synthesis in rat aortic smooth muscle cells that were either left untreated or were treated with 100 ng/ml OP-1 at 3, 4, 5 and 6 days post- confluence.
  • Figure 7B shows the amount of Type I collagen synthesis by rat aortic smooth muscle cells that were either left untreated or were treated with 100 ng/ml OP-1 at 3, 4, 5 and 6 days post-confluence.
  • Figure 7C shows the amount of Type III collagen synthesis by rat aortic smooth muscle cells that were either left untreated or were treated with 100 ng/ml OP-1 at 3, 4, 5 and 6 days post-confluence.
  • Figure 8 A shows the effect of OP-1 treatment on the uptake of 3 H-thymidine by rat aortic smooth muscle cells that have undergone injury by mercuric chloride.
  • Figure 8B shows the effect of OP-1 treatment on the uptake of 3 H-thymidine by rat aortic smooth muscle cells that have undergone injury by Antimycin-A.
  • Figure 9 shows the effect of OP-1 on the production of I-CAM in rat aortic smooth muscle cells exposed to 1 ng ml, 5 ng/ml or 10 ng/ml of ⁇ nterleuk ⁇ n-1 and either no OP-1 or 200 ng/ml of OP- 1
  • compositions according to the invention comprise a morphogenic protein or nucleic acid
  • the invention comprises administe ⁇ ng to a patient a composition including an adenoviral vector contaimng a DNA insert encoding a morphogemc protein
  • Other vectors may be used, including, for example, other viral vectors or posomes
  • admimstration of a mo ⁇ hogenic protein, or a vector comprising a DNA encoding a morphogenic protein inhibits the proliferation of smooth muscle cells, particularly vascular smooth muscle cells
  • Methods for maintaining smooth muscle cell phenotype and protecting the cells against injury include use of compositions comprising
  • Example 1 compares the effect of TGF- ⁇ 1 on the proliferation of vascular smooth muscle cells in vitro to the effect of BMP-2 or OP-1 on the same cells
  • Example 2 compares the amount of collagen synthesis resulting from the admimstration of either BMP-2 or TGF- ⁇ 1 to rat aortic smooth muscle cells m vitro.
  • Example 3 details the production of an adenoviral vector contaimng a DNA insert encoding BMP-2, and also details the production of a control adenoviral vector contaimng a DNA insert encoding ⁇ -galactosidase
  • Example 3 further describes the effects of admimstration of an adenoviral vector contaimng a DNA insert encoding either BMP-2 or ⁇ - galactosidase on the proliferation of vascular smooth muscle cells in vitro
  • Example 4 compares the effects of in vivo treatment with an adenoviral vector contaimng a DNA insert encoding BMP- 2 with one contaimng ⁇ -galactosidase on vascular smooth muscle cells after undergoing a balloon angioplasty
  • Example 5 describes the effects of in vivo treatment with OP-1 delivered to the site of a vascular occlusion after performance of balloon angioplasty at that site
  • Example 6 desc ⁇ bes the effect of OP-1 admims
  • Rat aortic smooth muscle cells were prepared from 8 week old Wistar rats by the explant method of Campbell, et al, Physiol. Rev. 59: 1-61 (1979), incorporated by reference herein. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Life Technologies, Inc., Tokyo Japan), 100 ⁇ g/ml streptomycin, and 100 U/ml penicillin.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • the amount of cell proliferation was determined by the rate of DNA synthesis in the cells. This was estimated based on the incorporation of 3 H-thymidine.
  • the cells were seeded into a 24- well culture plate, grown to subconfluence, and serum-deprived by culturing in DMEM without FCS for 60 hours. The cells were then stimulated by addition of a 1% solution of FCS and pulse- labeled with 37 kBq/ml of [6- 3 H]thymidine in 500 ⁇ l of a 1% FCS solution for 2 hours.
  • the amount of 3 H-thymidine inco ⁇ oration into the cells was determined by a trichloroacetic acid precipitation method. See, Y. Takuwa, et al, Bioche . Biophys. Res. Commun. 77- ⁇ :96-101 (1991), inco ⁇ orated by reference herein.
  • the stimulated cells were either left untreated or were treated with either 0.3 pM of BMP-2 or 100 pM of TGF- ⁇ 1. Cell proliferation was monitored for 48 hours. The results are shown in Fig. 1 A. As shown in that figure, cells treated with BMP-2 had low cell proliferation at all times over the 48 hour test period.
  • Untreated cells began to proliferate at approximately 14 hours after stimulation, reached peak proliferation at approximately 24 hours after stimulation, and then declined.
  • the TGF- ⁇ 1 treated cells showed 57% and 30% less cell proliferation at 19 and 24 hours, respectively. They reached peak proliferation at 32 hours. TGF- ⁇ 1, therefore, only delayed the peak of cell proliferation.
  • Figure IB shows the effect on DNA synthesis of varying concentrations of BMP -2 or TGF- ⁇ 1.
  • BMP-2 inhibited the growth of smooth muscle cells in a dose-dependent manner from the threshold concentration of 0.03 pM to 0.3 pM.
  • the effect of adding BMP-2 at varying times after serum stimulation is shown in Fig. l C.
  • BMP-2 was capable of inhibiting cell proliferation even when added after the cells had entered S phase (i.e., it inhibited cell growth even when added 20 hours after serum stimulation).
  • TGF- ⁇ 1 was unable to inhibit cell growth when added either directly before (i.e., 13 hours after serum stimulation) or after (i.e., 24 hours after serum stimulation) S phase entry.
  • BMP-2 treatment also resulted in a 62% decrease in cell number compared to cells grown without treatment over a 72 hour period.
  • the cells treated with TGF- ⁇ 1 showed only a marginally lower incidence of cell growth as compared to untreated cells over the same 72 hour period.
  • BMP-2 was, therefore, found to be superior in inhibiting smooth muscle cell proliferation in vitro as compared to TGF- ⁇ 1.
  • Rat aortic smooth muscle cells were prepared as previously described. The cells were plated (4xl0 4 cells per well of a 24 well plate) in growth medium containing 5% FCS. After standing overnight, the medium was replaced with medium containing no serum in order to initiate cell starvation and to synchronize subsequent cell growth. After 60 hours of starvation, cell growth was stimulated by addition of 1% FCS. OP-1 was also added at concentrations of 0.1 ng/ml, 1 ng/ml, 10 ng/ml and 40 ng/ml, and the cells were incubated for 24 hours.
  • the amount of cell proliferation was determined by the rate of DNA synthesis in the cells, which was estimated based on the amount of 3 H-thymidine inco ⁇ orated. Twenty-two hours after the induction of cell growth, the cells were pulse-labeled with [6- 3 H]thymidine for 2 hours. The amount of 3 H-thymidine inco ⁇ oration into the cells was determined as previously described. The results of OP-1 treatment on the cells is shown in Figure IE. Similarly to BMP-2, OP-1 inhibited the proliferation of smooth muscle cells in a dose-dependent manner.
  • Rat aortic smooth muscle cells were prepared as described in Example 1. These cells were tested for collagen synthesis after stimulation and treatment with either BMP-2 or TGF- ⁇ 1. Confluent rat aortic smooth muscle cells were seeded into a 12-well plate and serum-deprived by maintenance in minimum essential medium ( ⁇ -MEM) for 24 hours.
  • ⁇ -MEM minimum essential medium
  • the cells were then treated with either BMP- 2 or TGF- ⁇ 1 in ⁇ -MEM containing 50 ⁇ g ascorbic acid and 50 ⁇ g ⁇ -aminopropylnitrile for 24 hours.
  • the cells were then labeled with 185 kBq/ml of l-[2,3- 3 H]proline in 1 ml of fresh ⁇ -MEM for 3 hours.
  • a vector containing a DNA insert encoding BMP-2 was constructed.
  • An adenoviral vector containing a DNA insert encoding BMP-2 was constructed.
  • a control adenoviral vector containing a DNA insert encoding ⁇ -galactosidase was also constructed.
  • the adenovirus AxCABMP2 containing a BMP-2 insert useful in transfecting smooth muscle cells was created by inserting a cDNA encoding BMP-2 into plasmid pCAGGS.
  • Plasmid pCAGGS is constructed by introducing CAG promoter and rabbit ⁇ -globin gene sequences, including a polyadenylation signal and a SV40 ori sequence, into vector pUC13. That vector is reported in Messing, Methods Enzymol, 101: 20-78 (1983), inco ⁇ orated by reference herein.
  • the £coO109 site of pUC13 is changed to aXhol site by insertion of aXho ⁇ linker.
  • the EcoOI09-£coRI region of pUC13 is then excised and the Xhol-EcoRl segment of pAGS-3, which includes the AG promoter, is inserted in its place.
  • the EcoRI-S ⁇ /I region of the plasmid is then replaced by the EcoRl-Xho fragment from pKCR-3 (See O'Hare, et al, Proc. Nail. Acad. Sci USA, 75: 1527-1531 (1981), incorporated by reference herein), which includes a polyadenylation signal and the 3 '-flanking sequence of the rabbit ⁇ -globin gene.
  • a BamBl linker is then used to insert the Bam fragment from pAGS-/ cZ.
  • the resulting vector contained CMV-I ⁇ enhancer, chicken ⁇ -actin promoter, an intron, the coding region of BMP-2 and rabbit ⁇ -globin poly- A. This vector was then blunt-end ligated into the Swa
  • the adenovirus thus obtained was isolated, screened for the BMP-2 insert, propagated, purified and titrated as described in Kanegae, et al, Jpn. J. Med. Sci. Biol, ⁇ */7:157-166 (1994), incorporated by reference herein.
  • a schematic illustration of the BMP-2 containing adenoviral vector AxCABMP2 is shown in Fig. 2.
  • Adenoviral vector containing a DNA insert encoding ⁇ -galactosidase was constructed in the same manner as described above, except that DNA encoding ⁇ -galactosidase was inserted into the Xhol site of pCAGGS.
  • a schematic illustration of AxC ALacZ is shown in Fig. 3.
  • the adenoviral vector AxCABMP2 was tested for its ability to cause expression of BMP-
  • FIG. 4A A Western blot analysis of these conditioned media is shown in Fig 4A
  • Lane 1 of the blot in Fig 4A contains 5 ng of recombinant BMP-2, purified from a Chinese Hamster Ovary cell line (control)
  • Lane 2 of the blot in Fig 4 A contains medium from the AxC ALacZ transfected cells
  • Lane 3 contains medium from the AxCABMP2 transfected cells
  • the conditioned media from cells transfected with AxCABMP2 were found to contain the BMP-2 protein
  • Conditioned media from cells transfected with AxCALacZ were not found to contain detectable amounts of BMP-2
  • the adenoviral vector AxCABMP2 was, therefore, able to induce BMP-2 expression in smooth muscle cells in vitro
  • adenoviral vector AxCABMP2 was then tested for its ability to inhibit the proliferation of serum-stimulated rat aortic smooth muscle cells in vitro
  • Subconfluent smooth muscle cells seeded onto a 24-well plate were incubated with 200 ⁇ l of DMEM contaimng 5% FCS for 2 hours and were then serum deprived for 60 hours The cells were then stimulated with a 1% FCS solution, and the degree of cell proliferation was determined by measuring the amount of 3 H-thymidine incorporated into the cells 24 hrs.
  • adenoviral vector AxCABMP2 showed less cell proliferation (i.e., showed less 3 H-thymidine inco ⁇ oration into their DNA) than those cells transfected with the control adenoviral vector AxCALacZ, which did not show any decrease in the amount of cell proliferation
  • the AxC ABMP2 and AxCALacZ adenoviral vectors were added at the same range of viral titer The adenoviral vector AxCABMP2 was, therefore, shown to inhibit smooth muscle cell proliferation in vitro
  • the ability of the adenoviral vector AxCABMP2 to inhibit smooth muscle cell proliferation in vivo was tested next
  • the left common carotid arteries of 10 week-old SPF Wistar rats were injured by embolectomy catheter General anesthesia consisting of 90 mg/kg of ketamine introduced intrape ⁇ toneally and 15 mg/kg of xylazine introduced intramuscularly was administered After intravenous administration of 75 U/kg of heparin, the external and internal carotid arteries were cross-clipped using a 2v-clip microc p (S &.
  • a 2F Fogarty embolectomy catheter (Baxter, Irvine, CA) was introduced into the left common carotid artery through an approximately 3 mm longitudinal aiteriotomy made in the external carotid artery
  • the left common carotid arteries were injured by six passes of the embolectomy catheter inflated with 0.2 ml of air.
  • the arteriotomy was closed by suturing with 10-0 nylon. After suture, blood flow was resumed by removal of the clips.
  • AxCABMP2 or AxCALacZ was administered into the artery on the fifth day after the balloon injury.
  • 50 ⁇ l of virus fluid at 1 x 10 10 plaque forming units (pfu)/ml of either AxCABMP2 or AxCALacZ virus was administered into a 1.5 cm length of the left common carotid artery.
  • the AxCABMP2 vector was administered to one group of rats, and the AxCALacZ vector was administered to another group.
  • the portion of the external carotid artery proximal to the incision was then threaded with 7-0 nylon.
  • a group of untreated rats was used as a control.
  • the vector was allowed to incubate for 40 minutes, and the cross-clipping was then released.
  • FIG. 5 A is a schematic illustration of an Elastica von Gieson-stained carotid artery after undergoing balloon injury, as described above, and receiving no further treatment.
  • Figure 5B is a schematic illustration of an Elastica von Gieson-stained carotid artery after undergoing balloon injury, as described above, and then being transfected with AxCABMP2 five days after balloon injury.
  • Figure 5C is a schematic illustration of an Elastica von Gieson-stained carotid artery after undergoing balloon injury, as described above, and then being transfected with AxCALacZ five days after balloon injury. The illustrations shown in Fig. 5A-5C are all shown at approximately x25 magnification.
  • AxCABMP2 was found to decrease the I M ratio. For each 1 cm of artery specimens, five round cross-sections were stained with Hematoxylin and Eosin and photographed. The cross-sectional areas of intimal and medial regions were analyzed using image analyzing software (NIH image). Administration of AxCABMP2 was found to reduce the intimal mass of injured arteries by 41% as compared to administration of AxCALacZ.
  • Figure 6 A shows the mean intimal mass areas for untreated, AxCABMP2 transfected, and AxCALacZ transfected specimens. The medial mass was similar between all three groups.
  • Figure 6B shows the mean medial mass areas for untreated, AxCABMP2 transfected and AxCALacZ transfected specimens As can be seen in Fig 6C, the I/M ratio was, therefore, substantially reduced in rats transfected with AxCABMP2
  • the left common carotid arteries of 10 week-old SPF Wistar rats are injured by embolectomy catheter General anesthesia consisting of 90 mg/kg of ketamine introduced intrape ⁇ toneally and 15 mg/kg of xylazine introduced intramuscularly is administered After intravenous administration of 75 U/kg of heparin, the external and internal carotid arteries are cross-clipped using a 2v-cl ⁇ p microchp (S & T, Inc , Germany)
  • a 2F Fogarty embolectomy catheter (Baxter, Irvine, CA) coated with a biologically compatible composition containing OP-1 is introduced into the left common carotid artery of one group of the rats through an approximately 3 mm longitudinal arteriotomy made in the external carotid artery.
  • the embolectomy catheter is not coated with protein
  • the left common carotid arteries are injured by six passes of the embolectomy catheter inflated with 0.2 ml of air After placement of a microchp on the proximal portion of the left common carotid artery, the arteriotomy is closed by suturing with 10-0 nylon After suture, blood flow is resumed by removal of the clips
  • Rat aortic smooth muscle cells were prepared as previously described The cells were plated (4xl 0 5 /100 mm dish) and were grown in medium containing 5% FCS overnight. The next day, the plates were divided into two sets. To one set, 100 ng/ml of OP-1 was added every day for six days. No OP-1 was added to the second set. One plate from each set was harvested on each of days 3, 4, 5 and 6 post-confluence and the RNA extracted.
  • RT-PCR was performed on the extracted RNA, and a Northern blot was performed to identify the amount of Type I collagen, Type III collagen, and ⁇ -actin RNA produced by the control cells (no OP-1 added) and the cells that received OP-1.
  • the results are shown in Figures 7A-7C.
  • Figure 7A illustrates the effect of OP-1 on the expression of ⁇ -actin in the cultured cells.
  • Lanes 1-4 illustrate the amount of ⁇ -actin RNA synthesis in the control cells on each of days 3, 4, 5, and 6 post-confluence.
  • Lanes 5-8 illustrate the amount of ⁇ -actin RNA synthesis in the cells treated with OP-1 on days 3, 4, 5, and 6 post-confluence. As is seen by the figure, the cells treated with OP-1 were able to maintain or increase their level of ⁇ -actin RNA production in the prolonged culture.
  • Type I and Type III collagen Similar results for Type I and Type III collagen can be seen in Figures 7B and 7C, respectively.
  • the level of Type I collagen RNA is maintained in cells treated with OP-1 as compared to the control cells.
  • the level of Type III collagen RNA is maintained or increased in cells treated with OP-1 as compared to the control cells, which showed a decrease in Type III collagen synthesis. Maintaining the normal balance of Type I and Type III collagen produced by these cells allows them to maintain their elasticity.
  • the OP-1 treated cells were, therefore, able to maintain their characteristic phenotypic markers as compared to cells not treated with OP-1.
  • OP-1 OP-1 to protect smooth muscle cells against toxic insult (e.g., exposure to toxic substances such as mercuric chloride (HgCl ) or Antimycin-A) was tested in vitro. Smooth muscle cells treated with OP-1, either before or after undergoing toxic insult, were able to survive as compared to cells left untreated.
  • toxic insult e.g., exposure to toxic substances such as mercuric chloride (HgCl ) or Antimycin-A
  • Rat aortic smooth muscle cells were prepared as previously described. The cells were plated (4x10 4 ) and treated with 0.1 mM HgCl 2 for three hours in growth medium containing 5% FCS. The plates were then washed exhaustively, and the cells were treated with serum-free growth medium and either no OP-1 or OP-1 at concentrations of 0.1 ng/ml, 1 ng/ml, 10 ng/ml. 40 ng/ml or 200 ng/ml. Forty-eight hours after treatment with OP-1, the cells were pulsed with [6- ⁇ ]thymidine to determine the amount of cell proliferation.
  • Figure 8A shows the results.
  • the addition of OP-1 at concentrations of 10 ng/ml or more increased the survival rate of cells exposed to HgCl 2 in a dose-dependent manner as compared to cells left untreated or treated with only small amounts of OP-1.
  • Rat aortic smooth muscle cells were prepared as described above. The cells were plated (4x10 4 ) and treated with either 5 ⁇ M, 10 ⁇ M, or 20 ⁇ M Antimycin-A solution for three hours in growth medium containing 5% FCS. After washing exhaustively, the cells were treated with serum-free medium and OP-1 at concentrations of either 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 40 ng/ml or 200 ng/ml. After 48 hours, the cells were pulse-labeled with [6- 3 H]thymidine to determine the amount of cell proliferation.
  • I-CAMs intercellular adhesion molecules
  • IL-1 interleukin-1
  • Rat aortic smooth muscle cells were prepared as previously described The cells were then plated (4x10 4 ) in growth medium containing 5% FCS overnight The growth medium was then replaced by a medium containing 0 5% FCS LL-1 was added to the cells at concentrations of either 0 ng/ml, 1 ng/ml, 5 ng/ml, or 10 ng/ml For each concentration of EL-1, the cells were also treated with 200 ng/ml of OP-1 or left untreated (control) After incubation for five days, the amount of I-CAM produced by the OP-1 treated and control cells at the various LL-1 concentrations was determined from cell extracts using I-CAM ELISA The results are shown in Figure 9 As can be seen by Figure 9, cells treated with OP-1 showed less I-CAM production than the control cells OP-1 therefore protects smooth muscle cells against the effects of inflammatory cytokines such as LL-1
  • Morphogenic protein can therefore be used to preserve smooth muscle integ ⁇ ty following various traumas Morphogenic protein is particularly useful in the treatment of vascular disorders, such as atherosclerosis and restenosis, because administration of mo ⁇ hogenic proteins not only reduces intimal thickening of blood vessels caused by excess collagen synthesis, inflammatory responses and cellular proliferation, but also maintains the phenotype of the cells

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Abstract

L'invention concerne des compositions et des procédés servant à entretenir l'intégrité de muscles lisses, en particulier de muscles lisses vasculaires. Les maladies vasculaires sont caractérisées par une accumulation excessive de cellules de muscles lisses vasculaires qui a pour conséquence l'occlusion d'un vaisseau sanguin, et/ou par une perte d'élasticité des vaisseaux sanguins. Des causes d'occlusion de vaisseaux sanguins comprennent notamment une prolifération de cellules de muscles lisses et des réactions inflammatoires. Une inhibition de la prolifération de cellules de muscles lisses ou de réactions inflammatoires constitue un traitement efficace de troubles vasculaires tels que l'athérosclérose et la resténose. Le traitement peut comporter l'administration d'une protéine morphogénique. La protéine elle-même peut être administrée au site d'occlusion vasculaire, ou peut être administrée par un vecteur, tel qu'un vecteur adénoviral contenant un insert d'ADN codant pour une protéine morphogénique. Ces compositions et procédés peuvent également inhiber les réactions de cellules de muscles lisses à divers traumatismes, telle qu'une exposition à des agents toxiques. Tous ces traitements servent à préserver le phénotype de la cellule par l'inhibition d'une augmentation de protéines de matrice extracellulaire, tel le collagène, ou par l'entretien de l'équilibre normal de protéines de matrice extracellulaire, tels les collagènes de type I et III.
EP98961838A 1997-12-04 1998-11-30 Entretien de l'integrite de muscles lisses a l'aide de proteines morphogeniques Withdrawn EP1037910A2 (fr)

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DK0448704T3 (da) * 1989-10-17 1999-04-06 Stryker Corp Osteogene anordninger
ATE287949T1 (de) * 1992-07-31 2005-02-15 Stryker Corp Löslicher komplex morphogener proteine und zusammensetzungen davon
US5906827A (en) * 1994-06-03 1999-05-25 Creative Biomolecules, Inc. Matrix for the manufacture of autogenous replacement body parts
WO1996026737A1 (fr) * 1995-03-01 1996-09-06 Creative Biomolecules, Inc. Regeneration de la dentine induite par un morphogene
WO1997007135A2 (fr) * 1995-08-14 1997-02-27 Creative Biomolecules, Inc. Liaison de la proteine oestrogene 1 (op-1) et de ses analogues avec le recepteur alk-1 de surface de cellule et ses analogues
DE69740089D1 (de) * 1996-03-22 2011-02-10 Stryker Corp Verfahren zur verbesserten funktionellen Erholung der motorischen Koordiination, der Sprache oder der Sinneswahrnehmung nach Trauma oder Ischämie des ZNS
US6498142B1 (en) * 1996-05-06 2002-12-24 Curis, Inc. Morphogen treatment for chronic renal failure
ATE239514T1 (de) * 1997-02-07 2003-05-15 Stryker Corp Matrixlose osteogene vorrichtungen und implantate und verfahren zu deren verwendung

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WO1999028341A2 (fr) 1999-06-10

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