CA2295040C - Method for coating stents with dna and expression of recombinant genes from dna coated stent in vivo - Google Patents
Method for coating stents with dna and expression of recombinant genes from dna coated stent in vivo Download PDFInfo
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- CA2295040C CA2295040C CA002295040A CA2295040A CA2295040C CA 2295040 C CA2295040 C CA 2295040C CA 002295040 A CA002295040 A CA 002295040A CA 2295040 A CA2295040 A CA 2295040A CA 2295040 C CA2295040 C CA 2295040C
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- implantable device
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
The present invention describes DNA coated stents and methods of using the same to treat or prevent vascular diseases, such as restenosis.
Description
METHOD FOR COATING STENTS WITH DNA AND EXPRESSION OF RECOMBINANT
GENES FROM DNA COATED STENT IN VIVO
Field of the Invention:
This invention provides an intravascular DNA coated stmt and methods for expressing recombinant genes in vivo using the DNA coated stmt. DNA coated stems are useful for treating coronary and peripheral vascular diseases, particularly restenosis.
Background of the Invention:
Coronary and peripheral angioplasty is routinely performed to treat obstructive atherosclerotic lesions in the coronary and peripheral blood vessels.
Following balloon dilation of these blood vessels, 30-40% of patients undergo restenosis.
Restenosis is the reclosure of a peripheral or coronary artery following trauma to that artery caused by efforts to open a stenosed portion of the artery, such as, for example, by balloon dilation, ablation, atherectomy or laser treatment of the artery. Restenosis is believed to be a natural healing reaction to the injury of the arterial wall. The healing reaction begins with the thrombotic mechanism at the site of the injury. The final result of the complex steps of the healing process can be intimal hyperpiasia, the uncontrolled migration and proliferation of medial smooth muscle cells, combined with their extracellular matrix production, until the artery is again stenosed or occluded. Thus, restenosis is characterized by both elastic recoil or chronic constriction of the vessel in addition to abnormal cell proliferation.
Currently restenosis must be treated with subsequent angioplasty procedures.
In an attempt to prevent restenosis, metallic intravascular stems have been permanently implanted in coronary or peripheral vessels. For example, U.S. 5,304,122 (Schwartz et al.) describe metal stems useful for treating restenosis after balloon angioplasty or other coronary interventional procedures.
The stmt is typically inserted by catheter into a vascular lumen and expanded into contact with the SUBSTITUTE SHEET (RULE 26) diseased portion of the arterial wall, thereby providing mechanical support for the lumen.
However, it has been found that restenosis can still occur with such stems in place; likely, because although the stmt prevents elastic recoil of the artery, it fails to prevent the cell proliferation which leads to intimal hyperplasia. In addition, the stmt itself can cause undesirable local thrombosis. To address the problem of thrombosis, persons receiving stems also receive extensive systemic treatment with anticoagulant and antiplatelet drugs.
Stems coated with various compositions have been proposed. For example, Dichek et al.
(Circulation 1989, 80:1347-1353) describe coating stainless steel stems with sheep endothelial cells that had undergone retrovirus-mediated gene transfer for either bacterial (3-galactosidase or human tissue-type plasminogen activator. The stems were studied cx nivo in tissue culture dishes only.
The feasibility of implanting the stents into arteries were not explored. This procedure of coating stems with cells is tedious, cumbersome and costly because cell have to be derived from a patient.
Other methods of providing therapeutic substances to the vascular wall by means of stems have also been proposed. For example, WO 91/12779, entitled "Intraluminal Drug Eluting Prosthesis," and WO 90/13332, entitled "Stmt With Sustained Drug Delivery,"
suggest coating stems with antiplatelet agents, anticoagulant agents, antimicrobial agents, anti-inflammatory agents.
antimetabolic agents and other drugs to reduce the incidence of restenosis.
Similarly, U. S.
5,571,166 and 5,554,182 (both to Dinh et al.) describe intraluminal stems coated with fibrin and heparin. The stmt is used to treat restenosis.
SUMMARY OF THE INVENTION
Accordingly, one object of this invention is to provide an intravascular DNA
coated stmt.
A second object of this invention is to provide methods for expressing recombinant genes in vivo using the DNA coated stems.
GENES FROM DNA COATED STENT IN VIVO
Field of the Invention:
This invention provides an intravascular DNA coated stmt and methods for expressing recombinant genes in vivo using the DNA coated stmt. DNA coated stems are useful for treating coronary and peripheral vascular diseases, particularly restenosis.
Background of the Invention:
Coronary and peripheral angioplasty is routinely performed to treat obstructive atherosclerotic lesions in the coronary and peripheral blood vessels.
Following balloon dilation of these blood vessels, 30-40% of patients undergo restenosis.
Restenosis is the reclosure of a peripheral or coronary artery following trauma to that artery caused by efforts to open a stenosed portion of the artery, such as, for example, by balloon dilation, ablation, atherectomy or laser treatment of the artery. Restenosis is believed to be a natural healing reaction to the injury of the arterial wall. The healing reaction begins with the thrombotic mechanism at the site of the injury. The final result of the complex steps of the healing process can be intimal hyperpiasia, the uncontrolled migration and proliferation of medial smooth muscle cells, combined with their extracellular matrix production, until the artery is again stenosed or occluded. Thus, restenosis is characterized by both elastic recoil or chronic constriction of the vessel in addition to abnormal cell proliferation.
Currently restenosis must be treated with subsequent angioplasty procedures.
In an attempt to prevent restenosis, metallic intravascular stems have been permanently implanted in coronary or peripheral vessels. For example, U.S. 5,304,122 (Schwartz et al.) describe metal stems useful for treating restenosis after balloon angioplasty or other coronary interventional procedures.
The stmt is typically inserted by catheter into a vascular lumen and expanded into contact with the SUBSTITUTE SHEET (RULE 26) diseased portion of the arterial wall, thereby providing mechanical support for the lumen.
However, it has been found that restenosis can still occur with such stems in place; likely, because although the stmt prevents elastic recoil of the artery, it fails to prevent the cell proliferation which leads to intimal hyperplasia. In addition, the stmt itself can cause undesirable local thrombosis. To address the problem of thrombosis, persons receiving stems also receive extensive systemic treatment with anticoagulant and antiplatelet drugs.
Stems coated with various compositions have been proposed. For example, Dichek et al.
(Circulation 1989, 80:1347-1353) describe coating stainless steel stems with sheep endothelial cells that had undergone retrovirus-mediated gene transfer for either bacterial (3-galactosidase or human tissue-type plasminogen activator. The stems were studied cx nivo in tissue culture dishes only.
The feasibility of implanting the stents into arteries were not explored. This procedure of coating stems with cells is tedious, cumbersome and costly because cell have to be derived from a patient.
Other methods of providing therapeutic substances to the vascular wall by means of stems have also been proposed. For example, WO 91/12779, entitled "Intraluminal Drug Eluting Prosthesis," and WO 90/13332, entitled "Stmt With Sustained Drug Delivery,"
suggest coating stems with antiplatelet agents, anticoagulant agents, antimicrobial agents, anti-inflammatory agents.
antimetabolic agents and other drugs to reduce the incidence of restenosis.
Similarly, U. S.
5,571,166 and 5,554,182 (both to Dinh et al.) describe intraluminal stems coated with fibrin and heparin. The stmt is used to treat restenosis.
SUMMARY OF THE INVENTION
Accordingly, one object of this invention is to provide an intravascular DNA
coated stmt.
A second object of this invention is to provide methods for expressing recombinant genes in vivo using the DNA coated stems.
2 SUBSTITUTE SHEET (RULE 26) A third object of this invention is to provide methods for treating coronary and peripheral vascular diseases, particularly restenosis and vein by-pass grafts, using the DNA coated stems.
The present inventors have now realized these and other objects through their discovery of methods for coating DNA on the outside surface of a stmt.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a restriction map of plasmid pCMV-CAT (VR1332).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
DNA coated stents Stents are devices which can be delivered percutaneously to treat coronary artery occlusions and to seal dissections or aneurysms of splenic, carotid, iliac and popliteal vessels.
Suitable stents useful in the invention are polymeric or metallic. Examples of polymeric stems include stents made with biostable or bioabsorbable polymers such as polyethylene terephthalate), polyacetal, poly(lactic acid), and polyethylene oxide)/poly(butylene terephthalate) copolymer.
Examples of metallic stems include stents made from tantalum or stainless steel. Stems are available in myriad designs; all of which can be used in the present invention and are either commercially available or described in the Literature. For example, a self expanding stmt of resilient polymeric material is described in WO 91/12779, entitled "Intraluminal Drug Eluting Prosthesis." Alternatively, U.S. Pat. 4,886,062 describes a deformable metal wire stent.
Commercial sources of stems include Johnson & Johnson, Boston Scientific, Cordis, Advanced Catheter Systems, and U. S. Catheter, Inc.
Suitable genes which encode for therapeutic proteins useful in the invention include genes which encode antiplatelet agents, anticoagulant agents, antimitotic agents, antioxidants, antimetabolite agents, and anti-inflammatory agents. Preferred genes which encode therapeutic
The present inventors have now realized these and other objects through their discovery of methods for coating DNA on the outside surface of a stmt.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a restriction map of plasmid pCMV-CAT (VR1332).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
DNA coated stents Stents are devices which can be delivered percutaneously to treat coronary artery occlusions and to seal dissections or aneurysms of splenic, carotid, iliac and popliteal vessels.
Suitable stents useful in the invention are polymeric or metallic. Examples of polymeric stems include stents made with biostable or bioabsorbable polymers such as polyethylene terephthalate), polyacetal, poly(lactic acid), and polyethylene oxide)/poly(butylene terephthalate) copolymer.
Examples of metallic stems include stents made from tantalum or stainless steel. Stems are available in myriad designs; all of which can be used in the present invention and are either commercially available or described in the Literature. For example, a self expanding stmt of resilient polymeric material is described in WO 91/12779, entitled "Intraluminal Drug Eluting Prosthesis." Alternatively, U.S. Pat. 4,886,062 describes a deformable metal wire stent.
Commercial sources of stems include Johnson & Johnson, Boston Scientific, Cordis, Advanced Catheter Systems, and U. S. Catheter, Inc.
Suitable genes which encode for therapeutic proteins useful in the invention include genes which encode antiplatelet agents, anticoagulant agents, antimitotic agents, antioxidants, antimetabolite agents, and anti-inflammatory agents. Preferred genes which encode therapeutic
3 SUBSTIME SHEET (RULE 26) proteins include proteins which can inhibit proliferation of cells (particular of vascular smooth muscle cells (vsmc), including:
HSV thymidine kinase (McKnight, 1980, Nucleic Acids Res. 8:5949; Mansour et al., 1988, Nature 336:348-352), (3-galactosidase, p 16 (Chap et al., 1995, Mol. Cell. Bioi. 15:2682-2688; Guan et al., Genes &
Dev. 8:2939-2952), p21 (Harper et al., 1993, Cell 75:805; Xiong et al., 1993, Nature 366:701 ), p27 (Toyoshima et al., 1994, Cell 78:67-74; Polyak et al., 1994, Cell 78:59-66), p57 (Lee et al., 1995, Genes & Dev. 9:639-649; Matsuoka et al., 1995, Genes &
Dev.
9:650-662), retinoblastoma (Rb) (see Chang et al., 1995, Science, 267:_518) or its mutants (see for example, Hamel et al., 1992, Mol. Cell. Biol. 12:3431 ), and cytosine deaminase (WO 9428143; Wang et al., 1988, Can. Soc. Petrol. Geol.
Mem., 1 5 14:71 ).
The sequences of these gene products are known in the literature. Any DNA
encoding these gene products can be used, including the cDNA sequences that are described in the literature.
Alternatively, fusion proteins of the above can be used. The preferred genes encode thymidine kinase (HSV-tk) or cytosine deaminase gene.
Any DNA encoding the above therapeutic proteins can be used. Preferably, the DNA
sequence of the human cDNA encoding those proteins are used. The DNA can be naked or can be incorporated into a vector. Suitable vectors include shuttle vectors, expression vectors. retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
Preferably a replication-defective adenovirus vector is used, such as pAd-BgIII as described by Davidson et al. ( 1993, Nature Genet. 3:219-223). These vectors have been demonstrated to program high levels of
HSV thymidine kinase (McKnight, 1980, Nucleic Acids Res. 8:5949; Mansour et al., 1988, Nature 336:348-352), (3-galactosidase, p 16 (Chap et al., 1995, Mol. Cell. Bioi. 15:2682-2688; Guan et al., Genes &
Dev. 8:2939-2952), p21 (Harper et al., 1993, Cell 75:805; Xiong et al., 1993, Nature 366:701 ), p27 (Toyoshima et al., 1994, Cell 78:67-74; Polyak et al., 1994, Cell 78:59-66), p57 (Lee et al., 1995, Genes & Dev. 9:639-649; Matsuoka et al., 1995, Genes &
Dev.
9:650-662), retinoblastoma (Rb) (see Chang et al., 1995, Science, 267:_518) or its mutants (see for example, Hamel et al., 1992, Mol. Cell. Biol. 12:3431 ), and cytosine deaminase (WO 9428143; Wang et al., 1988, Can. Soc. Petrol. Geol.
Mem., 1 5 14:71 ).
The sequences of these gene products are known in the literature. Any DNA
encoding these gene products can be used, including the cDNA sequences that are described in the literature.
Alternatively, fusion proteins of the above can be used. The preferred genes encode thymidine kinase (HSV-tk) or cytosine deaminase gene.
Any DNA encoding the above therapeutic proteins can be used. Preferably, the DNA
sequence of the human cDNA encoding those proteins are used. The DNA can be naked or can be incorporated into a vector. Suitable vectors include shuttle vectors, expression vectors. retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
Preferably a replication-defective adenovirus vector is used, such as pAd-BgIII as described by Davidson et al. ( 1993, Nature Genet. 3:219-223). These vectors have been demonstrated to program high levels of
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~'~~ ~,.r Plasmid Description CMVp27Aftk CMVp27tk with the Avall-Fspl fragment deleted (that contains -the region of p27 between the cdk2 binding site and the putative N LS) CMVp27SNtk CMVp27citetk with the Sacll-Ncol fragment deleted (containing the C-terminus of p27) CMVp27Sp21 Ftk CMVp27tk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Sacll and Fspl sites CMVp27Np21 Ftk CMVp27tk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Narl and Fspl sites CMVp27Sp21 FcitetkCMVp27citetk with the Hindlll-Ncol fragment from 1012-p21 N
(containing the N-terminal part of p21 coding sequence) inserted between the Sacll and Fspl sites in the p27 coding region CMVp27Np21 FcitetkCMVp27citetk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Narl and Fspl sites CMVp27Sp21 Clal-Sacll fragment from CMVp27citetk fused to the Ncol-Clal fragment of VR 1012-p21 N (giving a fusion between p27N and p21 N) CMVp27Np21 Clal-Narl fragment from CMVp27 citetk fused to the Ncol-Clal fragment of VR 1012-p21 N (giving a fusion between p27N and p21 N) CMVp27Dkcitetk CMVp27citetk with all K mutated to R between ATG
and Sacll of p27. There is an additional 'c' before the Sacll site CMVp27Ncitetk CMVp27citetk with a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27NLScitetk CMVp27citetk with a NLS (GRRRRA = ATF2 NLS) and a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27DKNcitetk CMVp27Dkcitetk with a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27DKNLScitetkCMVp27Dkcitetk with a NLS (GRRRRA = ATF2 NLS}
and a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) The stmt can optionally be coated with other therapeutic proteins such as heparin, hirudin, angiopeptin, ACE inhibitors, growth factors (such as IL2_»~), nitric oxide or with DNA encoding the same.
Suitable polymerizable matrix useful for binding the DNA to the stmt include any monomeric biocompatible material which can be suspended in water, mixed with DNA and
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~'~~ ~,.r Plasmid Description CMVp27Aftk CMVp27tk with the Avall-Fspl fragment deleted (that contains -the region of p27 between the cdk2 binding site and the putative N LS) CMVp27SNtk CMVp27citetk with the Sacll-Ncol fragment deleted (containing the C-terminus of p27) CMVp27Sp21 Ftk CMVp27tk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Sacll and Fspl sites CMVp27Np21 Ftk CMVp27tk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Narl and Fspl sites CMVp27Sp21 FcitetkCMVp27citetk with the Hindlll-Ncol fragment from 1012-p21 N
(containing the N-terminal part of p21 coding sequence) inserted between the Sacll and Fspl sites in the p27 coding region CMVp27Np21 FcitetkCMVp27citetk with the Hindlll-Ncol fragment from 1012-p21 N
inserted between the Narl and Fspl sites CMVp27Sp21 Clal-Sacll fragment from CMVp27citetk fused to the Ncol-Clal fragment of VR 1012-p21 N (giving a fusion between p27N and p21 N) CMVp27Np21 Clal-Narl fragment from CMVp27 citetk fused to the Ncol-Clal fragment of VR 1012-p21 N (giving a fusion between p27N and p21 N) CMVp27Dkcitetk CMVp27citetk with all K mutated to R between ATG
and Sacll of p27. There is an additional 'c' before the Sacll site CMVp27Ncitetk CMVp27citetk with a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27NLScitetk CMVp27citetk with a NLS (GRRRRA = ATF2 NLS) and a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27DKNcitetk CMVp27Dkcitetk with a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) CMVp27DKNLScitetkCMVp27Dkcitetk with a NLS (GRRRRA = ATF2 NLS}
and a stop codon between Sacll and Xbal in p27 (only the N-terminus of p27 remains) The stmt can optionally be coated with other therapeutic proteins such as heparin, hirudin, angiopeptin, ACE inhibitors, growth factors (such as IL2_»~), nitric oxide or with DNA encoding the same.
Suitable polymerizable matrix useful for binding the DNA to the stmt include any monomeric biocompatible material which can be suspended in water, mixed with DNA and
6 SUBSTITUTE SHEET (RULE 26) subsequently polymerized to form a biocompatible solid coating. Thrombin polymerized fibrinogen (fibrin) is preferred.
The stmt is preferably coated with about 50 pg to about S mg of DNA. The thickness of the polymerizable matrix containing the DNA is typically about 5-500 pm. The matrix preferably covers the entire surface of the stent.
Methods for coating a stent with DNA
Methods for coating surfaces are well known in the art and include, for example, spray coating, immersion coating, etc. Any of these methods can be used in the invention. For example, a liquid monomeric matrix can be mixed with the DNA and polymerization initiated. The stmt can then be added to the polymerizing solution. such that polymer forms over its entire surface. The coated stent is then removed and dried. Multiple application steps can be used to provide improved coating uniformity and improved control over the amount of DNA applied to the stmt.
In a preferred embodiment, an aqueous mixture of DNA and human thrombin is added to an aqueous suspension of fibrinogen. The fibrinogen concentration of the suspension is typically between about 10-50, preferably about 20-40, more preferably about 30 mg/ml.
The concentration of the DNA in the aqueous mixture is typically about 1-20, preferably about 5-15, more preferably about 10 p,g/ml. The amount of human thrombin in the aqueous mixture about 0.5 to S, preferably about 1 U. The DNA and human thrombin are first added together to form a mixture and that mixture is then added to the fibrinogen suspension. Thereafter, a stmt is dipped into the polymerizing solution. After the mixture solidifies, the stent is removed.
Methods for placing the DNA coated stent within the vasculature The stmt can be placed onto the balloon at a distal end of a balloon catheter and delivered by conventional percutaneous means (e.g. as in an angioplasty procedure) to the site of the SUBSTITUTE SHEET (RULE 261 is:ai:, ''3t;:~.'lia~'s ~ ~'~_,..A.,;~;.~;. v ari y ~3~;
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Method of treating coronary and peripheral vascular diseases with the DNA
coated stems Coronary and peripheral diseases, including restenosis, atherosclerosis, coronary artery bypass graft stenosis, vein bypass graft stenosis or restenosis, arterio-venous fistula stenosis or restenosis, peripheral artery stenosis or restenosis, can be treated by implanting the DNA
coated stent of the present invention, into a coronary or peripheral artery or vein of a patient.
Suitable patients include mammals such as dogs, horses, cattle, humans, etc. Humans are preferred patients.
In an alternate embodiment, the DNA coated stent is implanted into the patient and an antiplatelet agent, anticoagulant agent, antimicrobial agent, anti-inflammatory agent, antimetabolic agent, antimitotic agent or other drug is administered to reduce the incidence of restenosis.
Suitable anticoagulant agents can include drugs such as heparin, coumadin, protamine, hirudin and tick anticoagulant protein. Suitable antimitotic agents and antimetabolite agents can include drugs such as colchicine, methotrexate, azathioprine, vincristine, vinblastine, fluorouracil, adriamycin and mutamycin. Ganciclovir or acyclovir is preferably administered.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
EXAMPLES
Procedure for coating the stents using thrombin polymerized fibrinogen (fibrin}
Human fibrinogen was dissolved in water at concentrations of 30 mg/ml. 100 ~1 of dif~'erent concentrations of fibrinogen were used in the preparation.
Fibrinogen was diluted in water when necessary and transferred to an Eppendorf tube.
Plasmid CAT (pCMV-CAT) was dissolved in water at concentrations of 10 mg/ml.
The DNA was diluted in water in an Eppendorf tube to a final volume of 100 ~.g/ml.
1 U of human SUBSTfTUTE SHEET (RULE 261 thrombin was added in the DNA solution and mixed gently.
The mixture of DNA and thrombin was added to the fibrinogen solution. After brief mixing, the mixture was loaded into Tygon tubing ( I /8" ID; 1 " to 1 I /4"
long, Formulation S-50-HL) which was sealed at one end. A Johnson & Johnson metallic stmt, 5.0 mm, was immediately inserted into the DNA / fibrinogen / thrombin mixture in the tubing, and incubated until the mixture solidified. The fibrin-coated stmt was removed and air dried.
The coated stmt was installed into the left and right pig iliac femoral arteries using routine surgical procedures.
Three days after installment of the stems, the arteries were excised, and homogenized using glass dowels. The protein extract was freeze-thawed 3x, heat-inactivated for I S minutes at 65°C and the supernatant was collected. 300 lxg of the soluble protein was used for CAT assays.
The results were read using a Betagen machine which measures the acetylation of CAT.
Implantation of the DNA coated stents in the vasculature 1 5 Juvenile domestic pigs (3 months, I 5-20 kg) of either sex are given aspirin ( I 0 mg/kg) orally two days prior to surgery and three times weekly for the duration of the study.
Pigs were anesthetized using Telazol (6.0 mg/kg IM) and xylazine (2.2 mg/kg IM) and intubated with an endotracheal tube. I% isofluane is administered throughout the surgical procedure. 150 units/kg of heparin were administered via IV prior to surgery.
Following prepping and draping, a midiine abdominal incision was made, extending caudally to the pubis through the skin and fascia, and the abdominal musculature was divided in the midline. The peritoneal cavity was opened and the intestines retracted cranially using a Balfour retractor. Using a combination of blunt and sharp dissection, each iliac and femoral artery was isolated from their cranial extent, caudally to beyond the bifurcation of the femoral artery.
SUBST~UTE SHEET tRULE 26) The internal iliac artery was ligated at its most caudal point with 2-0 silk.
Ties were looped around the proximal iliac and femoral arteries, then temporarily secured. An arteriotomy of the internal iliac artery was made just proximal to the ligature. The balloon-expandable stmt was advanced to the iliac artery and the balloon inflated using an inflation device at pressure of 6 atmospheres. The balloon was deflated and the balloon catheter removed, then the internal iliac artery was ligated followed by release of the loops. Restoration of arterial blood flow was confirmed. The peritoneum and the muscle were closed with 1-0 vicryl continuous sutures, and the fasciai layer closed with I -0 vicryl interrupted sutures. The skin was closed with staples.
Results 'I 0 The following data demonstrate the expression of the reporter gene, CAT, in porcine arteries in vivo following implantation of the DNA coated stmt.
Fibrinogen Reporter DNA % CAT activitydays after (mg) stem placement 1 15 100 8.4, 23 . I 3 500 , 6.2 3 15 1000 7.5, 3.9 3 2. 0 2 15 100 3.4 7 3 IS 100 2.54 10 4 10 100 2.8 3 5 10 100 0.9 10 The above data was used to determine the optimal dose of DNA and fibrinogen.
This data supports the principle that DNA coated stems can be implanted in a patient, the gene is expressed 'I 5 as a protein, and sufficient quantities of protein are produced to allow measurement thereof.
Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.
SUBSTITUTE SHEET (RULE 26)
The stmt is preferably coated with about 50 pg to about S mg of DNA. The thickness of the polymerizable matrix containing the DNA is typically about 5-500 pm. The matrix preferably covers the entire surface of the stent.
Methods for coating a stent with DNA
Methods for coating surfaces are well known in the art and include, for example, spray coating, immersion coating, etc. Any of these methods can be used in the invention. For example, a liquid monomeric matrix can be mixed with the DNA and polymerization initiated. The stmt can then be added to the polymerizing solution. such that polymer forms over its entire surface. The coated stent is then removed and dried. Multiple application steps can be used to provide improved coating uniformity and improved control over the amount of DNA applied to the stmt.
In a preferred embodiment, an aqueous mixture of DNA and human thrombin is added to an aqueous suspension of fibrinogen. The fibrinogen concentration of the suspension is typically between about 10-50, preferably about 20-40, more preferably about 30 mg/ml.
The concentration of the DNA in the aqueous mixture is typically about 1-20, preferably about 5-15, more preferably about 10 p,g/ml. The amount of human thrombin in the aqueous mixture about 0.5 to S, preferably about 1 U. The DNA and human thrombin are first added together to form a mixture and that mixture is then added to the fibrinogen suspension. Thereafter, a stmt is dipped into the polymerizing solution. After the mixture solidifies, the stent is removed.
Methods for placing the DNA coated stent within the vasculature The stmt can be placed onto the balloon at a distal end of a balloon catheter and delivered by conventional percutaneous means (e.g. as in an angioplasty procedure) to the site of the SUBSTITUTE SHEET (RULE 261 is:ai:, ''3t;:~.'lia~'s ~ ~'~_,..A.,;~;.~;. v ari y ~3~;
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Method of treating coronary and peripheral vascular diseases with the DNA
coated stems Coronary and peripheral diseases, including restenosis, atherosclerosis, coronary artery bypass graft stenosis, vein bypass graft stenosis or restenosis, arterio-venous fistula stenosis or restenosis, peripheral artery stenosis or restenosis, can be treated by implanting the DNA
coated stent of the present invention, into a coronary or peripheral artery or vein of a patient.
Suitable patients include mammals such as dogs, horses, cattle, humans, etc. Humans are preferred patients.
In an alternate embodiment, the DNA coated stent is implanted into the patient and an antiplatelet agent, anticoagulant agent, antimicrobial agent, anti-inflammatory agent, antimetabolic agent, antimitotic agent or other drug is administered to reduce the incidence of restenosis.
Suitable anticoagulant agents can include drugs such as heparin, coumadin, protamine, hirudin and tick anticoagulant protein. Suitable antimitotic agents and antimetabolite agents can include drugs such as colchicine, methotrexate, azathioprine, vincristine, vinblastine, fluorouracil, adriamycin and mutamycin. Ganciclovir or acyclovir is preferably administered.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
EXAMPLES
Procedure for coating the stents using thrombin polymerized fibrinogen (fibrin}
Human fibrinogen was dissolved in water at concentrations of 30 mg/ml. 100 ~1 of dif~'erent concentrations of fibrinogen were used in the preparation.
Fibrinogen was diluted in water when necessary and transferred to an Eppendorf tube.
Plasmid CAT (pCMV-CAT) was dissolved in water at concentrations of 10 mg/ml.
The DNA was diluted in water in an Eppendorf tube to a final volume of 100 ~.g/ml.
1 U of human SUBSTfTUTE SHEET (RULE 261 thrombin was added in the DNA solution and mixed gently.
The mixture of DNA and thrombin was added to the fibrinogen solution. After brief mixing, the mixture was loaded into Tygon tubing ( I /8" ID; 1 " to 1 I /4"
long, Formulation S-50-HL) which was sealed at one end. A Johnson & Johnson metallic stmt, 5.0 mm, was immediately inserted into the DNA / fibrinogen / thrombin mixture in the tubing, and incubated until the mixture solidified. The fibrin-coated stmt was removed and air dried.
The coated stmt was installed into the left and right pig iliac femoral arteries using routine surgical procedures.
Three days after installment of the stems, the arteries were excised, and homogenized using glass dowels. The protein extract was freeze-thawed 3x, heat-inactivated for I S minutes at 65°C and the supernatant was collected. 300 lxg of the soluble protein was used for CAT assays.
The results were read using a Betagen machine which measures the acetylation of CAT.
Implantation of the DNA coated stents in the vasculature 1 5 Juvenile domestic pigs (3 months, I 5-20 kg) of either sex are given aspirin ( I 0 mg/kg) orally two days prior to surgery and three times weekly for the duration of the study.
Pigs were anesthetized using Telazol (6.0 mg/kg IM) and xylazine (2.2 mg/kg IM) and intubated with an endotracheal tube. I% isofluane is administered throughout the surgical procedure. 150 units/kg of heparin were administered via IV prior to surgery.
Following prepping and draping, a midiine abdominal incision was made, extending caudally to the pubis through the skin and fascia, and the abdominal musculature was divided in the midline. The peritoneal cavity was opened and the intestines retracted cranially using a Balfour retractor. Using a combination of blunt and sharp dissection, each iliac and femoral artery was isolated from their cranial extent, caudally to beyond the bifurcation of the femoral artery.
SUBST~UTE SHEET tRULE 26) The internal iliac artery was ligated at its most caudal point with 2-0 silk.
Ties were looped around the proximal iliac and femoral arteries, then temporarily secured. An arteriotomy of the internal iliac artery was made just proximal to the ligature. The balloon-expandable stmt was advanced to the iliac artery and the balloon inflated using an inflation device at pressure of 6 atmospheres. The balloon was deflated and the balloon catheter removed, then the internal iliac artery was ligated followed by release of the loops. Restoration of arterial blood flow was confirmed. The peritoneum and the muscle were closed with 1-0 vicryl continuous sutures, and the fasciai layer closed with I -0 vicryl interrupted sutures. The skin was closed with staples.
Results 'I 0 The following data demonstrate the expression of the reporter gene, CAT, in porcine arteries in vivo following implantation of the DNA coated stmt.
Fibrinogen Reporter DNA % CAT activitydays after (mg) stem placement 1 15 100 8.4, 23 . I 3 500 , 6.2 3 15 1000 7.5, 3.9 3 2. 0 2 15 100 3.4 7 3 IS 100 2.54 10 4 10 100 2.8 3 5 10 100 0.9 10 The above data was used to determine the optimal dose of DNA and fibrinogen.
This data supports the principle that DNA coated stems can be implanted in a patient, the gene is expressed 'I 5 as a protein, and sufficient quantities of protein are produced to allow measurement thereof.
Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.
SUBSTITUTE SHEET (RULE 26)
Claims (80)
1. An implantable device comprising an intravascular stent coated with a polymer matrix and DNA encoding a therapeutically useful protein, said DNA
being uniformly dispersed within said matrix.
being uniformly dispersed within said matrix.
2. The implantable device of claim 1, wherein the stent is a polymeric or metallic stent.
3. The implantable device of claim 2, wherein the stent is stainless steel.
4. The implantable device of claim 1, wherein the therapeutically useful protein is an antiplatelet agent, anticoagulant agent, antimitotic agent, antioxidant, antimetabolite agent, or anti-inflammatory agent.
5. The implantable device of claim 1, wherein the therapeutically useful protein inhibits the proliferation of cells.
6 The implantable device of claim l, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma or cytosine deaminase.
7. The implantable device of claim 6, wherein the therapeutically useful protein is thymidine kinase or cytosine deaminase.
8. The implantable device of claim 1, wherein the stent is coated with about 50 µg to about 5 mg of DNA.
9 The implantable device of claim 1, wherein the polymer matrix comprises fibrin.
10. ~~The use of an implantable device for expressing therapeutically useful amounts of recombinant genes in vivo, wherein the device comprises an intravascular stent coated with a polymer matrix and DNA
encoding a therapeutically useful protein, said DNA being uniformly-dispersed within said matrix.
encoding a therapeutically useful protein, said DNA being uniformly-dispersed within said matrix.
11. ~~The use of claim 10, wherein the therapeutically useful protein is an antiplatelet agent, anticoagulant agent, antimitotic agent, antioxidant, antimetabolite agent, or anti-inflammatory agent.
12. ~~The use of claim 10, wherein the therapeutically useful proteins inhibits the proliferation of cells.
13. ~~The use of claim 10, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma or cytosine deaminase.
14. ~~The use of claim 13, wherein the therapeutically useful protein is thymidine kinase or cytosine deaminase.
15. ~~The use of an implantable device for treating or preventing a vascular disease, wherein the device comprises an intravascular stent coated with a polymer matrix and DNA encoding a protein therapeutically useful for treating the vascular disease, said DNA being uniformly dispersed within said matrix.
16. ~The use of claim 15, wherein the therapeutically useful protein is an antiplatelet agent, anticoagulant agent, antimitotic agent, antioxidant, antimetabolite agent, or anti-inflammatory agent.
17. ~The use of claim 15, wherein the therapeutically useful protein inhibits the proliferation of cells.
18. ~The use of claim 15, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma or cytosine deaminase.
19. ~The use of claim 15, wherein the therapeutically useful protein is thymidine kinase or cytosine deaminase.
20. ~The use of clam 15, wherein the vascular disease is restenosis, atherosclerosis, coronary artery bypass graft stenosis or restenosis, arterio-venous fistula stenosis or restenosis, or peripheral artery stenosis or restenosis.
21. ~The implantable device of claim 2, wherein the stent is a polymeric stent comprising poly(ethylene terephthalate), polyacetal, poly(lactic acid), and poly(ethylene oxide)/poly(butylene terephthalate) copolymer.
22.The implantable device of claim 1, wherein the DNA is naked DNA.
23. The implantable device of claim 1, wherein the DNA is incorporated into a vector.
24. The implantable device of claim 23, wherein the vector is selected from the group consisting of shuttle vectors, expressions vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
25. The implantable device of claim 1, wherein the polymer matrix is formed from an aqueous suspension of DNA and liquid monomeric matrix.
26. The implantable device of claim 1, wherein the DNA comprises an sm 22.alpha. promoter operatively linked to the DNA encoding the therapeutically useful protein.
27. The use of claim 10, wherein the DNA is naked DNA.
28. The use of claim 10, wherein the DNA is incorporated into a vector.
29. The use of claim 28, wherein the vector is selected from the group consisting of shuttle vectors, expression vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
30. The use of claim 10, wherein the therapeutically useful protein is a fusion protein.
31.~~The use of claim 10, wherein the DNA comprises an sm 22 .alpha. promoter operatively linked to the DNA encoding the therapeutically useful protein.
32.~~The use of claim 15, wherein the DNA is naked DNA.
33.~~The use of claim 15, wherein the DNA is incorporated into a vector.
34.~~The use of claim 33, wherein the vector is selected from the group consisting of shuttle vectors, expression vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
35.~~The use of claim 15, wherein the therapeutically useful protein is a fusion protein.
36.~~The use of claim 15, wherein the DNA comprises an sm 22 .alpha. promoter operatively linked to the DNA encoding the therapeutically useful protein.
37.~~The use of claim 10, wherein the stent is biostable.
38.~~The use of claim 10, wherein the polymer matrix is formed from an aqueous suspension of DNA and liquid monomeric matrix,
39. The use of claim 10, wherein the stent is a polymeric or metallic stent.
40. The use of claim 10, wherein the stent is stainless steel.
41. The use of claim 10, wherein the stent is coated with about 50 µg to about 5 mg of DNA.
42. The sue of claim 10, wherein the polymer matrix comprises fibrin.
43. The use of claim 10, wherein the stent is a polymeric stent comprising poly(ethylene terephthalate), polyacetal, poly(lactic acid), and poly(ethylene oxide)/poly(butylene terephthalate) copolymer.
44. The sue of claim 15, wherein the stent is biostable.
45. The use of claim 15, wherein the polymer matrix is formed from an aqueous suspension of DNA and liquid monomeric matrix.
46. The use of claim 15, wherein the stent is a polymeric or metallic stent.
47. The use of claim 15, wherein the stent is stainless steel.
48. The use of claim 15, wherein the stent is coated with about 50 pg to about 5 mg of DNA.
49. The use of claim 15, wherein the polymer matrix comprises fibrin.
50. The user of claim 15, wherein the stent is a polymeric stent comprising poly(ethylene terephthalate), polyacetal, poly(lactic acid), and poly(ethylene oxide)/poly(butylene terephthalate) copolymer.
51. the implantable device of claim 1, wherein the stent is biostable.
52. The implantable device of claim 1, wherein the therapeutically useful protein is a fusion protein.
53. An implantable device comprising an intravascular stent comprising a biostable material and coated with a polymer matrix and DNA encoding a therapeutically useful protein.
54. The implantable device of claim 53, wherein the stent is a polymeric or metallic stent.
55. The implantable device of claim 54, wherein the stent is stainless steel.
56. The implantable device of claim 53, wherein the therapeutically useful protein is an antiplatelet agent, anticoagulant agent, antimitolic agent.
antioxidant, antimetabolite agent, or anti-inflammatory agent.
antioxidant, antimetabolite agent, or anti-inflammatory agent.
57. The implantable device of claim 53, wherein the therapeutically useful protein inhibits the proliferation of cells.
58. The implantable device of claim 53, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma or cytosine deaminase.
59. The implantable device of claim 58, wherein the therapeutically useful protein is thymidine kinase or cytosine deaminase.
60. The implantable device of claim 53, wherein the stent is coated with about 50 µg to about 5 mg of DNA.
61. The implantable device of claim 53, wherein the polymer matrix comprises fibrin.
62. The implantable device of claim 54, wherein the stent is a polymeric stent comprising poly(ethylene terephthalate), polyacetal, poly(lactic acid), and poly(ethylene oxide) poly(butylene terephthalate) copolymer.
63. The implantable device of claim 53, wherein the DNA is naked DNA.
64. The implantable device of claim 53, wherein the DNA is incorporated into a vector.
65. The implantable device of claim 64, wherein the vector is selected from the group consisting of shuttle vectors, expression vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors and liposomes.
66. The implantable device of claim 53, wherein the polymer matrix is formed from an aqueous suspension of DNA and liquid monomeric matrix.
67. The implantable device of claim 53, wherein the DNA comprises an sm 22oc promoter operatively linked to the DNA encoding the therapeutically useful protein.
68. The implantable device of claim 53, wherein the therapeutically useful protein is a fusion protein.
69. An implantable device comprising an intravascular stent coated with a polymer matrix and DNA encoding a therapeutically useful protein, said DNA being in contact with said polymer matrix.
70. The implantable device of claim 69, wherein the stent is a polymeric or metallic stent.
71. The implantable device of claim 70, wherein the stent is stainless steel.
72. The implantable device of claim 69, wherein the therapeutically useful protein is an antiplatelet agent, anticoagulant agent, antimitotic agent, antioxidant, antimetabolite agent or anti-inflammatory agent.
73. The implantable device of claim 69 wherein the therapeutically useful protein inhibits the proliferation of cells.
74. The implantable device of claim 69, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57 retinoblastoma, protein or cytosine deaminase.
75. The implantable device of claim 74, wherein the therapeutically useful protein is thymidine kinase or cytosine deaminase.
76. The implantable device of claim 69, wherein the stent is in contact with about 50 µg to about 5 mg of DNA.
77. The implantable device of claim 69, wherein the polymer matrix comprises fibrin.
78. The use of an implantable device comprising an intravascular stent coated with a polymer matrix and DNA encoding a therapeutically useful protein, said DNA being in contact with said matrix, for expressing therapeutically useful amounts of recombinant genes in vivo.
79. The use of claim 78, wherein the therapeutically useful protein inhibits the proliferation of cells.
80. The use of claim 78, wherein the therapeutically useful protein is thymidine kinase, p16, p21, p27, p57, retinoblastoma, protein or cytosine deaminase.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/884,352 US6818016B1 (en) | 1997-06-27 | 1997-06-27 | Methods for coating stents with DNA and expression of recombinant genes from DNA coated stents in vivo |
| US08/884,352 | 1997-06-27 | ||
| PCT/US1998/013301 WO1999000071A1 (en) | 1997-06-27 | 1998-06-26 | Method for coating stents with dna and expression of recombinant genes from dna coated stent in vivo |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2295040A1 CA2295040A1 (en) | 1999-01-07 |
| CA2295040C true CA2295040C (en) | 2005-08-23 |
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| CA002295040A Expired - Fee Related CA2295040C (en) | 1997-06-27 | 1998-06-26 | Method for coating stents with dna and expression of recombinant genes from dna coated stent in vivo |
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| US (3) | US6818016B1 (en) |
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| DE (1) | DE69841041D1 (en) |
| WO (1) | WO1999000071A1 (en) |
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| CA2178541C (en) * | 1995-06-07 | 2009-11-24 | Neal E. Fearnot | Implantable medical device |
| US6335029B1 (en) | 1998-08-28 | 2002-01-01 | Scimed Life Systems, Inc. | Polymeric coatings for controlled delivery of active agents |
| US8088060B2 (en) | 2000-03-15 | 2012-01-03 | Orbusneich Medical, Inc. | Progenitor endothelial cell capturing with a drug eluting implantable medical device |
| US9522217B2 (en) | 2000-03-15 | 2016-12-20 | Orbusneich Medical, Inc. | Medical device with coating for capturing genetically-altered cells and methods for using same |
| AU2001238518B2 (en) * | 2000-04-04 | 2006-09-07 | Boston Scientific Limited | Medical devices suitable for gene therapy regimens |
| US6812217B2 (en) * | 2000-12-04 | 2004-11-02 | Medtronic, Inc. | Medical device and methods of use |
| US8038708B2 (en) * | 2001-02-05 | 2011-10-18 | Cook Medical Technologies Llc | Implantable device with remodelable material and covering material |
| DE60106962T2 (en) | 2001-12-12 | 2005-04-28 | Hehrlein, Christoph, Dr. | Porous metallic stent with a coating |
| US7491234B2 (en) | 2002-12-03 | 2009-02-17 | Boston Scientific Scimed, Inc. | Medical devices for delivery of therapeutic agents |
| EP1444995A1 (en) * | 2003-02-06 | 2004-08-11 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | FGF-2 derived proteins for the preparation of biomaterials or medical devices such as stents |
| DE10311729A1 (en) * | 2003-03-18 | 2004-09-30 | Schultheiss, Heinz-Peter, Prof. Dr. | Endovascular implant with an at least sectionally active coating of ratjadon and / or a ratjadon derivative |
| WO2004100836A1 (en) * | 2003-05-12 | 2004-11-25 | Cook Incorporated | Stent graft |
| WO2007016251A2 (en) * | 2005-07-28 | 2007-02-08 | Cook Incorporated | Implantable thromboresistant valve |
| US20070196423A1 (en) * | 2005-11-21 | 2007-08-23 | Med Institute, Inc. | Implantable medical device coatings with biodegradable elastomer and releasable therapeutic agent |
| CN100358483C (en) * | 2005-12-28 | 2008-01-02 | 中国医学科学院生物医学工程研究所 | Implanting device carried with plasmid DNA nanometer particle and its prepn. method |
| US8642063B2 (en) * | 2008-08-22 | 2014-02-04 | Cook Medical Technologies Llc | Implantable medical device coatings with biodegradable elastomer and releasable taxane agent |
| EP3495488A1 (en) | 2008-10-27 | 2019-06-12 | Baxalta GmbH | Models of thrombotic thrombocytopenic purpura and methods of use thereof |
| CA2902209A1 (en) | 2013-03-15 | 2014-09-18 | The Regents Of The University Of California | Peptides having reduced toxicity that stimulate cholesterol efflux |
| CN104825249B (en) * | 2015-04-28 | 2017-11-07 | 温州医科大学 | A kind of surface mediated gene therapeutic type intraocular lens and preparation method thereof |
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| US5698531A (en) * | 1989-03-31 | 1997-12-16 | The Regents Of The University Of Michigan | Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor |
| US5439446A (en) * | 1994-06-30 | 1995-08-08 | Boston Scientific Corporation | Stent and therapeutic delivery system |
| US5190540A (en) | 1990-06-08 | 1993-03-02 | Cardiovascular & Interventional Research Consultants, Inc. | Thermal balloon angioplasty |
| CA2082411A1 (en) | 1991-06-28 | 1992-12-29 | Robert D. Rosenberg | Localized oligonucleotide therapy |
| WO1993006792A1 (en) | 1991-10-04 | 1993-04-15 | Scimed Life Systems, Inc. | Biodegradable drug delivery vascular stent |
| US5500013A (en) | 1991-10-04 | 1996-03-19 | Scimed Life Systems, Inc. | Biodegradable drug delivery vascular stent |
| US5464450A (en) | 1991-10-04 | 1995-11-07 | Scimed Lifesystems Inc. | Biodegradable drug delivery vascular stent |
| US5336615A (en) * | 1992-01-06 | 1994-08-09 | Yale University | Genetically engineered endothelial cells exhibiting enhanced migration and plasminogen activator activity |
| US5599352A (en) * | 1992-03-19 | 1997-02-04 | Medtronic, Inc. | Method of making a drug eluting stent |
| US5571166A (en) * | 1992-03-19 | 1996-11-05 | Medtronic, Inc. | Method of making an intraluminal stent |
| US5591224A (en) * | 1992-03-19 | 1997-01-07 | Medtronic, Inc. | Bioelastomeric stent |
| US5383928A (en) * | 1992-06-10 | 1995-01-24 | Emory University | Stent sheath for local drug delivery |
| US5464650A (en) | 1993-04-26 | 1995-11-07 | Medtronic, Inc. | Intravascular stent and method |
| US5443827A (en) * | 1993-05-03 | 1995-08-22 | President And Fellows Of Harvard College | Fibrin-targeted inhibitors of thrombin |
| AU7019494A (en) | 1993-05-20 | 1994-12-20 | Baylor College Of Medicine | Genetic therapy for cardiovascular disease |
| US5886026A (en) * | 1993-07-19 | 1999-03-23 | Angiotech Pharmaceuticals Inc. | Anti-angiogenic compositions and methods of use |
| US6780406B1 (en) | 1994-03-21 | 2004-08-24 | The Regents Of The University Of Michigan | Inhibition of vascular smooth muscle cell proliferation administering a thymidine kinase gene |
| US5588962A (en) * | 1994-03-29 | 1996-12-31 | Boston Scientific Corporation | Drug treatment of diseased sites deep within the body |
| DK0754054T3 (en) * | 1994-04-08 | 2002-10-14 | Viron Therapeutics Inc | Antirestenosis protein |
| WO1997009006A1 (en) * | 1995-09-01 | 1997-03-13 | Emory University | Endovascular support device and method of use |
| US5863904A (en) | 1995-09-26 | 1999-01-26 | The University Of Michigan | Methods for treating cancers and restenosis with P21 |
| US6090618A (en) * | 1996-10-07 | 2000-07-18 | Arch Development Corporation | DNA constructs and viral vectors comprising a smooth muscle promoter |
| US5833651A (en) | 1996-11-08 | 1998-11-10 | Medtronic, Inc. | Therapeutic intraluminal stents |
| ZA9710342B (en) * | 1996-11-25 | 1998-06-10 | Alza Corp | Directional drug delivery stent and method of use. |
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1998
- 1998-06-26 AU AU82676/98A patent/AU8267698A/en not_active Abandoned
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| US20080112997A1 (en) | 2008-05-15 |
| EP1023005A4 (en) | 2006-06-14 |
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| WO1999000071A1 (en) | 1999-01-07 |
| EP1023005B1 (en) | 2009-08-05 |
| CA2295040A1 (en) | 1999-01-07 |
| JP2007301402A (en) | 2007-11-22 |
| US6818016B1 (en) | 2004-11-16 |
| EP1023005A1 (en) | 2000-08-02 |
| ATE438361T1 (en) | 2009-08-15 |
| US20050038499A1 (en) | 2005-02-17 |
| AU8267698A (en) | 1999-01-19 |
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