EP1355659A2 - Methodes et compositions permettant de stimuler l'angiogenese - Google Patents

Methodes et compositions permettant de stimuler l'angiogenese

Info

Publication number
EP1355659A2
EP1355659A2 EP02714757A EP02714757A EP1355659A2 EP 1355659 A2 EP1355659 A2 EP 1355659A2 EP 02714757 A EP02714757 A EP 02714757A EP 02714757 A EP02714757 A EP 02714757A EP 1355659 A2 EP1355659 A2 EP 1355659A2
Authority
EP
European Patent Office
Prior art keywords
pdgf
fgf
vegf
angiogenesis
tissue
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.)
Ceased
Application number
EP02714757A
Other languages
German (de)
English (en)
Inventor
Robert Pawliuk
Philippe Leboulch
Renhai. Cao
Yihai. Cao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bluebird Bio Inc
Original Assignee
Genetix Pharmaceuticals Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Genetix Pharmaceuticals Inc filed Critical Genetix Pharmaceuticals Inc
Publication of EP1355659A2 publication Critical patent/EP1355659A2/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
    • 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
    • A61K38/1825—Fibroblast growth factor [FGF]
    • 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
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • 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
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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

  • tissue ischemias including myocardial ischemia, ischaemic bowel disease, and peripheral ischemia.
  • the alleviation of tissue ischemia is critically dependent upon angiogenesis, the process by which new capillaries are generated from existing vasculature and tissue.
  • the spontaneous growth of new blood vessels provide collateral circulation in and around an ischemic area, improves blood flow, and alleviates the symptoms caused by the ischemia.
  • surgery or angioplasty may help to revascularize ischemic regions in some cases, the extent, complexity and location of the arterial lesions which cause the occlusion often prohibits such treatment.
  • the present invention provides novel methods and compositions for promoting angiogenesis to treat a variety of tissue ischemias, including peripheral and myocardial ischemia.
  • Selected angiogenic factors or synergistic combinations of factors, functional analogues of such factors or combinations of factors, or nucleic acids encoding such factors or combinations of factors are delivered to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.
  • the invention further includes improved methods and vehicles for delivering such factors or combinations of factors, functional analogues of such factors or combinations of factors, and nucleic acids encoding such factors or combinations of factors.
  • the invention provides a method of promoting angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.
  • the PDGF-BB can be delivered either alone or in combination with another angiogenesis-promoting factor, particularly bFGF and/or NEGF-A.
  • the angiogenesis-promoting factor or combination of factors can be administered in the form of a protein composition or an expression plasmid encoding the protein(s).
  • the angiogenesis-promoting factor or combination of factors can also be administered in the form of functional analogues of the factor or combination of factors.
  • anti-idiotypic antibodies of PDGF-BB, NEGF-A and/or bFGF can be administered in accordance with the invention.
  • suitable vectors include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, R ⁇ A vectors, liposomes, cationic lipids, lentiviral vectors and transposons.
  • the invention provides a method for promoting angiogenesis by delivering angiogenic factors, such as those described above, to a localized area of tissue using heparin sepharose-containing microcapsules in an amount effective to induce angiogenesis within the area of tissue.
  • the angiogenic factors or expression plasmids encoding the factors are incorporated into the microcapsules as described in the working examples provided below for slow, sustained release into localized areas of tissue.
  • the microcapsules are made up of uncoated heparin sepharose beads, heparin sepharose beads coated with a single layer of alginate polymer, heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or heparin sepharose beads coated with alternating layers of alginate and PEG.
  • the microcapsules range in size from 1-200 microns.
  • Suitable angiogenic factors for incorporating into the microcapsules include, for example, M-CSF, GM-CSF, NEGF-A, VEGF-B, VEGF-C, NEGF-D, VEGF-E, neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, PDGF-BB, PDGF- AA, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF- beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1, and combinations thereof or an expression vectors encoding such angiogenic factors.
  • the angiogenic factors can be purified from their native sources or produced by recombinant expression.
  • the microcapsules are contacted with the localized area of tissue generally by injection or surgical implantation.
  • injection can be performed using a catheter based trans-myocardial injection technology, such as the ⁇ OGA technology.
  • the present invention provides a method for promoting angiogenesis by contacting a localized area of tissue with a gradient of one or more angiogenic factors or a nucleic acid encoding one or more angiogenic factors, such that directed vascular growth along the gradient is achieved.
  • Such directed vascular growth can be used to achieve interconnection and/or intraconnection of blood vessels (e.g., to circumvent blood flow around a blockage within a blood vessel).
  • the angiogenic factor or nucleic acid is released in a gradient using a biocompatible material which is contacted with (e.g., implanted within) the localized area of tissue.
  • the angiogenic factor is associated with the biocompatible material (e.g., absorbed onto the biocompatible material) such that it is released onto surrounding tissue. This can be achieved by treating the biocompatible material with the angiogenic factor prior to contact with (e.g., implantation into) a selected area of tissue.
  • the angiogenic factor is then released from the biocompatible material onto the surrounding tissue in a directed gradient determined by the placement of the biocompatible polymer
  • Suitable biocompatible materials include, for example, polymers or threads which incorporate the angiogenic factor.
  • the biocompatible material is an absorbable thread, such as polyglyconate monofilament, poliglecaprone 25-(Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid, Biodyn glycomer 631, chromic surgical gut or plain surgical gut.
  • Figure 1 is a graph comparing levels of angiogenesis in the Matrigel model using a low dose of transduced cells encoding GFP alone (control), NEGF-A, NEGF-C, NEGF-D bFGF or PDGF-BB.
  • C57B1/10 mice were each injected subcutaneously into the abdominal with a low dose of 3 x 10 5 retrovirally transduced autologous myoblast cells, suspended in 0.4ml of Matrigel. Mice were sacrificed 13 days later and the matrigel pellet and a section of the abdominal muscle adjacent to the pellet was removed. Samples were sectioned and the number of microvessels in the abdominal muscle was quantified by visual inspection of sections under the microscope.
  • FIG. 2 is a graph comparing levels of angiogenesis in the Matrigel model using a high dose of cells transduced to express bFGF, NEGF-A and PDGF-BB. C57B1/10 mice were each injected with a high cell dose of 2 x 10 6 retrovirally transduced autologous myoblast cells suspended in 0.4ml of Matrigel.
  • PDGF-BB was as potent as either bFGF or NEGF-A at stimulating angiogenesis.
  • Figure 3 shows photographs of mouse corneas 6 days following the implantation of pellets coated with control saline (A), PDGF-BB (B), NEGF-A (C) or bFGF (D) alone.
  • Bottom panels Quantification of the angiogenic effect elicited by each factor. Vessel length (E), clock hours (F) and area (G) are shown.
  • Figure 4 shows photographs of mouse corneas 6 days following the implantation of pellets coated with VEGF-A alone (left panel), bFGF (middle panel) or both factors combined (right panel).
  • (B) shows the quantification of the angiogenic effect elicited by each growth factor in terms of clock hours (left panel), vessel length (middle panel) and area (right panel).
  • Figure 5 shows photographs of mouse corneas 6 days post- transplantation of pellets coated with bFGF alone (left panel) or bFGF combined with PDGF-BB (middle and right panels). Bottom panels show photographs of mouse corneas 6 days post-transplantation of pellets coated with either VEGF-A alone (left panel) or VEGF-A combined with PDGF-BB (right panel).
  • Figure 6 is a graph comparing the quantification of angiogenesis in the mouse cornea model using PDGF-BB, VEGF-A or bFGF either alone or in combination.
  • Corneal micropockets were created with a cataract knife in the eyes of 8-week old C57B1/6 mice.
  • aluminum sulfate pellets coated with between 80 and 160ng of recombinant human PDGF-BB, VEGF-A, bFGF or combinations thereof were implanted and mice were monitored daily. A total of 5 mice were transplanted per group. The area of newly grown vessels was assessed 5 days post implantation. Mice implanted with control pellets showed no evidence of angiogenesis.
  • bFGF stimulated the highest level of angiogenesis followed by VEGF-A and PDGF-BB.
  • the level of angiogenesis stimulated by VEGF-A in combination with PDGF-BB was equivalent to that observed for bFGF alone.
  • the most potent combination was PDGF-BB and bFGF.
  • PDGF-BB and bFGF together stimulated the greatest level of angiogenesis, significantly greater than that observed for VEGF-A and bFGF.
  • FIG. 7 is a schematic illustration of the experimental strategy to make heparin sepharose/alginate microcapsules.
  • Heparin sepharose beads (Pharmacia: 50- 150 Dm in size) are mixed with a solution of sodium alginate to a final concentration of 200mg/ml.
  • the heparin sepharose/alginate solution is then loaded into a 5ml syringe and slowly injected into a coaxial airflow system constructed at Genetix.
  • the coaxial air flow creates a mist of the heparin sepharose/alginate solution which drops into a 1.5% calcium chloride bath.
  • the alginate hits the calcium solution the alginate becomes cross-linked, forming a solid gel capsule roughly in the shape of a sphere.
  • the size of the microcapsules can vary greatly from 50 - 400 Dm. Large microcapsules (greater than 200 Dm in size) are removed from the capsule mixture using a 200 Dm sieve. Once formed the capsules are washed twice in sterile water and stored in buffer composed of 0.9% sodium chloride and ImM calcium chloride. Capsules are loaded with recombinant human PDGF-BB by incubation in binding buffer (0.9% NaCl, ImM CaC12 and 0.05% gelatin) at 4°C overnight (-16 hours) with gentle shaking. The next day the capsules are removed, washed twice in binding buffer and either cultured in vitro to determine the kinetics of PDGF-BB release or injected in vivo to assess angiogenesis. The efficiency of PDGF-BB uptake is quantified by ELISA of the binding buffer following removal of the capsules.
  • Figure 8 is a graph showing that heparin sepharose/alginate capsules bind large amounts of recombinant human PDGF-BB. Shown is the amount of PDGF-BB absorbed by 3000 capsules following incubation with various quantities of growth factor. The amount of PDGF-BB remaining in the binding buffer following incubation with capsules was quantified by ELISA. Three thousand capsules were able to absorb at least 35 ⁇ g of PDGF-BB representing 13 ng of PDGF-BB per capsule.
  • Figure 9 is a graph showing that heparin sepharose/alginate microcapsules provide slow, high level and long term release of bound PDGF-BB in vitro.
  • Ten ⁇ g of recombinant human PDGF-BB was incubated with three different types of test samples. The first test sample was composed of non-encapsulated heparin sepharose beads while the second and third groups were composed of alginate encapsulated heparin sepharose beads made using either a 1.2% or a 1.6% alginate solution.
  • Three thousand beads/microcapsules were incubated with PDGF-BB at 4°C overnight with gentle shaking.
  • ELISA analysis of the binding buffer the next day showed absorption of 90% (9 ⁇ g) of the PDGF by the capsules.
  • the beads/microcapsules were washed, resuspended in 5mls of serum free medium and incubated at 37°C. Every 24 hours the medium was changed and the amount of PDGF- BB present in the medium quantified by ELISA. A slow, sustained release of approximately 0.5-3% of the total bound PDGF-BB (representing 125-250ng) was detected each day for a minimum of 14 days, the longest time point tested. Importantly, the proportion of PDGF-BB released per day is equivalent to the amount of PDGF-BB that was estimated to be secreted by muscle cells transduced with the PDGF-BB retro virus in the foregoing Matrigel experiments. The release kinetics for non- encapsulated heparin sepharose beads was better than those observed for the alginate encapsulated heparin sepharose.
  • Figure 10 is a graph showing that PDGF-BB microcapsules potently stimulate angiogensis in vivo in the stringent Matrigel model.
  • Three thousand microcapsules loaded with l ⁇ g or lO ⁇ g of PDGF-BB were mixed with 400 ⁇ l of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. The results showed that the number of microvessels in mice receiving microcapsules loaded with lOmg of PDGF-BB was 2.5- fold greater than that of control mice.
  • FIG 11 is a graph showing that PDGF-BB microcapsules stimulate angiogenesis in infarcted rat hearts 3 weeks post-injection.
  • Infarcted rat hearts were injected with 1600 microcapsules containing ⁇ g (control) or 18 ⁇ g of PDGF-BB in a volume of 20 ⁇ l.
  • Three weeks post injection rats were sacrificed, hearts were removed, fixed, sectioned and the number of microvessels within the infarct region quantified by visual inspection under a microscope. Shown is the number of microvessels per 5 high power fields for recipients of control and PDGF-BB microcapsules.
  • Rats injected with PDGF-BB microvessels showed an approximate 2-fold increase in the number of microvessels as compared to control rats.
  • Figure 12 shows an analysis of cardiac function in rats injected with control vs. PDGF-BB microcapsules following myocardial infarction.
  • Left ventricular pressure (LVP), dP/dT, neg dP/dT and tau were measured prior to sacrifice at 3 weeks post injection.
  • Left ventricular pressure (LNP) is the maximum pressure in the left ventricle during contraction.
  • the dP/dT variable is the first derivative of the pressure wave and is separately viewed for the upstroke (dP/dT) and the downstroke (neg dP/dT).
  • the upstroke (dP/dT) is a measure of contractility and reflects the condition of the muscle independent of the pressure.
  • Neg dP/dT reflects the relaxation of the muscle, which together with the relaxation constant, tau, provides information on the stiffness of the ventricular wall following infarction.
  • a significant improvement in all parameters was detected in rats injected with PDGF-BB microcapsules. Rats injected with PDGF-BB microcapsules showed a 25% increase in left ventricular pressure, a 2-3 fold increase in cardiac contractility/relaxation and a 2.5-3 fold decrease in the relaxation constant tau.
  • Figure 13 is a graph showing that PDGF-BB and bFGF delivered by slow release microcapsules potently synergize to stimulate angiogensis in vivo in the stringent Matrigel model.
  • Three thousand microcapsules loaded with l ⁇ g of bFGF were mixed with 400 ⁇ l of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. The results showed that the number of microvessels in mice receiving bFGF + PDGF-BB microcapsules was 4-fold greater than that of mice implanted with either growth factor alone.
  • Figure 14 is a schematic illustration of the structure of various angiogenic expression plasmids. All vectors were constructed using the pCI vector backbone from Promega. All vectors contained the Cytomegalovirus immediate-early enhancer/promoter region, a chimeric intron and the late poly adenylation signal from SV40. The cDNA encoding either human PDGF-BB, NEGF-A or bFGF was inserted into this vector downstream of the chimeric intron.
  • a cD ⁇ A encoding for the mature PDGF-BB protein was cis-linked to the secretory signal from the murine IgGkappa immunoglobulin light chain gene while the NEGF-A cD ⁇ A utilized its endogenous secretory signal. Since endogenous bFGF is not secreted by the usual Golgi pathway and prior groups have had difficulty in obtaining high level secretion of bFGF from cells transduced with the bFGF cD ⁇ A, the bFGF cD ⁇ A was linked in cis to the secretory signal from the human Interleukin-2 cD ⁇ A.
  • test animals are anesthetized and intubated. The chest wall is opened and a myocardial infarct is created by tying off the anterior descending artery. 180 ⁇ g of control or test expression plasmid is injected into the heart wall in a volume of 20 ⁇ l. Cardiac function is assessed 3 weeks post injection. Animals are sacrificed, the heart is removed and efficiency of plasmid uptake is assessed by staining with X-gal. The size of the infarct and the extent of angiogenesis is quantified.
  • the present invention provides, for the first time, methods for promoting angiogenesis and treating such ischemia using particular angiogenic factors, such as PDGF-BB, as well as particular combinations of factors (e.g., combinations which include PDGF-BB).
  • angiogenic factors such as PDGF-BB
  • combinations of factors e.g., combinations which include PDGF-BB
  • the present invention also provides improved methods for delivering the factors to increase their efficacy, for example, by enabling directed and/or controlled release of the angiogenic factors onto surrounding tissue (e.g., ischemic myocardium).
  • angiogenic factor refers to any known protein factor capable of promoting growth of new blood vessels from existing vasculature ("angiogenesis"). Suitable angiogenic factors for use in the invention include, for example, PDGF-BB, PDGF-AA, M-CSF, GM-CSF, NEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof.
  • angiogenic factor also refers to functional analogues of the above- mentioned factors.
  • Such functional analogues include, for example, functional peptides or portions of the factors.
  • Functional analogues also include anti-idiotypic antibodies which bind to the receptors of the factors and, thus, mimic the activity of the factors in promoting angiogenesis. Methods for generating such anti-idiotypic antibodies are well known in the art and are described, for example, in WO 97/23510, the contents of which are incorporated by reference herein.
  • Antigens have specific epitopes to which certain antibodies will bind.
  • the region of the antibody that specifically interacts with the epitopes on the antigen is called the antigen combining site.
  • the antigen combining site is composed of a collection of idiotopes which are unique sequences on the antibody that specifically interact with the epitopes on the antigen.
  • the specific collection of idiotopes that interact with the epitopes on the antigen is defined as an antibody's "idiotype".
  • an anti- idiotype antibody is an antibody that is directed against the antigen combining site of the first antibody. Anti-idiotype antibodies combine with those specific sequences and may resemble or act as the epitope to which the first antibody reacts.
  • This anti-idiotype antibody may then mimic the biological activity of bFGF itself by binding to the bFGF receptor and activating it.
  • anti-idiotype antibodies as growth factor analogues
  • antibodies have much greater half lives and therefore their potency is maintained for a greater length of time in vivo.
  • the levels of growth factors that are required to achieve a biological effect can, in certain instances, produce adverse reactions such as toxicity and hypotension. Since lower levels of anti-idiotype antibodies may be required to produce the same biological effect these adverse side effects may be prevented.
  • Angiogenic factors used in the present invention can be purified from their native sources or produced by recombinant expression and subsequently administered to patients as a protein composition. Alternatively, the factors can be administered in the form of an expression plasmid encoding the factors, as is described in further detail below.
  • suitable expression plasmids is well known in the art. Particular angiogenic expression plasmids for use in the invention are shown in Figure 14 and are described in Example 1.
  • Suitable vectors for constructing expression plasmids include, for example, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, RNA vectors, liposomes, cationic lipids, lentiviral vectors and transposons.
  • the present invention provides novel methods and compositions for promoting angiogenesis to promote angiogenesis and to treat a variety of tissue ischemias.
  • Selected angiogenic factors or synergistic combinations of factors, functional analogues of such factors or combinations of factors, or nucleic acids encoding such factors or combinations of factors, are delivered to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.
  • the invention provides a method of promoting angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.
  • the PDGF-BB can be delivered either alone or in combination with another angiogenesis-promoting factor.
  • Particularly preferred combinations include PDGF-BB combined with bFGF and/or VEGF-A. DELIVERY VEHICLES FOR ANGIOGENIC FACTORS
  • the present invention provides a means for delivering angiogenic factors to a localized area of tissue in a controlled, sustained fashion.
  • One problem in using purified (e.g., recombinant) angiogenic proteins to stimulate angiogenesis for the treatment of myocardial and peripheral tissue ischemia can be the short half-life of the protein upon injection in vivo. This problem is addressed by way of the present invention using slow-release heparin sepharose-containing microcapsules in an amount effective to induce angiogenesis within the area of tissue.
  • One property that many angiogenic factors share is the ability to bind to heparin, a highly sulfated glycosaminoglycan that plays a role in anti-coagulation in vivo.
  • the microcapsules are injected or surgically implanted into localized areas of tissue.
  • the microcapsules are delivered to localized areas of tissue using the NOGA system (Biosense).
  • NOGA is a 3 dimensional catheter based transmyocardial injection system. A catheter is inserted into a major vein/artery and is snaked up into either of the ventricals of the heart. The end of the catheter contains a needle and a space in which therapeutic agents can be inserted and subsequently injected intra-myocardially into the damaged areas of the heart muscle. This delivery method is much easier and safer for the patient and is much more efficacious than other methods of delivery including intracoronary injections or placing therapeutic agents against the wall of the heart.
  • microcapsules of the present invention can be used with the system.
  • the microcapsules are composed of single heparin sepharose beads which optionally can be coated with a thin layer of alginate polymer.
  • the microcapsules are made up of uncoated heparin sepharose beads, heparin sepharose beads coated with a single layer of alginate polymer, heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or heparin sepharose beads coated with alternating layers of alginate and PEG.
  • PEG poly-ethylene glycol
  • the microcapsules can also be made small enough for use in the NOGA delivery system.
  • the microcapsules typically range from 1-200 microns in size.
  • they are able to absorb large quantities of angiogenic factors, such as FGF-2, VEGF-A and PDGF-BB, and slowly release the factors over extended periods of time at levels which are able to stimulate the growth of new blood vessels in vivo.
  • the present invention provides a means for delivering angiogenic factors to a localized area of tissue using a gradient of one or more angiogenic factors or a nucleic acid encoding one or more angiogenic factors, such that directed vascular growth along the gradient is achieved.
  • Angiogenic factors such as those described in the preceding examples, work by providing a gradient of angiogenic factor that stimulates the chemotaxis and proliferation of endothelial cells, and their supporting cells towards the source of the factor. Regulating the growth of new vessels from existing vasculature (angiogenesis) that effectively bypass an arterial lesion requires strict spatial and temporal control. Accordingly, by forming a directed gradient of angiogenic factors in accordance with the present invention, interconnection and/or intraconnection of blood vessels (e.g., to circumvent blood flow around a blockage within a blood vessel) can be achieved.
  • the angiogenic factor (or a nucleic acid encoding the factor) is released in a gradient using a biocompatible material which contains the factor such that the factor is released onto surrounding tissue when the biocompatible material is contacted with (e.g., implanted within) the tissue.
  • a biocompatible material which contains the factor such that the factor is released onto surrounding tissue when the biocompatible material is contacted with (e.g., implanted within) the tissue.
  • This can be achieved by treating the biocompatible material with the angiogenic factor prior to contact with (e.g., implantation into) a selected area of tissue in a manner which allows for release of the factor from the material in vivo.
  • the biocompatible material is then contacted with (e.g., implanted into) a localized area of tissue in a configuration which provides a directed gradient of the angiogenic factor once it is released from the material.
  • Suitable biocompatible materials for use in the invention include, for example, polymers or threads which incorporate the angiogenic factor.
  • the biocompatible material is an absorbable thread, such as polyglyconate monofilament, poliglecaprone 25-(Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid, Biodyn glycomer 631, chromic surgical gut or plain surgical gut.
  • the biocompatible material can be coated with one or several angiogenic factors to allow delivery of growth factor or growth factor combinations that provide the optimal angiogenic stimulus.
  • the biocompatible material can also be engineered to release certain growth factors at specific rates and at specific times that may help mimic the natural angiogenic process more closely.
  • EXAMPLE 1 Construction of Retroviral Vectors Containing Angiogenic cDNAs , Analysis of Virus Titer and Assessment of Stable Gene Transfer
  • PDGF-BB In order to provide stable, high-level delivery of PDGF-BB in the Matrigel model, primary myoblasts from C57B1/10 mice were transduced with retroviral vectors encoding human PDGF-BB. To compare the angiogenic potential of PDGF-BB to other known angiogenic agents, retroviral vectors encoding human VEGF-A165, VEGF-C, VEGF-A, VEGF-D, PDGF-BB, or bFGF also were constructed and tested. All vectors are shown schematically in Figure 14.
  • VEGF-C, VEGF-D and PDGF-BB cDNAs encode proteins which are inactive (or less active in the case of PDGF-BB) in their non- processed form
  • vectors containing cDNAs encoding the mature forms of the aforementioned proteins linked to the powerful secretory signal from the murine IgG kappa immunoglobulin gene were constructed.
  • All vectors also contained the gene encoding the green fluorescent protein (GFP) to enable the fast efficient and non-toxic selection of transduced target cells by fluorescence activated cell sorting (FACS).
  • FACS sorting is used to isolate the brightest 10% of GFP positive retrovirally transduced cells. Since both GFP and the angiogenic cDNA are translated from the same mRNA molecule, this ensures that the sorted cells also express high levels of the angiogenic protein. A strong correlation between the fraction of top GFP expressing cells and the amount of angiogenic protein secreted by the sorted cells exists. This result, in combination with data from Southern blot analysis of transduced cells which showed 3-6 proviral copies per genome in sorted GFP positive cells, indicates that the levels of angiogenic protein production and secretion are at their maximal level using this system.
  • Vectors were tested for stability of gene transfer and virus titer.
  • Vectors demonstrated a virus titer ranging from approximately 5 x 10 5 - 1.2 x 10 6 infectious virus particles per ml and all vectors showed stable transfer of the angiogenic cDNA to target primary skeletal muscle myoblasts from C57B1/10 mice.
  • all myoblast samples were also marked by infection with a retroviral vector encoding a ⁇ - galactosidase/neomycin ( ⁇ -GEO) resistance fusion gene.
  • ⁇ -GEO ⁇ - galactosidase/neomycin
  • the potency of the angiogenic proteins secreted from the transduced myoblasts described in Example 1 was assessed in vivo using the stringent Matrigel assay.
  • 3 x 10 5 - 2 x 10 6 transduced myoblasts were suspended in Matrigel and injected subcutaneously into the dorsal abdominal region of C57B1/10 mice.
  • the Matrigel pellets in addition to a section of abdominal muscle adjacent to the Matrigel pellet, were recovered 13 days post-injection. Following harvesting of the Matrigel pellet and the adjacent abdominal muscle 13 days post-injection, Matrigel pellets were stained with X- gal and analyzed for the presence of blue, retrovirally transduced myoblasts. In addition, the number of microvessels in the adjacent abdominal muscle was quantified by visual inspection.
  • mice Corneal micropockets were created with a cataract knife in the eye of 8-week old C57B1/6 mice. Into this pocket, a 0.34mm X 0.34mm sucrose aluminum sulfate pellet coated with hydron polymer containing 160ng of recombinant human PDGF-BB, 160ng of human VEGF-A, or 80 ng of human bFGF was implanted and mice were monitored daily. While those mice implanted with control pellets showed no evidence of angiogenesis, all mice receiving PDGF-BB coated pellets showed evidence of potent angiogenesis (see Figure 3).
  • recombinant PDGF-BB protein can potently stimulate the growth of new vessels in both the mouse Matrigel and corneal assays.
  • PDGF-BB by itself, although inducing a clear and potent angiogenic response, was less potent than either VEGF-A or bFGF ( Figure 3).
  • Figure 3 The differences observed for the Matrigel and mouse corneas models could be explained by the production of additional endogenous angiogenic factors by transduced myoblasts that synergize more readily with PDGF-BB compared to VEGF-A.
  • the levels of recombinant VEGF-A and PDGF- BB produced in vivo may differ to the levels produced in vitro.
  • This polymer is able to form gels through chemical cross-linking with multivalent cations such as calcium.
  • the capsules were forced through a 250 ⁇ m sieve, washed twice in sterile water and stored in buffer composed of 0.9% sodium chloride and ImM calcium chloride. Visual analysis of the capsules under the microscope showed that the vast majority of microcapsules were composed of individual heparin sepharose beads coated with a thin layer of alginate. Heparin-sepharose/alginate microcapsules were incubated overnight at 4 degrees
  • mice The ability of the heparin-sepharose/alginate microcapsules to stimulate angiogenesis in vivo was assessed using the stringent Matrigel assay.
  • Three thousand microcapsules loaded with 1 ⁇ g or 1 O ⁇ g of PDGF-BB were mixed with 400 ⁇ l of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. As shown in Figure 10, the number of microvessels in mice receiving microcapsules loaded with lOmg of PDGF-BB was 2.5-fold greater than that of control mice.
  • PDGF-BB and bFGF delivered by slow release microcapsules synergize to stimulate angiogensis in vivo in the stringent Matrigel model.
  • Three thousand microcapsules loaded with l ⁇ g of bFGF were mixed with 400 ⁇ l of Matrigel and subcutaneously injected into the abdominal region of
  • mice C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope.
  • Figure 13 shows that the number of microvessels in mice receiving bFGF + PDGF-BB microcapsules was 4-fold greater than that of mice implanted with either growth factor alone.
  • PDGF-BB microcapsules were also tested for their ability to stimulate angiogenesis in infarcted rat hearts 3 weeks post-injection.
  • Infarcted rat hearts were injected with 1600 microcapsules containing ⁇ g (control) or 18 ⁇ g of PDGF-BB in a volume of 20 ⁇ l.
  • Three weeks post injection rats were sacrificed, hearts were removed, fixed, sectioned and the number of microvessels within the infarct region quantified by visual inspection under a microscope (i.e., number of microvessels per 5 high power fields for recipients of control and PDGF-BB microcapsules).
  • rats injected with PDGF-BB microvessels showed an approximate 2-fold increase in the number of microvessels as compared to control rats.
  • heparin-sepharose/alginate microcapsules were also assessed using the ischemic rat heart model as follows.
  • Adult male rats were anesthetized, intubated and ventilated with a Harvard respirator. Under sterile conditions, a left lateral thoractomy was performed. The heart was exposed and the left descending coronary artery was ligated with a 8-0 Prolene suture.
  • each heart was injected twice intramyocaridally with lO ⁇ l of a buffer suspension containing approximately 800 heparin sepharose/alginate microcapsules with 9 ⁇ g of absorbed recombinant human PDGF-BB protein.
  • a total of 1600 microcapsules containing 18 ⁇ g of human PDGF-BB protein were injected into each rat heart.
  • the lungs were then inflated and the wound was closed in layers.
  • cardiac function was assessed using a variety of parameters including left ventricular pressure (LVP), dP/dT (a measure of cardiac contractility), negative dP/dT (a measure of relaxation of the cardiac muscle) and tau (the relaxation constant) (see Figure 12).
  • LVP left ventricular pressure
  • dP/dT a measure of cardiac contractility
  • negative dP/dT a measure of relaxation of the cardiac muscle
  • tau the relaxation constant
  • angiogenesis regulates the growth of new vessels from existing vasculature that effectively bypass an arterial lesion requires strict spatial and temporal control.
  • Angiogenic factors such as those described in the preceding examples, work by providing a gradient of angiogenic factor that stimulates the chemotaxis and proliferation of endothelial cells, and their supporting cells towards the source of the factor.
  • biocompatible absorbable threads containing angiogenic factors can be employed to provide a small, highly localized and orderly gradient of angiogenic factors in the appropriate and crucial areas. This enables the creation of a
  • molecular road map that directs the growth of new vessels from around the site of the arterial occlusion to join again at a point below or downstream of the blockage.
  • absorbable surgical threads can be coated with the appropriate angiogenic factors (e.g., PDGF-BB, FGF-2, VEGF-A and PDGF-B). Such threads can then be surgically placed at the site of arterial occlusion such that they provide a clear spatial direction and gradient of angiogenic factor(s) to direct the generation of new vessels around the block.
  • the biocompatible threads can be coated with one or several angiogenic factors to allow delivery of growth factor or growth factor combinations that provide the optimal angiogenic stimulus. Moreover, such threads can be engineered to release certain growth factors at specific rates and at specific times that may help mimic the natural angiogenic process more closely.
  • Figure 14 shows a variety of expression plasmids encoding angiogenic factors that can be administered directly to localized areas of tissue to promote angiogenesis.
  • the plasmids encode biologically active PDGF-BB protein
  • supernatant from 293T cells transiently transfected with the PDGF-BB expression plasmid was added to NIH 3T3 cells growing under serum free conditions. Seventy two hours later cells were trypsinized, spun down and counted using a hemocytometer. The results showed that the PDGF-BB supernatant specifically and potently induced the proliferation of NIH3T3 cells.
  • test animals e.g., rats
  • control vs. angiogenic plasmids e.g., PDGF-BB expression plasmids
  • test animals can be anesthetized and intubated.
  • the chest wall is opened and a myocardial infarct is created by tying off the anterior descending artery.
  • 180 ⁇ g of control or test expression plasmid is injected into the heart wall in a volume of 20 ⁇ l.
  • Cardiac function is assessed 3 weeks post injection. Animals are sacrificed, the heart is removed and efficiency of plasmid uptake is assessed by staining with X-gal. The size of the infarct and the extent of angiogenesis is quantified.

Abstract

L'invention concerne des méthodes permettant de stimuler l'angiogenèse par l'administration de facteurs angiogéniques. Ces facteurs peuvent être administrés sous forme de gradient dans une zone de tissu localisée afin de diriger la croissance vasculaire.
EP02714757A 2001-01-26 2002-01-18 Methodes et compositions permettant de stimuler l'angiogenese Ceased EP1355659A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US26445701P 2001-01-26 2001-01-26
US264457P 2001-01-26
PCT/US2002/001666 WO2002058718A2 (fr) 2001-01-26 2002-01-18 Methodes et compositions permettant de stimuler l'angiogenese

Publications (1)

Publication Number Publication Date
EP1355659A2 true EP1355659A2 (fr) 2003-10-29

Family

ID=23006154

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02714757A Ceased EP1355659A2 (fr) 2001-01-26 2002-01-18 Methodes et compositions permettant de stimuler l'angiogenese

Country Status (5)

Country Link
EP (1) EP1355659A2 (fr)
JP (1) JP4434584B2 (fr)
AU (1) AU2002247007B2 (fr)
CA (1) CA2436076A1 (fr)
WO (1) WO2002058718A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020072489A1 (en) * 1998-10-13 2002-06-13 Whitehouse Martha J. Angiogenically effective unit dose of FGF-2 and method of use
JP2005168654A (ja) * 2003-12-09 2005-06-30 Kissei Pharmaceut Co Ltd 増殖因子結合低分子ヘパリン修飾体
FR2891149B1 (fr) * 2005-09-26 2007-11-30 Biodex Sarl Composition pharmaceutique a action cicatrisante comprenant un derive de dextrane soluble et un facteur de croissance derive des plaquettes.
JP5025646B2 (ja) * 2005-11-08 2012-09-12 公益財団法人先端医療振興財団 虚血性心疾患の治療方法
FR2914191A1 (fr) * 2007-03-29 2008-10-03 Proteins & Peptides Man Composition angiogenique.
RU2498820C2 (ru) 2008-09-26 2013-11-20 Адосиа Комплекс, образованный полисахаридом и нвр
WO2010078624A1 (fr) * 2009-01-07 2010-07-15 Vegenics Limited Matériaux et méthodes pour le traitement de l’hypertension
GB201504665D0 (en) * 2015-03-19 2015-05-06 Cell Therapy Ltd Composition

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4889919A (en) * 1986-08-13 1989-12-26 Zymogenetics, Inc. Biologically active PDGF derived A-chain homodimers
US5705485A (en) * 1987-09-18 1998-01-06 Ethicon, Inc. Gel formulations containing growth factors
US4950483A (en) * 1988-06-30 1990-08-21 Collagen Corporation Collagen wound healing matrices and process for their production
NZ237832A (en) * 1990-04-17 1994-05-26 Curative Tech Inc Coating a prosthetic surface with mammalian cells
AU6778794A (en) * 1993-04-29 1994-11-21 Washington University Use of platelet-derived growth factor to improve collateral circulation
WO2000041732A1 (fr) * 1999-01-19 2000-07-20 The Children's Hospital Of Philadelphia Compositions hydrogel pour l'administration controlee de vecteurs viraux, et leurs procedes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02058718A2 *

Also Published As

Publication number Publication date
JP2005506279A (ja) 2005-03-03
JP4434584B2 (ja) 2010-03-17
CA2436076A1 (fr) 2002-08-01
WO2002058718A2 (fr) 2002-08-01
WO2002058718A3 (fr) 2003-04-17
AU2002247007B2 (en) 2006-12-07

Similar Documents

Publication Publication Date Title
AU2001268149B2 (en) Methods and compounds for controlled release of recombinant parvovirus vectors
Winn et al. Gene therapy approaches for modulating bone regeneration
ES2206342T3 (es) Dispositivo medico que comprende una superficie sintetica que tiene acido nucleico para la induccion in vivo de su endotelializacion.
US20030139333A1 (en) Methods and compositions for promoting angiogenesis
US20080031923A1 (en) Biologic Replacement for Fibrin Clot
JP2006524691A (ja) 心臓導電系の標的化された領域の遺伝子修飾
CA2433936A1 (fr) Procede d'injection myocardiale localisee dans le traitement du myocarde ischemique
CN1198675A (zh) 含有hgf基因的药物
AU2002247007B2 (en) Use of compositions containing PDGF-BB for promoting angiogenesis
US20080102059A1 (en) Treatment for arthritis
AU772857B2 (en) Method of inducing angiogenesis
AU2002247007A1 (en) Use of compositions containing PDGF-BB for promoting angiogenesis
CN103638002A (zh) 包囊的肝细胞组合物
WO1989007944A1 (fr) Procede et dispositif de neovascularisation a localisation controlee
EA019099B1 (ru) Способ направленной доставки трансгена в миокард пациента с ишемией миокарда
CN1630538A (zh) 用vegf－b刺激血管生成
ES2434257T3 (es) Coágulos de células hematopoyéticas no poliméricos para suministro de agentes activos
JP2014218509A (ja) 遺伝子治療用アデノ随伴ウイルスベクターの長期順行性の心外膜冠動脈注入
WO2001087312A1 (fr) Utilisation de polymeres polyethyleneglycols (peg) dans la stimulation de l'angiogenese
Vranckx et al. 15 Gene Transfer of Growth Factors for Wound Repair
Wang et al. In vitro circadian ANP secretion by gene transferring cells encapsulated in polycaprolactone tubes: gene chronotherapy
WO2007059303A2 (fr) Compositions pour l'expression coordonnee de vegf et de pdgf, et methodes d'utilisation
Davidson 13 Gene Therapy of Wounds
WO2005086684A2 (fr) Procede de modulation, de regulation et/ou de stabilisation de l'angiogenese
EP1556494A2 (fr) Boite d'expression, vecteur de plasmide a deux cistrons, produit pharmaceutique et leur utilisation en therapie genique angiogenique

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030801

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20060421

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20081110