WO1998007415A2 - Procedes pour la prevention de la proliferation cellulaire et de la restenose - Google Patents

Procedes pour la prevention de la proliferation cellulaire et de la restenose Download PDF

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WO1998007415A2
WO1998007415A2 PCT/EP1997/004503 EP9704503W WO9807415A2 WO 1998007415 A2 WO1998007415 A2 WO 1998007415A2 EP 9704503 W EP9704503 W EP 9704503W WO 9807415 A2 WO9807415 A2 WO 9807415A2
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staurosporine
antisense molecule
inhibitor
restenosis
revascularization
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PCT/EP1997/004503
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WO1998007415A3 (fr
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Margaret Forney Prescott
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Novartis Ag
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Publication of WO1998007415A3 publication Critical patent/WO1998007415A3/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0014Skin, i.e. galenical aspects of topical compositions
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y207/00Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013Protein kinase C (2.7.11.13)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers

Definitions

  • the invention relates to a method for the prevention and treatment of diseases involving enhanced cellular proliferation and/or decreased cellular apoptosis, particularly smooth muscle cell proliferation leading to restenosis of blood vessels following revascularization intervention.
  • the method comprises administration of protein kinase C inhibitors, particularly local administration of staurosporine derivatives.
  • the present invention relates to the use of protein kinase C inhibitors for the preparation of a medicament for the treatment or prevention of restenosis following revascularization.
  • Atherosclerosis is a common form of biologically mediated vascular injury leading to stenosis.
  • mechanical injuries can also lead to vascular stenosis. Such mechanical injuries can often result following balloon angioplasty, vascular surgery, transplantation surgery and other similar invasive processes that disrupt vascular integrity. Consequently, arterial restenosis often occurs in human patients following revascularization intervention.
  • Percutaneous transluminal angioplasty is one well established revascularization intervention to reduce symptoms resulting from stenotic atherosclerotic plaques. Both angioplasty and coronary bypass surgery provide revascularization of stenotic arteries, particularly coronary arteries. Angioplasty offers a lower rate of procedure-related death or serious complications, as well as lower cost and reduced hospitalization time due to the minimally invasive nature of angioplasty.
  • Angioplasty of coronary arteries involves insertion of an angioplasty catheter in the femoral artery, threading the catheter through the vascular system to the site of the stenotic occlusion and dilation of the angioplasty balloon to crack open the stenotic plaque.
  • Angioplasty of other arteries involves similar techniques with variations depending on the type of artery.
  • angioplasty has a primary success rate of 90-95%
  • late restenosis or vessel reclosure occurs in approximately 30-50% of patients within three to six months of the procedure. Late restenosis causes little increase in mortality, however it causes a substantial increase in morbidity in the form of angina, mycardial infarction, or need for repeat revascularization via angioplasty or bypass. More than half of the patients with angiographically defined restenosis require an additional revascularization procedure within six months and 80% with restenosis require repeat revascularization within six years.
  • the methods involve administration of therapeutically effective amounts of protein kinase C (PKC) inhibitors, particularly staurosporine derivatives and antisense molecules. Additionally, local administration of PKC inhibitors is provided.
  • PKC protein kinase C
  • the methods inhibit smooth muscle cell proliferation, stimulate apoptosis and inhibit stenosis following revascularization.
  • the present invention relates to the use of protein kinase C inhibitors for the preparation of a medicament for the treatment or prevention of restenosis following revascularization.
  • compositions also find use as antineoplastic agents for the treatment of cancer, particularly in methods for local delivery.
  • the invention provides a method of preventing or treating restenosis in hollow tubes, increased cell proliferation or decreased apoptosis in a mammal in need thereof by administering a therapeutically effective amount of a composition comprising protein kinase C (PKC) inhibitors.
  • PKC protein kinase C
  • Prevention of restenosis includes inhibition of restenosis so that it occurs at a lesser rate or results in less restenosis than would occur without treatment.
  • the administration may be by one or more of the following routes: orally, parenterally, intravascularly, intranasally, intrabronchially, interperitoneally, esophageal, transdermally, or locallv.
  • Restenosis includes the reocclusion of hollow tubes after mechanical dilation or ablation. particularly revascularization.
  • Reocclusion can occur as a result of cellular proliferation or local deposition of cellular products.
  • Hollow tubes include circulatory system vessels such as blood vessels (both arteries and veins), tissue lumen, lymphatic pathways, digestive tract including alimentary canal, respiratory tract, excretory system tubes, reproductive system tubes and ducts, body cavity tubes, tissue lumen, etc.
  • hollow tubes are circulatory vessels.
  • Dilation or ablation include the restoration of access to hollow tubes following an occlusion.
  • Revascularization usually refers to the restoration of flow to circulatory vessels, particularly arteries following angioplasty or other physical intervention. Revascularization for purposes of this invention usually does not include regrowth and repair of circulatory tissue following injury such as bruising.
  • Local administration of the PKC inhibitors are a preferred administration route.
  • Local application of the drug affords concentrated delivery of the drug, achieving tissue levels in target tissues not otherwise obtainable through other administration routes.
  • drug release occurs via diffusion, a time-dependent process is involved and delivery to the target site will be sustained for days to weeks or beyond, depending upon the delivery system utilized.
  • local delivery reduces systemic drug exposure, thereby limiting systemic side effects.
  • Local delivery allows for delivery agents which might otherwise be difficult or impossible to deliver via oral or intravenous routes due to problems of solubility or formulation.
  • Local administration also provides the possibility of utilizing agents which might not otherwise be administratable because of concerns or toxicity encountered with conventional routes of administration.
  • Means for local drug delivery to hollow tubes can be by physical delivery of an active ingredient either internally or externally to the hollow tube.
  • Local internal drug delivery includes balloon catheter delivery systems, endovascular polymer coated stents, facilitated diffusion, polymeric endoluminal paving, controlled release matrices, cell targeting such as affinity based delivery, receptor mediated cell toxicity, and the like. See, Eccleston et al. (1995) Interventional Cardiology Monitor 1:33-40-41 and Slepian, N.J. (1996) Intervente. Cardiol. 1:103-116, which disclosures are herein incorporated by reference.
  • Local external drug delivery includes external patches around the hollow tube, hollow tube cuff, external paving, external stent sleeve, etc. Moreover, delivery could be by genetic therapy of appropriate tissue cells.
  • biocompatible polymers are applied via catheter to the endoluminal surface following angioplasty.
  • Polymers are applied to the surface of an organ or organ-component and custom-contoured in situ to yield a layer or film of polymer in intimate contact with the underlying tissue surface.
  • Particularly preferred polymers include polymeric hydrogels or colloidal systems which can be utilized as the applied polymeric material.
  • Hydrogels are polymeric materials that are highly swollen but not dissolved by water. See, for example Slepian (1993) Transactions of the Society for Biomaterials XVI:233; Slepian et al. (1993) Circulation 88 :l-660 (abstract), Slepian et al.
  • endoluminal hydrogels function as local, direct contact, arterial wall drug delivery reservoirs. Additionally, the hydrogels provide drug delivery for a few days to several weeks following application. Furthermore, hydrogels readily allow incorporation and subsequent release of entrapped drugs, materials, or cells.
  • Gel materials for use in the invention include poly(oxalkylene) polymers, i.e., poly(Ecaprolactone), polyethylene glycol (PEG) - lactice polymers, etc.
  • drug diffusion can be regulated by the porosity of the hydrogel or the method by which the active ingredient is dispersed within the hydrogel.
  • hydrogels of greater porosity can be utilized.
  • the length of drug delivery and treatment can be regulated by the porosity of the hydrogel.
  • a slow release gel which delivers the compounds for at least about 2 days to about 1 year, preferably about 2 to about 30 days, more preferably about 8 to about 16 days, most preferably from about 10 to about 14 days is utilized.
  • a slow release gel which delivers the compounds for at least about 2 days to about 1 year, preferably about 10 to about 30 days, more preferably about 15 to about 25 days
  • Means for delivery or application of the compounds in a slow release gel are available in the art.
  • catheter systems have been designed for localized in situ formation of both thin and thick hydrogel paving layers.
  • the catheter systems may vary depending upon the nature of the flowable baseline material utilized for hydrogel formation as well as the chemical or physical means utilized to locally convert the pre-gel constituents to a gel locally. See, for example, Slepian et al. (1993), supra, and Hill et al. (1993) Circulation 33:1-319, herein incorporated by reference. Delivery or application of the compounds can also occur using stents or sleeves.
  • An intraluminal stent composed of or coated with polymer or other materials into which the active drug has been impregnated can be used.
  • Such stents can be biodegradable or can be made of metal or another stable substance when intended for permanent use.
  • lumenal and/or ablumenal coating or external sleeve made of polymer or other materials that contain the active drug can also be used for localized delivery.
  • PKC inhibitors are available in the art for use in the invention. These include bryostatin (U.S. Patent 4,560,774), safinogel (WO 9617603), fasudil (EP 187371), 7- hydoxystaurosporin (EP 137632B), various diones described in EP 657458, EP 657411 and WO9535294, phenylmethyl hexanamides as described in WO9517888, various indane containing benzamides as described in WO9530640, various pyrrolo [3,4-c]carbazoles as described in EP 695755, LY 333531 (IMSworld R & D Focus 960722, July 22, 1996 and Pharmaprojects Accession No.
  • bryostatin U.S. Patent 4,560,774
  • safinogel WO 9617603
  • fasudil EP 187371
  • 7- hydoxystaurosporin EP
  • antisense molecules can be used as PKC inhibitors. Although such antisense molecules inhibit mRNA translation into the PKC protein, such antisense molecules are considered PKC inhibitors for purposes of this invention.
  • Such antisense molecules against PKC inhibitors include those described in published PCT patent applications WO 93/19203, WO 95/03833 and WO 95/02069, herein incorporated by reference.
  • Such inhibitors can be used in formulations for local delivery to prevent cellular proliferation. Such inhibitors find particular use in local delivery for preventing rumor growth and restenosis.
  • PKC inhibitors of particular interest include staurosporine derivatives, particularly N- substituted staurosporine compounds, more particularly, N-benzoyl staurosporine. See, U.S. Patent No. 5,093,330, herein incorporated by reference.
  • Other preferred inhibitors are the antisense molecules against PKC, preferably those as mentioned herein, particularly ISIS 3521 and ISIS 9606.
  • Staurosporine derivatives inhibit a variety of protein kinase C isoforms which phosphorylate serine and threonine residues in a variety of target proteins critically involved in cell proliferation and differentiation.
  • PKC enzymes have also been shown to regulate apoptosis (programmed cell death) and extra cellular matrix deposition.
  • N-benzoyl staurosporine has additional activity against the platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) tyrosin kinases. Both PDGF and FGF are released at the time of angioplasty and both have potent affects on proliferation and matrix formation.
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • an antisense molecule according to the present invention which has a “chimeric” structure.
  • the term “chimeric” structure with respect to antisense molecules is well known by those of skill and will therefore be explained in the following only for completeness.
  • a “chimeric” structure also termed a “chimera” is to be understood as meaning an antisense molecule (an oligonucleotide) which contains 2 or more chemically different regions which are in each case synthesized from one type of nucleic acid building block.
  • Such chimeric antisense molecules typically comprise at least one region of modified nucleic acid building blocks which confer one or more advantageous property/properties (for example increased resistance to nucleases, increased binding affinity or diminished occurrence of sequence-independent side-effects) on the antisense molecule, the so-called "wing", and a region which enables RNAse H-mediated cleavage of the target nucleic acid to take place, i.e. the so-called "RNAse H window”.
  • the affinity of an antisense molecule customarily determined by measuring the T m value of the antisense molecule/target nucleic acid hybrid.
  • the T m value is the temperature at which the antisense molecule and the target nucleic acid dissociate from a previously formed hybrid.
  • the dissociation is determined spectrophotometrically.
  • Methods for determining the T m value belong to the state of the art (cf., for example, Fritsch and Maniatis, "Molecular Cloning -A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989).
  • increased resistance to nucleases denotes decreased or slowed-down degradation of the oligonucleotide derivatives according to the invention by exonucleases or endonucleases which are present in a cell.
  • RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of the enzyme therefore leads to cleavage of the target RNA and consequently increases the efficacy of the antisense mechanism. Cleavage of the target RNA can customarily be demonstrated by gel electrophoresis. Since, in a chimera, different advantageous properties are present in one and the same molecule, chimeric antisense molecules according to the invention possess a pronounced antisense effect with regard to inhibiting the expression of protein kinase C.
  • the present invention relates to the use of a PKC inhibitor for the preparation of a medicament for the treatment or prevention of restenosis following revascularization.
  • the PKC inhibitor is a staurosporine derivative, in particular N-benzoyl staurosporine.
  • the PKC inhibitor is an antisense molecule.
  • such an antisense molecule has a nucleotide base sequence as set forth in SEQ:ID:NO. 1, like e.g. ISIS 3521. More preferably, the antisense molecule has a nucleotide base sequence as set forth in SEQ ID NO:l and has a chimeric structure, like, in particular, ISIS 9606.
  • PKC inhibitors and staurosporine derivatives can be utilized to inhibit late restenotic lesion development in humans and other animals.
  • early lesions are those which occur between 7 and 14 days post-injury.
  • SMC smooth muscle cell
  • Late lesions in preclinical animal models such as rat, rabbit and pig, are lesions which occur more than 3 weeks post-injury (usually measured at 3, 4, 6, or 8 weeks).
  • late lesions occur over a prolonged period of time compared to non-humans (usually 3-9 months post-injury).
  • Compounds which only inhibit SMC migration usually do not inhibit late lesion size.
  • inhibition of late lesions requires inhibition of intimal SMC proliferation (and possibly the stimulation of intimal SMC apoptosis, and/or inhibition of extracellular matrix and constrictive remodeling, as well).
  • Post-injury for the present invention usually means revascularization intervention.
  • the compounds can be utilized to inhibit intimal smooth muscle proliferation, matrix formation and constrictive remodeling and stimulation of apoptosis.
  • the present invention is also drawn to local application of the compounds for their antitumor activity. Such local delivery of the compounds provides the benefits of local administration discussed above.
  • N-benzoyl staurosporine Solubility characteristics of N-benzoyl staurosporine appear to make it an ideal candidate for incorporation into a slow-release formulation for local delivery, particularly a slow release gel.
  • the local delivery of the present invention allows for high concentration of compound at the disease site with extremely low concentration of circulating compound, obviating concerns that systemic administration at high dosages may be associated with effects on proliferating tissue, such as in the intestine, testes and bone marrow.
  • the amount of compound used for local delivery applications will vary according to the indication being treated, etc.
  • a therapeutically effective amount will be administered.
  • therapeutically effective amount is intended an amount sufficient to inhibit cellular proliferation and resulting in the prevention and treatment of the disease state.
  • local delivery gel formulation requires much less compound than systemic administration.
  • about 200 to about 10,000 ⁇ g is utilized, specifically about 400 to about 5000 ⁇ g, more specifically about 500 to about lOOO ⁇ g per deposition site.
  • a deposition site is typically from about 10 to about 1000 mm squared in size.
  • the compounds can be locally applied at rates of about 200 to about 10,000 g, specifically about 400 to about 5000 ⁇ g, more specifically about 500 to about lOOO ⁇ g per day for from about 2 days to about 1 year.
  • the active ingredient is an antisense molecule
  • the amount used is typically lower than when other PKC inhibitors such as staurosporine derivatives.
  • Such amounts can be in the nM range, preferably from about 50 nM to 1000 nM, more preferably concentrations of at least 200 nM.
  • the compounds of the invention may be used individually or alternatively more than one compound can be formulated into the gel or other local delivery device for delivery.
  • This drug's high partition coefficient (log P>5.48) and near insolubility in aqueous media (.068 mg/1) may lead to improved tissue retention and reduced loss to blood flow following local deposition in the blood vessel.
  • N-benzoyl staurosporine has demonstrated extended stability in physiologic buffers (pH 7-9). The compound has the ability to produce a micronized form ( ⁇ 5 ⁇ m). Therefore, for purposes of the invention, the compound can be used as a particulate suspension or encapsulation to control release (slow dissolution or diffusion) from a hydrogel polymer.
  • organic solubility methylene chloride, 1,2 propylene glycol
  • Solubility in polyethylene glycol (PEG) improves the changes for successful incorporation within a gel polymer, a PEG based material.
  • Analytical methods for detection of active substance and metabolites have been developed.
  • Staurosporine derivatives are available in sufficient quantity for gel formulation release kinetics study.
  • Rat aortic A10 SMC were purchased from American Type Culture Collection (Rockville, MD). Human coronary arteries were purchased from Clonetics Corp. (San Diego, California). Primary porcine SMC were isolated from the coronary artery of porcine hearts from small pigs (55-77 kg) which were obtained from a local abattoir. The left anterior descending artery and left circumflex were dissected from each heart and excess muscle tissue cut away. The vessels were opened longitudinally and the endothelial layer was scraped away with a cotton-tipped applicator.
  • the intimal layer of SMC was peeled away and incubated, with magnetic stirring, in an enzyme solution containing collagenase (460 U/ml), papain (25 U/ml) and DL-dithiothreitol (0.4 mg/ml) in Dulbecco's phosphate-buffered saline for 55 minutes at 37°C.
  • the supernatant was discarded and the pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 20% FBS, 5% penicillin- streptomycin and the resulting floculent mass was shaken to release the cells. Any remaining debris was removed by filtration through a fine mesh.
  • DMEM Dulbecco's modified Eagle's medium
  • Cells were plated in 25 cm 2 Falcon Primaria flasks at a density of 2 X IO 6 cells. Cells were allowed to grow for 5 days before medium was removed and replaced with DMEM containing 10% FBS, 1% penicillin- streptomycin. Cultures were identified as SMC by their characteristic hills-and valleys growth pattern and immunofluorescence with anti- ⁇ smooth muscle actin monoclonal antibody.
  • Rat (passages 18-21) and porcine SMC (passage 2-6) were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin- streptomycin at 37°C in a 95% air/5% CO 2 humidified atmosphere.
  • Human SMC (passage 3- 7) were grown in Smooth Muscle Cell Growth Media (SMGM)(Clonetics Corp., San Diego California) supplemented with 0.5 ⁇ g/ml hEGF, 5 ⁇ g/ml insulin, 1 ⁇ g/ml hFGF, 5% FBS and 50 ⁇ g/ml gentamycin at 37°C in a 95% air/5% CO 2 humidified atmosphere. Cells were subcultured by aspiration of the growth medium followed by a 30-sec rinse with a solution of 0.5 mM EDTA/0.05% trypsin.
  • SMGM Smooth Muscle Cell Growth Media
  • VSMC VSMC, grown in 96-well microtiter plates (5,000 cells/well) to 80% confluence were labeled overnight with 5-bromo-2'-deoxyuridine (10 ⁇ M) at 37°C. After labeling, the cells were centrifuged (10 min, 250 g) and resuspended into culture medium. Cells were incubated with PKC inhibitors for varying time periods. Cells were then lysed, fragmented DNA was separated from intact chromatin by centrifugation, and internucleosomal DNA fragmentation was quantitatively determined using a DNA fragmentation photometric ELISA kit (Boehringer Mannheim). The absorbance at 30 nm is recorded using a microplate reader (Molecular Devices Corporation).
  • DNA fragmentation was assessed quantitatively by DNA agarose gel electrophoresis.
  • Cells were removed from 75cm 2 flasks by gentle scraping after the indicated treatments. The cell suspension was transferred by a conical centrifuge tube, and the cells were pelleted by centrifugation at 750 x g for 5 min. The supernatant was removed, and the cells were resuspended in 5ml PBS.
  • N-benzoyl staurosporine (CGP 41251) potently inhibited serum-induced proliferation in cultured rat, porcine and human vascular smooth muscle cells with corresponding IC 50 values of 303, 78, 100 nM, respectively, whereas the inactive analog CGP 42700, was without effect.
  • Staurosporine demonstrated antiproliferative IC50 values of 34, 7 and 0.8 nM in serum-stimulated rat, porcine, and human smooth muscle cells, respectively.
  • CGP 41251 was tested in the rat carotid balloon-injury model of restenosis.
  • mice Male Sprague Dawley rats weighing 350 to 500 gm were housed individually and allowed to acclimate prior to surgery. All animals received standard rat chow and water ad libitum. Group size was normally 12 animals per group.
  • Dosin ⁇ CGP 41251 or analogues were administered per os (p.o.) once per day, either as a suspension of active ingredient in comstarch or, in the case of CGP 41251, gelucire formulation suspended in distilled water. Doses of the compound varied per experiment and ranged between 25 mg/kg/day and 100 mg/kg/day of active ingredient. In all but the "window therapy” studies, compound was administered once daily 5 days prior to surgery and daily post-balloon injury throughout the entire study. Study length varied from 4 to 56 days post- balloon-injury. For "window therapy” studies, compound was given once daily 5 days pre- ballooning and daily post-ballooning for time courses ranging from 5 to 14 days post- ballooning. An all studies, control animals were dosed with appropriate vehicle using the same dosing schedule as compound-treated animals.
  • CGP 41251 For local delivery studies using analogues of CGP 41251, such as CGP 75139 A, compound administration was perivascular. A segment of ballooned carotid was encircled with a 1 cm length of silastic tubing (0.25 inch inside diameter, .047 inch outside diameter) to which was attached a catheter which feeds into an osmotic pump containing either compound or vehicle. This delivery system provided continuous, local delivery to the adventitia of the wrapped portion of vessel. Local compound administration ranges between 5 ug and 10 mg, locally, per day, depending on the solubility characteristics of the individual compounds.
  • the balloon was inserted into the common carotid artery via the left external branch, inflated with saline, and pulled back three times through the lumen with a rotating motion to ensure maximal endothelial denudation.
  • the catheter was then removed, the external carotid artery was ligated and the wound was closed.
  • Each animal was given an injection of the antibiotic Bacillin (200,000 units/kg) and the analgesic Buprenophine (0.06 mg/kg) immediately following surgery.
  • Termination. Histology and Morphometr Animals were killed at the following time periods raging between 4 and 56 days post-balloon injury. One half hour before termination, blood was collected, centrifuged, and stored at -20 degrees Centigrade for analysis of circulating levels of compound. 5% Evans Blue was then injected intravenously to allow discrimination of re-endothelialized areas at the time of histologic processing. Animals were killed by administration of an overdose of ketamine and rompun, The carotid arteries were pressure-perfusion fixed using STF (Streck Labs) at 100 mmHg. For local delivery studies, the osmotic pumps were recovered and the volume of remaining content was recorded to ensure that pump failure had not occurred.
  • Carotid arteries were excised and immersion fixed in STF for 1-5 days, then transferred to Ringer's solution. Two samples from control blue region of each left carotid artery were imbedded in paraffin. A minimum of six carotid sections, 20 ⁇ M apart were cut per animal and stained with Verhoff Elastic stain to produce a modified Verhoff stain. Intimal and medial area measurements were performed with a digitizing tablet interfaced to a VIDAS imaging system. Any sections in which disruption of the internal elastic lamina was observed were excluded from measurement. Additional cross sections were cut from 3 week and 6 week animals and stained with Verhoff -Van Geisen to allow visual determination of the extent of connective tissue deposition.
  • Table 1 shows the results of various PKC inhibitors and the effect on early and late restenosis after balloon angioplasty. 50 mg/kg p.o. reduced both lesion size and intimal SMC proliferation at 9 days (d) post-injury (70% and 43%, respectively). Late lesions at 3 weeks were inhibited dose-dependently (25, 50, and 100 mg/kg/d inhibited 26%, 43% and 66% respectively). This inhibition of late lesions was demonstrated to be unique; numerous compounds including matrix metal loprotease (MMP) inhibitors, angiotensin converting enzyme (ACE) inhibitors, angiotensin II antagonists, calcium channel blockers and heparin inhibit early but not late lesion formation in this model.
  • MMP matrix metal loprotease
  • ACE angiotensin converting enzyme
  • II antagonists calcium channel blockers
  • heparin inhibit early but not late lesion formation in this model.
  • CGP 41241 Efficacy of CGP 41241 on late lesions appears due to its combined ability to reduce intimal SMC division, inhibit matrix deposition, and stimulate SMC apoptosis. Constrictive remodeling may also be reduced as external elastic lamina perimeters were larger in treated animals.
  • Dogs and rats were given CGC 41251 p.o. for extended periods of time. Tissue samples from the animals were analyzed for toxicological effects. The no adverse effect level in a three month toxicity study was 10 mg/kg in the rat and 1 mg/kg in the dog and in the 6-month (interim) study in the rat was 3 mg kg.
  • Treatment with CGP 41251 at doses of and above 3 mg/kg in dogs (up to 3 months) and rats (up to 6 months) was associated with effects on proliferating tissues. The most sensitive tissues were the intestine, testes and bone marrow. Effects on bone marrow were accompanied by hematological changes.
  • the methods of the present invention offer advantages not currently available in the art.
  • Local application of drugs via a gel polymer offers therapeutic advantage not provided by systemic administration or physical anti restenotic devices, e.g. metal stenting.
  • the gel-drug polymer functions as a synthetically interposed tissue barrier.
  • restenosis, matrix formation, smooth muscle cell proliferation and dysfunctional apoptosis is prevented and/or treated as well as the prevention of the adhesion of platelets to the surface.

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Abstract

L'invention concerne des procédés pour la prévention et le traitement de maladies impliquant une prolifération cellulaire et une apoptose dysfonctionnelle. Ces procédés consistent à administrer des inhibiteurs de la protéine kinase C et leurs dérivés. En outre, l'invention traite de l'administration locale d'inhibiteurs de la protéine kinase C pour traiter la resténose et le cancer.
PCT/EP1997/004503 1996-08-20 1997-08-18 Procedes pour la prevention de la proliferation cellulaire et de la restenose WO1998007415A2 (fr)

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AU42055/97A AU4205597A (en) 1996-08-20 1997-08-18 Methods for prevention of cellular proliferation and restenosis

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US2421996P 1996-08-20 1996-08-20
US60/024,219 1996-08-20
US2507296P 1996-08-30 1996-08-30
US60/025,072 1996-08-30

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US6190869B1 (en) 1999-10-26 2001-02-20 Isis Pharmaceuticals, Inc. Antisense inhibition of protein kinase C-theta expression
US6641611B2 (en) 2001-11-26 2003-11-04 Swaminathan Jayaraman Therapeutic coating for an intravascular implant
JP2005507412A (ja) * 2001-10-30 2005-03-17 ノバルティス アクチエンゲゼルシャフト Flt3レセプター・チロシン・キナーゼ活性インヒビターとしてのスタウロスポリン誘導体
DE102004019413A1 (de) * 2004-04-19 2005-11-24 Phenos Gmbh Hemmung der Proteinkinase C epsilon zur Behandlung von Krankheiten
US7867988B2 (en) 2006-09-13 2011-01-11 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US8088789B2 (en) 2006-09-13 2012-01-03 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US10695327B2 (en) 2006-09-13 2020-06-30 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190869B1 (en) 1999-10-26 2001-02-20 Isis Pharmaceuticals, Inc. Antisense inhibition of protein kinase C-theta expression
JP2005507412A (ja) * 2001-10-30 2005-03-17 ノバルティス アクチエンゲゼルシャフト Flt3レセプター・チロシン・キナーゼ活性インヒビターとしてのスタウロスポリン誘導体
JP4959918B2 (ja) * 2001-10-30 2012-06-27 ノバルティス アーゲー Flt3レセプター・チロシン・キナーゼ活性インヒビターとしてのスタウロスポリン誘導体
US7973031B2 (en) 2001-10-30 2011-07-05 Novartis Ag Staurosporine derivatives as inhibitors of FLT3 receptor tyrosine kinase activity
US6641611B2 (en) 2001-11-26 2003-11-04 Swaminathan Jayaraman Therapeutic coating for an intravascular implant
DE102004019413A1 (de) * 2004-04-19 2005-11-24 Phenos Gmbh Hemmung der Proteinkinase C epsilon zur Behandlung von Krankheiten
US8088789B2 (en) 2006-09-13 2012-01-03 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US7867988B2 (en) 2006-09-13 2011-01-11 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US8367081B2 (en) 2006-09-13 2013-02-05 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US8404641B2 (en) 2006-09-13 2013-03-26 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US9149470B2 (en) 2006-09-13 2015-10-06 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US10123996B2 (en) 2006-09-13 2018-11-13 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use
US10695327B2 (en) 2006-09-13 2020-06-30 Elixir Medical Corporation Macrocyclic lactone compounds and methods for their use

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AU4205597A (en) 1998-03-06
ID18301A (id) 1998-02-26

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