WO2011071692A1 - Modulation de l'électrophysiologie cellulaire - Google Patents

Modulation de l'électrophysiologie cellulaire Download PDF

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Publication number
WO2011071692A1
WO2011071692A1 PCT/US2010/057793 US2010057793W WO2011071692A1 WO 2011071692 A1 WO2011071692 A1 WO 2011071692A1 US 2010057793 W US2010057793 W US 2010057793W WO 2011071692 A1 WO2011071692 A1 WO 2011071692A1
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blocking agent
channel
cells
ion
channel blocking
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PCT/US2010/057793
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English (en)
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Vinrod Sharma
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Medtronic, Inc.
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Publication of WO2011071692A1 publication Critical patent/WO2011071692A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane of all living cells by allowing the flow of ions down their electrochemical gradient. They are present in the membranes that surround all biological cells and regulate the flow of ions across the membrane. Ion channels are key components in a wide variety of biological processes that involve rapid changes in cells, such as cardiac, skeletal, and smooth muscle contraction, epithelial transport of nutrients and ions, T-cell activation and pancreatic beta-cell insulin release. Genetic engineering approaches that result in the expression of exogenous ion channels have therapeutic potential to modify cellular electrophysiology and automaticity.
  • the heart is an electromechanical organ with considerable electrical heterogeneity and this electrical heterogeneity is conferred by the differential expression of a variety of ion channels in various regions of the heart.
  • the expression of exogenous ion channels to change the underlying electrophysiology of the heart is an attractive therapeutic approach to modify cardiac cellular electrophysiology in variety of cardiac dysfunctions, including, but not limited to, ventricular tachycardia, arrhythmia, atrial fibrillation, bradycardia, and heart failure.
  • the present invention presents a method of increasing the viability of cells and tissues expressing exogenous nucleotide sequences that encode membrane polypeptides regulating the flow of ions across the cell membrane.
  • the present invention includes a method of increasing the viability of cells expressing an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane, the method including contacting the cells expressing the exogenous polynucleotide with an ion channel blocking agent.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is an ion channel.
  • the cells comprise nonexcitable cells.
  • the cells comprise excitable cells.
  • the viability of nonexcitable cells in the heterologous tissue is improved.
  • the present invention includes a method of increasing the viability of nonexcitable cells in a heterologous tissue expressing an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane, the method including contacting the cells expressing the exogenous polynucleotide with an ion channel blocking agent.
  • the membrane polypeptide that regulates flow of ions across a cell membrane is an ion channel.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • the present invention includes a method of increasing the viability of cells in a heterologous tissue expressing an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane, the method including contacting the cells expressing the exogenous polynucleotide with an ion channel blocking agent.
  • the membrane polypeptide that regulates flow of ions across a cell membrane is an ion channel.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • the present invention includes a method of modifying the electrophysiological function of a heterologous tissue including excitable cells and nonexcitable cells, the method including contacting the heterologous tissue with one or more ion channel blocking agents before, after, and/or coincident to transfection or transduction of the heterologous tissue with an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane.
  • the membrane polypeptide that regulates flow of ions across a cell membrane is an ion channel.
  • the viability of nonexcitable cells in the heterologous tissue is improved.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • the present invention includes a method of modifying the electrophysiological function of a heterologous tissue including excitable cells and nonexcitable cells, the method including transfecting or transducing said heterologous tissue with an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane; and contacting said heterologous tissue with an ion channel blocking agent.
  • the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • the membrane polypeptide that regulates flow of ions across a cell membrane is an ion channel.
  • the heterologous tissue is contacted with an amount of ion channel blocking agent effective to increase viability of the nonexcitable cells within the heterologous tissue.
  • a heterologous tissue includes excitable cells and nonexcitable cells. In some embodiments, the method increases the viability of nonexcitable cells. In some embodiments, the method increases the viability of excitable cells. In some embodiments, the heterologous tissue is cardiac tissue.
  • cells or heterologous tissue may be contacted with the ion channel blocking agent before, after, and/or coincident to the expression of the polynucleotide encoding the polypeptide.
  • an ion channel blocking agent may be formulated in composition for controlled or sustained release.
  • an ion channel includes a gap junction channel, calcium channel, a sodium channel, a chloride channel, a hyperpolarized activated cyclic nucleotide (HCN) channel, SERCA2a, a non-specific leak channel, or a potassium channel.
  • the ion channel includes a potassium channel.
  • the potassium channel includes a member of the Kvl-9 family. In some embodiments, the potassium channel includes Kvl .3. In some embodiments, the membrane polypeptide that regulates the flow of ions across a cell membrane is not a hyperpolarized activated cyclic nucleotide (HCN) channel.
  • HTN hyperpolarized activated cyclic nucleotide
  • the ion channel blocking agent includes a calcium channel blocking agent, a sodium channel blocking agent, a gap junction channel blocking agent, a chloride channel blocking agent, a hyperpolarized activated cyclic nucleotide (HCN) channel blocking agent, a SERCA2a blocking agent, a nonspecific leak channel blocking agent, and/or a potassium channel blocking agent.
  • the ion channel blocking agent comprises a potassium channel blocking agent.
  • the ion channel blocking agent comprises charybdotoxin.
  • the ion channel blocking agent is not a hyperpolarized activated cyclic nucleotide (HCN) channel blocking agent.
  • contacting the cells or the heterologous tissue includes intermittent and/or continuous delivery of the ion channel blocking agent.
  • the present invention includes a composition including an ion channel blocking agent, wherein the composition is in a formulation for controlled or sustained release.
  • the present invention includes a kit including an ion blocking agent and a delivery device for delivery of the ion blocking agent to a cell or tissue expressing an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane.
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • Figure 1 shows electrical heterogeneity of various regions of the heart.
  • Figure 2 shows Kvl .3 as a model ion channel.
  • Figure 3 shows viability of cardiac myocytes in the presence of Kvl .3.
  • Fig. 3 A brightfield imaging confirms expression of AdV-Kvl .3-GFP.
  • Fig. 3B fluorescent imaging reveals non-apoptotic nuclei.
  • Figure 4 shows that Kvl .3 induces degradation of cardiac fibroblasts by blebbing, rounding, and detachment.
  • Fig. 4A brightfield imaging confirms expression of AdV- Kvl .3-GFP.
  • Fig. 4B Hoechst dye staining reveals apoptotic nuclei as indicated by the arrows.
  • Figure 5 shows dose-dependent reduction of Cx43.
  • Figure 6 shows deterioration of cardiac fibroblasts in a dose-dependent manner.
  • Figs. 6A-D phase contrast microscopy shows cell morphology and decreasing number of fibroblasts with increasing Kvl .3.
  • Figs. 6E-H fluorescence microscopy shows expression of Kvl .3 following transfection with AdV-Kvl .3-GFP.
  • Fig. 6A and 6E are non-transfected control fibroblasts. Increased Kvl .3 (Figs. 6E-H) correlates with a loss of cell number and disruption in cellular morphology (Figs. 6B-D).
  • Figure 7 shows an increase in apoptosis in cardiac fibroblasts in a dose-dependent manner.
  • Fig. 7A represents the addition of Charybdotoxin alone and
  • Fig. 7B represents the addition of Charybdotoxin and 5% serum results in partial rescue of cells from Kvl .3 induced apoptosis.
  • Figure 8 shows cell viability is rescued in cardiac fibroblasts expressing Kv3.1 with the addition of Charybdotoxin.
  • phase contrast microscopy shows cellular morphology of the fibroblasts.
  • Fig. 8C and 8D fluorescent microscopy confirms expression of Kvl .3 following transfection with 10 7 PFU/ml AdV-Kvl .3 -GFP.
  • Fig. 8A shows Kvl .3 induces degradation of cardiac fibroblasts by blebbing, rounding, and detachment.
  • Fig. 8B shows the addition of Charbdyotoxin restores normal fibroblast morphology.
  • the present invention relates to compositions, apparatus, and methods for improving the viability of cells and tissues expressing exogenous polynucleotides encoding membrane proteins that regulate the flow of ions across the cell membrane.
  • the viability of the cells and tissues is improved by contacting the cells or tissue with one or more ion channel blocking agents.
  • Membrane proteins that regulate the flow of ions across the cell membrane which are encoded by an exogenously administered
  • polynucleotide include, but are not limited to, transmembrane ion channel proteins, active ion transport proteins and ion exchange proteins.
  • a membrane protein that regulates the flow of ions across the cell membrane is an ion channel protein.
  • an ion channel is an assembly of several proteins.
  • Such "multi-subunit" assemblies usually involve a circular arrangement of identical or homologous proteins closely packed around a water-filled pore through the plane of the membrane or lipid bilayer.
  • the pore-forming subunit(s) are called the a-subunit, while the auxiliary subunits are denoted ⁇ , ⁇ , and so on. While some channels permit the passage of ions based solely on charge, the archetypal channel pore conducts a specific species of ion, such as sodium or potassium, and conveys them through the membrane single file, nearly as quickly as the ions move through free fluid.
  • Ion channels of the present invention include voltage-gated ion channels, which activate/inactivate depending on the voltage gradient across the plasma membrane.
  • Voltage-gated ion channels include, but are not limited to, voltage-gated sodium channels, voltage-gated calcium channels, voltage-gated potassium channels (KV),
  • hyperpolarization-activated cyclic nucleotide-gated channels HCN
  • voltage-gated proton channels HCN
  • the ion channel is not a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel.
  • Ion channels of the present invention include ligand-gated ion channels, which activate/inactivate depending on binding of a ligand to the channel.
  • Ligand-gated ion channels also known as ionotropic receptors, open in response to specific ligand molecules binding to the extracellular domain of the receptor protein. Ligand binding causes a conformational change in the structure of the channel protein that ultimately leads to the opening of the channel gate and subsequent ion flux across the plasma membrane.
  • Examples of such channels include the cation-permeable "nicotinic" acetylcholine receptor, ionotropic glutamate-gated receptors and ATP-gated P2X receptors, and the anion-permeable ⁇ -aminobutyric acid-gated GABAA receptor.
  • Ion channels of the present invention include chloride ion channels, sodium ion channels, calcium ion channels, proton channels, and potassium ion channels.
  • Ion channels of the present invention include general ion channels that are relatively nonspecific for ions and thus let many types of ions through the channel.
  • a potassium ion channel of the present invention may belong to any of the following major classes of ion channels.
  • a calcium-activated potassium channel which is open in response to the presence of calcium ions or other signaling molecules, also known as a Ca2 + -activated K + channel.
  • An inwardly rectifying potassium channel passes current (positive charge) more easily in the inward direction, into the cell.
  • a tandem pore domain potassium channel which is constitutively open or possess high basal activation, such as the "resting potassium channels” or “leak channels” that set the negative membrane potential of cardiac cells. When open, they allow potassium ions to cross the membrane at a rate which is nearly as fast as their diffusion through bulk water.
  • Such "leak" K + channels are so termed because of the apparent lack of gating control.
  • a voltage-gated potassium channel which opens or closes in response to changes in the transmembrane voltage.
  • a potassium ion channel of the present invention may be one of a large family of mammalian potassium channels, such as for example, Kvl (shaker), Kv2, Kv3 (Shaw), Kv4 (Shal), Kv5, Kv6, Kv7, Kv8, or Kv9.
  • the potassium ion channel is Kvl .3.
  • any of a wide variety of available ion channel blocking agents may be administered.
  • An ion channel blocking agent may inhibit the movement of ions through the ion channels.
  • blocking or “block” of an ion channel means any block or inhibition of current through that ion channel.
  • An ion channel blocking agent includes, but is not limited to, a calcium channel blocking agent, a sodium channel blocking agent, a gap junction channel blocking agent, a chloride channel blocking agent, a hyperpolarized activated cyclic nucleotide (HCN) channel blocking agent, a SERCA2a blocking agent, a non-specific leak channel blocking agent, and/or a potassium channel blocking agent.
  • a calcium channel blocking agent includes, but is not limited to, a calcium channel blocking agent, a sodium channel blocking agent, a gap junction channel blocking agent, a chloride channel blocking agent, a hyperpolarized activated cyclic nucleotide (HCN) channel blocking agent, a SERCA2a blocking agent, a non-specific leak channel blocking agent, and/or a potassium channel blocking agent.
  • HCN hyperpolarized activated cyclic nucleotide
  • the ion channel blocking agent is not a hyperpolarized activated cyclic nucleotide (HCN) channel blocking agent.
  • a sodium channel blocking agent inhibits the movement of sodium ions through a sodium channel.
  • sodium channel blockers include, but are not limited to, tetrodotoxin (TTX) (used by puffer fish and some types of newts for defense), saxitoxin (produced by a dinoflagellate, also known as red tide), lidocaine and novocaine (local anesthetics which block sodium ion channels), quinidine, procainamide, disopyramide, phenytoin, mexiletine, tocainide, encainide, flecainide, propafenone, and moricizine.
  • TTX tetrodotoxin
  • saxitoxin produced by a dinoflagellate, also known as red tide
  • lidocaine and novocaine local anesthetics which block sodium ion channels
  • quinidine quinidine
  • procainamide disopyramide
  • phenytoin mexiletine
  • tocainide
  • a calcium channel blocking agent inhibits the movement of calcium ions through calcium channels.
  • Examples include, but are not limited to, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, diltiazin, efonidipine, felodipine, gallopamil, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, and verapamil.
  • a chloride channel blocking agent inhibits the movement of chloride ions through chloride channels.
  • sodium channel blockers examples include, but are not limited to, 4,4'-diisothiocyanatosilbene (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), N-phenylanthracillic acid (NPA), g-amino-camptothecin (9-AC), 5-nitro-2- (3-phenylpropylamino)-benzoate (NPPB), flufenamate and diphenylamine-2-carboxylate (DPC), and niflumic acid.
  • DIDS 4,4'-diisothiocyanatosilbene
  • SITS 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid
  • NPA N-phenylanthracillic acid
  • NPPB g-amino-camptothecin (9-AC)
  • NPPB 5-nitro-2- (3-phenylpropy
  • Hyperpolarization-activated cyclic nucleotide-gated channels serve as ion channels across the plasma membrane of heart and brain cells and are sometimes referred to as "pacemaker channels” because they help to generate rhythmic activity within groups of heart and brain cells.
  • HCN channels are encoded by four genes (HCN 1-4) and are widely expressed throughout the heart and the central nervous system.
  • HCN channel blocking agents include, but are not limited to, ivabradine, L-cis diltiazem, tetracaine, calmodulin antagonists, 6-(phenylamino)-5,8-quinolinedone (LY83583), H-8 [N-2-(methylamino)ethyl-5-isoquinolinesulfonamide, and cesium chloride (CsCl).
  • a potassium channel blocking agent that inhibits the movement of potassium ions through the potassium channels.
  • Examples include, but are not limited to, charydotoxin (a 37 amino acid neurotoxin from the venom of the scorpion Leiurus quinquestriatus hebraeus), dendrotoxin (produced by mamba snakes), iberiotoxin (produced by the Buthus famulus), heteropodatoxin (produced by Heteropoda venatoria), amiodarone, bretylium, sotalol, dofetilide, sotalol, ibutilide, azimilide, clofilium, nifekalant, tedisamil, and sematilide.
  • an ion blocking agent increases the viability of cells expressing an exogenous polynucleotide encoding an ion channel.
  • Cells, tissues, and organs may be contacted with one or more ion blocking agents before, after, and/or coincident to the expression of an exogenous polynucleotide encoding a membrane protein that regulates the flow of ions.
  • a polynucleotide may also be referred to herein as "polynucleotide sequence,” “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” and similar terms.
  • the terms “encodes,” “encoding,” “coding sequence,” and similar terms refer to a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under control of appropriate regulatory sequences. Expression includes the transcription of a DNA sequence into a m NA.
  • Expression may be accomplished by a variety of methods, including, but not limited to, expression by transfection or transduction procedures.
  • Transfection and transduction refer to the acquisition by a cell of new genetic material by incorporation of added nucleic acid molecules. Transfection can occur by physical or chemical methods. Transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus.
  • Ion channels of the present invention include full-length wild-type channels and functional variants or fragments thereof.
  • An ion channel may be of mammalian origin. In various embodiments, the ion channel may be of human origin.
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding a membrane protein, including an ion channel, may be inserted into a vector for expression.
  • a vector for expression Various vectors are publicly available.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures.
  • DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a
  • transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to those of skill in the art.
  • a vector including a polynucleotide encoding an ion channel can be delivered into a cell by, for example, transfection or transduction procedures.
  • Transfection and transduction refer to the acquisition by a cell of new genetic material by incorporation of added nucleic acid molecules. Transfection can occur by physical or chemical methods.
  • transfection techniques are well known to those of ordinary skill in the art including, without limitation, calcium phosphate DNA co-precipitation, DEAE-dextrin DNA transfection, electroporation, naked plasmid adsorption, and cationic liposome -mediated transfection.
  • Transduction refers to the process of transferring nucleic acid into a cell using a
  • Suitable viral vectors for use as transducing agents include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and Semliki Forest viral vectors.
  • Expression vectors containing DNA encoding an ion channel may be administered in vivo in any known or future developed manner.
  • the expression vectors are packaged into viruses, such as adenoviruses, and are delivered in proximity to targeted cells, tissue or organs.
  • the expression vectors are packaged into adenoviruses, such as helper-dependent adeno viral vector (HDAd) or adeno-associated virus pseudo-type 9 (AAV2/9).
  • HDAd virus packaging typically elicits less of an immunogenic response in vivo compared to some other adenoviruses and thus allows for longer term expression.
  • AAV2/9 packaging can result in cardiac tropism as well as a prolonged expression time frame.
  • viruses of clinical relevance include lentiviruses.
  • Replication deficient lentiviruses are RNA viruses, which can integrate into the genome and lead to long-term functional expression.
  • Viral vectors systems in addition to lentiviral vectors, AAV vectors, and HD AdV may also be used for the delivery of a polynucleotide encoding an ion channel.
  • non- viral delivery systems may be employed.
  • liposomes, DNA complexes, plasmid, liposome complexes, naked DNA, DNA-coated particles, or polymer based systems may be used to deliver the desired sequence to the cells.
  • An exogenous nucleotide sequence encoding an ion channel may be administered to cardiac cells, such as cardiomyocytes, Purkinje cells, or conductive tissue, SAN or
  • An exogenous nucleotide sequence encoding an ion channel may be administered to non-cardiac cells, such as, for example, skeletal, and smooth muscle cells, epithelial cells, and nerve cells. Delivery of a nucleotide sequence encoding an ion channel to myocardial cells, such as, for example, cardiac atrial cells, Purkinje fiber cells or ventricular cells, can be carried out according to any method known or developed in the art. For example, the delivery or administration may be accomplished by injection, catheter and other delivering vehicle known or developed in the art.
  • Exogenous polynucleotides encoding membrane proteins regulating the flow of ions across a cell membrane can be made by traditional PCR-based amplification and known cloning techniques.
  • a polynucleotide of the invention can be made by automated procedures that are well known in the art.
  • a polynucleotide of the invention may include a start codon to initiate transcription and a stop codon to terminate translation.
  • Suitable polynucleotides for use with the invention can be obtained from a variety of public sources including, without limitation, GenBank (National Center for Biotechnology Information (NCBI)), EMBL data library, SWISS-PROT (University of Geneva, Switzerland), the PIR-International database; and the American Type Culture Collection (ATCC).
  • GenBank National Center for Biotechnology Information (NCBI)
  • EMBL data library EMBL data library
  • SWISS-PROT Universality of Geneva, Switzerland
  • PIR-International database Universal Type Culture Collection
  • ion channel blockers may be used to increase the viability of cells, tissues, and organs that express an exogenous polynucleotide that encodes a membrane polypeptide that regulates the flow of ions across the cell membrane.
  • Cell viability may be monitored by any of a variety of methods, including, but not limited to, any of those described herein.
  • the methods of the present invention may increase the viability of nonexcitable and/or non-excitable cells.
  • the viability of fibroblast is improved.
  • the viability of myocytes is improved.
  • the viability of fibroblasts and myocytes is improved.
  • ion channel blockers may be used to prevent apoptosis in cells that express an exogenous polynucleotide that encodes a membrane polypeptide that regulates the flow of ions across the cell membrane.
  • the extent of apoptosis in cells may be monitored by any of a variety of methods, including, but not limited to, any of those described herein.
  • the methods of the present invention may prevent apoptosis in nonexcitable and/or non-excitable cells.
  • apoptosis in fibroblast is prevented.
  • apoptosis of myocytes is prevented.
  • apoptosis in fibroblasts and myocytes is prevented.
  • ion channel blockers may be used to modulate the electrophysiological function of cells, tissues, and organs that express an exogenous polynucleotide that encodes a membrane polypeptide that regulates the flow of ions across the cell membrane. Modulating means that the activity of the flow of ions across the cell membrane in the cells or tissues may be either increased or decreased.
  • Electrophysiological function may be assayed by any of a variety of methods including, but not limited to, voltage clamp electrophysiology (in particular patch clamp), immunohistochemistry, histological analysis of a biopsy sample, and RT-PCR.
  • the methods of the present invention may be applied to any of a variety of cells, including, but not limited to, cardiac myocytes, fibroblasts, neuronal cells, skeletal muscle cells, smooth muscle cells, epithelial cells, endothelial cells, and immune cells, such as T lymphocytes.
  • Such cells may be excitable cells or nonexcitable cells, wherein an excitable cell demonstrates electrical activity and a nonexcitable cell demonstrates no obvious electrical activity.
  • the methods of the present invention may be applied to any of a variety tissue, including, but not limited to cardiac tissue, nervous tissue, skeletal muscle, smooth muscle, secretory epithelial tissue, and beta cells of the pancreas.
  • a tissue may be heterologous tissue, including both excitable cells and nonexcitable cells.
  • the methods of the present invention may affect the excitable cells and/or the nonexcitable cells within a heterologous tissue.
  • Some embodiments of the present invention may affect fibroblasts and/or myocytes within a heterologous tissue.
  • the methods of the present invention may be applied to any of a variety of organs, including, but not limited to the heart, the brain, the spine, nerves, lung, bladder, and blood vessels, including veins and arteries.
  • treatment is an approach for obtaining beneficial or desired results, including and preferably clinical results.
  • Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.
  • an "effective amount” or a “therapeutically effective amount” of a substance is that amount sufficient to affect a desired biological effect, such as beneficial results, including clinical results.
  • more than one ion channel blocker may be administered.
  • compositions of ion channel blockers include, for example, buffering agents to help to maintain the pH in an acceptable range or preservatives to retard microbial growth.
  • Such compositions may also include a pharmaceutically acceptable carrier.
  • compositions of the present invention refers to one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • the compositions of the present invention are formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration.
  • a composition of the present invention may include a mixture or cocktail of two, three, four, five, or more blockers.
  • one or more additional therapeutic agents may be administered, in addition to the administration of an ion channel blocker.
  • An ion channel blocker may be administered before, after, and/or coincident to the administration of one or more additional therapeutic agents.
  • An ion channel blocker and one or more additional therapeutic agents may be administered separately or as a part of a mixture or cocktail.
  • agents of the present invention can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including
  • transdermal transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical, or the injection or application into or around the site of the exogenously expressed polynucleotide encoding a membrane protein that regulates the flow of ions across the cell membrane.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, intracardiac, and intrapericardial administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. Such preparation may be pyrogen-free.
  • the inhibitor may be administered in a tablet or capsule, which may be enteric coated, or in a formulation for controlled or sustained release.
  • Ion channel blockers may be formulated for controlled or sustained release locally at the site of the cells or tissue that are to express exogenous polynucleotides encoding a membrane proteins that regulates the flow of ions across the cell membrane.
  • Many suitable formulations are known, including polymeric or protein microparticles encapsulating the ion blocking agent, gels, or solutions which can be used locally to administer drug. These can take the form of implants.
  • Such an implant may be implanted within the tissue that is to express the exogenous polynucleotide encoding a membrane protein. Delivery of a blocker may also be via an osmotic implanted at the delivery site. Such a pump may be programmed to deliver a predetermined rate.
  • Delivery of an ion channel blocker may be acute (as a single administration), subacute (delivered over days or weeks), or a long term, chronic delivery. Delivery may be continuous or may be intermittent. Intermittent delivery may occur at predetermined timed intervals over the entire twenty-four hour day, for example, once a day, twice a day, three times a day, four times a day, six times a day, eight times a day, twelve times a day, or twenty- four times a day.
  • a baseline dosage delivered continuously or intermittently at specified intervals may be supplemented with a bolus dosage. Such a bolus dosage may be delivered in response to the determination of a difference between a currently measured physiological state in a subject and a baseline physiologic state.
  • an ion channel blocking agent may be delivered to the heart. Any of the wide variety of mechanisms for delivering to the heart available to one skilled in may be used, ranging, for example, from a simple injection, to the use of a catheter, to the use of a drug pump.
  • Such a pump may be, for example, an external drug pump, such as, for example, the Medtronic® MiniMed® pump, an implantable drug pump, such as, for example, the Medtronic® SynchroMed Infusion System®, or am osmotic pump.
  • Systems for monitoring heart function may be directly or indirectly (via wireless technology) coupled to the drug pump.
  • the present invention and/or one or more portions thereof may be implemented in hardware or software, or a combination of both.
  • the functions described herein may be designed in conformance with the principles set forth herein and
  • CMOS complementary metal-oxide-semiconductor
  • the present invention may be implemented using one or more computer programs executing on programmable computers, such as computers that include, for example, processing capabilities, data storage (e.g., volatile and nonvolatile memory and/or storage elements), input devices, and output devices.
  • Program code and/or logic described herein are applied to input data to perform functionality described herein and generate desired output information.
  • the output information may be applied as an input to one or more other devices and/or processes, in a known fashion.
  • Any program used to implement the present invention may be provided in a high level procedural and/or object orientated programming language to communicate with a computer system.
  • programs may be implemented in assembly or machine language.
  • the language may be a compiled or interpreted language.
  • Any such computer programs may preferably be stored on a storage media or device (e.g., ROM or magnetic disk) readable by a general or special purpose program, computer, or a processor apparatus for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer readable storage medium, configured with a computer program, where the storage medium so configured causes the computer to operate in a specific and predefined manner to perform functions described herein.
  • the present invention and/or one or more portions thereof include circuitry that may include a computer system operable to execute software to provide for the determination of a physiological state, e.g., heart failure, bradycardia, etc.
  • a physiological state e.g., heart failure, bradycardia, etc.
  • the circuitry may be implemented using software executable using a computer apparatus, other specialized hardware may also provide the functionality required to provide a user with information as to the physiological state of the individual.
  • the term circuitry as used herein includes specialized hardware in addition to or as an alternative to circuitry such as processors capable of executing various software processes.
  • the computer system may be, for example, any fixed or mobile computer system, e.g., a personal computer or a minicomputer.
  • Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the compounds in known in vitro and in vivo systems, such as those described herein. Dosages for humans or other animals may then be extrapolated therefrom. With the methods of the present invention, the efficacy of the administration of one or more agents may be assessed by any of a variety of parameters well known in the art.
  • an ion blocking agent of the present invention may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.
  • a subject includes, but is not limited to, humans and non- human vertebrates.
  • a subject is a mammal, particularly a human.
  • a subject may be an individual.
  • a subject may be a patient.
  • Non-human vertebrates include livestock animals, companion animals, and laboratory animals.
  • Non- human subjects also include non-human primates as well as rodents, such as, but not limited to, a rat or a mouse.
  • Non-human subjects also include, without limitation, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.
  • the methods of the present invention include in vitro, ex vivo, and in vivo methods.
  • in vitro is in cell culture and “in vivo” is within the body of a subject.
  • isolated refers to material that has been either removed from its natural environment (e.g., the natural environment if it is naturally occurring), produced using recombinant techniques, or chemically or enzymatically synthesized, and thus is altered “by the hand of man” from its natural state.
  • kits for the delivery of an ion blocking agent may include an ion blocking agent and a delivery device for the delivery of the ion blocking agent to a cell or tissue expressing an exogenous polynucleotide encoding a membrane polypeptide that regulates the flow of ions across a cell membrane.
  • a kit may also include written direction for use of the kits in methods including, but not limited to, any of the methods described herein.
  • exogenous ion channels is an attractive approach to modify cardiac cellular electrophysiology for several translation applications (e.g. arrhythmia and heart failure therapy). It is important to understand what other changes the externally introduced channel might confer to the heterogeneous cell population constituting cardiac tissue.
  • This example uses Kvl .3, a voltage-gated K + channel, as a model system, to determine its effect on cardiac fibroblasts and cardiac myocytes.
  • the heart is an electromechanical organ with considerable electrical heterogeneity. This electrical heterogeneity of the heart is conferred by differential expression of ion channels in various regions of the heart (Fig. 1). Several applications are being attempted wherein exogenous ion channels are expressed to change the underlying electrophysiology of the heart.
  • Ventricular tachycardia, atrial fibrillation, bradycardia, and heart failure all present examples of ion channel therapies for cardiac dysfunctions.
  • the over expression of gap junction channels may be used to repair conduction in the infarcted regions of the myocardium.
  • remnant connexin 43 may be completely knocked out to eliminate slow conduction (i.e. akin to molecular ablation).
  • the expression of ion channels may be used to slow conduction in the AV node, thus enabling to control ventricular rate during atrial fibrillation.
  • expression of exogenous channels primarily hyperpolarization activated cyclic nucleotide channel; HCN
  • HCN hyperpolarization activated cyclic nucleotide channel
  • SERCA2a may normalize calcium handling in the failing heart.
  • Kvl .3 can serve as a model ion channel in ion channel therapies for cardiac dysfunction.
  • Kvl .3 belongs to the Kv family of channels of which 9 types (Kvl -9) have been described.
  • the Kvl subfamily has 9 different a-subunits of which Kvl .3 is a member.
  • Ion channels are expressed ubiquitously in both non-excitable and excitable tissues (e.g. expressed in T-lymphocytes, kidney, liver, CNS, skeletal muscle).
  • Kvl .3 is a voltage gated ion channel with rapid activation and very slow inactivation (Fig. 2).
  • Kvl .3 can be inhibited by ⁇ -subunit KCNE4.
  • Kvl .3 is small enough to be efficiently packaged into various types of viral vectors.
  • HBSS Hank's balanced salt solution
  • Cell Viability and apoptosis Cells were assayed for viability and apoptosis with Hoechst nuclei stain.
  • Immunostaining Cells were fixed and immunostained for Connexin-43 (Cx43), a- actinin, and vimentin.
  • FIG. 4 Cellular blebbing, rounding and detachment are all clearly visible (Fig. 4). Fibroblasts were transfected with 10 8 PFU/ml AdV-Kvl .3 -GFP and fluorescence microscopy confirmed expression of Kvl .3 (Fig. 4A). The induction of apoptotis is demonstrated by Hoechst dye staining. Punctate DNA within the nuclei of cells (indicated by the arrows) is indicative of apoptosis (Fig. 4B).
  • Cx43 expression was highly disrupted and down regulated in the transfected myocytes. Fluorescent imaging of Cx43 alone exhibited reduced expression in the transfected cells (Fig. 5D) when compared to imaging of Cx43 alone in the non- transfected cells (Fig. 5B).
  • Kvl.3 transfected fibroblasts showed a dose-dependent deterioration: Cardiac fibroblasts transfected with AdV-Kvl .3-GFP demonstrated dose-dependent deterioration.
  • An ion channel construct including, but not limited to the AdV-Kvl .3-GFP and AdV-GFP constructs described in Example 1 will be transfected in vivo into the cardiac tissue of an animal, including, for example, a rat or pig.
  • the viability of cardiac myocytes and fibroblasts will be assayed with and without the administration of an ion channel blocking agent, such as, for example, Charybdotoxin.

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Abstract

La présente invention concerne des compositions, un équipement et des méthodes d'amélioration de la viabilité des cellules y compris, sans limitation, des cellules et des tissus non excitables exprimant des polynucléotides exogènes qui codent pour des protéines de la membrane qui régulent la circulation des ions à travers la membrane cellulaire. La viabilité des cellules et des tissus peut être améliorée en mettant les cellules ou le tissu en contact avec un ou plusieurs bloqueurs de canaux ioniques. Les protéines membranaires qui régulent la circulation des ions à travers la membrane cellulaire comprennent, sans limitation, des canaux ioniques.
PCT/US2010/057793 2009-12-11 2010-11-23 Modulation de l'électrophysiologie cellulaire WO2011071692A1 (fr)

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