Classifications

• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
  • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
  • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • 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
  • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• • 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
  • A61P9/06—Antiarrhythmics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0657—Cardiomyocytes; Heart cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

• the technology described herein relates to methods of treating cardiovascular disease.
• Cardiovascular disease remains the leading cause of death for both men and women worldwide, with a rapidly growing impact on developing nations.
• Cardiomyocyte replacement therapy is an area of active investigation for the treatment of cardiovascular disease, and can restore heart function after myocardial infarction.
• Human stem cells cultured in vitro can serve as a starting material for producing human cardiomyocytes for engraftment into an injured heart.
• cardiac engraftment there are complications involved with cardiac engraftment, one of which is the lack of maturity of the in vitro- differentiated cardiomyocytes which can lead to the development of transient cardiac arrhythmias.
• a method of preventing and treating arrhythmias caused by the cardiac graft is needed to improve patient outcomes following cardiomyocyte replacement therapy.
• the methods described herein are related, in part, to the discovery that the combination of amiodarone and ivabradine treatment improves survival and reduces arrhythmia burden in subjects receiving cardiac grafts.
• the methods described herein reduce the transient graft-associated arrhythmias and significantly improve the safety and survival of subjects treated with both anti -arrhythmic agents.
• described herein is a method of treating or ameliorating an engraftment arrhythmia in a subject recipient of a cardiac graft of cardiomyocytes, the method comprising administering to the subject an effective amount of amiodarone and an effective amount of ivabradine.
• the cardiac graft of cardiomyocytes comprises in vi t ro -di ffc re n t i ate d cardiomyocytes.
• the in vi tro -d i ffc re n t i ate d cardiomyocytes are differentiated from induced pluripotent stem (iPS) cells or from embryonic stem (ES) cells.
• the cardiac graft of cardiomyocytes is derived from stem cells autologous to the subject.
• the cardiac graft of cardiomyocytes is derived from stem cells allogeneic to the subject.
• the amiodarone and ivabradine are administered concurrently with the cardiac graft of cardiomyocytes.
• administration of amiodarone is initiated prior to administration of the graft of cardiomyocytes.
• administration can be, for example 1 day before, 2 days before, 3 days before, 4 days before, 5 days before, 6 days before, or 7 days or more prior to the graft.
• administration of ivabradine is initiated prior to administration of the graft of cardiomyocytes.
• administration can be, for example 1 day before, 2 days before, 3 days before, 4 days before, 5 days before, 6 days before, or 7 days or more prior to the graft.
• administration of both amiodarone and ivabradine is initiated prior to administration of the cardiac graft of cardiomyocytes.
• administration can be, for example 1 day before, 2 days before, 3 days before, 4 days before, 5 days before, 6 days before, or 7 days or more prior to the graft.
• amiodarone can be administered sooner than ivabradine, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days sooner.
• the administration of ivabradine is initiated concurrently with or after administration of the cardiac graft of cardiomyocytes.
• the administration of amiodarone is initiated concurrently with or after administration of the cardiac graft of cardiomyocytes.
• the administration of amiodarone is a single bolus administration.
• the administration is continuous or repeated administration.
• the administration is oral administration and/or intravenous injection.
• the amiodarone is administered orally at a dose of 100-800 mg, three times per day.
• the amiodarone is administered by IV bolus at a dose of 100-300 mg.
• the amiodarone is administered to a serum concentration of 1.5 to 2.5 pg/ml.
• the ivabradine is orally administered at a dose of 5 to 15 mg, twice per day. [0022] In another embodiment of this or any other aspect, the ivabradine is administered when there is tachycardia.
• ivabradine is administered to maintain a resting heart rate of less than or equal to 150 beats per minute (bpm). In another embodiment, ivabradine is administered to maintain a resting heart rate of 60 to 150 bpm, e.g., 60-140 bpm, 60-130 bpm, 60-120 bpm, 60-110 bpm, 60-100 bpm, 60-90 bpm or 60-80 bpm.
• the administration of amiodarone and ivabradine reduces post-graft accelerated heart rate experienced by the graft recipient by at least 10% relative to a subject receiving a graft of the same type of cells in the absence of amiodarone and ivabradine administration.
• the administration of amiodarone and ivabradine reduces the proportion of time in which the subject experiences engraftment arrhythmia by at least 10% relative to a subject receiving a graft of the same type of cardiomyocytes in the absence of amiodarone and ivabradine administration.
• the proportion of time in which the subject experiences engraftment arrhythmia is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
• a method of cardiomyocyte transplant comprising: a) administering in wVro-diffcrentiated cardiomyocytes to cardiac tissue of a subject in need thereof; and b) administering to the subject an amount of amiodarone and an amount of ivabradine effective to reduce engraftment arrhythmia in the subject.
• engraftment arrhythmia is reduced in the subject relative to a subject receiving in vi iro -d i ffc re n t i ate d cardiomyocytes without receiving amiodarone and ivabradine.
• the in wVro-diffcrentiated cardiomyocytes are differentiated in vitro from embryonic stem cells or from iPS cells.
• the iPS cells are autologous to the subject.
• the iPS cells are allogeneic to the subj ect.
• the amiodarone and ivabradine are administered concurrently with the in vi iro -d i ffc rc n t i atcd cardiomyocytes.
• administration of amiodarone is initiated prior to administration of the in vi iro -d i ffc rc n t i ate d cardiomyocytes.
• administration of ivabradine is initiated prior to administration of the in vi iro -d i ffc rc n t i ate d cardiomyocytes.
• administration of both amiodarone and ivabradine is initiated prior to administration of the in vi iro -d i ffc rc nt i atcd cardiomyocytes.
• administration of ivabradine is initiated concurrently with or after administration of the in wVro-diffcrentiated cardiomyocytes.
• the administration of amiodarone is initiated concurrently with or after administration of the in wVro-diffcrentiated cardiomyocytes.
• the administration of ivabradine is a single bolus administration.
• the administration is continuous or repeated administration.
• the administration of amiodarone and ivabradine is short-term.
• the administration of amiodarone and ivabradine is terminated after engraftment arrhythmia burden reaches zero, without recrudescence of the arrhythmia.
• the administration is oral administration and/or intravenous (IV) injection.
• the amiodarone is administered orally at a dose of 100-800 mg, three times per day.
• the amiodarone is administered by IV bolus at a dose of 100-300 mg.
• the amiodarone is administered to a serum concentration of 1.5 to 2.5 pg/ml.
• the ivabradine is orally administered at a dose of 5 to 15 mg, twice per day.
• the ivabradine is administered when there is tachycardia.
• ivabradine is administered to maintain a resting heart rate of less than or equal to 150 beats per minute (bpm).
• the subject is a human.
• the subject has or is at risk for having a cardiovascular disease or a cardiac event.
• the cardiovascular disease or the cardiac event is selected from the group consisting of: atherosclerotic heart disease, myocardial infarction, cardiomyopathy, cardiac arrhythmia, valvular stenosis, congenital heart disease, chronic heart failure, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.
• described herein is a composition comprising in vi iro -d i ffe re n t i ated cardiomyocytes, amiodarone and ivabradine.
• FIGs. 1A-1B demonstrate that amiodarone and ivabradine therapy reduces heart rate and arrhythmia burden in pigs that received cardiac grafts compared with untreated pigs that received cardiac grafts.
• FIG. 1A shows the average heart rate for pigs that received
• FIGs. 3-14 show the heart rate and % arrhythmia for each individual pig that received cardiac graft in the presence and absence of amiodarone/ivabradine treatment. Heart rate, reported as beats per minute, is indicated in grey on the left vertical axis in the figures.
• Arrhythmia burden reported as percent time in arrhythmia compared to normal sinus rhythm, is indicated by the black circles on the right vertical axis.
• FIGs. 3-8 The pigs treated with anti -arrhythmic agents are shown in FIGs. 3-8. Amiodarone treatment is indicated by the black bar at the top of each graph, while ivabradine treatment is indicated by the checked bar.
• FIG. 15 shows an embodiment of anti-arrhythmic regimen doses for pigs (swine) and humans.
• FIG. 16 shows a flowchart of the study design.
• Phase 1 consisted of nine total subjects, four used to study the natural history of engraftment arrythmia (EA) and five used to screen seven candidate antiarrhythmic agents.
• Amiodarone and ivabradine were found to have promising signs of effect and advanced for further study.
• Phase 2 consisted of 19 total subjects: nine assigned to treatment with amiodarone and ivabradine, eight to no treatment and two to infarct with sham transplant and no anti-arrhythmic drug treatment.
• FIG. 17 shows a study timeline for Phase 2 drug trial of chronic amiodarone and adjunctive ivabradine therapy.
• Myocardial infarction (MI) was induced by 90-minute balloon occlusion of the mid- left anterior descending artery two weeks prior to human embryonic stem cell-derived cardiomyocyte transplantation (day 0). All subjects received multi-drug immunosuppression.
• Treatment cohort received rate and rhythm control with combined oral amiodarone and adjunctive oral ivabradine.
• FIG. 18 shows plasma amiodarone levels in pigs. Amiodarone levels were measured in plasma by a liquid chromatography-mass spectrometry assay. Chronic oral amiodarone in six pigs was discontinued after achieving electrical maturation and stabilization of engraftment arrythmia. Serum concentrations of amiodarone were assayed weekly, including 3-4 weeks after discontinuation.
• FIG. 19 shows variable morphologies of engraftment arrhythmia (EA) in a single pig.
• EA engraftment arrhythmia
• NSR normal sinus rhythm
• AJR accelerated junctional rhythm
• VT ventricular tachycardia
• AIVR accelerated idioventricular rhythm
• a continuous rhythm recording exhibits multiple foci of impulse generation from hESC-CM grafts interacting at various levels of the host conduction system to induce EA. No sustained arrythmias were noted in surgical sham controls.
• FIG. 20 shows hESC-CM graft histology and location.
• Left panel Histological sections stained with picrosirius red to identify collagen (infarct) and fast green to identify viable myocardium.
• Adjacent sections labeled with human cTnT identify transplanted hESC-CM graft within unstained porcine myocardium and scar tissue. Both treatment and no treatment sections were obtained on post transplantation day 42.
• Right panel Transplanted hESC-CM grafts were located similarly between treatment (closed square) and no treatment (open circle) and successfully targeted the infarct and peri- infarct regions of the anterior wall.
• FIG. 21A-21B demonstrates the acute effects of amiodarone and ivabradine on engraftment arrhythmia.
• Amiodarone was effective as an intravenous bolus to cardiovert engraftment arrythmia to normal sinus or a lower heart rate (FIG. 21 A) .
• FIG. 22A-22B shows antiarrhythmic treatment with amiodarone and ivabradine for engraftment arrhythmia in pig.
• FIG. 23A-23F demonstrates the effect of antiarrhythmic treatment on heart rate and arrhythmia burden.
• Peak heart rate (FIG. 23E) and peak arrythmia burden (FIG. 23F) were significantly reduced with treatment (black) compared to no treatment (light gray). * p ⁇ 0.05, ** p ⁇ 0.005.
• FIG. 24A-24B shows that transplanted hESC-CM graft interacts with a diffuse Purkinje conduction system in the porcine myocardium.
• Purkinje fibers are distributed in a mesh-like network throughout the native porcine myocardium (FIG. 24A).
• FIG. 24B Further magnified view of white boxed regions show Cx40 localizes to gap junctions of Purkinje cells that display lower sarcomere content (F-Actin) (i.) and lack T-Tubules (WGA) (ii.) in contrast to surrounding cardiomyocytes, scale bar 20 pm.
• F-Actin sarcomere content
• WGA T-Tubules
• High magnification of boxed regions show example of Purkinje-transitional cell-graft (i., white arrowhead) and direct Purkinje -graft (ii., white arrow) interactions, scale bar 500 pm (top) or 50 pm (bottom).
• FIG. 25 demonstrates that connexin 40 specifically stains Purkinje fibers.
• Connexin 40 (Cx40) marks Purkinje fibers (PFs) and localizes to gap junctions of PFs that display lower sarcomere content (F-Actin) and lack T-Tubules (WGA) in contrast to surrounding cardiomyocytes, scale bars 20 pm.
• ES and iPS cardiomyocytes lack the functional maturity to fully integrate with the native heart tissue.
• graft-induced arrhythmias appear shortly after the in wVro-diffcrentiated cardiomyocytes are transplanted, during which time the recipient is at risk for sudden cardiac death and heart failure.
• subjects that do survive the engraftment procedure have an observed return to normal sinus rhythm that reflects the in vivo maturation of the transplanted cardiomyocytes. In order for this to occur, sufficient electrical integration with the host myocardium is necessary to avoid arrhythmogenicity and improve survival following cardiac engraftment.
• the methods described herein are related, in part, to the discovery that the combination of amiodarone and ivabradine treatment improves survival, prevents tachycardia and reduces arrhythmia burden in subjects that receive cardiac grafts.
• the methods described herein reduce the transient graft- associated arrhythmias and significantly improve the safety and survival of subjects treated with both anti -arrhythmic agents. Definitions:
• “Treatment” of a cardiac disorder, a cardiac disease, event, or a cardiac injury refers to therapeutic intervention that enhances cardiac function, reduces engraftment arrhythmia, and/or enhances cardiomyocyte engraftment and/or enhances cardiomyocyte transplant or graft vascularization in a treated area, thus improving the function of e.g., the heart. That is, cardiac “treatment” is oriented to the function of the heart (e.g., enhanced function within an infarcted area), and/or other site treated with the compositions described herein.
• a therapeutic approach that improves the function of the heart for example as assessed by measuring heart rate and/or the frequency of arrhythmias reduced by at least 10%, and preferably by at least 20%, 30%, 40%, 50%, 75%, 90%, 100% or more, e.g., 2-fold, 5 -fold, 10-fold or more, up to and including full function, relative to an appropriate control is considered effective treatment.
• Effective treatment need not cure or directly impact the underlying cause of the heart disease or disorder to be considered effective treatment.
• the term “short term,” when applied to treatment with amiodarone and ivabradine for engraftment arrhythmia, means treatment only so long as engraftment arrhythmia continues to occur. It is demonstrated herein that treatment with this combination of drugs reduces engraftment arrhythmia burden, and can be safely withdrawn after engraftment arrhythmia is resolved, without recrudescence of arrhythmia.
• short term treatment will be on the order of weeks (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks or fewer) to months (e.g., 1 month, 2 months, 3 months, 4 months or fewer) post graft.
• weeks e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks or fewer
• months e.g., 1 month, 2 months, 3 months, 4 months or fewer
• prevention when used in reference to a disease, disorder or symptoms thereof, refers to a reduction in the likelihood that an individual will develop a disease or disorder, e.g., heart failure following myocardial infarction, as but one example.
• the likelihood of developing a disease or disorder is reduced, for example, when an individual having one or more risk factors for a disease or disorder either fails to develop the disorder or develops such disease or disorder at a later time or with less severity, statistically speaking, relative to a population having the same risk factors and not receiving treatment as described herein.
• the failure to develop symptoms of a disease, or the development of reduced (e.g., by at least 10% on a clinically accepted scale for that disease or disorder) or delayed (e.g., by days, weeks, months or years) symptoms is considered effective prevention.
• the terms “patient”, “subject” and “individual” are used interchangeably herein, and refer to an animal, particularly a human, to whom treatment, including prophylactic treatment is provided.
• the term “subject” as used herein refers to human and non-human animals.
• the term “non-human animals” and “non-human mammals” are used interchangeably herein includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc.
• the subject is human.
• the subject is an experimental animal or animal substitute as a disease model.
• the subject is a domesticated animal including companion animals (e.g., dogs, cats, rats, pigs, guinea pigs, hamsters etc.).
• companion animals e.g., dogs, cats, rats, pigs, guinea pigs, hamsters etc.
• a subject can have previously received a treatment for a cardiovascular disease or cardiac event, or has never received treatment for a cardiovascular disease or a cardiac event.
• a subject can have previously been diagnosed with having a cardiovascular disease, or has never been diagnosed with a cardiovascular disease.
• the term "human stem cell” refers to a human cell that can self-renew and differentiate to at least one cell type.
• human stem cell encompasses human stem cell lines, human-derived induced pluripotent stem (iPS) cells, human embryonic stem cells, human pluripotent cells, human multipotent stem cells, amniotic stem cells, placental stem cells, or human adult stem cells.
• iPS induced pluripotent stem
• human embryonic stem cells human embryonic stem cells
• human pluripotent cells human multipotent stem cells
• amniotic stem cells placental stem cells
• placental stem cells or human adult stem cells.
• in vi tro -d i ffc re n t i ate d cardiomyocytes refers to cardiomyocytes that are generated in culture, typically via step-wise differentiation from a precursor cell such as a human embryonic stem cell, an induced pluripotent stem cell, an early mesoderm cell, a lateral plate mesoderm cell or a cardiac progenitor cell.
• anti-arrhythmic or “anti-arrhythmic agent” refers any agent (e.g. , small molecule or pharmaceutical composition) that reduces the onset, frequency, and/or severity of a cardiac arrhythmia. Anti-arrhythmics can be used to treat irregular cardiac rhythm, to reduce heart rate in tachycardias, increase heart rate in bradycardias, or otherwise promote a normal sinus rhythm and prevent sudden cardiac death.
• the term “derived from,” used in reference to a stem cell means the stem cell was generated by reprogramming of a differentiated cell to a stem cell phenotype.
• the term “derived from,” used in reference to a differentiated cell means the cell is the result of differentiation, e.g., in vitro differentiation, of a stem cell.
• iPSC-CMs or “induced pluripotent stem cell-derived cardiomyocytes” are used interchangeably to refer to cardiomyocytes derived from an induced pluripotent stem cell.
• phrases "pharmaceutically acceptable” is employed herein to refer to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount.
• “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
• “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
• the terms “increased,” “increase,” “increases,” or “enhance” or “activate” are all used herein to generally mean an increase of a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold
• a “reference level” refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition disclosed herein).
• a normal, otherwise unaffected cell population or tissue e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition disclosed herein.
• an “appropriate control” refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that was not contacted by an agent or composition described herein) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment.
• an appropriate control can be a subject or a tissue that has not been administered amiodarone and ivabradine.
• statically significant or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
• compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
• the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
• a cardiovascular disease is a disease that affects the heart and/or circulatory system of a subject.
• cardiac diseases include atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.
• Symptoms of cardiovascular disease can include but are not limited to syncope, fatigue, shortness of breath, chest pain, and palpitations.
• a cardiovascular disease is generally diagnosed by a physical examination, blood tests, and/or an electrocardiogram (EKG). An abnormal EKG is an indication that the subject has an abnormal cardiac rhythm or cardiac arrhythmia. Methods of diagnosing arrhythmias are known in the art.
• cardiac event refers to an incident of a myocardial injury, myocardial infarction, ventricular fibrillation, stenosis, arrhythmia, or the like.
• Cardiac electrophysiological and contractile function is a tightly controlled process. When ion channel regulation or contractile function is disrupted in a cardiac cell or tissue, this can result in cardiac arrhythmias that can sometimes be deadly. Cardiac diseases remain a leading cause of death worldwide.
• described herein is a method of treating a cardiovascular disease.
• described herein is a method of treating or ameliorating an engraftment arrhythmia in a subject recipient of a cardiac graft of cardiomyocytes, the method comprises: administering to the subject an effective amount of amiodarone and an effective amount of ivabradine.
• the subject has or is at risk for having a cardiovascular disease or a cardiac event.
• the subject having a cardiovascular disease is in need of or has received a cardiac graft.
• the subject has or is diagnosed with an engraftment arrhythmia.
• described herein is a method of preventing or reducing an engraftment arrhythmia.
• An engraftment arrhythmia is a novel and aberrant cardiac rhythm that occurs following administration of a graft of cardiac cells or cardiomyocytes. Engraftment arrhythmias are observed after cardiac graft transplantation and generally persist transiently for days to weeks. Engraftment arrhythmia can cause sudden cardiac death and heart failure in the subject.
• Cardiac engraftment adminsters cardiomyocytes to a site of cardiac injury in the heart.
• a skilled physician can determine the site of injury by methods known in the art.
• a primary goal of cardiac engraftment is to provide electrical and mechanical stability to the injured myocardium that cannot be achieved by pharmaceutical treatments alone.
• Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into more specialized cell types.
• Three broad types of mammalian stem cells include: embryonic stem (ES) cells that are found in blastocysts, induced pluripotent stem cells (iPSCs) that are reprogrammed from somatic cells, and adult stem cells that are found in adult tissues.
• ES embryonic stem
• iPSCs induced pluripotent stem cells
• Other sources of pluripotent stem cells can include amnion-derived or placental -derived stem cells.
• Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.
• Cardiomyocytes useful in the compositions and methods described herein can be differentiated from both embryonic stem cells and induced pluripotent stem cells, among others.
• the compositions and methods provided herein use human cardiomyocytes differentiated from embryonic stem cells.
• the compositions and methods provided herein do not encompass generation or use of human cardiogenic cells made from cells taken from a viable human embryo.
• Embryonic stem cells Embryonic stem cells and methods for their retrieval are well known in the art and are described, for example, in Trounson A O Reprod Fertil Dev (2001) 13: 523, Roach M L Methods Mol Biol (2002) 185: 1, and Smith A G Annu Rev Cell Dev Biol (2001) 17:435.
• the term "embryonic stem cell” is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see e.g., US Patent Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer ⁇ see, for example, US Patent Nos. 5,945,577, 5,994,619, 6,235,970).
• Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from a stem cell bank or other recognized depository institution.
• Other means of producing stem cell lines include methods comprising the use of a blastomere cell from an early stage embryo prior to formation of the blastocyst (at around the 8-cell stage). Such techniques correspond to the pre implantation genetic diagnosis technique routinely practiced in assisted reproduction clinics. The single blastomere cell is co-cultured with established ES-cell lines and then separated from them to form fully competent ES cell lines.
• Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli.
• the human cardiomyocytes described herein are not derived from embryonic stem cells or any other cells of embryonic origin.
• the compositions and methods described herein utilize cardiomyocytes that are differentiated in vitro from induced pluripotent stem cells.
• An advantage of using iPSCs to generate cardiomyocytes for the compositions described herein is that, if so desired, the cells can be derived from the same subject to which the desired human cardiomyocytes are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then re-differentiated into a human cardiomyocyte to be administered to the subject (e.g., autologous cells).
• the cardiomyocytes are essentially derived from an autologous source, the risk of engraftment rejection or allergic responses is reduced compared to the use of cells from another subject or group of subjects. While this is an advantage of iPS cells, in alternative embodiments, the cardiomyocytes useful for the methods and compositions described herein are derived from non-autologous sources (e.g., allogenic). In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one embodiment, the stem cells used to generate cardiomyocytes for use in the methods and compositions described herein are not embryonic stem cells.
• Reprogramming is a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Stated another way, reprogramming is a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics.
• differentiated cells do not render these cells non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells.
• the transition of a differentiated cell to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character when differentiated cells are placed in culture.
• Reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.
• the cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming.
• cells can be terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells.
• reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state. In some embodiments, reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming.
• reprogramming of a differentiated cell causes the differentiated cell to assume an undifferentiated state with the capacity for self-renewal and differentiation to cells of all three germ layer lineages.
• differentiated cell e.g., a somatic cell
• iPSCs induced pluripotent stem cells
• iPSCs are generated from somatic cells by introducing a combination of reprogramming transcription factors.
• the reprogramming factors can be e.g., nucleic acids, vectors, small molecules, viruses, polypeptides, or any combination thereof.
• Non-limiting examples of reprogramming factors include Oct4 (Octamer binding transcription factor-4), Sox2 (Sex determining region Y)-box 2, Klf4 (Kruppel Uike Factor-4), and c-Myc.
• Factors e.g., UIN28 + Nanog, Esrrb, Pax5 shRNA, C/EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 UT(T), hTERT
• chemicals e.g., BIX-01294, BayK8644, RG108, AZA, dexamethasone, VPA, TSA, SAHA, PD025901 + CHIR99021(2i), A-83-01
• pluripotent stem cells from somatic cells e.g., any cell of the body with the exclusion of a germ line cell; fibroblasts, etc.
• somatic cells e.g., any cell of the body with the exclusion of a germ line cell; fibroblasts, etc.
• any method that re-programs a somatic cell to the pluripotent phenotype would be appropriate for use in the methods described herein.
• the efficiency of reprogramming i.e., the number of reprogrammed cells derived from a population of starting cells can be enhanced by the addition of various small molecules as shown by Shi, Y., el al. (2008) Cell-Stem Cell 2:525-528, Huangfu, D., el al. (2008) Nature Biotechnology 26(7):795-797, and Marson, A., el al. (2008) Cell-Stem Cell 3: 132-135.
• agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX- 01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
• isolated clones can be tested for the expression of one or more stem cell markers.
• stem cell markers can include but are not limited to SSEA3, SSEA4, CD9, Nanog, Oct4, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl, among others.
• a cell that expresses Oct4 or Nanog is identified as pluripotent.
• Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry.
• the pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate to cells of each of the three germ layers.
• teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
• the cells are introduced to nude mice and histology and/or immunohistochemistry is performed on a tumor arising from the cells.
• the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
• Adult Stem Cells are stem cells derived from tissues of a post-natal or post-neonatal organism or from an adult organism.
• An adult stem cell is structurally distinct from an embryonic stem cell not only in markers it does or does not express relative to an embryonic stem cell, but also by the presence of epigenetic differences, e.g. differences in DNA methylation patterns.
• cardiomyocytes differentiated from adult stem cells can also be used for cardiac grafts as described herein. Methods of isolating adult stem cell are known in the art. See for example, U.S. Patent No. 9,206,393 B2; and US Application No. 2010/0166714 Al; which are incorporated herein by reference in their entireties.
• the step-wise differentiation of ESCs or iPSCs to cardiomyocytes proceeds in the following order: ESC or iPSC > cardiogenic mesoderm > cardiac progenitor cells > cardiomyocytes (see e.g.. Lian el al. Nat Prot (2013); US Applicant No. 2017/0058263 Al; 2008/0089874 Al; 2006/0040389 Al; US Patent No. 10,155,927 B2; 9,994,812 B2; and 9,663,764 B2, the contents of each of which are incorporated herein by reference their entireties).
• a number of protocols for differentiating ESCs and iPSCs to cardiomyocytes are known in the art.
• agents can be added or removed from cell culture media to direct differentiation to cardiomyocytes in a step-wise fashion.
• factors and agents that can promote cardiomyocyte differentiation include small molecules (e.g., Wnt inhibitors, GSK3 inhibitors), polypeptides (e.g., growth factors), nucleic acids, vectors, and patterned substrates (e.g., nanopattems).
• the addition of growth factors necessary in cardiovascular development including but not limited to fibroblast growth factor 2 (FGF2), transforming growth factor b (TGF ) superfamily growth factors- Activin A and BMP4, vascular endothelial growth factor (VEGF), and the Wnt inhibitor DKK-1, can also be beneficial in directing differentiation along the cardiac lineage.
• FGF2 fibroblast growth factor 2
• TGF transforming growth factor b
• VEGF vascular endothelial growth factor
• DKK-1 vascular endothelial growth factor
• Additional examples of factors and conditions that help promote cardiomyocyte differentiation include but are not limited to
• a method of cardiomyocyte graft or transplant comprises: a) administering cardiomyocytes to cardiac tissue of a subject in need thereof; and b) administering to the subject an amount of amiodarone and an amount of ivabradine effective to reduce engraftment arrhythmia in the subject.
• the term “transplanting” or “transplant” is used in the context of the placement of cells, e.g. cardiomyocytes, as described herein into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced.
• the cells e.g. cardiomyocytes, or their differentiated progeny (e.g. cardiac fibroblasts etc.) and cardiomyocytes can be implanted directly or into the cardiac tissue of the recipient, e.g., at or near a site, or into cardiac tissue of a subject with a cardiac disease.
• cardiomyocytes generally do not proliferate to an extent that the heart can heal from an acute injury comprising cell death.
• the cells are optionally transplanted on or within a scaffold or biocompatible material that supports viability of the implanted cardiomyocytes, and/or, for example, assists with keeping administered cells in the desired location for engraftment or promotes integration with native cardiac cells in a subject.
• the cardiomyocytes are human stem cell derived- cardiomyocytes or in vriro-diffcrcntiatcd cardiomyocytes as described herein.
• the in vitro-differentiated cardiomyocytes are differentiated in vitro from embryonic stem cells or from iPS cells.
• a scaffold is a structure, comprising a biocompatible material including but not limited to a gel, sheet, or lattice that can contain the cells in a desired location but permit the entry or diffusion of factors in the environment necessary for survival and function.
• a biocompatible material including but not limited to a gel, sheet, or lattice that can contain the cells in a desired location but permit the entry or diffusion of factors in the environment necessary for survival and function.
• a number of biocompatible polymers suitable for a scaffold are known in the art.
• an effective amount of human cardiomyocytes can comprise at least 1 X 10 7 , at least 1.1 X 10 7 , at least 1.2 X 10 7 , at least 1.3 X 10 7 , at least 1.4 X 10 7 , at least 1.5 X 10 7 , at least 1.6 X 10 7 , at least 1.7 X 10 7 , at least 1.8 X 10 7 , at least 1.9 X 10 7 , at least 2 X 10 7 , at least 3 X 10 7 , at least 4 X 10 7 , at least 5 X 10 7 , at least 6 X 10 7 , at least 7 X 10 7 , at least 8 X 10 7 , at least 9 X 10 7 , at least 1 X 10 8 , at least
• the methods described herein further comprise administering to the subject an effective amount of amiodarone and an effective amount of ivabradine.
• amiodarone an effective amount of amiodarone
• ivabradine an effective amount of ivabradine
• Amiodarone is a class III antiarrhythmic agent prescribed for the treatment of cardiac arrest, ventricular tachycardia, and atrial fibrillation.
• Analogs and derivatives of amiodarone are known in the art, see e.g., US Patent Nos. 7,799,799 B2; 9,018,250 B2; and Carlsson et al. J Med Chem. 2002 Jan 31 ;45(3):623-30, which are incorporated herein by reference in their entireties. It is contemplated that amiodarone analogs and derivatives can also be beneficial for treating engraftment arrhythmias.
• amiodarone inhibits voltage-gated potassium channels and voltage-gated calcium channels, which in turn prolongs phase 3 of the cardiac action potential.
• amiodarone inhibits the pore-forming subunit of the potassium ion channel, K V 11.1 (encoded by the KCNH2 gene/ and inhibits the voltage-gated calcium channel (encoded by the CACNA2D2 gene).
• K V 11.1 encoded by the KCNH2 gene/
• the voltage-gated calcium channel encoded by the CACNA2D2 gene
• amiodarone has also been shown to inhibit voltage-gated sodium channel activity, which can contribute to the drug’s pro-arrhythmic potential.
• amiodarone exhibits beta-blocker like activity, in that it reduces heart rate when administered to a subject.
• the clinical effects of amiodarone include the prolongation of the QT interval due to the increased refractory periods of the ventricles, bundles of His, and Purkinje fibers. While this effect is beneficial for the treatment of arrhythmias such as atrial fibrillation, this effect can become pro-arrhythmic. It is well known in the art that amiodarone and other anti-arrhythmic agents can lead to drug-induced QT prolongation depending on the dose and treatment regimen. This can be exacerbated when combined with other drugs or anti-arrhythmic agents.
• Ivabradine is a class I f anti-arrhythmic agent that is used for the treatment of angina, tachycardia, and heart failure.
• Analogs and derivatives of ivabradine are known in the art. See for example, US Patent Nos. 7,879,842; 7,361,650; 7,867,996; and 7,361,649, which are incorporated herein by reference in their entireties. It is contemplated that certain ivabradine analogs and derivatives can also be beneficial for the methods described herein.
• Ivabradine is known to inhibit funny channels (also known as HCN channels) in the heart.
• funny channels also known as HCN channels
• the major function of funny current (I f ) in the heart is to maintain the pacemaker activity in the SA node. Blocking funny channels with ivabradine results in an overall reduction in heart rate.
• described herein are methods for treating or ameliorating a cardiac disease, disorder, event, or injury comprising administering cardiomyocytes to a subject in need thereof and administering an effective amount of amiodarone and ivabradine.
• methods described herein prevent an anticipated disorder e.g., an engraftment arrhythmia.
• the anti-arrhythmic agent described herein can be administered by any appropriate route which results in a reduction in arrhythmic burden in the subject.
• the term “administering” refers to the administration of a pharmaceutical composition comprising one or more agents.
• the administering can be done by direct injection (e.g., directly administered to a target cell or tissue), subcutaneous injection, intramuscular injection, oral, or nasal delivery, or a combination thereof to the subject in need thereof.
• Anti- arrhythmic agents e.g., amiodarone and ivabradine
• Administration of amiodarone and ivabradine can be IV or oral for both drugs.
• both drugs or analog thereof
• both drugs can be administered orally, both IV, or one orally and the other IV in either combination.
• both drugs are administered orally.
• an “effective amount” as used herein refers to the amount of amiodarone and/or ivabradine or analog thereof needed to alleviate an engraftment arrhythmia.
• an “effective amount” refers to the amount of amiodarone and/or ivabradine or analog thereof needed to alleviate an engraftment arrhythmia.
• engraftment and integration of grafted cells can be facilitated and the clinical outcome for the subject can be improved, including reduced risk for heart failure or sudden cardiac death.
• arrhythmia burden can be calculated as described in the working examples herein or as known in the art.
• Arrhythmic burden without the drug regimen described can be 75% or more. This can be reduced by at least 10% with the administration of amiodarone and ivabradine. It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
• the administration of amiodarone and ivabradine or analog thereof reduces post-graft accelerated resting heart rate experienced by the graft recipient by at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more relative to a subject receiving a graft of the same type of cells in the absence of amiodarone and ivabradine administration or analog thereof.
• the administration of amiodarone and ivabradine or analog thereof reduces engraftment arrhythmia burden, i.e., the proportion of time in which the subject experiences engraftment arrhythmia, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or more relative to a subject receiving a graft of the same type of cardiomyocytes in the absence of amiodarone and ivabradine administration. While at least 10% reduction is considered effective treatment, it is contemplated that administration of amiodarone and ivabradine permit up to complete cessation of engraftment arrhythmia.
• the effective dose can be estimated initially from cell culture assays, and a dose range can be formulated in animals (e.g., pig). Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such anti-arrhythmic agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of use or administration utilized.
• compositions are administered so that the amiodarone is used or given at a dose from at least 50 mg, at least 100 mg, at least 150 mg, at least 200 mg, at least 250 mg, at least 300 mg, at least 350 mg, at least 400 mg, at least 450 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, at least 800 mg, at least 850 mg, at least 900 mg, to about 1000 mg.
• the amiodarone or analog thereof is administered orally at a dose of 100-800 mg, three times per day. In some embodiments of any of the aspects, the amiodarone is administered by IV bolus at a dose of 100-300 mg. In some embodiments of any of the aspects, the amiodarone is administered to a serum concentration of 1.5 to 2.5 pg/ml.
• ivabradine administered at a dose from at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg, at least 9 mg, at least 10 mg, at least 11 mg, at least 12 mg, at least 13 mg, at least 14 mg, at least 15 mg, at least 16 mg, at least 17 mg, at least 18 mg, at least 19 mg, to about 20 mg.
• the ivabradine or analog thereof is orally administered at a dose of 5 to 15 mg, twice per day.
• the administration of amiodarone is a single bolus administration. In some embodiments, the administration is continuous or repeated administration. In some embodiments, the administration is oral administration and/or intravenous injection. In some embodiments, the amiodarone is administered orally at a dose of 100-800 mg, three times per day. In some embodiments, the amiodarone is administered by IV bolus at a dose of 100-300 mg. In some embodiments, the amiodarone is administered to a serum concentration of 1.5 to 2.5 pg/ml. In some embodiments, the ivabradine is orally administered at a dose of 5 to 15 mg, twice per day.
• the ivabradine is administered when there is tachycardia. In some embodiments, the ivabradine is administered to maintain a resting heart rate of less than or equal to 150 beats per minute (bpm). In some embodiments, the administration of amiodarone and ivabradine is short-term. In some embodiments, the administration of amiodarone and ivabradine is terminated after engraftment arrhythmia burden reaches zero, without recrudescence of the arrhythmia.
• the anti-arrhythmic agents, amiodarone and ivabradine or analog thereof described herein, are used in combination for treating an engraftment arrhythmia and/or a cardiovascular disease.
• Administered "in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder (a cardiovascular disease or an engraftment arrhythmia) and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration.
• the delivery of one treatment ends before the delivery of the other treatment begins.
• the treatment is more effective because of combined administration.
• the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
• delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
• the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
• the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
• the anti-arrhythmic agents described herein and/or at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially.
• the amiodarone described herein can be administered first, and the ivabradine can be administered second, or the order of administration can be reversed.
• the agent and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder (e.g. , during an engraftment arrhythmia), or during a period of remission or less active disease (e.g., before or after engraftment).
• the anti-arrhythmic agents can be administered before cardiac grafting, concurrently with the treatment, post-treatment, or during a flare of a cardiovascular disease following engraftment.
• the amiodarone and the ivabradine can be administered in an amount or dose that is higher, lower, or the same as the amount or dosage of each anti-arrhythmic agent used individually, e.g., as a monotherapy.
• the administered amount or dosage of the amiodarone, ivabradine, or both is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each anti-arrhythmic agent used individually.
• the amount or dosage of amiodarone, ivabradine, or both, that results in a desired effect is lower (e.g. , at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent individually required to achieve the same therapeutic effect.
• the amiodarone and ivabradine or an analog thereof are administered concurrently with the graft of cardiomyocytes.
• administration of amiodarone is initiated prior to administration of the graft of cardiomyocytes.
• administration of ivabradine is initiated prior to administration of the graft of cardiomyocytes.
• administration of both amiodarone and ivabradine is initiated prior to administration of the graft of cardiomyocytes.
• administration of ivabradine is initiated concurrently with or after administration of the graft of cardiomyocytes.
• administration of amiodarone is initiated concurrently with or after administration of the graft of cardiomyocytes.
• the amiodarone is administered beginning at least 1 hour, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, prior to engraftment.
• the ivabradine is administered beginning at least 1 hour, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, prior to engraftment.
• ivabradine is administered as needed to control heart rate, i.e., to limit or control tachycardia as commonly occurs with engraftment arrhythmia.
• amiodarone is administered either before (e.g., beginning 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day) or concurrently with graft administration and continues after grafting.
• the amiodarone is administered at least once per day, twice per day, three times per day, or more.
• the ivabradine is administered at least once per day, twice per day, three times per day, or more.
• anti-arrhythmics can be administered concurrently or in addition to amiodarone and ivabradine to aid in treatment of the subject.
• amiodarone and/or ivabradine or analogs thereof can be administered as needed for rate control in the subject and to alleviate at least one symptom of an engraftment arrhythmia.
• the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
• the methods described herein comprise administering an effective amount of amiodarone and ivabradine to a subject in order to alleviate at least one symptom of the engraftment arrhythmia and/or cardiovascular disease.
• "alleviating at least one symptom of the cardiovascular disease” or “alleviating at least one symptom of an engraftment arrhythmia” is ameliorating any condition or symptom associated with the cardiovascular disease (e.g., fatigue, shortness of breath, syncope, chest pain) and includes, for example, reduction of the arrhythmia itself. As compared with an equivalent untreated control, such reduction is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
• the treatments can be administered on a less frequent basis. For example, administration daily for three weeks, administration can be reduced to every other day, every three days, weekly, or less frequently as warranted by the subject’s incidence of arrhythmia. Alternatively, dosing can remain as frequent but be reduced in amount(s).
• the subject is first diagnosed as having a cardiovascular disease or disorder prior to administering a cardiomyocyte graft as described herein. In some embodiments, the subject is first diagnosed as being at risk of developing a cardiac disease (e.g., myocardial injury) or disorder prior to administering the cells.
• a cardiac disease e.g., myocardial injury
• the ivabradine or an analog thereof is administered when there is tachycardia.
• Adult human resting heart rate is generally 60 to 100 beats per minute (bpm).
• Tachycardia is a resting heart rate greater than this.
• patients can typically manage with a rate of less than 150 bpms.
• ivabradine can be administered for tachycardia after grafting with a goal of maintaining resting heart rate between 100 and 150 bpms, preferably less than 140 bpm, less than 130bpm, less than 120 bpm, or less than 110 bpm
• all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
• this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary.
• the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.
• a method of treating or ameliorating an engraftment arrhythmia in a subject recipient of a cardiac graft of cardiomyocytes comprising administering to the subject an effective amount of amiodarone and an effective amount of ivabradine.
• in vitro-differentiated cardiomyocytes are differentiated from induced pluripotent stem (iPS) cells or from embryonic stem (ES) cells. 4. The method of any of paragraphs 1-3, wherein the cardiac graft of cardiomyocytes is derived from stem cells autologous to the subject.
• iPS induced pluripotent stem
• ES embryonic stem
• a method of cardiomyocyte transplant comprising: a) administering in wVro-diffcrentiated cardiomyocytes to cardiac tissue of a subject in need thereof; b) administering to the subject an amount of amiodarone and an amount of ivabradine effective to reduce engraftment arrhythmia in the subject.
• cardiovascular disease or the cardiac event is selected from the group consisting of: atherosclerotic heart disease, myocardial infarction, cardiomyopathy, cardiac arrhythmia, valvular stenosis, congenital heart disease, chronic heart failure, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.
• a composition comprising in vi tro -d i ffc re n t i ated cardiomyocytes, amiodarone and ivabradine.
• EXAMPLE 1 AMIODARONE/IVABRADINE THERAPY REDUCES GRAFT- ASSOCIATED ARRHYTHMIAS
• Cardiomyocyte replacement therapy is an area of active investigation, and can restore heart function after myocardial infarction.
• Human pluripotent stem cells hPSC
• hPSC-derived cardiomyocytes are electrophysiologically immature, and elicit cardiac arrhythmias in large animal models (ibid., Romagnuolo et al, 2019).
• graft-induced arrhythmias appear shortly after the hPSC-derived cardiomyocytes are transplanted, and persist transiently for 3-4 weeks, during which the recipient is at risk for sudden cardiac death and heart failure.
• the observed return to normal sinus rhythm is hypothesized to reflect in vivo maturation of the transplanted hPSC-derived cardiomyocytes and suggests complete electrical integration with the host myocardium.
• the treatment group (n 6), received a combination anti- arrhythmic drug therapy consisting of loading and maintenance dosing of amiodarone for target serum levels of 1.5 and 2.5 pg/ml and 5-15 mg twice a day of oral ivabradine.
• EXAMPLE 2 ANTI-ARRHYTHMIC DRUGS TESTED TO FOR THE TREATMENT OF ENGRAFTMENT ARRHYTHMIAS
• Yucatan miniature pigs weighing 30-40 kg were used to model myocardial infarction.
• animals were subjected to a 90-minute occlusion of the left anterior descending coronary artery using a percutaneous transluminal coronary angioplasty balloon.
• An implanted electrocardiography device allowed continuous remote monitoring of the heart for the duration of the study.
• the hPSC-derived cardiomyocytes were administered two weeks after infarction, and the pigs were immunosuppressed to prevent immune rejection of the transplanted cells. In total, 500 million cardiomyocytes were delivered via percutaneous transendocardial injections.
• the control groups did not receive any anti-arrhythmic drugs while the treated groups were administered an anti-arrhythmic indicated in TABLE 1.
• a number of anti -arrhythmic therapies were tested to determine their efficacy in treating and preventing engraftment arrhythmias. TABLE 1 summarizes these results (below).
• amiodarone When administered alone, amiodarone produced a moderate effect on reducing heart rate and provided cardioversion. Ivabradine was able to slow heart rate but had no major effect on reducing arrhythmia burden in the engraftment models. The additional anti-arrhythmics tested had moderate or no significant effects on heart rate or reducing arrhythmia burden in engraftment animals.
• EXAMPLE 3 PHARMACOLOGIC THERAPY FOR ENGRAFTMENT ARRHYTHMIA INDUCED BY TRANSPLANTATION OF HUMAN CARDIOMY OC YTES
• EAs Background Engraftment arrhythmias
• hPSC-CM human pluripotent stem cell-derived cardiomyocytes
• Methods hPSC-CM were transplanted into the infarcted porcine heart by surgical or percutaneous delivery to induce EA. Following a screen of antiarrhythmic agents, a prospective study was conducted to determine the effectiveness of amiodarone plus ivabradine in preventing cardiac death and suppressing EA.
• MI Myocardial infarction
• hPSCs Human pluripotent stem cells
• ESCs embryonic stem cells
• iPSCs induced pluripotent stem cells
• hPSC-derived cardiomyocytes hPSC-derived cardiomyocytes
• hPSC-CMs infarcted myocardium of small animals-mice, rats, guinea pigs-has shown stable engraftment (3-7).
• Remuscularization and functional benefit in infarcted non-human primates (NHP) following transplantation of human pluripotent stem cell hPSC-CM have been described (8-10).
• the human graft vascularizes and electromechanically couples with the host myocardium within one-month post-transplant and remains durable up to at least three months.
• EAs engraftment arrhythmias
• NHPs (8-10) and pigs (11) which are termed engraftment arrhythmias (EAs) herein.
• EAs are generally transient, occurring within a week of transplantation and generally resolving spontaneously after one month. Based on electrical mapping, overdrive pacing, and cardioversion studies, EAs appear to originate focally in the graft or peri-graft myocardium and function as automatons foci rather than reentrant pathways (9,11).
• the Laflamme group (11) reported that EA can be lethal in some pigs. For this reason, EA has emerged as the biggest impediment to clinical translation of human cardiomyocyte transplantation (12).
• the pig shows heightened sensitivity to EAs, and because it is a well-established model in cardiovascular research (13) and cell therapy (14), whose larger size permits use of percutaneous delivery catheters, approaches for mitigating the risk and sequellae of EA were tested in this large animal model.
• a panel of anti-arrhythmic agents were screened. Amiodarone and ivabradine emerged independently as the most promising agents for control of rhythm and rate, respectively.
• a second phase to test was performed to determine the effect of combined amiodarone and ivabradine treatment. Interestingly, this regimen reduced sudden cardiac death, as well as suppressed tachycardia and arrhythmia.
• suspension-adapted pluripotent aggregates were induced to differentiate in RPMI-1640 (Gibco), MCDB-131 (Gibco), or M199 (Gibco) supplemented with B-27 (Gibco) or serum albumin by timed use of small molecule GSK-3 inhibitors and Wnt/ -catenin signal pathway inhibitors (Tocris).
• RUES2 hESC-CMs were heat-shocked to enhance their survival after harvest, cryopreservation, thaw, and transplantation.
• Cardiomyocyte aggregates were dissociated by treatment with Liberase TH (Fisher) and TrypLE (Gibco) and were cryopreserved in CryoStor CS10 (Stem Cell Technologies) supplemented with 10 mM Y-27632 (Stem Cell Technologies) using a controlled-rate liquid nitrogen freezer. Approximately 3 h before transplantation, cryopreserved hESC-CM were removed from cryogenic storage (-150 ° C to -196 ° C) and thawed in a 37 °C water bath (2 min ⁇ 30 s).
• RPMI-1640 supplemented with B-27 and >200 Kunitz Units/mL DNase I (Millipore) was added to the cell suspension to dilute the cryopreservation media. Subsequent wash steps were done using RPMI- 1640 basal media in progressively smaller volumes in order to concentrate the cell suspension. For the last centrifugation step, the cell pellet was resuspended in a sufficient volume of RPMI-1640 to achieve a target cell density for injection of ⁇ 0.3xl0 9 cells/mL in 1.6mL. In one case a larger dose of cells was resuspended in RPMI supplemented with serum albumin to a density of 0.43xl0 9 cells/mL in approximately 2.3mL. The final volume of the cell suspension was determined by the results of a count sampled before the final centrifugation step. Cell counts were performed as described previously to achieve a final total dose of 500xl0 6 live cells (9).
• the objective of this study was to identify a pharmacological regimen to attenuate arrhythmias following cardiac remuscularization therapy.
• This study was designed in two phases: the first, to observe the natural history of EA and screen various antiarrhythmic agents for efficacy and the second, test for efficacy (FIG. 16). All subjects were 30-40 kg castrated male Yucatan minipigs between 6-13 months of age (S&S Farms).
• Phase 1 nine subjects underwent cardiac remuscularization therapy with 500xl0 6 hESC-CM delivered by direct surgical transepicardial injections or later, by percutaneous transendocardial injections.
• Phase 2 The first four subjects (one non-infarcted and three infarcted) were followed to leam the natural history of EA and establish clinical endpoints and parameters for the Phase 2 drug trial.
• the subsequent five subjects underwent systematic dosing with antiarrhythmic agents with continuous electrocardiography (ECG) monitoring to determine effect on rhythm and rate (Table 2).
• ECG electrocardiography
• VF ventricular fibrillation
• tachycardia-induced heart failure requiring euthanasia.
• VF ventricular fibrillation
• VF tachycardia-induced heart failure requiring euthanasia.
• VF was associated with frequent non-sustained episodes of unstable EA >350 beats per minute (bpm) and heart failure with chronically elevated heart rate >150 bpm, which can be tachycardia-induced.
• Phase 2 began as a prospective pilot drug trial to prevent EA-related mortality.
• Sotalol (III) 1 mg/kg PO, 2 mg/kg PO, 4 mg/kg PO No response on HR or EA burden Metoprolol 5 mg IV, 25 mg PO BID, 50 mg PO Moderate HR effect (IV only), no (PiAR) BID, 75 mg PO BID response on EA burden
• Ivabradine (I f ) 2.5 mg PO, 5 mg PO BID, 10 mg PO
• iAR b ⁇ -adrenergic receptor
• BID twice daily
• HR heart rate
• EA engraftment arrhythmia
• I f funny current
• PO oral
• RyRi ryanodine receptor 1
• VT ventricular tachycardia
• Phase 2 a two-drug antiarrhythmic study was conducted with amiodarone and ivabradine and enrolled an additional 15 subjects (seven treatments, six untreated and two sham transplant) who underwent MI and percutaneous hESC-CM remuscularization at two weeks post-MI. Two additional subjects underwent MI with sham vehicle injection to serve as sham transplant controls (FIG. 16, FIG. 17).
• the primary endpoint was prespecified as combined cardiac death (either spontaneous death from arrhythmia or heart failure, or clinically directed euthanasia necessitated by tachycardia >350 bpm or signs of heart failure).
• Prespecified secondary endpoints were suppression of tachycardia, percent time in arrhythmia (arrhythmia burden) and resolution of arrhythmia, termed electrical maturation and defined as arrhythmia burden ⁇ 25% for 48 consecutive hours.
• Antiarrhythmic therapy was discontinued after electrical maturation or post-transplantation day 30, whichever was earlier.
• treatment was titrated to maintain target heart rate and arrhythmia burden at ⁇ 150 bpm and ⁇ 25%, respectively. Based on early experience that tachycardia >350 bpm often degenerated to VF, subjects were euthanized humanely if heart rates >350 bpm were reached.
• Continuous telemetric ECG was monitored for eight weeks total (two weeks post-MI and six weeks’ post-transplantation).
• subjects 1 and 2 (no treatment) and 3 and 4 (treatment) received H7 hESC-CM and subjects 1, 2 and 3 were transplanted surgically prior to adopting percutaneous delivery.
• Subject 5 was euthanized on day 37 as a prespecified endpoint following electrical maturation, prior to extending the study duration to 6 weeks’ post-transplantation for extended treatment washout and monitoring.
• Porcine myocardial infarction model [00195] Percutaneous ischemia/reperfusion injury was induced as previously described in NHP (9) with modification for the porcine model. A 5-8 cm incision was made in the femoral triangle and the femoral artery was exposed by blunt dissection. Prior to obtaining vascular access, heparin was administered to achieve therapeutic anticoagulation (activated coagulation time >250 sec). A 5 -French guidewire/introducer sheath system (Terumo Medical) was placed into the femoral artery and secured. Continuous ECG, invasive arterial blood pressure, pulse-oximetry and capnography were monitored throughout the procedure.
• Intravenous amiodarone 150 mg and lidocaine 100 mg were administered as single boluses prior to ischemia to minimize the risk of arrhythmia.
• fluoroscopic guidance OEC 9800 Plus, GE Medical Systems
• a 5-French Judkins right 2 or hockey stick guide catheter (Boston Scientific) was advanced into the ascending aorta to selectively engage the ostium of the left main coronary artery.
• Coronary angiography was performed using hand injections of contrast (Visipaque) and a 0.035” coronary guidewire (Runthrough NS Extra Floppy, Terumo Medical) was placed into the distal left anterior descending coronary artery (LAD).
• angioplasty balloon catheter was then positioned into the mid-LAD distal to the first diagonal branch artery and inflated to the minimum pressure required for total obstruction of distal perfusion as confirmed by angiography. Ischemia was confirmed by ST-segment elevation on the ECG. Animals were maintained under anesthesia with ventilatory and hemodynamic support for 90 minutes, after which the balloon was deflated to restore distal perfusion, again confirmed by fluoroscopy and ECG. The animal was observed for reperfiision arrhythmias and externally cardioverted if ventricular fibrillation occurred. Prior to recovery, all subjects received implantable telemetry units and central venous catheter placement.
• the external jugular vein in the jugular furrow was exposed and a 5-French central venous catheter (Access Technologies) was inserted and tunneled out to the dorsal prescapular area.
• the telemetry transmitter (EMKA easyTEL+) was implanted in a subcutaneous pocket using the same incision in the jugular furrow, and subcutaneous leads were tunneled to capture the cardiac apex to base.
• the overall procedural mortality including the infarct was ⁇ 10%.
• the cyclosporine A trough level was decreased to >300 ng/ml and the methylprednisolone reduced to 1.0 mg/kg for subjects 7-19 without histologic evidence of rejection.
• Prophylactic oral cephalexin was administered for all subjects to prevent infection of the indwelling central venous catheter.
• Prophylactic sulfamethoxazole/trimethoprim was added after subject 3 developed Pneumocystis pneumonia.
• Prophylactic valganciclovir and probiotics were added after activation of endogenous porcine cytomegalovirus was found in subject 6.
• Treatment subjects were loaded with oral amiodarone 1000-1200 mg orally twice daily starting seven days prior to cell transplantation followed by maintenance dose of 400-1000 mg orally twice daily to maintain a steady-state plasma level of 1.5-4.0 pg/ml (FIG. 18).
• Ivabradine was started at 2.5 mg orally twice daily when sustained tachycardia reached >150 bpm and titrated every 3 days up to 15 mg twice daily for goal heart rate ⁇ 125 bpm. All but one subject (subject 1) required adjunctive ivabradine for additional heart rate control.
• Antiarrhythmics were discontinued after electrical maturation was achieved or post-transplantation day 30, whichever was earlier, to allow for treatment washout and assess for recrudescence of arrhythmia. All subjects tolerated the antiarrhythmic regimen without complication. Untreated and sham transplant control subjects did not receive antiarrhythmic agents following the MI procedure, but otherwise received all immunosuppression and standard care.
• Amiodarone drug monitoring were discontinued after electrical maturation was achieved
• a novel liquid chromatography-mass spectrometry assay was established for amiodarone to monitor steady state serum levels in the porcine model and guide oral dosing to ensure efficacy and avoid dose-related toxicity.
• a target serum level of 1.5-4.0 pg/ml was extrapolated from prior human pharmacokinetic studies (16,17). Elimination kinetics after discontinuation of oral amiodarone therapy were also studied by obtaining weekly trough concentrations in 6 pigs (subjects 6, 7, 8, 13, 14, 16) (FIG. 18)
• ECG Telemetric ECG was continuously monitored in real-time from the time of myocardial infarction to detect the primary endpoint of cardiac death or unstable EA.
• Automated quantification of heart rate and arrhythmia burden was performed offline by a board-certified cardiologist using the ecgAUTO 3.3.5.10 software package (EMKA Technologies).
• Arrhythmia was defined as an ectopic beat (e.g. premature ventricular contraction) or rhythm (e.g. idioventricular rhythm, ventricular tachycardia).
• EA was typically observed as sustained and non-sustained ventricular tachyarrhythmia of varying rates and morphologies but also included slow and narrow complex ectopic rhythms (FIG. 19).
• Heart rate and arrhythmia burden were quantified for two continuous minutes every five minutes (40% of total rhythm was counted) and presented as daily averages.
• infarct regions were identified by picrosirius red staining; human graft was identified by anti-human cardiac troponin T, stained using avidin-biotin reaction (ABC Kit, VectorLabs) followed by chromogenic detection via diaminobenzidine (Sigmafast, Sigma Life Science) (FIG. 20).
• the slides were digitized using a whole slide scanner (Nanozoomer, Hamamatsu), and the images were viewed and exported with NDP.view 2.6.13 (Hamamatsu). Areas of infarct and graft were analyzed using a custom-written algorithm in the Image J open source software platform (18).
• hESC-CM successfully targeted the peri-infarct border zone and central ischemic regions as intended and resulted in discrete hPSC-CM grafts transplanted into host myocardium as previously reported (8-11). All grafts were located in the anterior, antero-septal and antero-lateral walls and appeared structurally immature at early time points before 2 weeks’ post-transplantation with increasing maturity up to the end of study as previously reported in pig (11).
• FIG. 16 A flow chart for all animals in the study is shown in FIG. 16. No significant arrhythmias were noted in the two sham transplant subjects (9 and 10) that underwent myocardial infarction and percutaneous intracardiac injection of vehicle. All subjects that received human cardiomyocyte grafts developed EA between 2-6 days following cell transplantation. Initiation of EA was characterized by salvos of non-sustained VT, and this typically progressed to periods of sustained VT with rates ranging from 110 to 250 bpm (FIG. 19). The VT was often polymorphic, with the same animal showing different electrical axes and both wide- and narrow-complex tachycardia at different times.
• EA was either fatal or necessitated euthanasia due to a prespecified endpoint of unstable tachycardia (defined as sustained heart rate >350 bpm).
• acute heart failure was noted clinically shortly after initiation of EA at a rate of 300 bpm and based on recommendations from veterinary staff, the subject was euthanized. Signs of heart failure were subsequently confirmed on necropsy.
• EA was noted with a rapid acceleration to >350 bpm (subjects 11 and 12) and, in two cases, deterioration to VF prior to euthanasia (subjects 1 and 2) (Table 3).
• bpm beats per minute
• EA engraftment arrhythmia
• hESC-CM human embryonic stem cell-derived cardiomyocytes
• HF heart failure
• HR heart rate
• Ml myocardial infarction
• surg surgery
• PCP pneumocystis pneumonia
• pCMV porcine cytomegalovirus
• perc percutaneous
• VF ventricular fibrillation
• lidocaine (lb, sodium channel inhibitor), flecainide (Ic, sodium channel inhibitor), propafenone (Ic, sodium channel inhibitor), amiodarone (III, potassium channel inhibitor), sotalol (III, potassium channel inhibitor) and metoprolol (b ⁇ -adrenergic receptor inhibitor) for effect on EA heart rate and rhythm.
• ivabradine the funny current/HCN4 channel antagonist, ivabradine, was tested (Tables 1 and 2). This series was not meant to be definitive but rather to rapidly identify candidate agents.
• two of the treatment subjects (3 and 6) experienced non-cardiac deaths at post-transplant days 19 and 26 due to immunosuppression-related complications (Pneumocystis pneumonia and porcine cytomegalovirus, respectively).
• FIGs. 23A-23B and 23C-23D Pooled and individual subject-level data of heart rate and arrythmia burden are provided in FIGs. 23A-23B and 23C-23D, respectively.
• the average heart rate was significantly lower with antiarrhythmic treatment compared to no treatment.
• FIG. 24 Microscopy of hESC-CM graft in porcine myocardium demonstrates interaction with the diffuse Purkinje conduction system of the host porcine heart (FIG. 24). Consistent with previous reports (20,21), a mesh-like network of intramural Purkinje fibers (PFs) throughout the left ventricle (FIG. 24A; video data not shown confirmed the mesh-like network throughout the native porcine myocardium) which localize with hESC-CM grafts in close proximity were observed (FIG. 24B; video data not shown confirmed that hPSC-cardiomyocytes, marked by slow skeletal troponin I (ssTnl), interact with connexin 40+ purkinje fibers). Connexin 40 (Cx40) specifically stains Purkinje cell gap junctions (20,22) where lower sarcomere content and lack of T-Tubules are observed as expected (FIG. 25).
• PFs intramural Purkinje fibers
• Intramyocardial transplantation of hPSC-CM is a promising strategy to remuscularize the infarcted heart and restore function (2).
• Such a therapy to prevent and treat heart failure would be a seminal advance in addressing a large unmet need.
• Studies in large animals have demonstrated long term efficacy but also defined a significant safety signal of transient but potentially fatal arrhythmias.
• EA is a predictable complication of cardiac remuscularization therapy for myocardial infarction (23).
• EA typically presents as a wide-complex tachycardia with a variable electrical axis (8,9), and this was reproduced in the pig recently by the Laflamme laboratory (11).
• EA is described herein as polymorphic due to the observed changes in electrical axis as ectopy originating from different graft foci.
• a narrow-complex VT was observed that alternates with wide-complex tachycardia, a pattern not seen in the NHP.
• histology of native and grafted porcine myocardium support the hypothesis that that the wide-complex beats originate from grafts contacting the working-type myocardium with slow conduction, and that the narrow complex beats originate when grafts contact the intramural Purkinje fibers that diffusely permeate the porcine heart (20,21).
• the mechanism of benefit for the antiarrhythmic treatment may be related to suppression of automaticity, reducing both heart rate and arrhythmia burden.
• the drugs were particularly beneficial during the early phase of EA, which carries the greatest risk of deterioration to VF.
• heart rates rapidly accelerate to >350 bpm, and the possibility that this escalation can be a distinct mechanism, e.g. automaticity leading to reentry, was not excluded.
• amiodarone Although classified principally as a K + channel blocker, amiodarone is well-known also to antagonize Na + channels, Ca 2+ channels, and b-adrenergic receptors (27). Thus, while in no way diminishing the importance the discovery of the effective drug combination disclosed herein, it is difficult to gain insights into the mechanism of EA from amiodarone ’s efficacy.
• the disappearance of EA coincides with maturation of the stem cell-derived graft (8,9,28) and it is contemplated that the window of arrhythmogenicity can reflect a period of in vivo graft maturation prior to reaching a state more similar to host myocardium (26,29-33).
• Additional strategies such as promoting maturation prior to transplanting, gene editing, and modulating host/cell interaction can provide additional means of arrhythmia control. Further investigation of the mechanism underlying EA would be accelerated by the development of higher throughput platforms to perform genetic, pharmacological and electrophysiological studies before phenotyping in large animal models.
• Engraftment arrhythmia is the most significant barrier to clinical translation of cardiac remuscularization therapy.
• the natural history of EA emerging from the NHP and more recent porcine data suggests that, once EA resolves, there is low risk for further arrhythmia.
• This study provides a proof-of-concept that clinically relevant antiarrhythmic drug treatment can successfully suppress fatal arrhythmias and control tachycardia to achieve electrical quiescence. This is an important step forward in regard to the clinical safety of cardiomyocyte transplantation for cardiac remuscularization therapy.
• This study demonstrates that EA is responsive to pharmacologic suppression. Clinically relevant doses of amiodarone and ivabradine were administered in the study.
• infarcted pigs underwent hESC-CM transplantation with all exhibiting stereotypic EA. Subjects were administered multiple trials antiarrhythmics and observed for acute response by continuous ECG monitoring. Intravenous agents were delivered as a bolus dose over 2 minutes. Oral agents were administrated by direct observation in a minimum of apples, apple sauce or pumpkin puree with daily feeding and dosed daily for dose escalation. A washout period of at least three days was provided between agents. Amiodarone was administered as the last agent fortesting given concern for prolonged half-life and elimination kinetics. All agents were tested in at least two subjects.
• tissue was cut and trimmed to 1 cm X 1 cm X 3 mm, snap frozen in isopentane, and embedded in OCT (TissueTek). 10 pm sections were immersed in 100% methanol at - 20°C for 15 minutes and stained with standard immunofluorescence technique using stains described below. Images were acquired on a Leica SP8 confocal microscope.
• Sections were stained the following reagents: Hoechst 33342 (DNA, Thermo Fisher Scientific, #62249), Wheat germ agglutinin-Oregon Green (WGA, Thermo Fisher Scientific, #W6748), Phalloidin-647 (F-Actin, Thermo Fisher Scientific, #A22287), anti-Connexin 40 (Cx40, Alpha Diagnostics, #CXN40A), or anti-slow skeletal troponin I (ss-Tnl, Novus, #NBP2-46170) with one of two anti-rabbit secondary antibodies (Alexa Fluor 555/647, Thermo Fisher Scientific, #A-31570/A - 31573).
• References [00220] 1. Collaborators GBDCoD. Global, regional, and national age -sex-specific mortality for
• Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias. Stem Cell Reports 2019;12:967-981.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Biomedical Technology (AREA)
• Cell Biology (AREA)
• Epidemiology (AREA)
• Cardiology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Developmental Biology & Embryology (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Heart & Thoracic Surgery (AREA)
• Virology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Rheumatology (AREA)
• General Engineering & Computer Science (AREA)
• Vascular Medicine (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Reproductive Health (AREA)
• Gynecology & Obstetrics (AREA)
• Nutrition Science (AREA)
• Physiology (AREA)
• Dermatology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (1)

Family

ID=77291628

Family Applications (1)

Country Status (11)

Cited By (3)

Families Citing this family (2)

Citations (3)

Family Cites Families (2)

• 2021
  • 2021-02-09 US US17/798,127 patent/US20230077983A1/en active Pending
  • 2021-02-09 BR BR112022015768A patent/BR112022015768A2/pt not_active Application Discontinuation
  • 2021-02-09 CN CN202180027573.1A patent/CN115484966A/zh active Pending
  • 2021-02-09 CA CA3166678A patent/CA3166678A1/en active Pending
  • 2021-02-09 MX MX2022009807A patent/MX2022009807A/es unknown
  • 2021-02-09 JP JP2022548412A patent/JP2023512843A/ja active Pending
  • 2021-02-09 IL IL295236A patent/IL295236A/en unknown
  • 2021-02-09 KR KR1020227029054A patent/KR20220140532A/ko not_active Withdrawn
  • 2021-02-09 EP EP21753964.2A patent/EP4103206A4/en active Pending
  • 2021-02-09 AU AU2021218653A patent/AU2021218653A1/en not_active Abandoned
  • 2021-02-09 WO PCT/US2021/017220 patent/WO2021163037A1/en not_active Ceased

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events