Classifications

• • 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
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • 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
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
  • A61J1/14—Details; Accessories therefor
  • A61J1/1406—Septums, pierceable membranes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
  • A61J1/14—Details; Accessories therefor
  • A61J1/1412—Containers with closing means, e.g. caps
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
  • A61J1/14—Details; Accessories therefor
  • A61J1/1468—Containers characterised by specific material properties
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
  • A61J1/14—Details; Accessories therefor
  • A61J1/20—Arrangements for transferring or mixing fluids, e.g. from vial to syringe
• • 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
  • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • 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
• • 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
  • C12N5/0662—Stem cells
• • 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
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)

Definitions

• the present invention relates to the treatment of heart disease disorders or predispositions of a disorder through delivery of a pharmaceutical composition to an individual in need.
• a pharmaceutical composition comprising cells committed to the generation of heart tissue and at least one pharmaceutically acceptable excipient, contained in a container in a way to allow cell survival and transportation to locations worldwide, convenient handling by staff for delivery to the recipient, wherein said cells are produced according to internationally recognized standards for pharmaceutical product manufacture.
• Regenerative cell therapies are particularly relevant for diseases in which organs are compromised in such a way that tissue reconstruction is required, e.g. to restore the morphology as well as the function of a diseased organ, or when physiological repair mechanisms are impaired.
• the heart is a terminally differentiated organ and massive loss of cardiomyocytes such as in heart attack causes irreversible damages, hence the need for repair.
• heart disease is a leading cause of mortality worldwide.
• Cell therapy for heart repair is a challenge of utmost importance.
• mESCs mouse embryonic stem cells
• the cardiopoietic cell is an intermediate cell phenotype where the cell is committed to the generation of heart tissue but not yet fully differentiated. Key milestones in basic research in the field of cardiac regeneration are disclosed in:
• the present invention solves this problem by disclosing a method for industrially producing a pharmaceutical composition containing cells committed to the generation of heart tissue and at least one pharmaceutically acceptable excipient according to internationally recognized standards for pharmaceutical product manufacture, a method for cell preservation and packaging that allows differed use whilst maintaining the features of cells committed to the generation of heart tissue, their survival, transportation to locations worldwide, convenient handling by staff for delivery to the recipient.
• 'BMMSC designates bone marrow mesenchymal stem cells. 'hBMMSC designates such BMMSCs of human origin.
• 'Cardiopoietic cells' are an intermediate cell phenotype, i.e. committed to the generation of heart tissue but not yet fully differentiated. Cardiopoietic cells are characterized by nuclear translocation of Nkx2.5 and MEF2C, combined to the absence of sarcomeric proteins (Behfar et al. 'Derivation of a cardiopoietic population from human mesenchymal stem yields progeny', Nature Clin. Pract., Cardiovasc. Med. (2006) 3: S78-S82).
• Cardiopoietic cells retain a proliferative capacity.
• Cardiopoietic cells can be derived from stems cells including for example, human adult mesenchymal stem cells, human embryonic stem cells (provided their production implies no human embryo destruction), embryonic-like stem cells, inducible pluripotent stem cells, or any other adapted source.
• a 'cardiogenic cocktail' or 'cocktail' designates a composition containing at least two cardiogenic substances.
• a 'cardiogenic substance' is a substance which, in contact with a cell, improves the ability of said cell to differentiate into a cardiopoietic cell.
• the 'effective amount' means a sufficient amount of the pharmaceutical composition to provide the desired therapeutic or physiological effect or outcome. Such an effect or outcome includes the repair, maintenance, regeneration, augmentation of heart tissue or improvement of heart function. Undesirable effects are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what an appropriate 'effective amount' is. This amount may vary from subject to subject, depending for example on the subject age, general condition, genetic and epigenetic variability and the like, and the mode of administration. Thus, it may not be possible to specify an exact 'effective amount'.
• an appropriate 'effective amount' in any individual case may be determined by one of ordinary skill in the art before or during the administration procedure of the pharmaceutical composition, for example by delivering the highest amount possible without undesirable effect during delivery of the pharmaceutical composition.
• 'Excipient' is an inactive substance used as a carrier for the active ingredients of a medication.
• an "active" substance may not be easily administered and absorbed by the human body; in such cases the substance in question may be dissolved into or mixed with an excipient.
• excipients can be used in the manufacturing process to aid in the handling of the active substance concerned.
• different excipients may be used.
• excipients are added, ensuring that the active ingredient stays "active", and, just as importantly, stable for a sufficiently long period of time that the shelf-life of the product makes it competitive with other products.
• the terms 'subject', 'recipient' and 'patient' are used interchangeably herein and refer unless explicitly stated to any human or mammal in need of treatment for a cardiac disease or disorder with the hereby disclosed pharmaceutical composition. Subjects also include those at risk of having such a cardiac disease or disorder.
• 'a stem cell' includes a single cell, as well as two or more cells
• reference to 'an agent' or 'a reagent' includes a single agent or reagent, as well as two or more agents or reagents
• reference to 'the invention' or 'an invention' includes single or multiple aspects of an invention
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising cells committed to the generation of heart tissue and at least one pharmaceutically acceptable excipient.
• Cells committed to the generation of heart tissue are preferably produced according to internationally recognized standards for pharmaceutical product manufacture.
• said pharmaceutically acceptable excipient is a preservation solution.
• the preservation solution is chosen in the group comprising preservation solutions which allow cryopreservation at temperatures between -196 ⁇ € and 0 ⁇ € and preservation solutions which allow preservation at temperatures between O'O and +40 ⁇ .
• the preservation solution is a preservation solution which may contain ions, pH buffers, impermeants, colloids and metabolites, dimethylsulfoxide (DMSO), glycerol, sucrose, serum albumin, trehalose, or any combination thereof.
• ions are selected from the group consisting of Na + , K + , Ca 2+ , Mg 2+ , Cl " and combinations of said ions.
• pH buffers are selected from the group consisting in H 2 PO 4 " , HCO 3 " , (4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid) (HEPES) and mixtures thereof.
• impermeants are selected from the group consisting of lactobionate, sucrose, mannitol, glucose and combinations thereof.
• the colloid is Dextran-40.
• the metabolites are selected from the group consisting of adenosine, glutathione, and combinations thereof.
• said at least one pharmaceutically acceptable excipient further comprises at least one component selected from the group consisting of growth factors, cytokines, proteins involved in organogenesis signalling, pharmaceuticals, platelet lysate, serum, isotopes, means for tracing cells in vivo, diluents, lubricants, matrix or scaffold materials, and combinations thereof.
• cells committed to the generation of heart tissue may be stem cells or cells committed to the generation of heart tissue derived from stem cells.
• stem cells are selected in the group consisting of adult stem cells, embryonic stem cells, induced pluripotent stem cells (IPS), marrow-isolated adult multilineage inducible cells (MIAMI), resident cardiac stem cells, vegetal stem cells, or any combination thereof.
• stem cells are mesenchymal stem cells harvested from a suitable tissue source selected in the group consisting of bone marrow, adipose tissue, umbilical cord blood, amniotic fluid, menstrual fluid, blood.
• cells committed to the generation of heart tissue are mammalian cells.
• mammalian cells are selected in the group consisting of humans, cats, dogs, pigs, horses, mice, rats, hamsters and other mammals.
• said cells are autologous cells, allogeneic cells, xenogenic cells, or any combination thereof.
• cardiopoietic cells are cardiopoietic cells.
• cardiopoietic cells can be derived from stems cells including for example, from human adult mesenchymal stem cells, human embryonic stem cells (provided their production implies no human embryo destruction), embryonic-like stem cells, inducible pluripotent stem cells, cardiac resident stem cells or any other adapted source or combination thereof.
• said composition contains no detectable non-cardiopoietic cells, including but not limited to cardiomyocytes, hematopoietic cells, endothelial progenitor cells, adipoblasts, adipocytes, chondroblasts, chondrocytes, osteoblasts osteocytes, neuroblasts and neurocytes.
• the contents of total non-cardiopoietic cells is between 0% and 50% of the total number of cells, preferably between 0% and 15% of the total number of cells.
• the contents of each category of non- cardiopoietic cells is between 0% and 50% of the total number of cells, preferably between 0% and 15% of the total number of cells.
• the invention also relates to a pharmaceutical composition for use for the treatment of ischemic cardiomyopathy, acute myocardial infarction, chronic myocardial infarction, heart failure of non-ischemic origin, heart failure of ischemic origin, congenital cardiomyopathy, or combination thereof.
• the invention also relates to a process for the manufacture of a pharmaceutical composition which comprises the following steps: - obtaining cells from which cells committed to the generation of heart tissue can derive;
• the process of the invention is conducted according to internationally recognized standards for pharmaceutical product manufacture, which comprises taking samples at any step of the process for the purpose of performing quality control operations.
• the process of the invention is conducted according to internationally recognized standards for pharmaceutical product manufacture, which comprises taking samples at the last step of cell culture for the purpose of performing quality control of the active substance.
• quality control criteria of the active substance comprise at least one test selected from the group consisting of identity test, homogeneity test, purity test and combinations thereof.
• identity of said cardiopoietic cells corresponds to an observed increase in expression level of at least one gene in the group consisting of Nkx2.5, Tbx5, MEF2C, GATA4, GATA6, Mespi , FOG1 , FOG2, FIkI and homologues thereof.
• the increase of gene expression is a minimum 2-fold as determined by qPCR method, as compared to a reference.
• cardiopoietic cells when cells committed to the generation of heart tissue are cardiopoietic cells, identity of said cardiopoietic cells is considered fulfilled by the observed presence of at least one polypeptide species chosen from the group consisting of Nkx2.5, Tbx5, MEF2C, GAT A4, GAT A6, Mespi , FOG1 , FOG2, FIkI and homologues thereof, as compared to a reference, and either Nkx2.5 or MEF2C or both being further translocated to the nuclei of the cardiopoietic cells.
• polypeptide species chosen from the group consisting of Nkx2.5, Tbx5, MEF2C, GAT A4, GAT A6, Mespi , FOG1 , FOG2, FIkI and homologues thereof, as compared to a reference, and either Nkx2.5 or MEF2C or both being further translocated to the nuclei of the cardiopoietic cells.
• the observed presence is shown by immuno-labelling with at least one antibody in the group consisting of anti-Nkx2.5, anti-Tbx5, anti-MEF2C, anti-GATA4, anti-GATA6, anti-Mesp1 , anti-FOG1 , anti-FOG2, anti-Flk1 , and homologues thereof.
• homogeneity is fulfilled when at least one antibody in the group consisting of anti-Nkx2.5, anti-Tbx5, anti-MEF2C, anti-GATA4, anti-GATA6, anti-Mesp1 , anti-FOG1 , anti-FOG2, anti-Flk1 , and homologues thereof.
• homogeneity is fulfilled when at least
• cardiopoietic cells 50% in a given sample are cardiopoietic cells.
• homogeneity is fulfilled when at least 50%, most preferably at least 85%, in a given sample are cardiopoietic cells.
• presence of mesenchymal stem cells amongst harvested cells is shown by positive immuno-labelling with an antibody against a surface marker selected in the group consisting of CD105, CD90, CD133, CD105, CD166, CD29, and CD44, and lack of detectable immune-labelling with an antibody against a surface marker selected in the group consisting of CD14, CD34, and CD45.
• purity is not fulfilled with increased in expression level of the CD34, FABP4, osteocalcin, nestin, Sox9, and MYH7 genes and homologues thereof two-fold or greater when compared to a reference.
• the increase of gene expression is determined by qPCR method, as compared to a reference.
• purity is not fulfilled with increased number of non-cardiopoietic cells, including but not limited to cardiomyocytes, hematopoietic cells, endothelial progenitor cells, adipoblasts, adipocytes, chondroblasts, chondrocytes, osteoblasts osteocytes, neuroblasts and neurocytes, as shown by immuno-labelling.
• said reference consists of non-cardiopoietic cells.
• said reference consists of cells cultured in the absence of any cardiogenic substance.
• culturing conditions include using a bioreactor which comprises immobilising or encapsulating said cells on particles or on a matrix and passing cell culture media through the bed of particles or matrix.
• the invention also relates to a method for the treatment of heart disease disorders or predisposition of a disorder wherein the pharmaceutical is delivered into an individual in an effective amount.
• the individual shows an insufficiency of the cardiovascular system.
• the individual suffers from ischemic cardiomyopathy, myocardial infarction, heart failure of ischemic origin, or heart failure of non-ischemic origin, congenital cardiomyopathy, or combination thereof.
• the pharmaceutical composition is delivered using a route of administration selected in the group consisting of intra-myocardial, intra-cardiac, intra- coronary, intra-muscular, sub-cutaneous, intra-peritoneal, in utero, parenteral, or systemic.
• the pharmaceutical composition is injected intramyocardially using a catheter, a syringe, or combination thereof.
• Cells from which cells committed to the generation of heart tissue can derive may be put into contact with at least one cardiogenic substance, which may be selected from the group consisting of TGF- ⁇ 1 , TGF- ⁇ 2, TNF- ⁇ , BMP-1 , BMP-2, BMP- 4, BMP-6, FGF-2, FGF-4, FGF-5, FGF-12, FGF-13, FGF-15, FGF-20, leukaemia inhibitory factor (LIF), VEGF-A, VEGF-C, insulin-like growth factor 1 (IGF-1 ), interleukin 6 (IL-6), Activin A, ⁇ thrombin, retinoic acid, cardiotrophin 1 , cardiogenol C, and combinations thereof.
• at least one cardiogenic substance which may be selected from the group consisting of TGF- ⁇ 1 , TGF- ⁇ 2, TNF- ⁇ , BMP-1 , BMP-2, BMP- 4, BMP-6, FGF-2, FGF-4, FGF-5, FGF-12, FGF-13, FGF-15,
• a great number of cardiogenic cocktails may be used. The list given below is not limitative.
• One may use for instance a cocktail of cardiogenic substances that comprises TGF ⁇ -1, BMP4, ⁇ -thrombin, a compound selected from the group consisting of Cardiotrophin and IL-6, and a compound selected from the group consisting of Cardiogenol C and retinoic acid.
• Another cocktail may comprise TGF ⁇ -1 , BMP4, ⁇ -thrombin, Cardiotrophin and Cardiogenol C.
• Still another cocktail may comprise at least one compound selected from the group consisting of FGF-2, IGF-1 , Activin-A, TNF- ⁇ , FGF-4, LIF, VEGF-A and combinations thereof.
• FGF-2 may also comprise FGF-2, IGF-1 and Activin-A.
• Other preferred cocktails comprise Activin-A, FGF-2, IL-6, IGF-1 and retinoic acid.
• Other cocktails can lack at least one compound chosen in the group consisting of TNF- ⁇ , FGF-4, LIF, and VEGF-A.
• one of the following compounds when one of the following compounds is present in a cocktail, it may be present in an amount of between 1 and 5 ng of said TGF ⁇ -1 per ml, between 1 and 10 ng of said BMP4 per ml, between 0.5 and 5 ng of said Cardiotrophin per ml, between 0.5 and 5 units of said ⁇ -thrombin per ml, and between 50 and 500 nM of said Cardiogenol C, between 1 and 10 ng of said FGF-2 per ml, between 10 and 100 ng of said IGF-1 per ml, between 1 and 50 ng of said Activin-A per ml, between 1 and 50 ng of said TNF- ⁇ per ml, between 1 and 20 ng of said FGF-4 per ml, between 10 and 100 ng of said IL-6 per ml, between 1 and 10 units of said LIF per ml, between 1 and 50 ng of said VEGF-A per ml, between 0.1 and 1.0 ⁇ M of said
• a particular cocktail type comprises recombinant TGF ⁇ -1(2.5 ng/ml), BMP4 (5 ng/ml), Cardiotrophin (1 ng/ml), Cardiogenol C (100 ⁇ M nM), used in a combinatorial fashion.
• Particularly preferred cocktails comprises such compounds and further comprise ⁇ -thrombin, (1 U/ml), FGF-2 (10 ng/ml), IGF-1 (50 ng/ml) and Activin- A (5 ng/ml).
• TGF ⁇ -1 (2.5 ng/ml), BMP4 (5 ng/ml), Activin-A (5 ng/ml), FGF-2 (10 ng/ml), IL-6 (100 ng/ml), Factor-lla (h ⁇ - thrombin, 1 U/ml), IGF-1 (50 ng/ml), and retinoic acid (1 ⁇ M) used in a combinatorial fashion.
• the cocktail may be diluted in a medium containing compounds selected from the group consisting of foetal calf serum, human serum, platelet lysate, platelet- derived growth factor, and mixtures thereof and compounds selected.
• the present invention also relates to a kit for the administration of a pharmaceutical composition which comprises a container containing said pharmaceutical composition.
• a container containing said pharmaceutical composition.
• said container is a biocompatible container that allows cell survival and transportation worldwide, convenient handling by staff for delivery to the 5 recipient.
• said container is hermetically closed.
• said container is compatible with the excipient and conditions of preservation.
• said container is a closed glass container.
• said container has a piercable septum cap.
• said piercable septum cap allows a vial adapter that includes a luer activated valve to draw fluid from the container.
• said piercable septum cap O can be accessed with a needle.
• said pharmaceutical composition is stored in a hermetically closed container suitable for cryopreservation.
• shelf-life of pharmaceutical composition in said container is at least 48 hours, preferably 72 hours.
• said kit further comprises at least one catheter.
• said kit further comprises at least one syringe. 5 Brief Description of the Drawing
• LVEDV Left Ventricular
• LVESV LV End Systolic Volume
• LVEF LV Ejection Fraction
• Adherent BMMSCs are washed with phosphate buffer saline (PBS), culture medium is added, and culture is resumed until initial passage PO with change of medium every four to six days.
• PO phosphate buffer saline
• An initial passage (PO) is performed to dissociate colonies and to allow them to expand and form a monolayer. Cells are seeded at a one-to-one ratio in 175-cm 2 flasks and allowed to expand and form a monolayer for up to 6 days. Confluence determines timing for the next passage. This step may be skipped if BMMSCs spontaneously form a monolayer with no detectable colonies.
• P1 - Start of cardiogenic cocktail treatment Cells are cultured for 5 days in culture medium and a cardiogenic cocktail.
• cardiogenic cocktails such as described in WO2006/015127, WO2009/151907 and Behfar et al. 'Derivation of a cardiopoietic population from human mesenchymal stem yields progeny, Nature Clinical Practice, Cardiovascular Medicine, March 2006 vol. 3 supplement 1 , pages S78-S82) may be used.
• P2 - End of cardiogenic cocktail treatment The medium containing the cardiogenic cocktail is discarded.
• the culture now contains cardiopoietic cells.
• the culture is passaged and seeded in new containers with culture medium to allow for additional expansion phases as needed.
• P3 - Expansion and harvest The following passages are numbered 'P3' followed by a sequential letter. Cells are passaged when optimal confluence is reached, and repeated until the number of cells obtained is between 600x10 6 and 1200x10 6 cells. When this criterion is met, the cells are harvested. This step involves a final trypsinization followed by washing and concentration steps by centrifugation. The final wash is performed in a cell preservation solution.
• the preservation solution employed may be similar to standard organ and biological tissue preservation aqueous cold storage solutions such as HypoThermosol-FRS ® from BioLifeSolutions (Bothell, Wash).
• the cell concentrate is then transferred into a biocompatible container (in this example glass bottle of Type I, Ph. Eur.) and preservation solution added to reach a total volume of 10 ml and a cellular concentration in the range of 60x10 6 to 120x10 6 cells/ml. This finalizes the manufacture of the pharmaceutical composition.
• a biocompatible container in this example glass bottle of Type I, Ph. Eur.
• preservation solution added to reach a total volume of 10 ml and a cellular concentration in the range of 60x10 6 to 120x10 6 cells/ml. This finalizes the manufacture of the pharmaceutical composition.
• composition release criteria typically include cellular identity, homogeneity and purity that are combined with manufacture parameters that confirm the absence of adventitious contamination (asepsis, low level of endotoxin, and mycoplasma not added by the process).
• the preservation medium is HypoThermosol-FRS ® from BioLifeSolutions (Bothell, Wash).
• HypoThermosol-FRS contains ions (10O mM Na+, 42.5 mM K + , 0.05 mM Ca2+, 5 mM Mg2+, 17.1 mM Cl-); pH buffers (10 mM H2PO4-, 5 mM HCO3-, 25 mM HEPES); impermeants to counteract cell swelling (10O mM lactobionate, 20 mM sucrose, 20 mM mannitol); colloid (6% Dextran-40); and metabolites (5 mM glucose, 2 mM adenosine, 3 mM glutathione).
• DMSO dimethylsulfoxide
• the shelf-life of the pharmaceutical product a container is at least 72 hours. It is particularly remarkable to observe that a total number of 1200x10 6 cells have a volume of about 8 millilitres, whereas the maximum desirable volume for intramyocardial injection is about 10 millilitres. This means that the quantity of HypoThermosol-FRS is small vis-a-vis the cellular volume and will be in the range of only a few millilitres. [0054] It has surprisingly been observed that even such a small quantity of HypoThermosol-FRS is sufficient to maintain such an important shelf-life of 72 hours for the pharmaceutical composition. This provides for sufficient time to confirm all release criteria are met, to ship the pharmaceutical composition anywhere in the world, and to deliver it to the recipient.
• Cardiopoietic cells are characterized by positive expression and, when applicable, nuclear translocation of several markers of early cardiac differentiation, including Nkx2.5, MEF2C and GATA- 4. Positive identity of cardiopoietic cells contained in the pharmaceutical composition is represented by a minimum 2-fold increase in expression level for MEF2C and/or Tbx5 compared to the reference standard, measured by real-time quantitative RT- PCR (qPCR) and maintenance thereof during shelf-life.
• qPCR real-time quantitative RT- PCR
• Table 1 , Table 2 and Table 3 below show the expression of early cardiac differentiation markers is maintained for as long as 14 days after the pharmaceutical composition is stored in its final container, and that viability and proliferation of such cells is maintained for at least 5 days. This demonstrates the unique capacity of the manufacturing process hereby described to obtain and maintain the identity of the cells exposed to the cardiogenic substances in a state suitable for their intended use.
• Table 2 Viability of the pharmaceutical composition at cellular concentration of 100 million total viable cells/ml.
• Table 3 Proliferative capacity of the pharmaceutical composition at cellular concentration of 100 million total viable cells/ml.
• Table 4 and Table 5 show that the pharmaceutical composition is not limited to a singly defined cell concentration yield. Indeed, maintenance of cell viability and proliferative capacity at different cellular concentrations are distinguishing features of the pharmaceutical composition hereby described. Table 4 Influence of cell concentration on percent viability
• composition release criteria - Homogeneity In order to determine the percentage of cardiopoietic cells in the pharmaceutical composition hereby described in this preferred embodiment, dual-immunolabelling is performed on an aliquot of cells with antibodies against MEF2C and CD105, followed by nuclear staining using DAPI. The goal is to determine the percentage of cardiopoietic cells (nuclear staining for MEF2C) and mesenchymal stem cells (CD105-positive), with the total number of counted cells given by the number of DAPI-stained nuclei.
• composition release criteria - Purity The purity test performed according to the preferred embodiment of this invention aim at determining that cell types different from cardiopoietic cells and BMMSCs are absent from the pharmaceutical composition.
• a method of choice to address purity criteria is qPCR.
• the approach taken to develop the qPCR method for purity testing included the identification of suitable markers, design of proprietary primer and probe sets, analysis of amplification curves and melting peaks, and identification of commercially available positive control RNAs. Absence of the hematopoietic phenotype and of the endothelial progenitor phenotype both normally present in bone marrow is determined by the absence of detectable levels of CD34-expressing cells in the pharmaceutical composition.
• Absence of adipoblasts, chondroblasts, osteoblasts or neuroblasts is determined in the pharmaceutical composition by the absence of detectable levels of FABP4-expressing cells, Sox9-expressing cells, osteocalcin-expressing cells and nestin-expressing cells, respectively. Exclusion of mature cardiomyocytes is evaluated by the absence of detectable levels of MYH7-expressing cells in the pharmaceutical composition.
• Subjects presenting with chronic heart failure secondary to ischemic cardiomyopathy were randomly assigned to the control or treated group.
• the control group received optimal standard of care.
• the treated group received the pharmaceutical composition containing cardiopoietic cells in addition to optimal standard of care.
• the pharmaceutical composition containing cardiopoietic cells was injected endoventricularly in the border zone of the infracted area using the MyoStar® Injection Catheter (Biologies Delivery Systems, California, USA). In a single injection procedure, up to 1 ,2 x 10 9 cells were injected in up to twenty injection sites surrounding the infarcted area.
• LV function Two-dimensional echocardiographic evaluation of left ventricular (LV) function was performed in 17 subjects enrolled in this trial (9 treated, 8 control) at baseline and at 6 months thereafter. Cardiac function was assessed by measuring the change in
• LVEDV LV End Diastolic Volume
• LVESV LV End Systolic Volume
• LVEDV LV End Diastolic Volume
• LVVESV LV End Systolic Volume
• the pharmaceutical composition described herein is only a preferred embodiment that depicts use of autologous cardiopoietic cells derived from bone marrow mesenchymal stem cells (BMMSCs), i.e. a pharmaceutical composition prepared from BMMSCs to be used in the same individual the BMMSCs were collected from.
• BMMSCs bone marrow mesenchymal stem cells
• the scope the present invention is not limited to autologous BMMSCs and includes use of any stem cells, irrespective of the source.
• Cells which allow to obtain cells committed to the generation of heart tissue may be allogeneic and xenogenic. Original cells may also be obtained by other means than fresh bone marrow procurement.
• Original cells may be embryonic stems cells provided that their procurement does not involve destruction of human embryos.
• Original cells can be embryonic-like stem cells such as Induced Pluripotent Stem (IPS) cells obtained by any means including transfection, cellular reprogramming or other method which renders IPS free of exogenous genes.
• IPS Induced Pluripotent Stem
• Original cells can also be Marrow- Isolated Adult Multilineage Inducible (MIAMI) cells, resident cardiac stem cells, vegetal stem cells, or any combination thereof.
• MIAMI Marrow- Isolated Adult Multilineage Inducible
• the subject invention is not limited to specific formulation components, manufacturing methods, biological materials or reagents, dosage regimens and the like, as such may vary.
• the pharmaceutical composition described herein may include additional components for example cardiogenic substances, growth factors such as fibroblast growth factors, placental growth factor or vascular endothelial growth factor, cytokines, or proteins involved in organogenesis signalling, molecular constructs, non-cardiopoietic cells altered ex vivo, pharmaceuticals, platelet lysate, scaffold materials such as collagen, laminin, or any other extracellular matrix proteins.
• growth factors such as fibroblast growth factors, placental growth factor or vascular endothelial growth factor, cytokines, or proteins involved in organogenesis signalling
• molecular constructs such as non-cardiopoietic cells altered ex vivo
• pharmaceuticals platelet lysate
• scaffold materials such as collagen, laminin, or any other extracellular matrix proteins.
• the kit described herein may include a catheter according to PCT/EP2010/055869, TW0991 13613, US 61/312371 , BE2009/0271 , PCT/EP2010/055856, TW0991 13627, or BE2009/0272.
• the kit described herein may include additional components for instance bags or media suitable for refrigeration, freeze, cryopreservation, lyophilisation, vitrification, thawing, rehydration, washing, sorting, concentration, filtering, lyophilisation, centrifugation, resuspension, sampling, or aliquoting the pharmaceutical composition.
• the kit described herein may include thermomonitoring, anti-tamper device, or radio-frequency identification device.

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