EP1165754A1 - Compositions pharmaceutiques renfermant des cellules de sang circulant, de preference des monocytes et leur utilisation - Google Patents

Compositions pharmaceutiques renfermant des cellules de sang circulant, de preference des monocytes et leur utilisation

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Publication number
EP1165754A1
EP1165754A1 EP00926832A EP00926832A EP1165754A1 EP 1165754 A1 EP1165754 A1 EP 1165754A1 EP 00926832 A EP00926832 A EP 00926832A EP 00926832 A EP00926832 A EP 00926832A EP 1165754 A1 EP1165754 A1 EP 1165754A1
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EP
European Patent Office
Prior art keywords
mcp
csf
composition
pharmaceutical
circulating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00926832A
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German (de)
English (en)
Inventor
Ivo Max-Planck-I. für Phy. u. Klin. F. BUSCHMANN
Wolfgang Max-Planck-I. für P. und K. F. SCHAPER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Priority to EP00926832A priority Critical patent/EP1165754A1/fr
Publication of EP1165754A1 publication Critical patent/EP1165754A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • compositions comprising circulating blood cells, preferably monocytes and uses thereof
  • the present invention relates to a (pharmaceutical) composition
  • a (pharmaceutical) composition comprising a circulating blood cell, preferably a monocyte loaded with a therapeutically active molecule and, optionally, a pharmaceutically acceptable carrier and/or diluent.
  • the present invention relates to the use of a circulating blood cell, preferably a monocyte loaded with a therapeutically active molecule for the preparation of a (pharmaceutical) composition for enhancing collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections and/or preventing and/or treating an occlusive disease.
  • the present invention also relates to a method for enhancing collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections, and/or preventing and/or treating an occlusive disease, said method comprising administering to a subject in need thereof an effective amount of circulating blood cells, preferably monocytes loaded with a therapeutically active molecule.
  • circulating blood cells preferably monocytes loaded with a therapeutically active molecule.
  • diagnostic compositions their preparation and use as well as diagnostic methods.
  • Preexisting collateral arteries are found in many regions of the body (hind limbs, heart, brain etc.). These vessels have the ability to markedly increase their lumen by growth so as to provide enhanced perfusion to the jeopardized ischemic regions following acute and chronic arterial occlusions. This process is called arteriogenesis and is not a process of passive dilatation but one of active proliferation and remodeling. Under normal flow conditions and depending on the pressure, gradient between the interconnecting arterial networks there is only minimal net forward flow, but small amounts of flow may oscillate within the network.
  • VCAM VCAM
  • ICAM increased endothelial production of several cytokines [MCP-1 , GM-CSF, TNF- ⁇ ]
  • MCP-1 cytokines
  • GM-CSF GM-CSF
  • TNF- ⁇ cytokines
  • attraction of circulating blood cells preferably monocytes along the chemotactic gradient to the activated endothelium
  • adhesion and invasion of circulating blood cells preferably monocytes and maturation to macrophages.
  • FGF-2 fibroblast growth factor-2
  • the invasion of circulating blood cells, preferably monocytes is soon followed by the first wave of mitosis of the endothelial- and smooth muscle cells (proliferating phase).
  • the technical problem underlying the present invention is to provide (pharmaceutical) compositions and methods for the enhancement of the growth of collateral arteries and/or other arteries from preexisting arteriolar connections.
  • the solution to this technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to a composition, preferably a pharmaceutical composition
  • a composition comprising a circulating blood cell, preferably a monocyte loaded with a therapeutically active molecule and, optionally, a pharmaceutically acceptable carrier and/or diluent.
  • monocytes descend from bone marrow derived myeloid progenitors and belong to the mononuclear phagocyte system. They circulate in the blood and have the ability to adhere to the endothelium. Surviving apoptosis they ultimately transmigrate through the endothelial layer and differentiate into macrophages (for details see, e.g., Roitt, I. et al., Immunology, Gower Medical Publishing Ltd., London, 1985). As used in accordance with the present invention, the term "monocyte” comprises monocytes as well as cells that display essentially the same biological properties/activities as monocytes.
  • circulating blood cells that are also involved in arteriogenesis
  • mast cells, lymphocytes, or granulocytes as well as cells that display essentially the same biological properties/activities as mast cells, lymphocytes, or granulocytes
  • circulating blood cells preferably monocytes when isolated from the blood of a subject and treated in vitro so as to take up a desired molecule retain their biological properties/activities when returned to said subject.
  • such circulating blood cells preferably monocytes keep the ability to be attracted by chemotactic molecules and, more importantly, that these loaded cells are able to transmigrate through the endothelium of proliferating collateral arteries. Rather, the person skilled in the art would have expected that the such loaded circulating blood cells would have been selected against in the spleen and then been discarded. Further, in vitro manipulation of cells often leads to adverse affection of membranes that often renders them unsuitable for therapeutic in vivo applications.
  • circulating blood cells preferably monocytes are used as vehicles for therapeutically active molecules, wherein said circulating blood cells, preferably monocytes can deliver said therapeutically active molecules selectively to a desired site.
  • said therapeutically active molecules are carried within the circulating blood cells, preferably monocytes and are only released therefrom at the desired site (due to the fact that circulating blood cells, preferably monocytes attach to and penetrate shear stress activated endothelium, for example, due to the upregulation of ICAM-1), the application of the (pharmaceutical) compositions of the present invention advantageously results in low systemic concentrations of said therapeutically active molecules but in very high local concentrations. Thereby it is possible to avoid systemic side effects of the applied substance in patients without decreasing efficacy of the applied substance at the place of interest.
  • the (pharmaceutical) composition of the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent.
  • suitable (pharmaceutical) carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods.
  • These (pharmaceutical) compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The .
  • dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the (pharmaceutical) composition should be in the range of 1 ⁇ g to 10 mg units per day.
  • the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 12 copies of the DNA molecule.
  • the compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the (pharmaceutical) composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the (pharmaceutical) composition.
  • the present invention relates to the use of a circulating blood cell, preferably a monocyte loaded with a therapeutically active molecule for the preparation of a (pharmaceutical) composition for enhancing collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections.
  • a circulating blood cell preferably a monocyte loaded with a therapeutically active molecule for the preparation of a (pharmaceutical) composition for enhancing collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections.
  • arteriogenesis is the in situ growth of arteries by proliferation of endothelial and smooth muscle cells from preexisting arteriolar connections supplying blood to ischemic tissue, tumor or sites of inflammation. These vessels largely grow outside the affected tissue but are much more important for the delivery of nutrients to the ischemic territory, the tumor or the site of inflammation than capillaries sprouting in the diseased tissue by angiogenic processes.
  • the present invention also relates to the use of a circulating blood cell, preferably a monocyte loaded with a therapeutically active molecule for the preparation of a (pharmaceutical) composition for preventing and/or treating an occlusive disease.
  • the present invention relates to a method for enhancing collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections, and/or preventing and/or treating an occlusive disease, said method comprising administering to a subject in need thereof an effective amount of circulating blood cells, preferably monocytes loaded with a therapeutically active molecule.
  • said occlusive disease is an arterial occlusive disease.
  • said arterial occlusive disease is a coronary artery disease, cerebral occlusive disease, peripheral occlusive disease, visceral occlusive disease, renal artery disease or mesenterial arterial insufficiency.
  • said therapeutically active molecule is a nucleic acid molecule encoding an arteriogenic polypeptide or functionally equivalent fragment thereof, or is an arteriogenic polypeptide or functionally equivalent fragment thereof or a lipopolysaccharide-like molecule.
  • lipopolysaccharide-like molecule is intended to comprise molecules that display the antigenicity but not the toxicity of LPS. Also comprised in certain embodiments of the invention is .
  • said nucleic acid molecule is DNA.
  • nucleic acid molecules like, e.g., RNA, peptide nucleic acid (PNA) or nucleic acid molecules comprising two or more of the above mentioned nucleic acid molecules may also be used in accordance with the present invention.
  • nucleic acid molecule is comprised in a vector.
  • Vectors that may be used in accordance with the present invention comprise, e.g., plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the nucleic acid molecule of the invention into targeted cell populations.
  • nucleic acid molecule of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vector comprising the nucleic acid molecule of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host.
  • calcium chloride transfection is commonly utilized for prokaryotic cells, whereas, e.g., calcium phosphate, liposome or DEAE-Dextran mediated transfection or electroporation may be used for other cellular hosts; see Sambrook, supra.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • said nucleic acid molecule is operatively linked to an expression control sequence.
  • Said expression control sequence allows expression in prokaryotic or preferably in eukaryotic cells.
  • Expression of said nucleic acid molecule comprises transcription of the nucleic acid molecule into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and, optionally, a poly-A signal ensuring termination of transcription and stabilization of the transcript, and/or an intron further enhancing expression of said polynucleotide. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions.
  • Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV- enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the nucleic acid molecule.
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the nucleic acid molecule of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI , pcDNA3 (In-vitrogene), pSPORTI (GIBCO BRL) ) or pCI (Promega).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • the vector of the present invention may also be a gene transfer or targeting vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications ot gene transfer.
  • Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911- 919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • nucleic acid molecule and vector of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • said expression control sequence is constitutively active or inducible.
  • Expression control sequences that are constitutively active and that may be used in accordance with the present invention have been discussed hereinabove. Such expression control sequence may be used if, e.g., expression of the arteriogenic polypeptide or functionally equivalent fragment thereof at sites other than the desired site is tolerable.
  • a “desired site” in accordance with the present invention may be, e.g., a site of vessel occlusion or, generally, a site where collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections shall be enhanced like, e.g., the collateral circulation of the brain or the arteries of the neck (stenosis of the carotid arteries).
  • inducible expression control sequences may advantageously be used that ensure expression only at the target site.
  • inducible expression control sequences include sequences that are sensitive to either a biological signal (such as increased levels of shear stress within collateral arteries or elevated concentrations of, e.g., a cytokine and/or chemokine) and/or are sensitive to the signal of an externally located device producing a physical, chemical, biological and/or other signal (see below).
  • a biological signal such as increased levels of shear stress within collateral arteries or elevated concentrations of, e.g., a cytokine and/or chemokine
  • said arteriogenic polypeptide attracts and/or activates circulating arteriogenic cells and/or influences the survival rate of circulating and resident arteriogenic cells or is VCAM, ICAM, FGF-2, J-309, or any other CC- chemokine or classical chemoattractant like N-farnesyl peptides, C5a, leukotrine B4 and/or Platelet-activating factor (PAF) or a functionally equivalent fragment thereof.
  • said arteriogenic polypeptide attracting circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 -alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM-CSF, G-CSF, CSF-1 , and/or M-CSF, or a functionally equivalent fragment thereof
  • said arteriogenic polypeptide activating circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 -alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM-CSF, G-CSF, CSF-1 , and/or M-CSF, Tumor Necrosis Factor alpha (
  • said therapeutically active molecule is reversibly coupled to a microsphere.
  • coupling of the therapeutically active molecule to a microsphere allows the selective transport of these molecules. Moreover, these molecules stay inactive as long as they are coupled to the microspheres.
  • An example of such a reversible coupling is a hydrophobic or van-der-Waals coupling reaction. Only after the release from said microsphere these molecules turn active.
  • the person skilled in the art is well aware of corresponding coupling methods.
  • the complex of therapeutically active molecules and microspheres is preferably taken up by, e.g., the monocytes by phagocytosis.
  • said microsphere is a latex particle, lipid-comprising structure, sponge-comprising structure, microbubble, polystyrene microsphere, biotinylated polystyrene microsphere, protein or dextran conjugate with different sizes and surface properties or any other substance that can bind said therapeutically active molecule.
  • loading of the circulating blood cell, preferably a monocyte is effected by phagocytosis, diffusion, transfection/transformation, receptor mediated uptake of molecules, electroporation and/or other mechanical methods of uptake.
  • a monocyte prior to said loading the lysosomal activity of said circulating blood cell, preferably a monocyte is inhibited.
  • This inhibition of activity can be achieved by several (pharmaceutical) and/or other substances decreasing the lyosomal activity like, e.g., gangliosides, protease- inhibitors, etc., that are well known in the art. Inhibition of the lysosomal activity may be necessary in cases where said therapeutically active molecule is prone to rapid degradation.
  • said therapeutically active molecule is released from the circulating blood cell, preferably a monocyte at the desired site.
  • said therapeutically active molecule is uncoupled from said microsphere.
  • this uncoupling is effected by an external device, which is located close to or directly on the skin in regions of proliferating collateral arteries and which influences the binding of the molecule, DNA etc. to the microsphere.
  • An externally controlled release further guarantees that only microsphere loaded circulating blood cells, preferably monocytes in the region of interest may release the molecule and/or DNA and/or other substance.
  • Other circulating blood cells, preferably monocytes, attaching, e.g., to atherosclerotic plaques etc. may invade these regions but cannot release the molecule.
  • the therapeutically active molecule after uncoupling is directly secreted from the circulating blood cell, preferably a monocyte and delivered to the desired target site.
  • the therapeutically active molecule is a nucleic acid molecule encoding an arteriogenic polypeptide or functionally equivalent fragment thereof, said acid molecule may be delivered to the described target cells where said arteriogenic polypeptide or functionally equivalent fragment thereof is expressed.
  • said nucleic acid molecule may be delivered to the nucleus of the circulating blood cell, preferably a monocyte and said arteriogenic polypeptide or functionally equivalent fragment thereof be expressed in the circulating blood cell, preferably a monocyte and secreted.
  • uncoupling is effected by a pH-change, echocardiographic signal, magnetic resonance signal, ultrasound, and/or thermographic signal.
  • said (pharmaceutical) composition is administered after local administration of a circulating blood cell monocyte chemotactic protein, preferably a monocyte chemotactic protein.
  • an effective amount of a circulating blood cell monocyte chemotactic protein prior to administering said effective amount of circulating blood ceils, preferably monocytes loaded with a therapeutically active molecule, an effective amount of a circulating blood cell monocyte chemotactic protein, preferably a monocyte chemotactic protein is administered.
  • the present invention further relates to a (pharmaceutical) kit comprising the (pharmaceutical) composition of the present invention and, in a different compartment, another (pharmaceutical) composition comprising a circulating blood cell, preferably a monocyte chemotactic protein.
  • said circulating blood cell monocyte chemotactic protein preferably a monocyte chemotactic protein attracts and/or activates circulating arteriogenic cells and/or influences the survival rate of circulating and resident arteriogluic cells or is a lipopolysaccharide-like molecule, J-309, or any other CC- chemokine or classical chemoattractant like N-famesyl peptides, C5a, leukotrine B4 and/or Platelet-activating factor (PAF).
  • PAF Platelet-activating factor
  • said monocyte chemotactic protein attracting circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 - alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM- CSF, G-CSF, CSF-1 , and/or M-CSF, or a functionally equivalent fragment thereof
  • said monocyte chemotactic protein activating circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 -alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM-CSF, G-CSF, CSF-1 , and/or M-CSF, Tumor Necrosis Factor al
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising a circulating blood cell, preferably a monocyte loaded with a detectable molecule.
  • the diagnostic composition optionally comprises suitable means for detection.
  • suitable means for detection There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.
  • the present invention also relates to the use of a circulating blood cell, preferably a monocyte loaded with a detectable molecule for the preparation of a diagnostic composition for detecting collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections.
  • the present invention relates to the use of a circulating blood cell, preferably a monocyte loaded with a detectable molecule for the preparation of a diagnostic composition for detecting a vessel occlusion.
  • the present invention relates to a method for detecting collateral growth of collateral arteries and/or arteries from preexisting arteriolar connections, and/or detecting and/or diagnosing a vessel occlusion, comprising the detection of microspheres originally contained in the circulating blood cells, preferably monocytes in said arteries or occlusions.
  • occlusion can then be measured by clinical parameters such as Doppler echo or contrast media echo etc.
  • said detectable molecule is a substance that attracts and/or activates circulating arteriogenic cells and/or influences the survival rate of circulating and resident arteriogluic cells or is a lipopolysaccharide-like molecule, J-309, or any other CC-chemokine or classical chemoattractant like N-famesyl peptides, C5a, leukotrine B4 or Platelet-activating factor (PAF) or an expressible nucleic acid molecule encoding one or more of the above mentioned (poly)peptides.
  • a lipopolysaccharide-like molecule J-309, or any other CC-chemokine or classical chemoattractant like N-famesyl peptides, C5a, leukotrine B4 or Platelet-activating factor (PAF) or an expressible nucleic acid molecule encoding one or more of the above mentioned (poly)peptides.
  • said substance attracting circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 -alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM-CSF, G-CSF, CSF-1 , and/or M-CSF, or a functionally equivalent fragment thereof
  • said substance activating circulating arteriogenic cells is selected from the group consisting of chemokines, preferably MCP-1 , MCP-2, MCP-3, MCP-4, MCP-5, RANTES, Fraktalkines, MIP-1 - alpha, and/or MIP-1 beta, interleukins, colony stimulating factors, preferably GM- CSF, G-CSF, CSF-1 , and/or M-CSF, Tumor Necrosis Factor alpha (TNF-alpha), or a functionally equivalent
  • said detectable molecule is coupled to a microsphere.
  • said microsphere is a latex particle, lipid-comprising structure, sponge-comprising structure, microbubble, polystyrene microsphere, biotinylated polystyrene microsphere, protein or dextran conjugate with different sizes and surface properties or any other substance that can bind said detectable molecule.
  • the use or the method of the present invention loading of the circulating blood cell, preferably a monocyte is effected by phagocytosis, diffusion or transfection.
  • the detectable molecule is a nucleic acid molecule, what has been discussed hereinabove with respect to expression of nucleic acid molecules, i.e. endogenous production of therapeutic substances, methods of introducing nucleic acid molecules into cells (e.g. calcium phosphate, liposome or DEAE-Dextran mediated transfection or electroporation), etc., also applies here.
  • kits such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container.
  • the surgical procedure was performed under sterile conditions. Femoral arteries were exposed and cannulated with a sterile polyethylene catheter (inner diameter: 1mm; outer diameter: 1.5mm) pointing upstream, with the tip of the catheter positioned distal to the branching of the arteria circumflexa femoris.
  • the catheter itself occludding the artery was connected to an osmotic minipump (2ML-2, Alza Corporation, Palo Alto, CA), which was implanted under the skin of the lower right abdomen.
  • osmotic minipump 2ML-2, Alza Corporation, Palo Alto, CA
  • Hematocrit and serum values of total protein, albumin, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase were not significantly changed by the treatment.
  • the animals were again anesthetized with an intramuscular injection of ketamine hydrochloride and xylazine for tracheostomy and artificial ventilation. Anesthesia was deepened with pentobarbital (12 mg/kg body weight per hour). The carotid artery was cannulated for continuous pressure monitoring.
  • the arteria saphena magna (anterior tibial artery in humans and main arterial supply to the lower limb and foot in the rabbit) was exposed just above the ankle and cannulated with sterile polyethylene heparinized tubing (inner diameter 0.58mm; outer diameter 0.96mm). These tubings were connected to a Statham P23DC pressure transducer (Statham, Spectramed) for measurement of peripheral pressures (PP). After heparinization with 5000 Units heparin, the left femoral artery was exposed and cannulated with sterile polyethylene catheter (inner diameter: 1 mm; outer diameter: 1.5mm) for the microsphere reference sample.
  • a pump-driven shunt was installed to ensure oxygenated blood flow from the carotid artery via the canula in the abdominal aorta into the right and left legs.
  • a flow probe was installed to measure total flow to both hindlimbs. The absence of any residual volume in the minipumps ( ⁇ 3%) after the experiment verified delivery of the contents.
  • the six perfusion pressures levels (40,50,60,70,80,90 mmHg) were generated in vivo with a roller pump (Stoeckert) installed in the above mentioned shunt between carotid artery and abdominal aorta.
  • Peripheral pressures and collateral flows were measured under maximal vasodilation (adenosine).
  • microspheres with a different fluorescent color either scarlet, crimson, red, blue-green, yellow-green or orange; diameter: 15 ⁇ m; Molecular Probes, Eugene, Oregon, U.S.
  • All recordings were stored on a computerized recordings system (MacLab, Macintosh) from which they were retrieved for further processing.
  • Example 3 Counting of Microspheres
  • the Quadriceps-, adductor longus-, adductor magnus-, gastrocnemius-, soleus, and peroneal muscles were dissected and each muscle was divided into 3 consecutive pieces from the proximal to the distal end. The whole muscle and afterwards each sample were weight and cut into small pieces (1-2g). The muscle samples were then packed loosely into 12mm x 75mm polystyrene tubes (Becton Dickinson & Co, Lincoln Park, NJ) containing 3 ml of SDS solution [SDS solution (Boehringer Mannheim Corp.): 1% SDS, 0.5% sodium azide (Sigma Chemical Company, St.
  • microspheres were counted using a flow cytometer (FACS-Calibur) equipped with a second laser and a detector for a fourth fluorescence.
  • Flows for each sample were calculated from the number of microspheres in the sample (m s ), the respective microspheres count in the reference sample (m rs ), the internal standard in the sample (IS S ), internal standard in the reference sample (IS rs ), the weight of the reference sample (W) and the time during which the reference sample was withdrawn using following equation:
  • SP systemic pressure
  • PP peripheral pressure
  • AP atmospheric pressure
  • Collateral flow is equal to the sum of flows to the tissue of the distal adductor plus the flow to the tissue of the lower leg.
  • Collateral resistance was defined as pressure difference between SP and PP divided by the flow going to the distal adductor an the lower leg. The reciprocal values of these resistances represent collateral-, peripheral-, and bulk conductance. Because a positive pressure intercept is observed even at maximal vasodilation, all conductances were calculated from the slope of pressure-flow relations.
  • Example 8 Evaluation of the kinetics of monocytes in collateral arteries via monocyte loading with fluorescent microspheres
  • the right femoral artery was ligated in 12 rabbits. Isolated rabbit monocytes were loaded with fluorescent microspheres (0 2 ⁇ m) and reinfused intravenously. 48 hours after reinfusion animals were killed and biopsies were taken of the adductor and quadriceps muscle in both hindlimbs whereby the biopsies of the left hindlimb served as control. In a second control group microspheres were directly infused. With FACS analysis the total number of monocytes per gram muscle tissue was quantified ( Figure 1).
  • Example 9 Attraction of loaded monocytes via MCP-1

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Abstract

L'invention concerne une composition (pharmaceutique) renfermant une cellule de sang circulant, de préférence un monocyte chargé d'une molécule thérapeutiquement active et, facultativement, un vecteur pharmaceutiquement acceptable et/ou un diluant. En outre, l'utilisation d'une telle cellule, de préférence un monocyte chargé d'une molécule thérapeutiquement active, permet de préparer une composition (pharmaceutique) destinée à renforcer la croissance collatérale d'artères collatérales et/ou d'artères à partir de branchements artériolaires préexistants et/ou à prévenir et/ou à traiter des occlusions. De plus, un procédé permet de renforcer la croissance collatérale d'artères collatérales et/ou d'artères à partir de branchements artériolaires préexistants et/ou à prévenir et/ou à traiter des occlusions, ledit procédé consistant à administrer au patient concerné une dose efficace de cellules de sang circulant, de préférence des monocytes chargés d'une molécule thérapeutiquement active. L'invention traite enfin de trousses (pharmaceutiques), de compositions diagnostiques, de leur préparation et utilisation, ainsi de méthodes diagnostiques.
EP00926832A 1999-04-06 2000-04-06 Compositions pharmaceutiques renfermant des cellules de sang circulant, de preference des monocytes et leur utilisation Withdrawn EP1165754A1 (fr)

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WO1999017798A1 (fr) 1997-10-02 1999-04-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procedes de modulation de la neovascularisation et/ou du developpement d'arteres collaterales et/ou d'autres arteres a partir de connexions arteriolaires existantes
WO2001087314A1 (fr) * 2000-05-18 2001-11-22 Genetix Pharmaceuticals, Inc. Procedes et compositions destines a promouvoir l'angiogenese au moyen de monocytes
US6875612B2 (en) 2001-03-30 2005-04-05 Greenville Hospital System Monocyte-specific particulate delivery vehicle
US20040191215A1 (en) * 2003-03-25 2004-09-30 Michael Froix Compositions for induction of a therapeutic response

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FR2709309B1 (fr) * 1993-08-25 1995-11-10 Centre Nat Rech Scient Compositions cellulaires, préparation et utilisations thérapeutiques.
DK0969877T3 (da) * 1997-04-04 2003-03-24 Max Planck Gesellschaft Fremgangsmåder til modulering af væksten af collaterale arterier og/eller andre arterier fra forudeksisterende arteriolære forbindelser
US20020068048A1 (en) * 1997-09-05 2002-06-06 Patrick A. Dreyfus Method for the treatment or diagnosis of human pathologies with disseminated or difficult to access cells or tissues
WO1999017798A1 (fr) * 1997-10-02 1999-04-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procedes de modulation de la neovascularisation et/ou du developpement d'arteres collaterales et/ou d'autres arteres a partir de connexions arteriolaires existantes

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See references of WO0060054A1 *

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