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
  • A61K35/34—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
  • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • 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
  • A61P9/10—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
• • 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
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

• the present invention relates to a cell preparation and to a use of this cell preparation as a revascularization stimulating agent.
• the targeted pathologies are here numerous, and overall it is the pathologies that led to denaturation or destruction of a vascular system. Such circumstances occur especially when the arterial blood supply in a tissue or organ decreases or stops.
• Cellular therapies consist precisely of regenerating altered tissue, whatever it is, from specific cells cultured in vitro, or not, and then transplanted into the altered tissue. Many advances have already been made to treat different pathologies using these cell therapies.
• the general principle is essentially based on the ability of certain types of cells at certain stages of their development, to multiply and to differentiate to produce specialized cells that acquire a morphology and a specific function of the tissue in which they are implanted.
• Embryonic stem cells in the early stages after fertilization, are undifferentiated and will be able to lead to the formation of all tissues of the body.
• Adult stem cells on the other hand, are already engaged in a specific tissue program and can only lead to the formation or regeneration of distinct tissues; they are said to be multipotent whereas embryonic stem cells are themselves, totipotent.
• precursor cells derived from stem cell divisions have already acquired, during their development, a certain degree of specialization and are physiologically functional.
• multipotent stem cell populations from skeletal muscle tissue.
• This method makes it possible to prepare multipotent stem cell populations aimed at the regeneration of many types of tissues, but it is relatively complex to implement.
• myocardial tissues lack stem cells capable of forming cardiomyocytes to regenerate, it has also been imagined to prepare relatively homogeneous cell populations whose dominant type has the characteristics of myoblastic cells, and to inject directly these populations in the myocardial tissue or indirectly in the arterial circulation.
• WO01 / 94555 in which such a method is described.
• the sorting of different cell populations requires the observation of specific cell markers.
• the cell populations obtained are specifically adapted to the regeneration of the heart muscle.
• both the first document analyzed above has a relatively broad spectrum of use of multipotent stem cell populations obtained, but does not provide convincing results for a pro-angiogenic activity, as the second document discloses populations specific cells with the characteristics of myoblastic cells and therefore of a more restricted use.
• a problem that arises and that the present invention aims to solve is to provide a cellular preparation for use as a revascularization stimulating agent, which allows the vascular system of a damaged mammalian tissue to be reconstituted and particular of human, when it is administered to him.
• a revascularization stimulating agent which allows the vascular system of a damaged mammalian tissue to be reconstituted and particular of human, when it is administered to him.
• the present invention provides a cellular preparation comprising endothelial cell precursors (EPCs, for Endothelial Precursor CeIIs in English language) and smooth muscle cell precursors (SMCs, for Smooth Muscle CeIIs, in English language). ) as a combination product for simultaneous administration, separate or spread over time, for use as a revascularization stimulating agent.
• EPCs Endothelial Precursor CeIIs in English language
• SMCs smooth muscle cell precursors
• a characteristic of the invention lies in the use of two types of cells, both endothelial cell precursors and smooth muscle cell precursors which then interact to stimulate the formation and development of blood capillaries from pre-existing blood vessels.
• said endothelial cell precursors are obtained by in vitro differentiation of progenitors from umbilical cord blood or hematopoietic marrow or circulating peripheral blood or any other tissue.
• EPCs endothelial cell precursors
• said endothelial cell precursors express a specific receptor capable of receiving a protein material for activating said endothelial cell precursors (EPCs).
• EPCs endothelial cell precursors
• said specific receptor is an Eph receptor with tyrosine kinase activity, for example of the EphB type and more specifically EphB4.
• the protein material preferably comprises a ligand specific for said marker, said ligand being associated with a binding polypeptide.
• the specific ligand is an ephrin ligand, for example ephrin-B or more precisely ephrin-B2 or ephrin-B1.
• the binding polypeptide it is preferably an Fc fragment of immunoglobulin.
• endothelial cells with specific receptors are obtained, to which specific receptors are associated a protein material consisting of a ligand and a binding polypeptide; such endothelial cell precursors are then activated.
• precursors of smooth muscle cells they are preferably obtained by in vitro differentiation of progenitors from umbilical cord blood or hematopoietic marrow or circulating peripheral blood or any other tissue.
• mononuclear cells are first isolated, for example from umbilical cord blood, and then differentiated into smooth muscle cell precursors. They are then recovered and then associated with endothelial cell precursors to be administered.
• said smooth muscle cell precursors are obtained from a biopsy of muscle tissue that is taken from a skeletal muscle of a human and that the cultured to harvest only the precursors of identified smooth muscle cells.
• These Smooth muscle cell precursors are indeed clearly identifiable through specific cell markers.
• the present invention provides the use of a cell preparation associating endothelial cell precursors and smooth muscle cell precursors for the preparation of a cellular composition for stimulating revascularization or ischemia tissue angiogenesis. mammals and especially in humans. Furthermore, it is also envisaged a combination of these precursors of endothelial cells and smooth muscle cell precursors for the preparation of a medicament for normalizing tumor revascularization.
• a cell preparation associating endothelial cell precursors and smooth muscle cell precursors for the preparation of a medicament for stimulating the revascularization of damaged tissues in the healing phase.
• pathological conditions such as: radiation or post-radiation dermatitis, burns or post-traumatic skin breakdown or any other origin.
• the cell preparation combining endothelial cell precursors and smooth muscle cell precursors is suitable for the preparation of a therapeutic composition intended for the prevention or treatment of cancers in prior or simultaneous administration to anti-cancer treatment by chemotherapy or radiotherapy. It is also adapted to treat vascular malformations and in particular angiomas.
• a cell preparation of the type according to the invention as a pro-angiogenic active ingredient, in association with a physiologically acceptable excipient, for the preparation of a composition for therapeutic use in the body.
• treatment of vascular insufficiency particularly in the revascularization of ischemic, cardiac, cerebral or peripheral tissues.
• FIG. 1 is a histogram showing the comparative efficacy of the pro-angiogenic activity of the cell preparations which are the subject of the present invention.
• EPCs endothelial cell precursors
• SMCs smooth muscle cell precursors
• samples of 30 to 50 ml each of human umbilical cord blood are first collected and placed in sterile tubes containing an anticoagulant solution of sodium heparin.
• the mononuclear cells were then isolated from umbilical cord blood by density gradient centrifugation using Pancoll (1.07 g per milliliter, sold by Miguel Dutscher S.A., Brumath, France).
• Pancoll (1.07 g per milliliter, sold by Dominique Dutscher S.A., Brumath, France).
• the isolated mononuclear cells are then separated from the adherent cells by culturing on plastic boxes for 24 hours at 37 ° C.
• a mixture of isolated mononucleated cells and freed from the adherent cells is then collected, which mixture is then placed in the wells of a six-well plate covered with a defined matrix.
• This defined matrix contains fibronectin, laminin, heparan sodium sulfate, type I and type IV collagen (these products are all supplied by Sigma-Aldrich) and a hVEGF growth factor (R & D Systems, Oxford UK).
• Squamous-type cells are in fact precursors of endothelial cells because they express the main markers of this type of cell, von Willebrand Factor, CD31, eNOS, VE-Cadherin, VEGF-R1 or VEGF-R2.
• fusiform-type cells are precursors of smooth muscle cells because they express as a marker, ⁇ SMA, calponin, SM22 ⁇ and SM-MHC.
• a first cell preparation then comprising both endothelial cell precursors and smooth muscle cell precursors is tested on batches of male Nude mice according to a first protocol defined below.
• the efficacy of the above preparation will be compared not only to a neutral control preparation with PBS (buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.84 mM KH 2 PO 4 ) without cell colony, but also with respect to a preparation containing only endothelial cell precursors and a preparation containing only smooth muscle cell precursors. Therefore, four batches of six animals will be reserved, and the four preparations will be administered to the animals of the four batches, respectively.
• PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.84 mM KH 2 PO 4
• the ligation of the right femoral artery of all seven-week-old mice is performed to simulate ischemia.
• the first cellular preparation of endothelial cell precursors and smooth muscle cell precursors was simultaneously injected intravenously into the retro-orbital sinus, at a rate of approximately 250,000 cells for both cell types and for each mouse of a first batch, and respectively the other preparations to the other three batches of animals.
• the other cell preparations tested will contain about 500,000 cells.
• 12 days after ligation the mice are sacrificed and the gastronecmius muscles of the ischemic leg and the non-ischemic leg are removed.
• the angiographic score measures the density of the vessels and is determined by micro-angiography (operating modalities in the article by Silvestre J. S et al., Cir Res., 2001; 89: 259-264). In this case, the density of the vessels is measured for each animal in the ischemia member and in the non-ischemic limb and the result obtained is presented in the form of a report, ischemic paw on non-ischemic leg.
• capillary density which represents the number of capillaries per square millimeter, it is measured by labeling sections of the gastronecmius muscle by means of an antibody directed against the endothelial-specific CD31 marker, as indicated above. and comparing these sections to sections of the same muscle of the non-ischemic limb. The results thus obtained are presented in the form of the ratio, ischemic paw on nonischemated paw.
• the cutaneous blood flow As for the cutaneous blood flow, it is evaluated quantitatively by the ratio, the blood flow measured on the ischemia limb and the blood flow measured on the non-ischemic limb. It is thus verified that the variation in the number of vessels corresponds to a functional adaptation and therefore to a variation in perfusion of the ischemia limb.
• Table I of the results The measurement of the above three parameters is recorded in the table below for each of the four groups of six individuals. The measurement is the result of the average of the six animals.
• endothelial cell precursors EPCs or precursors of smooth muscle cells SMCs causes a slight increase, respectively of approximately 60% and 35%, of the density of the vessels compared to that of the group of control animals, to which the control PBS (or, multiplied by 1.60 and 1.35, respectively) was administered.
• the density of vessels is multiplied substantially by 2.4 relative to that of the "control" group of animals.
• the animals to which the endothelial precursors and the smooth muscle cell precursors have been simultaneously administered have a capillary density that is substantially 50% greater than the capillary density of the animals to which it has been subjected. administered either precursors of endothelial cells alone or precursors of smooth muscle cells alone.
• a cell preparation according to the invention incorporating endothelial cell precursors and smooth muscle cell precursors can be administered as a medicament, in particular for treating vascular insufficiency, in particular in the revascularization of cardiac, cerebral or cerebral ischemic tissues. peripheral devices.
• a preparation is also indicated to normalize the tumor vasculature and thus allow drugs administered in chemotherapy to spread better in the tumor.
• the tumor vasculature is not consistent with the normal vasculature of healthy tissue and it is accompanied in particular by hemostatic and chemical disorders.
• the drugs administered reach more difficult the heart of the tumor which thus decreases their activity and their effectiveness.
• the aforementioned cell preparation leads to restore the tumor normal blood supply and therefore normally receive drugs circulating in the blood.
• the cell preparation according to the invention may be packaged in unit dose form containing both smooth muscle cell precursors and endothelial cell precursors or even in the form of separate doses.
• the two cellular compositions are administered, either simultaneously, or separately or even spread over time.
• endothelial cell precursors and smooth muscle cell precursors can be independently, frozen and stored at minus 80 ° C. Thus, when necessary, they are then thawed and then cultured for a period of about one week to be administered simultaneously or separately.
• the cell preparation is particularly suitable for the preparation of a composition intended for the treatment of arteritis, insufficiency coronary vascular, or cardiac, and cerebrovascular insufficiency.
• a composition intended for the treatment of arteritis, insufficiency coronary vascular, or cardiac, and cerebrovascular insufficiency On the aforementioned animal model, it has provided good results in the treatment of critical ischemia of the lower limbs; and consequently the hope is great of being able to obtain, in man in such circumstances, a cure to avoid amputation.
• the cell composition according to the invention can therefore be administered in the mammal and in particular in humans according to a procedure known per se.
• the cell composition is capable of being injected at or near the vascular lesion, into the blood, or delivered directly to the lesion site by means of a suitable vector.
• the endothelial cell precursors, on the one hand, and the smooth muscle cell precursors, on the other hand may be separately administered by injection.
• a cellular composition according to the invention comprising 5 to 10 9 cells.
• activated EPCs endothelial cell precursors and SMC smooth muscle cell precursors are associated.
• endothelial cell precursors having a specific cell marker are provided on the surface of the outer membrane of the cell, said cellular marker being chosen from the group consisting of Eph, in particular EphB4 or EphB1; and a protein material of structure LK is associated therewith.
• the protein material consists of a ligand (L) specific for said marker, and a binding peptide (K), in particular an Fc fragment of immunoglobulin or antibodies.
• EPCs cells comprising the Eph marker are activated by the specific L ligand belonging to the ephrin family.
• the ligand L may also consist of a peptide fragment of an ephrin, for example ephrin-B2, which would then have the same biological activity.
• the endothelial cell precursors are activated via an EphB4 cell marker to which an ephrin-B2 ligand is attached, the ligand being itself associated to an antibody Fc fragment.
• activated endothelial cell precursors of the form: EPC-EphB4-ephrin-B2-Fc are obtained.
• squamous cell colonies derived from the cell preparation obtained according to the above-mentioned first preparation method are collected.
• This cell preparation contains, inter alia, endothelial cell precursors provided with the EphB4 marker.
• These cell colonies are then treated with 3 ⁇ g / ml of Ephrin-B2-Fc fusion protein, for an incubation period of 30 minutes at 37 ° C. Rinse with PBS is separated off each unbound fusion protein (at least two rinses are needed).
• An endothelial precursor composition of structure EPC-EphB4-ephrin-B2-Fc is then obtained.
• the above-mentioned cellular preparation obtained according to the first method of preparation contains precursors of smooth muscle cells identifiable by their fusiform type. Therefore, these smooth muscle cell precursors will be taken in such a preparation, and will be associated with EPC-EphB4-ephrin-B2-Fc endothelial cell precursors.
• a cellular composition is collected at the end of the first part of the aforesaid first method of preparation at the height of the first embodiment of the invention.
• the mononuclear cells of the umbilical cord blood were isolated by centrifugation and were then separated from the adherent cells by culture on plastic dishes for 24 hours at 37 ° C. A cell mixture is thus obtained containing mononuclear cells expressing the EphB4 marker and mononuclear cells not expressing said marker.
• the CD34 + labeled cells are isolated and purified from non-adherent cells by a standard immunomagnetic separation technique, in particular by means of the "CD34 isolation kit” material (marketed by MILTENYI BIOTECH, Paris, France), which comprises a monoclonal antibody. anti-CD34.
• the cell mixture thus obtained which contains 1.5 x 10 6 to 3.5 x 10 6 CD34 + cells, can be placed in the wells of a 6-well plate coated with a matrix containing fibronectin, laminin, heparan sodium sulphate, and type I and IV collagen (products sold by the aforementioned SIGMA-ALDRICH company) and in a culture medium containing hVEGF, bFGF and IGF1 (products marketed by the so-called R & D company). SYSTEMS INC., Oxford, United Kingdom). After 15 days of culture, a cell mixture enriched in EPC-EphB4 is collected.
• This cell mixture is then treated identically to the first embodiment, with 3 ⁇ g / ml of Ephrin-B2-Fc fusion protein.
• a cell composition is thus obtained, predominantly comprising EPC-EphB4-ephrin-B2-Fc endothelial cell precursors.
• this cell composition is associated with smooth muscle cell precursors taken in a manner similar to the first embodiment, in a cell preparation according to the first preparation method.
• a third cell preparation according to the invention will then be obtained.
• the precursors of smooth muscle cells can also be obtained according to the second aforementioned method of obtaining.
• the second cell preparation comprising both endothelial cell precursors of EPC-EphB4-ephrin-B2-Fc structure and smooth muscle cell precursors is tested according to a second protocol substantially identical to the first protocol above, on batches of male Nude mice. It will be noted that the third cell preparation mentioned above would lead to the same result as the second preparation.
• the efficacy of the second preparation will be compared to a control preparation (PBS), and also to a preparation containing only EPC-EphB4-ephrin-B2-Fc endothelial cell precursors. Three sets of six animals will then be reserved, and the three preparations will be administered to the animals of these three lots respectively.
• mice are sacrificed and the gastronecmius muscles of the ischemic leg and the non-ischemic leg are removed.
• the three parameters already met above, the angiographic score, the capillary density, and the cutaneous blood flow are then measured.
• Table II of the results The measurement of the above three parameters is recorded in the table below for each of the three groups of six individuals. The measure is the average of the results observed on the six animals.
• endothelial cell precursors activated via their EphB4 marker associated with ephrin-B2 ligand and Fc antibody fragment, and in combination with smooth muscle cell precursors. , cause an increase in vessel density equivalent to 3.5 times the density of the vessels obtained with the control composition which is devoid of cells.
• the cell composition containing only endothelial cells activated via their EphB4 marker associated with the ephrin-B2 ligand and the antibody Fc fragment already causes an increase in the density of the vessels twice that of the control composition.
• the second cell preparation according to the invention incorporating activated endothelial cell precursors and in combination, smooth muscle cell precursors, can be administered as a drug, in order to also treat vascular insufficiencies, in particular in the revascularization of cardiac, cerebral or peripheral ischemic tissue.
• this second preparation is suitable for producing a medicament for normalizing tumor vascularization.

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