WO2006059329A1 - Utilisation therapeutique de microparticules d'origine plaquettaire - Google Patents

Utilisation therapeutique de microparticules d'origine plaquettaire Download PDF

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WO2006059329A1
WO2006059329A1 PCT/IL2005/001283 IL2005001283W WO2006059329A1 WO 2006059329 A1 WO2006059329 A1 WO 2006059329A1 IL 2005001283 W IL2005001283 W IL 2005001283W WO 2006059329 A1 WO2006059329 A1 WO 2006059329A1
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pmp
administration
ischemia
organ
pathological condition
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PCT/IL2005/001283
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English (en)
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David Varon
Alexander Brill
Julia Rivo
Olga Dashevsky
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Hadasit Medical Research Services & Development Limited
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    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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

Definitions

  • the present invention concerns platelet-derived microparticles (PMP) and their use in therapy.
  • PMP platelet-derived microparticles
  • Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. J Biol Chem 263:18205-18212.
  • Platelets are accumulated at the sites of vascular injury or disturbances in blood flow, where they are activated and release various granular mediators.
  • the platelet-derived biologically active substances recruit additional platelets to the forming thrombus, thus contributing to its growth.
  • platelets might participate in other processes, such as wound or ulcer healing.
  • Platelets are known to produce microparticles in response to various activatory stimuli (5),(6). These particles are microvesicles of 0.05 - 1 ⁇ m in size, released from the plasma membrane blebs of almost all cell types upon stress conditions, reflecting both in vitro as well as in vivo cell stimulation and/or tissue degeneration under a variety of physiologic conditions (4, 3).
  • PMP platelet microparticles
  • stimulation of blood coagulation with thromboplastin
  • possible participation in pathogenesis of atherosclerosis and vascular injury in inflammation (7)
  • promotion of bone cell proliferation (8)
  • others at least one clinical syndrome has been described whose pathogenesis is directly based on the lack of platelet ability to form microparticles in response to different activatory stimuli (Scott's syndrome) (9).
  • PMP were demonstrated to bind neutrophils, mediate neutrophil aggregation, and activate their phagocytic properties, which might be important in immune and inflammatory responses in which neutrophils are involved (10).
  • PMP also mediate leukocyte-leukocyte and leukocyte-endothelial cell interactions (13). Binding of PMP to cells is capable of modifying cell functional properties. For example, it was demonstrated that PMP can bind hematopoietic progenitors and stimulate their engraftment (1 1).
  • PMP are known to participate in thrombotic events due to their ability to interact with endothelium, subendothelium and blood cells and due to their ability to transfer pro-coagulant substances like tissue factor and others.
  • Intra-venous (i.v.) or intra-arterial (i.a.) introduction of PMP may lead to local (as well as distant) thrombotic events with potential vessel occlusion. Indeed, it was found that when PMP were injected into a mouse tail vein, in most cases an immediate death of the animal was exhibited, apparently due to thrombo-embolic events (cardiac, pulmonary or brain events) (2).
  • PMP fulfilling a beneficial, hemostatic function under physiologic conditions have been classified as "good” microparticles. On the other hand, their production in excess leads to the possible pathological deviation toward thrombosis, thus acting as "bad” microparticles (3),(4).
  • the invention is described in the following detailed description with reference to therapeutic methods for inducing or promoting angiogenic processes, as well as for treating pathological conditions where it is desirable for said treatment to induce or promote an angiogenic process by the use of platelet derived microparticles (PMP) as the active ingredient.
  • PMP platelet derived microparticles
  • PMP for the preparation of a composition for administration to a subject having a pathological condition and being in need of angiogenesis induction or promotion for treatment.
  • a platelet derived microparticle includes one or more such particles, and thus also equivalently denotes platelet derived microparticles or "PMP".
  • compositions consisting essentially of PMP as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.
  • the invention is based on the finding that when PMP are in situ applied to the myocardium, an angiogenic process was initiated, leading to the sprouting and propagation of blood vessels, while in the absence of PMP, this process was not promoted.
  • the invention is based on the finding that while intravascular administration of PMP may lead to undesired thrombosis, administration by other means (generally termed herein " extravascular administration") results in increased vascularization, with no detectable evidence for the formation of thrombosis.
  • the present invention provides a method of in situ inducing or promoting angiogenesis at a target tissue or organ, the method comprising administering to said target tissue or organ an amount of PMP, the amount being effective to induce or promote an angiogenic process.
  • In situ means that induction or promotion of the angiogenic process occurs when the tissue or organ are at their natural location within the body.
  • the method is for treating a subject having a pathological condition where it is desirable for said treatment to induce or promote angiogenesis, the method comprising extravascular administration to a damaged tissue or organ associated with said pathological condition an amount of PMP, the amount being effective to induce or promote angiogenesis at or in proximity with said damaged tissue or organ.
  • the invention also provides the use of PMP for the preparation of a composition for in vivo inducing or promoting angiogenesis.
  • the invention provides the use of PMP for the preparation of a composition for treating a subject having a pathological condition where it is desirable for said treatment to induce or promote angiogenesis at a damaged tissue or organ.
  • compositions comprising as active ingredient an amount of PMP, the amount being effective to induce or promote in vivo an angiogenic process.
  • compositions for treating a subject having a pathological condition where it is desirable for said treatment to induce or promote angiogenesis comprising as active ingredient an amount of PMP, the amount being effective to induce or promote an angiogenic process in said subject.
  • a preferred pathological condition in accordance with the invention is ischemia or a condition related to ischemia.
  • the pathological condition is a condition associated with endothelial cell hypoxia (e.g. as a result of damage to a tissue or organ).
  • the invention particularly provides a method and composition for treating a pathological condition as defined herein above and below, where the PMP are administered to the subject in need by a route other than intravascular administration. It has been realized by the inventors that administration of PMP intravascularly may cause the undesired development of thrombosis. Without wishing to be limited thereby, it is believed that intravenous or intraarterial injection of PMP may supply large amounts of thromboplastin to the flowing blood, leading to intravascular blood coagulation and thrombus formation leading to obstruction of the vessel and possible severe complications, including death. Thus, the route of administration of PMP has now been determined to be crucial regarding its piO-thrombotic activity and an alternative administration route has now been suggested in order to overcome this effect. Thus, in accordance with a preferred embodiment of the invention, there is provided a method and composition wherein PMP is administered extravascularly.
  • FIGS 2A-2G Effect of cytokine inhibitors on P ⁇ dP-induced angiogenesis.
  • FIG. 3A Signaling pathways involved in the pro-angiogenic effect of PhIP.
  • PMP were seeded to the aortic rings in the presence or absence of signaling kinase inhibitors. The following samples are presented: negative control (Fig. 3A);PMP 50 ⁇ g/ml (Fig. 3B); PMP + PP2 (Src kinase inhibitor) (Fig. 3C); PMP + SB203580 (inhibitor of p38 kinase) (Fig. 3D); PMP + LY294002 (PI3- kinase inhibitor) (Fig. 3E); PMP + Calphostin C (PKC inhibitor) (Fig.
  • FIGS 4A-4C PMP adhere to EC. PMP adhered to endothelial cells are shown as white spots and marked with arrows in images obtained by confocal microscopy (Fig. 4A, bar 20 ⁇ m), and electron microscopy (Fig. 4B (bar 5 ⁇ m) and Fig. 4C (bar 10 ⁇ m)).
  • FIGS. 5A-5G PMP induce EC invasion through matrigel layer.
  • the number of migrated cells per membrane was determined from photograph images (Figs. 5A-5F) and converted to a bar graph (Fig. 5G) showing the corresponding bars (below each image) with the first bar corresponding to the negative control (no PMP), the second bar corresponding to PMP alone, and the remaining bars corresponding to PMP, each with the inhibitor as indicated.
  • FIGs 6A-6H PMP induce angiogenesis in vivo.
  • Agarose beads without cytokine inhibitors Fig. 6A
  • Fig. 6B vascular endothelial growth factor
  • Fig. 6C PMP
  • Fig. 6D immunostaining with anti-vWF niAb was performed (Fig. 6D, Fig. 6E and Fig. 6F, respectively).
  • Area of capillaries surrounding the beads was measured using Software (Fig. 6G.) and vessels were counted in 5 occasional view fields in each slide shown in Figs. 6D-F (Fig. 6H).
  • FIG. 7A-7D Paraffin-embedded sections from Sabra rats' hearts treated as described in the M&M were photographed (Fig. 7A-7C) and the number of vessels per view field was determined by the use of Software (Fig. 7D).
  • FIGs 8A-8B Heart echocardiography in rats after LAD ligation, and in particular percent of ejection fraction (Fig. 8A) and percent of fraction shortening (Fig. 8B).
  • Figure 9 Mortality rate of rats injected with PMPs vs. control animals during 1 month after operation.
  • Angiogenesis is a fundamental process required for the normal growth and development of tissues, and involves the proliferation of new capillaries from preexisting blood vessels. Further, angiogenesis is a prerequisite for the development and differentiation of the vascular tree, as well as for a wide variety of fundamental physiological processes including embryogenesis, somatic growth, tissue and organ repair and regeneration, cyclical growth of the corpus luteum and endometrium, and development and differentiation of the nervous system.
  • angiogenesis occurs in the follicle during its development, in the corpus luteum following ovulation and in the placenta to establish and maintain pregnancy.
  • Angiogenesis additionally occurs as part of the body's repair processes, e. g. in the healing of wounds and fractures.
  • Angiogenesis is also a factor in tumor growth, since a tumor must continuously stimulate growth of new capillary blood vessels in order to grow.
  • Endothelial cells and pericytes surrounded by a basement membrane, form capillary blood vessels.
  • Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes.
  • the endothelial cells which line the lumen of blood vessels, then protrude through the basement membrane.
  • Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
  • the migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate.
  • the endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
  • Promoting angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangiitis obliterans. Enhancing angiogenic activity may also be useful in treating ischemic conditions, including cardiovascular and limb ischemia. Finally, materials or methods that initiate or increase angiogenesis could potentially also be used to create research models with greater-than-normal angiogenesis.
  • the present invention is based on the following novel findings:
  • PMP induce human endothelial cell (EC) invasion through a layer of Matrigel, an in vitro model which mimics in vivo conditions.
  • PMP induce angiogenesis in an in vivo model, in which agarose beads containing the substances under study were transplanted subcutaneously into mice. Immunohistochemical staining of the skin in the vicinity of the beads showed the presence of von Willebrand factor, the marker of endothelial cells. This finding expands the possible importance of PMP to the clinical situations whose pathogenesis is dependent upon development of blood vessels de novo.
  • the present invention provides a method of in vivo inducing or promoting angiogenesis at a target tissue or organ comprising administering to said target tissue or organ an amount of platelet derived microparticles (PMP), the amount being effective to induce or promote an in vivo angiogenic process.
  • PMP platelet derived microparticles
  • induction or promotion of angiogenesis denotes any measurable effect PMP has on endothelial cells resulting in either the initiation of an angiogenic process or the promotion or acceleration of an already occurring angiogenic process.
  • the induction or promotion should be such that it results in the sprouting of vessels, preferably, the development of new blood vessels (vascularization or revascularization).
  • induction or “promotion” may be used herein interchangeably with the terms initiation, acceleration, increase or the like, to denote the above meaning.
  • angiogenesis refers to the manifestation of the angiogenic cascade as a whole, resulting in vessel sprouting, at minimum. It is notable that the presented results, for the first time, demonstrate the in vivo stimulatory effect of PMP on blood vessel growth as an integral process in ischemic region in contrast to their in vitro effects on various differential responses of endothelial cells that were described before (12).
  • the angiogenic process in the context of the present invention also concerns the in situ application of PMPs to promote organ specific angiogenesis (blood vessel growth, sprouting and/or development) to achieve improvement in the organ's function, e.g.
  • the PMP is used to initiate or promote angiogenesis where it is desired for the purpose of treating a pathological condition.
  • pathological condition in the context of the present invention denotes any condition, disease or disorder which may benefit from angiogenesis, vascularization or revascularization.
  • a pathological condition may be characterized by reduction or abolition of blood supply within an organ or part of an organ, which may be caused by the constriction or obstruction of a blood vessel.
  • ischemia or "ischemia related conditions” or "condition related to ischemia”.
  • ischemia In heart disease, ischemia is often used to describe the heart muscle that is not getting the proper amount of oxygen- rich blood because of narrowed or blocked coronary arteries. The symptoms of ischemia depend on the organ that is "ischemic". With the heart, ischemia often results in angina or "chest pain” that can be an early warning of an impending heart attack. In the brain, ischemia can result in a stroke.
  • Non-limiting examples for pathological conditions which relate to ischemia include myocardial ischemia, limb ischemia, tissue ischemia, ischemia-reperfusion injury, angina, coronary artery disease, peripheral vascular disease, peripheral arterial disease, ischemic stroke, chronic wound, diabetic wound, myocardial infarction, congestive heart failure, pulmonary infarction, fractured bone healing, stenosis, restenosis, and thromboangiitis obliterans (Buerger's disease).
  • a pathological condition in the context of the invention may be characterized by damage or dysfunction of endothelial cells, i.e. wound.
  • wounds which may be treated by the use of PMP in accordance with the invention are chronic wound, diabetic wound, ulcer, and burns.
  • pathological condition will be considered equivalent to the terms "disease” or "disorder”.
  • the PMP are administered to a subject in need by any route available in the art, other than by vascular (intravascular or intra-arterial) administration.
  • extravascular administration is preferably employed to achieve the desired therapeutic effect.
  • extravascular administration denotes, without being limited thereto, any one of the following: subcutaneous, intramuscular, intra-peritoneal, or administration into parenchymal tissue or organs (intra-parenchymal) such as lung, heart or brain.
  • the PMP are administered to or in proximity to a target tissue or organ, i.e. directly to the area at which the induction or promotion of an angiogenic process is of interest.
  • the administration of PMP concerns local administration by any method known to those versed in the art.
  • the PMP may be implanted at the area of interest.
  • the "target tissue or organ” is any biological tissue or organ being associated with a pathological condition as defined above. It is typically a damaged tissue or organ suffering from inadequate blood (oxygen) supply. It is to be understood that for achieving a therapeutic effect on the pathological condition (i.e. new vessel formation and thereby improvement of blood (oxygen) supply), an angiogenic process at the target tissue or organ or in its proximity is required.
  • the target tissue or organ is the heart muscle that is not getting the proper amount of oxygen-rich blood because of narrowed or blocked coronary arteries and for achieving a therapeutic effect, the formation of new blood vessels around the infarcted area in the heart muscle to provide the required amount of oxygen to the heart may be beneficial.
  • Endothelial cells proliferation is involved in the initial stages of blood vessel sprouting and growth of blood vessels.
  • it is preferable that endothelial cells are present at the vicinity of the site of treatment (e.g. the site of injection).
  • the amount of PMP administered to the subject in need is such that treatment of the pathological condition is achieved.
  • treatment denotes treatment of a condition, per se, as well as prevention of a condition from developing. These terms refer to the administering of a therapeutically effective amount of PMP in a manner effective to achieve a meaningful induction or promotion of an angiogenic cascade at or in proximity to the target tissue or organ, thereby resulting in a desired therapeutic effect.
  • the desired therapeutic effect may include, without being limited thereto, the proliferation of endothelial cells, the sprouting of blood vessels, the growth of blood vessels, the reconstruction of damaged blood vessels (revascularization), protective effect on ischemic tissue diminishing its need for oxygen and thus reducing the inflicted injury all of which result in the amelioration of undesired symptoms associated with the pathological condition, e.g. chest pain, in case of heart ischemia, the slowing down of progression of the pathological condition or the deterioration of symptoms associated with the condition (as may be evident from, for example, the decrease in chest pains associated with heart ischemia), slowing down any irreversible damage caused in a progressive chronic stage of the condition, e.g. damage to the heart, lessening the severity or curing the condition, the improvement in survival rate or more rapid recoveiy from the condition, or the prevention of the disease form occurring or a combination of two or more of the above.
  • the pathological condition e.g. chest pain
  • the amount of PMP must be effective to achieve the desired therapeutic effect.
  • the amount, or "effective amount” is used herein to mean an amount sufficient to initiate or increase to some beneficial degree, preferably to increase by at least about 30 percent, more preferably by at least 40 percent, more preferably by at least 50 percent, more preferably by at least 60 percent, more preferably by at least 70 percent, more preferably by at least 80 percent, most preferably by at least 90 percent, angiogenesis as compared to untreated controls.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, or until cure is effected or diminution of the disease state is achieved.
  • the amount of PMP per se, or a composition comprising PMP, to be administered will depend upon the subject being treated, the severity of the pathological condition, the manner of administration, the judgment of the prescribing physician, and other factors as known to those versed in the art.
  • An effective amount is typically determined through appropriate dose-fmding clinical studies.
  • the manner of determining an effective dose is within reach of a person versed in the art of clinical development.
  • the dosing schedule will typically be determined on the basis of the pharmacokinetic (PK) properties of the PMP.
  • PK pharmacokinetic
  • the PMP can be stored frozen at -8O 0 C, then thawed once and applied in the target tissue (e.g. ischemic area). Alternatively, the PMP can be lyophilized, kept at 4 0 C and re-suspended before use. In any case, the PMP may be used in combination with a physiologically acceptable carrier or excipients, to form a pharmaceutical composition.
  • a pharmaceutical composition of the invention is to facilitate administration of the PMP as an active ingredient to an individual in need.
  • the phrase "physiologically acceptable carrier” refers to any acceptable vehicle suitable for delivery of the PMP as active ingredient.
  • the vehicle may be a physiological solution, a polymer-based particle including nanopsheres (defined as polymeric spherical matrices) and nanocapsules (defined as tiny oil cores surrounded by a distinct wall polymer) wherein the PMP may be encapsulated in the core of particle or adsorbed at the surface of the particle, implantable pellets, and others.
  • an “excipient” refers to an inert substance added to the PMP to further facilitate administration thereof, as known to those versed in the art.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent, inter alia, upon the route of administration chosen.
  • the PMP are formulated in a form suitable for topical application or administration.
  • the PMP may be formulated in forms suitable for subcutaneous administration, intramuscular administration, intra-organ administration, intra-peritoneal administration, and intra-parenchymal administration.
  • the carrier may at times have the effect of the improving the delivery of PMP to the endothelial cells in vicinity to the target tissue or organ.
  • the carrier may also be a substance that stabilizes the formulation (e.g. a preservative).
  • a preservative e.g. a preservative
  • carriers, stabilizers and adjuvants see E. W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co (June, 1990).
  • compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the choice of carrier will be determined in part by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable pharmaceutical compositions of the present invention.
  • the PMP may be combined with a physiologically and pharmaceutically acceptable (biocompatible) carrier, such as a polymeric carrier, a vesicle, (e.g. liposome), beads (e.g. immunobeads), as known in the art of drag delivery.
  • a physiologically and pharmaceutically acceptable (biocompatible) carrier such as a polymeric carrier, a vesicle, (e.g. liposome), beads (e.g. immunobeads), as known in the art of drag delivery.
  • a physiologically and pharmaceutically acceptable (biocompatible) carrier such as a polymeric carrier, a vesicle, (e.g. liposome), beads (e.g. immunobeads), as known in the art of drag delivery.
  • biocompatible carrier such as a polymeric carrier, a vesicle, (e.g. liposome), beads (e.g. immunobeads), as known in the art of drag delivery.
  • a biocompatible polysaccharide e.
  • the biocompatible polymeric carrier may be a naturally occurring, semisynthetic, or synthetic polymer.
  • the polysaccharide when using a polysaccharide as the carrier, the polysaccharide may be a naturally occurring, semi-synthetic, or synthetic polysaccharide.
  • Platelet units ( ⁇ 50 ml) were obtained from Blood Bank in Hadassah Medical Center, Jerusalem. Platelet rich plasma (PRP) was isolated by centrifugation (12O x g). Platelets (20 ml PRP, 6-12 x 10 6 / ⁇ l) were washed twice at 750 x g in the presence of 5 mM citric acid, resuspended ill 0.5 ml PBS containing calcium and magnesium, and 5 U/ml human thrombin was added. After 5 min incubation with slow shaking, platelet aggregates were removed by 5 min centrifugation at 1500 x g.
  • PRP Platelet rich plasma
  • Microparticles were isolated as described elsewhere (8). Supernatant was collected, centrifuged at 100 000 x g for 1 h at 4°C, and the pelleted microparticles were resuspended in 400 ⁇ l of PBS. Upon isolation, PMP were characterized using a flow cytometer with mAb against CD41, and the presence of whole platelets in the suspension was ruled out. The amount of PMP applied in the experiments was expressed as total protein concentration, determined using the Bradford method. PMP were stored at -80 0 C until used in experiments.
  • PMP 100 - 500 ⁇ g/ml protein
  • B16 murine melanoma cell line 4 x 10 5 cells
  • PBS PBS
  • Melanoma cells injected in the absence of PMP served as a control.
  • the animals were sacrificed and the lungs were excised and fixed with Bouen solution (Sigma, Israel) for 24 h. The number of metastases was counted under microscope and calculated statistically using Student's t-test.
  • Nicosia and Ottinetti [Nicosia, R.F., and Ottinetti, A. 1990. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest 63:115-122.] was used with some modifications, as described elsewhere (2).
  • the thoracic aorta was isolated from Sabra rats (weight 160-190 g), placed into warm Bio-MPM medium, and cleaned from the surrounding tissues by fine microdissecting forceps and scissors. The aorta was sliced into rings 1 mm wide.
  • the rings were then washed three times in sterile warm Bio-MPM medium containing penicillin and streptomycin, and embedded in collagen gel obtained from rat tail tendons, as described by Mintesano et al. [Montesano, R., et al. 1983. In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J Cell Biol 97:1648-1652.].
  • the collagen solution was prepared by mixing 7 parts of collagen with 1 part of MEM x 10 medium and 2 parts of 0.3 M NaHCO 3 .
  • the ring-containing collagen gel was prepared in 24-well tissue culture plates in quadruplicate and Bio-MPM (500 ⁇ l; supplemented with 1% glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) was added. PMP, with or without blocking mAbs and inhibitory compounds, were added to the medium in the appropriate wells. Wells containing only medium were used as a control. The plates were maintained for 1 week (37°C, 8% CO 2 , humidified atmosphere), and the medium with all constituents was changed eveiy two days.
  • the cultures were fixed with 4% formalin for 24 h and stained with 0.02% crystal violet solution in ethanol (Sigma).
  • the stained samples were photographed with a Nikon (Coolpix 990, Japan) camera connected to the microscope (Olympus CK40, Japan; magnification x 20). Measurement of the area covered by blood vessels (in mm") was performed using the software ImagePro 4.5 (Media Cybernetics, USA).
  • the first aim was to determine whether PMP affect vessel development in vitro.
  • the aortic ring assay was employed, in which an intensive angiogenic response to human cytokines (e.g., VEGF, FGF 5 endostatin) and platelets was previously shown (2).
  • cytokines e.g., VEGF, FGF 5 endostatin
  • PMP in increasing concentrations (1 - 100 ⁇ g/ml) strongly induced sprouting. This was statistically significant starting from a concentration as low as 30 ⁇ g/ml (sprouts area of 2.94 ⁇ 0.2 mm" in the control).
  • Angiogenesis is a multi-step process controlled by different signal transduction pathways.
  • PI3-kinase is known to play a pivotal role in mediating EC survival, proliferation, cytoskeleton reorganization and cell motility, all of these features being critically important for vessel growth [Brader, S., and Eccles, S.A. 2004. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tiimori 90:2-8].
  • neutralization of PI3-kinase led to the complete inhibition of not only sprouting, but also of cell proliferation and movement in the ring vicinity.
  • PI3 -kinase is activated by such angiogenesis-related cytokines as VEGF and FGF [Qi, J.H., et al. 1999. Phosphoinositide 3 kinase is critical for survival, mitogenesis and migration but not for differentiation of endothelial cells. Angiogenesis 3:371-380]. Activation of PI3-kinase may occur by several mechanisms. VEGF receptor FIk-I /KDR on EC has been shown to be constitutively associated with the regulatory subunit of PI3-kinase p85 [Thakker, G.D., et al. 1999.
  • PI3-kinase The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J Biol Chem 274: 10002-10007] and to directly activate PI3-kinase upon binding to its ligand, VEGF. Furthermore, FGF receptor- 1 is known to activate PI3-kinase in a tyrosine-766-dependent fashion. In addition, activation of PI3 -kinase may proceed via Src kinase family members [Schlessinger, J. 2000. Cell signaling by receptor tyrosine kinases. Cell 103:211- 225].
  • PKC protein kinase Calpha
  • PI3-kinase seems to be important for both sprouting and cell movement since blocking of PI3 -kinase results in a "naked ring" without even one cell in the vicinity of the ring.
  • no effect on angiogenesis was observed following blocking of PKC and p38 kinase (5.6 ⁇ 2.7 mm 2 and 4.87 ⁇ 1.7 mm 2 , respectively; Fig. 3).
  • a mutual action of the cytokines inside PMP triggers very specific pathways in endothelial cells, leading to cell proliferation, movement, and angiogenesis, whereas other routes remain intact.
  • Endothelial cells EAhy.926 were grown on watch glasses in DMEM with 15% FCS until a monolayer was formed.
  • PMP 100 ⁇ g/ml
  • integrilin, tirofibane or EDTA 2 mM
  • ISh 37 0 C, 8% CO 2 , humidified atmosphere.
  • the cells were gently washed 3 times with PBS 5 fixed with cold methanol, and incubated with the anti-CD41 mAb 3.2 mg/ml for Ih at room temperature (RT).
  • the watch glasses were mounted to the slides and analysed using a Zeiss LSM-410 confocal laser scanning system attached to a Zeiss Axiovert microscope.
  • Bovine aortic endothelial cells were maintained at 37 0 C, 8% CO 2 , humidified atmosphere, in DMEM low glucose, supplemented with 10% heat- inactivated fetal calf serum, L-glutamine, 1 % penicillin/streptomycin and 1 ng/ml bFGF for at least 3 days until a 80% confluent monolayer was formed. The cells were then separated from the surface by trypsine, washed in the medium without serum, and seeded into upper compartment of blind well Boy den chambers (210 ⁇ l, 2 x 10 5 cells). The same medium, with or without PMP and the inhibitors, was placed into the lower compartment.
  • a membrane (Whatman, pore size 8 ⁇ m) coated with matrigel (50 ⁇ l, dissolved 1 :3 in PBS), and allowed to dry in a hood, separated each compartment.
  • the system was incubated for 18 h (37°C, 8% CO 2 , humidified atmosphere), after which the membranes were fixed with ice-cold methanol (5 min) and stained with Diff-Quik Staining Kit (Dade Behring Inc, Newark, DE, USA) according to the manufacturer's instructions. Cells from the upper side of the membrane were removed by gentle wiping with cotton. Endothelial cells that filtered to the lower outlet of the membrane were photographed and counted over the whole membrane.
  • Agarose gel (4%, 30 ⁇ l) was mixed with PBS, VEGF, bFGF, or PMP (8 ⁇ l) and allowed to polymerize for 30 seconds. The resulting beads were placed in a Petri dish on ice for up to 1 h. Sabra mice were narcotized with ketamine and xylazine. The dorsal skill was incised and the agarose beads introduced into the subcutaneous space. After 72 h, the mice were re-opened and the beads with surrounding skin photographed.
  • vWF von Willebrand factor
  • EC endothelial cells
  • Paraffin-embedded skin sections (4 ⁇ m) were deparaffinized by heating (30 min, 60 0 C) and rinsing with xylene (3 x 10 min), and dehydrated by immersing in increased concentrations of ethanol.
  • Intrinsic peroxidase activity was blocked by H 2 O 2 (3% solution in methanol, 15 min). The samples were treated with pronase (Sigma; 0.1%, 30 min, at room temperature (RT)) and incubated with anti-vWF antibody (1 :200, 18 h, 4°C). A Histostain-Plus Kit (Zymed Laboratories, San Francisco, CA) was used for staining, according to the manufacturer's instructions.
  • Figs. 6G-6H represent quantitative evaluation of the results obtained in three independent experiments performed in duplicate. Consequently, PMP are also capable of inducing vessel growth also in in vivo conditions.
  • Sabra rats were anesthetized with intraperitoneal ketamine and xylasine, intubated by venflon 18 GA and ventilated with positive pressure ventilation.
  • a left thoracotomy was performed in the fourth intercostal space and the pericardium opened to expose the heart.
  • a 7 — 0 silk suture was passed around a prominent branch of the left coronaiy artery with a taper needle and ligated.
  • a cyanotic region was delineated on the surface of middle to apical portion of the left ventricle, corresponding to the area of severe ischemia. The development ischemia was confirmed by ECG changes.
  • Rats constituted each experimental group; with a mortality rate after experimental myocardial infarction of up to 40-50%, at least 18 rats were treated or operated on for each group. Rats were maintained for three months following operation. Heart echocardiography was performed on days 0 (before operation, baseline), 1 (24 h after operation), 7, 14, 30, 60 and 90. Thereafter, rats were sacrificed and their hearts excised for histological evaluation of angiogenesis using BSA-FITC (5 rats from each group) and for whole-heart staining for myocardial zone assessment (5 rats from each group).
  • Sham-operated rats were anesthetized and a sham chest operation, but no manipulation of the heart was made. Functional tests were performed, the rats were sacrificed, and hearts were excised for analysis.
  • a control group (PBS only) were given an injection of 50 ⁇ l phosphate buffer saline (PBS) directly into cardiac muscle upon operation, without LAD ligation.
  • An infarcted-PMP group underwent LAD ligation followed by four injections of PMP (250 ⁇ g/ml protein totally) in PBS (20 ⁇ l per each injection) at a distance of 2 mm from the center of the cyanotic region in 4 directions oriented 90 degrees apart from one another.
  • Fig. 8A and Fig. 8B show that as soon as 24 hours after on-set of ischemia both parameters characterizing cardiac muscle functioning were higher in PMP- injected animals i.e., after PMP injection myocardium operated more effectively.
  • ejections fraction remained elevated in PMP-given mice; fraction shortening without PMP failed to recover whereas in the presence of PMP this parameter was continuously improving during the observation period.
  • the mortality rate of rats injected with PMPs vs. control animals as described above was determined (Fig. 9).
  • PMP Treatment may show Advantages Over Other Platelet Products and VEGF
  • Rats are fed for 8 weeks with a high-cholesterol diet (3% cholesterol, 0.5% cholic acid, 0.2% 6-propyl 2-thiouracil, 5% sucrose, 10% lard, and 81.3% regular rat chow).
  • Rats are sacrificed, and specimens of thoracic and abdominal aorta are excised and fixed in Bouin's solution, dehydrated, embedded in paraffin and cut in 5 ⁇ m sections. The sections are stained with hematoxylin-eosin-safran (HES), Masson's trichrome (MT) and orcein.
  • HES hematoxylin-eosin-safran
  • MT Masson's trichrome
  • the level of atherosclerosis in the aorta are scored on a 4-point scale: 0, normal; I 5 widening of elastic fibers with few foam cells; 2, fragmentation of elastic lamellae with numerous foam cells and fibrosis; 3, smooth muscle cell proliferation, medial lipid infiltration and fibrosis; and 4, lipid-calcic plaque or ulcerated plaque.

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Abstract

La présente invention concerne des méthodes propres à induire ou à favoriser un angiogenèse in situ, selon lesquelles on administre des microparticules d'origine plaquettaire (PMP) à un tissu ou à un organe cible, ou bien à proximité dudit tissu ou organe. Les méthodes de l'invention peuvent s'utiliser pour le traitement d'affections en rapport avec l'hypoxie tels que l'ischémie. L'invention concerne également des compositions contenant le principe actif PMP pour induction ou promotion in situ de l'angiogenèse. Un groupe particulier de pathologies à traiter au moyen des méthodes et des compositions de la présente invention est constitué par les affections cardiaques, en particulier les maladies cardio-vasculaires, et singulièrement les infarctus du myocarde.
PCT/IL2005/001283 2004-12-01 2005-12-01 Utilisation therapeutique de microparticules d'origine plaquettaire WO2006059329A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI489986B (fr) * 2013-06-07 2015-07-01
EP3397260A1 (fr) * 2015-11-30 2018-11-07 Flagship Pioneering Innovations V, Inc. Procédés et compositions se rapportant à des chondrisomes provenant de produits sanguins
EP3612193A4 (fr) * 2017-04-20 2020-12-30 North Carolina State University Cellules modifiées par des vésicules de plaquettes et vésicules extracellulaires pour la réparation ciblée de tissus
WO2021232015A1 (fr) * 2020-05-15 2021-11-18 Cellphire, Inc. Vésicules extracellulaires dérivées de plaquettes
US11491480B2 (en) 2014-06-13 2022-11-08 Children's Medical Center Corporation Products and methods to isolate mitochondria
US11529587B2 (en) 2019-05-03 2022-12-20 Cellphire, Inc. Materials and methods for producing blood products
US11701388B2 (en) 2019-08-16 2023-07-18 Cellphire, Inc. Thrombosomes as an antiplatelet agent reversal agent
US11767511B2 (en) 2018-11-30 2023-09-26 Cellphire, Inc. Platelets as delivery agents
US11903971B2 (en) 2020-02-04 2024-02-20 Cellphire, Inc. Treatment of von Willebrand disease
US11965178B2 (en) 2018-11-30 2024-04-23 Cellphire, Inc. Platelets loaded with anti-cancer agents

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024419A2 (fr) * 2001-09-18 2003-03-27 Vasogen Ireland Limited Vesicules naturelles simulant l'apoptose et utilisation associee dans un traitement medical
WO2003024422A1 (fr) * 2001-09-18 2003-03-27 Vasogen Ireland Limited Procede permettant d'accelerer la guerison d'un traumatisme au moyen d'entites synthetiques ou naturelles simulant l'apoptose

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024419A2 (fr) * 2001-09-18 2003-03-27 Vasogen Ireland Limited Vesicules naturelles simulant l'apoptose et utilisation associee dans un traitement medical
WO2003024422A1 (fr) * 2001-09-18 2003-03-27 Vasogen Ireland Limited Procede permettant d'accelerer la guerison d'un traumatisme au moyen d'entites synthetiques ou naturelles simulant l'apoptose

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRILL A ET AL: "Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization", CARDIOVASCULAR RESEARCH, vol. 67, no. 1, 1 July 2005 (2005-07-01), pages 30 - 38, XP004931736, ISSN: 0008-6363 *
DASHEVSKY OLGA ET AL: "Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization.", BLOOD, vol. 104, no. 11, Part 2, November 2004 (2004-11-01), & 46TH ANNUAL MEETING OF THE AMERICAN-SOCIETY-OF-HEMATOLOGY; SAN DIEGO, CA, USA; DECEMBER 04 -07, 2004, pages 65B, XP009062405, ISSN: 0006-4971 *
KIM H K ET AL: "Platelet microparticles induce angiogenesis in vitro", BRITISH JOURNAL OF HAEMATOLOGY 2004 UNITED KINGDOM, vol. 124, no. 3, February 2004 (2004-02-01), pages 376 - 384, XP002369293, ISSN: 0007-1048 *
NOMURA S ET AL: "Function and clinical significance of pletelet-derived microparticles", INTERNATIONAL JOURNAL OF HEMATOLOGY, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 74, no. 4, 2001, pages 397 - 404, XP002991725, ISSN: 0925-5710 *
RATAJCZAK M Z ET AL: "Platelet- and tumor-cell-derived microparticles: New-found regulators of angiogenesis", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 43, March 2002 (2002-03-01), & 93RD ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH; SAN FRANCISCO, CALIFORNIA, USA; APRIL 06-10, 2002, pages 28, XP001246502, ISSN: 0197-016X *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI489986B (fr) * 2013-06-07 2015-07-01
US11491480B2 (en) 2014-06-13 2022-11-08 Children's Medical Center Corporation Products and methods to isolate mitochondria
EP3397260A1 (fr) * 2015-11-30 2018-11-07 Flagship Pioneering Innovations V, Inc. Procédés et compositions se rapportant à des chondrisomes provenant de produits sanguins
EP3397260A4 (fr) * 2015-11-30 2019-10-16 Flagship Pioneering Innovations V, Inc. Procédés et compositions se rapportant à des chondrisomes provenant de produits sanguins
US11903975B2 (en) 2015-11-30 2024-02-20 Flagship Pioneering Innovations V, Inc. Methods and compositions relating to chondrisomes from blood products
US11903974B2 (en) 2015-11-30 2024-02-20 Flagship Pioneering Innovations V, Inc. Methods and compositions relating to chondrisomes from cultured cells
EP3612193A4 (fr) * 2017-04-20 2020-12-30 North Carolina State University Cellules modifiées par des vésicules de plaquettes et vésicules extracellulaires pour la réparation ciblée de tissus
US11767511B2 (en) 2018-11-30 2023-09-26 Cellphire, Inc. Platelets as delivery agents
US11965178B2 (en) 2018-11-30 2024-04-23 Cellphire, Inc. Platelets loaded with anti-cancer agents
US11752468B2 (en) 2019-05-03 2023-09-12 Cellphire, Inc. Materials and methods for producing blood products
US11813572B2 (en) 2019-05-03 2023-11-14 Cellphire, Inc. Materials and methods for producing blood products
US11529587B2 (en) 2019-05-03 2022-12-20 Cellphire, Inc. Materials and methods for producing blood products
US11701388B2 (en) 2019-08-16 2023-07-18 Cellphire, Inc. Thrombosomes as an antiplatelet agent reversal agent
US11903971B2 (en) 2020-02-04 2024-02-20 Cellphire, Inc. Treatment of von Willebrand disease
WO2021232015A1 (fr) * 2020-05-15 2021-11-18 Cellphire, Inc. Vésicules extracellulaires dérivées de plaquettes

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