WO2018109525A1 - Nouvelles vésicules extracellulaires anti-angiogéniques - Google Patents

Nouvelles vésicules extracellulaires anti-angiogéniques Download PDF

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WO2018109525A1
WO2018109525A1 PCT/IB2016/057608 IB2016057608W WO2018109525A1 WO 2018109525 A1 WO2018109525 A1 WO 2018109525A1 IB 2016057608 W IB2016057608 W IB 2016057608W WO 2018109525 A1 WO2018109525 A1 WO 2018109525A1
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mscs
evs
anyone
angiogenesis
angiogenic
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PCT/IB2016/057608
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English (en)
Inventor
Antonella Viola
Maurizio Muraca
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Università Degli Studi Di Padova
Fondazione Città Della Speranza - Onlus
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Priority to PCT/IB2016/057608 priority Critical patent/WO2018109525A1/fr
Priority to EP17823225.2A priority patent/EP3554513A1/fr
Priority to AU2017374947A priority patent/AU2017374947B2/en
Priority to CA3052739A priority patent/CA3052739A1/fr
Priority to PCT/IB2017/057928 priority patent/WO2018109700A1/fr
Priority to CN201780086372.2A priority patent/CN110312515B/zh
Publication of WO2018109525A1 publication Critical patent/WO2018109525A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells

Definitions

  • the present invention discloses new anti-angiogenic extracellular vesicles (extracellular vesicles, EV) formed from stem/ stromal mesenchymal cells (also known as mesenchymal stem cells, MSC), said EVs for the treatment of diseases characterized by an increased vascularisation, pharmaceutical compositions comprising the same and methods for the preparation of said EVs.
  • extracellular vesicles extracellular vesicles, EV
  • stem/ stromal mesenchymal cells also known as mesenchymal stem cells, MSC
  • pharmaceutical compositions comprising the same and methods for the preparation of said EVs.
  • Angiogenesis is the process that leads to the formation of new blood/lymphatic vessels from a pre-existing vascular system, plays a key role in many physiological processes (e.g. during foetal development and in tissue regeneration) as well as in many pathological conditions such as ischemic, inflammatory, autoimmune diseases and in cancer.
  • angiogenesis consists in the succession of finely regulated different phases that are characterized by endothelial and extra-cellular matrix modifications.
  • an increase in the permeability of the vessels with decreased connection between endothelial cells is observed.
  • the disruption of the capillary basal membrane which is essential to enable the tissue invasion by new vessels.
  • the endothelial cells organize by forming new vascular lumens.
  • the newly formed capillary is stabilized with the construction of the basal membrane and intercellular junctions. This process can be altered when vascular insufficiency occurs (such as in stroke) or, on the opposite, by excess proliferation, such as in haemangiomas, in tumours and in retinopathies.
  • angiogenesis and inflammation are two closely interconnected processes.
  • angiogenesis Upon inflammation, if the inflammatory stimulus persists, angiogenesis is initiated by the migration of endothelial cells lining the venules into the tissue. The generation of new blood vessels is required for the survival of inflammatory cells within the tissue, and thus inhibition of factors that promote angiogenesis may reduce inflammation and prevent its pathological consequences such as inflammatory tissue damage, autoimmunity, fibrosis or tumour growth.
  • angiogenesis is a very interesting therapeutic approach for various pathologies.
  • a positive control is desired when for all the pathologies wherein the formation of new vessels has a therapeutic effect
  • a negative control is desired for all the pathologies in which angiogenesis plays a key role in the triggering and/or maintenance of the disease such as in inflammatory diseases, proliferating diseases (retinopathies, cancers, neoplastic diseases), autoimmune diseases, fibrosis, transplant rejections and the like.
  • Many molecules that interfere with angiogenesis have been developed and tested both in preclinical and clinical stages, but their effectiveness is generally limited.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • pericytes display similar morphological and functional features, although the two cell types are likely to have different functions in vivo. While pericytes regulate capillary homeostasis and architecture, the in vivo functional role of MSC is less clear and it is likely to be tissue-specific.
  • MSC contribute to the formation of the "niche" for the hematopoietic stem cells (HSCs), thus providing an appropriate microenvironment for haematopoiesis.
  • HSCs hematopoietic stem cells
  • MSCs may be involved in homeostatic control and tissue repair.
  • MSCs have a potent stabilizing effect on the vascular endothelium, having the capacity of inhibiting endothelial permeability after traumatic brain injury and in haemorrhagic shock. Therefore, the vascular endothelium seems to be a specific target of MSC biological activity.
  • Zanotti et al Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1, Leukemia (2016) 30, 1143-1154) the effect being mediated by soluble factors.
  • MSCs may cause tumour formation or aberrantly differentiate after ectopic engraftment.
  • MSCs may form aggregates that could cause pulmonary emboli or infarctions in patients.
  • E-MSCs Encapsulation of MSCs
  • the authors of the present invention provide a highly effective tool for the inhibition of angiogenesis that does not present the drawbacks known to be associated with the use of MSCs.
  • EVs MSCs isolated from MSCs cells cultured in the presence of pro-inflammatory cytokines exhibit an anti-angiogenic activity thereby maintaining the anti-angiogenic activity exhibited by the MSCs cells due to proteins secreted by said cells.
  • the present invention provides, for the first time, Extracellular Vesicles released by Mesenchymal stem/ stromal Cells (EVs MSCs) said EVs MSCs exhibiting angiogenic inhibitory activity.
  • EVs MSCs Mesenchymal stem/ stromal Cells
  • EVs have already been used as therapeutic tools in the art, they are complex biological particles that transmit a series of signals, and may therefore interfere at various levels with both the angiogenic process that with that inflammatory, on the contrary of the drugs currently in use, which act selectively on a specific street or metabolic stage.
  • the new EVs MSCs of the invention provide a new, safe and powerful tool that can be used for the therapy of diseases in which angiogenesis plays a pathogenic role.
  • EVs MSCs Mesenchymal stem/ stromal Cells
  • said EVs MSCs for use in therapy, a pharmaceutical composition comprising said EVs MSCs) wherein said EVs MSCs, and at least one pharmaceutically acceptable carrier; said pharmaceutical composition for use in therapy; a medical device comprising said EVs MSCs; a process for the preparation of said EVs MSCs, EVs MSCs obtainable or obtained by said process, a pharmaceutical composition comprising the same, their use in therapy and, the use of the EVs MSCs of the invention, in any embodiment described for the preparation of a medicament and a medical treatment comprising the step of administering to a subject in need thereof a therapeutically effective amount of the EVs MSCs of the invention.
  • Figure 1 EVs isolated from primed MSC inhibit angiogenesis in vitro.
  • the figure shows the results of a comparative test on the effects of MSC EVs obtained from MSC cells stimulated with inflammatory agents according to the process of the invention and of MSC EVs obtained from unstimulated MSCs.
  • the figure reports the quantification of segment length of tubes produced by SVEC4-10 cells cultured either with EVs obtained from MSC stimulated with inflammatory agents according to the process of the present invention (EV st MSC- CM) or with EVs obtained from MSC that were not stimulated with inflammatory agents (EV unst MSC-CM).
  • EV st MSC-CM EVs obtained from MSC stimulated with inflammatory agents according to the process of the present invention
  • EV unst MSC-CM EV unst MSC-CM
  • Figure 2 EVs isolated from primed MSC affect angiogenesis in vivo.
  • the figure shows the results obtained on vascularization model on a matrigel plug. The experiment is described in detail in the examples section.
  • Anesthetized 12-week-old male C57BL/6N mice were subcutaneously injected in the dorsal back either with 5xl0 5 unst- or st-MSC, or EVs obtained from the same cells, mixed with 500 ⁇ 1 Matrigel.
  • Matrigel plus 50 ng/mL VEGF and lOO ng/mL bFGF was used as positive control.
  • Bare Matrigel was injected as negative control. The plug was removed after 10 days and the vascularization of each plug was evaluated by haemoglobin quantification.
  • the figure shows that the plugs supplemented with EV st MSC-CM or with st MSCs showed a vascularization that was even below the one observed in the negative control thereby demonstrating the in vivo antiangiogenic effect of the EVs MSC of the invention.
  • Fig. 3 EVs isolated from primed MSC affect angiogenesis in the developing mouse retina.
  • the figure shows the results obtained using a model of vascularization in the developing mouse retina upon systemic treatment with EVs obtained from stimulated or unstimulated MSC. The experiment is described in detail in the examples section.
  • the retinal vascular expansion was calculated as the ratio between the vascular radius (v) and the retinal radius (R).
  • the figure shows how systemic administration of the EVs of the invention has a direct effect on the retinal vascularization.
  • MSC Mesenchymal stem cells
  • MSC Mesenchymal stem cells
  • MSCs When derived from fetal membranes, such as chorionic and amniotic membranes, MSCs are considered an intermediate between human embryonic stem cells (hESCs) and adult stem cells. When the meaning is referred to human MSCs, non-embryonic MSCs are considered as a possible preferred embodiment of the invention.
  • Extracellular Vesicles released by Mesenchymal stem/stromal Cells are Extracellular Vesicles secreted by Mesenchymal stem/stromal Cells.
  • EVs MSCs include all kind of EVs secreted/released by MSCs.
  • MSCs secrete a wide range of extracellular vesicles (EVs) of different size, morphology, content and function that interact with target cells and modify their phenotype and function.
  • EVs can be classified according to their size, origin, and isolation methods, into three main classes: (i) Microvesicles or shedding vesicles (size between 50 and 1000 nm, budding from the plasma membrane, and enriched in CD40); (ii) Apoptotic bodies (size between 800 and 5000 nm, derived from fragments of dying cells, and enriched in histones and DNA); and (iii) Exosomes, which are small (-30-120 nm) membrane vesicles from endocytic origin (enriched in late endosomal membrane markers, including TsglOl, CD63, CD9, and CD81).
  • Microvesicles or shedding vesicles size between 50 and 1000 nm, budding from the plasma membrane, and enriched in CD40
  • Apoptotic bodies size between 800 and 5000 nm, derived from fragments of dying cells, and enriched in histones and DNA
  • Exosomes which are small (-30-120 nm) membrane
  • angiogenesis may encompass the formation of new blood vessels and/or lymphatic vessels from preexisting vessels (also known in the art, respectively as angiogenesis and lymphangiogenesis).
  • blood vessels angiogenesis and lymphatic vessels angiogenesis are encompassed by the more general angiogenesis.
  • the term may refer to the formation of new blood vessels, to the formation of new lymphatic vessels or to the formation of both.
  • anti-angiogenic or "exhibiting angiogenesis inhibitory activity” are considered as synonyms and indicate a compound, a cell, a vesicle, a composition, a molecule, a mixture, a moiety, a substance, a product, inhibiting angiogenesis in vitro and/or in vivo as defined above.
  • the inhibition may be at least partial i.e. a reduced angiogenesis when compared to a positive control that is not treated with the anti-angiogenic compound, cell, vesicle, composition, molecule, mixture, moiety or substance tested, or total i.e.
  • a therapeutically effective amount is an amount sufficient to exert in a patient or in a disease model assayed, an inhibition of the angiogenic activity as defined above in the site of interest, thereby provoking at least a reduction of the symptoms of the disease treated.
  • therapeutically effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
  • subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
  • EVs MSCs Mesenchymal stem/stromal Cells
  • angiogenic inhibitory activity or anti-angiogenic activity as a possible synonym in the whole description as stated above.
  • the EVs MSCs of the present invention exhibit said angiogenic inhibitory activity is exhibited by said EVs MSCs in vitro and/or in vivo. In a preferred embodiment said activity is exhibited both in vitro and in vivo.
  • the anti-angiogenic EVs MSCs claimed exhibit a detectable and/or measurable angiogenic inhibitory activity.
  • said activity can be assayed in standard in vitro tube formation assay.
  • Tube formation assay is a standard test, known in the art and well reported in literature, to measure/assess the angiogenic (negative or positive) activity of a product.
  • any tube formation assay disclosed in the art is suitable for measuring/detecting the anti-angiogenic activity of the EVs MSCs of the invention.
  • the tube formation assay can be carried out as described in Ponce, 2009. Methods Mol Biol. 2009;467: 183-8. doi: 10.1007/978-l-59745-241-0_10.Tube formation: an in vitro matrigel angiogenesis assay.
  • the tube formation assay according to the present description can be carried out as detailed in the experimental section.
  • the angiogenic inhibitory activity of the EVs MSCs of the invention can be assessed an in vivo matrigel plug vascularisation assay.
  • the matrigel plug vascularisation assay is a standard assay to assess the angiogenic activity of a product.
  • any matrigel plug vascularisation assay disclosed in the art is suitable for measuring/detecting the anti-angiogenic activity of the EVs MSCs of the invention.
  • the matrigel plug vascularisation assay can be carried out as described in Brown et al, 2016. Methods Mol Biol. 2016; 1430: 149-57. doi: 10.1007/978-l-4939-3628-l_9.Tube-Forming Assays.Brown RM1, Meah CJ2, Heath VL2, Styles IB3, Bicknell R4.
  • matrigel plug vascularisation assay according to the present description can be carried out as detailed in the experimental section.
  • angiogenesis encompasses blood vessels angiogenesis and/or lymphatic vessels angiogenesis, therefore, according to an embodiment of the invention, the EVs MSCs described above and in anyone of the following embodiments may exhibit inhibitory activity of blood vessels angiogenesis and/or of lymphatic vessels angiogenesis
  • EVs MSCs of anyone of the embodiments herein disclosed is suitable for use in therapy.
  • angiogenesis is a feature of various diseases. Inflammatory diseases are characterised by angiogenic activity, proliferative diseases are characterised by angiogenic activity, autoimmune diseases are also characterised by angiogenic activity as well as transplant rejection.
  • the EVs MSCs of the invention are particularly useful in the treatment of diseases, and or condition wherein the inhibition of angiogenesis results in a therapeutic effect.
  • the EVs MSCs of the invention are suitable for use in therapies wherein inhibition of angiogenesis is indicated.
  • the EVs MSCs of the invention are particularly suitable for use in the treatment of pathologies or conditions characterised by the pathological formation of new blood and/or lymphatic vessels by angiogenesis.
  • said pathologies or conditions are selected from proliferative, inflammatory and autoimmune diseases or conditions, ocular and periodontal diseases, obesity- and aging-related conditions.
  • said proliferative diseases are selected from cancers; said inflammatory/autoimmune diseases are selected from psoriasis, arthritis, endometriosis, Crohn's disease, inflammatory bowel disease; said ocular diseases are selected from diabetic retinopathy, retinopathy of prematurity, radiation and solar retinopathies, macular degeneration; said periodontal diseases are selected from gingivitis and periodontitis; said obesity-related conditions are selected from obesity-driven neovascularization and obesity-driven inflammation; said aging- related conditions are selected from wrinkle and cutaneous aging.
  • Another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising Extracellular Vesicles released by Mesenchymal stem/stromal Cells (EVs MSCs) wherein said EVs MSCs exhibit angiogenic inhibitory activity and at least one pharmaceutically acceptable carrier.
  • EVs MSCs Extracellular Vesicles released by Mesenchymal stem/stromal Cells
  • the pharmaceutical composition of claim 11 characterised in that said angiogenic inhibitory activity is exhibited by said EVs MSCs both in vitro and in vivo and may be assayed via an in vitro tube formation assay and/or an in vivo matrigel plug vascularisation assay.
  • the pharmaceutical composition will exert anti angiogenic activity over blood vessels angiogenesis and/or a lymphatic vessels angiogenesis.
  • composition of the invention will be prepared in a form suitable for both systemic (enteral and parenteral) and topical administration.
  • Non limiting examples of said administration modes are intravenous, intramuscular, intra-organ, transdermal, rectal, eyedrops, intravitreal and others commonly used in the art.
  • the pharmaceutical composition of can be in the form of a solution, a suspension, a cream, a foam, an emulsion, a paste, a gel, a lotion, a shake lotion, an ointment, a transdermal patch, a powder, a sponge, eye drops, a tape, a suppository, an enema.
  • Solutions and emulsions can be suitable either for systemic either for topical administration or both.
  • the EVs MSCs of the invention directly be administered alone and is usually preferably made into various pharmaceutical preparations.
  • the pharmaceutical preparations can be produced by a routine method of pharmaceutics by mixing the active ingredient with one or two or more pharmacologically acceptable carriers.
  • a carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic or topical administration or parenteral injection.
  • the carrier will usually comprise sterile water, at least In large part, though other ingredients, for example, to aid solubility, may be included.
  • injectable solutions for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
  • injectable suspensions may also be prepared in which case appropriate liquid carriers and suspending agents may be employed.
  • the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause a significant deleterious effect to the skin.
  • suitable additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
  • These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.
  • composition of the invention will be suitable for use in therapy.
  • the composition will be useful for the treatment of any of the diseases or condition already described above.
  • the composition is useful for the treatment
  • said inflammation-associated pathologies can be selected from proliferative diseases, inflammatory diseases, autoimmune diseases, transplant rejections proliferative, inflammatory and autoimmune diseases or conditions, ocular and periodontal diseases, obesity- and aging-related conditions.
  • said proliferative diseases are selected from cancers; said inflammatory/autoimmune diseases are selected from psoriasis, arthritis, endometriosis, Crohn's disease, inflammatory bowel disease; said ocular diseases are selected from diabetic retinopathy, retinopathy of prematurity, radiation and solar retinopathies, macular degeneration; said periodontal diseases are selected from gingivitis and periodontitis; said obesity-related conditions are selected from obesity-driven neovascularization and obesity-driven inflammation; said aging- related conditions are selected from wrinkle and cutaneous aging.
  • the invention relates to a medical device comprising the EVs MSCs or the pharmaceutical composition as described herein, in a particular embodiment said medical device is in the form of a transdermal patch, in another embodiment said medical device is in the form of a dispenser for intranasal administration.
  • the present invention also relates to a process for the preparation of MSCs EVs comprising the following steps:
  • said one or more pro-inflammatory cytokine can be selected from IL- ⁇ , IL-6, TNFa and chemokines . Therefore, during step a) of the process of the invention, one or more of the pro-inflammatory cytokines listed above can be used to complement the growth medium used.
  • said pro-inflammatory cytokines are at least two or at least three.
  • said pro-inflammatory cytokines are represented by a mixture of IL- ⁇ , IL-6 and TNFa.
  • a suitable amount of pro-inflammatory cytokines for carrying out step a. can be represented by a total amount of cytokines of about 30-70 ng/ml.
  • the concentration can be concentration between from 15 ng/ml to 40 ng/ml of each cytokine in case two cytokines are used or of between from between from 15 ng/ml to 30 ng/ml of each cytokine in case three cytokines are used.
  • any known medium commonly used for growing MSCs is suitable for carrying out steps a. and b. of the invention.
  • the medium will be further complemented with standard additional compounds/sub stances commonly used for the cultivation of MSCs such as bovine foetal serum (FBS) in suitable amounts, antibiotics, suitable amino acids and the like.
  • FBS bovine foetal serum
  • FBS can be from 5 to 15%, e.g. about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.
  • suitable antibiotics can be any antibiotic commonly used for the culture of MSCs such as streptomycin, penicillin, altri or a mixture thereof. Commonly used amounts of antibiotics can be used in the process of the invention.
  • a non-limiting example is an overall antibiotic amount of 80-150 U/ml (eg. About 100 U/ml). In a non-limiting example about 100 U/ml of penicillin/streptomycin can be used.
  • Further additional complementing substances can be one or more amino acid.
  • about 2 mM glutamine can be added to the growth medium.
  • Suitable media for step a. or b. of the process of the invention are represented by any medium commonly used for culturing MSCs.
  • a non-limiting example of said media is represented by DMEM low glucose or similar media.
  • said MSCs can be cultured from 18 to 30 hours during step a. hence, said MSCs can be cultured about 18, 19, 20, 21, 22, 23, 24, 25, 25 ,27, 28, 29, 30 hours.
  • said MSCs are cultured during step a. for about 22-26 hours, e.g. for about 22, 23, 24, 25 or 26 hours.
  • the MSCs cells can be cultured from 12 to 24 hours during step b.
  • said MSCs can be cultured from 12 to 24 hours during step b. hence, said MSCs can be cultured about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours.
  • said MSCs are cultured during step b. for about 16-20 hours, e.g. for about 16, 17, 18, 19 or 20 hours.
  • the isolation of EVs from culture medium surnatant is known in the art. Any suitable method can be used for carrying out the process of the invention. In a non- limiting embodiment the EVs of the invention can be collected by ultrafiltration of the surnatant.
  • Another object of the invention is represented by the anti-angiogenic EVs MSCs obtainable from the process described above.
  • the invention also encompasses the use in therapy of said EVs MSCs, pharmaceutical compositions comprising said EVs MSCs and uses thereof, and medical devices comprising said anti-angiogenic EVs MSCs and uses thereof.
  • the invention also refers to the medical treatment of all the diseases and conditions described above wherein a therapeutically effective amount of the EVs MSCs of the invention or of the pharmaceutical composition of the invention is administered to a subject in need thereof.
  • MSC were plated and let grow until confluence in ventilated cap flask in DMEM low glucose supplemented with 20% FBS, 2mM glutamine, 100 U/ml penicillin/streptomycin.
  • Culture medium was then substituted with DMEM low glucose supplemented with 10% FBS, 2mM glutamine, 100 U/ml penicillin/streptomycin, with or without 25ng/ml mILIb, 20ng/ml mIL6, 25ng/ml mTNFa for 24 hours.
  • the medium was then changed with DMEM low glucose supplemented with 2mM glutamine, 100 U/ml penicillin/streptomycin for the following 18 hours.
  • conditioned media from unstimulated (unst MSC-CM) or stimulated MSC (st MSC-CM) were obtained.
  • EVs were isolated from unst or st MSC-CM by ultrafiltration using Amicon® Ultra 15 mL Filters (Merck Millipore). Tube formation assay.
  • l,3xl0 4 SVEC4-10 cells (ATCC® CRL-2181TM) were seeded in a Matrigel coated well (80 ⁇ 1 Matrigel/well) in ⁇ of either unst or st MSC-CM and their EV (EV unst or st MSC-CM).
  • DMEM low glucose with 10% heat-inactivated FBS was used as positive control.
  • EVs isolated from primed MSC according to the invention affect angiogenesis in vivo.
  • Anesthetized 12-week-old male C57BL/6N mice were subcutaneously injected in the dorsal back either with 5x105 unst- or st-MSC, or EVs obtained from the same cells, mixed with 500 ⁇ 1 Matrigel.
  • Matrigel plus 50 ng/mL VEGF and lOO ng/mL bFGF was used as positive control.
  • Bare Matrigel was injected as negative control.
  • matrigel was supplemented with Heparin (50 units/ml). After 10 days, mice were sacrificed, and the Matrigel plugs were harvested, weighed and photographed (A).
  • EVs isolated from primed MSC according to the invention affect angiogenesis in the developing mouse retina.
  • the retinal radius (R, indicated with the black arrow from the optic nerve to the edge of the retina) and the vascular radius (v, indicated with the grey arrow from the optic nerve to the vascular front) of each petal of the retina were measured.
  • the retinal vascular expansion was calculated as the ratio between the vascular radius (v) and the retinal radius (R).
  • Tube formation an in vitro matrigel angiogenesis assay.

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Abstract

La présente invention concerne de nouvelles vésicules extracellulaires anti-angiogéniques (vésicules extracellulaires, EV) formées à partir de cellules mésenchymateuses souches/stromales (également connues sous le nom de cellules souches mésenchymateuses, CSM), lesdites EV étant destinées au traitement de maladies caractérisées par une vascularisation accrue. L'invention concerne également des compositions pharmaceutiques les contenant et des procédés de préparation desdites EV.
PCT/IB2016/057608 2016-12-14 2016-12-14 Nouvelles vésicules extracellulaires anti-angiogéniques WO2018109525A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
PCT/IB2016/057608 WO2018109525A1 (fr) 2016-12-14 2016-12-14 Nouvelles vésicules extracellulaires anti-angiogéniques
EP17823225.2A EP3554513A1 (fr) 2016-12-14 2017-12-14 Nouvelles vésicules extracellulaires anti-angiogéniques
AU2017374947A AU2017374947B2 (en) 2016-12-14 2017-12-14 New anti-angiogenic extracellular vesicles
CA3052739A CA3052739A1 (fr) 2016-12-14 2017-12-14 Nouvelles vesicules extracellulaires anti-angiogeniques
PCT/IB2017/057928 WO2018109700A1 (fr) 2016-12-14 2017-12-14 Nouvelles vésicules extracellulaires anti-angiogéniques
CN201780086372.2A CN110312515B (zh) 2016-12-14 2017-12-14 新的抗血管生成细胞外囊泡

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EP3943597A4 (fr) * 2020-05-25 2022-08-17 CK-Exogene Co., Ltd. Méthode de préparation d'un exosome dérivé d'une cellule souche mésenchymateuse et culture cellulaire préparée à partir dudit exosome

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AU2017374947A8 (en) 2020-01-16
CN110312515A (zh) 2019-10-08
AU2017374947A1 (en) 2019-08-01
WO2018109700A1 (fr) 2018-06-21
EP3554513A1 (fr) 2019-10-23
AU2017374947B2 (en) 2023-08-24
CN110312515B (zh) 2023-05-16

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