WO2018109700A1 - Nouvelles vésicules extracellulaires anti-angiogéniques - Google Patents

Nouvelles vésicules extracellulaires anti-angiogéniques Download PDF

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WO2018109700A1
WO2018109700A1 PCT/IB2017/057928 IB2017057928W WO2018109700A1 WO 2018109700 A1 WO2018109700 A1 WO 2018109700A1 IB 2017057928 W IB2017057928 W IB 2017057928W WO 2018109700 A1 WO2018109700 A1 WO 2018109700A1
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evs
mscs
anyone
angiogenesis
angiogenic
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PCT/IB2017/057928
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English (en)
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Antonella Viola
Maurizio Muraca
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Università Degli Studi Di Padova
Fondazione Città Della Speranza - Onlus
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Priority to EP17823225.2A priority Critical patent/EP3554513A1/fr
Priority to CN201780086372.2A priority patent/CN110312515B/zh
Priority to AU2017374947A priority patent/AU2017374947B2/en
Priority to CA3052739A priority patent/CA3052739A1/fr
Publication of WO2018109700A1 publication Critical patent/WO2018109700A1/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 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 rumour formation or aberrantly differentiate after ectopic engrafbnent.
  • 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 5x10 s unst- or st-MSC, or EVs obtained from the same cells, mixed with 500 ⁇ 1 Matrigel.
  • Matrigel plus 50ng/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.
  • Fig.4 - EVs from MSC-CM fully mimic the whole conditioned medium
  • SVEC4-10 cells were seeded on the top of a matrigel layer in the presence of appropriate stimuli. After 6 hours, images were acquired with a phase contrast inverted microscope at 4* objective magnification.A) Representative pictures of the experiment; scale bar corresponding to 100 ⁇ m .
  • 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. no detectable angiogenesis when compared to a positive control that is not treated with the anti-angiogenic compound, cell, vesicle, composition, molecule, mixture, moiety, product or substance tested.
  • 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- 1-59745-241-0 J O.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.
  • Each form can be prepared by the skilled person according to the best practice of pharmaceutic preparation, therefore, following the common technical knowledge, the skilled person will know how to select the suitable carriers and other useful ingredients for the preparation
  • 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- 1 ⁇ , 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
  • 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 is any known medium commonly used for growing
  • MSCs is suitable for carrying out steps a. and b. of the invention.
  • 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, ami 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 raM 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 sumatant 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 sumatant.
  • Another object of the invention is represented by the anti-angiogenic EVs MSCs obtainable from the process described above.
  • the anti-angiogenic EVs MSCs obtainable by the process of the invention are characterised by the expression of TIMP1, CD73 and CD39.
  • the EVs obtainable by the process of the invention express each of said markers at least in a two-fold ratio when compared to EVs obtained from the same MSC-CM without stimulation during cell culturing as disclosed in the present description (herein referred also as EVs obtained from unstimulated MSC-CM).
  • the EVs MSCs of the invention i.e. the EVs obtainable from stimulated medium
  • the EVs MSCs of the invention are characterized by an expression of TUMP 1 of at least two to tenfold higher in comparison to the expression of TIMP1 of the EVs MSCs from unstimulated medium as described herein.
  • the expression of TIMPl by the EVs of the invention is at least two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold higher in comparison to the EVs MSCs from unstimulated medium as described herein.
  • the EVs MSCs of the invention are characterized by an expression of CD73 of at least two to three-fold higher in comparison to the expression of CD73 of EVs MSCs obtainable from unstimulated medium as described herein.
  • 1 fold is the amount of the same marker expressed by the same amount of EVs obtained by the same population of cells, in the same culturing conditions except that the medium is not complemented with one or more inflammatory cytokine, as disclosed in the present description.
  • the comparison was carried out on 3ug of total proteins of EVs MSCs extracted from each kind of EVs obtained from unstimulated and stimulated medium and submitted to western blot for the marker quantification.
  • 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 mTNFct 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.
  • 1,3x10* 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).
  • Extracellular vesicles recapitulate the phenotype of MSC-CM from which they are isolated
  • EVs were obtained from unstimulated or stimulated murine MSC-CM by ultrafiltration. Their quality and quantity were validated by Nanosight analysis (data not shown).
  • a tube formation assay with SVEC4-10 cells was carried out and the effects of EVs isolated from unstimulated or stimulated MSCs (EVs unst- or st- MSC-CM, respectively) with whole medium (CM) was compared.
  • stimulated MSC- CM decreases the tube length, in contrast to control and unstimulated MSC-CM.
  • the same anti-angiogenic effect was exerted by EVs from st-MSC-CM, which strongly inhibit the ability of SVEC4-10 to form capillary-like, tubular structures ( Figure 14 A, B).
  • TIMP1 has a known prominent role as anti-angiogenic mediator of st-MSCs
  • T1MP1 is involved in the anti-angiogenic effect exerted by EVs derived from st-MSC-CM.
  • TIMP1 is highly enriched in EVs st-MSC- CM, while the expression of the extracellular vesicles' markers CD63 and CD9 was unaffected ( Figure 4 C).
  • the tube formation assay was repeated using TIMP1 blocking antibody ( Figure 4 D), the result show that the antiangiogenic effect displayed by EVs isolated from st-MSC-CM is TIMP1 -dependent
  • the scratch wound healing assay reveals a second anti-angiogenic mechanism of EVs st- MSC-CM
  • Angiogenic and antiangiogenic activities can be assessed by using several in vitro assays, which are typically exploited to investigate different crucial steps of the in vivo process 124.
  • the matrix digestion and the endothelial morphogenesis during the development of the tubular network was evaluated.
  • the assembly of endothelial cells into vessel tubes in vivo also requires the coordinated migration of endothelial cells.
  • the scratch wound healing in vitro assay based on the ability of cells to move in a free space in response to a pro- angiogenic factor 126 was used.
  • SVECA4-10 cells were seeded to form a monolayer and, by scratching, a cell-free gap in the confluent layer was generated. Stimulation with the pro angiogenic factor VEGF induced the subsequent cell migration. EVs derived from unst- or st-MSCs were added. The distance covered by migrating cells was measured after 6 hours of incubation at 37°C 10% C02 ( Figure 5 A). As expected, in response to VEGF, the migratory ability of SVEC4-10 cells is increased in comparison to unstimulated cells (vehicle without VEGF). Treatment with EVs from unst-MSC-CM did not affect the migration of endothelial cells ( Figure 5 B). In contrast, EVs isolated from st-MSC- CM completely abolished the SVEC4-10 migratory ability in response to VEGF ( Figure 5 B).
  • CD39 and CD73 confer anti-angiogenic activity to EVs st-MSC-CM
  • Adenosine is a purine nucleoside that can be generated by the hydrolysis of extracellular ATP. This reaction is exerted by two enzymes confined on the cellular plasma membrane, the ectonucleotidases CD39 and CD73, catalysing, respectively, the hydrolysis of ATP to AMP, and of AMP to adenosinel25.
  • ROS reactive oxygen species
  • adenosine in the regulation of the reactive oxygen species (ROS) production.
  • Extracellular adenosine interacts with four subtypes of G protein-coupled cell surface receptors (AIR, A2AR, A2BR, and A3R).
  • AIR G protein-coupled cell surface receptors
  • A2AR G protein-coupled cell surface receptors
  • A3R G protein-coupled cell surface receptors
  • NADPH nicotinamide adenine dinucleotide phosphate
  • NOX oxidase
  • Oxidative stress is recognized as a potent inducer of senescence in endothelial cells and of dysfunctional cytoskeletal rearrangements two mechanisms potentially responsible for altered migratory response.
  • EVs st-MSC-CM anti-angiogenic EVs
  • ROS levels in migrating endothelial SVEC4-10 cells were detected ( Figure 7).

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Abstract

La présente invention concerne de nouvelles vésicules extracellulaires (vésicules extracellulaires, VE) anti-angiogéniques formées à partir de cellules souches/stromales mésenchymateuses (également connues sous le nom de cellules souches mésenchymateuses, CSM). L'invention concerne également l'utilisation desdits VE pour le traitement de maladies caractérisées par une vascularisation accrue, des compositions pharmaceutiques les comprenant et des procédés de préparation desdits VE.
PCT/IB2017/057928 2016-12-14 2017-12-14 Nouvelles vésicules extracellulaires anti-angiogéniques WO2018109700A1 (fr)

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EP17823225.2A EP3554513A1 (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
AU2017374947A AU2017374947B2 (en) 2016-12-14 2017-12-14 New anti-angiogenic extracellular vesicles
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2020214100A3 (fr) * 2019-04-18 2020-11-19 Agency For Science, Technology And Research Composition
EP3909594A1 (fr) * 2020-05-14 2021-11-17 Bernat Soria Escoms Cellules souches mésenchymateuses pour le traitement de covid-19 et autres maladies inflammatoires, auto-immunes et dégénératives

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113384597A (zh) * 2020-03-13 2021-09-14 西比曼生物科技（上海）有限公司 含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用
WO2021207025A1 (fr) * 2020-04-06 2021-10-14 University Of Georgia Research Foundation, Inc Exosomes des cellules mésenchymateuses augmentant la différenciation des lymphocytes t en lymphocytes t régulateurs
KR102184428B1 (ko) * 2020-05-25 2020-11-30 주식회사 씨케이엑소젠 중배엽줄기세포 유래의 엑소좀 생산방법 및 이로부터 제조된 배양액
TW202346572A (zh) * 2022-05-16 2023-12-01 台灣粒線體應用技術股份有限公司 用於減緩口腔損傷的組合物、其用途及其製備方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090202479A1 (en) * 2008-01-31 2009-08-13 Yufang Shi Method for modulating immune responses using stem cells and cytokines
WO2009105044A1 (fr) * 2008-02-22 2009-08-27 Agency For Science, Technology And Research (A*Star) Particules de cellules souches mésenchymateuses
WO2014013029A1 (fr) * 2012-07-18 2014-01-23 Universität Duisburg-Essen Utilisation de préparations, comprenant des exosomes dérivés de cellules souches mésenchymateuses (csm), dans la prévention et la thérapie d'affections inflammatoires
WO2015076717A2 (fr) * 2013-11-21 2015-05-28 Isletone Ab Csm utilisées dans le traitement de troubles cardiaques

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160199413A1 (en) * 2013-08-01 2016-07-14 Isletone Ab Mscs in the treatment of inflammatory pulmonary diseases
CN105861430B (zh) * 2016-04-29 2019-07-23 南京大学 一种外泌体、外泌体的制备方法及其在制备治疗脓毒症药物或者制剂中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090202479A1 (en) * 2008-01-31 2009-08-13 Yufang Shi Method for modulating immune responses using stem cells and cytokines
WO2009105044A1 (fr) * 2008-02-22 2009-08-27 Agency For Science, Technology And Research (A*Star) Particules de cellules souches mésenchymateuses
WO2014013029A1 (fr) * 2012-07-18 2014-01-23 Universität Duisburg-Essen Utilisation de préparations, comprenant des exosomes dérivés de cellules souches mésenchymateuses (csm), dans la prévention et la thérapie d'affections inflammatoires
WO2015076717A2 (fr) * 2013-11-21 2015-05-28 Isletone Ab Csm utilisées dans le traitement de troubles cardiaques

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BROWN ET AL., METHODS MOL BIOL., vol. 1430, 2016, pages 149 - 57
CONSUELO MERINO-GONZÁLEZ ET AL: "Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Angiogenesis: Potencial Clinical Application", FRONTIERS IN PHYSIOLOGY, vol. 7, 9 February 2016 (2016-02-09), CH, XP055398494, ISSN: 1664-042X, DOI: 10.3389/fphys.2016.00024 *
MAZZEO AURORA ET AL: "Molecular mechanisms of extracellular vesicle-induced vessel destabilization in diabetic retinopathy", ACTA DIABETOLOGICA, SPRINGER INTERNATIONAL, BERLIN, DE, vol. 52, no. 6, 19 August 2015 (2015-08-19), pages 1113 - 1119, XP035579481, ISSN: 0940-5429, [retrieved on 20150819], DOI: 10.1007/S00592-015-0798-9 *
NINA HELDRING ET AL: "Therapeutic Potential of Multipotent Mesenchymal Stromal Cells and Their Extracellular Vesicles", HUMAN GENE THERAPY, vol. 26, no. 8, 1 August 2015 (2015-08-01), US, pages 506 - 517, XP055398495, ISSN: 1043-0342, DOI: 10.1089/hum.2015.072 *
PONCE, METHODS MOL BIOL., vol. 467, 2009, pages 183 - 8
STEFANIA BRUNO ET AL: "Effects of Mesenchymal Stromal Cell-Derived Extracellular Vesicles on Tumor Growth", FRONTIERS IN IMMUNOLOGY, vol. 5, 11 August 2014 (2014-08-11), XP055161795, DOI: 10.3389/fimmu.2014.00382 *
SZE SIU KWAN ET AL: "Elucidating the secretion proteome of human embryonic stem cell-derived mesenchymal stem cells", MOLECULAR & CELLULAR PROTEOMICS, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 6, no. 10, 1 October 2007 (2007-10-01), pages 1680 - 1689, XP002531526, ISSN: 1535-9476, DOI: 10.1074/MCP.M600393-MCP200 *
ZANOTTI ET AL.: "Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-l", LEUKEMIA, vol. 30, 2016, pages 1143 - 1154

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020214100A3 (fr) * 2019-04-18 2020-11-19 Agency For Science, Technology And Research Composition
EP3909594A1 (fr) * 2020-05-14 2021-11-17 Bernat Soria Escoms Cellules souches mésenchymateuses pour le traitement de covid-19 et autres maladies inflammatoires, auto-immunes et dégénératives

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