WO2019197442A1 - Combinaison d'ingrédients actifs pour le traitement d'une tumeur - Google Patents

Combinaison d'ingrédients actifs pour le traitement d'une tumeur Download PDF

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WO2019197442A1
WO2019197442A1 PCT/EP2019/059033 EP2019059033W WO2019197442A1 WO 2019197442 A1 WO2019197442 A1 WO 2019197442A1 EP 2019059033 W EP2019059033 W EP 2019059033W WO 2019197442 A1 WO2019197442 A1 WO 2019197442A1
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tki
evs
tumor
tyrosine kinase
extracellular vesicle
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PCT/EP2019/059033
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Giovanni Camussi
Benedetta Bussolati
Valentina Fonsato
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Unicyte Ev Ag
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a combination of active ingredients for use in the therapeutic treatment of tumor diseases.
  • TKI tyrosine kinase inhibitors
  • the anticancer activity of TKIs is related to the inhibition of growth factor receptors overexpressed in several tumors and co-responsible for tumor angiogenesis and cell proliferation.
  • tyrosine kinase inhibitors Sunitinib, and Sorafenib have conferred a good clinical outcome of patients in term of response rate, progression-free survival and overall survival.
  • TKI therapies are not without limitations, including several adverse effects such as hand-foot syndrome, mucosal inflammation, hypothyroidism and fatigue together with hematological adverse events like anemia, leukopenia and thrombocytopenia. Moreover, in the vast majority of cases, TKI long-term anti-tumor effect leads to the development of resistance.
  • CSCs Cancer Stem Cells
  • RCC renal cell carcinoma
  • CD105 surface endoglin
  • EVs extracellular vesicles
  • HLSC human liver stem cells
  • stromal cell population isolated from human adult liver that inhibits liver carcinomas as well as gliomas and lymphoblastomas.
  • W02009050742 discloses the use of microvesicles derived from cells of the endothelial cell lineage, preferably from endothelial progenitor cells, in the treatment of type I or type II diabetes by pancreatic islet transplantation.
  • WO2011107437 discloses the use of microvesicles derived from adult stem cells for the therapeutic treatment of tumors.
  • the anti-tumor treatment may additionally comprise the administration of a cytotoxic agent, such as e.g. a TKI compound.
  • a cytotoxic agent such as e.g. a TKI compound.
  • WO2011107437 does not provide any specific indication on how the combined therapy should be administered in order to be clinically effective.
  • WO2011107437 is silent whether the micro vescicle and the cytotoxic agent should be administered as a physical mixture or as separate ingredients.
  • the object of the present invention is to provide a medicament having activity against tumor proliferation and growth, which is effective in the eradication of Cancer Stem Cells (CSCs), thereby achieving a long-lasting clinical response as well as preventing tumor relapse.
  • CSCs Cancer Stem Cells
  • EVs extracellular vesicles
  • an aspect of the present invention is a combined pharmaceutical preparation comprising an extracellular vesicle (EV) derived from an adult stem cell and a tyrosine kinase inhibitor (TKI), for simultaneous or sequential use in the therapeutic treatment of a tumor disease and/or in the prevention of tumor relapse in a patient, wherein the sequential use is performed by first administering the tyrosine kinase inhibitor (TKI) and then administering the extracellular vesicle (EV).
  • EV extracellular vesicle
  • TKI tyrosine kinase inhibitor
  • the extracellular vesicle (EV) is administered at least 40 hours, preferably at least 48 hours, after the administration of the tyrosine kinase inhibitor (TKI).
  • TKI tyrosine kinase inhibitor
  • the results of the apoptosis analysis conducted by the present inventors clearly indicate that the combined preparation of the invention enhances considerably the chemosensitivity of tumor cells to TKI through the pro-apoptotic effect exerted by the EVs.
  • the inventors believe that the increased tumor chemosensitivity seen with the combined preparation of the invention may be linked to EV-dependent enhancement of cellular mechanisms induced in target cancer cells upon TKI treatment rather than to epigenetic changes induced by EVs, leading to increased TKI sensitivity.
  • the adult stem cell is a human liver stem cell or a human mesenchymal stem cell.
  • a preferred human liver stem cell is the human non-oval liver stem cell (HLSC) expressing both mesenchymal and embryonic stem cell markers.
  • HLSCs are disclosed e.g. in W02006126236.
  • the human mesenchymal stem cell is derived from human adult bone marrow (BM-MSC).
  • the extracellular vesicle (EV) expresses a marker selected from CD63 and CD81.
  • the tyrosine kinase inhibitor (TKI) is selected from the group consisting of Gefitinib, Erlotinib, Lapatinib, Vandetanib, Afatinib, Sorafenib, Sunitinib, Pazopanib, Axitinib, Regorafenib, Nintedanib, Levantinib, Cabozantinib, Trametinib, and any combination thereof.
  • the combined preparation of the invention is suitable for use in the therapeutic treatment of tumor diseases and/or in the prevention of tumor relapse in a patient, preferably for the treatment of solid tumors, more preferably for the treatment of a solid tumor selected from the group consisting of renal cancer, breast cancer, liver cancer and gastrointestinal stromal tumor (GIST).
  • a solid tumor selected from the group consisting of renal cancer, breast cancer, liver cancer and gastrointestinal stromal tumor (GIST).
  • the present inventors have demonstrated that the co-administration of TKI and EVs, either simultaneous or sequential, is particularly effective against cancer stem cells by inducing activation of cell death.
  • the targeted solid tumor comprises one or more cancer stem cells.
  • CSCs stem cell markers expressed by CSCs vary according to the type of solid tumor. Consequently, the specific CSC phenotype may assist in the characterization of tumors which are more likely to be responsive to a particular therapeutic treatment as well as in the design of optimal therapeutic regimens.
  • the combined preparation of the invention has been found to be particularly effective against cancer stem cells expressing at least one stem cell marker selected from the group consisting of CD105, ALDH1, OCT4, SSEA4 and CD24YCD44 + .
  • the exact dose of the combined administration of TKI and EVs according to the invention may vary depending on the targeted tumor as well as on the specific components of the combined preparation, i.e. the TKI compound and the type of extracellular vesicle, and on the patient’s characteristics (e.g. sex, age, weight, etc.).
  • the daily dosage of the tyrosine kinase inhibitor (TKI) is comprised between 0.5 and 2.0 mg per kilogram body.
  • the extracellular vesicle (EV) may be administered in an amount ranging from 5 x 10 9 to 5 x 10 12 EVs per kilogram body per day.
  • the therapeutic and/or prophylactic treatment of the invention comprises administering to a patient a dose of the tyrosine kinase inhibitor comprised between 0.7 and 1.5 mg/kg/die of the tyrosine kinase inhibitor (TKI) and a dose of the extracellular vesicles comprised between 1 x l0 lo and 1 x l0 12 /kg/die of the extracellular vesicles (EV).
  • TKI tyrosine kinase inhibitor
  • EV extracellular vesicles
  • the TKI and EVs may also be effectively administered in the form of a pharmaceutical composition, i.e. of a physical mixture of the two active ingredients.
  • a second aspect of the present invention is a pharmaceutical composition for use in the therapeutic treatment of a tumor disease and/or in the prevention of tumor relapse in a patient, comprising an extracellular vesicle (EV) derived from an adult stem cell, a tyrosine kinase inhibitor (TKI), and optional pharmaceutically acceptable vehicles, excipients and/or diluents.
  • EV extracellular vesicle
  • TKI tyrosine kinase inhibitor
  • extracellular vesicles As drug vehicles, wherein drug loading strategies may involve, for example, direct incorporation of the drug substance into the extracellular vesicle or, alternatively, its binding on the external surface of the EV’s membrane.
  • drug loading strategies may involve, for example, direct incorporation of the drug substance into the extracellular vesicle or, alternatively, its binding on the external surface of the EV’s membrane.
  • the present inventors have found that the pro-apoptotic effect exerted on cancer stem cells by extracellular vesicles (EVs) loaded with a tyrosine kinase inhibitor (TKI) is comparable with the rate of cell death measured in the same cellular culture following co-administration of EVs and TKI as separate compounds (see Figure 5).
  • TKI tyrosine kinase inhibitor
  • the pharmaceutical composition comprises TKI-loaded extracellular vesicles, wherein the TKI is incorporated into the extracellular vesicle (EV) or it is bound to the external surface of the EV.
  • the extracellular vesicles are loaded with a number of TKI molecules of at least 1 x l0 3 /EV, preferably with a number of TKI molecules ranging from 1 x l0 3 /EV to 1 x l0 7 /EV, more preferably with a number of TKI molecules ranging from 1 x l0 4 /EV to 1 x l0 6 /EV.
  • composition of the invention is suitable to be administered as a cancer therapy to any mammal, including human beings.
  • composition of the invention is suitable for administration e.g. via the topical, enteral or parenteral route.
  • a yet further aspect of the present invention is an in vitro method of promoting apoptosis of cancer stem cells (CSC) in a cell culture, comprising contacting the cell culture first with a tyrosine kinase inhibitor (TKI) and subsequently with extracellular vesicles (EVs) derived from adult stem cells.
  • CSC cancer stem cells
  • the cancer stem cells (CSC) in the cell culture are contacted with extracellular vesicles (EVs) following incubation with a tyrosine kinase inhibitor (TKI).
  • EVs extracellular vesicles
  • TKI tyrosine kinase inhibitor
  • the period of incubation in the presence of TKI is of at least 40 hours, preferably of at least 48 hours. In another embodiment, the period of incubation in the presence of EV s is of at least 6 hours, preferably for at least 8 hours.
  • Figure 1 shows the characterization of G7 renal CSCs.
  • Figure 2 shows the characterization of EVs isolated from HFSCs.
  • C) Representative electron microscopy of HFSC-EVs (scale bar 100 nm).
  • Figure 3 A is a graph showing that incubation of G7 renal CSCs with HFSC-EVs induces a significant dose-dependent apoptotic effect compared to control. Apoptosis was evaluated by Muse Annexin V & Dead Cell Assay as the percentage of apoptotic cells after 48 hours incubation with different doses of HFSC-EVs.
  • Figure 3B is a graph showing that incubation of G7 renal CSCs with ImM Sunitinib (Sun) in combination with different doses of HFSC- EVs (5 x 10 3 , 10 x 10 3 , and 50 x 10 3 EV s/target cells) for 48 hours significantly inhibits proliferation compared to CSCs stimulated with ImM Sunitinib alone.
  • Figures 3C and 3D show that incubation for 48 hours of G7 renal CSCs (Figure 3C) and C10 breast CSCs ( Figure 3D) with HLSC-EVs (50 x 10 3 EV s/target cells) in combination with ImM Sunitinib (HLSC-EVs+Sun), 5mM Sorafenib (HLSC-EVs+Sor) or 2mM Cabozantinib (HLSC- EVs+Cabo) significantly inhibits cell proliferation compared to controls and CSCs treated with ImM Sunitinib, 5mM Sorafenib or 2mM Cabozantinib alone.
  • Figure 4A is a schematic representation of the sequential administration of TKIs and EVs. The entire incubation period is of 48 hours.
  • G7 renal CSCs were first incubated with HLSC-EVs for 8 hours, and then stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for additional 40 hours.
  • the post-EVs scheme G7 renal CSCs were initially stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for 40 hours, and then incubated with HLSC-EVs for additional 8 hours.
  • Figure 4B is a graph showing the pro-apoptotic effects on G7 renal CSCs exerted by TKI and EVs administered in pre-EVs or post-EVs sequential order as depicted in figure 4A.
  • a significant increase of the percentage of apoptotic cells is observed following post-EVs sequential administration, compared to pre-EVs.
  • Figure 5 is a graph showing the results of apoptosis analysis on G7 renal CSCs after incubation with HLSC-EVs loaded with Sunitinib (EV-SUN), HLSC-EVs loaded with Sorafenib (EV-SOR), Sunitinib or Sorafenib alone.
  • the supernatants (sum-SUN and surn- SOR) recovered after ultracentrifugation in the loading experiments were used as negative controls.
  • the results are expressed as mean ⁇ SD of three different experiments.
  • Renal cell carcinoma stem cells were obtained from specimens of renal cell carcinomas from patients undergoing radical nephrectomy according to the Ethics Committee of the S. Giovanni Battista Hospital of Torino, Italy (168/2014). Cells were isolated, using anti-CD 105 Ab coupled to magnetic beads, by magnetic cell sorting using the magnetic- activated cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA) from renal carcinomas (histological types: 3 clear-cell type and 2 undifferentiated carcinomas).
  • MCS magnetic- activated cell sorting
  • cells were labelled with the anti-CD 105 mAh for 20 min, washed twice and resuspended in MACS buffer (PBS without Ca 2 and Mg 2 , supplemented with 1% BSA and 5 mM EDTA) at a concentration of 2xl0 7 cells. After washings, cells were separated on a magnetic stainless steel wool column (Miltenyi Biotec), according to the manufacturer’s recommendations.
  • Magnetically sorted CDl05 + CSCs were cultured in the presence of the expansion medium, consisting of DMEM LG (Invitrogen), with insulin-transferrin- selenium, 10 9 M dexamethasone, 100 U penicillin, 1000 U streptomycin, 10 ng/ml EGF (all from Sigma- Aldrich) and 5% fetal calf serum (FCS) (Sigma- Aldrich).
  • the expansion medium consisting of DMEM LG (Invitrogen), with insulin-transferrin- selenium, 10 9 M dexamethasone, 100 U penicillin, 1000 U streptomycin, 10 ng/ml EGF (all from Sigma- Aldrich) and 5% fetal calf serum (FCS) (Sigma- Aldrich).
  • FCS fetal calf serum
  • Single cells were plated at 1000 cells/ml in serum- free DMEM-F12 (Cambrex BioScience, Venviers, Belgium), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), 5 (pg/ml insulin and 0.4% bovine serum albumin (all from Sigma).
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • bovine serum albumin all from Sigma.
  • C10 breast cell carcinoma stem cell line (C10 breast CSCs) was selected and used for all the experiments.
  • HLSC human cryopreserved normal hepatocytes obtained from Lonza (Basel, Switzerland, www.lonza.com).
  • Human hepatocytes were plated in the presence of alfa minimum essential medium/endothelial cell basal medium 1 (expansion media: aMEM/EBM in the ratio 3: 1, Lonza), supplemented with antibiotics (100 U penicillin and l,000U streptomycin; both from Sigma, St. Louis) and 10% Foetal Calf Serum (FCS, Sigma). After 2 week HLSC colonies that were evident were expanded.
  • aMEM/EBM in the ratio 3: 1, Lonza
  • antibiotics 100 U penicillin and l,000U streptomycin; both from Sigma, St. Louis
  • FCS Foetal Calf Serum
  • MSCs Bone marrow-derived mesenchymal stem cells
  • HLSC-EVs and MSC-EVs were resuspended in RPMI supplemented with 1% dimethyllsulfoxide (DMSO) and frozen at -80 °C for later use.
  • DMSO dimethyllsulfoxide
  • EVs Concentration and size distribution of EVs were determined by the Nanosight LM10 system(NanoSight, Wiltshire, UK). Briefly, EV preparations were diluted (1:200) in sterile saline solution and analyzed by the Nanoparticle Analysis System using the NTA 1.4 Analytical Software. To evaluate the internalization of EVs in G7 renal CSCs by fluorescent microscopy, EVs were labelled with 1 mM Dil dye (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, purified EVs were resuspended in PBS supplemented with 1 pM Dil dye and ultracentrifuged at 100,000 g for 1 h at 4 °C. Following labelling, the EVs were washed with PBS by ultracentrifugation as mentioned above. The pellet obtained was then resuspended in RPMI with 1% DMSO and frozen for subsequent studies.
  • EVs were loaded with 10 pM of Sunitinib or 50 pM of Sorafenib by incubating together for 15 minutes at 37 °C and then ultracentrifuged at 100,000 g for 1 h at 4 °C to remove the unloaded drug.
  • EVs were resuspended in RPMI with 1% DMSO and named EV-SUN those loaded with Sunitinib or EV-SOR those loaded with Sorafenib.
  • the supernatant (sum-SUN and surn-SOR) was recovered and used in the experiments as negative control.
  • the dose of Sunitinib and Sorafenib used was chosen on the base of preliminary experiments showing 8-10% of drug incorporation. Spectrum analysis was used to evaluate the effective drug loading within EVs and it revealed the presence of 1.8 mM Sunitinib and 10 pM for Sorafenib in EV-SUN or EV-SOR.
  • Apoptosis was evaluated by MuseTM Annexin V and Dead Cell Assay (Millipore, Merck KGaA, Darmstadt, Germany) according to manufacturer’s instructions.
  • the assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells, using fluorescently labeled Annexin V in combination with the dead cell marker, 7- AAD.
  • PS phosphatidylserine
  • C10 breast CSCs were seeded at the concentration of 2xl0 3 cells/well and, after cell attachment, were stimulated with HLSC-EVs or Sunitinib or Sorafenib alone or in combination and cultured for 48 hours.
  • the supernatant containing dead cells and cells were recovered, incubated for 20 minutes with Annexin V/7-AAD reagent and read at Muse. The results were showed as the percentage of total apoptotic cells.
  • Intracellular phosphoproteins were evaluated in the lysates of renal G7 CSCs by the magnetic bead-based_immunoassays Bio-Plex Pro cell- signaling assay according to manufacturer’s instruction (BIoRad, Hercules, California, US).
  • cells were treated or not with Sunitinib (1 pM) or Sorafenib (5 pM) or HLSC-EVs (1 x 10 3 EV/target cell) or with the co-administration of HLSCEVs/Sunitinib or HLSC- EVs/Sorafenib for three hours. Then, cells were lysed and lysates were_incubated with capture antibodies coupled to the beads. Coupled beads react with the sample containing the analyte of interest. After a series of washes to remove unbound protein, a_biotinylated detection antibody was added to create a sandwich complex. The final detection_complex was formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate and submitted to Bio-Plex system with Bio-Plex Manager software analysis. 1.6 Western blot analysis
  • G7 renal CSCs were stimulated for 3 hours with HLSC-EVs alone or in combination with Sunitinib_or Sorafenib.
  • cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail and PMSF (Sigma- Aldrich). Aliquots of the cell lysates_containing 30 pg proteins form cells or 10 pg from EVs, as determined by the PierceTM BC A Protein method (Thermo Scientific, Rockford, IL, USA), were run on 4-20% SDS-PAGE under_reducing conditions and blotted onto PVDF membrane filters using the iBLOT system (Life_Technologies).
  • the membranes were blocked in Tris-buffered saline-Tween (TBS-T; 25 mM Tris,_pH 8.0, 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) non-fat dried milk for 1 h. After blocking, membranes were probed overnight with primary antibody. Anti-vinculin (Santa Cruz Biotechnology), anti-AKT or anti p-AKT (Ser473), anti-PTEN or anti-pPTEN, anti-mTOR or antip-mTOR, anti-CREB or anti-pCREB and anti-Erk 1/2 (all from Cell Signalling) primary Abs were used.
  • the blots were incubated with appropriate peroxidase conjugated secondary antibodies for 1 h at room temperature. Goat anti-Rabbit IgG and goat antimousejgl HRP conjugated secondary antibodies (Thermo Scientific, Rockford, IL, USA) were used. Following incubation, the membranes were washed extensively with TBS- T, probed with_ClarityTM Western ECL substrate (Bio-rad, CA, USA), and detected by the Chemidoc system (Biorad, CA, USA).
  • Renal CSCs were isolated from renal carcinoma by magnetic cell sorting using selection for the_CDl05 surface antigen.
  • the G7 clone were used for all experiments. Immunophenotypic analysis showed the positivity for CD105, expression of the mesenchymal stem cell marker CD73, SSEA4 and the absence of CD 133 and CD24, known to be marker of normal renal progenitor cells and EPCAM ( Figure 1A).
  • Figure 1B When cultured in non-adhesive culture conditions, G7 renal CSCs were able to growth and form spheres that could be propagated for several passages.
  • HFSC-EVs were isolated by ultracentrifugation from HFSC and analysed in term of size and_distribution by NTA ( Figure 2A). EVs were characterized by Western blot analysis for the_expression of their characteristic markers CD63 and CD81 and by electron microscopy for their morphology ( Figure 2A).
  • HFSC-EV s labelled with DIF dye were internalized by tumor cells after 1 hour of_incubation at 37°C, as shown in Figure 2B. These characteristics are similar to those described for EVs derived by mesenchymal stromal cells (MSC-EVs).
  • MSC-EVs mesenchymal stromal cells
  • the inventors first evaluated the effect of EVs from bone marrow and from liver mesenchymal stromal cells, at different doses (5, 10, 50 x 10 3 EV/target cell) on G7 renal CSCs. As shown in Figure 3, HLSCEVs exerted a dose dependent pro-apoptotic effect on G7 renal CSC that was statistically significant at the dose of 50x10 3 EV/target cell (Figure 3A). Similar results were observed following incubation of G7 renal CSCs with different doses of MSC-EVs (data not shown).
  • the inventors In order to evaluate a possible combinatory effect of EVs with Sunitinib, the inventors first performed dose-response experiments of the TKI alone on G7 renal CSCs. The lower significant dose with an apoptotic effect was shown to be 2 mM Sunitinib. The inventors therefore planned combinatory experiments with a dose minimally affecting renal CSCs (1 mM).
  • Figure 3 shows that both the Sorafenib/HLSC-EVs and Cabozantinib/HLSC-EVs co administration induced an enhancement of apoptotic cells, with a similar effect of the Sunitinib/HLSC-EVs coadministration. Moreover, this increment was substantial not only in respect to control cells, but also to cells stimulated with HLSC-EVs or TKIs alone (Figure 3C).
  • the inventors performed experiments of sequential administration of HLSC-EVs and TKIs.
  • the inventors first incubated G7 renal CSCs with HLSC-EVs for 8 hours and then stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for additional 40 hours, to reach 48 hour incubation used in co-administration experiments ( Figure 4A).
  • the inventors incubated G7 renal CSCs with Sunitinib or Sorafenib for 40 hours and then stimulated with HLSC-EVs for additional 8 hours (Figure 4A).
  • the inventors generated HLSC-EVs loaded with Sunitinib or Sorafenib. As these TKIs are lipophilic, EVs were co-incubated with TKIs for 15 minutes followed by ultracentrifugation to wash out the unbound drugs. The EVs obtained were called EV-SUN or EV-SOR to indicate EVs loaded with Sunitinib (10 pM) or Sorafenib (50 pM), respectively. G7 renal CSCs were then incubated with EV-SUN or EV-SOR in the amount needed to reach the same TKI concentration used in experiments above.
  • the inventors performed a Bio-Plex Pro cell signaling assay for the detection of intracellular phosphoproteins. The results obtained were then validated by Western blot analysis. As shown in Figure 6, the inventors found that TKIs and HLSC-EVs co-administration induced a synergistic effect in respect to the use of TKI or EVs alone on specific pathways. In particular, the co-administration of Sunitinib and HLSC-EVs was able to reduce Akt activity and enhance the oncosuppressor PTEN trough decrease of its phosphorylated form in respect to treatments alone (Fig. 6 A and B).
  • Akt/PTEN pathways was inhibited also by co-administration of Sorafenib and HLSC-EVs, even if the reduction of pPTEN/PTEN ratio did not reach significance ( Figure 6 A and B).
  • the inventors found that the PTEN protein was directly expressed by HLSCEVs (Fig. 6 B, inset).
  • HLSC-EVs alone significantly reduced the active phosphorylated form of mTOR ( Figure 6 C) and the activation of the Creb transcription factor ( Figure 7 A and B).

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Abstract

La présente invention concerne le domaine du traitement thérapeutique de tumeurs. L'invention concerne une combinaison d'ingrédients actifs, comprenant une vésicule extracellulaire (EV) dérivée d'une cellule souche adulte et d'un inhibiteur de tyrosine kinase (TKI). La combinaison de l'invention est efficace contre la prolifération de cellules souches cancéreuses (CSC).
PCT/EP2019/059033 2018-04-12 2019-04-10 Combinaison d'ingrédients actifs pour le traitement d'une tumeur WO2019197442A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111494417A (zh) * 2020-02-10 2020-08-07 寇晓星 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126236A1 (fr) 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progenitrices du foie
WO2009050742A1 (fr) 2007-10-15 2009-04-23 Fresenius Medial Care Deutschland Gmbh Utilisation de microvésicules (mvs) pour préparer un médicament ayant une activité d'adjuvant sur la transplantation de cellules endothéliales, en particulier dans le traitement du diabète par une transplantation d'îlots pancréatiques, et procédé associé
EP2363136A1 (fr) * 2010-03-02 2011-09-07 Fresenius Medical Care Deutschland GmbH Microvésicules dérivées de cellules souches adultes pour une utilisation dans le traitement thérapeutique d'une maladie tumorale
WO2018011191A1 (fr) * 2016-07-12 2018-01-18 Evox Therapeutics Ltd Administration à médiation par des vésicules extracellulaires de conjugués protéine de liaison-petite molécule
WO2018035204A1 (fr) * 2016-08-16 2018-02-22 Henry Ford Health System Compositions pour le traitement de la douleur neuropathique et la sensibilisation des tumeurs aux chimiothérapies

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126236A1 (fr) 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progenitrices du foie
WO2009050742A1 (fr) 2007-10-15 2009-04-23 Fresenius Medial Care Deutschland Gmbh Utilisation de microvésicules (mvs) pour préparer un médicament ayant une activité d'adjuvant sur la transplantation de cellules endothéliales, en particulier dans le traitement du diabète par une transplantation d'îlots pancréatiques, et procédé associé
EP2363136A1 (fr) * 2010-03-02 2011-09-07 Fresenius Medical Care Deutschland GmbH Microvésicules dérivées de cellules souches adultes pour une utilisation dans le traitement thérapeutique d'une maladie tumorale
WO2011107437A1 (fr) 2010-03-02 2011-09-09 Fresenius Medical Care Deutschland G.M.B.H. Microvésicules (mvs) dérivées de cellules souches adultes destinées à être utilisées dans le cadre d'un traitement thérapeutique d'une maladie tumorale
WO2018011191A1 (fr) * 2016-07-12 2018-01-18 Evox Therapeutics Ltd Administration à médiation par des vésicules extracellulaires de conjugués protéine de liaison-petite molécule
WO2018035204A1 (fr) * 2016-08-16 2018-02-22 Henry Ford Health System Compositions pour le traitement de la douleur neuropathique et la sensibilisation des tumeurs aux chimiothérapies

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRUNO S. ET AL., STEM CELLS DEV, vol. 22, no. 5, 2013, pages 758 - 71
CAMUSSI G. ET AL., KIDNEY INT., vol. 78, no. 9, 2010, pages 838 - 48
G. RAPOSO ET AL: "Extracellular vesicles: Exosomes, microvesicles, and friends", NATURE REVIEWS CANCER, vol. 9, no. 1, 18 February 2013 (2013-02-18), pages 40 - 383, XP055147456, ISSN: 1474-175X, DOI: 10.1083/jcb.201211138 *
GUOHUA LOU ET AL: "Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma", JOURNAL OF HEMATOLOGY & ONCOLOGY, vol. 8, no. 1, 1 December 2015 (2015-12-01), XP055494433, DOI: 10.1186/s13045-015-0220-7 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111494417A (zh) * 2020-02-10 2020-08-07 寇晓星 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用
CN111494417B (zh) * 2020-02-10 2024-04-09 寇晓星 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用

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