WO2019072885A1 - Nanoparticules magnétiques pour le traitement du cancer - Google Patents

Nanoparticules magnétiques pour le traitement du cancer Download PDF

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WO2019072885A1
WO2019072885A1 PCT/EP2018/077547 EP2018077547W WO2019072885A1 WO 2019072885 A1 WO2019072885 A1 WO 2019072885A1 EP 2018077547 W EP2018077547 W EP 2018077547W WO 2019072885 A1 WO2019072885 A1 WO 2019072885A1
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cells
cancer
milh
caspase
amf
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PCT/EP2018/077547
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Véronique GIGOUX
Pascal CLERC
Daniel FOURMY
Julian Carrey
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Institut National Des Sciences Appliquées
Centre National De La Recherche Scientifique (Cnrs)
Université Paul Sabatier Toulouse Iii
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Publication of WO2019072885A1 publication Critical patent/WO2019072885A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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 methods and pharmaceutical compositions for the treatment of cancer in a subject in need thereof.
  • LMP lysosome membrane permeabilization
  • MILH magnetic intra-lysosomal hyperthermia
  • AMF high frequency alternating magnetic field
  • MNPs magnetic nanoparticles
  • MILH differs from standard magnetic hyperthermia whereby tumor eradication is achieved with large doses of MNPs which cause a temperature elevation of the whole tumor (9).
  • MILH MNPs, even of low heating power (Specific Absorption Rate), generate a local temperature rise causing LMP (4-6,10).
  • LMP 4-6,10
  • lysosomal cell death through apoptosis was demonstrated to involve the release of lysosomal hydrolases into the cytosol, especially Cathepsin-D and Cathepsin-B (CathB) which digest apoptosis-regulated proteins, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptotic caspases activation (3,14).
  • MOMP mitochondrial outer membrane permeabilization
  • the inventors investigated the cellular and molecular mechanisms involved in cancer cell death induced by MILH.
  • the inventors report that, under an AMF, lysosome- accumulated MNPs enhanced reactive oxygen species (ROS) production through the Fenton reaction within lysosomes causing lipid peroxidation of lysosome membrane, LMP and subsequent cell death by a non-conventional mechanism which is dependent of Caspase-1 and CathB but independent of apoptotic Caspase-3.
  • ROS reactive oxygen species
  • the present invention relates to methods and pharmaceutical compositions for the treatment of cancer in a subject in need thereof.
  • MILH Magnetic Intra-Lysosomal Hyperthermia
  • MNPs magnetic nanoparticles
  • Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers, the inventors provide evidences that MILH causes cell death through a non-apoptotic signaling pathway.
  • the mechanism of cell death involves temperature elevation at the nanoparticle periphery which enhances the production of reactive oxygen species through the lysosomal Fenton reaction.
  • MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3.
  • Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3.
  • the invention relates to a magnetic nanoparticle grafted with a tumor targeting agent and application of a high frequency alternating magnetic field (AMF) for use in a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof.
  • AMF high frequency alternating magnetic field
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent for use in a method for inducing non-apoptotic signaling of resistant cancer cell in a subject afflicted with cancer in need thereof.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent for use in the treatment of cancer by inducing non-apoptotic signaling of cancer cell in a subject in need thereof.
  • a subject denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a cancer.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a pancreatic endocrine cancer, pancreatic exocrine cancer or gastric cancer.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the subject suffers from a cancer selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, tracheal cancer, head and neck cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers.
  • a cancer selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, tracheal cancer, head and neck cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma,
  • the subject suffers from cancer resistant to anti-cancer treatment.
  • cancer resistant to anti-cancer treatment denotes cancer resistant to conventional treatments like chemotherapy and/or immunotherapy.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • magnetic nanoparticle or “MNP” has its general meaning in the art and refers to magnetic nanoparticle used in nanotherapy by inducing Magnetic Intra-Lysosomal Hyperthermia (MILH).
  • MILH Magnetic Intra-Lysosomal Hyperthermia
  • magnetic nanoparticle also refers to magnetic nanoparticle inducing Lysosomal Cell Death.
  • magnetic nanoparticle also refers to iron oxide magnetic nanoparticles (MNPs) but not limited to iron oxide magnetic nanoparticles (MNPs) coated with PEG-COOH and iron oxide magnetic nanoparticles (MNPs) coated with PEG- amine.
  • magnetic nanoparticle also refers to iron oxide magnetic nanoparticles (MNPs) coated with PEG-Amine such as described in the example.
  • MNPs iron oxide magnetic nanoparticles
  • magnetic nanoparticle also refers to magnetic nanoparticle comprised an iron-oxide core such as magnetite-Fe304 or maghemite-y-Fe203 and magnetic nanoparticle such as described in WO2012078745; Sanchez et aL , 2014; Domenech et aL, 2013; Creixell et aL , 201 1 ; Fourmy et aL, 2015; Silva et aL , 201 1 ; Cole et aL , 201 1 ; Kim et aL, 2012; Yang et aL, 2012.
  • tumor targeting agent has its general meaning in the art and refers to ligand of receptor overexpressed or specifically expressed in cancer cell.
  • tumor targeting agent also refers to ligands, agents or antibodies interacting with tumor membrane-bound and intracellular targets overexpressed or specifically expressed in cancer cell.
  • Tumor targeting agent include but are not limited to gastrin, the ligand of gastrin receptors (CCK2R), antibodies directed against the EDB domain of fibronectin, antibodies or agents binding Vascular endothelial growth factor receptor 2, antibodies or molecules binding fibroblast growth factor receptor- 1 , antibodies or agents that interact with CD31 , antibodies or agents interacting with tumor lymphatic endothelium (Podoplanin, Lyve-1), or antibodies or agents binding to ⁇ 3 integrin such as RGD peptides.
  • CCK2R gastrin receptors
  • the "tumor tageting agent” is gastrin, the ligand of gastrin receptors (CCK2R) overexpressed in pancreatic endocrine cancer cells.
  • non-apoptotic signaling has its general meaning in the art and refers to non- apoptotic cell death.
  • non-apoptotic signaling also refers to cell death by the non- conventional mechanism which is dependent of Caspase-1 and Cathepsin B but independent of apoptotic Caspase-3.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention in combination with one or more compound selected from the group consisting of:
  • Compound inducing iron excess or iron enrichment (such as FeC13, transferrin), Compound increasing the acidification of the lysosomal pH (such as Artesunate, activator of ATPase proton pump),
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention in combination with one or more compound selected from the group consisting of:
  • Compound inducing iron excess or iron enrichment (such as transferrin),
  • Compound inducing iron excess or iron enrichment has its general meaning in the art and refers to compound inducing iron overload such as transferrin, ferrous sulphate such as Ferrosanol®, FeC13, and iron composition for iron-enriched diet.
  • Compound increasing the acidification of the lysosomal pH has its general meaning in the art and refers to compounds reducing lysosomal pH such as Artesunate, activator of ATPase proton pump, epinephrine, norepinephrine, cAMP (Cyclic adenosine monophosphate), ⁇ -adrenergic receptor agonist such as isoproterenol, adenosine receptor agonist 5' -(N-ethylcarboxamido)-adenosine (NECA), A2A adenosine receptors agonist such as CGS21680, cell-permeable analogues chlorophenylthio-cAMP (cpt-cAMP) and 8-bromo- cAMP, 3-isobutyl-l-methylxanthine (IBMX) and forskolin such as described in Liu et ah, 2008 and WO2012149285.
  • HSP70 has its general meaning in the art and refers to the heat shock protein 70 involved in assisting protein folding, preventing protein aggregation and transporting proteins across membranes (Li et al, 2016; Assimon et al, 2013; Reikvam et al, 2014; Patury et al, 2009).
  • HSP70 inhibitor has its general meaning in the art and refers to a compound that selectively blocks or inactivates the HSP70.
  • HSP70 inhibitor also refers to a compound that selectively blocks the binding of HSP70 to its molecular chaperones (such as HSP40 (DNAJ) (Heat shock protein 40); Auxilin (DNAJ6); HSP110 (HSPH) Heat shock protein 110; BAG (Bcl-2-associated athanogene); GrpE; Bap; HIP HSP70-interacting protein; CHIP (Carboxyl terminus of HSP70-interacting protein); HOP (HSP70/HSP90-organizing protein); and Tom70 (Translocases of outer membrane 70).
  • HSP40 DNAJ
  • Auxilin DNAJ6
  • HSP110 HSP110
  • BAG Bcl-2-associated athanogene
  • GrpE GrpE
  • Bap HIP HSP70-interacting protein
  • CHIP Carboxyl terminus of HSP70-interacting protein
  • HSP70 inhibitor also refers to a compound able to prevent the action of HSP70 for example by inhibiting the activity of HSP70 or the HSP70 chaperone pathway.
  • selectively blocks or inactivates refers to a compound that preferentially binds to and blocks or inactivates HSP70 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the HSP family.
  • Compounds that block or inactivate HSP70, but that may also block or inactivate other HSP sub-types, as partial or full inhibitors, are contemplated.
  • the term “HSP70 inhibitor” also refers to a compound that inhibits HSP70 expression.
  • a HSP70 inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, intra-antibody, an oligonucleotide, a ribozyme or a CRISPR.
  • HSP70 inhibitors are well-known in the art as illustrated by Li et al, 2016; Assimon et al, 2013; Reikvam et al, 2014; Patury et al, 2009; WO2015130922; and US20150025052.
  • the HSP70 inhibitor is selected from the group consisting of inhibitors of the N-terminal ATP -binding domain such as MKT-077, YM- 1 , VER- 155008, NSC 630668, MAL3-101, MAL2-11B, Apoptozole, Myricetin; inhibitors of the C- terminal peptide-binding domain such as 2-phenylethynesulfonamide/Pifithrin ⁇ (PES), ADD70; Inhibitory blocking antibodies such as cmHSP70.1 mAb; Inhibitors of HSF-1 such as Quercetin, KNK437, Triptolide, KRIBBl l; inhibitors of the HSP40 co-chaperone such as Phenoxy-N-arylacetamides (Reikvam et al., 2014; Li et al., 2016; Patury et al., 2009); YK5 (Li et al, 2016); epigallocatechin gallate (
  • a gene product can be the direct transcriptional product of a gene (e.g. , mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., HSP70) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • proteins e.g., HSP70
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • HSP70 expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of HSP70 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of HSP70 proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding HSP70 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Small inhibitory RNAs can also function as expression inhibitors for use in the present invention.
  • Gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that HSP70 expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al.
  • Ribozymes can also function as expression inhibitors for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as HSP70 inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing HSP70.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors.
  • These plasmids however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • HSP70 expression inhibitors include but are not limited to siRNAs and compounds such as described in Patury et al, 2009; Reikvam et al, 2014; Guo et al, 2005.
  • Cathepsin-B has its general meaning in the art and refers to a lysosomal cysteine protease linked to general protein turnover in lysosomes (Aggarwal and Sloane, 2014).
  • Cathepsin-B activator refers to any compound that can directly or indirectly stimulate the signal transduction cascade related to the Cathepsin-B.
  • Cathepsin-B activator also refers to a compound that selectively activates the Cathepsin-B.
  • selectively activates refers to a compound that preferentially binds to and activates Cathepsin-B with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the Cathepsin family.
  • Cathepsin-B activator is a small organic molecule or a peptide.
  • a Cathepsin-B activator is a small organic molecule, a peptide, a modified Cathepsin-B or an activator of Cathepsin-B expression.
  • the Cathepsin-B activator is selected from the group consisting of Compounds described in CN 106220735; WO2005060663.
  • Caspase-1 has its general meaning in the art and refers to a cysteine protease implicated in cell death by pyroptosis, known to occur in cells of the immune system during inflammatory processes.
  • the term “Caspase-1” also refers to caspase implicated in apoptosis and non-apoptotic cell death pathway (32; 44-49).
  • Caspase-1 activator refers to any compound that can directly or indirectly stimulate the signal transduction cascade related to the Caspase-1.
  • the term “Caspase-1 activator” also refers to a compound that selectively activates the Caspase-1.
  • selectively activates refers to a compound that preferentially binds to and activates Caspase-1 with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the Caspase family.
  • Compounds that prefer Caspase-1, but that may also activate other Cathepsin sub-types, as partial or full activators, and thus that may have multiple Caspase activities, are contemplated.
  • a Caspase-1 activator is a small organic molecule or a peptide.
  • a Caspase-1 activator is a small organic molecule, a peptide, a modified Caspase-1 or an activator of Caspase-1 expression.
  • an “activator of expression” refers to a natural or synthetic compound that has a biological effect to activate the expression of a gene.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention in combination with one or more anticancer compound for use in a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention which sensitizes cancer cells to anticancer compound.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention for use in a method for enhancing therapeutic efficacy of anti-cancer compound such as doxorubicin in a subject in need thereof.
  • the present invention relates to the magnetic nanoparticle grafted with a tumor targeting agent according to the invention in combination with doxorubicin for use in a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof.
  • anti-cancer compound has its general meaning in the art and refers to compounds used in anti-cancer therapy such as anti-angiogenic compound, tyrosine kinase inhibitors, tyrosine kinase receptor (TKR) inhibitors, Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway inhibitors, interferon therapy, anti-HER2 compounds, anti- EGFR compounds, alkylating agents, anti-metabolites, immunotherapeutic agents, Interferons (IFNs), Interleukins, and chemotherapeutic agents such as described below.
  • anti-angiogenic compound has its general meaning in the art and refers to compounds used in anti-angiogenic therapy such as tyrosine kinase inhibitors, anti-angiogenic tyrosine kinase receptor (TKR) inhibitors, anti-angiogenics targeting the Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway such anti-VEGF antibody bevacizumab (Avastin) and VEGF receptor tyrosine kinase inhibitor (TKI) compounds such as sunitinib (Sutent), vandetanib (Zactima), pazopanib (Votrient), sorafenib (Nexavar) and cediranib, interferon therapy and anti-HER2 compounds such as Trastuzumab (herceptin) and pertuzumab.
  • TKI Vascular Endothelial Growth Factor Receptors
  • TKI VEGF receptor tyrosine kinas
  • anti-angiogenic compound refers to Sunitinib (Sutent), an anti-angiogenic TKR inhibitor of VEGFRs, platelet-derived growth factor receptors (PDGF-Rs), and c-kit.
  • tyrosine kinase inhibitor has its general meaning in the art and refers to any of a variety of therapeutic agents or drugs such as compounds inhibiting tyrosine kinase, tyrosine kinase receptor inhibitors (TKRI), EGFR tyrosine kinase inhibitors, EGFR antagonists.
  • TKRI tyrosine kinase receptor inhibitors
  • EGFR tyrosine kinase inhibitors
  • EGFR antagonists EGFR antagonists.
  • tyrosine kinase inhibitor or “TKI” has its general meaning in the art and refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases.
  • Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase.
  • tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to Erlotinib, sunitinib (Sutent; SU11248), dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazol
  • the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication.
  • inhibitors include, but are not limited to Erlotinib, Gefitinib, Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KR -633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP- 547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, K N-951, Dovitinib, Seliciclib, SNS- 032, PD-0332991, MKC-I (Ro-317453; R-440), Sorafenib, ABT-869
  • EGFR tyrosine kinase inhibitors as used herein include, but are not limited to compounds selected from the group consisting of but not limited to Erlotinib, lapatinib, Rociletinib (CO- 1686), gefitinib, Dacomitinib (PF-00299804), Afatanib, Brigatinib (AP26113), WJTOG3405, NEJ002, AZD9291, HM61713, EGF816, ASP 8273, AC 0010.
  • antibody EGFR inhibitors examples include Cetuximab, panitumumab, matuzumab, zalutumumab, nimotuzumab, necitumumab, Imgatuzumab (GA201, RO5083945), and ABT- 806.
  • the MNP and the combination of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent such as chemotherapeutic or radiotherapeutic.
  • the MNP and the combination of the present invention is administered with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its a
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem lntl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyr
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
  • the MNP and the combination of the present invention is administered with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs", “molecularly targeted therapies”, “precision medicines”, or similar names.
  • the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor as defined above.
  • the MNP and the combination of the present invention is administered with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non- cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Nonspecific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN- ⁇ ) and IFN-gamma (IFN- ⁇ ). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs Interferons
  • IFN-a IFN-alpha
  • IFN- ⁇ IFN-beta
  • IFN-gamma IFN-gamma
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Colony-stimulating factors contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy.
  • CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used.
  • Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin).
  • immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e.
  • Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor.
  • Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins.
  • Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22.
  • Other examples include anti-CTLA4 antibodies (e.g.
  • antibodies include B cell depleting antibodies.
  • Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g.
  • the immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC.
  • the immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E.
  • NK cells circulating lymphocytes
  • the activated lymphocytes or NK cells are most preferably be the subject's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro.
  • chemotherapeutics include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, epipodophyllotoxins such as etoposide and teniposide, camptothecins such as irinotecan and topotecan, bleomycin, doxorubicin, idarubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil and 5-fluorouracil combined with leucovorin, taxanes such as docetaxel
  • additional therapeutic active agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional therapeutic active agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi- specific antibodies, monobodies, polybodies.
  • Further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
  • the antiemetic agent is granisetron or ondansetron.
  • the further therapeutic active agent can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or non- opioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, buprenorphine, meperidine, loperamide, ethoheptazine, betaprodine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazone, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, clorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the MNP and the combination of the present invention is administered with a radiotherapeutic agent.
  • radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • said additional active compounds may be contained in the same composition or administrated separately.
  • the MNP and the combination according to the invention as described above are administered to the subject in a therapeutically effective amount.
  • a “therapeutically effective amount” of the MNP and the combination of the present invention as above described is meant a sufficient amount of the MNP and the combination for treating cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the MNP and the combination of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific MNP and the combination employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific MNP and the combination employed; the duration of the treatment; drugs used in combination or coincidental with the specific MNP and the combination employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the MNP and the combination at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the MNP and the combination of the present invention for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the MNP and the combination of the present invention, preferably from 1 mg to about 100 mg of the MNP and the combination of the present invention.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the MNP and the combination according to the invention may be used in a concentration between 0.01 ⁇ and 20 ⁇ , particularly, the MNP and the combination of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 ⁇ .
  • the MNP and the combination of the present invention is administered to the subject in the form of a pharmaceutical composition.
  • the MNP and the combination of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, sulfate, a pharmaceutically acceptable, or pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Solutions comprising MNP and the combination of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the MNP and the combination of the present invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the MNP and the combination of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof.
  • the present invention relates to a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof, comprising the steps of administering to said subject the magnetic nanoparticle grafted with a tumor targeting agent, and application of a high frequency alternating magnetic field (AMF).
  • AMF high frequency alternating magnetic field
  • high frequency alternating magnetic field has its general meaning in the art and refers to AMF frequency of 100 to 350 kHz and AMF amplitude of 20 to 60 mT.
  • high frequency alternating magnetic field also refers to AMF of frequency of 100, 150, 200, 250, 300 or 350 kHz and amplitude of 20, 30, 40, 50 or 60 mT.
  • high frequency alternating magnetic field also refers but not limited to AMF of frequency and amplitude of 275 kHz-52 mT, 275 kHz-50 mT, 275 kHz-40 mT and 300 kHz- 53mT (Sanchez et al., 2014; Domenech et al., 2013; Creixell et al., 2011; Silva et al., 2011).
  • kits comprising the MNP and the combination of the invention. Kits containing the MNP and the combination of the invention find use in therapeutic methods.
  • FIGURES are a diagrammatic representation of FIGURES.
  • MILH increases cellular ROS production.
  • AMF application to cells containing MNPs would generate a nanoscale temperature elevation and enhance ROS production within lysosomes. Lysosomes are well-known major sites of ROS production through the Fenton reaction (Fe 2+ +H 2 0 2 ⁇ Fe 3+ +OH ⁇ +OH') which catalyzes the transformation of hydrogen peroxide to hydroxyl radicals.
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were incubated in the presence or absence of 1 mM DFO, 1 nM BafAl, 20 ⁇ ART or 120 ⁇ FeC13, exposed or not to AMF and incubated with CellROX reagent. Quantification of ROS production was performed by analyzing the fluorescence intensity from confocal microscopy images. Results are expressed as fold change of fluorescence intensity over control cells (in absence of Gastrin-MNP and AMF) and c) MILH increases lysosomal ROS production.
  • INR1 G9-CCK2R cells having or not internalized Gastrin- MNP were incubated in the presence or absence of 1 mM DFO, 1 nM BafAl, 20 ⁇ ART or 120 ⁇ FeC13. Cells were incubated with CellROX reagent during the 30-min of AMF exposure. Quantification of ROS production colocalizing with Gastrin-MNP was performed by analyzing the % of relative fluorescence intensities of ROS production over Gastrin-MNP from confocal microscopy images, d) Absence of Gastrin-MNP degradation after AMF exposure. INR1G9-CCK2R cells were incubated with Gastrin-MNP for 24h and exposed or not to AMF for 2h.
  • FIG. 2 MILH-induced ROS generation causes lysosome lipid peroxydation, lysosome membrane permeabilization and cell death
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were incubated in the presence or absence of 1 mM DFO or 1 nM BafAl and incubated with Image-IT ® lipid peroxidation reagent during the 30-min of AMF exposure. Quantification was performed by measuring the % of fluorescence intensity of lipid peroxidation over that of Gastrin-MNP from confocal microscopy images, b) LMP induced by MILH is dependent on ROS production.
  • INR1G9-CCK2R transiently expressing GFP-CathB and RFP-Lampl were incubated with Gastrin-MNP and exposed to AMF in the presence or not of NAC.
  • LMP was evaluated by analyzing the co localization between GFP-CathB and RFP-Lampl on confocal microscopy images using Pearson's coefficient (left panel) and quantified by measuring the % of fiuorescence intensity of GFP-CathB over that of RFP-Lampl from confocal microscopy images (right panel), c) Monitoring of LMP during AMF exposure.
  • INR1G9-CCK2R transiently expressing GFP-CathB and RFP-Lampl were incubated with Gastrin-MNP and exposed or not to AMF. Different positions were marked inside (+AMF) and outside (-AMF) the gap of the electromagnet allowing to visualize the effects of AMF under a confocal microscope. Before and during AMF application for 60-min, LMP was analyzed by measuring the % of GFP-CathB colocalized with RFP-Lampl . Results are expressed as % of CathB/Lam l co localization over control cells (in absence of Gastrin-MNP and AMF). d) MILH-induced cell death is dependent on lysosomal ROS production.
  • INR1G9-CCK2R cells were incubated with Gastrin-MNP, exposed to AMF in presence or not of 5 mM NAC, 1 mM DFO, 1 nM BafAl, 20 ⁇ ART or 120 ⁇ FeC13.
  • Dead cells were counted 4h after AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histogram. Results are the mean ⁇ SEM of at least 3 separate experiments.
  • FIG. 3 Cell death induced by MILH is dependent on CathB activity
  • INR1G9- CCK2R cells having or not internalized Gastrin-MNP were incubated with or without 10 ⁇ CathB inhibitor CA-074-Me and exposed or not to AMF.
  • Dead cells were counted 4h after the end of AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histograms.
  • INR1 G9-CCK2R transiently expressing GFP-CathB-C29A enzymatically inactive mutant (B) or GFP-CathB wild-type enzyme (C) were incubated with Gastrin-MNP and exposed or not to AMF.
  • Dead cells were counted 4h after the end of AMF exposure by confocal microscopy analysis and expressed as the % of cells labeled with annexinV among transfected and non- trans fected cells. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histograms. Results are the mean ⁇ SEM of at least 4 separate experiments.
  • FIG. 4 Cell death induced by MILH is mediated through a non-apoptotic Caspase-1 signaling pathway which is dependent on CathB activity, a) MILH-induced cell death is independent of Caspase-3.
  • INR1G9-CCK2R cells having or not internalized Gastrin- MNP were exposed or not to AMF in the presence of 10 ⁇ Caspase-3 inhibitor. Dead cells were counted 4h after the end of AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide.
  • INR1G9-CCK2R cells were incubated with 1 ⁇ staurosporine for 4h.
  • Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells is indicated above the histogram, b) MILH does not induce Caspase-3 activation. Cells having or not internalized Gastrin-MNP were exposed or not to AMF in the presence or absence of 10 ⁇ Caspase-3 inhibitor. As positive and negative controls of Caspase-3 activation, INR1G9- CCK2R cells were stimulated with 1 ⁇ staurosporine in the absence or in the presence of Caspase-3 inhibitor.
  • MILH-induced cell death is dependent of Caspase-1.
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were exposed or not to AMF in presence of 2.5 ⁇ Caspase-1 inhibitor.
  • Dead cells were counted 4h after the end of AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in the absence of Gastrin-MNP and AMF).
  • the % of dead cells is indicated above the histogram, e) INR1G9-CCK2R transiently expressing GFP-Caspl-C284A enzymatically inactive mutant were incubated with Gastrin-MNP and exposed or not to AMF. Dead cells were counted 4h after AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells is indicated above the histogram, f) MILH induces Caspase-1 activation.
  • MILH triggers a non-apoptotic cell death mechanism related to pyroptosis.
  • MILH does not induce 111 ⁇ secretion.
  • INR1G9-CCK2R cells were treated or not with 500 ng/ml of LPS, Gastrin-MNP and exposed or not to AMF.
  • Thpl macrophages were treated with 500 ng/ml LPS.
  • ⁇ release in culture supernatant was assayed with an ELISA kit.
  • MILH induces DNA fragmentation. INR1G9-CCK2R cells having or not internalized Gastrin-MNP were exposed or not to AMF.
  • INR1G9-CCK2R, AR4-2J and AGS-CCK2R cells were incubated or not with Gastrin-MNP and exposed or not to AMF.
  • Dead cells were counted 4h after the end of AMF exposure by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF) for each cell line.
  • Figure 7 a) Cellular ROS production in the absence of AMF exposure. INR1G9- CCK2R cells having or not internalized Gastrin-MNP were incubated in the presence or absence of ImM DFO, InM BafAl or 120 ⁇ FeCb and incubated with CellROX Green reagent. Quantification of ROS production was performed by analyzing the fluorescence intensity from confocal microscopy images. Results are expressed as fold change of fluorescence intensity over control cells (in absence of Gastrin-MNP) and are the mean ⁇ SEM of at least 3 separate experiments, b) Analysis of lysosomal acidity.
  • INR1G9-CCK2R cells were incubated with 1 nM BafAl or 20 ⁇ ART for 3h, then with 5 ⁇ acridine orange for 15-min at 37°C and washed before microscope observation. Quantitative analysis of red fluorescence of acridine orange from microscope images of 2-3000 cells. Results are expressed as the % of Red fluorescence relative to control cells and are the mean ⁇ SEM of at least 3 separate experiments.
  • FIG. 8 MILH induces ROS production through NADPH oxidases, but not through mitochondrial complex of respiratory chain.
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were incubated or not in the presence of 1 ⁇ rotenone (inhibitor of mitochondrial complex of respiratory chain) or DPI (inhibitor of NAPDH oxidases) and exposed or not to AMF for 2h. After AMF application, cells were incubated with CellROX Green reagent. Quantification of ROS production was performed by analyzing the intensity of CellROX Green reagent labeling of confocal microscopy images. Results are expressed as fold change of basal fluorescence intensity over control cells (in the absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 4 separate experiments.
  • FIG. 10 MILH-induced cellular ROS production is inhibited by NAC.
  • INR1G9- CCK2 cells having or not internalized Gastrin-MNP were incubated in the presence or absence of 5mM NAC, exposed or not to AMF for 2h and incubated with CellROX Green reagent. Quantification of ROS production was performed by analyzing the fluorescence intensity of confocal microscopy images. Results are expressed as fold change of fluorescence intensity over control cells (in absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 3 separate experiments.
  • Figure 11 Cell death determination in the presence of drugs modifying ROS production.
  • INR1G9-CCK2R cells were incubated with Gastrin-MNP for 24h and then with 5 mM NAC, 1 mM DFO, 1 nM BafAl or 120 ⁇ FeCb for 7-10h.
  • Dead cells were counted by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 3 separate experiments. The % of dead cells are indicated above the histograms.
  • Figure 12 Analysis of MILH-induced cell death in presence of rotenone or DPI.
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were incubated or not in the presence of 1 ⁇ rotenone (inhibitor of mitochondrial complex of respiratory chain) or DPI (inhibitor of NAPDH oxidases) and exposed or not to AMF for 2h.
  • Dead cells were counted by confocal microscopy analysis of cells labeled with annexinV and/or propidium iodide. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 3 separate experiments. The % of dead cells are indicated above the histograms.
  • FIG. 13 MILH induces lysosomal rupture and CathB leakage from lysosomes.
  • INR1G9-CCK2R transiently expressing GFP-CathB and RFP-Lampl were incubated with Gastrin-MNP for 24h and exposed to AMF.
  • FIG. 14 MILH does not induce Pro-Caspase-3 maturation.
  • INR1G9-CCK2R cells having internalized Gastrin-MNP were exposed or not to AMF.
  • INR1G9-CCK2R cells were treated with 1 ⁇ staurosporine.
  • Pro-Caspase-3 expression was analyzed by Western blot of total cell lysates. Results of quantification of pro-Caspase-3 expression represent mean ⁇ SEM of 4 independent experiments and are expressed as fold control with control expression set to 1.
  • FIG. 15 MILH does not induce mitochondrial outer membrane permeabilization (MOMP).
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were exposed or not to AMF.
  • INR1G9-CCK2R cells were stimulated with 1 ⁇ staurosporine for 4h. 24h after AMF exposure or staurosporine treatment, Cells were incubated with JC-10 reagent and were analyzed by confocal microscopy. Quantification of MOMP was performed by analyzing the green/red fiuorescence ratio of confocal microscopy images. Results are the mean ⁇ SEM of 3 separate experiments.
  • FIG. 16 MILH-induced Caspase-1 activation is dependent on CathB activity.
  • INR1G9-CCK2R cells having or not internalized Gastrin-MNP were exposed or not to AMF in the presence or absence of 2.5 ⁇ Caspase-1.
  • Caspase-1 activation was analyzed by confocal microscopy of cells labeled with FAM-FLICA-Caspl. Results are expressed as % of total cells labeled with fluorescent Caspase substrate and are the mean ⁇ SEM of 4 separate experiments.
  • FIG. 17 MILH induces the death of HEK-CCK2R cells and activates Caspase- 1.
  • HEK-CCK2R were incubated or not with Gastrin-MNP for 24h.
  • a) Cells were exposed or not to AMF for 2h at 37°C. Cell survival was determined by MTT assay 24h after AMF exposure. Results are expressed as the % of cell survival relative to control cells (in absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 5 separate experiments,
  • Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF) and are the mean ⁇ SEM of at least 4 separate experiments. The % of dead cells are indicated above the histograms, c) Gastrin-MNP uptake was measured by colorimetric assay (total Fe) or by particle electron paramagnetic resonance (magnetic Fe) and expressed as the mean ⁇ SEM of at least 4 separate experiments, d-e) 4h after AMF exposure, Caspase-3 and -1 activation was analyzed by confocal microscopy of cells labeled with FAM-FLICA-Casp3 or FAM- FLICA-Caspl .
  • INR1G9-CCK2R cells were stimulated with 1 ⁇ staurosporine. Results are expressed in % of total cells labeled with fluorescent Caspase substrate and are the mean ⁇ SEM of 4 separate experiments.
  • FIG. 18 Absence of Caspase-3 activation following MILH treatment of different cancer cells.
  • INR1G9-CCK2R, AGS-CCK2R and AR4-2J cells were incubated with Gastrin- MNP for 24h and exposed to AMF. 4h after AMF exposure, Caspase-3 activation was analyzed by confocal microscopy of cells labeled with FAM-FLICA-Casp3.
  • INR1G9-CCK2R cells were stimulated with 1 ⁇ staurosporine. Results are expressed in % of total cells labeled with fluorescent Caspase substrate and are the mean ⁇ SEM of 4 separate experiments.
  • FIG. 19 Effect of combination of magnetic intra-lysosomal hyperthermia and doxorubicin treatment on cell death.
  • the impact of the treatments was determined 4 hours after AMF exposure by counting cells labeled with FITC-tagged annexin V (AnnV) and/or propidium iodide (IP) which identified dead cells. The % of induction of cell death are indicated above the histogram. Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNPs in absence of AMF exposure and doxorubicin treatment was indicated above histogram bar. Statistical significances between other conditions are also indicated. 2,000-3,000 cells/experiments were analyzed and results are the mean ⁇ SEM of five separate experiments.
  • HSP70 inhibition on cell death INR1G9-CCK2R cells were pre-incubated with 16 ⁇ Gastrin-MNP for 24 hours, allowing their internalization and lysosome accumulation, washed to eliminate unbound and non-internalized nanoparticles, before the addition of different concentrations of HSP70 inhibitor, PES, for 1 hour and submitted or not to a high frequency alternating magnetic field (40 mT, 275 kHz). The impact of the treatments was determined 4 or 24 hours after magnetic field application by counting cells labeled with FITC-tagged annexin V (AnnV) and/or propidium iodide (PI), which identified dead cells using flow cytometry.
  • AnnV FITC-tagged annexin V
  • PI propidium iodide
  • Results represent the mean ⁇ sem of eight independent experiments, a) Analysis of cell death in presence of 5 ⁇ of PES 4 hours after AMF exposure, b) Analysis of cell death in presence of 7.55 ⁇ of PES 4 hours after AMF exposure, c) Analysis of cell death in presence of 5 ⁇ of PES 24 hours after AMF exposure.
  • Figure 21 Effect of combination of magnetic intra-lysosomal hyperthermia with HSP70 down-regulation on cell death.
  • INR1G9-CCK2R cells were transfected or not with SiRNA directed against rat HSP70 (Dharmacon)for 48 hours, incubated or not with 16 ⁇ g/ml Gastrin-MNP for 24 hours, allowing their internalization and lysosome accumulation, washed to eliminate unbound and non-internalized nanoparticles, and exposed or not to a high frequency alternating magnetic field (40 mT, 275 kHz).
  • a high frequency alternating magnetic field 40 mT, 275 kHz
  • Figure 22 Effect of combination of magnetic intra-lysosomal hyperthermia with HSP70 inhibition on lysosome membrane permeabilization.
  • INR1G9-CCK2R cells having internalized Gastrin-MNP were exposed or not to AMF (40 mT, 275 kHz) for 2 hours in the presence or absence of 5 ⁇ of HSP70 inhibitor PES. Cells were then incubated with RPMI 1640 0.5% FBS containing 75 nM LysoTraker Red (Molecular probes, excitation wavelength: 543 nm) for 15 minutes and rinsed with incubation medium. Lysosome membrane permeabilization was analyzed by flow cytometry by quantifying the fluorescence intensity of Lysotraker and normalized over control cells.
  • HSP70 inhibition on Caspase-1 (a) and Caspase-3 (b) activation Cells having or not internalized Gastrin-MNP were exposed or not to AMF in the presence or absence of 5 ⁇ of HSP70 inhibitor PES. 4h after AMF exposure, Caspase-1 or Capsase-3 activation was analyzed by flow cytometry of cells labeled with FAM-FLICA-Caspl or FAM-FLICA-Casp3. Results are expressed as fold change of caspase-1 activation over control cells (in absence of Gastrin- MNP and AMF).
  • the synthesis method and characterization of the magnetic nanoplatform used in the present article have been previously described 4 .
  • the nanoplatform (Gastrin-MNP) is composed of commercial iron oxide magnetic nanoparticles (MNPs) coated with PEG-Amine (Gecco Dots, Sweden) and decorated with 100 molecules of a synthetic replicate of gastrin (Covalab) plus 20 molecules of the fluorescent label DY647-PEG1 (Dyomics GmbH, Germany).
  • the size of the nanoparticle core determined by transmission electron microscopy was 8.7 ⁇ 1.6 nm.
  • the specific absorption rate of these MNPs is 13 W/g at 275kHz and 40mT.
  • N-acetyl-cystein (NAC), Desferoxamine (DFO), bafilomycine A (BafAl), FeCb and Doxorubicin were purchased from Sigma- Aldrich.
  • CA-074-Me and Caspase-3 inhibitors were from ApexBio.
  • Caspase-1 inhibitor was from Santa Cruz Biotechnologies.
  • the HEK293 cells (Flp-In system, Invitrogen) stably expressing the CCK2R, named HEK-CCK2R, and the clone B13 of AR4-2J cells (kindly provided by Prof.
  • Timo Otonkoski University of Helsinki, Finland, with the permission of Dr Itaru Kojima, Gunma University, Maebashi, Japan
  • DMEM medium containing 10% fetal bovine serum (FBS) and lOOIU/ml penicillin/streptomycin (Life technologies).
  • the AGS gastric adenocarcinoma cell line was permanently transfected with CCK2R (AGS-CCK2R) driven by the EFl-a promoter under puromycin selection and grown in DMEM/F-12 medium supplemented with 10% FBS and lOOIU/ml penicillin/streptomycin 16 .
  • the cells were grown in a humidified atmosphere at 95% air and 5% CO2 at 37°C.
  • Cells were seeded onto 6-well plates at a density of 250 to 500xl0 3 cells/well and grown for 24h.
  • Cells were incubated with Gastrin-MNP (16 ⁇ g FesC ml) in medium buffered with lOmM HEPES buffer pH7.4 containing 0.5% FBS and lOOIU/ml penicillin-streptomycin for 24h, at 37°C in a 5%> CO2 atmosphere, washed twice with ice-cold PBS. The number of cells was counted on an aliquot and cellular uptake of Gastrin-MNP was evaluated using a Prussian blue staining assay.
  • Cells were seeded onto 6-well plates at a density of 250 to 500xl0 3 cells/well and grown for 24h. Cells were incubated with Gastrin-MNP (16 ⁇ g FesC ml) in medium buffered with lOmM HEPES buffer pH7.4 containing 0.5% FBS and lOOIU/ml penicillin-streptomycin for 24h, at 37°C in a 5% CO2 atmosphere, washed twice with ice-cold PBS. The number of cells was counted on an aliquot and the electron paramagnetic resonance analysis was performed as described previously (17).
  • Gastrin-MNP 16 ⁇ g FesC ml
  • lOmM HEPES buffer pH7.4 containing 0.5% FBS and lOOIU/ml penicillin-streptomycin
  • Cell treatment by alternalting magnetic field was performed as follows. Cells were seeded onto 4-compartment Cellview dishes (Greiner Bio-One) at a density of 60 to 120xl0 3 cells/compartment, grown overnight and incubated with Gastrin-MNP (16 ⁇ g Fe304/ml) for 24h at 37°C in medium buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% FBS and 100 IU/ml penicillin-streptomycin to allow Gastrin-MNP internalization and accumulation in lysosomes.
  • Gastrin-MNP 16 ⁇ g Fe304/ml
  • Incubation medium was withdrawn and cells were rinsed twice with incubation medium and then incubated with 5mM NAC, ImM DFO, InM BafAl, 10 ⁇ CA-074 Me, 2.5 ⁇ Caspase-1 inhibitor or 10 ⁇ Caspase-3 inhibitor for lh or 120 ⁇ FeC13 for 4h and exposed to AMF (275kHz, 40mT) for 2h using a commercial magnetic inductor (Fives Celes, Lautenback, France) as previously described (4).
  • the temperature of the Cellview dish was maintained at 37.0 ⁇ 0.2°c and controlled using a thermal probe (Reflex, Neoptix, Canada) placed in the incubation medium of the cells.
  • a humidified atmosphere at 5% C02 at 37°C.
  • AMF treatment was investigated as follows: first, cell survival was determined 24h post- AMF treatment using a MTT viability assay. Secondly, dead cells labeled with AnnexinV-iFluor488 and propidium iodide (excitation: 488and 540nm respectively, AAT Bioquest) were counted 4h after the end of AMF exposure as described before (4).
  • INR1 G9- CCK2R cells were seeded at a density of 250xl0 3 cells onto 35mm dishes and transfected with 3 ⁇ g of pCathB-eGFP, pCathB-C29A-eGFP (given by Klaudia Brix, Jacobs University Bremen, Germany) or pMSCV2.2-IRES-GFP-Caspase-l-C284A (given by Petr Broz, University of Basel, Switzerland) using Lipofectamine 2000 reagent (Life technologies).
  • INR1G9-CCK2R cells were seeded 24 h before the experiments onto 4-compartment Cellview dishes (Greiner Bio-One) at a density of 60 to 120xl0 3 cells/compartment and incubated with Gastrin-MNP (16 ⁇ g Fe304/ml) for 24h. Cells were rinsed twice with incubation medium and incubated with 5mM NAC, ImM DFO, InM BafAl for lh or 120 ⁇ FeC13 for 4h.
  • INR1G9-CCK2R cells were seeded 24 h before the experiments onto 4-compartment
  • Lysosomal lipid peroxidation were quantified by fluorescence intensity ratio of lipid peroxidation reagent to Gastrin-MNP labelings of confocal microscopy images (LSM780 confocal microscope, Zeiss) using Image J software. 20-30 cells/experiment were analyzed from at least 4 independent experiments.
  • INR1G9-CCK2R cells were seeded at a density of 250xl0 3 cells onto 35-mm dishes and transfected with O. ⁇ g of YFP-Lampl (Addgene, plasmid #1816) and 2 ⁇ g of pcDNA3 using Lipofectamine 2000 reagent (Life technologies). 24h post-transfection, cells were incubated with Gastrin-MNP-DY549 (16 ⁇ g Fe304/ml) for 24h, included in 0.5% agarose gel, poured into the electromagnet placed in a CELLView dish and covered with RPMI containing 0.5% FBS, as previously described (18). Different positions were marked inside and outside the gap. Cells were exposed to the AMF (53mT, 300kHz).
  • fluorescence intensities of DY549 (excitation: 540nm) and YFP-Lampl (excitation: 510nm) were measured from confocal microscopy images LSM780 confocal microscope, Zeiss) using ImageJ software. Twenty to thirty cells/experiment were analyzed from 4 independent experiments.
  • INR1G9-CCK2R cells were seeded at a density of 250xl0 3 cells onto 35-mm dishes and transfected with O. ⁇ g of RFP-Lampl (Addgene, plasmid#1816), pCathB-eGFP (given by Klaudia Brix, Jacobs University Heidelberg, Germany) and 2 ⁇ g of pcDNA3 using Lipofectamine 2000 reagent (Life technologies).
  • INR1 G9-CCK2R cells were seeded at a density of 250xl0 3 cells onto 35-mm dishes and transfected with O. ⁇ g of pCathB-eGFP (given by Klaudia Brix, Jacobs University Heidelberg, Germany), O. ⁇ g RFP-Lampl (Addgene, plasmid #1817) and 2.% ⁇ g of pcDNA3 using Lipofectamine 2000 reagent (Life technologies). 24h post- transfection, cells were incubated with Gastrin-MNP (16 ⁇ g FesC ml) for 24h.
  • INR1G9- CCK2R cells expressing or not GFP-CathB and having or not internalized Gastrin-MNP were included in 0.5% agarose gel, poured into the electromagnet placed in a CELL View dish and covered with RPMI containing 0.5%> FBS, as previously described ⁇ Conord, 2015 #200 ⁇ . Different positions were marked inside and outside the gap. Cells were exposed to the AMF (53mT, 300kHz) for 60min.
  • INR1G9-CCK2R cells were incubated with 1 ⁇ staurosporine for 4h.
  • cells were pretreated with 2.5 ⁇ Caspase-1 inhibitor, 10 ⁇ Caspase-3 inhibitor or 10 ⁇ CA-074-Me CathB inhibitor for lh.
  • Inhibitors were washed from the cells before adding FLICA.
  • cells were washed and analyzed by confocal micrsocopy (LSM510 confocal microscope, Zeiss). After Caspase-1 staining, cells were labeled with AnnexinV-iFluor555 according to manufacturer's instruction (AAT Bioquest). Counting of labeled cells was carried out by analyzing confocal microscopy images representing populations of 2-3000 cells/experiment using ImageJ software.
  • Mitochondrial Membrane Potential Assay For fluorometric measurement of mitochondrial outer membrane permeabilization (MOMP), INR1G9-CCK2R cells were seeded 24h before the experiments onto four- compartments Cellview dishes (Greiner Bio-One) at a density of 60x10 3 cells/compartment and incubated with Gastrin-MNP (16 ⁇ FesC ml) for 24h, exposed to AMF for 2h and stained, 4 or 24h after AMF application, with ⁇ JC10 for 30-min as manufacturer's instruction (AAT Bioquest). Next, 2-3000 cells/experiments were washed and analyzed by confocal micrsocopy (LSM780 confocal microscope, Zeiss). Quantification of MOMP was performed by analyzing the green/red fluorescence ratio of confocal microscopy images.
  • MOMP mitochondrial outer membrane permeabilization
  • INR1G9-CCK2R cells were seeded 24h before the experiments onto four-compartment
  • DAPI 4,6-diamidino-2-phenylindole
  • 2-3000 cells/experiments were analyzed under a fluorescence confocal microscope (LSM780 confocal microscope, Zeiss) using 550 or 385nm excitation filter, and 590 or 420nm band pass filter for TUNEL and DAPI assays, respectively. Images of 10 microscopic fields at 40-fold magnification were captured randomly, and the number of TUNEL-positive cells and DAPI-positive nuclei was counted manually to calculate the percentage of cells presenting DNA fragmentation.
  • INR1G9-CCK2R cells were seeded 24h before the experiments onto 35-mm dishes, incubated with Gastrin-MNP (16 ⁇ g Fe304/ml) for 24h, exposed or not to AMF for 2h. Cells were scraped and centrifuged (lOOOxg, 5-min) in PBS at 4°C. The pellet was resuspended in 40 ⁇ 1 phosphate-citrate buffer consisting of 192 parts of 0.2M Na 2 HP04 and 8 parts of 0.1M citric acid (pH7.8), incubated at room temperature for at least 30-min and centrifuged lOOOxg for 5 min.
  • INR1G9-CCK2R or Th l macrophages were seeded 24h before the experiments onto 35 -mm dishes, primed or not with ultrapure lipopolysaccharide (LPS, 500 ng/mL) (Invivogen) for 24h, incubated with Gastrin-MNP ( ⁇ g FesC ml) for 24h, exposed or not to AMF for 2h. Supernatants were recovered and assayed for II- 1 ⁇ secretion with an ELISA kit according to the manufacturer's instructions (Becton Dickison).
  • LPS ultrapure lipopolysaccharide
  • Gastrin-MNP ⁇ g FesC ml
  • INR1G9-CCK2R or Thpl macrophages were seeded 24h before the experiments onto 35-mm dishes, primed or not with ultrapure LPS (500 ng/mL) (Invivogen) for 4h, incubated with Gastrin-MNP (16 ⁇ g FesC ml) for 24h, exposed or not to AMF for 2h.
  • Cells were lysed in lOmM HEPES (pH7.5) buffer containing lOmM KC1, O.lmM EDTA, O.lmM EGTA, ImM DTT, 2% Complete protease inhibitor cocktail® (Roche, 1 tablet/ml), ImM NasVC and 1% Nonidet P-40.
  • Proteins were separated by SDS-PAGE followed by Western-blot assays using anti-11- ⁇ (1/500, sc-7884 Santa Cruz Biotechnologies), anti-Caspase-1 (1/200, sc-514 M20 Santa Cruz Biotechnologies) or anti-Caspase-3 (1/200, sc-271759 C6 Santa Cruz Biotechnologies) followed by reprobing with anti-GAPDH (1/1000, sc-25778 FL335, Santa Cruz Biotechnologies) for loading control.
  • the protein expression signal was detected with Pierce SuperSignal Western blotting substrate.
  • INR1G9-CCK2R cells were seeded 24h before the experiments onto four-compartments Cellview dishes (Greiner Bio-One) at a density of 60x 10 3 cells/ compartment and incubated with 1 nM BafAl for lh at 37°C, stained with 5 ⁇ g/ml acridine orange for 15-min and rinsed in complete medium. Cells were observed under a fluorescence confocal microscope (LSM780, Zeiss) using 550nm excitation filter and 590nm band pass filter. Measurements of lysosomal red AO-fluorescence were analyzed by determining the intensity of red fluorescence over that of control cells.
  • Results are expressed as the mean ⁇ SEM of at least 3 independent experiments. Statistical analysis was performed using AN OVA test. Differences were considered significant when p ⁇ 0.05.
  • Magnetic intra-lysosomal hyperthermia enhances ROS production through the Fenton reaction within lysosomes
  • Gastrin-grafted iron oxide MNPs are internalized specifically by the pancreatic endocrine tumoral cells INR1G9 overexpressing the CCK2R (INR1 G9-CCK2R cells) through a CCK2R-dependent physiological process, and are then trafficked to lysosomes where they accumulated (Fig. lA).
  • Gastrin-MNPs killed 32.3 ⁇ 2.7% of INR1 G9-CCK2R tumoral cells by a phenomenon that we termed "Magnetic IntraLysosomal Hyperthermia" (MILH) (4) (Fig. lA).
  • MILH Magnetic IntraLysosomal Hyperthermia
  • Fig.lA propidium iodide labelings
  • lysosomes are well-known major sites of ROS production through the Fenton reaction (Fe 2+ +H 2 0 2 ⁇ Fe 3+ +OH ⁇ +OH') which catalyzes the transformation of hydrogen peroxide to hydroxyl radicals.
  • This reaction is dependent on low-molecular- weight iron derived from degraded iron-containing proteins, lysosomal lumen acidity and temperature (16-18).
  • LMP lysosome membrane permeabilization
  • MILH also generates ROS by activating, for instance, the mitochondrial respiratory chain complex or NADPH oxidases which are transmembrane proteins mostly present at the plasma membrane, in endosomes and in endoplasmic reticulum (21).
  • rotenone an inhibitor of the mitochondrial respiratory chain complex
  • DPI diphenyleneiodonium
  • MILH initially triggers ROS production through the Fenton reaction within lysosomes and, likely secondary, through NADPH oxidases.
  • ROS production was initiated through the Fenton reaction within lysosomes and, likely secondary, through NADPH oxidases.
  • no significant variation in ROS level was observed between the different conditions in the absence of AMF exposure (Fig.7 A, 8).
  • thermometers were composed of the fluorescent moieties DY549 attached to MNPs via PolyEthyleneGlycol of 7nm-length or the Yellow Fluorescent Protein (YFP) linked to Lysosome- Associated Membrane Protein 1 (Lampl) (4). Intensity of fluorescence emission of the two probes was previously shown to depend on the temperature (25). This relationship was confirmed by the calibration curve showing linearity of fluorescence intensity decrease as a function of temperature increase (Fig.9).
  • Quantitative fluorescence imaging was then carried out on cells exposed to AMF for a limited duration ( ⁇ 10-min) and incubated with ROS scavenger NAC in order to preserve lysosome integrity and avoid possible chemical quenching of the fluorescent molecular thermometers by ROS.
  • fluorescence intensity of DY549 grafted to MNPs declined with the time of AMF exposure.
  • the percentage of decrease of fluorescence intensity observed after ⁇ 7-min of treatment corresponds to a temperature increase of 14.1 ⁇ 1.4°C, indicating that the temperature actually reached 51°C at 7nm-distance from the MNP core.
  • MILH-enhanced ROS production within lysosomes causes lysosomal membrane permeabilization and cell death
  • the decrease of CathB content in lysosomes was chosen to represent lysosomal enzyme leakage (Fig.2B).
  • INR1G9-CCK2R cells having internalized Gastrin-MNPs presented a high level of GFP-CathB/RFP-Lampl co localization which was reduced by the application of AMF (Pearson coefficient: 0.54 ⁇ 0.03 vs 0.69 ⁇ 0.02 in presence vs absence of AMF).
  • the effect of MILH on GFP-CathB/RFP-Lampl co localization was cancelled in the presence of the NAC scavenger (Pearson coefficient: 0.74 ⁇ 0.03, Fig. 2B, 10).
  • the lysosomal CathB level decreased significantly by 25.4 ⁇ 4.7% and 43.9 ⁇ 6.3% after 30 and 60-min of MILH treatment respectively, demonstrating that leakage of lysosome content is an early event occurring concomitantly with the generation of lysosomal ROS.
  • RFP-Lampl and GFP-CathB fluorescence intensities decreased respectively by 20 ⁇ 2.4% and 16.5 ⁇ 6.6% from 30-min of AMF exposure, strongly suggesting that the changes in RFP-Lampl and GFP-CathB fluorescence represent respectively lysosomal rupture and CathB leakage from lysosome into the cytosol and that both events occur early in the signaling cascade leading to cell death.
  • MILH-induced cell death was not inhibited by rotenone or DPI (Fig.12), indicating that neither the mitochondria respiratory chain complex nor NADPH oxidases were involved in cell death, further supporting the idea that ROS generated by NADPH oxidases are secondary to lysosomal stress caused by MILH treatment. All together, these results demonstrate that lysosomal ROS production, which is enhanced by MILH through the Fenton reaction, is at the origin of cancer cell death.
  • MILH-induced cell death depends on lysosomal cysteine cathepsins which include cathepsins B, C, H, K, L, S and X (4).
  • CathB which is involved in lysosomal cell death, abundant in lysosomes, overexpressed in cancers and maintains its activity at neutral pH (27-29).
  • MILH experiments were performed in the presence of the selective inhibitor of CathB CA-074-Me (30) or by overexpressing the enzymatically inactive mutant GFP-CathB-C29A.
  • both the inhibitor CA-074-Me and the inactive mutant GFP-CathB-C29A prevented MILH-induced cell death (1.2 ⁇ 0.1 and 1.5 ⁇ 0.1-fold over basal value vs 2.5 ⁇ 0.1-fold).
  • overexpression of wild-type CathB (GFP-CathB-WT) augmented the efficiency of MILH to eradicate cancer cells (4.1 ⁇ 0.2 vs 3.0 ⁇ 0.3-fold over basal, Fig.3C) reaching 34.8% of dead cells comparatively to 17.3% of dead cells in the absence of CathB-WT overexpression.
  • MILH induces cell death through a Caspase-l-dependent but Caspase-3 independent mechanism.
  • MILH did not modify mitochondrial outer membrane permeabilization (MOMP), which critically relies on apoptotic cell death, as demonstrated with staurosporine treatment (Fig.4C, 15), lh or 4h after AMF exposure (31). Hence, cell death induced by MILH does not involve an apoptotic pathway.
  • MOMP mitochondrial outer membrane permeabilization
  • Caspase-1 An important function of Caspase-1 consists to process the precursor of the inflammatory cytokines, interleukine- ⁇ (II 1 ⁇ ) and interleukine-18 (11-18) into their active forms (33). Furthermore, activation of Caspase-1 in cells is a hallmark of pyroptosis, which is a form of regulated cell death, usually defined by several additional biochemical features such as independence of apoptotic caspases, Ann/PI positive labeling, DNA fragmentation (34). The current study demonstrates that MILH-induced cell death is dependent on Caspase-1 activation and featured by Ann/PI labeling.
  • MILH induces Caspase-1 -dependent cell death without activating the Caspase-1 pro-inflammatory response in the endocrine tumoral INR1G9-CCK2R cells.
  • proinflammatory response could be activated by MILH in cancer cells expressing higher level of Pro-ILip.
  • Gastrin-grafted MNPs induces the death of cells expressing the CCK2R by MILH.
  • the preceding experiments analyzed the effects and mechanisms of MILH applied to the pancreatic endocrine tumor cells INR1G9-CCK2R.
  • the gastric cell line AGS and the pancreatic cell line AR4-2J, which express the CCK2R at low levels ( ⁇ 100 fmol/10 6 cells) (36).
  • Cell survival data show that, 24h after AMF exposure, MILH has eradicated 22.0 ⁇ 4.5% of AGS-CCK2R cells and 23.1 ⁇ 1.3% of AR4-2J cells presenting Gastrin-MNPs mainly in their lysosomes (Fig.6 A).
  • HEK-CCK2R cells were killed using ⁇ g/ml or 1 ⁇ of Gastrin-MNP respectively (Fig.17A).
  • Ann/PI labeling confirmed that MILH-induced cell death was detectable 4h post-AMF (Fig.6B) and dead cells were more often labeled with PI in AGS-CCK2R and AR4-2J cells than in INR1G9-CCK2R cells.
  • MILH also triggered cell death in the non-tumoral embryonic kidney cell overexpressing the CCK2R (HEK-CCK2R: 2.0 ⁇ 0.2 pmol/10 6 cells) previously used to characterize uptake and cellular trafficking of Gastrin-MNP (Fig. l7B) (4,37).
  • AGS-CCK2R, AR4-2J and HEK-CCK2R cells was similar to that identified in INR1G9- CCK2R. Firstly, no significant Caspase-3 activation could be detected in the 3 cell lines, similarly to INR1G9-CCK2R cells (Fig. l7D, 18). Secondly, MILH increased Caspase-1 activation, 4h after AMF exposure, by 3.5- ⁇ 0.1, 3.6- ⁇ 0.2-fold and 6.2 ⁇ 0.6-fold in AGS- CCK2R, AR4-2J and HEK-CCK2R respectively, similarly to INR1G9-CCK2R cells (Fig.6D, 17E).
  • MILH is effective in inducing the death of cancer cells from three different types of cancer (pancreatic endocrine, pancreatic exocrine and gastric) even if the targeted receptor, is expressed at low levels and if minute amounts of Gastrin-MNPs are internalized.
  • MILH-induced cell death occurs through activation of Caspase-1 and without activation of Caspase-3, indicating that MILH triggers a non-apoptotic cell death pathway following lysosome damage which is dependent on Caspase-1 activation.
  • MILH first upregulates the catalysis of ROS production through the Fenton-type reaction within lysosomes which subsequently causes lipid peroxidation, followed by LMP and the leakage of lysosomal content into the cytosol.
  • CathB plays a critical role in cell death and activates Caspase-1.
  • MILH-induced tumor cell death can be related to pyroptosis mainly described in macrophages, but rarely in epithelial cells (32,38,39).
  • Caspase-1 is synthesized as a cytosolic, inactive, monomeric zymogen (pro-Caspase-1) that is thought to be activated by dimerization and autoproteo lytic processing, resulting in generation of large and small subunits (called p20 and pi 0) of the catalytically active enzyme (44,45).
  • This activation step is preceded by recruitment of Pro-Caspase-1 into inflammasomes, which are multiprotein signaling complexes.
  • a direct processing of Caspase-1 by CathB (46,47) or an indirect mechanism of activation through CathB-induced activation of inflammasome complexes (38,47-49) were reported.
  • CathB activation is critical for Caspase-1 activation and tumor cell death indicating that one of these two mechanisms may function, but this remains to be determined more precisely.
  • MILH Magnetic intra-lysosomal hyperthermia
  • the strategy developed in the team is in the field of targeted nanotherapy of cancers by magnetic hyperthermia.
  • This strategy aims at developing a new therapeutic approach for cancer eradication by tumor targeting using magnetic iron oxide nanoparticles on which the ligand of a receptor overexpressed in cancers is grafted.
  • the ligand allows the specific binding of the nanoparticles to the receptors located on the surface of cancer cells and then the activation of these receptors triggers the process of internalisation of the "cargo" composed of the receptor, its ligand and the magnetic nanoparticles.
  • This internalization is followed by intracellular transport within vesicles of endocytosis to the lysosomes, vesicles of degradation of "cellular waste”.
  • MILH magnetic intra-lysosomal hyperthermia
  • MILH Magnetic Intra-lysosomal Hyperthermia
  • MILH catalyzes ROS production by the Fenton reaction within the lysosomes. This reaction is at the origin of the signaling cascade leading to lysosome permeabilization and to cell death.
  • an iron-complexing agent prevents these events and that, conversely, the culture of cells in an iron-ion enriched medium increases the treatment efficiency (death level increased by 40%) ( Figure 2D).
  • a pH-neutralizing agent (bafilomycin A) of the lysosomes inhibits these effects, whereas an agent increasing the acidity of the lysosomal pH (Artesunate) increases them.
  • MILH has been tested and validated by targeting the gastrin receptor (G-protein coupled transmembrane receptor, named CCK2R) overexpressed on the surface of pancreatic endocrine tumor cells, pancreatic exocrine or gastric cancer.
  • CCK2R G-protein coupled transmembrane receptor
  • Targeting of the iron oxide nanoparticles (size 10 nm) was obtained by grafting, on the surface of these nanoparticles, a peptide analogue of natural gastrin.
  • Cysteine protease involved in MILH-induced death is Cathepsin-B.
  • the mechanism of death induced by MILH is not apoptosis and does not involve Caspase-3. It is a mechanism involving Caspase-1 which could be related to cell death by pyroptosis, known to occur in cells of the immune system during inflammatory processes.
  • activation of Caspase-1 occurs without proteolysis of pro-Caspase 1 and without secretion of interleukin- ⁇ (IL 1 ⁇ ) in the cell lines used in this study.
  • IL 1 ⁇ interleukin- ⁇
  • Pro-Caspase- 1 proteolysis and II 1 ⁇ secretion may happen in other cell lines, depending on the level of their expression.
  • Lysosome permeabilization can be induced in apoptotic signaling initiated from death receptors or using lysosomotropic agents. To date, these agents do not specifically target tumor cells. Magnetic intra-lysosomal hyperthermia (MILH) induces cell death by a different mechanism, although initiated by permeabilization of the lysosome membrane. The leakage of lysosomal enzymes and more particularly of Cathepsin-B activates the pro-inflammatory and non-apoptotic Caspase-1 which then triggers a mechanism of cell death by pyroptosis.
  • MILH Magnetic intra-lysosomal hyperthermia
  • MILH uses the newly emerging lysosomal death pathway and is described as a promising way to circumvent the apoptosis resistance phenomena that cancer cells develop before and during anti-cancer treatments ( ⁇ 1).
  • this therapeutic strategy could also be used in combination with conventional treatments to increase the efficacy of eradication of tumor cells while decreasing the doses of the latter in order to limit their side effects (see Example 1 And ⁇ 2).
  • MILH-induced death pathway uses the Fenton reaction taking place within the lysosomes. Several approaches could be used to increase this one:
  • iron enrichment could also be envisaged by a pretreatment with transferrin.
  • Magnetic nanoparticles are capable of transforming electromagnetic energy into heat when exposed to a high frequency alternating magnetic field. The heating of the magnetic particles depends on their size and their magnetic properties.
  • HSP70 heat shock protein inhibits lysosomal membrane permeabilization and MILH-induced cell death (data not shown). It is therefore not excluded that HSP70, localized in lysosomes, play a protective role in the efficiency of HMIL on the leakage of the lysosome membrane. It should be noted that HSP proteins are often overexpressed in tumor cells. Their activity is modulable by means of pharmacological inhibitors. Pretreatment with HSP70 inhibitors could increase the effectiveness of the HMIL strategy (see example 3: HMIL and HSP70 inhibitors).
  • lysosomal enzymes including Cathepsin-B are released into the cytosol.
  • Cathepsin-B has a primordial role in the cell death triggered by MILH since it activates Caspase-1, the enzyme executing cell death.
  • MILH does not induce apoptosis but a Caspase-1 -dependent pathway that could be related to pyroptosis.
  • MILH could be an alternative to eradicate tumor cells resistant to conventional treatments and cell death by apoptosis.
  • cells dying by pyroptosis present a "porous" plasma membrane allowing the passage of pro-inflammatory molecules, such as ATP.
  • pyroptosis has the power to stimulate inflammation, which from the point of view of anti-cancer therapies is an advantage.
  • the level of Pro-Caspase 1 (the pro-form of Caspase-1) can be stimulated by cytokines such as TNFa or interleukin l ; it can therefore be envisaged to increase the effectiveness of an anti-cancer treatment by MILH by first administering a cytokine, such as TNFa, to the patients.
  • MILH strategy has several advantages:
  • the inventors also investigate the MILH in a transgenic murine model of endocrine tumors (mouse MEN1 +/-).
  • INR1G9-CCK2R INR1G9-CCK2R.
  • HSP70 inhibition PES inhibitor or SiRNA
  • PES inhibitor or SiRNA PES inhibitor
  • Other drug inhibiting HSP70 are also used; preliminary results show identical results.
  • the effects of the MILH/HSP70 inhibition are analyzed in vivo on different murine models of cancer: tumor growth, cell death, cell survival and proliferation, study of signaling pathways associated with cell death and survival.
  • Heat shock protein 70 the next chaperone to target in the treatment of human acute myelogenous leukemia?

Abstract

La présente invention concerne des méthodes et des compositions pharmaceutiques pour le traitement du cancer chez un sujet nécessitant un tel traitement. Des stratégies thérapeutiques faisant appel à des médicaments qui provoquent la mort des cellules lysosomales ont été proposées pour l'éradication des cellules cancéreuses résistantes. Une nanothérapie basée sur l'hyperthermie intra-lysosomale magnétique (MILH) générée par des nanoparticules magnétiques (MNP) qui sont greffées avec des ligands de récepteurs surexprimés dans des tumeurs semble être une option thérapeutique très prometteuse. Les inventeurs ont examiné les mécanismes grâce auxquels la MILH induit la mort cellulaire à l'aide des MNP greffées avec la gastrine administrées spécifiquement à des lysosomes de cellules tumorales de différents cancers. Les inventeurs démontrent que la MILH provoque la mort cellulaire par l'intermédiaire d'une voie de signalisation non apoptotique. Le mécanisme de mort cellulaire implique l'élévation de la température à la périphérie des nanoparticules qui améliore la production d'espèces réactives de l'oxygène par la réaction de Fenton lysosomale. Par la suite, la MILH induit une peroxydation lipidique, une perméabilisation de la membrane lysosomale et une fuite d'enzymes lysosomales dans le cytosol, y compris de la cathepsine-B qui active la caspase-1 mais pas la caspase-3. Ainsi, l'invention concerne une nanoparticule magnétique greffée avec un agent de ciblage des tumeurs destinée à être utilisée dans une méthode permettant d'induire une signalisation non apoptotique d'une cellule cancéreuse chez un sujet atteint d'un cancer nécessitant un tel traitement.
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