EP3654937A1 - Injizierbare wasser-in-öl-emulsionen und verwendungen davon - Google Patents

Injizierbare wasser-in-öl-emulsionen und verwendungen davon

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Publication number
EP3654937A1
EP3654937A1 EP18740230.0A EP18740230A EP3654937A1 EP 3654937 A1 EP3654937 A1 EP 3654937A1 EP 18740230 A EP18740230 A EP 18740230A EP 3654937 A1 EP3654937 A1 EP 3654937A1
Authority
EP
European Patent Office
Prior art keywords
oil
emulsion
nanoparticles
emulsion according
emulsions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18740230.0A
Other languages
English (en)
French (fr)
Inventor
Frédéric Deschamps
Thierry DE BAERE
Lambros TSELIKAS
Thomas ISOARDO
Nicolas HUANG
Laurence Moine
Nicolas Tsapis
Elias Fattal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Universite Paris Saclay
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Universite Paris Saclay
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut Gustave Roussy (IGR), Universite Paris Saclay filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP3654937A1 publication Critical patent/EP3654937A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to injectable and stable water-in-oil therapeutic emulsions. It also relates to the use of said emulsions, in particular for the treatment of cancer.
  • Hepatic arterial chemoembolization is the standard treatment for certain liver tumors, such as inoperable hepatocellular carcinoma and hypervascular metastases.
  • This treatment consists of a combination of chemotherapy injection (usually doxorubicin) and ischemic arterial embolization directly into the tumor arteries.
  • Chemotherapy is released slowly and prolonged into the tumor arteries through the use of a vector in which chemotherapy is previously "loaded". This vector should ideally have diagnostic properties (visibility in imaging) and therapeutic (controlled and prolonged release of chemotherapy).
  • none of the vectors currently used Lipiodol® or loaded beads has this theranostic property (diagnostic and therapeutic).
  • the loaded beads are microspheres based on polyvinyl alcohol which can be loaded with chemotherapy (ion exchange mechanism) and are available in different sizes ranging from 70 to 700 ⁇ . These beads have already demonstrated a significant increase in exposure to tumor chemotherapy and a decrease in systemic toxicity but they do not contain contrast agents. Thus, it is impossible to accurately visualize the administration of chemotherapy during the procedure and to quantify the concentration of chemotherapy in the tumor after the procedure. In addition, only doxorubicin and irinotecan can be loaded into beads so far.
  • Lipiodol is used for its hydrophobic radiopaque contrast agent (oil) properties under X-ray fluoroscopy and for its selectivity to tumor arteries.
  • the realization of chemotherapy emulsions with Lipiodol thus makes it possible to hope for a selective and visible delivery of chemotherapy in liver tumors.
  • the theranostic properties of these lipiodolated emulsions are limited by their low stability: the phase shift between chemotherapy and lipiodol is done in a few minutes. Very quickly, the two phases of the emulsion separate and almost all chemotherapy that is soluble in water disappears from the tumor.
  • the present invention therefore aims to provide a new therapeutic water-in-oil emulsion, stable over time, especially for at least 24 hours.
  • the present invention also aims to provide a new stable emulsion preferably having theranostic properties.
  • the present invention also aims to provide a new stable emulsion comprising at least one anticancer agent, and having a tumor selectivity greater than that of conventional emulsions.
  • the present invention also aims to provide a new emulsion that can be loaded with different therapeutic agents and potentially visible magnetic resonance imaging (MRI).
  • MRI visible magnetic resonance imaging
  • the present invention also aims to provide a new biodegradable emulsion, biocompatible and less toxic or irritating than usual emulsions stabilized with synthetic surfactants or mineral particles.
  • the present invention relates to a water-in-oil emulsion comprising a continuous oily phase and a dispersed aqueous phase in the form of drops, said aqueous phase comprising polyester-based nanoparticles, and at least one therapeutic agent.
  • Oily phase
  • the emulsion according to the invention comprises a continuous oily phase (or fatty phase).
  • the oily phase according to the invention comprises at least one oil.
  • the oily phase may comprise a single oil or a mixture of several different oils.
  • Any suitable pharmaceutical oil or oil can be used to form the oil phase.
  • oil is meant a non-aqueous compound, immiscible with water, liquid at room temperature (25 ° C) and atmospheric pressure (760 mm Hg).
  • oils adapted according to the invention mention may in particular be made of fatty acids, esters of fatty acids and mineral oils (especially like squalene).
  • marine oils especially fish oils, and in particular salmon oil.
  • ethyl icosapentate which is one of the polyunsaturated fatty acids contained in fish oil.
  • the oily phase according to the invention preferably comprises injectable oils, preferably injectable vegetable oils.
  • injectable oils mention may be made of those well known to those skilled in the art, especially as described in the article by Hippamgaonkar et al. (2010) AAPS Pharm Sci Tech, 11 (4), p.1526-1540.
  • the oily phase according to the invention comprises long-chain triglycerides (LCT) and / or medium-chain triglycerides (MCT).
  • LCT long-chain triglycerides
  • MCT medium-chain triglycerides
  • the LCTs there may be mentioned triolein, soybean oil, safflower oil, sesame oil and castor oil.
  • the MCT include oil fractionated coconut as triglycerides caprylic / capric acids, such as MIGLYOL 810 ® or 812 products ®, Neobee ® M-5 or Captex ® 300.
  • the oily phase comprises at least one vegetable oil, in particular castor oil, sesame oil, carnation oil, olive oil, soybean oil, coconut oil, triolein and mixtures thereof.
  • the oily phase comprises at least one oil that has been modified with iodine to render it radiopaque.
  • oils mention may be made of flaxseed oil, linseed oil (see J Pharm Sci, 102: 4150, 2013), Labrafac WL1349 (Attia et al., Macromol Biosci, 2017), castor oil (ACS Nano, 8 (10), 10537, 2014) or vitamin E.
  • the oily phase comprises ethyl esters of iodized fatty acids of the carnation oil, and in particular consists of Lipiodol.
  • Lipiodol consists of the ethyl esters of the iodized fatty acids of the carnation oil. It contains 43 to 53% iodine. It is prepared by saponification of the carnation oil which releases the fatty acids in the form of soaps which are subsequently iodinated with iodine chloride and finally esterified with ethanol. The carnation oil is extracted from black poppy seeds (Papaver somniferum). The main fatty acids included in this oil are linoleic acid and linolenic acid. Lipiodol is also used as a contrast agent in radiological investigations.
  • the oily phase comprises triglycerides of average chain length, in particular comprising between 8 and 12 carbon atoms, or a mineral oil composed mainly of squalene.
  • the oily phase of the emulsion according to the invention further comprises a compound, comprising for 100% of its mass, from 10% to 95% of a mineral oil comprising:
  • the oily phase of the emulsion according to the invention may comprise at least one compound chosen from MONTANIDE products marketed by SEPPIC such as those described in application FR 2 955 776.
  • the oily phase of the emulsion according to the invention may further comprise at least one surfactant.
  • the ratio between the volume of oily phase and the volume of aqueous phase is between 4: 1 and 3: 3.
  • this ratio is 4: 1, 3: 1, 2: 1, 3: 2, or 3: 3 and is preferably 3: 1.
  • the emulsion according to the invention comprises from 40% to 80%, preferably from 60% to 80%, by weight of oily phase relative to the total weight of said emulsion.
  • the emulsion according to the invention comprises a dispersed aqueous phase.
  • the aqueous phase according to the invention comprises at least water.
  • the emulsion according to the invention comprises from 20% to 60%, preferably from 20% to 40%, by weight of aqueous phase relative to the total weight of said emulsion.
  • the aqueous phase according to the invention is in the form of drops, sizes greater than one micron.
  • the droplet size of the aqueous phase is between 10 ⁇ and 100 ⁇ , preferably between 20 ⁇ and 50 ⁇ .
  • the aqueous phase of the invention comprises at least one nanoparticle based on polyester.
  • the nanoparticles according to the invention are biodegradable.
  • the biodegradable polyester-based nanoparticles are used as solid particles to stabilize the water / oil interface in the emulsions. These nanoparticles have low toxicity and a limited inflammatory response is observed in the presence of these nanoparticles.
  • the emulsions according to the invention have the advantages of being biodegradable, biocompatible and potentially less toxic or irritating than usual emulsions stabilized with synthetic surfactants or mineral particles.
  • nanoparticles according to the invention are polymeric nanoparticles well known to those skilled in the art.
  • these nanoparticles are solid particles having at least two dimensions less than 1 ⁇ .
  • these nanoparticles are nanospheres with a matrix core of average size less than 1 ⁇ (when measured by light scattering).
  • the nanoparticles have an average size of 200 nm. They are generally between 50 nm and 400 nm and more precisely between 100 nm and 300 nm.
  • size refers to the diameter
  • the emulsions of the invention comprise between 5 and 25 mg / ml, preferably 15 mg / ml, of nanoparticles as defined above.
  • the nanoparticles according to the invention are based on polyester. As mentioned above, they are preferably solid and therefore consist of at least one polyester. According to one embodiment, the polyester-based nanoparticles are chosen from the group consisting of polyacetic acid polylactide-based nanoparticles, poly glycolic acid (polyglycolide), lactide-glycolide copolymers (with different ratios of lactide / glycolic), copolymers of lactide-glycolide-co-polyethylene glycol, polyorthoesters, polyanhydrides, polybutylacetone, polyvalerolactone, poly malic acid, polylactones and mixtures thereof.
  • polyacetic acid polylactide-based nanoparticles poly glycolic acid (polyglycolide), lactide-glycolide copolymers (with different ratios of lactide / glycolic), copolymers of lactide-glycolide-co-polyethylene glycol, polyorthoesters, polyanhydr
  • the nanoparticles based on polyester further comprise iron oxide particles, preferably of size between 5 nm and 30 nm, and preferably equal to 10 nm.
  • An advantage of using Pickering emulsions for injection is the possibility of making them detectable by MRI, thanks to the oily nature of the vector and / or through the incorporation of iron oxide particles.
  • MRI is increasingly used in patients because of a significantly higher accuracy and sensitivity for diagnosis in the detection of hepatocellular carcinoma compared to CT.
  • an imaging agent has been introduced into the stabilizing nanoparticles used according to the invention.
  • the iron oxide particles are considered effective T2 contrast agents. Therefore, according to one embodiment, the therapeutic agent is encapsulated within the water droplets, while the iron oxide particles are incorporated into the nanoparticles stabilizing these droplets. Having the therapeutic agent with the iron oxide nanoparticles in a single injectable form makes it possible to follow the fate of the therapy after administration.
  • the emulsion according to the invention comprises at least one therapeutic agent.
  • said therapeutic agent is encapsulated in the drops of aqueous phase.
  • This encapsulation is advantageous in that it makes it possible to protect and thus stabilize certain types of therapeutic agents, in particular fragile molecules such as monoclonal antibodies.
  • Monoclonal antibodies de facto of their protein structure can be degraded when exposed to heat, light, pH, or with strong agitation, in the presence of certain metals and oils / organic solvents. These are fragile molecules whose handling may present risks of aggregation even denaturing. They may be altered and denatured when incorporated into a vector except to provide a protection system. Current emulsions do not allow the transport of these active ingredients and their controlled release. By introducing a physical barrier via the stabilizing nanoparticles between the water droplets containing the therapeutic agent and the oil, the antibody remains stable.
  • the therapeutic agent is chosen from immunomodulators, anticancer drugs, anti-angiogenic drugs, anti-infective drugs, anti-inflammatory drugs, imaging contrast agents, radioactive agents and agents. infectious.
  • the term “immunomodulator” designates a compound capable of modulating the immune response.
  • tumor antigen refers to a molecule specifically present on the surface of cells (for example: vascular endothelial growth factor, CTLA-4, PD1 or PDL-1).
  • anti-angiogenic drug refers to a medicament for inhibiting the growth process of new blood vessels (neovascularization) from preexisting vessels (eg Bevacizumab, Sunitinib or Sorafenib).
  • anti-infective drug refers to a medicament for the treatment of infections of microbial origin (antibiotics, anti-virals or anti-fungal, for example).
  • antibiotics there may be mentioned for example arnoxicillin or cefazolin.
  • anti-inflammatory drug refers to a medicament for the treatment of inflammations (steroidal and non-steroidal anti-inflammatory drugs).
  • steroids and non-steroidal anti-inflammatory drugs for example, there may be mentioned methylprednisolone or ketoprofen.
  • the term "imaging contrast agent” refers to a substance that artificially increases the contrast to visualize an anatomical structure or naturally little or no contrast. It is more particularly possible to mention iodinated contrast products, MRI contrast products or radio-elements.
  • the term “radio-elements” denotes a chemical element that emits ⁇ , ⁇ - or ⁇ radiation often accompanied by the emission of high energy photons. These elements are used in nuclear medicine for low-dose diagnostic purposes, and for high-dose therapeutic purposes to treat cancers ( 99m Technetium, 18 Fluor, 123 lode, 90 Yttrium, 131 Iodine or 166 Holmium for example).
  • infectious agent refers to a biological agent responsible for an infectious disease (such as bacteria, viruses, prions, yeasts and parasites).
  • the emulsion according to the invention comprises, as therapeutic agent, at least one anticancer drug.
  • the anticancer drug is selected from the group consisting of alkylating agents, platinum derivatives, cytotoxic antibiotic agents, antimicrotubule agents, anthracyclines, group I and II topoisomerase inhibitors, fluoropyrimidines, cytidine analogues. , adenosine analogs, methotrexate, folinic acid, enzymes, antivascular agents, anti-angiogenic agents, antimitotic agents, including spindle poisons, kinase inhibitors, hormones, antibodies monoclonal antibodies, radioelements, oncolytic viruses and their mixtures.
  • alkylating agents examples include cyclophosphamide, melphalan, ifosfamide, chlorambucil, busulfan, thiotepa, prednimustine, carmustine, lomustine, semustine, steptozotocine, decarbazine, temozolomide, procarbazine and hexamethylmelamine.
  • platinum derivatives mention may in particular be made of cisplatin, carboplatin and oxaliplatin.
  • cytotoxic antibiotic agents there may be mentioned, for example, bleomycin, mitomycin and dactinomycin.
  • antimicrotubule agents there may be mentioned vinblastine, vincristine, vindesine, vinorelbine and taxoids (paclitaxel and docetaxel).
  • anthracyclines mention may be made of doxorubicin, daunorubicin, idarubicin, epirubicin, mitoxantrone and losoxantrone.
  • topoisomerase inhibitors of groups I and II mention may be made, for example, of etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex.
  • Cytidine analogues include 5-azacytidine, cytarabine, gemcitabine, 6-mercaptomurine and 6-thioguanine.
  • adenosine analogues such as pentostatin, cytarabine or fludarabine phosphate
  • L-asparaginase hydroxyurea
  • trans-retinoic acid hydroxyurea
  • suramin hydroxyurea
  • dexrazoxane amifostine
  • herceptin as well as estrogenic and androgenic hormones.
  • combretastatin derivatives for example CA4P
  • chalcones or colchicine for example ZD6126
  • Anti-angiogenic agents include bevacizumab, sorafenib or sunitinib malate.
  • Therapeutic tyrosine kinase inhibitors include imatinib, gefitinib, sunitinib, sorafenib, vandetanib and erlotinib.
  • the poisonous spindle agents there may be mentioned vincristine, vinblastine, taxol and taxotere.
  • radioelements 99m Technetium, 18 Fluor, 123 lode, 32 Phosphorus, 89 Strontium, 90 Yttrium, 131 lode, 166 Holmium, 18 ⁇ Rhenium and 169 Erbium.
  • Oncolytic viruses include T-VEC.
  • the therapeutic agent is an anticancer drug selected from the group consisting of doxorubicin, irinotecan, oxaliplatin and mixtures thereof.
  • the emulsion according to the invention comprises, as therapeutic agent, at least one antibody targeting the antigens, and more particularly at least one monoclonal antibody.
  • the antibody is an antibody targeting the tumor antigens selected from the group consisting of the anti-human monoclonal antibodies. angiogenic, anti-CTLA-4 monoclonal antibodies, anti-PD-1 monoclonal antibodies, anti-PD-L1 monoclonal antibodies, and mixtures thereof.
  • anti-CTLA-4 include ipilimumab and tremelimumab.
  • monoclonal antibodies anti-PD-1 include nivolumab and pembrolizumab.
  • Atezolizumab and Avelumab.
  • the emulsion according to the invention comprises between 5 and 25 mg / ml of therapeutic agent.
  • the therapeutic agent concentration is between 10 and 25 mg / mL, and is preferably 20 mg / mL.
  • the therapeutic agent concentration is 20 mg / ml.
  • the therapeutic agent concentration is 5 mg / ml.
  • the therapeutic agent concentration is 5 mg / ml.
  • the present invention also relates to a medicament, characterized in that it comprises an emulsion as defined above.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an emulsion as defined above, as well as at least one pharmaceutically acceptable excipient.
  • compositions contain an effective dose of at least one emulsion according to the invention (containing at least one therapeutic agent), as well as at least one pharmaceutically acceptable excipient.
  • Said excipients are chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients which are known to those skilled in the art.
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, intra-arterial, topical, local, intratracheal, intranasal, transdermal or rectal, the emulsion can be administered in unit dosage form, in admixture with conventional pharmaceutical excipients, to animals and humans for the treatment of disorders or disorders. diseases above.
  • the dosage appropriate to each patient is determined by the physician according to the mode of administration, the weight and the response of said patient.
  • the present invention also relates to an emulsion as defined above for use as a medicament.
  • the present invention also relates to an emulsion as defined above for its use for the treatment of cancer.
  • the present invention also relates to a method of treating cancer comprising administering to a patient an effective dose of an emulsion according to the invention.
  • Figure 1 shows optical microscopy images of the emulsions obtained at different concentrations of PLGA nanoparticles.
  • Figure 2 shows optical microscopy images of the emulsions obtained at different Lipiodol / physiological saline ratios.
  • Figure 3 shows 24-hour turbiscan creaming monitoring of a 3/1 emulsion stabilized with 15 mg / mL of nanoparticles.
  • FIG. 4 represents the 15-minute turbiscan phase shift monitoring of a non-stabilized 3/1 emulsion by nanoparticles (before emulsion and after 15 minutes of follow-up).
  • Figure 5 relates to the injectability of emulsions.
  • Figure 5A relates to the injectability of emulsions through a Progreat 2.4F catheter at different ratios at a rate of 2 mm / s (the curve 'a' corresponds to an emulsion with an oil / water ratio of 8: 3 the curve 'b' corresponds to an emulsion with an oil / water ratio of 3: 1, the curve 'c' corresponds to an emulsion with an oil / water ratio of 6: 1 and the curve 'd' corresponds to Lipiodol alone
  • Figure 5B relates to the injectability of a 3/1 emulsion at different injection speeds (a: 6 mm / s, b: 4 mm / s, c: 2 mm / s and d: 1 mm / s).
  • Figure 5C relates to the injectability of emulsions through a 18G needle at different ratios at a rate of 2 mm / s (a: Lipiodol alone, b: ratio 3: 2, c: ratio 6: 1 and d : 8: 3 ratio).
  • Figure 6 shows 24-hour turbiscan creaming of a 3/1 emulsion stabilized with 15 mg / mL of nanoparticles at a concentration of 20 mg / mL of doxorubicin.
  • Figure 7 shows the 4-hour turbiscan phase shift monitoring of a 4/1 unstabilized emulsion by nanoparticles at a concentration of 20 mg / mL of doxorubicin.
  • FIG. 8 relates to the in vitro release of doxorubicin from lipiodolated emulsions of ratio 3/1 loaded at a concentration of 20 mg / ml of doxorubicin without nanoparticles (curve with diamonds) and with nanoparticles at 15 mg / mL (curve with triangles) and charged balls (curve with squares).
  • Figure 9 relates to the in vitro release of irinotecan with nanoparticle (NP) stabilized emulsions at an irinotecan concentration of 20 mg / mL (round), at a ratio of 3/1 compared to an unstabilized emulsion (triangles). ), to balls loaded with irinotecan (diamonds) and free irinotecan (squares).
  • NP nanoparticle
  • Figure 10 relates to the in vitro release of irinotecan with NP stabilized emulsions at a concentration of 15 mg / mL at a ratio of 3/1 (triangles), 3/2 (squares) and 1/1 (diamonds). ).
  • Figure 1 1 represents the percentage of in vitro release of platinum with emulsions made with different ratios of Lipiodol and oxaliplatin, with or without stabilizing nanoparticles.
  • the curve with the diamonds corresponds to a ratio of 3/1 without nanoparticles
  • the curve with the squares corresponds to a ratio of 3/1 with nanoparticles
  • the curve with the triangles corresponds to a 2/1 ratio with nanoparticles
  • the curve with the crosses corresponds to a 1/1 ratio with nanoparticles.
  • Figure 12 represents the amount of platinum released in vitro with emulsions made with different ratios of Lipiodol and oxaliplatin, with or without stabilizing nanoparticles.
  • the curve with the diamonds corresponds to a ratio of 3/1 without nanoparticles
  • the curve with the squares corresponds to a ratio of 3/1 with nanoparticles
  • the curve with the triangles corresponds to a 2/1 ratio with nanoparticles
  • the curve with the crosses correspond to a 1: 1 ratio with nanoparticles.
  • Figure 13 relates to the in vitro release of ipilimumab of lipiodolated emulsions of ratio 3/1 stabilized by NP at a concentration of 15 mg / ml.
  • Figure 14 shows the plasma pharmacokinetics of oxaliplatin after injection of lipiodolated Pickering emulsions (dotted line) or conventional lipiodol emulsions (solid line) into the left hepatic arteries.
  • PLGA nanoparticles were prepared according to the emulsion-evaporation process already described in the literature (Astete, CE, Sabliov, CM Synthesis and Characterization of PLGA Nanoparticles, J. Biomater, Sci Polym Ed, 2006, 77 (3)). , 247-289). 100 mg of PLGA were dissolved in 5 ml of dichloromethane and emulsified by sonication (VibraCell sonicator, Fisher Scientific, France) at a power of 40% for 1 minute with 20 ml of an aqueous solution containing 2.5 mg / ml of PVA. The organic solvent was then evaporated at room temperature with magnetic stirring for 2 h.
  • the NPs were purified by ultracentrifugation (Beckman Coulter Optima TM LE-80K Ultracentrifuge) at 4 ° C, 37,000 g for 1 h. After removal of the supernatant, the NPs were resuspended in an aqueous solution containing 50 mg / mL of trehalose (cryoprotectant). Then, the suspension of the NPs was lyophilized. Prior to use, the lyophilized NPs were redispersed in MilliQ water at the desired concentration.
  • the emulsions were formulated with a lipiodol ratio (Guerbet, France) / physiological saline solution of 3/1 (v / v) by repetitive pump (70 round trip) of 2 10 ml syringes through a 3-way stopcock. 70 seconds.
  • the first syringe contains lipiodol, the second is empty and saline arranged in a 3 rd syringe is gradually introduced into the system via a pump with a flow rate of 1 mL / min.
  • the aqueous phase is a suspension of nanoparticles at different concentrations in physiological saline (see Figure 1).
  • Example 3 Obtaining a water-in-oil emulsion of physiological serum stabilized with PLGA nanoparticles at different oil / water ratios
  • Example 4 Determination of the direction of the emulsion by a drop test
  • the type of emulsion was determined by a colorimetric test using two solutions, one containing Lipiodol (previously colored with Sudan red) and the other. other containing physiological saline (previously stained with methylene blue).
  • a droplet of one of the solutions was added to one drop of the tested emulsion.
  • the continuous phase of the emulsion was revealed by observing the possible miscibility of the solution droplets with the emulsion drop. The test was carried out immediately after emulsification.
  • the emulsion was analyzed using a Turbiscan® MA 2000 (Formulation, L'Union, France).
  • the tube containing the emulsion was not removed or even touched from the instrument until the end of the measurement to avoid any disturbance of the system.
  • the measurements were made at predetermined times according to the evolution of the system. This monitoring was carried out until the change in intensities was negligible.
  • Turbiscan is used to measure the reversible (creaming and sedimentation) and irreversible (coalescence and segregation) destabilization phenomena in the sample without dilution ( Figures 3 and 4).
  • the emulsions were formulated according to the same protocol as Example 2.
  • the aqueous phase is composed of doxorubicin hydrochloride reconstituted at a concentration of 10 or 20 mg / ml in a saline solution (Adriblastine, Pfizer, USA) for the formulation of emulsions.
  • the emulsions were made using different Lipiodol / doxorubicin ratios, different concentrations of doxorubicin and different concentrations of nanoparticles (see Table 1 below).
  • the therapeutic emulsions obtained were all in the water-in-oil direction, that is to say reversed, whatever the conditions used.
  • Example 8 Stability of lipiodolated water-in-oil emulsions loaded with doxorubicin stabilized with nanoparticles
  • the emulsions were formulated according to the same protocol as Example 2.
  • the aqueous phase is composed of an antibody (5 mg / ml Ipilimumab, Yervoy, Bristol Myers Squibb).
  • the emulsions were carried out at a Lipiodol / ipilimumab ratio of 3/1 with a concentration of 15 mg / ml of nanoparticles.
  • the emulsions obtained were in the water-in-oil direction and stable over several weeks. No aggregation of the antibodies was observed contrary to the emulsions prepared without nanoparticles.
  • the integrity of the antibodies was verified by western blot in denaturing condition. The migration was made on polyacrylamide gel and the samples were prepared with 2% SDS (method known to those skilled in the art). Electrophoresis is performed at 120V for 90 minutes and membrane transfer at 100V for 45 minutes. The membrane is washed with ethanol and the revelation of the antibodies was made with Ponceau red.
  • the nanoparticles have shown their effectiveness in maintaining the integrity of the therapeutic agent, in this example ipilimumab.
  • TRIS buffered saline
  • the emulsions were formulated according to the same protocol as Example 2.
  • the aqueous phase is composed of irinotecan (irinotecan hydrochloride trihydrate, 20 mg / ml, Campto®, Pfizer).
  • the released amounts of irinotecan were quantified by 370 nm UV spectroscopy (FIGS. 9 and 10).
  • the emulsions were formulated according to the same protocol as Example 2.
  • the aqueous phase is composed of oxaliplatin (Eloxatin®, 5 mg / ml, Sanofi-Aventis).
  • the released amounts of platinum were quantified by ICP mass spectrometry (FIGS. 11 and 12).
  • Example 13 Water-in-oil emulsions stabilized with nanoparticles formulated from different oils
  • the emulsions were formulated at a ratio of 3/1 according to the same protocol as Example 2.
  • the aqueous phase is composed of either physiological saline or doxorubicin at a concentration of 20 mg / ml.
  • the oil phase is composed of either olive oil, sesame oil, castor oil, carnation oil or miglyol. All emulsions are formulated with nanoparticles at a concentration of 15 mg / ml.
  • the emulsions are water-in-oil type according to the colorimetric test and stable over more than one week.
  • Example 14 Preparation of Biodegradable Nanoparticles of PLGA
  • the PLGA-Iron nanoparticles were prepared according to the same process described in Example 1. 500 ⁇ of solution of Fe 3 O 4 nanoparticles decorated with oleic acid (25 mg ml -1 , size 10 nm, Ocean, USA) were added to 100 mg of PLGA previously dissolved in 5 ml of dichloromethane and emulsified by sonication ( VibraCell sonicator, Fisher Scientific, France) at a power of 40% for 1 minute with 20 ml of an aqueous solution containing 2.5 mg / ml of PVA The organic solvent was then evaporated at room temperature with magnetic stirring for 2 minutes.
  • NP were purified by ultracentrifugation (Beckman Coulter Optima TM LE-80K Ultracentrifuge) at 4 ° C., 37000 g for 1 h After removal of the supernatant, the NPs were resuspended in an aqueous solution containing 50 ml. mg / mL trehalose (cryoprotectant) and the NP suspension was lyophilized. Prior to use, the lyophilized NPs were redispersed in MilliQ water at the desired concentration.
  • Example 15 Obtaining a water-in-oil lipiodolated emulsion of physiological serum stabilized with various concentrations of PLGA-Iron nanoparticles
  • the emulsions were formulated according to the same protocol as Example 2 at different concentrations of PLGA-Iron nanoparticles (20, 15 or 10 mg / mL).
  • the emulsions are of water-in-oil type according to the colorimetric test.
  • Example 16 Obtaining a lipiodolated water-in-oil emulsion of doxorubicin stabilized with PLGA-Iron nanoparticles
  • the emulsion was formulated according to the same protocol as Example 2 at a concentration of 20 mg / ml of doxorubicin and 15 g / ml of PLGA-Fer nanoparticles. A water-in-oil emulsion was obtained.
  • the microscopic structure of the emulsions was observed using a confocal laser scanning microscope (Leica TCS SP8 - STED, Germany) equipped with a WLL laser (488 and 563 nm excitation waves) and an immersion goal CS2 63x / 1, 40.
  • a confocal laser scanning microscope Leica TCS SP8 - STED, Germany
  • WLL laser 488 and 563 nm excitation waves
  • immersion goal CS2 63x / 1, 40 To prevent deformation of the emulsion droplets, the sample was placed on a curved glass slide.
  • PLGA-Iron nanoparticles have been used to formulate emulsions in order to visualize them in transmission (avoids interference with the emission spectrum of doxorubicin).
  • the fluorescence of doxorubicin was observed with a 600-710 nm filter under 590 nm laser illumination. The red fluorescence emissions were collected in a sequential mode.
  • the emulsions were evaluated in MRI to confirm that this imaging modality made it possible to quantify the ratio of oil contained in a tumor ghost.
  • Table 2 MRI Quantification of the Percent Lipiodol in Emulsions with Decreasing Levels of Lipiodol.
  • the emulsions were formulated according to the same protocol as Example 2.
  • the aqueous phase is composed of active ingredient previously reconstituted at a therapeutic concentration recommended by the supplier or directly from an active ingredient in aqueous solution to the concentration of the commercial form. .
  • the emulsions were made at a Lipiodol / active ingredient ratio in solution of 3/1 with a concentration of 15 mg / ml in nanoparticles.
  • the aqueous phase consists of either gemcitabine (40 mg / mL), fludarabine (25 mg / mL), clamoxyl (200 mg / mL), cefazolin (330 mg / mL), sunitinib (0.5 mg / mL), and mL), methylprednisolone (10 mg / mL) or ketoprofen (25 mg / mL).
  • the therapeutic emulsions obtained were all in the water-in-oil direction according to the colorimetric test.
  • the drop size measurement was performed with a particle counter by image analysis technique (Flowcell FC200S + HR, Occhio, Belgium).
  • the emulsion is first diluted 20 times in the oil and then 0.5 ml of the diluted emulsion are introduced through a spacer of 400 ⁇ for analysis.
  • Each sample was measured at least 4 times on day 7 and on different days (day 0, day 7, day 35) for samples containing saline and ipilimumab.
  • the calculations were done on at least 500 drops.
  • the emulsions were formulated according to the same protocol as Example 9.
  • the in vitro release of ipilimumab from a lipiodolated emulsion was evaluated.
  • 0.8 ml of emulsion corresponding to 1 mg of ipilimumab were deposited in tubes containing 20 ml of buffered saline solution (PBS, pH 7.4) and placed in an incubator at 37 ° C. at a speed of 150 rpm .
  • PBS buffered saline solution
  • aliquots 300 ⁇
  • the released amounts of ipilimumab were quantified by the BCA method: colorimetric protein assay based on bicinchonic acid ( Figure 13).
  • VX2 tumors of the liver were implanted percutaneously in New Zealand white rabbits under general anesthesia (intramuscular injections of Ketamine 20-40mg / kg and xylazine 3-5mg / kg, isoflurane 3-5% for induction and 1, 5-3% in a mixture with 0 2 at 1 L / min for the procedure).
  • hepatic intra-arterial injections were performed by an interventional radiologist. This was done under general anesthesia and under fluoroscopic guidance in a room equipped with an X-ray angiography table.
  • the femoral artery was surgically exposed and catheterized with a 4F vascular angiography catheter.
  • a 2.4F micro-catheter was used for selective catheterization of the left branch of the hepatic artery and to inject 0.5 ml of emulsion (ie 0.625 mg of oxaliplatin).
  • Venous blood samples (2 mL) were taken at 5, 10, 20, 30 and 60 minutes post injection to determine the plasma oxaliplatin concentration. 4 groups of rabbits were made:
  • the pharmacokinetics of oxaliplatin after injection of both types of emulsion are summarized in Table 4 and Figure 14.
  • the plasma peak of oxaliplatin (Cmax) is significantly lower after injection of the Pickering emulsion compared to conventional emulsion (0.49 ⁇ 0.14 ng / mL vs. 1.08 ⁇ 0.41 ng / mL, p ⁇ 0.01).
  • Table 4 Average concentration of oxaliplatin (ng / mg) in the liver tissues according to the type of emulsion injected and the sacrifice time.
  • Tissue concentrations of oxaliplatin are shown in Tables 4 & 5.
  • Table 5 Average concentration of oxaliplatin (ng / mg) in liver tumors according to the type of emulsion injected and the sacrifice time.

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