US20110059180A1 - Method for Preparing Nanoparticles Based on Functional Amphiphilic Molecules or Macromolecules, and the Use Thereof - Google Patents

Method for Preparing Nanoparticles Based on Functional Amphiphilic Molecules or Macromolecules, and the Use Thereof Download PDF

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US20110059180A1
US20110059180A1 US12/745,670 US74567008A US2011059180A1 US 20110059180 A1 US20110059180 A1 US 20110059180A1 US 74567008 A US74567008 A US 74567008A US 2011059180 A1 US2011059180 A1 US 2011059180A1
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Philippe Barthelemy
Michel Camplo
Nathalie Campins
Bruno Chauffert
Florence Bouyer
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    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A—HUMAN NECESSITIES
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    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4816—Wall or shell material
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals

Definitions

  • the invention relates to a process for the preparation of nanoparticles based on functional amphiphilic molecules or macromolecules and the use thereof for the transport or vectorization of therapeutic agents, in particular of anti-neoplastic agents.
  • cis-platin is an anti-neoplastic agent that is widely used, in particular for the treatment of solid tumours.
  • its use is limited by its toxicity as well as the onset of an acquired resistance.
  • the U.S. Pat. No. 7,908,160 relates to cis-platin derivatives bound to ligands, the activity of which is reversible as a function of the bond to the ligand.
  • compositions of cis-platin encapsulated in lipophilic nanoparticles obtained by repeated heating and freezing cycles based on negatively charged natural lipids, in particular dioleyl-phosphatidylserine. It is indicated in this application that cis-platin forms, in water, positively charged aggregates having a higher solubility than the non-charged species, which allows their interaction with the negatively charged lipid membranes and the reorganization of the lipid membranes around the cis-platin aggregates.
  • a means is sought which allows transport of the therapeutic agents (in particular cis-platin and/or its derivatives) rapidly to the inside of the tumorous cells with a high pharmacological activity, whilst preserving the healthy cells, i.e. by reducing the neurological, renal, auditory, digestive, etc. toxicity, by simultaneously limiting the phenomena of the appearance of resistance to this therapeutic agent.
  • the therapeutic agents in particular cis-platin and/or its derivatives
  • nanoparticles formed from functional amphiphilic molecules or macromolecules allows the effective and rapid intracellular delivery of therapeutic agents and exhibited improved stability properties, in particular at 37° C., allowing a sustained vectorization of said therapeutic agents over time.
  • therapeutic agents is meant, for example, a natural or synthetic molecule used for the prevention or treatment of a pathology or the restoration of a biological function, in vitro or in vivo, in particular in animals, including human beings, or also on isolated cells, with the exception of nucleic acids or fragments thereof.
  • Such a molecule can be chosen, for example, from the active ingredients of medicaments, in particular from anti-neoplastic agents such as, for example:
  • nanoparticles formed from functional amphiphilic molecules or macromolecules of formula (I) for encapsulating these compounds and ensuring their intracellular delivery makes it possible to limit the phenomena of resistance to these compounds.
  • platinum complexes in particular cis-platin, are preferred therapeutic agents for the purposes of the invention.
  • Inorganic complexes based on ruthenium II and/or III can be, for example, the complexes called NAMI-A, RAPTA-C, KP1019.
  • Such non-platinum complexes are described in Ott I. and Gust R., Arch. Pharm. Chem. Life Sci. 2007, 340, 117-126; Reedijk J., Curr Opin Chem. Biol., 1999, 3, 236-40; Haimei Chen et al., J. Am. Chem. Soc., 2003, 125, 173-186.
  • the invention therefore relates to a process for encapsulating a therapeutic agent, preferably an anti-neoplastic agent, comprising the stages consisting of:
  • the nanoparticles obtained in this way have a structure which allows an effective and rapid intra-cell delivery of the encapsulated active ingredients, in particular anti-neoplastic agents.
  • said nanoparticles also have a life span compatible with their use as a vector for a therapeutic agent.
  • n is advantageously comprised between 1 and 500, preferably comprised between 1 and 100, in particular comprised between 1 and 50, quite particularly comprised between 1 and 10.
  • linear or branched C 1 -C 5 alkyl is meant for example a methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, tert-butyl radical, preferably methyl or ethyl.
  • the purine or pyrimidine base, or the non-natural heterocyclic base can be substituted by at least one substituent chosen, for example, from a halogen, an amino group, a carboxy group, a carbonyl group, a carbonylamino group, a hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl group etc.
  • substituent chosen, for example, from a halogen, an amino group, a carboxy group, a carbonyl group, a carbonylamino group, a hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl group etc.
  • non-natural heterocyclic base is meant a base other than uracil, adenine, guanine, cytosine, thymine or hypoxanthine, which does not exist in nature.
  • heteroaryl group containing 1 to 4 nitrogen atoms is meant a monocyclic or bicyclic, aromatic or partially unsaturated, carbocyclic group containing 5 to 12 atoms, interrupted by 1 to 4 nitrogen atoms, in particular the pyrazole, triazole, tetrazole or imidazole groups.
  • the process according to the invention can comprise the stages implemented under the following general conditions:
  • the solution obtained is centrifuged. The supernatant is separated. After several centrifugations, the pellet is dried.
  • the organic solvent can be chosen, for example, from the usual organic solvents in the field, such as, for example, chloroform, an alcohol such as methanol or ethanol, etc.
  • the heating is carried out, preferably, to a temperature of the order of 20° C. to 80° C., and the cooling to a temperature of the order of ⁇ 190° C. (liquid nitrogen) to 0° C. (ice).
  • An appropriate heating/cooling cycle can, for example, be 45° C. for the heating and ⁇ 78° C. for the cooling.
  • the therapeutic agent is chosen from the platinum complexes (cis-platin, carboplatinum, etc.), cis-platin being particularly preferred, or ruthenium capable of binding to platinum complexes, or also the abovementioned inorganic complexes without platinum based on ruthenium II or III, titanium, gallium, cobalt, iron or gold.
  • platinum complexes cis-platin, carboplatinum, etc.
  • cis-platin being particularly preferred, or ruthenium capable of binding to platinum complexes, or also the abovementioned inorganic complexes without platinum based on ruthenium II or III, titanium, gallium, cobalt, iron or gold.
  • the nanoparticles obtained can be optionally extruded on polycarbonate filter having, for example, a pore diameter of the order of 100 or 200 nm.
  • solid nanoparticles which are constituted by a core rich in therapeutic agent (active ingredient) surrounded by one or more lipid layers constituted by the functional amphiphilic compound of formula (I) as defined above, with or without co-lipid.
  • Said solid nanoparticles constituted by a core rich in therapeutic agent, preferably an anti-neoplastic agent, in particular a platinum complex, surrounded by one or more lipid layers constituted by the functional amphiphilic compound of formula (I) as defined above, with or without co-lipid, represent a subsequent subject of the invention.
  • all the lipid layers are constituted by the same lipids (compound of formula (I) with or without co-lipid).
  • At least one co-lipid will be used in the lipid mixture, in addition to the compound of formula (I).
  • co-lipid a compound used in combination with the compound of formula (I), which contributes to the production of the structure of the lipid layers(s) of the nanoparticle.
  • a zwitterionic co-lipid will be used.
  • Said co-lipid can be, for example, chosen from dioleylphosphatidylcholine (DOPC) or dioleylphosphatidyluridine phosphatidylcholine (DOUPC), in combination with the compound of formula (I) in order to form the lipid layer(s) of the nanoparticle.
  • DOPC dioleylphosphatidylcholine
  • DOUPC dioleylphosphatidyluridine phosphatidylcholine
  • These compounds can play the role of co-lipid when they are used in mixture with a compound of formula (I).
  • they can be included in formula (I), such as, for example, dioleylphosphatidyluridinephosphatidylcholine (DOUPC).
  • DOUPC dioleylphosphatidyluridinephosphatidylcholine
  • they will either play the role of compound of formula (I) or, in combination with another compound of formula (I), the role of co-lipid.
  • said lipid mixture contains solely at least one compound of formula (I) and does not contain co-lipid.
  • the therapeutic agent will be used at a concentration of the order of 0.1 ng/mL to 10 mg/mL in the aqueous phase, so that the intracellular delivery of the active ingredient is significant.
  • the preferred compounds of formula (I) are those in which X represents oxygen.
  • the above compounds are new compounds which represent a subject of the invention, as well as the nanoparticles comprising these compounds and a therapeutic agent, in particular an anti-neoplastic agent, in particular the platinum complexes (such as, for example cis-platin, carboplatin, oxaliplatin, nedaplatin, lobaplatin,), or ruthenium capable of binding to the platinum complexes, or also the abovementioned inorganic complexes without platinum based on ruthenium, titanium, gallium, cobalt, iron or gold.
  • Cis-platin is a preferred anti-neoplastic agent for the purposes of the invention.
  • the compounds of formula (I) can comprise purine or pyrimidine base derivatives having an anti-neoplastic activity, such as, for example, aracytosine (AraC), 5-fluorouracil (5-FU), Iododeoxyuridine (IdU), 2′-deoxy-2′-methylidenecytidine (DMDC) or 5-chloro-6-azido-5,6-dihydro-2′-deoxyuridine.
  • AraC aracytosine
  • 5-FU 5-fluorouracil
  • IdU Iododeoxyuridine
  • DMDC 2′-deoxy-2′-methylidenecytidine
  • 5-chloro-6-azido-5,6-dihydro-2′-deoxyuridine such as, for example, aracytosine (AraC), 5-fluorouracil (5-FU), Iododeoxyuridine (IdU), 2′-deoxy-2′-methylidenecytidine
  • a subject of the invention is also the use of the nanoparticles capable of being obtained by the process described above, for the transport or vectorization of therapeutic agents, in particular anti-neoplastic agents.
  • the invention relates to the use of the nanoparticles capable of being obtained by the process described above, for the intracellular delivery of therapeutic agents, in particular anti-neoplastic agents.
  • the invention also relates to the use of the nanoparticles capable of being obtained by the above process, for the preparation of anti-neoplastic medicaments.
  • the invention also relates to the nanoparticles capable of being obtained by the above process, for the treatment of tumour diseases, in particular cancers, such as, for example, cancers of the ovary, testicle, colon, cervix, lung, or adenosarcoma etc.
  • tumour diseases in particular cancers, such as, for example, cancers of the ovary, testicle, colon, cervix, lung, or adenosarcoma etc.
  • the yield is 75%.
  • the yield is 63%.
  • the yield is 60%.
  • the yield is 60%.
  • the yield is 65%.
  • NMR 13 C (75.47 MHz, CDCl 3 ): NMR 13 C (75.47 MHz, CDCl 3 ): ⁇ 14.2 (CH 2 ), ⁇ 22.7 (CH 2 ), ⁇ 26.1 (CH 2 ), ⁇ 29.4 (CH 2 ), ⁇ 29.5 (CH 2 ), ⁇ 29.6 (CH 2 ), ⁇ 32.0 (CH 2 ), ⁇ 58.0 (CH 2 ), ⁇ 70.4 (CH 2 ), ⁇ 74.1 (CH), ⁇ 80.1 (C).
  • the yield is 27%.
  • the yield is 36%.
  • the yield is 39%.
  • the mixture is then oxidized by the addition of 43 mL of a 0.02M solution of diiodine in THF/Pyr/H 2 O. After 12 hours at ambient temperature, the solvent is evaporated off under vacuum. The residue is dissolved in 8 mL of dichloromethane. Then, 0.2 mL of 1,5-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.3 eq, 0.87 mmol) is added to the reaction medium over 5 hours. The reaction medium is washed with a 0.1 N solution of HCl then with a saturated solution of Na 2 S 2 O 7 . The organic phase is concentrated under vacuum. The compound is obtained after purification by flash chromatography (381 mg) using an elution gradient (MeOH/DCM 9:1 to 1:1).
  • DBU 1,5-diazabicyclo[5.4.0]undec-7-ene
  • the yield is 69%.
  • the reaction medium is magnetically stirred for 24 hours at ambient temperature and under nitrogen.
  • the mixture is then oxidized by the addition of 43 mL of a 0.02M solution of diiodine in THF/Pyr/H 2 O. After 12 hours at ambient temperature, the solvent is evaporated off under vacuum and dried under P 2 O 5 overnight using a pump. The residue is dissolved in 8 mL of dichloromethane. Then, 0.2 mL of 1,5-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.3 eq, 0.87 mmol) is added to the reaction medium over 5 hours. The reaction medium is washed with a 0.1 N solution of HCl then with a saturated solution of Na 2 S 2 O 3 . The organic phase is concentrated under vacuum. The compound is obtained after purification by flash chromatography (180 mg) using an elution gradient (MeOH/DCM 98:2 to 1:1).
  • the yield is 24%.
  • the yield is 42%.
  • the yield is 35%.
  • the yield is 78%.
  • NMR 13 C (75.47 MHz, DMSO d 6 ): ⁇ 12.5 (CH 3 ), ⁇ 14.4 (CH 3 ), ⁇ 22.6 (CH 2 ), ⁇ 25.1, ⁇ 31.7 (CH 2 ), ⁇ 38.4 (CH 2 ), ⁇ 51.6 (CH 2 —N), ⁇ 66.0 (CH—O), ⁇ 71.3 (C), ⁇ 84.4, ⁇ 84.5, ⁇ 110.3 ( ⁇ C—N), ⁇ 122.8, ⁇ 136.4, ⁇ 150.9, ⁇ 152.4, ⁇ 164.1.
  • the yield is 46%.
  • the yield is 41%.
  • the yield is 72%.
  • the yield is 30%.
  • the yield is 59%.
  • the yield is 59%.
  • the yield is 76%.
  • the yield is 78%.
  • the yield is 80%.
  • the compound thymidine 3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di C16 dT) prepared in Example 2 was used as a compound of formula (I) and dioleylphosphatidylcholine (DOPC) as a co-lipid.
  • Solution A 20 mg of diC16dT are solubilized in 2 mL of chloroform (10 mg/mL). This sample is stored at ⁇ 20° C.
  • Solution B DOPC: solution at 20 mg/mL in chloroform, stored at ⁇ 20° C.
  • the chloroform is evaporated off under nitrogen in order to obtain a homogeneous lipid film.
  • 1.2 mL of the cis-platin solution pre-incubated at 37° C. are used to rehydrate the lipid film prepared beforehand.
  • the mixture is incubated at 37° C. for 30 min.
  • a series of 10 heating (water bath at 45° C.) and freezing (dry ice/methanol ⁇ 78° C.) cycles is carried out.
  • the tube is centrifuged at 2100 rpm at 4° C. for 5 min. After elimination of the supernatant the pellet is re-suspended in 1 mL of milli-Q water. A second centrifugation is carried out (2100 rpm at 4° C. for 5 min), then the supernatant is removed and the pellet is dried.
  • the concentration (ICP/Optical) of the pellet re-suspended in 1 mL of milli-Q water is 2.844 mM equivalent in cis-platin (852.2 mg/L). This concentration corresponds to 47.4% of the cis-platin initially used.
  • the compound thymidine 3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di C16 dT) prepared in Example 2 is used as a compound of formula (I) and dioleylphosphatidylcholine (DOPC) as a co-lipid.
  • Solution A 20 mg of diC16dT are solubilized in 2 mL of dichloromethane (10 mg/mL). The solution is stored at ⁇ 20° C.
  • Solution B DOPC: solution at 20 mg/mL in dichloromethane, stored at ⁇ 20° C.
  • the dichloromethane is evaporated off with compressed nitrogen in order to obtain a homogeneous lipid film.
  • the suspension is stirred and placed in a glass haemolysis tube, then subjected to sonication for 5 min. After sonication, the suspension is centrifuged at 1000 rpm/2.5 min/20° C. in order to remove the large capsules which are to be found in the pellet which will be eliminated. The supernatant is again centrifuged at 10,000 rpm/5 min/20° C. The nanoparticles are to be found in the pellet. The latter is resuspended in 1 mL of milli-Q water and a second centrifugation is carried out. The pellet is suspended in 1 mL and subjected to an Inductively Coupled Plasma (ICP) Optical assay.
  • ICP Inductively Coupled Plasma
  • the nanoparticles prepared according to the protocol of Example 16 are assayed by ICP optical spectrometry (the measured value corresponds to the total concentration).
  • the suspension of the nanoparticles is aliquoted into 5 Eppendorf® tubes (150 ⁇ L). The latter are incubated at 37° C. under stirring (300 rpm) for different periods of time (0, 2.5, 5, 10 and 24 hours).
  • the tube is centrifuged at 14,000 rpm/10 min/20° C. and 50 ⁇ L of supernatant (recovered carefully so as not to resuspend the pellet) are assayed.
  • Nanoparticles based on 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a co-lipid are prepared according to the same protocol as a comparison.
  • Cx concentration found at a given time (x).
  • C0 concentration found in the supernatant before incubation.
  • CT total concentration found without incubation and without centrifugation.
  • the curve of the release of cis-platin as a function of the incubation time is represented in FIG. 1 .
  • the nanoparticles of Example 16 are represented by the symbol —O— and the nanoparticles based on DOPC/DOPS by the symbol
  • nanoparticles prepared according to the protocol of Example 16 were analyzed with a MALVERN zetasizer.
  • the nanoparticles are suspended in 2 mL of milli-Q water (volume necessary for the size measurement). The concentration is approximately 0.5 mM (the suspension must be cloudy in order to diffuse the light). Single-use cuvettes (1 cm/1 cm) are used for the measurement.
  • the results show that more than 95% of the nanoparticles have a size between 100 and 250 nm with a polydispersity of 0.228.
  • IGROV1 cells (ovarian adenocarcinoma line) at 80% confluence (dish 10 cm in diameter) are treated with 100 ⁇ M of free cis-platin or cis-platin encapsulated in the nanoparticles of Example 16 for 2, 4 or 6 h. On completion of this treatment two washings with PBS are carried out. The cells are treated with trypsin and resuspended in PBS. Two washings of the cell suspensions with PBS are carried out (centrifugation 1000 rpm/1 min). The cells are suspended in 1 mL of PBS and counted.
  • 10 6 cells are lysed with 500 ⁇ L of the cell lysis solution (lysis buffer from SIGMA). The volume is topped up to 5 mL with milli-Q water with 1% HNO 3 acid.
  • FIG. 2 shows the concentration of cis-platin released after cell lysis as a function of time, corresponding to the concentration of cis-platin internalized in the treated cells.
  • the hatched column corresponds to the free cis-platin and the dotted column (on the right) to the nanoparticles containing cis-platin.
  • the test implemented uses the human cancer cell line HCT8 (colorectal adenocarcinoma), known for its intrinsic resistance to platinum derivatives.
  • HCT8 colonal adenocarcinoma
  • the protocol is as follows:
  • the survival of the cells was determined, and the overall percentage of death (or cytotoxicity) obtained at the different test formulation concentrations was compared with that obtained with the negative controls (untreated cells).
  • the cytotoxicity results were presented in the form of dose-effect curves characterized by their inhibitory concentration 50 (IC 50 ), i.e. the concentration at which 50% dead cells are observed.
  • the nanoparticles were prepared according to the process described in Example 15, but with a single heating/cooling cycle for the compound of Example 12.
  • IGROV1 cells 1000 to 5000 IGROV1 cells are incubated in 100 ⁇ L of medium with serum per well (96-well plate). After 24 h the medium is removed and the cells are treated for 1 to 3 days with the formulations according to the invention and/or free cis-Pt (at the desired concentrations). The cells are incubated at 37° C. in 200 ⁇ L of medium with serum. The cell viability is revealed by an MTS colorimetric assay at the end of the treatment.
  • the survival of the cells was determined, and the overall percentage of death (or cytotoxicity) obtained at the different concentrations of formulations tested was compared with that obtained with the negative controls (untreated cells).
  • the cytotoxicity results were presented in the form of dose-effect curves characterized by their inhibitory concentration 50 (IC 50 ), i.e. the concentration at which 50% dead cells are observed.
  • 2500 cells (IGROV1, SKOV3, ovarian adenocarcinoma lines) per well are incubated in 100 ⁇ L of the medium with serum in a 96-well plate. After 24 hours the medium is aspirated and the cells are treated with free cis-platin or cis-platin encapsulated in the nanoparticles of Example 16 in 100 ⁇ L of the medium without serum at different concentrations (500, 100, 10, 1, 0.1, 0.01, 0.001 ⁇ M). After treatment for 24 hours, the medium is removed and the cells are washed twice with 100 ⁇ L of PBS then incubated with 100 ⁇ L of the medium with serum.
  • IGROV1, SKOV3, ovarian adenocarcinoma lines 2500 cells (IGROV1, SKOV3, ovarian adenocarcinoma lines) per well are incubated in 100 ⁇ L of the medium with serum in a 96-well plate. After 24 hours the medium is aspirated and the cells are treated
  • the cell viability is revealed by adding 20 ⁇ L of MTS.
  • the absorbance at 490 nm is measured after incubation for 2 to 4 hours at 37° C. The absorbance is proportional to the cell viability.
  • FIG. 3 shows the concentration necessary to obtain 50% cell death (IC50) with free cis-platin (column A) or the nanoparticles containing cis-platin (column B).
  • FIG. 3A relates to the cell line IGROV1 and FIG. 3B relates to the cell line SKOV3.
  • Example 16 containing cis-platin are more effective than the free cis-platin in the two cell lines, IGROV1 (sensitive to cis-platin) and SKOV3 (resistant to cis-platin).
  • the nanoparticles containing cis-platin are respectively 9 and 8 times more effective than the free cis-platin on the lines IGROV1 and SKOV3.

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WO2021026647A1 (fr) * 2019-08-12 2021-02-18 Integrated Nanotherapeutics Inc. Lipides pour l'administration d'un matériau chargé, leurs formulations et leur procédé de fabrication
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs

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FR2945946B1 (fr) * 2009-05-29 2011-08-26 Univ Victor Segalen Bordeaux 2 Formulations a compartiments multiples a base de molecules ou macromolecules amphiphiles fonctionnelles
EP3085360A1 (fr) 2015-04-20 2016-10-26 Universite De Bordeaux Compositions de nanovecteurs à base de lipide chargées avec des nanoparticules métalliques et agent thérapeutique
EP3502684A1 (fr) 2017-12-22 2019-06-26 Universite De Bordeaux Procédé de détection d'ions métalliques dans des solutions aqueuses au moyen de composés nucléolipides
EP3505178A1 (fr) 2017-12-29 2019-07-03 Universite De Bordeaux Formulations de gel de bio-encres comprenant un composé à base de nucléolipides
FR3078064A1 (fr) 2018-02-22 2019-08-23 Universite de Bordeaux Procede de decontamination d'un milieu liquide aqueux contenant des micropolluants

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US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
WO2021026647A1 (fr) * 2019-08-12 2021-02-18 Integrated Nanotherapeutics Inc. Lipides pour l'administration d'un matériau chargé, leurs formulations et leur procédé de fabrication

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