WO2020174060A1 - Redox-sensitized drug delivery with imaging ability - Google Patents

Redox-sensitized drug delivery with imaging ability Download PDF

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Publication number
WO2020174060A1
WO2020174060A1 PCT/EP2020/055182 EP2020055182W WO2020174060A1 WO 2020174060 A1 WO2020174060 A1 WO 2020174060A1 EP 2020055182 W EP2020055182 W EP 2020055182W WO 2020174060 A1 WO2020174060 A1 WO 2020174060A1
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Prior art keywords
membered heteroaryl
picolinium
probe according
probe
anyone
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PCT/EP2020/055182
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French (fr)
Inventor
Peter Dalko
Laurence MOTTE
Yoann Lalatonne
Erwann Guenin
Didier Letourneur
Anna BAROSI
Hamid DHIMANE
Petra Dunkel
Original Assignee
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Universite Paris 13 - Paris Nord
Universite De Paris
Centre National De La Recherche Scientifique
Assistance Publique - Hopitaux De Paris
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Application filed by Institut National De La Sante Et De La Recherche Medicale (Inserm), Universite Paris 13 - Paris Nord, Universite De Paris, Centre National De La Recherche Scientifique, Assistance Publique - Hopitaux De Paris filed Critical Institut National De La Sante Et De La Recherche Medicale (Inserm)
Publication of WO2020174060A1 publication Critical patent/WO2020174060A1/en

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    • 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
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/559—Redox delivery systems, e.g. dihydropyridine pyridinium salt redox systems
    • 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

Definitions

  • the present invention relates to drug delivery and more particularly to system enabling a control of the drug delivery in space and time.
  • Drug delivery is the process that refers to all approaches, formulations, technologies and systems that ensures the safe transport of therapeutics in the body, permitting to achieve the desired therapeutic effects. Ideally, only the pharmacological target should be exposed to the drug, in order to minimize the collateral effects.
  • traditional drug administration made the drug accessible to the blood stream, producing systemic side effects attributable to the non-specific distribution and uncontrollable drug delivery characteristics.
  • advanced nanoscale systems have been developed to achieve time and space controlled release of actives at the target sites. These include liposomes, polymersomes, polymer nanoparticles, micelles, dendrimers and inorganic nanoparticles made of iron oxide, quantum dots, gold or metal oxide frameworks.
  • the drug-loaded nanoplatforms ensure that the drug will not be freely released during the blood circulation, but delivered only when the target is reached by active or passive targeting strategy.
  • Current surface functionalization methodologies can help the nanocarriers to control their pharmacokinetics and biodistribution.
  • the carrier should retain the drug during the biodistribution process, but, once the target reached, the drug has to be released ideally in totality and in a short time. Also, the drug should escape from all metabolism and clearance process during the transport into the body, but strongly interact with the target cells/tissues/organs.
  • the alternative to the traditional drug delivery offered by nanocarriers is the on-demand release, allowing good spatial, temporal and dosage control. On demand delivery is possible with the design of stimuli-responsive systems that are able to recognize their microenvironment and react in a dynamic way. This approach is difficult, as requires biocompatible materials that could undergo chemical modifications by protonation, hydrolytic cleavage or conformational changes in response to an applied stimulus.
  • the objective of the invention is to provide a vehicle for drug release that enables a spatial and temporal control of the release of the drug.
  • Another objective of the invention is to provide such a vehicle and activation method that enables a depth penetration in tissues.
  • this probe comprising:
  • N-comprising (5 to 14)-membered heteroaryl wherein at least one, preferably one, of the N atom is quaternarized and one of the carbon atom is substituted with an organic function, said reactive function being adapted to be cleaved by the action of electron and is chosen among alcohol, ester, carbamate, amide, aryl- ether, quaternary ammonium, amine, phosphate and phosphonate functions;
  • the probe according to the invention is preferably a probe for X-rays or beta/gamma triggered drug delivery.
  • the support (S) is preferably inorganic particles responsive to X-rays or beta/gamma radiations.
  • the support (S) is a metal, or metal oxide (inorganic) particle.
  • the support (S) is more preferably chosen among iron oxide, hafnium oxide, gadolinium oxide and titanium oxide, silver or gold.
  • the support (S) is iron oxide, hafnium oxide or gold.
  • Iron oxide can be chosen among FeeC , Fe2C>3 (preferably y- Fe2C>3) and hafnium oxide can be HfC>2.
  • the support (S) can be defined as an immobilization platforms and is biocompatible.
  • the probe according to the invention is susceptible to be used to deliver a molecule of interest, for example a drug, to a person in need thereof.
  • a molecule of interest for example a drug
  • Said molecule of interest, e.g drug will be covalently linked to the probe according to the invention by reaction with the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
  • Molecules of interest such as drugs can be linked to the probes via the reactive function.
  • probes are developed as redox sensors. They are able harnessing electrons and undergo irreversible fragmentation, of the reactive function of the N-comprising (5 to 14)-membered heteroaryl, liberating the covalently linked drug.
  • Redox enzymes beta/gamma/X-ray-emitting radionuclides and high-energy electromagnetic waves such as X-ray, or, gamma-ray can be used as electron sources.
  • the source can be a radioactive source of Cs-137.
  • the N-comprising (5 to 14)-membered heteroaryl is chosen among groups enabling delocalization of electrons.
  • the N-comprising (5 to 14)-membered heteroaryl is chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium.
  • the reactive function of the N-comprising (5 to 14)-membered heteroaryl is able to covalently link a molecule of interest, preferably a drug, to be delivered.
  • this reactive function corresponds to a function of formula -CH(R 2 )-X-R 1 , wherein X is a heteroatom, preferably O, NR 3 , S or P(0)R 3 , P(0)XR 3 , R 2 and R 3 , identical or different are chosen among FI, (C1 -C6) linear or branched alkyl, (6 to 14)-membered aryl, preferably phenyl, or (5 to 14)-membered heteroaryl, preferably thiophene, pyrrol, imidazole, triazole, oxazole, thiazole, furane; R 1 is chosen among H, carbonate, carbamate, urea each optionally activated with N-hydroxysuccinimide or imidazole.
  • the linker has to be adapted to the support (S) in order to optimize the release of the drug.
  • the N-comprising (5 to 14)-membered heteroaryl is picolinium, quinolinium, isoquinolinium or acridinium and the length of the linker is comprised between 10 and 20 A (between 10.10 10 and 20.10 10 m).
  • the linker comprises two parts, a first part enabling a linking to the particle through a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function and a second part enabling a covalent linking to the N-comprising (5 to 14)-membered heteroaryl via the N quaternary ammonium.
  • the linker is of the following formula L 1 -L 2 -L 3 -L 4 wherein L 1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L 2 and L 4 , identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L 3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L 3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole.
  • the probe according to the invention responds to the following structure:
  • S, R 2 , X and R 1 are as defined above is the N-comprising (5 to 14)-membered heteroaryl defined above and is preferably chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium;
  • L is of the following formula L 1 -L 2 -L 3 -L 4 wherein L 1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L 2 and L 4 , identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L 3 is a donor group improving the electron transfer to the N- comprising (5 to 14)-membered heteroaryl, preferably L 3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole.
  • L 1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate
  • L 2 and L 4 identical or different represent linear or branched (C1 -C6) alkyl, preferably ethy
  • S is iron oxide, gold or hafnium oxide; and/or is picolinium or quinolinium, preferably picolinium; and/or
  • L 1 comprises phosphonate, preferably bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)- P(0)(0H) 2 ; and/or
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are (C1 -C6) alkyl, preferably ethyl; and/or
  • R 2 is (C1 -C6) linear or branched alkyl, preferably methyl;
  • R 1 is H
  • X is O.
  • S is iron oxide, gold or hafnium oxide; and/or is picolinium or quinolinium, preferably picolinium; and/or
  • L 1 comprises phosphonate, preferably bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)- P(0)(0H) 2 ; and/or
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are ethyl
  • R 2 is methyl
  • R 1 is H
  • X is O.
  • S is iron oxide, gold or hafnium oxide; is picolinium or quinolinium, preferably picolinium;
  • L 1 comprises bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)-P(0)(0H) 2 ;
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are (C1 -C6) alkyl, preferably ethyl;
  • R 2 is (C1 -C6) linear or branched alkyl, preferably methyl
  • R 1 is H
  • S is iron oxide, gold or hafnium oxide; is picolinium or quinolinium, preferably picolinium;
  • L 1 comprises bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)-P(0)(0H) 2 ;
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are ethyl
  • R 2 is methyl
  • R 1 is H
  • X is O.
  • S is iron oxide, gold or hafnium oxide; picolinium;
  • L 1 comprises bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)-P(0)(0H) 2 ;
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are ethyl
  • R 2 is methyl
  • R 1 is H
  • X is O.
  • the present invention also relates to a process for the preparation of a probe according to the invention.
  • Such process comprises the following steps: 1 ) provide or synthesize support (S) by preparing the inorganic material for example as disclosed in the below examples; 2) functionalize support (S) with the linker (L) by convenient surface coating methods for example as disclosed in the below examples and 3) covalent coupling of the N-comprising (5 to 14)-membered heteroaryl to the linker preferably by “click-type” reactions.
  • the click-type reaction is copper(l) catalyzed azide/alkyne cycloaddition.
  • the click reaction is generally performed in sub-stoichiometric amount of heterocycle to avoid saturation and subsequent particles aggregation.
  • the support (S) can be for example produced by classical non-aqueous sol-gel synthesis and microwave activation. This enables precise control of size and reproducibility.
  • probes can be advantageously used, as a delivery mean for molecules of interest, preferably for molecules having therapeutic effect, such as drug.
  • the molecule of interest can be covalently linked via the reactive function of the N- comprising (5 to 14)-membered heteroaryl.
  • redox enzymes beta/gamma/X-ray-emitting radionuclides and high-energy electromagnetic waves such as X-ray, or, gamma-ray can be used as electron sources.
  • X-ray, or, gamma-ray activates the support (S), generating electrons; the probe is able harnessing electrons triggering irreversible fragmentation of the organic function of the N-comprising (5 to 14)-membered heteroaryl, liberating the covalently linked drug.
  • the biodistribution / accumulation of the nanoparticles can be followed by dedicated imaging modalities, such as MRI, or, CTI or photonic modalities (IR, NIR). It is thus advantageously possible to spatially determine and control the action of the drug delivery.
  • the present invention thus relates to probes according to the invention for use for delivering, e.g to a person in need thereof, molecules of interest, preferably molecules having therapeutic effect, such as drug.
  • the present invention also relates to a process for delivering, e.g to a person in need thereof, molecules of interest, preferably molecules having therapeutic effect, such as drug, comprising the preparation of a probe according to the invention to which the molecule of interest is linked and the administration to the person in need thereof.
  • the delivering can be controlled in space and time.
  • the present invention thus also relates to probes according to the invention for its use for the controlled delivery in space and time of molecules of interest, preferably for molecules having therapeutic effect, such as drug, said molecules comprising at least a heteroatom, covalently linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
  • the present invention also relates to probes according to the invention further comprising a molecule of interest, preferably molecules having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
  • the molecule of interest carrying probe comprises:
  • a support (S) that is chosen among inorganic particles
  • Such probes can be called molecule of interest carrying probe.
  • the support (S), the N-comprising (5 to 14)-membered heteroaryl and the linker (L) being as defined above.
  • the N-comprising (5 to 14)-membered heteroaryl carries a substituent of formula -CH(R 2 )-X-R 4 , wherein X is a heteroatom, preferably O, NR 3 , S or P(0)R 3 , P(0)XR 3 , R 2 and R 3 , identical or different are chosen among H, (C1 -C6) linear or branched alkyl, (6 to 14)-membered aryl, preferably phenyl, or (5 to 14)-membered heteroaryl, preferably thiophene, pyrrol, imidazole, triazole, oxazole, thiazole, furane; R 4 is the molecule of interest.
  • X is a heteroatom, preferably O, NR 3 , S or P(0)R 3 , P(0)XR 3 , R 2 and R 3 , identical or different are chosen among H, (C1 -C6) linear or branched alkyl, (6 to 14)-member
  • the molecule of interest carrying probe according to the invention responds to the following structure:
  • N-comprising (5 to 14)-membered heteroaryl defined above and is preferably chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium;
  • L is of the following formula L 1 -L 2 -L 3 -L 4 wherein L 1 is, or comprises, a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L 2 and L 4 , identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L 3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L 3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole.
  • L 1 is, or comprises, a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate
  • L 2 and L 4 identical or different represent linear or branched (C1 -C6)
  • S is iron oxide, gold or hafnium oxide; and/or is picolinium or quinolinium, preferably picolinium; and/or
  • L 1 comprises a phosphonate, preferably bisphosphonate, preferably L 1 is P(0)(0H) 2 - C(0H)-P(0)(0H) 2 ; and/or
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are ethyl
  • R 2 is methyl
  • X is O.
  • S is iron oxide, gold or hafnium oxide; is picolinium or quinolinium, preferably picolinium;
  • L 1 comprises a bisphosphonate, preferably L 1 is P(0)(0H) 2 -C(0H)-P(0)(0H) 2 ;
  • L 3 is carbazole or 1 ,2,3-triazole
  • L 2 and L 4 are ethyl
  • R 2 is methyl
  • X is O.
  • the molecule of interest carrying probe is obtained by reacting the probe according to the invention with a molecule of interest, for example a drug by esterification, condensation, substitution, addition, cycloaddition, organometallic coupling reaction.
  • a molecule of interest for example a drug by esterification, condensation, substitution, addition, cycloaddition, organometallic coupling reaction.
  • the drug or molecule of interest can be for examples drug for the treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases, for example the drug can be Sorafenib,® Doxorubicin, Monomethyl auristatin E (MMAE) or Combretastatine® preferably with the N-comprising heterocycle being quinolinium.
  • MMAE Monomethyl auristatin E
  • Combretastatine® preferably with the N-comprising heterocycle being quinolinium.
  • the present invention also relates to probes according to the invention further comprising molecules of interest, preferably molecules having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl, for its use for the treatment of diseases in preference for the treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases.
  • molecules of interest preferably molecules having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl, for its use for the treatment of diseases in preference for the treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases.
  • Such probes can be called molecule of interest carrying probe.
  • the present invention also relates to a method of treatment of disease, for example treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases, comprising the administration to the person in need thereof of a therapeutically amount of a molecule of interest carrying probe according to the invention wherein the molecule of interest is adapted to treat the concerned disease (for example cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases).
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one probe according to the invention with at least one pharmaceutically acceptable excipient.
  • the present invention also relates to a kit comprising:
  • - at least one molecule of interest preferably a drug.
  • the drug is advantageously adapted to be linked to the probe.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one probe according to the invention carrying a molecule of interest, preferably molecules having therapeutic effect, such as drug, linked thanks to the reactive function of the N- comprising (5 to 14)-membered heteroaryl.
  • the pharmaceutical composition also comprises at least one pharmaceutically acceptable excipient.
  • alkyl refers to an aliphatic-hydrocarbon group, which may be straight or branched, having 1 to 3 carbon atoms in the chain unless specified otherwise.
  • Preferred alkyl groups have 1 or 2 carbon atoms in the chain.
  • Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl.
  • the alkyl group is methyl or ethyl.
  • Aryl relates to an aromatic mono or bicycle comprising from 6 to 10 carbon atom.
  • An example of aryl is phenyl, naphtyl, preferably phenyl.
  • heterocyclic refers to a monocyclic saturated or partially unsaturated non-aromatic ring containing from 4 to 10 atom, of which at least one atom, preferably 1 or 2 atom, of the ring is a heteroatom such as N, O, S, S(O) or S(0) .
  • the heterocycle is a monocyclic saturated or partially unsaturated non-aromatic ring containing from 4 to 6 atom of which at least one atom, preferably 1 or 2 atom, of the ring is a heteroatom such as N, O, S, S(O) or S(0) 2 .
  • the carbon atoms of the heterocycle can also be oxidized to form a C(O) group.
  • Suitable heterocycles are also disclosed in the Handbook of Chemistry and Physics, 76 th Edition, CRC Press, Inc., 1995-1996, pages 2-25 to 2-26.
  • heterocyclyl groups include but are not limited to azetidinyl, oxetanyl, oxazolyl, oxazolidinyl, oxadiazolyl, pyrrolyl, pyrrolidinyl, pyridyl, dihydropyridinyl, tetrahydropyridinyl, piperidinyl, morpholinyl, pyrazolyl, pyrimidinyl, pyrazinyl, tetrazolyl, imidazolyl, thienyl, thiazolyl, furanyl, thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyrazolyl, isoxazolyl, 2-pyrrolidinonyl, imidazol-2,4-dione, 1 ,2,4-oxadiazol-5-one, 1 ,5-dihydropyrrolyl-2-one, pyrazinone
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” is employed for any excipient, solvent, dispersion medium, absorption retardant, diluent or adjuvant etc., such as preserving or antioxidant agents, fillers, binders, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents and the like, that does not produce a secondary reaction, for example an allergic reaction, in humans or animals.
  • excipients include mannitol, lactose, magnesium stearate, sodium saccharide, talcum, cellulose, sodium crosscarmellose, glucose, gelatine, starch, lactose, dicalcium phosphate, sucrose, kaolin, magnesium carbonate, wetting agents, emulsifying agents, solubilizing agents, sterile water, saline, pH buffers, non-ionic surfactants, lubricants, stabilizing agents, binding agents and edible oils such as peanut oil, sesame oils and the like.
  • excipients commonly used in the art may be included.
  • Pharmaceutically acceptable carriers or excipients are well known to a person skilled in the art, and include those described in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985) or Remington 23rd Edition; The Science and Practice of Pharmacy 2015, Merck Index (Merck & Company, Rahway, N.J.), Gilman et al (Eds. The pharmacological basis of therapeutics, 8 th Ed., Pergamon press, 1990) or Goodman & Gilman's the Pharmacological Basis of Therapeutics 13th edition, 2017. Except insofar as any conventional media or adjuvant is incompatible with the active ingredient according to the invention, its use in the therapeutic compositions is contemplated.
  • the present invention also relates to a process for delivering a drug to a person in need thereof comprising the following steps:
  • Figure 1 represents the TEM image and size distribution of USPIO NPs synthesized with non aqueous solgel process at 250 °C and coated with HMBPyne ligand.
  • Figure 2 represents the IR spectrum of USPIO NPs coated with HMBPyne.
  • Figure 3 represents the IR spectra of USPIO 0.2 (A) and 191 (B)
  • Figure 4 represents the transmission electron microscopy image of hafnium oxide particles synthesized at 250 °C and coated with HMBPyne ligand.
  • Figure 5 represents the IR spectrum of Hf0 2 nanoparticles coated with HMBPyne
  • Figure 6 represents the size distribution of HfC HMBPyne NPs after coupling with 0.2 or 0.5 equiv. of 191 , respectively.
  • Figure 7 represents the IR spectra of redox probe 191 (B), Hf0 2 0.2 (A)
  • HMBPyne (1 -hydroxy-1 -phosphonopent-4-ynyl)phosphonic acid
  • HMBPyne ligand was added to the solution of nanoparticles at acidic pH (pH 2); under these conditions, nanoparticles are positively charged and bisphosphonates are negatively charged.
  • the mass ratio HMBPyne to nanoparticles is equal to 2.
  • Surface functionalization was realized by stirring the mixture at room temperature for 2 h. Then the excess of free ligands is eliminated by repeating 5 times ultrafiltration with Dl water. After washing, coated nanoparticles were re-dispersed in water at neutral pH.
  • HMBPyne-coated iron oxide or hafnium oxide NPs nanoparticles
  • picolinium 191 having an azido end-group
  • the reaction conditions were optimized for the synthesis of covalently linked picolinium-carbazole via copper(l)-catalyzed azide/alkyne cycloaddition (CuAAC), by using catalytic amount of Cul (0.1 eq.) at room temperature for 24 h.
  • CuAAC copper(l)-catalyzed azide/alkyne cycloaddition
  • the reaction was performed in an acetonitrile / water 1/1 mixture. In a 0.2 molar ratio of alkyne and azide.
  • NPs were washed several times with deionized water and then re-dispersed at pH 7.4 in distilled water for various physicochemical characterizations. From this reaction, 2 coupled NPs batches were obtained that will be referred as follows:
  • the iron concentration was determined by a colorimetric assay as described in ACS Chem.Biol, 2016, 1 1 , 2812.
  • the hydrodynamic size and zeta potential of the NPs were investigated by dynamic laser light scattering (DLS), using a Nano-ZS (Red Badge) ZEN 3600 device (Malvern Instruments, Malvern, UK).
  • TEM images were obtained using a FEI Tecnai 12 (Philips), and samples were prepared by depositing a drop of NP suspension on carbon-coated copper grids placed on a filter paper.
  • the grafting of the HMBPyne to the surface of the NPs and coumpound 191 were studied by Fourier transform infra-red (FTIR) analysis.
  • FTIR Fourier transform infra-red
  • HMBP-yne coating was evaluated by thermogravimetric analysis (TGA) using a LabsSys evo TG-DTA-DSC 16000 device from Setaram Instrumentation and/or by Energy-dispersive X-ray (EDX) microanalysis using a TM 3000 tabletop microscope equipped with a Swift EDX-ray 3000 microanalysis system (Oxford Instruments). Samples were deposited as powder on a copper surface, and data were collected using a 15 kV accelerating voltage. Quantification of coating was evaluated by studying ratio of iron or hafnium vs phosphorus and knowing the average number of iron (hafnium) atoms/ particles.
  • the UV spectra of (191 ) was recorded on a Varian Cary 50 Scan UV-vis spectrophotometer.
  • the coupling efficiency of the (191 ) conjugation onto NP was investigated qualitatively after hydrolysis of pyrene fluorophore using fluorescence measurement recorded on a SpectroFluorimeter Spex FluoroMax (HORIBA Jobin-Yvon, France with a Hamamatsu 98 photomultiplier).
  • USPIO Iron
  • NPs with an average diameter of 9 nm were synthesized using microwave non aqueous sol-gel synthesis in a two-step process, following a procedure described in Richard et al. ( Nanomedicine (Lond) 2016. DOI 10.2217/nnm-2016-0177).
  • the NPs were surface passivated with hydroxyl methylene bisphosphonate bearing alcyne as terminal functionality.
  • the USPIO-HMBPyne NPs were characterized using several physicochemical methods. Transmission Electronic Microscopy (TEM) images show well-dispersed NPs with spherical morphology ( Figure 1 ).
  • TEM Transmission Electronic Microscopy
  • the NPs have a hydrodynamic diameter of 17 nm, which confirms a low state of aggregation and colloidal stability at physiological pH, Table 1.
  • the negative zeta potential at pH 7.4 clearly shows efficient binding of the HMBP-yne to the NP surface, Table 1.
  • FTIR Fourier-Transform Infra Red
  • Figure 2 The IR spectrum (KBr pellets) of USPIO NPs after functionalization shows strong stretching bands from 1200 to 900 cnr 1 assigned to P-0 stretches of phosphonic acid groups.
  • the wide band at 600 cnr 1 corresponds to the vibration of the iron-oxygen bond at the surface.
  • HMBP-yne molecules per particle was determined by ThermoGravimetric Analysis (TGA) and EDX analysis. An average of 440 ⁇ 40 HMBP-yne molecules per NP was determined.
  • the picolinium 191 with an azido end-group (0.2 eq.) was coupled to USPIO-HMBPyne (1 equiv.) using copper(l)-catalyzed azide/alkyne cycloaddition (CuAAC) (as disclosed in Langmuir; 2013, 29, 14639-14647), called USPIO 0.2.
  • USPIO 0.2 particles displayed equal hydrodynamic size and zeta potential before and after coupling, Table 1 , indicating at least low aggregation.
  • the number of grafted molecules on the NPs surface was evaluated using fluorescence quantification after pyrene fluorophore hydrolyzis in basic solution. Control NP solutions that did not undergo alkaline composition displayed almost no fluorescence, confirming that all measured fluorescence was a result of the pyrene release.
  • Hf0 2 NPs were synthesized using microwave non-aqueous sol-gel synthesis adapted from USPIO synthesis as previously described.
  • the NPs were surface passivated with hydroxyl methylene bisphosphonate bearing alcyne as terminal functionality.
  • the Hf0 2 -HMBPyne NPs were characterized using several physicochemical methods.
  • Transmission Electronic Microscopy (TEM) images show well-dispersed NPs with ellipsoid shape with a 4.5 nm (minor axis) and a 2.7 nm (major axis) sizes ( Figure 4).
  • the NPs have a hydrodynamic diameter of 1 1 nm, suggesting a low state of aggregation and colloidal stability at pH 7, with a negative zeta potential (-32), due to the presence of negatively charged bisphosphonate coating at pH 7, table 1 .
  • HMBP-yne molecules per particle was determined by EDX analysis. An average of 167 HMBPyne molecules for NP was determined.
  • HfC>2 0.2 Dynamical light scattering measurement shows the presence of two particle populations, Figure 6. The first one at around 30 nm and the second one at 130 nm related to the presence of aggregates.
  • the number of grafted molecules on the NPs surface was evaluated using fluorescence quantification after pyrene fluorophore hydrolysis in basic solution. Control NP solutions that did not undergo alkaline composition displayed almost no fluorescence, confirming that all measured fluorescence was a result of the pyrene release.

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Abstract

Surface-modified metal / metal (oxide) nanoparticles, such as iron oxide (USPIO), hafnium oxide as well as gold nanoparticles were used as a support for redox-gated drug delivery. Immobilized picolinium, or, quinolinium-derived probes were developed as redox sensors. Probes are able harnessing electrons and undergo irreversible fragmentation, liberating the covalently linked ligands. Redox enzymes, beta/gamma/X-ray-emitting radionuclides and high-energy electromagnetic waves such as X-ray, or, gamma-ray can be used as electron sources. The biodistribution / accumulation of the nanoparticles can be followed by dedicated imaging modalities, such as MRI, or, CTI.

Description

Redox-Sensitized Drug Delivery with Imaging Ability
The present invention relates to drug delivery and more particularly to system enabling a control of the drug delivery in space and time.
Drug delivery is the process that refers to all approaches, formulations, technologies and systems that ensures the safe transport of therapeutics in the body, permitting to achieve the desired therapeutic effects. Ideally, only the pharmacological target should be exposed to the drug, in order to minimize the collateral effects. However, traditional drug administration made the drug accessible to the blood stream, producing systemic side effects attributable to the non-specific distribution and uncontrollable drug delivery characteristics. To overcome these limitations, advanced nanoscale systems have been developed to achieve time and space controlled release of actives at the target sites. These include liposomes, polymersomes, polymer nanoparticles, micelles, dendrimers and inorganic nanoparticles made of iron oxide, quantum dots, gold or metal oxide frameworks. They are characterized by a small size (from a few tenths to a few hundreds of nanometers) and specific bio-functions providing benefits and new opportunities for the smart drug delivery system. The drug-loaded nanoplatforms ensure that the drug will not be freely released during the blood circulation, but delivered only when the target is reached by active or passive targeting strategy. Current surface functionalization methodologies can help the nanocarriers to control their pharmacokinetics and biodistribution.
In the last years this field received a considerable attention, a large number of smart drug nanocarriers were developed with promising results in preclinical animal models. However, few of them have only been finally translated into clinics. To ensure the clinical potential of those carriers, there are some key issues that should be addressed:
(i) biocompatibility and biodegradability;
(ii) stability in physiological conditions;
(iii) high drug loading capacity;
(iv) low toxicity.
The careful analysis of these requirements reveals, however, some contradictions. Indeed, the carrier should retain the drug during the biodistribution process, but, once the target reached, the drug has to be released ideally in totality and in a short time. Also, the drug should escape from all metabolism and clearance process during the transport into the body, but strongly interact with the target cells/tissues/organs. The alternative to the traditional drug delivery offered by nanocarriers is the on-demand release, allowing good spatial, temporal and dosage control. On demand delivery is possible with the design of stimuli-responsive systems that are able to recognize their microenvironment and react in a dynamic way. This approach is difficult, as requires biocompatible materials that could undergo chemical modifications by protonation, hydrolytic cleavage or conformational changes in response to an applied stimulus.
Despite the impressive progresses made, only few nanoparticle-based treatment have reached the market. This fact can be attributed to the poor drug loading capacity, which is usually less than 5% (weight %), or to the too rapid release of the encapsulated drug.
There is thus a need to provide new systems enabling a spatial and temporal control of the release of drugs.
The objective of the invention is to provide a vehicle for drug release that enables a spatial and temporal control of the release of the drug.
Another objective of the invention is to provide such a vehicle and activation method that enables a depth penetration in tissues.
Other objectives of the invention will appear by reading the description of the invention that follows.
These objectives are achieved by the present invention which relates to probe for drug delivery, this probe comprising:
i. a support (S) that is chosen among inorganic particle;
ii. a N-comprising (5 to 14)-membered heteroaryl wherein at least one, preferably one, of the N atom is quaternarized and one of the carbon atom is substituted with an organic function, said reactive function being adapted to be cleaved by the action of electron and is chosen among alcohol, ester, carbamate, amide, aryl- ether, quaternary ammonium, amine, phosphate and phosphonate functions;
iii. a linker (L) linking the support (S) to the N-comprising (5 to 14)-membered heteroaryl, the linker (L) being linked to the N-comprising (5 to 14) membered heteroaryl thanks to the quaternary N atom.
The probe according to the invention is preferably a probe for X-rays or beta/gamma triggered drug delivery. The support (S) is preferably inorganic particles responsive to X-rays or beta/gamma radiations.
In a preferred embodiment, in the probes according to the invention, the support (S) is a metal, or metal oxide (inorganic) particle. The support (S) is more preferably chosen among iron oxide, hafnium oxide, gadolinium oxide and titanium oxide, silver or gold. Preferably, the support (S) is iron oxide, hafnium oxide or gold. Iron oxide can be chosen among FeeC , Fe2C>3 (preferably y- Fe2C>3) and hafnium oxide can be HfC>2.
The support (S) can be defined as an immobilization platforms and is biocompatible.
The probe according to the invention is susceptible to be used to deliver a molecule of interest, for example a drug, to a person in need thereof. Said molecule of interest, e.g drug, will be covalently linked to the probe according to the invention by reaction with the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
Molecules of interest such as drugs can be linked to the probes via the reactive function.
Without to be bound by any theory, probes are developed as redox sensors. They are able harnessing electrons and undergo irreversible fragmentation, of the reactive function of the N-comprising (5 to 14)-membered heteroaryl, liberating the covalently linked drug. Redox enzymes, beta/gamma/X-ray-emitting radionuclides and high-energy electromagnetic waves such as X-ray, or, gamma-ray can be used as electron sources. For example, the source can be a radioactive source of Cs-137.
Preferably, the N-comprising (5 to 14)-membered heteroaryl is chosen among groups enabling delocalization of electrons. Preferably, the N-comprising (5 to 14)-membered heteroaryl is chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium.
The reactive function of the N-comprising (5 to 14)-membered heteroaryl is able to covalently link a molecule of interest, preferably a drug, to be delivered. Preferably, this reactive function corresponds to a function of formula -CH(R2)-X-R1 , wherein X is a heteroatom, preferably O, NR3, S or P(0)R3, P(0)XR3, R2 and R3, identical or different are chosen among FI, (C1 -C6) linear or branched alkyl, (6 to 14)-membered aryl, preferably phenyl, or (5 to 14)-membered heteroaryl, preferably thiophene, pyrrol, imidazole, triazole, oxazole, thiazole, furane; R1 is chosen among H, carbonate, carbamate, urea each optionally activated with N-hydroxysuccinimide or imidazole.
Advantageously, the linker has to be adapted to the support (S) in order to optimize the release of the drug. Preferably, the N-comprising (5 to 14)-membered heteroaryl is picolinium, quinolinium, isoquinolinium or acridinium and the length of the linker is comprised between 10 and 20 A (between 10.10 10 and 20.10 10 m).
Preferably, the linker comprises two parts, a first part enabling a linking to the particle through a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function and a second part enabling a covalent linking to the N-comprising (5 to 14)-membered heteroaryl via the N quaternary ammonium.
Preferably, the linker is of the following formula L1-L2-L3-L4 wherein L1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L2 and L4, identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole.
Preferably, the probe according to the invention responds to the following structure:
Figure imgf000005_0001
wherein
S, R2, X and R1 are as defined above
Figure imgf000005_0002
is the N-comprising (5 to 14)-membered heteroaryl defined above and is preferably chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium;
L is of the following formula L1-L2-L3-L4 wherein L1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L2 and L4, identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L3 is a donor group improving the electron transfer to the N- comprising (5 to 14)-membered heteroaryl, preferably L3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole. Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide; and/or
Figure imgf000006_0001
is picolinium or quinolinium, preferably picolinium; and/or
L1 comprises phosphonate, preferably bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)- P(0)(0H)2; and/or
L3 is carbazole or 1 ,2,3-triazole; and/or
L2 and L4 are (C1 -C6) alkyl, preferably ethyl; and/or
R2 is (C1 -C6) linear or branched alkyl, preferably methyl; and/or
R1 is H; and/or
X is O.
Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide; and/or
Figure imgf000006_0002
is picolinium or quinolinium, preferably picolinium; and/or
L1 comprises phosphonate, preferably bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)- P(0)(0H)2; and/or
L3 is carbazole or 1 ,2,3-triazole; and/or
L2 and L4 are ethyl; and/or
R2 is methyl; and/or
R1 is H; and/or
X is O.
Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide;
Figure imgf000006_0003
is picolinium or quinolinium, preferably picolinium;
L1 comprises bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)-P(0)(0H)2;
L3 is carbazole or 1 ,2,3-triazole;
L2 and L4 are (C1 -C6) alkyl, preferably ethyl;
R2 is (C1 -C6) linear or branched alkyl, preferably methyl;
R1 is H; and
X is O. Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide;
Figure imgf000007_0001
is picolinium or quinolinium, preferably picolinium;
L1 comprises bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)-P(0)(0H)2;
L3 is carbazole or 1 ,2,3-triazole;
L2 and L4 are ethyl;
R2 is methyl;
R1 is H; and
X is O.
Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide;
Figure imgf000007_0002
picolinium;
L1 comprises bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)-P(0)(0H)2;
L3 is carbazole or 1 ,2,3-triazole;
L2 and L4 are ethyl;
R2 is methyl;
R1 is H; and
X is O.
The present invention also relates to a process for the preparation of a probe according to the invention. Such process comprises the following steps: 1 ) provide or synthesize support (S) by preparing the inorganic material for example as disclosed in the below examples; 2) functionalize support (S) with the linker (L) by convenient surface coating methods for example as disclosed in the below examples and 3) covalent coupling of the N-comprising (5 to 14)-membered heteroaryl to the linker preferably by “click-type” reactions. For example, when the N-comprising (5 to 14)-membered heteroaryl is picolinium, the click-type reaction is copper(l) catalyzed azide/alkyne cycloaddition. The click reaction is generally performed in sub-stoichiometric amount of heterocycle to avoid saturation and subsequent particles aggregation.
The support (S), can be for example produced by classical non-aqueous sol-gel synthesis and microwave activation. This enables precise control of size and reproducibility. According to the invention probes can be advantageously used, as a delivery mean for molecules of interest, preferably for molecules having therapeutic effect, such as drug. Indeed, the molecule of interest can be covalently linked via the reactive function of the N- comprising (5 to 14)-membered heteroaryl.
Without to be bound by any theory, redox enzymes, beta/gamma/X-ray-emitting radionuclides and high-energy electromagnetic waves such as X-ray, or, gamma-ray can be used as electron sources. X-ray, or, gamma-ray activates the support (S), generating electrons; the probe is able harnessing electrons triggering irreversible fragmentation of the organic function of the N-comprising (5 to 14)-membered heteroaryl, liberating the covalently linked drug. The biodistribution / accumulation of the nanoparticles can be followed by dedicated imaging modalities, such as MRI, or, CTI or photonic modalities (IR, NIR). It is thus advantageously possible to spatially determine and control the action of the drug delivery.
The present invention thus relates to probes according to the invention for use for delivering, e.g to a person in need thereof, molecules of interest, preferably molecules having therapeutic effect, such as drug.
The present invention also relates to a process for delivering, e.g to a person in need thereof, molecules of interest, preferably molecules having therapeutic effect, such as drug, comprising the preparation of a probe according to the invention to which the molecule of interest is linked and the administration to the person in need thereof. The delivering can be controlled in space and time.
The present invention thus also relates to probes according to the invention for its use for the controlled delivery in space and time of molecules of interest, preferably for molecules having therapeutic effect, such as drug, said molecules comprising at least a heteroatom, covalently linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
The present invention also relates to probes according to the invention further comprising a molecule of interest, preferably molecules having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl.
Preferably, the molecule of interest carrying probe comprises:
i. a support (S) that is chosen among inorganic particles; ii. a N-comprising (5 to 14)-membered heteroaryl wherein at least one, preferably one of the N atom is quaternarized and one of the carbon atom is substituted with the molecule of interest notably via a -CH(R2)-X- chain;
iii. a linker (L) linking the support (S) to the N-comprising (5 to 14)-membered heteroaryl, the linker (L) being linked to the N-comprising (5 to 14) membered heteroaryl thanks to the quaternary N atom.
Such probes can be called molecule of interest carrying probe.
The support (S), the N-comprising (5 to 14)-membered heteroaryl and the linker (L) being as defined above.
Preferably, the N-comprising (5 to 14)-membered heteroaryl carries a substituent of formula -CH(R2)-X-R4, wherein X is a heteroatom, preferably O, NR3, S or P(0)R3, P(0)XR3, R2 and R3, identical or different are chosen among H, (C1 -C6) linear or branched alkyl, (6 to 14)-membered aryl, preferably phenyl, or (5 to 14)-membered heteroaryl, preferably thiophene, pyrrol, imidazole, triazole, oxazole, thiazole, furane; R4 is the molecule of interest.
Preferably, the molecule of interest carrying probe according to the invention responds to the following structure:
Figure imgf000009_0001
wherein
S, R2, X and R4 are as defined above
N )
is the N-comprising (5 to 14)-membered heteroaryl defined above and is preferably chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium;
L is of the following formula L1-L2-L3-L4 wherein L1 is, or comprises, a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L2 and L4, identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole. Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide; and/or
Figure imgf000010_0001
is picolinium or quinolinium, preferably picolinium; and/or
L1 comprises a phosphonate, preferably bisphosphonate, preferably L1 is P(0)(0H)2- C(0H)-P(0)(0H)2; and/or
L3 is carbazole or 1 ,2,3-triazole; and/or
L2 and L4 are ethyl; and/or
R2 is methyl; and/or
X is O.
Preferably, in the structure (I):
S is iron oxide, gold or hafnium oxide;
Figure imgf000010_0002
is picolinium or quinolinium, preferably picolinium;
L1 comprises a bisphosphonate, preferably L1 is P(0)(0H)2-C(0H)-P(0)(0H)2;
L3 is carbazole or 1 ,2,3-triazole;
L2 and L4 are ethyl;
R2 is methyl; and
X is O.
The molecule of interest carrying probe is obtained by reacting the probe according to the invention with a molecule of interest, for example a drug by esterification, condensation, substitution, addition, cycloaddition, organometallic coupling reaction. The drug or molecule of interest can be for examples drug for the treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases, for example the drug can be Sorafenib,® Doxorubicin, Monomethyl auristatin E (MMAE) or Combretastatine® preferably with the N-comprising heterocycle being quinolinium.
The present invention also relates to probes according to the invention further comprising molecules of interest, preferably molecules having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl, for its use for the treatment of diseases in preference for the treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases.
Such probes can be called molecule of interest carrying probe. The present invention also relates to a method of treatment of disease, for example treatment of cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases, comprising the administration to the person in need thereof of a therapeutically amount of a molecule of interest carrying probe according to the invention wherein the molecule of interest is adapted to treat the concerned disease (for example cancer, inflammation, diabetes, bacterial and/or viral infections, orphan diseases).
The present invention also relates to a pharmaceutical composition comprising at least one probe according to the invention with at least one pharmaceutically acceptable excipient.
The present invention also relates to a kit comprising:
- at least one probe according to the invention with at least one pharmaceutically acceptable excipient.
- at least one molecule of interest, preferably a drug.
The drug is advantageously adapted to be linked to the probe.
The present invention also relates to a pharmaceutical composition comprising at least one probe according to the invention carrying a molecule of interest, preferably molecules having therapeutic effect, such as drug, linked thanks to the reactive function of the N- comprising (5 to 14)-membered heteroaryl. The pharmaceutical composition also comprises at least one pharmaceutically acceptable excipient.
The term’’alkyl”, as used herein, refers to an aliphatic-hydrocarbon group, which may be straight or branched, having 1 to 3 carbon atoms in the chain unless specified otherwise. Preferred alkyl groups have 1 or 2 carbon atoms in the chain. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl. Preferably, the alkyl group is methyl or ethyl.
Aryl relates to an aromatic mono or bicycle comprising from 6 to 10 carbon atom. An example of aryl is phenyl, naphtyl, preferably phenyl.
The term "heterocyclic", as used herein and without contrary definition specifically mentioned, either alone or in combination with another radical, refers to a monocyclic saturated or partially unsaturated non-aromatic ring containing from 4 to 10 atom, of which at least one atom, preferably 1 or 2 atom, of the ring is a heteroatom such as N, O, S, S(O) or S(0) . Preferably, the heterocycle is a monocyclic saturated or partially unsaturated non-aromatic ring containing from 4 to 6 atom of which at least one atom, preferably 1 or 2 atom, of the ring is a heteroatom such as N, O, S, S(O) or S(0)2. The carbon atoms of the heterocycle can also be oxidized to form a C(O) group. Suitable heterocycles are also disclosed in the Handbook of Chemistry and Physics, 76th Edition, CRC Press, Inc., 1995-1996, pages 2-25 to 2-26. Exemplary heterocyclyl groups include but are not limited to azetidinyl, oxetanyl, oxazolyl, oxazolidinyl, oxadiazolyl, pyrrolyl, pyrrolidinyl, pyridyl, dihydropyridinyl, tetrahydropyridinyl, piperidinyl, morpholinyl, pyrazolyl, pyrimidinyl, pyrazinyl, tetrazolyl, imidazolyl, thienyl, thiazolyl, furanyl, thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyrazolyl, isoxazolyl, 2-pyrrolidinonyl, imidazol-2,4-dione, 1 ,2,4-oxadiazol-5-one, 1 ,5-dihydropyrrolyl-2-one, pyrazinone, pyridazinone, pyridone, pyrimidone, dioxanyl, pyrrolidinyl, imidazolidinyl, pyranyl, tetrahydrofuranyl, dioxolanyl, dihydropyranyl, tetrahydropyranyl. Without contrary definition specifically mentioned, the heterocycle can be carbon or nitrogen linked.
The expression“optionally substituted” means“non-substituted or substituted by chemical groups that are further defined” or“unsubstituted or substituted chemical groups that are further defined”.
The term "pharmaceutically acceptable carrier” or "pharmaceutically acceptable excipient” is employed for any excipient, solvent, dispersion medium, absorption retardant, diluent or adjuvant etc., such as preserving or antioxidant agents, fillers, binders, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents and the like, that does not produce a secondary reaction, for example an allergic reaction, in humans or animals. Typical, non-limiting examples of excipients include mannitol, lactose, magnesium stearate, sodium saccharide, talcum, cellulose, sodium crosscarmellose, glucose, gelatine, starch, lactose, dicalcium phosphate, sucrose, kaolin, magnesium carbonate, wetting agents, emulsifying agents, solubilizing agents, sterile water, saline, pH buffers, non-ionic surfactants, lubricants, stabilizing agents, binding agents and edible oils such as peanut oil, sesame oils and the like. In addition, various excipients commonly used in the art may be included. Pharmaceutically acceptable carriers or excipients are well known to a person skilled in the art, and include those described in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985) or Remington 23rd Edition; The Science and Practice of Pharmacy 2015, Merck Index (Merck & Company, Rahway, N.J.), Gilman et al (Eds. The pharmacological basis of therapeutics, 8th Ed., Pergamon press, 1990) or Goodman & Gilman's the Pharmacological Basis of Therapeutics 13th edition, 2017. Except insofar as any conventional media or adjuvant is incompatible with the active ingredient according to the invention, its use in the therapeutic compositions is contemplated.
The present invention also relates to a process for delivering a drug to a person in need thereof comprising the following steps:
i. providing a probe according to the invention
ii. covalently liking the drug to the probe
iii. injecting a pharmaceutical composition comprising the probe obtained at step ii) to the person in need thereof
iv. Imaging the body of the person to show the accumulation of probe on the desired tissue, or organ
v. Emission of X-ray, b-ray or gamma-ray at the specific location of the desired tissue or organ.
The present invention will be now described based on the following non limiting examples.
Figure 1 represents the TEM image and size distribution of USPIO NPs synthesized with non aqueous solgel process at 250 °C and coated with HMBPyne ligand.
Figure 2 represents the IR spectrum of USPIO NPs coated with HMBPyne.
Figure 3 represents the IR spectra of USPIO 0.2 (A) and 191 (B)
Figure 4 represents the transmission electron microscopy image of hafnium oxide particles synthesized at 250 °C and coated with HMBPyne ligand.
Figure 5 represents the IR spectrum of Hf02 nanoparticles coated with HMBPyne
Figure 6 represents the size distribution of HfC HMBPyne NPs after coupling with 0.2 or 0.5 equiv. of 191 , respectively.
Figure 7 represents the IR spectra of redox probe 191 (B), Hf02 0.2 (A)
Example 1 : Preparation of nanoparticle coated with HMBPvne (S-U-L2)
Preparation of hvdroxyl methylene bisphosphonates (HMBPyne)
(1 -hydroxy-1 -phosphonopent-4-ynyl)phosphonic acid (HMBPyne) was synthesized from the condensation of pent-4-ynoyl chloride with tris(trimethylsilyl) phosphite on a gram scale, following a procedure described in J.Nanopart.Res, 2012, 14:965 according to the following scheme: 70%
Tris(trimethylsilyl)phosphite (6.7 mL, 20 mmol) was added dropwise at -20 °C to the previously prepared pent-4-ynoyl chloride without solvent and under inert atmosphere. When the addition was completed, the reaction mixture was allowed to stand at room temperature for 4 h. The evolution of the reaction was monitored by 31 P NMR {1 H}. Then, volatile fractions were evaporated under reduced pressure (0.1 Torr) before being hydrolyzed with methanol. After methanol evaporation the product was dissolved in water at pH = 2.3 and lyophilized. The product was then precipitate twice in a water/ methanol mixture [1 :9]. The sodium salt of HMBPyne was obtained as a white powder (1 .9 g, 70 %). 1 H NMR (400 MHz, D20) d 2.37 (m, 2H), 2.18 (t, J4 H-H = 2.6, 1 H), 2.08 (m, 2H). 13C NMR (100.63 MHz, CDCI3)d 85.8, 73.1 (t, JP-CH = 137.0), 69.2, 32.7, 13.3. 31 P
NMR (161 .98 MHz, CDCI3) d 17.8. IR (KBr, pH = 7) 3260.8, 2955.4, 21 1 1 .2, 1471 .7, 1448.0, 1379.0, 1329.7, 1268.6, 1 164.1 , 1057.7, 945.3, 909.8, 754.6, 738.3, 655.5, 545.1 , 450.5 crrv1. HR-MS (ESI-Q Tof) C5H9O7P2 : m/z (M-H)-: 242.9824; calc:
242.9830.
Preparation of iron oxide nanoparticles (S)
The iron oxide nanoparticles synthesis was performed by heating iron (III) acetyl aceton ate (1 .8 mmol) and benzylalcohol (15 ml) at 250 °C for 30 minutes under microwave irradiation. The resulting suspension was separated using a magnet and the precipitate was washed several times with dichloromethane and ethanol. The solid was re suspended in water at pH = 2.
Preparation of hafnium oxide nanoparticles (S)
The hafnium oxide nanoparticles synthesis was performed by heating hafnium (IV) ethoxide (0.7 mmol) and benzyl alcohol (10 ml) at 250 °C for 180 minutes under microwave irradiation. The resulting suspension was separated using centrifugation and the precipitate was washed several times with dichloromethane and ethanol. The solid was re-suspended in water at pH = 2. Preparation of nanoparticles coated with HMBPvne (S-L1-L2)
Surface functionalization was performed following similar procedure for iron and hafnium oxide particles; HMBPyne ligand was added to the solution of nanoparticles at acidic pH (pH 2); under these conditions, nanoparticles are positively charged and bisphosphonates are negatively charged. The mass ratio HMBPyne to nanoparticles is equal to 2. Surface functionalization was realized by stirring the mixture at room temperature for 2 h. Then the excess of free ligands is eliminated by repeating 5 times ultrafiltration with Dl water. After washing, coated nanoparticles were re-dispersed in water at neutral pH.
Figure imgf000015_0002
Example 2 : Synthesis wherein R4 is a compound of interest and is derived from pyrene butyric acid
Preparation of an azido-alkylated picolinium (191)
Figure imgf000015_0001
2-Chloroethanol was heated at 1 10 °C in presence of NaN3 to obtain 2-azidoethanol
(188). The latter was then converted into triflate (189) by reaction with triflic anhydride at room temperature. On the other hand, 1 -(pyridin-4-yl)ethan-1 -ol, (72), was esterified with pyrene butyric acid in presence of EDC and DMAP, (CH2CI2, room temperature). Ester (190) was obtained in 63 % yield and was then quaternarized with 2-azidoethyl triflate
(189) in DCM at room temperature, giving rise to (191 ).
Compound (191 ) was characterized by IR spectroscopy (KBr pellets). Distinctive peaks were assigned as the azide stretching (2150 cm-1), ester bond stretching (1750 cm-1), and pyrene aromatics (845 cm-1), respectively. The UV spectra of (191 ), measured in a 1/1 acetonitrile / H20 solution, shows a first absorption maximum situated at 338 nm, attributed to the presence of pyrene, with molar extinction smax = 32560 M 1cnr1 at the absorption maximum.
Coupling with the HMBPyne-coated iron oxide and hafnium oxide nanoparticles of example 1
The coupling between HMBPyne-coated iron oxide or hafnium oxide NPs (nanoparticles) and picolinium 191 , having an azido end-group, was realized by using similar procedures for both types of Nanoparticles (NPs). The reaction conditions were optimized for the synthesis of covalently linked picolinium-carbazole via copper(l)-catalyzed azide/alkyne cycloaddition (CuAAC), by using catalytic amount of Cul (0.1 eq.) at room temperature for 24 h. The reaction was performed in an acetonitrile / water 1/1 mixture. In a 0.2 molar ratio of alkyne and azide. A 1 mg/ml solution of yFe2C>3 and / or HfC>2 alkynes (1 equiv.) was reacted with 0.2 equiv. of azide 191. Compound 191 was used in sub-stoichiometric amount in order to avoid saturation and subsequent particles aggregation that may be driven by the tt-p interaction of the pyrene moieties. Reaction mixtures were stirred at room temperature for 24 h in the dark.
Figure imgf000016_0001
The obtained NPs were washed several times with deionized water and then re-dispersed at pH 7.4 in distilled water for various physicochemical characterizations. From this reaction, 2 coupled NPs batches were obtained that will be referred as follows:
Name Description
USPIO 0.2 USPIO- HMBPyne NPs coupled with 0.2 eq. of 191
Hf02 0.2 Hf02- HMBPyne NPs coupled with 0.2 eq. of 191
Example 3: Phvsico-chemical characterization
The iron concentration was determined by a colorimetric assay as described in ACS Chem.Biol, 2016, 1 1 , 2812. The hydrodynamic size and zeta potential of the NPs were investigated by dynamic laser light scattering (DLS), using a Nano-ZS (Red Badge) ZEN 3600 device (Malvern Instruments, Malvern, UK). TEM images were obtained using a FEI Tecnai 12 (Philips), and samples were prepared by depositing a drop of NP suspension on carbon-coated copper grids placed on a filter paper. The grafting of the HMBPyne to the surface of the NPs and coumpound 191 were studied by Fourier transform infra-red (FTIR) analysis. The FTIR spectra were recorded as thin films on KBr pellets on a Thermo Scientific Nicolet 380 FTIR. Quantification of HMBP-yne coating was evaluated by thermogravimetric analysis (TGA) using a LabsSys evo TG-DTA-DSC 16000 device from Setaram Instrumentation and/or by Energy-dispersive X-ray (EDX) microanalysis using a TM 3000 tabletop microscope equipped with a Swift EDX-ray 3000 microanalysis system (Oxford Instruments). Samples were deposited as powder on a copper surface, and data were collected using a 15 kV accelerating voltage. Quantification of coating was evaluated by studying ratio of iron or hafnium vs phosphorus and knowing the average number of iron (hafnium) atoms/ particles.
The UV spectra of (191 ) was recorded on a Varian Cary 50 Scan UV-vis spectrophotometer.
The coupling efficiency of the (191 ) conjugation onto NP was investigated qualitatively after hydrolysis of pyrene fluorophore using fluorescence measurement recorded on a SpectroFluorimeter Spex FluoroMax (HORIBA Jobin-Yvon, France with a Hamamatsu 98 photomultiplier). The saponification of the ester bond was obtained by alkalinization of NP solutions with NaOH (aq, 1 N), for 24 h and adjustement to pH =7. The average number of (191 ) was deduced from a calibration curve reporting the fluorescence intensity versus the pyrene butyric acid s (pH = 7) concentration at hex = 341 nm and Aem = 376 nm.
80 keV X-ray irradiation was realized at the European Synchrotron Radiation Facility (ESRF) of Grenoble, France, using NP suspension (5 mhi and 50 mhi) in HsO/EtOH 1/1 .
Results
USPIO nanoplateform
Iron (USPIO) NPs with an average diameter of 9 nm were synthesized using microwave non aqueous sol-gel synthesis in a two-step process, following a procedure described in Richard et al. ( Nanomedicine (Lond) 2016. DOI 10.2217/nnm-2016-0177). The NPs were surface passivated with hydroxyl methylene bisphosphonate bearing alcyne as terminal functionality. The USPIO-HMBPyne NPs were characterized using several physicochemical methods. Transmission Electronic Microscopy (TEM) images show well-dispersed NPs with spherical morphology (Figure 1 ). The NPs have a hydrodynamic diameter of 17 nm, which confirms a low state of aggregation and colloidal stability at physiological pH, Table 1. The negative zeta potential at pH 7.4, clearly shows efficient binding of the HMBP-yne to the NP surface, Table 1. This result was corroborated considering Fourier-Transform Infra Red (FTIR) spectrum of USPIO-HMBP-yne NPs (Figure 2). The IR spectrum (KBr pellets) of USPIO NPs after functionalization shows strong stretching bands from 1200 to 900 cnr1 assigned to P-0 stretches of phosphonic acid groups. The wide band at 600 cnr1 corresponds to the vibration of the iron-oxygen bond at the surface. The number of HMBP-yne molecules per particle was determined by ThermoGravimetric Analysis (TGA) and EDX analysis. An average of 440 ± 40 HMBP-yne molecules per NP was determined. The picolinium 191 with an azido end-group (0.2 eq.) was coupled to USPIO-HMBPyne (1 equiv.) using copper(l)-catalyzed azide/alkyne cycloaddition (CuAAC) (as disclosed in Langmuir; 2013, 29, 14639-14647), called USPIO 0.2. USPIO 0.2 particles displayed equal hydrodynamic size and zeta potential before and after coupling, Table 1 , indicating at least low aggregation. Qualitatively, the coupling efficiency was accessed with infrared spectroscopy, Figure 3. 191 presents characteristic peaks: the azide stretching (2150 cnr 1), ester bond stretching (1750 cnr1), and pyrene aromatics (845 cm 1 , Figure 3 part B). Comparing free picolinium 191 and USPIO 0.2, the 2100 cnr1 band, representative of the presence of the azide function of 191 involved in the click coupling, disappear, whereas the ester bond stretching at 1750 cnr1 and the pyrene aromatic peak at 845 cnr1 are still observable.
The number of grafted molecules on the NPs surface was evaluated using fluorescence quantification after pyrene fluorophore hydrolyzis in basic solution. Control NP solutions that did not undergo alkaline composition displayed almost no fluorescence, confirming that all measured fluorescence was a result of the pyrene release. The grafting degree is calculated using calibration curve and corresponds to [191] = 2.70 x 106 mol.L-1 for USPIO 0.2.
Since USPIO NPs are negative MRI contrast agents, we used local changes in intensity on T2 weighted MRI to evaluate the USPIO-0.2 NP biodistribution in B6 mice using 7T- MRI. Shortly after injection (t = 15 min) a large negative contrast enhancement was observed in organs rich in macrophages, such as in liver, kidneys and spleen. Moreover, the contrast in liver and spleen stays constant up to 45 min after injection whereas intensity signal decreases steadily in kidney. This fact suggests NP accumulation in the liver and spleen and also NP clearance from the kidneys. Hence, compared to previous results obtained with gadolinium (III) complex, the USPIO-0.2 NP residence time in the body is greatly improved up to 24 h, notably in liver and spleen.
The USPIO 0.2 were X-ray irradiated at various doses, Table 2. Approximately 500 mI_ of samples, c = 3-12 mM of the immobilized pyrenebutyric ester according to the invention in water was irradiated at the ID17 biomedical beamline of the ESRF synchrotron (Grenoble, France) Conditions: Beam: 80 keV, Bunch length: 48 ps, Bunch repetition rate 5.68 MHz, Doses: up to 30 Gy or, a conventional Cs-137 source (BIOBEAM 8000, Gamma-Service Medical GmbH; activity 80.29 TBq ± 20%; dose rate: 5.86 Gy/min). Samples were diluted by equivolume of ethanol for analysis and the released amount of pyrene-butyric acid was quantified by fluorescence (Aex = 341 nm; Aem max = 376 nm) and reported in Table 2.
Tablel :
OH (nm) Zeta potential (mV)
Iron oxide nanoparticles (USPIO) - HMBPyne 17 -30
USPIO 0.2
Figure imgf000019_0001
- 29
Table2: % release pyrene (measured by fluorescence)
Figure imgf000019_0002
HfOå nanoplateform
Hf02 NPs were synthesized using microwave non-aqueous sol-gel synthesis adapted from USPIO synthesis as previously described. The NPs were surface passivated with hydroxyl methylene bisphosphonate bearing alcyne as terminal functionality.
The Hf02-HMBPyne NPs were characterized using several physicochemical methods. Transmission Electronic Microscopy (TEM) images show well-dispersed NPs with ellipsoid shape with a 4.5 nm (minor axis) and a 2.7 nm (major axis) sizes (Figure 4). The NPs have a hydrodynamic diameter of 1 1 nm, suggesting a low state of aggregation and colloidal stability at pH 7, with a negative zeta potential (-32), due to the presence of negatively charged bisphosphonate coating at pH 7, table 1 .
Fourier-Transform Infra Red (FTIR) spectrum of Hf02-HMBP-yne NPs, confirms the efficiency of the Np surface functionalization by HMBP-yne (Figure 5). The IR spectrum (KBr pellets) of Hf02 NPs after functionalization shows strong stretching bands, assigned to P-0 stretches of phosphonic acid groups (of HMBPyne ligand) in the 1200 to 900 cnr1 area and the main peaks at 767, 605, 519 cnr1 due to the formation of Hf-0 bonds (A.Ramadoss, K.Krishnamoorthy, S. J. Kim, Mat. Res. Bulletin, 2012, 47, 2680-2684).
The number of HMBP-yne molecules per particle was determined by EDX analysis. An average of 167 HMBPyne molecules for NP was determined.
Picolinium 191 with an azido end-group (0.2 eq.) was coupled to HfC HMBPyne (1 equiv.) using copper(l)-catalyzed azide/alkyne cycloaddition (CuAAC, Langmuir, 2013, 29, 14639-14647) called HfC>2 0.2. HfC>2 0.2 Dynamical light scattering measurement shows the presence of two particle populations, Figure 6. The first one at around 30 nm and the second one at 130 nm related to the presence of aggregates.
Qualitatively, the coupling efficiency was accessed with infrared spectroscopy, Figure 7. Comparing free picolinium 191 (Figure 7 B) and Hf02 0.2 (Figure 7 A), the 2100 cnr1 band, representative of the presence of the azide function of 191 involved in the click coupling, disappear, whereas the ester bond stretching at 1750 cnr1 and the pyrene aromatic peak at 845 cnr1 are still observable.
The number of grafted molecules on the NPs surface was evaluated using fluorescence quantification after pyrene fluorophore hydrolysis in basic solution. Control NP solutions that did not undergo alkaline composition displayed almost no fluorescence, confirming that all measured fluorescence was a result of the pyrene release. The grafting degree is calculated using calibration curve and corresponds to [191]= 1.21 x 10-5 mol.L1 for Hf02 0.2.
The USPIO 0.2 were X-ray irradiated at various doses, Table 4. Approximately 500 mI_ of samples, c = 3-12 mM (Fe concentration) of the immobilized pyrenebutyric ester according to the invention in water was irradiated at the ID17 biomedical beamline of the ESRF synchrotron (Grenoble, France) Conditions: Beam: 80 keV, Bunch length: 48 ps, Bunch repetition rate 5.68 MHz, Doses: up to 30 Gy. Samples were diluted by equivolume of ethanol for analysis and the released amount of pyrene-butyric acid was quantified by fluorescence (Aex = 341 nm; Aem max = 376 nm) and reported in Table 4.
Table 3:
DH (nm) Zeta potential(mV)
Hf02 - HMBPyne 1 1 -32
HfO20.2 30- 130 -25
Figure imgf000020_0001
Table 4: % release pyrene (measured by fluorescence)
Figure imgf000020_0002

Claims

1. Probe for drug delivery, preferably Probe for X-rays or beta/gamma triggered drug delivery comprising:
i. a support (S) that is chosen among inorganic particles, preferably inorganic particles responsive to X-rays or beta/gamma radiations;
ii. a N-comprising (5 to 14)-membered heteroaryl wherein at least one, preferably one, of the N atom is quaternarized and one of the carbon atom is substituted with an organic function, said reactive function being adapted to be cleaved by the action of electron and is chosen among alcohol, ester, carbamate, amide, aryl- ether, quaternary ammonium, amine, phosphate and phosphonate functions;
iii. a linker (L) linking the support (S) to the N-comprising (5 to 14)-membered heteroaryl, the linker (L) being linked to the N-comprising (5 to 14) membered heteroaryl thanks to the quaternary N atom.
2. Probe according to claim 1 , wherein the support (S) is a metal, or metal oxide (inorganic) particle, the support (S) is more preferably chosen among iron oxide, hafnium oxide, gadolinium oxide and titanium oxide, silver or gold.
3. Probe according to claim 1 or 2, wherein the N-comprising (5 to 14)-membered heteroaryl is chosen among groups enabling delocalization of electrons. Preferably, the N-comprising (5 to 14)-membered heteroaryl is chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium.
4. Probe according to anyone of claims 1 to 3, wherein the reactive function corresponds to a function of formula -CH(R2)-X-R1, wherein X is a heteroatom, preferably O, NR3, S or P(0)R3, P(0)XR3, R2 and R3, identical or different are chosen among H, (C1 -C6) linear or branched alkyl, (6 to 14)-membered aryl, preferably phenyl, or (5 to 14)-membered heteroaryl, preferably thiophene, pyrrol, imidazole, triazole, oxazole, thiazole, furane; R1 is chosen among H, carbonate, carbamate, urea each optionally activated with N-hydroxysuccinimide or imidazole.
5. Probe according to anyone of claims 1 to 4, wherein the linker is of the following formula L1-L2-L3-L4 wherein L1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L2 and L4, identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for example carbazole or 1 ,2,3-triazole.
6. Probe according to anyone of claims 1 to 5 of the following structure:
Figure imgf000022_0001
wherein
S, R2, X and R1 are as defined in anyone of claims 1 to 6
NT)
is the N-comprising (5 to 14)-membered heteroaryl defined in anyone of claims 1 to 6 and is preferably chosen among picolinium, quinolinium, isoquinolinium or acridinium, preferably the N-comprising (5 to 14)-membered heteroaryl is picolinium or quinolinium, preferably picolinium;
L is of the following formula L1-L2-L3-L4 wherein L1 is a phosphonate, amine, aniline, ammonium, carboxylic acid or thiol function, preferably phosphonate, more preferably bisphosphonate; L2 and L4, identical or different represent linear or branched (C1 -C6) alkyl, preferably ethyl; L3 is a donor group improving the electron transfer to the N-comprising (5 to 14)-membered heteroaryl, preferably L3 is an aromatic monocyclic or polycyclic (5 to 14)-membered heteroaryl, for examples carbazole or 1 ,2,3-triazole.
7. Probe according to claim 6, wherein
S is iron oxide, gold or hafnium oxide; and/or
NT)
is picolinium or quinolinium, preferably picolinium; and/or
L1 comprises phosphonate, preferably bisphosphonate, preferably is P(0)(0H)2- C(0H)-P(0)(0H)2; and/or
L3 is carbazole or 1 ,2,3-triazole; and/or L2 and L4 are ethyl; and/or
R2 is methyl; and/or
R1 is H; and/or
X is O.
8. Probe according to claim 6, wherein
S is iron oxide, gold or hafnium oxide;
N )
is picolinium or quinolinium, preferably picolinium;
L1 comprises bisphosphonate, preferably is P(0)(0H)2-C(0H)-P(0)(0H)2;
L3 is carbazole or 1 ,2,3-triazole;
L2 and L4 are ethyl;
R2 is methyl;
R1 is H; and
X is O.
9. Process for the preparation of a probe according to anyone of claims 1 to 8 comprises the following steps:
1 ) provide or synthesize support (S);
2) functionalize support (S) with the linker (L)
3) covalent coupling of the N-comprising (5 to 14)-membered heteroaryl to the linker.
10. Probe according to anyone of claims 1 to 8 for use for delivering molecules of interest, preferably molecules having therapeutic effect, such as drug, the molecule of interest being covalently linked via the reactive function of the N- comprising (5 to 14)-membered heteroaryl.
1 1 . Probes according to anyone of claims 1 to 8 for its use for the controlled delivery in space and time of molecules of interest, preferably for molecules having therapeutic effect, such as drug, said molecules comprising at least a heteroatom, covalently linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl of the probe.
12. Probe obtained by the coupling of a probe according to anyone of claims 1 to 8 with a molecule of interest, preferably molecule having therapeutic effect, such as drug, linked to the reactive function of the N-comprising (5 to 14)-membered heteroaryl of the probe.
13. Pharmaceutical composition comprising at least one probe according to claim 12 with at least one pharmaceutically acceptable excipient.
14. Method for delivering a molecule of interest to a person in need thereof comprising the administration of the probe according to claim 12.
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