WO2011073473A1 - Systèmes lipidiques fonctionnalisés avec des vinylsulfones, synthèse et utilisations de ces derniers - Google Patents

Systèmes lipidiques fonctionnalisés avec des vinylsulfones, synthèse et utilisations de ces derniers Download PDF

Info

Publication number
WO2011073473A1
WO2011073473A1 PCT/ES2010/000525 ES2010000525W WO2011073473A1 WO 2011073473 A1 WO2011073473 A1 WO 2011073473A1 ES 2010000525 W ES2010000525 W ES 2010000525W WO 2011073473 A1 WO2011073473 A1 WO 2011073473A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound according
protein
compound
group
general formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2010/000525
Other languages
English (en)
Spanish (es)
Inventor
Francisco SANTOYO GONZÁLEZ
Antonio Osuna Carrillo De Albornoz
Julia Morales Sanfrutos
Alicia MEGÍA FERNÁNDEZ
Teresa Cruz Bustos
Gloria Maribel GONZÁLEZ GONZÁLEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad de Granada
Original Assignee
Universidad de Granada
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad de Granada filed Critical Universidad de Granada
Publication of WO2011073473A1 publication Critical patent/WO2011073473A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • 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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6907—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 colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1268—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules host-guest, closed hollow molecules, inclusion complexes, e.g. with cyclodextrins, clathrates, cavitates, fullerenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C315/00—Preparation of sulfones; Preparation of sulfoxides
    • C07C315/04—Preparation of sulfones; Preparation of sulfoxides by reactions not involving the formation of sulfone or sulfoxide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/02—Sulfones; Sulfoxides having sulfone or sulfoxide groups bound to acyclic carbon atoms
    • C07C317/08—Sulfones; Sulfoxides having sulfone or sulfoxide groups bound to acyclic carbon atoms of an acyclic unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/16—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C317/18—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton with sulfone or sulfoxide groups bound to acyclic carbon atoms of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C317/32—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C317/34—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring
    • C07C317/38—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring with the nitrogen atom of at least one amino group being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/64—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton
    • C07C323/65—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton containing sulfur atoms of sulfone or sulfoxide groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS

Definitions

  • the present invention relates to a new compound of general formula (I) which comprises a molecule of a lipid nature and a vinyl sulfone group that allows the lipidation of biomolecules to be carried out in a highly efficient and simple manner.
  • the present invention also relates to its methods of obtaining and its uses. More particularly, it refers to the use of these compounds in the development of two new applications of ISCOMs based on nanoencapsulation capacity and the incorporation of antibodies to their membrane: a) Their use in fluorescent immunomarking; b) Development of systems for the targeted transport of drugs.
  • lipidomics (Wenk, MR Nature Reviews Druq Discovery (2005), vol. 4 (7), pp 594-610.) Has emerged as a new field of research that advances rapidly and which is of great importance since, like genes and proteins, lipids also play crucial functions in cells. Lipidomics is dedicated to the study and characterization of all cellular lipid species, molecules and macromolecules with which they interact and their biological functions.
  • lipids are not defined by a common structural characteristic but by a physical-chemical behavior: their insolubility in water.
  • technological advances have revealed the existence of thousands of different lipid species in the human body, suggesting the existence of functions not yet explored, such as cellular signaling, the targeting of proteins to their cellular destiny, the anchoring of proteins to membranes and the entry of toxins, viruses and bacteria.
  • membrane lipids through their interactions with integral proteins or associated with membrane modulate their function, anchor and traffic.
  • diseases associated with a defective lipid balance such as atherosclerosis, obesity, diabetes and Alzheimer's disease.
  • lipid network including lipid mediators for metabolic and gene regulation and their integration into non-lipid signaling systems.
  • Protein activity is not only controlled by the speed of synthesis and degradation but also by specific and selective processes of covalent modification or post-transduction modification that modulate molecular interactions, protein localization and stability.
  • post-transductional modifications that proteins can undergo, one of them is lipidation, the most relevant being fatty acid acylation.
  • the acylation of proteins with fatty acids mainly involves two of them palmitic and myristic. Protein binding can occur through the formation of different types of bonds:
  • the incorporation of peptides and proteins into liposomes can be carried out by two different strategies: through the inclusion of proteins in the liposome during formation of the same or by binding the protein to the lipid bilayer of the liposome. In this last option it is necessary that the protein contains a hydrophobic region so that the association is established and for this, in many cases, the chemical incorporation of fatty acids by covalent binding on the structure of the protein has been carried out.
  • ISCOMs Barr, IG et al. Immunoloqy and Cell Bioloqy (1996), vol. 74 (1), pp 8-25
  • immunostimulatory complex derives from the ability that these particles present to act as immunostimulatory complexes when They are used in vaccines for animals. Research on this type of systems exploits its ability to act as an adjuvant (compounds that act in a non-specific way to increase immunity against an antigen).
  • ISCOM Classic ISCOM, consisting of saponin, cholesterol, phospholipids and proteins.
  • ISCOMs The basis of the ISCOMs architecture is the interactions that are established between saponin and cholesterol. From a structural point of view they are spherical, hollow and box-shaped particles, with a heterogeneous size around 40 nm in diameter and with a negative charge. Each ISCOM consists of 20 or more globular subunits assembled in a pentagonal dodecahedron, and once formed they are tremendously stable.
  • ISCOMs can be prepared by various procedures, many of which are adaptations of liposome preparation methods. To date, five different methods have been described in literature: dialysis, centrifugation, hydration of lipid film, ethanol injection and ether injection. Of these, the most widespread are dialysis and centrifugation due to its simplicity and greater ease of scaling.
  • ISCOMs have focused, above all, on their use in vaccines (Sanders, M. T. et al. Immunoloqy and Cell Bioloqy (2005), vol. 83 (2), pp. 1 19-128).
  • the incorporation of antigens into the structure of ISCOM leads to a greater immune response, possibly due to the sum of the effect of the antibody and the immunostimulation produced by saponin.
  • they have also found application as vehiculization systems for amphotericin B and other drugs of a lipid nature (Morein, B. et al. Proceedinqs of the International Symposium on Controlled Relay of Bioactive Materials (1997), 24th, 1 18).
  • they have been used as antigens in immunoassays (Bjorkman, C. et al. Parasite Immunoloqy (1994), vol. 16 (12), pp 643-648).
  • a new compound of general formula (I) comprising a molecule of a lipid nature and a vinyl sulfone group that allows the lipidation of biomolecules to be carried out in a highly efficient and simple manner.
  • These compounds constitute an alternative to the derivatizations used in lipidomics for the incorporation of lipid moieties in biomolecules.
  • the use of these compounds is provided in the development of two new applications of ISCOMs based on their nanoencapsulation capacity and the incorporation of antibodies to the ISCOMs membrane: a) Their use in fluorescent immunomarking; b) Development of systems for the targeted transport of drugs.
  • a first aspect of the present invention relates to compounds of general formula (I) (hereafter compounds of the invention): where:
  • Y is a group -CH 2 -S0 2 R 1 - or -CH 2 -; preferably Y is -CH 2 - R 1 is a radical selected from the group comprising a (C1-C10) alkyl, a (C 1 -C 10 ) alkenyl, an alkynyl (CC 10 ), a dialkylaryl (Ci-C 0 ) Ar (CrCi 0 ) or a group (CH 2 CH 2 0) n CH 2 CH 2 ; preferably R 1 is a group (CH 2 CH20) nCH 2 CH2;
  • n takes values from 1 to 20; preferably n takes values between 2 to 10, more preferably n is 2 to 5 and even more preferably n is 2.
  • X is a group -CO-NH-R 2 -Z-CH 2 -, -Z-CH 2 - or -CH 2 -;
  • Z is S or O
  • R 2 is a radical selected from the group comprising an alkyl (CC 10 ), an alkenyl (C Ci 0 ), an alkynyl (C 1 -C 10 ), a dialkylaryl (C 1 -Ci 0 ) Ar (C 1 -C 10 ) or a group (CH 2 CH 2 0) m CH 2 CH 2 ;
  • n takes values from 1 to 20; preferably m takes values from 2 to 10, more preferably m is from 2 to 5 and even more preferably m is 2; Y
  • ⁇ ⁇ - ⁇ represents a lipid
  • lipid is meant in the present invention a molecule of a non-polar nature, such as saturated or unsaturated hydrocarbons, for example but not limited to alkyl groups (Ci-C 30 ), alkenes (C 2 -C 30 ) , alkynes (C 2 -C 30 ) or sterols. Most of these types of molecules are biomolecules, composed mainly of carbon and hydrogen and to a lesser extent oxygen, although they can also contain phosphorus, sulfur and nitrogen, which have as their main characteristic being hydrophobic or insoluble in water and yes in organic solvents.
  • the lipid is a steral or a saturated or unsaturated aliphatic hydrocarbon.
  • aliphatic hydrocarbon refers, in the present invention, to organic molecules consisting of carbon and hydrogen, in which the carbon atoms form linear or branched chains, saturated or unsaturated. That is, they can be both alkyl (saturated) and alkenyl or alkynyl (unsaturated) groups.
  • the lipid can be selected from a hydrocarbon (C 4 -C 30 ), saturated or unsaturated. And more preferably the carbon number is between 10 and 20.
  • steral refers in the present invention to spheroids with 27 to 29 carbon atoms. Its chemical structure derives from cyclopentaneperhydrophenanthrene or esterano, a 17-carbon molecule consisting of three hexagonal and one pentagonal rings. It can be selected from the list comprising, but not limited to cholesterol, epicolesterol, stigmasterol, lanosterol, ergosterol and coprostenol. More preferably the steral is cholesterol.
  • alkyl refers in the present invention to aliphatic, linear or branched chains, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc.
  • lipids they would be aliphatic chains having 1 to 30 carbon atoms, more preferably 4 to 30 carbon atoms and even more preferably 10 to 20 carbon atoms.
  • R 2 independently, they would preferably be aliphatic chains having 1 to 10 carbon atoms, more preferably 2 to 5 carbon atoms and even more preferably an ethyl.
  • alkenyl in the present invention refers to an alkyl radical, described above, and having one or more unsaturated bonds, specifically has at least one double bond, although it can also have at least one triple bond.
  • Alkenyl radicals may be optionally substituted by one or more substituents such as an aryl, halogen, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, nitro, etc.
  • alkynyl refers in the present invention to an alkyl radical, described above, and having one or more unsaturated bonds, specifically has at least one triple bond, although it can also have at least one double bond.
  • Alkenyl radicals may be optionally substituted by one or more substituents such as an aryl, halogen, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, nitro, etc.
  • dialkylaryl is meant in the present invention an aryl group that is substituted with two alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably having 1 to 5 carbon atoms.
  • the alkyl, alkenyl or alkynyl groups may be the same or different, preferably they are the same.
  • aryl means in the present invention an aromatic or heteroaromatic system having 6 to 12 carbon atoms or some other atom, such as O, N, S, etc., can be single or multiple ring , separated and / or condensed.
  • Typical aryl groups contain 1 to 3 separate or condensed rings and from about 6 to 10 about 18 ring carbon atoms, such as phenyl, naphthyl, indenyl, phenanthryl or anthracil radicals
  • X is a group -CO-NH- 2 -Z-CH 2 -
  • the compounds of the invention have the general formula (II):
  • R, Z, Y and ⁇ " ' are defined above.
  • the compound of the invention would be of the general formula (I), except for the compound 1 - (vinylsulfonyl) octadecane, that is, when the lipid is a hexadecanyl, X and Y cannot be -CH 2 -.
  • the compound of the invention is selected from the list comprising:
  • a second aspect of the present invention relates to a method of obtaining the compounds of the invention of general formula (II) and (III) and comprising:
  • the bis-vinylsulfone is the DVS divinylsulfone (ie, Y is -CH 2 -).
  • the method of obtaining the compounds of the invention comprises reacting: a compound of the general formula (IX) with a bis-vinylsulfone of the formula (VIII):
  • the bis-vinylsulfone is the DVS divinylsulfone (ie, Y is -CH 2 -).
  • Lipidation agents containing vinyl sulfones can be linked to any biomolecule containing complementary functional groups (such as, for example, the amino, thiol and imidazole groups) present therein naturally or artificially through of a Michael type addition reaction.
  • complementary functional groups such as, for example, the amino, thiol and imidazole groups
  • the compounds are compatible with the biological nature of the biomolecules and the lipidation reaction does not require any activation strategy.
  • the reactions can be carried out under physiological conditions, aqueous medium, narrow pH range, mild temperatures.
  • another aspect of the present invention relates to the use of a compound of general formula (I) as a lipidating agent for molecules, and more preferably for biomolecules.
  • the compounds of formula (I) that are used as molecule lipidation agents can be selected from the list comprising:
  • the selected biomolecules are proteins.
  • the protein selected is the usual protein A (42kDa polypeptide constituent of the Staphilococcus aureus wall that has affinity for antibodies through the Fe portion) or protein G (polypeptide of between 30000 and 35000 Daltons isolated from the cell wall of beta-hemolytic streptococcus of strains C or G. Like protein A it has affinity for antibodies).
  • lipidation of proteins, or of the molecules in general is carried out in a solution thereof in a buffer that does not contain free amines such as, but not limited to, phosphate, HEPES or carbonate. , of moderate ionic strength (50 - 200 mM) and basic pH (7.5 - 8.7) and reaction with an excess of the labeling reagent of general formula (I) for a sufficient time (usually for a few hours at room temperature or at 4 ° C) the excess of reagent being removed by dialysis.
  • a buffer that does not contain free amines such as, but not limited to, phosphate, HEPES or carbonate.
  • Another aspect of the present invention is the incorporation of the modified biomolecules with the lipidating agents of general formula (I), forming the covalent bioconjugates, into a nanobox, for example but not limited to the ISCOMs.
  • the selected biomolecules are proteins.
  • the protein selected is protein A or protein G.
  • nucle is understood in the present invention to a type of system such as those described in publications B. Morein K.L. Bengtsson, Methods 19, 94-102 (1999) and H.-X Sun, Y. Xie, Y.-P. Ye, Vaccine 27, 4388-4401 (2009).
  • Another aspect of the present invention relates to the use of the compounds of general formula (I) in the preparation of nanoboxes, preferably ISCOMs functionalized with viniisulfone groups and the use of these viniisulfone groups to bind any biomolecule containing complementary functional groups (amino groups , thiols and imidazoles) present therein naturally or artificially through a Michael type addition reaction.
  • a complementary functional groups amino groups , thiols and imidazoles
  • the selected biomolecules are proteins and compound of general formula (I) contains a cholesterol molecule as a molecule of a lipid nature.
  • the protein selected is protein A or protein G.
  • Another aspect of the present invention relates to the incorporation of antibodies to the membrane of the lipid nanocases, more preferably to the ISCOMs, through their union with the biomolecule, preferably protein A or protein G previously incorporated (by any of the methodologies previously described).
  • the antibodies are IgG against a parasite such as Trypanosoma cruzi.
  • Another aspect of the present invention relates to the use of these systems, lipid-antibody nanocages, more preferably ISCOMs-antibody, in the development of two new applications of ISCOMs or similar systems, taking advantage of their nanoencapsulation capacity: a) Their use in fluorescent immunomarking; b) Development of systems for the targeted transport of drugs.
  • the encapsulated fluorescent system contains a fluorescent molecule, preferably phycocyanin.
  • the lipid-antibody nanocajas system also contains active ingredient.
  • active ingredient is meant in the present invention a drug or a molecule of biological origin, for example proteins or nucleic acids, and which are capable of acting by blocking an enzymatic reaction or affecting the biosynthesis of macro-molecules by the cell.
  • active ingredient it can be added to the previous system, for example actinomycin-D.
  • FIGURE 1 Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with phycocyanin.
  • FIGURE 2 Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with ISCOMs-phycocyanin.
  • FIGURE 3 Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with ISCOMs-phycocyanin-protein A modified with 4.
  • FIGURE 4 Micrograph taken with confocal laser microscopy.
  • Immunofluoroassay epimastigotes of Trypanosoma cruzi with ISCOMs-phycocyanin-protein A modified with 4-lgG against Trypanosoma cruzi.
  • FIGURE 5 Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with ISCOMs-phycocyanin-protein A modified with 25.
  • FIGURE 6 Micrograph taken with confocal laser microscopy.
  • Immunofluoroassay epimastigotes of Trypanosoma cruzi with ISCOMs-phycocyanin-protein A modified with 25-lgG against Trypanosoma cruzi.
  • FIGURE 7. Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with ISCOMs obtained with compound 15 (1: 1 ratio) -phycocyanin-protein A.
  • FIGURE 8 Micrograph taken with confocal laser microscopy.
  • Immunofluoroassay epimastigotes of Trypanosoma cruzi with ISCOMs obtained with compound 15 (1: 1 ratio) -phycocyanin-protein A- IgG against Trypanosoma cruzi.
  • FIGURE 9 Micrograph taken with confocal laser microscopy. Negative control of immunofluoroassay: epimastigotes of Trypanosoma cruzi with ISCOMs obtained with compound 15 (ratio 2: 3) -phycocyanin-protein A.
  • FIGURE 10 Micrograph taken with confocal laser microscopy.
  • Immunofluoroassay epimastigotes of Trypanosoma cruzi with ISCOMs obtained with compound 15 (ratio 2: 3) -phycocyanin-protein A- IgG against Trypanosoma cruzi.
  • FIGURE 11 Survival rate in the different cytotoxicity tests: A.- Epicastigotes of T. cruzi treated with Actinomycin D. (0.05 pg); B.- Epimastigotes of 7. cross / treated with ISCOM-lgG against T. cruzi containing a total of 2.88x10 "5 pg of Actinomycin D; C- Epimastigotes of T. cruzi treated with ISCOM-lgG against T. cruzi containing a total of 5.76X10 "5 pg of Actinomycin D; D.- Epicastigotes of T. cruzi treated with ISCOM-lgG versus T. cruzi containing a total of 1 1.52 X10 "5 pg of Actinomycin D
  • EXAMPLE 5.1 Lipidation of protein A with lipidation agents 4 and 25.
  • Compounds 4 and 25 are solubilized in methanol and 0.125 M carbonate buffer, pH 8 until reaching a final concentration of 20mg / ml.
  • the compounds are added to a solution of protein A (100mg / mL) in a 5: 1 ratio. The final solution is incubated 12 hours at 4 ° C. After this time, 20 ⁇ of 0.125M carbonate buffer-1 M glycine is added and incubated for 3 hours at 4 ° C.
  • EXAMPLE 6 Incorporation of antibodies in the ISCOMs membrane and use in fluorescent immunomarkers. a) Preparation of ISCOMATRIX.
  • a solution in 2 ml of distilled water containing cholesterol and phosphatidylcholine (1: 1) is prepared at a final concentration of 15mg / ml and 0.400mg of Mega-10.
  • a solution of Quil A is prepared at a concentration of 100mg / ml.
  • the appropriate amount is taken to obtain a 1: 1: 5 ratio of cholesterol: phosphatidylcholine: Quil A in a final volume of 12 milliliters. It is incubated one hour at room temperature and concentrated to a volume of approximately 1/5 of the initial.
  • the concentrate is dialyzed for 40 hours in PBS with four changes. Once the dialysate has been collected, the ISCOMs formed by a sucrose gradient centrifugation at 50000g for 18 hours are purified and finally dialyzed again b) Phycocyanin binding to ISCOMs.
  • the solution obtained in the previous section is mixed in a 1: 3 ratio with the IgG vs. T. cruzi (457 ⁇ g / mL). The resulting solution is incubated at least 1 hour at 4 ° C with stirring. d) Direct immunofluorescence assays.
  • the slides must be clean and free of grease, for which they are immersed in acetone for 24 hours and then allowed to dry at room temperature.
  • Trypanosoma cruzi epismastigotes were cultured in MTL medium supplemented with 10% fetal bovine serum at 28 ° C for 5 days.
  • EXAMPLE 7 Obtaining functionalized ISCOMs with vinyl sulfone groups, incorporation of antibodies in the ISCOMs membrane and use in fluorescent immunomarkers. a) Obtaining functionalized ISCOMs with vinyl sulfone group through compound 15
  • ISCOMATRIX are prepared in the same manner described above, but the composition of cholesterol is varied using different proportions of cholesterol: 15. It is prepared on the one hand ISCOMATRIX with the normal formulation that will serve as a positive control and on the other hand, maintaining the final concentration of 15mg / ml of Cholesterol, 15 and phosphatidylcholine, we will vary the composition using 1: 1 cholesterol: 15 and 3: 2 cholesterol: 15. b) Binding of protein A to ISCOMs obtained with compound 15.
  • the solution obtained in the previous section is mixed in a 1: 3 ratio with the IgG against T. cruzi.
  • the resulting solution is incubated at least 1 hour at 4 ° C with stirring.
  • d) Direct immunofluorescence assays.
  • EXAMPLE 8 Use of ISCOMs in the development of targeted drug transport systems.
  • a solution is prepared in 2 ml of distilled water containing cholesterol and phosphatidylcholine (1: 1) at a final concentration of 15mg / ml in addition to 0.400mg of Mega-10.
  • a solution of Quil A is prepared, at a concentration of 100mg / ml.
  • Quil A in a final volume of 10 mL and 2 mL of a stock solution of Actinomycin D is added (5mg / ml). Incubate for one hour at room temperature and concentrate in a protein concentrator up to 1/5 of the initial volume.
  • the concentration of antibiotic trapped in the nanoparticles (ISCOM-actinomycin D-IgG ⁇ 0.192 mg / mL suspension) was determined spectrophotometrically. e) Cytotoxicity test.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Nanotechnology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Steroid Compounds (AREA)

Abstract

La présente invention concerne des systèmes lipidiques fonctionnalisés avec des vinylsulfones, leur synthèse et leurs utilisations. Le composé selon l'invention comprend une molécule de nature lipidique et un groupe vinylsulfone qui permet d'effectuer la lipidation de biomolécules d'une forme hautement efficace et simple. L'invention concerne également ses procédés d'obtention et ses utilisations. Plus concrètement, l'invention porte sur l'utilisation de ces composés dans le développement de deux nouvelles applications des ISCOM qui sont basées sur la capacité de nanoencapsulation et l'incorporation d'anticorps dans la membrane de ces derniers: a) leur utilisation dans l'immunomarquage fluorescent; b) la mise au point de systèmes pour le transport dirigé de médicaments.
PCT/ES2010/000525 2009-12-16 2010-12-15 Systèmes lipidiques fonctionnalisés avec des vinylsulfones, synthèse et utilisations de ces derniers Ceased WO2011073473A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200902389A ES2337226B2 (es) 2009-12-16 2009-12-16 Sistemas lipidicos funcionalizados con vinilsulfonas. sintesis y usos.
ESP200902389 2009-12-16

Publications (1)

Publication Number Publication Date
WO2011073473A1 true WO2011073473A1 (fr) 2011-06-23

Family

ID=42077483

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2010/000525 Ceased WO2011073473A1 (fr) 2009-12-16 2010-12-15 Systèmes lipidiques fonctionnalisés avec des vinylsulfones, synthèse et utilisations de ces derniers

Country Status (2)

Country Link
ES (1) ES2337226B2 (fr)
WO (1) WO2011073473A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5414135A (en) * 1991-12-30 1995-05-09 Sterling Winthrop Inc. Vinyl sulfone coupling of polyoxyalkylenes to proteins
WO2001005999A1 (fr) * 1999-07-14 2001-01-25 Nen Life Science Products, Inc. Element d'analyse et son procede de fabrication
WO2002049676A2 (fr) * 2000-12-19 2002-06-27 California Institute Of Technology Compositions contenant des complexes d'inclusion
FR2912410A1 (fr) * 2007-02-12 2008-08-15 Specific Polymers Sarl Silicones sulfones formant des elastomeres par autoassemblage,procedes de preparation de tels silicones et leurs utilisations.
WO2008156327A2 (fr) * 2007-06-20 2008-12-24 Lg Household & Health Care Ltd. Lipide présentant un groupe fonctionnel et composition pour soins personnels comprenant le lipide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5414135A (en) * 1991-12-30 1995-05-09 Sterling Winthrop Inc. Vinyl sulfone coupling of polyoxyalkylenes to proteins
WO2001005999A1 (fr) * 1999-07-14 2001-01-25 Nen Life Science Products, Inc. Element d'analyse et son procede de fabrication
WO2002049676A2 (fr) * 2000-12-19 2002-06-27 California Institute Of Technology Compositions contenant des complexes d'inclusion
FR2912410A1 (fr) * 2007-02-12 2008-08-15 Specific Polymers Sarl Silicones sulfones formant des elastomeres par autoassemblage,procedes de preparation de tels silicones et leurs utilisations.
WO2008156327A2 (fr) * 2007-06-20 2008-12-24 Lg Household & Health Care Ltd. Lipide présentant un groupe fonctionnel et composition pour soins personnels comprenant le lipide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOREIN, B. ET AL.: "Immunomodulation by Iscoms, Immune Stimulating Complexes", METHODS, vol. 19, 1999, pages 94 - 102 *
SUN, H.-X. ET AL.: "ISCOMs and ISCOMATRIXTM", VACCINE, vol. 27, 2009, pages 4388 - 4401 *

Also Published As

Publication number Publication date
ES2337226A1 (es) 2010-04-21
ES2337226B2 (es) 2011-09-14

Similar Documents

Publication Publication Date Title
CN117098558A (zh) 聚噁唑啉-脂质缀合物及包含其的脂质纳米颗粒和药物组合物
JP3907662B2 (ja) 自己構築ポリヌクレオチド送達システム
ES2913626T5 (en) Compounds and compositions for intracellular delivery of agents
US20220370624A1 (en) Lipid compositions comprising peptide-lipid conjugates
CN1863558B (zh) 具有向核内转移能力的聚精氨酸修饰的脂质体
JPH11511757A (ja) 治療用分子を細胞内投与するためのカチオン両親媒性物質と補助脂質とを含む組成物
AU2023317702A1 (en) Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions
WO2007102481A1 (fr) Vecteur pour le transport nucléaire d'une substance
JP5241711B2 (ja) 両親媒性分子、それを含む分子集合体及びその用途
US20250325491A1 (en) Polyvalent molecule based lipid nanoparticles for nucleic acid delivery
US20220378702A1 (en) Peptide-lipid conjugates
PT1833465E (pt) Liofilização de virossomas
Makowska et al. Impact of lipidation site on the activity of α-helical antimicrobial peptides
Miura et al. Identification and evaluation of the minimum unit of a KALA peptide required for gene delivery and immune activation
JP5069920B2 (ja) マンノース６−リン酸−ポリエチレングリコール結合体
ES2431316T3 (es) Sistema de nanotransporte con arquitectura dendrítica
JP2023515608A (ja) アミノ脂質化合物、その調製方法、およびその用途
US20250332275A1 (en) Branched Polypeptide Carrier for Efficient Nucleic Acid Delivery and Its Variants
ES2337226B2 (es) Sistemas lipidicos funcionalizados con vinilsulfonas. sintesis y usos.
WO2014016460A1 (fr) Composés dendritiques carbosilanes homo et hétéro-fonctionnalisés
JP2005515990A (ja) 化合物
JPWO2006077857A1 (ja) 粒子を脂質膜で被覆する方法
HK40103124A (zh) 聚恶唑啉-脂质缀合物及包含其的脂质纳米颗粒和药物组合物
Larson Development of anionic liposomal carriers containing listeriolysin O and red blood cell ghosts as plasmid DNA delivery platforms
JP2005535666A (ja) 薬剤のデリバリーシステム

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10837074

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10837074

Country of ref document: EP

Kind code of ref document: A1