WO2012101309A1 - Nanoliposomes fonctionnalisés avec des peptides - Google Patents

Nanoliposomes fonctionnalisés avec des peptides Download PDF

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Publication number
WO2012101309A1
WO2012101309A1 PCT/ES2012/070037 ES2012070037W WO2012101309A1 WO 2012101309 A1 WO2012101309 A1 WO 2012101309A1 ES 2012070037 W ES2012070037 W ES 2012070037W WO 2012101309 A1 WO2012101309 A1 WO 2012101309A1
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Prior art keywords
nanoliposome
peptide
glycerol
peg
distearoyl
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PCT/ES2012/070037
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English (en)
Spanish (es)
Inventor
David POZO PÉREZ
Rebecca KLIPPSTEIN MARTIN
Ricardo GONZÁLEZ CAMPORA
Inmaculada TRIGO SÁNCHEZ
María Teresa VARGAS DE LOS MONTEROS
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Fundación Progreso Y Salud
Universidad De Sevilla
Servicio Andaluz De Salud
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Priority claimed from ES201130072A external-priority patent/ES2389347B1/es
Priority claimed from ES201130185A external-priority patent/ES2390147B1/es
Application filed by Fundación Progreso Y Salud, Universidad De Sevilla, Servicio Andaluz De Salud filed Critical Fundación Progreso Y Salud
Publication of WO2012101309A1 publication Critical patent/WO2012101309A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K47/6911—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 liposome

Definitions

  • the present invention is within the field of medicine, chemistry, biochemistry and immunology, and refers to the use of nanoliposomes functionalized with peptides on its surface that allow the identification of tissues and / or target cells and the release of drugs selectively, and in particular, to the functionalization of nanoliposomes with the VIP peptide.
  • the present invention also relates to the compositions, the preparation process and the uses of said nanoliposomes.
  • nanoliposomes are of special relevance because they are the best clinically established nanometric systems for the transport and shipment of drugs because they are not cytotoxic, they are biocompatible and biodegradable, and also their synthesis is relatively cheap and Easy scaling in industrial processes. Liposomes in general have been used in therapies to treat cancer for more than a decade since they have been shown to systematically reduce side effects, toxicity and facilitate drug elimination. (Torchlin, 2005. Nat Rev. Drug Discovery 4, 145).
  • Nanoliposomes can be used as carriers of various substances both outside and inside and for a variety of biomedical applications such as gene therapy or for drug delivery, so that acids nucleic or the drug are protected inside avoiding its enzymatic degradation and its direct contact with other healthy cells.
  • they allow the sending of biologically active molecules of lipophilic character and sizes greater than 500 Da, two of the most important problems that an active molecule can present to end up as an active ingredient of a medicine.
  • Nanoliposomes of various sizes can quickly enter areas where tumors exist since the vascular endothelial wall is fenestrated. In contrast, nanoliposomes remain in the bloodstream of healthy tissue through the wall of the non-fenestrated vascular endothelium.
  • the functionalization of the nanoliposomes with peptides that would recognize with greater selectivity the therapeutic targets would allow addressing specifically in those cases in which it is known that said targets overexpress the receptors that recognize said peptides.
  • liposomes functionalized with peptides on their surface There are currently functionalized liposomes with peptides on their surface.
  • liposomes functionalized with tuftsin have allowed increasing the efficacy of sodium stibogluconate (Agrawal & Gupta, 2000. Adv. Drug Deliv. Rev. 41: 135-146; Gupta & Haq, 2005. Met ods Enzymol. 391: 291- 304) and amphotericin B (Gupta & Haq, 2005. Methods Enzymol. 391: 291-304, Agrawal et al., 2002. J. Drug Target. 10: 41-45).
  • peptide ligands bind to the terminal end of a PEG chain.
  • the first method involves incorporating PEG-lipid conjugates with their functionalized end into the liposomes and then conjugate it with the peptide ligands (Zalipsky et al., Bioconjug. Chem., 1995, 6, 705-8).
  • the terminal group of the functionalized PEG is conjugated to the peptide ligands, non-homogeneous conjugation can occur if there is more than one reactive group in the ligand.
  • the second method is to directly incorporate the peptide-PEG-lipid conjugate into the liposomal membranes (Zalipsky et al., Bioconjug. Chem., 1997, 8, 1 1 1-8).
  • the synthesis of these conjugates is difficult, since the chemical properties of the side chains in the peptides are diverse, the molecular mass of the PEG is heterogeneous and the nature of the lipids is amphiphilic. These properties make it difficult to protect the side chains in the process of synthesis, purification, and reaction. In fact, very few peptide-PEG-lipid conjugates have been synthesized.
  • Application US2007 / 0106064 describes the preparation of this type of conjugates through the use of a peptide resin in which the amino groups are initially protected.
  • the biological effects of the VIP neuropeptide have a growing interest in its modulating capacity in pathologies in which there is an inflammatory and / or autoimmune component [Grimm, M. C. et al. (2003) J. Immunol. 171, 4990-4994; Pozo, D. (2003) Trends Mol. Med. 9, 21 1-217; Ganea, D., and Delgado, M. (2002). Crit. Rev. Oral. Biol. Med. 13, 229-237; Delgado, M. et al. (2003). Trends Immunol. 24, 221-224; Pozo et al. (2009). J Immunol 183, 4346-4359].
  • the process of nanoliposome functionalization with this peptide faces the added problem of designing a procedure in which the carboxyl end of VIP is free, since it is at this end that it interacts with its specific membrane receptors. It is necessary, therefore, to develop a method of functionalization of nanoliposomes with peptides, leaving the carboxyl end of said peptide free to interact with its receptor.
  • nanoliposomes functionalized with peptides on their surface, stable, non-toxic, soluble in water, and compatible with biological systems, as well as a procedure for obtaining them, which are useful for vehiculating active ingredients and / or pharmaceutical compositions
  • the procedure that leads to these nanoliposomes allows functionalization with the peptide so that the carboxyl-terminal end is free, thus leaving intact its ability to interact with its specific receptors, which allows formulating in vivo drug detection and selective release strategies. on cells that are interested in eliminating (as is the case with tumor cells), or expanding (through the administration of trophic factors) depending on the pathology that is intended to be addressed.
  • nanoliposomes with the VIP peptide increases the cytotoxic capacity of antitumor drugs encapsulated inside the nanoliposome, such as doxorubicin, improving the effectiveness of this active ingredient, which allows the incorporation of the drug into the liposomal composition at lower concentrations than those used in other state-of-the-art formulations, such as the case of non-functionalized liposomes or in solution.
  • a first aspect of the invention relates to a nanoliposome functionalized with a peptide on its surface, hereinafter, nanoliposome of the invention.
  • the carboxyl-terminal end of the peptide is free, being able to interact with its receptor through its carboxyl-terminal end.
  • the nanoliposome of the invention comprises:
  • the peptide is covalently bound to the nanoliposome through the reaction between the maleimide of the conjugate and the sulfhydryl group of the modified peptide.
  • the nanoliposome further comprises an active ingredient.
  • Said active ingredient can be hydrophilic, being incorporated into the aqueous core of the nanoliposome, or it can be hydrophobic, in which case it is incorporated into the lipid bilayer of the nanoliposome.
  • the present invention relates to a procedure for the preparation of a nanoliposome as previously defined.
  • a third aspect of the invention is a functionalized nanoliposome obtainable according to the procedure described above.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a nanoliposome as previously defined and an active ingredient capable of diagnosing, curing, mitigating, treating or preventing a disease.
  • a final aspect of the invention relates to the use of the nanoliposome as previously defined for the preparation of a medicament for the treatment of diseases that occur with cell proliferation.
  • FIG. 1 Structure of the vasoactive intestinal peptide neuropeptide (VIP).
  • Figure 2 Determination of VIP peptide on the surface of the nanoliposome loaded with a biologically relevant substance, in this case doxorubicin.
  • FIG. 3 Characterization of the size of the naoliposomes by Dynamic Light Scattering (DLS).
  • DLS Dynamic Light Scattering
  • Figure 4 Functional characterization of the interaction with VPAC receptors (cAMP production, second intracellular messenger of the VIP receptor / effector system) on tumor lines of prostate cancer.
  • Figure 5 Optical microscopy (20X) after 48 hours of treatment of DU-145 cells.
  • A untreated cells;
  • C and
  • E show the cells after being treated with nanoliposomes and nanoliposomes functionalized with VIP respectively.
  • B cells treated with soluble doxorubicin, doxorubicin encapsulated in the nanoliposome, and doxorubicin encapsulated in the nanoliposome functionalized with VIP are shown.
  • Figure 6 Histogram representing the percentage of lactate dehydrogenase (LDH) release in the DU-145 cell line after exposure to the drug doxorubicin (10 g / mL) for 24 hours.
  • LDH lactate dehydrogenase
  • the invention relates to a nanoliposome functionalized with a peptide on its surface, preferably where the carboxyl-terminal of the peptide is free, being able to interact with its receptor through its carboxyl-terminal end.
  • nanoliposomes are understood as essentially spherical aqueous compartments, surrounded by at least one closed lipid double layer with an average diameter of less than 1000 nm. In the present invention, preferably less than 500 nm, more preferably less than 400 nm and even more preferably less than 200 nm.
  • Medium diameter means the average diameter of the population of nanoliposomes dispersed in an aqueous medium.
  • the average diameter of these systems can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the experimental part below.
  • the nanoliposomes have an average diameter between 75 and 125 nm, preferably between 85 and 1 nm, more preferably between 95-105 nm.
  • the nanoliposomes of the invention preferably comprise a lipid-spacer-maleimide conjugate.
  • conjugate should be understood as the product resulting from the covalent bond between the three constituent components of the conjugate, that is, the lipid, the spacer and the maleimide.
  • lipid is a natural or synthetic amphipathic molecule that has a hydrophilic part and a hydrophobic part in the same molecule and that can spontaneously form bilayer vesicles in an aqueous medium or can be stably incorporated in lipid bilayers.
  • lipid comprises acylglycerides, cerids, phospholipids, lysophospholipids and glycolipids (cerebrosides and gangliosides).
  • these lipids include stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerol ricinolate, hexadecyl myristate, isopropyl myristate, amphoteric acrylic polymer, fatty acid amides, cholesterol, cholesterol ester, diacylglycerolsuccinate, fatty acid glycerol and similar fatty acid.
  • cationic lipids consisting of a positively charged terminal group, such as an amine, polyamine or polylysine, may be employed attached to a portion lipophilic neutral character, such as a sterol, a hydrocarbon chain or two hydrocarbon chains.
  • Examples of these cationic lipids include 1,2-dioleyloxy-3- (trimethylamino) propane (DOTAP); N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxy) propyl] -N, N - dimethyl-N-hydroxyethylammonium (DORIE), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dimethylammonium bromide (DDAB).
  • DOTAP 1,2-dioleyloxy-3- (trimethylamino) propane
  • DMRIE N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide
  • DORIE N- [1
  • the term lipid refers to a phospholipid.
  • phospholipid comprises phosphoaccylglycerols, that is, compounds that are composed of a glycerol molecule, two of whose hydroxyl groups are esterified by fatty acids (saturated or partially unsaturated straight chain monocarboxylic acids with 8 to 28 atoms of carbon), the third hydroxyl group being esterified by a phosphate group that binds glycerol to another organic molecule through a phosphodiester bond, which usually contains nitrogen, such as choline, serine or ethanolamine, and which may have an electric charge.
  • phosphoacylglycerols examples include phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine and phosphatidylserine. Also included within the term phospholipids are those complex mixtures extracted from natural products that essentially comprise phosphoacylglycerols such as lecithin.
  • the phospholipid is selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof.
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DSPE 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine
  • DPPC 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine
  • DPPE 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine
  • a linear hydrophilic polymer with functional terminal groups capable of binding to the lipid and the amino group of the maleimide can be used.
  • Suitable spacers in the present invention include, but are not limited to, polyglycine, polyethylene glycol, polypropylene glycol, polymethyl acrylamide, polydimethyl acrylamide, polyhydroxyethyl acrylate, polyhydroxypropyl methacrylate and polyoxyalkene.
  • the spacer is polyethylene glycol (PEG).
  • the conjugation of the lipid with the PEG requires that both the PEG and the lipid have a suitable functional terminal group.
  • the PEG can be functionalized with a halide or a sulphonate so that it is coupled with an amino terminal group of the lipid.
  • the PEG can be functionalized by an activated carbonate (-C (O) -imidazolyl, -OC (0) -para-nitrophenyl, - OC (0) -succinimide, -OC (0) -2,4,4-trichlorophenyl) such that it is coupled with an amino terminal group of the lipid.
  • activated carbonate -C (O) -imidazolyl, -OC (0) -para-nitrophenyl, - OC (0) -succinimide, -OC (0) -2,4,4-trichlorophenyl
  • the PEG can be functionalized by an activated carboxyl group (carboxyl group activated by DCC (dichlorohexylcarbodimide) / HOBt (N-hydroxybenzotriazole), DCC / DMAP (dimethylaminopyridine), DIPCDI (1,3-diisopropylcarbodiimide) / HOBt, EDC (1 - (3-dimethylaminopropyl) -3-ethyl-carbodiimide)) / NHS (N-hydroxysuccinimide)) so that it is coupled with an amino terminal group of the lipid.
  • DCC dichlorohexylcarbodimide
  • HOBt N-hydroxybenzotriazole
  • DCC / DMAP dimethylaminopyridine
  • DIPCDI 1,3-diisopropylcarbodiimide
  • EDC (1 - (3-dimethylaminopropyl) -3-ethyl-carbodiimi
  • the phospholipid binding with the PEG is carried out through an amide bond, resulting from the reaction between the carboxyl group with which the PEG is functionalized and the amino terminal group of the organic phospholipid molecule.
  • the conjugation of the PEG with the maleimide molecule also requires that the PEG at the other end have a suitable functional terminal group capable of reacting with the maleimide amino group.
  • a suitable functional terminal group capable of reacting with the maleimide amino group.
  • any of the aforementioned functional groups can be used.
  • the lipid-spacer-maleimide conjugate has the following structure:
  • Said conjugate is commercially available.
  • the peptide is a synthetic or natural compound peptide by a chain of amino acids.
  • the peptide is selected from the group consisting of hormones, cytokines, toxins, quemotaxins and peptides of the extracellular matrix for cell adhesion.
  • Said peptide can bind to a receptor such as somatostatin receptors, vasoactive intestinal peptide receptors, integrin receptors or growth factor receptors, among others.
  • a receptor such as somatostatin receptors, vasoactive intestinal peptide receptors, integrin receptors or growth factor receptors, among others.
  • the peptide is selected from a list comprising: glucagon, gastric inhibitory polypeptide (GIP), secretin, growth hormone, somatoliberin, somatotropin, PHI peptide (peptide histidine isoleucine), PHM peptide (peptide histidine- methionine), PACAP (pituitary adenylate cylase-activating peptides), adrenomedulin, corticostatin and vasoactive intestinal peptide (VIP).
  • the peptide is a neuropeptide, and more preferably it is the vasoactive intestinal peptide (VIP) whose structure is represented in Figure 1.
  • a neuropeptide refers to small protein-like molecules of a peptide bond of two or more amino acids and which differ from proteins by their length, and because they originate from cerebral synaptic transduction. Its size can vary from two amino acids, such as carnosine, to more than forty such as CRH (corticotropin releasing hormone). They have both stimulating and inhibitory brain function, producing effects such as analgesia, appetite or sleep, among others.
  • vasoactive intestinal peptide is understood as a polypeptide hormone formed by 28 amino acid residues and produced by many structures of the human body such as the digestive system, the pancreas and the suprachiasmatic nucleus of the hypothalamus in the brain. It is characterized by its vasodilator property and its activity in the peripheral nervous system (for example, VIP relaxes the lungs, trachea and gastric musculature). It inhibits the secretion of gastric enzymes and stimulates the secretion of glucagon, insulin and somatostatin, increases adenyl cyclase, as well as bile secretion in the liver.
  • VIP vasoactive intestinal peptide
  • the VIP is also referred to as PHM27 or MGC13587. It is encoded by a gene found on chromosome 6 (6q25). Its amino acid sequence is found in SEQ ID NO: 1.
  • VIP is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
  • nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) are nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the VIP peptide.
  • VPAC1 Three VIP receivers known as VPAC1, VPAC2 and PAC1 are known.
  • the VPAC1 receptor is expressed in malignant epithelial neoplasms, lung cancer and other cancers such as stomach, colon, breast, prostate, liver and urinary bladder, while VPAC2 has only been found in a few tumors.
  • PAC1 is expressed primarily in tumors originating in the neural and endocrine systems, such as glial tumors (glioblastomas, neuroblastomas, astrocinomas, etc.), or pituitary adenomas.
  • the nanoliposome with the peptide is modified by incorporating a terminal sulfhydryl group by methods known in the state of the art.
  • the peptide is reacted with the Traut reagent (2-iminothiolane hydrochloride) according to the following scheme:
  • AR-S is Traut's reagent
  • the addition of EDTA to the reaction helps prevent oxidation of the sulfhydryl group by preventing the formation of disulfide bonds.
  • the peptide binds to the nanoliposome by reaction of the sulfhydryl group with the maleimide molecule of the conjugate present in the nanoliposome and described above, according to the following scheme:
  • the peptide is modified with a cysteine molecule, so that the free -SH group is also left to react with the conjugate maleimide.
  • the nanoliposomes may further comprise other phospholipids that are not conjugated but free, such as those mentioned above.
  • these phospholipids are selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl- sn-glycerol-3-phosphocholine (DPPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof.
  • these phospholipids are 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) represented in the formula (I), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) represented in the formula (II) or any of its salts, derivatives or analogs, or any of its combinations of the compounds of formula (I) and (II).
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine
  • the nanoliposomes comprise 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG).
  • the nanoliposomes of the invention comprise, in addition to the lipid-spacer-maleimide conjugate and the modified peptide, 1,2-distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG), 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC).
  • DSPE-PEG 1,2-distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)]
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine
  • the nanoliposomes of the invention comprise:
  • the peptide is covalently bound to the nanoliposome through the reaction between the maleimide of the conjugate and the sulfhydryl group of the modified peptide.
  • the peptide is the VIP peptide modified with a cysteine molecule.
  • the DPPC and the DSPC are in a mass ratio of approximately 6: 4.
  • the DSPE-PEG and the DSPE-PEG-Maleimide conjugate are at a final concentration of about 5 and 10%, respectively.
  • the lipid bilayer of the nanoliposomes may also contain one or more sterols such as cholesterol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, stigmasterol, sitosterol, campesterol and brasicasterol, sugars such as glycerol and sucrose, esters of glycerin fatty acids such as triolein and trioctanoin.
  • antioxidant substances such as tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene, compounds that provide positive charge such as stearylamine and oxylamine, compounds that provide negative charge such as di-methyl phosphate, membrane proteins.
  • the invention relates to a process for the preparation of the nanoliposomes defined above, comprising:
  • lipid film for example by reverse phase evaporation, of a compound comprising phospholipids, preferably
  • DSPC and DPPC and at least one pegylated phospholipid
  • the lipid film of step (a) comprises a lipid-spacer-maelimide conjugate as previously defined; More preferably, the lipid-spacer-maleimide conjugate is in a proportion of about 10% by weight with respect to the total weight of the lipid film.
  • the lipid film further comprises at least one phospholipid and / or at least one phospholipid conjugated to a PEG molecule.
  • the lipid film further comprises 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2- dipalmitoyl-sn-glycer-3-phosphocholine (DPPC) and 1, 2- distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG).
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DPPC 2- dipalmitoyl-sn-glycer-3-phosphocholine
  • DSPE-PEG 2- distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)]
  • the mass ratio of the DPPC: DSPC phospholipids is approximately 6: 4.
  • the proportion of DSPE-PEG is around 5% by weight with respect to the total weight of the lipid film.
  • the buffer solution is a solution of HEPES (4- (2-hydroxyethyl) -1 -piperazinethanesulfonic acid) of pH 7.4.
  • the organic solvent is a mixture of diethylether and chloroform in an approximate ratio of 1: 1 by volume. More preferably, the volume ratio of organic phase: aqueous phase is about 4: 1.
  • step c) is performed in a sonic bath until the liposomes form spontaneously and the sample appears dispersed in a single phase.
  • this sonication stage is carried out for 3 to 5 minutes.
  • the removal of the organic solvent is carried out by evaporation, for example with rotary evaporator, at a temperature between 35 and 55 ° C, more preferably at 40 ° C, and at a rotation speed between 100 and 150 rpm, more preferably at 120 rpm.
  • the sonication of step (f) is performed in a sonic bath at a temperature higher than that of its transition phase, preferably at more than 60 ° C, to control the size of the nanoliposome.
  • the nanoliposome is adjusted to an average size between 75 and 125 nm, preferably between 85 and 1 nm, more preferably about 95-105 nm.
  • the functionalization of the nanoliposome with the peptide is carried out by adding at least 100 ⁇ g of the modified peptide, more preferably at least 125 ⁇ g and even more preferably at least 150 ⁇ g, to the sample of Nanoliposomes kept under stirring and room temperature for more than 10 hours, and more preferably between 12 and 16 hours.
  • the peptide is dissolved in a buffer solution at a pH of around 6-6.5.
  • Said peptide is modified with a sulfhydryl group at one end thereof.
  • the maleimide present in the lipid-spacer-maleimide conjugate reacts with the peptide through the sulfhydryl -SH group.
  • Said reaction is preferably carried out under nitrogen bubbling for at least one hour to prevent sulfhydryl groups from being oxidized.
  • the sample is incubated at room temperature and with stirring.
  • excess mercaptoethanol or free cysteine can be added, subsequently dialyzing to remove the excess of unreacted ⁇ -mercaptoethanol and peptide.
  • the peptide is a neuropeptide and even more preferably it is the VIP peptide.
  • the nanoliposomes obtained after step (f) can be purified by, for example, a Sephadex column, preferably G-50.
  • the functionalized nanoliposomes obtained after step (g) can be purified by, for example, dialysis against deionized water for at least 15 hours, preferably for at least 20 hours, and much more preferably for 24 hours.
  • nanoliposomes of the invention can also incorporate an active ingredient.
  • active ingredient means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body. of man or other animals.
  • the term includes those components that promote a chemical change in processing of the drug and are present therein in a modified form intended to provide the specific activity or effect.
  • the active substance can be both hydrophilic and hydrophobic. In the first case it is incorporated into the aqueous nucleus of the liposome, while in the second case it is incorporated into the lipid bilayer of the nanoliposome.
  • the active ingredient has an antitumor activity. In another preferred embodiment of this aspect of the invention, the active ingredient has anti-inflammatory activity. More preferably, the active substance is doxorubicin.
  • the encapsulation of the active substance in the nanoliposome can be performed using the osmotic gradient technique as described in the experimental part.
  • the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising a nanoliposome as previously defined and an active ingredient capable of diagnosing, curing, mitigating, treating or preventing a disease.
  • the composition further comprises a pharmaceutically acceptable carrier. More preferably, the pharmaceutical composition of the invention further comprises another active ingredient.
  • compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art.
  • they can be, without limitation, in sterile aqueous solution or in biological fluids, such as serum.
  • Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals.
  • compositions may be combined with various inert vehicles or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch, etc.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch, etc.
  • compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, ... of the mammal.
  • the term “therapeutically effective amount” refers to the amount of active ingredient, or its salts, prodrugs, derivatives or analogs, or combinations thereof, that produce the desired effect and, in general, It will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogues and the therapeutic effect to be achieved.
  • the "adjuvants” and “pharmaceutically acceptable carriers” that can be used in said compositions are the vehicles known to those skilled in the art.
  • excipient refers to a substance that aids the absorption, distribution or action of any of the active ingredients of the present invention, stabilizes said active substance or aids in the preparation of the medicament in the sense of giving it consistency or providing flavors. Make it more enjoyable.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
  • pharmaceutically acceptable excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
  • the excipient must be pharmaceutically suitable, that is, an excipient that allows the activity of the active ingredient or of the active ingredients, that is, that is compatible with the active ingredient, in this case, the active ingredient is any of the compounds of the present invention.
  • a “pharmaceutically acceptable carrier” refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.
  • the vehicle like the excipient, is a substance that is used in the medicament to dilute any of the compounds of the present invention to a certain volume or weight.
  • the pharmaceutically acceptable carrier is an inert substance or action analogous to the active ingredients of the present invention.
  • the function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition.
  • the pharmaceutically acceptable carrier is the diluent.
  • Another aspect of the invention relates to the use of the nanoliposome of the invention, in the preparation of a medicament for the treatment of diseases that occur with cell proliferation.
  • the disease that occurs with cell proliferation is selected from the list comprising: malignant epithelial neoplasms, lung cancer and other cancers such as stomach, colon, breast, prostate, liver and urinary bladder.
  • the disease that occurs with cell proliferation is prostate cancer.
  • a lipid film was obtained by reverse phase evaporation of a mixture containing 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2- dipalmitoyl-sn-glycerol-3-phosphocholine ( DPPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanol amine-N- [amino (polyethylene glycol)] (DSPE-PEG) and 1,2-distearoyl-sn-glycerol-3- phosphoethanolamine-N- [amino (polyethylene glycol) -maleimide] or (DSPE-PEG-maleimide).
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DPPC 1, 2- dipalmitoyl-sn-glycerol-3-phosphocholine
  • DSPE-PEG 1,2-distearoyl-sn-glycerol-3-phosphoethanol amine-N
  • the film formed was rehydrated with a HEPES buffer solution at pH 7.4 and a mixture of diethyl ether and chloroform was added in a 1: 1 volume ratio, such that the volume ratio of the organic phase: aqueous phase was 4 :one .
  • a two-phase system was formed.
  • This system was subjected to a sonication stage for about 3-5 minutes until the sample appeared dispersed in a single phase.
  • the organic solvent was removed by rotoevaporation at a temperature of 40 ° C and at a speed of 120 rpm, until a gel phase was reached. Then the gel became an aqueous suspension where the liposomes were dispersed.
  • liposomes were subjected to an additional stage of sonication to reduce their size and thus convert them into nanoliposomes.
  • the sonication was performed in a sonic bath at a temperature above 60 ° C.
  • the nanoliposomes were purified using a Sephadez G-50 column.
  • the functionalization of the nanoliposomes with the VIP was carried out by adding 150 ⁇ g of said modified peptide with a cysteine molecule on the aqueous suspension of the nanoliposomes, while stirring at room temperature for 12 hours.
  • the nanoliposomes were purified by dialysis against deionized water for 24 hours.
  • the surface VIP was quantified using a commercial system called LavaPep - peptide quantification kit TM (Fluorotechnics) based on the fluorescence detection of the peptide / fluorophore complexes. The results related to this quantification are shown in Figure 2.
  • the functionalized nanoliposomes were characterized by their size using the Dynamic Light Scattering (DLS) technique, obtaining values of around 100 nm as they were shown in figure 3.
  • DLS Dynamic Light Scattering
  • Example 2 Specificity and effectiveness of nanoliposomes functionalized with VIP in the specific recognition of cells expressing their receptors.
  • CAMP production was measured by a bioluminescence assay using the Promega cAMP-GLO TM assay according to the manufacturer's instructions. This assay is based on the decrease in luciferase-coupled light production when increased concentrations of cAMP produce a protein activation. kinase A and a concomitant decrease in ATP available for the detection reaction indicated above.
  • Figure 4 shows the results obtained in cells and demonstrates a specifically increased cAMP production after stimulation of the cells with the functionalized nanoliposomes.
  • Doxorubicin encapsulation is performed using the osmotic gradient technique, in which two different pH buffers are used.
  • the first consists of 250 mM ammonium sulfate pH 5.5, which is the aqueous phase in the synthesis of reverse evaporation, so this buffer is inside and outside the nanonanoliposome.
  • the nanonanoliposome sample is passed through a column of Sephadex G-50 previously washed with saline HEPES buffer pH 7.4 to collect the nanoliposomes dispersed in this same buffer. This difference in osmolarity inside and outside the nanoliposome creates an osmotic gradient across the membrane.
  • doxorubicin dissolved in water is added to the nanoliposome sample and allowed to incubate for 90 minutes at a temperature higher than the transition, in our case at 60 ° C, since the transition temperature of our nanoliposome It is 46 ° C. This temperature causes the nanoliposomes to become more fluid, gaps form in their membrane, and doxorubicin penetrates. The movement of water into the nanoliposomes is due to the osmotic gradient and the gaps in the membrane that facilitates the encapsulation of doxorubicin and allows to reach encapsulation efficiency values close to 90%.
  • the sample is subsequently purified by means of a Sephadex G-50 column to eliminate unincorporated doxorubicin and the encapsulation efficiency is measured by the fluorescence spectrophotometer, measuring the sample before and after being purified and also after adding 1% triton (In this way the nanoliposome is broken and the drug inside is released).
  • Encapsulation efficiency (A-B / C) X 100
  • A Intensity of the purified sample after adding 1% triton.
  • B Intensity of the purified sample without 1% triton.
  • DU-145 cells (human prostate cancer cell line) were treated for 48 hours with soluble doxorubicin, with doxorubicin encapsulated in liposomes and with doxorubicin encapsulated in VIP-functionalized nanoliposomes.
  • Figure 5 shows the optical microscopy photographs obtained in all cases and their comparison with a sample of untreated cells. The increased cytotoxic effect of doxorubicin is observed in the presence of nanoliposomes functionalized with the VIP peptide.
  • LDH lactate dehydrogenase

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Abstract

La présente invention concerne des nanoliposomes fonctionnalisés avec des peptides bioactifs au niveau de leur surface qui permettent d'effectuer l'identification de tissus et/ou de cellules cible et la libération de médicaments de manière sélective et en particulier au moyen de nanoliposomes fonctionnalisés avec le peptide VIP. La présente invention porte également sur des compositions pharmaceutiques qui comprennent lesdits nanoliposomes, sur un procédé de préparation de ces derniers ainsi que sur leurs utilisations médicales.
PCT/ES2012/070037 2011-01-24 2012-01-24 Nanoliposomes fonctionnalisés avec des peptides WO2012101309A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
ES201130072A ES2389347B1 (es) 2011-01-24 2011-01-24 Nanoliposomas funcionalizados con peptidos.
ESP201130072 2011-01-24
ES201130185A ES2390147B1 (es) 2011-02-11 2011-02-11 Nanoliposomas funcionalizados con péptidos bioactivos como sistemas para mejorar la citotoxicidad de fármacos antitumorales.
ESP201130185 2011-02-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014167126A3 (fr) * 2013-04-13 2015-01-08 Universidade De Coimbra Plateforme pour l'administration ciblée à des cellules souches et des cellules tumorales et ses procédés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009142525A2 (fr) * 2008-05-22 2009-11-26 Universidade De Coimbra Administration ciblée à destination d'affections et troubles humains

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Publication number Priority date Publication date Assignee Title
WO2009142525A2 (fr) * 2008-05-22 2009-11-26 Universidade De Coimbra Administration ciblée à destination d'affections et troubles humains

Non-Patent Citations (3)

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Title
DAGAR, S. ET AL.: "VIP grafted sterically stabilized liposomes for targeted imaging of breast cancer: in vivo studies", JOURNAL OF CONTROLLED RELEASE., vol. 91, no. 1-2, 28 August 2003 (2003-08-28), pages 123 - 133 *
NALLAMOTHU, R. ET AL.: "A tumor vasculature targeted liposome delivery system for Combrestatin A4: design, characterization and in vitro evaluation", AAPS PHARM SCI TECH., vol. 7, no. 2, 2006, pages E1 - E10 *
SCHIFFELERS, R.M. ET AL., ANTI-TUMOR EFFICACY OF TUMOR VASCULATURE-TARGETED LIPOSOMAL DOXORUBICIN, vol. 91, no. 1-2, 28 August 2003 (2003-08-28), pages 115 - 122 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014167126A3 (fr) * 2013-04-13 2015-01-08 Universidade De Coimbra Plateforme pour l'administration ciblée à des cellules souches et des cellules tumorales et ses procédés

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