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WO2012066171A1 - Nanoparticules destinées à prévenir et/ou à traiter des maladies des muqueuses - Google Patents

Nanoparticules destinées à prévenir et/ou à traiter des maladies des muqueuses Download PDF

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Publication number
WO2012066171A1
WO2012066171A1 PCT/ES2011/070780 ES2011070780W WO2012066171A1 WO 2012066171 A1 WO2012066171 A1 WO 2012066171A1 ES 2011070780 W ES2011070780 W ES 2011070780W WO 2012066171 A1 WO2012066171 A1 WO 2012066171A1
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Prior art keywords
nanoparticles
mucosa
muc5ac
disease
mucin
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English (en)
Spanish (es)
Inventor
Jenny PÁRRAGA MENESES
Giovanni Konat Zorzi
Patrizia Paolicelli
Begoña Seijo Rey
Alejandro SÁNCHEZ BARREIRO
Laura Contreras Ruiz
Yolanda Diebold Luque
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Universidade de Santiago de Compostela
Universidad de Valladolid
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Universidade de Santiago de Compostela
Universidad de Valladolid
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Publication of WO2012066171A1 publication Critical patent/WO2012066171A1/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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/60—Vectors comprising a special origin of replication system from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention is within the field of biotechnology, and more specifically of biomedicine. Specifically, it refers to nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end, or a plasmid or a genetic construct containing it, and its use for the elaboration of a medicine for the prevention and / or treatment of a disease that has a mucin deficiency.
  • the mucous membranes of the organism are one of the most sensitive elements to both inflammatory and infectious conditions. Within these mucous membranes, one of the most important elements are the mucins. These mucins are a heterogeneous group of high molecular weight glycoproteins and are the fundamental element of the body's mucous secretions. Within this group there are described at least 17 different proteins with different characteristics of both expression and functional.
  • mucins are especially relevant in the case of the ocular mucosa, where the mucins have high importance in the homeostasis of the mucous tissue, as well as in the lubrication of both corneal and conjunctival epithelia during flickering.
  • the mucins act at the ocular level as stabilizers of the tear film preventing the desiccation of the epithelium and as a barrier against the entry of both foreign agents and pathogens. Due to the importance of these mucins, the deficiency in them carries several ocular pathological problems.
  • ocular surface which is considered an established functional unit (Stern et al, 1998. Cornea. 17: 584-589).
  • This ocular surface is a mucous tissue similar to other mucous membranes in the body such as the mucosa of the gastro-intestinal tract, the mucosa of the respiratory tract, the urinary mucosa and the vaginal mucosa.
  • diseases that occur with alteration in the regulation of mucin production are, for example, autoimmune diseases that occur with inflammation. In these cases it can lead, depending on the disease, an overproduction or a subproduction of the mucins characteristic of the ocular surface. This variation of the levels of the mucins, cause that the homeostasis produced by them does not occur and therefore gives rise to serious pathological processes that can influence the visual function.
  • mucin MUC5AC which is specific to the goblet cells of the conjunctiva.
  • the production of mucin by these cells is strongly diminished (up to 90% reduction) in various eye diseases such as for example keratoconjunctivitis sicca, Sjógren's syndrome, pemphigoid, Stevens Johnson's syndrome (Kunert et al, 2001. Invest Ophthalmol Vis Sci. 42: 2483-2489), in atopic patients with corneal ulcers (Dogru et al, 2005. Cornea 24: S18-S23; Dogru et al, 2005. Curr Eye Res.
  • chitosan is a polymer cited as essential in the formation of nanoparticles by ionic crosslinking. Chitosan nanoparticles are being questioned as there are studies that question their usefulness against simple solutions of bioactive molecules with the polymer (Dyer et al, 2002. Pharm Res. 19: 998-1008). In addition, the possible cytotoxicity of chitosan nanoparticles has also been noted (Loretz and Bernkop-Schnürch, 2007. Nanotoxicology. 1: 139-148).
  • the present invention relates to nanoparticles useful for the preparation of a medicament that allows the recovery of mucin production.
  • Said nanoparticles are associated with a polynucleotide encoding a modified MUC5AC protein, a genetic construct or a vector containing said polynucleotide.
  • This composition allows the recovery of mucin production, so it is useful for the development of a drug for the treatment of diseases of the associated mucous membranes to a mucin deficiency.
  • the present invention demonstrates how the inventors have developed a plasmid that includes a polynucleotide capable of encoding a mucin, which can be associated with nanoparticles.
  • the nanoparticles act as vehicles of the genetic material, without presenting cytotoxic activity, and allow the cells to produce the mucin encoded by the plasmid.
  • the present invention demonstrates the ability of the composition to recover tear production in mice with induced dry eye, demonstrating the usefulness of said composition for the development of medicaments for the treatment of mucosal diseases that occur with mucin deficiency .
  • the composition of the present invention would be used for the treatment of mucosal diseases that occur with mucin deficiency.
  • a plasmid has been associated that integrates a sequence encoding the modified MUC5AC protein (whose sequence is SEQ ID NO: 1), which corresponds to the C and N terminal ends of the native MUC5AC protein.
  • This nucleotide sequence (SEQ ID NO: 2) coding for the modified mucin is substantially shorter (7.7 kilobases) than the coding sequence of the complete mucin (16 kilobases), which has the advantage of greater ease of cloning in plasmids, genetic constructs or vectors.
  • This shorter coding sequence encodes the MUC5AC mucin that has fused the C-terminal and N-terminal ends, and that maintains the most important domains of the original protein such as the dimerization domain (present at the C-terminal end ), the polymerization domain, some of the different glycosylation points in the complete protein, and the signal peptide for its correct conduction through the secretory pathway.
  • This protein therefore, has the N-terminal end of the MUC5AC (amino acids 1-2488) attached to the dimerization domain present at the C-terminal (amino acids 5.220-5.333), eliminating the central region where highly repeated sequences are found. rich in proline, threonine and serine (PTS).
  • said modified MUC5AC protein is useful for treating diseases that occur with mucin deficiency such as dry eye syndrome.
  • a first aspect of the invention relates to polymeric nanoparticles, hereinafter nanoparticles of the invention, comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end.
  • modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end refers to a protein that has the N-terminal end of the MUC5AC that includes both the polymerization domain, as the signal peptide for its correct conduction by the secretory pathway, with the dimerization domain of the C-terminal end of the MUC5AC protein.
  • An example of a modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end is, for example, but not limited to, the sequence protein SEQ ID NO: 1, which has the amino acids 1-2488 of the MUC5AC protein (corresponding to the N-terminal end) attached to amino acids 5,220-5,333 (corresponding to the dimerization domain of the C-terminal end).
  • SEQ ID NO: 1 which has the amino acids 1-2488 of the MUC5AC protein (corresponding to the N-terminal end) attached to amino acids 5,220-5,333 (corresponding to the dimerization domain of the C-terminal end).
  • a polypeptide could have variations in the amino acid sequence with respect to that described by way of example without a substantial alteration in the protein. This means that this variation would not be determinant in the polypeptide neither for its structure nor for its activity.
  • variations are generated by substitutions, deletions or additions.
  • substitutions occur between amino acids that have similar characteristics such as polarity, size or charge, and include, but are not limited to, substitutions between glutamic acid (Glu) and aspartic acid (Asp), between Lysine (Lys) and Arginine (Arg), between asparagine (Asn) and glutamine (Gln), between serine (Ser) and threonine (Thr), and between the amino acids that make up the alanine (Ala), leucine (Leu), valine (Val) and isoleucine group (lie)
  • the variations can be artificially generated variations such as by mutagenesis or direct synthesis.
  • the modified MUC5AC protein having the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end has at least 90%, preferably 95 %, more preferably 98% identity of with respect to SEQ ID NO: 1.
  • the modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end is SEQ ID NO: 1.
  • a polynucleotide that codes for a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end would be any polynucleotide that codes for a protein or polypeptide included under the designation " modified MUC5AC protein presenting the N- end MUC5AC terminal linked to the dimerization domain located at the C-terminal end.
  • nucleotide sequence refers to a polymeric form of nucleotides of any length that may be or no, chemically or biochemically modified. They therefore refer to any polyiribonucleotide or polydeoxyribonucleotide, both chain s match like double strand.
  • the polynucleotide of the invention can be found in nature and obtained by isolation by methods known in the state of the art, or obtained artificially by conventional cloning and selection methods, or by sequencing.
  • the polynucleotide in addition to the coding sequence, can carry other elements such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides can additionally also include coding sequences for additional amino acids that may be useful, for example, but not limited to increasing the stability of the peptide generated from it or allowing a better purification thereof.
  • the polynucleotide has at least 90%, preferably 95%, more preferably 98% identity with respect to SEQ ID NO: 2. In a more preferred embodiment the polynucleotide is SEQ ID NO: 2.
  • peptide refers to a Polymeric form of amino acids of any length, which may or may not be chemically or biochemically modified.
  • identity refers to the proportion of identical nucleotides or amino acids between two nucleotide or amino acid sequences that are compared. Sequence comparison methods are known in the state of the art and include, but are not limited to, the GAG program, including GAP (Devereux et al. 1984, Nucleic Acids Research 12: 287 Genetics Computer Group University of Wisconsin, Madison , (Wl); BLAST or BLASTN, and FASTA (Altschul et al. 1999, J. Mol. Biol. 215: 403-410) Additionally, the Smith Waterman algorithm should be used to determine the degree of identity of two sequences.
  • the polynucleotide encoding the modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end may be included in gene constructs or vectors. Therefore, a preferred embodiment of the first aspect of the invention relates to nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end, where the polynucleotide It is included in a genetic construct or vector.
  • the vector in which the polynucleotide is included is a plasmid.
  • control sequence refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked.
  • control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous Examples of control sequences are, for example, but not limited to, promoters, transcription initiation signals, transcription termination signals, polyadenylation signals or transcriptional activators.
  • operably linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended manner.
  • a control sequence "operably linked" to a polynucleotide is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • promoter refers to a region of DNA, generally “upstream” or “upstream” of the transcription start point, which is capable of initiating transcription in a cell.
  • This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters. Control sequences depend on the origin of the cell in which the nucleic acid is to be expressed. Examples of prokaryotic promoters include, for example, but not limited to, promoters of the trp, recA, lacZ, lacl, tet, gal, trc, or tac genes of E. coli, or the promoter of the ⁇ -amylase gene of B.
  • subtilis For the expression of a nucleic acid in a prokaryotic cell, the presence of an upstream ribosomal binding site of the coding sequence is also necessary.
  • Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.
  • Both the polynucleotide of the invention and the genetic construct of the invention comprising the polynucleotide can be introduced, for example, into a cloning vector or an expression vector to allow replication or expression.
  • said vector is an appropriate vector for expression of the peptide of the invention.
  • cloning vector refers to a DNA molecule in which another DNA fragment can be integrated, without losing the capacity for self-replication.
  • expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes.
  • expression vector refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a host cell. Said nucleic acid is generally operatively linked to control sequences.
  • expression refers to the process by which a polypeptide is synthesized from a polynucleotide. It includes transcription of the polynucleotide into a messenger RNA (mRNA) and the translation of said mRNA into a protein or polypeptide. Expression can take place in a host cell, but also by any process of protein expression in vivo.
  • mRNA messenger RNA
  • the nanoparticles can be formed by an ionic interaction mechanism that causes the joint precipitation of the components in the form of nanoclusters as a result of the addition of a cross-linking agent that has an electric charge.
  • the ionic interaction procedure has advantages that do not require the use of chemical elements other than the components of the nanoparticles themselves that can cause toxicity or incompatibility of the system.
  • the use of an ionic crosslinking agent allows the cross-linking of the ionic polymer giving rise to the nanoparticles. This procedure is a fast and reliable, economical and easily scalable process for industrial production.
  • the nanoparticles are formed by:
  • polymers useful for the formation of nanoparticles are, for example, but not limited to, hyaluronic acid or salts thereof, colominic acid or derivatives, glucomannan or derivatives, chondroitin sulfate, queratane sulfate, dextran sulfate, dermatan sulfate, heparin, carrageenan, gellan, albumin, collagen or gelatin.
  • These polymers also include polymers on which modifications have been made such as enzymatic fragmentation, chemical fragmentation or derivatization by, for example, but not limited to, the anionization or cationization of the polymer prior to the preparation of the particles.
  • polymers of (i) can have a natural, semi-synthetic or synthetic origin. It is interesting that the polymers used are natural or semi-synthetic polymers since this allows to minimize the possible rejection of the treated individuals to the nanoparticles. Therefore, in a more preferred embodiment of this aspect of the invention, the polymer of (i) is a natural or semi-synthetic polymer.
  • the naturally occurring polymer with an electric charge (i) is cationized gelatin.
  • ionic crosslinking agents are, for example, but not limited to, phosphates, sulfates, citrates or salts thereof, spermine or salts thereof, or spermidine or salts thereof. These agents have different electrical charges (phosphates, sulfates and citrates anionic charge; spermine and spermidine cationic charge). Among these, one of the best characterized and the best result is tripolyphosphates. Therefore, in another more preferred embodiment of this aspect of the invention, the ionic crosslinking agent (ii) is a tripol phosphate. In an even more preferred embodiment, the ionic crosslinking agent is sodium tripolyphosphate.
  • the nanoparticles may comprise an additional element, which is an ionic polymer of electric charge opposite to the first polymer of the nanoparticles.
  • an ionic polymer of electric charge opposite to the first polymer of the nanoparticles allows a better ionic gelation, and allows to modify the characteristics of the nanoparticles obtained both in size, as in structural characteristics and surface electric charge. Therefore, in a more preferred embodiment of the first aspect of the invention, the nanoparticles are further formed by an element (iii) which is at least one polymer of charge opposite to the polymer (i).
  • polymers may be, for example, but not limited to, hyaluronic acid or salts thereof, colominic acid or derivatives, glucomannan or derivatives, chondroitin sulfate, queratane sulfate, dextran sulfate, dermatan sulfate, heparin, carrageenan, gelane, albumin, collagen or gelatin.
  • the polymer (iii) of opposite charge to the polymer (i) is dextran sulfate or chondroitin sulfate.
  • the polynucleotide Due to the formation of the nanoparticles by ionic crosslinking, the polynucleotide, either independently or included in a vector or a gene construct, can be retained inside them by purely physical entrapment, due to the difference in its size and that of the mesh formed in said crosslinking process. On the other hand, due to the ionic character of the polymers used, either in (i) or in (iii), the polynucleotide can also be attached to the nanoparticles as a result of an electrostatic interaction between it and said polymers.
  • nanoparticle is understood as a stable system, with homogeneous reproducible and modulable characteristics, perfectly distinguishable from self-assembled systems, which are formed as a consequence of a controlled process of ionotropic cross-linking of the polymer provided with the constituent charge thereof mediated by ionic crosslinking agents.
  • the electrostatic interaction that results between the different components of the nanoparticles in the crosslinking process generates characteristic physical entities, which are independent and observable, whose average size is less than 1 ⁇ .
  • Average size in the present invention is understood as the average diameter of the nanoparticles, which can be measured using standard procedures known to a person skilled in the art, such as, but not limited to, the one used in the examples herein. invention.
  • the nanoparticles of the invention have an average size between 1 and 999 nm, preferably between 50 and 600 nm, and more preferably between 100 and 400 nm. This average particle size is influenced by their composition, as well as by the environmental conditions in which they are formed.
  • the nanoparticles can have different surface electric charges (potential Z) depending on their composition, and the proportions of each of the elements.
  • This charge may have positive, negative or 0 values, preferably, the electrical charge of the nanoparticles is between +50 mV and -50mV, more preferably between +45 and OmV, and even more preferably between +40 and + 10mV.
  • the measurement of the electric charge can be carried out by methods known in the state of the art, such as that used in the examples of the present invention.
  • the nanoparticles once they have formed and present the associated polynucleotide, can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes.
  • This lyophilization may slightly increase the crosslinking of the particles. Freeze-drying requires the prior addition of molecules that act as cryoprotectants or lioprotectors such as, but not limited to, glycols, or sugars such as glucose, sucrose or trehalose at a concentration of between 1 and 5%. Therefore, in another preferred embodiment, the nanoparticles of the invention are in lyophilized form.
  • Another aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament or alternatively to the nanoparticles of the invention for use as a medicament.
  • the nanoparticles of the invention have utility in recovering mucin production by body cells. Therefore, another aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, or alternatively to the nanoparticles. of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency.
  • mucin deficiency diseases that affect the ocular mucosa and / or other mucous membranes of the body, such as limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, burn thermal, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, infectious, immune and irritative-toxic-drug conjunctivitis, Sjógren Syndrome primary or secondary to other autoimmune diseases (such as scleroderma), rodermatitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, infectious, immune and irritative-toxic-drug conjunctivitis, Sj
  • nanoparticles comprising a plasmid carrying a polynucleotide encoding the modified MUC5AC protein presenting the N-terminal end of MUC5AC bound to the domain dimerization located at the C-terminal end, allows the expression of this mucin.
  • a preferred embodiment of this aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa, or alternatively to the nanoparticles for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa.
  • the nanoparticles of the invention allow the recovery of tear production in a disease that is deficient in mucins, and more specifically with a deficiency in the mucin MUC5AC. Therefore, another preferred embodiment relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency, where the mucin is MUC5AC, or alternatively to the nanoparticles for use as a medicine for the prevention and / or treatment of a mucosal disease that presents with a mucin deficiency, where the mucin is MUC5AC.
  • the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that is present with a mucin deficiency, where the mucosa is The ocular mucosa and the mucin is MUC5AC, or alternatively to the nanoparticles of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, where the mucosa is the mucosa eyepiece and the mucin is MUC5AC.
  • Another preferred embodiment relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency
  • the disease of a mucosa that has a Mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkaline burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid , eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, infectious, immune and irritative-toxic-drug conjunctivitis, primary or secondary Sjógren syndrome (such as scleroderma, mesenupus disease) rheumatoi
  • a more preferred embodiment of this aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency.
  • the disease of a mucosa that is present with a mucin deficiency is dry eye syndrome
  • the nanoparticles of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that courses with a Mucin deficiency, where the disease of a mucosa that has a mucin deficiency is dry eye syndrome.
  • compositions hereinafter pharmaceutical composition of the invention, comprising the nanoparticles of the invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises another active ingredient.
  • the pharmaceutical composition further comprises another active ingredient and a pharmaceutically acceptable carrier.
  • active substance means any component that potentially provides a pharmacological activity, a metabolic effect, an immunological effect or another different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of the human being or other animals.
  • the term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
  • compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including humans, in a variety of ways known in the state of the art. Thus, they can be, without being limited, in aqueous or non-aqueous solutions, in emulsions or suspensions.
  • non-aqueous solutions are, for example, but not limited to, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters, such as ethyl oleate.
  • aqueous solutions are, for example, but not limited to, water, alcoholic solutions in water, or saline media.
  • Aqueous solutions may be buffered or not, and may have additional active or inactive components.
  • Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamins and minerals.
  • preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamins and minerals.
  • nutrients including glucose, dextrose, vitamins and minerals.
  • the compositions can be prepared for administration in solid form.
  • compositions may be combined with various inert carriers 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; lubricants, such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint or methyl salicylate.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch
  • lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • flavoring agents such as peppermint or methyl salicylate.
  • the composition of the invention is especially advantageous for constituting
  • compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to the human being, in a variety of ways, including, but not limited to, oral, oral, sublingual, ocular, nasal, pulmonary, otic, intrauterine, parenteral, intraperitoneal, intravenous, intradermal, intrastromal, intraarticular, intrasinovial, intrathecal, intralesional, intraarterial, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, topical, through transdermal patches, rectal, vaginal, by administering a suppository, percutaneous, spray nasal, surgical implant, internal surgical paint, infusion pump or catheter.
  • the nanoparticles of the invention due to their usefulness in the mucous membranes, have an advantageous administration by different mucosal routes such as, but not limited to, ocular mucosa, mucosa of the entire gastro-intestinal tract including the buccal or oral, mucosa of the respiratory tract , including the nasal and bronchial mucosa, the urinary mucosa and the vaginal mucosa. Therefore, a preferred embodiment of this aspect of the invention relates to a pharmaceutical composition comprising the nanoparticles of the invention for administration via ocular mucosa, mucosa of the entire gastro-intestinal tract, mucosa of the respiratory tract, urinary mucosa and the vaginal mucosa.
  • the nanoparticles can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes. Therefore, in another preferred embodiment the pharmaceutical composition is in lyophilized form.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the mammal.
  • the term "therapeutically effective amount” refers to the amount of the pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said pharmaceutical composition and the therapeutic effect to be achieved.
  • compositions are the vehicles known in the state of the art.
  • Another aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament or alternatively to the pharmaceutical composition of the invention for use as a medicament.
  • Another aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, or alternatively the pharmaceutical composition. of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency.
  • a preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, wherein the Mucosa is the ocular mucosa, or alternatively to the pharmaceutical composition for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa.
  • Another preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, wherein the Mucin is MUC5AC, or alternatively to the pharmaceutical composition for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucin is MUC5AC.
  • the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency, where the mucosa it is the ocular mucosa and the mucin is MUC5AC, or alternatively to the pharmaceutical composition of the invention for use as a medicine for the prevention and / or treatment of a disease of a mucosa that is present with a mucin deficiency, where the mucosa is the ocular mucosa and the mucin is MUC5AC.
  • Another preferred embodiment relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency
  • the disease of a mucosa that has a a mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, membrane pemphigoid mucous membranes, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, keratoconjunctivitis sicca or dry eye syndrome, infectious, immune and irritative-toxic-medicative conjunctivitis, primary or secondary Sjógren's syndrome to other autoimmune diseases (such as scleroderma, mesenchyderma
  • a more preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency where the A mucosal disease that occurs with a mucin deficiency is dry eye syndrome, or alternatively to the pharmaceutical composition of the invention for use as a medicament for the prevention and / or treatment of a mucosal disease that courses with a deficiency of mucin, where the disease of a mucosa that has a mucin deficiency is dry eye syndrome.
  • Another aspect of the present invention relates to a process for the preparation of nanoparticles comprising:
  • This production method is a fast, economical, reproducible and scalable method, which allows its industrial implementation.
  • the absence of chemical elements other than the constituents of the nanoparticles, as well as the use of mild conditions for production allows greater safety of the resulting particles.
  • the DNA molecule In order to carry out the association of the DNA molecule, it is dissolved in the dissolution of the polymer or the crosslinking agent, depending on the electrical charge of each of these components. The DNA is incorporated into the solution that has a negative charge.
  • a polymer with a charge opposite to that of step (1) could be incorporated, either in step (3), or subsequently after forming the nanoparticles.
  • the incorporation of the polymers is carried out by means of an aqueous solution thereof at a concentration between 0.1 and 6 mg / mL, preferably between 0.1 and 5 mg / mL.
  • the crosslinking agent is incorporated by an aqueous solution thereof at a concentration of between 0.0625 and 1.5 mg / mL, preferably between 0.25 and 1.5 mg / mL.
  • the nanoparticles After the formation of the nanoparticles, they can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes. This lyophilization can increase the crosslinking of the particles.
  • the prior addition of molecules that act as cryoprotectants or lioprotectors is necessary, for example, although sugars such as glucose, sucrose or trehalose are not limited to a concentration of between 1 and 5%.
  • FIG. 1 Scheme of plasmids A: plRES2-AcGFP1-MUC5AC-280; and B: pMUCIN5AC-29.
  • MA0151 and MA0153 primers; PMCMV: promoter of cytomegalovirus; pUC ori: origin of pUC replication; HSV TK poly A: thymidine kinase polyadenylation signal from herpex simplex virus; KanR / NeoR: resistance to kanamycin and neomycin; SV40 ori / p: SV40 origin of replication; f1 ori: origin of replication f1; AcGFPI: Aequorea coerulescens green fluorescent protein; IRES: internal ribosome entry site; MUC5AC ORF: reading frame of MUC5AC; SacI: cut-off point of the SacI restriction enzyme; EcoRV: cut-off point of the EcoRV restriction enzyme.
  • Figure 2 Image of agarose gel. Results of the digestion of 0.5 microliters of the preparation of pMUCIN5AC-29 with the SacI and EcoRV enzymes and their respective negative controls (-) without digesting with the enzyme.
  • FIG. 1 Morphological characterization of nanoparticles by transmission electron microscopy.
  • A GCspm137 nanoparticles: TPP;
  • B GCspm137: CS: TPP nanoparticles;
  • C GCspm137 nanoparticles: DS: TPP.
  • Figure 4 Image of the 1% agarose electrophoresis gel loaded with: (A) Free plasmid pMUC5AC-280; (B) GCspm137: TPP nanoparticles, (C) GCspm137: CS: TPP nanoparticles; (D) GCspm nanoparticles: DS: TPP.
  • FIG. 1 Cell viability values obtained using the XTT test in A: human cornea cells (HCE) or B: conjunctiva (IOBA-NHC).
  • C Control not treated;
  • C + positive control of cell death (treated with 1% benzalkonium chloride solution);
  • FIG. 6 Expression of MUC5AC relative to the control, at the mRNA level in 72h post-transfection cells in A: human cornea cells (HCE) or B: human conjunctiva cells (IOBA-NHC).
  • Control Control not treated;
  • Figure 8 Production of tear in animals not subjected to induction of dry and untreated eye (Control), in animals with induced dry eye and untreated (Dry Eye Control), in animals with induced dry eye and treated with free pMUC5AC (Eye Dry + free pMUC5AC), in animals with induced dry eye and treated with white nanoparticles (without associated plasmid) (Dry Eye + white NPs), in animals with induced dry eye and treated with nanoparticles associating pMUC5AC (Dry Eye + pMUC5AC-NPs) and in animals with induced dry eye and treated with commercially available formulation for the treatment of dry eye (Dry Eye + Restasis®).
  • the nanoparticles have been characterized in terms of size, zeta potential (or surface charge) and encapsulation efficiency.
  • the determination of the isoionic or isoelectric point is to measure the concentration of hydrogen ions of a solution of the polymer that has been demineralized by contact with ion exchange resins (Commission on Methods for Testing Photographic Gelatin. PAGI METHOD 10th Ed. 2006).
  • the process consists in placing a 1% solution of the cationized protein in contact with a mixture of cationic acid and anionic basic resins in 1: 2 proportions.
  • the Zeta particle potential has been determined by the laser dispersion anemometry (LDA) technique and using a Zeta Sizer (Zeta Sizer, Nano series, Nano-ZS, Malvern Instruments, UK). For this, the samples were conveniently diluted in a millimolar solution of KCI.
  • TEM transmission electron microscopy
  • the efficiency of association of genetic material with nanoparticles has been determined by the agarose gel electrophoresis technique.
  • a 1% agarose gel was prepared in TAE buffer (Tris-Acetate-EDTA, 40mM Tris, 1% acetic acid, 1 mM EDTA) pH 8 with ethidium bromide (10 mg / mL, 5 ⁇ _) and used a loading buffer and migration marker composed of glycerin (30%).
  • a potential difference of 100 V was applied for 120 minutes and free genetic material was used as a control.
  • the Picogreen® method (Invitrogen, ES) was used, in accordance with the manufacturer's instructions, in supernatants of formulations previously centrifuged in Microfuge 22R centrifuge (Beckman Coultern, US ) at 12,000 rpm at 4 ° C for 30 minutes.
  • Two cell lines were used for cell culture experiments: human corneal HIT (Human Corneal Epithelium) and human IOBA-NHC (Normal Human Conjunctive) conjunctiva cells.
  • HCE cells were cultured in DMEM / F-12 medium supplemented with 15% fetal bovine serum (FBS), 0.5% DMSO, cholera toxin (1 mg / mL), endothelial growth factor (EGF; 100 ⁇ g / mL), insulin (4 mg / mL), streptomycin (0.1 mg / mL) and penicillin (100 U / mL) (Invitrogen, ES).
  • FBS fetal bovine serum
  • DMSO cholera toxin
  • EGF endothelial growth factor
  • insulin 4 mg / mL
  • streptomycin 0.1 mg / mL
  • penicillin 100 U / mL
  • IOBA-NHC cells were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS), cholera toxin (1 mg / mL), endothelial growth factor (EGF; 2 ng / mL), insulin ( 10 mg / mL), hydrocortisone (50 ⁇ g / mL), fungizone (250 ⁇ g / mL), streptomycin / penicillin (5000 U / mL) (Invitrogen, ES). The cells were maintained at 37 ° C under a humidified atmosphere of 5% CO2.
  • FBS fetal bovine serum
  • cholera toxin 1 mg / mL
  • EGF endothelial growth factor
  • insulin 10 mg / mL
  • hydrocortisone 50 ⁇ g / mL
  • fungizone 250 ⁇ g / mL
  • streptomycin / penicillin 5000 U / mL
  • the absorbance measurement was carried out at 450 nm with the reference wavelength of 620 nm, in a SpectraMax M5 plate multilector (Molecular Devices, US), expressing the results in% of viability relative to the control.
  • RT-PCR was used, using the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) gene as a reference .
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • the mRNA was extracted and purified with RNeasy Mini Kit (Qiagen, Germany) and quantified with Quant-it RNA Assay Kit (Invitrogen, US) according to the manufacturer's instructions.
  • the cDNA was constructed with SuperScript® Villo cDNA Synthesis Kit (Invitrogen, US) following the manufacturer's instructions.
  • the parameters of the RT-PCR were: an initial denaturation step at 95 ° C for 10 minutes; a second denaturation and extension step, with 40 cycles, from 15 seconds at 95 ° C and 60 seconds at 60 ° C, using 7500 RT-PCR System (Applied Biosystems, US).
  • the MUC5AC was quantified by ELISA.
  • Analog B of the MUC5AC (AnaSpec, US) was used as standard. For this, 50 [L cell lysates were incubated in 96-well plates with 50 ⁇ of 0.05M bicarbonate buffer pH 9.6 at 37 ° C over night.
  • the plates were washed three times with PBS and blocked with 2% bovine serum albumin (BSA fraction V) (Sigma-Aldrich, Spain) for 1 h at room temperature.
  • BSA fraction V bovine serum albumin
  • the plates were again washed three times with PBS and incubated with 100 ⁇ of anti-MUC5AC mouse antibody (Chemicon, US) (1: 500) which was diluted in PBS containing 0.05% Tween 20. After one hour, the Wells were washed three times with PBS and then 100 ⁇ of anti-mouse donkey IgG conjugated with diluted peroxidase (1: 10,000) in PBS containing 0.05% Tween 20 and 0.1% of BSA After one hour, the plates were washed three times with PBS.
  • BSA fraction V bovine serum albumin
  • the color reaction was developed with the 3,3 ' , 5,5 ' -tetramethylbenzidine peroxide reagent (TMB), as described by the corresponding manufacturer (Invitrogen, ES).
  • TMB 3,3 ' , 5,5 ' -tetramethylbenzidine peroxide reagent
  • the absorbance was read at 450 nm in SpectraMax M5 plate reader (Molecular Devices, US). The values obtained were normalized with respect to the total amount of protein and the results were expressed as mucin production relative to the control.
  • the sequence of the plasmid construct encoding MUC5AC was verified by sequencing.
  • the identification of the mucin encoding plasmid exogenously administered, alone or associated with the nanoparticles, inside human cells of the ocular surface was done by fluorescence microscopy, evaluating the expression of the green GFP fluorescent protein.
  • the assessment of a possible toxic effect on human cells as a result of this administration was carried out by means of a standard toxicity measurement test (XTT).
  • the pEGFP DNA plasmid was purchased from Elim Biopharmaceuticals (US).
  • the DNA plasmid plRES2-AcGFP1 -Mucin-5AC-280 was synthesized in Biomedal (Spain). Sodium tripolyphosphate and sodium citrate were purchased from Sigma Aldrich (Spain).
  • Example 1 Development of a plasmid DNA encoding a mucin and an easily identifiable protein.
  • Plasmid plRES2-AcGFP1 - Mucin-5AC-280 was obtained from a culture of a transformant of strain DH5 alpha. The DNA was extracted and purified using Sigma's "GenElute TM HP Plasmid Megaprep" kit, and finally lyophilized.
  • Figure 1A shows a scheme of said plasmid. The plasmid obtained, plRES2-AcGFP1-Mucin-5AC-280, was tested by transfecting human corneal epithelium (HCE) cells (Araki-Sasaki et al, 1995. Invest Ophthalmol Vis Sci.
  • HCE human corneal epithelium
  • Transfection efficiency was analyzed by fluorescence microscopy, observing the expression of the green fluorescent protein.
  • the production of MUC5AC was studied by ELISA and RT-PCR in real time.
  • Both HCE and IOBA-NHC cells are successfully transfected with plasmid plRES2-AcGFP1 -Mucin-5AC-280, observing cells with green fluorescence, corresponding to GFP expression.
  • MUC5AC expression is detected in transfected HCE and IOBA-NHC cells, by immunofluorescence and Western blotting. This expression is greater in corneal epithelial cells than conjunctival epithelium.
  • the amount of MUC5AC detected by ELISA in transfected HCE cells is -2500% higher than in untransfected cells. In the case of IOBA-NHC cells, this percentage is -1500%.
  • the real-time RT-PCR data support those obtained at the protein level, with an expression of MUC5AC of the order of 10,000 times higher in cells transfected with JetPei-RGD than in control cells, in both cell lines. In the case of lipofectamine, the expression of MUC5AC was still higher. Therefore, the HCE and IOBA-NHC eye cells, after transfection with plasmid plRES2-AcGFP1-MUC5AC-280, are capable of expressing the plasmid, of synthesizing the transduced protein (MUC5AC) and of secreting it.
  • FIG. 1 B shows a scheme of said plasmid.
  • the procedure consisted of the removal of the IRES-GFP region from the plRES2-AcGFP1 -MUC5AC-280 construction by digestion and religation.
  • the deleted region was that between the Sacl site located from the base at position 8370, and the Notl site located from position 9728 of the sequence of plRES2-AcGFP1 -MUC5AC-280.
  • the strategy used was the one described below:
  • Plasmid DNA from clone pMUCIN5AC-29 (SEQ ID NO: 7) was prepared, which was eluted in distilled and sterilized water, and digested separately with the Sacl and EcoRV enzymes, obtaining digestion patterns compatible with those expected (cut with Sacl: 1 fragment of 1,1702 base pairs; cut with EcoRV: 3 fragments of 6,692 base pairs, 3,135 base pairs and 1,875 base pairs, respectively).
  • Photograph 2 shows the results of digesting 0.5 microliters the preparation of pMUCIN5AC-29 with these enzymes (negative controls of the undigested plasmid are included).
  • Example 3 Preparation of nanoparticles associating plasmid plRES2-AcGFP1 -MUC5AC-280 made from cationized gelatin and combinations with anionic polymers.
  • the nanoparticles of plasmid plRES2-AcGFP1-MUC5AC-280 were associated. It is a negatively charged macromolecule, so it was incorporated together with the anionic crosslinker, to avoid the appearance of interactions prior to the formation of the particles.
  • the crosslinking agent used was the anionic tripolyphosphate molecule (TPP).
  • TPP anionic tripolyphosphate molecule
  • aqueous solutions were prepared in milli-Q water of gelatin previously cationized with spermine (1.0 mg / mL).
  • the crosslinking agent was added by using an aqueous solution of TPP (0.125-0.25 mg / mL) in milli-Q water.
  • the anionic polymers of dextran sulfate (DS) (0.1 mg / mL) or chondroitin sulfate (CS) (0.125 mg / mL) were incorporated into the composition.
  • the corresponding genetic material was incorporated in a concentration of 0.05 [Q / [L resulting in a mass ratio between 7.3% and 9% with respect to the constituents of the particles).
  • the bioactive molecule was incorporated into the crosslinker solution and the resulting solution was mixed with the cationized gelatin solution under magnetic stirring, which was maintained for half an hour allowing the complete evolution of the system towards a stable nanoparticular form.
  • the formulations developed and their physical-chemical characteristics (average diameter of the nanoparticles obtained as well as surface electric charge) are shown in Table 1.
  • Example 4 The nanoparticular systems associating plasmid DNA do not exhibit significant cytotoxicity in human cornea and conjunctiva cells.
  • the viability evaluation of cells in contact with nanoparticles made from cationized gelatin and combinations with anionic polymers was carried out in human cornea and conjunctiva cells.
  • the cells were seeded in 96-well plates Costar ® (Corning, US) at a confluence of 10,000 cells per well and were allowed to grow for 24 hours before testing.
  • the cells were incubated for 4h with 20 ⁇ of formulation (nanoparticles), either with 20 ⁇ of 1% benzalkonium chloride solution (positive cell death control) or with 20 ⁇ _ of culture medium (negative control), completing said volumes with DMEM / F-12 medium up to a total volume of 200 ⁇ _.
  • the cells were washed and 200 ⁇ of the respective culture medium was added.
  • the cells were then incubated with 200 ⁇ _ of RPMI medium without phenol red with XTT (0.2 mg / mL) for 15 h.
  • the results were expressed as a percentage of cell viability relative to the untreated control. As can be seen in Figure 5, the systems do not show significant toxicity in any of the cell lines studied.
  • Example 5 The nanoparticular systems associating plasmid DNA are sufficient and necessary to give rise to the production of MUC5AC mRNA in human cornea (HCE) and conjunctiva (IOBA-NHC) cells.
  • HCE human cornea
  • IOBA-NHC conjunctiva
  • cells were plated in 24-well plates Costar ® (Corning, US) at a density between 50,000 and 80,000 cells per well and allowed to grow for 24 hours before transfection. They were then incubated with nanoparticles made from cationized gelatin and combinations with anionic polymers.
  • JetPEI-RDG complexes Polyplus-transfections, US
  • plRES2-AcGFP1-MUC5AC-280 (1/10 ratio) or with pEGFP (1/5 ratio) were used, according to the manufacturer's instructions.
  • the cells were incubated with the systems and controls (1-5 ⁇ g of plasmid per well) for 4 hours in pure DMEM / F-12 medium.
  • the relative quantification of the MUC5AC mRNA according to the previously described procedure, was expressed, expressing the results in MUC5AC mRNA log, normalized with GAPDH, relative to the untransfected control.
  • Figure 6 in both the cell lines and with all the formulations tested, significant levels of MUC5AC mRNA were detected, indicating a successful transfection.
  • Example 6 The nanoparticular systems associating plasmid DNA are sufficient and necessary to give rise to the production by human cornea and conjunctiva cells of the mucin MUC5AC.
  • cells were plated in 24-well plates Costar ® (Corning, US) at a density between 50,000 and 80,000 cells per well and allowed to grow for 24 hours before transfection. They were then incubated with nanoparticles made from cationized gelatin and combinations with anionic polymers.
  • JetPEI-RDG complexes Polyplus-transfections, US
  • plRES2-AcGFP1-MUC5AC-280 (1/10 ratio) or with pEGFP (1/5 ratio) were used, according to the manufacturer's instructions.
  • the cells were incubated with the systems and controls (1-5 ⁇ g of plasmid per well) for 4 hours in pure DMEM / F-12 medium.
  • MUC5AC The expression of MUC5AC at the protein level was evaluated 72h post-transfection. How can appreciate in Figure 7 for both cell lines a significant expression of MUC5AC was obtained, confirming that the systems are effective for transfection in cornea and conjunctiva cells.
  • Example 7 The nanoparticular systems associating plasmid DNA give rise to a biological response capable of restoring functionality in tear production in animals with induced dry eye, similar to that achieved with commercially available formulation for this purpose.
  • Dry eye was induced in C57BL / 6 mice using a technique called drying stress.
  • the animals were treated with scopolamine hydrobromide subcutaneously (0.5mg / 0.2ml_; Sigma-Aldrich, US) three times a day alternating administrations between the right and left leg and subjected to a low air flow humidity.
  • the animals were kept in cages with perforated side walls to allow a flow of air provided by fans (one on each side of the cage). These cages were kept inside a flow hood (AirClean Systems, US) in which the air humidity was maintained below 40%.
  • the tear production in animals was determined by using a sterile cotton thread with a red phenol pH indicator (Zone-Quick, Lacrimedics, US).
  • the contact time of the thread with the eye was thirty seconds.
  • the migration distance of the tear was measured in millimeters, based on the color change that said thread experiences when the tear rises by capillarity. Dry eye induction began five days before administration of the formulations and processing of the controls, and was maintained during the five days of treatment.
  • the F2 formulation (GC137spm: CS: TPP) was concentrated 4 times by centrifugation (10,000g, 30 min, 4 ° C, CR22 Beckman Coulter, US) to a final concentration of 0.2 [ig / ⁇ iL from pMUCIN5AC-29.
  • the nanoparticles were administered in a volume of 5 ⁇ _, 4 times a day for 5 days, in each of the eyes, with an interval of 1 h between administrations ⁇ g per day of pMUCIN5AC-29; 20 ⁇ 9 pMUCIN5AC-29 in total).
  • the animals were immobilized manually for 1 min.
  • the tear production was determined according to the procedure previously described, quantifying the basic tear production values (zero time), those obtained after induction of dry eye (day 5) and those obtained at the end of the treatment (day 10). The results were expressed as mean ⁇ standard error of the mean tear migration in the thread (mm) of experimentally independent groups of 4 animals (total of 8 animals). For the statistical treatment of the results, a one-way variance analysis (ANOVA) was used with repeated measures. The differences were considered significant when (p ⁇ 0.05).

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Abstract

La présente invention concerne des nanoparticules polymères qui comprennent un polynucléotide codant pour une protéine MUC5AC modifiée de manière à présenter l'extrémité N-terminale de la MUC5AC reliée au domaine de dimérisation situé au niveau de l'extrémité C-terminale, ou un plasmide ou une construction génétique le contenant, et leur utilisation dans l'élaboration d'un médicament destiné à prévenir et/ou à traiter une maladie qui présente une carence en mucine. L'invention concerne également une composition pharmaceutique qui comprend ces nanoparticules et son utilisation dans l'élaboration d'un médicament destiné à prévenir et/ou à traiter une maladie qui présente une carence en mucine.
PCT/ES2011/070780 2010-11-15 2011-11-14 Nanoparticules destinées à prévenir et/ou à traiter des maladies des muqueuses Ceased WO2012066171A1 (fr)

Applications Claiming Priority (2)

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ESP201031678 2010-11-15
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WO2004069136A2 (fr) * 2003-02-05 2004-08-19 Applied Research Systems Ars Holding N.V. Nouveaux polypeptides de type mucine
WO2010049562A1 (fr) * 2008-10-28 2010-05-06 Universidade De Santiago De Compostela Systèmes nanoparticulaires élaborés à base de polymères anioniques

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WO2004069136A2 (fr) * 2003-02-05 2004-08-19 Applied Research Systems Ars Holding N.V. Nouveaux polypeptides de type mucine
WO2010049562A1 (fr) * 2008-10-28 2010-05-06 Universidade De Santiago De Compostela Systèmes nanoparticulaires élaborés à base de polymères anioniques

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