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WO2010020703A1 - Inhibiteurs de la mek 1/2 pour le traitement de la fibrose péritonéale - Google Patents

Inhibiteurs de la mek 1/2 pour le traitement de la fibrose péritonéale Download PDF

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Publication number
WO2010020703A1
WO2010020703A1 PCT/ES2009/070348 ES2009070348W WO2010020703A1 WO 2010020703 A1 WO2010020703 A1 WO 2010020703A1 ES 2009070348 W ES2009070348 W ES 2009070348W WO 2010020703 A1 WO2010020703 A1 WO 2010020703A1
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Prior art keywords
mek
seq
cells
expression
activity
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Spanish (es)
Inventor
Miguel Ángel DEL POZO BARRIUSO
Manuel LÓPEZ CABRERA
Raffaele Strippoli
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CENTRO NACIONAL DE INVESTIGACIONES CARDIOVASCULARES (CNIC) (75%)
Consejo Superior de Investigaciones Cientificas CSIC
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CENTRO NACIONAL DE INVESTIGACIONES CARDIOVASCULARES (CNIC) (75%)
Consejo Superior de Investigaciones Cientificas CSIC
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Publication of WO2010020703A1 publication Critical patent/WO2010020703A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/12—Dual-specificity kinases (2.7.12)
    • C12Y207/12002—Mitogen-activated protein kinase kinase (2.7.12.2), i.e. MAPKK or MEK1 or MEK2
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense

Definitions

  • the present invention falls within the field of molecular biology, biotechnology, and medicine, and specifically refers to the prevention and treatment of fibrosis of the peritoneal membrane (peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome) that occurs as a result of long-term peritoneal dialysis. It also refers to a method of identifying drugs useful in the treatment of peritoneal fibrosis and a method for obtaining useful data in the diagnosis of said disease.
  • peritoneal membrane peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome
  • Peritoneal dialysis is an alternative to hemodialysis for the treatment of kidney diseases in their last stage.
  • the peritoneal membrane acts as a permeability barrier through which filtration and diffusion take place.
  • the MP consists of a simple layer of mesothelial cells (CM) covering a submesothelial region composed of connective tissue and a small number of fibroblasts, mast cells, macrophages and blood vessels.
  • CM mesothelial cells
  • the epithelial-mesenchymal transition is characterized by the acquisition by epithelial cells of mesenchymal characteristics that allow them to detach from neighboring cells, reorganize their cytoskeleton, degrade the basal lamina and the underlying extracellular matrix, and acquire the ability to migrate through the latter.
  • Examples of epithelial-mesenchymal transitions are the formation of the mesoderm through the primitive line, the differentiation of the neural crest, the disintegration of the dermomiotome or the formation of the valvuloseptal mesenchyme in the endocardial cushions, and is a mechanism involved in tissue fibrosis.
  • TEM has been described as a fundamental process in tumor progression and in chronic inflammatory pathologies that can affect organs such as kidneys, liver, lung and peritoneum of patients undergoing peritoneal dialysis.
  • TEM can play an important role in the development and progression of peritoneal fibrosis, leading to a functional failure of the membrane (Yanez-Mo et al., 2003. N Eng J Med 2003; 348: 403- 13; Margetts et al., 2005. J Am Soc Nephrol 16: 425-36; Aroeira et al., 2005. Am J Kidney Dis 2005; 46: 938-48).
  • the TEM in the peritoneum is linked to a dysfunction of the peritoneal membrane, and its establishment limits the effectiveness of peritoneal dialysis.
  • the molecular regulation of TEM in the peritoneum is of interest, therefore, from the clinical point of view.
  • Kidney International, 60: 996-1008 also use PD98059 ( 2'- amino-3 ' -methoxyflavone) (20 ⁇ mol / L, Calbiochem), a specific MEK inhibitor, to determine if the modification of the protein kinase activity has any effect on The junctional intercellular communications gap (GJIC) and / or Cx43, concluding that MEK inhibitors do not restore GJIC and there is no significant change in phosphorylation of Cx43 in the presence of these inhibitors.
  • GJIC junctional intercellular communications gap
  • TGF- ⁇ 1 the growth factor ⁇ 1
  • IL-1 ⁇ interleukin-1 ⁇
  • TGF- ⁇ 1 the growth factor ⁇ 1
  • IL-1 ⁇ interleukin-1 ⁇
  • TGF- ⁇ 1 the growth factor ⁇ 1
  • IL-1 ⁇ interleukin-1 ⁇
  • TGF- ⁇ 1 induces biochemical and morphological changes in the omental mesothelial cells that are reminiscent of the one that takes place during the TEM.
  • the mechanisms that control the establishment and progression of TEM appear to be multiple and cell-specific.
  • one of the regulators is the SnaiM transcription factor, a potent transcriptional repressor of cadherin-E.
  • SnaiM's expression integrates a complex network of intracellular signals, including integrin-bound kinase (ILK), phosphatidyl inositol 3 kinase (PI3-K) and mitogen-activated protein kinase (MAP), GSK-3 ⁇ , and nuclear transcription factor kappa B (NF- ⁇ B). It has recently been shown that NF- ⁇ B plays a major role in inducing TEM in the cancer cell model transformed by Ras (Huber et al. 2004. J. Clin. Invest. 114 (4): 569-581).
  • NF- ⁇ B is complexed by the kappaB inhibitor (IKB), which under activation is phosphorylated and subsequently degraded, allowing the release and nuclear translocation of NF- ⁇ B and subsequent expression of specific genes.
  • IKB kappaB inhibitor
  • a first aspect of the invention refers to a composition, of now the composition of the invention, which comprises an agent modulating the activity of MEK 1/2 for the prevention and treatment of peritoneal fibrosis. It also refers to the use of a composition comprising a modulating agent of the activity of MEK 1 and / or MEK 2, from now on the composition of the invention, for the preparation of a medicament for the prevention and treatment of peritoneal fibrosis .
  • MEK 1/4 or “MEK 1” and “MEK 2” also called in the literature "MAPKK 1/2", “MEKIa”, “MAPKK1”, “MKKIa”, “PRKMK1”, “MAP kinase kinase 1 “or” and “” or “MAP2K2”, “MAPKK2”, “MEK2”, “MKK2”, “PRKMK2”, “ERK activator kinase 2", “MAP kinase kinase 2" or “FLJ26075”, refers to Ser / Thr / Thr-kinase enzyme that phosphorylates the MAPK enzyme in its Thr and Tyr residues of the Thr-XTyr sequence 185/187, unless otherwise indicated, when the term “MEK” is used herein 1/2 "or” MAPKK 1/2 ", refers to both the gene and the human MEK 1 and / or MEK 2 protein (enzyme).
  • ERK1 and ERK2 The downstream targets of MEK 1 identified so far are ERK1 and ERK2. After phosphorylation, ERKs activate nuclear, membrane, cytosolic and cytoskeleton targets, which mediate multiple signal cascades.
  • MEK 1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the MEK 1 protein, and which would comprise various variants from:
  • MEK 2 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the MEK 2 protein, and which would comprise various variants from:
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98 % or 99% with SEQ ID NO: 2. in which the polypeptide encoded by said nucleic acids has the activity and structural characteristics of the MEK 2 protein.
  • the sequence of the MEK 2 protein is substantially homologous to that of MEK 1, except in its amino terminal part, and for a proline-rich region inserted between the conserved kinase subdomains 9 and 10.
  • the activity of MEK 1/2 can be modulated by the modification of the levels and / or the activity of MEK 1/2, or by the modification of the levels at which the genes encoding MEK 1 and MEK 2 are transcribed. that the activity levels of the MEK 1 and MEK 2 proteins in the cell is modulated.
  • the modulating agents can also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, preferably a decrease in the activity of MEK 1 and / or MEK 2), as antagonists (substances that not only do not activate the receiver, but actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.
  • the modulating agents included in the composition of the invention are selected from a list comprising: a) an organic molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) an antibody, or e) a ribozyme.
  • the modulating agent of the composition specifically inhibits the activity of MEK 1.
  • organic molecules that can specifically bind to MEK 1/2 without binding to other polypeptides or proteins.
  • the organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1000 to 10,000 daltons.
  • Organic molecules libraries They are commercially available.
  • the route of administration may be, without limitation, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • first-generation MEK inhibitors such as U0126
  • others such as PD98059, CI-1040 (PD 184352), PD184161, Ro092210 , L783277, LLZ16402, Anthrax LF, ARRY-438162 and other orally bioavailable inhibitors AZD6244 (ARRY-142886), PD0325901 and RDEA119.
  • another preferred embodiment of the invention refers to the use of the composition of the invention for the preparation of a medicament for the treatment of peritoneal fibrosis, where the modulating agent is selected from the list comprising: U0126, PD98059, CI -1040 (PD 184352), PD184161, Ro092210, L783277, LLZ16402, Anthrax LF, ARRY-438162, AZD6244 (ARRY-142886), PD0325901, RDEA119 or any combination thereof.
  • the modulating agent is selected from the list comprising: U0126, PD98059, CI -1040 (PD 184352), PD184161, Ro092210, L783277, LLZ16402, Anthrax LF, ARRY-438162, AZD6244 (ARRY-142886), PD0325901, RDEA119 or any combination thereof.
  • the modulating agent is U0126 (formula I) or any of its salts, prodrugs, derivatives or the like, or combinations thereof.
  • a preferred embodiment refers to the use of a composition comprising the compound U0126 or any of its salts, prodrugs, derivatives or the like, or combinations thereof, for the preparation of a medicament for the treatment of peritoneal fibrosis, or alternatively, to a composition comprising the compound U0126 or any of its salts, prodrugs, derivatives or the like, or combinations thereof for use in the treatment of peritoneal fibrosis.
  • its formula is (formula I):
  • U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene) is a highly selective inhibitor of both MEK 1 and MEK 2. It has been found that functionally antagonize the transcriptional activity AP - 1 by means of the non-competitive inhibition of the MEK dual kinase with an IC 5 O of 72 nM for MEK1 and 58 nM for MEK2.
  • the modulating agent is CI-1040 (PD 184352) (formula II) or any of its salts, prodrugs, derivatives or the like, or combinations thereof.
  • a preferred embodiment refers to the use of a composition comprising the compound CI-1040 (PD 184352) or any of its salts, prodrugs, derivatives or the like, or combinations thereof, for the preparation of a medicament for the treatment of the peritoneal fibrosis, or alternatively, to a composition comprising the compound CI-1040 (PD 184352) or any of its salts, prodrugs, derivatives or the like, or combinations thereof for use in the treatment of peritoneal fibrosis.
  • Its formula is (formula II):
  • MAPK activation ERK kinase 1; MEK1
  • CM pretreatment with PD98059 inhibits the downregulation of the expression of cytokine-mediated cadherin E mRNA at all times analyzed in cells in which TGF transdifferentiation has been induced - ⁇ 1 and IL-1 ⁇ . Therefore, in a more preferred embodiment the modulating agent is PD98059 (formula III) or any of its salts, prodrugs, derivatives or analogs, or combinations thereof.
  • a preferred embodiment refers to the use of a composition comprising the compound PD98059 or any of its salts, prodrugs, derivatives or the like, or combinations thereof, for the preparation of a medicament for the treatment of peritoneal fibrosis, or alternatively, to a composition comprising the compound PD98059 or any of its salts, prodrugs, derivatives or the like, or combinations thereof for use in the treatment of peritoneal fibrosis.
  • Its formula is (formula III):
  • PD 98059 (2- (2-amino-3-methoxyphenyl) -4H-1-benzopyran-4-one), with CAS number 167869-21-8 is an inhibitor that has been shown to be active in vivo as a highly selective inhibitor of The activation of MEK1 and the cascade of MAP kinases. Prevents the activation of MEK1 induced by c-Raf. Inhibits MEK1 and MEK2 with IC50 values of 4 ⁇ M and 50 ⁇ M.
  • derivative includes both pharmaceutically acceptable compounds, that is, derivatives of the compounds of formula (I), (II) or (III), respectively, which can be used in Ia Preparation of a medicament, as pharmaceutically acceptable derivatives, since these may be useful in the preparation of pharmaceutically acceptable derivatives.
  • prodrugs of the compounds of formula (I), (II) or (III) are the prodrugs of the compounds of formula (I), (II) or (III).
  • prodrug includes any compound derived from a compound of formula (I), (II) or (III), for example, esters, including carboxylic acid esters, amino acid esters, phosphate esters , sulphonate esters of metal salts, etc., carbamates, amides, etc., which, when administered to an individual, is capable of providing, directly or indirectly, said compound of formula (I), (II) or (III) in that individual.
  • said derivative is a compound that increases the bioavailability of the compound of formula (I), (II) or (III) when administered to an individual or that enhances the release of the compound of formula (I), (II) or ( III) in a biological compartment.
  • the nature of said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I), (II) or (III) in a biological compartment of an individual.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation.
  • interference RNA interfering RNA or RNAi
  • MEK 1/2 proteins would therefore constitute the inhibition of their biological activity, and in particular, of the activity that is contributing to peritoneal fibrosis (peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome), angiogenesis and hyalinizing vasculopathy that occurs as a consequence of peritoneal dialysis.
  • antisense polynucleotides are meant ribonucleotide or deoxyribonucleotide chains that can inhibit MEK 1 and / or MEK 2 by one of these three mechanisms:
  • Antisense oligonucleotides capable of modulating the activity of MEK 1 or MEK 2 are known in the state of the art. For example, and without limiting our, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the MEK 1 or MEK 2 protein.
  • siRNA small interfering RNA
  • small interfering RNA or silencing RNA capable of inhibiting the genetic expression of the MEK 1 or MEK 2 protein.
  • siRNA small interfering RNA or small interfering RNA
  • siRNA small interfering RNA or small interfering RNA
  • this specific gene is MEK 1 or MEK 2.
  • the present invention includes by way of illustration, but not limited to, the antisense nucleotides included in US Patent 6,096,543 (corresponding to the sequences SEQ ID NO: 5 to SEQ ID NO: 42), and the siRNAs collected in Ussar & Voss, 2004.
  • the modulating agent is an antisense oligonucleotide that is selected from the list comprising: the sequence SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 10; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO:
  • siRNA capable of hybridizing a nucleic acid molecule that encodes the human MEK 1 and / or MEK 2 protein that is collected in SEQ ID NO: 1 and SEQ ID NO: 2.
  • They could also be an RNA construct that at least it contains any one of the possible nucleotide sequences of siRNA capable of inhibiting the expression of MEK 1 and / or MEK 2, and notwithstanding that any of the RNA sequences and constructions of the invention are additionally part of the present invention previously described that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and thereby greater efficiency.
  • RISC complex RNA- / nduced silencing complex
  • activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex.
  • the resulting ribonucleoproteic complex binds to the target mRNA (messenger RNA of MEK 1 or MEK 2, which is collected in SEQ ID NO: 3 and SEQ ID NO: 4).
  • target mRNA messenger RNA of MEK 1 or MEK 2
  • siRNA sequences of the invention or of the RNA constructs of the invention would be evident to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications both in the different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends.
  • the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.
  • the design of the nucleotide sequences of the siRNA of the invention would also be evident to one skilled in the art. Thus, it could be done through a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information that it has in the transcript.
  • Another non-limiting alternative of Ia The present invention would be the conventional design by simple parameters developed by the pioneers of the technique (Calipel et al., 2003. J Biol Chem. 278 (43): 42409-42418) completed with a BLAST nucleotide analysis.
  • Another possibility could be a rational design, in which a computer procedure is used to identify the optimal siRNA targets in an mRNA.
  • the target sequences are analyzed in groups of 19 nucleotides at the same time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.
  • a genetic DNA construct could also be part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising, at least, the sequence encoding the siRNA of the invention or the RNA construct of the invention for transcription, or, b) nucleotide sequence of DNA, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for the transcription and its adequate regulation in time and place, for example, start signals and termination, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
  • compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro.
  • the transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of the siRNA inside the cell.
  • vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates.
  • the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs can be conjugated with release peptides or other compounds to favor the transport of these siRNAs into the cell.
  • Antibodies capable of binding to MEK 1 and / or MEK 2 proteins can be used to inhibit the activity of said proteins.
  • Such antibodies are available in the state of the art (such as, but not limited to, antibodies marketed by Santa Cruz Biotechnology, Inc.
  • the modulating agent is selected from the above antibodies, or fragments thereof, or any combination thereof.
  • the antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen).
  • the monoclonal antibody can be biochemically altered, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, of portions that are not necessary for the recognition of MEK 1 and / or MEK 2 proteins and being substituted by others that communicate additional advantageous properties to the antibody.
  • the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with proteins.
  • portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.
  • a "recombinant antibody or polypeptide” is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.
  • rAc can be expressed and directed to specific cellular sub-compartments when the appropriate sequences for intracellular traffic are incorporated.
  • These antibodies are called intrabodies, and have proven effective not only to divert proteins from their usual compartment or block interactions between proteins involved in signaling pathways, but also to activate intracellular proteins.
  • Also part of the invention are genetic DNA constructs capable of transcribing to a peptide, antibody or antibody fragment, for use in the treatment of peritoneal fibrosis (peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome ), angiogenesis and hyalinizing vasculopathy that occurs as a result of peritoneal dialysis.
  • peritoneal fibrosis peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome
  • angiogenesis and hyalinizing vasculopathy that occurs as a result of peritoneal dialysis.
  • Said genetic construction of DNA would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises, at least, one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, which it comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for its transcription in vitro, or intracellular, b) DNA nucleotide sequence, preferably double stranded, corresponding to a system or vector of gene expression comprising the sequence coding for the antibody sequence or antibody fragment of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its proper regulation in time and place, for example, start and end signals, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
  • peritoneal fibrosis peritoneal opacification, bronchial peritoneum syndrome, fibrosis mural and sclerosing peritonitis syndrome
  • angiogenesis and hyalinizing vasculopathy that occurs as a result of peritoneal dialysis.
  • compositions provided by this invention can be provided by any route of administration to an animal, more preferably to a mammal, and even more preferably human, for which said composition will be formulated in the pharmaceutical form suitable to the route of administration chosen.
  • 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 may include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals.
  • the compositions can be prepared for administration in solid form.
  • 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; 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.
  • Pharmaceutically acceptable vehicles are known in the state of the art.
  • compositions or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety in ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • composition of the invention for the preparation of a medicament for the treatment of peritoneal fibrosis, where the composition of the invention also comprises a pharmaceutically acceptable carrier.
  • the composition of the invention further comprises another active principle.
  • active substance means any component that potentially provides a pharmacological activity or other different effect on 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 the preparation of the drug and are present therein in a modified form provided that provides the specific activity or effect.
  • ribozyme refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and fragment it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
  • RNA a catalytic polynucleotide
  • Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
  • medication refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • it refers to a composition comprising an agent modulating the activity of MEK 1 and / or MEK 2, or any of its salts, derivatives and / or the like, for the prevention or treatment of fibrosis peritoneal
  • peritoneal fibrosis is defined as the replacement of the peritoneum with fibrous tissue secondary to peritonitis. It is characteristic in patients in peritoneal dialysis programs that have recurrent peritonitis and loss of the dialysate efficacy of the peritoneum. It has a progressive character and four syndromes are distinguished: 1) opacification and loss of brightness; 2) tanned peritoneum with absence of mesothelium and presence of hyalinized collagen; 3) mural fibrosis with clinical manifestations; 4) encapsulating sclerosing peritonitis, with intense periintestinal fibrosis that prevents its normal functioning.
  • Treatment refers to both therapeutic and prophylactic treatment or preventive measures. Those necessary for treatment include those already associated with alterations as well as those in which the alteration is prevented. An “alteration” is any condition that would benefit from treatment with the composition of the invention, as described herein.
  • the modulating agents of the activity of MEK 1 and / or MEK 2 of said compositions are in a therapeutically effective amount.
  • the expression “therapeutically effective amount” refers to the amount of modulating agents (prodrugs, derivatives or analogs, or genetic constructions that allow the intracellular expression of said agents) calculated to produce the desired effect and, in general, it will be determined, among other causes, by the characteristics of these agents (and constructions) and the therapeutic effect to be achieved.
  • the pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
  • the invention provides methods for identifying compounds that can be used for the treatment of diseases related to the activity of MEK 1/2, and in particular those useful for the treatment of peritoneal fibrosis. These methods allow the identification of candidates, compounds to be tested or agents (for example, peptides, peptidomimetics, organic molecules, antisense oligonucleotides or other molecules) that can bind to MEK 1 and / or MEK 2, and have an inhibitory effect on the activity Biological MEK 1 and / or MEK 2 or its expression, and thus determine whether these compounds would have an effect on the diseases in which MEK 1 and / or MEK 2 is involved, and in particular the peritoneal fibrosis (peritoneal opacification, syndrome of the bronzed peritoneum, mural fibrosis and sclerosing peritonitis syndrome), angiogenesis and hyalinizing vasculopathy that occurs as a result of peritoneal dialysis.
  • agents for example, peptides, peptidomime
  • the tests to identify these molecules, compounds or agents that modulate the activity of MEK 1 and / or MEK 2 can use cells that express MEK 1 and / or MEK 2, or in tests with the isolated MEK 1 and / or MEK 2 enzymes ( or with its variants, such as biologically active fragments or fusion proteins that include a portion or part of MEK 1 and / or MEK 2).
  • another aspect of the invention consists in a method of identifying therapeutic agents useful in the treatment of peritoneal fibrosis comprising: a) contacting the compound to be analyzed with the MEK 1 and / or MEK 2 polynucleotide and / or polypeptide, b) detecting the binding of said compound to be analyzed with the MEK 1 and / or MEK 2 polynucleotide and / or polypeptide.
  • the compounds that bind to the MEK 1 and / or MEK 2 polypeptide would be identified as potential therapeutic agents against peritoneal fibrosis (peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome), angiogenesis and hyalinizing vasculopathy that occurs as a consequence of peritoneal dialysis.
  • peritoneal fibrosis peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome
  • angiogenesis and hyalinizing vasculopathy that occurs as a consequence of peritoneal dialysis.
  • these assays may involve the complete MEK 1 and / or MEK 2 polypeptide, a biologically active fragment thereof, or a fusion protein that involves all or a portion of the MEK 1 and / or MEK 2 polypeptide.
  • Determine Ia The ability of a compound to modulate the activity of MEK 1 and / or MEK 2 can be carried out, for example, by determining the ability of MEK 1 and / or MEK 2 to bind or interact with a target molecule of said compound, directly or indirectly. . They can also be activity tests, directly or indirectly measuring the activity of MEK 1 and / or MEK 2.
  • assays can also be combined with an in vivo test measuring the effect of a test compound on the symptoms of diseases related to MEK 1 and / or MEK 2, and in particular peritoneal fibrosis (for example, but not limited, on animal models or other model systems known in the art).
  • the compounds to be tested used in the method of identification of therapeutic agents are not limited to low-weight organic molecules molecular, but also proteins (including antibodies), peptides, oliogonucleotides, etc. and can be natural and / or synthetic.
  • antibodies capable of binding to an epitope of MEK 1 and / or MEK 2 which can be used therapeutically, as discussed above, can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical tests known in the state of the art.
  • the MEK 1 and / or MEK 2 polypeptides can be used to immunize an animal, to obtain polyclonal antibodies.
  • Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cultured cell lines, which include, but are not limited to, hybridomas, human B cell hybridomas. Techniques for producing chimeric, humanized or synthetic antibodies are known in the state of the art.
  • the therapeutic agents identified by the identification method described herein can be used in an animal or other model to determine the mechanism of action of said agent. Moreover, the therapeutic agents selected by the method described herein would be used in the treatment of peritoneal fibrosis (peritoneal opacification, bronzed peritoneum syndrome, wall fibrosis and sclerosing peritonitis syndrome), angiogenesis and hyalinizing vasculopathy that occurs as a consequence of Ia peritoneal dialysis.
  • peritoneal fibrosis peritoneal fibrosis
  • bronzed peritoneum syndrome bronzed peritoneum syndrome
  • wall fibrosis and sclerosing peritonitis syndrome wall fibrosis and sclerosing peritonitis syndrome
  • angiogenesis hyalinizing vasculopathy that occurs as a consequence of Ia peritoneal dialysis.
  • a method of identifying therapeutic agents useful in the treatment of peritoneal fibrosis comprising: a) determine the activity of MEK 1 and / or MEK 2 at an established concentration of the compound to be analyzed or in the absence of said compound, and b) determine the activity of MEK 1 and / or MEK 2 at a concentration of the compound to be analyzed different from Ia of (a).
  • the determination of the activity of MEK 1 and / or MEK 2 is carried out at an established concentration of the compound to be analyzed.
  • Compounds that give rise to a different activity of MEK 1 and / or MEK 2 would be identified as potential therapeutic agents against peritoneal fibrosis (peritoneal opacification, bronzed peritoneal syndrome, wall fibrosis and sclerosing peritonitis syndrome), angiogenesis and hyalinizing vasculopathy that It is produced as a consequence of peritoneal dialysis.
  • Another aspect of the invention relates to a method of identifying therapeutic agents useful in the treatment of peritoneal fibrosis which comprises: a) determining the activity of MEK 1 and / or MEK 2 at a certain concentration of the compound to be analyzed, and b) determine the activity of MEK 1 and / or MEK 2 in the presence of a compound known to modulate the activity of MEK 1 and / or MEK 2.
  • the diseases in which the alteration of the activity of MEK 1 and / or MEK 2 can be diagnostic, and in particular, peritoneal fibrosis, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) that encode for MEK 1 and / or MEK 2, or the amount of MEK 1 and / or MEK 2 protein that is expressed, compared to normal cells.
  • the detection of oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT-PCR).
  • Procedures for detecting the expression of the MEK 1 and / or MEK 2 protein are also well known in the state of the art, such as, for example, poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
  • a method for obtaining useful data in the diagnosis and / or prognosis of peritoneal fibrosis comprising:
  • MEK 2 obtained in a) with the standard values in healthy or diseased mammals.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.
  • peptide refers to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.
  • Fig. 1 Mesothelial cells derived from the omentum undergo TEM under Ia peritonitis effluent exposure
  • A Photomicrographs of the confluent monolayers of the untreated primary omental human mesothelial cells (NT) or treated for 72 hours with the peritoneal effluent (diluted) 1: 1 with the culture medium) of a patient undergoing peritoneal dialysis who has suffered peritonitis.
  • B Confocal (red) immunofluorescence of treated and untreated mesothelial cells stained with monoclonal antibodies against pan-cytokeratin (72 hours of stimulation). The core was dyed with Hoechst 33342 (blue).
  • Fig. 2 Mesothelial cells derived from omentum undergo TEM under the stimulation of TGF- ⁇ 1 and IL-1 ⁇ .
  • A Photomicrographs of the confluent monolayers of untreated primary omental human mesothelial cells (NT) or treated with TGF- ⁇ 1 (0.5 ng / ml) in combination with IL-1 ⁇ (2 ng / ml) for 24 hours (T / l).
  • B Confocal (red) immunofluorescence of treated and untreated mesothelial cells stained with monoclonal antibodies against cadherin E (24 hours of stimulation) and with pan-cytokeratin (56 hours of stimulation). The core was dyed with Hoechst 33342 (blue).
  • Fig. 3 The nuclear translocation of NF- ⁇ B is inhibited by the expression of the super IKB repressor.
  • A Confocal immunofluorescence of the expression and localization of NF- ⁇ B.
  • Primary human peritoneal CMs were co-stimulated with TGF- ⁇ 1 and IL-1 ⁇ during the indicated times; NT, untreated controls.
  • Fixed and permeabilized cells were stained with a polyclonal antibody against p65 NF- ⁇ B.
  • the histogram shows the average fluorescence intensities of the nuclear staining of NF- ⁇ B, quantified using the Leica LAS-AS software. The bars represent SEM. A total of 50 cells were analyzed per condition; AU: arbitrary units.
  • A Primary omental mesothelial cells were infected with the retrovirus labeled with Cop Green encoding the IKB super repressor (IKB, lower panels) or with the virus - empty labeled with Cop Green (empty vector, upper panels). The cells were subsequently co-stimulated with TGF- ⁇ 1 and IL-1 ⁇ for 30 minutes. The fixed and permeabilized cells were stained for p65 NF- ⁇ B (yellow in the superimposed image. The green fluorescence indicates infected cells. The confocal spectral technology allows the discrimination between the yellow and green fluorescence emitted. The superimposed images show the Hoechst 33342 stains, anti-NF- ⁇ B and Cop green.The data are representative of three independent experiments.
  • Fig. 4 The downregulation of epithelial markers in cytokine-stimulated mesothelial cells requires the signal activator of NF- ⁇ B.
  • A Western blots showing the expression of cadherin E and IKB in the total cell lysate of mesothelial cells.
  • the primary omental mesothelial cells were infected with the Green-tagged Cop retrovirus encoding the super repressor IKB (IKB) OR with the empty virus labeled with Cop Green, and stimulated for 24 hours with TGF- ⁇ 1 and IL-1 ⁇ , as has been indicated (T / l); NT, untreated cells.
  • the expression of ⁇ -tubulin was used as a control.
  • the histogram shows the intensity ratios of cadherin E / ⁇ -tubulin band of a representative experiment.
  • B Effect of the inhibition of NF- ⁇ B on the expression of cadherin E mRNA in mesothelial cells.
  • MeT-5A mesothelial cells were infected as described above, with the retrovirus encoding the super IKB repressor (IKB), or with the empty control plasmid (CopG). The cultures of MeT-5A were selected after infection by green fluorescence to obtain almost pure cultures of infected cells.
  • ERK controls the downregulation of cadherin E and cytokeratin during the TEM of mesothelial cells stimulated by cytokines.
  • A Western blot showing the expression of phosphorylated (active) ERK (upper part) in the total cell lysate of the mesothelial cells.
  • Primary omental CMs were left untreated (NT) or co-stimulated with TGF- ⁇ 1 and IL-1 ⁇ for the indicated time (T / l). As a control, the samples were tested for total ERK expression (part lower).
  • C Western blot showing the expression of cadherin E and Phosphorylated (active) ERK in the total mesothelial cell lysate.
  • Omental mesothelial cells were pretreated with DMSO or U0126 (20 ⁇ m) and treated for 24 hours with TGF- ⁇ 1 and IL-1 ⁇ as indicated. The detection of the total ERK expression was used as a control.
  • D confocal immunofluorescence analysis of cytokeratin expression.
  • E effect of the inhibition of ERK on the expression of cadherin E mRNA in mesothelial cells.
  • the cells were pretreated with DMSO (gray bars) or U0126 (black bars) and co-stimulated during the times indicated with TGF- ⁇ 1 and IL-1 ⁇ (T / l); NT, non-stimulated controls.
  • a quantitative RT-PCR of the total RNA was carried out. Histone H3 mRNA levels were used for normalization. The bars represent the mean ⁇ SEM of the duplicate determinations in three independent experiments.
  • ERK controls the nuclear translocation of NF- ⁇ B and the transcriptional activity induced by the stimulation of TGF- ⁇ 1 and IL-1 ⁇ in mesothelial cells.
  • A Analysis by confocal immunofluorescence of the expression and localization of NF- ⁇ B. Omental mesothelial cells were pretreated with DMSO or U0126 (20 ⁇ m) and co-stimulated during the times indicated with TGF- ⁇ 1 and IL-1 ⁇ ; NT, untreated. Fixed and permeabilized cells were stained with a polyclonal antibody against p65 NF- ⁇ B. The results of three representative independent experiments are shown.
  • B effect of the inhibition of ERK on the transcriptional activity of NF- ⁇ B.
  • MeT-5A cells were transiently transfected with the KBF-luc reporter plasmid together with Renilla luciferase. The cells were pretreated as indicated with U0126 (20 ⁇ m) and untreated (NT, gray bars) or co-stimulated for the times indicated with TGF- ⁇ 1 and IL-1 ⁇ (T / l, black bars). The bars represent The mean ⁇ SEM of the determinations of three independent experiments carried out in duplicate. P ⁇ 0.05 compared to cells treated with DMSO.
  • C Mesothelial omental cells were pretreated with DMSO or U0126 (20 ⁇ m) and treated as indicated. Qualitative RT-PCR was carried out with specific primers for COX-2 and Histone 3.
  • ERK and NF- ⁇ B controls the expression of SnaiH induced by the costimulation of TGF- ⁇ 1 and IL-1 ⁇ in mesothelial cells.
  • A Effect of the inhibition of the expression of SnaiM mRNA in mesothelial cells. Omental mesothelial cells were pretreated with DMSO (gray bars) or 20 ⁇ m of U0126 (black bars) and co-stimulated with TGF- ⁇ 1 and IL-1 ⁇ (T / l); NT, untreated. The quantitative RT-PCR of the total mRNA was carried out. The mRNA expression of Histone 3 was used for normalization. The bars represent the mean ⁇ SEM of the determinations of three independent experiments.
  • Omental mesothelial cells were pretreated with DMSO or U0126 (20 ⁇ m) and treated as indicated for 24 hours. LiCI (40 mM) and MG132 (10 ⁇ m) were added to the cells 4 hours before the end of the stimulation. The cells were fixed, permeabilized, stained with a polyclonal antibody against SnaiM and subjected to confocal microscopy analysis. The panels show the staining of the cell nucleus with Hoechst 33342 (nuclei) and the expression of SnaiM stained by immunofluorescence. The results shown are from three independent representative experiments.
  • Fig. 8 Inhibition of ERK and NF- ⁇ B can reverse TEM in transdifferentiated mesothelial cells of peritoneal patients undergoing continuous ambulatory peritoneal dialysis (CAPD).
  • CAPD continuous ambulatory peritoneal dialysis
  • A Western blot showing the expression of pERK in monolayer cultures of mesothelial cells derived from peritoneal effluent from patients undergoing continuous ambulatory peritoneal dialysis (CAPD) or normal omentum. Confluent monolayers of omental mesothelial cells from control donors or DPAC patients were used, and those used were faced with monoclonal antibodies against pERK. The total ERK expression was detected as a control. The histogram shows the pERK ratios / total ERK band intensity. * , P ⁇ 0.05 control donors compared with DPAC patients.
  • C Photomicrographs of confluent monolayers of non-epithelioid CM derived from the effluent treated for 48 hours with DMSO (NT) or U0126 (20 ⁇ M).
  • C Left. Western blot showing the expression of cadherin E in monolayer cultures of CM derived from effluent or CM control of normal omentum (O). Confluent monolayers of mesothelial cells, treated with DMSO or with U0126 (20 ⁇ M). Alternatively, the cells were infected with the retrovirus encoding for super IKB repressor (IKB) OR with the empty virus (CopGreen).
  • IKB super IKB repressor
  • Confluent monolayers of effluent-derived mesothelial cells (patient 10), showing non-epithelioid morphology, were treated with DMSO or U0126 for 56 h.
  • the cells were stained with polyclonal anti-p65NF-KB and monoclonal anti-ketoratin antibodies.
  • the cores were stained with Hoechst 33342 (blue). Lower left.
  • the histograms show the fluorescence intensities of the nuclear NF- ⁇ B stained from treated cells as in the upper part of the panel (patients analyzed 1-3, 9, 10) quantified with the Leica LAS-AS software.
  • the bars represent SEM. A total of 250 cells were analyzed per condition; AU: arbitrary units.
  • Lower - center Western blots showing the expression of cytokeratin and cadherin E in non-epithelioid CM derived from effluent treated with DMSO (D) or U0126 (patients 11-13). Bottom - right. Western blot quantification shown in the middle of the panel.
  • Fig. 9 Effect of U0126 and Cl 1040 on the reversal of the mesenchymal epithelial transition (TEM) in mesothelial cells from effluent from patients undergoing peritoneal dialysis (PD).
  • TEM mesenchymal epithelial transition
  • AB Mesothelial cells (A, patient A; B, patient B) were treated with DMSO, with U0126 (20 ⁇ M) or with Cl 1040 (2.5 ⁇ M) for 56 hours. Then, the cells were fixed, permeabilized and stained with an anti-cytokeratin monoclonal antibody and with Hoechst to visualize the nuclei. These cells have been analyzed with confocal microscopy.
  • C Mesothelial cells with different phenotype mixed phenotype; epithelial phenotype; non-epithelial phenotype
  • E Mesothelial cells from a patient were treated with DMSO or Cl 1040 (2.5 ⁇ M) for 56 hours. Then, the cells were fixed, permeabilized and stained with an anti- ⁇ -SMA monoclonal antibody and with Hoechst to visualize the nuclei. These cells were analyzed by confocal microscopy.
  • Fig. 10 Effect of Cl 1040 on the inhibition of TEM induced by TGF ⁇ + IL-1 ⁇ in primary mesothelial cells of human omentum.
  • B Human primary mesothelial cells were pretreated with Cl 1040 (2.5 ⁇ M) or with DMSO for 1 hour. Then the cells were treated with TGF ⁇ (0.5 ng / ml) + IL-1 ⁇ (2 ng / ml) for 24 hours. Then, the cells were fixed, permeabilized and stained with an anti- ⁇ -SMA monoclonal antibody and with Hoechst to visualize the nuclei. These cells were analyzed by confocal microscopy.
  • Fig. 11 Effect of Cl 1040 and U0126 on the inhibition of TEM induced by TGF ⁇ + IL-1 ⁇ in primary mesothelial cells from mouse peritoneum.
  • C Primary mesothelial cells from mouse peritoneum were pretreated with U0126 (20 ⁇ M) or with DMSO for 1 hour. The cells were then left untreated or treated with TGF ⁇ (0.5 ng / ml) + IL-1 ⁇ (2 ng / ml) for 24 hours. These cells were used and the used cell phones were analyzed by Western Blotting (WB). Monoclonal antibodies anti-fibronectin, -E-cadherin, -tubulin have been used to reveal the expression of said proteins.
  • D Primary mesothelial cells from mouse peritoneum pretreated with Cl 1040 (2.5 ⁇ M) or with DMSO for 1 hour.
  • the cells were then left untreated or treated with TGF ⁇ (0.5 ng / ml) + IL-1 ⁇ (2 ng / ml) for 24 hours.
  • the cells were fixed, permeabilized and stained with an anti-cytokeratin monoclonal antibody and with Hoechst to visualize the nuclei. These cells have been analyzed by confocal microscopy.
  • E Primary mesothelial cells from mouse peritoneum pretreated with Cl 1040 (2.5 ⁇ M) or with DMSO for 1 hour.
  • the cells were then left untreated or treated with TGF ⁇ (0.5 ng / ml) + IL-1 ⁇ (2 ng / ml) for different times.
  • a quantitative PCR assay was performed to detect the expression of SnaiM on total RNA. Histone H3 mRNA levels have been used for normalization.
  • CM Human mesothelial cells
  • CMs were treated with a combination of recombinant human Transforming Growth Factor (TGF) - ⁇ 1 (0.5 ng / mL) and Interleukin (IL) -1 ⁇ (2 ng / mL) (R&D Systems, Minneapolis, MN) described in Ya ⁇ ez Mo et al., 2003. N. Engl. J. Med. 30: 403-413.
  • TGF- ⁇ 1 and IL-1 ⁇ can separately induce phenotypic changes characteristic of TEM
  • the combined stimulation induces a genuine TEM (Ya ⁇ ez Mo et al., 2003. N. Engl. J. Med. 30: 403-413).
  • the doses used to induce TEM are those found in peritoneal dialysis fluids in the presence of peritonitis, and are similar to the doses used in previous studies (Ya ⁇ ez Mo et al. 2003. N. Engl. J. Med. 30: 403- 413).
  • CM derived from peritoneal effluent from clinically stable patients were isolated using a method already described (López Cabrera et al., 2006. Perit Dial Int. 26: 26-34). The cells were cultured according to the method indicated above, and after 10 to 15 days of culture they reach confluence and divide 1: 2. The morphological characteristics of these cells remain stable for two to three passes.
  • the confluent CMs that come from effluents from peritoneal dialysis have two main phenotypes, epithelial (E) or non-epithelial (NE).
  • CM cultures derived from effluents evaluated six had the epithelial type phenotype, and seven had the non-epithelial phenotype.
  • the purity of the omentum and of the CM cultures derived from the effluent was determined by means of the evaluation of the standard markers intercellular adhesion molecule (ICAM) -I and cytokeratins.
  • CMs expressed high levels of ICAM-1 and low levels of the specific fibroblast marker.
  • CM cultures were also negative for the CD31 endothelial marker and the CD45 macrophage marker.
  • MeT-5A human mesothelial cell line (ATCC, Rockville, MD) was cultured in Earle's M199 medium and stimulated with the same doses of TGF- ⁇ 1 and IL-1 ⁇ .
  • Met-5A is a line of non-transformed mesothelial cells commonly used in the investigation of peritoneal mesothelial cells; the data obtained with this cell line show concordance with the data obtained with primary cells.
  • BD monoclonal antibodies against BD (Becton-Dickinson Laboratories, Mountain View, CA) were purchased; Monoclonal antibodies against tubulin, pan-cytokeratin and ⁇ SMA were obtained from Sigma (Saint Louis, MO). Monoclonal anti fibronectin antibodies have been purchased from Zymed (Invitrogen, Carlsbad, CA). Compound U0126 was purchased from Calbiochem (EMD, Darmstad, Germany). Cl 1040 has been purchased at Alexis (Axxora, Bingham, UK).
  • CM monolayers were used in modified RIPA buffer (50 mM Tris-HCI, pH 7.4; 1% NP-40; 0.1% SDS; 0.25% Sodium-deoxylated; 150 mM NaCI; 1 mM EDTA; 1 mM EGTA; 1 mM PMSF ; 1 ⁇ g / ml of aprotinin, leupeptin and pepstatin; and 25 mM NaF (all obtained from Sigma) Similar amounts of used proteins were separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore, Bedford, VA) and incubated with antibodies using standard procedures. antibodies attached to PVDF were detected by chemiluminescence with ECL (Amersham Life Sciences, Little Chalfont, UK).
  • the cells were fixed for 20 minutes in 3% formaldehyde in PBS, permeabilized in 0.2% Triton X-100 / PBS for 5 min, and blocked with 2% BSA for 20 min.
  • the cells were fixed and permeabilized in cold methanol for 10 minutes.
  • Snail staining the cells were pretreated with LiCL and MG132 to block Snail phosphorylation, ubiquitination or subsequent degradation.
  • Secondary antibodies conjuggated to Alexa-647, -488) and Hoechst 33342 were purchased from Pierce Chemical Co. (Rockford, IL). Confocal miscroscopy images were obtained using a spectral Leica SP5 Confocal Microscope.
  • the spectral technology allows the discrimination between yellow and green fluorescence.
  • the quantification of the NF- ⁇ B nuclear intensity was carried out using the Leica LAS-AF software. Briefly, the nucleus was delimited using the Hoechst marker, and the measurement of the fluorescence intensity of the labeled NF- ⁇ B was quantified. A minimum of 3 different fields per condition were acquired, with a number of cells between 30 and 50 per quantified condition.
  • OmeT-derived MeT5A and CM cells were infected with the retroviral vector pRV-IRES-CopGreen (Genetrix, Madrid, Spain) encoding the super mutant l ⁇ B ⁇ repressor, with mutations S32A and S36A, or with the empty pRV-IRES-CopGreen vector as control.
  • a critical step in the activation of NF- ⁇ B is the phosphorylation of IKB by the IKB kinase complex High molecular weight (IKK).
  • the mutations S32A and S36A make l ⁇ B ⁇ insensitive to phosphorylation by IKK.
  • the CM were seeded in 6 plates (2 x 10 5 cells per plate) and the retrovirus producing 293T cells were seeded in 3 x 10 6 per 10 cm of plaque.
  • the supernatants of 293T cells were filtered through a 0.45- ⁇ m filter (Whatman, Dassel, Germany), and 5 ⁇ g / ml polybrene (Sigma-Aldrich, St. Louis, Missouri, USA) was added to the filtrate.
  • the medium was removed from the CM and replaced with 293T cells containing the retrovirus. This process was repeated twice in a 24 hour interval.
  • the transcriptional activity of NF- ⁇ B was measured by transient infection of MeT5A cells with the KBF-luc reporter plasmid and the subsequent luciferase activity assay.
  • 2 x 10 5 cells were transfected with 2 ⁇ g of the KBF-luc reporter plasmid together with 500 ng of the reporter plasmid pRL-null, with a Renilla Luciferase gene without promoter (Promega, Madison, Wl). Transfection was carried out by incubating the cells for 4 hours with a mixture of DNA and lipofectamine and a ration of 1: 2.5 (Lipofectamine 2000; Invitrogen, Carlsbad, California, USA) in a serum-free medium.
  • the cells were pretreated overnight with a vehicle (DMSO) or U0126 (20 ⁇ M).
  • DMSO fetal calf serum
  • U0126 20 ⁇ M
  • the cells were stimulated with TGF- ⁇ 1 and IL-1 ⁇ for the indicated times.
  • Luiciferase activity was measured with Dual-Luciferase Repórter Assay System (Promega), according to the manufacturer's instructions, and determined in a Sirius Single Tube luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). All experiments were carried out in duplicate.
  • the hybridization temperature for the amplification of cadherin E and H3 was 62 ° C, and the fluorescence was measured at the end of each elongation cycle.
  • the hybridization temperature was 55 0 C and the fluorescence was measured at 88 0 C after each elongation cycle. All experiments were carried out in duplicate.
  • the PCR products were confirmed by the analysis of the melting curve, and gel electrophoresis. COX-2 mRNA levels were estimated by 35 qualitative PCR cycles with an alignment temperature of 63 0 C and the following primers SEQ ID NO: 57 and .SEQ ID NO: 58.
  • EXAMPLE 1- Reversion to epithelial morphology of mesenchymal cells (CM) treated with U0126.
  • cytokeratins a highly expressed epithelial marker in mesothelial cells
  • cadherin E another epithelial marker
  • the effluent of peritonitis induced an increase in the expression of proteins such as fibronectin, cadherin E, whose expression is associated with EMT (Fig. 1C and 5B).
  • NF- ⁇ B The downregulation of cadherin E and cytokeratin in CM, induced by TGF- ⁇ 1 and IL-1 ⁇ , is mediated by NF- ⁇ B.
  • the super repressor l ⁇ B ⁇ blocks the nuclear translocation of NF-kB induced by TGF- ⁇ 1 and IL-1 ⁇ , while infection with the retroviral vector pRV-IRES-CopGreen had no effect (Fig. 3C). Infection with the vector with the super repressor l ⁇ B ⁇ also interfered with the biochemical changes associated with TEM. In cells stimulated with TGF- ⁇ 1 plus IL-1 ⁇ for 24 hours, the expression of the super repressor l ⁇ B ⁇ , limits the regulation of the lowering of the expression of the cadherin E protein, while this was not affected in cells infected with the control vector pRV-IRES-CopGreen (Fig 4A).
  • the expression of the l ⁇ B ⁇ super repressor in primary CM also limits the inhibition of cytokine-induced cytokeratin, as demonstrated by confocal immunofluorescence analysis after 56 hours of treatment (Fig. 4C).
  • the route of activation of NF- ⁇ B therefore controls both the downregulation of cadherin E and cytokeratin during TEM in primary CM.
  • the downregulation of cadherin E and cytokeratin during TEM in CM is controlled by the ERK route.
  • CMs Upstream events that take place for the activation of NF- ⁇ B in this experimental system have been analyzed. Recently it has been reported in several cell models that have documented a role for MAP kinases, particularly ERKs, in establishing the TEM. The treatment of CM with TGF- ⁇ 1 plus IL-1 ⁇ induced a strong ERK phosphorylation (Fig. 5A), and the effluent of peritonitis has a similar effect (Fig. 5B). To examine the role of ERK activation in TEM, CMs were pretreated with U0126, a pharmacological inhibitor of MEK-1, the upstream activator of ERK.
  • U0126 suppressed markedly the down-regulation of cadherin E and cytokeratin mediated by cytokines Fig. 5C and D). U0126 also inhibits the downregulation of the expression of cytokine-mediated mRNA of cadherin E at all times analyzed (Fig. 5E). Similar results were obtained with the MEK-1 PD98059 inhibitor (20 ⁇ M). These results strongly suggest that the activation of the ERK pathway mediates the downregulation of cadherin E and cytokeratin induced by TGF- ⁇ 1 and IL-1 ⁇ .
  • ERK regulates the nuclear localization of NF- ⁇ B and the transcriptional activity during TEM induced by cytokines.
  • CM pretreatment of CM with U0126 reduced the intensity and persistence of the nuclear staining of NF- ⁇ B under the combined treatment of cytokines (Fig. 6A).
  • cytokines Fig. 6A
  • CM transfected with a luciferase reporter construction with multiple NF- ⁇ B binding sites (KBF-luc).
  • Pretreatment of these cells with U0126 markedly reduced the luciferase activity induced by cytokine (Fig. 6B).
  • Snail 1 is the best known member of the family of zinc finger transcriptional regulators involved in the induction of TEM.
  • SnaiM directly blocks the transcription of the cadherin E promoter and appears to be involved in the cytokeratin gene repression.
  • Quantitative RT-PCR showed that the stimulation of primary CM with TGF- ⁇ 1 and IL-1 ⁇ induces an increase in the expression of SnaiM mRNA, increasing the nuclear expression and location (Fig. 7B, top). Similar to its effect on the expression of SnaiM mRNA, pretreatment with U0126 markedly decreased the expression of SnaiM protein in cells stimulated by cytokines (Fig. 7B, below).
  • CMs were infected with the retrovirus encoding the super repressor l ⁇ B ⁇ or the empty vector pRV-IRES-CopGreen, and stimulated with TGF- ⁇ 1 and IL-1 ⁇ .
  • Exogenous expression of the super repressor l ⁇ B ⁇ partially reduced the expression of the cytokine-induced SnaiM mRNA (Fig 7C, left).
  • this experiment was repeated in cultures of retrovirally infected MeT5A cells and purified by FACS.
  • ERK or NF- ⁇ B restores the epithelial phenotype in CMs transdifferentiated by the peritoneal effluent.
  • the TEM of peritoneal CM in patients undergoing PD is linked to the dysfunction of the peritoneal membrane (MP), that is, to a failure in ultrafiltration.
  • MP peritoneal membrane
  • CM was obtained from the effluents of 13 patients undergoing PD (Table 1).
  • the parameters used to evaluate the different stages of transdifferentiation of these cells were both morphological (epithelioid and non-epithelioid) and biochemical (reduced levels of cadherin E and cytokeratins, high expression of VEGF and vimentin), as already described in others. studies.
  • the analysis by Western Blot showed that, compared to the control samples of the omentum, CM not treated with the effluent derived from the DP presented a significant increase in the levels of active ERK (Fig. 8A).
  • CM treated with the effluent derived from the DP were treated with U0126 or infected with the retrovirus encoding the super repressor l ⁇ B ⁇ .
  • Microscopic analysis of non-epithelioid CM derived from effluents showed that treatment with U0126 reversed the cells to epithelial morphology (Fig. 8B).
  • the analysis by Western Blot showed that the expression of cadherin E, both in CM derived from both epithelioid and non-epithelioid effluent, was restored both in cells treated with U0126 and in those expressing the super repressor l ⁇ B ⁇ (Fig. 8C).
  • EXAMPLE 2 Reversal of the mesenchymal epithelial transition (TEM) in mesothelial cells from effluent from patients undergoing peritoneal dialysis (PD) with Cl 1040 and U0126.
  • TEM mesenchymal epithelial transition
  • FIG. 9C shows the inhibition of MEK1 / 2 in mesothelial cells from patients undergoing peritoneal dialysis.
  • Fig. 9D it shows how MEK1 / 2 inhibitors increase levels of E-cadherin in mesothelial cells from patients undergoing peritoneal dialysis.
  • Fig. 9E it is shown how Cl 1040 reduces the levels of ⁇ SMA in mesothelial cells from patients undergoing peritoneal dialysis.
  • Fig. 10A it is observed how Cl 1040 increases the levels of E-cadherin and limits the reduction of its levels after treatment with TGF ⁇ + IL-1 ⁇ in human primary mesothelial cells.
  • Fig. 10B it is observed how Cl 1040 blocks the acquisition of an elongated form and reduces the levels of ⁇ SMA in primary mesothelial cells of human omentum treated with TGF ⁇ + IL-1 ⁇ .
  • EXAMPLE 3 Assays with compound CI-1040 in mouse mesothelial cells (ex vivo) in which transdifferentiation with T / l has been induced.
  • FIG. 10B the quantitative analysis of morphological alterations in mouse primary mesothelial cells pretreated with U0126 and stimulated with TGF ⁇ + IL-1 ⁇ is observed.
  • Fig. 11C it is observed how Cl 1040 blocks the induction of TEM measured by TGF ⁇ + IL-1 ⁇ in primary mouse mesothelial cells.
  • Fig. 11 D it is shown how treatment with Cl 1040 increases the expression of cytokeratin in primary mouse mesothelial cells.
  • Fig. 11 E it is observed how Cl 1040 inhibits the expression of SnaM induced by TGF ⁇ + IL-1 ⁇ in primary mouse mesothelial cells.

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Abstract

L'invention concerne des médicaments utiles dans la prévention et le traitement de la fibrose de la membrane péritonéale (opacification péritonéale, syndrome du péritoine bronzé, fibrose murale et syndrome de péritonite sclérosante) qui se produit en conséquence d'une dialyse péritonéale pratiquée sur le long terme. L'invention concerne également un procédé de sélection de ces médicaments et un procédé pour la collecte de données utiles dans le diagnostic de cette maladie.
PCT/ES2009/070348 2008-08-20 2009-08-20 Inhibiteurs de la mek 1/2 pour le traitement de la fibrose péritonéale Ceased WO2010020703A1 (fr)

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Cited By (2)

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CN104168954A (zh) * 2012-03-14 2014-11-26 泰拉克利昂公司 用于治疗性处理的装置和用于控制处理装置的方法
WO2016130917A1 (fr) * 2015-02-12 2016-08-18 Memorial Sloan Kettering Cancer Center Traitement contre le cancer à action synergique

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TWI454287B (zh) * 2012-11-21 2014-10-01 Taipei Veteran General Hospital 治療纖維化之透析液

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Title
ENGLISH J.: "COBB M. Pharmacological inhibitors of MAPK pathways.", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 23, no. 1, 2002, pages 40 - 45 *
SELGAS, R. ET AL.: "Epithelial-to-mesenchymal transition of the mesothelial cell-its role in the response of the peritoneum to dialysis.", NEPHROLOGY DIALYSIS TRANSPLANTATION, vol. 21, 2006, pages II2 - II7 *
SELGAS, R. ET AL.: "Transición epitelio- mesenquimal en procesos fibrosantes. Células mesoteliales obtenidas ex vivo de pacientes tratados con diálisis peritoneal como modelo de transdiferenciacion.", NEFROLOGIA, vol. XXIV., no. 1., 2004, pages 34 - 39 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104168954A (zh) * 2012-03-14 2014-11-26 泰拉克利昂公司 用于治疗性处理的装置和用于控制处理装置的方法
CN104168954B (zh) * 2012-03-14 2017-10-27 泰拉克利昂公司 用于治疗性处理的装置和用于控制处理装置的方法
WO2016130917A1 (fr) * 2015-02-12 2016-08-18 Memorial Sloan Kettering Cancer Center Traitement contre le cancer à action synergique
US10792284B2 (en) 2015-02-12 2020-10-06 Memorial Sloan Kettering Cancer Center Synergistic cancer treatment

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