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WO2010037878A1 - Utilisation de nanoparticules de métaux nobles en tant qu'immunomodulateurs et composition immunomodulatrice - Google Patents

Utilisation de nanoparticules de métaux nobles en tant qu'immunomodulateurs et composition immunomodulatrice Download PDF

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Publication number
WO2010037878A1
WO2010037878A1 PCT/ES2009/000476 ES2009000476W WO2010037878A1 WO 2010037878 A1 WO2010037878 A1 WO 2010037878A1 ES 2009000476 W ES2009000476 W ES 2009000476W WO 2010037878 A1 WO2010037878 A1 WO 2010037878A1
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WO
WIPO (PCT)
Prior art keywords
nanoparticles
tlr2
immunomoductor
preparation
thiopronin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2009/000476
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English (en)
Spanish (es)
Other versions
WO2010037878A9 (fr
Inventor
David POZO PÉREZ
Rafael FERNÁNDEZ MONTESINOS
Juan Luis Herrera Cabello
José Antonio MEJÍAS ROMERO
Paula Margarita CASTILLO HERNÁNDEZ
Ana Paula ZADERENKO GARCÍA
Pedro Pablo GARCÍA LUNA
José Luis PEREIRA CUNILL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fundacion Publica Andaluza Para La Gestion de la Investigacion En Salud En Sevilla
Universidad de Sevilla
Universidad Pablo de Olavide
Original Assignee
Fundacion Publica Andaluza Para La Gestion de la Investigacion En Salud En Sevilla
Universidad de Sevilla
Universidad Pablo de Olavide
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Publication date
Application filed by Fundacion Publica Andaluza Para La Gestion de la Investigacion En Salud En Sevilla, Universidad de Sevilla, Universidad Pablo de Olavide filed Critical Fundacion Publica Andaluza Para La Gestion de la Investigacion En Salud En Sevilla
Publication of WO2010037878A1 publication Critical patent/WO2010037878A1/fr
Publication of WO2010037878A9 publication Critical patent/WO2010037878A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • 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
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/38—Silver; Compounds thereof
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • One aspect of the present invention is the use of the immunomodulatory effects of silver metal nanoparticles functionalized with thiopronin [iV- (2-mercaptopropionyl) glycine].
  • a second aspect is an immunomodulatory composition that includes silver nanoparticles functionalized with thiopronin.
  • nanoparticles those that have a nucleus formed by a noble metal are especially interesting, mainly due to their plasmonic properties, which allow it to act as molecular markers [Gong, JL et al. Ag / SiO2 core-shell nanoparticle-based surface-enlianced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools. Biosens Bioelectron 22, 1501-7 (2007)], together with its signal amplification effects in RAMAN and SEIR spectroscopy. They are also used as contrast elements in electron microscopy [Murphy, CJ. et al. Anisotropic metal nanoparticles: Synthesis, assembly, and optical applications.
  • ToIl receptors recognize what is known as pathogen-associated patterns or PAMPs (from pathogen-associated molecular patterns [Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. CeIl 124, 783-801 ( 2006)]
  • PAMPs pathogen-associated molecular patterns
  • These TLRs thus form the main system of detection of what is known as innate immunity and in this sense they are fundamental to recognize the own thing of the alien in the organism human. For this reason, the modulation of responses induced by the activation of TLRs is suggested as a therapeutic target in infectious diseases, sepsis, inflammatory and / or autoimmune diseases or in the development of vaccines [Romagne, F.
  • nanoparticles have a silver core of size between 1 and 100 nm coated with a thiopronin monolayer.
  • the nanoparticles have a silver core of size between 2 and 10 nm and even more preferably the nanoparticles have a 5nm silver core.
  • the immunomodulatory composition acts on the TLR2, TLR2 / 6, TLR3 and TLR9 receptors and is used for the treatment of inflammatory pathologies produced by:
  • the immunomodulatory composition can be used ex vivo in immune cell therapies where cell transfer occurs and also as adjuvants in vaccination protocols.
  • Another aspect of the present invention is an immunomodulatory composition for the treatment of pathologies mediated by the TLR2, TLR2 / 6, TLR3 and TLR9 receptors.
  • Said composition comprises nanoparticles with a silver core of a size between 1 and 100 nm coated with a thiopronin monolayer.
  • the nanoparticles have a silver core of size between 2 and 10 nm and even more preferably the nanoparticles have a 5nm silver core.
  • FIG. 1 A. Electron microscopy images of Ag @ nanoparticles obtained with the Philips-FEI CM200 miscroscope.
  • B Scheme of a thiopronin molecule adsorbed to an Ag nanoparticle (not to scale) indicating the atoms of the thiopronin molecule used in the interpretation of NMR spectra.
  • Figure 2 Effect of Ag @ on the viability of Raw 256.7 measured as release of LDH (membrane integrity) or mitochondrial function (reduction of MTT) after 24 hours of cultivation at the indicated conditions.
  • MG-32 is a high toxicity proteasome inhibitor that we use as a positive control.
  • FIG. 3 Differential regulation of IL-6 production stimulated by different TLR ligands in Raw 264.7 in the absence or in the presence of Ag @.
  • A. Shows the location TLRs on the cell surface.
  • B. Shows the TLRs located in the endocytic compartment.
  • FIG. 4 The previous exposure of Ag @ modulates the subsequent response of IL-6 production after stimulation with TLR ligands in Raw 264.7 A. It shows the localization TLRs on the cell surface. B. Shows the TLRs located in the endocytic compartment.
  • the TLRs can be divided into two groups according to their cellular location: TLRs 1, 2, 4, 5, 6 are mainly located on the cell surface and primarily recognize components of the battery wall, on the contrary the TLRs 3, 1, 8, and 9 are found in endocytic compartments and primarily recognize viral products. There are 13 paralogs identified to date in mouse and human genomes, however the ligands of some of them are not known for now. Binding of the ligand to TLR produces a secretion of pro-inflammatory cytokines such as the IL-6 chosen in the present invention [Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. CeIl 124, 783-801 (2006)].
  • IL-6 pro-inflammatory cytokines
  • Silver nitrate (AgNO 3 , 99.8%, Panreac, Lyon, France), _V- (2- mercaptopropionyi) glycma (thiopronin,> 98%) and NaBH 4 , 98% come from Sigma-Aldrich, St. Louis, MO, USES. Water grade Milli-Q, Millipore, Biller ⁇ ca, MA, USA.
  • Ag @ thiopronin are prepared by reduction of AgNO 3 using NaBH 4 as a reducing agent, in an aqueous solution containing thiopronin (thiopronin / Ag molar ratio of 3: 1), according to procedures described previously [Song, Y ., Huang, T. & Murray, RW Heterophase ligand exchange and metal transfer between monolayer protected clusters. J Am Chem Soc 125, 11694-701 (2003) and. Huang, LW & Murray, RW Luminescence of tiopronin monolayer-protected silver clusters changes to that of gold clusters upon galvanic core metal exchange. J Phys. Chem. B 107, 7434-7440 (2003)].
  • NMR spectra are obtained at 500 MHz on a Bruker AMX-500 spectrometer at room temperature in deuterated water.
  • the HMBC (Heteronuclear Multiple Bond Coherence) studies are optimized for a J H1C -S HZ and the TOCSY (Total Correlation Spectroscopy) studies are carried out with a DPFGSE sequence, 50 ms selective pulses and a 120 ms mixing time .
  • a Bruker IFS 66 / s spectrometer with DTGS detector (Billerica, MA, USA) was used. 150 scans were acquired with a frequency of 2.5 Hz and resolution of 1 cm "1. All spectra are obtained in KBr tablets with 10 mg of nanoparticles.
  • TLR Toll-like receptors
  • the cell line comes from the European Collection (ECACC, Gate Down, Wiltshire, UK). Cultivation conditions are the standards and previously used. Cultures are carried out in 12-well plates in a final volume of 2 mL. Macrophages are treated for 24 hours with specific TLR ligands at the concentration previously optimized in the absence or in the presence of 10 ppm of Ag @. The list of ligands and final concentrations used are indicated below: Lipopolysaccharide (1 ⁇ g / mL LPS. E. coli serotype 0127: B8, Sigma, St. Louis, MO, USA), Oligodeoxynucleotides with unmethylated CpG motifs (1 ⁇ g / mL CpG +.
  • CpG-DNA 1668 5'- TCCATGACGTTCCTGATGCT-3 ', TIB MolBiol, Berlin, Germany
  • polyinosinic acid polycycidyl (50 ⁇ g / mL poly I: C)
  • lipoteic acid 10 ⁇ g / mL LTA
  • lipopeptide synthetic bacterial Pam 3 CSK4 300 ng / mL
  • bacterial DNA 10 ⁇ g / mL DNA
  • synthetic mycoplasma lipoprotein (1 ⁇ g / mL FSL-I)
  • imiquimod (10 ⁇ g / mL IMQ) and ssRNA40 (0.25 ⁇ g / mL) were obtained from InvivoGen, San Diego, CA, USA.
  • Supernatants are stored after 24 hours of treatment and the production of IL-6 is measured by conventional ELISA according to the manufacturer's instructions (OptEI
  • LDH lactate dehydrogenase
  • Cytotoxic effects were characterized by quantifying the release of LDH in the medium and quantifying the MTT reducing capacity. Both parameters measure, respectively, the integrity of the cell membrane and the metabolic capacity of the cells and are compromised when the cell viability is reduced.
  • Figure 2 shows that Ag @ (1-100 ppm) have no cytotoxic effects on Raw 264.7 cells, therefore the effects observed by
  • Ag @ are not due to a compromise of the viability of Raw 264.7 cells, but to a specific alteration of them in the TLR signaling system.
  • Ag @ are not pro-inflammatory agents since the baseline levels of IL-6 production were not affected ( Figure 3). It is interesting to assure that Ag @ are a better option compared to other metal or ceramic nanoparticles that do produce an increase in the secretion of pro-inflammatory cytokines [Lucarelli, M. et al. Innate defense functions of macrophages can be biased by nano-sized ceramic and metallic particles. Eur Cytokine Netw 15, 339-46 (2004)]. However, Ag @ differently inhibit IL-6 secretion mediated by TLRs located on the cell surface ( Figure 3A) or in the endocytic compartments ( Figure 3B).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Nanotechnology (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne l'utilisation des effets immunomodulateurs des nanoparticules métalliques d'argent fonctionnalisées avec de la tiopronine [N-(2-mercaptopropionyl) glycine]. Un second aspect de cette invention concerne une composition immunomodulatrice contenant des nanoparticules d'argent fonctionnalisées avec de la tiopronine.
PCT/ES2009/000476 2008-10-02 2009-09-25 Utilisation de nanoparticules de métaux nobles en tant qu'immunomodulateurs et composition immunomodulatrice Ceased WO2010037878A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200802831 2008-10-02
ES200802831A ES2335852B1 (es) 2008-10-02 2008-10-02 Utilizacion de nanoparticulas de metales nobles como inmunomoduladores y composicion inmunomoduladora.

Publications (2)

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WO2010037878A1 true WO2010037878A1 (fr) 2010-04-08
WO2010037878A9 WO2010037878A9 (fr) 2010-06-10

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928958A (en) * 1994-07-27 1999-07-27 Pilgrimm; Herbert Superparamagnetic particles, process for their manufacture and usage
WO2007025274A2 (fr) * 2005-08-25 2007-03-01 Boston Scientific Scimed, Inc. Structures endovasculaires auto-assemblees
WO2008008483A2 (fr) * 2006-07-12 2008-01-17 The Regents Of The University Of Michigan Compositions basées sur dendrimères et procédés d'utilisation correspondants
WO2009095516A1 (fr) * 2008-01-31 2009-08-06 Universidad Pablo De Olavide Nanoparticules métalliques fonctionnalisées avec le neuropeptide vip et procédé de préparation de celles-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928958A (en) * 1994-07-27 1999-07-27 Pilgrimm; Herbert Superparamagnetic particles, process for their manufacture and usage
WO2007025274A2 (fr) * 2005-08-25 2007-03-01 Boston Scientific Scimed, Inc. Structures endovasculaires auto-assemblees
WO2008008483A2 (fr) * 2006-07-12 2008-01-17 The Regents Of The University Of Michigan Compositions basées sur dendrimères et procédés d'utilisation correspondants
WO2009095516A1 (fr) * 2008-01-31 2009-08-06 Universidad Pablo De Olavide Nanoparticules métalliques fonctionnalisées avec le neuropeptide vip et procédé de préparation de celles-ci

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CASTILLO P. M. ET AL.: "Tiopronin monolayer- protected silsee nanoparticles modulate IL-6 secretion mediated by Toll-like receptor ligands", NANOMEDICINE., vol. 3, no. 5, October 2008 (2008-10-01), pages 627 - 635 *
ZHENG M. ET AL.: "Nanoparticles comprising a mixed monolayer for specific bindings with biomolecules", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 126, no. 38, 29 September 2004 (2004-09-29), pages 12047 - 12054 *

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Publication number Publication date
WO2010037878A9 (fr) 2010-06-10
ES2335852A1 (es) 2010-04-05
ES2335852B1 (es) 2011-02-28

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