EP1722816A2 - Systeme porteur sous la forme de nanoparticules a base proteique et destine a l'enrichissement a specificite cellulaire de principes actifs pharmaceutiques - Google Patents

Systeme porteur sous la forme de nanoparticules a base proteique et destine a l'enrichissement a specificite cellulaire de principes actifs pharmaceutiques

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Publication number
EP1722816A2
EP1722816A2 EP05715659A EP05715659A EP1722816A2 EP 1722816 A2 EP1722816 A2 EP 1722816A2 EP 05715659 A EP05715659 A EP 05715659A EP 05715659 A EP05715659 A EP 05715659A EP 1722816 A2 EP1722816 A2 EP 1722816A2
Authority
EP
European Patent Office
Prior art keywords
nanoparticles
antibody
carrier system
cell
specific
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.)
Withdrawn
Application number
EP05715659A
Other languages
German (de)
English (en)
Inventor
Sabine Balthasar
Hagen Von Briesen
Norbert Dinauer
Jörg KREUTER
Klaus Langer
Heidrun Wartlick
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.)
LTS Lohmann Therapie Systeme AG
Original Assignee
LTS Lohmann Therapie Systeme AG
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Filing date
Publication date
Application filed by LTS Lohmann Therapie Systeme AG filed Critical LTS Lohmann Therapie Systeme AG
Publication of EP1722816A2 publication Critical patent/EP1722816A2/fr
Withdrawn legal-status Critical Current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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6927—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 solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • 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
    • A61K9/5192—Processes
    • 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

Definitions

  • Carrier system in the form of protein-based nanoparticles for cell-specific enrichment of pharmaceutically active substances. drugs
  • the invention relates to a carrier system for pharmaceutically active substances which is suitable for cell-specific enrichment of the pharmaceutically active substances and in the form of avidin-modified nanoparticles based on: protein, preferably based on gelatin and / or ode-t serum albumin, in particular human serum albumin (HSA ), to which biotinylated antibodies are bound by the formation of a stable avidin-biotin complex and in which an additional binding of pharmaceutically active substances can take place either covalently or in a complexing manner via the avidin-biotin system as well as incorporated or adsorptively.
  • protein preferably based on gelatin and / or ode-t serum albumin, in particular human serum albumin (HSA )
  • HSA human serum albumin
  • Nanoparticles are particles between 10 and 1000 nm in size made from artificial or natural macromolecular substances to which drugs or other biologically active material are covalently, ionically or adsorptively bound or in which this material can be incorporated.
  • EP 1 392 255 discloses nanoparticles based on human serum albumin, to which apolipoprotein E is coupled covalently or via an avidin / biotin system in order to overcome the blood-brain barrier.
  • a particular goal of pharmacotherapy is not only the specific enrichment of a pharmacologically active ingredient or therapeutically active drug to be achieved in a specific tissue or organ, as disclosed in EP 1 392 255, but moreover even in specific cells.
  • Unmodified nanoparticles enable passive "drug targeting", which is characterized in that the particles are taken up by cells of the mononuclear phagocyte system (MPS) after intravascular application. An accumulation of such nanoparticles has been found in macrophages of the liver, spleen, and bone marrow as well as in circulating monocytes. Active "drug targeting” is distinguished from passive “drug targeting", in which the active ingredient is to be specifically enriched with the aid of modified nanoparticles, even in primarily inaccessible body compartments or cell systems. This is necessary for this To use nanoparticles with hydrophilic surface structures that minimize non-specific interactions with non-target cells and to equip them with ligands that enable cell-specific enrichment of the nanoparticles.
  • MPS mononuclear phagocyte system
  • Such ligands are also referred to as “drug targeting ligands”.
  • drug targeting ligands The use of cell-specific nanoparticles as a carrier for medicinal products enables a pharmacologically active ingredient to be accumulated in target cells under controlled conditions or to be brought to its target location in the body. Most drugs do not achieve this goal without a suitable dosage form and show cellular enrichment or body distribution, which is due to the physico-chemical properties of the active ingredient itself. Only a part of the applied drug reaches the desired destination, whereas the remaining part is responsible for undesirable side effects or toxic effects. Cell-specific nanoparticles therefore help to reduce unwanted side effects and toxic properties of active ingredients.
  • hydrophilic latex particles were used which were produced by the copolymerization of hydroxyethyl methacrylate, methacrylic acid and methyl ethacrylate. An antibody against rabbit ⁇ -globulin was bound to these particles. In comparison to unmodified particles, binding of the antibody-modified preparation to lymphocytes was observed, which were pre-incubated with an antiserum obtained from the rabbit against these lymphocytes.
  • Corresponding particle systems based on polyacrylates with additionally integrated iron oxide were subsequently used to carry out a magnetic separation of lymphocytes and erythrocytes.
  • adsorptive binding of ligands is not suitable for cell-specific drug targeting in biological systems.
  • Nanoparticle systems can be seen in the fact that they are based on non-biodegradable polymer materials such as latex and polyacrylates.
  • nanoparticles which do not have the disadvantages of the nanoparticle systems described, but instead have a high cell specificity when used in biological systems in order to be able to specifically enrich pharmacologically active substances in selected target cells, and that are based on a biodegradable material.
  • the task is surprisingly achieved by a carrier system in the form of avidin-modified protein-based nanoparticles to which biotinylated antibodies are attached Formation of a stable avidin-biotin complex are bound.
  • Gelatin and / or serum albumin, particularly preferably human serum albumin are preferably used as proteins.
  • pharmacologically active substances can be additionally bound to the nanoparticles both covalently, complexing via the avidin-biotin system and also incorporated or adsorptively.
  • FIG. 1 shows the structure of an avidin-modified nanoparticle based on gelatin or HSA, with an antibody bound via an avidin-biotin complex.
  • Figure 2 is a bar graph showing the cellular uptake of antibody (trastuzumab) -modified gelatin A manoparticles in various breast cancer cell lines as determined by FACS analysis.
  • the antibody-modified nanoparticles were compared with the unmodified nanoparticles under the same incubation conditions. Untreated cells served as controls.
  • an aqueous gelatin solution was converted into nanoparticles by a double desolvation process and these were subsequently stabilized by crosslinking.
  • the functional groups (amino groups, carboxyl groups, hydroxyl groups) on the surface of these nanoparticles can be converted into reactive thiol groups by suitable reagents.
  • Functional proteins can have bifunctional spacer molecules that are both reactive towards amino groups as well as free thiol groups, to which thiol group-modified nanoparticles are bound. These functional proteins include, in particular, avidine derivatives or cell-specific antibodies.
  • the antibodies were either acquired biotinylated or biotinylated by reaction with NHS-biotin (N-hydroxysuccinimidobiotin) and the avidin-modified nanoparticles were added.
  • NHS-biotin N-hydroxysuccinimidobiotin
  • the avidin-modified nanoparticles were added.
  • As a result of the avidin-biotin complex formation described antibody-modified nanoparticles based on gelatin were obtained (FIG. 1).
  • Corresponding antibody-modified nanoparticles can also be based on Serum albumin, preferably human serum albumin.
  • the present invention thus comprises a carrier system for cell-specific, intracellular enrichment of at least one pharmacologically active substance, which is in the form of protein-based nanoparticles and has structures coupled via reactive groups which enable cell-specific attachment and cellular uptake of the nanoparticles.
  • Gelatin and / or serum albumin, particularly preferably human serum albumin, are preferably used as the protein base.
  • the reactive group is preferably an amino thiol, carboxyl group or an avidin derivative and the coupled structure is an antibody, particularly preferably an onoclonal antibody.
  • the invention also comprises a corresponding carrier system which additionally contains at least one pharmaceutically active substance which is bound to the carrier system or the nanoparticles by adsorption, incorporation or covalent or complexing bond via the reactive groups.
  • the invention also includes the use of a carrier system according to the invention for the production of a medicament for the enrichment of a pharmaceutically active substance on or in specific cells.
  • the invention further comprises a method for producing a carrier system in the form of protein-based nanoparticles for cell-specific enrichment, at least a pharmacologically active substance which comprises the following steps: desolvation of an aqueous protein solution, stabilization of the nanoparticles formed by desolvation by crosslinking, conversion of some of the functional groups on the surface of the stabilized nanoparticles to reactive thiol groups, covalent attachment of functional proteins, preferably of avidin, by means of bifunctional spacer molecules, optionally biotinylation of the antibody, loading of the avidin-modified nanoparticles with biotixiylated antibody, - loading of the avidin-modified nanoparticles with biotinylated and pharmaceutically or biologically active substance.
  • gelatin and / or serum albumin, in particular serum albumin, of human origin is preferred.
  • Desolvation is preferably carried out by stirring and adding a water-miscible non-solvent for proteins or by salting out.
  • the water-miscible non-solvent for proteins is preferably selected from the group comprising ethanol, methanol, isopropanol, and acetone.
  • Thermal processes or bifunctional aldehydes in particular glutaraldehyde, or formaldehyde are preferably used to stabilize the nanoparticles.
  • a bifunctional spacer molecule is preferably one
  • Protein nanoparticles were dissolved 500 mg of gelatin A in 10.0 ml of purified water with heating and precipitated into a sediment by adding 10.0 ml of acetone. The precipitated gelatin was separated off, redissolved in 10.0 ml of water while heating, and the pH of the solution was adjusted to pH 2.5. Nanoparticles were obtained from this solution by dropwise addition of 30 ml acetone (desolvation process). The nanoparticles were stabilized by adding 625 ⁇ l of 8% glutaraldehyde and stirring overnight. The nanoparticles were purified in 2.0 ml aliquots by 5 cycles of centrifugation and redispersion using ultrasound treatment.
  • a further purification of the now covalently FITC-NeutrAvidin TM -modified nanoparticles was carried out as described above.
  • the supernatants obtained from the particle purification were examined photometrically for unbound NeutrAvidin TM and the proportion of covalently bound NeutrAvidin TM was calculated therefrom.
  • the NeutrAvidin TM -modified nanoparticles (20 mg / ml) were mixed with 500 ⁇ l of the biotinylated antibody (25 ⁇ g / ml) and incubated at 10 ° C. for 90 min. After the incubation, the particles were cleaned again by centrifugation and redispersion. The particle supernatants obtained were examined by Western blot analysis for unbound antibody. It was shown that more than 80% of the antibody used was connected to the particle system.
  • cell-specific particle accumulations in target cells were found in various cell culture experiments which carry the surface antigen recognized by the antibody.
  • the following cell culture models were used:
  • Nanoparticles were loaded with the approved antibody Trastuzu ab (Herceptin ® ), which had previously been biotinylated. The cultured cells were incubated with the nanoparticle system in concentrations between 100 and 1000 ⁇ g / l and after 4 h of incubation, unbound nanoparticles were separated by washing the cells. The cells were examined using flow cytometry (FACS) and confocal microscopy (CLSM) with regard to nanoparticle uptake.
  • FACS flow cytometry
  • CLSM confocal microscopy
  • Jurkat T cells were seeded at a density of 1 ⁇ 10 6 cells per well on a 24-well microtiter plate and RPMI medium cultured. The medium was supplemented with 10% (v / v) fetal calf serum (FCS), 2 ⁇ L-glutamine and 1% penicillin / streptoycin.
  • FCS fetal calf serum
  • the nanoparticles modified with the antibody were mixed with the cells in a concentration of 1000 ⁇ g / ml
  • HER2 overexpressing cells (BT474 and SK-Br-3) were used in a density of 2 X 10 5 and 1 X 10 5 cells per Well on a 24 well Microtiter plate sown and cultivated in RPMI medium or M ⁇ Coy's 5 A.
  • the medium of the BT474 was supplemented with 20% (v / v) fetal calf serum (FCS), 2% L-glutamine, 1 penicillin / streptomycin and 100 U insulin.
  • the medium of SK-Br-3 was supplemented with 10% (v / v) fetal calf serum (FCS), 2% L-glutamine and 1% penicillin / streptomycin.
  • FCS fetal calf serum
  • the nanoparticles modified with the antibody were incubated with the cells at a concentration of 100 ⁇ g / ml over a period of 3 h.
  • various comparative studies were carried out. On the one hand, nanoparticles were used that were not loaded with a specific antibody.
  • the investigations were carried out with MCF-7 cells (normal HER2 expression).
  • Antibody also did not result in uptake in the target cells. Control experiments were also carried out with breast cancer cells (MCF-7 cells) that do not have the CD3 surface antigen. In these control experiments, all were chosen
  • the cells used showed an expression of the HER2 surface antigen to varying degrees, which was used as a target for cellular uptake of the antibody-modified nanoparticles.
  • the expression of the cells was determined by Western blot analysis before incubation with the nanoparticles (Table 1).
  • Table 1 Expression of the HER2 surface antigen on the surface of various tumor cells determined by Western blot analysis. Expression was calculated relative to the values of the "normally expressing" MCF-7 cells. Both FACS and CLSM were able to show that nanoparticles were taken up in cells, which were used modified with the cell-specific antibody trastuzumab (FIG. 2). The cellular uptake of the specific nanoparticles could be prevented if the cells were treated with the free specific antibody before the particle addition. Nanoparticles of the same production approach, which were not used modified with the biotinylated antibody, showed only a low cellular enrichment under the chosen conditions. The extent of the cellular uptake of the antibody-modified nanoparticles could be correlated with the extent of the expression of the HER2 surface antigen.
  • Enable target cells Under comparable conditions, the particle systems are only taken up in the corresponding target cells but not by control cells. The pre-incubations with free specific antibody clearly demonstrate that particle uptake occurs through a process of receptor-mediated endocytosis.
  • the developed nanoparticulate drug carrier system thus offers the possibility of transporting drugs specifically to diseased cells, provided that these target cells differ from healthy cells in their surface properties.
  • a well-characterized, particulate carrier system is provided which, with a functional drug targeting ligand which carries it on its surface, enables cell-specific uptake and enrichment, possibly also by the carrier system Adsorption, incorporation or pharmaceutically active substances bound by covalent or complexing bond enables.

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Abstract

L'invention concerne un système excipient se présentant sous la forme de nanoparticules à base protéique et destiné à l'enrichissement intracellulaire à spécificité cellulaire au moins d'un principe pharmacologiquement actif qui présente des structures couplées par des groupes réactifs. Ces structures permettent une addition à spécificité cellulaire et une absorption cellulaire des nanoparticules. L'invention concerne également son procédé de production.
EP05715659A 2004-03-09 2005-03-02 Systeme porteur sous la forme de nanoparticules a base proteique et destine a l'enrichissement a specificite cellulaire de principes actifs pharmaceutiques Withdrawn EP1722816A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004011776A DE102004011776A1 (de) 2004-03-09 2004-03-09 Trägersystem in Form von Nanopartikeln auf Proteinbasis zur zellspezifischen Anreicherung von pharmazeutisch aktiven Wirkstoffen
PCT/EP2005/002185 WO2005089797A2 (fr) 2004-03-09 2005-03-02 Systeme porteur sous la forme de nanoparticules a base proteique et destine a l'enrichissement a specificite cellulaire de principes actifs pharmaceutiques

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US (1) US20080095857A1 (fr)
EP (1) EP1722816A2 (fr)
JP (1) JP2007527881A (fr)
KR (1) KR20070006828A (fr)
CN (1) CN1993145A (fr)
AU (1) AU2005223986B2 (fr)
BR (1) BRPI0508134A (fr)
CA (1) CA2558730A1 (fr)
DE (1) DE102004011776A1 (fr)
IL (1) IL177879A0 (fr)
NZ (1) NZ549355A (fr)
RU (1) RU2388463C2 (fr)
WO (1) WO2005089797A2 (fr)

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DE102006011507A1 (de) 2006-03-14 2007-09-20 Lts Lohmann Therapie-Systeme Ag Wirkstoffbeladene Nanopartikel auf Basis hydrophiler Proteine
JP2008162981A (ja) * 2006-12-28 2008-07-17 Japan Science & Technology Agency ビオチン化ないしホーミングペプチド提示型バイオナノカプセル
GB0724360D0 (en) * 2007-12-14 2008-01-23 Glaxosmithkline Biolog Sa Method for preparing protein conjugates
US9125949B2 (en) * 2008-12-30 2015-09-08 University Of North Texas Direct utilization of plasma proteins for the in vivo assembly of protein-drug/imaging agent conjugates, nanocarriers and coatings for biomaterials
US9211283B2 (en) * 2009-12-11 2015-12-15 Biolitec Pharma Marketing Ltd Nanoparticle carrier systems based on human serum albumin for photodynamic therapy
RU2542417C2 (ru) * 2013-05-17 2015-02-20 Александр Александрович Кролевец Способ биоинкапсуляции лекарственных препаратов группы цефалоспоринов
CA2949092A1 (fr) * 2014-05-16 2015-11-19 Dana-Farber Cancer Institute, Inc. Particules a base de proteines permettant d'administrer un medicament
WO2015018380A2 (fr) 2014-07-03 2015-02-12 Cspc Zhongqi Pharmaceutical Technology(Shijiazhuang)Co., Ltd. Nanoparticules thérapeutiques et leurs procédés de préparation
CA3039195A1 (fr) * 2016-10-10 2018-04-19 Abraxis Bioscience, Llc Formulations nanoparticulaires et leurs procedes de production et d'utilisation
WO2020241562A1 (fr) * 2019-05-24 2020-12-03 ユーハ味覚糖株式会社 Nanoparticules et leur procédé de production
US20220387338A1 (en) * 2019-10-04 2022-12-08 Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec Hydrogel-like particles, methods and uses thereof
CN112451679A (zh) * 2020-11-25 2021-03-09 天津医科大学第二医院 结合了纳米药物载体的卡介苗复合体及其制备方法
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NZ549355A (en) 2009-09-25
RU2006130260A (ru) 2008-02-27
WO2005089797A3 (fr) 2006-11-23
CA2558730A1 (fr) 2005-09-29
KR20070006828A (ko) 2007-01-11
IL177879A0 (en) 2006-12-31
BRPI0508134A (pt) 2007-07-17
JP2007527881A (ja) 2007-10-04
US20080095857A1 (en) 2008-04-24
AU2005223986A1 (en) 2005-09-29
CN1993145A (zh) 2007-07-04
RU2388463C2 (ru) 2010-05-10
WO2005089797A2 (fr) 2005-09-29
DE102004011776A1 (de) 2005-11-03
AU2005223986B2 (en) 2010-12-23

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