WO2012054564A2 - Nanoparticules d'or conjuguées avec un anticorps anti-vegf/fragment, méthodes de fabrication et méthodes thérapeutiques - Google Patents

Nanoparticules d'or conjuguées avec un anticorps anti-vegf/fragment, méthodes de fabrication et méthodes thérapeutiques Download PDF

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WO2012054564A2
WO2012054564A2 PCT/US2011/056825 US2011056825W WO2012054564A2 WO 2012054564 A2 WO2012054564 A2 WO 2012054564A2 US 2011056825 W US2011056825 W US 2011056825W WO 2012054564 A2 WO2012054564 A2 WO 2012054564A2
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derived therefrom
vegf
fragment derived
bevacizumab
gold
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WO2012054564A3 (fr
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Ravi Shukla
Dean P. Hainsworth
Kattesh V. Katti
Raghuraman Kannan
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The Curators Of The University Of Missouri
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A61K9/0051—Ocular inserts, ocular implants
    • 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
    • A61K47/6845—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 cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • 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
    • A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • a field of the invention is anti-VEGF (vascular endothelial growth factor) treatments.
  • Example applications of the invention include a therapeutic method for slow responsive release of anti-VEGF antibodies and fragments thereof for treatment of diseases comprising neovascularisation, e.g. cancers and 5 macular degeneration.
  • vascular endothelial growth factor vascular endothelial growth factor
  • Bevacizumab is an example humanized monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A), which is a chemical signal that stimulates angiogenesis.
  • VEGF-A vascular endothelial growth factor A
  • US FDA United States Food and Drug Administration
  • An early approved use was for combined use with standard chemotherapy for metastatic colon cancer and non-small cell lung cancers.
  • a once approved but currently withdrawn use by the US FDA was for breast cancer, but other countries still list bevacizumab for breast cancer. It is also prescribed "off-label" in the US for breast cancer.
  • effective delivery of bevacizumab or another anti-VEGF agent should target the location of the cancer.
  • Bevacizumab is commercially available as Avastin 1 and has also been recognized as an effective treatment for eye diseases, such as age-related macular degeneration.
  • Clinical studies and off-label prescriptions have been conducted to evaluate bevacizumab for treatment of macular degeneration, an eye disease also characterized by proliferation of blood vessels in the retina.
  • One difficulty that remains concerns delivery Free bevacizumab in the eye has a short duration of efficacy, which requires that a treatment procedure include multiple injections over a period of time. The practice of multiple injections creates additional risks that can outweigh the potential benefit of bevacizumab delivery to the eye.
  • bevacizumab has been reported to have an intraocular half life of 4.3 days following intravitreal injection.
  • Ranibizumab (trade name Lucentis) is a monoclonal antibody fragment (Fab) derived from the same antibody as bevacizumab. Ranibizumab is offered commercially tinder the tradename of Lucentis , M . It also binds to VEGF- A. Ranibizumab has been approved to treat the "wet" type of age-related macular degeneration, but the intraocular injection regime is like that of bevacizumab and presents the same risks.
  • Fab monoclonal antibody fragment
  • Gold nanoparticles have been investigated for various diagnostic and therapeutic methods including molecular imaging, cancer targeting and drug delivery. See, e.g., Chan CP. et al, "New Trends in Immunoassays," Adv Biochem Eng Biotechnol 2008;109:123-54; Hainfeld J.F., "Gold Nanoparticles: A New X-ray Contrast Agent," Br J Radiol 2006;79:248-53; Huang X. J.P., et al. "Gold Nanoparticles and Nanorods in Medicine: From Cancer Diagnostics to Photothermal Therapy," Nanomedicine 2007; 2:4. Gold nanoparticles are typically in the range of 1-100 nra in size.
  • nanoparticles When gold nanoparticles are used in testing and therapeutic methods in the art, the nanoparticles are formed and then any ligands are chemically attached. Molecules being attached must withstand the procedure of attachment. Unfortunately, the chemical attachment of monoclonal antibodies can destroy the biological activity of the antibody. Other delivery vehicles have therefore been the focus of research and investigation.
  • Preferred embodiments of the invention include gold nanoparticles, preferably less than about 20nm, that are conjugated with biologically active anti- VEGF antibody or a fragment derived therefrom.
  • the particles can be formed by a "one-pot" synthesis method of the invention, in which the biologically active anti-VEGF antibody or a fragment derived therefrom acts to stabilize formed gold nanoparticles while simultaneously retaining biological activity.
  • the method uses a reducing agent of nontoxic trimeric alanine compound that is benign toward the biological activity of the anti-VEGF antibody or a fragment derived therefrom.
  • Therapeutic methods of the invention include injection of a solution of conjugated gold nanoparticles of the invention at vascularisation sites such as cancer sites and intraocular injection.
  • Preferred embodiments of the invention include gold nanoparticles, preferably less than about 20nm, conjugated with a biologically active anti-VEGF antibody or fragment derived therefrom.
  • the particles can be formed by a "one- pot" synthesis method of the invention, in which the biologically active anti- VEGF antibody or fragment derived therefrom acts to stabilize formed gold nanoparticles while simultaneously retaining biological activity.
  • the synthesis method in which the biologically active anti-VEGF antibody or fragment derived therefrom acts as a stabilizing agent is benign to the biological activity of the biologically active anti-VEGF antibody or fragment derived therefrom, which is therefore retained after conjugation.
  • the synthesis methods of the invention have been demonstrated to not affect biological activity of the anti-VEGF humanized monoclonal antibody bevacizumab when it is used as a stabilizing agent in the synthesis method.
  • Ranibizumab the monoclonal antibody fragment (Fab) derived from the same antibody as bevacizumab, has also been successfully conjugated when to gold nanoparticles via the method of the invention when used as a stabilizing agent in the same manner.
  • Animal testing had not yet been completed on ranibizumab at the time of this application's filing, however, the inventors believe that biological activity will be retained because the preferred synthesis methods utilize a reducing agent of nontoxic trimeric alanine compound.
  • This reducing agent has been demonstrated to not affect the biological activity of bevacizumab and its benign nature is believed, based upon the results, to be benign toward the biological activity of anti-VEGF antibodies or fragments thereof, toward anti-VEGF monoclonal antibodies or fragments thereof, and toward the specific anti-VEGF humanized monoclonal antibody bevacizumab and its fragment ranibizumab.
  • the testing of gold nanoparticles of the invention conjugated with biologically active bevacizumab has shown that the formation process does not affect biological activity of the bevacizumab. Tests have also shown that the biological activity and release can be retained for exceptionally long periods that would permit less frequent injections in a therapeutic method of the invention.
  • the release is triggered by biological events (e.g., intraocular neovascularization) in the eye over a period of months, instead of weeks. The longevity and long dwell time in the eye have been demonstrated with animal studies.
  • the preferential binding to new blood vessels provided by the gold nanoparticles also provides benefits not available with other delivery vehicles.
  • the therapeutic methods and preferential of the invention include the injection at cancer sites where the anti-VEGF antibody or fragment derived therefrom conjugated gold nanoparticles will preferentially bind at the cancer site.
  • bevacizumab nanoparticles For the fabrication of bevacizumab nanoparticles in experiments, the precursors were obtained from commercial vendors: NaAuCl 4 (Alfa-Aesar); AvastinTM (Genentech); THPAL (Nanoparticles Biochem Inc) grids and other chemicals through Fisher Scientific.
  • Preferred embodiment gold nanoparticles with non-covalently bonded bevacizumab were formed by the reduction of gold precursors (a source of Au J r ions) by a reducing agent.
  • sodium tetrachloro aurate (NaAuCl 4 ) with a nontoxic trimeric alanine compound, trimetric alanine peptide, (P[CH 2 NHCH(CH) 3 COOH]3;THPAL(Trimeric Alanine Phosphine conjugate)) reducing agent in the presence of bevacizumab, which acted as a stabilizing solution.
  • a nontoxic trimeric alanine compound trimetric alanine peptide
  • P[CH 2 NHCH(CH) 3 COOH]3;THPAL(Trimeric Alanine Phosphine conjugate) reducing agent
  • bevacizumab which acted as a stabilizing solution.
  • a stabilizing solution Specifically, to a glass vial was added 4 ml of sterile de-ionized water followed by the addition of 80 ⁇ of Avastin solution (25 mg/ niL).
  • the UV-Vis spectrum of gold nanoparticles showed a strong absorption band at ca. 530 nm is observed that corresponds to surface plasmon resonance of the gold nanoparticles.
  • the presence of a ca. 530 peak is an indicative of the bevacizumab gold nanoparticle synthesis.
  • the nanoparticles were further characterized by TEM imaging, which showed that the average morphology of the nanoparticles to be quasi-spherical and that the particles appear to be nearly monodispersed in size distribution histogram.
  • the core particle size as determined by TEM was 9.4 nm (SD 1.1 nm).
  • Hydrodynamic size as measured by zeta seizer nano S90 is 19 ⁇ 1 nm, indicating the protein covering the nanoparticle surface is 8-10 nm (including its hydrodynamic environment.
  • Zeta potential of nanoparticles as measured ranges from +32.5 ⁇ 1 mV.
  • the solution of bevacizumab gold nanoparticles has a dark reddish appearance that remains in suspension for more than 6 months, indicating a very long shelf life of nanoparticles. Synthesis and stability of all gold nanoparticles was monitored by U V-vis spectroscopy on a Varian Cany 50 UV-Vis- spectrophotometer operated at a resolution of 5 nm.
  • Physicochemical properties, such as size, charge, and morphology of bevacizumab gold nanoparticles were determined by two independent techniques; transmission electron microscopy (TEM) and dynamic light scattering (DLS). Dynamic light scattering method was employed to calculate the hydrodynamic size of bevacizumab gold nanoparticles. Zeta Potential ( ⁇ ), provides information on the stability and shelf life of the nanoparticle dispersion. TEM was used to determine the core size of bevacizumab gold nanoparticles and DLS was used to evaluate the hydrodyanamic size of the bevacizumab gold nanoparticles.
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • Samples were prepared by drop-coating fiims of the bevacizuraab gold nanoparticles solution on 300 mesh carbon-coated copper TEM grids followed by measurements on a JEOL model 1400 instrument, JOEL Ltd., Tokyo Japan, operated at an accelerating voltage of lOO kV
  • Bevacizumab gold nanoparticle solution was added to 0.25 mL aliquots of 2.5 % NaCl, 0.2 m histidine, 0.5 % BSA or 0.5 % HSA respectively.
  • MTT assay was performed as described by the manufacturer (ATCC, USA) to evaluate the cytotoxicity of the bevacizumab gold nanoparticle Human fibroblasts primary cultures were obtained as a gift from Prof. Cris Lorson, Bond Life Science Centre, University of Missouri-Columbia. The cells were mantained in DMEM (Gibco BRL) supplemented with 10% donor bovine serum, 10 pgmL "1 phenol red, 100 units mL "1 penicillin, 100 pgmL "1 streptomycin and 10 mm HEPES.
  • the plates were kept for 18 h in dark at 25 °C to dissolve all the crystals and the intensity of color development was measured by micro plate reader (Dynastic MR 5000, USA) operating at 570 nm. Wells with complete medium, nanoparticles, and MTT, but without cells were used as blanks. Untreated cells were considered 100 % viable.
  • bevacizumab gold nanoparticles The biological activity of bevacizumab gold nanoparticles was measured through highly specific sandwiched ELISA assay. This assay is critical in the sense that it provides the estimates of VEGF binding sites available on conjugated bevacizumab gold nanoparticles. Briefly, wells of maxisorp ELISA plate (Nunc) were coated with 50 ⁇ / well predetermined concentrations of free bevacizumab, bevacizumab gold nanoparticles, human IgG conjugated gold nanoparticles and PEG (polyethylene glycol to increase the aqueous solubility) conjugated gold nanoparticles as controls in 0.1 M sodium carbonate coating buffer (pH 9.5) at 4 °C for 16h.
  • 0.1 M sodium carbonate coating buffer pH 9.5
  • the plates were washed twice with wash buffer provided with BD-OptEIA reagent set followed by blocking in Assay diluent (BD) for 2 h at room temperature. After incubation the well were washed 5 times with wash buffer and biological activity of bevacizumab in nanoparticlulate suspension was targeted by reincubation of plate with biotinylated Fluorikine VEGF (R&D systems) for 30 min at room temperature. Following VEGF incubation the wells were futher washed for 5 times with wash buffer and 100 ⁇ / well Strepatvidin HRP (Sigma)( 1 :1000 dilution) added in dark for color development.
  • BD Assay diluent
  • the assay was terminated by addition of 1 M phosphoric acid and plates were read at 450 nm in Powerwave plate reader (Bioteck Instruments, USA). A standard graph of bevacizumab concentration was plotted against OD and bound bevacizumab concentrations were measured.
  • Rat fundus photography was performed immediately and seven days after intravitreal Bevacizumab gold nanoparticle injection using a Carl Zeiss 450 digital camera modified with a Volk 2.2 condensing lens (Volk Ophthalmic,).
  • Angiography was performed 10 minutes after intraperitoneal injection of 0.1ml of 2.5% fluorescein sodium solution.
  • rat eyes were carefully enucleated and placed in modified Davidson's fixative for at least 24 hours. Fixed eyes were processed for histological evaluation using H & E staining.
  • Slides were deparafiinized by placing them in Xylene for 10 min by two changes of xylene, 2 changes of absolute alcohol and 2 changes of 95% alcohol and then rinsed in distilled water.
  • the slides were kept in wash buffer (Dako, Carpinteria, CA cat# S3006) for 5 min followed by 20 min incubation with FITC conjugated affinity purified goat anti human IgG, F(ab') (Jackson Immuno Research Labs, Inc, PA, Cat# 109-095-097) in dark at 25 °C. Since, bevacizumab is a humanized monoclonal antibody, antibody specific for human JgG allowed the presence of bevacizumab to be determined in rat eyes.
  • the retinas were detached from the sclera and both sides of the retina were processed for scanning electron microscopy (SEM).
  • SEM scanning electron microscopy
  • the samples after 24 h of fixation were washed with sodium cacodylate buffer followed by secondary fixation in osmium tetraoxide for 1 h on rocking platform at 25 °C.
  • the samples were washed thrice with ultrapure distilled water and dehydrated in graded acetone series. After acetone dehydration samples were subjected to critical point drying (Tousimis Auto-Samidri 815) and mounted on SEM stub.
  • New Zealand white rabbits were injected with 50 ⁇ of
  • Bevacizumab gold nanoparticle solution intravitreally Four eyes were injected immediately after sacrifice and then enucleated to avoid any possible systemic absorption or metabolism of the Bevacizumab gold nanoparticle. Four eyes were enucleated for each time point at seven days and 21 days post injection and frozen at -70 degrees. The frozen eyes were dissected, separating the tissue into three groups; 1) retina, choroid and sclera, 2) vitreous, and 3) lens, iris, aqueous and cornea.
  • Neutron activation analysis was used to determine whether bevacizumab gold nanoparticle remained localized to the eye after intravitreal injection or was absorbed systemically. NAA was performed to determine the gold content of each group in each eye.
  • Retina, sclera and choroid of the dissected eye were analyzed as a single fraction. It was assumed that the majority of the gold in this fraction was within the retina, with minimal penetration to the sclera. Accordingly, an average rabbit retinal mass of 150 mg for analysis was decided from the available literature.
  • Intravitreal bevacizumab gold nanoparticle injection results in a dark reddish vitreous opacity.
  • bevacizumab gold nanoparticle injection results in a dark reddish vitreous opacity.
  • Immediately following bevacizumab gold nanoparticle injection into rabbit and rat eyes little retinal detail can be seen, although more detail was seen with fluorescein angiography.
  • the diffuse vitreous haze cleared with only fine reddish clumping of bevacizumab gold nanoparticle remains to be seen with retinal examination.
  • These clumps are manifested as focal blockage on fluorescein angiography.
  • These clumps represent aggregates of bevacizumab gold nanoparticles since individual nanoparticles are not discernable without electron microscopy. Testing showed that the vast majority of the bevacizumab gold nanoparticles have migrated out of the vitreous by the first week, resulting in remarkably clear ocular media.
  • the gold measured in the retina/choroid/sclera combined tissue was calculated per tissue weight as if only in the retina.
  • SEM images with associated backscattering confirmed the presence of bevacizumab gold nanoparticle within the retina.
  • the presence of the bevacizumab antibody coating was seen on SEM images surrounding the bevacizumab gold nanoparticles seen on backscatter images.
  • Bevacizumab gold nanoparticle was tested for its immunoreactivity toward human VEGF and long term biological activity of bevacizumab gold nanoparticles was measured through ELISA assay.
  • a highly specific in vitro ELISA assay was used to measure biological activity of bevacizumab in bevacizumab gold nanoparticle using biotinylated VEGF as a detection.
  • Freshly prepared bevacizumab was compared with two different 6 months old batches of Bevacizumab gold nanoparticle stored at 4°C. Human IgG conjugated gold nanoparticles, and gum arabic glycoprotein conjugated nanoparticles were used as negative control. These results demonstrated long term VEGF immunorecativity of bevacizumab gold nanoparticles. The results did not make clear how much amount of bevacizumab is conjugated pr unit of bevacizumab gold nanoparticle, but it the amount of conjugated nanoparticles exhibited a long term stability and preservation of biological activity of bevacizumab towards human VEGF.
  • the synthesis process was a one-pot synthesis that is scalable, and does not require or produce any toxic by-products.
  • the bevacizumab gold nanoparticles clear from the vitreous by dispersing into the retina.
  • the studies showed that after one week, the majority of bevacizumab gold particles remained within the retina. This provides a reservoir of bevacizumab gold particles that can release bevacizumab that can function as a sustained release in response to retinal neovascularisation.
  • bevacizumab which is a humanized IgG antibody, is recognized as foreign in a rat model and a resultant immunological reaction is being mounted against this foreign protein, which would explain the degradation of bevacizumab by two weeks.
  • a therapeutic method of the invention involves initially injecting a combination of free bevacizumab and bevacizumab gold nanoparticles, so that the free bevacizumab is present to inhibit retinal neovascularization and the bevacizumab gold nanoparticles to maintain this inhibition for an extended period of time.
  • Bevacizumab is non-covalently bound to the gold nanoparticles in the invention. This allows for dissociation in the presence of high levels of VEGF in the local intraocular environment; this dissociation being driven by the concentration gradient of increased levels of VEGF. This dissociation of bevacizumab can likely decrease as local VEGF levels decrease, allowing a store of bevacizumab to be maintained until needed by increased activity of retinal neovascularization.
  • the ability of bevacizumab gold nanoparticles to inhibit retinal neovascularization, both initially and after recurrent activity can provide an important therapeutic benefit.
  • the ability of gold nanoparticles to bind preferentially to neovascularization makes it a useful platform for targeted delivery in many retinal diseases.

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Abstract

Cette invention concerne, dans des modes de réalisation préférés, des nanoparticules d'or, de préférence de moins de 20 nm environ, qui sont conjuguées avec un anticorps anti-VEGF biologiquement actif ou avec un fragment dérivé de ce dernier. Les particules peuvent être obtenues par une méthode de synthèse monotope selon l'invention dans laquelle l'anticorps anti-VEGF biologiquement actif ou son fragment dérivé agissent de manière à stabiliser les nanoparticules d'or formées, tout en conservant simultanément l'activité biologique. La méthode utilise un agent réducteur d'un composé alanine trimère non toxique qui est neutre vis-à-vis de l'activité biologique de l'anticorps anti-VEGF ou de son fragment dérivé. Les méthodes thérapeutiques selon l'invention consistent à injecter une solution de nanoparticules d'or conjuguées de l'invention dans des sites de vascularisation, par exemple les sites d'un cancer, et à procéder à une injection par voie intraoculaire.
PCT/US2011/056825 2010-10-19 2011-10-19 Nanoparticules d'or conjuguées avec un anticorps anti-vegf/fragment, méthodes de fabrication et méthodes thérapeutiques WO2012054564A2 (fr)

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