WO2014002108A1 - Nanostructure noyau-coquille basée sur des protéines avec des agents thérapeutiques correspondants - Google Patents

Nanostructure noyau-coquille basée sur des protéines avec des agents thérapeutiques correspondants Download PDF

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WO2014002108A1
WO2014002108A1 PCT/IN2013/000141 IN2013000141W WO2014002108A1 WO 2014002108 A1 WO2014002108 A1 WO 2014002108A1 IN 2013000141 W IN2013000141 W IN 2013000141W WO 2014002108 A1 WO2014002108 A1 WO 2014002108A1
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protein
therapeutic
core
composition
shell
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PCT/IN2013/000141
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Koyakutty MANZOOR
Retnakumari ARCHANA
V. Nair SHANTIKUMAR
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Amrita Vishwa Vidyapeetham University
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Publication of WO2014002108A1 publication Critical patent/WO2014002108A1/fr
Priority to US14/585,013 priority Critical patent/US9707186B2/en
Priority to US14/732,716 priority patent/US9545382B2/en
Priority to US15/374,176 priority patent/US10143700B2/en
Priority to US15/619,559 priority patent/US20170333365A1/en

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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
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • 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
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the invention is related to a -protein-protein composite or core-shell nanoparticle formulation capable of delivering multiple therapeutic molecules in a controlled fashion and the methods for the preparation of the same.
  • An example of an application wherein such a formulation is especially beneficial is cancer and its associated manifestations, wherein drug ' resistance can require the administration of multiple drugs
  • the nanomedicine can be designed to target specific cells over-expressing specific receptors Each protein carries a different drug and the design of the composite architecture determines the delivery modalities
  • a core-shell structure is capable of delivering one drug first followed by delivery of the second drug in a sequential fashion, whereas a composite nanoparticle containing both proteins that are uniformly distributed throughout the particle can deliver both drugs simultaneously but at different rates of release.
  • the therapeutic molecules can be selected from the class of synthetic chemical agents such as cytotoxic drugs, small molecule kinase inhibitors, or phytochemicals or nucleic acid drugs which can be derivatives of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) such as small interfering RNA (siRNA), microRNA (miRNA), peptide nucleic acids (PNAs). smaii hairpin RNA (shRNA) etc.
  • RNA small interfering RNA
  • miRNA microRNA
  • PNAs peptide nucleic acids
  • shRNA smaii hairpin RNA
  • the therapeutic molecules can target aberrant protein kinase over-expressed/activated/mutated in cancer cells such as mTOR, PI3-Akt, BCR-ABL, EGFR, VEGF, SRC, STAT5, MAPK, HER2, PDGFR etc
  • the therapeutic molecules can be selected from the class of demethylation agents, retinods, antimetabolites, antimicrotubule agents, anti- angiogenesis agents, alkylating agents, biologic response modifiers, antitumor antibiotics, proteasome inhibitors, topoisomerase I inhibitors, hormones, immunomodulators.
  • the current invention is aimed at the simultaneous targeting of multiple pathways responsible for the survival of diseased cells.
  • the composite nanomedicine can be targeted to the diseased cells in a receptor specific fashion, which makes it biocompatible to the normal healthy cells.
  • the double hitting strategy ensures the destruction of diseased cells, which otherwise may evade the inhibitory effects of a single therapeutic agent.
  • the current invention provides a safe, biocompatible and efficient delivery of multiple therapeutic molecules simultaneously, where the interference from the carriers is negligible in terms of the cytotoxic side effects.
  • nanodelivery of therapeutic molecules helps to improve their aqueous solubility in the biological fluids, thereby improving the absorption characteristics and hence therapeutic outcome
  • the current invention holds promise in terms of better stability, good tissue specificity, high reproducibility and ease of synthesis
  • the nanomedicine is further conjugated to a targeting ligand. which specifically aims at the diseased cells, thus rendering the healthy cells unharmed
  • the carrier proteins possess intrinsic targeting capability as well, such as in the case of transferrin, albumin, etc for targeting cancer cells
  • Design of drug delivery systems involve generally encapsulation of the drug within the nanoparticle either within a hollow shell or distributed within a solid nanoparticle.
  • Successful management of diseases requires development of drug delivery systems with maximum therapeutic benefits Most of the diseases including cancer are associated with deregulation of multiple signaling pathways.
  • An essentia; requirement of drug delivery systems is the controlled delivery of a therapeutic molecule to the diseased site at therapeutically relevant concentrations.
  • the site-specific delivery of multiple therapeutic molecules to the diseased site using a single carrier vehicle in a specified steady concentration for prescribed time duration improves the efficacy of the therapeutic molecule and thus reduces the possible side effects, thus improving the therapeutic index.
  • the release kinetics of the therapeutic molecule is often dependeni upon the encapsulating material/carrier properties, drug-particle interactions or through some other trigger mechanisms, which assist in the drug release.
  • the advantages of such encapsulation is the control over release kinetics, giving the ability for slow release over a long period of time, and protection of the drug from a potentially degrading biological environment Recent advances in nanotechnology have revolutionized the field of drug delivery.
  • nanoparticles over conventional systems of drug delivery include, high surface area to volume ratio enabling better cellular uptake, thereby affecting intracellular pathways of action compared to that of free molecules and the ability to efficiently biofunctionalize the particulate surface with cell-specific targeting ligands for specific attachment to particular cells which require drug action
  • Protein based drug delivery systems are ideal platforms for the delivery of multiple therapeutics for in vivo applications due to their amphiphilic nature, biocomapatbility and biodegradabiiUy coupled with low toxicity.
  • the degradation products of the carrier system will be amino acids, which are well tolerated by the human body Depending upon the nature of the molecules to be encapsulated, wide choices of preparation are available such as desolvation, heat denaturation, coacervation, cross-linking, nanoprecipiaticn emulsification etc
  • the particle size of the system can be fine tuned with slight changes in the synthesis parameters such as temperature, pH etc
  • the protein nanoparticles possess greater stability during storage, stability of the therapeutic molecules in vivo after administration, availability of surface functional groups for conjugation to cancer targeting ligands and also they are suitable for different routes of administration. Different protein nanoparticles carrying different therapeutic molecules can either undergo spontaneous complexation mediated by electrostatic interactions or can be cross-linked using protein cross-linking agents to produce protein-protein composite or core-shell nanoparticles
  • this particular invention there are two phases to the nanopartide based carrier system, each encapsulating a different therapeutic molecule.
  • this invention proposes the use of two separate protein phases is envisioned rather than synthetic materials as in engineered nanoparticles. Use of proteins has been considered previously because of trie reduced toxicity of such natural materials tout the use of a protein-protein core shell/composite structure for deliver multiple lrugs is a new invention not envisioned before.
  • a second part of this invention is the modification of one or more of the proteins by doping/embedding them with metallic nanoclusters of gold (Au) or other suitable metals nanoclusters for imparting specific characteristic properties to the nanocarner such as optical/magnetic/x-ray contrast
  • Au gold
  • suitable metals nanoclusters for imparting specific characteristic properties to the nanocarner such as optical/magnetic/x-ray contrast
  • Tumorigenesis is a multi-step process, where the genetic alterations enable the cancer cells to acquire properties such as self-sufficiency of growth signals, insensitivity to anti-growth signals evasion of apoptosis, limitless replicative potential, sustained angiogenesis which further lead to tissue invasion and metastasis
  • the conventional chemotherapy regimen in an attempt to reduce the tumor volume, do not make discrimination between rapidly dividing normal cells and tumor cells, thus leading to severe side-effects.
  • protein kinase inhibitors target specifically the protein kinases, which are deregulated (constitutively activated/mutated/over-expressed) in cancer cells.
  • kinase inhibitors have been found to have low levels of undesirable side effects in clinical and preclinical studies compared to cytotoxic drugs. Yet, for highly aggressive and metastatic cancers, cytotoxic drugs present an immediate effect compared to kinase inhibitors. Resistance to kinase inhibitors in the long run due to point mutations in the drug-binding domain of kinases eventually distorts the conformation of drug binding domain and hence prevents the drug from binding to it In certain cases, kinase inhibition of the primary oncoprotein can lead to the activation or over-expression of a secondary survival signal in the oncogenic network. In those cases monotherapy with a single therapeutic agent remains ineffective.
  • Nanomedicine Therapeutic molecule loaded in to earner in the nanoscale size regime
  • Protein-protein composite nanomedicine Combination of two proteins in the form of nanoparticles possessing different amino acid sequences and chemical characteristics carrying two different therapeutic molecules
  • Protein-protein core-shell nanomedicine Combination of two proteins in the form of nanoparticles, wherein, one protein carrying a therapeutic molecule forms the core and the second protein carrying another therapeutic molecule forms the shell.
  • Therapeutic molecule synthetic drugs including ototoxic drugs and small molecule inhibitors, phytochemicals or nucleic acid drugs such as siR As shRNAs, miRNAs, PNAs, DNA. DNAzymes, nbozymes etc.
  • US 2005/0037989A1 describes the inhibition of gene function by delivery of polynucleotide based gene expression inhibitors to mammalian cells in vivo.
  • US 7, 947, 653 B1 describes a method of treating EGFR tyrosine kinase inhibitor resistant cancer by administering an EGFR inhibitor along with an IL-6 siRNA or antibody.
  • US 7.838,51282 dated Nov 23, 2010, describes methods and compositions for enhancing cancer cell death using therapeutically effective amounts of DNA damaging agent(s) that act in combination to enhance cancer cell death.
  • US 0196933A1 dated Aug .6, 2009 describes compositions and methods for preparation of poorly water soluble drugs with increased stability using nanoparticles.
  • US01 122077A1 dated May 6 201 ⁇ describes combination therapy methods of treating proliferative diseases like cancer with a first therapy comprising of effective amount of a taxane in a nanoparticle composition, with second " therapy such as radiation, surgery, administration of chemotherapeutic agents such as anti-VEGF antibody or combinations thereof.
  • second " therapy such as radiation, surgery, administration of chemotherapeutic agents such as anti-VEGF antibody or combinations thereof.
  • protein-protein core-sheH/composite nanomedicines wherein each protein carries a therapeutic molecule and where one or both proteins may carry dopants such as gold or other suitable metal nanodusters for the purpose of tracking the nanoparticles in vivo.
  • the art method and manner of designing and synthesizing a protein-protein composite or core-shell nanomedicine formulation carrying two different therapeutic molecules The proteins are derived from natural sources including human serum derived proteins
  • the nanoformulation is intended for a combination therapy, where monotherapy with a single therapeutic agent fails.
  • the protein-protein composite nanomedicine is further conjugated to a cell .targeting ligand so that the nanomedicine is specifically accumulated in the diseased cells in a target specific fashion.
  • the carrier proteins may themselves possess intrinsic targeting capability such as in the case of transferrin (specificity against Transferrin receptor/TfR/CD71 ), albumin (specificity against albondin/GP60 receptor)
  • transferrin specificity against Transferrin receptor/TfR/CD71
  • albumin specificity against albondin/GP60 receptor
  • the two therapeutic molecules are compactly packed individually in separate protein carriers and later complexed with each other using wet chemical techniques, to generate a protein-protein composite/core-shell nanoparticles During the entire process, the two therapeutic molecules are not mixed with each other and hence one does not interfere with the molecular activity of another Thus the chemical identity of the therapeutic • molecule is retained throughout the synthesis procedure.
  • the selection of the protein nanocarriers including the targeting agents as well as the therapeutic molecules require detailed molecular characterization of the diseased cells.
  • the targeting ligands include ligands specific to cell surface receptors or cell surface glyco-profeins, antibodies or antibody fragments.
  • the targeting of the protein-protein nanomedicine can be either passive utilizing the enhanced permeability and retention (EPR) effect of tumors having leaky vasculature or active targeting, wherein, the nanomedicine interacts and accumulates in cancer cells with the help of specific ceil surface receptors.
  • the targeting of nanomedicine can subsequently increase the drug concentration in the diseased tissue compared to normal tissue.
  • the protein encapsulation will further enhance the solubility of the t erapeutic molecule in the biological fluids and also improve the chemical stability of the therapeutic molecule preventing its degradation.
  • the enhanced dissolution of the pharmaceutical components can further enhance their cellular uptake and also improve the therapeutic outcome.
  • the combination of multiple therapeutic molecules can cause enhanced cytotoxic effect even at relatively fewer concentrations than that of t e therapeutic molecutes when used individually.
  • the invention provides compositions comprising of protein-protein composite/core-shell nanomedicines for simultaneously carrying more than one therapeutic molecule
  • the two therapeutic molecules are compactly packed individually in separate protein nanocarners and later complexed to form a single entity without significant cross talks among each other to form protein-protein composite nanoparticles or core-she!i nanoparticles
  • the interactions between the therapeutic molecules with the protein carriers are preferably non-covalent so that the protein nanodelivery will not affect the chemical characteristics and hence the activity of the therapeutic molecules significantly
  • the protein nanocarrier is selected from different types of proteins derived from natural sources and is highly biocompatible and also biodegradable The amphiphilic nature c the proteins enables them to deliver both hydrophilic as well as hydrophobic molecules.
  • the therapeutic molecules can also be a salt in the crystalline phase, -semi-crystaiiine phase, amorphous phase or a combination.
  • the formulation can be administered as a parenteral injection (intravenous, intramuscular or subcutaneous), oral administration as liquid, solid or aerosol.
  • the proteins modified or unmodified for preparing nanoformulations include but are not limited to transferrin, albumin, casein, soy protein, protamine etc. the proteins can be doped with metallic nanoclusters to impart specific characteristics such as alteration of zeta potential and/or to induce optical contrast.
  • the development of nanomedicine was performed after detailed molecular characterization of cancer cells. Based on these specific molecular features, we designed the protein- protein composite/core-shell nanomedicines.
  • Targeting of the therapeutic molecules at therapeutically relevant concentrations to the diseased cells in a cell-specific manner can enhance the therapeutic outcome manifold.
  • the targeting can be achieved either by active targeting or passive targeting as in the case of inflamed tissues or solid tumors Due to the less frequent and intimate association of tumor blood vessels with pericytes and the vascular basement membrane, tumor vasculature is highly permeable. Due to this enhanced permeability, nanoparticles in the range of 10-100nm accumulate within the tumors, where the payload is released.
  • the therapeutic molecules and their salts incorporated in the protein-protein composite nanoparticles can be conjugated to cell specific targeting ligands and monoclonal antibodies; which enable the active targeting of the therapeutic molecules to the diseased cells without affecting the normal cells.
  • the targeting can be done usinc the protein nanocarrier itself, which possesses an intrinsic targeting capability or another molecule, which can be either a specific ligand or a monoclonal antibody, conjugated to the protein
  • the targeting of the therapeutic molecules specifically to the diseased cells can significantly improve the therapeutic efficacy of the therapeutic molecules.
  • Enhanced solubility of the therapeutic molecules Most of the synthetic drugs including small molecule inhibitors as well as phytochemicals have poor solubility owing to their hydrophobic nature. This drastically curtails their dissolution in biophysical fluids and hence absorption in the target tissues Hence the therapeutic outcome will be reduced drastically than what expected.
  • the solubility of the therapeutic molecules can be improved significantly by encapsulating them in the protein nanocarrier.
  • the amphophilic nature of the proteins enable the encapsulation of the therapeutic molecules in their hydrophobic pockets, whereas the hydrophilic side chains provide aqueous solubility and hence enhanced dissolution properties in the bio-fluids.
  • a cytotoxic chemodrug is favorably combined with a small molecule inhibitor or small interfering RNA targeting a deregulated kinase contributing to carcinogenesis
  • the inhibitors preferably kinase inhibitors are targeted specifically to an aberrant protein kinase over- expressed/activated/mutated in cancer cells such as mTOR, PI3-Akt, BCR-ABL, EGFR, VEGF, SRC, STAT5, STAT3, JAK, MAPK, HER2, PDGFR etc.
  • the therapeutic molecules can be selected from the class of demethylation agents, retinods, antimetabolites, antimicrotubule agents, anti-angiogenesis agents, alkylating agents, biologic response modifiers, antitumor antibiotics, proteasome inhibitors, topoisomerase I inhibitors, hormones, immunomodulators, monoclonal antibodies, hormones, aromatase inhibitors, glucocorticosteroids, cytokines, enzymes, anti-androgen molecules etc. More specifically, the current invention is aimed at the simultaneous targeting of multiple pathways responsible for the survival of malignant cells.
  • Example 1 the current invention is aimed at the simultaneous targeting of multiple pathways responsible for the survival of malignant cells.
  • protamine-rapamycin nanocore was - prepared- usingr aqueous " wet chemical route The cationic peptide, protamine ( 10kDa) was dissolved at a concentration of 1 1 mg/ml in nuclease and endotoxin free water Rapamycin was dissolved in DMSO as per the manufacturer s instructions Rapamycm was added to aqueous solution of protamine. The complexation of protamine and rapamycin resulted in a turbid solution, which was vortexed vigorously and incubated at room temperature for 30min to enable the effective complexation of rapamycin with protamine. Thus formed protamine-rapamycin nanocore was purified by dialysis using 2kDa cut off dialysis membrane
  • the reaction was continued for ⁇ 2 h at room temperature.
  • the solution was further subjected to dialysis using dialysis cassettes with 2kDa molecular weight cut off and lyophilized for 48 h.
  • the percentage entrapment of the drug was determined from standard graph of rapamycin and dasatinib.
  • Protamine-rapamycin nanocore was prepared using aqueous wet chemical route The cationic peptide, protamine ( lOkDa) was dissolved at a concentration of 1 tmg/ml in nuclease and endotoxin free water Imatimb was dissolved in DMSO as per the manufacturer ' s instructions Imatinib was added to aqueous solution of protamine The solution, was vortexed vigorously and incubated at room temperature for 30mm to enable the effective complexation of imatinib with protamine
  • Protamine-siRNA nanoconjugates were prepared using aqueous wet chemical route, with an N/P ratio of 12.
  • the cationic peptide, protamine (10kOa) was dissolved at a concentration of 1 1 mg/ml in nuclease and endotoxin free water.
  • Lyophilized powder of siRNA targeted to BCR-ABL fusion kinase transcript in chronic myeloid leukemia (CML) was dissolved in RNase free water as per the manufacturer's instructions to prepare a stock solution of 350nM.
  • Protamine solution was added drop-wise to siRNA solution at the respective N/P ratio
  • the complexation of protamine and siRNA resulted in a turbid solution, which was vortexed vigorously and incubated at room temperature for 30min to enable the effective complexation of siRNA with protamine.
  • Synthesis of albumin sorafenib nanoconjugates Albumin-sorafenib nanoparticles were prepared using an aqueous wet chemical route Sorafenib, which is a multi-kinase inhibitor targeting STAT5 kinase in drug resistant CML was prepared in DMSO and aliquots were stored at -20°C.
  • Bioconjugation of nAlb-Soraf with Transferrin targeting tigand The bioconjugation of nAlb-Soraf with iron-saturated transferrin was performed using EDC-Sulfo NHS coupling chemistry. 5mg/ml transferrin was prepared in conjugation buffer [100 mM 2( - morpholino) ethanesulfonic acid (MES), 500 mM sodium chloride (NaCI), pH 6 0] Transferrin was activated by the addition of 2mg EDC and 6mg sulfo-NHS.
  • conjugation buffer [100 mM 2( - morpholino) ethanesulfonic acid (MES), 500 mM sodium chloride (NaCI), pH 6 0] Transferrin was activated by the addition of 2mg EDC and 6mg sulfo-NHS.
  • the transferrin-conjugated nAlb-Soraf is represented as Tf-nAlb-Soraf.
  • Complexation of protamine siRNA wit Tf-nAlb-Soraf Tf-nAlb-Soraf was mixed with protamine-siR A solution and kept at 4 J C for 30min
  • the two protein nanoconjugates undergo complexation mediated by electrostatic interactions to form protem-protein composite nanomedicine
  • the nanomedicine was resuspended in culture medium to suitable concentrations before addition to cells
  • protamine-siRNA nanoconjugates are done as described in example 3
  • the earner protein itself possesses cancer cell specific targeting capability
  • Transferrin used for encapsulating the small molecule kinase inhibitor is pre-doped with metallic nanoclusters of gold, platinum, silver etc for imparting specific characteristics such as optical contrast, magnetic contrast or and/or catiomc zeta potential.
  • the precursors of metals are added to protein solution at 37°C at 10m concentration kept under stirring
  • the reduction of the metal ions to metallic nanoclusters is aided by reducing agents such as NaOH . ascorbic acid etc.
  • the resulting solution is purified using desalting columns and further used for preparing transferrin-sorafenib nanoconjugates as described in example 3, which is further used for complexation with protamine-siRNA nanoconjugates to form protein-protein conmposite nanomedicine.

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Abstract

L'invention concerne la conception et la synthèse d'une nanomédecine comprenant un composite protéine-protéine ou une nanoparticule noyau-coquille, une protéine portant un type de molécule thérapeutique et une seconde protéine portant un autre type de molécule thérapeutique. La formulation de nanomédecine est destinée à traiter des maladies, notamment le cancer. Pour être spécifique, l'invention est conçue pour administrer deux types différents de molécules thérapeutiques en séquence ou en combinaison au moyen d'une seule entité de nanoparticules formées par deux protéines différentes qui portent séparément deux molécules thérapeutiques.
PCT/IN2013/000141 2012-02-21 2013-03-12 Nanostructure noyau-coquille basée sur des protéines avec des agents thérapeutiques correspondants WO2014002108A1 (fr)

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US14/585,013 US9707186B2 (en) 2012-02-21 2014-12-29 Core-shell particle formulation for delivering multiple therapeutic agents
US14/732,716 US9545382B2 (en) 2012-02-21 2015-06-06 Nanoparticle formulations for delivering multiple therapeutic agents
US15/374,176 US10143700B2 (en) 2013-02-19 2016-12-09 Nanoparticle formulations for delivering multiple therapeutic agents
US15/619,559 US20170333365A1 (en) 2012-02-21 2017-06-12 Core-shell particle formulation for delivering multiple therapeutic agents

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IN2550/CHE/2012 2012-06-27
IN2550CH2012 2012-06-27

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PCT/IN2013/000108 Continuation-In-Part WO2013124867A1 (fr) 2012-02-21 2013-02-19 Polymer - polymer or polymer - protein core - shell nano medicine loaded with multiple drug molecules
US14/585,013 Continuation-In-Part US9707186B2 (en) 2012-02-21 2014-12-29 Core-shell particle formulation for delivering multiple therapeutic agents
US14/732,716 Continuation-In-Part US9545382B2 (en) 2012-02-21 2015-06-06 Nanoparticle formulations for delivering multiple therapeutic agents
US15/374,176 Continuation-In-Part US10143700B2 (en) 2013-02-19 2016-12-09 Nanoparticle formulations for delivering multiple therapeutic agents

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WO2017011618A1 (fr) * 2015-07-15 2017-01-19 The Curators Of The University Of Missouri Conjugué de nanoparticules ciblé et procédé d'administration conjointe d'arnic et de médicament
CN111840254A (zh) * 2020-08-28 2020-10-30 湖南省肿瘤医院 纳米雄黄复合药物及其制备方法和应用
CN113041354A (zh) * 2021-03-30 2021-06-29 广州中医药大学(广州中医药研究院) 一种特异性水解模板蛋白分子的纳米粒及其制备方法与应用
CN113521034A (zh) * 2021-08-16 2021-10-22 中新国际联合研究院 一种抗皮肤光老化的复合纳米颗粒及其制备方法
CN114015094A (zh) * 2022-01-07 2022-02-08 中国农业大学 一种高强度大麦醇溶蛋白壳聚糖复合膜及其制备方法
CN114129571A (zh) * 2021-11-30 2022-03-04 福州大学 一种基于金属-有机共组装的无载体纳米药物及其制备与应用
CN115350283A (zh) * 2022-03-22 2022-11-18 四川大学 一种碳水化合物功能化纳米颗粒及其制备方法与应用

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CN113041354A (zh) * 2021-03-30 2021-06-29 广州中医药大学(广州中医药研究院) 一种特异性水解模板蛋白分子的纳米粒及其制备方法与应用
CN113041354B (zh) * 2021-03-30 2022-12-23 广州中医药大学(广州中医药研究院) 一种特异性水解模板蛋白分子的纳米粒及其制备方法与应用
CN113521034A (zh) * 2021-08-16 2021-10-22 中新国际联合研究院 一种抗皮肤光老化的复合纳米颗粒及其制备方法
CN114129571A (zh) * 2021-11-30 2022-03-04 福州大学 一种基于金属-有机共组装的无载体纳米药物及其制备与应用
CN114129571B (zh) * 2021-11-30 2023-11-14 福州大学 一种基于金属-有机共组装的无载体纳米药物及其制备与应用
CN114015094A (zh) * 2022-01-07 2022-02-08 中国农业大学 一种高强度大麦醇溶蛋白壳聚糖复合膜及其制备方法
CN114015094B (zh) * 2022-01-07 2022-03-11 中国农业大学 一种高强度大麦醇溶蛋白壳聚糖复合膜及其制备方法
CN115350283A (zh) * 2022-03-22 2022-11-18 四川大学 一种碳水化合物功能化纳米颗粒及其制备方法与应用
CN115350283B (zh) * 2022-03-22 2024-01-26 四川大学 一种碳水化合物功能化纳米颗粒及其制备方法与应用

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