WO2020030954A1 - Theranostics-like protein sanps conjugated to integrin and pmsa targeting peptides and therapy of prostate cancer - Google Patents

Theranostics-like protein sanps conjugated to integrin and pmsa targeting peptides and therapy of prostate cancer Download PDF

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Publication number
WO2020030954A1
WO2020030954A1 PCT/IB2018/055990 IB2018055990W WO2020030954A1 WO 2020030954 A1 WO2020030954 A1 WO 2020030954A1 IB 2018055990 W IB2018055990 W IB 2018055990W WO 2020030954 A1 WO2020030954 A1 WO 2020030954A1
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sanps
composition according
nanoparticles
composition
ligand
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PCT/IB2018/055990
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French (fr)
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Igor GENNADIEVICH SIVOV
Tamara SERGEEVNA GALAKTIONOVA
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Integrative Medicine Clinic, Sia
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Priority to PCT/IB2018/055990 priority Critical patent/WO2020030954A1/en
Priority to PCT/IB2019/056768 priority patent/WO2020031136A1/en
Publication of WO2020030954A1 publication Critical patent/WO2020030954A1/en

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    • 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
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001195—Prostate specific membrane antigen [PSMA]
    • 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
    • A61K47/62—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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10023—Virus like particles [VLP]
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32141—Use of virus, viral particle or viral elements as a vector
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18123—Virus like particles [VLP]
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18141—Use of virus, viral particle or viral elements as a vector

Definitions

  • the invention applies to the field of medicine and molecular biology and may be used for long-lasting therapy of prostate cancer with adjuvant effect using SANPs-theranostics (SANPs - self assembled nano particles).
  • SANPs-theranostics SANPs-theranostics
  • researchers should develop perspective technologies of creation of new SANPs-theranostics and hold their pre-clinical and clinical trials, results of theranostics usage prove it.
  • Cancer therapy largely depends on the introduction of cytotoxic drugs, which affect both cancerous and healthy cells due to limited selectivity of the drugs and wide spread of cytotoxic molecules throughout the body. Integration of targeted therapy and diagnostics have created a new field of treatment called theranostics.
  • the main task of the discipline is the potential ability to track the progress of target delivery of drugs or other pharmaceutically acceptable derivatives using molecular imaging in the study subjects.
  • SANPs "theranostic-alike" drug was held as as AbraxaneTM or TaxolTM which are used in medical diagnostics, but the cargo SANPs are salts of monovalent thallium isotopes.
  • a composition of self assembled nanoparticles comprising: a) a ligand of conjugation binding to membrane antigen of prostate (PSMA); b) a ligand of conjugation binding to integrin anb3, c) a biologically active agent; and d) nanoparticles.
  • PSMA membrane antigen of prostate
  • the ligand of conjugation binding to PSMA is attached to a nanoparticle, wherein a biologically active agent is encapsulated in the nanoparticle or wherein the ligand of conjugation binding to integrin anb3 is attached to the nanoparticle.
  • the biologically active agent is monovalent thallium compound, preferably thallium salt.
  • the biologically active agent may be selected from a group consisting of: actinomycin D; ametantrone; 9-aminocamptothecin; aminoglutethimide; amsacrine; anastrozole; antagonists of purine and pyrimidine; anthracyclines; aromatase linkers; asparaginase; antiestrogens; bendamustine; bexarotene; biolimus A9; bleomycin; buserelin; busulfan; calicheamicin; camptothecin; derivatives of camptothecin; capecitabine; carboplatin; carmustin; chlorambucil; cisplatin; cladribine; cyclophosphamide; cytarabine; cytosine- arabinoside; alkylating cytostatics; dacarbazine; dactinomycin;
  • the ligand of conjugation binding to PSMA is an organic molecule from class of inhibitors of enzymatic activity, and the nanoparticle contains a radioactive probe in concentration from 10 7 to 10 9 .
  • the ligand of conjugation binding to PSMA is heterodimeric glutamate urea or N -acetyl -L-aspartyl -L-glutamate .
  • the nanoparticles have a diameter in the range from about 30 nm to about 50 nm.
  • the composition of the nanoparticles may has a value of IC50 in the range of from about 10 6 to about 10 9 .
  • the compositions of the nanoparticles may have a value of IC50 in the range from about 5 x 10 6 to about 5 x 10 9 .
  • a method for the treatment or prevention of tumor of a subject including the stage of introduction to the subject an aforementioned composition, so that the introduction of the composition of the nanoparticles is effective for treatment or prevention of the tumor.
  • the tumor is a prostate cancer or metastasis of prostate cancer.
  • composition is administered to the subject for therapy purposes.
  • the composition is administered simultaneously or part thereof consecutively.
  • the subject may be a human.
  • a pharmaceutical composition may contain the aforementioned composition and a pharmaceutically suitable excipient.
  • the present invention provides a modification of any SANPs to obtain the SANPs-"theranostic-alike" medication with adjuvant effect and enhancing of the effectiveness of therapy and diagnosis of prostate cancer.
  • the invention provides the ability of long-lasting usage of SANPs- theranostics in therapy and diagnosis of prostate cancer.
  • this invention provides the ability of long-lasting usage of any described to date SANPs-theranostics in gene therapy.
  • any described embodiment of this invention unless otherwise noted or required is a SANPs-theranostic, including drugs with display that has different nature, for example, from low molecular weight compounds or fragments of nucleic acids (aptamers).
  • this invention provides the ability of long-lasting usage of any described to date SANPs-theranostics in oncotherapy and their usage in combination with gene therapeutic SNAPs-theranostics which have similar structure.
  • this invention provides antigens which have biological activity after application of SANPs-theranostics, which use antigens specific to prostate cancer (e.g. but not exclusively, mutant ras-peptides, mucin 1 or p53).
  • compositions e.g., but not only vaccines Sipuleucel-T (Provenge) or PROSTVAC
  • compositions including pharmaceutical compositions, containing SANPs- theranostics.
  • this invention provides compositions of SANPs-theranostics with antigens specific to prostate cancer (e.g. but not exclusively, mutant ras-peptides, mucin-l or p53 or vaccine Sipuleucel-T (Provenge) or PROSTVAC).
  • This invention also provides kits, arrays containing any product in combination with SANPs- theranostics and antigens specific to prostate cancer (e.g. but not exclusively, mutant ras- peptides, mucin 1 or p53).
  • this invention provides a method of treating prostate cancer or suppressing tumor growth foreseeing the introduction of individual compositions that contain SANPs-theranostics with directing ligands and filled with cytostatic compounds and isotopes: technetium-99, iodine-l23 or -131, thallium-20l, gallium-67, fluorine-l8, indium- 111, but not exclusively.
  • this invention provides the therapeutic method and composition for the treatment or alleviate prostate cancer or inflammatory processes which are mediated by viruses and expressing cells of prostate.
  • this invention provides a method of diagnosis of stated above pathological processes using SANPs-theranostics foreseeing "carrier” of quantum dots (compounds of monovalent thallium with selenium).
  • this invention provides a diagnostic or prognostic kit that contains the components described above using the SANPs-theranostics filled with diagnostic metabolites that are specific for tumor cells of prostate cancer and isotopes (radionuclide theranostics using technicia-99, iodine-l23 and -131, thallium-20l, gallium-67, fluorine-l8, indium-l 11, but not exclusively) or quantum dots (compounds of monovalent thallium with selenium).
  • this invention provides a method of testing possible antitumor agents or medications for prostate cancer therapy by trial which indicates ability or its lack of the medication or agent to inhibit intracellular pathological processes.
  • Fig. 1 The scheme for obtaining the structure of S ANPs, which, by way of example, are chemically modified MW-particles with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) and GRFLTGGTGRLLRIS .
  • Fig. 2 Forming of antibodies of different specificity in rabbits in response to the introduction of two drugs of bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV) under designation A in Fig. 2, PSA146 (P-2 KLQCVDLHV) under designation B in Fig. 2 and PSA154 (PSA-3 VISNDVCAQV) under designation C in Fig. 2, and phage MS2 with GRFLTGGTGRLLRIS under designation D in Fig. 2, but only one of which is filled with Tl + (average concentration is 2000 ions Tl + ).
  • PSA141 PSA-l FLTPKKLQCV
  • PSA146 P-2 KLQCVDLHV
  • PSA154 PSA-3 VISNDVCAQV
  • phage MS2 with GRFLTGGTGRLLRIS under designation D in Fig. 2, but only one of which is filled with Tl + (average concentration is 2000 ions Tl + ).
  • Fig. 3 Forming of reaction of delayed-type hypersensitivity as redness diameter in rabbits in response to introduction of two drugs of bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) and phage MS2 with GRFLTGGTGRLLRIS, but only one of which is filled with approximate 2000 ions Tl + .
  • the photo of Fig. 3 shows the measure method for the area of allergic inflammation.
  • the schematic record of the area of the allergic reaction of delayed hypersensitivity skin relationship ratios (DHS; in mm) to the modifying BSA (bovine serum albumin) by the specified peptide epitope injection as well-known to those skilled is art.
  • DHS delayed hypersensitivity skin relationship ratios
  • Fig. 4 Forming of antibodies of different specificity in rabbits in response to introduction of two drugs: one is bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV) under designation E in Fig. 4, PSA146 (P-2 KLQCVDLHV) under designation F in Fig. 4 and PSA154 (PSA-3 VISNDVCAQV) filled with TINO 3 (average concentration is 2000 ions Tl + ) under designation G in Fig. 4, and the other are particles of ferritin with GRFLTGGTGRLLRIS under designation H in Fig. 4.
  • PSA141 PSA-l FLTPKKLQCV
  • PSA146 P-2 KLQCVDLHV
  • PSA154 PSA-3 VISNDVCAQV
  • TINO 3 average concentration is 2000 ions Tl +
  • G in Fig. 4 and the other are particles of ferritin with GRFLTGGTGRLLRIS under designation H in Fig. 4.
  • Figure 5 Forming of the reaction of delayed-type hypersensitivity in rabbits in response to introduction of two drugs: one is bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKK-LQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) filled with TINO3 (average concentration is 2000 ions Tl + ) and the other are particles of ferritin with GRFLTGGTGRLLRIS.
  • DHS delayed hypersensitivity skin relationship ratios
  • BSA bovine serum albumin
  • Fig. 6 The formation of antibodies of different specificity in rabbits in response to introduction of drugs: one are particles of bacteriophage MS2, filled with TINO3 (average concentration is 2000 ions Tl + ) under designation I in Fig. 6 and the other particles of ferritin with PSA141 peptides (PSA-l FLTPKK-LQCV) under designation J in Fig. 6, PSA146 (P- 2 KLQCVDLHV) under designation K in Fig. 6, PSA154 (PSA-3 VISNDVCAQV) under designation L in Fig. 6 and GRFLTGGTGRLLRIS under designation M in Fig. 6.
  • PSA-l FLTPKK-LQCV the other particles of ferritin with PSA141 peptides
  • PSA146 P- 2 KLQCVDLHV
  • K in Fig. 6 PSA154 (PSA-3 VISNDVCAQV) under designation L in Fig. 6
  • GRFLTGGTGRLLRIS under designation M in Fig. 6.
  • Fig. 7 Forming of reaction of delayed-type hypersensitivity in rabbits in response to introduction of two drugs: one is particle of a bacteriophage MS2, filled with TINO3 (average concentration is 2000 ions Tl + ) and the other are particles of ferritin with PSA141 peptides (PSA-l FLTPKK-LQCV), PSA146 (P-2 KLQCVDLHV), PSA154 (PSA-3 VISNDVCAQV) and GRFLTGGTGRLLRIS.
  • DHS delayed hypersensitivity skin relationship ratios
  • BSA bovine serum albumin
  • Fig. 8 Pharmacodynamics of chemically modified MS2 bacteriophage particles (1), VLP FMDV (3), AVV virus (5) or ferritin (7) with covalently attached to the surface peptide GRFLTGGTGRLLRIS (2, 4, 6 and 8 commensurate to the deliveries) in Nude PC3- xenograft mice.
  • the ratio tumor sizes measurement with Leica Application Suite Live Image Builder for Nude mouse are well-known to those skilled in the art.
  • This invention provides new methods of obtaining SANPs-theranostics which can be long term used for treatment of prostate cancer with adjuvant effect. But adjuvant effect is only to the antigens of the tumor.
  • As a filling can be used different monovalent thallium compounds known as salts of metal, including radionuclides and also, perhaps, quantum dots, primarily, the complexes TlSe or Tl 2 Se.
  • the principle of creating SANPs-theranostics can be applied for diagnostics, for example, reservoirs of viral infections caused by a variety of viruses - causative agents.
  • AAV is an abbreviation for adeno-associated virus.
  • VLP FMDV is an abbreviation for virus-like particles (VLP) of foot-and-mouth disease virus (FMDV).
  • SANPs Self- Assembled Nano Particles
  • SANPs Self- Assembled Nano Particles
  • peptides which can form numerous nanostructures because there are many different types of interactions between links of the polypeptide chain, for example, electrostatic, hydrophobic, or due to forming of hydrogen bonds.
  • Peptide nanomaterials that are the result of self-assemblage into stable structures have potential application as tools for medication delivery and/or encapsulation.
  • SANPs of this invention are natural reservoirs that represent theirselves as natural virions of viruses and virus-like particles formed like virions but without nucleic acid which are empty virions and reservoirs formed by known natural proteins.
  • MMT-particles is SANPs, such as phage MS2, virus AAV, VLP FMDV and the particles of ferritin, but not only, filled with the pharmaceutically acceptable derivatives, such as salts of thallium, but not only.
  • iRGD is cyclic polypeptide containing L-arginine, glycine and L-aspartic acid (order in the polypeptide is shown).
  • this sequence (RGD sequence or RGD-peptide) is a common recognition element and protein-protein interaction of cellular proteins with integrins a n b3 and aib 5 .
  • RGD-containing peptides are often used in cell biology and biotechnology due to the property to specifically bind with the specified integrins.
  • Tl is the chemical symbol of thallium.
  • thallium (Tl) is referred as a salt of monovalent thallium or radioactive isotope or a component of complex compounds with selenium (Se).
  • Ras is a superfamily of Ras-proteins which are small GTPhases and it includes Ras, Rho, Arf, G-protein, Rab and Ran.
  • the Ras-superfamily includes more than a hundred structurally similar human proteins, there are more than ten human proteins of Ras. Ras are membrane- bound proteins engaged in one of the first steps of signal transmission from outside of the cell and, as a rule, regulate the reproduction of cells.
  • P53 is a transcription factor that regulates cell cycle and functions as a suppressor of malignancy.
  • PSMA is a specific membrane antigen of prostate. It is carboxypeptidase and is relatively unique in its ability to function as N-acetylated a-related dipeptidase and hydrolase g- glucamine (i.e. folate).
  • Polypeptide and “protein” are used interchangeably and mean any compound by a peptide bond the chain of amino acids, regardless of length. According to accepted in the biochemistry of designations of amino acids, peptide structure "KLQCVDLHV” means heteropolymer with the sequence of amino acid residues NH 2 - lysyl - leucyl - glutamyl - cysteyl - valyl - aspartyl - leucyl - histidyl - valyl - COOH.
  • A“covalent bond” is called compound atoms by using a common (shared between them) electron pairs. In this case, refers to the ability of atoms connect with other atoms.
  • atoms combine their electrons as if in common "piggy Bank" which is formed by atomic shells of individual atoms. This new wrapper has possibly completed the number of electrons and replaces the atoms of their own incomplete atomic shell.
  • carrier is SANPs nanoparticle with chemically modified surface and containing in its volume a cytostatic, or a pharmaceutically acceptable derivative.
  • ligand refers to PSMA acceptor.
  • a ligand is a chemical compound (often, but not always, a small molecule) that forms a complex with a particular biomolecule (most often a cell receptor, integrin). This binding occurs with forming of the so-called “coordination" donor-acceptor bond with polypeptides on the surface of SANPs-nanoparticles act as Lewis base, so they are donors of electronic pairs.
  • ligands may also be suitable targets for the SANPs-theranostic and such targets are also covered by the present invention.
  • enzymes PSMA like.
  • pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, solvate, prodrug and/or their compounds according to the invention which when administered to the recipient, can provide (directly or indirectly) the cytotoxic action or its consequences.
  • pharmaceutically acceptable derivatives are recognized by the person skilled in the art without undue experimentation. Nevertheless, we make reference to the teaching of medicinal chemistry and the cure Burger's 5th Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent to which such derivatives.
  • Preferred pharmaceutically acceptable derivatives are salts of heavy metals and containing organic compounds. Particularly preferred pharmaceutically acceptable derivatives are salts of thallium or platinum.
  • “Pharmaceutical compositions” are compositions that include a quantity (e.g., a unit dosage) of one or more described compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be obtained by standard methods of pharmaceutical compositions, such as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19 edition).
  • Therapeutically effective dosages can be determined stepwise by combinations of approaches such as: (i) characterization of effective doses of the composition or compounds in the analyses carried out on cell cultures in vitro criteria of tumor growth and/or survival of cells as a testimony, followed by
  • subject means any animal, including mammals and particularly humans.
  • standard conditions of conjugation refers to the conditions of joining of the peptide (-s) that allow you to create a covalent bond between the peptide and the surface of the SANPs.
  • conjugation conditions with higher efficiency can be used to provide at least 85% of points on the surface of the carrier.
  • Quantum dots are nano-sized (less than 10 nm) probes with high quantum yield, high photostability and fluorescence emission ability. Energy of quantum dots can be transferred to the surrounding molecules of 0 2 that leads to formation of superoxide anion (radical) having a high cytotoxicity. Quantum dots can be synthesized in aqueous solutions and are obtained for specific targets in the pathological region.
  • Example 1 Obtaining of preparative quantities of SANPs.
  • IB Receiving of adeno-associated virus (AAV) and virus-like particles of FMDV (VLP FMDV) carried out in the laboratory of diseases of pigs (Federal Centre for animal health, Russia) according to protocols RF patents N°N° 2335542 and 2332233, respectively.
  • AAV adeno-associated virus
  • VLP FMDV virus-like particles of FMDV
  • Example 2 Filling SANPs (bacteriophage MS2, virus AAV, VLP FMDV and ferritin particles) of thallium salts (receiving MBT-particles) and control of these processes.
  • SANPs bacteria MS2, virus AAV, VLP FMDV and ferritin particles
  • 2A Filling SANPs by method of drying, when the drying was carried out for the preparation of phage MS2, or virus AAV, or VLP FMDV, or particles of ferritin in a mixture with solutions of thallium salts.
  • 2B Filling SANPs by method of disassembly/assembly, when it was carried out according to the protocol known by specialists (US Patent 8987173). Preparation of MS2 phage were kept for 24 hours in ST buffer (50 mM Tris, 100 mM NaCl) in presence of 0.25 M TMAO and 0.1 M thallium salt. Then the solution was centrifuged (lOOOOg, 10 minutes).
  • the supernatant was mixed with PEG6000 and NaCl to final concentration of 12.5% and 0.5 M, respectively. After 2 hours, the solution was precipitated by centrifugation (l7800g, 45 minutes) at 4°C. The precipitate was dissolved in minimum quantity of ST buffer and re- precipitated (lOOOOg, 10 minutes). The supernatant fractionated and the fraction corresponding to the intact capsule of the virus MS2 were collected and stored at 4°C in ST buffer.
  • the grid was subjected to contrast staining in uranyl acetate and analyzed by for 35 min and washed three times by immersing the grids in lead citrate for 7 min.
  • the Mesh was observed on transmission electron microscope JEM-1400 operating at an accelerating voltage of 80 kV using magnification > ⁇ 8000.
  • the peptide was removed from resin by treatment with a mixture of trifluoroacetic acid, tri-(isopropyl)-silane, 3,6- dioxa-l,8-octanedithiol and water (in volume ratio of 94: 1 :2,5:2.5) and precipitated methyl tert-butyl ether.
  • Precipitate of the peptide was dissolved in 10% aqueous acetonitrile with 0.1% trifluoroacetic acid, and the resulting solution was subjected to purification by HPLC on the column Zorbax SB-C8, 21,2x250 mm, 7 pm in a concentration gradient of acetonitrile in 0.1% aqueous solution of trifluoroacetic acid.
  • the fraction containing the target peptide were collected and evaporated under vacuum, and then carried out the removal of the protective Acm groups of cysteine residues with simultaneous disulfide bridge formation by a known method (Fernando Albert cio et al.
  • Example 5 The definition of sensitivity of endothelial cell cultures and cultures of prostate cancer cells to modified MBT-particles (e.g., nitrate, but not exclusively), and agglutination of the cells of the modified virions of the phage MS2 and VLP FMDV.
  • modified MBT-particles e.g., nitrate, but not exclusively
  • Example 6 Determination of the sensitivity of animals to MBT-particles with modified surface.
  • mice The study was performed on 310 white noninbred mice and 160 white noninbred rats.
  • weight of mice was 18.0-22.0 g weight rats 170-250 g.
  • Cages with animals were in a separate room.
  • Light regime 12 hours light, 12 hours darkness.
  • Mode of ventilation 100%, without recirculation, with a shift of about 15 volumes of the room per hour, C0 2 concentration ⁇ 0, 15% ammonia ⁇ 0,001 mg/1.
  • the animals received ad libitum a high-grade specialized food for rats and mice, 2 hours before the introduction of the MBT- particles, the animals were deprived of food.
  • Composition of surface-modified MBT- particles with radioactive 201 Tl was administered orally by adding sterile drug particles in drinking water.
  • the remaining conditions of animals consistent with good laboratory practices (GLP, General Guidelines for Submitting a Proposal to ECVAM for the Evaluation of the Readiness of a Test Method to Enter the ECVAM Prevalidation and/or Validation Process, and "OECD series on principles of good laboratory practice and compliance monitoring").
  • GLP General Guidelines for Submitting a Proposal to ECVAM for the Evaluation of the Readiness of a Test Method to Enter the ECVAM Prevalidation and/or Validation Process, and "OECD series on principles of good laboratory practice and compliance monitoring”
  • Example 7 Determination of the effectiveness of the compositions of the nanoparticles on model of xenograft mice, according to the Xenograft Tumor Assay Protocol, Iruela-Arispe Lab (University of California, Los Angeles).
  • Cell line PC3 was administered to 128“nude” healthy adult female mice (line NCRNU- F). In order to strengthen the relevant activity of the cells of PC3 line, pre-grown under conditions known to specialists. Cells were collected and injected into mice subcutaneously. Mixture for injection contained 5-l5xl0 6 cells in the buffer with penicillin and streptomycin, and the combination of 17P-valerate-estradiol (15 mg) and 300 m ⁇ Matrigel. In a period of 2 months, the surviving mice were selected for analysis of the engraftment of the tumor. The resulting model was considered positive according to histological analysis, immunofluorescence and PCR, in comparison with the control.
  • composition of surface-modified MBT-particles with radioactive 201 Tl in the amount of 10 10 particles were orally administered by adding sterile drug particles in drinking water.
  • the medication was administered to three groups of mice with imbedded tumors (tumor sizes was 5, 7 and 10 mm 3 ), two control groups with tumors treated with cisplatin at therapeutic dose, and intact animals.
  • tumor sizes was 5, 7 and 10 mm 3
  • two control groups with tumors treated with cisplatin at therapeutic dose
  • intact animals were administered to three groups of mice with imbedded tumors (tumor sizes was 5, 7 and 10 mm 3 ), two control groups with tumors treated with cisplatin at therapeutic dose, and intact animals.
  • the disappearance of the cell line PC3 in xenograft mice treated with MBT-particles was observed by 12 days after the first dose.
  • Example 8 Determination of the titer of antibodies to antigens of nanoparticles and tumor (PSMA) in mice treated MBT-particles according to protocol E. Kolsch, A. J. S. Davies, E. Leuchars.

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Abstract

The invention relates to the field of medicine and molecular biology and may be used for long-lasting therapy of prostate cancer with adjuvant effect using SANPs-theranostics (SANPs – self assembled nano particles). A composition of self assembled nanoparticles comprising: a) a ligand of conjugation binding to membrane antigen of prostate (PSMA); b) a ligand of conjugation binding to integrin αvβ3, c) a biologically active agent; and d) nanoparticles.

Description

THERANOSTICS-LIKE PROTEIN SANPS CONJUGATED TO INTEGRIN AND PMSA TARGETING PEPTIDES AND THERAPY OF PROSTATE CANCER
Field of the Invention
The invention applies to the field of medicine and molecular biology and may be used for long-lasting therapy of prostate cancer with adjuvant effect using SANPs-theranostics (SANPs - self assembled nano particles). In order to provide progress in the therapy of prostate cancer researchers should develop perspective technologies of creation of new SANPs-theranostics and hold their pre-clinical and clinical trials, results of theranostics usage prove it.
Prior-art
Despite large progress in diagnostics and therapy of prostate cancer, this disease is still an longstanding problem for national health and economy in different countries. It’ s well known that androgen receptor (AR) plays the key role in appearance and development of prostate cancer. Researches in the sphere of D2D (targeted) delivery of therapeutic agents brought to a new therapy of prostate cancer (Elix C. et al. The role of peroxisome proliferator-activated receptor gamma in prostate cancer//Asian Journal of Andrology (2017) v.19, pp.1-6), which doesn’t allow to hold diagnostic while curing the patient. Cancer therapy largely depends on the introduction of cytotoxic drugs, which affect both cancerous and healthy cells due to limited selectivity of the drugs and wide spread of cytotoxic molecules throughout the body. Integration of targeted therapy and diagnostics have created a new field of treatment called theranostics. The main task of the discipline is the potential ability to track the progress of target delivery of drugs or other pharmaceutically acceptable derivatives using molecular imaging in the study subjects.
The term "theranostics" has been introduced for medications used for the treatment of various diseases, combining diagnostic and therapeutic capabilities in a single agent (US Patents 9687569, 9655980; Gupta M. K. et al. 2017). In the application to therapy and diagnosis of prostate cancer the first“theranostic”-alike delivery methods used the well- known radioactive labels to detect the accumulation of the drug (US Patent 9579408), then this approach was improved by the introduction of isotopes in nanoparticles (US Patents 9687573; 9550000), in consequence, the isotopic labels were replaced with fluorescent dyes (US Patent 9724035) and/or quantum dots (US Patent Application 20150194577; Probst C. E. 2013; Gobbo O. L. et al. 2015).
Currently, the efforts of research groups (e.g. Institute of Johns Hopkins in the USA or Oxford University in England) focused on the most precise direction of the target drug ("theranostic") or other pharmaceutically acceptable derivatives to cells of prostate cancer which can be reached by a combination of the two ligands (J. L. Crisp et al. Dual targeting of integrin anb3 and matrix metalloproteinase-2 for optical imaging of tumors and chemotherapeutic delivery//Mol Cancer Ther. 2014), one of which is targeted to integrin anb3 (Ruoslahti E. 2002), which was called iRGD peptide (US Patents 7488792; 8367621; 9441026; US Patent Application 20130343989; Pasqualini R. et al. 1996), and the second was called ligand for cancer membrane antigen PSMA (US Patents 9468672; 9446157; US Patent Applications 20100322862; 20170226141; 20170066719). But therapeutically acceptable doses of the drugs or their pharmaceutically acceptable derivatives should be taken into account.
One of the main problems of target nanotechnology medications is overcoming non-specific toxicity which can be eliminated by packing therapeutic drugs in SANPs with homotropic allosteric cooperative effect which is achieved by chemical modification of surface of the particles by peptides of the same structure. As an example, we can mention ferritin (apoferritin) (Heger Z. et al. 2014 u He D. et al. 2015), encapsulin (Moon H. et al 2014) or phage MS2 (Ashley C.E. et. al 2011; WO/2017/052419).
The last cited patent application discloses a method of cell apoptosis of malignant solid tumors by using chemically modified bacteriophage MS2 particles filled with salts of monovalent thallium (201Tl thallium isotope used for diagnostic purposes: US Patent 9745631; US Patent Application 20160339113).
The development was to provide the production of SANPs "theranostic-alike" drug was held as as Abraxane™ or Taxol™ which are used in medical diagnostics, but the cargo SANPs are salts of monovalent thallium isotopes.
Summary of the invention
This invention relates to the method of making SANPs-theranostics with adjuvant effect turned to therapy and diagnostic of prostate cancer and possible ways of usage. A composition of self assembled nanoparticles comprising: a) a ligand of conjugation binding to membrane antigen of prostate (PSMA); b) a ligand of conjugation binding to integrin anb3, c) a biologically active agent; and d) nanoparticles.
The ligand of conjugation binding to PSMA is attached to a nanoparticle, wherein a biologically active agent is encapsulated in the nanoparticle or wherein the ligand of conjugation binding to integrin anb3 is attached to the nanoparticle.
The biologically active agent is monovalent thallium compound, preferably thallium salt. The biologically active agent may be selected from a group consisting of: actinomycin D; ametantrone; 9-aminocamptothecin; aminoglutethimide; amsacrine; anastrozole; antagonists of purine and pyrimidine; anthracyclines; aromatase linkers; asparaginase; antiestrogens; bendamustine; bexarotene; biolimus A9; bleomycin; buserelin; busulfan; calicheamicin; camptothecin; derivatives of camptothecin; capecitabine; carboplatin; carmustin; chlorambucil; cisplatin; cladribine; cyclophosphamide; cytarabine; cytosine- arabinoside; alkylating cytostatics; dacarbazine; dactinomycin; daunorubicin; 5'-deoxy-5- fluorouridine;, docetaxel; doxorubicin (adriamycin); epirubicin(4-EPI-adriamycin); estramustine; etoposide; exemestane; fludarabine; fluorouracil; folate antagonists; fortesta; gemcitabine; corticosteroids; goserelin; hormones and hormone antagonists; Hycamtin; hydroxy carbamide (hydroxyurea); idarubicin; ifosfamide; imatinib; irinotecan; leuprorelin; lomustine; maytansinoid; melphalan; mercaptopurine; mitofusin; mitomycin; metabolized, antimitotic agents; mitoxantrone; nimustine; oxaliplatin; oxazaphosphorine; paclitaxel; pentostatin; derivatives of podophyllotoxin; procarbazine; rapamycin (sirolimus); radomized D, tamoxifen; temozolomide; teniposide; testolactone; thiotepa; thioguanine; topoisomerase linker; topotecan; Tretinoin; triptorelin; trofosfamide; the Vinca alkaloids vinblastine, vincristine, vindesine, vinorelbine; cytostatically active antibiotics; chloramine; treosulfan; mitomycin C; , methotrexate; 5 -fluorouracil; 6-thioguanine; 6-mercaptopurine; azathioprine, raltitrexed; alkaloids; podophyllotoxins; Taxol®; miltefosin®; glucocorticoids; prednisone; estrogens; fosfestrol; LHRH; flutamide; cyproterone acetate; toremifene; formestane; letrozole; Cu/Zn SOD (Super Oxide Dismutase), glutathione.
The ligand of conjugation binding to PSMA is an organic molecule from class of inhibitors of enzymatic activity, and the nanoparticle contains a radioactive probe in concentration from 107 to 109. The ligand of conjugation binding to PSMA is heterodimeric glutamate urea or N -acetyl -L-aspartyl -L-glutamate .
The nanoparticles have a diameter in the range from about 30 nm to about 50 nm. The composition of the nanoparticles may has a value of IC50 in the range of from about 106 to about 109. The compositions of the nanoparticles may have a value of IC50 in the range from about 5 x 106 to about 5 x 109.
A method for the treatment or prevention of tumor of a subject including the stage of introduction to the subject an aforementioned composition, so that the introduction of the composition of the nanoparticles is effective for treatment or prevention of the tumor. The tumor is a prostate cancer or metastasis of prostate cancer.
The composition is administered to the subject for therapy purposes. The composition is administered simultaneously or part thereof consecutively. The subject may be a human.
A pharmaceutical composition may contain the aforementioned composition and a pharmaceutically suitable excipient.
Thus, in one embodiment the present invention provides a modification of any SANPs to obtain the SANPs-"theranostic-alike" medication with adjuvant effect and enhancing of the effectiveness of therapy and diagnosis of prostate cancer.
In other embodiments the invention provides the ability of long-lasting usage of SANPs- theranostics in therapy and diagnosis of prostate cancer.
In another embodiments this invention provides the ability of long-lasting usage of any described to date SANPs-theranostics in gene therapy.
It is understood that any described embodiment of this invention unless otherwise noted or required is a SANPs-theranostic, including drugs with display that has different nature, for example, from low molecular weight compounds or fragments of nucleic acids (aptamers). In other aspects this invention provides the ability of long-lasting usage of any described to date SANPs-theranostics in oncotherapy and their usage in combination with gene therapeutic SNAPs-theranostics which have similar structure.
In other embodiments this invention provides antigens which have biological activity after application of SANPs-theranostics, which use antigens specific to prostate cancer (e.g. but not exclusively, mutant ras-peptides, mucin 1 or p53).
This invention also provides compositions (e.g., but not only vaccines Sipuleucel-T (Provenge) or PROSTVAC), including pharmaceutical compositions, containing SANPs- theranostics.
In other embodiment this invention provides compositions of SANPs-theranostics with antigens specific to prostate cancer (e.g. but not exclusively, mutant ras-peptides, mucin-l or p53 or vaccine Sipuleucel-T (Provenge) or PROSTVAC). This invention also provides kits, arrays containing any product in combination with SANPs- theranostics and antigens specific to prostate cancer (e.g. but not exclusively, mutant ras- peptides, mucin 1 or p53).
In another embodiment, this invention provides a method of treating prostate cancer or suppressing tumor growth foreseeing the introduction of individual compositions that contain SANPs-theranostics with directing ligands and filled with cytostatic compounds and isotopes: technetium-99, iodine-l23 or -131, thallium-20l, gallium-67, fluorine-l8, indium- 111, but not exclusively.
In another embodiment this invention provides the therapeutic method and composition for the treatment or alleviate prostate cancer or inflammatory processes which are mediated by viruses and expressing cells of prostate.
In another embodiment this invention also provides a method for detection and diagnosis of tumor of prostate providing stages:
(i) selection of relevant sample from the body of the subject who is suspected of the presence of pathological process tumor.
(ii) detecting presence or absence of complex which is formed by corresponding SANPs- theranostic with filling which is used in modem diagnostics such as, but not limited to, a radioactive isotope (radionuclide theranostic using technicia-99, iodine-l23 or -131, thallium-20l, gallium-67, fluorine-l8, indium-l 11, but not exclusively).
In another embodiment this invention provides a method of diagnosis of stated above pathological processes using SANPs-theranostics foreseeing "carrier" of quantum dots (compounds of monovalent thallium with selenium).
In next embodiment this invention provides a diagnostic or prognostic kit that contains the components described above using the SANPs-theranostics filled with diagnostic metabolites that are specific for tumor cells of prostate cancer and isotopes (radionuclide theranostics using technicia-99, iodine-l23 and -131, thallium-20l, gallium-67, fluorine-l8, indium-l 11, but not exclusively) or quantum dots (compounds of monovalent thallium with selenium).
In next embodiment, this invention provides a method of testing possible antitumor agents or medications for prostate cancer therapy by trial which indicates ability or its lack of the medication or agent to inhibit intracellular pathological processes. Brief description of the drawings
Fig. 1. The scheme for obtaining the structure of S ANPs, which, by way of example, are chemically modified MW-particles with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) and GRFLTGGTGRLLRIS .
Fig. 2. Forming of antibodies of different specificity in rabbits in response to the introduction of two drugs of bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV) under designation A in Fig. 2, PSA146 (P-2 KLQCVDLHV) under designation B in Fig. 2 and PSA154 (PSA-3 VISNDVCAQV) under designation C in Fig. 2, and phage MS2 with GRFLTGGTGRLLRIS under designation D in Fig. 2, but only one of which is filled with Tl+ (average concentration is 2000 ions Tl+). The results of typical IFA titer determinations of the corresponding IgG antibodies reacting with peptide epitopes indicated or known to the special sheets are graphically presented.“X” - the 6-day periods of sampling of blood for determination of antibodies number,“ Y” - the staining of fluid in the cells value intensity (OD450).
Fig. 3. Forming of reaction of delayed-type hypersensitivity as redness diameter in rabbits in response to introduction of two drugs of bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) and phage MS2 with GRFLTGGTGRLLRIS, but only one of which is filled with approximate 2000 ions Tl+. The photo of Fig. 3 shows the measure method for the area of allergic inflammation. The schematic record of the area of the allergic reaction of delayed hypersensitivity skin relationship ratios (DHS; in mm) to the modifying BSA (bovine serum albumin) by the specified peptide epitope injection as well-known to those skilled is art.
Fig. 4. Forming of antibodies of different specificity in rabbits in response to introduction of two drugs: one is bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKKLQCV) under designation E in Fig. 4, PSA146 (P-2 KLQCVDLHV) under designation F in Fig. 4 and PSA154 (PSA-3 VISNDVCAQV) filled with TINO3 (average concentration is 2000 ions Tl+) under designation G in Fig. 4, and the other are particles of ferritin with GRFLTGGTGRLLRIS under designation H in Fig. 4. The results of typical IFA titer determinations of the corresponding IgG antibodies reacting with peptide epitopes indicated or known to the special sheets are graphically presented.“X” - the 6-day periods of sampling of blood for determination of antibodies number,“ Y” - the staining of fluid in the cells value intensity (OD450).
Figure 5. Forming of the reaction of delayed-type hypersensitivity in rabbits in response to introduction of two drugs: one is bacteriophage MS2 with covalently attached peptides PSA141 (PSA-l FLTPKK-LQCV), PSA146 (P-2 KLQCVDLHV) and PSA154 (PSA-3 VISNDVCAQV) filled with TINO3 (average concentration is 2000 ions Tl+) and the other are particles of ferritin with GRFLTGGTGRLLRIS. The schematic record of the area of the allergic reaction of delayed hypersensitivity skin relationship ratios (DHS; in mm) to the modifying BSA (bovine serum albumin) by the specified peptide epitope injection as well- known to those skilled is art.
Fig. 6. The formation of antibodies of different specificity in rabbits in response to introduction of drugs: one are particles of bacteriophage MS2, filled with TINO3 (average concentration is 2000 ions Tl+) under designation I in Fig. 6 and the other particles of ferritin with PSA141 peptides (PSA-l FLTPKK-LQCV) under designation J in Fig. 6, PSA146 (P- 2 KLQCVDLHV) under designation K in Fig. 6, PSA154 (PSA-3 VISNDVCAQV) under designation L in Fig. 6 and GRFLTGGTGRLLRIS under designation M in Fig. 6. The results of typical IFA titer determinations of the corresponding IgG antibodies reacting with peptide epitopes indicated or known to the special sheets are graphically presented.“X” - the 6-day periods of sampling of blood for determination of antibodies number,“Y” - the staining of fluid in the cells value intensity (OD450).
Fig. 7. Forming of reaction of delayed-type hypersensitivity in rabbits in response to introduction of two drugs: one is particle of a bacteriophage MS2, filled with TINO3 (average concentration is 2000 ions Tl+) and the other are particles of ferritin with PSA141 peptides (PSA-l FLTPKK-LQCV), PSA146 (P-2 KLQCVDLHV), PSA154 (PSA-3 VISNDVCAQV) and GRFLTGGTGRLLRIS. The schematic record of the area of the allergic reaction of delayed hypersensitivity skin relationship ratios (DHS; in mm) to the modifying BSA (bovine serum albumin) by the specified peptide epitope injection as well- known to those skilled is art.
Fig. 8. Pharmacodynamics of chemically modified MS2 bacteriophage particles (1), VLP FMDV (3), AVV virus (5) or ferritin (7) with covalently attached to the surface peptide GRFLTGGTGRLLRIS (2, 4, 6 and 8 commensurate to the deliveries) in Nude PC3- xenograft mice. Fig. 9. Therapeutic efficacy of chemically modified bacteriophage MS2 bacteriophage particles, VLP FMDV, AVV virus or ferritin with covalently attached peptides PSA141 (PSA-l FLTPKK-LQCV), PSA146 (P-2 KLQCVDLHV), PSA154 (PSA-3 VISNDVCAQV) and GRFLTGGTGRLLRIS filled with TINO3 (average concentration is 2000 ions Tl+) in Nude PC3 -xenograft mice. The ratio tumor sizes measurement with Leica Application Suite Live Image Builder for Nude mouse are well-known to those skilled in the art.
Brief description of the Invention
This invention provides new methods of obtaining SANPs-theranostics which can be long term used for treatment of prostate cancer with adjuvant effect. But adjuvant effect is only to the antigens of the tumor. As a filling can be used different monovalent thallium compounds known as salts of metal, including radionuclides and also, perhaps, quantum dots, primarily, the complexes TlSe or Tl2Se.
Medications obtained on the basis of SANPs-theranostics filled with monovalent thallium compounds known as salts of metals and radionuclides and quantum dots.
The principle of creating SANPs-theranostics can be applied for diagnostics, for example, reservoirs of viral infections caused by a variety of viruses - causative agents.
Definitions
“AAV” is an abbreviation for adeno-associated virus.
“VLP FMDV” is an abbreviation for virus-like particles (VLP) of foot-and-mouth disease virus (FMDV).
"SANPs" (Self- Assembled Nano Particles) means ‘self-assembling nanoparticles of polypeptide nature’. But it’s known that self-assemblage of nanoparticles is also possible from peptides which can form numerous nanostructures because there are many different types of interactions between links of the polypeptide chain, for example, electrostatic, hydrophobic, or due to forming of hydrogen bonds. Peptide nanomaterials that are the result of self-assemblage into stable structures have potential application as tools for medication delivery and/or encapsulation. SANPs of this invention are natural reservoirs that represent theirselves as natural virions of viruses and virus-like particles formed like virions but without nucleic acid which are empty virions and reservoirs formed by known natural proteins.
"MBT-particles" is SANPs, such as phage MS2, virus AAV, VLP FMDV and the particles of ferritin, but not only, filled with the pharmaceutically acceptable derivatives, such as salts of thallium, but not only.
"iRGD" is cyclic polypeptide containing L-arginine, glycine and L-aspartic acid (order in the polypeptide is shown). In a protein this sequence (RGD sequence or RGD-peptide) is a common recognition element and protein-protein interaction of cellular proteins with integrins anb3 and aib5. RGD-containing peptides are often used in cell biology and biotechnology due to the property to specifically bind with the specified integrins.
"Tl" is the chemical symbol of thallium. In the description of this invention thallium (Tl) is referred as a salt of monovalent thallium or radioactive isotope or a component of complex compounds with selenium (Se).
"Ras" is a superfamily of Ras-proteins which are small GTPhases and it includes Ras, Rho, Arf, G-protein, Rab and Ran. The Ras-superfamily includes more than a hundred structurally similar human proteins, there are more than ten human proteins of Ras. Ras are membrane- bound proteins engaged in one of the first steps of signal transmission from outside of the cell and, as a rule, regulate the reproduction of cells.
"P53" is a transcription factor that regulates cell cycle and functions as a suppressor of malignancy.
"PSMA" is a specific membrane antigen of prostate. It is carboxypeptidase and is relatively unique in its ability to function as N-acetylated a-related dipeptidase and hydrolase g- glucamine (i.e. folate).
"Polypeptide" and "protein" are used interchangeably and mean any compound by a peptide bond the chain of amino acids, regardless of length. According to accepted in the biochemistry of designations of amino acids, peptide structure "KLQCVDLHV" means heteropolymer with the sequence of amino acid residues NH2 - lysyl - leucyl - glutamyl - cysteyl - valyl - aspartyl - leucyl - histidyl - valyl - COOH.
A“covalent bond” is called compound atoms by using a common (shared between them) electron pairs. In this case, refers to the ability of atoms connect with other atoms. During forming of covalent bonds atoms combine their electrons as if in common "piggy Bank" which is formed by atomic shells of individual atoms. This new wrapper has possibly completed the number of electrons and replaces the atoms of their own incomplete atomic shell.
In used here meaning "carrier" is SANPs nanoparticle with chemically modified surface and containing in its volume a cytostatic, or a pharmaceutically acceptable derivative.
Used here term "ligand" refers to PSMA acceptor. In biochemistry and pharmacology, a ligand is a chemical compound (often, but not always, a small molecule) that forms a complex with a particular biomolecule (most often a cell receptor, integrin). This binding occurs with forming of the so-called "coordination" donor-acceptor bond with polypeptides on the surface of SANPs-nanoparticles act as Lewis base, so they are donors of electronic pairs. When attaching ligands to the central atom chemical properties of the complexing agent and ligands themselves often undergo significant conformational changes. Other ligands may also be suitable targets for the SANPs-theranostic and such targets are also covered by the present invention. As a non-limiting example can be given enzymes, PSMA like.
Used here term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, solvate, prodrug and/or their compounds according to the invention which when administered to the recipient, can provide (directly or indirectly) the cytotoxic action or its consequences. Such derivatives are recognized by the person skilled in the art without undue experimentation. Nevertheless, we make reference to the teaching of medicinal chemistry and the cure Burger's 5th Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent to which such derivatives. Preferred pharmaceutically acceptable derivatives are salts of heavy metals and containing organic compounds. Particularly preferred pharmaceutically acceptable derivatives are salts of thallium or platinum.
"Pharmaceutical compositions" are compositions that include a quantity (e.g., a unit dosage) of one or more described compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be obtained by standard methods of pharmaceutical compositions, such as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19 edition).
Therapeutically effective dosages can be determined stepwise by combinations of approaches such as: (i) characterization of effective doses of the composition or compounds in the analyses carried out on cell cultures in vitro criteria of tumor growth and/or survival of cells as a testimony, followed by
(ii) characterization in animal trials using the inhibition of tumor growth and/or survival of animals as a testimony, followed by
(iii) characterization in human studies with usage of enhanced inhibition of tumor growth and/or increased frequency of survival of patients as evidence.
As used herein, the term "subject" means any animal, including mammals and particularly humans.
As used herein, "standard conditions of conjugation" refers to the conditions of joining of the peptide (-s) that allow you to create a covalent bond between the peptide and the surface of the SANPs. In accordance with a particular embodiment of the conjugation conditions with higher efficiency can be used to provide at least 85% of points on the surface of the carrier.
"Quantum dots" are nano-sized (less than 10 nm) probes with high quantum yield, high photostability and fluorescence emission ability. Energy of quantum dots can be transferred to the surrounding molecules of 02 that leads to formation of superoxide anion (radical) having a high cytotoxicity. Quantum dots can be synthesized in aqueous solutions and are obtained for specific targets in the pathological region.
The common methods
The practice of this invention will use, unless otherwise indicated, conventional techniques of molecular biology (including recombinant DNA techniques), Microbiology, cell biology, biochemistry and immunology, which are within the competence in this area. Such techniques are explained fully in the literature, such as: "Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Wei & C. C. Blackwell, eds.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991). Examples
Below are examples of this invention, which are given for illustrative purposes only and not to limit the present invention.
The present invention will be further described with reference to the following examples, however, it should be understood that this invention is not limited to such examples.
Example 1. Obtaining of preparative quantities of SANPs.
IA. Obtaining of preparative amounts of phage MS2 was carried out by the previously described method (Knyazhev V.A. Sivov, I.G., Sergienko V.I. RNA-transduction of non- infectious virions of phage MS2: Molecular genetics, Microbiology and Virology 2002, No. 3, pp. 56-63). Verification of the result was carried out by sequencing cDNA obtained from the RNA preparation. Titer determination of phage particles was carried out by the method of agar layers (Gratia A. Numerical Relations between Lysogenic Bacteria and Particles of Bacteriophage//Ann. Inst. Pasteur 1936, v.57, p. 652). The concentration of the phage preparation was performed as known in the art: a clarification of the cell lysate by centrifugation (15,000 rpm), followed by deposition PEG6000 in the presence of NaCl and dehydration of dry Sephadex G10.
IB. Receiving of adeno-associated virus (AAV) and virus-like particles of FMDV (VLP FMDV) carried out in the laboratory of diseases of pigs (Federal Centre for animal health, Russia) according to protocols RF patents N°N° 2335542 and 2332233, respectively.
IC. Getting a ferritin solution that doesn’t form nanoparticles was carried out according to protocols known in the art (Heger Z. et al. Apoferritin applications in nanomedicine// Nanomedicine (Lond.) 2014, 9 (14), 2233-2245).
Example 2. Filling SANPs (bacteriophage MS2, virus AAV, VLP FMDV and ferritin particles) of thallium salts (receiving MBT-particles) and control of these processes.
2A. Filling SANPs by method of drying, when the drying was carried out for the preparation of phage MS2, or virus AAV, or VLP FMDV, or particles of ferritin in a mixture with solutions of thallium salts. 2B. Filling SANPs by method of disassembly/assembly, when it was carried out according to the protocol known by specialists (US Patent 8987173). Preparation of MS2 phage were kept for 24 hours in ST buffer (50 mM Tris, 100 mM NaCl) in presence of 0.25 M TMAO and 0.1 M thallium salt. Then the solution was centrifuged (lOOOOg, 10 minutes). The supernatant was mixed with PEG6000 and NaCl to final concentration of 12.5% and 0.5 M, respectively. After 2 hours, the solution was precipitated by centrifugation (l7800g, 45 minutes) at 4°C. The precipitate was dissolved in minimum quantity of ST buffer and re- precipitated (lOOOOg, 10 minutes). The supernatant fractionated and the fraction corresponding to the intact capsule of the virus MS2 were collected and stored at 4°C in ST buffer.
2C. Filling SANPs was carried out by change of pH (2.5 to 8.0) to obtain particles of ferritin in solution with the salts of thallium.
2D. Estimation of the amount of included thallium was carried out by previously described method (DI Zhang et al. A Thallium Transport FLIPR-Based Assay for the Identification of KCC2-Positive Modulators// Journal of Biomolecular Screening 2010, V. l, pp. 177-184). It is fluorescence with PTSA (quaternary sodium salt of pyrene-l,3,6,8-tetrasulfonic acid). Samples were obtained after washing of phage particles or ferritin filled with thallium as well as after thermal denaturation of precipitate of these particles and subsequent processing of the heated sample by RNase (in case of a bacteriophage).
2E. Estimation of aggregation of MBT-particles was carried out on the device Horiba SZ- 100, method known in the art.
Example 3. Comparison of phage MS2 and its modified variants.
3A. Comparison of phage MS2 and its modified variants was carried out in accordance to "Methods in Enzymology" (Academic Press, Inc.) by electrophoresis in 2% polyacrylamide gel under conditions known in the art.
3B. Comparison of phage MS2 and its modified variants was performed using equilibrium centrifugation in density gradient of sucrose 5-50% (weight/volume), formed in tubes (Beckman Instruments, Inc., Fullerton, CA) according to the method (Brakke M. K. Density- gradient centrifugation. Methods in Virology Volume 2. Edited by: K Maramorosch and H Koprowski. New York, Academic Press; 1967: 93-118). A sample of phage was suspended in 1 ml of cold phosphate buffer pH=9.0 and was applied to the sucrose gradient and centrifuged (85000g, 6 hours).
Table 1
The results of filling of TINO3 into ferritin particles, bacteriophage MS2, VLP FMDV, and particles of adeno-associated virus (AAV).
Figure imgf000016_0001
Notes:
- the size of the particles before and after filling was measured on the instrument Horiba SZ-
100;
- the sedimentation rate was measured only for VLP FMDV (data are given in parentheses);
- if necessary, introduction to MBT-particle 201Tl its nitrate salt was injected into a solution of particles in a ratio of 1 : 1000.
3C. Comparison of phage MS2 and its modified variants was performed using transmission electron microscopy (TEM). To visualization the MBT-particles used contrasting silver (45 min, Aurion, UK). Fixation was performed in 1% (weight/volume) solution of Os04 in phosphate buffer for 1 hour and the filters were washed in phosphate buffer for 10 min. The filters were removed from the insert and randomly cut into 2 segments (3-5 mm x 2 mm). These segments were dehydrated in 30-100% ethanol and placed in Epon resin. Diamond knife Diatome did ultrathin sections of 70-80 nm thickness which then were placed on a copper grid coated with Pioloform. The grid was subjected to contrast staining in uranyl acetate and analyzed by for 35 min and washed three times by immersing the grids in lead citrate for 7 min. The Mesh was observed on transmission electron microscope JEM-1400 operating at an accelerating voltage of 80 kV using magnification ><8000.
3D. Comparison of phage MS2 and its modified variants was performed by SPR-method which is study using surface plasmon resonance (SPR) was performed on the device Eva 2.0 (http://www.pcbiosensors.com, RF patents 2341785, 2442142). The solution of anti-MS2 IgG (10 pg/ml) in the mixture with the dye N-hydroxysuccinimidyl ester 5- carboxytetramethylrhodamine (0.1 pm, kex=575 nm; kem=605 nm) was stored in 0.1 mm sodium acetate (pH=5,5). The standard procedure of binding IgG with glass was performed using 3,3-diethoxypropene triethoxysilane by previously described protocol (patent RF 681837). ETnreacted groups on the glass surface had been inactivated by treatment with 1 M ethanolamine pH 8.5. All studies were performed three times with a volume rate of 300 pl/min of Sample containing phage were diluted 1 : 100 and/or 1 :50 in "phage" buffer. After each measurement, the surface was restored with a solution of 100 mM NaOH and subsequent running rinse "phage" buffer. The molar ratio between IgG and dye allows to estimate the maximum amount of protein associated with the surface of the chip, the fluorescence of the dye (Patent US 5800996) associated with glass, thanks to the anti-MS2 IgG.
Example 4. Modification of SANPs surface by peptides.
4A. Synthesis of a peptide with the structure (NH2)-GRFLTGGTGRLLRIS-(COOH). Other peptides were synthesized in a similar way. The peptide was synthesized using method of solid state peptide synthesis based on Fmoc amino acids for automated peptide synthesizer Applied Biosystems 433 A on the resin with the attached residue of Fmoc-Cys(Acm) using amino acid derivatives: Fmoc-Cys(Acm), Fmoc-Arg(Pbf), Fmoc-Asp(OtBu), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Pro. Removal of Fmoc-protective group from N-terminal alpha- amino group of the growing peptide chain was carried out in 22% solution of 4-demerol in N-dimethylformamide (Aleshina E. Yu., Pyndyk N. In. Moisa A. A., Sanzhakov M. A., Harybin O. N., Nikolaev E. N., Kolesanova E. F. Synthesis of a fragment of P-amyloid 5RHDSGY10 and its isomers. Biomed. chemistry, 2008, vol. 54, No. 2, 154-166). The joining of amino acids to a growing peptide chain (except Fmoc-Cys(Acm)) were carried out with pre-activated Fmoc amino acid hexaflurophosphate 2-(lH-benzotriazole-l-yl)- l,l,3,3-tetramethylene in the presence of 1 hydroxybenzo-triazole and 2,4,6-collidine, according to the FastMoc procedure described in the instructions to the synthesizer. Fmoc- Cys(Acm) joins with activation in situ, using diisopropylcarbodiimide as an activator in the presence of l-hydroxybenzotri azole. At the end of the synthesis the peptide was removed from resin by treatment with a mixture of trifluoroacetic acid, tri-(isopropyl)-silane, 3,6- dioxa-l,8-octanedithiol and water (in volume ratio of 94: 1 :2,5:2.5) and precipitated methyl tert-butyl ether. Precipitate of the peptide was dissolved in 10% aqueous acetonitrile with 0.1% trifluoroacetic acid, and the resulting solution was subjected to purification by HPLC on the column Zorbax SB-C8, 21,2x250 mm, 7 pm in a concentration gradient of acetonitrile in 0.1% aqueous solution of trifluoroacetic acid. The fraction containing the target peptide were collected and evaporated under vacuum, and then carried out the removal of the protective Acm groups of cysteine residues with simultaneous disulfide bridge formation by a known method (Fernando Albert cio et al. Preparation and handling of peptides containing methionine and cysteine // In: Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Eds. W. C. Chang and P. D. White. Oxford University Press, 2000). The peptide was subjected to re-purification by HPLC on the same column, the fraction of the target peptide was evaporated under vacuum.
4B. The purity of synthesized peptide was confirmed by mass spectrometric analysis by electrospray ionization and detection by ion trap and analytical HPLC in accordance with the protocols described earlier (M. H. V. Van Regenmortel, S. Muller. Synthetic peptides as antigens. Elsevier, 1999, pp.88-90).
4C. Conjugation of the peptide with SANPs (virions of phage MS2, or virus AAV, or VLP FMDV, or ferritin particles) was performed using dimethylacetamide in accordance with standard procedure known in the art (M. H. V. Van Regenmortel, S. Muller. Synthetic peptides as antigens. Elsevier, 1999, pp. 88-90).
Example 5. The definition of sensitivity of endothelial cell cultures and cultures of prostate cancer cells to modified MBT-particles (e.g., nitrate, but not exclusively), and agglutination of the cells of the modified virions of the phage MS2 and VLP FMDV.
Cell cultures were obtained from collection and were propagated according to methods known, for example, from the RF patents 2359030 and 2493251. Induction of integrins appearance on the surface of endothelial cells and cells of prostate cancer conducted endothelial growth factor (VEGF), according to the procedure described (RF patent 2377017). Agglutination of cells was performed according to the described procedure (US Patent 5401636; US Patent 5541417). The penetration of virions of the MS 2 phage, filled with thallium, recorded using fluorescence microscopy with the PTSA dye according to the procedure described previously (patent RF 2305270; Fig. 9). The calculation of the LD50 (number of particles per cell, which causes death in 50% of cases) was performed using the program Probit Analysis. The following results were obtained: HepG2 LD50 = 5,9; HBL100 LD50 =11.4 and for PC3 LD50 =3.3.
Example 6. Determination of the sensitivity of animals to MBT-particles with modified surface.
6 A. The study was performed on 310 white noninbred mice and 160 white noninbred rats. By the beginning of the introduction of the MBT-particles weight of mice was 18.0-22.0 g weight rats 170-250 g. Cages with animals were in a separate room. Light regime: 12 hours light, 12 hours darkness. The temperature: 2l-23°C, relative humidity of 55-70%. Mode of ventilation: 100%, without recirculation, with a shift of about 15 volumes of the room per hour, C02 concentration <0, 15% ammonia < 0,001 mg/1. The animals received ad libitum a high-grade specialized food for rats and mice, 2 hours before the introduction of the MBT- particles, the animals were deprived of food. Composition of surface-modified MBT- particles with radioactive 201Tl was administered orally by adding sterile drug particles in drinking water. The remaining conditions of animals consistent with good laboratory practices (GLP, General Guidelines for Submitting a Proposal to ECVAM for the Evaluation of the Readiness of a Test Method to Enter the ECVAM Prevalidation and/or Validation Process, and "OECD series on principles of good laboratory practice and compliance monitoring"). After triple introduction, at 1010 described above MBT-particles to each of the animals of dead animals was not.
6B. An experiment with newborn animals showed no toxicity in the same dose. Example 7. Determination of the effectiveness of the compositions of the nanoparticles on model of xenograft mice, according to the Xenograft Tumor Assay Protocol, Iruela-Arispe Lab (University of California, Los Angeles).
7A. Cell line PC3 was administered to 128“nude” healthy adult female mice (line NCRNU- F). In order to strengthen the relevant activity of the cells of PC3 line, pre-grown under conditions known to specialists. Cells were collected and injected into mice subcutaneously. Mixture for injection contained 5-l5xl06 cells in the buffer with penicillin and streptomycin, and the combination of 17P-valerate-estradiol (15 mg) and 300 mΐ Matrigel. In a period of 2 months, the surviving mice were selected for analysis of the engraftment of the tumor. The resulting model was considered positive according to histological analysis, immunofluorescence and PCR, in comparison with the control.
7B. Composition of surface-modified MBT-particles with radioactive 201Tl in the amount of 1010 particles were orally administered by adding sterile drug particles in drinking water. The medication was administered to three groups of mice with imbedded tumors (tumor sizes was 5, 7 and 10 mm3), two control groups with tumors treated with cisplatin at therapeutic dose, and intact animals. In preliminary experiments established that the effect only MBT-particles, but not particles without filling or with filling, but without the "target" ligand. The disappearance of the cell line PC3 in xenograft mice treated with MBT-particles was observed by 12 days after the first dose. However in the group with a large tumor (10 mm3) animals were killed by the deadline in 25% of cases. The size of the tumor was significantly reduced in all dead animals, but the dead animals with a tumor of large size found peritoneal exudate biochemical parameters which were consistent with laboratory values of tumor lysis syndrome (TLS). Biochemical characteristics of exudate were: uric acid > 1 mg/ml, sodium > 0.5 mg/ml, phosphorus > 0.5 mg/ml.
Table 2
Figure imgf000021_0001
Note: the quantity of animals with TLS signs, smaller values of biochemical parameters.
Example 8. Determination of the titer of antibodies to antigens of nanoparticles and tumor (PSMA) in mice treated MBT-particles according to protocol E. Kolsch, A. J. S. Davies, E. Leuchars.
The immune response to phage fd in normal and thymus-deprived animals of a low responding inbred strain and in genetically thymusless mice// Eur. Journal of Immunology 1971 Jun;l(3):2l0-2l3 Jose E. Belizario. Immunodeficient Mouse Models: An Overview The Open Immunology Journal, 2009, 2, 79-85). Detection of antibodies was performed 30 days after the start of observation, by measuring particle size (Hariba SZ-100, see table 1; example 2E) by dynamic light scattering. The results are presented in Table 3.
Table 3
Figure imgf000021_0002

Claims

1. A composition of self assembled nanoparticles comprising: a) a ligand of conjugation binding to membrane antigen of prostate (PSMA); b) a ligand of conjugation binding to integrin anb3, c) a biologically active agent; and d) nanoparticles.
2. The composition according to claim 1, wherein the ligand of conjugation binding to PSMA is attached to a nanoparticle, wherein a biologically active agent is encapsulated in the nanoparticle or wherein the ligand of conjugation binding to integrin anb3 is attached to the nanoparticle.
3. The composition according to claims 1 or 2, wherein the biologically active agent is monovalent thallium compound, preferably thallium salt.
4. The composition according to any one of claims 1 to 3, wherein the biologically active agent is selected from a group consisting of: actinomycin D; ametantrone; 9- aminocamptothecin; aminoglutethimide; amsacrine; anastrozole; antagonists of purine and pyrimidine; anthracyclines; aromatase linkers; asparaginase; antiestrogens; bendamustine; bexarotene; biolimus A9; bleomycin; buserelin; busulfan; calicheamicin; camptothecin; derivatives of camptothecin; capecitabine; carboplatin; carmustin; chlorambucil; cisplatin; cladribine; cyclophosphamide; cytarabine; cytosine- arabinoside; alkylating cytostatics; dacarbazine; dactinomycin; daunorubicin; 5'-deoxy- 5-fluorouridine;, docetaxel; doxorubicin (adriamycin); epirubicin(4-EPI-adriamycin); estramustine; etoposide; exemestane; fludarabine; fluorouracil; folate antagonists; fortesta; gemcitabine; corticosteroids; goserelin; hormones and hormone antagonists; Hycamtin; hydroxy carbamide (hydroxyurea); idarubicin; ifosfamide; imatinib; irinotecan; leuprorelin; lomustine; maytansinoid; melphalan; mercaptopurine; mitofusin; mitomycin; metabolized, antimitotic agents; mitoxantrone; nimustine; oxaliplatin; oxazaphosphorine; paclitaxel; pentostatin; derivatives of podophyllotoxin; procarbazine; rapamycin (sirolimus); radomized D, tamoxifen; temozolomide; teniposide; testolactone; thiotepa; thioguanine; topoisomerase linker; topotecan; Tretinoin; triptorelin; trofosfamide; the Vinca alkaloids vinblastine, vincristine, vindesine, vinorelbine; cytostatically active antibiotics; chloramine; treosulfan; mitomycin C; , methotrexate; 5 -fluorouracil; 6-thioguanine; 6-mercaptopurine; azathioprine, raltitrexed; alkaloids; podophyllotoxins; Taxol®; miltefosin®; glucocorticoids; prednisone; estrogens; fosfestrol; LHRH; flutamide; cyproterone acetate; toremifene; formestane; letrozole; Cu/Zn SOD (Super Oxide Dismutase), glutathione.
5. The composition according to any one of claims 1 to 4, wherein the ligand of conjugation binding to PSMA is an organic molecule from class of inhibitors of enzymatic activity, and the nanoparticle contains a radioactive probe in concentration from 107 to 109.
6. The composition according to any one of claims 1 to 5, wherein the ligand of conjugation binding to PSMA is heterodimeric glutamate urea or N-acetyl-L-aspartyl-L- glutamate.
7. The composition according to any one of claims 1 to 6, where the nanoparticles have a diameter in the range from about 30 nm to about 50 nm.
8. The composition according to any one of claims 1 to 7, characterized in that the composition of the nanoparticles has a value of IC50 in the range of from about 106 to about 109.
9. The composition any one of claims 1 to 8, characterized in that the compositions of the nanoparticles have a value of IC50 in the range from about 5 x 106 to about 5 x 109.
10. A method for the treatment or prevention of tumor of a subject including the stage of introduction to the subject a composition according to any one of claims 1 to 9, so that the introduction of the composition of the nanoparticles is effective for treatment or prevention of the tumor.
11. The method according to claim 10, wherein the tumor is a prostate cancer or metastasis of prostate cancer.
12. The method according to claim 10 or 11, wherein the composition according to any one of claims 1 to 9 is administered to the subject for therapy purposes.
13. The method according to claim 12, wherein the composition is administered simultaneously or part thereof consecutively.
14. The method according to any one of preceding claims 10 to 13, wherein the subject is a human.
15. A pharmaceutical composition containing a composition according to any one of claims 1 to 9 and a pharmaceutically suitable excipient.
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