WO2022050376A1 - 光を用いた薬物伝送方法、及び標的集積型複合体 - Google Patents

光を用いた薬物伝送方法、及び標的集積型複合体 Download PDF

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WO2022050376A1
WO2022050376A1 PCT/JP2021/032432 JP2021032432W WO2022050376A1 WO 2022050376 A1 WO2022050376 A1 WO 2022050376A1 JP 2021032432 W JP2021032432 W JP 2021032432W WO 2022050376 A1 WO2022050376 A1 WO 2022050376A1
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target
drug
disease
antibody
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和秀 佐藤
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Tokai National Higher Education and Research System NUC
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    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
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Definitions

  • the present invention relates to a target-accumulated complex that transmits a drug to a target cell or organ and then releases the drug using light.
  • the present invention relates to a complex in which a drug and a photosensitizer are bound to a specific binding substance such as an antibody, a substance accumulating in a tissue or an organ, or a carrier substance that can be administered to a living body, and its use.
  • the target-integrated complex of the present invention specifically binds to or circulates in the vicinity of the target, and can release the drug at the light-irradiated site by near-infrared light (Near Infrared, NIR) irradiation.
  • NIR near-infrared light
  • a drug exerts its medicinal effect by reaching a target organ or cell along the internal circulation by an administration method such as oral administration, intravenous injection, intramuscular injection, or subcutaneous injection. Since the administered drug is distributed and diluted throughout the body due to internal circulation, it is necessary to administer a certain concentration or higher in order to exert the drug effect at the target site, which often leads to side effects at sites other than the target. rice field. Due to its side effects (toxicity), many drugs have had to give up development. As a device to avoid this, a drug delivery system (DDS) and a target protein-specific antibody have been developed.
  • DDS drug delivery system
  • target protein-specific antibody have been developed.
  • DDS is a drug transmission system that controls the pharmacokinetics of drugs by controlling sustained release, absorbability, and target orientation.
  • the DDS technology in which a drug is encapsulated in high molecular weight micelles or liposomes and used as a drug carrier is well known, but it has low targetability and the liposomes administered intravenously interact with blood components, resulting in an enclosed drug. It also has the disadvantage that it leaks out and the liposomes disintegrate, and that it is captured by the reticuloendothelial tissue and quickly disappears from the blood.
  • caged compounds have been known that can protect (modify) a drug with a protecting group that can be deprotected by light and generate an active substance by irradiation with light anywhere inside or outside the tissue or cell (patented).
  • Document 1 Patent Document 2.
  • short-wavelength ultraviolet light is often used to deprotect these caged compounds, there are drawbacks such as damage to cells, limited usable range in living organisms, and non-target specificity.
  • an antibody drug conjugate in which a drug having cytotoxic activity to an antibody is bound to the antibody via a linker has been developed and has already been used for cancer treatment. It is used.
  • the synergistic effect of the biological activity as an antibody drug and the effect of the drug carried by the antibody not only exerts a selective effect on the lesion, but also enables the ADC to deliver the therapeutic drug only to the lesion site. Therefore, it is possible to expand the safety range and it is expected as a next-generation antibody drug (Non-Patent Document 1).
  • the mechanism of action is as follows. The antibody portion of the ADC binds to the antigen on the cell surface and is then incorporated into the cell by internalization.
  • the linker of the internalized ADC is cleaved in the cell by the action of intracellular lysosomes, and the small molecule drug is released.
  • the released small molecule drug exerts its medicinal effect in the cell and acts. Since the compound is bound to the antibody, the drug is delivered to the cells to which the antibody is bound, and the cells are specifically removed, the drug can be selectively delivered to the target cells. Therefore, even a smaller amount of the compound is effective in the target cells and has little effect on normal cells, so that it is possible to achieve a wider therapeutic dose range as compared with conventional chemotherapy.
  • DDS which controls the target of action of the drug by changing the physical properties such as particle size and hydrophilicity of the drug using high molecular weight micelles and liposomes
  • DDS which is a caged compound
  • have low target orientation so side effects are completely eliminated. It is difficult to suppress it.
  • DDS by a caged compound is deprotected by using ultraviolet rays, cell damage due to ultraviolet rays is also a problem.
  • ADC using antibody acts selectively on target cells, so it is a very effective method with higher target directivity than the above-mentioned DDS.
  • a target molecule exposed on the cell surface is required and the target does not exist on the entire cell surface of the affected tissue such as an inflammatory disease or an infectious disease, even if it is used, only a limited effect can be obtained. .. Since ADC is taken up into cells and then the linker is cleaved by intracellular enzymes to release the drug, it is said that it was applied to inflammatory diseases or infectious diseases in which inflammation is seen in the entire tissue. Can only be expected to have a limited effect.
  • An object of the present invention is to provide a drug complex that acts on a subject that cannot be applied by conventional DDS, and a therapeutic method.
  • the present invention relates to a method for releasing and treating a drug by administering a target-accumulated complex in which a drug and a photosensitizer are bound to a carrier and irradiating the diseased site with light.
  • a target-directed molecule such as an antibody or a molecule that accumulates at the disease site, accumulating it in the target tissue, and then releasing the drug with light.
  • the complex used in the conventional ADC is a complex in which a compound is bound to an antibody, and is a complex that specifically acts on a target cell.
  • the present invention can also use an antibody as a carrier, but its concept and treatment target are significantly different from those of conventional ADCs. Therefore, in order to distinguish it from other antibody-drug conjugates, a complex in which a drug and a photosensitizer are bound to the carrier of the present invention is referred to as a target-accumulated conjugate. Furthermore, since the drug is released by light irradiation, even when a substance that does not have target directivity and circulates in the body, such as albumin, is used as a carrier, it acts locally only at the place where light irradiation is performed. Is possible.
  • the target-accumulated complex exerts its medicinal effect only when it is irradiated with light, it can be made to act locally, and there is a possibility that a compound that could not be used due to side effects can be used.
  • the following target integrated complex is provided.
  • the carrier is a binding molecule exhibiting binding property to a target molecule or a molecule accumulating at a disease site.
  • the target-accumulated complex according to (1) or (2), wherein the disease to be treated is any of cancer, inflammation, infectious disease, collagen disease, and organ-specific disease. ..
  • Complex The target-accumulated complex according to any one of (2) to (5), wherein the binding molecule is an antibody, an antigen-binding antibody fragment of an antibody, or an aptamer.
  • (12) A technique for transmitting a drug, in which a drug is irradiated by irradiating a target-integrated complex having a structure in which a near-infrared light-sensitive substance and a drug are linked to a previously administered carrier. Photodrug transmission technology that liberates from binding molecules.
  • (123) A therapeutic method comprising administering the pharmaceutical composition according to (11) to a subject having a disease, irradiating it with near-infrared light after a lapse of a predetermined time, and releasing the drug.
  • the irradiation of near-infrared light is characterized by direct irradiation from outside the body or by using a device introduced into a disease stem to irradiate near-infrared light (13) or (14). Treatment method.
  • the figure which shows the synthetic pathway of a dexamethasone derivative The figure which shows the synthesis process of IgG-DEX-IR700.
  • A shows the change of the antibody until the dexamethasone is bound to the antibody and the dexamethasone derivative is synthesized.
  • B is a figure confirming that IR700 was bound to the antibody-dexamethasone complex.
  • (A) is blank, (B) is 16J / cm 2 for T-DM1-IR700, (C) is ⁇ MFc-NC-DM1 not irradiated with near infrared light, and (D) is 16J for ⁇ MFc-NC-DM1.
  • the result of mass spectrometry analysis by irradiating near infrared light at / cm 2 is shown.
  • the figure which shows that T-DM1-IR700 specifically binds to HER2-positive cells.
  • the figure which shows that the HER2-negative cell alone which does not bind an antibody has no effect of a target accumulation type complex.
  • the figure which shows that the target accumulation type complex is effective in the in vitro disease model system using various HER2-positive cells.
  • the present inventor combines an ADC with a light-sensitive substance and irradiates it with light to selectively bind to target cells and exert an effect, which is the same as the effect of the ADC, but does not express an antigen molecule. It was found that it is possible to give a therapeutic effect to the disease. Furthermore, if this is further developed and a drug and a photosensitizer are bound to a molecule having high biostability, using any substance as a carrier, not only an antibody, the drug will be released only at the site irradiated with light, and the drug will be released. Local action can be expected.
  • a target-accumulated complex in which a drug and a photosensitizer are bound to a carrier having high biostability it can be applied to more diseases than ADC.
  • the target-accumulated complex can act locally, there is a possibility that compounds that could not be used due to side effects can be used.
  • the target accumulation type complex a complex in which a drug and a near-infrared photosensitive substance are linked to an antibody is shown, but any molecule having high biostability is used.
  • an antibody or a molecule that accumulates at a disease site can be preferably used.
  • the present inventors analyzed the mechanism of action of the antibody-IR700 complex used in near-infrared immunotherapy (NIR-PIT), which is a type of ADC, to induce necrosis.
  • NIR-PIT near-infrared immunotherapy
  • Photochemical reaction by irradiation with near-infrared light cleaves the ligand of IR700 from the antibody-IR700 complex, resulting in physical changes in the antibody-IR700 complex, which instantly induces cell death. It was found and reported (Non-Patent Document 2). Since the antibody-IR700 complex undergoes a structural change due to a photochemical reaction, the complex in which a drug is bound to the antibody-IR700 complex may also undergo a structural change.
  • the disease that is the subject of the present invention is not limited to the diseases shown in the following examples, and any disease can be applied.
  • a disease in which a cell surface marker is specified, or a disease in which a molecule specifically present at a disease site or a molecule that accumulates is specified can be a more suitable target. Since the drug can be accumulated at the diseased site, a therapeutic effect can be expected for the diseased tissue or the organ itself even when the target molecule is not expressed in the entire diseased tissue. It is also possible to release the drug only to the diseased site by binding the drug and the light-sensitive substance to a carrier having no target directivity and irradiating the diseased site with light.
  • target diseases include, but are not limited to, infectious diseases, inflammatory diseases, collagen diseases, cancers, and organ-specific diseases.
  • Conventional antibody drugs require that the target molecule is expressed on the cell surface in order to exert a therapeutic effect.
  • the target accumulation type complex carries a wide range of substances such as a molecule specific to a disease, a molecule having a binding property to a molecule expressed at a disease site, a molecule accumulating at a disease site, or a substance circulating in the body. Can be used as.
  • ADC an antibody having a binding property to a molecule expressed at a disease site is used, but the antigen is not always uniformly expressed at the disease site, and there is a problem that a therapeutic effect cannot be obtained.
  • the target-accumulated complex differs from conventional antibody drugs in that the therapeutic effect of the drug extends to cells that do not express the antigen if there are cells that express even a small amount of the antigen molecule in the surrounding area. It is an epoch-making drug and a cure.
  • the effect is produced even if a molecule that accumulates at the disease site is used, it is possible to deal with a wider range of diseases.
  • the effect is obtained only in the area irradiated with light, it is possible to use a substance circulating in the body as a carrier.
  • the target-accumulated complex has a structure in which a drug and a photosensitizer are bound to a carrier.
  • a carrier Any substance may be used as the carrier, but as described above, a substance having high biostability is preferable.
  • a binding molecule that exhibits binding property to the target molecule or a molecule that accumulates at the disease site can be preferably used.
  • the binding molecule is a molecule that is expressed in a diseased cell and exhibits a binding property to a molecule expressed on the cell membrane thereof.
  • a target molecule of the binding molecule a molecule expressed in association with a disease or a molecule expressed in a diseased organ may be used.
  • a molecule that binds the drug to the photosensitizer may be selected as appropriate.
  • the binding molecule preferably used include an antibody, an aptamer and the like, and an antibody can be preferably used.
  • the antibody may be one that specifically recognizes the epitope of the antigen, and is not only an intact antibody, but also a Fab fragment, a Fab'fragment, an F (ab') 2 fragment, an Fv fragment, and a disulfide-stabilized V region fragment (dsFv). ),
  • An IgG fragment, or a peptide containing CDR which may contain a functional fragment of the antibody, and may include a portion of the antibody having binding to an epitope.
  • Functional fragments of antibodies that bind to these antigens are referred to as antigen-binding antibody fragments.
  • the antibody was produced by genetic manipulation such as a single chain antibody (scFv), a low molecular weight antibody (Fv-Casp), a bispecific antibody, a chimeric antibody such as a humanized antibody, or a humanized CDR transplanted antibody. Also includes antibodies. All of these binding molecules can be prepared by using known methods.
  • target molecules for binding molecules such as antibodies include molecules exposed on the cell surface.
  • the target molecule not only the molecule related to the disease and appearing on the cell surface but also any molecule expressed at the disease site may be used. That is, the target molecule is a molecule that causes a disease, a molecule that is expressed in relation to a disease, or a molecule that is expressed at a disease site even if it is not directly related to the disease. You may. Alternatively, it may be a molecule derived from a pathogen.
  • Specific examples of the target molecule of the binding molecule include so-called cancer antigens that are specifically expressed on the cell surface of cancer cells, and surface antigens that are expressed on specific immune cells in the case of collagen disease.
  • pathogen-derived molecules derived from pathogens such as viruses, bacteria, fungi, and parasites and appearing on the cell surface, and substances secreted by the pathogens can be mentioned.
  • pathogens such as viruses, bacteria, fungi, and parasites and appearing on the cell surface, and substances secreted by the pathogens
  • it may be a molecule of a host cell that appears on the cell surface due to a disease caused by a pathogen.
  • the target molecule include molecules on the cell surface composed of peptides, proteins, lipids, polysaccharides, proteoglycans, lipopolysaccharides, nucleic acids and the like.
  • a drug and a photosensitizer may be combined with a molecule that accumulates at a disease site.
  • molecules that accumulate at the disease site include cytokines, chemokines, and growth factors (FGF, EGF, BTC, PEDF, SCF, HER2, HER3) that accumulate in the inflamed region in the case of inflammatory diseases.
  • Angiogenic factors VEGF, PIGF
  • growth factors LIF, OSM, TGF, BMP, IGF, CNTF
  • inflammatory mediators chemical mediators (bradykinin, serotonin, histamine), prostanoids (prostagrandin, leukotriene), chloride
  • renal diseases such as tarium and citric acid, creatinine, albumin, low molecular weight albumin, peptide, diethylenetriamine compound such as MAG3 (mercaptoacetylglycylglycylglycine), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid)
  • Feritin phosphate, Ca compound, bisphosphonic acid compound, transferase, HMDP (hydroxymethylene diphosphonate), MDP (methylene diphosphonic acid) in the case of myeloid disease, platelets and coagulation factors in the case of bleeding site (I-XIII), thrombins, growth
  • VEGF vascular endothelial growth factor
  • surfactent surfacten
  • KL-6 phosphatidylcholine
  • choline choline
  • sphingosine ACE
  • MAA MAA coarse aggregation
  • lung diseases Albumin
  • iodine for thyroid disease sodium hypertechnetiumate, cardiac myosin for heart disease, MIBG (methiodobenzylguanidin), BMIPP ( ⁇ -methyl-p-iodophenylpentadecanoic acid), tetrophosmin, MIBI (methoxy) Isobutylisonitrile), tallium chloride, tin colloid for liver disease, GSA, PMT, tin colloid for lymph node diseases, futinic acid, etc., Proton pump target for digestive diseases, tin colloid, partlyctate, HSA-D, PMT, A substance that accumulates in each organ, such as GSA, can also be used as a carrier.
  • Substances other than those exemplified here for example, substances that bind a radioisotope such as technetium as a tracer in nuclear medicine can be suitably used as a carrier. As long as it is a substance known to accumulate at a diseased site, a substance other than those exemplified above can be treated by binding a drug and a light-sensitive substance as a carrier.
  • the target-accumulated complex can also be used for the treatment of organ-specific diseases such as heart disease, renal disease, liver disease, lung disease, thyroid disease, digestive organ disease, and neuromuscular disease.
  • organ-specific diseases such as heart disease, renal disease, liver disease, lung disease, thyroid disease, digestive organ disease, and neuromuscular disease.
  • Diseases by binding and administering a drug used for the treatment of each disease and a photosensitizer to a binding molecule that binds to a molecule that is specifically expressed in an organ such as the heart or kidney, or a molecule that accumulates specifically in an organ.
  • Drugs can be accumulated and treated in an organ-specific manner. Since the purpose is to accumulate target-accumulated complexes in diseased organs, organ-specifically expressed molecules such as myocardial troponin in the heart, aquaporin in the kidney, and lung, even if they are not related to the disease.
  • ACE receptor for thyroid
  • neuropeptide for neuromuscular disease
  • neurotransmitter for neuropeptide
  • myosin for neuromuscular disease
  • transferase ferritin for bone marrow
  • incretin gastrin
  • glp-1 for digestive organs, etc.
  • molecules known to be expressed and accumulated in association with the disease such as ⁇ -synuclein and ⁇ , which are known to accumulate in the brain nervous system in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.
  • -Amyloid for heart diseases such as heart failure and myocardial infarction, ⁇ 1 receptor, AT1 (angiotensin receptor), CRHR2 (corticotropin-releasing hormone receptor 2), which are highly expressed in the heart, for renal diseases.
  • a substance without target directivity can be used as a carrier.
  • a drug and a light-sensitive substance By binding a drug and a light-sensitive substance to a carrier, circulating the drug in the body, and irradiating the diseased site with light, the drug can be selectively acted on the diseased site.
  • the substance used as a non-target-oriented carrier may be any substance such as compounds, peptides, proteins, lipids, polysaccharides, proteoglycans, lipopolysaccharides, nucleic acids, extracellular vesicles, exosomes, and biocompatible nanomaterials. Although not, it is preferably a substance having biostability.
  • the drug even if a drug and a photosensitizer are combined with a highly biostable substance such as albumin or dextran and used, the drug will not be released unless it is irradiated with light, so even if it circulates in the blood. , The drug has no side effects. By irradiating light at the diseased site, the drug is released and the drug is effective only at the diseased site.
  • a highly biostable substance such as albumin or dextran
  • the substance having biostability refers to a molecule that is physically stable for a period of time from administration to light irradiation.
  • the time from administration to light irradiation differs depending on the target cells, tissues, and organs, and the time required for the target accumulation type complex to accumulate differs, and depending on the disease, multiple light irradiations may be performed. It cannot be decided unconditionally.
  • any compound having a blood half-life of 10 minutes or more and 6 months or less, preferably 1 hour or more and 12 weeks or less may be used.
  • an already approved antibody drug may be used. More than 70 antibody drugs have already been approved in Japan, the United States and Europe, and these antibody drugs can be used by binding drugs and photosensitizers. In addition, a target-accumulated complex may be produced using an antibody drug approved in the future.
  • the main drugs approved for use in cancer as antibody drugs that are binding molecules are rituximab, offalmumab, obinuzumab, HER2 targeting trastuzumab, pertuzumab, and EGFR targeting cetuximab, which are approved for use in cancer.
  • the target-accumulated complex using the above-mentioned antibody drug is effective for the treatment of cancer, particularly solid cancer.
  • Solid cancers generally exhibit so-called heterogeneity, in which cells expressing the target molecule and cells not expressing the target molecule are mixed in one tumor.
  • conventional antibody drugs are effective only on cells expressing the target molecule
  • the target-accumulated complex is also effective against cancer cells existing around the cells expressing the target molecule. Play.
  • Conventional DDS and ADC could not cope with tumors with heterogeneity, but the target-accumulated complex also has a therapeutic effect on tumors with heterogeneity.
  • a cytotoxic drug is linked as a payload to the antibody, which is a binding molecule.
  • the drug may be linked to the antibody in the form of a prodrug.
  • cytotoxic drugs include alkylating agents, platinum preparations, antimetabolites, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors and the like.
  • the target molecule selectively binds to the cells existing on the cell surface, and the effect of the drug extends only to the surrounding cells, so that even a cytotoxic drug has side effects. It is also effective against cancer cells that are few and do not express the target molecule on the cell surface.
  • the targets of diseases other than cancer include collagen disease, infectious disease, and inflammatory disease.
  • the main antibody drugs are tosirizumab and salilumab, which target IL-6R, for rheumatoid arthritis, which is a collagen disease, and CD11, which is an inflammatory disease, for psoriasis.
  • examples include efarizumab, bladrumab targeting IL-17R, vedrizumab targeting ⁇ 4 ⁇ 7 integrin for Crohn's disease, and dupilumab targeting IL-4R ⁇ for atopic dermatitis.
  • a steroid or the like may be linked as a payload to the carrier, and in the case of an inflammatory disease, an anti-inflammatory drug may be linked as a drug.
  • compounds already used for the treatment of each disease may be linked as a payload.
  • Specific steroids include dexamethasone acetate, prednisolone, hydrocortisone acetate, prednisolone acetate, prednisolone valerate, triamcinolone acetonide, clobetazone butyrate, hydrocortisone butyrate, dexamethasone propionate, dexamethasone valerate, halcinonide.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs anti-rheumatic drugs
  • steroids JAK inhibitors, PDE4 inhibitors, etc.
  • anti-inflammatory drugs, steroids, immunomodulators, etc. can be used as the payload.
  • drugs used for treating each infectious disease such as antibiotics, antibacterial agents, and antiviral agents, may be loaded as a payload.
  • MCTD Mixed arteritis
  • polyarteritis nodosa Wegener's granulomatosis
  • eosinophilic polyangulomatosis with granulomatosis Charg-Strauss syndrome
  • microscopic polyarteritis Takayasu's arteritis (Aortitis Syndrome), Giant Cell Arteritis (Temporal Arteritis)
  • Rheumatic Polyarteritis nodosa Eosophilamyositis, Adult Still's Disease, Tonic Dermatomyositis, Psoriatic Arthritis, Recurrence Polyarteritis , Bechet's disease and sarcoidosis.
  • viruses that can cause infectious diseases include various coronaviruses, specifically SARS-CoV-2, SARS-CoV, and MARS-CoV.
  • Ebola virus decavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T-cell leukemia virus, simple herpesvirus type 1, simple herpesvirus type 2, varicella / herpes zoster Virus, Cytomegalovirus, Human herpesvirus 6, Human herpesvirus 7, Epstein bar virus, Human herpesvirus 8 (capoic sarcoma-related herpesvirus), influenza virus, adenovirus, norovirus, rotavirus, RS virus, measles virus , Mumps virus, rhinovirus, dengue virus, papillomavirus, poliovirus and mad dog disease virus.
  • bacteria that can cause infectious diseases include pathogenic Escherichia coli, Shigella spp. (Shigella dysenteriae, S. frexneri, S. sonei, etc.), Salmonella spp. .Typhimurium, S. enteritidis, etc.), Enterobacter aerogenes, E. cloacae, etc.), Klebsiella pneumoniae, K. oxytoca, etc., Proteus mirabilis, P. vulgaris, etc. Bacteria of the genus (Yersinia pestos, Y. enterocolitica, etc.), Bacteria of the genus Vibrio (Vivrio chorelae, V.
  • parahaemolytucs etc.
  • Bacteria of the genus Hemophilus Bacteria of the genus Hemophilus (Haemophilus influenzae, H. parainfluenzae, H. Capacia, P. putida, etc.), Acinetobacter calcoaceticus, A. baumannii, A. lowffii, etc., Legionella pneumophila, etc., Bordetella. Melitensis, B. abortus, B. Etc.), Rabbit bacteria (Francisella tularensis), Bacteroides fragilis, B. melaninogenicus, etc., Neisseria gonorrhoeae, N.
  • meningitidis, etc. Staphylococcus aureus, S. epider saprophyticus, etc.), Enterococcus faecalis, E. faecium, E. avium, etc., Bacillus subtiris, B.anthracis, B. cereus, etc.), Clostridium difficiole, C .Botulinum, C. perfringens, C. tetani, etc.), Corynebacterium diphtheirae, etc., Mycobacterium tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulare, M. .Kansasii, M.
  • fungi that can cause infectious diseases include Candida albicans, C. krusei, C. glabrata, C. tropicalis, etc., Cryptococcus neoformans, and Aspergillus fumigatus. , A. niger, etc.), Mucorethrix fungi (Mucor circinelloides, Lichtheimia corymbifera, Rhizopus), Sporothrix schenkii, blastis Examples include Paracoccidiides brasiliensis, Coccidiides immitis, and Histoplasma capsulatum.
  • Examples of parasites that can cause infectious diseases include diarrhea amoeba parasites, colon baranchidium, Naegleria fowleri, Acanthamoeba, Giardia lamblia, and Cryptosporidium spp. ), Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosomabrucei, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma cruzi, Ryuschmania mania Toxoplasma gondii) and Brazilian pneumocystis (Ancylostoma braziliense) can be mentioned.
  • drugs and near-infrared light-sensitive molecules are bound to molecules that specifically appear on the cell surface with the disease or that bind to substances secreted by pathogens as targets.
  • the target integrated complex may be prepared by linking the above. If infected cells can be specifically killed, virus carriers such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and human T-cell leukemia virus may be able to completely eliminate the virus from the body. ..
  • the payload may use one kind of drug or two or more kinds of drugs.
  • a target-accumulated complex in which the drugs are individually loaded as a payload may be mixed and used, or a plurality of types of drugs may be linked to one carrier and used.
  • the drug antibody ratio (DAR) is high, but in the target integrated complex, the drug carrier ratio (corresponding to DAT) is 1 to 10. All you need is.
  • the target-accumulated complex is applied not only to cancer but also to various diseases such as inflammatory diseases, collagen diseases, and infectious diseases. Since it is considered that the amount of drug to be treated is different, it is desirable to optimize it as appropriate.
  • the method of the present invention is a therapeutic method that can be applied locally. Therefore, even drugs that have been abandoned due to off-target effects or systemic toxicity may be usable. In addition, by acting locally, it becomes possible to use it at a lower dose than before, so that side effects can be reduced.
  • linkers / spacers are maleimide caproyl, maleimide caproyl-polyethylene 20 glycol (MC (PEG) 6-OH), p-aminobenzylcarbomile (PAB), valine-citrulin (vc), N-methyl-valine.
  • N-succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB), N-succinimidyl 4- (2-pyridyldithio) 2-Sulfobutanoate (sulfo-SPDB), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP), 2-iminothiolane and anhydrous acetyl Sulfonic acid can be exemplified.
  • SCC N-maleimidemethyl) cyclohexane-1-carboxylate
  • SPDB N-succinimidyl 4- (2-pyridyldithio) butanoate
  • SPDB N-
  • the drug is usually linked to the antibody by a conjugated reaction via lysine or cysteine.
  • a conjugated reaction via lysine or cysteine.
  • Selective bioconjugation reaction by incorporation of unnatural amino acids, introduction of free cysteine by gene modification (THIOMAB method), exposure of aldehyde from sequences containing N-terminal and free cysteine, and conjugation for the purpose of producing more homogeneous ADC.
  • Techniques such as a method for carrying out a reaction (SMARTag method) and a ligation method using an enzyme can also be applied.
  • near-infrared photosensitizers are linked to the target-accumulated complex.
  • high-energy photons that is, light with a short wavelength.
  • a near-infrared light-sensitive substance is usually selected.
  • a phthalocyanine-based photosensitizer As a compound suitable for this condition, there is a phthalocyanine-based photosensitizer.
  • IR700 IRDye (registered trademark) 700DX, LI-COR Biosciences
  • NHS ester of IR700 it can be conjugated to a specific binding molecule such as an antibody by covalent bonding.
  • the target-accumulated complex can be administered locally or systemically using any method. Specifically, it is administered by intramuscular administration by injection into the affected area, subcutaneous administration, intradermal administration, intravenous administration, or intraperitoneal administration, inhalation, ointment, application, application, nasal instillation, or parenteral means by instillation.
  • oral administration may be used.
  • parenteral formulations are injectable fluids that include pharmaceutically acceptable and physiologically acceptable fluids such as water as a vehicle, saline solution, equilibrium salt solution, aqueous dextrose, or glycerol.
  • pharmaceutically acceptable and physiologically acceptable fluids such as water as a vehicle, saline solution, equilibrium salt solution, aqueous dextrose, or glycerol.
  • Non-toxic solid carriers for solid compositions may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. can.
  • the administered pharmaceutical composition is a small amount of non-toxic, such as a wetting agent or emulsifier, a preservative, and a pH buffer, such as sodium acetate or sorbitan monolaurate. May contain auxiliary substances.
  • the therapeutically effective dose of the target-accumulated complex depends on the target disease and what kind of compound is bound as a drug, but can be appropriately set according to the disease and symptoms. For example, it can be appropriately set between 0.1 mg and 1000 mg per 60 kg of body weight.
  • the dose varies depending on the administration method such as intravenous administration, local administration, and intraperitoneal administration.
  • the administration may be selected according to the disease, symptom, etc., such as daily administration or single administration. In addition, it can be administered in the presence of other therapeutic agents.
  • an LED, an LED laser, a light beam that has passed through a filter, or the like may be used to irradiate a therapeutic dose having an appropriate wavelength.
  • the treatment dose may be 1 to 1000 Jcm -2 , and the irradiation time may be appropriately set between 5 seconds and 1 hour.
  • Direct irradiation may be performed from outside the body, but as a device to be introduced and irradiated to the diseased trunk, a light guide catheter, an endoscopic light guide fiber, a puncture irradiation fiber, a blood vessel light guide catheter, a drain indwelling light guide device, or an implantable body can be used.
  • a light irradiation device such as a mold, a sticking mold, or a bracelet mold can be used.
  • the target-accumulated complex When the target-accumulated complex is systemically administered as a pharmaceutical composition by intravenous injection, the target-accumulated complex may be irradiated with near-infrared light after accumulating in the lesion in consideration of the time for accumulating in the lesion.
  • the time for the target accumulation type complex to accumulate in the lesion portion varies depending on the carrier used and the like, but is, for example, about 5 minutes to 48 hours. In the case of local administration, light irradiation can be performed in a shorter time after administration than in the case of systemic administration.
  • the irradiation interval is not particularly limited.
  • various irradiation schedules can be set, such as irradiation multiple times on the same day at predetermined intervals of about 5 minutes to 10 hours, irradiation every day, every other day, or every few days to several weeks. ..
  • the administration schedule when setting a multiple irradiation schedule is not particularly limited, but if time has passed since the previous administration of the target accumulation type complex, the administration may be performed again before the irradiation, and the target accumulation may occur. It can be determined in consideration of the pharmacokinetics of the type complex.
  • Example 1 Preparation of target-accumulated complex in which dexamethasone (DEX) and IR700 were bound to mouse IgG A target-accumulated complex in which dexamethasone and IR700 were bound to an antibody was prepared and its effect was examined. First, a dexamethasone derivative having an SH group was synthesized, bound to an antibody, and finally IR700 was bound to prepare a target-integrated complex. First, a method for synthesizing a dexamethasone derivative will be described (FIG. 1).
  • Step 2 Add 4M HCl ethyl acetate (4.5 mL) under an ice bath to a solution of intermediate product 1 (601.6 mg) in methylene chloride (9 mL), stir at room temperature for 30 minutes, distill off the solvent under reduced pressure, and whiten.
  • Intermediate product 2 (Int. 2) (530.7 mg, crude yield 93%) was obtained as a solid. Confirmation was performed by mass spectrometry.
  • Step 3 Add DIPEA (307 ⁇ L) under an ice bath to a solution of Intermediate Product 2 (371.1 mg), 3- (triphenylmethylthio) propionic acid (256.7 mg) and HATU (330.7 mg) in DMF (7 mL). , Stirred for 1 hour at room temperature. The reaction solution examined under the same conditions was combined with this reaction solution using Intermediate Product 2 (51.3 mg), and ethyl acetate and 5% aqueous sodium hydrogen carbonate were added to separate the layers. The organic phase was washed twice with 5% saline, then anhydrous sodium sulfate was added and dried.
  • Milli-Q water was added and the mixture was centrifuged again at 4000 g and 4 ° C. for 15 minutes to remove the reagent residue from the antibody solution.
  • the maleimided antibody was recovered from the filter unit, and Milli-Q water was added to prepare a 500 ⁇ L solution. This solution was used as it was for the next reaction.
  • LC / MS TIC total ion chromatogram
  • the peak seen after dexamethasone modification indicates a peak derived from a dexamethasone derivative in which an SH group is added to dexamethasone.
  • the peak derived from the dexamethasone derivative disappeared (FIG. 2 (A) after two extrafiltrations), and it can be seen that only the antibody to which the dexamethasone was added was purified.
  • IR700 conjugation was bound to the dexamethasone-conjugated antibody synthesized and purified by the above step. 145 ⁇ L of 0.1 M phosphate buffer (pH 7.4) was added to the dexamethasone conjugated antibody (50 ⁇ L, 290 ⁇ g). 80 ⁇ L of Milli-Q water was added, and an additional 14.5 ⁇ L of DMSO was added. 0.77 ⁇ L of a separately prepared IR-700 NHS ester / DMSO solution was added and incubated at 25 ° C. for 1 hour.
  • the reaction solution was transferred to a centrifugal filter unit (Amicon Ultra-4, cutoff 30K, Merck Millipore), and Milli-Q water (containing 5% DMSO) was further added so that the liquid volume became 4 mL. After centrifuging at 4000 g and 4 ° C. for 15 minutes to remove the filtrate, Milli-Q water was added and the mixture was centrifuged again at 4000 g and 4 ° C. for 15 minutes. This centrifugal filtration operation was repeated twice more to remove the reagent residue from the antibody solution. The dexamethasone-IR-700 conjugated antibody was recovered from the filter unit, and Milli-Q water was added to prepare a 200 ⁇ L solution (antibody concentration 170 ⁇ g / mL, 34 ⁇ g).
  • FIG. 2 The left figure shows a CBB stained image, and the right figure shows an image in which IR700 is visualized by fluorescence. Only in IgG-SMCC-DEX-IR700, there was a band visualized by fluorescence, and it was confirmed that IR700 was bound to the antibody-dexamethasone complex.
  • IgG-SMCC-DEX-IR700 was irradiated with light and analyzed whether dexamethasone was released.
  • the liquid that had permeated the membrane was recovered, and analysis was performed using an evaluation system (Non-Patent Document 3) that detects phosphorylation of Rb protein that changes with the addition of dexamethasone. It is known that the addition of dexamethasone increases the phosphorylation of Rb protein, and this evaluation system is a system for evaluating the release of dexamethasone by a bioassay using the same.
  • a fraction of human lung adenocarcinoma-derived cell line A549 cells 1 ⁇ 105 was seeded in a 6 -well plate and the antibody component was removed from the IgG-SMCC-DEX-IR700 complex that had been irradiated or unirradiated the next day. 2 ⁇ l of each was added, and the cells were cultured for 2 days. No addition was added as a negative control, dexamethasone was added so as to be 2 ⁇ 10 -7 M as a positive control, and the cells were similarly cultured for 2 days. The cells were collected and analyzed by Western blotting (Fig. 3).
  • Antibodies to detect Rb protein Cell Signaling Technology, Rb (4H1) mouse monoclonal antibody
  • antibodies to detect phosphorylated Rb protein Cell Signaling Technology, Phospo-Rb (Ser807 / 811) (D20B12) XP rabbit monoclonal antibody
  • Western blotting was performed using an antibody that detects ⁇ -actin (Fuji Film Wako Junyaku Co., Ltd., anti- ⁇ -actin monoclonal antibody) (Fig. 3, left).
  • dexamethasone that is, a drug component
  • the antibody component is removed after light irradiation for analysis, but in vivo, the antibody was bound by accumulating the antibody at the target using the antibody and releasing the drug by light irradiation. It is shown that the surrounding cells and tissues can also be treated with the drug.
  • Example 2 1. Effect of light irradiation on T-DM1 (trastuzumab-emtancin) -IR700 From Example 1 above, it was shown that the drug was cleaved from the carrier while maintaining its medicinal effect by near-infrared light irradiation. For confirmation, analysis was performed using a system in which IR700 was bound to an existing ADC.
  • T-DM1 (Kadosila TM, 1.0 mg, 6.6 nM) and IRDye700DX NHS (66.8 ⁇ g, 34.2 nM) in 0.1 M phosphate buffer (Na 2 HPO 4 , pH 8.5). After incubating for 1 hour at room temperature, unreacted reagents and T-DM1-IR700 complex were separated by Sephadex G50 column (PD-10; GE healthcare). The protein concentration was measured by the CBB method, and the recovered T-DM1-IR700 concentration was determined. In addition, the IR700 concentration was determined by measuring the absorbance at 698 nm, and the number of fluorescent molecules bound to the antibody was confirmed. Here, three times as many fluorescent molecules are bound to T-DM1, but the ratio of the fluorescent molecules to the ADC can be about 1 to 20.
  • the adjusted T-DM1-IR700, 2 ⁇ g was irradiated with near-infrared light at 0, 1, 4, 8, 16 J / cm 2 using an LED having an emission wavelength of 690 nm, and analyzed by SDS-PAGE (FIG. 4). ).
  • the protein was visualized by CBB staining (Fig. 4, left), and IR700 was detected by fluorescence (Fig. 4, right).
  • the same amount of T-DM1-IR700 was run in each lane (Fig. 4, left), but as the irradiated light energy became stronger, the fluorescence intensity decreased, and near-infrared light was irradiated at 16 J / cm 2 . Almost no fluorescence was observed in the sample (Fig. 4, right).
  • T-DM1 (Kadosila) is a complex in which trastuzumab is linked to trastuzumab with the microtubule polymerization inhibitor emtansine, which is a maitansine derivative, by a non-cleaving SMCC linker (Fig. 5).
  • emtansine which is a maitansine derivative
  • Fig. 5 a non-cleaving SMCC linker
  • T-DM1 is taken up into cells by internalization after trastuzumab binds to HER2 on the cell surface.
  • emtancin is cleaved from the antibody moiety by an enzyme in the cell, and it has a medicinal effect as a microtubule polymerization inhibitor.
  • the T-DM1-IR700 which is a target-accumulated complex, does not change in the T-DM1 portion by light irradiation, the T-DM1-IR700 is a HER2-positive cell to which trastuzumab can bind, similar to Kadcyla. Will only act selectively. Further, if the maytansine derivative is subjected to a structural change by light irradiation and a large structural change is given, the effect as a microtubule polymerization inhibitor may be lost.
  • the maitansine derivative which is a drug
  • the structure of DM1 is maintained, that is, the medicinal effect as a microtubule polymerization inhibitor.
  • Analysis was performed by mass spectrometry to determine whether the cells were cut while being maintained.
  • the T-DM1-IR700 was irradiated with near-infrared light at 0 or 16 J / cm 2 , the antibody component was separated and removed by centrifugation, and the filtrate was mass-analyzed for analysis. Mass spectrometry was performed using QTRAP6500 (SCIEX) and an HPLC system using Prominence (Shimadzu Corporation) under the following conditions. Detection: Cation mode Column: L-column2 ODS 3 ⁇ m, 1.5 ⁇ 150 mm Column temperature: 40 ° C Mobile phase: A 0.1% formic acid, 5% acetonitrile: B 0.1% formic acid, acetonitrile gradient Monitoring time: 20 minutes Injection volume ( ⁇ l): 10
  • the ⁇ MFc-NC-DM1-IR700 (FIG. 8 (D)) irradiated with near-infrared light 16 J / cm 2 was compared with the unirradiated ⁇ MFc-NC-DM1-IR700 (FIG. 8 (C)). Peaks were observed at different retention times. Further, the retention time of the peak observed by irradiating the ⁇ MFc-NC-DM1-IR700 with light for 13.68 minutes is the retention of the peak observed by irradiating the T-DM1-IR700 with light (FIG. 8 (B)). The time was 13.58 minutes, which was almost the same, and it is considered that S-Me-DM1 was released in both cases. Note that FIG. 8A shows a blank.
  • HER2-positive and negative cells were mixed was prepared, and the effect of the target-accumulated complex was analyzed using T-DM1-IR700. Analysis was performed using HER2-positive cells 3T3 / HER2 in which the HER2 gene was introduced into mouse fibroblasts and MDA-MB-468 luc in which luciferase was introduced into HER2-negative human breast cancer cells.
  • T-DM1-IR700 specifically binds to HER2-positive cells. After blocking 3T3 / HER2 cells (Fig. 9 left) or MDA-MB-468 luc cells (Fig. 9 right) with 10 ⁇ g / ml T-DM1-IR700 or 100 ⁇ g / ml T-DM1 in advance, 10 ⁇ g / ml T-DM1-IR700 was added and analyzed by flow cytometry. As a result, T-DM1-IR700 specific binding was observed in 3T3 / HER2 cells, whereas in MDA-MB-468 luc cells, cells blocked by T-DM1 and T-DM1-IR700 alone. No difference was observed from the cells incubated in 1 and it was confirmed that the binding of T-DM1-IR700 did not occur.
  • HER2-positive cells antigen-positive cells
  • HER2-negative cells antigen-negative cells
  • T-DM1-IR700 was 1 ⁇ g / ml, 5 ⁇ g / ml, 10 ⁇ g / ml, and Tra-IR700 was 10 ⁇ g / ml. Processing was performed (FIG. 11). Even when the T-DM1-IR700-treated cells were treated at 1 ⁇ g / ml, a decrease in luciferase activity was observed in the near-infrared light-irradiated cells. In contrast, no decrease in luciferase activity was observed in Tra-IR700-treated cells. As shown in FIG.
  • MDA-MB-468 luc cells into which luciferase has been introduced are HER2-negative and do not bind to T-DM1-IR700. Therefore, suppression of luciferase activity is considered to be the effect of DM1 on cells that do not express HER2.
  • the luciferase activity is increased in the cells irradiated with near-infrared light as compared with the cells not irradiated. This indicates that even if the HER2-positive cells die, the HER2-negative cells into which luciferase has been introduced are not affected.
  • MDA-MB-468 luc cells were seeded alone, treated with T-DM1-IR700, and irradiated with near-infrared light to confirm the effect of the target-accumulated complex (FIG. 12).
  • MDA-MB-468 luc cells which are HER2-negative cells, were seeded alone, and after 24 hours, they were replaced with T-DM1-IR700 or a medium supplemented with PBS as a control, and after 6 hours, using an LED having an emission wavelength of 690 nm. Irradiate near-infrared light at 4 J / cm 2 . After culturing for 4 days after light irradiation, the luciferase activity of each group was analyzed (Fig. 12).
  • T-DM1-IR700 binds to HER2-positive cells due to the coexistence of HER2-positive cells, and DM1 is released by near-infrared light irradiation, which is effective against HER2-negative cells. It shows that it is.
  • HER2-positive cells Using human HER2-positive cells, it was confirmed whether the same event occurs not only in 3T3 / HER2 cells, which are cells into which the HER2 gene has been introduced, but also in cancer cell lines established from human tumors (Fig. 13).
  • human lung cancer cell line H2170 or human breast cancer cell line SK-BR-3 mixed culture with MDA-MB-468 luc cells was performed in the same manner as above to prepare an in vitro model system with heterogeneity, and T. -The action of DM1-IR700 was analyzed.
  • T-DM1-IR700 when T-DM1-IR700 was allowed to act and irradiated with near-infrared light, a decrease in luciferase activity was observed, and MDA-MB-468 luc of HER2-negative cells was observed. It was found that the cells were dead. That is, it is shown that the effect of T-DM1-IR700 also has an effect on cells to which trastuzumab, which is an antibody moiety, does not bind. By comparing this result with the result of mass spectrometry, it can be concluded that DM1 is released from the antibody by near-infrared light irradiation and has an effect on cells to which the antibody does not bind. These results indicate that the drug affects not only the cells to which the target-accumulated complex is directly bound, but also the cells in the vicinity thereof. The result clearly shows that the treatment with the target-accumulated complex is a different treatment method from the conventional ADC and NIR-PIT.
  • IR700 can be added to an already approved ADC such as ADCETRIS (trademark, generic name: brentuximab vedotin). Further, IR700 may be added to the ADC to be developed in the future and used.
  • the irradiation start date was set to Day 0.
  • T-DM1-IR700 was administered by tail intravenous injection at 3.6 ⁇ g per 1 g of mouse body weight. Since it is a treatment using near-infrared light using an antibody drug, a treatment using a target-specific complex is also referred to as NIR-PIT.
  • NIR-PIT was performed by irradiating Day 0 with a laser at 15 J / cm 2 and Day 1 at 30 J / cm 2 only on the right side.
  • the therapeutic effect was achieved by measuring the estimated tumor volume and the luciferase activity of the tumor.
  • the estimated tumor volume was calculated as (major axis x minor axis 2 x 1/2) by measuring the major axis and minor axis of the tumor.
  • Luciferase activity was measured by intraperitoneal administration of D-luciferin (7.5 mg / ml, 200 ⁇ l) and using IVIS® imaging system. The unit of light emission to be measured was radiance, and the analysis was performed using Living Image Software (registered trademark) (FIG. 14).
  • drugs can be bound to cytokines, albumin, etc., as well as antibodies, and the drugs can be released by light at the diseased site to act, so diseases that could not be targeted for DDS until now. Can also be targeted.
  • Photodrug transmission technology using a target-accumulated complex in which a drug and IR700 are bound to a specific binding substance is effective for cells other than the target cells with conventional near-infrared spectroscopy (NIR-PIT) and ADC. It is a completely different technology in that it gives.
  • NIR-PIT near-infrared spectroscopy
  • the present inventor prepared a target-accumulated complex in which a drug and a photosensitizer were bound to an antibody, and analyzed its effect and mechanism of action. As a result, they have found that the effect of the drug extends not only to the cells to which the antibody is bound but also to the cells surrounding the cells that do not express the antigen, and completed the present invention.
  • the conventional treatment with NIR-PIT or ADC is effective only on the cells to which the antibody binds, but the target-accumulated complex is characterized by having an effect on the cells not expressing the antigen.
  • the target accumulation type complex can be produced by binding a drug and a photosensitive substance not only to a specific binding substance such as an antibody but also to a substance accumulating at a disease site or a substance having high blood stability. Therefore, it can be applied to a wider range of diseases than the conventional ADC.

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WO2020179749A1 (ja) * 2019-03-05 2020-09-10 国立大学法人東海国立大学機構 標的特異的複合体及びその用途

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