Antimicrobial Phototherapy

Abstract

Provided is a novel application of near-infrared ray immunological therapy. Specifically, provided are: a target-specific complex having a structure in which a near-infrared light sensitive substance is linked to IgY specific to targets classified as bacteria, fungi, molds, viruses, parasitic organisms, parasitic bugs, or Rickettsia; a therapeutic method using the target-specific complex; and the like.

Description

[Technical field]

[0001] The present invention relates to photoantimicrobial therapy, specifically to a target-specific complex that exhibits target-selective inhibitory activity by utilizing near-infrared light irradiation, and uses thereof. [Background technology]

[0002] Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment method in which a monoclonal antibody bound to a photosensitive substance (e.g., IRdye700DX (IR700)) is reacted with cancer cells, and then near-infrared light is irradiated to selectively destroy the cancer cell membrane. Clinical trials have also been conducted on head and neck cancer targeting EGFR, with good results, and phase 3 clinical trials are currently underway. Recently, it has been elucidated that the mechanism by which NIR-PIT exerts its antitumor effect is a photochemical reaction that is completely different from existing antitumor treatments (see Non-Patent Document 1). Patent Documents 1 to 3 propose the use of NIR-PIT in tumor treatment, etc. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2014 / 0120119 [Patent Document 2] US Patent Application Publication No. 2018 / 0150405 [Patent Document 3] US Patent Application Publication No. 2014 / 0120119 [Non-patent literature]

[0004] [Non-Patent Document 1] Sato K et al. ACS Cent. Sci., 2018, 4 (11), pp 1559-1569. [Non-Patent Document 2] Gadde U et al. Anim Health Res Rev. 2015 Dec;16(2):163-76. Summary of the Invention [Problem to be solved by the invention]

[0005] NIR-PIT is a technology with high potential due to its unique mechanism of action, etc., and further applications are expected. Therefore, an objective of the present invention is to provide new uses for NIR-PIT. [Means for solving the problem]

[0006] In conventional NIR-PIT targeting cancer cells, IgG monoclonal antibodies are usually used as the antibodies that bind photosensitizers, but in exploring applications of NIR-PIT, the inventors focused on IgY (immunoglobulin Y: chicken egg yolk immunoglobulin). IgY is an antibody unique to birds. Chicken IgY, which can be said to be a representative IgY, is the main immunoglobulin in egg-laying chickens and migrates from serum to egg yolk to confer passive immunity. Chicken IgY can be collected from chicken eggs, so it can be easily collected at high concentrations. In other words, it can be prepared cheaply and in large quantities. In contrast to antibiotics, IgY does not show side effects, resistance, or toxic residues. Therefore, IgY is attracting attention as a new means of suppressing infectious diseases that is different from antibiotics. So far, the inhibitory effect of IgY against pathogens such as Escherichia coli, Salmonella enteritidis, Salmonella enterocolitica, and Gallibacterium analis has been reported (see, for example, Non-Patent Document 2). However, the effect is not sufficiently high, and there are many practical problems, such as the need for continuous administration to enhance the effect. In light of this situation, the present inventors thought that combining NIR-PIT and IgY would be an effective treatment for infectious diseases, and prepared a target-specific structure in which a near-infrared light-sensitive substance was linked to a chicken-derived IgY polyclonal antibody, and verified its effect and practicality. As a result of detailed verification experiments, the antibacterial effect of NIR-PIT using IgY polyclonal antibody was very high and was rapidly exerted. The fact that it has a rapid effect can be said to be a particularly favorable feature as an attacking means against bacteria and fungi that grow quickly. Furthermore, the target-specific construct reacted not only with a specific Candida fungus (C. albicans), which is the original antigen of the IgY used, but also with closely related species, but did not react with human cells. These facts mean that it can be effective against a wide range of bacterial species while ensuring a high level of safety, confirming its high practicality. In addition, the fact that a bactericidal effect was confirmed against Candida fungus, which has a strong cell wall structure, indicates that the target-specific construct has strong inhibitory activity and a wide range of applications.

[0007] As described above, it has become clear that photoantibacterial targeted therapy (PAT) using IgY in NIR-PIT (hereinafter, this targeted photoantibacterial therapy may be referred to as "IgY-PAT therapy") is targeted without any effect on the human body and is extremely effective as a means of combating infectious diseases such as fungal and bacterial infections (treatment and prevention of infectious diseases, prevention of the spread of infection, etc.). Based on this result, the following invention is provided. [1] A target-specific complex in which a near-infrared light receptor is linked to IgY specific for a target classified as a bacterium, fungus, mold, virus, parasite, helminth, or rickettsia. [2] The target-specific complex described in [1], wherein the target is a bacterium, a fungus, or a mold. [3] The target-specific complex described in [2], wherein the bacterium is a species selected from the group consisting of Pseudomonas, Acinetobacter, Staphylococcus, Streptococcus, Enterococcus, Escherichia coli, Shigella, Salmonella, Enterobacter, and Klebsiella, and the fungus or mold is a species selected from the group consisting of Candida, Aspergillus, Mucor, and Cryptococcus. [4] The target-specific complex according to any one of [1] to [3], wherein the IgY is a polyclonal antibody. [5] The target-specific complex according to any one of [1] to [4], wherein the near-infrared light sensitive substance is a phthalocyanine dye. [6] The target-specific conjugate according to [5], wherein the phthalocyanine dye is IR700. [7] A composition comprising the target-specific complex described in any one of [1] to [6]. [8] The composition described in [7], which is used for treating or preventing an infectious disease caused by the target. [9] A treatment method comprising the following steps (1) and (2): (1) administering the composition according to [8] to a subject to be treated, and allowing the target-specific complex to bind to the target; (2) illuminating the target with near-infrared light.

[10] The method of treatment described in [9], wherein the wavelength of the near-infrared light is 650 to 740 nm.

[11] The method of treatment described in [9], wherein the wavelength of the near-infrared light is 670 to 720 nm.

[12] The irradiation dose of the near-infrared light is 1 J cm -2 The above-mentioned treatment method is described in any one of [9] to

[11] .

[13] The irradiation dose of the near-infrared light is 2 J cm -2 ~500J cm -2 The method for treatment according to any one of [9] to

[11] ,

[14] The composition described in [7], which is used for disinfecting or decontaminating a contaminated object by the target. [Brief description of the drawings]

[0008] [Figure 1] Preparation and quality confirmation of Candida albicans-IgY (CA-IgY)-IR700. Binding of CA-IgY to IR700 was confirmed by protein staining (left) and fluorescent staining (right). [Diagram 2] Binding of CA-IgY-IR700 to C. albicans. CA-IgY-IR700 was added at various concentrations to C. albicans seeded in a tube, and after incubation at 37°C for 1 hour, the fluorescence intensity was measured by flow cytometry. [Diagram 3] Binding of CA-IgY-IR700 to C. albicans and related species, and A431. [Figure 4] In vitro NIR-PAT experimental method. [Diagram 5] In vitro NIR-PAT experimental results. The relationship between antibody concentration and therapeutic effect (left), and the relationship between radiation dose and therapeutic effect (right) were evaluated by colony count. [Figure 6] In vitro NIR-PAT experimental results. The effect of IgY-PAT was evaluated using a confocal microscope (dead cell staining) (left) and a scanning electron microscope (right). [Figure 7] In vivo NIR-PIT experimental method. [Figure 8] In vivo NIR-PIT experimental results. Fluorescence at the treatment site was observed over time for the CA-IgY-IR700 group (applied with CA-IgY-IR700 and not irradiated with near-infrared light) and the IgY-PAT group (applied with CA-IgY-IR700 and then irradiated with near-infrared light). [Figure 9] In vivo NIR-PIT experimental results. Tumor area was compared among the germ-free group (no C. albicans application), CA group (only C. albicans), CA-IgY-IR700 group (CA-IgY-IR700 application without near-infrared light irradiation), Light group (near-infrared light irradiation only), and IgY-PAT group (CA-IgY-IR700 application followed by near-infrared light irradiation). [Figure 10] In vivo NIR-PIT experimental results. Purulent discharge from the ulcer area was compared and evaluated among the germ-free group (no C. albicans applied), CA group (only C. albicans), CA-IgY-IR700 group (applied CA-IgY-IR700 and no near-infrared light irradiation), Light group (only near-infrared light irradiation), and IgY-PAT group (applied CA-IgY-IR700 and then near-infrared light irradiation). [Figure 11] In vivo NIR-PIT experimental results. Colony counts were compared among a germ-free group (no C. albicans applied), a CA group (only C. albicans), and an IgY-PAT group (applied with CA-IgY-IR700 and then irradiated with near-infrared light). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] 1. Target-specific complex A first aspect of the present invention relates to a "target-specific complex", which is a structure that exhibits specific binding to a target (subject of attack) and can exert damaging activity. The target-specific complex of the present invention has a structure in which a near-infrared light sensitive substance is linked to an antibody against the target. Damaging activity is the action or effect of causing damage to the target, resulting in the death, inhibition of growth, detoxification, removal, etc. of the target. If the target is a bacterium or fungus / mold, the term "damaging activity" can be replaced with "antibacterial activity".

[0010] In the target-specific complex of the present invention, the use of an antibody provides target-specificity. IgY is used as the antibody. IgY is an antibody characteristic of birds. For example, IgY from poultry such as chickens and quails can be used. IgY is abundantly contained in serum as well as egg yolk, and is also called egg yolk antibody. Since it is abundant in egg yolk, it is possible to relatively easily prepare target-specific IgY in large quantities from the egg yolk of birds (typically chickens) sensitized with an antigen. The target-specific IgY may be prepared by a conventional method. An example of a method for preparing target-specific IgY is shown below.

[0011] First, birds are immunized with an antigen (target bacteria, fungi, etc.) (sensitization with antigen). Parts of bacteria, fungi, etc. may be used as antigens. Chickens, for example, are used as birds. Immunization is repeated as necessary, and eggs are collected when the antibody titer has risen sufficiently. It is advisable to check the antibody titer of the serum and determine the timing of egg collection. Usually, an increase in the IgY concentration in the egg yolk is observed 3 to 7 days after the IgY concentration in the serum reaches its peak. Therefore, it is advisable to collect eggs 10 to 20 days after the final sensitization (immunization) and extract and purify IgY. For the extraction and purification of IgY from eggs, methods such as ultracentrifugation, delipidation using organic solvents, and lipoprotein separation using dextran sodium sulfate can be used (see, for example, Jensenius JC et al., J Immunol Methods 46: 63-68, 1981; Hatta H. et al., Agric Biol Chem. 54: 2531-2535, 1990; Polson A. Immunological Investigations 19: 253-258, 1990). In addition, affinity columns (e.g., HiTrap IgY Purification HP Column from Global Life Science Technologies Japan Co., Ltd.) and kits for purifying IgY from egg yolk are commercially available, and these may also be used.

[0012] In addition to the polyclonal IgY prepared as described above, monoclonal IgY can also be used. Preparation of monoclonal IgY can also be performed by a conventional method (see, for example, Nishinaka, S. et al. Int Arch Allergy Appl Immunol, 89, 416(1989); Nishinaka, S. et al. J Immunol Methods, 139, 217(1991); Nishinaka, S. et al. J Vet Med Sci, 58, 1053(1996)). An example of a method for preparing monoclonal IgY is shown below. First, an immunization procedure is performed in the same manner as above. Immunization is repeated as necessary, and when the antibody titer has risen sufficiently, antibody-producing cells are extracted from the immunized bird. Next, the obtained antibody-producing cells are used to obtain hybridomas by cell fusion. Next, the hybridomas are monoclonalized, and then clones that produce antibodies with high specificity to the antigen are selected. The culture medium of the selected clone is purified to obtain the desired antibody. On the other hand, after the hybridomas have been multiplied to a desired number or more, they can be transplanted into the abdominal cavity of an animal (e.g., a mouse), grown in the ascites, and the ascites can be purified to obtain the desired antibody. Affinity chromatography with an antigen immobilized is preferably used for purifying the above culture solution or ascites. Affinity chromatography with an antigen immobilized can also be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

[0013] The targets of attack by the target-specific complex of the present invention are bacteria, fungi, viruses, parasites, worms, and rickettsia. In particular, typical targets are pathogens (bacteria, fungi, viruses, parasites, worms, and rickettsia) that are infectious and have harmful effects on humans or animals under the control of humans (pets / companion animals, industrial animals such as livestock and poultry, laboratory animals, and exhibit animals kept and kept in zoos and parks, etc.), that is, pathogens that can cause infectious diseases. The present invention targets the pathogens themselves, not the cells that the pathogens infect.

[0014] By using IgY, which can be prepared relatively easily and in large quantities, it is possible to accommodate a large number / amount of targets (bacteria, fungi, molds, etc.) and rapid target proliferation.

[0015] The target bacteria are not particularly limited, and various bacteria can be targeted, such as gram-negative bacilli, gram-negative cocci, gram-positive bacilli, and gram-positive cocci. Specific examples of bacteria that can be targeted include Escherichia coli, Shigella (S. dysenteriae, S. frexneri, S. sonnei, etc.), Salmonella (S. typh, S. paratyphi-A, S. schottmuelleri, S. typhimurium, S. enteritidis, etc.), Enterobacter bacteria (E. aerogenes, E. cloacae, etc.), Klebsiella (K. pneumoniae, K. oxytoca, etc.), Serratia (S.mmarcescens), Morganella (M.morganii, etc.), Providencia (P.rettgeri,m P.alcalifaciens, P.stuartii, etc.), Hafnia (H.alvei, etc.), Proteus (P. mirabilis, P. vulgaris, etc.), Yersinia (Y. pestis, Y. enterocolitica, etc.), Vibrio (V. cholerae, V. parahaemolyticus, etc.), Haemophilus (H. influenzae, H. parainfluenzae, H. ducreyi, etc.), Pseudomonas (P. aeruginosa, P. cepacia, P. putida, etc.), Aeromonas (A. hydrophilia, etc.), Acinetobacter bacteria (A. calcoaceticus, A. baumannii, A. lwoffii, etc.), Legionella (L. pneumophila, etc.), Bordetella (B. pertussis, B. parapertussis, B.bronchiseptica, etc.), Brucella (B. melitensis, B. abortus, B. suis, etc.), Francisella tularensis, Bacteroides (B. fragilis, B. melaninogenicus, etc.), Citrobacter (C. freundii, etc.), Xanthomonas (X. maltophilia), Flavobacterium (F. meningosepticum, etc.), Neisseria (N. gonorrhoeae, N. meningitidis, etc.), Staphylococcus (S. aureus, S. epidermidis, S. saprophyticus, etc.), Streptococcus (S. pyogenes, S. agalactiae, S. viridans, S. pneumoniae, S. mutans, S. sobrinus, etc.), Enterococcus (E. faecalis, E. faecium, E. avium, etc.), Bacillus (B. subtilis, B. anthracis, B. cereus, etc.), Listeria (L. monocytogenes, etc.), Clostridium (C. difficile, C. botulinum, C. perfringens, C. tetani, etc.), Corynebacterium (C. diphtheriae, etc.), Branhamella (B. catarrhalis, etc.), Mycobacterium (M. tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulare, M. kansasii, M. ulcerans, etc.), Peptcoccus (P.anaerobius, etc.), Peptostreptococcus (P. anaerobius, etc.), Eubacterium (E. lentum, etc.), Propionibacterium (P. .acnes), Lactobacillus (L. plantarum, etc.), Bacteroides (B. fragilis, B. melaninogenicus, etc.), Fusobacterium (F. gonidiaformans, F. necrophorum, F. nucleatum, etc.), Mycoplasma (Mycoplasma, etc.), Borrelia (B. recurrentis, B. burgdoferi, etc.), Treponema pallidum (B. burgdoferi, etc.) palidum, Campylobacter bacteria (C. coli, C. jejuni, C. fetus, etc.), Helicobacter bacteria (H. pylori, H. heilmannii, etc.), Rickettsia bacteria (R. prowazekil, R. mooseri, R. tsutsugamushi, etc.), Chlamydia bacteria (C. trachomatis, C. psittaci, etc.), Porphyromonas bacteria (P. gingivalis, etc.), Prevotella bacteria (Prevotella intermedia, etc.), Aggregatibacter bacteria (A. actinomycetemcomitans, etc.), and Treponema denticola bacteria (T. denticola, etc.).

[0016] Similarly, examples of fungi and molds that can be targets include Candida (C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, etc.), Aspergillus (A. fumigatus, A. flavus, A. niger, etc.), Cryptococcus (C. neoformans, etc.), Mucor (M. circinelloides, etc.), Rhizopus (R. oryzae, R. microsporus, etc.), Cunninghamella (C. bertholletiae, etc.), Apophysomyces (A. elegans), Sakseneae (S. vasiformis), Coccidioides (C. immitis, etc.), Histoplasma (H. capsulatum, etc.), Paracoccidioides (P. brasiliensis), Penicilium (P. marneffei), Blastomyces (B. dermatitidis), Sporothix (S. schenckii, etc.), Chromomycosis (Fonsceaea, etc.), Phialophora (P. verrucosa, etc.), Cladophialophora (C. carrinoii, etc.), Malassezia (M. furfur, M. globasa, etc.), and Pneumocystis (P. jirovecii, etc.).

[0017] Examples of viruses that can be targeted include smallpox virus, vaccinia virus, contagious wart virus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (HHV-3), cytomegalovirus (HHV-5), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7), Epstein-Barr virus (HHV-4), human herpes virus 8 (HHV-8, also known as Kaposi's sarcoma-associated herpes virus (KSHV)), adenovirus, human papillomavirus, parvovirus B19, Zika virus, and type B. Examples of such viruses include hepatitis virus, influenza virus, measles virus, mumps virus, respiratory syncytial virus, human immunodeficiency virus (HIV), human T-lymphotropic virus type 1 (HTLV-1), coronavirus, Lassa virus, rubella virus, Japanese encephalitis virus, yellow fever virus, dengue virus, hepatitis C virus, hantavirus, poliovirus, coxsackievirus, echovirus, rhinovirus, hepatitis A virus, hepatitis E virus, Norwalk virus, human astrovirus, rabies virus, Marburg virus, Ebola virus, reovirus, and rotavirus.

[0018] On the other hand, examples of parasites that can be targeted include Plasmodium (P. falciparum, P. vivax, P. malariae, etc.), Leishmania (L. donovani, L. braziliensis, etc.), Cryptosporidium (C. parvum, etc.), Trypanosoma (T. brucei, T. cruzi, etc.), Trichomonas (T. vaginalis, etc.), Toxoplasma (T. gondii, etc.), Babesia (B. microti, etc.), Entamoeba (E. hitsolytica, etc.), Gicardia (G. intestinalis, G. muris, etc.), and Cryptosporidium (C. parvum, etc.).

[0019] On the other hand, examples of parasites that can cause infectious diseases include Ascaris (Ascaris lumbricoides, etc.), Ancylostomoa duodenale, Necator americanus, Enterobius vermicularis, Strongyloides stercoralis, Anisakis (A. simplex, A. physeteris, etc.), Pseudoterranova (P. decipiens), Wunchereri bancroft, Brugia malayi, Gnathostoma (Gnathostoma nipponicum, G. spinigerum, etc.), Trichinella (T. spiralis, T. pseudospiralis, etc.), and Angiostrongylus (A. cantonesis. A. constaricensis), Clonorchis sinensis, Metagonimus yokokawai, Paragonimus (P. westermani etc.), Diphyllobothrium latum, Echinococcus (E. granulosus, E. multilocularis), Hymenolepis diminuta), Hymenolepis nana, Taenia multiceps, Taenia asiatica, Taenia saginata and Taenia solium.

[0020] On the other hand, examples of Rickettsia that can cause infectious diseases include the genus Rickettsia (R. rickettsia, R. prowazekii, R. typhi, etc.), Orientia (O. tsutsugamushi, etc.), Ehrlichia (E. chaffeensis, etc.), Anaplasma (A. phafocytophilum, etc.), and Coxiella (C. burnetii, etc.).

[0021] It is also possible to target multiple pathogens with similar antigenicity (e.g., multiple closely related species) at once. When targeting a relatively wide range of pathogens in this way, polyclonal IgY is preferably used. By using polyclonal IgY, it is possible to exhibit inhibitory activity against a relatively wide range of targets while being pathogen-selective and not binding to the human body. On the other hand, monoclonal IgY is suitable for constructing a target-specific structure focused on a specific pathogen (e.g., a specific bacterial species or strain), in other words, when selectivity or specificity needs to be increased (however, polyclonal IgY may also be used in this case).

[0022] The present invention utilizes the principle of photoimmunotherapy (PIT). To this end, a near-infrared light sensitizer is linked to target-specific IgY. Typically, a phthalocyanine dye is used as the near-infrared light sensitizer. Phthalocyanine dyes are a group of photosensitizer compounds that have a phthalocyanine ring system. For the synthesis and use (applications) of various phthalocyanine dyes, see, for example, WO 2005 / 099689 and U.S. Patent No. 7,005,518.

[0023] Preferably, a phthalocyanine dye having an absorption peak in the near infrared (NIR) region and emitting fluorescence by strongly absorbing near infrared rays is used. More specifically, a phthalocyanine dye having an absorption peak preferably in the range of 600 nm to 950 nm, more preferably in the range of 660 nm to 740 nm, and even more preferably in the range of 680 nm to 720 nm is used.

[0024] A particularly preferred phthalocyanine dye is IR700 (IRDye® 700DX). IR700 is commercially available from LI-COR Biosciences. Amino-reactive IR700 is a relatively hydrophilic dye and can be covalently conjugated to IgY using, for example, an NHS ester of IR700.

[0025] The near-infrared light sensitizer is directly or indirectly linked to the target-specific IgY via covalent or non-covalent bonds. Non-covalent bonds can be achieved, for example, by electrostatic interactions, van der Waals forces, hydrophobic interactions, π-effects, ionic interactions, hydrogen bonds or halogen bonds. In the case of indirect linkage, a linker is usually used.

[0026] 2. Pharmaceutical compositions and their uses The target-specific complex of the present invention can be formulated to prepare a pharmaceutical composition. In general, a pharma- ceutical acceptable carrier (carrier, vehicle) is used for formulation. Examples of carriers include water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, mannitol, lactose, starch, and magnesium stearate. For information on pharma- ceutical acceptable carriers and their usage, see, for example, Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995).

[0027] In addition to the carrier, the pharmaceutical composition may contain diluents (such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose), excipients (such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc.), lubricants (such as magnesium stearate, calcium stearate, talc, etc.), pH adjusters (such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.), emulsifiers, solubilizers, isotonicity agents, preservatives, preservatives, etc.

[0028] The dosage form / shape of the formulation is not particularly limited. Examples of dosage forms include aerosols, liquids, suspensions, injections, syrups, emulsions, jellies, tablets, pills, powders, fine granules, granules, capsules, external preparations (ointments, patches, poultices, lotions, liniments, suppositories), inhalants, nasal drops, and eye drops.

[0029] The pharmaceutical composition of the present invention contains an active ingredient in an amount (i.e., a therapeutically effective amount) necessary to obtain the expected therapeutic effect (or preventive effect). The amount of the active ingredient in the pharmaceutical composition of the present invention generally varies depending on the dosage form, but is set, for example, within the range of about 0.001% by weight to about 99% by weight so as to achieve the desired dosage.

[0030] A further aspect of the present invention relates to the use of the pharmaceutical composition. Typically, the pharmaceutical composition of the present invention is used for the treatment, prevention, or amelioration of a disease or pathology. "Treatment" includes alleviating (alleviating) symptoms characteristic of a target disease or associated symptoms, preventing or delaying the worsening of symptoms, and the like. "Prevention" refers to preventing or delaying the onset / manifestation of a disease (injury) or its symptoms, or reducing the risk of onset / manifestation. On the other hand, "improvement" refers to alleviating (alleviating), improving, remission, or curing (including partial cure) a disease (injury) or its symptoms. Thus, treatment, prevention, and improvement are partially overlapping concepts, and it is difficult to clearly distinguish between them, and there is little practical benefit in doing so. In this specification, treatment for the purpose of prevention or improvement is also included in the concept of the term "treatment method".

[0031] The pharmaceutical composition of the present invention is typically applied to the treatment, prevention, or amelioration of infectious diseases caused by targets (bacteria, fungi, viruses, parasites, helminths, rickettsia). In other words, the pharmaceutical composition of the present invention is used in IgY-PAT therapy.Examples of infectious diseases that may be the subject of treatment include contagious impetigo (bullous impetigo, crusted impetigo), erysipelas, cellulitis, folliculitis, furuncles, carbuncles, possible hidradenitis, keloid folliculitis, chronic buttock pyoderma, bacterial paronychia, infantile multiple hidradenitis abscesses, staphylococcal scalded skin syndrome, toxic shock syndrome, scarlet fever, necrotizing fasciitis, gas gangrene, sepsis, Osler's nodes, yellow fungus hair, erythrasma, cat scratch disease, actinomycosis, external dental fistula, nocardiosis, MRSA infection, Pseudomonas aeruginosa infection, Serratia infection, enterohemorrhagic Escherichia coli infection, bacterial meningitis, tuberculosis, nontuberculous mycobacteria, cholera, plague, diphtheria infection, bacterial dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionnaires' disease, leptospirosis, Lyme disease, tularemia, gonorrhea, chlamydia infection (chlamydia pneumonia, trachoma, genital chlamydia infection, psittacosis, etc.), carbapenem-resistant Enterobacteriaceae infection, dental caries, periodontal disease, endophthalmitis, candidiasis, aspergillosis, blastomycosis, cryptococcosis, muco Coccidioidomycosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, ringworm, smallpox, viral warts, herpes labialis, herpes stomatitis, corneal herpes, genital (genital) herpes, shingles, Kaposi's sarcoma, hepatitis (types A, B, C, and E), HIV, influenza, cold, norovirus infection, rotavirus infection, respiratory syncytial virus infection, rubella, measles, mumps, Japanese encephalitis, yellow fever, dengue fever, Ebola hemorrhagic fever, genital warts, pharyngeal papilloma, polio, and rabies. The medicinal diseases that can be caused by the following diseases are particularly useful: malaria, leishmaniasis, cryptosporidiosis, trypanosomiasis, trichomoniasis, toxoplasmosis, babesiosis, amebiasis, intestinal diccardiosis, cryptosporidiosis, ascariasis, hookworm disease, enterobiasis, strongyloidiasis, anisakiasis, pseudoterraniobasis, filariasis, gnathostomiasis, trichinosis, angiostrongyliasis, clonorchiasis, yokogawa fluke disease, paragonimiasis, diphyllobothriasis, echinococcosis, taeniasis, rickettsia infection, tsutsugamushi disease, Japanese spotted fever, typhus, ehrlichiosis, anaplasmosis, and Q fever. The medicinal composition of the present invention is particularly useful for local infections of the skin, mucous membrane, etc. The medicinal composition of the present invention can also be applied to the treatment of abscesses caused by bacterial infections, etc.

[0032] In a treatment method using the pharmaceutical composition of the present invention, the following steps (1) and (2) are carried out. (1) administering the pharmaceutical composition of the present invention to a subject to be treated, thereby allowing the target-specific complex of the present invention to bind to the target. (2) illuminating the target with near-infrared light.

[0033] In step (1), the pharmaceutical composition of the present invention is administered to a subject to be treated. The route of administration may be selected depending on the dosage form of the pharmaceutical composition, the treatment plan, etc. Either oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, application to the affected area, patch, spray, etc.) can be used. Furthermore, these administration routes are not mutually exclusive, and any two or more of them can be used in combination (for example, intravenous injection at the same time as oral administration or after a certain time has elapsed). Either systemic administration or local administration (for example, application, patch, spray, etc. to the infected area or the surrounding area) can be used. Not only humans, but also animals under human (person) control (pet / companion animals, industrial animals such as livestock and poultry, laboratory animals, and animals kept and exhibited in zoos and parks, etc. Specific examples include various monkeys, chimpanzees, gorillas, orangutans, cows, pigs, goats, sheep, horses, donkeys, camels, ostriches, chickens, quail, ducks, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits, elephants, giraffes, bears, zebras, hippos, rhinos, and penguins) may be subjects for treatment.

[0034] The dosage of the pharmaceutical composition is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dosage, the symptoms, age, sex, and body weight of the patient are generally taken into consideration. Those skilled in the art can set an appropriate dosage taking these factors into consideration. Examples of dosages (as the amount of active ingredient, i.e., the target-specific complex) are, for example, 0.1 to 1000 mg, 0.2 to 500 mg, 0.5 to 100 mg, or 1 to 20 mg per 60 kg body weight in the case of systemic administration, and 100 mg per 1 cm of the application site in the case of local administration. 2For example, the dose is 0.01 to 50 mg, 0.03 to 30 mg, or 0.05 to 10 mg per dose. In creating an administration schedule, the patient's condition, duration of effect of the active ingredient, and the like can be taken into consideration.

[0035] After the target-specific complex, which is the active ingredient of the pharmaceutical composition, is bound to the target by administration, the target is irradiated with near-infrared light (step (2)). Without being bound by theory, irradiation with near-infrared light damages the surface structure of the target (for example, the cell wall in the case of bacteria), and exerts its effect (such as killing the target or inhibiting its proliferation). This mechanism of action differs from that of photodynamic therapy (PDT), which exerts its effect by causing oxidative stress in mitochondria, and brings about a rapid effect.

[0036] For example, LED, LED laser, light beams through a filter, etc. may be used for irradiation with near-infrared light. In addition to direct irradiation, devices such as light-guiding catheters, endoscopic light-guiding fibers, puncture irradiation fibers, vascular light-guiding catheters, drain-indwelling light-guiding devices, internally embedded devices, adhesive devices, and bracelet-type devices may be used, but are not limited to these. The irradiation conditions for near-infrared light are not particularly limited as long as the damaging activity based on the principle of NIR-PIT is obtained, but the wavelength of the near-infrared light used is, for example, 650 to 740 nm, preferably 670 to 720 nm, and more preferably 680 to 710 nm. The irradiation dose is, for example, at least 1 J cm. -2 , at least 2 J cm -2 , at least 5 J cm -2 , at least 10 J cm -2 , at least 20 J cm -2 , at least 30J cm -2 , at least 40J cm -2 , at least 50J cm -2 , at least 60J cm -2 , at least 70J cm -2 , at least 80J cm -2 , at least 90J cm -2or at least 100 J cm -2 More specifically, for example, 1 to 1000 J cm -2 , 2~500J cm -2 , 5~300J cm -2 or 10 to 100 J cm -2 The irradiation dose is set to 100 μg / kg. The irradiation time is, for example, 5 seconds to 1 hour, 5 seconds to 30 minutes, or 5 seconds to 15 minutes. The irradiation time is preferably 10 seconds or more, more preferably 1 minute or more, and even more preferably 3 minutes or more. In addition, although not limited to this example, when the pharmaceutical composition is administered systemically by intravenous injection or the like, the near-infrared light is irradiated for, for example, 5 minutes to 48 hours, preferably 10 minutes to 24 hours, and even more preferably 15 minutes to 12 hours after the administration of the pharmaceutical composition. In the case of local administration, it is preferable to set the interval between the administration of the pharmaceutical composition and the irradiation of the near-infrared light shorter than in the case of systemic administration (for example, the near-infrared light is irradiated for 1 minute to 12 hours after the administration of the pharmaceutical composition).

[0037] Instead of a single irradiation, multiple irradiations may be performed. In the case of multiple irradiations, the intervals are not particularly limited. For example, various irradiation schedules can be set, such as multiple irradiations on the same day with a predetermined interval (e.g., 5 minutes to 10 hours), daily irradiation, irradiation every other day or every few days, irradiation every week or several weeks, and irradiation every month or several months. The administration schedule of the pharmaceutical composition in the case of multiple irradiations is not particularly limited. For example, when the interval between the first irradiation and the second irradiation is short, typically the pharmaceutical composition is administered only before the first irradiation. As another example, when a long time has passed since the previous irradiation (e.g., when one day to several months have passed), the pharmaceutical composition may be administered again and then irradiation may be performed.

[0038] In parallel with the treatment or prevention with the pharmaceutical composition of the present invention, treatment with other medicines, for example, antibacterial drugs (e.g., penicillin antibacterial drugs, cephalosporin antibacterial drugs, carbapenem antibacterial drugs, penem antibacterial drugs, tetracycline antibacterial drugs, β-lactamase inhibitors, fosfomycin, vancomycin, aminoglycoside antibacterial drugs, macrolide antibacterial drugs) may be administered. By combining the pharmaceutical composition of the present invention with a medicine having a different mechanism of action, a composite action / effect can be obtained, and the therapeutic effect can be enhanced.

[0039] 3. Disinfection and decontamination compositions The target-specific complex of the present invention can also be used for disinfecting or decontaminating objects (contaminated objects) contaminated with pathogens (bacteria, fungi, molds, viruses, parasites, worms, rickettsia) that can cause infectious diseases. That is, a disinfection / decontamination composition (hereinafter, for convenience of explanation, referred to as the "disinfectant of the present invention") can be prepared using the target-specific complex of the present invention. The disinfectant of the present invention can be used for disinfecting or decontaminating various facilities, instruments, and fixtures (examination or surgical equipment, instruments or devices, urinals, floors, walls, curtains, furniture, doors, windows, bedding, clothing, etc.) and spaces (hospital rooms, operating rooms, clean rooms, clean benches, etc.) in medical institutions (hospitals, clinics, nursing homes for the elderly, visiting care stations, midwifery clinics, pharmacies, etc.), osteopathic clinics, chiropractic clinics, acupuncture clinics, etc., and for disinfecting or decontaminating various facilities, instruments, and fixtures (manufacturing equipment, instruments, and devices) in pharmaceutical, cosmetic, or medical device manufacturing plants or research institutes. It can be used for disinfection or decontamination of surfaces (workbenches, floors, walls, curtains, furniture, doors, windows, work clothes, gloves, etc.) and spaces (inside factories, clean rooms, clean benches, etc.), disinfection or decontamination of kitchens, toilets, washrooms or bathrooms, disinfection or decontamination of tableware, cutlery (knives, forks, spoons, etc.) or cooking utensils (kitchen knives, knives, pots, mixers, microwave ovens, ovens, etc.), disinfection or decontamination of hands, fingertips or the oral cavity (for example, for the treatment or prevention of periodontal disease and dental caries, or sterilization after implant surgery).

[0040] The disinfectant of the present invention may be in a liquid form (e.g., a spray, lotion), gel form, or solid form (e.g., powder) and may be used by application, spraying (atomization), scattering, dropping, or the like. When using the disinfectant of the present invention, for example, the disinfectant of the present invention is applied to a contaminated object by application, etc., and then irradiated with near-infrared light to exert the damaging activity. The irradiation conditions for near-infrared light are similar to those for treatment using a pharmaceutical composition. EXAMPLES

[0041] <Development of new photoantibacterial therapy> 1. Preparation and quality confirmation of Candida albicans-IgY (CA-IgY)-IR700 1-1. Experimental method First, CA-IgY-IR700 was synthesized. CA-IgY (1.0 mg, 5.4 nmol) and IR700 (47.8 μg, 24.5 nmol) were incubated with 0.1 M Na2HPO4 (pH 8.5) at room temperature for 1 hour, and then the mixture was passed through a Sephadex G50 column (PD-10; GE Healthcare) to recover the mixture (CA-IgY-IR700 solution). After Coomassie staining, the absorbance (wavelength 595 nm) was measured to determine the concentration of CA-IgY-IR700 (protein concentration). The concentration of IR700 was also determined by measuring the absorbance (wavelength 698 nm), and the number of fluorescent molecules bound to the antibody was confirmed. Meanwhile, the above mixture was subjected to SDS-PAGE to confirm the binding of the antibody to IR700. Diluted CA-IgY was used as a control in SDS-PAGE, and images were taken with a Pearl imager (LICOR).

[0042] 1-2. Results and Discussion At the height of the band in the protein staining (Figure 1, left), fluorescence was observed only for CA-IgY-IR700 (Figure 1, right), confirming that CA-IgY and IR700 were bound (conjugated).

[0043] 2. Evaluation of Candida albicans (C. albicans) antigen binding of CA-IgY-IR700 2-1. Experimental method C. albicans (JCM1542) at 1 × 10 5 The cells were seeded in tubes at 10 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL, and incubated with the prepared CA-IgY-IR700 (10 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL) at 37°C for 1 h, after which the fluorescence intensity was measured by flow cytometry. In addition, four other C. albicans species (FC18, IFO579, IFO1060, and IFO1385), their closely related species Candida tropicalis (IFO1402), Candida guilliermondii (IFO838), Candida krusei (IFO1162), and A431 (human skin cancer cell line) were similarly measured using 200 μg / mL CA-IgY-IR700.

[0044] 2-2. Results and Discussion Fluorescence increased in proportion to the antibody concentration of CA-IgY-IR700 (Figure 2). In previous reports of conventional NIR-PIT, a concentration of 10 μg / mL was used, but 10 μg / mL was considered insufficient for C. albicans. One of the reasons for this was thought to be that the proliferation rate of C. albicans was fast, and the number of C. albicans increased during the 1-hour incubation, resulting in a relative lack of antibodies. Fluorescence was increased for all C. albicans and its related species, but not for A431 (Figure 3). Therefore, it is believed that CA-IgY-IR700 bound to Candida in general, but not to human cells.

[0045] 3. In vitro NIR-PAT 3-1. Experimental method To evaluate antibody concentration and therapeutic efficacy, C. albicans (JCM1542) was cultured in liquid medium for 24 hours and then diluted with 1 × 10 5 Each tube was seeded with 100 μL of medium, and CA-IgY-IR700 was added (10 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL). After incubation at room temperature for 4 hours, the cells were exposed to near-infrared light (256 J / cm ) using an LED with an emission wavelength of 690 nm. 2After the treatment, 300 bacteria were immediately seeded on a 10 cm dish, and after 24 hours of incubation, the number of colonies was counted to evaluate the number of live bacteria. The control was one that did not receive either antibody or near-infrared light irradiation.

[0046] Next, to evaluate the irradiation dose and therapeutic effect, CA-IgY-IR700 was adjusted to 200 μg / mL, and after 4 hours of incubation at room temperature, near-infrared light (32 J, 64 J, 128 J, and 256 J / cm) was applied using an LED with an emission wavelength of 690 nm. 2 ) was irradiated with near-infrared light (256 J / cm) without adding CA-IgY-IR700. 2 After treatment, the number of colonies was counted in the same manner to evaluate the viable bacterial count (Figure 4). To measure the viable bacterial count visually, the same method was used to react with 200 μg / mL of CA-IgY-IR700 and expose the specimens to near-infrared light at 256 J / cm. 2 One hour after irradiation, the cells were observed using a confocal microscope and a scanning electron microscope with a dead cell stain (Propiodium Iodide: PI stain).

[0047] 3-2. Results and Discussion In assessing antibody concentration and therapeutic effect, the viable cell count of C. albicans was reduced in a concentration-dependent manner with CA-IgY-IR700 (Figure 5, left). At 10 μg / mL, the therapeutic effect was poor, but was effective at 50 μg / mL or higher. In assessing irradiation dose and therapeutic effect, the viable cell count was reduced in a concentration-dependent manner, while near-infrared light irradiation alone did not change the viable cell count (Figure 5, right). The reduction in viable cell count with CA-IgY-IR700 alone is thought to be due to the inherent antibacterial action of IgY. IgY-PAT is thought to significantly enhance the antibacterial effect of IgY.

[0048] In addition, in observations using a confocal microscope, fluorescence at 700 nm was observed when only CA-IgY-IR700 reacted, whereas in those treated with IgY-PAT, dead cell staining was positive and the fluorescence at 700 nm was weakened (Figure 6, left). This was presumed to be because IR700 was decomposed by near-infrared light irradiation, destroying the bacterial cells. In scanning electron microscope images, no changes were observed in the control or with CA-IgY-IR700 alone, but holes were observed on the surface of the bacterial cells and deformation of the bacterial cells was observed in those treated with IgY-PAT (Figure 6, right). This deformation is thought to be due to the surface of the bacterial cells being destroyed by IgY-PAT, causing the cells to burst.

[0049] 4. In vivo NIR-PIT 4-1. Experimental method (Figure 7) After removing the hair from both sides of the lower back of 8-10 week-old BALC slc mice, two skin defects were created on the left and right sides using a 6 mm skin biopsy punch. C. albicans (JCM1542) was washed with PBS and then diluted with 1x10 9 100 μL of the concentrated solution was applied to the skin defect at 200 μg / mL, and the ulcer was protected with a bandage (designated as Day -1). The following day (Day 0), 100 μL of 200 μg / mL CA-IgY-IR700 diluted with water-soluble jelly was applied to the ulcer, and the ulcer was protected with a bandage. The following day (Day 1), the bandage was removed, and the ulcer was evaluated using a Pearl imager (LICOR) to see whether CA-IgY-IR700 could be confirmed. After that, near-infrared light (256 J / cm 2) was irradiated with a laser. The ulcer area on the day of irradiation was set as 100%, and the transition of the ulcer area was quantitatively evaluated. The ulcer area was photographed and calculated using Image J (open source). The treatment group was the IgY-PAT group, and the controls were the germ-free group without C. albicans application, the CA group with C. albicans only, the CA-IgY-IR700 group with CA-IgY-IR700 application and no near-infrared light irradiation, and the Light group with only near-infrared light irradiation. The ulcer area was evaluated up to Day 14. To evaluate the bacterial count, the subcutaneous tissue of the ulcer area of ​​the germ-free group, CA group, and IgY-PAT group was excised with a sterilized 6 mm skin biopsy punch on Day 2, the tissue was stirred, and seeded on a medium to count the number of colonies. The number of colonies in the CA group was set as 100%.

[0050] 4-2. Results and Discussion In the group where CA-IgY-IR700 was applied, fluorescence was observed at the application site, and the fluorescence weakened after near-infrared light irradiation (Figure 8). This is presumably because antibodies were attached to the ulcer surface, and CA-IgY-IR700 was decomposed by reacting with near-infrared light as in vitro. The ulcer area was smallest in the sterile group, followed by the IgY-PAT group and the CA-IgY-IR700 group, and was almost the same in the CA group and the Light group. There was no significant difference between the sterile group and the IgY-PAT group, but there was a significant difference between the IgY-PAT group and the CA-IgY-IR700 group. There was also a significant difference between the CA-IgY-IR700 group and the CA group (Figure 9). In addition, the amount of purulent discharge from the ulcer area was small in the sterile group, CA-IgY-IR700 group, and IgY-PAT group (Figure 10). From these results, as in the in vitro results, CA-IgY-IR700 alone had an antibacterial effect, but the antibacterial effect was enhanced by IgY-PAT, and it was speculated that the course of the disease was similar to that of the germ-free group. In addition, there were no adverse events such as burns, and it is considered that the overall results contributed to the healing of the ulcer. The number of colonies was significantly lower in the germ-free and IgY-PAT groups compared to the CA group (Figure 11). Colonies were observed in the germ-free group due to the contamination with resident bacteria. From these results, it was speculated that IgY-PAT also showed an antibacterial effect in vivo, and that the reduction in ulcer area was related to the number of bacteria.

[0051] 5. Summary IgY-PAT, which uses a complex of CA-IgY and IR700 (conjugate), demonstrated a high antibacterial effect. This innovative treatment can be used to treat many intractable infections and drug-resistant bacteria. [Industrial Applicability]

[0052] The target-specific complex of the present invention (a structure in which IgY is linked to a near-infrared light-sensitive substance) exhibits target-specific inhibitory activity by the principle of NIR-PIT and exerts a therapeutic effect. The present invention, which utilizes IgY, which is inexpensive and easy to prepare in large quantities, is a new therapeutic means that can deal with the explosive increase in targets in infectious diseases caused by bacteria, fungi, mold, etc., and is expected to be applicable to various infectious diseases. The present invention is an innovative technology that is different from conventional antibacterial therapies that use IgY alone, and can provide high therapeutic effects. In addition, due to its unique mechanism of action, rapid onset of effects can be expected. The fact that the bactericidal effect was confirmed against Candida, which has a strong cell wall structure, supports the idea that the present invention can be an effective means of attack not only against various gram-positive bacteria, but also against various gram-negative bacteria and various fungi.

[0053] This invention is not limited to the above-mentioned embodiments and examples. Various modified embodiments are also included in this invention within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art. For example, target-directed substances include commonly considered antibodies (IgG, IgM, antibody fragments, minibodies, diabodies, etc.), peptides, aptamers, and other binding substances, which can be used in place of IgY. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all hereby incorporated by reference.

Claims

1. A target-specific complex having a structure in which a near-infrared light sensitizer is linked to IgY specific for a target classified as a bacterium, fungus, mold, virus, parasite, helminth, or rickettsia.

2. The target-specific complex of claim 1 , wherein the target is a bacterium or a fungus or mold.

3. The target-specific complex according to claim 2, wherein the bacterium is a species selected from the group consisting of Pseudomonas, Acinetobacter, Staphylococcus, Streptococcus, Enterococcus, Escherichia coli, Shigella, Salmonella, Enterobacter and Klebsiella, and the fungus or mold is a species selected from the group consisting of Candida, Aspergillus, Mucor and Cryptococcus.

4. The target-specific complex of any one of claims 1 to 3, wherein the IgY is a polyclonal antibody.

5. The target-specific complex according to any one of claims 1 to 4, wherein the near infrared light sensitizer is a phthalocyanine dye.

6. The target-specific conjugate of claim 5, wherein the phthalocyanine dye is IR700.

7. A composition comprising the target-specific complex according to any one of claims 1 to 6.

8. The composition of claim 7 for use in treating or preventing an infection by said target.

9. A method of treatment comprising the following steps (1) and (2): (1) administering the composition of claim 8 to a subject (excluding humans) to allow the target-specific complex to bind to the target; (2) illuminating the target with near-infrared light.

10. The method of claim 9, wherein the wavelength of the near-infrared light is 650 to 740 nm.

11. The method of claim 9, wherein the wavelength of the near-infrared light is 670 to 720 nm.

12. The near-infrared light is irradiated at a dose of 1 J cm -2 The method for treatment according to any one of claims 9 to 11.

13. The near-infrared light is irradiated at a dose of 2 J cm -2 ~500J cm -2 The method of any one of claims 9 to 11,

14. 8. The composition of claim 7 for use in disinfecting or decontaminating a contaminant by said target.

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