WO2021112174A1 - Optical antimicrobial therapeutic method - Google Patents

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

Photoantibacterial therapy

The present invention relates to photoantibacterial therapy. More specifically, the present invention relates to a target-specific complex that exhibits target-selective damaging activity using near-infrared light irradiation, its use, and the like.

Near-infrared ray immunotherapy (NIR-PIT) is a monoclonal antibody bound to a photosensitizer (eg IRdye700DX (IR700)) that reacts with cancer cells and then is selectively irradiated with near-infrared light. It is a new cancer treatment that destroys the cancer cell membrane. Clinical trials have also been conducted for head and neck cancers targeting EGFR, with good results, and Phase 3 clinical trials are currently underway. Recently, it has been clarified that the mechanism showing the antitumor effect of NIR-PIT is a photochemical reaction completely different from the existing antitumor treatment (see Non-Patent Document 1). It should be noted that Patent Documents 1 to 3 propose that NIR-PIT is used for treatment of tumors and the like.

U.S. Patent Application Publication No. 2014/01/2019 U.S. Patent Application Publication No. 2018/015405 U.S. Patent Application Publication No. 2014/01/2019

NIR-PIT is a technology with high potential due to its unique mechanism of action, and further application can be expected. Therefore, it is an object of the present invention to provide a new use of NIR-PIT.

In conventional NIR-PIT that targets cancer cells, IgG monoclonal antibody is usually used as an antibody that binds a photosensitizer. However, while exploring the application of NIR-PIT, the present inventors have IgY (immunoglobulin). Y: Tori egg yolk immunoglobulin) was focused on. IgY is an antibody peculiar to birds. Chicken IgY, which can be said to be a representative of IgY, is the main immunoglobulin in spawning chickens and transfers from serum to egg yolk in order to impart passive immunity. Since chicken IgY can be collected from chicken eggs, it can be easily collected in high concentration. In other words, it can be prepared inexpensively and in large quantities. Also, IgY shows no side effects, resistance or toxic residues in contrast to antibiotics. Therefore, IgY is attracting attention as a new means of controlling infectious diseases, which is different from antibiotics. So far, it has been reported that IgY is suppressed against pathogens such as Escherichia coli, Salmonella typhimurium enterocolitica, Salmonella typhimurium enterocolitica, and Galibacterium analyst (see, for example, Non-Patent Document 2). ). However, it cannot be said that the effect is sufficiently high, and there are many practical problems such as the need for continuous administration in order to enhance the effect. Based on this situation, the present inventors considered that the combination of NIR-PIT and IgY would be an effective therapeutic means for infectious diseases, etc., and a target in which a near-infrared photosensitizer was linked to a chicken-derived IgY polyclonal antibody. A specific structure was prepared and its effect and practicality were verified. As a result of detailed verification experiments, the antibacterial effect of NIR-PIT using IgY polyclonal antibody was exhibited very high and promptly. It can be said that obtaining a rapid effect is a particularly preferable feature as a means of attacking bacteria and fungi whose growth speed is high. In addition, the target-specific structure is reactive with related species in addition to the specific Candida albicans, which is the original antigen of IgY used, but not with human cells. It was. These facts mean that they can be effective against a wide range of bacterial species while ensuring high safety, and support their high practicality. In addition, the fact that a bactericidal effect was confirmed against Candida, a fungus having a tough cell wall structure, indicates that the target-specific structure has a strong damaging activity and its application range is wide.

As described above, Photoantibacterial target therapy (PAT) using IgY for NIR-PIT (hereinafter, the target photoantibacterial therapy may be referred to as "IgY-PAT therapy") is applied to the human body. It has been clarified that it is extremely effective as a countermeasure against infectious diseases such as fungi and bacteria (treatment / prevention of infectious diseases, prevention of spread of infection, etc.). Based on this result, the following inventions are provided.
[1] A target-specific complex having a structure in which a near-infrared light-sensitive substance is linked to a target-specific IgY classified as a bacterium, fungus or mold, virus, parasite, parasite or rickettsia.
[2] The target-specific complex according to [1], wherein the target is a bacterium or fungus or mold.
[3] Bacteria selected from the group consisting of Pseudomonas, Asinetobacter, Dactophila, Streptococcus, Enterococcus, Escherichia coli, Shigera, Salmonella, Enterobacter and Klebsiella. The target-specific complex according to [2], wherein the fungus or mold is a bacterial species selected from the group consisting of Enterobacter, Aspergillus, Mucor and Cryptococcus fungi.
[4] The target-specific complex according to any one of [1] to [3], wherein 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 pigment.
[6] The target-specific complex according to [5], wherein the phthalocyanine pigment is IR700.
[7] A composition containing the target-specific complex according to any one of [1] to [6].
[8] The composition according to [7], which is used for treating or preventing an infectious disease caused by the target.
[9] A treatment method including the following steps (1) and (2):
(1) A step of administering the composition according to [8] to a therapeutic subject and binding the target-specific complex to the target.
(2) A step of irradiating the target with near-infrared light.
[10] The treatment method according to [9], wherein the wavelength of the near infrared light is 650 to 740 nm.
[11] The treatment method according to [9], wherein the wavelength of the near infrared light is 670 to 720 nm.
[12] The treatment method according to any one of [9] to [11], wherein the irradiation dose of the near infrared light is 1 J cm ^{-2 or more.}
[13] The treatment method according to any one of [9] to [11], wherein the irradiation dose of the near infrared light is ² J cm -2 to 500 J cm ^-2.
[14] The composition according to [7], which is used for disinfecting or decontaminating contaminants by the target.

Preparation and quality confirmation of Candida albicans-IgY (CA-IgY) -IR700. The binding between CA-IgY and IR700 was confirmed by protein staining (left) and fluorescence staining (right). C. Binding of CA-IgY-IR700 to albicans. CA-IgY-IR700 was added to C. albicans seeded in tubes at each concentration, incubated at 37 ° C. for 1 hour, and then the fluorescence intensity was measured by flow cytometry. C. Albicans and its relatives, as well as the binding of CA-IgY-IR700 to A431. In vitro NIR-PAT experimental method. Experimental results of In vitro NIR-PAT. The relationship between the antibody concentration and the therapeutic effect (left) and the relationship between the irradiation dose and the therapeutic effect (right) were evaluated by the number of colonies. Experimental results of In vitro NIR-PAT. The effect of IgY-PAT was evaluated with a confocal microscope (dead cell staining) (left) and a scanning electron microscope (right). In vivo NIR-PIT experimental method. Experimental results of In vivo NIR-PIT. For the CA-IgY-IR700 group (CA-IgY-IR700 is applied and not irradiated with near-infrared light) and the IgY-PAT group (CA-IgY-IR700 is applied and then irradiated with near-infrared light), the treatment site Fluorescence was observed over time. Experimental results of In vivo NIR-PIT. Sterile group (without applying C. albicans), CA group (only with C. albicans), CA-IgY-IR700 group (with applying CA-IgY-IR700 and not irradiating near infrared light), Light group (near red) The tumor area was compared and evaluated between the IgY-PAT group (irradiating near-infrared light after applying CA-IgY-IR700) and the IgY-PAT group (irradiation with external light only). Experimental results of In vivo NIR-PIT. Sterile group (without applying C. albicans), CA group (only with C. albicans), CA-IgY-IR700 group (with applying CA-IgY-IR700 and not irradiating near infrared light), Light group (near red) The purulent excrement of the ulcer was compared and evaluated between the IgY-PAT group (irradiating near-infrared light after applying CA-IgY-IR700) and the IgY-PAT group (irradiation with external light only). Experimental results of In vivo NIR-PIT. Comparative evaluation of the number of colonies among the sterile group (without applying C. albicans), CA group (only C. albicans), and IgY-PAT group (irradiating near infrared light after applying CA-IgY-IR700) did.

1. 1. Target-Specific Complex The first aspect of the present invention relates to a "target-specific complex", which is a structure that exhibits specific binding to a target (target 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. The damaging activity is an action or effect that impairs (damages) the target, and brings about death of the target, suppression of proliferation, detoxification, removal, and the like. If the target is a bacterium or a fungus / mold, the term "damage activity" can be replaced with "antibacterial activity".

In the target-specific complex of the present invention, directivity toward the target is imparted by using an antibody. IgY is used as the antibody. IgY is an antibody characteristic of birds. For example, IgY of poultry such as chickens and quails can be adopted. IgY is abundantly contained in egg yolk in addition to serum, and is also called egg yolk antibody. Due to the high abundance in egg yolk, it is possible to relatively easily and in large quantities prepare target-specific IgY from the egg yolk of antigen-sensitized birds (typically chickens). Target-specific IgY may be prepared by a conventional method. The following is an example of a method for preparing target-specific IgY.

First, immunize birds with antigens (target bacteria, fungi, etc.) (sensitization by antigens). A part of bacteria, fungi and the like may be used as an antigen. For example, chickens are used as birds. Immunization is repeated as necessary, and eggs are collected when the antibody titer rises sufficiently. It is advisable to check the antibody titer of serum and determine the time 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 the peak. Therefore, it is advisable to collect eggs 10 to 20 days after the final sensitization (immunity) and extract and purify IgY. For extraction and purification of IgY from eggs, methods such as ultracentrifugation, degreasing using an organic solvent, and lipoprotein separation using sodium dextran sulfate can be used (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 Investments 19: 253-258, 1990. Etc.). Affinity columns for purifying IgY from egg yolk (for example, HiTrap IgY Purification HP Column of Global Life Science Technologies Japan Co., Ltd.) and kits are commercially available, and these may be used.

In addition to polyclonal IgY prepared as described above, monoclonal IgY can also be used. Monoclonal IgY can also be prepared by conventional methods (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 the preparation method of monoclonal IgY is shown. First, the immune operation is performed in the same procedure as described above. Immunization is repeated as necessary, and when the antibody titer rises sufficiently, antibody-producing cells are removed from the immunized birds. Next, using the obtained antibody-producing cells, a hybridoma is obtained by a cell fusion method. Subsequently, after monoclonalizing this hybridoma, a clone that produces an antibody having high specificity for the antigen is selected. The antibody of interest is obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can also be obtained by growing a desired number of hybridomas, transplanting them into the abdominal cavity of an animal (for example, a mouse), and growing them in ascites to purify the ascites. Affinity chromatography on which an antigen is immobilized is preferably used for purification of the culture solution or ascites. Affinity chromatography in which an antigen is 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 are used alone or in any combination.

The targets of attacks by the target-specific complex of the present invention are bacteria, fungi / molds, viruses, parasites, parasites and rickettsia. Infectious to humans or animals under human control (pet / companion animals, industrial animals such as livestock and poultry, laboratory animals, exhibited animals bred and stored in zoos and parks, etc.) Typical targets are those that show and have harmful effects, namely pathogens that can cause infectious diseases (bacteria, fungi / molds, viruses, parasites, parasites, liquettia). The present invention targets the pathogen itself, not the cells infected with the pathogen.

By using IgY, which can be prepared in large quantities relatively easily, it is possible to cope with the large number / amount of targets (bacteria, fungi, molds, etc.) and the rapid growth speed of the targets.

The target bacteria are not particularly limited, and various bacteria such as gram-negative bacilli, gram-negative cocci, gram-positive cocci, and gram-positive cocci can be targeted. Specific examples of potential target bacteria include Escherichia coli, Shigella (S. dysenteriae, S. frexneri, S. sunnei, 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, mP.alcalifaciens, P.stuartii, etc.), Hafnia Bacteria of the genus Hafnia (H.alvei, etc.), Bacteria of the genus Proteus (P. mirabilis, P. vulgaris, etc.), Bacteria of the genus Yersinia (Y. pestis, Y. Vibrio) (V. cholerae, V. parahaemolyticus, etc.), Hemophilus (Haemophilus) (H. influenzae, H. parainfluenzae, H. ducreyi, etc.), Pseudomonas (P. aeruginosa, P. cepacia, P. .Putida, etc.), Aeromonas (A.hydrophilia, etc.), Acinetobacter (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.), Rabbit fungus (Franc) isella tularensis), Bacteroides (B. fragilis, B. melaninogenicus, etc.), Citrobacter (C. freundii, etc.), Xanthomonas (X. maltophilia), flavobacterium 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.), Mycobacteria (B. catarrhalis, etc.) Mycobacterium) (M. tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulare, M. kansasii, M. ulcerans, etc.), Peptcoccus (P. anaerovius, etc.), Peptostreptococcus Bacteria (Peptostreptococcus) (P. anaerobius, etc.), Eubacterium (E. lentum, etc.), Propionibacterium (Propi, etc.) onibacterium (P.acnes), Lactobacillus (L.plantarum, etc.), Bacteroides (B.fragilis, B.melaninogenicus, etc.), Fusobacterium (F.gonidiaformans, etc.) F. necrophorum, F. nucleatum, etc.), Mycoplasma (Mycoplasma, etc.), Borrelia (B. recurrentis, B. burgdoferi, etc.), Treponema pyridum, Campylobacter (C. coli , C. jejuni, C. fetus, etc.), Helicobacter (H. pylori, H. heilmannii, etc.), Rickettsia (R. prowazekil, R. mooseri, R. tsutsugamushi, etc.), Chlamydia Bacteria of the genus (Chlamydia) (C. trachomatis, C. psittaci, etc.), Bacteria of the genus Porphyromonas (P. gingivalis, etc.), Bacteria of the genus Prevotella (Prevotella, intermediate, etc.) Aggregatibacter) (A. actinomycetemcomitans, etc.), Treponema genus Bacteria (Treponema, T. denticola, etc.) can be mentioned.

Similarly, examples of fungi and fungi that can be targeted are 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, etc.) R. microsporus, etc.), Cunninghamella (C. bertholletiae, etc.), Apophysomyces (A. elegans), Sakseneae (S. vasiformis), Coccidioides fungi (Coccidioides) (C. immitis, etc.), Histoplasma (H. capsuleum, etc.), Paracoccidioides (P. brasiliensis), Penicilium (P. marneffei), Blast Mrs. Blastomyces (B. dermatitidis), Sporothix (S. schenckii, etc.), Chromomycosis fungi (Fonsceaea) (F. pedrosoi, etc.), Phialophora (P. verrucosa, etc.) Etc.), Cladophialophora (C. carrinoii, etc.), Malassezia (M. furfur, M. globasa, etc.), Pneumocystis (P. jirovecii, etc.) Can be done.

In addition, examples of viruses that can be targeted include natural pox virus, vactinia virus, infectious irritation virus, herpesvirus 1 (HSV-1), herpesvirus 2 (HSV-2), and varicella / herpesvirus 6 (HSV-2). HHV-3), Cytomegalovirus (HHV-5), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Epstein Bar Virus (HHV-4), Human Herpesvirus 8 (HHV-4) HHV-8, also known as Kaposi sarcoma-related herpesvirus (KSHV)), adenovirus, human papillomavirus, parvovirus B19, dicavirus, hepatitis B virus, influenza virus, measles virus, mumpsvirus, RS virus, human immunodeficiency virus (HIV), human T lymphophilic virus (HTLV-1), coronavirus, lassavirus, eczema virus, Japanese encephalitis virus, yellow fever virus, dengue fever virus, hepatitis C virus, huntervirus, poliovirus, coxsackie virus, echo Viruses, rhinoviruses, hepatitis A virus, hepatitis E virus, nowalk virus, human astrovirus, mad dog disease virus, Marburg virus, ebora virus, leovirus, rotavirus can be mentioned.

On the other hand, examples of parasites that can be targeted include malaria (P. falciparum, P. vivox, P. malariae, etc.), Leishimania (L. donovani, L braziliensis, etc.), and 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.), Cryptosporidium (C.parvum, etc.).

On the other hand, examples of parasites that can cause infectious diseases are roundworms (Ascaris) (Ascaris lumbricoides, etc.), Zubini worms (Ancylostomoa duodenale), Necator americanus, worms (Enterobius vermicularis), and threadworms (Strongyloides stercoralis). , Anisakis (A. simplex, A. physeteris, etc.), Pseudoterranova (P. decipiens), Wunchereri bancroft, Brugia malayi, Gnathostoma ) (G. nipponicum, G. spinigerum, etc.), Trichinella (T. spiralis, T. pseudospiralis, etc.), Angiostrongylus (A. cantonesis. A. constaricensis), liver sucking insect (Clonorchis sinensis) ), Yokokawa worm (Metagonimus yokokawai), lung worm (Paragonimus) (P. westernmani, etc.), brugia malayi (Diphyllobothrium latum), Echinococcus (E. granulosus, E. multilocularis), reduced worm (Hymenolepis) ), Small worms (Hymenolepis nana), multi-headed worms (Taniea multiceps), Asian worms (Taenia asiatica), worms (Taniea saginata), and brugia malayi (Taenia solium).

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

It is also possible to collectively target multiple pathogens with similar antigenicity (for example, multiple closely related species). When targeting a relatively wide range of pathogens in this way, polyclonal IgY may be adopted. By using polyclonal IgY, it does not bind to the human body and can exhibit pathogen-selective yet damaging activity against a relatively wide range of targets. On the other hand, monoclonal IgY is suitable when constructing a target-specific structure targeted to a specific pathogen (for example, a specific strain or strain), in other words, when it is necessary to enhance selectivity or specificity ( However, even in this case, polyclonal IgY may be used).

The present invention utilizes the principle of photoimmunotherapy (PIT). Therefore, a near-infrared light-sensitive substance is linked to the target-specific IgY. Typically, a phthalocyanine dye is used as a near-infrared light sensitive substance. Phthalocyanine pigments are a group of photosensitizer compounds having a phthalocyanine ring system. For example, WO 2005/099689 and US Pat. No. 7,005,518 can be referred to for the synthesis method and usage (use) of various phthalocyanine pigments.

Preferably, a phthalocyanine pigment having an absorption peak in the near infrared (NIR) region and strongly absorbing near infrared rays to emit fluorescence is used. More specifically, a phthalocyanine dye having an absorption peak at 600 nm to 950 nm, more preferably 660 nm to 740 nm, and even more preferably 680 nm to 720 nm is used.

IR700 (IRDye® 700DX) can be mentioned as a particularly preferable phthalocyanine pigment. IR700 is commercially available from LI-COR (LI-COR Biosciences). Amino-reactive IR700 is a relatively hydrophilic dye that can be covalently bound to IgY using, for example, the NHS ester of IR700.

The near-infrared light-sensitive substance is directly or indirectly linked to the target-specific IgY via a covalent bond or a non-covalent bond. Non-covalent bonds are achieved, for example, by electrostatic interactions, van der Waals forces, hydrophobic interactions, π effects, ionic interactions, hydrogen bonds or halogen bonds. For indirect concatenation, a linker is usually used.

2. Pharmaceutical composition and its use The target-specific complex of the present invention can be formulated to prepare a pharmaceutical composition. Generally, a pharmaceutically acceptable carrier (carrier, vehicle) is used for formulation. Examples of carriers include water, saline, balanced salt solution, aqueous dextrose, glycerol, mannitol, lactose, starch and magnesium stearate. For pharmaceutically acceptable carriers and their usage, for example, Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995) can be referred to.

In addition to carriers, pharmaceutical compositions include diluents (lactorose, sucrose, dicalcium phosphate, or carboxymethyl cellulose, etc.), excipients (starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, etc.) Sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc.), lubricants (magnesium stearate, calcium stearate, talc, etc.), pH regulators (acetate, citrate, etc.) Sodium acid, cyclodextrin derivative, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.), emulsifier, solubilizer, isotonic agent, preservative, preservative, etc. may be contained.

The dosage form / shape when formulating is not particularly limited. Examples of dosage forms are aerosols, liquids, suspensions, injections, syrups, emulsions, jellies, tablets, pills, powders, fine granules, granules, capsules, external preparations (ointments, patches). , Paps, lotions, pills, suppositories), inhalants, nasal drops and eye drops.

The pharmaceutical composition of the present invention contains an amount (that is, a therapeutically effective amount) of the active ingredient necessary for obtaining 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 the amount of the active ingredient is set in the range of, for example, about 0.001% by weight to about 99% by weight so that a desired dose can be achieved.

A further aspect of the present invention relates to the use of the pharmaceutical composition. Typically, the pharmaceutical compositions of the present invention are used for the treatment, prevention or amelioration of diseases and conditions. "Treatment" includes alleviating (mitigating) the symptomatology or concomitant symptom characteristic of the target disease, preventing or delaying the worsening of the symptomatology, and the like. "Prevention" means preventing or delaying the onset / delay of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, "improvement" means that the disease (disorder) or its symptoms are alleviated (mild), improved, ameliorated, or cured (including partial cure). Thus, treatment, prevention, and improvement are some overlapping concepts, which are difficult to distinguish and capture, and the benefits of doing so are small. As used herein, treatment for the purpose of prevention or improvement is also included in the concept of the term "therapeutic method".

The pharmaceutical composition of the present invention is typically applied to the treatment, prevention or amelioration of infectious diseases caused by targets (bacteria, fungi / molds, viruses, parasites, parasites, rickettsia). In other words, the pharmaceutical composition of the present invention is utilized for IgY-PAT therapy. Examples of infectious diseases that can be treated include infectious pyoderma (impetigo vesicular, impetigo crust), tan poison, bee folliculitis, folliculitis, furuncle, yo (furuncle). carbuncle), possible sweat adenitis, keroid impetigo, gluteal chronic pyoderma, bacterial impetigo, infant multiple sweat gland abscess, staphylococcal burn-like skin syndrome, toxic shock syndrome, red fever, necrotizing muscle Membranitis, gas necrosis, sepsis, Osler nodule, yellow fungus hair, erythroma, cat scratch disease, actinomycosis, external fistula, nocardiosis, MRSA infection, impetigo infection, seratia infection , Intestinal hemorrhagic Escherichia coli infection, bacterial meningitis, tuberculosis, non-tuberculous mycobacteriosis, cholera, pesto, diphtheria infection, bacterial erythema, erythema, charcoal, syphilis, tetanus, Hansen's disease, regionera pneumonia, leptspira Diseases, Lime's disease, rabbit disease, gonococcal infection, chlamydia infection (chlamydia pneumonia, tracoma, genital chlamydia infection, parrot disease, etc.), carbapenem-resistant enterobacteriaceae bacterial infection, rodent, periodontal disease, endophthalmitis , Candida disease, Aspergillosis, Blast Mres disease, Cryptococcus disease, Mucor disease, Coccidioides disease, Histoplasmosis, Paracoccidioides disease, Sporotricum disease, Scalyx, Natural pox, Viral irritation, Lip herpes, Herpes stomatitis, Corneal herpes (Genital) Herpes, herpes zoster, impetigo sarcoma, hepatitis (types A, B, C, E), HIV, influenza, sickness, norovirus infection, rotavirus infection, RS virus infection, wind rash, measles, Epidemic parotid adenitis (Otafukukaze), Japanese encephalitis, yellow fever, dengue fever, Ebola hemorrhagic fever, apex condyloma, pharyngeal papilloma, polio, mad dog disease, malaria, leash mania, cryptospolidium disease, tripanosomosis, trichomonas disease, toxoplasmosis , Babesia disease, erythema amoeba disease, intestinal dicardiosis, cryptospolidium disease, roundworm disease, worm disease, worm disease, fecal nematode disease, anisakis disease, pseudoteranova disease, filamentous worm disease, jaw worm disease, curly worm disease, Impetigo, hepatic impetigo, Yokokawa impetigo, pulmonary impetigo, fissure impetigo, echinocox disease, impetigo, liquettia infection, tsutsugamushi disease, Japanese erythema fever, rash typhoid, erikiosis, anaplasmosis , Q fever. The pharmaceutical composition of the present invention is particularly useful for local infections such as skin and mucous membranes. The pharmaceutical composition of the present invention can also be applied to the treatment of abscesses caused by bacterial infections and the like.

In the therapeutic method using the pharmaceutical composition of the present invention, the following steps (1) and (2) are performed.
(1) A step of administering the pharmaceutical composition of the present invention to a therapeutic subject and binding the target-specific complex of the present invention to the target (2) A step of irradiating the target with near-infrared light.

In step (1), the pharmaceutical composition of the present invention is administered to the therapeutic subject. The route of administration may be selected according to the dosage form of the pharmaceutical composition, the treatment policy, and the like. Both oral and parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, application to affected areas, application, spray, etc.) can be adopted. Is. In addition, these administration routes are not mutually exclusive, and two or more arbitrarily selected administration routes can be used in combination (for example, intravenous injection or the like is performed at the same time as oral administration or after a lapse of a predetermined time). Both systemic administration and topical administration (for example, application, application, spray, etc. to the infected area and the like) can be adopted. Not limited to humans, animals under the control of humans (pets / companion animals, industrial animals such as livestock and poultry, experimental animals, exhibited animals bred and stored in zoos and parks, etc. Specific examples are Various monkeys, chimpanzees, gorillas, orautans, cows, pigs, goats, sheep, horses, donkeys, camels, ostriches, chickens, quail, ducks, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits, elephants, giraffes, bears. , Shimauma, hippo, rhinoceros, and penguins) can also be treated.

The dose of the pharmaceutical composition is set so as to obtain the expected therapeutic effect. Symptoms, patient age, gender, weight, etc. are generally considered in the setting of therapeutically effective doses. Those skilled in the art can set an appropriate dose in consideration of these matters. For example, in the case of systemic administration, for example, 0.1 to 1000 mg, 0.2 to 500 mg, 0.5 to 100 mg or 1 per 60 kg of body weight. In the case of topical administration, it is, for example, 0.01 to 50 mg, 0.03 to 30 mg, or 0.05 to 10 mg per ^{1 cm 2 of the application site.} In addition, in creating the administration schedule, the medical condition of the patient and the duration of effect of the active ingredient can be taken into consideration.

By administration of the pharmaceutical composition, the target-specific complex, which is the active ingredient thereof, is bound to the target, and then the target is irradiated with near-infrared light (step (2)). Although not 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 effects (such as killing the target and suppressing its growth). .. This mechanism of action is different from the mechanism of action of photodynamic therapy (PDT) (which exerts its effect by causing oxidative stress in mitochondria), and brings about a rapid effect.

For irradiation of near-infrared light, for example, an LED, an LED laser, a light beam through a filter, or the like may be used. Other than direct irradiation, the devices include, but are not limited to, 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, an implantable type, and a sticking type. , A bracelet type device, etc. can be considered. The irradiation conditions of near-infrared light are not particularly limited as long as the damaging activity based on the principle of NIR-PIT can be obtained, but the wavelength of near-infrared light used is, for example, 650 to 740 nm, preferably 670 to 720 nm, and further. It is preferably 680 to 710 nm. Also, the irradiation doses are, 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 30 J cm ^-2 , at least 40 J cm ^-2. , at least 50 J cm ^-2, at least 60 J cm ^-2, at least 70 J cm ^-2, at least 80 J cm ^-2, at least 90 J cm ^-2, or at least 100 J cm ^-2. More specifically, for example, ^{1 ~ 1000J cm -2, 2 ~} 500J cm -2, the irradiation dose of 5 ~ 300 J cm ^-2 or 10 ~ 100J cm ^-2. 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 longer, more preferably 1 minute or longer, and even more preferably 3 minutes or longer. Further, although not limited to this example, when the pharmaceutical composition is systemically administered by intravenous injection or the like, after administration of the pharmaceutical composition, for example, for 5 minutes to 48 hours, preferably 10 minutes to 24 hours. Irradiation with near-infrared light is carried out over time, more preferably between 15 minutes and 12 hours. In the case of topical administration, it is preferable to set the interval between administration of the pharmaceutical composition and irradiation of near-infrared light shorter than in the case of systemic administration (for example, 1 minute to 12 hours after administration of the pharmaceutical composition). Irradiate near-infrared light during).

It is possible to perform multiple irradiations instead of a single irradiation. In the case of multiple irradiations, the interval is not particularly limited. For example, multiple irradiations on the same day at predetermined intervals (for example, 5 minutes to 10 hours), daily irradiation, every other day or every few days, one week or every few weeks, one month or number. Various irradiation schedules can be set, such as irradiation every month. The administration schedule of the pharmaceutical composition in the case of multiple irradiations is not particularly limited. For example, if the interval between the first and second irradiations is short, the pharmaceutical composition is typically administered only prior to the first irradiation. To give another example, if the elapsed time from the previous irradiation is long (for example, one day to several months have passed), it is advisable to administer the pharmaceutical composition again and then perform irradiation. ..

In parallel with the treatment or prevention with the pharmaceutical composition of the present invention, other medicines such as antibacterial agents (for example, penicillin antibacterial agents, cephem antibacterial agents, carbapenem antibacterial agents, penem antibacterial agents, tetracycline antibacterial agents, β Treatment with lactamase inhibitors, phosphomycin, vancomycin, aminoglycoside antibacterial agents, macrolide antibacterial agents) may be used. When the pharmaceutical composition of the present invention and a drug having a different mechanism of action are used in combination, a complex action / effect can be obtained and the therapeutic effect can be increased.

3. 3. Disinfectant / Decontamination Composition The target-specific complex of the present invention is contaminated with pathogens (bacteria, fungi / molds, viruses, parasites, parasites, rickettsia) that can cause infectious diseases (contaminants). ) Can also be used for disinfection or decontamination. That is, a composition for disinfection / decontamination (hereinafter, referred to as "disinfectant of the present invention" for convenience of explanation) can be prepared using the target-specific complex of the present invention. The disinfectant of the present invention is, for example, various facilities / instruments / equipment in medical institutions (hospitals, clinics, nursing homes for the elderly, home-visit nursing care stations, midwifery centers, pharmacies, etc.), osteopathic clinics, osteopathic clinics, acupuncture and moxibustion clinics, etc. (Inspection or surgical equipment, instruments or devices, urinary organs, floors, walls, curtains, furniture, doors, windows, bedding, clothing, etc.) and spaces (in hospital rooms, operating rooms, clean rooms, clean benches, etc.) Disinfection or decontamination, various equipment, utensils, equipment, etc. at manufacturing plants or research institutions of pharmaceuticals, cosmetics or medical equipment (manufacturing equipment, worktables, floors, walls, curtains, furniture, doors, windows, work clothes, gloves, etc.) Disinfection or decontamination of spaces (in factories, clean rooms, clean benches, etc.), disinfection or decontamination of kitchens, toilets, washrooms or bathrooms, tableware, cutlery (knives, forks, spoons, etc.) or cooking utensils (knives, forks, spoons, etc.) Disinfection or decontamination of kitchenware, knives, pots, mixers, microwaves, ovens, etc., disinfection or decontamination of hands, fingertips or oral cavity (eg, treatment or prevention of periodontal disease and rodents, or sterilization after implant), It is available for.

The disinfectant of the present invention is composed of, for example, a liquid (for example, a spray agent, a lotion), a gel form, or a solid form (for example, a powder), and is used by coating, spraying (spraying), spraying, dropping, or the like. In using the disinfectant of the present invention, for example, after applying the disinfectant of the present invention to a contaminated object by application or the like, the disinfectant of the present invention is irradiated with near-infrared light to exert a damaging activity. The irradiation conditions of near-infrared light are the same as in the case of treatment using a pharmaceutical composition.

<Development of new photoantibacterial therapy>
1. 1. Preparation of Candida albicans-IgY (CA-IgY) -IR700 and confirmation of quality 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 Na 2} HPO ₄ (pH 8.5) 0.1 M for 1 hour at room temperature, followed by a Sephadex G50 column (PD-10; The mixture was collected through GE healthcare) (CA-IgY-IR700 solution). After Coomassie staining, the absorbance (wavelength 595 nm) was measured to determine the concentration (protein concentration) of CA-IgY-IR700. In addition, the concentration of IR700 was determined by measuring the absorbance (wavelength 698 nm), and the number of fluorescent molecules bound to the antibody was confirmed. On the other hand, the above mixed solution was subjected to SDS-PAGE, and the binding between the antibody and IR700 was confirmed. Diluted CA-IgY was used for SDS-PAGE control, and imaging was performed with a Pearl imager (LICOR).

1-2. Results and discussion Fluorescence was observed only in CA-IgY-IR700 at the band height of protein staining (Fig. 1, left) (Fig. 1, right), and it was confirmed that CA-IgY and IR700 were bound (conjugated).

2. Evaluation of Candida albicans (C. albicans) antigen binding of CA-IgY-IR700 2-1. Experimental method C. albicans (JCM1542) ^{was seeded in tubes in 1 × 10 5} increments, and the prepared CA-IgY-IR700 (10 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL) and 1 at 37 ° C. After time incubation, the fluorescence intensity was measured by flow cytometry. In addition, four other C. albicans species (FC18, IFO579, IFO1060, and IFO1385), and related species such as Candida tropicalis (IFO1402), Candida guilliermondii (IFO838), Candida krusei (IFO1162), and A431 (human skin cancer cells). ) Were measured in the same manner using CA-IgY-IR700 of 200 μg / mL.

2-2. Results and discussion Fluorescence was enhanced in proportion to the antibody concentration of CA-IgY-IR700 (Fig. 2). Previously reported NIR-PIT used 10 μg / mL, but C. albicans was considered to be insufficient at 10 μg / mL. It was considered that one of the reasons for this was that the growth rate was high, C. albicans increased during the 1-hour incubation, and the antibody was relatively deficient. Fluorescence enhancement was observed for all C. albicans and related species, but no fluorescence enhancement was observed for A431 (Fig. 3). Therefore, it is considered that CA-IgY-IR700 bound to Candida in general, but not to human cells.

3. 3. In vitro NIR-PAT
3-1. Experimental method In order to evaluate the antibody concentration and therapeutic effect, C. albicans (JCM1542) was cultured in a liquid medium for 24 hours, then seeded in a tube with 100 μL of medium in ^{1 × 10 5 each, and CA-IgY-IR700 was added.} After incubation at room temperature for 4 hours (10 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL), near-infrared light (256 J / cm ² ) was irradiated using an LED with an emission wavelength of 690 nm. Immediately after the treatment, 300 bacterial cells were inoculated on a 10 cm dish, and after culturing for 24 hours, the number of colonies was measured to evaluate the viable cell count. The control was one in which neither antibody addition nor near-infrared light irradiation was performed.

Next, in order to evaluate the irradiation dose and therapeutic effect, CA-IgY-IR700 was adjusted to 200 μg / mL, and after incubation at room temperature for 4 hours, near-infrared light (32J, 64J,) using an LED with an emission wavelength of 690 nm was used. It was irradiated with 128J and 256J / cm ^2). Those subjected to near-infrared light irradiation (256 J / cm ² ) without adding CA-IgY-IR700 were also evaluated. After the treatment, the number of colonies was measured by the same method to evaluate the viable cell count (Fig. 4). For macroscopic measurement, CA-IgY-IR700 200 μg / mL was reacted in the same manner, and ^{1 hour after irradiation with near infrared light 256 J / cm 2} 1 hour, dead cell staining (Propiodium Iodide: PI staining) was used. It was observed with a confocal microscope and a scanning electron microscope.

3-2. Results and discussion In the evaluation of antibody concentration and therapeutic effect, the viable cell count of C. albicans decreased in a CA-IgY-IR700 concentration-dependent manner (Fig. 5, left). The therapeutic effect was poor at 10 μg / mL, and the effect was observed at 50 μg / mL or higher. In the evaluation of the irradiation dose and the therapeutic effect, the viable cell count decreased depending on the irradiation dose, and the viable cell count did not change only by near-infrared light irradiation (Fig. 5, right). The decrease in the viable cell count with CA-IgY-IR700 alone is considered to be due to the original antibacterial action of IgY. IgY-PAT is thought to significantly enhance the antibacterial effect of IgY.

In addition, observation with a confocal microscope revealed fluorescence at 700 nm when only CA-IgY-IR700 reacted, and death cell staining was positive when IgY-PAT was performed, and fluorescence at 700 nm was attenuated (). FIG. 6 left). It was speculated that this was because IR700 was decomposed by near-infrared irradiation and destroyed the bacterial cells. In the scanning electron microscope image, no change was observed only with the control and CA-IgY-IR700, but with IgY-PAT, holes and deformation of the cells were observed on the surface of the cells (Fig. 6). right). This is considered to be the deformation caused by the destruction of the surface of the cells by IgY-PAT and the rupture of the cells.

4. In vivo NIR-PIT
4-1. Experimental method (Fig. 7)
After hair removal from both lumbar regions of 8- to 10-week-old BALC slc mice, two skin defect sites were created on the left and right with a 6 mm skin biopsy punch. C. albicans (JCM1542) was washed with PBS, ^{then concentrated to 1x10 9} / mL with PBS, 100 μL was applied to the skin defect, and the ulcer was protected with an emergency bond (set as Day -1). The next 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 by an emergency bond. The next day (Day 1), the emergency bond was removed, and it was evaluated by Pearl imager (LICOR) whether CA-IgY-IR700 could be confirmed in the ulcer area. Then, near infrared light (256 J / cm ² ) was irradiated with a laser. The transition of the ulcer area was quantitatively evaluated with the ulcer area on the irradiation day as 100%. The ulcer area was photographed and calculated using Image J (open source). The treatment group is the IgY-PAT group, which is a sterile group without C. albicans, a CA group with only C. albicans, and a CA-IgY-IR700 group with CA-IgY-IR700 applied and not irradiated with near-infrared light. , Light group with only near-infrared light irradiation. The ulcer area was evaluated until Day 14. To evaluate the number of bacteria, on Day 2, the subcutaneous tissue of the ulcer part of the sterile group, CA group, and IgY-PAT group was removed with a sterilized 6 mm skin biopsy punch, and the tissue was stirred and seeded in the medium to inoculate the number of colonies. Was measured. The number of colonies in the CA group was set to 100%.

4-2. Results and Discussion In the group coated with CA-IgY-IR700, fluorescence of the coated part was observed, and the fluorescence was attenuated after irradiation with near-infrared light (Fig. 8). It is presumed that this is because the antibody adhered to the ulcer surface and reacted with near-infrared light to decompose CA-IgY-IR700 as in vitro. The ulcer area was the smallest in the sterile group, followed by the IgY-PAT group and the CA-IgY-IR700 group, and the CA group and the Light group were almost the same. No significant difference was observed between the sterile group and the IgY-PAT group, and a significant difference was observed between the IgY-PAT group and the CA-IgY-IR700 group. A significant difference was also observed between the CA-IgY-IR700 group and the CA group (Fig. 9). In addition, the amount of purulent excrement in the ulcer part was small in the sterile group, CA-IgY-IR700 group, and IgY-PAT group (Fig. 10). From these results, it was presumed that the antibacterial effect was observed by CA-IgY-IR700 alone as in the in vitro result, but the antibacterial effect was enhanced by IgY-PAT, and the course was similar to that of the sterile group. .. In addition, there were no adverse events such as burns, and it was considered that it contributed to the overall healing of the ulcer. The number of colonies was significantly smaller in the sterile group and the IgY-PAT group than in the CA group (Fig. 11). Colonies are also observed in the sterile group due to the contamination of indigenous bacteria. From this result, it was speculated that IgY-PAT also showed an antibacterial effect in vivo, and that the decrease in ulcer area was associated with the number of bacteria.

5. Summary IgY-PAT using a complex that combines CA-IgY and IR700 exhibited a high antibacterial effect. This innovative treatment can combat many refractory infections and drug-resistant bacteria.

The target-specific complex of the present invention (a structure in which a near-infrared light-sensitive substance is linked to IgY) exhibits target-specific damaging activity according to the principle of NIR-PIT and exerts a therapeutic effect. The present invention using IgY, which is inexpensive and easy to prepare in large quantities, provides a new therapeutic means capable of coping with the explosive increase in targets in infectious diseases such as bacteria, fungi, and molds, and is effective against various infectious diseases. It is expected to be applied or applied. The present invention is an innovative technique that is different from the conventional antibacterial therapy using IgY alone, and a high therapeutic effect can be obtained. In addition, due to its unique mechanism of action, rapid onset of effects can be expected. The fact that the bactericidal effect on Candida having a tough cell wall structure has been confirmed means 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. To support.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. For example, examples of target-directing substances include commonly considered binding substances such as antibodies (IgG, IgM, antibody fragments, Minibody, Diabody, etc.), peptides, aptamers, etc., which can be used in place of IgY. .. The contents of the articles, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (14)

1. A target-specific complex with a structure in which a near-infrared light-sensitive substance is linked to a target-specific IgY classified as a bacterium, fungus or mold, virus, parasite, parasite or rickettsia.
2. The target-specific complex according to claim 1, wherein the target is a bacterium or a fungus or a mold.
3. The bacterium is a bacterial species selected from the group consisting of Pseudomonas genus, Acinetobacter genus, Bacillus subtilis, Streptococcus, Enterococcus bacterium, Escherichia coli, Shigera bacterium, Salmonella bacterium, Enterobacter bacterium and Klebsiella bacterium. The target-specific complex according to claim 2, wherein the bacterium or mold is a bacterial species selected from the group consisting of Enterobacter, Aspergillus, Mucor and Cryptococcus.
4. The target-specific complex according to 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-sensitive substance is a phthalocyanine pigment.
6. The target-specific complex according to claim 5, wherein the phthalocyanine pigment is IR700.
7. A composition containing the target-specific complex according to any one of claims 1 to 6.
8. The composition according to claim 7, which is used for treating or preventing an infectious disease caused by the target.
9. Therapeutic methods, including the following steps (1) and (2):
   (1) A step of administering the composition according to claim 8 to a therapeutic subject and binding the target-specific complex to the target.
   (2) A step of irradiating the target with near-infrared light.
10. The treatment method according to claim 9, wherein the wavelength of the near-infrared light is 650 to 740 nm.
11. The treatment method according to claim 9, wherein the wavelength of the near-infrared light is 670 to 720 nm.
12. The treatment method according to any one of claims 9 to 11, wherein the irradiation dose of the near-infrared light is 1 J cm- ^{2 or more.}
13. The treatment method according to any one of claims 9 to 11, wherein the irradiation dose of the near-infrared light is ² J cm -2 to 500 J cm ^-2.
14. The composition according to claim 7, which is used for disinfecting or decontaminating contaminants by the target.

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