Fungicides

Abstract

This bactericide contains a conjugate (10) in which a photosensitizer (12) is bound to an antibody (11). In the conjugate (10), the antibody (11) causes an antigen‐antibody reaction with Staphylococcus aureus (16). Moreover, the absorption wavelength range of the photosensitizer (12) overlaps with the wavelength range from red rays to near infrared rays. In the usage of the bactericide, the surface (14) of a nasal cavity coated with the bactericide is irradiated with excitation light (20) in the wavelength range from red rays to near infrared rays. In another usage thereof, the surface of a navel coated with the bactericide is irradiated with excitation light in the wavelength range from red rays to near infrared rays. In still another usage thereof, the bactericide is administered to a cow udder, and the udder is irradiated with excitation light in the wavelength range from red rays to near infrared rays.

Description

[Technical field]

[0001] The present invention relates to a germicidal agent, in particular an agent for photoimmunotherapy that targets bacteria. [Background technology]

[0002] The "Summary of Research Results" in Non-Patent Document 1 describes the selective sterilization of Staphylococcus aureus by photoimmunotherapy (PIT). In this method, a compound obtained by binding a photosensitizer to a monoclonal antibody is targeted and specifically bound to Staphylococcus aureus, and the Staphylococcus aureus is irradiated with near-infrared light. This monoclonal antibody specifically recognizes peptidoglycan, which is the main cell wall component of Staphylococcus aureus. Non-Patent Document 2 describes the peptidoglycan of Staphylococcus aureus.

[0003] The method of Non-Patent Document 1 was able to induce a bactericidal effect even against drug-resistant Staphylococcus aureus such as MRSA (methicillin-resistant Staphylococcus aureus) and VISA (vancomycin-intermediate S. aureus). This bactericidal effect is due to the specific binding of anti-peptidoglycan monoclonal antibodies to Staphylococcus aureus. It was suggested that photoimmunotherapy using this compound may be clinically applicable in the future as a target-specific treatment for infectious diseases.

[0004] Example 15 of Patent Document 1 discloses that an antibody conjugate was evaluated for its ability to kill bacterial cells. Paragraph

[0550] describes the use of an antibody directly conjugated to a phthalocyanine photosensitizer such as IRDye 700DX. Here, it was evaluated whether an antibody bound to Protein A, a protein presented on the cell surface, would kill bacterial cells when exposed to laser irradiation. Protein A is a protein that binds to the Fc region of an antibody and is presented on the cell surface of Staphylococcus aureus.

[0005] Also, paragraph

[0553] of Patent Document 1 describes that PIT-mediated cell killing of Staphylococcus aureus occurred in the presence of an antibody-IR700 conjugate that binds to Protein A. Only bacterial cells incubated with cetuximab-IRDye 700DX and then irradiated with a laser had a statistically significant reduction in CFU compared to the other three groups. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2018-528267 [Non-patent literature]

[0007] [Non-Patent Document 1] Masato Mitsunaga, “Development of photoimmunochemical therapy to overcome drug-resistant bacterial infections”, [online], May 10, 2017, Japan Society for the Promotion of Science, [Retrieved March 14, 2019], Internet,<https: / / kaken.nii.ac.jp / report / KAKENHI-PROJECT-26670487 / 26670487seika / > ,2015 Research Report Issue No. 26670487; Makoto Mitsunaga, “Development of bacteria-targeted photoimmunotherapy”, [online], May 10, 2017, The Japan Society for the Promotion of Science (JSPS), [searched on March 14, 2019], internet,<https: / / kaken.nii.ac.jp / en / report / KAKENHI-PROJECT-26670487 / 26670487seika / > , 2015 Fiscal Year Final Research Report, Project / Area Number 26670487 [Non-Patent Document 2] Akiko Shiratsuchi, “Induction and regulation of innate immune responses by cell-wall components of Staphylococcus aureus”, The Journal of Biochemistry (JB), Japanese Biochemical Society, September 9, 2012, Volume 84, Number 9,pp.737-752 [Non-Patent Document 3] Hanaki H, Hiramatsu K. [Evaluation of reduced vancomycin susceptibility of MRSA strain Mu50 with various conditions of antibiotic susceptibility tests]. Jpn J Antibiot. 1997 Sep;50(9):794-8. [Non-Patent Document 4] Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S, Fukuchi Y, Kobayashi I. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet. 1997 Dec 6;350(9092):1670-3. [Non-Patent Document 5] QED Bioscience Inc., "Staphylococcus aureus Monoclonal Antibodies, ORDERING INFORMATION", [online], [Retrieved May 29, 2019], Internet<URL:https: / / www.qedbio.com / product / staphylococcus-aureus / > Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a beneficial use for the clinical application of agents for photoimmunotherapy targeting bacteria. [Means for solving the problem]

[0009] <1> A bactericide comprising a conjugate in which a photosensitizer is bound to an antibody, In the conjugate, the antibody undergoes an antigen-antibody reaction with Staphylococcus aureus, and the absorption wavelength range of the photosensitizer overlaps with a wavelength range from red light to near infrared light, In the method, the surface of the nasal cavity to which the disinfectant has been applied is irradiated with excitation light having a wavelength range from red light to near infrared light. Disinfectant. <2> The antibody is an anti-peptidoglycan antibody. <1> The fungicide described in . <3> The photosensitizer has a silicon phthalocyanine complex moiety. <2> The fungicide described in . <4> The silicon phthalocyanine complex moiety is IRDye 700DX represented by the following formula: [ka] In the formula, the two oxygen atoms coordinated to the Si atom at the center of the complex may be in trans positions across the phthalocyanine skeleton, or may be in cis positions on one side of the phthalocyanine skeleton. <3> The fungicide described in . <5> In its usage, The surfaces of the nasal cavity to which the disinfectant is applied include the surfaces of the nasal vestibule. <1> The fungicide described in . <6> In its usage, the nasal cavity being that of a patient to be treated; The application and irradiation are performed before the patient receives the treatment; The treatment includes treatment for the nose and treatment for areas other than the nose, but does not include the treatment by application and irradiation itself. <5> The fungicide described in . <7> In its usage, the patient is or is suspected of being a carrier of antibiotic-resistant Staphylococcus aureus, <6> The fungicide described in . <8> In its usage, The antibiotic-resistant Staphylococcus aureus is MRSA. <7> The fungicide described in . <9> In its usage, Preliminarily testing whether the patient is a carrier of MRSA; If the test result indicates that the patient is a carrier or is suspected of being a carrier of MRSA, the application and irradiation are performed. <6> The fungicide described in . <10> A bactericide comprising a conjugate in which a photosensitizer is bound to an antibody, In the conjugate, the antibody undergoes an antigen-antibody reaction with Staphylococcus aureus, and the absorption wavelength range of the photosensitizer overlaps with a wavelength range from red light to near infrared light, In this method, the surface of the navel to which the disinfectant has been applied is irradiated with excitation light having a wavelength range from red light to near infrared light. Disinfectant. <11> A bactericide comprising a conjugate in which a photosensitizer is bound to an antibody, In the conjugate, the antibody undergoes an antigen-antibody reaction with Staphylococcus aureus, and the absorption wavelength range of the photosensitizer overlaps with a wavelength range from red light to near infrared light, In the method, the disinfectant is administered to the udder of a cow, and the udder is irradiated with excitation light having a wavelength range from red light to near infrared light. A disinfectant for the prevention or treatment of bovine mastitis. Effect of the Invention

[0010] The present invention can provide a beneficial method for the clinical application of agents for photoimmunotherapy targeting bacteria. [Brief description of the drawings]

[0011] [Figure 1] Schematic diagram of the conjugate and bacterial flora. [Diagram 2] Cross section of the nasal cavity. [Diagram 3] Dependence of the bactericidal effect of antimicrobial photoimmunotherapy (aPIT) on the amount of conjugate. [Figure 4] Dependence of the bactericidal effect of aPIT on irradiation energy. [Diagram 5] Controlled studies of the bactericidal effect of aPIT. [Figure 6] Bactericidal effect of aPIT against methicillin-resistant strains. [Figure 7] Specificity for Staphylococcus aureus in aPIT. [Figure 8] Evaluation of aPIT against bacteria colonizing cotton rats. [Figure 9] Microscopic image of a cotton rat nasal tissue section. [Figure 10] Evaluation of aPIT for log-phase bacteria. [Figure 11] Evaluation of aPIT after culturing bacteria on various nonselective media. [Figure 12] Evaluation of aPIT after suspension of bacteria in defibrinated equine blood. [Figure 13] Evaluation of aPIT after co-culture of bacteria and fibroblasts. [Figure 14] Comparison of antibody clones in aPIT. [Figure 15] Assessment of the interaction between Protein A and Fc. [Figure 16] No effect on bacterial flora including colonizing bacteria, no irradiation. [Figure 17] Impact on bacterial flora including colonizing bacteria, with irradiation. [Figure 18] Survival curves of mice infected with S. aureus. [Figure 19] Evaluation of aPIT after allowing bacteria to form biofilms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <1. Fungicides and conjugates>

[0013] The disinfectant according to this embodiment contains a conjugate. Fig. 1 shows a schematic diagram of a conjugate 10. The conjugate 10 is an antibody-drug conjugate (ADC) consisting of an antibody 11 and a photosensitizer 12.

[0014] In Fig. 1, the antibody 11 may be a monoclonal antibody specific to the target bacterium 16. The target bacterium 16 is Staphylococcus aureus. The antibody 11 undergoes an antigen-antibody reaction with Staphylococcus aureus.

[0015] In the antibody 11 shown in Fig. 1, the antibody class may be any of IgM, IgD, IgG, IgA, and IgE. The antibody 11 in the figure is IgG. The IgG subclass may be any of 1 to 4. The antibody 11 may be a chimeric antibody, a humanized antibody, or a fully human antibody. The antibody may be a hybridoma antibody or a recombinant antibody.

[0016] The antibody 11 shown in Figure 1 may be a full-length immunoglobulin or a variant, or a partial fragment. The partial fragment may be a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain Fv protein, so-called scFv, or a disulfide-stabilized Fv protein, so-called dsFv. The antibody 11 in the figure is a full-length IgG.

[0017] In Figure 1, antibody 11 is specific for an antigenic determinant carried by target molecule 17 that is unique to target bacterium 16. Target bacterium 16 has target molecule 17 on its surface. In one embodiment, target molecule 17 is peptidoglycan of Staphylococcus aureus. Antibody 11 is an anti-peptidoglycan antibody. Antibody 11 is specific for the surface of Staphylococcus aureus.

[0018] According to Non-Patent Document 2, peptidoglycan is known to account for a large proportion of the cell wall components of Staphylococcus aureus. In its structure, sugar chains formed by alternating bonds between N-acetylglucosamine and N-acetylmuramic acid are cross-linked with oligopeptides consisting of pentaglycine. D-amino acids are added to the oligopeptide portion. The lysine residues contained in the oligopeptide serve as the bridging moiety of pentaglycine. For this reason, it is also called lysine-type peptidoglycan (Lys-type PGN). The following chemical formula is quoted from Non-Patent Document 2.

[0019] [ka]

[0020] <2. Imparting photosensitivity>

[0021] As shown in FIG. 1, in the conjugate 10, a photosensitizer 12 is bound to an antibody 11. In another aspect, the conjugate 10 is an antibody modified with a photosensitizer 12. The antibody 11 and the photosensitizer 12 are covalently bound. In the example shown in the figure, the photosensitizer 12 is bound to the Fc region of the antibody 11 via a linker 13. More specifically, the photosensitizer 12 is bound to the heavy chain constant region (Cc) of the antibody 11 via the linker 13.H Area) C H2 is bound to.

[0022] 1, the covalent bond between antibody 11 and photosensitizer 12 may be replaced with a non-covalent bond. For example, photosensitizer 12 may be bound to a site-specific antibody-binding peptide, and the conjugate of the antibody-binding peptide and photosensitizer may then be bound to a specific site on antibody 11.

[0023] In Fig. 1, one or two or more photosensitizers 12 are bound to one antibody 11. Any of 3, 4, 5, 6, 7, 8, 9, 10, and 11 or more photosensitizers 12 may be bound to one antibody 11. The conjugate 10 may be a mixture of conjugates having different numbers of bound photosensitizers 12. The average number of bound photosensitizers 12 may be 1 to 10, 2 to 5, or 2.5 to 4.

[0024] In Fig. 1, when viewed from the perspective of the entire conjugate 10, which is a single molecule, the photosensitizer 12 portion is interpreted as an atomic group. The conjugate 10 itself can also be interpreted as a photosensitizer. However, for ease of explanation, the scope of this specification is limited to this atomic group portion, and this will be simply referred to as a photosensitizer.

[0025] The photosensitizer 12 shown in Fig. 1 has a predetermined absorption wavelength range. This absorption wavelength range overlaps with the wavelength range from red light to near infrared light. The wavelength range from red light to near infrared light is preferably a wavelength range of 650 to 850 nm.

[0026] The reason why this wavelength range is selected is due to the substances in the body. There are light absorbing substances such as water on the epidermis and in bacterial flora. The light in the above wavelength range is absorbed by these substances at a lower rate than light in other wavelength ranges. This is sometimes referred to as the "NIR window."

[0027] In some technical fields, the wavelength range of 650 to 850 nm may be interpreted as including not only near-infrared light but also visible light. This is because this wavelength range is a connecting region between near-infrared light and visible light. However, the strict distinction of whether light in such a wavelength range is infrared light or visible light is not strongly related to the essence of the invention. In this embodiment, when the conjugate exerts a photosensitizing effect by being irradiated with excitation light, the excitation light may contain red visible light as a component in addition to near-infrared light.

[0028] The photosensitizer 12 shown in FIG. 1 may be a fluorophore or a chromophore. In this embodiment, even if the photosensitizer 12 emits fluorescence in the wavelength region of 650 to 850 nm, such fluorescence is not actively used. It is sufficient that the photosensitizer 12 has a photosensitizing effect 21 and can convert the light energy of the excitation light 20 into damage to the target bacteria 16. The higher the rate at which the photosensitizer 12 can convert the light energy into damage to the target bacteria 16, the better. The photosensitizer may be selected from these viewpoints.

[0029] The photosensitizer 12 shown in Fig. 1 has a silicon phthalocyanine complex portion (atomic group). The photosensitizer 12 is preferably IRDye 700DX, abbreviated as IR700, represented by the following formula. Note that "IRDye" is a trademark.

[0030] [ka]

[0031] IR700 is, for example, provided by LI-COR as the NHS ester shown in the formula below. NHS esters can easily label, for example, amino groups located in the constant region of an antibody. As long as stereochemistry allows, the two oxygen atoms coordinated to the Si at the center of the complex may be in trans positions across the phthalocyanine skeleton, or in cis positions on one side of the phthalocyanine skeleton. In the chemical formula, the oxygen atoms are shown as being in cis positions.

[0032] [ka]

[0033] Other photosensitizers or structures of photosensitizers that can be applied to the photosensitizer 12 shown in FIG. 1 include porphyrins or derivatives having a porphyrin skeleton, phthalocyanines or derivatives having a phthalocyanine skeleton, and naphthalocyanines having a structure similar to that of IR700. The photosensitizer may be a porphyrin derivative used in photodynamic therapy (PDT). Examples of porphyrin derivatives include chlorin e6, protoporphyrin, and hematoporphyrin derivative (HpD).

[0034] 3. Binding mode between antibodies and target bacteria

[0035] In Figure 1, antibody 11 and target molecule 17 are bound by an antigen-antibody reaction. There are known binding modes between Staphylococcus aureus and antibodies other than the antigen-antibody reaction. That is, protein A is expressed on the surface of Staphylococcus aureus. As described in the Background Art, protein A specifically binds to the Fc region of IgG antibodies.

[0036] In Figure 1, conjugate 10 specifically binds to target bacterium 16, Staphylococcus aureus, by utilizing the antigen recognition ability of antibody 11, without relying on the binding between protein A and the Fc region. In one example, antibody 11 does not have an Fc region. In one example, antibody 11 binds to photosensitizer 12 at a region other than the Fc region. In one example, the Fc region of antibody 11 binds to photosensitizer 12, and furthermore, the affinity of the Fc region for protein A is reduced due to steric hindrance of photosensitizer 12.

[0037] <4. Formulation of fungicide>

[0038] The bactericide may be a liquid containing the conjugate. The bactericide includes a pharma- ceutically acceptable carrier. Pharmaceutically acceptable fluids and physiologically acceptable fluids may be used as vehicles to prepare the liquid. Examples of vehicles are water, saline, balanced salt solutions, aqueous dextrose, or glycerol. Wetting agents, emulsifiers, preservatives, and pH buffering agents may also be added. Examples of additives include sodium acetate and sorbitan monolaurate.

[0039] The dosage form of the bactericide is not limited to the above liquid formulation. The bactericide may be any of solid formulations for external use including powders for external use, liquid formulations for external use including liniments and lotions, spray formulations for external use including aerosols and pump sprays, ointments, creams, gels, patches, tapes, and poultices. When administered to the nasal cavity including the nasal vestibule, spray formulations are preferred.

[0040] <5. How to use the disinfectant>

[0041] Bactericides are suitable for use in PIT for bacterial infections, especially near-infrared photoimmunotherapy (NIR-PIT). First, the bactericide is applied to the inside of the patient's nose. Application can be by swabs or sprays. Other methods of administration can also be used.

[0042] 2 shows a cross section of a nose. The nasal vestibule 24 is located at the entrance of the external nares 26 of the nasal cavity 25. Nose hairs usually grow in the nasal vestibule. The epidermis and nose hairs of the nasal vestibule 24 may be home to a bacterial flora including Staphylococcus aureus.

[0043] In Fig. 2, a bactericide is applied to the nasal vestibule 24. The nasal vestibule 24 to which the bactericide has been applied is irradiated with excitation light using a light source such as an infrared therapy device 27. The light source of the excitation light may be a laser or an LED. The infrared therapy device 27 may be a diffuser-type laser fiber. If necessary, a bactericide may be further applied to the mucous membrane in the nasal cavity 25, and excitation light may be further irradiated. For irradiation, the infrared therapy device 27 may be inserted into the nasal cavity 25 through the external nostril 26.

[0044] Returning to Figure 1, the target bacteria 16 live within the bacterial flora 15. The bacterial flora 15 is a flora occurring on the epidermis 14. Here, the epidermis 14 is the epidermis of the nasal vestibule. The bacterial flora 15 also includes bacteria other than the target bacteria 16.

[0045] In Fig. 1, if a bactericide with a wide antibacterial spectrum is used instead of the bactericide containing the conjugate 10, there is a possibility that all bacteria other than the target bacteria 16 in the bacterial flora 15 will be killed. In contrast, by activating the conjugate 10 with the excitation light 20 as in this embodiment, the target bacteria 16 in the bacterial flora 15 can be targeted and attacked. Therefore, photoimmunotherapy is less likely to generate new resistant bacteria from bacteria other than the target bacteria 16. However, this embodiment does not exclude the use of a bactericide in addition to photoimmunotherapy.

[0046] In Fig. 1, the photosensitizer 12 is excited by receiving excitation light 20. The excited photosensitizer 12 exerts a photosensitizing effect 21, thereby damaging the target bacterium 16. The photosensitizing effect 21 involves energy, but is not necessarily electromagnetic waves.

[0047] 1, light in the wavelength range from red light to near infrared light is used as the excitation light 20. Light having a wavelength of 650 to 900 nm, preferably 660 to 740 nm, and more preferably 660 to 710 nm is irradiated as the excitation light 20. The wavelength may be 680 nm.

[0048] The irradiation dose of the excitation light 20 shown in FIG. 2 ) or more, and more preferably 10 to 500 (J / cm 2 The exposure doses were 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 and 400 (J / cm 2 ) is also acceptable.

[0049] After one dose of the fungicide, irradiation may be performed once or more than once. Irradiation may be performed 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The fungicide may be administered two or more times. The number of irradiations after the second or subsequent doses of the fungicide may also be one or more than once.

[0050] <6. Effective amount for sterilization>

[0051] It is necessary to estimate the effective amount of the conjugate for disinfection. The effective amount is the amount of the conjugate sufficient to achieve the desired effect in the patient's body or body part being treated. The effective amount may depend on several factors, such as the patient or affected part being treated, the type of conjugate, and the method of administration. Regardless of the general effective amount determined in advance, it is considered that the effective amount in an individual patient varies depending on the condition of the patient. In order to administer an effective amount of the disinfectant, the disinfectant may be administered in a single dose or in multiple doses.

[0052] The effective amount of the conjugate is, for example, at least 0.5 mg / kg, at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg per 60 kilograms of body weight. For intravenous administration, it is, for example, 0.5 to 50 mg / 60 kg. The amount used may be 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg.

[0053] The effective amount of the conjugate is at least 10 μg / kg, at least 100 μg / kg, at least 500 μg / kg or at least 500 μg / kg based on body weight. For intraperitoneal administration, for example, 10 μg / kg to 1000 μg / kg. The amount used may be, for example, 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg.

[0054] <7. Preventive Measures>

[0055] The bactericide of this embodiment may be used on a patient prior to other main treatment to prevent the patient from being infected with Staphylococcus aureus after the treatment. As an example, the bactericide is applied to the nasal vestibule of a patient who is scheduled to undergo surgery as treatment. Then, the nasal vestibule is irradiated with excitation light as described above. The application and irradiation are performed before the patient undergoes surgery. The surgery here includes surgery on areas other than the nose. Such application and irradiation are also an example of the application of PIT. They may be performed on patients who are undergoing treatment other than surgery. The patient may be an inpatient. Treatments performed after PIT include treatment on the nose and treatment on areas other than the nose, but do not include the application and irradiation of the bactericide itself. In other words, the application and irradiation of the bactericide are performed on the premise that the main treatment will be performed.

[0056] Patients who undergo PIT include those who have already been found to be carriers of antibiotic-resistant Staphylococcus aureus, so-called resistant bacteria. In this embodiment, the term "antibiotic" does not include the bactericide of this embodiment. PIT may also be applied when a patient is suspected of being a carrier of resistant bacteria. In one example, the resistant bacteria is a multidrug-resistant bacteria. In one example, the resistant bacteria is MRSA. In one example, the MRSA further has complete or incomplete resistance to vancomycin and mupirocin. Vancomycin and mupirocin are types of antibacterial drugs used against MRSA. In one example, the resistant bacteria is VISA or mupirocin-resistant MRSA.

[0057] In one example, a patient may be pre-tested to determine whether or not they are carriers of resistant bacteria, and if the test results indicate that the patient is or is suspected of being a carrier of antibiotic-resistant S. aureus, PIT may be performed.

[0058] For example, a method for testing hospitalized patients is universal surveillance, which tests all hospitalized patients. This is also called "search and destroy" and is carried out in countries such as Finland, Denmark, and the Netherlands. Another method for testing is active surveillance, which tests patients at high risk of carrying resistant bacteria. Another method for testing is passive surveillance, which tests only patients who show signs or symptoms of infection. After any surveillance method is carried out, the disinfectant of this embodiment may be used to perform PIT on carriers or those suspected of being carriers.

[0059] <8. Variations>

[0060] The present invention is not limited to the above embodiment, and can be modified as appropriate without departing from the spirit and scope of the present invention. In the above embodiment, the nasal cavity, particularly the nasal vestibule, of a human patient is used as an example. The site to be sterilized may be the upper respiratory tract including the pharynx or a wound site of the patient.

[0061] MRSA that is present in the umbilicus of patients can also be targeted for sterilization. A disinfectant can be applied to the umbilicus, followed by exposure to light. This method is effective in preventing MRSA infections during and after surgery.

[0062] The application and irradiation treatment of the disinfectant is also useful for preventive and therapeutic applications in non-human animals, including mammals. For example, treatment may be performed on cows to prevent or treat mastitis. When the conjugate is used as a preventive agent for mastitis, it is preferable to apply the conjugate to the surface of the udder and irradiate the udder with light. When the conjugate is used as a therapeutic agent for mastitis, it is preferable to apply the conjugate to the inside of the udder and irradiate the udder with light. EXAMPLES

[0063] We investigated the effect of photoimmunotherapy (aPIT) on bacteria. In the following, regardless of whether the experiment is in vitro or in vivo, the procedure of applying the conjugate to bacteria, irradiating the bacteria with excitation light, and allowing the photosensitizer to act specifically on the bacteria is referred to as aPIT.

[0064] 9. Bacterial strains

[0065] The JKmsSA1 strain was used as the MSSA (methicillin-susceptible Staphylococcus aureus). The JKmsSA1 strain is a preserved strain provided from the bacterial library of the Basic Research Facility of the Center for Integrated Medical Sciences, The Jikei University School of Medicine. The effect of aPIT may be confirmed using other strains, such as the JCM2874 strain described in Non-Patent Document 1, instead of the JKmsSA1 strain.

[0066] The MRSA used was the JKmrSA1 strain, a preserved strain provided from the bacterial library at the Basic Research Facility of the Center for Integrated Medical Sciences, The Jikei University School of Medicine.

[0067] The MU50 strain was used as the VISA. The history and characteristics of the MU50 strain are described in Non-Patent Documents 3 and 4.

[0068] The mupirocin-resistant MRSA strain used was the JKmmrSA1 strain, a preserved strain provided from the bacterial library of the Basic Research Facility at the Center for Integrated Medical Sciences, The Jikei University School of Medicine.

[0069] <10. Conjugates>

[0070] A conjugate was prepared according to the method described in Non-Patent Document 1. First, anti-peptidoglycan mouse monoclonal antibody mAb15704 (clone number Staph12-569.3, IgG3 antibody, QED bioscience) and IR700DX NHS ester (LI-COR) were mixed in a predetermined ratio. It has been confirmed that mAb15704 also reacts with Protein A-negative Staphylococcus aureus. See the product data sheet of QED bioscience in Non-Patent Document 5. A conjugation reaction was performed to obtain the compound mAb15704-IR700. Hereinafter, mAb15704-IR700 may be referred to as SA-IR700. Next, the compound was purified and extracted by gel filtration. The purified extract was subjected to absorbance measurement using a spectrophotometer. The measurement results confirmed that about three molecules of IR700 were bound to one molecule of antibody.

[0071] <11. Colony counting method>

[0072] The bacterial suspension was reacted with mAb15704-IR700. The cell suspension was then irradiated with near-infrared light with a wavelength peak of 690 nm using an LED light as excitation light. After irradiation, the cell suspension sample was vortexed. The cell suspension was serially diluted, and each concentration of the cell suspension was dropped onto a plate medium. The bacteria were cultured at 37°C for 24 hours. The bactericidal effect of aPIT was evaluated based on the number of colonies on the plate medium. Unless otherwise noted, the plate medium was nutrient agar medium.

[0073] <12.Results>

[0074] Figure 3 shows the results of colony counting after aPIT on the MSSA JKmsSA1 strain using 0.01-1 μg of conjugate per test. The excitation light dose was 5 J / cm. 2The number of bacteria that formed colonies decreased as the amount of conjugate increased. This indicates that the bactericidal effect increases depending on the amount or concentration of the conjugate. ** indicates P<0.01. cfu stands for colony forming unit. The graph shows the average of three independent trials. Error bars indicate standard deviation. The same applies below.

[0075] Figure 4 shows that for MSSA, the maximum allowable range is 5-45 J / cm per test. 2 aPIT was performed with an excitation light irradiation dose of 100 μg. The results of subsequent colony counting are shown below. The amount of conjugate was 0.3 μg. The number of bacteria that formed colonies decreased as the irradiation dose increased. This shows that the bactericidal effect increases depending on the irradiation dose of the excitation light.

[0076] Figure 5 shows the results of aPIT performed on MSSA under various control conditions, followed by colony counting. The second graph from the left shows the results when 1 μg of conjugate was reacted with a bacterial suspension after adding a large excess of 10 μg of unconjugated antibody. Compared to the sixth graph in Figure 3, the irradiation dose of excitation light was increased to 15 J / cm. 2 No bactericidal effect was observed, even when the concentration was increased to 100 mM. This is believed to be the result of antagonism between the conjugate and the antibody before conjugation, indicating that the bactericidal effect is dependent on the antibody moiety of the conjugate.

[0077] The third graph from the left in Figure 5 shows the results when 4 μg of conjugate was added but no excitation light was applied. The fourth graph from the left shows the results when 100 J / cm 2 The results are shown for the case where the cells were irradiated with excitation light of 1000 nm but no conjugate was added. In either case, no bactericidal effect was observed. This shows that the bactericidal effect cannot be obtained unless the addition of the conjugate to the cell suspension and the irradiation of the excitation light are synchronized.

[0078] Figure 6 shows the results of aPIT performed on resistant bacteria MRSA, VISA, and mupirocin-resistant MRSA, followed by colony counting. 2 μg of conjugate was added per test for each resistant bacteria. In addition, 90 J / cm 2 The conjugate and excitation light were then irradiated with an excitation light of 1000 nm. No colonies were formed by the conjugate and excitation light. This indicates that the bactericidal effect is exerted not only on sensitive bacteria but also on resistant bacteria.

[0079] FIG. 7 shows the results of aPIT performed on a mixture of MRSA and heterogeneous bacteria, and then culturing the mixture on a plate medium. The heterogeneous bacteria used was the JCM2414 strain of Staphylococcus epidermidis. 2 μg of the conjugate was added per test. MSA selection medium and OPA II selection medium were used as the plate medium. Both MRSA and Staphylococcus epidermidis formed colonies on the MSA selection medium. Only MRSA formed colonies on the OPA II selection medium, but Staphylococcus epidermidis did not form colonies. According to the SPECIFICATION SUMMARY of the product data sheet, the anti-peptidoglycan mouse monoclonal antibody mAb15704 is also said to recognize the peptidoglycan of Staphylococcus epidermidis. However, according to the inventors' observations, the conjugate synthesized from mAb15704 did not show sufficient binding to Staphylococcus epidermidis compared to Staphylococcus aureus, and did not show the same bactericidal effect against Staphylococcus epidermidis as against Staphylococcus aureus even when irradiated with light.

[0080] In the upper photographs of Figure 7, the light and dark are inverted. From the left, the plates are irradiated with excitation light at 0, 5 and 40 J / cm. 2 The bacteria subjected to aPIT formed colonies on MSA selective medium even when the dose of irradiation was increased, but formed fewer colonies on OPA II selective medium as the dose of irradiation was increased.

[0081] In the lower part of Figure 7, evaluation was performed using the colony counting method. The irradiation dose of excitation light was set to 0, 5, and 40 J / cm. 2MRSA treated with aPIT was less likely to form colonies as the dose of aPIT increased. The number of colonies of Staphylococcus epidermidis treated with aPIT did not change even with increasing doses of aPIT. Therefore, it was shown that the bactericidal effect is specific to Staphylococcus aureus.

[0082] In Figures 8 and 9, MRSA was colonized in the nasal cavity of cotton rats. Cotton rats are an animal species with low resistance to infection. Therefore, cotton rats are a suitable model organism for creating a bacterial carriage state in the nasal cavity. aPIT was performed on the nasal cavity of cotton rats.

[0083] In Figure 8, 5 μg of SA-IR700 per mouse was administered by dropwise application to the colonization site, and then 0, 10, and 50 J / cm were applied to the colonization site. 2 The cotton rat's nose was small, so near-infrared light was irradiated from the outside of the nose toward the nasal cavity. Bacteria were removed from the animals by cutting the nasal cavity. Bacterial flora was removed from the nasal cavity and cultured on OPA II selective medium. The efficacy of aPIT was evaluated by colony counting.

[0084] The bottom row of Figure 8 shows the number of MRSA colonies, and the top row shows photographs of the bacterial cultures corresponding to each graph. The light and dark are inverted in the photographs. As the amount of irradiation to the colonized area increased, the number of colonies decreased. This shows that aPIT can also achieve a bactericidal effect against the bacterial flora on the surface of the body.

[0085] The bottom row of Figure 9 shows microscopic images of nasal tissue sections after aPIT was performed on cotton rats colonized with MRSA. The top row shows a control experiment in which aPIT was not performed. In both cases, the nasal mucosa tissue was healthy.

[0086] In Figure 10, aPIT was performed on MSSA using 1 μg of SA-IR700 conjugate per test. The excitation light dose was 15 J / cm. 2However, in the experiment shown in Figure 10, MSSA was in the logarithmic phase of growth, i.e., at the plateau. In contrast, in the experiments shown in Figures 3 to 5, it was in the growth phase. Colony counting was performed after aPIT. The bactericidal effect of aPIT was observed even against MSSA in the logarithmic phase.

[0087] In Figure 11, aPIT was performed on MSSA during culture in various media using 1 μg of SA-IR700 conjugate per test. From the left, the media are TSB (tryptone soy broth) medium, TSB medium supplemented with glucose (TSBG medium), BHI (brain heart infusion) medium, and LB (Luria-Bertani medium). Colony counting was performed after aPIT. The bactericidal effect of aPIT was observed even after culture in various media.

[0088] In Figure 12, MSSA was suspended in defibrinated horse blood and then subjected to aPIT with SA-IR700 conjugate at a dose of 4 μg per test. Colony counting was performed after aPIT. The bactericidal effect of aPIT was observed even in nutrient-rich blood.

[0089] In Figure 13, 1x10 per test 7CFU of MSSA were treated with 30 μg of SA-IR700 conjugate per test. S. aureus was added to 3T3 fibroblast cells and co-cultured at 37°C under 5% CO2 atmosphere for 30 min. 3T3 fibroblast cells were preliminarily grown to confluence on a 35 mm diameter culture dish. Cells were rinsed three times with 1 ml of RPMI medium (Roswell Park Memorial Institute medium). Extracellular S. aureus was lysed with 1 ml of 1 mg / ml lysostaphin solution (Fujifilm Wako Pure Chemicals) for 30 min. Cells were rinsed five times with 1 ml of RPMI medium and then irradiated with near infrared light. Cells were homogenized and serially diluted, then cultured on agar medium and colony counting was performed. The graph on the left shows the case where excitation light was not irradiated. It shows that S. aureus treated with SA-IR700 conjugate was taken up by cells and escaped eradication by lysostaphin. This indicates that treatment with the SA-IR700 conjugate alone is not enough to prevent S. aureus from parasitising cells. In contrast, S. aureus was eradicated when exposed to excitation light. This indicates that even if S. aureus escapes into cells after treatment with the SA-IR700 conjugate, the killing effect of excitation light reaches inside the cells.

[0090] In Figure 14, another conjugate was prepared using anti-peptidoglycan mouse monoclonal antibody mAb15702 (clone number Staph11-232.3, IgG3 antibody, QED bioscience). (a) The second and fourth graphs from the left show the results of aPIT performed with the antibody of clone number Staph12-569.3 mentioned above.

[0091] In Figure 14, another anti-peptidoglycan mouse monoclonal antibody, clone number Staph11-232.3, IgG3 antibody, QED bioscience, was used. (b) The third and fifth graphs from the left show the results of aPIT performed with the antibody clone number Staph11-232.3. There were slight differences in the bactericidal effect of aPIT depending on the antibody clone. However, when the irradiation dose was 15 J / cm2 from 45 J / cm 2 The bactericidal effect of aPIT was observed for all antibody clones by increasing the concentration of IgG1 to IgG1. *** indicates P<0.001.

[0092] In Figure 15, the interaction between Protein A and Fc was evaluated. aPIT was performed with 2 μg of Tra-IR700 conjugate per test. Tra-IR700 is a conjugate in which IR700 is bound to the CH2 region of trastuzumab. Trastuzumab is a humanized monoclonal antibody that contains a framework region derived from human IgG1 and a complementary antigen recognition region of a mouse monoclonal anti-HER2 antibody. Since Tra-IR700 is an antibody specific to human HER2, it is considered that Tra-IR700 does not undergo an antibody-antigen reaction with bacteria.

[0093] In FIG. 15, the wild-type RN4220 strain was used as Staphylococcus aureus. A protein A-deficient strain (Δspa) was also used. For other points, aPIT and colony counting were performed as described above. Regardless of the presence or absence of protein A, the bactericidal effect of aPIT via the Fc region of trastuzumab was not observed. In contrast, PIT due to the interaction between protein A and the Fc region was successful in Patent Document 1. However, the results shown in FIG. 15 suggest that aPIT due to the interaction between protein A and the Fc region may not be a reliable method. In addition, the results of the experiment on the protein A-deficient strain indicate that the aPIT of this embodiment is effective against protein A-negative Staphylococcus aureus.

[0094] In Figure 16, cotton rats were colonized with MRSA in the same manner as in Figure 8, and then the colonized site was irradiated with near-infrared light only without administering the conjugate. In Figure 17, 2 μg of conjugate was administered per test, and the colonized site was irradiated with near-infrared light. Unlike Figure 8, the bacteria in the bacterial flora were cultured on a non-selective plate medium and the colony count method was performed. This bacterial flora is thought to be the original bacterial flora that the cotton rats had before they were infected with MRSA.

[0095] As shown in Figure 8, MRSA was eradicated by aPIT. However, as shown in Figure 17, other bacteria in the bacterial flora were not killed by aPIT. This indicates that aPIT using SA-IR700 acts specifically on Staphylococcus aureus and does not affect the growth of other bacteria in the bacterial flora. Furthermore, as shown in Figure 16, irradiation with excitation light did not reduce the number of live bacteria in the bacterial flora.

[0096] In Figure 18, mice were treated with 10 8 CFU of MRSA was administered intraperitoneally. The MRSA was treated with 50 μg of SA-IR700 per mouse prior to intraperitoneal administration. Then, 100 J / cm 2 Mice were irradiated with near-infrared light from outside the body. Those not irradiated were designated aPIT(-). All mice that received aPIT survived. All mice that did not receive aPIT died. It is believed that these mice developed peritonitis. This shows that contact between SA-IR700 and S. aureus alone is not enough to prevent disease. It has been shown that a combination of administration of SA-IR700 and irradiation with excitation light is necessary for the treatment and prevention of diseases caused by S. aureus.

[0097] In Figure 19, MRSA was statically cultured for 16 hours in tryptic soy culture medium with 1% glucose added. This allowed MRSA to form a biofilm on the culture dish. The biofilm was removed from the culture dish with a scraper, and the biofilm was collected along with MRSA. This biofilm was washed with RPMI medium. MRSA was treated with SA-IR700 and then irradiated with near-infrared light. The irradiated MRSA was suspended in RPMI medium and serially diluted. MRSA was then seeded on an agar medium to allow MRSA to form colonies. The number of colonies was counted.

[0098] The graph in Figure 19 shows the percentage of viable bacteria. In the figure, both (-) and (+) samples were treated with 4 μg of SA-IR700 per test. Only the (+) sample was treated with 30 J / cm 2We performed aPIT by irradiating the area with near-infrared light.

[0099] As shown in Figure 19, a bactericidal effect was observed in the irradiated (+) sample compared to the non-irradiated (-) sample. From the above, it was found that aPIT can achieve a bactericidal effect even when antibiotic-resistant Staphylococcus aureus forms a biofilm.

[0100] This application claims priority based on Japanese Patent Application No. 2019-103573, filed on June 3, 2019, the disclosure of which is incorporated herein in its entirety. [Explanation of symbols]

[0101] 10 Conjugates 11 Antibodies 12 Photosensitizers 13. Linker 14 Epidermis 15 Bacterial flora 16 Target bacteria 17 Target molecules 20 Excitation Light 21 Photosensitization 24 Nasal vestibule 25 Nasal cavity 26 Nostrils 27 Infrared Therapy Device

Claims

1. A bactericide comprising a conjugate in which a photosensitizer is bound to an antibody specific for Staphylococcus aureus, In the conjugate, the Staphylococcus aureus-specific antibody targets peptidoglycan of Staphylococcus aureus, and the absorption wavelength range of the photosensitizer overlaps with a wavelength range from red light to near infrared light; In the method, the site to which the disinfectant is applied is irradiated with excitation light having a wavelength range from red light to near infrared light. Disinfectant.

2. The photosensitizer has a silicon phthalocyanine complex moiety. The disinfectant according to claim 1.

3. The silicon phthalocyanine complex moiety is IRDye 700DX represented by the following formula: 【Chemistry 1】 In the formula, the two oxygen atoms coordinated to the Si atom at the center of the complex may be in trans positions across the phthalocyanine skeleton, or may be in cis positions on one side of the phthalocyanine skeleton. The disinfectant according to claim 2.

4. In its usage, The site to which the disinfectant is applied is at least one selected from the group consisting of the nasal cavity, the upper respiratory tract, the navel, a wound site, and an inflammation site; The disinfectant according to claim 1.

5. In its usage, the site is a site on a patient to be treated; The application and irradiation are performed before the patient receives the treatment; The treatment includes treatment for the area and treatment for areas other than the area, but does not include the treatment by application and irradiation itself. The disinfectant according to claim 4.

6. In its usage, The patient is or is suspected of having an infection with Staphylococcus aureus, including antibiotic-resistant strains, or is or is suspected of being a carrier of Staphylococcus aureus, including antibiotic-resistant strains. The disinfectant according to claim 5.

7. In its usage, the antibiotic resistant strain is MSSA, MRSA, VISA, or mupirocin resistant Staphylococcus aureus; The disinfectant according to claim 6.

8. In its usage, The antibiotic-resistant Staphylococcus aureus forms a biofilm. The disinfectant according to claim 6.

9. In its usage, Preliminarily testing whether the patient is a carrier of an antibiotic-resistant strain; performing said application and irradiation if the test results in the patient being or being suspected of being infected with an antibiotic-resistant strain, or being or being suspected of being a carrier of an antibiotic-resistant strain; The disinfectant according to claim 5.

10. In its usage, the site to which the disinfectant is applied is a site on a patient receiving treatment; The bactericide is applied in an amount of 1 μg / kg or more per patient; The excitation light is 1 J / cm ２ More than 300J / cm ２ The irradiation is performed at a dose of The disinfectant according to claim 1.

11. A bactericide comprising a conjugate in which a photosensitizer is bound to an antibody specific for Staphylococcus aureus, In the conjugate, the Staphylococcus aureus-specific antibody targets peptidoglycan of Staphylococcus aureus, and the absorption wavelength range of the photosensitizer overlaps with a wavelength range from red light to near infrared light; In the method, the disinfectant is applied to an economic animal including a cow, and the economic animal is irradiated with excitation light having a wavelength range from red light to near infrared light. Bactericides for the prevention or treatment of infectious diseases in economic animals, including cattle.

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