Pharmaceutical composition for treatment and / or prevention of pulmonary aspergillosis

Abstract

The present invention addresses the problem of providing a pharmaceutical composition for treating and / or preventing pulmonary aspergillosis, and use of a compound for enhancing the antifungal effect of a triazole-based antifungal agent.　This pharmaceutical composition for treating and / or preventing pulmonary aspergillosis according to the present invention is obtained by combining a triazole-based antifungal agent and luliconazole.

Description

Pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis

[0001] The present invention relates to a pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis.

[0002] Pulmonary aspergillosis is caused by infection with the fungus Aspergillus, and is roughly classified into invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis. Among these, chronic pulmonary aspergillosis is a chronic infectious disease that mainly occurs against the background of immunodeficiency and respiratory underlying diseases. More than 3 million people worldwide are affected, and it has a poor prognosis with a 5-year survival rate of 50-60%. The main causative bacterium of pulmonary aspergillosis is Aspergillus fumigatus (A. fumigatus), and the increase in A. fumigatus strains resistant to triazole antifungal drugs, which are the first-choice drugs, has become a problem in recent years. In infectious diseases caused by resistant strains, there are reports that the lethality rate exceeds 90%. In the Fungal Priority Pathogens List published by the WHO (World Health Organization) in 2022, A. fumigatus is ranked in the Critical Priority Group and is positioned as having the highest priority for research and development. Patent Document 1 discloses a chimeric antigen receptor (CAR) polypeptide that includes an antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and at least one co-stimulatory signaling region, and the antigen-binding domain binds to an ASP antigen, for the purpose of providing a novel therapy effective for preventing or treating Aspergillus-related diseases and disorders. Further, Patent Document 2 discloses a compound having at least two fucose moieties for the purpose of providing a compound to be used as a drug for the prevention and / or treatment of infectious diseases caused by Aspergillus species.

[0003] Triazole antifungal agents exert their antifungal activity by targeting 14-α-demethylase (CYP51) in the sterol biosynthesis pathway and inhibiting ergosterol synthesis. A. fumigatus has two isotypes of 14-α-demethylase, encoded by CYP51A and CYP51B, which act complementaryly in the ergosterol pathway. Itraconazole, voriconazole, and fluconazole bind to both CYP51A and CYP51B, but their affinity for CYP51B is reported to be higher than that for CYP51A (Non-Patent Literature 1).

[0004] Non-patent document 2 reports that oral or intravenous administration of luliconazole extended the survival period in a rat model infected with A. fumigatus. Non-patent document 3 reports that luliconazole monotherapy showed antifungal activity against azole-resistant A. fumigatus at concentrations of 0.001–0.02 μg / mL, and that this effect was almost equivalent across various azole-resistant A. fumigatus strains, regardless of the azole resistance mechanism. Furthermore, non-patent document 4 reports that the combined use of itraconazole and luliconazole showed a synergistic antifungal effect against dermatophytes with point mutations in the squalene epoxidase gene.

[0005] Patent Document 3 discloses compounds as defined herein that are useful in the treatment of mycoses, compositions containing the same, and their use in therapeutic methods, and describes a pharmaceutical in which a triazole compound is combined with a second active ingredient. Patent Document 4 describes an antifungal composition comprising at least one antifungal agent, at least one fatty acid or ester thereof, and optionally one or more excipients, wherein the fatty acid has a carbon chain length in the range of C-1 to C-14, and the composition has synergistic antifungal activity. Non-Patent Document 5 investigates the drug susceptibility of luliconazole to triazole-resistant A. fumigatus strains, disclosing that many (13 / 16) of the triazole-resistant A. fumigatus strains have a CYP51A mutation, and that luliconazole exhibits strong antibacterial activity against resistant strains. Non-patent document 6 discloses that the expression of the Cyp51A protein increases in the absence of Cyp51B, and vice versa, suggesting that one protein has the ability to compensate for the deficiency of the other at the protein level, and that cyp51A deletion is more sensitive to voriconazole than cyp51B deletion, and that voriconazole is more active against Cyp51B.

[0006] Special table No. 2023-520343 Publication Special table No. 2020-507660 Publication Special table No. 2017-536382 Publication Special table No. 2019-521993

[0007] Warrilow A. et al., Antimicrob Agents Chemother., 54(10), 4225-4234, 2010. Niwano Y., et al., Int J Antimicrob Agents, 12(3), 221-228, 1999. Furnica D., et al., J Fungi (Basel), 8(4), 350, 2022. Sardana K., et al., Antimicrob Agents Chemother., 65(8), e0032121. Japanese Journal of Chemotherapy 66, Supp-A, 273, O2-104, 2018. Roundtree et al. Antimicrob Agents Chemother. 64 (10) (2020)

[0008] Although Patent Documents 1 and 2 describe new therapeutic agents, their effectiveness against triazole-resistant strains has not been investigated. The present invention aims to provide pharmaceutical compositions for the treatment and / or prevention of pulmonary aspergillosis, and for the use of compounds to enhance the antifungal effect of triazole antifungal agents.

[0009] As a result of diligent research, the present inventors have found that the above problems can be solved by a pharmaceutical composition comprising a triazole antifungal agent and luliconazole. The present invention includes the following embodiments.

[0010] <1> A pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis, comprising a triazole antifungal agent and luliconazole. <2> The pharmaceutical composition according to <1>, wherein the triazole antifungal agent is at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole, as well as their salts and solvates. <3> The pharmaceutical composition according to <1> or <2>, wherein the pulmonary aspergillosis is chronic pulmonary aspergillosis. <4> The pharmaceutical composition according to any one of <1> to <3>, wherein the pulmonary aspergillosis is treatment-resistant to the triazole antifungal agent. <5> The pharmaceutical composition according to any one of <1> to <4>, wherein the causative agent of pulmonary aspergillosis is Aspergillus fumigatus. <6> Use of luliconazole to enhance the antifungal effect of a triazole antifungal agent. <7> A method for treating and / or preventing pulmonary aspergillosis, comprising the step of administering a pharmaceutical composition comprising a triazole antifungal agent and luliconazole to a target subject as needed. <8> The method for treating and / or preventing pulmonary aspergillosis according to <7>, wherein the triazole antifungal agent is at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole, as well as their salts and solvates. <9> The method for treating and / or preventing pulmonary aspergillosis according to <7> or <8>, wherein the pulmonary aspergillosis is chronic pulmonary aspergillosis. <10> The method for treating and / or preventing pulmonary aspergillosis according to any one of <7> to <9>, wherein the pulmonary aspergillosis is treatment-resistant to triazole antifungal agents. <11> The method for treating and / or preventing pulmonary aspergillosis according to any one of <7> to <10>, wherein the causative agent of the pulmonary aspergillosis is Aspergillus fumigatus.<12> Use of a combination of a triazole antifungal agent and luliconazole for the treatment and / or prevention of pulmonary aspergillosis. <13> Use according to <12>, wherein the triazole antifungal agent is at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole, as well as their salts and solvates. <14> Use according to <12> or <13>, wherein the pulmonary aspergillosis is chronic pulmonary aspergillosis. <15> Use according to any one of <12> to <14>, wherein the pulmonary aspergillosis is treatment-resistant to a triazole antifungal agent. <16> The use according to any one of <12> to <15>, wherein the causative agent of pulmonary aspergillosis is Aspergillus fumigatus. <17> The use of a combination of a triazole antifungal agent and luliconazole for the manufacture of a pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis. <18> The use according to <17>, wherein the triazole antifungal agent is at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole, as well as their salts and solvates. <19> The use according to <17> or <18>, wherein the pulmonary aspergillosis is chronic pulmonary aspergillosis. <20> The use according to any one of <17> to <19>, wherein the pulmonary aspergillosis is pulmonary aspergillosis resistant to triazole antifungal agents. <21> The use according to any one of <17> to <20>, wherein the causative agent of the pulmonary aspergillosis is Aspergillus fumigatus.

[0011] The present invention provides pharmaceutical compositions for the treatment and / or prevention of pulmonary aspergillosis, and the use of compounds for enhancing the antifungal effect of triazole antifungal agents.

[0012] Figure 1 shows the results of silkworm infection experiment-1. Figure 1 shows the results of silkworm infection experiment-2.

[0013] The present invention will be described in detail below using exemplary embodiments as examples, but the present invention is not limited to the embodiments described below. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in the practice of the present invention. Furthermore, all publications and patents cited herein in connection with the invention described herein constitute part of this specification, for example, as indicating methods, materials, and other things that can be used in the present invention.

[0014] In this specification, the notation "A to B" indicating a numerical range means a numerical range that includes the endpoints A and B. The same applies to "A to B".

[0015] [Pharmaceutical Composition for the Treatment and / or Prevention of Pulmonary Aspergillosis] The pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis of this embodiment (hereinafter also simply referred to as "the pharmaceutical composition of this embodiment") comprises a combination of a triazole antifungal agent and luliconazole. The pharmaceutical composition of this embodiment is effective in the treatment and / or prevention of pulmonary aspergillosis, and in particular, it has been found that a superior therapeutic and / or preventive effect is exhibited when a triazole antifungal agent and luliconazole are combined against pulmonary aspergillosis resistant to triazole antifungal agents (pulmonary aspergillosis with resistance to triazole antifungal agents). Triazole antifungal agents are known to exert antifungal activity against A. fumigatus by inhibiting Cyp51A and Cyp51B, which contribute to the intermediate reaction of the ergosterol production pathway of A. fumigatus. After diligent research, the inventors discovered that in A. fumigatus strains resistant to triazole antifungal agents, the inhibitory effect of triazole antifungal agents on Cyp51A was insufficient, potentially leading to the acquisition of resistance. The inventors searched for compounds with inhibitory activity against Cyp51A and found that luliconazole, an imidazole antifungal agent, possesses this inhibitory activity. Combining a triazole antifungal agent with luliconazole showed antifungal activity against azole-sensitive A. fumigatus strains at doses lower than those of triazole antifungal agents alone or luliconazole alone. Surprisingly, the combination of triazole antifungal agents and luliconazole showed synergistic antifungal activity against a variety of A. fumigatus strains resistant to triazole antifungal agents. These results indicate that the combination of triazole antifungal agents and luliconazole is effective in treating and / or preventing pulmonary aspergillosis, particularly pulmonary aspergillosis resistant to triazole antifungal agents.

[0016] <Triazole Antifungal Agents> The triazole antifungal agents are preferably at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isavuconazole, and posaconazole, as well as their salts and solvates. More preferably, they are at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, and fosfluconazole, as well as their salts and solvates. Even more preferably, they are at least one selected from the group consisting of itraconazole and voriconazole, as well as their salts and solvates. Currently, the standard treatment involves using itraconazole or voriconazole as the first-line drug, with voriconazole being particularly frequently used. The salts mentioned above are pharmaceutically acceptable salts, and examples include acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, mineral salts such as nitrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, trifluoromethanesulfonate, organic salts such as oxalate, tartrate, citrate, maleate, succinate, acetate, trifluoroacetate, benzoate, mandelate, ascorbate, lactate, gluconate, and malate; amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate; or base addition salts such as inorganic salts or ammonium salts such as lithium salt, sodium salt, potassium salt, calcium salt, and magnesium salt, or salts with organic bases such as triethylamine salt, diisopropylamine salt, cyclohexylamine salt, and N-methyl-D-glucamine salt. Note that hydrated salts are included in the definition of salt.Solvates are pharmaceutically acceptable solvates, and there are no particular restrictions on their selection; they can be appropriately chosen depending on the purpose, such as hydrates and ethanolates.

[0017] The structures of itraconazole, voriconazole, fluconazole, phosfluconazole, phosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole are as follows.

[0018]

[0019] <Luliconazole> Luliconazole is an imidazole antifungal agent having the following chemical formula. Currently, no pharmaceutically acceptable salts and solvates are known, but salts and / or solvates may be used.

[0020]

[0021] The pharmaceutical composition of this embodiment comprises a triazole antifungal agent and luliconazole. The pharmaceutical composition may be a single preparation containing the triazole antifungal agent and luliconazole, or it may be separate preparations containing the triazole antifungal agent and luliconazole, respectively, and is not particularly limited. When these compounds are formulated separately to form two preparations, they may be administered simultaneously or at very short intervals. For example, it is preferable to include a note in the package insert or sales brochure of the commercially available drug stating that they should be used in combination. It is also preferable to formulate these active ingredients separately to form a kit consisting of two preparations. The triazole antifungal agent and luliconazole may be administered simultaneously or separately, and are not particularly limited. Furthermore, the administration routes of triazole antifungal agents and luliconazole are not particularly limited; they may be administered orally, or parenterally (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, transdermal, pulmonary, or respiratory). The administration routes of triazole antifungal agents and luliconazole may be the same or different.

[0022] The dosage form of the pharmaceutical composition in this embodiment is not particularly limited, and examples include oral dosage forms such as tablets, pills, powders, granules, capsules, liquids, emulsions, syrups, and aerosols; inhalants such as aerosols; topical preparations; patches, suppositories, and injections. Furthermore, for dosage form, it may be administered together with pharmaceutically or pharmaceutically acceptable additives. As such additives, carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions may be used, and if necessary, commonly used binders, disintegrants, lubricants, coatings, sugar coatings, pH adjusters, solvents, or aqueous or non-aqueous solvents may be used. Specifically, examples include water, lactose, dextrose, fructose, sucrose, sorbitol, mannitol, polyethylene glycol, propylene glycol, potato starch, corn starch, gum, gelatin, alginate, calcium silicate, calcium phosphate, cellulose, liquid sugar, methylcellulose, polyvinylpyrrolidone, alkyl p-hydroxybenzoate, talc, stearic acid, magnesium stearate, agar, pectin, acacia gum, glycerin, sesame oil, olive oil, soybean oil, cocoa butter, ethylene glycol, low viscosity hydroxypropylcellulose (HPC-L), microcrystalline cellulose, carboxymethylcellulose (CMC), sodium carboxymethylcellulose (CMC-Na), dextrin, cyclodextrin, and other commonly used substances. Hydrates of the above compounds may also be used. Triazole antifungal agents and luliconazole may be prepared as liposomal formulations.

[0023] The target of administration of the pharmaceutical composition of this embodiment is preferably a mammal that is suffering from pulmonary aspergillosis or is highly likely to suffer from pulmonary aspergillosis. The mammals include humans; non-human primates including chimpanzees, other apes and monkey species; livestock such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; and small animals or laboratory animals including rodents such as mice, rats and guinea pigs. Among these, humans are preferred. Patients at high risk of developing pulmonary aspergillosis include those with hematological malignancies, those receiving immunosuppressive therapy for autoimmune diseases, immunocompromised patients such as HIV-infected individuals (especially those at high risk of developing invasive pulmonary aspergillosis or chronic pulmonary aspergillosis), those with underlying lung diseases such as chronic obstructive pulmonary disease (COPD), old pulmonary tuberculosis, and cystic fibrosis (especially those at high risk of developing chronic pulmonary aspergillosis), and adult patients with bronchial asthma (especially those at high risk of developing allergic bronchopulmonary aspergillosis). In such cases, the drug is administered to prevent invasive pulmonary aspergillosis.

[0024] The disease targeted for treatment and / or prevention by the pharmaceutical composition of this embodiment may be any pulmonary aspergillosis selected from the group consisting of invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis, but chronic pulmonary aspergillosis is particularly preferred. Chronic pulmonary aspergillosis may be treated with triazole antifungal agents over a long period of time, and many patients tend to acquire resistance to triazole antifungal agents. Therefore, in one embodiment, the patients targeted for treatment with the pharmaceutical composition of this embodiment are patients with pulmonary aspergillosis (preferably chronic pulmonary aspergillosis) that is resistant to triazole antifungal agents and has a history of treatment with triazole antifungal agents. Furthermore, while Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terresu, and Aspergillus tubingensis are among the causative agents of pulmonary aspergillosis, this treatment is particularly useful for the treatment and / or prevention of pulmonary aspergillosis caused by Aspergillus fumigatus, which accounts for the largest number of patients. Treatment resistance to triazole antifungal agents in pulmonary aspergillosis occurs because the causative agent acquires resistance to triazole antifungal agents. The mechanisms of triazole resistance in Aspergillus fumigatus include, but are not limited to, 1) cyp51A point mutations: decreased affinity of the cyp51A protein to triazole antifungal drugs; 2) cyp51A tandem repeats (TR): increased expression of cyp51A; 3) hmg1 point mutations; and 4) enhanced efflux pump activity (Hagiwara et al., Frontiers in microbiology 2016; 7: 1382). In Japan, cyp51A point mutations have been reported to be the most common mechanism of resistance (Takeda et al., Med Mycol 2021; 59(4): 327-334).Many cyp51A and hmg1 point mutations that confer triazole resistance have been reported, and the point mutations in the cyp51A and hmg1 mutant strains used in the examples described later can be cited as examples. Furthermore, it has been reported that approximately 80% of Aspergillus tubingensis strains are poorly susceptible to existing triazoles (see A. Hashimoto, et al. Antimicrob Agents Chemother. 2017; 61 (8). DOI: 10.1128 / aac.02583-16), and the pharmaceutical composition of the present invention may also be useful for the treatment and / or prevention of pulmonary aspergillosis caused by Aspergillus tubingensis. Moreover, it is preferable that the pulmonary aspergillosis targeted for treatment and / or prevention is pulmonary aspergillosis resistant to triazole antifungal agents. According to the present invention, by using a triazole antifungal agent in combination with luliconazole, excellent therapeutic and / or preventive effects can be observed even for pulmonary aspergillosis that is resistant to triazole antifungal agents.

[0025] "Treatment" is used to mean delaying or stopping the progression of a disease or condition, as well as improving and curing the disease or condition. "Prevention" is used to mean suppressing the onset of a disease or condition, and reducing its incidence. Since "pulmonary aspergillosis" is a disease that is difficult to definitively diagnose, administering the drug to a person suspected of having pulmonary aspergillosis also constitutes "treatment." Because it takes a long time to isolate the causative fungus from patients with pulmonary aspergillosis resistant to triazole antifungal drugs and to identify the mechanism of triazole resistance, clinically, it is necessary to "treat" patients with pulmonary aspergillosis or those suspected of having pulmonary aspergillosis by administering the pharmaceutical composition of this embodiment before the causative fungus or the mechanism of triazole resistance can be identified.

[0026] There are no particular restrictions on the aforementioned dosage, and it can be appropriately selected considering various factors such as the method of administration, dosage form, type of causative fungus, age, sex, weight, constitution, severity of the disease, and whether or not other pharmaceuticals or drugs containing other active ingredients are being administered. For example, when administering triazole antifungal drugs to humans, for example, it can be administered within the range of 0.5 mg / kg to 100 mg / kg per adult per day. The same applies to luliconazole; the preferred dosage is the same. There are no particular restrictions on the timing, interval, or duration of administration, and these can be appropriately selected according to the purpose.

[0027] For adult human patients with chronic pulmonary aspergillosis, voriconazole is administered, for example, 6 mg / kg twice daily by intravenous infusion on the first day, and 4 mg / kg twice daily by intravenous infusion or oral administration from the second day onward. Itraconazole is administered, for example, 200 mg twice daily by intravenous infusion or oral administration for the first two days, and 200 mg once daily by intravenous infusion or oral administration from the third day onward. Furthermore, for maintenance therapy in adult human patients with chronic pulmonary aspergillosis, voriconazole is administered, for example, 200 mg twice daily (between meals) by oral administration, and itraconazole is administered, for example, 200 mg of itraconazole solution once daily (on an empty stomach) by oral administration, or 200 mg of itraconazole capsules twice daily (immediately after meals) by oral administration. The dosage should be adjusted according to body weight.

[0028] In addition to the pharmaceutical compositions described above, the following embodiments are also disclosed in the present invention. [Use of Luliconazole] This embodiment also discloses the use of luliconazole to enhance the antifungal effect of triazole antifungal agents. It is preferably used to enhance the antifungal effect of triazole antifungal agents used to treat pulmonary aspergillosis, particularly chronic pulmonary aspergillosis, and more preferably to enhance the antifungal effect of triazole antifungal agents used to treat pulmonary aspergillosis resistant to triazole antifungal agents. The antifungal effect can be evaluated by the method described in the examples. It is also used to enhance the therapeutic and / or preventive effect on the target.

[0029] Furthermore, the following embodiments are also included: • A method for treating and / or preventing pulmonary aspergillosis using a pharmaceutical composition comprising a triazole antifungal agent and luliconazole. • A method for treating and / or preventing pulmonary aspergillosis, comprising the step of administering a pharmaceutical composition comprising a triazole antifungal agent and luliconazole to a target recipient as needed. • Use of a triazole antifungal agent and luliconazole in the manufacture of therapeutic and / or prophylactic agents for pulmonary aspergillosis. • Use of a combination of a triazole antifungal agent and luliconazole for the treatment and / or prevention of pulmonary aspergillosis. • A combination of a triazole antifungal agent and luliconazole for use in the treatment and / or prevention of pulmonary aspergillosis. • Luliconazole for use in a pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis in combination with a triazole antifungal agent.

[0030] The present invention will be described in more detail by the following examples, but these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0031] [A. fumigatus, A. niger, A. tubingensis strains] All clinical strains are stored at the National BioResource Project (NBRP) at the Chiba University Medical Mycology Research Center (http: / / www.nbrp.jp / ). Each strain was pre-cultured in Potato Dextrose Agar medium (Beckton Dickinson and Company, Sparks, MD, USA) for the experiments described below, and spores were collected and diluted to the required concentration for use.

[0032] [Antifungal Drugs] The itraconazole and voriconazole powders used in the checkerboard method described below were purchased from Sigma (St. Louis, MO, USA). The itraconazole powder used in the silkworm infection experiment was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. In addition, luliconazole used in both the checkerboard method and the silkworm infection experiment was purchased from Tokyo Chemical Industry Co., Ltd. Each drug was dissolved in dimethyl sulfoxide (DMSO) and stored frozen as a stock solution.

[0033] [Checkerboard Method] Drug interactions were determined using the checkerboard method based on the CLSI (Clinical and Laboratory Standards Institute) M38-Ed3 protocol. Specifically, itraconazole (ITCZ) or voriconazole (VRCZ) (50 μL, 4x concentration) was placed at the specified concentration in each well of 96-well round-bottom microtiter plates (Violamo, Osaka, Japan), and luliconazole (LLCZ) (50 μL, 4x concentration) was mixed into each well at the specified concentration. For ITCZ ​​and VRCZ, a 2x dilution series was prepared within the concentration range of 0.12 to 16 μg / mL, and each concentration of the drug, including a control (solvent only), was added to an 8x9 cell. Spore suspension (100 μL) of each bacterial strain was added to each well, and the final spore concentration was 2.5 × 10⁶. ４ The concentration was adjusted to CFU / mL. After culturing this microplate at 35°C for 48 hours, the synergistic effect of both agents on each strain was determined using the following fraction inhibitory combination index (FICI) value. The formula for calculating FICI is as follows.

[0034]

[0035] In the above formula, MIC stands for Minimal Inhibitory Concentration. A synergistic effect was determined when FICI ≤ 0.5, no correlation when 0.5 < FICI ≤ 4, and an antagonistic effect when 4 < FICI. Each experiment was performed three times.

[0036] [Creation of cyp51A and cyp51B knockout strains] We transformed A. fumigatus using the method reported by Umeyama T, et al. (Umeyama T, et al. Antimicrob Agents Chemother. 2018;62(9)). Specifically, after the protoplast-polyethylene glycol method for the laboratory strain AfS35, we used CRISPR / Cas9 genome editing technology to perform homologous recombination with the hygromycin resistance gene, a drug resistance marker, to create knockout strains of either cyp51A or cyp51B.

[0037] [MIC measurement of cyp51A and cyp51B deficient strains] MICs for ITCZ, VRCZ, and FLCZ were determined based on the Clinical and Laboratory Institute (CLSI) M38-E3 broth microdilution method protocol, with minor modifications (Arai T, et al. Antimicrobial Agents and Chemotherapy. 2020;64(4):e02271-19). Specifically, spores from the bacterial strains were collected and their concentrations were measured, resulting in a value of 2.5 × 10⁻⁶. ４ The solution was added to RPMI 1640 medium (pH 7.0) to a concentration of CFU / mL. Then, 100 μL was added to each well of a dried plate for antifungal susceptibility testing (Eiken Chemicals, Tokyo, Japan), incubated at 35°C for 48 hours, and the MIC was determined.

[0038] [Silkworm Infection Experiment] Silkworm larvae were purchased from Ehime Silkworm Seed Co., Ltd., reared at 30°C for two days, and then infected with silkworm larvae at 3 days of the 5th instar. A spore suspension of A. fumigatus was applied in 5 × 10⁻⁶ units. ７Diluted to / mL, 50 μL was injected into silkworms as body fluid using a 1 mL syringe with a 29-gauge needle (Terumo Medical Corporation, Tokyo, Japan). The spore concentration is the amount of bacteria that causes silkworms to die in approximately 48 hours. Subsequently, an antifungal agent was administered once by body fluid injection. That is, each stock solution of luliconazole and itraconazole was diluted with DMSO to a specified concentration and then further diluted 10-fold with 0.6% NaCl solution to obtain the final concentration. In the silkworm infection experiment - 1, five silkworms were used in each group, and the drug effect was verified based on the number of survivors from 48 hours to 72 hours after inoculation with the bacterial solution. Also, in the silkworm infection experiment - 1, strain IFM64301 was used as the A. fumigatus for infection. In the silkworm infection experiment - 2, five silkworms were used in each group, and the drug effect was verified based on the number of survivors from 48 hours to 88 hours, and the same experiment was conducted twice. Also, in the silkworm infection experiment - 2, IFM65548, a resistant strain to triazole antifungal drugs, was used as the A. fumigatus for infection.

[0039] Table 1 shows the evaluation results of the antifungal effects of triazole antifungal drugs (itraconazole (ITCZ), voriconazole (VRCZ), fluconazole (FLCZ)) against the cyp51A - deficient strain and the cyp51B - deficient strain. Also shown are the evaluation results of the antifungal effect of luliconazole (LLCZ) against the cyp51A - deficient strain and the cyp51B - deficient strain.

[0040]

[0041] As shown in Table 1, for triazole antifungal drugs, the MIC in the cyp51B - deficient strain was equivalent to that of the parental strain (AfS35), but the MIC in the cyp51A - deficient strain was lower than that of the parental strain. On the other hand, for luliconazole, the MIC in the cyp51B - deficient strain was significantly lower than that of the parental strain, while the MIC in the cyp51A - deficient strain was slightly lower than that of the parental strain. From these results, it was suggested that triazole antifungal drugs have high specificity for Cyp51B and luliconazole has high specificity for Cyp51A. Based on the above results, the combined use of triazole antifungal drugs and luliconazole was investigated.

[0042] Table 2 shows the results of evaluating the antifungal activity of the combination of luliconazole and various triazole antifungal drugs (itraconazole (ITCZ), voriconazole (VRCZ), fluconazole (FLCZ)) against the triazole-sensitive wild strain (AfS35) by the checkerboard method.

[0043]

[0044] When a triazole antifungal drug and luliconazole were used in combination, they showed an antifungal effect against the azole-sensitive strain (AfS35) at concentrations lower than the effective concentrations when each of the triazole antifungal drug or luliconazole was used alone. This AfS35 strain is naturally resistant to fluconazole, but when fluconazole and luliconazole were used in combination, the growth of the AfS35 strain was completely inhibited.

[0045] Table 3 shows the measurement results of drug interaction by the checkerboard method between itraconazole (ITCZ) and luliconazole (LLCZ). Also, Table 4 shows the measurement results of drug interaction by the checkerboard method between voriconazole (VRCZ) and luliconazole (LLCZ).

[0046]

[0047]

[0048] As shown in Tables 3 and 4, in strains resistant to triazole antifungal drugs, especially in all strains with a MIC of 16 μg / mL or more against triazole antifungal drugs, the FICI was 0.5 or less when combined with luliconazole, and a synergistic effect was observed when combined with luliconazole.

[0049] Table 5 shows the results of measuring the drug interaction by the checkerboard method between itraconazole (ITCZ) and luliconazole (LLCZ) against A. niger and A. tubingensis.

[0050]

[0051] As shown in Table 5, in A. niger and A. tubingensis strains with low susceptibility to itraconazole (MIC of 4 μg / mL or higher), the FICI was 0.5 or less with the combined use of itraconazole and luliconazole, demonstrating a synergistic effect with the combined use of luliconazole. In the diagnosis of pulmonary aspergillosis, there are many cases where the causative organism cannot be identified, and there is a need for a treatment method that can broadly cover bacteria that can be causative. The pharmaceutical composition of the present invention shows high antibacterial efficacy not only against triazole-resistant A. fumigatus but also against triazole-resistant A. niger and A. tubingensis, so it can be expected to be effective with a high probability even in triazole-resistant pulmonary aspergillosis where the causative organism cannot be identified.

[0052] Figure 1 shows the results of silkworm infection experiment-1. In Figure 1, "mock" refers to the group that was not infected with A. fumigatus, "NT" refers to the group that was not treated with antifungal agents, "ITCZ" refers to the group administered 6 μg / silkworm with itraconazole, "LLCZ" refers to the group administered 0.0469 μg / silkworm with luliconazole, and "ITCZ+LLCZ" refers to the group administered 6 μg / silkworm with itraconazole and 0.0469 μg / silkworm with luliconazole. As a result, all five silkworms in the mock group survived after 72 hours. In the NT group, there was one surviving silkworm after 48 hours and zero surviving silkworms after 52 hours. In the ITCZ ​​group, there were zero surviving silkworms after 48 hours, and in the LLCZ group, there was one surviving silkworm after 48 hours and zero surviving silkworms after 72 hours. In contrast, in the ITCZ+LLCZ group, there were 3 surviving silkworms after 48 hours, 2 after 52 hours, and 0 after 72 hours. This demonstrates that the combined use of itraconazole and luliconazole can extend the lifespan of silkworms.

[0053] Figure 2 shows the results of silkworm infection experiment-2. In Figure 2, "mock" refers to the group that was not infected with A. fumigatus, "NT" refers to the group that was not treated with antifungal agents, "ITCZ" refers to the group administered 6 μg / silkworm itraconazole, "LLCZ" refers to the group administered 1.5 μg / silkworm luliconazole, and "ITCZ+LLCZ" refers to the group administered 6 μg / silkworm itraconazole and 1.5 g / silkworm luliconazole. As a result, the survival rate at 88 hours was 100% in the mock group. The survival rate at 48 hours was 0% in the NT group. In the ITCZ ​​group, the survival rate at 48 hours was 10%, and the survival rate at 64 hours was 0%. In the LLCZ group, the survival rate at 64 hours was 100%, but the survival rate at 72 hours was 50%, and the survival rate at 88 hours was 0%. In the ITCZ+LLCZ group, the survival rate at 72 hours was 100%, and the survival rate at 88 hours was 10%. It was also shown that the combined use of itraconazole and luliconazole can extend the life of silkworms even in triazole antifungal strains (IFM65548).

[0054] The combination of triazole antifungal agents and rilconazole has been shown to improve pulmonary aspergillosis. This is particularly promising as an effective treatment for pulmonary aspergillosis resistant to triazole antifungal agents.

Claims

1. A pharmaceutical composition for the treatment and / or prevention of pulmonary aspergillosis, comprising a triazole antifungal agent and luliconazole.

2. The pharmaceutical composition according to claim 1, wherein the triazole antifungal agent is at least one selected from the group consisting of itraconazole, voriconazole, fluconazole, fosfluconazole, fosravuconazole, efinaconazole, ravuconazole, isabconazole, and posaconazole, as well as salts thereof and solvates thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pulmonary aspergillosis is chronic pulmonary aspergillosis.

4. The pharmaceutical composition according to claim 1 or 2, wherein the pulmonary aspergillosis is pulmonary aspergillosis resistant to triazole antifungal agents.

5. The pharmaceutical composition according to claim 1 or 2, wherein the causative agent of pulmonary aspergillosis is Aspergillus fumigatus.

6. Use of luliconazole to enhance the antifungal effect of triazole antifungal agents.

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