JP7365674B2 - Aspergilloma model non-human animal - Google Patents

Description

The present invention relates to chronic aspergillosis model non-human mammals, and more specifically, aspergilloma model non-human mammals, methods for producing these model non-human mammals, and screening for therapeutic or preventive agents for chronic aspergillosis. Regarding methods etc.

Aspergillosis, a deep-seated mycosis caused by the genus Aspergillus, can be divided into three types: invasive aspergillosis, chronic aspergillosis, and allergic bronchopulmonary aspergillosis. Among these, chronic aspergillosis has a poor prognosis, with a 5-year survival rate of 50%, and a clear treatment strategy has not yet been established, making it one of the most common intractable infectious diseases of today (non-patent Reference 1). The present inventors have so far clarified the clinical significance and interpretation of Aspergillus in clinical specimens obtained from patients (Non-patent Document 2), and have conducted a detailed analysis of the pathology of chronic aspergillosis. He proposed a new disease concept called "Pulmonary Aspergillosis" (Non-Patent Document 3), and demonstrated that clinical differentiation is difficult using existing classification methods (Non-Patent Document 4), and has developed a new disease concept for the treatment of chronic aspergillosis. Various attempts have been made to improve the strategy. The present inventors have disseminated treatment strategies based on these results overseas (Non-patent Document 5), and in Japan, the results of the present inventors have been reflected in various guidelines (Non-patent Documents 6, 7). It has made a certain contribution to clinical practice. Furthermore, in recent years, the present inventors have clarified the importance of pulmonary nontuberculous mycobacterial disease as a complication of chronic aspergillosis (Non-Patent Document 8). Pulmonary non-tuberculous mycobacterial infections are still on the rise, and secondary chronic aspergillosis is expected to increase.

Aspergilloma is a bacterial ball formed by the pathogenic fungus Aspergillus in the cavity of the human body. Typical examples include aspergilloma in the lung cavity seen in chronic pulmonary aspergillosis (Figure 1) and bacterial masses in the paranasal sinuses observed in sinonasal mycosis (Figure 2). Chronic pulmonary aspergillosis, a typical disease that causes aspergilloma, is estimated to affect more than 300,000 people worldwide, and more than 7,900 people in Japan, and the 5-year survival rate is approximately It is one of the refractory infections with a poor prognosis of 50%. However, there is still no radical treatment other than surgical removal, and no radical treatment with existing antifungal drugs can be expected.

In addition, in recent years, there has been concern about the increase in drug-resistant Aspergillus (Non-Patent Document 9). Chronic aspergillosis requires long-term antifungal treatment, and there is concern that Aspergillus may develop drug resistance during the course of treatment. The present inventors epidemiologically clarified the actual situation of drug-resistant Aspergillus fumigatus in Japan for the first time, and not only discovered for the first time a case where A. fumigatus became resistant in a patient due to long-term antifungal administration, but also discovered the mechanism behind the resistance. It was found that the mechanism is caused by the G54 mutation of the target protein CYP51A (Non-patent Document 10). In addition, we revealed for the first time that there is a correlation between the duration of antifungal drug administration and the development of drug resistance in A. fumigatus isolated from patients, proving that long-term administration of antifungal drugs induces resistance. (Non-Patent Document 11). As drug-resistant Aspergillus is spreading worldwide, the development of new treatment strategies that do not rely on conventional antifungal treatments has become an urgent global issue.

The main antifungal drugs used to treat aspergillosis are limited to three classes: polyenes, azoles, and echinocandins. Although each drug has a different mechanism of action, there are few drugs that are both highly safe and highly effective. Furthermore, the oral drugs used for chronic aspergillosis are limited to azole antifungals. The development of new antifungal drugs, like all other antimicrobial drugs, has stalled, and over the past few years, it has been difficult to expect any new antifungal drugs to be put into practical use. Chronic aspergillosis, one of the most difficult to treat of modern infectious diseases, requires breakthroughs in new therapeutic strategies. A detailed understanding of the pathology is essential for generating innovative treatment strategies. However, no progress has been made in the analysis of the pathogenesis of chronic aspergillosis, and there is currently an overwhelming lack of basic information.

The reason why progress has not been made in the analysis of the pathology of chronic aspergillosis is the lack of basic research, and the reason for this is the complex process by which the pathology is established. As shown in FIGS. 1 and 2, chronic aspergillosis develops based on physical cavities such as destroyed lungs and sinuses. In other words, clinical cases involve two stages: cavity formation and fungal ball formation, and the pathological condition is formed over many years. Although it is not impossible to create models for basic research over a similar period of time, it is not efficient, and it is difficult to confirm the reproducibility of experimental results and conduct scientific studies. The lack of an appropriate model that reproduces the pathology of chronic aspergillosis is the biggest factor hindering the progress of basic research.

Chronic aspergillosis models can be broadly classified into in vitro models and animal models. The present inventors have also been working on the development of an in vitro model, and have clarified that the anaerobic environment within the cavity plays an important role in the formation of pathological conditions (Non-Patent Document 13). However, in vitro models have limitations in reproducing long-term interactions between bacterial cells and living organisms, and the establishment of animal models is essential.

To date, there is no established animal model for chronic aspergillosis either in Japan or abroad. Only in 2015, Urb et al. reported in the journal Infection and Immunity that they encapsulated Aspergillus conidia in small particles made from agar medium called Agar beads and administered them intratracheally. reported a mouse model in which bacteria were not eliminated (Non-Patent Document 14). They claim that this is the first analysis of the biological response to Aspergillus hyphal colonization, but in this model Aspergillus disappeared within a month, so it cannot be called a chronic aspergillosis model in the true sense. In other words, it is not a model that reproduces the long-term interaction between a large amount of bacterial mass and a living body.

Masato Tashiro, Journal of the Japanese Respiratory Society 5 (Special Issue) 28-28 March 2016 Tashiro T, Tashiro M, et al. Med Mycol. 49: 581-587, 2011 Tashiro T, Tashiro M, et al. Jpn J Infect Dis. 66: 312-316, 2013 Izumikawa K, Tashiro M, et al. J Infect Chemother. 20: 208-212, 2014 Izumikawa K, Tashiro M, et al. Ann N Y Acad Sci. 1272: 40-48, 2012 Diagnosis and treatment guidelines for deep-seated mycoses 2014, edited by the Deep-seated mycoses guideline creation committee. Diagnosis and treatment guidelines for deep-seated mycoses 2014: Kyowa Kikaku; 143-150, 2014 Edited by the Japanese Society of Medical Mycology, Diagnosis and Treatment Guidelines for Aspergillosis 2015, Japanese Society of Medical Mycology; 39-46, 2015 Takeda K, Tashiro M, et al. Med Mycol. 54: 120-127, 2016 Tashiro M, et al. Med Mycol J. 57: J103-J112, 2016 Tashiro M, et al. Antimicrob Agents Chemother. 56: 584-587, 2012 Tashiro M, et al. Antimicrob Agents Chemother. 56: 4870-4875, 2012 Masataka Yoshida, Masato Tashiro, et al., Medical Mycology Journal (2185-6486) Volume 56 Suppl. 1 Page 106 (2015.09) Hidetoshi Ikeda, Masato Tashiro, Program/Abstract Collection May 2016 Page 54, ISSN 2186-8425 Urb M, et al. Infection and Immunity, 83: 3590-3600, 2015

The object of the present invention is to provide a model non-human mammal for chronic aspergillosis, that is, a model non-human mammal in which fungal balls exist in a physical cavity within a living body, and which reproduces the long-term interaction between the living body and fungal balls. An object of the present invention is to provide a method for producing a drug, and a method for screening a therapeutic or preventive drug for chronic aspergillosis.

The genus Aspergillus is a filamentous fungus that takes two forms: conidia (asexual spores) and hyphae. Animal models based on continuous exposure to conidia have already been established in the research field of allergic diseases, but no animal model in which hyphae exist in vivo for a long period of time has been established. As shown in Figure 3, it is a well-known fact that conidia and hyphae differ greatly in cell surface structure and antigenicity, and their immunological responses are markedly different.
The present inventors have made extensive studies to solve the above problems, and have succeeded in creating a cavity in a mouse with high experimental efficiency by creating a cavity under the skin that is easy to approach. In addition, humans become sensitized during the process of aspergilloma formation over a long period of time, and become positive for anti-Aspergillus antibodies, but in this model, because the artificially prepared fungal ball is placed, In this case, sensitization has not been established. Therefore, by sensitizing the mice at the time of indwelling the fungal balls, they succeeded in creating a pathology similar to that in humans. In fact, we succeeded in sensitizing the mice by intraperitoneal administration of mycelium solution twice a week for 4 weeks, and confirmed that anti-Aspergillus precipitated antibodies became positive. Furthermore, in order to shorten the process of aspergilloma formation, we decided to artificially prepare and indwell the fungal balls first. If a fungus ball made only of live bacteria is placed, it will cause strong tissue invasion and the result will be different from the clinical picture, so the key point is to artificially create a fungus ball made up of dead and live bacteria. They discovered something and completed the present invention.

That is, the present invention is as follows.
[1] A chronic aspergillosis model non-human mammal, which has a cavity between the dermis and the subcutaneous tissue, and the cavity contains a fungal ball containing killed bacteria of the genus Aspergillus.
[2] The non-human mammal according to [1], wherein the model non-human mammal is an Aspergilloma model non-human mammal.
[3] The non-human mammal according to [1] or [2], wherein the fungal ball is in contact with the subcutaneous tissue.
[4] The non-human mammal according to any one of [1] to [3], in which fungal balls are maintained for three months or more.
[5] The non-human mammal according to any one of [1] to [4], wherein the fungal ball contains dead and live Aspergillus bacteria.
[6] The non-human mammal according to any one of [1] to [5], wherein the hyphae of Aspergillus bacteria do not invade the subcutaneous tissue.
[7] The non-human mammal according to any one of [1] to [6], in which the Splendore-Hoeppli phenomenon is observed on the surface or inside of the fungal ball.
[8] The non-human mammal according to any one of [1] to [7], wherein the subcutaneous tissue is a dorsal subcutaneous tissue.
[9] The non-human mammal according to any one of [1] to [8], wherein the non-human mammal is a rodent.
[10] The non-human mammal according to [9], wherein the fungal ball has a diameter of 1 mm to 50 mm.
[11] The non-human mammal according to any one of [1] to [10], wherein the Aspergillus bacterium is Aspergillus fumigatus.
[12] A method for producing a chronic aspergillosis model non-human mammal, comprising:
(a) a step of injecting air between the dermis and subcutaneous tissue of a non-human mammal to form a cavity;
(b) a step of culturing Aspergillus bacteria to produce a fungal ball containing dead bacteria; and
(c) placing the Aspergillus bacteria sphere obtained in step (b) in the cavity formed in step (a);
including methods.
[13] The method according to [12], wherein the model non-human mammal is an Aspergilloma model non-human mammal.
[14] The following steps before step (a):
(a') further comprising the step of sensitizing the non-human mammal to Aspergillus bacteria, and injecting air into the sensitized non-human mammal in step (a) to form a cavity, [12] or [ 13].
[15] The method according to [14], wherein the sensitization in step (a') is performed by intraperitoneally administering a mycelial fluid of Aspergillus.
[16] The method according to any one of [12] to [15], wherein the fungal ball is in contact with subcutaneous tissue.
[17] The method according to any one of [12] to [16], further comprising maintaining the fungal balls for 3 months or more.
[18] The method according to any one of [12] to [17], wherein the fungal ball contains dead and live Aspergillus bacteria.
[19] The method according to any one of [12] to [18], further comprising the step of confirming the Splendore-Hoeppli phenomenon on the surface or inside of the fungal ball when it is placed in the cavity.
[20] The method according to any one of [12] to [19], wherein the subcutaneous tissue is dorsal subcutaneous tissue.
[21] The method according to any one of [12] to [20], wherein the non-human mammal is a rodent.
[22] The method according to [21], wherein the size of the fungal ball is 1 mm to 50 mm in diameter.
[23] The method according to any one of [12] to [22], wherein the Aspergillus bacterium is Aspergillus fumigatus.
[24] A method for screening a therapeutic or preventive drug for chronic aspergillosis, comprising:
(A) Measuring the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball of the chronic aspergillosis model non-human mammal according to any one of [1] to [11];
(B) administering a test substance to the non-human mammal;
(C) measuring the size of the fungal ball of the non-human mammal and/or the survival rate of Aspergillus bacteria in the fungal ball after administration;
(D) Comparing the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball before and after administration; and
(E) Selecting a test substance that reduces the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball, based on the comparison results;
including methods.
[25] The method according to [24], wherein the size measurement is performed non-invasively using computed tomography.
[26] A method for evaluating drug migration of a test substance to aspergilloma, comprising:
(i) administering a test substance to the chronic aspergillosis model non-human mammal according to any one of [1] to [11];
(ii) measuring the amount of the test substance in the fungal ball after administration of the test substance; and
(iii) a step of evaluating the drug transferability of the test substance to aspergilloma based on the measured values in (ii);
including methods.
[27] A method for evaluating the drug treatment period necessary for disappearance of aspergilloma, comprising:
(i') administering a therapeutic agent for chronic aspergillosis to the chronic aspergillosis model non-human mammal according to any one of [1] to [11];
(ii') measuring the period until the fungal balls disappear after administration of the therapeutic agent; and
(iii') A step of evaluating the medication treatment period required for aspergilloma disappearance based on the measured values in (ii');
including methods.

The non-human mammal model of the present invention will reproduce a completely new phenomenon, and there are great expectations not only for the academic originality and creativity of creating a new animal model, but also for its expandability. Specifically, it will become possible to discover new therapeutic targets by elucidating pathological conditions, conduct diagnostic and therapeutic research using animal models, and in particular explore research into therapeutic and/or preventive drugs, which will lead to the development of new therapeutic strategies. Significant progress is expected in the necessary research. In other words, the present invention is world-class and innovative, and can be expected to serve as a foundation for future basic research into chronic aspergillosis.

FIG. 1 is a diagram showing a lung CT image of a human patient with chronic aspergillosis. It can be confirmed that a part of the lung cavity (the black part) is filled with fungal balls (§). FIG. 2 is a diagram showing a CT image of sinus aspergillosis. The sinus cavities are filled with calcified bacterial masses, indicating the formation of aspergilloma (§). The interior of the reverse sinus cavity is normal and filled with gas (†). FIG. 3 is a diagram showing differences in cell surface structure depending on the morphology of Aspergillus (Nat Rev Microbiol 14:163-176 (2016)). FIG. 4 is a diagram showing an image of a mouse with fungal balls indwelled in the subcutaneous cavity. Air is injected into the subcutaneous tissue of the mouse's back to artificially create a subcutaneous cavity. A fungal ball prepared in a test tube is placed there. FIG. 5 is a diagram showing the manufacturing procedure of a non-human mammal model of chronic aspergillosis. FIG. 6 is a diagram showing fungal balls of a non-human mammal model of chronic aspergillosis. The left side shows fungal balls produced in a test tube. The upper right shows the appearance of a mouse with indwelling fungal balls, and the lower right shows a CT sagittal image of a mouse with indwelling fungal balls. FIG. 7 is a diagram showing the relationship between fungal balls and tissues within a cavity in a clinical example. The boundary between the fungal ball inside the cavity and the tissue is clear, and no tissue invasion is observed. Lung pathological image, GMS staining, ×100. FIG. 8 is a diagram showing fungal balls and biological reactions within a cavity in a clinical example. Deposition of eosinophilic non-structured material is observed around the fungal bulb (→). Lung pathological image, HE staining, ×200. FIG. 9 is a diagram showing the pathological image of aspergilloma in a non-human mammal model of chronic aspergillosis. More than 3 months passed in the Aspergilloma model mouse. Aspergilloma (HE staining). FIG. 10 is a diagram showing the pathological image of aspergilloma in a non-human mammal model of chronic aspergillosis. Left: clinical pathological image of aspergilloma obtained from a human, right: pathological image of aspergilloma obtained from an aspergilloma model mouse (both HE stained).

The present invention will be explained in detail below.

1. Chronic aspergillosis model non-human mammal The present invention relates to a chronic aspergillosis model non-human mammal, which has a cavity between the dermis and subcutaneous tissue, and the cavity contains dead Aspergillus bacteria. A non-human mammal comprising a sphere is provided.

The model non-human mammal may be an Aspergilloma model non-human mammal.

As used herein, "Aspergilloma" refers to a pathological condition in which Aspergillus bacteria saprophytically parasitizing the body form aggregates as a result of proliferation, exhibiting a spherical shape or the like. It is a characteristic proliferation of tangled hyphal masses, typically encapsulated by fibrous tissue, with fibrin exudates and a few inflammatory cells.

In this specification, "aspergillosis" is an opportunistic infection caused by inhaling the spores of Aspergillus fungi, which are filamentous fungi that are ubiquitous in the environment. It causes sexual necrosis and infarction. May present with symptoms of asthma, pneumonia, sinusitis, or rapidly progressive systemic disease. Aspergillosis can be distinguished into three types: invasive aspergillosis, chronic aspergillosis, and allergic bronchopulmonary aspergillosis. Invasive aspergillosis usually occurs by inhalation of spores and sometimes by direct entry through broken skin.

"Chronic aspergillosis" refers to a pathological condition in which aspergilloma is formed in a cavity within an organism, such as a cavity in the lungs, an external auditory canal, or a sinus cavity. In one embodiment, "aspergilloma" in the present invention is aspergilloma in chronic aspergillosis. Chronic aspergillosis is characterized by the aspergilloma being maintained for one month or more, usually three months or more. Furthermore, the aspergilloma is characterized by the Splendore-Hoeppli phenomenon observed on the surface or inside. There are often no symptoms and it is often first detected on a chest X-ray, but symptoms such as hemoptysis may develop. Some statistics show that the 5-year survival rate is 50%, but it is unclear whether chronic aspergillosis is the direct cause of death. At present, there is no effective treatment method, and even direct spraying through an endoscope has almost no effect. Ultimately, the surrounding tissue is removed, but this causes great damage to the patient. Patients with structural abnormalities such as pulmonary tuberculosis/pneumothorax are more likely to be infected, and even normal people are occasionally infected, but the relationship with immunity is unclear. It is estimated that more than 300,000 people worldwide are infected with the disease, and more than 7,900 people in Japan.

The "dermis" is a layer of skin between the epidermis and subcutaneous tissue, consisting of the papillary layer and the reticular dermis layer, and is composed of fibrous connective tissue. Approximately 70% of the dermis is made up of collagen, and other fibers include elastic fibers (elastin), extracellular matrix, and hyaluronic acid.

"Subcutaneous tissue" refers to the part that connects the skin and underlying tissues such as fascia, and is made of connective tissue with a lower fiber density than the dermis. The subcutaneous tissue layer contains a lot of fat tissue called subcutaneous fat, which has the function of storing nutrients and keeping the body warm.

In one embodiment, the non-human mammal of the present invention does not have subcutaneous tissue invaded by Aspergillus hyphae. Fungal balls are present in the subcutaneous cavities of the non-human mammals of the present invention, and the tissue is exposed to large amounts of hyphae. If the non-human mammal of the present invention is not sufficiently sensitized to Aspergillus bacteria at the time when the fungal ball is placed in the subcutaneous cavity of the non-human mammal of the present invention, the hyphae will invade the tissue. . Humans become sensitized during the long-term process of aspergilloma formation and become positive for anti-Aspergillus antibodies. In actual patients with chronic aspergillosis, even though a large amount of hypha exists in the body as hyphae, the hyphae do not invade tissues. The non-human mammal of the present invention is fully sensitized to Aspergillus bacteria and exhibits pathological conditions similar to humans. In this specification, "the hyphae of Aspergillus bacteria do not invade the subcutaneous tissue" refers to the case where the hyphae of Aspergillus bacteria do not invade the subcutaneous tissue at all, as well as the case where the hyphae of Aspergillus bacteria substantially invade the subcutaneous tissue. This includes cases where the disease does not invade the body. "The hyphae of Aspergillus bacteria do not substantially invade the subcutaneous tissue" means that the invasion of the subcutaneous tissue by Aspergillus bacteria occurs in a very localized area within the range of contact between the subcutaneous tissue and Aspergilloma. means. "A very localized area within the area of contact between the subcutaneous tissue and aspergilloma" refers to, for example, 10% or less, more preferably 5% or less, of the area of contact between the subcutaneous tissue and aspergilloma. It is more preferably 1% or less, most preferably 0.1% or less.

In said non-human mammal, the fungal ball is preferably in contact with the subcutaneous tissue. "Contact" refers to a state in which at least a portion of the Aspergillus bacteria constituting the fungal ball is in contact with at least a portion of the subcutaneous tissue components.

The subcutaneous tissue is distributed throughout the skin of the body, and although the specific site is not particularly limited, it is usually the back subcutaneous tissue or the abdominal subcutaneous tissue, and preferably the back subcutaneous tissue.

As used herein, "fungal ball" refers to an aggregate of filamentous fungi. As used herein, the fungal ball of Aspergillus bacteria includes at least dead Aspergillus bacteria, and preferably includes both live and dead Aspergillus bacteria. In the present specification, the volume or weight ratio of live and dead Aspergillus bacteria in the Aspergillus bacteria sphere is usually 0:1 to 9:1, preferably 2:8 to 8:2. , more preferably 3:7 to 7:3, most preferably 4:6 to 6:4.

The size of the "bacteria ball" is not particularly limited and is set appropriately depending on the size of the non-human mammal used. If the non-human animal used is a rodent, it is usually 1 mm to 50 mm in diameter, and is preferably has a diameter of 5 mm to 30 mm, more preferably a diameter of 10 mm to 15 mm.

The period during which fungal balls are maintained in the model non-human mammal of the present invention is not particularly limited, but is usually 1 month or longer, preferably 2 months or longer, and more preferably 3 months or longer.

In the present specification, the Aspergillus bacteria include Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus lentulus, Aspergillus fumisynnematus, Aspergillus udagawae, Aspergillus fumigatiaffinis, Aspergillus novofumigatus, Aspergillus viridinutans, Aspergillus brevipes, Aspergillus duricaris (Aspergillus duricaulis), Aspergillus unilateralis, Aspergillus oryzae, Aspergillus terreus, Aspergillus nidulans, etc., but are not particularly limited. The Aspergillus bacteria are preferably Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus, more preferably Aspergillus fumigatus. As these Aspergillus bacteria, in addition to the wild type, any mutant type, cell line, transformant, etc. can be used.

Examples of non-human mammals used in the invention include mammals other than humans such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, sheep, cows, horses, and monkeys. Among them, mice, rats, Rodents (Rodentia) such as hamsters and guinea pigs are preferred, more preferably mice or rats, and even more preferably mice.

When a mouse is used as a non-human mammal, its age is not particularly limited, but it is preferably 5 to 81 weeks old, more preferably 6 to 68 weeks old, still more preferably 6 to 54 weeks old.

The Splendore-Hoeppli phenomenon is preferably observed on the surface or inside of the fungal ball in the model non-human mammal of the present invention. Splendore-Hoeppli phenomenon is a phenomenon in which eosinophilic (red by HE staining) substances (antigen-antibody complexes containing IgG, etc., and plasma components) are observed surrounding filamentous fungi, actinomycetes, parasitic larvae, etc. Say something. The eosinophilic substances are attributed to the deposition of antigen-antibody complexes and debris from host inflammatory cells. The model non-human mammal of the present invention is produced, for example, by " 2. Method for producing a chronic aspergillosis model non-human mammal " described below.

2. Method for producing a chronic aspergillosis model non-human mammal The present invention provides a method for producing a chronic aspergillosis model non-human mammal, comprising:
(a) a step of injecting air between the dermis and subcutaneous tissue of a non-human mammal to form a cavity;
(b) a step of culturing Aspergillus bacteria to produce a fungal ball containing dead bacteria; and
(c) placing the Aspergillus bacteria sphere obtained in step (b) in the cavity formed in step (a);
(hereinafter also referred to as the manufacturing method of the present invention).

The model non-human mammal may be an Aspergilloma model non-human mammal.

(a) Step of injecting air between the dermis and subcutaneous tissue of a non-human mammal to form a cavity.Air can be a gas with the same composition as the ground air, or contain 80% or more nitrogen, 85% or more. % or more, 90% or more, 95% or more, or 99% or more can also be used. Injection can be performed by injecting the air into an appropriate site of the non-human mammal using a syringe or the like. The site of inoculation is not particularly limited as long as it is a site that is likely to form a cavity, but it is preferable to inject subcutaneously into a non-human mammal because it is easy to inject and it is easy to form a cavity. Specifically, it is subcutaneous on the back or abdomen, preferably subcutaneous on the back. Although it varies depending on the type and size of the non-human mammal, the site of inoculation, etc., for example, when subcutaneously inoculating mice, about 0.5 ml to 30 ml of air can be injected using a syringe. After the cells are injected, the animal is housed normally, but since the air gradually escapes, it is reinjected as necessary to maintain the cavity.

(b) Step of cultivating Aspergillus bacteria to produce fungal balls containing dead bacteria of the bacteria The medium used for culturing Aspergillus bacteria in the production method of the present invention is not particularly limited, and is suitable for culturing filamentous fungi. A wide variety of materials can be used. Examples include CD minimal medium, YPD medium, TSB medium, malt medium, PDA medium, SD minimal medium, and the like. Glucose, starch, cellulose, xylan, mannan, etc. may be added to the above medium as a carbon source. The amount of the carbon source added is not particularly limited, but can be appropriately set, for example, in the range of 0.5 to 10%, more preferably 1 to 4%. The culture temperature is not particularly limited and can be appropriately set in the range of 20 to 45°C, more preferably 25 to 37°C. The culture time is also not particularly limited, but can be appropriately set, for example, in the range of 12 to 96 hours, more preferably 24 to 72 hours.

The Aspergillus bacteria obtained as a result of the culture may be conidia or hyphae, but preferably hyphae. The fungal ball preferably contains hyphae, and more preferably, 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the Aspergillus bacteria contained therein are hyphae. , most preferably 100% mycelium.

In one embodiment, the cultured Aspergillus bacteria are killed by a method such as starvation treatment, fungicide treatment, autoclaving, etc., to produce a fungal ball made of the killed bacteria.

In one embodiment, a part of the cultured Aspergillus bacteria is left as viable bacteria, and the rest is killed by a method such as starvation treatment, fungicide treatment, autoclaving, etc., and these are mixed to form live bacteria and dead bacteria. Create a fungal ball consisting of.

Fungal balls that are naturally formed by culturing Aspergillus hyphae may be used, or formation and shape maintenance may be promoted by adding a polymeric substance (agar, etc.) to the Aspergillus culture solution. Fungal balls may also be used.

A "fungal ball" refers to an aggregate of filamentous fungi. As used herein, the fungal ball of Aspergillus bacteria includes at least dead Aspergillus bacteria, and preferably includes both live and dead Aspergillus bacteria. In the present specification, the volume or weight ratio of live and dead Aspergillus bacteria in the Aspergillus bacteria sphere is usually 0:1 to 9:1, preferably 2:8 to 8:2. , more preferably 3:7 to 7:3, most preferably 4:6 to 6:4.

The size of the "bacteria ball" is not particularly limited, but if the non-human animal used is a rodent, it is usually 1 mm to 50 mm in diameter, preferably 5 mm to 30 mm in diameter, more preferably 10 mm to 15 mm in diameter. It is.

(c) Step of placing the Aspergillus bacteria sphere obtained in step (b) in the cavity formed in step (a) This can be carried out by transferring the bacteria sphere to the non-human mammal. The transfer can be carried out by incising a site where a cavity exists in the subcutaneous tissue, transferring the fungal ball prepared in step (b), and suturing it. The fungal ball is preferably placed in contact with subcutaneous tissue. After the fungus balls are transferred, the fungus balls can be left in the animal's body by normally raising the animal under sterile conditions.

In said non-human mammal, the fungal ball is preferably in contact with the subcutaneous tissue.

The period during which fungal balls are maintained in the model non-human mammal of the present invention is not particularly limited, but is usually 1 month or longer, preferably 2 months or longer, and more preferably 3 months or longer. The production method of the present invention further includes maintaining the fungal balls for 3 months or more. In one embodiment, the period of time that fungal balls are maintained in the model non-human mammal of the invention can be one year or more.

The manufacturing method of the present invention includes the following steps before step (a):
The method may further include the step of (a') sensitizing the non-human mammal to Aspergillus bacteria, and in step (a), air is injected into the sensitized non-human mammal to form a cavity.

(a') Step of sensitizing a non-human mammal to Aspergillus bacteria In the method for producing a chronic aspergillosis model non-human mammal of the present invention, the non-human mammal is first sensitized to Aspergillus bacteria. When fungal balls are placed in the subcutaneous cavity of a non-human mammal, the tissue is exposed to a large amount of hypha in a short period of time, and there is a possibility that the tissue may be invaded before acquired immunity to hyphal antigens is induced. This is because there is. Humans become sensitized during the long-term process of aspergilloma formation and become positive for anti-Aspergillus antibodies. In actual patients with chronic aspergillosis, even though a large amount of hyphae exists in the body as fungal balls, the hyphae do not invade tissues. In the production method of the present invention, since the artificially produced fungal ball is indwelled in the body of a non-human mammal, the non-human mammal is not sensitized at the time of indwelling the fungal ball, but the sensitized animal is By using this, the condition is similar to that of actual chronic aspergillosis patients.

As a practical method, for example, sensitization can be achieved by administering a fungal fluid of Aspergillus to a non-human mammal. Administration routes include oral administration, parenteral administration such as intravenous, intramuscular, intradermal, subcutaneous, intraarticular, intraperitoneal, and intrarectal administration, with intraperitoneal administration being preferred.

Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Examples include formulations, emulsions, suspensions, and the like. Such compositions may be manufactured by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used, and injections include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. may also include. Such injections can be prepared according to known methods. Injections can be prepared, for example, by dissolving, suspending, or emulsifying the antibody or low-molecular-weight compound of the present invention, or a salt thereof, in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., It may be used in combination with a nonionic surfactant (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), propylene glycol, polyethylene glycol), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into suitable ampoules. Suppositories used for rectal administration may be prepared by mixing the above-described antibody or salt thereof with a conventional suppository base.

The dosage of the above composition varies depending on the subject, target disease, symptoms, administration route, etc., but for example, the size of fungal balls grown in a liquid medium is usually about 1 mm to 30 mm, preferably 3 mm to 15 mm. Crush the bacteria to about 5 mm to 6 mm, more preferably about 5 mm to 6 mm, and add 200 μl of the 10-fold diluted bacterial solution 3 times a day to once every two weeks, preferably 7 times a week to once a week, most preferably. Advantageously, the administration is twice weekly. Similar amounts can be administered in other cases of parenteral administration and oral administration. In order to prove the effectiveness of the dose, it is desirable to confirm the positivity of the precipitated antibody using an anti-Aspergillus precipitated antibody measurement kit.

The administration period is usually 1 day to 1 year, preferably 1 week to 6 months, more preferably 2 weeks to 6 weeks, even more preferably 3 weeks to 5 weeks, and most preferably 4 weeks.

The number of administrations of the composition is at least once, but preferably at least twice from the viewpoint of effectiveness. Further administration is sometimes referred to as booster immunization, and this can provide a more effective infection-preventing effect. Booster intervals of at least 1 week are recommended, and intervals of 1 to 4 weeks are preferred.

In the most preferred embodiment, a mouse is used as the non-human mammal, and the bacteria balls grown in a liquid medium are crushed to a size of about 5 mm, and 200 μl of a 10-fold diluted bacterial suspension is added twice a week. It is to be administered for 4 weeks.

The above composition may further contain an adjuvant to enhance its effect. Examples of the adjuvant include aluminum hydroxide gel, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly(I,C), CpG-DNA, and the like.

When a mouse is used as a non-human mammal, its age at the time of production is not particularly limited, but for example, it is preferably 5 to 10 weeks old, more preferably 6 to 9 weeks old, still more preferably 6 to 8 weeks old. be.

In the present specification, the Aspergillus bacteria include Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus lentulus, Aspergillus fumisynnematus, Aspergillus udagawae, Aspergillus fumigatiaffinis, Aspergillus novofumigatus, Aspergillus viridinutans, Aspergillus brevipes, Aspergillus duricaris (Aspergillus duricaulis), Aspergillus unilateralis, Aspergillus oryzae, Aspergillus terreus, Aspergillus nidulans, etc., but are not particularly limited. The Aspergillus bacteria are preferably Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus, more preferably Aspergillus fumigatus. As these Aspergillus bacteria, in addition to the wild type, any mutant type, cell line, transformant, etc. can be used. The Aspergillus species used for sensitization is preferably the same as the Aspergillus species to be placed.

3. Method for screening a therapeutic or preventive drug for chronic aspergillosis The present invention provides a method for screening a therapeutic or preventive drug for chronic aspergillosis, comprising:
(A) Measuring the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball of the chronic aspergillosis model non-human mammal described in "1. Chronic aspergillosis model non-human mammal "above.;
(B) administering a test substance to the non-human mammal;
(C) measuring the size of the fungal ball of the non-human mammal and/or the survival rate of Aspergillus bacteria in the fungal ball after administration;
(D) Comparing the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball before and after administration; and
(E) Selecting a test substance that reduces the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball, based on the comparison results;
(hereinafter also referred to as the screening method of the present invention).

(A) Measuring the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball of the chronic aspergillosis model non-human mammal described in "1. Chronic aspergillosis model non-human mammal " above. The method for measuring the size of fungal balls is not particularly limited. For example, a method may be used in which the length, width, and height of the fungal ball are measured to calculate the volume of the fungal ball. Furthermore, the method for measuring the survival rate of Aspergillus bacteria in fungal balls is not particularly limited. For example, the survival rate can be determined by removing a sample of the bacterial ball through biopsy and measuring ATP levels, a marker of cell viability, using bioluminescence. Here, "survival rate" means not only the ratio of living cells to the total number of cells, but also a value proportional to the number of living cells. Additionally, in embodiments in which the transplanted viable filamentous fungal cells have been modified to emit fluorescence or luminescence, or in embodiments in which they have been labeled with other detectable substances, the fluorescence or luminescence can also be measured in this step. It can be used for.

In the above embodiment, a gene encoding a fluorescent protein or a luminescent protein is integrated into the genome and/or a plasmid and introduced into the cell so that it can function in the living Aspergillus genus bacterial cell. Cells modified to stably express the protein are used. If a model animal is produced using such cells according to the production method of the present invention, the size of the fungal sphere of Aspergillus cells can be observed noninvasively, in real time, and in vivo using the fluorescence or luminescence as an indicator. It becomes possible to do so. As the fluorescent protein and the luminescent protein, any one commonly used in the technical field can be used. For example, fluorescent proteins include green fluorescent protein (GFP), DsRed, and improved versions thereof, and luminescent proteins include firefly luciferase, Renilla luciferase, jellyfish aequorin, and improved versions thereof. . These are commercially available from suppliers well known to those skilled in the art.

In the above embodiment, the size of the fungal ball can be measured by detecting, for example, radionuclides, MRI enhancers (e.g., paramagnetic ions), radiopaque substances, contrast agents, fluorescent substances, etc. Label the aspergilloma with substances such as radiography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintigraphy, positron emission tomography (PET), and single photon radiation. This can be done by visualizing and imaging using methods such as tomography (SPECT), endoscopy, and laparoscopy.

Radionuclides useful for PET include, for example, ¹⁸ F, ⁵¹ Mn, ^52m Mn, ⁵² Fe, ⁵⁵ Co, 62 Cu, ⁶⁴ Cu, ⁶⁸ Ga, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ^{82m Rb} , ⁸³ Sr, Examples include ⁸⁶ Y, ⁸⁹ Zr, ^94m Tc, ¹¹⁰ In, ¹²⁰ I, and ¹²⁴ I. Radionuclides useful for gamma ray detection include, for example, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu, ⁶⁷ Ga, ⁷⁵ Se, ⁹⁷ Ru, ^99m Tc, ¹¹¹ In, ^114m In, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁶⁹ Yb, ¹⁹⁷ Hg, ²⁰¹ Tl and the like.

Suitable paramagnetic ions include, for example, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), Examples include samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III), with gadolinium being particularly preferred. Metals such as lanthanum (III), gold (III), lead (II), and bismuth (III) are also useful in applications such as X-ray imaging.

Examples of radiopaque substances and contrast agents include iodine compounds (e.g., organic iodine acids such as iodocarboxylic acids, iodoform, triiodophenol, tetraiodoethylene, etc.), barium compounds (e.g., barium sulfate, etc.), gallium Examples include compounds (eg, gallium citrate, etc.), thallium compounds (eg, thallium chloride, etc.), and the like.

Examples of the fluorescent substance include rhodamine, fluorescein (FITC), Cy dye, Alexa (registered trademark) Fluor, phycoerythrin (PE), allophycocyanin (APC), and derivatives thereof. Further, near-infrared fluorescent reagents such as indocyanine are also exemplified as preferred fluorescent substances.

In one embodiment, the measurement of fungal ball size is performed non-invasively with computed tomography.

(B) Step of administering the test substance to the non-human mammal The test substance to be subjected to the screening method may be any known compound or new compound, for example, a nucleic acid, a carbohydrate, a lipid, a protein, Peptides, organic low-molecular compounds, compound libraries created using combinatorial chemistry technology, random peptide libraries created by solid-phase synthesis or phage display methods, or natural ingredients derived from microorganisms, animals, plants, marine organisms, etc. Can be mentioned.

The method of administering the test substance is not particularly limited. For example, the test substance is administered orally or parenterally (e.g., intravenously, intramuscularly, intraperitoneally, intraarterially, subcutaneously, intradermally, in the respiratory tract, etc.) in the form of solid, semisolid, liquid, aerosol, etc. can do. The dose of the test substance varies depending on the type of compound, animal species, body weight, administration form, etc., and can be appropriately selected from the range of 0.01 to 1000 mg/kg/day, and the dose is It can be administered in one or several divided doses. The period of administration is also not particularly limited, but it can be administered, for example, every day for 1 to 14 days or every 2 to 4 days. It is preferable to provide a negative control group (for example, to which a solvent is administered) in addition to the test substance administration group. Furthermore, a positive control group may be provided in which a known chronic aspergillosis therapeutic agent is administered.

(C) Step of measuring the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball of the non-human mammal after administration The size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball The rate may be measured at any time after the start of administration of the test substance. The test may be performed during the administration period of the test substance, at the end of the administration period, or after an arbitrary period of time has elapsed from the end of the administration period. The method for measuring the size of fungal balls is not particularly limited. For example, a method may be used in which the length, width, and height of the fungal ball are measured to calculate the volume of the fungal ball. Furthermore, the method for measuring the survival rate of Aspergillus bacteria in fungal balls is not particularly limited. For example, the survival rate can be determined by removing a sample of the bacterial ball through biopsy and measuring ATP levels, a marker of cell viability, using bioluminescence. Here, "survival rate" means not only the ratio of living cells to the total number of cells, but also a value proportional to the number of living cells. Additionally, in embodiments in which the transplanted viable filamentous fungal cells have been modified to emit fluorescence or luminescence, or in embodiments in which they have been labeled with other detectable substances, the fluorescence or luminescence can also be measured in this step. It can be used for.

In the above embodiment, a gene encoding a fluorescent protein or a luminescent protein is incorporated into a plasmid so that it can function in the cell as a viable Aspergillus genus bacterial cell, and the protein is stably produced by introducing the gene into the plasmid. Cells modified to express this are used. If a model animal is produced using such cells according to the production method of the present invention, the size of the fungal sphere of Aspergillus cells can be observed noninvasively, in real time, and in vivo using the fluorescence or luminescence as an indicator. It becomes possible to do so. As the fluorescent protein and the luminescent protein, any one commonly used in the technical field can be used. For example, fluorescent proteins include green fluorescent protein (GFP), DsRed, and improved versions thereof, and luminescent proteins include firefly luciferase, Renilla luciferase, jellyfish aequorin, and improved versions thereof. . These are commercially available from suppliers well known to those skilled in the art.

In the above embodiment, the size of the fungal ball can be measured by detecting, for example, radionuclides, MRI enhancers (e.g., paramagnetic ions), radiopaque substances, contrast agents, fluorescent substances, etc. Label the aspergilloma with substances such as radiography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintigraphy, positron emission tomography (PET), and single photon radiation. This can be done by visualizing and imaging using methods such as tomography (SPECT), endoscopy, and laparoscopy.

(D) Step of comparing the size of the fungal ball before and after administration and/or the survival rate of Aspergillus bacteria in the fungal ball Size of Aspergilloma before and after administering the test substance and/or compare the survival rate of Aspergillus bacteria in the fungal spheres.

(E) A step of selecting a test substance that reduces the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball based on the comparison results.The size of the Aspergilloma before administering the test substance. If the size of aspergilloma after administering the test substance is smaller compared to (e.g., extinction) and/or have a preventive effect. A test substance that preferably reduces the size of aspergilloma to 50% or less, more preferably a test substance that reduces the size of aspergilloma to 10% or less is determined to be a therapeutic agent and/or a therapeutic agent for aspergilloma.

If the survival rate of Aspergillus bacteria in the fungal sphere after administering the test substance has decreased compared to the survival rate of Aspergillus bacteria in the bacterial sphere before administering the test substance, then the test It can be determined that the test substance has a therapeutic effect and/or a preventive effect on chronic aspergillosis. Preferably, a test substance that reduces the survival rate of Aspergillus bacteria in fungal balls by 50% or more, more preferably 80% or more, is determined to be a therapeutic agent and/or therapeutic agent for chronic aspergillosis.

4. Method for evaluating the drug transferability of a test substance to aspergilloma The present invention is a method for evaluating the drug transferability of a test substance to aspergilloma, comprising:
(i) administering a test substance to the chronic aspergillosis model non-human mammal described in "1. Chronic aspergillosis model non -human mammal"above;
(ii) measuring the amount of the test substance in the fungal ball after administering the test substance; and
(iii) a step of evaluating the drug transferability of the test substance to aspergilloma based on the measured values in (ii);
(hereinafter also referred to as the method for evaluating drug transferability of the present invention) including:

Administration of the test substance in step (i) can be performed in the same manner as in step (B) of the screening method of the present invention.

The measurement of the concentration of the test substance in step (ii) is carried out during a specific period (usually 1 year or more, preferably 6 months or more, more preferably 6 weeks or more, even more preferably 6 weeks or more) after the administration of the therapeutic agent in step (i'). After 3 weeks or more), fungal balls are removed from a chronic aspergillosis model non-human mammal, purified to obtain a test substance fraction, and analysis can be carried out using analytical techniques (such as HPLC) well known to those skilled in the art.

The drug migration of the test substance to Aspergilloma in step (iii) is evaluated, for example, by determining the ratio of the amount of the test substance in the fungal ball measured in step (ii) to the total dose of the test substance. It can be done based on

5. Method for evaluating the period of drug treatment necessary for the disappearance of aspergilloma The present invention provides a method for evaluating the period of drug treatment necessary for the disappearance of aspergilloma, comprising:
(i') administering a therapeutic agent for chronic aspergillosis to the chronic aspergillosis model non-human mammal described in " 1. Chronic aspergillosis model non-human mammal "above;
(ii') measuring the period until the fungal balls disappear after administration of the therapeutic agent; and
(iii') A step of evaluating the medication treatment period required for aspergilloma disappearance based on the measured values in (ii');
(hereinafter also referred to as the method for evaluating the duration of drug treatment of the present invention) comprising:

Administration of the therapeutic agent in step (i') can be carried out in the same manner as administration of the test substance in step (B) of the screening method of the present invention.

The period in step (ii') is measured for a specific period after administration of the therapeutic agent in step (i') (usually 1 year or more, preferably 6 months or more, more preferably 6 weeks or more, even more preferably 3 weeks). (above) can be carried out at suitable intervals in the same manner as the measurement of the size of fungal spheres in step (C) of the screening method of the present invention.

The evaluation in step (iii') is that from the time of administration of the therapeutic agent in step (i'), in the measurement of step (ii'), the size of the fungal balls is below the detection limit, and disappearance of the fungal balls is confirmed. This can be done by setting the period up to this point as the period until aspergilloma disappears.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples in any way.

[Sensitization of mice]
Inoculate frozen Aspergillus fumigatus into PDA medium and culture at 30°C for 4-7 days. Spores formed on the colony surface were collected and a suspension of 1x10 ⁵ cells/ml was prepared using PBS supplemented with 1% Tween20. 200 μl of the prepared suspension was inoculated into 20 ml of PDB medium, and cultured with shaking at 30° C. and 250 rpm for 72 to 96 hours. One of the formed bacterial spheres of approximately 5-6 mm was placed in a tube together with 1 ml of saline and a stainless steel sphere, and crushed at 1500 rpm for 1 minute using a BMS BMS-M10N21 Shake Master Neo. The prepared mycelial fungus solution was diluted 10 times with saline to prepare a fungal solution for administration. The bacterial solution was intraperitoneally administered to mice (ICR female, 6 weeks old) 8 times in total, 1 ml each time, twice a week for 4 weeks. It was confirmed that anti-Aspergillus precipitated antibodies became positive using this method.
[Preparation of fungus balls to be placed]
Aspergillus fumigatus that had been frozen was inoculated into PDA medium and cultured at 30°C for 4-7 days. Spores formed on the colony surface were collected and a suspension of 1x10 ⁵ cells/ml was prepared using PBS supplemented with 1% Tween20. 200 μl of the prepared suspension was inoculated into 20 ml of PDB medium, and cultured with shaking at 30° C. and 250 rpm for 72 to 96 hours. One grown bacterial ball of about 5-6 mm was inoculated into 20 ml of fresh PDB medium, and cultured with shaking at 30°C and 250 rpm for 24-72 hours. The formed fungal ball was sterilized in an autoclave to make a dead fungal ball. A dead bacterial sphere was placed in a PDB medium containing 10 ⁵ cells/ml of Aspergillus fumigatus spores, and cultured with shaking at 30°C and 250 rpm for 6 hours to obtain an indwelling bacterial sphere consisting of dead bacteria and live bacteria.
[Creation and maintenance of subcutaneous cavity]
Air was injected subcutaneously into the back of the mouse, which had been sensitized a total of eight times, and the subcutaneous tissue was peeled off. Thereafter, the skin was incised and the fungal balls prepared above were placed. After placement, the skin was sutured using suture clips and air was injected to create a subcutaneous cavity. Thereafter, if the air in the subcutaneous cavity decreased, additional air was injected as appropriate to maintain the cavity.
[Preparation of Aspergilloma]
After more than 3 months, aspergilloma was formed from the indwelling fungal ball. The Splendore-Hoeppli phenomenon was pathologically confirmed in the excised aspergilloma, and the presence of viable bacteria was confirmed by culture.

[Example 1]
1) Establishment of a mouse model with subcutaneous cavity Aspergillus sphere placement Chronic aspergillosis forms lesions not only in the lung cavities but also in the paranasal sinus cavities as shown in Figure 2. Furthermore, when a lesion is formed in a lung cavity, the cavity wall surface becomes erosive, and the lung-specific epithelium has disappeared. In other words, it is suggested that the physical feature of "cavity" is more important than organ-specific features in the pathological process. The gas-filled cavities are one of the reasons why inflammatory cells infiltrate the fungal spheres inefficiently, making them difficult to treat, so it is important to reproduce cavities in animal models. In the studies conducted by the present inventors to date, it has been difficult to establish a mouse model of chronic aspergillosis after lung cavity formation due to the restriction of lung size. Although we considered using other larger animal species, we established a mouse model from the standpoint of efficiency.

As mentioned above, if the presence of organ-specific cells is not an absolute condition for reproducing the pathological condition, we can create a ``cavity of a certain size that is filled with gas'' anywhere in the body. It would be a good thing if possible. Therefore, they focused on the subcutaneous region as the site where the largest cavity could be formed without affecting the survival of the mouse. Specifically, air was injected subcutaneously into the back of the mouse to create a persistent subcutaneous cavity as shown in Figure 4, and Aspergillus bacteria balls prepared in a test tube were placed in the cavity (Figure 5). , 6).

[Example 2]
2) Technique for maintaining Aspergillus fungi within the subcutaneous cavity As shown in Figure 7, in actual patients with chronic aspergillosis, despite the presence of a large amount of hyphae as fungi within the cavity, tissue spreads beyond the cavity wall. has not invaded. The reason for this may be that the hyphal elongation reaction is suppressed by the deposition of eosinophilic non-structures around the fungal ball (Splendore-Hoeppli phenomenon) as shown in FIG. This structure is composed of an antigen-antibody complex, fibrin, etc., and patients with chronic aspergillosis actually test positive for anti-Aspergillus precipitated antibodies. When fungal balls are placed in the subcutaneous cavity of mice, they are exposed to a large amount of hyphae over a short period of time, and there is a possibility that they may invade tissues before the acquired immunity to hyphal antigens is induced. Therefore, we adopted a method in which mice were sensitized with Aspergillus hyphal antigens in advance, and fungal balls were indwelled after inducing acquired immunity.

[Example 3]
3) Evaluation as a mouse model of chronic aspergillosis The clinical definition of chronic aspergillosis is an Aspergillus infection that lasts for more than 1 month, and more precisely, it is considered to be more than 3 months. Therefore, after artificially creating a subcutaneous cavity and indwelling the fungus balls, we continued non-invasive evaluation using CT and confirmed that the fungus balls could be maintained within the subcutaneous cavity for more than 3 months (Fig. 9). The fungal balls were removed from the mice after three months and cultured to prove that Aspergillus was still alive. Furthermore, the fungal balls were pathologically evaluated, and it was confirmed that the Splendore-Hoeppli phenomenon was reproduced on the fungal ball surface (Figure 10). If all of these requirements are met, we believe that the conditions as a chronic aspergillosis mouse model are fully met.

By using the non-human mammal model of chronic aspergillosis of the present invention, the effectiveness of candidate therapeutic agents for chronic aspergillosis and the feasibility and usefulness of chronic aspergillosis treatment can be appropriately evaluated in vivo in non-human mammals. It becomes possible to do so. The production method of the present invention enables the production of a chronic aspergillosis model in a non-human mammal that has characteristics that are histologically and pathologically highly similar to human chronic aspergillosis. Therefore, the chronic aspergillosis model non-human mammal of the present invention can serve as an effective preclinical model and can contribute to the treatment of chronic aspergillosis.

Claims (19)

Chronic aspergillosis model non-human mammal, which has a cavity between the dermis and the subcutaneous tissue of the back , contains a fungal ball containing killed Aspergillus bacteria in the cavity, and has a surface of the fungal ball. or a non-human mammal in which the Splendore-Hoeppli phenomenon is observed , where the model non-human mammal is an Aspergilloma model non-human mammal, and the human mammal is a rodent. 2. The non-human mammal of claim 1 , wherein the fungal ball is in contact with subcutaneous tissue. 3. The non-human mammal according to claim 1 or 2 , wherein the fungal ball is maintained for 3 months or more. The non-human mammal according to any one of claims 1 to 3 , wherein the fungal ball contains dead and live Aspergillus bacteria. The non-human mammal according to any one of claims 1 to 4 , wherein the hyphae of Aspergillus bacteria do not invade subcutaneous tissue. The non-human mammal according to any one of claims 1 to 5 , wherein the fungal ball has a diameter of 1 mm to 50 mm. The non-human mammal according to any one of claims 1 to 6 , wherein the Aspergillus bacterium is Aspergillus fumigatus. A method for producing a chronic aspergillosis model non-human mammal, the method comprising:
(a) a step of injecting air between the dermis and the dorsal subcutaneous tissue of a non-human mammal to form a cavity;
(b) a step of culturing Aspergillus bacteria to produce a fungal ball containing dead bacteria; and
(c) placing the Aspergillus bacteria sphere obtained in step (b) in the cavity formed in step (a);
wherein the Splendore-Hoeppli phenomenon is observed on the surface or inside of the fungal ball during indwelling, wherein the model non-human mammal is an Aspergilloma model non-human mammal, and the human mammal is a rodent . The following steps before step (a):
Claim 8 , further comprising the step of (a') sensitizing the non-human mammal to Aspergillus bacteria, and injecting air into the sensitized non-human mammal in step (a) to form a cavity. the method of. 10. The method according to claim 9 , wherein the sensitization in step (a') is performed by intraperitoneally administering a mycelial fluid of Aspergillus. 11. The method according to any one of claims 8 to 10 , wherein the fungal ball is in contact with subcutaneous tissue. The method according to any one of claims 8 to 11 , further comprising maintaining the fungal spheres for 3 months or more. The method according to any one of claims 8 to 12 , wherein the fungal ball contains dead and live Aspergillus bacteria. The method according to any one of claims 8 to 13 , wherein the size of the fungal sphere is 1 mm to 50 mm in diameter. The method according to any one of claims 8 to 14 , wherein the Aspergillus bacterium is Aspergillus fumigatus. A screening method for a therapeutic or preventive drug for chronic aspergillosis, the method comprising:
(A) measuring the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball of the chronic aspergillosis model non-human mammal according to any one of claims 1 to 7 ;
(B) administering a test substance to the non-human mammal;
(C) measuring the size of the fungal ball of the non-human mammal and/or the survival rate of Aspergillus bacteria in the fungal ball after administration;
(D) Comparing the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball before and after administration; and
(E) Selecting a test substance that reduces the size of the fungal ball and/or the survival rate of Aspergillus bacteria in the fungal ball, based on the comparison results;
including methods. 17. The method of claim 16 , wherein the size measurements are performed non-invasively with computed tomography. A method for evaluating drug migration of a test substance to aspergilloma, comprising:
(i) administering a test substance to the chronic aspergillosis model non-human mammal according to any one of claims 1 to 7 ;
(ii) measuring the amount of the test substance in the fungal ball after administering the test substance; and
(iii) a step of evaluating the drug transferability of the test substance to aspergilloma based on the measured values in (ii);
including methods. A method for evaluating the drug treatment period required for disappearance of aspergilloma, the method comprising:
(i') administering a therapeutic agent for chronic aspergillosis to the chronic aspergillosis model non-human mammal according to any one of claims 1 to 7 ;
(ii') measuring the period until the fungal balls disappear after administration of the therapeutic agent; and
(iii') A step of evaluating the medication treatment period required for aspergilloma disappearance based on the measured values in (ii');
including methods.