CZ20001476A3 - Materials for treating and prevention of mucous tissue inflammation - Google Patents

Abstract

Materials for the treatment and prevention of non-invasive fungus-induced mucositis, for non-invasive fungal diagnosis induced rhinosinusitis and for the cultivation of non-invasive fungi from a mammalian mucus sample as well as specific antifungal preparations and medical tools for treatment and prevention non-invasive fungus-induced rhinosinusitis. Solution it further provides materials for the treatment and prevention of other conditions non-invasive fungus-induced mucositis such as chronic otitis media, chronic colitis and Crohn's disease and for the treatment and prevention of chronic asthma.

Description

The invention relates to materials involved in the treatment and prevention of non-invasive fungal-induced mucosal inflammation as well as asthma symptoms.

BACKGROUND OF THE INVENTION

Mucositis, an inflammation of mucosal tissue, is a serious medical problem that affects millions of people worldwide. For example, conservative estimates indicate that between 20-40 million Americans suffer from chronic rhinosinusitis, an inflammation of the nasal cavity and / or paranasal sinuses.

Mostly the cause of chronic sinusitis is unknown. However, in a small percentage of patients, non-invasive fungal organisms living in mucosa appear to be involved. Patients with these conditions, now known as allergic fungal sinusitis (AFS), were first described in the early 1980s (Miller JW et al., Prod. Scot. Thor. Soc. 36: 710 (1981) and Katzenstein ALA et al. , J. Allergy Clin Immunol., 72: 89-93 (1983)). Specifically, 3 to 8 percent of chronic rhinosinusitis requiring surgery to block nose by polyp formation was classified as AFS. In short, AFS is diagnosed by the presence of densified mucus in the nasal / paranasal cavity. Typically, this mucus comprises clusters or coatings of necrotic eosinophils, Charcot-Leyden crystals and non-invasive fungal hyphae. In addition, AFS patients typically have nasal / paranasal polyposis in their past and may have had several operations. Inflammation can affect all nasal / paranasal sinuses, but can also be asymmetric, affecting only one side. Computer tomography images have a typical appearance in AFS patients and often reveal bone damage in adjacent structures. Indeed, it has been shown that bone destruction adjacent to cavities and nasal areas ranges from 19 to 80 percent.

Although fungal organisms appear to be a causative agent in AFS, successful treatment is lacking.

Currently, AFS patients as well as with chronic rhinosinusitis are undergoing surgical treatment with or without steroid therapy. The operation helps to clean the nasal / paranasal sinuses when polyps are blocked and steroid therapy helps control inflammatory responses that are believed to cause tissue and bone destruction. Unfortunately, patients treated with surgery only almost always experience recurrent symptoms of rhinosinusitis and further polyp growth. In addition, the long-term use of steroids is associated with serious side effects and the omission of steroid therapy leads to recurrent episodes of rhinosinusitis. For these reasons, people suffering from chronic rhinosinusitis typically experience repeated cycles of intense inflammation, surgery, and steroid therapy followed by recurrent intense inflammation. Therefore, neither surgery nor steroid therapy is particularly effective or acceptable as a long-term treatment for chronic rhinosinusitis.

Summary

The present invention generally relates to methods and materials for treating and preventing non-invasive fungal-induced mucositis. The term "mucositis" as used herein means inflammation, as opposed to infection, of the mucous membrane. The present invention is based on the discovery that a condition known as AFS can be successfully treated by the use of antifungal agents in an amount, frequency, and for a period of time to effectively reduce inflammation induced by the presence of fungal organisms in nasal / paranasal mucus. In addition, the present invention is based on the discovery that the use of antifungal agents in an amount, frequency, and for a period of time while effectively maintaining a reduced level of fungal organisms in nasal / paranasal mucus may prevent the symptoms of AFS. In particular, the invention comprises administering an antifungal agent to a mammal such that the antifungal agent is in contact with the mammalian mucus and reduces the presence of the fungal organisms in the mucus. In addition, being the only known method for successful treatment and prevention of AFS, the use of an antifungal agent is particularly advantageous for patients as compared to other currently available medical treatments for AFS such as surgery and steroid therapy. Such medical procedures have side effects, can be expensive and can be inconvenient for patients.

The present invention is also based on the discovery that most, if not all, cases of chronic rhinosinusitis have a fungi etiology and that most, if not all, cases of chronic rhinosinusitis can be treated using antifungal agents in an amount, frequency and for a period of time to effectively reduced the presence of fungal organisms in nasal / paranasal mucus. In addition, the use of anti-fungal agents in an amount, frequency, and for a period of time that effectively maintains a reduced level of fungal organisms in nasal / paranasal mucus may prevent the symptoms of AFS.

This discovery is against current understanding of chronic rhinosinusitis and has far-reaching implications for medicine. For example, a number of medical research articles show that about three to eight percent of cases of chronic rhinosinusitis requiring surgery are AFS, a condition of AFS. • Rhinosinusitis having a non-invasive fungal etiology. In fact, less than 150 cases of AFS have been reported in the literature over the past fifteen years. It is noted that the lack of understanding of the non-invasive fungal etiology of chronic rhinosinusitis could be due to the fact that patients are often affected by bacterial infections (i.e. invasive bacteria). Probably tissue damage by inflammation induced by non-invasive fungi results in a higher incidence of bacterial infections in these damaged areas. Therefore, an overlapping bacterial infection in affected patients could mask the root cause, a fungal organism in the mucus.

For the purposes of this invention, the term "non-invasive fungal-induced rhinosinusitis" includes AFS as well as other nasal-paranasal mucositis conditions having a non-invasive fungal etiology.

Treatment and prevention of non-invasive fungal-induced rhinosinusitis, whether diagnosed as AFS or any other rhinosinusitis condition having a non-invasive fungal etiology, by using antifungal agents bypasses the need for surgical treatment and steroid therapy that induce significant pain and suffering to the patient. In addition, the use of antifungal agents to treat and prevent non-invasive fungal-induced rhinosinusitis actually targets treatment against an etiological agent (ie, fungus), as opposed to surgical treatment, steroid therapy, and antibacterial therapy.

As used herein, the term "chronic" refers to problems that persist for at least three months. It is to be understood that conditions that are treated as described herein and become asymptomatic may be classified as chronic. Thus, chronic complaints may be symptomatic or asymptomatic.

The present invention is also based on another equally serious discovery that chronic asthma symptoms can be successfully treated and prevented by the use of antifungal agents in an amount, frequency and time effective to reduce the presence of fungal organisms in the airway mucosa. It is also evident from the present inventions that antifungal agents can be administered directly into the airways of the lungs to treat chronic asthma. Again, these inventions conflict with current understanding of chronic asthma and have far-reaching clinical implications. Together, these important scientific discoveries can enable a large population to experience a happier, healthier and more productive life.

In particular, the invention provides methods and materials for treating and preventing a wide variety of inflammatory mucosal diseases using antifungal agents. The use of antifungal agents is a safe and highly effective treatment approach that involves the mucosal administration of antifungal agents in amounts, frequencies, and for an effective period of time to reduce, prevent, or eliminate them. Non-invasive fungal-induced mucositis. The term "mucosal administration" as used herein refers to any type of administration where the administered substance is placed in contact with the mucus. The present invention also provides specific antifungal formulations that can be applied to different parts of a mammal having mucus. In addition, the invention provides medical instruments that can be used to administer antifungal formulations. These tools are particularly advantageous as they can be used individually to deliver an effective dose of an antifungal formulation to a suitable area of the body. Further, the invention provides improved methods and materials for collecting and culturing fungal organisms of saccharose samples. These culture techniques can be used to monitor the number of fungal species of tastes during a particular antifungal treatment regimen. In addition, these fungal collection and culture methods are useful for identifying the genotype and phenotype of a specific fungal organism that induces non-invasive fungal-induced mucositis. The identification and characterization of non-invasive fungal organisms found in a particular individual mucus can help clinicians to identify appropriate treatments and prophylactic procedures. For example, this information can help determine the specific antifungal agent, the amount and mode of administration, and the number of applications to be used, as well as possible combination therapies that may include other medications and procedures such as steroids, antibacterial agents and operations.

In general, the invention features methods of treating a mammal (eg, a human) having non-invasive fungal-induced rinosinusitis. This method involves direct mucosal administration of the formulation to a portion of the mammalian nasal-paranasal regions in an amount, frequency, and for a time effective to reduce or eliminate non-invasive fungal-induced rhinosinusitis. The formulation comprises an antifungal agent or a plurality of antifungal agents and may be in a solid, liquid or aerosol form (eg, powder, crystalline substance, gel, paste, ointment, cream, solution, suspension, partial liquid, spray, spray, substance nebula) , atomized steam, aerosol and tincture). In addition, the formulation may be in a form suitable for mucosal administration by humans.

Further, the formulation may comprise a pharmaceutically acceptable aqueous vehicle (eg, saline and water). For example, the liquid form of the formulation may contain about 0.00001 percent to 20 percent of the antifungal agent as determined by the weight of the antifungal agent relative to the volume of the vehicle. In addition, the formulation may contain about 0.01 ng to 1000 mg of an antifungal agent (eg, amphotericin B) per liter in some embodiments of the invention, or about 1 ng to 500 mg of an antifungal agent per liter in other embodiments of the invention. In addition, an effective amount of such aqueous formulations may be about 0.01 ml to 1 liter of formulation per 4 4 4 4 4 4 4 4 4

The nostril in some embodiments of the invention, or about 5 ml to 100 ml of the nostril formulation in yet other embodiments of the invention. Alternatively, an effective amount of the formulation may be about 0.01 ng to 1000 mg of antifungal agent per kg body weight in some embodiments of the invention, or 1 ng to 500 mg of antifungal agent per kg body weight in other embodiments of the invention. The effective amount of the formulation may change or remain the same during the effective duration. The effective frequency of direct mucosal administration may range from four times a day to once every two weeks in some embodiments of the invention, or twice a day in yet other embodiments of the invention. In addition, the effective frequency for direct mucosal administration may be greater than once per day or greater than once per week. The effective duration may be greater than 7, 14, 30, 60 or 90 days.

The mammal may be atopic or non-atopic and may be immunocompetent or immunocompromised. In addition, non-invasive fungal-induced rhinosinusitis can be characterized by polyp formation or polypoid changes. Non-invasive fungi-induced rhinosinusitis can also be a chronic condition. Mucosal administration may be by irrigation of at least a portion of the nasal-paranasal region with a liquid formulation. Alternatively, mucosal administration may include administering an aerosol form of the formulation to at least a portion of the nasal paranasal anatomy. The antifungal agent may be in solid, liquid or aerosol form. In addition, the antifungal agent may be a polyene macrolide, tetraene macrolide, pentaene macrolide, fluorinated pyrimidine, imidazole, azole, triazole, halogen phenol ether, thiocarbamate, allylamine, sterol inhibitor and a substance that disrupts fungal cell wall components. Such antifungal agents include amphotericin B, blucytosine, ketoconazole, miconazole, itraconazole, fluconazole, grisofulvin, clotrimazole, econazole, terconazole, butoconazole, oxiconazole, sulconazole, saperconazole, voriconazole, ciclopiroxine naphthin, morfinoline, haloproxinine, haloproxinine, haloproxinine, haloproidine , natamycin, butenafine, undecylene acid, Whitefieled ointment, propionic acid and caprylic acid. In addition to the antifungal agent, the composition may also include, without limitation, a pharmaceutically acceptable aqueous vehicle, a pharmaceutically acceptable solid vehicle, a steroid, a mucolytic agent, an antibacterial agent, an anti-inflammatory agent, an immunosuppressant, dilator, vasoconstrictor, decongestive agent, leukotriene inhibitor, anticholinergic, antihistaminic. and combinations thereof.

The method may also include administering a second composition comprising, without limitation, an antifungal agent, an antibacterial agent, a steroid, a mucolytic agent, an anti-inflammatory agent, an immunosuppressant, a dilatant, a vasoconstrictor, a decongestant, a leukotriene inhibitor, an anticholinergic, an antihistamine, their combination. The method may also include the next step after direct mucosal administration. This additional step may be prophylactic mucosal administration of the prophylactic preparation to a mammal in an amount, frequency and for a period effective for preventing non-invasive fungal-induced rhinosinusitis. The prophylactic formulation also contains an antifungal agent and may be in a solid, liquid or aerosol form (eg, powder, crystalline substance, gel, paste, ointment, cream, solution, suspension, partial liquid, spray, spray, atomized nebula, atomized steam, aerosol, tincture, pill, capsule, tablet and gel capsule). In addition, prophylactic mucosal administration may be by direct or indirect mucosal administration. For example, prophylactic mucosal administration may be by flushing at least a portion of the nasal paranasal anatomy with a liquid form of the prophylactic composition, administering an aerosol form of the prophylactic composition to at least one portion of the nasal-paranasal anatomy, or orally administering the prophylactic composition to a mammal in solid or liquid form.

It is to be understood that any other feature of the invention described above may be applied to the following other embodiments and aspects of the invention. For example, methods for the prophylactic treatment of mammals at risk of developing non-invasive fungal-induced rhinosinusitis, and methods for treating asthma may utilize a formulation in which the antifungal agent is 0.00001 percent to 20 percent by weight or volume of the formulation, and so on.

In another embodiment, the invention provides a method of prophylactic treatment of a mammal that is at risk of developing non-invasive fungal-induced rhinosinusitis. The method comprises mucosal administration of the composition to a mammal in an amount, frequency and for a period of time that is effective in preventing non-invasive fungal-induced rhinosinusitis. This product contains an antifungal agent.

Another embodiment of the invention provides a method for treating a mammal having non-invasive fungal-induced rinosinusitis. The method comprises the steps of identifying (eg, diagnosing) a mammal, and direct mucosal administration of the composition to at least a portion of the mammalian nasal-paranasal anatomy in an amount, frequency, and for a time effective to reduce or eliminate non-invasive fungal-induced rinosinusitis. This product contains an antifungal agent.

Another embodiment of the invention is a method of prophylactic treatment of a mammal at risk of developing non-invasive fungal-induced rhinosinusitis. The method comprises the steps of identifying (eg, diagnosing) a mammal and mucosal administering the composition to at least a portion of the nasal paranasal anatomy of the mammal in an amount, frequency, and for a time effective to prevent non-invasive fungal-induced rhinosinusitis. This product contains an antifungal agent.

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In another embodiment, the invention provides a method for treating a mammal suffering from asthma. The method includes direct mucosal administration of the composition to at least a portion of the airways (e.g., nasal-paranasal and pulmonary airways) of a mammal in an amount, frequency, and for a time effective to reduce or eliminate asthma symptoms. This product contains an antifungal agent. Direct mucosal administration may be by flushing the mammalian nasal-paranasal anatomy with a liquid formulation. Alternatively, direct mucosal administration may be by inhalation of the formulation through the mammalian mouth or nose. In addition, the method may comprise an additional step after direct mucosal administration. This additional step may be prophylactic mucosal administration of the prophylactic preparation to the mammal in an amount, frequency and for a time effective to prevent the symptoms of asthma. This prophylactic preparation also contains an antifungal agent.

In another embodiment, the invention provides a method for the prophylactic treatment of a mammal at risk of developing asthma. The method comprises mucosal administration of the composition to at least a portion of the airways (e.g., nasal-paranasal airways and lung airways) of a mammal in an amount, frequency, and for a time effective to prevent the symptoms of asthma. This product contains an antifungal agent.

Another embodiment of the invention is a method of treating a mammal suffering from asthma. The method comprises the steps of identifying (eg, diagnosing) a mammal and direct mucosal administration of the composition to at least a portion of the airways (e.g., nasal-paranasal and airways) of the mammal in an amount, frequency and for a time effective to reduce or eliminate asthma symptoms. This product contains an antifungal agent.

Another embodiment of the invention provides a method for the prophylactic treatment of a mammal at risk of developing asthma. The method comprises the steps of identifying (eg, diagnosing) a mammal and mucosal administering the composition to at least a portion of the airways (e.g., nasal-paranasal and airways) of the mammal in an amount, frequency and for a time effective to reduce or eliminate symptoms. asthma. This product contains an antifungal agent.

In another embodiment, the invention provides a method for treating a mammal suffering from non-invasive fungi induced by intestinal mucositis (e.g., chronic colitis and Crohn's disease). The method involves mucosal administration of the composition to a mammal in an amount, frequency and for a time effective to reduce or eliminate the symptoms of non-invasive fungal-induced intestinal mucositis. This composition contains an antifungal agent and may be in the form of a controlled release capsule (eg, a pH-controlled or time-regulated release capsule). Mucosal administration may be by oral administration to the mammalian gastrointestinal tract. Alternatively, the mucosal administration of the composition to the mammalian gastrointestinal tract may be by enema.

In another embodiment, the invention provides a method for the prophylactic treatment of a mammal at risk of developing non-invasive fungal-induced intestinal mucositis (e.g., chronic colitis and Crohn's disease). The method involves mucosal administration of the composition to a mammal in an amount, frequency and for a period of time that is effective in preventing the symptoms of non-invasive fungal-induced intestinal mucositis. This product contains an antifungal agent.

Another embodiment of the invention provides a method for treating a mammal suffering from non-invasive fungi induced by otitis media. The method includes mucosal administration of the composition to a mammal in an amount, frequency, and for a time effective to reduce or eliminate non-invasive fungal-induced otitis media. This product contains an antifungal agent. The mucosal administration may be application of the preparation to the middle ear of a mammal. For example, a liquid formulation can be used to flush the middle ear when the tympanic membrane is raised or intact. Alternatively, the formulation may be injected into the middle ear or myringotomy may be used to penetrate the tympanic membrane. In addition, myringotomy tubes can be used to bridge the tympanic membrane. Further, the preparation may be mucosally administered to the middle ear through the nose and the Eustachian tube.

In another embodiment, the invention provides a method for the prophylactic treatment of a mammal at risk of developing non-invasive fungal-induced otitis media. The method includes mucosal administration of the composition to a mammal in an amount, frequency, and for a time effective to prevent the symptoms of non-invasive fungal-induced otitis media. This product contains an antifungal agent.

In another aspect, the invention features an article of manufacture that includes packaging material (eg, boxes, containers, vials, and other containers) and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material comprises a label or package insert indicating that the composition can be directly mucosally administered to at least a portion of the nasal-paranasal anatomy of a mammal suffering from non-invasive fungi-induced rinosinusitis in an amount, frequency and for a time effective to reduce or eliminate non-invasive fungi-induced. rinosinusitis.

In another embodiment, the invention features an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material comprises a label or package insert showing that the composition can be directly mucosal administered to at least a portion of the nasal-paranasal anatomy of a mammal suffering from non-invasive fungi-induced rinosinusitis in an amount, frequency and for a time effective to prevent non-invasive fungi-induced rinosinusitis.

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In another embodiment, the invention features an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material comprises a label or package insert indicating that the composition may be directly mucosal administered to at least a portion of the airways of a mammal suffering from asthma in an amount, frequency, and for a time effective to reduce or eliminate asthma symptoms.

In another embodiment, the invention features an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material comprises a label or package insert indicating that the composition may be mucosal administered to at least a portion of the mammalian airway at risk of developing asthma in an amount, frequency, and for a time effective to prevent asthma symptoms.

Another embodiment of the invention is an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent.

The packaging material comprises a label or package insert indicating that the composition may be mucosal administered to a mammal suffering from non-invasive fungal-induced intestinal mucositis in an amount, frequency and for a time effective to reduce or eliminate the symptoms of non-invasive fungal-induced intestinal mucositis.

Another embodiment of the invention is an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material comprises a label or package insert indicating that the composition may be mucosal administered to a mammal at risk of developing non-invasive fungal-induced intestinal mucositis in an amount, frequency, and for a time effective to prevent the symptoms of non-invasive fungal-induced intestinal mucositis.

Another embodiment of the invention is an article of manufacture comprising a packaging material and a formulation included in the packaging material. This product contains an antifungal agent. The packaging material includes a label or package insert indicating that the composition may be mucosally administered to a mammal suffering from non-invasive fungus-induced otitis media in an amount, frequency and for a time effective to reduce or eliminate the symptoms of non-invasive fungal-induced otitis media.

In another aspect, the invention features the use of an antifungal agent in the manufacture of a medicament for the treatment or prevention of non-invasive fungal-induced rhinosinusitis.

In another aspect, the invention features the use of an antifungal agent for the manufacture of a medicament for the treatment or prevention of asthma symptoms.

In another aspect, the invention features the use of an antifungal agent for the manufacture of a medicament for treating or preventing non-invasive fungal-induced intestinal mucositis.

In another aspect, the invention features the use of an antifungal agent for the manufacture of a medicament for treating or preventing non-invasive fungal-induced otitis media.

In another aspect, the invention provides an antifungal composition comprising an antifungal agent, a flavoring agent and water. The water constitutes at least about 50% of the formulation. For example, the water may comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the composition.

In another embodiment, the invention provides an antifungal preparation comprising itraconazole and water. Itraconazole is dissolved in the formulation at a concentration of greater than about 25 pg per ml. For example, itraconazole may be dissolved in the formulation at a concentration of greater than about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 µg per ml. In addition, water constitutes at least about 50% of the composition. For example, the water may comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the composition. The composition may also comprise polyethylene glycol (eg, PEG-200, PEG-400, PEG-800, etc.). The composition may also contain a flavoring agent (eg, peppermint oil, cherry flavor, syrup, and the like).

In another embodiment, the invention provides an antifungal preparation comprising itraconazole and water. Itraconazole is present in the formulation in suspension at a concentration of greater than about 25 pg per ml. For example, itraconazole may be in a suspension formulation at a concentration of greater than about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 µg per ml. In addition, water constitutes at least about 50% of the composition. For example, the water may comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the composition. The composition may also comprise polyethylene glycol (eg, PEG-200, PEG-400, PEG-800, etc.). The composition may also contain a flavoring agent (eg, peppermint oil, cherry flavor, syrup, and the like).

In another embodiment, the invention provides an antifungal composition comprising an antifungal agent, a flavoring agent and water. The water constitutes at least about 50% of the formulation. For example, the water may comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the composition. In addition, the antifungal agent may be amphotericin B, ketoconazole, saperconazole, voriconazole, flucytosine, miconazole, fluconazole, griseothulvin, clotrimazole, econazole, terconazole, butcononazole, oxiconazole, sulconazole, ciclopirox olamine, haloproginine, terpine, tolpine, tolpine, tolopine, tolopine, , natamycin, butenafm, undecylene acid, Whitefield ointment, propionic acid and caprylic acid.

In another aspect, the invention provides a method of making an antifungal preparation. It contains itraconazole and water. Itraconazole is dissolved in the formulation at a concentration of greater than about 25 pg per ml. For example, itraconazole may be dissolved in the formulation at a concentration of greater than about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 or 180 pg * * Φ

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Ml Φ · · • • φ φ φ • • • • φ φ φ φ φ φ φ φ φ φ · φ · · · per per φ · · · ml ml ml ml ml ml ml ml ml ml ml ml ml In addition, water constitutes at least about 50% of the composition. For example, the water may comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the composition. The method involves adding water to a stock solution containing itraconazole.

In another aspect, the invention provides a method for cultivating mammalian mucus fungi. The method comprises 1) contacting the mucus with a mucolytic agent to reduce the viscosity of the mucus, 2) separating the fungi from the reduced viscosity mucus, 3) contacting the separated fungi with a mushroom growth medium to form a mushroom culture, 4) incubating the mushroom culture to the separated culture grew.

In another aspect, the invention provides a method for obtaining a fungal antigen. The method comprises 1) contacting the mucus with a mucolytic agent to reduce the viscosity of the mucus, 2) separating the fungi from the reduced viscosity mucus, 3) contacting the separated fungi with a mushroom growth medium to form a mushroom culture, 4) incubating the mushroom culture to the separated culture was grown, and 5) isolating the antigen from the cultured fungi.

In another aspect, the invention provides a method for producing fungal-specific antibodies. The method comprises 1) contacting the mucus with a mucolytic agent to reduce the viscosity of the mucus, 2) separating the fungi from the reduced viscosity mucus, 3) contacting the separated fungi with a mushroom growth medium to form a mushroom culture, 4) incubating the mushroom culture to the separated culture was grown, 5) isolating the antigen from cultured fungi, and 6) immunizing the animal with a fungal antigen to generate antibodies.

In another aspect, the invention features a nasal mucus collecting device. The apparatus comprises a material collection container, a collection tube and a connecting portion. The container of collected material is suitable for collecting mucus. The collecting tube extends the collection container and determines the distal end and the lumen so that the mucus can pass the lumen from the distal end of the collection tube to the collection container. The collecting tube is usually flexible at least over a portion of the length of the tube so that a professional can selectively manipulate the collecting tube into the desired configuration during the material collection process. Further, the collecting tube is generally moldable, which means that it typically retains the desired configuration until the professional has further manipulated the collecting tube to achieve a different configuration. The connecting portion begins at the collected material container and determines a second lumen by which it communicates with the interior of the collected material container. The coupling portion is adapted to be able to withstand a vacuum source. In addition, the apparatus may further comprise a flap that adjusts the opening of the second lumen. The container of collected material may be removable from the collection tube and the connecting portion.

In another aspect, the invention provides a pharmaceutical composition comprising an antifungal agent.

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In another embodiment, the invention provides a pharmaceutical composition comprising an antifungal agent and a mucolytic agent.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and a steroid.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and a decongestant agent.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and an antibiotic.

Another embodiment provides a pharmaceutical composition comprising an antifungal agent and an anti-inflammatory agent.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and an antihistamine.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and an anticholinergic agent.

Another embodiment is a pharmaceutical composition comprising an antifungal agent and a leukotriene inhibitor.

In another aspect, the invention provides a composition for affecting a mammalian immune response to fungi, characterized by a substance designed for direct mucosal administration to mammalian mucus and having antifungal means for eliminating or reducing fungi below a threshold level where the fungus no longer activates eosinophil migration into the affected area.

In another aspect, the invention provides a pharmaceutical composition for treating a fungal-related condition in a nasal-sinus, pulmonary, ear, or intestinal area of a mammalian patient, said composition comprising an effective dose of an antifungal agent as described herein.

In another aspect, the invention provides a pharmaceutical composition for treating a fungal-related condition in a nasal-sinus, pulmonary, ear, or intestinal area of a mammalian patient, said composition comprising an effective dose of an antifungal agent and at least one other agent or inhibitor described herein.

In another aspect, the invention provides a pharmaceutical composition for treating a fungal-related condition in a nasal-sinus, pulmonary, ear, or intestinal area of a mammalian patient, said composition comprising an effective dose of an antifungal agent suitable for long-term use in nasal-paranasal areas.

In another aspect, the invention provides a medicament comprising a mucolytic agent and an antifungal agent, as described herein, for the treatment of sinusitis, asthma, otitis media or patient colitis.

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In another aspect, the invention provides a lavage medication for treating an inflamed nasal region, a lung region, an ear region, or an intestinal region of a patient, wherein the inflamed nasal region, lung region, ear region, and intestinal region is caused by a fungus, and the medicament comprises an effective dose of an antifungal agent. a steroid as described herein.

In another aspect, the invention provides a lavage medication for treating an inflamed nasal region, a lung region, an ear region, or an intestinal region of a patient, wherein the inflamed nasal region, lung region, ear region, and intestinal region is caused by a fungus, and the medicament comprises an effective dose of an antifungal agent. a mucolytic agent as described herein.

In another aspect, the invention provides a lavage medication for treating an inflamed nasal region, a lung region, an ear region, or an intestinal region of a patient wherein the inflamed nasal region, lung region, ear region, and intestinal region is caused by a fungus, and the medicament comprises an effective dose of steroid and mucolytic. an agent as described herein.

In another aspect, the invention provides a lavage medication for treating an inflamed nasal region, a lung region, an ear region, or an intestinal region of a patient, wherein the inflamed nasal region, lung region, ear region, and intestinal region is caused by a fungus, and the medicament consists of effective doses of an antifungal agent. a steroid and a mucolytic agent as described herein.

In another aspect, the invention provides a rinsing medication for treating an inflamed nasal region, a lung region, an ear region, or an intestinal region of a patient wherein the inflamed nasal region, lung region, ear region, and intestinal region is caused by a fungus, and the medicament is an effective dose of at least one medicament. selected from the group consisting of an antifungal agent, a steroid, a mucolytic agent, or any combination thereof, as described herein.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods similar or identical to those described herein can be used in the practice and testing of the present invention, suitable methods and materials are described below. All publications, patent applications, and other references mentioned herein are incorporated by reference in their entirety.

In case of conflict, the present specification, including definitions, will be reviewed. In addition, the materials, methods, and examples are illustrative only and not deliberately limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

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DESCRIPTION OF THE PICTURE

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Figure 1 is a CT scan of a patient with bilateral chronic rhinosinusitis.

Figure 2 is a CT scan of the patient of Figure 1, 4 months after treatment with anti-fungal washes.

Figure 3 is a diagram showing a mucus collection apparatus.

Figure 4 is a diagram showing a mucus collection apparatus.

SUMMARY OF THE INVENTION

DETAILED DESCRIPTION

The invention includes methods and materials for treating and preventing non-invasive fungal-induced mucositis. In particular, the invention encompasses the mucosal administration of an antifungal agent in an amount, frequency and for a time effective to prevent, reduce or eliminate chronic non-invasive fungal-induced rhinosinusitis. The invention also provides methods and materials for diagnosing chronic non-invasive fungal-induced rhinosinusitis and for culturing non-invasive fungi from a mammalian mucus sample as well as specific antifungal preparations and medical instruments for treating and preventing non-invasive fungal-induced rinosinusitis. In addition, the invention provides methods and materials for treating and preventing other conditions of non-invasive fungal-induced mucositis such as chronic otitis media, chronic colitis and Crohn's disease. Further, the invention includes methods and materials for treating and preventing the symptoms of chronic asthma.

Although not limited to any particular mode of action, the present invention, including the treatment and prevention of non-invasive fungal-induced mucosal tissue inflammation using an antifungal agent, is based on the following putative disease progression mechanisms, which are derived from the disclosures herein. In general, most if not all individuals have fungal organisms living in their mucus. Normally, most individuals tolerate these non-invasive organisms and live a normal, disease-free life. For unknown reasons, some individuals do not tolerate these fungal organisms and begin to develop an immune response against them. As the immune response increases, eosinophils accumulate in local tissue. This accumulation of eosinophils may contribute to the formation of blocking tissue mass (eg polyps and polypoid structures) as well as to the transmigration of activated eosinophils from tissues (inside

ΦΦ «« φ φ φ φ φ · · φ φ φ φ φ φ · · · · · · · · · φ • • • • φ φ φ φ · ♦ * · φ φ φ »of the body) into the mucus (outside the body). These tissue mass barriers appear to prevent normal purging of the cavity and may thus facilitate further fungal growth. Once inside the mucus, eosinophils may release their granule contents probably after activation of surface Fc receptors. Eosinophil granules contain many toxic molecules such as eosinophil cationic protein (ECP), eosinophilic peroxidase (EPO), and major basic protein (MBP). Upon release, these toxic molecules can damage both the target foreign microorganism (e.g., the fungus) as well as the tissues themselves. The degree of damage induced by eosinophil accumulation and eosinophil degranulation varies considerably, ranging from mild inflammatory pain and discomfort to significant structural abnormalities such as tissue and bone destruction and the formation of polyps, polypoid structures and other swelling. Once the tissue itself is damaged, the individual may also have increased sensitivity to bacterial infections. Therefore, most, if not all, characteristic inflammatory responses, resulting damage and subsequent bacterial infection are actually triggered by non-invasive fungal organisms. It is known that fungal organisms can be observed in tissue under extreme conditions of mucositis in damaged tissue and bone simply because the barrier (eg, epithelium) between the inner and outer parts of the body has been destroyed or damaged. In these situations, simply observing the presence of a small number of fungal organisms in a localized area of tissue damage does not prevent the fact that the disease is non-invasive fungal-induced mucositis and not infection.

The discovery that most, if not all, cases of chronic rhinosinusitis are caused by non-invasive fungal organisms assume that other chronic inflammatory conditions such as chronic middle ear inflammation, chronic colitis and Crohn's disease are most likely caused by non-invasive fungal organisms living in mucus. In addition, the discovery that non-invasive fungal-induced rhinosinusitis can be successfully treated and limited by antifungal agents after mucosal administration in effective amounts, at an effective frequency, and for an effective period of time suggests that these other non-invasive fungal-induced mucositis conditions can also be treated and reduced by antifungal agents, when used appropriately as described herein. Furthermore, the discovery that symptoms of chronic asthma can be treated and reduced by antifungal agents when administered directly to the airways via nasal-paranasal flushing suggests that antifungal agents may be effective when directly injected into the airways by inhalation through the nose or mouth. Therefore, the methods and materials described herein represent a potential treatment for millions of people suffering from chronic rhinosinusitis, chronic otitis media, chronic colitis, Crohn's disease and non-invasive fungal-induced mucositis as well as chronic asthma.

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As described above, non-invasive fungal-induced mucositis is inflammation, not infection. In general, inflammations are fundamentally and clinically different from infections. Infection is defined as the growth of an organism in a tissue. In addition, infection is characterized as an invasive disease, meaning that the infectious organism enters host tissue and then triggers the host's immune response and / or induces damage. Therefore, the role of the infectious organism is typical of an invasive pathogen. In addition, the infected host may be immunocompetent or immunocompromised. If the infected host is immunocompromised, the infection is often called an "opportunistic" infection. Further, the infection may be acute or chronic depending on many factors such as the competence of the immune system of the infected individual, the nature of the invasive pathogen, and the availability of medical treatment.

In contrast, inflammation is characterized as a localized protective response that serves to destroy, alleviate, and / or remove a damaging agent. In addition, the typical inflammatory response causes redness, swelling, heat and pain. In the case of non-invasive fungal-induced mucositis, the localized protective response is directed against non-invasive fungal organisms living outside the tissue (e.g., mucus). Typically, some individuals suffering from non-invasive fungi induced by mucositis are atopic and / or immunocompetent. Moreover, the role of the damaging agent (i.e., the fungus) is the same as that of the non-invasive allergen. Therefore, non-invasive fungal-induced mucositis is an allergic reaction triggered by the individual's immune system against a fungal organism living outside the individual's tissue.

As described herein, the invention provides methods and materials that reduce the presence of fungal organisms in the mucus to a level and for a period such that the characteristic inflammatory response and the resulting mucositis-related damage are stopped, treated or reduced. For the sake of clarity, reducing the fungal organism in mucus to treat or prevent mucositis is similar to removing an allergen (eg, pollen) from the reach of an individual suffering from an allergic reaction (eg, hay fever). Again, an allergic reaction against, for example, pollen is not an infection but an inflammation. Moreover, the simplicity of the present invention highlights the far-reaching clinical implications that follow these discoveries. For example, once clinicians understand that most, if not all, cases of chronic rhinosinusitis are caused by non-invasive fungal organisms in nasal-paranasal mucus using the methods and materials described herein, millions of people will live healthier, happier, and more productive lives.

Identification of non-invasive fungal-induced mucositis «9

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Non-invasive fungal-induced mucositis is defined as inflammation of mucosal tissue induced by a non-invasive fungal organism. Examples of mucosal tissue include, but are not limited to, mucosa of the mouth, intestine, nasal passages, paranasal sinuses, lung airways, middle ear trachea, Eustachian tube, vagina, and urethra. In general, mucosal inflammation can be determined using any method commonly known to those skilled in the art. For example, mucosal inflammation may be identified in an individual after a tissue biopsy examination, as well as by visual examination, endoscopic analysis, and imaging techniques (eg, X-rays, CT scans and magnetic resonance imaging (MRI)), since different mucosal areas with inflammation tend to show abnormal characteristics.

Many diagnostic methods can be used to determine whether a particular mucositis is a non-invasive fungus-induced mucositis. In general, such diagnostic methods include, without limitation, assessing the medical history of individuals prior to medical treatment, discussing and reviewing the patient, collecting and analyzing biological samples from the affected individual.

Assessing the individual's medical history may be helpful in determining whether a particular mucositis is a non-invasive fungus-induced mucositis, since such inflammations are typically recurrent and chronic. Thus, signs of previous episodes of non-invasive fungus-induced mucositis would suggest that current mucositis is also non-invasive fungus-induced mucositis. Other useful information in the medical history of an individual could include, without limitation, allergies, surgery, and other diseases such as cystic fibrosis and ciliary dismotility syndromes.

Discussion and evaluation of the affected individual may also help to identify non-invasive fungal-induced mucositis. For example, individuals suffering from chronic mucositis symptoms such as airway blockages, olfactory loss, hearing loss, shortness of breath, shortness of breath, coughing, headache and facial pressure may have mucositis-induced non-invasive fungi. In addition, non-invasive fungal-induced mucositis may be excluded in individuals who exhibit symptoms of infection such as fever, fungal dissemination, fungemia, polymorphonuclear leukocyte elevation and acute onset. It is known that recurrent bacterial infections may indicate underlying conditions by non-invasive fungal-induced mucositis, since chronic inflammation may lead to epithelial destruction and thereby increase the individual's sensitivity to bacterial infection. In addition, various diagnostic tests can be performed to assist in the identification of non-invasive fungal-induced mucositis. For example, common allergic tests using a panel of fungal and non-fungal antigens may do this to this to this to this. to be used to determine whether an individual is atopic, since some cases of non-invasive fungal-induced mucositis include atopic individuals. In addition, immunoassays for the presence of antibodies against fungal antigens, tests for abnormal pulmonary function or without methacholine, audiograms, and tympanograms may be used to identify non-invasive fungal-induced mucositis.

Collection and analysis of biological samples from affected subjects can help identify non-invasive fungal-induced mucositis. In general, biological samples such as mucus, stool, urine, sputum, and blood can be collected and analyzed for features that indicate the involvement of non-invasive fungal organisms. Such features may include, without limitation, the presence of an antigenic marker for non-invasive fungal-induced mucositis; the presence of eosinophils, eosinophilic products (eg, MPB and ECP), antibodies, fungal antigens or fungal organisms in samples of mucus, stool, urine, sputum; and the absence of fungal organisms in blood samples. For example, the identification of allergic mucus (i.e., mucus that contains evidence of the presence of eosinophils) may indicate non-invasive fungal-induced mucositis. Such evidence of the presence of eosinophils includes, without limitation, the presence of intact eosinophils, necrotic eosinophils, and eosinophil products. Many methods are known for detecting the presence of these various traits and markers in biological samples that can be utilized. For example, the presence of eosinophils in allergic mucus can be detected using hematoxylin / eosin labeling followed by microscopic examination.

In addition, a tissue biopsy can be collected and analyzed for the absence of invasive fungi. As mentioned above, fungal organisms can be observed under extreme mucositis conditions in tissue or bone destruction when a tissue biopsy is monitored, simply because the barrier (i.e., epithelium) between the internal and external environment is destroyed or damaged. In these situations, the mere presence of small amounts of fungal organisms in a localized area of tissue does not necessarily mean that the disease is not a non-invasive fungus-induced mucositis.

In addition, immunoassays can be used to detect the presence of various features of non-invasive fungal-induced mucositis in biological samples. Many immunological-based assays are known including, but not limited to, the ELISA method and the radioallergosorbent assay (RAST). Methods using RAST are described, for example, in McRury J et al. (Clin Exp Immunol 65: 631-638 (1986)), Mabry RL and Manning S (Otolaryngol Head Neck Surg. 113: 721-723 (1995)) and Lynch NR et al. (Int Arch Allergy Immunol 114: 59-67 (1997)). Immunological-based tests may use polyclonal 9 99 99 99 * J * 99 * J * 99 Antibodies, monoclonal antibodies or fragments thereof having antigen specificity that can be used as a diagnostic marker for non-invasive fungal-induced mucositis. For example, monoclonal antibodies having specificity for fungal organisms known to induce non-invasive fungal-induced mucositis can be produced and used to screen biological samples. Such antibodies can be produced using methods published elsewhere (Zeidan et al., Experimental Approaches in Biochemistry and Molecular Biology, William C. Brown Publisher (1996) and Seaver, Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up, Marcel Dekker Inc). , New York, NY (1987)). Briefly, the mouse can be immunized with a sample of fungal organism isolate. After a few weeks, lymphocytes from the spleen of immunized mice can be obtained and fused with smeloma cells to form hybridoma cells. Hybridomas exhibiting specificity for an immunizing fungal isolate may then be isolated and monoclonal antibodies may be prepared.

Since the specific methods and materials used to identify non-invasive fungal-induced mucositis may vary depending on the particular location of the mucositis, a more detailed description is provided below for several examples of mucosal tissue.

1. Nasal-paranasal cavities

The external skeleton of the nose is formed by two elongated nasal bones. One nasal bone is placed on each side of the central bar with two bones forming an arched cross section. The nasal septum divides the nasal cavity in half. The lateral nasal wall has three spirals that enlarge the mucosal surface of the nasal cavity and vestibules. The nasal vestibule is bounded by the nasal septum and lateral wall. This large surface area of the spirals and nasal septum provides extensive contact with the inspiratory air, thereby facilitating humidification, particulate removal, and temperature control of the inspiratory air.

Paranasal sinuses are spaces that are in contact with air and connect the nasal cavity through openings and orifices. Although paired, paranasal sinus is usually asymmetric in shape and location and includes maxillary, frontal, etmoidal, and wedge sinus. The presumed functions of the paranasal sin include relieving the bones of the skull, providing the mucus for the nasal cavity and acting as resonant chambers for sound generation. Maxillary sinus is the largest of the paranasal sinus. Each maxillary sine is placed in the maxilla and opens into the middle passage. The frontal sinus is located in the frontal bone and is above and at the level of the eye socket. Frontal sin also opens into the middle passage. The etmoid sinus is numerous with irregular shape of air spaces, which open to middle and higher passages.

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The wedge sinus is in the wedge and is posterior to both the eye and the upper part of the nasal cavity. The wedge sine opens into the upper passage. Mucosal tissue (mucosa) flank both the nasal cavity and paranasal sinus and is generally made up of epithelial layer, connective tissue and mucosal glands. The mucus layer normally covers the mucosa. Mucus, which is secreted from mucosa, serves to trap particles and prevents dehydration of nasal and paranasal tissues that are otherwise exposed to air. The mucus is normally transported by the cilia towards the nasopharynx and then swallowed.

Subjects suffering from rhinosinusitis can be identified using methods generally known in the art. Symptoms of rhinosinusitis include, without limitation, nasal airway obstruction, olfactory loss, facial pain, headache, post-nasal leakage, and rhinorrhea. The presence of dense mucus or visual identification of nasal or paranasal obstruction with mucus or polyps after examination often indicates rhinosinusitis. Nasal polyps are growths from nasal paranasal mucosa that are typically smooth, gelatinous, semi-transparent, round or pear-shaped, and faded. Generally, nasal polyps are located in the lateral wall of the nose, usually in the middle passage or along the middle and upper spirals. Most nasal polyps originate from etmoid sinus, but some polyps originate in maxillary wedge sinus. The mass of nasal polyps consists mainly of edematous fluid with scattered fibrous cells and sparse mucous glands. The superficial epithelium of nasal and paranasal polyps generally reveals squamous metaplasia. Eosinophils are usually present in polyps in moderate or high numbers, and nasal polyp fluid is known to contain greater than normal levels of IgA, IgE, IgG and IgM antibodies as well as abnormally high concentrations of IL-5, a cytokine that contributes to activation and survival eosinophils. As shown herein, the presence of nasal polyps is not a risk factor for rinosinusitis, but rather a terminal stage of chronic inflammation.

The following methods and materials can be used to identify subjects suffering from rhinosinusitis-induced non-invasive fungi. As described above, the condition known as AFS is a condition of non-invasive fungal-induced rhinosinusitis. Therefore, any method known in the art that is used to identify AFS can be used to identify non-invasive fungal-induced rhinosinusitis (Cody DT et al., Laryngoscope 104: 1074-1079 (1994) and Kupferberg SB et al., Otolaryngol. Head Neck Surg. 117: 35-41 (1997)). For example, non-invasive fungal-induced rhinosinusitis can be detected by the presence of a thickened mucus that contains clusters or coatings of necrotic eosinophils, CharcotLeyden crystals, and non-invasive jungal hyphae. In addition, image analyzes such as MRI and CT can be used to identify non-invasive fungal-induced rhinosinusitis,

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99 because these conditions typically exhibit a characteristic appearance and often cause bone breakdown in adjacent structures (Quiraishi et al., Otolaryngol. Head Neck Surg. 117: 29-34, (1997); Manning et al., Laryngoscope 107. 170-176 ( 1997); Kinsella et al., Head and Neck 18: 211-217, (1996); Allbery et al., RadioGraphics 15: 1311-1327 (1995); Roth MR, Ear, Nose and Throat J. 73: 928- 930 (1994) and Bartynski et al., Otolaryngol. Head Neck Surg. 103: 32-39 (1990)). Furthermore, subjects with non-invasive fungi induced by rhinosinusitis may have had nasal-paranasal polyposis in history and could undergo numerous operations.

Results using currently available diagnostic methods indicate that about three to eight percent of chronic rhinosinusitis cases requiring surgery are AFS cases.

In general, these current AFS diagnostic methods include criteria such as the presence of a characteristic appearance in CT images, the presence of allergic mucus, and the presence of fungal organisms in mucus samples as confirmed either histologically or by fungal growth in culture. The present invention demonstrates that more than 90% of all cases of chronic rhinosinusitis have a fungi etiology based on a better understanding of chronic rhinosinusitis, improved diagnostic procedures and the remarkable degree of successful antifungal therapy approaches described herein. In addition, the present invention shows that the ability to grow fungal organisms from mucus samples is not a useful criterion for the diagnosis of non-invasive fungal-induced rhinosinusitis, such as AFS, since most, if not all, human subjects have fungal organisms in their nasal-paranasal mucus. Example 1). However, it is known that the collection, analysis and / or cultivation of fungal organisms from nasal-paranasal mucus samples can provide useful diagnostic information. Such information may include, but is not limited to, the level of the fungal organisms and the number of different fungal species present in a particular mucosal sample.

There seems to be a loss of understanding for the non-invasive fungal etiology of chronic rhinosinusitis, for several reasons. First, it seems that reliance on inadequate collection of mucus and mushroom cultivation techniques has led to misinterpretation of negative fungal growth results. As shown herein, these negative results were most likely false-negative, since the fungal organisms can be grown from nasal-paranasal mucus samples taken by most, if not all, humans. Consequently, the ability to cultivate fungal organisms from nasal-paranasal mucus samples as a diagnostic criterion for a non-invasive fungal-induced rhinosinusitis condition, including AFS, is essentially nonsensical. Second, clinicians routinely wash or nullify the nasal-paranasal cavity mucus during surgery before removing and examining polyps for the presence of allergic mucus. Therefore, the failure to find 9 · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 Allergic mucus is most likely caused by failure to remove the appropriate medium for examination. This, in turn, could lead to a widely accepted medical theory that polyposis is caused by certain inflammatory conditions in the nasal-paranasal cavity. As discussed above, polyposis may be considered to be the result of end-stage chronic inflammation. Third, chronic inflammatory conditions as described herein may have led to recurrent bacterial infections that could mask the underlying condition by non-invasive fungal-induced rhinosinusitis. In addition, any temporary relief observed after antibacterial treatment could complicate the diagnosis of a condition having a non-invasive fungal etiology.

Despite everything, the present invention teaches that special care must be taken to protect the mucus for analysis and that the presence of allergic mucus can be used to identify non-invasive fungi-induced rhinosinusitis. In addition, recurrent bacterial infections in the nasal-paranasal sinuses may indicate non-invasive fungal-induced rinosinusitis.

Any individual who had a previous event with rhinosinusitis is at risk of developing non-invasive fungi-induced rhinosinusitis. In addition, elderly individuals having cystic fibrosis, asthma and, in family history, nasal problems or allergies may be at risk of developing non-invasive fungal-induced rhinosinusitis. Furthermore, individuals who are exposed to a significant level of allergens (eg, fungal spores, pollen, and chemicals) may be at risk of developing non-invasive fungal-induced rhinosinusitis.

2. Middle ear

The ear can be divided into three parts: the outer ear, the middle ear, and the inner ear. The middle ear is a cavity that is connected to the nasopharynx through the Eustachian tube. In addition, the middle ear is separated from the opening of the outer ear by a tympanic membrane and comprises a chain of three small bones that connect the tympanic membrane to the inner ear. Mucous tissue flank most of the middle ear.

Subjects suffering from otitis media mucosal tissue inflammation can be identified based on the medical history of middle ear involvement, visual examination, tissue biopsy and symptoms such as hearing loss, ear discharge and middle ear effusion.

In general, non-invasive fungal-induced otitis media can be identified by 1) collecting and analyzing a medium or mucus sample from the middle ear for fungal organisms and the presence of eosinophils, 2) analyzing a tissue biopsy for non-invasive fungal organisms, 3) an audiogram consistent with conductive hearing loss; ) by a flat tympanogram. In contrast to nasal-paranasal mucus, the identification of the presence of a fungal organism in samples taken from the middle ear may indicate non-invasive fungal-induced inflammatory conditions because the middle ear is normally very sterile.

Any subject who has had a previous case of otitis media is at risk of developing non-invasive fungal-induced otitis media. In addition, young subjects (eg infants and toddlers) as well as subjects having family history of ear problems or allergies may be at risk of developing non-invasive fungal-induced otitis media.

3. Intestines

The mucous tissue is flanked by both the small and large intestine. Individuals suffering from inflammatory bowel disease (eg, ulcerative colitis and Crohn's disease) can be identified using methods and materials generally known in the art. For example, tissue biopsy analysis as well as endoscopic analysis can be used to identify a state of intestinal mucositis such as intestinal polyposis. In addition, inflammatory bowel disease may indicate symptoms such as diarrhea, abdominal cramps, flatulence and nausea.

Generally, non-invasive fungal-induced intestinal mucositis can be identified by the presence of fungal organisms, eosinophils or eosinophil products in stool samples. In addition, tissue biopsies revealing the presence of non-invasive fungal organisms may indicate the condition of non-invasive fungal-induced intestinal mucositis.

Any individual who has had a previous case of intestinal inflammation is at risk of developing non-invasive fungal-induced intestinal mucositis. In addition, elderly individuals having digestive problems in family history, intestinal polyposis or allergies may be at risk of developing non-invasive fungal-induced intestinal mucositis.

Functional organisms

Any fungal organism living in a mammal's mucus may be a non-invasive fungal organism that is capable of inducing mucositis because the mere presence of the organism in the mucus of an intolerant individual causes inflammation. For example, all previously identified fungal organisms in mucus samples from AF patients may be non-invasive fungal organisms capable of inducing non-invasive fungal-induced mucositis, including, but not limited to, Absidia, Aspergillus flavus, Aspergillus fumatus, Aspergillus glaucus, Aspergillusemaria, Aspergillusemaria, Aspergillusemidaria, , Bipolaris, Candida albicans, Candida lypolytica, Candida parapsilosis, Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschler, Exserohilum, Fusarium, Malbranchia, Paecilomvces, Penicillium, Pseudallescheria, Rhizopus, Rhizopus. In addition, fungal organisms that have not yet been identified in mucus samples from patients diagnosed with AFS may be non-invasive fungal organisms capable of inducing non-invasive fungal-induced mucositis including, without limitation, Acremonium, Arachniotus citrinus, Aurobasidioum, Beauveria, Chaospomium, Chaetomium Exophilia jeanselmei, Geotrichum, Oidiodendron, Phoma, Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala, Scolebasidium, Scopulariopsis, Ustilago, Trichoderma, and Zygomycete. A list of other fungal organisms that may be non-invasive fungal organisms capable of inducing non-invasive fungal-induced mucositis can be found in most taxonomic mycological books.

Collection of mucus samples

Generally, the mucus may be collected from the surface of any mucosal tissue using a collection solution to wash the cavity containing the mucus. Proper mucus collection techniques should maximize recovery of the mucus-containing collection solution by allowing sufficient penetration into the correct anatomical cavities and minimize absorption of the collection solution by the individual. Vasoconstrictors can be used to maximize mucus collection, and mucolytic agents can be used to dissolve the obstructive mucus so as to increase penetration of the collection solution.

Therefore, prior to collection of the mucosal sample, the subject may be treated with a vasoconstrictor and / or mucolytic agent so as to induce sufficient vasoconstriction and / or mucolytic action in a particular region. Suitable vasoconstrictors may include, without limitation, phenylephrine hydrochloride (NEO-SYNEPHRINE®; Sanofi Pharmaceuticals), cocaine, and epinephrine. A mucolytic agent is any substance that dissolves the mucus so that it can be removed from the patient. Suitable mucolytic agents may include, without limitation, N-acetyl-Lcysteine (MUCOSIL ™; Dey Laboratories) and recombinant humanase (PULMOZYME®; Genentech, Inc). Any administered vasoconstrictor or mucolytic agent should be allowed sufficient time to act upon administration, such as two to five minutes.

The following methods and materials can be used to collect nasal andaranasal mucus samples. First, the individual is prepared to receive the collection solution in at least one nostril or nasal-paranasal cavity by instructing to inhale and lower the chin, or otherwise prevent the escape of fluids out of the mouth down into the esophagus. For vertically sitting or standing individuals, these maneuvers tend to minimize the loss or swallowing of the collection solution. Other maneuvers are also possible provided that this goal is achieved. Secondly, an injection and collection system is provided. Generally, the configuration is such that the collection solution can be administered to the subject's nostrils and then effectively withdrawn into the container. The injection system may be, without limitation, a syringe with a curved blunt needle or an assembly tube. The container may be any type of container that retains the liquid. In addition, the reservoir may be, without limitation, a reservoir that is suitable for use as a transport or sealing apparatus so that the sample taken can be handled or transported. These containers may also contain substances such as preservatives or antibacterials depending on the desired use of the sample taken. Third, the collection solution is administered to the subject's nostril and is collected. Prior to administration, the subject may be instructed to expel the collection solution after sensing that the fluid is in the nasal-paranasal cavity. During administration, the collection solution may be forcibly injected into at least one nostril or half of the nasal-paranasal anatomy. The volume of the collection solution may vary depending on the individual or the condition of the mucositis. For example, the volume of the fluid may be, without limitation, between 0.1 ml to 100 ml or more, and in particular between 0.1 ml and 25 ml. The collection solution may be, without limitation, saline, water, or any other suitable solution suitable for contact with mucosal tissue. In addition, the collection solution may contain other substances which may be useful for mucus collection such as a mucolytic substance.

One objective of the collection solution is to release and remove the mucus from the cavities containing the mucus. In addition to acting as a natural washing agent, the penetrating effect of the mucolytic agent in the collection solution can help liquefy thick obstructive mucus. Further, the combination of feed force along with almost simultaneous extrusion under pressure can help release and collect the mucus. Typically, the collection solution may be administered over a period of less than 5 seconds to one side. In addition, the collection solution may be administered over a period of less than 3 seconds to one side. Alternatively, the time period for administering the collection solution may be extended to more than 5 seconds depending on specific factors such as the degree of inflammation, the presence of obstruction, and the size of the individual. In addition, longer than 5 seconds administration can be utilized when very small volumes or strands of collection solution are required.

Other collection procedures may also be used to collect mucosal samples, especially if the individual is unable to accommodate or manage the liquid collection procedure. Such other procedures are well known in the art and include, without limitation, surgical removal of the mucus, swab or mechanical mucus recovery procedure, and pressure or vacuum systems that extract mucus. In addition, these additional collection procedures, as well as the methods and materials described herein, may be modified or modified to obtain biological fluids from other areas of the body, such as the middle ear or intestine.

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After the mucus is collected, the sample can be analyzed for the presence of markers that indicate involvement of non-invasive fungal-induced mucositis. For example, a mucus sample may be examined for the presence of allergic mucus. In addition, fungal organisms can be cultured and analyzed from a mucosal sample using the techniques described herein as well as techniques known in the art.

Figures 3 and 4 show an example of a tool 10 for aspirating and collecting mucus and other fluids. The tool 10 is comprised of an upper member 12, a sample container 14 and a collection tube 16. The upper member 12 then comprises a central portion 22, a screw portion 24, a connecting portion 26, and a tube receiving component 28. The central portion may generally define an opening 29 therein. The flap 30 is operatively provided in the aperture 29. The screw portion 24 may extend downward from the central position 22. The connecting portion 26 extends radially from the central position 22, is generally circular in cross-section in this embodiment, and defines a channel 32. The channel 32 connects external the tool part 10 with its internal part. The component receiving tube 28 generally extends radially from the central portion 22. The component receiving tube 28 is disposed generally on the opposite side to the connecting portion 26 in this embodiment. The tube receiving the component 28 defines channel 34. As with channel 32, channel 34 connects the external part of the tool 10 to its internal part.

In this embodiment, the cartridge 14 is mounted on the screw portion 24. However, the cartridge 14 may be secured to the central position 22 in any known manner. Although a conically shaped bottom of the configuration is shown, the cartridge 14 may have different configurations and will be valid within the scope of the present invention. The collecting tube 16 extends away from and is disposed in the channel 34. The length of the tube 16 may be positioned within the interior of the container 14 to facilitate insertion of the collected material. The collecting tube 16 defines a lumen 36 through which the collected mucus travels. In one embodiment, the tube 16 is a flexible memory diameter to facilitate adaptation to a variety of patient anatomy. That is, the tube remains in the desired and flexible configuration, for example as shown by the dashed line in Figure 3. Other means to facilitate passage of the mucus through the collection tube and the tool 10 may include a tube or device having a material of such a nature that adherence is minimized. torment to him.

In one embodiment, the tool 10 is disposable. The tool 10 can be made of many materials, but synthetic rubbers such as polyethylene can be used. The coupling portion 26 connects the apparatus 10 to the vacuum hose 38. Thus, the coupling portion 26 may have an external configuration such that the result is a tight fit to the vacuum hose. The flap 30 adjusts the amount of vacuum that is in contact through the lumen 36. The flap 30 may be provided

increasing or decreasing amount of vacuum applied. In this example, the shutter 30 typically includes a tapered slot having a sliding portion. The sliding portion may be modified by another user to expose all, no, or only a portion of the tapered slit, thereby adjusting the vacuum passing through the lumen 36. However, other means for adjusting the vacuum control are also applicable to the present invention. Another example includes a "IV" type of flap that uses a cylindrical flap to adjust the thrust or collection tube.

In contrast to other tools, the tube receiving component 28 and the collecting tube 16 extend generally perpendicularly from the longitudinal axis of the cartridge 14. This allows users to better adjust the position of the collecting tube 16 when mucus or other fluids are obtained. It is known that other collecting containers that comfortably conform to the patient's facial anatomy and which can be held in place by the patient or health care provider are valid within the scope of the present invention. Such embodiments may depend on vacuum, severity, or other collection mechanisms, provided that they allow rapid entry for the fluids to be injected to the patient, while simultaneously allowing drainage or removal of fluids and mucus from the patient.

In this embodiment, the lumen 36 has a diameter of 1 mm to 10 mm and a total length of 5 to 50 cm. The cartridge 14 of the example generally has a diameter of 1 "to 3" and a length of 3 "to 6", although various other sizes may be useful.

The instrument 10 is preferably used to obtain mucus and fluid samples from a patient's nasal, paranasal, or pulmonary anatomy. To obtain mucus or fluid samples, the tool 10 is connected to a vacuum source and the flap 30 is adapted as desired. Tube 16 is configured to the desired position. Then, the tube 16 is inserted into a portion of the patient's anatomy from which the mucus or fluid is to be removed. The flap 30 is further adapted as needed to obtain a sample while ensuring patient safety. The obtained mucus or fluid is collected in the container 14. Once the collection is complete, the container 14 can be released from the upper portion 12 for storing or transporting the obtained mucus or fluid sample.

Cultivation of fungal organisms from mucus samples

A mucus sample can be prepared for culturing fungal organisms by treating the sample with a mucolytic agent such as N-acetyl-L-cysteine or dithiothreitol (DTT) to enhance or facilitate further liquefaction of the mucus. After addition of the mucolytic substance, the mucus sample may be mixed and incubated at room temperature. This liquefaction allows the release of fungal organisms present in the mucus. Once liquefied, the mucus can be isolated by centrifugation or other means, since the mucus typically forms a layer separated from the remainder of the solution ( i.e. from the collection solution). Once isolated, the mucus may be mixed and part of it placed in a suitable fimgant growth medium such as agar growth medium dishes. A fungal growth medium is any medium that promotes the growth of fungal organisms, including, without limitation, RPMI-1649, Dulbeccos modified eagles medium (DMEM), inhibitory fungal agar (IMA), and Bay agar. The fungal growth medium may contain antibacterial agents (e.g., chloramphenicol and ciprofloxacin) to prevent bacterial growth.

Once the liquified mucus is contacted with a suitable fungal growth medium, the cultures are incubated at an optimum temperature, for example between 20 ° C and 37 ° C and in some cases between 25 ° C and 35 ° C. The optimum temperature can be determined by placing duplicate cultures with the same level of growth at different temperatures. Typically, the cultures are incubated for about 35 days at 30 ° C. Once fungal growth is observed, methods known in the art determine the fungus species and characterize the phenotype and genotype of each fugue isolate. For example, the fungal isolate can be examined to determine drug resistance or drug sensitivity properties.

Treatment and prevention of non-invasive fungal-induced mucositis

Antifungal agents can be administered to a mammal mucosally in an amount, frequency, and for a period effective for the treatment or prevention of non-invasive fungal-induced mucositis. An "antifungal agent" is any agent that is effective against fungal organisms. For example, the antifungal agent is any agent that prevents or kills fungal organisms, such as antifungal polyene macrolides, tetraene macrolides, pentaene macrolides, fluorinated pyrimidines, imidazoles, triazoles, azoles, halogenated phenol ethers, thiocarbamates, and allylamines. In addition, the antifungal agent may be such agents that are inserted between the fungal cell wall components or act as sterol inhibitors. Specific antifungal agents within the scope of this invention include, but are not limited to, amphotericin B, flucytosine, ketoconazole, miconazole, itraconazole, fluconazole, griseofulin, clotrimazole, econazole, terconazole, butoconazole, oxiconazole, sulconazole, saperconazole, voriconazole, tolcophotol, ciclopiopiopi naphthifine, nystatin, natamycin, terbinafine hydrochloride, butenafine, undecylene acid, Whitefield's ointment, propionic acid and caprylic acid as well as those which can be identified as antifungal agents by methods known in the art. It is known that a particular patient can catch a fungal organism that is resistant to a particular antifungal agent. In such a case, an important aspect of the invention comprises treating such a patient with an effective antifungal agent (e.g.

this antifungal agent that inhibits growth or kills a fungal organism acting as an etiological agent). Such fungal organisms acting as etiological agents can be identified using the collection or culture methods described herein.

The term "mucosal administration" as described herein refers to any type of administration in which the administered substance is placed in contact with the mucus. Therefore, any intravenous substance that does not exit the bloodstream is not considered a mucosal substance because the substance is not in contact with the mucus. In addition, the term "mucosal administration" may be divided into "direct" and "indirect" mucosal administration. The term "direct mucosal administration" as used herein means any type of administration in which the administered substance is placed in direct contact with the target mucus prior to passing through the epithelium. For purposes of this invention, it is understood that injection of a substance into a cavity containing a mucus is considered to be a direct mucosal administration when the substance is in contact with the mucus, although an injection device (eg a needle, tube or catheter) can be used to pass through the epithelium. Thus, the use of a needle to bypass the tympanic membrane and inject the substance into the middle ear is considered to be a direct mucosal administration that targets the middle ear mucus.

It follows that any intravenous substance that subsequently exits the bloodstream, permeates the epithelium and comes into contact with the mucus is not considered to be directly mucosal, because the substance passes through the epithelium before it is in contact with the mucus. In this case, however, the administered substance is considered to be an indirectly mucosal administered substance because the term "indirect mucosal administration" means any type of administration that places the administered substance in contact with the target mucus only after the substance has passed through the epithelium. Again, the use of injectables such as a needle, tube or catheter to deliver a substance across the epithelium does not necessarily mean that administration is indirect mucosal administration.

It also follows that oral administration can be either direct or indirect mucosal administration depending on the target mucus. For example, the substance may be swallowed and then travels through the esophagus, stomach and small intestine to come into direct contact with the mucus in the large intestine without passing through the epithelium (i.e., direct mucosal administration). Similarly, the orally administered substance may be absorbed in the intestine, accumulate systemically, and permeate through the nasal epithelium to come into contact with the nasal mucus (i.e., indirect mucosal administration). Therefore, the direct and indirect nature of mucosal administration depends on the specific route of administration as well as the specific location of the target mucus. A typical mode of direct and indirect mucosal administration for various mammalian mucus locations is described below.

The effective amount of the antifungal agent or composition comprising the antifungal agent can be any amount that reduces, prevents, or eliminates non-invasive fungi

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Induced mucositis after mucosal administration to a mammal without causing significant toxicity in the mammal. Typically, the effective amount may be any amount greater than or equal to the minimum inhibitory concentration (MIC) for a fungal organism or isolate present in a particular mucus of an individual and which does not induce significant toxicity in the individual after mucosal administration. Some antifungal agents may have a relatively large concentration range that is effective, while others may have a relatively narrow concentration range. In addition, the effective amount may vary depending on the specific antifungal organism or isolate, since certain organisms and isolates are more or less sensitive to a particular antifungal agent. Such effective amounts may be determined for individual antifungal agents using generally available or readily detectable information including antifungal effective concentration, toxic concentration in animals, and degree of tissue permeability. For example, non-toxic antifungal agents may typically be administered, directly or indirectly, mucosal in any amount that exhibits antifungal activity in the mucus. In addition, antifungal agents that do not cross the mucosal epithelium can typically be administered directly mucosal in any amount that exhibits antifungal activity in the mucus. Using the information provided herein, these effective amounts can also be determined by routine in vitro and i vivo experiments. For example, a patient exhibiting a non-invasive fungal-induced mucositis condition may receive directly a mucosally administered antifungal agent in an amount close to the MIC calculated from in vitro analysis. If the patient does not respond, then the amount may be increased, for example, ten times. Upon receiving this higher dose, both patient responsiveness and signs of toxicity can be monitored and the dosage adjusted accordingly.

For amphotericin B, an effective amount of 0.01 ng to 1000 mg per kg of mammal body weight per administration may be effective for direct mucosal administration. When used as a nasal wash solution, the effective amount may be 0.01 ml to 1 L per nostril per administration of a solution containing 0.01 mg amphotericin B per liter to 1000 mg amphotericin B per liter. Alternatively, an effective amount may be 20 ml per nostril per administration (eg, two to four times daily) of a flushing solution containing 100 mg amphotericin B per liter of saline or water. Typically, saline or water is sterile. The effective amount may remain constant or may be adjusted as a moving scale or variable dose depending on the individual response to the treatment. Effective amounts for other antifungal agents can be ascertained by one of ordinary skill in the art using routine experimentation with respect to the numerous teachings described herein.

Typically, an effective amount of any directly mucosal antifungal agent (eg, itraconazole, ketoconazole, and voriconazole) may be 0.01 ng to 1000 mg per kg body weight of a mammal per administration. The MIC values for voriconazole are in the range of 0.003 pg / ml to 4 pg / ml depending on the specific fungal organism or isolate being tested. For fluconazole, the MIC values range from 0.25 pg / ml to more than 64 pg / ml.

In order to help determine the effective amount of the various antifungal agents, it may be useful to refer to the equivalent of the effective amount based on the effective amount of the conventional antifungal. For example, direct mucosal administration of about 20 mL of amphotericin B flushing solution containing 100 mg amphotericin B per liter to the nostril for administration (eg, twice a day) is shown herein as an effective amount. The effects achieved by this effective amount can be used as a reference point to compare the effects observed with other antifungal agents used at various concentrations. Once a consistent effect is observed, a specific effective amount for that particular antifungal can be determined. In this case, this amount will be named equivalent to an effective amount of amphotericin B.

Various factors may influence the actual effective amount used for a particular application. For example, the frequency of mucosal administration, duration of treatment, combination with other antifungal agents, and anatomical formation of the area to be treated may require an increase or decrease in the effective effective mucosal amount administered.

The frequency of mucosal administration may be any frequency that reduces, prevents, or eliminates non-invasive fungal-induced mucositis in a mammal without producing significant mammalian toxicity. For example, the frequency of mucosal administration may be from twice daily to once weekly. In addition, the frequency of mucosal administration may remain constant or may change during treatment. Like the effective amount, various factors may affect the frequency of mucosal administration used for a particular application. For example, an effective amount, duration of treatment, combination with other antifungal agents, site of administration, degree of inflammation, and anatomical formation of the area to be treated may require an increase or decrease in the frequency of mucosal administration.

The effective duration of the mucosal administration of the antifungal agent may be any duration that reduces, prevents or eliminates non-invasive fungal-induced mucositis in a mammal without causing significant mammalian toxicity. Thus, the effective duration may be in the range of several days, weeks, months or years. Usually, the effective duration for treatment with non-invasive fungal-induced mucositis is in the range of several days to several months. However, once the antifungal application is stopped, non-invasive fungal-induced mucositis can return. Therefore, the effective duration period for the prevention of non-invasive fungal-induced mucositis may in some cases be throughout the life of the individual.

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For those anatomical regions that are less sensitive to repopulation factors of fungal organisms (eg, human middle ear with intact tympanic membrane), the effective duration may be in the range of 10 to 30 days. In less sterile environments such as the nasal paranasal region, the effective duration can range from 30 days to more than 80 days. In the respiratory or digestive tract, the effective duration can range from 10 days to 30 days, or even more than 90 days. Again, prophylactic treatment typically takes a longer course and may last for the lifetime of the individual.

Numerous factors can influence the actual effective duration used for a particular treatment or prophylactic regimen. For example, the effective duration may vary with the frequency of administration of the antifungal agent, the effective amount of the antifungal agent, the combination of the various antifungal agents, the site of administration, the degree of inflammation, and the anatomical formation of the area to be treated. Furthermore, the specific antifungal agent used may affect the actual effective duration. For example, an effective duration for treatment with non-invasive fungi-induced rhinosinusitis may be 30 days for amphotericin B and about 7 days for itraconazole.

It is understood that diagnostic algorithmic methods may be devised to determine or express the effective dose, duration and frequency of administration.

Compositions comprising at least one antifungal agent Compositions comprising at least one antifungal agent may be in any form provided that the composition can be administered mucosally in amounts, frequencies, and for a time effective to prevent, reduce or eliminate non-invasive fungal-induced mucositis. For example, the composition of the present invention may be in solid, liquid and / or aerosol form, including forms such as, but not limited to, powder, crystalline substance, gel, paste, ointment, cream, solution, suspension, partial liquid, spray, spray , a fabric nebula, atomized steam, tincture, pill, capsule, tablet, and gel capsule. In addition, the composition may contain a cocktail of antifungal agents. For example, the composition of the present invention may include, without limitation, one, two, three, four, five or more different antifungal agents. Further, the composition of the present invention may contain other ingredients including, but not limited to, pharmaceutically acceptable aqueous ingredients, pharmaceutically acceptable solid ingredients, steroids, mucolytic agents, antibacterial agents, anti-inflammatory agents, immunosuppressants, dilatants, vasoconstrictors, decongestants, leukotriene inhibitors , anticholinergic agents, antihistamines, therapeutic agents and combinations thereof. In addition, the composition may contain any one or more substances known to be effective in suppressing the mammalian reflexes reflex.

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For example, the pharmaceutically acceptable aqueous additive may be any liquid solution that is capable of dissolving the antifungal agent and is not toxic to the particular individual receiving the formulation. Examples of pharmaceutically acceptable aqueous additives include, without limitation, saline, water, and acetic acid. Typically, the pharmaceutically acceptable aqueous ingredients are sterile. Pharmaceutically acceptable solid additives may be formulated such that the antifungal agent is suitable for oral administration. For example, capsules or tablets may contain:

An antifungal agent in enteric form. The dose provided by each capsule or tablet may vary because an effective amount may be achieved by administering either one or more capsules or tablets. Any well known pharmaceutically acceptable material such as gelatin and cellulose derivatives can be used as pharmaceutically acceptable solid ingredients. In addition, the pharmaceutically acceptable solid additive may be a solid carrier, including, without limitation, starch, sugar or bentonite. Further, the tablet or pill formulation of the antifungal agent may be followed by conventional procedures using solid carriers, lubricants and the like.

Steroids can be any substances containing a hydrocyclopentanophenanthrene ring structure. Examples of steroids include, without limitation, prednisone, dexamethasone and hydrocortisone. The mucolytic substances can be any substances that liquefy the mucus. Suitable mucolytic agents may include, but are not limited to, N-acetyl-L-cysteine (MUCOSIL ™; Dey Laboratories) and recombinant humanase (PULMOZYME®; Genentech, Inc). The antibacterial agents can be any agents that are effective against bacteria such as penicillin, erythromycin, neomycin, gentamicin, and clindamycin. The anti-inflammatory agent may be any anti-inflammatory agent such as ibuprofen and salicylic acid. The immunosuppressant may be any agent that suppresses or interferes with normal immune functions such as cyclosporin. The dilating agent may be any substance that causes an aperture such as albuterol. The vasoconstrictor may be any substance that contracts or constricts blood vessels such as phenylephrine hydrochloride (NEOSYNEPHRINE®; Sanofi Pharmaceuticals), cocaine and epinephrine. The decongestant may be any substance that acts to reduce nasal-paranasal congestion or swelling such as pseudoephedrine hydrochloride, phenylpropanolamine and oxymetazoline. The leukotriene inhibitor may be any substance that inhibits the function or synthesis of leukotrienes, such as Azelastin®. The anticholinergic agent may be any agent that blocks parasympathetic nerve impulses, such as ipratropium bromide. The antihistamine may be any substance that acts against the action of histamine or against its release from cells (eg, mast cells), such as terfenadine and astemizole.

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The therapeutic agent may be any agent that has a therapeutic effect upon administration. For example, the therapeutic agent may be any agent that blocks or interferes with the interaction of eosinophils with an immunoglobulin bound to a fungal antigen, for example, by targeting the interaction of the Fc receptor or the S-type lectin factor receptor (eg, galectin-3). Such agents may include, but are not limited to, antibodies such as IgE, IgA, IgG, IgM, and IgD, as well as antibody fragments such as Fab, F (ab ') 2, FcγRI, FcγRII, FcαR, FcεRII and FcεRI.

Mucosal administration directed to the nasal-paranasal area

The mucosal administration of the agent to the nasal-paranasal region can be any type of administration that places the agent in contact with the nasal-paranasal mucus. Direct mucosal administration to the nasal-paranasal region may include, without limitation, nasal wash, nasal spray, nasal inhalation, and nasal pouch with, for example, saturated gauze, provided that the administered substance contacts the nasal-paranasal musculature before passing through epithelium. In addition, injection into the nasal-paranasal cavity using, for example, a needle or catheter is considered to be a direct mucosal administration provided that the administered substance comes into contact with the nasal-paranasal mucus upon leaving the needle or catheter but before passing through the epithelium. Any instrument, including, without limitation, a syringe flask, inhaler, can, spray, nebulizer, and mask, can be utilized for direct mucosal administration of the substance to the nasal-paranasal region. For example, a 20 ml flask may be used to moisten the nasal-paranasal area with a liquid formulation of the antifungal agent. Such a liquid formulation can be stored at -20oC, 0oC or at room temperature. When stored at a temperature below room temperature, the formulation is typically warmed to nasal - paranasal cavities prior to administration.

Indirect mucosal administration to the nasal - paranasal regions may include, without limitation, oral, intravenous, intradermal and intraperitoneal administration, provided that the administered substance is in contact with the nasal - paranasal mucus. In addition, any instrument including, without limitation, a syringe and a controlled release capsule may be used for indirect mucosal administration to the nasal, paranasal areas.

It is known that the route of administration may affect the effective amount and duration of antifungal treatment, as well as the frequency of mucosal administration. E.g. an orally mucosal antifungal may require higher concentrations to deliver an effective amount to the nasal-paranasal mucus than direct mucosal administration via nasal washes.

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Mucosal delivery directed to the pulmonary airways The airway is any part of the mammalian anatomy through which air passes during breathing, including the mouth, nasal passages, trachea, bronchi, and bronchial tubes. The pulmonary respiratory tract is any area of the lungs that passes through the air lined with mucosa, including the bronchi and bronchial tubes. The mucosal administration of the agent to the pulmonary airways may be any type of administration that places the agent in contact with the pulmonary airway mucus. Direct mucosal administration to the pulmonary airways may include, without limitation, nasal spray inhalation and nasal lavages, provided that the administered substance comes into contact with pulmonary airway muscles before passing through the epithelium. In addition, injection into the pulmonary airways using, for example, a needle or catheter is considered to be a direct mucosal administration provided that the administered substance comes into contact with pulmonary airway muscles after leaving the needle or catheter and before passing through the epithelium. Any instrument including, without limitation, a syringe, flask, inhaler, nebulizer, aerosol can, spray, and mask can be used for direct mucosal administration of the agent to the pulmonary airways.

Indirect mucosal administration to the pulmonary airways may include, without limitation, oral, intravenous, intradermal, and intraperitoneal administration, provided that the administered substance is in contact with the pulmonary airways after passing through the epithelium. In addition, any instrument, including but not limited to a controlled release syringe and capsule, may be used to indirectly mucosal deliver the substance to the pulmonary airways.

It is known that a particular route of administration may affect the effective amount and duration of antifungal treatment as well as the frequency of mucosal administration. For example, enteric mucosal administration of an antifungal agent may require a higher concentration to deliver an effective amount to pulmonary airway mucus than direct mucosal administration by inhalation through the mouth or nose.

It is understood that direct and indirect mucosal administration to the airways, including trachea, nasal passages and mouth, may be by the methods and materials described herein for the pulmonary airways.

Mucosal administration directed to the middle ear Mucosal administration of the substance to the middle ear can be any type of administration that places the substance in contact with the middle ear mucus. Direct mucosal administration to the middle ear may include, without limitation, ear drops or ear lavages, provided that the administered substance contacts the middle ear mucus before passing through the epithelium. For example, if the tympanic membrane is damaged or otherwise punctured, then the ear irrigation will be «« · · »» · ···. , ·. ··· ·· ί ϊ. . It is considered to be a direct mucosal administration, provided that the substance administered is given by the following: · · * · ován ován ován považ považ ován ován in contact with the middle ear mucus. In addition, injection into the middle ear using, for example, a needle or myringotomy tube is considered to be a direct mucosal administration, provided that the administered substance comes into contact with the middle ear mucus after leaving the needle or tube and before passing through the epithelium. Any instrument can be used to direct mucosal administration of the substance to the middle ear including, without limitation, a syringe and a flask.

Indirect mucosal administration to the middle ear includes, without limitation, oral, intravenous, intradermal and intraperitoneal administration, provided that the administered substance contacts the middle ear mucus after passing through the epithelium. In addition, any instrument can be used for indirect mucosal administration of the agent to the middle ear including, without limitation, a syringe and a controlled release capsule.

It is known that a particular route of administration may affect the effective amount and duration of antifungal treatment, as well as the frequency of mucosal administration. E.g. an orally mucosal antifungal may require higher concentrations to deliver an effective amount to the middle ear mucus than direct mucosal administration by the middle ear.

Mucosal administration directed to the intestine

The mucosal administration of the agent to the gut may be any type of administration that places the agent in contact with the gut mucus. Direct mucosal administration to the intestine may include, without limitation, oral or enema administration, provided that the administered substance contacts the intestinal mucus prior to passing through the epithelium. In addition, injection into the gastrointestinal tract using, for example, a needle or catheter is considered direct mucosal administration, provided that the administered substance comes into contact with the intestinal mucus after leaving the needle or catheter and before passing through the epithelium. Any instrument can be used to direct mucosal administration of the agent to the intestine including, without limitation, a syringe and a controlled release capsule. For example, an antifungal agent may be formulated into a controlled release capsule such that the antifungal agent is released after passing through, for example, the stomach (eg, a pH controlled release and time controlled release capsule).

Indirect intestinal mucosal administration includes, without limitation, oral, intravenous, intradermal and intraperitoneal administration, provided that the administered substance contacts the intestinal mucus after passing through the epithelium. In addition, any instrument can be used to indirectly mucosal deliver the substance to the intestine including, without limitation, a syringe.

It is known that a particular route of administration may affect the effective amount and duration of antifungal treatment, as well as the frequency of mucosal administration. E.g. an orally mucosal antifungal agent may require higher concentrations to deliver an effective amount to the intestinal mucus than direct mucosal administration by enema.

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Other treatments

Other treatments may be used in combination with formulations containing an antifungal agent to help enhance the treatment or prevention of non-invasive fungal-induced mucositis conditions. Such other treatments include, without limitation, surgery and administration of a second formulation. Surgical procedures may include, without limitation, removal of polypoid growth or other swelling, physical opening of the cavity and insertion of a catheter, and the like. The second formulation may include, without limitation, antifungal agents, mucolytic agents, antibacterial agents, anti-inflammatory agents, immunosuppressants, dilatants, vasoconstrictors, decongestants, steroids, anticholinergic agents, leukotriene inhibitors, antihistamines, therapeutic agents, and combinations thereof. In addition, the second composition may be administered to the mammal by any means. For example, the second composition may be delivered to a mammal orally, intraperitoneally, intradermally, intravenously, subcutaneously, intramuscularly, locally, intranasally, and intrabronchially

Treatment and prevention of asthma

Asthma can be characterized by a paradoxical narrowing of the bronchi (lung passages) so that breathing becomes difficult. Subjects suffering from asthma exhibit symptoms such as whistling, difficulty breathing (especially air displacement), shortness of breath and tightness in the chest. Factors that may worsen asthma include rapid changes in temperature or humidity, allergies, upper respiratory tract infections, exercise, stress and smoking. Subjects suffering from asthma can be identified using any of the well-known techniques. In general, asthma can be diagnosed, without limitation, by a pulmonary function test (increased airway resistance) or without airway provocation (eg, a methacholine challenge test), lung x-ray, and chest listening.

Subjects at risk of developing asthma may include, without limitation, those who have had asthma attacks in the past. In addition, elderly individuals, individuals with cystic fibrosis, chronic rhinosinusitis with or without nasal paranasal polyps, aspirin sensitivity or a family history of respiratory problems or allergies, and individuals exposed to significant levels of allergens (eg fungal spores, pollen and chemicals) or irritants.

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Subjects with chronic asthma may be treated by direct mucosal administration of the antifungal agent to at least a portion of the airways in an amount, frequency, and duration that is effective in reducing or eliminating asthma symptoms. Such direct mucosal administration may be similar to the methods and materials described herein in the treatment and prevention of non-invasive fungal-induced rhinosinusitis, since the nasal-paranasal sinuses are the airways. For example, nasal sprays and nasal washes may be used to direct mucosal administration of an antifungal to the airway mucus. In addition, subjects with chronic asthma may be treated by direct mucosal administration of an antifungal agent to at least a portion of the pulmonary airways in an amount, frequency, and duration that is effective in reducing or eliminating asthma symptoms. For example, aerosol or powder forms of the antifungal may be directly mucosal administered to the pulmonary airway by inhalation through the mouth or nose.

Further, individuals at risk of chronic asthma may be prophylactically treated by mucosal administration of an antifungal agent to at least a portion of the airways in an amount, frequency and duration that is effective in preventing asthma symptoms. Again, such prophylactic treatment may be similar to the methods and materials described herein in the treatment and prevention of non-invasive fungal-induced rhinosinusitis.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES OF EMBODIMENTS OF THE INVENTION

Example I - Collection and analysis of mucus samples

The following methods and materials were used to collect and analyze mucus from 202 patients. Prior to collection of the mucus, each patient was directed to inhale and then lower the chin towards the breasts to minimize or prevent the collection solution from flowing through nasal paranasal passages through normal drainage at the back of the throat. The collection solution was either sterile saline or sterile water. In addition, each patient was positioned to minimize or prevent collecting fluid from flowing through the nasal passages. Some patients have been given a vasoconstrictor such as phenylephrine hydrochloride (1-2 injections in the nostril) or cocaine (local fluid or powder, less than four mg per kg body weight). Some patients received about 3 ml of a 20% solution of N-acetyl-L-cysteine in a spray.

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Patients receiving both agents were first given a vasoconstrictor and then after two minutes N-acetyl-L-cysteine.

Once the patient was ready, the collection container was placed under the nostril (s) from which the mucus sample was to be taken. An injection device such as a syringe-like instrument with a tubular assembly or a blunt bent needle was then placed into one of the patient's nostrils or the paranasal area so that the collection solution can be expelled through the patient's nasal-paranasal area. In some cases, 5 to 30 ml of the collection solution was then injected into the nostril over a time interval of 0.5 to 5 seconds. In most cases, 10 to 20 ml of the collection solution was injected into the nostril over a time interval of 0.5 to 3 seconds.

Mostly, each patient blew out or abruptly squeezed the collection solution either simultaneously as it was injected or after sensing their entry into the nostril. This rapid displacement of the injected collection solution significantly contributed to the release of the mucus in the patient's nasal-paranasal lumen. In addition, special care was taken to reduce or prevent the loss of sample volume. Once the mucus-containing collection solution has been expelled from the patient's nostril, it would be collected in a collection container located below the nostril. After the nasal paranasal mucus was removed, the mucus was cultured using the following two methods. In the first method, 1 ml of a 20% N-acetyl-L-cysteine solution was added to about 10 ml of the obtained mucus collection solution. The mixture was then shaken for 30 seconds and incubated for 15 minutes at room temperature. After incubation, the mixture was centrifuged in a 50 ml tube for 5 minutes at 4800 rpm. After separation, the supernatant was removed and the remaining mucus was shaken for 30 seconds. 0.5 ml of isolated mucus was added to each culture dish, one IMA dish containing chloramphenicol and one IMA dish containing ciprofloxacin. The plates were then incubated at 30 ° C and read as a routine fungal culture. Growth of individual isolates was observed after 2 to 35 days.

In the second method, 10 ml of DTT was diluted with 90 ml of sterile distilled water. An equal amount of this freshly diluted DTT solution was added to the obtained mucus collection solution and incubated for 15 minutes at room temperature. After incubation, the mixture was centrifuged in a 50 ml tube at 3000 rpm for 10 minutes. After separation, the supernatant was removed and the remaining mucus was shaken for 30 seconds. 0.5 ml of isolated mucus was added to each culture dish, one IMA dish containing chloramphenicol and one IMA dish containing ciprofloxacin. The plates were then incubated at 30 ° C and read as a routine fungal culture. Growth of individual isolates was observed after 2 to 35 days.

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Once fungal growth was observed, the organism was identified using standard mycological techniques including visual, histological and immunological techniques. Identified fungal genera and species included many fungal organisms previously isolated from AFS patients such as Absidia, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus versicolor, Alternaria, Basidiobolus, Bipolaris, Candida albicans, Candida lypolytica, Candida lypolytica, Candida lypolytica, Conidiobolus, Cunninahamella, Curvularia, Dreschler, Exserohilum, Fusarium, Malbranchia, Paecilomvces, Penicillium, Pseudallescheria, Rhizopus, Schizophylum and Sporothrix. In addition, fungal organisms that have not yet been identified in mucus samples from patients diagnosed with AFS may be non-invasive fungal organisms capable of inducing non-invasive fungal-induced mucositis including, without limitation, Acremonium, Arachniotus citrinus, Aurobasidioum, Beauveria, Chaospomium, Chaetomium Exophilia jeanselmei, Geotrichum, Oidiodendron, Phoma, Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala, Scolebasidium, Scopulariopsis, Ustilago, Trichoderma, and Zygomycete.

To determine the optimal temperature for the cultivation of fungal organisms inducing non-invasive fungal-induced mucositis, liquified mucus samples from two patients were cultured on IMA dishes containing either chloramphenicol or ciprofloxacin. Two dishes (one containing chloramphenicol and one containing ciprofloxacin) for each sample were incubated at 25, 28, 30, 32, 33, 35 and 37 ° C. Each dish was visually inspected for fungal growth and development every other day within two to 35 days of the day of culture. The readings were averaged at each temperature to give an estimate of the optimal temperature for germination of spores and subsequent growth and development of fungal organisms. The results showed that the optimal temperature for fungal growth varied depending on the particular fungal species or isolate. In general, a temperature of 30 ° C promoted the growth of the largest number of fungal species and isolates.

The following procedure was used to determine the effective antifungal agent as well as the effective concentration of the antifungal agent so that the fungal organism isolated from the patient does not grow or is killed.

Seventeen fungal isolates were collected from eight patients with rhinosinusitis and tested for susceptibility to amphotericin B, ketoconazole and itraconazole. Each antifungal agent was tested on these fungal isolates using a macroboril dilution technique according to the protocol of the National Commission for Clinical Laboratory Standards (NCCLS). MIC readings after 48 hours were recorded and interpreted according to NCCLS guidelines by indicating that the culture was susceptible, transient, and antifungal resistant in the test subjects. · · - ···· concentrations. The results of this procedure provided estimates of the efficacy of the antifungal agent against a particular fungal isolate in vitro. In general, the MIC values for these antifungal agents for each isolate were found to be in a wide range from 0.03 to 100 pg / ml (Table I).

Table I. Functional organisms isolated from 64 of the 66 rhinosinusitis patients studied, including 17 MIC values for amphotericin B, ketoconazole and itraconazole in selected isolates from eight rhinosinusitis patients.

Fungální organismismus Number of species / isolates Amphotericin B MIC (pg / ml) Ketoconazole MIC (pg / ml) Itraconazole MIC (pg / ml) Acremonium 1 0.2 1.56 Neg * Altemaiia 40 0.8 3.13 Neg > 5 12.5 Neg Aiachniotus citrinus 2 Aspergillus 21 Aurobasidium 2 Candida 13 0.2 50 0.03 0.4 1.56 neg 0.1 0.39 neg 0.2 > 100 neg Cladosporium 21 3 1.56 neg 0.4 0.05 neg 0.1 0.05 0.125 Chryosporium 1 Epicoccum 6 Exophilium Jeanselmei 2 Fusarium 18 > 5 > 100 35 ° C ** 2 100 ALIGN! 1 > 5 12.5 > 16 Geotrichum 5 0.1 0.05 Deň: 32 ° C Mucor 2 Oididendron 1 Paecilomyces lilacinus 2 Papularia 1 Penicillium 30 0.4 3.13 0.5 1 1.56 0.25 Phoma 1 Pithomyces 2 Rhodoturula 1 Scolebasidium 1 Trichoderma 3 4 50 Deň: 32 ° C Ustilago 2 Unidentified (2 monila, 7 3 dermatous)

*, neg means that the organism did not grow in the test medium (PEG 400); ** 37 ° C means the organism did not grow at 37 ° C.

··· · · · · · ⁴ · · · · · · · · · · · · ·

The following study was performed to determine the frequency of rhinosinusitis with non-invasive fungal etiology. For this study, the following criteria were used to determine whether a patient had rhinosinusitis induced by non-invasive fungi: 1) presence of observable disease in the nasal-paranasal region, 2) presence of allergic mucus, and 3) presence of fungal organisms in nasal-paranasal mucus. Each patient underwent a CT scan using standard procedures to detect the presence of observable disease in their nasal-paranasal area. To detect the presence of allergic mucus, a surgical sample was taken from each patient and examined histologically. It is noted that special care has been taken to collect each surgical specimen to ensure that the mucus specimens have not been washed off. To detect the presence of fungal organisms in nasal-paranasal mucus, the methods and materials described herein were used to collect and cultivate fungal organisms from the patient's mucus.

Seventy-three patients entered the study. The age of these patients ranged from 13 to 73 years, with an average of 50.1 years. 39 out of 73 patients were women and 34 were men. The number of previous rhinosinusitis-related surgeries for each patient ranged from 0 to 25, with an average of 3.41 surgeries per patient. 70 of the 73 patients had previously had recurrence of polyposis and rhinosinusitis.

Seven patients were subsequently excluded from the study due to lack of an acceptable mucus sample. Of the remaining 66 patients, 66 (100%) were diagnosed as CT-scan positive, 62 (94%) were diagnosed positive for allergic mucus and 64 (97%) had positive fungal cultures. Overall, 60 (91%) of the 66 rhinosinusitis cases had all three criteria. In other words, 91% of the 66 rinosinusitis patients evaluated had rinosinusitis induced by non-invasive fungi based on the above criteria. These 91% represent a dramatic increase in the number of cases of rhinosinusitis involving non-invasive fungal organisms. For example, numerous medical research articles report that about 3-8% of chronic rhinosinusitis cases requiring surgery are AFS cases, rhinitis states having a non-invasive fungal etiology. Thus, the results presented here show that the involvement of non-invasive fungal organisms in rhinosinusitis is much more widespread than originally recognized.

A total of 25 different fungal species were identified from mucus samples of patients with this non-invasive fungus induced by rhinosinusitis. 16 organisms that were never described in association with AFS were detected from 64 mucus specimens that showed fungal growth. The range ranged from 1 to 7 fungal organisms per patient with an average of about 2.9 fungal species per patient. In 63% of cultures Alternaria was in 47%

9 • 9

9 *

• · • 9 ♦ ·

Penicillium, 33% Cladosporium, 33% Aspergillus, 28% Fusarium and 20%

Candida.

In a separate study, the mucus of 12 control subjects (i.e., subjects without chronic rhinosinusitis) was collected and analyzed as described herein. All 12 (100%) had positive fungal cultures. Specifically, seven different fungal organisms were cultivated with an average of 2.25 different fungal organisms per person and range from 1 to 4. In 50% of cultures, Cladosporium, 42% Altemaria, 33% Geotrichum, 33% Aspergillus, 25% Penicillium , 8% Acremonium and 8% Candida. These results show that fungal organisms live in the nasal-paranasal mucus of most, if not all, humans.

Example 2 - Treatment and prevention of non-invasive fungi-induced rhinosinusitis

132 patients with rhinosinusitis were enrolled in the study to evaluate the use of an antifungal agent to treat non-invasive fungi-induced rhinosinusitis. After diagnostic analysis, 125 (95%) out of 132 patients had the following criteria: 1) presence of observable disease in the nasal-paranasal region as evidenced by CT scan, 2) presence of allergic mucus as evidenced by histological examination of the surgical specimen and 3) organism in nasal-paranasal mucus, as it has been able to cultivate a fungal organism from a mucus sample. 125 patients with rhinosinusitis-induced non-invasive fungi received antithungal treatment with 20 ml of amphotericin B solution per nostril, two to four times daily for at least 3 months. The concentration of amphotericin B in the solution was 100 mg per liter of saline or water. The patient used a 20 ml flask to mucosally administer the amphotericin B solution to the nasal-paranasal area of the patient. The data were processed for 53 patients who returned for analysis of their 3 month follow-up. In addition to patient questioning, CT scan analysis, visual examination, and fungal culture analysis, two types of assessments were used to assess the success of treatment: endoscopic and patient symptom assessments. These assessments were assessed as follows:

Endoscopic evaluation:

Grade 0: no evidence of disease

Grade 1: polypoid changes / polyps observed only by endoscopy Grade 2: polyps in the middle meat Grade 3: polyps filling the nasal cavity

Assessment of patient symptoms:

ί · · · ::. . · · · ·

· .. · .. · ..........

Grade - 2: very bad / much worse

Grade - 1: poor / worse

Grade 0: basal level / unchanged

Stage 1: good / improved

Grade 2: very good / no symptoms

Endoscopic evaluation revealed that 33 of 53 patients had recovered from grade 2 or grade 3 to grade 3 after 3 months. 6 of these 33 non-disease cases were confirmed by CT scan. For example, one recent non-steroid non-surgical patient was diagnosed with bilateral rhinosinusitis and a CT scan revealed bilateral involvement (Figure)

1). The patient then received 20 ml of amphotericin B solution (10 mg / l) per nostril as a treatment. After 4 months of continuous antifungal treatment, a CT scan was performed and showed complete disappearance of opacity and symptoms characteristic of rinosinusitis (Figure 2).

out of 53 patients recovered from condition 2 or 3 according to endoscopic evaluation to condition 1 after 3 months. The remaining 9 patients did not respond to treatment. Five of the 9 non-responders had previously collected mucus samples available for evaluation. Analysis of these five available samples showed that all 5 patients had fungal organisms in their mucus that were resistant to amphotericin B, an antifungal agent used in treatment.

Patient symptom assessment revealed that 44 out of 53 patients had a grade 2, 3 out of 53 grade 1 and 6 out of 53 grade 0 treatment after treatment. 9 patients who had grade 1 or 0 were the same as 9 patients who had endoscopic evaluation showed no response, 5 of which were shown to have fungal organisms resistant to amphotericin Β. A subsequent review of other patients showed that several mismatched patients did not have fungal organisms resistant to amphotericin B.

In addition, several patients had mucus collected and analyzed before and after antifungal therapy. Comparing the results from the evaluation of mucus samples before and after antifungal treatment, it was found that the number of different jungal species was remarkably reduced in these patients after antifungal treatment, as determined by cultivation techniques of the jungal organisms. Thus, patients with rhinosinusitis were asymptomatic and had fewer fungi in their mucus after treatment with an antifungal agent.

In a separate unique study, a patient with rhinosinusitis in the left paranasal sinus was diagnosed because the CT scan showed an inflammatory disease characterized by turbidity associated with rhinosinusitis in the left paranasal sinus. The Alternative RAST test showed 6.23 pounds of units per liter (KU / 1) and bilateral fungal cultures confirmed the growth of Altemaria in each nostril. However, only the left nasal-paranasal side was operated together with an intra-operative and post-operative treatment of 20 ml amphotericin B (100 mg / L) solution two to four times daily. At each post-operative visit, the left paranasal sinus of the patient was free of disease. However, the RAST reading taken 8-10 weeks after the disappearance of rhinosinusitis symptoms in the patient's left sinus was 7.16 KU / L. This represents an increase compared to the first RAST reading. 6 months after the operation, the patient was diagnosed with rhinosinusitis in the right paranasal sinus based on a CT scan and a RAST reading of 10.0 KU / 1 on Altemaria. After surgery of the patient's right paranasal sinus and after antifungal treatment on both sides with 20 ml amphotericin B (100 mg / l) solution to the nostril two to four times daily for 7 weeks, the patient remained symptom free and had a RAST reading of 4.47 KU / 1. . Six months after the last operation, the patient remained disease-free as evidenced by a CT scan.

Together, these results indicate that appropriate flushing with a properly administered antifungal agent on one side results in the prevention of an inflammatory process on that side. In addition, the previously detected fimgal load on the originally untreated side (right side) was sufficient to eventually induce the presence of visual or palpable symptoms of rhinosinusitis on that originally untreated side. Furthermore, fungal organisms present on this originally untreated side (right side) induced high titers of IgE, as shown in IgE readings from RAST assays, independent of concurrent reduction of fungal organisms due to antifungal treatment applied on the left side. In this case, a reduction in IgE readings from RAST tests was observed only after flushing both sides with an antifungal agent.

Thus, IgE reduction and prevention of disease symptoms coincided with treatment with an antifungal agent on both sides.

To further evaluate the use of the antifungal agent in the treatment of non-invasive fungal-induced rhinosinusitis, patient information was collected for each patient returning to the doctor's office after a week period. Only those patients who were previously seen and instructed in the use of nasal washes with antitungal amphotericin B were admitted to this study.

During the one-week period, 20 patients returned to the doctor's office (Table II). The mean age of returning patients was 47 years (range 16-74 years). Patients used amphotericin B washes for an average of both 6 months (range 1-16 months). Some patients have had a nasal surgery in the last month, others have never had such a surgery. In addition, some patients used topical or systemic steroid therapy. In addition, some patients have taken antibiotic nasal washes in addition to anti-fungal washes. The antibacterial solution contained 80 mg gentamicin per liter of saline (Wilson's solution).

• Φ · · · · · · «·« · «·« · «

Some patients mixed the antibacterial solution with the antifungal solution and then performed a nasal wash, while others took each of the solutions separately in sequential order. Some patients also had other diseases including asthma (15 out of 20 patients) and colitis (2 out of 20 patients).

After endoscopic evaluation, most patients had a noticeable improvement in their condition by non-invasive fungal-induced rhinosinusitis. These observable improvements correlated with the improvement in symptom scores reported by patients. One patient stopped amphotericin B nasal washouts after two months. After 8 months, the patient showed recurrent symptoms of non-invasive fungal-induced rhinosinusitis. Two other patients jumped from amphotericin B solution (duration: 3 months; frequency: twice daily) to itraconazole solution (duration: 1 month; frequency: twice daily). One felt better after only seven days of taking itraconazole solution. Together, these results indicate that antifungal agents can be effectively used in the treatment of non-invasive fungal-induced rhinosinusitis.

Table II: Patient data obtained during the weekly period

Age Duration (Amfo) Frequency Last operation Steroid therapy Other diseases Endoscopic score Score flags (B) AND (B) AND 59 3 months ¹ 2 times a day 2 months No asthma ON ON -1 1 40 1 month (s) 2 times a day No Yes ⁸ Asthma Colitis 2 1 -1 +2 63 4 months 1x daily 4 months No Colitis 1 0 -1 +1 16 12 months ² 1 x daily 26 měs Local Asthma 2 0 -1 +2 44 12 months ² 2 times a day 9 months No Asthma 3 0 -1 +1 40 2 months ³ 2 times a day 12 months No No 3 3 -1 0 23 16 měs 2 times a day 10 months No Asthma 3 0 -1 +1 48 4 months 2 times a day > 10 years Local No 2 0 -1 0 50 4 months 2 times a day 4 months No Asthma 1 0 -1 +1 45 2 months 2 times a day No Local Asthma 1 ON -1 0 74 2 months 2 times a day > 4 years Local No 2 ON -1 +1 57 2 months 2 times a day 1 month (s) system local Asthma 2 0 -1 +2 16 12 months ⁴ 1 x daily 12 months No Asthma ¹² 3 0 -1 +2 71 7 months 2 times a day > 6 years No Asthma 3 3 -2 -2 38 12 months 2 times a day > 6 years Yes ⁵ Asthma 3 3 -1 0 38 5 months 2 times a day 28 měs No Asthma 0 0 -2 +2 66 13 měs 2 times a day > 4 years Local No 3 3 -2 0 70 3 months ⁵ 2 times a day > 2 years System. ¹⁰ Local Asthma 3 3 -2 +1 32 3 months ⁶ 2 times a day 5 months No Asthma 3 1 -2 +2 47 2 mes' 2 times a day > 3 years System. ¹¹ local Asthma 3 0 -2 0

B, prior to antifungal treatment; A, after antifungal treatment • 9 · *

9 »♦

9 9 ·

9 · ·

99

Also washed with Wilson's solution (80 mg gentamicin per liter of saline) twice daily also washed with Wilson's solution stopped nasal washes 8 months ago and the disease returned also washed with Wilson's solution (80 mg gentamicin per liter of saline) ) He felt better once a day seven days after switching from amphotericin B flushing solutions (duration: 3 months; frequency: twice daily) to itraconazole (duration: 1 month; frequency: twice daily).

changed rinses to itraconazole (duration: 1 month; frequency: twice daily) after 3 months on amphotericin also washed with Wilson's solution (duration 1 year) received 1 month ago Kenalog 40IM Medval Dose Pack received 6 months ago Kenalog Shot got Prednison for 1 week had systemic steroid treatment for 3 years stopped taking theophylline and tylade after the onset of antifungal washouts

Example 3: Treatment and prevention of non-invasive fungal-induced rhinosinusitis in patients without previous nasal surgery

The following 3 patients with non-invasive fungi induced by rhinosinusitis did not undergo previous nasal surgery.

A 61 year old woman was diagnosed with non-invasive fungi-induced rhinosinusitis and the patient was instructed to flush amphotericin B twice daily. Endoscopic analysis prior to initiation of treatment revealed that her nasal cavity was filled with polyps (endoscopic score 3) and the patient evaluated herself for a symptom score of -1. After taking amphotericin B washes for 14 months, endoscopic analysis revealed no evidence of disease (endoscopic score 0) and the patient achieved a symptom score of +2.

A 64 year old woman was diagnosed with non-invasive fungi-induced rhinosinusitis and the patient was instructed to flush amphotericin B twice daily, which was later changed to 4 times daily. Endoscopic analysis prior to initiation of treatment revealed polypoid changes (endoscopic score 1) and the patient evaluated herself for symptom score of -1. After taking amphotericin B washes for 16 months, endoscopic analysis revealed no evidence of disease (endoscopic score 0) and the patient achieved a symptom score of +2.

A 54-year-old man was diagnosed with non-invasive fungi-induced rhinosinusitis and the patient was instructed to flush amphotericin B twice daily. The patient received an intramuscular steroid injection every 3 to 8 months, with the last injection given about 7 months before amphotericin B flushes. Endoscopic analysis prior to treatment did not reveal disease evidence (endoscopic score 0), but the patient evaluated himself for a symptom score of -1. After amphotericin B washes for 4 months, endoscopic analysis revealed no evidence of disease (endoscopic score 0), but the patient achieved a symptom score of +1.

Example 4 - Reduction of eosinophilia using antifungal therapy

A 67 year old woman was diagnosed with non-invasive fungi-induced rhinosinusitis and the patient was instructed to flush amphotericin B twice daily. After 9 months of amphotericin B washes, the patient underwent sinus surgery for further improvement. During the operation, mucosal biopsies were taken and the number of eosinophils was compared with the numbers from the biopsies obtained from the patients during surgery but before antifungal treatment.

The number of eosinophils in all mucosal biopsies of all sinus, except frontal, was reduced (<5%) after antifungal treatment. The number of eosinophils in the frontal sinus biopsy was 10%. In addition, allergic mucus was present in the frontal sinus, probably because amphotericin B lavage did not enter the frontal sinus due to obstruction of the frontal sinus. Thus, previously observed hypereosinophilia decreased to normal in all treated areas of the sinus.

Example 5 - Treatment and prevention of chronic asthma symptoms of 53 patients in the study described in Example 2 had previously diagnosed chronic asthma. After 3 months of antifungal therapy, 28 out of 37 asthmatic patients reported improvement or complete resolution of asthma symptoms. Four patients out of these 28 were analyzed using a pulmonary function test after antifungal therapy, since they performed a similar test before antifungal therapy. Comparing the results before and after antifungal therapy, it was confirmed that all four of these asthmatic patients had improved pulmonary function. In addition, 26 of the 28 patients who no longer had asthma symptoms discontinued their asthma medication. 23 of these 26 patients used systemic steroids before antifungal therapy for asthma, but none subsequently took steroids after antifungal therapy was started.

In a separate study, 7 asthmatic patients were sampled from lung sputum. Culture analysis of these samples revealed the presence of fungal organisms in each sample. In particular, Candida albicans, Penicillium, Fusarium, Scopulariopsis, Cryptococcus, this were cultivated to this to.

Cladosporium, Aspergillus, Aspergillus fumigatus, Aspergillus nidulans, and yeast. The number of different fungal species cultivated from each sputum ranged from 1 to 5.

Example 6 - Itraconazole preparations

Itraconazole preparations were made by dissolving itraconazole in polyethylene glycol (PEG) to obtain a stock solution of itraconazole. Itraconazole was obtained from 100 mg itraconazole capsules (Janssen Pharmaceutica, Inc). Typically, PEG 400 was used to dissolve itraconazole. Once dissolved, the stock solution was filtered to remove any insoluble material. The stock solution was then ready for use by dilution with sterile water.

Specifically, twenty 100 mg itraconazole capsules were opened and the itraconazole beads were placed in a calibrated cylinder. One liter of heated (70 ° C) PEG-400 was added to a calibrated cylinder containing itraconazole. The mixture was then placed on a stirred heated plate and continued at 70 ° C for 30 minutes. After 30 minutes, the hot mixture was filtered into a glass vial and allowed to cool to room temperature. Once cooled, 100 ml of the filtered solution was placed in an empty plastic vial. Then 900 ml of sterile water was added and the mixture was shaken. After shaking, one drop of flavoring (peppermint oil) was added. This procedure typically resulted in a solution containing from 98.8 µg to 111 µg of itraconazole per ml.

The following itraconazole concentrations were determined by HPLC for each of the above solutions (Table III).

Table III. Itraconazole concentration in solution.

Solution Concentration of soluble itraconazole (pg / ml) 2000 mg itraconazole from capsules to 11 PEG-400 (stock solution) 1839 100 ml stock solution diluted with 900 ml sterile water 113 500 mg itraconazole powder 'AS' into 250 ml PEG-400 (stock 1951 solution) 100 ml stock solution diluted with 900 ml sterile water 85 2000 mg itraconazole from capsules to 11 PEG-400 (stock solution) 100 ml stock solution + 150 ml PEG-400 diluted with 750 ml sterile water 179 2000 mg itraconazole from capsules to 11 PEG-400 (stock solution) 100 ml stock solution + 25 ml PEG-400 diluted with 875 ml sterile water 155

• tt • ttt tt

Tt tt

Tttt tttt tt

Itraconazole containing steroid was also produced. Specifically, the content of two PULMICORT 200 µg inhalers (total of about 91 µg budesonide) was added to the itraconazole PEG-400 stock solution at 70 ° C for 15 minutes. Budesonide was added about 5 minutes after itraconazole powder had dissolved in PEG-400. After cooling to room temperature some precipitation occurred. Insoluble material was removed by filtering the solution through a fine paper filter under vacuum. The filter was dried and the collected precipitate was weighed (36-40 µg). Thus, between 5 and 54 pg of steroids remained in the solution / fine suspension.

Example 7- Treatment and prevention of non-invasive fungi-induced rhinosinusitis with itraconazole

Three patients with non-invasive fungi induced by rhinosinusitis (a man aged 33 years and 70 years and a woman aged 57 years) were instructed to perform nasal washes with itraconazole solution. The itraconazole solution contained about 100 mg itraconazole per liter of solution (10% PEG-400 in sterile water) and was prepared as described herein. Two patients were instructed to perform itraconazole flushes because they did not respond to amphotericin B flushes. Each patient reported a significant improvement in symptoms within two weeks of the onset of itraconazole flushes (symptom scores: -1 to +2 and -1 to +1). 16 days after the onset of itraconazole flushes, one of the two patients showed an improvement that was revealed by endoscopic analysis (endoscopic score: from 1 to 0 for the right side and from 1 to 1 for the left side). In addition, the patient reported that she had significantly improved asthma symptoms and reduced asthma medications (Flovent and Servent) from twice daily to once daily.

A third patient was instructed to flush itraconazole because of an adverse local reaction to amphotericin B (burning sensations). After treatment with itraconazole, the patient reported an improvement in symptoms (symptom score: from -1 to 0). In addition, the patient had no adverse local reactions or problems with itraconazole flushes.

Example 8 - Treatment and Prevention of Symptoms of Chronic Asthma with Itraconazole A year old Caucasian without history or symptoms of chronic rhinosinusitis exhibited severe asthma symptoms despite medical therapy with systemic and local steroids despite frequent use of bronchodilators. Samples of sputum and nasal-paranasal mucus were collected and analyzed. Cultivation analysis revealed the presence of Candida albicans in · t · t · t · t · t · t · t

Sput and Penicillium, Geotrich, Alternaria and Cladosporium species in nasal-paranasal mucus.

The patient began antifungal treatment with 20 ml of itraconazole solution per nostril twice a day. The itraconazole concentration in the solution was 100 mg / L. After a few weeks, the patient received his last treatment with systemic steroids. Two months after stopping systemic steroids, the patient also stopped taking bronchodilators. The patient's symptoms have dramatically improved since stopping all steroid therapy. Specifically, the patient reported not experiencing any events related to shortness of breath and wheezing during the entire five months following complete cessation of steroid therapy.

Objective analysis also showed a dramatic improvement. In a study performed prior to antifungal therapy, the patient exhibited abnormal pulmonary function. After seven months of continuous antifungal flushing as described, with the last 4-5 months without steroid therapy, the patient showed improved pulmonary function. Specifically, the forced lung vital capacity (FVC) improved from 3.99 liters before treatment to 4.80 liters after treatment, corresponding to an increase in

20.3%; forced expiratory volume per second (FEV1), a marker for lower airway resistance levels, improved from 3.34 liters before treatment to 4.27 liters after treatment, corresponding to a 27.84% increase; maximum forced expiration (FEVmax) improved from 9.1 liters per second before treatment to 12.6 liters after treatment, corresponding to an increase of 38.46%, and maximum intentional ventilation (MW) improved from 119 liters per minute before treatment to 156 liters per minute after treatment, which corresponds to an increase of 31.90%.

In summary, objective markers showed an improvement in pulmonary function between 20.3% and 38.46%, despite the fact that no medical therapy other than antifungal nasal washes has been used in the last 4-5 months. These results suggest that the symptoms of chronic asthma can be treated and prevented by the mucosal administration of antifungal agents to the airways.

After some time when the patient's symptoms of asthma improved, the patient stopped flushing itraconazole. Symptoms of asthma returned after 4-6 weeks when the patient did not flush itraconazole. At this point, the patient inhaled steroids only to control the symptoms of asthma. After 4-6 weeks, he changed the use of the steroid inhaler to an itraconazole powder inhaler. Specifically, the patient was instructed to inhale about 400 µg of pure itraconazole per day using a powder inhaler. Itraconazole powder was an authentic "AS" substance (Janssen Pharmaceutica, Inc.). The inhaler was Pulmicort 200 pg TURBOHALER® manufactured by ASTRA pharmaceuticals. This inhaler was designed to deliver budesonide inhalation powder, but was adapted for the administration of itraconazole.

φφ ·· ·· ··. , · · · »Φ *; Φ · · φ φ * φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ · · · φ · • φ

The patient was asymptomatic for 4 weeks and continued to take itraconazole powder therapy. The patient also initially had at least one nasal polyp. The life span of this polyp visibly decreased after the second week of treatment.

Another asthmatic patient was also instructed to inhale 200 μg of pure itraconazole per day using a modified Pulmicort 200 pg TURBOHALER® inhaler. After about two weeks, the patient's condition improved significantly. The patient continued treatment.

An asthmatic patient with non-invasive fungi induced by rhinosinusitis was treated with itraconazole with a nebulizer. In particular, about 2 ml of itraconazole solution (10 mg itraconazole per ml PEG-400) was applied per day in a nebulizer. It was an air-pressure sprayer PULMO-MATE from DeVillbis. After about two weeks, the patient showed an improvement in both asthma and non-invasive fungal-induced rhinosinusitis, as evidenced by an overall improvement in symptom scores. Improvements were also noted after one week.

Example 9 - Identification of non-invasive fungal-induced otitis media Mucosal middle ear samples were collected from three patients diagnosed with chronic otitis media using a suction well. Culture analysis of the samples revealed the presence of fungal organisms. Specifically, the mucus sample from the first patient was positive for Candida and Trichophyton rubrum species, the mucus sample from the second patient was positive for Penicillium species, and the mucus sample from the third patient was positive for Aspergillus species. In addition, microscopic analysis revealed a large number of degenerating eosinophils in each mucus sample. The results therefore show that chronic otitis media is very likely to be caused by non-invasive fungal organisms. In addition, chronic otitis media appears to be non-invasive fungi induced by mucositis that can be treated and prevented by the methods described herein.

Example 10 - Treatment of non-invasive fungal-induced intestinal mucositis Three out of five patients with chronic rhinosinusitis reported having colitis. Two patients began antifungal treatment with one capsule of itraconazole (provided by Janssen Pharmaceutica, Inc.) per day. The capsule contained 100 mg of itraconazole. Each patient was instructed to take the capsule at bedtime, at least two hours after the last meal and without cola. Food and beverages from cola increase drug absorption. When used as described, about 50% of itraconazole should remain in the intestinal lumen to treat the symptoms of non-invasive fungal-induced intestinal mucositis.

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OTHER EMBODIMENTS

It is to be understood that while the invention has been described in accordance with the detailed description thereof, the following description is intended to illustrate and not limit the scope of the invention, which is defined by the options in the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (68)

1. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be directly mucosal administered at least in part nasal-paranasal anatomy of a mammal with non-invasive fungi-induced rhinosinusitis in an amount, at a frequency and for a time effective to reduce or eliminate said non-invasive fungi-induced rinosinusitis. 2. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert indicating that said composition may be mucosally administered to at least a portion of the nasal paranasal anatomy of a mammal at risk of non-invasive fungi-induced rinosinusitis in an amount, at a frequency and for a time effective to prevent said non-invasive fungi-induced rinosinusitis. 3. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be directly mucosal administered at least in part of a mammal with asthma in an amount, frequency, and for a time effective to reduce or eliminate the symptoms of said asthma. 4. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be mucosally administered to at least a portion of the respiratory tract. in a mammal at risk of developing asthma in an amount, frequency, and for a time effective to prevent symptoms of said asthma. 5. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that said composition may be mucosally administered to a mammal with non-invasive fungi. induced intestinal mucositis in an amount, at a frequency and for a time effective to reduce or eliminate said non-invasive fungal-induced intestinal mucositis. 6. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be mucosally administered to a mammal at risk of developing non-invasive fungal-induced intestinal mucositis in an amount, at a frequency and for a time effective to prevent said non-invasive fungal-induced intestinal mucositis. 7. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be mucosally administered to a mammal with non-invasive fungi. induced otitis media in an amount, at a frequency and for a time effective to reduce or eliminate said non-invasive fungal-induced otitis media. 8. An article of manufacture comprising packaging material and a composition contained in said packaging material, said composition comprising an antifungal agent and said packaging material comprising a label or package insert that states that said composition may be mucosally administered to a mammal at risk of developing non-invasive fungal-induced otitis media in an amount, frequency and time effective to prevent said non-invasive fungal-induced otitis media. Use of an antifungal agent in the manufacture of a medicament for treating or preventing non-invasive fungal-induced rhinosinusitis. The use of claim 9, wherein said medicament is mucosally administered to at least a portion of the mammalian nasal-paranasal anatomy in an amount, at a frequency and for a time effective to reduce, eliminate or prevent said non-invasive fungal-induced rhinosinusitis. The use of claim 10, wherein said mucosal administration is direct mucosal administration. The use of claim 11, wherein the direct mucosal administration comprises flushing said nasal-paranasal anatomy with a liquid form of said medicament. The use of claim 11, wherein said direct mucosal administration is the administration of an aerosol form of said medicament to said nasal-paranasal anatomy. The use of claim 11, wherein said direct mucosal administration is the administration of a powdered form of said medicament to said nasal-paranasal anatomy. The use of claim 10, wherein said effective amount comprises 0.01 ml to 1 liter of said composition per nostril of said mammal. The use of claim 10, wherein said effective amount of drug remains constant during said effective duration. Use according to claim 10, wherein the effective frequency of mucosal administration ranges from four times daily to once every two weeks. The use of claim 10, wherein said effective duration is greater than 7 days. Use according to claim 9, wherein the non-invasive fungal-induced rhinosinusitis is characterized by polyp formation or polypoid changes. The use of claim 9, wherein the non-invasive fungal-induced rhinosinusitis is chronic. Use according to claim 9, wherein the medicament is in solid, liquid or gaseous form. The use of claim 9, wherein the medicament is in a form selected from the group consisting of powder, crystalline substance, gel, paste, ointment, cream, solution, suspension, partial liquid, spray, nebula, mist, atomized vapor, aerosol and tincture. . The use of claim 9, wherein the antifungal agent comprises a macrolide. The use of claim 9, wherein the antifungal agent comprises azole. The use of claim 9, wherein the antifungal agent comprises an antifungal agent selected from NEt of the group consisting of amphotericin B, ketoconazole, itraconazole, saperconazole, voriconazole, NN M / n Ni &lt; A &gt; / 'Νι butoconazole, oxiconazole, sulconazole, ciclopirox olamine, haloprogin, tolnaphthate, naftifine, thiophene i / iN? mmu terbinafine hydrochloride, morpholines, nystatin, natamycin, butenafine, undecylene acid, Whitefield ointment, propionic acid and caprylic acid. The use of claim 9, wherein the medicament comprises a pharmaceutically acceptable aqueous vehicle. The use of claim 26, wherein said medicament comprises 1 ng to 500 mg of said antifungal agent per liter. The use of claim 26, wherein the medicament comprises 100 mg of said antifungal agent per liter. The use of claim 9, wherein the medicament comprises 0.01 ng to 1000 mg of said antifungal agent per liter. The use of claim 9, wherein the medicament comprises a variety of antifungal agents. The use of claim 9, wherein the medicament comprises a substance selected from the group consisting of a pharmaceutically acceptable aqueous vehicle, a pharmaceutically acceptable solid vehicle, a mucolytic agent, an antibacterial agent, an anti-inflammatory agent, an immunosuppressant, a dilatant, a vasoconstrictor, steroids and a therapeutic agent. Use of an antifungal agent in the manufacture of a medicament for the treatment or prevention of asthma. The use of claim 32, wherein said medicament is mucosal administered to at least a portion of the airways of said mammal in an amount, at a frequency and for a time effective to reduce, eliminate or prevent the symptoms of said asthma. Use of an antifungal agent in the manufacture of a medicament for treating or preventing non-invasive fungal-induced intestinal mucositis. *) The use of claim 34, wherein the medicament is mucosally administered to a mammal in an amount, at a frequency and for a time effective to reduce, eliminate or prevent said non-invasive fungal-induced intestinal mucositis. Use of an antifungal agent in the manufacture of a medicament for the treatment or prevention of non-invasive fungal-induced otitis media. The use of claim 36, wherein the medicament is mucosally administered to the mammal in an amount, at a frequency and for a time effective to reduce, eliminate or prevent the symptoms of said non-invasive fungal-induced otitis media. 38. An antifungal composition comprising an antifungal agent, a flavoring agent and water, said water constituting at least 50% of said composition. 39. The antifungal preparation of claim 38, wherein said water comprises at least 75% of said preparation. 40. The antifungal preparation of claim 38, wherein said water comprises at least 85% of said preparation. 41. An antifungal agent comprising an antifungal agent, a flavoring agent and water, said water comprising at least 50% of said preparation and said antifungal agent comprising an antifungal agent selected from the group consisting of amphotericin B, ketoconazole, itraconazole, saperconazole, voriconazole, flucytosine, miconazole, fluconazole, griseofulvin, clotrimazole, econazole, terconazole, butoconazole, oxiconazole, sulconazole, ciclopirox olamine, haloprogin, tolnaphthate, naftifine, terbinafmine hydrochloride, morpholines, nystatin, natamycin, natamycin, natamycin, natamycin, natamycin, natamycin, and caprylic acid. 42. An antifungal composition comprising itraconazole and water, wherein itraconazole is dissolved in said composition at a concentration of greater than 25 pg per ml and said water constitutes at least 50% of said composition. φ · · · · · · φ · · · φ · · · · · · 43. The antifungal preparation of claim 42, comprising polyethylene glycol. 44. An antifungal preparation according to claim 42, comprising an aromatic substance. 45. An antifungal preparation comprising itraconazole and water, wherein itraconazole is suspended in said formulation at a concentration of greater than 25 pg per ml and said water constitutes at least 50% of said formulation. 46. A method of making an antifungal preparation, said preparation comprising itraconazole and water, and itraconazole is dissolved in said preparation at a concentration of greater than 25 pg per ml and the water comprises at least 50% of said preparation, said method comprising adding said water to a storage a solution comprising said itraconazole. 47. A method of culturing a mammalian mucus fungus comprising: (a) applying a mucolytic agent to said mucus to reduce the viscosity of said mucus b) separating said fungus from said reduced viscosity mucus c) contacting said separated fungus with a fungal growth medium to form a fungal culture; and d) incubating said fungal culture such that said separated fungus grows. 48. A method of obtaining a fungal antigen comprising: (a) applying a mucolytic agent to said mucus to reduce the viscosity of said mucus b) separating said fungus from said reduced viscosity mucus c) contacting said separated fungus with a fungal growth medium to form a fungal culture d) incubating said fungal culture such that said separated fungus grows; and e) isolating said antigen from said cultured fungi. 49. A method for producing fungal-specific antibodies comprising: (a) applying a mucolytic agent to said mucus to reduce the viscosity of said mucus b) separating said fungus from said reduced viscosity mucus c) contacting said separated fungus with a fungal growth medium to form a fungal culture d) incubating said fungal culture such that said separated fungus grows e) isolating said antigen from said cultured fungi, and f) immunizing the animal with said antigen to produce said antibody. 50. A nasal mucus collection tool comprising: (a) a collection container, said collection container suitable for storing mucus, b) a collecting tube extending from said collecting vessel, said collecting tube defining a distal end and a lumen such that the mucus can pass through the lumen from the distal end of said collecting tube to said collecting vessel, the collecting tube being generally flexible at least about a certain length of said collecting vessel the tube so that a professional can selectively manipulate said collection tube into a desired configuration during collection, the collection tube further being deformable such that said collection tube generally retains said desired configuration until the professional begins to manipulate said collection tube to obtain a different configuration, and c) a connecting portion extending from said collection container, said connecting portion defining a secondary lumen that communicates with the interior of said collection container, said connecting portion being adapted to attach a vacuum source thereto. 51. The tool of claim 50 including a flap that regulates opening of said secondary lumen. 52. The tool of claim 50, wherein the collection container is removable from said collection tube and said coupling portion. 53. A pharmaceutical composition comprising an antifungal agent. 54. A pharmaceutical composition comprising an antifungal agent and a mucolytic agent. 55. A pharmaceutical composition comprising an antifungal agent and a steroid. tt · · · · · · · · 56. A pharmaceutical composition comprising an antifungal agent and a decongestant agent. 57. A pharmaceutical composition comprising an antifungal agent and an antibiotic. 58. A pharmaceutical composition comprising an antifungal agent and an anti-inflammatory agent. 59. A composition for treating an immune response to a fungus in a mammal, comprising a substance designed for direct mucosal administration to a mammalian mucus having antifungal means for eliminating or reducing the fungus to a subliminal level, wherein the fungus stops activating eosinophil migration to the affected area. 60. A pharmaceutical composition for treating a fungal-related condition in the nasal sinus, pulmonary, ear, or intestinal region of a mammalian patient, comprising an effective dose of an antifungal agent as described herein. 61. A pharmaceutical composition for treating a fungal-related condition in the nasal-sinus, pulmonary, ear, or intestinal region of a mammalian patient, comprising an effective dose of an antifungal agent and at least one additional agent or inhibitor as described herein. . 62. A pharmaceutical composition for treating a fungal-related condition in the nasal sinus, pulmonary, ear, or intestinal region of a mammalian patient, comprising an effective dose of an antifungal agent suitable for long-term use in the nasal-sinus region. 63. A medicament for the treatment of a patient sinusitis, asthma, otitis media or colitis, comprising a mucolytic agent; and an antifungal agent as described herein. 64. A medicament for irrigation for treating an inflamed nasal, lung, ear, and intestinal region of a patient, wherein the inflamed nasal, lung, ear, and intestinal region is caused by the presence of a fungus, wherein the medicament comprises effective doses of an antifungal agent. and steroids as described herein. 65. A medicament for irrigation for treating an inflamed nasal, lung, ear, and intestinal area of a patient, wherein the inflamed nasal, lung, ear, and intestinal area is caused by the presence of a fungus, wherein the medicament comprises effective doses of an antifungal agent. and mucolytic agents as described herein. 66. A medicament for flushing for treating an inflamed nasal, lung, ear, and intestinal area of a patient, wherein the inflamed nasal, lung, ear, and intestinal area is caused by the presence of a fungus, wherein the medicament comprises effective doses of a mucolytic agent. and a steroid as described herein. 67. A medicament for rinsing for treating an inflamed nasal, lung, ear, and intestinal area of a patient, wherein the inflamed nasal, lung, ear, and intestinal area is caused by the presence of a fungus, wherein the medicament comprises effective doses of an antifungal agent. , a steroid and a mucolytic agent as described herein. 68. A medicament for flushing for treating an inflamed nasal region, a lung region, an ear region, and an intestinal region of a patient, wherein the inflamed nasal region, lung region, ear region and intestinal region is caused by a fungus, wherein the medicament comprises an effective dose of at least one drugs selected from the group consisting of an antifungal agent, a steroid, a mucolytic agent, and any combination thereof as described herein.