JPH092970A - Antifungal agent - Google Patents

Abstract

PURPOSE: To provide antifungal agent showing strong antifungal action at a low concentration. CONSTITUTION: This antifungal agent contains an azole-based antifungal substance and lactoferrin as active ingredients. The azole-based antifungal substance is clotrimazole, ketoconazole, lanoconazole, itoraconazole or their mixture. The amount of lactoferrin contained is at least 100 pts.wt. based on 1 pt.wt. of the azole-based antifungal substance. Consequently, since the antifungal agent has strong antifungal effect with a small amount, it is useful as a therapeutic agent for various mycoses. The antifungal agent is safely usable as a means for suppressing abnormal breeding of a fungus of the genus Candida in a digestive organ. The antifungal agent has slight adverse effect and reduces the frequency of appearance of a resistant fungus. The antifungal agent means can extremely reduce the amount of the azole-based antifungal substance by using it together with lactoferrin at a low concentration level not showing antifungal action.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antifungal agent containing an azole antifungal substance and lactoferrin as active ingredients and exhibiting a strong antifungal action at low concentrations.
More specifically, the present invention uses an azole antifungal substance in combination with lactoferrin, which is a physiologically active protein that is mainly present in milk of mammals, so that the active ingredient thereof is more effective than conventional azole antifungal agents. The present invention relates to a novel antifungal agent capable of significantly reducing the amount used, having a strong antifungal effect at a small dose, having few side effects, and reducing the frequency of appearance of resistant bacteria.

[0002]

BACKGROUND ART In superficial mycosis, invasion of the causative bacteria is caused by epidermis,
It is defined as a disease limited to keratinized tissues such as hair and nails, and mucosal sites adjacent to the skin such as the oral cavity and vagina, and is the most frequently occurring disease. The incidence of dermatophytosis (tinea unguium), known as one of the representative superficial mycosis, is one of the total population.
There are many cases in which the recurrence or reinfection reaches 0% or more, and there are many cases in which the pathological conditions are tinea pedis, tinea corporis,
Various pathological conditions such as tinea cruris are known.

[0003] As another typical superficial mycosis, candidiasis is known. Regarding candidiasis, for example, diseases such as oral candidiasis, esophageal / intestinal candidiasis, and vulvovaginal candidiasis are caused by the invasion of fungi of the genus Candida. , Candidiasis such as paronychia and cystitis are also known. It has also been reported that abnormal growth of Candida fungi in the intestinal tract causes atopic dermatitis (Clinic of Allergy, Vol. 11, pp. 768-772, 1991).

On the other hand, in recent years, serious deep-seated mycosis has been rapidly increasing. Behind that, there are many drugs such as broad-spectrum antibiotics, anticancer agents, immunosuppressants for organ transplantation, oral contraceptives, etc. There are problems such as administration and high calorie infusion, use of indwelling catheters, etc., and a rapid increase in the number of patients who are easily infected, such as AIDS disease, which are major clinical problems.

Conventionally, antifungal agents used for the treatment of mycosis as internal antifungal agents include polyene-based amphotericin B, fluoropyridine-based flucytosine, imidazole-based (azole-based) miconazole and triazole-based (azole-based). 4) of fluconazole, but in September 1993, an azole type itraconazole was marketed.

The types of pathogenic fungi targeted by these antifungal agents and the drug sensitivities differ depending on the respective antifungal agents. The types of pathogenic fungi targeted by these antifungal agents are, for example,
Candida, Cryptococcus, Aspergillus,
The Trichophyton genus, Malassezia genus, Kochidioides genus, etc. are typical.

[0007] Antifungal substances, which are generically referred to as azoles, have an imidazole ring or a triazole ring and exhibit similar properties in biological activity. These antifungal agents are common in that the impaired response to sterol / C-14 demethylation in the ergosterol synthetic pathway localized in the endoplasmic reticulum of fungal cells is the mechanism of action of their antifungal activity. are doing.

[0008] At present, the use of such an antifungal agent for internal use, which has been exclusively used as a single preparation, has a large occupancy rate of an azole agent. The reason why azole agents are commonly used in this manner is that they are relatively safe compared to amphotericin B and flucytosine, and that resistant bacteria are less likely to appear. However, it has been reported that fuchconazole (an azole antifungal agent), which has the highest occupancy rate, has a low efficacy rate for aspergillosis compared to a high efficacy rate for candidiasis and cryptococcosis (chem Area of therapy, first
0, pp. 17-26, 1994).

Since azole agents generally act bacteriostatically, it is said that it is difficult to respond when the infection resistance of the patient is remarkably reduced or when invasive serious infection is induced. (Area of Chemotherapy, Volume 10, 17-2
6 pages, 1994). Thus, current azoles do not have a bactericidal effect, and thus require relatively long-term administration of a large amount of the drug (in the area of chemotherapy,
10: 17-26, 1994). Therefore, with regard to such antifungal agents, the occurrence of side effects such as liver damage, renal damage, diarrhea, and vomiting due to long-term large-dose administration thereof is considered to be a clinical problem, and further, long-term administration thereof causes them. There is concern about the problem of drug resistance of fungal pathogens.

On the other hand, lactoferrin is an antibacterial protein possessed by the living body and is known to exert antibacterial properties against fungi [Infection and Immunity, Vol. 60, Vol. 46
04-4611, 1992]. Lactoferrin is known to play an important role as a biological protein that is synthesized in large quantities in vivo during acute inflammation caused by infection with pathogenic bacteria. For example, bovine lactoferrin is a physiological protein that is contained in a very small amount in milk. It is one of the active proteins, and its antibacterial activity is at the level of physiological activity in vivo, and therefore, when compared with known antibacterial agents such as conventional azole antifungal agents, It is no exaggeration to say that the antibacterial activity is of a low level that is almost incomparable. Conventionally, as a combination of an antibacterial agent and lactoferrin, a pharmaceutical composition containing a beta-lactam antibiotic and bovine lactoferrin has been reported (JP-A-3-181421).
As far as beta-lactam antibiotics are concerned, there have been no cases where azole antifungal agents were previously discussed in relation to lactoferrin, and by combining azole antifungal agents with lactoferrin, antifungal activity was It has not been known to date that there will be an increase in the, and there have been no reports. From such circumstances of the prior art, with respect to the azole antifungal agent and the like, the development of an antifungal agent having a bactericidal action even when used in a small amount, that is, an antifungal agent having few side effects and a strong antifungal action has been developed. Long-awaited.

[0011]

DISCLOSURE OF THE INVENTION As a result of intensive studies on novel antifungal agents in view of the above prior art, the present inventors have found that by adding lactoferrin to an azole antifungal substance, We have found that the fungal agent exerts a strong antifungal activity at a dose much lower than the conventional dose, and completed the present invention.

An object of the present invention is to provide an antifungal agent which is effective in a small dose, has few side effects, and has a low frequency of appearance of resistant bacteria.

[0013]

The present invention for solving the above problems is an antifungal agent containing an azole antifungal substance and lactoferrin as an active ingredient, wherein the azole antifungal substance is clotrimazole, ketoconazole, It is also a desirable embodiment that it is ranoconazole, itraconazole or a mixture thereof, and that lactoferrin is contained in a ratio of at least 100 parts (weight) to 1 part (weight) of the azole antifungal substance.

Next, the present invention will be described in detail. The azole antifungal substance used in the present invention may be any of the azole antifungal substances conventionally used as an azole antifungal agent, and in particular, clotrimazole, ketoconazole or itraconazole is exemplified as a preferable one. It

The lactoferrin (lact) used in the present invention
As forerrin), milk, human milk, or other milk prepared by a conventional method, that prepared from a genetically engineered lactoferrin homologous preparation, or a salt thereof can be used. Is preferably 90% (by weight; the same applies unless otherwise indicated below unless otherwise specified), and particularly preferably 95% or more in purity. Further, apolactoferrin having no metal bonded thereto, hololactoferrin having a metal bonded thereto such as iron or zinc may be used, and the type thereof is not particularly limited as long as it is lactoferrin.

As is clear from the test examples described below, the antifungal agent of the present invention is at least 100 parts, preferably 2, based on 1 part (by weight, the same applies hereinafter) of an azole antifungal substance.
When lactoferrin is blended at a ratio of 000 to 32,000 parts, the combined effect of lactoferrin is exhibited. If the content of lactoferrin is less than 100 parts, the antifungal effect is not enhanced by the combined use of lactoferrin, and if it exceeds 32,000 parts, the antifungal effect is not enhanced by the increase of lactoferrin.

The antifungal agent of the present invention can be obtained by a conventional method in the form of tablets,
It can be formulated into granules, liquids, powders, etc. and can be processed into oral preparations, injections, vaginal preparations, troches, ointments, lotions, etc. by known methods, and these are used. In this case, it can be administered in the same manner as the known administration method of the antifungal agent contained therein. Further, the dose of the antifungal agent of the present invention is, as is clear from the test examples described later, 1 / the dose of the conventional azole antifungal agent.
Even if it is 4 or 1/16 or less, a particularly remarkable combined effect, which is unexpected from the prior art, is obtained which has an antifungal activity equal to or higher than that of a conventional azole antifungal agent. The content of the active ingredient in the antifungal agent of the present invention is at least 3.1 ng / ml of azole antifungal substance and at least 10 μg / ml of lactoferrin. Lactoferrin is one component of milk or the like that is ingested as a food, and is also used as a food, which itself does not show any toxicity, and according to the present invention, by using the lactoferrin in combination. It is possible to significantly reduce the amount of the conventional antifungal agent used, which causes side effects, and thereby to provide a safe antifungal agent. The effect of reducing the amount of the antifungal agent used is unique to the azole antifungal substance obtained only when lactoferrin is allowed to coexist, and is a novel one that has not been reported so far.

Next, the present invention will be described in more detail with reference to test examples. Test Example 1 1) Preparation of sample Ketoconazole (manufactured by Sigma) was used as an azole antifungal substance, and this was dissolved in dimethyl sulfoxide (manufactured by Nacalai Tesque). Bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) was dissolved in distilled water and sterilized by filtration.

2) Test method 1 ml of Sabouraud dextrose broth (1% peptone, 2% glucose) as a test medium was weighed in a test tube, and ketoconazole was added at a concentration shown in Table 1 per 1 ml of the medium, and a control sample ( (Single use). On the other hand, 1 ml of the broth was used as a test medium, lactoferrin was added as a test agent at a ratio of 100 μg per 1 ml of the medium, and ketoconazole was added at the concentration shown in Table 1 to give a test sample (combination).

Candida albicans (Ca
ndida albicans) TIMM1768 strain (obtained from Teikyo University Medical and Fungal Center) is scraped from the agar slant and added to each of the samples at a final bacterial concentration of about 10 ⁵ / ml, and cultured at 37 ° C. for 17 hours, After that, the absorbance was measured at 630 nm to determine the growth rate of the bacteria. To determine the growth rate of bacteria, the absorbance of a test tube containing no test drug was determined as 1
When it was set to 00%, a test tube having an absorbance of more than 25% was judged to be proliferative (+), and a test tube having an absorbance of 25% or less was judged to be non-proliferative (-).

3) Test Results The results of this test are shown in Table 1. As is clear from Table 1, 100 μg / ml bovine lactoferrin
The growth of the bacteria could not be suppressed by itself. On the other hand, ketoconazole alone suppressed bacterial growth at 50 ng / ml, but coexistence of 100 μg / ml bovine lactoferrin resulted in an amount of ketoconazole required to suppress bacterial growth of 3.1 ng / ml (target sample). 1/16). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced.
In addition, the fungi and lactoferrin were tested with different types, but almost the same results were obtained.

[0022]

[Table 1]

Test Example 2 1) Preparation of Sample A sample was prepared in the same manner as in Test Example 1 except that clotrimazole (manufactured by Sigma) was used as the azole antifungal substance.

2) Test Method A test was conducted in the same manner as in Test Example 1.

3) Test Results The results of this test are shown in Table 2. As is clear from Table 2, 100 μg / ml bovine lactoferrin
The growth of the bacteria could not be suppressed by itself. On the other hand, clotrimazole alone suppressed the growth of bacteria at 50 ng / ml, but the co-presence of 100 μg / ml of bovine lactoferrin, the amount of clotrimazole required to suppress the growth of bacteria was 12.5 ng / ml. It was reduced to ml (1/4). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0026]

[Table 2]

Test Example 3 1) Preparation of sample Itrizole (registered trademark) as an azole antifungal substance.
A sample was prepared in the same manner as in Test Example 1 except that itraconazole purified from capsule 50 of Janssen Kyowa Co., Ltd. was used.

2) Test method The test was carried out by the same method as in Test Example 1.

3) Test Results The results of this test are shown in Table 3. As is apparent from Table 3, 100 μg / ml bovine lactoferrin
The growth of the bacteria could not be suppressed by itself. On the other hand, itraconazole alone suppressed the growth of the bacterium at 50 ng / ml, but the coexistence of 100 μg / ml of bovine lactoferrin, the amount of itraconazole required to suppress the growth of the bacterium was 12.5 ng / ml (1 / It was reduced to 4). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0030]

[Table 3]

Test Example 4 1) Preparation of sample Using lanoconazole purified from an Astat (registered trademark, manufactured by Tsumura Co., Ltd.) solution as an azole antifungal substance, and using a concentration of ranoconazole of 20 per 1 ml of the test medium.
A sample was prepared in the same manner as in Test Example 1, except that the sample was adjusted to 0 μg.

2) Test Method A test was conducted in the same manner as in Test Example 1.

3) Test Results The results of this test are shown in Table 4. As is clear from Table 4, 100 μg / ml bovine lactoferrin
The growth of the bacteria could not be suppressed by itself. On the other hand, lanoconazole alone suppressed the growth of bacteria at 200 ng / ml, but the coexistence of 100 μg / ml of bovine lactoferrin, the amount of lanoconazole required to suppress the growth of bacteria was 50 ng / ml (1/4). It has been reduced to.
This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0034]

[Table 4]

Test Example 5 1) Preparation of sample Same as Test Example 1 except that clotrimazole (manufactured by Sigma) and human lactoferrin (manufactured by Sigma) were used as azole antifungal substances. A sample was prepared by the method described in 1.

2) Test Method A test was conducted in the same manner as in Test Example 1.

3) Test Results The test results are shown in Table 5. As is clear from Table 5, human lactoferrin 100 μg / ml alone could not suppress the growth of bacteria. On the other hand, clotrimazole alone suppressed bacterial growth at 50 ng / ml, but the co-existence of human lactoferrin at 100 μg / ml resulted in an amount of clotrimazole required to suppress bacterial growth of 12.5 ng / ml. It was reduced to ml (1/4). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0038]

[Table 5]

Test Example 6 1) Preparation of Sample Test Example 1 except that clotrimazole (manufactured by Sigma) was used as an azole antifungal substance and bovine apolactoferrin (manufactured by Morinaga Milk Industry) was used. Samples were prepared by the same method.

2) Test Method A test was conducted in the same manner as in Test Example 1.

3) Test Results The results of this test are shown in Table 6. As is clear from Table 6, 100 μg of bovine apolactoferrin /
ml alone could not suppress the growth of bacteria. On the other hand, clotrimazole alone suppressed the growth of bacteria at 50 ng / ml, but the co-presence of 100 μg / ml of bovine apolactoferrin, the amount of clotrimazole required to suppress the growth of bacteria was 3.1 ng. / Ml (1/16). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced.
I tested different types of fungi and lactoferrin,
Almost the same results were obtained.

[0042]

[Table 6]

Test Example 7 1) Preparation of Sample A sample was prepared in the same manner as in Test Example 6 except that bovine iron-saturated lactoferrin (manufactured by Morinaga Milk Industry) was used.

2) Test Method A test was conducted in the same manner as in Test Example 1.

3) Test Results The results of this test are shown in Table 7. As is clear from Table 7, 100 μg of bovine iron saturated lactoferrin
/ Ml alone could not suppress the growth of bacteria. On the other hand, clotrimazole alone suppressed bacterial growth at 50 ng / ml, but bovine iron saturated lactoferrin 100 μg / m
The amount of clotrimazole required to suppress the growth of bacteria due to the coexistence of 1 is 12.5 ng / ml (1 /
It was reduced to 4). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0046]

[Table 7]

Test Example 8 1) Preparation of sample The use of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) and the concentration of clotrimazole were 20 per 1 ml of the test medium.
A sample was prepared in the same manner as in Test Example 6 except that the sample was adjusted to 0 μg.

2) Test method Spores of Trichophyton mentagrophytes TIMM1189 strain (obtained from Teikyo University Medical and Fungal Center) were scraped from the agar slant as a test bacterium, and the final spore concentration was about 10 ⁴ / m
The test was performed in the same manner as in Test Example 1 except that the test medium was added at a ratio of 1 and cultured at 27 ° C. for 4 days.

3) Test Results The results of this test are shown in Table 8. As is clear from Table 8, bovine lactoferrin 100 μg / ml
The growth of the bacteria could not be suppressed by itself. On the other hand, clotrimazole alone suppressed the growth of the bacteria at 200 ng / ml, but the co-presence of 100 μg / ml of bovine lactoferrin, the amount of clotrimazole required to suppress the growth of the bacteria was 50 ng / ml ( It has been reduced to 1/4). This indicates that the coexistence of lactoferrin can enhance the antifungal activity to the extent that the amount of the azole antifungal agent used can be significantly reduced. Different types of fungi and lactoferrin were tested and similar results were obtained.

[0050]

[Table 8]

[0051]

EXAMPLES Next, the present invention will be described more specifically by way of examples, but the present invention is not limited to the following examples. Example 1 1 mg of ketoconazole (manufactured by Sigma) was mixed with 10 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.),
An antifungal agent for oral administration was manufactured.

Example 2 1 mg of ketoconazole (manufactured by Sigma), 1 g of human lactoferrin (manufactured by Sigma) and 100 mg of sodium chloride (manufactured by Wako Pure Chemical Industries) in 10 ml of water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.)
Dissolve in mg ratio, adjust pH to 7, sterilize by filtration,
Ampoules were filled in 1 ml portions to obtain 10 antifungal agents for injection.

Example 3 10 g of bovine apolactoferrin (manufactured by Morinaga Milk Industry), methyl paraoxybenzoate (manufactured by Nacalai Tesque) 0.1
g, propyl paraoxybenzoate (manufactured by Nacalai Tesque) 0.1 g, propylene glycol (manufactured by Nacalai Tesque) 12 g, and purified water 27.8 g were dissolved with stirring. Separately, clotrimazole (manufactured by Sigma) 5
mg, white petrolatum (manufactured by Nacalai Tesque) 25 g, stearyl alcohol (manufactured by Nacalai Tesque) 20 g, polyoxyethylene hydrogenated castor oil (manufactured by Nacalai Tesque)
4g, 1g of glyceryl monostearate (manufactured by Nacalai Tesque Co., Ltd.) were added to the above solution while stirring, and emulsified using a homomixer to prepare O / W type cream. The tube was filled to obtain 10 topical antifungal agents.

[0054]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention is an antifungal agent containing an azole antifungal substance and lactoferrin as active ingredients. The effects of the present invention are as follows. 1) Since the antifungal agent of the present invention has a strong antifungal effect in a small dose, it can be used as a therapeutic agent for various fungal diseases. 2) The antifungal agent of the present invention can be safely used as a means for preventing abnormal growth of Candida in the digestive tract. 3) The antifungal agent of the present invention has few side effects and the appearance frequency of resistant bacteria is reduced. 4) According to the present invention, the use amount of the azole antifungal agent can be remarkably reduced by the combined use of lactoferrin at a low concentration level which does not show an antifungal effect.

Claims (3)

[Claims] 1. An antifungal agent comprising an azole antifungal substance and lactoferrin as active ingredients. 2. The antifungal agent according to claim 1, wherein the azole antifungal substance is clotrimazole, ketoconazole, lanoconazole, itraconazole, or a mixture thereof. 3. The antifungal agent according to claim 1, wherein lactoferrin is contained at a ratio of at least 100 parts (weight) to 1 part (weight) of the azole antifungal substance.