WO2006049477A1

WO2006049477A1 - Antifungal pharmaceutical composition

Info

Publication number: WO2006049477A1
Application number: PCT/KR2005/003772
Authority: WO
Inventors: Hae-Jun Park
Original assignee: Bio Dreams Co. Ltd.
Priority date: 2004-11-08
Filing date: 2005-11-08
Publication date: 2006-05-11

Abstract

Disclosed is an antifungal pharmaceutical composition including nanosized silica-silver particles 0.5 to 30 nm in size, in which nano-silver is bound to silica molecules and a water-soluble polymer, as an effective ingredient. Also disclosed is a method of disinfecting a pathogenic fungus using the composition.

Description

ANTIFUNGAL PHARMACEUTICAL COMPOSITION

Technical Field

The present invention relates to an antifungal pharmaceutical composition comprising nαnosized silica- silver particles 0 5 to 30 nm in size and a method of disinfecting a pathogenic fungus using the same

Background Art

Antifungal agents m current use are mainly obtained from fungi and others or are chemically synthesized

Chemically synthesized antifungal agents are polyene and azole derivatives and allyamine-thiocarbamates which all interact with ergosterol, a fungal cell membrane component, or inhibit the synthesis of ergosterol Of these, polyenes increase membrane permeability and allow outward leakage of intracellular components, leading to death of fungal cells

Despite many side effects of polyene antibiotics, only amphotericin B is currently available for the treatment of systemic fungal infections Azole compounds are mostly synthetic products and a number of new azoles are available Azoles block ergosterol synthesis by inhibiting

C-14 demethylase m the ergosterol biosynthetic pathway

However, some imidazole compounds (clotrimazole, miconazole, etc.) are highly toxic because they directly interact with cell membranes and damage the membrane, and are thus used topically (D. P. Hanger, et al. , Antimicrob. Agents Chemother., 32, 646, 1988) . Recently developed azole compounds (ketoconazole, itraconazole, etc.) have less serious toxicity but are reported to having side effects of blocking the synthesis of steroid hormones in the endocrine system by inhibiting cytochrome P-450-dependent enzymes. In addition, other antifungal agents, such as the allyamme compounds, naftifme and terbinafme, and the thiocarbamate compound, tolnaftate, are known to block erogosterol synthesis by inhibiting squalene epoxidase and oxidosqualene cyclase and have broad spectrums of in vitro antifungal activity and less toxicity than azoles. However, these compounds exhibit poor pharmacokinetic performances and are thus used mainly in skin diseases.

In addition, synthetic antifungal agents are problematic in requiring high manufacturing cost and being difficult to purify. Thus, there is a need for the development of antibiotics exhibiting desired antifungal activity with less side effects than currently developed antifungal agents.

Silicon (Si) , which is the second most abundant material in the earth, is taken up by plants and enhances resistance to diseases and stress therein (Role of Root hairs and Lateral Roots in Silicon Uptake by Rice J. F. Ma et al. Ichii Plant Physiology (2001) 127: 1773-1780, etc.) . In particular, when plants are treated with an aqueous solution of silicate, silicate displays excellent preventive effects on major plant diseases including powdery mildew and downy mildew. Also, silicate promotes physiological activity of plants and improves plant growth while providing resistance to diseases and stress (Suppressive effect of potassium silicate on powdery mildew of strawberry m hydroponics T. Kanto et al. J GenPlant Pathol (2004) 70. 207 211), etc.) . However, since silica does not have direct disinfecting effects on plant pathogens, it does not exhibit positive effects when diseases develop xn plants.

Silver (Ag) , which is known as a strong bactericidal agent, destroys unicellular microorganisms through its antimicrobial activity against enzymes performing metabolic functions in microbes (T N. Kim, Q. L. Feng, et al. , J. Mater. Sci. Mater. Med., 9, 129 (1998)) . Heavy metals such as copper and zinc also have the same function as silver However, silver has the strongest bactericidal effect and also has excellent effects on algae. Silver has been studied as a substitute for chloride or other toxic microbicides To date, a variety of inorganic antimicrobial agents using silver have been developed

Silver-based inorganic antimicrobial agents in current use are commercially available m the form of sliver-supported inorganic powder, silver colloids, metal silver powder, and the like. Of them, the silver-supported inorganic powder form makes up the largest part of this demand, and this form is generally referred to as an inorganic antimicrobial agent.

When silver exists in an ion state, it has good antimicrobial activity. However, silver is unstable due to its high reactivity and is easily oxidized or reduced to a metal according to the surrounding atmosphere, thereby spontaneously changing in color or causing other materials to be changed in color. These phenomena lead to a reduction in the duration of the antimicrobial action of silver. When present in a metal or oxidized form, silver is stable in the environment but must be used in relatively large amounts due to its low antimicrobial activity.

Silver, having the advantages and drawbacks as noted above, is spotlighted in the form of nanoparticles. Nanoparitcles are synthesized by a variety of methods including mechanical grinding, coprecipitation, spraying, sol-gel processing, electrolysis and reverse-phase microemulsion processing. However, these methods are problematic in terms of being difficult to control the size of formed particles or requiring high production costs for micro metal particles. For example, the coprecipitation method is incapable of controlling the size, shape and size distribution of particles because it is based on forming particles in an aqueous solution. The electrolysis and sol- gel techniques require high production costs and have difficulty producing nanoparticels in a large scale. The reverse-phase microemulsion processing easily controls the size, shape and size distribution of particles, but provides a very complicated manufacturing process and thus does not have practical use.

On the other hand, a method of preparing nanometer- sized particles by irradiation with radiation rays has the following advantages: it easily controls the size, shape and size distribution of particles; it can form nanoparticles at room temperature; and it provides a simple manufacturing process and thus makes mass production thereof possible with low costs.

Korean Pat. Registration No. 0425976 discloses a method of preparing nanometer-sized silver colloids by irradiation with radiation rays and nanometer-sized silver colloids. The method of preparing silver colloids comprises dissolving a silver salt m triple distilled water, adding sodium dodecyl sulfate (SDS) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) and others as colloid stabilizers to the solution, carrying out nitrogen purging, and irradiating the resulting solution with radiation rays. However, since this method produces silver colloids having a particle size greater than 100 nm, high concentrations of silver colloids should be produced for use as an antimicrobial agent against microbes, especially fungi.

In addition, Korean Pat. Laid-open Publication No. 2003-0082065 (Application No. 10-2002-0020594) discloses a method of preparing stable silver colloids using polymer stabilizers, including PVP used in Korean Pat. Registration No 0425976 as a polymer stabilizer, (1-vmylpyrrolxdone) - acrylate copolymer, and (1-vmylpyrrolidone) -vinyl acetate copolymer

In addition to the aforementioned methods, many efforts have been made to provxde nano-silver applicable to a broad range of fields with purposes including anti- bacteπa, cleaning and deodoπzation Despite these efforts, there is still a need for the development of a more simple process for preparing cheaper and more stable nano-silver

Based on this background, the present inventors prepared nanosized silica-silver particles, in which nano- failver is bound to silica molecules and a water-soluble polymer, by mixing a silver salt, silicate and a water- soluble polymer and irradiating the resulting mixture with radiation rays, and found that the nanosized silica-silver particles thus prepared are uniform m size, are stable, and have excellent antifungal effects even in very low concentrations, thus leading to the present invention

Disclosure of the Invention

It is therefore an object of the present invention to provide an antifungal pharmaceutical composition comprising nanosized silica-silver particles 0 5 to 30 nm m size, m which nano-silver is bound to silica molecules and a water- soluble polymer, as an effective ingredient It is another object of the present invention to provide a method of disinfecting a pathogenic fungus by administrating nanosized silica-silver particles, m which nano-silver is bound to silica molecules and a water- soluble polymer

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which

Fig Ia is a flowchart of a process of preparing nanosized silica-silver, and Fig Ib shows TEM images of nanosized silica-silver formed after irradiation with gamma rays,

Fig 2 shows the colloidal stability of nanosized silica-silver m water,

Fig 3 shows the absorption spectrum of nanosized silica silver at 403 nm compared with the absorption spectra of water and silver ions,

Fig 4 shows the change of nanosized silica-silver m absorbance at 403 nm according to concentrations of sodium silicate (Na₂SiO₃) ,

Fig 5 shows the absorption spectra at 403 nm of nanosized silica-silver prepared with varying concentrations of polyvinylpyrrolidone (PVP) ,

Fxgs 6a and 6b show the absorption spectra at 403 nra of nanosized silica silver prepared with other water- soluble polymers (high levan and corn starch, respectively) ,

Fig 7 shows the absorption spectra at 403 nm of nanosized silica-silver according to radiation doses,

Figs 8a to 8c show the antifungal effects of nanosized silica silver on pathogenic fungi, Rhizoctonia solani (Fig 8a) , Botrytis cmerea (Fig 8b) , and Pythium ultimum and Magπaporthe grisea (Fig 8c) , and

Fig 9 shows the antifungal effect of nanosized silica-silver on Botrytis cmerea

Best Mode for Carrying Out the Invention

In one aspect, the present invention relates to an antifungal pharmaceutical composition comprising nanosized silica-silver particles 0 5 to 30 nm in size, in which nano-silver is bound to silica molecules and a water soluble polymer, as an effective ingredient The term "nanosized silica-silver", as used herein, refers to a composite m which nano-sized silver particles and silica molecules are bound to a water soluble polymer According to a detailed aspect, the nanosized silica silver may be prepared by irradiating a solution containing a silver salt, silicate and a water-soluble polymer with radiation rays A form of the composite is exemplified by a structure m which nano-sized silver particles, formed from silver ions, and silica molecules, formed from silicate, are individually or together surrounded by a water-soluble polymer by irradiation with radiation rays. The nanosized silica-silver thus prepared is present m a form m which nanoparticles are dissociated from each other at a colloidal state or assembled into loose spherical aggregates (Fig. Ib) . The aggregates are disassembled into dispersed nanoparticles when temperature increases Nano- silver particles in which nano-silver is coated with silica particles were conventionally developed However, these particles, unlike the nanosized silica-silver particles contained in the antifungal pharmaceutical composition of the present invention, do not include a water-soluble polymer m the particle composition. Also, a water-soluble polymer was conventionally used to form nano-silver particles However, in this case, the water-soluble polymer was used not as a component of nano-silver particles but as a dispersing agent for forming a colloidal solution

The nanosized silica-silver contained m the present composition, as demonstrated from the absorption spectrum of Fig. 3, absorbs light at 403 nm, characteristic for nano-silver, and as shown in Fig. Ib has a uniform nanoparticle size. The nanosized silica silver has a particle size of preferably 0.5 to 30 nm, more preferably 1 to 20 nm, and most preferably 1 to 5 nm The nanosized silica silver is prepared by preparing a solution containing a silver salt, silicate and a water-soluble polymer and irradiating the solution with radiation rays This method may further include bubbling (or purging) with inert gas before, after, or before and after irradiation with radiation rays. The inert gas is exemplified by nitrogen and argon, and nitrogen gas is preferred. The bubbling is preferably carried out for 10 mm to 30 min. In the method, the solution containing a silver salt, silicate and a water-soluble polymer may further include a radical scavenger for scavenging radicals generated by irradiation with radiation rays The radical scavenger is exemplified by alcohols, glutathione, vitamin E, flavonoid and ascorbic acid Available alcohols may include methanol, ethanol, nor-propanol, isopropanol (IPA) and butanol. Of them, isopropanol is preferred The alcohol may be used in an amount of 0.1 to 20%, and preferably 3 to 10% based on the total amount of the solution containing a silver salt, silicate and a water-soluble polymer The silver salt available m the preparation of nanosized silica-silver may be exemplified by siver nitrate (AgNO₃) , silver perchlorate (AgClO₄) , silver chlorate (AgClO₃) , silver chloride (AgCl) , silver iodide (AgI) , silver fluoride (AgF) , and silver acetate (CH₃COOAg) . A highly water-soluble silver salt (e g , silver nitrate) is preferred. The water soluble polymer used m the preparation of nanosized silica-silver may be exemplified by polyvinylpyrrolidone (PVP) , polyvinyl alcohol (PVA) , polyacrylic acid and derivatives thereof, levan, pullulan, gellan, water-soluble cellulose, glucan, xanthan, water soluble starch, and corn starch Of them, polyvinylpyrrolidone (PVP) is preferred The silicate used m the preparation of nanosized silica silver may be exemplified by sodium silicate, potassium silicate, calcium silicate, and magnesium silicate Of them, sodium silicate is preferred The use of silicate for preparing nano-silver was not reported prior to the present invention The present inventors are the first to describe the use of silicate, not a silica form, m the reaction with a silver salt m order to provide nanosized silica silver havxng excellent antibacterial effects, in which silica molecules and a water soluble polymer are bound to nano silver In the preparation of nanosized silica silver, the silver salt and silicate are reacted in a weight ratio of 1 0 5 to 1 3 (silver salt silicate) Preferably, the reaction is carried out m a weight ratio of 1 1 The particle size of nanosized silica silver may be controlled according to the amount of silicate The use of silicate in a small amount results in increased size of particles In contrast, when an excess amount of silicate compared to the silver salt is used, particles do not form In the nanosized silica silver preparation, the silver salt and water-soluble polymer are reacted in a weight ratio of 1 0 5 to 2 5 (silver salt water soluble polymer) Preferably, the reaction is carried out in a weight ratio of 1 1 For the preparation of nanosized silica silver, radiation rays may be used, which include beta rays, gamma rays, X rays, ultraviolet and electron rays A gamma ray dose of 10 to 30 kGy is preferred Generally, nano sized particles are able to penetrate the plasma membrane, and silica is well taken up by fungi The nanosized silica silver is taken up by fungal cells, in which the nanosized silica-silver exhibits increased antimicrobial activity mediated by silver nanoparticles, and forms a physical barrier against pathogenic fungi due to the property of silica to increase resistance by inducing dynamic resistance to diseases, thereby preventing recurrence of diseases for a considerable period of time after pathogens are disinfected The term "antifungal", as used herein, includes both growth inhibition of pathogenic fungi and disinfection of pathogenic fungi through survival inhibition

The nanosized silica-silver contained in the present composition exhibits excellent antimicrobial activity against fungi including Candida, Cryptococcus, Aspergillus, Trichophyton, Trichomonas, Malassezia and Mucor Thus, the present composition is useful for the treatment of diseases caused by the fungi, specifically dermatophytosis, tinea, aspergillosis (mycotic pneumonia), candidiasis, mycotoxicosis, favus, dandruff, histoplasmosis and cryptococcosis

In a detailed practice, the present composition dxsplayed excellent fungxcxdal actxvxty agaxnst Candida, Cryptococcus, Aspergillus and Malassezia furfur even m a minimal xnhibxtory concentration (MIC) of less than 2 μt,/ml Also, a study of acute toxxcxty of nanosized silxca-sxlver in rats on dermal application resulted in no side effects and no death of rats, revealing that the present composition xs stable

Conventional antifungal agents are problematic in terms of having serious side effects despite their high fungicidal activity, or having only fungistatic activity with no fungicidal activity Thus, there is a need of fungicidal agents having higher fungicidal activity with no side effects Under this situation, the above results, indicating that silver, widely known as an antimicrobial agent with no side effects, is useful as an antifungal agent having excellent fungicidal effects when processed into the form of nanoparticles bound to silica molecules and a ^ater soluble polymer, has economical importance, because the present invention provides a simple process for producing nanosized silica silver with low costs by a simple process, as well as having medical importance

For antifungal action, the nanosized silica-silver contained in the antifungal composition of the present invention may be used alone or may be administered m combination with a conventionally known antifungal substance

The nanosized silica silver particles contained m the antifungal composition of the present invention have a particle size of 0.5 to 30 nm, preferably 1 to 20 nm, and more preferably 1 to 5 nm.

According to the intended therapeutic purpose, the present composition may be formulated into pharmaceutical preparations common in the pharmaceutical field. For example, the formulations include tablets, capsules, powders, granules, suspensions, emulsions, syrups, plasters, ointments, sprays, oils, gels, spirits, tinctures, baths, liniments, lotions, patches, pads and creams. Topical formulations are preferably used for direct application of the composition to a desired area of the external surface of the skin. Preferred topical formulations include ointments, lotions, sprays and gels. Topical formulations may be also contained in a support base or matrix directly applicable to a desired area of the skin. Examples of the support base include gauze or bandages. The nanosized silica-silver may be used in a colloidal or dried powder form in the formulations. For ointment formulation, taking into consideration various factors including temperature of the skin surface, pH of the skin, transdermal water loss levels and total lipid levels of the epidermis, the present composition may be mixed with oligmous bases, which are exemplified by Vaseline, liquid paraffin, paraffin, plastibase, silicon, lard, vegetable oils, waxes and purified lanolin, water-soluble bases, emulsion bases, suspension bases, and the like. The ointments may be supplemented with an antioxidant (e.g , tocoperol, BHA, BHT, NDGA, etc.), an antiseptic (e.g., phenolic compounds, chlorobutanol, benzylalcohol, parabens, benzoic acid, etc.), a humectant (e.g., glycerin, propylene glycol, sorbitol, etc.), a solution adjuvant (e.g., ethanol, propylene glycol, etc.), a softening adjuvant (e.g., liquid paraffin, glycerin, propylene glycol, surfactants, etc.), and other additives.

For lotion formulation, the present composition may be formulated into various lotion forms including solutions, suspensions and emulsions. For lotions to be applied to the skin, the present composition may be formulated into lotions, for example, with a viscosity of 200 cps to 500 cps, and may be preferably supplemented with a humectant such as glycerin or propylene glycol to give a soft feeling upon application to the skin.

For spray formulation, the additives may be mixed with a propellant to disperse a water-dispersed concentrate or humidified powder. For patch formulation, a permeation stimulator may be used to increase the permeation of compounds through the skin.

In another aspect, the present invention relates to a method of disinfecting a pathogenic fungus by administering the composition.

The present composition may be administered by various routes, for example, orally, parenterally, topically mtrarectally and xntranasally For example solid formulations for oral administration may include, in addition to active substances, a diluent (e g , lactose, dextrose, sucrose, cellulose, corn starch, potato starch etc ) , a lubricant (e g , silica, talc, stearic acid, magnesium or calcium stearate, polyethylene glycol, etc ) , a binder (e g , starch, arable gum, methylcellulose gelatin carboxymethylcellulose, polyvinylpyrrolidone, etc ) , a disintegrator (e g , starch, arginic acid, argmate, sodium starch glycolate, etc ) , a formal mixture, a dye, a sweetener a humectant (e g , lecitin, polysorbate, laurylsulfate, etc ) , and other pharmacokinetically inactive materials which are commonly used m pharmaceutical preparations Preparations for topical administration may include an excipient (e g , lactose, starch, cellulose, polyethylene glycol, etc ) , a lubricant (e g , magnesium stearate, stearic acid, glyceryl behenate, talc, etc ) , and a preservative (e g , benzalkonium chloride) The parental administration includes injection bringing about systemic effects or injection based on direct administration to a painful area Examples of parental administration include subcutaneous intravenous, intramuscular, intradermal, intrathecal and intraocular injection, and general infusions

The topical administration includes the treatment of infection regions or organs which are easily approachable by topical application, for example, to the ear, the vagina, opened and sutured wounds or closed wounds and the skin as well as to infections of the eye, external ear and middle ear. Also, topical administration includes transdermal delivery bringing about systemic effects.

Formulations for rectal administration include suppositories.

The intranasal administration includes nasal aerosol or inhalation applications. The present composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount", as used herein, refers to an amount sufficient for treatment or prevention of diseases, which is commensurate with a reasonable benefit/risk ratio applicable for medical treatment or prevention. An effective dosage of the present composition may be determined depending on the patient's diseases and severity of the diseases; drug activity; the patient's age, body weight, health state and gender; the patient's drug sensitivity; administration time, administration routes and excretion rates of a used extract; duration of treatment; drugs used in combination with or simultaneously used with a used extract; and other factors known in medical fields. Typically, the present composition may be administered m a dosage of 0.01 to 100 mg/kg/day.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention

EXAMPLE 1 Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

1 g of sodium silicate (Na₂SiOs) , 1 g of silver nitrate (AgNO₃) , 1 g of polyvinylpyrrolidone (PVP) and 12 ml of isopropylalcohol (IPA) were dissolved in distilled water at a total volume of 200 ml Nitrogen gas was injected into the resulting solution for 20 mm After bubbling, the solution was irradiated with gamma rays of 25 kGy, thereby preparing nanosized silica-silver

Fig Ia is a flowchart of a method of preparing nanosized silica silver bound to silica molecules and a water-soluble polymer according to one embodiment of the present invention After irradiation with gamma rays, the solution appeared yellow, characteristic for nano-silver particles This result indicates the formation of stable nano sized silica silver particles through linkage of silica molecules, the water-soluble polymer and silver particles by the above reactions

The particles formed by the above reactions were examined to determine if they were nano-silver particles Test samples were prepared according to the compositions described m Table 1, below, and were allowed to stand for 24 hrs at room temperature Thereafter, test samples were examined for color change .

TABLE 1

^*■ Solution prepared in this example

Test samples A and B were the prepared solutions irradiated with radiation rays, and test samples C and D were the prepared solutions containing Ag^* ions but not irradiated with radiation rays. Test samples SW and DW were used as controls, not containing silver ions or silver particles.

Silver is easily oxidized in an ionic state. In the presence of Cl^" ions, silver ions are precipitated as a brown precipitate, AgCl, wherein they turn brown. Based on this fact, the state of silver was investigated using tap water containing Cl ions. Silver forms precipitates in an ionic state (Ag⁺) , and appears yellow when present as stable nano-silver particles. The results are given in Table 2, below.

TABLE 2

As shown in Table 2, test samples SW, D and DW were colorless with no change in color after incubation for 24 hrs, indicating that silver ions, chloride ions, or neither silver ions nor chloride ions were in existence In contrast, test sample C changed from colorless to reddish brown This is because silver ions bonded to chloride ions contained xn tap water to form a precipitate of AgCl Test samples A and B appeared yellow with no change in color indicating that the irradiation with radiation rays formed stable nano silver particles, bound to silica molecules and a water-soluble polymer with no formation of AgCl precipitates even m the presence of chloride ions The color changes are also photographically shown m Fig 2

Fig 3 shows the absorption spectrum of the nanosized silica silver of the present invention, prepared as described above The absorption spectrum of the nanosized silica silver was compared with absorption spectra of test samples DW, B and D, described in Table 2 Only test sample B absorbed light at 403 nm, characteristic for nano silver Test samples DW and D did not absorb light at the same wavelength

As revealed from the results obtained after incubation for 24 hrs and the absorption spectra, the irradiation of a solution containing sodium silicate, silver nitrate and PVP with radiation rays forms stable nanosized silica silver bound to silica molecules and a water soluble polymer Fig. Ib shows TEM (Transmission Electron Microscope) images of the nanosized silica-silver prepared as described above. As shown in Fig. Ib, nanosized silica-silver particles have a uniform particle size distribution with a particle size less than 20 nm, specially, ranging from 1 nm to 5 nm. The nanosized silica-silver particles are dissociated from each other or assemble into loose spherical aggregates by intermolecular attractive forces. The aggregates are easily disassembled by heating.

EXAMPLE 2: Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that sodium silicate (NajSiO₃) was used in varying amounts of 0.5 to 2 g. Various test samples, described in Table 3, below, were prepared with varying amounts of sodium silicate and examined.

TABLE 3

Fig. 4 shows the changes in absorbance and color of nanosized silica-silver according to varying concentrations of sodium silicate, described in Table 3

As shown in Fig 4, the highest absorbance was observed m a sodium silicate to silver nitrate ratio of 1 1 The absorbance decreased when sodium silicate was used in a 1 5-fold hxgher amount than silver nitrate Also, when sodium silicate was used in a 0 5-fold lower amount than silver nitrate, orange-gold color was observed, indicating that silver particles increased m size

The above results indicate that the added amount of sodium silicate is an important factor upon preparation of nanosized silica-silver, and that the particle size of nanosized silica-silver can be controlled by varying the amount of sodium silicate

EXAMPLE 3 Preparation of nanosized silica silver bound to silica molecules and water soluble polymer

Nanosized silica silver was prepared according to the same method as m Example 1, except that polyvinylpyrrolidone (PVP) was used m varying amounts of 0 5 to 2 g The changes in absorbance and color of nanosized silica silver according to varying concentrations of polyvinylpyrrolidone (PVP) are given m Table 4, below, and Fig 5

TABLE 4

As shown xn Table 4 and Fxg 5, when sodium silicate was used in an equal ratio to silver nitrate polyvinylpyrrolidone (PVP) can be used m a concentration 0 5 to 2-fold higher than sodium silicate (or silver nitrate)

EXAMPLE 4 Preparation of nanosized silica silver bound to silica molecules and water soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that high levan or corn starch was used instead of polyvinylpyrrolidone (PVP)

The absorbance and absorption spectra of the prepared nanosized silica silver are given m Table 5, below, and Figs 6a and 6b

TABLE 5

Test samples Ab at 403 nm

High levan 0 208

Corn starch 0 211

As shown in Table 5 and Figs 6a and 6b, polysaccharides such as levan or corn starch are available for preparation of nanosized silica-silver although the use of lcvan or corn starch resulted m decreased absorbance at 403 nm

EXAMPLE 5 Preparation of nanosized silxca-sxlver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that varying radiation doses were used

The absorbance and absorption spectra of the prepared nanosized silica-silver are given in Table 6, below, and Fig 7

TABLE 6

As shown m Table 6 and Fig 7, the absorbance at 403 nm occurred even in a gamma-ray dose of 10 kGy, and increased with increasing gamma-ray doses These results indicate that nanosized silica-silver can be prepared using a radiation dose higher than 10 kGy

EXAMPLE 6 Evaluation of antifungal effects of nanosized silica-silver bound to silica molecules and water-soluble polymer TEST EXAMPLE 1 Antifungal activity against Rhizoctonia.

A culture tnedxum for the growth of microorganisms PDA medium (Difco) , was autoclaved and aliquotted xn 25 ml xnto petri dishes Before the medium was hardened (at about 40^°C) , xt was mixed with silica molecules for test sample A, the nanosized silica-silver prepared in Example 1 for test sample B, silver partxcles 20 nm xn size for test sample C, and sxlver particles 100 nm in size for test sample D Then, the medium was allowed to cool to give PDA plates Each PDA plate was inoculated with a pathogenic fungus, Rhizoctonia solani, which was sufficiently grown on a solid medium and excised as a circle 5 mm m diameter Thereafter, each plate was incubated for 2 days at room temperature to examine whether the growth of R solani would be inhibited The additionally supplemented materials m the test samples were used in concentrations of 6 ppm and 0 3 ppm

As shown in Fig 8a, test sample A mixed with silica molecules exhibited the same results as m a control in both concentrations of silica molecules Test samples C and D, mixed with 20 nm silver and 100 nm silver, respectively, displayed the same results as m the control at 0 3 ppm of silver In contrast, test sample B, mixed with the nanosized silica silver of the present invention, exhibited remarkably inhibited growth of Rhizoctonia solani even m a very low concentration of 0 3 ppm of nanosized silica silver

TEST EXAMPLE 2 Antifungal activity against Botrytis

A culture medium for the growth of microorganisms, PDA medium (Difco) , was autoclaved and aliguotted m 25 ml into petri dishes. Before the medium was hardened (at about 40T¹) , it was mixed with silica molecules for test sample A, the nanosized silica-silver prepared m Example 1 for test sample C, silver particles 20 nm m size for test sample B, and silver particles 100 nm in size for test sample D Then, the medium was allowed to cool to give PDA plates Each PDA plate was inoculated with a pathogenic fungus, Botrytis cmerea, which was sufficiently grown on a solid medium and excised as a circle 5 mm in diameter Thereafter, each plate was incubated for 2 days at room temperature to determine whether the growth of B. cmerea would be inhibited The additionally supplemented materials in the test samples were used m concentrations of 6 ppm, 3 ppm and 0 3 ppm

As shown in Fig. 8b, test sample A mixed with silica molecules exhibited the same results as in a control in all concentrations of silica molecules Test samples B and D, mixed with 20 nm silver and 100 nm silver, respectively, displayed the same results as in the control at 0 3 ppm of silver In contrast, test sample C, mixed with the nanosized silica-silver of the present invention, exhibited remarkably inhibited growth of Botrytia oiπeiea even xn a very low concentration of 0 3 ppm of nanosized silica silver in comparison with the treatment of B cmerea with 20 iim silver and 100 nm silver In addition, as shown m Fig 9, the nanosized silica-silver inhibited the germination of spores of Botrytis cmerea in a low concentration of 3 ppm

TEST EXAMPLE 3 Antifungal activity against Pythium and Magnaporthe

A culture medium for the growth of microorganisms, PDA medium (Difco) , was autoclaved and aliquotted m 25 ml into petri dishes Before the medium was hardened (at about 40 C) , it was mixed with the nanosized silica silver, prepared xn Example 1, of 0, 0 3, 1 6 and 3 ppm Then, the medium was allowed to cool to give PDA plates The PDA plates were inoculated with Pythium ultimum and Magnaporthe grisea, which were sufficiently grown on a solid medium and excised as circles 5 mm m diameter Thereafter, each plate was incubated for 2 days at room temperature to determine whether the growth of P ultimum and M grisea would be lnhxbxted

As shown m Fig 8c, the nanosized silica silver exhibited remarkable growth inhibitory effects on Pythium ultimum and Magnaporthe grisea at 3 ppm TEST EXAMPLE 4 Antifungal activity against Candida, Cryptococcus and Aspergillus

Minimal inhibitory concentrations (MICs) of nanosized silica-silver, tolnaftate, amphotericin B and itraconazole agaxnst various human pathogenic fungi were measured The pathogenic fungi included Candida lusj.tanj.ae, Candida tropicalis, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Cryptococcus neoformans, Mucor ramosissmus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus The MICs were measured using a standard procedure proposed by the AFST EUCAST (Anitifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing, Rodriguez Tudela et al , (2003) Method for the determination of minimum inhibitory concentration by broth dilution of fermentative yeasts, Clinical Microbiology and Infection, 9, I-VIII) This standard is based on the reference procedure of the National Committee for Clinical Laboratory Standards (NCCLS) , which is described in the literature (National Committee for Clinical Laboratory Standards (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast-Second Edition Approved Standard M27-A2 NCCLS, Wayne, PA, USA)

In detail, of the pathogenic fungi, Candida species, Cryptococcus neoformans and Mucor ramosissmus were cultured using SDA (Sabouraud Dextrose Agar) medium at 35^°C for 24 hrs for Candida species and for 48 hrs for C. neoformans and M. ramosissmus. About five colonies of less than 1 mm were picked, suspended in 5 ml of 0.85% saline (8.5 g/L NaCl) , and adjusted to a final density of 2XlO³ cells/ml with RPMI 1640 medium to give an inoculum. Also, Aspergillus species were sufficiently cultured at 35^"C for 7 days using PDA (Potato Dextrose Agar) medium. After 5 ml of sterile distilled water and one drop of Tween 20 were poured onto the PDA plate, spores were scratched with a sterile micropipette tip and placed into a test tube. After the test tube was allowed to stand for 3 to 5 min, the supernatant was recovered and adjusted to a density of 2XlO⁴ CFU/ml to give an inoculum. Of the nanosized silica- silver prepared in the above Examples and demonstrated to have antifungal activity, the nanosized silica-silver prepared in Example 1 was used in this test and was two¬ fold serially diluted with RPMI 1640 medium. Also, as controls, tolnaftate, amphotericin B and itraconazole were dissolved in DMSO (dimethyl sulfoxide) and two-fold serially diluted with RPMI 1640 medium. The final concentration of DMSO was 2.5%. 100 ιΛ of each dilution and 100 βi of each inoculum were aliquotted into 96-well plates, thereby giving a final concentration of 128 μg/wl to 0.0313 μg/mH. for antifungal agents contained in the two-fold serial dilutions. 96-well plates seeded with Candida species and Aspergilles fumigatus were incubated at 35^°C for 48 hrs. Cryptococcus neoformans and Mucor ramosissmus were cultured at 35^°C for 72 hrs. After cultivation, the culture was observed with the naked eye, and the lowest concentration inhibiting visible fungal growth was considered a minimum growth inhibitory concentration (MIC; l≥μg/urf) . The results are given in Table 7, below.

TABLE 7

(Unit, μg/ml)

As shown in Table 7, the nanosized silica-silver exhibited antifungal activity against pathogenic fungi, Candida, Cryptococcus, Mucor and Aspergillus.

TEST EXAMPLE 5: Antifungal activity against Malassezia furfur Minimal inhibitory concentrations (MICs) of nanosized silica-silver and amphotericin B against Malassezia furfur, often associated with the formation of dandruff, were measured Tests were carried out according to the same procedure as in Test Example 4, except that Malassezia furfur was two-fold serially diluted with M429 broth instead of RPMI 1640 medium used m Test Example 4 The results are given m Table 8, below

TABLE 8

As shown in Table 8, the nanosized silica-silver exhibited antifungal activity against the dandruff causing fungus, Malassezia furfur

TEST EXAMPLE 6 Toxicity test of nanosized silica-silver on dermal application m rats

An acute toxicity test of the nanosized silica-silver on dermal application was conducted with specific pathogen- free (SPF) SD female rats (219 1 to 231 g) and male rats (257 9 to 285 2 g) (Oπentbio Inc , Korea) Female rats and male rats were independently grouped into two groups, each consisting of five rats As a control, injectable sterile distilled water (Choongwae Pharma Corporation, Korea) was dermally administered to one of the female or male rat groups m a do&age of 2 m-C/kg/day The nanosized silica- sxlver prepared in Example 1 was dermally administered to the other group in a dosage of 2 mC/kg/day

Dermal application was carried out as follows At the day before dermal application, animals were shaved on an abdominal region of 6X8 πif The injectable sterile distilled water or nanosized silica-silver was evenly put onto two sheets of gauze of 4x4 πn in a dosage of 2 mC/kg/day The gauze was placed onto the shaved abdominal region and closely contacted therewith using a vinyl piece and a medical adhesive plaster After 24 hrs, the gauze and the adhesive plaster were removed, and residual materials were washed off with physiological saline For 8 days after dermal application, the animals were observed for side effects or mortality, as follows

The rats were observed for mortality and signs of toxicity 1, 3 and 6 hrs after application on the day of dermal application and once or more every day for a period of the next day to 8 days after application Body weights were recorded immediately before application and 2, 4 and 8 days after application 8 days after application, the rats were anesthetized with CO₂, and the abdomen was opened The rats were sacrificed by dissecting the abdominal artery and bleeding Abnormality of all organs was visually observed In the acute dermal toxicity test, in which the pharmaceutical composition of the present invention was administered in a dosage of 2 m4/kg/day, no effects were observed on viability, gross clinical signs, body weight change and autopsy findings.

Industrial Applicability

As described hereinbefore, the antifungal pharmaceutical composition containing nanosized silica- silver according to the present invention has broad applications for treating fungal infections due to its strong antifungal activity, with almost no side effects.

Claims

1 An antifungal pharmaceutical compositxon comprising nanosized silica-silver particles 0 5 to 30 nm in size, m which nano silver is bound to silica molecules and a water-soluble polymer, as an effective ingredient, the nanosized silica silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water-soluble polymer with radiation rays

2 The composition as set forth m claim 1, which is effective against a fungus, Candida, Cryptococcus, Mucor,

Aspergillus or Malassezia

3 A topical formulation comprising the composition of claim 1

4 The topical formulation as set forth in claim 3, which is m a form of ointments, lotions, sprays, patches, creams, powders, suspensions or gels