RU2749481C1 - Method for surface mycosis therapy - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine. The method of treating superficial mycoses consists in exposing the skin surface to a transdermal preparation containing an antifungal compound, while a transfollicular preparation is used as a transdermal preparation, which is an antifungal compound immobilized in vaterite particles, the effect of a transfollicular preparation is carried out by applying its alcohol suspension with a concentration of 100-150 mg / ml on the surface of the skin in the area of ​​pathology until a dose of 4-5 mg of particles / cm2containing 10-20 wt.% of an antifungal compound is reached, after which the application area is exposed to ultrasound with a frequency of 0.88-1 MHz with an intensity of 0.4-0, 5 W / cm2for 2-3 minutes, while the treatment procedure is carried out no more than 1 time in 3 days.EFFECT: invention provides increased effectiveness of antifungal agents for treatment of superficial mycoses while reducing the number of procedures for applying an antifungal drug, as well as providing possibility of topical application of drugs that previously did not have a topical form due to poor solubility and low bioavailability.1 cl, 18 dwg, 6 tbl, 6 ex

Description

The invention relates to medicine and is directed to the treatment of superficial mycoses using an antifungal composition for topical use, which has the ability to penetrate and accumulate in hair follicles. The composition contains vaterite carrier particles loaded with an antifungal agent. The invention provides effective intradermal accumulation and prolonged intake of the active substance, thereby reducing the frequency of application of the antifungal agent and the course dose without loss of antifungal efficacy.

Superficial fungal infections are one of the most common infectious diseases of the skin and its appendages and cover 20-25% of the world's population, and their distribution has grown significantly over the last quarter of a century, due to the growth of tourist activity, changes in the nature of migration, as a result of changes in the socio-economic nature [Ameen M. Clinics in dermatology. 2010, 28, 197-201; Aggarwal NJ Pharmaceutics. 2012, 437, 277-287; Bottonae EJJ Dermatology. 2006, 2, 41-50] . This pathology is highly contagious in the human population, in particular, in the children's audience.

Despite the existence of a large number of effective antifungal agents, to date, the urgency of improving antifungal therapy remains, due to the need to reduce the risk of side effects on the body, increase the bioavailability of drugs, reduce the duration of the course of treatment and increase the effectiveness of external therapy for fungal diseases in general [Medvedeva T V. et al. J. Skin and Venereal Diseases, 2007, 4, 54-57; Lewis RE Mayo Clinic Proceedings. 2011, 86, 805-817] . In this regard, in recent decades, the development of approaches associated with the immobilization of antimycotic agents in various nanoscale and submicron systems, such as liposomes, micelles, emulsions, polymer or lipid containers [Kumar L. et al. Artificial Cells, Nanomedicine, and Biotechnology, 2014, 42, 32-46; Basak A. et al. Recent trends in antifungal agents and Antifungal Therapy, Springer India; 2016; Scorzoni L. et al. Frontiers in Microbiology, 2017, 8, 36; Bangia R. el al. Expert Opinion on Drug Delivery, 2019, 16, 377–96; Nicola AM et al. Pharmacology & Therapeutics, 2019, 195, 21-38] .

However, despite numerous attempts to create an effective system for the delivery of antifungal agents, today there are only three commercially available antifungal medicines based on nanoparticles: the liposomal form of Amphotericin B - the drug “Ambizome” (AmBisome®), the lipid complex of Amphotericin B “Abelcet” (Abelcet ®), colloidal disk-like particles of Amphotericin B "Amphocyl" or "Amphotec", depending on the country of registration (Amphotec® / Amphocil®), as well as liposomal Nystatin “Niotran” (Nyotran®). Moreover, these drugs are used in the treatment of systemic fungal infections and are intended for intravenous infusion.

A known method for the treatment of superficial mycoses using the dosage form of Amphotericin B based on cholesterol-containing sediment with a diameter of 20-200 nm (RF patent No. 2481100, according to class IPC A61K 9/127, publ. 05/10/2013). A distinctive feature of this invention is the possibility of resuspending such a dosage form in saline, which made it possible to replace the latter with dextrose during intravenous infusions and, thus, reduce the nephrotoxicity of intravenously administered Amphotericin B. At the same time, the suspension of cholesterol-containing sediments in saline was stable.

Since superficial mycoses, as a rule, affect the surface layers of the skin, as well as its appendages (hair and nails), without involving other organs and tissues in the pathological process [Aggarwal NJ Pharmaceutics. 2012, 437, 277-287] , then it is advisable to develop approaches to improve the effectiveness of external use of antimycotics [Kaur IP et al. Expert opinion on drug delivery, 2010, 7, 1303-1327] . The advantages of external use are the reduction of side effects and load on the gastrointestinal tract, compared with their systemic use, due to the possibility of localizing the therapeutic effect in the pathology zone [Kaur IP et al. Expert opinion on drug delivery, 2010, 7, 1303-1327 ; Güngör SJ Cosmetics, Dermat. Sc. and App., 2013, 3, 56-65] .

With topical application of a cream based on the above-mentioned cholesterol-containing sediment Amphotericin B, the possibility of more effective accumulation of the drug in the skin was demonstrated, compared to the standard Amphotericin B cream. However, this result was achieved only 2 hours after applying the cream, and the efficiency of accumulation of the proposed sediment in the skin still inferior to other existing systems of intra- and transdermal drug delivery, the possibility of penetration into hair follicles is not discussed by the authors. In addition, for the present invention, an assessment of the antifungal efficacy in the treatment of mycoses of the skin and appendages in vivo has not been shown, although a good antifungal activity has been demonstrated in vitro against a wide range of fungi, as well as in vivo for ophthalmic use, as exemplified by the Aspergillus fumigatu keratitis model in rabbits.

There are also known methods of treating superficial mycoses using topical compositions, the action of which is aimed at ensuring long-term contact of the drug with the affected area by forming a coating containing an antifungal agent. So, for example, there is a known method of treatment by aerosol exposure to the affected area with an agent containing the antimycotic ketoconazole, an emulsion carrier, emulsifiers, castor oil, water, as well as targeted additives - a stabilizer and a preservative (RF patent No. 2337689 according to class IPC A61K 31/496 , publ. 10.11.2008). The invention is capable of providing a longer contact of a medicinal substance with the affected area simultaneously with its protection from external influences and, as a result, reducing the risk of transmission of mycosis from an infected person to other people. In addition, such a dosage form has increased hygiene of use and sedimentation resistance. The fungicidal activity of the composition against the strains of Candida albicans and Aspergillus nige has been demonstrated in vitro , however, the effectiveness of the therapy for dermatomycosis in vivo of the present invention has not been discussed.

There is also known a method of treating fungal infections on the basis of a liquid composition for topical application (see application WO 2007/039533 by class IPC A61K47 / 32, publ. 12.04.2007) containing an antifungal agent, a film-forming agent and a solvent. In this case, the antifungal agent is selected from the group consisting of imidazoles, triazoles and allyalmines. This form is intended for application to the infected area of the skin, where it forms an antifungal film that ensures the supply of the active substance. In the treatment of patients with epidermophytosis of the feet, it was found that with a single application of the composition of the invention, a treatment efficacy is achieved that is equivalent to or exceeds the efficacy of treatment achieved when performing multiple application regimens of classical antifungal pharmaceutical products for one to four weeks. The use of this composition provided penetration of the antifungal agent into the stratum corneum of the skin of patients, however, the possibility of its penetration and deposition in such skin appendages as hair follicles has not been considered.

The development of approaches aimed at ensuring intra- and transdermal targeting and deposition of antifungal agents in the skin when applied externally is a significant task in the treatment of superficial mycoses.

The closest to the claimed technical solution is a method of treating fungal infections using a transdermal absorbable preparation containing an antifungal compound and an enhancer for its percutaneous penetration (RF patent No. 2576612 according to IPC class A61K 31/445, publ. 03/10/2016). As such an enhancer, the present invention proposes the use of compounds such as higher alcohols, higher monocarboxylic acids or their esters, aromatic / non-aromatic monoterpenes, either individually or in combination. It is worth noting that the use of various chemical enhancers is one of the classic approaches to increasing the efficiency of transdermal delivery of biologically active substances, which has repeatedly demonstrated its effectiveness [Prausnitz MR et al. Dermatology. 2012, 2065–2070; Haque T. et al. Advanced Pharmaceutical Bulletin. 2018, 8, 169-179] . The penetration study of various variations of the aforementioned invention was carried out in vitro on the skin excised from the peritoneum of a bald rat, placed in a Franz diffusion cell. The antifungal activity of this technical solution has not been investigated by the authors. In addition, the authors again did not consider the possibility of penetration and deposition of the antifungal compound in the hair follicles. Moreover, the use of the skin of a hairless rat as a model excluded the possibility of studying the contribution of transfollicular transfer to the accumulation of substances in the skin due to differences in the anatomy and physiology of hair follicles of specially bred lines of hairless and standard animals [Ishii Y. et al. Journal of Anatomy. 1997, 191 (1), 99-106] .

However, the possibility of increasing the efficiency of intrafollicular penetration of antifungal agents is of particular interest in the framework of external therapy for dermatomycosis, since the space of the sebaceous hair follicle itself is capable of serving as a reservoir for storing the drug substance, ensuring its localization in the affected area [Knorr FJ Pharm. Biopharm. 2009, 71, 173-180] . The use of carrier matrices for the immobilization of the active substance is one of the significant approaches to ensure their efficient transfollicular drug delivery [Lademann J. Opt. Health Sci. 2015, 8, 1-7] .

The technical problem of our claimed invention is the development of a method for local therapy of superficial mycoses, involving intrafollicular delivery of an antifungal drug using carrier containers and characterized by high therapeutic efficacy and the absence of pronounced toxic effects.

Technical result claimed invention consists in increasing the effectiveness of antifungal agents for the treatment of superficial mycoses while reducing the number of procedures for applying an antifungal drug, as well as providing the possibility of topical application of drugs that previously did not have a topical form due to poor solubility and low bioavailability.

This technical result is achieved by the fact that in the method of treating superficial mycoses, which consists in exposing the skin surface to a transdermal preparation containing an antifungal compound, according to the invention, a transfollicular preparation is used as a transdermal preparation, which is an antifungal compound immobilized in vaterite particles, the effect of the transfollicular preparation is carried out by application of its alcohol suspension with a concentration of 100-150 mg / ml on the surface of the skin in the area of pathology until a dose of 4-5 mg of particles / cm ² containing 10-20 wt.% of the antifungal compound is reached, after which the application area is exposed to ultrasound with a frequency of 0.88- 1 MHz with an intensity of 0.4-0.5 W / cm ² for 2-3 minutes, while the treatment procedure is carried out no more than 1 time in 3 days.

As an antifungal compound, naftifine hydrochloride or griseofulvin is used.

EFFECT: transportation of particles with an antifungal agent immobilized in them to the depth of the hair follicle, thus ensuring its improved intradermal accumulation. The gradual degradation of particles in the hair follicles contributes to an increase in the duration (prolongation) of the contact of the antimycotic with the affected area.

The invention is illustrated by tables and drawings, the description of which is given below.

Table 1. The values of the thickness of the fluorescent near-surface region (d) and the average intensity of its fluorescence (I), calculated from CLSM images of cryosections of rat skin from the intact area and the area of introduction of calcium carbonate particles loaded with griseofulvin. Values are presented as (mean ± standard error). Statistics were collected for 3 slices for each area, 15-20 fluorescence profiles from each slice.

Table 2. Experimental groups of laboratory animals (guinea pigs) with a model of dermatomycosis, participating in an experiment to study the therapeutic effect of topical application of an antifungal composition based on vaterite particles.

Table 3. The intensity of manifestation of clinical signs of an infectious process before starting treatment in laboratory animals from an experiment to study the therapeutic effect of topical application of an antifungal composition based on vaterite particles. The table shows the scores for each animal, as well as the average values for the group (M ± m), presented as (mean ± standard deviation).

Table 4. The intensity of manifestation of clinical signs of the infectious process on the 14th day after infection (after 1 week of treatment, ie in the middle of the study) in laboratory animals from an experiment to study the therapeutic effect of topical application of an antifungal composition based on vaterite particles. The table shows the scores for each animal, as well as the average values for the group (M ± m), presented as (mean ± standard deviation).

Table 5. Intensity of manifestation of clinical signs of an infectious process at the end of treatment in laboratory animals from an experiment to study the therapeutic effect of topical application of an antifungal composition based on vaterite particles. The table shows the scores for each animal, as well as the average values for the group (M ± m), presented as (mean ± standard deviation).

Table 6. Summary table with the results of assessing the intensity of manifestation of clinical signs of an infectious process during a study in laboratory animals from an experiment to study the therapeutic effect of topical application of an antifungal composition based on vaterite particles. The table shows the average values of the sum of points for each group of animals, presented as (mean ± standard deviation).

FIG. 1. Scanning electron microscopy image of vaterite particles containing the antifungal agent naftifine hydrochloride.

FIG. 2. Image obtained by confocal laser scanning microscopy, cryosection of rat skin after in vivo introduction of vaterite particles loaded with the antifungal agent naftifine hydrochloride and labeled with a fluorescent dye cyanine-7. The blue fluorescent channel corresponds to autofluorescence of the hair follicle tissues in the range of 420-490 nm, in the red channel the intradermal distribution of the Cy7 dye is demonstrated in the range of 750-790 nm. The dye is delivered to the entire depth of the hair follicle, as evidenced by the clear outlines of the hair follicles.

FIG. 3. Scanning electron microscopy image of vaterite particles containing the antifungal agent griseofulvin.

FIG. 4. Images obtained by confocal laser scanning microscopy of vaterite particles loaded with the antifungal substance griseofulvin. Fluorescence and transmission microscopy overlay (a) and fluorescence only image (b). The blue fluorescent signal corresponds to the fluorescence of griseofulvin in the range 420-490 nm when excited at a wavelength of 405 nm.

FIG. 5. Images obtained by the method of confocal laser scanning microscopy, cryosections of rat skin from the intact area (a) and the area of introduction of vaterite particles loaded with the antifungal substance griseofulvin (b). The blue signal corresponds to autofluorescence of hair follicle tissues, as well as fluorescence of griseofulvin in the range of 420-490 nm when excited at a wavelength of 405 nm.

FIG. 6. Images obtained by the method of scanning electron microscopy of the hair follicles destroyed by the blade, pulled out after the introduction of vaterite particles, loaded with an antifungal agent naftifine hydrochloride. The images illustrate the gradual degradation of containers in mouse follicles in vivo . The bottom row of images represents the enlarged areas selected in the corresponding images of the top row.

FIG. 7. Typical results of microscopic (a, b) and cultural (c) studies of biomaterial (scales of skin and hair of animals) taken from the lesion before starting treatment of animals with a trichophytosis model: fluorescent (a) and bright-field images (b) obtained with magnification x400; growth of a colony of a fungus after sowing (c).

FIG. 8. Photos of the focus of guinea pig trichophytosis from the control group without treatment (group 1): 6th (a), 10th (b), 13th (c), 16th (d), 19th (e ) and 21st (e) days after infection.

FIG. 9. Photos of the focus of guinea pig trichophytosis from the control group with the introduction of unloaded vaterite particles (group 2): a - before the first introduction of particles (7th day after infection before the introduction of particles); b - before the second introduction of particles (10th after infection); c - before the third introduction of particles (13th after infection); d - before the fourth introduction of particles (16th after infection); e - before the fifth introduction of particles (19th after infection); f - at the end of the experiment (21st day after infection).

FIG. 10. Photos of the focus of guinea pig trichophytosis from the group with the introduction of vaterite particles loaded with an antifungal agent naftifine hydrochloride (group 3): a - before the first introduction of particles (7th day after infection before the introduction of particles); b - before the second introduction of particles (10th after infection); c - before the third introduction of particles (13th after infection); d - before the fourth introduction of particles (16th after infection); e - before the fifth introduction of particles (19th after infection); f - at the end of the experiment (21st day after infection).

FIG. 11. Photos of the focus of guinea pig trichophytosis from the group with the introduction of vaterite particles loaded with the antifungal substance griseofulvin (group 4): a - before the first introduction of particles (7th day after infection before the introduction of particles); b - before the second introduction of particles (10th after infection); c - before the third introduction of particles (13th after infection); d - before the fourth introduction of particles (16th after infection); e - before the fifth introduction of particles (19th after infection); f - at the end of the experiment (21st day after infection).

FIG. 12. Photos of the focus of guinea pig trichophytosis from the group treated with the reference drug "Mikoderil" (group 5): a - before the start of treatment (7th day after infection before the introduction of particles); b - after 3 days of daily treatment (10th after infection); c - after 6 days of daily treatment (13th after infection); d - after 9 days of daily treatment (16th after infection); e - after 12 days of daily treatment (19th after infection); f - at the end of the experiment (15 days of daily treatment, 21 days after infection).

FIG. 13. Macrophotographs of intact (a) and control areas of rabbit skin (b-i) from an experiment on the study of in vivo toxicity of topical application of an antifungal composition based on vaterite particles: the area of alcohol application after the first application (b) and at the end of the experiment (f); the zone of application of an alcohol suspension of unloaded vaterite particles after the first application (c) and at the end of the experiment (g); the zone of application of the alcohol suspension of the antifungal substance grisofulvin after the first application (d) and at the end of the experiment (h); the area of application of an alcoholic suspension of vaterite particles loaded with the antifungal agent griseofulvin after the first application (e) and at the end of the experiment (i). The application of all solutions and suspensions was accompanied by ultrasonic treatment.

FIG. 14. Histological preparation of the skin in the control group of animals (group 1) from an experiment to study the in vivo toxicity of topical application of an antifungal composition based on vaterite particles. The sections were stained according to the standard scheme using hematoxylin and eosin.

FIG. 15. Histological preparation of the skin with the application of alcohol (group 2) to animals from an experiment to study the in vivo toxicity of topical application of an antifungal composition based on vaterite particles. The sections were stained according to the standard scheme using hematoxylin and eosin.

FIG. 16. Histological preparation of skin with application of unloaded vaterite particles (group 3) to animals from an experiment on in vivo toxicity study of topical application of an antifungal composition based on vaterite particles. The sections were stained according to the standard scheme using hematoxylin and eosin.

FIG. 17. Histological skin preparation with application of the antifungal agent griseofulvin (Group 4) animals from the experimental study of in vivo toxicity topical antifungal compositions based vaterite particles. The sections were stained according to the standard scheme using hematoxylin and eosin.

FIG. 18. Histological preparation of skin with application of vaterite particles loaded with an antifungal agent griseofulvin (group 5) to animals from an experiment to study the in vivo toxicity of topical application of an antifungal composition based on vaterite particles. The sections were stained according to the standard scheme using hematoxylin and eosin.

Disclosure of invention

The proposed method for the treatment of superficial mycoses involves the use of an antifungal substance immobilized in vaterite particles.

Vaterite particles have a porous structure, as well as a number of such advantages as biocompatibility, biodegradability, solubility at pH <6.5, ease of preparation, low cost [DV Volodkin et al. Langmuir. 2004, 20, 3398; Yu. Svenskaya et al. Biophysical chemistry. 2013, 182, 11-15; Yu. Svenskaya et al. ACS applied materials & interfaces. 2019, 11 (19), 17270-17282] . The porosity of vaterite particles determines the efficiency of incorporating dosage forms into their volume. The possibility of using vaterite containers for encapsulating drugs is also due to their low cytotoxicity and lack of effect on the viability of cell cultures [Yu. Svenskaya et al. Colloid Surf. B. 2016, 146, 171-179; B. Parakhonskiy et al. Biomaterials Science. 2013, 1 (12), 1273-1281] .

Synthesis particles is accomplished by crystallization from solution at draining equivolume equimolar salt solutions of calcium chloride (CaCl ₂₎ and sodium carbonate (Na ₂ CO ₃₎ followed by washing three times with 95% ethanol and drying the precipitate formed at ⁶⁰ ° C in an oven or by lyophilisation at a temperature of -50 ^o C.

Immobilization of antimycotic in vaterite particles is carried out by the method of its co-precipitation with particles in the process of their formation [ Yu. Svenskaya et al. Colloid Surf. B. 2016, 146, 171-179] or by the method of crystallization-induced adsorption [S. German et al. Scientific reports 2018, 8 (1), 17763] . In the first case, 2 ml of an aqueous-alcoholic solution of antimycotic is added to 40 mg of dry vaterite containers. The test tube with the reaction mixture is kept in a freezer at a temperature of -20 ° C for 2 hours until completely frozen with constant stirring. Next, the particles are defrosted at room temperature, followed by a single rinsing with a water-alcohol solution. The freeze / thaw cycle can be repeated several times until the required amount of active ingredient is immobilized. The sample is then freeze-dried. In the second case, solutions of Na ₂ CO ₃ and CaCl ₂ salts are simultaneously poured into a solution of antimycotic in a solvent in a ratio of 1: 5. The process of mixing solutions is carried out using a magnetic stirrer at a speed of 700 rpm for 2-2.5 hours at room temperature. The resulting particles are washed three times with 95% ethyl alcohol, then the sample is freeze-dried.

The use of vaterite carriers of an antifungal agent for the treatment of superficial mycoses is associated with the possibility of ensuring their effective delivery into and through the appendages of the skin (in particular, into the hair follicles) to the sites of the direct localization of pathological processes. This method of treatment is distinguished by the localization and targeting of the therapeutic effect, since it is known that with dermatomycosis, first of all, the appendages of the skin are damaged, without the involvement of other organs and tissues in the pathological process [G. Ginter-Hanselmayer et al. Clinically Relevant Mycoses. Springer, Cham, 2019, 145-176; NJ Aggarwal. Pharmaceutics. 2012, 437, 277-287] .

It is assumed that a portioned portion of dry vaterite particles loaded with an antifungal compound are dispersed in a solvent (70% ethyl alcohol) immediately before use. Transfollicular delivery of containers is carried out by application of this suspension to the surface of the skin in the area of pathology and their subsequent introduction using therapeutic ultrasound.

Particles are applied no more than once every three days. Such a frequency of application is due to the biodegradability of the containers: a noticeable decrease in the intrafollicular concentration of particles is observed only 72 hours after their introduction.

Examples of implementation of the method.

Immobilization and intrafollicular delivery of antifungal agents using vaterite containers.

Example 1. Confirmation of intrafollicular delivery of vaterite particles with antifungal agent.

The example considers the surface application of a suspension of vaterite particles containing an antifungal agent from the group of allylamines (naftifine hydrochloride), labeled with a fluorescent dye cyanine-7 (Cy7), in order to confirm the possibility of their intrafollicular delivery, as well as to assess the depth of their percutaneous penetration. The study is carried out in vivo on the back of a male white laboratory rat.

Immobilization of naphthyfine hydrochloride (Nf) is carried out by the method of its adsorption from solution onto the surface of previously obtained vaterite particles by the method of crystallization-induced adsorption. For this, 2 ml of an aqueous-alcoholic solution of NF with a concentration of 1 mg / ml is added to 30 mg of particles. The test tube with the reaction mixture is kept in a freezer at a temperature of -20 ° C for 2 hours until completely frozen, constantly stirring. After that, the samples are thawed at room temperature and washed once with water. The freeze / thaw cycle is repeated twice.

Using this technique, containers with a diameter of 0.9 ± 0.2 μm were obtained, containing 10 wt% NP (wt% expresses the ratio of the mass of the immobilized antimycotic to the mass of particles). A scanning electron microscopy (SEM) image of the particles is shown in FIG . 1.

In order to further study the penetration of particles into the skin of rats using confocal laser scanning microscopy (CLSM), they are labeled with a Cy7 fluorescent dye. For this, a weighed portion of dry vaterite particles containing Hf is kept in a dye solution with continuous shaking using a shaker for 30 minutes. Then, a single wash with 70% ethanol is carried out.

The day before the experiment, on a 4x4 cm section of the back of the animals, the hair is shaved with a machine to a length of 1 mm without damaging the skin. The rat is anesthetized immediately prior to testing. A weighed portion of dried particles weighing 30 mg is dispersed in 300 μl of 70% ethanol (suspension concentration 100 mg / ml) and applied to a prepared area with a diameter of 3 cm.Then, the action is carried out using a low-intensity therapeutic ultrasound with a frequency of 0.88 MHz and an intensity of 0.4 W / cm ² in within 2 minutes on the application area. Upon completion of the implantation procedure, excess particles are removed from the skin surface using a cotton pad soaked in water.

The depth of percutaneous penetration of particles is investigated by analyzing histological sections of biopsy material taken from laboratory animals (without killing them) immediately after insertion. Subsequent freezing of samples and preparation of histological sections with a thickness of 20 μm is carried out using a cryostat. The study of the obtained sections is carried out by the CLSM method. A representative image obtained when evaluating the depth of penetration of particles into the skin is shown in Fig . 2. The blue fluorescent signal in these images corresponds to autofluorescence of hair follicle tissues in the ultraviolet range (in particular, to the hair shaft), red - to the fluorescence of the Cy7 dye in the range of 750-795 nm.

As a result, the introduction of particles to the entire depth of the hair follicle was demonstrated, which is confirmed by the presence of a fluorescent signal in the red channel, clearly outlining the hair follicles and propagating along the hair shaft.

Example 2: Accumulation in the skin of an antifungal substance that does not have a commercially available topical form, when used in a form immobilized in vaterite particles.

Example 2 discusses the creation of a form for surface application of an antifungal agent with low solubility and bioavailability (griseofulvin), by immobilizing it in vaterite particles. Study of the possibility of intradermal accumulation of the proposed form. This study is carried out in vivo on the back of a male white laboratory rat.

The synthesis of containers is carried out by co-precipitation of the antifungal substance griseofulvin (GF) with vaterite particles during their formation in an ethylene glycol medium at 60 ° C. For this, a sample of GF is added to the ethylene glycol medium to obtain a solution with a concentration of 4 mg / ml. Then, aqueous solutions of salts Na ₂ CO ₃ and CaCl ₂ are mixed with a suspension of GF in ethylene glycol in a ratio of 1: 5. The process of mixing the solutions is carried out using a magnetic stirrer at a speed of 700 rpm for 2.5 hours. The resulting particles are washed three times with 95% ethyl alcohol, then the sample is lyophilized.

Using this technique, containers with a diameter of 1.1 ± 0.4 μm were obtained, containing 20 wt% GF. The SEM image of the resulting particles is shown in Fig . 3.

In order to confirm the percutaneous transportation of GF with the help of vaterite particles, as well as its accumulation in the hair follicles and adjacent tissues, a study of histological sections of biopsy material by the CLSM method is carried out.

Intrafollicular delivery of particles, as well as sampling, preparation and examination of biopsy material is performed according to the scheme described in example 1 , with the difference that CLSM visualization of cryosections is carried out in the range 420-490 nm with excitation by a laser with a wavelength of 405 nm. Although these parameters of GF fluorescence excitation are not optimal (the optimum corresponds to the parameters Ex = 292 / Em = 412), however, they make it possible to detect the fluorescent signal from the drug-loaded vaterite particles. This fact is confirmed by the presence of a bright, well-defined fluorescent signal from the containers themselves in the CLSM images shown in Fig . 4.

The study of the possibility of intradermal accumulation of GF with the superficial application of its form immobilized in vaterite particles is carried out in comparison with the intact area of the skin (without application of particles). Representative fluorescent images of the obtained skin cryosections are shown in Fig . 5.

Despite the fact that tissue autofluorescence is observed with the used shooting parameters, the contribution of GF fluorescence introduced into the skin is obvious. The accumulation of the drug in the skin of rats after application of the loaded vaterite particles is evidenced by an increase in the intensity of the fluorescent signal in the surface layer of the skin, as well as a noticeable increase in the thickness of the near-surface fluorescent region.

For a numerical assessment of these changes, a study of the fluorescence profiles of the near-surface layer on CLSM images of the studied samples is carried out. For this, the CLSM method is used to examine 3 independent cryosections obtained from the biopsy material of the intact area and the area of introduction of particles. The obtained images are analyzed for the distribution of fluorescence intensity in depth using the Gwyddion software and the thickness of the fluorescent region in the marked area (d) and the average intensity of the fluorescent signal in it (I) are calculated. The data obtained are shown in Table. 1 .

It was found that the intensity of the fluorescent signal of the near-surface region after the introduction of vaterite particles loaded with the antifungal agent GF increased 2.6 times compared to the intact region, and the thickness of the fluorescent near-surface region increased by 1.6. The analysis of variance (ANOVA) of the obtained data confirmed that the indicated differences between the two studied samples are statistically significant at p <0.05.

Thus, the CLSM method demonstrated the accumulation of the antifungal substance griseofulvin in rat skin after topical application of vaterite particles loaded with it.

Gradual degradation of vaterite particles loaded with antifungal agent in hair follicles

Example 3: Confirmation of the gradual biodegradation of vaterite particles with an antifungal agent in hair follicles.

Intrafollicular delivery of vaterite particles containing an antifungal agent from the group of allylamines (naftifine hydrochloride) is carried out in order to study the duration of their degradation in hair follicles. The study is carried out in vivo on the back of a laboratory Balb / c mouse.

The synthesis of containers and their transdermal introduction are carried out according to the protocol given in example 1, without preliminary anesthesia of laboratory animals. To assess the state of vaterite carriers containing the antifungal agent Nf, in hair follicles, hair is extracted together with the bulb from the area of their application immediately after the introduction of the particles, and then 3.5, 24, 48, 72, 120 hours after the introduction. Hair extraction is carried out in an amount of 5 pieces for each sample. After that, the tissue of the removed hair follicle of each hair is destroyed with a blade and the obtained material is examined by the SEM method. The results of this study are presented in Fig.6 .

It was found that the integrity of vaterite particles containing the antifungal agent naftifine hydrochloride in follicles is maintained for 48 hours after implantation. After 72 hours, a noticeable thinning of the containers in the follicles is observed, after 120 hours the containers are completely resorbed in the follicles of the mouse. In this regard, it is advisable to take an interval of no more than 1 time in 3 days for the frequency of application of the proposed surface form. Thus, the gradual degradation of particles in the hair follicles contributes to an increase in the duration of contact of the antimycotic with the affected area.

Therapeutic efficacy of the proposed method for the treatment of dermatomycosis

Example 4. Study of the therapeutic effect of topical application of an antifungal composition based on vaterite particles with the ability to penetrate and accumulate in hair follicles in an in vivo model of mycotic disease.

As part of the study of the antifungal activity of the developed form of antimycotic, an in vivo study is carried out on a model of mycotic skin lesions (trichophytosis caused by Trichophyton mentagrophytes ) of guinea pigs. The surface application of vaterite particles containing the antifungal agent naftifine hydrochloride and vaterite particles containing the antifungal agent griseofulvin is investigated in comparison with the commercially available preparation Mikoderil® in the form of a topical ointment.

For the experiment, the following samples are prepared:

- vaterite containers with immobilized naftifine hydrochloride (VkNf);

- vaterite containers with immobilized griseofulvin (VkGf);

- unloaded vaterite containers (Vk).

The synthesis of containers is carried out according to the methods described in examples 1 and 2 . The synthesis of control containers (unloaded vaterite particles) is carried out according to a similar protocol, but without the addition of antimycotic. The containers are prepared under aseptic conditions, then additionally disinfected with 70% ethanol.

The dose of containers for surface application is determined based on the requirements for the optimal dose for the active substance for a particular antimycotic. In this case, the application dose corresponds to 30 mg vaterite containers (i.e. 3 mg naftifine hydrochloril and 6 mg griseofulvin).

The strain T. mentagrophytes RKPG F 1457 was taken for the study from the Russian collection of pathogenic fungi of the N.I. P.N. Kashkin. To prepare a test culture for infection, it was grown for 14 days at 28 ° C on agar Sabouraud's medium in Petri dishes.

Experimental animals (guinea pigs, Cavia porcellus ) were taken from FGUP PLZh "Rappolovo" of the Russian Academy of Medical Sciences. The age of the animals was 30 days, the weight of the animals by the beginning of the experiment was 250 - 300 g. The total number of animals in the study was 18.

Before testing, animals are acclimatized in the laboratory for 7 days. During this period, a daily examination of the external condition of the animals is carried out. After quarantine, the animals are examined in order to exclude sick and injured individuals from the experiment. Then they are randomized into experimental groups according to the scheme shown in Table 2. The conditions for keeping animals comply with the standards of GOST R 53434 - 2009 "Principles of Good Laboratory Practice" and "Sanitary Rules for the Design, Equipment and Maintenance of Experimental Biological Clinics (Vivariums)", approved by the USSR Ministry of Health on 07/06/1973.

The animals are infected with the grown test culture of the fungus. Then, on the 5th and 21st days after infection (after the development of characteristic signs of the disease), skin scales, hair, crusts are taken from the lesion into sterile Petri dishes with a scalpel and tweezers for microscopic and cultural studies. Every day, the infected animals are examined with photofixation of the lesion, its size is measured and described, as well as the presence and degree of erythema, crust formation and peeling in the focus of infection. Observation of the animals continues for 21 days. Infection parameters are assessed in points on a scale:

0 - no observed feature,

1 - a slight presence of a sign in the lesion focus,

2 - a noticeable presence of a sign in the lesion focus,

3 - the presence of a pronounced feature on a wider area of the focus,

4 - the presence of a pronounced sign on the entire lesion focus.

On the day before the start of treatment, clinical manifestations in the control and experimental groups for four out of five observed signs were characterized as uniform and did not differ between groups ( Table 3 ). It was noted that in experimental group 5, ulceration in the focus of infection was more pronounced. Microscopic examination of biomaterial (skin scales, wool) taken before starting treatment on the 6th day after infection in animals in the control and experimental groups, confirmed the presence of septic micromycete mycelium. When sowing on a solid nutrient medium, the growth of T. Mentagrophytes was obtained in all samples. Images obtained for animals of group 1 are shown in Fig . 7. A similar picture was observed in all groups of animals with infection (groups 1-5).

Treatment of animals in groups 2-4 began 6 days after infection with a culture of micromycete, when the clinical picture of infection was pronounced.

Treatment of animals with the use of VkNf, VkGf and Vk was carried out by applying a suspension of containers to the studied surface of the skin and subsequent exposure to therapeutic ultrasound with a frequency of 1 MHz at an intensity of 0.5 W / cm ² for 2 minutes. The procedure for applying containers was carried out once every three days for 14 days (5 procedures), i.e. on the 7th, 10th, 13th, 16th and 19th days after infection.

Suspensions were prepared directly on the skin at the site of infection. For this, before applying the containers, 50 μL of a solvent (70% ethanol) was applied to the lesion, then a weighed portion of the test containers (one dose) was added, after which another 50 μL of solvent was added. After one minute of exposure to ultrasound, 100 μl of the solvent was added to a test tube with the remains of the test object, and the resulting suspension was applied to the lesion, continuing the exposure to the ultrasonic apparatus for another minute.

Treatment of animals with the reference drug (Mikoderil® 1%, ZAO Lekko) in group 5 was started 6 days after infection and was performed daily, once for 14 days, i.e. from the 7th to the 20th days after infection, by applying 0.3 ml (i.e. 3 mg of naftifine hydrochloride) of the reference drug to the affected skin of guinea pigs.

Observation of infected animals was carried out within 21 days after infection.

For animals in the control group without treatment, the entire list of studies was carried out, as well as for animals in the groups with treatment.

Photographs of the lesion in dynamics for groups 1-5 are shown in Fig. 8-12 . Indicators of the infectious process in points recorded in the middle at the end of the observation are shown in Tables 4-6 .

As the animals of control groups 1 and 2 were observed after their infection with trichophytes, the disease progressed, accompanied by an intensification of the infectious process and an increase in the severity of clinical signs during 19 and 16 days of observation, respectively. Then the signs of the manifestation of the disease (redness, peeling and crusting) weakened, but remained pronounced by the end of the experiment ( Figures 8 and 9 ). When using BK, a change in the clinical picture was noted compared to animals in the group without treatment. A slight decrease in the indicators of the infectious process and the acceleration of its weakening is associated with the use of 70% ethanol as a medium for preparing a suspension and particles when applied to the lesion, since ethanol has antifungal activity against dermatomycetes.

When treating animals with the use of VkNf and VkGf, as well as the comparison drug - cream "Mikoderil 1%" (active ingredient - naftifine hydrochloride) (groups 3-5), a pronounced therapeutic effect was observed, expressed in a noticeable weakening of the clinical manifestations of the disease a week after the onset treatment ( Fig. 10-12 ).

Microscopic examination of the material taken from the lesions on the 21st day after infection confirmed the absence of fungus both in the skin scales and in the hair of animals from groups 3 and 5. Cultures of biomaterial from animals of these groups on a dense nutrient also gave a negative result, confirming the effectiveness of the therapy. In group 4, the elements of the fungus were also not found in the hair of animals. Microscopic examination of material from animals from control group 1 (without treatment) revealed spores and septate mycelium of micromycete in hair and scales from the focus of infection.

Thus, at the end of the experiment, with direct microscopy of hair from the lesions, mycelium and fungal spores were found only in group 1 (infection without treatment). At the same time, the frequency of application of the reference drug (preparation "Mikoderil" ^® ) was 15 applications, and the frequency of application of vaterite particles containing the antifungal agent naftifine hydrochloride and vaterite particles containing the antifungal agent griseofulvin was 5. That is, the number of treatment procedures was reduced to 3 times. Thus, it has been demonstrated that the application of the claimed invention provides an optimization of the treatment of superficial mycoses.

Assessment in vivo toxicity of the proposed method for the treatment of dermatomycosis.

Example 5: Evaluation of the in vivo pathological effect of topical application of an antifungal composition based on vaterite particles with the ability to penetrate and accumulate in hair follicles.

In order to assess the local irritant effect and its reversibility during the infollicular introduction of the developed form of antimycotic, an in vivo study is carried out on rabbits of the Californian breed. The surface application of vaterite particles containing the antifungal substance griseofulvin is investigated in comparison with controls (including the surface application of a suspension of unimmobilized griseofulvin).

For the experiment, the following samples are prepared:

- vaterite containers with immobilized griseofulvin (VkGf);

- alcoholic suspension of griseofulvin (GF);

- unloaded vaterite containers (Vk).

The synthesis of containers is carried out according to the method described in example 2. The synthesis of control containers (unloaded vaterite particles) is carried out according to a similar protocol, but without the addition of antimycotic. The containers are prepared under aseptic conditions, then additionally disinfected with 70% ethanol.

The dose of containers for surface application is determined based on the requirements for the optimal dose for the active substance for a particular antimycotic. In this case, the application dose corresponds to 30 mg vaterite containers. This sample of dried particles is dispersed in 200 μl of 70% ethyl alcohol (suspension concentration 150 mg / ml) immediately before application to the skin in groups with application of both BK and BKGF.

The weighed amount of free GF required to prepare a suspension for comparative application corresponds to the mass of GF contained in one dose of VkGf, i.e. in this case is 6 mg. This weighed portion of the antifungal agent is dispersed in 200 μl of 70% ethyl alcohol immediately before application to the skin.

The age of the experimental animals (rabbits of the Californian breed) in the study was 6 months, the weight of the animals at the beginning of the experiment was 2.0 ± 0.5 kg. The total number of animals in the study was 24.

Before testing, animals are acclimatized in the laboratory for 7 days. During this period, a daily examination of the external condition of the animals is carried out. After quarantine, the animals are examined in order to exclude sick and injured individuals from the experiment. Then they are randomized into 5 groups. The day before the start of the experiment, the hair is removed from the rabbits by shaving, avoiding cuts and abrasions, on the area of the skin in the back, with an area of 8 × 9 cm.

The study is conducted for the following groups:

1. Control group, no exposure, intact skin.

2. Comparison group 1 - surface application of ethanol 70%.

3. Comparison group 2 - application of alcohol suspension Vk.

4. Experimental group 1 - application of alcohol suspension Gf.

5. Experimental group 2 - application of an alcohol suspension of VkGf.

The application of substances (200 μl of alcohol, as well as alcohol suspensions of VkGf, Gf and Vk) to animals from groups 2-5 is carried out, followed by exposure to therapeutic ultrasound with a frequency of 0.89 MHz at an intensity of 0.5 W / cm ² for 3 minutes.

All samples are applied three times, once a week, for three weeks.

For two months, the general condition of the animals, the peculiarities of their behavior, coordination of movements, consumption of food and water, are recorded daily. The skin reaction is recorded immediately after the application of the test substances and then every day for 60 days. Photo fixation of research foci is carried out immediately after the first application of the substances and after the last (third) application. The results of photofixation in such an experiment are shown in FIG. 13 .

One week, two weeks and a month after the last application of the test substances, with preliminary anesthesia with zoletil (40 mg / kg), skin samples are taken from the animals for histological examination.

As a result of the application of the test preparations to the skin of rabbits, no symptoms of intoxication were noted. In the study of the irritating effect of the drugs, it was found that with a single exposure to the drugs on the skin, a short-term slight reddening appeared, since the drugs were prepared with alcohol ( Fig. 13 ). Upon further examination of the treated skin area in comparison with the initial period and control animals, no signs were observed that would indicate a pronounced irritating effect of the tested drugs.

Thus, it was found that suspensions of GF and VKGF did not cause visible functional disorders of the skin, such as severe erythema, edema, cracks, ulceration and changes in local skin temperature.

When histological examination of the control group of animals (group 1) at all stages of the study noted the normal structure of the skin and subcutaneous fat ( Fig. 14 ).

In group 2, one week after the first application of alcohol, acanthosis and signs of mild hyperkeratosis were noted ( Fig. 15 ). After 2 weeks, the severity of hyperkeratosis increased, the epidermis itself thickened, infiltration of the dermis with lymphocytes appeared, and the severity of capillary plethora increased. After a month, the structure of the skin and subcutaneous tissue approached normal, papillomatous outgrowths were noted. The data of histological examination indicated the drying effect of the alcohol solution on the skin, but this reaction is reversible.

In group 3, a week after the first application of VK in the skin, moderate edema of the dermis, papillomatous outgrowths of the epidermis (FIG. sixteen). Inflammatory infiltration was observed in the dermis. After 2 weeks, the severity of edema in the dermis increased, and a slight thickening of the epidermis was observed. A month later observed only moderate swelling of the dermis. Thus, a nonspecific inflammatory reaction was observed, caused by the body's immune response to the introduction of calcium carbonate. However, the reaction was also reversible.

In group 4, a week after the first application of the GF suspension, slight acanthosis and thickening of the epidermis, hyperkeratosis were noted; edema and plethora of blood vessels were observed in the dermis ( Fig. 17 ). After 2 weeks, only vascular congestion remained. After a month, the structure of the epidermis and dermis approached normal. Thus, there was no inflammatory response to GF administration, and the observed changes were reversible.

In group 5 with the application of VkGf one week after the introduction of the particles in the dermis, edema and plethora of blood vessels were noted, papillomatosis was observed ( Fig. 18 ). After 2 weeks, the edema of the dermis increased, the epidermis became thinner. After a month the edema decreased. Lymphoid infiltration, which develops in response to the introduction of HKGF, may indicate the activation of the immune system in response to the administration of the studied drugs.

Thus, it was demonstrated that the investigated substances did not cause visible damage to all layers of the skin, but histological examination revealed the development of a nonspecific inflammatory reaction in the dermis in response to the introduction of BK and the appearance of lymphoid infiltration in the dermis in response to the introduction of HKGF. After 2 weeks, an increase in the severity of morphological changes was noted, primarily a violation of blood filling - plethora, edema of the dermis, as well as the reaction of the epidermis itself and changes in its thickness. However, after a month, the structure of the skin approached the norm, which confirms the reversibility of the described changes.

The detected response of the lymphatic system in healthy animals to intraflycular injection of vaterite containers with antimycotics is considered a positive phenomenon, since such a nonspecific stimulation of the immune response can play a special role in the therapy of dermatomycosis.

Example 6. Investigation of the effect of topical application of an antifungal composition based on vaterite particles with the ability to penetrate and accumulate in hair follicles on the biochemical parameters of animal urine.

In order to assess the pathological effect of the infollicular introduction of the developed form of antimycotic to white laboratory rats, their urine samples are analyzed for 10 main physiological indicators: sugar level, bilirubin, specific gravity, levels of ketones, erythrocytes, pH, protein, urobilinogen, nitrates and leukocytes. The surface application of vaterite particles containing the antifungal substance griseofulvin is being investigated.

The synthesis of containers is carried out according to the method described in example 2.

The dose of containers for surface application in this experiment is chosen deliberately higher than the optimal one (1.67 times). Since the optimal dose for surface application of vaterite containers with GF on an area with a diameter of 3 cm was previously taken to be a sample corresponding to 30 mg (which in terms of unit area corresponded to 4 mg particles / cm ² ), this study investigates the effect of a 50 mg sample containers. For this, it was also necessary to increase the area of the region to 10 cm ² in order to ensure efficient introduction of the particles, thus, in terms of unit area, the dose was ultimately 5 mg particles / cm ² . In terms of the active substance, this corresponds to 10 mg griseofulvin applied per 1 rat.

The experimental group included three male white laboratory rats aged from 6 to 8 months weighing 800 ± 50 g.

The day before the start of the experiment, the lower back of each rat was shaved to avoid damaging the integrity of the skin. Then the animals are placed in individual metabolic cages (OpenScience, Russia). One day later, control urine samples were collected for biochemical studies. Then carry out the introduction of particles of vaterite containing HF, according to the method described in example 5 , with ultrasonic exposure at a frequency of 0.88 MHz with an intensity of 0.4 W / cm ² for 3 minutes. The remainder of the suspension of particles is carefully removed from the surface of the skin of the rats with a cotton pad after the completion of the embedding procedure.

A urine sample is taken every day for 12 days after the introduction of the particles (every 24 hours for 288 hours). After centrifuging the samples for 5 minutes at 3000 rpm, the main indicators are measured using urine test strips (UroColorTM10, Standard Diagnostics, Korea).

In addition, a study of urine of a control group of animals is carried out, which also includes 3 individuals of a white laboratory rat, which do not carry out the introduction of particles. However, the rest of the manipulations (shaving, landing in boxes, studying the parameters of urine for 288 hours) are carried out.

As a result, such an assessment did not reveal any deviations in the indicators in the animals of the experimental group, which indicated the absence of pathological processes associated with the state of carbohydrate metabolism, kidney and liver function, acid-base balance and urinary tract infection.

Based on these observations, it can be concluded that the proposed delivery system did not have a pronounced toxic effect on the animal organism.

Claims (2)

1. A method of treating superficial mycoses, which consists in the effect on the skin surface of a transdermal preparation containing an antifungal compound, characterized in that a transfollicular preparation is used as a transdermal preparation, which is an antifungal compound immobilized in vaterite particles, the effect of a transfollicular preparation is carried out by application of its alcohol suspension concentration of 100-150 mg / ml on the skin surface in the area of pathology until a dose of 4-5 mg of particles / cm ² containing 10-20 wt.% of an antifungal compound is reached, after which the application area is exposed to ultrasound with a frequency of 0.88-1 MHz with an intensity 0.4-0.5 W / cm ² for 2-3 minutes, while the treatment procedure is carried out no more than 1 time in 3 days. 2. The method according to claim 1, characterized in that naftifine hydrochloride or griseofulvin is used as the antifungal compound.

Images