EA040511B1 - COMPOSITIONS FOR TREATMENT AND PREVENTION OF DISEASES OF HOOVES AND CLAWS - Google Patents

Description

The present invention relates to a composition containing antigenic material of keratinophilic fungi and/or keratinophilic yeast, for use in a method for the treatment and/or prevention of diseases of the hooves and claws in animals, and to a new strain of Trichophyton verrucosum, which, for example, can be used in such a method of treatment and/or warnings.

Digital dermatitis (DD for short, also called Mortellaro's disease or Italian foot rot), which was first described in 1974 by Cheli and Mortellaro, is a major problem in dairy cows and beef cattle and is present on many dairy farms. DD is highly contagious and difficult to treat and prevent. It is also difficult to control its removal from the herd. This disease causes significant economic losses worldwide. Various types of bacteria are responsible for skin lesions in the interdigital region, which are characterized by red or strawberry-like, hairless and painful ulcers with epithelial hyperplasia and swelling in the affected areas. They are typically located on the back of the foot in the interdigital region. Inactive or treated lesions can be very difficult to distinguish from other warty digital lesions. DD must be distinguished from interdigital dermatitis (ID), which results in necrosis or necrobacillosis of the distal skin between the fingers. Studies have identified spirochete bacteria on DD tissue sections. In additional analysis, many types of bacteria were identified in DD lesions. In particular, Treponema species such as T. phagedenis, T. vincentii, and T. denticola can develop clinical signs of DD. A very complex complex of different etiological factors was discovered.

Interdigital dermatitis (ID) is commonly found in dairy cattle and is one of the main infectious causes of lameness. Exudative dermatitis, moist and foul-smelling erosions with scabs or crusts and solitary ulcers characterize the clinical manifestation of skin lesions in the heel and interdigital region. There may also be clinical symptoms on the dorsal surface of the fingers. The heels are painful. Then hyperplasia (corns, fibroma) is often observed with chronic irritation of the interdigital region, leading to lameness. ID causes a mixture of different bacteria. Dichelobacter nodosus plays an important role in the manifestation of clinical symptoms. This microorganism is anaerobic with strong photolytic properties. This infection is mainly caused by bacteria from infected cows, where D. nodosus is spread from infected to uninfected animals. Although these bacteria cannot survive long in the ground, they can survive in the claws. These bacteria destroy the epidermis, but do not penetrate into the dermal layers. Destruction of the border between the skin and the soft horny layer of the heel gives erosion and ulcers. These lesions cause a feeling of discomfort.

Interdigital phlegmon (IP for short, also called panaritium or foot rot) is a necrotic inflammation of the skin in the interdigital region. This disease usually manifests itself in an acute or subacute form. Lesions, erosions, and breaks in the skin between the fingers serve as a gateway for infection. In addition, bacterial microflora can enter the subcutaneous tissue and infect it. Infected animals become lame, have cellulitis and swelling. The clinical symptoms of IP are accompanied by decreased milk production, reduced appetite, and fever. Microorganisms Dichelobacter nodosus, Staphylococcus aureus, Escherichia coli, Arcanobacterium pyogenes and others are mainly involved in IP. The main bacterium involved in IP is Fusobacterium necrophorum, which is a Gram-negative, non-spore-forming, non-flagellated, non-motile, pleomorphic anaerobic bacterium. This bacterium produces lipopolysaccharide endotoxin, which has necrotizing activity and causes necrosis of the skin and subcutaneous tissue.

DD, ID and IP, which are the most common infectious diseases of the hoof and claw, spread sporadically around the world but may be endemic, particularly in intensive beef or dairy cattle complexes. The frequency, among other factors, depends on the weather, time of year, periods of grazing and management system. IP usually results in lameness and significant weight loss, loss of fertility, and decreased milk production. The frequency can be from 5 to 30%. At the first epidemic cases, approximately 30 to 80% of animals may show clinical signs of the disease. However, on average, IP accounts for only up to 15% of claw diseases.

It was surprising that many researchers suggest that the etiological factors of DD, ID and IP are the same microorganisms, such as Dichelobacter nodosus, Fusobacterium necroforun and Fusobacterium spp., which first destroy the epidermis and allow spirochetes from Treponema spp., such like T. phagedenis, T. vincentii, and T. denticola access deeper tissues to develop clinical signs of DD. Other bacterial species isolated from pathological material from tissues affected by DD, ID and IP are Campylobacter spp., Staphylococcus aureus, Escherichia coli, Arcanobacterium pyogenes and Prevotella spp. It was also suggested that the virus plays an important role in the pathogenesis of these diseases.

A typical treatment strategy for DD, ID, and IP is the use of antibiotics, antibacterials, and topical pads with antibiotics, antiseptics, and blood-stopping solutions.

Intramuscular applications of penicillin or oxytetracellin for 3 days show good results for the treatment of IP, but are not effective for the treatment of ID and DD.

For the treatment of IP, ID, and DD, topical application of soluble oxytetracycline, lincomycinspectinomycin, nitrofurazone, or sulfa preparations, as well as a mixture of sulfamethazine powder and anhydrous copper sulfate, may provide sufficient results. However, before this treatment, the lesion must be thoroughly cleaned and the necrotic tissue must be removed.

For preventive use, you can use a foot bath with 7-10% copper or zinc sulfate, or a 3-5% formaldehyde solution. You can also use foot baths with oxytetracycline or lincomycin-spectinomycin. However, these solutions may pollute the environment and are prohibited in some areas or countries. This method has been adopted to control IP and ID, but is less effective for the treatment of DD.

For the treatment of DD, ID and IP, a vaccine based on the bacterin Treponema spp. has been developed in the United States. It has been shown that immunization with said vaccine could reduce the clinical symptoms of DD in cattle. But in clinical trials, it has been found that herd-specific pathogens, including Treponema spp., cannot provide sufficient protection against DD. Cows given the bacteria during the dry period had no reduction in lesions compared to unvaccinated controls. Similar results were obtained in Germany. It was concluded that this treatment would probably not be an effective therapeutic, control or preventive strategy for DD in the future.

There is no corresponding commercially available vaccine on the market.

Many studies have been carried out to induce protection against F. necrophorum through the use of different types of antigenic components. Cattle injected with F. necrophorum culture supernatant containing leukotoxin have been reported to have a low incidence of F. necrophorum interdigital phlegmon. Supernatant stimulation of a high leukotoxin-producing F. necrophorum strain mixed with adjuvant resulted in a high titer of anti-leukotoxin antibody when injected into steers and conferred significant protection against experimentally induced liver abscesses. F. necrophorum bacteria has been used as an immunization agent for cattle and sheep against liver necrosis. In addition, cells from a culture of virulent F. necrophorum isolates were inactivated with 0.4% formalin. Mice immunized with these inactivated cells and live F. necrophorum had no detectable bacteria in the liver, lung or spleen for up to 28 days. It was concluded that immunization of formalin-killed F. necrophorum mice provided protection against infection. Injection of endotoxin with leukotoxic activity into immunized animals also prevents F. necrophorum infection.

In order to produce such a leukotoxoid-based vaccine, F. necrophorum bacteria were cultured in such a way as to enhance the production of leukotoxin in the supernatant. Bacterial growth and release of the leukotoxin was stopped, and the vaccine was obtained by inactivating at least the supernatant containing the leukotoxin. This was done by separating the supernatant containing the leukotoxin from the bacteria and then by inactivating with formalin, β-propiolactone, heat, radiation, or any other known method of inactivation. Alternatively, the entire culture could be inactivated to produce a vaccine.

Another formalin-inactivated leukocidin-exotoxin is also known, obtained as a result of the production of the Fusobacterium necrophorum 0-1 strain (Russian Research Institute of Experimental Veterinary Medicine), containing 1-10% of the inactivated bacterial mass of the specified strain, containing 7.0-7.7 billion cells per 1 cm, glycerin, a mixture of mineral oil Markol-52, an emulsifier, taken in a weight ratio of 1: (0.9-1.1), a suspension of live spores of anthrax vaccine strain 55 (Russian Research Institute of Veterinary Virology and Microbiology) in physiological solution and adjuvant. Thus, immunization of animals against F. necrophorum leukotoxin can prevent diseases associated with F. necrophorum infection, such as liver abscesses in cattle and sheep, and ID as well as IP in cattle.

The known vaccine for the prevention of necrobacteriosis in cattle contains endotoxin and exotoxin antigens derived from necrobacteriosis pathogens (F. necrophorum), a formalin inactivator, saline and an adjuvant based on mineral oil and lanolin. Known vaccine against necrobacillosis of farm animals contains a complex of soluble antigens from strains of F. necrophorum serotypes I and II, formalin, latex and adsorbent adjuvant. However, these known vaccines contain antigens of one type of pathogen and do not provide sufficient prophylactic activity against necrobacillosis - diseases involving anaerobic microorganisms and purulent wound microflora.

Also known are at least several inactivated commercially available vaccines for the prevention and treatment of ID and IP. For example, a polyvalent vaccine is known to prevent necrobacteriosis in cattle. Said vaccine contains formalin-inactivated antigens of Fusobacterium necrophorum, Staph. aureus, Corynebac. pyogenes, Clostridium perfringes type A toxoid, Clostridium perfringes type A, aluminum hydroxide adjuvant and immunopotentiating agent GMDP (N-acetylglucosaminyl-(1-4)-N-acetimuromil-Z-alanine-D-isoglutamine). Also known is the Nekovac vaccine, which contains inactivated cultures of Fusobacterium necrophorum VGNKI N Kp-1 DEP and/or Fusobacterium necrophorum VGNKI N Tula DEP, Actinomyces (Corybacterium) pyogenes VGNKI No. 4/2334 DEP, Staphylococcus aureus VGNKI No. 7315 DEP and Clostridium perfringens type A VGNKI No. 28 DEP. This vaccine also contains toxoid from Clostridium perfringens type A VGNKI No. 28 DEP and aluminum hydroxide adjuvant. In addition, this vaccine contains a saline solution such as saline.

All known vaccines often fail to induce a sufficient immune response and protect animals against interdigital dermatitis and interdigital phlegmon. There are no effective vaccines for digital dermatitis.

Approaches are known in the art regarding the use of inactivated dermatophytes as vaccines against ringworm. For example, active immunization against Trichophyton purpureum infection in rabbits using an inactivated suspension of Trichophyton rubrum hyphae has been described. Also in EP 393371 and WO 9307894 inactivated vaccines against ringworm containing dermatophytes of the genus Trichophyton and/or Microsporum are disclosed. In addition, WO 98/15284 describes vaccines against fungal infections that contain homogenized inactivated dermatophyte microconidia and inactivated homogenized yeast blastospores.

Dermatomycosis in animals are anthropozoonotic diseases of the skin and related tissue. Clinical symptoms are hair loss in the affected area, flushing, scaling, and asbestos-like scabs. Calves are more susceptible to ringworm infection than older animals. Ringworm is often also characterized by localized skin infection. In cattle, the lesions are most commonly found on the neck, head, and consist of gray-white crusts that rise on the skin. In addition, ringworm can lead to alopecia. The transmission of ringworm-causing organisms from infected animals to humans has been described. Dermatomycosis in animals has a significant socioeconomic impact. Sick animals require long-term treatment and can spread the infection to both animals and humans. So far, ringworm has been treated with different types of drugs applied topically to the affected areas of the skin. They include a range of ointments, rubbings, solutions and other substances containing fungicides and fungistatics. The disadvantages of such treatments are that they are not very effective, they require quarantine measures and disinfection of areas where animals were kept (shedding boxes, vivariums, farms, zoos, circuses, etc.). In addition, they require significant expenditures on drugs and veterinary treatment, and they create difficulties in immobilizing animals (for captive wild animals). Inactivated vaccines have been developed for the treatment of trichophytosis in cattle, dermatophytosis in horses, cats and dogs, as, for example, described in WO 93/07894, and live vaccines for the treatment of fur animals and rabbits (see USSR patent No. 835446), camels (see USSR patent No. 1190574) and other animals. A vaccine has also been developed for the prevention and treatment of mycosis in mammals (see WO 98/15284).

Thus, it is an object of the present invention to provide novel compositions for use in a method for the treatment and/or prevention of diseases of the hooves and claws in animals. Another object of the present invention is to provide compositions for use in a method for the treatment and/or prevention of digital and/or interdigital dermatitis and/or interdigital phlegmon in animals, in particular bovids and pigs. Another object of the present invention is to propose a new strain of Trichophyton verrucosum. Another object of the present invention is to provide compositions for use in a method for the treatment and/or prevention of dermatophytosis.

These tasks are solved by means of the subject matter of the invention defined in the claims.

The use of the word in the singular when used in conjunction with the term containing in the claims and/or description may mean one, but it is also consistent with the meaning of one or more than one, at least one and one or more than one.

The term roughly means that the stated value plus or minus 5% of the declared value is considered, or the standard error for measurements of that value.

The term containing, as used here, should not be construed as being limited to the meaning of consisting of (i.e., excluding the presence of additional other items). Rather, containing implies that additional items may possibly be present. The term containing encompasses, as specifically contemplated, embodiments falling within its scope into consisting of (i.e., excluding the presence of an additional other item) and containing but not consisting of (i.e., requiring the presence of an additional other item), the former being more preferred.

The term hoof and claw disease, as used herein, refers in particular to infectious diseases of the hoof and claw in bovids and/or pigs. These diseases are in particular caused by bacteria, fungi and/or viruses. In particular, the term hoof and claw diseases refers to digital dermatitis, interdigital dermatitis, and interdigital cellulitis.

The term bovid as used here refers specifically to artiodactyl ruminant mammals, including bison, African buffalo, Asiatic buffalo, antelope, gazelles, sheep, goats, muskox, and cattle.

The term lameness as used here refers specifically to lameness resulting from infection and tissue damage. In particular, the term lameness refers to lameness due to diseases of the hooves and claws, more specifically digital dermatitis, interdigital dermatitis and interdigital phlegmon.

The term chitosan as used herein refers to a copolymer of 2-amino-2-deoxy-D-glucopyranose and 2-acetamido-2-deoxy-10-glucopyranose wherein the degree of deacetylation is greater than 50%, preferably greater than 60 , 70, 80 or 90%. Chitosan can be chemically obtained from chitin, which is poly-1,4-s-N-acetyl-O-glucosamine, more specifically N-acetyl-1,4-s-O-glucopyranosamine, by deacetylation. Typical chitosan preparations have varying molecular weights, depending on the method of manufacture.

It has now surprisingly been found that vaccines containing keratinophilic or yeast antigenic material give good resistance, preventive and curative effects against digital dermatitis, interdigital dermatitis and interdigital phlegmon in animals. In particular, it has been unexpectedly found that vaccines containing homogenized inactivated dermatophyte microconidia and/or inactivated homogenized yeast blastospores give good resistance, preventive and curative effects against digital dermatitis, interdigital dermatitis and interdigital phlegmon in animals.

The present invention relates to a composition containing antigenic material of keratinophilic fungi or keratinophilic yeast, for use in a method for the treatment and/or prevention of diseases of the hooves and claws in animals. Preferably, these animals are mammals, more preferably bovids and/or pigs, most preferably cattle.

The keratinophilic or yeast antigenic material may be derived from any parts of the keratinophilic fungi or yeast containing antigens, such as mycelium, arthrospores, dermatophyte microconidia, yeast blastospores, or others. These antigens are preferably polysaccharides and/or glycopeptides. Preferably, the keratinophilic or yeast antigenic material is selected from the group consisting of homogenized inactivated dermatophyte microconidia, homogenized inactivated yeast blastospores, yeast blastospore antigenic material, and dermatophyte microconidia antigenic material. Thus, the present invention more specifically relates to a composition comprising homogenized inactivated dermatophyte microconidia and/or homogenized inactivated yeast blastospores and/or yeast blastospore antigenic material and/or dermatophyte microconidia.

In particular, diseases of the hooves and claws lead to lameness. More specifically, hoof and claw diseases are digital dermatitis and/or interdigital dermatitis and/or interdigital cellulitis. Hoof and claw diseases and digital dermatitis, interdigital dermatitis, interdigital phlegmon, respectively, can be caused by Dichelobacter nodosus, Fusobacterium necroforun, Fusobacterium spp, Treponema spp, such as T. phagedenis, T. vincentii and T. denticola, Campylobacter spp, Staphylococcus aureus , Escherichia coli, Arcanobacterium pyogenes and Prevotella spp., and/or a virus.

The antigenic material of keratinophilic yeasts, in particular yeast blastospores, of the composition for use in the present invention preferably belongs to the genus Candida and more preferably to the species Candida albicans. The antigenic material of keratinophilic dermatophytes, in particular dermatophyte microconidia, preferably belongs to the genera Trichophyton and/or Microsporum. More preferably, the dermatophyte microconidia belong to the species Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton equinum, Trichophyton sarkisovii, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and/or Microsporum gypseum. In particular, the species of Microsporum canis may be Microsporum canis var. obesum and/or Microsporum canis var. distortum.

In a preferred embodiment of the present invention, yeast blastospores and dermatophyte microconidia are obtained from strains of the aforementioned species that have been obtained by directional selection based on spore production and/or attenuation. It is highly preferred to use a strain that grows faster in a nutrient medium, produces more microconidia and blastospores, respectively, has less virulence and/or no adverse reactions after its intramuscular application, compared to any epizootic strain from which it is derived. Examples of such strains are Trichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton ru

- 4 040511 brum DSM-9471, Trichophyton rubrum DSM-9472, Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458 and Candida albicans DSM-9459. Thus, in particularly preferred embodiments of the present invention, yeast blastospores and dermatophyte microconidia are obtained from the strains Trichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM-9471, Trichophyton rubrum DSM -9472, Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458 and Candida albicans DSM-9459.

The strains of Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM-9471, Trichophyton rubrum DSM-9472, Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458 and Candida albicans DSM-9459 were deposited according to the Budapest Agreement in Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) (DSM), Mascheroder Weg 1B, W-38124 Braunschweig, Germany (modern name and address of which is Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DMSZ), Inhoffenstraβe 7B, 38124 Braunschweig, Germany) October 05, 1994 Basotherm GmbH, Eichendorfweg 5, 88396 Biberach an der Riss. The current depositors of these strains are applicants, namely Dr. Igor Polyakov and PhD Ludmila Ivanova, Eberhardtstr. 40, 89073 Ulm. TRICHOPHYTON RUBRUM, No. DSM-9469

This strain was deposited with DSM on 10/05/1994 under serial no. DSM-9469. This strain was obtained by directed selection based on spore production and attenuation of epizootic strain No. 533, which was identified on human skin in 1985. This strain was identified using the Rebell-Taplin identifier (Rebell G., Taplin D.: Dermatophytes, their recognition and identification, 3rd Print, University of Miami Press, Coral Gables, Florida, USA, 1978). The biological properties of this strain are described in table. A. Strain No. DSM-9469 differs from the epidemic strain in its faster growth in the nutrient medium, huge production of microconidia and less virulence.

Table A

properties and characteristics strains Strain No. DSM-9469 Epidemic strain No. 533 Description culture Mature 15-day colony on Sabouraud agar: white, velvety, flat, edge of the colony 20-day colony on Sabouraud agar: white, fluffy, raised, edge of the colony

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fringed, yellow under the surface, dark purple in the center, colony diameter 6063 mm. smooth, purple under the surface, colony diameter - 3035 mm. Morphologists cal characteristics Mature 15-day culture with divided septa branched hyphae 1-3 µm wide, numerous obovate oval microconidia 2-3 x 3-5 microns in size, macroconidia are long club-shaped pencil-shaped with 4-5 transverse walls, having a size of 4-6 x 15-40 microns. 20 day culture with partitioned branched hyphae 1-3 µm wide, microconidia from club-shaped to round in small open clusters and along hyphae 2-3 x 3-6 µm, macroconidia sparse, long and pencil-shaped with 3-5 transverse walls, having a size of 4-7 x 15-50 microns. Pathogenic characteristics The strain is weakly virulent. After 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² scales are formed on the injured skin of guinea pigs. Spontaneous recovery after 18-20 days. The strain is virulent. 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² thin necrotic scabs are formed on the wounded skin of guinea pigs. Spontaneous recovery after 25-30 days. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes

immunity. immunity.

TRICHOPHYTON RUBRUM, No. DSM-9470

This strain was deposited with DSM on 05.10.1994 under serial no. DSM-9470. This strain was obtained by directed selection based on spore production and attenuation of epizootic strain No. 535, which was identified on human skin in 1990. This strain was identified using the Rebell-Taplin identifier (Rebell G., Taplin D.: Dermatophytes, their recognition and identification, 3rd Print, University of Miami Press, Coral Gables, Florida, USA, 1978). The biological properties of this strain are described in table. B. Strain No. DSM-9470 differs from the epidemic strain in its faster growth in the nutrient medium, huge production of microconidia and less virulence.

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Table B

properties and characteristics strains Strain No. DSM-9470 Epidemic strain No. 535 Description culture A mature 15-day-old colony on Sabouraud agar: white, velvety fluffy in the center, folded, the edge of the colony is even, colorless or pink under the surface, the diameter of the colony is 25-30 mm. A 20-day colony on Sabouraud agar: white, fluffy, the edge of the colony is even, under the surface - yellow, diameter - 20 mm. Morphologists cal characteristics Mature 15-day culture with divided septa branched hyphae 1-3 µm wide, round-oval purulent microconidia 2-3 x 3-7 µm in size. 20 day culture with partitioned hyphae 1-3 µm wide, microconidia club-shaped to round in small open clusters and along hyphae 2-3 x 3-6 µm, no macroconidia. Pathogenic characteristics The strain is weakly virulent. 9-10 days after applying a dose of 500- The strain is virulent. 9-10 days after applying a dose of 500-600 thousand cells

600 thousand cells of fungal material per cm ² necrotic scabs are formed on the injured skin of guinea pigs. Spontaneous recovery after 22-25 days. fungal material per cm ² thin necrotic scabs form on the wounded skin of guinea pigs. Spontaneous recovery after 25-30 days. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity. Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity.

TRICHOPHYTON RUBRUM, No. DSM-9471

This strain was deposited with DSM on 10/05/1994 under serial no. DSM-9471. This strain was obtained by directed selection based on spore production and attenuation of epizootic strain No. 620, which was identified on the human nail in 1989. This strain was identified using the Rebell-Taplin identifier (Rebell G., Taplin D.: Dermatophytes, their recognition and identification, 3rd Print, University of Miami Press, Coral Gables, Florida, USA, 1978). The biological properties of the strain are described in table. B. Strain No. DSM-9471 differs from the epidemic strain in its faster growth in the nutrient medium, huge production of microconidia and less virulence.

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Table B

properties and characteristics strains Strain No. DSM-9471 Epidemic strain No. 620 Description culture Mature 15-day colony on Sabouraud agar: white, velvety, 20 day colony on agar Saburo: white, fluffy, raised, edge of the colony smooth, yellow under the surface, dark purple in the center, colony diameter 32-35 mm. raised, edge of the colony smooth, purple under the surface, colony diameter - 2025 mm. Morphologists cal characteristics Mature 15-day culture with divided septa branched hyphae 1-3 µm wide, round-oval purulent microconidia 2-3 x 3-7 µm in size. 20 day culture with partitioned branched hyphae 1-3 µm wide, microconidia club-shaped to round in small open clusters and along the hyphae 2-3 x 3-6 µm in size; macroconidia are rare, long and pencil-shaped with 3-5 transverse walls, having a size of 4-7 x 15-50 microns. Pathogenic characteristics The strain is weakly virulent. After 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² scales are formed on the injured skin of guinea pigs. Spontaneous recovery after 18-20 days. The strain is virulent. 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² thin necrotic scabs are formed on the wounded skin of guinea pigs. Spontaneous recovery after 25-30 days. Reciprocal reaction Result of intramuscular injection of inactivated particulate antigens from cultures: no observed changes in the clinical condition of the animals. The result of intramuscular injection of inactivated corpuscular antigens from cultures: inflammation at the injection point, edema. Immunogenic answer Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity. immunity.

TRICHOPHYTON RUBRUM, No. DSM-9472

This strain was deposited with DSM on 05.10.1994 under serial no. DSM-9472. This strain was obtained by directed selection based on spore production and attenuation of epizootic strain No. 754, which was identified on the human nail in 1990. This strain was identified using the Rebell-Taplin identifier (Rebell G., Taplin D.: Dermatophytes, their recognition and identification, 3rd Print, University of Miami Press, Coral Gables, Florida, USA, 1978). The biological properties of the strain are described in table. D. Strain No. DSM-9472 differs from the epidemic strain in its faster growth in the nutrient medium, huge production of microconidia and less virulence.

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Table D

properties and characteristics strains Strain No. DSM-9472 Epidemic strain No. 754 Description culture A mature 15-day-old colony on Sabouraud agar: white, velvety, folded in the center, the edge of the colony is even, yellow under the surface, purple in the center, the diameter of the colony is 35-40 mm. 20-day colony on Sabouraud's agar: white-pink, fluffy, the edge of the colony is even, purple under the surface, colony diameter - 20-25 mm. Morphologists cal characteristics Mature 15-day culture with divided septa branched hyphae 1-3 µm wide, round-oval purulent microconidia 2-3 x 3-7 µm in size. 20 day culture with partitioned branched hyphae 1-3 µm wide, microconidia club-shaped to round in small open clusters and along hyphae 2-3 x 3-6 µm, macroconidia sparse, long and pencil-shaped with 3-5 transverse walls, having a size of 4-7 x 15-50 microns. Pathogenic The strain is The strain is virulent. characteristics weakly virulent. After 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² scales are formed on the injured skin of guinea pigs. Spontaneous recovery after 18-20 days. 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² thin necrotic scabs are formed on the wounded skin of guinea pigs. Spontaneous recovery after 25-30 days. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity. Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity.

CANDIDA ALBICANS, No. DSM-9456

This strain was deposited with DSM on 10/05/1994 under serial no. DSM-9456. This strain was obtained by directional selection based on the stabilization of cultural and morphological characteristics and the weakening of the epidemic strain No. 008-L, which was identified in humans in 1990. This strain was identified using the Lodder identifier (Lodder, J: The yeast: A Taxonomic Study. North-Holland Publ. Co., Amsterdam-London (1970) The biological properties of this strain are described in Table D. Strain No. DSM-9456 differs from the epidemic strain in its faster growth in nutrient medium, stable biological properties, huge biomass production and less virulence.

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Table D

properties and characteristics strains Strain No. DSM-9456 Epidemic strain No. 008-L Description 10-day one-spore colony 10-day one-spore colony culture on Sabouraud agar: creamy, smooth and pasty, shiny, raised, edge of the colony even, colony diameter - 20-30 mm. on Sabouraud agar: creamy, soft and smooth with fluffy branches at the edges, the diameter of the colony is 10-15 mm. Morphologists cal characteristics 10 day culture with spherical oval blastospores 3.5-6 x 610 µm in size, chlamydospores 12-15 µm wide, pseudohyphae 58 µm wide, hyphae 1.5-3 µm wide. 10-day one-spore culture on Sabouraud agar with spherical oval buds blastospores 3-5 x 5-8 µm in size, chlamydospores 10-15 µm in diameter, pseudohyphae 5-8 µm wide, hyphae 1.5-3 µm wide. Pathogenic characteristics The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells in white mice, a granuloma is formed in the abdominal organs of 50% of the animals. No lethal effect was observed. The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells to white mice, a granuloma is formed in the abdominal organs of 80-100% of the animals. The lethal effect was observed in 50-70%. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity. Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity.

CANDIDA ALBICANS, No. DSM-9457

This strain was deposited with DSM on 05.10.1994 under serial no. DSM-9457.

This strain was obtained by directional selection based on the stabilization of cultural and morphological characteristics and the weakening of epidemic strain No. 012, which was identified in humans in 1992. This strain was identified using the Lodder identifier (Lodder, J: The yeast: A Taxonomic Study. North-Holland Publ Co., Amsterdam - London (1970) The biological properties of this strain are described in Table E. Strain No. DSM-9457 differs from the epidemic strain in its faster growth in nutrient media, stable biological properties, huge biomass production and less virulence.

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Table E

properties and characteristics strains Strain No. DSM-9457 Epidemic Strain No. 012 Description culture A 10-day one-spore colony on Sabouraud agar: creamy, rough, raised, the edge of the colony is lobed, the diameter of the colony is 20-23 mm. A 10-day one-spore colony on Sabouraud agar: creamy, rough, raised, the edge of the colony is fringed and lobed, the diameter of the colony is 15-20 mm. Morphologists cal characteristics A 10-day single-spore culture with spherical oval blastospores 3.5-5 x 510 µm in size, chlamydospores 12-15 µm wide, pseudohyphae 47 µm wide, hyphae 2-3 µm wide. 10-day one-spore culture on Sabouraud agar with spherical oval buds blastospores with a size of 3-5 x 5-8 microns, chlamydospores with a diameter of ΙΟΙ 5 microns, pseudohyphae 5-8 microns wide, hyphae 1.5-3 microns wide. Pathogenic characteristics The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells in white mice, a granuloma develops in the abdominal organs in 30% of the animals. No lethal effect was observed. The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells in white mice, a granuloma is formed in the abdominal organs of 50% of the animals. A lethal effect of no more than 50% was observed. Reciprocal The result of intramuscular The result of intramuscular reaction injections of inactivated particulate antigens from cultures: no observed changes in the clinical condition of the animals. injections of inactivated particulate antigens from cultures: no observed changes in the clinical condition of the animals. Immunogenic answer Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity. Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity.

CANDIDA ALBICANS, No. DSM-9458

This strain was deposited with DSM on 10/05/1994 under serial no. DSM-9458. This strain was obtained by directional selection based on the stabilization of cultural and morphological characteristics and the weakening of the epidemic strain No. 047, which was identified in humans in 1989. This strain was identified using the Lodder determinant (Lodder J.: The yeast: A Taxonomic Study. North- Holland Publ. Co., Amsterdam - London (1970) The biological properties of this strain are described in Table G. Strain No. DSM-9458 differs from the epidemic strain in its faster growth in nutrient medium, stable biological properties, huge biomass production and less virulence.

- 11 040511

Table G

properties and characteristics strains Strain No. DSM-9458 Epidemic strain No. 047 Description culture 10-day-old single-spore colony on Sabouraud agar: creamy, smooth and pasty, shiny, raised, edge of the colony even, colony diameter - 16-18 mm. 10-day-old single-spore colony on Sabouraud agar: creamy, soft and smooth with fluffy branches at the edges, the diameter of the colony is 10-15 mm. Morphologists cal characteristics 10 day culture with spherical oval blastospores measuring 3.6-6 x 6- 10-day one-spore culture on Sabouraud agar with spherical oval buds 11 microns, chlamydospores 12-15 microns wide, pseudohyphae 48 microns wide, hyphae 1.5-3 microns wide. blastospores with a size of 3-5 x 5-8 microns, chlamydospores with a diameter of ΙΟΙ 5 microns, pseudohyphae 5-8 microns wide, hyphae 1.5-3 microns wide. Pathogenic characteristics The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells to white mice, a granuloma is formed in the abdominal organs of 5 ΟΙ 00% of the animals. A lethal effect was observed in 50%. The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells to white mice, a granuloma is formed in the abdominal organs of 80-100% of the animals. The lethal effect was observed in 70-100%. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity. Results of immunization of a group of white mice with inactivated antigen from cultures (repeated at least 10 times): establishes immunity.

CANDIDA ALBICANS, No. DSM-9459

This strain was deposited with DSM on 10/05/1994 under serial no. DSM-9459. This strain was obtained by directional selection based on the stabilization of cultural and morphological characteristics and the weakening of the epidemic strain No. 158, which was identified in humans in 1990. This strain was identified using the Lodder identifier (Lodder, J: The yeast: A Taxonomic Study. North- Holland Publ. Co., Amsterdam - London (1970) The biological properties of this strain are described in Table 3. Strain No. DSM-9459 differs from the epidemic strain in its faster growth in nutrient medium, stable biological properties, huge biomass production and less virulence.

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Table H

properties and characteristics strains Strain No. DSM-9459 Epidemic strain No. 158 Description of culture Morphological characteristics Pathogenic characteristics Responsiveness Immunogenic response 10-day one-spore colony on Sabouraud agar: creamy, smooth, pasty, shiny, raised, edge of the colony even, colony diameter - 16-18 mm. 10 day culture with spherical oval blastospores 3.6-6 x 611 µm in size, chlamydospores 12-15 µm wide, pseudohyphae 48 µm wide, hyphae 1.5-3 µm wide. The strain is weakly virulent. 30 days after intraperitoneal injection of a dose of 10-100 million fungal cells to white mice, a granuloma is formed in the abdominal organs of 40% of the animals. No lethal effect was observed. The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. Results of immunization of a group of white mice with inactivated antigen from cultures (repeated no less than 10 times): establishes immunity. A 10-day one-spore colony on Sabouraud agar: creamy, smooth, pasty, the edge of the colony is lobed and with fluffy branches on the edges, the diameter of the colony is 10-15 mm. 10-day one-spore culture on Sabouraud agar with spherical oval buds blastospores with a size of 3-5 x 5-8 microns, chlamydospores with a diameter of ΙΟΙ 5 microns, pseudohyphae 5-8 microns wide, hyphae 1.5-3 microns wide. The strain is weakly virulent. 30 days after an intraperitoneal injection of a dose of 10-100 million fungal cells in white mice, a granuloma is formed in the abdominal organs of 50% of the animals. A lethal effect was observed in 20-50%. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Results of immunization of a group of white mice with inactivated antigen from cultures (repeated no less than 10 times): establishes immunity.

The Trichophyton mentagrophytes strain DSM-7279 was deposited under the Budapest Agreement with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM), Mascheroder Weg 1B, W38124 Braunschweig, Germany (modern name and address of which is Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( DMSZ), Inhoffenstraβe 7B, 38124 Braunschweig, GERMANY) October 1, 1992 Boehringer Ingelheim Vetmedica GmbH, 6507 Ingelheim am Rhein (whose current address is Boehringer Ingelheim Vetmedica GmbH, 55216 Ingelheim am Rhein). The current depositors of said strain are the applicants, namely Dr. Igor Polyakov and PhD Ludmila Ivanova, Eberhardtstr. 40, 89073 Ulm.

Trichophyton mentagrophytes No. VKPGF-930/1032, No. DSM-7279

This strain was deposited with the DSM on October 1, 1992 under serial no. DSM-7279. The strain was obtained by directional selection based on spore production and attenuation of epizootic strain No. 1032, which was found in the horse in 1985. This strain was identified as described above by Rebel, Taplin, loc. cit., and Kashkin, loc. cit.). Biological properties are described in table. I. Strain No. VKPGF-930/1032, DSM-7279, differs from the epizootic strain in its faster growth in a nutrient medium, its huge production of microconidia, its lower virulence, and the absence of any reaction with its antigens.

Trichophyton verrucosum No. DSM-28406

The Trichophyton verrucosum BINO 348 strain was deposited by Binomed GmbH (Einsteinstraβe 59, 89077 Ulm) under the Budapest Agreement with the Leibniz-Institut DSMZ - Deutsche Sammlung von Mi

- 13 040511 croorganism en und Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Braunschweig, Germany under serial no. DSM-28406 February 12, 2014 that the deposited material be made available to the public in accordance with Rule 31 of the EPC (European Patent Agreement). This strain was obtained by directional selection based on spore production and attenuation of epizootic strain No. 348, which was isolated from cattle in 1997. This strain was identified using the Rebell-Taplin identifier (Rebell G., Taplin D.: Dermatophytes, their recognition and identification, 1978) and according to Kashkin P.N. et al. (Determinant of pathogenic, toxic, harmful fungi for humans, 1979). The biological properties of this strain are described in table. I. The BINO 348-DSM 28406 strain differs from the epizootic strain in its faster growth in nutrient medium, huge production of microconidia, less virulence, and the absence of any deleterious reactions after intramuscular application of antigens.

Table I

Properties and characteristics of the strain Strain No. VKPGF-930/1032 Epizootic strain No. 1032

Culture description Mature 10-15 day old colony in agar/wort: Mature 25-30 day old colony in agar/wort:

creamy, velvety/powdery, white, flat, narrow, growing edge, sub-flat with slight flat elevation in surface reddish brown, center, narrow growing edge, fringed, colony diameter 15-20 mm. light brown under the surface, colony diameter 25-30 mm.

Morphological characteristics Branched septate hyphae Branched septate 1-3 µm wide, numerous straight and spiral hyphae 1-3 µm wide pus-like, oval microconidia, round, flattened pus-like 1 to 3 x 2 to 6 µm in size, no microconidia ranging in size from 1 to 3 x 2 to 6

macroconidia. µm, slightly elongated oval macroconidium with 2-5 septa, 2 to 6 x 15 to 25 µm in size. Pathogenic characteristics Necrotic scabs. 9 to 10 days after applying a dose of 500- Thick, asbestos-like scabs.

600 thousand cells of fungal material per cm ² on the wounded skin of a rabbit.

Spontaneous recovery after 22-25 days Response No observed changes 30-35 days in clinical Inflammation at the injection point, edema

able

Results of subcutaneous and intramuscular injection of inactivated particulate antigens from cultures Antigenic response

20 to 25 days after injection of particulate antigens into rabbits, antibody titers observed in serum Determined PHRs 1:320 to 1:640

Defined ELISA (enzyme-linked immunosorbent assay) from 1:400 to 1:1600

Immunogenic response Establishes immunity

Immunization of a group of rabbits with inactivated antigens from cultures (repeated at least 5 times) 1:320 to 1:640 1:400 to 1:1600

Sets immunity

- 14 040511

Table Y

properties and characteristics strains Strain No. DSM-28406 Epidemic Strain No. 348 Description culture 20-day colony on malt extract agar: white or light yellow, velvety, furrowed, colony diameter - 1520 mm. 25-30-day colony on malt extract agar: light yellow, creamy, velvety, folded, colorless under the surface, colony diameter - 10-12 mm. Morphologists cal characteristics Mature 20-day culture with numerous oval, purulent microconidia size 1.5-3 x 3-5 microns. Mature 25-30 day culture with partitioned branched mycelium, few oval, purulent microconidia ranging in size from 1 to 3 microns x from 3 to 6 microns, macroconidia with 2-6 septa, few arthrospores and chlamydospores 9-11 microns. Pathogenic characteristics The strain is weakly virulent. After 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² scales are formed on the injured skin of guinea pigs. Spontaneous recovery after 15-20 days. The strain is virulent. 9-10 days after applying a dose of 500-600 thousand cells of fungal material per cm ² thin necrotic scabs are formed on the wounded skin of guinea pigs. Spontaneous recovery after 25-30 days. Reciprocal reaction The result of intramuscular injections of inactivated particulate antigens from cultures: no observed changes in clinical the condition of the animals. The result of intramuscular injections of inactivated corpuscular antigens from cultures: inflammation at the point of injection, edema. Immunogenic answer Results of immunization of a group of guinea pigs with inactivated Results of immunization of a group of guinea pigs with inactivated antigen from cultures (repeated at least 5 times): establishes immunity against dermatophytosis caused by T. verrucosum. antigen from cultures (repeated at least 5 times): establishes immunity against dermatophytosis caused by T. verrucosum.

In a preferred embodiment of the composition for use according to the present invention, the composition comprises homogenized inactivated dermatophyte microconidia of one type of microconidia or a mixture of microconidia from two, three, four, five, six, seven, eight, nine or ten of the dermatophyte strains listed above. In another preferred embodiment, the composition comprises a mixture of homogenized inactivated dermatophyte microconidia from one, two, three, four, five, six, seven, eight, nine or ten of the above dermatophytes and homogenized, inactivated yeast blastospores from one, two, three or four of yeast listed above. In another preferred embodiment, the composition comprises homogenized inactivated dermatophyte microconidia from one or a mixture of two, three or four of the yeasts listed above. These compositions may further comprise dermatophyte microconidial antigenic material and/or yeast blastospore antigenic material, as described herein. Alternatively or additionally, the compositions may further comprise chitosan modified with an organic carboxylic acid or a salt thereof.

In another preferred embodiment, the composition comprises a single dermatophyte microconidial antigenic material or a mixture of dermatophyte microconidial antigenic material from two, three, four, five, six, seven, eight, nine or ten of the above dermatophyte strains. In another preferred embodiment, the composition comprises a mixture of dermatophyte microconidia antigenic material from one, two, three, four, five, six, seven, eight, nine or ten of the above dermatophytes and yeast blastospore antigenic material from one, two, three

- 15 040511 or four of the yeasts listed above. In another preferred embodiment, the composition comprises yeast blastospore antigenic material of one or a mixture of two, three or four of the yeasts listed above. These compositions may further comprise homogenized inactivated dermatophyte microconidia from one, two, three, four, five, six, seven, eight, nine or ten of the dermatophytes listed above and/or homogenized inactivated yeast blastospores from one, two, three or four of the listed above yeast. Alternatively or additionally, the compositions may further comprise chitosan modified with an organic carboxylic acid or a salt thereof.

In a preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated dermatophyte microconidia Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Trichophyton sarkisovii, Microsporum canis, Microsporum canis var. obesum, Microsporum canis var. distortion and Microsporum gypseum. For example, the vaccine Polivak-TM (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) corresponds to this embodiment and can be used as a composition for use according to the present invention. Polivak-TM is a vaccine developed for animals such as cats, dogs, horses and others. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophytes Trichophyton mentagrophytes, Trichophyton verrucosum and Trichophyton sarkisovii. For example, the Polivak-T vaccine (manufacturer: Vetbiokhim LLC, Moscow; distributor: Prostor LLC, Moscow) corresponds to this embodiment and can be used as a composition for use according to the present invention. Polivak-T is a vaccine specially developed for cattle. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated dermatophyte microconidia Trichophyton mentagrophytes, Trichophyton verrucosum and Trichophyton sarkisovii, and homogenized inactivated yeast blastospores Candida albicans. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains homogenized inactivated microconidia of the dermatophyte Trichophyton mentagrophytes, in particular Trichophyton mentagrophytes DSM-7279. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains homogenized inactivated microconidia of the dermatophyte Trichophyton verrucosum, in particular Trichophyton verrucosum DSM - 28406. This composition may additionally contain an antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophyte Trichophyton verrucosum, in particular Trichophyton verrucosum DSM-28406, and homogenized inactivated Candida albicans yeast blastospores, in particular Candida albicans DSM-9456. This composition may additionally contain antigenic material, as described here, chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophytes Trichophyton mentagrophytes, Trichophyton verrucosum and Trichophyton sarkisovii. For example, a variant of the Polivak-TM vaccine, which does not contain, compared to the classic Polivak-TM, Trichophyton equinum and Microsporum strains (manufacturer: Vetbiokhim LLC, Moscow; distributor: Prostor LLC, Moscow), corresponds to this embodiment and can be used as a composition for use in this invention. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophytes Trichophyton mentagrophytes, Microsporum canis, Microsporum canis var. obesum, Microsporum canis var. distortum and Microsporum gypseum. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated dermatophyte microconidia Trichophyton men- 16 040511 tagrophytes, Trichophyton verrucosum, Trichophyton equimim, Trichophyton sarkisovii, Microsporum canis,

Microsporum canis var. obesum, Microsporum canis var. distortion and Microsporum gypseum. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophytes Trichophyton mentagrophytes, Trichophyton verrucosum and Trichophyton sarkisovii. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains homogenized inactivated Candida albicans yeast blastospores, in particular Candida albicans DSM-9456. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated microconidia of the dermatophytes Trichophyton mentagrophytes, Trichophyton verrucosum and Trichophyton sarkisovii. This composition may additionally contain antigenic material as described here, and/or chitosan modified with an organic carboxylic acid or its salt.

In another preferred embodiment, the composition for use according to the present invention comprises yeast blastospore and/or dermatophyte microconidia antigenic material. Particularly preferred is a composition containing the antigenic material of the microconidia of the dermatophyte Trichophyton verrucosum, in particular the strain Trichophyton verrucosum DSM-28406.

In another preferred embodiment, the composition for use according to the present invention contains a mixture of homogenized inactivated dermatophyte microconidia from one, two, three, four, five, six, seven, eight, nine or ten of the above dermatophytes and antigenic material from one, two, three, four, five, six, seven, eight, nine or ten of the dermatophytes listed above and/or one, two, three or four of the yeasts listed above.

In another preferred embodiment, the composition for use of the present invention comprises a mixture of antigenic material of dermatophyte microconidia from one, two, three, four, five, six, seven, eight, nine or ten of the above dermatophytes with homogenized inactivated yeast blastospores of one, two, three or four strains of Candida, in particular Candida albicans, more specifically Candida albicans DSM-9456, Candida albicans DSM9457, Candida albicans DSM-9458 or Candida albicans DSM-9459.

In another preferred embodiment, the composition for use according to the present invention comprises a mixture of blastospore antigenic material from one, two, three or four strains of Candida, in particular Candida albicans, more specifically Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458 or Candida albicans DSM-9459, with homogenized inactivated dermatophyte microconidia from one, two, three, four, five, six, seven, eight, nine, or ten of the dermatophytes listed above.

The antigenic material of yeast blastospores and/or dermatophyte microconidia preferably contains polysaccharides and/or glycopeptides isolated from keratinophilic fungi or yeasts. The antigenic material containing such polysaccharides and/or glycopeptides may be insoluble antigenic material (ANMP), soluble antigenic material (ASMP), or exogenous antigenic material (AEMP). The keratinophilic fungi are preferably Trichophyton or Microsporum species, more preferably Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton equinum, Trichophyton sarkisovii, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis, and the keratinophilic yeast is preferably Candida species, more preferably Candida albicans. Especially preferred is antigenic material derived from Trichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM9471, Trichophyton rubrum DSM-9472, Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458 and Candida albicans DSM-9459. This antigenic material can be obtained, for example, by the method disclosed in WO 97/07232.

In general, to obtain ANMP, fungal cells belonging to the group of keratinophilic fungi or yeasts are treated under aqueous alkaline conditions, the solid and liquid phases of the preparation are separated, and after separation, the solid phase is treated with a mineral or organic acid. Treatment under aqueous alkaline conditions is preferably carried out with about 0.1 to 5% (w/v) KOH or NaOH at about 20 to 150° C. for up to 30 hours. The solid phase is preferably treated with 0.2- 1.5 M organic acid or 0.05-1 M mineral acid and washed with an aqueous solution. More specifically, keratinophilic fungi or yeasts are preferably cultured on agar plates. One preferred medium is, for example, Malt Extract Agar from Oxoid. You can also use other media that will allow the growth of keratinophilic fungi or yeast. The resulting fungal biomass was torn off and treated with an aqueous solution of alkali. Then the solid and liquid phases of the drug are separated, for example, by centrifugation, filtration or precipitation. Preferably, the separation is carried out by centrifugation, for example at 3500 g, which provides a good separation of fragments of fungal cells. Both the treatment under aqueous alkaline conditions and the separation step can be repeated several times. After alkaline treatment, the resulting supernatant is treated under aqueous acidic conditions as described above. For example, HCl or acetic acid can be used. The acid treatment is preferably carried out for from about 0.5 to about 3 hours. The temperature is preferably in the range from about 70 to about 100°C. The aqueous washing solution is preferably distilled water. Advantageously, the washing is repeated about five times. Finally, the solid phase is separated and homogenized in water for injection or in an aqueous solution of 0.1-0.9% chitosan modified with an organic carboxylic acid or a salt thereof. Homogenization is preferably carried out in a volume of from about 100 to about 500 ml. The particle concentration is then preferably adjusted to about 30 to 90 million particles per ml. Finally, the preparation containing the antigenic material can be lyophilized and stored under dry conditions.

ASMP can usually be prepared as follows: keratinophilic or yeast fungal cells are treated under aqueous alkaline conditions, the solid and liquid phases of the preparation are separated, after separation, the supernatant is treated with mineral or organic acid, and after separation, ASMP is precipitated from the supernatant. More specifically, keratinophilic fungi or yeasts are cultured on agar plates, for example as described in EP 0564620. One preferred medium is, for example, Oxoid Malt Extract Agar. You can also use other media that will allow the growth of keratinophilic fungi or yeast. The resulting fungal biomass is torn off and treated with an aqueous solution of alkali. Preferred aqueous alkaline solutions are NaOH or KOH at preferred concentrations of 0.1-5% (w/v). Alkaline treatment is preferably carried out at about 20-150°C for up to 30 hours After treatment under aqueous alkaline conditions, the solid and liquid phases of the drug are separated, for example, by centrifugation, filtration or precipitation. Preferably, separation is achieved by centrifugation, which ensures good separation of fungal cell debris, for example, at forces of about 3500 g. The treatment under aqueous alkaline conditions as well as the separation step can be repeated several times. After alkaline treatment and separation, the resulting supernatant is treated under acidic aqueous conditions, for example with 0.2-1.5M organic acid or 0.05-1M mineral acid. For example, HCl or acetic acid can be used, preferably at pH values from about pH 2.5 to pH 4.5. Preferably, the treatment under aqueous acidic conditions is carried out for about 2 to 4 hours at temperatures of about 4 to 8° C., where the solid and liquid layers are then separated. The treatment under aqueous acidic conditions as well as the separation step can be repeated several times, preferably under the above conditions. The supernatant from the separation step then undergoes a precipitation step. Preferably the precipitation is carried out by adding a suitable organic solvent, for example an alcohol such as a lower alcohol, for example methanol or ethanol. A ratio of one volume of supernatant to 2-5 volumes of alcohol will result in good precipitation of antigenic material. Other non-alcohol precipitation techniques known to those skilled in the art may also be used, such as precipitation with ammonium sulfate or other salts. The solid phase is then subjected to a further separation step, preferably under the conditions described above. The resulting solid is isolated and, if desired, dissolved in an aqueous solution, preferably distilled water, typically in a volume of about 25 to 100 ml. Finally, the ASMP preparation can be lyophilized and stored for extended periods of time under dry conditions.

AEMP can usually be prepared as follows: fungal cells of keratinophilic fungi or yeast are cultured in a liquid medium, the solid phase and liquid phases of the preparation are separated, and after separation, AEMP is precipitated from the supernatant. More specifically, keratinophilic fungi or yeast can be incubated in an aqueous solution or cultured in a liquid medium. Cultivation can be carried out for up to about 240-250 hours. The volume of the solution or culture is defined here as the primary volume (PV). You can use distilled water, as well as media described in EP 0564620. After incubation or cultivation, fungal cells are separated, for example, by centrifugation, filtration or precipitation, preferably centrifugation, under the conditions described above. The supernatant that may form is then lyophilized and then dissolved in an aqueous solution, preferably water. Preferably, the volume of water is about 0.1 to 0.2 volumes of primary volume (PV). The resulting solution or the resulting supernatant obtained after separation is then subjected to a precipitation step. Preferably the precipitation is carried out by adding a suitable organic solvent, for example an alcohol such as

- 18 040511 lower alcohol, such as methanol or ethanol. A ratio of one volume of supernatant to about 1-5 volumes of alcohol will result in good precipitation of antigenic material. Other non-alcohol precipitation techniques known to those skilled in the art may also be used, such as precipitation with ammonium sulfate or other salts. The precipitate formed is isolated and, if desired, dissolved in an aqueous solution, preferably distilled water. Preferably, about 0.5 to 50 mg of the precipitate is dissolved in 1 ml of an aqueous solvent. Finally, the AEMP solution can be lyophilized and stored for long periods of time under dry conditions, preferably at about 2-10°C.

In a particularly preferred embodiment of the present invention, the composition for use according to the present invention comprises the strain Trichophyton verrucosum BINO 348-DSM 28406, its antigenic material and/or its homogenized inactivated dermatophyte microconidia. This composition may further comprise an antigenic material as described herein and/or chitosan modified with an organic carboxylic acid or a salt thereof, or a hydrocolloid according to the present invention.

In other particularly preferred embodiments of the present invention, the microconidia of the dermatophyte Trichophyton verrucosum of the embodiments described above are microconidia of the dermatophyte Trichophyton verrucosum BINO 348-DSM 28406. However, embodiments of the present invention as described above may further comprise microconidia of the dermatophyte strain Trichophyton verrucosum BINO 348-DSM 28406 .

In another particularly preferred embodiment, the composition for use according to the present invention comprises a mixture of homogenized inactivated microconidia of the dermatophyte Trichophyton verrucosum BINO 348-DSM 28406 and homogenized inactivated yeast blastospores of Candida albicans. This composition may further comprise an antigenic material as described herein and/or chitosan modified with an organic carboxylic acid or a salt thereof, or a hydrocolloid according to the present invention.

Thus, the present invention also relates to a strain of Trichophyton verrucosum DSM28406. In another aspect, the present invention relates to homogenized inactivated microconidia of the dermatophyte Trichophyton verrucosum DSM-28406. In addition, the present invention relates to an antigenic material comprising ANMP, AEMP and ASMP microconidia of the dermatophyte Trichophyton verrucosum DSM-28406.

In addition, the present invention relates to a strain of Trichophyton verrucosum DSM-28406, homogenized inactivated dermatophyte microconidia Trichophyton verrucosum DSM28406 and an antigenic material comprising ANMP, AEMP and ASMP dermatophyte microconidia Trichophyton verrucosum DSM-28406, for use in human and/or veterinary medicine. In particular, the present invention relates to a strain of Trichophyton verrucosum DSM-28406, homogenized inactivated microconidia of the dermatophyte Trichophyton verrucosum DSM-28406 and an antigenic material of microconidia of the dermatophyte Trichophyton verrucosum DSM-28406, for use in a method for the treatment and/or prevention of diseases of the hooves and claws in animals, lameness, digital dermatitis, interdigital dermatitis, interdigital phlegmon and dermatophytosis in animals, and warts in humans. The animals are preferably mammals, more preferably bovids and/or pigs, most preferably cattle.

In addition, the present invention relates to a composition containing homogenized inactivated microconidia of the dermatophyte strain Trichophyton verrucosum DSM-28406. The present invention also relates to a composition containing antigenic material of dermatophyte microconidia of Trichophyton verrucosum strain DSM-28406. The compositions of the present invention are preferably pharmaceutical compositions. The present invention also relates to a composition containing homogenized inactivated microconidia of the dermatophyte strain Trichophyton verrucosum DSM-28406 for use in human and/or veterinary medicine, in particular for use in a method for the treatment and/or prevention of diseases of the hooves and claws in animals, and lameness, digital dermatitis, interdigital dermatitis, interdigital phlegmon, dermatophytosis in animals, and warts in humans. The present invention also relates to a composition containing antigenic material of dermatophyte microconidia of Trichophyton verrucosum DSM28406 strain for use in human and/or veterinary medicine, in particular for use in a method for the treatment and/or prevention of hoof and claw diseases in animals, and lameness, digital dermatitis, interdigital dermatitis, interdigital phlegmon, dermatophytosis in animals, and warts in humans. The animals are preferably mammals, more preferably bovids and/or pigs, most preferably cattle.

Antigenic material comprising ANMP, ASMP and AEMP dermatophyte microconidia of Trichophyton verrucosum strain DSM-28406 can be obtained in the same manner as described generally above for yeast blastospore and/or dermatophyte microconidia antigenic material.

The composition for use in the present invention and the composition of the present invention are summarized below as the compositions of the present invention. Compositions of the present invention containing only one strain of dermatophyte microconidia or only one strain of yeast blastospores can be prepared as follows:

(a) growing the dermatophyte and yeast, respectively, on a suitable solid medium, collecting and homogenizing the dermatophyte, and (b) inactivating the homogenate obtained in step (a).

Compositions of the present invention containing a mixture of dermatophyte microconidia and/or yeast blastospores can be prepared as follows:

(a) growing one strain of dermatophyte and two, three, four, five, six, seven, eight, nine or ten different strains of dermatophytes, respectively, separately on a suitable solid medium, selecting each culture and homogenizing each culture separately, (b) possibly growing one yeast strain and two, three or four different yeast strains, respectively, separately on a suitable solid medium, selecting each culture and homogenizing each culture separately, and (c) combining and inactivating the homogenates obtained in step (a), and optionally obtained in step (b).

The cultivation of dermatophytes according to the above preparation method is preferably carried out on agar and wort in culture flasks. Preferably, the culture is carried out for about 15 to about 30 days. Preferably the cultivation is carried out at a temperature of from about 26 to about 28°C.

The cultivation of the yeast according to the preparation methods described above is preferably carried out on malt extract agar or on Sabouraud agar in culture flasks. Preferably, the culture is carried out for about 4 to about 7 days. Preferably the cultivation is carried out at a temperature of from about 28 to about 37°C.

After cultivation, the dermatophytes and yeasts, respectively, are homogenized to obtain a fine suspension. Preferably, homogenization is carried out in deionized water, in an aqueous solution containing about 0.1 to 0.3% fermented hydrolysed muscle protein or about 0.1 to 1% soy or porcine peptone in combination with about 5-6% glucose and about 0.1-1% yeast extract, or in an aqueous solution containing 0.1-0.9% (w/v) chitosan modified with organic carboxylic acid or its salt.

Suitable volumes for homogenization are from about 100 to 500 ml. Preferably, the concentration of microconidia and blastospores is adjusted to about 30 to about 90 million microconidia and blastospores, respectively, per ml, or to about 250 to about 500 thousand, more preferably from about 250 to about 400 thousand microconidia and blastospores, respectively. , per ml The suspension can then optionally be further adjusted to about 40, 50, or 60 million microconidia and blastospores, respectively, per ml, or to about 250 to about 500 thousand, more preferably to about 250 to about 400 thousand microconidia and blastospores, respectively, per ml. ml with distilled water, physiological saline such as sodium chloride or other suitable solution.

In the case of a mixture, the single suspensions are preferably adjusted to the same number of microconidia and blastospores, respectively, per ml, and equal volumes of each culture in the suspension are mixed in one container.

The inactivation is preferably carried out using thiomersal, formaldehyde and/or 2 propiolactone. Agents for inactivation can be added directly to the cell suspension. Preferred is inactivation by adding thiomersal in a ratio of about 1:11,000 to about 1:25,000 (w/v). Also preferred is inactivation by adding formaldehyde to achieve a final concentration of from about 0.2 to about 0.4% (v/v). This mixture is then preferably incubated. Incubation can be carried out for about 1 to 30 days at a temperature of from about 20 to about 37°C. Incubation is preferred for about 1-3 days at room temperature, for about 5-7 days at 37°C, for about 30 days at room temperature, or for about 30 days at about 26-28°C.

In a preferred embodiment, the microconidia of the composition of the present invention are swollen and/or have germ tubes. More preferably, at least 50% of the blastospores and/or microconidia are swollen and/or have germ tubes.

The swollen state and/or germ tubes of the dermatophytes can be obtained, for example, by means of a second incubation step. Said second incubation step is preferably carried out after homogenization and before inactivation as described above. For the second stage of cultivation, the suspension of microconidia is placed in a separate vessel containing the same medium as in the first stage of incubation. The second culture step is preferably carried out for about 10 to about 48 hours. The second culture step is preferably carried out at a temperature of about 28°C. Preferably, the second culture step is continued until

50% of the microconidia will not show a swollen or sprouting state, and no more than about 7 to 10% of the cells will not show a second mycelial branch. The diameter of swollen and germinating microconidia increases by about 1.2 times or more compared to normal microconidia.

Chitosan modified with an organic carboxylic acid or a salt thereof may also be referred to as a variant of chitosan or a derivative of chitosan. It can preferably be obtained by bringing chitosan into contact with an organic carboxylic acid or a salt thereof. Said contact is preferably carried out by incubating the chitosan in an aqueous solution of an organic carboxylic acid or salt thereof, more preferably by incubating the chitosan in an aqueous solution of valeric acid, lactic acid, para-aminobenzoic acid or glucuronic acid or a salt thereof, in particular valeric acid chloride. Preferably, said incubation is carried out by mixing and/or with stirring.

Thus, chitosan modified with an organic carboxylic acid or a salt thereof can preferably be produced by a method including:

(a) incubation of chitosan in an aqueous solution of an organic carboxylic acid or its salt.

In a preferred embodiment, the chitosan is first dissolved under aqueous acid conditions and then precipitated by increasing the pH to a pH of about 8.0 to about 8.5 before incubating it in an aqueous solution of an organic carboxylic acid or salt thereof as described above.

Thus, chitosan modified with an organic carboxylic acid or a salt thereof can preferably be produced by a method including:

(i) dissolving the chitosan in an aqueous acid solution, (ii) increasing the pH until the chitosan precipitates, (iii) isolating the precipitated chitosan, and (a) incubating the isolated chitosan from step (iii) in an aqueous organic carboxylic acid solution, or her salt.

The organic carboxylic acid or salt thereof from step (a) preferably has a pK of about 2 to about 5, more preferably about 2.3 to about 4.9. More preferably said organic carboxylic acid is valeric acid, lactic acid, para-aminobenzoic acid or glucuronic acid or a salt thereof, in particular valeric acid chloride. Said organic carboxylic acid or salt thereof is preferably used at a concentration of from about 0.2 to about 22.5 M. (a) results in a solution of the chitosan, modification of the chitosan, and/or formation of a gel. Preferably, the pH of the aqueous solution in step (a) is from about 5 to about 6, or about 5.0; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9 or 6.0. Preferably, the modification of chitosan occurs in an aqueous solution containing from about 1 to about 100 mm organic carboxylic acid or its salts, more preferably from about to about 10 mm.

Preferably, step (a) is carried out until the chitosan is modified and dissolved. It is preferably carried out by mixing the chitosan with an aqueous solution of an organic carboxylic acid or a salt thereof, or by suspending the chitosan under aqueous conditions and adding the organic carboxylic acid to the suspension. It is preferably carried out with stirring for about 1 to about 72 hours, more preferably for about 24 to about 48 hours. Step (a) may include the addition of additional acid or may be carried out in the presence of additional acid. Said additional acid is preferably a mineral acid, an organic acid or a salt of said mineral acid or organic acid. Preferably the mineral acid is HCl or H2SO4 and the organic acid is glutamic acid, para-aminobenzoic acid or lactic acid. The mineral or organic acid is preferably added or present in an amount to adjust the pH of the mixture from step (a) to a pH of about 5 to about 6, or about 5.0; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9 or 6.0. The addition of additional acid may support the dissolution and/or modification of the chitosan, for example by reducing the time it takes for the modified chitosan to dissolve.

The concentration of chitosan in step (a), or if the process includes step (i), for step (i) is preferably from about 1 to about 20 grams of chitosan per liter, more preferably from about 5 grams to about 15 grams of chitosan per liter , most preferably from about 8 to about 10 grams of chitosan per liter.

The chitosan used for step (a), or if the method includes step (i), for step (i), may be commercially available chitosan or chitosan isolated from any natural source containing chitosan, such as biomass containing chitosan. Alternatively, you can use chitin, which is deacetylated to obtain chitosan up to stage (a), or, if the method includes stage (i), up to stage (i). Said chitin may be chitin,

- 21 040511 commercially available, or it can be isolated from a natural source containing chitin, such as biomass containing chitin. The biomass for the isolation of chitin and/or chitosan is preferably a biomass of fungi, insects and/or crustaceans. The deacetylation of chitin can be carried out by methods known in the art, for example, by using sodium hydroxide (NaOH) in excess as a reagent and water as a solvent, or by enzymatic methods. The isolation of chitosan and/or chitin from natural sources can also be carried out by methods known in the art.

The chitosan used for step (a) or, if the process includes step (i) for step (i), preferably has a deacetylation degree of from about 62% to about 95%, more preferably from about 80% to about 94%, more preferably from about 89 to about 93%, or about 93 to about 98%, about 93 to 95%, about 95 to about 98%, or at least 60%, more preferably at least 70, 80, 90, or 95% , or the degree of deacetylation from about 60% to about 100%, more preferably from about 80% to about 95%, even more preferably from about 90% to about 95%, most preferably from about 77% to about 80%. The chitosan used for step (a) or, if the method includes step (i) for step (i), preferably has a molecular weight or an average molecular weight of from about 50 to about 700 kDa, in particular from about 15 a to about 500 kDa, more specifically from about 15 to about 150 kDa, or from about 80 to about 200 kDa, from about 150 to about 300 kDa, from about 100 to about 250 kDa, or from about 300 to about 700 kDa.

The chitosan used for step (a) or, if the process includes step (i) for step (i), preferably has a viscosity of from about 50 to 400 MPa-s, more preferably from about 70 to about 150 MPa-s, or from about 151 to about 350 MPa-s.

Before using the chitosan in step (a), or if the method includes step (i) in step (i), the chitosan can be sterilized by autoclaving. Said sterilization may result in the modified chitosan being less toxic, better tolerated by the subject, and/or resulting in fewer unintended side effects.

Preferably step (i) is carried out by using an aqueous solution of a weak acid, preferably an organic acid or a salt thereof, more preferably acetic acid, valeric acid, lactic acid, para-aminobenzoic acid or glucuronic acid, or a salt thereof, in particular valeric acid chloride. This acid is preferably used at a concentration of from about 0.8 to about 2%. Step (i) is preferably carried out with stirring. Stirring can be carried out for from about 2 to about 24 hours. Preferably, step (i) is carried out until a gel or gel suspension is obtained. Undissolved particles can be removed, for example, by filtration. For example, for such filtering, a metal mesh with a cell from 200 to 300 microns can be used.

Step (ii) is preferably carried out by increasing the pH of the gel or gel suspension obtained in step (i) until a precipitate forms. It is preferably carried out with stirring. It is preferably carried out by treating the chitosan under aqueous alkaline conditions, more preferably under aqueous alkaline conditions comprising from about 0.1% to about 25.0% alkali. In a preferred embodiment, the alkali is NaOH. Preferably, said step is carried out at a temperature of from about 4 to about 55°C. Preferably, the treatment is carried out for from about 20 minutes to about 2 hours, more preferably for from about 30 to about 70 minutes, but it can also take up to about 24 hours. (i). Preferably the pH is increased to give a pH of about 8.0 to about 8.5. Step (ii) may result in additional deacetylation of the chitosan. It may also result in the modified chitosan being less toxic, better tolerated by any subject, and/or resulting in fewer unintended side effects.

Step (iii) is preferably carried out by centrifuging the mixture or suspension obtained in step (ii). Centrifugation is preferably carried out at about 4000 to about 6000 rpm, more preferably at about 5000 rpm. Centrifugation is preferably carried out for up to 60 minutes.

Methods by which modified chitosan can be obtained may include additional steps. For example, the product obtained in step (ii) may be homogenized. Preferably the homogenization step is carried out in a closed sterile homogenizer. Alternatively or additionally, the product obtained in step (a) may be dialyzed. Dialysis is preferably carried out in a closed system to remove free salt ions and low molecular weight compounds. Preferably, dialysis is carried out by tangential filtration for about 1 to about 6 hours or by membrane filtration against distilled water for about 24 to about 48 hours.

Alternatively or additionally, methods by which the modified

- 22 040511 bath chitosan may include an additional step for obtaining the final product. Obtaining the final product may include dilution of the resulting product. Preferably the product is reconstituted with water, more preferably with sterile water for injection. However, the product may also be diluted with any other suitable aqueous solution. Alternatively or additionally, obtaining the final product may include the addition of one or more additional compounds, such as diluents or preservatives. Suitable preservatives are, for example, chlorocresol, thiomersal and formalin. Finally, the final product can be sterilized. Preferably, sterilization is carried out by heating, preferably for about 40 to 50 minutes at a temperature of from about 65 to about 80°C. Preferably said sterilization is repeated one, two, three, four or five times.

Preferably the final product has a concentration of from about 0.02 to about 2 grams of modified chitosan per liter, more preferably from about 0.04 to about 1 g of modified chitosan per liter.

In a preferred embodiment, the methods by which modified chitosan can be obtained include the steps described in the examples. For example, these methods may include the following steps: possibly sterilizing the chitosan, for example by autoclaving, (i) dissolving the chitosan in an aqueous acid solution, in particular in the presence of acetic acid, possibly removing undissolved particles, for example by filtration, (ii) increasing the pH value, until the chitosan precipitates, (iii) isolating the precipitated chitosan, possibly homogenizing the isolated chitosan under aqueous conditions, (a) incubating the isolated chitosan from step (iii) or the homogenized isolated chitosan in an aqueous solution of an organic carboxylic acid or salt thereof, possibly in the presence of additional mineral acid or organic acid, it is possible to carry out dialysis of the product obtained in step (a), it is possible to add additional compounds such as diluents or preservatives, and it is possible to sterilize the final product, for example by heating.

Preferably, the order of steps as described above is in the order listed above. However, as one skilled in the art would know, the order of the single steps may vary, as long as the same effects are achieved. For example, diluents such as water may be added at various stages of the process as described above.

In a preferred embodiment of the present application, the modified chitosan, chitosan derivative or chitosan variant is a polyamino sugar colloid, preferably a hydrocolloid. In another preferred embodiment of the present application, the hydrocolloid is a chitosan-glucuronic acid hydrocolloid or a chitosan-p-aminobenzoic acid hydrocolloid, or a chitosan-valeric acid hydrocolloid. In another preferred embodiment of the present application, the chitosan-glucuronic acid hydrocolloid has the chemical formula: (C ₆ H11O ₄ N) _x (C ₈ H ₁₃ O ₅ N _y (C ₆ H ₁₀ O7) _z (H ₂ O)m. Preferably, the chitosan hydrocolloid β-glucuronic acid has the following molecular weight: x*(161)+y*(203)+z*(194.14)+m*(18) In another preferred embodiment of the present application, the chitosan-p-aminobenzoic acid hydrocolloid has the following chemical formula: (C ₆ H11O ₄ N) _x (C ₈ H ₁₃ O ₅ N) _y (C7H7O2N) _z (H ₂ O) _m Preferably, the chitosan-paminobenzoic acid hydrocolloid has the following molecular weight: x*(161)+y* (203)+z*(137.14)+m*(18) In another preferred embodiment of the present application, the chitosan-valeric acid hydrocolloid has the following chemical formula: (C ₆ H _n O ₄ N) _x (C ₈ H ₁₃ O ₅ N) _y (C ₅ H ₁₀ O ₂ ) _z (HCl) _z (H ₂ O) _m Preferably, the chitosan-valeric acid hydrocolloid has the following molecular weight: x*(161)+y*(203)+z* (102)+z*(36.5)+m*(18).

In another preferred embodiment of the present application, the modified chitosan, chitosan derivative or chitosan variant is a naturally white to yellowish viscous liquid. Preferably the modified chitosan, chitosan derivative or chitosan variant has a typical carboxylic acid flavor, preferably a typical valeric acid flavor.

In another preferred embodiment, the modified chitosan, chitosan derivative, or chitosan variant contains about 0.2% pentanoyl chloride, or 0.2% glucuronic acid, or 0.2% p-aminobenzoic acid.

In another preferred embodiment, the modified chitosan, chitosan derivative, or chitosan variant contains about 1.0% pentanoyl chloride.

In another preferred embodiment, the modified chitosan, chitosan derivative, or chitosan variant contains 1.0% of the chitosan residue from the dried chitosan.

In another preferred embodiment, the modified chitosan, chitosan derivative or chitosan variant has from about 10 to about 1000 mOsmol, preferably from about 10 to about 200 mOsmol, most preferably about 100 mOsmol.

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) 0.1 to 5% (w/v) chitosan and 0.001 to 5% (w/v) valeric acid or a salt thereof,

- 23 040511 preferably valeric acid chloride, or (ii) 0.1 to 5% (w/w) chitosan and 0.001 to 5% (w/w) glucuronic acid or paminobenzoic acid, or salts thereof.

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) from 0.1 to 3% (w/v) of chitosan and from 0.001 to 2% (w/v) of valeric acid or a salt thereof, preferably valeric acid chloride, or (ii) from 0.1 up to 3% (w/w) chitosan; and from 0.001 to 2% (w/w) glucuronic acid or paminobenzoic acid, or salts thereof.

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) 0.1 to 1.2% (w/v) chitosan and 0.001 to 1% (w/v) valeric acid or a salt thereof, preferably valeric acid chloride, or (ii) from 0 1 to 1.2% (w/w) of chitosan; and 0.001 to 1% (w/w) of glucuronic acid or paminobenzoic acid, or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) 0.1 to 1.2% (w/v) chitosan and (ii) 0.01 to 0.44% (w/v) valeric acid or a salt thereof, preferably valeric acid chloride .

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) 0.1 to 1.2% (w/w) chitosan; and (ii) 0.001 to 0.6% (w/w) glucuronic acid or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosan derivative or variant of chitosan is a hydrocolloid containing:

(i) 0.1 to 1.2% chitosan; and (ii) 0.006 to 1% (w/w) p-aminobenzoic acid or a salt thereof.

Preferably, the chitosan of this hydrocolloid is a compound of formula [X]n, wherein n is an integer from about 1 to about 5000, in particular an integer from about 300 to about 4000, and X has the following formula (1):

iCHpOH

where from about 2% to about 38%, more preferably from about 5% to about 20% of the X residues constituting said compound are modified by acetylation, and where all or part of the X residues constituting said compound are modified with an organic carboxylic acid or a salt thereof.

In a preferred embodiment, the remaining percentage of the hydrocolloid according to the present invention is provided by a dispersion medium, preferably water or water and hydrochloric acid (HCl).

In a preferred embodiment, the hydrocolloid of the present invention is used as a dilution, preferably at a 0 to 10 fold dilution.

In a typical embodiment, the modified chitosan can be obtained by the following method:

As a raw material, chitosan with deacetylation, for example 67%, with a viscosity of 50200 MPa-s, can be used. 40 g of chitosan is sterilized by autoclaving and added to 3.5 l of water for injection with stirring. 40 ml of acetic acid is added to the resulting suspension. The final volume is adjusted to a volume of 4 liters with water for injection. This mixture is stirred in a sterile container for about 24 hours until a gel suspension is obtained. Undissolved particles are removed by filtration through a metal mesh with a cell size of 200-300 microns. 4 n. sodium hydroxide (NaOH) is added dropwise to the resulting suspension to give a final pH of 8.0. This precipitates white flakes that contain chitosan. The suspension is stirred for about 30 minutes. 4 ml of valeric acid chloride is added dropwise to the suspension with constant stirring. The resulting suspended material is stirred for one hour. Flakes and undissolved particles are separated and then resuspended in 4 L of sterile water for injection. Add with stirring 4 N. hydrochloric acid to obtain a pH of 5.0. The resulting gel is dialyzed in a closed system to remove free salt ions and low molecular weight compounds. After dialysis, the final product is obtained. To obtain the final product, 3 l of the modified chitosan gel is adjusted to a volume of 25 l by adding sterile water for injection with stirring. Then 500 ml of a chlorocresol solution containing 30 g of chlorocresol is added to the mixture. The resulting suspension is brought to a volume of 30 liters. The resulting sterile product is dispensed into vials under sterile conditions.

- 24 040511

Unless specific temperature ranges are given for the steps of the method as described herein, these steps are preferably carried out at room temperature and/or in the range of from about to about 40°C, more preferably in the range of from about 20 to about 30°C.

In a preferred embodiment, the compositions of the present invention have a concentration of from about 40 to about 90 million blastospores per ml, very preferably a concentration of from about 40 to about 60 million spores per ml. These concentrations are particularly preferred when the compositions are intended for intramuscular administration. In another preferred embodiment, the compositions of the present invention have a concentration of from about 0.2 to about 0.4 million spores per ml, very preferably a concentration of from about 0.25 to about 0.3 million spores per ml. These concentrations are particularly preferred if the compositions are intended for intradermal, intramuscular administration.

In another preferred embodiment, the compositions of the present invention have a concentration of from about 40 to about 60 million particles of dermatophyte microconidia and/or yeast blastospore antigenic material per ml.

In another preferred embodiment, the concentration of chitosan modified with an organic carboxylic acid or salt thereof is preferably in the range of about 0.1 to about 0.9% (w/v), more preferably in the range of about 0.1 to about 0 3% (w/v), more preferably in the range of from about 0.1 to about 0.3% (w/v).

Surprisingly, it has been found that if the composition of the present invention further comprises chitosan modified with an organic carboxylic acid, or a salt thereof, homogenized inactivated dermatophyte microconidia and/or inactivated homogenized yeast microspores can be used at about 50 times lower dose.

The compositions of the present invention may further contain a pharmaceutically acceptable carrier, excipients and/or supporting agents.

The compositions of the present invention are capable of modulating the immune system, ie. they have immunostimulating properties. They can be used as a vaccine to prevent a subject from the diseases described above. Alternatively or additionally, they can be used to treat and cure the subject's diseases as described above. These compositions can be administered by known routes of administration, for example parenterally, by intramuscular injection, by intradermal injection, by transdermal injection and/or topically, preferably dermally. They can be administered in the absence or presence of one or more additional immunostimulatory substances. In one embodiment, said one or more additional immunostimulatory substances are administered separately to the compositions of the present invention. In another embodiment, one or more additional immunostimulatory substances are contained in the compositions of the present invention or added to the compositions of the present invention.

Said one or more immunostimulatory substances is preferably an adjuvant, preferably selected from the group consisting of vitamin E acetate, oil-in-water emulsion, aluminum phosphate, aluminum oxide, aluminum hydroxide/methylcellulose gel, oil emulsion, muramyl dipeptides, adjuvants Freund and saponins, and/or at least one cytokine, preferably selected from the group consisting of IL-2 (interleukin-2), IL-12 and INF-gamma.

In a more preferred embodiment, the compositions of the present invention further comprise chitosan modified with an organic carboxylic acid, or a salt or hydrocolloid thereof, of the present invention.

In a preferred embodiment, the compositions of the present invention are in the form of a vaccine and/or are used as a vaccine.

The present invention also relates to a pharmaceutical composition containing antigenic material of keratinophilic fungi or keratinophilic yeast, as described above. Such a pharmaceutical composition may further contain a pharmaceutically acceptable diluent, excipient or carrier.

In another aspect, the present invention relates to a composition comprising keratinophilic fungal or keratinophilic yeast antigenic material as described above for use in a method of treating an animal organism through therapy. Such a method typically comprises administering to the subject an effective amount of a keratinophilic fungal or keratinophilic yeast antigenic material as described above, or a composition or pharmaceutical composition as described above. The subject may be, for example, an animal, in particular a mammal, more preferably a bovid and/or a pig, most preferably a bovine animal. In particular, a keratinophilic fungal or keratinophilic yeast antigenic material as described above, or a composition or pharmaceutical composition as described above, can be used in methods of treating or preventing the above described hoof and claw diseases as described above. This method of treatment may include the treatment and/or prevention of bacterial, fungal and/or

- 25 040511 viral infections of the skin, leg, hoof, claw, back of the foot and/or interdigital area.

These infections can be caused by Dichelobacter nodosus, Fusobacterium necroforun, Fusobacterium spp, Treponema spp, such as T. phagedenis, T. vincentii and T. denticola, Campylobacter spp, Staphylococcus aureus, Escherichia coli, Arcanobacterium pyogenes and Prevotella spp., and /or a virus.

The dosage and route of administration used in the method of treatment (and/or prevention) according to the present invention depends on the specific disease/site of infection being treated. The route of administration may be, for example, parenteral, intramuscular injection, intradermal injection, transdermal injection, topically, cutaneously, or any other route of administration.

For example, the dosage and route of administration used in a method of treating and/or preventing diseases of the hoof and claw, in particular ID, DD and/or IP, in cattle may be as follows:

path introductions concentration [millions of blastospores/microconidia/mL] frequency of drug administration number sites interval between the introduction yami medicinal funds [day] dose [ml] inside we- cervical 10-150, more preferably 30- 90, over preferably 40-60 or 20, 30, 40, 50, 60, 70, 80, 90, or 100 1, 2, 3, 4 or 5 1 or 2 5-21 days, more preferably 7- 10 days or 5, 6, 7, 8, 9, 10, I, 12, 13, or 14 days 0.5 - Yu, more pre- reverently 1-5 or 0.5, 1, 2, 2.5, 3, 4, 5 or 6 inside- dermal 0.1 - 0.6, more preferably 0.250.4 or 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, or 0.6 1, 2, 3, 4 or 5 1, 2 or 3 5-21 days, more preferably 7-10 days or 5, 6, 7, 8, 9, 10, I, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days 0.2 - 0.6, more pre- reverently 0.3-0.5 or 0.2, 0.3, 0.4, 0.5 or 0.6

The following examples illustrate the present invention, but should not be construed as limiting.

Example 1

Polivak-TM vaccine (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) against animal dermatophytosis was used for the prevention and treatment of digital and/or interdigital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 2

Polivak-T vaccine (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) against bovine dermatophytosis was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 3

The culture of the dermatophyte species Trichophyton mentagrophytes DSM - 7279 is cultivated on agar/wort, for example in 4 Roux flasks. Each culture is cultivated for 18 days at 28°C.

Fungal masses are torn off and homogenized in 500 ml of deionized water. The suspension of microconidia is then adjusted to 60 million microconidia per ml with saline - sodium chloride. The homogenate is inactivated by adding formaldehyde at the end to 0.4% (v/v) directly into the cell suspension. This mixture is incubated for 5-7 days at 37°C. The resulting composition is bottled, tested for sterility, safety and microconidia count according to conventional known methods, and can be stored refrigerated at 4-10°C. The vaccine prepared according to this method was used to prevent and treat interdigital, digital dermatitis, and/or interdigital phlegmon in cattle, as described below.

Example 4

A culture of the dermatophyte species Trichophyton verrucosum DSM-28406 is cultured on agar/wort, for example in 10 Roux flasks. Each culture is cultivated for 25 days at 26°C.

Fungal masses are torn off and homogenized in 300 ml of deionized water. The concentration of microconidia is adjusted to 80 million per ml for each homogenate. The microconidia suspension is then adjusted to 40 million microconidia per ml with distilled water. The homogenate is inactivated by adding formaldehyde to reach 0.5% (v/v) directly into the cell suspension. This mixture is incubated for 5 days at 37°C. The resulting composition is bottled, tested for sterility, safety and microconidia count according to conventional methods, and stored at 48°C. The vaccine prepared according to this method was used for the prevention and treatment of interdigital, digital dermatitis and interdigital phlegmon in cattle, as described below.

Example 5

A culture of the dermatophyte species Trichophyton verrucosum DSM-28406 is cultured on agar/wort, for example in 8 Roux flasks. Each culture is cultivated for 30 days at 27°C.

Fungal masses are torn off and homogenized in 400 ml of deionized water. The concentration of microconidia is adjusted to 70 million per ml for each homogenate. The microconidia suspension is then adjusted to 60 million microconidia per ml with distilled water. The homogenate is inactivated by adding formaldehyde to reach 0.3% (v/v) directly into the cell suspension. This mixture is incubated for 7 days at 37°C. The resulting composition is bottled, tested for sterility, safety and microconidia count according to conventional methods, and stored at 48°C. The vaccine prepared according to this method was used for the prevention and treatment of interdigital, digital dermatitis and interdigital phlegmon in cattle, as described below.

Example 6

A culture of the dermatophyte species Trichophyton verrucosum DSM-28406 is cultured on agar/wort, for example in 9 Roux flasks. Each culture is cultivated for 25 days at 28°C.

The fungal mass of the dermatophyte is torn off and homogenized in 500 ml of an aqueous solution of 0.3% fermented hydrolyzed muscle protein in combination with 5% glucose and 0.1% yeast extract. The concentration of microconidia is adjusted for the homogenate to 40 million per ml. The microconidia suspension is then fermented for 1 day at 28° C. until 65% of the microconidia have germ tubes. After fermentation, the cell suspension is washed with physiological sodium chloride solution. The homogenate is inactivated by adding thiomersal in a ratio of 1:25,000 (w/v) directly into the cell suspension. This mixture is incubated for 30 days at room temperature. The resulting composition is bottled, tested for sterility, safety and immunogenicity according to accepted methods, and can be stored refrigerated at 4-10°C. The composition prepared according to this method was used for the prevention and treatment of interdigital, digital dermatitis, interdigital phlegmon and dermatophytosis in cattle, as described below.

Example 7

A variant of the Polivak-TM vaccine against animal dermatophytosis, which, compared to the classic Polivak-TM, does not contain Trichophyton equinum and Microsporum strains (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow), was used for the prevention and treatment of interdigital and/or digital dermatitis , and/or interdigital phlegmon in cattle, as described below.

Example 8

Polivak-TM vaccine against animal dermatophytosis (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 9

Polivak-TM vaccine against animal dermatophytosis (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) was used for the prevention and treatment of digital and/or interdigital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 10

Polivak-T vaccine against bovine dermatophytosis (manufacturer: LLC Vetbiokhim, Moscow; distributor: LLC Prostor, Moscow) was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

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Example 11.

A culture of the dermatophyte species Trichophyton verrucosum DSM - 28406 is cultivated on agar/wort, for example in 10 Roux flasks. The culture is cultivated for 28 days at 28°C.

The fungal mass is torn off and homogenized in 500 ml of deionized water. The concentration of microconidia in the homogenate is adjusted to 50 million per ml. The microconidia suspension is then adjusted to 50 million microconidia per ml with distilled water.

Candida albicans species DSM-9456 is cultured on malt extract agar or Sabouraud agar, for example, in 3 Roux flasks. The culture is cultivated for 4 days at 30°C. Blastospores are washed with physiological sodium chloride solution. The concentration of blastospores in the suspension is adjusted to 60 million per ml. Equal volumes of each culture in suspension are mixed in one container. The homogenates are inactivated by adding formaldehyde to achieve 0.4% (v/v) in the cell suspension. This mixture is incubated for 6 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 12.

The species Candida albicans DSM-9456 is cultured on Sabouraud agar, for example in 7 Roux flasks. The culture is cultivated for 7 days at 37°C. Blastospores are washed with sterile water. The concentration of blastospores in the suspension is adjusted to 40 million per ml. The homogenate is inactivated by adding formaldehyde to reach 0.5% (v/v) in the cell suspension. This mixture is incubated for 7 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 13

A solution of chitosan modified with valeric acid chloride was added to the Polivak-T vaccine against bovine dermatophytosis (manufacturer: OOO Vetbiokhim, Moscow; distributor: OOO Prostor, Moscow) to achieve a final concentration of 0.1% (w/v). The concentration of microconidia was 40 million per ml. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 14

A culture of the dermatophyte species Trichophyton verrucosum DSM-28406 is cultured on agar/wort, for example in 7 Roux flasks. This culture is cultivated for 27 days at 26°C.

Fungal masses of the dermatophyte are torn off and homogenized in 500 ml of an aqueous solution of 0.2% chitosan modified with para-aminobenzoic acid. The concentration of microconidia is adjusted to 50 million per ml for each homogenate. The homogenate is inactivated by adding formaldehyde to a final concentration of 0.5% (v/v) directly into the cell suspension. This mixture is incubated for 7 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon and/or dermatophytosis in cattle, as described below.

Example 15

Candida albicans species DSM-9456 is cultured on malt extract agar for 6 Roux flasks. The culture is cultivated for 7 days at 35°C. Blastospores are washed with physiological sodium chloride solution.

Fungal masses are torn off and homogenized in 500 ml of an aqueous solution of 0.1% chitosan modified with valeric acid chloride. The concentration of microconidia is adjusted in the homogenate to 80 million per ml. The homogenate is inactivated by adding formaldehyde directly to the cell suspension, reaching 0.2% (v/v) at the end. This mixture is incubated for 7 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 16

The fraction obtained according to this method consists of insoluble antigenic material containing polysaccharide and/or glycopeptides (ANMP), as disclosed in WO 97/07232. A culture of the dermatophyte species Trichophyton verrucosum DSM-28406 is cultured on agar/wort, for example in 10 Roux flasks. This culture is cultivated for 28 days at 28°C.

The resulting fungal biomass is torn off and treated with an aqueous solution of alkali. Preferred aqueous alkaline solutions are NaOH at a concentration of 3% (v/v). The alkaline treatment is preferably carried out at 80° C. for up to 6 hours. After the treatment under aqueous alkaline conditions, the solid and liquid phases of the preparation are separated by centrifugation at about 3500 g. After alkaline treatment, the solid phase is treated with 0.2 M acetic acid added to the solid phase for 1 hour at 70°C. After acid treatment, the solid phase is washed with distilled water. Washing is repeated three times. Finally, the solid phase is torn off and homogenized in 500 ml of water for injection. The concentration of particles is adjusted to 60 million per ml of the final product. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 17

The fraction obtained according to this method consists of insoluble antigenic material containing polysaccharide and/or glycopeptides (ANMP), as disclosed in WO 97/07232. The culture of the dermatophyte species Trichophyton verrucosum DSM -28406 is cultivated on agar/wort in 9 Roux flasks. This culture is cultivated for 25 days at 27°C. The resulting fungal biomass is torn off and treated with an aqueous solution of alkali. Preferred aqueous alkaline solutions are KOH at preferred concentrations of 2% (w/v). The alkaline treatment is preferably carried out at 60° C. for up to 10 hours. After the treatment under aqueous alkaline conditions, the solid and liquid phases of the formulation are separated by centrifugation at about 3500 g. The treatment under aqueous alkaline conditions was repeated twice as well as the separation step by centrifugation at about 3500 g. After alkaline treatment, the solid phase is treated with 0.5 M HCl added to the solid phase for 1.5 hours at 80°C. After acid treatment, the solid phase is washed with distilled water twice. Finally, the solid phase is separated off and homogenized in 500 ml of an aqueous solution containing 0.3% chitosan modified with valeric acid chloride. The particle concentration for each homogenate is adjusted to 60 million per ml. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 18.

The product is obtained from chitosan. This product is obtained in two stages. At the first stage, a concentrated gel of modified chitosan is obtained, at the second stage - the final product, which can be used for administration.

First stage. The raw material used is chitosan with deacetylation 80%, viscosity 27513250 MPa-s. 40 grams of polysaccharide is sterilized by autoclaving, and 3.5 liters of water for injection are added with stirring. 40 ml of acetic acid is added to the resulting suspension. The final volume is adjusted with water for injection to 4 liters. The suspended polysaccharide is stirred in a sterile container for 24 hours to form a gel suspension. Undissolved particles are removed by filtration through a metal mesh with a pore size of 200-300 μm. 4 n. sodium hydroxide (NaOH) is added dropwise to the resulting suspension until the suspension has a pH of 8.0. In this case, white flakes containing chitosan precipitate. The suspension is stirred for 30 minutes. 8 ml of lactic acid are added dropwise to the suspension with constant stirring. The resulting suspended substance is stirred for another hour. The flakes and undissolved particles are separated from the suspension and resuspended in 4 liters of water for injection. Add with stirring 4 N. hydrochloric acid until pH 5.6 is reached. The resulting suspension is dialyzed in a closed system to remove free salt ions and low molecular weight compounds. The resulting suspension is a concentrated gel of modified chitosan. The concentration of modified chitosan is from about 0.8 to about 1%. After dialysis, this polysaccharide is used to obtain the final product.

Second stage. To obtain the final product containing the modified chitosan obtained in the first stage, the resulting suspension of the first stage is adjusted to a volume of 25 l by adding sterile water for injection with stirring. Then, 500 ml of a chlorocresol solution containing 30 g of chlorocresol is added to this mixture as a preservative. The resulting suspension is brought to a volume of 30 liters. The resulting sterile product is dispensed into vessels under aseptic conditions. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle, as described below.

Example 19.

The culture of the dermatophyte species Trichophyton verrucosum DSM - 28406 is cultivated on agar/wort, for example in 8 Roux flasks. The culture is cultivated for 30 days at 28°C.

The fungal masses of the dermatophyte are torn off and homogenized in 500 ml of an aqueous solution of 0.1% chitosan modified with valeric acid chloride. The concentration of microconidia is adjusted to 400 thousand per ml for each homogenate. The homogenates are inactivated by adding formaldehyde directly to the cell suspension, reaching 0.4% (v/v) at the end. This mixture is incubated for 7 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon and/or dermatophytosis in cattle, as described below.

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Example 20.

The culture of the dermatophyte species Trichophyton mentagrophytes DSM-7279 is cultivated on agar/wort in Roux flasks. The culture is cultivated for 20 days at 26°C.

Fungal masses of the dermatophyte are torn off and homogenized in 400 ml of an aqueous solution of 0.2% chitosan modified with para-aminobenzoic acid. The concentration of microconidia is adjusted to 250 thousand per ml for each homogenate. The homogenates are inactivated by adding formaldehyde directly to the cell suspension, reaching 0.3% (v/v) at the end. This mixture is incubated for 6 days at 37°C. The composition prepared according to this method was used for the prevention and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon and/or dermatophytosis in cattle, as described below.

Example 21.

Cows with clinical evidence of lameness and interdigital lesions that are typical of DD, ID and IP were treated with different compositions. The composition was administered by intramuscular injection twice with an interval of 10 days. The dose for the composition was 5 ml for each application.

The results are shown in table. 2.

group number The composition in the form in which it is obtained in the example Number of animals Frequency of drug administration Number of healthy animals 30-35 days after the first application At 53-55 days after the first application 1 1 10 2 4 5 2 2 10 2 4 5 3 3 10 2 5 5 4 4 10 2 4 5 5 5 15 2 7 7 6 6 13 2 6 6 7 7 10 2 4 5 8 8 AND 2 5 6 9 9 12 2 5 6 10 10 13 2 6 6 AND AND 14 2 7 7 12 12 13 2 6 7 13 13 10 2 5 7 14 14 12 2 5 7 15 15 14 2 6 8 16 16 10 2 5 5 17 17 12 2 6 6 18 18 14 2 7 9

No general or local reactions were observed after application of these compositions. The therapeutic efficiencies of treatment with different compositions obtained as described in examples 1-18 were 40-64%.

Example 22.

Cows with clinical evidence of lameness and interdigital lesions that are typical of DD, ID and IP were treated with different compositions. The composition was administered by intradermal injection twice with an interval of 7-10 days. The dose of the composition was a total of 0.4 ml, which was injected into the animal in two places.

The results are shown in table. 3.

group number The composition in the form in which it was obtained in example Number of animals Frequency of drug administration Number of healthy animals 30-35 days after the first application At 53-55 days after the first application 1 19 100 2 60 85 2 20 100 2 58 87

No general or local reactions were observed after application of these compositions. The therapeutic efficacy of treatment with different compositions obtained according to examples 19 and 20 was approximately 85-87%.

Example 23.

Cows with clinical evidence of lameness and interdigital lesions that are typical of DD, ID, and IP were treated with different drugs. The composition obtained according to example 15 was administered as follows: 2 times intramuscularly at a dose of 5 ml in one place with an interval of 10 days.

Clinical manifestation of the disease:

+ convalescent or grey, no pain ++ cured, less than 2 cm, yellow, mild pain +++ convalescent, or greater than 2 cm, yellow, moderate pain ++++ acute disease, greater than 2 cm, red, severe pain

The results are shown in table. 4.

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Before treatment 30 days after treatment 58 days after treatment 12 animals++ 4 animals - healthy 4 animals + 6 animals++ 11 animals - healthy 3 animals + 4 animals +++ 2 animals +++ 3 animals++ 4 animals ++++ 2 animals ++++ only 2 animals were selected for slaughter only 3 animals were selected for slaughter

No general or local reactions were observed after application. The effectiveness of the treatment was approximately 70%.

Example 24 Dose Titration Study

Cows with clinical evidence of lameness, interdigital lesions that are typical of DD, ID and IP were treated with different drugs. Therapeutic application of the composition obtained according to example 14: 3 times intramuscularly in one place with an interval of 7 days.

Clinical manifestation of the disease:

+ convalescent or grey, no pain ++ cured, less than 2 cm, yellow, mild pain +++ convalescent, or greater than 2 cm, yellow, moderate pain ++++ acute disease, greater than 2 cm, red, severe pain

The results are shown in table. 5.

Before treatment 51 days after Treatment Dose - 5 ml Before treatment 51 days after treatment Dose - 3 ml 19 animals +++ 5 animals - healthy 7 animals + 18 animals 1 animal - healthy 4 animals + 5 animals++ 6 animals++ 2 animals +++ 2 animals were selected for slaughter 5 animals were selected for slaughter

No general or local reactions were observed after application. The effectiveness of treatment with a dose of 5 ml was approximately 63%, and with a dose of 3 ml - 28%.

Example 25.

Cows with clinical evidence of lameness and interdigital lesions that are typical of DD, ID, and IP were treated. Therapeutic application of the composition obtained according to example 14: intramuscularly, 2 times in one place at a dose of 5 ml with an interval of 10 days.

Research Summary:

Before treatment Number of animals/number of lame limbs 52 to 54 days after last treatment 100/138 Number of animals/number of lame limbs 29/29 Number of healthy animals 71 Reducing the number of lame limbs 78.99%

Example 26 Dose Titration Study

Cows were treated against DD, ID and IP. Prophylactic use of the composition obtained according to example 14: intramuscularly, 2 times with an interval of 10 days.

We investigated the clinical manifestation of the disease:

+ convalescent or grey, no pain ++ cured, less than 2 cm, yellow, mild pain +++ convalescent, or greater than 2 cm, yellow, moderate pain ++++ acute disease, greater than 2 cm, red, severe pain

The results are shown in table. 6.

73 days after application Dose 1 ml 100 animals Dose 2.5 ml 100 animals Control of 215 animals 7 animals + 7 animals + 21 animals + 18 animals++ 6 animals +++

No general or local reactions were observed after application. The effectiveness of treatment with doses of 1 ml

- 31 040511 and 2.5 ml was approximately 93%. 45 animals (approximately 21%) from the control group were with clinical symptoms of DD, ID and IP.

The results are shown in table. 7.

107 days after application Dose 1 ml 100 animals Dose 2.5 ml 100 animals Control of 215 animals 7 animals + 9 animals + 11 animals + 12 animals++ 4 animals - selected for slaughter 4 animals - selected for slaughter 9 animals +++ 27 animals - selected for slaughter

The effectiveness of treatment with doses of 1 and 2.5 ml was approximately 87-89%. 59 animals (approximately 27%) from the control group were with clinical symptoms of DD, ID and IP.

The results are shown in table. 8.

170 days after application of the composition Dose 1 ml 100 animals Dose 2.5 ml 100 animals Control of 215 animals 25 animals + 41 animals + 112 animals + 6 additional animals were selected for slaughter 3 additional animals were selected for slaughter 42 additional animals were selected for slaughter

The effectiveness of treatment with doses of 1 ml was approximately 70%, and with doses of 2.5 ml - approximately 53%. 154 animals (approximately 72%) from the control group were with clinical symptoms of DD, ID and IP. This study demonstrates the prophylactic treatment of animals with a dose of 1.0 ml. The duration of immunity was approximately 5.5 months.

Example 27.

Cows were treated against DD, ID and IP. Prophylactic use of the composition obtained according to example 14: intramuscularly, 2 times at a dose of 5 ml with an interval of 10 days.

Research Summary:

Treated animals in group 1

Observation before treatment

Number of animals/clinical manifestations DD, ID and IP 100/100

160 to 175 days after last treatment

Number of animals/number of lame limbs 100/10

Number of healthy animals 90

Treatment efficiency 90%

Animals in group 2 were treated with placebo (control)

Observation before processing

Number of animals/clinical manifestations DD, ID and IP 100/100

160 to 175 days after last placebo

Number of animals/number of animals showing clinical symptoms DD, W and IP 100/48

Number of healthy animals 52

The number of sick animals during the observation period 48%

All animals in the control group (placebo) with a clinical symptom of diseases were treated with topical application of aseptic drug or antibiotics. In the case of IP, intramuscular injection of antibiotics was used.

Example 28.

Cows with clinical evidence of lameness and interdigital lesions that are typical of DD, ID and IP were treated with different compositions. The composition was administered by intramuscular injection twice in one place with an interval of 10 days. The dose of the composition was 5 ml for each application.

The results are shown in table. 9.

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group number Composition, in the form in which it is obtained in the example Number of animals Frequency of drug administration Number of healthy animals 30-35 days after the first application At 53-55 days after the first application 1 14 10 2 10 10 2 15 10 2 9 9 3 18 10 2 10 10 4 Control - treated with conventional methods 10 3 5

Example 29 Hydrocolloids

Chemical nomenclature: chitosan-valeric acid hydrocolloid Subtitle: polyaminosugar-valeric acid hydrocomplex Structural formula:

Chemical formula: (C6H _n NO4)x(C8H13NO5)y (C5H10O2K (HCl)z (HO^

General properties:

Molecular weight: x*(161)+y*(203)+z*(102)+z*(36.5)+m*(18)

Appearance: Natural white to yellowish viscous liquid with a typical odor

Solubility: soluble in water

Smell: typical, similar to valeric acid

Density: 1.002

pH value: 5.5

Storage: store protected from light; store in an air-tight container in a refrigerator at 4°C-8°C

Stability: 36 months under the conditions described above

Chemical nomenclature: chitosan-4-aminobenzoic acid hydrocolloid

Subtitle: polyaminosugar-p-aminobenzoic acid hydrocomplex

Structural formula:

Chemical formula: (C6H _n NO4)x(C8H13NO5)y (C7H7O2)z (H2O) _m

General properties:

Molecular weight: x*(161)+y*(203)+z*(137.14)+m*(18)

Appearance: yellowish to yellow viscous liquid

Chemical nomenclature: chitosan-glucuronic acid hydrocolloid Subtitle: polyaminosugar-glucuronic acid hydrocomplex Structural formula:

Chemical formula: (C6HnNO4)x(C8H _B NO5)y (C6H10O7K (HO)m

General properties:

Molecular weight: x*(161)+y*(203)+z*(194.14)+m*(18)

Appearance: yellowish to yellow viscous liquid

Example 30 Preparation of Chitosan-Valeric Acid Hydrocolloid

Purification of Chitosan 80/100 and 80/200, AS-No.: 9012-76-4

Amino-N-acetyl-D-glucosamine is sterilized in a separate vessel and manipulated to

- 33 040511 obtaining pharmaceutical grade chitosan.

Reagent solution

Sterile amino-N-acetyl-D-glucosamine is resuspended with stirring for 15 minutes in this sterile water. Add 400 ml of acetic acid to the suspension with stirring (24

h) until a clear solution is obtained.

Purification stage

To this solution (carefully) add 4 n. sodium hydroxide solution to obtain a pH of 8.0 to 8.5. The resulting solution precipitated to a white mass. The resulting suspension is stirred for at least 30 minutes. The precipitate is separated from the liquid phase by filtration.

Resuspension

The pellet is resuspended in an equal amount of purified (sterile) water (Water for Injection (EP)) (40 L, original amount). Measure out 80 ml of pentanoyl chloride. Pentanoylchloride is added drop by drop to the suspension under stirring conditions. The resulting suspension is stirred until the solution becomes clear. Add 1.6 g of thiomersal (40 μg/ml). This clear solution is the active ingredient (hydrocolloid). The resulting polysaccharide colloid (CVHC) is stored at 4-8°C. For the final product, an aqueous solution with a certain biological activity is prepared.

Overview of fabrication reaction steps

1. Chitosan + water ^ suspension

Suspension + HAs (24 h) → chitosan-HAs solution

2. Purification stage

2.1. Chitosan-HAc solution + 4 n. NaOH ^ (pH 8-8.5) chitosan + NaAc + H ₂ O

2.2. Chitosan + NaAc + H ₂ O ^ H ₂ O + NaAc ^ chitosan (solid, purified)

3. Production

Chitosan (solid) + H ₂ O + pentanoyl chloride ^

Chitosan + valeric acid + H ₂ O + HCl ^ CVHC (chitosan-valeric acid hydrocolloid)

The production is a combination of the stage of purification of the initial substance of chitosan and the reaction during this process with the second reagent - pentanoyl chloride.

This first critical step is the precipitation of the chitosan to obtain a total amount of pharmaceutical grade purified chitosan.

Monitoring during the process: the reaction time and the pH value are monitored to obtain a quantitative precipitation.

Analysis of the pharmaceutical quality of chitosan:

Analysis of the quality of the intermediate (pharmaceutical grade chitosan).

Solubility in water: Approximately 250 mg of a chitosan precipitate sample is resuspended in 1 ml of purified water.

Purpose: Solubility cannot be obtained.

Quality: Performed if no reduction in solids can be detected.

Solubility in stronger acids: in parallel, the same amount of precipitate is suspended in 1 ml of HCl (3N).

Goal: complete dissolution

Quality: Passed if a solution of total solids can be detected.

The second critical step is the process of dissolution of the active ingredient. The control is carried out visually: the total amount of sediment must be solubilized.

Example 31 Identity Verification Using UV (Ultraviolet)/Visible Spectroscopy

Used the method of analysis according to the European Pharmacopoeia 2.2.25.

Instrument: Jasco 7800 spectrophotometer

Measurement conditions:

Bandwidth 2 nm

Interval 200-600 nm

Solvent blank correction

Temperature: 25°C

UV cell: 12.5 x 45 mm semi-micro, 10 mm path length, UV grade silica

Solvent: H ₂ O

Test solution: an adequate sample of chitosan-HCl, chitosan-HAc, chitosan, chitosan-valeric acid hydrocolloid and valeric acid, respectively, was dissolved in the above solvent. This mixture was shaken and then sonicated in an ultrasonic bath for 5 minutes.

According to the general principles of spectroscopy and chemical structure with specific chromophore groups and substituents for chitosan-HCl, chitosan-HAc, chitosan-valeric acid hydrocolloid, and valeric acid, respectively, one can expect absorption maxima at 200 nm.

UV maximum Chitosan HCI Chitosan Us Chitosan CVHC Valerian acid nm 200 200 - 200 211

Chitosan absorption maxima could not be analyzed because chitosan is a water insoluble solid which also cannot be solubilized in typical organic solvents.

Comparison of all spectra shows no significance or structural modification like aromatic bonds etc. Based on the measured spectra and literature data on raw materials, the measured spectrum corresponds to the sought spectra. Thus, the above measured data confirm the identity of the desired structure.

Example 32 Absorption IR (Infrared) Spectrophotometry

Used the method of analysis according to the European Pharmacopoeia 2.2.24.

To identify the active principle of the chitosan-valeric acid hydrocolloid, a series of IR spectra of various chitosan derivatives are compared with the spectrum of the product and valeric acid.

1. Method and parameters

Instrument: infrared spectrometer FT/IR 410 Jasco

Range: 4000 to 600 cm ^-1 .

Sample to be analyzed: a mixture of 4.8 mg of chitosan or a mixture of 4 mg of chitosan-HCl, or a mixture of 3.8 mg of chitosan acetate and 100 mg of KBr is thoroughly ground and pressed into a suitable potassium bromide-based disk or a chitosan-valeric acid hydrocolloid film or plate based on NaCl for valeric acid.

Measurement conditions:

Background correction: current

Temperature: 20°C.

The measured spectrum directly corresponds to the literature spectra from the database.

Result: The above measured data confirm the identity of the tested compounds.

2. Data for different IR spectra

chitosan base Chitosan -HC1 Chitosan -NAS Dried colloid chitosan valeric acid Valerian acid 3398 3365 3424 3426 2919/2875 2887 2926/ 2960-2872 2960-2875 2673 2018 2092 2130 1708 1717 1665 1596 1606 1562 1561 1569 1509

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Comparison with literature data: Based on the measured spectra and literature data on the raw material, the measured CVHC spectrum corresponds to the desired spectrum.

Result: The above measured data confirm the identity of the proposed structure.

Example 33 ^.sup.13 C-NMR (Nuclear Magnetic Resonance) Spectroscopy Analysis

Used the method of analysis according to the European Pharmacopoeia 2.2.33.

a) ¹³ C-NMR spectrum of chitosan

1. Method and parameters

Bruker AMX 500 AVANCE instrument

Measurement conditions

Scanning frequency: 125 MHz for chitosan, chitosan-HCl, chitosan-HAc, glucosamine-HCl, Nacetylglucosamine, chitosan-valeric acid hydrocolloid, valeric acid.

Temperature: 300 K for chitosan, chitosan-HCl, chitosan-HAc, chitosan-valeric acid hydrocolloid, valeric acid; 301 K for glucosamine-HCl and N-acetylglucosamine.

Solvent: D ₂ O for chitosan, chitosan-HCl, chitosan-HAc, glucosamine-HCl, Nacetylglucosamine;

DMSO (dimethyl sulfoxide)-D6 for chitosan-valeric acid hydrocolloid

CDCl ₃ for valeric acid.

Concentration: for chitosan, chitosan-HCl, chitosan-HAc, chitosan-valeric acid hydrocolloid; approximately 15 mg/0.5 ml for glucosamine-HCl, N-acetylglucosamine and valeric acid.

Calibration: for chitosan, chitosan-HCl, chitosan-HAc, glucosamine-HCl, N-acetylglucosamine

DMSO-D6 for chitosan-valeric acid hydrocolloid

CDCl ₃ for valeric acid.

2. Results

a) Analysis of chitosan by ¹³ C-NMR spectroscopy

Measurement in solution: Measurement in solution is not possible due to the lack of solubility in neutral solvents.

Solid state measurement: Solid state measurement was not possible. Also, after long measurement conditions (time), acceptable signals did not appear.

Result: identification of chitosan by NMR is not possible.

b) Analysis of chitosan-HCl ¹³ C-NMR spectroscopy

results [d] Classification (carbon number) 97.67 C1 76.41 / 74.80 C5 70.28 NW 64.41 C4 60.11 C6 56.06 C2

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Target The following characteristic chemical shifts [million' ¹ ] according to the general provisions of spectroscopy and the chemical skeleton with substituents can be expected at: 100 70 56 General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Result: The above measured data confirm the identity of the tested substance.

c) Analysis of chitosan-HAc ¹³ C-NMR spectroscopy

results [d] Classification (carbon number) Skeleton of glucosamine 98.39 C1 74.79 C5 - NW - C4 - C6 - C2 Acetic acid 23.82 SNz 180.31 >c=o Target The following characteristic chemical shifts, according to the general principles of spectroscopy and the chemical skeleton with substituents, can be expected at: [million' ¹ ] 98.39 23.82 180.31 General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Result: The above measured data confirm the identity of the tested substance.

d) Analysis of glucosamine-HCl ¹³ C-NMR spectroscopy

results [d] Classification (carbon number) 92.94 / 89.34 C1 76.25 C5 72.28/71.69 NW 69.85 /69.77 C4 60.66/60.51 C6 54.62/57.08 C2

Target The following characteristic chemical shifts, according to the general principles of spectroscopy and the chemical skeleton with substituents, can be expected at: [million' ¹ ] 92.94/ 89.34 60.66/ 60.51 54.62/ 57.08 General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Comparison with literature data: the measured spectrum directly corresponds to the literature spectra from the database.

Result: The above measured data confirm the identity of the tested substance.

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e) Analysis of N-acetylglucosamine ¹³ C-NMR spectroscopy

results [d] Classification (carbon number) 95.06/90.95 C1 76.01 /74.08 C5 71.64/70.86 NW 70.22 / 69.99 C4 60.89 / 60.74 C6 56.90 / 54.26 C2 22.29 / 22.03 CH ₃ 3 174.85/ 174.59 c=o Target The following characteristic chemical shifts, according to the general principles of spectroscopy and the chemical skeleton with substituents, can be expected at: 95.06/ 90.95 22.29/ 22.03 174.85/ 174.59 General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Result: The above measured data confirm the identity of the tested substance.

f) Analysis of chitosan-valeric acid hydrocolloid ^{by 13} C-NMR spectroscopy

results [d] Classification (carbon number) Skeleton of glucosamine 100.95 C1 78.57 C5 76.10 NW 73.10 C4 61.40 C6 57.57 C2 Valeric acid 180.66 C5' 29.23 C4' 24.85 NW' 23.37 C2' 14.87 CU

[million]

The following characteristic chemical shifts according to the general principles of spectroscopy and

chemical skeleton with substituents

100.95

61.40

57.57 expected at:

180.66

14.87

General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Result: The above measured data confirm the identity of the proposed structure.

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g) Analysis of valeric acid ^{by 13} C-NMR spectroscopy

results TO Classification (carbon number) 180.5 C5 33.8 C4 26.7 sz 22.2 C2 10.6 C1

Target The following characteristic chemical shifts, according to the general principles of spectroscopy and the chemical skeleton with substituents, can be expected at: [million' ¹ ] 180 10.6 General literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5.Auflage 1995

Comparison with literature data: the measured spectrum directly corresponds to the literature spectra from the database.

Result: The above measured data confirm the identity of the tested substance.

3. Comparison of NMR spectra

ChitosanHCl Chitosan-NAS Glucosamine- HC1 N-Acetyl Glucosamine Hydrocolloid chitosanvaleric acid Valeric acid Classification (carbon number) 97.7 98.4 92.9 / 89.3 95.0/91.0 101.0 C1 56.1 54.6/57.1 54.3 / 56.9 57.6 C2 70.3 71.7/72.3 71.6/70.9 76.1 NW 64.4 69.9 / 69.8 70.2 / 70.0 73.1 C4 76.4 / 74.8 74.8 76.3 76.0/74.1 78.6 C5 60.1 60.5 / 60.7 60.9 / 60.7 61.4 C6 22.7 23.8 180.3 174.9/ 174.6 14.9 13.06 23.4 22.02 24.9 26.07 29.2 33.8 108.7 180.5 Literature - - X - - X

Comparison with literature data: not available or based on measured spectra and literature data on raw materials, with the measured spectrum directly corresponding to the spectra sought.

Result: The above measured data confirm the identity of the proposed structure.

Example 34 TLC (Thin Layer Chromatography) Method for Analyzing Chitosan and Impurities in a New Product Chitosan-Valeric Acid Hydrocolloid (CVHC)

Used the method of analysis according to the European Pharmacopoeia 2.2.27.

This section presents thin layer chromatography techniques and data for the identification of CVHC, along with Rf values (retention factor) in the solvent mixtures used and spot color when detected under UV light (365 nm and 254 nm), visible light and using typical reagents for visualization.

The initial main raw material for any type of glucosamine is the natural substance chitin from insects or crabs. The monomeric structure of these biopolymers is N-acetylglucosamine. For pharmaceutical and other applications, deacetylated chitin is in most cases typical. This resulting biopolymer is the so-called chitosan, which can be modified to water-soluble ionic compounds. The monomeric structure of this chitosan should theoretically be glucosamine. Since the deacetylation step does not go completely, chitosan has a mixed structure of N-acetylglucosamine (acetylated) and glucosamine (deacetylated) units. Hydrocolloid chitosan-valeric acid is a new polyaminosugar-valeric acid hydrocomplex. Therefore, positive analytical results for N-acetylglucosamine and glucosamine should not be possible. When monomeric fragments are interspersed in the form of residual impurities, it should be possible to identify chitosan in the form of its water-soluble ionic compounds chitosan-HCl and chitosan-HAc.

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1. Method

device Camag Chromatography Tank System Record for TLC Pre-coated Merck Si 60 F 254 Conditions Protected from sunlight and with camera saturation Temperature 20 - 25°C Manifestation: Vertical development

Chromatographic Conditions

sample solution See single analytes Application 30 µl Drying At least 2 minutes in a jet of air Travel interval 80 mm

mobile phase

Solvents Acetone Water 25% aq. ammonium - Mixture 20 10 5 -

2. Analysis and reagents

a) Chitosan

Sample preparation

Sample: chitosan suspended in water

1) Appliance: reflux condenser

Conditions: heating for about 30 minutes at reflux (145°C)

2) Appliance: ultrasonic bath

Conditions: sonication for about 30 minutes at 45°C

3) Appliance: reflux condenser

Conditions: heating for about 30 minutes at reflux (145°C)

4) Filtration: 0.45um filter

A clear filtrate was used for analysis.

Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulphuric Iodine acids Connection signal No No No No impurities No No No No

Group-specific reagent 1: Naturstoff reagent / DT / 366 nm

Group specific reagent 2: 5% ninhydrin/EtOH

RF value The signal for chitosan could not be identified - Non-specific impurities: Not identified

Alternative: solubilization in organic solvents shows the same results due to the lack of solubility of chitosan.

Result: an acceptable solution of chitosan in aqueous or organic solvents like methanol, etc. not possible. Suitable solubilization of chitosan is only possible in stronger acids like HCl or HAc in the production of chitosan-HCl or chitosan-HAc.

b) Chitosan-HCl

5 mg chitosan-HCl/mL _H2O was used for analysis.

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Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal No No gray spot Brown spot impurities No No No No Group-specific reagent 1: Naturstoff reagent / DT / 366 nm Group specific reagent 2: 5% ninhydrin/EtOH RF value Chitosan-HC1 oh oh Non-specific impurities: Not identified Target Connection purity One main spot The anisaldehyde-sulfuric acid reagent and iodine, respectively, as non-selective reagents for the detection of non-specific impurities, should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described for such a polymer can be expected at: 0.0 - -

Result: purity of the compound; one major spot.

c) Chitosan-HAs

5 mg of chitosan-HAc/mL H2O were used for analysis.

Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Specific in respect group reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal impurities specific specific No No singing group reac singing group reac No No tive 1: reagent no. tive 2: 5% ningid] gray spot No iturstoff / DT / 366] ein / EtOH brown stain No nm Rf value |Chitosan-HAc 0.0 | Non-specific impurities: Not identified

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Meaning from literature Not available data from - Target Connection purity One main spot The anisaldehyde-sulfuric acid reagent and iodine, respectively, as non-selective reagents for the detection of non-specific impurities, should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described for such a polymer can be expected at: 0.0 - -

Result: purity of the compound; one major spot.

d) Glucosamine-HCl

For analysis, 5 mg glucosamine-HCl/ml H2O was used.

Detection using UV fluorescence and in the visible region.

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal blue spot red spot gray spot Brown spot impurities No No No No

Group-specific reagent 1: Naturstoff reagent / DT / 366 nm Group specific reagent 2: 5% ninhydrin/EtOH RF value Glucosamine-HC1 0.67 Non-specific impurities: Not identified Value from literature Not available data from - Target Connection purity One main spot ^anisaldehyde -sulfuric acid eactive and iodine, respectively, as non-selective reagents for the detection of non-specific impurities should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described can be expected at: 0.6 And 0.8

Result: purity of the compound; one major spot.

e) N-acetylglucosamine

5 mg N-acetylglucosamine/ml H2O was used for analysis.

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Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Specific in respect group reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal blue spot No gray spot Brown spot impurities No No No No Group-specific reagent 1: Naturstoff reagent / DT /366 nm Group specific reagent 2: 5% ninhydrin/EtOH

RF value N-acetylglucosamine 0.72 Non-specific impurities: Not identified Target Connection purity One main spot The anisaldehyde-sulfuric acid reagent and iodine, respectively, as non-selective reagents for the detection of non-specific impurities, should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described can be expected at: 0.6 And 0.8

Result: purity of the compound; one main spot

f) Hydrocolloid chitosan-valeric acid (CVHC)

CVHC is a highly viscous watery gel. Two drops of CVHC were used for analysis.

Detection using UV fluorescence and in the visible region.

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal No No gray spot Brown spot impurities No No No No Group-specific reagent 1: Naturstoff reagent / DT / 366 nm Group specific reagent 2: 5% ninhydrin/EtOH RF value Hydrocolloid chitosan-valeric acid 0.0 Non-specific impurities: Not identified

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Value from literature Not available data from - Target Connection purity One main spot The anisaldehyde-sulfuric acid reagent and iodine, respectively, as non-selective reagents for the detection of non-specific impurities, should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described for such a polymer can be expected at: 0.0 - -

Result: purity of the compound; one major spot.

g) Valeric acid

For analysis, 1 μl of valeric acid (pure) was used.

Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent 1 Group specific reagent 2 Reagent based on anisaldehyde-sulfuric acid Iodine Connection signal No No No yellow

spot impurities No No No No Group-specific reagent 1: Naturstoff reagent / DT / 366 nm Group specific reagent 2: 5% ninhydrin/EtOH

RF value Valeric acid oh oh Non-specific impurities: Not identified Value from literature See available data from - Target Connection purity One main spot The anisaldehyde-sulfuric acid reagent and iodine, respectively, as non-selective reagents for the detection of non-specific impurities, should not show a higher content of impurities. The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described can be expected at: 0.0 - -

Result: purity of the compound; one major spot.

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3. Comparison of TLC analysis results

Chitosan Chitosan- HCl Chitosan- Us Glucosamine- HC1 N-acetylglucosamine -HC1 Chitosanvaleric acid colloid Valeric acid RF value Not Maybe 0 0 0.67 0.72 0 0 Revealing Connection signal UV 254 nm - - - - - - - UV 365 nm - - - - - - - visible light - - - - - - -

Reagent Naturstoff - - - blue spot blue stain - - Ninhydrin reagent - - - Red spot - - - Reagent based on anisic aldehyde-sulphuric acid Gray spot gray spot gray spot gray spot Gray spot - Reagent for based on iodine brown- new spot brown- new spot Brown spot Brown spot brown- new spot yellow spot

The above TLC results indicate that there is no evidence of the presence of a monomeric or dimeric structure that could be detected using the specific derivative reagents tested above. The detection and Rf 0 value show the similarity of the chitosan-valeric acid hydrocolloid with the related compounds chitosan-HCl and chitosan-HAc. Specific identification of valeric acid using this TLC system has failed. The chitosan-valeric acid hydrocolloid can only be a polyamino sugar colloid, but not a solution of chitosan or a chitosan derivative with valeric acid in water.

Example 35 TLC Method for Analytical Detection of Valeric Acid in Chitosan-Valeric Acid Hydrocolloid

1. Method and parameters

Created a new TLC system for the identification and analysis of the purity of the constituent component - valeric acid.

device Camag Chromatography Tank System Record for TLC Pre-coated Merck Si 60 F 254 Conditions Protected from sunlight and with camera saturation Temperature 20-25°C Manifestation: Vertical development

Chromatographic Conditions

Drying At least 2 minutes in a jet of air Travel interval 80 mm

mobile base

Solvents ethyl acetate - - - Mixture 100 - - -

2. Results

a) Valeric acid (pure)

For analysis, 2 μl of valeric acid (pure) was used.

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Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent Reagent based on anisic aldehyde-sulphuric acid Iodine Connection signal Yellow spot/blue background pink spot yellowish spot impurities No No No Group-Specific Reagent: Bromocresol Green/Bromophenol Blue/Potassium Permanganate Reagent [Jork et al.]

RF value Valeric acid 0.56 Non-specific impurities: Not identified Meaning from literature Not available data from -

Threshold for detection of valeric acid using this imaging reagent after TLC chromatography: 0.03 μg.

b) Chitosan-valeric acid hydrocolloid (CVHV) 45 µl of chitosan-valeric acid hydrocolloid, pure (this is about 850 times the amount of valeric acid compared to the analyzes given earlier) was used for the assay.

Detection using UV fluorescence and in the visible region.

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No Detection with imaging reagents Visible light ι 1 Connection signal Group specific reagent Blue spot/blue background Anisaldehyde-sulphuric acid reagent Gray spot Iodine brown spot impurities No No No Group-Specific Reagent: Bromocresol Green/Bromophenol Blue/Potassium Permanganate Reagent [Jork et al.]

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Target Connection purity One main spot Reagents based on anisaldehyde-sulphuric acid and iodine, respectively, as non-selective reagents for the detection of non-specific impurities should not show a higher content of impurities. A group-specific reagent based on bromocresol green/bromophenol blue/potassium permanganate should show typical results for compounds The relative retention factor (Rf) of a given compound according to the chemical skeleton under the given chromatographic conditions described can be expected at: oh oh for chitosan derivatives approx. 0.6 for valeric acid, if available

RF value | Hydrocolloid chitosan-valeric acid oh oh | Non-specific impurities: Not identified RF value [Valeric acid Not identified Non-specific impurities: Not identified Value from literature available data from -

Threshold for detection of valeric acid using this imaging reagent after TLC chromatography: 0.03 μg.

Pure valeric acid can be identified using this TLC system. Colloidal integrated valeric acid cannot be detected in pure chitosan-valeric acid hydrocolloid. The detection and Rf 0 value show the similarity of the chitosan-valeric acid hydrocolloid with other related chitosan compounds. The chitosan-valeric acid hydrocolloid can only be a polyamino sugar colloid, but not a solution of chitosan or a chitosan derivative with valeric acid in water. The above results confirm the identity of the proposed structure.

Example 36 Elimination of Valeric Acid from Chitosan-Valeric Acid Hydrocolloid Using High Vacuum and Elevated Temperature

Method: loss on drying estimation (special method)

Appliance: high-speed circulating vacuum concentrator

Conditions: 500 Pa

Temperature: 60°C

Time: 1 week

Expected result: constant mass

Appearance: vitreous mass

Resulting odor: no typical valeric acid odor

Sample preparation

Redissolving, partly with water

Appearance: high viscosity gel

TLC Analysis

Instrument: Camag flask system

Record for TLC: pre-coated records Merck Si 60 F 254

Conditions: protected from sunlight and with chamber saturation

Temperature: 20-25°C

Appearance: vertical manifestation

Chromatographic Conditions

Sample solution: see above

Application: 5 µl

Drying: at least 2 minutes in a stream of air

Travel interval: 80 mm

mobile phase

Solvents ethyl acetate - - - Mixture 100 - - -

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Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent Reagent based on anisic aldehyde-sulphuric acid Iodine Connection signal Blue spot/blue background gray spot brown spot impurities No No No Group-Specific Reagent: Bromocresol Green/Bromophenol Blue/Potassium Permanganate Reagent [Jork et al.] RF value spot 0

Threshold for detection of valeric acid using this imaging reagent after TLC chromatography: 0.03 μg.

Result: when using a strong vacuum and high temperature, there is a disproportion of the chitosan-valeric acid hydrocolloid. The elimination of valeric acid can be demonstrated by the absolute absence of the typical odor of valeric acid. Elimination of valeric acid can be demonstrated by TLC analysis: by the absence of the typical spot of free valeric acid at an Rf value of 0.56. Chitosan or chitosan compounds can be identified at an Rf value of 0. The chitosan-valeric acid hydrocolloid can only be a polyamino sugar colloid, not a solution of chitosan or a valeric acid derivative of chitosan in water.

Example 37 Disproportion of chitosan-valeric acid hydrocolloid when using solvents

The structure of the hydrocolloid chitosan-valeric acid decomposes in ethyl acetate to valeric acid and the chitosan compound.

Sample preparation

Apparatus: separating funnel, evaporator

Liquid-liquid distribution: 20 ml chitosan-valeric acid hydrocolloid and 10 ml ethyl acetate.

Conditions: shaking for about 5 minutes and waiting for phase separation.

Phase separation: the ethyl acetate phase was taken.

Concentration step: Approximately 10 ml was concentrated to a liquid residue (aqueous) using an evaporator.

Redissolve: in 1 ml methanol.

Homogenization: centrifugation step for approx. 5 minutes at 12,000 rpm

Phase separation: upper phase - clear methanol solution.

Lower phase: highly viscous gel.

TLC analysis of the upper and lower phases (see above).

a) Upper phase analysis (clear methanol solution).

TLC Analysis

Instrument: Camag flask system

Record for TLC: pre-coated records Merck Si 60 F 254

Conditions: protected from sunlight and with chamber saturation

Temperature: 20-25 °С

Appearance: vertical manifestation

Chromatographic Conditions

Sample solution: clear methanol solution

Application: 5 µl

Drying: at least 2 minutes in a stream of air

Travel interval: 80 mm

mobile phase

Solvents ethyl acetate - - - Mixture 100 - - -

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Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent Reagent based on anisaldehyde-sulphuric acid Iodine Connection signal Yellow spot/blue background No Light yellowish spot impurities No No No Group-Specific Reagent: Bromocresol Green/Bromophenol Blue/Potassium Permanganate Reagent [Jork et al.]

RF value Valeric acid 0.57 Non-specific impurities: -

Detection threshold using imaging reagent: 0.03 μg.

Result: The upper phase is a clear methanol solution. Valeric acid can be identified after the breakdown of the hydrocolloid in a given solution using TLC. By means of TLC, chitosan or a chitosan compound cannot be detected.

b) Lower phase analysis (highly viscous gel)

TLC Analysis

Instrument: Camag flask system

Record for TLC: pre-coated records Merck Si 60 F 254

Conditions: Protected from sunlight and using a camera

Temperature: 20-25 °C

Appearance: vertical manifestation

Chromatographic Conditions

Sample solution: highly viscous gel, completely redissolved in water Application: 30 µl

Drying: at least 2 minutes in a stream of air

Travel interval: 80 mm

mobile phase

Solvents ethyl acetate - - - Mixture 100 - - -

Detection using UV fluorescence and in the visible region

Fluorescence wavelength 254 nm 365 nm Visible area Connection signal No No No impurities No No No

Detection with imaging reagents

visible light Group specific reagent Reagent based on anisic aldehyde-sulphuric acid Iodine Connection signal Blue spot/blue background gray spot brown stain impurities No No No Group-Specific Reagent: Bromocresol Green/Bromophenol Blue/Potassium Permanganate Reagent [Jork et al.] RF value Spot 0 Non-specific impurities: -

Detection threshold using imaging reagent: 0.03 μg.

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Results: The lower phase is a highly viscous gel, soluble in water. Valeric acid cannot be detected in this phase by TLC. Chitosan or a chitosan compound can be identified in the lower phase (gel) by TLC.

Results from TLC analysis Possible disproportion of chitosan-valeric acid hydrocolloid using typical solvents like ethyl acetate and then methanol Redissolution from disproportionated chitosan-valeric acid hydrocolloid can be carried out using methanol

The decomposition of chitosan-valeric acid hydrocolloid in ethyl acetate shows two phases Upper phase Lower phase Ethyl acetate phase Aqueous colloidal residue After concentration, the ethyl acetate phase was redissolved in methanol and this also resulted in two phases Upper phase Lower phase clear methanol solution high viscosity gel This gel can be completely redissolved in water can be identified Can't be identified Cannot detect chitosan or chitosan compound Chitosan or chitosan compound may be detected

Summary of results

Pure Valeric Acid Chitosanvaleric acid hydrocolloid (pure CVHC) Elimination from CVHC using high vacuum Chitosanvaleric acid hydrocolloid (degraded with ethyl acetate) Upper phase (methanol) Lower phase (water) RF value 0.56 + - - + - RF value 0 - + + - + Revealing Connection signal UV 254 nm - - - - - UV 365 nm - - - - - visible light - - - - - Reagent based on anisaldehyde-sulfuric acid Pink spot gray spot gray spot gray spot Iodine reagent yellowish Brown Brown Light Brown spot spot spot yellowish spot spot Bromocresol Green Bromophenol Blue Reagent yellow spot blue stain blue spot yellow spot blue stain

Result: chitosan-valeric acid hydrocolloid can only be a polyamino sugar colloid, but not a solution of chitosan or a chitosan derivative with valeric acid in water. The above results confirm the identity of the proposed structure.

Example 38 Relative Density Estimation

Due to the high viscosity of the chitosan-valeric acid hydrocolloid, density estimation is not possible using the density vial/pycnometer according to the analysis method of Euro-50 040511 Pharmacopoeia 2.2.5.

1. Method of analysis by weight

Instrument: 250 ml volumetric flask

Balance: Sartorius MS 1 LC 2200S

Thermometer: thermometer with scale graduation (minimum 0.5°C) and measurement interval not greater than 60°C.

Results 1,001 [d ₂₀ ²⁰ ]

The active principle is a hydrogel, so the theoretical density must be greater than 1.0. The measured data confirm the identity of the proposed substance.

2. Method of analysis using a hydrometer

Used the method of analysis according to the European Pharmacopoeia 2.2.5.

Instrument: 250 ml volumetric flask

Hydrometer: Widder 1573°, 20°C-M100-DIN 12791 class H

Thermometer: thermometer with scale graduation (minimum 0.5°C) and measurement interval not greater than 60°C.

Measurement conditions: temperature 20 plus/minus 0.5°C with electronic thermostat.

Results 1.002 [d20 ²⁰ ]

The active principle is a hydrogel, so the theoretical density must be greater than 1.0. The measured data confirm the identity of the proposed substance.

Example 39 Sulphated Ash

Used the method of analysis according to the European Pharmacopoeia 2.4.14.

Conducting an analysis

device A suitable crucible (porcelain or platinum) was heated at 600+/- 50°C for 30 min in a muffle furnace Allow to cool in a desiccator over silica gel or other suitable drying agent. Estimation of the mass of the crucible Weight Crucible weighing 1: 52.0120 [g] Crucible weighing 2: 57.6055 [g] Method 2 (acid-insoluble ash) An additional concentration to dryness step for this hydrogel was carried out by drying at 105° C. in a normal oven. Sample: 25ml CVHC hydrogel, typically 1-2g Sample weight: typically 1-2 g, or sufficient to obtain a residue of at least 1 g. Moisten the sample with a small amount of sulfuric acid R [95-97% w/w] (usually 1 ml) and heat at as low a temperature as is practical until the residue is charred After cooling, moisten the residue with a small amount of sulfuric acid R [95-97% w/w] (usually 1 ml) Heat until no more white fumes are emitted Heat at 600+/- 50°C for 30 minutes until the residue is completely ashed. Flames are not allowed at any time during this procedure. Allow to cool in a desiccator over silica gel or other suitable drying agent. Weigh and calculate percentage of balance Definition of general masses Total mass of crucible 1: 52.0668 g Total mass of crucible 2: 57.6612 g Sulphated ash content Value 1: 0.0548 g Value 2: 0.0557 g Average: 0.05525 g/25 ml

Calculation of sulfated ash content

0.05525 g/25.05 g = 0.0022055 g/g = 2.2055 mg/g

0.22%

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Example 40 Loss on Drying

Based on this, Phytochem® has developed suitable methods for determining loss on drying.

1. Method and parameter for the analysis of chitosan-HCl, chitosan and chitosan-HAc

Sample preparation

Pre-treatment of the container: the substance is placed in a suitable weighing bottle, previously dried under the conditions used subsequently.

Filling: the substance is filled no higher than 5 mm.

Transport: Close the weighing bottle with a suitable cap.

Method PC: under high vacuum - modified method of Pharmacopoeia 2.2.32 (EP) under vacuum in a desiccator.

Appliance: desiccator.

Drying time: to constant weight.

Drying temperature: 25°C plus/minus 2°C.

Vacuum: constant 400-800 Pa obtained using special pumps

Desiccant: Diphosphoropentoxide (freshly prepared)

2. Estimation of loss during drying of chitosan in chitosan-valeric acid hydrocolloid (special method).

The content of chitosan in the chitosan-valeric acid hydrocolloid is estimated using a gravimetric measurement.

Device: high-speed circulating vacuum concentrator. Method: Estimation of loss on drying (special method). Measurement conditions Pressure: 500 Pa Temperature: 60°C

Time: 1 week

Expected result: constant mass

Appearance: vitreous mass

Measurement: experimental solution - 4 ml of chitosan-valeric acid hydrocolloid

Repeatability: 10 times

Forming odor: no typical valeric acid odor

Weighing

1 40.20 mg 6 40.10 mg 2 39.80 mg 7 39.90 mg 3 40.20 mg 8 39.60 mg 4 39.90 mg 9 40.20 mg 5 40.10 mg 10 40.40 mg

Mean 40.04 mg

Standard deviation 0.236643191

Relative standard deviation 0.591016961

Dispersion 0.056

Results: Weighing of the dried matter shows good similarity. Based on the measurement data, the content of chitosan in the chitosan-valeric acid hydrocolloid is 1%.

3. Comparison of results

Chitosan, solid Chitosan-HCl, solid Chitosan-HAs, solid Hydrocolloid chitosanvaleric acid Loss on drying 7.2% 7.9% 20.3% - Drying residue - - - 1% Target EP - <10% - -

The active principle should be a hydrocolloid gel. The measured data confirm the structure of the compound.

Example 41 Estimating Osmolarity

An estimate of osmolarity that could be made was an indirect measurement of the decrease in the melting point of the solution.

Instrument: Halbmicro Osmometer Knauer.

Measuring conditions: external cooling system

Range: 0-1600 mOsmol

-

Claims (10)

Method: freezing Analysis Method Calibration with a standard solution of 400 mOsmol/kg: 12.687 g NaCl in 1 liter of water at 20°C. Repeatability: 2 times. Vessel: bottle made of special glass Sample: chitosan-valeric acid hydrocolloid Experimental solution: 1 - no dilution 2 - dilution 1:5 Quantity: 150 µl each. Calibration Sample Specification Set value Measured value Calibration 1 Bi-distilled 0 mOsmol 0 mOsmol water Calibration 2400 mOsmol/kg 400 mOsmol 400 mOsmol Number Measurement Sample measured value 1a Hydrocolloid chitosan-valeric acid 100 mOsmol 16 Hydrocolloid chitosan-valeric acid 110 mOsmol 2a Hydrocolloid chitosan-valeric acid 1:5 20 mOsmol 26 Hydrocolloid chitosan-valeric acid 1:5 20 mOsmol Results: Measurement of the osmolarity of the hydrocolloid chitosan-valeric acid shows a relatively low content. The measured content of osmotically active components can be so low only if there is no solution or suspension of chitosans and valeric acid. The highly viscous gelling compound can only be a hydrocolloid. Result: The above measured data confirm the identity of the proposed substance. CLAIM 1. The use of a composition containing keratinophilic fungi and/or keratinophilic yeast antigenic material for the treatment and/or prevention of hoof and claw diseases in bovids and/or pigs, where the hoof and claw diseases are selected from the group consisting of digital dermatitis, interdigital dermatitis and /or interdigital phlegmon, and the antigenic material contains antigens of keratinophilic fungi and/or keratinophilic yeast. 2. Use of a composition according to claim 1, wherein the bovids are cattle. 3. The use of a composition according to claim 1 or 2, where the antigenic material of keratinophilic fungi and / or yeast contains homogenized inactivated dermatophyte microconidia and / or homogenized inactivated yeast blastospores, and / or antigenic material of dermatophyte microconidia, and / or antigenic material of yeast blastospores, in in particular, where the antigenic material of dermatophyte microconidia and/or the antigenic material of yeast blastospores contains an antigenic material insoluble in aqueous solutions containing a polysaccharide and/or glycopeptide (ANMP), an antigenic material soluble in aqueous solutions containing a polysaccharide and/or glycopeptide (ASMP ), or antigenic exogenous material containing a polysaccharide and/or glycopeptide (AEMP). 4. Use of a composition according to any one of claims 1 to 3, wherein the antigenic material of the keratinophilic fungi and/or keratinophilic yeast originates from Trichophyton species, in particular Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton equinum, Trichophyton sarkisovii, Trichophyton rubrum or Trichophyton mentagrophytes, Microsporum , in particular Microsporum gypseum, Microsporum canis, in particular Microsporum canis var. obesum or Microsporum canis var. distortum, and/or Candida, in particular Candida albicans, in particular, where the antigenic material of keratinophilic fungi is derived from strains of Trichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM28406, Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM- 9471 or Trichophyton rubrum DSM-9472 and/or the keratinophilic yeast antigenic material is derived from Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM9458 or Candida albicans DSM-9459. 5. Use of a composition according to any one of claims 3, 4, wherein the microconidia are swollen and/or have germ tubes. 6. Use of a composition according to any one of claims 3 to 5, wherein the blastospores and/or microconidia have been inactivated with thiomersal, formaldehyde and/or 2-propiolactone. 7. The use of a composition according to any one of claims 1 to 6, wherein the composition further comprises one or more immunomodulatory substances. - 53 040511 8. Use of a composition according to any one of claims 1 to 7, wherein the composition further comprises chitosan modified with valeric acid, lactic acid, para-aminobenzoic acid, glucuronic acid, or valeric acid chloride, or a hydrocolloid containing: (i) from 0.1 to 5% (w/v) of chitosan and from 0.001 to 5% (w/v) of valeric acid or a salt thereof, preferably valeric acid chloride, or (d) from 0.1 up to 5% (wt./wt.) chitosan and from 0.001 to 5% glucuronic acid or (wt./wt.) paminobenzoic acid, or their salts. 9. Use of a composition according to any one of claims 1 to 8, wherein the composition is a pharmaceutical composition, preferably a vaccine. 10. Use of Trichophyton verrucosum DSM-28406 strain, homogenized inactivated microconidia of Trichophyton verrucosum DSM-28406 dermatophyte microconidia or antigenic material containing antigens of Trichophyton verrucosum DSM-28406 strain, including ANMP, AEMP and ASMP of Trichophyton verrucosum DSM-28406 dermatophyte microconidia, for treatment and/or or preventing diseases of the hooves and claws in animals, where the diseases of the hooves and claws are selected from the group consisting of digital dermatitis, interdigital dermatitis and/or interdigital phlegmon. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2