Compositions for the treatment and prevention of hoof and claw diseases

Abstract

The present invention relates to a composition comprising antigenic material of keratinophilic fungi and/or keratinophilic yeasts for use in a method of treating and/or preventing hoof- and claw diseases in animals and a new Trichophyton verrucosum strain which can e.g. be used in such a method of treatment and/or prevention.

Claims

1. A method of treating digital dermatitis, interdigital dermatitis or interdigital phlegmon in a cow comprising administering to said cow an effective amount of antigenic material comprising an antigen of a keratinophilic fungus and/or a keratinophilic yeast.

2. The method of claim 1, wherein the antigenic material of said keratinophilic fungus and/or yeast comprises homogenised inactivated dermatophyte microconidia and/or homogenised inactivated yeast blastospores and/or antigenic material of dermatophyte microconidia and/or antigenic material of yeast blastospores.

3. The method of claim 2, wherein the antigenic material of dermatophyte microconidia and/or antigenic material of yeast blastospores comprise antigenic material, not soluble in aqueous solutions, comprising polysaccharide and/or glycopeptide (ANMP), antigenic material, soluble in aqueous solutions, comprising polysaccharide and/or glycopeptide (ASMP) or antigenic exogenous material comprising polysaccharide and/or glycopeptide (AEMP).

4. The method of claim 1, wherein the antigenic material of keratinophilic fungi and/or keratinophilic yeasts are derived from Trichophyton, Microsporum, and/or Candida.

5. The method of claim 1, wherein the antigenic material of keratinophilic fungi is derived from the strains Trichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM-9471, or Trichophyton rubrum DSM-9472 and/or the antigenic material of keratinophilic yeasts is derived from the strains Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458, or Candida albicans DSM-9459.

6. The method of claim 2, wherein the microconidia are in a swollen condition and/or have germ tubes.

7. The method of claim 2, wherein the blastospores and/or microconidia have been inactivated with thiomersal, formaldehyde and/or 2-propiolactone.

8. The method of claim 1, wherein the composition additionally comprises one or more immunomodulatory substances.

9. The method of claim 1, wherein the composition comprises additionally chitosan modified by valeric acid, lactic acid, para-aminobenzoic acid, glucuronic acid or chloride of valeric acid or a hydro colloid comprising: (i) 0.1% to 5% (w/v) chitosan and 0.001 to 5% (w/v) valeric acid, or a salt thereof, or (ii) 0.1% to 5% (w/w) chitosan and 0.001 to 5% (w/w) glucuronic acid or p-aminobenzoic acid or a salt thereof.

10. The method of claim 1, wherein the composition is a pharmaceutical composition.

11. A composition comprising Trichophyton verrucosum strain DSM-28406, homogenised inactivated dermatophyte microconidia of Trichophyton verrucosum DSM-28406 or antigenic material comprising antigens of Trichophyton verrucosum strain DSM-28406 including ANMP, AEMP and ASMP of dermatophyte microconidia of Trichophyton verrucosum DSM-28406.

12. A vaccine formulation comprising Trichophyton verrucosum strain DSM-28406, homogenised inactivated dermatophyte microconidia of Trichophyton verrucosum DSM-28406 or antigenic material comprising antigens of Trichophyton verrucosum strain DSM-28406 including ANMP, AEMP and ASMP of dermatophyte microconidia of Trichophyton verrucosum DSM-28406.

13. A method of treating digital dermatitis, interdigital dermatitis, or interdigital phlegmon in a cow comprising administering to said cow an effective amount of Trichophyton verrucosum DSM-28406, homogenised inactivated dermatophyte microconidia of Trichophyton verrucosum DSM-28406 or antigenic material comprising antigens of Trichophyton verrucosum strain DSM-28406 including ANMP, AEMP and ASMP of dermatophyte microconidia of Trichophyton verrucosum DSM-28406.

14. The method of claim 4, wherein the antigenic material of keratinophilic fungi and/or keratinophilic yeasts are derived from Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton equinum, Trichophyton sarkisovii, Trichophyton rubrum Trichophyton mentagrophytes, Microsporum gypseum, Microsporum canis, and/or Candida albicans.

15. The method of claim 14, wherein the antigenic material of keratinophilic fungi and/or keratinophilic yeasts are derived from Microsporum canis var. obesum or Microsporum canis var. distortum.

16. The method of claim 11, wherein the valeric acid, is chloride of valeric acid.

Description

EXAMPLE 1

(1) Vaccine Polivac-TM (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) against dermatophytosis of animals was used for the prophylaxis and treatment of digital and/or interdigital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 2

(2) Vaccine Polivac-T (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) against dermatophytosis of cattle was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 3

(3) Dermatophyte culture of the 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.

(4) The fungal masses are lifted off and homogenised in 500 ml deionized water. Then suspension of microconidia is adjusted to 60 million of microconidia per ml with physiological sodium chloride salt solution. The homogenate is inactivated by adding formaldehyde to 0.4% (v/v) in end directly to the cell suspension. The mixture is incubated for 5-7 days at 37° C. The resulting composition is bottled, checked for sterility, safety and amount of microconidia in accordance with usual known methods and can be stored refrigerated at 4-10° C. Vaccine obtainable according to this method was used for the prophylaxis and treatment of interdigital digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 4

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

(6) The fungal masses are lifted off and homogenised in 300 ml deionized water. The concentration of microconidia is adjusted to 80 million per ml for each homogenate. Then suspension of microconidia is adjusted to 40 million of microconidia per ml with distilled water. The homogenate is inactivated by adding formaldehyde to reach 0.5% (v/v) directly to the cell suspension. The mixture is incubated for 5 days at 37° C. The resulting composition is bottled, checked for sterility, safety and amount of microconidia in accordance with usual methods and stored at 4-8° C. Vaccine obtainable according to this method was used for the prophylaxis and treatment of interdigital, digital dermatitis and interdigital phlegmon in cattle as described below.

EXAMPLE 5

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

(8) The fungal masses are lifted off and homogenised in 400 ml deionized water. The concentration of microconidia is adjusted to 70 million per ml for each homogenate. Then suspension of microconidia is adjusted to 60 million of microconidia per ml with distilled water. The homogenate is inactivated by adding formaldehyde to reach 0.3% (v/v) directly to the cell suspension. The mixture is incubated for 7 days at 37° C. The resulting composition is bottled, checked for sterility, safety and amount of microconidia in accordance with usual methods and stored at 4-8° C. Vaccine obtainable according to this method was used for the prophylaxis and treatment of interdigital, digital dermatitis and interdigital phlegmon in cattle as described below.

EXAMPLE 6

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

(10) The fungal mass of the dermatophyte are lifted off and homogenised in 500 ml 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 to 40 million per ml for homogenate. Then the suspension of microconidia is fermented for 1 day at 28° C., until 65% of the microconidia have germ tubes. After fermentation the cell suspensions is washed with physiological solution of sodium chloride. The homogenate is inactivated by adding thiomersal in a ratio of 1:25000 (w/v) directly to the cell suspension. The mixture is incubated for 30 days at room temperature. The resulting composition is bottled, checked for sterility, safety and immunogenic properties in accordance with accepted methods and can be stored refrigerated at 4-10° C. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital, digital dermatitis, interdigital phlegmon and dermatophytosis in cattle as described below.

EXAMPLE 7

(11) A variation of the vaccine Polivac-TM against dermatophytosis of animals comprising in comparision to the classical Polivac-TM no Trichophyton equinum and Microsporum strains (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 8

(12) Vaccine Polivac-TM against dermatophytosis of animals (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 9

(13) Vaccine Polivac-TM against dermatophytosis of animals (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was used for the prophylaxis and treatment of digital and/or interdigital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 10

(14) Vaccine Polivac-T against dermatophytosis of cattle (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 11

(15) Dermatophyte culture of the 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.

(16) The fungal mass is lifted off and homogenised in 500 ml deionized water. The concentration of microconidia is adjusted to 50 million per ml in homogenate. Then suspension of microconidia is adjusted to 50 million of microconidia per ml with distilled water.

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

EXAMPLE 12

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

EXAMPLE 13

(19) A solution of chitosan modified by valeric acid chloride was added to the vaccine Polivac-T against dermatophytosis of cattle (manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) to reach a final concentration of 0.1% (w/v). The concentration of microconidia was 40 million per ml. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 14

(20) Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 is cultivated on agar/wort, for example in 7 Roux flasks. The culture is cultivated for 27 days at 26° C.

(21) The fungal masses of the dermatophyte is lifted off and homogenised in 500 ml an aqueous solution of 0.2% of chitosan modified by paraaminobenzoic acid. The concentration of microconidia is adjusted to 50 million per ml for each homogenate. The homogenate is inactivated by adding formaldehyde to reach an end concentration of 0.5% (v/v) directly to the cell suspension. The mixture is incubated for 7 days at 37° C. The composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon and/or dermatophytosis in cattle as described below.

EXAMPLE 15

(22) The species Candida albicans DSM-9456 is cultivated on malt extract-agar for 6 Roux flasks. Culture is cultivated for 7 days at 35° C. The blastospores are washed off with a physiological solution of sodium chloride.

(23) The fungal masses is lifted off and homogenised in an 500 ml aqueous solution of 0.1% solution of chitosan modified by valeric acid chloride. The concentration of microconidia is adjusted to 80 million per ml in homogenate. The homogenate is inactivated by adding formaldehyde to reach 0.2% (v/v) in end directly to the cell suspension. The mixture is incubated for 7 days at 37° C. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 16

(24) The fraction obtainable according to this process consists of antigenic nonsoluble material comprising polysaccharide and/or glycopeptides (ANMD) as disclosed in WO 97/07232. Dermatophyte culture of the 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.

(25) The resulting fungal biomass is lifted off and treated with an aqueous solution of alkali. Preferred aqueous alkaline solutions are NaOH at concentration of 3% (w/v). Alkaline treatment is preferably performed at 80° C. for up to 6 h. Following the processing under aqueous alkaline conditions, the solid and liquid phases of the preparation are separated by centrifugation at forces of about 3500 g. After alkaline treatment, the solid phase is treated with 0.2 M acetic acid are added to the solid phase for 1 hour at temperatures of 70° C. After acidic treatment the solid phase is washed with distilled water. The washing is repeated three times. Finally, the solid phase is lifted off and homogenised in 500 ml water for injection. The concentration of the particles is adjusted to 60 million per ml end product. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 17

(26) The fraction obtainable according to this process consists of antigenic nonsoluble material comprising polysaccharide and/or glycopeptides (ANMP) as disclosed in WO 97/07232. Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 is cultivated on agar/wort in 9 Roux flasks. The culture is cultivated for 25 days at 27° C. The resulting fungal biomass is lifted off and treated with an aqueous solution of alkali. Preferred aqueous alkaline solutions are KOH at preferred concentrations of 2% (w/v). Alkaline treatment is preferably at 60° C. for up to 10 h. Following the processing under aqueous alkaline conditions, the solid and liquid phases of the preparation are separated by centrifugation at forces of about 3500 g. The treatment under aqueous alkaline conditions was repeated two times, as well as the separation step by centrifugation at forces of about 3500 g. After alkaline treatment, the solid phase is treated with 0.5M HCl is added to the solid phase for 1.5 hours at temperatures of 80° C. After acidic treatment the solid phase is washed with distilled water two times. Finally, the solid phase is lifted off and homogenised in 500 ml an aqueous solution comprising 0.3% chitosan modified by valeric acid chloride. The concentration of particles is adjusted to 60 million per ml for each homogenate. The composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 18

(27) The product is prepared from Chitosan. The product is prepared in two stages. In the first stage a concentrated modified chitosan gel is obtained, in the second stage the final product, which can be used for administration.

(28) The First Step.

(29) Chitosan with deacetylation of 80%, a viscosity of 2751-3250 mPas is used as raw material. 40 grams of the polysaccharide is sterilized by autoclaving and 3.5 liter of water for injection is added under stirring. In the obtained suspension 40 ml of acetic acid are added. The final volume is adjusted with water for injection to 4 liter. Suspended polysaccharide is stirred in a sterile container for 24 hours until a gel suspension is obtained. Unsolved particles are removed by filtration through a metal grid with a pore size of 200 μm-300 μm. 4 N sodium hydroxide (NaOH) is added dropwise to the obtained suspension until the suspension has a pH of 8.0. Upon that white flakes precipitate comprising the chitosan. The suspension is stirred for 30 minutes. 8 mL lactic acid is added dropwise to the suspension under constant stirring. The obtained suspended material is stirred for another hour. Flakes and unsolved particles are separated from the suspension and resuspended in 4 liter water for injection. 4N hydrochloric acid is added under stirring until a pH of 5.6 is reached. The resultant suspension is dialysed in a closed system to remove free ions of salts and low molecular weight compounds. The resultant suspension is a concentrated modified chitosan gel. The modified chitosan concentration is about 0.8% to about 1%. After the dialysis, the polysaccharide is used to prepare the final product.

(30) The Second Step.

(31) For obtaining the final product comprising the modified chitosan obtained in the first step the resultant suspension of the first step is adjusted to a volume of 25 liter by adding sterile water for injection under stirring. Then, 500 ml of chlorocresol solution containing 30 grams chlorocresol are added to the mixture as preservative. The resultant suspension is adjusted to a volume of 30 liter. The resultant sterile product is dispensed into vials under aseptic conditions. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 19

(32) Dermatophyte culture of the 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.

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

EXAMPLE 20

(34) Dermatophyte culture of the species Trichophyton mentagrophytes DSM-7279 is cultivated on agar/wort in 6 Roux flasks. The culture is cultivated for 20 days at 26° C.

(35) The fungal masses of the dermatophyte is lifted off and homogenised in 400 ml an aqueous solution of 0.2% chitosan modified by paraaminobenzoic acid. The concentration of microconidia is adjusted to 250 thousand per ml for each homogenate. The homogenates are inactivated by adding formaldehyde to reach 0.3% (v/v) in end directly to the cell suspension. The mixture is incubated for 6 days at 37° C. Composition obtainable according to this method was used for the prophylaxis and treatment of interdigital and/or digital dermatitis and/or interdigital phlegmon and/or dermatophytosis in cattle as described below.

EXAMPLE 21

(36) Cows with clinical evidence of lameness and lesions of the interdigital space, which are typical for DD, ID and IP, were treated with various compositions. The composition was administered by intramuscular injection two times with an interval of 10 days. The dose for composition was 5 ml for each application.

(37) The results are shown in Table 2.

(38) TABLE-US-00012 Amount of healthy animals Composition Frequency of In 30-35 days In 53-55 days No of as prepared in drug after the first after the first group example No of animals administration application 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 11 2 5 6 9 9 12 2 5 6 10 10 13 2 6 6 11 11 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

(39) No common or local reactions after application of the compositions were observed. Therapeutic efficacies of treatment with different compositions produced as described in Examples 1 to 18 were 40% to 64%.

EXAMPLE 22

(40) Cows with clinical evidence of lameness and lesions of the interdigital space, which are typical for DD, ID and IP, were treated with various compositions. The composition was administered by intracutaneous injection two times with an interval of 7-10 days. The composition dose was 0.4 ml in total which was injected into two sites of the animal.

(41) The results are shown in Table 3.

(42) TABLE-US-00013 Amount of healthy animals Composition as Frequency of In 30-35 days In 53-55 days No of prepared in drug after the first after the first group example No of animals administration application application 1 19 100 2 60 85 2 20 100 2 58 87

(43) No common or local reactions after application of the compositions were observed. Therapeutic efficacy of treatment with different compositions produced according to examples 19 and 20 were about 85% to 87%.

EXAMPLE 23

(44) Cows with clinical evidence of lameness and lesions of the interdigital space, which are typical for DD, ID and IP, were treated with various drugs. The composition prepared in accordance with Example 15 was administered as follows: 2 times intramuscular in a dose of 5 ml at one site with an interval of 10 days.

(45) Clinical Manifestation of Disease:

(46) + Recovering, or gray, no pain

(47) ++ in healing, <2 cm, yellow, light pain

(48) +++ Recovering, or >2 cm, yellow, moderate pain

(49) ++++ acute disease>2 cm, red, significant pain

(50) The results are shown in Table 4.

(51) TABLE-US-00014 Before treatment 30 days after treatment 58 days after treatment 12 animals ++ 4 animals-healthy 11 animals-healthy 4 animals + 3 animals + 6 animals ++ 4 animals +++ 2 animals +++ 3 animals ++ 4 animals ++++ 2 animals ++++ in total 2 animals were in total 3 animals were culled culled for slaughter for slaughter

(52) No common or local reactions after application were observed. Efficacy of treatment was about 70%.

EXAMPLE 24: DOSE TITRATION STUDY

(53) Cows with clinical evidence of lameness, lesions of the interdigital space, which are typical for DD, ID and IP, were treated with various drugs. Therapeutic application of the composition prepared in accordance with Example 14: 3 times intramuscular at one site with an interval of 7 days.

(54) Clinical Manifestation of Disease:

(55) + Recovering, or gray, no pain

(56) ++ In healing, <2 cm, yellow, light pain

(57) +++ Recovering, or >2 cm, yellow, moderate pain

(58) ++++ acute disease>2 cm, red, significant pain

(59) The results are shown in Table 5.

(60) TABLE-US-00015 51 days Before after treatment Before 51 days after treatment treatment Dose - 5 ml treatment Dose - 3 ml 19 5 animals-healthy 18 1 animal-healthy animals +++ 7 animals + animals +++ 4 animals + 5 animals ++ 6 animals ++ 2 animals +++ 2 animals were 5 animals were culled culled for slaughter for slaughter

(61) No common or local reactions after application were observed. Efficacy of treatment with dose 5 ml was about 63% and 3 ml-28%.

EXAMPLE 25

(62) Cows with clinical evidence of lameness and lesions of the interdigital space, which are typical for DD, ID and IP, were treated. Therapeutic application of the composition prepared in accordance with Example 14: intramuscular, 2 times at one site in dose of 5 ml with an interval of 10 days.

(63) Summary of Investigation:

(64) TABLE-US-00016 Before treatment Amount of animals/amount of limbs with lameness 100/138 52 to 54 days after last treatment Amount of animals/amount of limbs with lameness 29/29 Amount of healthy animals 71 Degrease of limbs with lameness 78.99%

EXAMPLE 26—DOSE TITRATION STUDY

(65) Treatment of cows against DD, ID and IP was done. Prophylactic application of a composition prepared in accordance with Example 14: intramuscular, 2 times with an interval of 10 days.

(66) Clinical Manifestation of Disease was Investigated:

(67) + Recovering, or gray, no pain

(68) ++ in healing, <2 cm, yellow, light pain

(69) +++ Recovering, or >2 cm, yellow, moderate pain

(70) ++++ acute disease>2 cm, red, significant pain

(71) The results are shown in Table 6.

(72) TABLE-US-00017 73 days after application Dose 1 ml Dose 2.5 ml Control 100 animals 100 animals 215 animals 7 animals + 7 animals + 21 animals + 18 animals ++ 6 animals +++

(73) No common and local reactions after application of the composition were observed. Efficacy of treatment with doses of 1 ml and 2.5 ml was about 93%. 45 animals (about 21%) from control group were with clinical symptoms of DD, ID and IP.

(74) The results are shown in Table 7.

(75) TABLE-US-00018 107 days after application of composition Dose 1 ml Dose 2.5 ml Control 100 animals 100 animals 215 animals 7 animals + 9 animals + 11 animals + 12 animals ++ 4 animals-culling for 4 animals-culling for 9 animals +++ slaughter slaughter 27 animals-culling for slaughter

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

(77) The results are shown in Table 8.

(78) TABLE-US-00019 170 days after application of composition Dose 1 ml Dose 2.5 ml Control 100 animals 100 animals 215 animals 25 animals + 41 animals + 112 animals + 6 additional animals were 3 additional animals 42 additional animals culled for slaughter were culled for were culled for slaughter slaughter

(79) The efficacy of treatment with doses of 1 ml was about 70% and with doses of 2.5 ml was about 53%. 154 animals (about 72%) from the control group were with clinical symptoms of DD, ID and IP. This investigation demonstrates prophylactic treatment of animals with a dose of 1.0 ml. Duration of immunity was about 5.5 month.

EXAMPLE 27

(80) Treatment of cows against DD, ID and IP was done. Prophylactic application of a composition prepared in accordance with Example 14: intramuscular, 2 times in a dose of 5 ml with an interval of 10 days.

(81) Summary of Investigation:

(82) TABLE-US-00020 Animals in-group 1 were treated Observation before the treatment Amount of animals/clinical manifestations of DD, ID and IP 100/100 160 to 175 days after last treatment Amount of animals/amount of limbs with lameness 100/10 Amount of healthy animals 90 Efficacy of treatment 90% Animals in group 2 were treated with placebo (control) Observation before treatment Amount of animals/clinical manifestations of DD, ID and IP 100/100 160 to 175 days after last application of placebo Amount of animals/amount of animals with manifestations of 100/48  Clinical symptoms of DD, ID and IP Amount of healthy animals 52 Amount of ill animals during of observation time 48%

(83) All animals of the control group (placebo) with clinical symptom of diseases were treated with local application of aseptic medicine or antibiotics. In case of IP the intramuscular injection of antibiotics was used.

EXAMPLE 28

(84) Cows with clinical evidence of lameness and lesions of the interdigital space, which are typical for DD, ID and IP, were treated with various compositions. The composition was administered by intramuscular injection two times at one site with an interval of 10 days. The dose of the composition was 5 ml for each application.

(85) The results are shown in Table 9.

(86) TABLE-US-00021 Amount of healthy animals Frequency of In 30-35 days In 53-55 days No of Composition as No of drug after the first after the first group prepared in example animals administration application application 1 14 10 2 10 10 2 15 10 2 9 9 3 18 10 2 10 10 4 Control - treated 10 — 3 5 by common methods

EXAMPLE 29. HYDRO-COLLOIDS

(87) Chemical nomenclature: Chitosan-Valeric acid-Hydro-Colloid

(88) Subtitle: Polyaminosugar-Valeric acid-Hydrocomplex

(89) Structural formula:

(90) ##STR00002##
(C.sub.6H.sub.11NO.sub.4).sub.x(C.sub.8H.sub.13NO.sub.5).sub.y(C.sub.5H.sub.10O.sub.2).sub.z(HCl).sub.z(H.sub.2O).sub.m  Chemical formula:

(91) General Properties

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

(93) Appearance: natural white to yellowish viscous liquid with typical odor

(94) Solubility: soluble in: Water

(95) Odor: typical, similar to Valeric acid

(96) Density: 1.002

(97) pH-value: 5.5

(98) Storage: Keep protected from light; store in a container protected from air in a refrigerator at 4°-8° C.

(99) Stability: 36 months under conditions described above

(100) Chemical nomenclature: Chitosan-4-Aminobenzoic acid-Hydro-Colloid

(101) Subtitle: Polyaminosugar-p-Aminobenzoic acid-Hydrocomplex

(102) Structural formula:

(103) ##STR00003##
(C.sub.6H.sub.11NO.sub.4).sub.x(C.sub.8H.sub.13NO.sub.5).sub.y(C.sub.7H.sub.7NO.sub.2).sub.z(H.sub.2O).sub.m  Chemical formula:

(104) General Properties

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

(106) Appearance: Yellowish to yellow viscous liquid

(107) Chemical nomenclature: Chitosan-Glucuronic acid-Hydro-Colloid

(108) Subtitle: Polyaminosugar-Glucuronic acid-Hydrocomplex

(109) Structural formula:

(110) ##STR00004##
(C.sub.6H.sub.11NO.sub.4).sub.x(C.sub.8H.sub.13NO.sub.5).sub.y(C.sub.6H.sub.10O.sub.7).sub.z(H.sub.2O).sub.m  Chemical formula:

(111) General Properties

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

(113) Appearance: Yellowish to yellow viscous liquid

EXAMPLE 30. MANUFACTURING OF CHITOSAN-VALERIC ACID-HYDRO-COLLOID

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

(115) Amino-N-acetyl-D-glucosamine is sterilized in a separate vessel and is carried out to obtain Chitosan in pharmaceutical quality.

(116) Reagent Solution

(117) Sterile Amino-N-acetyl-D-glucosamine is resuspended under stirring for 15 minutes in this sterile water. 400 ml of Acetic acid is added to suspension under stirring (24 h) until a clear solution is obtained.

(118) Purification Step

(119) To this solution the 4 N Sodium hydroxide solution is added drop by drop (carefully) to obtain a pH 8.0 to 8.5. The resulting solution precipitates to a white mass. The obtained suspension is stirred not less than 30 minutes. The residue is separated from the liquid phase by filtration.

(120) Resuspension

(121) The precipitate is resuspended in an equal amount of purified (sterile) water (water for injection (Pharm. Eur.)) (401, initial amount). 80 ml of Pentanoyl chloride is measured. Under stirring conditions the Pentanoyl chloride is added drop by drop to the suspension. The obtained suspension is stirred until the solution is clear. 1.6 g Thiomersal is added (40 m/mL). The clear solution is the active ingredient (Hydro-Colloid). The obtained polysaccharide colloid (CVHC) is stored under 4° C. to 8° C. For an end product a aqueous solution is done with defined biological activity.

(122) Overview of the Reaction Steps of Manufacturing

(123) 1. Chitosan+water.fwdarw.suspension

(124) suspension+HAc (24 h).fwdarw.Chitosan-HAc-solution

(125) 2. Purification step

(126) 2.1. Chitosan-HAc-solution+4N NaOH.fwdarw.(pH 8-8.5) Chitosan+NaAc+H.sub.2O

(127) 2.2. Chitosan+NaAc+H.sub.2O.fwdarw.H.sub.2O+NaAc

(128) .fwdarw.Chitosan (solid, purified)
3. Production
Chitosan (solid)+H.sub.2O+Pentanoylchlorid.fwdarw.
Chitosan+Valeric acid+H.sub.2O+HCl.fwdarw.CVHC (Chitosan-Valeric acid-Hydro-Colloid)

(129) The production is a combination of a purification step of the basic material Chitosan and in process reaction with the second reagent Pentanoyl chloride.

(130) This first critical step is the precipitation of Chitosan to obtain the total amount of the purified chitosan in pharmaceutical quality.

(131) In process control: The reaction time and the pH-value are monitored to get a quantitative precipitation.

(132) Test for the pharmaceutical quality of chitosan:

(133) Test for the quality of the intermediate (Chitosan pharm quality)

(134) Solubility in water: A sample of about 250 mg of the precipitate of Chitosan is resuspended in 1 ml of purified water

(135) Target: No solubility can be obtained

(136) Quality: is fulfilled if no reduction of the amount of the solid material can be detected.

(137) Solubility in stronger acids: In parallel same amount of precipitate is suspended in 1 ml HCl (3N)

(138) Target: Total solution

(139) Quality: is fulfilled if a solution of the total amount of the solid material can be detected.

(140) The second critical step is the dissolution process to the active ingredient. The control is done visually: The total amount of the precipitate should be solubilized.

EXAMPLE 31. EXAMINATION ON IDENTITY BY USING UV/VIS-SPECTROSCOPY

(141) Test method according to EUROPEAN PHARMACOPOEIA 2.2.25 was used.

(142) Apparatus: Spectrophotometer Jasco 7800

(143) Conditions of measurement: Bandwidth 2 nm Range 200-600 nm Blank correction with solvent Temperature: 25° C. UV-Cell: 12.5×45 mm semi-micro, 10 mm path length UV-grade silica

(144) Solvent: H.sub.2O

(145) Test solution: An adequate sample of Chitosan HCl, Chitosan-HAc, Chitosan, Chitosan-Valeric acid-Hydro Colloid and valeric acid, respectively was dissolved in the solvent above. This mixture was shacked and afterwards sonified in an ultrasonic bath for 5 min.

(146) The absorption maxima according to the general fundamentals of spectroscopy and the chemical structure with specific chromophore groups and substituents can be expected at: 200 nm for Chitosan HCl, Chitosan-HAc, Chitosan-Valeric acid-Hydro Colloid and valeric acid, respectively

(147) TABLE-US-00022 UV- Chitosan Chitosan Maximum HCI HAc Chitosan CVHC Valeric acid nm 200 200 — 200 211

(148) The absorption maxima of Chitosan could not be analysed since Chitosan is a water insoluble solid, which can also not be solubilized in typical organic solvents.

(149) The comparison of all spectra show no significance or structural modification like aromatic bonds etc. Based on the measured spectra and literature data of the raw materials the measured spectrum corresponds to prospected spectra. Thus, the measured data above confirm the identity of the prospected structure.

EXAMPLE 32: IR-ABSORPTION SPECTROPHOTOMETRY

(150) Test method according to EUROPEAN PHARMACOPOEIA 2.2.24 was used.

(151) For identification of the active principle Chitosan-Valeric acid-Hydro-Colloid a series of IR-spectra of different Chitosan-Derivates are compared with the spectrum of the product and of Valeric acid.

(152) 1. Method and Parameters

(153) Apparatus Infrared-Spectrometer FT/IR 410 Jasco

(154) Range: 4000 cm.sup.−1 to 600 cm.sup.−1

(155) Test sample: 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 KBr is carefully grinded and pressed to a suitable potassium bromide disk, or a film of Chitosan-Valeric acid-Hydro Colloid or NaCl plate for valeric acid

(156) Conditions of Measurement:

(157) Background correction: actual

(158) Temperature 20° C.

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

(160) Result: The measured data above confirms the identity of the tested substances.

(161) 2. Data of the Different IR-Spectra

(162) TABLE-US-00023 Dried Chitosan- Chitosan- Chitosan- valeric acid Base HCl Chitosan-HAc Colloid Valeric 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 1467/1456 1421 1410 1408 1424 1413 1377 1380 1381 1320 1320 1336 1315 1279 1256 1246 1254 1236 1215 1154 1155 1155 1154 1079/1032 1084 1089 1076-1013 1109  897 896  890  926  940

(163) The IR signals of Valerie acid in the active principle are very small to not visible.

(164) Comparison to literature data: Based on the measured spectra and literature data of the raw materials, the measured spectrum of CVHC corresponds to prospected spectrum.

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

EXAMPLE 33. .SUP.13.C-NMR-SPECTROSCOPY ANALYSIS

(166) Test method according to EUROPEAN PHARMACOPOEIA 2.2.33 was used.

(167) a) 13C-NMR-Spectrum of Chitosan

(168) 1. Method and Parameters

(169) Apparatus Bruker AMX 500 AVANCE

(170) Conditions of measurement Scan frequency: 125 MHz for Chitosan, Chitosan HCl, Chitosan HAc, Glucosamin HCl, N-Acetylglucosamin, Chitosan-Valeric acid-Hydro-Colloid, Valerie acid Temperature: 300 K for Chitosan, Chitosan HCl, Chitosan HAc, Chitosan-Valeric acid-Hydro-Colloid, Valerie acid; 301 K for Glucosamin HCl and N-Acetylglucosamin Solvent: D.sub.2O for Chitosan, Chitosan HCl, Chitosan HAc, Glucosamin HCl, N-Acetylglucosamin; DMSO-D6 for Chitosan-Valeric acid-Hydro-Colloid CDCl.sub.3 for Valerie acid Concentration: —for Chitosan, Chitosan HCl, Chitosan HAc, Chitosan-Valeric acid-Hydro-Colloid; approx. 15 mg/0.5 ml for Glucosamin HCl, N-Acetylglucosamin and Valerie acid Calibration: —for Chitosan, Chitosan HCl, Chitosan HAc, Glucosamin HCl, N-Acetylglucosamin DMSO-D6 for Chitosan-Valeric acid-Hydro-Colloid CDCl.sub.3 for Valerie acid
2. Results
a).sup.13C-NMR-Spectroscopy Analysis of Chitosan

(171) Measurement in solution: According to the missing solubility in neutral solvents a measurement in solution is not possible.

(172) Measurement in solid state: A measurement in solid state was not possible. Also after long measurement conditions (time) no acceptable signals appeared.

(173) Result: NMR-Identification of Chitosan is not possible.

(174) b).sup.13C-NMR-Spectroscopy Analysis of Chitosan HCl

(175) TABLE-US-00024 Classification Results [d] (Carbon number) 97.67 C1 76.41/74.80 C5 70.28 C3 64.41 C4 60.11 C6 56.06 C2 [ppm] Target The following characteristic chemical shifts 100 according to the general fundamentals of 70 spectroscopy and the chemical skeleton with 56 substituents can be expected at: General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(176) Result: The measured data above confirms the identity of the tested substance.

(177) c).sup.13C-NMR-Spectroscopy Analysis of Chitosan HAc

(178) TABLE-US-00025 Classification Results [d] (Carbon number) Glucosamine 98.39 C1 skeleton 74.79 C5 — C3 — C4 — C6 — C2 Acetic Acid 23.82 CH.sub.3 180.31 >C═O [ppm] Target The following characteristic chemical shifts 98.39 according to the general fundamentals of spe spectroscopy and the chemical skeleton with 23.82 substituents can be expected at: 180.31 General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(179) Result: The measured data above confirms the identity of the tested substance.

(180) d).sup.13C-NMR-Spectroscopy Analysis of Glucosamin HCl

(181) TABLE-US-00026 Classification Results [d] (Carbon number) 92.94/89.34 C1 76.25 C5 72.28/71.69 C3 69.85/69.77 C4 60.66/60.51 C6 54.62/57.08 C2 [ppm] Target The following characteristic chemical shifts 92.94/89.34 according to the general fundamentals of 60.66/60.51 spectroscopy and the chemical skeleton with 54.62/57.08 substituents can be expected at: General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(182) Comparison to literature data: The measured spectrum corresponds directly to the literature spectra from database.

(183) Result: The measured data above confirms the identity of the tested substance.

(184) e).sup.13C-NMR-Spectroscopy Analysis of N-Acetylglucosamin

(185) TABLE-US-00027 Classification Results [d] (Carbon number) 95.06/90.95 C1 76.01/74.08 C5 71.64/70.86 C3 70.22/69.99 C4 60.89/60.74 C6 56.90/54.26 C2 22.29/22.03 CH.sub.3 174.85/174.59 C═O Target The following characteristic chemical shifts 95.06/90.95 according to the general fundamentals of 22.29/22.03 spectroscopy and the chemical skeleton with 174.85/174.59 substituents can be expected at: General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(186) Result: The measured data above confirms the identity of the tested substance.

(187) f).sup.13C-NMR-Spectroscopy Analysis of Chitosan-Valeric Acid-Hydro-Colloid

(188) TABLE-US-00028 Classification Results [d] (Carbon number) Glucosamine 100.95 C1 skeleton 78.57 C5 76.10 C3 73.10 C4 61.40 C6 57.57 C2 Valeric acid 180.66  C5{acute over ( )} 29.23  C4{acute over ( )} 24.85  C3{acute over ( )} 23.37  C2{acute over ( )} 14.87  C1{acute over ( )} [ppm] Target The following characteristic chemical shifts 100.95 according to the general fundamentals of 61.40 spectroscopy and the chemical skeleton with 57.57 substituents can be expected at: 180.66 14.87 General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(189) Result: The measured data above confirms the identity of the proposed structure.

(190) g).sup.13C-NMR-Spectroscopy Analysis of Valeric Acid

(191) TABLE-US-00029 Classification Results [d] (Carbon number) 180.5 C5 33.8 C4 26.7 C3 22.2 C2 10.6 C1 [ppm] Target The following characteristic chemical shifts 180 according to the general fundamentals of 10.6 spectroscopy and the chemical skeleton with substituents can be expected at: General Literature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

(192) Comparison to literature data: The measured spectrum corresponds directly to the literature spectra from database.

(193) Result: The measured data above confirms the identity of the tested substance.

(194) 3. Comparison of the NMR Spectra

(195) TABLE-US-00030 Gluco- N- Chitosan- Classification Chitosan Chitosan samin Acetyl- Valeric acid- Valeric (Carbon HCl HAc HCl glucosamin Hydro- acid number) 97.7 98.4 92.9/89.3 95.0/91.0 101.0 C 56.1 54.6/57.1 54.3/56.9 57.6 C 70.3 71.7/72.3 71.6/70.9 76.1 C 64.4 69.9/69.8 70.2/70.0 73.1 C 76.4/74.8 74.8 7 76.0/74.1 78.6 C 60.1 60.5/60.7 60.9/60.7 61.4 C 22.7 23.8 180.3 174.9/174.6 14.9 13.0 23.4 22.0 24.9 26.0 29.2 33. 108.7 180. Literature — — X — — X

(196) Comparison to literature data: Not available or Based on the measured spectra and literature data of the raw materials, the measured spectrum corresponds directly to prospected spectra.

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

EXAMPLE 34. TLC-METHOD FOR THE ANALYSIS OF CHITOSAN AND IMPURITIES IN THE NEW PRODUCT CHITOSAN-VALERIC ACID-HYDRO-COLLOID (CVHC)

(198) Test method according to EUROPEAN PHARMACOPOEIA 2.2.27 was used.

(199) This part presents the procedures and data of thin layer chromatography for the identification of CVHC along with the Rf values in the used solvent mixtures and spot colors when detected under UV-light (365 nm and 254 nm), visible light and with typical visualisation reagents.

(200) The original based raw material for any kind of glucosamines is the natural material Chitin from insects or crabs. The monomeric structure of these biopolymers is N-Acetyl-Glucosamine.

(201) For pharmaceutical and other use in most cases deacetylated Chitin is typical. This resulting biopolymer is the so called Chitosan, which can be modified into water soluble ionic compounds. The monomeric structure of this Chitosan should be theoretically Glucosamine. Because the deacetylation step does not run totally, Chitosan has a mixed structure of N-Acetyglucosamine (acetylated) and Glucosamine (deacetylated) units. Chitosan-Valeric acid-Hydro-Colloid is a new Polyaminosugar-valeric acid hydro-complex. Therefore no positive analytical test results for N-Acetyl-Glucosamine and Glucosamine should be possible. If monomeric fragments are embedded as residual impurities, it should be possible to identify Chitosan in form of its water soluble ionic compounds Chitosan HCl and Chitosan HAc.

(202) 1. Method

(203) TABLE-US-00031 Apparatus Camag Chromatographic Tank System TLC-plate Merck Si 60 F 254 precoated plates Conditions Protected from sunlight and with chamber saturation Temperature 20-25° C. Development: Vertical development
Chromatographic Conditions

(204) TABLE-US-00032 Sample-solution See the single analytes Application 30 μl Drying Min. 2 minutes in an air-stream Motion range 80 mm
Mobile Phase

(205) TABLE-US-00033 Solvents Acetone Water 25% aq. Ammonia — Mixture 20 10 5 —

(206) 2. Analysis and Results

(207) a) Chitosan

(208) Sample Preparation

(209) Sample: Chitosan suspended in water

(210) 1) Apparatus: reflux condensor

(211) Conditions: heating for about 30 minutes under reflux (145° C.)

(212) 2) Apparatus: Ultra sonic bath

(213) Conditions: Sonification for about 30 minutes at 45° C.

(214) 3) Apparatus: reflux condensor

(215) Conditions: heating for about 30 minutes under reflux (145° C.)

(216) 4) Filtration: 0.45 μm filter

(217) The clear filtrate was used for analysis.

(218) Detection with UV-Fluorescence and VIS

(219) TABLE-US-00034 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(220) Detection with Visualisation Reagents

(221) TABLE-US-00035 Group Group Anisaldehyde- specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound signal No No No No Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value No signal for chitosan can be identified — Non specified impurities: Not detected

(222) Alternative: Solubilization in organic solvents show equal results because of the missing solubility of Chitosan.

(223) Result: An acceptable solution of Chitosan in waterish or organic solvents like Methanol etc. is not possible. A suitable solubilization of Chitosan is only possible in stronger acids like HCl or HAc under production of Chitosan HCl or Chitosan HAc.

(224) b) Chitosan HCl

(225) 5 mg Chitosan HCl/ml H.sub.2O was used for analysis.

(226) Detection with UV-Fluorescence and VIS

(227) TABLE-US-00036 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(228) Detection with Visualisation Reagents

(229) TABLE-US-00037 Group specific Group Anisaldehyde- reagent 1 specific Sulfuric acid Visible light reagent 2 reagent Iodine Compound No No Grey spot Brown spot signal Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value Chitosan HCl 0.0 Non specified impurities: Not detected Target Compound purity One main spot Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impurities should show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromato-graphic conditions for such an polymer can be expected at: 0.0 — —

(230) Result: Compound purity; One main spot.

(231) c) Chitosan HAc

(232) 5 mg Chitosan HAc/ml H.sub.2O was used for analysis.

(233) Detection with UV-Fluorescence and VIS

(234) TABLE-US-00038 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(235) Detection with Visualisation Reagents

(236) TABLE-US-00039 Group Group Anisaldehyde- specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound signal No No Grey spot Brown spot Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value Chitosan HAc 0.0 Non specified impurities: Not detected Literature Value Not available data from — Target Compound purity One main spot Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non- selective reagents for detection of non-specified impurities should show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromatographic conditions for such an polymer can be expected at: 0.0 — —

(237) Result: Compound purity; One main spot.

(238) d) Glucosamine HCl

(239) 5 mg Glucosamine HCl/ml H.sub.2O was used for analysis.

(240) Detection with UV-Fluorescence and VIS

(241) TABLE-US-00040 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(242) Detection with Visualisation Reagents

(243) TABLE-US-00041 Group Anisaldehyde- Group specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound Blue spot Red spot Grey spot Brown spot signal Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value Glucosamine HCl 0.67 Non specified impurities: Not detected Literature Not data from — Value available Target Compound One main spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impuritiesshould show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromato-graphic conditions can be expected between 0.6 and 0.8

(244) Result: Compound purity; One main spot.

(245) e) N-Acetylglucosamine

(246) 5 mg N-Acetylglucosamine/ml H.sub.2O was used for analysis

(247) Detection with UV-Fluorescence and VIS

(248) TABLE-US-00042 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(249) Detection with Visualisation Reagents

(250) TABLE-US-00043 Group Anisaldehyde- Group specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound Blue spot No Grey spot Brown spot signal Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value N-Acetylglucosamine 0.72 Non specified impurities: Not detected Target Compound One main spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impurities should show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromato-graphic conditions can be expected between 0.6 and 0.8

(251) Result: Compound purity; One main spot.

(252) f) Chitosan-Valeric acid-Hydro-Colloid (CVHC)

(253) CVHC is a high viscous waterish gel. Two drops of CVHC was used for analysis.

(254) Detection with UV-Fluorescence and VIS

(255) TABLE-US-00044 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(256) Detection with Visualisation Reagents

(257) TABLE-US-00045 Group Anisaldehyde- Group specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound No No Grey spot Brown spot signal Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value Chitosan-Valeric acid-Hydro-Colloid 0.0 Non specified impurities: Not detected Literature Not available data from — Value Target Compound One main spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impurities should show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromato-graphic conditions for such an polymer can be expected at: 0.0 — —

(258) Result: Compound purity; One main spot.

(259) g) Valeric Acid

(260) 1 μl Valeric acid (pure) was used for analysis.

(261) Detection with UV-Fluorescence and VIS

(262) TABLE-US-00046 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(263) Detection with Visualisation Reagents

(264) TABLE-US-00047 Group Anisaldehyde- Group specific specific Sulfuric acid Visible light reagent 1 reagent 2 reagent Iodine Compound No No No Yellow spot signal Impurities No No No No Group specific reagent 1: Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5% Ninhydrine/EtOH Rf-value Valeric acid 0.0 Non specified impurities: Not detected Literature Not available data from — Value Target Compound One main spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impurities should show no greater impurities A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromato-graphic conditions can be expected between 0.0 — —

(265) Result: Compound purity; One main spot.

(266) 3. Comparison of the Results of the TLC Analysis

(267) TABLE-US-00048 Gluco- N-Acetyl- Chitosan- Chitosan Chitosan samine glucoamine Valerie Valerie Chitosan HCl HAc HCl HCl colloid acid Rf-value Not 0 0 0.67 0.72 0 0 possible Detection Compound signal UV 254 nm — — — — — — — UV 365 nm — — — — — — — Visible light — — — — — — — Naturstoff-Reagent — — — Blue spot Blue spot — — Ninhydrine — — — Red spot — — — Reagent Anisalde- — Grey Grey Grey Grey Grey — hyde-Sulfuric spot spot spot spot spot acid reagent Iodine — Brown Brown Brown Brown Brown Yellow Reagent spot spot spot spot spot Spot

(268) The results above from TLC show that there is no evidence of monomeric or dimeric structure which could be detected with the specific derivation reagents tested above. The detection and the Rf value of “0” show the similarity of Chitosan-Valeric acid-Hydro-Colloid to the related compounds Chitosan HCl and Chitosan HAc. A specific identification of Valeric acid with this TLC-System failed. Chitosan-Valeric acid-Hydro-Colloid can only be a Poly-Amino-sugar-colloid, but not a solution of Chitosan or a Chitosan derivate with Valeric acid in water.

EXAMPLE 35. TLC-METHOD FOR THE ANALYTICAL DETECTION OF VALERIC ACID IN CHITOSAN-VALERIC ACID-HYDRO-COLLOID

(269) 1. Method and Parameters

(270) A new TLC system was established for a identification and purity testing of the constituent Valeric acid.

(271) TABLE-US-00049 Apparatus Camag Chromatographic Tank System TLC-plate Merck Si 60 F. 254 precoated plates Conditions Protected from sunlight and with chamber saturation Temperature 20-25° C. Development: Vertical development

(272) Chromatographic Conditions

(273) TABLE-US-00050 Drying Min. 2 minutes in an air-stream Motion range 80 mm

(274) Mobile Phase

(275) TABLE-US-00051 Solvents Ethyl Acetate — — — Mixture 100 — — —
2. Results
a) Valeric Acid (Pure)

(276) 2 μl of Valeric acid (pure) was used for analysis.

(277) Detection with UV-Fluorescence and VIS

(278) TABLE-US-00052 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(279) Detection with Visualisation Reagents

(280) TABLE-US-00053 Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagent Iodine Compound signal Yellow spot/blue Pink spot Yellowish background spot Impurities No No No Group specific reagent: Bromcresol Green/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.] Rf-value Valeric acid 0.56 Non specified impurities: Not detected Literature Value Not available data from —

(281) Detection limit: of valeric acid with this visualisation reagent after TLC-chromatography: 0.03 μg

(282) b) Chitosan-Valeric Acid-Hydro-Colloid (CVHV)

(283) 45 μl Chitosan-Valeric acid-Hydro-Colloid, pure (this is an about 850 times higher amount of valeric acid, compared with the tests before) was used for analysis.

(284) Detection with UV-Fluorescence and VIS

(285) TABLE-US-00054 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(286) Detection with Visualisation Reagents

(287) TABLE-US-00055 Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagent Iodine Compound signal Blue spot/blue Grey spot Brown spot background Impurities No No No Group specific reagent: Bromcresol Green/Bromphenol Blue/Potassium Permanganate Reagent [Pork et al.] Target Compound One main spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selective reagents for detection of non-specified impurities should show no greater impurities The Group specific reagent Bromcresol Green/ Bromphenol Blue/Potassium Permanganate Reagent should show typical results for the compounds A relative retardation factor (Rf) of this compound according to the chemical skeleton under this described chromatographic conditions can be expected at 0.0 for Chitosan-derivatives appr. 0.6 for valeric acid if available Rf-value Chitosan-Valeric acid-Hydro-Colloid 0.0 Non specified impurities: Not detected Rf-value Valeric acid Not detected Non specified impurities: Not detected Literature Value Not available data from —

(288) Detection limit of valeric acid with this visualisation reagent after TLC-chromatography: 0.03 μg

(289) Pure Valeric acid can be identified with this TLC-System. Colloidal integrated Valeric acid can not be detected in the pure compound Chitosan-Valeric acid-Hydro-Colloid. The detection and the Rf value of “0” show the similarity of Chitosan-Valeric acid-Hydro-Colloid to other related Chitosan compounds. Chitosan-Valeric acid-Hydro-Colloid can only be a Poly-Amino-sugar colloid, but not a solution of Chitosan or a Chitosan derivate with Valeric acid in water. The results above confirm the identity of the proposed structure.

EXAMPLE 36. ELIMINATION OF VALERIC ACID FROM CHITOSAN-VALERIC ACID-HYDRO-COLLOID WITH HIGH VACUUM AND HIGHER TEMPERATURE

(290) Method: Estimation of the loss on drying (special method)

(291) Apparatus: Speed circulating vacuum concentrator

(292) Conditions: 5 mbar

(293) Temperature: 60° C.

(294) Time: 1 week

(295) End point: Constant mass

(296) Appearance: Glassy mass

(297) Result Odor: No typical odor from valeric acid

(298) Sample Preparation

(299) Redissolution partly with water

(300) Appearance: High viscous gel

(301) TLC-Analysis

(302) Apparatus: Camag Chromatographic Tank System

(303) TLC-plate: Merck Si 60 F 254 precoated plates

(304) Conditions: Protected from sunlight and with chamber saturation

(305) Temperature: 20-25° C.

(306) Development: Vertical development

(307) Chromatographic Conditions

(308) Sample-solution See above

(309) Application: 5 μl

(310) Drying: Min. 2 minutes in an air-stream

(311) Motion range: 80 mm

(312) Mobile Phase

(313) TABLE-US-00056 Solvents Ethyl Acetate — — — Mixture 100 — — —

(314) Detection with UV-Fluorescence and VIS

(315) TABLE-US-00057 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(316) Detection with Visualisation Reagents

(317) TABLE-US-00058 Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagent Iodine Compound signal Blue spot/blue grey spot brown spot background Impurities No No No Group specific reagent: Bromcresol Green/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.] Rf value spot 0

(318) Detection limit: of Valeric acid with this visualization reagent after TLC-chromatography: 0.03 μg

(319) Result: With high vacuum and higher temperature a disproportion of Chitosan-Valeric acid-Hydro-Colloid takes place. The elimination of Valeric acid can be shown by absolutely no typical odor from Valeric acid. The elimination of Valeric acid can be shown by TLC analysis: no typical spot of free valeric acid at Rf-value 0.56. Chitosan or Chitosan compounds can be identified at Rf-value 0. Chitosan-Valeric acid-Hydro-Colloid can only be a Poly-Amino-sugar-colloid, but not a solution of Chitosan or a Chitosan derivate with valeric acid in water.

EXAMPLE 37. DISPROPORTION OF CHITOSAN-VALERIC ACID-HYDRO-COLLOID WITH SOLVENTS

(320) The structure of Chitosan-Valeric acid-Hydro-Colloid is decomposed in Ethyl acetate to Valeric acid and a Chitosan compound.

(321) Sample Preparation

(322) Apparatus: separating funnel, evaporator Liquid-liquid distribution: 20 ml Chitosan-Valeric acid-Hydro-Colloid and 10 ml Ethyl acetate Conditions: Shaking for about 5 minutes and wait for phase separation Separation of phases: The ethyl acetate phase was collected Concentration step: The about 10 ml were concentrated to liquid residue (waterish) with an evaporator Resolubilization: in 1 ml Methanol Homogenization: Centrifugation step about 5 min 12.000 rpm Phase separation: Upper phase: clear methanolic solution Lower phase: high viscous gel
TLC Analysis of Upper and Lower Phase (See Above)
a) Analysis of upper phase (clear methanolic solution)
TLC-Analysis

(323) Apparatus: Camag Chromatographic Tank System

(324) TLC-plate: Merck Si 60 F 254 precoated plates

(325) Conditions: Protected from sunlight and with chamber saturation

(326) Temperature: 20-25° C.

(327) Development: Vertical development

(328) Chromatographic Conditions

(329) Sample-solution: clear methanolic solution

(330) Application: 5 μl

(331) Drying: Min. 2 minutes in an air-stream

(332) Motion range: 80 mm

(333) Mobile Phase

(334) TABLE-US-00059 Solvents Ethyl Acetate — — — Mixture 100 — — —

(335) Detection with UV-Fluorescence and VIS

(336) TABLE-US-00060 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(337) Detection with Visualisation Reagents

(338) TABLE-US-00061 Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagent Iodine Compound signal Yellow spot/blue No Light background yellowish spot Impurities No No No Group specific reagent: Bromcresol Green/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.] Rf-value Valeric acid 0.57 Non specified impurities: —

(339) Detection limit with visualisation reagent: 0.03 μg

(340) Result: The upper phase is a clear methanolic solution. Valeric acid can be identified after decomposition of the Hydro-Colloid in this solution with TLC. No Chitosan or Chitosan compound can be detected with TLC.

(341) b) Analysis of Lower Phase (High Viscous Gel)

(342) TLC-Analysis

(343) Apparatus: Camag Chromatographic Tank System

(344) TLC-plate: Merck Si 60 F 254 precoated plates

(345) Conditions: Protected from sunlight and with chamber

(346) Temperature: 20-25° C.

(347) Development: Vertical development

(348) Chromatographic Conditions

(349) Sample-solution: high viscous gel, totally redissolved in water

(350) Application: 30 μl

(351) Drying: Min. 2 minutes in an air-stream

(352) Motion range: 80 mm

(353) Mobile Phase

(354) TABLE-US-00062 Solvents Ethyl Acetate — — — Mixture 100 — — —

(355) Detection with UV-Fluorescence and VIS

(356) TABLE-US-00063 Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No No Impurities No No No

(357) Detection with Visualisation Reagents

(358) TABLE-US-00064 Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagent Iodine Compound signal Blue spot/blue Grey spot Brown spot background Impurities No No No Group specific reagent: Bromcresol Green/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.] Rf-value Spot 0 Non specified impurities: —

(359) Detection limit with visualisation reagent: 0.03 μg

(360) Results: The lower phase is a high viscous gel, soluble in water. No Valeric acid can be detected in this phase by TLC. Chitosan or a Chitosan compound can be identified in the lower phase (gel) by TLC.

(361) TABLE-US-00065 Results A disproportion of Chitosan-Valeric acid-Hydro-Colloid is from TLC possible with typical solvents like Ethyl acetate and analysis afterwards with Methanol A re-solubilization of from disproportioned Chitosan-Valeric acid-Hydro- Colloid can be realized with Methanol The decomposition of Chitosan-Valeric acid-Hydro-Colloid in Ethyl acetate shows two phases Upper phase Lower phase Ethyl acetate phase Aqueous Colloid residue After concentration the Ethyl acetate phase was redissolved in Methanol and results also two phases Upper phase Lower phase clear methanolic solution high viscous gel This gel can be re-dissolved totally in water can be identified No can be identified No Chitosan or Chitosan Chitosan or a Chitosan compound can be detected compound can be detected

(362) Summary of the Results

(363) TABLE-US-00066 Chitosan-Valeric acid- Chitosan- Hydro-Colloid Valeric Elimination (decomposed with Ethyl Acid-Hydro- of from acetate) Colloid CVHC with Upper Lower Valeric acid (pure high phase phase pure CVHC) vacuum (Methanol) (Water) Rf-value 0.56 + − − + − Rf-value 0 − + + − + Detection Compound signal UV 254 nm — — — — — UV 365 nm — — — — — Visible light — — — — — Anisaldehyde-Sulfuric Pink spot Grey spot Grey spot — Grey spot acid reagent Iodine reagent Yellowish Brown Brown Light Brown spot spot spot yellowish spot spot Bromcresol Green Yellow spot Blue spot Blue spot Yellow spot Blue spot Bromphenol Blue reagent

(364) Result: Chitosan-Valeric acid-Hydro-Colloid can only be a Poly-Amino-sugar-colloid, but not a solution of Chitosan or a Chitosan derivate with valeric acid in water. The results above confirm the identity of the proposed structure.

EXAMPLE 38. ESTIMATION OF THE RELATIVE DENSITY

(365) Because of the high viscosity of Chitosan-Valeric acid-Hydro-Colloid the estimation of the density is not possible with a density bottle/pycnometer according to Test method according to EUROPEAN PHARMACOPOEIA 2.2.5.

(366) 1. Test Method by Weighing

(367) Apparatus: 250 ml volumetric flask Balance: Sartorius MC 1 LC 2200S

(368) Thermometer: Thermometer with graduation (min 0.5° C.) and a range not more than 60° C.

(369) Results 1.001 [d.sub.20.sup.20]

(370) The active principle is a hydrogel, so the theoretical density should be higher than 1.0. The measured data confirms the identity of the proposed substance.

(371) 2. Test Method with Hydrometer

(372) Test method according to EUROPEAN PHARMACOPOEIA 2.2.5 was used.

(373) Apparatus: 250 ml volumetric flask

(374) Hydrometer: Widder 1573°, 20° C.-M100-DIN 12791 Klasse H

(375) Thermometer with graduation (min 0.5° C.) and a range not more than 60° C.

(376) Conditions of measurement: Temperature 20+/−0.5° C. with electronic thermostate

(377) Results 1.002 [d.sub.20.sup.20]

(378) The active principle is a hydrogel, so the theoretical density should be higher than 1.0. The measured data confirms the identity of the proposed substance.

EXAMPLE 39. SULPHATED ASH

(379) The Test method according to EUROPEAN PHARMACOPOEIA 2.4.14 was used.

(380) Testing

(381) TABLE-US-00067 Apparatus Suitable crucible (porcelain or platinum) were ignited at 600 +/− 50° C. for 30 min in a “Muffel”-oven allow to cool in a desiccator over silica gel or other suitable desiccant Estimation of crucible weight Weight Weight of crucible 1: 52.0120 [g] Weight of crucible 2: 57.6055 [g] Method 2 Additional for this Hydrogel a concentration step to dryness (acid insoluble ash) was done by drying at 105° C. in an normal oven Sample: 25 ml_ of Hydrogel CVHC Usually 1-2 g Sample weight: usually 1-2 g or sufficient amount to obtain a residue of minimum 1 g. Moisten the sample with a small amount of sulfuric acid R [95-97% m/m] (usuallyl ml_) and heat at as low temperature as practicable until the residue is charred. After cooling, moisten the residue with a small amount of sulfuric acid R [95-97% m/m](usuallyl ml_) Heat until white fumes are no longer evolved Ignite at 600 +/− 50° C. for 30 min until the residue is completely incinerated. Flames are not allowed to be produced at any time during the procedure allow to cool in a desiccator over silica gel or other suitable desiccant Weigh and calculate the percentage of residue Weighting of total Total Weight of crucible 1: 52.0668 g weight Total Weight of crucible 2: 57.6612 g Sulphated ash Value 1: 0.0548 g content Value 2: 0.0557 g Average: 0.05525 g/25 ml.sub.— Calculation of content of 0.05525 g/25.05 g = 0.0022055 g/g = sulphated ash 2.2055 mg/g 0.22%

EXAMPLE 40. LOSS ON DRYING

(382) Based on this Phytochem® established appropriate methods for the determination of loss on drying.

(383) 1. Method and Parameter for Test of Chitosan HCl, Chitosan and Chitosan HAc

(384) Sample Preparation

(385) Pretreatment of container: The substance is placed in a suitable weighing bottle, previously dried under the conditions used afterwords

(386) Filling: the material is filled not higher than 5 millimeter

(387) Transport: The weighing bottle is closed with a suitable cover

(388) PC-method: A “under higher vacuum” modified Pharmacopoeia-method 2.2.32 (EP) “in vacuum in a desiccator”

(389) Apparatus: desiccator

(390) Drying time: to constant weight

(391) Drying temperature: 25° C.±2° C.

(392) Vacuum: permanent 4-8 mbar with specific pumps

(393) Drying reagent: Diphosporuspentoxide (freshly)

(394) 2. Estimation of the Loss on Drying of Chitosan in Chitosan-Valeric Acid-Hydro-Colloid (Special Method)

(395) The content of Chitosan in Chitosan-Valeric acid-Hydro-Colloid is estimated with a gravimetric measurement.

(396) Apparatus: Speed circulating vacuum concentrator

(397) Method: Estimation of the loss on drying (special method)

(398) Conditions of Measurement

(399) Pressure: 5 mbar

(400) Temperature: 60° C.

(401) Time: 1 week

(402) End point: Constant mass

(403) Appearance: Glassy mass

(404) Measurement: Test solution 4 ml Chitosan-Valeric acid-Hydro-Colloid

(405) Repetition: 10 times

(406) Result Odor: No typical odor from valeric acid

(407) Weighing

(408) TABLE-US-00068 1 40.20 mg 2 39.80 mg 3 40.20 mg 4 39.90 mg 5 40.10 mg 6 40.10 mg 7 39.90 mg 8 39.60 mg 9 40.20 mg 10 40.40 mg

(409) Average 40.04 mg

(410) Standard deviation 0.236643191

(411) Relative standard deviation 0.591016961

(412) Variance 0.056

(413) Results: The weighing of the dried substance shows good similarity. Based on this measurements the content of Chitosan in Chitosan-Valeric acid-Hydro-Colloid is 1%.

(414) 3. Comparison of the Results

(415) TABLE-US-00069 Chitsoan Chitsoan Chitosan- Chitsoan HCI HAc Valeric acid- solid solid solid Hydro-Colloid Loss on drying 7.2%  7.9% 20.3% . Residue from . . . 1% drying Target EP . <10% . .

(416) The active principle should be a Hydro Colloid gel. The measured data confirm the structure of compound.

EXAMPLE 41. ESTIMATION OF THE OSMOLARITY

(417) The estimation of the Osmolarity can be done was an indirect measurement of the decrease of the melting point of a solution.

(418) Apparatus: Halbmicro Osmometer Knauer

(419) Conditions of measurement: External cooling system

(420) Range: 0-1600 mOsmol

(421) Method: Freezing

(422) Test Procedure

(423) Calibration with Standard solution 400 mOsmol/Kg: 12,687 g NaCl in 1 l Wasser at 20° C.

(424) Repetition: 2 times

(425) Vessel: Specific glass vial

(426) Sample: Chitosan-Valeric acid-Hydro-Colloid

(427) Test solution: 1 without dilution 2 Dilution of 1:5

(428) Quantity 150 μl each

(429) Calibration

(430) TABLE-US-00070 Sample Spezification Setpoint Measured value Calibration 1 Bidest. water  0 mOsmol  0 mOsmol Calibration 2 400 m Osmol/kg 400 mOsmol 400 mOsmol

(431) Measurement

(432) TABLE-US-00071 Number Sample Measured value 1a Chitosan-Valeric acid-Hydro-Colloid 100 mOsmol 1b Chitosan-Valeric acid-Hydro-Colloid 110 mOsmol 2a Chitosan-Valeric acid-Hydro-Colloid 1:5 20 mOsmol 2b Chitosan-Valeric acid-Hydro-Colloid 1:5 20 mOsmol

(433) Results: The measurement of the Osmorarity of Chitosan-Valeric acid-Hydro-Colloid show a relatively low content. The measured content of osmolar reacting components can only be so low, if there is no solution or suspension of chitosans and valeric acid. The high viscous gelling compound can only be a Hydro-Colloid.

(434) Result: The measured data above confirms the identity of the proposed substance.