JPH07233086A - Agent for treatment of animal dermatosis - Google Patents

Abstract

PURPOSE:To obtain an agent for the treatment of animal dermatosis, free from side action and safely handleable by the user. CONSTITUTION:This agent for the treatment of animal dermatosis contains at least 3wt.% of lactoferrins, hydrolyzed lactoferrins, peptides separated from the hydrolyzates, synthetic peptides having the same amino acid sequence as these peptides or a mixture of two or more of the above substances and at least 1wt.% of lactoperoxidase as active components.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a therapeutic agent for animal skin. More specifically, the present invention relates to a safe and effective therapeutic agent for external use against diseases that occur on the skin of companion mammals, livestock and the like.

[0002]

2. Description of the Related Art Lactoferrin is a natural iron-binding protein with a molecular weight of about 80,000, which is contained in tears, saliva, peripheral blood, milk (about 200 mg / l for milk), etc. in vivo, and is used in Escherichia coli and Candida. Exhibiting antibacterial action against harmful microorganisms such as bacteria and Clostridium (Cited document 18),
And for Staphylococcus and Enterococcus 0.5-30m
It is known to have an antibacterial effect at a concentration of g / ml (Cited document 19).

Further, lactoferrin has an antibacterial action and
Cell growth promotion, lipid peroxide production suppression, function control of immune related cells, iron absorption promotion, histamine release suppression, anti-inflammatory, inhibition of intestinal adherence of Escherichia coli, antiviral action (cited reference 20) and antioxidant action (cited It is known to have reference 21). In addition, peptides having various physiological effects are known, and many peptides having antibacterial activity against microorganisms have been reported (Cited Documents 23 to 3).
9).

The inventors of the present invention have also found that a degradation product obtained by hydrolyzing lactoferrin with a protease, a peptide isolated from this degradation product, and a synthesized peptide having the same amino acid sequence as these peptides, It has been found that it has antibacterial activity against various microorganisms, and has already applied for a patent (References 40 to 45). On the other hand, peroxidase, which is an enzyme that catalyzes the reaction of passing one oxygen atom from hydrogen peroxide to its acceptor molecule, is contained in wasabi, fig juice, mammalian leukocytes, milk, etc. Especially, the one that is present in milk (about 30 mg / l in milk) is called lactoperoxidase. Lactoperoxidase is a heme protein having a molecular weight of about 82,000 and having a porphyrin-like structure, and is known to exhibit an antibacterial action in the presence of thiocyanate ion and hydrogen peroxide (or its precursor) (cited document Two
2), called LP system or LPO system.

Due to the above antibacterial action and the like, lactoferrin is used in foods and drinks, cosmetics, feed and the like (Cited documents 4 to 12). Similarly, lactoperoxidase is also used for food and drink, feed, etc. (Cited document 1).
3-16). Further, the lactoperoxidase is
It has been found that lactoferrin enhances the enzymatic activity (Cited document 17), and JP-A-61-83131 discloses that lactoferrin and an LP system are contained at the same time, and they have an antibacterial action in the gastrointestinal tract. A product or a feed is disclosed, and Japanese Patent Application Laid-Open No. 5-92927 discloses that lactoferrin and lactoperoxidase can be used for the prevention and treatment of pathogenic bacteria infection in cultured aquatic animals.

However, it has not hitherto been known that a mixture of lactoferrin and lactoperoxidase is effective for treating skin diseases in animals, and there is no publication describing such a fact. Diseases that occur on the skin of animals include eczema, urticaria, allergic dermatitis, pruritic dermatitis, purulent dermatitis, dermatophytosis, scabies, otitis externa,
There are ear dermatitis, trichophyton, and parasitic skin diseases. Conventionally, the following drugs have been used to treat these diseases. Disinfectant for outer skin containing salicylic acid, phenol, benzalkonium chloride, etc. as active ingredients (References 1 and 2). An antihistamine preparation containing diphenhydramines as an active ingredient (Cited document 1). An external steroid hormone agent containing dexamethasone, prednisolones, fluocinolone acetonide as an active ingredient (Cited document 1). Camphor, dibucaine hydrochloride, ethyl aminobenzoate,
An analgesic, antipruritic, astringent, and anti-inflammatory agent containing zinc oxide or the like as an active ingredient (Reference 1). An agent for parasitic skin disease containing as an active ingredient bithionol, hexachlorophene, malathion, nitrofurazone, sulfur and the like (Cited document 1). A drug for dogs with skin diseases, which contains γ-linolenic acid as an active ingredient (Cited document 3).

[0007]

However, the above-mentioned conventional techniques for treating diseases occurring on the skin of animals have the following problems. That is, the non-steroidal skin external preparation has a very low therapeutic effect, and the steroidal skin external preparation has a high pharmacological effect, but the site of application has an increased susceptibility to infection and thinning of the skin. In some cases, side effects such as weakening of the blood vessel wall and abnormal activation of the pilosebaceous system may be caused, and systemic side effects may be observed due to the drug transdermally absorbed.

From the current state of the art, there has been a strong demand for a drug for effectively and safely treating diseases that occur on the skin of animals. The present invention has been made in view of the above circumstances, and an object thereof is to provide a safe and effective external therapeutic agent for diseases that occur on the skin of animals.

[0009]

The inventors of the present invention have
The inventors have conducted extensive studies on the uses of lactoferrin and lactoperoxidase, and have found that these substances are extremely effective in treating diseases that occur on the skin of animals, and completed the present invention. That is, this invention is
To solve the above problems, at least 3% (by weight) of lactoferrins, hydrolysates of lactoferrins, peptides separated from these hydrolysates, and synthesized peptides having the same amino acid sequence as these peptides There is provided a therapeutic agent for animal skin diseases, which comprises, as an active ingredient, a derivative of these peptides, or a mixture of two or more thereof, and at least 1% (weight) of lactoperoxidase.

Further, in the therapeutic agent for animal skin diseases of the present invention, lactoferrins, hydrolysates of lactoferrins, peptides separated from the hydrolysates, synthetic peptides having the same amino acid sequence as these peptides, It is a preferred embodiment that the derivative of these peptides, or a mixture of two or more thereof, is contained in an amount of 4 to 10% (by weight), and / or the above lactoperoxidase is included in an amount of 2 to 5% (by weight).

Further, regarding the peptide, SEQ ID NO: 1
~ It also exemplifies the peptide having the sequence set forth in SEQ ID NO: 31. Hereinafter, the present invention will be described in detail. Animals to which the therapeutic agent of this invention can be applied include
Pets such as dogs and cats, cows, horses, goats, sheep,
Examples include livestock such as pigs. Also,
Skin diseases on which the therapeutic agent of the present invention effectively acts include, for example, dermatophytosis, atopic dermatitis, pyoderma, allergic dermatitis, contact dermatitis, purulent traumatic dermatitis, otitis tinea, Otitis externa.

The lactoferrins used in this invention are
Commercially available lactoferrin, mammalian (eg, human, bovine, goat, sheep, horse, etc.) colostrum, transitional milk, normal milk, end-stage milk, etc., or non-fat milk, which is a processed product of these milks, whey, etc. Lactoferrin separated by a method (for example, ion exchange chromatography), apolactoferrin deironed with hydrochloric acid, citric acid, etc., metal saturated lactoferrin chelated with metals such as iron, copper, zinc and manganese, various It may be any of lactoferrin saturated with a metal at a saturation degree or any mixture of two or more thereof (hereinafter, these may be collectively referred to as lactoferrins), but a commercially available lactoferrin separated from milk However, it is preferable since it can be obtained at the lowest cost. The hydrolyzate of lactoferrin used in the present invention is a mixture of peptides obtained by hydrolyzing lactoferrin with a protease or an acid, and is produced, for example, by the invention of JP-A-5-320068 as follows. can do.

When the hydrolysis is carried out with an acid, lactoferrins are made into an aqueous solution, an inorganic acid or an organic acid is added thereto, and the mixture is heated at a predetermined temperature for a predetermined time for hydrolysis. When the enzyme is used for hydrolysis, lactoferrins are made into an aqueous solution, the pH of the enzyme used is adjusted to an optimum pH, enzymes such as pepsin and trypsin are added, and the mixture is kept at a predetermined temperature for a predetermined time for hydrolysis. After hydrolysis, the enzyme is inactivated by a conventional method. The degradation product obtained by hydrolysis with an acid or enzyme is a mixture of degradation products containing peptides having antibacterial properties and having various molecular weights, and the degree of degradation calculated as follows is 6 to 20%. The range of is desirable.

The above-mentioned degree of decomposition is calculated by the following formula from the formol nitrogen measured by the formol titration method with respect to the total nitrogen measured by the Kjeldahl method: Decomposition rate = (formal nitrogen amount / total nitrogen amount) × It is a value calculated by 100. The above-mentioned reaction solution obtained by hydrolysis with an acid or an enzyme (a solution of a decomposed product of lactoferrins) is cooled by a conventional method, and if necessary, neutralized, desalted and decolorized by a conventional method, and further required. The solution thus obtained can be fractionated by a conventional method, and the resulting solution can be used as it is, or concentrated to be in a liquid state, or concentrated and dried to be in a powder state.

The peptide used in the present invention is a peptide separated from the hydrolyzate of lactoferrin described in the Sequence Listing, a synthetic peptide identical to these peptides, or a derivative thereof, for example, JP-A-78392. JP, JP-A-92994, JP-A-1
It can be manufactured as follows according to JP-A-48295, JP-A-148296 and JP-A-148297.

Lactoferrins are hydrolyzed with an acid or an enzyme, and a mixture of the decomposed products is subjected to liquid chromatography.
A method for obtaining a fraction containing a peptide by a separation means such as
Alternatively, the amino acid sequence of the peptide obtained as described above is determined by a known method (for example, a method using a gas phase sequencer), and a peptide containing the peptide having the above amino acid sequence is known by a known method (for example, It can be prepared by a method of obtaining a desired peptide by synthesizing it by a method using an automatic peptide synthesizer). These peptides are, for example, peptides having the amino acid sequences described in the sequence listing, and specifically, SEQ ID NO: 1,
Peptides having amino acid sequences of 2 and 27 (JP-A-5-78392), peptides having amino acid sequences of SEQ ID NOs: 3, 4, 5 and 6 (JP-A-5-148).
297), peptides having the amino acid sequences of SEQ ID NOs: 7, 8, 9 and 31 (JP-A-5-148296), SEQ ID NOs: 10, 11, 12, 13, 14, 1
Peptides having the amino acid sequences of 5, 16, 17, 18, 19, 20 and 21 (JP-A-5-148295), SEQ ID NOs: 22, 23, 24, 25, 26, 28,
It is a peptide having the amino acid sequences of 29 and 30 (JP-A-5-92994).

The lactoperoxidase used in the present invention is commercially available lactoperoxidase, colostrum of mammals (eg, human, bovine, goat, sheep, horse, etc.), transitional milk, normal milk, terminal milk, or the like, or These may be either lactoperoxidase separated from skim milk, which is a processed product of these milks, whey, etc., by a conventional method (for example, ion exchange chromatography etc.), or an arbitrary mixture of two or more thereof. Commercially available lactoperoxidase separated from milk is preferable because it can be obtained at the lowest cost.

At least 3% of said lactoferrin,
Desirably, 4 to 10%, at least 1%, preferably 2 to 5% of the above-mentioned lactoperoxidase is mixed, and the animal skin disease therapeutic agent of the present invention can be obtained by the following known method. Further, the dosage form of the therapeutic agent for animal skin diseases of the present invention is not particularly limited, and examples of the carrier or excipient include white petrolatum, beeswax, liquid paraffin, glycerin, cellulose derivatives, polyethylene glycol, ointment bases. (Plastic base), silicone, bentonite, alcohol, water, olive oil, etc. are used alone or in a suitable combination of two or more kinds, and formulated into various forms such as cream, paste, gel, ointment, emulsion, lotion or solution by a conventional method. Can be done.

The animal skin disease therapeutic agent of the present invention produced as described above is applied to the affected area once or several times a day, depending on the degree of the disease. In addition, since the main component of the therapeutic agent for animal skin diseases of the present invention is a milk component, it is safe without side effects even if the animal stabs the affected area, and is safe for the user. Next, the present invention will be described in more detail by showing test examples. Test Example 1 This test was conducted to examine the effect on skin diseases of pet dogs. 1) Test Subject 67 dogs suffering from various skin diseases were used. 2) Test method Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) for the above dogs
Was dissolved in water at a ratio of 50 mg / ml and lactoperoxidase (manufactured by Morinaga Milk Industry Co., Ltd.) at a rate of 30 mg / ml, and the solution was absorbed on a cotton swab or absorbent cotton and applied to the affected area. 3) Test results The results of this test are shown in Table 1. As is clear from Table 1, 11 out of 16 cases in atopic dermatitis, 10 out of 15 cases in pyoderma, and 1 in dermatophytosis.
11 out of 3 cases and 5 out of 7 cases with flea allergic dermatitis
In the case of otitis externa, all cases were cured. Although the number of cases is small, 2 out of 3 cases of allergic contact dermatitis, all cases of purulent traumatic dermatitis, 1 case of 2 in seborrheic dermatitis, and 2 cases of eczema. In 1 case, all cases were healed by hair loss and redness.

It is clear that 70% of all cases of administration of the therapeutic agent for animal skin disease of the present invention were cured and 20% were improved, and the therapeutic agent for animal skin disease of the present invention is extremely effective for animal skin disease. It was found to have Similar results were obtained when other lactoferrins and peptides were used.

[0021]

[Table 1]

Test Example 2 This test was conducted to examine the effect of pet cats on skin diseases. 1) Test subjects 16 cats suffering from various skin diseases were used. 2) Test method 50 mg / ml of the same lactoferrin as in Test Example 1 and 30 lactoperoxidase were added to the above-mentioned cat.
A swab containing a therapeutic solution dissolved in water at a ratio of mg / ml.
Absorbed with absorbent cotton and applied to the affected area. 3) Test results The results of this test are shown in Table 2. As is clear from Table 2, 4 out of 5 cases of dermatophytosis, 2 out of 3 cases of otitis scabies, and 3 of flea allergic dermatitis.
Two of the cases were cured respectively.

It is apparent that 69% of all cases of administration of the therapeutic agent for animal skin disease of the present invention were cured and 19% were improved, and the therapeutic agent for animal skin disease of the present invention is significantly effective for skin disease of animals. It was found to have Similar results were obtained when other lactoferrins and peptides were used.

[0024]

[Table 2]

Test Example 3 This test was conducted to examine the therapeutic effect of a mixture of lactoferrin hydrolyzate or peptides and lactoperoxidase. 1) Test Subject Twenty dogs suffering from a skin disease were used. 2) Test method The above dogs were randomly divided into 2 groups of 10 dogs, and one group was treated with 50 mg / ml of the lactoferrin hydrolyzate produced by the same method as in Reference Example 2 and the same as in Test Example 1. A therapeutic solution prepared by dissolving lactoperoxidase in water at a rate of 30 mg / ml was added to the other group, and the peptide of SEQ ID NO: 26 (separated from lactoferrin hydrolyzate by the same method as in the above-mentioned separation example) was added at 50 mg / ml. The therapeutic solution prepared by dissolving lactoperoxidase in water at a rate of 30 mg / ml was absorbed on a cotton swab, absorbent cotton, etc., and applied to the affected area. 3) Test results The results of this test are shown in Table 3. As is clear from Table 3, 8 out of 10 cases using the lactoferrin hydrolyzate and 10 cases using the peptides were also observed.
It was clear that 7 of the cases were cured, and it was confirmed that the therapeutic agent for animal skin diseases of the present invention had a remarkable effect on animal skin diseases. Similar results were obtained when other lactoferrin hydrolysates or peptides were used.

[0026]

[Table 3]

Test Example 4 This test was conducted to determine the effective amount of lactoferrin. 1) Test Subject 70 dogs suffering from a skin disease were used. 2) Test method The dogs were divided into 7 groups of 10 dogs each, and a therapeutic solution containing 30 mg / l of the same lactoperoxidase as in Test Example 1 and the same lactoferrin as in Test Example 1 at the following ratios, respectively. Absorbed with a cotton swab or absorbent cotton, and applied to the affected area. Group 1: Lactoferrin 20 mg / ml Group 2: Lactoferrin 30 mg / ml Group 3: Lactoferrin 40 mg / ml Group 4: Lactoferrin 50 mg / ml Group 5: Lactoferrin 100 mg / ml Group 6: Lactoferrin 200 mg / ml 7th Group: lactoferrin 300 mg / ml 3) Test results The results of this test are shown in Table 4. As is clear from Table 4, in the first group, there were 4 cases of improvement in 10 cases, whereas in the second group, healing and improvement were 3 cases out of 10 cases respectively, and in groups 3 to 5 More than 8 out of 10 cases were cured or improved. Further, in the 6th and 7th groups, 7 out of 10 cases were cured or improved, but no increase in the healing effect due to an increase in the lactoferrin content was observed.

From these results, it was confirmed that an animal skin disease therapeutic agent containing lactoferrin in an amount of 3% or more, preferably 4 to 10%, has a remarkable effect on animal skin diseases. In addition,
Similar results were obtained when the hydrolyzate or peptide of lactoferrin was changed and when the lactoperoxidase content was changed.

[0029]

[Table 4]

Test Example 5 This test was conducted to determine the effective amount of lactoperoxidase. 1) Test Subject 70 dogs suffering from a skin disease were used. 2) Test method The dogs were divided into 7 groups of 10 dogs each, and the same lactoferrin as in Test Example 1 was added to the therapeutic solution containing 50 mg / ml and the same ratio as in Test Example 1 in the following lactoperoxidase. Absorbed with a cotton swab or absorbent cotton, and applied to the affected area. Group 1: Lactoperoxidase 5 mg / ml Group 2: Lactoperoxidase 10 mg / ml Group 3: Lactoperoxidase 20 mg / ml Group 4: Lactoperoxidase 30 mg / ml Group 5: Lactoperoxidase 50 mg / ml Group 6: Lactoperoxidase 100 mg / ml Group 7: Lactoperoxidase 200 mg / ml 3) Test results The results of this test are shown in Table 5. As is clear from Table 5, in the first group, there were 2 cases of improvement in 10 cases, whereas in the second group, there were 5 cases of cure and 1 case of improvement in 10 cases, and in group 3 of 10 cases. Healing in 6 cases and improvement in 2 cases
In each of the groups 4 and 5, 9 out of 10 cases were cured or improved. Also, in the sixth and seventh groups, 10
Eight of the cases were cured or improved, but no increase in the healing effect due to an increase in the lactoperoxidase content was observed.

From these results, it was confirmed that the therapeutic agent for animal skin diseases containing lactoperoxidase in an amount of 1% or more, preferably 2 to 5%, has a remarkable effect on animal skin diseases. When a lactoferrin hydrolyzate or peptide was used, almost the same results were obtained even when the lactoferrin content was changed.

[0032]

[Table 5]

Reference Example 1 1 kg of lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) was dissolved in 20 liters of deionized water, placed in a tube, and 4 liters were added to 400 liters of 0.1 M citric acid solution (pH 2.2).
Dialyze at 36 ° C for 36 hours and to remove further citric acid, 2
It was dialyzed against 0 times amount of deionized water at 4 ° C. for 24 hours (the deionized water was changed twice during the process), and the dialyzed solution was freeze-dried to obtain about 950 g of apolactoferrin. Reference Example 2 50 g of commercially available lactoferrin (Bergi made by Oleofina) as it was separated from milk was dissolved in 950 g of purified water, and 1N hydrochloric acid was added to the obtained solution to adjust the pH to 2, 120 Heat at 15 ° C. for 15 minutes, cool, and then lactoferrin degradation product solution (lactoferrin degradation product concentration: 5
%) About 1000 g was obtained. The degradation degree of this lactoferrin degradation product was 9%.

The above-mentioned lactoferrin decomposition product solution was concentrated under reduced pressure and freeze-dried to obtain about 49 powders of the lactoferrin decomposition product.
g was obtained. Reference Example 3 1 kg of commercially available lactoferrin (Bergi made by Oleofina) as it was separated from milk was dissolved in 9 kg of purified water, and 2 molar citric acid was added to adjust the pH to 2.5. 30 g of porcine pepsin (1: 10,000, manufactured by Wako Pure Chemical Industries, Ltd.) was added, mixed uniformly, kept at 37 ° C. for 180 minutes, heated at 85 ° C. for 10 minutes to inactivate the enzyme, and then cooled. , Lactoferrin hydrolyzate about 10
I got kg. The degree of decomposition of this lactoferrin hydrolyzate was 11.3%.

The lactoferrin hydrolyzate is concentrated,
Lyophilized and powdered lactoferrin hydrolyzate about 960
g was obtained. Reference Example 4 50 mg of commercially available bovine lactoferrin (manufactured by Sigma) was dissolved in 0.9 ml of purified water, pH was adjusted to 2.5 with 0.1 N hydrochloric acid, and then 1 mg of commercially available porcine pepsin (manufactured by Sigma) was added. Added and hydrolyzed at 37 ° C. for 6 hours. Then, adjust the pH to 7.0 with 0.1 N sodium hydroxide, heat at 80 ° C. for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 15,000 rpm for 30 minutes,
A clear supernatant was obtained. 100 μl of this supernatant was added to TSK gel O
It was subjected to high performance liquid chromatography using DS-120T (manufactured by Tosoh Corporation) and eluted with 20% acetonitrile containing 0.05% TFA (trifluoroacetic acid) for 10 minutes after injecting the sample at a flow rate of 0.8 ml / min. , Then 30 minutes 0.0
Elution was carried out with a gradient of 20 to 60% acetonitrile containing 5% TFA, and the fractions eluted during 24 to 25 minutes were collected and dried under vacuum. This dried product was dissolved in purified water at a concentration of 2% (W / V), and the TSK gel ODS-120T was dissolved again.
(Manufactured by Tosoh Corporation), and subjected to high performance liquid chromatography at a flow rate of 0.8 ml / min for 10 minutes after injection of the sample.
Elute with 24% acetonitrile containing 05% TFA, then elute with a gradient of 24-32% acetonitrile containing 0.05% TFA for 30 minutes, 33.5-35.
Fractions eluting during 5 minutes were collected. The above operation was repeated 25 times, and vacuum drying was performed to obtain about 1.5 mg of the peptide.

The peptide was hydrolyzed with 6N hydrochloric acid, and the amino acid composition was analyzed by an ordinary method using an amino acid analyzer. The same sample was subjected to Edman degradation 25 times using a gas phase sequencer (manufactured by Applied Biosystems) to determine the sequence of 25 amino acid residues. In addition, a disulfide bond analysis method using DTNB (5,5-dithio-bis (2-nitrobenzoic acid)) [Analytical Biochemistry
emistry), Vol. 67, p. 493, 1975], it was confirmed that a disulfide bond exists.

As a result, this peptide consists of 25 amino acid residues, the 3rd and 20th cysteine residues are disulfide-bonded, and the 3rd cysteine residue is N
It was confirmed to have the amino acid sequence of SEQ ID NO: 26 in which two amino acid residues were bound to the -terminal side and 5 amino acids were bound to the C-terminal side from the 20th cysteine residue, respectively. . Reference Example 5 Using an automatic peptide synthesizer (Pharmacia LKB Biotechnology, LKBBiolynx4170), Shepard et al. [Journal of Chemical Society Parkin I (Journal of Chemical Societ
y Perkin I), p. 538, 1981], and synthesized as follows.

N, N-dicyclohexylcarbodiimide was added to an amino acid whose amine functional group was protected by a 9-fluorenylmethoxycarbonyl group to produce an anhydride of the desired amino acid, and this Fmoc-amino acid anhydride was synthesized. Using. In order to produce a peptide chain, Fmoc-lysine anhydride corresponding to the C-terminal lysine residue was reacted with ultrocin A resin (Pharmacia LKB Biotechnology-
Fixed). The resin is then washed with dimethylformamide containing piperidine to remove the amine functional group protecting group of the C-terminal amino acid. Later C of amino acid sequence
The penultimate-corresponding Fmoc-lysine anhydride was coupled via the C-terminal amino acid residue to the deprotected amine functional group of lysine immobilized on the resin. Similarly, methionine, arginine, tryptophan,
Glutamine, tryptophan, arginine, arginine, threonine, and lysine were fixed. After completion of coupling of all amino acids and formation of a peptide chain having a desired amino acid sequence, 94% TFA, 5% pheno-
Of the protective group other than acetamidomethyl and elimination of the peptide with a solvent consisting of 1% ethanol and 1% ethanediol, the peptide was purified by high performance liquid chromatography, and the solution was concentrated and dried, Peptide powder was obtained.

The peptide thus obtained was analyzed by the same method as in Reference Example 4 above and confirmed to have the amino acid sequence of SEQ ID NO: 27. Next, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples.

[0040]

【Example】

Example 1 50 l of lactoferrin (manufactured by Morinaga Milk Industry) in 10 l of purified water
0 g and lactoperoxidase (Morinaga Milk Industry Co., Ltd.) 3
Dissolve 00g, filter with a sterilization filter (manufactured by Japan Millipore Industrial Co., Ltd.), and aseptically fill 100ml each into a container,
98 therapeutic agents for animal skin diseases were obtained. Example 2 To 840 g of a hydrophilic ointment base (manufactured by Yoshida Pharmaceutical Co., Ltd.), 100 g of lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) and 60 g of lactoperoxidase (manufactured by Morinaga Milk Industry Co., Ltd.) were added and uniformly kneaded to prepare a therapeutic agent for animal skin diseases. About 1 kg was obtained. Example 3 98 animal skin disease therapeutic agents were obtained by the same method as in Example 1 except that apolactoferrin obtained by the same method as in Reference Example 1 was used. Example 4 In 840 g of hydrophilic ointment base (manufactured by Yoshida Pharmaceutical Co., Ltd.), Reference Example 3
100 g of lactoferrin hydrolyzate (manufactured by Morinaga Milk Industry Co., Ltd.) and 60 g of lactoperoxidase (manufactured by Morinaga Milk Industry Co., Ltd.) produced by the same method as in Example 1 were added and uniformly kneaded to obtain about 1 kg of a therapeutic agent for animal skin diseases. Example 5 46 g of a hydrophilic ointment base (manufactured by Yoshida Pharmaceutical Co., Ltd.) and 2 g of the peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 4
And 2 g of lactoperoxidase (manufactured by Morinaga Milk Industry Co., Ltd.) were added and kneaded uniformly to obtain about 50 g of a therapeutic agent for animal skin diseases. Example 6 About 50 g of a therapeutic agent for animal skin diseases was obtained by the same method as in Example 5, except that the peptide of SEQ ID NO: 27 produced by the same method as in Reference Example 5 was used.

[0041]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, animal skin diseases can be treated effectively and without causing side effects, and can be safely handled by the user. An agent is provided.

<References> 1) Minoru Sawada, “Handbook of veterinary drug products for 1991 edition”, Japan Animal Pharmaceutical Association, 1991 2) JP 62-106022 A 3) JP 4 -290820 gazette 4) Unexamined-Japanese-Patent No. 3-330130 5) Unexamined-Japanese-Patent No. 61-83131 6) Japan Food Science, 27th volume, 1st.
No. 25 to 34, 1988 7) Science, 197, 263 to 265, 1977 8) AF Williams & JD Baum (AF Williams & JD) Baum), "Human Milk Banking", Nestle Nutrition Workshop Series, Part 5
Volume (Nestle Nutrition Workshop Series Volume 5),
Pages 133-143, Leven Press
Books, Inc. (Reven Press Books Ltd., 1984, 9) Dairy Sci.
ence Abstracts), Vol. 30, No. 9, page 500 [3210], 1968 10) Journal of the Japanese Society of Pediatrics, Vol. 88, No. 7, No. 15
81 to 1582, "A-43", 198
4th year 11) The Journal of Infectious Diseases, Volume 153, Issue 2, Pages 232-237.
1986 12) French Patent No. 2,596,986,
1987 13) Journal of Food Protection (Jou
rnal of Food Protection), Volume 51, No. 7, 55
Pp. 8 to 561, 1988 14) Milchwissenschaft,
Volume 38, Issue 4, Pages 203-206
1983 15) Dairy Cooperative Material, Volume 31, No. 5, 2nd to 10th pages, 1982 16) JP-A-4-81417 17) The Journal of Immunology
al of Immunology), Vol. 128, No. 2, pages 726-731, 1982 18) Journal of Pediatrics (Journal).
of Pediatrics), Volume 94, Page 1, 1979 19) Journal of Dear Science (Jour
nal of Dairy Science) Vol. 67, p. 606, 1
984 20) Dairy Science and Food Research, Volume 38, A277, 1989 21) Food Industry, Volume 35, No. 6, Page 39, 1
1992 22) Protides of the Biological Floods, Proceedings of the Colloquium (Prot
ides of the Biological Fluids, Proceedings of the
Colloquium), Volume 32, 111-114, 1
1985 23) JP-A-57-106689 24) JP-A-58-13594 25) JP-A-58-213744 26) JP-A-59-51247 27) JP-A-60-130599 28) JP-A-60-172998 29) JP-A-61-251699 30) JP-A-63-44598 31) JP-A-62-22798 32) JP-A-62-51697 33) Japanese Patent Laid-Open No. 63-17897 34) Japanese Patent Laid-Open No. 2-35799 35) Japanese Patent Laid-Open No. 2-500804 36) Japanese Patent Laid-Open No. 3-261717 37) Proceedings of the National Academy of the Science of United
States of America (Proceedings of the Nationa
l Academy of the Science of United States of Ameri
ca), Vol. 84, pp. 5449-p. 5453, 1987 38) Fimiya Prilord Neuf Soejenieni (K
himiya Prirodnykh Soedinenij), No. 1, pp. 130 to 133, 1978 39) JP-A-2-53799 40) JP-A-2-234684 41) JP-A-5-92994 42) JP-A-5-78392 43) JP-A-5-148295 44) JP-A-5-148296 45) JP-A-5-148297

[0043]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0044] SEQ ID NO: 2 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0045] SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0046] SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the following sequence, Xaa represents any amino acid residue except Cys.

[0047] SEQ ID NO: 5 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0048] SEQ ID NO: 6 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0049] SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0050] SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0051] SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0052] SEQ ID NO: 10 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

[0053] SEQ ID NO: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

[0054] SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

Sequence: Gln Trp Gln Arg 1 SEQ ID NO: 13 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Includes this peptide and this peptide as a fragment peptide.

Sequence: Trp Gln Arg 1 SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment .

Sequence: Arg Arg Trp Gln Trp 15 Sequence number: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide are fragments As a peptide.

Sequence: Arg Arg Trp Gln 1 SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and this peptide as a fragment peptide SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment SEQ ID NO: 18 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 22 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded.

Sequence: Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val 20 SEQ ID NO: 23 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear Type of sequence: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond.

Sequence: Lys Cys * Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys * Val 20 SEQ ID NO: 24 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear sequence type: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded.

Sequence: Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys Ile 20 SEQ ID NO: 25 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear Type of sequence: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond.

Sequence: Lys Cys * Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys * Ile 20 SEQ ID NO: 26 Sequence Length: 25 Sequence Type: Amino Acid Topology: Linear sequence type: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment. In the sequence below, Cys 3 and 20 are disulfide-bonded.

Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25 SEQ ID NO: 27 Sequence Length: 11 Sequence Type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 28 Sequence length: 38 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. 16 in the sequence below
Cys No. 33 and Cys No. 33 have a disulfide bond.

Sequence: Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Glu Trp Phe Lys 1 5 10 15 Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser 20 25 30 Ile Thr Cys Val Arg Arg Ala Phe 35 SEQ ID NO: 29 Sequence length: 32 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. 10 in the sequence below
No. 27 Cys and No. 27 Cys are disulfide-bonded.

Sequence: Thr Ile Ser Gln Pro Glu Trp Phe Lys Cys Arg Arg Trp Gln Trp 1 5 10 15 Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys Val Arg Arg 20 25 30 Ala Phe SEQ ID NO: 30 of the sequence Length: 47 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the following sequence, in the peptide having a sequence length of 36 and having Cys at positions 9, 26, and 35, the 9th Cys and the 26th Cys form a disulfide bond, and 35 of peptides
No. 10 Cys has a sequence length of 11 and has a disulfide bond with No. 10 Cys of a peptide having Cys at No. 10.

Sequence: Val Ser Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn 1 5 10 15 Met Arg Lys Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp 20 25 30 Ser Pro Ile Gln Cys Ile 35 Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10 SEQ ID NO: 31 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide Peptides included as fragments. Xaa in the sequence below
Indicates any amino acid residue except Cys.

[0067]

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Claims (4)

[Claims] 1. At least 3% (by weight) of lactoferrins, hydrolysates of lactoferrins, peptides isolated from these hydrolysates, synthesized peptides having the same amino acid sequence as these peptides, these peptides Or a mixture of two or more thereof and at least 1% (weight) of lactoperoxidase as an active ingredient. 2. A lactoferrin, a hydrolyzate of lactoferrin, a peptide separated from this hydrolyzate,
The therapeutic agent for animal skin diseases according to claim 1, which comprises 4 to 10% (by weight) of a synthetic peptide having the same amino acid sequence as these peptides, a derivative of these peptides, or a mixture of two or more thereof. 3. 2-5% of lactoperoxinase
(Weight) The therapeutic agent for animal skin diseases according to claim 1 or 2. 4. The peptides are SEQ ID NO: 1 to SEQ ID NO: 31.
The therapeutic agent for animal skin diseases according to claim 1, 2 or 3 having the sequence described in 1.