AU2014226865B2

AU2014226865B2 - Anti-inflammatory agent

Info

Publication number: AU2014226865B2
Application number: AU2014226865A
Authority: AU
Inventors: Ken Katoh; Toshiya Kobayashi; Yuki Mizuno; Tomoki Takahashi; Hiroshi URAZONO; Koichiro YOSHIOKA
Original assignee: Megmilk Snow Brand Co Ltd
Current assignee: Megmilk Snow Brand Co Ltd
Priority date: 2013-03-08
Filing date: 2014-03-07
Publication date: 2018-09-13
Anticipated expiration: 2034-03-07
Also published as: AU2014226865A1; TW201511769A; CN105025915A; JP2014172861A; HK1211843A1; WO2014136932A1

Abstract

This anti-inflammatory agent contains, as an active component, lactadherin and/or a decomposition product obtained by treating lactadherin with a proteolytic enzyme. Having mammalian milk as the raw ingredient, this anti-inflammatory agent can be easily and economically produced and can be safely ingested on a daily basis.

Description

The present invention relates to an anti-inflammatory agent which exhibits an excellent effect production of inflammatory cytokines, excessive inflammation in living organisms, prevention diseases, infectious safety.

of inhibiting the which ameliorates which is useful for or treatment of, for example, connective tissue allergic diseases, metabolic diseases, diseases, and

The present anti-inflammatory anti-inflammatory anti-inflammatory anti-inflammatory and food, which has excellent stability invention also relates to an anti-inflammatory drink, nutritional feed, each agent.

and an an composition, and an of which contains the

BACKGROUND ART [0002]

Inflammatory cytokines are produced from lymphocytes or macrophages and are involved in inflammatory reactions associated with bacterial or viral infection, tumor, or tissue damage. Known inflammatory cytokines include interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) , and GM-CSF. Inflammatory cytokines act on the liver in a living

SNOW-217 organism, to induce the synthesis and secretion of acute-phase proteins or to enhance adhesion of neutrophils to endothelial cells. In addition, inflammatory cytokines induce chemokines, such as interleukin-8 (IL-8) and MCP-1, to mediate the chemotaxis of neutrophils, monocytes, or lymphocytes to inflamed sites, resulting in initiation of inflammation. Excessive production of inflammatory cytokines cause pathological conditions, such as systemic inflammatory response syndrome, connective tissue diseases (e.g., chronic rheumatoid arthritis), allergic diseases, metabolic diseases (e.g., arteriosclerosis, insulin resistance, and diabetes), and infectious diseases (e.g., multiple sclerosis, graft-versus-host disease, viral hepatitis, and HIV infection) . Thus, inhibition of the production of inflammatory cytokines has been reported to be very useful for prevention, treatment, or amelioration of such a pathological condition, or for prevention of recurrence thereof. Although antihistamines or steroids are known as drugs for inhibiting excessive inflammation in living organisms, such a drug may cause side effects, including tinnitus, headache, and anorexia. In addition, such a substance cannot be added to foods and drinks in view of safety or production cost. Under these circumstances, a demand has arisen for development of a food, drink, or feed which can be safely ingested routinely and can inhibit excessive production of inflammatory cytokines.

SNOW-217 [0003]

Lactadherin is a glycoprotein present in milk fat globule membrane, and accounts for 10% of proteins contained in bovine milk fat globule membrane. Lactadherin has a molecular weight of 43 kDa to 53 kDa and an isoelectric point of 7.0, and includes two epidermal growth factor-like domains. Lactadherin contained in bovine milk is also called PAS-VI-VII (PAS6/7), and lactadherin contained in murine milk is also called MFG-E8 . Lactadherin is considered to play a role in regulating the gastrointestinal function of newborns, and is incorporated into infant powdered milk for the purpose of preventing newborns from rotavirus infection.

Traditional food ingredients capable of inhibiting excessive production of inflammatory cytokines include sphingomyelin, lactoperoxidase, and acidic xylooligosaccharides. Unfortunately, lactadherin and/or a decomposed product thereof has not yet been known to have an excellent effect of inhibiting the production of inflammatory cytokines, or to ameliorate excessive inflammation in living organisms .

RELATED ART [0004]

Patent Document 1: Japanese Patent Application Laid-Open No. 2007-112793

2014226865 22 Aug 2018

Patent Document 2: Japanese Patent Application Laid-Open No. 2007-223910

NON-PATENT DOCUMENT [0005]

Non Patent Document 1: Journal of Nutritional Science and

Vitaminology, 56, 54-59, 2010

SUMMARY OF INVENTION [000 6]

Embodiments of the present invention provide an anti-inflammatory agent which exhibits a remarkable effect of inhibiting the production of inflammatory cytokines, which ameliorates excessive inflammation in living organisms, and 15 which is useful for prevention or treatment of, for example, connective tissue diseases, allergic diseases, metabolic diseases, and infectious diseases. Another embodiment provides an anti-inflammatory food, an anti-inflammatory drink, an anti-inflammatory nutritional composition, and an 20 anti-inflammatory feed which contain the anti-inflammatory agent.

[0006a]

According to a first aspect, the present invention provides use of a decomposition product of lactadherin in the manufacture

2014226865 22 Aug 2018 of a medicament for treating or preventing inflammation, wherein the medicament is for oral administration to a subject in need thereof at a dose of from 5mg/kg body weight to 50 mg/kg body weight of the decomposition product, and wherein the decomposition product is prepared by decomposition of lactadherin with at least one protease.

[0006b]

According to a second aspect, the present invention provides an anti-inflammatory food, drink, nutritional composition, or feed comprising as an active ingredient a decomposition product of lactadherin, wherein the decomposition product is prepared by decomposition of lactadherin with at least one protease.

[0006c]

According to a third aspect, the present invention provides a method of treating, preventing or ameliorating inflammation in a subject, comprising orally administering to the subject an anti-inflammatory agent comprising a decomposition product of lactadherin at a dose of from 5mg/kg body weight to 50 mg/kg body weight of the decomposition product, wherein the decomposition product is prepared by decomposition of lactadherin with at least one protease.

[0007]

The present inventors have conducted extensive studies

4A

SNOW-217 for solving the above-described problems, and have consequently found that lactadherin and/or a decomposed product thereof has an excellent effect of inhibiting the production of inflammatory cytokines.

The present invention includes the following aspects: Aspect (1). An anti-inflammatory agent containing lactadherin and/or a decomposed product thereof as an active ingredient. Aspect (2). The anti-inflammatory agent according to Aspect (1), wherein lactadherin and/or a decomposed product thereof is derived from bovine milk.

Aspect (3). The anti-inflammatory agent according to Aspect (1) or (2), wherein the decomposed product of lactadherin is prepared by decomposition of lactadherin with a protease.

Aspect (4). The anti-inflammatory agent according to Aspect (3) , wherein the protease is at least one selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, and papain .

Aspect (5) . An anti-inflammatory food, drink, nutritional composition, or feed comprising the anti-inflammatory agent according to any one of Aspects (1) to (4) .

Aspect (6) . A method of ameliorating inflammation in a living organism, comprising orally ingesting the anti-inflammatory agent according to any one of Aspects (1) to (4).

Aspect (7) . A method of using an anti-inflammatory agent containing lactadherin and/or a decomposed product thereof as

SNOW-217 an active ingredient, the method comprising applying the anti-inflammatory agent to a patient with inflammation.

ADVANTAGEOUS EFFECTS OF INVENTION [0008]

The anti-inflammatory agent of the present invention exhibits a remarkable effect of inhibiting the production of inflammatory cytokines, and is useful for prevention or treatment of, for example, connective tissue diseases, allergic diseases, metabolic diseases, and infectious diseases.

DESCRIPTION OF EMBODIMENT [0009]

Lactadherin, which is an active ingredient of the anti-inflammatory agent, can be prepared by desaltation or concentration of component (A), (B), or (C) with an ultrafiltration (UF) membrane, a microfiltration (MF) membrane, a reverse osmosis (RO) membrane, or an ion-exchange resin, wherein component (A) is buttermilk, which is an aqueous phase component prepared during production of butter from raw milk; component (B) is an aqueous phase component discharged through phase inversion of high-fat cream having a fat content of 60% or more by a heating or shearing process, the high-fat cream being separated from cream having a fat content of 40 to 50% and being separated from raw milk with a separator; and

SNOW-217 component (C) is butter serum, which is an aqueous phase component separated from heat-melted butter. Alternatively, lactadherin can be prepared by the following procedure: hydrochloric acid is added to butter serum, into a pH of 4.4, calcium chloride is added to the resultant mixture and the mixture is held at 50°C for 30 minutes, followed by separation, and the resultant precipitate is dissolved or suspended in water, followed by desaltation or concentration of the solution or the suspension with an ultrafiltration (UF) membrane, a microfiltration (MF) membrane, a reverse osmosis (RO) membrane, or an ion-exchange resin. A lactadherin decomposed product, which is an active ingredient of the anti-inflammatory agent, can be prepared by decomposition of lactadherin with any protease. Examples of the protease include commercially available protease products for food and industrial uses, such as Protease A Amano SD (trade name), Thermoase PC10F (trade name), and Protin SD-AY10 (trade name), trypsin, pancreatin, chymotrypsin, pepsin, and papain. These proteases may be used in combination. The above-prepared lactadherin and/or a decomposed product thereof may be freeze-dried or spray-dried. [0010]

Lactadherin and/or a decomposed product thereof can be prepared from the milk of a mammal, such as human, cow, buffalo, goat, or sheep, produced by a genetic engineering technique, or purified from the blood or organs of such a mammal.

SNOW-217

Lactadherin and/or a decomposed product thereof can be produced from such a material by a simple process at low cost, and the resultant product can be safely ingested routinely. In particular, lactadherin and/or a decomposed product thereof is preferably derived from bovine milk. Lactadherin and/or a decomposed product thereof may be a commercially available purified reagent.

[0011]

Lactadherin and/or a decomposed product thereof may be used as an anti-inflammatory agent without any treatment, or may optionally be prepared into a powdery, granular, tablet, capsular, or drink product by a common process. Freeze-dried or spray-dried lactadherin and/or a decomposed product thereof may be used as an anti-inflammatory agent without any treatment, or may be prepared into any product by a common process.

The resultant lactadherin product may be incorporated into nutrients, foods and drinks (e.g., yogurt, beverages, and wafer), nutritional compositions, or feeds.

[0012]

The anti-inflammatory food, drink, nutritional composition, or feed of the present invention may contain, in addition to lactadherin and/or a decomposed product thereof, any material which is commonly contained in any other food, drink, or feed. Examples of such materials include stabilizers, saccharides, lipids, flavors, vitamins, minerals, flavonoids,

SNOW-217 and polyphenols. The anti-inflammatory food, drink, nutritional composition, or feed may contain any component exhibiting an anti-inflammatory effect, such as sphingomyelin, lactoperoxidase, or acidic xylooligosaccharide in combination with lactadherin and/or a decomposed product thereof, which is an active ingredient.

[0013]

The anti-inflammatory food, drink, nutritional composition, or feed may contain lactadherin and/or a decomposed product thereof in any amount. In order to achieve an effect of inhibiting the production of inflammatory cytokines in the present invention, the amount of lactadherin and/or a decomposed product thereof incorporated into a drug, food, drink, or feed is preferably adjusted such that the daily oral dose of lactadherin and/or a decomposed product thereof is 5 mg or more per adult.

[0014]

The anti-inflammatory agent may be prepared into any drug product by addition of an appropriate auxiliary agent to lactadherin and/or a decomposed product thereof, which is an active ingredient. Preparation of the drug product may involve use of a common excipient or diluent, such as a filler, an extender, a binder, a disintegrant, a surfactant, or a lubricant. Examples of the excipient includes sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride,

SNOW-217 magnesium aluminate, synthetic aluminum silicate, magnesium metasilicate aluminate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, and carboxymethyl cellulose calcium. These excipients may be used alone or in combination .

[0015]

Although the present invention has been described above with reference to embodiments, the embodiments, which constitute a part of this disclosure, should not be construed as limiting the invention thereto. Various alternative embodiments, examples, and operational techniques will be apparent to those skilled in the art from this disclosure.

For example, a method of using the anti-inflammatory agent is provided, wherein the anti-inflammatory agent, which contains lactadherin and/or a decomposed product thereof described in the embodiments as an active ingredient, is applied to a patient with inflammation. Lactadherin and/or a decomposed product thereof used in this method may be isolated or purified from a raw material as described below in Examples. The anti-inflammatory agent may also be applied to non-human mammals, including domestic animals such as dog, monkey, cat, cattle, horse, pig, chicken, and sheep.

EXAMPLES [0016]

SNOW-217

The present invention will now be described in detail by way of Examples and Test Examples, which should not be construed as limiting the invention thereto.

Example 1 [0017]

Acetone (100 kg) was added to freeze-dried, non-sterilized buttermilk powder (10 kg), and the mixture was treated with a quark separator, to completely remove the precipitate. Acetone was removed from the resultant supernatant with an evaporator, and the residue was dissolved in deionized water. Subsequently, the solution was treated with an ultrafiltration membrane having a cut-off molecular weight of 60 kDa, to recover a filtrate. The filtrate was then treated with an ultrafiltration membrane having a cut-off molecular weight of 30 kDa, to remove impurities, followed by desaltation and concentration. Thereafter, the resultant product was freeze-dried, to prepare lactadherin powder (Example sample 1) (68 g) . The lactadherin powder had a lactadherin content of 78%. The thus-prepared lactadherin can be used as an anti-inflammatory agent without any treatment. Example 2 [0018]

The lactadherin powder prepared in Example 1 (500 mg) was dissolved in purified water (100 mL) , and the pH of the solution was adjusted to 8 with sodium bicarbonate . Thereafter, trypsin

SNOW-217 (manufactured by Sigma) (i.e., a protease) was added to the solution, into a final lactadherin concentration of 0.01%, and the mixture was treated with the enzyme at 37°C for one hour. The mixture was then thermally treated at 85°C for 10 minutes, to inactivate the enzyme. The resultant mixture was freeze-dried, to prepare powder of a lactadherin decomposed product (Example sample 2) (463 mg). The thus-prepared lactadherin decomposed product had a molecular weight of 5 kDa or less. The lactadherin decomposed product can be used as an anti-inflammatory agent without any treatment.

Example 3 [0019]

The lactadherin powder prepared in Example 1 (10 g) was dissolved in purified water (200 mL) , and the solution was then held at 45°C. Protease A Amano SD (manufactured by Amano Enzyme Inc.) (2 g) was added to the solution, and the mixture was treated with the enzyme for two hours . The mixture was then thermally treated at 80°C for 10 minutes, to inactivate the enzyme. The resultant mixture was freeze-dried, to prepare powder of a lactadherin decomposed product (Example sample 3) (8.2 g) . The thus-prepared lactadherin decomposed product had a molecular weight of 5 kDa or less . The lactadherin decomposed product can be used as an anti-inflammatory agent without any treatment.

[0020]

SNOW-217 [Test Example 1]

The lactadherin prepared in Example 1 or the lactadherin decomposed product prepared in Example 2 was evaluated for the effect of inhibiting the production of IL-6 and IL-8, which are inflammatory cytokines. Human colon adenocarcinoma cells Caco-2, which have small intestinal epithelial cell-like characteristics, were inoculated into 24-well microtiter plates and cultured in a Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum for two weeks. The lactadherin powder prepared in Example 1 (final concentration: 0.5, 5, 50, or 500 qg/mL in terms of lactadherin) or the lactadherin decomposed product powder prepared in Example 2 (final concentration: 0.5, 5, 50, or 500 qg/mL in terms of lactadherin decomposed product) was added to a Caco-2-cultured microtiter plate, followed by culturing for 12 hours. Lactoperoxidase (final concentration: 5, 50, or 500 qg/mL in terms of lactoperoxidase), serving as a positive control, was added to another Caco-2-cultured microtiter plate, followed by culturing for 12 hours. Subseguently, IL- 1β (final concentration: 0.2 ng/mL) was added to each of the microtiter plates, followed by culturing for 24 hours. Thereafter, the culture supernatant was recovered, and the IL-6 and IL-8 concentrations of the supernatant were determined by ELISA. Tables 1 and 2 show the results.

SNOW-217 [0021] [Table 1]

IL-6 concentration (pg/mL)
Control (no addition)		0.2 ± 0.05
IL-Ιβ		172.1 ±6.52
Lactadherin	0.5 μθ/ηΊ + IL-1 β	168.7 ±7.43
Lactadherin	5 μθ/ηΊ + IL-1 β	149.2 ±3.67*
Lactadherin	50 μθ/ηΊ + IL-1 β	132.4 ±2.76*
Lactadherin	500 μθ/ηΊ + IL-1 β	119.5 ±2.38*
Lactadherin decomposed product	0.5 μ9/ηΊ + IL-1 β	170.5 ±4.89
Lactadherin decomposed product	5 μ9/ηΊ + IL-1 β	151.1 ±7.54*
Lactadherin decomposed product	50 μ9/ΐϊ1 + IL-1 β	129.3 ±6.34*
Lactadherin decomposed product	500 μ9/ηΊ + IL-1 β	118.8 ±3.67*
Lactoperoxidase	5 μ9/ηΊ + IL-1 β	169.5 ±9.02
Lactoperoxidase	50 μ9/ΐϊ1 + IL-1 β	152.1 ±2.11
Lactoperoxidase	500 μ9/ηΊ + IL-1 β	135.4 ±3.09

Numerals: average ± standard deviation (n=8).

*: significantly lower than the case of addition of lactoperoxidase of the same concentration (p<0.05).

SNOW-217 [0022] [Table 2]

IL-8 concentration (pg/mL)
Control (no addition)		0.2 ± 0.01
IL-Ιβ		785.1 ±11.04
Lactadherin	0.5 μθ/ηΊ + IL-1 β	779.7 ±9.89
Lactadherin	5 μθ/ηΊ + IL-1 β	702.3 ±12.67*
Lactadherin	50 μθ/ηΊ + IL-1 β	658.9 ±8.72*
Lactadherin	500 μθ/ηΊ + IL-1 β	567.5 ±10.73*
Lactadherin decomposed product	0.5 μ9/ηΊ + IL-1 β	783.5 ±14.12
Lactadherin decomposed product	5 μ9/ηΊ + IL-1 β	710.2 ±13.26*
Lactadherin decomposed product	50 μ9/ιτ1 + IL-1 β	646.4 ± 7.81 *
Lactadherin decomposed product	500 μ9/ηΊ + IL-1 β	559.1 ±8.52*
Lactoperoxidase	5 μ9/ηΊ + IL-1 β	781.2 ±11.63
Lactoperoxidase	50 μ9/ιτ1 + IL-1 β	701.9 ±7.84
Lactoperoxidase	500 μ9/ηΊ + IL-1 β	656.7 ±12.18

Numerals: average ± standard deviation (n=8).

[0023]

With reference to Tables 1 and 2, addition of lactadherin or the lactadherin decomposition compound (final 10 concentration: 5 pg/mL or more) significantly reduced the IL-6 and IL-8 concentrations of the culture supernatant. This effect was significantly higher than that obtained by addition of lactoperoxidase. These results indicate that lactadherin or a decomposed product thereof exhibits an anti-inflammatory 15 effect.

[0024]

SNOW-217 [Test Example 2]

The anti-inflammatory effect of lactadherin or a decomposed product thereof was evaluated in accordance with the method described in Japanese Patent Application Laid-Open No. 2007-112793 by use of a carrageenan-induced rat paw edema model. Specifically, the lactadherin powder prepared in Example 1 or the lactadherin decomposed product powder prepared in Example 3 was suspended in 0.5 wt% agueous carboxymethyl cellulose solution, and the suspension was orally administered to Wister male rats (body weight: 110 to 130 g, 10 rats per group) at a dose of lactadherin or lactadherin decomposed product of 5 or 50 mg/kg body weight. In the same manner as described above, lactoperoxidase was orally administered at a dose of 50 mg/kg body weight (i.e., positive control) . One hour thereafter, 1 wt% suspension of λ-carrageenan (i.e., inflammatory substance) in saline was subcutaneously injected (0.1 mL) to the right hindlimb footpad of a rat for initiation of edema. Before injection of λ-carrageenan and five hours after injection thereof, the area of the right hindlimb footpad was measured, to determine a percent increase in footpad volume (Vl). For a negative control, 0.5 wt% agueous carboxymethyl cellulose solution containing neither lactadherin, a decomposed product thereof, nor lactoperoxidase was orally administered to a rat, 1 wt% suspension of λ-carrageenan (i.e., inflammatory substance) in saline was subcutaneously injected (0.1 mL) to

SNOW-217 the right hindlimb footpad of the rat, and the percent increase in footpad volume (VO) of the rat was determined. The percent inhibition of carrageenan-induced edema was calculated by the following expression: { (VO-VI)/VO}xl00. Thus, a higher percent inhibition of edema indicates a higher anti-inflammatory effect. Table 3 shows the results.

[0025] [Table 3]

Test substance	Dose (mg/kg body weight)	Percent inhibition of carrageenaninduced edema (%)
Lactadherin	5	25.310.9
Lactadherin	50	41.211.2*
Lactadherin decomposed product	5	27.910.6
Lactadherin decomposed product	50	39.711.0*
Lactoperoxidase	50	29.111.3

Numerals: average 1 standard deviation (n=10).

*: significantly higher than the case of administration of 50 mg of lactoperoxidase (p<0.05).

[0026]

With reference to Table 3, oral administration of lactadherin or a decomposed product thereof at a dose of 5 mg/kg body weight or more increased a percent inhibition of carrageenan-induced edema, which was significantly higher than that in the case of oral administration of lactoperoxidase. These results indicate that lactadherin or a decomposed product thereof exhibits an anti-inflammatory effect.

Example 4

SNOW-217 [0027] (Preparation of anti-inflammatory capsule)

Raw materials were mixed in proportions shown in Table

4, and the mixture was granulated by a common process and then encapsulated, to prepare an anti-inflammatory capsule.

[0028] [Table 4]

Example sample 1	2.0 (wt%)
Lactose	33.5
Soluble starch	64.0
Magnesium stearate	0.5

Example 5 [0029] (Preparation of anti-inflammatory tablet)

Raw materials were mixed in proportions shown in Table

5, and the mixture was formed into a compact (1 g) by a common process, followed by tableting, to prepare an anti-inflammatory tablet.

[0030] [Table 5]

Aqueous crystalline glucose 88.5 (wt%)

Example sample 25.0

Mineral mixture5.0

Sugar ester1.0

Flavor0.5

SNOW-217

Example 6 [0031] (Preparation of anti-inflammatory liquid nutritional composition)

Example sample 1 (50 g) was dissolved in deionized water (4,950 g), and the solution was heated to 50°C. Thereafter, the solution was agitated with a TK homomixer (TK ROBO MICS, manufactured by PRIMIX Corporation) at 6, 000 rpm for 30 minutes, to prepare a lactadherin solution having a lactadherin content of 39 g/5 kg. The lactadherin solution (5.0 kg) was mixed with casein (5.0 kg), soybean protein (5.0 kg), fish oil (1.0 kg), perillaoil (3.0 kg), dextrin (17.0 kg), a mineral mixture (6.0 kg), a vitamin mixture (1.95 kg), an emulsifier (2.0 kg), a stabilizer (4.0 kg), and a flavor (0.05 kg). The mixture was placed into 200-mL retort pouches and sterilized with a retort sterilizer (Type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Hisaka Works, Ltd.) at 121°C for 20 minutes, to prepare an anti-inflammatory liquid nutritional composition (50 kg).

Example 7 [0032] (Preparation of anti-inflammatory beverage)

Powdered skim milk (300 g) was dissolved in deionized water (409 g) , and Example sample 3 (1 g) was dissolved in the solution. The solution was heated to 50°C and agitated with

SNOW-217 an ultra-disperser (ULTRA-TURRAX T-25, manufactured by IKA Japan) at 9, 500 rpm for 30 minutes. The resultant mixture was mixed with maltitol (100 g) , an acidulant (2 g) , reduced starch syrup (20 g), a flavor (2 g), and deionized water (166 g). Thereafter, the mixture was placed into 100-mL glass bottles and sterilized at 95°C for 15 seconds and then sealed, to prepare 10 anti-inflammatory beverages (100 mL each).

Example 8 [0033] (Preparation of anti-inflammatory feed for dog)

Example sample 1 (2 kg) was dissolved in deionized water (98 kg), and the solution was heated to 50°C. Thereafter, the solution was agitated with a TK homomixer (MARK II 160, manufactured by PRIMIX Corporation) at 3, 600 rpm for 40 minutes, to prepare a lactadherin solution having a lactadherin content of 1.56 g/100 g. The lactadherin solution (10 kg) was mixed with soybean lees (12 kg), powdered skim milk (14 kg), soybean oil (4 kg), corn oil (2 kg), palm oil (23.2 kg), corn starch (14 kg), flour (9 kg), bran (2 kg), a vitamin mixture (5 kg), cellulose (2.8 kg), and a mineral mixture (2 kg), and the mixture was sterilized at 120°C for four minutes, to prepare an anti-inflammatory feed (100 kg).

2014226865 22 Aug 2018 [0034]

Throughout this specification and the claims which follow, unless the context requires otherwise, the wordcomprise, and variations such as comprises or comprising, will be 5 understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0035]

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which 15 this specification relates.

20A

2014226865 22 Aug 2018

Claims

The claims defining the invention are as follows:

[Claim 1]

Use of a decomposition product of lactadherin in the

5 manufacture of a medicament for treating or preventing inflammation, wherein the medicament is for oral administration to a subject in need thereof at a dose of from 5mg/kg body weight to 50 mg/kg body weight of the decomposition product, and wherein the decomposition product of lactadherin is prepared 10 by decomposition of lactadherin with at least one protease.
[Claim 2]

The use according to claim 1, wherein the decomposition product of lactadherin is derived from bovine milk.
[Claim 3]

15 The use according to claim 1 or 2, wherein the at least one protease is selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, and papain.
[Claim 4]

An anti-inflammatory food, drink, nutritional

20 composition, or feed comprising as an active ingredient a decomposition product of lactadherin, wherein the decomposition product of lactadherin is prepared by decomposition of lactadherin with at least one protease. [Claim 5]

25 A method of treating, preventing or ameliorating

2014226865 22 Aug 2018 inflammation in a subject, comprising orally administering to the subject an anti-inflammatory agent comprising a decomposition product of lactadherin at a dose of from 5mg/kg body weight to 50 mg/kg body weight of the decomposition product, 5 wherein the decomposition product of lactadherin is prepared by decomposition of lactadherin with at least one protease.

[Claim 6]

The method of claim 5, wherein the subject has inflammation .

10 [Claim 7]

The method of claim 5 or 6, wherein the decomposition product of lactadherin is derived from bovine milk.

[Claim 8]

The method of any one of claims 5 to 7, wherein the at

15 least one protease is selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, and papain.