AU2014226864B2

AU2014226864B2 - Infection protection agent

Info

Publication number: AU2014226864B2
Application number: AU2014226864A
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: CN105025914B; AU2014226864A1; CN105025914A; JP2014172862A; JP6346405B2; NZ711985A; MY179921A; TWI646969B; WO2014136931A1; TW201446257A; HK1210942A1

Abstract

This infection protection 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 infection protection agent can be easily and economically produced and can be safely ingested on a daily basis.

Description

The present invention relates to an infection phylactic agent (prophylactic and/or therapeutic agent) which exhibits an excellent protective effect against influenza virus infection, which is useful for prevention or treatment of influenza, and which has excellent stability and safety. The present invention also relates to an infection phylactic food, an infection phylactic drink, an infection phylactic nutritional composition, and an infection phylactic feed, each of which contains the infection phylactic agent.

BACKGROUND ART [0002]

Influenza viruses cause epidemics in humans through airborne infection every year. Recent sanitary improvements and medical advances have reduced the threat of influenza epidemics, but influenza viruses could still cause death. Influenza viruses are classified into three types A, B, and C. Among these, influenza A virus is likely to mutate and to cause pandemics. Vaccination is a common technique for preventing influenza virus infection. Unfortunately, vaccination is not necessarily satisfactory for prevention of the infection,

SNOW-216 because an epidemic influenza virus, which is likely to undergo antigenic shift or antigenic drift, may often be inconsistent with the antigen for vaccination. Thus, preventive vaccination in children is not currently mandatory in Japan. Influenza therapeutics, such as amantadine, oseltamivir, and zanamivir, require taking into account side effects (e.g., hallucination and sleep disorder) and emergence of resistant bacteria, and thus such a drug must be used with great care. Under these circumstances, a demand has arisen for development of a food, drink, or feed which can be safely ingested routinely and is expected to be useful for prevention or treatment of influenza .

Traditional food ingredients having a protective effect against influenza virus infection include sphingomyelin, lactoferrin, and κ-casein glycomacropeptide, which is produced by the action of rennet or pepsin on κ-casein. It has also been known that an extracellular polysaccharide produced by Lactobacillus bulgaricus 1073R-1, which is one of lactobacilli contained in fermented milk, acts on the intestinal tract to increase the amount of IgA produced, and enhances the NK activity via Peyer's patches to exhibit a protective effect against influenza virus infection.

[0003]

Lactadherin is a glycoprotein present in milk fat globule membrane, and accounts for 10% of proteins contained in bovine

SNOW-216 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. Unfortunately, lactadherin and/or a decomposed product thereof has not yet been known to have a protective effect against influenza virus infection.

RELATED ART [0004]

Patent Document 1: Japanese Patent Application Laid-Open No. 2008-37788

NON-PATENT DOCUMENT [0005]

Non Patent Document 1: Bioscience, Biotechnology and Biochemistry, 57, 1214-1215, 1993

Non Patent Document 2: International Immunopharmacology, 11, 2246-2250, 2011

SUMMARY OF INVENTION

2014226864 22 Aug 2018 [0006]

Embodiments of the present invention provide an infection phylactic agent which exhibits a protective effect against influenza virus infection, and is useful for prevention or 5 treatment of influenza. Another embodiment provides an infection phylactic food, an infection phylactic drink, an infection phylactic nutritional composition, and an infection phylactic feed, each of which contains the infection phylactic agent.

[0006a]

According to a first aspect, the present invention provides use of purified lactadherin and/or a decomposition product thereof in the manufacture of a phylactic agent for protection against influenza virus infection.

[0006b]

According to a second aspect, the present invention provides an infection phylactic food, drink, nutritional composition, or feed comprising a phylactic agent protecting against influenza virus infection, wherein the phylactic agent 20 comprises purified lactadherin or a decomposition product thereof .

2014226864 22 Aug 2018 [0006c]

According to a third aspect, the present invention provides a method of preventing or treating influenza virus infection in a subject comprising administering to the subject a phylactic agent comprising purified lactadherin and/or a decomposition product thereof.

[0007]

The present inventors have conducted extensive studies for solving the above-described problems, and have consequently found that lactadherin and/or a decomposed product thereof has an excellent protective effect against influenza virus infection .

The present invention includes the following aspects:

Aspect (1) . An infection phylactic agent against influenza viruses infection, containing lactadherin and/or a decomposed product thereof as an active ingredient.

Aspect (2) . The infection phylactic agent against influenza viruses a infection acording to Aspect (1) , wherein lactadherin 20 and/ora decomposed product thereof is derived from bovine milk.

Aspect (3) . The infection phylactic agent against influenza

4A

SNOW-216 viruses infection according to Aspect (1) or (2), wherein the decomposed product of lactadherin is prepared by decomposition of lactadherin with a protease.

Aspect (4). The infection phylactic agent against influenza viruses infection 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 infection phylactic food, drink, nutritional composition, or feed comprising the infection phylactic agent against influenza viruses infection according to any one of Aspects (1) to (4).

Aspect (6) A method of protecting against influenza virus infection comprising orally ingesting the infection phylactic agent infection according to any one of Aspects (1) to (4).

Aspect (7) A method of using an infection phylactic agent containing lactadherin and/or a decomposed product thereof as an active ingredient, the method comprising applying the infection phylactic agent to a patient with influenza virus infection.

EFFECTS OF INVENTION [0008]

The infection phylactic agent ( prophylactic and/or therapeutic agent ) of the present invention exhibits a remarkable protective effect against influenza virus infection,

SNOW-216 and is useful for prevention or treatment of influenza.

DESCRIPTION OF EMBODIMENT [0009]

Lactadherin, which is an active ingredient of the infection phylactic 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 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

SNOW-216 microfiltration (MF) membrane, a reverse osmosis (RO) membrane, or an ion-exchange resin. A lactadherin decomposed product, which is an active ingredient of the infection phylactic 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. 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

SNOW-216 used as an infection phylactic 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 infection phylactic 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 infection phylactic 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, and polyphenols. The infection phylactic food, drink, nutritional composition, or feed may contain any component exhibiting an infection phylactic effect, such as sphingomyelin or lactoferrin in combination with lactadherin and/or a decomposed product thereof, which is an active ingredient. [0013]

The infection phylactic food, drink, nutritional composition, or feed may contain lactadherin and/or a

SNOW-216 decomposed product thereof in any amount. In order to achieve a protective effect against influenza virus infection, 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 0.1 mg or more per adult. [0014]

The infection phylactic 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, adisintegrant, a surfactant, or a lubricant. Examples of the excipient includes sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, 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

SNOW-216 embodiments, examples, and operational techniques will be apparent to those skilled in the art from this disclosure.

For example, a method of using the infection phylactic agent is provided, wherein the infection phylactic agent, which contains lactadherin and/or a decomposed product thereof described in the embodiments as an active ingredient, is applied to a patient with influenza virus infection. 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 infection phylactic 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]

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

SNOW-216 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 infection phylactic 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 (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) (4 63 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 infection phylactic agent without any treatment.

SNOW-216

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 infection phylactic agent without any treatment.

[0020] [Test Example 1] (Determination of protective effect against influenza virus infection)

Mice (Balb/c, male, 6 weeks old) were nasally infected with A/Guinzhou virus (i.e., influenza A virus) or B/Ibaraki virus (i.e., influenza B virus). A solution of Example sample 1 (lactadherin content: 100 pg/mL) and a solution of Example sample 2 (lactadherin decomposition compound content: 100 μρ/mL) were prepared. Each of the solutions was nasally administered at a dose of 1 μΕ/ηηεηΙ cavity (dose: 0.1 μρ) , and

SNOW-216 the protective effect against influenza virus infection was determined by the virus titer in nasal cavity washes. For comparison, a group of mice nasally infected with each of the influenza viruses was provided as a control. The plaque method using MDCK cells was used for determination of the protective effect. Table 1 shows the results.

[0021] [Table 1]

Sample Nasal cavity virus titer (n) (PFU/mL; 10ⁿ)

A/Guinzhou virus	Control	3.12 ± 0.16
	Example sample 1	1.8610.10*
	Example sample 2	2.01 ±0.11 *
B/lbaraki virus	Control	2.98 ±0.09
	Example sample 1	2.6710.12*
	Example sample 2	2.7510.06*

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

*: significantly lower than the corresponding control (p<0.05).

[0022]

With reference to Table 1, nasal administration of lactadherin or the lactadherin decomposition compound exhibited a protective effect against infection with influenza A and B viruses, particularly against infection with influenza A virus .

[0023] [Test Example 2] (Determination of protective effect against influenza virus infection through oral administration)

SNOW-216

Mice (Balb/c, male, 6 weeks old) were infected with influenza virus PR8 (H1N1) (lxlO³ pfu) . Before the viral infection, mice were divided into a group of oral administration of lactadherin (Example sample 1) or the lactadherin decomposed product (Example sample 3) (dose: 0.1 mg/kg body weight), and a group of oral administration of lactadherin (Example sample 1) or the lactadherin decomposed product (Example sample 3) (dose: 10 mg/kg body weight). The protective effect against influenza virus infection was determined by the virus titer in nasal cavity washes three days after the viral infection.

For comparison, a group of mice with oral administration of lactoferrin (10 mg/kg body weight) before the viral infection was provided as a positive control, and a group of mice with oral administration of a solvent (only water) before the viral infection was provided as a control. The plaque method using MDCK cells was used for determination of the protective effect. Table 2 shows the results.

[0024] [Table 2]

Sample

Nasal cavity virus titer (n) (PFU/mL; 10ⁿ)

Control

Lactadherin 0.1 mg

Lactadherin decomposed product 0.1 mg

Lactadherin 10 mg

Lactadherin decomposed product 10 mg

Lactoferrin 10 mg

3.06 ± 0.08

2.75 ±0.12*1

2.83 ±0.11 *1

2.10 ±0.04*1, *2

2.13 ±0.06*1, *2

2.29 ±0.10*1

Numerals: average ± standard deviation (n=10)

SNOW-216 *1: significantly lower than control (p<0.05) *2: significantly lower than lactoferrin 10 mg (p<0.05) [0025]

With reference to Table 2, oral administration of lactadherin or the lactadherin decomposed product significantly reduced the nasal cavity virus titer, as compared to the control. The protective effect of lactadherin or the lactadherin decomposed product was significantly higher than that of orally administered lactoferrin. These results indicate that lactadherin or a decomposed product thereof exhibits an excellent protective effect against influenza virus infection. The results also indicate that this effect is achieved through oral administration of the infection phylactic agent at a dose of 0.1 mg/kg mouse body weight or more. Example 4 [0026] (Preparation of capsule for protection against influenza virus infection)

Raw materials were mixed in proportions shown in Table 3, and the mixture was granulated by a common process and then encapsulated, to prepare a capsule for protection against influenza virus infection.

SNOW-216 [0027] [Table 3]

Example sample 2	2.0 (wt%)
Lactose	33.5
Soluble starch	64.0
Magnesium stearate	0.5

Example 5 [0028] (Preparation of tablet for protection against influenza virus infection)

Raw materials were mixed in proportions shown in Table

4, and the mixture was formed into a compact (1 g) by a common process, followed by tableting, to prepare a tablet for protection against influenza virus infection.

[0029] [Table 4]

Aqueous crystalline glucose 88.5 (wt%)

Example sample 35.0

Mineral mixture5.0

Sugar ester1.0

Flavor0.5

Example 6 [0030] (Preparation of liquid nutritional composition for protection against influenza virus infection)

SNOW-216

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 a liquid nutritional composition for protection against influenza virus infection (50 kg).

Example 7 [0031] (Preparation of beverage for protection against influenza virus infection)

Powdered skim milk (300 g) was dissolved in deionized water (409 g) , and Example sample 2 (1 g) was dissolved in the solution. The solution was heated to 50°C and agitated with 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

SNOW-216 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 beverages for protection against influenza virus infection (100 mL each).

Example 8 [0032] (Preparation of dog feed for protection against influenza virus infection)

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 a feed for protection against influenza virus infection (100 kg).

2014226864 22 Aug 2018 [0033]

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.

[0034]

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.

18A

2014226864 22 Aug 2018

Claims

The claims defining the invention are as follows: [Claim 1]

Use of purified lactadherin and/or a decomposition

5 product thereof in the manufacture of a phylactic agent for protection against influenza virus infection.
[Claim 2]

The use according to claim 1, wherein the lactadherin and/or a decomposition product thereof is derived from bovine milk.

10 [Claim 3]

The use according to claim 1 or 2, wherein the decomposition product of lactadherin is prepared by decomposition of lactadherin with at least one protease . [Claim 4] 15 The use according to claim 3, wherein the at least one

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

[Claim 5]

An infection phylactic food, drink, nutritional

20 composition, or feed comprising a phylactic agent protecting against influenza virus infection, wherein the phylactic agent comprises purified lactadherin or a decomposition product thereof .

[Claim 6]

25 A method of preventing or treating influenza virus

2014226864 22 Aug 2018 infection in a subject comprising administering to the subject a phylactic agent comprising purified lactadherin and/or a decomposition product thereof.

[Claim 7]

5 The method of claim 6, comprising orally administering the phylactic agent to the subject.

[Claim 8]

The method of claim 6 or 7, wherein the subject has an influenza virus infection.

10 [Claim 9]

The method of any one of claims 6 to 8, wherein the lactadherin and/or a decomposition product thereof is derived from bovine milk.

[Claim 10]

15 The method of any one of claims 6 to 9, wherein the decomposition product of lactadherin is prepared by decomposition of lactadherin with at least one protease. [Claim 11]

The method of claim 10, wherein the at least one protease

20 is selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, and papain.