NZ519957A - Antigen-specific IgE antibody production inhibitors - Google Patents

Abstract

A mammalian antigen specific IgE antibody production inhibitor and immunomodulatory compositions, each of which comprises as an active ingredient lactoferrin or a hydrolyzate thereof. This invention is also concerned with IFN-g production potentiators, each of which comprises LF or its hydrolyzate and a ceramide.

Description

1 DESCRIPTION ANTIGEN-SPECIFIC IGE ANTIBODY PRODUCTION INHIBITORS Technical Field This invention relates to IgE production inhibitors effective for the prevention or amelioration of allergic diseases such as atopic dermatitis. This invention is also concerned with compositions which stimulate the immune systems in living bodies to potentiate immunity.

Background Art Lactoferrin (LF) is an iron-binding glycoprotein contained not only in milk but also in exocrine fluids such as saliva, tear, bile juice and pancreatic juice. It is also released by neutrophils activated at an inflammation site.

LF has a broad spectrum of biological activities. For example, antimicrobial activity, antiviral activity, antitumor activity, cancerous metastasis inhibitory activity and the like are included.

Further, LF affects cytokine excretion. For example, LF promotes excretion of interleukin-8 (IL-8) from human neutrophils, controls excretion of IL-1, IL-6 and TNFa, and inhibits gene-level expression of GM-CSF.

Selective production of cytokine plays a protective role or etiologic role in various morbidities. For example, a majority of allergies are type I allergies with which IgE antibody is associated. This IgE production is induced by a process involving IL-4, and on the other hand, is controlled 5 by a process involving interferon-y (IFN-y). Production of IFN-y is induced by its epistatic IL-12 and IL-18.

IFN-y is also shows antiviral activity, antimicrobial activity and antitumor activity, which rely upon various immunomodulating functions (J. Immunol., 130, 2011-2013, 1983; Proc. Natl. Acad. Sci. USA, 85, 4874-4878, 1988).

Nakajima et al. reported that, when splenocytes of a mouse were incubated in the presence of mitogen (Con A) subsequent to oral administration of bovine LF (bLF), production of IFN-y was enhanced but production of IL-4 was 15 not enhanced (Biomedical Research, 29(1), 27-33, 1999).

As mentioned above, LF takes part in the production and control of diverse biological activities and cytokine. An object of the present invention is, therefore, to uncover a new biological activity of LF and based on the activity, to 20 provide pharmaceuticals and functional foods which contain LF as an active ingredient. Another object of the present invention is to find an orally-ingestable substance which has an effect to enhance biological activities of LF when combined with LF.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common gene* I -ACTUAL PROPERTT New Zealand as at the priority date of any of the claims. | office of n.z - 3 APR 2003 RECEIVED Disclosure of the Invention The present inventors have found that, when a mouse which has orally ingested LF, heat-treated LF or an LF hydrolyzate is immunized with ovalbumin (OVA) , a principal food allergen, production of an OVA-specific IgE antibody is inhibited and production of an antigen-specific IL-4 in a splenocyte incubation system is also inhibited. They have also found that, when macrophages are incubated in the presence of LF, production of IL-12 by the macrophage is enhanced. In addition, they have also found that combined use of LF with a ceramide pronouncedly enhances production of IFN-y in the splenocyte culture system. The inhibition of the production of the antigen-specific IgE antibody by the oral ingestion of LF is considered to result from inhibition of activation of IgE antibody producing B lymphocytes by the inhibition of IL-4 production and also from a shift toward Thl dominance in the Thl/Th2 balance by the enhancement of production of IL-12. Namely, oral ingestion of LF can prevent a reduction in cellular immunity, which would otherwise take place by a shift toward Th2 response, and can prevent or ameliorate nephritis resulting from an allergic disease or an antibody reaction. The pronounced enhancement of IFN-y production by the combination of LF and the ceramide indicates that the combination of LF and a ceramide is effective against viral diseases, bacterial infections or tumors.

Described specifically, the present invention provides a mammalian (including human) IgE antibody production inhibitor comprising as an active ingredient LF or a hydrolyzate thereof.

The present invention also provides a composition for controlling immunity by inhibiting production of a mammalian (including human) IgE antibody, which comprises as an active ingredient LF or a hydrolyzate thereof.

The present invention also provides an IFN-y production potentiator composition, antiviral agent composition, antibacterial agent composition or antitumor agent composition for mammals including humans, which comprises LF or a hydrolyzate thereof and a ceramide.

The present invention also provides an immunopotentiator composition for mammals including humans, which comprises LF or a hydrolyzate thereof and a ceramide.

The present invention also provides use of LF or a hydrolyzate thereof for the preparation of a mammalian (including human) IgE antibody production inhibitor.

The present invention also provides use of LF or a hydrolyzate thereof for the preparation of a composition for controlling immunity by inhibiting production of a mammalian (including human) IgE antibody.

The present invention further provides use of LF or a hydrolyzate thereof and a ceramide for the preparation of an IFN-y production potentiator composition, antiviral agent composition, antibacterial agent composition or antitumor agent composition for mammals including humans.

The present invention further provides use of LF or a 5 hydrolyzate thereof and a ceramide for the preparation of an immunopotentiator composition for mammals including humans.

The present invention still further provides a method for inhibiting production of an IgE antibody in a mammal including a human, which comprises administering an effective 10 amount of LF or a hydrolyzate thereof.

The present invention still further provides a method for modulating production of an IgE antibody in a mammal including a human, which comprises administering an effective amount of LF or a hydrolyzate thereof. 15 The present invention still further provides a method for enhancing production of IFN-y in amammal including a human, which comprises administering effective amounts of LF or a hydrolyzate thereof and a ceramide.

The present invention still further provides a method 20 for immunopotentiating a mammal including a human, which comprises administering effective amounts of LF or a hydrolyzate thereof and a ceramide.

Brief Description of the Drawings FIG. 1 is a diagrammatic representation of overall IgE concentrations in serum (mean±SD; *p<0.05) after OVA antigen sensitization of mice in a 1% bovine LF oral ingestion group (■: n=7) and a control group (□: n=8).

FIG. 2 is a diagrammatic representation of OVA-specific IgE antibody levels in the serum (mean ± SD; *p<0.05).

FIG. 3 is a diagrammatic representation of IL-4 producing abilities of splenocytes from mice (cells were pooled in the respective groups) . • : 1% bovine LF oral ingestion group, ■: control group.

FIG. 4 is a diagrammatic representation of total IgE antibody concentrations in mouse serum after sensitization with OVA antigen (three times) , for a 2 . 2% heat-treated bovine LF oral ingestion group (n=19) and a control group (n=17) (*p<0.05) .

FIG. 5 is a diagrammatic representation of OVA-specific IgE antibody levels in serum (*p<0.05).

FIG. 6 is a diagrammatic representation of production of OVA-specific IFN-y by splenocytes from mice (bovine LF oral ingestion group: n=ll, control group: n=10).

FIG. 7 is a diagrammatic representation of production of OVA-specific IL-4 by splenocytes from mice (*<0.05).

FIG. 8 is a diagrammatic representation of production of OVA-specific IFN-y by splenocytes from mice after sensitization with OVA antigen (four times), for a 2.2% heat-treated bovine LF oral ingestion group (n=8) and a control 7 group (n=7).

FIG. 9 is a diagrammatic representation of productions of OVA-specific IL-4 by splenocytes from mice (control group: 7 mice, 2.2% bLF: 8 mice).

FIG. 10 is a diagrammatic representation of total IgE antibody concentrations in mouse serums after sensitization with OVA antigen, for a 1% bovine LF hydrolyzate oral ingestion group (n=7) and a control group (n=8) (*p<0.05).

FIG. 11 is a diagrammatic representation of effects on 10 OVA-specific IgE antibody levels in the serum.

FIG. 12 is a diagrammatic representation of productions of OVA-specific IFN-y by splenocytes from mice.

FIG. 13 is a diagrammatic representation of productions of OVA-specific IL-4 by splenocytes from mice. 15 FIG. 14 is a diagrammatic representation of productions of IL-12 in culture supernatants after mouse peritoneal macrophages were incubated in the presence of 100 pg/mL of bovine LF (**p<0.01).

FIG. 15 is a diagrammatic representation of production 20 of IFN-y in culture supernatants when mouse splenocytes after peritoneal administration of lactosylceramide and splenocytes after non-administration of lactosylceramide were incubated in the presence or absence of 100 jjg/mL of bovine LF.

Best Modes for Carrying Out the Invention In many allergic diseases, allergen-specific IgEs are known to take part in their onset. As a matter of fact, allergen-specific IgEs are often observed in the serum of allergic patients (Allergy Clin. Immunol., 16, 161, 1996). Inhibition of IgE production is, therefore, considered to be an effective method for the prevention and treatment of allergy.

In the production of IgE, a cytokine produced by CD4 + helper T cells (Th cells) plays an important role. Depending upon the produced cytokine and its function, Th cells are roughly classified into two subgroups, Thl cells associated with cellular immunity and Th2 cells associated with humoral immunity, and it is Th2 cells that take part in the production of IgE (J. Immunol., 136, 2348, 1986).

Thl cells and Th2 cells are correlated with each other such that they inhibit each other in differentiation and functional expression by which cytokines they produce. Described specifically, Th2 cells produce IL-4, IL-5 and IL-6 to control IgE production. Conversely, IFN-y produced by Thl cells inhibits IgE production by inhibiting IgE class switch and Th cell response. Accordingly, IgE production and allergic diseases are enhanced by Th2 cell response (Nature, 383, 787, 1996). Their prevention and amelioration are, therefore, feasible by inducing a Thl cell response to return the Thl/Th2 balance to normal. Attempts have, hence, been made to screen food components for factors which induce Thl response. Certain types of lactic acid bacteria (Int. Arch. Allergy Immunol., 115, 278, 1998), nucleotides (Int.. Arch. Allergy Immunol. , 122, 33, 2000) and the like have heretofore been found to show this effect.

In recent years, biological defense effects by oral administration of LF have been reported on various animal models. Using mice, the present inventors, therefore, first conducted a study on the production of total IgE and an antigen-specific IgE in serum when the mice were allowed to orally ingest LF ad libitum. As a result, it has been found that ad libitum oral ingestion of LF inhibits the production of total IgE antibody and the production of an antigen-specific IgE.

The physiological activities of LF are known to drop by heat treatment (J. Pediatr., 90, 29, 1977). It was, therefore, postulated that the ability of LF ,to inhibit the production of total IgE and the production of antigen-specific IgE would be lowered by subjecting LF to heat treatment. A study was, hence, conductedon possible effects of heat-treated LF on the production of total IgE antibody and the production of antigen-specificIgE. Further, splenocyteswere incubated in the presence of OVA and the levels of cytokines (IFN-y, IL-4) in the culture supernatant was determined. As a result, it has been found that the inhibitory activities of LF against the production of an antigen-specific IgE antibody and the production of IL-4 are not lost even when LF is heated.

It has been found that, when LF or heat-treated LF is orally ingested, the production of total IgE antibody and the production of an antigen-specific IgE are inhibited significantly compared with a control group. It has also been found that, when splenocytes sensitized with an antigen are incubated in the presence of the antigen, the production of IL-4 is lowered significantly compared with a control group.

A study was then conducted on possible effects on the production of total IgE antibody and the production of antigen-specific IgE when hydrolyzed LF is orally ingested ad libitum. In addition, splenocytes sensitized with an antigen were incubated in the presence of the antigen and the levels of cytokines (IFN-y, IL-4) in the supernatant were determined. As a result, the production of total IgE antibody and the production of the antigen-specific IgE were observed to have a tendency to lower in the LF hydrolyzate ingestion group. On the other hand, the production of IFN-y was demonstrated to have a tendency to increase in the presence of OVA irrespective of its concentration in the LF hydrolyzate ingestion group, but concerning IL-4, no difference was observed between the LF hydrolyzate ingestion group and the control group. 11 IL-12 is a cytokine produced by antigen presenting cells such as monocytes, macrophages and dendrintic cells, and plays an important role in the production of IFN-y and the induction of differentiation of helper T cells into Thl (Blood, 84, 4008, 5 1994; J. Leukoc. Biol., 55, 280, 1994). Gazzinelli et al. reported that infection of mice with Toxoplasma gondii leads to an increase in the production of IL-12 by cells in the spleen or the peritoneal cavity and subsequent administration of an anti-IL-12 antibody to the mice leads to a decrease in the 10 production of IL-12 by splenocytes and to an increase in the production of IL-4 and IL-10 (J. Immunol., 153, 2533, 1994) . Further, Nastala et al. have found that administration of IL-12 to cancer-transplanted mice inhibits growth of the cancer and increases in the concentration of IFN-y in blood (J. Immunol. , 15 153, 1697, 1994) .

Production of IL-12 was, thus, studied by incubating mouse peritoneal macrophages, which had been induced in a thioglycolate medium, together with LF. As a result, it has been found that LF induces production of IL-12 by macrophages . 20 A atopic dermatitis was previously considered to be a mild form of dermatitis, but is now regarded as an intractable diseases observed in all ages (infants to adults), and has become a serious problem to the society. A metabolic disorder of sphingolipid in the epidermis is thought to be a possible 25 cause. In fact, a decrease in the amount of ceramides in the epidermis is observed in many atopic dermatitis patients. Ceramides are also finding utility as nutraceuticals and functional foods. The present inventors, therefore, came to a postulation that oral ingestion of LF and a ceramide in combination enhances the effects of LF over LF alone in the prevention and amelioration of allergic diseases.

The present inventors, therefore, conducted a study on possible effects of the combination of LF and a ceramide on the production of IFN-y. As a result, it has been found that the combination of LF and a ceramide pronouncedly enhances the production of IFN-y in splenocytes . Among sphingolipids, sphingolipids (sphingosine, sphingomyelin, lysophophatidylcholine) in kefir (fermented milk) are known to promote production of IFN-3 (Biotherapy, 115-123, 1994) . It was also reported that blood IFN-3 and IFN-y in infected mice increased by administration of sphingosine (Osada, K. et al., Animal Cell Technology: Development towards the 21st Century, 1067-1071, 1995).

From these results, the inhibition of production of an antigen-specific IgE antibody by oral ingestion of LF is considered to be attributable to the inhibition of activation of IgE-bearing B lymphocytes by the inhibition of IL-4 production and also to a shift towards Thl response in the Thl/Th2 balance by the enhancement of IL-12 production. Therefore, oral ingestion of LF is effective for the prevention 13 and amelioration of allergic inflammatory diseases. Accordingly, addition of LF in an effective amount as a supplement to foods makes it possible to prevent and ameliorate allergic diseases through diet, and LF can also be used as 5 a pharmaceutical for these diseases. Illustrative of allergic inflammatory diseases are chronic bronchial asthma, atomic dermatitis, pollinosis (allergic rhinitis), allergic vaculitis, allergicconjunctivitis, allergic gastroenteritis, allergic hepatopathy, allergic urocystitis, and allergic 10 purpura. LF can also be expected to prevent and ameliorate nephritis or the like caused by antibodies.

As production of IFN-y is pronouncedly enhanced by combined use of LF and a ceramide, it is appreciated that combined use of LF and a ceramide inhibits production of IgE 15 antibodyby a shift towards Thl dominance in the Thl/Th2 balance. IFN-y is known to display antiviral activity, antitumor activity, cytotoxic T lymphocyte inducing effect, natural killer cell (NK cell) activity inducing effect, neutrophil activating effect, macrophage activating effect, MHC class 20 II expression promoting effect, IL-2 receptor expression promoting effect, Fc receptor expression promoting effect, and the like. IFN, on the other hand, is basically a biosubstance which takes part in the defense mechanisms of a living body against foreign materials, is extremely high 25 in selective toxicity, and has high safety to the living body.

When a food or the like which contains a composition of LF and a ceramide in combination is orally ingested, the composition stimulates cells of the living body to promote excretion of IFN-y and to draw the above-described physiological activities of IFN-y. Therefore, a composition with LF and a ceramide contained in combination therein can be used as an antiviral agent, an antitumor agent and/or a antibacterial agent. Concerning the safety of LF, absolutely no abnormality was observed even by administration of bovine LF at a maximum dose, 2,000 mg/kg/body weight/day, in both the acute toxicity test and the subacute toxicity test of bovine LF, which were conducted using rats (Milk Science, 48(3), 227-232, 1999) . As to the safety of ceramides, LD5o of Oryza Ceramide (a ceramide derived from the seed of Oryza Sativa Linne) for mice is 5,000 mg/kg or more (FOOD Style, 4(10), 99-105, 2000) .

Examples of LF usable in the present invention can include commercially-available LF, LFs isolated by a method known per se in the art (for example, ion-exchange chromatography or the like) from colostrum, transitional milk, normal milk, late lactation milk and the like of mammals (for example, humans, cows, sheep, goats, horses, or the like) or skimmilks or wheys derived from these milks, their hydrolyzate by an acid or an enzyme, apoLF obtained by removing iron with hydrochloric acid, citric acid or the like, andmetal saturated or partially saturated LFs obtained by chelating apoLF with metals such as iron, copper, zinc and manganese.

For the preparation of LF hydrolyzate in the present invention, a known hydrolytic method can be used. When an 5 enzyme is used for the hydrolysis, enzymes other than trypsin, for example, hepsin and papain are usable although trypsin was used in the present invention. No limitation is imposed on the enzyme insofar as the hydrolyzate has the activities according to the present invention. The hydrolyzate may be 10 subjected to concentration by inactivating the enzyme under heat and then conducting fractionation by a method known per se in the art such as ultrafiltration. The resulting liquid hydrolyzate may be used as is, or may be used after lyophilization.

Recombinant LFs include polypeptides having substantially the same amino acid sequence as that disclosed by Orla M. Conneely et al. (U.S. Patent 5,766,939). Also included are naturally occurring alleles and LFs modified by insertion, substitution or depletion of one or more amino acids 20 compared with natural LF. Recombinant LFs also include recombinant LFs expressed in transgenic animals, for example, bovine. Their glycosylation patterns can be different from that of natural LF obtained from human milk.

Ceramides usable in the present invention include 25 ceramide-related substances. For example, sphingolipids, 16 especially sphingoglycolipids are preferred. Examples of sphingoglycolipids can include cerebroside found as the most simple sphingoglycolipid in milk, brain and kidney, sulfatide with a sulfate group added to cerebroside, ceramide 5 oligohexoside.with several neutral sugar molecules added to cerebroside, and gangliosides with cyanuric acid added to cerebroside. There are more than 100 sphingoglycolipids the structures of which have been ascertained relying upon differences in sphingoglycolipid sugar chains. 10 Sphingoglycolipids are all included insofar as they have effects in the present invention. Preferred examples can include lactosylceramide, galactosylceramide and glycosylceramide. Further, sphingomyelin (which is contained in milk), a phospholipid, is also preferred. In 15 addition, asymmetrical synthesis technology or the like has made it possible to chemically synthesize natural ceramides, and development of optically active ceramides is under way. They include ceramide2, and ceramide3 (of Cosmo Farm Co. , Ltd. ) . These ceramides are usable in the present invention insofar 20 as they have effects in the present invention. Moreover, plant-derived ceramides are attracting attention, and are finding utility. In a rice-derived sphingolipid, for example, a hydrophobic ceramide with a fatty acid bonded through an acid-amide structure to a long-chain basic sphingosine is a 25 basic skeleton like animal sphingolipids. The rice-derived 17 sphingolipid has diversity in molecular varieties depending upon differences in the number of carbon atoms of the long-chain basic sphingosine and fatty acid and existence or non-existence of hydroxyl groups or double bonds. According to a report 5 by Professor Fujino of Obihiro University of Agriculture and Veterinary Medicine, there are at least more than 20 molecular varieties of sphigolipid. The present invention includes these ceramides.

Concerning the content of LF (including all LFs, LF 10 derivatives having equivalent physiological activities to the LFs, and hydrolyzates of the LFs and LF derivatives) and the ceramide in the present invention, the content of the ceramide is estimated to range preferably from 0.0001 to 0.1 wt.%, more preferably from 0.0005 to 0.01 wt.%, particularly preferably 15 from 0.0001 to 0.05 wt.%, all based on the total weight of the composition, and that of LF is estimated to range preferably from 0.005 to 10 wt.%, particularly preferably from 0.01 to 5 wt.%, most preferably from 0.01 to 3 wt.%, all based on the total weight of the composition. A person skilled in the art 20 is, however, believed to be able to readily determine their optimal contents in each target composition, for example, by the below-described experiment. Incidentally, the content of ceramides in breast milk is known and is believed to serve to at least some extent as a standard for the amount of a ceramide 25 to be added. 18 a» « a J It has been clarified by the present invention that oral ingestion of LF inhibits production of an antigen-specific IgE antibody. It is, therefore, possible for a person having ordinary skill in the art to choose, based on the present invention, an LF of a preferred form applicable to the present invention from the above-mentioned various LFs and its effective amount, for example, by using the production of an IgE antibody as an index; and in view of a correlation between clinical data, clinical symptoms or the like and the serum IgEs of patients, to. determine the shape and effective dose of the LFupon using it in foods, supplements, foods for invalids, modified milk powders for babies, health foods, health claim foods, functional foods, specific health foods, pharmaceuticals or the like. Accordingly, the types of LFs and their effective dosages determined as described above are embraced in the present invention. Technology to add or process LF into foods and the like is well known and is in common use. Upon using LF as a pharmaceutical in an effective amount, it can be used by formulating it into various preparation forms known to those skilled in the art.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

Examples The present invention will hereinafter be described based on Tests and Examples. It should however be borne in mind that the present invention is not limited by these Tests 19 and Examples.

In the following Tests, male immature BALB/c mice of 3 weeks old (Japan SLC, Inc., Shizuoka, Japan) were used in Tests 1-4 and 6, and female BALB/c mice of 8 weeks old (Japan 5 SLC, Inc., Shizuoka, Japan) were used in Test 5. A test of significance between each control group and its corresponding experiment group was conducted by Student's t test.

Test 1 IgE antibody production inhibiting activity of bovine LF (bLF) During the period of the experiment, mice in an LF administration group (n=7) were allowed to ingest ad libitum a feed (AIN 76 based), which contained casein as a protein source, and water which contained 1% bovine LF (bLF, product of DMV Japan K. K.) . Mice in a control group (n=8) were allowed 15 to ingest ad libitum the above-described feed and bLF-free water.

On the 5th day and 19th day after initiation of the experiment, the mice were each intraperitoneally immunized with 10 )ag of ovalbumin (OVA, product of SEIKAGAKU CORPORATION) 20 together with 4 mg of aluminum hydroxide. On the 2.6th day, blood was drawn from the orbital cavity, and the overall IgE antibody level and OVA-specific IgE antibody level in the serum were assayed by ELISA. The total IgE antibody level (FIG. 1) and OVA-specific IgE antibody level (FIG. 2) in the serum 25 were both lower in the mice of the bLF group than those in the control group (*p<0.05).

Test 2 IL-4 production inhibiting activity of bLF In Test 1, the spleens were excised to prepare splenocyte suspensions. Subsequent to hemolyzation, splenocytes were incubated at various concentrations (5, 10, 50 and 100 jjg/mL) in the presence of OVA for 72 hours. After the incubation, the IL-4 level in each culture supernatant was assayedby ELISA. In the bLF administration group (n=7) , the IL-4 levels at the individual OVA concentrations were all lower than the corresponding IL-4 levels in the control group (n=8).

Test 3 IgE antibody and cytokine production inhibiting activity of heat-treated bLF A feed [bLF( + )], which had been obtained by adding 2 . 2% of bLF to the feed of Test 1, was heated at 70°C for 1 hour. As a control, a feed not added with bLF [bLF (-) ] was similarly heated at 7 0°C for 1 hour.

Mice were allowed to ingest ad libitum bLF ( + ) (experiment group) or bLF(-) (control group) during the period of the experiment. On the 5th day, 14th day, 23rd day and 33rd day after the initiation of the experiment, each mouse was intraperitoneally immunized with ovalbumin (OVA, product of SEIKAGAKU CORPORATION) (10 jag) together with aluminum hydroxide (4 mg) . On the 8th day after the third peritoneal immunization (the 30th day) (7 weeks old), blood was drawn from the tail, and antibodies in the serum were assayed by 21 ELISA. Further, on the 11th day after the third peritoneal immunization (the 33rd day) and on the 8th day after the fourth peritoneal immunization (the 40th day), splenocytes were collected separately depending upon the individual mice. The 5 splenocytes were incubated together with OVA at various concentrations (0, 50, 100, 200 and 400 jag/mL) for 72 hours in RPMI 1640 medium which contained 10% of FCS, 100 U/mL of penicillin G, 100 pg/mL of streptomycin and 5 x 10"5 M of 2-mercaptoethanol. After the incubation, the IFN-y level and 10 IL-4 level in each culture supernatant were assayed by ELISA.

When the immunization was conducted three times, the total IgE antibody level and OVA-specific IgE antibody level in the serum of the experiment group lowered significantly compared with the corresponding ones in the control group (FIGS. 15 4 and 5) . Concerning the production of IFN-y by splenocytes at the respective OVA concentrations (0, 50, 100 and 200 yg/mL) , no substantial difference was observed between the experimental group and the control group (FIG. 6). The production of IL-4 by splenocytes at the respective OVA 20 concentrations, on the other hand, was generally lower in the experimental group than in the control group, and under stimulation by 200 |jg/mL of OVA, the production lowered more significantly in the experimental group than in the control group (FIG. 7).

When the immunization was conducted four times, the 22 production of IFN-y by splenocytes at the respective OVA concentrations (0, 50, 100, 200 and 400 jjg/mL) resulted in no substantial difference between the groups as in the example with three immunizations (FIG. 8). The production of IL-4 at the respective OVA concentrations, on the other hand, generally loweredmore significantly in the experimental group than in the control group (FIG. 9).

From the above results, it has been found that the antigen-specific IgE antibody production inhibiting activity and IL-4 production inhibiting activity of bLF are not lost by heating.

Test 4 IgE antibody and cytokine production inhibiting activity of hydrolyzed bLF (1) Preparation of hydrolyzed bLF Trypsin ("207-09891", product of Wako Pure Chemical Industries, Ltd., for biochemical use, produced from swine spleens, 4,500 USP trypsin units/mg) was dissolved in sterilized PBS toprovide it as a trypsin stock (x500, 50mg/mL) . bLF ("127-04122", product of Wako Pure Chemical Industries, Ltd., lot. KSG7724) (10 g) was dissolved in 25 mM CaCl2_0.1 M Tris-HCl (pH 8.2) to form a 1% solution. After 1 L of the bLF solution was heated to 37°C, the trypsin stock (2 mL) was added, followed by reaction at 37°C for 4 hours. Subsequent to the reaction, heating was conducted at 80°C for 30 minutes to inactivate the enzyme. After centrifugation at 1,700 x 23 g for 20 minutes, the supernatant was collected and filter-sterilized by a 0 . 4 5-jam filter. The sample was treated by "Micro Acilyezer SI" (an electrical dialysis machine manufacturedbyAsahi ChemicalIndustryCo., Ltd.) to eliminate 5 salts from the sample. After the sample was filter-sterilized again by a 0.4 5-yim filter, it was stored at -20°C until it was provided for an experiment. The yield of bLF hydrolyzate was substantially 100%. The concentration of the bLF hydrolyzate was 1%.

Mice were allowed to ingest ad libitum the feed of Test 1 and a solution which contained the bLF hydrolyzate at 1%. A control group was allowed to ingest ad libitum a similar solid feed and bLF-free water. On the 5th day and 19th day after the initiation of the experiment, each mouse was 15 intraperitoneally immunized with ovalbumin (OVA, product of SEIKAGAKU CORPORATION) (10 jjg) together with aluminum hydroxide (4 mg) . On the 8th day after the second immunization (the 26th day) , blood was drawn, and antibodies in the serum were assayed by ELISA. At that time, splenocytes were also 20 collected separately depending upon the individual mice. The splenocytes were incubated together with OVA (0 to 400 pg/mL) for 72 hours, and the IFN-y and IL-4 levels in each culture supernatant were assayed by ELISA.

The total IgE antibody level and OVA-specific IgE level 25 in the serum of the experimental group were observed to become lower compared with the corresponding levels in the control group (FIGS. 10 and 11).

The production of IFN-y by splenocytes at the various OVA concentrations was recognized to have a general tendency to be higher in the experimental group than in the control group (FIG. 12) . The production of IL-4, on the other hand, displayed no substantial difference between the experimental group and the control group (FIG. 13).

Test 5 Effect of LF on the production of IL-12 Although IL-12 is a cytokine found relatively recently, it is known to act on T cells and NK cells to induce production of IFN-y (J- Exp. Med., 173, 869-879, 1991; J. Exp. Med., 177, 1199-1204, 1993). For the induction of IL-12 production, stimulation by a cell component such as LPS is effective, and cells so produced include a wide variety of cells such as macrophages, B cells and neutrophils.

The present inventors incubated peritoneal macrophages in a medium with bLF (product of Wako Pure Chemical Industries, Ltd.) added therein, and assayed the level of IL-12 in the culture supernatant by ELISA.

Mice, which had been allowed to ingest ad libitum a feed "MF" (product of Oriental Yeast Co., Ltd.), were each intraperitoneally injected with a thioglycolate medium (product of DIFCO Laboratories) (2.5mL) to activate peritoneal macrophages. Four days later, Dulbeccos' phosphate buffer ("Dulbeccos' PBS", product of NISSUI PHARMACEUTICAL CO. , LTD.) with 1% FCS contained therein was injected into the peritoneal cavity to collect peritoneal cells. After the cells were washed twice, they were suspended again in "Dulbeccos' PBS" 5 and subsequent to addition to a 96-well plate (2 x 105 cells/0.2 mL well) , the plate was incubated for 2 hours in a C02 incubator. After non-adsorbed cells were removed, each well was filled with RPMI 1640 medium (200 yaL) which contained 10% of FCS, 100 U/mL of penicillin G, 100 pg/mL of streptomycin, 5 x 10"5 10 M of 2-mercaptoethanol and 100 ug/mL of bLF (product of Wako Pure Chemical Industries, Ltd.). As a control, the above-described medium was used without bLF. Each plate was incubated for 18 hours in a CO2 incubator. After the incubation, the IL-12 level in each supernatant was assayed by ELISA. The 15 results are shown in FIG. 14 . The addition of bLF to the medium pronouncedly enhanced the production of IL-12 by macrophages.

From the above results, lactoferrin has been found to induce production of IL-12 by macrophages.

Test 6 Effect of a ceramide on- the production of IFN-y 20 BALB/c mice (3 weeks old, male) (Japan SLC, Inc.) were provided for the experiment. The mice were divided into a lactosylceramide administration group (experimental group) and lactosylceramide non-administration group (control group) (3 mice per group) such that both of the groups had 25 substantially equal mean and deviation of mouse body weights. 26 Employed as a ceramide was lactosylceramide ("Biochemical Reagent 126-04491", product of Wako Pure Chemical Industries, Ltd., derived from cow butter milk, lot ELK6191). Lactosylceramide was dispersed at 200 pg/mL 5 concentration in a 0.9% NaCl solution which contained 0.5% of Tween20, and the resulting dispersion was stored at -20°C until use. At the time of use, the dispersion was thawed, to which an equiamount of sterilized PBS was added. Subsequent to thorough mixing, the lactosylceramide solution (200 ]iL per 10 mouse) was intraperitoneallv administered to the mice everyday for 14 days except for Sundays and Saturdays (20 jig/mouse) . To the control group, only a solvent (0.9% NaCl solution containing 0.5% of Tween20 and added with an equiamount of sterilized PBS) was administered.

Splenocytes obtained from each mouse on the 14th day after the administration were inoculated to a 96-well microplate ("No. 3072", manufactured by Becton Dickinson and Company) at an inoculum size of 4 x 105 cells per well. Employed for incubation was RPMI 1640 medium ("No. 11875-093", product 20 of Gibco BRL) which contained 10% of fetal calf serum (product of Japan Biotest Laboratory, lot 10086-1), 100 U/mL of penicillin, 100 pg/mL of streptomycin and 5 x 10"5 M of 2-mercaptoethanol. The incubation was conducted in four runs, and the splenocytes were incubated for 20 hours in the presence 25 of bLF (product of Meggle GmbH, 0 or 100 pg/mL) . After the 27 incubation, IFN-y in each culture supernatant was assayed by an ELISA kit (product of ENDOGEN Inc. ) . The data were analyzed by two-way classification variance analysis (2x2, significance level: 5%) while taking as factors the 5 administration of lactosylceramide andthe stimulationby bLF.

The results are shown in FIG. 15. When the ceramide was not administered, production of IFN-y was not observed no matter whether bLF existed or not. When the ceramide was administered beforehand, production of IFN-y was observed to 10 significantly increase both in the absence and presence of bLF. As an interaction was observed between these two factors of the ceramide and bLF (p=0.0016), administration of lactosylceramide has been found to enhance bovine-LF-stimulated production of IFN-y. 28 Example 1 Bovine LF was added at 0.01% to "EPITOLESS" (product of MEIJI MILK PRODUCTS CO., LTD.) a milk for milk-allergy sufferers, which did not use milk serum proteins as a raw material and had the below described formula. 29 Ingredient Per 100 gram-product Proteins (N x 6.38) g 14.5 Fats g O o CM Hydrocarbons g 60. 0 (Lactose) g (sucrose 8.0) (Soluble polysaccharides) g (52.0) Ash g 2.5 Water g 3.0 Energy kcal 478 Vitamin A IU 2, 000 Vitamin B1 mg 0.6 Vitamin B2 mg 0.9 Vitamin B6 mg 0.3 Vitamin B12 ix g 4 Vitamin C mg 50 Vitamin D IU 370 Vitamin E (as a-tocopherol) mg 6 Vitamin K ix g Pantothenic acid mg 2 Niacin mg 6 Folic acid mg 0.2 j3 -carotene ix g Inositol mg Linoleic acid g 1.7 a-linolenic acid g 0.34 Taurin mg 27 Calcium mg 400 Magnesium mg 42 Potassium mg 525 Sodium mg 150 Phosphorus mg 230 Chlorine mg 320 Iron mg 6.5 Copper ix g 320 Zinc mg 2.8 Example 2 Bovine LF was added at 0. 01% to a hydrolyzed milk "MEIJI NOBIYAKA" (product of MEIJI MILK PRODUCTS CO., LTD.), which contained milk serum proteins as a raw material. 31 Ingredient Per 100 gram-product Proteins (N x 6.38) g 12.2 Fats g .0 Hydrocarbons g 57.5 (Lactose) g (29.5) (Soluble polysaccharides) g (28.0) Ash g 2.2 Water g 3.0 Energy kcal 504 Vitamin A IU 2, 000 Vitamin B1 mg 0.6 Vitamin B2 mg 0.9 Vitamin B6 mg 0.3 Vitamin B12 li g 4 Vitamin C mg 50 Vitamin D IU 375 Vitamin E (as a-tocopherol) mg 6 Vitamin K H g Pantothenic acid mg 2 Niacin mg 6 Folic acid mg 0.2 j3 -carotene M g 70 Inositol mg Linoleic acid g 2.6 a-linolenic acid g 0. 66 Taurin mg 27 Calcium mg 360 Magnesium mg 40 Potassium mg 510 Sodium mg 170 Phosphorus mg 200 Chlorine mg 300 Iron mg 6.5 Copper H g 320 Zinc mg 2.8 32 Industrial Applicability By the present invention, it has been found that in vivo production of IgE antibody is inhibited by oral ingestion of LF. 33

Claims (21)

1. Amammalian (including human) antigen-specific IgE antibody production inhibitor comprising as an active ingredient lactoferrin or a hydrolyzate thereof.

2. An inhibitor according to claim 1, further comprising a ceramide as an active ingredient.

3 . Acomposition for controlling immunityby inhibiting production of a mammalian (including human) antigen-specific IgE antibody, comprising as an active ingredient lactoferrin or a hydrolyzate thereof.

4. A composition according to claim 3, further comprising a ceramide as an active ingredient.

5. An IFN-y production potentiator composition for mammals (including human), which comprises as active ingredients lactoferrin or a hydrolyzate thereof and a ceramide.

6 . An antiviral agent composition f ormammals including humans, comprising as active ingredients lactoferrin or a hydrolyzate thereof and a ceramide.

7. An antibacterial agent composition for mammals including humans, comprising as active ingredients lactoferrin or a hydrolyzate thereof and a ceramide.

8 . An antitumor agent composition f ormammals including humans, comprising as active ingredients lactoferrin or a hydrolyzate thereof and a ceramide. 34

9. An immunopotentiator composition for mammals including humans, comprising as active ingredients lactoferrin or a hydrolyzate thereof and a ceramide.

10. Use of lactoferrin or a hydrolyzate thereof for the preparation of a mammalian (including human) antigen-specific IgE antibody production inhibitor.

11. Use according to claim 10, further adding a ceramide .

12. Use of lactoferrin or a hydrolyzate thereof for the preparation of a composition for controlling immunity by inhibiting production of a mammalian (including human) antigen-specific IgE antibody.

13. Use according to claim 12, further adding a ceramide.

14. Use of lactoferrin or a hydrolyzate thereof and a ceramide for the preparation of an IFN-yproduction potentiator composition for mammals including humans.

15. Use of lactoferrin or a hydrolyzate thereof and a ceramide for the preparation of an antiviral agent composition for mammals including humans.

16. Use of lactoferrin or a hydrolyzate thereof and a ceramide for the preparation of an antibacterial agent composition for mammals including humans.

17. Use of lactoferrin or a hydrolyzate thereof and a ceramide for the preparation of an antitumor agent composition for mammals including humans.

18. Use of lactoferrin or a hydrolyzate thereof and a 35 r 7 ceramide for the preparation of an immunopotentiator composition for mammals including humans.

19. An inhibitor according to claim 1 substantially as hereinbefore described with reference to any of the figures and/or examples.

20. A composition according to any one of claims 2 to 9 substantially as hereinbefore described with reference to any of the figures and/or examples.

21. Use according to any one of claims 10 to 18 substantially as hereinbefore described with reference to any of the figures and/or examples. DATED: 1 April, 2003 PHILLIPS ORMONDE & FITZPATRICK Attorneys for: MEIJI DAIRIES CORPORATION INTELLECTUAL PROPERTY OFFICE- OF N.i? 37 ABSTRACT This invention relates to mammalian (including human) IgE antibody production inhibitors and immunomodulatory compositions, each of which comprises lactoferrin (LF) or its hydrolyzate as an active ingredient. This invention is also concerned with IFN-y production potentiators, each of which comprises LF or its hydrolyzate and a ceramide.