WO2007097502A1

WO2007097502A1 - Composition for reducing the exudation of serum proteins

Info

Publication number: WO2007097502A1
Application number: PCT/KR2006/003344
Authority: WO
Inventors: Seong-Hyun Choi
Original assignee: Kt & G Corporation
Priority date: 2006-02-23
Filing date: 2006-08-24
Publication date: 2007-08-30

Abstract

Disclosed is a composition for reducing the exudation of serum proteins. More specifically, the composition of the present invention relates to a composition capable of improving conditions by reducing the exudation of the serum proteins in skins and mucosae, wherein the conditions includes atopic dermatitis, atopic eczema, pruritus cutaneous, atopic rhinitis, atopic erythema, atopic erythroderma, contact dermatitis, asthma, chronic obstructive pulmonary diseases, etc.

Description

COMPOSITION FOR REDUCING THE EXUDATION OF SERUM PROTEINS

TECHNICAL FIELD

The present invention relates to a composition for reducing the exudation of serum proteins, and more particularly to a composition capable of improving conditions

such as atopic dermatitis, atopic eczema, pruritus cutaneous, atopic rhinitis, atopic

erythema or erythroderma, contact dermatitis, asthma, chronic obstructive pulmonary

diseases, etc. by reducing the exudation of serum proteins in skins and mucosae.

BACKGROUND ART

For the purpose of the academic understandings of atopic diseases and the

improvement and the treatment of their symptoms, there are many attempts by

researchers to understand the immunoregulatory mechanisms in a living body or the cell

signaling mechanisms and the methods employing the mechanisms along with the

development of immunology and cytology. On the other hand, some studies have been

going on by some researchers to elucidate the causes of the diseases on the changes with the development of modern civilization, that is, the changes in diet or the exposure to

pollutants.

The common fact found in the studies on the atopic diseases in these various

findings is that the serum proteins exude from skins or mucosae of patients with atopic diseases, regardless of the understanding that the atopic diseases would be caused by the immunological reasons. However, there has been no study to conclude clearly whether the serum proteins exude from the skins or mucosae as a result of the development of the atopic diseases or the exudation of the serum proteins in the skins or mucosae

triggers the atopic diseases.

It is known that the exudation of the serum proteins in outer skins or mucosae

are observed mainly using serum albumin as a marker protein. It has been known that

an exudation level of serum albumin in the skin diseases is closely correlated with the severity of the diseases. Hypoalbuminemia in the blood is induced as the serum

proteins severely exuded from the skin (Worm et al., 1981. Br. J. Dermatol. 104:

389-396; and Worm and Rossing, 1980. J. Invest. Dermatol. 75: 302-305). Patients are

suffering from allergic asthma and rhinitis developed symptoms such as intestinal

lymphangiectasia that resulted by severe loss of albumin through the inner wall of the

intestines (Esenberh, 1976. Ann. Allergy, 36: 342-350). In case of children, the loss of

albumin by severe skin diseases makes them retard their growth (Abrahamov, 1986. Eur.

J. Pediatr. 145: 223-226), and is also accompanied with oiguria and acrocyanosis along

with the lymphangiectasia (Capulong et al. 1996. Pediatr. Allergy Immunol. 7: 100-102).

It was reported that an exudation level of albumin in diseased skin area is closely

related to the severity of the conditions even in the case of atopic eczema and contact dermatitis (David et al., 1990. Br. J. Dermatol. 122: 485-489; and Wijsbek et al., 1991.

Int. J. Microcirc. Clin. Exp. 10: 193-204).

It was reported that the exudation of albumin-including serum proteins was

found even in sputa of the patients suffering from symptoms of asthma and chronic obstructive pulmonary disease (Schoonbrood et al. 1994. Am. J. Respir. Crit. Care Med.

150:1519-1527; and Anderson & Persson 1988. Agents Actions Suppl. 23: 239-260),

and it was also reported that the exudation of albumin occurs without a sign of eosinophilia in the case of chronic cough (Pizzichini et al., 1999. Can. Respir. J.

6:323-330).

The exudation of the serum proteins is induced by the chemicals including

materials such as methyl salicylate, phenol, croton oil, benzalkonium, etc. (Patrick et al.

1985. Toxicol. Appl. Pharmacol. 81: 476-490); and toluene, m-xylene, cyclohexane, etc.

(Iyadomi et al., 1998. Ind. Health 36:40-51). 2,4-dinitrofluorobenzne is used in the

animal models to conduct a skin irritation study, and also induces the exudation of the

serum proteins (Nakamura et al., 2001. Toxical. Pathol. 29:200-207). It was reported

that all organic solvent materials do not induce the exudation of the serum proteins, and

the exudation levels are also varied according to the structure of chemicals. The

exudation of the serum proteins is easily induced by the chemicals having aromatic rings,

but nearly affected by organic solvents such as acetone.

Keeping in mind that the atopic diseases are caused by reason of the westernized

food habits (Weiland et al., 1999. Lancet 353:2040-2041; von Mutis et al., 1998. Lancet

351:862-866; and Dunder et al., 2001. Allergy 56:425-428), the researchers have paid

attention to the changes of the components and the content of lipids ingested by the

people. With the advent of 1970s, a few of researchers have found that the imbalance

of the human metabolism is caused by the excessive ingestion of unsaturated fatty acid

and trans oil which are main components of vegetable oil, which results in the

augmentation of the atopic diseases.

Unlike other tissues and organs, there are various phospholipids having very high contents of di-saturated fatty acids in lungs of mammals including human, and the surfactants present on a surface of the lung have, in particular, a very high content of dipalmitoylphosphatidylcholine (DPPC). It has been known that the component

functions to reduce a surface tension in lungs. The reduction of the surface tension by

the surfactant on the surface of lung can be made easy to breathe when air is inhaled into the lungs, and prevents alveolar walls from being attached to each other or their

structure from being collapsed when exhaled out (Reviews: Jon Goerke 1998. Biochem.

Biophy. Acta. 1408:79-89).

The lipid components of the surfactant, present on a surface of the lung, are

composed of about 90 % of phospholipid and about 10 % of neutral fat (a majority of

the neutral fat is cholesterol). The phospholipid components are composed of about 80 % of phosphatidylcholine (PC), and the PC components are composed of about 50 %

of dipalmitoyl phosphatidylcholine. The phospholipids include phosphatidylglycerol,

phosphatidylinositol, phosphatidylserine and phsophatidylethanolamine, whose contents

are 9.1 %, 2.6 %, 0.9 % and 12.3%, respectively (Veldhuizen et al., 1998. Biochim.

Biophys. Acta. 1408: 90-108). One of the scientific results that should be further considered in addition to the

facts as described above is a pathway for synthesizing the surfactant lipids in the lungs.

One point is that most cholesterol present in the human lung is supplied from the blood.

Cholesterol synthesized in the lung accounts for only about 1 %, and the rest of

cholesterol should be supplied from the blood (Hass and Longmore, 1979. Biochim.

Biophys. Acta 573:166-174). It has been known that phospholipids constituting most of the surfactant of the lung are synthesized in and secreted from Type II epithelial cells, but most of the fatty acid constituting the phospholipids are supplied from VLDL (very

low density lipoprotein) in the blood (Rama et al., 1997. J. Clin. Invest. 99: 2020-2029). SP-A (surfactant protein A) is one of the major proteins present in the surfactant

in lung that has been studied for the last 30 years. The SP-A protein is synthesized in

and secreted from Type II epithelial cells present in the alveoli, and also found in a

surface of the small intestine, Eustachian tube of ear, tears, etc. Its formula weight is

approximately 700 kDa (measured by a gel-filtration assay), and its 18 identical units,

each having a formula weight of 32 kDa, are gathered to form a mature protein having

the peculiar function. It has been known that the protein has various biological

functions, but they may be mainly categorized into two groups. The first is an

immunological function in which SP-A protects the lungs by binding to bacteria or

viruses, as well as a house dust mite, anther dust, etc. which enter the lungs while breathing in. The second function is taking part in maintaining the homeostasis of the

surfactant in lung (Tino and Wright 1998. Biochim. Biophys. Acta. 1408: 241-263;

Haagsman, Biochim. Biophys. Acta. 1408: 264-277; Crouch & Wright, 2001. Annu.

Rev. Physiol. 63:521-524; and Haagsman and Diemel, 2001 Compar. Biochem. Physiol.

129: 191-108).

It have been widely known that lung tissues and lung functions are damaged by

the excessive intake of unsaturated fatty acid (Wolfe et al., Nutrition 2002 18: 647-653).

Also, intravascular administration of oleic acid has been used as a respiratory distress

syndrome model (Kate et al., Am. J. Physiol-Lung cell and mol. Physiol.2000 279:1091-1102). The damage of the lung function by the unsaturated fatty acid is caused since most of the lipids required for synthesis of pulmonary surfactants is supplied from blood, as described above, and therefore it was assumed that the lung tissue requiring a large amount of saturated phospholipids is more sensitive to the damage than other organs.

In order to cope most effectively with the tissue damage caused by excessive

unsaturated fatty acid, the present invention is designed to recover rapidly the functions

of the tissues by directly supplying saturated phospholipids to the tissues to dilute a

concentration of the unsaturated fatty acid.

Also, the composition may protect skins from external stimuli since di-saturated

phospholipid forms the lipid layer as the surface of the lung when applied to the skins,

and some extra di-saturated phospholipid is supplied into the epidermal cells, and then

utilized in the lipid metabolism. DPPC (dipalmitoyl phosphatidylcholine) in the

di-saturated phospholipids may be directly used as a building block of cell membranes,

and also used as a source of supply of palmityl-CoA in the cells. The Palmityl-CoA

and serine in cytoplasm are used as the starting materials in a metabolism for

synthesizing ceramide. It has been considered that the ceramide plays a very important

role in atopic skin diseases. It has been known that patients with the atopic dermatitis

have a very low level of ceramide in their epidermis. This result comes from the facts

that staphylococcus aureus-specific lipase (in particular, sphingomyelin deacylase)

present in the atopic skin diseases may lower an amount of the ceramide (Higuchi et al.,

Biochem. J. 2000 350:747-756), but, on the other hand, an intracellular amount of the palmityl-CoA, necessarily required for intracellular lipid synthesis, is relatively deficient

due to the excessive intake of the unsaturated fatty acid. As a result, the patients with atopic diseases may have a lower level of the ceramide in a lesional-skin area of the

atopic dermatitis as well as a non-lesional skin area (Imokawa et al., J. Invest. Dermatol. 1991 96:523-526). In order for an epidermal cell to facilitate the synthesis of the ceramide required in epidermis, the present invention is designed to supply

di-saturated phospholipid containing palmitic acid (16:0) (phospholipid having two

palmitic acids, particularly dipalmitoyl phosphatidylcholine (DPPC)) to the epidermal

cell, the di-saturated phospholipid being extracted from healthy animal lungs. It is

considered that supplying palmitic acid directly to the tissue has a disadvantage that the

palmitic acid may reduce an adverse effect on stability of the cell membrane in the

substantially same manner as in the detergent. In the present invention, organic acids

(particularly, citrate/citric acid), employed with the di-saturated phospholipid, may also

serve to facilitate the easy absorption of the phospholipid molecules into the epidermis

by preventing the di-saturated phospholipid from being aggregated in the presence of

Ca²⁺ ions, as well as act as a positive regulator of the lipid synthesis pathway after the

organic acids are taken up into the cytoplasm of the epidermal cell. In the lipid

metabolism in the cells, citrate in the cytoplasm facilitates the activity of acetyl-CoA

carboxylase to accelerate the synthesis of malonyl-CoA (Triscari and Sullivan Lipid.

1977 12:357-363). The malonyl-CoA is a key material in the synthesis pathway of

palmitoyl-CoA, and also inhibits the palmitic acids, synthesized in the cytoplasm, from

being transported into mitochondria. The present invention is designed to supply saturated fatty acid isolated from healthy animal to tissue cells with atopic diseases, affected by the excessive intake of the vegetable unsaturated fatty acid, in a safe and easy manner, and also to minimize an adverse effect caused by the excessive intake of vegetable unsaturated fatty acid and to facilitate the endogenous lipid synthesis in the

cells. DISCLOSURE OF INVENTION

Accordingly, the present invention is designed to solve the problems of the prior

art, and therefore it is an object of the present invention to provide a composition

capable of relieving, preventing and/or treating diseases that are associated with the

exudation of serum proteins.

In order to accomplish the above object, the present invention provides a

composition for relieving or treating diseases which are associated with the exudation of

serum proteins, comprising di-saturated phospholipid as an effective component.

The term "di-saturated phospholipid", used herein, means a phospholipid whose

fatty acid-derived aliphatic substituents are all saturated with lipids having at least one phosphate group (mono or diester type), wherein the fatty acid-derived aliphatic

substituents are attached to each other by means of ester bonds between carbons 1 and 2

of phosphoglyceride. The carbon atoms of the aliphatic substituent preferably ranges

from 14 to 20, and a palmitoic acid-derived substituent having 16 carbon atoms is most

preferred.

The di-saturated phospholipid of the present invention is preferably extracted

from animals, more preferably from a cattle or a pig, and most preferably from a

bronchial alveolar lavage or lung tissue homogenate.

Also, the di-saturated phospholipid of the present invention preferably includes dipalmitoylphosphatidylcholine and/or dipalmitoylphosphatidylinositol, and is most preferably dipalmitoylphosphatidylcholine and/or dipalmitoylphosphatidylinositol.

Also, the composition of the present invention preferably further includes calcium and organic acid containing a carboxyl group(s), and the organic acid including a carboxyl group(s) is most preferably metabolizable organic acid, namely organic acid

including, but is not limited to, at least one selected from the group consisting of lactic

acid, succinic acid, fumaric acid and citric acid. Also, the composition of the present invention may further include an additive, for example gylcerol, if necessary.

Also, the present invention provides a composition for treating skin diseases

including the composition of the present invention. Also, the present invention

provides a cosmetic composition including the composition of the present invention.

The diseases associated with the exudation of the serum proteins of the present

invention are selected from the group consisting of, but are not limited to, atopic

dermatitis, atopic eczema, pruritus cutaneous, atopic rhinitis, atopic erythema or

erythroderma, contact dermatitis, asthma and chronic obstructive pulmonary diseases.

The composition of the present invention includes, for example, at least one

selected from the group consisting of an acceptable dilute, an additive and a carrier.

The composition of the present invention includes the di-saturated phospholipid

in a suitable pharmaceutically available composition for delivery or administration into

in vivo or ex vivo tissues or organs.

The composition may be administered in various routes including, but is not

limited to, parenteral, enteral, topical administrations or inhalations. The parenteral

administration means any administration that is not administered through a digestive tract, including, but is not limited to, injections (namely, intravenous, intramuscular and other injections as described later). The enteral administration means any form for the

parenteral administration including, but is not limited to, tablet, capsules, oral solution, suspension, spray and derivatives thereof. For this purpose, the route of enteral administration means a route of transrectal and intravaginal administration. The route

of topical administration means any route of administration including, but is not limited

to, creams, ointments, gels and parenteral patches (also see Remington's Pharmaceutical

Sciences, 18^th eds. Gennaro, et al., Mack Printing Company, Easton, Pennsylvania,

1990).

The parenteral pharmaceutical compositions of the present invention may be

administered, for example, venously (intravenously), arterially (intraarterially), muscularly (intramuscularly), under the skin (subcutaneously or into depot composition),

into the pericardium, by injection to coronary arteries, or with solutions for delivery to

tissues or organs.

Injectable compositions may be pharmaceutical compositions that are suitable

for the routes of administration by injection including, but is not limited to, injections into the veins, the arteries, the coronary vessels, into the mesothelioma, around the

blood vessels, into the muscles, and subcutaneous and articular administrations. The

injectable pharmaceutical compositions may be pharmaceutical compositions for direct

administration into the heart, the pericardium or the coronary arteries.

For the oral administration, the pharmaceutical formulations may be ingested in a form of tablet or capsule prepared in the conventional methods, for example, with pharmaceutically available additives such as binders (for example, pregelled corn starch,

polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen-phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium

starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets may be coated using the methods known in the art (see Remington's Pharmaceutical Sciences, 18^th eds. Gennaro et al., Mack Printing Company, Easton, Pennsylvania, 1990).

The oral composition may be ingested in a form of, for example, solution, syrup

or suspension, or be dried products that may be mixed with water or other suitable

solvents before its use. The composition solution may be manufactured, using the

conventional methods, with pharmaceutically available additives such as suspensions

(for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats);

emulsions (for example, lecithin or acacia); insoluble carriers (for example, ethyl

alcohol or long-chain alcohol); and preservatives (for example, methyl or propyl

p-hydroxybenzoate or sorbic acid).

The pharmaceutical compositions may also include a buffer salt, a spice, a

pigment and a sweetener, if necessary.

The enteral compositions may be suitable for oral administration in a form of,

for example, a tablet, troches or a lozenge. The di-saturated phospholipid of the present invention may be manufactured with solutions (rectal cream), suppositories or

ointments for the routes of transrectal and intravaginal administrations. The enteral

compositions may be suitable for a mixed solution of a total parenteral nutrition (TPN)

mixture or an intake mixture such as a solution for delivery by an intake tube (see

Dudrick et al., 1998, Surg. Technol. Int. VII: 174-184; Mohandas et al., 2003, Natl.

MedJ. India 16 (1): 29-33; Bueno et al., 2003, Gastrointest.Endosc. 57 (4): 536-40;

Shike et al., 1996, Gastrointest.Endosc . 44 (5): 536-40).

For the administration by inspiration, the di-saturated phospholipid of the present invention may be generally delivered in the presence of aerosol spray or in a

form of a nebulizer in a container pressured with suitable propellants such as, for

example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of the pressured aerosol, its capacity

may be determined depending on a valve for conveying its weighed amount. A capsule

and, for example, a gelatine cartridge may be formulated to be used for an inhaler or an

insufflator including suitable powder bases such as lactose or starch, and a powder mix

of the compounds.

The compositions of the present invention may include a suitable topical vehicle

for applying to the skins. The suitable topical vehicle, which may be used in the

present invention, may include a cream, a lotion, a solution, a hydroalcohol solution, a

pack, a powder and a skin patch.

The composition of the present invention is administered to the subject in need

of treatment of reducing the exudation of serum proteins. Toxicity and therapeutic

efficiency of the composition may be determined according to the standard

pharmaceutical procedure for experimental animals, such as cell culture or LD₅₀ (50 %

lethal density of one group) measurement and ED₅₀ (50 % effective density of one group) measurement. A ratio of the added composition between the toxic effect and the therapeutic effect is referred to as a therapeutic index, and the therapeutic index may

be represented by a LD_5O/ED₅o ratio. The composition having a high therapeutic index

is preferred.

In one embodiment, the data obtained from cell culture analyses and animal studies may be used to determine a dosage for application to humans. The dose of the composition according to the present invention is preferably within the range of

circulating density including an ED₅₀ value in which the composition is not toxic or

hardly toxic. The dose is varied depending on the formulations applied within the

range, and the routes of administration used herein. In the composition used in the method of the present invention, a therapeutically available dose may be measured from

cell culture analysis at the very beginning. The dose is designed in an animal model in

order to obtain a plasma density range including an IC₅₀ value (namely, a density of a

test material showing a half of the maximum inhibition), as determined in the cell

culture. The information may be used to more correctly determine an effective dose

for humans. A level of the test material in plasma may be, for example, determined by

high performance liquid chromatography.

In the composition of the present invention, the di-saturated phospholipid is preferably present at a concentration of 1 to 700 mg/ml. The range of the di-saturated

phospholipid is most preferred since effects of the composition on treatment or relief of

the diseases are poor if the di-saturated phospholipid is present in a concentration of less

than 1 mg/ml, while the suspending solution is not dispersed homogeneously due to its

high viscosity if the di-saturated phospholipid is present in a concentration of 700 mg/ml or more.

Hereinafter, preferred embodiments of the present invention will be described in

detail.

In the present invention, on the basis that the atopic diseases have a common symptom that the serum proteins exude from skins or mucosae because a function of an endothelial-epithelial barrier is lowered by excessive intake of unsaturated fatty acid and trans oil, a novel animal model was employed. It was found that, when

saline-emulsified plant-extracted oil is intratracheally introduced into rat lung, the

animal showed the exudation of the serum proteins in the lung. It is also found that the

symptom of the exudation of the serum proteins can be reduced or suppressed by the

intratracheal administration of the stable biocomponents - a composition invented here -

that are completely metabolized in vivo. And, it is also found that the composition, containing the di-saturated phospholipids isolated from animal's lung, a certain

concentration of calcium ions and organic acid which contains (a) carboxyl group(s), act

successfully on the affected skin area of patients with the atopic diseases.

In the present invention, it has been found that a unique sedimentary layer

containing an ideal combination of the di-saturated phospholipids is formed when the SP-A protein is aggregated in the presences of divalent cations and then spun in

bronchial alveolar lavage or lung homogenate of the healthy animal. The present

invention includes a method including the steps eliminating the surfactant proteins

(especially, organic solvent-soluble proteins SP-B and SP-C) and hydrophobic peptides

which may induce immune responses in human, cholesterol and unsaturated phospholipids from the sedimentary layer, and then obtaining an ideal combination of

the di-saturated phospholipid fractions existing in healthy animal lung. On the basis of

the findings that the SP-A protein exhibits a strong non-specific hydrophobic interaction

in the absence of divalent cations, a certain concentration of calcium ions is required when a composition containing the di-saturated phospholipid fraction is applied to skin or mucosa of the patients with an atopic disease to suppress or to reduce the exudation of the serum proteins, as well as the organic acids having (a) carboxyl group(s) which can be metabolized in cell, for example citrate or citric acid are further added to the

composition to prevent the di-saturated phospholipids from being aggregated by a

certain concentration of calcium ions.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of

the present invention will be more fully described in the following detailed description,

taken accompanying drawings. In the drawings:

Fig. 1 is a diagram showing that total lipids obtained from animal lung are visualized using TLC. In Fig. 1, the * indicator represents the di-saturated phospholipid fraction used in the present invention. This photograph shows all

fractions, which are collected after passing the total lipids in the lung through a silica

column. Analysis of lipid profile is carried out using silica gel 60 (Merck), and a

developing solvent is used as a mixture of chloroform: methanol: water (volume ration

of 65:25:4). Each of the lipids are developed by spraying 99 % ethanol containing 5 %

sulfuric acid to TLC and carbonizing at 190 ^°C .

FIG. 2 is a diagram showing results determined by mass spectrometry. Two

spots, which are show in fractions as represented in a section which is limited by the *

indicator in the right arrow of TLC in FIG. 1, were scraped together from the TLC plate, eluted with a chloroform:methanol (2:1, v/v) solvent, and then analyzed using a Waters Micromass ZQ system. It was confirmed that a DPPC peak and a DPPI peak are

observed from the lipids eluted from the upper spot and the lower spot shown in the photograph of FIG. 1, respectively, wherein the DPPC peak has a formula weight of 734.68 and the DPPI peak has a formula weight of 826.64.

A left section of FIG. 2 shows a lipid profile determined by mass spectrometry

of the upper spot in TLC, and a right section of FIG. 2 shows a lipid profile determined

by mass spectrometry of the lower spot in TLC.

FIG. 3 is a diagram showing normal protein profiles analyzed after the exudation

of serum proteins is induced in rat lungs and suppressed by the composition. In FIG. 3,

Section (1) shows a result obtained by inducing the serum proteins in the rat lungs,

Section (2) shows a result obtained by adding the composition to the rat in which the

exudation of serum proteins is induced, and Section (3) shows a result obtained from a normal rat. In the protein profiles, "A" represents a peak containing SP-A (~700KDa)

protein, and "B" represents a peak containing serum albumin (~70KDa), and therefore

an effect on suppression of the exudation was observed by comparing two peak

magnitudes.

FIG. 4 is a diagram showing results obtained after applying the composition to

the atopic dermatitis (2~4 weeks). Left sections of these photographs are obtained before applying the composition. Right sections of these photographs are obtained

after applying the composition. The application of the composition was carried out for

the period of 2 to 4 weeks, and the top of the photograph shows a patient suffering from

atopic eczema, the second photograph from the top shows a patient suffering from

atopic dermatitis including skin ulcer, the third photograph from the top shows a patient suffering from atopic erythema, and the bottom of the photograph shows a patient

suffering from atopic dermatitis.

FIG. 5 is a photograph showing the result before/after the composition was applied. These result shows photographs taken before the application of the

composition to an atopic skin disease developed in the back of a 6-year-old female (left)

and after application of the composition to the atopic skin disease for 4 weeks (right).

FIG. 6 is a photograph showing the result before/after the composition was

applied. These result shows photographs taken before application of the composition

to an atopic skin dermatitis, including skin ulcer, developed in a popliteal region of the

knee of a 6-year-old female (left) and after the application of the composition to the atopic skin dermatitis for 5 weeks (right).

FIG. 7 is a photograph showing the result before/after the composition was

applied. These result shows photographs taken before the application of the

composition to a common atopic skin disease developed in a popliteal region of the

knee of a 16-year-old female (left) and after the application of the composition to the

atopic skin disease for 3 weeks (right).

FIG. 8 is a photograph showing the result before/after the composition was

applied. These result shows photographs taken before the application of the composition to an early-stage atopic skin disease developed in the face of a

10-month-old female (left) and after the application of the composition to the atopic

skin disease for 3 weeks (right).

FIGs. 9 to 13 show the effects of citric acid/citrate components, added to the

composition, on treatment of the atopic skin diseases. The composition [Basic composition (All)] of the present invention including the di-saturated phospholipid, calcium and citrate was applied to left arms or left legs of test volunteers, and a control composition [Composition 1] including the di-saturated phospholipid and calcium was applied to right arms or right legs of the test volunteers. It was revealed that effects of

the two compositions are slightly different to each other in the case of slight atopic skin

diseases, but the composition of the present invention has a more excellent therapeutic

effect than that of the control composition in the case of severe atopic skin diseases.

FIG. 9 is a diagram showing results obtained by applying the Basic composition

(left leg) and the Composition 1 (no-citrate) to slight atopic conditions, respectively:

11 -year-old male, the upper photograph: before the application, and the lower

photograph: 4 days after the application.

FIG. 10 is a diagram showing results obtained by applying the Basic

composition (left arm) and the Composition 1 (no-citrate, right arm) to slight atopic

conditions, respectively: 13-year-old male, the upper photograph: before the application,

and the lower photograph: 4 days after the application.

FIG. 11 is a diagram showing results obtained by applying the Basic

composition (left leg) and the Composition 1 (no-citrate, right leg) to slight atopic

conditions, respectively: 7-year-old male, the upper photograph: before the application,

and the lower photograph: 5 days after the application.

FIG. 12 is a diagram showing results obtained by applying the Basic

composition (left leg) and the Composition 1 (no-citrate, right leg) to severe atopic

and the lower photograph: 7 days after the application.

FIG. 13 is a diagram showing results obtained by applying the Basic

conditions, respectively: 11 -year-old female, the upper photograph: before the application, and the lower photograph: 4 days after the application.

BEST MODES FOR CARRYING OUT THE INVENTION

detail with reference to the accompanying drawings. However, the description

proposed herein is just a preferable example for the purpose of illustrations only, not

intended to limit the scope of the invention.

Example 1: Extraction of the di-saturated phospholipid fraction from Bronchial

alveolar lavage of pig

(1) A method for obtaining a bronchial alveolar lavage

Lung taken from a healthy pig was lavaged by administering an osmotic saline

solution, containing 5mM calcium chloride (CaCl₂), into the lung. The lavaging was

carried out at a room temperature by administering about 0.6L of a solution to one of the

lungs using a pressure pump, followed by collecting a lavage that exudes from the lung. The lavaging was carried out with 15 L of a saline solution per lung. The collected

bronchial alveolar lavage was immediately transferred to an ice-water bath and cooled

down.

(2) A method for obtaining a precipitate of the surfactant from a bronchial alveolar lavage using an aggregation phenomenon. As the bronchial alveolar lavage including calcium ion (5 mM) was kept in an ice-water bath for 1 hour, then the SP-A protein begins to aggregates with the lipids in the surfactant. This solution was spun in a centrifuge (Component-R, Hanil, Republic

of Korea) having a low-acceleration spinning function. The solution was centrifuged at 4 ^°C for 35 minutes under a centrifugal force of 4,50Og. The precipitate forms

three distinctive layers after centrifugation. A small amount of a bottom layer contains

cells and insoluble and/or denatured proteins, and the largest amount of an intermediate layer and a small amount of a top layer contains both the surfactant lipids and surfactant

proteins whish are required in this invention. The intermediate and top layers of the

precipitate was collected together and proceeded to the organic solvent extraction to

obtain a di-saturated phospholipid fraction.

(3) Primary organic solvent extraction

A 2-fold volume of distilled water was added to the mixture of the surface-active

protein and the surfactant lipid prepared in the previous step, and the then mixture was

suspended. A mixed solvent (2:1 volumetric ratio) of chloroform and methanol was

added to the sample at the same volume, and shaken at 30 ^"C for 30 minutes. The

sample was centrifuged for 15 minutes under a centrifugal force of 1,000 g, and then the

organic solvent layer was collected for the further isolation procedure.

(4) Extraction of the di-saturated phospholipid fraction using silica column

chromatography

A solvent was evaporated from the sample obtained by the primary organic solvent extraction using a rotary vacuum evaporator (Eyela, Japan) and then the sample

was re-dissolved in chloroform. The sample was loaded into a column having a

diameter to height ratio of 1:5, filled with silica (Merck, 230-400 mesh). The silica column was pre-washed with pure chloroform before the application of the sample onto

the column. To get rid of cholesterol and the surface-active proteins, dissolved in the organic solvent, from samples and to obtain a di-saturated phospholipids fraction, the solvent systems were employed in the following order: after a silica column was washed

with pure chloroform, pure chloroform - pure acetone - pure chloroform - a mixed

solvent of chloroform:methanol (9:1 volumetric ratio) - a mixed solvent of

chloroform:methanol (4:1 volumetric ratio) and - a mixed solvent

chloroform:methanol: water (8:6:1 volumetric ratio) as a final solvent were applied.

The organic solvent-soluble surface-active proteins and hydrophobic peptides were

eluted with the solvents of pure acetone and pure chloroform, and a fraction of

di-saturated phospholipids, which is target components required for the present

invention, was eluted with the last solvent system. The phospholipid components of the obtained fractions were identified by comparing Rf values using the standard lipids

(commercially available from Sigma), the Rf values being obtained when a developing

solvent of chloroform: methanol: water (65:25:4) was used (Fig. 1). Main components

of the extracted di-saturated phospholipids were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylinositol, as shown in the results by the thin layer

chromatography (TLC).

Two spots observed after developing in TLC were scraped together from the

TLC plate, eluted with a chloroform:methanol (2:1, v/v) solvent, and then analyzed using a Mass spectrometry. The Mass spectrometry was carried out using a Waters Micromass ZQ system. It was confirmed that a DPPC peak (dipalmitoyl

phosphatidylcholine) and a DPPI (dipalmitoyl phosphatidylinositol) peak are observed from the lipids eluted from the upper spot and the lower spot shown in the photograph

of FIG. 1, respectively, wherein the DPPC peak has a formula weight of 734.68 and the

DPPI peak has a formula weight of 826.64 (FIG. 2). (5) Post-treatment process of the extracted di-saturated phospholipid fraction

The final sample extracted from the column was passed through a 3 -layer

Whatman Filterpaper No.l, and then the solvent was removed using a rotary vacuum

evaporator. Pure ethanol was added to the solvent-free sample, and then filtered using

a PVDF (polyvinylidene fluoride, 0.22 μ m pore-size, Millipore) filter. Ethanol was

evaporated again using an evaporator. Washing the sample with ethanol was carried

out 2 times. A trace amount of the remaining ethanol was removed using a freeze-drier.

A powder form of the di-saturated phospholipids was stored at -70 ^°C in an air-tight

storage container filled with nitrogen gas.

Example 2: Extraction of the di-saturated phospholipid fraction from bronchial

alveolar lavage of cattle

The method of the steps (1) to (5) described in Example 1 was repeated to obtain

the di-saturated phospholipid fraction from a bronchial alveolar lavage of cattle, except that a cattle was used instead of a pig.

Example 3: Method for obtaining the di-saturated phospholipid fraction from a

lung tissue homogenate of healthy pig

(1) Primary homogenization of the animal lung tissue 150 g of a lung tissue taken from a healthy pig was cut into a size of

approximately 2 cm X 2 cm. 700 ml of a cooled osmotic-saline solution containing

3mM divalent positive ions (calcium chloride) was added to the diced lung pieces, and then homogenized with a metal-blade blander. Homogenization was carried out 6 times at a low rotational speed for 20 seconds each. During the homogenization, the

blander was often cooled using an ice-water bath so as to prevent temperature from

being increased. The lung tissues were cut at about a bean size without being

completely homogenized at an early stage.

(2) A method for removing serum from a primary homogenate without a loss of

surfactant lipids

The primary homogenate prepared thus was kept for 1 hour in an ice-water bath

to allow serum to easily exude from the homogenized tissue having a large surface area,

and simultaneously to allow the surfactant components to be aggregated in the presence

of divalent ions (calcium ion, 3mM). The homogenate was kept for 1 hour, and then

centrifuged at 4 ^°C for 40 minutes under a centrifugal force of 4,50Og using a

centrifuge having a low-acceleration spinning function. A solution containing serum

except for a precipitate and floating matters (some small bean sizes of the lung tissue

that is not completely homogenized floats because it includes the air in them) was

removed after centrifugation.

(3) Secondary homogenization of the lung tissue and the precipitation of the

surfactant lipids

500 ml of a 75mM sodium chloride solution (a half concentration of a saline

solution) containing 5mM divalent positive ion (calcium ion) was added to the

serum-free primary lung tissue homogenate, and then the tissue was minutely homogenized with a metal-blade blander. In this procedure, a hypotonic solution was employed to obtain easily multilamellar structures that have function as a reservoir of the surfactant lipids existing inside Type II epidermal cells in alveoli. Homogenization was carried out 6 to 8 times at a high rotational speed for 40 seconds each, and the

being increased during the homogenization procedure. The secondarily homogenized

sample was kept at 4 ^°C for 1 hour to prevent the blood coagulation proteins from

being coagulated and at the same time, to allow the SP-A protein to aggregate with the

surfactant lipids in the presence of calcium ions. The sample was centrifuged at 4 °C

for 40 minutes under a centrifugal force of 4,50Og using a centrifuge having a

low-acceleration spinning function. The sample forms two distinctive layers that are

clearly different in colors and textures after centrifugation. A supernatant of the

precipitate layer was separately taken and subject to an organic solvent extraction

procedure so as to fractionate the di-saturated phospholipids. A certain amount of the

surfactant lipids integrated into the bottom layer in the centrifuge tubes could be

extracted as the saline solution containing 5mM calcium ions was added to the bottom

layer again and suspended it again, and then centrifuged in the same manner as

described above. The reformed top layer of the precipitate was collected and combined

with the sample prepared previously, and then subject to the organic solvent extraction

procedure.

(4) A method for obtaining the di-saturated phospholipid fraction

The method of the steps (3) to (5) described in Example 1 was repeated to obtain a di-saturated phospholipid fraction from the lung tissue homogenate.

Example 4: A method for obtaining a di-saturated phospholipid fraction from a

lung tissue homogenate of healthy cattle The method described in Example 3 was repeated to obtain the di-saturated phospholipid fraction from a bronchial alveolar lavage of cattle, except that a cattle was used instead of a pig.

Experimental example 1; Animal experiment

1-1. Induction of the exudation of the serum proteins in animal lung

Corn oil (commercially available from Sigma Co.) and a saline solution were

sterilized, respectively, using an autoclave. 500 fd (microliter) of the corn oil and

1500 μl of the saline solution were mixed, and then vortexed very vigorously to give a

homogeneous suspension. The suspension was intratracheally administered to the

lungs of grown-up Sprague-Dawley rats to induce the exudation of the serum proteins in

the lung. In order to study a placebo effect, only the sterile saline solution was intratracheally administered to the control rats. 2 weeks after application, the groups of

rats were anesthetized using an anesthetic Ketamine (Yuhan Co. Ltd., republic of Korea),

and then immediately euthanized using carbon dioxide gas. Lung was taken out right

after the experimental animal was killed, and the lung was lavaged with total 50 ml of a

saline solution (containing 3mM calcium chloride) to collect a bronchial alveolar lavage

in each animal.

1-2. Test of an effect on the suppression or reduction of the serum protein

exudation and the component of the composition applied

The exudation of the serum proteins was induced in animals in the same method described above, and then the compositions containing the di-saturated phospholipid fraction prepared in Examples 1 to 4 were administered intratracheally 1 week after the induction of the serum protein exudation procedure. The lung extraction and bronchial alveolar lavage from the experimental animals were carried out 1 week after the

compositions were administered intratracheally.

The composition applied to the rats to suppress or to reduce the exudation of the

serum proteins in lung was a concentration of 30 mg/ml of di-saturated phospholipids,

1.5 mM calcium chloride, 7.5 mM citrate (citrate trisodium salt) and 125 mM sodium

chloride, and pH of the solution was set to 6.0. The solution was vigorously vortexed

to form a homogeneous suspension before it was administered to the animal.

1-3. Analysis of the induction and suppression of the exudation of serum

proteins in the tested animals

(1) Preparation of the samples from each experimental animal

The bronchial alveolar lavages obtained from each experimental animal group

were centrifuged at 4 ^°C for 30 minutes under a centrifugal force of 4,50Og using a

centrifuge having a low-acceleration spinning function. Only a layer of the surfactant,

aggregated in the presence of divalent ion (calcium ion) with SP-A, was collected from

the precipitate. Approximately 3-fold volume of a saline solution (including 3mM

calcium chloride) was added to the collected precipitate and the precipitate was suspended again. The suspension was centrifuged under the same centrifugal force to

form a pellet. The washing procedure described above was repeated twice to clean up

the pellet using the same solution. A suspension of the final precipitate was divided

into ImI miniature centrifuge tubes, and the volume of the precipitate was adjusted to

200,ut

ImI of 1OmM Tris buffered saline (pH 7.4, 1OmM Tris-HCl, 140 mM NaCl)

containing 1OmM EDTA (ethylenediamineteteraacetic acid) was added to 200 μ£ of the surfactant precipitate described above, and the precipitate was then suspended in the

solution. A zwitter-ionic detergent CHAPS (Amresco, USA) was added to the solution

drop by drop with gentle shaking until the suspension was solubilized by CHAPS. In

order to solubilize the sample, a concentration of a 10 % (w/v, approximately 162 mM)

CHAPS stock solution was employed and the final concentration of CHAPS in the

sample was about 65 mM.

(2) Analysis

1 ml of the samples were taken from each sample (2 ml) prepared in the

procedure described above, and applied to a chromatography column packed with

Sephacry S-400HR (1.5 cm x 20 cm, Amersham Bioscience) resin to analyze the elution

pattern of the proteins. The size-exclusion chromatography was carried out using

GradiFrac (Pharmacia). At this time, the buffer solution used in the chromatography

work as a running buffer was a 10 mM Tris buffer solution (pH 7.4) including 5 mM

EDTA, 1 mM CHAPS and 140 mM NaCl. An exudation level of the serum proteins

and a suppression level of the proteins were determined, respectively, by comparing a

size (quantity) of a peak containing SP-A around a molecular weight of 700 kDa with a

size of a peak containing albumin around a molecular weight of 70 kDa from a UV absorption graph of the proteins eluted from the column (see Fig. 2).

1-4. Animal experiment results

From the graph of the protein elution patterns of the samples obtained from the group of the tested rats in which the exudation of the serum proteins was induced, the samples of the rats in which exudation of serum proteins was suppressed by the administration of the composition, the samples obtained from the rats into which only a saline solution was administered, and the sample obtained from the untreated control

rats, the ratios of the protein peak sizes around a molecular weight of 700 kDa to the

protein peak sizes around a molecular weight of 70 kDa were calculated respectively.

The ratios of two peak sizes (quantity) obtained from the groups of rats were listed in

the following Table 1.

Table 1

From and the obtained results shown in the Table 1, it was discovered that the

compositions containing the di-saturated phospholipid fraction as a main component

reduces, extracted from the bronchial alveolar lavage and from the lung homogenate of

the healthy animal, the serum protein exudation in the tested rats was reduced. A peak size of the proteins containing albumin as a major protein was reduced by approximately

50 % when the composition was administered intratracheally only one time, compared to the results of the serum protein exudation-induced rats. Such a reduced amount showed in Table 1 represents a value calculated simply from the criterion set in the

size-exclusion chromatography. However, the actual reduced amount of the exudates

is highly larger than the calculated value shown in Table 1. The reason is that 2-3 ml

of a precipitate (volume/head) is generally obtained on average when the bronchial alveolar lavage samples are obtained from normal rats, but the amount of precipitate

obtained from the serum protein exudation-induced rats were 3 to 5 times larger than

that of the normal rats although a quantity of the precipitate may be widely varied

depending on subjects. However, the amounts of the precipitates from the rats in

which the serum protein exudation were suppressed by the composition of the present

invention were almost the same or more than, at most, about 2 times those of the

precipitates obtained from the control rats. That is, it was shown that the composition

of the present invention significantly reduced a total amount of the precipitates, when

they were compared to those obtained from the serum protein exudation-induced rats.

However, these values are not shown in here quantitatively since the quantities of the precipitates obtainable from the tested rats may be greatly varied by the experimental

factors such as the experiment temperature, the compliancy of lung tissues, examiner's

dexterity, etc. Therefore, the reductions of the serum proteins shown in Fig. 2 and

Table 1 did not exhibit the total amount of the proteins. They only show the ratios

between the peak sizes containing SP-A and the peak sizes containing serum albumins

as major protein the fraction. As the total amounts of the serum proteins reduced by the composition of the present invention (see B of Fig. 2(2)) are measured, it might be

found that the reduced levels are very significant in its effect.

From the results shown above, it was found that the most ideal composition of di-saturated phospholipids for suppressing or reducing the serum protein exudation was

the one isolated from the bronchial alveolar lavage of healthy animal. However, the

di-saturated phospholipid fraction obtained from the de-blooded lung tissue homogenate also exhibited the almost same suppression level of the exudation of the serum proteins in the results.

Experimental example 2: Test on volunteers with atopic skin disease

2-1. Application of composition used in skin

(1) Composition

The di-saturated phospholipid fraction obtained from the pig lung

homogenization method exploiting an aggregation phenomenon of the surfactant by

divalent positive ion (calcium ion) and filtered through a PVDF (polyvinylidenefluoride,

0.2 μm pore-size, Millipore) filter having a pore size of 0.22 μm was used in the test.

The fraction was freeze-dried. A certain concentration of the dried di-saturated

phospholipids was emulsified in iso-osmotic saline and then autoclaved. A calcium

chloride stock solution, a citrate (citrate trisodium salt) stock solution and glycerol were

also autoclaved, respectively. Each sterilized component (solution) was mixed under

an aseptic condition to a concentration of 15 mg/ml of di-saturated phospholipids, 1.5

mM of calcium ions, 7.5 mM of citrate (citrate trisodium salt) and 10 %

(volume/volume ratio) glycerol to pH 6.0. Glycerol was added at 10 % of the total concentration to give viscosity (or stickiness) to the solution so that the di-saturated phospholipids can be easily spread on skin with a mechanical force during the application on the skin. The mixed components were vigorously vortexed to form a homogeneous suspension, and then used hereinafter.

(2) Application

The application on skin was carried out by falling a drop (approximately 30 μl)

of the solution (described above) on every 2 cm X 2 cm of the ailing skin area and then

by rubbing the solution very softly until it is absorbed completely to the skin. It was

recommended that the solution be applied at least 2 times daily. It was also

recommended that cosmetics or soaps with strong scent, which may contain the

chemicals having an aromatic ring(s) be not used during the application period of the

composition.

2-2-1. Experiment results on primary volunteers (observation with the naked

eye)

The tests on volunteers were carried on 15 skin sites of 8 volunteers (5 to 40

years old, average age of 33 years old, 4 males and 4 females) suffering from atopic skin

diseases. The application of the composition on the skin was carried out for a period

of 2 to 4 weeks in which the period was determined by both the severity and the

improvement of skin conditions. It was found that, during the period of the application,

the symptoms of the atopic skin diseases were improved in all of the test volunteers to

the extent that it is difficult to distinguish the boundaries of the previous diseased skin

area. It was observed that skin crusts, formed since the skin exudates are dried due to the atopic skin diseases, are not formed again in the skin surface about 3 days after the application of the composition. In the case of the volunteers with the atopic erythema, the symptom disappeared so that the rim of the affected skin area by atopic skin disease could not be distinguished 1 week after the application of the composition. It was

confirmed that the wounds were completely recovered 3.5 weeks (approximately 25

days) after the application of the composition in the case of the patients having

complications of skin gangrene (skin ulcer) (see Fig. 4).

2-2-2. Experiment results on secondary volunteers (observation with the naked eye)

Another tests on volunteers were carried on 27 volunteers (0.8 to 42 years old,

average age of 23.4 years old, 13 males and 14 females) suffering from atopic skin

diseases.

The application of the composition on the skin was carried out for a period of 2

to 5 weeks. The symptoms of the atopic skin diseases were improved in 25 volunteers

except 5 volunteers to the extent that it is difficult to distinguish the boundaries of the

originally diseased skin area. Two patients who had a complication of atopic skin disease and severe acne symptom on the face and the back, respectively, did not show

the significant improvement of the condition. Three volunteers who had a very thick

cornification of skin as a consequence of the very long atopic skin diseases sufferings

showed the slight improvement of the skin condition during the period of the application.

3 out of 25 volunteers whose symptoms were clearly improved had to be treated with an antibacterial ointment during the application of the composition because a skin infection was observed and the patients could not overcome the infection for themselves. The photographs showing the results of the skin condition improvement are shown in

Figs 5 to 8. Experimental example 3: Effects of calcium and organic acid added to the composition of the present invention

In order to determine the effects of calcium ions and an organic acid in the

composition on the treatment of atopic diseases, the compositions having different

components were prepared and applied to affected parts of atopic patients. Patients

whose severity of the atopic skin diseases are similar on both arms or both legs were

selected and subject to a therapeutic test, and the affected parts of atopic patients were

observed with naked eyes.

3-1: Preparation of compositions having different combination

A solution in which the finally freeze-dried di-saturated phospholipid fraction

was emulsified in distilled water, a citrate tri-sodium salt/citric acid standard solution

(stock solution, pH 6), a CaCl₂ standard solution and glycerol were autoclaved,

respectively. After autoclave, each of the components was mixed to give compositions

having the following concentrations. Basic composition (All) of the present invention: 20mg/ml of dried phospholipid,

1.5mM CaCl₂, 7.5mM citrate/ citric acid, 10% (v/v) glycerol

Control 1 (no-citrate): 20mg/ml of dried phospholipid, 1.5mM CaCl₂, 10% (v/v)

glycerol

Control 2 (no-Ca): 20mg/ml of dried phospholipid, 7.5mM citrate/ citric acid,

10% (v/v) glycerol.

Each of the compositions was adjusted to a pH value of 6.0.

3-2: Application method and Subjects

Patients whose severity of atopic conditions are similar on their arms or both legs were selected, and then the Control 1 (no-citrate) or the Control 2 (no-Ca) was

applied to the right affected parts of the patients, and the composition (All) of the

present invention was applied to the left affected parts of the patients. One drop (about

30 μ 1) of the compositions was administered to the affected parts (2cm x 2cm), and

rubbed on the skins until the compositions were smeared completely. It is

recommended to apply the composition to the skins twice to four times daily, and if other infections are accompanied with it, each of the compositions was applied to the

skins and then fusicidic acid (Product name: Fusidin, Dong-Wha Pharmaceutical

Industrial Co Ltd) was applied on it.

Seven patients participated in a comparison test of Control 1 (no-Citrate), and

they were 10.4 years old in average (5~15 years old, five males and two females). Three patients participated in a comparison test of Control 2 (no-Ca), and they were

11.6 years old in average (7~15 years old, two males and one female). The comparison

tests on efficiencies of the components were carried out up to one week.

3-3: Result and Conclusion

(1) Comparison between efficiencies of Control 1 (no-citrate) and Composition

(All) of the present invention

The citric acid/citrate-free control composition [Composition 1] was applied to a

right region in the affected part of the atopic skin disease, and the calcium and citric

acid/citrate-containing composition of the present invention was applied to a left region in the affected part of the atopic skin disease to compare efficiencies of the two compositions to each other. As a result, it was revealed that the therapeutic effects of

the two compositions are more prominent with the naked eye as the atopic skin diseases get more severe. In the case of the slight atopic dermatitis, the two compositions all

had good therapeutic effects by appearance, but the test volunteers (6 out of 7 patients)

complained of irritations (itches) in the right region to which the Control composition

[Composition 1] is applied (see FIGs. 9 to 13).

(2) Comparison between efficiencies of Control 2 (no-Ca) and Basic

composition (All) of the present invention

The Control composition [Composition 2], obtained by adding phospholipid and

citric acid except for calcium ions to the Basic composition, was applied to a right

region in the affected part of the atopic skin disease, and the Basic composition was

applied to a left region in the affected part of the atopic skin disease. As a result, it

was revealed that the Composition 2 has a strong irritation to the skins. 2~3 hours

after its application to the skins, all the test volunteers (3 out of 3 patients) complained

of severe itches. The comparison tests between efficiencies of the Control 2 (no-Ca)

and the Basic composition (All) were immediately suspended due to the severe itches of

the test volunteers.

(3) Conclusion

If the di-saturated phospholipid was directly applied to tissues in the expectation of a good therapeutic effect on the atopic skin diseases, the organic acids containing a carboxyl group such as citrate are essentially required so that the di-saturated

phospholipid cannot be aggregated by calcium and easily applied to the tissues without changing a calcium concentration which is inherently present in the tissues. In particular, it is considered that citrate plays an important role in relieving pathological

conditions caused by the intake of excessive unsaturated fatty acid since the citrate

prevents the phospholipid molecules from being aggregated by calcium ions, and also

regulates the synthesis of fatty acid in the cytoplasm.

INDUSTRIAL APPLICABILITY

As described above, the composition including di-saturated phospholipid

according to the present invention may be useful to relieve or treat atopic diseases, etc.

which are caused by the exudation of the serum proteins.

Claims

What is claimed is;

1. A composition for relieving or treating diseases which are associated with the exudation of serum proteins, comprising di-saturated phospholipid as an

effective component.

2. The composition according to claim 1, wherein the di-saturated phospholipid is extracted from animals.

3. The composition according to claim 1, wherein the di-saturated

phospholipid is extracted from a cattle or a pig.

4. The composition according to claim 2 or 3, wherein the di-saturated

phospholipid is extracted from a bronchial alveolar lavage or lung tissue homogenate.

5. The composition according to any one of claims 1 to 3, wherein the

di-saturated phospholipid includes dipalmitoylphosphatidylcholine and/or

dipalmitoylphosphatidylinositol as major constituents.

6. The composition according to claim 1, further comprising calcium ions

and/or organic acid containing a carboxyl group(s).

7. The composition according to claim 1, wherein the organic acid is selected from the group consisting of lactic acid, succinic acid, fumaric acid and citric

acid.

8. A composition for treating skin diseases, comprising the composition as defined in claim 1.

9. A cosmetic composition comprising the composition as defined in claim 1.

10. The composition according to claim 1, wherein the di-saturated

phospholipid is present at a concentration of 1 mg/ml to 700 mg/ml.

11. The composition according to claim 1, wherein the disease associated

with the exudation of the serum proteins is selected from the group consisting of atopic

dermatitis, atopic eczema, pruritus cutaneous, atopic erythema, atopic erythroderma and

contact dermatitis.

12. The composition according to claim 1, wherein the disease associated

rhinitis, asthma, and chronic obstructive pulmonary diseases.

13. The composition according to claim 6, wherein the disease associated with the exudation of the serum proteins is selected from the group consisting of atopic dermatitis, atopic eczema, pruritus cutaneous, atopic erythema, atopic erythroderma and

contact dermatitis.

14. The composition according to claim 6, wherein the disease associated

rhinitis, asthma, and chronic obstructive pulmonary diseases.