U.S. patent application number 09/731830 was filed with the patent office on 2001-05-03 for method of supressing immune responses by reducing intracellular content of glutathione in macrophages and monocytes.
This patent application is currently assigned to AJINOMOTO CO. INC.. Invention is credited to Hamuro, Junji, Murata, Yukie.
Application Number | 20010000784 09/731830 |
Document ID | / |
Family ID | 27338952 |
Filed Date | 2001-05-03 |
United States Patent
Application |
20010000784 |
Kind Code |
A1 |
Hamuro, Junji ; et
al. |
May 3, 2001 |
METHOD OF SUPRESSING IMMUNE RESPONSES BY REDUCING INTRACELLULAR
CONTENT OF GLUTATHIONE IN MACROPHAGES AND MONOCYTES
Abstract
A method of suppressing immune responses, comprising
administering to a patient in need thereof an effective amount of a
composition comprising a substance capable of reducing the content
of reductive glutathione in macrophages.
Inventors: |
Hamuro, Junji;
(Kanagawa-ken, JP) ; Murata, Yukie; (Kanagawa-ken,
JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO. INC.
15-1, Kyobashi 1-Chome
Tokyo
JP
|
Family ID: |
27338952 |
Appl. No.: |
09/731830 |
Filed: |
December 8, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09731830 |
Dec 8, 2000 |
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09334647 |
Jun 17, 1999 |
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6197749 |
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09334647 |
Jun 17, 1999 |
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09181881 |
Oct 29, 1998 |
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Current U.S.
Class: |
424/278.1 ;
514/16.6; 514/21.6; 514/4.3; 514/5.5; 514/550; 514/6.9 |
Current CPC
Class: |
A61K 31/225 20130101;
A61K 47/62 20170801; A61K 31/22 20130101; A61K 38/05 20130101; A23L
33/175 20160801; A61K 38/063 20130101; A61K 31/198 20130101 |
Class at
Publication: |
514/18 ;
514/550 |
International
Class: |
A61K 038/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 1997 |
JP |
312727 |
Oct 29, 1998 |
JP |
308300 |
Claims
What is claimed is:
1. A method of suppressing immune responses, comprising
administering to a patient in need thereof an effective amount of a
composition comprising a substance capable of reducing the content
of reductive glutathione in macrophages.
2. The method of claim 1, wherein the substance has a
intramolecular disulfide bond.
3. The method of claim 2, wherein the substance is a cystine
derivative.
4. The method of claim 3, wherein the cystine derivative is
represented by formula (I): 2wherein R.sup.1 and R.sup.2,
independently from each other, represent an alkyl group, and
R.sup.3 and R.sup.4, independently from each other, represent an
acyl group or a peptidyl group.
5. The method of claim 4, wherein R.sup.1 and R.sup.2,
independently from each other, represent an alkyl group having 1 to
12 carbon atoms.
6. The method of claim 4, wherein R.sup.3 and R.sup.4,
independently from each other, represent an acyl group having 1 to
12 carbon atoms.
7. The method of claim 6, wherein R.sup.1 and R.sup.2,
independently from each other, represent an alkyl group having 1 to
12 carbon atoms.
8. The method of claim 4, wherein R.sup.3 and R.sup.4,
independently from each other, represent an peptidyl group having 1
to 10 amino acid residues.
9. The method of claim 8, wherein R.sup.1 and R.sup.2,
independently from each other, represent an alkyl group having 1 to
12 carbon atoms.
10. The method of suppressing immune responses according to claim
1, wherein the substance is capable of selectively removing
reductive macrophages which contain an increased amount of
intracellular reductive glutathione.
11. The method of suppressing immune responses according to claim
10, wherein said substance is a compound in which a cytotoxic DNA
alkylating agent having is conjugated with glutathione, or one
which shows a cytotoxicity after being incorporated into
macrophages as a precursor.
12. The method of suppressing immune responses according to claim
1, wherein the patient is in a cachectic condition caused by
cancers, or suffering from diabetes, gastrointestinal inflammatory
diseases, chronic rheumatoid arthritis, hepatitis, hepatic
cirrhosis, hypersensitive interstitial pneumonia, pulmonary
fibrosis or autoimmune inflammatory diseases.
13. A food, a nutrient or an infusion, containing a cystine
derivative represented by formula (I): 3wherein R.sup.1 and
R.sup.2, independently from each other, represent an alkyl group,
and R.sup.3 and R.sup.4, independently from each other, represent
an acyl group or a peptidyl group.
14. The food, nutrient or infusion of claim 13, wherein R.sup.1 and
R.sup.2, independently from each other, represent an alkyl group
having 1 to 12 carbon atoms.
15. The food, nutrient or infusion of claim 13, wherein R.sup.3 and
R.sup.4, independently from each other, represent an acyl group
having 1 to 12 carbon atoms.
16. The food, nutrient or infusion of claim 13, wherein R.sup.1 and
R.sup.2, independently from each other, represent an alkyl group
having 1 to 12 carbon atoms.
17. The food, nutrient or infusion of claim 13, wherein R.sup.3 and
R.sup.4, independently from each other, represent an peptidyl group
having 1 to 10 amino acid residues.
18. The food, nutrient or infusion of claim 13, wherein R.sup.1 and
R.sup.2, independently from each other, represent an alkyl group
having 1 to 12 carbon atoms.
19. The food, nutrient or infusion of claim 13, which is a food.
Description
BACKGROUND OP THE INVENTION
1. 1. Field of the Invention
2. The present invention relates to a novel immunomodulator. More
specifically, the present invention relates to an immunomodulator
(e.g., immunoenhancer and immunosuppressant) capable of oral intake
which has a novel suppressive function on macrophages (hereinafter
sometimes abbreviated as "M.PHI.") or monocytes and which may be
used, for example, for the treatment, improvement and prevention of
human immunological diseases such as hepatic cirrhosis, hepatitis,
diabetes, gastrointestinal inflammatory diseases such as
inflammatory bowel diseases (ulcerative colitis, Crohn disease,
etc.), auto-immune diseases and allergic diseases such as
hypersensitive interstitial pneumonia, pulmonary fibrosis, chronic
rheumatoid arthritis, asthma and cutaneous atopy, and cancers, and
to a drug, a food (including a food for medical care, a health food
or a special sanitary food), a nutrient and an infusion containing
the same.
3. 2. Description of the Related Art
4. As used herein, the term immune system refers to a system in an
organism for defending itself from exogenous infection with virus,
bacteria or the like, or from invasion of a human body with
transformed cells (tumor cells and the like) formed by
transformation of autologous cells. However, the immune system
occasionally behaves abnormally, i.e., it functions excessively and
acts to reject autologous components, or, on the other hand it
sometimes functions deficiently, resulting in an immunocompromised
state. Diseases resulting from these abnormal responses are
generally called immunological diseases. Examples thereof include
diverse diseases, for example, acute or chronic inflammatory
diseases such as atopic cutaneous inflammatory diseases,
pollinosis, asthma and sarcoidosis; autoimmune diseases such as
allergic diseases, chronic rheumatoid arthritis, diabetes (IDDM),
SLE and chronic fatigue syndrome; hepatitis, hepatic cirrhosis,
inflammatory bowel diseases (IBD) such as ulcerative colitis and
Crohn disease; and cancer cachexia. These immunological diseases
originate from complex pathological causes. Systemic
immunodeficiency and functional deficiency originate from
pathological inflammation accompanied by cell proliferation,
differentiation or cell necrosis through local production of
cytokines or inflammatory mediators.
5. As cells that participate in immunity, T lymphocytes and B
lymphocytes are well known. These cells exhibit a wide variety of
functions as cells playing roles in cellular immunity and humoral
immunity respectively. Meanwhile, macrophages and monocytes are
cells that are intimately involved in both cellular immunity and
humoral immunity, and they participate significantly in rejection
of non-self foreign bodies, for example, in immunological diseases
such as allergy and rheumatism, cancers and bacterial
infection.
6. The functions of macrophages and monocytes are classified into
four different types, a secretary function, an immunoregulatory
function (mainly antigen presentation), treatment of foreign bodies
and waste matters, a phagocytic function and a cytotoxic/cytostatic
activity against target cells. It is widely accepted that these
cells produce diverse inflammatory mediators; for example cytokines
such as TNF, IL-12, IL-1, IL-6, TGF.beta. and IL-8 and so on;
hormonal molecules such as neopterine (NPT) and
dihydroxyepiandrosterone (DHEA); arachidonic acid metabolites such
as PGE2 and LTB4; complement and related molecules such as C5a and
C3; such as reactive oxygen and reactive nitrogen intermediates. It
has not been clarified whether these diverse functions are
exhibited by one kind of macrophage or monocyte or by distinctive
groups of macrophages or monocytes having different functions.
While lymphocytes are classified into distinctive subsets according
to their cell surface markers and the distinctive functional
markers uniquely correspond to each subset of lymphocyte, the
correspondence between the wide variety of functions of macrophages
is less clear. Monocytes have not been classified into cellular
subsets. For this reason, although macrophages and monocytes play
quite important roles in the triggering and the pathological
progression of the above mentioned inflammatory, allergic and
immunological diseases, the functional classification of
macrophages and monocytes subsets has not yet been applied at all
to therapeutic, prophylactic and preventive treatment of human
diseases, with the assumption of the presence of macrophage and
monocyte subsets, and even the hypothesis thereof has not yet been
given.
7. In recent years, in the patients suffering from allergic
diseases, autoimmune- diseases such as chronic rheumatoid arthritis
and cancer, the inclination of helper T cell subsets in the
peripheral blood has been pointed out and has been linked to the
pathology of these diseases. Helper T lymphocytes which are a
subset of T lymphocytes have been further classified into two
subsets, namely Th1 and Th2, and it is currently proving that the
ratio of these two types is an relevant index of immunological
functions of patients. Attempts are being made to establish a more
appropriate therapeutic treatment by diagnosis of the ratio or by
improvement of the ratio based on this index. That is, it is known
that when the amount of Th2 inducing IgE production from B cells is
higher than that of Th1 (Th1<Th2), allergic diseases are
worsened. Attempts are being made to suppress allergy upon
measuring a Th1/Th2 ratio to examine an immunological response of
patients or to provide Th1 response superior to Th2 responses. On
the contrary, the presence of diseases caused by a predominance of
Th1 has been successively indicated also in chronic rheumatoid
arthritis or an asthmatic inflammatory disease at the chronic
stage.
8. Even when the Th1/Th2 balance is measured using biological
materials and the functions of the two subsets are modulated, this
modulation has not successfully been utilized currently in the
examination or the diagnosis of local chronic inflammatory diseases
or allergic diseases. The terms such as Th1 diseases and Th2
diseases have been used lately. However, these terms cannot
necessarily be distinguished clearly.
9. The Th1/Th2 presence ratio is only an index of lymphocyte
subsets. Since the in vivo dynamism of the lymphocyte subsets is
actually regulated by the cell group called accessory cells
including macrophages in the present invention, it is difficult to
appropriately diagnose the progression of diseases with only the
Th1/Th2 presence ratio and to treat the same on the basis of this
index. As will be described below, the Th1/Th2 balance is
controlled by the distinct macrophage/monocytes functions. Even if
a skewing to Th1>Th2 is intended, this is hardly effective for
therapy of immunological diseases, due to the presence of a complex
cytokine network, and a new index for diagnosis and therapy has
been in demand.
10. It has been clarified that in macrophages participating in the
inflammatory reactions, the functions of the cells are variable
depending on environmental factors such as oxidative stress,
cytokine stimulation, infection with virus or bacteria and the
like. However, the correspondence between the functions and the
classification of cell subsets of macrophages is highly uncertain.
New findings are required in the above-mentioned classification of
functions and subsets, and these findings will lead to the
development of quite useful new therapeutic methods. Under such
circumstances, the development of excellent agents for modulating
immunity, namely, immunomodulators, has been in demand.
SUMMARY OF THE INVENTION
11. It is an object of the present invention to provide a method of
suppressing immune responses.
12. It is another object of the present invention to provide
compositions, especially food compositions, which may be used to
suppress immune responses.
13. The present inventors have conducted investigations to solve
the above-mentioned problems, and have consequently found the
following findings. That is, they have attempted to distinguish
macrophages (including monocytes) which share with an
immunosuppressive activity, a cachexia inducing activity, an
activity of inducing malignant progression and an activity of
prolonging inflammation from immunoregulatory macrophages in view
of a difference in a redox state (potential) of macrophages, and
have then succeeded in this attempt. The reductive glutathione
(GSH) content in macrophages is employed as an index thereof.
14. Accordingly, the objects of the present invention, and others,
may be accomplished with method of suppressing immune responses by
administering to a patient in need thereof an effective amount of a
composition comprising a substance capable of reducing the content
of reductive glutathione in macrophages.
BRIEF DESCRIPTION OF THE DRAWINGS
15. A more complete appreciation of the invention and many of the
attendant advantages thereof will be readily obtained as the same
becomes better understood by to the following detailed description
when considered on combination with the accompanying drawings,
wherein:
16. FIG. 1 is a diagrammatic view showing a relationship of a
difference in a function of macrophages, with respect to the Th1
and Th2 balance, immunosuppression, malignant progression, cancer
cachexia, and local inflammatory responses.
17. FIG. 2 illustrates that the presence ratio of oxidative and
reductive macrophages controls the immunological functions through
the skewed generation of Th1 and Th2 cytokines. This is based on
the new findings of the present inventors, showing that the redox
condition of macrophages plays an important role in amplifying the
inclination of the in vivo responses between humoral and cellular
immunity.
18. FIG. 3 is a view showing the results of the examination of
functions of both macrophages, namely the functional differences
between oxidative macrophages and reductive macrophages.
19. FIG. 4 is a view showing the results of examining whether there
is a difference in the IL-12 production between Lentinan (LNT)
induced M.PHI. and Lipopolysaccharide (LPS) induced M.PHI.. It
indicates that there is a great difference in the amount of IL-12
(Th1 inducing cytokine) produced between oxidative and reductive
macrophages and IL-12 is produced only from reductive macrophages
with the high intracellular reductive glutathione content.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
20. Glutathione is present in all mammalian cells, and well known
as an intrinsic antioxidant. It is a tripeptide having a wide
variety of functions in cells, such as removal of radicals or
peroxides, metabolism of eicosanoids such as prostaglandin,
detoxication of biologically foreign materials, amino acid
transport and the like. Glutathione includes reductive glutathione
(GSH) and oxidative glutathione (GSSG), and these form a coupled
cycle. In normal cells, the content of reductive glutathione (GSH)
is higher, and it acts defensively on oxidative stress, especially
on H.sub.2O.sub.2.
21. Ruede et al. have already reported that with respect to
macrophages differentiated in the presence of GM-CSF and
macrophages differentiated in the presence of M-CSF from monocytes,
the cellular GSH content of the former is higher than that of the
latter, so that the difference in the GSH content in cells seems
likely to participate in the function of macrophages (Germann, T.,
Mattner, F., Partenheimer, A. et al.: Different accessory function
for Th1 cells of bone marrow derived macrophages cultured in
granulocyte-macrophage colony-stimulating factor or macrophage
colony-stimulating factor. Int. Immunol., 4:755, 1992: Frosch, S.,
Bonifas, U., Eck, H. P. et al. : The efficient bovine insulin
presentation capacity of bone marrow-derived macrophages activated
by granulocyte-macrophage colony-stimulating factor correlates with
a high level of intracellular reducing thiols. Eur. J. Immunol. ,
23; 430, 1993). The present inventors have measured the reductive
GSH content in macrophages, and have found that there is a great
difference in an immunological function between macrophages having
different GSH contents (refer to FIG. 1); they have tested the
immune responses with regards to the cellular GSH content, and have
found that the redox states can artificially be modulated with an
orally administerable low-molecular weight substance, and that
these substances capable to modulate intracellular GSH content can
widely be applied to treatment of wide variety of diseases and the
substance can be also used as a food (refer to FIG. 1). These
findings have led to the completion of the present invention.
22. FIG. 1 is a diagrammatic view showing a linkage between a
difference in a function of macrophages or monocytes (both are
referred to as "macrophages" in the present invention), and an
effect on a Th1/Th2 balance, a mechanism of immunosuppression,
cachexia induction and induction of malignant tumor progression
caused by a functional difference of macrophages and local
inflammatory diseases. For example, according to the tumor
progression, the local Th1/Th2 balance is skewed, an inclination to
humoral immunity appears, the structure and the function of the
cytokine receptor complex are changed, oxidative macrophages with a
low intracellular GSH content are increased, the production of
active oxygen or inflammatory mediators such as PGE2, IL-6, IL-10
and IL-8 are increased to cause systemic immunosuppression or
induction of cachexia and to prolong chronic inflammation
accompanied by allergic reactions or tissue injury.
23. The present inventors have conducted further investigations on
the basis of the above-mentioned findings, and have consequently
found that heterogeneous macrophages, which play important roles in
the inflammation reactions can be classified into two groups,
namely, oxidative macrophages and reductive macrophages by
determining the intracellular content of oxidative glutathione and
the intracellular content of reductive glutathione in macrophages.
The oxidative macrophages induce local chronic inflammatory
diseases or an allergic reaction in immunological diseases and the
Th1/Th2 balance controlling the balance of humoral and cellular
immunity is regulated with the redox state of macrophages, that the
redox state of the macrophages plays an important role in
immunological diseases, and this redox state is monitored and
artificially controlled or modified which is useful in the
diagnosis or the therapy of these immunological diseases, and that
this control can easily be conducted using low-molecular weight
substances capable of oral intake.
24. With respect to the definition of the oxidative macrophage and
the reductive macrophage in the present invention, macrophages are
reacted with monochlorobimane which is a chemical reagent specific
to reductive glutathione (GSH) to determine the GSH content in
cells. A macrophage having a GSH content which is higher in
comparison with the resident macrophages is defined as a reductive
macrophage, and a macrophage having a GSH content which is lower is
defined as an oxidative macrophage. Further, a macrophage in which
the GSH content becomes more than 2 nmoles/5.times.10.sup.5
macrophages by bringing a low-molecular weight substance capable of
oral intake into contact with the macrophage for from 2 to 24 hours
is defined as the reductive macrophage (or monocyte), and a
macrophage in which the GSH content becomes less than 0.1
nmoles/5.times.10.sup.5 macrophages is defined as the oxidative
macrophage. Alternatively, a macrophage of which the GSH content is
two or more times than that of the resident macrophage is defined
as a reductive macrophage, and a macrophage of which the GSH
content is 1/5or less that of the resident macrophage is defined as
an oxidative macrophage.
25. At present, it is considered that the Th1/Th2 balance is
regulated by the ratio of IL-6 or IL-4 and IL-12 produced in vivo.
It is known that Th2 participating in humoral immunity is induced
by the former two and Th1 by IL-12 respectively. It is clarified
that IL-6 and IL-12 are produced from macrophages. However,
assuming that the same macrophages produce both IL-6 and IL-1 2,
one type of a macrophage participating in both the Th1 induction
and the Th2 induction comes to be present. Thus, there is a great
contradiction in considering the host immune responses.
26. The present inventors have found that IL-12 is produced from
only the reductive macrophage having the high intracellular GSH
content to act on the Th1 induction and that the IL-6 production is
increased in the oxidative macrophage to induce Th2. They have
further found that when the macrophage is inclined to the oxidative
type in spite of the production of IFN.gamma., a typical Th1
cytokine, IL-6 skewing the balance to Th2 is produced in a large
amount by IFN.gamma. stimulation. On the contrary, it has also been
found that IFN.gamma., a typical Th1 cytokine, increases the
phenotype of the reductive macrophage further by acting on the
reductive macrophage. When IL-4, the typical Th2 cytokine, acts on
the oxidative macrophage, the oxidative macrophage phenotypes are
further increased. These knowledge indicate that the balance
between humoral immunity and cellular immunity is unequivocally
defined by the redox state of macrophages, and they are relevant
new findings innovating the basic concept of immunology (refer to
FIG. 2). On the basis of these findings, the quite useful, original
invention overcoming the conventional confused immunological
disease therapy was already completed with respect to the diagnosis
and the therapy of the immunological diseases. Investigations have
been assiduously conducted on the basis of the above-mentioned
findings. Consequently, the present invention has been newly
completed.
27. That is, the present invention is an immunomodulator containing
substances having an activity of changing a content of glutathione
in macrophages. In the present invention, the macrophage also
includes monocytes. This substance is preferably one which provide
M.PHI. with productivity of interleukin 12 by increasing the
content of reductive glutathione in macrophages. More preferable
examples thereof include low-molecular weight substances, for
example, a GSH precursor metabolized into GSH within cells, such as
N-acetylcysteine (NAC), .gamma.-glutamylcysteine,
.gamma.-glutamylcysteine dimethyl ester, .gamma.-glutamylcysteine
diethyl ester and N-acetylcysteine nitroxybutyl ester; glutathione
derivatives such as glutathione monoester, glutathione diester and
glutathione nitroxybutyl ester; lipoic acid and derivatives
thereof; gliotoxin and derivatives thereof having two SH groups or
more in the molecule; and ortene. These can be administered orally
or percutaneously. It is also possible to use antioxidants such as
flavonoid and derivatives thereof which raise the GSH content,
increase the production of IL- 12 and decrease the production of
IL-6 by contact with macrophages. Further, high-molecular weight
substances which are used in combination therewith, such as
.beta.(1-3) glucan and cytokine, are preferably used in the
intravenous administration and the administration using DDS (drug
delivery system). Preferable examples of the cytokine include IL-4,
IL-2, IL-12, TGF.beta. and IFN.gamma.. When it is required to
increase cellular immunity, IL-2 and/or IFN.gamma. is especially
preferable. When it is required to decrease cellular immunity, IL-4
and/or TGF.beta. is especially preferable. These substances can be
contained either solely or in combination, and a higher effect is
expected by a combination of a low-molecular weight orally
administerable immunomodulator and a high-molecular weight
immunomodulator suited for intravenous administration.
28. Further, the present invention also includes an immunomodulator
containing a substance which can selectively remove either of two
types of macrophages, reductive macrophages or oxidative
macrophages which are different in the intracellular content of
reductive glutathione. Examples of the substance include a
substance in which a cytotoxic DNA alkylating agent is conjugated
with glutathione, and a substance in which an oxidative or
reductive macrophage-specific antibody is conjugated directly or
through a linker with a low-molecular weight compound having a
cytotoxicity to macrophages or with a material showing a
cytotoxicity after being incorporated into a macrophage. Examples
of the alkylating agent include cyclophosphamide, nimustine (ACNU),
mitomycin C and melphalan. In an oxidative macrophage in which
glutathione S-transferase is activated, an DNA alkylating agent,
bound to glutathione directly or through a linker, is deconjugated
by the action of this enzyme and can remove the reductive
macrophage by specifically killing the same. Further, a substance
which has no cell-killing property in vitro but comes to show the
cell-killing property with the action of an enzyme increased either
in oxidative or reductive macrophages can also be used as a
prodrug.
29. The present inventors have conducted further investigations
especially on gastrointestinal inflammatory diseases based on these
findings, have looked for substances of reducing a content of
reductive glutathione in macrophages, and have found substances
which exhibit the effect at quite low doses. They have produced
animal models having gastrointestinal inflammatory diseases similar
to those of humans, and have promoted their investigations for
development of an immunosuppressant as a drug, and a food such as a
food for medical care, a health food or a special sanitary food, a
nutrient and an infusion which have an immunosuppressive activity.
They have focused on cystine derivatives, and have widely examined
an effect of inhibiting an immunological activity in vitro and an
immunosuppressive effect in the administration to animals using a
compound represented by the following structural formula (1), as
well as a pharmaceutical effect of candidate substances using gene
knock out mice which are spontaneously accompanied with
gastrointestinal inflammatory diseases. Consequently, they have
found that cystine derivatives, especially those represented by the
following structural formula (1) are effective as pharmaceutical
substances having an activity of reducing the content of reductive
glutathione in macrophages and monocytes. 1
30. wherein R.sup.1 and R.sup.2, independently from each other,
represent an alkyl group or a substituted alkyl group such as the
nitroxybutyl group, and R.sup.3 and R.sup.4, independently from
each other, represent an acyl group or a peptidyl group.
31. Especially, it is quite useful as an immunosuppressant against
inflammatory bowel diseases such as ulcerative colitis and Crohn
disease, and gastrointestinal inflammatory diseases such as
hepatitis and hepatic cirrhosis.
32. Further, the present invention includes a food (including a
food for medical care, a health food or a special sanitary food), a
nutrient or an infusion containing the above-mentioned
immunomodulator. The food includes ordinary foods and those which
are put into the mouth, such as a toothpaste, a chewing gum and the
like. It is especially preferable to incorporate the
immunomodulator in health foods. Further, it may be used as an
additive which is added to a food. As the nutrient, vitamin
preparations and calcium preparations are available. As the
infusion, a high calorie infusion, a physiological saline solution
and blood preparations are available.
33. In addition, the immunomodulator, the food, the nutrient and
the infusion of the present invention is preferably used for
improvement of the cachectic condition of patients suffering from
cancers and for diabetes, gastrointestinal inflammatory diseases,
chronic rheumatoid arthritis, hepatitis, hepatic cirrhosis, above
stated autoimmune inflammatory diseases and/or chemoprevention of
cancers.
34. Especially, an immunomodulator having an activity of reducing a
content of reductive glutathione in macrophages of humans can
widely be used in not only drugs but also the foods as an
immunosuppressant of the autoimmune inflammatory diseases in the
form of a single compound or a mixture. Thus, it is useful as the
food, the nutrient and the infusion having the immunosuppressive
activity.
35. The present invention is described in even more detail
below.
36. The present invention provides an immunomodulator useful for
therapy of patients suffering from immunological diseases in which
macrophages are classified into oxidative macrophages and reductive
macrophages having different functions by determining the contents
of oxidative glutathione and/or reductive glutathione in
macrophages using a body fluid or a cell sample separated and
collected from humans, and the ratio of these macrophages present
is artificially controlled with a substance capable of oral intake
or either oxidative macrophages or reductive macrophages are
artificially removed, as well as to a food, a nutrient and an
infusion which are useful for improvement of diseases. With respect
to the body fluid/cell samples separated and collected from humans,
there are, for example, cells separated from the peripheral blood,
the peritoneal cavity, the thoracic cavity, the solid carcinoma
local tissue, the articular cavity and various organs.
37. With respect to a method of measuring a content of glutathione,
a content of oxidative or reductive glutathione is directly
measured biochemically by an enzymatic recycling method [refer to
Protocol of Active Enzyme Experiments (Saibo Kogaku, separate
volume), Shujunsha, pp. 84-88, 1994, Analytical Chemistry, vol.
106, pp. 207-212, 1980, and Cellular Immunology, vol. 164, pp.
73-80, 1995]. Each of these publications is incorporated herein by
reference. Further, it can indirectly be measured using a
monoclonal antibody or a polyclonal antibody specific to oxidative
or reductive macrophages or using a reagent which is specifically
reacted with GSH to form a complex and emits fluorescent light
through laser excitation, such as monochlorobimane.
38. Glutathione in the present invention is also called
L-glutamyl-L-cysteinylglycine. It is an SH compound which is mostly
present in vivo, and generally referred to as "GSH". Glutathione is
classified into reductive glutathione and oxidative glutathione.
Reductive glutathione refers to the above-mentioned glutathione
(GSH). Oxidative glutathione is also called glutathione disulfide,
and is referred to as "GSSG".
39. The macrophage in the present invention also includes the
above-mentioned monocyte. Macrophage-related cells called dendritic
cells and Kupffer cells are also included. The macrophage is known
to secrete or release various mediators such as cytokines and
inflammatory mediators from cells thereof. Whether they are
secreted or not is determined depending on its activated or
differentiated condition, and the amount released varies depending
thereon. In the present invention, an attention is directed to the
contents of oxidative glutathione and reductive glutathione in
macrophages. Macrophages are monitored by the ratio of oxidative
macrophages and reductive macrophages, and the immunological state
is identified. The balance of these macrophages is modulated with
the immunomodulator and the like of the present invention to
improve the in vivo immunological state and to treat or prevent
various diseases usefully.
40. In the reductive macrophage, the content of reductive
glutathione is relatively higher than that in oxidative macrophage.
In the oxidative macrophage, the content of reductive glutathione
is relatively lower than that in reductive macrophage. Further, the
reductive macrophage and the oxidative macrophage are different in
activation of a transcriptional factors due to the difference in
the reductive GSH content. Consequently, there occurs a difference
in the gene expression of cytokines or inflammatory mediators, so
that the type and the amount of the resulting inflammatory
cytokines or inflammatory mediators are changed and the quality of
inflammation is changed.
41. With the oxidative macrophage, inflammatory cytokines and
mediators such as IL-6, IL-1, IL-8, IL-10, TNF, hydrogen peroxide,
superoxide and PGE2 are produced. With the reductive macrophage,
nitrogen monoxide (NO), IL-12 and LTB4 are produced. Further, the
oxidative macrophage and the reductive macrophage are
inter-converted through stimulation or the like. The reductive
macrophage can be converted to the oxidative macrophage through
artificial stimulation using LPS or PMA inducing inflammatory or
ischemic shock and cytokines such as IL-4 and TGF.beta.. On the
contrary, the oxidative macrophage can be converted to the
reductive macrophage with the addition of IFN.gamma., IL-2,
lentinan (LNT) which is an antitumor polysaccharide, or lipoic acid
with an antioxidant nature. This can be applied to therapy of
immunological diseases.
42. The amounts of the oxidative macrophage and the reductive
macrophage vary depending on the pathological state of the said
diseases. The amount of the oxidative macrophage contained in the
body fluid or the cell sample collected from patients suffering
from allergic diseases or advanced cancers is relatively larger
than that in the healthy person. This can be used in the
examination for diagnosis of immunological diseases and tumor
cachexia and the subsequent therapy thereof.
43. Further, it has been clarified through image analysis using an
adherent cell analyzing system (ACAS) or biochemical determination
using an enzyme recycling method that a content of reductive
glutathione in macrophages harvested from model animals suffering
from gastrointestinal inflammatory diseases (hepatitis, Crohn
disease and ulcerative colitis) is relatively lower than that in
normal animals. This indicates that macrophages are inclined to be
oxidative in intestinal inflammatory bowel diseases or
gastrointestinal inflammatory diseases, and that oxidative
macrophages participate in a mechanism of preventing the
progression of diseases. When oxidative macrophages participate in
the disease progression, these have to be converted to reductive
ones. When oxidative macrophages participate as a defense mechanism
of preventing disease progression, the oxidative state of
macrophages has to be maintained through administration of a drug.
The inventors have assiduously conducted studies as to which of
these two possibilities is the essence of diseases. Consequently,
they have found for the first time in the world that the oxidative
macrophages have the defense mechanism of preventing the
progression of chronic inflammatory diseases and autoimmune organ
specific inflammatory diseases mainly in the digestive tract, and
that it is useful to reduce the content of reductive glutathione in
the macrophages as an approach of improving and treating the
gastrointestinal inflammatory diseases.
44. In accordance with the present invention, the low molecular
weight compound which has an activity of changing the content of
reductive glutathione in the macrophage cell after measuring the
same by the above-mentioned method and which maintains the activity
even through the oral intake is formulated into a drug in a usual
manner, and this drug can be taken in the patient every day or at
fixed intervals upon monitoring the condition of the disease. At
the chronic stage, the marked effect is brought forth by the
long-term administration.
45. With respect to the definition of the oxidative macrophage and
the reductive macrophage in the present invention, the reductive
glutathione (GSH) content in the cell is determined through the
reaction with monochlorobimane which is a chemical reagent specific
to GSH. The macrophage of which the GSH content is increased in
comparison with the resident macrophage is defined as the reductive
macrophage, while the macrophage of which the GSH content is
decreased is defined as the oxidative macrophage. Preferably, the
macrophage of which the GSH content is more than 2
nmoles/5.times.10.sup.5 macrophages by being brought into contact
with the low-molecular weight substance capable of oral intake for
from 2 to 24 hours is defined as the reductive macrophage, and the
macrophage of which the GSH content is less than 0.1
nmoles/5.times.10.sup.5 macrophages is defined as the oxidative
macrophage. Alternatively, the macrophage of which the GSH content
is at least twice that of the resident macrophage is defined as the
reductive macrophage, while the macrophage of which the GSH content
is at most 1/5that of the resident macrophage is defined as the
oxidative macrophage.
46. As the substance having the activity of changing the content of
reductive glutathione in macrophages, any substance will do if
macrophages (or monocytes) are incubated at concentrations of
5.times.10.sup.5 cells/200 .mu.l/well using a 96-well microplate,
from 0.01 .mu.M to 5 mM of a substance to be tested are added
thereto and incubated at 37.degree. C. in a 5% CO.sub.2 incubator
and the reductive GSH content is increased or decreased relative to
the control group after from 2 to 24 hours. A substance that can
increase the GSH content to 2 nmoles/5.times.10.sup.5 macrophages
or more or decrease the same to 0.1 nmoles/5.times.10.sup.5
macrophages or less is preferable. Examples thereof include
antioxidants, for example, a precursor of GSH which is metabolized
into GSH in cells, such as N-acetylcysteine (NAC),
-glutamylcysteine diethyl ester, glutathione derivatives such as
glutathione monoester and glutathione diester, lipoic acid and
derivatives thereof, ortene, and flavonoid and derivatives thereof.
They are substances having an activity of changing the content of
glutathione in cells by the incubation with macrophages in vitro
for a few hours. These agents can be used either singly or in
combination. The effect thereof can be measured by collecting
monocytes from an body fluid of local inflammatory sites or a
peripheral blood after the intake or the administration and
determining the change in the content of reductive glutathione in
cells relative to that before the treatment by the above-mentioned
method. The usefulness as the immunomodulator is clearly evaluated
by this procedure, and the agents are effective for the treatment
of the patients. When a cysteine derivative is used as an
immunosuppressant, it is included by the above-stated
immunomodulator and as a matter of course, it can be used in the
same way as stated above.
47. The diseases to which the present invention is applied include
cachectic conditions of patients suffering from cancers; autoimmune
diseases such as diabetes, chronic rheumatoid arthritis, SLE and
pulmonary fibrosis; inflammatory diseases such as hepatitis,
hepatic cirrhosis and inflammatory bowel diseases, centering on
gastrointestinal inflammatory diseases; and allergic diseases such
as hypersensitive interstitial pneumonia, asthma, atopic cutaneous
inflammatory diseases, sarcoidosis, etc.
48. Especially, as diseases to which the immunosuppressant
according to the present invention is applicable, it is expected
that it is effective against autoimmune inflammatory diseases.
Above all, it is desirable to apply the immunosuppressant to
chronic inflammatory diseases caused in digestive organs, including
a group of diseases called inflammatory gastrointestinal diseases
such as hepatitis, hepatic cirrhosis and inflammatory bowel
diseases such as ulcerative colitis and Crohn disease.
49. The agents can widely be applied to diseases associated with
the abnormal Th1/Th2 balance or the functional deficiency of
macrophages, for example, cachexia of patents suffering from
cancers, diabetes, chronic rheumatoid arthritis, autoimmunological
diseases such as SLE, chronic inflammatory diseases such as
hepatitis, hepatic cirrhosis, inflammatory bowel diseases, and
allergic diseases such as asthma, cutaneous atopy and sarcoidosis.
The agents are also effective for chemoprevention of cancers as the
immunomodulator. This makes it clear that during the period in
which one normal cell undergoes transformation and carcinogenesis
in the human body and then reaches to 10.sup.9 cells where the
presence of cancer tissues is clinically detected, the cancerous
tissue is profitably present in the reductive condition. That is,
it is scientifically verified that active oxygen or the like which
is produced by inflammatory responses in vivo contributes to
malignant progression.
50. The immunomodulator used in the present invention can be
administered singly in the actual medical care. The
immunomodulators capable of oral intake which are included in
present invention can also be used in combination. Further,
immunomodulator of the present invention can be mixed with, or used
in combination with, the other immunomodulator in capable of oral
intake but charging the content of reductive glutathione in
macrophages with the different function, for example, exogenous and
endogenous substances such as .beta.(1-3) glucan typified by
lentinan and cytokines typified by interleukin 2(IL-2). Especially
when it is required to increase cellular immunity, IL-2 or
.gamma.-interferon (.gamma.IFN) is used in combination whereby
interleukin 12 (IL-12) is produced in vivo in a large amount from
the reductive macrophage to more increase the effect the present
invention. On the other hand, when the therapeutic effect is
intended by decreasing cellular immunity, production of IL-12 is
decreased with the combined use of interleukin 4 (IL-4) or
TGF.beta. to increase the effect. It has been found in the present
invention that these cytokines change themselves the content of
reductive glutathione in macrophages, increasing the usefulness and
the scope of the present invention.
51. When substances which inhibit the production or the function of
IL-12, other than antibodies, are used in combination as in vitro
substances, a synergistic effect is further expected.
52. It is also included in the present invention that either of the
macrophages which are different in the content of reductive
glutathione in cells, namely, the macrophage (oxidative macrophage)
having the low reductive GSH content and macrophage (reductive
macrophage) having the high reductive GSH content is selectively
removed. The substance used in this case may be a low-molecular
weight compound or a high-molecular hat compound. Among others,
antibodies and derivatives thereof are effective.
53. As already stated, the correspondence of a variety of functions
of macrophages/monocytes to their subsets has been to date totally
unknown. Accordingly, although macrophages/monocytes play quite an
important role in the triggering and the progression of
inflammatory diseases, allergic diseases and immunological
diseases, the functional classification on the basis of the
presence of distinct macrophage/monocyte sub-sets has not been
applied at all to the therapy, improvement and prevention of human
diseases, and this application has not been even imagined. Before
the completion the present invention, the reductive GSH content of
the macrophage was measured, and it was discovered for the first
that there is a great difference in an effect of macrophages having
different GSH contents on the immunological functions. Further, the
contents of oxidative glutathione and reductive glutathione in
macrophages which play an important role in the inflammatory
reaction were measured to classify heterogeneous macrophages into
the two types, namely, oxidative macrophages and reductive
macrophages. Then, it was found that the oxidative macrophages
induce local chronic inflammatory diseases or allergic reaction
accompanied by immunological diseases, that the Th1/Th2 balance
controlling the balance of humoral immunity and cellular immunity
is regulated by the redox state of macrophages, end that the redox
state of the macrophages plays an important role in the progression
of immunological diseases. In order to artificially control the
presence ratio of these two macrophages, the above-mentioned
low-molecular weight substance capable of oral intake is used as a
drug, and the selective removal of either of these macrophages is
also quite useful. This is also understandable from the fact that
various monoclonal antibodies to lymphocytes are on the market as
an immunosuppressor. It is easily conceivable to those skilled in
the art that antibodies to either of these macrophages or to
markers expressed in larger amounts in either of these macrophages
can be used.
54. Further, substances having toxicity to cells or derivatives
thereof can be used. However, since there is a great difference in
intracellular enzymatic activities between reductive macrophages
and oxidative macrophages, substances which can be converted to
those having a selective cytotoxicity within either of reductive
macrophages or oxidative macrophages are most appropriate prodrugs
in the present invention. For example, the use of a pyrimidine
nucleotide phosphorylase enzymatic activity or a
glutathione-S-transferase enzymatic activity which is increased in
the oxidative macrophages is mentioned. There is a product in which
an alkylating agent having a cytotoxicity is conjugated with
glutathione.
55. That the immunomodulator of the present invention can be
applied to a wide variety of immunological diseases is clearly seen
from the fact that it controls the secretion of an inflammatory
mediator from macrophages at the very beginning stage of the
production. For example, non-steroidal acidic anti-inflammatory
drug (aspirin or the like) is said to exhibit the pharmaceutical
effect by controlling production or secretion of prostaglandin.
Meanwhile, an antioxidant such as vitamin E exhibits the
pharmaceutical effect by controlling production of active oxygen.
Thus, the function is only to control one of various properties of
macrophages which are inflammatory cells. For this reason, its
effect is not workable, and almost no effect is exhibited to
chronic inflammatory diseases in particular. On the other hand, the
immunomodulator of the present invention controls the redox
condition of macrophages, and can suppress the production of a
large number of harmful inflammatory mediators all at once. In this
context, the conventional concept to date of anti-inflammatory
drugs is said to be fundamentally changed.
56. As stated above, the useful pharmaceutical effect of the
immunomodulator of the present invention in the actual medical care
is self-evident from its profitable immunological activity. It is
useful for both the acute and chronic stages of diseases.
Especially, it can widely be applied to diseases associated with
the abnormal Th1/Th2 balance or the functional deficiency or the
abnormality of macrophages, for example, cachexia of patients
suffering from cancers; autoimmunological diseases such as
diabetes, chronic rheumatoid arthritis, SLE and pulmonary fibrosis;
chronic inflammatory diseases such as hepatitis, hepatic cirrhosis
and gastrointestinal inflammatory diseases centering on
inflammatory bowel diseases (ulcerative colitis and Crohn disease);
and allergic diseases such as hypersensitive interstitial
pneumonia. asthma, cutaneous atopy and sarcoidosis . It is also
effective for chemoprevention of cancers. With respect to the
cachectic condition of patients suffering from cancers, an effect
to increase the survival rate is expected, and the immunomodulator
is considered to be also useful especially in the improvement of
quality of life (QOL) of the patient.
57. Especially, with respect to the immunomodulator of the
invention, the use of the cystine derivatives as an
immunosuppressant is described in detail below.
58. The substances having the activity of reducing the content of
reductive glutathione are as mentioned above. The cystine
derivatives represented by the structural formula (1) and having
the activity of reducing the content of reductive glutathione in
the macrophages are all included in the cystine derivatives used in
the invention. Examples thereof include
N,N'-diacetylcystine-[(NAC).sub.2], N,N'-dipropylcystine
[(NPC).sub.2], N,N'-diacetylcystinedimethyl ester
[(NAC-OMe).sub.2], N,N'-diacetylcystinediisopropyl ester
[(NAC-OiPr).sub.2] and N,N'-di-L-alanylcystinedimethyl ester
[(NAlaC-OMe).sub.2.
59. With respect to R.sup.1 and R.sup.2 in the structure of the
compounds, a wide variety of substituents can be used so long as
reductive glutathione in the macrophages is oxidized into oxidative
macrophages through the disulfide linkage. For example, R.sup.1 and
R.sup.2, independently from each other, represent an alkyl group
having from 1 to 12 carbon atoms (inclusive of all specific values
and subranges therebetween, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11
carbon atoms) or a substituted alkyl group such as the nitroxybutyl
group, and R.sup.3 and R.sup.4, independently from each other,
represent an acyl group having from 1 to 12 carbon atoms (inclusive
of all specific values and subranges therebetween, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 and 11 carbon atoms) or a peptidyl group. The
peptidyl group means an amino acid residue or peptide residue
consisting of plural amino acids, which is bound through its
carboxyl group. The number of amino acid may vary widely, for
example, from 1 to 10 amino acids. Preferably, the acyl groups are
derived from hydrocarbon carboxylic acids.
60. These agents can be used either singly or in combination. The
effect thereof can be estimated by harvesting monocytes from
inflammatory parts or peripheral blood after intake or
administration, examining the change in the content of reductive
glutathione in cells relative to that before treatment using the
method and measuring the change in the immunological activity in
vivo. This clearly proves the usefulness as the immunosuppressant
in particular, and these agents are effective against diseases.
61. The administration form is not particularly limited, and it
includes administration by injection and oral administration.
62. However, the oral administration is advantageous. The dose of
the substance having an activity of changing the content of
reductive glutathione as an active ingredient is selected depending
on the conditions of patients or the like to which the substance is
administered or the use purpose. In the case of patients suffering
from serious diseases, for example, a advanced gastric cancer, the
dose is between 1 and 5,000 mg (oral drug), preferably between 10
and 500 mg/day. It is not particularly difficult to produce
preparations, and preparations can be produced in the form of an
oral agent, an injection, a percutaneous agent and the like as
required in a usual manner.
63. It has been described above that the immunomodulator of the
present invention is quite useful and quite new as a drug im a
narrow sense. Since the immunomodulator of the present invention
contains a substance capable of oral intake as a main ingredient,
its use is not limited to drugs in the actual medical care. That
is, the immunomodulator of the present invention can also be
provided in the form of a food (including all that are put into the
mouth, such as a chewing gum, a tooth paste the like), as a food
for medical care, a health food or a special sanitary food
containing a substance having an activity of changing the content
of reductive glutathione in human macrophages (including monocytes,
Kupffer cells and dendritic cells) either singly or as a mixture,
as well as in the form of a nutrient an infusion. These are also
included in the present invention. It can also be contained in a
liquid component or take the form of a solid food.
64. The food, the nutrient and the infusion can be applied to the
same diseases as those to which the drugs are applied.
65. The immunomodulator of the present invention can be provided in
the form of the food, the nutrient and the infusion having an
immunomodulatory function for improvement of the cachectic
condition of patients suffering from cancers, diabetes,
inflammatory bowel diseases, chronic rheumatoid arthritis,
hepatitis, hepatic cirrhosis, gastrointestinal inflammatory
diseases centering on inflammatory bowel diseases (ulcerative
colitis and Crohn disease), autoimmune inflammatory diseases, and
for chemoprevention of cancer. The dose of the active ingredient
may be determined according to what has been described in the
above-mentioned drugs. It can be applied not only to patients
suffering from attacked or chronic diseases but also to high-risk
persons suffering from adult diseases or the like.
66. Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Example 1
Test for functions of oxidative macrophages and reductive
macrophages
67. Oxidative macrophages were induced by administering 20 .mu.g of
LPS (lipopolysaccharide) to an abdominal cavity of a mouse, and
reductive macrophages were induced by administering 100 .mu.g of
lentinan to an abdominal cavity of a mouse three times every two
days. These were clarified by adhering peritoneal exudate cells to
the plastic surface, then reacting the same with 10 .mu.M of
monochlorobimane at 37.degree. C. for 30 minutes and conducting
analysis with Adherent Cell Analyzing System (ACAS). The increase
in the amounts of oxidative macrophages can easily be measured
visually from the fact that almost no reaction product is observed,
that is, gray or blue image is obtained, and the increase in the
amounts of reductive macrophages from the fact that the red or
yellow image is obtained, respectively.
68. Accordingly, NO, IL-6 and PGE2 produced by inducing the
peritoneal exudate cells into oxidative end reductive cells were
measured.
69. (1) Materials
70. Cells: The peritoneal exudate cells obtained by the
above-mentioned stimulation, namely, the macrophages were added to
a 96-well microplate in an amount of 1.times.10.sup.5 cells/200
.mu.l each.
71. Medium: Phenol red-free RPM 11640: 200 .mu.l/well
72. LPS: Lipopolysaccharide (made by Sigma Co.) (origin: E. coli)
100 ng/ml
73. IFN.gamma.: 100 units/ml
74. (2) Incubation
75. Incubated in a 5% CO.sub.2 incubator at 37.degree. C. for 48
hours.
76. (3) Measuring method
77. After the completion of the above-mentioned incubation, the
culture supernatant was recovered. The amount of IL-6 was measured
by the proliferation assay using an IL-6-dependent cell strain,
MH60, the amount of PGE2 was measured using an ELISA-kit, and the
amount of NO was measured using a Griess-Romijn reagent. These
measurements were conducted by a method which those skilled in the
art usually employ.
78. (4) Results:
79. The results are shown in FIG. 3. As is clear from FIG. 3, there
are differences in the concentration and the type among
inflammatory cytokine IL-6, inflammatory mediator PGE2 and NO
produced between oxidative macrophages and reductive macrophages.
That is, with the oxidative macrophages, the production of IL-6, a
Th2 cytokine and the production of PGE2, which is immunosuppressive
to suppress the Th1 induction are increased, and the production of
NO is decreased. On the contrary, with the reductive macrophages,
the production of NO is increased, and the production of PGE2 and
the production of IL-6 are suppressed. Thus, there is a functional
difference seen between both macrophages.
Example 2
Test using animal disease models which are immunologically
deficient by knocking out a gene
80. In order to clarify a mechanism underlying the conversion of an
acute to a chronic phase and progression of inflammatory diseases,
it is important to analyze molecularly why there is a difference in
the production of an inflammatory mediator or a cytokine between
oxidative M.PHI. and reductive M.PHI.. Generally, extracellular
stimulation (ligand or the like) of the all is signaled into cells
through a receptor present on the cell surface. Various kinases are
activated with signals from the receptor, and transcriptional
factors are also activated in cytoplasm. The activated
transcriptional factors are translocated into the nucleus, and
bound to target genes to conduct gene expression. According to the
recent studies, it is being clarified that the intracellular redox
system regulates activation of transcriptional factors,
translocation thereof into the nucleus and binding with genes
(Annual Rev. Immunology, vol. 8, pp. 453-475, 1990, Embo J., 10,
2247-2251, 1991). It is currently unknown how the intracellular
redox system participates in the gene expression system after the
receptor triggering in M.PHI.. As a method of clarifying the same,
M.PHI. was harvested from a knock out mouse deficient in a molecule
participating in a signal transduction system from a receptor, and
the function of the redox state was analyzed. Specifically, a
common .gamma. chain (.gamma.c) which is commonly used as a
receptor constituting molecule of IL-2, IL-4, IL-7, IL-9 and IL-5
and Jak3 which is a molecule present downstream thereof and
transducing a signal from .beta.c were used as target
molecules.
81. Cytokine and stimulator:
82. As mouse IFN.gamma., a recombinant supplied by Genzyme was
used. As human IL-2 and human IL-6, recombinants supplied by
Ajinomoto Co., Inc. were used. As human IL-12, a recombinant
supplied by Pharningen was used.
83. As LPS, a substance derived from E. coli 055:B5 supplied by
Difco was used. As lentinan, a preparation produced by Ajinomoto
Co., Inc. was used.
84. Mice used:
85. .gamma.c Knock out mice were obtained from Professor Sugamura,
Tohoku University Medical School. Jak3 knock out mice were obtained
from Professor Saito, Chiba University Medical School.
86. As wild mice used for mating and as a control, C57BL/6 obtained
from Charles River Japan (CRJ) was used.
87. Harvest of peritoneal M.PHI.:
88. Peritoneal cells were harvested by injecting 5 ml of a phenol
red-free DMEM medium (supplied by Nikken Seibutsusha) ice-cooled
into a peritoneal cavity of a mouse which had been put to
sacrificial death with ether using an injection cylinder fitted
with a 22-gauge needle, squeezing the same and pulling out the
medium.
89. Determination of the amount of IL-6:
90. A stimulator was added to 1.times.10.sup.6 M.PHI., and the
incubation was conducted at 37.degree. C. for 2 days in a CO.sub.2
incubator. After centrifugation, the culture supernatant was
collected.
91. The amount of IL-6 was determined using IL-6 dependent mouse
hybridoma MH60 cells (J. Eur. Immunol., vol. 18, p. 951, 1988). One
hundred microliters of the culture supernatant were added to 100
.mu.l of the MH60 cell suspension adjusted to 1.times.10.sup.5
cells/ml in a 10% FCS-containing RPMI medium, and the mixed
solution was incubated at 37.degree. C. for 2 days in a CO.sub.2
incubator. Subsequently, 10 .mu.l of MTT (supplied by Sigma Co.)
solution adjusted to a concentration of 5 mg/ml in the same medium
were added thereto, and the reaction was conducted at 37.degree. C.
for 5 hours. After the completion of the reaction, the
centrifugation was conducted. The supernatant (160 .mu.l) was
removed, and 100 .mu.l of a mixture of hydrochloric acid and
propanol were added to the residue. The suspension was conducted
using a pipetman to dissolve the cells. Immediately after the
dissolution, an absorbance of 570 nm was measured with an
immunometer (supplied by Bio-Rad).
92. Measurement of a concentration of NO.sub.2:
93. A stimulator was added to 1.times.10.sup.5 M.PHI., and the
incubation was conducted at 37.degree. C. for 2 days in a CO.sub.2
incubator. After the completion of the centrifugation, the culture
supernatant was collected.
94. One hundred microliters of a Griess-Romijn reagent (supplied by
Waco Pure Chemical Industries, Ltd.) adjusted to a concentration of
50 mg/ml in distilled water were added to 100 .mu.l of the culture
supernatant, and the reaction was conducted at room temperature for
15 minutes. After the completion of reaction, an absorbance of 540
nm was measured. NaNO.sub.2 was used as a standard.
95. Determination of GSH in cells with ACAS:
96. Three-hundred microliters of a cell suspension adjusted
concentration of 3.times.10.sup.5 cells/ml in an RPMI 1640 medium
(phenol red-free) were charged into a chambered coverglass
(#136439, supplied by Nunc), and incubated at 37.degree. C. for 2
hours using a CO.sub.2 incubator. The culture solution was washed
with the medium, and 300 .mu.l of monochlorobimane (supplied by
Molecular Plobe) adjusted to 10 .mu.M in the same medium were added
thereto. The mixture was charged into a CO.sub.2 incubator of
37.degree. C., and the reaction was conducted for 30 minutes. The
fluorescent intensity was measured with ACAS. In ACAS, a UV laser
was used.
97. Determination of an amount of IL-12:
98. The amount of IL-12 was determined through bioassay using cells
of human T cell strain 2D6 (J. Leukocyte Biology, vol. 61, p. 346,
1997).
99. 2D6 cells which had been incubated in an RPMI 1640 medium
containing 500 pg/ml of recombinant human IL-12, 50 .mu.M of
2-mercaptoethanol and 10% FCS (fetal calf serum) were moved to a
tube, and centrifugally washed three times with the above-mentioned
medium without IL-12 and cell density was adjusted to
1.times.10.sup.5/ml. The cell suspension was added in an amount of
100 .mu.l each to a 96-well flat bottom plate containing a sample
serially diluted in advance with an RPMI 1640 medium containing 50
.mu.M of 2-mercaptoethanol and 10% FCS in an amount of 100 .mu.l
each. Subsequently, the mixture was charged into a 5% O.sub.2
incubator of 37.degree. C., and incubated for 48 hours. For final 6
hours, .sup.3H-TdR was pulsed (a substance adjusted to 370 kBq/ml
in an RPMI 1640 medium containing 50 .mu.M of 2-mercaptoethanol and
10% FCS was added in an amount of 50 .mu.l each). The cells a
harvested, and the radioactivity was measured using a .beta.
counter (Matrix 96, supplied by Packard).
100. Measurement of the GSH content in M.PHI. produced from knock
out mice:
101. Peritoneal cells were produced from knock out mice, and the
GSH content in cells was analyzed by ACAS using an MCB reagent. The
content of reductive glutathione was clearly decreased in any mice
compared with control mice (C57BL/6).
102. Function of M.PHI. produced from knock out mice:
103. Peritoneal cells were produced from wild mice (C57BL/6) knock
out mice, and stimulated with LPS, IL-2, IFN.gamma. and a
combination thereof. The NO production, the IL-6 production the IL-
12 production were measured. Almost no NO production was observed
in any mice derived M.PHI. in the absence of stimulation. In the
stimulation with the combination of LPS IFN.gamma., almost no
additive effect was observed in the .gamma.c knock out mice, and
the NO production was decreased to less than half that in control
mice. The same results as in .gamma.c were provided in Jak3 knock
out mice. Further, the IL-6 production was analyzed. In the LPS
stimulation, an increase in the IL-6 production was observed in
.gamma.c knock out mice (962 pg/ml relative to 81 pg/ml of a
control). In the IFN.gamma. stimulation, an increase in the IL-6
production was observed in .gamma.c knock out mice. The results
were the same with the suppression pattern of the NO production.
Still further, the IL-12 production with the LPS stimulation and
the IFN.gamma. stimulation was examined. No production was observed
at all in any mice derived M.PHI.. This proves that in the sick
animals of the gene knock out mice used herein, the amount of the
oxidative macrophages is increased to increase the humoral immunity
or the allergic reaction mainly caused by Th2 and to decrease the
cellular immunity supported by Th1. In the animal disease models,
it is clearly shown that the diagnosis of immunological diseases
required for the immunomodulator of the present invention is
original and significant.
Example 3
Determination of the amount of reductive glutathione in M.PHI. of
advanced tumor-bearing mice
104. Method:
105. Oxidative and reductive macrophages collected from peritoneal
cavities of advanced tumor-bearing cachectic mice (COLON 26) and
normal mice were determined. The COLON 26 transplantable tumor well
known to induce a cancer cachexia was implanted subcutaneously in
the back portion of CDF1 mice at a density of 5.times.10.sup.5
cells/mouse. On day 21 after the tumor implantation, the cachectic
condition was provided. Five milliliters of a physiological saline
solution were intraperitoneally injected into the mice which became
resistant to a therapeutic treatment. Peritoneal macrophages were
collected, and suspended in a phenol red-free RPMI 1640 medium
containing 10% fetal calf serum to a density of 3.times.10.sup.5
cells/ml. One hundred microliters of the suspension were charged in
a Lab-Tek Chamber Slide (#136439, supplied by Nunc), and incubated
in 5% CO.sub.2 at 37.degree. C. for 3 hours. After the nonadherent
cells were removed, 200 .mu.l of the above-mentioned medium free
from serum were added thereto, and monochlorobimane (MCB) was added
thereto in an amount of 10 .mu.M. The reaction was conducted for 30
minutes, and the image analysis was conducted on the basis of the
UV absorption using an ACAS device (supplied by Meridien).
106. Results:
107. The content of reductive glutathione was determined by ACAS.
As a result, in the advanced tumor-bearing mice, the amount of the
macrophage of which the reductive glutathione content was
decreased, namely, the oxidative macrophage was relatively
increased in comparison with that in the normal mice. Since the
amount of the oxidative macrophage was increased, the amount of
IL-6 in the above-mentioned macrophage culture supernatant was
markedly increased (600 pg/ml relative to 120 pg/ml in control
mice). Further, the amount of PGE 2 was 32 ng/ml relative to 7.6
ng/ml in control mice, and it was increased tp 5 times or more. It
was found that the immunosuppressive state or the cachectic state
at the advanced tumor-bearing stage is based on the excessive
production of these mediators. In addition, the increase in an
amount of active oxygen produced was also observed. It shows that
the redox state of macrophages is measured upon determination of
the glutathione content without measuring a large number of
parameters whereby the examination for diagnosis of the
pathological state and the immunological function of patients
suffering from cancers can be conducted easily and exactly.
Accordingly, the above-mentioned classification of macrophages
enables the examination for diagnosis of diseases and the
immunological function of patients suffering from cancers.
Example 4
Induction of reductive macrophages by oral administration of
glutathione ethyl ester to advanced tumor-bearing mice
108. The COLON 26 transplantable tumor was implanted subcutaneously
in the back portion of CDF1 mice at a density of 5.times.10.sup.5
cells/mouse. On day 21 after the tumor implantation, the mice were
proved to be in the cachectic condition. Glutathione ethyl ester
was orally administered to the mice every day in a dosage of 1
mg/0.5 ml/h. This oral administration was continued for 10 days.
The peritoneal cells were collected from the mice in the same
manner as in Example 3. Peritoneal macrophages were collected, and
suspended in a phenol red-free RPMI 1640 medium containing 10%
fetal calf serum to a density 3.times.10.sup.5 cells/ml. The
suspension was charged into a Lab-Tek Chamber Slide (#136439,
supplied by Nunc) in an amount of 100 .mu.l, and the incubation was
conducted in 5% CO.sub.2 at 37.degree. C. for 3 hours. After the
nonadherent cells were removed, 200 .mu.l of above-mentioned medium
free from serum were added thereto, and 10 .mu.M of
monochlorobimane were then added thereto. The reaction was
conducted for 30 minutes, and the image analysis was conducted
based on the UV absorption using an ACAS device (supplied by
Meridien).
109. Results:
110. The content of reductive glutathione was determined by ACAS
method. Consequently, in the advanced tumor-bearing model mice to
which glutathione ethyl ester had been administered, the amount of
the macrophage of which the reductive glutathione content was
decreased, namely, the oxidative macrophage was relatively
decreased in comparison with that in control mice to which the
physiological saline solution had been administered. Since the
amount of the reductive macrophage was increased, the amount of
IL-6 in the above-mentioned macrophage culture supernatant was
decreased (642 pg/ml relative to 5,200 pg/ml in control mice).
Further, the amount of PGE2 was also much decreased to 6.5 ng/ml
relative to 32 ng/ml in control mice. It was thus clarified that
the immunosuppressive state or the cachectic state at the advanced
tumor-bearing stage can be improved by the oral administration of
glutathione ethyl ester. Accordingly, the average number of
survival days of mice in the treated group increased from 42 (in
control mice) to 148.
Example 5
Examination of macrophages collected from the patient suffering
from sarcoidosis and conversion of oxidative macrophages to
reductive macrophages
111. The amounts of oxidative and reductive monocytes macrophages
contained in monocytes preparation separated and collected in a
usual manner from the peripheral blood and the thoracic cavity of
the patient suffering from sarcoidosis were examined by
biochemically measuring the contents of reductive glutathione (GSH)
end oxidative glutathione (GSSG) by the enzyme recycling method.
The peripheral blood of the healthy person was used as a
control.
112. Materials:
113. The peripheral blood of the healthy person and the peripheral
blood of the patient suffering from sarcoidosis were collected with
heparin. Or 150 ml of a physiological saline solution was injected
into the bronchia of the patient using a bronchofiber, and 75 ml of
bronchoalveolar lavaged fluid were recovered. Monocytes obtained by
separating and purifying both of them using Ficoll-Hypaque
(LYMPHOPREP) were suspended in an RPMI 1640 medium containing 10%
fetal calf serum, and washed three times to obtain
macrophage/monocyte preparation adherent to a glass petri dish for
30 minutes. Subsequently, a group incubated for 3 hours with the
addition of 5 mM N-acetylcysteine (NAC) and a group of a medium
component alone were prepared. A rubber policeman was used to
recover adherent cells from the petri dish. With respect to
5.times.10.sup.5 macrophages, the following examination was
conducted.
114. Method:
115. The contents of reductive glutathione and oxidative
glutathione were measured by the above-mentioned enzyme recycling
method.
116. Production of samples:
117. One hundred microliters of Triton X-100 prepared with a 0.1 M
phosphate buffer (pH 7.5) containing 5 mM EDTA ice-cooled were
added to cell pellets which had been washed with PBS, and the
mixture was allowed to stand at room temperature for 5 minutes to
dissolve the cells. Fifteen microliters of 0.1 M HCl were added
thereto, and 15 .mu.l of a 50% sulfosalicylic acid (SSA) solution
were further added thereto. The mixture was centrifuged at 12,000
rpm for 5 minutes, and the supernatant was collected [*] to form a
measuring sample having a total glutathione concentration
(GSH+GSSG).
118. Measuring method:
119. A 10 mM phosphate buffer (590 .mu.l, pH 7.5) containing 0.5 mM
EDTA, 100 .mu.l of glutathione reductase (supplied by Boehlinger
Mannhein) adjusted to a concentration of 6 u/ml in the same buffer,
50 .mu.l of 4 mM NADPH (supplied by Sigma Co.) prepared with 5%
NaHCO.sub.3 and 10 .mu.l of the sample were mixed. The mixture was
incubated at 37.degree. C. for 5 minutes. Fifty microliters of a 10
mM 5,5'-dithio-bis(2-nitroben- zoic acid) (DTNB, supplied by Sigma
Co.) prepared with a 0.1 M phosphate buffer (pH 7.5) containing 5
mM EDTA were added thereto. The change in the absorbance of 412 nm
at 37.degree. C. over the course of time was measured using a
spectrophotometer. As a standard sample, GSH (supplied Sigma Co.)
prepared in the same manner as the above-mentioned sample was used.
Separately, the content of oxidative glutathione (GSSG) alone was
measured,--2 .mu.l of 2-vinylpyridine (supplied by Tokyo Kaseisha)
were added thereto after the above-mentioned procedure [*] and were
mixed at room temperature for 1 minute, and after the pH was
adjusted to 7.5, reaction mixture was allowed to stand at room
temperature 60 minutes to form a measuring sample, and the
measurement conducted in the above-mentioned manner.--and the
content of reductive glutathione (GSH) was obtained by the
subtraction from the total glutathione content.
120. Results:
121. With respect to the contents of reductive glutathione and
oxidative glutathione in the peripheral blood of the patient, the
GSSG content was 5.29 .mu.M, and the GSH content was 20.45 .mu.M.
Thus, the ratio of reductive GSH was approximately 80%, and still
higher (the ratio of reductive GSH was 90% or more in the healthy
person). In the macrophages within the thoracic cavity, the content
of reductive GSH was 1.45 .mu.M, and the content of oxidative GSSG
was 15.85 .mu.M. Thus, the ratio of oxidative GSSG was
approximately 86%, and the presence ratio thereof was completely
inverted. In the NAC addition group, content of reductive GSH was
20.45 .mu.M, and the content of oxidative GSSG was 4.32 .mu.M.
Thus, the content of oxidative GSSG was largely decreased, and the
ratio of reductive GSH exceeded 80%. In this manner, the peripheral
blood level was recovered. It shows that in this disease, the
oxidative macrophages play a great role in the progression of the
disease and this progression can be improved through NAC
administration. Thus, the usefulness of the present invention is
clarified not only in animal disease models but also in patients
suffering from rheumatoid arthritis or diabetes and people with a
high risk of these diseases.
Example 6
Induction of reductive macrophages by oral administration of NAC
and GSH-OEt
122. Macrophages (M.PHI.) were prepared from knock out mice
deficient in a molecule participating in a signal transduction
system from a receptor, and the function of the redox system was
analyzed. Specifically, a common .gamma. chain (.gamma.c) which is
commonly used as a receptor constituting molecule of IL-2, IL-4,
IL-7, IL-9 and IL-15 and JaK3, a molecule present downstream
thereof and transducing a signal from .gamma.c were gene targeted
molecules. Methods applied in Example 2 was repeated. JaK3 knock
out mice were divided into three groups. A control group was a
group of usual city water free-intake. An NAC group was a group of
free intake of city water containing 1 mg/ml of NAC. A GSH-OEt
group was a group of free intake of city water containing 1 mg/ml
of GSH-OEt. Breeding was continued under SPF condition for 24 days,
and peritoneal exudate cells, namely, macrophages were likewise
obtained.
123. Cytokines and stimulator:
124. A recombinant supplied by Genzyme was used as mouse IFN.
Recombinants supplied by Ajinomoto Co., Inc. were used as human
IL-2 and human IL-6. A recombinant supplied by Pharmigen was used
as human IL-12.
125. A product derived from E. coli. 055; B5 as supplied by Difco
was used as LPS. A preparation supplied by Ajinomoto Co. Inc. was
used as lentinan.
126. Determination of an amount of IL-6:
127. Measurement of an NO.sub.2 concentration:
128. Determination of a GSH content in cells by ACAS:
129. Determination of an amount of IL-12:
130. These were all conducted in the same manner as in Example
2.
131. Measurement of a GSH content in M.PHI. prepared from knock out
mice:
132. Peritoneal cells of knock out mice which had undergone the
respective treatments were harvested, and the GSH content in the
cells was analyzed using ACAS with MCB. In any of the mice in the
groups of free intake of city waters containing NAC and GSH-OEt,
the content of reductive glutathione in M.PHI. was markedly
increased in comparison with that in control mice (city or free
intake group). The image of reductive M.PHI. derived through
intraperitoneal administration of NAC in normal mice was shown.
133. Function of M.PHI. harvested from knock out mice:
134. Peritoneal cells were harvested from three groups of knock out
mice, and stimulated with LPS, IL-2, IFN.gamma. and a combination
thereof. The NO production, the IL-6 production and the IL-12
production were measured. With respect to the NO production, almost
no NO production was observed in any mice derived M.PHI. in the
absence of stimulation. Then, the IL-6 production was analyzed. In
LPS stimulation, the amount was detected as 962 pg/ml in the knock
out mice derived M.PHI. culture, 122 pg/ml in the NAC group, and 82
pg/ml in the GSH-OEt group. In view of the function, it was
identified that the conversion to reductive macrophages was
possible. In consideration of the fact that IL-6 is a main cytokine
of inducing Th2, it is clearly shown that the biological Th1/Th2
balance can be controlled by the oral intake of these substances.
This result was inversely related with the suppression of the NO
production and the recovery pattern with medications. Next, the
IL-12 production by stimulation of LPS or IFN.gamma. was examined.
No production was observed at all in the control group. This shows
that in the animals disease models, the JaK3 gene knock out mice
used here, the amount of the oxidative macrophage is increased,
humoral immunity or an allergic reaction mainly caused by Th2 is
increased, and cellular immunity caused by Th1 is decreased. On the
other hand, it was identified that in the NAC and GSH-OEt
administration groups, the amounts of IL-12 are 420 pg/ml and 610
pg/ml respectively. This proves that in the animal disease models,
the immunomodulator of the present invention is also useful in the
improvement of the immunological diseases, and is original and
significant.
Example 7
Difference in the IL-12 production between reductive and oxidative
macrophages
135. When there are defects in differentiation, selection and
functional expression steps of T cells, the host immune system
becomes deficient. From this fact, it is considered that T cells
play an central role in the host immune system. Helper T cells (Th)
which are one subset of T cells produce lymphokines to regulate
immunocytes or inflammatory cells. Recently, the following concept
has been proposed. That is, Th is further classified into two
types, Th1 and Th2 depending on the types of the lymphokines
produced, and these cells have the different immunological
functions (J. Immunol., vol. 136, pp. 2348, 1986). That is, Th1
produces IL-2 or IFN.gamma., and is a main cell to modulate
cellular immunity. Th2 produces IL-4, IL-5, IL-6 and IL-10, and is
a main cell to modulate humoral immunity. The homeostasis of the in
vivo immunity is maintained by the Th1/Th2 balance. Usually, when
the Th1/Th2 balance is inclined to either of Th1 and Th2, the host
correspond to correct the skewing and tend to maintain the
homeostasis. However, it is considered that when the imbalance is
not corrected for some reasons, immunological diseases will occur.
Th1 and Th2 are differentiated from the precursor of them, namely
Th0. In the differentiation of Th0 to Th1, IL-12 produced by M.PHI.
is important (Immunology Today, vol. 335, p 14, 1993). In the
differentiation of Th0 to Th2, IL-4 produced by NKT cells is
important (J. Exp. Medicine, vol. 179, pp. 1285, 1994).
136. In the above-mentioned Example, it is clarified that the
M.PHI. function differs depending on the difference in the redox
state of M.PHI.. With respect to M.PHI., there are two types of
M.PHI., oxidative M.PHI. and reductive M.PHI. based on the
difference in the GSH content, and these two distinctive M.PHI.
behave differently in the NO or IL-6 production. The main producer
of IL-12, which induces differentiation of Th0 to Th1 and which is
a key molecule of controlling the Th1/Th2 balance, is considered to
be M.PHI.. However, the detailed analysis has not yet been
reported. In view of the clarification of attack mechanism of
immunological diseases, it is quite interesting to know whether or
not the IL-12 production is different between oxidative M.PHI. and
reductive M.PHI.. The present inventors have found that IL-12 is
produced from only reductive M.PHI., and that IL-4 considered to
control the Th1/Th2 balance like IL-12 acts on oxidative M.PHI. and
reductive M.PHI. whereby the Th1/Th 2 balance is skewed to the Th2
side. On the basis of the findings which were obtained prior to the
completion of the present invention, it is shown that the redox
state of M.PHI. regulates the Th1/Th2 balance, and the usefulness
of the present invention in the diagnosis of immunological diseases
is described.
137. IL-12 is produced from reductive M.PHI.:
138. In Example 1, it was indicated that M.PHI. produced by
injecting lentinan (LNT) intraperitoneally was reductive M.PHI.
with the high GSH content and that M.PHI. induced by injecting LPS
intraperitoneally was oxidative M.PHI. with the low GSH content. It
was examined whether there is a difference in the IL-12 production
between LNT-induced M.PHI. and LPS-induced M.PHI.. By stimulation
with LPS and INF.gamma., the remarkable IL-12 production (1,312
pg/ml) was observed in the LNT-induced M.PHI.. However, no IL-12
production was observed in LPS-induced M.PHI. and control resident
M.PHI. (FIG. 4). Next, the same analysis was conducted using M.PHI.
induced by intraperitoneally injecting substances for changing the
GSH content in cells. With respect to M.PHI. induced by
administering glutathione monoethyl ester (GSH-OEt), a substance
increasing the GSH content in cells and diethyl maleate (DEM), a
substance decreasing the same, IL-12 (3,570 pg/ml) was produced
only in M.PHI. derived from GSH-OEt administered mice through
stimulation with LPS and IFN.gamma.. These results show that IL-12
is produced only by reductive M.PHI. having the high GSH content in
cells.
139. The IL-12 production from reductive M.PHI. is suppressed by
decreasing the GSH content in cells:
140. As mentioned above, IL-12 was produced only in reductive
M.PHI. having the high GSH content in cells. It was examined
whether this production is suppressed by converting M.PHI. to
oxidative M.PHI.. That is, it was analyzed whether the IL-12
production is suppressed by exposing lentinan-induced M.PHI. with
DEM. As a result, it was clarified that the IL-12 production (828
pg/ml) from lentinan-induced M.PHI. is completely suppressed (0
pg/ml) with the addition of DEM. That is, it was suggested that
reductive glutathione in cells is deprived through DEM treatment
and reductive M.PHI. is converted to oxidative M.PHI. to suppress
the IL-12 production.
141. IL-4 suppresses the IL-12 production by reductive M.PHI.:
142. IL-4 is a cytokine which acts on M.PHI. suppressively. IL-4 is
considered to have an opposite function to IL-12 in the Th1/Th2
balance as well. Accordingly, it was examined whether IL-4 acts
suppressively on the IL-12 production by reductive M.PHI.. It was
clarified that the IL-12 production by LNT-induced M.PHI. and the
IL-12 production by GSH-OEt administered mouse M.PHI. are
remarkably suppressed by the pretreatment with IL-4 (from 1, 580
pg/ml to 370 pg/ml and from 490 pg/ml to 258 pg/ml). That is, it
was suggested that there is a possibility that IL-4 acts on M.PHI.
to suppress the IL-12 production whereby the Th1/Th2 balance is
skewed to the Th2 side. At this time, it was clarified from the
image analysis by ACAS that IL-4 markedly decreases the content of
reductive glutathione in M.PHI..
143. IL-4 suppresses the NO production and increases the IL-6
production:
144. Reductive M.PHI. increases the NO production by the IFN.gamma.
stimulation in comparison with oxidative M.PHI., and rather
suppresses the IL-6 production. IFN.gamma. is known to be a
cytokine produced from Th1 cells. What function IL-4 shows in the
NO production and the IL-6 production with IFN.gamma. was analyzed
using respective M.PHI.'s. IFN.gamma. NO production from M.PHI.
pretreated with IL-4 was significantly suppressed in comparison
with M.PHI. untreated with IL-4. Further, M.PHI. of which the GSH
content in cells was increased by the stimulation with GSH-OET and
M.PHI. of which the GSH content in cells was decreased by the
stimulation with DEM were pretreated with IL-4, and IFN.gamma.
thereafter and LPS stimulation was carried out to induce NO
production. As a result, the NO production was remarkably
suppressed in IL-4-treated M.PHI. in comparison with IL-4-untreated
M.PHI. in both subsets of M.PHI..
145. Meanwhile, with respect to the IL-6 production, the production
with IFN.gamma. was markedly increased by pretreatment with IL-4 in
resident M.PHI., LPS-induced M.PHI. and LNT-induced M.PHI..
146. Further, M.PHI. of which the GSH content in cells was
increased by the stimulation with GSH-OEt and M.PHI. of which the
GSH content in cells was decreased by the stimulation of DEM were
pretreated with IL-4, and IFN.gamma. was exposed thereon to induce
the IL-6 production. Consequently, the IL-6 production was
increased in IL-4-treated M.PHI.'s in comparison with
IL-4-untreated M.PHI.. These results revealed that IL-4 induces
oxidative macrophages by decreasing the content of reductive
glutathione in cells, suppressing the NO production by the
stimulation with IFN.gamma. and increasing the IL-6 production.
This indicates that IL-4 suppresses the NO production by
IFN.gamma., namely, Th1 type response, increases the IL-6
production by IFN.gamma., and has an activity of enhancing Th2 type
response. These findings scientifically prove the usefulness of the
immunomodulator according to the present invention.
Example 8
Enhancement of IL-12 production with a combination of NAC orally
taken in and IL-2 infusion
147. Two groups, namely, a group of 8-week-old DBA/2 female mice
which were caused to freely drink city water as in Example 6, and a
group of the same female mice which were caused to freely drink
city water containing 1 mg/ml of NAC, were prepared. Further, the
two groups of the-above-mentioned mice to which human recombinant
IL-2 in an amount of 2 .mu.g/0.5 ml/h was intraperitoneally
administered twice a day, every two days for two weeks were
provided. On day 14, the IL-12 production from M.PHI. was measured
in the same manner as in Example 6.
148. Measurement of the GSH content in M.PHI. prepared:
149. The peritoneal cells were harvested from the mice which had
undergone the respective treatments, and the GSH content in cells
was analyzed by ACAS using an MCB reagent. In comparison with
control mice (group caused to freely drink city water), the content
of reductive glutathione was markedly increased in the group caused
to freely drink NAC-containing city water and the IL-2
administration group, showing the image of reductive M.PHI..
150. The content of reductive glutathione was more increased in the
group which had undergone the combination of the free-drinking of
NAC-containing city water and the IL-2 administration than in any
of the group of the free drinking of NAC-containing city water and
the IL-2 administration group. Thus, the effect brought forth by
the combination of the treatments in the induction of reductive
M.PHI. was clearly observed in the ACAS image analysis. In the
group which had undergone the combination of the treatments, the
increase in the content of reductive glutathione was observed in
all M.PHI.'s (in contrast with the fact that the increase in the
content thereof in the group of the sole treatment was observed in
from 40 to 50% of M.PHI.).
151. Function of M.PHI. produced from each group:
152. Peritoneal cells were harvested from four groups of the mice,
and stimulated with LPS and lFN.gamma.. Then, the NO production,
the IL-6 production and the IL-12 production were measured. Since
the content of reductive macrophage was increased in three groups
of the sole administration and the combined administration in
comparison with the control group, the amount of IL-6 in the
macrophage culture supernatant was decreased (relative to 1,240
pg/ml in control mice, 320 pg/ml in the group caused to freely
drink NAC-containing city water, 520 pg/ml in the IL-2
administration group, and 67 pg/ml in the group which had undergone
the combination of the free-drinking of NAC containing city water
and the IL-2 administration). In consideration of the fact that
IL-6 is a main cytokine inducing Th2, the combination of the NAC
oral intake and the injection of IL-2, the cytokine can control the
Th1/Th2 balance more strongly. The increase pattern of the NO
production was inversely related with the IL-6 production. With
respect to the IL-12 production, the amount of IL-12 was 620 pg/ml
in the group caused to freely drink NAC-containing city water, 946
pg/ml in the IL-2 administration group, and 2,386 pg/ml in the
group which had undergone the free drinking of NAC-containing city
water and the IL-2 administration in comparison with 0 pg/ml in
control mice. Thus, the remarkable effect was observed by the
combination of the treatments. It shows that the present invention
provides the immunomodulator which is useful for the remarkable
improvement of the immunological diseases such as rheumatoid
arthritis in combination with the cytokines, and is therefore
original and significant.
Example 9
Induction of oxidative macrophages through administration of
(NAC-OMe).sub.2, (NAC).sub.2 or acetylgliotoxin
153. Oxidative macrophages were induced by intraperitoneally
administering 20 .mu.g/0.5 ml/h of (NAC-OMe).sub.2 or (NAC).sub.2,
or 10 .mu.g/0.5 ml/h of acetylgliotoxin in DBA/2 mice on Day 1 and
Day 2, and reductive macrophages were induced by intraperitoneally
administering 2 mg/0.5 ml/h of NAC in DBA/2 mice on Day 1 and Day
2. This was clarified by harvesting peritoneal exudate cells 20
hours after the completion of the administration, adhering the
cells to the plastic surface, then reacting the cells with 10 .mu.M
of monochlorobimane at 37.degree. C. for 30 minutes, and analyzing
the reaction product with ACAS. The increase in the amounts of
oxidative macrophages can easily be determined visually from the
fact that almost no reaction product with monochlorobimane is
observed, namely, a gray or blue image is provided, and the
increase in the amounts of reductive macrophages from the fact that
a red or yellow image is provided respectively. It was found that
macrophages induced 20 hours after subcutaneously administering 40
.mu.g/0.1 ml/h of dexamethasone, a typical steroid having an
immunosuppressive activity as well known on the back of a mouse on
Day 1 and Day 2 gave almost a gray image, namely, oxidative
macrophages were strongly induced. Meanwhile, the same examination
was conducted by harvesting peritoneal exudate cells after 20 hours
of the administration of 2 mg of N-acetylcystine (NAC).
Consequently, a red or yellow image was obtained, and it was
identified that reductive macrophages were induced. Similarly, the
induction of oxidative macrophages was confirmed on ACAS, by
intraperitoneally administering cystine derivatives, i.e.,
N,N'-diacetylcystine nitroxybutyl ester, N,N'-diacetylcystine
dimethyl ester (NAC-OMe).sub.2), N,N'-diacetylcystine diisopropyl
ester (NAC-OiPr).sub.2) and N,N'-di-L-alanylcystine dimethyl ester
(NAlaC-OMe).sub.2) (administering 20 .mu.g/0.5 ml/h each, twice a
week for three weeks, 9-11 weeks of age). <Production of NO and
IL-6 from macrophages induced through administration of
(NAC-OMe).sub.2, (NAC).sub.2 or acetylgliotoxin>
154. Thus, the peritoneal exudate adherent cells were incubated in
the following manner, and the amounts of NO and IL-12 produced in
the culture supernatant were measured. With respect to the amount
of IL-6, the amount of IL-6 produced spontaneously in the absence
of a stimulator was measured.
155. (1) Materials
156. Cells: The peritoneal exudate adherent cells obtained by the
stimulation, namely, the macrophages were added to a 96-well
microplate in an amount of 1.times.10.sup.5 cells/200 .mu.l
each.
157. Medium: Phenol red-free RPMI 1640: 200 .mu.l/well
158. LPS: Lipopolysaccharide (made by Sigma Co.) (origin: E. coli)
100 ng/ml
159. IFN.gamma.: 100 units/ml
160. (2) Measuring method
161. Harvest of peritoneal M.PHI.:
162. Peritoneal cells were harvested by injecting 5 ml of a phenol
red-free DMEM medium (supplied by Nikken Seibutsusha) ice-cooled
into a peritoneal cavity of a mouse which had been put to
sacrificial death with ether using an injection cylinder fitted
with a 22-gauge needle, squeezing the same and pulling out the
medium.
163. Determination of the amount of IL-6:
164. Conducted as in Example 2.
165. Measurement of a concentration of NO.sub.2:
166. Conducted as in Example 2.
167. Determination of GSH in cells with ACAS:
168. Conducted as in Example 2.
169. Determination of an amount of IL-12:
170. Conducted as in Example 2.
171. Results:
172. The effects of inhibiting production of NO, IL-6 and IL-12
from macrophages are shown in Table 1.
1 TABLE 1 (Amount of produced relative to Sample NO IL-6 IL-12
(NAC-OMe).sub.2 12 320 11 (NAC).sub.2 18 340 15 Acetylgliotoxin 22
450 14 NAC 172 42 920 Dexamethasone 45 1020 5
173. As is apparent from Table 1, the amounts of inflammatory
cytokines IL-6, NO and IL-12 produced are changed with the
oxidative macrophages induced through the administration of
(NAC-OMe).sub.2, (NAC).sub.2 and acetylgliotoxin. That is, with the
oxidative macrophages obtained through the administration of the
agent, the IL-6 production is increased, and the production of NO,
which induces tissue injury, and the production of IL-12, which
increases the cellular immunity, are both decreased. This effect is
the same as, or higher than, that of dexamethasone, a typical
steroid-type immunosuppressant. On the contrary, with the reductive
macrophages induced by N-acetylcysteine (NAC), the NO production
and the IL-12 production are increased, and the IL-6 production is
reduced.
Example 10
Effect of inhibiting delayed type hypersensitivity reaction to
ovalbumin
174. 20 g/0.5 ml/h of (NAC-OMe).sub.2, 20 g/0.5 ml/h of
(NAC).sub.2, 2 mg/0.5 ml/h of NAC and 30 g/0.5 ml/h (Day 1) of
dexamethasone were continuously administered from Day 1 to Day 5 as
in Example 9. With respect to an antigen, 100 .mu.l of a suspension
of ovalbumin and Complete H37Ra Adjuvant (Difco) at a ratio of 1:1
(containing 250 .mu.g of ovalbumin) were administered
subcutaneously to the back as a sensitization antigen on Day 2 and
to the left ear as an induction antigen on Day 8. After 24 hours,
the thickness on the left ear was compared with that of the right
ear.
175. The results of the effect of inhibiting the delayed type
hypersensitivity reaction to the ovalbumin antigen are shown in
Table 2. The administration of (NAC-OMe).sub.2 and (NAC).sub.2
remarkably inhibited the delayed type hypersensitivity reaction to
the ovalbumin antigen. This reveals that the administration of
these substances inhibits the cellular immunity.
2 TABLE 2 Increase in the ear Sample thickness (mm) Control group
15.1 (NAC-OMe).sub.2 9.75 (NAC).sub.2 9.88 NAC 16.5 Dexamethasone
4.75
Example 11
Function of macrophages in an animal in which a gene is knocked
out
176. For clarifying a mechanism of chronic or advanced
inflammation, it is important to analyze, at a molecular level, why
there is a difference in production of an inflammatory mediator or
cytokine between oxidative M.PHI. and reductive M.PHI.. Generally,
extracellular stimulation (ligand) is transferred into cells
through a receptor present on cell surf aces. Various kinases are
activated with a signal from a receptor, and transcriptional
factors are also activated, translocated to the nucleus, and bound
to target genes for expression. According to the recent studies, it
is being clarified that a redox system in cells participates in
activation of transcriptional factors, shifting of the same into
the nucleus and binding of the same to genes (refer to Annual Rev.
Immunology, vol. 8, pp. 453-457, and Embo J., 10, pp. 2247-2251,
1991). At present, it is unknown how a redox system in cells
participates in a gene expression system through a receptor in
M.PHI.. As one approach for clarifying it, M.PHI.'s were produced
from knock out mice deficient in a molecule participating in a
signal translocation system from a receptor, and the function of
the redox system was analyzed. Specifically, a common .gamma. chain
(.gamma.c) used in common as a receptor constituting molecule of
IL-2, IL-4, IL-7, IL-9 and IL-15 was used as a target molecule.
177. Cytokine and stimulator:
178. As mouse IFN.gamma., a recombinant supplied by Genzyme was
used. As human IL-2 and human IL-6, recombinants supplied by
Ajinomoto Co. Inc. were used. As human IL-12, a recombinant
supplied by Pharmingen was used.
179. As LPS, a substance derived from E. coli 055:B5 supplied by
Difco was used. As lentinan, a preparation produced by Ajinomoto
Co. Inc. was used.
180. Mice used:
181. .gamma.c knock out mice were obtained from Professor Sugamura,
Tohoku University Medical School.
182. As wild mice used for mating and as a control, C57BL/6
obtained from CRJ was used.
183. Measurement of the GSH content in M.PHI. produced from knock
out mice:
184. Peritoneal cells were harvested from .gamma.c knock out mice,
and the GSH content in cells was analyzed by ACAS using an MCB
reagent. The content of reductive glutathione in M.PHI. derived
from .gamma.c knock out mice was remarkably decreased in comparison
with that in control mice (C57BL/6).
185. Function of M.PHI. harvested from knock out mice:
186. Peritoneal cells were produced from wild mice (C57BL/6) and
.gamma.c knock out mice, and stimulated with IPS, IL-2, IFN.gamma.
and a combination thereof. The NO production, the IL-6 production
and the IL-12 production were measured. Almost no NO production was
observed in any mice in the absence of stimulation. In the
stimulation with the combination of LPS and IFN.gamma., the
decreased NO production in .gamma.c knock out mice derived M.PHI.
was observed to less than half that in control mice. Further, the
IL-6 production was analyzed. In the LPS stimulation, an increase
in the IL-6 production was observed in .gamma.c knock out mice (962
pg/ml relative to 81 pg/ml of a control). In the IFN.gamma.
stimulation, an increase in the IL-6 production was observed in
.gamma.c knock out mice. Still further, the IL-12 production with
the LPS stimulation and the IFN.gamma. stimulation was examined. No
production was observed at all in any mice. This proves that in the
gene knock out mice used herein, the amount of the oxidative
macrophages is increased to increase the humoral immunity or the
allergic reaction mainly caused by Th2 and to decrease the cellular
immunity supported by TH1. In the animal disease models, it is
clearly shown that the invention is original and significant in the
diagnosis of immunological diseases.
Example 12
Spontaneous progression of inflammatory bowel disease in .gamma.c
knock out mice
187. In wild-type normal littermates of .gamma.c knock out mice
(genetic phenotypes +/+, +/Y), no inflammatory bowel disease is
observed at all under ordinary SPF breeding conditions. However, in
.gamma.c knock out mice (genetic phenotypes, -/-, -/+ and -/Y),
inflammatory bowel diseases occur very of ten. With respect to
homo-knock out mice with -/- and -/Y phenotypes, approximately 70%
thereof are spontaneously accompanied with inflammatory bowel
diseases within 4 months. Also with respect to mice with +/-
phenotype, approximately 60% thereof are spontaneously accompanied
therewith within 6 months. That is, intestinal shortening, bloody
stool, diarrhea, loose passage, anal prolapse and colonal
hypertrophy are observed.
188. With respect to an intestinal inflammation model, the oral
intake of dextran sulfate is well known. However, an inflammatory
image of .gamma.c knock out mice is by far similar to that observed
in inflammatory bowel diseases of humans. In the histochemical
analysis using pathologic samples, the following fact was
clarified.
189. Analysis of pathologic specimens was conducted with respect to
formalin-immobilized HE dyed specimens of the large intestine
corresponding to the colon in a portion which was from 2 to 3 cm
from the anus. The evaluation was conducted with respect to five
points, 1) longitudinal spreading of inflammatory cell invasion in
a direction of a mucous epithelium--lamina propria mucosae--lamina
muscularis mucosae--submucous layer--internal circular layers of
muscular tunics--external longitudinal layers of muscular
tunics--serous membrane, 2) lateral spreading thereof, 3) types of
invaded cells, 4) degree of neovascularization and 5) hypertrophy
of a submucous layer. Invasion of inflammatory cells was scarcely
observed in +/Y normal samples, wild-type littermates of .gamma.c
knock out mice, and the mucous structure was retained almost intact
in both goblet cells and mucous epithelium cells. In the group of
the free drinking of 1% dextran sulfate water, drop of neutrophils
and inflammatory cells in the gland cavity was observed, and
degeneration and disappearance of goblet cells and metaplasia and
degeneration of the mucous epithelium were identified at high
levels. Invasion of lymphocytes, M.PHI. and neutrophils,
neovascularization and vasodilation occurred at high levels. It was
rated as grade 4.
190. In untreated samples of .gamma.c knock out mice with
genotypes, the mucous epithelium was nearly intact, but hyperplasia
was observed. Drop of inflammatory cells into the gland cavity did
not occur. No edema was observed in the submucous layer, nor did
invasion in the muscular tunics and the submucous layer occur.
Invasion of M.PHI. and lymphocytes was observed only in the lamina
propria mucosae, and unlike dextran sulfate-induced models,
invasion of neutrophils was not observed. It was different from a
mere acute inflammation image, and close to a human inflammatory
bowel disease pathology image.
191. In untreated samples of .gamma.c knock out mice -/Y, the
mucous epithelium was 2 or 3 times that of wild-type mice, and
invasion of inflammatory cells was observed only in the lamina
propria mucosae as a local cluster. The mucosa in contact with the
cluster in the untreated samples of .gamma.c knock out mice +/- was
close to the normal one compared with that of mice -/- and -/Y. The
invasion cluster of inflammatory cells was observed in the bottom
of the lamina propria mucosae only at a low level.
192. Accordingly, the intestinal inflammation of .gamma.c knock out
mice with -/- and -/Y genotypes is similar to that of humans, and
the dextran sulfate-induced model is considered different in
mechanism from the .gamma.c knock out mouse spontaneous
inflammatory bowel disease model.
Example 13
Effect of inhibiting spontaneous inflammatory bowel diseases in
.gamma.c knock out mice
193. On the basis of the findings mentioned above, the effect of
inhibiting spontaneous progression of inflammatory bowel disease in
.gamma.c knock out mice was examined with respect to human Crohn
disease and ulcerative colitis models. Saline, (NAC-OMe).sub.2 or
(NAC).sub.2 (20 .mu.g/h) was administered twice a week, 5 times in
total, to each of 6 .gamma.c knock out male mice with +/Y genotype
and 6 .gamma.c knock out male mice with -/Y genotype which were
inclined to have oxidative M.PHI.. It was examined whether or not
the intestinal inflammation spontaneously progressed in -/Y was
inhibited. In case of (NAC-OMe).sub.2, on Day 14 of the
administration, the occurrence rate of the non-administration -/Y
group and the administration -/Y group was 83% to 25%; on Day 31,
the occurrence rate was 83% to 25%, and the survival rate was 33%
to 100%; and on Day 45, the occurrence rate was 100% to 25%, and
the survival rate was 33% to 75%. In case of (NAC).sub.2, it was
30, 40, 100, 40, 75% respectively. Thus, it was identified that the
substance used in the invention clearly exhibited the effect to
prolong survival of life and the effect of preventing the disease
progression in the intestinal inflammation spontaneously induced.
By the way, in the non-administration +/Y group and the
administration +/Y group, the disease progression did not occur,
and there was no difference therebetween.
Example 14
Effect of inhibiting dextran sulfate-induced inflammatory bowel
disease in .gamma.c knock out mice
194. To 3-week-old .gamma.c knock out mice with +/- genotypes were
administered 20 .mu.g/h of (NAC-OMe).sub.2 three times a week for 2
weeks. On day 16, the free drinking of 1% dextran sulfate water was
started. On Day 30, the occurrence rate of the inflammatory bowel
disease was 100% in the non-administration group and 16.6% in the
administration group, and the effect of the administration of this
agent was observed. Further, the survival rate was 57% in the
non-administration group and 100% in the administration group.
Thus, the remarkable effect was observed. With respect to the +/+
mice, the occurrence rate was 60% in the administration group and
80% in the non-administration group. Therefore, the effect of the
administration was also observed. The .gamma.c knock out mice and
their wild-type male mice (+/Y- and -/Y) and female mice (+/+, +/-
and -/-) were caused to freely drink water containing 1% dextran
sulfate as a inflammatory bowel disease model. The female mice
showed a resistance, and the order of the resistance was found to
be -/Y and +/Y in male mice, and -/-, +/- and +/+ in female mice.
After the start-up of the administration of dextran sulfate, the
survival rate was 80, 0, 100, 100 and 40% on Day 13, showing that
the mice mainly having oxidative M.PHI. were more resistant to the
dextran sulfate-induced gastrointestinal inflammation. This
indirectly proved the invention to be reasonable.
Example 15
Functions of macrophages in synovial cells from rheumatoid
arthritis patients
195. So as to elucidate the mechanism of chronic exacerbation of
inflammation, it is significant to analyze at a molecular level why
the difference in the generation of an inflammatory mediators and
cytokines emerges between oxidized M.PHI. and reduced M.PHI..
Generally, extracellular stimuli (ligands triggering) are
intracellularly transmitted via specific receptors present on cell
surface. Intracellular signals from the receptors activate various
kinases and additionally activate transcriptional factors, so that
the transcriptional factors translocate into nuclei and bind to the
enhancer/promoter sequence of a target gene therein, leading to the
expression thereof. Recent research works are under way of
elucidating that intracellular redox systems are participating in
activation, nuclear translocation and DNA binding of
transcriptional factors [ANNUAL REV. IMMUNOLOGY, Vol. 8, 453-475
(1990); EMBO J., 10, 2247-2251 (1991)]. Up to now, however, it has
not yet been elucidated how the intracellular redox systems are
involved in the receptor-mediated gene expression systems of
inflammatory mediators or cytokines in macrophages.
196. Cytokine and stimulator:
197. As mouse IFN.gamma., a recombinant supplied by Genzyme was
used. As human IL-2 and human IL-6, recombinants supplied by
Ajinomoto Co. Inc. were used. As human IL-12, a recombinant
supplied by Pharmingen was used.
198. As LPS, a substance derived from E. coli 055:B5 supplied by
Difco was used. As lentinan, a preparation produced by Ajinomoto
Co. Inc. was used.
199. Joint-derived macrophages:
200. Synovial tissue was aseptically sampled from tissues resected
during joint surgery.
201. The sampled synovial tissue was rinsed in phosphate buffer,
from which the surface was peeled off in a petri dish containing
the same phosphate buffer. Then, the resulting synovial tissue was
cut into pieces with a pair of scissors. To the pieces were added
2% hyaluronidase (manufactured by Sigma), 0.2% DNase (derived from
bovine pancreas) and 5% collagenase, for enzymatic treatment at
37.degree. C. for 2 hours. After discarding the resulting debris, a
cell fraction recovered by centrifugation was adjusted to
5.times.10.sup.5 cells/ml in a phenol red-free DMEM culture medium
supplemented with 10% FCS (manufactured by Nikken Biology Co.). For
3 hours, the cells were allowed to adhere to the plastic surface.
Plastic surface adherent macrophage-like cells harvested with a
rubber policeman were then subjected to rinsing in combination with
centrifugation three times using the culture medium under ice
cooling. The resulting cells were designated as synovial
tissue-derived macrophages and were then subjected to the following
experiments.
202. Determination of the amount of IL-6:
203. A stimulator was added to 1.times.10.sup.6 M.PHI., and the
incubation was conducted at 37.degree. C. for 2 days in a CO.sub.2
incubator. After centrifugation, the culture supernatant was
collected.
204. The amount of IL-6 was determined using IL-6 dependent mouse
hybridoma MH60 cells (J. Eur. Immunol., vol. 18, p. 951, 1988). One
hundred microliters of the culture supernatant were added to 100
.mu.l of the MH60 cell suspension adjusted to 1.times.10.sup.5
cells/ml in a 10% FCS-containing RPMI medium, and the mixed
solution was incubated at 37.degree. C. for 2 days in a CO.sub.2
incubator. Subsequently, 10 .mu.l of MTT (supplied by Sigma Co.)
solution adjusted to a concentration of 5 mg/ml in the same medium
were added thereto, and the reaction was conducted at 37.degree. C.
for 5 hours. After the completion of the reaction, the
centrifugation was conducted. The supernatant (160 .mu.l) was
removed, and 100 .mu.l of a mixture of hydrochloric acid and
propanol were added to the residue. The suspension was conducted
using a pipetman to dissolve the cells. Immediately after the
dissolution, an absorbance of 570 nm was measured with an
immunometer (supplied by Bio-Rad).
205. Measurement of a concentration of NO.sub.2:
206. A stimulator was added to 1.times.10 .sup.5 M.PHI., and the
incubation was conducted at 37.degree. C. for 2 days in a CO.sub.2
incubator. After the completion of the centrifugation, the culture
supernatant was collected.
207. One hundred microliters of a Griess-Romijn reagent (supplied
by Waco Pure Chemical Industries, Ltd.) adjusted to a concentration
of 50 mg/ml in distilled water were added to 100 .mu.l of the
culture supernatant, and the reaction was conducted at room
temperature for 15 minutes. After the completion of the reaction,
an absorbance of 540 nm was measured. NaNO.sub.2 was used as a
standard.
208. Determination of GSH in cells with ACAS:
209. Three-hundred microliters of a cell suspension adjusted to a
concentration of 3.times.10.sup.5 cells/ml in an RPMI 1640 medium
(phenol red-free) were charged into a chambered coverglass
(#136439, supplied by Nunc), and incubated at 37.degree. C. for 2
hours using a CO.sub.2 incubator. The culture solution was washed
with the same medium, and 300 .mu.l of monochlorobimane (supplied
by Molecular Plobe) adjusted to 10 .mu.M in the same medium were
added thereto. The mixture was charged into a CO.sub.2 incubator of
37.degree. C., and the reaction was conducted for 30 minutes. The
fluorescent intensity was measured with ACAS. In ACAS, a UV laser
was used.
210. Determination of an amount of IL-12:
211. The amount of IL-12 was determined through bioassay using
cells of human T cell strain 2D6 (J. Leukocyte Biology, vol. 61, p.
346, 1997).
212. 2D6 cells which had been incubated in an RPMI 1640 medium
containing 500 pg/ml of recombinant human IL-12, 50 .mu.M of
2-mercaptoethanol and 10% FCS (fetal calf serum) were moved to a
tube, and centrifugally washed three times with the above-mentioned
medium without IL-12 and cell density was adjusted to
1.times.10.sup.5/ml. The cell suspension was added in an amount of
100 .mu.l each to a 96-well flat bottom plate containing a sample
serially diluted in advance with an RPMI 1640 medium containing 50
.mu.M of 2-mercaptoethanol and 10% FCS in an amount of 100 .mu.l
each. Subsequently, the mixture was charged into a 5% CO.sub.2
incubator of 37.degree. C., and incubated for 48 hours. For final 6
hours, .sup.3H-TdR was pulsed (a substance adjusted to 370 kBq/ml
in an RPMI 1640 medium containing 50 .mu.M of 2-mercaptoethanol and
10% FCS was added in an amount of 50 .mu.l each). The cells were
harvested, and the radioactivity was measured using a counter
(Matrix 96, supplied by Packard).
Example 16
GSH concentration in M.PHI. prepared from synovial tissue from
rheumatoid arthritis patients
213. By ACAS with an MCB reagent, the GSH concentration in the
macrophage-like cells prepared by the aforementioned method was
determined. The macrophages collected were suspended and adjusted
in a phenol red-free RPMI 1640 culture medium supplemented with 10%
fetal calf serum to 3.times.10.sup.6 cells/ml; the suspension was
divided in 100-.mu.l portions on Lab-Tek Chamber Slide (#136439;
manufactured by NLNC, CO.), for culturing in 5% CO.sub.2 at
37.degree. C. for 3 hours; after discarding the nonadherent cells,
the serum-free culture medium of 200 .mu.l was fed to the resulting
culture, followed by addition of MCB (abbreviation for
monochlorobimane) to a final concentration of 10 .mu.M, for
reaction for 30 minutes; and the amount of intracellular GSH was
assayed by imaging analysis with an ACAS system (manufactured by
Meridian Co.).
214. Results:
215. Reduced glutathione was quantitatively assayed by the ACAS
method. In the macrophages derived from tissues of rheumatoid
arthritis patients at active stage, compared with the macrophages
derived from tissues of patients with osteoarthritis, the relative
ratio of oxidative macrophages, namely macrophages with the reduced
amount of glutathione, was increased. Because of the increase of
oxidative macrophages, IL-6 in the culture supernatant of the
macrophages was prominently increased. It is thus indicated that
the identified redox state of macrophages on the basis of the
glutathione content assay can suggest the pathological state and
immune function of a rheumatoid arthritis patient in a simple and
appropriate manner, with no need of determination of numerous
functional parameters of macrophages. For the administration of the
inventive anti-rheumatoid agent, thus, the pathological state and
immune function of a patient can be examined and diagnosed
according to the aforementioned macrophage classification
method.
216. Function of M.PHI. prepared from joint cavities of rheumatoid
arthritis patients:
217. Macrophages were prepared from synovial tissues of patients
with osteoarthritis and rheumatoid arthritis patients at active
stage by the same method as described above, which were then
stimulated with LPS, IFN.gamma. and a combination thereof for
assaying the abilities of NO and IL-12 generation. With no
stimulation, almost no IL-12 generation was observed in the
macrophages from any of the origins; by stimulation with a
combination of LPS and IFN.gamma., no such generation was observed
therein. The level of NO generation in the macrophages from
patients with active-stage rheumatoid arthritis was decreased
1/2-1/4-fold the level in the control, which indicates that
oxidative macrophages predominant in the rheumatoid arthritis
patients at active stage potentiate humoral immune response driven
by Th2, while cellular immune response for which Th1 is responsible
is declined in these patients. The findings described above suggest
that the pathological state of rheumatoid arthritis patients can be
diagnosed by assaying the GSH content in macrophages, which is
beneficial to determine the way for the administration and
application of the inventive anti-rheumatoid agent. Thus, the
creativeness and significance of the invention can thus be
exemplified.
Example 17
Modification of macrophage redox function with addition of chemical
agents to culture system of macrophage-like cells from tissues of
rheumatoid arthritis patients
218. By the same manner as in Example 3, macrophage-like cells were
collected from tissues of rheumatoid arthritis patients, and were
then suspended and adjusted in a phenol red-free RPMI 1640 culture
medium supplemented with 10% fetal calf serum to 3.times.10.sup.6
cells/ml. The suspension was divided in 100-.mu.l portions on
Lab-Tek Chamber Slide (#136439; manufactured by NUNC, CO.), for
culturing in 5% CO.sub.2 at 37.degree. C. for 3 hours; after
discarding the nonadherent cells, the 200-.mu.l serum-free culture
medium further containing any of the following chemical agents was
fed to the resulting culture, for reaction for 3 hours; and then,
the resulting culture was rinsed three times, followed by addition
of the serun-free culture medium of 200 .mu.l and addition of
monochlorobimane to a final concentration of 10 .mu.M, for reaction
for 30 minutes. The amount of intracellular GSH was assayed by
imaging analysis with an ACAS system (manufactured by Meridian
Co.).
219. Results:
220. Reduced glutathione was quantitatively assayed by the ACAS
method. In the glutathione ethyl ester (2 mM)-added group compared
with the control physiological saline-added group, the relative
ratio of oxidative macrophages, namely macrophages with the reduced
amount of intracellular glutathione, was decreased. Because of the
increase of reductive macrophages, IL-6 in the culture supernatant
of the macrophages was prominently decreased. The same action was
also observed in groups with addition of .gamma.-glutamylcysteine
dimethyl ester, N-acetylcysteine nitroxybutyl ester, glutathione
monoethyl ester, glutathione nitroxybutyl ester and glutathione
diethyl ester (all added at 2 mM). The induction of oxidative
macrophage with the reduced amount of intracellular glutathione was
confirmed in groups with addition of cystine derivatives such as
N,N'-diacetylcystine nitroxybutyl ester, N, N'-diacetylcystine
dimethyl ester [(NAC-OMe).sub.2], N,N'-diacetylcystine diisopropyl
ester [(NAC-OiPr).sub.2], and N,N'-di-L-alanylcystine dimethyl
ester [(NAlaC-OMe).sub.2] (all added at 2 .mu.M).
Example 18
Effects of chemical agents administered to rats with
adjuvant-induced arthritis (AA)
221. Adjuvant-induced arthritis was triggered in Lewis rats in a
conventional manner; and the effects of chemical agents were
examined in the animals. Adjuvant-induced arthritis is well known
as an experimental arthritis inducible in rats [Taurog et al.,
Meth. Enzymol. 162 339-355 (1988)]. It is suggested that clinical
efficacy of non-steroidal acid anti-inflammatory agents and
immunosuppressive agents such as cyclosporin, cyclophosphamide and
methotrexate as antirheumatoid agents can be reflected in the
model. An adjuvant is administered at 1 mg/0.1 ml per rate at a
site of caudal root. The edema reaches its peak about 2 to 3 weeks
after the administration of the adjuvant. The edema is triggered by
inflammatory infiltration of monocytes. Swollen joints are measured
with a micrometer every day; comparing the diameters of the joints
prior to and after adjuvant administration and after initiation of
the treatment with chemical agents with the diameters of the joints
in the control group, the effects of the chemical agents were
expressed as the ratio in % of joint swelling in the experimental
groups to joint swelling in the control group. The effects were
determined on day 15 after adjuvant administration. The chemical
agents were given, starting the day next to the day of adjuvant
administration, and every 3 days thereafter. The results are shown
in Table 3.
3TABLE 3 Ratio in % of joint swelling in experimental Chemical
Agents groups to joint swelling in control group Physiological
saline 100 (NAC-OMe).sub.2 50 (NAC).sub.2 96 NAC 132 Lentinan
52
222. The results based on the experiments of drug efficacy
apparently indicate that adjuvant-induced arthritis can be
suppressed by the administration of a substance inducing oxidative
macrophages. A single dose is 1 mg/kg for (NAC).sub.2 and
(NAC-OMe).sub.2; 100 mg/kg for NAC; and 0.1 mg/kg for lentinan.
Example 19
Effects of administration of chemical agents, including oral
administration thereof
223. Adjuvant-induced arthritis was induced in Lewis rats in a
conventional manner. In the same manner as in Example 11, the
effects of chemical agents were examined in these rats. The
conditions were the same as described above. The results are shown
in Table 4.
4TABLE 4 Ratio in % of joint swelling in experimental groups
Chemical Agents to joint swelling in control group Physiological
saline 100 Intravenous (NAC-OMe).sub.2 42 Intravenous
(NAlaC-OMe).sub.2 45 Intravenous (NAC).sub.2 95 Oral NAC 142
Intraperitoneal 120 .gamma.-glutamylcysteine dimethyl ester
Intraperitoneal 132 glutathione diethyl ester Intravenous lentinan
50
224. The doses were similar to those in Example 11; the doses of
(AlaC-OMe).sub.2, .gamma.-glutamylcysteine dimethyl ester, and
glutathione diethyl ester were all 100 mg/kg.
225. The above results apparently indicate that compounds
represented by the structural formula 1, including
N,N'-diacetylcystine [(NAC).sub.2], N,N'-dipropylcystine
[(NPC).sub.2], N,N'-diacetylcystine dimethyl ester
[(NAC-OMe).sub.2], N,N'-diacetylcystine diisopropyl ester
[(NAC-OiPr).sub.2] and N,N'-di-L-alanylcystine dimethyl ester
[(NAlaC-OMe).sub.2], and nitroxybutyl esters thereof and exerting
actions to decrease the content of reduced glutathione in
macrophages, suppress delayed type hypersensitivity reactions, and
suppress the generation of IFN.gamma. or IL-12, are
pharmacologically effective when administered to animals with
adjuvant-induced arthritis.
226. Even when the compounds were administered 3 times per week for
3 weeks, the effects on the suppression of joint swelling were also
observed in model mice with the spontaneous onset of rheumatoid
arthritis. In the model mice, swelling of foreleg finger bone
joints was started on age 2 months, together with pannus formation
in the synovial tissue of the joints under observation; by the
dosing of the compounds with actions to decrease the content of
reduced glutathione in macrophages, suppress delayed type
hypersensitivity reactions, and suppress the generation of
IFN.gamma. or IL-12 to the animals, however, the joint score 4.5
months later was suppressed to 40 to 50% of the score in the
physiological saline-dosed control group. Alternatively, it is
revealed that joint swelling is enhanced by reductive
macrophage-inducing substances including glutathione precursors
such as .gamma.-glutamylcysteine, .gamma.-glutamylcysteine dimethyl
ester, and N-acetylcysteine nitroxybutyl ester, and glutathione
derivatives such as glutathione monoester, glutathione nitroxybutyl
ester and glutathione diester.
Example 20
Functions of macrophages in NOD mice with spontaneous diabetes
mellitus
227. So as to elucidate the mechanism of chronic exacerbation of
inflammation, it is significant to analyze at a molecular level why
the difference in the generation of an inflammatory mediators and
cytokines emerges between oxidized M.PHI. and reduced M.PHI..
Generally, extracellular stimuli (ligands triggering) are
intracellularly transmitted via specific receptors present on cell
surface. Intracellular signals from the receptors activate various
kinases and additionally activate transcriptional factors, so that
the transcriptional factors translocate into nuclei and bind to the
enhancer/promoter sequence of a target gene therein, leading to the
expression thereof. Recent research works are under way of
elucidating that intracellular redox systems are participating in
activation, nuclear translocation and DNA binding of
transcriptional factors [ANNUAL REV. IMMUNOLOGY, Vol. 8, 453-475
(1990); EMBO J., 10, 2247-2251(1991)]. Up to now, however, it has
not yet been elucidated how the intracellular redox systems are
involved in the receptor-mediated gene expression systems of
inflammatory mediators or cytokines in macrophages.
228. Cytokine and stimulator:
229. As mouse IFN.gamma., a recombinant supplied by Genzyme was
used.
230. As human IL-2 and human IL-6, recombinants supplied by
Ajinomoto Co. Inc. were used. As human IL-12, a recombinant
supplied by Pharmingen was used.
231. As LPS, a substance derived from E. coli 055:B5 supplied by
Difco was used. As lentinan, a preparation produced by Ajinomoto
Co. Inc. was used.
232. Mice used:
233. NOD mice as model animals of insulin-dependent diabetes
mellitus were purchased from Nippon Clair, Co. Among them, female
mice were mainly used. Wild-type ICR mice purchased from Nippon
Charles River, Co. (CRJ) were used as controls.
Insulin-non-dependent diabetes mellitus-diseased animals were db/db
mice purchased from Nippon Clair, Co.
234. Harvest of peritoneal M.PHI.:
235. Peritoneal cells were harvested by injecting 5 ml of a phenol
red-free DMEM medium (supplied by Nikken Seibutsusha) ice-cooled
into a peritoneal cavity of a mouse which had been put to
sacrificial death with ether using an injection cylinder fitted
with a 22-gauge needle, squeezing the same and pulling out the
medium.
236. Determination of the amount of IL-6:
237. Measurement of a concentration of NO.sub.2:
238. Determination of GSH in cells with ACAS:
239. Determination of an amount of IL-12:
240. These were conducted in the same manner as in Example 15.
241. Measurement of the GSH content in M.PHI. produced from NOD
mice:
242. Peritoneal cells were prepared according to the method stated
above, and the GSH content in cells was analyzed by ACAS using an
MCB reagent. The content of reductive glutathione was clearly
decreased in the NOD mice at the age of 3-5 weeks, and was clearly
increased in those accompanied with the disease (diabetes), as
compared with control mice.
243. Function of M.PHI. produced from NOD mice:
244. Peritoneal cells were prepared from wild mice and NOD mice,
and stimulated with LPS, IL-2, IFN.gamma. and a combination
thereof. The NO production and the IL-12 production were measured.
Almost no IL-12 production was observed in any mice derived M.PHI.
in the absence of stimulation. In the stimulation with the
combination of LPS and IFN.gamma., no IL-12 production was observed
in the NOD mice at the age of 3-5 weeks, but the IL-12 production
was observed in those accompanied with the disease. The NO
production was decreased to from one third to one fourth in the NOD
mice at the age of 3-5 weeks, but was increased to from two to
three times in those accompanied with the disease, as compared with
control mice. This proves that in the NOD mice used herein as a
model of spontaneous diabetic mellitus, the oxidative macrophages
are predominant at the stage of infiltration of inflammatory cells
in pancreatic islet, thereby increasing the humoral immune response
mainly caused by Th2 and decreasing the cellular immune response
supported by Th1. On the other hand, reductive macrophages are
predominant at the stage of occurrence of diabetes and deficient
insulin secretion by destruction of islets of Langerhans, thereby
increasing the cellular immune response mainly caused by Th1 and
decreasing the humoral immune response supported by Th2. In the
animal disease models, it is clearly shown that the anti-diabetes
agent provided by the present invention and the diagnosis of
diseases required for applying the agent is original and
significant.
Example 21
Redox states of macrophage in NOD mice
245. Method:
246. Macrophages were sampled from the abdominal cavities of the
NOD mice and the control mice, to identify whether the macrophages
were of an oxidative form or a reductive form. After physiological
saline of 5 ml was administered intraperitoneally into these mice,
intraperitoneal macrophages were collected, which were then
suspended and adjusted in a phenol red-free RPMI 1640 culture
medium supplemented with 10% fetal calf serum to 3.times.10.sup.6
cells/ml. The suspension was divided in 100-.mu.l portions on
Lab-Tek Chamber Slide (#136439; manufactured by NUNC, Co.), for
culturing in 5% CO.sub.2 at 37.degree. C. for 3 hours; after
discarding the nonadherent cells, the serum-free culture medium of
200 .mu.l was fed to the resulting culture, followed by addition of
MCB (abbreviation for monochlorobimane) to a final concentration of
10 .mu.M for reaction for 30 minutes; and based on UV absorption,
GSH was assayed by imaging analysis with an ACAS system
(manufactured by Meridien Co.).
247. Results:
248. Reduced type of glutathione was quantitatively assayed by the
ACAS method. In the NOD mice of age 3 to 5 weeks compared with the
control mice, the relative ratio of oxidative macrophages, namely
macrophages with the reduced amount of intracellular glutathione,
was increased. Because of the increase of oxidative macrophages,
IL-6 in the culture supernatant of the macrophages was prominently
increased (at 430 pg/ml vs at 120 pg/ml in the control mice). In
the mice with the onset of the diabetes, macrophages with increased
amount of intracellular reduced glutathione, namely reductive
macrophages, were relatively increased. It is thus indicated that
the identified redox state of macrophages on the basis of the
glutathione content assay can suggest the pathological conditions
and immune function of a diabetic patient in a simple and
appropriate manner, with no need of determination of numerous
functional parameters of macrophages. For the administration of the
inventive anti-diabetes mellitus agent, thus, the pathological
conditions and immunological state of a diabetic patient can be
examined and diagnosed according to the aforementioned macrophage
classification method.
Example 22
Induction of reductive macrophages by chemical agents administered
to NOD mice of age 3 to 5 weeks
249. Through a probe, glutathione ethyl ester was orally given at 1
mg/0.5 ml/h/day to the NOD mice of age 4 weeks on alternate days,
in total five times. By the same method as in Example 3,
intraperitoneal cells were collected from the mice to sample
intraperitoneal macrophages, which were then suspended and adjusted
in a phenol red-free RPMI 1640 culture medium supplemented with 10%
fetal calf serum to 3.times.10.sup.6 cells/ml. The suspension was
divided in 100-.mu.l portions on Lab-Tek Chamber Slide (#136439;
manufactured by NUNC, Co.), for culturing in 5% CO.sub.2 at
37.degree. C. for 3 hours; after discarding the nonadherent cells,
the serum-free culture medium of 200 .mu.l was added to the
resulting culture, followed by addition of MCB (abbreviation for
Monochlorobimane) to a final concentration of 10 .mu.M for reaction
for 30 minutes; and based on UV absorption, intracellular GSH was
assayed by imaging analysis with an ACAS system (manufactured by
Meridian Co.).
250. Results:
251. Reduced glutathione was quantitatively assayed by the ACAS
method. In the NOD mice dosed with glutathione ethyl ester,
compared with the control NOD mice administered with physiological
saline, the relative ratio of oxidative macrophages, namely
macrophages with the decrease in the content of reduced
glutathione, was decreased. Because of the increase of reductive
macrophages, IL-6 in the culture supernatant of the macrophages was
prominently decreased (at 460 pg/ml in the NOD mice vs at 3800
pg/ml in the control group). It is thus indicated that the redox
state of macrophages can be improved by glutathione ethyl ester
orally administered. The same action is also observed for
intraperitoneal dosing of .gamma.-glutamylcysteine dimethyl ester
(2 mg/0.5 ml/animal, administered on alternate days, in total five
times, starting from the age 4 weeks), intraperitoneal dosing of
N-acetylcysteine nitroxybutyl ester (0.5 mg/0.5 ml/animal,
administered on alternate days in total five times, starting from
the age 4 weeks), oral dosing thereof (1 mg/0.5 ml/animal,
administered on alternate days, in total five times, starting from
the age 4 weeks), intraperitoneal or oral dosing of glutathione
monoethyl ester (2 mg/0.5 ml/animal, administered on alternate
days, in total five times, starting from the age 4 weeks),
intraperitoneal or oral dosing of glutathione nitroxybutyl ester
(0.5 mg/0.5 ml/animal, administered on alternate days, in total
five times, starting from the age 4 weeks), intraperitoneal or oral
dosing of glutathione diethyl ester (2 mg/0.5 ml/animal,
administered on alternate days, in total six times, starting from
the age 3 weeks), and intraperitoneal dosing of lipoic acid (4
mg/0.5 ml/animal, administered on alternate days, in total five
times, starting from the age 4 weeks).
Example 23
Effects of chemical agents administered to NOD mice aged 3 to 6
weeks at a stage of macrophages at oxidized state and with
potential occurrence of inflammatory cell infiltration in
pancreatic islet
252. NOD mice purchased from Nippon Clair, Co. were naturally mated
together. Among the off spring mice, a colony of NOD mice with a
high frequency of the spontaneous onset of insulin-dependent
diabetes mellitus was established. Female NOD mice from the colony
were used for the present experiment. Chemical agents were orally
or intraperitoneally administered to the NOD mice aged 3 to 6 weeks
three times per week, in total nine times; and based on the test
results of urine glucose, positive or negative, the onset of
diabetes mellitus was followed once per week. Urine glucose was
detected, using Uropaper (BM Test Glucose 5000, manufactured by
Yamanouchi Pharmaceuticals, Co. Ltd.).
253. The results are shown in Table 5.
5TABLE 5 Frequency (%) of onset Frequency (%) of onset Chemical
Agents of diabetes mellitus *a of diabetes mellitus *b
Physiological saline 50 80 (NAC-OMe).sub.2 70 80 (NAC).sub.2 60 80
Oral NAC 30 40 Intraperitoneal NAC 10 20 Lentinan 10 20 GSHOEt 0 5
*a. on age 18 weeks *b. on age 22 weeks
254. Chemical agents were administered intraperitoneally, unless
otherwise stated.
255. The aforementioned results based on the experiments of drug
efficacy apparently indicate that reductive macrophage-inducing
substances including glutathione precursors such as
.gamma.-glutamylcysteine, .gamma.-glutamylcysteine dimethyl ester,
and N-acetylcysteine nitroxybutyl ester, glutathione derivatives
such as glutathione monoethyl ester, glutathione nitroxybutyl ester
and glutathione diethyl diester, and .beta.(1-3)-linked glucans
such as lentinan can suppress the spontaneous onset of diabetes
mellitus in NOD mice of age 3 to 6 weeks at a stage of macrophages
at oxidized state and with potential occurrence of inflammatory
cell infiltration in pancreatic islet. (NAC).sub.2 and
(NAC-OMe).sub.2 were at a single dose of 20 .mu.g/animal; and NAC
and GSHOEt were at a single dose of 2 .mu.g/animal; and a single
dose of lentinan was 0.1 mg/kg.
Example 24
Effects of chemical agents administered to NOD mice of age 3 to 6
weeks at a stage of macrophages at oxidized state and with
potential occurrence of inflammatory cell infiltration in
pancreatic islet and to age 9 toll weeks
256. NOD mice purchased from Nippon Clair, Co. were naturally mated
together. Among the off spring mice, a colony of NOD mice with a
high frequency of the spontaneous onset of insulin-dependent
diabetes mellitus was established. Female NOD mice from the colony
were used for the present experiment. Chemical agents were orally
or intraperitoneally administered to the NOD mice of age 3 to 6
weeks three times per week and to the NOD mice of age 9 to 11 weeks
twice per week, in total 15 times; and based on the test results of
urine glucose, positive or negative, the onset of diabetes mellitus
was followed once per week. Urine glucose was detected, using
Uropaper (BM Test Glucose 5000, manufactured by Yamanouchi
Pharmaceuticals, Co. Ltd.).
257. The results are shown in Table 6.
6 Frequency (%) of onset of diabetes Chemical agents mellitus
Physiological saline 60 Intraperitoneal 70 (NAC-OMe).sub.2 (20
.mu.g/h) Oral NAC (2 mg/h) 40 Intraperitoneal 10
.gamma.-glutamylcystein dimethyl ester (1 mg/h) Oral
.gamma.-glutamylcysteine 20 dimethyl ester (1 mg/h) Intraperitoneal
glutathione monoethyl 0 ester (1 mg/h) Intraperitoneal glutathione
diethyl 0 ester (1 mg/h) Lentinan (0.1 mg/kg) 0 Oral glutathione
diethyl diester 0 (5 mg/h)
258. The onset was judged on week 22.
259. The aforementioned results based on the experiments of drug
efficacy apparently indicate that reductive macrophage-inducing
substances including glutathione precursors such as
.gamma.-glutamylcysteine, .gamma.-glutamylcysteine dimethyl ester,
and N-acetylcysteine nitroxybutyl ester, glutathione derivatives
such as glutathione monoethyl ester, glutathione nitroxybutyl ester
and glutathione diester and .beta.(1-3)-linked glucans such as
lentinan, can suppress the spontaneous onset of inflammation of
pancreatic islet and that of diabetes mellitus owing to the
suppression of inflammatory cell infiltration into pancreatic
islet, when these substances are administered to NOD mice of age 3
to 6 weeks at a stage of macrophages at oxidized state and with
potential occurrence of inflammatory cell infiltration in
pancreatic islet, and to age 9 to 11 weeks at a stage of induction
of reductive macrophages.
Example 25
Effects of chemical agents administered to NOD mice aged 9 to 11
weeks at a stage of macrophages at reduced state
260. NOD mice purchased from Nippon Clair, Co. were naturally mated
together. Among the off spring mice, a colony of NOD mice with a
high frequency of the spontaneous onset of insulin-dependent
diabetes mellitus was established. Female NOD mice from the colony
were used for the present experiment. Chemical agents were orally
or intraperitoneally administered to the NOD mice aged 9 to 11
weeks three times per week, in total nine times; and based on the
test results of urine glucose, positive or negative, the onset of
diabetes mellitus was followed once per week. Urine glucose was
detected, using Uropaper (BM Test Glucose 5000, manufactured by
Yamanouchi Pharmaceuticals, Co. Ltd.).
261. The results are shown in Table 7.
7TABLE 7 Frequency (%) Chemical Agents of onset of diabetes
mellitus Physiological Saline 67 Intraperitoneal (NAC-OMe).sub.2 11
(NAlaC-OMe).sub.2 11 (NAC).sub.2 60 Oral NAC 60 Intraperitoneal 55
.gamma.-Glutamylcysteine dimethyl Ester Intraperitoneal 67
glutathione diethyl ester Lentinan 50
262. The onset was judged on week 22; the doses were the same as in
Example 24.
263. The aforementioned results apparently indicate that compounds
represented by the structural formula 1, including
N,N'-diacetylcystine [(NAC).sub.2], N,N'-dipropylcystine
[(NPC).sub.2], N,N'-diacetylcystine dimethyl ester
[(NAC-OMe).sub.2], N,N'-diacetylcystine diisopropyl ester
[(NAC-OiPr).sub.2] and N,N'-di-L-alanylcystine dimethyl ester
[(NAlaC-OMe).sub.2], and nitroxybutyl esters thereof and exerting
actions to decrease the content of reduced glutathione in
macrophages, suppress delayed type hypersensitivity reactions, and
suppress the production of IFN.gamma. or IL-1 2, can exert drug
efficacy when these compounds are administered to the NOD mice aged
9 to 11 weeks at a stage of macrophages at reduced state.
Example 26
Effects of chemical agents in db/db mice
264. db/db mice purchased from Nippon Clair, Co. were naturally
mated together. Among the off spring mice, a colony of db/db mice
with a high frequency of the spontaneous onset of
insulin-non-dependent diabetes mellitus was established. Male db/db
mice from the colony were used for the present experiment. Chemical
agents were intraperitoneally administered to the db/db mice aged 4
to 9 weeks three times per week, in total 18 times; and based on
the level of blood sugar in the mice at satiation, the effects of
the chemical agents on the improvement of the diseased conditions
of insulin-non-dependent diabetes mellitus were followed once per
week.
265. The results are shown in Table 8.
8 TABLE 8 Chemical Agents 6 weeks 7 weeks 8 weeks Physiological
saline 100 100 100 (NAC-OMe).sub.2 94 92 93 (NAC).sub.2 95 100 98
.gamma.-Glutamylcysteine 85 65 60 dimethyl ester Glutathione
diethyl ester 82 62 60 Lentinan + glutathione 70 55 50 diethyl
ester
266. The doses were the same as in Example 24.
267. The aforementioned results based on the experiments of drug
efficacy apparently indicate that when administered to the db/db
mice at a high frequency of the spontaneous onset of
insulin-non-dependent diabetes mellitus, these reductive
macrophage-inducing substances including glutathione precursors
such as .gamma.-glutamylcysteine, .gamma.-glutamylcysteine dimethyl
ester, and N-acetylcysteine nitroxybutyl ester, glutathione
derivatives such as glutathione monoester, glutathione nitroxybutyl
ester and glutathione diester, and .beta.(1-3)-linked glucans such
as lentinan, significantly decrease the blood sugar level and
effectively ameliorate the diseased conditions of
insulin-non-dependent diabetes mellitus with the etiology of poor
glucose incorporation into muscle and fat cells. The action
mechanism is not yet elucidated, but the pharmacological effects
may possibly be ascribed to the improvement of liver function or
the inhibitory action against phosphatase.
268. Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
269. U.S. patent application Ser. No. 09/181,881, filed on Oct. 29,
1998, is incorporated herein by reference.
270. Japanese Patent Application Serial No. 308300, filed on Oct.
29, 1998, is incorporated herein by reference.
271. Japanese Patent Application Serial No. 9-312727, filed on Oct.
27, 1997, is incorporated herein by reference.
* * * * *