U.S. patent application number 11/596674 was filed with the patent office on 2009-01-15 for pharmaceutical composition and therapeutic method.
This patent application is currently assigned to KEIO UNIVERSITY. Invention is credited to Naoki Aikawa, Seitaro Fujishima, Kazuhiko Sekine.
Application Number | 20090016987 11/596674 |
Document ID | / |
Family ID | 32338400 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090016987 |
Kind Code |
A1 |
Aikawa; Naoki ; et
al. |
January 15, 2009 |
Pharmaceutical composition and therapeutic method
Abstract
The object of the present invention is, based on the finding of
a novel effect of IL-18, to provide pharmaceutical compositions
containing IL-18 and therapeutic methods, for preventing,
improving, or treating inflammatory diseases such as systemic
inflammatory response syndrome (SIRS). Inflammatory diseases, such
as SIRS, can be prevented, improved, or treated by administering
the pharmaceutical compositions according to the present invention
containing IL-18 in an amount which does not bring the decreased
interleukin 18 level in either plasma or liver to a higher level
than normal.
Inventors: |
Aikawa; Naoki; (Tokyo,
JP) ; Fujishima; Seitaro; (Tokyo, JP) ;
Sekine; Kazuhiko; (Tokyo, JP) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
KEIO UNIVERSITY
MINATIO-KU TOKYO
JP
|
Family ID: |
32338400 |
Appl. No.: |
11/596674 |
Filed: |
May 17, 2005 |
PCT Filed: |
May 17, 2005 |
PCT NO: |
PCT/JP05/08944 |
371 Date: |
September 9, 2008 |
Current U.S.
Class: |
424/85.2 ;
530/351 |
Current CPC
Class: |
G01N 21/8986 20130101;
G01N 21/8806 20130101; A61P 29/00 20180101; G01B 11/04
20130101 |
Class at
Publication: |
424/85.2 ;
530/351 |
International
Class: |
C07K 14/54 20060101
C07K014/54; A61K 38/21 20060101 A61K038/21; A61P 29/00 20060101
A61P029/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 2004 |
FI |
20040694 |
Claims
1. A pharmaceutical composition for preventing, improving or
treating an inflammatory disease, comprising interleukin 18
(IL-18).
2. The pharmaceutical composition of claim 1, wherein the
inflammatory disease is systemic inflammatory response syndrome
(SIRS).
3. The pharmaceutical composition of claim 2, wherein the
inflammatory disease is sepsis syndrome.
4. The pharmaceutical composition of claim 3, wherein the
inflammatory disease is septicemia.
5. The pharmaceutical composition of claim 1, wherein the
inflammatory disease is an acute lung injury or a multi organ
dysfunction syndrome, secondary to SIRS.
6. The pharmaceutical composition of claim 1, wherein said
composition acts independently of interferon .gamma.
(IFN-.gamma.).
7. The pharmaceutical composition of claim 1, wherein the
inflammatory disease decreases the interleukin 18 level in either
plasma or liver.
8. The pharmaceutical composition of claim 7, wherein said
composition does not bring the interleukin 18 level in either
plasma or liver having decreased in the inflammatory disease to a
higher level than normal.
9. A therapeutic method for treating a non-human vertebrate
individual suffering from an inflammatory disease, comprising
administering a pharmaceutical composition comprising interleukin
18 to the vertebrate individual.
10. The therapeutic method of claim 9, wherein the inflammatory
disease decreases the interleukin 18 level in either plasma or
liver.
11. The therapeutic method of claim 10, comprising the step of
administering the pharmaceutical composition comprising IL-18 in an
amount which does not bring the interleukin 18 level in either
plasma or liver having decreased in the inflammatory disease to a
higher level than normal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to a
Japanese Patent Application No. 2004-145971, filed on May 17, 2004,
which is incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to pharmaceutical compositions
and therapeutic methods for preventing, improving or treating
inflammatory diseases.
BACKGROUND ART
[0003] Interleukin 18 (IL-18) was identified as an interferon
.gamma. (IFN-.gamma.) production-inducing factor, which appears in
the blood when a small dose of lypopolysaccharide (LPS) has been
intravenously injected into a mouse pretreated with heat-killed
Propionibacterium acnes (P. acnes), a gram-positive bacillus.
IFN-.gamma. enhances cellular immunity by activating macrophages
and NK cells, thereby elevating resistance to infection with
pathogens such as bacteria. At present, it has been clarified that
IL-18 induces IFN-.gamma. production by activating CD8 cells, NK
cells, and TH-1 cells.
[0004] IL-18, unlike many cytokines, is observed to be expressed at
a low level even under normal conditions. However, its expression
is enhanced upon infection with pathogens (viruses, prokaryotes, or
eukaryotes), thereby conferring resistance to pathogens on cells.
For example, administration of IL-18 into mice prior to infection
with HSV remarkably improves the survival rate of the mice.
Administration of IL-18 also suppresses proliferation of a fungus
Cryptococcus neoformans in cryptococcosis, thereby conferring
resistance to Candida infection. Further, treatment of a mouse with
anti-IL-18 antibody impairs its resistance to Salmonella
typhimurim, an intracellular microorganism, whereas administration
of IL-18 to a mouse infected with a toxic strain of Salmonella at a
lethal dose decreases the number of the bacteria in the tissue,
thereby reducing lethality ("Cytokine Reference: A Compendium of
Cytokines and Other Mediators of Host Defense, edited by Joost J.
Oppenheim, Marc Feldmann, and Scott K. Durum; Academic Press,
U.S.A, 2000; pp. 338-350.). IL-18 is known to induce cytokines
other than IFN-.gamma., and chemokines, among which are
inflammatory cytokines such as IL-8, TNF, and IL-6. In addition,
from the fact that IL-18-knockout mice have resistance to
administration of LPS, which induces inflammatory cytokines, it is
evident that IL-18 is a factor indispensable to LPS-induced
inflammatory cytokines.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0005] As described above, IL-18 is known to be a mediator of
immune response and has been considered to play an important role
in the induction of the inflammatory cytokines.
[0006] However, the inventors of the present application has
discovered a novel effect of IL-18, i.e. suppression of
inflammation. Accordingly, based on the finding of this novel
effect of IL-18, the object of the present invention is to provide
pharmaceutical compositions containing IL-18 and therapeutic
methods for preventing, improving or treating inflammatory diseases
such as systemic inflammatory response syndrome (SIRS).
Means for Solving the Problems
[0007] IL-18 is observed to be expressed at a low level even under
normal conditions. The inventors found that severe burn injury
decreases the IL-18 expression level, and that administration of a
small dose of IL-18 to severely burned mice remarkably improves the
survival rate. The inventors further found that, although splenic
cells isolated from those burned mice secrete inflammatory cytokine
in response to LPS under culture condition, this secretion is
suppressed in the IL-18-administered mice, and thus accomplished
the present invention.
[0008] The pharmaceutical composition according to the present
invention is a pharmaceutical composition for preventing, improving
or treating an inflammatory disease, which contains interleukin 18
(IL-18) . The inflammatory disease may be SIRS, sepsis syndrome, or
septicemia. The inflammatory disease may be acute lung injury or
multi organ dysfunction syndrome, secondary to SIRS. The
inflammatory disease may act independently of interferon
.gamma..
[0009] The inflammatory disease to be treated by the pharmaceutical
composition may decrease the interleukin 18 level in the liver. In
this case, the pharmaceutical composition according to the present
invention preferably does not bring the liver interleukin 18 level
which has decreased in the aforementioned inflammatory disease to a
higher level than normal. "Normal" as used herein refers to a
measured value in a healthy individual, which typically has a
certain extent of a range.
[0010] In the therapeutic method according to the present
invention, an effective dose of IL-18 is administered to a patient
suffering from an inflammatory disease. The inflammatory diseases
may be SIRS, sepsis syndrome, or septicemia. The inflammatory
disease may be acute lung injury or multi organ dysfunction
syndrome, secondary to SIRS. The inflammatory disease may act
independently of interferon .gamma..
[0011] Further, the inflammatory disease to be treated by the
pharmaceutical composition may be a disease which decreases
interleukin 18 level in plasma or liver. In this case, the
pharmaceutical composition preferably does not to bring the
interleukin 18 level in plasma or liver which has decreased in the
inflammatory disease to a higher level than normal.
[0012] The therapeutic method according to the present invention is
a therapeutic method for treating a human or nonhuman vertebrate
individual suffering from an inflammatory disease, including
administering a pharmaceutical composition containing interleukin
18 to the vertebrate individual. The inflammatory disease may
decrease interleukin 18 level in plasma or liver. It is preferable
to administer the pharmaceutical composition containing interleukin
18 in an amount which does not bring the interleukin 18 level in
plasma or liver which has decreased in the aforementioned
inflammatory disease to a higher level than normal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph showing a time course of the IL-18 content
in livers and lungs after burn injury in the mice of the control
group and the burn group in an embodiment of the present
invention.
[0014] FIG. 2 is a graph showing a time course of the plasma IL-18
level after burn injury in humans in an embodiment of the present
invention.
[0015] FIG. 3 is a graph showing the survival rates of the mice of
the control group, the non-IL-18-administered group, and the
IL-18-administered group after LPS administration in an embodiment
of the present invention.
[0016] FIG. 4 is a graph showing images of the histological
sections of lungs after the LPS administration into the mice of the
control group, the non-IL-18-administered group, and the
IL-18-administered group in an embodiment of the present
invention.
[0017] FIG. 5 shows the secreted amounts of various cytokines in
the splenic cells isolated from the mice of the control group, the
non-IL-18-administered group, and the IL-18-administrated group,
which were stimulated with anti-CD3 antibody in vitro in an
embodiment of the present invention.
[0018] FIG. 6 shows the secreted amounts of various cytokines in
the splenic cells isolated from the mice of the control group, the
non-IL-18-administered group, and the IL-18-administrated group,
which were stimulated with LPS in vitro in an embodiment of the
present invention.
[0019] FIG. 7 shows the secreted amounts of cytokines in the lungs
of the mice of the control group, the untreated group, and the
IL-18 administrated group in an embodiment of the present
invention.
PREFERRED EMBODIMENT OF THE INVENTION
[0020] Embodiments of the present invention accomplished based on
the above-described findings are hereinafter described in detail by
giving Examples. Unless otherwise explained, methods described in
standard sets of protocols such as J. Sambrook and E. F. Fritsch
& T. Maniatis (Ed.), "Molecular Cloning, a Laboratory Manual
(3rd edition), Cold Spring Harbor Press and Cold Spring Harbor,
N.Y. (2001); and F. M. Ausubel, R. Brent, R. E. Kingston, D. D.
Moore, J. G. Seidman, J. A. Smith, and K. Struhl (Ed.), "Current
Protocols in Molecular Biology," John Wiley & Sons Ltd., or
alternatively, modified/changed methods from these are used. When
using commercial reagent kits and measuring apparatus, unless
otherwise explained, attached protocols to them are used.
[0021] The object, characteristics, and advantages of the present
invention as well as the ideas thereof will be apparent to those
skilled in the art from the descriptions given herein. It is to be
understood that the embodiments and specific examples of the
invention described hereinbelow are to be taken as preferred
examples of the present invention. These descriptions are for
illustrative and explanatory purposes only and are not intended to
limit the invention to these embodiments or examples. It is further
apparent to those skilled in the art that various changes and
modifications may be made based on the descriptions given herein
within the intent and scope of the present invention disclosed
herein.
Diseases to be Dealt with the Present Invention
[0022] The pharmaceutical composition according to the present
invention is a pharmaceutical composition containing IL-18 for
preventing, improving or treating an inflammatory disease.
[0023] The inflammatory disease is exemplified by systemic
inflammatory response syndrome (SIRS). SIRS is a concept of
inflammatory response put forward by a collaboration of the
American College of Chest Physicians and the Society of Critical
Care Medicine in 1991, and defined to be diagnosed when two or more
of the following four conditions are present: 1) body temperature
is 38.degree. C. or higher, or 36.degree. C. or lower; 2) heart
rate is 90 beats/min or more; 3) respiratory rate is 20
breaths/min, or PaCO.sub.2 is less than 32 torr; and 4) leukocyte
count is 12,000 cells/mm.sup.3 or more, or 4,000 cells/mm.sup.3 or
less, or immature leukocytes are 10% or more. SIRSs are categorized
into the systemic inflammatory conditions upon trauma, burns,
pancreatitis, highly invasive surgery, etc., which are not
accompanied by infection, and the systemic inflammatory conditions
accompanied by infection (sepsis syndrome). The sepsis syndromes
are further categorized into the septicemia, a condition
accompanied by the infection by bacteria in the bloodstream, and
other conditions. All these inflammatory diseases can be considered
to be treated with the pharmaceutical composition according to the
present invention.
[0024] For example, upon severe burn injury, the affected area is
infected with bacteria and the multiplied bacteria invade the
bloodstream, thereby causing septicemia. The septicemia results in
acute lung injury (ALI), which can sometimes lead to multi organ
dysfunction syndrome (MODS). The pharmaceutical composition
according to the present invention can treat such severe injury as
well by exerting an anti-inflammatory effect.
[0025] The studies by the present inventors have revealed that
severe burn injury, septicemia, acute lung injury, etc is
accompanied with a decrease in the level of IL-18 in plasma or
liver. An administration of IL-18 against these diseases does not
increase the level of IFN-.gamma. that is secreted from splenic
cells in response to inflammatory stimuli. Therefore, it is
considered that IL-18 administration against these diseases has the
effect of suppressing inflammation independently of IFN-.gamma..
However, excessive doses may induce IFN-.gamma., therefore it is
preferable to administer the IL-18 so as not to bring the IL-18
level to a higher level than the normal IL-18 level.
Dosage Form and Dosage Regimen of the Pharmaceuticals According to
the Present Invention
[0026] When administered to a human, IL-18 or its pharmacologically
acceptable salt may be administered intravenously at a dose of, for
example, about 8 mg/kg (body weight) daily, in a single dose or
divided doses, and the dose and the number of administration may be
suitably changed depending on symptoms, age, dosage regimen,
etc.
[0027] IL-18 and its pharmacologically acceptable salts may be
orally administered in preparations such as tablets, capsules,
granule, powder, syrups, ointments, etc.; directly applied to the
affected area; or parenterally administered by intraperitoneal,
intravenous, intra-arterial, intramuscular, subcutaneous or other
injections, in preparations such as injectable formulations,
suppositories, etc. Alternatively, it may be applied or sprayed to
the skin, mucosa, the affected area, etc., in liquid form, or may
be inhaled from the nasal mucosa, throat mucosa, etc.
[0028] The content of IL-18 or its pharmacologically acceptable
salt (an active ingredient) in a preparation may vary between 1 to
90% by weight. For example, when the preparation is in the form of
a tablet, a capsule, a granule, a powder, etc., the content of the
active ingredient is preferably 5 to 80% by weight. In the case of
a liquid preparation such as syrup, the content of the active
ingredient is preferably 1 to 30% by weight. In the case of an
injectable preparation for the parenteral administration, the
content of the active ingredient is preferably 1 to 10% by
weight.
[0029] IL-18 and its pharmacologically acceptable salts may be
formulated by known methods by using formulation additives such as:
excipients (sugars such as lactose, sucrose, glucose, and mannitol;
starches such as potato, wheat, and corn; inorganic substances such
as calcium carbonate, calcium sulfate, and sodium hydrocarbonate;
cellulose crystal; etc.), binders (starch-paste liquid, gum arabic,
gelatin, sodium alginate, methylcellulose, ethylcellulose,
polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylcellulose,
carmellose, etc.), lubricants (magnesium stearate, talc,
hydrogenated vegetable oil, macrogol, and silicone oil),
disintegrators (starch, agar, gelatin powder, cellulose crystal,
sodium carboxymethylcellulose, calcium carboxymethylcellulose,
calcium carbonate, sodium bicarbonate, sodium alginate, etc.),
correctives (lactose, sucrose, glucose, mannitol, fragrant
essential oils, etc.), solvents (injectable water, sterile purified
water, sesame oil, soybean oil, corn oil, olive oil, cottonseed
oil, etc.), stabilizers (inert gases such as nitrogen and carbon
dioxide; chelating agents such as EDTA and thioglycolic acid;
reducing substances such as sodium bisulfite, sodium thiosulfate,
L-ascorbic acid, rongalite, etc.), preservatives (paraoxybenzoate
esters, chlorobutanol, benzyl alcohol, phenol, benzalkonium
chloride, etc.), detergents (hydrogenated castor oil, polysorbates
80 and 20, etc.), buffers (sodium salts of citric acid, acetic
acid, and phosphoric acid; boric acid; etc.), and diluents.
[0030] It should be noted that examples of pharmacologically
acceptable salts of IL-18 that may be used according to the present
invention include, but not limited to, organic salts such as
quaternary ammonium salts, and metal salts such as alkali metals,
etc.
EXAMPLES
[0031] To confirm immune response to inflammatory diseases resulted
from burn, as well as effects of IL-18 administration in humans and
nonhuman vertebrates, the following experiments were performed.
Example 1
Preparation of a Mouse Model of Burn Injury
[0032] Seven-week-old male Balb/c mice (body weight 23 to 28 g)
were caged and supplied with water and food ad libitum for about a
week, and then the mice were divided into a control group and a
burn group. The mice were shelved over the dorsum under
pentobarbital anesthesia (50 mg/kg) one day before the experiment
(Day 0). On the next day (day 1), all mice were anesthetized with
ether. For the mice of the burn group, their shelved dorsal area
was exposed to hot steam for 5 seconds to cause full-thickness burn
covering about 15% of total body surface area. Control mice were
not exposed to hot steam. All the mice were immediately
resuscitated with 4 ml of physiological saline.
Example 2
Measurement of IL-18 and IL-12 Levels in Lung and Liver
[0033] IL-12 as well as IL-18 is the cytokine classified as Th-1
cytokines involved in Th-1 cell differentiation. To examine the
behaviors of these Th-1 cytokines after burn injury, the IL-18 and
IL-12 contents in lung and liver of the mice in the burn group and
the control group prepared according to Example 1 were
measured.
[0034] Twenty-four hours after the burn treatment, mice were
anesthetized by intraperitoneal injection of pentobarbital, and
their lungs and livers were resected. The resected lungs and livers
were homogenized with 10 or 50 times by weight of phosphate
buffered saline (PBS), centrifuged, and the supernatants were
removed. Cytokine levels in the supernatants of lungs and livers
were determined for IL-18 and IL-12 by the sandwich ELISA method
using an ELISA kit (BD Biosciences). The results are shown in FIG.
1. The cytokine levels were compared by an analysis of variance
(ANOVA), followed by a Scheffe's comparison test (A significant
difference was judged by p<0.05. The levels of the following
cytokines were compared in the same manner.).
[0035] The Th-1 cytokine levels in the lung were significantly
lower than those in the liver. In the liver of the burn group mice,
the IL-18 content remarkably decreased on day 7, and the IL-12
level was generally lower until day 11. These findings revealed
that, after severe burn injury, the Th-1 cytokine level decreased
in the liver.
Example 3
Measurement of IL-18 Levels After Burn Injury in Humans
[0036] To observe changes in the IL-18 level after burn injury in
humans, plasma IL-18 levels of the severely-burned human subjects
were measured at some time points after burn injury, and the
results were divided into four subgroups: day 1, day 2 to 3, day 6
to 8, and day 9 to 11, after the burn injury. The measurements were
performed by using the ELISA kit (BD Biosciences) as in Example 2.
Mean values for each subgroup were calculated from the plurality of
measurements of the IL-18 levels in each subgroup. The IL-18 level
in each subgroup was subjected to an analysis of variance by using
these mean values, and then statistically treated by Fisher's PLSD
method (a significant difference was judged by p<0.05).
[0037] As shown in FIG. 2, the IL-18 levels showed a significant
decrease on day 2 to 3 after the burn injury, and this decrease
reached its peak on day 6 to 8. Such behavior of the IL-18 level is
the same as the changes in the IL-18 content in the liver of the
burn group mice described in Example 2. It was thus confirmed that
the IL-18 level decreased after burn injury in humans as well.
Example 4
Effect of IL-18 Administration on Survival Rate After Endotoxin
Treatment
[0038] The results from Examples 2 and 3 suggested that the
decrease in the Th-1 cytokine levels after burn injury is
critically involved in inflammatory diseases such as septicemia and
acute lung injury resulted from severe burn, etc. Thus, by using an
inflammation model mouse that has developed acute inflammation due
to administration of endotoxin (LPS) after burn injury, the effect
of administering a small dose of IL-18 was examined on the survival
rate after the LPS administration as a marker.
[0039] Mice in the burn group prepared according to Example 1 were
divided into two groups: the IL-18-administered group and the
non-IL-18-administered group. The mice in the IL-18 administered
group received 0.2 .mu.g of recombinant IL-18 (MBL Co. Ltd.) every
other day for 10 days after the burn injury, whereas mice in the
non-IL-18-administered group received nothing. On the eleventh day
(day 12) after the burn treatment, inflammation was induced in all
the mice of the three groups by intravenous injections of 10 ml/kg
body weight of physiological saline containing 3 mg/kg body weight
of LPS (Escherichia coli O111:B4 endotoxin; Sigma Corp.). Then, the
number of mice survived in each group was examined at every 12
hours until 72 hours after the induction. FIG. 3 is a graph showing
the results. The survival rates were compared between the groups by
using the Wilcoxon-Gehan test (a significant difference was judged
by p<0.05).
[0040] At 72 hours after the burn, the survival rate of the control
group was 100%, and the rate of the non-IL-18-administered group
was 42.9% whereas the rate of the IL-18-administered group was
91.7%, indicating that while the survival rate of the non-IL-18
administered group is considerably lower compared with that for the
control group, the IL-18 administration remarkably improves the
survival rate.
[0041] These results indicate that IL-18 has an anti-inflammatory
effect, suggesting that it is useful for a pharmaceutical
composition for preventing, improving, or treating inflammatory
diseases, such as systemic inflammatory response syndromes,
including septicemia, etc.
Example 5
Effect of IL-18 Administration on Pulmonary Edema
[0042] Using the inflammation model mice described in Example 4,
their lungs were resected, and their upper lobes were fixed with 4%
formaldehyde solution, embedded in paraffin, sectioned, and
histopathogically examined. As shown in FIG. 4, unlike the control
group (FIG. 4A), in the non-IL-18-administered group (FIG. 4B) a
severe pulmonary edema was induced, and the number of neutrophils
increased. Being a pathological condition similar to that in acute
lung injury, this can be a model of the acute lung injury.
Meanwhile, in the IL-18-administrated group (FIG. 4C), improvements
of conditions such as pulmonary edema and the increased number of
neutrophils were observed.
[0043] It is thus suggested that the administration of IL-18 is
useful for preventing, improving, or treating diseases such as
acute lung injury and multi organ dysfunction syndrome, secondary
to SIRS, etc.
Example 6
Cytokine Secretion by Activated Splenic Cells
[0044] In this Example, the amount of secreted cytokine was
measured by isolating splenic cells from the inflammation model
mice in Example 4 and then activating the splenic cells by
administering anti-CD3 antibody or LPS under the culture condition.
A stimulation of splenic cells with anti-CD3 antibody activates
immune response mediated by T cells, and a stimulation of splenic
cells with LPS activates mainly macrophages, resulting in secretion
of inflammatory cytokines.
[0045] First, the resected spleens were gently minced in RPMI 1640
supplemented with 5% fetal calf serum (FCS), and filtered through
nylon mesh strainer. The splenic cells were treated with 0.84%
ammonium chloride hemolyzing solution to hemolyze contaminated red
blood cells. After centrifugation, the splenic cells were
resuspended in PRMI-1640/5% FCS, added with 1 .mu.g/mL anti-CD3
antibody (mAb clone I45-2C11, BD Biosciences) or 10 .mu.g/mL LPS,
and incubated in 5% CO.sub.2 at 37.degree. C. for 24 or 48 hours.
For the culture supernatant, the levels of the inflammatory
cytokines TNF-.alpha. and MIP-2, the Th-1 cytokine IFN-.gamma., and
the Th-2 cytokines IL-4 and IL-10, were determined by using ELISA
kits (BD Biosciences).
[0046] When the splenic cells were stimulated with anti-CD3
antibody (FIG. 5), unlike the control group, the secreted amounts
of the inflammatory cytokines, the Th-1 cytokine, and the Th-2
cytokines all decreased. The secreted amounts of these cytokines in
the IL-18-administered group also decreased. Taken together, it is
evident that IL-18 cannot contribute to the decrease in cytokine
secretion by T cell activation.
[0047] In contrast, when the spleen cells were stimulated with LPS,
the secreted amounts of the inflammatory cytokines increased,
whereas the secreted amount of the Th-1 cytokine markedly
decreased, and the secreted amount of IL-10, a Th-2 cytokine,
increased (IL-4 secretion was undetectable in either case). In the
IL-18-administered group, unlike the non-IL-18-administered group,
the secretion of MIP-2, one of the inflammatory cytokines,
decreased, and that of IL-10, one of the Th-2 cytokines, increased
compared with the non-IL-18-administered group. This indicates that
IL-18 has an anti-inflammatory effect at the cytokine level.
Further, from the fact that the amount of IFN-.gamma. secretion did
not increased, it is considered that the effect of IL-18
administration on individual mice does not depend on IFN-.gamma.,
and has a different mechanism from the known effects of IL-18 when
IL-18 is enhanced against pathogenic infection.
Example 7
Cytokine Secretion After Endotoxin Treatment
[0048] The results of Example 4 revealed that the IL-18
administration markedly improves survival rates of the inflammation
model mice. Thus, the following experiment was performed to examine
the relationship between IL-18 and the secreted amounts of
cytokines in inflammation model mice.
[0049] In this Example, the lungs of the inflammation model mice
were isolated at 12 hours after the LPS administration, homogenized
with 10 or 50 times by weight of phosphate buffered saline (PBS),
and centrifuged, and the supernatants were removed. The levels of
TNF.alpha., IFN-.gamma., IL-4, IL-10, IL-12, IL-18, and MIP-2 in
the lung supernatants were determined by the sandwich ELISA method
using ELISA kits (BD Biosciences or Wako Pure Chemical Industries,
Ltd.) . The results are shown in FIG. 7. Cytokine levels were
compared by an analysis of variance (ANOVA), followed by a
Scheffe's comparison test (a significant difference was judged by
p<0.05).
[0050] As shown in FIG. 7, the secreted amounts of the inflammatory
cytokines, the Th-1 cytokine, and the Th-2 cytokines in the
non-IL-18-administered group all markedly increased compared with
the control group, whereas in the IL-18-administered group, the
secreted amounts of the inflammatory cytokines (TNF.alpha. and
MIP-2) significantly decreased. Therefore, this indicates that
IL-18 has an anti-inflammatory effect at the cytokine level.
INDUSTRIAL APPLICABILITY
[0051] The present invention provides pharmaceutical compositions
containing IL-18 for preventing, improving or treating inflammatory
diseases such as SIRS.
* * * * *