Note: Descriptions are shown in the official language in which they were submitted.
CA 02352614 2001-05-25
SPECIFICATION
LAK ACTIVITY-SCREENING MATERIALS
CONTAINING LENTINUS EXTRACT OF EDODES MYCELIUM AND
LAK ACTIVITY-SCREENING METHODS USING THE EXTRACT
FIELD OF THE INVENTION
The present invention relates to the field of tumor
immunology. Specifically, the present invention relates to
materials and methods for screening immunotherapeutic
agents having an antitumor and/or anticancer activity.
More specifically, the present invention relates to
screening materials and methods for determining in vitro
whether or not an LAK (Lymphokine Activated Killer) cell
activity-enhancing effect can be obtained in vivo.
PRIOR ART
It is well known in the field of tumor immunology,
that tumor cells contain tumor antigens. Tumor antigens
expressed by tumor cells include tumor-specific antigens
(TSA) which are expressed in tumor cells but not in normal
cells, and tumor-associated antigens (TAA) which are
expressed in normal cells as well as tumor cells, but at
very low levels unless they are upregulated by malignant
transformation. These tumor antigens are newly expressed
when genetic alteration takes place as a result of
malignant transformation of normal cells or as a result of
variation in expression regulation resulting from such
genetic alteration. The most commonly employed therapy for
treating cancer in which tumor cells exist which have an
- 1 -
CA 02352614 2001-05-25
altered antigenic expression is immunotherapy. This kind
of therapy may involve either the immunization of a patient
with a tumor antigen or the use of a drug which enhances
the patient's immune function. It is now accepted that
among the various cells functional in the immune system, Nk
(natural killer) cells exhibit a particularly potent anti
tumor-cell effect. I tis also recognized that NK cell
activity can be enhanced by employing immunotherapy. NK
cells are non-T/non-B cytotoxic lymphocytes present in
normal individuals, and they are known to have a MHC
antigen-nonrestricted cytotoxicity effect against not only
tumor cells, but also virus-infected cells and other cells
which do not express or decrease expressing MHC class I
molecules. However, there have now been identified tumor
cells resistant to even NK cells.
Dr. S. Rosenberg of the National Cancer Institute
(NCI) in the US found that incubation of peripheral
lymphocytes with interleukin 2 (IL-2) can induce the
production of killer cells showing cytotoxicity against a
wide range of target cancer cells including autologous
cancer cells and that these killer cells can kill even NK
cell-resistant cancer cells, (see Japanese Patent Public
Disclosure No. 116518/87). These killer cells were named
lymphokine activated killer (LAK) cells. LAK cells do not
consist of a cytologically homogeneous population and are
known to include NK cells and killer T-cells. Recently,
adoptive immunotherapy has been attempted wherein
peripheral lymphocytes from a subject are activated with
- 2 -
CA 02352614 2001-05-25
IL-2 in a cell culture system and then LAK cells showing
antitumor activity are reinfused into the subject (LAK
therapy). It has been reported that remission from
terminal cancer has been achieved or tumor growth-inhibited
by the use of adoptive immunotherapy involving repeated
administration of such LAK cells. However, LAK therapy
exerts different effects in different individuals and
sometimes has almost no effect. It also involves a number
of problems such as the physical stress imposed on the
subject associated with the isolation of a great number of
leukocytes from the patient, the high cost of performing
mass culture of isolated leukocytes, etc, and moreover, LAK
therapy involving direct administration of IL-2 causes
serious side effects due to the administration of IL-2 at a
high concentration.
Specifically, it is known that LAK adoptive
immunotherapy using IL-2 causes side effects such as
general prostration, chills, fever, hypoalbuminemia,
anemia, eosinophilia and that these side effects are more
serious than those caused by administration of IL-2 alone.
More notably, some important side effects are associated
with the cytotoxicity of LAK cells against normal cells.
It is also reported that such cytotoxicity of LAK cells
against hematopoietic stem cells induces anemia and
thrombocytopenia, as well as causing in vitro damage to
lymphocytes, macrophages and vascular endothelial cells.
Moreover, IL-2 administered via the oral route is poorly
absorbed and must therefore mainly be administered via
- 3 -
CA 02352614 2001-05-25
injection for direct administration at the present time.
Thus, it would be desirable to determine in vitro
whether or not LAK activity can be enhanced by direct
administration and to avoid the application of LAK therapy
which is liable to cause side effects because of the
uncertainty of the effects. However, screening in vitro
using IL-2 has the disadvantage of being too expensive.
Some bacteria, foods and other naturally occurring
substances are known to have anticancer properties.
Bacteria and food-type substance are preferential for use
as anti-cancer agents due to their generally benign nature
and low sideeffect profile. Many attempts have been made
to cure cancer by using bacteria, as shown in reports
relating to Coley's toxin consisting of a culture filtrate
of Serratia marcesens and Streptococcus pyogenes (1964);
treatment of leukemia with BCG (Mathe, G., Adv. Cancer Res.,
14, 1, 1971); tumor regression in guinea pigs (Zbar, B., et
al., J. Natl. Cancer Inst., 48, 831, 1971); and
effectiveness of administration of yeast cell wall
polysaccharide against transplanted tumor cells such as
sarcoma 180, for example.
Especially, a great amount of research has been
conducted into the anticancer effect of polysaccharides
derived from yeast such as yeast glucan and yeast mannan,
from other bacteria, from lichens and from basidiomycetes.
Among them, commercial products currently available on the
market as anticancer immunopotentiators include Krestin
derived from the cultured mycelia of kawaratake (Coriolus
- 4 -
CA 02352614 2001-05-25
versicolor, Basidiomycetes: Polyporaceae) (booster of
immune function of hosts, Kureha Chemical Industry and
Sankyo Co.Ltd.), a polysaccharide derived from shiitake
(Lentinus edodes) called lentinan and a polysaccharide
derived from suehirotake (Schizophyllum Commune).
Lentinus edodes (Shiitake) is a common edible
mushroom found in Japan and China, and has been cultivated
in Japan for about 300 years. It has been recently
elucidated for its pharmacological effects and effective
ingredients and reported to have various effects, such as
the growth inhibition effects on transplanted tumor cells
in the large bowel and liver in rats and mice (Sugano N. et
al., Cancer Letter, 27:1, 1985; Suzuki Y. et al., Journal
of the Japan Society of Coloproctology,. 43:178, 1990);
mitogenic effect (Tabata T. et al., Immunopharmacology,
24:57, 1992; Hibino et al., Immunopharmacology, 28:77,
1994), etc.
The present inventors researched the LAK activity-
enhancing effect (antitumor and/or anticancer activity) of
Lentinus edodes with a view to providing materials and
methods for screening the LAK activity-enhancing effect in
vivo demonstrated by direct administration of an extract of
Lentinus edodes mycelium.
In a conventional method, the LAK activity-enhancing
effect was tested by actually administering an LAK activity
enhancer to the host or reinfusing into the host the
activated lymphocytes which are prepared by isolating a
large amount of autologous lymphocytes followed by
- 5 -
CA 02352614 2001-05-25
activating them in vitro with an LAK activity enhancer.
This method involves a number of problems such as the
physical stress imposed on the subject, the high cost of
the therapy. Therefore, the establishment of an in vitro
screening method to confirm whether or not an LAK activity
enhancer actually has an effect in vivo may make it
possible to reduce physical stress and the high cost.
DISCLOSURE OF THE INVENTION
The inventors of the present invention found that
an extract of the mycelium, which is a precursor to the
edible fruiting body of Lentinus edodes, has a'far higher
immunopotentiating activity, antitumor activity and/or
anticancer activity than the fruiting body. We also found
that said extract can be used as an alternative to IL-2 to
induce a LAK activity in vitro. We accomplished the
present invention on the basis of the finding that the
antitumor effect in vivo and/or anticancer effect,
especially LAK activity-enhancing effect shown by direct in
vivo administration of said extract can be screened in
vitro.
More specifically, the present inventors found that
the in vivo cytotoxicity, which is exerted by the direct
administration of an antitumor or anticancer agent,
especially an LAK activity enhancer containing Lentinuis
edodes mycelium extract, has a positive correlation with
the cytotoxicity which is exerted when lymphocytes prepared
from a subject are activated with said LAK activity
- 6 -
CA 02352614 2001-05-25
enhancer. The present invention provides a method for
determining in vitro a material having an LAK activity-
enhancing effect suitable for a subject, comprising the
steps of:
(a) isolating peripheral blood from the subject to
prepare lymphocyte fractions,
(b) preparing an LAK-induced sample, which is
produced by treating said lymphocyte fractions with a
screening material of the present invention, and a control
sample, which is produced in the absence of the screening
material, and
(c) measuring and comparing the LAK activities of
said induced sample and said control sample to determine
the in vitro LAK activity-enhancing effect of the screening
material for said subject. The present invention also
provides a screening material containing the extract of
Lentinus edodes mycelium which can be used in said in vitro
screening method to screen whether or not the in vivo LAK
activity can be enhanced. Therefore, the present invention
relates to screening materials containing the extract of
Lentinus edodes mycelium and screening methods using said
materials capable of determining in vitro before
administration of the LAK activity enhancer whether or not
the in vivo LAK activity-enhancing effect can be expected
from an LAK activity enhancer. Screening materials and
screening methods of the present invention can be applied
to humans as well as to domestic animals.
As used herein, "LAK activity" means the anti-tumor
- 7 -
CA 02352614 2008-03-12
cytotoxic activity of cytotoxic T-lymphocytes, which
attack tumors unrecognizable by lymphocytes having NK
activity, but which have little influence on autologous
normal cells. "LAK activity-enhancing" refers to the
effect of enhancing this LAK activity, that is inducing
the production of LAK cells from lymphocytes or further
enhancing the antitumor activity of existing LAK cells.
Enhancement of LAK activity increases antitumor
activity of LAK cells, which leads to an improvement in
the function of the cell-mediated immune system. Thus, the
present invention can be applied not only to treatments
for improving antitumor activity but also to treatments
for improving the immune function.
One aspect of the invention relates to a screening
material containing an extract of Lentinus edodes mycelium
for use in the in vitro screening method of the invention
to determine the in vitro LAK activity-enhancing effect of
the extract of Lentinus edodes mycelium in a subject.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a bar graph corresponding to the data of
Table 1 showing the results of screening for LAK activity
enhancement with the extract of Lentinus edodes mycelium of
the present invention.
THE MOST PREFERRED EMBODIMENTS OF THE INVENTION
In one embodiment, there is provided a method for
-8-
CA 02352614 2008-03-12
determining whether an extract of Lentinus edodes mycelium
has an in vitro Lymphokine Activated Killer (LAK) activity-
enhancing effect suitable for a subject, comprising the
steps of:
(a) isolating peripheral blood from the subject to
prepare lymphocyte fractions,
(b) preparing an LAK-induced sample, which is
produced by treating said lymphocyte fractions with the
extract of Lentinus edodes mycelium, and a control sample,
which is produced in the absence of the extract of Lentinus
edodes mycelium, and
(c) measuring and comparing the LAK activity of said
LAK induced sample and said control sample to determine the
in vitro LAK activity-enhancing effect of the extract of
Lentinus edodes mycelium for said subject.
The present invention provides screening materials
containing an extract of Lentinus edodes mycelium and
screening methods using said materials capable of in vitro
determining before administration of said extract whether
or not an LAK activity-enhancing effect can be expected by
direct in vivo administration of an antitumor agent or an
anticancer agent, especially an LAK activity-enhancing
-8a-
CA 02352614 2001-05-25
formulation containing the extract of Lentinus edodes
mycelium. As used herein, "screening materials" refers to
materials used for testing in vitro the LAC activity-
enhancing effect obtained by in vivo administration. "An
extract of Lentinus edodes mycelium" used as a screening
material in the present invention refers to an extract
prepared by crushing and decomposing mycelia grown from
Lentinus edodes cultured on a solid medium or a solid
medium itself containing said mycelia in the presence of
water and an enzyme.
The extract of Lentinus edodes mycelium as used
herein is preferably obtained by, but not limited to, the
following process. Lentinus edodes spawn is inoculated on
a solid medium based on bagasse (sugar cane residue) and
defatted rice bran to grow mycelia, and then the solid
medium containing the grown mycelia is delignified to
enable about 30% by weight or less to pass through a 12-
mesh sieve. To this delignified solid medium are added
water and one or more enzymes consisting of a carbohydrase,
protease or combination thereof, and said solid medium is
maintained at a temperature of around 30-55 C to crush and
grind. The enzymes used in this step include, but not
limited to, cellulase, protease or glucosidase. The solid
medium crushed and ground in said step is adapted so that
at least 70% by weight of bagasse fiber is able to pass
through a 12-mesh sieve, and then the solid medium is
heated to a temperature of 95 C to ensure inactivation of
the enzyme and sterilization. Finally, the resulting
- 9 -
CA 02352614 2001-05-25
suspension is filtered to give an extract of Lentinus
edodes mycelium.
The extract of Lentinus edodes mycelium may be
directly used in screening materials or immunotherapeutic
agents of the present invention, but conveniently can be
concentrated and freeze-dried into a powder to be stored
and used in various forms. The freeze-dried product is a
brown powder with hygroscopic characteristics and has a
peculiar taste and odor.
The extract of Lentinus edodes mycelium of the
present invention can be directly added to lymphocyte
fractions prepared from peripheral blood. When the extract
of Lentinus edodes mycelium is directly added to lymphocyte
fractions, they are contained in screening materials of the
present invention preferably at a concentration of 1 ng/ml
- 100 mg/ml, more preferably 1 g/ml - 100 g/ml, most
preferably 10 g/ml - 50 g/ml. The extract of Lentinus
edodes mycelium of the present invention is preferably
sterilized with acetone before it is added to cultured
cells or directly added to peripheral blood.
Screening methods using screening materials of the
present invention can be performed according to the method
of Takagi et al. (Clinical Immunology, 19:245-249, 1987)
with exception that a screening material such as the
extract of Lentinus edodes mycelium of the present
invention is used in place of IL-2.
Accordingly, LAK activity enhancement-screening
methods of the present invention are methods for.
- 10 -
CA 02352614 2001-05-25
determining in vitro a material having an LAK Activity-
enhancing effect suitable for a subject, comprising the
steps of:
(a) isolating peripheral blood from the subject to
prepare lymphocyte fractions,
(b) preparing an LAK-induced sample, which is
produced by treating said lymphocyte fractions with a
screening material of the present invention, and a control
sample, which is produced in the absence of the screening
material, and
(c) measuring and comparing the LAK activities of
said induced sample and said control sample to determine
the in vitro LAK activity-enhancing effect of the screening
material for said subject.
To induce LAK cells, lymphocytes are isolated from
peripheral blood of the subject. Heparin is added to
peripheral blood from the subject, and monocytes at the
interface are separated by density gradient centrifugation
on Ficoll-Conray solution (s.g. = 1.077). The separated
monocytes are washed with PBS (pH 7.4, without Ca and Mg)
2-3 times and then suspended in a culture medium
(preferably, RPMI 1640 medium (Gibco) containing FBS
(inactivated fetal bovine serum) and/or antibiotics, if
desired) at a density of 1 x 106 cells /ml. This
suspension is transferred to a Petri dish which has been
precoated with autoserum (plasma) at 37 C for 15 minutes,
and incubated at 37 C for 1 hour. Unattached cells are
recovered as lymphocyte fractions.
- 11 -
CA 02352614 2001-05-25
LAK-induced samples are, for example, prepared by the
following procedure. Lymphocyte fractions prepared by the
procedure above are suspended in a culture medium at a
final concentration of 1 x 105 - 1 x 106 cells/ml and 100
l of the medium containing suspended cells is added to
each well at a density of 1 x 104 to 1 x 105 cells/well.
The number of cells per well can be appropriately
determined by those skilled in the art on the basis of the
activity of effector cells used, and the sensitivity of
target cells to effector cells, etc. Said suspending
solution contains the extract of Lentinus edodes mycelium
as a screening material at a final concentration in a range
of 1 ng/ml - 100 mg/ml in accordance with the experimental
design.
Thus, lymphocytes of the subject are cultured in the
presence of the extract of Lentinus edodes mycelium of the
present invention at various concentrations (including
zero) to prepare effector cells. As used herein, the term
effector cells refer to cells treated under the culture
condition for 3 days and include both lymphocytes
cocultured with an LAK activity enhancer (lymphocytes
treated with an LAK activity enhancer) and lymphocytes
cultured in the medium alone without LAK activity enhancer
(lymphocytes treated under the extract-free condition).
Control samples are prepared by the same procedure as
for LAK-induced samples with exception that sterilized
recombinant IL-2 (rIL-2; 2000 U/ml) is added in place of
the screening material.
- 12 -
CA 02352614 2001-05-25
LAK activity can be determined by 51Cr release assay,
[3H] uridine assay or the like. In terms of convenience
and objectivity, the 51Cr release assay is preferably used
in the present invention. The51Cr release assay is one of
methods for determining in vitro the cytotoxicity against
target cells of LAK cells induced from lymphocytes treated
with an LAK activity enhancer. The51.Cr release'assay is a
method.for determining the cytotoxicity of effector cells
against target cells, which comprises the steps of:
(i) adding 51Cr-labeled sodium chromate to the target
cells to label the target cells,
(ii) reacting the target cells with effector cells
(such as killer T cells or LAK cells) stimulated with a
screening material or rIL-2 as a control, and
(iii) measuring the amount of 51Cr released into the
cell culture supernatant from the target cells bursted by
the effector cells.
Subcultured cells used as target cells in the 51Cr
release assay are preferably Daudi cells or Raji cells.
Target cells cultured in a culture flask are recovered,
labeled with 51Cr and then divided into each well of a
microtiter plate after labeling. It is preferable to use a
culture media suitable for the growth of the cells as a
culture media for the target cells. The liquid media
include, for example, RPMI 1640 appropriately supplemented
with serum, antibiotics, etc.
Target cells are labeled by adding 100-150 Ci 51Cr-
sodium chromate per 106 cultured target cells followed by
- 13 -
CA 02352614 2001-05-25
stirring thoroughly and incubating at 37 C for 1-2 hours.
Cultured cells are washed with PBS three times, and then
suspended in RPMI 1640 medium containing 10% FBS at 1 x 106
cells/mi. The labeled cells are washed with the medium
which is used for culture or phosphate buffer (PBS), and
adjusted to a final concentration of 1 x 106 cells/ml in
the medium containing 10% fetal bovine serum (FBS) or fetal
calf serum (FCS) for assay. Target cells at a density of 5
x 104 cells/well are added to each well of a microtiter
plate in an amount of 50 i.
In the assay for determining cytotoxicity, each well
containing said target cells is further supplied with 100
l of 1N HC1 for maximum dissociation, 100 l of the medium
alone for natural dissociation, or effector cells in 100 l
of the medium at a density of 1 x 105 - 1 x 106 cells/mi
stimulated with the extract of Lentinus edodes mycelium of
the present invention at various concentrations or 2000
U/mi rIL-2 as a control for experimental dissociation.
Then, the microtiter plate is centrifuged at 800 rpm for 5
minutes on a plate centrifuge to collect cells at the
bottom of the well, and then incubated in a 5% CO2
incubator at 37 C for 3.5 hours.
Cytotoxicity to target cells in the 51Cr release assay
is calculated by the equation below.
Experimental dissociation(cpm)-Natural dissociation(cpm)
LAK activity% = x 100
Maximum dissociation(cpm)-Natural dissociation(cpm)
In vitro LAK activity-inducing ability of the
- 14 -
..,.._ _
CA 02352614 2001-05-25
screening material can be determined by comparing the LAK
activities of the induced sample and control sample
calculated by the equation above.
At the step of determining maximum dissociation,
natural dissociation and experimental dissociation, target
cells are incubated under 5% CO2 at 37 C. Those skilled in
the art may appropriately determine a culture period in
accordance with the purpose of the experiment, the number
of cells used or other conditions; for example, 3.5 hours
in the present invention.
The radioactivity of 51Cr released in the culture
supernatant can be measured using a scintillation counter
or the like.
In a preferred embodiment of the present invention,
various steps are performed as follows, though it will be
appreciated by those skilled in the art that suitable
changes and modifications can be made.
The culture supernatant in each well is collected
from the incubated plate to measure radioactivity in a
scintillation counter.
The extract of Lentinus edodes mycelium which
exhibited an in vitro LAK-inducing activity in the above
screening method was administered at 3600 mg/day for 7 days
to induce LAK activity in vivo. Lymphocyte fractions
collected by the above lymphocyte recovery method were used
to determine LAK activity % under the same conditions as
for said LAK-induced samples, showing that the in vivo LAK
activity-enhancing effect has a positive correlation with
- 15 -
CA 02352614 2001-05-25
the in vitro result.
The following examples further illustrate the present
invention but should not be taken as limiting the scope of
the invention.thereto. It will be appreciated by those
skilled in the art that various changes and modifications
can be made without departing from the spirit of the
present invention.
EXAMPLES
Example 1: Preparation of an extract of Lentinus edodes
mvicelium
A solid medium consisting of 90 parts by weight of
bagasse and 10 parts by weight of rice bran was soaked with
an appropriate amount of pure water, and then inoculated
with Lentinus edodes spawn and allowed to stand in an
incubator at controlled temperature and humidity to grow
mycelia. After mycelia spread over the solid medium, the
bagasse base was delignified to enable 24% by weight or
less to pass through a 12-mesh sieve. To 1.0 kg of this
delignified medium were added 3.5 L of pure water and 2.0 g
of purified cellulase while maintaining the solid medium at
40 C to prepare a medium-containing mixture.
Then, the medium-containing mixture was circulated by
a variable speed gear pump, during which the solid medium
was crushed and ground at the gears for about 200 minutes
so that about 80% by weight of bagasse fiber is able to
pass through a 12-mesh sieve. The medium-containing
mixture was crushed and ground while the temperature of
- 16 -
CA 02352614 2001-05-25
said mixture was gradually increased. Then, the medium-
containing mixture was further heated to 90 C to ensure
deactivation of the enzyme and sterilization and allowed to
stand at 90 C for 30 minutes. The resulting medium-
containing mixture was filtered through a 60-mesh filter
cloth to give an extract solution of Lentinus edodes
mycelium, which was concentrated and then converted into a
freeze-dried powder.
The extract of Lentinus edodes mycelium as prepared
above contained 25.3% (w/w) carbohydrates determined by the
phenol-sulfuric acid method, 19.7% (w/w) proteins
determined by the Lowry method and 2.6% (w/w) polyphenols
determined by the Folin-Denis method using gallic acid as
standard. The extract of Lentinus edodes mycelium further
contains 8% crude fat, 22% crude ash and about 20% soluble
nitrogen-free materials other than carbohydrates.
The extract of Lentinus edodes mycelium had a sugar
composition (%) as follows: Xyl 15.2, Ara 8.2, Man 8.4, Gul
39.4, Gal 5.4, G1cN 12.0, G1uUA 11.3.
Example 2= Determination of LAK activitv
Initially, peripheral blood was collected from
subjects A, B and C before administration of the extract of
Lentinus edodes mycelium and after oral administration of
1200 mg the extract of Lentinus edodes mycelium three times
daily for one week to each subject. Lymphocyte fractions
isolated from these peripheral bloods by the method below
can be screened for the correlation between the in vivo
- 17 -
CA 02352614 2001-05-25
lymphocyte-activating ability of the extract of the present
invention and the in vitro lymphocyte-activating ability of
the extract.
Heparin was first added to the peripheral bloods, and
monocytes at the interface were separated by density
gradient centrifugation on Ficoll-Conray solution (s.g. _
1.077), then the separated monocytes were washed with PBS
(pH 7.4, without Ca and Mg) twice and then suspended in
RPMI 1640 medium (Gibco) containing 10% FBS (inactivated
fetal bovine serum) at a density of l x 106 cells /ml. The
cells isolated by the method above were transferred to a
culture dish which had been precoated with autoserum
(plasma) at 37 C for 15 minutes followed by incubation at
37 C for 1 hour, and then unattached cells were recovered
as lymphocyte fractions.
Target cells (Daudi cells) subcultured in RPMI 1640
medium containing 10% FBS were recovered by centrifugation,
and incubated with 100-150 Ci/106 cells of 51Cr=sodium
chromate (New England Nuclear) in a 5% COZ incubator at
37 C for 1 hour. Cultured cells labeled with 51Cr were
washed with PBS three times, and then suspended in RPMI
1640 medium containing 10% FBS at 1 x 106 cells/ml.
A 50 l aliquot (5 x 104 cells/well) of target cells
labeled as above was added to each well of a microtiter
plate, and 100 Rl of 1N HC1 was further added to each well
of the maximum dissociation group (positive control), 100
l of RPMI 1640 medium containing 10% FBS was further added
to each well of the natural dissociation group (negative
- 18 -
CA 02352614 2001-05-25
control), and effector cells stimulated with 10 g/ml of
the extract of Lentinus edodes mycelium of the present
invention or 2000 U/ml of rIL-2 as a control were further
added to each well of the experimental dissociation group
(each 100 l (1 x 104 cells/well)). The plate was
centrifuged at 800 rpm for 5 minutes on a plate centrifuge
to collect cells at the bottom of the well, and then
incubated in a 5% COZ incubator at 37 C for 3.5 hours.
The culture supernatant in each well was collected by
SOKEN-PET E-96 from the incubated plate, and the
radioactivity was measured in a y-scintillation counter.
LAK activity was calculated by the equation below.
Experimental dissociation(cpm)-Natural dissociation(cpm)
LAK activity% = x 100
Maximum dissociation(cpm)-Natural dissociation(cpm)
The results are shown in Table 1 and Fig. 1.
Table 1:
LAK activity
Subject
Test No. A B C
1 Before administration 13% 27% 14%
2 Screening using extract 21% 34% 15%
(final concentration: 10 g/ml)
3 After administration of extract 40% 43% 15%
INDUSTRIAL APPLICABILITY
The extract of Lentinus edodes mycelium was orally
administered to subjects A and B to exhibit an enhancement
- 19 -
..~.am.~.,~..~ ._ ~..~....~ .. -_ _._.~. __
CA 02352614 2001-05-25
of LAK activity in vivo(see Table 1, Test No. 3). The in
vitro screening with the extract of the present invention
exhibited an LAK activity-enhancing effect when lymphocytes
isolated from peripheral blood of subjects A and B were
stimulated with the extract of the present invention and
said effect had a positive correlation with the LAK
activity-enhancing effect obtained by directly orally
administering the extract of the present invention to
subjects A and B (see Table 1, Test No. 2). Thus, it was
found that the LAK activity-enhancing effect.of the extract
of Lentinus edodes mycelium of the present invention
obtained by oral administration can be predicted by in
vitro screening.
The extract of Lentinus edodes mycelium was actually
orally administered to subject C to exhibit unenhancement
of LAK activity even in vivo (see Table 1, Test No. 3).
The in vitro screening with the extract of the present
invention showed no LAK activity-enhancing effect even when
lymphocytes collected from peripheral blood of subject C
were stimulated with the extract of the present invention
(see Table 1, Test No. 2). This example also demonstrated
that the LAK activity-enhancing effect of the extract of
Lentinus edodes mycelium of the present invention obtained
by oral administration can be predicted by in vitro
screening.
Therefore, it was found that the LAK activity-
enhancing effect of LAK activity enhancers of the present
invention obtained by oral administration can be exactly
- 20 -
CA 02352614 2001-05-25
predicted from in vitro screening results. Thus, the in
vivo LAK activity-enhancing effect of the direct
administration of LAK activity enhancers containing the
extract of Lentinus edodes mycelium of the present
invention can be conveniently determined in vitro before
said LAK activity enhancers are actually directly
administered. As a result, LAK activity enhancers can be
effectively and rapidly administered to subjects promising
for LAK activity-enhancing effect and, moreover, useless
administration of said LAK activity enhancers to subjects
unpromising for LAK activity-enhancing effect can be
prevented.
Moreover, physical stress on the subject is greatly
lessened in methods of the present invention because the in
vivo therapeutic effect of LAK activity enhancers can be
screened in vitro without the need to collecting a large
number of lymphocytes from the blood of the subject.
- 21 -
