Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITION CONTAINING DECURSIN
The present application has been divided out of Canadian Patent
Application Serial No. 2,347,750, Canadian national phase of International
Application Serial No. PCT/KR99/00632 filed internationally October 21, 1999
and published internationally as W02000/23074 on April 27, 2000.
Technical Field
The present invention relates to a pharmaceutical composition
containing decursin and its pharmaceutically acceptable carrier.
Background Art
As a great number of the pharmaceutical compositions used in the
prevention or treatment of various diseases have a variety of toxicities
including nephrotoxicity (i.e., toxicity on the kidney), due care should be
exercised in their clinical use. In particular, when using antineoplastic or
antibiotic composition, since its side effect such as nephrotoxicity is very
serious, its clinical use has been extremely restricted. For example,
cisplatin is
an antineoplastic agent used in the treatment of various tumors of the testis,
esophagus, stomach, bladder, prostate, lung, neck of uterus and osteosarcoma,
notably of genital tumors. However, its serious toxicities on the kidney,
ears,
gastro-intestine and bone marrow have been reported including allergies.
Among these, nephrotoxicity becomes so severe as to lead renal failure with
high dose of cisplatin for a long-term period, and thus clinical use of
cisplatin
has been extensively restricted. Under this situation, it is clinically
important
to reduce the toxicity of cisplatin on the bone marrow, gastrointestinal
tract,
and particularly on the kidney in the treatment of various cancers using
cisplatin.
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la
Intensive researches have been focused on the alleviation of
nephrotoxicity associated with the use of cisplatin. The conventional methods
designed to reduce the nephrotoxicity are as follows: One is to synthesize
platinum derivatives less toxic than cisplatin; one example being carboplatin.
However, its toxicity on the bone marrow is very severe even though its
nephrotoxicity is less than cisplatin. Another is to facilitate the excretion
of
drug for reducing the toxicity. To this end, mannitol or hypertonic saline
solution is concurrently used. However, this method may shorten the half-life
of cisplatin, resulting in decreasing the antineoplastic effect. A third is to
administer an antineoplastic agent
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together with antidotes via a two-route chemotherapy (TRC) so as to
reduce the toxicities of cisplatin. To this end, bismuthsubnitrate or
selenium is concurrently used. Holxiever, this method may cause some
adverse effect such as accumulation of heavy metals in the body.
In the meantime, all of the normal cells in the human body are
formed by proliferation to a certain extent and a subsequent differentiation
as an indispensable process. It is believed that such proliferation and
differentiation process are controlled at the genetic level. Unlike the
normal cells, howevet, cancer cells consist of immature cells that freely
proliferate without any differentiation, despite the fact that the cells are
derived from normal tissue. As a result, cancer cells differ from normal
cells in terms of metabolism, enzyme patterns and surface structure of
cells (Raymond W. Ruddon, Cancer:: A disease of abnormal
differentiation, Cancer Biology, 2nd edition: 69, 1987).
The development mechanism of such undifferentiated cancer cells
has yet to be reported. However, the main debate on the development of
cancer cells has been centered on whether the differentiation-completed
adult cells are dedifferentiated or the undifferentiated cells lose their
differentiation capacity. According to the report up to the present, the
transformation into cancel cells occurs only in the normal cells capable of
proliferation; while the differentiation of cells in which its short-term fate
to a certain path way is already programmed is irreversible, the terminal
differentiation itself is reversible (D. Yaffe, Cellular aspects of muscle
differentiation in vitro. Current Topics in Developmental Biology; 4:39,
1969).
Pierce et al. reported that normal tissue stem cells with renewal
capacity are origins of malignant tumor (G.B. Pierce, Differentiation of
normal and malignant cells, Fed. Proc.29: 1248, 1970). The stem cells
coincide with the cancer cells in that they are products of undifferentiated
cells with sustained proliferation capacity. However, recent studies revealed
that the abnormality in cancer cell is not completely irreversible.
The conventional method that is frequently applied to leukemia,
hepatocyte and fibroblast cells, is to induce differentiation of cancer cells
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into the normal cells or their similar cells by way of differentiation
induction agent (Alphonse Krystosek and Leo Sachs, Control of lysozyme
induction in the differentiation of myeloid leukemia cells, Cell, 9:675:684,
1976; Shinichi Murao, M. Anne Gemmell, Michael F. Callaham, N. Leigh
Anderson and Eliezer Huberman, Control of macrophage cell
differentiation in human promyelocytic U-937 leukemia cells by
1,25-dihydroxy vitamin D3 and phorbol-12 myri state- 13 -acetate, Cancer
Research, 43:4989-4996, 1983). Unlike the conventional antineoplastic
agents based on the cytotoxic mechanism, this method is significant in
that it is designed to treat the neoplastic tumors via new pathway.
Moreover, some of the conventional antineoplastic agents are reported to
have differentiation induction function at a concentration lower than
cytotoxic concentration, which is very stimulating in that side effect of the
antineoplastic agent can be alleviated by using at a very low
concentration.
In fact, active vitamin A, which is known as a differentiation
induction agent, is clinically employed to the treatment of acute
promyelocytic leukemia (APL) where the combined therapy of multi-drug
using daunomycin has been mainly conducted hitherto. According to the
Journal of American Blood Association published on November 1988, it
is reported that when a large dose of retinoic acid was administered to 22
APL patients, 96% of them were in complete remission. Thereafter, this
method was adopted in many medical institutions in the United States,
France, Japan, etc., and achieved an average remission rate of more than
80% with an average remission time of 29 days. In addition, there
showed side effects including skin dryness and gastric disturbance, but
their severity was milder than other antineoplastic agents. Thus, it can
be said that the differentiation induction agent has been significantly
recognized as a beneficial antineoplastic agent from the worldwide
medical arena.
The therapeutic mode of action in conventional antineoplastic
agents is related to the cytotoxicity to kill the rapid growing cancer cells
via inhibition of DNA replication, reduction of intracellular metabolism
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and biosynthesis or generation of free radicals. Therefore, a very high dose
of the
agent has been required. In this case, adverse effects are expected in various
organs
where cell proliferation is fairly rapid such as bone marrow, as well as in
the organs
where the agent is metabolized and excreted such as gastrointestinal tract,
liver,
kidney and cardiovascular tissues. In fact, administration of such
antineoplastic
agent is much limited due to such severe side effects. Accordingly, there has
been a
strong need for the development of a novel antineoplastic agent with less
toxicity,
i.e., the agent with cancer-cell differentiation induction function.
Decursin is a natural product that was firstly isolated from Angelica
decursiva (Fr. et Sav.), in Japan in 1966. It was reported in 1967 and 1969
that
Angelica gigas Nakai contains a large amount of decursin (J. Pharm. Soc.
Korea,
11: 22-26, 1967 and 13: 47-50, 1969). In addition, decursin was also isolated
from
the fruit of Peucedanum terebinthaceum (Fisher et Turcz) (Korea Pharmacology
Journal 30(2): 73-78, 1986). As for the pharmacological effect of decursin,
the
present inventors discovered in 1993 that decursin has toxicity on various
cell lines
of uterine cancer, leukemia, hepatoma or large intestine cancer at
concentration of
more than 10ppm (See Korean Patent Application No. 93-17935, published as 10-
1995-0007844 on April 15, 1995).
Disclosure of the Invention
Through intensive research for a long time, the present inventors discovered
various use of decursin to complete the present invention.
An object of the present invention is to provide a cancer-cell differentiation
induction agent composition comprising decursin as an active ingredient and a
pharmaceutically acceptable carrier.
Another object of the present invention is to provide a nephrotoxicity
inhibitor composition comprising decursin as an active ingredient and a
pharmaceutically acceptable carrier.
Another object of the present invention is to provide an antineoplastic
composition comprising decursin as a nephrotoxicity inhibitor, an
antineoplastic
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agent and a phannaceutically acceptable carrier.
A further object of the present invention is to provide an antidiabetic
composition comprising decursin as a nephrotoxicity inhibitor, an antidiabetic
agent
and a pharmaceutically acceptable carrier.
5 The present invention relates to a pharmaceutical composition containing
decursin.
According to the present invention, the pharmaceutical composition
containing decursin as a nephrotoxicity inhibitor can effectively inhibit the
nephrotoxicity associated with the pharmaceuticals. In particular, as can be
seen in
the following examples, decursin can effectively inhibit the nephrotoxicity of
cisplatin, a representative antineoplastic agent, as well as that of alloxan,
a
representative diabetes-inducing material.
Therefore, the pharmaceutical composition of the present invention may be
in the form of nephrotoxicity inhibitor composition containing decursin as an
active
ingredient.
Furthermore, the pharmaceutical composition of the present invention may
be in the form of antineoplastic composition containing decursin as a
nephrotoxicity
inhibitor. In this case, 1-5 moles of decursin may be employed to 1 mole of
antineoplastic agent, and the examples of antineoplastic agent may include
cisplatin.
Furthermore, the pharmaceutical composition of the present invention may
be in the form of antidiabetic composition containing decursin as a
nephrotoxicity
inhibitor. In this case, 0.2-10 moles of decursin may be employed to 1 mole of
antidiabetic agent.
The dose of the composition containing decursin as a nephrotoxicity
inhibitor may vary depending on its purposes; the composition may be
administered
at a dose of 1-500 mg per kg of body weight.
Although the mechanism of inhibiting nephrotoxicity by decursin has not
been proved yet, it appears to be caused by the scavenger effect of decursin
owing
to its powerful antioxidation property. For example, the nephrotoxicity of
cisplatin
can be explained as a typical inflammation
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process, including that cisplatin, which is already distributed throughout
the body, is absorbed in the proximal renal tubules of kidney while being
rapidly excreted, then neutrophils are induced and stimulated by
chemotaxis and finally the surrounding normal tissue is damaged by the
oxygen radicals secreted from the neutrophils. Therefore, antioxidants =
acting as a scavenger for free radicals can be thought to alleviate the
nephrotoxicity of cisplatin. For this purpose, however, the following
requirements should be met. Firstly, the antioxidants should be
adequately distributed in the body during the above process. Secondly,
the antioxidants should not be the bio-substances that are produced in the
body and needed by the whole body tissues including kidney so that
cancer cells cannot absorb the antioxidants and thus protect themselves.
Thirdly, the antioxidants should not be easily metabolized to be inactive
or have a strong affinity to the liver, which may result in delaying the
distribution of the antioxidants in the kidney or decreasing the amount to
be distributed. In other words, it is thought that decursin has the above
three requirements so as to effectively act as a scavenger of free radical
and thus effectively inhibit the nephrotoxicity.
Next, the pharmaceutical composition of the present invention
containing decursin as a cancer-cell differentiation induction agent may
vary in its dose depending on the purpose; the pharmaceutical
composition may be administered at a dose of 1-300 mg per kg of body
weight.
The composition of the present invention comprising decursin. as
an active ingredient can effectively induce the cancer-cell differentiation.
In particular, as can be seen in the following examples, decursin can
effectively induce the differentiation of human leukemia cells. In other
words, the composition of the present invention can be used as a novel
antineoplastic composition since decursin can induce the differentiation of
the cancer-cell, particularly of human leukemia cell, at much lower
concentration than cytotoxicity-producing concentration.
The pharmaceutical composition of the present invention
containing decursin may be formulated in various dosage forms using
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pharmaceutically acceptable carriers, excipients and/or additives. More
specifically, the pharmaceutical composition of the present invention may
include excipients such as lactose, starch, etc., lubricants such as
magnesium stearate, emulsifiers, suspension agents and isotonic agents,
and if desired, sweetening agents and/or flavors. Furthermore, the
pharmaceutical composition of the present invention may be formulated in
oral or peripheral dosage form; the oral dosage fonns include tablet,
capsule and liquid, while the peripheral dosage forms include intravenous,
intraperitoneal, and subcutaneous injection.
The invention is explained in detail in the following examples but is not
limited by those examples.
Example 1: The effect of decursin on;, alleviating nephrotoxicity
associated with the use of cisplatin
To determine the inhibitory effect of decursin on the side effects
associated with the use of cisplatin, such as weight loss, nephrotoxicity
and hepatotoxicity, a mixture of cisplatin and decursin was administered
to normal SD rats while changing the dose amount, dose period and dose
frequency. The detailed test methods were summarized hereinafter.
BUN (blood urea nitrogen) and creatinine as a marker of nephrotoxicity in
the blood, sGPT as a marker of hepatotoxicity, occult blood in the urine,
bilirubin, urobilinogen, ketone, protein, nitrite, glucose, pH and specific
gravity were determined as follows.
(1) Measurement of BUN
BUN value was measured using the BUN measurement kit
(Youngdong Pharm. Co. Ltd., Korea) in the following procedure.
0.1 ml of urease was added to 20ml of buffer solution to obtain
enzymatic buffer solution. The solution was put into two test tubes
respectively; 0.02m1 of a serum sample to be tested was added in one tube,
while 0.02m1 of reference solution (containing urea-N 60mg/100ml) as
control was added to the other tube. The two test tubes were incubated
at 37 1C for 15 minutes. Then 2m1 of color-forming solution was added
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into each test tube and incubated again at 37 C for 5 minutes. The
absorbance was measured at 570nm to determine the amount of BUN.
(2) Measurement of creatinine
Creatinine value was measured using the creatinine measurement
kit (Youngdong Pharm. Co. Ltd., Korea) in the following procedure.
4ml of tungsten solution was added in 0.5m1 of serum sample to be
tested, and stirred vigorously and then left for 10 minutes. Then the
mixture was centrifuged at 1500xg for 10 minutes to isolate the
supematant. Every 3m1 of the supematant, reference solution of
creatinine and distilled water (for blank test) was put to the separate test
tubes, followed by the addition of picrate solution (1 ml each). Then
0.5m1 of 1.4N NaOH was.added in each tube and stirred to measure the
absorbance at 515nm exactly after the correct lapse of 15 minutes.
(3) Measurement of sGPT
sGPT value was measured using the sGPT measurement kit
(Youngdong Pharm. Co. Ltd., Korea) in the following procedure.
GPT substrate solution (1 ml each) was incubated at 37 C for 3
minutes. 0.2m1 of serum sample was added to the substrate solution and
incubated again at 37 C for 30 minutes. 1 ml of 2,4-dinitrophenol was
added into the cultured solution, left for 20 minutes, then 0.4N NaOH
(10m1) is added and stirred well. The absorbance was measured at
505nm.
(4) Urinary test
TM
The urinary test was performed using urine test strips (Gen 9,
Youngdong Pharm. Co. Ltd., Korea) in the following procedure.
Immediately after collecting the urine from rats, the test strips were
stained with the urine was used for observing the color within 1 minute.
A total 10 of male SD rats (body weight: 200g) were used for this
experiment. The animals were divided into two groups each containing
4 rats, while one of the remaining two rats was selected as control (a
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group where cisplatin only was intraperitoneally administered at a dose of
5.8mg/kg) and the other as a normal group (a group with no drug
administration). To the first group (pre-treated group), decursin was
administered intraperitoneally three times at a dose of 17.4mg/kg in the
molar ratio of decursin to cisplatin (3:1) at the time intervals of 1, 24, and
48 hours prior to the intraperitoneal administration of cisplatin (5.8mg/kg).
To the second group (post-treatment group), decursin was administered
intraperitoneally three times at a dose of 5.8mg/kg in the molar ratio of
decursin to cisplatin (3:1) at the time intervals of 1, 24, and 48 hours after
the intraperitoneal administration of cisplatin (17.4mg/kg). The body
weights of rats were weighed at the beginning of the experiment (0 day).
The rats were sacrificed 4 days after the administration, and then their
weights were weighed. The urinary te~t was performed from the
administration of cisplatin to observe the nephrotoxicity induced by
cisplatin. After 4 days from the commencement of experiments, all rats
were sacrificed. The collected blood samples were left for 30 minutes to
isolate the sera. The isolated sera were used to determine BUN and
creatinine values as a marker of nephrotoxicity including sGPT value as a
marker of hepatotoxicity by the methods as previously described. The
results are shown in the following Tables 1 to 3.
Table 1.1Vleasurement of GPT, BUN and creatinine
Group G P T B U N Creatinine
Pre-treated 10.8 4.9 17.5t2.8 mg/dl 0.74f0.29mg/dl
Post-treated 19.0 5.3 55.2 2.1 mg/dl 2.08 0.52mg/di
Normal 17 15.58mg/dl 0.19mg/dl
Control 18 69.50mg/dl 3.68mg/dl
As seen in Table 1, the cisplatin-treated group (control) showed
severe nephrotoxicity as BUN and creatinine values as a marker of
nephrotoxicity were determined to be 69.5mg/di and 3.68mg/dl
respectively. By contrast, in the decursin-treated group, BUN value was
17.5 2.8mg/dl (normal value: less than 20) from the pre-treatment group,
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while BUN value was 55.2f2.lmg/dl from the post-treatment group.
BUN value in the pre-treatment group was completely recovered to the
normal level, but significant differences in the post-treatment group were
observed even though the nephrotoxicity was slightly alleviated.
5 Creatinine value in the pre-treatment group was 0.74t0.29mg/dl
(normal value: less than 1), showing complete recover to the normal level.
By contrast, creatinine value in the post-treatment group was
2.08 0.52mg/dl, showing the reduction of nephrotoxicity by about 40%
compared with the control group receiving cisplatin.
10 Further, changes in sGPT value in the blood was observed to
identify whether the alleviation of nephrotoxicity after the administration
of decursin is owing to the transfer of cisplatin accumulation from the
kidney to liver. Both of.::the pre-treatmerit and post-treatment groups
showed normal levels as 10.8t4.9 and 19.0t5.3, respectively. As a
result, it was revealed that there was no hepatotoxicity in the
administration of cisplatin, which coincides with those published in other
literatures.
Table 2. Urinary test
Pre-treatment group Post-treatment group
Nonnal Contml
172 3 4 1 2 3 4
Occult + + + + + ++ ++
Bilirubin + + + + + + +
LhubffinTm + + + + +
Ketone t
Protein -
Nitrite + +
Glucose + + + ++ + + + + ++
PH f
Specific
+ + +++
gravity
As confirmed from the above Table 2, there was a significant
increase in control in terms of various parameters such as occult blood in
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urine, bilirubin, urobilinogen, ketone, protein, glucose and specific gravity.
By contrast, the above parameters in the pre-treatment group were
reduced to the normal level. However, the nephrotoxicity in the
post-treatment group was alleviated depending on some parameters
compared with cisplatin-treatment group.
Table 3. Body weight
Pre-treatment group Post-treatinent group Nomial Cont.rol
1 2 3 4 1 2 3 4
Weight (g) 184 190 180 180 190 185 180 180 210 160
As confirmed from the above Table 3, the body weights were
reduced in both of the pre-treatment and post-treatment group when rats
were given decursin three times at the intervals of 1, 24 and 48 hours.
However, the weight loss was smaller than in the group treated with
cisplatin only.
The above results showed that when decursin was given to the rats
at the intervals of 1, 24 and 48 hours prior to the treatment of cisplatin, it
effectively inhibited nephrotoxicity activity without inducing any
hepatotoxicity.
Example 2: The effect of decursin on inhibiting renal failure
associated with diabetic complications
2 days after ICR mice were given alloxan (75mg/kg, i.v.), their
glucose levels at fasting were measured to select high-glucose mice. The
high-glucose mice were divided into two groups; physiological saline
solution was given to one group, while decursin was orally administered
to the other group at a dose of 50mg/kg for 10 days. The alloxan-free
group was also orally given physiological saline solution for 10 days.
The body weighs were checked periodically, together with urinary tests.
To isolate their sera and kidney, mice were slightly anesthetized with
ether and their abdomens were dissected. The blood samples were
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~ collected from heart to isolate the sera. The kidney was perfused with
0.9% NaCI and isolated after removal of the blood. Then the weight of
right kidney was measured. Further, to measure the protein amount from
the kidney tissues, these tissues were assayed according to the Bradford
method using Bio-RadT" protein assay kit in the presence of bovine serum
albumin as a reference standard. To measure the MDA in the kidney
tissues, 0.4m1 of 10% sodium dodecylsulfate was added to 0.5m1 of 10%
kidney-tissue homogenization solution and left in water bath at 37 C , for
30 minutes. With the subsequent addition of 0.1N-HCl (2ml) and 0.67%
TBA (imi), the mixture was voltexed and left in boiling water bath for 30
minutes. The mixture was further cooled in ice bath for 2-3 minutes to
stop the reaction. Then 2m1 of n-butanol was added to the mixture, stirred
well and centrifuged at 3000 rpm for 10 minutes. The supematant was
collected to measure ~ the absorbance at 532nm. l 0,ug/ml
tetraethoxypropane (TEP) was employed as a reference standard. To
measure the MDA in sera, 0.8% thiobarbituric acid (TBA) was added to
0.5m1 of sera and left in boiling water bath for 30 minutes. The mixture
was further cooled in ice bath. for2-3 minutes tostop the reaction. The
supernatant was collected to measure the absorbance at 532nm. 10/ug/ml
:L
TEP was employed as a reference standard.
Mice were orally given decursin at a dose of 50mg/kg for 10 days
continuously. Then various parameters such as BUN and. MDA in the
blood as a marker of renal failure were measured, together with urinary
test. When MDA value was measured by isolating the kidney tissues
(table 4), the BUN value of single alloxan-treated group, 17.4 3.7 mg/dl
-was recovered to the normal level (4.8 0.6 mg/dl) by the treatment of
decursin. The MDA value of single alloxan-treated group, 0.51 0.07,
was reduced to 0.36 0.03 by the treatment of decursin, which was similar
to a normal control group. Thus it was noted that the damage in the
kidney was completely recovered. Since the reduction of both MDA
values in the blood and kidney tissues may be clinically reasonable and
desirable, the MDA value in the kidney tissues was measured. As a
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result, the MDA value in the kidney tissues of a single alloxan-treated
group, 13.24 5.93, was reduced to 5.63 0.99 by the treatment of
decursin, which was lower than that of normal control group (8.44 1.01).
Thus it was revealed that decursin was quite effective in protecting the
kidney tissues.
Table 4. BUN and MDA values in blood, and 1VIDA values in renal tissue
MDA ( g/mi) MDA (pg/g)
Group BUN (mg/dl) in blood _ in renal tissue
Normal 5.4 0.8 0.38t0.04 8.44 1.01
alloxan 17.4 3.7 0.51 0.07 13.24t5.93
Alloxan + decursin 4.8f0.6 0.36t0,03 5.63t0.99
As confirmed from the above Table 5, urinary test,showed that in
the treatment of alloxan, the excreting amounts of occult blood, bilirubin,
urobilinogen and glucose were significantly increased as well as the
visible increase of specific gravity thereto. By contrast, all parameters
were nearly normal in a decursin-treated group. The results coincided
with the test result of BUN and MDA in the blood. The weight loss of the
alloxan-
treated group was significantly reduced by decursin treatment.
Table 5. Urinary test
Category Nonnal Alloxan Alloxan + decursin
+++ ++ + - +++ ++ + ++ + -
Occult blood 10 8 2 2 8
Bilirubin 1 9 2 6 2 2 8
Urobilinogen 10 10 1 9
Ketone 10 3 7 10
Protein 10 10 10
Nitrite 10 4 6 10
Glucose 9 10 1 9
pH 10 10 10
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Specific 10 9 1 l0
gravity
Example 3: The cell differentiation induction activity of decursin
U-937cells, optioned from Korea Cell Line Bank, was cultured in
RPMI 1640 medium (Sigma Co., R-6504) supplemented with 10% fetal
bovine serum (FBS). The medium was exchanged every 5 day by 70%.
U-937 cells were inoculated into a cell culture dish at concentration of
2X 105 cells/ml. Then decursin added into the dish at concentration of
10"g_ l0-6M, then the cells were cultured for 4 days. Trypan blue
exclusion test was performed to determine the number of live cells using a
hemacytometer, and the percentage of cancer-cell growth inhibition was
calculated. Then, the differentiation was induced by treating with 10"2 M
diluted ethanol solution containing various amount of decursin in the
medium.
U-937 cells were plated on a cell culture dish at a concentration of
2X 105 cells/ml in a medium containing 10-7M decursin and incubated in a
5% CO2 incubator at 37 C .
In the nitrobluetetrazolium (NBT) reduction test, cell solution
(about 1 x 106 cells/ml) was well stirred and pelleted by centrifugation at
2000 rpm for 5 minutes. The pellets were suspended in 0.5m1 of
medium, followed by the addition of a mixing solution (0.5m1) containing
10-6M formyl-methionine leucine phenylalanine (flVII.,P) and NBT
1.Omg/DPBSmI (1:9). The mixture was incubated at 37 C for 30
minutes. The reaction was stopped in ice bath and centrifuged at 2000
rpm for 5 minutes. The supernatant was removed and then 200,r.ce of
DPBS was added. The solution was lightly swayed and checked under a
microscope to observe a minimum number of 200 cells. The number of
NBT (+) cells was calculated by percentage.
In the phagocytosis test, 106 cells were well stirred and pelleted by
centrifugation at 2000 rpm for 5 minutes. I ml of 0.2% serum free
medium solution in polystyrene particle were added to the pellets, swayed
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and incubated at 37 C for 4 hours. After being washed with phosphate
buffered saline (PBS) three times, cell pellets were harvested and then
about 200,r.c( of DPBS was added. The solution was lightly swayed and
checked under a microscope to observe a minimum number of 200 cells.
5 The number of cells containing the particles was calculated by percentage.
To determine a-naphthyl acetate esterase activity in the esterase
activity test, the cultured solution of U-937 cells was pelleted by
centrifugation at 2000 rpm for 5 minutes, followed by the addition of
1001t,2 PBS to prepare a cell-concentrated solution. A drop of the cell
10 solution was put on a slide glass and dried for more than 1 hour. Then
40m1 of deionized water was preheated at 37 C . 1 ml of sodium nitrate
TM
solution was mixed with lml of Fast Blue BB base solution and left for 2
minutes. The mixture, being turned to yellow color with thick brown,
was added to 40m1 of the preheated deionized water. After the addition
TM
15 of 5m1 of TRIZMAL TM 7.6 buffer solution (Sigma Co. 870-2) and 1 ml
of a-naphthyl acetate solution, the resulting solution, which was turned to
green color, was poured into a petri dish. The cells on a slide glass were
fixed with citrate-acetone-formaldehyde solution (CAF solution) at room
temperature. The cells were soaked in the previously prepared solution
and incubated at 37 C for 30 minutes at a dark place. The cells were
completely washed with deionized water for more than 2 minutes and
counterstained with hematoxylin solution for 2 minutes, washed with tap
water and dried in the air. After completely dried, the cells were
observed under a microscope to determine the blackish cell numbers.
To determine naphthol AS-D chloroacetate esterase activity, the
cultured solution of U-937 cells was pelleted by centrifugation at 2000
rpm for 5 minutes, followed by the addition of 1000 PBS to prepare a
cell-concentrated solution. A drop of the cell solution was put on a slide
glass and dried for more than 1 hour. Then 40m1 of deionized water was
preheated at 371C. 1 ml of sodium nitrate solution was mixed with 1 ml
TM
of Fast Red Violet LB base solution and left for 2 minutes. The mixture
was added to 40m1 of preheated deionized water. After the addition of
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5ml of TRIZMAL TM 6.3 buffer solution (Sigma Co. T-3128) and 1 ml of
naphthol AS-D chloroacetate solution, the resulting solution, which was
turned to red color, was poured on a petri dish. The cells on a slide glass
were fixed with citrate-acetone-formaldehyde solution (CAF solution) at
room temperature. The cells were soaked in the previously prepared
solution and incubated at 37 C for 30 minutes at a dark place. The
cells were completely washed with deionized water for more than 2
minutes and counterstained with. hematoxylin solution for 2 minutes,
washed with tap water and dried in the air. After completely dried, the
cells were observed under a microscope to calculate the reddish cell
numbers by percentage.
(1) Growth inhibition test
As confirmed from' the following Table 6, when measuring the
growth inhibition rate of decursin on U-937 cells after 96-fhours, decursin
showed 30-50% of inhibitory effect at a concentration of 10-g -10~M.
Table 6. Growth inhibition test
Conc. Growth inhibition rate (%)
Vitamin D3 Decursin
1x10-6M 54.0t6.7 52.0t2.8
5x10"7M 48.0t6.0 48.0f7.1
1x10"7M 42.5f6.4 42.0t6.4
5x101gM 33.5 0.7 35.5f7.4
1x10"gM 26.0t5.7 31.0 7.8
(2) NBT reduction test
The differentiation was investigated by using the fact that the
differentiated leukocyte cells may generate superoxide anion by
stimulation. The cells were treated with decursin or vitamin D3 at 10"7M
and incubated for 4 days. The cultured mixture, which was reacted with
superoxide, was reduced to form a precipitate. Then NBT was added to
the precipitate (Table 7). As confirmed from the following Table 7, 14%
CA 02594186 2007-08-01
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of positive cells showed NBT reduction power in control, while positive
cells treated with vitamin D3 at 10-7 M and decursin at the same
concentration were increased by 39% and 43%, respectively.
Table 7. NBT reduction test
Conc. Untreated Decursin Vitamin D3
1x10" M 14.2f2.1% 43.1f4.0% 39.0t2.1%
(3) Phagocytosis test
U-937 cells were treated with decursin or vitamin D3 at 10'7M, and
then incubated for 4 days. The results of phagocytosis test designed to
assess the functional change according to differentiation of U-937 cells
was shown in the following Table 8. As confirmed from the following
Table 8, about 5% of control cells showed the phagocytosis activity, while
cells treated with vitamin D3 and decursin at the same concentration were
increased by 25.8% and 28.6%, respectively.
Table 8. Phagocytosis test
Conc. Untreated Decursin Vitamin D3
1x10-7M 4.8 0.8% 28.6t1.2% 25.8f1.3%
(4) Esterase activity test
Table 9 showed an enzyme activity test results of naphthol AS-D
chloroacetate esterase that is developed via differentiation into
granulocyte like cells and a-naphthyl acetate esterase which is developed
via differentiation into macrophage like cells. As confirmed from Table
9, none of the cells showed naphthol AS-D chloroacetate esterase activity
in all drugs. However, about 10% of cells showed la-naphthylacetate
esterase activity in control cells, while it demonstrated that 80% in
vitamin D3-treated group and 83% in decursin-treated group showed
a-naphthylacetate esterase activity. Thus it was considered that the cells
were differentiated into macrophage like cells by the action of decursin.
CA 02594186 2007-08-01
Table 9. Esterase activity test
Activity (%)
Enzyme
Control Vitamin D3 Decursin
a-Naphtylacetate esterase 10.5 0.7 79.3 4.5 83.3 3.0
Naphtol AS-D chloroacetate esterase 0.0 0.0 0.0
From the above results, it was confirmed that decursin is effective
5 in inducing the cancer-cell differentiation. More specifically, as a result
of determining the cytotoxicity of decursin and vitamin D3 at a
concentration of 10"8M-10-6M on the human-derived JU-937 cells, U-937
cells exhibited a very high cytotoxicity. Decursin showed a higher
cytotoxicity than vitamin D3 at a concentration of 5 X l 0"'M and I 0-6M.
10 To investigate the differentiation-inducing activity of decursin on U-937
cells at 10-7M, which showed about 83% of cytotoxicity, the cells were
treated with decursin for 4 days, with vitamin D3 as comparative material.
After performing NBT -reduction test and phagocytosis test, decursin at
10"7 M showed more potent differentiation power than vitamin D3 in both
15 of reduction test and phagocytosis test and the cells were differentiated
into macrophage like cell. Therefore, it was confirmed that decursin
effectively induces the cancer-cell differentiation.
Example 4: The toxicity test of decursin on normal cells
To determine the cytotoxicity of decursin on normal cells, they
were sub-cultured in a medium-199 containing 3% fetal bovine serum
(FBS), penicillin (1 OOU/ml) and streptomycin ( l 00,ug/ml) every 3 to 4 day.
Then cells were treated with trypsin to prepare a cell suspension. 1 ml of
the cell suspension was seeded on 24-well plate at a concentration of
2 X l 0' cells/ml and attached to the medium for one day. After the
supematant was removed, 1 ml of decursin was added to the cell culture
medium at appropriate concentrations and incubated for 3 days to count
the cell numbers. The results were shown in the following Table 10.
As confirmed in Table 10, the cytotoxicity on nor,nal cells (LLC-PKI)
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WO 00/23074 PCT/KR99/00632
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was not shown down to 10-6M.
Table 10. Cytotoxicity test on normal cells
Growth inhibition rate (%)
Conc.
Vitamin D3 Decursin
1x10-6M 9.0 0.7 0.0 0.0
5x10'7M 4.0 0.1 0.0 0.0
1 x 10"7M 4.0 0.2 0.0 0.0
5x10-8M 0.0 n.0 0.0 0.0
1x10"8M 0.0 0.0 0.0 0.0
As described above, decursin is an effective nephrotoxicity
inhibitor to alleviate the nephrotoxicity. Especially, decursin can be
effectively used in the antineoplastic composition or antidiabetic
composition. In addition, decursin _ can be used as a cancer-cell
differentiation induction agent to effectively induce cancer-celt
differentiation, especially as a human leukemia-cell differentiatiori
induction agent to cure human leukemia.
