U.S. patent application number 12/527302 was filed with the patent office on 2010-07-01 for herbal composition phy906 and its use in chemotherapy.
This patent application is currently assigned to YALE UNIVERSITY. Invention is credited to Yung-Chi Cheng, Zaoli Jiang, Shwu-Huey Liu.
Application Number | 20100166892 12/527302 |
Document ID | / |
Family ID | 39690520 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100166892 |
Kind Code |
A1 |
Liu; Shwu-Huey ; et
al. |
July 1, 2010 |
HERBAL COMPOSITION PHY906 AND ITS USE IN CHEMOTHERAPY
Abstract
This invention provides herbal compositions useful for
increasing the therapeutic index of chemotherapeutic compounds.
This invention also provides methods useful for improving the
quality of life of an individual undergoing chemotherapy.
Furthermore, this invention improves the treatment of disease by
increasing the therapeutic index of chemotherapy drugs by
administering the herbal composition PHY906 to a mammal undergoing
such chemotherapy.
Inventors: |
Liu; Shwu-Huey; (Madison,
CT) ; Jiang; Zaoli; (Woodbridge, CT) ; Cheng;
Yung-Chi; (Woodbridge, CT) |
Correspondence
Address: |
COOLEY LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
YALE UNIVERSITY
New Haven
CT
PHYTOCEUTICA, INC.
New Haven
CT
|
Family ID: |
39690520 |
Appl. No.: |
12/527302 |
Filed: |
February 14, 2008 |
PCT Filed: |
February 14, 2008 |
PCT NO: |
PCT/US08/53965 |
371 Date: |
January 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60901310 |
Feb 15, 2007 |
|
|
|
Current U.S.
Class: |
424/741 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 36/65 20130101; A61K 31/4412 20130101; A61K 31/7068 20130101;
A61K 36/725 20130101; A61K 36/484 20130101; A61K 36/539 20130101;
A61K 36/484 20130101; A61K 2300/00 20130101; A61K 36/539 20130101;
A61K 2300/00 20130101; A61K 36/65 20130101; A61K 2300/00 20130101;
A61K 36/725 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/741 |
International
Class: |
A61K 36/539 20060101
A61K036/539; A61K 36/725 20060101 A61K036/725; A61K 36/484 20060101
A61K036/484; A61P 35/00 20060101 A61P035/00 |
Claims
1. A composition comprising: i) a pharmaceutically acceptable
carrier; ii) an herbal preparation comprising Scutellaria,
Glycyrrhiza, Ziziphus, and Paeonia; and iii) one or more cancer
chemotherapeutic compounds.
2. The composition of claim 1 wherein the plant species comprises
Scutellaria baicalensis, Glycyrrhiza uralensis, Ziziphus jujuba,
and Paeonia lactiflora.
3. (canceled)
4. The composition of claim 1 wherein the cancer chemotherapeutic
compound is selected from the group consisting of capecitabine and
sorafenib.
5. A method of increasing the therapeutic index or enhancing
therapeutic effectiveness of a cancer therapeutic compound for the
treatment of cancer comprising administering to a mammal in need
thereof, a therapeutically effective amount of a composition
comprising a pharmaceutically acceptable carrier, and an herbal
preparation comprising Scutellaria, Glycyrrhiza, Ziziphus, and
Paeonia.
6. A method of treating cancer in a mammal in need thereof
comprising administering to the mammal a therapeutically effective
amount of a composition comprising: i) a pharmaceutically
acceptable carrier; ii) an herbal preparation comprising
Scutellaria, Glycyrrhiza, Ziziphus, and Paeonia; and iii) a
chemotherapeutic formulation comprising a cancer chemotherapeutic
compound.
7. A method of relieving side effects of a cancer chemotherapeutic
compound in a mammal comprising administering a composition
comprising: i) a pharmaceutically acceptable carrier; ii) an herbal
preparation comprising Scutellaria, Glycyrrhiza, Ziziphus, and
Paeonia; and iii) a chemotherapeutic formulation comprising the
cancer chemotherapeutic compound.
8-10. (canceled)
11. A method inhibiting the growth of tumors comprising
administering a composition comprising: iv) a pharmaceutically
acceptable carrier; v) an herbal preparation comprising
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia; and vi) a
chemotherapeutic formulation comprising a cancer chemotherapeutic
compound.
12. The method of claim 11, wherein the tumors are present in a
mammal or in vitro cells.
13-15. (canceled)
16. The method of claim 5, 6, 7, 8, or 11, wherein the cancer
chemotherapeutic compound is selected from the group consisting of
capecitabine and sorafenib.
17. The method of claim 5 or 6, wherein the cancer is a
gastrointestinal cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit to U.S. Provisional Patent
Application Ser. No. 60/901,310, the contents of which are herein
incorporated by reference in their entirety for all purposes. This
application is also related to the following applications and
patent: U.S. patent application Ser. No. 09/522,055 filed Mar. 9,
2000; International Application No. PCT/US2001/007353 filed Mar. 8,
2001; U.S. patent application Ser. No. 10/220,876 filed Dec. 30,
2002 and issued as U.S. Pat. No. 7,025,993 on Apr. 11, 2006; U.S.
Provisional Patent Application Ser. No. 60/625,943 filed Nov. 9,
2004; U.S. patent application Ser. No. 11/100,433 filed Apr. 7,
2005; and International Application No. PCT/US2005/040605 filed
Nov. 9, 2005, the contents of which are herein incorporated by
reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to herbal compositions and the
use of them for enhancing the therapeutic effects of
chemotherapeutic compounds.
BACKGROUND OF THE INVENTION
[0003] Cancer remains one of the major cause of death around the
world. Specifically, cancer is the second overall cause of death in
the United States. Gastrointestinal cancers, including colorectal,
liver, and pancreatic cancers, are of particular concerns not only
because of their high incidence rates, but also because of their
high mortality rate, especially in pancreatic and liver cancer
patients (1-4). From years 1992-1999, studies revealed that the
five-year relative survival rate of colorectal cancer was 62.3%
while that of liver cancer was 6.9% and 4.4% for pancreatic cancer.
The median survival of liver cancer was 3.5 weeks to 6 months while
it was 4 to 6 months for pancreatic cancer (3). With only very poor
chemotherapeutic regimens available, pancreatic cancer has the
highest mortality rate among all cancers in the United States, with
a less than 5% survival rate 5 years from diagnosis (3). Although
several regimens are currently used in clinical trials for
hepatocellular carcinoma, there is no FDA-approved chemotherapeutic
agent available. The low survival rates for both pancreatic and
hepatocellular cancers are attributed to many factors including
diagnosis is difficult, the tumor growth is highly aggressive,
surgical removal of tumor is of low probability, and the tumor has
a high rate of chemotherapy resistance.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 shows the effect of PHY906 (500 mg/kg, bid, D1-4 and
8-11) on tumor growth in Sorafenib (30 mg/kg, po, bid,
D1-14)-treated BDF-1 mouse bearing mouse colon 38 tumors. Sorafenib
(30 mg/kg) was given orally twice a day for a consecutive 14 days.
PHY906 (500 mg/kg) was given orally 30 min before sorafenib twice a
day on days 1-4 and days 8-11 (N=5 in each group).
[0005] FIG. 2 shows the effect of PHY906 (500 mg/kg, bid, D1-4,
8-11 and 15-18) on tumor growth in Sorafenib (30 mg/kg, po, bid,
D1-20)-treated nude mice bearing human HepG2 tumors. Sorafenib (30
mg/kg) was given orally twice a day for a consecutive 20 days.
PHY906 (500 mg/kg) was given orally 30 min before sorafenib twice a
day on days 1-4, 8-11 and 15-18 (N=5 in each group).
[0006] FIG. 3 shows the impact of PHY906 and Sorafenib on blood
vessels from the liver of NCr-nude mice bearing human HepG2
xenografts. Tissue sections were prepared from formalin-fixed,
paraffin-embedded liver cancer specimens. Immunohistochemical
staining was done using specific antibodies against CD31 (brown)
and nuclear DNA (blue).
[0007] FIG. 4 shows impact of PHY906 and Sorafenib on VEGF level
from the liver of NCr-nude mice bearing human HepG2 xenografts.
Tissue sections were prepared from formalin-fixed,
paraffin-embedded liver cancer specimens. Immunohistochemical
staining was done using specific antibodies against VEGF (brown)
and nuclear DNA (blue)
[0008] FIG. 5 shows the impact of PHY906 and Sorafenib on
HIF-1.alpha. level from the liver of NCr-nude mice bearing human
HepG2 xenografts. Tissue sections were prepared from
formalin-fixed, paraffin-embedded liver cancer specimens.
Immunohistochemical staining was done using specific antibodies
against HIF-1.alpha. (brown) and nuclear DNA (blue).
[0009] FIG. 6 shows the effect of PHY906 on the tumor growth in
Capecitabine-treated NCr-nude mice bearing human Panc-1 tumor;
Capecitabine (720 mg/kg) was given orally twice a day on days 1-7,
15-21 and 29-32 days. PHY906 was given orally 30 min before
capecitabine twice a day on days 1-4, 8-11, 15-18, 22-25 and 29-32
at 500 mg/kg (N=5 in each group)
SUMMARY OF THE INVENTION
[0010] In one aspect, the present invention provides a composition
comprising: i) a pharmaceutically acceptable carrier; ii) an herbal
preparation comprising Scutellaria, Glycyrrhiza, Ziziphus, and
Paeonia; and iii) one or more chemotherapeutic compounds.
[0011] In another aspect, the present invention provides a method
of treating a disease in a mammal in need thereof comprising
administering a therapeutically effective amount of a composition
comprising: i) a pharmaceutically acceptable carrier; ii) an herbal
preparation comprising Scutellaria, Glycyrrhiza, Ziziphus, and
Paeonia; and iii) one or more chemotherapeutic compounds.
[0012] In another aspect, the present invention provides a method
of increasing the therapeutic index of cancer therapeutic compounds
for the treatment of cancer by administering to a mammal in need
thereof, a therapeutically effective amount of a composition
comprising a pharmaceutically acceptable carrier, and an herbal
preparation comprising Scutellaria, Glycyrrhiza, Ziziphus, and
Paeonia.
[0013] In yet another aspect, the present invention provides a
method of relieving side effects of a chemotherapeutic compound in
a mammal comprising administering a composition comprising: i) a
pharmaceutically acceptable carrier, ii) an herbal preparation
comprising Scutellaria, Glycyrrhiza, Ziziphus, and Paeonia; and
iii) one or more chemotherapeutic compounds.
[0014] In yet another aspect, the present invention provides a
method of improving the quality of life of a mammal undergoing
chemotherapy which comprises administering a therapeutically
effective amount of one or more chemotherapeutic compounds and a
composition comprising: i) a pharmaceutically acceptable carrier;
ii) an herbal preparation comprising Scutellaria, Glycyrrhiza,
Ziziphus, and Paeonia; and iii) one or more chemotherapeutic
compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Gemcitabine is the only clinically approved chemotherapeutic
agent for pancreatic cancer; however, the response rate in patients
to gemcitabine is only 6-11% and the overall survival time is
generally 4-6 months. Gemcitabine is a nucleoside analog with two
mechanisms of action, including the inhibition of ribonucleotide
reductase, an enzyme that converts nucleotide diphosphate to
deoxynucleotide triphosphate and that is required for DNA synthesis
and that competes with deoxycytidine triphosphate as a fraudulent
base in DNA synthesis (3,5-10). With the low response and survival
rates of gemcitabine monotherapy, several gemcitabine-combination
drug regimens have been tested clinically for improving therapeutic
efficacy. These trials include gemcitabine with other commonly used
and FDA-approved anti-cancer drugs including CPT-11, capecitabine,
and oxaliplatin (11-14). Unfortunately, no satisfactory combination
drug regimens have been discovered and an effective regimen for
pancreatic cancer is urgently needed.
[0016] Capecitabine (Xeloda), an oral fluoropyrimidine, is a
rationally designed oral prodrug efficiently absorbed from the
gastrointestinal tract and converted to 5-FU, preferentially in
neoplastic tissues. It has been approved by the FDA as a first-line
chemotherapy for the treatment of colorectal and breast cancers
with reduced toxicities (15-17). Capecitabine has also shown
promising antitumor activity as a single agent in pancreatic cancer
(18) and liver cancer (19).
[0017] Hepatocellular carcinoma (HCC) is currently treated by
surgical procedures and chemotherapy. Surgical removal and
postoperative therapies may improve the outlook for some patients.
Unfortunately, the vast majority of patients with hepatocellular
carcinoma will have unresectable cancers. In late 2007, sorafenib
became the first FDA-approved chemotherapeutic agent for HCC.
Published clinical studies indicate significant anti-tumor effects
(20,21). Oral multikinase inhibitor sorafenib (BAY 43-9006) has a
dual-action on Raf kinase and vascular endothelial growth factor.
Sorafenib prevents tumor growth by combining inhibition in tumor
cell proliferation and tumor angiogenesis. Preclinical studies
suggest that sorafenib may offer therapeutic benefits in HCC by
blocking Raf-1 signal transduction pathway.
[0018] Colorectal cancer has been reported to be the third most
common cause of death from cancer in the United States (22).
Recently, the FDA approved the triple combination use of
Oxaliplatin/5-FU/LV as the first-line treatment for patients with
advanced colorectal cancer. Oxaliplatin is a synthesized
diaminocyclohexane platinum compound, which like cisplatin, causes
platinum-DNA adduct formation and destroys the integrity of DNA
(23). Other types of chemotherapeutic agents, such as 5-FU, CPT-11,
are common chemotherapeutic agents used in the treatment of
colorectal cancer. Unfortunately, severe diarrhea has been
identified as one of the dose-limiting toxicities among patients
treated with chemotherapy.
[0019] Our studies showed that PHY906, an herbal composition, not
only reduced chemotherapy-induced toxicities, including body weight
loss and mortality, but it also enhanced the antitumor efficacy of
a broad-spectrum of anticancer agents including, but not limited to
CPT-11, 5-FU, CPT-11/5-FU/LV, VP-16, L-OddC and oxaliplatin/5-FU/LV
in colorectal cancer; sorafenib, capecitabine, thalidomide, and
CPT-11 in liver cancer; and capecitabine, oxaliplatin, gemcitabine
and gemcitabine/oxaliplatin in pancreatic cancer in vivo animal
models. The positive results from these preclinical studies
demonstrate that PHY906 can be used as an adjuvant for a
broad-spectrum of different types of chemotherapeutic agents in
anti-cancer therapy. These chemotherapeutic agents include, but are
not limit to, capecitabine and sorafenib. The cancers include, but
are not limited to, colorectal, liver, and pancreatic cancers. The
methods of the present invention can be used to improve the quality
of life of patients including mammals under chemotherapy.
Specifically, this invention relates to the dosing and scheduling
of PHY906 in potentiating the therapeutic index of a broad-spectrum
of cancer chemotherapeutic agents by the herbal composition
PHY906.
[0020] In one embodiment, the present invention provides a
composition comprising a pharmaceutically acceptable carrier,
materials or chemicals from a plant species of each of the
following genera of herbs: Scutellaria, Glycyrrhiza, Ziziphus and
Paeonia, and one or more chemotherapeutic compounds. In another
embodiment, the materials or chemicals from a plant species is in a
form of a herbal composition comprising Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia. In yet another embodiment, the herbal
composition consists essentially of Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia.
[0021] In one embodiment, the plant species comprise Scutellaria
baicalensis, Glycyrrhiza uralensis, Ziziphus jujuba, and Paeonia
lactiflora. In another embodiment of the invention one or more
chemotherapeutic compounds are cancer chemotherapeutics. In one
embodiment of the invention the cancer chemotherapeutics are
selected from the group consisting of capecitabine, sorafenib, and
a combination thereof.
[0022] In one embodiment of the invention, a therapeutically
effective amount of a composition comprising a pharmaceutically
acceptable carrier, materials or chemicals from a plant species of
each of the following genera of herbs: Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia, and one or more chemotherapeutic compounds is
used to treat a disease in a mammal in need thereof. In another
embodiment, the materials or chemicals from a plant species is in a
form of a herbal composition comprising Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia. In yet another embodiment, the herbal
composition consists essentially of Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia.
[0023] In one embodiment, the present invention provides a method
of treating a disease in a mammal. The method comprises
administering to the mammal in need thereof a therapeutically
effective amount of a composition comprising a pharmaceutically
acceptable carrier, materials or chemicals from a plant species of
each of the following genera of herbs: Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia, and one or more chemotherapeutic compounds.
In another embodiment, the materials or chemicals from a plant
species is in a form of a herbal composition comprising
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia. In yet another
embodiment, the herbal composition consists essentially of
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia.
[0024] In one embodiment, the present invention provides a method
of relieving the side effects of a chemotherapeutic compound in a
mammal. The method comprises administering to the mammal in need
thereof a composition comprising a pharmaceutically acceptable
carrier, materials or chemicals from a plant species of each of the
following genera of herbs: Scutellaria, Glycyrrhiza, Ziziphus and
Paeonia, and one or more chemotherapeutic compounds.
[0025] In one embodiment of the invention, a composition comprising
a pharmaceutically acceptable carrier, materials or chemicals from
a plant species of each of the following genera of herbs:
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia, and one or more
chemotherapeutic compounds is administered to a mammal to enhance
the therapeutic effectiveness of chemotherapeutic compound. In
another embodiment, the materials or chemicals from a plant species
is in a form of a herbal composition comprising Scutellaria,
Glycyrrhiza, Ziziphus and Paeonia. In yet another embodiment, the
herbal composition consists essentially of Scutellaria,
Glycyrrhiza, Ziziphus and Paeonia.
[0026] In one embodiment of the invention, a composition comprising
a pharmaceutically acceptable carrier, materials or chemicals from
a plant species of each of the following genera of herbs:
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia, and one or more
chemotherapeutic compounds is administered to a mammal to enhance
the antitumor activity of a chemotherapeutic compound. In another
embodiment, the materials or chemicals from a plant species is in a
form of a herbal composition comprising Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia. In yet another embodiment, the herbal
composition consists essentially of Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia.
[0027] In one embodiment of the invention, a therapeutically
effective amount of a composition comprising a pharmaceutically
acceptable carrier, materials or chemicals from a plant species of
each of the following genera of herbs: Scutellaria, Glycyrrhiza,
Ziziphus and Paeonia, and one or more chemotherapeutic compounds is
administered to a mammal to treat tumors. In another embodiment,
the materials or chemicals from a plant species is in a form of a
herbal composition comprising Scutellaria, Glycyrrhiza, Ziziphus
and Paeonia. In yet another embodiment, the herbal composition
consists essentially of Scutellaria, Glycyrrhiza, Ziziphus and
Paeonia.
[0028] In one embodiment of the invention, a composition comprising
a pharmaceutically acceptable carrier, materials or chemicals from
a plant species of each of the following genera of herbs:
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia, and one or more
chemotherapeutic compounds is administered to a mammal to inhibit
the growth of tumors in mammals. In another embodiment, the
materials or chemicals from a plant species is in a form of a
herbal composition comprising Scutellaria, Glycyrrhiza, Ziziphus
and Paeonia. In yet another embodiment, the herbal composition
consists essentially of Scutellaria, Glycyrrhiza, Ziziphus and
Paeonia.
[0029] In one embodiment of the invention, a composition comprising
a pharmaceutically acceptable carrier, materials or chemicals from
a plant species of each of the following genera of herbs:
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia, and one or more
chemotherapeutic compounds is used to inhibit the growth of tumors.
In another embodiment, the materials or chemicals from a plant
species is in a form of a herbal composition comprising
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia. In yet another
embodiment, the herbal composition consists essentially of
Scutellaria, Glycyrrhiza, Ziziphus and Paeonia. In one embodiment,
the tumors are present in a mammal or in vitro cells.
[0030] In one embodiment, the present invention provides a method
of improving the quality of life of a mammal undergoing
chemotherapy. The method comprises administering a therapeutically
effective amount of one or more chemotherapeutic compounds and a
composition comprising: i) a pharmaceutically acceptable carrier;
ii) materials or chemicals from a plant species of each of the
following genera of herbs: Scutellaria, Glycyrrhiza, Ziziphus and
Paeonia; and iii) one or more chemotherapeutic compounds. In
another embodiment, the materials or chemicals from a plant species
is in a form of a herbal composition comprising Scutellaria,
Glycyrrhiza, Ziziphus and Paeonia. In yet another embodiment, the
herbal composition consists essentially of Scutellaria,
Glycyrrhiza, Ziziphus and Paeonia. Preferably, the mammal as
referenced herein is a human.
[0031] The above-referenced chemotherapeutic agents or compounds,
genera of herbs, and other terms and phrases have been described
and defined with details in the following patent applications and
patent: U.S. patent application Ser. No. 09/522,055 filed Mar. 9,
2000; International Application No. PCT/US2001/007353 filed Mar. 8,
2001; U.S. patent application Ser. No. 10/220,876 filed Dec. 30,
2002 and issued as U.S. Pat. No. 7,025,993 on Apr. 11, 2006; U.S.
Provisional Patent Application Ser. No. 60/625,943 filed Nov. 9,
2004; U.S. patent application Ser. No. 11/100,433 filed Apr. 7,
2005; and International Application No. PCT/US2005/040605 filed
Nov. 9, 2005, the content of which are herein incorporated by
reference in their entirety for all purposes.
EXAMPLES
Materials and Methods
[0032] Drug: Sorafenib (Nexavar) was purchased from Bayer
HealthCare (Leverkusen, Germany). Capecitabine (Xeloda.RTM., CAP)
was purchased from Roche Laboratories Inc. (Nutley, N.J.). The
clinical drug substance of PHY906 (PHY906-6, FDA 165542) with 10%
excipient was prepared by Sun Ten Pharmaceutical, Inc. (Taipei,
Taiwan). The PHY906 formula is composed of four herbs: Scutellaria
baicalensis Georgi, Paeonia lactiflora Pall., Ziziphus jujuba Mill
and Glycyrrhiza uralensis Fisch., with a relative weight ratio of
3:2:2:2.
[0033] Mice: Female BDF-1 mice with body weights between 16 and 20
g (4-6 weeks old) were purchased from Charles River Laboratories
(Wilmington, Mass.). Male NCr athymic nude mice with body weights
between 16 and 20 g (4-6 weeks old) were purchased from Taconic
Farms (Germantown, N.Y.).
[0034] Preparation of Sorafenib solution: Sorafenib (200 mg/tablet)
was dissolved in 5% gum arabic as the vehicle. The final solution
contains 30 mg/ml of sorafenib.
[0035] Preparation of capecitabine solution from capecitabine
tablet: Capecitabine (150 mg/tablet) was dissolved in 40 mM citrate
buffer (pH 6.0) containing 5% gum arabic as the vehicle. The final
solution contains 36 mg/ml of capecitabine.
[0036] Preparation of herbal extract from dry powder: The
preparation of the herbal extract followed SOP#HERB-001-PHY906.
Briefly, one gram of PHY906 dry powder, containing 10% starch
excipient, was added to 10 ml of 80.degree. C. H.sub.2O and
incubated at 80.degree. C. for 30 minutes. The supernatant was
separated from the debris by centrifugation (12000 rpm, 10 min) at
room temperature. The concentration of PHY906 supernatant was
calculated as 90 mg/ml of PHY906 (1 g/10 ml.times.0.9), based on
the dry weight of the dry powder. The herbal extract was stored at
room temperature and used within 24 hours. Any residual precipitant
that occurred upon standing was vortexed into a suspension and used
to treat the animals.
[0037] Tumor cells: The human hepatocellular carcinoma HepG2, human
PANC-1 pancreatic cancer, and mouse Colon 38 colorectal cancer cell
lines were purchased from the American Type Culture Collection
(Rockville, Md.). The HepG2 and Colon 38 cell lines were routinely
grown in MEME media while the PANC-1 cell line was grown in DMEM
media, supplemented with 10% fetal bovine serum (FBS). The cells
were implanted into the left flank of mice. Tumor transplantation
from mice to mice was performed when the tumor reached 1500-2000
mm.sup.3.
[0038] Mouse tumor model: Tumor cells (5.times.10.sup.6 cells in
0.1 ml PBS) were transplanted subcutaneously into the left flank of
mice. After 14 days, tumor ranging in size from 300-500 mm.sup.3
was selected for drug studies. The length and width of the each
tumor was measured with sliding calipers. The tumor size was
estimated according to the following formula:
Tumor size (mm.sup.3)=length (mm).times.width (mm).sup.2/2.
[0039] The studies were conducted and the animals were maintained
at the Yale Animal Facility.
[0040] Antitumor activity of chemotherapeutic agents in the
presence or absence of PHY906: A total of 20 tumor-bearing mice
were divided into 4 groups (N=5 mice/group): [0041] 1. Vehicle
[0042] 2. PHY906 [0043] 3. Chemotherapeutic agent [0044] 4.
PHY906+Chemotherapeutic agent
[0045] The first day of drug treatment was defined as day 1. PHY906
(500 mg/kg, bid) was administrated orally to the mice 30 min before
chemotherapeutic agents at the days indicated. Chemotherapeutic
agents were given either intraperitoneally or orally at the dose
and schedule indicated. The tumor size, body weight, and mortality
of the mice were monitored daily. Mice were sacrificed when the
tumor size reached 10% of body weight.
[0046] Immunohistochemistry: Formalin-fixed paraffin-embedded liver
tissue was freshly cut into slices of 4 mm. The sections were
mounted on Superfrost slides, dewaxed with xylene, and gradually
hydrated. Antigen retrieval was achieved by 0.05% citraconic
anhydride buffer (pH 7.4) at 94.degree. C. for 1 h. The primary
HIF-1.alpha., CD31 or VEGF antibodies was diluted 1:75 using
Tris-HCl buffer containing 1% BSA and 0.5% Tween-20. The primary
antibody was incubated at room temperature for 1 hour. As a
negative control, two slides were processed without primary
antibody. Detection took place by the conventional labeled
streptavidin-biotin method with alkaline phosphatase as the
reporting enzyme according to the manufacturer's instructions. DAB
(3,3'-diaminobenzidine tetrahydrochloride, purchased from
Sigma-Aldrich, St Louis, Mo.) served as chromogen. Afterwards, the
slides were briefly counterstained with hematoxylin and aqueously
mounted.
[0047] Statistical analysis and statistical power of the study
(24): A random effects model was employed to analyze data from
similar dosing animal trials. The PROC MIXED procedure in SAS was
used to take into account the correlation among observations
collected from the same mouse.
[0048] The following model was used to analyze the longitudinal
data:
y.sub.ijk=.mu.+.alpha.t.sub.k+.beta.(I.sub.Dt.sub.k)+.gamma.(I.sub.Pt.su-
b.k)+.delta.(I.sub.DI.sub.Pt.sub.k)+e.sub.ijk,
[0049] where y.sub.ijk is the relative tumor size of the jth
individual with the ith group (no treatment, drug alone, PHY906
alone, and drug+PHY906) at the kth time point, t.sub.k is the kth
time point, .alpha. is the baseline time effect (no treatment
group), I.sub.D and I.sub.P are indicator variables for having the
drug treatment and the PHY906 treatment, .beta. is the
drug-specific linear time effect, .gamma. is the PHY906-specific
linear time effect, .delta. is the drug-PHY906 synergistic linear
time effect, and e.sub.ijk is the residual (error) term. We assumed
that the errors from different individuals are independent, and
errors from the same individual at different time points follow the
autoregressive model, AR(1), to take into account the fact the
observations from the same individual within the same treatment
group are more correlated, and the responses from closer time
points are more correlated within the same individual. The PROC
MIXED in SAS 8.01 was used to perform the statistical analysis.
Results
(1) Sorafenib
Effect of PHY906 in Antitumor Activity of Sorafenib in Murine Colon
38 Bearing BDF-1 Mice
[0050] To determine whether the combinational use of PHY906 and
sorafenib in order to improve anti-tumor activity of sorafenib.
Sorafenib at dose of 30 mg/kg (BID, D1-14), in combination with a
fixed dose of PHY906 at 500 mg/kg (BID, D1-4 and 8-11), were
studied in BDF-1 mice bearing Colon 38 murine colorectal cancer. As
shown in FIG. 1, PHY906 significantly enhanced the antitumor
activity of sorafenib in Colon 38 bearing mice. Indeed, the tumor
growth was suppressed when mice received the combination of PHY906
and sorafenib.
Effect of PHY906 in (a) Antitumor Activity, (b) Blood Vessels, (c)
VEGF Level and (d) HIF-1.alpha. of Sorafenib in Human HepG2
Xenografts
[0051] PHY906 (500 mg/kg, BID, D1-4, 8-11 and 15-18) was tested on
the antitumor activity of sorafenib (30 mg/kg, BID, D1-20) in human
HepG2 bearing nude mice. As shown in FIG. 2, the combination of
sorafenib and PHY906 shrank the tumor size approximately 60% after
the first week of combination drug treatment while mice treated
with sorafenib alone did not have the shrinkage in tumor.
[0052] The immunohistochemical stainings on mouse liver indicate
that the integrity of tumor blood vessels are destroyed with the
combination treatment of PHY906 and sorafenib, as shown in FIG. 3.
The expressions of VEGF and HIF-1.alpha. are suppressed by the
combination treatment of PHY906 and sorafenib, as shown in FIGS. 4
and 5, respectively. The data also suggests that the combination
treatment of PHY906 and sorafenib affects the Fos/Juk
transcription.
(2) Capecitabine
Effect of PHY906 on the Antitumor Activity of Capecitabine in Human
Panc-1 Tumor-Bearing Nude Mice
[0053] PHY906 was previously found to potentiate the antitumor
activity of capecitabine in human HepG2 xenografts. An experiment
was therefore conducted to study whether PHY906 could enhance the
antitumor activity of capecitabine in human Panc-1 xenografts.
Total 20 NCr nude mice transplanted with Panc-1 human pancreatic
carcinoma cells were divided into 4 groups (N=5 mice/group): Group
(A) vehicle control; Group (B) treated with PHY906 (500 mg/kg, bid,
day 1-4, 8-11, 15-18, 22-25 and 29-32); Group (C) treated with
capecitabine (720 mg/kg, bid, day 1-7, 15-21, and 29-32); and Group
(D) treated with PHY906 (500 mg/kg, bid, days day 1-4, 8-11, 15-18,
22-25 and 29-32) plus capecitabine (720 mg/kg, bid, day 1-7, 15-21,
and 29-32). PHY906 was found to enhance the antitumor activity of
capecitabine, as shown in FIG. 6. A similar observation was found
with lower doses of capecitabine (data not shown).
[0054] All applications, patent, and publications referenced herein
are incorporated by reference to the same extent as if each
individual application, patent, and publication was specifically
and individually indicated to be incorporated by reference.
Specifically, the disclosures of WO 01/66123, WO 06/053049, U.S.
Pat. No. 7,025,993, US 2005/0196473, and US 2003/0211180 are
incorporated herein by reference in their entirety for all
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