U.S. patent application number 12/984042 was filed with the patent office on 2011-07-07 for anticancer formulation.
This patent application is currently assigned to National Dong Hwa University. Invention is credited to Tzyy-Wen Chiou, Horng-Jyh Harn, Shinn-Zong Lin.
Application Number | 20110165201 12/984042 |
Document ID | / |
Family ID | 43801814 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110165201 |
Kind Code |
A1 |
Chiou; Tzyy-Wen ; et
al. |
July 7, 2011 |
ANTICANCER FORMULATION
Abstract
This invention relates to a pharmaceutical formulation
containing z-butylidenephthalide and a polymer, e.g., a
polyanhydride. Also disclosed is use of this formulation to treat
tumor.
Inventors: |
Chiou; Tzyy-Wen; (Hualien,
TW) ; Harn; Horng-Jyh; (Taipei, TW) ; Lin;
Shinn-Zong; (Taichung, TW) |
Assignee: |
National Dong Hwa
University
Shoufeng
TW
|
Family ID: |
43801814 |
Appl. No.: |
12/984042 |
Filed: |
January 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61292311 |
Jan 5, 2010 |
|
|
|
Current U.S.
Class: |
424/400 ;
514/470 |
Current CPC
Class: |
A61K 31/343 20130101;
A61K 47/34 20130101; A61P 35/02 20180101; A61P 35/00 20180101; A61K
9/0024 20130101 |
Class at
Publication: |
424/400 ;
514/470 |
International
Class: |
A61K 31/343 20060101
A61K031/343; A61K 9/00 20060101 A61K009/00; A61P 35/00 20060101
A61P035/00 |
Claims
1. A pharmaceutical formulation comprising z-butylidenephthalide,
and a polymer admixed with the z-butylidenephthalide.
2. The pharmaceutical formulation of claim 1, wherein the polymer
is selected from the group consisting of poly(lactic-co-glycolic
acid), a chitosan, a collagen, a hydrogel, a polyanhydride, and a
mixture thereof
3. The pharmaceutical formulation of claim 2, wherein the
polyanhydride is prepared from bis(p-carboxyphenozy)propane,
bis(p-carboxyphenoxy)butane, bis(p-carboxyphenoxy) pentane,
bis(p-carboxyphenoxy)heptane, bis(p-carboxyphenoxy)hexane,
bis(p-carboxyphenoxy)octane, isophthalic acid, 1,4-phenylene
dipropionic acid, dodecanedioic acid, oxalic acid, malonic acid,
succinic acid, glutaric acid, adipic acid, pimelic acid, suberic
acid, azelaic acid, sebacic acid, phthalic acid, isophthalic acid,
terephthalic acid, or a mixture thereof.
4. The pharmaceutical formulation of claim 3, wherein the
polyanhydride is prepared from bis(p-carbodyphenozy)propane,
bis(p-carboxyphenoxy)hexane, isophthalic acid, 1,4-phenylene
dipropionic acid, dodecanedioic acid, sebacic acid, or a mixture
thereof
5. The pharmaceutical formulation of claim 4, wherein the
polyanhydride is prepared from a mixture of
bis(p-carboxyphenoxy)propane and sebacic acid.
6. The pharmaceutical formulation of claim 5, wherein the ratio
between the bis(p-carboxyphenoxy)propane and the sebacic acid is
1:2 to 1:10.
7. The pharmaceutical formulation of claim 6, wherein the ratio
between the bis(p-carboxyphenoxy)propane and the sebacic acid is
about 1:4 to 1:5.
8. The pharmaceutical formulation of claim 7, wherein the weight
percentage of the z-butylidenephthalide is 3%-20% of the
formulation
9. The pharmaceutical formulation of claim 8, wherein the weight
percentage of the z-butylidenephthalide is about 10% of the
formulation.
10. The pharmaceutical formulation of claim 1, wherein the
formulation is in form of powders, wafers, sheets, rods,
microspheres, nanospheres, paste, or glue.
11. The pharmaceutical formulation of claim 1, wherein the weight
percentage of the z-butylidenephthalide is 3%-20% of the
formulation.
12. A method of treating tumor comprising administering to a
subject in need thereof an effective amount of a pharmaceutical
formulation containing z-butylidenephthalide and a polymer admixed
with the z-butylidenephthalide.
13. The method of claim 12, wherein the polymer is selected from
the group consisting of poly(lactic-co-glycolic acid), a chitosan,
a collagen, a hydrogel, a polyanhydride, and a mixture thereof
14. The method of claim 12, wherein the tumor is glioblastoma
multiforme, lung cancer, hepatocellular carcinoma, colon cancer,
melanoma, breast cancer, neuroblastoma, teratoma, or human
leukemia.
15. The method of claim 13, wherein the polyanhydride is prepared
from bis(p-carbodyphenozy) propane, bis(p-carboxyphenoxy)hexane,
isophthalic acid, 1,4-phenylene dipropionic acid, dodecanedioic
acid, sebacic acid, or a mixture thereof
16. The method of claim 15, wherein the polyanhydride is prepared
from a mixture of bis(p-carboxyphenoxy)propane and sebacic
acid.
17. The method of claim 16, wherein the ratio between the
bis(p-carboxyphenoxy) propane and the sebacic acid is 1:2 to
1:10.
18. The method of claim 17, wherein the ratio between the
bis(p-carboxyphenoxy) propane and the sebacic acid is about 1:4 to
1:5.
19. The method of claim 12, wherein the weight percentage of the
z-butylidenephthalide is 3%-20% of the formulation.
20. The method of claim 19, wherein the weight percentage of the
z-butylidenephthalide is about 10% of the formulation.
21. The method of claim 12, wherein the pharmaceutical formulation
is subcutaneously implanted, interstitially implanted, or
intracranially implanted in the subject.
22. The method of claim 12, wherein the cancer is glioblastoma
multiforme.
23. The method of claim 22, wherein the z-butylidenephthalide
inhibits Ax1-mediated proliferation, migration or invasion of tumor
cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/292,311, filed Jan. 5, 2010, the content of
which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] n-Butylidenephthalide (Bdph) is a chemical compound isolated
from Angelica sinensis. It can be used to treat various tumors,
e.g., gliobastoma multiforme and breast cancer. See, e.g., Tsai et
al., Clin. Cancer Res. 2005, 11(9): 3475-3484 and Tsai, et al., J
Neurochem. 2006, 99(4): 1251-62. However, delivering
n-butylidenephthalide to the cancer site in a selective and
sustained manner is critical for its use in effective cancer
therapy. This is especially important for treating brain cancer,
where the drug is difficult to reach the disease area because of
the blood brain barrier. There is a need of developing effective
ways for delivering the drug.
SUMMARY
[0003] This invention is based on a discovery that a pharmaceutical
formulation, from which n-butylidenephthalide, in particular, the
Z-from (i.e. (Z)-n-butylidenephthalide, z-butylidenephthalide, and
z-Bdph), can be gradually released over a long period, e.g., more
than 30 days, and that z-Bdph, rather than E-from (i.e.,
(E)-n-butylidenephthalide, e-butylidenephthalide, and e-Bdph), has
antitumor effects.
[0004] In one aspect, this invention features a pharmaceutical
formulation, which contains (i) z-butylidenephthalide and (ii) a
polymer, which are admixed together.
[0005] The polymer can be poly(lactic-co-glycolic acid), a
chitosan, a collagen, a hydrogel, or a polyanhydride, e.g., a
polyanhydride prepared from bis(p-carboxyphenozy) propane,
bis(p-carboxyphenoxy)butane, bis(p-carboxyphenoxy)pentane,
bis(p-carboxyphenoxy)heptane, bis(p-carboxyphenoxy)hexane,
bis(p-carboxyphenoxy) octane, isophthalic acid, 1,4-phenylene
dipropionic acid, dodecanedioic acid, oxalic acid, malonic acid,
succinic acid, glutaric acid, adipic acid, pimelic acid, suberic
acid, azelaic acid, sebacic acid, phthalic acid, isophthalic acid,
terephthalic acid, or a mixture thereof.
[0006] An example of the formulation is a mixture of
z-butylidenephthalide and polyanhydride p(CPP-SA), which is
prepared from bis(p-carboxyphenoxy)propane (CPP) and the sebacic
acid (SA). In the polyanhydride, the ratio between the
bis(p-carboxyphenoxy) propane and the sebacic acid is preferably
1:2 to 1:10 (e.g., 1:4). The weight percentage of the
z-butylidenephthalide is 3%-50% (e.g., 3%-20%, 10%, and 15%) of the
formulation. The formulation can in form of powders, wafers,
sheets, rods, microspheres, nanospheres, paste, or glue.
[0007] In another aspect, this invention features use of the
above-described pharmaceutical formulation to treat tumor. Examples
of tumor to be treated include, but are limited to, glioblastoma
multiforme, lung cancer, hepatocellular carcinoma, colon cancer,
melanoma, breast cancer, neuroblastoma, teratoma, and human
leukemia.
[0008] Also within the scope of this invention is use of the
above-described formulation in manufacturing medicament useful for
treating tumor.
[0009] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0010] One aspect of this invention relates to a pharmaceutical
formulation containing n-butylidenephthalide, in particular, its z
form, z-butylidenephthalide, and a polymer. The formulation can be
used in inhibiting growth of tumors, such as glioblastoma
multiforme.
[0011] z-butylidenephthalide used to practice this invention is
commercially available, e.g., from Lancaster Synthesis Ltd. (UK).
It can also be isolated from a chloroform extract of Angelica
sinensis. See, e.g., Tsai et al., Clin. Cancer Res. 2005, 11(9):
3475-3484. The z-butylidenephthalide compound, either purchased or
isolated, can be further purified by flash column chromatography,
high performance liquid chromatography, crystallization, or any
other suitable methods.
[0012] The polymer used to practice this invention either is
commercially available or can be prepared by known methods in the
art. For example, one can reflux a diacid compound in acetic
anhydride to obtain a polyanhydride.
[0013] The polymer may be a copolymer. As an example, such a
copolymer can be prepared from two different polyanhydride moieties
using the melt polycondensation process. See, e.g., Domb et al.,
Journal of polymer science, 1987, 25: 3373-3386.
[0014] The obtained polymer can be purified by any suitable method
and characterized by NMR, MS, or FT-IR.
[0015] To prepare the formulation of this invention, one can mix
the z-butylidenephthalide and the polymer, e.g., a polyanhydride,
at the desired ratio (e.g., 10 parts by weight the
z-butylidenephthalide and 90 parts by weight the polyanhydride). As
another example, one can dissolve the butylidenephthalide and
polyanhydride in a solvent (e.g., methylene chloride) and then
remove the solvent to provide a dry powder.
[0016] The thus obtained mixture can be further processed into
various forms such as wafers, sheets, rods, microspheres,
nanospheres, paste, or glue. For example, one can use a mold to
compress the mixture into wafers.
[0017] The term "pharmaceutical formulation" is used herein to mean
a composition which (i) is suitable for administration to a human
being or other mammal or which can be treated, e.g. sterilized, to
make it suitable for such administration, and (ii) comprises at
least one drug (e.g., z-butylidenephthalide) and at least one of
the above-mentioned polymers. The formulation can be part or all of
any device that can deliver a drug, including pills, capsules,
gels, depots, medical implantable devices (e.g., stents, including
self-expanding stents, balloon-expandable stents, drug-eluting
stents and stent-grafts, grafts (e.g., aortic grafts), artificial
heart valves, cerebrospinal fluid shunts, pacemaker electrodes,
endocardial leads, bioerodable implants and the like), and
externally manipulated devices (e.g. drug devices and catheters,
including catheters which can release a drug, e.g. as a result of
heating the tip of the catheter). The pharmaceutical formulation
may also include one or more other additives, for example
pharmaceutically acceptable excipients, carriers, penetration
enhancers, stabilizers, buffers or other materials physically
associated with the drug and/or the polymer to enhance the
deliverability of the dosage form and/or the effectiveness of the
drug. The formulation may be, for example, a liquid, a suspension,
a solid (such as a tablet, pill, and capsule, including a
microcapsule), emulsion, micelle, ointment, gel, emulsion, depot
(including a subcutaneously implanted depot), or coating on an
implanted device, e.g. a stent or the like. The formulation can for
example be applied externally, e.g. as a patch, or a device applied
partly externally and partly implanted, or completely implanted or
injected subcutaneously.
[0018] The term "drug" means a material which is biologically
active in a human being or other mammal, locally and/or
systemically. Examples of drugs are disclosed in the Merck Index,
the Physicians Desk Reference, and in column 11, line 16, to column
12, line 58, of U.S. Pat. No. 6,297,337, and in paragraph 0045 of
US 2003/0224974, the entire disclosures of which are incorporated
by reference herein for all purposes. Drugs can for example be
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness, including vitamins and mineral
supplements; substances which affect the structure or the function
of a mammal; pro-drugs, which are substances which become
biologically active or more active after they have been placed in a
physiological environment; and metabolites of drugs. Examples of
diagnostic agents are imaging agents containing radioisotopes,
contrasting agents containing for example iodine, enzymes,
fluorescent substances and the like.
[0019] The formulation of this invention may also contain suitable
additives. These additives can be included in the formulation at
any stage of the preparation of the formulation. The desired
concentrations of the additives in the formulation for conferring
the intended effect, as recognized by those skilled in the art, can
be assayed using conventional methods.
[0020] The formulation of this invention, upon contact with fluid,
releases z-butylidenephthalide--an antitumor agent. Thus, this
invention also relates to a method of treating tumor by
administering an effective amount of the formulation to a subject
in need thereof. The butylidenephthalide in the formulation is
slowly and continuously released into the adjacent tissue with in a
certain period of time, e.g., 20, 30, 35, 40, 50, 60 days.
[0021] As used herein, the term "treating" or "treatment" is
defined as the administration of an effective amount of the
formulation to a subject, who has tumor, a symptom of tumor, a
disease or disorder secondary to tumor, or a predisposition toward
tumor, with the purpose to cure, alleviate, relieve, remedy, or
ameliorate the tumor, the symptom of the tumor, the disease or
disorder secondary to the tumor, or the predisposition toward the
tumor.
[0022] A "subject" refers to a human and a non-human animal.
Examples of a non-human animal include all vertebrates, e.g.,
mammals, such as non-human primates (particularly higher primates),
dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals,
such as birds, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model. A subject to be treated for a tumor, cancer, or other
cellular proliferative disorder can be identified by standard
diagnosing techniques for the disorder.
[0023] Tumor is a swelling or lesion formed by an abnormal growth
of cells. It can be benign tumor or malignant tumor (i.e., cancer).
Cancer refers to a class of diseases in which a group of cells
display uncontrolled growth, invasion, and sometimes metastasis.
Examples of cancer to be treated include, but are not limited to
glioblastoma multiforme, lung cancer, hepatocellular carcinoma,
colon cancer, melanoma, breast cancer, neuroblastoma, teratoma, and
human leukemia.
[0024] The term "an effective amount" refers to an amount of a
formulation or a compound which confers a therapeutic effect on the
subject to be treated. The treatment method can be performed in
vivo or ex vivo, alone or in conjunction with other drugs or
therapy. In an in vivo approach, a compound or a formulation is
administered to a subject. Generally, the compound or formulation
is prepared in a pharmaceutically-acceptable carrier (e.g.,
physiological saline) and administered orally or by intravenous
infusion, or injected or implanted subcutaneously, intramuscularly,
intrathecally, intraperitoneally, intrarectally, intravaginally,
intranasally, intragastrically, intratracheally, or
intrapulmonarily.
[0025] Preferably, the formulation can be subcutaneously,
intramuscularly, intravenously, interstitially or intracranially
implanted in a cancer patient. In one embodiment, the formulation,
in various forms, can be implanted to the cancer site or in its
proximity with or without the tumor tissue being removed.
[0026] The dosage required depends on the choice of the route of
administration; the nature of the formulation; the nature of the
patient's illness; the subject's size, weight, surface area, age,
and sex; other drugs being administered; and the judgment of the
attending physician. It can be adjusted by one skilled in the art,
e.g., a nutritionist, dietician, or treating physician, in
conjunction with the subject's response. Suitable dosages are in
the range of 0.01-100 mg/kg. Variations in the needed dosage are to
be expected in view of the variety of compounds available and the
different efficiencies of various routes of administration.
[0027] The formulation can be used together with surgery or
radiotherapy. It can also be used in combination with one or more
other chemotherapeutic agents. The chemotherapeutic agents may be,
for example, camptothecins such as topotecan, anthracycline
antibiotics such as doxorubicin, alkylating agents such as
cyclophosphamide, or antimicrotubule agents such as paclitaxel,
temozolomide, or carmustin.
[0028] Without further elaboration, it is believed that the above
description has adequately enabled the present invention. The
following examples are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. All of the publications cited herein are
hereby incorporated by reference in their entirety.
EXAMPLE 1
Synthesis of Polymers
[0029] SA monomer was recrystallized twice from alcohol. 2.7 g SA
monomer was refluxed in 60 ml acetic anhydride for 30 minutes
(mins) at 135-140.degree. C. under vacuum (10.sup.-4 torr). The
unreacted acetic anhydride was removed. The SA prepolymer was dried
under vacuum at 60.degree. C. and then dissolved in dry toluene.
The solution was added to a 1:1 v/v mixture of dry ethyl ether and
petroleum ether at a volume ratio of 1:10 and allowed to sit
overnight to precipitate out the SA prepolymer (10:1 v/v). After
the ethyl ether and petroleum ether were removed, the SA prepolymer
was dried under vacuum.
[0030] 3 g CPP monomers were refluxed with 50 ml acetic anhydride
for 30 mins at 150.degree. C. under vacuum (10.sup.-4 torr). After
cooling, the reaction mixture was filtered. The filtrate was
concentrated by removing some of unreacted acetic anhydride, and
the CPP prepolymer was crystallized at 0.degree. C. The remaining
unreacted acetic anhydride was removed. The CPP prepolymer was
washed with ether and dried under vacuum. DMF and dry ether (DMF:
dry ether=1:9) were sequentially added to the CPP prepolymer. After
about 12 hours, DMF and ether were removed and the CPP prepolymer
crystal was dried under vacuum again.
[0031] CPP prepolymer and SA prepolymer at a ratio of 20:80 were
charged into a glass tube (2.times.20 cm) and heated at 180.degree.
C. in an oil bath for 1 min. The pressure was reduced to 10.sup.-4
mmHg. The vacuum was eliminated at every 15 min throughout
polymerization. The tube was washed with dichloromethane and then
petroleum ether was added to precipitate out p(CPP-SA) copolymer,
which was washed with anhydrous ether and dried under vacuum.
[0032] The p(CPP-SA) copolymer was characterized by IR and .sup.1H
NMR. In the IR spectroscopy, the characteristic signal of anhydride
bond was observed at 1812.76 cm.sup.-1. In the .sup.1H NMR
spectroscopy, the characteristic signals of aromatic protons of CPP
were observed at 6.9-8.2 ppm, and the characteristic signal of
methylene protons of SA was measured at 1.3 ppm. Further, the ratio
of CPP and SA in the copolymer was identified as 1:4.about.1:5
according to characteristic peak intensity of CPP and SA in the
.sup.1H NMR spectroscopy.
[0033] p(CPP-SA) polymer was mixed with z-Bdph to provide a mixture
containing 3% or 10% by weight z-Bdph. A mixture containing 97%
p(CPP-SA) and 3% 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was
also prepared. Each mixture was dissolved in methylene chloride at
the concentration of 10% (w/v). The solution was dried under vacuum
for 72 h. The thus-obtained dry powder was compressed to form
z-Bdph p(CPP-SA) discs (100 mg/disc) using a stainless steel mold
(internal diameter, 13 mm) under light pressure from a Carver Press
at 200 psi as described in Walter et al., Cancer Res. 1994, 54(8):
2207-12; Leong et al., J Biomed Mater Res. 1985, 19(8): 941-55; and
Storm et al. J Neurooncol. 2002, 56(3): 209-17. BCNU p(CPP-SA)
discs of the same size were also prepared by compression
molding.
EXAMPLE 2
Release Kinetics
[0034] z-Bdph -p(CPP-SA) discs were placed in scintillation vials
having 1.0 ml of 0.1 M phosphate-buffered saline (pH 7.4) and 1.0
ml n-octanol and incubated at 37.degree. C. The solution was
replaced by fresh buffer at various time points. Absorption at the
wavelength of 310 nm was measured using a spectrophotometer to
determine the concentration of z-Bdph in the buffer as described in
Weingart et al., Int. J. Cancer. 1995, 62(5): 605-9. Sustained
release of z-Bdph was observed.
EXAMPLE 3
In Vitro Controlled Release and Cytotoxic Effects
[0035] Assays were conducted to examine the growth inhibitory
effects of p(CPP-SA)-10% z-Bdph on RG2 rat glioblastoma multiforme
(GBM) cells. RG2 cells were treated with 10% Bdph-wafer for 24 hrs.
Cell viability were determined by MTT assay. It was found that the
growth inhibitory effects of p(CPP-SA)-10% z-Bdph were 50% compared
with control. In addition, the morphology of GBM cells gradually
changed and detaching of the cells from the bottom of culture plate
was observed after treatment. Compared to untreated cells, most of
the detached GBM cells were apoptotic after p(CPP-SA)-10% z-Bdph
treatment.
[0036] Note that data were expressed as the mean.+-.SD or SE
(standard deviation and standard error, respectively) from three
independent experiments. Statistical significance was analyzed by
Student's t-test and Mantel-Cox test. The survival analysis was
performed using the Kaplan-Meier method. A P value of <0.05 was
considered significant.
EXAMPLE 4
Apoptotic Pathways And Nurr Translocattion Induced by
p(CPP-SA)-z-Bdph
[0037] To confirm the results of the oligodeoxynucleotide-based
microarray analysis, expression of the orphan receptors NOR-1,
Nurr1, and Nur77 was examined in z-Bdph-treated RG2 cells by
RT-PCR.
[0038] RG2 cells were incubated with IC.sub.50 concentration of
z-Bdph for various time periods (0, 0.5, 1, 3, and 6 h). After
incubation, cells were collected and total RNA isolated. Expression
of GADPH was used as an internal control.
[0039] After treatment with z-Bdph, the mRNA expression of Nurr77
was induced in the cells in a time-dependent manner. Nurr77 mRNA
expression was significantly induced from half hour after z-Bdph
treatment to 6 hours after the treatment. Nur77, which was highly
induced after z-Bdph treatment, has been implicated in growth
inhibition and apoptosis, suggesting that Nur77 induction could be
an early event of z-Bdph-induced apoptosis in GBM cells.
[0040] Whether translocation of Nur77 occurred in response to
z-Bdph was examined. DBTRG-05NG cells (human GBM cells) were
treated with Bdph (100 .mu.g/mL) for 24 hours, then immunostained
with anti-Nur77 antibody followed by corresponding
Rhodamine-conjugated anti-IgG secondary antibody. Simultaneously,
cells were stained with DAPI to display the nuclei. The fluorescent
images were visualized with a fluorescence microscope. The result
showed that, Nur77 was much more abundant in the nucleus than in
the cytosol. After treatment of z-Bdph for 24 hours, Nur77 was
translocated from the nucleus to the cytoplasm.
[0041] For further confirmation, cytosolic and nucleus fractions of
cells were examined by Western blot analysis. RG2 cells were plated
on 10 cm dishes and incubated to 90% confluence. The cells were
treated with Bdph (100 .mu.g/ml) for different time periods (0, 6,
12, 24 and 48 hours). The cells were harvested, and nuclear and
cytoplasmic fractions were isolated. Western blot analysis showed
that Nur77 was predominantly localized in the nucleus in the
absence of z-Bdph treatment.
[0042] Finally, signaling pathways involved in z-Bdph-induced Nur77
gene expression were investigated. RG2 cells were treated with Bdph
(100 .mu.g/m) for various time periods (0, 15, 30, 60, and 180
mins). Western blot analysis showed that JNK, AKT, ERK were
significantly phosphorylated after z-Bdph treatment for 1 hour.
Moreover, MTT assay results showed that cell viability increased
after the cells were pretreated with a pJNK inhibitor at 5-20 nM
and treated with z-Bdph.
EXAMPLE 5
Animal Studies
[0043] Male F344 rats (230-260 g) and male Foxn1 nu/nu mice (10-12
weeks) were obtained from National Laboratory Animal Center
(Taipei, Taiwan). All procedures were performed in compliance with
the standard operation procedures of the Laboratory Animal Center
of National Tau Hwa University (Hualien, Taiwan) and China Medical
University Hospital (Taichung, Taiwan). RG2 cells and DBTRG-05MG
cells were used in animal experiments to monitor the anti-tumor
activities of p(CPP-SA)-3% or 10% z-Bdph formulations and
p(CPP-SA)-3% BCNU.
[0044] Syngeneic F344 rats received subcutaneous back implants of
RG2 cells. Animals were treated by subcutaneous implant with
p(CPP-SA)-3%, p(CPP-SA)-10% z-Bdph formulations, p(CPP-SA)-3% BCNU,
or polymer alone at least 1.5 cm removed from the original
injection site after the tumor cell implantation.
[0045] In addition, Foxn1 nu/nu mice received subcutaneous
implantation of DBTRG-05MG cells, and subcutaneous implantation of
p(CPP-SA)-3%, p(CPP-SA)-10% z-Bdph formulations, p(CPP-SA)-3% BCNU,
or polymer alone at least 1.5 cm removed from the original
injection site after the tumor cell implantation.
[0046] Tumor sizes were measured by using a caliper and the volume
was calculated as L.times.H.times.X.times.0.5236. Animals were
sacrificed when the volumes of tumor exceeded 25 cm.sup.3 in rat
and 1000 mm.sup.3 in mice. That date was used to calculate the
final survival date for the rats and mice.
EXAMPLE 6
Therapeutic Effects of p(CPP-SA)-z-Bdph in Animal Model
[0047] RG2 cells (5.times.10.sup.6) were implanted subcutaneously
into the hind flank region of F344 rats. After five days of RG2
cell transplantation, the rats were treated subcutaneously with
p(CPP-SA)-3% z-Bdph, p(CPP-SA)-10% z-Bdph, p(CPP-SA) alone, or
p(CPP-SA)-3% BCNU. Significant tumor growth inhibition was observed
for the p(CPP-SA)-10% z-Bdph treated group, as compared with the
p(CPP-SA)-3% z-Bdph treated group, p(CPP-SA)-3% BCNU treated group,
and p(CPP-SA) treated group (p<0.005).
[0048] Average tumor sizes at day 30 were 2070.79.+-.784.90
mm.sup.3 for the control (untreated) group, 1586.30.+-.243.69
mm.sup.3 in the p(CPP-SA) treated group, 346.71.+-.521.68 mm.sup.3
in the p(CPP-SA)-3% z-Bdph treated group, 87.89.+-.167.44 mm.sup.3
in the p(CPP-SA)-10% z-Bdph treated group, and 357.48.+-.27.30
mm.sup.3 in the p(CPP-SA)-3% BCNU treated group.
[0049] The immunohistochemical stain of ki-67, indicating cell
proliferation, showed a significant decrease of cell proliferation
in the p(CPP-SA)-10% z-Bdph treated group. In addition, the
immunohistochemical stain of caspase, indicating cell apoptosis,
showed a significant increase of cell apoptosis in the
p(CPP-SA)-10% z-Bdph treated group. Finally, no drug related
toxicities, as evaluated by the body weights and the histological
analyses of various organs, were observed in the animals in the
p(CPP-SA)-10% z-Bdph treated group. In contrast, significant body
weight loss was observed in the p(CPP-SA)-3% BCNU treated
group.
EXAMPLE 7
Therapeutic Effects of p(CPP-SA)-z-Bdph in Xenograft Tumor
Growth
[0050] Foxn1 nude mice were inoculated with human DBTRG-05MG cells
(2.times.10.sup.6) and implanted with p(CPP-SA)-z-Bdph (0%, 3% 10%)
at day 5. Significant suppressions of tumor growth in the 3% and
10% z-Bdph-wafer treated groups was observed. The mean values of
tumor sizes at day 39 were 1098.46.+-.170.11 in the control group,
605.8.+-.98.8 mm.sup.3 in p(CPP-SA)-3% z-Bdph treated group, and
504.4.+-.38.9 mm.sup.3 in p(CPP-SA)-10% z-Bdph treated group
(p<0.05).
EXAMPLE 8
z-Bdph Inhibits Migration and Invasion of Human Glioblastoma
Multiformis
[0051] The invasion of DBTRG-05MG cells was examined using a
BioCoat matrigel invasion chamber system (BD Bioscience, Bedford,
Mass.). The BD matrigel Matrix is composed of laminin, collagen IV,
nidogen/entrctin, and proteoglycan on polyethylene terephthalate
(PET) membranes containing 8 .mu.m pores. In the in vitro migration
assay, low pore density PET track-etched membrane on Falcon culture
insert (BD Bioscience) was applied. The membrane was placed between
the upper and lower wells of a Matrigel chamber or Falcon culture
inserts. The cells were first resuspended in PRMI 1640 containing
10% fetal bovine serum and seeded into the upper wells of the
chamber (50,000 cells per well). After incubating for 24 hours at
37.degree. C., the cells that invaded or migrated through the
membrane were stained with Liu stain (Handsel Technologies, Inc.,
Taipei, Taiwan) and counted under a microscopy. Each experiment was
repeated thrice.
[0052] The above-described system was used to examine the effects
of z-Bdph on the migration and invasion of DBTRG-05MG cells (human
GBM). It was found that z-Bdph inhibited migration and invasion of
DBTRG-05MG cells in a dose dependent manner.
Example 9
z-Bdph Inhibits Tumor Migration and Invasion Via Repressing Ax1
[0053] Reverse transcriptase-polymerase chain reaction (RT-PCR) was
used to examine the effect of z-Bdph on the gene expression profile
in GBM cells to elucidate the possible mechanisms of z-Bdph's
inhibition of malignant brain tumor. It was found that the mRNA
expressions of Ax1 receptor tyrosine kinase (RTK) were
down-regulated in the presence of z-Bdph.
[0054] Further, the over-expression of Ax1 (i.e., by transfecting a
pcDNA3.0-Ax1 plasmid into the GBM cells) could reverse the
inhibitory effect of z-Bdph on Ax1 mediated proliferation,
migration and invasion of the GBM cells.
[0055] Western Blot assays were also carried out to examine the Ax1
protein level of the GBM cells in the presence of z-Bdph. The
results show that the Ax1 protein level was reduced.
[0056] It is now well established that protein tyrosine kinases
play an important role in the regulation of cellular proliferation
and differentiation and in the genesis of many neoplasias including
human glioma. Ax1 was reported to be involved in tumor migration
and invasion. The above results suggested that z-Bdph inhibited the
protein expression of Ax1 receptor tyrosine kinase and thereby
inhibited migration and invasion of the GBM cells.
OTHER EMBODIMENTS
[0057] All of the features disclosed in this specification may be
combined in any combination. An alternative feature serving the
same, equivalent, or similar purpose may replace each feature
disclosed in this specification. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0058] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the scope of the following claims.
* * * * *