U.S. patent application number 13/559737 was filed with the patent office on 2012-11-22 for treatment of lung cancer.
This patent application is currently assigned to RAMOT AT TEL-AVIV UNIVERSITY LTD.. Invention is credited to Roni Haklai, Yoel Kloog, Adi Zundelevich.
Application Number | 20120294957 13/559737 |
Document ID | / |
Family ID | 39203260 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120294957 |
Kind Code |
A1 |
Kloog; Yoel ; et
al. |
November 22, 2012 |
TREATMENT OF LUNG CANCER
Abstract
Disclosed are methods of treating lung cancer by administering
to a human in need thereof effective amounts of FTS, or various
analogs thereof, or a pharmaceutically acceptable salt thereof,
optionally, in combination with a chemotherapeutic agent.
Chemotherapeutic agents, and combinations thereof, for use with
FTS, its analogs, or its salts are also disclosed.
Inventors: |
Kloog; Yoel; (Herzliya,
IL) ; Zundelevich; Adi; (Kiryat Ono, IL) ;
Haklai; Roni; (Ramat Gan, IL) |
Assignee: |
RAMOT AT TEL-AVIV UNIVERSITY
LTD.
Tel Aviv
IL
|
Family ID: |
39203260 |
Appl. No.: |
13/559737 |
Filed: |
July 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12519578 |
Nov 13, 2009 |
8232253 |
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PCT/IL2007/001556 |
Dec 17, 2007 |
|
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13559737 |
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60875915 |
Dec 19, 2006 |
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Current U.S.
Class: |
424/649 ;
514/110; 514/265.1; 514/27; 514/283; 514/34; 514/449; 514/492;
514/568 |
Current CPC
Class: |
A61K 31/282 20130101;
A61K 31/519 20130101; A61K 31/555 20130101; A61K 31/555 20130101;
A61K 31/704 20130101; A61K 31/337 20130101; A61P 35/00 20180101;
A61K 31/7068 20130101; A61K 31/7068 20130101; A61K 31/282 20130101;
A61K 31/704 20130101; A61K 31/192 20130101; A61K 31/192 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 31/337 20130101 |
Class at
Publication: |
424/649 ;
514/568; 514/283; 514/34; 514/449; 514/492; 514/265.1; 514/27;
514/110 |
International
Class: |
A61K 31/192 20060101
A61K031/192; A61K 31/4745 20060101 A61K031/4745; A61K 31/704
20060101 A61K031/704; A61K 31/664 20060101 A61K031/664; A61K 33/24
20060101 A61K033/24; A61K 31/282 20060101 A61K031/282; A61K 31/519
20060101 A61K031/519; A61K 31/7048 20060101 A61K031/7048; A61P
35/00 20060101 A61P035/00; A61K 31/337 20060101 A61K031/337 |
Claims
1. A method of treating a human afflicted with lung cancer,
comprising administering to the human an effective amount of FTS or
an analog thereof as represented by the formula: ##STR00004##
wherein R.sup.1 represents farnesyl or geranyl-geranyl; R.sup.2
represents the groups COOR.sup.7, CONR.sup.7R.sup.8, wherein
R.sup.7 and R.sup.8 are each independently hydrogen, alkyl or
alkenyl, and COOM wherein M is a cation; R.sup.3, R.sup.4, R.sup.5
and R.sup.6 are each independently hydrogen, alkyl, alkenyl,
alkoxy, halo, trifluoromethyl, trifluoromethoxy, or alkylmercapto;
and X represents S; or a pharmaceutically acceptable salt thereof,
and a chemotherapeutic agent.
2. The method of claim 1, wherein the human afflicted with lung
cancer is administered FTS.
3. The method of claim 1, wherein the human afflicted with lung
cancer is administered an analog of FTS which is GGTS.
4. The method of claim 1, wherein FTS or its analog or a
pharmaceutically acceptable salt thereof is administered
orally.
5. The method of claim 1, wherein the chemotherapeutic agent is
administered intravenously.
6. The method of claim 1, wherein the chemotherapeutic agent is
vinorelbine.
7. The method of claim 1, wherein the chemotherapeutic agent is
doxorubicin.
8. The method of claim 1, wherein the chemotherapeutic agent is
paclitaxel.
9. The method of claim 1, wherein the chemotherapeutic agent is
docetaxel.
10. The method of claim 1, wherein the chemotherapeutic agent is a
platinum based drug or an analog thereof.
11. The method of claim 10, wherein the platinum based drug is
cisplatin.
12. The method of claim 10, wherein the platinum based drug is
carboplatin.
13. The method of claim 1, wherein the chemotherapeutic agent is
pemetrexed.
14. The method of claim 1, wherein the chemotherapeutic agent is
doxorubicin.
15. The method of claim 1, wherein the chemotherapeutic agent is
etoposide.
16. The method of claim 1, wherein the chemotherapeutic agent is
topotecan.
17. The method of claim 1, wherein the chemotherapeutic agent is
vinblastine.
18. The method of claim 1, wherein the chemotherapeutic agent is
vindesine.
19. The method of claim 1, wherein the chemotherapeutic agent is
ifosfamide.
20. The method of claim 1, wherein the chemotherapeutic agent is
mitomycin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 12/519,578, filed on Jun. 17, 2009, which
application is a national phase entry under 35 U.S.C. .sctn.371 of
International Application No. PCT/IL2007/001556, filed Dec. 17,
2007, which claims priority from U.S. Provisional Patent
Application No. 60/875,915, filed Dec. 19, 2006, all of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Lung cancer is the leading cause of cancer-related deaths in
the world [Greenlee et al., CA Cancer J Clin 51:15-36 (2001)]. Only
one in ten patients diagnosed with this disease will survive the
next five years. Although lung cancer was previously an illness
that affected predominately men, the lung cancer rate for women has
been increasing in the last few decades, which has been attributed
to the rising ratio of female to male smokers. More women die of
lung cancer than any other cancer, including breast cancer, ovarian
cancer and uterine cancers combined. [American Cancer Society.
Cancer Facts and FIGS. 2006. Atlanta: American Cancer Society
(2006)]. Despite advances in surgery, chemotherapy, and radiation
therapy, survival rates have barely changed in the last decade, and
long-term survival remains dramatically poor.
[0003] Lung cancers can arise in any part of the lung. Ninety to
95% of cancers of the lung are thought to arise from the
epithelial, or lining cells of the larger and smaller airways
(bronchi and bronchioles); for this reason lung cancers are
sometimes called bronchogenic carcinomas. Cancers can also arise
from the pleura (the thin layer of tissue that surrounds the
lungs), called mesotheliomas, or rarely from supporting tissues
within the lungs, for example, blood vessels.
[0004] It has been established that lung cancer arises as a
consequence of the accumulation of multiple genetic changes
involving critical genes controlling cell motility, proliferation,
differentiation, and apoptosis. [Sekido et al., Biochimica et
Biophysica Acta 1378:F21-F59 (1998)].
[0005] According to the American Cancer Society, there are two
major types of lung cancer: small cell lung cancer (SCLC) and
non-small cell lung cancer (NSCLC). SCLC comprises about 15% of all
cancers. NSCLC, however, comprises about 85% of all lung cancers
and is divided into three distinct sub-types: squamous cell
carcinoma (about 25-30% of the cases), large cell carcinomas (about
10-15%), and adenocarcinomas (about 40%). The cells in these
sub-types differ in size, shape, and chemical make-up. These lung
cancers are inclusive of bronchogenic carcinoma, bronchial
carcinoids, chondromatous hamartoma, solitary pulmonary nodules,
pulmonary sarcomas, undifferentiated small cell carcinoma,
undifferentiated large cell carcinoma, and bronchioloalveolar
carcinomas.
[0006] Current research indicates that the factor with the greatest
impact on risk of lung cancer is long-term exposure to inhaled
carcinogens. The most common means of such exposure is tobacco
smoke.
[0007] Treatment and prognosis depend upon the histological type of
cancer and the stage (degree of spread). Possible treatment
modalities include surgery, chemotherapy, and/or radiotherapy.
SUMMARY OF THE INVENTION
[0008] A first aspect of the present invention is directed to a
method of treating lung cancer. The method comprises administering
to a human in need thereof an effective amount of
S-farnesylthiosalicylic acid (FTS) or an analog thereof, or a
pharmaceutically acceptable salt thereof.
[0009] Another aspect of the present invention is directed to a
method of treating lung cancer. The method comprises administering
to a human in need thereof effective amounts of
S-farnesylthiosalicylic acid (FTS) or an analog thereof, or a
pharmaceutically acceptable salt thereof, and a chemotherapeutic
agent.
[0010] The results of a first set of experiments described herein
showed that in five human cell lines commonly used in the study of
lung cancer [non-small cell lung carcinoma cell lines (NSCLC), a
human lung squamous cell carcinoma cell line, and a lung epidermoid
carcinoma cell line)], FTS inhibited cancer cell growth.
[0011] The results of a further set of experiments described herein
showed that in a human lung carcinoma A549 cell line, FTS reversed
the transformed morphology of the cells, altered the cytoskeletal
organization of the cells, and inhibited the anchorage-independent
growth of cancer cell colonies.
[0012] The results of another set of experiments described herein
showed that the combined treatment of FTS with a chemotherapeutic
agent in vitro caused greater cell death with both drugs than
treatment with either drug alone in a human lung epithelial
carcinoma A549 cell line.
[0013] The results of an additional set of experiments described
herein showed that administering FTS i.p. to a lung cancer cell
nude mouse model inhibited A549 and HTB-58 (SK-MES-1) tumor cell
growth.
[0014] Yet another set of experiments described herein showed that
the combination of FTS with a chemotherapeutic agent in vivo caused
greater cell death with the combined treatment than with either
drug alone in a nude mouse model.
[0015] The results of another set of experiments described herein
showed that in four human lung cancer (NSCLC) cell lines (H1734,
H2030, H1975, and H3255) FTS sensitized the cells resulting in cell
death.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a bar graph illustrating the inhibition of BrdU
into the DNA of A549 cells (NSCLC) after incubation of the
FTS-treated cells (75 .mu.M) for 48 h, expressed as a percentage of
control.
[0017] FIG. 2 are photomicrograph images of vehicle-treated (left)
and FTS-treated (right) A549 cells (NSCLC) and further depicts the
reduction in number of FTS-treated cells.
[0018] FIG. 3 is a bar graph illustrating the dose dependent
inhibition of A549 cell (NSCLC) growth at increasing concentrations
of FTS (.mu.M), expressed as a percentage of control.
[0019] FIG. 4 illustrates the results of a FACS analysis describing
FTS induced cell-cycle arrest in A549 cells (NSCLC).
[0020] FIG. 5 is a bar graph illustrating the dose dependent
inhibition of H-1299 cells (NSCLC) at increasing concentrations of
FTS (.mu.M) as determined by direct cell counting.
[0021] FIG. 6 is a bar graph illustrating the dose dependent
inhibition of lung squamous cell carcinoma cell line HTB-58
(SK-MES-1) cells at increasing concentrations of FTS (.mu.M) as
determined by direct cell counting.
[0022] FIG. 7 is a bar graph illustrating the dose dependent
inhibition of H23 cells (NSCLC) at increasing concentrations of FTS
(.mu.M) as determined by direct cell counting.
[0023] FIG. 8 is a bar graph illustrating the dose dependent
inhibition of HTB54 lung epidermoid carcinoma cells at increasing
concentrations of FTS (.mu.M) as determined by direct cell
counting.
[0024] FIG. 9 is a table summarizing the half maximal inhibitory
concentration (IC.sub.50) of FTS (.mu.M) in each of the human lung
cancer cell lines [A549, H23, HTB54, H-1299, HTB-58
(SK-MES-1)].
[0025] FIG. 10 is a series of six fluorescent microscopic images
illustrating FTS-induced alterations in stress fiber (F-Actin) and
focal adhesion (.alpha.-Vinculin) formation on the cytoskeleton of
A549 cells (NSCLC).
[0026] FIGS. 11A-11C are typical immunoblots and quantitative
analyses of the results (means.+-.SD of four experiments), as
determined by densitometry and normalized to the level of
expression of each protein. (A) illustrates the reduction in levels
of K-Ras-GTP (upper panels) and of phospho-ERK and phospho-Akt
(lower panels) by FTS. (B) illustrates the unaffected levels of
Rac1-GTP by FTS. (C) illustrates the induced increase in RhoA-GTP
by FTS (*P<0.05 compared to vehicle-treated control).
[0027] FIGS. 12A-12B illustrates the inhibition of the
anchorage-independent growth or transformation of A549 cells
(NSCLC) in soft agar by FTS. Photomicrograph images (A) illustrate
the DMSO-treated (control) cells and colony formation before and
after treatment with FTS (50 .mu.M and 100 .mu.M). The bar graph
(B) illustrates the inhibition of A549 cell colony formation at
increasing concentrations of FTS (0 .mu.M, 50 .mu.M, and 100
.mu.M).
[0028] FIGS. 13A-13D are bar graphs illustrating (A) the effects of
the combination of FTS (40 .mu.M) and gemcitabine (0, 100, and 200
nM) on A549 cell (NSCLC) death; (B) the effects of the combination
of FTS (40 .mu.M) and doxorubicine (0, 50, and 100 nM) on A549 cell
(NSCLC) death; (C) the effects of the combination of FTS (40 .mu.M)
and cisplatin (0, 5.0, and 10.0 nM) on A549 cell (NSCLC) death; (D)
the effects of the combination of FTS (40 .mu.M) and paclitaxel (0,
2.5, and 5.0) on A549 cell (NSCLC) death.
[0029] FIGS. 14A-14D are bar graphs illustrating (A) the effects of
i.p. administration of FTS alone (10 mg/kg) in A549-cell-implanted
nude mouse models; (B) the effects of i.p. administration of FTS
alone (10 mg/kg) in HTB58-cell-implanted nude mouse models; (C) the
effects of oral administration of FTS alone (50 mg/kg) in
A549-cell-implanted nude mouse models; and (D) the effects of oral
administration of FTS alone (60 mg/kg), the effects of oral
administration of gemcitabine alone, and the combined effects of
oral administration of FTS and gemcitabine in A549-cell-implanted
nude mouse models.
[0030] FIG. 15 is a graph illustrating the effects of increasing
concentrations of FTS on human NSCLC cell lines H1734 and H2030
(KRAS mutations) and H1975 and H3255 (EGFR mutations).
DETAILED DESCRIPTION
[0031] Ras proteins act as on-off switches that regulate
signal-transduction pathways controlling cell growth,
differentiation, and survival. [Reuther, G. W., Der, C. J., Curr
Opin Cell Biol 12:157-65 (2000)]. They are anchored to the inner
leaflet of the plasma membrane, where activation of cell-surface
receptors, such as receptor tyrosine kinase, induces the exchange
of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on
Ras and the conversion of inactive Ras-GDP to active Ras-GTP.
[Scheffzek, K., Ahmadian, M. R., Kabsch, W., et al. Science
277:333-7 (1997)]. The active Ras protein promotes oncogenesis
through activation of multiple Ras effectors that contribute to
deregulated cell growth, differentiation, and increased survival,
migration and invasion. [See, e.g., Downward, J., Nat. Rev. Cancer
3:11-22 (2003); Shields, J. M., et al., Trends Cell Biol 10:147-541
(2000); and Mitin, N., et al., Curr Biol 15:R563-74 (2005)].
[0032] FTS is a potent Ras inhibitor that acts in a rather specific
manner on the active, GTP-bound forms of H-, N-, and K-Ras
proteins. [Weisz, B., Giehl, K., Gana-Weisz, M., Egozi, Y.,
Ben-Baruch, G., Marciano, D., Gierschik, P., Kloog, Y., Oncogene
18:2579-2588 (1999); Gana-Weisz, M., Halaschek-Wiener, J., Jansen,
B., Elad, G., Haklai, R., Kloog, Y., Clin. Cancer Res. 8:555-65
(2002)]. FTS competes with Ras-GTP for binding to specific
saturable binding sites in the plasma membrane, resulting in
mislocalization of active Ras and facilitating Ras degradation.
[Haklai, et al., Biochemistry 37(5):1306-14 (1998)]. This
competitive inhibition prevents active Ras from interacting with
its prominent downstream effectors and results in reversal of the
transformed phenotype in transformed cells that harbor activated
Ras. As a consequence, Ras-dependent cell growth and transforming
activities, both in vitro and in vivo, are strongly inhibited by
FTS. [Weisz, B., et al., supra.; Gana-Weisz, M., et al.,
supra.].
[0033] FTS and its analogs useful in the present invention are
represented by formula I:
##STR00001##
wherein [0034] R.sup.1 represents farnesyl, geranyl or
geranyl-geranyl;
[0035] R.sup.2 is COOR.sup.7, or CONR.sup.7R.sup.8, wherein R.sup.7
and R.sup.8 are each independently hydrogen, alkyl or alkenyl;
[0036] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently
hydrogen, alkyl, alkenyl, alkoxy, halo, trifluoromethyl,
trifluoromethoxy, or alkylmercapto; and [0037] X represents S.
[0038] The structure of FTS is as follows:
##STR00002##
[0039] FTS analogs embraced by formula I, and which may be suitable
for use in the present invention, include 5-fluoro-FTS,
5-chloro-FTS, 4-chloro-FTS, S-farnesyl-thiosalicylic acid methyl
ester (FTSME), and S-geranyl,geranyl-thiosalicylic acid (GGTS).
Structures of these compounds are set forth below.
##STR00003##
[0040] In some embodiments, GGTS is administered in an amount
effective to treat a patient diagnosed with lung cancer.
[0041] Methods for preparing the compounds of formula I are
disclosed in U.S. Pat. No. 5,705,528 (RE39,682) and U.S. Pat. No.
6,462,086. See also, Marom, M., Haklai, R., Ben-Baruch, G.,
Marciano, D., Egozi, Y., Kloog, Y., J Biol Chem 270:22263-70
(1995).
[0042] Pharmaceutically acceptable salts of the Ras antagonists of
formula I may be useful. These salts include, for example, sodium
and potassium salts. Other pharmaceutically acceptable salts may be
selected in accordance with standard techniques as described in
Berge, S. M., Bighley, L. D., and Monkhouse, D. C., J. of Pharm.
Sci. 66(1):1-19 (1977). In preferred embodiments, however, FTS and
its analogs are not administered in the form of a salt (i.e., they
are administered in non-salified form).
[0043] In some embodiments, treatment also includes administering
an anti-cancer therapy which includes, for example, chemotherapy,
radiation therapy, immunotherapy or gene therapy, and combinations
thereof.
[0044] In some embodiments, treatment includes administering a
chemotherapeutic agent to a patient diagnosed with lung cancer.
Chemotherapeutic agents are those medications that are used to
treat various forms of cancer and, particularly, lung cancer and
its various forms and associated manifestations. Generally, these
medications are given in a particular regimen over a period of
weeks. In some cases, combination chemotherapy may be recommended.
Methods of preparing and using chemotherapeutic agents are
well-known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy (21st Edition), Lippincott, Williams &
Wilkins, (2005).
[0045] Chemotherapeutic agents may be administered as the first
line of treatment or it may be started after a tumor is surgically
resected, for example. The agents may be administered by various
methods including, oral (by mouth), injection (intramuscular or
subcutaneous), intravenous (IV), intra-arterial (into the arteries,
intralesional (directly into the tumor), intraperitoneal (into the
peritoneal cavity), intrathecal (into the spinal fluid), and
topical (applied to the skin). A variety of factors, including the
overall health, size and weight of the patient, the patient's
tolerance to the treatment, and the type and stage of the cancer,
will determine the type of chemotherapy used and the mode and
duration of administration. Optimally, dosages for each of the
chemotherapeutic agents are prescribed in accordance with current
labeling instructions. Dosages, however, may be adjusted to satisfy
a patient's needs.
[0046] Examples of chemotherapeutic agents include, but are not
limited to, paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.),
cisplatin, carboplatin (Paraplatin.RTM.), gemcitabine hydrochloride
(Gemzar.RTM.), doxorubicin hydrochloride, etoposide
(Etopophos.RTM., Vepesid.RTM.), pemetrexed (Alimta.RTM.), topotecan
(Hycamtin.RTM.), vinblastine (Velbe.RTM.), Vindesine
(Eldisine.RTM.), vinorelbine (Navelbine.RTM.), ifosfamide
(Mitoxana.RTM.), and Mitomycin. Those most commonly used agents to
treat lung cancer include: gemcitabine, cisplatin, carboplatin,
vinorelbine, paclitaxel, docetaxel, and doxorubicin. These agents
may be given in combination, for example, vinorelbine and cisplatin
or carboplatin; gemcitabine with cisplatin or carboplatin or
paclitaxel; MIC (mitomycin, ifosfamide and cisplatin); MVP
(mitomycin, vinblastine and cisplatin); and EC (etoposide and
carboplatin).
[0047] In some embodiments, the chemotherapeutic agent is
paclitaxel (Taxol.RTM.)
[5,20-Epoxy-1,2,4,7,10,13-hexahydroxytax-11-en-9-one 4,10-diacetate
2-benzoate 13-ester with (2R,3S)--N-benzoyl-3-phenylisoserine], an
anti-neoplastic agent isolated from the bark of the Pacific yew
tree, Taxus brevifolia. Paclitaxel is an antimicrotubule
antineoplastic agent. Paclitaxel promotes microtubule assembly by
enhancing the polymerisation of tubulin, the protein subunit of
spindle microtubules, even in the absence of the mediators normally
required for microtubule assembly (e.g., guanosine triphosphate
(GTP)), thereby inducing the formation of stable, nonfunctional
microtubules. It is a colorless to slightly yellow viscous
solution.
[0048] In one example, combination chemotherapy using Taxol.RTM.
and cisplatin is indicated. The recommended regimen, given every 3
weeks, is Taxol.RTM. administered intravenously over 24 hours at a
dose of 135 mg/m.sup.2 followed by cisplatin at 75 mg/m.sup.2.
[0049] In some embodiments, the chemotherapeutic agent is docetaxel
(Taxotere.RTM.) [(2R,3S)--N-carboxy-3-phenylisoserine,N-tert-butyl
ester, 13-ester with
5.beta.-20-epoxy-1,2.alpha.,4,7.beta.,10.beta.,13.alpha.-hexahydroxytax-1-
1-en-9-one 4-acetate 2-benzoate, trihydrate], an antineoplastic
agent belonging to the taxoid family. It is prepared by
semisynthesis beginning with a precursor extracted from the
renewable needle biomass of yew plants. Docetaxel differs from
paclitaxel at two positions in its chemical structure. It has a
hydroxyl functional group on carbon 10, whereas paclitaxel has an
acetate ester and a tert-butyl substitution exists on the
phenylpropionate side chain. The carbon 10 functional group change
causes docetaxel to be more lipid soluble than paclitaxel. [Clarke,
S. J., Rivory, L. P., Clin Pharmacokinet 36(2):99-114 (1999)]. The
main mode of therapeutic action of docetaxel is the suppression of
microtubule dynamic assembly and disassembly. [Lyseng-Williamson,
K. A., Fenton, C., Drugs 65(17):2513-31 (2005); Yvon, A. C.,
Wadsworth, P., Jordan, M. A., The American Society for Cell Biology
10:947-959 (1999)]. The docetaxel injection concentrate is a clear
yellow to brownish-yellow viscous solution.
[0050] When used as a single agent therapy, a recommended dose
regimen of docetaxel for patients is 75 mg/m.sup.2 administered
intravenously over 1 hour every 3 weeks.
[0051] In some embodiments, the chemotherapeutic agent is a
platinum-based drug. The platinum-based drugs useful in the
practice of the present invention include cisplatin
[cis-diamminedichloroplatinum(II)] and its analogs, e.g.,
carboplatin [diammine(1,1-cyclobutanedicarboxylato)-platinum(II)].
These drugs are known to inflict damage on cellular nucleic acids,
including DNA. Cisplatin acts by cross-linking DNA in various
different ways, making it impossible for rapidly dividing cells to
duplicate their DNA for mitosis. The damaged DNA sets off DNA
repair mechanisms, which activate apoptosis when repair proves
impossible. Methods of preparing and using cisplatin as an
anti-cancer agent are described in, for example, U.S. Pat. No.
5,562,925 and Inorg Synth 7:239 (1963).
[0052] Carboplatin differs from cisplatin in that it has a closed
cyclobutane dicarboxylate moiety on its leaving group in contrast
to the readily leaving chloro groups. This results in very
different DNA binding kinetics. Methods of preparing and using
carboplatin as an anti-cancer agent are described in, for example,
U.S. Pat. No. 4,657,927 and Inorg Chem Acta 46:L15 (1980). Both
cisplatin and carboplatin are indicated for combination
chemotherapy.
[0053] A recommended dosage of cisplatin for adults and children
when used as single agent therapy is 50-100 mg/m.sup.2 as a single
IV infusion every 3-4 weeks, or 15-20 mg/m.sup.2 as a daily IV
infusion for 5 days every 3-4 weeks.
[0054] A recommended dosage of carboplatin in previously untreated
adult patients with normal kidney function is 400 mg/m.sup.2 as a
single IV dose administered by short-term (15 to 60 minutes)
infusion. Therapy should not be repeated until four weeks after the
previous carboplatin course, and/or until the neutrophil count is
at least 2000 cells/mm.sup.3 and the platelet count is at least
100,000 cells/mm.sup.3.
[0055] In some embodiments, the chemotherapeutic agent is
gemcitabine hydrochloride (Gemzar.RTM.)
[2'-deoxy-2',2'-difluorocytidine monohydrochloride]. The cytotoxic
effect of gemcitabine is attributed to a combination of two actions
of the diphosphate and the triphosphate nucleosides, which leads to
inhibition of DNA synthesis. It is a white powder, which forms a
clear solution. Gemcitabine, alone or in combination with
cisplatin, is indicated for the first line treatment of patients
with locally advanced or metastatic non-small cell lung cancer.
[See, e.g., FDA REVISED LABEL--VERSION 082598; 010603; 051904;
042005; 042605 for Gemzar.RTM.]. Combination chemotherapy for
treatment of lung cancer (NSCLC) with gemcitabine also includes
carboplatin [See, e.g., Tassarini, D., et al., Tumori 90:54-59
(2004)] and paclitaxel [See, e.g., Kosmidis, P., J Clin Oncol.
20(17):3578-85 (2002)].
[0056] A recommended adult dose of gemcitabine (Gemzar.RTM.) as a
single agent for lung cancer (NSCLC) is 1000 mg/m.sup.2, given by
30-minute intravenous infusion. This should be repeated once weekly
for three weeks, followed by a one-week rest period. This four-week
cycle is then repeated. Dosage reduction with each cycle or within
a cycle may be applied based upon the amount of toxicity
experienced by the patient.
[0057] A recommended adult dose of gemcitabine for combination
therapy using cisplatin, for example, has been investigated using
two dosing regimens. One regimen used a three-week schedule and the
other used a four-week schedule. The three-week schedule used
gemcitabine 1250 mg/m.sup.2, given by 30-minute intravenous
infusion, on days 1 and 8 of each 21-day cycle. Cisplatin should be
administered intravenously at 100 mg/m.sup.2 on day 1 after the
infusion of Gemzar.RTM.. Dosage reduction with each cycle or within
a cycle may be applied based upon the amount of toxicity
experienced by the patient.
[0058] The four-week schedule used gemcitabine 1000 mg/m.sup.2,
given by 30-minute intravenous infusion, on days 1, 8, and 15 of
each 28-day cycle. Cisplatin at a dose of 100 mg/m.sup.2 should be
administered intravenously after the infusion of Gemzar.RTM. on Day
1. Dosage reduction with each cycle or within a cycle may be
applied based upon the amount of toxicity experienced by the
patient.
[0059] In some embodiments, the chemotherapeutic agent is
doxorubicin hydrochloride. Doxorubicin [5,12-Naphthacenedione,
10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-te-
trahydro-6,8,11-trihydroxy-8-(hydroxylacetyl)-1-methoxy-,
hydrochloride (8S-cis)-] is a cytotoxic anthracycline antibiotic
isolated from cultures of Streptomyces peucetius var caesius (U.S.
Pat. No. 3,590,028). Doxorubicin intercalates the base pairs of the
DNA double helix, thus inhibiting nucleic acid synthesis,
inhibiting topoisomerase II, and producing oxygen radicals. It is a
red-orange, crystalline powder, which dissolves easily in
water.
[0060] When doxorubicin is administered as a single agent, a
recommended dose per cycle is 60-75 mg/m.sup.2 every three weeks.
The drug is generally given as a single dose per cycle; however, it
is possible to give the drug dosage per cycle in divided
administrations (e.g., day 1 through 3, or days 1 and 8).
Administration of doxorubicin in a weekly regimen has been shown to
be as effective as the tri-weekly schedule. The recommended weekly
dosage is 10-20 mg/m.sup.2. In combination chemotherapy, the
recommended dose per three-week cycle is in the 30-60 mg/m.sup.2
range.
[0061] The frequency of administration, dosage amounts, and the
duration of treatment of each of the active agents may be
determined depending on several factors, e.g., the overall health,
size and weight of the patient, the severity of the disease, the
patient's tolerance to the treatment, and the particular treatment
regimen being administered. For example, duration of treatment with
FTS or the combination of FTS and the chemotherapeutic agent may
last a day, a week, a year, or until remission of the disease is
achieved. Thus, relative timing of administration of these active
agents is not critical (e.g., FTS may be administered before,
during, and after treatment with the chemotherapeutic agent).
[0062] As used herein, the term "effective amount" refers to the
dosage(s) of FTS alone or in combination with the chemotherapeutic
agent that is effective for the treating, and thus includes dosage
amounts that ameliorate symptom(s) of the disorder and its
associated manifestations, diminish extent of disease, delay or
slow disease progression, or achieve partial or complete remission
or prolong survival. The average daily dose of FTS generally ranges
from about 50 mg to about 2000 mg, and in some embodiments, ranges
from about 200 mg to about 1200 mg. The average dose of paclitaxel
according to its prescribed regimen generally ranges from about 10
mg to about 300 mg, and in some embodiments about 10 mg to about
200 mg. The average dose of docetaxel generally ranges from about
10 mg to about 130 mg, and in some embodiments about 10 mg to about
100 mg. The average dose of cisplatin generally ranges from about
10 mg to about 170 mg, and in some embodiments about 10 mg to about
120 mg. The average dose for carboplatin generally ranges from
about 30 mg to about 620 mg, and in some embodiments about 30 mg to
about 400 mg. The average dose of gemcitabine generally ranges from
about 50 mg to about 1700 mg, and in some embodiments about 50 mg
to about 1000 mg. The average dose of doxorubicin generally ranges
from about 10 mg to about 130 mg, and in some embodiments about 10
mg to about 100 mg.
[0063] In some embodiments, FTS is administered on a daily basis,
e.g., each in single once-a-day or divided doses, while the
chemotherapeutic agent is administered in accordance with its
approved dosing schedule. In some embodiments, both drugs may be
administered at the same or at different times.
[0064] The methods of the present invention may be used for the
treatment of cancer in mammals, particularly humans. The actives
may be administered in accordance with standard methods. In
preferred embodiments, FTS is administered orally. In an oral
dosage form, the FTS is typically present in a range of about 50 mg
to about 500 mg, and in some embodiments, from about 100 mg to
about 300 mg.
[0065] In some embodiments, FTS may be administered by dosing
orally on a daily basis for three weeks, followed by a one-week
"off period", and repeating until remission is achieved. In another
embodiment, FTS may be administered by dosing twice daily and
continuing the treatment until remission is achieved. Parenteral
administration may also be suitable.
[0066] In preferred embodiments, the chemotherapeutic agent, e.g.,
paclitaxel, docetaxel, cisplatin, carboplatin, gemcitabine, and
doxorubicin, is administered intravenously. The agent is typically
administered as a drip infusion into the vein through a cannula.
Agents may also be given through a central line, which is inserted
under the skin into a vein near the collarbone, or into a PICC line
which is inserted into a vein in the crook of the arm.
[0067] In some embodiments, the administration of FTS with the
chemotherapeutic agent may be cyclic and repeated until remission
is achieved. For example, in one treatment regimen, FTS (200 mg) is
administered twice daily for a period of three weeks followed by a
one-week interval without FTS ("off period") while the
chemotherapeutic agent, e.g. gemcitabine (Gemzar.RTM.), is
administered once weekly (1500 mg) for a period of three weeks,
followed by a one-week rest period. The treatment regimen is
repeated as many times as needed, e.g., until remission is
achieved. Under this regimen, gemcitabine and FTS are administered
in three-week cycles (with increasing or decreasing dose amounts as
needed) each separated by a one-week "off period". Dosage reduction
with each cycle or within a cycle may be applied based upon the
amount of toxicity experienced by the patient. Combination
chemotherapy may also be administered in accordance with standard
procedures while dosing with FTS.
[0068] In another embodiment, the treatment regimen may entail
administration with oral FTS (e.g., a capsule or a tablet)
continuously without interruption (i.e., without an "off period")
and intravenous cisplatin as a daily infusion for five days every
three to four weeks until remission is achieved. Dosing regimens
for administering the chemotherapeutic agent or agents may be
administered according to standard procedures or may be adjusted to
meet the particular needs of the patient.
[0069] Oral compositions for FTS and its analogs for use in the
present invention can be prepared by bringing the agent(s) into
association with (e.g., mixing with) a pharmaceutically acceptable
carrier. Suitable carriers are selected based in part on the mode
of administration. Carriers are generally solid or liquid. In some
cases, compositions may contain solid and liquid carriers.
Compositions suitable for oral administration that contain the
active are preferably in solid dosage forms such as tablets (e.g.,
including film-coated, sugar-coated, controlled or sustained
release), capsules, e.g., hard gelatin capsules (including
controlled or sustained release) and soft gelatin capsules, powders
and granules. The compositions, however, may be contained in other
carriers that enable administration to a patient in other oral
forms, e.g., a liquid or gel. Regardless of the form, the
composition is divided into individual or combined doses containing
predetermined quantities of the active ingredient or
ingredients.
[0070] Oral dosage forms may be prepared by mixing the active
pharmaceutical ingredient or ingredients with one or more
appropriate carriers (optionally with one or more other
pharmaceutically acceptable additives or excipients), and then
formulating the composition into the desired dosage form e.g.,
compressing the composition into a tablet or filling the
composition into a capsule or a pouch. Typical carriers and
excipients include bulking agents or diluents, binders, buffers or
pH adjusting agents, disintegrants (including crosslinked and super
disintegrants such as croscarmellose), glidants, and/or lubricants,
including lactose, starch, mannitol, microcrystalline cellulose,
ethylcellulose, sodium carboxymethylcellulose,
hydroxypropylmethylcellulose, dibasic calcium phosphate, acacia,
gelatin, stearic acid, magnesium stearate, corn oil, vegetable
oils, and polyethylene glycols. Coating agents such as sugar,
shellac, and synthetic polymers may be employed, as well as
colorants and preservatives. See, Remington's Pharmaceutical
Sciences, The Science and Practice of Pharmacy, 20th Edition,
(2000).
[0071] Liquid form compositions include, for example, solutions,
suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active ingredient or ingredients, for example,
can be dissolved or suspended in a pharmaceutically acceptable
liquid carrier such as water, an organic solvent (and mixtures
thereof), and/or pharmaceutically acceptable oils or fats. Examples
of liquid carriers for oral administration include water
(particularly containing additives as above, e.g., cellulose
derivatives, preferably in suspension in sodium carboxymethyl
cellulose solution), alcohols (including monohydric alcohols
(including monohydric alcohols and polyhydric alcohols, e.g.,
glycerin and non-toxic glycols) and their derivatives, and oils
(e.g., fractionated coconut oil and arachis oil). The liquid
composition can contain other suitable pharmaceutical additives
such as solubilizers, emulsifiers, buffers, preservatives,
sweeteners, flavoring agents, suspending agents, thickening agents,
colorants, viscosity regulators, stabilizers or osmoregulators.
[0072] Carriers suitable for preparation of compositions for
parenteral administration include Sterile Water for Injection,
Bacteriostatic Water for Injection, Sodium Chloride Injection
(0.45%, 0.9%), Dextrose Injection (2.5%, 5%, 10%), Lactated
Ringer's Injection, and the like. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols and mixtures thereof, and
in oils. Compositions may also contain tonicity agents (e.g.,
sodium chloride and mannitol), antioxidants (e.g., sodium
bisulfite, sodium metabisulfite and ascorbic acid) and
preservatives (e.g., benzyl alcohol, methyl paraben, propyl paraben
and combinations of methyl and propyl parabens).
[0073] In order to fully illustrate the present invention and
advantages thereof, the following specific examples/experiments are
given, it being understood that the same is intended only as
illustrative and in no way limitative.
EXAMPLE 1
Experimental Design
[0074] The purpose of these in vitro and in vivo experiments was to
assess the ability of FTS, alone and in combination with a
chemotherapeutic agent, to impact lung cancer cell integrity and
survival. Here, the effects of the Ras inhibitor FTS on growth of
non-small cell lung carcinoma (NSCLC) cell lines H-1299 [American
Type Culture Collection ("ATCC"), CRL-5803), H23 (ATCC, CRL-5800,
K-Ras mutation), HTB54 (ATCC, K-Ras mutation), A549 (ATCC, K-Ras
mutation) and on the growth of lung squamous cell carcinoma cell
line SK-MES-1 (ATCC, HTB-58) were examined. FTS on tumor cell
growth in a nude mouse model was also examined. In addition, the
combination of FTS and a chemotherapeutic agent on tumor cell
growth inhibition was examined. The primary goal was to determine:
(I) whether FTS induced cell-cycle arrest in A549 cells and also
whether FTS induced growth inhibition in all five human lung cancer
cell lines; (II) whether FTS altered cytoskeleton organization in
A549 cells; (III) whether FTS inhibited active K-Ras-GTP and
inhibited anchorage-independent growth of lung cancer cells in A549
cells; (IV) whether A549 cells were resistant to apoptosis after
exposure to a chemotherapeutic agent in the presence of FTS; (V)
whether FTS administered i.p. inhibited tumor growth in both A549
and HTB-58 (SK-MES-1) nude mouse models and whether oral FTS,
alone, and in combination with a chemotherapeutic agent inhibited
tumor growth in the A549 lung cancer cell nude mouse model; and
(VI) whether increasing concentrations of FTS sensitized human
NSCLC cell lines H1734 and H2030 (KRAS mutations) and H1975 and
H3255 (EGFR mutations) to cell death.
[0075] The results of the first set of experiments (I) demonstrated
that FTS induced cell cycle arrest in A549 cells. In addition, FTS
caused dose-dependent inhibition in A549, HTB54, and H23 cell lines
(which harbor activated K-Ras) and in H-1299 and HTB-58 (SK-MES-1)
cell lines (neither of which harbors mutated Ras). Thus, FTS
inhibited the growth of tumor cells even when the cells did not
harbor mutated Ras genes. Results also indicated that the
half-maximal inhibitory concentration (IC.sub.50) of FTS ranged
between 30 to 75 .mu.m depending on the cell line.
[0076] The second set of experiments (II) revealed that A549 cells
treated with FTS showed strong actin stress fibers and focal
adhesions as compared with the control cells. Thus, FTS altered
cytoskeleton organization and cell morphology in the A549 cell
line.
[0077] In the third set of experiments (III), FTS inhibited the
development of A549 human lung cancer cell colonies. Thus, FTS
inhibited the anchorage-independent growth of A549 cells. In
addition, FTS reduced the amount of K-Ras-GTP in a dose-dependent
manner.
[0078] The results of the fourth set of experiments (IV) revealed
that FTS increased sensitivity of A549 cells to cytotoxic drugs.
Results showed that the combination of FTS and the chemotherapeutic
agent demonstrated that the combined treatment with both drugs was
more effective than treatment with either drug alone in A549
cells.
[0079] In the fifth set of experiments (V), i.p. administration of
FTS inhibited tumor growth in A549 and HTB-58 (SK-MES-1) cell nude
mouse models. Thus, FTS (i.p.) inhibited tumor growth as elicited
by A549 and SK-MES-1 cells in vivo. In addition, oral
administration of FTS inhibited tumor growth in the A549 lung
cancer cell nude mouse model. Results also indicated that the
combinations of FTS and gemcitabine (oral) were more effective than
treatment with either drug alone.
[0080] The results of a sixth set of experiments showed that FTS at
increasing concentrations sensitized human NSCLC cell lines H1734
and H2030 (KRAS mutations) and H1975 and H3255 (EGFR mutations) to
cell death.
Materials and Methods
Cell Culture
[0081] FTS was provided by Concordia Pharmaceuticals, Inc. (Ft.
Lauderdale, Fla.). All cell lines were obtained from American Type
Culture Collection ("ATCC") (Manassas, Va.). A549 cells,
non-small-cell lung carcinoma (CCL, ATCC) cells, were cultured in
Kaighn's modification of Ham's F-12 medium containing 1.5 g/l
sodium bicarbonate, 10% fetal calf serum (FCS), 100 U/ml
penicillin, and 100 .mu.g/ml streptomycin. HTB54 lung carcinoma
cells were cultured in McCoy's 5A medium with 10% FCS, 100 U/ml
penicillin, and 10 .mu.g/ml streptomycin. HTB-58 (SK-MES-1, ATCC),
a human lung squamous cell carcinoma cell line, was cultured in
Eagle's minimum essential medium with 2 mM L-glutamine and Earle's
BSS, 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids,
1 mM sodium pyruvate, 10% FCS, 100 U/ml penicillin, and 100
.mu.g/ml streptomycin. H23 (NCI-H23, ATCC), a human non-small-cell
lung adenocarcinoma cell line, was cultured in RPMI 1640 medium
with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose,
mM HEPES, 1 mM sodium pyruvate, 10% FCS, 100 U/ml penicillin, and
100 .mu.g/ml streptomycin. H1299 (NCI-H1299, ATCC), a
non-small-cell lung carcinoma cell line, was cultured in RPMI 1640
medium with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l
glucose, 10 mM HEPES, 1 mM sodium pyruvate, 10% FCS, 100 U/ml
penicillin, and 100 .mu.g/ml streptomycin. The cells were plated in
24-well plates in 1 ml of medium at a density of 5000 cells/well
(or 2500 cells/well, HTB54) and incubated at 37.degree. C. in a
humidified atmosphere of 95% air and 5% CO.sub.2. Cells were
treated with the indicated concentrations of FTS (Concordia
Pharmaceuticals, Sunrise FL) or with 0.1% Me.sub.2SO.sub.4 (DMSO)
(vehicle) 24 h after plating and were counted 5 days later. Dead
cells were counted after addition of Hoechst 33258 dye
(Sigma-Aldrich, St. Louis, Mo.); 1 .mu.g/ml) to vehicle-treated
control cultures or to cultures treated for 24 or 48 h with 75
.mu.M FTS. Fluorescence images were collected 5 min after the dye
was added.
[0082] In drug combination experiments, cells were grown for 2 days
in the absence or in the presence of 40 .mu.M FTS and were then
treated for 4 h with gemcitabine (100 or 200 nM), cisplatin (50 or
100 nM), doxorubicin (50 or 100 nM), or paclitaxel (2.5 or 5 nM).
Live cells were counted after a further 3 days of incubation with
or without FTS. Experiments were performed twice in
quadruplicate.
BrdU Incorporation into DNA
[0083] A549 cells were plated on glass cover slips
(1.2.times.10.sup.5 cells/well in 6-well plates) and incubated for
24 h in medium containing 5% FCS. The cells were then incubated for
24 h with or without 75 .mu.M FTS and then for 24 h with
5-bromo-2-deoxyuridine (BrdU) (Zymed BrdU labeling kit, 1:100
dilution). Cells were fixed with 4% paraformaldehyde, permeabilized
with 0.2% Triton X-100 (BDH, Poole, UK), washed with PBS, blocked
with TBS Tween (TBST; 50 mM Tris, pH 7.4, 100 mM NaCl, 0.1% Tween
20) containing 1% bovine serum albumin (BSA), treated sequentially
with 2 N HCl and 0.1 M sodium borate pH 8.5, and then blocked with
goat .gamma.-globulin and washed with TBST-BSA (described above).
The cells were then labeled successively with mouse anti-BrdU
antibody (Ab) (Zymed kit; 1:50 dilution), biotinylated rabbit anti
mouse IgG (5 .mu.g/ml), and Cy3-streptavidin (1.5 .mu.g/ml). Cells
with BrdU-stained nuclei were counted under a fluorescence
microscope.
FACS Analysis
[0084] A549 cells were plated (9.times.10.sup.5 cells) in 10-cm
plates, incubated for 24 h in medium containing 5% FCS, and then
incubated for 24 or 48 h with or without 75 .mu.M FTS. The cells
were collected, resuspended in PBS containing propidium iodide (50
.mu.g/ml; Sigma) and 0.05% Triton X-100, and subjected to analysis
by a fluorescence-activated cell sorter (FACSCalibur; Becton
Dickinson, Los Angeles, Calif.).
Immunofluorescence and Confocal Microscopy
[0085] A549 cells were plated on glass cover slips
(2.times.10.sup.4 cells/well in 6-well plates), incubated for 24 h
in medium containing 5% FCS, and then incubated for 48 h with or
without .mu.M FTS. The cells were fixed and permeabilized at room
temperature by successive incubations with 3.7% formaldehyde (20
min) and 0.2% Triton X-100 in PBS (5 min), then washed for 5 min
with UB buffer (150 mM NaCl, 10 mM Tris pH 7.6, and 0.2% sodium
azide in PBS) and blocked with 2% BSA in UB (UBB, 5 min). The fixed
cells were incubated successively with naive goat IgG for 30 min
(200 .mu.g/ml, Jackson ImmunoResearch Laboratories, West Grove,
Pa.), anti-vinculin Ab for 1 h (1:400, Sigma-Aldrich), goat
anti-mouse Cyt-conjugated Ab for 1 h (1:200, Jackson), and
rhodamine-labeled phalloidin for 1 h (1:1000, Sigma-Aldrich).
Between each of the above steps the cells were washed for 30 min
with UBB. Lastly, the cover slips were washed with UB, dried, and
mounted onto the slides with Muviol. F-actin (red) and vinculin
(green) were visualized with a Zeiss LSM 510 confocal microscope
fitted with non-leaking green and red fluorescence filters.
Co-localization was assessed using the co-localization function of
the LSM 510 software.
Hoechst Staining Procedures
[0086] Hoechst 33258 dye, an ultraviolet light-excitable dye that
demonstrates increased fluorescence when bound to the condensed
chromatin of apoptotic cells, was used to quantify apoptotic cells
in cell culture after FTS treatments. In tissue culture, cells were
seeded at a density of 20.times.10.sup.4 cells in 6-well plates for
24 h. Once cells had reached 70% confluence in normal FCS, the
media was changed to low serum media (0.5% FCS for) and FTS was
added. Control cells were treated with 0.1% DMSO. Hoechst solution
was added to each well for 5-10 min and three pictures from each
well were taken while using fluorescence microscopy.
Anchorage-Independent Colony Formation Assay in Soft Agar
[0087] Noble agar (2% and 0.6%; Difco, Detroit, Mich.) was prepared
in water and autoclaved. The 2% agar was melted in a microwave
oven, mixed 1:1 with medium (.times.2 Kaighn's modification of
Ham's F-12 medium with 20% FCS, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin) and poured onto 96-well plates (50 .mu.l per well) to
provide the 1% base agar. The 0.6% agar (5 ml) was mixed with 5 ml
of medium (.times.2), containing 8.times.10.sup.4 A549 cells, and
the mixture (50 .mu.l) was plated on top of the base agar. The
cells were incubated for 19 days at 37.degree. C. with or without
the indicated concentrations of FTS (6 wells for the control and
for each treatment) and colonies were stained with MTT (1 mg/ml for
4 h). The colonies were then visualized by light microscopy,
imaged, and counted using the ImagePro software.
Ras, Rac and Rho Pull-Down Assays and Immunoblotting Procedures
[0088] A549 cells were incubated for 24 or 48 h with or without
FTS, as described above, and then lysed with lysis buffer as
described in Haklai, R., Gana-Weisz, M., Elad, G., et al.,
Biochemistry 37:1306-14 (1998). The apparent amounts of K-Ras-GTP
in 0.5 mg protein of total cell lysates were determined by the
glutathione-S-transferase (GST)-RBD (Ras-binding domain of Raf)
pull-down assay, as described in (Elad-Sfadia, G., Haklai, R.,
Ballan, E., Gabius, H. J., Kloog, Y., J Biol Chem 277:37169-75
(2002). The apparent amounts of Rac1-GTP and of RhoA-GTP, each in 2
mg protein of total cell lysates, were determined, respectively, by
pull-down assays with GST-PBD (Rac1-binding domain of
PAK1)-conjugated and GST-Rhotekin BD (Rho-binding domain of
Rhotekin)-conjugated beads [Benard, V., Bohl, B. P., Bokoch, G. M,
J Biol Chem 274:13198-204 (1999); Fiordalisi, J. J., Keller, P. J.,
Cox, A. D., Cancer Res 66:3153-61 (2006). The pulled-down GTPases
were subjected to SDS-PAGE followed by immunoblotting with the
appropriate antibodies: anti K-Ras (1:30; Calbiochem, La Jolla,
Calif.), anti Rac-1 (1:2500; Santa Cruz Biotechnology, Santa Cruz,
Calif.), or anti RhoA (1:700; Upstate Biotechnology, Lake Placid,
N.Y.). Immunoblots were exposed to 1:2500 peroxidase-goat
anti-mouse IgG. Levels of phospho-ERK and phospho-Akt were
determined by immunoblotting [Haklai, R., Gana-Weisz, M., Elad, G.,
et al., supra.] using rabbit anti phospho-ERK1/2 Ab (Santa Cruz
Biotechnology, Santa Cruz, Calif.) and rabbit anti phospho-Akt Ab
(Cell Signaling, Beverly, Mass.). Protein bands were visualized by
enhanced chemiluminescence and quantified by densitometry using
ImageJ computer software (National institutes of Health, Bethesda,
Md.).
Animal Studies
[0089] Nude mice (6 weeks old) were housed in barrier facilities on
a 12-h light/dark cycle. Food and water were supplied ad libitum.
On day zero, A549 or HTB-58 cells (5.times.10.sup.6 cells in 0.1 ml
PBS) were implanted subcutaneously (s.c.) just above the right
femoral joint. After 5 or 11 days the mice were separated randomly
into control groups that had received only the vehicle and
FTS-treated groups. Daily FTS treatments were administered either
intraperitoneally (i.p.) or orally. Tumor volumes or weights were
determined as described in Barkan, B., Starinsky, S., Friedman, E.,
Stein, R., Kloog, Y., Clin Cancer Res 12:5533-42 (2006).
Gemcitabine treatment (36 mg/kg, i.p.) was administered every 4
days.
Results
I. FTS Inhibited the Growth of A549, H-1299, H23, HTB54, and HTB-58
(SK-MES-1) Human Lung Cancer Cells.
[0090] The tumor cell lines of the present study were originally
derived from human lung epithelial cells and are representative of
lung cancers and its associated manifestations. Here, we examined
the impact of Ras inhibitor FTS on growth of non-small cell lung
carcinoma cell lines A549 (K-Ras mutation), H23 (K-Ras mutation),
and H-1299. We also examined the impact of FTS on the growth of
HTB23 lung epidermoid carcinoma cell line and on lung squamous cell
carcinoma cell line HTB-58 (SK-MES-1).
[0091] To investigate the effect of FTS on lung cancer cell
proliferation, we first incubated A549 cells that harbor the
activated K-ras gene mutated at codon 12. A549 cells are commonly
used as a model for drug screening. Incubation of the cells with 75
.mu.M FTS for 48 h inhibited the incorporation of BrdU into their
DNA by 56.7.+-.17.4% relative to vehicle-treated control cells
(P<0.05) (FIG. 1). Typical photomicrographs of control and 75
.mu.M FTS-treated A549 cells (72 h) showed that FTS induced a
reduction in cell number and altered the morphology of the cells
(FIG. 2). Increasing concentration of FTS inhibited A549 cell
growth at a dose dependent rate, with a decrease of 50% at 40 .mu.M
FTS. The number of cells in the FTS-treated cultures was determined
by direct counting of A549 cells grown for 6 days in the presence
of FTS and was expressed as a percentage of the number recorded in
the controls. Data were means of 12 counts .+-.SD. *P<0.01,
**P<0.0005, compared to control (FIG. 3). In another set of
experiments, cells were also treated for 24 and 48 h with FTS and
collected for FACS analysis (FIG. 4). The apoptotic population of
cells (indicated in the FACS analysis as sub-G1) was 3.8% at 24 h
and 8.4% at 48 h in cells treated with 75 .mu.M FTS, compared to
1.0% and 2.8%, respectively, in control cells (FIG. 4). The results
of these experiments also showed that FTS caused a reduction in the
G1 population of cells but not in that of G2/M cells. Cells treated
with FTS for 24 h and 48 h showed reductions in G1 of 5.6% and 19%,
respectively (FIG. 3). Thus, FTS induced cell-cycle arrest in A549
cells, resulting in inhibition of cell growth.
[0092] The growth-inhibitory effects of FTS were not limited to the
A549 human lung cancer cells. Similar growth inhibition curves were
obtained for H-1299 cells (FIG. 5) and SK-MES-1 cells (FIG. 6),
which express relatively large amounts of EGF and insulin-like
growth factor (IGF) receptors which activate Ras, and for H23 cells
(FIG. 7) and HTB54 cells (FIG. 8), which harbor oncogenic K-Ras.
The IC.sub.50 values ranged between 30-75 .mu.M FTS, depending on
the cell line (FIG. 9).
II. FTS Altered Cytoskeleton Organization of A549 Cells.
[0093] Next, to determine the effects of FTS on the cytoskeleton of
A549 cells, the cells were incubated, treated with 75 .mu.M FTS,
and stained with rhodamine-labeled phalloidin, which associates
with polymeric F-actin, and with anti-vincullin, which associates
with focal adhesions. Typical fluorescence images of control and of
FTS-treated cells are shown in FIG. 10. Cells treated with FTS
showed strong actin stress fibers and focal adhesions as compared
with the control cells. The untreated cells exhibited short, thin
actin stress fibers and relatively few focal adhesions, whereas the
FTS-treated cells exhibited long, thick stress fibers and a
relatively large number of focal adhesions that looked larger than
those observed in the control cells. Statistical analysis indicated
that more than 80% of the cells in the FTS-treated cultures had
undergone changes in cell morphology. These results combined with
the growth-inhibitory effects of FTS observed in lung cancer cell
lines suggested that the FTS had, at least, partially reversed the
transformed phenotype of the cells. Moreover, these results are
consistent with the previous experiments that demonstrated an
observed change in A549 cell morphology (FIG. 1).
III. FTS Inhibited Anchorage-Independent Growth of A549 Cells.
[0094] To determine whether active K-Ras-GTP and its prominent
downstream signals to ERK and Akt were inhibited in A549 cells, and
if so, whether the anchorage-dependent growth of the cells was also
affected, two experiments were performed. First, A549 cells were
incubated in the absence and in the presence of various
concentrations of FTS and K-Ras-GTP, phospho-ERK and phospho-Akt
levels were measured. FTS reduced the amount of K-Ras-GTP in a
dose-dependent manner with no significant effect on the total
amount of Ras (FIG. 11A). The reduction in K-Ras-GTP (mean.+-.SD)
was 23.+-.15.3%, 37.+-.3.7% (P<0.01), and 46.+-.1.9%
(P<0.002), respectively, in cells treated with 25 .mu.M, 50
.mu.M, and 75 .mu.M FTS. The effective concentration range (50-75
.mu.M) for the reduction in K-Ras-GTP (FIG. 11A) was similar to
that required for the inhibition of cell growth (FIG. 3). FTS also
reduced the levels of phospho-ERK and phospho-Akt causing 33.+-.2%
and 58.+-.6% inhibition, respectively (FIG. 2).
[0095] The effect of FTS appeared to be specific to the Ras
protein, since it had no effect on the amount of the prenylated
active Racl-GTP protein as determined by a specific Racl-GTP
pull-down assay (FIG. 11B). Moreover, using a specific pull-down
assay for prenylated active RhoA-GTP, FTS induced a significant
increase of 2.+-.0.2 fold (P<0.002) in RhoA-GTP (FIG. 11C).
Thus, while FTS did not reduce the total amounts of the three
GTPases (K-Ras, Rac-1, and RhoA), it clearly had a selective
inhibitory effect on active K-Ras. The observed increase in
RhoA-GTP is consistent with the observed increase in stress-fiber
formation and focal adhesion assembly (FIG. 10).
[0096] Next, to determine the effect of FTS on the
anchorage-independent growth of A549 cells, a soft agar assay was
performed. The cells were seeded in soft agar and treated with
increasing concentrations of FTS 0 .mu.M, 50 .mu.M and 100 .mu.M
(FIGS. 12A-12B). Control cells were treated with 0.1%
Me.sub.2SO.sub.4 (DMSO). FTS inhibited A549 cell growth in soft
agar by 27.+-.5.5% and 58.+-.21% at 50 .mu.M and 100 .mu.M FTS,
respectively. Thus, FTS inhibited the anchorage-independent growth
of A549 cells.
IV. Combining FTS With a Chemotherapeutic Agent Enhanced Cell Death
in Human Lung Cancer A549 Cells.
[0097] To determine whether A549 cells were resistant to apoptosis,
an experiment to examine the survival of human lung cancer A549
cells after exposure to a chemotherapeutic agent in the presence of
FTS was performed. Thus, to determine whether treatment with FTS
can increase the sensitivity of A549 cells to cytotoxic drugs, A549
cells were incubated for 48 h with DMSO (control) or with 40 .mu.M
FTS, then for 4 h with gemcitabine, cisplatin, doxorubicin, or
paclitaxel at the indicated concentrations. The cells were then
washed and incubated for a further 72 h with DMSO or with 40 .mu.M
FTS. Live cells were collected and counted. The numbers of cells in
the drug-treated cultures, expressed as percentages of the numbers
in the vehicle-treated control, are shown in FIGS. 13A-13D. Values
are means.+-.SD. *P<0.05, **P<0.01, compared to
vehicle-treated control.
[0098] As shown in FIG. 13A, the effects of gemcitabine in the
presence of FTS caused an enhanced increase in cell death that was
measurably more effective than treatment with either drug alone in
A549 cells. As shown, FTS alone caused a 25.+-.6.3% reduction in
cell numbers (mean.+-.SD) at 40 .mu.M, while gemcitabine alone at
100 and 200 nM had no effect (<11%). The combinations of FTS and
gemcitabine at 100 and 200 nm enhanced cell number reductions of
45.+-.5.3% and 60.+-.5.7%, respectively.
[0099] As shown in FIG. 13C, the effects of cisplatin in the
presence of FTS caused an increase in cell death that was
measurably more effective than treatment with either drug alone in
A549 cells. As shown, FTS alone caused a 33.+-.9.5% reduction in
cell numbers (mean.+-.SD) at 40 .mu.M, while cisplatin alone at 50
and 100 nM caused reductions of 11.+-.11% and 30.+-.12.9%,
respectively. The combinations of FTS and cisplatin at 50 and 100
nm caused cell number reductions of 47.+-.6.9% and 63.+-.12.7%,
respectively.
[0100] As with cisplatin, the observed effects of the combinations
of doxorubicin (FIG. 13B) and of paclitaxel (FIG. 13D) in the
presence of FTS caused an increase in cell death that was
measurably more effective than treatment with either drug alone in
A549 cells.
V. FTS Alone and in Combination With a Chemotherapeutic Agent
Inhibited Tumor Growth in Lung Cancer Cell Nude Mouse Models.
[0101] To determine whether FTS inhibited tumor growth in vivo,
experiments were conducted using a nude mouse model. The lung
cancer cells were implanted s.c. above the right femoral joint and
the mice were then treated with FTS. In a first experiment, the
effect of i.p. administration of FTS on tumor growth in A549 cells
was assessed. Treatment was started 5 days after cell implantation,
by which time the tumors were palpable. Tumor volumes were
determined 24 days after implantation in two groups of mice (n=8)
that had received daily i.p. administration of either the vehicle
(control) or 10 mg/kg FTS. Significant inhibition of tumor growth
relative to the control (53.8%, P<0.05) was recorded in the
FTS-treated group (FIG. 14A).
[0102] In a second experiment carried out with mice implanted s.c.
with HTB-58 cells (n=7 per group), significant inhibition of tumor
growth (76.4.+-.48.8%) was observed in the group treated daily with
10 mg/kg FTS i.p. (FIG. 14B). Tumor volume measured 14 days after
cell implantation in that group was 0.02.+-.0.045 cm.sup.3 compared
to 0.09.+-.0.08 cm.sup.3 in the vehicle-treated controls
(P<0.05).
[0103] In an additional set of experiments, the A549-cell-implanted
nude mouse model was used to examine the effect of orally
administered FTS on tumor growth. First, cells were implanted as
described above and daily oral treatment with FTS (50 mg/kg; n=6)
or vehicle (n=5) was started either 11 days (FIG. 14C) or 6 days
(FIG. 14D) after implantation. As shown in FIG. 14C, after 16 days
of treatment the tumor weights (mean.+-.SD) in FTS-treated and
control mice were 0.4.+-.0.19 g and 0.9.+-.0.39 g, respectively,
representing a significant inhibition of 53.7.+-.19.1% in tumor
growth (P<0.025) in the FTS-treated mice. Next, the effects of
orally administered FTS, alone or in combination with gemcitabine,
on A549-cell tumor growth was examined (FIG. 14D). Six days after
cell implantation, mice were divided into four groups (n=8 per
group) and treated orally with vehicle alone (control), FTS alone
(60 mg/kg), vehicle and gemcitabine (36 mg/kg, i.p. every 4 days),
or FTS and gemcitabine. Treatments with gemcitabine began 1 week
after FTS treatment was started. Consistent with the results of the
first experiment (FIG. 14C), oral FTS treatment caused a
significant inhibition in tumor growth; tumor weights in the mice
treated with vehicle only (control) and with FTS only (mean.+-.SD)
were 0.90.+-.0.40 g and 0.49.+-.0.15 g, respectively (46.2.+-.16.3%
inhibition, P<0.02; FIG. 14D). A significant reduction in tumor
weight (P<0.015) was also observed in a fifth group of mice
treated with gemcitabine alone (FIG. 14D). The combined effect of
gemcitabine and FTS treatments were more effective than the effect
of each treatment alone. Thus, the result reinforces the results of
the in vitro experiments indicating that combined treatment with
the two drugs was more effective than treatment with either of the
drugs alone.
VI. FTS Alone Sensitized Human NSCLC Cell Lines H1734, H2030,
H1975, and H3255 to Cell Death.
[0104] To determine whether FTS sensitized other human NSCLC cell
lines to cell death, experiments were conducted on cell lines H1734
and H2030 (KRAS mutations) and H1975 and H3255 (EGFR mutations).
The four cell lines were grown in increasing concentrations of FTS
(dissolved in DMSO). After 96 hours, viable cells were quantified
using an Alamar blue assay. Results are the mean.+-.standard error
of three independent experiments, in which there were 3 replicates
of each condition, as shown in FIG. 15.
[0105] The publications cited in the specification, patent
publications and non-patent publications, are indicative of the
level of skill of those skilled in the art to which this invention
pertains. All of these publications are herein incorporated by
reference to the same extent as if each individual publication were
specifically and individually indicated as being incorporated by
reference.
[0106] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *