U.S. patent application number 17/390292 was filed with the patent office on 2022-03-31 for methods of treating cancer using compositions comprising perillyl alcohol derivative.
The applicant listed for this patent is NeOnc Technologies, Inc.. Invention is credited to Thomas CHEN.
Application Number | 20220096464 17/390292 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096464 |
Kind Code |
A1 |
CHEN; Thomas |
March 31, 2022 |
METHODS OF TREATING CANCER USING COMPOSITIONS COMPRISING PERILLYL
ALCOHOL DERIVATIVE
Abstract
A method for treating brain metastases of a cancer in a mammal
includes administering to the mammal a therapeutically effective
amount of a perillyl alcohol carbamate, such as TMZ-POH. The brain
metastases can be originated or spread from breast cancer. The
perillyl alcohol derivative may be perillyl alcohol conjugated with
a therapeutic agent, such as a chemotherapeutic agent. The
chemotherapeutic agents that may be used in the present invention
include a DNA alkylating agent, a topoisomerase inhibitor, an
endoplasmic reticulum stress inducing agent, a platinum compound,
an antimetabolite, an enzyme inhibitor, and a receptor
antagonist.
Inventors: |
CHEN; Thomas; (La Canada,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NeOnc Technologies, Inc. |
Los Angeles |
CA |
US |
|
|
Appl. No.: |
17/390292 |
Filed: |
July 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16722816 |
Dec 20, 2019 |
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17390292 |
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15916549 |
Mar 9, 2018 |
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16722816 |
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15026649 |
Apr 1, 2016 |
9913838 |
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PCT/US14/59600 |
Oct 8, 2014 |
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15916549 |
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14455293 |
Aug 8, 2014 |
9663428 |
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15026649 |
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13566731 |
Aug 3, 2012 |
8916545 |
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14455293 |
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PCT/US11/49392 |
Aug 26, 2011 |
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13566731 |
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61471402 |
Apr 4, 2011 |
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61377747 |
Aug 27, 2010 |
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61888253 |
Oct 8, 2013 |
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International
Class: |
A61K 31/495 20060101
A61K031/495; A61K 47/55 20060101 A61K047/55; A61K 47/54 20060101
A61K047/54 |
Claims
1. A method for treating a brain metastasis of a cancer in a
mammal, comprising administering to the mammal a therapeutically
effective amount of a compound comprising perillyl alcohol (POH)
conjugated with temozolomide (TMZ).
2. The method of claim 1, wherein the compound is 3-methyl
4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamic
acid-4-isopropenyl cyclohex-1-enylmethyl ester (TMZ-POH).
3. The method of claim 1, wherein the brain metastasis originates
from a primary cancer selected from the group consisting of a
systemic cancer, lung cancer, prostate cancer, breast cancer,
hematopoietic cancer, ovarian cancer, bladder cancer, germ cell
tumors, kidney cancer, leukemia, lymphoma, and melanoma.
4. The method of claim 3, wherein the brain metastasis originates
from breast cancer.
5. The method of claim 1, wherein the compound is administered by
inhalation, intranasally, orally, intravenously, subcutaneously or
intramuscularly.
6. The method of claim 1, wherein the administering comprises
administering the compound intranasally using a nasal delivery
device selected from the group consisting of an intranasal inhaler,
an intranasal spray device, an atomizer, a nebulizer, a metered
dose inhaler (MDI), a pressurized dose inhaler, an insufflator, a
unit dose container, a pump, a dropper, a nasal spray bottle, a
squeeze bottle and a bi-directional device.
7. The method of claim 1, further comprising treating the mammal
with radiation before, during, or after the administration of the
compound.
8. The method of claim 1, further comprising delivering to the
mammal an additional chemotherapeutic agent.
9. A method for treating metastatic breast cancer in a mammal that
has spread to the brain of the mammal, comprising administering to
the mammal a therapeutically effective amount of 3-methyl
4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamic
acid-4-isopropenyl cyclohex-1-enylmethyl ester (TMZ-POH).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/916,549 filed Mar. 9, 2018, which is a
continuation of U.S. patent application Ser. No. 15/026,649 filed
Apr. 1, 2016, now U.S. Pat. No. 9,913,838; which is a 371 U.S.
National Stage of International Patent Application No.
PCT/US2014/059600 filed Oct. 8, 2014, which claims priority to U.S.
patent application Ser. No. 14/455,293 filed Aug. 8, 2014, now U.S.
Pat. No. 9,663,428, which is a continuation of U.S. patent
application Ser. No. 13/566,731 filed Aug. 3, 2012, now U.S. Pat.
No. 8,916,545. U.S. patent application Ser. No. 13/566,731 is a
continuation of International Patent Application No.
PCT/US2011/049392, which claims benefit of U.S. Provisional
Application Nos. 61/471,402 (filed Apr. 4, 2011) and 61/377,747
(filed Aug. 27, 2010). International Patent Application No.
PCT/US2014/059600 claims benefit to U.S. Provisional Application
No. 61/888,253 filed Oct. 8, 2013.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions of perillyl
alcohol (POH) derivatives such as POH carbamates, as well as the
use thereof for treating cancers.
BACKGROUND OF THE INVENTION
[0003] Malignant gliomas, the most common form of central nervous
system 20 (CNS) cancers, is currently considered essentially
incurable. Among the various malignant gliomas, anaplastic
astrocytomas (Grade III) and glioblastoma multiforme (GBM; Grade
IV) have an especially poor prognosis due to their aggressive
growth and resistance to currently available therapies. The present
standard of care for malignant gliomas consists of surgery,
ionizing radiation, and chemotherapy. Despite 25 recent advances in
medicine, the past 50 years have not seen any significant
improvement in prognosis for malignant gliomas. Wen et al.
Malignant gliomas in adults. New England J Med. 359: 492-507, 2008.
Stupp et al. Radiotherapy plus concomitant and adjuvant
temozolomide for glioblastoma. New England J Med. 352: 987-996,
2005.
[0004] The poor response of tumors, including malignant gliomas, to
various types of chemotherapeutic agents are often due to intrinsic
drug resistance. Additionally, acquired resistance of initially
well-responding tumors and unwanted side effects are other problems
that frequently thwart long-term treatment using chemotherapeutic
agents. Hence, various analogues of chemotherapeutic agents have
been prepared in an effort to overcome these problems. The
analogues include novel therapeutic agents which are hybrid
molecules of at least two existing therapeutic agents. For example,
cisplatin has been conjugated with Pt-(II) complexes with cytotoxic
codrugs, or conjugated with bioactive shuttle components such as
porphyrins, bile acids, hormones, or modulators that expedite the
transmembrane transport or the drug accumulation within the cell.
(6-Aminomethylnicotinate) dichloridoplatinum(II) complexes
esterified with terpene alcohols were tested on a panel of human
tumor cell lines. The terpenyl moieties in these complexes appeared
to fulfill a transmembrane shuttle function and increased the rate
and extent of the uptake of these conjugates into various tumor
cell lines. Schobert et al. Monoterpenes as Drug Shuttles:
Cytotoxic (6-minomethylnicotinate) dichloridoplatinum(II) Complexes
with Potential To Overcome Cisplatin Resistance. J. Med. Chem.
2007, 50, 1288-1293.
[0005] Metastasized cancer, such as breast cancer, that has spread
to the brain poses a similarly serious therapeutic challenge as
malignant gliomas. This challenge once was a late aspect of disease
progression, but increasingly is becoming a first site of disease
progression after otherwise successful treatment of primary tumor
and metastases outside the cranium. Traditional breast cancer
therapeutics, such as paclitaxel or doxorubicin, only reach brain
metastases at concentrations that are far lower than needed to be
therapeutically active. P. R. Lockman, et al. Heterogeneous
blood-tumor barrier permeability determines drug efficacy in
experimental brain metastases of breast cancer, Clin Cancer Res 16
(2010) 5664-5678. The most critical barrier to effective entry of
chemotherapeutics into the brain is the blood brain barrier (BBB),
and very few anticancer drugs are able to overcome this obstacle.
E. Fokas, J. P. Steinbach, C. Rodel, Biology of brain metastases
and novel targeted therapies: time to translate the research,
Biochim Biophys Acta 1835 (2013) 61-75.
[0006] Perillyl alcohol (POH), a naturally occurring monoterpene,
has been suggested to be an effective agent against a variety of
cancers, including CNS cancer, breast cancer, pancreatic cancer,
lung cancer, melanomas and colon cancer. Gould, M. Cancer
chemoprevention and therapy by monoterpenes. Environ Health
Perspect. 1997 June; 105 (Suppl 4): 977-979. Hybrid molecules
containing both perillyl alcohol and retinoids were prepared to
increase apoptosis-inducing activity. Das et al. Design and
synthesis of potential new apoptosis agents: hybrid compounds
containing perillyl alcohol and new constrained retinoids.
Tetrahedron Letters 2010, 51, 1462-1466.
[0007] The alkylating agent temozolomide (TMZ) is able to cross the
BBB after oral dosing and has become the chemotherapeutic standard
of care for patients with glioblastoma multiforme (GBM). Zhang et
al. Temozolomide: mechanisms of action, repair and resistance. Curr
Mol Pharmacol 5 (2012) 102-114. TMZ acts as a prodrug. Its
mechanism of activation involves hydrolytic opening of its
tetrazinone ring, which takes places spontaneously in aqueous
solution at 37.degree. C., and does not require the participation
of cellular enzymes. The resulting product, the unstable monomethyl
MTIC (5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide), reacts
with water to liberate AIC (4-amino-5-imidazole-carboxamide) and
the highly reactive methyldiazonium cation, which methylates DNA
purine residues.
[0008] When TMZ was tested for activity against brain metastatic
breast cancer in heavily pretreated patients, it revealed mixed
outcomes that ranged from "encouraging activity" and "disease
control" to "well-tolerated, but no objective responses". C.
Christodoulou et al., Phase II study of temozolomide in heavily
pretreated cancer patients with brain metastases, Annals Oncol 12
(2001) 249-254; L. E. Abrey et al., A phase II trial of
temozolomide for patients with recurrent or progressive brain
metastases, J Neurooncol 53 (2001) 259-265; M. E. Trudeau et al.,
Temozolomide in metastatic breast cancer (MBC): a phase II trial of
the National Cancer Institute of Canada--Clinical Trials Group
(NCIC-CTG). Annals Oncol 17 (2006) 952-956; R. Addeo et al. Phase 2
trial of temozolomide using protracted low-dose and whole-brain
radiotherapy for nonsmall cell lung cancer and breast cancer
patients with brain metastases, Cancer 113 (2008) 2524-2531; S.
Siena et al., Dose-dense temozolomide regimen for the treatment of
brain metastases from melanoma, breast cancer, or lung cancer not
amenable to surgery or radiosurgery: a multicenter phase II study.
Annals Oncol 21 (2010) 655-661; R. Addeo et al., Protracted low
dose of oral vinorelbine and temozolomide with whole-brain
radiotherapy in the treatment for breast cancer patients with brain
metastases, Cancer Chemother Pharmacol 70 (2012) 603-609. The
underlying basis for these inconsistent results was not
investigated, but it is conceivable that these differences may have
been due to variable expression levels of O6-methylguanine-DNA
methyltransferase (MGMT; also called O6-alkylguanine-DNA
alkyltransferase, AGT), a DNA repair enzyme that removes alkyl
groups located on the O6-position of guanine. A. E. Pegg,
Multifaceted roles of alkyltransferase and related proteins in DNA
repair, DNA damage, resistance to chemotherapy, and research tools,
Chem Res Toxicol 24 (2011) 618-639; M. Christmann et al.,
O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues
and tumors: enzyme activity, promoter methylation and
immunohistochemistry, Biochim Biophys Acta 1816 (2011) 179-190.
Because the primary toxic DNA lesion set by TMZ is alkylation of
O6-guanine, high expression levels of MGMT protect tumor cells from
the cytotoxic impact of TMZ and provide treatment resistance. J. R.
Silber et al., O(6)-methylguanine-DNA methyltransferase in glioma
therapy: promise and problems, Biochim Biophys Acta 1826 (2012)
71-82; A. V. Knizhnik et al., Survival and death strategies in
glioma cells: autophagy, senescence and apoptosis triggered by a
single type of temozolomide-induced DNA damage, PLoS One 8 (2013)
e55665. When MGMT expression was investigated in breast cancer
metastases to the brain, it was found that over half of the
intracranial lesions analyzed were strongly positive for MGMT
immunoreactivity. B. Ingold et al., Homogeneous MGMT
immunoreactivity correlates with an unmethylated MGMT promoter
status in brain metastases of various solid tumors, PLoS One 4
(2009) e4775.
[0009] MGMT activity is unusual in that it represents a "suicide"
mechanism, whereby acceptance of the alkyl group from DNA
irreversibly inactivates the enzyme and leads to its rapid
degradation. This feature is exploited by the use of specific MGMT
inhibitors, such as O6-benzylguanine (O6-BG), which act as
pseudosubstrates. B. Kaina, et al. Targeting O(6)-methylguanine-DNA
methyltransferase with specific inhibitors as a strategy in cancer
therapy, Cell Mol Life Sci 67 (2010) 3663-3681. Benzylation of MGMT
via reaction with O6-BG causes the same structural change in the
enzyme as that seen after alkylation following DNA repair, and
therefore also leads to rapid degradation of the protein. A. E.
Pegg, et al., Use of antibodies to human O6-alkylguanine-DNA
alkyltransferase to study the content of this protein in cells
treated with O6-benzylguanine or
N-methyl-N'-nitro-N-nitrosoguanidine, Carcinogenesis 12 (1991)
1679-1683. Ablation of MGMT activity after treatment of
MGMT-positive cells with O6-BG generally increases their
sensitivity to killing by TMZ, and this has been well established
in numerous in vitro and in vivo tumor models. However, a recent
phase-II clinical trial yielded mixed outcomes when O6-BG and TMZ
were administered to brain cancer patients with TMZ-resistant
tumors: while the addition of the MGMT inhibitor restored
TMZ-sensitivity in a fraction (16%) of patients with anaplastic
glioma, there was no significant effect (3%) in patients with GBM.
J. A. Quinn, et al., Phase II trial of temozolomide plus
o6-benzylguanine in adults with recurrent, temozolomide-resistant
malignant glioma, J Clin Oncol 27 (2009) 1262-1267. While the
underlying reasons for this disappointing outcome remain to be
established, the limited response documented in this trial does not
generate enthusiasm for the potential study of this drug
combination in brain metastatic breast cancer patients.
[0010] There is a need to prepare effective therapeutic agents and
methods of use thereof in the treatment of cancers such as
malignant gliomas and other cancers metastasized in the brain.
SUMMARY OF THE INVENTION
[0011] The invention provides for a method for treating brain
metastases of a cancer in a mammal, comprising delivering to the
mammal a therapeutically effective amount of a perillyl alcohol
derivative, such as a perillyl alcohol carbamate. The invention
also provides for a method for treating a metastatic cancer of a
mammal that has spread to the brain by delivering to the mammal a
therapeutically effective amount of a perillyl alcohol derivative,
such as a perillyl alcohol carbamate.
[0012] The perillyl alcohol derivative may be perillyl alcohol
conjugated with a therapeutic agent, such as a chemotherapeutic
agent. The chemotherapeutic agents that may be used in the present
invention include a DNA alkylating agent, a topoisomerase
inhibitor, an endoplasmic reticulum stress inducing agent, a
platinum compound, an antimetabolite, an enzyme inhibitor, and a
receptor antagonist. In certain embodiments, the therapeutic agent
can be temozolomide (TMZ). The perillyl alcohol carbamate may be
3-methyl
4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamic
acid-4-isopropenyl cyclohex-1-enylmethyl ester (TMZ-POH).
[0013] The method may further comprise treating the mammal with
radiation before, during, or after the administration of the
pharmaceutical composition, and/or further comprise delivering to
the mammal another chemotherapeutic agent. The brain metastasis or
metastases to be treated can originate or spread from a cancer such
as a systemic cancer, lung cancer, prostate cancer, breast cancer,
hematopoietic cancer, ovarian cancer, bladder cancer, germ cell
tumors, kidney cancer, leukemia, lymphoma, and melanoma. In one
embodiment, the brain metastases originate or are spread from
metastatic breast cancer.
[0014] The routes of administration of the perillyl alcohol
derivative include inhalation, intranasal, oral, intravenous,
subcutaneous or intramuscular administration. In some embodiments,
the perillyl alcohol derivative can be administered intranasally
using a nasal delivery device selected from the group consisting of
an intranasal inhaler, an intranasal spray device, an atomizer, a
nebulizer, a metered dose inhaler (MDI), a pressurized dose
inhaler, an insufflator, a unit dose container, a pump, a dropper,
a nasal spray bottle, a squeeze bottle and a bi-directional
device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of dimethyl celecoxib (DMC) in killing
U87, A172 and U251 human glioma cells.
[0016] FIG. 2 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the POH-DMC conjugate in killing U87,
A172 and U251 human glioma cells according to the present
invention.
[0017] FIG. 3 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of temozolomide (TMZ) in killing U87,
A172 and U251 human glioma cells.
[0018] FIG. 4 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the TMZ-POH conjugate in killing U87,
A172, and U251 human glioma cells according to the present
invention.
[0019] FIG. 5 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the POH-Rolipram conjugate and
Rolipram in killing A172 human glioma cells.
[0020] FIG. 6 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the POH-Rolipram conjugate and
Rolipram in killing U87 human glioma cells.
[0021] FIG. 7 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the POH-Rolipram conjugate and
Rolipram in killing U251 human glioma cells.
[0022] FIG. 8 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the POH-Rolipram conjugate and
Rolipram in killing L229 human glioma cells.
[0023] FIGS. 9A and 9B show the inhibition of tumor growth by
butyryl-POH in mouse models. FIG. 9A shows the images of
subcutaneous U-87 gliomas in nude mice treated with butyryl-POH,
purified (S)-perillyl alcohol having a purity greater than 98.5%
("Purified POH"), POH purchased from Sigma chemicals ("Sigma"), or
phosphate buffered saline ("PBS"; negative control). FIG. 9B shows
average tumor growth over time (total time period of 60 days).
[0024] FIG. 10 shows the results of a Colony forming Assay (CFA)
demonstrating the cytotoxic effect of TMZ and TMZ-POH on TMZ
sensitive (U251) and TMZ resistant (U251TR) U251 cells.
[0025] FIG. 11 shows the results of a Colony forming Assay (CFA)
demonstrating the cytotoxic effect of POH on TMZ sensitive (U251)
and TMZ resistant (U251TR) U251 cells.
[0026] FIG. 12 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the TMZ-POH conjugate in killing U251
cells, U251TR cells, and normal astrocytes.
[0027] FIG. 13 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of the TMZ-POH conjugate in killing
normal astrocytes, brain endothelial cells (BEC; confluent and
subconfluent), and tumor brain endothelial cells (TuBEC).
[0028] FIG. 14 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of TMZ and the TMZ-POH conjugate in
killing USC-04 glioma cancer stem cells.
[0029] FIG. 15 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of POH in killing USC-04 glioma cancer
stem cells.
[0030] FIG. 16 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of TMZ and the TMZ-POH conjugate in
killing USC-02 glioma cancer stem cells.
[0031] FIG. 17 shows the results of the MTT cytotoxicity assays
demonstrating the efficacy of POH in killing USC-02 glioma cancer
stem cells.
[0032] FIG. 18 shows a western blot demonstrating that TMZ-POH
induces ER stress (ERS) in TMZ sensitive ("U251-TMZs") and
resistant ("U251-TMZr") U251 glioma cells.
[0033] FIG. 19 shows survival of breast cancer cells after drug
treatment, where various breast cancer cell lines were exposed to
increasing concentrations of TMZ or TMZ-POH for 48 hours, and
survival was determined via colony formation assay (CFA). Shown is
the fraction of colony-forming cells, where colony formation by
control cells (treated with DMSO vehicle only) is set at 1. Graphs
with error bars display mean (.+-.SD) from .gtoreq.3 independent
experiments; graphs without error bars show the average from two
independent experiments.
[0034] FIGS. 20A-20B show cytotoxic potency of TMZ-POH and its
individual components, where survival of drug-treated MDA-MB-231
cells was determined by CFA. In FIG. 20A, cells were exposed for 48
hours to increasing concentrations of TMZ (diamonds), TMZ-POH
(circles), POH (triangles), or equimolar concentrations of TMZ plus
POH (squares). Colony formation by control cells (treated with
vehicle only) is set at 1; graphs display mean (.+-.SD) from
.gtoreq.3 independent experiments. In FIG. 20B, cells were exposed
to 10 .mu.M TMZ-POH, TMZ, or POH, or to 10 .mu.M TMZ-POH or TMZ
combined with 10 .mu.M POH. Shown is a photo of one representative
CFA.
[0035] FIGS. 21A-21C show MGMT expression levels in various cell
lines, where all parts show Western blot analysis of MGMT protein
levels with actin as the loading control. FIG. 21A shows MGMT basal
levels in the six breast cancer cell lines used in this study. FIG.
21B shows MGMT basal levels in three GBM cell lines compared to
MCF7 breast cancer cells. In FIG. 21C, MDA-MB-468 cells were
treated with the indicated concentrations of TMZ-POH, TMZ, or O6-BG
for 17 hours before harvest of cellular lysates. vh.=cells treated
with vehicle only.
[0036] FIGS. 22A-22B show drug sensitivity of MGMT-transfected
cells, where MDA-MB-231 cells were stably transfected with MGMT
cDNA. In FIG. 22A, two individually selected clones, 231-MGMT-1 and
-2, were analyzed by Western blot for basal level MGMT protein
expression in comparison to parental cells. In FIG. 22B, 231-MGMT-1
and -2 were treated with increasing concentrations of TMZ-POH and
TMZ for 48 hours, and cell survival was analyzed by CFA. Graph with
231-MGMT-1 cells displays mean (.+-.SD) from 3 independent
experiments; graph with 231-MGMT-2 cells shows the average from two
independent experiments.
[0037] FIGS. 23A-23C show effect of inclusion of O6-BG, where cells
were exposed to TMZ or TMZ-POH for 48 hours in the presence or
absence of O6-BG, and cell survival was determined by CFA. FIG. 23A
shows colony survival of MDA-MB-231 cells; FIG. 23B shows
MGMT-transfected 231-MGMT-2 cells, and FIG. 23C shows MDA-MB-468
cells. Shown is mean number of colonies (+SD) from .gtoreq.3 wells
treated in parallel.
[0038] FIGS. 24A-24D show drug effects on DNA damage marker, where
cells were treated with different concentrations of TMZ-POH or TMZ
and analyzed by Western blot analysis for expression levels of
.gamma.-H2AX, a marker for double-strand DNA damage. Actin was used
as a loading control. MDA-MB-231 cells were treated with 50 .mu.M
TMZ-POH for the indicated time periods (FIG. 24A); MDA-MB-231 cells
were treated with 50 .mu.M TMZ-POH or 50 .mu.M TMZ for the
indicated time periods (FIG. 24B); MDA-MB-231 cells were treated
with TMZ-POH, TMZ, POH, or TMZ combined with POH (all at 10 .mu.M
each) for 24 hours (FIG. 24C); MCF7 cells were treated with or
without 50 .mu.M TMZ-POH in the presence or absence of 30 O6-BG for
48 hours (FIG. 24D).
[0039] FIGS. 25A-25B show DNA damage and cell death marker
analysis, where MDA-MB-231 cells were used for Western blot
analysis of expression levels for markers of DNA damage
(.gamma.-H2AX) and cell death (activated caspase 7 and cleaved
PARP). In FIG. 25A, cells were treated with 15 .mu.M TMZ-POH and
harvested every 24 hours up to 6 days; control cells remained
untreated, or received vehicle (vh.) only. In FIG. 25B, cells were
treated with 20 .mu.M of either TMZ-POH, TMZ, or POH individually,
or with 20 .mu.M TMZ combined with 20 .mu.M POH (TMZ+POH) and
harvested after 24 hours or 5 days; control cells remained
untreated, or received vehicle (vh.) only. In the case of caspase
7, only the activated (cleaved) form is shown (cl. C-7). In the
case of PARP, the top panel shows both full-length and
proteolytically cleaved forms of the protein, whereas the bottom
panel only shows faster-migrating, cleaved PARP.
[0040] FIGS. 26A-26C depicts determination of drug stability, where
MDA-MB-231 cells were analyzed in colony formation assays. In FIG.
26A, cells were treated with 15 .mu.M TMZ-POH or 30 .mu.M TMZ for
30 min or 1, 2, 4, and 24 hours. Thereafter, drug-containing medium
was removed, fresh medium (without drug) was added, and cells
remained undisturbed until colony staining 12 days later. In FIG.
26B, cells were exposed to supernatant (i.e., the drug-containing
medium removed from cells shown in FIG. 26A). The arrows indicate
which cells received which supernatant. After 24 hours of
incubation, all drug-containing medium was removed, fresh medium
(without drug) was added, and cells remained undisturbed until
colony staining 12 days later. FIG. 26C shows a representative
6-well plate with stained colonies. Left panel (untreated): control
cells without drug treatment. Middle panel (0-24 h): Cells received
15 .mu.M TMZ-POH or 30 .mu.M TMZ for 24 hours. Right panel (1-25
h): TMZ-POH and TMZ were incubated in neutral buffer at 37.degree.
C. for 1 hour before addition to cells to a final concentration of
15 .mu.M TMZ-POH and 30 .mu.M TMZ for 24 hours.
[0041] FIGS. 27A-27B show drug effects on intracranial tumor
growth, where luciferase-positive D3H2LN cells were implanted into
the brains of 24 nude mice. Ten days later, tumor take was
confirmed via bioluminescent imaging, and treatment was initiated
with vehicle only (control group), 25 mg/kg TMZ-POH, or 25 mg/kg
TMZ, once daily over the course of 10 days. In FIG. 27A, all
surviving animals were imaged again on days 21, 28, and 36. The top
panel shows one representative mouse from the vehicle-only treated
group. Note 12-fold increased ROI radiance (representative of tumor
growth) from 1.65E7 to 1.92E8 between days 10 and 21. The bottom
panel shows a representative mouse from the group of
TMZ-POH-treated animals. Here, radiance increased only 1.7-fold
(from 1.11E7 to 1.92E7) between days 10 and 21, but reached 1.88E8
(similar to control mouse on day 21) by day 43. Heat bar to the
right shows scale of radiance. FIG. 27B shows Kaplan-Meier survival
plot of all animals carrying intracranial tumors. Arrow labeled Rx
indicates the time period of treatment. Statistical difference
between groups of TMZ-treated and TMZ-POH-treated animals:
p<0.001.
DETAILED DESCRIPTION
[0042] The present invention provides for a derivative of
monoterpene or sesquiterpene, such as a perillyl alcohol
derivative. The present invention also provides for a
pharmaceutical composition comprising a derivative of monoterpene
or sesquiterpene, such as a perillyl alcohol derivative.
[0043] For example, the perillyl alcohol derivative may be a
perillyl alcohol carbamate. The perillyl alcohol derivative may be
perillyl alcohol conjugated with a therapeutic agent such as a
chemotherapeutic agent. The monoterpene (or sesquiterpene)
derivative may be formulated into a pharmaceutical composition,
where the monoterpene (or sesquiterpene) derivative is present in
amounts ranging from about 0.01% (w/w) to about 100% (w/w), from
about 0.1% (w/w) to about 80% (w/w), from about 1% (w/w) to about
70% (w/w), from about 10% (w/w) to about 60% (w/w), or from about
0.1% (w/w) to about 20% (w/w). The present compositions can be
administered alone, or may be co-administered together with
radiation or another agent (e.g., a chemotherapeutic agent), to
treat a disease such as cancer. Treatments may be sequential, with
the monoterpene (or sesquiterpene) derivative being administered
before or after the administration of other agents. For example, a
perillyl alcohol carbamate may be used to sensitize a cancer
patient to radiation or chemotherapy. Alternatively, agents may be
administered concurrently. The route of administration may vary,
and can include, inhalation, intranasal, oral, transdermal,
intravenous, subcutaneous or intramuscular injection. The present
invention also provides for a method of treating a disease such as
cancer, comprising the step of delivering to a patient a
therapeutically effective amount of a derivative of monoterpene (or
sesquiterpene).
[0044] The compositions of the present invention may contain one or
more types of derivatives of monoterpene (or sesquiterpene).
Monoterpenes include terpenes that consist of two isoprene units.
Monoterpenes may be linear (acyclic) or contain rings. Derivatives
of monoterpenoids are also encompassed by the present invention.
Monoterpenoids may be produced by biochemical modifications such as
oxidation or rearrangement of monoterpenes. Examples of
monoterpenes and monoterpenoids include, perillyl alcohol (S(-))
and (R(+)), ocimene, myrcene, geraniol, citral, citronellol,
citronellal, linalool, pinene, terpineol, terpinen, limonene,
terpinenes, phellandrenes, terpinolene, terpinen-4-ol (or tea tree
oil), pinene, terpineol, terpinen; the terpenoids such as p-cymene
which is derived from monocyclic terpenes such as menthol, thymol
and carvacrol; bicyclic monoterpenoids such as camphor, borneol and
eucalyptol.
[0045] Monoterpenes may be distinguished by the structure of a
carbon skeleton and may be grouped into acyclic monoterpenes (e.g.,
myrcene, (Z)- and (E)-ocimene, linalool, geraniol, nerol,
citronellol, myrcenol, geranial, citral a, neral, citral b,
citronellal, etc.), monocyclic monoterpenes (e.g., limonene,
terpinene, phellandrene, terpinolene, menthol, carveol, etc.),
bicyclic monoterpenes (e.g., pinene, myrtenol, myrtenal, verbanol,
verbanon, pinocarvcol, carene, sabinene, camphene, thujene, etc.)
and tricyclic monoterpenes (e.g. tricyclene). See Encyclopedia of
Chemical Technology, Fourth Edition, Volume 23, page 834-835.
[0046] Sesquiterpenes of the present invention include terpenes
that consist of three isoprene units. Sesquiterpenes may be linear
(acyclic) or contain rings. Derivatives of sesquiterpenoids are
also encompassed by the present invention. Sesquiterpenoids may be
produced by biochemical modifications such as oxidation or
rearrangement of sesquiterpenes. Examples of sesquiterpenes include
farnesol, farnesal, farnesylic acid and nerolidol.
[0047] The derivatives of monoterpene (or sesquiterpene) include,
but are not limited to, carbamates, esters, ethers, alcohols and
aldehydes of the monoterpene (or sesquiterpene). Monoterpene (or
sesquiterpene) alcohols may be derivatized to carbamates, esters,
ethers, aldehydes or acids.
Chloroformate
[0048] Carbamate refers to a class of chemical compounds sharing
the functional group
##STR00001##
based on a carbonyl group flanked by an oxygen and a nitrogen.
R.sup.1, R.sup.2 and R.sup.3 can be a group such as alkyl, aryl,
etc., which can be substituted. The R groups on the nitrogen and
the oxygen may form a ring. R.sup.1--OH may be a monoterpene, e.g.,
POH. The R.sup.2--N--R.sup.3 moiety may be a therapeutic agent.
[0049] Carbamates may be synthesized by reacting isocyanate and
alcohol, or by reacting chloroformate with amine. Carbamates may be
synthesized by reactions making use of phosgene or phosgene
equivalents. For example, carbamates may be synthesized by reacting
phosgene gas, diphosgene or a solid phosgene precursor such as
triphosgene with two amines or an amine and an alcohol. Carbamates
(also known as urethanes) can also be made from reaction of a urea
intermediate with an alcohol. Dimethyl carbonate and diphenyl
carbonate are also used for making carbamates. Alternatively,
carbamates may be synthesized through the reaction of alcohol
and/or amine precursors with an ester-substituted diaryl carbonate,
such as bismethylsalicylcarbonate (BMSC). U.S. Patent Publication
No. 20100113819.
[0050] Carbamates may be synthesized by the following approach:
##STR00002##
Suitable reaction solvents include, but are not limited to,
tetrahydrofuran, dichloromethane, dichloroethane, acetone, and
diisopropyl ether. The reaction may be performed at a temperature
ranging from about -70.degree. C. to about 80.degree. C., or from
about -65.degree. C. to about 50.degree. C. The molar ratio of
perillyl chloroformate to the substrate R--NH.sub.2 may range from
about 1:1 to about 2:1, from about 1:1 to about 1.5:1, from about
2:1 to about 1:1, or from about 1.05:1 to about 1.1:1. Suitable
bases include, but are not limited to, organic bases, such as
triethylamine, potassium carbonate, N,N'-diisopropylethylamine,
butyl lithium, and potassium-t-butoxide.
[0051] Alternatively, carbamates may be synthesized by the
following approach:
##STR00003##
Suitable reaction solvents include, but are not limited to,
dichloromethane, dichloroethane, toluene, diisopropyl ether, and
tetrahydrofuran. The reaction may be performed at a temperature
ranging from about 25.degree. C. to about 110.degree. C., or from
about 30.degree. C. to about 80.degree. C., or about 50.degree. C.
The molar ratio of perillyl alcohol to the substrate
R--N.dbd.C.dbd.O may range from about 1:1 to about 2:1, from about
1:1 to about 1.5:1, from about 2:1 to about 1:1, or from about
1.05:1 to about 1.1:1.
[0052] Esters of the monoterpene (or sesquiterpene) alcohols of the
present invention can be derived from an inorganic acid or an
organic acid. Inorganic acids include, but are not limited to,
phosphoric acid, sulfuric acid, and nitric acid. Organic acids
include, but are not limited to, carboxylic acid such as benzoic
acid, fatty acid, acetic acid and propionic acid, and any
therapeutic agent bearing at least one carboxylic acid functional
group Examples of esters of monoterpene (or sesquiterpene) alcohols
include, but are not limited to, carboxylic acid esters (such as
benzoate esters, fatty acid esters (e.g., palmitate ester,
linoleate ester, stearate ester, butyryl ester and oleate ester),
acetates, propionates (or propanoates), and formates), phosphates,
sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl).
[0053] A specific example of a monoterpene that may be used in the
present invention is perillyl alcohol (commonly abbreviated as
POH). The derivatives of perillyl alcohol include, perillyl alcohol
carbamates, perillyl alcohol esters, perillic aldehydes,
dihydroperillic acid, perillic acid, perillic aldehyde derivatives,
dihydroperillic acid esters and perillic acid esters. The
derivatives of perillyl alcohol may also include its oxidative and
nucleophilic/electrophilic addition derivatives. U.S. Patent
Publication No. 20090031455. U.S. Pat. Nos. 6,133,324 and
3,957,856. Many examples of derivatives of perillyl alcohol are
reported in the chemistry literature (see Appendix A: CAS Scifinder
search output file, retrieved Jan. 25, 2010).
[0054] In certain embodiments, a POH carbamate is synthesized by a
process comprising the step of reacting a first reactant of
perillyl chloroformate with a second reactant such as dimethyl
celocoxib (DMC), temozolomide (TMZ) and rolipram. The reaction may
be carried out in the presence of tetrahydrofuran and a base such
as n-butyl lithium. Perillyl chloroformate may be made by reacting
POH with phosgene. For example, POH conjugated with temozolomide
through a carbamate bond may be synthesized by reacting
temozolomide with oxalyl chloride followed by reaction with
perillyl alcohol. The reaction may be carried out in the presence
of 1,2-dichloroethane.
[0055] POH carbamates encompassed by the present invention include,
but not limited to, 4-(bis-N,N'-4-isopropenyl
cyclohex-1-enylmethyloxy carbonyl [5-(2,5-dimethyl
phenyl)-3-trifluoromethyl pyrazol-1-yl] benzenesulfonamide,
4-(3-cyclopentyloxy-4-methoxy
phenyl)-2-oxo-pyrrolidine-1-carboxylic acid 4-isopropenyl
cyclohex-1-enylmethyl ester, and (3-methyl
4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)carbamic
acid-4-isopropenyl cyclohex-1-enylmethyl ester. The details of the
chemical reactions generating these compounds are described in the
Examples below.
[0056] In certain embodiments, perillyl alcohol derivatives may be
perillyl alcohol fatty acid esters, such as palmitoyl ester of POH
and linoleoyl ester of POH, the chemical structures of which are
shown below.
##STR00004##
Hexadecanoic Acid 4-isopropenyl-cyclohex-1-enylmethyl Ester
(Palmitoyl Ester of POH)
##STR00005##
[0057] Octadeca-9, 12-dienoic Acid
4-isopropenyl-cyclohex-1-enylmethyl Ester (Linoleoyl Ester of
POH)
[0058] The monoterpene (or sesquiterpene) derivative may be a
monoterpene (or sesquiterpene) conjugated with a therapeutic agent.
A monoterpene (or sesquiterpene) conjugate encompassed by the
present invention is a molecule having a monoterpene (or
sesquiterpene) covalently bound via a chemical linking group to a
therapeutic agent. The molar ratio of the monoterpene (or
sesquiterpene) to the therapeutic agent in the monoterpene (or
sesquiterpene) conjugate may be 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1,
or any other suitable molar ratios. The monoterpene (or
sesquiterpene) and the therapeutic agent may be covalently linked
through carbamate, ester, ether bonds, or any other suitable
chemical functional groups. When the monoterpene (or sesquiterpene)
and the therapeutic agent are conjugated through a carbamate bond,
the therapeutic agent may be any agent bearing at least one
carboxylic acid functional group, or any agent bearing at least one
amine functional group. In a specific example, a perillyl alcohol
conjugate is perillyl alcohol covalently bound via a chemical
linking group to a chemotherapeutic agent.
[0059] According to the present invention, the therapeutic agents
that may be conjugated with monoterpene (or sesquiterpene) include,
but are not limited to, chemotherapeutic agents, therapeutic agents
for treatment of CNS disorders (including, without limitation,
primary degenerative neurological disorders such as Alzheimer's,
Parkinson's, multiple sclerosis, Attention-Deficit Hyperactivity
Disorder or ADHD, psychological disorders, psychosis and
depression), immunotherapeutic agents, angiogenesis inhibitors, and
anti-hypertensive agents. Anti-cancer agents that may be conjugated
with monoterpene or sesquiterpene can have one or more of the
following effects on cancer cells or the subject: cell death;
decreased cell proliferation; decreased numbers of cells;
inhibition of cell growth; apoptosis; necrosis; mitotic
catastrophe; cell cycle arrest; decreased cell size; decreased cell
division; decreased cell survival; decreased cell metabolism;
markers of cell damage or cytotoxicity; indirect indicators of cell
damage or cytotoxicity such as tumor shrinkage; improved survival
of a subject; or disappearance of markers associated with
undesirable, unwanted, or aberrant cell proliferation. U.S. Patent
Publication No. 20080275057.
[0060] Also encompassed by the present invention is admixtures
and/or coformulations of a monoterpene (or sesquiterpene) and at
least one therapeutic agent.
[0061] Chemotherapeutic agents include, but are not limited to, DNA
alkylating agents, topoisomerase inhibitors, endoplasmic reticulum
stress inducing agents, a platinum compound, an antimetabolite,
vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor
antagonists, tyrosine kinase inhibitors, boron radiosensitizers
(i.e. velcade), and chemotherapeutic combination therapies.
[0062] Non-limiting examples of DNA alkylating agents are nitrogen
mustards, such as Cyclophosphamide (Ifosfamide, Trofosfamide),
Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine
and Estramustine; nitrosoureas, such as Carmustine (BCNU),
Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and
Streptozocin; alkyl sulfonates, such as Busulfan (Mannosulfan,
Treosulfan); Aziridines, such as Carboquone, Triaziquone,
Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as
Dacarbazine and Temozolomide (TMZ); Altretamine and
Mitobronitol.
[0063] Non-limiting examples of Topoisomerase I inhibitors include
Campothecin derivatives including SN-38, APC, NPC, campothecin,
topotecan, exatecan mesylate, 9-nitrocamptothecin,
9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan,
diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as
described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and
U.S. Patent Publication No. 200510250854; Protoberberine alkaloids
and derivatives thereof including berberrubine and coralyne as
described in Li et al. (2000) Biochemistry 39(24):7107-7116 and
Gatto et al. (1996) Cancer Res. 15(12):2795-2800; Phenanthroline
derivatives including Benzo[i]phenanthridine, Nitidine, and
fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.
11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as
described in Xu (1998) Biochemistry 37(10):3558-3566; and
Anthracycline derivatives including Doxorubicin, Daunorubicin, and
Mitoxantrone as described in Foglesong et al. (1992) Cancer
Chemother. Pharmacol. 30(2):123-]25, Crow et al. (1994) J. Med.
Chem. 37(19):31913194, and Crespi et al. (1986) Biochem. Biophys.
Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but
are not limited to Etoposide and Teniposide. Dual topoisomerase I
and II inhibitors include, but are not limited to, Saintopin and
other Naphthecenediones, DACA and other Acridine-4-Carboxamindes,
Intoplicine and other Benzopyridoindoles, TAS-I03 and other
7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR 11576 and
other Benzophenazines, XR 5944 and other Dimeric compounds,
7-oxo-7H-dibenz[f,ij]Isoquinolines and
7-oxo-7H-benzo[e]pyrimidines, and Anthracenyl-amino Acid Conjugates
as described in Denny and Baguley (2003) Curr. Top. Med. Chem.
3(3):339-353. Some agents inhibit Topoisomerase II and have DNA
intercalation activity such as, but not limited to, Anthracyclines
(Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,
Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones
(Mitoxantrone and Pixantrone).
[0064] Examples of endoplasmic reticulum stress inducing agents
include, but are not limited to, dimethyl-celecoxib (DMC),
nelfinavir, celecoxib, and boron radiosensitizers (i.e. velcade
(Bortezomib)).
[0065] Platinum based compounds are a subclass of DNA alkylating
agents. Non-limiting examples of such agents include Cisplatin,
Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin,
Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J.
Clin. Oncol. 201:1232-1237 and in general, CHEMOTHERAPY FOR
GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in
the Series Basic and Clinical Oncology, Angioli et al. Eds.,
2004).
[0066] "FOLFOX" is an abbreviation for a type of combination
therapy that is used to treat colorectal cancer. It includes 5-FU,
oxaliplatin and leucovorin. Information regarding this treatment is
available on the National Cancer Institute's web site, cancer.gov,
last accessed on Jan. 16, 2008.
[0067] "FOLFOX/BV" is an abbreviation for a type of combination
therapy that is used to treat colorectal cancer. This therapy
includes 5-FU, oxaliplatin, leucovorin and Bevacizumab.
Furthermore, "XELOX/BV" is another combination therapy used to
treat colorectal cancer, which includes the prodrug to 5-FU, known
as Capecitabine (Xeloda) in combination with oxaliplatin and
bevacizumab. Information regarding these treatments are available
on the National Cancer Institute's web site, cancer.gov or from 23
the National Comprehensive Cancer Network's web site, nccn.org,
last accessed on May 27, 2008.
[0068] Non-limiting examples of antimetabolite agents include Folic
acid based, i.e. dihydrofolate reductase inhibitors, such as
Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase
inhibitors, such as Raltitrexed, Pemetrexed; Purine based, i.e. an
adenosine deaminase inhibitor, such as Pentostatin, a thiopurine,
such as Thioguanine and Mercaptopurine, a
halogenated/ribonucleotide reductase inhibitor, such as Cladribine,
Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such
as Thioguanine; or Pyrimidine based, i.e. cytosine/cytidine:
hypomethylating agent, such as Azacitidine and Decitabine, a DNA
polymerase inhibitor, such as Cytarabine, a ribonucleotide
reductase inhibitor, such as Gemcitabine, or a thymine/thymidine:
thymidylate synthase inhibitor, such as a Fluorouracil (5-FU).
Equivalents to 5-FU include prodrugs, analogs and derivative
thereof such as 5'-deoxy-5-fluorouridine (doxifluroidine),
1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine
(Xeloda), S-I (MBMS-247616, consisting of tegafur and two
modulators, a 5-chloro-2,4-dihydroxypyridine and potassium
oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337),
LY231514 and ZD9331, as described for example in Papamicheal (1999)
The Oncologist 4:478-487.
[0069] Examples of vincalkaloids, include, but are not limited to
Vinblastine, Vincristine, Vinflunine, Vindesine and
Vinorelbine.
[0070] Examples of taxanes include, but are not limited to
docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An
example of an epothilone is iabepilone.
[0071] Examples of enzyme inhibitors include, but are not limited
to farnesyltransferase inhibitors (Tipifarnib); CDK inhibitor
(Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib);
phosphodiesterase inhibitor (Anagrelide; rolipram); IMP
dehydrogenase inhibitor (Tiazofurine); and lipoxygenase inhibitor
(Masoprocol). Examples of receptor antagonists include, but are not
limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and
a sex steroid (Testolactone).
[0072] Examples of tyrosine kinase inhibitors include, but are not
limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib,
Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib,
Neratinib); RTK class III: C-kit (Axitinib, Sunitinib, Sorafenib),
FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and
VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib);
bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus
kinase 2 (Lestaurtinib).
[0073] "Lapatinib" (Tykerb.RTM.) is an dual EGFR and erbB-2
inhibitor. Lapatinib has been investigated as an anticancer
monotherapy, as well as in combination with trastuzumab,
capecitabine, letrozole, paclitaxel and FOLFIRI (irinotecan,
5-fluorouracil and leucovorin), in a number of clinical trials. It
is currently in phase III testing for the oral treatment of
metastatic breast, head and neck, lung, gastric, renal and bladder
cancer.
[0074] A chemical equivalent of lapatinib is a small molecule or
compound that is a tyrosine kinase inhibitor (TKI) or alternatively
a HER-1 inhibitor or a HER-2 inhibitor. Several TKIs have been
found to have effective antitumor activity and have been approved
or are in clinical trials. Examples of such include, but are not
limited to, Zactima (ZD6474), Iressa (gefitinib), imatinib mesylate
(STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib (CI
1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib
(BAY 43-9006), sutent (SUI 1248) and lefltmomide (SU101).
[0075] PTK/ZK is a tyrosine kinase inhibitor with broad specificity
that targets all VEGF receptors (VEGFR), the platelet-derived
growth factor (PDGF) receptor, c-KIT and c-Fms. Drevs (2003) Idrugs
6(8):787-794. PTK/ZK is a targeted drug that blocks angiogenesis
and lymphangiogenesis by inhibiting the activity of all known
receptors that bind VEGF including VEGFR-I (Flt-1), VEGFR-2
(KDR/Flk-1) and VEGFR-3 (Flt-4). The chemical names of PTK/ZK are
1-[4-Chloroanilino]-4-[4-pyridylmethyl] phthalazine Succinate or
1-Phthalazinamine,
N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-butanedioate (1:1).
Synonyms and analogs of PTK/TK are known as Vatalanib, CGP79787D,
PTK787/ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TK
inhibitor, ZK 222584 and ZK.
[0076] Chemotherapeutic agents that can be conjugated with
monoterpene or sesquiterpene may also include amsacrine,
Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide,
asparagine depleter Asparaginase/Pegaspargase), Celecoxib,
Demecolcine, Eleselomol, Elsamitrucin, Etoglucid, Lonidamine,
Lucanthone, Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and
Vorinostat.
[0077] The monoterpene or sesquiterpene derivative may be
conjugated with angiogenesis inhibitors. Examples of angiogenesis
inhibitors include, but are not limited to, angiostatin, angiozyme,
antithrombin III, AG3340, VEGF inhibitors, batimastat, bevacizumab
(avastin), BMS-275291, CAI, 2C3, HuMV833 Canstatin, Captopril,
carboxyamidotriazole, cartilage derived inhibitor (CDI), CC-5013,
6-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin,
combretastatin A4 Phosphate, Dalteparin, EMD 121974 (Cilengitide),
endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginone
hydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11
Inducible protein 10, interferon-alpha, interleukin 12, lavendustin
A, LY317615 or AE-941, marimastat, mspin, medroxpregesterone
acetate, Meth-1, Meth-2, 2-methoxyestradiol (2-ME), neovastat,
oteopontin cleaved product, PEX, pigment epithelium growth factor
(PEGF), platelet factor 4, prolactin fragment, proliferin-related
protein (PRP), PTK787/ZK 222584, ZD6474, recombinant human platelet
factor 4 (rPF4), restin, squalamine, SU5416, SU6668, SU11248
suramin, Taxol, Tecogalan, thalidomide, thrombospondin, TNP-470,
troponin-1, vasostatin, VEG1, VEGF-Trap, and ZD6474.
[0078] Non-limiting examples of angiogenesis inhibitors also
include, tyrosine kinase inhibitors, such as inhibitors of the
tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2),
inhibitors of epidermal-derived, fibroblast-derived, or platelet
derived growth factors, MMP (matrix metalloprotease) inhibitors,
integrin blockers, pentosan polysulfate, angiotensin II
antagonists, cyclooxygenase inhibitors (including non-steroidal
anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, as
well as selective cyclooxygenase-2 inhibitors such as celecoxib and
rofecoxib), and steroidal anti-inflammatories (such as
corticosteroids, mineralocorticoids, dexamethasone, prednisone,
prednisolone, methylpred, betamethasone).
[0079] Other therapeutic agents that modulate or inhibit
angiogenesis and may also be conjugated with monoterpene or
sesquiterpene include agents that modulate or inhibit the
coagulation and fibrinolysis systems, including, but not limited
to, heparin, low molecular weight heparins and carboxypeptidase U
inhibitors (also known as inhibitors of active thrombin activatable
fibrinolysis inhibitor [TAFIa]). U.S. Patent Publication No.
20090328239. U.S. Pat. No. 7,638,549.
[0080] Non-limiting examples of the anti-hypertensive agents
include angiotensin converting enzyme inhibitors (e.g., captopril,
enalapril, delapril etc.), angiotensin II antagonists (e.g.,
candesartan cilexetil, candesartan, losartan (or Cozaar), losartan
potassium, eprosartan, valsartan (or Diovan), termisartan,
irbesartan, tasosartan, olmesartan, olmesartan medoxomil etc.),
calcium antagonists (e.g., manidipine, nifedipine, amlodipine (or
Amlodin), efonidipine, nicardipine etc.), diuretics, renin
inhibitor (e.g., aliskiren etc.), aldosterone antagonists (e.g.,
spironolactone, eplerenone etc.), beta-blockers (e.g., metoprolol
(or Toporol), atenolol, propranolol, carvedilol, pindolol etc.),
vasodilators (e.g., nitrate, soluble guanylate cyclase stimulator
or activator, prostacycline etc.), angiotensin vaccine, clonidine
and the like. U.S. Patent Publication No. 20100113780.
[0081] Other therapeutic agents that may be conjugated with
monoterpene (or sesquiterpene) include, but are not limited to,
Sertraline (Zoloft), Topiramate (Topamax), Duloxetine(Cymbalta),
Sumatriptan (Imitrex), Pregabalin (Lyrica), Lamotrigine (Lamictal),
Valaciclovir (Valtrex), Tamsulosin (Flomax), Zidovudine (Combivir),
Lamivudine (Combivir), Efavirenz (Sustiva), Abacavir (Epzicom),
Lopinavir (Kaletra), Pioglitazone (Actos), Desloratidine
(Clarinex), Cetirizine (Zyrtec), Pentoprazole (Protonix),
Lansoprazole (Prevacid), Rebeprazole (Aciphex), Moxifloxacin
(Avelox), Meloxicam (Mobic), Dorzolamide (Truspot), Diclofenac
(Voltaren), Enlapril (Vasotec), Montelukast (Singulair), Sildenafil
(Viagra), Carvedilol (Coreg), Ramipril (Delix).
[0082] Table 1 lists pharmaceutical agents that can be conjugated
with monoterpene (or sesquiterpene), including structure of the
pharmaceutical agent and the preferred derivative for
conjugation.
TABLE-US-00001 TABLE 1 Brand Generic Preferred Name Name Activity
Structure Derivative Zoloft Sertraline Depression ##STR00006##
Carbamate Topamax Topiramate Seizures ##STR00007## Carbamate
Cymbalta Duloxetine Depression ##STR00008## Carbamate Imitrex
Sumatriptan Migraine ##STR00009## Carbamate Lyrica Pregabalin
Neuropathic pain ##STR00010## Carbamate or Ester Lamictal
Lamotrigine Seizures ##STR00011## Carbamate Valtrex Valaciclovir
Herpes ##STR00012## Carbamate Tarceva Erlotinib Non-small cell lung
cancer ##STR00013## Carbamate Flomax Tamsulosin Benign prostatic
Cancer ##STR00014## Carbamate Gleevec Imatinib Leukemia
##STR00015## Carbamate Combivir Zidovudine HIV infection
##STR00016## Carbamate Combivir Lamivudine HIV infection
##STR00017## Carbonate Sustiva Efavirenz HIV infection ##STR00018##
Carbamate Epzicom Abacavir HIV infection ##STR00019## Carbamate
Kaletra Lopinavir HIV infection ##STR00020## Carbamate Actos
Pioglitazone Type-2 diabetes ##STR00021## Carbamate Clarinex
Desloratidine Allergic rhinitis ##STR00022## Carbamate Zyrtec
Cetirizine Allergic ##STR00023## Ester Protonix Pentoprazole
Gastrointestinal ##STR00024## Carbamate Prevacid Lansoprazole
Gastrointestinal ##STR00025## Carbamate Aciphex Rebeprazole
Gastrointestinal ##STR00026## Carbamate Diovan Valsartan
Hypertension ##STR00027## Carbamate Cozaar Losartan Hypertension
##STR00028## Carbamate Avelox Moxifloxacin Bacterial infection
##STR00029## Carbamate or Ester Mobic Meloxicam Osteoarthritis
##STR00030## Carbamate Truspot Dorzolamide Intraocular pressure
##STR00031## Carbamate Voltaren Diclofenac Osteoarthritis &
rheumatoid arthritis ##STR00032## Carbamate or Ester Vasotec
Enlapril Hypertension ##STR00033## Carbamate or Ester Singulair
Montelukast Asthma ##STR00034## Ester Amlodin Amlodipine
Hypertension ##STR00035## Carbamate Toporol Metoprolol Hypertension
##STR00036## Carbamate Viagra Sildenafil Erectile dysfunction
##STR00037## Carbamate Coreg Carvedilol Hypertension ##STR00038##
Carbamate Delix Ramipril Hypertension ##STR00039## Carbamate or
Ester Sinemet (Parcopa, Atamet) L-DOPA Neurological disorders
##STR00040## Carbamate or Ester
[0083] The purity of the monoterpene (or sesquiterpene) derivatives
may be assayed by gas chromatography (GC) or high pressure liquid
chromatography (HPLC). Other techniques for assaying the purity of
monoterpene (or sesquiterpene) derivatives and for determining the
presence of impurities include, but are not limited to, nuclear
magnetic resonance (NMR) spectroscopy, mass spectrometry (MS),
GC-MS, infrared spectroscopy (IR), and thin layer chromatography
(TLC). Chiral purity can be assessed by chiral GC or measurement of
optical rotation.
[0084] The monoterpene (or sesquiterpene) derivatives may be
purified by methods such as crystallization, or by separating the
monoterpene (or sesquiterpene) derivative from impurities according
to the unique physicochemical properties (e.g., solubility or
polarity) of the derivative. Accordingly, the monoterpene (or
sesquiterpene) derivative can be separated from the monoterpene (or
sesquiterpene) by suitable separation techniques known in the art,
such as preparative chromatography, (fractional) distillation, or
(fractional) crystallization.
[0085] The invention also provides for methods of using
monoterpenes (or sesquiterpenes) derivatives to treat a disease,
such as a cancer or other nervous system disorders. A monoterpene
(or sesquiterpene) derivative may be administered alone, or in
combination with radiation, surgery or chemotherapeutic agents. A
monoterpene or sesquiterpene derivative may also be co-administered
with antiviral agents, anti-inflammatory agents or antibiotics. The
agents may be administered concurrently or sequentially. A
monoterpene (or sesquiterpene) derivative can be administered
before, during or after the administration of the other active
agent(s).
[0086] The monoterpene or sesquiterpene derivative may be used in
combination with radiation therapy. In one embodiment, the present
invention provides for a method of treating tumor cells, such as
malignant glioma cells or brain metastases, with radiation, where
the cells are treated with an effective amount of a monoterpene
derivative, such as a perillyl alcohol carbamate, and then exposed
to radiation. Monoterpene derivative treatment may be before,
during and/or after radiation. For example, the monoterpene or
sesquiterpene derivative may be administered continuously beginning
one week prior to the initiation of radiotherapy and continued for
two weeks after the completion of radiotherapy. U.S. Pat. Nos.
5,587,402 and 5,602,184.
[0087] In one embodiment, the present invention provides for a
method of treating tumor cells, such as malignant glioma cells or
brain metastases, with chemotherapy, where the cells are treated
with an effective amount of a monoterpene derivative, such as a
perillyl alcohol carbamate, and then exposed to chemotherapy.
Monoterpene derivative treatment may be before, during and/or after
chemotherapy.
[0088] Monoterpene (or sesquiterpene) derivatives may be used for
the treatment of nervous system cancers, such as a malignant glioma
(e.g., astrocytoma, anaplastic astrocytoma, glioblastoma
multiforme), retinoblastoma, pilocytic astrocytomas (grade I),
meningiomas, metastatic brain tumors, neuroblastoma, pituitary
adenomas, skull base meningiomas, and skull base cancer. As used
herein, the term "nervous system tumors" refers to a condition in
which a subject has a malignant proliferation of nervous system
cells.
[0089] Cancers that can be treated by the present monoterpene (or
sesquiterpene) derivatives include, but are not limited to, lung
cancer, ear, nose and throat cancer, leukemia, colon cancer,
melanoma, pancreatic cancer, mammary cancer, prostate cancer,
breast cancer, hematopoietic cancer, ovarian cancer, basal cell
carcinoma, biliary tract cancer; bladder cancer; bone cancer;
breast cancer; cervical cancer; choriocarcinoma; colon and rectum
cancer; connective tissue cancer; cancer of the digestive system;
endometrial cancer; esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer; intra-epithelial neoplasm; kidney
cancer; larynx cancer; leukemia including acute myeloid leukemia,
acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid
leukemia; liver cancer; lymphoma including Hodgkin's and
Non-Hodgkin's lymphoma; myeloma; fibroma, neuroblastoma; oral
cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian
cancer; pancreatic cancer; prostate cancer; retinoblastoma;
rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the
respiratory system; sarcoma; skin cancer; stomach cancer;
testicular cancer; thyroid cancer; uterine cancer; cancer of the
urinary system, as well as other carcinomas and sarcomas. U.S. Pat.
No. 7,601,355.
[0090] The present monoterpene (or sesquiterpene) derivatives can
be used for treating brain metastases that originate or spread from
a primary cancer such as a systemic cancer, lung cancer, prostate
cancer, breast cancer, hematopoietic cancer, ovarian cancer,
bladder cancer, germ cell tumors, kidney cancer, leukemia,
lymphoma, and melanoma. In some embodiments, the present invention
provides for a method for treating a mammal having a metastatic
cancer, such as metastatic breast cancer that has spread to the
brain, by administering to the mammal a monoterpene (or
sesquiterpene) derivative described herein, e.g., a POH carbamate,
such as TMZ-POH.
[0091] The present invention also provides methods of treating CNS
disorders, including, without limitation, primary degenerative
neurological disorders such as Alzheimer's, Parkinson's,
psychological disorders, psychosis and depression. Treatment may
consist of the use of a monoterpene or sesquiterpene derivative
alone or in combination with current medications used in the
treatment of Parkinson's, Alzheimer's, or psychological
disorders.
[0092] The present invention also provides a method of improving
immunomodulatory therapy responses comprising the steps of exposing
cells to an effective amount of a monoterpene or sesquiterpene
derivative, such as a perillyl alcohol carbamate, before or during
immunomodulatory treatment. Preferred immunomodulatory agents are
cytokines, such interleukins, lymphokines, monokines, interfereons
and chemokines.
[0093] The present composition may be administered by any method
known in the art, including, without limitation, intranasal, oral,
transdermal, ocular, intraperitoneal, inhalation, intravenous, ICV,
intracisternal injection or infusion, subcutaneous, implant,
vaginal, sublingual, urethral (e.g., urethral suppository),
subcutaneous, intramuscular, intravenous, rectal, sub-lingual,
mucosal, ophthalmic, spinal, intrathecal, intra-articular,
intra-arterial, sub-arachinoid, bronchial and lymphatic
administration. Topical formulation may be in the form of gel,
ointment, cream, aerosol, etc; intranasal formulation can be
delivered as a spray or in a drop; transdermal formulation may be
administered via a transdermal patch or iontorphoresis; inhalation
formulation can be delivered using a nebulizer or similar device.
Compositions can also take the form of tablets, pills, capsules,
semisolids, powders, sustained release formulations, solutions,
suspensions, elixirs, aerosols, or any other appropriate
compositions.
[0094] To prepare such pharmaceutical compositions, one or more of
monoterpene (or sesquiterpene) derivatives may be mixed with a
pharmaceutical acceptable carrier, adjuvant and/or excipient,
according to conventional pharmaceutical compounding techniques.
Pharmaceutically acceptable carriers that can be used in the
present compositions encompass any of the standard pharmaceutical
carriers, such as a phosphate buffered saline solution, water, and
emulsions, such as an oil/water or water/oil emulsion, and various
types of wetting agents. The compositions can additionally contain
solid pharmaceutical excipients such as starch, cellulose, talc,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk and the like. Liquid
and semisolid excipients may be selected from glycerol, propylene
glycol, water, ethanol and various oils, including those of
petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,
soybean oil, mineral oil, sesame oil, etc. Liquid carriers,
particularly for injectable solutions, include water, saline,
aqueous dextrose, and glycols. For examples of carriers,
stabilizers and adjuvants, see Remington's Pharmaceutical Sciences,
edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).
The compositions also can include stabilizers and
preservatives.
[0095] As used herein, the term "therapeutically effective amount"
is an amount sufficient to treat a specified disorder or disease or
alternatively to obtain a pharmacological response treating a
disorder or disease. Methods of determining the most effective
means and dosage of administration can vary with the composition
used for therapy, the purpose of the therapy, the target cell being
treated, and the subject being treated. Treatment dosages generally
may be titrated to optimize safety and efficacy. Single or multiple
administrations can be carried out with the dose level and pattern
being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents can be readily
determined by those of skill in the art. For example, the
composition are administered at about 0.01 mg/kg to about 200
mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to
about 50 mg/kg. When the compounds described herein are
co-administered with another agent or therapy, the effective amount
may be less than when the agent is used alone.
[0096] Transdermal formulations may be prepared by incorporating
the active agent in a thixotropic or gelatinous carrier such as a
cellulosic medium, e.g., methyl cellulose or hydroxyethyl
cellulose, with the resulting formulation then being packed in a
transdermal device adapted to be secured in dermal contact with the
skin of a wearer. If the composition is in the form of a gel, the
composition may be rubbed onto a membrane of the patient, for
example, the skin, preferably intact, clean, and dry skin, of the
shoulder or upper arm and or the upper torso, and maintained
thereon for a period of time sufficient for delivery of the
monoterpene (or sesquiterpene) derivative to the blood scrum of the
patient. The composition of the present invention in gel form may
be contained in a tube, a sachet, or a metered pump. Such a tube or
sachet may contain one unit dose, or more than one unit dose, of
the composition. A metered pump may be capable of dispensing one
metered dose of the composition.
[0097] This invention also provides the compositions as described
above for intranasal administration. As such, the compositions can
further comprise a permeation enhancer. Southall et al.
Developments in Nasal Drug Delivery, 2000. The monoterpene (or
sesquiterpene) derivative may be administered intranasally in a
liquid form such as a solution, an emulsion, a suspension, drops,
or in a solid form such as a powder, gel, or ointment. Devices to
deliver intranasal medications are well known in the art. Nasal
drug delivery can be carried out using devices including, but not
limited to, intranasal inhalers, intranasal spray devices,
atomizers, nasal spray bottles, unit dose containers, pumps,
droppers, squeeze bottles, nebulizers, metered dose inhalers (MDI),
pressurized dose inhalers, insufflators, and bi-directional
devices. The nasal delivery device can be metered to administer an
accurate effective dosage amount to the nasal cavity. The nasal
delivery device can be for single unit delivery or multiple unit
delivery. In a specific example, the ViaNase Electronic Atomizer
from Kurve Technology (Bethell, Wash.) can be used in this
invention (http://www.kurvetech.com). The compounds of the present
invention may also be delivered through a tube, a catheter, a
syringe, a packtail, a pledget, a nasal tampon or by submucosal
infusion. U.S. Patent Publication Nos. 20090326275, 20090291894,
20090281522 and 20090317377.
[0098] The monoterpene (or sesquiterpene) derivative can be
formulated as aerosols using standard procedures. The monoterpene
(or sesquiterpene) derivative may be formulated with or without
solvents, and formulated with or without carriers. The formulation
may be a solution, or may be an aqueous emulsion with one or more
surfactants. For example, an aerosol spray may be generated from
pressurized container with a suitable propellant such as,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen,
carbon dioxide, or other suitable gas. The dosage unit can be
determined by providing a valve to deliver a metered amount. Pump
spray dispensers can dispense a metered dose or a dose having a
specific particle or droplet size. As used herein, the term
"aerosol" refers to a suspension of fine solid particles or liquid
solution droplets in a gas. Specifically, aerosol includes a
gas-borne suspension of droplets of a monoterpene (or
sesquiterpene), as may be produced in any suitable device, such as
an MDI, a nebulizer, or a mist sprayer. Aerosol also includes a dry
powder composition of the composition of the instant invention
suspended in air or other carrier gas. Gonda (1990) Critical
Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et
al., (1992) Pharmacol. Toxicol. Methods 27:143-159.
[0099] The monoterpene (or sesquiterpene) derivative may be
delivered to the nasal cavity as a powder in a form such as
microspheres delivered by a nasal insufflator. The monoterpene (or
sesquiterpene) derivative may be absorbed to a solid surface, for
example, a carrier. The powder or microspheres may be administered
in a dry, air-dispensable form. The powder or microspheres may be
stored in a container of the insufflator. Alternatively the powder
or microspheres may be filled into a capsule, such as a gelatin
capsule, or other single dose unit adapted for nasal
administration.
[0100] The pharmaceutical composition can be delivered to the nasal
cavity by direct placement of the composition in the nasal cavity,
for example, in the form of a gel, an ointment, a nasal emulsion, a
lotion, a cream, a nasal tampon, a dropper, or a bioadhesive strip.
In certain embodiments, it can be desirable to prolong the
residence time of the pharmaceutical composition in the nasal
cavity, for example, to enhance absorption. Thus, the
pharmaceutical composition can optionally be formulated with a
bioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g.,
highly purified cationic polysaccharide), pectin (or any
carbohydrate that thickens like a gel or emulsifies when applied to
nasal mucosa), a microsphere (e.g., starch, albumin, dextran,
cyclodextrin), gelatin, a liposome, carbamer, polyvinyl alcohol,
alginate, acacia, chitosans and/or cellulose (e.g., methyl or
propyl; hydroxyl or carboxy; carboxymethyl or hydroxylpropyl).
[0101] The composition containing the purified monoterpene (or
sesquiterpene) can be administered by oral inhalation into the
respiratory tract, i.e., the lungs.
[0102] Typical delivery systems for inhalable agents include
nebulizer inhalers, dry powder inhalers (DPI), and metered-dose
inhalers (MDI).
[0103] Nebulizer devices produce a stream of high velocity air that
causes a therapeutic agent in the form of liquid to spray as a
mist. The therapeutic agent is formulated in a liquid form such as
a solution or a suspension of particles of suitable size. In one
embodiment, the particles are micronized. The term "micronized" is
defined as having about 90% or more of the particles with a
diameter of less than about 10 .mu.m. Suitable nebulizer devices
are provided commercially, for example, by PARI GmbH (Starnberg,
Germany). Other nebulizer devices include Respimat (Boehringer
Ingelheim) and those disclosed in, for example, U.S. Pat. Nos.
7,568,480 and 6,123,068, and WO 97/12687. The monoterpenes (or
sesquiterpenes) can be formulated for use in a nebulizer device as
an aqueous solution or as a liquid suspension.
[0104] DPI devices typically administer a therapeutic agent in the
form of a free flowing powder that can be dispersed in a patient's
air-stream during inspiration. DPI devices which use an external
energy source may also be used in the present invention. In order
to achieve a free flowing powder, the therapeutic agent can be
formulated with a suitable excipient (e.g., lactose). A dry powder
formulation can be made, for example, by combining dry lactose
having a particle size between about 1 .mu.m and 100 .mu.m with
micronized particles of the monoterpenes (or sesquiterpenes) and
dry blending. Alternatively, the monoterpene can be formulated
without excipients. The formulation is loaded into a dry powder
dispenser, or into inhalation cartridges or capsules for use with a
dry powder delivery device. Examples of DPI devices provided
commercially include Diskhaler (GlaxoSmithKline, Research Triangle
Park, N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus
(GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519; Turbuhaler
(AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No.
4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat.
No. 4,353,365). Further examples of suitable DPI devices are
described in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 and
references therein.
[0105] MDI devices typically discharge a measured amount of
therapeutic agent using compressed propellant gas. Formulations for
MDI administration include a solution or suspension of active
ingredient in a liquefied propellant. Examples of propellants
include hydrofluoroalklanes (HFA), such as
1,1,1,2-tetrafluoroethane (HFA 134a) and
1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227), and
chlorofluorocarbons, such as CCl.sub.3F. Additional components of
HFA formulations for MDI administration include co-solvents, such
as ethanol, pentane, water; and surfactants, such as sorbitan
trioleate, oleic acid, lecithin, and glycerin. (See, for example,
U.S. Pat. No. 5,225,183, EP 0717987, and WO 92/22286). The
formulation is loaded into an aerosol canister, which forms a
portion of an MDI device. Examples of MDI devices developed
specifically for use with HFA propellants are provided in U.S. Pat.
Nos. 6,006,745 and 6,143,227. For examples of processes of
preparing suitable formulations and devices suitable for inhalation
dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and
6,221,398, and WO 99/53901, WO 00/61108, WO 99/55319 and WO
00/30614.
[0106] The monoterpene (or sesquiterpene) derivative may be
encapsulated in liposomes or microcapsules for delivery via
inhalation. A liposome is a vesicle composed of a lipid bilayer
membrane and an aqueous interior. The lipid membrane may be made of
phospholipids, examples of which include phosphatidylcholine such
as lecithin and lysolecithin; acidic phospholipids such as
phosphatidylserine and phosphatidylglycerol; and
sphingophospholipids such as phosphatidylethanolamine and
sphingomyelin. Alternatively, cholesterol may be added. A
microcapsule is a particle coated with a coating material. For
example, the coating material may consist of a mixture of a
film-forming polymer, a hydrophobic plasticizer, a surface
activating agent or/and a lubricant nitrogen-containing polymer.
U.S. Pat. Nos. 6,313,176 and 7,563,768.
[0107] The monoterpene (or sesquiterpene) derivative may also be
used alone or in combination with other chemotherapeutic agents via
topical application for the treatment of localized cancers such as
breast cancer or melanomas. The monoterpene (or sesquiterpene)
derivative may also be used in combination with narcotics or
analgesics for transdermal delivery of pain medication.
[0108] This invention also provides the compositions as described
above for ocular administration. As such, the compositions can
further comprise a permeation enhancer. For ocular administration,
the compositions described herein can be formulated as a solution,
emulsion, suspension, etc. A variety of vehicles suitable for
administering compounds to the eye are known in the art. Specific
non-limiting examples are described in U.S. Pat. Nos. 6,261,547;
6,197,934; 6,056,950; 5,800,807; 5,776,445; 5,698,219; 5,521,222;
5,403,841; 5,077,033; 4,882,150; and 4,738,851.
[0109] The monoterpene (or sesquiterpene) derivative can be given
alone or in combination with other drugs for the treatment of the
above diseases for a short or prolonged period of time. The present
compositions can be administered to a mammal, preferably a human.
Mammals include, but are not limited to, murines, rats, rabbit,
simians, bovines, ovine, porcine, canines, feline, farm animals,
sport animals, pets, equine, and primates.
[0110] The invention also provides a method for inhibiting the
growth of a cell in vitro, ex vivo or in vivo, where a cell, such
as a cancer cell, is contacted with an effective amount of the
monoterpene (or sesquiterpene) derivative as described herein.
[0111] Pathological cells or tissue such as hyperproliferative
cells or tissue may be treated by contacting the cells or tissue
with an effective amount of a composition of this invention. The
cells, such as cancer cells, can be primary cancer cells or can be
cultured cells available from tissue banks such as the American
Type Culture Collection (ATCC). The pathological cells can be cells
of a systemic cancer, gliomas, meningiomas, pituitary adenomas, or
a CNS metastasis or brain metastasis from a systemic cancer, lung
cancer, prostate cancer, breast cancer, hematopoietic cancer,
ovarian cancer, bladder cancer, germ cell tumors, kidney cancer,
leukemia, lymphoma, and melanoma. The cells can be from a
vertebrate, preferably a mammal, more preferably a human. U.S.
Patent Publication No. 2004/0087651. Balassiano et al. (2002)
Intern. J. Mol. Med. 10:785-788. Thorne, et al. (2004) Neuroscience
127:481-496. Fernandes, et al. (2005) Oncology Reports 13:943-947.
Da Fonseca, et al. (2008) Surgical Neurology 70:259267. Da Fonseca,
et al. (2008) Arch. Immunol. Ther. Exp. 56:267-276. Hashizume, et
al. (2008) Neuroncology 10:112-120.
[0112] In vitro efficacy of the present composition can be
determined using methods well known in the art. For example, the
cytoxicity of the present monoterpene (or sesquiterpene) and/or the
therapeutic agents may be studied by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
cytotoxicity assay. MTT assay is based on the principle of uptake
of MTT, a tetrazolium salt, by metabolically active cells where it
is metabolized into a blue colored formazon product, which can be
read spectrometrically. J. of Immunological Methods 65: 55 63,
1983. The cytoxicity of the present monoterpene (or sesquiterpene)
derivative and/or the therapeutic agents may be studied by colony
formation assay. Functional assays for inhibition of VEGF secretion
and IL-8 secretion may be performed via ELISA. Cell cycle block by
the present monoterpene (or sesquiterpene) derivative and/or the
therapeutic agents may be studied by standard propidium iodide (PI)
staining and flow cytometry. Invasion inhibition may be studied by
Boyden chambers. In this assay a layer of reconstituted basement
membrane, Matrigel, is coated onto chemotaxis filters and acts as a
barrier to the migration of cells in the Boyden chambers. Only
cells with invasive capacity can cross the Matrigel barrier. Other
assays include, but are not limited to cell viability assays,
apoptosis assays, and morphological assays.
[0113] The following are examples of the present invention and are
not to be construed as limiting.
EXAMPLES
Example 1: Synthesis of Dimethyl Celecoxib bisPOH Carbamate
(4-(bis-N,N'-4-isopropenyl cyclohex-1-enylmethyloxy Carbonyl
[5-(2,5-dimethyl phenyl)-3-trifluoromethyl pyrazol-1-yl]
benzenesulfonamide) (also Referred to as POH-DMC or DMC-POH
Herein)
[0114] The reaction scheme is the following:
##STR00041##
[0115] Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added to a
mixture of perillyl alcohol (2.0 grams, 13.1 mmol) and potassium
carbonate (5.4 grams, 39.1 mmol) in dry toluene (30 mL) over a
period of 30 minutes while maintaining the temperature between
10.degree. C. to 15.degree. C. The reaction mixture was allowed to
warm to room temperature and stirred for 8.0 hours under N.sub.2.
The reaction mixture was quenched with water (30 mL) and the
organic layer was separated. The aqueous layer was extracted with
toluene (20 mL) and the combined organic layer was washed with
water (50 mL.times.2), brine (15%, 30 mL) and dried over sodium
sulfate (20 grams). The filtered organic layer was concentrated
under vacuum to give perillyl chloroformate as an oil. Weight: 2.5
grams; Yield: 89%. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.5
(m, 1H), 1.7 (s, 3H), 1.8 (m, 1H), 2.0 (m, 1H), 2.2 (m, 4H), 4.7
(dd, 4H); 5.87 (m, 1H).
[0116] Perillyl chloroformate (0.11 grams, 0.55 mmol) was added
slowly to a mixture of dimethyl celecoxib (0.2 grams, 0.50 mmol)
and potassium carbonate (0.13 grams, 1.0 mmol) in dry acetone (10
mL) over a period of 5 minutes under N.sub.2. The reaction mixture
was heated to reflux and maintained for 3 hours. Since TLC analysis
indicated the presence of dimethyl celecoxib (>60%), another 1.0
equivalent of perillyl chloroformate was added and refluxed for an
additional 5 hours. The reaction mixture was cooled and acetone was
concentrated under vacuum to give a residue.
[0117] The resulting residue was suspended in water (15 mL) and
extracted with ethyl acetate (3.times.15 mL). The combined organic
layer was washed with water (20 mL) followed by brine (15%, 20 mL)
and dried over sodium sulfate. The filtered organic layer was
concentrated under vacuum to give a residue which was purified by
column chromatography [column dimensions: diameter: 1.5 cm, height:
10 cm, silica: 230-400 mesh] and eluted with hexanes (100 mL)
followed by a mixture of hexanes/ethyl acetate (95:5, 100 mL). The
hexane/ethyl acetate fractions were combined and concentrated under
vacuum to give a gummy mass.
[0118] The product POH carbamate exhibited a weight of 120 mg and a
yield of 31%. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 0.9 (m,
2H), 1.4 (m, 2H), 1.7 (m, 7H*), 1.95 (m, 8H*), 2.1 (m, 4H), 2.3 (s,
3H), 4.4 (d, 2H), 4.7 (dd, 2H), 5.6 (br d, 2H), 6.6 (s, 1H), 7.0
(br s, 1H), 7.12 (d, 1H), 7.19 (d, 1H), 7.4 (d, 2H), 7.85 (d, 2H);
MS, m/e: 751.8 (M 3%), 574.3 (100%), 530.5 (45%), 396 (6%). *N.B.
further 2H overlapping from presumed impurity discounted in NMR
integration.
Example 2: In Vitro Cytotoxicity Studies of POH-DMC Carbamate
(POH-DMC)
[0119] First cytotoxicity assays were carried out after cells were
treated with dimethyl-celecoxib (DMC) alone. FIG. 1 shows the
results of the MTT cytotoxicity assays performed on human malignant
glioma cells U87, A172 and U251 with DMC alone.
[0120] Then U87, A172 and U251 cells were treated with dimethyl
celecoxib bisPOH carbamate (POH-DMC) (e.g., synthesized by the
method in Example 1), and the MTT cytotoxicity assays performed
(FIG. 2). The results suggest that POH carbamate POH-DMC exhibited
much better cytotoxicity than DMC alone.
Example 3: Synthesis of Temozolomide POH Carbamate (3-methyl
4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamic
acid-4-isopropenyl cyclohex-1-enylmethyl Ester) (also Referred to
as TMZ-POH or POH-TMZ Herein)
[0121] The reaction scheme is the following:
##STR00042##
[0122] Oxalyl chloride (0.13 grams, 1.0 mmol) was added slowly to a
mixture of temozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol)
in 1,2-dichloroethane (10 mL) over a period of 2 minutes while
maintaining the temperature at 10.degree. C. under N.sub.2. The
reaction mixture was allowed to warm to room temperature and then
heated to reflux for 3 hours. The excess of oxalyl chloride and
1,2-dichloroethane were removed by concentration under vacuum. The
resulting residue was re-dissolved in 1,2-dichlorethane (15 mL) and
the reaction mixture was cooled to 10.degree. C. under N.sub.2. A
solution of perillyl alcohol (0.086 grams, 0.56 mmol) in
1,2-dichloroethane (3 mL) was added over a period of 5 minutes. The
reaction mixture was allowed to warm to room temperature and
stirred for 14 hours. 1,2-dichloroethane was concentrated under
vacuum to give a residue, which was triturated with hexanes. The
resulting yellow solid was filtered and washed with hexanes.
Weight: 170 mg; Yield: 89%. .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta. 1.4-2.2 (m, 10H), 4.06 (s, 3H), 4.6-4.8 (m, 4H), 5.88 (br
s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H); MS, no molecular ion peak
was observed. m/e: 314 (100%), 286.5 (17%), 136 (12%).
[0123] Alternatively, temozolomide POH carbamate was synthesized
according to the following procedure. Oxalyl chloride (0.13 grams,
1.0 mmol) was added slowly to a mixture of temozolomide (OChem
Incorporation, 0.1 grams, 0.5 mmol) in 1,2-dichloroethane (10 mL)
over a period of 2 minutes while maintaining the temperature at
10.degree. C. under N.sub.2. The reaction mixture was allowed to
warm to room temperature and then heated to reflux for 3 hours. The
excess of oxalyl chloride and 1,2-dichloroethane were removed by
concentration under vacuum. The resulting residue was re-dissolved
in 1,2-dichlorethane (15 mL) and the reaction mixture was cooled to
10.degree. C. under N.sub.2. A solution of perillyl alcohol (0.086
grams, 0.56 mmol) in 1,2-dichloroethane (3 mL) was added over a
period of 5 minutes. The reaction mixture was allowed to warm to
room temperature and stirred for 14 hours. 1,2-Dichloroethane was
concentrated under vacuum to give a residue, which was purified by
a short silica-plug column (column dimensions: diameter: 2 cm,
height: 3 cm, silica: 230-400 mesh) and eluted with a mixture of
hexanes/ethyl acetate (1:1, 100 mL). The hexane/ethyl acetate
fractions were combined and concentrated under vacuum to give a
white solid residue which was triturated with heptanes and filtered
to obtain a white solid. Weight: 170 mg; Yield: 89%. .sup.1H-NMR
(400 MHz, CDCl3): 1.4-2.2 (m, 10H), 4.06 (s, 3H), 4.6-4.8 (m, 4H),
5.88 (br s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H); MS, no molecular
ion peak was observed, m/e: 314 (100%), 286.5 (17%), 136 (12%).
Example 4: In Vitro Cytotoxicity Studies of TMZ-POH
[0124] First cytotoxicity assays were carried out after cells were
treated with temozolomide (TMZ) alone, the standard alkylating
agent used in the treatment of malignant gliomas. FIG. 3 shows the
results of the MTT cytotoxicity assays performed on human malignant
glioma cells U87, A172 and U251 with TMZ alone. Increasing
concentrations of TMZ had minimal cytotoxicity towards the cell
lines tested.
[0125] Then TMZ-resistant glioma cell lines U87, A172 and U251
cells were treated with TMZ-POH (e.g., synthesized by the method in
Example 3). The MTT assay results (FIG. 4) showed that TMZ-POH
exhibited substantially higher kill rates of the various human
glioma cells compared to TMZ alone.
Example 5: Synthesis of Rolipram POH Carbamate
(4-(3-cyclopentyloxy-4-methoxy
phenyl)-2-oxo-pyrrolidine-1-carboxylic acid 4-isopropenyl
cyclohex-1-enylmethyl Ester) (also Referred to as Rolipram-POH or
POH-Rolipram Herein)
[0126] The reaction scheme is the following:
##STR00043##
[0127] Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added to a
mixture of perillyl alcohol (2.0 grams, 13.1 mmol) and potassium
carbonate (5.4 grams, 39.1 mmol) in dry toluene (30 mL) over a
period of 30 minutes while maintaining the temperature between
10.degree. C. to 15.degree. C. The reaction mixture was allowed to
warm to room temperature and stirred for 8.0 hours under N.sub.2.
The reaction mixture was quenched with water (30 mL) and the
organic layer separated. The aqueous layer was extracted with
toluene (20 mL) and the combined organic layer washed with water
(50 mL.times.2), brine (15%, 30 mL) and dried over sodium sulfate
(20 grams). The filtered organic layer was concentrated under
vacuum to give perillyl chloroformate as an oil. Weight: 2.5 grams;
Yield: 89%. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.5 (m, 1H),
1.7 (s, 3H), 1.8 (m, 1H), 2.0 (m, 1H), 2.2 (m, 4H), 4.7 (dd, 4H);
5.87 (m, 1H).
[0128] Butyl lithium (2.5 M, 0.18 mL, 0.45 mmol) was added to a
solution of rolipram (GL synthesis, Inc., 0.1 grams, 0.36 mmol) in
dry THF at -72.degree. C. over a period of 5 minutes under N.sub.2.
After the reaction mixture was stirred for 1.0 hours at -72.degree.
C., perillyl chloroformate (dissolved in 4 mL THF) was added over a
period of 15 minutes while maintaining the temperature at
-72.degree. C. The reaction mixture was stirred for 2.5 hours and
quenched with saturated ammonium chloride (5 mL). The reaction
mixture was allowed to warm to room temperature and extracted with
ethyl acetate (2.times.15 mL). The combined organic layer was
washed with water (15 mL), brine (15%, 15 mL), and then dried over
sodium sulfate. The filtered organic layer was concentrated to give
an oil which was purified by column chromatography [column
dimensions: diameter: 1.5 cm, height: 10 cm, silica: 230-400 mesh]
and eluted with a mixture of 8% ethyl acetate/hexanes (100 mL)
followed by 12% ethyl acetate/hexanes (100 mL). The 12% ethyl
acetate/hexanes fractions were combined and concentrated under
vacuum to yield a gummy solid. Weight: 142 mg; Yield: 86%.
.sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.5 (m, 1H), 1.6 (m,
2H), 1.7 (s, 3H), 1.9 (m, 6H), 2.2 (m, 5H), 2.7 (m, 1H), 2.9 (m,
1H), 3.5 (m, 1H), 3.7 (m, 1H), 3.8 (s, 3H), 4.2 (m, 1H), 4.7 (m,
6H), 5.8 (br s, 1H), 6.8 (m, 3H); MS, m/e: 452.1 (M.sup.+1 53%),
274.1 (100%), 206.0 (55%).
Example 6: In Vitro Cytotoxicity Studies of Rolipram POH
Carbamate
[0129] To compare the cytotoxicity of Rolipram POH Carbamate
(POH-Rolipram) (e.g., synthesized by the method in Example 5) with
rolipram, a type IV phosphodiesterase inducing differentiation and
apoptosis in glioma cells, A172, U87, U251 and LN229 human glioma
cells were treated with either POH-Rolipram or rolipram for 48
hours. The MTT assay results are shown in FIGS. 5 to 8.
POH-Rolipram exhibited substantially higher kill rates compared to
rolipram alone for each of the several different human glioma cell
types. FIG. 5 shows the MTT assay for increasing concentrations of
rolipram and POH-rolipram for A-172 cells. Rolipram alone
demonstrates an IC50 of approximately 1000 uM (1 mM). In the
presence of POH-rolipram, IC50 is achieved at concentrations as low
as 50 uM. FIG. 6 shows the MTT assay for increasing concentrations
of rolipram with U-87 cells. IC50 is not met at 1000 uM. On the
other hand, IC50 iss achieved at 180 uM with POH-rolipram. FIG. 7
shows that IC50 for rolipram alone for U251 cells is achieved at
170 uM; plateau cytotoxicity is reached at 60%. POH-rolipram
achieves IC50 at 50 uM, with almost 100% cytoxicity at 100 uM. FIG.
8 shows that IC50 for rolipram alone for LN229 cells is not
achieved even at 100 uM. On the other hand, IC50 for POH-rolipram
is achieved at 100 uM, with almost 100% cytotoxicity at 10 uM.
Example 7: In Vivo Tumor Growth Inhibition by POH Fatty Acid
Derivatives
[0130] Inhibition of tumor growth by butyryl-POH was studied in a
nude mouse subcutaneous glioma model. Mice were injected with U-87
glioma cells (500,000 cells/injection) and allowed to form a
palpable nodule over two weeks. Once palpable nodule was formed,
the mice were treated with local application of various compounds
as indicated in FIGS. 9A and 9B via a Q-tip (1 cc/application/day)
over a period of 8 weeks. FIG. 9A shows the images of subcutaneous
U-87 gliomas in nude mice treated with butyryl-POH, purified
(S)-perillyl alcohol having a purity greater than 98.5% ("purified
POH"), POH purchased from Sigma chemicals, or phosphate buffered
saline (PBS; negative control). FIG. 9B shows average tumor growth
over time (total time period of 60 days). Butyryl-POH demonstrated
the greatest inhibition of tumor growth, followed by purified POH
and Sigma POH.
Example 8: In Vitro Cytotoxicity Studies of TMZ and TMZ-POH on TMZ
Sensitive and Resistant Glioma Cells
[0131] Colony forming assays were carried out after cells were
treated with TMZ alone, POH alone, and the TMZ-POH conjugate. The
colony forming assays were carried out as described in Chen T C, et
al. Green tea epigallocatechin gallate enhances therapeutic
efficacy of temozolomide in orthotopic mouse glioblastoma models.
Cancer Lett. 2011 Mar. 28; 302(2):100-8. FIG. 10 shows the results
of the colony forming assays performed on TMZ sensitive (U251) and
TMZ resistant (U251TR) U251 cells with TMZ or TMZ-POH. TMZ
demonstrated cytotoxicity towards TMZ sensitive U251 cells, but had
minimal cytotoxicity towards TMZ resistant U251 cells. TMZ-POH
demonstrated cytotoxicity towards both TMZ sensitive and TMZ
resistant U251 cells.
[0132] FIG. 11 shows the results of the colony forming assays
performed on TMZ sensitive (U251) and TMZ resistant (U251TR) U251
cells with POH. POH demonstrated cytotoxicity towards both TMZ
sensitive and TMZ resistant U251 cells. TMZ-POH (FIG. 10) exhibited
substantially greater potency compared to POH alone (FIG. 11) in
the colony forming assays.
Example 9: In Vitro Cytotoxicity Studies of TMZ-POH on U251 Cells,
U251TR Cells, and Normal Astrocytes
[0133] MTT cytotoxicity assays were carried out after cells were
treated with the TMZ-POH conjugate. The MTT cytotoxicity assays
were carried out as described in Chen T C, et al. Green tea
epigallocatechin gallate enhances therapeutic efficacy of
temozolomide in orthotopic mouse glioblastoma models. Cancer Lett.
2011 Mar. 28; 302(2):100-8. FIG. 12 shows the results of the MTT
cytotoxicity assays performed on TMZ sensitive cells (U251), TMZ
resistant cells (U251TR) and normal astrocytes. TMZ-POH
demonstrated cytotoxicity towards both TMZ sensitive and TMZ
resistant U251 cells, but not towards normal astrocytes.
Example 10: In Vitro Cytotoxicity Studies of TMZ-POH on BEC, TuBEC,
and Normal Astrocytes
[0134] MTT cytotoxicity assays were carried out after cells were
treated with the TMZ-POH conjugate. The MTT cytotoxicity assays
were carried out as described in Chen T C, et al. Green tea
epigallocatechin gallate enhances therapeutic efficacy of
temozolomide in orthotopic mouse glioblastoma models. Cancer Lett.
2011 Mar. 28; 302(2):100-8. FIG. 13 shows the results of the MTT
cytotoxicity assays performed on normal astrocytes, brain
endothelial cells (BEC; confluent and subconfluent), and tumor
brain endothelial cells (TuBEC). TMZ-POH did not induce significant
cytotoxicity on normal astrocytes, confluent BEC, or TuBEC. Mild to
moderate cytotoxicity was demonstrated in subconfluent BEC at high
concentrations of TMZ-POH.
Example 11: In Vitro Cytotoxicity Studies of TMZ and TMZ-POH on
USC-04 Glioma Cancer Stem Cells
[0135] MTT cytotoxicity assays were carried out after cells were
treated with the TMZ alone, POH alone, or the TMZ-POH conjugate.
The MTT cytotoxicity assays were carried out as described in Chen T
C, et al. Green tea epigallocatechin gallate enhances therapeutic
efficacy of temozolomide in orthotopic mouse glioblastoma models.
Cancer Lett. 2011 Mar. 28; 302(2):100-8. FIG. 14 shows the results
of the MTT cytotoxicity assays performed on USC-04 glioma cancer
stem cells. TMZ did not induce significant cytotoxicity with
increasing concentrations (0-400 uM). TMZ-POH demonstrated evidence
of cytotoxicity with IC50 at 150 uM. FIG. 15 shows the results of
the MTT cytotoxicity assays performed on USC-04 glioma cancer stem
cells treated with POH. POH demonstrated cytotoxicity on USC-04
with increasing concentrations (0-2 mM).
Example 12: In Vitro Cytotoxicity Studies of TMZ and TMZ-POH on
USC-02 Glioma Cancer Stem Cells
[0136] MTT cytotoxicity assays were carried out after cells were
treated with the TMZ alone, POH alone, or the TMZ-POH conjugate.
The MTT cytotoxicity assays were carried out as described in Chen T
C, et al. Green tea epigallocatechin gallate enhances therapeutic
efficacy of temozolomide in orthotopic mouse glioblastoma models.
Cancer Lett. 2011 Mar. 28; 302(2):100-8. FIG. 16 shows the results
of the MTT cytotoxicity assays performed on USC-02 glioma cancer
stem cells. TMZ did not induce significant cytotoxicity with
increasing concentrations (0-400 uM). TMZ-POH demonstrated evidence
of cytotoxicity with IC50 at 60 uM. FIG. 17 shows the results of
the MTT cytotoxicity assays performed on USC-02 glioma cancer stem
cells treated with POH. POH demonstrated cytotoxicity on USC-02
with increasing concentrations (0-2 mM).
Example 13: In Vitro Studies of ER Stress by TMZ-POH on TMZ
Sensitive and Resistant Glioma Cells
[0137] Western blots were performed after TMZ sensitive and
resistant glioma cells were treated with the TMZ-POH conjugate for
18 hr. FIG. 18 shows a western blot demonstrating that TMZ-POH
induces ER stress (ERS) in TMZ sensitive and resistant U251 glioma
cells. Activation of the proapoptic protein CHOP was shown at
concentrations as low as 60 uM of TMZ-POH.
Example 14: In Vitro and In Vivo Studies of TMZ-POH on Certain
Breast Cancer Cells
Pharmacological Agents
[0138] TMZ was obtained from the pharmacy at the University of
Southern California (USC) and dissolved in ethanol to a
concentration of 50 mM. TMZ-POH, which is also referred to as T-P
in this example, was provided by NeOne Technologies Inc. and was
dissolved in DMSO at 100 mM. Perillyl alcohol (POH) and
O6-benzylguanine (O6-BG) were purchased from Sigma-Aldrich (St.
Louis, Mo.) and diluted with DMSO to make stock solutions of 100
mM. DMSO was from Sigma-Aldrich. In all cases of cell treatment,
the final DMSO concentration in the culture medium never exceeded
0.5%. Stock solutions of all drugs were stored at -20.degree.
C.
Cell Lines
[0139] The human cancer cell lines were obtained from the American
Tissue Culture Collection (ATCC; Manassas, Va.), except for
HCC-1937, which was provided by Dr. Michael Press. Cells were
propagated in DMEM (provided by the Cell Culture Core Lab of the
USC/Norris Comprehensive Cancer Center and prepared with raw
materials from Cellgro/MediaTech, Manassas, Va.) supplemented with
10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin,
and 0.1 mg/mL streptomycin in a humidified incubator at 37.degree.
C. and a 5% CO.sub.2 atmosphere.
Colony Formation Assay
[0140] Depending on the cell line (and plating efficiency), 200-350
cells were seeded into each well of a 6-well plate. After cells had
fully attached to the surface of the culture plate, they were
exposed to drug treatment (or DMSO solvent alone) for various times
up to 48 hours. Thereafter, the drugs were removed, fresh growth
medium was added, and the cells were kept in culture undisturbed
for 12-16 days, during which time the surviving cells spawned
colonies of descendants. Colonies (defined as groups of >50
cells) were visualized by staining for 4 hours with 1% methylene
blue (in methanol), and then were counted.
[0141] In the case of O6-BG treatment, cells were pretreated with
10 .mu.M O6-BG for one hour before addition of TMZ or TMZ-POH.
After 24 hours, another 10 .mu.M O6-BG was added to the medium.
Another 24 hours later, drug-laced medium was removed, and fresh
medium without drugs was added. Thereafter, cells remained
undisturbed until staining with methylene blue.
Stable Transfections
[0142] MDA-MB-231 cells were co-transfected in 6-well plates with
the use of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.)
according to manufacturer's instructions. 2 .mu.g pSV2MGMT
(containing the human MGMT cDNA) was combined with 0.2 .mu.g
pSV2neo (containing the neomycin gene for selection of cells in
G418). Both plasmids were provided by Bernd Kaina (Mainz, Germany).
Individual clones of transfected cells were selected in medium
containing 750 .mu.g/mL G418 and propagated in 250 .mu.g/mL G418.
G418 was obtained as G418 disulfate salt from Sigma-Aldrich and
dissolved in PBS at 75 mg/mL. Selection medium was removed from
cells several days before experimental drug treatment.
Immunoblots
[0143] Total cell lysates were analyzed by Western blot analysis as
described in P. Pyrko et al. Downregulation of survivin expression
and concomitant induction of apoptosis by celecoxib and its
non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC),
in tumor cells in vitro and in vivo. Mol Cancer 5 (2006) 19. The
primary antibodies were purchased from Cell Signaling Technology
(Beverly, Mass.) or Santa Cruz Biotechnology, Inc. (Santa Cruz,
Calif.) and used according to the manufacturers' recommendations.
All immunoblots were repeated at least once to confirm the
results.
In Vivo Model
[0144] All animal protocols were approved by the Institutional
Animal Care and Use Committee (IACUC) of University of Southern
California, and all rules and regulations were followed during
experimentation on animals. Athymic mice (Harlan, Inc.,
Indianapolis, Ind.) were implanted intracranially with
2.times.10.sup.5 cells. A subline of MDA-MB-231 cells called D3H2LN
was used, which was transfected with the firefly luciferase gene
and had been selected for aggressive growth and metastasis in vivo.
Ten days after intracranial implantation, efficient tumor take was
confirmed in all animals via non-invasive whole-body bioluminescent
imaging. For this purpose, mice were intravenously injected with 50
mg/kg D-Luciferin (Perkin Elmer, Waltham, Mass.) and imaged using
the Xenogen IVIS-200 Imaging System (Caliper/Perkin Elmer). Images
were analyzed by region-of-interest (ROI) analysis using the Living
Image software package (Caliper/Perkin Elmer) to quantitate light
output (radiance, i.e., photons per second per square centimeter
per steradian).
[0145] Animals were distributed into three groups so that each
group contained animals with comparable radiance within the ROI
(i.e., area of the head) and drug treatment was initiated. Group 1
was the control group that received vehicle only (45% glycerol, 45%
ethanol, 10% DMSO) via subcutaneous injection. Group 2 was the
experimental group that received 25 mg/kg TMZ-POH via subcutaneous
(s.c.) injection. Group 3 was the comparison group and animals
received 25 mg/kg TMZ via gavage. Treatment was once per day for a
period of 10 days (i.e., 10 treatments total). Thereafter, all
surviving animals were imaged again, once per week.
Statistical Analysis
[0146] All parametric data were analyzed using the Student t-test
to calculate the significance values; a probability value
(p)<0.05 was considered statistically significant.
Results
[0147] The cytotoxic potency of TMZ-POH, was analyzed by colony
formation assay (CFA) in a variety of human breast cancer cell
lines and compared to the cytotoxicity of TMZ. We used estrogen
receptor positive cells MCF7 and T47D, the triple-negative lines
MDA-MB-231, MDA-MB-468, and HCC-1937, and a brain-seeking variant
of the 231 cell line, MDA-MB-231-br. As shown in FIG. 19, low
micromolar concentrations of TMZ-POH prevented colony formation in
all six cell lines, and in all instances TMZ-POH's potency was
substantially stronger than that of TMZ.
[0148] Previous studies showed that POH is able to exert cytotoxic
effects in cancer cells, although concentrations approaching the
millimolar range were required. Thus, we tested whether simply
mixing the two compounds TMZ and POH could mimic the effects of the
TMZ-POH conjugate. MDA-MB-231 cells were treated with the
individual compounds (TMZ-POH, TMZ or POH) alone, or with an
equimolar mix of TMZ plus POH, and cell survival was analyzed by
CFA. As shown in FIGS. 20A and 20B, TMZ-POH was much more potent
than a mix of TMZ plus POH, i.e., mixing TMZ with POH was unable to
achieve the strong cytotoxic potency of TMZ-POH, and in fact, the
addition of equimolar concentrations of POH to TMZ did not increase
the potency over TMZ alone. For instance, 10 .mu.M TMZ reduced
colony formation by about 50%, and the combination of 10 .mu.M TMZ
with 10 .mu.M POH also caused a 50% reduction; in comparison, 10
.mu.M TMZ-POH caused about 95% fewer colonies (FIG. 20A).
Consistent with earlier reports, POH by itself required
concentrations well above 100 .mu.M in order to become cytotoxic,
and its IC50 in MDA-MB-231 cells was about 700 .mu.M (FIG.
20A).
[0149] FIG. 20B shows a representative example of an individual
CFA. It illustrates that 10 .mu.M blocks colony formation
substantially more potently than TMZ, and that the addition of
equimolar concentrations of POH to either TMZ or TMZ-POH is unable
to enhance toxicity any further. Altogether, the above results
shows that TMZ-POH has with increased potency over TMZ that cannot
be matched by merely mixing its individual parts, TMZ and POH.
[0150] Because the DNA repair protein MGMT is known to play a key
role in cellular resistance to TMZ, we investigated how it would
impact the cytotoxic potency of TMZ-POH. We first determined its
basal level of expression in the six breast cancer cell lines we
used above. FIG. 21A shows that three cell lines (MDA-MB-468,
HCC-1937, MCF7) were strongly positive, whereas the others (T47D,
MDA-MB-231, MDA-MB-231-br) had undetectable levels of MGMT protein,
as determined by Western blot analysis. For comparison purposes, we
also assessed MGMT protein levels in three commonly used GBM cell
lines known to be MGMT negative (U251, LN229) and positive (T98G).
This side-by-side evaluation revealed that MGMT protein levels in
the positive breast cancer lines were similar to the levels found
in the T98G brain cancer line.
[0151] MGMT expression was aligned with the cytotoxic potency of
TMZ-POH in comparison to TMZ. As summarized in Table 2, the IC50 of
TMZ-POH (i.e., the concentration required to decrease colony
formation by 50%) was noticeably higher in all three MGMT-positive
breast cancer cell lines. Whereas the IC50 in MGMT-negative cell
lines ranged from 1.2 to 4.6 .mu.M, it increased to 31 to 33 .mu.M
in the three MGMT-positive lines. Nonetheless, these IC50 values
still were substantially lower than the corresponding 1050s of TMZ
for each cell line. Noteworthy as well is the differential (fold
increase in potency) between TMZ-POH and TMZ shown in Table 2: The
fold-increase in cytotoxic potency of TMZ-POH, as compared to TMZ,
is consistently greater in each of the MGMT-positive cell lines
(6.3 to 15.5-fold) as compared to the MGMT-negative cell lines (3.2
to 4.3-fold). This latter finding suggests that the increased
potency of TMZ-POH over TMZ, although apparent in all cell lines
analyzed, might become particularly advantageous in the context of
therapeutically targeting MGMT-positive cells.
TABLE-US-00002 TABLE 2 Drug Sensitivities of Various Breast Cancer
Cell Lines MGMT IC50 TMZ IC50 T-P Differential Cell Line status
(.mu.M) (.mu.M) (-fold) MDA-MB-231-br - 3.8 1.2 3.2 MDA-MB-231 -
9.9 2.3 4.3 T47D - 20 4.6 4.3 HCC-1937 + 186 31 6.0 MDA-MB-468 +
195 31 6.3 MCF7 + 513 33 15.5
[0152] The major cytotoxic DNA lesion set by TMZ is methylation of
O6-guanine, and it is well known that removal of this methyl group
by MGMT leads to rapid degradation of the DNA repair protein. As
well, the pseudosubstrate O6-BG also activates the suicide
mechanism of MGMT, which is confirmed in FIG. 21C, showing that
treatment of cells with O6-BG strongly decreases MGMT protein
levels. Treatment of cells with TMZ also down-regulates MGMT
levels, but the effect is fairly weak and high concentrations of
the drug are required. In comparison, TMZ-POH affects MGMT levels
more potently than TMZ; for instance, while 50 .mu.M TMZ has no
effect, 50 .mu.M TMZ-POH causes a significant decrease (FIG. 21C).
Together, these results indicate that TMZ-POH's superior potency
over TMZ may involve more extensive methylation of O6-guanine
targets.
[0153] While the above results suggested that TMZ-POH's mechanism
of action might be due to the drug's increased efficacy of setting
cytotoxic DNA lesions, there was also a possibility that covalently
conjugating POH might have conferred additional mechanistic
features to the new molecule. Additional experiments were performed
to characterize the significance of DNA damage, and in particular
O6-guanine methylation, caused by TMZ-POH.
[0154] While the experiments summarized in Table 2 revealed a
correlation of MGMT positivity with decreased TMZ-POH toxicity,
they did not establish cause and effect. To investigate the latter,
we stably transfected MGMT-negative MDA-MB-231 cells with MGMT cDNA
and isolated individual clones. FIG. 22A shows elevated expression
of MGMT protein in two different clones (231-MGMT-1 and 231-MGMT-2)
of transfected cells. Both clones were treated with increasing
concentrations of TMZ-POH and TMZ and analyzed by CFA. As shown in
FIG. 22B and Table 3, resistance of cells to drug treatment clearly
increased for both TMZ-POH and TMZ, as compared to parental cells.
Intriguingly however, similar to what was noted in Table 2,
resistance to TMZ-POH increased less than resistance to TMZ
(summarized in Table 3).
TABLE-US-00003 TABLE 3 Drug Sensitivities of Cells Transfected with
MGMT cDNA MGMT 1050 TMZ 1050 T-P Differential Cell Line status
(.mu.M) (.mu.M) (-fold) MDA-MB-231 - 9.9 2.3 4.3 231-MGMT-1 + 202
27 7.5 231-MGMT-2 + 212 34 6.2
[0155] CFAs were also performed with the addition of the MGMT
inhibitor O6-BG. Cells were pre-treated with O6-BG for 60 minutes
before addition of TMZ-POH or TMZ. As shown in FIG. 23A, O6-BG had
no effect on the survival of drug-treated MDA-MB-231 cells,
consistent with their MGMT-negative status that does not provide a
target for O6-BG. In contrast, O6-BG greatly enhanced toxicity of
TMZ-POH and TMZ in 231-MGMT-1 (FIG. 23B) and 231-MGMT-2 cells (not
shown). Similarly, O6-BG also increased the cytotoxic outcome of
TMZ-POH and TMZ treatment in MGMT-positive MDA-MB-468 (FIG. 23C)
and MCF7 cells (not shown). Altogether, these results indicate that
the key trigger for cell death caused by TMZ-POH is methylation of
O6-guanine, which appears to be achieved much more effectively by
TMZ-POH as compared to TMZ.
[0156] The above conclusion was further confirmed by studying H2AX
protein. Phosphorylation of H2AX, noted as y-H2AX, is a marker for
double strand breaks in DNA. MDA-MB-231 cells treated with TMZ-POH
over a time course of 72 hours revealed substantially increased
levels of .gamma.-H2AX (FIG. 24A), and this effect of TMZ-POH was
much stronger as compared to TMZ (FIG. 24B). As well, the mere
combination of TMZ with POH was unable to mimic the strong
induction of y-H2AX caused by conjugated TMZ-POH (FIG. 24C),
consistent with the CFA results shown in FIG. 20 and the notion
that TMZ-POH represents a chemical entity different from the mix of
TMZ plus POH.
[0157] The same concentration of TMZ-POH that was applied to
MDA-MB-231 cells was also added to MGMT-positive MCF-7 cells.
However, in this case, there was no increased phosphorylation of
H2AX, consistent with the established model that MGMT rapidly
repairs O6-methyl-guanine lesions; however, when these cells were
pre-treated with O6-BG, increased levels of .gamma.-H2AX became
readily apparent (FIG. 24D). In sum, the above results indicate
TMZ-POH as an alkylating agent with cytotoxic mechanism similar to
TMZ, but with potency that is substantially greater than the
original compound.
[0158] It is known that GBM cells treated with physiological
concentrations of TMZ (<100 .mu.M) in vitro can survive for
several (5-7) days seemingly unaffected before substantial cell
death becomes apparent. We observed a similar phenotype when breast
cancer cell lines were treated with TMZ-POH, i.e., cell cultures
only began to deteriorate approximately a week after the onset of
drug treatment. In order to characterize TMZ-POH-induced cell death
in greater detail, we treated MDA-MB-231 cells with 15 .mu.M of the
drug and collected cell lysates daily over the course of 6 days.
The lysates were analyzed by Western blot for the presence of two
apoptosis markers, cleaved (i.e. activated) caspase 7 and cleaved
PARP-1 (poly ADP-ribose polymerase-1), along with the DNA damage
marker y-H2AX. As above, TMZ-POH treatment resulted in pronounced
increase in y-H2AX expression levels, which except for an
unexplained dip at 3 days continued to increase over time (FIG.
25A). Both active caspase 7 and cleaved PARP started to increase at
day 3 and remained elevated for several more days until day 6 (FIG.
25A), which is about the time when microscopic examination of
treated cells reveals increasing deterioration of the monolayer.
These results indicate that TMZ-POH-induced cell death, similar to
what has been reported for physiological concentrations of TMZ, is
a slow process and involves apoptotic mechanisms.
[0159] As shown in FIGS. 20A-20B above, an equimolar combination of
TMZ+POH was unable to achieve the same potency in blocking colony
survival as the TMZ-POH conjugate. Having established TMZ-POH's
impact on DNA damage and its activation of apoptosis, we next
determined whether TMZ-POH's superior effect would also be
reflected at the molecular level of these marker proteins. We
treated cells with the same concentration (20 .mu.M) of TMZ-POH,
TMZ, POH, or TMZ combined with POH (TMZ+POH), and analyzed the
induction of .gamma.-H2AX, activated caspase 7, and cleaved PARP.
As shown in FIG. 25B, all three indicator proteins were induced
quite prominently by TMZ-POH after 5 days of treatment, whereas TMZ
or TMZ+POH exerted noticeably weaker effects and POH alone was
inactive in these measurements. Thus, the results from the cell
survival assay (FIGS. 20A-20B) correlated closely with the effects
of these compounds on DNA damage and apoptosis markers (FIG. 25B),
and in all cases TMZ-POH clearly generated the strongest anticancer
impact.
[0160] TMZ is a prodrug, and it is well known that its activation
takes place spontaneously in aqueous solution at 37.degree. C.
(i.e., no cellular functions are required for this conversion). As
well, the half-lives of both prodrug and active product are fairly
short, where all cytotoxic triggers are set within the first few
hours of treatment. To evaluate whether TMZ-POH and TMZ differed in
their half-lives, we determined how quickly, and for how long, the
drugs exhibited cytotoxic activity in cell culture. First, we
exposed cells to variably short periods of drug treatment, washed
off the drug, and then continued to keep cells in medium without
drug to determine survival and colony-forming ability. For most of
these experiments, we used 15 .mu.M TMZ-POH and 30 .mu.M TMZ,
because these concentrations are approximately equipotent in the
>90% cytotoxicity range (when measured by CFAs and a drug
exposure time of 24 hours).
[0161] As shown in FIG. 26A (right two bars), exposure of cells to
15 .mu.M TMZ-POH or 30 .mu.M TMZ resulted in about 3% and 8% colony
survival, respectively, when drugs remained in the medium for 24
hours. Yet, despite TMZ-POH unfolding slightly more potency over
the course of 24 hours, TMZ displayed noticeably greater efficacy
when cells were exposed for shorter time periods. As shown in FIG.
26A, a one-hour exposure to TMZ reduced colony formation by
>50%, whereas during the same time period TMZ-POH reduced it by
only 20%; similarly, a two-hour exposure to TMZ had more than
double the cytotoxic impact (23% survival) than TMZ-POH (51%).
Thus, TMZ acted more quickly than TMZ-POH; it required only 4 hours
to exert maximum toxicity, whereas TMZ-POH had not yet reached its
maximum impact at this time point.
[0162] We next modified this experiment as follows. After cells had
been exposed to drug treatment for the specific times shown in FIG.
26A, we removed the medium containing the drug from the cells, and
transferred this supernatant to fresh cells, which were then
exposed for 24 hours. As shown in FIG. 26B (right two bars), when
supernatant was transferred after prior 24-hours of incubation, no
cytotoxic activity remained, i.e., there was no reduction in
colony-forming ability of the receiving cells. In contrast, when
supernatant was transferred after prior one-hour incubation,
colony-forming ability of receiving cells was 48% in cells
receiving TMZ-containing supernatant, and 22% in TMZ-POH-containing
supernatant. Even more strikingly, TMZ-containing supernatant had
lost all of its activity when transferred after 4 hours, whereas
TMZ-POH-containing supernatant still contained nearly 50% of its
cytotoxic activity (FIG. 26B). Together, these results demonstrate
that TMZ-POH retained its cytotoxic potency substantially longer
than TMZ.
[0163] To exclude the involvement of cellular enzymes in the
turnover of TMZ-POH, we incubated TMZ-POH (and TMZ) in
phosphate-buffered saline at 37.degree. C. for one hour (in the
absence of cells). After this pre-incubation, TMZ-POH and TMZ were
added to cells for 24 hours, and survival was determined by CFA. As
a control, both drugs were also added to cells without prior
incubation in aqueous solution. A representative CFA is shown in
FIG. 26C, where the middle panel confirms that both drugs were used
at approximately equipotent concentrations; i.e., when added
straight to cells, they reduced survival by .about.95%. However,
pre-incubation in aqueous solution for only one hour preempted the
cytotoxic potency of TMZ by about 50%, but that of TMZ-POH much
less (80% remaining; see right panel). These results establish that
TMZ-POH is more stable than TMZ, suggesting that its increased
potency over TMZ might be due to longer half-life, which may
provide for extended opportunity to inflict cytotoxic DNA
damage.
[0164] We also investigated whether TMZ-POH would be able to exert
its anticancer effects in vivo as well, and whether it would be
able to do so with a mouse tumor model representing breast cancer
spread to the brain. We used D3H2LN cells, which are a
bioluminescent variant of the MDA-MB-231 cell line with aggressive
tumor growth in mice. These cells were implanted into the brains of
nude mice, and 10 days later all animals were imaged for luciferase
expression in order to confirm efficient tumor take.
[0165] Animals were distributed into three groups and treated once
daily for 10 days with vehicle alone (control), 25 mg/kg TMZ-POH,
or 25 mg/kg TMZ. Another whole-body imaging after this 10-day
treatment period showed (FIG. 27A) that all vehicle-only treated
animals exhibited much increased bioluminescent radiance
(indicative of vigorous intracranial tumor growth), some of which
had conspicuously spread along the spine. Most of these animals
also exhibited behavioral signs of neurological problems and
reduced body weight, which necessitated euthanasia. In stark
contrast, all animals in the TMZ-POH-treated group seemed to
thrive, and their imaging analysis showed only small changes in
radiance (FIG. 27A). In comparison, all animals in the TMZ-treated
group showed clearly increasing bioluminescence over time,
indicating that tumor growth had continued throughout the 10-day
treatment period, and had begun to include the spine in some of the
animals. Overall, the TMZ-treated group seemed to have fared
somewhat better than the vehicle-treated group, but clearly worse
than the animals treated with TMZ-POH.
[0166] All animals were cared for and observed in the absence of
any further drug treatment. As summarized in FIG. 27B,
vehicle-treated animals were moribund by day 20 and had to be
euthanized within the following four days (median survival: 22
days). TMZ-treated animals survived somewhat longer (median
survival: 28 days). Remarkably, by day 36, when all TMZ-treated
animals had succumbed to disease, all TMZ-POH-treated animals were
still alive with no obvious signs of distress. Median survival of
TMZ-POH-treated animals turned out to be 50 days, i.e., they
survived an additional 30 after the termination of treatment, as
compared to TMZ-treated animals, which survived only an additional
8 days after treatment. Altogether, these results demonstrate
potent anticancer effects of TMZ-POH that are considerably stronger
than those of TMZ in vitro and in vivo.
Discussion
[0167] A landmark phase III trial completed 10 years ago
established a significant survival benefit for the alkylating agent
temozolomide when added to radiotherapy (plus surgery when
possible) for newly diagnosed glioblastoma. R. Stupp et al.,
Radiotherapy plus concomitant and adjuvant temozolomide for
glioblastoma. N Engl J Med 352 (2005) 987-996. TMZ prolonged median
survival from 12.1 to 14.6 months, and increased 5-year overall
survival 5-fold from 1.9 to 9.8%. R. Stupp et al., Effects of
radiotherapy with concomitant and adjuvant temozolomide versus
radiotherapy alone on survival in glioblastoma in a randomised
phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet
Oncol 10 (2009) 459-466. Altogether, these positive outcomes have
cemented TMZ plus radiotherapy as the current standard of care for
most patients with GBM. As would be expected, this approach was
also evaluated for activity against intracranial metastases
secondary to primary tumors of the lung, breast, and other
extracranial sites. However, the results of several phase II trials
in heavily pretreated patients were not impressive enough to
establish this regimen as a standard of care for instances of
metastatic spread to the brain from cancers such as breast
carcinoma. We therefore sought to create a novel analog of TMZ with
superior activity against brain metastases.
[0168] In the past, extensive molecular modeling studies of
antitumor imidazotetrazines including TMZ, showed that the initial
activating ring-opening reaction, involving nucleophilic addition
at C-4 of the tetrazinone ring, is not affected by bulky moieties
at C-8. J. Arrowsmith, S. A. Jennings, A. S. Clark, M. F. Stevens,
Antitumor imidazotetrazines, 41. Conjugation of the antitumor
agents mitozolomide and temozolomide to peptides and lexitropsins
bearing DNA major and minor groove-binding structural motifs, J Med
Chem 45 (2002) 5458-5470; A. S. Clark et al., Antitumor
imidazotetrazines. 32. Synthesis of novel imidazotetrazinones and
related bicyclic heterocycles to probe the mode of action of the
antitumor drug temozolomide, J Med Chem 38 (1995) 1493-1504; E.
Lunt et al. Antitumor imidazotetrazines. 14. Synthesis and
antitumor activity of 6- and 8-substituted
imidazo[5,1-d]-1,2,3,5-tetrazinones and 8-substituted
pyrazolo[5,1-d]-1,2,3,5-tetrazinones, J Med Chem 30 (1987) 357-366.
Therefore, irrespective of the nature of the targeting group
conjugated at C-8, the final step in the activation process
releases the electrophilic methyldiazonium ion that methylates
nucleophilic sites in DNA. Based on these earlier structural and
bioactivity studies, we expected that TMZ-POH would preserve the
release of the reactive methyldiazonium, and therefore that the
cytotoxic activity of TMZ-POH would involve DNA methylation,
similar to its parental molecule TMZ.
[0169] Our data are consistent with the above mechanistic model.
For instance, we show that the presence of MGMT, which highly
specifically repairs O6-methylguanine and provides profound
protection against TMZ, minimizes DNA damage caused by TMZ-POH
(FIG. 24D) and increases cellular resistance to this agent (FIG.
22B). Conversely, the presence of O6-BG, a specific inhibitor of
MGMT, substantially enhances DNA damage caused by TMZ-POH (FIG.
24D) and increases this agent's cytotoxic potency exclusively in
MGMT-positive cells (FIG. 23). As well, TMZ-POH treatment of cells
leads to a reduction in MGMT protein levels (FIG. 21C), which is a
well-established effect in the case of TMZ, due to the DNA repair
enzyme's "suicide" mechanism of action, whereby acceptance of the
alkyl group from 06-methylguanine leads to the protein's rapid
degradation.
[0170] While our data establish DNA alkylation by TMZ-POH as a key
mechanism by which this agent exerts its cytotoxic effect, we
cannot exclude the possibility that its POH moiety may contribute
additional functions. POH is known to affect several intracellular
processes. For instance, it has been shown to inhibit the activity
of telomerase and of sodium-potassium pump (Na+/K+-ATPase) [52;
53]. As well, it has been described as a farnesyl-transferase
inhibitor that results in the blockage of ras oncoprotein activity
(I. R. et al., Inhibition of protein prenylation by metabolites of
limonene. Biochem Pharmacol 57 (1999) 801-809; P. L. Crowell et
al., Selective inhibition of isoprenylation of 21-26-kDa proteins
by the anticarcinogen d-limonene and its metabolites. J Biol Chem
266 (1991) 17679-17685), although this has been challenged (J.
Karlson et al Inhibition of tumor cell growth by monoterpenes in
vitro: evidence of a Ras-independent mechanism of action,
Anticancer Drugs 7 (1996) 422-429; R. J. Ruch et al., Growth
inhibition of rat liver epithelial tumor cells by monoterpenes does
not involve Ras plasma membrane association, Carcinogenesis 15
(1994) 787-789.). Importantly, in all these cases relatively high
concentrations of POH (well above 100 .mu.M) are required to
achieve 50% inhibition of target activity (see also FIG. 20A). In
comparison, TMZ-POH is active in the range of 1-5 .mu.M in
MGMT-negative cells (Table 2). Notably as well, when POH is mixed
with TMZ and applied as a separate agent, this combination is
unable to replicate the high potency of conjugated TMZ-POH (FIGS.
20A-20B, 24C, 25B), indicating that the mere presence of
non-conjugated POH is unable to provide additional potency over
TZM. These considerations, combined with TMZ-POH's notable
sensitivity to MGMT and O6-BG as detailed above, diminish the
likelihood for involvement of functions other than DNA damage.
[0171] If conjugation of POH indeed does not provide additional
pro-apoptotic mechanisms over TMZ alone, why is TMZ-POH
significantly more potent than TMZ? It has been well established
that TMZ (and its active degradation product) exhibits rapid
turnover in vitro and in vivo, with a half-life in the range of 1-2
hours. Consistent with these characteristics, we find that after 4
hours of incubation in medium, nearly 100% of TMZ's cytotoxic
activity has been spent (FIGS. 26A-26C). In contrast, TMZ-POH
appears significantly longer-lived, where after 4 hours about 50%
activity remains (FIGS. 26A-26C). Thus, while not wishing to be
bound by any particular theory, we propose that the extended
presence of TMZ-POH may provide for greater opportunity to set DNA
lesions, resulting in increased cytotoxicity.
[0172] While the extended half-life of TMZ-POH may suffice to
explain its greater potency in vitro, it remains to be established
whether it also contributes to its substantially increased in vivo
potency in our brain metastasis model (FIGS. 27A-27B). Because the
lipophilicity of TMZ-POH is increased over TMZ (data not shown), it
is also possible that TMZ-POH may cross the BBB more efficiently
than TMZ. In the case of TMZ, it is known that drug levels achieved
in the cerebrospinal fluid (CSF) are 80% lower than drug levels in
the systemic circulation, i.e., in plasma. It is therefore
conceivable that TMZ, despite its established therapeutic benefit,
would exert even greater activity, if only higher intracranial
concentrations could be achieved. In this regard, TMZ-POH might be
the vehicle to achieve this.
[0173] It is quite intriguing that TMZ displayed only minor
activity in our intracranial in vivo model (FIGS. 27A-27B). The
breast cancer cell line we used, a variant of MDA-MB-231, does
exhibit exquisite in vitro sensitivity to TMZ (IC50<10 .mu.M),
and therefore is more sensitive to TMZ than most MGMT-negative GBM
cell lines reported in the literature and inclusive of several GBM
cell lines we analyzed in parallel (data not shown). As well, the
TMZ dosage used (25 mg/kg) is well within the range of dosages
shown to exert potent activity in GBM mouse models, where even 5
mg/kg has significant activity. T. C. Chen et al. Green tea
epigallocatechin gallate enhances therapeutic efficacy of
temozolomide in orthotopic mouse glioblastoma models, Cancer Lett
302 (2011) 100-108. We therefore speculate that this
triple-negative 231 cell line might harbor intrinsic mechanisms of
resistance to TMZ that emerge only in the in vivo environment, and
perhaps are reflective of the unimpressive responses that were
noted when breast cancer patients with brain metastases were
treated with TMZ. While this conjecture remains hypothetical at
this time, it is obvious from our studies that TMZ-POH provides far
superior therapeutic benefit than TMZ in our intracranial tumor
model (FIGS. 27A-27B), which may bode well for the clinical
setting.
[0174] The scope of the present invention is not limited by what
has been specifically shown and described hereinabove. Those
skilled in the art will recognize that there are suitable
alternatives to the depicted examples of materials, configurations,
constructions and dimensions. Numerous references, including
patents and various publications, are cited and discussed in the
description of this invention. The citation and discussion of such
references is provided merely to clarify the description of the
present invention and is not an admission that any reference is
prior art to the invention described herein. All references cited
and discussed in this specification are incorporated herein by
reference in their entirety. Variations, modifications and other
implementations of what is described herein will occur to those of
ordinary skill in the art without departing from the spirit and
scope of the invention. While certain embodiments of the present
invention have been shown and described, it will be obvious to
those skilled in the art that changes and modifications may be made
without departing from the spirit and scope of the invention. The
matter set forth in the foregoing description and accompanying
drawings is offered by way of illustration only and not as a
limitation.
* * * * *
References